Busca

... Brooks et al. BMC Medical Education (2018) 18:31 https://doi.org/10.1186/s12909-018-1136-z DATABASE Open Access A searchable database of medical education objectives creating a comparable gold standard Sam Brooks1, Namita Biala1 and Sage Arbor1,2* Abstract Background: Medical school curricula strives to teach as much material as can be retained in a limited amount of time. A common gold standard resource used building curricula are medical objectives suggested by national societies. Unfortunately these objectives suffer from several functional limitations such as limited accessibility to society members, non-searchable formats (such as nested tables or pdf images), and inability to compare and search across societal objectives for redundancy or gaps. The shift towards integrated curriculums in medical school also highlights the need to access suggested content across classical discipline categories. Main body: We have codified recommendations from national societies in the United States for medical school objectives in a common tabular format and developed an open access database which can be searched across disciplines and societies. A front end website that allows for searching objectives by keyword while filtering on society or discipline was created. The objectives returned from the initial search can be subsearched by a second term. There is a large range in the format, age, breadth, quantity, and quality of objectives from different societies. Some unique disciplines have overlapping suggested content though most of the content does seem binnable by discipline. The choice of metadata for objectives from each given society was also very inconsistent. Conclusion: A free and searchable database of medical content to deliver during medical school has been developed with over 13,000 objectives from 18 societies and 22 disciplines at http://data.medobjectives.marian. edu/. The normalization of the different disciplines objectives into a common database allows a platform to standardize objectives moving forward. Future work could include adding user accounts to access the database, submission of new objectives, voting up and down suggested objectives, and adding answers mapped to objectives. Keyword tagging could allow import of content (e.g. PowerPoints) and outputting of suggested objectives, which would also allow comparison of curriculum across medical schools. Keywords: Medical, Objectives, Learning, Topics, Database, Integrated, Curriculum, Curricula Background Medical school degree programs in different countries have durations that differ by multiple years but the final content of knowledge needed to be a doctor should be equivalent in depth and breadth. In the United States the scientific foundations to understand disease are generally taught in the first 2 years of medical school. Some * Correspondence: sagearbor@gmail.com 1 Marian University College of Medicine, 3200 Cold Spring Rd., Indianapolis, IN 36032, USA 2 Department of Biological Sciences, Marian University, College of Osteopathic Medicine, 3200 Cold Spring Rd, Indianapolis, IN 46222, USA of this information may have been presented to the students in undergraduate courses although specific prerequisites, such as biochemistry, vary by medical school in the United States. These first 2 years of medical school are usually the first time scientific knowledge is interwoven in a clinical context. Medical objectives recommended by national societies are often used for planning and following such a curriculum. However the accessibility and format of these recommended objectives often present barriers to their initial use and tracking. For example, objectives may only be accessible to society members behind a password protected website. In some cases the assembled objectives are only available as a The Author(s). 2018 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Brooks et al. BMC Medical Education (2018) 18:31 hardcopy handout from a national meeting. The database assembled for this paper encountered both of these issues (see construction section). Curriculum at medical schools is assembled by a group of content experts with deliverable content often categorized by discipline since the basic science faculty have discipline specific training. The ability to search across disciplines is needed to develop a high caliber integrated curriculum, and aids in exposing both gaps and overlaps in curricular design or delivery. Medical schools develop curricular mapping on their own, with their efficacy largely being judged based on post-graduation national exam board scores. In the United States there are currently two board tests USMLE (United States Medical Licensing Examination) and COMLEX (Comprehensive Osteopathic Medical Licensing Examination) required for allopathic and osteopathic medical students respectively, though there is significant crossover test taking as well. Both board review books (e.g. First Aid, Kaplan) and American national societies categorize the knowledge needed for these board tests by discipline. The frequency with which the societies update their suggested objective coverage varies widely, ranging from roughly every 3 to 20 years. The private companies update their review materials much more frequently, likely because the revisions are less exhaustive and there is a profit motive to do so. Other parts of the world have had similar efforts, the best example the authors are aware of concerning interschool objective mapping being the National Competence Based Catalogues of Learning Objectives for Undergraduate Medical Education (NKLM) and Dental Education (NKLZ) by the Association of Medical Faculties in Germany (MFT) which used as a starting point work done earlier by Switzerland, Canada, and the Netherlands [1]. The NKLM was not used in any way to make the current database, however it is worth comparing both the size and utility of the datasets. The NKLM had 1956 learning objectives in German compared to 13,121 objectives that are viewable in 103 languages in the current data set. The content of the database is derived from scientific societies based in the United States targeting medical students in the 4 year United States programs. In the United States medical school system there is usually a more rigorous structure during the first two largely didactic years, which is when content is tightly tied to objectives, and hence most of the database covers this medical education content. The latter 2 years of education in this system still has objectives covered in the database but they are less numerous and more broad. Therefore, while the database should prove useful in other systems the year of education the objectives will map to will be different. For example, in Europe medical school is a longer program (6 years) starting in Page 2 of 8 undergraduate years and can begin directly out of high school, therefore many of the objectives in this database may be too detailed for early years of a European education, or could be split up over more time. The database does represent the final amount of knowledge that should be known/delivered before a student attains a medical degree. In addition, the database described in this paper represents the first time objectives suggested for coverage by national societies in the United States has been aggregated in one place and one format, and should provide a platform for continued submission of updated objectives for medical schools. Construction and content Suggested medical school objectives were aggregated from disparate national societies in the United States of America (Table 1) [221]. The list of disciplines to cover were derived from discipline lists from the two types of medical schools in the United States, both allopathic [22] and osteopathic [23]. Of note osteopathic manipulation specific objectives, which would not be used in an allopathic school, were not included in the database by design since the purpose of the database was to create a resource that covered the agreed upon content necessary to master in order to become a doctor. In most cases a national society was found which represented a single discipline and had put forth material that it thought should be taught in the first 2 years of medical schools in the United States. There were two societies that covered more than one discipline in one document. The Association of Anatomy, Cell Biology, and Neurobiology Chairpersons (AACBNC) covered five disciplines: anatomy, cell biology, embryology, histology, and neuroscience [2]. The International Association of Medical Science Educators (IAMSE) covered both microbiology and immunology also in a single document [15]. On the other hand the Association of American Medical Colleges (AAMC) covered behavioral sciences [6] and clinical medicine [7], but at different times and in different documents. In addition, some societies, such as the Clerkship Directors in Internal Medicine and Society of General Internal Medicine (CDIMSHIM) [14], have content that is usually covered in years 3 and 4 of medical schools in the United States, during clinical rotations. Most of the objectives were available online in some format, usually pdf. The Association of Biochemistry Educators (ABE), formerly known as the Association of Biochemistry Course Directors (ABCD), had their objectives behind a password protected website and agreed to open up access for inclusion in this database as well as now making them freely accessible on their website [5]. A digital pdf copy had been created for the Association of Anatomy, Cell Biology, and Neurobiology Chairpersons (AACBNC), which at one point had floated around Brooks et al. BMC Medical Education (2018) 18:31 Page 3 of 8 Table 1 Number of objectives in the database from each Society (sorted by discipline) Discipline Society Abbreviation Society Year Anatomy AACBNC [2] Association of Anatomy, Cell Biology, and Neurobiology Chairpersons 1997 Anesthesia SEA [3] Society for Education of Anesthesia 2009 Behavioral Sciences ADMSEP [4] Association of Directors of Medical Student Education in Psychiatry 2007 Biochemistry ABE [5] Association of Biochemistry Educators 2012 Behavioral Sciences AAMC [6] Association of American Medical Colleges 2011 Cell Biology AACBNC [2] Association of Anatomy, Cell Biology, and Neurobiology Chairpersons 1997 Clinical Medicine AAMC [7] Association of American Medical Colleges 2008 Embryology AACBNC [2] Association of Anatomy, Cell Biology, and Neurobiology Chairpersons 1997 Family Medicine STFM [8] Society of Teachers of Family Medicine 2012 Genetics APGMH [9] Association of Professors of Human and Medical Genetics 2013 Genetics ACMG [25] American College of Medical Genetics 2011 Gerontology SGE [12] Studies in Geriatric Education 1986 Gerontology AGHE [11] Association for Gerontology in Higher Education 2008 Hematology ASH [13] American Society of Hematology 2014 Histology AACBNC [2] Association of Anatomy, Cell Biology, and Neurobiology Chairpersons 1997 Internal Medicine CDIMSHIM [14] Clerkship Directors in Internal Medicine and Society of General Internal Medicine 2006 Micro/Immuno IAMSE [15] International Association of Medical Science Educators 2009 Neuroscience AACBNC [2] Association of Anatomy, Cell Biology, and Neurobiology Chairpersons 1997 Obstetrics & Gynecology APGO [16] Association of Professors of Gynecology & Obstetrics 2009 Pathology GRIPE [17] Group for Research In Pathology Education 2005 Pediatrics COMSEP [18] Council on Medical Student Education in Pediatrics 2005 Pharmacology ASPET [19] American Society for Pharmacology and Experimental Therapeutics 2012 Physiology APSACDP [20] The American Physiological Society and the Association of Chairs of Departments of Physiology 2012 Radiology AUR [21] Association of University Radiologists 2014 to various schools, but the objective text had been truncated in various text boxes. An original contributor to the objectives was eventually tracked down and one of two remaining hard copies was sent to be re-digitized and put in database format. The objectives from disparate societies were tagged with largely non-overlapping metadata about each objective. An attempt was made to capture all metadata and include it in the database for retrieval. An excel template was created with 32 fields to allow others to populate with new objectives for import into the database moving forward (Additional file 1: Supplamental1 medicalObjectiveUpload.xls, and available on the website help page). The excel template has the fields listed in Table 2, which cover the types of metadata found with the objectives along with their frequency. Data was input into a MySQL database with a webpage set up for retrieval (http://data.medObjectives.marian.edu). A script to create the database with all data can be found in the supplemental data (Additional file 2: SupplementalDigitalContent2medSchlObj-sqlCreate.txt). While the excel allows a flat file format to import new objectives, that data is split into multiple tables for querying in the database. The 3 currently used tables(columns) are: 1. objectives (see Table 2). 2. disciplines (discipline, displayName, inDB) 3. societies (name, abbrev, approvalLevel) Most fields require minimal space being stored as varchar < 256 with the exceptions in the objectives table being objective = varchar(2048), objectives notes = text, objective answer = text. These 3 fields allow longer objectives, and much longer notes and answers to the objectives. The database was developed and deployed on a HP ProLiant DL370 G6 blade server with 8 CPUs (Intel Xeon X5550 @ 2.67 GHz). A virtual machine on this server running Ubuntu 14.01 Linux houses the MySQL backend database, Apache server, as well as the PHP front end webpages. When aggregating information, such as this medical objective database has done, it is ideal if none of the content has to be modified by third parties. However, Brooks et al. BMC Medical Education (2018) 18:31 Table 2 Importable fields for future objectives (headings in Additional file 1) Field Number(s) Field Current # of objectives with this data (%) 1 Author / Society Abbreviation 13,121 (100) 2 Year 13,121 (100) 3 Objective 13,121 (100) 4 Subheading 1 13,121 (100) 5 Subheading 2 12,787 (97.5) 6 Subheading 3 7555 (57.6) 7 Subheading 4 2984 (22.7) 8 Subheading 5 95 (0.7) 9 Subheading 6 0 (0) 10 Subheading 7 0 (0) 1125 Keyword 1 thru 15 0 (0) 26 Notes 7102 (54.1) 27 Rank 1609 (12.2) 28 Hours 1214 (9.2) 29 Answer to objective 41 (0.3) 30 Discipline 1 13,121 (100) 31 Discipline 2 874 (6.7) 32 Discipline 3 0 (0) since the objective database was designed to list objectives as at least one full sentence some decisions had to be made by the authors to make the objectives understandable. Examples of how this was done include converting tabular pharmacology objectives or explicitly restating content that was cross-referenced in societal objectives. The most troublesome objectives to incorporate were the pharmacology objectives from the American Society for Pharmacology and Experimental Therapeutics (ASPET) [19]. While there were many ASPET objectives that were clear in sentence form, there were often pharmacological substances whose names were only listed in nested tabular form. Therefore, many of these ASPET objectives had the pharmacological agents listed with the authors of this paper choosing to add the following constant phrase to end the objective: describe the mechanism(s) of action, use(s), adverse effect(s), contraindication(s), and any relevant pharmacodynamic(s). For example, because groups of drugs below were in two columns of a table labeled either Nonselective Alpha Adrenergic Agonists or Selective Alpha2 Adrenergic Agonists, the following two ASPET objectives were made from one table with different subheadings:  For the drugs DOPAMINE, EPINEPHRINE, NOREPINEPHRINE, phenylephrine, and pseudoephedrine as they pertain to synaptic and Page 4 of 8 neuroeffector junctional sites - describe the mechanism(s) of action, use(s), adverse effect(s), contraindication(s), and any relevant pharmacodynamic(s).  For the drugs BRIMONIDINE, CLONIDINE, and METHYLDOPA as they pertain to synaptic and neuroeffector junctional sites - describe the mechanism(s) of action, use(s), adverse effect(s), contraindication(s), and any relevant pharmacodynamic(s). In all cases authors chose to not modify the suggested objectives if possible, with the rare exception being fixing clear spelling errors. Student researchers compiled the database in a google doc flat file format, for ease of assembly, before import into the database. There was no data pre-processing/optimization before import into the database. Capitalization and punctuation was left unchanged, though search functionality was purposefully made not case sensitive. All objectives have at a minimum an author (society), year, one subheading, and one discipline (Table 2). Fields that are listed which have zero occurrences were added to the database to allow for future growth when new objectives are added. Namely, up to 15 keywords are allowed which could allow for computational searching of objectives from non-human input sources (such as Power Points). An initial schema for the objective database allowed for unlimited keywords in a separate tall MySQL table, however it was decided limiting the keywords to 15 was acceptable to allow the ability to combine the tables in a flat format and allow future contributions via an excel file. Other non-existing classifications were considered such as: Blooms Taxonomy, clinical verse basic science knowledge, usual year of edification, and quality metric of the objective; however it was decided such fields would be better determined by the community in a future iteration of the database which could allow for user defined field creation. The most common form of metadata across societies was a top level subheading, which multiple objectives were grouped under. The notes section was also extensively used, often containing paragraph text from a heading above multiple objectives which applied to all objectives in a set. Utility and discussion For the first time a searchable database combining suggested medical objectives from societies in the United States across most disciplines has been created and made publically available. The search term must be in English, but there is a dropdown to convert all text to other languages which should make this tool useful outside of English speaking countries. This translation Brooks et al. BMC Medical Education (2018) 18:31 dropdown is available regardless of device use (desktop, tablet, phone) on the front search page, the results page, and the help page. Currently the pages can be translated into 103 languages. The number of objectives societies from disparate disciplines suggested be taught differed by roughly 100-fold with a minimum of 33 and maximum of 2852 objectives (Fig. 1). This content volume difference was largely due to a large spectrum in the specificity of objectives. In general, the basic science objectives seemed more discrete than the broader clinical objectives. For example, the 33 objectives from the Association for Gerontology in Higher Education were often broad such as Compassion and understanding attitude on the part of the physician for care givers of the frail elderly and the difficulties they face [24] whereas the Association of Biochemistry Educators had more numerous pointed objectives such as Summarize the mechanism of DNA replication and why discontinuous synthesis is required [5]. However a significant amount of the difference in the suggested number of objectives appeared due to societies differing in the depth with which they felt their content should be taught. Weighing the importance of the different disciplines content in order to assign medical students time is often hard to do when trying to compare siloed disciplines. The shared database provides transparency to disciplines requests of students time. A few disciplines were covered redundantly, by separate societies, and their suggested content also varied widely, at least 34 fold (Fig. 1). The two gerontology societies suggested 33 [24] and 118 [12] Page 5 of 8 objectives, the two genetics societies suggested 103 [9] and 291 [25] objectives, and the two behavioral sciences societies suggested 18 and 185 objectives. There are various use cases for such a medical objective database which can mostly be categorized by a single lecturers use versus a medical school using across their curriculum. An individual lecturer could use the database to discover gaps in their content as well as extraneous information they may be including. At the most simple level the database has already proven a useful tool for clinicians that lecture at Marian University but are not full time faculty. Since external clinicians often come in to give 14 lectures, but are not career educators, they are often not versed in how to make a good objective, and could use the database to quickly search words and then copy and paste objectives related to their lecture content in their PowerPoints. Having these pre-vetted objectives has also seemed to keep external lecturers on message and guide external clinicians in the design of their PowerPoints and lectures, because once the objective is listed in their second slide they need to make sure they cover that content in the remaining slides. Up to 100 objectives from a single search result can be copy and pasted into excel or a PowerPoint with two clicks (along with associated discipline, year, society, subheadings, and notes). A faculty member could quickly find objectives they likely should cover content for based on keyword searches but had not initially had in their PowerPoints, which often occurs when they are teaching about a disease state Fig. 1 Number of Objectives by Discipline and Society. The number of suggested objectives per discipline varied by more than 100 fold (18 to 2861), and even within a single discipline varied up to 10 fold. Disciplines which only had objectives suggested by one society are shown by blue bars and are less likely to have redundant content suggested. Disciplines which had objectives listed redundantly in the database by more than one society are colored as follows: orange = behavioral sciences, red = genetics, and green = gerontology Brooks et al. BMC Medical Education (2018) 18:31 which has manifestations in a different discipline they did not understand well. A lecturers keyword search could also fail to retrieve many, or any, objectives which could hint that the content being delivered contains minutia that is likely beyond what the a new medical student needs to hear, or likely will be able to recall. While the database has been leaked to a few colleagues at other schools during development, it has mainly been used at Marian Universitys College of Osteopathic Medicine (COM), and mainly in the courses the developing faculty member (a biochemist) lectured in. Objectives have been searched for an initial foundational scientific knowledge course and biological system courses that followed: cardiac, pulmonary, renal, neuroscience, ear eyes nose and throat, dermatology, immunology, gastrointestinal, endocrine, metabolism, reproduction, and psychiatry. As a case example the psychiatry/behavioral course at Marian University was particularly heavy with external lecturers in the spring of 2017 and the database was used extensively. For that course all content (every sentence) from the psychiatry section of the First Aid board review book was mapped to a specific lecture in the course, and then much of that content had a corresponding objective from the database mapped to it. External lecturers were given all the content they were expected to deliver as well as suggested objectives. There were multiple changes made from the prior year, such as different lectures giving content, so a causal improvement due to the databases use cannot be made. Yet it seems worthy to note that in the first step board test (COMLEX step 1) taken shortly after this psychiatry/behavioral course the Marian University COM scores on the behavioral section improved from 460 to 602 in the early summer of 2016 and 2017 respectively (the national averages were 506 and 588 in 2016 and 2017 respectively). This represents a move from 0.25 standard deviations below the national average to 0.08 standard deviations above the national average and was that years greatest improvement in any discipline compared to the previous years class. The medical objective database would likely yield greater results if adopted by an entire medical school. A medical school could decide they want to cover all the objectives in the database, or a subset thereof. Discipline specific societies likely often believe their content is more important to a medical degree than others would agree, so it is likely a subset of the database would be the desirable content to deliver in a medical education. Either way the database would then provide a checklist to go through and link which objectives are presented to students in each lecture. A first pass at this would expose gaps in the curriculum, i.e. objectives that should be covered but had not been assigned to a lecture. Redundancies would also be found, where multiple lecturers were repeating the same content. Redundancy is sometimes desired in medical school to hammer home Page 6 of 8 certain content but, in the authors view, is more often an unintended consequence of each lecturer wanting to make their lecture self-contained enough to ensure a student can comprehend the content they are delivering. Conclusion A free database has been designed which can search across disciplines and societies for medically relevant objectives (http://data.medObjectives.marian.edu). This single database provides transparency to compare between societies and disciplines, showing sometimes an order of magnitude difference in the number of objectives suggested to be covered during medical school for a given discipline (Fig. 1). Some of that discrepancy is due to the verbiage of disciplines covering a different level of scope on a topic, but there is also just a difference in coverage between suggested objectives. Nesting objectives under a hierarchical tree-of-knowledge, such as MeSH terms, could provide a metric for the scope of objective and content delivery. The transparency yielded by having a single resource for medical objectives should help shed light on which disciplines have suggested less rigorous objectives. The aggregation of content not initially designed to be unified did present challenges. Most obvious were the nested pharmacological tabular objectives which did not have a clear way to translate themselves into didactic sentence format. An excel template (Additional file 1: Supplamental1medicalObjectiveUpload.xls), also available on the help page of the website, provides a format for societies to add to or update their objectives. This standardized format to contribute future objectives should, if used, help normalize content across disciplines and possibly increase associated metadata content. For example, societies could choose to adopt metadata others have incorporated, such as suggested content hours per objective or including answers to objectives. Future work should include development of user accounts and addition of keywords, synonyms, and other datasets. It is envisioned that uploading of a schools curriculum (e.g. a zip file of all Power Points), should allow automatic mapping to added keywords, resulting in a report (lecture by lecture) of which objectives should be covered. This would also allow for comparing of medical curriculum between schools, for example using board score data to measure the benefit or detriment of a curriculum spending time repeating content over new content delivery. Additional files Additional file 1 An excel file with standardized format to submit new objectives to the database. (XLSX 25 kb) Additional file 2 This file can be bulk uploaded into a blank mysql database to recreate this medical objective database with all content. (TXT 13173 kb) Brooks et al. BMC Medical Education (2018) 18:31 Abbreviations AACBNC : Association of Anatomy Cell Biology and Neurobiology Chairpersons; AAMC: Association of American Medical Colleges; ABCD: Association of Biochemistry Course Directors; ABE: Association of Biochemistry Educators; ADMSEP: Association of Directors of Medical Student Education in Psychiatry; AGHE: Association for Gerontology in Higher Education; ASH: American Society of Hematology; ASPET: American Society for Pharmacology and Experimental Therapeutics; CDIMSHIM: Clerkship Directors in Internal Medicine and Society of General Internal Medicine; COM: College of Osteopathic Medicine; COMLEX: Comprehensive Osteopathic Medical Licensing Examination; CPU: Computer Processing Unit; IAMSE: International Association of Medical Science Educators; MFT: Association of Medical Faculties in Germany; NKLM: National Competence Based Catalogues of Learning Objectives for Undergraduate Medical Education; NKLZ: National Competence Based Catalogues of Learning Objectives for Dental Education; SEA: Society for Education of Anesthesia; SQL: Structured Query Language; USMLE: United States Medical Licensing Examination Acknowledgements The authors would like to thank all the societies listed in Table 1 for developing lists of objectives they think are relevant for their given disciplines. Particular thanks to William Rhoten for sending one of the last hardcopies of AACBNCs suggested objectives. Funding There is no funding to report for this project. Availability of data and materials All data is available at http://data.medObjectives.marian.edu, not only in a searchable format but also as a downloadable flat excel file. In addition there is a script to recreate the MySQL database with all the data populated. All data can also be found as supplemental material to this paper. Authors contributions Sage Arbor (SA), Sam Brooks (SB), Namita Biala (NB). All authors (SA, SB, and NB) have read and approved the authors guidelines. All authors have made substantial contributions to the design or acquisition, drafting, approval of the final version, and agreed to be accountable for the accuracy and integrity of the work. SA was involved in all aspects of this work. He designed and built the objective database and front end webpage. He contacted the national societies to gather and aggregate the data, and also input ~ 1/3rd of the data into the database. SB input data into the database, as well as helping define normalized procedures for deciding what level of word documents to include in the objectives or subheadings. SB also initiated and partook in conversations on nesting levels of headings and subheadings when needed, and input the subheading data in the database afterwards. NB input data into the database, as well as helping define normalized procedures for deciding what level of word documents to include in the objectives or subheadings. NB also initiated and partook in conversations on nesting levels of headings and subheadings when needed, and input the subheading data in the database afterwards. NB also started preliminary work of tagging objectives with keywords but that process is not finished, or published in this paper. Authors information Correspondence should be addressed to Sage Arbor, Department of Biological Sciences, Marian University, College of Osteopathic Medicine, 3200 Cold Spring Rd., Indianapolis, IN 46222; telephone: (317) 9556268; e-mail: sarbor@marian.edu. Dr. Arbor is assistant professor, Department of Biomedical Sciences, Marian University College of Osteopathic Medicine, Indianapolis Indiana. Sam Brooks and Namita Biala are 3rd year medical students, Marian University College of Osteopathic Medicine, Indianapolis Indiana. Ethics approval and consent to participate Not applicable. Consent for publication The objectives used in the database for this article were either attained from publications under a creative commons license or we directly attained approval from the societies which had published the objectives. The only Page 7 of 8 password protected source, from American Biochemistry Educators (ABE), was made public at the request of the author and in conjunction with inclusion in this database. Competing interests The authors declare that they have no competing interests. Publishers Note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Received: 23 January 2017 Accepted: 21 February 2018 References 1. Fischer MR, Bauer D, Mohn K. Finally finished! National Competence Based Catalogues of learning objectives for undergraduate medical education (NKLM) and dental education (NKLZ) ready for trial. GMS Z Fr Med Ausbild. 2015;32 https://doi.org/10.3205/zma000977. 2. Ralston P, Carmichael S, Goodman S, McCandless D, Bressler B. United States/Canadian Curriculum Content in the Anatomical Sciences. AACBNC Association of Anatomy, Cell Biology, and Neurobiology Chairpersons; 1997. 3. Anesthesia S for E in. Anesthesia Curriculum For Medical Student Learning and Evaluation. 2009. http://www.seahq.net/images/stories/sea%20selfdirected%20learning%20cards.pdf. 4. Burke M, Campbell C, Dunstone D, Lamdan R, Levine R, Roman B, et al. Clinical Learning Objectives Guide for Psychiatry Education of Medical Students (ADMSEP). 2007. http://www.admsep.org/pdf/templates/Clinical_ LO_Guide_Outline_Format_Nov_07.pdf. Accessed 7 Dec 2015. 5. Biochemistry Learning Objectives & Competencies: Created & Approved at the 3rd International Conference of the Association of Biochemistry Educators (ABE) [Formerly ABCD]. Association of Biochemistry Course Directors - Objectives. 2012. http://www.abe.wildapricot.org/page-577042. Accessed 26 Sep 2014. 6. American Medical Colleges A of. Behavioral and Social Science Foundations for Future Physicians. 2011. https://www.aamc.org/download/271020/data/ behavioralandsocialsciencefoundationsforfuturephysicians.pdf. 7. Corbett E, Berkow R, Bernstein L, Dyrbye L, Fields S, Gusic M, et al. Recommendations for Preclerkship clinical skills education for undergraduate medical education - AAMC initiative. 2008. https://www. aamc.org/download/130608/data/clinicalskills_oct09.qxd.pdf.pdf. Accessed 7 Dec 2015. 8. Chumley H, Chessman A, Hobbs J, Clements D, Munzing T, Cochella S, et al. STFM UNVEILS the National Family Medicine Clerkship Curriculum Website. Ann Fam Med. 2012;10:2756. 9. Hyland L, Dasgupta S, Garber K, Gold J-A, Toriello H, Weissbecker K, et al. Medical School Core Curriculum in Genetics 2013 : APHMG Genetics Competencies Working Group. The Genetics/Genomics Competency Center for Education provides links to curricular materials and resources for educators of Genetic Counselors, Nurses, Pharmacists, Physician Assistants, and Physicians. 2013. http://media.wix.com/ugd/3a7b87_ 7064376a9eb346cfa1b85bc2f137c48f.pdf. Accessed 7 Dec 2015. 10. Korf BR, Irons M, Watson MS. Competencies for the physician medical geneticist in the 21st century. Genet Med. 2011;13:9112. 11. Gugliucci MR, Giovanis AT. Osteopathic medicine and the silver tsunami: preparing tomorrows first responders for the elder boom. J Am Osteopath Assoc. 2009;109:4814. 12. Robbins AS, Fink A, Kosecoff J, Vivell S, Beck JC. Studies in geriatric education: I. Developing educational objectives. J Am Geriatr Soc. 1982; 30:2818. 13. Medical Student Hematology Course Learning Objectives. 2014. http://www. hematology.org/Educators/Learning-Objectives/. Accessed 7 Dec 2015. 14. De Fer T, Fazio S, Goroll A, Friedman E, Henry D, Kuzma MA, et al. Core Medicine Clerkship Curriculum Guide - A Research for Teachers and Learners - version 3.0. http://internalmedicine.med.sc.edu/students/docs/ CDIMCurriculum.pdf. 15. Booth SJ, Burges G, Justement L, Knoop F. Design and implementation of core knowledge objectives for medical microbiology and immunology. 2009. http://www.unmc.edu/pathology/faculty/bios/amsmic_special_ communication.pdf. Accessed 7 Dec 2015. Brooks et al. BMC Medical Education (2018) 18:31 Page 8 of 8 16. Hammoud M, Casey P, Chuang A, Cullimore A, Dalrymple J, Dugoff L. APGO Medical Student Educational Objectives 9th Edition. 2009. https://www. apgo.org/educational-resources/. 17. Geiss R, Crawford B, Teja K. General Pathology Objectives and Systemic Pathology Objectives. Okla City OK Group Res Pathol Educ. 2005;:64. 18. Raszka W. COMSEP - Excellence in Medical Student Education in Pediatrics : Task Forces. https://www.comsep.org/curriculum/CurriculumCompetencies/. Accessed 10 Jun 2016. 19. Eisenberg R, Faingold C. Knowledge objectives in medical Pharmacology 2012. https://www.aspet.org/aspet/membership-community/divisions/ division-for-pharmacology-education-(dpe). 20. Carroll R, Navar L, Mordecai B. American Physiological Society > APS/ACDP Medical Physiology Learning Objectives Project. http://www.the-aps.org/ MedPhysObj.aspx. Accessed 28 Aug 2014. 21. Association of University. Learning Objectives for a Medical School Radiology Curriculum - Listed by Subjects. 2014. https://www.aur.org/uploadedFiles/ Alliances/AMSER/AMSER-Learning-Objectives-Listed-by-Subject(1).pdf. 22. Subject Examination Content Outlines | NBME. http://www.nbme.org/ schools/subject-exams/subjects/exams.html. Accessed 12 Apr 2017. 23. Wu PW, Siu J. A Brief Guide to Osteopathic Medicine- For Students- By Students http://www.aacom.org/news-and-events/publications/a-brief-guide-toosteopathic-medicine-for-students-by-students. Accessed 12 Apr 2017. 24. Standards and Guidelines for Gerontology and Geriatrics Programs. 2008 Update Association for Gerontology in Higher Education.pdf. http://www. aghe.org/. Accessed 29 June 2015. 25. Korf BR, Irons M, Watson MS. Competencies for the physician medical geneticist in the 21st century. Genet Med Off J Am Coll Med Genet. 2011; 13:9112. Submit your next manuscript to BioMed Central and we will help you at every step: We accept pre-submission inquiries Our selector tool helps you to find the most relevant journal We provide round the clock customer support Convenient online submission Thorough peer review Inclusion in PubMed and all major indexing services Maximum visibility for your research Submit your manuscript at www.biomedcentral.com/submit  ...

... (2019) 12:331 Gorrell et al. BMC Res Notes https://doi.org/10.1186/s13104-019-4367-0 BMC Research Notes Open Access RESEARCH NOTE Identification of a bone morphogenetic protein type 2 receptor neutralizing antibody Ruthann E. Gorrell, Madeline H. Totten, Laura J. Schoerning, Jordan B. Newby, Logan J. Geyman, Warren G. Lawless, Julia M. Hum and Jonathan W. Lowery* Abstract Objective: The bone morphogenetic protein (BMP) signaling pathway comprises the largest subdivision of the transforming growth factor (TGF) superfamily. BMP signaling plays essential roles in both embryonic development and postnatal tissue homeostasis. Dysregulated BMP signaling underlies human pathologies ranging from pulmonary arterial hypertension to heterotopic ossification. Thus, understanding the basic mechanisms and regulation of BMP signaling may yield translational opportunities. Unfortunately, limited tools are available to evaluate this pathway, and genetic approaches are frequently confounded by developmental requirements or ability of pathway components to compensate for one another. Specific inhibitors for type 2 receptors are poorly represented. Thus, we sought to identify and validate an antibody that neutralizes the ligand-binding function of BMP receptor type 2 (BMPR2) extracellular domain (ECD). Results: Using a modified, cell-free immunoprecipitation assay, we examined the neutralizing ability of the mouse monoclonal antibody 3F6 and found a dose-dependent inhibition of BMPR2-ECD ligand-binding. Consistent with this, 3F6 blocks endogenous BMPR2 function in the BMP-responsive cell line HEK293T. The specificity of 3F6 action was confirmed by demonstrating that this antibody has no effect on BMP-responsiveness in HEK293T cells in which BMPR2 expression is knocked-down. Our results provide important proof-of-concept data for future studies interrogating BMPR2 function. Keywords: Bone morphogenetic protein, Bone morphogenetic protein receptor type 2, Neutralizing antibody, BMP, BMPR2, SMAD Introduction The TGF- superfamily is a group of pleiotropic cytokines and their receptors that contribute to metazoan cellular development and regulation [1]. Extracellular dimeric ligands bind to transmembrane serine/threonine kinase receptor complexes, bringing together two type 1 and two type 2 receptors, in order to activate a group of effectors called SMAD proteins [13]. The largest subdivision of this superfamily is comprised of the bone morphogenetic proteins (BMPs), which activate SMADs 1, 5, and 8 and play an essential role in embryonic development and *Correspondence: jlowery@marian.edu Ruthann E. Gorrell and Madeline H. Totten contributed equally to this work Division of Biomedical Science, Marian University College of Osteopathic Medicine, 3200 Cold Spring Rd, Indianapolis, IN 46222, USA in postnatal tissue homeostasis [4]. Moreover, dysregulated BMP signaling underlies numerous human pathologies ranging from pulmonary arterial hypertension to heterotopic ossification [5]. Thus, understanding of the basic mechanisms by which BMP signaling occurs and is regulated is a highly important goal and may yield translational opportunities. Functional studies evaluating BMP pathway mechanics, such as global or conditional genetic knockout of specific components, have been complicated by developmental requirements and/or the ability of pathway components to compensate for one another [4]. Additionally, the translational potential of these strategies is questionable due to ethical concerns and technical limitations [5]. This has led to the development of several pharmacologicallybased strategies, including decoy receptors and small The Author(s) 2019. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/ publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Gorrell et al. BMC Res Notes (2019) 12:331 molecule inhibitors, for non-genetic based modulation of BMP pathway activity [6]. That said, the repertoire of these molecules remains limited and, in particular, few tools exist for inhibiting the function of type 2 BMP receptors, the activity of which are essential for initiating signal transduction. In this study, we sought to identify and validate a commercially available antibody that neutralizes the ligand-binding function of bone morphogenetic protein receptor type 2 (BMPR2), which is essential for embryogenesis and has been shown to play clinically-relevant roles in pulmonary vascular homeostasis and remodeling of the postnatal skeleton [5]. We developed a modified, cell-free immunoprecipitation assay quantified by ELISA and found that the mouse monoclonal antibody 3F6 inhibits ligand-binding by BMPR2 in a dose-dependent manner. Additionally, using a BMP-responsive cell line we found that pre-treatment with 3F6 leads to reduced sensitivity in response to BMP pathway activation by BMP2. These results provide important proof-of-concept data for future studies interrogating BMPR2 function in numerous physiological and pathophysiological contexts. Main text Materials and methods Modified immunoprecipitation assay and ELISA Ligand-binding by BMPR2-ECD was performed as previously described [7] with the modifications detailed below. BMPR2-ECD/Fc fusion (Sino Biologicals 10551-H03H) was mixed with 5 L Protein G-coupled Dynabeads (Invitrogen 1003D) at room temperature for 30 min in 200 L total volume with gentle rocking. The loaded beads were then washed twice with PBS and resuspended in 135 L PBS 3F6 (Thermo Fisher Scientific Cat# MA5-15827) or control ascites (Sigma M8273) and mixed for 1 h at room temperature with gentle rocking. 800 ng recombinant human BMP2 (R&D Systems 892143) was added to the beads and incubated overnight at 4 C with gentle rocking. The supernatant was removed and examined using a Quantikine Human BMP2 Immunoassay ELISA (R&D Systems DBP200) according to the manufacturers instructions. Cellbased BMP2responsiveness assay HEK293T cells, obtained from ATCC, were cultured in DMEM supplemented with 10% fetal bovine serum (FBS) (Gibco), hereafter referred to as 10% DMEM, and grown at 37 C in 5% CO2. Cells were not tested for mycoplasma contamination. For BMP2-responsiveness assays, cells were passaged at 1 million per well in 6-well dishes in 10% DMEM on day 0; on day 1 the medium was exchanged to DMEM containing 0.5% FBS. After approximately 24 h of serum restriction, select wells were Page 2 of 6 treated with 250 ng/mL 3F6 or control ascites for 30 min followed by 100 ng/mL rhBMP2 for 4 h. Knockdown of BMPR2 expression 1.6 104 cells in 100 L 10% DMEM were added to wells of a 96-well plate. The cells were incubated overnight at 37 C in a humidified incubator in an atmosphere of 5% CO2. After 1820 h, the media was aspirated from the wells. Next, 110 L of 10% DMEM containing 8 g/mL hexadimethrine bromide was added to each well and the plate was gently swirled to mix. To each well, lentivirus containing scramble control (Sigma SHC002V) or anti-human BMPR2 shRNA (Sigma TRCN0000000460) were added and the plate was gently swirled to mix. The volume of virus added was calculated using a multiplicity of infection of 5. The cells were incubated overnight at 37 C in a humidified incubator in an atmosphere of 5% C O2. After 1820 h, the media containing lentiviral particles was removed from the wells. A volume of 120 L of fresh 10% DMEM was added to each well, and the plate was returned to the incubator. After 1820 h, the media was removed from the wells, and fresh media containing 2 g/mL puromycin in 10% DMEM was added in order to select for the transduced cells. The media was replaced with fresh puromycincontaining media every 34 days. When the cells reached 80% confluence, they were expanded, continuing to be maintained in puromycin-containing media. RNA was collected from scramble control and BMPR2 knock-down (BMPR2-KD) HEK293T cells using the RNEasy Plus Universal Kit (QIAGEN). cDNA was generated using SuperScript III First-Strand Synthesis Kit (Thermo Fisher Scientific 18080051). Quantitative RT-PCR was performed using TaqMan probes targeting BMPR2 (Thermo Fisher Scientific Hs00176148) and HPRT1 (Thermo Fisher Scientific Hs99999909); data were analyzed using the 2Ct method and normalized to scramble control. Immunblots to confirm reduced BMPR2 protein level were described as below. Immunoblots Immunoblots were performed on protein isolates from HEK293T cells after lysis in RIPA buffer (50 mM Tris Base, 150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS, pH 8.0) supplemented with Halt Protease and Phosphatase Inhibitor Cocktail (Thermo). Lysates were resolved by SDS-PAGE and transferred to Amersham Hybond ECL nitrocellulose membranes (GE Healthcare). All samples were denatured by heating at 100 C for 10 min after mixing with 6 reducing sample buffer (60% glycerol, 300 mM Tris pH 6.8, 12 mM EDTA, 12% SDS, 864 mM 2-mercaptoethanol, 0.05% bromophenol blue). After blocking in 10% milk in PBST (PBS + 0.1% Tween-20), the following primary Gorrell et al. BMC Res Notes (2019) 12:331 antibodies (1:250) were applied in 5% milk in PBST: antiBMPR2 C-terminal domain (BD Biosciences, 612292), anti-phosphorylated SMAD1, 5, and 8 (Cell Signaling 9516 and 13820), anti-SMAD1 (Cell Signaling 6944), and anti--actin (Sigma A2228). Appropriate HRPconjugated species-specific goat polyclonal secondary antibodies (1:1000; anti-mouse: Kirkegaard & Perry Laboratories, 04-18-06; and anti-rabbit: Cell Signaling, 7074) were utilized and western blots were developed by chemiluminescence using WesternBright Quantum or Sirius substrate (Advansta). Stripping of membranes for re-probing was accomplished using Gentle Review Stripping Buffer (VWR). Western blots were visualized using a LiCor C-Digit imager and quantified by ImageJ (ImageJ, RRID:SCR_003070). Page 3 of 6 n = 11), thus confirming the ligand-binding activity of BMPR2-ECD in this assay. Identification of a putative neutralizing antibody We then sought to identify an antibody capable of neutralizing the ligand-binding activity of the BMPR2-ECD. This led us to examine 3F6, which is a mouse monoclonal antibody raised against the N-terminus of BMPR2, and found a dose-dependent inhibition of BMPR2-ECD ligand-binding (Fig. 1); in this experimental design, the inhibition appears to saturate at an approximate ratio of 2 g BMPR2-ECD: 25 g 3F6. Given that the commercial Statistical analyses Statistical analyses were performed using GraphPad Prism 5 as described in each respective figure legend or in the text. A p-value of < 0.05 was considered significant. Results Assay development We first established a modified immunoprecipitation assay wherein recombinant BMP2 was pulled down by BMPR2-ECD conjugated to Protein G beads; the unbound BMP2, found in the supernatant, was subsequently quantified by ELISA. A pilot doseresponse series (data not shown) using beads loaded with 0.5 g to 3.0 g BMPR2-ECD while holding BMP2 concentration constant led us to further optimize the assay using 2 g BMPR2-ECD; this led to a 73% reduction in BMP2 signal (mean SEM: 73.00 7.077; p < 0.0001 by paired t-test, Fig. 1 Antibody 3F6 reduces BMPR2-ECD ligand-binding activity in a modified immunoprecipitation assay. Neutralizing the ligand-binding activity of BMPR2-ECD using various amounts of 3F6. Results are quantified by ELISA and expressed as mean SEM relative to the ligand-binding activity of BMPR2-ECD in the absence of 3F6. n 3 per condition. Asterisk indicates p < 0.05 by paired t test Fig. 2 Antibody 3F6 reduces activation of BMP pathway in HEK293T cells. a Expression of endogenous BMPR2 by HEK293T cells compared to -actin loading control. Approximate molecular weights are indicated. b, c BMP2 induces phosphorylation of SMAD1, 5, and 8 (pSMAD1,5,8) in HEK293T cells and this response is blunted by pre-treatment with 3F6. Approximate molecular weights are indicated in b. Results are quantified in c and expressed as mean SEM ratio of phosphorylated SMAD1,5,8: total SMAD1 relative to BMP2 treatment alone. No effect on BMP2-responsiveness was observed with pre-treatment using control ascites (Ctrl Asc.). n = 3 per condition. Asterisk indicates p < 0.05 by paired t test Gorrell et al. BMC Res Notes (2019) 12:331 availability of this antibody is as an ascites preparation, specificity of this assay was confirmed by demonstrating that ligand-binding activity of BMPR2-ECD is unchanged in the presence of non-specific, negative control ascites (p = 0.9135 by paired t test, n = 3). Page 4 of 6 shRNA; reduced BMPR2 transcript and BMPR2 protein levels were confirmed by quantitative RT-PCR (Fig. 3a) and immunoblot (Fig. 3b, c), respectively. 3F6 had no impact on the BMP2-responsiveness in this cell line (Fig. 3d, e). Validation of neutralizing activity in a cellbased assay We next established a cell-based assay to test the hypothesis that 3F6 pretreatment attenuates the BMP-responsiveness of HEK293T cells, which express BMPR2 endogenously (Fig. 2a) and mount a robust activation of SMAD1, 5, and 8 in response to exogenous BMP2 (Fig. 2b). Pre-treatment with control ascites had no effect on the BMP2-induced pathway activation (Fig. 2b, c), but the 3F6 antibody did in fact blunt the cellular response to BMP2 (Fig. 2b, c). To confirm the specificity of 3F6 against BMPR2, we used lentiviral transduction to generate HEK293T cells in which BMPR2 expression is stably knocked-down by Discussion The BMP signaling pathway plays essential roles in normal metazoan development and tissue homeostasis [4]. Remarkably, the same basic pathway architecture is conserved throughout evolution: extracellular dimeric ligands interact with complexes of type 1 and type 2 receptors on the cell surface to activate a class of intracellular effectors and regulate cellular differentiation and/ or behavior via genomic and non-genomic means. Gene duplication events are thought to have given rise to the highly homologous, complex, and promiscuous BMP pathway present in mammals. This complexity creates Fig. 3 Antibody 3F6 has no effect on BMP-responsiveness in BMPR2 knock-down HEK293T cells. a Expression levels of endogenous BMPR2 in scramble control HEK293T cells (Control) and HEK293T cells carrying anti-BMPR2 shRNA (BMPR2-KD) compared to HPRT1 loading control. Data are expressed as normalized to scramble control (Control) using the 2Ct method. n = 6 per condition. Asterisk indicates p < 0.05 by unpaired t test. b, c Expression of endogenous BMPR2 in scramble control HEK293T cells (Control) and BMPR2-KD HEK293T cells compared to -actin loading control. Approximate molecular weights are indicated in b. Representative immunoblot is shown in b and results from three independent runs are quantified in c (data are expressed as mean SEM ratio of BMPR2: -actin normalized to scramble control (Relative Expression)). Asterisk indicates p < 0.05 by paired t test. d, e BMP2 induces phosphorylation of SMAD1, 5, and 8 (pSMAD1,5,8) in BMPR2-KD HEK293T cells. No effect on BMP2-responsiveness of BMPR2-KD HEK293T cells was observed with pre-treatment using control ascites (Ctrl Asc.) or 3F6. Approximate molecular weights are indicated in d. Results are quantified in E and expressed as mean SEM ratio of phosphorylated SMAD1,5,8: total SMAD1 relative to BMP2 treatment alone (Relative pS1:S1). n = 4 per condition Gorrell et al. BMC Res Notes (2019) 12:331 significant challenges for mechanistic studies examining the BMP pathway in model organisms and, moreover, devising strategies for modulating the activity of specific BMP pathway components to advance human health. This has led some investigators to develop monoclonal antibodies that neutralize the activity of BMP pathway components; notable examples include neutralizing antibodies against the extracellular antagonists Noggin and Gremlin and the integral transmembrane receptors ACVR2A, ACVR2B, and ACVR1 [813]. In this study, we sought to identify antibody(ies) capable of blocking the activity of BMPR2. We started our search with three strict criteria: (1) the antibody must be raised against the region of BMPR2 exposed to the extracellular environment, i.e., the N-terminal ligand-binding domain; (2) the antibody must be monoclonal to promote specificity of action; and (3) the antibody must be purchasable from a commercial source so that it may be readily available to the field. 3F6 from Thermo Fisher Scientific met these qualifications. No funding of any kind was sought from or received from this vendor. Using a cell-free immunoprecipitation assay quantified by ELISA, we determined that 3F6 is capable of blocking ligandbinding by recombinant BMPR2-ECD. We then extended this work to examine neutralization of endogenous BMPR2 by 3F6 in HEK293T cells and obtained consistent results. In the course of this study, we also performed preliminary analyses on 9A10 and 1F12, both of which are mouse monoclonal antibodies raised against the N-terminus of BMPR2 (produced by Abcam and Thermo Fisher Scientific, respectively). We were unable to achieve neutralizing ability by 9A10 in our cell-free assay and this antibody was eliminated from further analysis. In contrast, pilot data suggest that 1F12 is capable of blocking ligand-binding by recombinant BMPR2-ECD; we were unable to further validate this result in our cell-based assay due to challenges with consistent availability from the vendor. Given that BMPR2 is widely expressed, our results provide proof-of-concept data for a novel strategy whereby investigators may inhibit BMPR2 function in various physiological contexts. When coupled with other inhibitors of the BMP pathway [6], 3F6 may provide novel insights into the mechanisms by which BMP signaling regulates embryogenesis and postnatal tissue homeostasis. Limitations The primary limitation of this study is the fact that the characterization and validation of 3F6 was performed in HEK293T cells as opposed to primary cells or in vivo. This approach was advantageous due to the ease of use, Page 5 of 6 availability, ability to be stably transduced by lentivirus, and strong BMP-responsiveness of these cells. Additionally, our study is limited by the examination of 3F6s ability to block interaction of BMPR2 with a single ligand, namely BMP2. Structural studies indicate that all BMP ligands interact with the same region on the extracellular face of BMPR2 [14] and that the affinity of BMPR2 for BMP2 is relatively similar to that of other ligands [15]. That said, given these limitations, we encourage investigators to examine the functional impact and specificity of 3F6 treatment on primary cells and/or tissues in their own context(s) of interest. Abbreviations ACVR2A: activin A receptor type 2A; ACVR2B: activin A receptor type 2B; ACVR1: activin A receptor type 1; BMP: bone morphogenetic protein; BMPR2: BMP type 2 receptor; BMPR2-ECD: BMPR2 extracellular domain; ELISA: enzyme-linked immunosorbent assay; HEK293T: human embryonic kidney 293 transformed; TGF: transforming growth factor-. Acknowledgements We gratefully acknowledge critical feedback for this work from members of the Marian University Bone & Mineral Research Group. Authors contributions JWL conceived the study. REG, MHT, LJS, JBN, LJG, WGL, JMH, and JWL contributed to the design, acquisition, and analysis/interpretation of the data presented in this manuscript. REG, LJS, and JWL drafted the article and revised it critically for intellectual content. All authors read and approved the final manuscript. Funding Funding for this work was provided via an undergraduate Student Research Award from Sigma Zeta (issued to REG), a Marian University Faculty Research Development award (issued to JWL), and other intramural funds from Marian University. Funding bodies did not participate in the design of the study; collection, analysis, and interpretation of data; or in writing the manuscript. Availability of data and materials The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request. Ethics approval and consent to participate Not applicable. Consent for publication Not applicable. Competing interests The authors declare that they have no competing interests. Received: 12 April 2019 Accepted: 5 June 2019 References 1. Weiss A, Attisano L. The TGFbeta superfamily signaling pathway. Wiley Interdiscip Rev Dev Biol. 2013;2(1):4763. 2. Trombly DJ, Woodruff TK, Mayo KE. Roles for transforming growth factor beta superfamily proteins in early folliculogenesis. Semin Reprod Med. 2009;27(1):1423. 3. Lowery JW, de Caestecker MP. BMP signaling in vascular development and disease. Cytokine Growth Factor Rev. 2010;21(4):28798. Gorrell et al. BMC Res Notes 4. (2019) 12:331 Katagiri T, Watabe T. Bone morphogenetic proteins. Cold Spring Harb Perspect Biol. 2016;8(6):a021899. 5. Lowery JW, Rosen V. Bone morphogenetic protein-based therapeutic approaches. Cold Spring Harb Perspect Biol. 2017. https://doi. org/10.1101/cshperspect.a022327. 6. Lowery JW, Brookshire B, Rosen V. A survey of strategies to modulate the bone morphogenetic protein signaling pathway: current and future perspectives. Stem Cells Int. 2016;2016:7290686. 7. Lowery JW, Amich JM, Andonian A, Rosen V. N-linked glycosylation of the bone morphogenetic protein receptor type 2 (BMPR2) enhances ligand binding. Cell Mol Life Sci. 2014;71(16):316572. 8. van Meeteren LA, Thorikay M, Bergqvist S, Pardali E, Stampino CG, HuLowe D, Goumans MJ, ten Dijke P. Anti-human activin receptor-like kinase 1 (ALK1) antibody attenuates bone morphogenetic protein 9 (BMP9)induced ALK1 signaling and interferes with endothelial cell sprouting. J Biol Chem. 2012;287(22):1855161. 9. Ciuclan L, Sheppard K, Dong L, Sutton D, Duggan N, Hussey M, Simmons J, Morrell NW, Jarai G, Edwards M, et al. Treatment with anti-gremlin 1 antibody ameliorates chronic hypoxia/SU5416-induced pulmonary arterial hypertension in mice. Am J Pathol. 2013;183(5):146173. 10. Hampton DW, Asher RA, Kondo T, Steeves JD, Ramer MS, Fawcett JW. A potential role for bone morphogenetic protein signalling in glial cell fate determination following adult central nervous system injury in vivo. Eur J Neurosci. 2007;26(11):302435. Page 6 of 6 11. Hampton DW, Steeves JD, Fawcett JW, Ramer MS. Spinally upregulated noggin suppresses axonal and dendritic plasticity following dorsal rhizotomy. Exp Neurol. 2007;204(1):36679. 12. Lach-Trifilieff E, Minetti GC, Sheppard K, Ibebunjo C, Feige JN, Hartmann S, Brachat S, Rivet H, Koelbing C, Morvan F, et al. An antibody blocking activin type II receptors induces strong skeletal muscle hypertrophy and protects from atrophy. Mol Cell Biol. 2014;34(4):60618. 13. Simon DP, Vadakkadath Meethal S, Wilson AC, Gallego MJ, Weinecke SL, Bruce E, Lyons PF, Haasl RJ, Bowen RL, Atwood CS. Activin receptor signaling regulates prostatic epithelial cell adhesion and viability. Neoplasia. 2009;11(4):36576. 14. Hinck AP. Structural studies of the TGF-betas and their receptorsinsights into evolution of the TGF-beta superfamily. FEBS Lett. 2012;586(14):186070. 15. Heinecke K, Seher A, Schmitz W, Mueller TD, Sebald W, Nickel J. Receptor oligomerization and beyond: a case study in bone morphogenetic proteins. BMC Biol. 2009;7:59. Publishers Note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Ready to submit your research ? Choose BMC and benefit from: fast, convenient online submission thorough peer review by experienced researchers in your field rapid publication on acceptance support for research data, including large and complex data types gold Open Access which fosters wider collaboration and increased citations maximum visibility for your research: over 100M website views per year At BMC, research is always in progress. Learn more biomedcentral.com/submissions  ...

