Pathology teaching in different undergraduate medical curricula within and outside the United States: a pilot study.

Pathology education is taught using different curricula in the United States (USA) and abroad. We evaluate and compare the hours spent in different forms of pathology teaching such as lectures, team-based learning (TBL), problem-based learning (PBL), and other methods taught in general and systemic pathology amongst different medical schools within the USA and outside the USA. The total number of lecture hours taught in general and systemic pathology combined was greater in outside schools than within the USA (141 h vs 97.8 h, respectively). Three subjects in general pathology and six subjects in systemic pathology had a significantly greater lecture hours in outside medical schools. The greatest difference was the hours spent in labs were longer for both general and systems pathology in schools outside the USA. The overall utilization of PBL in general and systemic pathology teaching combined was much greater outside the USA compared to within the USA (average overall hours PBL - 97.2 outside vs 16.5 in the USA), however, the reverse was observed for using TBL (average overall hours TBL - 59.5 outside vs 84.5 in USA). Average hours used with other methods of teaching was also greater in outside medical schools compared to USA medical schools (80.8 h vs 44 h, respectively). Pathology teaching in both general and systemic pathology has more extensive lecture hours, laboratory hours, PBL, and other methods of teaching pathology in outside medical schools with different curricula than USA medical schools. TBL is utilized more extensively in USA medical schools.

Loss of claudin-3 expression increases colitis risk by promoting Gut Dysbiosis.

Dysregulation of both the gut barrier and microbiota (dysbiosis) promotes susceptibility to and severity of Inflammatory Bowel Diseases (IBD). Leaky gut and dysbiosis often coexist; however, potential interdependence and molecular regulation are not well understood. Robust expression of claudin-3 (CLDN3) characterizes the gut epithelium, and studies have demonstrated a positive association between CLDN3 expression and gut barrier maturity and integrity, including in response to probiotics. However, the exact status and causal role of CLDN3 in IBD and regulation of gut dysbiosis remain unknown. Analysis of mouse and human IBD cohorts helped examine CLDN3 expression in IBD. The causal role was determined by modeling CLDN3 loss of expression during experimental colitis. 16S sequencing and in silico analysis helped examine gut microbiota diversity between Cldn3KO and WT mice and potential host metabolic responses. Fecal microbiota transplant (FMT) studies were performed to assess the role of gut dysbiosis in the increased susceptibility of Cldn3KO mice to colitis. A significant decrease in CLDN3 expression characterized IBD and CLDN3 loss of expression promoted colitis. 16S sequencing analysis suggested gut microbiota changes in Cldn3KO mice that were capable of modulating fatty acid metabolism and oxidative stress response. FMT from naïve Cldn3KO mice promoted colitis susceptibility in recipient germ-free mice (GFM) compared with GFM-receiving microbiota from WT mice. Our data demonstrate a critical role of CLDN3 in maintaining normal gut microbiota and inflammatory responses, which can be harnessed to develop novel therapeutic opportunities for patients with IBD.

Repeated Social Defeat Stress Induces an Inflammatory Gut Milieu by Altering the Mucosal Barrier Integrity and Gut Microbiota Homeostasis.

Background: Posttraumatic stress disorder (PTSD) is a mental health condition triggered by exposure to traumatic events in an individual's life. Patients with PTSD are also at a higher risk for comorbidities. However, it is not well understood how PTSD affects human health and/or promotes the risk for comorbidities. Nevertheless, patients with PTSD harbor a proinflammatory milieu and dysbiotic gut microbiota. Gut barrier integrity helps to maintain normal gut homeostasis and its dysregulation promotes gut dysbiosis and inflammation. Methods: We used a mouse model of repeated social defeat stress (RSDS), a preclinical model of PTSD. Behavioral studies, metagenomics analysis of the microbiome, gut permeability assay (on mouse colon, using an Ussing chamber), immunoblotting, and immunohistochemical analyses were performed. Polarized intestinal epithelial cells and 3-dimensional crypt cultures were used for mechanistic analysis. Results: The RSDS mice harbor a heightened proinflammatory gut environment and microbiota dysbiosis. The RSDS mice further showed significant dysregulation of gut barrier functions, including transepithelial electrical resistance, mucin homeostasis, and antimicrobial responses. RSDS mice also showed a specific increase in intestinal expression of claudin-2, a tight junction protein, and epinephrine, a stress-induced neurotransmitter. Treating intestinal epithelial cells or 3-dimensional cultured crypts with norepinephrine or intestinal luminal contents (fecal contents) upregulated claudin-2 expression and inhibited transepithelial electrical resistance. Conclusions: Traumatic stress induces dysregulation of gut barrier functions, which may underlie the observed gut microbiota changes and proinflammatory gut milieu, all of which may have an interdependent effect on the health and increased risk of comorbidities in patients with PTSD.

