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... -~- \ () I Role of peroxisome proliferator-activated receptor y (PPARy) in Coxiel/a burnetii infection I~ Celina Spencer, Adelaide Calhoun, Minal Mulye ~ Marian University College of Osteopathic Medicine, Indianapolis, IN Introduction Coxiel/a burnetii . G ram negative bacterium . Causative agent of human Q fever 3. Disease ~ - Pneumonitis ~ - Endocarditis - Granulomas 4. Exit Usually none in man f 1. Entry Aerosol transmission Infectious dose (< 10 organisms) Potential bio-terror \~~ agent 71 ID ~ I 'I 1 ~ ,I I; t,l'. o :\( l r, l Lipid droplets are storage organelles important for cellular metabolism . Neutral lipid storage organelles . Store esterified cholesterol and free fatty acids (triacyglycerol} Biogenesis from ER . Functions - Energy homeostasis, membrane trafficking, signaling . Preferably infects alveolar macrophages . Found in lipid droplet-rich foam cells in endocarditis patients . Parasitophorous vacuole (PV) is essential for bacterial growth . Uses Type 4 Secretion System (T4SS) to manipulate host cells . Lipid droplets are important for Coxiella intracellular survival ... . /4-- la' !.f': ......,. A'""""" ti, n: - e v.ttkle ~=: ---- 0.,0 Dy 2 0.,4 Mt"d MH-S cells Infected with wikMype Coxiefla were treated with vehk:fe 2. Spread Hematogenous (through blood) Pathogenesis of Coxiel/a Figure 2: Blocking lipid droplet breakdown results in reduced Coxiella growth (OMSO) and 20uM atglistatin. Bacterial growth was determined at day 4 by FFU Assay (n=3) =p<0.os:- =p<0.0001 compare<:1 to vehicletreated cells two-way ANOVA with Bonferronl post~oc test. Scale bar= Overall Question Does Coxiel/a infection affect PPARy expression and activity to induce anti-inflammatory immune response? Results Figure 3: Coxiella infection upregulates PPARy expression in infected alveolar macrophages 10 11m. "C ~~ MAG. DAG. TAG- Mono, Di and Tnacytglycerol. OGAT - Oiacyl glycerol acyt trsnsferese, ACA"J 1 - Acyt coenzyme A acetyl transferase, ATGL-Adipose trig'tyoeride lipase , HSL- Hormone sensitive lipase Preliminary Data I Are lipid droplets important for Coxiel/a intracellular pathogenesis? Figure 1: Lipid droplet accumulation is dependent on the Coxiella T4SS 200 .... .--, = 150 . .. - Lipid droplets and PPARy - 3 QI :, Cl 0 C: ... ., "C .. ~a. 2 ,:;: QI (,) o E IL 0 (,) 0 + Coxiella Day 1 + Day2 wtr .... ""'"' s, t t.~ - t-~ t,....t: t~jj" " ~ 100 C: - C: 4 Wt-$ e.lnftettd W'f o:.ua.l. ANA waa COl&Cled Oty- 1 .-Id 2 poei:-rftclion. r.....,,.IJ8rea'ibecl IOcONA end(lll'leexpressk,nwat~--0~ Reei 11me (~-PCR). Fdd CNl'9I .,.,,. ca:""80 ~ e d IO ~ ...,,__ UllirO GNl0li ~ a t ~ ' ""s)<0.01 M oetMnned by 0n&,wey NKNA 'Nth TLM)"I f)061 .--, .--, - a] a. C: . Lipid droplet breakdown releases free fatty acids (FFAs) . FFAs are PPARy agonists . Activation of PPARy induces antiinflammatory immune response . Example: Mycobacterium tuberculosis, Mycobacterium leprae I Conclusions I ..J so ., . i -, : .,. . .: . . . . - - - - - - --Oay2 andKt:Nlty ... Wild.fypt T4SSmutent Oay1 . f PPAR ....,...don .., :,;: ~ ."":.. Day Wild-type and T4SS mutant Coxle/19-tnfected mouse alveolar maaophages (MHS) cens were stained for Plin2 and Coxiella. Nun-ber of lipid droplets were counted by fluorescence mlaoscopy. Graph represents number or lipid droplets/cell in uninfected and infected cells. fn=3l .." ..=p<0.0001 determined bv two-wav ANOVA. t Antl-4"n-o,y -~~ffp,o,'IH c.,,,-- 6, u 0 ~~ . IWI f !kaldatlon N)1:DH,1, . ,..,.,..,,..1n Coxiella breaks down lipid droplets in presence or lhe enzyme ATGL to release Free Fatty Acids (FFAs) which act as PPAR.v agonists. The activated PPARy receptor then heterodimerizes with Retinoid X Receptor (RXR) and translocates to the nucl01Js, binds to PPAR response elements (PPRE) and regulates expression of several genes influencing cellular 13-oxidalion, host Immune response etc. Coxiel/a infection upregulates PPARy gene expression in alveolar macrophages suggests Coxiella might manipulate PPARy expression and activity to induce an anti-inflammatory immune response to promote intracellular survival.  ...

