Bacteriophage targeting of gut bacterium attenuates alcoholic liver disease.

Chronic liver disease due to alcohol-use disorder contributes markedly to the global burden of disease and mortality1-3. Alcoholic hepatitis is a severe and life-threatening form of alcohol-associated liver disease. The gut microbiota promotes ethanol-induced liver disease in mice4, but little is known about the microbial factors that are responsible for this process. Here we identify cytolysin-a two-subunit exotoxin that is secreted by Enterococcus faecalis5,6-as a cause of hepatocyte death and liver injury. Compared with non-alcoholic individuals or patients with alcohol-use disorder, patients with alcoholic hepatitis have increased faecal numbers of E. faecalis. The presence of cytolysin-positive (cytolytic) E. faecalis correlated with the severity of liver disease and with mortality in patients with alcoholic hepatitis. Using humanized mice that were colonized with bacteria from the faeces of patients with alcoholic hepatitis, we investigated the therapeutic effects of bacteriophages that target cytolytic E. faecalis. We found that these bacteriophages decrease cytolysin in the liver and abolish ethanol-induced liver disease in humanized mice. Our findings link cytolytic E. faecalis with more severe clinical outcomes and increased mortality in patients with alcoholic hepatitis. We show that bacteriophages can specifically target cytolytic E. faecalis, which provides a method for precisely editing the intestinal microbiota. A clinical trial with a larger cohort is required to validate the relevance of our findings in humans, and to test whether this therapeutic approach is effective for patients with alcoholic hepatitis.

Virtual learning allows for adaptation of study strategies in a cohort of U.S. medical students.

BACKGROUND: In response to the COVID-19 pandemic, most U.S. medical schools acutely transitioned from regimented in-person learning to highly flexible virtual asynchronous learning. This transition at our medical school provided a unique opportunity to evaluate if and how students adapted their academic and personal lives in response. METHODS: Medical students in a single class that made this transition were retrospectively provided with 24-hour diaries for three periods - one shortly before the transition, a second early in the transition, and third several months into the transition - and asked to select the academic or personal activities done in each hour. The percentage of medical students performing each activity each hour was analyzed, as was the time spent on each activity per day, and per morning, afternoon, per evening within the day. RESULTS: Overall study time did not change in either virtual period but shifted significantly to the morning (6 AM to 12 PM). Time spent studying in groups fell significantly during both virtual periods, concordant with a significant increase in alone study time in the early virtual period. Early in the transition to virtual learning, students replaced in-person didactics with online faculty lectures; several months later in virtual learning, they had replaced online faculty lectures with commercial products. There was no significant change in time spent on specific personal activities. CONCLUSIONS: Consistent with extensive constraints imposed by the heavy cognitive load of a medical school curriculum, students did not significantly change their overall study time and any self-care-related activities in the transition to virtual learning. However, transitioning to virtual learning allowed our students to adapt their study strategies, i.e. reducing group study time and increasing lone studying time. Furthermore, students shifted studying time to the morning to optimize the management of the cognitive task-load they faced.

Relationships between preadmission variables and academic outcomes for postbaccalaureate students in medical school.

There is currently little guidance for medical school admissions committees regarding how to weigh postbaccalaureate program grades relative to undergraduate grades. This study was designed to address this issue. Admissions data, preclerkship course performance and United States Medical Licensing Exam (USMLE) Step 1 results were analyzed over three years for University of California, San Diego (UCSD) postbaccalaureate premedical (PBPM) students (n = 25), students who participated in other postbaccalaureate programs (n = 34), and for the remainder of the medical students who did not participate in any postbaccalaureate programs (n = 329). UCSD PBPM program alumni did not significantly differ in their cumulative academic performance on exams in preclerkship courses and USMLE Step 1 pass rates compared to the rest of the class despite their significantly lower GPA, lower Biology, Chemistry, Physics and Math (BCPM) GPA, and Medical College Admissions Test (MCAT) percentiles. For students who participated in the PBPM programs, PBPM program GPA was a significant predictor of preclerkship academic performance and USMLE Step 1 performance. When assessing academic readiness of applicants who have completed postbaccalaureate programs, admissions committees might closely consider the postbaccalaureate program GPA in addition to other academic metrices such as BCPM GPA and MCAT score.

