bluuebunny/crossref_metadata_2025 huggingface dataset:
Allergen‐Induced Endoplasmic Reticulum Stress Regulates Lung Inflammation and Fibrosis
Authors: Mariswamy Siddesha Jalahalli , Nakada Emily M , Mihavics Bethany , Hoffman Sidra , Chapman David , Irvin Charles G , Dixon Anne , Poynter Matthew , Anathy Vikas
Published: November 2023

Background

The endoplasmic reticulum (ER) stress is known to play a critical role in many inflammatory diseases including asthma. One of the canonical responders of the ER stress, ATF6α is known to regulate inflammation in asthma. We have previously demonstrated that endobronchial biopsies from asthmatics and non‐asthmatics revealed a predominant increase in markers of ER stress in the asthmatic epithelium. These Increases positively correlated with higher eosinophil counts and bronchodilator response in asthmatics (Hoffman, S. et al., JACI, 2015‐in press). However, the mechanistic role of ER stress and ATF6α in asthma is not well understood.

Objective

We sought to examine the contribution of ER stress in the pathogenesis of allergic asthma, and the effect of a chemical chaperone tauroursodeoxycholic acid (TUDCA) in mitigating house dust mite (HDM)‐induced airway ER stress, inflammation and fibrosis.

Methods

We examined the markers of ER stress in asthmatic airway epithelium and utilized murine models of allergic asthma to evaluate the significance of upregulation of ER stress and evaluated the efficacy of TUDCA in mitigating allergic asthma.

Results

HDM‐challenge in mice resulted in robust increases in markers of ER stress including cleaved ATF6α as compared to Ovalbumin challenged mice. Wild type (WT) mice challenged with HDM showed a significantly higher influx of eosinophils and lymphocytes into the airways and to a lesser extent neutrophils and macrophages as compared to ATF6α−/− mice challenged with HDM. AHR measurements revealed that central airway resistance was significantly decreased in ATF6α−/− mice as compared to WT mice challenged with HDM. We also found increases in markers of fibrosis in HDM‐challenged WT mice as compared to HDM‐challenged ATF6α−/− mice.

TUDCA administered during the HDM‐challenge phase (a prophylactic regimen) significantly decreased HDM‐induced markers of ER stress, airways inflammation, AHR and fibrosis as compared to control mice. Further, administration of TUDCA after the establishment of allergic airways disease, efficiently decreased HDM‐induced airways inflammation, mucus metaplasia, AHR and markers of UPR but not airway fibrotic remodeling.

Conclusion

Our novel results demonstrate that the markers of ER stress is increased in the airway epithelium of asthmatics and in mice with allergic airway disease. Specifically, deletion of ATF6, decreased pro‐inflammatory, fibrotic mediators, and AHR. We also demonstrate that a chemical chaperone mitigates the effects of allergen in a mouse model of asthma. These results suggest that inhibiting specific components of ER stress and strategies to utilize chemical chaperones may provide an opportunity to alleviate allergic asthma.

Support or Funding Information

Funding: NIH R01 HL122383, Parker B. Francis Fellowship and Department of Pathology & Laboratory Medicine.
Dissection And Reconstitution Of Autophagic Membrane Fusion
Authors: Zhong Qing , Diao Jiaojie , Liu Rong , Rong Yueguang
Published: November 2023

