The Philadelphia College of Osteopathic Medicine (PCOM) offers a masters level course enrolling 90–100 students per year as part of the School of Health Sciences, Biomedical Graduate Program (Biomed) for students who may continue onto allied‐health‐professions, not restricted to a clinical setting. The course historically utilized only models and prosected cadavers. Dissection is rare for an undergraduate or masters program and therefore lends an opportunity to improve student skill sets including 3D spatial reasoning as a tool for knowledge retention. In Fall 2019, PCOM Biomed‐Anatomy opted to include special dissections for every enrolled student, which required a new format for teaching. We hypothesized that standardizing the Teaching Assistants’ (TAs) Program would have a positive impact on both the TA experience and student outcomes. Further, by adding opportunities to peer teach and dissect, we hypothesized that it would have a positive impact on student experience and satisfaction. Noting opportunities for course enhancement and developing a pathway for medical education to interested graduates, two students who completed Biomed‐Anatomy were identified to serve as Lead TAs in a pilot program. Lead TAs collaborated with the course director to formalize the TA system. Students who excelled in the course in prior years were invited to apply to volunteer as general pool TAs and we recruited 57 students for gross anatomy and 11 students for histology labs. Lead TAs helped to support both general pool TA’s and students by creating a 1‐to‐6 TA‐to‐student ratio structure. Lead TAs would schedule general population TAs to labs. For the first time, Lead TAs would provide feedback during peer teaching preparation sessions, prepare active original special dissections, deliver clinical vignettes in lab, and generate and disseminate supplemental material. Throughout the course, we solicited weekly feedback from general pool TA’s and enrolled students, which helped us identify strengths and opportunities to refine multiple programs using anatomy at PCOM. In regard to student feedback, students trust TAs to provide information that is high yield for the course. Students agree and strongly agreed that TAs were an asset to their learning style. Students valued preparation sessions for peer teaching and the experience of the peer teaching component was a mechanism to keep pace with material. In PCOM Biomed‐Anatomy, we have made adjustments for the future to optimize TA and student experience based on the evaluations. For example, we will start every lab with a one hour review on prior material facilitated by TAs followed by brief clinical vignettes more suitable as faculty presentations. Lab will culminate with new material and preparation of special dissection under the guidance of Lead TAs and faculty. As a result of implementing this new format, we found an overall positive impact on TA experience and enrolled students performed higher than the year prior. Furthermore, faculty satisfaction has improved with the addition of these well qualified Lead‐TAs teaching in the DO program.

Our previous studies of brain tissues obtained at autopsy have established an association of Chlamydia pneumoniae (Cpn) infection with late‐onset Alzheimer’s disease (AD). Given these findings, we have designed a study using electron microscopy (EM) to determine if various forms of Cpn from geriatric patient’s blood samples could be identified and correlated with cognitive decline associated with AD.Patients, >65 years old, and who participated in this IRB approved study, met well‐defined exclusion criteria prior to being evaluated for cognitive status using the SLUMS and FAST testing assessments. After psychometric testing, blood was drawn from the patients and these samples were prepared for EM and assessed for the presence of Cpn.Our ultrastructural analysis revealed evidence for the presence of Cpn in peripheral blood cells from 12 patients correlated with MCI and AD. Intravacuolar reticulate bodies, elementary bodies, and intermediate bodies characteristic of Cpn were observed in leukocytes from the buffy coat. More specifically, we observed the classical “pear‐shaped” elementary bodies characteristic of Cpn. Immuno‐ EM was performed for positive identification of these various forms of Cpn.Our data indicate that screening for Cpn from blood samples may prove to be a viable biomarker in differentiating cognitive change consistent with stages associated with MCI and AD. Investigating the distinct morphologies of Cpn in human blood samples from the geriatric population by EM, with other frontline diagnostic procedures, may clarify the diagnosis of MCI and late onset AD.Grant Funding Source: Center for Chronic Disorders of Aging