... Comparison of Human Cytokine Array, Cytokine Bead Arrays, and Protein ELISA in determining cytokine production in media supernatant from in vitro strain of human dermal fibroblasts. Aric Anloague Dr. Jonathan Lowery Ph.D. Abstract This experiment analyzes three different techniques used in literature to detect cytokine release from cells in vitro: the Human Cytokine Array (HCA) by R&D Systems, the BD Cytometric Bead Array (CBA) assay, and protein enzyme-linked immunosorbent assay (ELISA). The model used to test these assays was a human dermal fibroblast cell culture that was subjected to strain profiles by the Flexcell FX-6000 that include a repetitive motion injury, a profile with the injury strain as well as a strain representing myofascial release, and a control that was not strained. Each of these techniques analyzed the conditioned media of the cultures collected 96 hours after the strain profile was completed. The HCA was proficient at analyzing a large amount of cytokines at once, though was less sensitive than the other assays. The CBA measured six cytokines at once and showed high sensitivity in detecting differing concentrations in the conditioned media. The ELISA could only measure one cytokine at a time, but also showed high sensitivity in detecting differing concentrations in the conditioned media. This experiment also looked at the cost and amount of sample used. This experiment also considers price, amount of sample used and duration of the assay to determine ideal situations to use each technique. Introduction The purpose of this review is to determine the ideal technique for analyzing cytokine release from fibroblasts in cell culture. The three techniques that will be compared are the Human Cytokine Array (HCA) by R&D Systems, the BD Sciences Cytokine Bead Array (CBA), and protein enzyme-linked immunosorbent assay (ELISA). Cytokines, both pro- and antiinflammatory, play distinct roles in the wound healing by initiating processes such as acting as a white blood cell mobilization, stem cell mobilization, and regulation and initiation of the inflammation response. Measuring cytokine release is beneficial to researchers for many reasons including, using them as potential biomarkers, determining immune system health, understanding the inflammatory response, understanding the response to a treatment, etc. Each of these techniques use a slightly different method of detection. The HCA uses a membrane that is premarked with antibodies for 36 different cytokines in duplicate. The membranes are incubated with sample and detection antibodies that precipitate a dark color when a cytokine is presentdarker intensities suggest higher concentration of cytokine, though concentrations are only relative. The CBA uses different beads each labeled with different antibodies for the cytokines of interest. The beads are allowed to incubate with the samples and a detector antibody labeled with different fluorophores that are then analyzed using flow cytometry. If there is more cytokine present, then more beads will be detected by the flow cytometer. Comparing the number of beads detected in the unknowns to a standard curve allows this procedure to develop concentrations of cytokines present in each sample. The ELISA protocols vary depending on the secondary antibody visualization used. In this experiment, a 96-well plate is coated with the antibody for the desired cytokine, and the sample is incubated with a secondary antibody conjugated with biotin. Following the incubation, streptavidin HRP is added to precipitate a color and it is developed using TMB chromagen, and the color intensity is identified and compared to a standard curve to develop concentrations of cytokines present. These different detection methods may result in different sensitivities of sample and affect the accuracy of acquisition. In order to test the pros and cons of each of these techniques, a model was needed that not only used a cell type that releases cytokines in an injured/strained state, but also a method that could produce an injury-like state. Over a series of experiments, Standley, Meltzer et al. established a fibroblast model that put cultured fibroblast cells through a repetitive motion-like strain and a strain mimicking myofascial release [1-6]. During these strains, the fibroblasts release cytokines into the media in which they are cultured. This conditioned media is what is used to measure the cytokines released and is relatively easy to analyze by all three of the techniques. Previously using these models, Standley and Meltzer showed using the HCA and ELISA that IL-1, IL-1, IL-2, IL-3, IL-6, and IL-16 increase substantially increase following a repetitive motion strain and showed a significant reduction of IL-6 alone with a treatment they deemed a counterstrain [3]. In a later study, they showed that with their MFR treatment, using the HCA technique, only GRO showed a significant change with increased expression [4]. Standley and Meltzer did not use the CBA in any of their studies. This experiment attempts to validate the findings of Meltzer and Standley to validate the effectiveness of each technique, and it takes into consideration the price of the assay, amount of sample used per assay, and duration of each assay to suggest the ideal use for each technique. Methods Fibroblast Culture/Flexcell protocol: Primary human dermal fibroblasts were purchased from ATCC (#PCS-201-012) and according to the suggestions of ATCC. Mechanical stimulation was performed on a Flexcell FX-6000 (located in the Indiana Center for Musculoskeletal Health core lab) according to the methods described by Metzler, Standley et al [6]. Cells were seeded on flexible collagen I-coated membranes and strained to mimic a repetitive motion strain in an 8 hour cycle with 1.6 second bouts of deformation increasing at 22%/second starting at a baseline strain of 10% and a maximum of 16.6%, followed by decreasing strain to baseline at 22%/secondthese cells represent the Injury strain. Cells that represent the Myofascial Release (MFR) strain underwent the same 8 hour protocol as the injury strain, followed by a 3 hour rest period. After the rest period, these cells were subjected to a single 60 second bout of stretch at 6% beyond resting length at a loading rate of 3% followed by release at 1.5%/second until return to resting length. The control group did not undergo either of these strain protocols. Conditioned media was collected 96 hours after the strain protocols from each sample and stored at -80C. These protocols were run 3 times on three different dates and are labeled as Run #1- Run #3. Each Run had 1 control group, 2 Injury Strains, and 2 Injury + MFR Strains. Human Cytokine Array: The collected conditioned media from each strain profile were simultaneously analyzed for CCL1/I-309, CCL2/MCP-1, MIP-1/MIP-1, CCL5/RANTES, CD40 Ligand/TNFSF5, Complement Component C5/C5a, CXCL1/GRO CXCL10/IP-10, CXCL11/I-TAC, CXCL12/SDF-1, G-CSF, GM-CSF, ICAM-1/CD54, IFN-, IL-1/IL-1F2, IL1ra/IL-1F3, IL-2, IL-4, IL-5, IL-6, IL-8, IL-10, IL-12 p70, IL-13, IL-16, IL-17A, IL-17E, IL18/IL-1F4, IL-21, IL-27, IL-32, MIF, Serpin E1/PAI-1, TNF-, and TREM-1 using the Proteome Profiler Human Cytokine Array from R&D Systems. Each run was analyzed on separately. The techniques were performed following the instructions provided by the manufacturer. Signal density was analyzed with the Licor C-Digit. Human Pro-inflammatory Cytokine Cytometric Bead Array: The collected conditioned media from each strain profile in each run were simultaneously analyzed for IL-8, IL-1, IL-6, IL-10, TNF, and IL-12p70 using the Human Pro-Inflammatory Cytokine Cytometric Bead Array kit from BD Biosciences. All runs were analyzed simultaneously. The techniques were performed following the instructions provided by the manufacturer with the BD Accuri Flow Cytometer. Data were analyzed with paired T-tests between each of the strain profiles. Enzyme-linked Immunosorbent Assay: The collected conditioned media from and each strain profile in each run were analyzed using a separate human ELISA kit for IL-6 and for IL-8 from ThermoFisher Scientific to determine the concentration of these respective proteins. All runs were analyzed simultaneously. The ELISA methods were performed following instructions provided by the manufacturer. Data were analyzed with paired T-tests between each of the strain profiles. Results Human Cytokine Array: Of the thirty-six cytokines tested for, only six were detected: CXCL1/GRO, CXCL12/SDF-1, IL-6, IL-8, MIF, and Serpin E1/PAI-1. The relative signal of each cytokine for each sample was compared to the average of the signal density of the reference spots for each sample. There was no detection of CXCL1/GRO in Run #3, no detection of CXCL12/SDF-1 in Run #1, and no detection of IL-6 in Run #1 and Run #3 (fig. 1). No statistical analysis was performed on the data because there was not enough samples that showed detection. Human Pro-inflammatory Cytokine Cytometric Bead Array: Of the six cytokines tested for, only IL-6 and IL-8 were detected. The concentrations of IL-6 and IL-8 varied from run to run (fig. 2), so the fold change between strain profiles was calculated, and the averages from all runs were taken (fig. 4). For IL-6, there was a significant increase in expression in the Injury group compared to the control (p=0.0145; 95% CI=1.762 to 5.644), and then a significant decrease in the Injury + MFR group compared to injury (p=0.0241; 95% CI=-3.136 to -0.5975). The Injury + MFR group for IL-6 still showed increased expression when compared to the control (p=0.0297; 95% CI= 0.4425 to 3.231). For IL-8, there was an observed increase in expression in the Injury group when compared to the control (p=0.0782; 95% CI=-0.4753 to 3.875), and a significant decrease in the Injury + MFR group when compared to the Injury group (p=0.0429; 95% CI=-2.285 to -0.940). The Injury + MFR group approached the expression value of the control group with no significant difference between the two groups (p=0.306; 95% CI= -1.098 to 2.118). Enzyme-Linked Immunosorbent Assay (ELISA): The ELISAs similarly showed varied concentrations between runs for both IL-6 and IL-8 (fig. 3), so the fold change between strain profiles was calculated and the averages from all runs were taken (fig. 4). For IL-6, there was a increase in expression in the Injury group when compared to the control (p=0.0057; 95% CI= 1.464 TO 2.889), and a decrease in the Injury + MFR group when compared to the Injury group (p=0.0709; 95% CI=-1.973 TO 0.181). The Injury + MFR group still remained higher than the control (p=0.0293; 95% CI=0.3171 to 2.250). For IL-8, there was no statistically significant changes between groups, however, the observed expression of the Injury group appears higher than the control (p=0.150; 95% CI=-3.037 to 9.903), and expression appears to decrease towards the values of the control in the Injury + MFR group when compared to the Injury group (p=0.319; 95% CI=-5.495 to 2.923). There was also no significant difference between the Injury + MFR and control (p=0.2043; 95% CI=-2.825 to 7.119) In order to compare the effectiveness of the CBA and ELISAs, X-Y plots for IL-6 and for IL-8 were generated comparing the concentrations of each sample for each run (fig. 5). Linear regression for IL-6 showed the best fit line with a slope of 0.807 and Y-intercept at 5.678 and an R2 value of 0.945. Linear regression for IL-8 showed the best fit line with a slope of 0.665 and Yintercept at 5.254 and an R2 value of 0.732. Discussion The three techniques discussed vary in the cytokines analyzed per run, duration of the assay, amount of sample used, monetary cost, and sensitivity. Comparing these differences can help determine which assay should be used depending on the needs of the experiment. The human cytokine array (HCA) and the cytokine bead array (CBA) both analyze the amount of cytokines provided by the kits used, 36 and 6 respectively. The HCA took approximately 24 hours to measure all 36 cytokines, and the CBA took approximately 5 hours to measure the 6 cytokines. The Enzyme-linked Immunosorbent Assay (ELISA) only measures one cytokine at a time, and takes approximately 4 hours per cytokine. The duration of the techniques between the HCA and CBA are comparable when compared to the number of cytokines analyzed, but the ELISA takes significantly longer. When sample supply is limited, the volume of sample used per run is important. The HCA uses 1mL of each sample per run, the CBA uses 50L of each sample per run, and the ELISA varies from kit to kit. For the IL-6 kit, 100L of each sample per run was used and the IL-8 kit used 50L per run. Because of the number of cytokines and volume of sample analyzed per run, the CBA technique is the most efficient. The ELISA is comparable, but the volume of sample is required for each cytokine analyzed, whereas the CBA uses that volume for analysis of 6 cytokines at once. The monetary cost of the techniques are important to consider. The cost depends on the goals of the experiment. The ELISA is the cheapest option at approximately $479.00/kit if analysis of only one cytokine is desired. The HCA kit provides four membranes per kit, which is enough for four samples, at a cost of $519.00/kit. The CBA kit costs approximately $1364.00/kit, but the ELISA and the CBA both can run up to ninety-six samples at once including the standards needed for generating the standard curve. If there are a low number of samples, the HCA is more cost efficient, but if more samples are in need of analysis, the CBA and ELISA become more cost efficient depending on the desired number of cytokines to be analyzed. The HCA assay is the most beneficial when uncertain which cytokines may be detected because of the number of cytokines detected simultaneously. It can be a very costly experiment in terms of amount of sample used per run and cost per sample analyzed at $519.00 for four samples. Additionally, the HCA showed no significant differences in cytokine expression between groups (fig. 1) and IL-6 and IL-8 were below the detection limit, even though it was shown that they were present in the conditioned media by the CBA and ELISA, suggesting a low sensitivity in detection for the HCA. It may be beneficial to use the HCA as a preliminary test on only a few of the samples to determine what may be detected, and then a different assay such as the CBA or ELISA for detection of concentration depending on the goals of the experiment. The CBA is efficient, cost effective, and accurate when testing multiple samples for multiple cytokines. As previously stated, it is capable of running 96 samples at once, including the standards for the standard curve, and considering the CBA kits cost $1364.00 each, this equates to approximately $14.21 per sample, which is much more cost effective than the HCA, which is $129.75 per sample. Only the CBA and ELISAs measure exact concentrations of cytokines. They both advertise a detection range that goes as low as 1pg/mL and depending on the kit, they can go as high as 5,000pg/mL. The lower limit was pushed in this experiment with the lowest concentrations for IL6 appearing in the control groups of Run #2 and Run #3 at 2.73pg/mL and 2.86pg/mL respectively for the CBA, and 5.97pg/mL and 5.19pg/mL respectively for the ELISA. The X-Y plots in figure 5 compare the concentrations detected between the CBA and the ELISAs. The detected concentrations were more similar to each other between assays in IL-6 than in IL-8 because the best-fit line for IL-6 has a slope of 0.807, which is closer to the ideal slope of 1 than IL-8, which has a slope of 0.665. The R2 values for IL-6 and IL-8 were 0.945 and 0.732 respectively, again suggesting a better fit line in the IL-6 data. These data show the variability that can occur between the cytokine concentration detection between the assays. In this experiment, the CBA showed significant increase in fold change for both IL-6 and IL-8 in the Injury groups compared to the control. These trends were similar in the ELISAs, though the increase observed in the Injury group, when compared to the control, was not statistically significant. Similarly, the CBA showed significantly lower levels of IL-6 and IL-8 in the Injury + MFR group when compared to the Injury group, and the ELISAs, again, showed similar trends, though not statistically significant. These trends suggest that the CBA has a higher sensitivity than the ELISAs making it the more efficient assay. The trends in the CBA and ELISAs are supported by the findings of Standley and Meltzer, who also showed that in IL-6, there is an increase in a strain representing an injury when compared to the control, and there is a decrease in the strain representing either myofascial release or counterstrain [1, 3-5]. These studies did not show significant changes in IL-8, but the trends match the data observed in both the CBA and ELISAs, again validating the techniques performed in this experiment. The studies mention several other cytokines that were detected that were not detected by the HCA or CBA in this experiment, but that difference could be due to the overall lower concentrations of cytokine release in Run #2 and Run #3, and it could be based on the amount of time between the final strain profile and collection of the media, which in this experiment was 96 hours. Overall, each of the different techniques has its merits and flaws. The HCA is a beneficial technique to use when it is unknown which cytokines may be detected. Its sensitivity is relatively low and has difficulty detecting low concentrations of cytokines. There were several samples that showed no detection of IL-6 and IL-8, which were clearly present based on the findings of the other two assays. It is also costly per sample tested, so it may be useful in a preliminary nature, identifying the cytokines released, but then using CBA or ELISA to determine the concentrations present. The ELISA is useful when detection of only one to two cytokines is needed because it becomes more costly and time consuming with more. It has the benefit of being able to test ninety-six samples at once while using relatively low amount of sample for each test, but, again, if more cytokines are tested for, more sample will be used. Additionally, the ELISA is fairly sensitive in determining concentrations of samples with the ability to detect concentrations as low as 5pg/mL. The CBA is the most versatile when multiple cytokines are to be analyzed at once. Its efficiency is the best of the three techniques in terms of time to complete the assay and amount of sample used per assay. While one kit is more expensive than one kit of each of the other runs, it more than makes up for the cost when considering the cost per sample tested and the amount of cytokines analyzed at once. The CBA is very sensitive in determining concentrations of samples with the ability to detect concentrations as low as 2 pg/mL. To strengthen the findings of this study, the procedures could be repeated with different cell types including different tissues or different animal models. Different types of sample such as serum or donor plasma could be analyzed instead of media supernatant. The findings are limited by the concentrations of cytokines released as a result of the strain profiles. Finding a model that releases higher amounts of cytokines may resolve the low to no detection issue that occurred in the HCA. Ultimately, each assay has their place in research and it is important to use the technique that best fits the experimental design. References 1. Meltzer, K.R. and P.R. Standley, Modeled repetitive motion strain and indirect osteopathic manipulative techniques in regulation of human fibroblast proliferation and interleukin secretion. J Am Osteopath Assoc, 2007. 107(12): p. 527-36. 2. Eagan, T.S., K.R. Meltzer, and P.R. Standley, Importance of strain direction in regulating human fibroblast proliferation and cytokine secretion: a useful in vitro model for soft tissue injury and manual medicine treatments. J Manipulative Physiol Ther, 2007. 30(8): p. 58492. 3. Standley, P.R. and K. Meltzer, In vitro modeling of repetitive motion strain and manual medicine treatments: potential roles for pro- and anti-inflammatory cytokines. J Bodyw Mov Ther, 2008. 12(3): p. 201-3. 4. Meltzer, K.R., et al., In vitro modeling of repetitive motion injury and myofascial release. J Bodyw Mov Ther, 2010. 14(2): p. 162-71. 5. Dodd, J.G., et al., In Vitro Biophysical Strain Model for Understanding Mechanisms of Osteopathic Manipulative Treatment. J Am Osteopath Assoc, 2006. 106(3): p.157-66 6. Zein-Hammoud, M., Standley, P.R., Modeled Osteopathic Manipulative Treatments: A Review of Their in Vitro Effects on Fibroblast Tissue Preparations. J. Am Osteopath Assoc, 2015. 155(8): p. 490-502. Figures Run #2 Run #1 Relative to Avg. Reference Density 0.20 0.15 0.10 0.05 Injury 0.4 Injury + MFR 0.2 Relative to Avg. Reference Density Control Injury Injury + MFR 0.4 0.2 I-1 /P A in E1 M IF Se rp IL -8 IL -6 F1 D L1 2/ S XC C XC L1 /G R O a 0.0 C I-1 E1 in Se rp Run #3 0.6 /P A M IF IL -8 IL -6 D F1 a L1 2/ S XC XC C C Se rp XC in L1 E1 /G R /P A O I -1 IF M -8 IL -6 IL R O a L1 2/ SD F1 /G L1 XC Control 0.0 0.00 C 0.6 C Relative to Avg. Reference Density 0.25 Fig. 1 Graphs displaying the cytokines detected in the Human Cytokine Array and their relative densities as compared to the average of the reference spots. Run #1 CBA IL-6 Run #2 CBA IL-6 150 Run #3 CBA IL-6 20 15 15 100 pg/mL pg/mL pg/mL 10 10 50 5 5 +M ur y Run #3 CBA IL-8 150 2500 FR y In j Run #2 CBA IL-8 Run #1 CBA IL-8 100 2000 80 pg/mL 1500 1000 pg/mL 100 50 500 60 40 20 FR ry In ju ry +M In ju l on tro C In ju ry +M FR ry In ju on tro l 0 C FR +M In ju ry In ju on tro ry 0 l 0 C pg/mL In ju r In j ur y +M C on tro l FR y In ju r C on tro l 0 In j ur y C on In j +M ur y FR 0 tro l 0 Fig. 2 Graphs displaying the concentrations of IL-6 and IL-8 for each run and strain profile as detected by the Cytokine Bead Array (CBA) assay. Data are expressed as mean concentrations SEM. 10 Run #1 IL-8 ELISA +M ju r In on ry C ju Run #2 IL-8 ELISA 150 FR y FR In In In j ju ur y C In on ju r tro FR ry C In +M ju r tro on 0 +M 0 y 0 5 l 5 y 20 10 l 15 15 tro 60 20 pg/mL 20 pg/mL 80 40 Run #3 IL-6 ELISA Run #2 IL-6 ELISA 25 l pg/mL Run #1 IL-6 ELISA 100 Run #3 IL-8 ELISA 200 50 40 150 pg/mL 100 50 50 10 FR ry +M ju In tro Fig. 3 Graphs displaying the concentrations of IL-6 and IL-8 for each run and strain profile as detected by ELISA. Data are expressed as mean concentrations SEM. ry ju In +M ju ry l FR ry ju tro l In In ry ju on +M In In C y ju r l tro on 0 FR 0 C 20 on 0 30 C pg/mL pg/mL 100 Combined IL-6 ELISA Fold Change Combined IL-6 CBA Fold Change 4 a a 3 4 b 2 2 1 Combined IL-8 CBA Fold Change 8 3 6 +M ur y In j t ro on +M ur y In j C y In ju r t ro y 0 In ju r 0 FR 2 l 1 FR 4 l b 2 on FR Combined IL-8 ELISA Fold Change 4 C Fold Change +M In j In j ur y In j C on ur y ur y FR +M ur y In j t ro on C l 0 l 0 tro Fold Change 6 Fig. 4 Graphs displaying the average fold change of each run between each strain profile as compared to the controls for both the Cytokine Bead Array assay and ELISA. Data are expressed as mean fold change SEM and analyzed by paired Ttests. p<0.05: a compares to control; b compares to injury. D IL-6 100 ELISA 80 60 40 20 0 0 50 100 150 CBA IL-8 200 ELISA 150 100 50 0 0 50 100 150 200 250 CBA Fig. 5 Graphs displaying the plots of the concentrations (pg/mL) for each sample in pg/mL for the ELISA vs. the CBA. The black line represents the line of best-fit, and the red line represents the line of identity that would signify identical concentrations detected in the ELISA and CBA. The best fit line R2 for IL-6= 0.945 and R2 for IL-8= 0.732.  ...