Autophagy, an important catabolic pathway implicated in a broad spectrum of human diseases, begins by forming double membrane autophagosomes that engulf cytosolic cargo and ends by fusing autophagosomes with lysosomes for degradation. Membrane fusion activity is required for early biogenesis of autophagosomes and late degradation in lysosomes. However, the key regulatory mechanisms of autophagic membrane tethering and fusion remain largely unknown. We recently reported that ATG14/Barkor/ATG14L, an essential autophagy‐specific regulator of the class III phosphatidylinositol 3‐kinase complex, promotes membrane tethering of protein‐free liposomes, and enhances hemifusion and full fusion of proteoliposomes reconstituted with the target (t)‐SNAREs (soluble N‐ethylmaleimide‐sensitive factor attachment protein receptors) syntaxin 17 (STX17) and SNAP29, and the vesicle (v)‐SNARE VAMP8. ATG14 binds to the SNARE core domain of STX17 through its coiled‐coil domain, and stabilizes the STX17‐SNAP29 binary t‐SNARE complex on autophagosomes. The STX17 binding, membrane tethering and fusion‐enhancing activities of ATG14 require its homo‐oligomerization by cysteine repeats. In ATG14 homo‐oligomerization‐defective cells, autophagosomes still efficiently form but their fusion with lysosomes is blocked. Recombinant ATG14 homo‐oligomerization mutants also completely lose their ability to promote membrane tethering and to enhance SNARE‐mediated fusion in vitro. Our data suggest an autophagy‐specific membrane fusion mechanism in which oligomeric ATG14 directly binds to STX17‐SNAP29 binary t‐SNARE complex on autophagosomes and primes it for VAMP8 interaction to promote autophagosome‐lysosome fusion. We are currently investigating protein factors and likely protein modifications required for full reconstitution of autophagosomal fusion with lysosomes.

Support or Funding Information

The work was supported by grants to Q. Z. from the Welch Foundation (I‐1864), CPRIT (RP140320), and NIH R01 (GM116908).
Finding Your Fit Among Career Options With A Phd
Authors: McGee Rick
Published: November 2023

Over the past decade, there has been a dramatic and rapid shift in the life sciences from viewing achieving a career as a faculty member as the norm that everyone should aspire to, toward acknowledging and enabling exploration of multiple career options with a PhD. Many tools have been developed and made available to assist career exploration, and NIH has funded 17 awards for universities to develop approaches for Broadening Experience in Scientific Training (BEST). However, for many individuals, sorting out the best options and the routes to getting there is still a very complicated task. Over the past almost 20 years, we have been studying various aspects of development of young scientist, including what leads individuals to start the PhD vs. other career options, and, more recently, the processes by which individuals choose options after they start the PhD. All of our work is based on in‐depth interviews conducted longitudinally throughout the PhD and beyond, with analysis using qualitative research methods. I will present some of our recent findings that reveal: the complexity of the process; how differing interests and perceptions of work and non‐work balance influences it; unique challenges that women and minorities face; and varying strategies people use to manage those challenges. I will also introduce the concepts of Communities of Practice and Cultural Capital to help reveal the processes by which young scientists find fit, alignment and success within different careers.
How Antibiotics Affect The Human Gut Microbiome: Lessons From The Extreme
Authors: Xavier Joao
Published: November 2023

The intestinal microbiota is a symbiotic ecosystem of microbes that plays essential roles in human health. Microbes complement our ability to digest food by providing the genes and enzymes to break down complex nutrients but also keep gut pathogens out. We investigate how bacteria in the microbiota function together to make a healthy microbial ecosystem. By employing mathematical modeling techniques form ecology we could identify microbes and microbe‐microbe interactions that protect against enteric pathogens such as Clostridum difficile and vancomycin‐resistant Enterococcus (VRE). Our work suggests that mathematical modeling can be a valuable tool towards the rational design of antibiotic and probiotic therapies that tailor the microbiome to improve human health.

Support or Funding Information

DP2OD008440/OD/NIH HHS/United States
MUSE: A New, Fast, Simple Microscopy Method for Slide‐Free Histology and Surface Topography
Authors: Levenson Richard M , Fereidouni Farzad
Published: November 2023

Introduction

Conventional brightfield or fluorescence microscopy works best with thin, stained specimens mounted on glass slides, but to prepare these requires hours of processing and the help of highly skilled technical personnel. We describe a new, inexpensive form of light microscopy that can generate high‐quality histology and histopathology images directly from cut surfaces of fresh (or fixed) tissue samples of any thickness, with less than 1 minute of preparation.

Background

While alternative microscopy‐scale methods, such as confocal or two‐photon, or optical coherence tomography approaches, can generate reasonable images from thick tissues, the instrumentation is typically complex, expensive and not widely available. We are developing an alternative technique, MUSE (Microscopy with UV Surface Excitation), that relies on simple properties of light, tissue, and fluorescent stains, and can be implemented at relatively low cost. Thick pieces of tissue obtained by biopsy, surgery or necropsy can be imaged directly. The method is non‐destructive, and eliminates requirement for conventional histology processing, formalin fixation, paraffin‐embedding, or thin‐sectioning.