Claudin-2 protects against colitis-associated cancer by promoting colitis-associated mucosal healing.

Patients with inflammatory bowel disease (IBD) are susceptible to colitis-associated cancer (CAC). Chronic inflammation promotes the risk for CAC. In contrast, mucosal healing predicts improved prognosis in IBD and reduced risk of CAC. However, the molecular integration among colitis, mucosal healing, and CAC remains poorly understood. Claudin-2 (CLDN2) expression is upregulated in IBD; however, its role in CAC is not known. The current study was undertaken to examine the role for CLDN2 in CAC. The AOM/DSS-induced CAC model was used with WT and CLDN2-modified mice. High-throughput expression analyses, murine models of colitis/recovery, chronic colitis, ex vivo crypt culture, and pharmacological manipulations were employed in order to increase our mechanistic understanding. The Cldn2KO mice showed significant inhibition of CAC despite severe colitis compared with WT littermates. Cldn2 loss also resulted in impaired recovery from colitis and increased injury when mice were subjected to intestinal injury by other methods. Mechanistic studies demonstrated a possibly novel role of CLDN2 in promotion of mucosal healing downstream of EGFR signaling and by regulation of Survivin expression. An upregulated CLDN2 expression protected from CAC and associated positively with crypt regeneration and Survivin expression in patients with IBD. We demonstrate a potentially novel role of CLDN2 in promotion of mucosal healing in patients with IBD and thus regulation of vulnerability to colitis severity and CAC, which can be exploited for improved clinical management.

Combination Therapy with Gallium Protoporphyrin and Gallium Nitrate Exhibits Enhanced Antimicrobial Activity In Vitro and In Vivo against Methicillin-Resistant Staphylococcus aureus.

There is a major need for the development of new therapeutics to combat antibiotic-resistant Staphylococcus aureus. Recently, gallium (Ga)-based complexes have shown promising antimicrobial effects against various bacteria, including multidrug-resistant organisms, by targeting multiple heme/iron-dependent metabolic pathways. Among these, Ga protoporphyrin (GaPP) inhibits bacterial growth by targeting heme pathways, including aerobic respiration. Ga(NO3)3, an iron mimetic, disrupts elemental iron pathways. Here, we demonstrate the enhanced antimicrobial activity of the combination of GaPP and Ga(NO3)3 against methicillin-resistant S. aureus (MRSA) under iron-limited conditions, including small colony variants (SCV). This therapy demonstrated significant antimicrobial activity without inducing slow-growing SCV. We also observed that the combination of GaPP and Ga(NO3)3 inhibited the MRSA catalase but not above that seen with Ga(NO3)3 alone. Neither GaPP nor Ga(NO3)3 alone or their combination inhibited the dominant superoxide dismutase expressed (SodA) under the iron-limited conditions examined. Intranasal administration of the combination of the two compounds improved drug biodistribution in the lungs compared to intraperitoneal administration. In a murine MRSA lung infection model, we observed a significant increase in survival and decrease in MRSA lung CFUs in mice that received combination therapy with intranasal GaPP and Ga(NO3)3 compared to untreated control or mice receiving GaPP or Ga(NO3)3 alone. No drug-related toxicity was observed as assessed histologically in the spleen, lung, nasal cavity, and kidney for both single and repeated doses of 10 mg Ga /Kg of mice over 13 days. Our results strongly suggest that GaPP and Ga(NO3)3 in combination have excellent synergism and potential to be developed as a novel therapy for infections with S. aureus.

P62/SQSTM1 binds with claudin-2 to target for selective autophagy in stressed intestinal epithelium.

Impaired autophagy promotes Inflammatory Bowel Disease (IBD). Claudin-2 is upregulated in IBD however its role in the pathobiology remains uncertain due to its complex regulation, including by autophagy. Irrespective, claudin-2 expression protects mice from DSS colitis. This study was undertaken to examine if an interplay between autophagy and claudin-2 protects from colitis and associated epithelial injury. Crypt culture and intestinal epithelial cells (IECs) are subjected to stress, including starvation or DSS, the chemical that induces colitis in-vivo. Autophagy flux, cell survival, co-immunoprecipitation, proximity ligation assay, and gene mutational studies are performed. These studies reveal that under colitis/stress conditions, claudin-2 undergoes polyubiquitination and P62/SQSTM1-assisted degradation through autophagy. Inhibiting autophagy-mediated claudin-2 degradation promotes cell death and thus suggest that claudin-2 degradation promotes autophagy flux to promote cell survival. Overall, these data inform for the previously undescribed role for claudin-2 in facilitating IECs survival under stress conditions, which can be harnessed for therapeutic advantages.