YIPF6 controls sorting of FGF21 into COPII vesicles and promotes obesity.

Fibroblast growth factor 21 (FGF21) is an endocrine hormone that regulates glucose, lipid, and energy homeostasis. While gene expression of FGF21 is regulated by the nuclear hormone receptor peroxisome proliferator-activated receptor alpha in the fasted state, little is known about the regulation of trafficking and secretion of FGF21. We show that mice with a mutation in the Yip1 domain family, member 6 gene (Klein-Zschocher [KLZ]; Yipf6KLZ/Y ) on a high-fat diet (HFD) have higher plasma levels of FGF21 than mice that do not carry this mutation (controls) and hepatocytes from Yipf6KLZ/Y mice secrete more FGF21 than hepatocytes from wild-type mice. Consequently, Yipf6KLZ/Y mice are resistant to HFD-induced features of the metabolic syndrome and have increased lipolysis, energy expenditure, and thermogenesis, with an increase in core body temperature. Yipf6KLZ/Y mice with hepatocyte-specific deletion of FGF21 were no longer protected from diet-induced obesity. We show that YIPF6 binds FGF21 in the endoplasmic reticulum to limit its secretion and specifies packaging of FGF21 into coat protein complex II (COPII) vesicles during development of obesity in mice. Levels of YIPF6 protein in human liver correlate with hepatic steatosis and correlate inversely with levels of FGF21 in serum from patients with nonalcoholic fatty liver disease (NAFLD). YIPF6 is therefore a newly identified regulator of FGF21 secretion during development of obesity and could be a target for treatment of obesity and NAFLD.

Dysregulation of serum bile acids and FGF19 in alcoholic hepatitis.

BACKGROUND & AIMS: The degree of cholestasis is an important disease driver in alcoholic hepatitis, a severe clinical condition that needs new biomarkers and targeted therapies. We aimed to identify the largely unknown mechanisms and biomarkers linked to cholestasis in alcoholic hepatitis. METHODS: Herein, we analyzed a well characterized cohort of patients with alcoholic hepatitis and correlated clinical and histological parameters and outcomes with serum bile acids and fibroblast growth factor 19 (FGF19), a major regulator of bile acid synthesis. RESULTS: We found that total and conjugated bile acids were significantly increased in patients with alcoholic hepatitis compared with controls. Serum FGF19 levels were strongly increased and gene expression of FGF19 was induced in biliary epithelial cells and ductular cells of patients with alcoholic hepatitis. De novo bile acid synthesis (CYP7A1 gene expression and C4 serum levels) was significantly decreased in patients with alcoholic hepatitis. Importantly, total and conjugated bile acids correlated positively with FGF19 and with disease severity (model for end-stage liver disease score). FGF19 correlated best with conjugated cholic acid, and model for end-stage liver disease score best with taurine-conjugated chenodeoxycholic acid. Univariate analysis demonstrated significant associations between FGF19 and bilirubin as well as gamma glutamyl transferase, and negative correlations between FGF19 and fibrosis stage as well as polymorphonuclear leukocyte infiltration, in all patients with alcoholic hepatitis. CONCLUSION: Serum FGF19 and bile acids are significantly increased in patients with alcoholic hepatitis, while de novo bile acid synthesis is suppressed. Modulation of bile acid metabolism or signaling could represent a promising target for treatment of alcoholic hepatitis in humans. LAY SUMMARY: Understanding the underlying mechanisms that drive alcoholic hepatitis is important for the development of new biomarkers and targeted therapies. Herein, we describe a molecule that is increased in patients with alcoholic hepatitis. Modulating the molecular pathway of this molecule might lead to promising targets for the treatment of alcoholic hepatitis.