Method

Ultraviolet light at about 280 nm from an LED arranged to provide oblique illumination, is used to excite just the surface layer of tissue that has been briefly (~10 seconds) stained with common and inexpensive fluorescent dyes. Unlike light of longer wavelength, 280‐nm light only penetrates to a depth of 10 microns or less, and thus excites fluorescent signals, conveniently in the visible range, only from the cut specimen surface. The images, which can be diffraction‐limited, are captured using conventional microscope optics and a standard color camera

Results

MUSE samples from fresh (or fixed) tissues stained with general tissue stains such as rhodamine and Hoechst generate high‐resolution fluorescence images that can be converted in real time from fluorescence to H&E‐like brightfield appearance, for ease of interpretation. However, fluorescence display mode preserves surface shading and depth cues that allow for appreciation of surface profiles important in understanding 3D organization of complex specimens. We have obtained high‐quality MUSE images from normal and diseased tissues from multiple organisms, as well as from plants, and manufactured materials. Moreover, surface profile information provides views beyond what can be seen with in standard thin‐sectioned material, generating results that can resemble those obtainable with scanning electron microscopy (SEM).

Conclusion

We have developed a new form of optical microscopy that generates diagnostic‐quality histological images, with enhanced content, from fresh or fixed, but unsectioned tissue, rapidly, with high resolution, simply and inexpensively. We anticipate that there could be widespread adoption in preclinical and clinical research facilities, as well as hospital‐based and stand‐alone medical settings.
Deterministic transfection and genetic manipulation of organotypic brain slice cultures for ex vivo imaging
Authors: Gygli Patrick Edwin , Byers Spencer , Higuita‐Castro Natalia , Czeisler Catherine , Gallego‐Perez Daniel , Otero Jose Javier
Published: November 2023

The organotypic brain slice culture has been used to great effect for many studies, from electrophysiology to cell fate determination. However, a major limitation of this technique is the difficulty and expense of reliable genetic manipulation using transfection and virus methodologies. To overcome this problem, we combined cutting‐edge nano‐engineering techniques with established brain slice culture methodologies. We used the technique of nanochannel electroporation (NEP) to deliver defined quantities of plasmids for co‐expression of different genes within a single cell, which is not possible with probabilistic transfection methodologies such as lipid‐mediated transfection. Using the NEP methodology, we transfected organotypic brain slices with defined mixtures of plasmids encoding different fluorescent proteins. We were able to transfect a variety of cell types including neurons. Using live confocal imaging, we observed a very high degree of co‐transfection with plasmids encoding both green and red fluorescent proteins. We further transfected a plasmid encoding channelrhodopsin and used an optogenetic approach to stimulate specific neurons within slice cultures. Our novel methodology greatly expands the utility of the organotypic brain slice culture, and reduces the need for costly and time‐consuming reagents such as viruses for genetic manipulation. The flexibility of our NEP methodology opens the door to many experiments that would otherwise be prohibitively costly and time consuming, and allows investigators to easily perform experiments that would not be possible using standard methodologies.
Identification of Lipocalin 2 in Human Hepatoblastoma
Authors: Bell Danielle Elizabeth , Tao Junyan , Singh Sucha , Chikina Maria , Ranganathan Sarangarajan , Monga Satdarshan P
Published: November 2023

