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Context Practicing osteopathic manual medicine (OMM) requires medical students to learn a unique psychomotor skill. OMM techniques are taught during hands-on laboratory sessions in osteopathic medical schools across the country. Determining the optimal delivery of OMM training in the first and second years of medical school is instrumental in maximizing student engagement and confidence for future use in practice. In the academic year of 2020–2021, public health guidelines for COVID-19 forced Des Moines University College of Osteopathic Medicine to restructure their OMM laboratory sessions to include in-person and live-stream demonstrations of somatic dysfunction diagnoses and treatments. Objective(s) To determine if there was a difference in students’ perception of the learning experience and exam performance between in-person and live-streamed osteopathic manual medicine (OMM) laboratory instruction. Methods An online 8-question survey was sent to 225 first year medical students from Des Moines University at the end of their first year. The survey contained a combination of Likert scale, dichotomous, and open-ended questions. Statistical analysis for the Likert scale questions included paired-t test given the nature of correlated responses by the same cohort of students. A non-parametric permutation test was used to compare Fall 2020 practical exam grades due to the heavy skewness and ties of the exam score distributions. All computations were also made using the statistical computing software R. Free text was qualitatively analyzed for recurrent themes. Results The survey response rate was 67.1% with 151 respondents. When students were asked to rate their learning experience (engagement, comfort asking questions, understanding of material, ability to retain and recall lab material) between in-person vs. live-stream delivery of OMM lab material on a 5-point Likert Scale, there was a statistically significant mean difference for all of the responses, indicating a preference for in-person delivery method. Comparisons of the mean practical exam scores revealed no statistically significant differences. When asked to choose between in-person vs. live-stream, 83% of students reported a preference for in-person OMM laboratory demonstrations. Conclusion The students’ perceptions suggest that in-person delivery of OMM was superior to live-stream instruction based on higher rankings of engagement, comfort in asking questions, understanding material to practice, and recall of material in preparation of practical exams.

Pediatric high-grade gliomas (pHGGs) are common malignant brain tumors without effective treatment and poor patient survival. Abnormal posttranslational modification at the histone H3 tail plays critical roles in tumor cell malignancy. We have previously shown that the trimethylation of lysine 4 at histone H3 (H3K4me3) plays a significant role in pediatric ependymoma malignancy and is associated with tumor therapeutic sensitivity. Here, we show that H3K4me3 and its methyltransferase WDR82 are elevated in pHGGs. A reduction in H3K4me3 by downregulating WDR82 decreases H3K4me3 promoter occupancy and the expression of genes associated with stem cell features, cell proliferation, the cell cycle, and DNA damage repair. A reduction in WDR82-mediated H3K4me3 increases the response of pediatric glioma cells to chemotherapy. These findings suggest that WDR82-mediated H3K4me3 is an important determinant of pediatric glioma malignancy and therapeutic response. This highlights the need for a more thorough understanding of the potential of WDR82 as an epigenetic target to increase therapeutic efficacy and improve the prognosis for children with malignant gliomas.

T and B cells are the two arms of the adaptive immune system that mediate cellular and humoral immunity, respectively, using highly diverse repertoires of antigen receptors. T cells recognize antigens using the T cell receptor (TCR), whereas B cells use the B cell receptor (BCR or surface immunoglobulin). Complementary determining regions (CDR3) of TCRs and BCRs are randomly generated through somatic VDJ recombination and nucleotide deletions and insertions at the V-D and D-J junctions. Contrary to this paradigm, here we describe two networks of millions of TCRβ and IGH clonotypes that are made from only two CDR3 sequences and associated with more 63 diseases. The TCRβ network members bore either the prototypic signature CDR3 sequence (CASSPGTEAFF), its N-terminal VD motif (CASSPGT) recombined with various Jβ segments (CASSPGT-Jβx) or its DJβ motif recombined with various Vβ (Vβx-PGTEAFF). The BCR network members exhibit one signature CDR3 sequence (CARx1-4DTAMVYYFYDW) made from an invariant DJH motif (DTAMVYYFDYW) combined with various VH genes. The prototypes of the two networks are apparently teleogically related as they were dually expressed on the rare population of dual expresser (DE) lymphocytes and molecular dynamic simulations show that they were interacting partners. We conclude that members of the two networks represent a core set of evolutionary-conserved primordial antigen receptors that play fundamental roles in host defense and autoimmune diseases.

It is postulated that the complexity and heterogeneity in cancer may hinder most efforts that target a single pathway. Thus, discovery of novel therapeutic agents targeting multiple pathways, such as miRNAs, holds promise for future cancer therapy. One such miRNA, miR-489, is downregulated in a majority of breast cancer cells and several drug-resistant breast cancer cell lines, but its role and underlying mechanism for tumor suppression and drug resistance needs further investigation. The current study identifies autophagy as a novel pathway targeted by miR-489 and reports Unc-51 like autophagy activating kinase 1 (ULK1) and lysosomal protein transmembrane 4 beta (LAPTM4B) to be direct targets of miR-489. Furthermore, the data demonstrate autophagy inhibition and LAPTM4B downregulation as a major mechanism responsible for miR-489–mediated doxorubicin sensitization. Finally, miR-489 and LAPTM4B levels were inversely correlated in human tumor clinical specimens, and more importantly, miR-489 expression levels predict overall survival in patients with 8q22 amplification (the region in which LAPTM4B resides).Implications: These findings expand the understanding of miR-489–mediated tumor suppression and chemosensitization in and suggest a strategy for using miR-489 as a therapeutic sensitizer in a defined subgroup of resistant breast cancer patients. Mol Cancer Res; 16(9); 1348–60. ©2018 AACR.