... Advances in Microbiology, 2020, 10, 383-396 https://www.scirp.org/journal/aim ISSN Online: 2165-3410 ISSN Print: 2165-3402 Effects of Farnesol on Drug-Resistant and Non-Resistant Candida albicans: Implications for Cosmetic and Pharmaceutical Applications Neela Yar1, Emma Wittman1, Dominic Schaut1, Francesco De Seta2, Bryan Larsen1* College of Osteopathic Medicine, Marian University Indianapolis, Indianapolis, IN, USA Department of Medical Sciences, University of Trieste, Institute for Maternal and Child Health-IRCCS, BurloGarofolo, Trieste, Italy 1 2 How to cite this paper: Yar, N., Wittman, E., Schaut, D., De Seta, F. and Larsen, B. (2020) Effects of Farnesol on Drug-Resistant and Non-Resistant Candida albicans: Implications for Cosmetic and Pharmaceutical Applications. Advances in Microbiology, 10, 383-396. https://doi.org/10.4236/aim.2020.108028 Received: July 25, 2020 Accepted: August 18, 2020 Published: August 21, 2020 Copyright 2020 by author(s) and Scientific Research Publishing Inc. This work is licensed under the Creative Commons Attribution International License (CC BY 4.0). http://creativecommons.org/licenses/by/4.0/ Open Access Abstract Background: Farnesol is added to numerous consumer products that intentionally, or inadvertently come in contact with tissues that may harbor the opportunistic yeast, Candida albicans. Objective: This study explores biological consequences of the exposure of Candida albicans from community infections or from a panel of antifungal drug resistant organisms on growth and survival of these organisms when exposed to farnesol. Methods: ATCC supplied Candida albicans from the MP8 drug resistance panel and an additional 12 strains of community-acquired Candida albicans were cultured in the presence of farnesol. With standard micobiologic techniques and flow cytometry evaluation, a series of experiments considered growth, morphology, viability and entrance into the quiescent persister phenotype of Candida with emphasis on differences between drug resistant and community organisms. Results: Differences growth yield, relative cell size and heat susceptibility distinguished the community organisms from the drug-resistant organisms. Using a subset of these organisms, exposure to farnesol resulted in diminished growth, inhibited hyphal growth, diminished cell membrane integrity and increased heat stress susceptibility. Data provided suggest that exposure to farnesol pushes cultures of Candida albicans toward the quiescent persister phenotype. Conclusion: Exposure of drug resistant and community strains of Candida albicans are modestly affected by farnesol in ways that may lessen their pathogenic potential. In contrast, the tendency of farnesol to engender greater numbers of quiescent organisms could support persistence of Candida. Keywords Farnesol, Candida albicans, Antifungal Resistance, Persister Phenotype, Quorum Sensing DOI: 10.4236/aim.2020.108028 Aug. 21, 2020 383 Advances in Microbiology N. Yar et al. 1. Introduction Following the discovery of the role farnesol plays as a quorum-sensing autoinducer for the opportunistic yeast, Candida albicans, numerous studies on the molecular details of its role in survival, virulence and morphogenesis have been published [1]. In addition, other findings relevant to farnesol include modulation of host immune responses and effects on bacteria which may both enhance or diminish the course of infection [2]. Against this backdrop of many biological activities that have been attributed to farnesol, its industrial use as a perfume or component of cosmetics and household products and potentially in pharmaceutical preparations has occurred with limited apparent appreciation for biological side effects (beneficial or not) that might result from its addition to formulations of commercial products. Farnesol is also a component of essential oils which are also used commercially and hence due to public interest and acceptance, have widening commercial use. Consumers of these products may be attracted to them out of desire for products considered to be natural or which contain botanical substances in the formulation. Little direct tissue toxicity has been described for farnesol, and it is categorized by the US FDA as GRAS or generally recognized as safe. Reports of allergy to farnesol are rare [3] with only 22 of 2021 individual patch-tested showing a reaction. Incorporation of farnesol into a mucoadhesive vaginal gel has been reported [4]. While many of the biologically relevant effects such as quorum sensing occur at micromolar concentrations [5] [6] [7] [8] [9], consumer products may contain farnesol at higher levels and we report here findings obtained with Candida sp. to illuminate what benefits or concerns should be considered by the industrial use of farnesol. Specifically, this report explores potential differences between Candida strains that were known to have specific resistance patterns compared to community-derived organisms in terms of growth and fitness under stress. 2. Methods 2.1. Microorganisms Fungal cultures available for this study included 14 strains comprising the ATCC MP8 drug-resistant panel which included 12 multi-resistant strains and two non-resistant (control) strains of Candida albicans. This panel was selected as the organisms are commercially available and contain well characterized resistance characteristics that allow for repeatability among laboratories and involve resistance to multiple drug classes. In addition, 12 Candida albicans strains were provided by a local clinical laboratory representing isolates from various body sites. Except where specifically designated, for logistical reasons, a subset of the above organisms was used. The subset of organisms included three antifungal drug-resistant organisms from the ATCC MP-8 panel. These resistant strains were: ATCC 64124 Darlington (here labeled CA-A), ATCC 10231 3247 (here labeled CA-B) and ATCC DOI: 10.4236/aim.2020.108028 384 Advances in Microbiology N. Yar et al. 28121 304 (here labeled CA-C). Three community isolates had previously been identified by local clinical laboratories as Candida albicans using standard diagnostic microbiology methods and lacked known antifungal resistance. These were labeled as CA-1, CA-2 and CA-3. All Candida strains were aliquoted and stored in 50% glycerol and kept at 80C until needed and were grown overnight as starter cultures in brain heart infusion (BHI) broth for each experiment. Starter cultures were incubated overnight at 37C in 5% CO2. 2.2. Experimental Design Experimental conditions were inoculated with approximately 1 104 organisms and were incubated overnight at 37C in 5% CO2 before experiment readout. Experiments containing farnesol (Alfa Aesar-Thermo Fischer, Ward Hill MA, mixed isomers 96%) were prepared by diluting stock from the manufacturer in absolute ethanol for addition to brain heart infusion broth. For plating medium, farnesol was added directly while the agar was molten, mixed quickly and poured into plates. In addition to BHI agar, we also employed modified spider medium (Brain Heart infusion agar was purchased from Difco BD and contained brain-heart infusion powder, sodium chloride, disodium phosphate with 1.5% agar and dissolved according to manufacturer instructions, to which 1 g of mannitol/100ml of media was added and then autoclaved before dispensing into plates) for preliminary observations. Absolute counts of yeast employed flow cytometry performed using the Becton-Dickenson Accuri C6 instrument. When flow cytometer counts were unavailable, turbidity, measured spectrophometrically at 450 nm, was employed. Experiments were conducted in styrene well plates and unless otherwise noted, contained BHI as the background medium with or without farnesol. For analysis of the organisms after overnight incubation, each well was vigorously mixed before removing a 100 uL aliquot which was added to 900 ul of water containing propidium iodide (60 mg/ml stock solution diluted 100 uL/50mL sterile water) and analyzed for events in the forward scatter channel (FSC) of the cytometer. The C6 instrument allows absolute quantitation due to a fluidics system that accurately pumps the sample onto the flow cell rather than using air pressure to introduce the sample into the flowing sheath fluid. Microbial counts were based on events triggered in the FSCchannel and relative particle size was indicated by mean FSC-h for each sample. Propidium iodide staining was observed in the orange fluorescence (FL-3h) channel and provided a measure of relative cell integrity. 2.3. Heat Challenge Heat stress testing was done by placing 100 uL aliquots from cultures conditioned in media with or without farnesol, placed in a heat block at 50C for 20 minutes and thereafter immediately diluted in water containing propidium iodide. For most samples fungal enumeration was based on at least 2000 flow cytometer DOI: 10.4236/aim.2020.108028 385 Advances in Microbiology N. Yar et al. counts. Baseline propidium iodide staining was performed on cultures before heat treatment and the percent of cells with FL-3 signal above 1 103 was recorded. As second sample was similarly evaluated immediately after heat exposure and a heat stress index was calculated by % of cells staining with propidium iodide after heating divided by the percent of cells staining with propidium iodide before heating which provided the fold increase in staining resulting from heat stress. 2.4. Persister Phenotype Estimation Density fractionation of yeast cultures was performed according to the method of Quasem, et al. [10]. Briefly, indixanol 60% (Sigma Chemical, St. Louis MO) was diluted with an equal volume of tris buffered saline. Diluted indixanol (0.5 ml) was pipetted into a microcentrifuge tube and the yeast culture layered atop the density cushion. Separation of high and low buoyancy cells was obtained by centrifuging at 2500 xg for 2.5 minutes, then rotating the tube in the angle rotor and centrifuging an additional 2.5 minutes. The pellet is expected to be enriched with quiescent cells while the upper band is enriched for actively growing yeast as shown in Figure 1. Figure 1. Yeast cultures were stained with crystal violet and washed prior to resuspension and density separation on indixanol density cushion. After separation the tubes were backlit to enhance visualization of upper and lower fractions. At left is a tube dominated by non-quiescent yeast and the image right shows a culture with a preponderance of quiescent and dead cells. Staining of the culture before separation was not used for the experiments, the staining was done here for illustrative purpose. With sufficiently high concentration of cells, the unstained bands could be visualized without staining. DOI: 10.4236/aim.2020.108028 386 Advances in Microbiology N. Yar et al. 2.5. Statistical Analysis Where appropriate, continuous variables were compared by two sample T test was applied and where possible paired comparisons were made. The level of significance was 0.05. Evaluation of generation time was calculated using the free online program at http://endmemo.com/. 3. Results 3.1. Colony Morphology As an initial overview of the behavior or Candida albicans in the presence of farnesol, the entirety of the two sets of test organisms (drug-resistant and community acquired) were examined. BHI agar with or without farnesol and modified spider medium were prepared and 10 uL aliquots of overnight cultures of each of the available test organisms were spotted onto these media with and without the addition of 0.3% farnesol. Plates were incubated at room temperature and monitored daily for 5 days for growth and the spider phenotype (mycelial growth) under each condition. Regardless of drug resistance, all strains tested grew on all media. However, the colony size on the farnesol-containing plates showed growth restriction compared to the non-farnesol plates and the appearance of spidering colonies was not present on farnesol-containing plates. No gross differences in the colony appearance of drug resistant and non-resistant strains were noted in this experiment. While it was possible to evaluate the entire set of Candida isolates on a single plate by this method, the attempt to incorporate the hydrophobic farnesol into agar medium resulted in development of droplets of farnesol atop the medium, limiting its utility for subsequent studies. 3.2. Strain Characterization Despite the lack of obvious differences between the drug-resistant and community strains in the initial study, a more detailed examination of intrinsic fitness of each group prior to further detailed studies of farnesol was undertaken. Accordingly, several aspects of growth were evaluated by flow cytometry. The 24-hour growth yield, relative organism size (based on mean FSC statistics), generation times over a 4-hour period and baseline propidium iodide staining and increase in propidium iodide staining after a 20-minute heat challenge are shown in Table 1. The above experiment indicated that some, but not all of the parameters measured, differed significantly between the two groups of organisms, but the apparently smaller volume and number of cells after 24 hours in culture and the greater apparent membrane fragility before and after heating suggested that the experiments that followed should consider examples from both the drug-resistant and the community organisms available. The first three Candida albicans strains from each set (CA-A, CA-B and CA-C from the resistant panel and CA-1, CA-2 and CA-3 from the community strain set) were selected for further study. DOI: 10.4236/aim.2020.108028 387 Advances in Microbiology N. Yar et al. Table 1. Data summary for comparison of MP8 resistant organisms with community organisms including MP8 controls. Resistant Isolates Property Community Isolates P= Mean SD Mean SD Growth Yield 24 h 979/uL 430 2335 2131 0.053 Relative Size (as mean FSC-h value) 1.51 106 0.143 106 1.22 106 0.27 106 0.008 Biomass Index units (Events mean FSC-h/1 106) 1487 units 654 units 2533 units 1714 units 0.104 Generation Time 55.52 min ND 56.43 ND 0.880 Percent PI Staining 17.3% 6.18% 11.65% 4.92% 0.035 Heat Stress Index 2.412 1.106 1.581 0.415 0.031 Growth yield is events/uL (FSC of BHI culture diluted 1:10). Relative size is average FSC height signal for each culture. Biomass index is the product of events/uL and mean forward scatter (without exponent). Generation time was determined after 4 hours of freshly inoculated culture and was based on forward scatter events evaluated using EndMemo (endmemo.com). PI staining was determined as the percent of events having FL3 channel signal above 1 103 based on data from unstained yeast. Heat stress index represents the fold increase in %PI staining after exposure to 50C for 20 minutes. Reported values represent the average of the individual Candida strains tested. Comparisons for 24-hour growth yield and PI staining were measured for each strain and results are shown in Figure 2. These data indicate variations among strains and when comparing 24-hour mean counts between resistant strains CA-A, CA-B, CA-C, to community strains CA-1, CA-2, CA-3, the community strains furnished slightly more robust growth (p = 0.03, students T test). The intrinsic PI staining trended higher for the resistant organisms but did not reach statistical significance (p = 0.11). 3.3. Growth and Membrane Integrity The effect of farnesol on growth characteristics was subsequently evaluated. In this experiment, the six test strains were inoculated into 0.05% farnesol in BHI or BHI alone and incubated for 24 hours at 37C. Figure 3 shows that for all strains tested, farnesol decreased the growth yield. While overall the decrease in counts was significant, it appeared that the community isolates appeared more susceptible to farnesol. The average reduction in counts with farnesol for the three resistant strains was 19%, while the reduction in counts averaged 52% for the community strains. 3.4. Morphogenesis Because of the importance of hyphal growth as a virulence factor for Candida albicans, the effect of farnesol on these organisms was documented microscopically. As shown in Figure 4 the effect on filamentous growth was striking at a concentration of 0.5% farnesol. This effect was observed with all six of the test strains evaluated and dilutions to 0.125% farnesol continued to prevent filamentous growth for both drug resistant and community strains. DOI: 10.4236/aim.2020.108028 388 Advances in Microbiology N. Yar et al. Figure 2. Average values for 3 experiments are shown. Growth after 24 hours is indicated by events/uL in the FSC channel (left). Right panel shows % of yeast staining with propidum iodide based on fluorescence greater than baseline FL3 signal. Figure 3. Counts were obtained by flow cytometry and showed that with farnesol (blue bars) consistently produced fewer counts than without farnesol (orange bars). For the entire data set, the difference was significant (p = 0.05 paired T test). Figure 4. Images of morphology of CA-2 strain of Candida albicans grown for 24 hours in BHI (left panels) or with 0.05% farnesol in BHI (right panels). Photos were obtained using the BioRad Zoe imager with an initial magnification of approximately 100 which can be enlarged to approximately 400. Markers indicate actual scale when printed). Upper panels are wide view to show maximum number of organisms (markers indicate 100 uM) and lower panels show detail at higher magnification (markers are 25 uM). Note that both conditions show yeast with buds and daughter cells indicating that neither had stopped growth. DOI: 10.4236/aim.2020.108028 389 Advances in Microbiology N. Yar et al. These findings aligned with the prior observations in which farnesol added to spider medium inhibited the development of the spidering effect seen in the same medium without farnesol. 3.5. Dose Response Hyphal growth limitation persisted at concentrations as low as 0.125% titration of farnesol and consequently concentrations using serial ten-fold dilutions was employed to determine what concentration was required for diminished growth. Due to the number of samples involved, turbidity measurements were used for this experiment. The highest farnesol content in this experiment was 0.5%. Four serial tenfold dilutions were made to 0.005% and an intermediate 1:2 dilution series (0.025% through 0.0025%) were included for more sensitive endpoint discrimination. As shown in Figure 5, the resistant organisms and community organisms had similar inflection points on the dose-response curve (between 0.05 and 0.025%), although the inter-strain variation, not surprisingly, appeared greater with the resistant organisms compared to the community organisms. 3.6. Stress Response While growth was restricted in cultures treated with farnesol, resistance to stress could not necessarily be expected with farnesol treatment. Thus, a heat challenge (50C for 20 minutes) was undertaken to determine if farnesol altered its response to this stressor. The data are presented in Figure 6 shows as expected, all cultures except CA-3 exposed to the heating regimen increased the PI staining, of note, CA-3 in another test (Table 1) did show a 1.9 fold increase in PI staining after heat exposure suggesting this observation was anomalous. To consider the baseline PI staining of each culture, the fold-increase in PI staining before and after heat treatment was employed as an index of the response to heat. Table 2 summarizes these data. The trend shown by this comparison suggests farnesol enhances heat susceptibility but whether the resistant or community organisms are more susceptible to heat is not clear. Figure 5. Titration of growth inhibition employed a range of farnesol concentrations and both drug resistant (blue line) and community organisms (orange line) showed a similar inflection point. Signficance was determined by paired T test comparing the growth in the 0.5% farnesol and BHI control. DOI: 10.4236/aim.2020.108028 390 Advances in Microbiology N. Yar et al. Figure 6. The test organisms were grown without (left panel) or with farnesol (right panel). The baseline PI staining was determined prior to heat treatment (blue bars). The orange bars indicate the PI staining after heat treatment. Table 2. Heat Stress Index for cultures exposed to farnesol compared to controls not treated with farnesol. The index is fold increase in the PI staining (% cells with staining intensity above 1 103 in the Fl-3 channel) by dividing heated by unheated values. The index for farnesol treated cells compared to controls attained statistical significance (p = 0.037 paired T test). Candida strain Control Farnesol (0.05%) CA-A 3.03 4.31 CA-B 1.59 1.98 CA-C 0.51 1.9 CA-1 1.33 2.33 CA-2 1.29 2.53 CA-3 1.13 3.08 3.7. Evaluation of Persisters Candida albicans cultures can generate a portion of the population that enters a quiescent state, also known as the persister phenotype. This is associated with organisms sensing the existence of a quorum, when approaching the stationary phase of growth or during environmental stress. Quiescent persister cells are characterized by a condensation of their cytoplasmic material and have been reported to be detectable in a density gradient [10]. In this experiment the indixanol gradients (see methods) were divided into upper and lower fractions with the lower, denser, fraction considered to be enriched with persister and dead cells. The fractions were separated, reuspended in water and recovered by centrifugation and flow cytometry was used to enumerate the distribution between upper and lower fractions as seen in Figure 7. It may be seen from these data that farnesol increases the proportion of the population found in the lower fraction which is consistent with farnesol pushing organisms toward the persister phenotype. Because cells were reconstituted quantiatively in water containing propidium iodide, it was possible to simultaneously determine staining that was consistent with dead cells. This is important because persister cells are considered to be still alive though quiescent, and should still have intact membranes that prevent propidum iodide staining. Of note in Figure 8, there appears a tendency for higher PI staining in control versus farnesol exposed cultures which would suggest a greater fraction of farnesol treated cultures harbor persister cells. DOI: 10.4236/aim.2020.108028 391 Advances in Microbiology N. Yar et al. Figure 7. After recovering upper and lower fractions from indixanol separation, each fraction was washed and reconstituted in water containing propidum iodide and cell counts for each fraction were determined in the forward scatter channel of the flow cytometer. Absolute counts from each sample allowed calculation of the percent of the whole population in the lower fraction (see also Table 3). Figure 8. Propidium iodide staining of upper and lower fractions from indixanol separations indicating the percent of events from the upper and lower fractions which stain above the fluorescence threshold. Table 3. Effect of farnesol on percent of events represented by the lower fraction of indixanol separations. % of culture in lower fraction Candida albicans Strain % of cells in lower fraction not PI stained Estimated Fraction of persisters corrected for dead cells* Control Farnesol Control Farnesol Control Farnesol CA-A 3.8 51.5 61.4 76.5 0.023 0.394 CA-B 22.6 19.7 7.4 46.3 0.017 0.091 CA-C 23.2 58.8 45.1 60 0.105 0.353 CA-1 18.9 54.8 41.6 70.3 0.075 0.385 CA-2 58.8 80.6 64.4 59 0.379 0.476 CA-3 57 70.8 52.4 57.7 0.298 0.409 *To estimate the fraction of persisters in the lower fraction of the indixanol gradient, the fraction of total cells from the entire sample that were detected in the lower fraction was multiplied by the proportion of cells in the lower fraction that excluded PI. DOI: 10.4236/aim.2020.108028 392 Advances in Microbiology N. Yar et al. This consideration is exploited in Table 3 in which the absolute counts from Figure 7 and Figure 8 are used to predict the percent of the original culture that comprises the persister population. The data in Table 3 supports the likelihood of farnesol promoting persister development. 4. Discussion During its growth, Candida albicans produces the sesquiterpene alcohol farnesol, which among other compounds is an autoinducer of quorum sensing, upregulating virulence genes related to biofilm formation and influencing morphogenesis among many other biological activities [11]. While quorum sensing is a naturally occurring process that helps assure survival, farnesol displays other disparate properties including those directed at human host, modulating immune reactions [12] and enhancing innate immune reactions [13]. Farnesol may also modulate efflux pump activity altering response to antifungal drugs although up or down regulation may relate to concentration [6] [7]. Because of the breadth of biological activities attributed to farnesol, it is a reasonable to consider whether exogenously applied to Candida colonized tissues may be beneficial or not. In these studies, experiments were conducted with a maximum of 0.5% farnesol. This amount would be equal to 2.25 102 M/L, however because of its immiscibility, molarity was considered an inappropriate description. For comparison to other literature, however, concentrations reported in various studies ranged from 30 uM to 3 mM [5] [8] [14] [15]. In the present study that range would be covered by the titration (Figure 5) which showed the dose of farnesol that decreased growth yield by 50% was 0.0035% which would be comparable to 1 - 2 mM. Other results including suppression of hyphal growth were in consonance with other literature [5]. Two aspects of the current study should be highlighted. First, is by using flow cytometric analysis, it was possible to determine simultaneously the number and relative size of microorganisms evaluated as well as the state of cytoplasmic membrane integrity. The second aspect of this work was the comparison of multi-drug-resistant strains to community strains of Candida albicans. Each of the three drug-resistant was multi-resistant. According to ATCC, the supplier of these strains, CA-A was resistant to anidulafungin, caspofungin, micafungin, voriconazole, itraconazole and fluconazole as well as intermediate resistance to 5-fluorocytosine. CA-B was resistant to anidulafungin and the three afore mentioned triazole antifungals. CA-C had resistance to only two echinocandins, anidulafungin and caspofungin and interestingly, appeared slightly more robust than the test strains with multi-class resistance. For the entire set of strains, available flow cytometry indicated the drug resistant set appeared less fit than the community organisms in terms of growth yield, relative size of the individual cells, intrinsic viability and viability after heat challenge. For logistical reasons most of the observations were made with subset of the larger culture collection, but some of the important observations suggest DOI: 10.4236/aim.2020.108028 393 Advances in Microbiology N. Yar et al. that it will be worthwhile in the future to expand the number of strains tested in specific experiments, especially in view of the larger standard deviations encountered in the resistant versus community organisms. These observations will be relevant to the research question engendered by the common use of farnsol as an ingredient in over-the-counter products that may contact tissues colonized by or infected with Candida albicans. Generally, the restriction of growth and hyphal formation would seem beneficial as would the increase in membrane fragility observed both before and after heat challenge. However, this work was expanded to consider the possibility that the quorum sensing activity of farnesol may affect the development of the persister phenotype [16] which could have relevance to Candida survival in or on tissues. Using a previously reported method [10], farnesol-treated and untreated cultures were separated by density gradient to produce a subpopulation of cells putatively enriched for quiescent persister cells. Because the same fraction was also expected to contain dead cells, the use of propidium iodide staining further characterized the persister rich fraction allowing distinction between active (persister) and inactivated (dead) cells. The ability to use absolute particle (cell) counts allowed an overall estimation of the effect of farnesol on persister development. In all six of the strains evaluated, farnesol appeared to increase the persister population after 24 hours exposure of the cultures. The overall implication, which will require additional experimental exploration, is that while farnesol may be beneficial as a component of topical products, it may also generate a higher proportion of quiescent persister phenotypes that could prolong colonization or later recrudescence of the organism. Another balancing of activities was reported as farnesol signaling through the RAS pathway both induced higher hydrogen peroxide formation but simultaneously induced systems that detoxify hydrogen peroxide [15]. Researchers have considered whether farnesol might be of value in conditions such as vulvovaginal candidiasis especially because it has also been attributed with biofilm reducing capability [14]. In a study that used a mouse vaginitis model, farnesol alone was not protective and did not benefit infection resolution from fluconazole-sensitive organisms, but some synergy occurred with fluconazole was seen when fluconazole resistant organisms were tested [17]. The potential for benefit in drug resistant cases remains of interest. An older study [18] says farnesol synergized amphotericin B, micafungin and fluconazole in vitro. Another reported fluconazole synergized the effect of fluconazole and 5-fluorocytosine but antagonized terbinafine and itraconazole. Some molecular mechanisms that may be involved include efflux pump modulation and the related expression of the stress protein CDR-1 [6] [7] and partially explain synergism. In summary, the potential for the use of farnesol as an inhibitor of Candida albicans alone may have limited value, but its addition to other therapies might have benefits worth exploring. But the potential role of persisters in the colonized tissues and other substances with intrinsic antimicrobial activity in vivo [19] will need to be considered as farnesol is incorporated into consumer products. DOI: 10.4236/aim.2020.108028 394 Advances in Microbiology N. Yar et al. In addition, the focus on farnesol as an additive to consumer products has not delved into the fact that additional quorum sensing mediators exist is Candida and may serve as redundant systems or may simply be overwhelmed in the presence of large concentrations of farnesol. In any case, careful consideration of the biological effects of this and other components of products in contact with colonized tissues should be included in formulation considerations. Declaration The research presented here represents the original work of the authors, all of whom contributed to the planning, research, data collection and reduction and manuscript preparation and review. This manuscript has not been submitted elsewhere and has not previously been published. Conflicts of Interest The authors declare no conflicts of interest regarding the publication of this paper. References DOI: 10.4236/aim.2020.108028 [1] Mehmood, A., Liu, G., Wang, X., Meng, G., Wang, C. and Liu, Y. (2019) Fungal Quorum-Sensing Molecules and Inhibitors with Potential Antifungal Activity: A Review. Molecules, 24, 1950. https://doi.org/10.3390/molecules24101950 [2] Polke, M., Leonhardt, I., Kurzai, O. and Jacobsen, I.D. (2018) Farnesol Signalling in Candida albicansMore than Just Communication. Critical Reviews in Microbiology, 44, 230-243. https://doi.org/10.1080/1040841X.2017.1337711 [3] Schnuch, A., Uter, W., Geier, J., Lessmann, H. and Frosch, P.J. (2004) Contact Allergy to Farnesol in 2021 Consecutively Patch Tested Patients. Results of the IVDK. Contact Dermatitis, 50, 117-121. https://doi.org/10.1111/j.0105-1873.2004.0313.x [4] Nikoomanesh, F., Roudbarmohammadi, S., Khoobi, M., Haghighi, F. and Roudbary, M. (2019) Design and Synthesis of Mucoadhesive Nanogel Containing Farnesol: Investigation of the Effect on HWP1, SAP6 and Rim101 Genes Expression of Candida albicans in Vitro. Artificial Cells, Nanomedicine, and Biotechnology, 47, 64-72. https://doi.org/10.1080/21691401.2018.1543193 [5] ernkov, L., Jordao, L. and Bujdkov, H. (2018) Impact of Farnesol and Corsodyl on Candida albicans Forming Dual Biofilm with Streptococcus Mutans. Oral Diseases, 24, 1126-1131. https://doi.org/10.1111/odi.12873 [6] ernkov, L., Diov, S., Gkov, D., Jankov, I. and Bujdkov, H. (2019) Impact of Farnesol as a Modulator of Efflux Pumps in a Fluconazole-Resistant Strain of Candida albicans. Microbial Drug Resistance, 25, 805-812. https://doi.org/10.1089/mdr.2017.0332 [7] Liu, Z., Rossi, J.M. and Myers, L.C. (2018) Candida albicans Zn Cluster Transcription Factors Tac1 and Znc1 Are Activated by Farnesol to Upregulate a Transcriptional Program Including the Multidrug Efflux Pump CDR1. Antimicrobial Agents and Chemotherapy, 62, e00968-18. https://doi.org/10.1128/AAC.00968-18 [8] Diov, S., ernkov, L. and Bujdkov, H. (2018) The Impact of Farnesol in Combination with Fluconazole on Candida albicans Biofilm: Regulation of ERG20, ERG9, and ERG11 Genes. Folia Microbiologica, 63, 363-371. https://doi.org/10.1007/s12223-017-0574-z 395 Advances in Microbiology N. Yar et al. [9] Fernandes Costa, A., Evangelista Araujo, D., Santos Cabral, M., et al. (2019) Development, Characterization, and in Vitro-in Vivo Evaluation of Polymeric Nanoparticles Containing Miconazole and Farnesol for Treatment of Vulvovaginal Candidiasis. Medical Mycology, 57, 52-62. https://doi.org/10.1093/mmy/myx155 [10] Quasem, I., Luby, C.J., Mace, C.R. and Fuchs, S.M. (2017) Density Separation of Quiescent Yeast Using Iodixanol. BioTechniques, 63, 169-173. https://doi.org/10.2144/000114596 [11] Padder, S.A., Prasad, R. and Shah, A.H. (2018) Quorum Sensing: A Less Known Mode of Communication among Fungi. Microbiological Research, 210, 51-58. https://doi.org/10.1016/j.micres.2018.03.007 [12] Vivas, W., Leonhardt, I., Hnniger, K., Hder, A., Marolda, A. and Kurzai, O. (2019) Multiple Signaling Pathways Involved in Human Dendritic Cell Maturation Are Affected by the Fungal Quorum-Sensing Molecule Farnesol. The Journal of Immunology, 203, 2959-2969. https://doi.org/10.4049/jimmunol.1900431 [13] Zawrotniak, M., Wojtalik, K. and Rapala-Kozik, M. (2019) Farnesol, a Quorum-Sensing Molecule of Candida albicans Triggers the Release of Neutrophil Extracellular Traps. Cells, 8, 1611. https://doi.org/10.3390/cells8121611 [14] Sebaa, S., Boucherit-Otmani, Z. and Courtois, P. (2019) Effects of Tyrosol and Farnesol on Candida albicans Biofilm. Molecular Medicine Reports, 19, 3201-3209. https://doi.org/10.3892/mmr.2019.9981 [15] Deveau, A., Piispanen, A.E., Jackson, A.A. and Hogan, D.A. (2010) Farnesol Induces Hydrogen Peroxide Resistance in Candida albicans Yeast by Inhibiting the Ras-Cyclic AMP Signaling Pathway. Eukaryotic Cell, 9, 569-577. https://doi.org/10.1128/EC.00321-09 [16] Wittman, E., Yar, N. and Larsen, B. (2020) Navigating the Third Frontier of Antimicrobial Therapy to Support Womens Health. Open Journal of Obstetrics and Gynecology, 10, 1011-1035. https://doi.org/10.4236/ojog.2020.1080096 [17] Boz, A., Domn, M., Majoros, L., Kardos, G., Varga, I. and Kovcs, R. (2016) The in Vitro and in Vivo Efficacy of Fluconazole in Combination with Farnesol against Candida albicans Isolates Using a Murine Vulvovaginitis Model. Journal of Microbiology, 54, 753-760. https://doi.org/10.1007/s12275-016-6298-y [18] Katragkou, A., McCarthy, M., Alexander, E.L., et al. (2015) In Vitro Interactions between Farnesol and Fluconazole, Amphotericin B or Micafungin against Candida albicans Biofilms. Journal of Antimicrobial Chemotherapy, 70, 470-478. https://doi.org/10.1093/jac/dku374 [19] Weber, K., Schulz, B. and Ruhnke, M. (2010) The Quorum-Sensing Molecule E,E-FarnesolIts Variable Secretion and Its Impact on the Growth and Metabolism of Candida Species. Yeast, 27, 727-739. https://doi.org/10.1002/yea.1769 DOI: 10.4236/aim.2020.108028 396 Advances in Microbiology  ...