Cancer is the leading cause of medical death in children. Hepatoblastoma is the most common type of pediatric liver malignancy. Although it is a rare tumor, it often presents in advanced stage and is associated with a poor prognosis. It is well known that signaling abnormalities in the beta catenin pathway are associated with 90% of these tumors. We have recently shown activation of Yes associated protein (Yap) in this tumor type. In fact b‐catenin‐Yap co‐activation was observed in 80% of hepatoblastoma patients. Co‐expression of Yap and b‐catenin also led to hepatoblastoma in mice. To elucidate downstream targets of Yap and b‐catenin that may contribute to tumorigenesis, we performed gene array analysis using mRNA isolated from mice tumors. The most upregulated genes were assessed for presence of classical transcription factors that bind b‐catenin (TCF4) and Yap (TEAD). This analysis revealed lipocalin 2 among the top 5 candidates. Lipocalin 2, or NGAL (neutrophil gelatinous associated lipocalin) is a small molecule protein with known antibacterial properties, and is also used as a biomarker in kidney injury. Recently, it has been found to be expressed in a variety of adult cancers including breast, colon, pancreatic, and hepatocellular carcinoma. We used a tissue microarray of 74 patient hepatoblastoma samples from a tertiary care Pediatric Hospital to evaluate the presence of lipocalin 2 by immunohistochemistry. The tumors were classified by histology type and the degree of lipocalin staining was graded on a 0–3 scale. Lipocalin 2, which is normally not expressed in human liver (except for Kupffer cells), was found to be present in the nucleus or cytoplasm of 70 (95%) of the pediatric hepatoblastoma samples. Most hepatoblastoma samples (69%) showed greater than 1 histological component within the tumor tissue. The degree of staining for lipocalin 2 varied within each sample based on tumor histology. All embryonal components were strongly positive for lipocalin 2 with staining scores of 2–3. Ninety‐Six percent of crowded fetal histology components had positive staining, although the staining ranged from 1–2 in intensity. All components of blastema histology were negative and small cell undifferentiated components had only 18% positive lipocalin 2 expression. Next, cell lysates from a human hepatoblastoma cell line (HepG2) were examined for lipocalin 2 and showed notable expression as well. Thus, we have identified increased expression of lipocalin 2 downstream of b‐catenin and Yap activation in hepatoblastoma cells and in patient tissues. Lipocalin 2 may be playing an important role in b‐catenin‐Yap driven hepatoblastoma development.
Lack of β‐catenin in Hepatocytes Impairs Proliferation and Leads to Increased Morbidity in Response to a Choline‐Deficient Ethionine‐Supplemented Diet
Authors: Russell Jacquelyn Olivia , Okabe Hirohisa , Singh Sucha , Poddar Minakshi , Monga Satdarshan Paul
Published: November 2023

Despite the liver's capacity for regeneration, liver disease is the 12th leading cause of death in the United States. Treatments for chronic liver disease are limited due to incomplete understanding of liver regeneration mechanisms. Although activation of liver stem cells (LSCs) is thought to contribute to liver regeneration when hepatocyte proliferation is impaired, the role and origin of these cells is still controversial. The choline‐deficient ethionine‐supplemented (CDE) diet model of liver injury is known to induce proliferation of LSCs. However, recent evidence has supported repair primarily driven by hepatocyte self‐duplication in the CDE diet model. As a member of the WNT signaling pathway, β‐catenin plays an important role in liver regeneration by promoting hepatocyte proliferation. However, the role of β‐catenin in liver regeneration after injury from the CDE diet has not been previously characterized. We show that lack of β‐catenin‐driven WNT signaling in mouse livers leads to a defect in hepatocyte proliferation and increased morbidity in response to the CDE diet. Mice with liver‐specific deletion of β‐catenin (β‐catenin KO) and control wild‐type littermates (WT) on the CDE diet showed comparable initial levels of liver injury and inflammation. After 14 days on the CDE diet, WT mice demonstrated significant hepatocyte proliferation and reduced liver injury markers. In contrast, β‐catenin KO mice displayed a lack of hepatocyte proliferation, sustained liver injury, and increased fibrosis. Mice lacking WNT signaling via liver‐specific deletion of co‐receptors LRP5 and LRP6 (LRP5‐6 DKO) also displayed sustained liver injury after 14 days on the CDE diet. Interestingly, we observed proliferation of LSCs in WT, β‐catenin KO, and LRP5‐6 DKO mice, suggesting liver regeneration occurs independently from LSC activation. Therefore our results indicate a role of β‐catenin in liver regeneration through hepatocyte proliferation and provide additional evidence to support a model of regeneration driven by hepatocyte self‐duplication rather than LSC amplification.