Previous studies from our laboratory of brain tissues obtained at autopsy have established an association of Chlamydia pneumoniae infection with late-onset Alzheimer's disease. Given these findings, we have designed a translational research study to identify biomarkers associated with Chlamydia pneumoniae infection in the blood of geriatric patients. These biomarkers may be useful as a diagnostic screen in the evaluation of patients for cognitive change consistent with mild cognitive impairment and Alzheimer's disease. This translational investigation was approved by the PCOM Institutional Review Board prior to patient recruitment and evaluation. In order to participate, identified patients had to be >65 years old and meet well-defined inclusion criteria. Once informed consent was obtained, these patients were evaluated for cognitive status using the SLUMS and FAST testing assessments. After psychometric testing, blood was drawn from the patients and the buffy coat was isolated and assessed by serology, immunofluorescence microscopy, and electron microscopy to identify biomarkers of infection. All methods revealed evidence for Chlamydia pneumoniae infection in a subgroup of our patient population. Current analysis of 16 patients by serology demonstrated 14/16 positive for at least one immunoglobulin class, IgM, IgA, or IgG. Analysis by immunofluorescence microscopy of peripheral blood revealed 13/16 patients positive for Chlamydia when labeled with a genus-specific anti-Chlamydial antibody. Of these 13 patients, 6 demonstrated a very strong positivity in monocyte populations. Limited infection was observed for the remaining positive 7 patients. For additional confirmatory studies to identify Chlamydia pneumoniae infection, a subpopulation of the 13 patient samples was prepared for electron microscopy. In these samples, elementary bodies, intra-vacuolar reticulate bodies, and intermediate bodies, characteristic of Chlamydia, were observed in leukocytes from the buffy coat. More specifically, we observed the classical “pear-shaped” elementary bodies characteristic of Chlamydia pneumoniae. Our data indicate that biomarkers for Chlamydia pneumoniae are present in blood samples from the geriatric patients who participated in this study. These data suggest that these biomarkers could be useful as a diagnostic screen for the geriatric population who may be at risk for developing cognitive decline.

Depressed medical students are more prone to exhibit several depression stigma attitudes than non-depressed students.1 Approximately 50% of medical students experience burnout at some point during medical school, and 10% experience suicide ideation compared to the estimated 6.9% of the general population in the same United States age group of 25-34 years old. While suicide is the extreme end of the personal distress continuum, medical schools have a responsibility to have a system in place that identifies students currently suicidal, students at risk for suicidal thoughts and implement student support and wellness programs that address events outside students’ control.2 It is worrisome that the hesitance towards seeking mental health care among medical students and ultimately health care providers might lead to poorer patient care. Jennifer Tija, an instructor in Internal Medicine at the University of Pennsylvania stated, “if people don’t know how to treat their own depression, it has a negative impact on how they treat patients.”3 In July 2011, the American Osteopathic Association House of Delegates approved Resolution 205 which aims to increase awareness of depression among medical students and knowledge of available treatment options. At Northwestern’s Feinberg School of Medicine, a Health Living Unit has been added to the second year curriculum. This is a direct response to the competency added in 2009 of , “Personal Awareness and Self-Care,” students must achieve by graduation. Feinberg’s approach requires all second-year medical students to complete a Behavior Change Plan (BCP) in which students identify one behavior such as exercise, nutrition, sleep, personal habits/hygiene, study/work habits or mental/emotional health habits, set a goal, track progress and self-assess their success.4 Dyrbye expands the responsibility of medical schools as he explained that interventions such as that initiated at Feinberg’s school of medicine, need to extend beyond teaching students self-care skills and also include establishing an appropriately structured culture and learning environment which endorses student health. “Achieving competency in self-care is the shared responsibility of the individual physician/resident/medical student and the organization’s environment in which he or she functions.” A key component in turning the tide in the culture of medicine is by addressing the “hidden curriculum” that is characterized as modeled cynicism by superiors in whom perpetuate the stigma to mental illness and convey the message that only the “weak” struggle or need help. One specific strategy suggested to medical schools is to create a curriculum that builds in personal time and promotes personal health.5 Emphasizing the need for a shift in culture, Sharon Bahrych, PA-C, MPH writes, “The culture of medicine is not geared towards allowing health care providers to de-stress, acquire emotional support, or discuss in an encouraging environment various conflictive work scenarios with their colleagues. The end result of this culture of medicine leads providers to either leave their chosen profession, have professional burnout, deal with work conflict and/or become emotionally broken (i.e. having a lack of integrity, honesty, emotional connectedness with others, etc.)”.6 At Duke University School of Medicine, an online forum was started to provide emotional support to students without revealing identities. This forum gave students a space to discuss personal issues and overcome the feelings of isolation associated with depression. The postings were reviewed by the school psychiatrist. The forum received more than 100 postings in a mere ten days.6 This study was modeled after UCSF medical school’s Mental Illness Among Us (MIAU) event held annually run by second year students for second year students. While data has not yet been released regarding the quantitative effects of the event, personal discussions with students and faculty have revealed an increased sense of community that deeply impacted the students and their perspective on medicine. The overwhelming data and the resolution approved by the AOA House point distinctly to immediate action taken within the medical education community. Our hope was that holding a student-run symposium would effectively address the issue by reducing feelings of isolation and stigma associated with mental illness while increasing the awareness about the prevalence of mental illness and the available resources provided by PCOM for those to seek help. Additionally, we strive for this symposium to be a step forward in changing the culture of medicine as we increase the amount of community within our class and create a united front against the stigma associated with mental illness amongst health care professionals and society as a whole. In this report, we describe PCOM’s activity and present the prevalence of self-reported mental health issues among the class of 2014, the associated stigma, and the effect on stigma the activity may have had. Abstract:The American Osteopathic Association House of Delegates Resolution 205 recommends “increased awareness of depression amongst U.S. Medical students” due to the increasing body of research describing the rise of depression, burn-out and suicide ideation among medical students. There is consequently a need to understand mental health issues as a component of professional development. Hypothesis: A student-led symposium addressing mental and emotional health topics relevant to medical students would reduce the stigma associated with mental illness. Materials and Methods: A 2-hour student-run “Patient Perspective” was held during the 2nd year neuroscience block at an osteopathic medical school in the northeastern United States. One week before the program, a student-developed, online Wellness Survey measured prevalence of mental illness, common feelings during medical school, coping mechanisms used for stress, and use of mental health resources. Immediately before and after the program, students were asked to report their familiarity with mental illness and their feelings regarding a vignette about a mentally ill woman using “Mental Illness Among Us” Pre and Post surveys provided by the University of California San Francisco School of Medicine and adapted for the event. During the program, data from the online survey were shared, student organizers discussed emotional wellness and positive coping mechanisms in the context of the profession, and student panelists shared their experiences with mental health issues. A faculty psychiatrist spoke about mental health resources, and attendees received pamphlets listing these resources. The event concluded with student-led breakout sessions at which stress during medical school and strategies for promoting positive coping mechanisms were discussed, followed by the Post survey. Results: 113 students completed the Pre survey, 89 of whom completed the Post survey. For these 89, differences between Post and Pre responses were universally in the direction of increasing acceptance and decreasing stigma of those with mental illness; all differences were statistically significant. The largest shift regarded students’ reluctance to disclose their own theoretical mental illness to colleagues. Conclusion: Incorporating an emotional health symposium into medical students’ training may increase understanding and acceptance of those who may have mental illness and reduce stigma associated with mental illness. Abstract