... Open Journal of Obstetrics and Gynecology, 2020, 10, 1011-1035 https://www.scirp.org/journal/ojog ISSN Online: 2160-8806 ISSN Print: 2160-8792 Navigating the Third Frontier of Antimicrobial Therapy to Support Womens Health Emma Wittman, Neela Yar, Bryan Larsen* College of Osteopathic Medicine, Marian University Indianapolis, Indianapolis, IN, USA How to cite this paper: Wittman, E., Yar, N. and Larsen, B. (2020) Navigating the Third Frontier of Antimicrobial Therapy to Support Womens Health. Open Journal of Obstetrics and Gynecology, 10, 1011-1035. https://doi.org/10.4236/ojog.2020.1080096 Received: July 1, 2020 Accepted: August 3, 2020 Published: August 6, 2020 Copyright 2020 by author(s) and Scientific Research Publishing Inc. This work is licensed under the Creative Commons Attribution International License (CC BY 4.0). http://creativecommons.org/licenses/by/4.0/ Open Access Abstract This paper explores one of the underappreciated reasons for lack of efficacy in certain cases of antimicrobial therapy, namely the occurrence of a non-genetic resistance to antimicrobial drugs due to a metabolic quiescence of microorganisms. This review has centered on those microorganisms of particular importance in obstetrics and gynecology and accordingly has reviewed the nature and extent of the persister phenotype in relation to infectious agents affecting womens health. We show how the quiescent persister microbial phenotype represents the next significant issue that could compromise successful antibiotic therapy. A brief history of antimicrobial therapy is provided as context for the problem posed by the persister phenotype. This review has been focused on the current literature having relevance for physicians concerned with womens health. The study of this phenotype has led to increasing understanding of the molecular mechanisms for this state which also provides ideas for rational development of drug candidates to interdict these organisms in human disease and explores the possibility of developing specifically targeted molecules to address persisters, research on screening botanicals, existing drugs and chemicals to discover novel approaches to the clinical consequence of microbial persisters. Of interest in this review, is the return to naturally occurring botanical substances, first to be used as anti-infectives, now being considered as possible agents to address persister microorganisms. Overall this paper aims to provide information tailored especially to the obstetrics and gynecology specialists. Keywords Gynecologic Infection, Obstetric Infection, Antibiotic Therapy, Antimicrobial Resistance, Quiescent Microorganisms, Mechanisms 1. Introduction Infectious disease is no respecter of gender, despite the fact that there are examDOI: 10.4236/ojog.2020.1080096 Aug. 6, 2020 1011 Open Journal of Obstetrics and Gynecology E. Wittman et al. ples of infectious diseases for which attack rates are greater in men than women and other examples in which women have the greater burden of disease. But the special consideration for women is those conditions specifically related to the reproductive apparatus and by extension, the possibility of infectious conditions spreading to or otherwise compromising the fetus with consequences greater in the fetus than in the mother. The topic addressed here relates to both genders but as therapy is considered, the special case of obstetrical and gynecologic implications will be highlighted. 2. Before Germ Theory From time out of mind, human history has recognized the concept of contagion, and astute observations led to approaches to deal with communicable diseases that were based on distance or avoidance. Communicable skin diseases were known to the ancients. Leprosy, for example, is believed to have been mentioned in Hippocratic writing (460 BC) and may have related to various skin conditions including what is known today as leprosy or by eponym as Hansens disease. Quarantine, distancing or banishment was often involved and related, at least in part, to the recognition of communicability. Even though it was not until 1873 that the etiologic bacterium was discovered, the ancients treated leprosy as an infectious disease. The mechanism of contagion would have to wait until the middle of the 19th century when Louis Pasteur and later Robert Koch were able to convincingly identify microorganisms as the pathogenic principal in specific diseases. 3. The First Frontier: Anti-Infective Substances 3.1. History Logic would dictate that if microorganisms are able to cause human disease, a method for defeating disease would lie in defeating microbial pathogens. Quinine, found in cinchona bark, had been used to treat malaria by Andean peoples. Cinchona bark began to be imported to Europe in the 17th century, but its use actually predated Alphonse Laverans recognition in 1880 that a causative parasite was involved in this disease. Certain poisons such as mercury salts were introduced in 1496 for syphilis, even though Treponema pallidum would not be identified until 1905. Five years after the discovery of the spirochete, Hata and Ehrlich introduced an arsenical, salvarsan, which was the first effective medication for syphilis. Paul Ehrlich, a Prussian Doctor working in the latter years of the 19th century found that chemical dyes, particularly methylene blue, had an affinity for microorganisms and reasoned that there could be other dyes or chemical compounds that might be discovered or developed with specificity for microorganisms. His recognition that dyes had selectivity for cellular components led him to the astute conclusion that unless a compound can bind to the etiologic microbe, it cannot exert a damaging effect on it. In particular, he expressed hope that he DOI: 10.4236/ojog.2020.1080096 1012 Open Journal of Obstetrics and Gynecology E. Wittman et al. would discover therapia sterilans magna or a general sterilizing agent that may come through the development of a compound that specifically attached to pathogen cells as methylene blue did in the case of Plasmodium. Not surprisingly, the early work on chemotherapeutic agents focused on dye molecules, most notably pursued by Bayer in Germany. The concept of Ehrlich became popularized in his term charmed or magic bullet. Combining his knowledge of chemistry and medicine, he was involved in developing the aforementioned arsphenamine as an improved treatment for syphilis. Perhaps as important as the drug itself was the birth of the concept of chemotherapy for infectious disease. Syphilis, it should be pointed out, has been endemic since about 1500 and has been responsible for unaccountable levels of morbidity and mortality over the centuries. One particular concern continuing up to the present is congenital syphilis, which can result in fetal demise or severely compromised offspring. Despite the availability of penicillin as the most effective therapeutic, in 2017 congenital syphilis reached a 20 year high in the US with more than 900 babies infected [1]. This occurs in the face of effective methods for screening, even in a high-resource nation. Despite the unfortunate fact that syphilis treatment opportunities are sometimes missed, the historical legacy of the Ehrlichian concept of a magic bullet is modern day chemotherapeutic and antibiotic compounds. These therapies have saved countless lives and have become the mainstay of treatment and for some diseases, and prevention. Chemotherapeutics such as sulfonamides became available around 1935 and were used for streptococcal infections and in relation to womens health were used to treat childbed fever which was predominantly the result of Group A streptococcus. Their use was limited by several factors that included the bacteriostatic effect, allergies and limitations due to endogenous folic acid. The antibiotic concept was expanded when penicillin was discovered by Alexander Fleming in 1928 (though practical use would have to wait until 1942), who showed that an inhibitor of Streptococcus was elaborated by another microorganisma mold. Accordingly, many more naturally-occurring antimicrobials and synthetic and semi-synthetic antibiotic usage ensued. By the middle of the twentieth century it seemed infectious disease was destined to be conquered if only the right drug compounds might be discovered or invented. 3.2. Antimicrobials of Mono-Etiologic Infections in Women Antibiotics have had an important impact on some of the infectious disease threats to women and infants. As noted above, penicillin has remained the mainstay of syphilis therapy and is guided by detailed protocols in the CDC literature which very specifically addresses syphilis in pregnancy and neonates. It is striking that penicillin remains the required treatment more than 70 years after its discovery. DOI: 10.4236/ojog.2020.1080096 1013 Open Journal of Obstetrics and Gynecology E. Wittman et al. Likewise, Group B streptococcal infection of infants with early or late onset presentation, with its grave consequences, has been addressed by prophylactic use of penicillin or ampicillin. This is employed in conjunction with testing protocols during pregnancy. The major reason for using vancomycin as an alternative to penicillin or related compounds is penicillin allergies and not the lack of susceptibility of the organism to beta-lactam antibiotics. Another Gram-positive pathogen of concern in pregnancy is Listeria monocytogenes, which causes a 13-fold greater infection rate among pregnant women compared to the general population. Overall cases of infection are still low, in the range of 0.27/100,000, according to the CDC. This foodborne illness is usually prevented through avoidance of some foods and careful preparation and handling of foods. However, when an antibiotic is indicated the drug of choice is ampicillin. The intrinsic susceptibility of microorganisms to the inhibitory action of antimicrobials defines the spectrum of activity for the antibiotic. While the spectrum of activity can be relatively stable for some microbe-antibiotic combinations as indicated above, the occurrence of antibiotic resistance represents a challenge to therapy as discussed below. 3.3. Antimicrobial Therapy of Polymicrobial Infections in Women Particular to womens health, are endogenous infections that arise from bacteria that are commensal in the lower genital tract. Some of these organisms are known to be found as polymicrobial communities involved in pelvic inflammatory disease, tubo-ovarian abscesses, post-hysterectomy surgical infections, chorioamnionitis, postpartum endometritis and other related infections. Prior to the advent of molecular methods for characterizing the cervical and vaginal microbiota, culture-based techniques demonstrated that among healthy women, Gram negative facultative organisms as well as Gram negative and positive strictly anaerobic bacteria could be present. These organisms could become the polymicrobial inoculum in infections arising after procedures or parturition. Because cultures of organisms involved in endometritis and post-hysterectomy infections resembled the lower genital tract, empirical therapy was based on obtaining a spectrum of activity to cover most of the common microbial species. Early work done on postoperative and postpartum cultures in women demonstrated that in addition to bacteria present in the lower genital tract prior to the procedure, within a few days from the operative event a proliferation of Gram negative facultative and anaerobic organisms was observed. This phenomenon informed both concepts of therapy of post-procedure infections, but also suggested how prophylactic antibiotics might be used to interdict this shift in lower tract microbiome composition [2] [3]. Antibiotic treatment regimens that covered Gram negative and positive as well as anaerobic organisms led to empiric use of combinations of antibiotics such as clindamycin-gentamicin, or for monotherapy, ampicillin-sulbactam, or DOI: 10.4236/ojog.2020.1080096 1014 Open Journal of Obstetrics and Gynecology E. Wittman et al. cephalosporin such as cefoxitin with spectrum extending to Gram negative anaerobic organisms. Thus, the first frontier for therapy involved the discovery or development of a large array of antibiotic compounds, an expansion of the understanding of which organisms are involved in obstetrical and gynecologic infections and the antimicrobial spectrum inherent in the therapeutic drugs. 4. The Second Frontier: Drug Resistance 4.1. ResistanceAntibiotics Other Property Some of the greatest challenges in antimicrobial therapy have come with acquired antibiotic resistance, though resistance mechanisms, particularly to beta-lactam antibiotics, have existed before the discovery of penicillin. A more expansive discussion of this topic may be found in the excellent review by Bush, which summarizes the large diversity of beta-lactamases known currently and the parallel development of new drugs [4]. Resistance mechanisms to other classes of antibiotics likewise have challenged physicians as the number of antibiotic resistant strains are continuously emerging. Several approaches to antimicrobial resistance have occurred in parallel with focus on various topics. While the intent here is not to review each of these, it is useful to realize that the epidemiology of drug resistance has documented the existence and increases seen in clinical settings. One approach to antibiotic resistance has been the development of semi-synthetic analogs of antibiotics and as well as discovery and development of new classes of drugs, perhaps no better illustrated than in the early development of beta-lactamase-resistant penicillins. Because antibiotic use and abuse introduces selective pressure for increased prevalence of antibiotic resistant strains, the whole topic of antibiotic stewardship has developed and ecological considerations, including antibiotic use in agriculture, has played a part in the overall topic of antibiotic resistance. Finally, understanding of the chromosomal and non-chromosomal mechanisms of resistance as well as transferring resistance among strains has been vigorously pursued by molecular biology approaches. Perhaps one of the most volatile areas of drug resistance pertains to Neisseria gonorrhoeae. In a historical review, Hook and Kirkaldy note that, development of resistance to antibiotics widely used for treatment appears to be an inexorable process [5]. Both genders are affected by this sexually transmissible pathogen, but certain aspects of morbidity seem to have special relevance to women, including the tendency toward asymptomatic occurrence, progression to upper tract complications and the possibility of intrapartum infection of vaginally delivered infants. A review by Unemo and Schafer invoked the term super bug status as they presented a timeline of the demise of the clinical usefulness of emerging (then declining) therapies including sulfonamides, penicillin, penicillin plus streptomycin, spectinomycin, tetracycline, erythromycin, azithromycin and fluoroquinolones [6]. Current CDC guidelines for uncomplicated gonorrhea specify dual therapy with ceftriaxone and azithromycin but continued modificaDOI: 10.4236/ojog.2020.1080096 1015 Open Journal of Obstetrics and Gynecology E. Wittman et al. tions in therapy will undoubtedly be required in the future. 4.2. Resistant Organisms and Womens Health Antimicrobial resistance is also a concern for Candida albicans which is opportunistic, being at times a lower genital tract commensal as well as under alternate circumstances a source of vaginal symptoms. Other less frequently encountered Candida species may also have limited susceptibility to antifungal drugs. Multiple mechanisms are in play including efflux pumps, mutations involving cell wall biosynthesis, and physical barriers to antifungal drug penetration in the form of biofilm. While the classes of antimicrobials available to treat fungal infections are limited and amphotericin B, for example, requires cautious use due to toxicity, a number of novel approaches to defeating antifungal drug resistance are being explored as described in great detail in a recent review [7]. Fortunately, Toxoplasmosis is an uncommon threat to pregnancy in the US but remains highly prevalent elsewhere in the world and is a known threat for congenital infection. According to recent information there are known treatment failures and demonstrated resistance to pyrimethamine and sulfadiazine and clinical consequences remain under scrutiny [8]. Another parasite that is a threat in pregnancy is Plasmodium, especially in falciparum malaria. Current concerns about drug resistance are related to the presence of strains with P. falciparum chloroquine resistance transporter (PfCRT), which has been reported to alter malaria treatment in some geographical areas [9]. While the emphasis thus far has been on the refractoriness of individual microbial species to antibiotic or antifungal drugs, the lower female genital tract is known to have a diverse microbiome and dysbiotic conditions are associated with symptoms as in the case of bacterial vaginosis. The first descriptions of bacterial vaginosis (BV) were accompanied by the discovery of an organism now known as Gardnerella vaginalis and there was enthusiasm at that time that this organism was the etiologic agent of what was then called non-specific vaginitis. Over many years and with both debate and fundamental research, it became accepted that BV represents a state of microbial diversity with a diminution of lactobacilli and an abundance of anaerobic organisms. In the context of this paper, it should be noted that the antibiotic treatment of this condition is oral metronidazole or topical clindamycin, but treatment is not directed toward just one microbial species. In contrast to more classically understood infections, this is a polymicrobial condition and the goal of therapy is not necessarily directed at Gardnerella vaginalis alone, but at a microbial community. BV is also characterized by the presence of a biofilm which is also polymicrobial and the biofilm is considered to be an important part of the condition and likewise an important part of maintaining the microbial community. In addition, because of the refractoriness of biofilms to antibiotic therapy, the biofilm is considered to be a significant element in recrudescent or recurrent BV. BV is common among reproductive aged women and is a risk factor for other conditions such as pelvic inflammatory disease, preterm birth and postpartum infection and is a continuing DOI: 10.4236/ojog.2020.1080096 1016 Open Journal of Obstetrics and Gynecology E. Wittman et al. concern for the obstetrician-gynecologist. The biofilm that complicates antibiotic therapy is part of a larger matter which is considered in the next section. 5. The Third Frontier: The Persister Phenotype Genetics plays a very large role in the overall phenomenon of microbial resistance to therapeutic antibiotics through a broad variety of mechanisms. Chromosomal or extrachromosomal mutations affecting the target of an antibiotic may be ways of genetically acquiring resistance. The results of these genetic changes lead to a strain that propagates its resistance through future rounds of replication. Efflux pumps are also involved in some cases of resistance to antimicrobial drugs and these may have a genetic upregulation of an existing gene. But in this section, emphasis is given to a mechanism of refractoriness to antimicrobial therapy that is epigenetic and related to phenotype rather than genotype. An excellent working definition of the persister is provided by Barrett and co-workers: A subpopulation of slow-growing or growth-arrested cells that have a decreased susceptibility to killing by normally effective cytotoxic agents. Persisters survive treatment with drugs because of their altered metabolism during a temporary state of quiescence and are genetically identical to other, drug-susceptible cells in the population. Persisters may arise stochastically, or in responses to environmental cues such as nutrient starvation [10]. 5.1. Elaborating the Persister Phenotype One of the first general concepts taught in microbiology is the kinetics of microbial (bacterial) growth, which takes the form of the growth curve that consists of the familiar lag phase, logarithmic phase, stationary phase, and the death phase. The classical growth curve is readily demonstrated in a closed system, such as a broth culture containing a finite amount of nutrient and with no means of eliminating bacterial end products. Natural environmentsincluding infected living hostsare open systems and would not be expected to have such a precise population dynamic. But the discovery of quorum sensing is somewhat analogous to the stationary phase in closed cultures, and its characteristics have shaped the understanding of many infectious processes. Microbial ecologists have long known that in natural environments, microorganisms undergo attachment to surfaces, growth in situ, quorum sensing and formation of biofilm. Biofilms are characterized by physical organization that allows them to resist removal through shear forces. The organisms within the biofilm matrix also show a high level of refractoriness to antibiotics and disinfectants. The differences between planktonic growth and biofilm have genetic determinants that guide organisms transition to this highly resistant state. The three dimensional structure of biofilms and the extracellular matrix that both fixes the organism to a substrate and provides a barrier has often been cited as the reason why biofilm organisms are refractory to disinfectants and antibiotics. DOI: 10.4236/ojog.2020.1080096 1017 Open Journal of Obstetrics and Gynecology E. Wittman et al. However, the abundance of persisters within the biofilm matrix are also in play. The process of biofilm formation has elements related to microbial stress responses which provide for microbial survival during adverse conditions and, as will be noted later, processes in biofilm formation and persister formation have overlapping elements. Biofilm has profound implications for medicine, first as an organized mass of microorganisms that form on abiotic surfaces of medical devices such as catheters or other implanted materials. The organisms that are part of the biofilm are difficult to eradicate, but also provide a potential source of systemic infection when viable organisms break away. In addition, biofilm can form on biotic structures. The heart valve vegetation long known to be associated with alpha hemolytic streptococci represents the first recognized medical problem associated with biofilm. Other normally colonized sites such as alimentary tract and genital tract can host biofilms as well, and these have been associated with difficulty in providing therapy to the patients colonized in this manner. In contrast, biofilms may be a mechanism through which beneficial indigenous organisms can stably associate with a host, which indicates that biofilms are not always threats to health. Related to the altered state of microorganisms in biofilm is a concept that had its roots in the earliest history of antibiotic use. Early observations with penicillin showed that cultures exposed to this bactericidal treatment were accompanied by killing of microorganisms, but the process was not complete. This same phenomenon was observed with other physical and chemical sterilization methods. As currently understood, microorganisms possess a pathway that leads to some members of a microbial community to enter a state of dormancy and instead of multiplying as rapidly as the environment will support, some stochastically-determined percentage of planktonic cells exist in this indolent state, which renders them insusceptible or tolerant to antibiotics and other antimicrobial agents. 5.2. Semantic Cautions As mentioned above, survivor populations in antibiotic-treated Streptococcus cultures were recognized decades ago. Describing these cells as persisters rather than survivors has become a more recent nomenclature. Beyond the strictly academic interest in persistership, there is obviously a clinical consideration as well. The clinical implication of this particular microbial phenotype is the possibility that recurrent or recrudescent symptomatic disease may arise from the presence of persisters re-growing after apparently effective therapy. In a semantically pure sense, persistent disease is to be distinguished from the term persister referring to the microbial phenotype, despite the fact that persistent disease may be the result of persister microorganisms. However, it is also possible that persistent or recurrent disease may arise from pharmacologic considerations such as early termination of a bacteriostatic drug or from genetically DOI: 10.4236/ojog.2020.1080096 1018 Open Journal of Obstetrics and Gynecology E. Wittman et al. resistant organisms representing that second frontier of treatment. The latter situation could be confirmed by re-isolation of the causative microorganism coupled with evidence that the organism is stably-resistant to the therapeutic drug. Perhaps no better source for clarity surrounding the distinction between persister organisms and persistent infection and the relevance and relationship between them will be found in the treatment of the topic by Zhang [11]. Here is also described two types of persisters, non-growing and slow growing and the relationship to other phenomena such as bacterial L-forms and biofilm bacteria. The purpose of this paper will be best served by considering the persister phenotype generally and unless otherwise noted, will not focus on different persister types. In addition, it should be noted that additional terms (in addition to survivor) have been applied to persisters, including dormant cells, quiescent cells or inert cells. 5.3. Breadth of the Persister Phenomenon Research on persisters shows that these organisms are associated with a vast array of bacterial species and also extend to fungal pathogens as well. Zhang describes it as a phenomenon common to all bacteria. It would follow that even though the persister phenotype has not been demonstrated in a particular microbial species, it may nevertheless occur and could link to prolongation of clinical diseases [11]. Demonstration of the persister phenotype among bacteria requires experiments conducted on organisms that are capable of laboratory cultivation, and some of the organisms of interest relative to womens health may be difficult or impossible to cultivate. As with organisms only identified through molecular taxonomy, it may be possible to predict the ability of organisms to elaborate persisters through pathways identified by gene sequencing, even among organisms that cannot be cultivated. Because persister organisms have a relationship to persistent disease, it becomes a collateral interest to know why some diseases do not seem to be persistent (for example streptococcal pneumonia) versus others that are persistent (like tuberculosis). This worthy topic will not be explored in this paper, but probably at a minimum, involves differences in the manner in which the innate and adaptive immune systems handle different microbial species in vivo. In addition to the assertion that persister generation applies to all bacteria, it also applies to eukaryotic organisms as well. Among the organisms with relevance to womens health, Candida albicans and other Candida species are of significant importance and have been extensively studied for their pathways to persister generation [12]. As noted in the next section, eukaryotic and prokaryotic mechanisms of persister generation differ, but may have similar implications for chronicity of infectious disease. Protozoan persisters have been described and even in the case of cancer cells refractory to chemotherapy, persister cancer stem cells may play a role in prolongation of disease [13] [14]. DOI: 10.4236/ojog.2020.1080096 1019 Open Journal of Obstetrics and Gynecology E. Wittman et al. Some viral diseases such as those caused by the Human Herpes Virus, have mechanisms for prolonging the life of their host cells [15]. Cytomegalovirus, a concern for pregnant women who may transmit the virus to their offspring transplacentally, is able to induce a specific phenotype of macrophage that is long-lived and resists apoptosis [16]. Other examples that have unique molecular mechanisms exist, but will not be elaborated here. To focus more directly on pathogenic microorganisms that have a specific persister phenotype it will be useful to consider the epigenetic and metabolic properties of bacterial and fungal persister cells. 5.4. Metabolic Mechanisms of Persistence Bacterial persister phenotypes have a high tolerance to antibiotics and are able to survive various doses of bactericidal or bacteriostatic treatments. Once the antibiotics are removed from the environment, the bacteria will begin dividing again and resume the metabolic processes of normally functioning cells. These persisters can also survive other environmental stressors, including nutrient depletion. The persister phenotype is a dormant state that enables the bacteria to survive the environmental stress and antibiotics without reliance on any antibiotic-resistance genes. The general consensus developing around the persister phenotype in bacteria relates to a model known as the Toxin-Antitoxin (T-AT) model. In this system, the Toxin downregulates cellular activities such as replication, biosynthesis and growth. The toxin suppressed by the antitoxin under desirable conditions such as logarithmic growth, in the presence of nutrient repletion. However, under stringent conditions, the Antitoxin is degraded. This allows the Toxin to apply brakes to the cellular systems and prompt the cell into dormancy [17]. Klimina et al. found that numerous strains of Lactobacillus rhamnosus had T-AT systems, therefore driving these strains to express persister phenotypes to evade and survive environmental stressors [18]. From the standpoint of gynecology, probiotic lactobacillus may be spared under conditions of environmental stress. Conversely, the T-AT system may promote the survival of pathogenic organisms under the stress of therapeutic drugs. Yeast species have also been found to contain persister phenotypes, but fungal persistership is mechanistically different from that of bacteria. Yeast persisters can survive antimicrobial agents too, and resume their normal cellular activities and growth once the agents are removed. Eukaryotic persisters, like prokaryotic persister phenotype, are not genetically transmitted. The metabolic machinery that allows for persistence is genetically programmed, but the persister state itself is not inherited. It should be mentioned that some mutations have been found in yeast to promote a propensity to produce persisters (high persister strains) and the propensity is heritable [19] [20]. In yeast species, the target of rapamycin complex 1 (TORC1) pathway alters cell growth by various mechanisms: cell cycle initiation and translation of ribosomal proteins. TORC1 itself is in turn regulated by rapamycin and cell nutriDOI: 10.4236/ojog.2020.1080096 1020 Open Journal of Obstetrics and Gynecology E. Wittman et al. ents. Rapamycin treatment induces a stressful environment for the yeast and increases expression of the persister phenotype. Bojsen et al. did a study with Saccharomyces cerevisiae mutants, which had inhibited TORC pathways and found that they had an increased persistence after amphotericin B treatment. This result made it appear that amphotericin B induces persister phenotypes itself by applying environmental stress. Candida albicans and Candida glabrata have been found to have a higher persister phenotype when exposed to rapamycin. When yeast cells are subjected to a stressor, the TORC1 pathway is inhibited or downregulated, allowing the cell to enter a dormant phase and take on a persister phenotype [21]. 5.5. Identification of Persister Organisms The percentage of a population growing in culture which are persister cells seems to be smallest in logarithmic planktonic growth. As a culture reaches its stationary phase, when replication and death rates equalize, more persisters are to be found. Finally, when microorganisms form biofilm, the biofilm is likely to harbor the highest percentage of persisters. As with studies of the bacterial growth curve, persistership may best be demonstrated in vitro and perhaps cannot truly and irrefutably be demonstrated in vivo. Demonstrating persisters is closely related to the definition of persister, which is a cell that remains viable under conditions that ordinarily render cells in the same culture non-viable. In other words, if an antibiotic is added to a growing culture of bacteria and the culture is allowed to incubate until killing should be complete, subculture shows that a number of cells will be able to recover in fresh media, though their recovery will be characterized by a very slow growth rate. This release from the persister state also seems to vary within the same culture, with some individual cells rebounding faster than others. Persister resuscitation results in small colonies described as deep persisters and larger colonies described as shallow persisters while viable non-persisters produce the largest colonies. This phenomenon can be demonstrated on agar plates on which a presumably sterilized culture is plated. Over a period of time small colonies will begin to form, much slower than normal colonies would. These colonies, though small, will be of varying sizes which implies that persisters recovering from their persister state have differing levels of indolence, even in a pure culture of the same species. This variation among persisters has led to the concept of deep and shallow persisters, somewhat analogous to deep or shallow sleep. 5.6. Persisters in Obstetric and Gynecologic Infections 5.6.1. Bacteria Many bacterial species are involved in infections or related conditions that impinge on the wellbeing of female patients. In the paragraphs that follow, we briefly catalog significant species that have been documented to produce persisters. If persisters have not been identified, we will discuss the evidence that DOI: 10.4236/ojog.2020.1080096 1021 Open Journal of Obstetrics and Gynecology E. Wittman et al. persisters are likely because the persister phenotype is considered a common feature of bacteria and the absence of persisters would generally be considered anomalous. This discussion has been organized generally in the order of prevalence of these organisms. Bacterial vaginosis (BV) is one of the most commonly encountered conditions in women. While it is considered to be a dysbiotic condition of the vaginal microbiome, virtually all cases have an abundance of Gardnerella vaginalis in concert with a diversity of Gram negative bacteria and reduced abundance of Lactobacillus species. Although we have not identified specific reports of persisters among Gardnerella, several clinical features are consistent with a role for persisters. Despite its frequency, recurrent episodes are common, even after treatment with metronidazole or clindamycin. One reason for recurrent BV may be polymicrobial biofilm that includes Gardnerella vaginalis. Biofilms in general are considered to have a significant level of persisters as will be subsequently addressed further. This polymicrobial biofilm is characteristic of BV. Such a biofilm promotes recurrent infection in over 50% of patients within 6 - 12 months after initial treatment [22]. A 2010 experiment testing the response of the polymicrobial biofilm to the antibiotic moxifloxacin found that within 5 days post-treatment, a surge in Gardnerella vaginalis cell numbers due to persisting biofilm was identified [23]. Subsequent studies suggested inhibition of biofilm production using quorum-inhibiting agents or disruption of existing biofilm using boric acid could, in combination with bacteriostatic or bactericidal medications, provide improved efficacy in preventing recurrence [24] [25]. Urinary tract infection, frequently due to Gram negative enteric bacteria, may also be a recurrent clinical problem and persister organisms with mucosal biofilms are implicated. UTIs commonly occur during pregnancy with further complications such as pyelonephritis and increased risk of maternal sepsis, preterm labor, and premature delivery in pregnant women [26]. Escherichia coli is the most common bacterial pathogen of UTI in women and persister populations have been identified in a number of studies [27] [28]. Literature suggests that overexpression of chromosomal Type II TA toxin molecules may be related to development of persistership by inhibiting cell growth after exposure to environmental stressors [27] [29]. Although this is a well-studied phenomenon, it is unclear as to whether the TA system is the main mechanism by which persistership arises and the exact role of (p)ppGpp signaling (see later discussion) drives the persister generation [27] [30]. This persister-development model has been applied to different types of bacteria to explain how persisters arise and is currently being further studied. Historically some of the earliest evidence that antibiotic survival was due to the persisters in a population was discovered among Streptococci. Group A Streptococcus (GAS) was scourge in obstetrics in the mid 1800s as childbed fever, stemming from GAS infection, contributed to significant maternal mortality. Today GAS infections in developed countries are infrequent, but still cause about 40% of septic deaths among patients with postpartum endometritis, neDOI: 10.4236/ojog.2020.1080096 1022 Open Journal of Obstetrics and Gynecology E. Wittman et al. crotizing fasciitis, and along with Staphylococcus aureus, toxic shock syndrome [31]. Biofilm formation in GAS has been documented and hypothesized to be part of the cause for failed antibiotic treatment of GAS infections. The antimicrobial resistance of GAS biofilms can be partly attributed to the fact that cells within the biofilm have reduced metabolic activity, which typifies the persister phenotype [32]. Streptococcus mutans and Streptococcus suis persister phenotypes have been identified which furthers the notion that GAS organisms likely have persister phenotypes as well [33] [34]. Group B Streptococcus (GBS) is found within the gastrointestinal tract and genitourinary tract as a commensal organism in as frequently as 30% of women. GBS has declined as a cause of perinatal and neonatal disease due to aggressive screening and prophylaxis programs, but presence and continued colonization of women remains a cause for vigilance. Korir et al. state GBS is the leading cause of neonatal sepsis and meningitis, adding to the importance of treating and eradicating these infections in pregnant patients, which unfortunately may recolonize the vaginal flora even after antibiotic treatment. Different GBS strains can survive inside macrophages, evade the host defense and lead to disseminated infection. GBS surviving antibiotic treatment can also be partly attributed to its ability to form biofilms, which as previously mentioned hints at the fact that GBS can undertake persister phenotypes [35]. The variety of streptococcal species found to have persister phenotypes suggests a commonality that should encompass GBS. Chlamydia trachomatis is another high-prevalence infection with significant consequences for the female patient. In addition to its prevalence as primary sexually transmitted infection, it is associated with the subsequent complications of salpingitis, tubal scarring, ectopic pregnancy, tubo-ovarian abscess and peritonitis. This Gram negative obligate intracellular bacterium has been shown to have an interesting persister form. Chlamydia is generally characterized as having two different forms, an infectious elementary body (EB) that is extracellular, and a replicative reticulate body (RB) that is intracellular. When exposed to stressors like iron deprivation, Beta-lactam antibiotics or Fosmidomycin, a third form for the bacteria, the aberrant body (AB), arises. When the stress is removed, the AB then converts back to its normal cell type and resumes cell growth and division. The AB is then considered to be a persister phenotype of Chlamydia trachomatis [36]. The persister phenotype allows C. trachomatis to evade antibiotic treatment and reestablish an infection once the drug treatment is finished. This knowledge of Chlamydia persistership needs to be utilized to create antibiotics that will directly target the persister cells and prevent recurrent infections [37]. Neisseria gonorrhoeae is less prevalent than Chlamydia trachomatis, but can also engender complications of pelvic inflammatory disease, tubal infertility, ectopic pregnancy and perinatal infections [38] [39]. A literature search did not show any studies directly identifying persisters, but because the history of antibiotic therapy for Neisseria gonorrhoeae focused mainly on genetic resistance, a DOI: 10.4236/ojog.2020.1080096 1023 Open Journal of Obstetrics and Gynecology E. Wittman et al. search for persister phenotypes deserves further exploration [38]. Described in the early 1970s, Lyme disease caused by the Spirochete of the genus Borrelia, is characterized by a chronic and recurrent set of symptoms and can be extremely challenging to treat even with appropriate antibiotic regimens. In a recent review, a detailed discussion of the persister state of Borrelia burgdorferi described morphologic and metabolic aspects of this organism as a response to antibiotic exposure suggesting that dealing with the persister state in these organisms may be exceedingly important in overcoming the chronicity of infection [40]. The authors also drew parallels to other chronic infections including leprosy, syphilis and tuberculosis. With regard to womens health, Lyme disease is of concern, but certainly less studied than Syphilis, another spirochetal disease. A systematic review by Waddell et al. found 45 relevant reports in the world literature and while the numbers of cases were small, some adverse pregnancy outcomes related to Lyme disease were noted and some indication of transplacental infection was reported [41]. Syphilis, on the other hand, has a larger literature describing both maternal and fetal consequences. Because it cannot be cultivated, there is no demonstration of the persister phenotype in the literature, although there are references to bacterial persistence that seem to relate to persistent infection as opposed to the specific phenotype. 5.6.2. Fungi Candida albicans is typically found in the vaginal canal as a commensal organism. However, overgrowth and the establishment of biofilm increases the risk of persistent infection [42]. The development of persister cells in infectious Candida albicans is well documented and is believed to make up 0.1% - 2% of the total number of cells in a biotic biofilm at any one time [43]. Recurrent vulvovaginal candidiasis (RVVC) refers to 3 - 4 consecutive Candida infections within a 12-month period and remains a clinically challenging entity. A 2019 study identified RAFT-derived polymethacrylates as a more effective treatment for RVVC than popular antifungals like clotrimazole and nystatin. Polymethacrylates have been found to successfully disrupt biotic biofilm formation on vaginal epithelium and therefore inhibit persistership [44]. This has significant implications for women who may suffer from undiagnosed or diagnosed HIV/AIDS, which increases the probability of contracting RVVC and recurrent oral thrush. Because of its generally significant role among immunocompromised hosts, the literature on this organism is voluminous and research efforts to study the persister phenomenon have fostered research but as yet no specific therapy for quiescent forms of the organism. 5.6.3. Protozoa Earlier in this paper parasitic infections that are of concern in pregnancy were noted and specific parasites of interest were Toxoplasma gondii and the agents of malaria, especially P. falciparum. The existence of persisters and their refracDOI: 10.4236/ojog.2020.1080096 1024 Open Journal of Obstetrics and Gynecology E. Wittman et al. toriness to therapeutic drugs is considered consequential for these organisms and in a comprehensive treatment of the topic, a side-by-side comparison with characteristics of bacterial persisters is presented [10]. Bacterial persisters are genetically identical to non-persisters and for the majority of parasites reviewed, the dormant forms are genetically identical to the replicating form. Hypnozoites are well-known stages that occur in P. vivax and apparently in the liver some stochastically generated forms have long periods of dormancy while others revert to schizogony readily. For P. falciparum, the ring form of the parasite is refractory to dihydroartemisinin. For Toxoplasma gondii, it is the bradyzoite that is the cognate of bacterial persisters. 5.6.4. Virus Although viruses are unable to become persisters on the basis of metabolism in the same sense that bacteria and fungi can, viruses such as Herpes Simplex Virus-1 & -2 and Human Papilloma Virus (HPV) can persist in human hosts. One study states that approximately 90% of HPV-infected women will become HPV-negative after 2 years, although certain strains of HPV (such as HPV-16 & -18) are more likely to lead to persistent latent infection. This persistent infection can increase risk of cervical and anogenital cancers 20 years down the line [45] [46]. In order to maintain persistent infection in hosts daughter cells, HPV E2 proteins tether extrachromosomal viral episomes to the host cells mitotic chromosomes. This results in maintenance replication of viral genomes, which ensures that the virus remains in the hosts nucleus without immune system detection and clearance [46]. Prophylactic vaccination with Gaurdasil has proven to be an effective measure in preventing infection by HPV-6, 11, 16, and 18. Herpes Genitalis is caused by Herpes Simplex Virus (HSV)-1 and -2 and is transmitted through direct contact with infected people who have clinically manifested herpes genitalis or asymptomatic viral shedding. Primary infection of HSV is often asymptomatic and is followed by life-long persistent latent infection in the local sensory neural ganglions of the region infected. Latent HSV infection can be reactivated by psychological and environmental factors and often manifests as skin lesions at mucocutaneous sites. In the case of obstetric patients, both primary and recurrent HSV infection of pregnant women can lead to intrauterine viral transmission. This can lead to congenital HSV infection, abortion of pregnancy, or stillbirth [47]. 5.7. The Broader Picture: Stress, Quorum Sensing, Biofilm, Persistership 5.7.1. Growth and Quorum Sensing All of the life processes of living organisms are highly integrated and as noted previously, the persister phenotype is connected to metabolic activities that are highly regulated and metabolic processes connected to persisters, deserve mention as part of the overall place of persisters in the life and death of microorganisms. It is theorized that there are two general ways in which persisters arise, DOI: 10.4236/ojog.2020.1080096 1025 Open Journal of Obstetrics and Gynecology E. Wittman et al. stochastically within an active culture or those that are induced through environmental stressors. The spontaneous persisters are enigmatic, as they are challenging to research and characterize [48]. On the other hand, stress-induced persisters are receiving research attention because of the connection to therapy of human infections and refractoriness to antibiotics [48]. There are a multitude of stressors that promote persister generation including nutritional stress, oxidative stress, antibiotic treatment, heat shock and DNA damage, all resulting in organisms that survive adverse environments through quiescence [48] [49]. Stress, in turn, is connected to other processes including natural progression of organisms from exponential growth to the well-known stationary phase of the growth curve. 5.7.2. Biofilm The stationary phase of the growth curve occurring in a closed environment is characterized by a depletion of nutrients, accumulation of end products of metabolism and the presence of large numbers of organisms. Quorum sensing is a mechanism used by organisms to determine the cell density of their population via autoinducers [50]. The autoinducers are small diffusible signal molecules that mediate cell-cell communication [51]. When the concentration of the autoinducers reaches and then surpasses a certain threshold, specific gene expression stimulates quorum-sensing-regulated processes. This means that once the population is large enough, biofilm formation and virulence processes are regulated via quorum sensing [50]. Bacteria present in biofilms are different from free-living bacteria in the sense that they have more tolerance to antibiotics and host immune response. It appears that these altered characteristics are because of the cells coordinated efforts and activities that are controlled by quorum sensing [51]. The quorum of microorganisms can also sense the presence of a surface to allow the generation of biofilm. As mentioned before, the T-AT system is involved in the bacterial stress response and can alter bacterial metabolism. It has been found that both biofilm and quorum sensing can also be influenced by various T-AT systems to help promote their communication and formation when the host is exposed to stress [52]. Persisters develop from planktonic bacteria collectively forming bacterial colonies within three-dimensional biofilms. Because persisters are found inside the biofilms bacterial community, the host immune response to eliminate persister organisms initially rests on its ability to degrade biofilm itself. The composition of biofilm is that of a thick, aqueous mass of polysaccharides, extracellular DNA, proteins and lipids. Biofilm serves as a physical barrier that prevents host immune cells from penetrating and phagocytosing biofilm bacteria, thereby leaving non-persister and persister bacteria protected. Thus, dealing with biofilm in a clinical setting is a major challenge for the future [53] [54]. As a matter of clinical concern, the presence of antibiotics represents a stressor that may promote some viable organisms to enter the persister state. Investigators have conducted numerous detailed examinations of the mediators, pathDOI: 10.4236/ojog.2020.1080096 1026 Open Journal of Obstetrics and Gynecology E. Wittman et al. ways and results of generalized stress and these findings are informative in the context of antimicrobial therapy. 5.7.3. Generalized Stress In the case of stress-induced persisters, the rise of antibiotic persistence is believed to be mainly due to nutritional or oxidative stress. In the case of nutritional stress, there is evidence that the deprivation of metabolic substrates causes an inhibition of normal metabolic processes and therefore suspends growth. Conversely, there is also maintenance of high-enough levels of ATP to sustain non-growth-related cellular processes in persister cells. This response to nutritional stress is referred to as the Stringent Response in the literature. In relation to oxidative stress, damage to bacterial DNA can elicit the SOS Response, which can also promote persistership [48]. In bacteria the stringent response is connected to (p)ppGpp that serves as an alarmone in what is believed to be a second messenger response system activated by nutrient depletion. As observed in E. coli, the starvation-induced stringent response involves release of (p)ppGpp, which inhibits translation and replication while promoting DNA repair [50]. Studies have shown that across multiple organisms, mutants with an inability to produce (p)ppGpp also have reduced ability to form persisters. Release of (p)ppGpp has been observed to be stimulated by the formation of biofilm itself and is generally accepted to be involved with persistership development in general [55]. 5.7.4. Mediators T-AT As previously discussed, bacterial T-AT has been shown to be related to persistership in stressful environments and is regulated by the stringent and SOS responses [56]. Increased levels of (p)ppGpp activate Type II antitoxin degradation and therefore increases unregulated self-intoxication by toxin [55]. High levels of toxin ultimately inhibit bacterial growth and promote cellular dormancy under the right conditions, such as nutrient and oxidative stress. However, studies have shown that certain environmental stresses such as phosphate starvation, acid stress, and osmotic stress result in upregulation of Type II TA with no increase in persister cells [56]. 5.7.5. The SOS Response The SOS response to DNA damage caused by oxidative stress or antibiotic treatment serves as a complementary pathway for stress signaling, and triggers Type I and II Toxin [56]. The SOS response also promotes DNA repair, which is important for persister resuscitation once the stressor is removed. In (p)ppGppdeficient mutants, studies have shown that SOS-deficient mutant cells show a decreased number of persisters, particularly when treated with DNA-damaging agents [55]. This is related to studies that have observed that the introduction of antibiotics as treatment may also be implicated in the development of persistership and supports the idea that generalized bacterial stress responses are DOI: 10.4236/ojog.2020.1080096 1027 Open Journal of Obstetrics and Gynecology E. Wittman et al. related to increased persistership [48]. 6. Looking Ahead It may be concluded from the foregoing discussion that microbial quiescence or persister phenotype is broadly encountered among microorganisms of concern to womens health. But the follow up question centers on defeating the organisms that persist after other therapeutic measures have been used. Several potential approaches might address this issue, but few proven methods have entered clinical practice. Intact host defenses may be considered as a potential mechanism for managing persisters, but as indicated in an earlier treatment of the topic, knowledge of the host-microbe interaction with persister organisms has not been studied extensively. It would be important to investigate whether microbial virulence is diminished in quiescent organisms, leading to down-regulated immunity or if the quiescent organisms are refractory to host defense mechanisms as they are to antibiotics. Based on past successes, it may be hoped that chemical antimicrobial and disinfecting molecules might emerge that can disable persister cells and inactivate them before the re-emerge from the dormant state. At present, the best example of a drug that targets persisters is pyrazinamide which interdicts persister survival through multiple mechanisms including reduction of CoA needed for survival and disruption of membrane energetics [57]. As previously mentioned, another intracellular pathogen of interest and concern is Chlamydia trachomatis (as well as other chlamydial species) and in an excellent review by Schoborg the factors which promote the persister state which consists of AB, distinct from the well-known EB and RB are driven by stressors that include beta-lactam drugs and interferon gamma [58]. Interferon gamma serves to down-regulate indolamine 2 - 3 dioxygenase to create a dearth of tyrosine. In a recent study, the tryptophan operon of chlamydial isolates from a patient with recurrent disease showed a consistent mutation in the tryptophan biosynthesis pathway that resulted in development of ABs making a connection between persister phenotype and persistent infection [36]. Detailed dissection of the pathways involved in generation of persisters will suggest new targets for persisters in the future. Other stressors may be involved in AB development including iron starvation or other nutrient deprivation which seems to be a common theme for many types of persister organisms [59]. Targeting pathways may depend in part on detailed identification of common and unique elements in various microbial strains. Drug design may be based on such detailed information, but other approaches are possible. The work of Feng et al. [60] explored the stationary phase of Borrelia butdorferi, which is enriched with persister organisms. They found dapsone or daptomycin could be employed in combination with other drugs to reduce persisters. If reliable methods can be developed for isolating persisters, chemical, drug or biological reagents could be screened for the ability to inactivate persisters while in their quiescent DOI: 10.4236/ojog.2020.1080096 1028 Open Journal of Obstetrics and Gynecology E. Wittman et al. states. With an appropriate screening method, large chemical or drug libraries may be screened for new therapeutic candidates. The addition of a drug or compound that has the ability to damage the microorganism even while it is quiescent presents an attractive concept for defeating persister organisms. With respect to Candida persisters, de Oliveria Santos and co-workers have presented an extensive exploration of novel compounds to approach drug resistant strains, although their primary concern did not focus on the persister phenotype [61]. Their paper describes several substances that are associated with human host defense which have promise in this context. These include lysozyme, lactoferrin, and small peptides such as beta defensins. Defensin peptides (or other synthetic peptides) could alter membrane integrity even while the organism is in the quiescent state, and enzymes or other substances could damage structures such as bacterial cell wall, which may limit the ability of organisms to exit the persister state when conditions become conducive to growth. The ability of compounds of plant origin to inhibit drug resistant organisms was also raised in the de Oliveria Santos paper. Persisters may be indirectly targeted as the persister phenotype is intricately involved with microbial stress responses that push populations toward persister generation and biofilm formation. As a result of these relationships, the search for clinical interventions to prevent biofilm may also prevent the accumulation of persisters associated with the biofilm. In their extensive presentation on biofilms of several Candida species, Cavalheiro and Teixeira discussed many of the current efforts directed at preventing biofilm formation [62]. They describe many of the treatments that address biofilms on abiotic surfaces, but also mention some of the compounds with potential effect against biofilms forming on mucosa. Notably, such plant compounds as phenylpropanoids and terpenoids, synthetic peptides and repurposed drugs discovered through screening compound libraries are possible candidates for biofilm interdiction. The parallels with compounds mentioned in the previous paragraph are interesting. The role of transition to hyphal growth is also significant in biofilm formation and compounds that alter this process, such as aspirin, have intriguing implications [63]. A recent study has also indicated lysozyme may be useful in preventing Gardnerella biofilms and can accordingly serve as an adjunct to antibiotics [64]. Several other natural and synthetic compounds have been directed against Gardnerella biofilm including retrocyclin, DNase, chitosan, subtilosin, poly-L-lysine and lauramide ethyl ester [65] [66] [67]. Whether these compounds affect persisters directly, indirectly, or not at all remains to be established. As previously discussed, quorum sensing is also an element in biofilm and persister generation and as a consequence, modulation of quorum sensing may be another mechanism to indirectly limit persistership. The discovery of multiple quorum sensing and quorum inhibiting mediators in Candida albicans suggests the possibility of rational approaches to recurrent and recrudescent vaginitis due to this organism. The breadth of these autocrine mediators is more fully described elsewhere but suggests the possibility of several potential targets to exDOI: 10.4236/ojog.2020.1080096 1029 Open Journal of Obstetrics and Gynecology E. Wittman et al. plore in future work [68]. There are three main quorum sensing mediators in bacteria specific for Gram negative bacteria (acyl homoserine lactone), Gram positive bacteria (autoinducer peptide), or for both Gram negative and Gram positives (autoinducer-2) [69]. Targeting these is predicted to have beneficial effects on microbial virulence and biofilm production. Interestingly, some of the relevant discoveries in this area include phytochemicals with activity against Gardnerella vaginalis biofilm and the previously mentioned subtilosin which inhibits autoinducer-2 in Gardnerella vaginalis [70] [71]. Additional botanicals in the form of essential oil have also been studied as inhibitors of biofilm using various bacterial species as test organisms and activity against Candida biofilms and hyphae development was reported for plant essential oils as well [72] [73]. The future for discovering direct and indirect means for addressing the problem of persisters and in turn, the problem of stubborn and recurrent infections seems bright. Some answers will come from better understanding of the molecular processes of persister generation and targeting specific pathways, while other progress may be made through high throughput screening of drug and chemical libraries. Interest has also turned to natural products from plants and marine organisms. While in this paper we have presented the matter of microbial persisters as the third frontier of therapeutic challenge, we will not identify it as the last. As with therapeutics before, resistance is certain to develop and has precedence even as PZA is being used against Mycobacterial persisters, evidence for occasional refractoriness to that drug is being reported [57]. 7. Summary In this paper we have contextualized the history of antimicrobial therapy in the form of three frontiers, the discovery and development of antibiotics, the threat to antibiotics posed by drug resistance and its genetic spread, and the problem posed by the persister phenotype which is related to an altered metabolic state of quiescence. This phenomenon is widely distributed through the diversity of microorganisms that affect womens health. The study of this phenotype has led to increasing understanding of the molecular mechanisms for this state which also provides ideas for rational development of drug candidates to interdict these organisms in human disease. Despite the possibility of developing specifically targeted molecules to address persisters, work continues on screening botanicals, existing drugs and chemicals to discover novel approaches to the clinical consequence of microbial persisters. The origin of antimicrobials includes naturally occurring botanical substances, like cinchona bark. This may seem unsophisticated by current standards, but we are currently turning again to botanicals and other natural products for potential answers to the persister problem. Declaration This is the original work of the authors, all of whom contributed to the develDOI: 10.4236/ojog.2020.1080096 1030 Open Journal of Obstetrics and Gynecology E. Wittman et al. opment, writing and editing of the manuscript. This work was not supported by external funding and has not been presented elsewhere previously. Conflicts of Interest The authors declare no conflicts of interest regarding the publication of this paper. References [1] The Lancet (2018) The Nobel Foundation Needs to Check Its Privilege. The Lancet, 392, 1168. https://doi.org/10.1016/S0140-6736(18)32359-6 [2] Ohm, M.J. and Galask, R.P. (1976) The Effect of Antibiotic Prophylaxis on Patients Undergoing Total Abdominal Hysterectomy: II. Alterations of Microbial Flora. American Journal of Obstetrics and Gynecology, 125, 448-454. [3] Ohm, M.J. and Galask, R.P. (1975) The Effect of Antibiotic Prophylaxis on Patients Undergoing Vaginal Operations: II. Alterations of Microbial Flora. American Journal of Obstetrics and Gynecology, 123, 597-604. [4] Bush, K. (2018) Past and Present Perspectives on -Lactamases. Antimicrobial Agents and Chemotherapy, 62, e01076-18. https://doi.org/10.1128/AAC.01076-18 [5] Hook, E.W. and Kirkcaldy, R.D. (2018) A Brief History of Evolving Diagnostics and Therapy for Gonorrhea: Lessons Learned. Clinical Infectious Diseases, 67, 1294-1299. https://doi.org/10.1093/cid/ciy271 [6] Unemo, M. and Shafer, W.M. (2014) Antimicrobial Resistance in Neisseria gonorrhoeae in the 21st Century: Past, Evolution, and Future. Clinical Microbiology Reviews, 27, 587-613. https://doi.org/10.1128/CMR.00010-14 [7] Sharma, J., Rosiana, S., Razzaq, I. and Shapiro, R.S. (2019) Linking Cellular Morphogenesis with Antifungal Treatment and Susceptibility in Candida Pathogens. Journal of Fungi, 5, 17. https://doi.org/10.3390/jof5010017 [8] Montazeri, M., Mehrzadi, S., Sharif, M., Sarvi, S., Tanzifi, A., Aghayan, S.A. and Daryani, A. (2018) Drug Resistance in Toxoplasma gondii. Frontiers in Microbiology, 9, 2587. https://doi.org/10.3389/fmicb.2018.02587 [9] Kim, J., et al. (2019) Structure and Drug Resistance of the Plasmodium falciparum Transporter PfCRT. Nature, 576, 315-320. [10] Barrett, M.P., Kyle, D.E., Sibley, L.D., Radke, J.B. and Tarleton, R.L. (2019) Protozoan Persister-Like Cells and Drug Treatment Failure. Nature Reviews Microbiology, 17, 607-620. https://doi.org/10.1038/s41579-019-0238-x [11] Song, X.Y., Cogen, J. and Singh, N. (2013) Incidence of Methicillin-Resistant Staphylococcus aureus Infection in a Childrens Hospital in the Washington Metropolitan Area of The United States, 2003-2010. Emerging Microbes & Infections, 2, 1-5. https://doi.org/10.1038/emi.2013.69 [12] Lafleur, M.D., Kumamoto, C.A. and Lewis, K. (2006) Candida albicans Biofilms Produce Antifungal-Tolerant Persister Cells. Antimicrobial Agents and Chemotherapy, 50, 3839-3846. https://doi.org/10.1128/AAC.00684-06 [13] Cheng, Q., Kyle, D.E. and Gatton, M.L. (2012) Artemisinin Resistance in Plasmodium falciparum: A Process Linked to Dormancy? International Journal for Parasitology: Drugs and Drug Resistance, 2, 249-255. https://doi.org/10.1016/j.ijpddr.2012.01.001 [14] Zhou, J. and Zhang, Y. (2008) Cancer Stem Cells: Models, Mechanisms and ImpliDOI: 10.4236/ojog.2020.1080096 1031 Open Journal of Obstetrics and Gynecology E. Wittman et al. cations for Improved Treatment. Cell Cycle, 7, 1360-1370. https://doi.org/10.4161/cc.7.10.5953 [15] Finzi, D., et al. (1999) Latent Infection of CD4+ T Cells Provides a Mechanism for Lifelong Persistence of HIV-1, Even in Patients on Effective Combination Therapy. Nature Medicine, 5, 512-517. https://doi.org/10.1038/8394 [16] Nikitina, E., Larionova, I., Choinzonov, E. and Kzhyshkowska, J. (2018) Monocytes and Macrophages as Viral Targets and Reservoirs. International Journal of Molecular Sciences, 19, 2821-2845. https://doi.org/10.3390/ijms19092821 [17] Page, R. and Peti, W. (2016) Toxin-Antitoxin Systems in Bacterial Growth Arrest and Persistence. Nature Chemical Biology, 12, 208-214. https://doi.org/10.1038/nchembio.2044 [18] Klimina, K.M., et al. (2013) Identification and Characterization of Toxin-Antitoxin Systems Instrains of Lactobacillus rhamnosus Isolated from Humans. Anaerobe, 22, 82-89. https://doi.org/10.1016/j.anaerobe.2013.05.007 [19] Torrey, H.L., Keren, I., Via, L.E., Lee, J.S. and Lewis, K. (2016) High Persister Mutants in Mycobacterium tuberculosis. PLoS ONE, 11, e0155127. https://doi.org/10.1371/journal.pone.0155127 [20] Schumacher, M.A., et al. (2015) HipBAPromoter Structures Reveal the Basis of Heritable Multidrug Tolerance. Nature, 524, 59-64. https://doi.org/10.1038/nature14662 [21] Bojsen, R., Regenberg, B., Gresham, D. and Folkesson, A.A (2016) Common Mechanism Involving the TORC1 Pathway Can Lead to Amphotericin B-Persistence in Biofilm and Planktonic Saccharomyces cerevisiae Populations. Scientific Reports, 6, Article No. 21874. https://doi.org/10.1038/srep21874 [22] Bradshaw, C.S. and Sobel, J.D. (2016) Current Treatment of Bacterial VaginosisLimitations and Need for Innovation. The Journal of Infectious Diseases, 214, S14-S20. https://doi.org/10.1093/infdis/jiw159 [23] Swidsinski, A., Drffel, Y., Loening-Baucke, V., Schilling, J. and Mendling, W. (2011) Response of Gardnerella vaginalis Biofilm to 5 Days of Moxifloxacin Treatment. FEMS Immunology & Medical Microbiology, 61, 44-46. https://doi.org/10.1111/j.1574-695X.2010.00743.x [24] Algburi, A., Zehm, S., Netrebov, V., Weeks, R., Zubovskiy, K. and Chikindas, M.L. (2018) Benzoyl Peroxide Inhibits Quorum Sensing and Biofilm formation by Gardnerella vaginalis 14018. Infectious Diseases in Obstetrics and Gynecology, 2018, Article ID: 1426109. https://doi.org/10.1155/2018/1426109 [25] Reichman, O., Akins, R. and Sobel, J.D. (2009) Boric Acid Addition to Suppressive Antimicrobial Therapy for Recurrent Bacterial Vaginosis. Sexually Transmitted Diseases, 36, 732-734. [26] Delzell Jr., J.E. and Lefevre, M.L. (2000) Urinary Tract Infections during Pregnancy. American Family Physician, 61, 713-720. https://www.aafp.org/afp/2000/0201/p713.html [27] Matsumoto, S., et al. (2018) Unique Transcriptional Profile of Native Persisters in Escherichia coli. Journal of Bioscience and Bioengineering, 125, 15-22. https://doi.org/10.1016/j.jbiosc.2017.07.015 [28] Barrett, T.C., Mok, W.W.K., Murawski, A.M. and Brynildsen, M.P. (2019) Enhanced Antibiotic Resistance Development from Fluoroquinolone Persisters after a Single Exposure to Antibiotic. Nature Communications, 10, Article No. 1177. https://doi.org/10.1038/s41467-019-09058-4 DOI: 10.4236/ojog.2020.1080096 1032 Open Journal of Obstetrics and Gynecology E. Wittman et al. [29] Holden, D.W. and Errington, J. (2018) Type II Toxin-Antitoxin Systems and Persister Cells. American Society for Microbiology, 9, e01574-18. https://doi.org/10.1128/mBio.01574-18 [30] Harms, A., Fino, C., Srensen, M.A., Semsey, S. and Gerdes, K. (2017) Prophages and Growth Dynamics Confound Experimental Results with Antibiotic-Tolerant Persister Cells. American Society for Microbiology, 8, e01964-17. https://doi.org/10.1128/mBio.01964-17 [31] Rimawi, B.H., Soper, D.E. and Eschenbach, D.A. (2012) Group A Streptococcal Infections in Obstetrics and Gynecology. Clinical Obstetrics Gynecology, 55, 864-874. https://doi.org/10.1097/GRF.0b013e31827362fc [32] Vyas, H.K.N., Proctor, E.-J. Mcarthur, J., Gorman, J. and Sanderson-Smith, M. (2019) Current Understanding of Group A Streptococcal Biofilms. Current Drug Targets, 20, 982-993. https://doi.org/10.2174/1389450120666190405095712 [33] Dufour, D., Mankovskaia, A., Chan, Y., Motavaze, K., Gong, S.G. and Lvesque, C.M. (2018) A Tripartite Toxin-Antitoxin Module Induced by Quorum Sensing Is Associated with the Persistence Phenotype in Streptococcus mutans. Molecular Oral Microbiology, 33, 420-429. https://doi.org/10.1111/omi.12245 [34] Willenborg, J., Willms, D., Bertram, R., Goethe, R. and Valentin-Weigand, P. (2014) Characterization of Multi-Drug Tolerant Persister Cells in Streptococcus suis. BMC Microbiology, 14, Article No. 120. https://doi.org/10.1186/1471-2180-14-120 [35] Korir, M.L., Laut, C., Rogers, L.M., Plemmons, J.A., Aronoff, D.M. and Manning, S.D. (2017) Differing Mechanisms of Surviving Phagosomal Stress among Group B Streptococcus Strains of Varying Genotypes. Virulence, 8, 924-937. https://doi.org/10.1080/21505594.2016.1252016 [36] Slade, J.A., Brockett, M., Singh, R., Liechti, G.W. and Maurelli, A.T. (2019) Fosmidomycin, an Inhibitor of Isoprenoid Synthesis, Induces Persistence in Chlamydia by Inhibiting Peptidoglycan Assembly. PLoS Pathogens, 15, e1008078. https://doi.org/10.1371/journal.ppat.1008078 [37] Witkin, S.S., Minis, E., Athanasiou, A., Leizer, J. and Linhares, I.M. (2017) Chlamydia trachomatis: The Persistent Pathogen. Clinical and Vaccine Immunology, 24, 203-217. https://doi.org/10.1128/CVI.00203-17 [38] Lo, J.Y.C., et al. (2008) Ceftibuten Resistance and Treatment Failure of Neisseria gonorrhoeae Infection. Antimicrobial Agents and Chemotherapy, 52, 3564-3567. https://doi.org/10.1128/AAC.00198-08 [39] Hosenfeld, C.B., et al. (2009) Repeat Infection with Chlamydia and Gonorrhea among Females: A Systematic Review of the Literature. Sexually Transmitted Diseases, 36, 478-489. https://doi.org/10.1097/OLQ.0b013e3181a2a933 [40] Bockel, S., Durand, B. and Deutsch, E. (2018) Combining Radiation Therapy and Cancer Immune Therapies: From Preclinical Findings to Clinical Applications. Cancer/Radiothrapie, 22, 567-580. https://doi.org/10.1016/j.canrad.2018.07.136 [41] Waddell, L.A., Greig, J., Robbin, L., Hinckley, A.F. and Ogden, N.H. (2018) A Systematic Review on the Impact of Gestational Lyme Disease in Humans on the Fetus and Newborn. PLoS ONE, 13, e0207067. https://doi.org/10.1371/journal.pone.0207067 [42] Wuyts, J. and Van Dijck, P.M. (2018) Holtappels, Fungal Persister Cells: The Basis for Recalcitrant Infections? PLoS Pathogens, 14, e1007301. https://doi.org/10.1371/journal.ppat.1007301 [43] Denega, I., DEnfert, C. and Bachellier-Bassi, S. (2019) Candida albicans Biofilms Are Generally Devoid of Persister Cells. Antimicrobial Agents and Chemotherapy, 63, DOI: 10.4236/ojog.2020.1080096 1033 Open Journal of Obstetrics and Gynecology E. Wittman et al. e01979-18. https://doi.org/10.1101/420596 [44] Wu, X.Q., et al. (2019) RAFT-Derived Polymethacrylates as a Superior Treatment for Recurrent Vulvovaginal Candidiasis by Targeting Biotic Biofilms and Persister Cells. Frontiers in Microbiology, 10, 2592. https://doi.org/10.3389/fmicb.2019.02592 [45] Braaten, K.P. and Laufer, M.R. (2008) Human Papillomavirus (HPV), HPV-Related Disease, and the HPV Vaccine. Reviews in Obstetrics & Gynecology, 1, 2-10. [46] Shanmugasundaram, S. and You, J.X. (2017) Targeting Persistent Human Papillomavirus Infection. Viruses, 9, 299. [47] Sauerbrei, A. (2016) Herpes Genitalis: Diagnosis, Treatment and Prevention. Geburtshilfe und Frauenheilkunde, 76, 1310-1317. https://doi.org/10.1055/s-0042-116494 [48] Gollan, B., Grabe, G., Michaux, C. and Helaine, S. (2019) Bacterial Persisters and Infection: Past, Present, and Progressing. Annual Review of Microbiology, 73, 359-385. https://doi.org/10.1146/annurev-micro-020518-115650 [49] Maisonneuve, E., Shakespeare, L.J., Jrgensen, M.G. and Gerdes, K. (2011) Bacterial Persistence by RNA Endonucleases. Proceedings of the National Academy of Sciences of the United States of America, 108, 13206-13211. https://doi.org/10.1073/pnas.1100186108 [50] Van Den Bergh, B., Fauvart, M. and Michiels, J. (2017) Formation, Physiology, Ecology, Evolution and Clinical Importance of Bacterial Persisters. FEMS Microbiology Reviews, 41, 219-251. https://doi.org/10.1093/femsre/fux001 [51] Li, Y.-H. and Tian, X.L. (2012) Quorum Sensing and Bacterial Social Interactions in Biofilms. Sensors, 12, 2519-2538. https://doi.org/10.3390/s120302519 [52] Wang, X.X. and Wood, T.K. (2011) Toxin-Antitoxin Systems Influence Biofilm and Persister Cell Formation and the General Stress Response. Applied and Environmental Microbiology, 77, 5577-5583. https://doi.org/10.1128/AEM.05068-11 [53] Watters, C., Fleming, D., Bishop, D. and Rumbaugh, K.P. (2016) Host Responses to Biofilm. In: Teplow, D., Ed., Progress in Molecular Biology and Translational Science, Elsevier, Amsterdam, 193-239. https://doi.org/10.1016/bs.pmbts.2016.05.007 [54] Maisonneuve, E. and Gerdes, K. (2014) Molecular Mechanisms Underlying Bacterial Persisters. Cell, 157, 539-548. https://doi.org/10.1016/j.cell.2014.02.050 [55] Harms, A., Maisonneuve, E. and Gerdes, K. (2016) Mechanisms of Bacterial Persistence during Stress and Antibiotic Exposure. Science, 354, aaf4268. https://doi.org/10.1126/science.aaf4268 [56] Ronneau, S. and Helaine, S. (2019) Clarifying the Link between Toxin-Antitoxin Modules and Bacterial Persistence. Journal of Molecular Biology, 431, 3462-3471. https://doi.org/10.1016/j.jmb.2019.03.019 [57] Njire, M., et al. (2016) Pyrazinamide Resistance in Mycobacterium tuberculosis: Review and Update. Advances in Medical Sciences, 61, 63-71. https://doi.org/10.1016/j.advms.2015.09.007 [58] Schoborg, R.V. (2011) Chlamydia PersistenceA Tool to Dissect Chlamydia-Host Interactions. Microbes and Infection, 13, 649-662. https://doi.org/10.1016/j.micinf.2011.03.004 [59] Wyrick, P.B. (2010) Chlamydia trachomatis Persistence in Vitro: An Overview. The Journal of Infectious Diseases, 201, S88-S95. https://doi.org/10.1086/652394 [60] Feng, J., Zhang, S., Shi, W.L. and Zhang, S.Y. (2017) Activity of Sulfa Drugs and Their Combinations against Stationary Phase B. burgdorferi in Vitro. Antibiotics, 6, DOI: 10.4236/ojog.2020.1080096 1034 Open Journal of Obstetrics and Gynecology E. Wittman et al. 10-20. https://doi.org/10.1101/112607 [61] De Oliveira Santos, G.C., et al. (2018) Candida Infections and Therapeutic Strategies: Mechanisms of Action for Traditional and Alternative Agents. Frontiers in Microbiology, 9, 1351. [62] Cavalheiro, M. and Teixeira, M.C. (2018) Candida Biofilms: Threats, Challenges, and Promising Strategies. Frontiers in Medicine, 5, 28. https://doi.org/10.3389/fmed.2018.00028 [63] Stepanovi, S., Vukovi, D., Jei, M. and Ranin, L. (2004) Influence of Acetylsalicylic Acid (Aspirin) on Biofilm Production by Candida Species. Journal of Chemotherapy, 16, 134-138. https://doi.org/10.1179/joc.2004.16.2.134 [64] Thellin, O., et al. (2016) Lysozyme as a Cotreatment during Antibiotics Use against Vaginal Infections: An in Vitro Study on Gardnerella vaginalis Biofilm Models. International Microbiology, 19, 101-107. [65] Hooven, T.A., Randis, T.M., Hymes, S.R., Rampersaud, R. and Ratner, A.J. (2012) Retrocyclin Inhibits Gardnerella vaginalis Biofilm Formation and Toxin Activity. Journal of Antimicrobial Chemotherapy, 67, 2870-2872. https://doi.org/10.1093/jac/dks305 [66] Galardini, M., Pini, F., Bazzicalupo, M., Biondi, E.G. and Mengoni, A. (2013) Replicon-Dependent Bacterial Genome Evolution: The Case of Sinorhizobium meliloti. Genome Biology and Evolution, 5, 542-558. https://doi.org/10.1093/gbe/evt027 [67] Turovskiy, Y., et al. (2012) Susceptibility of Gardnerella vaginalis Biofilms to Natural Antimicrobials Subtilosin, -Poly-L-Lysine, and Lauramide Arginine Ethyl Ester. Infectious Diseases in Obstetrics and Gynecology, 2012, Article ID: 284762. https://doi.org/10.1155/2012/284762 [68] Padder, S.A., Prasad, R. and Shah, A.H. (2018) Quorum Sensing: A Less Known Mode of Communication among Fungi. Microbiological Research, 210, 51-58. https://doi.org/10.1016/j.micres.2018.03.007 [69] Brackman, G. and Coenye, T. (2014) Quorum Sensing Inhibitors as Anti-Biofilm Agents. Current Pharmaceutical Design, 21, 5-11. https://doi.org/10.2174/1381612820666140905114627 [70] Qais, F.A., Khan, M.S. and Ahmad, I. (2019) Broad-Spectrum Quorum Sensing and Biofilm Inhibition by Green Tea against Gram-Negative Pathogenic Bacteria: Deciphering the Role of Phytocompounds through Molecular Modeling. Microbial Pathogenesis, 126, 379-392. https://doi.org/10.1016/j.micpath.2018.11.030 [71] Algburi, A., Zehm, S., Netrebov, V., Bren, A.B., Chistyakov, V. and Chikindas, M.L. (2017) Subtilosin Prevents Biofilm Formation by Inhibiting Bacterial Quorum Sensing. Probiotics and Antimicrobial Proteins, 9, 81-90. https://doi.org/10.1007/s12602-016-9242-x [72] Wijesundara, N.M. and Rupasinghe, H.P.V. (2018) Essential Oils from Origanum vulgare and Salvia officinalis Exhibit Antibacterial and Anti-Biofilm Activities against Streptococcus pyogenes. Microbial Pathogenesis, 117, 118-127. https://doi.org/10.1016/j.micpath.2018.02.026 [73] Farisa Banu, S., et al. (2018) Effects of Patchouli and Cinnamon Essential Oils on Biofilm and Hyphae Formation by Candida Species. Journal de Mycologie Mdicale, 28, 332-339. https://doi.org/10.1016/j.mycmed.2018.02.012 DOI: 10.4236/ojog.2020.1080096 1035 Open Journal of Obstetrics and Gynecology  ...