A summer prematriculation program to help students succeed in medical school.

Medical schools with a diverse student body face the challenge of ensuring that all students succeed academically. Many medical schools have implemented prematriculation programs to prepare students from diverse backgrounds; however, evidence on their impact is largely lacking. In this study, we analyzed participants' demographics as well as the impact of the prematriculation program on Year 1 performance. Predictive validity of the program was assessed and compared to other traditional predictors, including grade point average (GPA) and Medical College Admission Test (MCAT) scores and subscores. Linear mixed effect models determined the impact of the prematriculation program, and linear regression analysis assessed the predictive value of the overall score in the prematriculation program and other traditional predictors. Demographics of students participating in the prematriculation program from 2013 to 2015 (n = 75) revealed a significantly higher prevalence of academically disadvantaged students including older students, students with lower GPA and MCAT scores and students of racial and ethnic populations that are underrepresented in medicine, compared to non-participants (n = 293). Participants performed significantly better in Year 1 courses that were covered in the prematriculation program compared to courses that were not covered. The overall performance in the prematriculation program correlated significantly with Year 1 performance and was found to be a strong predictor for Year 1 performance. This study suggests that a prematriculation program can help students to succeed in the first year of medical school. The results have implications for medical schools seeking to implement or evaluate the effectiveness of their prematriculation program.

Gut-liver axis at the frontier of host-microbial interactions.

Liver and intestine are tightly linked through the venous system of the portal circulation. Consequently, the liver is the primary recipient of gut-derived products, most prominently dietary nutrients and microbial components. It functions as a secondary "firewall" and protects the body from intestinal pathogens and other microbial products that have crossed the primary barrier of the intestinal tract. Disruption of the intestinal barrier enhances microbial exposure of the liver, which can have detrimental or beneficial effects in the organ depending on the specific circumstances. Conversely, the liver also exerts influence over intestinal microbial communities via secretion of bile acids and IgA antibodies. This mini-review highlights key findings and concepts in the area of host-microbial interactions as pertinent to the bilateral communication between liver and gut and highlights the concept of the gut-liver axis.

Intestinal microbiota and nonalcoholic steatohepatitis.

PURPOSE OF REVIEW: Nonalcoholic fatty liver disease (NAFLD) is a liver disease with high prevalence in western countries. Progression from NAFLD to nonalcoholic steatohepatitis (NASH) occurs in 10-20%. NASH pathogenesis is multifactorial including genetic and environmental factors. The gut microbiota is involved in disease progression and its role is complex. RECENT FINDINGS: NASH is associated with changes in the intestinal microbiota, although findings in recent studies are inconsistent. Dysbiosis can trigger intestinal inflammation and impair the gut barrier. Microbial products can now reach the liver, induce hepatic inflammation and contribute to NAFLD and NASH progression. As the gut microbiota is also involved in the regulation of metabolic pathways, metabolomic approaches identified unique metabolomic profiles in patients with NASH. Altered metabolite patterns can serve as biomarkers, whereas specific metabolites (such as ethanol) have been linked with disease progression. Modifying metabolic profiles might serve as new therapeutic microbiome-based approaches. SUMMARY: In this review, we will highlight findings from the recent literature important to the gut-liver axis. We will predominantly focus on human studies with NASH.

Student evaluation team focus groups increase students' satisfaction with the overall course evaluation process.