Figure S1. miR-489 modulates autophagy. Figure S2. Western blot analysis of autophagy flux in breast cancers. Figure S3. Validation of microarray using real-time RT-PCR analysis. Figure S4. miR-489 restoration in HCC1954 and T47D cells under starvation. Figure S5. Endogenous expression level of miR-489 in different classes of breast cancer cell lines. Figure S6. Drug sensitivity assay with cisplatin and doxorubicin. Figure S7. ULK1 restoration rescues MDA-MB-231 cells from cytotoxic effect of miR-489. Figure S8. Characterization and validation of nanoparticles in vitro and in vivo. Figure S9. Correlation of miR-489 and ULK1 mRNA expression in breast cancers. Supplemental Experimental Procedures Supplementary Table 1: Primer list and sequences. Supplementary Table 2: Demographic and histopathology data of the patient samples.

Supplementary methods and References - Supplementary methods describing key reagents, plasmids, cell culture, CRISP/Cas9 system, HER2 agonist preparation, RT-PCR, western blot, flow-cytometry, tumorsphere culture, ChIP assay, animal experiments, Immunohistochemically staining and microarray analysis.

Supplementary Figures with legends- Supplementary Figures S1 to S14. S1. Shows gene ontology enrichment analysis between MCF10A-HER2 and MCF10A-vector cells. S2 shows western blot analysis of HER2 signaling after IL-1β treatment. S3, S4 and S9 show knockout of IL1A and IL6 using CRISPR/Cas9 system. S5 shows western blot analysis of HER2-signalign pathways in cells treated with IRAK1 and JAK2 inhibitors. S6 shows that activation of NF-κB and STAT3 is not mediated by HER2-immediate downstream molecules. S7 and S10 shows western blot analysis of total HER2, phosphorylated p65, STAT3 and AKT in mammary gland tissues and primary tumors. S8 shows the effects of blockades of HER2-downstream signaling pathways on cell viability. S11 shows representative images of Immunohistochemical staining of HER2, IL-1α and IL6 in primary breast cancer tissues. S12 shows Kaplan-Meier plots of distant metastasis-free survival of patients. S13 shows combination drug effects of IRAK inhibitor (IRAKi) plus paclitaxel (PCL) or cisplatin (CPT). S14 shows tumor volume growth curves for individual mice after last treatment.

Supplementary Table S2 shows the clinicopathological characteristics of clinical samples.

Systemic inflammation in breast cancer correlates with poor prognosis, but the molecular underpinnings of this connection are not well understood. In this study, we explored the relationship between HER2 overexpression, inflammation, and expansion of the mammary stem/progenitor and cancer stem–like cell (CSC) population in breast cancer. HER2-positive epithelial cells initiated and sustained an inflammatory milieu needed to promote tumorigenesis. HER2 induced a feedforward activation loop of IL1α and IL6 that stimulated NFκB and STAT3 pathways for generation and maintenance of breast CSC. In mice, Il1a genetic deficiency delayed MMTV-Her2–induced tumorigenesis and reduced inflammatory cytokine expression as well as CSC in primary tumors. In clinical specimens of human breast tumor tissues, tissue microarray analysis revealed a strong positive correlation between IL1α/IL6 expression and CSC-positive phenotype. Pharmacologic blockade of IL1α signaling reduced the CSC population and improved chemotherapeutic efficacy. Our findings suggest new therapeutic or prevention strategies for HER2-positive breast cancers.Significance: IL1α signaling driven by HER2 promotes chronic inflammation needed to support cancer stem-like cell maintenance in HER2-positive breast cancers. Cancer Res; 78(8); 2040–51. ©2018 AACR.

Supplementary Tables - Supplementary Tables S1, S3 and S4. S1 shows frequency of CSCs after transplantation of the indicated cells. S3 shows multivariate Cox regression data. S4 Positive correlation between IL-1α and HER2 or IL6 expression in breast cancer tissues.

Table S1. The clinicopathological characteristics of clinical samples. Table S2. Primer sequences used in the cloning and quantitative PCR analysis. Table S3. Primer sequences used for the CHIP assay.

Immune escape is an important reason why the immune system cannot control tumor growth, but how escape variants emerge during immunotherapy remains poorly understood. Here, we identify a new mechanism of tumor immune escape using an in vivo selection strategy. We generated a highly immune-resistant cancer cell line (P3) by subjecting a susceptible cancer cell line (P0/TC-1) to multiple rounds of in vivo immune selection. Microarray analysis of P0 and P3 revealed that vascular cell adhesion molecule-1 (VCAM-1) is up-regulated in the P3-resistant variant. Retroviral transfer of VCAM-1 into P0 significantly increased its resistance against a vaccine-induced immune response. Analysis of tumors showed a dramatic decrease in the number of tumor-infiltrating cluster of differentiation 8+ (CD8+) T cells in the tumors expressing VCAM-1. In vitro transwell migration assays showed that VCAM-1 can promote the migration of CD8+ T cells through its interaction with the α4β1 integrin. Site-directed mutagenesis of VCAM-1 at amino acid residues required for interaction with α4β1 integrin completely abolished the immune resistance conferred by VCAM-1 in vivo. Surface staining showed that most renal cell carcinomas (RCC) express VCAM-1, whereas an RCC that responded to vaccination was VCAM-1 negative. These data provide evidence that tumor expression of VCAM-1 represents a new mechanism of immune evasion and has important implications for the development of immunotherapy for human RCC. [Cancer Res 2007;67(4):1832–41]

Supplementary Figure 3 from Ectopic Expression of Vascular Cell Adhesion Molecule-1 as a New Mechanism for Tumor Immune Evasion

Supplementary Figure 2 from Ectopic Expression of Vascular Cell Adhesion Molecule-1 as a New Mechanism for Tumor Immune Evasion

Supplementary Tables 1-2 from IFN-Producing Killer Dendritic Cells Are Antigen-Presenting Cells Endowed with T-Cell Cross-Priming Capacity

Supplementary Figure 1 from Ectopic Expression of Vascular Cell Adhesion Molecule-1 as a New Mechanism for Tumor Immune Evasion

Supplementary Methods from IFN-Producing Killer Dendritic Cells Are Antigen-Presenting Cells Endowed with T-Cell Cross-Priming Capacity

Supplementary Figures 1-8 from IFN-Producing Killer Dendritic Cells Are Antigen-Presenting Cells Endowed with T-Cell Cross-Priming Capacity