Anti-miR-96 and Hh pathway inhibitor MDB5 synergistically ameliorate alcohol-associated liver injury in mice.

Alcohol-associated liver disease (ALD) and its complications are significant health problems worldwide. Several pathways in ALD are influenced by alcohol that drives inflammation, fatty acid metabolism, and fibrosis. Although miR-96 has become a key regulator in several liver diseases, its function in ALD remains unclear. In contrast, sonic hedgehog (SHH) signaling has a well-defined role in liver disease through influencing the activation of hepatic stellate cells (HSCs) and the inducement of liver fibrosis. In this study, we investigated the expression patterns of miR-96 and Hh molecules in mouse and human liver samples. We showed that miR-96 and Shh were upregulated in ethanol-fed mice. Furthermore, alcoholic hepatitis (AH) patient specimens also showed upregulated FOXO3a, TGF-ß1, SHH, and GLI2 proteins. We then examined the effects of Hh inhibitor MDB5 and anti-miR-96 on inflammatory and extracellular matrix (ECM)-related genes. We identified FOXO3 and SMAD7 as direct target genes of miR-96. Inhibition of miR-96 decreased the expression of these genes in vitro in AML12 cells, HSC-T6 cells, and in vivo in ALD mice. Furthermore, MDB5 decreased HSCs activation and the expression of ECM-related genes, such as Gli1, Tgf-ß1, and collagen. Lipid nanoparticles (LNPs) loaded with the combination of MDB5, and anti-miR-96 ameliorated ALD in mice. Our study demonstrated that this combination therapy could serve as a new therapeutic target for ALD.

Elevated PAF1-RAD52 axis confers chemoresistance to human cancers.

Cisplatin- and gemcitabine-based chemotherapeutics represent a mainstay of cancer therapy for most solid tumors; however, resistance limits their curative potential. Here, we identify RNA polymerase II-associated factor 1 (PAF1) as a common driver of cisplatin and gemcitabine resistance in human cancers (ovarian, lung, and pancreas). Mechanistically, cisplatin- and gemcitabine-resistant cells show enhanced DNA repair, which is inhibited by PAF1 silencing. We demonstrate an increased interaction of PAF1 with RAD52 in resistant cells. Targeting the PAF1 and RAD52 axis combined with cisplatin or gemcitabine strongly diminishes the survival potential of resistant cells. Overall, this study shows clinical evidence that the expression of PAF1 contributes to chemotherapy resistance and worse clinical outcome for lethal cancers.

MicroRNA-1 attenuates the growth and metastasis of small cell lung cancer through CXCR4/FOXM1/RRM2 axis.

BACKGROUND: Small cell lung cancer (SCLC) is an aggressive lung cancer subtype that is associated with high recurrence and poor prognosis. Due to lack of potential drug targets, SCLC patients have few therapeutic options. MicroRNAs (miRNAs) provide an interesting repertoire of therapeutic molecules; however, the identification of miRNAs regulating SCLC growth and metastasis and their precise regulatory mechanisms are not well understood. METHODS: To identify novel miRNAs regulating SCLC, we performed miRNA-sequencing from donor/patient serum samples and analyzed the bulk RNA-sequencing data from the tumors of SCLC patients. Further, we developed a nanotechnology-based, highly sensitive method to detect microRNA-1 (miR-1, identified miRNA) in patient serum samples and SCLC cell lines. To assess the therapeutic potential of miR-1, we developed various in vitro models, including miR-1 sponge (miR-1Zip) and DOX-On-miR-1 (Tet-ON) inducible stable overexpression systems. Mouse models derived from intracardiac injection of SCLC cells (miR-1Zip and DOX-On-miR-1) were established to delineate the role of miR-1 in SCLC metastasis. In situ hybridization and immunohistochemistry were used to analyze the expression of miR-1 and target proteins (mouse and human tumor specimens), respectively. Dual-luciferase assay was used to validate the target of miR-1, and chromatin immunoprecipitation assay was used to investigate the protein-gene interactions. RESULTS: A consistent downregulation of miR-1 was observed in tumor tissues and serum samples of SCLC patients compared to their matched normal controls, and these results were recapitulated in SCLC cell lines. Gain of function studies of miR-1 in SCLC cell lines showed decreased cell growth and oncogenic signaling, whereas loss of function studies of miR-1 rescued this effect. Intracardiac injection of gain of function of miR-1 SCLC cell lines in the mouse models showed a decrease in distant organ metastasis, whereas loss of function of miR-1 potentiated growth and metastasis. Mechanistic studies revealed that CXCR4 is a direct target of miR-1 in SCLC. Using unbiased transcriptomic analysis, we identified CXCR4/FOXM1/RRM2 as a unique axis that regulates SCLC growth and metastasis. Our results further showed that FOXM1 directly binds to the RRM2 promoter and regulates its activity in SCLC. CONCLUSIONS: Our findings revealed that miR-1 is a critical regulator for decreasing SCLC growth and metastasis. It targets the CXCR4/FOXM1/RRM2 axis and has a high potential for the development of novel SCLC therapies. MicroRNA-1 (miR-1) downregulation in the tumor tissues and serum samples of SCLC patients is an important hallmark of tumor growth and metastasis. The introduction of miR-1 in SCLC cell lines decreases cell growth and metastasis. Mechanistically, miR-1 directly targets CXCR4, which further prevents FOXM1 binding to the RRM2 promoter and decreases SCLC growth and metastasis.