CONTEXT: Most medical schools use online systems to gather student feedback on the quality of their educational programmes and services. Online data may be limiting, however, as the course directors cannot question the students about written comments, nor can students engage in mutual problem-solving dialogue with course directors. We describe the implementation of a student evaluation team (SET) process to permit course directors and students to gather shortly after courses end to engage in feedback and problem solving regarding the course and course elements. METHODS: Approximately 16 students were randomly selected to participate in each SET meeting, along with the course director, academic deans and other faculty members involved in the design and delivery of the course. An objective expert facilitates the SET meetings. SETs are scheduled for each of the core courses and threads that occur within the first 2 years of medical school, resulting in approximately 29 SETs annually. SET-specific satisfaction surveys submitted by students (n = 76) and course directors (n = 16) in 2015 were used to evaluate the SET process itself. Survey data were collected from 885 students (2010-2015), which measured student satisfaction with the overall evaluation process before and after the implementation of SETs. RESULTS: Students and course directors valued the SET process itself as a positive experience. Students felt that SETs allowed their voices to be heard, and that the SET increased the probability of suggested changes being implemented. Students' satisfaction with the overall evaluation process significantly improved after implementation of the SET process. CONCLUSIONS: Our data suggest that the SET process is a valuable way to supplement online evaluation systems and to increase students' and faculty members' satisfaction with the evaluation process.

Small group activities within academic communities improve the connectedness of students and faculty.

BACKGROUND: The University of California, San Diego, School of Medicine implemented a curriculum change that included reduction of lectures, incorporation of problem-based learning and other small group activities. Six academic communities were introduced for teaching longitudinal curricular content and organizing extracurricular activities. METHODS: Surveys were collected from 904 first- and second-year medical students over 6 years. Student satisfaction data with their sense of connectedness and community support were collected before and after the implementation of the new curriculum. In a follow-up survey, medical students rated factors that contributed to their sense of connectedness with faculty and students (n = 134). RESULTS: Students' perception of connectedness to faculty significantly increased following implementation of a curriculum change that included academic communities. Students ranked small group clinical skills activities within academic communities significantly higher than other activities concerning their sense of connectedness with faculty. Students' perception of connectedness among each other was high at baseline and did not significantly change. Small group activities scored higher than extracurricular activities regarding students' connectedness among themselves. CONCLUSIONS: The implementation of a new curriculum with more small group educational activities including academic communities enhanced connectedness between students and faculty and resulted in an increased sense of community.

Deficiency of intestinal mucin-2 protects mice from diet-induced fatty liver disease and obesity.

Nonalcoholic fatty liver disease (NAFLD) and obesity are characterized by altered gut microbiota, inflammation, and gut barrier dysfunction. Here, we investigated the role of mucin-2 (Muc2) as the major component of the intestinal mucus layer in the development of fatty liver disease and obesity. We studied experimental fatty liver disease and obesity induced by feeding wild-type and Muc2-knockout mice a high-fat diet (HFD) for 16 wk. Muc2 deficiency protected mice from HFD-induced fatty liver disease and obesity. Compared with wild-type mice, after a 16-wk HFD, Muc2-knockout mice exhibited better glucose homeostasis, reduced inflammation, and upregulated expression of genes involved in lipolysis and fatty acid ß-oxidation in white adipose tissue. Compared with wild-type mice that were fed the HFD as well, Muc2-knockout mice also displayed higher intestinal and plasma levels of IL-22 and higher intestinal levels of the IL-22 target genes Reg3b and Reg3g. Our findings indicate that absence of the intestinal mucus layer activates the mucosal immune system. Higher IL-22 levels protect mice from diet-induced features of the metabolic syndrome.

Writing on the board as students' preferred teaching modality in a physiology course.

The introduction of PowerPoint presentation software has generated a paradigm shift in the delivery of lectures. PowerPoint has now almost entirely replaced chalkboard or whiteboard teaching at the undergraduate and graduate levels. This study investigated whether undergraduate biology students preferred to have lectures delivered by PowerPoint or written on the board as well as the reasons behind their preference. Two upper-division physiology courses were surveyed over a period of 7 yr. A total of 1,905 students (86.7%) indicated they preferred lectures delivered by "writing on the board" compared to 291 students (13.3%) who preferred PowerPoint. Common themes drawn from explanations reported by students in favor of writing on the board included: 1) more appropriate pace, 2) facilitation of note taking, and 3) greater alertness and attention. Common themes in favor of PowerPoint included 1) increased convenience, 2) focus on listening, and 3) more accurate and readable notes. Based on the students' very strong preference for writing on the board and the themes supporting that preference, we recommend that instructors incorporate elements of the writing on the board delivery style into whatever teaching modality is used. If instructors plan to use PowerPoint, the presentation should be paced, constructed, and delivered to provide the benefits of lectures written on the board. The advantages of writing on the board can be also incorporated into instruction intended to occur outside the classroom, such as animated narrated videos as part of the flipped classroom approach.