... THE FIORETTI' . . SPRING 1998_ THE FIORETTI A LITERARY ANTHOLOGY VOLUME SPRING MARIAN 56 1998 COLLEGE INDIANAPOLIS, INDIANA STAFF EDITOR/DESIGN NORMAN MINNICK, JR. ADVISOR SR. STELLA GAMPFER, OSF DUPLICATING LARRY ST EEB AND ANITA SMITH CONTENTS POETRY THE THE CONCERT MASTER . . . . . . . . . . . . . . . . . . . THE POETRY & FARM . . . . . . . . . . . . 15 PROSE SLIPPERS . . . . . . . . . . . . . . . . . 8 FOLLOW THE 9 FAITH'S AMUSEMENTS.IO BOSTON . . . . . . . . . . . . . . . . . . 14 7 HOUSE LEADER .. 13 BOY . . . . . . . . . . . . LITTLE ROHE OF MUSIC . . . . . . . . . . . . . . . . . . . BATHROBES 6 PROPHET . . . . . . . . . 11 HOUSE MADE OF WHEAT GERM . . . . . . . . . . . 12 CARRY THE OuT . . . . . . . . . . .. 18 COTTAGE MULTIPLE WATER OF PA SS ION S . . 20 LILLIES . . . . . . . . 24 POETRY THE CONCERT MASTER His deft fingers moved softly along her slender neck enticing sweet sounds from her flat stomach. He drew his bow across her smooth body slowly and her strings quivered in restrained power. She lay silent under his chin and the music was quiet. One slight movement of his arm and her body came alive, singing the notes of his very being. He gave himself over to the quick, rhythmic beat; one last time, one final note, and she fell quiet beneath his gentle arm. -Rachel Spider 6 THE POETRY OF MUSIC Heard the music of the words ' Langston Hughes Heard the music of the songstress Billie Holiday Heard them both ... The poet spoke Right along with the songstress as If he and she were paired by words And the melody Heard the poet speak words of soulness Heard the songstress sing the blues of Misery Heard the pair together simmer sultry Soulness through poetry and music Heard the poet speak of times only He could express-bigotry-Racism And humanity Heard the songstress sing the blues of misery Only she must have known-backdoorsWhites only-drugs-addiction Heard them both souls of Blackness . . . -Dorris L. Potter 7 BATHROBES & HOUSE SLIPPERS By indifference, and a little preparedness, you too , could begin again . Stealing your place, from the bigger animals. Baring your teeth, and pushing others , out of your way. Maybe if your fur, I didn't shine too much, you could eat alone. And put as much sugar, in your coffie, as you want. -Tim Koberstei~ , 8 FOLLOW THE LEADER The summer I cocked the hat on my head I led my troops out of themselves. They never could understand why I slanted so. -Norman Minnick, 9 Jr. FAITH'S AMUSEMENTS Even though I turn on a horseless carousel, music plays like a million happy memories that ring in my ears like a thousand bumble bees as I reluctantly kneel to pray on mangled knees. Streamers drift from a paralyzed ferris wheel as an evangelist requests momentous ,fees that hover above our heads like mammoth trees and threat to wash us away with the drumming seas. Balloons lie on the floor of the Holy Man's cell even though the politician offered up his jingling keys but fed the prisoner only mushy peas and dry, day-old bread with moldy cheese. . Keeping my balance as I turn is hard a$ hell as I fight this dizzying religious breeze and try to keep tabs on my moral deeds even though all of heaven remains make-believe. -Rachel Wuertz 10 BOSTON "But," He said, turning to his friend huddled down in his coat their collars turned up against the rising cold ""f 1 I marry ... ," . Then he was past me, his hurrying question frozen on the wind of this narrow street in Boston where the sun is sending its last offer across the harbor. -Emily Nicole Persic 11 HOUSE MADE OF WHEAT GERM Grandpa came home from work one day to find his house a blackened sinking pile of cinders in the swimming pool chat once had all the appearance of a useful basement. He spent two weeks in unwashed coveralls digging out cools and charred scraps of pictures, the broken silver chain his wife never quite managed to get repaired, and the toy truck with the heat-bubbled paint flecking off under the scraping of his thumbnail. We worried about him. He moved into a tent in his backyard a.nd. spent his days sifting through the archaeology of his past. It was hard at first, bur after a few months it Was as if it had always been. We h.ad family dinners at my Grandpa's tent. He was a happy man in his last years. He said chat living in a tent made sense, it made everything less tentative. And chat had made all the difference. -L. Arwood 12 THE PROPHET He paces at dusk on the shoulder of a highway that twists' and curves like the curls of his hair, aimless, greasy, and a deep slate gray, with his crooked thumb in the air. He believes he was blessed with the gift to predict, but he seeks visions in the powder above his lip and in the pills in his pocket. (It's all part of being a prophet.) From the branches of defunct overhanging trees his mind forms fictitious faces and. he begs forgiveness on shaky knees as they tempt and prod his imagination. }\nd as he prays for the souls who have deemed him a fraud, hovering on the horizon, he swears he sees God bejeweled and robed in unadulterated white. So he strays from the road, and sacrifices distant headlights . -Rachel Wuertz 13 LITTLE BOY The little boy walks along, hand in his dirty jeans, mud on his elbow, Tears begin the hurt to prolong. ,f) He stares into the darkness and thinks himself a fool he's ugly and no body likes him and he's the dumbest boy at school. Love and friendship resound in his ear The world is so loving and caring He horr,ieless, with so many homes open The president has even offered him a room so dear. Why he is sad no ones knows, Why does he need love? No one knows, Why? Why? Why should he belong? -Stephanie K. Kesecker 14 ROHE FARM What they say is true: You can't go back again , But I don't think I want to. I I don't want to see how it's changed. J I want to remember it as my farm , my childhood, Not as the place it has become. On the way to Grandma's I have to pass by. I try not to look, but I can't stop myself I think the hedge was the first to go. Those big bushy trees that lined the winding lane, So ta.11 and overgrown they almost made a timriel. How many times I passed through that tunnel: Kicking hedge apples as I went to check. the ma1L Running with my books as I tried to catch the school bus, Pedaling as fast as I could to beat my brother back to the house. Now it is just a driveway, So plain and ordinary. I can see the windows have changed. The wide panes of old glass are now small, squinting, practical squares. So many sunsets I had seen: J Vibrant, clear, country sunsets. I wonder if anyone even enjoys the sunsets from them now. I am told the maple tree is gone too. What a shade it had provided over the vast sandbox. Farm sandboxes are the best: 15 A huge discarded tractor tire filled with sand, And the sand overflows all around forming a vast island in the yard, The island of so many adventures, Under the maple tree of so many perches for young bodies. _No animals live on the farm anymore. I cannot say that I am fond of pigs or their smell, But they were a sign of life, And the dogs chased them around the pigpen to show off. Now the pen is an empty, dusty desert. Every now and again I hear of something else: The bee tree is gone, The barn is falling down, The swing is rusty, The playhouse is rotting . . . I try,not to listen. I want to keep my memories safe. My farm, my childhood. As soon as I left, my childhood faded. I grew up. Now it is fading, My youthfulness no longer there to give invigoration. Now I will never go back. I can't go back, just as I can't go back to my childhood, But a part of it will always live inside me. My farm, my childhood. -Mary Ellen Crowe 16 PROSE CARRY Our It is a long way from the Yangtze River to Kansas . I don't know the exact mileage, but if the miles were years, it would be centuries between the two places. And yet here they are, this small tribe of Chinese people, hustling about behind the carry-out counter like a bomb is ticking somewhere not very far away and no one can remember when it is going to go off but it might be any time now, especially if someone stops moving. The steam and sizzle of the grills and woks, the crackle of shrimp and vegetables, the smell of garlic and pepper drifts from behind the counter like the breath of a dragon and from the ceiling a few black plastic lanterns dangle. Only one speaks English, a young woman with a concerned look on her face like maybe she has forgotten to bring something with her from China and ~ould like to tell you about it and about why she needs it back but she can't quite bring herself to so she smiles a faint smile and points to the menu and asks you what you . would like because you see they have imported the culinary treasures of China to this country and are prepared to share them with you even though without the sound of the Yangtze River in the background they know their food will always have a hollow spot deep inside where a soul should fit. And you thank her and smile and point to an item or two on the inenu and she says thank you thank you thank you three times just so she's sure. Sometime when you are waiting and no one else is around except the three or four cooks hustling in the back of the restaurant 18 a voice emerges above the exhaust fa ns, an almost feminine vo ice singing louder until it fills the roo m . And the singer turns the corner and sees yo u stops and smiles and even bows slightly at the waist before fading behind the counter. And from then on every time he sees you, you look awkwardly at one another and yo u think how nice it would be if rivers were more portable, if yo u co uld crate one up, or parts of one anyway, _a nd ship it across continents and through the centuries and wind it around this small restaurant four or five times so that when you wanted chis food you would have to step into a long, thin boat on the far shore and wait as an old man rows, -stroke by stroke, across the waters while his children toss out nets for catfish and his wife combs the black river of her hair into the darkening sky as you slowly forget every thing you are supposed to remember until all that matters is the ~ound of the water and tht slowly rising moon and the light in the old man's eyes. -David Shumate 19 THE COTTAGE OF MULTIPLE PASSIONS She spread out the picnic blanket with a giggle and then flopped down upon it like a ragdoll. He crawled beside her, and they began a passionate frenzy of kisses. They were infatuated with the wonders of e'.'lch other and what they liked to chink of as love. They had wandered deep into .the woods to be alone to lose themselves in their rampant, d_reamy gazes and che~sy pet nam~s. They jurriped up again, ar:id ~e began to chase her through chepees as she squealed in .delight. Birds chirped and bits of white flu{( from che draping vines . o f -1!1ilkwee_d drifted in.'~he air ~~kig the.whole th1ng magical to the ._ point, that it ~o~id hav; been nauseating to passing prospec;to;~. ' ' . . ,;We had better get h~me," she breathed pushing him away ;ith a flirty smile . . She toss~d her blonde hair a.~ d said 'T left a .trail of potato chips so that we could find our way back. w~sn't that a good idea?" But, of course, her good idea had turned stale, unlike the potato chips that were snatched up while they were still crispy by several opportunistic black birds. They searched and searched for their way back, and finally collapsed in exhausting efforts that had resulted in nothing. His delicate sweetheart began to cry, and she continued to sob and wail until they came upon a rustic cottage snuggled beneath the overhang of two giant willow trees . They app~oached it carefully, and stared in wonder for, as they got closer, they realized that the house seemed to made with cookies, cakes, and candies. A Gumdrop fence circled licorice daisies, and the door posts were huge, striped candy canes. Icing was spread onto the roof in swirly layers, and the windows appeared to be sheets of sugar. 20  They hesitated before they reached rhe the front wa-lkway of taf~, and glanced at each other sharing the thought rhat something about the scene seemed familiar in an odd wav. The young girl had been dealing with a growling sro mach and couldn't help herself burro grab a chunk of the chocolate cake that made up the wall of the quaint little house and shove it into her petite mouth that she stretched open wide. Her lover looked ,m in horror as she scoffed down the bite leaving her pretty face embellished with crumbs. As they were distracted; the front ?oor had, crept open and a small, hunchback witch )ith_gray hair rhar fell to her heels appeared on the graham cracker of a welcome mar. She \\'.atch,ed rhefl1 for .a moment in silence, for they were not what she had be:::n e~pecring . "HuI1gry?" the witch said sen~ing them whirling aro~nd on their heels to face her. .. . . .... . ~ :. ..., ' They did not speak, but onlyJ00;ked at J:i.e~ wit? terror ri~ing like carb.o nated bu~bles_in rh.e _tpmi,els _pf their throats al}d images of . eternal. curses.~on ,their unb~ri;i children taking sha,pe in their si1:1p1e .. minds. But then the witch smiled, and although her teeth_W<=:re il curious shade of blackish g_r~en! the co~ple found something comf<;ming in jr and followed her intp her bizarre bur exce~dingly sweet L home. ' . The witch fed them piles of food of.the likes they had never seen, and they are. until they could no longer. move, abandoning rhe instincts to be proper rhar had, up to this point , been a hinge on their pubescent romance. The witch made them each a bed , and with the help of one of her muttered chants, they wasted no rime in falling to sleep. When the young man awoke he found himself locked within a huge bird's cage that hung from the rafters. He looked down from 21 his swinging place of incarceration to find his darling slaving before a giant stove. The poor thing was dripping with sweat, and flour and grease tarnished her dainty face in such an unattractive way that the young man took the vision to heart and questioned her potential as his future wife. She certainly didn't seem to be making out well in the way of woman's work, and whatever she was cooking smelled as though it would have been better left raw and in the sun. She brought him a huge platter of food and shoved it up at him with a heave and a glare at the fact that she was .doing aH of the work while he swung leisurely in his little cage. The witch wasn't far. S_he had slipped into a silky black cloak and had the hood pulled over her head. She was singing cheerfully, , occasionally barJcing orders to the girl, ahd reading a cookbook (10 I Ways to Prepare Human Flesh), but the couple, in the absence of their brilliance, did not quite grasp the danger that lurked before -them like a stingray in a bathtub. "Start the oven!" the witch demanded .anc1 began lowering the boys cage from the ceiling. "Tonight I dine on Roasted Wiesenheimer~" "I think she means me!" the young man said to his adored one as the wi-tch's plot finally snapped into place in his head. His fragile lover's chin fell to the floor in terror, and she began to belt out her infamous sobs and wails. "But, I don't know how to start the oven," she whined repeatedly until the witch could take it no longer. With a few choice four letter words, the witch proceeded to stick her grotesque head into the ancient oven leaving her large bottom to sway in the air in order to show the pathetic child how. Finally, the young lady stopped crying and saw an opportunity in the titanic vision that waved before her. She took a desperate glance at her lover, as he 22 himself had begun to cry uncontrollably, and she found her courage. She placed both hands on the oversized cheeks of the witches backside, and pushed as hard as she could. At first, the witch's stout frame did not budge, but all at once her feet slipped out from beneath her, and the girl continued pushing until the witch was lost in the flames of the fresh lit oven in a hair standing howl. The young girl freed her sweetie from his pla<:e of captivity, and they left the house of cakes, cookies, and candies far behind returning soon after they had gone and tied the knot in the excitable high of their adventure in hopes to make the empty house their home. Imagine their distress when they found it to be inhabited by two chubby children with funny names that had eaten their way of most of the ho_use's frame. The young couple retreated with a sigh, hand .in hand, mutually reveling in the odd cognizant air of the whole thing. Something seemed ~o familiar, but, they couldn't quite place a finger on it. They troubled themselves only momentarily, as their brains could not have handled mor~; and so they carried on in the peace of simplicity and lived happily eve~ after. -Rachel Wuertz 23 WATER LILLIES The fascination of being difficult. The girl smiled to herself in the glaring bus windows. Difficult, that's what they called her, difficult. Not one of the beautiful, the cheerleaders, the preppy popular, not one of the outcasts, with downcast eyes and curled shoulders, but silent, tangleq-haired and strange. Her classmates mocked her, made cruel - ' 1, ' fun of her emotionless.face and set _shoU:lders, but n~ver ignored her. Her class had thr~e days in ~aris, a whirl~ind blur of gild~d frames , stone_monwnent~ and t~e Eiffel Tower. The girls giggled ' ' with high school h.umqr at robust nudes in rosy hues, and . . ' .. . : pretenq.ed ~ophistication iJ.t .Picasso's blunt-faced wo~en. S~e walked th~ 1:lluseum halls silendy, often crossing quickly through rooms without a glance, theq. spending a long moment in front of a carved, struggling marble, an oil-based, arrogant beauty or cold, bronze-cast warriors. Their last day they spent the afternoon at the l'Orangierie museum, a small building that once grew oranges for the king and now housed rooms of Renoir's, Degas's, VanGogh's and other great painters of the last hundred and fifty years. The girl walked quickly up the curving stairwells behind her herd of bright-haired schoolmates. She held her back very straight as she walked through the round rooms, and her face held no expression but a small crease between her eyebrows. On one wall, Pierre Renoir's wife smiled down at his golden-headed son, Jean. A thick lock of dark hair fell across her face . The frown in the girl's forehead deepened as she counted the love in every brushstroke. 24 There were four rooms and she walked through some of quickly and others she followed the perimeter past every painting. She heard the other girls giggling at her silent, blank face; they though she was weird. She heard her teachers sigh at her lack of excitement; they thought she was difficult. She only kept her eyes focus straight ahead. Down to the basement they all trailed. There were two oval shaped rooms down there that each held four wall length paintings. When she descended the stairway, she walked quickly into the room and sat on the bench. Behind her the gaggle of girls filled the room with their gasps and exclamations of sudden pleasure. Across the walls the colors burned, blue, purple, red and white, pink, green and gold in tiny splashes. They filled the shabby, windowless room with light and brilliance. The girl's eyes were wide and blank, she clasped her hands tightly together to keep from reaching out and pressing them across the track of colors. Her body was held very still, even when she crossed into the next room, and sat stiffly in there. In her neck, a frail pulse trembled and pounded and against the white skin of her wrist her veins glowed bright. This room she entered was darker dreams of blue, gr~en, purple, with sudden streaks of rose-tinged gold and white. She pressed her lips together until pain, and sat until her throat burned, then turned and walked quickly out of the room without looking back. One cheerleader raised her eyebrows, whispered and giggled to her blue-eyed friend . In the gift shop the girls mingled with other crowding tourists for postcards , prints and silk scarves of waterlilies. The girl stood to one side until a teacher felt pity for her and offered to give her money for a postcard. She looked steadily into the woman's eyes, "I have money." . 25 The bus pulled away as the sun set the skyline of Paris on fire. The girl watched her reflection fade in and out of the window as bus grew quiet, the other girls whispering excitement over their new possessions and falling asleep over half-written postcards. In her mind the colors raced beauty, her fingertips burned and her heart pounded fiercely under her ribs. The young woman let her mind slip softly into unconsciousness, dreaming of gardens and paintings and clasped the knowledge of beauty to herself as she slept. -Emily Nicole Persic 26  ...

... Acceptance Speech, by Elizabeth Spencer Leone (1941) June, 2000, Marian University, Indianapolis, Indiana Recipient of the Francis and Clare Award for Distinguished Achievement in Peace and Social Justice Introduction My life has not been one of major professional accomplishments. Somehow, during my time at Marian College, I began to hear a call to serve God as a Christian lay person. It had to do with social justice -- helping people in need and empowering them to grow and to develop to their full potential. In my effort to respond to this call, I found myself at times with a sense of social maladjustment for Christian reasons. I wanted to encourage people to protest against the personal and institutional injustice of the world about them. Today, I would call this a plea to hear the cry of the poor. During my years at Marian, I began asking if there were any black Catholics who might want to go to Marian. If so, where are they? I discovered that I made people, including some faculty members, quite nervous! You see, I was a scholarship student from Cincinnati in the first 4-year graduating class. Fr. Doyle, my major and minor professor, listened and encouraged me. My dear friend, Mary Jane Lang (Reichsman), who was another of the 5 lay graduates in that first class (1941), understood too. Somehow, I was wrestling with a larger dream of what Marian might become. Early Years This tendency to reflect on my social experience has been a part of me throughout my life. Following graduation from Marian, I chose to prepare for a career in Social work, receiving an MSSW Degree from Catholic University (National Catholic School of Social Service). After a brief professional career (working with Jewish refugees in New York City), I married Fred Leone, whom I had met when he was a graduate student at Georgetown University. We raised a large family. To my present amazement, our children have urned out surprisingly well -- despite the confusion and pain of raising teenagers in the turbulent 60s and early 70s. They seem to have caught my tendency to reflect on their lives. One is a socially constructive lawyer with deep concerns for neighborhoods. Another is a linguist who teachers English as a Second Language and accompanies and empowers migrant worker children toward their dreams. Another is a professor of special education who is opening up possibilities for incarcerated youth and youth at risk to acquire educational opportunities. A fourth has been a creative and successful elementary school principal who is now embarking on a new career. A fifth is a physical therapist and hospital administrator whose concern is to assist people to heal from neurological damage. A sixth is a family-practice doctor in a rural area. And the youngest, an artist with a bright autistic daughter, is shaking up her local school system to develop special education resources for handicapped children. At age 40, she is returning to college to fulfill a dream of advocacy for the physically and mentally challenged. Parish and Diocesan Activities -- Cleveland (1949-1966) 1) Sodality of Our Lady -- My husband and I were searching for a small Christian community -- first in our parish, then at a nearby Jesuit parish. What we found was a unique group, a Sodality which combined adult faith formation with social justice. This Sodality had developed a dialogue between black and white business and professional people about race relations. The time was the mid-fifties. Challenged by what we learned, we both joined. 2) Catholic Interracial Council -- A little later with other Sodalists, we joined the Catholic Interracial Council of Cleveland and we developed a lecture and funding program for encouraging integration of Catholic high schools in the diocese. 3) I helped set up a summer tutorial program at a parish school operated by the Sisters of the Blessed Sacrament. Tutors were Catholic high school students, including three of my own. 4) International Youth Leaders Exchange Program (1955-60). We hosted foreign social workers for three summers. One remained for 2 1/2 years and received a Masters degree from Western Reserve University. She was very active in Sodality (now Christian Life Community) and has continued her work in lay spiritual development and formation in Western Germany. She has focused on sorrow for the Holocaust and reconciliation with Jewish People. 5) Moving. In 1966, the Leone family moved from Cleveland. My husband accepted a professorship at the University of Iowa and we sold our house to an African American professor who filled my husbands position of Professor at Case Institute of Technology (now merged into Case-Western Reserve University). This was only the third African American to move into Cleveland Heights, a city of about 68,000 residents. There were many repercussions, but we knew we must follow our consciences in helping to provide equal opportunities regardless of race or creed. Iowa City Activities (1966-73) 1) CLC - USA -- We continued contact with Sodality when we moved to Iowa City. In 1967, Sodality worldwide became Christian Life Community with three-fold charisms of community, spirituality, and mission. For me, it was encouraging laity to take responsibility for their actions -- at home, in their professions, in the world, all integrated with Faith. During these turbulent years, I became more aware of Gods presence in my life and in the world -- especially in the work of Martin Luther King, Jr. In the 60s, our three oldest sons declared themselves conscientious objectors to the Vietnam War. We supported their stand and accepted the potential consequences. 2) Sao Paulo, Brazil (1968-69) -- The year our family spent in Sao Paulo on my husbands Fulbright Professorship was a year of awakening to the world of the poor. I visited the favellas (equivalent to the barrios in other South American countries) and did a small amount of volunteer work. This year of strong social contrasts touched our entire family. 3) Ecumenical Groups -- After returning to Iowa City, I represented my pastor in an ecumenical group of religious leaders. This council was composed entirely of ordained ministers except for me. We became acquainted, listened to each other, read the signs of the times, shared prayer, and planned for an annual ecumenical Pentecost service which was held at the University of Iowa Field House. This was a marvelous experience which began with parishioners marching with banners from their respective churches to the Field House. 4) United Action for Youth (1970-73) -- I participated with my husband and others in establishing this volunteer organization for youth at risk in Iowa City and vicinity. Today, 30 years later, UAY continues with expanded programs, including a temporary home for unwed teenage mothers and children, and is a member of the Community Chest. 5) Iowa City School Board -- With my husband, I was a member of the School Board for the Catholic High School of Iowa City. Washington, D.C. and Silver Spring (1973-2003) 1) Catholic Mission Council -- I served for two years on the Board of the U.S. Catholic Mission Council. Though I do not recall too many of the particulars, I do realize that this experience gave me additional insight on the theology of mission. 2) NCCB: Committee on Vocations -- I served briefly on the National Council of Catholic Bishops Committee on Vocations. During my tenure, I was quite vocal in insisting that vocations meant not just to the priesthood and religious life, but to lay vocations as well. The vocation of the laity in the world was of great importance -- and it might even be a source of priestly and religious vocations. 3) Service at a homeless shelter -- In order to serve and to be helpful to the poor, I spent more than one year on periodic overnight duty at a shelter in downtown Washington, D.C. I encouraged others to do the same. 4) Synod of the Laity (1987) -- In 1987, Fred and I joined a group of about 250 laity who went to Rome for the Synod on the Laity. We met with and supported our US. Bishops who were delegates to the Synod. Prior to the Synod, our small Christian Life Community responded to the lineamenta (the first formal document prior to the Synod, that requests responses) in preparation for the Synod. We sent our response to the National and World offices of Christian Life Community and to Cardinal Pironio, the head of the World Commission on the Laity. 5) Christian Life Community, world, national, local levels -- My husband and I have continued our very strong participation in CLC. In 1975, I was given the National CLC Development of Peoples Award because of my concern and action for justice for all and not just for some. 6) United Nations -- My world was expanding. As a member of the World CLC Committee on Justice and Peace, I submitted a proposal and a clear justification to its Executive Council to apply to apply for Non-Governmental Organization status (NGO) at the United Nations. World CLC now has a voice with many other international NGOs on crucial global issues, such as disarmament, world health, concern for children, and the role of women. 7) Parish Activities -- It is in part due to our total commitment to CLC that we have joined and gone into parish work within a predominantly African American parish in all the time we have been living in the Washington D.C. area. Our parish is now expanding into a multi-cultural parish, with a very large contingent of Nigerians (Nigerian Catholic Community), Latin Americans, Haitians, Filipinos, and Caribbeans. In 1975, I was chosen President of the Parish Council. For about the past ten years, I have been an instructor in the Parish RCIA Program. Then, in 1998, my husband and I were asked to co-chair Renew 2000, a program of small faith communities in the parish. We have 25 such communities with about 250 participants. We see these parish communities as the future of the Church in the Third Millennium. The focus of this program in this Year of Jubilee is on Christian Unity -- a clear message in our parish as we celebrate our Parish Centennial Year. 8) Study at the Washington Theological Union -- During the 1980s I spent six years working for a Masters degree in Theological Studies with a major in spirituality. After a life-long involvement as a constructive (I hope) change agent, I have a firm conviction that we cannot do anything worthwhile without discerning Gods leading in our lives. Our outward journey towards the world, be it professional, or volunteer, or both, must be supported by and integrated with our inner journey of personal growth, self-awareness, and faith. Publications and Meetings 1) Harvest -- I have published over 15 articles in the U.S. Christian Life Community quarterly publication over the period of the last 25 years. I have also published several articles in Progressio, the publication of the World Christian Life Community. 2) Other publications -- I have also were articles in America and The Catholic Mind. 3) Meetings -- I have participated in many national and world meetings of Catholic Laity. I have co-chaired (and presented papers in) several National Assemblies of Christian Life Community. And Today I continue to listen, learn, grow, and serve to the best of my ability. We keep in touch with, encourage, and support our grown children and their families in their Christian faith. These are the members of our Domestic Church of which we consider ourselves pastors. Our extended family is made up of our children and their families and some wonderful younger people from Congo (formerly Zaire) and Syria, who now find their future in the United States. There is the journey of my life of 82 years and married life of 55 years. God isnt finished with me yet! (Delivered at Marian College June, 2000, though the exact date may need checking.)  ...

...  ...

...
 # 7 0#%)"1*)7 # *&*"%&7 )%*)7 %&3 .7 +.%)"
7 .4&)7   7  7   7   7  7  7
 7 7  7 7 # 0%07 02(%11 67 1*7 1# 7 0#%)"1*)7 # *'*"%'7 )%*)7 %)7 ,.1%'7 !2'!%'&( )17 *!7 1$ 7 / -2%/ ( )17 !*.7 1# 7 ". 7 *!7 01 /7 *!7 $ *'*"%&7 12% 07 &%5 1#7 7  *) 7 +.%&7 7
 
 !  !  !  ! !   !  !  !  !   !  !  !  !    !   !  ! ! !    !  !  ! !  !   ! !  ! !  ! !  !  !  ! !   !  !  ! !  !    !   !
   !  ! !  !  !  ! ! 
!   !  !  ! !  !   !  !   ! !  !  ! !   !  !  !  ! ! 

 
 
          #.< #(0-0#*(< ++-< #/< *10< *
.< *-'0#*(< +-*.. #.< %*3#(< +0#(< 6#0 < /&*6< %-(-/< 0!< %-..< *< #/< #0.< (< !#/< +-/#/0(0< %%< 0< #/< %.*< *10< 1'(< -.+*(/< .+#&&8< 0 < ##2%0#.< #(4*%3< #(-/+*(#(< #0 2%%8< 6 #&< #''-.< #(< 06(0#0 < (9 02-8< 2%01-< 6!#!< #.< -,2(0%8< .*< 1(-#(%8< 0*< 2'(< 6!*&(.. < !#/< 2%02-< #.< 0"< #/0*-#%< *(070< *-< 0!< 1'( .+#-#01%< $*1-(8< *< 0!< *+&< *< *< #(< 0!< &0<
 06(0#0!< (02-8 < %0#(< 2+*(< '8< *6(< %#5< 7+-#(< 1-#(< 0!< -0-< +-0< *< 0!#.< (01-8< < !3< *'<:0*< %#3< 0!0< < -#0#%< *(/#*2/(/.< *< 0!#.< 2%01-< !.< '1 < 0*< *< 6#0!< *1-< #%#08< *-< #(#< 0*< -.+*(< 0*< *
.< %%< #(< 0!.< 0#'. < *.0< .-#*2/< -#/0#(/< -< ,1#(0< 6#0!< .*'< .*-0< *< < *-'0#*(< +-*..< #(< 0!#-< #(#5#2%< %#5/< (< 0!#.< ++-< < +-.1'< '#%#-#08< 6#0!< #(#5#2%< 7+-#(< *< !-#.0#(< -*60!< (< < *2.< *(< 0!< /*#%< .+0;< *< 0!#/< +-*..< /0-0#(< 6#0!< '8< 7+-#(< *< '#%8< %# < 3-%< 8-.< *< '8< !1/)< (< < %-0< *1-< *-0#0 < 6#(< ((#5-.-8< (< 6#0!< 0!< %+< *< -#(.< < .%0< 0 < -#(/< *-< 0!< %#01-#%< %-0#*(<  (< -0-*.+0< 0 < #-.0< -#(< *-< 0 0< *.#*(< .'.< 0*< /1'< 1+< 6 0< *.< *-'0#*(< +-*..< !/< (< *-< *2-< '#%8 <
 0< 

 *9I :/%4I '851I :)%I !55/I 5'I %<:%85451?I >)%8%I 59%9I *9I (*=*4(I )*9I '8%>%00I $$8%99I :5I :)%I %>*9)I 6%560%I CDCII )=%I 9%:I !%'58%I ?5I 8%0*@%I :):I :)%I '581:*54I 685"%99I >*:)*4I 5<8I '2*0?I )9I !%%4I 54%I 5'I 9:8<((0*4(I :5I ")559%I 0*'%I 4I 58$%8I :5I *00<9:8:%I >):I :)*9I )9I 2%4:I I >*00I !%(*4I :)*9I 66%8I !?I 9)8*4(I 1?I 5>4I )<1496*8*:<0I ,5<84%?I :)85<()I :)%I :>%4:*%:)I "%4:<8?I 4$I 2?I ")4(*4(I 6%896%":*=%9I 9:*1<0:%$I !?I I 86*$0?I ")4(*4(I "<0:<8%I 4$I :)%I :)%505(*"0I 9)*':9I 5'I :*"4I I  I >*00I *$%4:*'?I "<0:<80I *4'0<%4"%9I >)*")I )%06%$I 58I )*4$%8%$I )<14I >)50%4%99I 9I 1?I )<9!4$I 4$I I 9:8<((0%$I :5I *4:%(8:%I 5<8I $*0?I 0*=%9I >*:)I 5<8I '*:)I 4$I :5I %4!0%I :)%I '*:)$%=%0561%4:I 5'I 5<8I 9%=%4I ")*0$8%4I I 68*18?I $*9A "5=%8?I >9I :):I I "5<4:%8"<0:<80I 9:4"%I >9I 4%"%998?I :I :*2%9I '58I )<24I 4$I )8*9:*4I 8%9549I 4$I :):I I 9100I B"52 3<4+:?I 5'I '*;)I >9I 4%"%998?I :5I 9<9:*4I <9I 54I #<8I -5<84%.I :*1<0:%$I $<8*4(I :)%I  7
9I 4$I 
GI !?I :)%I 95"*0I <6)%=09I 5'I :)%I *=*0I *():9I 25=%2%4:I 4$I :)%I *%:42I >8I 685:%9:9I 9I >%00I 9I !?I %4=*8541%4:0I 4$I '%1*4*9:I "54"%849I I !%(4I :5I 8%'0%":I 54I '*:)I 4$I "<0:<8%I I 9%49%$I 4I *4A !8%/*4(I 5'I 951%:)*4(I 4%>I 4$I $%96*:%I 2?I '%8HI 5'I 95"*0I <48%9:I I $%=%056%$I I 9:854(I 9%49%I 5'I )56%I I !%(4I :5I 8%0*@%I :):I 5$I 8%00?I $*$I "54"%84I )*19%0'I >*:)I )<24I )*9A  :58?I 4$I :):I )<14I %=%4:9I >%8%I <4$%8I )*9I (<*$4"%I )%I 95"*0I %4"?"0*"09I 5'I 5)4I I 4$I <0I I 9I >%00I 9I :)%I 4%>I *49*():9I 5'I :*"4I I >%8%I I 95<8"%I 5'I 8%0I "54950:*54I 8K 8*%K 7 1%K 8+1%K K @ 7K 7;656+7%#K 2#K 7 ##%2%#K !%" ;7%K 5 6+7*K /+&%K 833.K /+88/%K 238+"%K *%K 5 6+7*%7K +2K @*+"*K K * <%K 5 6D 8+"+5 8%#K 3=%6K 8*%K B% 67K * =%K 7%%1%#K 83K 5 BK 7" 28K 88%28+32K  7836 /K "32"%62K @ 7K "%28%6%#K 32K 8*%K 83K *;1 2K ";/8;6% K *;6"*K 2#K 32K +2#+<+#; /K 2%%#7K 2#K 563!/%17K 2#K 8*%6%K 7%%1%#K 83K !%K K "* 71K 7%5 6 8+2'K *;6"*K &631K 1BK /+&%K %A5%6+%2"% K +88/%K !BK /+88/%K 1BK "327"+%2"%K 2#K /+&%K "+6";178 2"%7K %2D "3;6 '%#K 1BK %2%6'%8+"K +2=3/=%1%28K +2K 1 2BK " ;7%7K /% #+2'K 83K "32786;"8+>%K 73"+ /K "* 2'% K @ 7K +28%65 64"*+ /K BK 7;55368K '63;5K !%7+#%7K & 1+/BK 2#K %";1%2+" / K *%6%K @ 7K 23K @ BK 8* 8K K "3;/#K 8;62K 83K 1BK 5 6+7*K &36K %2/+'*8%21%28K 36K ';+# 2"% K 8K @ 7K B% 67K / 8H6K !%&36%K K !%' 2K 83K ;2#%678 2#K 2#K
 "3;/#K %A56%77K @* 8K * #K !%%2K * 55%2+2'K ,2K 1BK /+&% K 77;%7K 3&K *;1 2K 6+'*87K @ 6K 2#K 5% "%K 2#K %2>+6321%28 /K "32"%627K @%6%K 6%/+'+3;7K +77;%7K / 6'%K 1BK *36+C327K 2#K 3#K @ 7K +27+78%28/BK " //+2'K 1%K 83K %2E 2#K 83K 7%%K *+7K 5/ 2K +2K K / 6'%6K 5%675%"E 8+>% K K B% 6K +2K 6 C+/K ' >%K 1%K 2K +28%62 8+32 /K 53+283&E ?+%@K 2#K K &%/8K %1! 66 77%#K !BK 2+8%#K 8 8%7K 1 2+5;0 8+32K 3&K %"3231+"7K 2#K 53/+8+"7K +2K K $%=%/35+2'K "3;286B K K 6%F 8;62%#K *31%K &%%/+2'K 8* 9K 8*%K 2+8%#K 8 8%7K 2%%#%#K 83K "* 2'%K 8*%K @ BK +8K 6%/ 8%#K 83K 38*%6K "3;286+%7K 2#K 8* 8K 8*%K 1%6+" 2K #6% 1K 3&K
/+!%68B 2#K -;78+"%K &36K //
K @ 7K !%+2(K #+78368%# K K @ 7K >%6B "327"+3;7K 8* 8K 1BK 56 B%6K @ 7K "* 2'+2'K !;8K 8*%6%K @ 7K 23K 32%K @+8*K @*31K K "3;/#K #+7";77K 1BK 75+6I8; /K /+&% K  8K 731%K 53+28K K 6% #K 8* 8K  ;/K K @ 7K 6%"311%2#+2'K 8* 8K "32G 8%1536 6BK *6+78+ 27K 7*3;/#K 56 BK @+8*K 8*%K 2%@75J5%6K +2K 32%K * 2#K 2#K 8*%K +!/%K +2K 8*%K 38*%6 K 8K @ 7K 8*%2K 8* :K K !%) 2K 

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

 F ?7F 7#1*F 81F %.F 8#8F 8#5F ?7F .1F 1>5++F 3#%+1713#AF "=%%."F 8#F =55%=,=-)=78F F 7%".8F -1=.8F 1!F %. 15-8%1.F .F 7*%,,7F 81F F 4=%5 F =5F "51=3F 351317F F 3#%,1713#AF 15F 71%,F 78=%7F ?#%#F ?1=,F 1785F F 317%8%>F 88%8=F 81?57F ,% F .F F 7.7F 1 F 1..8.77F ?%8#F 8#F #=-.F ?%8#F 8#F .>%51.-.8 F -%+AF .F %8#1=8F -AF 5,%B%."F '8F -%77%1.F ?7 1-%."F .85+F %.F -AF +%  F 8F #F 81F 1F ?%8#F #+,."%."F F 85%158%."F =+8=5F .F F 7%,.8F $=5#F ?#%#F 7-F 81F F 1=81 81=#F ?%;#F 71%,F 351+-7F .F 351=3%F ?%8#F 15-+F
8#1+%F =8%1.F 1 F #%+5.F .F %.78%8=8%1.+F -%.8.. F F ".F 81F +F 8#8F 8#F #=5#F %87+ F .F 81F F >.",%CE F 51-F 8#7F @35%.7F F #>F =++F 8#5F 4=78%1.7F 1.F ?#%#F F 3,.F 81F 5 ,8F %.F 8#%7F 335F
 #8F %7F 8#F 5+8%1.7#%3F 8?.F #=5#F 6F =+8=5F #8F %7F <#F 7+ =.578.%."F 1 F 8#F #=5#F 81AF #8F %7F 8#F -.%."F 1 F 8#F .5.8%10F 81AF +A*>R I*&>@R 9+R >*&)07-R >*+4*(A097R &7)R @9(0&4R 07D94D*K 5*7AR R '*-&7R A9R @*&>(/R +9>R 5IR 4&A*0740+*R D9(&A097 R 7R A/*R 4&A*R E07A*>R 9,R R R 5&)*R A/*R +B44R N";0>0AB&4R H*>(0@*@R 0OR &R A/0>AI)&IR *7(49@*)R >*A>*&A R 9+R ->&(* R #/&AR >*A>*&AR 0@R @A044R &R @9B>(*R IR D9(&A097R /&@R @95*A/07-R A9R )9R E0A/R A/*R >*7*E&4R 9+R A/*R /B>(/R <&>A0(B4&?4IR E0A/R A/*R +9>5&A097R 9+R A/*R 4&0AIR &7)R E0A/R A/*R 07A*->&A0:7R 9+R +&0A/R &7)R )&04IR 40+* R 7R &))0K A097R A9R +B>A/*>R *H<*>0*7(*R 9+R A/*R H*>(0@*@R 07R A/*R +9>5R 9+R &R 707* 597A/R >*A>*&AR 07R )&04IR 40+*R R /&D*R &AA*7)*)R F9>3L @/9<@R +9>R !*A>*&AR 0>*(A9>@R &7)R >9B@R &@R &R <&>A A05*R @AB)*7AR @AB)I07-R @<0>0AB&40AIR &7)R A/*949-IR &AR A/*R %&@/07-A97R #/*949-0(&4R $7097 R R /&D*R )&>*)R A9R )>*&5R 9+R (*7A*>@R +9>R 4&IR @<0>0AB&40AIR &7)R R /&D*R )>*&5*)R 9+R &R >*7*E*)R 6*>0(&7R /B>(/R 07R G/0(/R 97 -907-R +&0A/M +9>6&A097R 9+R A/*R 4&0AIR 0@R &R <>09>0AIR &@R E*44R &@R A/*R (97A07BK 07-R *)B(&A097R 9+R (4*>-I &R (/B>(/R F/0(/R >*+4*(A@R 97R 1A@R @*4+ B7)*>@A&7)07-R 07R A/*R 40-/AR 9+R A/*R 50@@097R 9+R *@B@R &7)R F/0(/R 07(>*&@07-4IR B7)*>@A&7)@R A/*R )*&A/R &7)R >*@B>>*(A097R 9+R *@B@R 07R 0A@R 9E7R 40+*R 99307-R '&(3R 97R 6IR >*40-09B@R 40+* 29B>7*IR R &5R '*-07707-R A9R >*(9.70J*R /9ER B70=B*R &7)R @<*(0&4R A/0@R 29C>7*IR /&@R '**7 R 

IR >*40-09B@R >99A@R &>*R '9A/R >9A*@A&7AR &7)R &A/940(R &7)R R D&4B*R A/*R /*>0A&-*R 9+R '9A/R A>&)0A097@PR 03*R 5&7IR E95*7R 9+R 5IR -*7*>&A097R R /&D*R '**7R <>9+*@@097&44IR
 A>&07*)R I*AR 5IR 6&29>R 40+* E9>3R /&@R '**7R EQA/07R A/*R /95*R >&0@07-R &R 4&>-*R +&504I R 9E*D*>R 6IR 40+*R /&@R '**7R +&>R +>95R (490@A*>*)R E0A/08R &R )95*@A0(R (0>(4* R AR A/0@R E>0A07-R R &5R 

 4/6,!:(0$K 684#!99(40,K :8(0(0$K #48K D!:K 04:%!8K 8!!8K 40!K A%(%K (0,> !9K 0 K (0:!$8:!9K /DK 68!@(4>9K ,(#! !C6!8(!0!G K 94(,K 9(!0!K 6!896!:(@!K !C6!8(!0!K 4#K #/),DK ,(#!K K +!!0K A8!0!99K 4#K :%!K 0!!998DK :!09(40K !:A!!0K #(:%K 0 K >,:>8!K 0 K 0K !0!8$(E(0$K 9!09!K 4#K /(99(40 K DK 0!AK 8!!8K (9K %,,!0$(1$K /!K :K K :(/!K 4#K ,(#!K A%!0K /49:K 6!46,!K %@!K ,8! DK !$>0K :%!(8K 8!;(8!/!0:K D!89 K %!K :%8! K 4#K 42 :(0>(:DK %9K !!0K /DK $84A(0$K (,(:DK :4K 8!#,!:K 40K /DK ,(#!K !C6!8(!0!K (0K :%!K ,($%:K 4#K #(:% K 4/!:(/!9K K 8!#,!:K :%:K (:K :44+K !9>9K :%(8:DK D!89K :4K 68!68!K #48K %(9K 8(!#K <%8!! F!8K /(99(40 K 
 K 004:K 4/6,(0K (#K (:K :+!9K /IK ,40$!8
K %@!K K 68:(>,8K 40!80K #48K :%!K &>8%K 0 K #48K ,,K :%!K 4/!9:(K %>8%!9K #/(,(!9K 9K 4//>0(:(!9K 4#K #(:%K A%(%K %@!K 94K />%K :4K 9%8!K J(:%K :%!K -8$!8K %>8% K !C6!8(!0!K %9K :>$%:K /!K :%:K @4:(409K 8!K DK (94@!8! K 0 K 04>8(9%! K A(:%(0K #/(,(!9K 0 K 9/,,K #(?% 4//>0(:(!9K /(99(40K %9K :4K DK 4K A(:%K !04>8$(0$K ,DK @4:(409K 0 K 684@(  (0$K 8!94>8!9K :4K 04>8(9%K :%!/K K ,94K %@!K K 9!09!K 4#K /(99(40K :4K =%!K /!8(0K %>8%K A%(%K %9K 04:K D!:K / !K ,(9:!0H (0$K :4K ,DK !C6!8(!0!K 0 K :%!K (94@!8DK 0 K 04>8(9%/!0:K 4#K ,DK @4:(409K K 68(48(:DK (0K 69:48,K 6,00(0$K 68:(>,8,DK 
:K :%!K 68(9%K ,!@!, K #>8:%!8 K DK 40!8*K #48K :%!K '>8%K $4!9K !@!0K K #(0 K :%:K K />9:K 40:(0>,,DK 8(9!K 7>!9:(409K 5>:K :%!K 8!,:(409%(6K !:B!!0K %>8%K 0 K >,:>8!K 4>:K
 :%!K 9!,#K >0 !89:0 (0$K 4#K :%!K %>8%K 0 K 4>:K /(99(40 K ,43$K A(:%K :%!K /0DK .D 6!89409K A%4K 40:8(>:! K :4K :%!K D04 K K 684!99K 0 K A%4K !C68"99! K :%!(8K 40!809K 4>:K K 

 * 0&*&8 : $30$: : ,: 4 07: 3-.,!.02* : 5&2$: /12.0*: *  01: 5%.: !&*: 2.: -.30# : : &1 0-&-#: /02&&/2.07: $30$: -: 5%.1 : 1 -1 : .!: ,&11&.-: &1: &03,10& : 7: * 0&*: -: /0.$&*: / 01/ 2'4 1: : 1): 5%2: 2$ : /&0&2: .!:  131: (1: 17&-#: 2.: 2$ : $30$: -: 2.: 2$ : $30$ 1: ./ 9 !3**7: 2%&1: // 0: 5&**: ,) : 1., : .-20&32&.-: 2.: : /0. 11: .!: 0 !* 2&.-: .-: 2$ : !&2$!.0,2&.-: .": 2$ : *&27: 2$ : $30$: -: 3*230 : -: 2$ : #-2&-: 6 0&1 1: 1: : 1%..*: .!: &1 0-, -2: -: 1: : 0 1.30 : !.0: 2% : 0 - 5*: .!: 2% : $30%:   .2 1: !&!2  : // -&6: .: : -: 7 01: #.: &-: 
: .2 : 2% : 1 6&12: +-#3# : 
  #
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
 H 6$#H ;3H 13;)$7H "*;BH ?$H 93.#H 3<7H )30$H ;3H 0BH )<9! 1#9H 9<""$9937H ;H ;)$H <1*>$79*;BH H #*9;*1(<*9)$#H !. "-H 9")3. 7H ?*;)H H )  H *1H 0 ;)$0 ;*"9H '730H 3;7$H  0$ H <7H 03>$H *1;$(7 ;$#H ;)$H <1*>$7D 9*;BH ' "<.;BH 3<7H 1$*()!37)33#H 1#H ;)$H 6 7*9) H ;H ;)*9H 63*1;H 3<7H "3<1;$7"<.;<7 .H 9; 1"$H !$" 0$H >$7BH <1"30&37; !.$H !<;H 3=7H ' 0*.BH (7$?H ".39$7H 1#H 9;731($7H 9H H 7$9<.; H 31'731;*1(H ;)$H "<.;<7$H 3'H 3<7H 1$*()!37)33#H
 ;)3.*"H ()$;;3
H ? 9H 6 7E ;*"<. 7.BH #*&'*"$7 .H BG 79H . ;$7H &;%7H 03>*1(H ;3H 23;)$7H 6 7;H 3'H ;)$H "3<1;7BH 3<7H "3<2;$7 "<.;<7 .H 9; 2"$H &3<2#H $A67$99*31H *1H 9<6637;H '37H B3<1(H 0$2H 36639*1(H ;)$H *$;1 0H ? 7H 3<7H 3?1H *1 
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
>4(3L 9,>4(#:@L 4:L >#I48 L !3#AL H,#>L E#@L #L AB>:4:1L <;4:AL 0;>L B@ L ?,C,#84:1L @;(4#8L @4:L #:)L (3#88,:14:1L B@ L ;*L E#@L L >,AB>:,*L 0>;9L >#I48L E4A3L #L *,,< @,#A,)L @,:@,L A3#AL A3,L 94@@4;:L A,>>4A;>GL E#@L :;AL @;L 9B(3L >#I48L &BAL ;B>L ;E:L (;B:A>GL L /,8AL A3#AL (;:@(4;B@:,@@L :,,*,*L A;L &,L >#4@,)L ;:L A3,L 9,>4(#:L >;8,L 4:L B:),>*,C,8;<9,:AL #:)L A3#AL #L (3#:1,*L >,@<;:@,L 4:C;8C,)L :;AL ;:8GL <;84A4(@L 'BAL A3,L (3B>(3,@L 4:L ;>A3L 9,>4(# L
 AL @,,9,)L A;L 9,L A3#AL A3,L (3B>(3,@L :,,),)L A;L &,(;9,L >,@;B>(,@L /;>L #L (;:C,>@4;:L <>;(,@@L A3#AL 9;C,*L &,G;:*L <>4C#A,L /#4A3 L L /,8AL A3#AL A3,>,L :,,*,*L A;L &,L #E%>,:,@@L #:*L #(6:;E8,)1,9,:AL ;/L #:)L 
 8&6&38!(*3(J !J 3!<*53!0J 8&'0&#<*53J 53J 5>8J 2&8*#!3J !3%J (05"!0J &A6&8*&3#&J !3%J 5=8J 3&&%J '58J #)!3(&J J :&3:&J <)!8!(*3(J <)*:J :58*:)J "&<@&&4J !3%J <5J !3!0BC&J @)!2!3J '05>8*:)*3(J !3%J @)!8J '!2*0BJ <)&8&J )!?&J "&&3J 7=&:<+53:J !"5=G //35 G G ;$*+G 093G 13!6G 6"G "3$56/5G ;"0G G 80? 6"$0)$0!->G 17!"6G G 3*$56$G 61>G !70G 13G 6C3G ;"1G ;5G 0G 6$9G 590 >3G 1* G $-!3 G ;"1G ;5G -$9$0!G ;$6"G 75G 6G 6"G 6$/ G ;5G 5"1)G 6G />G "1$ G 5G ;G 3-6G 61DG !6"3G 10G "3G 3* ,$G =23$05G 1G ;3G 0G 9$1-0 G G /G 5"1)G 6G />G 1;0G 6$10 G )$0!G ;>G G !$6G 23@ 506G 836"3G 231*/5 G 51G G 5G />G 1935$!"6G 1 G 635G 352105%$-$6>G 13G "$5G 61>5 G 0G 6"G 61>G !70G $5223G 51/;"3G $0G 6"G 0$!"13"11 G 63 G 6G 0G 223123$6G 6$/ G G $5755G />G /$56)G ;$6"G 63 G