Fig. S1. TGF-β/SMAD3 signaling mediates the promotion of migration and invasion by HOXB7. Fig. S2 Effects of HOXB7 overexpression and TGF-β2 knockdown on cellular morphology and proliferation rate. Fig. S3 Effects of HOXB7 overexpression on cellular morphology and proliferation rate of Her2 mouse mammary tumor cell line, H605

Context: Predoctoral Osteopathic Manual Medicine Fellowships (pOMMFs) are an additional year of medical training that frequently involve direct patient care, educating medical students, and research. Research has supported that a pOMMF can increase student satisfaction with, and understanding of, Osteopathic Manual Medicine (OMM) curriculum at their respective medical institution. In the interest of identifying programs that improve OMM utilization in practice and promote osteopathic leaders in medicine, pOMMFs might play a significant role. Overall, there is little known research on pOMMFs. Objective(s): To investigate the impact of the Des Moines University (DMU) pOMMF on the medical and professional careers of its graduates. Methods: A 26-question survey to be completed online was sent to 88 graduates of the DMU pOMMF who represented graduating classes from 1979 to 2020. The survey contained a combination of Likert scale, yes-no, and free text questions. Statistical analysis included descriptive statistics, Chi-square test of goodness-of-fit, and simultaneous 95% confidence intervals. Free text was qualitatively analyzed for recurrent themes. Results: Out of 61 respondents, 90.2% of participants reported that the pOMMF significantly improved their ability to teach medical students and residents, along with 77.1% of participants teaching residents/attendings OMM during residency. Of those eligible, 79.2% of alumni held at least one of the following leadership positions: chief resident, clinical preceptor, department chair, medical director. Most participants (83.6%) report providing OMT to their patients, along with 80.7% of respondents attributing the fellowship to significantly improving their ability to incorporate OMT into their medical practice. Conclusion: The study supports that the DMU pOMMF may create positive downstream effects in the medical careers of its graduates by providing abundant teaching opportunities, encouraging leadership roles, and promoting the utilization of OMT within residency and future practice. Graduates drew from the DMU pOMMF experiences to promote their medical and professional careers. Limitations include potential selection bias and inability to establish causal effect without a control population of non-pOMMF graduates from DMU or other institutions. Further research could corroborate these findings by investigating other pOMMFs and compare responses to non-pOMMF osteopathic medicine graduates.

Introduction: Osteopathic manipulative medicine (OMM) practical examinations (PEs) are important for assessing osteopathic medical students’ cognitive knowledge and psychomotor skills required for osteopathic manipulative treatment (OMT). In the response to the COVID-19 pandemic, first-year medical students at Des Moines University (DMU) were no longer allowed to participate in a standard in-person PE (sPE) in December 2020. A novel virtual PE (vPE) over Zoom was developed to assess the students’ understanding of OMM learned in lab to replace the sPE. Objective: To determine if the vPE was perceived by the students as a successful and efficacious alternative to the sPE. Method: After the graduating class of 2024 completed their vPE, an administrative email from the DMU OMM department was sent to gather anonymous, voluntary student feedback evaluating their perception and experience of the vPE. The survey consisted of 5 Likert scale questions that asked students to determine the extent they agree to 5 distinct statements and the survey contained a free-text response question asking for suggestions to improve the vPE experience. A Chi-square test of goodness-of-fit was used to assess for equal distributions of responses. Simultaneous 95% confidence intervals (CI) for multinomial proportions were created. The free text was qualitatively analyzed based on themes. Results: Of the 224 first-year osteopathic students, 207 students responded to the Likert scale questions and 113 responded to the free text. Students strongly agree or somewhat agree that the vPE was a fair assessment of their knowledge of OMM lab material (90.82%) and optimally assessed their procedural OMM skills (86.96%). Students strongly agree that the vPE better assessed their OMM lab knowledge than a written multiple-choice examination (73.91%) and they received helpful feedback during the vPE (77.78%). Conclusion: Survey results support that the vPE was perceived by students to be an effective and fair alternative to the sPE. The creative use of a pair of pants and a prop sacrum was validated, as students confirmed that the vPE optimally assessed their procedural skills given the practical’s limitations.

Contact-based education, offering meaningful contact with individuals living in recovery with mental illness, reduces stigma. This study evaluated the effectiveness of the National Alliance on Mental Illness Provider Education Program (NAMI PEP) when implemented as a curricular requirement across two cohorts of third-year osteopathic medical students, comparing traditional, passive learning and active, online delivery formats.Participants were two cohorts of third-year medical students (Cohort 1 n = 186; Cohort 2 n = 139; overall N = 325) who completed questionnaires measuring affect, beliefs, and behaviors toward patients with mental illness at pre-program, 1-week follow-up, and 6-month follow-up. For Cohort 1, the existing community-based NAMI PEP was implemented. For Cohort 2, the program was adapted to an online, active learning format tailored to medical students, and an additional 3-month follow-up assessment was added to better identify intermediate-term effects.The NAMI PEP was associated with longitudinal improvements in target outcomes, with enhanced effects with the adapted curriculum in Cohort 2. At 6-month follow-up, students reported less stigma, fewer stereotyping negative attitudes, and lower anxiety treating patients with mental illness. They also reported increased confidence integrating psychiatry into routine care and increased competence in principles of collaborative mental health treatment.This study demonstrates the longitudinal effectiveness of the NAMI PEP across two cohorts of medical students, with strengthened effects observed when the program is tailored to contemporary medical education.

We have recently identified a novel lymphocyte that is a dual expresser (DE) of TCRαβ and BCR. DEs in T1D patients are predominated by a public BCR clonotype (clone-x) that encodes a potent autoantigen that cross-activates insulin-reactive T cells. Betts and colleagues were able to detect DEs but alleged to not detect high DE frequency, clone-x, or similar clones in T1D patients. Unfortunately, the authors did not follow our methods and when they did, their flow cytometric data at two sites were conflicting. Moreover, contrary to their claim, we identified clones similar to clone-x in their data along with clones bearing the core motif (DTAMVYYFDYW). Additionally, their report of no increased usage of clone-x VH/DH genes by bulk B cells confirms rather than challenges our results. Finally, the authors failed to provide data verifying purity of their sorted DEs, making it difficult to draw reliable conclusion of their repertoire analysis. This Matters Arising Response paper addresses the Japp et al. (2021) Matters Arising paper, published concurrently in Cell.