... ml INDIANA UNIVERSITY Ii.ii SCHOOL OF MEDICINE Analysis of Incident DKA in the Indiana New Onset T1 D Patient Population Brianna Beer, Marian University College of Osteopathic Medicine Kelly Moors, Indiana University School of Medicine Carmella Evans-Molina, PhD, MD, Indiana University School of Medicine Emily K. Sims, MD, Indiana University School of Medicine Indiana University School of Medicine, Indianapolis Indiana ABSTRACT RESULTS BLOCK 1 COVID-19 RESULTS TABLE Diabetic ko1oacidosis (OKA) is a lifo,threaloning complication of type 1 d iabetes {T10) resulting from ketone body production and metabolic acidosis occurring due to insulin deficiency, We sought to define the occurrence of OKA amongst pediatric pationts presenting with new onset Tt O in Indiana and 10 dolermine whether patterns of OKA were affected by the COVID 19 pandemic. This was a. retrospective chart review for podie,lric patients admitted to RIiey Children's Hospil.al wilh a clink:al d iagnosis of new onHt T1D who had available chemistry values. Patients diagnosed from March 23 June 30, 2020 and O\ler the same period in 2019 were included. OKAwa:s ciassified as mild (bicarbonate 10-15 mmol/L) Of severe (bic!llbonate <10 mmol/l). Ninely-four paIienIs me! incius.On crlleria. Thete was no significant d ifference in rates of OKA (21 in 2019 vs. 25 in 2020; p>0.05). OKA was present in nearly half of all new onset pediatric T1 D cases in Indiana in 2019 and 2020. There was. no observed impact ol the COVI0- I9 pandemic on T10 or OKA. 48 patienIs diagnosed witrl T1 Din 2019 vs 46 In 2020 46194 (49%) patients met criteria lor OKA Age, yea,., mean. (t SD) HbAlc, o/,, mean,(t SD) BMI, mean, (tSO) Gender 36/46 (79%) we,e severe 10/46 (21 %) WOIO mild No signffic:ant difference in rates of OKA 18 28 Female 21/48 (44%) in 2019 vs. 25146 {54%) in 2020; p:,,0.05 Male OKA Cla:uification In T10 In 2019 and 2020 OKA (n46) No OKA (n48) 9.7(t4.75) 10.1 (t3.59) 12.3 (t1.80) 11.6 (2.47) 19.5 (S.16) 19.7 (t7.07) p value 0.67 0.058 0.59 0.06 27 19 T1 D is oflen triggered by infection Concern of Increased incidence of T1 D during the pandemic OKA can be precipitated by Infection Concern that patients would delay seeking care during pandemic Possible ineteased OKA incidence 0.48 Race/Ethnicity While, non-Hispanic 34 No individuals admitted f-or OKA had a positive diagnosis 39 of SARS-CoV-2 While, Hispanic Black/African American Unknown BACKGROUND Type I diabe1es {Tl 0) is an autoimmune disoase charaeterized by insulin deficiency and hyperglycemia due to destruction of pancreatic bota cells. It was kmg though! to bo solely autoimmune, but further studies have found it lo be a complex interaction between Ihe environment, gene6c lae1ort, bola cells, and lho immune system. Diabetic koloacidosis (OKA) is a potenlialty fatal complication of T1 O. Decreased insulin loads lo poor gtucose ulilization and hypergtycomla. Tho body then begin5 lipotysis and releases frae fatty acid5 from adipose tissue. Th

... Investigating the Role of Ca2 + and the Acto-myosin Mechanism on the Human MCF-7 Breast Cancer Cell Line Bri Scherer Bhupal Bhetwal Ph.D. - Mentor CELL SURVIVAL PATHWAYS 4JI.SI:BAiJ: Canrulhba,n of Ab lhroli&li pho,$phocylauoa ot rt4JBPb) PlJ lcinue. l Tbt.tG t-ocritic'&I ccll ,urviva! ~)'Sbl\"C b UltllSh'dy AIJdicd DI (be (tC-ldofbft.,g ~ r ec.II reM:utb aod my IQ(#t'b i.J pn:rwily fiix-u:icd Ofl i11vcstip1iqthc rok.olVGCC.MLCK IDd PI-J ticwc on MCP.7 U viability Ca 2 PLCf3 by IJWIC ptwma,roklp&I izlhib1000 IOWC:U ,1nDC tbao&cs ia ~wtuUwn"ivalril~aad~ITT&Uly1. WORTMAN NIN CamK l AIMS & ffYPODIESES bn,:-.s1i,11C lhc tok: ofC12-dc-pcodmt paib-ay kadill; 10.:to-lll)'Olia Pl3,4,5 Tris-phosphate n.c 1A!ubJLio11 ol1hc eatdcpc':wkn1 pa-..., 1111n ckfftuc viability I Cell surviva l & division ----- N1kdiplnt k I VGCC lnhlb11or, aftd U lbf: ~ t111limls lntttau:, !k ttll vulH.111)' dtm-Htl. Tbt~t~11hl!W;moa1cruf,c-amctl'1 11 -40W'III ~ Actin~mvosin crossbridge '' NlfWIPJSE BESl rlTS _..,..,.,......._.. u, ...,.._ ,.,,......,..,. -....__.. i Akt activat ion ~ MCf.7 Us 'll"(l'C ~ IIW(dori 96 "t"D plUc:, witfl 100 ul of media (compnwd oi DME.1\1, tctail bo'V"IAC Jffllm,IM ant1bioodari11m.yc-o11<") LIi cacti \lf(IJ aQd -ctt ndt'clfcd 24 lmaff to IMl6w tht tc:lli 1odlt-rc: ltl lk pl.Ille. Stlrl.tltJOM or ~~tflltlbtllflll oflbc dnlp irt~ pre:~ ud ldtrufliarttd 16 lhc wdli att(lf"dlfl.,ty. with 1 BME (l,rlld mtloo u.trace) bc1.11& tbc posill\"C C'OQlfOI u,d EtOlt bc11lg 1bc iacpu,-c CGOtrot CtU1 -m dlctl allowed 10cul1urc fo, 41.71 bol.n. Ccll \'llbilhy WU UkUC'd via ~m may UNI NircdlpiDc- \~s;ltcdCrbltcr ML-7 l M LC--->MLC-P l of MCF-7 ccl4 WC tO Ole p,revcntiOQ ol 1Ct11H11)'0t-1acroubrid,ec (Ol'Tl\lbOII, lnvc.s1i111e 1hr role of PI-J kiflaSC pa&hay(ccll 1..-vi,"11 path11o-ay) UI MCF,7cclb o The:: iahil!itki11 olPI-J kh:i&K)Wbway ill dttrusc riabilicyof MCF-7 lb dlx to lhc inbibiuocl of Ab acti,ltioo n"mlfY for ccU RO'hallDCldaYlSK'III mlltpbolor.y\'llmk-ros:.top)'Md~tothtl!uttN'LlMh. f-- MLCK cnm,t,ndr,e formadon Ill MCF-7 Us o Cell attachment, cell viability, cell migration TARGETED HYPOTHESES OF PHARMACOWGICALAGENTS Nifedipine is a VGCC inhibitor. Application of Nifedipine with varying doses will decrease cell viability because calcium is necessary for the ronnaiion or the actin-myosin crossbridge and closing the channels deprives the cells or 1he necesS-ar)1 calcium for cell attachment. viability. and migration. ML-7 is an MLCK inhibitor Application or Ml,-7 with varying doses will decrease rell viabilil)' through the inhibition or MLCK preventing the phospoorylation of myosin light chain necessary for the actin-myosin crossbridge. K C! is an indire