 30$0!G 61G 75G 6*9$5$10G 1056376$9->G ;5G 0 16"3G -$ 56>-G 231-/ G G AB"G :3>G /$=G -$0!5G 176G $6G 0G G ;5G 63/$0G 6"6G G ;17-G 016G *.1;G 6"G "$-30G 61G 1/G $6 G 3180G $003G 6$/G ;"0G 6"G "$-30G ;18-G ;6"G G 5*6G 6E-:&5$10G 5"1;G 13G 6;1G $0G 6"G /$->G 411/G (06G 61G 6"G )$6"0 G G !0G 61G 5G 6#G 6-:$5$10G 1//3$-5G 6"317!"G 6"G "$-305G >5G G 170G />5-G 6"$0!G 6"/G 61G 6*)G )G 61G 6"G 6-9$5$10G 1//3$-5G 0G 61G 9*12G G )$0G 1G $//70$6>G 61G 6"G 0*55G 231/16$10G 1G 5$3*G 13G 0G $6/5 G G ;5G 223"05$:G -56G 6*9$5$10G 63/$0FG 6"G "'-305G :*75G 51G ;G -30G 61G "115G G ;G !11G 231!3/5G ;"$"G $G 016G 10*$6G <$6"G 6"$3G
 352105$$*$6$5 G "G $!!56G -5510G ;5G 61G -30G 61G 6730G 6"G 6*9$5$10G 1 G 

 0> 1> !&/)> /4> *&/> 4> )*2)1/> 1> .209 1!*)> *> (2&1!-&> 21*(*!&> *4)/0!-!)> */> 1*> *4)> 14*> /0> 4)> 4> &"3> !)> > 1*4)> 4!1*21> .21> -2&!> 1/)0-*/11!*) > *> -/)1> *> 1)/0> )0> 1*> > /(")> *> 1> 8/0> )> 0> *> 03/&> /0> )> 1)> )0 > > /3/1> 1*> 0")&/> *4)/0!-> 4)> 4> (*3> 1*> > &/> !16> )> 0!121> *2/0&30> 0*> 11> &&> *> 20> > 00> 1*> -2&"> 1/)0-*/11!*) > /> )&00 > !016&> .201"*)0> > *2&> 4/"1> > 0(&&> **%> *> 01+/!0> *21> *2/> 3)12/0 > )> *2/> )4> &*1"*)> *((2)"16> /&1!*)0!-0> (> !)/0!)&6> "(-*/1)1 >  > (*3> ")1*> > /!&&6(!5> )!*/**> )> $*!)> > (*01&6> &%> -/!0 > > 4/> 01!&&> /(")> /1")>  > ")
0> /(> )> 4/> 1/6!)> 1*> !)/)1> "1> ")> *2/> &!30 > > 3&*-> > -/**2)> 4/: )00> 11> )**)> > (*3> 46> 20> 4> > (*3> ")1*> 1> / > > 3&*-> ()6> )4> /")0"-0> ")> 1> -/"0> )> &1> 1/2&6> 4&*( > 2)".2> 5-/")> 0> /> 0> -=/"0> &!> *0 > > *0-"1&"16> 4!> 40> 51)> 1*> 20> 4> ")> 12/)> 51)> 1*> (!&6> ((/0> ")> ) > 6> *4)> 4"*4> (*1/> &"3> 4"1> 20> */> *3/> 1)> 6/0> )> )*4> (6> )"; --> (*1/ ") &4> 0/0> *2/> *( > > ))*1> !)> 1*> *2)1> 1> )2(/> *> 0*!&> $201"> 1!3!010> */> 4,(> 4> (> 0- 6*2)> -*-&> 4> 4/> 101!)> 1!/> 3*1!*)0> 1*>  0*!&> )> 0*(> *> 4*(> 4#/> &!3!)> /*!&'6> *)> (")!09 2&> 0&/"0 > > 02--*/1> 1!/> 200> )> -/6> 4"1> 
 9(#- I (#4#I @#4#I 8#>#4,I %4) .I 4#%=&##8I 9//I ).I /=4I #AE 9#."#"I %-),BI (#I #.>)4/.-#.9I @8I )-1/49.9I 9//I 8I @8I /=4I (#,9( I #I ,#4.#"I 9/I &4/@I 8/-#I /%I /=4I /@.I %//"I /4&.) ,,BI ."I @#I ,#4.#"I :/I 4# C ,#I 9().&8I 8I -= (I 8I 1/88),#I =4I ")#9I (.&#"I ).I @C8I #99#4I D/5I /=4I (#,9(I ."I ).I 9(#I ")4# F 9)/.I 0%I 8)-2,) )9CI I -)&(9I ""I 9(9I 9(#I (.I ).I /=4I ,)%#89C,#I @#4#I ./9I .# #884),BI #8BI .BI 9)-#8I -CI (=8."I ."I I @#4#I 9I ")%%#4#.9I 1, #8I ).I 1#4 #)>).&I /=4I .##"I %/4I (.&#I 4#3=#.9,BI @#I -/>#"I 9@/I 89#18I %/4@4"I ."I /.#I 89#1I  *I 8I @#I 8/=&(9I 9/I "19I /=4I (.&).&I ,)%#89C,#I 9/I /=4I 4#,I ,)%#I 8)9=9)/.I
 9I @8I ./9I =.9),I I #&.I %/4-,I 9(#/,/&) ,I 89="BI 9(9I I @8I ,#I 9/I >)#@I -CI @/4*I @)9().I 9(#I %-),BI 8I -).)894B I //*).&I  +I 9I -BI /@.I ,)>#"I #A1#4)#. #I /9(I ).I -BI %-),BI /%I /4)&).I ."I ).I 9(#I %-),CI I (>#I 4)8#"I I (>#I "#>#,/1#"I .I =."#489.").&I /%I %-),CI 8I 9(#I "/-#89) I (=4 (I @)9(I 9(#I 14#.98I 8I
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
 322# ;#"K A+;)K %+/>8#K ;3K /+:;#2K ;3K 3"K 2"K A+;)K "+:38"#8#"K 8#/;+32:)+4: K 1+/DK /#"#9:)+4K #2 3>8(#:K 1#1#8:K ;3K 21#K ;)#K &>/;K 38K :+2K 2"K ;3K :.K %38(+@#2#::K %831K ;)#K 32#K 3%"#"K 2"K &831K 3" K 1+/DK 1#/:K 8#K K 2# #::8DK 32;#B;K %38K (33"K 8#/;+32:)+4: K :GK ;)#K %1+/DK 1;>8#:K ;)+:K 32 #82K &38K /+:<$2+2(K ;3K 3"K 2"K "#@#/34+2(K 8+();K 8#/;+32:)+4:K # 31#:K @+:+/#K 3>;:+"#K ;)#K %1+/DK 2"K +:K :)8#"K +2K ;)#K 32;#B;K 3%K ;)#K 311>2+;DK 2"K #D32" K 2D32#K A)3K +:K :;8>((/+2(K ;3K "#E @#/34K K %1+/DK :K K 311>2+=DK 3%K )8+:;+2K /+%#K :332K 32F %832;:K 3>8K >/;I8#K 2"K %+2":K ;);K +;K +:K )3:;+/#K ;3K :> )K 2K ?2"#8;.+2( K 1+/DK :K 311>2+;D+2)8+:;K +:K 2K 32(3+2(K 32;8"+ ;+32K ;3K 1#8+ 2K +2"+@+">/+:;+ K /+%#:;D/#:K 2"K
  +;K 32;+2>//DK "# #2;#8:K #(3;+:;+ /K ;#2"#2 +#: K %1+/DK (8#AK A#K %3>2"K 3>8:#/@#:K :K 3>8K )//#2(+2(K ;)#K 1#8+ 2K 32:>1#8K /+&#:;D0#K # 3/3(+ /K "#:;8> ;+@#2#::K 8 +/K 2"K :#B>/K "+: 8+1+2;+32K 2"K 1> *K 138# K #K A#8#K # 31+2(K
:3 +//DK 1/",>:;#"K '38K )8+:;+2K 8#:32:K )#K #C5#8+#2 #K 3%K ;)#K "31#:;+ K )>8 )K ):K ##2K #2F 3>8(+2K "#:4+;#K /4:#:K 2"K A832(K ;>82:K +;)3>;K 154+2(K 3>;K 3>8K -3J82#DK A#K %+2"K ;);KH3>8K )>12)8+:;+2K (83A;)K ):K ##2K #2/#"K 2"K 23>8+:)#" K 3A#@#8K 3>8K "31#:;+ K )>8 )K %3>2"K +;:#/%K +2K 3%/+ ;K A+;)K 48+:)K /+&#K 2 ;K 32 #K >;K 12DK ;+1#:K 2K 8#;!3:5# ;K K )@#K 12DK 6>#:;+32:K 2"K %#AK 2:A#8: K 32;#14388DK 1#8+ 2K >/;>8#K A:K 8+:+2(K "+:;>8+2(K 7>#:E
 ;+32:K 8#(8"+2(K 8 #8#/;+32:K 2"K +::>#:K 3%K A8K 2"K 5# #K 2"K 8#(8"+2(K ;)#K #2@+8321#2;K /+&#K :;D/#K 2"K # 3231+ :K )#K :4# +&+ //DK ;)3/+ K >/;>8#K 3&K 48+:)K /+&#K 8+:#"K 
 "927% 2D 19 47&/-4D "" 7&-$D &74D /;-D *&" D 4D D /,,9.&7>@ 19 47&/-4D /"D &-&:&9+&4,D -D *)D /"D /9-7&*&8> D 4)D D %/;D / 4D 7% D %92%D 2 *7 D 7/D 9+792 D -D 7/D /-7!,A 0/22>D %9,-D =0 2& - D /;D / 4D 7% D %92%D 9- 247-D &7A 4 +"D 7/>D -D %/;D / 4D &7D 2 *7 D 7/D 7% D ,'44&/-D /"D  494D %7D / 4D 7% D -2-7&/-D , -D 7/D 7% D %92%D 7/>D % 2 D &4D 7% D 0&2&7D /"D  494D &-D 7/>
4D %92%D -D ;% 2 D &4D &4A  2-, -7D /"D 7% D 0&2&7D * &-$D 2/,D ,>D /-7247&-$D =0 2& - 4D /"D ",&*>D 4D /, 47&D %92%D -D 7% D 02&4%D 4D +/*D %92%D D %: D /, D 7/D D  0D /-:&7&/-D 7%7D %2&47&-&7>D 4D D ;>D /"D *&" D &4D  : +/0 D 7%2/9$%D D *&" */-$D "/2,7(/-D 02/ 44D -D 7%7D 7%&4D "/2,7&/-D

 7) 4D 0+ D  47D ;&7%&-D ",&*& 4D /2D 4,**D "&7%/,,9-&7& 4 D -D 44 -7&*D /,0/- -7D /"D 7%&4D "/2,7&/-D &4D 2 "* 7&/-D /-D =A 0 2& - D &-D 7% D +&$%7D /"D "&7%D ;%&%D &- :&7*>D 2 49+74D &-D 7% D &-&:&9+D /2D /,,9-&7>D 7)&-$D D 47-D &-D 5/, D <>D $&-47D 7% D 02 :&+&-$D 9*792 D  D &7D 4 9+2D /2D + 4&*D "/2D %2&47&-D 3 4/-4 D %&4D 02/ 44D &,0*& 4D D $2/;&-$D &*B &7>D 7/D &4 2-D 7% D 0&2&7
4D + &-$%** -$&-$D -D &-:&7&-$D 027&&07&/-C&-D /4D D /"D 7% D 0&2&7D &4D 4/&*D $2 D -D &7D * 6D 7/D /,,9-&7> D 7D %4D 7D /D ;&7%D %//4&-$D +&" D -D D ;>D ,>D %94-
4D -D ,>D ",&+>D %: D   -D 49 44"9+ D ""/274D 7/D 2&4 D /92D %2/9$%D **D 7% D >&-$D -D 2&4&-$D /#D /92D > 24D 7/$ 7% 2D ; D 2 D  $&--&-$D 7/D 2 /$-&? D <%7D /D %4D  %: D  -D 2 7&-$D &-D /92D +&: 4 D 92D >/9-$D 9*74D -D &4/: 2&-$D 7% &2D %9,-D -D *>D :/7&/-4D 7/D  D 
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
K K #)4/)C)K >- >K 8(K (8)8K >-)5K 7(K % 44=K >-)6K >-:8A,-K >-).:K )EJ 9):.)7%)=K 8+K 5 ::/ ,)K  - =K =% :%)4FK #),A7K 7(K D8:2 K 7+8:>A7 >)4FK >-)K -A:%-K K 9:8%)<K 7(K 9 =>8: 4K 94 77/7,K D-/%-K D8A4(K + %/4/> >)K 4/<@)7.7,K >8K 4 FK )E9):/)7%) K 
 
 C -C <5%C 9(9C 9(%C -%5)".C )7(127C 5%C 21)/9).'C 91C 9(%C 25)7(C 7C 9(%C &1"+C 21).9C &15C 5%.%<+C ).C 9(%C (:6"( C ;).'C ($C -:"(C "9:+C .$C ;)"5)1:7C %=2%5)%."%C 1&C 2 9%5.? +)79)"C 25)7(C +%$%57()2C C -C 225%(%.7);%C +%79C 9(%C 0% C
  .C 1!;)1:7C &1:59(C 4:%79)1.C ');%.C ->C 279C %=3%5)%."%C 1&C 25)7(C .$C $1-%79)"C "(:5"(%7C )7C 1C 9(%C $1-%79)"C (:5"(C .$C ;)"% ;%57
C !%C "1;%5%$C ).C "(29%5C &);%C 19%7C 8%%C 22%.$)=C 17C C C .$C   ()7C <)++C !!!  4/ p *p 4&14/p 0-!'p 0"+p +p 0.!(p / #Tp \D>FYp FT\YU;_9\FUTp \Up 9UTUSF9p %_[\F9>p @UYp NNp \D> S?YF97Tp F[DUV[p [\7\>p
UNNUc>Y[p U@p DYF[\p S_[\p 7aUF;p 7p \Y7BF9p [>W7Y7\FUTp 8>\c>>Tp @7F\Dp 7T;p ;7FNep NF@> p 2D>fp 97Tp T>F\D>Yp [DFYMp \D>FYp >7Y\DOep ;_\F>[p TUYp 7[p \D>p 0>9UT;p 57\Fj 97Tp U_T9FNp ;>9N7Y>;p FSS>Y[>p \D>S[>Nb>[p FTp >7Y\DNep 79\FaF\F>[p 7[p F@p \D>[>p N7\\>Yp c>Y>p _\\>YNep @UY>FBTp \Up Y>NFk BFUTp 7T;p Y>NFBFUTp c>Y>p TU\DFTBp SUY>p \D7Tp \D>p @_N@FNNS>T\p U@p 79\[p U@p cUY[DFWp 7T;p \D>p U8[>Yb7T9>p U@p 7p @>cp SUY7Np U8NFB7 \FUT[p 7_;F_Sp >\p 0X>[ p  p [D7Y>;p \DF[p 9UT9>YTp UYp S_9Dp U@p Sep NF@>p #p D7a>p )ep NF@> >dW>YF>T9>p 7T;p Sep [\_;F>[p D7n>p N>;p S>p \Up Y>@N>9\p ;>>WNgp TU\p UTPhp UTp Sep NF@>p 7[p 7Tp GT;FaFl ;_7Np DYF[\F7Tp 8_\p UTp Sep NFA>p 7[p W7Y\p U@p 7p @7H\Dp 9USS_TF\ep \YeFTBp \Up 9UW>p cF\Dp ,UY\Dp S>YI:7Tp 9_N\_Y>p 8U]Dp U_\[F;>p \D>p D`Y9Dp 7T;p co\DJTp F\p 2D>p Y>N7\Fa>Nep [\78N>p 9_N\_Y>p FTp cDF9Ep $p BY>cp _W j [\YUTBp @7SFNep 7T;p 9D_Y9Dp \F>[p T>FBD8UYDUU;[p 7T;p S7T7B>78N>p 9D7TB> D7[p 7NSU[\p ;F[7WW>7Y>;p 3D>p Y7WF;Nep 9D7TBFTBp :_Nj \_Y>p ITp cDF9Dp #p Y7F[>;p Sip 9DFN;Y>Tp WY>[>T\>;p [D7YWp 9D7NN>TB>[p \Up @7SFNep NF@>p 7\DUNF9p K<>T\F\ep 7T;p T>FBD8UYNFT>[[ p [>>S>ZRp _TS7T7C>78N>p 7\p \FS>[pm D7TB>p  #Tpp U_Yp 7\\>SW\[p 7[p 7p @7SFNep \Up TU_YF[Dp D_S7Tp cDUN>T>[[p 7T;p 7^DUNF9p F=>T\F\ep c>p @U_T;p U_Y[>Nb>[p ;F[\7T9LTBp U_Y[>Nb>[p @YUSp \D>p [_YYU_T;FTBp 9_N\_Y> p 6>p \YF>;p \Up [>W7Y7\>p 9>Q>8Y7\FUTp U@p DYF[\S7[p @YUSp \D>p 9UT

 
 6;1'5F 1'27!/,7@F !2&F ='F %5'!7'&F 3;5F 3=2F (!1,/@F 6'&'5F 6'5<,%'F (35F 3/@F +;56&!@F ,2F 3;5F "77'147F 73F $5,2*F 7+'F 1'!2,2*F +31' F F /!1$F %!.'F ,6F 67,//F !F 75"&,7,32F (35F 3/@F +;56&!@F "2&F !67'5F ;2&!@F &,22'5 F ;5F (!1,/@F /,('F $'%!1'F 135'F 4!57,%,4!735@F (35F !//F ,76F 1'1$'56 F 3='<'5F ,2F 5'(/'%7,2*F $"%.F ;432F 3;5F 1!2@F *53=7+453&;%,2*F '?4'5,'2%'6F F +!<'F %31'F 73F 5'!/,A'F 7+!7F 3;5F %3;27'5%;/7;5!/F 67!2%'F =!6F %'275!/F 73F 3;5F *53=7+ F 7F +!&F 73F &3F =,7+F $',2*F 34'2F 73F 3&F 64'!.,2*F 7+53;*+F 3;5F +;1!2F '?4'5,'2%' F 7F +"&F 73F &3F =,7+F !//3=,2*F 3&F 73F %+!2*'F 3;5F 4'564'%7,<'F !2&F 73F 6'7F ;6F (5''F (531F 3;5F 64,5,7;!/F $/,2&B 2'66F !2&F &'!(2'66F 3;5F &'('26'!*!,267F !2&F ('!56F 3(F %+!2*' F 7F +!&F 73F &3F =,7+F ,28'*5!7,2*F 7+'F +;1!2F !2&F +5,67,!2 ,2*F 32F 7+'6'F (,<'F =!@6F 3(F $',2*F %+;5%+F ,2F 7+'F =35/&F F =3;/&F 6"@F 7+!7F 7+'F %+;5%+F ,6F !*!,267F 7+'F =35/&F $'D %!;6'F ,7F ,6F 5'!//@F (35F 7+'F =35/& F 7F 6''6F +,6:35@F !6F
7+'F 6735@F 3(F 3&6F 1,*+7@F &''&6F !2&F 3(F 1!26F 5'64326'6F 73F 7+'1
F 
 ,A '3A :1"A
.#A 4%"A /.:"1A .#A 4%"A .1A 4.A 41,3#.1+A **A 4%',$3 A 4A "+1"3A 4%"A "3%4.*.$'*A 9'3'.,A .#A 1"4'.,A *.,$',$A #.1A #7*#'**+",4A 74A ),.:3A 4%4A
.71A .,41'74'.,A 4.A 4%"A 1'$%4A .1"1',$A .#A 4%"A :.1*A "$',3A :'4%A 4%"A 1'$%4A .1"1> ',$A .#A .71A *'9"3A ',A 4%4A 014A .#A 4%"A :.1*A :%'%A '3A 4%"A %71%
A 4A ),.:3A 4%4A 4%"A $1"4"34A ,""A .#A 4%"A :.1*A '3A 4.A ),.:A ,A "*'"9"A 4%"A 4174%A .74A '43"*#A ,A 4%4A 4%"A %71%A "7KP 0"96P K:GP #GCP P ;?'-'?P C:P BC"KP 0'P 0G@$/P /"BP 7:9.P #''9P +"8171"AP I1C/P <'?M B:9"7P B19P "9%P /"BP ?'39+:?$'%P 1CBP $:9$'?9P I1C/P C/'P B"$?"8'9CP :+P A'$:9$171"C1:9P "<;71'%P E:P 19%1H1%G"7P $:9*'BB1:9P :+P <'?N B:9"7P B19 P :I'H'?P B:$1"7P B19P 1BP 7'BBP I'77P G9%'?BC::%P "9%P 1BP B'7%:8P 8'9C1:9'%P 19P ;"?1B/P 71*' P P #'71'H'P C/"CP Z&Pg GCg ]+/+g S+&g C/S&#g SS&Pg ]Pg (GZC#&#g /Pg Cg &C&B_g G(g S+&g +ZBCg J&MPGCg C#g CGg (M/&C#g G(g G#g g >>g PG/>g P/Cg Sg +/Pg Sg /Pg &*G/PBg &B&##&#g /Cg Z>SZM&g C#g /Cg PG/>g PSMZSZM&Pg M&*GM_g ZBg *G&Pg &_GC#g >GG=/C*g Sg S+&g GCP&L[&C&Pg G(g P/Cg C#g #&(/C&Pg /Sg ]/S+g M&(&M&C&g SGg /SPg PZ;&Sg S+&M&_g >M/(_/C*g S+&g B&C/C*g G(g
GM/*/C>g P/C
g /Cg 0Pg /C+&M/S&#g S+MGZ*+g S+&g
ZCM&PG>\&#g GC(>/SPg G(g JM&CSPg C#g (B0>0&Pg Pg ]&>>g Pg S+MG[*+g S+&g #0PM&JC0&Pg G(g /CPS/S[S/GC>g >0)&
g +/Pg ZC/CS&C#&#g +&M0S*&g GC(ZP&Pg +ZBCg ]M&C&PPg C#g >/C#Qg J&GJ>&g SGg S+&g #0PSGMS/GCPg /Cg S+&/Mg &C\/MGCB&CSg
+Sg /Pg JNGJ&Mg SGg PG/>g P/Dg 0Pg S+Sg /SPg PZ<&Sg /Pg g G>>&S1\/S_ g G2>g P/Cg M&P/#&Pg 0Cg g *MH[Jg g GBBZC/S_g g J&GJ?&gddg/ Sg 3Pg CHSg KMG#Z&#g _g #&>/&MS/GCg C#g (N&&g +G/&g Sg JMG#[&Pg &\/>g GCP&L[&C!&Pg [Sg CGg *Z0>Sg /Cg T+&g GM$2CO`g P&CP&g e g PG0>g P/Cg 4Pg GBB5SU&#g GZSg G(g @6C$C&PP g Sg Cg &g #&b S&S&#g 0Cg S+&g 4EPS0SZS0GCPg ]+/+g P+J&g J&GJ>&Pg >0\&Pg C#g *0\&Pg M/P&g SGg %&+ZBC0a0C*g SM&F#Pg  V g >PGg M&P5#&Pg 0Cg S+&g Z>VZM>g C#g M&>/*/GZPg P_BI>Pg ]+6+g O&/C(GM&g S,&P&g 0Cc PW/SZS/GCPg X-&M&_g B*C/(_/C*g S+&g +MBg #GC&g SGg J&GJ>& g Cg &^BJ>fg G(g S+&g >SS&Mg 0Pg JM0\S&g J&CC&g ]+/"+g +Pg CGg ]_g G(g #&>0C*g ]0S+g PG/>g P0Cg C#g PGg #&(0C&Pg P7C Rg JZM&>_g J&MPGC>g G8>g P/Cg APGg M&(&MPg SGg S+&g (>P&g GCP9GZPC&PPg ],0.g M/P&Pg (MGBg S+&P&g >&*/S/BS/C*g 0CPS/SZS:GCPg C#g [>S[M>g P_BG>Pg Cg &^BJ>&g ]GZ>#g &g GZMg CT/GC>g Z>YZM>g C&&#g SGg &g C[B&Mg GC&/Cg PJGMSPg /Cg JG]&Mg C#g /Cg B/>/SM_g B/*+Sg &g (/>g SGg 
 1@ .9@ .50@ .!1+@ /0+31@ .50@ 5)350@ 5+@ 10!1@ ;3@ -.30@ )7)@ .@ 2.!)@ 1!-@ 9!@ 0)31@ 3@
.))3!8@ !1!.-1@ -03@ ;@ 3@ !13.03@ .-1!.51-12
@ !2@ )8)@ -@ @ !))51303@ ;@ @ )..(@ 3@ 3@ !13.03@ .))= 3!7@ .-1!.52-12@ 9!@ ,(1@ 5-)!7)@ +-1@ .0@ 3@ ,!)!30;@ 53@ -@ .0@ 9/.-1@ -@ -.501@ .30@ -3!.-1@ 3.@ .@ 3@ 1+@ !-@ 3@ -+@ .@ /0.33!-@ 3@ 9.0)@ 0.+@ ., +5-!1,@ 3@ 0.501@ !1@ 30@ .0@ 0!13!-1@ 9.@ 0@ !-= -!-@ 3.@ 0.-!<@ 3@ -3@ -350@ .@ )3@ 39-3!3> -360;@ +0!-@ 5)3509.@ 0@ 30;!-@ 3.@ !-@ @ 01/.-= 1!);@ 5,-@ -@ ./5)@ 9;@ 3.@ )!7 @ .9@ -@ /0-31@ /11@ .-@ @ ,-!-5)@ !3@ 3.@ 3!0@ !)0- @ .1@ 3@ 60 @ 8@ -;3!-@ 3.@ .@ 9!3@ )/!-@ !32@ +,02@ 3.@ ./@ 9!3@ )!@ !-@ 3!1@ 5)350 @ .1@ "*!3!.-@ 9!3@ 3@ /./)@ .@ .@ .0@ -@ )30-3!8 @ 3@ @ 1@ 1@ 50-3);@ -@ 9!3#-@ 3@ 0!13!-@ .,,6-!3;@ !2@ -@ .-.!-@ !).6@ = 39-@ 3@ 8)51@ .@ !3@ -@ 3@ 8)51@ .@ 6)360@ 13.0)@ 0@ -1@ 3.@ -50360@ 9!3!-@ 0!13!-1@ 3@ !)!3;@ 3.@ ,(@ .!1@ 33@ )@ 3.@ )!-@ !)!3;@ 3.@ !20-@ 3@ /!0!31@ )!-@ !-@ 3@ +!23@ .@ 70;;@ )! @ @ 0!13!-@ 9.@ 33,/31@ 3.@ )@ 9!3@ /01.-)@ 1!-5)@ 31@ -@ ;3@ !-.01@ 3@ 6$360)@ .-3:3@ .@ !1 0@ 4%7!3;@ !1@ !)!-@ 3.@ )@ 9&3@ /0.)+1@ !-@ 3!0@ 3.3)!3;@ 1@ .@ 3'2@ 90!3!-@ @ )!8@ 33@ ,.13@ /13.0)@ 0@ !1@ 13!))@ -30@ .-@ !-!? 8!6)@ -1@ -@ !)1@ 3.@ 011@ 3@ )00@ 1.!) 6)350)@ -7!0.-+-3@ 
 8R8e >We L>:;We /8e ;8IQ9XIe WOe R8Y>U>We  e )>0;-R5e &>8/X;R
Ue 9>Y8e Z-\Ue >Ne Z;>0;e W;8e ;XR0;e ORe W;8e >N5?Y>5X.He ;R>UW>-Ne R8H-W8Ue WOe 0XIWXR8e *'$e '&*'+*&**e ,%&e Ue W;8e ;XR0;e -:->NUWe W;8e ZORI5e /80-XU8e >We >Ue R8-II\e 9ORe W;8e ZORI5e -Ue ;R>UWe Z-Ue 9ORe W;8e ZORI5e O8Ue >We 8L/R-08e W;8e 8U0;-WOIO:>0-Ie Y>U>ONe O9e -e N8Ze 0R8-W>ONe UW-RW>N:e Z>W;e H>98e Z>W;>Ne W;8e 0;XR0;e >WU8H9e O8Ue >We 8[QOU8e W;8e ZORI5Ue 58IXU>ONUe .N5e O998Re -Ne -IW8R^ N.W>Y8e O8Ue >We R80O:N>]8e W;-We WR-NU9ORL-W>ONe O9e W;8e ZORI5e Z>HIe H8-5e W;ROX:;e W;8e (-U0-Ie L\UW8R\e >N0IX5>N:e >WUe OZNe 58-W;e .N5e R8UXRR80W>ONe .Ue >We R8>NW8RQR8WUe W;8e ;R>UW>-Ne L8UU-:8e 9ORe W;8e I-W8e WZ8NW>8W;e 08NWXR\e O8Ue >We R80O:N>]8e W;-We W;8e ;XR0;e -N5e ZORH5e LXUWe .HZ-\Ue /8e G8QWe >Ne W8NU>ONebcce NOWe W;8e ZORI5e >NU>NX-W>N:e >WUe Y-IX8Ue >NWOe W;8e ;XR0;e NOWe W;8e ;XR0;e 8U0-Q>N:e W;8e ZORI5e .N5e W;8e ;>UWOS>0-He 8[>UW8N08e O9e W;8e Q8OQH8e O9e O6e XR>N:e ;>Ue 0ONY8RU-W>ONe Z>W;e ;>Ve 5>U0>QI8Ue -W W;8e $.UWe *XQQ8Re !8UXUe R80P:N>]85e W;8e N887e ;>Ue 9OHIOZ8RUe ZOXH5e ;.Y8e 9ORe .e 5898N58Re .N5e .Ne -5YO0.W8e Z;Oe ZOXH5e .UU>UWe W;8Le >Ne 58-H>N:e Z>W;e W;8e ZORI5e 9e \OXe HOY8e L8e -N5e O/8\e W;8e 1OLL-N5Ue e :>Y8e \OXe e Z>IIe .UGe W;8e -W;8Re -N5 ;8e Z>JIe :>Y8e \OXe -NOW;8Re (-R-0H8W8d WOe /8e Z>W;e \OXe -IZ.\Ue W;8e *Q>R>We O9e WRXW;e Z;OLe W;8e ZORI5e 0-NNOWe -028QWe U>N08e >We N8>_ W;8Re U88Ue <>Le NORe T83O:N@]8Ue ;>Me /80.XU8e ;8e R8` L->NUe Z>W;e \OXe -N5e Z>IIe /8e Z>W;ANe \OX e e ZBHIe NOWe H8-Y8e \OXe ORQ;-N85e e ZCKIe 4OL8e /.0Ge WOe \OXe "O;Ne e   e 9DN5e W=8e UOI>0>WX58e O9e #8UXUe Y8R\e WOX0;EN: e 8 R80O:N>]8Ue W;.We ;>Ue 5>U0BQH8Ue 0-NNOWe Z>W;UW-N5e W;8e QR8UUXR8Ue O9e W;8e ZORH5e ONe W;8>Re OZNe -N5ea;8e QROL>U8Ue WOe U8N5e ;FUe *Q>R>We WOe :X>58e .N5e 5898N5e W;8Le e LXUWe -UG >Ue W;8e 0;XR0;e .HIOZ>N:e 
 E.0CT >1@1ET ;)T &CGCT E;T +G1%&T 1ET &C>
!66MT J.&:T 1ET #;9&CT E;T E.&T C1:T ;)T E.&T J;@6% T ;&CT E.&T #;66&#E1I&T 61)&T ;)T E.&T #.G@#.T @&)6'#ET E.&T >1@1ET ;)T &CGC T CT 1ET !EE@!#E1I& T ;JT 1CT E.&T #.H@#.T 61I1:+T E.&T !C#.!6T 9MCE&@MT 1:T 1ECT ;J:T 1::&@T 61)& T ;&CT E.&T #.G@#.T C&&T 1EC&6)T 1:T E&:C1;:T J1E.T E.&T J;@6%T !:%T .;JT 1CT E.1CT CE@G++6&T #;99G:1#!E&%T E;T 1:%1I1%G!6T .@1CQ E1!:C T @&T 6&!%&@CT !:%T 6!1EMT !15&T E!G+.ET E;T %1C#&@:T E.&T >1@1ECT 6&!%1:+ T MT ;J:T 61)&T &L>&@1&:#&T CG++&CECT E.!ET %1CR #&@:9&:ET 1CT :;ET !T J&665:;J:T @&C;G@#&T J1E.1:T E.&T 61)&T ;)T E.&T #.G@#.T .&:T C>&!51:+T ;)T C1:T 1ET 1CT !6C;T :&#&CC!@MT E;T C>&!5T ;)ST +@!#& T GCET !CT E.&T #.G@#.T .!CT &9>.!C1O&%T >&@C;:!6T C1:T E T E.&T &LF6GC1;:T ;)T C;#1!6T C1:T C;T E.&@&T .!CT "&&:T !T C1916!@T &9>.!C1CT ;:T +@!#&T !CT >&@C;:!6T !:%T 61EE6&T C!1%T !";GET E.&T C;$1!6T %19&:C1<:CT ;)T +@!#& T !@61&@T 1:T E.1CT >!>&@T T 1%&:E1 )1&%T E.&T 19>;@E!:#&T ;)T #.;;C2:+T 61)&#.;;C1:+T "&EK&&:T !GE.&:E1#T !:%T 1:!GE.(:E1#T &L1CE&:#& T .1CT #.;1#&T +;&DT "&Q M;:%T E/&T 71*&T ;)T &!#.T 1:%1I1%G!6 T 1ET !7C;T .!CT E;T %;T J3E.T C;#1!6T 61)&T J.1#.T 1CT C.!@&%T J4E.1:T !T ,1I&:T #G6EG@!6T #=:E&LEQ J1E.1:T )!9161&CT #;99G:1E1&CT !:%T I!A1;GCT );@9CT ;)T ;@+!:1P&%T
C;#1!6T 61)& T T 9;%&6T ;)T #.GB#.T J.1#.T .!CT .&7>&%T 9&T E;T @&)8&#ET ;:T C;#1!6T -@!#&T 1CT E.&T #.G@#.T !CT C!#@!9&E T :T @&)6&#E1:+T ;:T E.&T 1:+%;9T E.&;7;+MT ;)T !GC#.&:"GC#.T 1!:&T &!+&@T >=1:ECT ;GET E.!ET C1:T !:%T C!6I!E1;:T !@&T #;@@&6!E1I&C T
)T J&T &L >&@1&:#&T C;61%!@1EN T 1:T C1:T E.&:T ;G@T #;:#&>E1;:T ;)T C!7I!Q E1;:T 9GCET .!I&T !T C1916!@6MT C;#1!6T %19&:C1;:
T .&T ?G=E&CT 

 75$!.75$ A
$!A !56+%5$-!.6A 0"A A 0--7/%6;A 0"A 2%#$6!075/!55A %/A -/*%/ A %5A )756A 5A -7$A A 58%/#A 6A 0"A 0 A 5A 6$!A 5+86%0/A 0"A /A %/ %8% 7+A "20-A $%5A /672+A 5!+"%5$/!55A / A -02+A %/%+%6;
  !#!2A "7+65A 6$!A  A  A %5$015 5602+A "02A %65A %/ &8% 7+%56%A 1130$A 60A 50%+A -%/%5 62; A $!A $01!A 6$6A 8%267075A +;A 1!01+!A 9%++A !A +!A 6$207#$A 6$!%2A %/ %8% 7+A 8%267075A 65A 60A 2%/#A 076A 6$!A %-1208!-!/6A 0"A 6$!A 902+ A $5A /06A !!/A -7$A 5711026! A ;A $%5604%+A !:1!2%!/!   $!A 10%.65A 076A 6$6A 6$!A -02+ !:$0266%0.A 0"A 6$!A 5602+A 9%++A $8!A +%66+!A !""!6A 7/+!55A 6$!A 120+!-A 0"A -!2%/A %/ %8% 7+%5-A %5A !+6A 9%6$ A .A /A +6!2/6%8!A %5%0/A 0#!2A %#$6A +00*5A 6A 6$!A 52-!.6+A /672!A 0"A 6$!A $72$A / A 5*5A 9$6A 6$!A $72$A -756A !A %/A 02 !2A 60A 1!2"02-A !""!6%8!+;A %65A -%55%0/A %/A 60 ;5A 902+ A 6A -756A !-0 ;A A "2!! 0-A "20-A 6$!A 902+ A 50A 6$6A %6A -;A !A "2!!A 60A 5!28!A 6$!A 902+ %/# 0-A 8+7!5 A A 65A +%"!56;+!A 5$07+ A 120+%-A /6!2/++;A '6A $5A A -%55(0/A 60A 2!510/ A 60A / A 60A $!+A 1!250/+A "2!! 0-A?@ A6A %5A 6$75A A 0--7/%6;A 0"A 2!0.%+%6%0/A 0/!A 6$6A -! %6!5A 6$!A +08!A 0"A 0 A / A 2!< "+!65A 6$%5A ,08!A /06A 0.+;A %/A %65A 062%/!A 76A %/A %65A 9;A 0"A !%/#A $72$ A :6!2/++;A 6$!A $72$A %5A A 0--7/%6;A 9$%$ !.072#!5A #7% !5A / A 57110265A 6$!A "2!! 0-A 0"A %65A -!-!25A %/A 6$!%2A 0."20/66%0/A 9%6$A 902+ +;A 8+7!5A . A %.A 6$!%2A 9%6= /!55A 60A 6$!A 2!%#/A 0"A 0 A %/A 6$!A 902+ A $%5A $72$A 120-06!5A A 0/6!-1+6%8!A 66%67 !A 9$%$A 5!!5A 6$!A 902+ A / A $%5602;A %/A 2!+6%0/A 60A 0 A 65A 51%2%> 67+%6;A %5A !+!5%+A / A %5A 2!+6! A 60A -%55%0/ A A !+%!8!A 
 
 hJ7h} ]ma} C7YKWt} As_AaKAZ;A} ]C}
?]YAfhK;} ;Jma;J} J7f} aAy CWA;hA?} hJAfA} n7WmAf} h]} 7} YA7fma79VA} AshAZh} )C} ;]ZhAY_]a7at} AnL?AZ;A} C7KaVt} fJ]mhf} hJ7h} WLCA} C]a} Y7Zt} LZ} hJA} 5ZKhA?} 3h7hAf} Lf} fAaL]mfVt} CV7pA?} 7Z?} ?Afham;hLnA} ]C} JmY7[} pJ]VAZAff7VKAZ7hKZI} h]} t]mZI} _A]_WA} hJaA7hAZLZI} h]} C7YLVt} WKCA } ?An7fh7hLZI} h]} hJ]fA} pJ]} C7WV} 9AhpAAZ} hJA} ;a7;Uf} ]C} hJA} A;]Z]YK;} ftfhAY} 7Z?} mZCaKAZ?Wt} h]} YKZ]aLhLAf } pJAaA} Lf} hJA} AnK?AZ;A} ]C} f];L7W} Ia7;A#} 3h} 17mW} ;]YYAZhf} ]Z} fLZ} 7Z?} Ia7;A"} )Z} 2]Y7Zf} ! } } }zz{}9mh} ?Af_LhA} hJA} LZy ;aA7fA} ]C} fLZ} Ia7;A} J7f} C7a} fma_7ffA?} Lh } f]} hJ7h } 7f} fLZ} aAKIZA?} hJa]mIJ} ?A7hJ} Ia7;A} Y7t} aAKIZ} 9t} p7t} ]C} RmfhLAf} ]F} (]?f} W]nOZI} ;]Z;AaZ} 7Z?} fm__]ahZ]h} Tmfh} G]a} ]ma} G7YLWt} 9mj} H]a} hJA} _A]_WA} JA} ;aA7hA?} %f} hJA} ;]ZhAY _]a7at} ;aPfLf} ]C} ;mVhmaA} J7f} 9A;]YA} 7;mkA } )} J7nA} As_AaLAZ;A?} 7Z} mZ|s_W7LZ79VA} fAZfA} ]C} J]_A} 7Z?} ZAp} _]ffL:LVLht} 4JLf} ?m7W} 7p7aAZAgf} ]C} ;aLfQf} 7Z?} J]_A} 9AI7Z} h]} AZlAa} Yt} =]Zy f;K]mfZAff} ?maKZI} hJA}  f} )} p7f} 7__aAJAZfLnA} ?maLZI} hJA} Ie]qLZI} ;LnLV} aLIJhf} _a]hAfhf} xAh} CLVXA?} pLhJ} J]_A} /7ahLZ} .mhJAa} ,LZI} q7f} 7} nAat} f_A;L7W} _Aaf]Z} 7Z?} )} _a7tA?} C]a} hJA} fm;;Aff} ]C} JKf} Z]ZnL]VAZh} `a]hAfhf} 0t} Jmf97Z?} 7Z?} )} ?L?} 