Introduction: This study aims to investigate if proprioception is the mechanism through which osteopathic manipulative treatment (OMT) corrects somatic dysfunctions. OMT focuses on resolving structural asymmetry in the body that may interfere with physiologic function, including proprioception. Multiple theories have been proposed regarding the physiologic mechanisms of OMT and the effect on proprioception, with few studies objectively establishing a connection. Hypothesis: Significant change in measurements of proprioception was expected in the treatment group (OMT) over the 4-week period. Methods: For the study, 35 adults recruited from the Des Moines University community were randomized equally into treatment and control groups. For 3 weeks, the treatment group received weekly osteopathic structural exams (OSE) and OMT, while the control group underwent the same OSE but without treatment. Proprioception was assessed using force plate measurements collected during two-leg and single-leg tests prior to the first intervention, immediately after the first intervention, and 1 week after last intervention. Measurements included anterior-posterior sway, medial-lateral sway, average velocity, area, path length and time, which were analyzed with a linear model of mixed effects for repeated measures to account for the fixed effects (time and treatments), interaction effects and the random subject effects. Results: Over the 4-week period, significant differences were found for 4 measurements: two-leg eyes-open-path-length (p=0.0342), two-leg eyes-open-average-velocity (p=0.0334), single-right-leg eyes-open-average-velocity (p=0.0145), and single-right-leg eyes-closed-medial-lateral-sway (p=0.0488). Conclusion: Certain measurements of proprioception changed in an adult population over a 4-week course with 3 weekly OMT treatments. These results indicate OMT may be altering proprioception through the correction of somatic dysfunctions. Our results suggest a need for further research investigating the effects of OMT on proprioception in symptomatic populations and larger sample sizes.

Context Asthma is a common chronic obstructive lung disease with increasing prevalence and economic burden. The effect of osteopathic manipulative treatment (OMT) has been studied in patients with several lung diseases, including asthma; however, no clinical trials have studied effects beyond the immediate time period in adults with asthma using spirometry. Objective To examine the immediate, intermediate, and long-term effects of OMT on objective pulmonary function and subjective quality of life in asthmatic adults. Methods Twenty-five adults with asthma were recruited from the Des Moines University community. Standardized Asthma Quality of Life Questionnaire (AQLQ(S)) surveys and spirometry measures including forced expiratory volume in one second (FEV1), forced vital capacity (FVC), the FEV1/FVC ratio, and peak expiratory flow (PEF) were collected at baseline. Spirometry testing was performed immediately after and 3 days after each of 3 weekly standardized OMT sessions. Spirometry and AQLQ(S) surveys were collected again 4 weeks after the final OMT session. Spirometry results were analyzed using a repeated measure, linear mixed-effect model, and survey results were analyzed using paired t-tests. Results The study demonstrated statistically significant changes to the spirometry results, including the PEF and FEV1/FVC ratio. The PEF measurements increased 3 days after the first treatment and remained elevated through the completion of the study. The FEV1/FVC ratio decreased by 0.01 at 4 weeks post-OMT compared to baseline. There were no significant differences observed in the immediate, intermediate or long-term FEV1 and FVC measurements post-OMT. However, there was a significant increase in the overall score and all 4 domains of the AQLQ(S), including Symptoms, Activity Limitations, Emotional Function, and Environmental Stimuli. Conclusion The results of this pilot study suggest that OMT may improve the quality of life in adults with asthma. Spirometry testing revealed a significant change in some measures of pulmonary function and participants reported an improvement in asthma-specific quality of life. The authors suggest that, in combination with preventive measures and pharmacologic therapy, OMT may offer additional benefit in the treatment of adults with asthma. The results also suggest a need for further study of the effects of OMT on respiratory function in asthmatic adults.

Pediatric high-grade gliomas (pHGGs) are common malignant brain tumors without effective treatment and poor patient survival. Abnormal posttranslational modification at the histone H3 tail plays critical roles in tumor cell malignancy. We have previously shown that trimethylation of lysine 4 at histone H3 (H3K4me3) plays a significant role in pediatric ependymoma malignancy and is associated with tumor therapeutic sensitivity. Here, we show that H3K4me3 and its methyltransferase WDR82 are elevated in pHGGs. Reduction of H3K4me3 by downregulating WDR82 decreases H3K4me3 promoter occupancy and expression of genes associated with stem cell features, cell proliferation, the cell cycle and DNA damage repair. Reduction of WDR82 mediated H3K4me3 increases the response of pediatric glioma cells to chemotherapy. These findings suggest that WDR82 mediated H3K4me3 is an important determinant of pediatric glioma malignancy and therapeutic response. This highlights the need for a more thorough understanding of the potential of WDR82 as an epigenetic target to increase therapeutic efficacy and improve prognosis for children with malignant gliomas.