Muc16 depletion diminishes KRAS-induced tumorigenesis and metastasis by altering tumor microenvironment factors in pancreatic ductal adenocarcinoma.

MUC16, membrane-bound mucin, plays an oncogenic role in pancreatic ductal adenocarcinoma (PDAC). However, the pathological role of MUC16 in the PDAC progression, tumor microenvironment, and metastasis in cooperation with KrasG12D and Trp53R172H mutations remains unknown. Deletion of Muc16 with activating mutations KrasG12D/+ and Trp53R172H/+ in mice significantly decreased progression and prolonged overall survival in KrasG12D/+; Trp53R172H/+; Pdx-1-Cre; Muc16-/- (KPCM) and KrasG12D/+; Pdx-1-Cre; Muc16-/- (KCM), as compared to KrasG12D/+; Trp53R172H/+; Pdx-1-Cre (KPC) and KrasG12D/+; Pdx-1-Cre (KC) mice, respectively. Muc16 knockout pancreatic tumor (KPCM) displays decreased tumor microenvironment factors and significantly reduced incidence of liver and lung metastasis compared to KPC. Furthermore, in silico data analysis showed a positive correlation of MUC16 with activated stroma and metastasis-associated genes. KPCM mouse syngeneic cells had significantly lower metastatic and endothelial cell binding abilities than KPC cells. Similarly, KPCM organoids significantly decreased the growth rate compared to KPC organoids. Interestingly, RNA-seq data revealed that the cytoskeletal proteins Actg2, Myh11, and Pdlim3 were downregulated in KPCM tumors. Further knockdown of these genes showed reduced metastatic potential. Overall, our results demonstrate that Muc16 alters the tumor microenvironment factors during pancreatic cancer progression and metastasis by changing the expression of Actg2, Myh11, and Pdlim3 genes.

Combining Gallium Protoporphyrin and Gallium Nitrate Enhances In Vitro and In Vivo Efficacy against Pseudomonas aeruginosa: Role of Inhibition of Bacterial Antioxidant Enzymes and Resultant Increase in Cytotoxic Reactive Oxygen Species.

Pseudomonas aeruginosa is a highly antibiotic-resistant opportunistic pathogenic bacteria that is responsible for thousands of deaths each year. Infections with P. aeruginosa disproportionately impact individuals with compromised immune systems as well as cystic fibrosis patients, where P. aeruginosa lung infection is a leading cause of morbidity and mortality. In previous work, we showed that a combination of gallium (Ga) nitrate and Ga protoporphyrin worked well in several bacterial infection models but its mechanism of action (MOA) is unknown. In the current work, we have investigated the MOA of Ga combination therapy in P. aeruginosa and its analysis in the in vivo model. In P. aeruginosa treated with Ga combination therapy, we saw a decrease in catalase and superoxide dismutase (SOD) activity, key antioxidant enzymes, which could correlate with a higher potential for oxidative stress. Consistent with this hypothesis, we found that, following combination therapy, P. aeruginosa demonstrated higher levels of reactive oxygen species, as measured using the redox-sensitive fluorescent probe, H2DCFDA. We also saw that the Ga combination therapy killed phagocytosed bacteria inside macrophages in vitro. The therapy with low dose was able to fully prevent mortality in a murine model of P. aeruginosa lung infection and also significantly reduced lung damage. These results support our previous data that Ga combination therapy acts synergistically to kill P. aeruginosa, and we now show that this may occur through increasing the organism's susceptibility to oxidative stress. Ga combination therapy also showed itself to be effective at treating infection in a murine pulmonary-infection model.