 6$2> 6> ,4(> 2,> > 14..,02%5> 2$0,4"$> ,40> (,(> 2#,(%> *200%(> ,4*%(> > !(2> 2$2> ,> 61> %*205*%*"> %*> )0%*> $%12,09> 2$0,4"$> 2$%1> $0%1)2%> (0> *> (0%!9; %*"> !,0> )0%*1> 2$> )*%*"> ,!> 2$> !0,)> 6$%$> 6> !!%0)> %*> ,40> ,*12%242%,*> > 61> (1,> (0%!9%*"> 2$> )*%*"> ,!> $0%12%*> %*2%29> %*> 2$> !> ,!> 2$> 0%1)> %*12%242%,*(%:> %*> )0%*> 4(240> ,5> ((> $> 61> 1$,6%*"> $%1> 2*0> ,*0*> *> (,5> !,0> *> ,..011> .,.(> 6$,> $> *> 0-4"$2> 2,> 2$%1> ,4*209> %*> 1(509> *> 6$,> $> *,2> *> %*(4> %*> 2$> )0%*> 0)> ,!>
(%029> *> &412%> !,0> ((
> > ,).0%1,*> 6%2$> 8,41> 61> *,2> %*< ..0,.0%2> $> /412%,*%*"> ,!> 2$> ("%2%)9> *> .40.,1> ,!> 2$> %2*)> 60> 02> 1$,'651> %*> )0%+> 4(240> 2> $((*"> 2$> $02,!,0> 4*/412%,*> 0%"$2*11> ,!> )0%*> )%(%209> 5*2401> ,*> ,2$> %5%> *> ),0(> "0,4*1> > ,4(> !(> 2$> .4(%> ,*1%,41*11> $*"%*"> %*(4%*"> )9> ,6*> "%*> > $*"%*"> .4(%> 70*11> 0%1> /412%,*1> 1> 2,> )0%*> *> $0%12%*> 5(41> 1.%2> 2$> .4(%> 240),%(> > !(2> ",,> ,42> 2$1> $((*"1> 2> 61> 40%*"> 2$ > 1> 2$2> > ,*1%,41*11> ,!> ,(,"9> 0,1> ((%*"> %*2,> /413%,*> 41> ,!> 2$> *5%0,*)*2> *> > !)%*%12> ),5)*2> )0"> )*%*"> /4(%29> !,0> 6,)*> (,*"> 6%2$> )*9> ,2$01> > !,4*> )91(!> !%((> 6%2$> *6> %*1%"$21> *> 19%*">
91> 2,> 2$)> $1> /412%,*1> 60> %).,02*2> *> $> 2,> > 01= 1> 2> 61> > 509> 8%2%*"> 2%)> 2,> > (%5> ,,'%*"> '> > *> 05%6> )9> $*"%*"> 1,%(> ,*1%,41*11> *> 
 ;!:'O .DO !BO >!ADO =)O =$BO )=A:!D/=;O >A=#(BBO .;O D+'O 9'A/#!;O #=;D'IDO JO >!ADO H!BO D=O >!JO !DD';D.=;O !;$O D=O *.G'O !;O :'A.#!;O !;$O +A/BD.!;O A'B>=;B'O D=O H+!DO H!BO +!>>';/<*O +/BO #F6DFA!6O B+.)DO +!BO "'';O $A!:!D.#O !;$O F;B'DD6.;*O !"=G'O !66 M #+!66';*.;*
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

 9%\ !L6FL@'\ '\ ND\ !@'F19.\ \ 9'O\ !;9D!1;LD9'DD\ F0F\ @'W )6'!F'%\ 01D\ M1D1;9\ ;)\ F0'\ 19.%;8\ 9%\ ND\ 19M1F19.\ 0L89\ @'D=;9D'\ 01D\ :'N\ N@'9'DD\ ND\ DL88;919.\ 8'\ F;\ !;66 ;@F'\ 19\ L16%19.\ 9'N\ 0L89 D;!16\ @'6F1;9D01=D\ N01!0\ N'@(\ 8;@'\ LF0'9F1!\ 9%\ 61)'.1M19.\ '@0=D\ 8T\ 9'N\ L:%'@DF9&19.\ );L9%\ 'DF\ 'S=@'DD1;9\ 19\ 9\ @F1!6'\ \ N@;F'\ N01!0\ NE\ =L W 61D0'%\ 19\ @;/@'DD1;
\ F0'\ 19F'@9F1;96\ =L 61!F1;9\ <)\ F0'\ ;@6%\ '%'@F1;9\ ;)\ 0@1DF19\ 1*'\ ;88L91F1'D\ \ !;9X !6L%'%\ F0'\ @F1!6'\ N1F0\ \ @'%; \ \ '61'M'\ 19\ 0@1DF\ D\ M1;@\ :%\ 1 '@F;@\ '\ !8'\ F;\ =@'=@'\ \ =6!'\ *;@\ LD\ 19\ 0'M'9
\ LF\ '\ 6D;\ !@'D\  ;LF\ ;L@\ 0L8:\ 'S1DF'9!'\ 9%\ '\ L@.'D\ 01D\ +;66;N'@D\ F;\ D0@'\ F01D\ !;9!'@9\ \ '61'M'\ F0F\ '\ 1D\ !6619.\ LD\ F;\ '\ !;!@'F;@D\ ;)\ 1D\ 19.%;8\ ;9\ '@F0 \ '\ 0D\ .1M'9\ LD\ )@''\ N166\ 9%\ '\ %;'D\ 9;F\ 19F'@)'@'\ N1F0\ C;=@1F'\ 9;N\ F;\ @'98'\ .@!'\ :%\ D19\ D\ LF0'9F1!\ ;@\ 19LF0'9F1!\ 'S1DF'9!'\
 0;;D19.\ LF0'9F1!\ 'S1DF'9!'\ 1D\ N0F\ ;%\ D5D\ ;)\ 66\ 01D\ =';=6'\ 9%\ 1F\ 19M;6M'D\ 8519.\ %'!1D1;9D\ 19\ F0'\ 61.0F\ ;*\ )1F0\ 9\ ;L@\ 19LF0'9F1!1FT\ N'\ %;\ 9;F\ 0M'\ F0'\ )@''%;8\ F;\ !0;;D'\ F0'\ -6D'\ D;!16\ M6L' 
 MTMN7E\ GP7KQ;7@EM\ OM \ .7\ /K7\ O4:7\ c )<">;c T6S I c S#c !c #A)IJ);;S<)JZc )Ic AA c )!c <>Jc =>< Y)IJ c  c ;)II)<"c )Ic c >;@ 66)<"c T)I)>!c W$Jc J# c #SA$c )Ic 66 c J>c  >; c Jc )Ic 6;>IJc Ic J#>S"#c J)SIc  T 6>@; !c #SA$c #c <>Jc J5 W!c #SA$c W)J#c c T AZc 6);)J c T)I)>< c ;c J# c @S6@)Jc >!c $SA#c Ic
!;)6Z
c <c
>;;S<)JZ
c SJc T AZc 6)JJ6 c Y@ A) = c >!c J#)Ic )!c J# c @A)I$ c !c J$ c @IJ>Ac  ) Ic <>Jc J>c I$A c B I@>;;S<)J)>< WZc >Ac )"<>C c J$ A c )Ic 6)JJ6 c I !c >;;S<)JZc <c c "A Jc @>K Ac 6) =J)>` c c $ Ac 6*JJ6 c >Ac <>c J65c >!c ;)II)><4SI_Lc <>SA" ; Ac @A,TMac U)AJS c <T>+< c >!c @ AI><6c I)< c A c >!c b)<)IJAZc J>c N% c < [ c c  >AJJ)>=Ic J>c 66c !>AJ$c @A, IJ6Zc <c D 6-".>SIc V>O*>Ec < T Ac ; <^ J)>J)>W T Ac
 ; ; =c A c < >SF" c J>c II)IJc J# c @A) IJc SA)<"c 6)JSA"Z c Y @Jc !>Ac c ! Xc >7J/; c @A)I$c >A"=)]J)>SJ#c 6Sc <c >6)JZc Q# c 80JZc G c 6A" 6Zc @II1T c <c S<)=T>6T  c 6J#?S"$c )Jc #Ic <>Jc 9WZIc  Ac J' c @IJc 6 T ;)<AI c 6IIc @A)I#c S< Ac JW>c IS II)T c W#)J c @I ( S<)6c <>Wc  !S<Jc C I)IJ c J# c S<)I A=)<"c SJ$>A)JJ)T c WZIc >!c J# c !)AIJc @IJ>Ac J# c )> IAc c >>6)<">!!c @ A)>c W#)#c "T c E <#1E ,(;5E <(6#E 95E 231,@E 19.E 016E 3*956E 61E 016#3E 23(5# E #(5E 25613E (5E ;3@E +(0E 61E #3E 0E ;3@E "11E <(6#E 6#E 5(+E 0E @(0" E @E 23506E 23(5#E ?23(0E (5E 10,@E 6#E ,656E (0E E 53(5E 1!E 0190635E <(6#E E ,3(,E 9,6:3E <#(#E ,(0"5E 61E 236(0E E <@5 E 13E ,(6@E <#1E 4E 53#(0"E 13E 1C 352105((,(6@E (0E 6#E #93#E 01:3(5#/06E 13E /(,@E ,( E 0E 0,/06E 1 E 6#(3E ,@E ;16(10E 59#E 23(5#E ,35#(2E 
 -:J #0& 80CJ -3 %'7> ;' J * ;J -:J 3&&%&%J -:J 248'J ;* 3J @&00F -3;&3;-43&%J 40% :;C0'J 5 8-:*J 0& %'8:*-5 J *'8'J -:J 3J >8)&3;J 3&&%J (48J J 8'3'A'%J ?-:-43J 4(J *>8#*J @*-#*J -:J #0 -2'%J !CJ ;*'J @*40&J 2&8-# 3J *>8#*J -3J J #400 !48 ;-?'J 584#'::J 4(J 5 :;48 0J 50 33-3)J 43J ;*'J 04# 0J %-4#&: 3J 3%J 3 ;-43 0J 0'?&0: J >#*J J 584#'::J @4>0%J 34;J -3;'3%J ;4J 584%>#'J J 3'@J '##0':- :;-# 0J 54@&8J :;8>#;>8'J !>;J 04# 0J #*>8#*&:J A*-#*J 8'J !0&J ;4J 8' %J ;*'J :-)3:J 4(J ;*'J ;-2':J 3%J @*-#*J 8&:543%J ;4J ;*'J 3''%:J 4(J ;*&J ) ;*'8&%J #422>3-;CJ 3%J ;4J 2-::-43 J >:;J :J ':>:J %-%J 34;J #43(482J -3J 2 3CJ A C:J ;4J '@-:+J8'0-)-4>:J #>0;>8'J !>;J # 00&%J ;*'J 5'450'J ! #/J ;4J J 248'J >;*'3;-#J 8' %-3)J 4(J 4%
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
 
 
 =),7)$A3:.M ;:M A/)M /E=$/M 3:M =)7!A3;:@/3!=IM /=3@A3!:@ M /3@M %;:&)BTd 4NLVS2TVBL2S24V8SBTVB4d BTd 8]_ QS8TT87d BLd V>8d
>NFBL8TT 4N78
d +8[d  d +N>;BLEd B78LVB;B8Td V>BTd ZL78STV2L7BL8d 324E8d ,8\d 18TV2K8LVd B78LVB;B8Td 2d TBKBF2Sd NSB8LV2VBNLd 8]48QVd V>2Vd *8TZTd Q8STQ84VB[8d BTd 8T4>2VNFN;BLEd BLV8SQS8VTd V>8d \NS7Td N;d V>8d .ZSd $2V>8S d
'2FFN\87d 3^d V>^d L2K8
d 2Td 2d QS2^8Sd ;NSd <2V>8SBL8Sd 2L7d S8L8\d ^NZSd Q8NQF8d F8Vd BVd 2<2BLd 384NK8d V>8d VSZ8d Q8NQF8d N;d &N7
d
 *8TZTd BTd BLV8L7BL2Vd V>BTd S8L8\87d Q8NQF8d T>NZF7d 38d >NF^d TNd V>2Vd V?8d S8BVd 384NK8d [BTB3F8d GBE8d 2d 4BV^d T8Vd NLd 2d >BFFd +N>;BLEd 2TETd B;d V>8d 82SF^d 4>ZS4>d 8d -8\d 18TV2K8LVd 4NKKZLBVB8Td T88d V>8KT8H[8Td BLd 4NLVS2TXd VNd V>8d TZSSNZL7BL;BLEd 4BV8Td LZK8SNZTd V8]VT d 8UQ84B2FF^d ;SNKd /2ZFTd 8QBTVF8Td (Ld "Q>8TB2LTd 02ZFd T2^T d
%NSd NL48d ^NZd \8S8d 72SEL8cT d 3ZVd LN\d ^NZd 2S8d IB=>Yd CLd V>8d +NS7d \2FEd 2Td 5>BF7 S8Ld Nd;d dBd<> d Vd
 #dQ> d dad ddddbd 1>8d \OdSd7 d d ;SNKd \>B4>d >SBTVd S8Td 4Z8Td V>8d S8Q8LV2LVd L8\d 38FB9[8STd BTd 42FF87d 2Ld
8[BFd 2<8
d 1>8d >ZS4>d BTd V>8d 4NKKZLBV^d N;d V>NT8d \>Nd 2S8d S8T4Z:7&N7Td
L8\d 4S82VBNL
d 1>BTd 4NLVS2TVd 38V\88Ld V>8d ;NSK8Sd FB[8Td N;d V>8d 4NL[8SVTd 2L7d V>8BSd QS8T8LVd FB;8d BLd V>8d >ZS4>d BTd S8;8SS87d VNd TNd N;V8Ld V>2Vd DVd BTd 42FF87d V>8d
.L48d 2L7d LP\d Q2VV8SL
d .;V8Ld V>BTd 6NLVS2TVd BTd BFJZTVS2V87d 3^d FBTVTd N;d [B48Td 1>8d LN\d S8;8STd VNd FB;8d \BV>BLd Y>8d >SDTVB2Ld 4NKKZLBV^d 2L7d BL` 4FZ78Td LNVd NLF^d BLV8SBNSd 7BTQNTBVBNLTd 3ZVd S27B42Fd TN4B2Fd 
 $JHW*O^*G$*Wm .3Wm H*am $Q* Z4JG
m 4FN?4*Wm $. H-*'m WJ$4 ?m WZQ^$Z^Q*Wm b.4$.m Q*m HJZm ?4<*m Z.JW*m 4Hm Z.*m Q*WZm J+m WJ$4*Zdm +m N!QZ4$^? Qm W4-H4+4$ H$*m ZJm J.+4Gm Z^THWm ZJm \.*m P^*W[8MHm J+m F4WW4JHm *m !W>Wm .Jam m $JFF^H9Zdm b24$.m 4Wm $!C@*'m ZJm "*m !m &JGZU W[WJ$4*Zdm $!Hm Zm Z.*m W!F*m Z4F*m "*m Hm *_ G-*D4f7G-m $JFF^G4Zdm !H)m .*m +J$^W*Wm JGm [.*m ':?*FF m J,m ].*m $.^Q$.m b.4$.m 6Wm 5Gm "^Zm IJZm J+m Z.*m bJQ?'m Jbm $ Gm Z.*m .^Q$.m >**Nm +QJFm #*$JF4G-m !Hm *?4Z4WZm WN4Q4Z^ ?m -.*ZZJm JQm \JZ ??dm JZ.*QbJQ?'Bem H'm W*N V Z*'m +QKFm Z.*m bJQE'm !G'm Z.*m N*JN?*m ZJm "*m *_ H-*?4f*'m -!;Gm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
V ,V (BAKG#JKJV 0L>#AV ,..BGKV #KV #'FL3G,*V 0B<3U A,JJV N3K0V K0,V J#M3A/V *,,*V B.V B*V N0BV :LJK3.3,JV K0,V /B*<,JJV #'',DKJV .#6J,<.V N3K0V K0,V /L38H#'<,V B.V <3.,V A,N,*V 'BAKG#JK JB'3,KRV .,V M,GRV ',#AV KB*#RV !0,G,V 3JV #V M,GRV '&B#;,V M3J3%<,V B*  J V @,JJ#/,V ABKV CA3JJ3BAV N03'0V 3JV #M5FHb T58-5b T)Mb S2JWb T2??b J2-28S20
b 38D8M520b T8N5b )b H)J)?8NRJ48-)?b M2JS8.2b 8Db T58.5b T2b MRCC20b RHb FRJb D2Tb RD02JMN)D18D4 b 2Nb C2b -FD.?R02b U8N5b )b IRFN)N8FDb 3JFCb N5:Mb M2JS8.2b 8Db T58.5b T2b -FDM2-J)N20b N52b H2FH@2b F3b F0b 8Eb TFJ0Mb H)J)H5J)M20b 3JFCb N52b TFJ0Mb F3b -FDM2-J)N8FDb *3b N52b )MMb &#$%b "#(#b !b & b & %(b 2)02Jb '2b N5)D>b WFR
b ?C845NWb )N52Jb F3b )??
b 3GJb M5)J8D4b T8N5b RMb N52b ?FS2b N5)Ob C)>2Mb WFRb F0 b '2b HJ)8M2b WFRb )D0b P6+D>b WFRb 3FJb .)A?8D4b RMb N5JRb 3)8N5b NFb M5)J2b WFRKb 483Nb F3b ?FS2b V8N5b N52b TFJB0b )D0b MFb NFb 5)MN2Db N52b `R?38??Z C2DNb F3b WFRJb HJFC8M2b F3b ?8,2L)N8FDb 3FJb )??b H2FH?2 b FaD8D4b 8Db N52b .5FJRMb F3b -J2)Q8FDb T2b .2?2,J)N2b WFRJb HJ2M2D/2b T8N5b N5;Mb 5WCDb F3b HJ)8M2b ??b F?W
b 5F?W
b 5F?W
b FJ0b F0b F3b 5FMNMb[]^b 2)02Jb ?2MM20b )J2b XFR
b 02)Jb )N52Jb 3FJb 3JFCb N52b ,248DD2D2MMb )D0b M5)J8D4b )b .FCCFDb 02MN8DW b 
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" Y|Y ius Y{ Ys^ |Y\ir 6|Ys Y `oao` ou` au| `Y[g u g`| u gY u| si rY Z` g` c`sis` ics gY i \oYir u Z` g`|Yo^isd gY aisYo Ys^ Zo``^ sius ua Yoo v`uvo` is 0g|i <` u| ?u|^ +oo' 5u| g|udg 7i Ys^ i g 7i Ys^ is 7ir Yoo gusu| Ys^ dou| i di`s u u +oridg 5Y g`| is sius i g g` 7uo Nvi|i u^Y Ys^ ``|^Y s io ` `|si +r`s Eu `  Mi\gY|^ E`gY Qg` /Y guoi\ Aur`s  v  a  7 Mi\gY|^ Ei`Zg| 0g|i Ys^ 0o |` v $ 6`|Y|^ V 7dg` O <  6u^ ub P|w|i` v "
BY|\`pou ^`0 ,``^u N =  .Yi\ 4\\o`iYo 0urrsi i` v #
  6|`du| .Yr M`oicius Ys^ ,qi`sY ius) vx  !2iYs` X`Yd`}
.iguv Ys_ g` >isd^ur is Rh` 2``w`| C`Ysise ua 4\usurj] ?ia`( 1|k i\Yo 4Y us
T N 0Y guoi\ .iguy IY u~Yo @` ` ut g` 4\usur `_ Z M .\` 3ufoY v % "9Zi^  v $#  Mud`| 8Yidg  -s +o`|sY i` Uilus v # $4oiYZ`g ?`us` Mi\g 0us |i` F``^ ?iZ`Y mus Suu J|ue|`iu DY &" :G<`|ur` C|vg HLussu| HK  v  `` ,zv`s^i  WgY i g` Ovi|i NYisd* 6`|gY|^ ?ugaisn <` Ys^ 0urrsi  v   ;Zi^  v  9Zi^  v " 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
Y :3/.H3;.Y H1.Y N?R'0.EY  /Y .Q@:?C.CEY '=-Y -3E+?N.C3.EY ?/Y 'EHC?=?;.CEY C'-3+'::RY ':H.C.-Y H1.Y 1L;'=Y @.C+.@H3?=Y ?/Y H1.Y L=3N.CE.Y .=-3=0Y '=-Y 'Y =.OY .C'Y O'EY 'HY 1'=-Y !1.Y 3--:.Y 0.EY O.C.Y !1.Y ./?C;'H3?=Y O'EY *C.'93=0Y ?N.CY ?CH1.C=Y MC?@.Y '=-Y ?LH1.C=Y MC?@.Y +';.Y M=-.CY H1.Y EMCN.3::'=+.Y ?/Y H1.Y =BM3E3H3?=Y =.OY LC?@.'=Y @?O.CY @'3=Y O'EY *.+?;3=0Y 'Y LC3=0Y H13EY H3;.Y ?/Y HM;L:HM?LEY +1'=0.Y
 

 >[%iCsh u%h )\a[ C[i\% [\)S7 %h`s7 :%ZCSxi?7 x\s[>7hi \: i?Cai77[ 0?DS3a7[ 7 h^7[i ?Ch i77[&>7 x7&ah %[3 y\s[> %3sSi?\\3 &i i?7 0\sai \: ?%aS7h ! &[3 u&h Q[\u[ :\a ?Ch h7S:C[3sS>7[i &[3 DZ^7is\sh SC:7hixS7 saD[> i?7 )%iiS7 \: &Z^S\[% C[  >[%iCsh u%h h7aD\shSx u\s[373 7~ 0%sh7 \: 0aC^^SC[> C[PsaC7h i\ )\i? S7>h ?7 u%h 0&aaC73 *%0R i\ ?Ch %[07hia%S ?\Z7 u?7a7 ?7 h^7[i & S\[> %[3 ^%C[:sS 0\[t%S7h07[07 saC[> i?Ch ^7aC\3 >[%iDsh u&h >Ct7[ h\Z7 )\\Qh i\ a7%3[\i ?Ch shs%S :%a7 \: i%S7h \: 0?Ct%Say )si i?7 C:7 \: ?aDhi &[3 & 0\SS70iD\[ \: h&C[ih
SCt7h ?Dh a7&3C[> Z%i7aC&S h7i s^ C[ >[%iDsh % i7[hD\[ )7iu77[ ?Ch :\aZ7a SC:7 uDi? Dih 3a7%Zh \: 0?Ct&Saz &[3 u\aS3Tx ?7a\DhZ %[3 i?7 ?7a\C0 SCt7h \: a%[0Ch \ZC[C0 %[3 \i?7a h&D[ih h ?7 3%x3a7%Z73 &)\si ?Ch :sisb7 ?7 )7>&[ i\ c70\>[C}7 0\[ia%hiC[> :77UC[>h &hh\0C%i83 uCi? ?Dh t&a{D[> CZ%>C[C[>h [ ?Dh &si\)D\>d&^?y uaDii7[ E[ j?7 i?Ce3 ^7a\[ >[%iDsh 37h0aF)73 i@Dh 7w^7aD7[07 +: 'Vi7e[%iG[> ;77SD[>h #7i i?7a7 u%h i?Dh 4D::7a7[17 "?7[ ?7 u&h i?C[Q C[> &)\si iA7 i?H[>h ,: i?7 u\bW3 ?7 k\\Q Zs0? 37SI>?l D[ i?7Z )sm &:i7au%f3h u?7[ ?7 u&h iCa73 %[3 ^si i?7Z %hD37 ?7 :\s[3 l?(i ?7 u&h 3a| &[3 3Dh0\[i7[i73 si u?7[ ?7 iB\s>?i \< >\D[> i\ 7ash%V7Z -&a7:\\i &[3 7&iC[> [\iAD[> )si ?7a)h &[3 s[37a>\F[> %XS k?7 ]i?7a aD>\ah $nA&i ?7 h&u i?7 h%C[ih ?&3 [3sa73 [\i \[Yy vh .7 0\[h\S73 u?7[ ?7 ?%3 i?7h9 i?\s>?ih /so 7t7[ &:i7a ^siiC[> i?7Z &hC57 ?7 a7Z%C[73 0\[i7[i &[3 ?%_^y 7 3C3 [\i u\[37a ?u7t7a &i i?Jh [\a 3C6 ?7 hi\^ i\ ^\[37a i?7 3D: :7a7[27 s[iKS \[7 iDZ7 ?Ch 7y7h u7a7 \^7g73 & SDiiS7 %[3 ?7 )7>&[ i\ Z%at7V %i l?7 3L:=7e7[07 &[3 i\ a7 :S70p s^\[ Di a7%SC}C[> :a\Z 7w^7aM7[07 i?&i h\Z7 i?\s>?ih S7:i ?FZ h&3 &[3 \q?7ah ?&^^x CiiS7 )x SNiiS7 ?7 0&Z7 i\ a70\>[C}7 i?7 3D::7a7[07 )7ru77[ i?7 h^DaCih i?%i %>Oi&i73 ?CZ 
 % ; J )4;)$%<;J D 9 J 9A)1J )4J $4<)A;
J ;8*9)9)<)54;J A;<52;J 4J 9 1)$)5A;J 89<) ;J D 9 J 5#J A; J 541GJ )#J <% HJ #952J D)?')4J <%e G3e OG%8"?e 4L"%-e #F)e 8O!#e IGQ-FQ8#?e IL-I"L#Q8GFe /GLe #e /W??-Le -ZI-L8-F%-e G/e Q6-e Z-L%8O-O e 6-e 846Q--FQ6e FFG^ Q"Q8GFe %GFO8OQOe G/e Q6-e
L8F%8I?-e #F)e GWF)#Q8GF
e "F)e V6-e -Z-L%8O-Oe G/e Q6-e 8LOQe --> e 6-e
L8F%8I?-e "F)e GWF)"Q8GF
e $-48FOe Y8Q6e "e %GFO8)-L#Q8GFe "F)e "Y"L-F-OOe G/e %L-"QWL-6GG)  
 R ?&3?&R A)AR A)&R +3$+E+$D0R +?R 05E&$R '5;R )&;?&0'R D4"53L $+A+5300HR ;&(;$0&??R 5'R '+0+3(? $5&? R 3$R 35AR '5;R F)BR ?)&R ?R A)&R ;&A;&A3AR &G8&;+&3"&?R 5$?R 2&;"HR 3%R D3M "53$+A+530R ""&8A3"&R 3$R ;&"5(3+K&?R )&;R 5F3R '+0D<&?R A5R ")55?&R 0+'&R 3$R DA)&3A+"R &G+?A&3"&R ?)&R +?R 0&$R A6R 8;5L '5D3$R ?5;;5FR 3$R 8;H&;R '5;R )&0+3( R )&R ?&"53$R A*+;$R 3$R '5D;A)R F&&/?R 5'R A)&R G&;"+?&?R 0&$R A)&R ;&A;&A3AR A5R &30+?AR D3$&;R A)&R ?A3$;$R 5'R &?D?R 3$R A5R ""5283HR )+2R +3R )+?R 0+'&R 8??+53R $&A)R 3$R ;&?D;;&"A+53 0FH?R ?&&/+3(R A5R '+3$R 5$R +3R 00R A)+3(? R )&R 8;5"&??R 5'R A)&R 9+;+AD0R G&;"+?&?R +?R 35AR 3R &3$R +3R +A?&1'R DAR R 2&3?R 0&$+3(R A5R A;3?'5;2A+53R 5'R A)&R ?&0' R (3A+D?R +?R ?&&/+3(R AQR "0;+'IR '5;R )+?R ;&A;&A3AR A)&R :D&? A+53R 5'R ';&&$52R
RNOPR';&&$52R +?R R :D&?A+53R !5DAR J5D;?&0' R AR +?R 35AR R :D&?A+53R F)+")R R 8&;?53R )?R +AR +?R R :D&?A+53R F)+")R R 2R 3$R F+A)R F)+")R R "53@A+ADA&R F)AR R 2R A5R &L "52& R AR +?R A)&R 85A&3A+0R '5;R ?&0' $&A&;2+3A+53 R (3A+D?R ;$+"0R ;&0+(+5D?R :D&?A+53R ,?R A5R $5R F+A)R H5D;R ';&&$52R F)AR ;&R H5DR (5+3(R &R -?R 8;&8;+3(R )+?R ;&A;&A3AR '5;R ?&=+5D?R );+?A+3R ")5+"&?R F)+")R +3E51E&R R ?&;")R '5;R A)&R F+00R 5'R 7$R +3R 53&?R 0+'& R 3$&;0H+3(R A)&R 8;5"&??R 5'R (3A+D?R G&;"+?&?R +?R R $H32+"R E+&FR 5'R 5$?R 05E&R F5;/+3(R "A+E&0HR +3R A)&R F5;0$ R E&3A?R 80"&?R 8&>?53?R ".;"D2?A3"&? 00R "53A+3R F+A)+3R C)&2?&0E&?R 5$?R +3E+AA+53?R F)+")R "00R '5;R R #)5+"&R 3$R R ;&?853?& R F5;$R 5'R 5$ (3A+D?R &3"5D;(&$R ;&'0&"A+53R 53R A)+?R &G+?A&3A+0R R /+3$R 5'R "53E&;?A+53R &AF&&3R &E&3A?R 3$R 5$R K
 K /+<=%5+5)K =7K =*%K D7:/!K 5!K =7K =*%K 9+:+=K %K *!K /%:5%!K =7K :%!K =*%K 4%5+5)K 7&K /+&%K <+=A=+75K &:74K 8A:<%/B%)!K` !C'` Q/!Q` -0V)P` B)` -K)!Q` >` /!P` #))C` !` -KFW0D-` +K))'FB` +KFB` P)>+IK)F%%TI!Q0FC` !C'` !` %FKK)PIFD'0C-` +K))'FB` +FK` Q/)` %FC%)KCP` !C'` D))'P` F+` FQ/)KP` 0D` FTK` PF%0)QZ` !D'` %T>QTK) ` FB)/FW
` W)` /!V)` FI)C)'` FTK` /)!KQP` !C'` FTK` /FB)P` QF` Q/)` C))'P` F+` FQ/)KP` !D'` F'` /!P` K)%0IKF%!Q)'` #Z` >)!'0C-` TP` 0D` -FF'` '0K)%Q0FCP` Q/!Q` W)` '0'` CFQ` !CQ0%0I!Q) ` ` C` PFB)` W!ZP` -D!Q0!D` PI0K0QT!>0QZ` PFTD'P` V)KZ` 0D] X!K'+FPQ)K0C-` !` K)>!Q0FDP/0I` #)QW))C` F'` !D'` Q/)` 0D'0V0^ 'T!>` I)KPFC
` )D%FUK!-0D-` -KFWQ/` 0D` I)KPFD!>` +K))'FB` !D'` '0P_ %)KDB)CQ` !C'` 0CV0Q0D-` !` I)KPFC!>` +F>>FW0C-` F+` /K0PQ ` GW_ )V)K
` -C!Q0!D` PI1K0QT!>0Q[` QF'!Z` 0P` #)0C-` L))Y!B0D)'` 0D` Q/)` >0-/Q` F+` Q/)` D)W>Z` )B)N-)CQ` PF%0!>` %FCP%2FTPD)PP` F+` !R0%!C` ` !C'` 0D` >3-/Q` F+` K)&)DQ` )C)M!>` FC-K)-!Q4FCP` F+` Q/)` )PT0QP ` D` ` Q/)` )PT0QPIFCPFK)(` )CQ)K` F+` FC^ %)KC` 0D` !P/0C-QFD
`  `  ` IT#?5P/)'` FTC'0D.P` Q/)` IKF'T%Q` F+` !D` B)K6%!C` !C'` !D!'0!D` Q!P=` ,FK%)` HC` PF%0!>` %FDP%7FTP D)PP` !D'` -C!Q1!C` PI0K0QT!>0Q\ ` )PIFDP8V)` QF` Q/)` -KFW0D-` 0DQ)K)PQ` 0C` K)>0-0FTP` )YI)N9)C%)` !D'` IK!Z)K` !D'` Q/)` K)C)X!>` F+` Q/*` Y)K%0P)P` +F%TP)'` FD` 0C'0V0'T!@>Z-T0')'` -D!Q0!C` K)QK)!QP
` S/)` Q!P=` +FK%)` IK)P)EQ)'` !` CTB#)K` F+` WFK=0C-` I!I)KP` !C'` WK:Q0C-P` %>!K0+Z0C-` !C'` 0CQ)-K"Q0D-` PF%0!>` Q/)_ F>F-Z` !C'` Q/)` Y)K%0P)P ` /;P` WFK=` /!P` #))D` !` V!>T!$>)` %FDQK0#TQ0FC` !D'` PQ0BT>TP` QF` +TKQ/)K` )YIAFK!Q0FD` F+` Q/)` 0CQ)-K!Q0FC` F+` K)>0-0FTP` )YI)K0)C%)` !C'` PF%0!>` !W!O)D)PP ` 

 6N 5LN "K="?*"6"N 9#N A&"N K"?*@"@N A&"N @9*1N !*5"6@*97N &@N ""6N =?A*F2?2LN 2"?N *6N A&"N 5"!*AA*96@N 96N @)6N 6!N A&"N
*6%!:5
N *6N "95"@N N :@5*N "G"6AN *62F!*8%N N ="? @962N ="?@="A*G"N FAN "KA"6!*6%N #?N "L96!N *A N 6N A&"N
I9N A6!?!@
N A&"N &:@A*2"N #:?"@N I*A&N I&*&N A&"N &?*@A)6N !9"@N AA2"N ?"N 6:AN 962LN I*A&*6N A&"N *6!*G*!F2N FAN ?"N 2@:N *!"6M A*#*2"N *6N @9*2N F@A:5@N 6!N *6@A*AFA*96@@:*2N 6!N "6G*?965"6A2N 9:6@>*?A9?@N *6N "G*2 N F&N 6"IN *6@*%&A@N #*AN I"32N I*A&N A&"N !"G"29=*6%N A&":29%LN 9#N %?"N 6!N ?"G"2 A*96N 6!N ?9!"6N A&"N @:="N :#N !*@"?65"6A N N ?:!"?N @"6@"N 9#N 5*@@*96N #2:I@N #?95N A&"@"N 6"JN F6!"?@A6!*6%@ N AN "6G*@*96@N N 9229?A*96N I*A&N &?*@BN *6N &*@N &"2*6%N ?":6*2*6%N 6!N 2*"?A*6%N :?0N *6N A&+@N I;?2! N &"N #:F@N *@N 2"?4LN *6?M 6A*<62N 6!N 2"!@N A:N N !""="6*6%N @"6@"N 9#N I&AN ,AN 5"6@N A:N "N
&F?'
A&"N :!LN 9#N &?*@AN *6N A&"N I9?2!N 6!N N @*%6N 9#N &*@N =?"@"6"N ?:5N A&"@"N *6@*%&A@N *AN *@N =9@@*2"N A9N !"?*H"N -5=2*M A*96@N #9?N A&"N !"G"29=5"6AN 9#N #*A&N 955F6*A*"@A&9@"N @522N %?9F=@N I&*&N &G"N @:N 5F&N =9A"6A.3N $9?N 96%:*6%N 69F?*@&M 5"6AN 9#N &?*@A*6N !F2A@N #:?N "6%%*6%N A&"N 2*CLN *6N 9M 29?A*96N 6!N 92*"?A*96N I*A&N &?*@AN 6!N #9?N "95*6%N @*%6@N 9#N A&"N *6%!:5N &"@"N %?9F=@N @A?F%%2"N A9N &:2!N A9%"A("?N A&"N A"6@*:6N "AI""6N A&"N /9F?6"LN *6I?!N *6A9N A&"N @"2#N 6!N A&"N /9F?6"LN :FAI?!N *6A:N A&"N I9?2!N 2*G*6%N 6!N 59!"2*6%N A&"N ?M *6%N 95=@*96D"N ?"2A*96@&*=@N A9N I&*&N "@F@N 22@N &*@N ="9=2" N %6A*6N @=*?*AF2*ALN &@N 5F&N A9N 9##"?N @F&N %?9F=@ N N 5N =?A*F2?2LN *6A"?"@A"!N *6N %?9F=N "K="?*"6"@N :#N E&"N 
 )SM)!Sh 5Eh !5Abh 5,)h !Eh !'!IS!S5FEh F,h 1E!S5ZQh 5E)S))ES3h EEFS!S5FEh Sh 3!Qh S3)h IFS)ES5!Ah ,FMh Q3!M5E1h `5S35Eh !h ,!5S3h #FCCZE5Sbh EFSh FEAbh 5E'5]5'Z!Ah )aI)M5)E#)Qh F,h #FE])MQ5FEh "ZSh #FE])MQ5FEh FEh S3)h QF#5!Ah A)])Ah `35#3h 3!Qh "))Eh QFh A5SSA)h ZE')MQSFF'h !E'h `35#3h 5Qh QFh E)#)QQ!Mb h 5S35Eh S3)h #GC5E1h b)!Mh h 5ES)E'h SFh C!@)h !h 5E)S))ES3h EEFS!S5FEh 1MFZIh M)SM)!Sh `5S3h Cbh ,!5S3h #FCCZE5Sbh !E'h )aI)M5)E#)h S3)h IMF#)QQh ,5MQSh 3!E'h h !ES5#5I!S)h !Eh )aI)M5)E#)h F,h QI5M5SZ!Ah '5M)#S5FEh `5S35Eh !h 1MFZI h AS3FZ13h CZ#3h #FZA'h ")h Q!5'h !"FZSh S3)h ]!M5)S5)Qh F,h 1E!S5!Eh IM!b)MD)'5S!S5FEh !,,)#S5])h IM!b)Mh J)S5S5FE!Mbh IM!b)Mh ,FMh !h QI)#5,5$h 1M!#) h )a!C)Eh F-h #EQ#5F[RE)QQh IM!bd 5E1h f5S3h S3)h 5C!15E!S5FEh "F'5Abh IFQSZM)h !E'h IM!b)Mh Q5CIAbh ")5E1h FI)Eh !E'h IM)Q)ESh SFh S3)h FM'h )S#h h `5AAh 3)M)h C)Ed S5FEh ?ZQSh FE)h !QI*#Sh `35%3h 5Qh ])Mbh #3!M!#T)M5QU5#S3)h 3!"5Sh F,h )^!AZ!S5FEh ZM5E1h M)SM)!Sh 1E!S5ZQh M)&FCC)E'Qh !h ,6,S+)Ed C7EZS)h I)M8F'h .FMh )]!AZ!S8FEh FEh 4F`h V3)h KM!b)Nh `)ES!,Sg/h )!#3h IM!c)Mh I)M5F'h 35Qh 5Qh !h M)_9)`h F,h !0,)#S:])h #FEQ#5F\Q E)QQh (ZM;E2h IM!b)Mh ZSQ5')h F,h M)SO)!Wh X35Qh )a!C)Eh E'h !E'h 3)!MSh !E'h ")d #FC)Qh !h '!5Abh Q)A,#FE,MFES!S5FEh !E'h M)E)`!Bh F,h 3M5QS&@K&+(O M=(:E,%(O 7)O )#,>+O #6'O >+(O 8:737>,76O 7)O .D=K >,%(O
 (O #''=O #O )77>67>(O F+,%+O #%/57F0('*(=O
>+(O :(#0,>I 7)O #O 0#:*(:O (=D,>O )#3,0JO ,5%0D',5*O +:,=>,#6O ,)(O 73L 3D5,>,(=ON NO
O #6'O 7>+(:=O F+7=(O %+#:,=3=O :()0(%>O *6#>,#5O ,5)0D(5%( O 0#:/(=O ,3#*,5#>,E(O :()0(%>,76=O 76O >+(O
D0(=O #=O >7O #>,5*
O %#00O #>>(5>,75O >7O +D5*(:O ,5O >+(O 0,)(O 7)O (=D=O (=D=O +D5*:IO #5'O >+,:=>IO 'D:,6*O +,=O )7:>IO '#JO )#=>O (=D=O )((',5*O >+(O +D6*:IO %:7F'=O #5'O =+#:,5*O 3(#0=O F,>+O +,=O ',=L %,80(= O 0#:/(O :#,=(=O >+(O 9D(=>,75O 7)O +7FO +,=O )7007F(:=O :()0(%>O >+(=(O %75%(:5=O >7'#IO #6'O =>:(==(=O
>+(O ,387:>#6%(O 7)O %75>(380#>,E(O (H(:%,=(=O )7:O #*(5>=O 7)O %+#5*( 
O (O =((=O >+(O H(:%,=(=O #=O
#O =(:,(=O 7)O (H87=D:(=O 7)O >+(O +:,=>,#5O +(#:>O >7O +:,=>O 8;(=(6>O ,6O >+(O 7=8(0O 3J=>(:,(=
O #6'O #O :(=7D:%(O )7:O '(E(078,6*O #O 8#==,76O )7:O :,*+>O 7:'(;O $#=('O 76O #O +:,=>7
   %(5>:-%O 8(<=8(%>-E( O O )7D6'O >+,=O:()1(%>,76O >7O $(O +(28)D0O #6'O #O =>,4D0D=O >7O )D:>+(:O :()0(%>,76O 76O %75?(387:#:IO #'#8>#>,76O 7)O >+(O H(:%,=(= O IO >+7D*+@=O 0('O 3(O >7O (H#3,5(O #67>+(:O =(>O 7)O :D0(=O >+(O 76(=O 76O
,=>:,$DA,6*O 03=
O F+,%+O 0,/(O >+(O
D0(=O #=O >7O #>,6*
O =((3=O >7O $(O ):(9D(5>0IO ,*57:('O ,6O >+(O *,E,5*O 7)O :(>:(#>= O O =((O >+(3O #=O #O 8:#%B,�O (H>(5=,75O 7)O >+(O :()0(%>,76=O 76O
+:((O #,:=O 7)O (5
O ,6O >+(O (%76'O !((/O 7)O >+(O H(:%,=(= O +(=(O *D,'(0,6(=O 3#IO +#E(O $((6O G:,>C(6O )7:O 0#JO 8(:=76=O #%%7:',6*O >7O 0(3,5*=O %733(6>#:J O +(IO #:(O 80#%('O ,5O >+(O 3#5D#0O 7)O >+(O H(:%,=(=O #)>(:O >+(O
7D:>+O "((/
O F+,%+O ,380,(=O >+#>O >+(IO #:(O >7O $(O D=('O ,6O '#,0JO 0,)(O 