Rationale: Cell therapy for myocardial infarction is promising but largely unsuccessful in part due to a lack of mechanistic understanding. Techniques enabling identification of stem cell-specific proteomes in situ in the injured heart may shed light on how the administered cells respond to the injured microenvironment and exert reparative effects. Objective: To identify the proteomes of the transplanted mesenchymal stem cells (MSCs) in the infarcted myocardium, we sought to target a mutant methionyl-tRNA synthetase (MetRSL274G) in MSCs, which charges azidonorleucine (ANL), a methionine analogue and non-canonical amino acid, to tRNA and subsequently to nascent proteins, permitting isolation of ANL-labeled MSC proteomes from ischemic hearts by ANL-alkyne based click reaction. Methods and Results: Murine MSCs were transduced with lentivirus MetRSL274G and supplemented with ANL; the ANL-tagged nascent proteins were visualized by bio-orthogonal non-canonical amino-acid tagging, spanning all molecular weights and by fluorescent non-canonical amino-acid tagging, displaying strong fluorescent signal. Then, the MetRSL274G-transduced MSCs were administered to the infarcted or Sham heart in mice receiving ANL treatment. The MSC proteomes were isolated from the left ventricular protein lysates by click reaction at days 1, 3, and 7 after cell administration, identified by LC/MS. Among all identified proteins (in Sham and MI hearts, three time-points each), 648 were shared by all 6 groups, accounting for 82±5% of total proteins in each group, and enriched under mitochondrion, extracellular exosomes, oxidation-reduction process and poly(A) RNA binding. Notably, 26, 110 and 65 proteins were significantly up-regulated and 11, 28 and 19 proteins were down-regulated in the infarcted vs. Sham heart at the three time-points, respectively; these proteins are pronounced in the GO terms of extracellular matrix organization, response to stress and regulation of apoptotic process and in the KEGG pathways of complements and coagulation cascades, apoptosis, and regulators of actin cytoskeleton. Conclusions: MetRSL274G expression allows successful identification of MSC-specific nascent proteins in the infarcted hearts, which reflect the functional states, adaptive response, and reparative effects of MSCs that may be leveraged to improve cardiac repair.

Study objective To investigate the frequency of Beers Criteria (BC) medication and opioid use in patients age 65 years and older arriving in the Emergency Department. Methods We performed a retrospective observational study of a convenience sample of 400 patients, age 65 years and older, arriving to and discharged solely from the Emergency Department. We examined 400 sequential patient charts with visit dates April–July 2017, for the presence of a Beers Criteria medication or opioid prescription. We also examined each chart for nine specific chief complaints, including return visits and subsequent admissions. Results Of the 400 patients included in this study, 304 patients (76%; 95% confidence interval [CI] 72% to 80%) had at least 1 prescription at the index ED visit for an “avoid” or “use with caution” Beers Criteria medication. Of these patients, 194 (64%; 95% CI 58% to 69%) had ≥2 Beers medication prescriptions and 122 patients (40%; 95% CI 35% to 46%) had ≥3 Beers medication prescriptions. We found no difference in the number of patients with a chief complaint of interest between the BC medication list (28%) and lacking a BC medication (29%) (p-value = 1). No patients returned in the next 7 days for a medication-related complaint. Conclusion The results of this study call into question the routine application of lists without high-quality evidence to critique the prescribing of certain medications. Further patient-oriented study of the relevance of the Beers Criteria list, especially in light of the changed face of medication profiles and populations, is called for.

Gastroesophageal junction (GEJ) cancer remains a clinically significant disease in Western countries due to its increasing incidence, which mirrors that of esophageal cancer, and poor prognosis. To develop novel and effective approaches for prevention, early detection, and treatment of patients with GEJ cancer, a better understanding of the mechanisms driving pathogenesis and malignant progression of this disease is required. These efforts have been limited by the small number of available cell lines and appropriate pre-clinical animal models for in vitro and in vivo studies. We have established and characterized a novel GEJ cancer cell line, GEAMP, derived from the malignant pleural effusion of a previously treated GEJ cancer patient. Comprehensive genetic analyses confirmed a clonal relationship between GEAMP cells and the primary tumor. Targeted next-generation sequencing identified 56 non-synonymous alterations in 51 genes including TP53 and APC, which are commonly altered in GEJ cancer. In addition, multiple copy-number alterations were found including EGFR and K-RAS gene amplifications and loss of CDKN2A and CDKN2B. Histological examination of subcutaneous flank xenografts in nude and NOD-SCID mice showed a carcinoma with mixed squamous and glandular differentiation, suggesting GEAMP cells contain a sub-population with multi-potent potential. Finally, pharmacologic inhibition of the EGFR signaling pathway led to downregulation of key downstream kinases and inhibition of cell proliferation in vitro. Thus, GEAMP represents a valuable addition to the limited number of bona fide GEJ cancer cell lines.

BACKGROUND: Ependymomas (EPNs) are the third most common brain tumor in children. These tumors are resistant to available chemotherapeutic treatments, therefore new effective targeted therapeutics must be identified. Increasing evidence shows epigenetic alterations including histone posttranslational modifications (PTMs), are associated with malignancy, chemotherapeutic resistance and prognosis for pediatric EPNs. In this study we examined histone PTMs in EPNs and identified potential targets to improve chemotherapeutic efficacy. METHODS: Global histone H3 lysine 4 trimethylation (H3K4me3) levels were detected in pediatric EPN tumor samples with immunohistochemistry and immunoblots. Candidate genes conferring therapeutic resistance were profiled in pediatric EPN tumor samples with micro-array. Promoter H3K4me3 was examined for two candidate genes, CCND1 and ERBB2, with chromatin-immunoprecipitation coupled with real-time PCR (ChIP-PCR). These methods and MTS assay were used to verify a relationship between H3K4me3 levels and CCND1 and ERBB2, and to investigate cell viability in response to chemotherapeutic drugs in primary cultured pediatric EPN cells. RESULTS: H3K4me3 levels positively correlate with WHO grade malignancy in pediatric EPNs and are associated with progression free survival in patients with posterior fossa group A EPNs (PF-EPN-A). Reduction of H3K4me3 by silencing its methyltransferase SETD1A, in primary cultured EPN cells increased cell response to chemotherapy. CONCLUSIONS: Our results support the development of a novel treatment that targets H3K4me3 to increase chemotherapeutic efficacy in pediatric PF-EPN-A tumors.

Genetic deletion of Src associated in mitosis of 68kDa (Sam68), a pleiotropic adaptor protein prevents high-fat diet-induced weight gain and insulin resistance. To clarify the role of Sam68 in energy metabolism in the adult stage, we generated an inducible Sam68 knockout mice. Knockout of Sam68 was induced at the age of 7-10 weeks, and then we examined the metabolic profiles of the mice. Sam68 knockout mice gained less body weight over time and at 34 or 36 weeks old, had smaller fat mass without changes in food intake and absorption efficiency. Deletion of Sam68 in mice elevated thermogenesis, increased energy expenditure, and attenuated core-temperature drop during acute cold exposure. Furthermore, we examined younger Sam68 knockout mice at 11 weeks old before their body weights deviate, and confirmed increased energy expenditure and thermogenic gene program. Thus, Sam68 is essential for the control of adipose thermogenesis and energy homeostasis in the adult.