Upregulation of Nox4 induces a pro-survival Nrf2 response in cancer-associated fibroblasts that promotes tumorigenesis and metastasis, in part via Birc5 induction.

BACKGROUND: A pro-oxidant enzyme, NADPH oxidase 4 (Nox4) has been reported to be a critical downstream effector of TGFß-induced myofibroblast transformation during fibrosis. While there are a small number of studies suggesting an oncogenic role of Nox4 derived from activated fibroblasts, direct evidence linking this pro-oxidant to the tumor-supporting CAF phenotype and the mechanisms involved are lacking, particularly in breast cancer. METHODS: We targeted Nox4 in breast patient-derived CAFs via siRNA-mediated knockdown or administration of a pharmaceutical inhibitor (GKT137831). We also determine primary tumor growth and metastasis of implanted tumor cells using a stable Nox4-/- syngeneic mouse model. Autophagic flux of CAFs was assessed using a tandem fluorescent-tagged ptfl-LC3 plasmid via confocal microscopy analysis and determination of the expression level of autophagy markers (beclin-1 and LC3B). Nox4 overexpressing CAFs depend on the Nrf2 (nuclear factor-erythroid factor 2-related factor 2) pathway for survival. We then determined the dependency of Nox4-overexpressing CAFs on the Nrf2-mediated adaptive stress response pathway for survival. Furthermore, we investigated the involvement of Birc5 on CAF phenotype (viability and collagen contraction activity) as well as the expression level of CAF markers, FAP and αSMA. CONCLUSIONS: We found that deletion of stroma Nox4 and pharmaceutically targeting its activity with GKT137831 significantly inhibited orthotopic tumor growth and metastasis of implanted E0771 and 4T1 murine mammary carcinoma cell lines in mice. More importantly, we found a significant upregulation of Nox4 expression in CAFs isolated from human breast tumors versus normal mammary fibroblasts (RMFs). Our in situ RNA hybridization analysis for Nox4 transcription on a human breast tumor microarray further support a role of this pro-oxidant in the stroma of breast carcinomas. In addition, we found that Nox4 promotes autophagy in CAFs. Moreover, we found that Nox4 promoted survival of CAFs via activation of Nrf2, a master regulator of oxidative stress response. We have further shown Birc5 is involved as a downstream modulator of Nrf2-mediated pro-survival phenotype. Together these studies indicate a role of redox signaling via the Nox4-Nrf2 pathway in tumorigenesis and metastasis of breast cancer cells by promoting autophagy and survival of CAFs.

Design and Evaluation of a Novel Health Security, Infectious Diseases, Health Systems Science, and Service Learning Course During the COVID-19 Pandemic.

During the COVID-19 pandemic, academic health centers suspended clinical clerkships for students. A need emerged for innovative virtual curricula to continue fostering professional competencies. In March 2020, a multidisciplinary team from the University of Nebraska Medical Center had 2 weeks to create a course on the impact of infectious diseases that addressed the COVID-19 pandemic in real time for upper-level medical and physician assistant students. Content addressing social determinants of health, medical ethics, population health, service learning, health security, and emergency preparedness were interwoven throughout the course to emphasize critical roles during a pandemic. In total, 320 students were invited to complete the survey on knowledge gained and attitudes about the course objectives and materials and 139 responded (response rate 43%). Students documented over 8,000 total hours of service learning; many created nonprofit organizations, aligned their initiatives with health systems efforts, and partnered with community-based organizations. Thematic analysis of qualitative evaluations revealed that learners found the greatest value in the emphasis on social determinants of health, bioethics, and service learning. The use of predeveloped, asynchronous e-modules were widely noted as the least effective aspect of the course. The COVID-19 pandemic introduced substantial challenges in medical education but also provided trainees with an unprecedented opportunity to learn from real-world emergency preparedness and public health responses. The University of Nebraska Medical Center plans to create a health security elective that includes traditional competencies for emergency preparedness and interrogates the social and structural vulnerabilities that drive disproportionately worse outcomes among marginalized communities. With further evaluation, many components of the curriculum could be broadly scaled to meet the increasing need for more public health and health security medical education.

Myristic Acid Supplementation Aggravates High Fat Diet-Induced Adipose Inflammation and Systemic Insulin Resistance in Mice.