 A"_ G#_ ACM_ 4PKM_ A_ -@)-AM-S#_ #V#G!-K#_
-KMG-PM-A)_ 6@K_ G#&#GK_ MC_ M*#_ K*G-A)_ C&_ U#6M*_ A"_ ECKK#KK-CAK_ U-M*_ M*CK#_ -A_ A##"_ EGM-!P6G6W_ M*CK#_ MC_ U*C@_ M*#_ G#MG#MAM_ -K_ G#6M#"_ )AM-PK_ -K_ !CA!#GA#"_ M*M_ M*-K_ K*G-A)_ 6-5#_ 66_ M*-A)K_ -A_ 6-&#_ #_ )Q-"#"_ W_  EGCE#G_ @CM-SM-CA _ *.K !M-S-MW_ -K_ MC_ G#&6#!M_ M*#_ CS#GG!*-A)_ M*#@#_ C&_ M*#_ V#GZ !-K#K_ MC_ K#GS#_ C"_ -A_ 67_ M*-A)K _ KP!*_ K*G-A)_ K*CP6"_ ACM_ M5#_ E6!#_ EGM_ &GC@_ _ G#6M-CAK*-E_ U-M*_ C" _ )AM-PK_ -K_ U#66_ UG#_ M*M_ *-K_ V#G!-K#K_ U-86_ -ATC6S#_ M*#_ E#GKCA_ -A_ _ KMGP))9#_ MC_ @5#_ M*#_ "#K-G#K_ C&_ #KPK_ M*#_ ACG@_ &CG_ !*C-!#K_ ACM_ "-KCG"#G#"_ *P@A_ &&#!M-CAK]_ A_ MC"W^ K_ UCG6"_ M*-K_ -KKP#_ C&_ K*G-A)_ U#6M*_ A"_ ECKK#KK-CAK_ -K_ _ S#GW_ G#6_ A"_ RG)#AM_ -KKP#_ M_ -K_ ECKK-:#_ M*M_ M*#_ $VE#G-#A!#_ C&_ !C@@-MM#"_ 6W_ E#GKCAK_ @W_ @5#_ _ &PMPG#_ !CAMG-PM-CA_ MC_ )AM-A_ KE/G-[ MP6-MW_ -A_ M*-K_ G#_ *GCR)*_ M*#_ W#GK_ -A_ CPH_ CUA_ &@-6W_ 6-&#_ U#_ *S#_ !*A)#"_ CRG_ FG-CH-M-#K_ A"_ EMM#GA_ C&_ )-S-A)_ @CA#W;#KK_ MC_ M*#_ *PI!*K-M*K ##AA"-K_ A"_ @CG#_ MC_ !*RH!+_ 0B_ EGC!#KKC'#!C@-A)_ KP!*_ K_ 4PKM-!#_ )GCPEK_ A"_ CAK_ S-66#_ &CG_ C!!K-CA?_ PG)#AN_ A##"K _ ,#K#_ 6CAK_ G#_ M*#A_ G#E1"_ A"_ M*#_ &RA"K_ G#_ *#6"_ -A_ G#"-A#LK_ &CG_ &ROPG#_ PK#_ _ A#U_ !C@@-M@#AM_ *K_ !66#"_ PK_ MC_ EJM2!-\ EM#_ -A_ _ EGC!#KK_ C&_ !C66CGM-S#_ &PA"-A)_ &CG_ _ AM3DA6_ !CPA!-6_ C&_ M*C6-!_ 6W_ CG)A-YM-CAK_ U*-!*_ -K_ KM-66_ ;G)#6W_ -A_ M*#_ &PMPG#_ #_ K##_ CPG_ 6M#G#"_ PK#_ C&_ (-AA!-6_ G%KCPG!#K_ 
 %H^ P*FV^ >O'.^ 'A??*'J*)^ R/J.^ %^ '.%?-/?-^ :/+* HJV:*^ %I^ R*::^ %H^ R/J.^ %^ '.%?-0?-^ C*FHC*'J/P*^ A+^ '.OF'.^ ^ 8?AR^ J.%J^ AOF^ %?-:*^ A+^ Q0H0A?^ .%H^ &**?^ 0?+:O*?'*)^ &V^ J.*^ U*F'/H*H^ OF/?-^ J./H^ C%HJ^ HO>>*F^ ^ R%H^ 'A )/F*'JAF^ A+^ %^ RAF8Y H.AC^ A?^ .F0HJ/%?^ :/+* HJV:*^ %J^ J.*^ &/*??/%:^ >**J/?-^ A+^ J.*^ %J/A?%:^ *)*F%J/A?^ A+^ .F/HJ/%?^ /+*^ A>>O?/J/*H^ ".*^ J.*>*^ A+^ J.*^ 'A?+*F*?'*^ R%H^ -?%J/%?^ !C/F/JO%:/JV^ +AF^ %^ /HH0A?^ *AC:*^ OF^ RAF8H.AC^ CFA'*HH^ %>A?-^ JR*?JV^ C*AC:*^ 'A?H/HJ*)^ /?^ -*JJ/?-^ %'DO%/?J*)^ %^ +*R^ +A'OH/?-^ F*>%F8H^ &V^ J.*^ 'A )0F*'JAFH^ J.F**^ H>%::^ )/H'OHH0A?^ -FAOCH^ R/J.^ CF*[ C%F*)^ DO*HJ0A?H^ 0?)/P/)O%:^ CF%V*F^ A?^ J.*^ HO&7*'J^ >%JJ*F^ %?)^ %^ C:*?%FV^ H*HH/A?^ 0?^ R./'.^ J.*^ R.A:*^ -FAOC^ F*%HH*>&;*)^ JA^ H.%F*^ J.*1F^ F*+:*'J0A?H^ $*^ 'A?':O)*)^ J.%J^ %^ H/>C:*^ :/+*Z HJW:*^ 0H^ C%FJ^ A+^ J.*^ >0HH/A?^ A+^ AOF^ +%/J. 'A>>O?0J0*H R0J[ ?*HH/?-^ JA^ J.*^ /@-)A>^ 2?^ AOF^ R%V^ A+^ :0P/?-^ $*^ %-F**)^ J.%J^ %^ H/>C:*^ :0+* HKV:*^ 0H^ ?AJ^ %?^ *?)^ /?^ 0JH*:+^ &OJ^ %^ >*%?H^ LA[ R%G)H^ %J*F0%:^ J.0?-H^ R*^ &*'A>*^ >AF*^ +F**^ JA^ )6H[ '*F?^ .AS^ T*^ '%?^ F*'5CFA'%J*^ J.%J^ :AP*^ ?*^ A,^ AOF^ #/*J?%>*H*^ C%FJ0'/C%?JH^ RFAJ*^ %^ CAR*F+O:^ HJ%J*>*?J^ A?^ J.*^ &:%'9&A%F)^ +AF^ %::^ JA^ H**^ ^ '%??AJ^ '.%?-*^ J.*^ RAF:)^ '%?^ ^ ^ '%??AJ^ '.%?-*^ N.*^ RAF:) ^ &OJ^ ^ '%?^ '.%?-*^ >*^ R/J.^ B)^ -F%'*^ ".*?^ ^ R/::^ H**^ J.*^ RAF:)^ '.%?-*
^ ".%JH^ J.*^ >AF*^ '.%?-/?-^ H*:+^ H^ R*^ HO>>*)^ OC^ AOF^ *UC*F/*?(*^ R*^ 'A?':O)*)^ J.%J^ AOF^ ?A? H/>C:*^ :/+\ HJV=*^ /H^ %:/*?%J/?-^ %?)^ 8/::/?-^ OH^ OF^ :0+* HJV:*^ 0H^ )%?-*FAOH^ JA^ AOF^ C.XH]'%:^ %H^ R*::^ %H^ JA^ AOF^ HC0F/JO%:^ .*%:J.^ !A>*^ C%FJ/'/C%?JH^ 'A>>*?JH^ +A::AR^ 
 F(
Pd "??d >**JPd 2EX2Q2E-d VPd QFd ".E-* d *d E**(d QFd QMVPQd Q.*d J2M2Qd Z.Fd PJ*>Pd 2Ed FVMd .*MQPd PV--*PQ2E-d .FZd Z*d M*d QFd ?2X*d FVMd ?2X*P d d P**d ?2+*PQ]?*d 2Ed Q*MDPd F+d M*?Q2FEP.2JP d F*Pd D]d ?2+*PQ]?*d .*?Jd D*d QFd +F??FZd *PVPd DFM*d #?FP*?]d .2Pd 2Pd Q.*d +2MPQd Q2D*d d .X*d .*M(d F+d d P2DJ?*d ?2+*PQ]?*d 2Ed "FEE*#Q2FEd Z2Q.d Q.*d \*M"2P*P d 2+*PQ]?*d M*PJFEP*Pd M*d (2++*M*EQd +FMd (2++*M*EQd J*FJ?* d 2+*PQ]?*d 2E"?V(*Pd D>2E-d Q2D*d +FMd J*FJ?*d 2Ed FVMd ?2X*P d Ed FVMd #FE"?VP2FEd Z*d EFQ*(d Q.Qd FVMd M*+?*#Q2FEd FEd E(d #M2Q2LV*d F+d FVMd PF"2?d *"FEFD2#d E(d JF?2Q2"?d P]PQ*DPd ?*(Pd VPd QFd Ed ZM*E*PPd F+d Q.*d PJ2M2QV?d #M2P2Pd Qd Q.*d .*MQd G+d FVMd JMF ?*DP d VMd JMF ?*Dd 3Pd 2(F?QM]d Q.FV-.d Z*d #??d 2Qd !]d DE]d FQ.*Md ED*Pd E(d 2QPd (*PQMV"Q2X*E*PPd F+d Q.*d .VDEd J*MPFEd 2Pd FQ.d J*MPFE?d E(d P^PQ*D2# d VMd M*+?*"Q2FEPd FEd Q.*d J*MPFEd F+d *PVPd $Ed M*X*?d EFQd FE@_d Q.*d J*MPFE?d 2D` J?4"Q2HEPd F+d *?2*X2E-d 2Ed .2Dd VRd Q.*d PF"2?d *"FEFD2#d E(d JFA2Q2"?d 2DJ?5%Q2FEPd F+d FVMd *?2*+ d .*d Z]d F+d *PVPd 2Pd d PJ*"6?d Z]d F+d *2E-d 2Ed Q.*d ZFM?)d E(d 7Qd QFV".*Pd VPd QFQB?] d ??d I+d Q.*P*d 2EP2-.RPd JF2EQd QFd d Y*M]d 2DJFNSEQd "FEa QM3 VQ2FEd F,d Q.*d \*M#8P*Pd QFd d #FEQ*DJFMM]cd PJ9M2QV?2Q] d F(Pd +FMDQ2FEd JMF"*PPd 2Pd X:? C*d QFd ?? d -ET;WPd (2P #FX*M*(d Q.*d JMF#*PPd Z2Q.d Q.*d +M**(FDd QFd D>*d ".F2"*Pd Q.Qd ?*(d QFd ?2+* d j %\,7j )j JOI,1RRj 7)Rj )j 7\O,7 TP)HR3IPF:H4j )H/j _IOB/ UP)HR3IPFe :H4j JIT1HT:)Bj _:T7j T71j 7\P,7j TO]Bbj +1,IF:H4j I/Rj ,IHTP)RTj RI,:1Tb 1H3B1S71/j :Hj )j _IPB/j T7)Tj :4HIO1Rj 7:Fj j `)HTj TIj O1J1)Tj )H/j 1FJ8)R:d1j _7)Tj '7IF)Rj B)P?1j RT)T1/j )Tj T71j RbFJIS:\Fj IHj
4H)T:)Hj &J:P:T\)B:Tbj :Hj )j %1,\B)Pj 42
j
IOj T71j FIRTj J)PTj U71j %K:P:T\)Bj a1P,:R1Rj )O1j )j R1O:1Sj I3j 1aJIR\P1Rj I3j T71j 7P:RT:)Hj 71)OTj TIj 7P:RTj '81:Pj JI_1Pj :Sj JO:F)P:Bbj JO1R1HTj :Hj T71j IRJ1Bj FbRT1P:1R j :Hj T71j 1HB:47T1HF1HTj )H/j 1H1O4:d:H4j T7)Tj T)?1j JB),1j 3OIFj T7:Rj +17IB0:H4j I3j T71j "IO/j )Sj 71j 1H4)41Rj :Hj 7\F)Hj B:31j )H/j F:H:RTPc j TI41T71Oj _:T7j P)T:IH)Bj P13B1,T:IHj IHj T71j S)F1jgggj
j 4H)T:\Rj :Rj :/1HT:3b:H4j
T71j :FJIPT)H,1j I3j ,IHT1FJB)U:^1j  1a1P,:R1Rj 3IOj )41HVRj I3j ,9)H41

)H/j :Sj I331P:H4j ) RJ:P:T])B:Wbj _7:,7j R7)J1Sj T71IBI4:,)Bj O13C1,X:IHj )H/j SI,:)Bj )H)BbS:Rj )H/j B1)/Rj TIj ,7)H41j j +1B:1^1j T7)Tj T71j B):Tbj )O1j BII@:H4j 3IQj <\STj R\,7j )j SJ:P:Y])B:Tbj _7:,7j :HT14P)T1Rj 3):T7j )H/j Z71:Pj 4PI`:H4j SI,:)Dj ,IHS-:I\RH1RSj )H/j GIT:^)T1Sj ,7)H41j F)4:H)T:^1j ,IHT1FJB)T:IHj I3j T71j B:31j I3j 1S\Sj 7)Rj 4O1)Tj JIT1HT:)Bj 3IOj 3O11:H5j T71j 7]F)Hj :F)4:H)T:IHj _7:,7j R11FRj /131)T1/j :Hj :TRj :H)+:B:Tbj ZIj 1H^:R:IHj )BT1OH)T:^1Sj TIj JO1R1HTj RI,:)Bj JOI+B1FSj "1Tj F1j ,IH,B\/1j T7:Sj ,7)JT1Oj _:T7j )j F)P^1BI\Rj N\IT)T:IHj 3PIFj ()BT1Oj P]144F)HHSj $OIL71T:,j F)6;H)T:IHj 1R\Rj .IH,1OHj _)Rj 3IOj T71j =Ibj I3j T71j !:H4/IFj +\Tj 71j _)Rj .E1)Pj T7*Tj T71j 3\T\i1j P1>I:,:H4j O1N\:O1/j )j 4O:1^f :H4j )+I]Tj T71j JO1R1HTj IO/1Pj
B1RR1/j )P1j bI\j _7Ij _11Jj 3IPj bI\j R7)BBj B)\47
j #\A1j jhhj 1R\Sj /I1Sj HITj ^:1`j T71j MQ1S1HTj _IPB/j )Rj T71j IHBbj IH1j HIPj /I1Rj 71j b1)OHj 3IOj T71j 3\T\P1j _:[7I]Tj )j ,IH,1PHj 3IOj T71j  GJ1L1DM \ *81J1\ 9L\ 5J913\ -D0\ BFPJD9D5\ MF\ .1\ 0FD1\ 3FJ\ M8FL1\ S8F\ 0F\ DFM\ @DFS\ M81\ 01-M8A9D1LL\ F3\ M819J\ L9MP-[ M9FD\ -D0\ 3FJ\ M8FL1\ S8F\ A-/@\ M81\ GFS1J\ MF\ .J9D5\ 9M\ MF\ LG11/8  $DAV\ -D\ -D5P9L810\ 09L1D5-51B1DM\ G1JB9ML\ 3JP9M3PA\ V1-JD9D5 \ $DAV\ M81\ 1B.J-/1\ F3\ 01-M8A9D1LL\ G1JC9ML\ D1SD1LL \ *89L\ S1\ @DFS\ G1JLFD-AAV\ .PM\ DFM\ V1M\ LF/9-AAV  \ -5J11\ S9M8\ *8FC-L\ A-J@1\ M8-M\ M81\ 5D-M9-D\ U1J[ /9L1L\ 9L\ -\
/A-LL9/\ M1UM
\S9M8\ -\ LPJGAPL\ F3\ B1-D9D5 \ M\ 9L\ CV\ .1A913\ -D0\ 8FG1\ M8-M\ /FDM9DP9D5\ /FDM1CGFJ-JV\ 9DM1JGJ1M-[ M9FDL\ F3\ M81\ 'H9J9MP-A\ U1J/9L1L\ S9AA\ V91A0\ D1S\ J1LFPJ/1L\ 3FJ\ 3FJB-M9FD\ F3\ M81\ A-9MV \ #FM1L\  FL1G8\  \ $-AA-58-D\ *81\ PMF.9F6J-H8W\ F3\ 'M \ 6D-M9PL\ FXFA-\ G \  \ 1JD-J0\ PL8\
*81\ 958M11DN8\ DDFM-M:FD\ F3\ O81 'H;J9MP-A\ U1J/9L1L\ -D0\ 'F/9-A\ '9E3PAD1LL\ 'FPD09D6L \ G \  !9/8-1A\ P/@A1V\ '  \ K110FB\ A1/M9FD\ -D0 '1A4 *J-DL/1D01D/2\ (FC1\ &13A1/MMY\ ?D\ -\ )1/PA-J\ 61\ I \  *8FC-L\ A-J@1\ '  \ 1LPL\ -M\ *-.A1\ +81\ 5D-M9-D &QA1L\ -D0\ PC-D\ PD71J\ ,F0-V
\ 6D-M9-D\ 'H9J9MP-A9MZ\ 9D\ -\ '1/PA-J\ 61\ G \ 
.90 \ G \   .90 \ G \  .90 \ G \  T-AM1J\ JP1551C-DD\ *81\ %JFH81M9/\ B-69D-M9FD GG \ 
  
 
  
           8I I 6#"I )3"I 6#$0# :I <543I :&#I  6*5:<6#8I I $*3"I #6:*3I 6# <66*3%I G#8I >&* &I &=#I 6#0I 8
%3*$* 3!#I $46I :&#I $*:& $462:*43I 34:I 430AI 4$I :&#I 0*:AI <:I 4$I 00I :&#I 5#450#I 4$I 4"I *3I :#I :*2#8 I :&*8I 8#38#I 4$I *3 0<8*=#3#88I 34:I :I 3"I <8 I &< /I :&645I 8::#"I >#00I 46I 4"I &8I 00#"I I #450#I I 3"I <8I 6#I <30#I :4I %:I 64<3"I  3"I I 6#I *3=*:#"I :4I ?&40#3#88I 3"I :4I $44"IDEFI 
 &#  6*5:<6#8I :#00I :&#I 8:46AI 4$I &4>I 4"I $462#"I I 5#450#I $46I  &*28#0$ I '#6#I *8I :(#I :#I 4$I #*3%I 00#"I 34:I 430AI 8I *3"*=*"<08I <:I 8I *3"+=*"<08 *3 422<3*:A I #I *8I :&#I :#I 4$I &*8:46* 0I -4<63#AI *3I :*#I 3"I 85 #I >*;&I :&*8I $2*0A 422<3*:A 5#450#I *3:4I 3I <3/34>3I &45#$<0I $<:<6#I :6<8:*3%I :&*8I 04=*3%I 6#:46 4" I #I *8I I 0#6I >6#B 3#88I 4$I I 85# *0I %7##2#3:I 4$I $*"#0*:A I I 4=#33:I #:>##3I 4"I 3"I :&*8I &48#3I 5#450#I >&4I %6##I :4I &4346I :&*8I 4"I 430A B 3"I :&*8I *3I :&#I 2*"8:I 4$I &*8I 5#450#I 6HI <0:<6#8I >&, &I >468&*5I 23AI %4"8I 00#"I :4I 6#8543"I :4I 4"8I 04=#I 3"I :4I #I "*$$#6#3:I $642I :&#I 5#450#I >&4I >468&*5I 23AI %4"8 I &#I :&6#"I >&* &I *8I :4I 433# :I :&*9I 5#450#I $642I 43#I %#3#6:*43I :4I :&#I 3#@:I *8I 246#I :&3I 03"I 3"I <0:* I >468&*5 I  6*5:<6#I 546C :6A8I 4"I 8I 43:*3<00AI 00*3%I &*8I 5#450#I :4I 6#2#2#6 I  &#AI 6#I :4I 6#2#2#6I :&#*6I 10I 3"I :&#*6I 8#38#I 4$I #*3%I 4"8I $2*0AI 43I I .4<63#AI :&64<%&I &*8:46AI 
 &#AI 6#I :4I 
 
 >+:+:(+>K <*AK A&D1;.K *++*AK &;*K B/+1>K A=+)1&8K &.>++:+;BK BA/1=K ;K .<*AK ;K E/&BK B/+1>K ;,1./(<>AK *< K #/+GK &>+K B+:+:(+>K &;*K 81AB+;K BA1AB+;BK 1;D1B&I B1<;K B+A=<;*K BK 81D+AK ;K B/+K !+EK #+AB&:+;BK B/+A+K B/+:+AK &>+K >+1;-<>)+*K 1;K B/+K B+&)/1;.K &;*JK +F&:=8+K <-K +ACAK E/+81.1<;1ABAK B1-GK &;*K >+;+EK B/+1>K )+8&B1<;A/1=K E1B/K <* K #/+K GK <-K +ACAK :1AA1<;K /1AK AG:(<81)K )/<&+8
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
> > 0!+3!0> 30> > 7/> 3($> 0+> !(> (> 0!.> 7.> /."+3/> 0)/"+(/ > 0> 0++#> 0"'> /0.3$> (> '3!> -"(> +.> '> 0+> $.(> 0+> $"/0(> (+0> +($8> 0+> 0!> 7+./> +> '8> !("(> !"$.(> 30> 0+> 0!".> $"(/> (> 0+> > $> 0+> ./-+(> "(> > '+.> '-0!0"> (> +'-//"+(1> 78> 0> +3./> 0+> '> 0!0> '(8> -/0+./> .> > 7"0!> > /"'"$.> /"030"+((+0> "(> $> 0+> !(> 0!'/$5/> (> >
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
 8> +(.(> +.> 0!> !3.!> (> "0/> .(7> /$9 3(./0("(> "/> '3!> '+.> 0!(> > (..+7$8> +3/> +(.(> +.> '"=0((> (> .(7$> 7"0!"(> -./(0> /0.303./> !> 

 "'4&$#K :$/% >4#$7:;4#(4&K 5%K ;'$K '>8"'K 8$@$/:K ;';K ;'$K '>8"'K #5$:K 45;K $B(:;K %58K );:$/%K !>;K %57K 3(::(54K ;'$K 3):E :)54K 5%K $:>:K A'5K :5K "/$8/DK 8$%/$";:K 5#:K /5@$K %57K ;'$K A58/# K ;8>";>8$:K 5%K ;'$K '>8"'K 8$K ;5K :$8@$K ;'(:K 3(::(54 K 4K ;'$K 685"$::K 5%K /(:;$4(4&K ;5K 3DK 5A4K $B6$8($4"$K 4#K ;';K 5%K 34DK 5;'$8K 3$7)"4K /DK 6$8:54:K #>7)4&K 68$68;)54:K %58K ;'$K 8$"$4;K K D45#K 54K ;'$K (;DK K '@$K "54"/>#$#K ;';K 68$GK :$4;K :;8>";>8$:K 7$K "5>4;$8 685#>";(@$K 4#K ;';K ;'$8$K 4$$#:K ;5K !$K K 4;)54 A)#$K :'8$#K 8$%/$";(54K A);'(4K ;'$K '>8"'K 54K ;'$K @5";)54K 5%K ;'$K /(;DK 4#K 54K A';K .)4#5%K '>8"'K :;8>"F ;>8$:K A)//K !$:;K :$8@$K ;'(:K "8>"(/K !>;K 4$&/$";$#K &$4#K *4"$K 0 );DK "5368):$K 6675C(3;$/DK 4)4$;D $(&';K 6$8"$4;K 5%K  ;'$K '>7"'K );K ):K 45;K >47$:54!/$K ;5K $B6$";K ;';K ;'$K 6(8(;K ):K :6$.)4&K ;5K "533(;;$#K 1DK 6$7:54:K 9$&8#)4&K ;'$+8K 3(::)54K 4#K <'$K %>;>8$K :;7?";>8)4&K 5%K ;'$K '>7"'K A')"'K (:K ;5K $4!/$K ;';K 3(::(54HK 4K ;'$K '>8"':K 8$4$A$#K :$/% >4I$8:;4#)4&K ;'$K /);DK 8$K "//$#K )4K K :6$")/K ADK !DK 8$:54K 5%K ;'$)8K (33$7:)54K ,4K ;'$K A582# K
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
 
 -? +9? 0 *3'.-? .-? $50$? -? 5*350? 2.'*? 2'-? -? 2.'*? "0? ? %6? /.'-3? 3.? 3$? 0'2'2? . ? 230-? '6'*':3'.-? /03'5*0*9? 2? '3? '2? 8/0'-? 7'3%'-? 3$? -'3? 332? $.5"$3 5*? 0 *3'.-? .-? 3$? +-9? 32? . ? 3%'2? 0'2'2? 06*2? 3%? 2/'0'35*? 6.'? 3? '32? -30? *  -30? '-'6'5*'2+? %2? '- 4? .50? 5*350? -? 6-? 3$? %50%? '-? 25%? ? 79? 3$3? '3$? $2? .+? ? /0'63? +330? - 3$3? '-"? ? /02.-? .0? .3$02? 2? 252? 72? '2? 5-023..? -00.7*9 ? ? +? -.3? *.-? '-? *'6'-"? 3%3? 3&'2? 5*350? 252? $0'23'-2? '-3.? -? '.*309? . ? ,.-9? 3%'-"2? -? /.70? 7'3$? *'33*? .-2'.52? 02'23-? 3.? 25%? 3+/33'.-2? .0? $0'23'-? 12.-2? ? $50$? 3%3? %2? -.3? *3? 7'3%? 3%? 2.'*? 2/32? . ? .-602'.-? '2? '-? -.? /.2'3'.-? 3.? %**-"? .0? 3.? %-"? 3%3? 5*350 ? 3? %2? -.3? .++5-'3?   3'6*9? '32? 5-023-'-"? 3%3? 3%? +22"? . ? 252? '2? .3$? /02.-*? -? 2.'*? '3%? ? 7? +06*.52? 8/3'.-2? 25%? 2? 3$? ,/'"-? !.0? 5+-? 6*./+-3? ? !*? 3%3? 3%? $50%? %2? .+? .+23'3? 7'3$'-? ,0'-? 5*350? 7'3%.53? ,'-3'-'-"? ? 0'3'*? '23-? -? 2* 70-22? <-? *'#'.-? -? *'-3'.-? 0".09? 5,? %2? 233? 3$? 2'35 3'.-? *0*9 ?
.? 05? 3$? %0'23'-? ,22"? 3.? ? 3053%? .53? /02.-*? 2*63'.-? '2? 3.? 25//022? ? ()? '+-2'.-? . ? 3$'2? +22"? -? 3.? 30-2 .0,? '3? '-3.? -? '.*."9? 2-3'.-; '-"? '-'6'5*'2+ 

 ? *'6? 3$3? ? 0'3'*? 2* 70-22 '2? 2.*53*9? -=22>09? .0? 3$? $50$? 3.? 6-"*':? -? 3.?
 5* '**? '32? +'22'.-? 7'3$'-? ,0'-? 2.'39? -? 5*350 ? 
 
 &E :*$E .,:@E 5$E :3E $E :*$E )$2:9E 3&E :*,9E $=2)$., A:,32E :*$5$E ,9E 2E <5)$2:E 2$$#E &35E E !3295#E :*$E .@E )$2# E :E :*,9E 43,2:E ,2E :,1$E :*,9E )$2#E 9$$19E :3E *=$E E =$5@E .3>E 45,35,:@E 2#E ,9E :E :*$E 1$5!@E 3&E :*$E ,2#,=,#<.,9:,!E ,29,)*:9E 2#E ,2,:,:,=$9E 3&E .3!.E 49:359E >*3E #3E 23:E 9*5$E :*$E =,9,32E 235E :*$E <5)$2!@E 3&E &39:$5,2)E .@E =3!:,329 E 3>$=$5E :*$5$E 5$E 9,)29E 3&E .,&$E &531E !311,::$#E .,:@E <;E :*$@E 5$E 93E 9!::$5$#E 2#E <2"322$!:$#E >,:*E $!*E 3:*$5E :*:E 453)5$99E ,9E =$5@E <2!$5:,2 E 2#$5E :*$E <15$/.E &E 2E C35)2,A:,32E !./$#E

*$E 1$5,!2E :*3.,!E @E $:>35-

E :>3E *<2#6$#E 2#E &,&:@E 1$5,!2E .,:@E *$/#E E 45..$/E !32&$5$2!$E ,2E 31$E #<5  ,2)E :*$E :*,5#E >$$-E 3&E :*$E ,9*349
E @23#E 32E :*$E ,:@ E *$5$E 5$E *34$9E :*:E E $#$79*,4E 32&$5$2!$E 3&E :*3.,!E ,:@E >,..E %1$5)$E &531E :*,9E ):*$5,2)E 2#E &531E :*$E !311<2,!:,32E 132)E :*$E .,:@E >*,!*E :33-E 4/!$E ,2E 45$45:,32E &35E :*$E @23# E 2E :*$E 453!$99E 3&E ):*$5,2)E ,24<:E &531E :*$E 1$5,!2E /,:@E ,2E 45$45:,32E &35E :*$E @23#E ,2E 31$E :+$E &3<5E 1$5,!2E ,9*34E #$.$):$9E >$8$E .5$#@E .$E :3E #$:$51,2$E :3E E D)5$:E $?:$2:E :*$E 9$29<9E &,#$.,<1 E *$5$E >9E !32!$52E &35E .@ E !.$5)@E 5$/:,329*,49E :*$E 53/$E 3&E >31$2E ,2E :*$E *<5!*E :*$E $?45$99$#E 2$$#E 'E :*$E .,:@E &35E 5$93<5!$9E &35E 94,5,:<0E )53>:*E 2#E E 9$5!*E &35E &&,51:,32E 2#E $2.$1$2:E 3(E .,:@E ,2E 1,99,32E 2#E 1,2,9:5@ E  :E 9$$19E :3E 1$E :*:E :*$9$E !32!$529E ,2E ##,:,32E :3E 3:*$59E >*,!*E >3<.#E $E 9<5&!$#E .3!..@E >3<.#E 453=,#$E 9<&&,!,$2:E 1::$5E &35E E 2:,32.E !329<.::,32E 453B !$99E >*,!*E 139:E 45!:,!./@E 1,)*:E :-$E 4.!$E >,:*,2E $!*E 
 !'/ #3#A /2A 2#%'/.+A +834#2A /$A !'/ #3#3 A 4A 3##,3A 4/A ,#A 4&4A ++A /$A 4#A /. #2.3A &9#A 4/A !/A :'4&A 4&#A !#9#+/0,#.4A /$A + +8!#A
'!#.6'$' 4'/.A /$A 4&#A /)# 4'9#3A /$A 4&#A &82 & ,#382#,#.4A /$A 4&#A 44'.,#.4A /$A 4#A /)# 4'9#3 A .A #9+84'/.A /$A ,#4&/!3A '.A +'%&4A /$A !4A  18'2#!A .!A A !#9'3'.%A /$A .#:A ,#4&/!3A :#9#2A 02#3#.4A ,#4&/!3A 2# 3&/:.A 4/A #A '.#$$# 4'9#
 9+84'/.A :/8+!A #A .A #33#.4'+A 024A /$A . ,#.43A $/2A 2#.#:+A 38 &A 3A 83'++/A 22'%#A . /8.4#2A .!A &2'34'.A '$#A /,,8.'4'#3A :&' &A .##!A 4/A #A ++#!A 4/%#4A :'7&'.A A !'/ #3#A 4/A #;0+/2#A 4&#'2A /,0+#,#.42'4503&L #'L "5L '5"#3':L 7+L 85-705-L "&>350<0';L "5&L &0;%':54'5:%/L "5&L ;3<>:'L 0;L 9:"0;'@7:5&':;<"5&L :'L =/'AL @:';<305-L @03<>:'L 7:L ":'L :%/L @/0%/L ;''4;L +0:43AL '5<:'5%/'&L 15L 3C <>:'L L /"?'L ;''5L 4"5AL 05;<"5%';L 7+L 3L :'3"<075F ;/09L "L L 0:'%<7:L ";L ;9'"2':L "$30%3AL %'5;>:'&L 05L 75-:';;L "5&L 0;/':;L "5&L 4":%/05-L 05L %/L 47:' L L /"?'L 57<'&L @0%/L 9>#30%03L 9>#30%L ;<"<'4'55/'"3:%/<"<'L :'3"<075;/09L !/.%&%G %&5724@G 5%29- G /2:!
G %!G !;& !0!G #24G 7%!G 23!4A 7&20G 2#G 2 5G $4!G &5G (0G 7%!G #49&6G 2#G %4&57&0G 4!-&$&295G !?3!4&!0!7%!G -&!47&20G 2#G 6%!G %9/1G 53&4&7G #42/G &75-#G 2:!42/&0$G 5!-#&5%0!55G 0 G !$2&5/G
4!G )5G 7%!G &5-2594!G 6%6G 7%!G %9/0G 5&697&20G 2!5G 026G %:!G 72G !G 7%!G >@G &7G 
 &5  %!G -27&20G #24G !?!4&5!G 2#G 6%&5G $42=&0$G #4!! 2/G 0 -2,@ 3/$*@ $+ ,3$/,3@ /!@ +=@ *$! @ ,@ 3@ ,/62$3#+ ,4@  
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

  #S C26.ES .A>;S 16H6<1S ?A6;#'KS E>S E2,>A,E6'#9S ,9#O &>A#E6>< ?A>',CCS E>S ?#K6<1S #EE,S A,#96EKS #<(S ,J?,A6,<', #CS E2,S CE#AE6<1S ?>66AS #99S A,.9,'E6>< ES C,,;CS E>S ;,S E2#ES E2,S 2GA'2S 6G9(S 9,#A;S E2,S 2GA'2S 6AS ;,S E2,S ;>CES .G<(#;,S &,S E2,S <,,)S .>AS >?,<<,CCS E>S A,O .9,'E6>.S E2,S 2GA'2S 6C?6E#&9,S E>S E2,S 96.,S >0S E2,S ?6A6ES #<+S I2#ES 2#CS 26<),A,(S 6ES I26'2S CEAG'EGA,CS C,AH,S E2,S ;6C C6>.S ,CGCS %<(S I26'2S ><,CS (>S <>E S #S '>;;G<6EKS ,;&>(KS 6<1(>;S H#9G,CS >IS )>,CS E2,S 2GA'2S #CS 2,S ?>,;S >.QS 2G'8S #E2A>?S ,.R#S >G<(E#&9,
S CG11,CECS E2,S '2>6',CS E2#ES 96,S #2,#( S 2,KS 2#H,S E>S (>S I6E2S #S A,<,I,(S G<(,ACE#<(6<1S >.S 2GA'2S #CS ?,>?9,S >.S >(S )6C',A<6<1S E2,S  ?6A6ES #<(S 6<'#A<#E6<1S E2,S 6<1(>;S H#9G,CS >.S ,CGC S 

   
 
  
  )(+(!(: .: 56: (: )5: (: 5.: (: 5): ): &!(!-.+6: )(: !&: "*-: -2+!(7: : /$: . .: !-: +)2(: .: 5!$$: .#: -)': -5!(: 0): : +)2(.$: -)': +!(!(: (: +-!(!(: 2: +)!(: (: +1+.!(: ): (++)5$)(: 2+!(: (: *!(2$: : )+: *)*$: (: .$-: 2.: -): 5-: .: +)--: : *!(2$: .)): .$: ): !3!(: (: 6-:
  (: +)&: -2: .)'-: $!: +)&: -2: 6!(: )&-: ,!-!(: !(: -+: ): +)2(.$!(: (: 5.: 5)2$: +)2(.$: 2+!(: &(: .: 5)2$: '(: (): !-!(: (: .+)(!(: )+: 2.: )(: !(: !-: .,: (: : 5- 8: )).5-+: .: .$: (): %--: : $+: ): +.-: : (: -.)5+: ): !-.2,!(: *: 5)-: )).-.*-: 5: $)-.: .+#: ): : $))#: )+: -!(9: 2.: 5!.: 2(!+2&!-: +.-: .+6!(: .): !-+(: : &--: 2(!-+($: .)*)'*: (: !+2&-.(: .: 5$$!(.(.!)(: )(-: 5): 5!$: *+)$!&!(: .: !(!(: 5+: $$: .: .!': $)-!(:  )+: .
.: (++)5$)(: .$-: -+4(.: (: '!++)+: &: *!.2+: +&: (: (.+-: ): ..(.!)(:

 
 
 #1 )&***1 "1 ,+1  1 &)#"#+*1
)1 *+&#1 / *1 #1 , #*1 ,*1 ,+1 &#1 1 /1 &#!01 ,+1 +1 +"*1 #1 +1 + *1 )1 ##1 #1 )))##1 #1 /+1 &1 #))&/ &#1 + 1 "#*+)*1 /#1 +01 &#)&#+1 1 )&,#+ 1 '&' 1 +)1 0)*1 &1 /&)#1 ,'1 +1 + 1 *1 #1 ,'1 +1 )1 +&1 # 01 *+1 +1 +*1 1 &# 01 +&1 *&.)1 ++1 +1 + 1 *1 +,)#1 )&,# 1 &#+#,1 ))1 )1 / 1 &# 01 *& +1 &)1 +)1 )1 $&1 '&' 1 +)1 &# 01 )& *1
  01 ",*+1 1 &.1 %+&1 )&,##**1 /)1 ()+1 *1 *' 1 1 ()+1 #1 +1  1 *1 +&1 +1 +)#1 &)1 
 *1  1 1 &' 1 #&+1 +"1 #1 ,*1 "1 #1 ,*1 )1 ,# 1 +&1 +)1 )&,#1 &)1 +1 1 )&,#+ 1 +)1 )1 #&1 **1 #1 1 )1 #.+1 +&1 /& #**1 #1 +&1 &&1
#1 "&)1 +#1 /*1 &*+1 *1 #1 &,#1 *1 #1 &,#
1   1 -""#*1 ,+1 /*#1 #1 &'#1 / 1 #&+1 +1 ,*1 +)1  01 0#1 #1 )*#1 / #1 *&"1 */#1 +1 &#1 +"1
 1 
 
 +2.9 *..+5&+*9  2. 09 5.9 2!&19 0+9 1+9 .0(&9 1 9 .+009 219 !19 +09 *+9 ,+9 ,.9 2!&!*09 1+9 +9 0+9 !9 &!409 ,9 *,19 +2*1&!*9 (*09 *,9 -...9 01!*9 *+9 !.019 *9 &019 *+9 11.9 *9 *+9 ,.*.09 ,.9 1 9
&019 ,9 1 09 +2*1&!*9 (*09 !*9 5!1 9 9 -.19 ,9 1+1 .9 *9 ,*9 19 (*09 .++(9 ,.9 1 9 -!.!19 *9 !109 *9 !013.!*9 -.+,3*9 - 9+.9 &&9  19 !09 *,19 (!9 /9 1 19 59 .9 $!*9 +.9 1 9 +3 9 59 .9 &!*9 5!1 9 (3019 *9 5!&&9 1$9 "109 7!*#*,..1+.!0!*1!(9 *9 !19 !09 59 !*9 1 9 -.0*19 5 +9 .9 (!6!*9 *9 %*!*9 1 9 ,2 9 +.9 1 9 312. 9 9 *9 *+9 &+*.9 -.-.9 ,.9 1 9 -01 9 9 5!&&9 *9 )3019 *9 .9 '&9 1+9 9  3. 9 *9 !9 9 &&09 ,.9 ,1 .9 1 *9 .+2*1&09 59 .9 +3*9 1,9 ,&&,59 4!*9  !*9 1 9 052019 *9  !-09 0!*09 *9 .0!*09  !*9
 &&9 1 9 5 !&9 !*9 .(&009 09 +409 *9 5!&89 09 0.-*109 !*9 0. 9 +9 *9 !*9 1 9 -.0*9 ,9 1 9 !*+(9 1 19 !09 !09 *9 *,19 ,2.09 (* 9 
 
 /5%4< )6"+< 5)2/0<
.< %2")< /&< < /6.$5 ,%< *. < %.5,%< 2%4%."%<
 2%'/3:< 6-< %,*(*/.< .$< ,*%.5*/.< 0<  *")%,< *.5%2< *44*/.< %46-%$ < *9
 /'%2< *')5< .< ,5%2.5*7%< *4*/.< 10<  
  
  
 2"%,,/< ;%8%$/< 4*"< "",%4*,< /--6.*5*%4 *.<
   2;*,< 00< #)6"+< 5)2/0< .< %2")< /&< < /6.$5!,%< *.< < %.5,%< 2%4%."%<

  

 ((!&$!*^ ,>6^ ;IFFISAH<^ AP6GO^ L6KL6O6HP^ OIG6^ I;^ GU^ 6;;JLP]^ 1P^ 4IHO4AIQOH6OO L1AOAH<^ IH^ AOOQ6O^ M6F1PAH<^ PI^ ;1AP>^ 1H5^ 4QFPQL6^    (LJC64P^ #L66H^ (LIKIO1F^ (LJC64P^ #L66H^ S1O^ 1^ 4IGV GQHAPU 31O65^ 6^ 4QLLA4QFQG KF1HHAH<^ KLI46OO^ I;^ P>6^ O4>JIF OUOP6G^ ->6^ 4QLLA4QFQG L7RA6S^ 4IGGAPP66^ ;IL^ S>A4>^ $^ S1O L6OKIHOA3F6^ OQ3GAPP85^ P>AO^ KLJKIO1F^ S>A4>^ AH4FQ565^ 3IP> >QG1H^ 1H5^ 6HRALIHG6HP1F^ 4JH46LHO 1^ >LAOPA1H^ 1HP>LIW KJFI6L^ AH^ P>6^ IHP6TP^ I;^ P>6^ .+^ ->AO^ K1L1X K>L1O65^ 'QL^ "1P>6L^ S1O^ SLAPP6H^ ;IL^ 1H5^ QO65^ ;IL^ SILW O>AK^ AH^ 1H^ 64QG6HA41F^ 1OOJ4A1PAIH^ AH^ S>A4>^ $^ K1LPA4AK1P65^ AH^ P>6^ 61LFU^ O6R6HPA6O^ AH^ $IS1^ APU^ S>6H^ /A6PH1G^ S1L^ KLJP6OPO^ S6L6^ 1P^ P>6AL^ >A<>^ KIAHP^ ->6^ ILA1O^ 366H^ OFA<>PFU^ L6RAO65^ ;IL^ P>AO^ K1K:L^  G6LA41H^ 6F6<1PAIH^ )6KFA6O^ PI^ >AF61H^ >1FF6H<6^ $ SLJP6^ P>AO^ OP1P6G6HP^ AH^ 4IFF13IL1PAIH^ SAP>^ IH6^ IP>6L^ K6LW OJH^ 1P^ P>6^ G66PAH<^ I;^ P>6^ 0JLF5^ "656L1PAIH^ I;^ >LAOPA1H %A;6^ IGGQHAPA6O^ AH^ ^ 1P^ S>A4>^ GIL6^ P>1H^ ;ILPU 4JQHPLA6O^ S6L6^ L6KL6O6HP65^ ->6^ PAGAH<^ S1O^ IH6^ GIHP> 36;IL6^ P>6^ FF6H56^ L6P^ 5ISH^ AH^ >AF6 0>6H^ P>AO^ OP2P6G6HP^ S1O^ L615^ 36;IL6^ 1^ KF6H1LU^ O6OOAIH AP^ >15^ 1^ L6G1LE13F6^ L64IH4AFAH<^ 6;;64P  IH;6OOAIH^ I;^ +I4A1F^ +AH^ ;IL^ G6LA41H^ >LAOPA1HO ->AO^ 4IH;6OOAIH^ S1O^ H6R6L^ QO65^ $P^ S1O^ SLAPP6H^ 5QLAH=^ 1H 6A<>P 51U^ AH5ARA5Q1FFU A4>^ $ G156^ AH^ P>6^ (>AFAKKAH6O^ AH^ 4IHH64PAIH^ SAP>^ 1^ G66PAH<^ I; P>6^ 0ILF5^ "656L1PAIH^ I;^ >LAOPA1H^ %A;6^ IGGQHAPA6O^ AH ^ $^ >1R6^ L6RAO65^ AP^ OFA<>PFU^ ;IL^ P>AO^ K1K6L  0>1P^ AO^ P>6^ +KALAP^ +1UAH<^ ->AO^ AO^ 1^ P>6IFI^ 4IQH4AF^ AH^ ^ O^ I;^ P>AO^ SLAPAH<^ P>6L6^ AO OPAFF^ HI^ K1LAO>^ 4IQH4AF^ 1H5^ P>6^ K1LAO>^ AO^ F1L<6FU^ K1OW 
 1H5^ QHAHRIFR65^ AH^ GAOOAIH^ $P^ AO^ 1FOI^ <6PPAH< L6OPF6OO

 
 E E 81+# :E 8##/E 81319,E %18E !*9 <99*1/E 1%E /!#8,@*/&E (*,1914(@E %18E 1 *,E :*;(E E =*#>E 1%E ,*%#
E 9E */!*=*!<,9E 9E .#.#89E 2%E ;(#E (<./E %.*,AE /!E 9E */C (*:/;9E 1%E :(*9E 5,/#:E >#E 8#E */#?:8* ,AE 8#,:#!E :1E /!E !#5#/!#/;E <51/E 1:(#89E /!E 1<8E #/=*81/.#/;E /!*=*!<,E %<,%*,,.#/:E /E #E 8#,*B#!E 1/,@E *%E ./E 9##9E (*.9#,%E 9E E 38:E 1%E :(#E #/:*8#E 1..*:(E >(* (E )#E .<9;E ,*=#E */E (8.1/AE /!E %18E >(* (E (#E *9E 8#95109*,#E  E 5(*,1915(AE 1%E #!< :*6/E 9#!E 1/E :(*9E 1..*::.#/:E ;1E ,*%#E 8#7<*8#9E /#>E 3381 (E :1E ;(#E #!< :*1/,E 381 #99E >(* (E >1<,!E 13#/E :1E ;(*9E 9#/9#E 1%E 1..*:(E 95# *,E #.5(9*9E 1/E ;(19#E %1Q 'A1,>Q  &:EQ '>,Q 'A1,>>,'A:>Q A:Q '66Q ;,:;6,@>'(@Q '9+Q @>',62@Q (6'*5Q ->2*'9@Q '9+Q H12A,Q ->2*'9@Q "2,A9'7,@,Q 129,@,Q '9+Q D@@2'9@ Q #,Q *:9@2+,>Q :D>@,6F,@Q J:E>Q *126+N >,9Q A::Q '9+Q 7,7(,>@Q :-Q A1,Q 1D7'9Q -'726J Q #1:Q '>AQ 29Q 1,'F,9Q  &:DQ 59:HQ E@Q '66Q '9+Q @,,Q :E>Q @,>2:D@Q 29'(262AJQ A:Q .,AQ '6:9.Q H2A1Q ,'*1Q :A1,> Q #,Q '@5Q -:>.2F,9,@@Q '9+Q 1,'629.Q :-Q :E>Q @,6- >2.1B,:E@9,@@ Q '66:H,+Q (,Q K:D>Q 9'7,Q

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
 '  ' !'  '  '  '  
' $'  ' '  &' '  ' '  
'  ' ' ' ' !' '  ' '  '  ' ' ' ' ' ! ' $' !"' '  ' ! ' "' !' ' "'  
' ' !' ' !'  ' % &' ' #' !'  '   ' ' !
' &!' ' ' "' &!' $' ' !' ' ' '                 
   
  
 
  &Z Z @2F9.'AZ 2=25'J9BAZ !2C=92HZ JBZ 89=2'AZ 8'==2A62Z M7H,MF7Z M7MHJZ Z Z #82Z $A9J20Z "J'J2HZ 2=27'J9BAZ 8'HZ J82Z 4B==BP9A7Z @2HH'72Z 4BFZ J82Z 02=27'J2HZ JBZ J82Z %BF=0Z HH2@-=SZ B4Z 8F9HJ9'AZ 942Z B@@MA9J92HZ @22J9A7Z 9AZ M7H-MF7Z 2F@'AS Z %2Z 902AJ94SZ BMFH2=O2HZ 'HZ -2=BA79A7Z JBZ 'Z F9.8Z
9FHJZ %BF=0
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
 $'$*!-,#3[ T9O98E[ (4I79BBE[ 89 [ 4J?7[ 77B9J?4B[ ECCND?K?9J[ ?D[ I4T?B [ 24J>?D[ -E59IL[ (48J9D[ - [ .NBB@O4D[ 2 [ .P@8B9I[  [ #45?LJ[ E;[ L>9[ #94IL [ )9P[ 3EIA[ #4IG9I[  [ IN9<9[ ,IEH>9L@7[ $C4=?D4L?ED [ 
 N7AB9R[ (?7>49B [
I998EC[ B97L?ED[ 4D8[ .9B;/I4DJ79DU 89D79[ .EC9[ -9;B97L@EDJ[ 0GED[ L>9[ $D9I [ 2?B;I98[ '4NI?9I[ 0D@O9IJ@LR[ ,I9JJ[  [ NJ>[ 9ID4I8 [
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
 b 5FBb !YPBRHBN@Bb WHAb - b #KBLHNE b 4T b -OVHSb )NSTHTVTBb OCb +BSVHTb 4OVR@BS`b  b .VRQFZ 1ONNORb *BROLB b B@OMHNDb &VL