... Stopping Pandemics in their Tracks Benjamin Abraham. B.S.1" . Aaron Schmid. Pharm.D. 1 lsrat Khan . M.P.H.1, Minal Mulye, Ph. D.12, Samina Akbar, Ph.D.12 1 = Marian University- College of Osteopathic Medicine 2 =Aaron Marian University-College of Osteopathic Medicine, Department of Microbiology and Immunology= Oral Presenter & Primary Correspondence la Introduction Figure 1 The proposed solution is a self-reported, symptoms-based syndromic surveillance system that is universal, interactive, integrative, and combined with artificial intelligence. Once developed, this framework has the potential to stop any future epidemics and pandemic in urban and rural areas worldwide. ~ l \.. We conducted a thorough literature review of existing short message service (SMS, text messaging) and interactive voice response (IVR, calling) surveillance systems, identified and addressed the shortcomings. We have concluded that many of the previous syndromic surveillance models suffer from data fragmentation, thus hindering their scalability to a global setting. 'I / _) \.. ~ J Rise in COVIO-hke symptoms: . 943 confirmed cases this week up from 700 tastweek. - 1200 suspected cases based on symptom 'I \.. reporting. 0 ,) Pro osed Model ~ l r Alerts Individuals The helpline can alert not only the individuals reporting symptoms but also other individuals within the same geographic location of a potential outbreak and can advise them to stay at home, avoid travel, watch for certain symptoms, or go to the doctor ) , 'I Health Officials Alerted The helpline can alert public health officials, governments. and other governing bodies about potential outbreaks and hot-spots so that those regions/countries can prepare for healthcare needs \.. - Machine Learning local guidelines COVID test k>catrons lb Artificial Intelligence In case ol an increased number of individuals reporting similar symptoms in a similar geographic location - the artificial intelligence with machine learning can recognize patterns based on previously researched diseases, and estimate risk and severity r scorecards and more. These are likely COVI019 symptoms. cllek here: How many COVlO cases are there in Indianapolis? l Physician Confirmed Findings If the patient is advised to see a healthcare provider, the signs and symptoms of infection can be confirmed and reported back into the system. These confirmed findings improve the artificial intelligence algorithm to better predict and identify syndromes and cases. Ory cough. lever. loss of taste Data Storage The helpline simultaneously stores data in a central database for monitoring and analysis ,) J i Global Spread of Infectious Disease Averted .... Analytics Advanced analytical and visualization tools help users quickly extract actionable insights from their data. Visualizations include fully customizable, continuously updating dashboards. interactive charts. time series and geospatial analyses. What symptoms are you experiencing? l 'I Details I ~ Response la The helpline responds to and interacts with the individual to provide appropriate ' next-steps" based on artificial4--' intelligence estimated risk and severity (ex: what other symptoms are you having; stay at home and quarantine for 14 days; contact us in 3 days if your symptoms are not getting better; you need to see a doctor right away) ,) r Methods Helpline lb 3:04 PM ( Messages TRACK Helpline Self-Reporting la Individuals report symptoms to the helpline via text message (SMS) or via calling (IVR) r _, m 4 Our analysis framework applies machine learning and other statistical techniques to generate accurate predictions and identify data anomalies. QR CODE Follow code fo r New York Academy of Science presentation: Top-10 Surveillance ,= Ii) .. I. . This framework (figure 1) outlines the concept of our proposed model. The Al will collect additional information that will hefp in triaging: Age, sex, racial or ethnic background, BMI, past medical history, fam ily history, surgical history, and/or social history (demographical information). Develop a demographical subtype in the Al' s database. Patients will be stratified into high and low-risk populations. Conclusion This proposal will allow decision-making officials and healthcare professionals to robustly identify local disease outbreaks, thus thwarting unchecked spread while preventing a breakdown in the supply chain. Automation of healthcare team reported findings will enable predictive diagnosis and prognosis, thereby allocating medical resources appropriately. Disease-specific databases will benefit from real-time refinement, then utilizable for prospective case mitigation, medical management, and pathogen containment.  ...