Saturated fatty acids (SFAs) are considered to be detrimental to human health. One of the SFAs, myristic acid (MA), is known to exert a hypercholesterolemic effect in mice as well as humans. However, its effects on altering adipose tissue (AT) inflammation and systemic insulin resistance (IR) in obesity are still unclear. Here, we sought to determine the effects of a high fat (HF) diet supplemented with MA on obesity-associated metabolic disorders in mice. Wild-type C57BL/6 mice were fed a HF diet in the presence or absence of 3% MA for 12 weeks. Plasma lipids, plasma adipokines, AT inflammation, systemic IR, glucose homeostasis, and hepatic steatosis were assessed. The body weight and visceral adipose tissue (VAT) mass were significantly higher in mice receiving the HF+MA diet compared to HF diet-fed controls. Plasma total cholesterol levels were marginally increased in HF+MA-fed mice compared to controls. Fasting blood glucose was comparable between HF and HF+MA-fed mice. Interestingly, the plasma insulin and HOMA-IR index, a measure of insulin resistance, were significantly higher in HF+MA-fed mice compared to HF controls. Macrophage and inflammatory markers were significantly elevated in the AT and AT-derived stromal vascular cells upon MA feeding. Moreover, the level of circulating resistin, an adipokine promoting insulin resistance, was significantly higher in HF+MA-fed mice compared with HF controls. The insulin tolerance test revealed that the IR was higher in mice receiving the MA supplementation compared to HF controls. Moreover, the glucose tolerance test showed impairment in systemic glucose homeostasis in MA-fed mice. Analyses of liver samples showed a trend towards an increase in liver TG upon MA feeding. However, markers of oxidative stress and inflammation were reduced in the liver of mice fed an MA diet compared to controls. Taken together, our data suggest that chronic administration of MA in diet exacerbates obesity-associated insulin resistance and this effect is mediated in part, via increased AT inflammation and increased secretion of resistin.

Preclinical Evaluation of a Humanized, Near-Infrared Fluorescent Antibody for Fluorescence-Guided Surgery of MUC16-Expressing Pancreatic Cancer.

Surgery remains the only potentially curative treatment option for pancreatic cancer, but resections are made more difficult by infiltrative disease, proximity of critical vasculature, peritumoral inflammation, and dense stroma. Surgeons are limited to tactile and visual cues to differentiate cancerous tissue from normal tissue. Furthermore, translating preoperative images to the intraoperative setting poses additional challenges for tumor detection, and can result in undetected and unresected lesions. Thus, pancreatic ductal adenocarcinoma (PDAC) has high rates of incomplete resections, and subsequently, disease recurrence. Fluorescence-guided surgery (FGS) has emerged as a method to improve intraoperative detection of cancer and ultimately improve surgical outcomes. Initial clinical trials have demonstrated feasibility of FGS for PDAC, but there are limited targeted probes under investigation for this disease, highlighting the need for development of additional novel biomarkers to reflect the PDAC heterogeneity. MUCIN16 (MUC16) is a glycoprotein that is overexpressed in 60-80% of PDAC. In our previous work, we developed a MUC16-targeted murine antibody near-infrared conjugate, termed AR9.6-IRDye800, that showed efficacy in detecting pancreatic cancer. To build on the translational potential of this imaging probe, a humanized variant of the AR9.6 fluorescent conjugate was developed and investigated herein. This conjugate, termed huAR9.6-IRDye800, showed equivalent binding properties to its murine counterpart. Using an optimized dye:protein ratio of 1:1, in vivo studies demonstrated high tumor to background ratios in MUC16-expressing tumor models, and delineation of tumors in a patient-derived xenograft model. Safety, biodistribution, and toxicity studies were conducted. These studies demonstrated that huAR9.6-IRDye800 was safe, did not yield evidence of histological toxicity, and was well tolerated in vivo. The results from this work suggest that AR9.6-IRDye800 is an efficacious and safe imaging agent for identifying pancreatic cancer intraoperatively through fluorescence-guided surgery.

Identification of Immunoglobulin G Autoantibody Against Malondialdehyde-Acetaldehyde Adducts as a Novel Serological Biomarker for Ulcerative Colitis.