BACKGROUND First proposed by Khachaturian in 1994, the calcium hypothesis postulates that sustained disturbance of intracellular calcium is the leading cause of neurodegenerative disorders. Studies showing alteration in calcium signaling in both sporadic and familial Alzheimer’s disease (AD) support this hypothesis. Intracellular calcium signaling is tightly regulated in time, intensity, and space, and is responsible for a variety of neuronal functions. Calcium influx from the extracellular environment modulates calcium levels, as do intracellular stores in the endoplasmic reticulum. The focus of this study was to test various calcium related genes in both monocytes and neuronal cells. Previous studies have shown that cells infected with Chlamydia pneumoniae (Cpn) exhibit altered protein processing, such as amyloid and tau modification, consistent with those found in AD. We expect to see significant alterations in calcium genes, as well as their protein products in Cpn infected cells. Every calcium gene has a unique function in the cell. Determining which genes are up or down regulated following infection may provide insight into how the neurodegeneration process observed in AD is initiated by Cpn infections. METHODS Using the AR39 strain of Cpn at a MOI of one, both THP-1 monocytes and SKNMC (ATCC) neuronal cells were infected for 48 hours. Cells were analyzed using Real-time PCR microarrays (SABiosciences) for calcium related genes. Protein regulation was recorded using fast western blotting with three calcium protein antibodies. Monocytes and neuronal cells were also labeled with 61C75 directly conjugated antibody to FITC (Fitzgerald, Inc.) for verification of infection with Cpn. RESULTS Calcium genes with a two fold increase or decrease in Cpn-infected cells were noted. Twelve genes exhibited this behavior in both THP-1 and SKNMC cells. Of the twelve genes, S100A12 (S100 Calcium Binding Protein) had the greatest degree of up-regulation (approx. 35x) in THP-1 monocytes. NF-1 (Neurofibromin-1) had the highest degree of down-regulation in both THP-1 and SKNMC cells. CHGA (Chromogranin A) was up-regulated two fold in THP-1 cells and down-regulated two fold in SKNMC. In western blot protein analysis, S100A12 was upregulated in SKNMC cells and showed no protein product in THP-1 monocytes; NF-1 was upregulated in both THP-1 and SKNMC cells; and CHGA was down-regulated in both THP-1 and SKNMC cells. Western blot analysis was confirmed by immunofluorescence for NF-1 and S100A12 in SKMNC neuronal cells. CONCLUSIONS Our data suggest that Cpn alters calcium-related gene regulation and levels of protein products of at least three calcium genes, perhaps to maintain an environment beneficial to its survival. These changes may be associated with the disturbances in intracellular calcium previously observed in AD, and may elucidate how Cpn may affect normal cellular processes, thereby contributing to the neuropathology seen in AD.