... Role of Peroxisome proliferator-activated receptors {PPARs} in autoimmunity, immunocompromised, and infectious states: a Review Aslesha S. Tingare1; Tyler Schenck1; Katherine Phelps 1; Minal Mulye, PhD1; Medhane Cumbay, PhD1 1 Marian Peroxisome proliferator-activated receptors (PPARs) are a family of nuclear receptors that activate a large array of genetic pathways. Different subtypes, including a, ~, and yl, y2, and y3 We continue to learn more about the involvement of PPARs in immune modulation. HIV have decreased PPAR-y expression and increased NADPH oxidases leading to enhanced oxidative stress impairment of the alveolar macrophages' ability to phagocytize and clear infection in the alveolar space and increased risk for lung infections. ~~lX,~ ;;~:- t Wol.m hNWlg resc,ons.n ....... "" t t.12 Maaopt\lg,N. t.lR{C0208) Argt H ROS Domon I lnllmrnalclfy IIIC)Of'N. NF-8. TNF-ci - L myocarditis and PPAR-a may play a more critical role after all Allergic rhinitis: type II immune responses in allergies (and nematode infections) are promoted by PPAR-y 1; 2 polymorphisms affect symptom severity TlDM: PPAR antagonists were shown to reduce the risk of development of tld in genetically predisposed line NOD3 University College of Osteopathic Medicine, Indianapolis, Indiana u ~bl~olPPAK...., andJ --------u L "'=-:JL Vl'AKJl,'6 JL u:::-- J Treatment of M.tb infected cells with Vitamin Bl showed increased expression levels of CD86 and MHC-11, but decreased expression levels of CD206 4 Treatment with PPAR-y agonist has been shown to attenuate the biofilm formation via the induction of paraoxoanse-2 (PON-2), an enzyme that degrades QS molecules involved in Influenza: PPAR-y decreases fibrotic remodeling and inflammation, and promotes host recovery TB: PPAR-y activation plays a role through induction of IL-10 and repression of pro-inflammatory cytokines; PPAR-a upregulates and translocates TFEB (Transcription factor EB) to the nucleus to perform its downstream effects of lysosomal biogenesis and autophagosomal antimicrobial responses against M.tb7 8 Pseudomonas aeruginosa (PA) infection: PPAR-a prevents excessive inflammatory response to PA infection by controlling NLP3; also has an inhibitory effect on TLR4, which recognizes pathogens and activates ro-inflammator c tokines9 10 Further understanding PPARs upstream pathways and interactions can contextualize pathophysiology in immunocompromised states There is a relationship between PPARs and infections Pharmacological evidence targeting these PPARs is prevalent to accelerate clearance of these pathogens but also assist in pharmacologic therapy for autoimmunity and immunocompromised states  ...

... ABSTRACT Brexanolone (ZULRESSO ) is a novel approach to treat postpartum depression in adult women as a neuroactive steroid similar in chemical composition to the hormone allopregnanolone. It was approved for use in adult women suffering from postpartum depression by the FDA in March 2019, with emphasis on its potential use in patients who are unresponsive to mainstay interventions. Brexanolone is the only drug currently on the market approved to treat PPD, use of Brexanolone requires patients to be enrolled in the REMS (Risk Evaluation and Mitigation Strategies) ZULRESSO program necessitating a 60-hour period of intravenous administration and close monitoring. The pharmacogenomics of this drug have yet to be studied but could potentially aide in determining if Brexanolone administration is an appropriate therapy for a patient. Additional clinical studies with larger sample sizes are necessary in order to evaluate the use of Brexanolone in women with PPD. PHARMACOLOGY Brexanolone: A Review of the Novel Drug Injection for Postpartum Depression Ashley Orr MS1, Samar Fakih MS2 , Julia Hum PhD1 Marian University School of Osteopathic Medicine, Indianapolis, IN; 2 Western University of Health Sciences College of Osteopathic Medicine of the Pacific-Northwest, Lebanon, OR NormalGABAA 1.. \ 1ll \ receptor signaling pathway ___,,,,,,, 82 ng,'ml (00 dose regimen) 5-4.04 ng/ml (80 OOH: reglfMf'I) ~ ng hrtmt. (ac> doH, regirMn) ~~~ --~- (~~~~~ 1ll \ Increased depression and anxiety 2M7 nghrfmt (e0 dose regimen) / 0 aUhrlkg inc:i.potn~ of doM Increased GABA A \ 1ll \ receptor signaling pathway ___,,,,,,, J1111-1111 ---- I I I HPA Axis The novel approach of Brexanolone has brought an approved therapy into a previously devoid space of postpartum depression in adult women. KEY REFERENCES =~ ~~ k.~ !!..~ ~'t:t1l~~~R.~~e;o,.AJ. Fritdlr. A. FMh. M.. HM1iM. -R~ & Dligiantm. K. M. (2011il) . Ph1~ p y cl Postpartum Depres.sion: Currene~ es .rod Novel Onig Devel09fl1enl CHS 3 C2()20). Increased Inhibition o l K@.t o-ffdi...ction, gluoof'ontdation. and sulflllltion Bic,l\e~ IMtn inititl rapod dt<:Wle httf..lif.e of 40 minutes lolowN by a l at..- tffminal Nllf~if11, of 12 hows No l ludi4tl conducted lo date. IJl'llike l~ th.el co&(fmif'li stration of CYP inhibitors or ind'~ B 'Mil al'ftct Bfuano4onea:q>Mi..ft in HAM-D scores when compared to placebo, thus supporting its use in treating PPD (Meltzer-Brody et. al, 2018). CONCLUSION receptor signahng pathway .__.-,/ 3 U'kg (baSM Oft a hfftthy indMdual) No eviOtnce showing PKreltted ne enhnctJ pht1,$ic nd tonic: inhibi,ion in the br9in thtough the po$itive allost@ric modulation of synaptic (left) and v:tn1syn, plic (right) GASA.c, receptors. CLINICAL TRIAL DATA ~ f C l f PO$q)-.,M ~ :ANovel~ltld 1 c,lto1 Cornprthtn$Nt Pos~ CM. ClinioM ~ i. 42(1). 231~5. : ~~~~ ~ ~ -~ ~ ~ WRainu. s.. Amold. R. Sohacedt. A. ~=/0.~ ~-CP;J~.~xi: ::.~~-~ 01~'::~E-547 q,!Clion) in p<>Sl-pa,timd~slon: /\rardomz!d ~ trial. Tb@ CAna!t Decreased depression and anxiety 3.f100Q3). 480-48\J. hl!pS:lfdoi.orp'10.10t ais01~731!(17Pl 2&4-3 Lldtl. L o.. OC-cnnd, M., & V#ldtn8tf9.A.(2010), ~ f\04ont b' Po i ~ ~ : Clinie:al EYidenotand PradiealCoMicSenifions.PhatmM'Jdllerapy Tl ~m,/o/ , 33(11).1105-1112. - ~ -!w~:~ ,'~  ...