It is postulated that the complexity and heterogeneity in cancer may hinder most efforts that target a single pathway. Thus, discovery of novel therapeutic agents targeting multiple pathways, such as miRNAs, holds promise for future cancer therapy. One such miRNA, miR-489, is downregulated in a majority of breast cancer cells and several drug-resistant breast cancer cell lines, but its role and underlying mechanism for tumor suppression and drug resistance needs further investigation. The current study identifies autophagy as a novel pathway targeted by miR-489 and reports Unc-51 like autophagy activating kinase 1 (ULK1) and lysosomal protein transmembrane 4 beta (LAPTM4B) to be direct targets of miR-489. Furthermore, the data demonstrate autophagy inhibition and LAPTM4B downregulation as a major mechanism responsible for miR-489–mediated doxorubicin sensitization. Finally, miR-489 and LAPTM4B levels were inversely correlated in human tumor clinical specimens, and more importantly, miR-489 expression levels predict overall survival in patients with 8q22 amplification (the region in which LAPTM4B resides). Implications: These findings expand the understanding of miR-489–mediated tumor suppression and chemosensitization in and suggest a strategy for using miR-489 as a therapeutic sensitizer in a defined subgroup of resistant breast cancer patients. Mol Cancer Res; 16(9); 1348–60. ©2018 AACR.

Systemic inflammation in breast cancer correlates with poor prognosis, but the molecular underpinnings of this connection are not well understood. In this study, we explored the relationship between HER2 overexpression, inflammation, and expansion of the mammary stem/progenitor and cancer stem–like cell (CSC) population in breast cancer. HER2-positive epithelial cells initiated and sustained an inflammatory milieu needed to promote tumorigenesis. HER2 induced a feedforward activation loop of IL1α and IL6 that stimulated NFκB and STAT3 pathways for generation and maintenance of breast CSC. In mice, Il1a genetic deficiency delayed MMTV-Her2–induced tumorigenesis and reduced inflammatory cytokine expression as well as CSC in primary tumors. In clinical specimens of human breast tumor tissues, tissue microarray analysis revealed a strong positive correlation between IL1α/IL6 expression and CSC-positive phenotype. Pharmacologic blockade of IL1α signaling reduced the CSC population and improved chemotherapeutic efficacy. Our findings suggest new therapeutic or prevention strategies for HER2-positive breast cancers. Significance: IL1α signaling driven by HER2 promotes chronic inflammation needed to support cancer stem-like cell maintenance in HER2-positive breast cancers. Cancer Res; 78(8); 2040–51. ©2018 AACR.

Medical students are constantly under stress caused by strenuous medical programs, which may exert persistent physical and psychological effects on their well-being. Using medical students as a model population, this work explores the gut microbiome as a potential contributing mechanism for why individuals exposed to similar stimuli react variably. We evaluated the relationship of gut microbiome composition of first year medical students and stress resilience over a period of 4 months. Our objective was to identify gut microbiome characteristics of individuals that showed long-term stress resilience. Students were voluntarily recruited and screened for lifestyle and environmental factors at 3 timepoints during the first semester. Fecal samples were also collected at each timepoint. In order to identify candidates with stress resilience, their perceived stress and depression levels were normalized and summed to produce a psychologic index score. The most notable finding is a correlation between psychologic resiliency of Bacteriodete:Firmicute abundance as well as a relationship between durable resiliency and microbiome stability. Phylogenetic assembly of participants by microbiome relatedness found that 100% of subjects who were resilient to stress across all timepoints (n=8) were phylogenetically clustered in adjacent positions, showing a high degree of temporal stability. Of participants who were not durably resilient to stress, only 62% of participants (n=8) showed microbiomes that were phylogenetically related across the same 4 month period. We identified 2,102 Operational Taxonomic Units (OTUs) which were unique to the durable resilience group and 94 OTUs which were unique to the susceptible group. Of the 4,794 observed OTUs, 6.1% (n=294) were significantly different between groups. These findings support that the gut microbiome may play an important role in stress resilience at a time scale of 4 months. A better understanding of the role of the gut microbiome in stress resilience may shed light on potential treatment to reduce stress/anxiety in general, as well as to promote wellbeing of our future health care providers and physicians.

T and B cells are the two known lineages of adaptive immune cells. Here, we describe a previously unknown lymphocyte that is a dual expresser (DE) of TCR and BCR and key lineage markers of both B and T cells. In type 1 diabetes (T1D), DEs are predominated by one clonotype that encodes a potent CD4 T cell autoantigen in its antigen binding site. Molecular dynamics simulations revealed that this peptide has an optimal binding register for diabetogenic HLA-DQ8. In concordance, a synthetic version of the peptide forms stable DQ8 complexes and potently stimulates autoreactive CD4 T cells from T1D patients, but not healthy controls. Moreover, mAbs bearing this clonotype are autoreactive against CD4 T cells and inhibit insulin tetramer binding to CD4 T cells. Thus, compartmentalization of adaptive immune cells into T and B cells is not absolute, and violators of this paradigm are likely key drivers of autoimmune diseases.