INTRODUCTION: Inflammatory bowel disease (IBD) is associated with immune responses with oxidative stress wherein high levels of malondialdehyde result in the formation of a highly stable and immunogenic malondialdehyde-acetaldehyde adduct (MAA). Thus, this study evaluated the status of MAA and anti-MAA antibody isotypes in IBD and their potential as novel serological biomarkers for differentiating ulcerative colitis (UC) from Crohn's disease (CD). METHODS: Levels of MAA and anti-MAA antibodies were examined in patients with IBD (171), non-IBD gastrointestinal diseases (77), and controls (83) from 2 independent cohorts using immunohistochemistry and enzyme-linked immunosorbent assay. Receiver operating characteristic curves and Youden cutoff index from logistic regression were used to determine the sensitivity and specificity. RESULTS: The MAA and blood immunoglobulin G (IgG) anti-MAA antibody levels were significantly elevated in IBD compared with non-IBD patients (P = 0.0008) or controls (P = 0.02). Interestingly, patients with UC showed higher levels of IgG anti-MAA (P < 0.0001) than patients with CD including those with colonic CD (P = 0.0067). The odds ratio by logistic regression analysis predicted stronger association of IgG anti-MAA antibody with UC than CD. Subsequent analysis showed that IgG anti-MAA antibody levels could accurately identify (P = 0.0004) UC in the adult cohort with a sensitivity of 75.3% and a specificity of 71.4% and an area under the curve of 0.8072 (0.7121-0.9024). The pediatric cohort also showed an area under the curve of 0.8801 (0.7988-0.9614) and precisely distinguished (P < 0.0001) UC with sensitivity (95.8%) and specificity (72.3%). DISCUSSION: Circulating IgG anti-MAA antibody levels can serve as a novel, noninvasive, and highly sensitive test to identify patients with UC and possibly differentiate them from patients with CD.

Teaching About Intergenerational Dynamics: An Exploratory Study of Perceptions and Prevalence in US Medical Schools.

PURPOSE: Prior studies suggest a role for promoting recognition of generational differences as a part of workplace ethics. To our knowledge, there is no published comprehensive analysis demonstrating how commonly or by what methods intergenerational dynamics are taught as structured coursework in medical school curricula. To address this gap, we carried out a survey of curriculum leaders of US medical schools to assess the current practices and attitudes toward content related to generational differences in medical school coursework. METHODS: A survey consisting of 23 closed- and open-ended questions that aimed to assess the presence, characteristics, and attitudes of participants towards intergenerational dynamics in medical school coursework was disseminated via email. Curriculum deans at 154 allopathic medical schools were invited to complete the survey and sent one reminder email. Quantitative responses were descriptively analyzed, and qualitative responses were thematically analyzed. RESULTS: The response rate was 38.3%, with the majority (58%) of responding institutions stating that their curriculum did not include coursework on intergenerational dynamics. When taught, the most frequent method of instruction was small-group activities. Most stated that the educational content for intergenerational dynamics has been part of their curriculum for fewer than five years. In total, 34% of respondents agreed that some form of education about intergenerational dynamics should be required during medical education as they felt that content could improve cultural competence. Those that were less supportive of inclusion of intergenerational material stated concerns about stereotyping and the value of generational descriptions. CONCLUSION: Our findings show a heterogeneity of responses on the perceptions and practice of curriculum leaders regarding inclusion of content related to intergenerational differences in medical school education. In summary, we present the first work assessing current practices and attitudes toward content related to the inclusion of material on intergenerational dynamics in undergraduate medical education in US allopathic medical schools.

Geranylgeranyl diphosphate synthase inhibitor and proteasome inhibitor combination therapy in multiple myeloma.

BACKGROUND: Multiple myeloma (MM) remains an incurable malignancy, despite the advent of therapies such as proteosome inhibitors (PIs) that disrupt protein homeostasis and induce ER stress. We have pursued inhibition of geranylgeranyl diphosphate synthase (GGDPS) as a novel mechanism by which to target protein homeostasis in MM cells. GGDPS inhibitors (GGSI) disrupt Rab geranylgeranylation, which in turn results in perturbation of Rab-mediated protein trafficking, leading to accumulation of intracellular monoclonal protein, induction of ER stress and apoptosis. Our lead GGSI, RAM2061, has demonstrated favorable pharmacokinetic properties and in vivo efficacy. Here we sought to evaluate if combination therapy with GGSI and PI would result in enhanced disruption of the unfolded protein response (UPR) and increase anti-MM efficacy. METHODS: MTT assays were conducted to evaluate the cytotoxic effects of combining RAM2061 with bortezomib in human MM cells. The effects of RAM2061 and/or PI (bortezomib or carfilzomib) on markers of UPR and apoptosis were evaluated by a combination of immunoblot (ATF4, IRE1, p-eIF2a, cleaved caspases and PARP), RT-PCR (ATF4, ATF6, CHOP, PERK, IRE1) and flow cytometry (Annexin-V). Induction of immunogenic cell death (ICD) was assessed by immunoblot (HMGB1 release) and flow cytometry (calreticulin translocation). Cell assays were performed using both concurrent and sequential incubation with PIs. To evaluate the in vivo activity of GGSI/PI, a flank xenograft using MM.1S cells was performed. RESULTS: Isobologram analysis of cytotoxicity data revealed that sequential treatment of bortezomib with RAM2061 has a synergistic effect in MM cells, while concurrent treatment was primarily additive or mildly antagonistic. The effect of PIs on augmenting RAM2061-induced upregulation of UPR and apoptotic markers was dependent on timing of the PI exposure. Combination treatment with RAM2061 and bortezomib enhanced activation of ICD pathway markers. Lastly, combination treatment slowed MM tumor growth and lengthened survival in a MM xenograft model without evidence of off-target toxicity. CONCLUSION: We demonstrate that GGSI/PI treatment can potentiate activation of the UPR and apoptotic pathway, as well as induce upregulation of markers associated with the ICD pathway. Collectively, these findings lay the groundwork for future clinical studies evaluating combination GGSI and PI therapy in patients with MM.