... Mechanical stimulation in vitro regulates pro-inflammatory cytokines: potential insight into soft tissue manual therapies for osteopenia and sarcopen1a Arie Anloague 1, 2, Aaron Mahoney 1, 2, Oladipupo Ogunbekun 1, 2, Taylor A. Hiland 1, 2, William R. Thompson 1, 3A, Bryan Larsen1, M. Terry Loghmani 1, 3A, Julia M. Hum 1, 2 and Jonathan W. Lowery 1, 2, 3 * 1 Division of Biomedical Science, Marian University College of Osteopathic Medicine, 2 Bone and Mineral Research Group, Marian University. 3 1ndiana Center for Musculoskeletal Health, School of Medicine, Indiana University, 4 Department of Physical Therapy, School of Health and Human Sciences, Indiana University Abstract Primary Dermal Fibroblasts Tibiae Explant Differentiation A Objecti ve: Soft tissue manual therapies are commonly utilized by osteopathic physicians, chiropractors, physical therapists and massage therapists. These techniques are predicated on subjecting tissues to biophysical mechanical stimulatio n but the cellular and molecular mechanism(s) mediating these effects are poorly understood. Previous studies established an in vitro model system for examini ng mechanical stimulation of dermal fibroblasts and established that cyclical strain, intended to mimic overuse injury, induces secretion of numerous pro-inflammatory cytokines. Moreover, mechanical strain intended to mimic soft tissue manual therapy reduces strain-induced secretion of pro-inflammatory cytokines. Here, we sought to partially confirm and extend these reports and provide independent corroboration of prior results. We have also begu n to extend these reports throug h the use of differentiation assays using osteoblastic precursors from both calvaria and long bo ne cells. We observe this differentiation into osteoblasts from the precursors when they are exposed to conditioned media containi ng the pro-inflammatory cytokines from the aforementioned mechanical strain and therapy studies Results: Using cultures of primary human dermal fibroblasts, primary human skeletal myocytes, and murine C2C12 satellite cells, we confirm, in fibroblasts, that cyclical mechanical strain increases levels of IL-6 and adding long-duration stretch, intended to mimic therapeutic soft tissue stimulation, after cyclical strain results in lower IL-6 levels. We also extend the prior work, reporting that longduration stretch results in lower levels of IL-8 in fibroblasts, as well as provide novel data showing cytokine changes in the myocytes and satellite cells. Although there are important limitations to this experimental model, these findings provide supportive evidence that therapeutic soft tissue stimulation may reduce levels of pro-inflammatory cytokines. In addition, we have exposed MC3T3E1 cells, an osteoblastic precursor from calvarial bone, and murine tibiae to the previously mentioned conditioned media containing these pro-inflammatory cytokines to observe its potential differentiation upon exposure. While this study did not provide any significant findings in the MC3T3E 1 line, it gave us key insight into our next steps for the differentiation assays. We also exposed another osteoblastic precursor, W-20-17, to the previously mentioned conditio ned media. Gene expression analyses suggest that soft tissue manipulation (STM) increases osteoblast differentiation, as determined by expression of the osteoblast marker Osteocalcin. However, these prelimi nary results were not statistically significant and will need to be replicated before confirming this preliminary analyses. The tibiae showed an increased expression of the osteoblastic marker, P1NP and a decrease in CTx- 1, a marker for bone resorption. One of these insights has led us to begin observing different cell lines, specifically cells originating from long bone. Future work is required to address these open questions and advance the mechanistic understanding of therapeutic soft tissue stimulation E3/4 : MIF A20 .6secoads~ 20% slope C13/ 14 : ll-8 .o 10 (1J g, O w ;: hk 5 0 --- A&B: Multi-analyte cytokine membrane arrays of the CSDS conditioned media collected at 24 hrs (A) and 96 hrs (B) detected the cytokines listed between the figures . C-E: Quantification of the anal ytes are represented as means SEM normalized to CSDS; n=4 per condition . F&G: Quantification of these analytes are from the cytometric bead array assay. They are represented as means SEM normalized to CSDS; n=3 per condition . *indicates p<0.05 against CSDS by paired T-Test g O 5 ~-~ Figure C: Human primary dermal fibroblasts, human skeletal muscle myocytes (HSkMC), and mouse C2C12 myoblasts were seeded separatel y onto fl ex plates coated with collagen I and subjected to a CSDS profile (A or B) alone, or a CSDS profile (A or B) followed by 3 hours of rest, then an acyclic long duration stimulation (ALDS) profile in w hich cells were elongated to 6% for 60 seconds with a loading rate of 3%/second and a release at 1.5%/second. Conditioned media was collected at 24 and 96 hours after treatment for fibroblasts , and 72 hours after treatment for C2C12 cells and HSkMCs. Conclusions The ALDS profile was developed to be representative of soft tissue manipulation (STM) techniques such as myofascial release . The fi broblast data and preliminary data for C2C 12 myoblasts and HSkMCs show decreased release of pro-inflammatory cytokines , IL-6 and IL-8, in particular, with the ALDS treatment. Chron ic IL-6 exposure has been show n to accelerate disease progression and have se veral effects on skeletal muscle, including decreased muscle protein synthesis and increased protein degradation , w hich ultimately results in muscle wasting . Reduction of these pathways by STM may reduce the effects of the muscle wasting that occurs in sarcopenia. 