PURPOSE: We have recently identified a previously unknown lymphocyte that is a dual expresser (DE) of productively rearranged and surface-expressed TCRαβ and BCR (surface immunoglobulin, Ig) (Ahmed et al, Cell, 2019: 177:11583). Importantly, a single immunoglobulin heavy-chain, IGHV clonotype (clone-x) predominates DEs that encodes a potent autoantigen (x-autoantigen) in its CDR3 region. The x-autoantigen (as a soluble intact x-mAb) cross-activate autoreactive T cells. The goal of this study is to investigate the properties of x-mAb reactive T cells and examining the mechanisms of how x-mAb recognizes and activates the tolerant autoreactive T cells in autoimmune diseases particularly in T1D. METHODS: We used EBV immortalized DE clone as a source of x-mAb and FACS-based protocols to identify x-mAb-responsive autoreactive T cells and their functional properties. ImmunoSEQ assay used to characterize TCR repertoires. RESULTS: Preliminary data show that x-mAb potentially binds and activates a subset of autoreactive T cells in T1D compared to HC subjects. Additionally, x-mAb-reactive T cells exhibits an activated and antigen experienced phenotype, including expression of CD45RO, CD44, and CD69. TCRVβ repertoire analysis shows that x-mAb reactive T cells are enriched for public clonally expanded TCRs in T1D patients. Further, x-mAb activates the autoreactive T cell through by crosslinking directly to their T cell receptor (TCR). CONCLUSIONS: DE cells in T1D patients secretes a public x-mAb that binds and activate specific subset of autoreactive T cells predominated by few clonotypes that express public TCRs. Our results are revealing previously unknown mechanism that appears to be a play critical role in pathogenesis of T1D.

Reactivation of latent human cytomegalovirus is a significant infectious complication of organ transplantation and current therapies target viral replication once reactivation of latent virus has already occurred. The specific molecular pathways that activate viral gene expression in response to transplantation are not well understood. Our studies aim to identify these factors, with the goal of developing novel therapies that prevent transcriptional reactivation in transplant recipients. Murine cytomegalovirus (MCMV) is a valuable model for studying latency and reactivation of CMV in vivo. We previously demonstrated that transplantation of MCMV-latently infected kidneys into allogeneic recipients induces reactivation of immediate early (IE) gene expression and epigenetic reprogramming of the major immediate early promoter (MIEP) within 48 h. We hypothesize that these events are mediated by activation of signalling pathways that lead to binding of transcription factors to the MIEP, including AP-1 and NF-κB. Here we show that transplantation induces rapid activation of several members of the AP-1 and NF-κB transcription factor family and we demonstrate that canonical NF-κB (p65/p50), the junD component of AP-1, and nucleosome remodelling complexes are recruited to the MIEP following transplantation. Proteomic analysis of recipient plasma and transcriptome analysis of kidney RNA identified five extracellular ligands, including TNF, IL-1β, IL-18, CD40L and IL-6, and three intracellular signalling pathways associated with reactivation of IE gene expression. Identification of the factors that mediate activation of these signalling pathways may eventually lead to new therapies to prevent reactivation of CMV and its sequelae.

While memory T-cells represent a hallmark of adaptive immunity, little is known about the genetic mechanisms regulating the longevity of memory CD4 T cells. Here, we studied the dynamics of gene expression in antigen specific CD4 T cells during infection, memory differentiation, and long-term survival up to nearly a year in mice. We observed that differentiation into long lived memory cells is associated with increased expression of genes inhibiting cell proliferation and apoptosis as well as genes promoting DNA repair response, lipid metabolism, and insulin resistance. We identified several transmembrane proteins in long-lived murine memory CD4 T cells, which co-localized exclusively within the responding antigen-specific memory CD4 T cells in human. The unique gene signatures of long-lived memory CD4 T cells, along with the new markers that we have defined, will enable a deeper understanding of memory CD4 T cell biology and allow for designing novel vaccines and therapeutics.

PURPOSE We have recently discovered a new lymphocyte that co-express BCR and TCR (Ahmed et al, Cell, 2019: 177:11583) and referred as X cell to denote its crossover phenotype. Importantly, X cells express a public BCR that also encodes a potent autoantigen in its CDR3 sequence that is 10 fold more potent than native insulin peptide (InsB:9–23) in binding to DQ8 and activating autologous CD4 T cells. The x-autoantigen cross-activate insulin specific CD4 T cells as a peptide in the context of HLA-DQ8 molecules or as a soluble intact mAb (x-mAb). The goal of this study is to characterize autoreactive CD4 T cells that are responsive to x-mAb to determine their phenotype, cytokine profile and TCR repertoire and whether they express public TCRs. METHODS We used EBV-lymphoblastoid X cell clone as a source of x-mAb (IgM) and FACS based protocol to identify IgM reactive CD4 T cells (referred as IgMpos) and their functional properties. ImmunoSEQ assay used to characterize TCR repertoires. RESULTS Preliminary data show that frequency of IgMpos CD4 T cells is significantly higher in T1D as compared to Healthy subjects. In addition, IgMpos CD4 T cells exhibit an activated phenotype as compared to autologous IgMneg CD4 T cells, including expression of CD45RO, CD44, and CD69. Analysis of TCRVβ repertoire shows that IgMpos CD4 T cells are enriched for public clonally expanded TCRs as compared to IgMneg counterparts. CONCLUSIONS X cells in T1D patients are predominated by a single public BCR and that the secreted version of this BCR (x-mAb) is autoreactive against a specific subset of CD4 T cells that predominated by few clonotypes that express public TCRs. Our results are revealing previously unknown mechanism that appears to be a play critical role in pathogenesis of T1D.

Objective To assess knowledge of the Zika virus (ZIKV), use of contraceptives, and sources of health information in rural communities in the Dominican Republic. Methods Over 4 days in March 2017, a research team traveled to four rural communities in the Dominican Republic to provide healthcare services. Overall, 90 men and women consented to a voluntary verbal 12-question survey. Results Of the participants, 55% were not certain whether ZIKV is transmitted sexually; 75% of participants were either not sure or thought ZIKV was not present in their community. Charlas (informal discussions led by community health workers) were cited as the most common source for public health information. Prevalence of contraceptive use was 26.6% hormonal and 1.1% long-acting reversible contraception (LARC); 30.0% cited no use of contraception. Conclusion Significant deficits in ZIKV knowledge, underutilization of LARCs, and socioeconomic factors exist that constrain the application of WHO recommendations for preventing ZIKV infection. Additional and more robust surveys are needed to assess public health education and interventions, critical for disease prevention in communities facing current and future epidemics.