Yip1 domain family, member 6 (Yipf6) mutation induces spontaneous intestinal inflammation in mice.

Using an environmentally sensitized genetic screen we identified mutations that cause inflammatory colitis in mice. The X-linked Klein-Zschocher (KLZ) mutation created a null allele of Yipf6, a member of a gene family believed to regulate vesicular transport in yeast, but without known functions in mammals. Yipf6 is a five transmembrane-spanning protein associated with Golgi compartments. Klein-Zschocher mutants were extremely sensitive to colitis induced by dextran sodium sulfate (DSS) and developed spontaneous ileitis and colitis after 16 mo of age in specific pathogen-free housing conditions. Electron microscopy, gene expression, and immunocytochemistry analyses provided evidence that impaired intestinal homeostasis stemmed from defective formation and secretion of large secretory granules from Paneth and goblet cells. These studies support a tissue- and organ-specific function for Yipf6 in the maintenance of intestinal homeostasis and implicate the orthologous human gene as a disease susceptibility locus.

Recombinant human annexin A5 inhibits proinflammatory response and improves cardiac function and survival in mice with endotoxemia.

OBJECTIVES: Annexin A5 is a 35-kDa protein with high affinity binding to negatively charged phospholipids. However, its effects on sepsis are not known. Our aim was to study the effects of annexin A5 on myocardial tumor necrosis factor-α expression, cardiac function, and animal survival in endotoxemia. DESIGN: Prospective experimental study. SETTING: University laboratory. SUBJECTS: Adult male C57BL/6 mice. INTERVENTIONS: Mice were challenged with lipopolysaccharide (4 or 20 mg/kg, i.p.) to induce endotoxemia with and without recombinant human annexin A5 treatment (5 or 10 µg/kg, i.v.). Cytokine expression and cardiac function were assessed, and animal survival was monitored. MEASUREMENTS AND MAIN RESULTS: Treatment with annexin A5 inhibited myocardial mitogen-activated protein kinase, and nuclear factor-κB activation in mice with endotoxemia. Furthermore, annexin A5-treated animals showed significant reductions in myocardial and plasma levels of tumor necrosis factor-α and interleukin-1ß while cardiac function was significantly improved during endotoxemia. Additionally, 5-day animal survival was significantly improved by either an immediate or a 4-hour delayed annexin A5 treatment after lipopolysaccharide challenge. Importantly, annexin A5 dose-dependently inhibited lipopolysaccharide binding to a toll-like receptor-4/myeloid differentiation factor 2 fusion protein. CONCLUSIONS: Annexin A5 treatment decreases cytokine expression and improves cardiac function and survival during endotoxemia. These effects of annexin A5 are mediated by its ability to inhibit lipopolysaccharide binding to toll-like receptor-4, leading to reductions in mitogen-activated protein kinase and Akt signaling. Our study suggests that annexin A5 may have therapeutic potential in the treatment of sepsis.

Deficiency of intestinal mucin-2 ameliorates experimental alcoholic liver disease in mice.