1.s[ l XCL j-5_ C * (J ~ 0.5 0.0 cl' cP~ Bi 1.su -1-alpha itll i C Osteoblastic Precursor Differentiation 1.0 Oen * 05 j 2.0i M 1.5 cl' if cP "C References & Acknowledgements ~ 0.5 1. 0.0 ~ CSDS CSDS 33.3% slope ALDS 24 hours 24, 72 or 96 hours A represents pilot data of HSkMC. B represents pilot data of C2C12 myoblasts. Quantification is by by multi-anal yte membrane array, and is represented by means SEM normalized to control ; n=2 per condition . *indicates p<0.05 against control by paired T-Test. -A, o-..-A,01-o,M 1oTA,..-..-~.l.a-l, L -- u,MJ, L 'fJW . lo oh ~ . . - -1...., 0~ Cells combined for seeding Preliminary differentiation assays have allowed us to see the affect of the conditioned media on bone explants and osteoblast precursors themsel ves. Original data from tibiae explants shows a statistically significant increase in bone formation marker P1NP and a statisticall y significant decrease in bone resorption marker CTx. Preliminary anal ysis of an osteoblastic precursor cell line, W-20-17, also suggests that w hen exposed to STM in fluenced conditioned media, there is an increase in osteoblast marker Osteocalcin. This preliminary data gives us a promising insight into potential treatments for osteopenia and osteoporosis, as we are observing the potential for bone resorption to be slowed dow n and bone formation to potentially be increased. Further experimental data is needed before any certainty can be con veyed. U 1.0 0.0 C w 0 Ail, ~ 1.0 1. 6seconds 210 [;] C2C12 & HSkMCs Figures A and B represent a cyclic short duration strain (CSDS) profile that previous researchers () have developed to represent a repetitive motion strain . Enzyme-Linked lmmunosorbent Assay (ELISA) results for bone formation marker P1 NP (A ) and bone resorption marker CTx (B) from neonatal tibiae explants exposed to osteogenic media +/- conditioned media (CM) from dermal fibroblasts subjected to cycl ic short duration strain (CSDS) followed by acyclic long duration strain (ALDS) or control. * indicates p<0.05 compared to control conditioned media B Cll/12 : ll-6 AlS/16 : CXCll/GROa Mechanical Strain Profiles ~ 15 CTx ES/6 :Serpin El/ PAl -1 0~ f}(i- c.P 0"' x~ v0'J~ qRT-PCR data for osteoblast marker Osteocalcin in W-20-17 cells exposed to osteogenic media +/conditioned media (C M) from dermal fibroblasts subjected to mechanical strain through CSDS + ALDS l . J. . 5. 6. 7. a. ~-a: ,.1..11 ll"t. 1 - 1 oo _ ... 1.,ra . . II C ~- . l lfllfA1 27;1]'.1): _ lt lot ! - - -1- - r .. t 1,.11 -n1-Ut -H 2 ._1 . ,.D: ll'-7&l' i ...,,T.l .,l.~. lol n,,a .. , .~.llo l--lllnO - ..r ro1,1ot1- - -1~1!n t ~ - : - - v h -1!-t oo .. - I - ht -lo.J o lot ,,_JOOlloro.,-7. :.;l): , . --&l' lolr ,l.~., ot a l, IIYll, -1111 o!ro,off .., ... .,..1.. lroi., ... J...,.. ovr.er,llf 1 t . 1"(2 ) : , . -71 . .,, .,, l.~ . ... , .~. . ....... - - ,.,..... - trO . .. - Ht- - . lof 1 - ... . ,.,,1or Ill- - - t ~l!nt . .. . 1.-.,-. NOfot .JMOt -tlMe,-l. 117(12):,. I ....... , .~. - l . , , .,, II Yh-lll1!ro"'"f -1 trO ... - I - . ht-lo: ,.1..1 lr- ! ~---t!.41 .. _ t....,~-... J . . . . . . .y l-----, .... ,...... M-,~,ot L, l trO , - t o 0 - - . . _ _ . _ , , . . . _ . ,, 1o1o1, . . - ,t -!!oro,t t .JA.,,_JOOllo(1 -),llf12. 11 ]'.J): ,.1-n lof : A JHroa l ! ,..,,..-,."".,..,,... l~ - -1 ,llf11. t 1 c,.,,,J .O.,ot a , .. .. er "" " t .. _ 1...., . alll1 or 1 Trau1a1, llt12. c111): , . 111u 1J We gratefully acknow ledge critical feedback for th is work from members of the Marian Un ivers ity Bone & Mineral Research Group and the consultation of Dr. TienMin Gabriel Chu {Ind iana Un ivers ity)  ...

... ~~ Assessment of bone healing agents for promoting bone regeneration in spaceflight _ ~ IN~lf Zachery R. campt,e111.2, Anane Zamanoh13, Ushash1 c. Dadwal1, Paul J. Childress1, and Melissa A. Kacena1 1 ~ d~ 9 lu" - ) ' _,.._..i:y _ ,.__ lnik...,_ uu.; 2 1&m1ant-.- . e , , Co11toQ9dO~ lr-obno,. U1A; laa..ci- ~ 1 1 - . ,io._-ordRot..1>1- , .- P n t t i - l M I -.u.-.-.c1aa0Paulo, Bnl,11; ~! ~N,IVEASITV Crollll.d INTRODUCTION Segm ental bone defects secondary lo high-energy trauma may not sponlaneousfy heal and orthopedic surgeons call on the use of bone graft substitutes. Bone morphogenetic prntein-2 (BMP-2) and thrombopoietin 1111 (TPO) are an FDA approved and a novel t herapeutic alternative, respectively, lo revitalize t hese bone g rafts t hus stimulating osleogenesis. With lhe growing global interest in space exploration and colo nization, these d rugs may be used in the absence of lo ading conditions, which may inlerfere with their mechanisms and actions. GOAL To evaluate the effects of BMP-2 and TPO on regenerating bone in a femoral bone defect model in m ice housed on the ground or in space. Figure 4: 1,1CT imilging of fra.C'lure femurs from ground grou p displayed no signs of bone regenera.fion were observed in the non4rea ted mice (SALINE). an abundant presence of ca.,s formation witfl bridging was seen in the BMPtfeated mice. Ahhough. TPO did not induce complete bone bridging. the trea1men1induced new bone formation a1a highly mineralized lewel. While in flight mi ce no signs o f bone regen era1ion were obselVl!d in the non-b'e ated mice (SALINE), an abundam1 presence of ca Dus forma1ion witfl bridgirlg was seen in the Br.tP-2 and TPO-treated mice. MATERIALS & METHODS AJ:hough SMP-2 led co a. larger calh.Js formation, TPO resuhs in ca.,s 1,rith better microiirchitecture. higher mineralization, and without the apparent side-effects observed wi1h BMP-2 treaunent Sixty, 9-\veek-Old, male , C 57BL/6 mice underwent a 2-mm femoral defeci surgery RESULTS G round mice were housed at Ke nnedy Space Station (5-day asynchronous cont rols to match conditions) and Spaceflight mice (Flight) were housed aboard the lnlem ational Space Station (15 5 ) for 4-week. Each group w as further subdivided into three groups according to the treatments: G