Disruption of FDPS/Rac1 axis radiosensitizes pancreatic ductal adenocarcinoma by attenuating DNA damage response and immunosuppressive signalling.

BACKGROUND: Radiation therapy (RT) has a suboptimal effect in patients with pancreatic ductal adenocarcinoma (PDAC) due to intrinsic and acquired radioresistance (RR). Comprehensive bioinformatics and microarray analysis revealed that cholesterol biosynthesis (CBS) is involved in the RR of PDAC. We now tested the inhibition of the CBS pathway enzyme, farnesyl diphosphate synthase (FDPS), by zoledronic acid (Zol) to enhance radiation and activate immune cells. METHODS: We investigated the role of FDPS in PDAC RR using the following methods: in vitro cell-based assay, immunohistochemistry, immunofluorescence, immunoblot, cell-based cholesterol assay, RNA sequencing, tumouroids (KPC-murine and PDAC patient-derived), orthotopic models, and PDAC patient's clinical study. FINDINGS: FDPS overexpression in PDAC tissues and cells (P < 0.01 and P < 0.05) is associated with poor RT response and survival (P = 0.024). CRISPR/Cas9 and pharmacological inhibition (Zol) of FDPS in human and mouse syngeneic PDAC cells in conjunction with RT conferred higher PDAC radiosensitivity in vitro (P < 0.05, P < 0.01, and P < 0.001) and in vivo (P < 0.05). Interestingly, murine (P = 0.01) and human (P = 0.0159) tumouroids treated with Zol+RT showed a significant growth reduction. Mechanistically, RNA-Seq analysis of the PDAC xenografts and patients-PBMCs revealed that Zol exerts radiosensitization by affecting Rac1 and Rho prenylation, thereby modulating DNA damage and radiation response signalling along with improved systemic immune cells activation. An ongoing phase I/II trial (NCT03073785) showed improved failure-free survival (FFS), enhanced immune cell activation, and decreased microenvironment-related genes upon Zol+RT treatment. INTERPRETATION: Our findings suggest that FDPS is a novel radiosensitization target for PDAC therapy. This study also provides a rationale to utilize Zol as a potential radiosensitizer and as an immunomodulator in PDAC and other cancers. FUNDING: National Institutes of Health (P50, P01, and R01).

Faculty Versus Resident Self-Assessment Using Pathology Milestones: How Aligned Are We?

Competent physicians must be able to self-assess skill level; however, previous studies suggest that medical trainees may not accurately self-assess. We utilized Pathology Milestones (PM) data to determine whether there were discrepancies in self- versus Clinical Competency Committee (CCC) ratings by sex, program year (PGY), time of evaluation, and question category (Patient Care, Medical Knowledge, Systems-Based Practice [SBP], Practice-Based Learning and Improvement [PBL], Professionalism [PRO], and Interpersonal and Communication Skills) and Residency In-Service Examination (RISE) score. We completed retrospective analyses of PM evaluation scores from 2016 to 2019 (n = 23 residents) 2 times per year. Discrepancies in evaluation scores were calculated by subtracting CCC scores from resident self-evaluation scores. There was no significant difference in discrepancy scores between male versus female residents (P = .94). Discrepancy scores among all PGYs were significantly different (P < .0001), with PGY1 tending to overrate the most, followed by PGY2. PGY3 and PGY4 underrated themselves on average compared to CCC ratings, with PGY4 having significantly lower self-ratings than CCC compared to any other PGY. In January, residents underscored themselves and in July residents overscored themselves compared to CCC (P < .0001 for both). Question types resulted in variable discrepancy scores, with SBP significantly lower than and PRO significantly higher than all other categories (P < .05 for both). Increases in RISE score correlated to increases in self- and CCC-scoring. These discrepancies can help trainees improve self-assessment. Discrepancies indicate potential areas for amelioration, such as curriculum adjustments or Milestone's verbiage.