Nearly 50% of all patients with lung cancer will need to receive radiation therapy (XRT) sometime during their course and some tumors will respond to XRT while others will not. Specific aims are to develop tumor mRNA and protein expression signatures predicting response to XRT before a patient is treated and to integrate this with signatures predicting response to chemotherapy (CTX) to ultimately facilitate selection of the best CTX regimen, XRT, and combined CTX + XRT for each individual patient ("personalized medicine"). We determined the XRT response phenotypes for a panel (up to 50) non-small cell lung cancers (NSCLCs) and small cell lung cancers (up to 30) (SCLCs) by performing radiation survival curves using clonogenic assays to determine the surviving fraction at 2 Gy (SF2). These values are integrated with our genome wide mRNA expression profiles (Affymetrix and Illumina arrays) on these same tumor lines with biostatistical approaches to develop mRNA expression signatures associated with sensitivity and resistance to radiation. Available SF2 values in NSCLCs are distributed over a range from 0.24 to 0.91. We also determined the repair kinetics of XRT induced DNA double strand breaks (DSBs) monitored by the disappearance of γH2AX and 53BP1 foci (determined by immunofluorescent staining with specific antibodies and scoring fluorescent foci). A striking correlation was found with increased radiosensitivity for NSCLC containing an EGFR oncogenic mutation and these tumor lines exhibited a deficiency in DNA DSB repair (see also Das Can Res 67:5267, 2007). Amundson et al., (Can Res, 68:415, 2008) recently reported a mRNA signature predictive of response to XRT in the NCI-60 panel of tumor cell lines. However, when we applied this signature to our mRNA array data we observed that their signature predicted that SCLC would be resistant to XRT which our previous studies (Carmichael Eur J Can Clin Oncol 25:527, 1989) and clinical experience disagree with their findings. NSCLCs display strikingly different radiation response phenotypes reflected in the corresponding SF2 values that will allow us to develop mRNA signatures predicting response to XRT; 2. mRNA signatures predictive of XRT response developed by other investigators in tumors other than lung cancer do not predict for lung cancer XRT response; 3. Radioresistant cell lines are more proficient in repairing the radiation-induced DSBs but radiosensitive cell lines show delayed repair kinetics and retain 30% of DSBs even 24 hr post irradiation.

BACKGROUND:Chlamydophila (Chlamydia) pneumoniae is an intracellular bacterium that has been identified within cells in areas of neuropathology found in Alzheimer disease (AD), including endothelia, glia, and neurons. Depending on the cell type of the host, infection by C. pneumoniae has been shown to influence apoptotic pathways in both pro- and anti-apoptotic fashions. We have hypothesized that persistent chlamydial infection of neurons may be an important mediator of the characteristic neuropathology observed in AD brains. Chronic and/or persistent infection of neuronal cells with C. pneumoniae in the AD brain may affect apoptosis in cells containing chlamydial inclusions.RESULTS:SK-N-MC neuroblastoma cells were infected with the respiratory strain of C. pneumoniae, AR39 at an MOI of 1. Following infection, the cells were either untreated or treated with staurosporine and then examined for apoptosis by labeling for nuclear fragmentation, caspase activity, and membrane inversion as indicated by annexin V staining. C. pneumoniae infection was maintained through 10 days post-infection. At 3 and 10 days post-infection, the infected cell cultures appeared to inhibit or were resistant to the apoptotic process when induced by staurosporine. This inhibition was demonstrated quantitatively by nuclear profile counts and caspase 3/7 activity measurements.CONCLUSION:These data suggest that C. pneumoniae can sustain a chronic infection in neuronal cells by interfering with apoptosis, which may contribute to chronic inflammation in the AD brain.