round-Saline; GroundTPO; GrouOO-BMP-2; Flight-Saline; Flighl-T PO; and Flight-BMP-2 (n=10fgroup) Figure 1 illustrates the maj or events leading up to and after launch Bo.ne regeneration in femurs was assessed by mk:r0-eomputed t omography (CT) Of the 30 mic,e housed on t he Earth (Figure 2), 5 (TPO) were euthanized early b ased on All statistical analyses were perfonned with RStudio by using two-way and three-way analysis of variance, followed by Tukey's posl hoc t est. p values less t han 0.05 w ere considered statistically significant l I I1 II I I I 1 .f I I I I II I .f I I I I L1d L!ld L5d L 10d Of the 30 mice housed on the ISS (Figure 2), 1 d ied (BMP-2) and 5 were eulhanized earfy based on NASA velerinarian recommend ation: 4 (BMP-2 ) and 1 (saline). L24d I ,/l I ....I , NASA vet erinarian recommendation due to observed aggressive behavior Zero hardware failures w ere observed w ithin t he spaceflight group In the g round-based group, one out of five scaffok:ls (20%) exhibited rupture during the study and w as excluded from the resuNs (Figure 3) No nei.v callus formation in the Ground-Saline or Flight-S aline groups (Figure 3), confirming the critical size of our segmental bone defect, which does not spont aneously heal. We detected callus format ion which resulted in bone bridging , for bolh G round-BMP-2 and Flight-BMP-2 mice. On grouOO, BMP-2 increased caJlus volume by three-fok:I (p=0.08) and SMI by 17% (p=<-0.01) w hen compared t o the Ground-Saline g roup (figure 4). Figur e 1: Tmeline deliiiling the o-..eraJI experimen!iil design including launch preparation/mou se acclimafon, launch. and mous.e euthana.sia. C row.I Bll'.P-2 group (a:l ) fOUlld moribund Bll'.P-2 groop(S=J)fOUJl..ddis!rffled - BllP-2 group (A=l) fo1Dd di!cmsed S1btgroap (a:l)foa1ddistresstd (Figure ). Comparing tt.e effects on g round or space, BMP-2 resulted in greater callus volume when used in flight (p=O.CJ03), but with lower d ensity (p=0.08), fewer trabeculae (p:0.03), and higher trabeculae separation (p:0.04) With TPO treatment, we detected bone bridging in our Flight group, but not on Ground (Figure 4). Here, in flight, TPO increased TV and BVITV by 35%, Coon.D by 173%, canus density by 61%, and Tb. N by 33%, when compared to Flight-Saline mice. "'"' TPO ~oup (~fouddutmstd BllP-2 ;row.p (a::3) mthmized. Aealtli!(oontrol stv.dr) In flight, BMP-2 incre ased callus volume by nearfy soc. fold (p<0.0001). BVffV by more t han two.fold (p=<-0. 001), and SM I by 50% (p:0.02)when compared to the Flight-Saline group Furthermore, TPO treatment in flight signifi cantly decreased trabecular separation when compared t o F lighl-BMP-2-trealed mice (p-=:0.05). Comparing t he effects on g round or sp ace, TPO resulted in a more d ense callus (p=0.05) and higher trabeculae conneciivily (p=0 .001), w he n applied in flight. L+l 2d DISCUSSION Fi gur e l: Microcorr,pu:ed (CT) ffla~es of femurs con1ainitg Figure, 2: Chronology of incidents occurred during the spacefligh t that led 10 unscheduled eu1hanasia. No hardware f ailures in the spaceflight g roup confirms the efficacy of our rodent model as an effective SBD model t o study bone regeneration in space S 6D approximately 4 weeks after surgery. d emonstrating nc BMP-2 has re markable bone regenerat ive properties bone bridging in ei1her the ground or ilight groups. Hardware failures (whi1e arrow) were onty obse,ved in ground mice While T PO appears to heal bone more slowly t han BMP-2, the formed caUus appears to be of better qualily and microarchitecture . Not ably, TPO' s mechanism of action does nol depend on CONCLUSIONS Non-union remains a challenge among patients with limited weight bearing because most options to induce osteogenesis are based on pathways that perform best with loading. Here, we show that TPO induces bone regeneration even under unloading co nditions. ACKNOWLEDGEMENTS This work was supported in piin by pos:doctoral NIHT32 DK00751g (UCD). NIH Training Grants T32 DK00751g (PJC) and T32 AR065fl 71 (KAM), Medic,a! S1udent Affairs Summer R~earcb Program in Academic Medicine, Indiana Uni-..ersity School of Medicne (JPF. JOR, OCS), 1he Ralph W. and G1ace t.l. Showalter Research Trust Fund (MAK), the Orthopaedic Trau ma. Association (MAK}. Nlli/NIAMS R01 AR060363 (MAK). and GA-2015-217 from the Center for the Advancement of Sciences in Sp.ace {MAK). This work was also supported by The Sio Paulo Research Foundation (FAPESP, #25606-4 to .AZ). This material is also i h e re-sutt cf wort stipported with resources and the use oft.:icilffies at the Richard L. Roudebush V A Medical Center. Indianapolis. IN: The presented cootents are solely the responsibility of the authors and d o no1n ecessar ily rep resent the off!CWI ,riews a f any o f the aforementioned aeenci,e,s. g ravily Furthermore, it is important t o highlight that the BMP-2 bone healing dat a are from the 5 surviving mice , where 50% of the spaceflown mice treated w ith BMP-2 died or we re euthanized early as NASA veterinarians deemed them lo show sufficient signs of distress. By contrast, a ll BMP-2 t reated mice on Earth survived to t he end-points (of not e all reagents used were f rom lhe same batch and there w ere a tota1 of 30 mice t reated with BMP-2 on t he g round: 10 b a.seline control mice, 10 vivarium co ntrol mice, and t he 10 identical ground control mice discussed here). While the exact cause of deat h/distress of spaceflight mice was not clear from posl-mortem pathological assessments (bul was not due to aggression), these findings highfighl lhe importaoce of further exploring this phenomenon as medical kits are currently being d esigned for treating huma ns for many medical problems, including f ractures, that w ill likety occur while living in space long-term such as that anticipated with colonization of t he moon or mars  ...