UNLABELLED: The intestinal mucus layer protects the epithelium from noxious agents, viruses, and pathogenic bacteria present in the gastrointestinal tract. It is composed of mucins, predominantly mucin (Muc) 2, secreted by goblet cells of the intestine. Experimental alcoholic liver disease requires translocation of bacterial products across the intestinal barrier into the systemic circulation, which induces an inflammatory response in the liver and contributes to steatohepatitis. We investigated the roles of the intestinal mucus layer, and in particular Muc2, in development of experimental alcohol-associated liver disease in mice. We studied experimental alcohol-induced liver disease, induced by the Tsukamoto-French method (which involves continuous intragastric feeding of an isocaloric diet or alcohol) in wild-type and Muc2(-/-) mice. Muc2(-/-) mice showed less alcohol-induced liver injury and steatosis than developed in wild-type mice. Most notably, Muc2(-/-) mice had significantly lower plasma levels of lipopolysaccharide than wild-type mice after alcohol feeding. In contrast to wild-type mice, Muc2(-/-) mice were protected from alcohol-associated microbiome changes that are dependent on intestinal mucins. The antimicrobial proteins regenerating islet-derived 3 beta and gamma were expressed at significantly higher levels in the jejunum of Muc2(-/-) mice fed the isocaloric diet or alcohol compared with wild-type mice. Consequently, Muc2(-/-) mice showed increased killing of commensal bacteria and prevented intestinal bacterial overgrowth. CONCLUSION: Muc2(-/-) mice are protected from intestinal bacterial overgrowth and dysbiosis in response to alcohol feeding. Subsequently, lower amounts of bacterial products such as endotoxin translocate into the systemic circulation, decreasing liver disease.

Vancomycin-resistant enterococci exploit antibiotic-induced innate immune deficits.

Infection with antibiotic-resistant bacteria, such as vancomycin-resistant Enterococcus (VRE), is a dangerous and costly complication of broad-spectrum antibiotic therapy. How antibiotic-mediated elimination of commensal bacteria promotes infection by antibiotic-resistant bacteria is a fertile area for speculation with few defined mechanisms. Here we demonstrate that antibiotic treatment of mice notably downregulates intestinal expression of RegIIIgamma (also known as Reg3g), a secreted C-type lectin that kills Gram-positive bacteria, including VRE. Downregulation of RegIIIgamma markedly decreases in vivo killing of VRE in the intestine of antibiotic-treated mice. Stimulation of intestinal Toll-like receptor 4 by oral administration of lipopolysaccharide re-induces RegIIIgamma, thereby boosting innate immune resistance of antibiotic-treated mice against VRE. Compromised mucosal innate immune defence, as induced by broad-spectrum antibiotic therapy, can be corrected by selectively stimulating mucosal epithelial Toll-like receptors, providing a potential therapeutic approach to reduce colonization and infection by antibiotic-resistant microbes.

MyD88-mediated signals induce the bactericidal lectin RegIII gamma and protect mice against intestinal Listeria monocytogenes infection.

Listeria monocytogenes is a food-borne bacterial pathogen that causes systemic infection by traversing the intestinal mucosa. Although MyD88-mediated signals are essential for defense against systemic L. monocytogenes infection, the role of Toll-like receptor and MyD88 signaling in intestinal immunity against this pathogen has not been defined. We show that clearance of L. monocytogenes from the lumen of the distal small intestine is impaired in MyD88(-/-) mice. The distal ileum of wild-type (wt) mice expresses high levels of RegIII gamma, which is a bactericidal lectin that is secreted into the bowel lumen, whereas RegIII gamma expression in MyD88(-/-) mice is nearly undetectable. In vivo depletion of RegIII gamma from the small intestine of wt mice diminishes killing of luminal L. monocytogenes, whereas reconstitution of MyD88-deficient mice with recombinant RegIII gamma enhances intestinal bacterial clearance. Experiments with bone marrow chimeric mice reveal that MyD88-mediated signals in nonhematopoietic cells induce RegIII gamma expression in the small intestine, thereby enhancing bacterial killing. Our findings support a model of MyD88-mediated epithelial conditioning that protects the intestinal mucosa against bacterial invasion by inducing RegIII gamma.