Studies have suggested that apoptosis may contribute to the neuronal cell loss observed in Alzheimer's disease (AD). Aβ 1–42 has been shown to induce apoptosis in neurons and may be an initiating factor in AD. Caspase is an effector in neuronal apoptosis that could also play a role in AD. However, the extent to which apoptosis contributes to cell death in AD has yet to be delineated. In an earlier study, we identified and isolated Chlamydia pneumoniae from brains of patients that had been diagnosed with sporadic AD. These in vitro studies suggested that neurons infected with C. pneumoniae are resistant to apoptosis, and that the processing or production of APP into Aβ1–42 was increased by the infection. In addition, we have developed a novel murine model in which non–transgenic mice infected with C. pneumoniae formed deposits of amyloid in areas of the brain most affected in Alzheimer's disease. Interestingly, some of the neurons in these areas showed a high level of Aβ 1–42 immunoreactivity, and these neurons did not appear to be undergoing apoptosis. The focus of the current studies was to delineate whether caspase is activated following a C. pneumoniae infection in neuronal cells. Apoptosis was experimentally induced by staurosporine in uninfected SK–N–MC cells and cells infected with C. pneumoniae. Caspase activity was analyzed using the Apo–ONE caspase 3/7 assay (Promega). We found that staurosporine induced an increase in caspase 3/7 activity in both infected and uninfected cells, however the staurosporine–treated infected cells had lower activity than even the basal activity of uninfected cells. These data were consistent with immunocytochemistry, which showed decreased labeling by antibodies that recognize cleaved (active) caspase 3 in the infected cells. These results suggest that inhibition of apoptosis by suppression of caspase 3/7 activity, and/or by decreasing levels of active caspase 3, may be mechanisms by which C. pneumoniae can sustain a persistent infection in the host and optimize its intracellular environment. In this way, C. pneumoniae may participate in the pathogenesis characteristic of Alzheimer's disease.

A genetically engineered cardiac TnC mutant labeled at Cys-84 with tetramethylrhodamine-5-iodoacetamide dihydroiodide was passively exchanged for the endogenous form in skinned guinea pig trabeculae. The extent of exchange averaged nearly 70%, quantified by protein microarray of individual trabeculae. The uniformity of its distribution was verified by confocal microscopy. Fluorescence polarization, giving probe angle and its dispersion relative to the fiber long axis, was monitored simultaneously with isometric tension. Probe angle reflects underlying cTnC orientation. In steady-state experiments, rigor cross-bridges and Ca2+ with vanadate to inhibit cross-bridge formation produce a similar change in probe orientation as that observed with cycling cross-bridges (no Vi). Changes in probe angle were found at [Ca2+] well below those required to generate tension. Cross-bridges increased the Ca2+ dependence of angle change (cooperativity). Strong cross-bridge formation enhanced Ca2+ sensitivity and was required for full change in probe position. At submaximal [Ca2+], the thin filament regulatory system may act in a coordinated fashion, with the probe orientation of Ca2+-bound cTnC significantly affected by Ca2+ binding at neighboring regulatory units. The time course of the probe angle change and tension after photolytic release [Ca2+] by laser photolysis of NP-EGTA was Ca2+ sensitive and biphasic: a rapid component approximately 10 times faster than that of tension and a slower rate similar to that of tension. The fast component likely represents steps closely associated with Ca2+ binding to site II of cTnC, whereas the slow component may arise from cross-bridge feedback. These results suggest that the thin filament activation rate does not limit the tension time course in cardiac muscle.

The kinetics of Ca2+-induced contractions of chemically skinned guinea pig trabeculae was studied using laser photolysis of NP-EGTA. The amount of free Ca2+ released was altered by varying the output from a frequency-doubled ruby laser focused on the trabeculae, while maintaining constant total [NP-EGTA] and [Ca2+]. The time courses of the rise in stiffness and tension were biexponential at 23°C, pH 7.1, and 200mM ionic strength. At full activation (pCa<5.0), the rates of the rapid phase of the stiffness and tension rise were 56±7s−1 (n=7) and 48±6s−1 (n=11) while the amplitudes were 21±2 and 23±3%, respectively. These rates had similar dependencies on final [Ca2+] achieved by photolysis: 43 and 50s−1 per pCa unit, respectively, over a range of [Ca2+] producing from 15% to 90% of maximal isometric tension. At all [Ca2+], the rise in stiffness initially was faster than that of tension. The maximal rates for the slower components of the rise in stiffness and tension were 4.1±0.8 and 6.2±1.0s−1. The rate of this slower phase exhibited significantly less Ca2+ sensitivity, 1 and 4s−1 per pCa unit for stiffness and tension, respectively. These data, along with previous studies indicating that the force-generating step in the cross-bridge cycle of cardiac muscle is marginally sensitive to [Ca2+], suggest a mechanism of regulation in which Ca2+ controls the attachment step in the cross-bridge cycle via a rapid equilibrium with the thin filament activation state. Myosin kinetics sets the time course for the rise in stiffness and force generation with the biexponential nature of the mechanical responses to steps in [Ca2+] arising from a shift to slower cross-bridge kinetics as the number of strongly bound cross-bridges increases.