Tractin is a novel member of the Ig-superfamily which has a highly unusual structure. It contains 6 Ig-domains, 4 FNIII-like domains, an acidic domain, 12 repeats of a novel prolineand glycine-rich motif with sequence similarity to collagen, a transmembrane domain, and an intracellular tail with an ankyrin and a PDZ-domain binding motif. By generating domain-specific antibodies we show that Tractin is proteolytically processed at two cleavage sites, one located in the third FTvTIII-domain, and a second located just proximal to the transmembrane domain. The alternative processing results in the formation of four fragments. The most NH2-terminal fragment, which is glycosylated with the Lan3-2, Lan42, and Laz2-369 glycoepitopes is secreted, and we propose a model in which the remaining fragments combine to form a secreted homodimer as well as a transmembrane heterodimer. The extracellular domain of the dimers is mostly made up of the collagen-like PG/YG-repeat *Department of Zoology and Genetics, Iowa State University, Ames, IA 50011 TDepartment of Biological Sciences, Western Michigan University, Kalamazoo, MI 49008 "•"Department of Physiology, Michigan State University, East Lansing, MI 48824

Ependymomas are the third most common form of brain tumors in children. These tumors are resistant to chemotherapy and despite genomic sequencing, there is a lack of effective molecular treatment targets. Efficient treatment targets need to be identified. Increasing evidence shows epigenetic alterations including posttranslational histone modifications (PTMs), associated with malignancy, chemotherapeutic resistance and prognosis in pediatric ependymomas. In this study we examined histone PTMs in ependymomas and identified potential targets to improve chemotherapeutic efficacy. Global levels of trimethylation at lysine 4 on histone H3 (H3K4me3), detected with immunohistochemistry and western blots, positively correlated with malignancy in pediatric primary ependymomas. Micro-array analysis of 22 pediatric ependymomoas identified upregulated candidate drug resistance genes. Promoter H3K4me3 occupancy was examined for two of these genes, CCND1 and ERBB2. Chromatin-immunoprecipitation with H3K4me3 coupled with real-time PCR, showed higher levels of H3K4me3 at these genes in grade III tumors, in comparison to grade II ones. When H3K4me3 levels were reduced in primary cultured ependymoma cells in vitro, cell response to chemotherapy increased. In summary, these results indicate that H3K4me3 levels are high at promoters of genes which confer chemotherapeutic resistance. Consequently, a novel treatment regimen which targets H3K4me3 in combination with traditional protocols may increase chemotherapeutic efficacy and improve prognosis for children who present with ependymoma.

Human epidermal growth factor receptor 2 (HER2 or ErBb2) is a receptor tyrosine kinase overexpressed in 20-30% of breast cancers and associated with poor prognosis and outcome. Dysregulation of several microRNAs (miRNAs) plays a key role in breast cancer progression and metastasis. In this study, we screened and identified miRNAs dysregualted in HER2-positive breast cancer cells. Our molecular study demonstrated that miR-489 was specifically downregulated by the HER2-downstream signaling, especially through the MAPK pathway. Restoration or overexpression of miR-489 in HER2-positive breast cancer cells significantly inhibited cell growth in vitro and decreased the tumorigenecity and tumor growth in xenograft mice. Mechanistically, we found that overexpression of miR-489 led to the decreased levels of HER2 and SHP2 and thus attenuated HER2-downstream signaling. Furthermore, we for the first time demonstrated that HER2 is a direct target of miR-489 and therefore HER2-SHP2-MAPK and miR-489 signaling pathways form a mutually inhibitory loop. Using quantitative real-time PCR analysis and Fluorescent in situ hybridization technique (FISH), we found that miR-489 was expressed at significantly lower level in tumor tissues compared to the adjacent normal tissues. Downregulation of miR-489 in breast cancers was associated with aggressive tumor phenotypes. Overall, our results define a double-negative feedback loop involving miR-489 and the HER2-SHP2-MAPK signaling axis that can regulate breast cancer cell proliferation and tumor progression and might have therapeutic relevance for HER2-positive breast cancer.

Invariant natural killer T (iNKT) cells recognize glycolipids as antigens and diversify into NKT1 (IFN-γ), NKT2 (IL-4), and NKT17 (IL-17) functional subsets while developing in the thymus. Mechanisms that govern the balance between these functional subsets are poorly understood due, partly, to the lack of distinguishing surface markers. Here we identify the heparan sulfate proteoglycan syndecan-1 (sdc1) as a specific marker of naive thymic NKT17 cells in mice and show that sdc1 deficiency significantly increases thymic NKT17 cells at the expense of NKT1 cells, leading to impaired iNKT cell-derived IFN-γ, both in vitro and in vivo. Using surface expression of sdc1 to identify NKT17 cells, we confirm differential tissue localization and interstrain variability of NKT17 cells, and reveal that NKT17 cells express high levels of TCR-β, preferentially use Vβ8, and are more highly sensitive to ɑ-GalCer than to CD3/CD28 stimulation. These findings provide a novel, noninvasive, simple method for identification, and viable sorting of naive NKT17 cells from unmanipulated mice, and suggest that sdc1 expression negatively regulates homeostasis in iNKT cells. In addition, these findings lay the groundwork for investigating the mechanisms by which sdc1 regulates NKT17 cells.

SummaryWe provide evidence that the Unc-51-like kinase 1 (ULK1) is activated during engagement of the type I interferon (IFN) receptor (IFNR). Our studies demonstrate that the function of ULK1 is required for gene transcription mediated via IFN-stimulated response elements (ISRE) and IFNγ activation site (GAS) elements and controls expression of key IFN-stimulated genes (ISGs). We identify ULK1 as an upstream regulator of p38α mitogen-activated protein kinase (MAPK) and establish that the regulatory effects of ULK1 on ISG expression are mediated possibly by engagement of the p38 MAPK pathway. Importantly, we demonstrate that ULK1 is essential for antiproliferative responses and type I IFN-induced antineoplastic effects against malignant erythroid precursors from patients with myeloproliferative neoplasms. Together, these data reveal a role for ULK1 as a key mediator of type I IFNR-generated signals that control gene transcription and induction of antineoplastic responses.