... Applications of Virtual Reality in Osteopathic Medical Student Education Anthony J. Perugini ID 12 ; Se1gej Stjepic12 ; Bradley J. Boget12 ; Garren P. Gebhardt, DO, MS2 3 ; David L. Dufeau, PhD 1.2 13D Visualization Laboratory, Marian University College of Osteopathic Mediciue, Indianapolis, IN, USA; 2Marian University College of Osteopathic Medicine, Indianapolis, IN, USA; 3 Department of Cliuical Affairs, Marian University College of Osteopathic Mediciue, Indianapolis, IN, USA; BACKGROUND CT SCANS & SIMULATION PRODUCTION Book, presentation, and video are the current most popular modalities used for grasping whal is conceptual in OMM; Students entering medical school Figure 1. Amira Data Analysis Package (F.E.I. Amira v 6.0.0) analyzing deidentified computerized tomography data to render 3D bones, tendons, and muscles. have a higher degree of technological literacy. Education technology reports have indicated that the current generation of students has shown great interest in interactive technology and web applications (apps) 1 Subsequently the gamiflcation of education has quiekty shown promise2. lhese ..games, virtual reality (VR), and augmented reality (AR) simulations have been shown to promote learning, engagement, clinical application, and immediate feedback l o the student3-. Integrative virtual reality and simulation based education is readily embraced as a standard for spetiahies such as surge,y, Figure 2. Camtasia Video Editor (Mac Version 2.10.08) editing software used to narrate, edit, and record the production of finalized instructional videos as addendum to simulation. urology, cardiology, basic procedural education, and more 5. We postulate that a movement towards a dynamic, lhreedim.ensional application, or website. that animates the techniques of OMM could be a highly beneficial development. The goal of lhis proof-of-concept is lo posil how we can come to understand whether technologies such as VR can be developed and integrated, and how their effects on osteopathic students can be measured. In order to achieve this goal we have developed a proof of concept technology al Marian University College of Osteopatl1ic Medicine (MUCOM) that models a number of basic dysfunctions, and that would be VR compatible. Figure 3. Sketchfab Online Open Source Forum; Visualization of 3D simulations. 3A - Emphasis on musculoskeletal anatomic position 38 -Anatomic Landmarks relevant to musculoskeletal pathology 3C - Referenced Information Pockets/ Drop Down for a given somatic dysfunction MATERIALS & METHODS Creating the Models These data for creating the anatomical simulations were sourced from an anonymized medical scan. The data used in this study are in the format of a Computerized Tomography (CT) imaging study. CT scans were uploaded to the Amira data analysis package (F.E.I. Amira v. 6.0.0) which was used to analyze volumetric data sets. These data were anatyzed and interpolated (slice-by-slice) to render individual three-dimensional bones and muscles. These data were used to construct educational simulations of kinetic threedimensional (3-0) movements. Narrated simulations were implemented as hyperlinks on a static human skeletal model. The narrated simulations were constructed and animated from volumetric data rendered on Amira (Fig ure 1). The rendered 30 objects were transferred from the Amira volume analysis software to the Camtasia video editor (Mac Version 2.10.8) (Figure 2). Camtasia is a video editing software that was used to narrate, record, ed~. and aid in the production of the final instructional videos that were the addendum to the original s ilent simulations. These simulations are viewable as interactive simulations on the Sketchfab online fon.im at the following address: httos://sldb.ly/606tl. Sketchfab is an open online forum that allows creators of three--dimensional simulations to upload their work unto its platfonn for public access. Sketchfab. allows educational and artistic models to be viewed and downloaded at the discretion of the creator of the simulated models (Figure 3). Figure 4. Sketchfab Interface; Example of Posterior lnnominate Somatic Dysfunction. PILOT STUDY Double-blinded case versus control study of incoming OMS-1 Participants asked via survey for willingness to participate Total number of students not exceeding 30 (n = 30) Students divided into two cohorts receiving either: - Simulations of Type I & II F,yette Somatic Dysfunctions Standard 50 n'inute Didactic Lecture on Type I & II F,yette Somatic Dysfunctions All Students take 30 Q Exam administered via lpad All Students go down to OMM Lab and asked to diagnose and treat a Fryette Type I or II Somatic Dysfunction previously diagnosed by Osteopathic Physician and faculty of MU-COM Students graded by same blinded faculty member using standard set criteria for MUCOM osteopathic physical exams and treatment. REFERENCES Physical Exam findings (with respect to R side): Positive Standing Flection Test lnnominate Compression Test, HigherASIS Higher Pubis Lower PSIS Deeper Sacral sulcus, Superior medial malleolus 1. McCoy L, Pettit RK, Lewis JH, et al. Oeveloping Technology.Enhanced Acti'we Learning for Medical Education; Challer,ges, Solutions, and Fu1ure Directions. JAOA 2 McCoy L, Lewis JH, Dalton O Gamification and Multime-dia for Medical Education: A Landscape Re'iew. The Journal of lhe American Osteopathic: Association. 2016;1-16(1):22-34. 3. A1<1 EA, Prelorius R:W, Sackett K, et al. The eflect of educational games on medical students' leamln:g outcomes: a Sy$Iemaric review: BEME Guide No 14. Med Teach. 2010;32{1):1$-27. 4 Kron FW, Gjerde CL, Sen A, Fetters MO Medical student attitudes toward video games and related new me-dia lecllnologies in medical education. BMC Medical Education. 2010;10 ( 1) 50. S. Shamim Kllan M, Ahmed K Fau . GavauiA, GavauiAFau. Gohil R, et al. Development and Implementation of een1ral&Zed simulation baining: evaluation of feuibility, ac:ceptabitity and col'l!truct validity. (1464--410X (Elecuonic)).  ...