Support or Funding Information

This research was funded by the National Institute of Biomedical Imaging and Bioengineering of the National Institutes of Health under award number T32EB0010216. Support was provided by the Cellular and Molecular Pathology Graduate Training Program of the University of Pittsburgh.
Hippo/YAP Signaling Induced by Endotoxin Promotes Kupffer Cell Activation and Hepatic Inflammation in Non‐Alcoholic Fatty Liver Disease (NFALD)
Authors: Song Kyoungsub , Kwon Hyunjoo , Han Chang , Zhang Jinqiang , Yao Lu , Ungerleider Nathan , Wu Tong
Published: November 2023

Recent studies show that the Hippo pathway and its downstream effector YAP play important roles in organ size control, liver fibrosis and hepatocarcinogenesis. However, it remains unknown whether Hippo/YAP signaling is implicated in the pathogenesis of non‐alcoholic fatty liver disease (NAFLD). This study was designed to investigate the potential role of Hippo/YAP signaling in a mouse model of high‐fat diet (HFD)‐induced NAFLD. Male C57BL/6 mice were fed a normal chow diet (NCD) or HFD (60% kcal fat) for 12 wks. Intraperitoneal injections of low‐dose lipopolysaccharide (LPS: 0.25mg/kg) were administered daily for 3 wks to NCD‐fed mice. Upregulation of YAP in Kupffer cells was observed in HFD‐fed mice and LPS‐treated mice. Our data indicated that LPS induced AP‐1 binding to their consensus site in the YAP gene promoter which leads to enhanced YAP gene transcription. In contrast, the protein level of MST1, an upstream kinase that inactivates YAP, was decreased by LPS treatment. We observed that overexpression of YAP significantly induced the expression of pro‐inflammatory cytokines (TNF‐α, IL‐6, MCP‐1, and IL‐1β) in primary Kupffer cells and in Raw264.7 cells (murine macrophage cell line). Pharmacological inhibition of YAP‐TEAD with vertepofin attenuated both YAP‐ and LPS‐induced pro‐inflammatory cytokine expressions. Moreover, the expression of pro‐inflammatory cytokines was markedly reduced in Kupffer cells isolated from vertepofin‐treated HFD‐fed mice compared to vehicle‐treated HFD‐fed mice. These results demonstrate a novel role of Kupffer cell Hippo/YAP signaling in endotoxin‐induced hepatic inflammation in NAFLD. Therefore, inhibition of YAP activation may represent a novel therapeutic approach for the treatment non‐alcoholic steatohepatitis.

Support or Funding Information

NIH R01 CA1023252 and R56DK077776
Giardia muris Protection against Citrobacter rodentium Colitis is Associated with Inflammasome Activation and Increased Release of Antimicrobial Peptides in the Gut
Authors: Manko Anna , Motta Jean‐Paul , Cotton James A , Oyeyemi Ayodele , Vallance Bruce A , Wallace John L , Buret Andre G
Published: November 2023

Background

Polymicrobial gastrointestinal (GI) infections are common in individuals living in areas with poor sanitation. In areas where Giardia sp. infections are found in association with enteropathogens causing diarrhea via gut inflammation, Giardia sp. appears to protect against diarrhea via mechanisms that remain unlcear. Intestinal epithelial anti‐microbial peptides (AMPs) and the canonical and non‐canonical inflammasome pathways are essential to host defense.

Objectives

The aim of this study was to determine whether Giardia muris infection may protect against Citrobacter rodentium colitis, and whether this involves inflammasome activation and/or enhanced AMP production.

Methods

Male C57BL/6 mice were co‐infected with G. muris trophozoites and GFP‐labeled C. rodentium. Animals were assessed for weight gain, fecal blood, histopathology, myeloperoxidase (MPO) assays, bacterial loads, immunofluorescence for microbial‐tissue interactions, as well as Western blotting and qPCR for caspase‐1 and ‐11 (markers of inflammasome activation), β‐Defensin‐2 and Trefoil‐Factor (TFF) 3.

Results

At 14 days post‐infection, mice infected with C. rodentium had increased macroscopic damage in colon, lost weight and rectal bleeding; G. muris co‐infection significantly inhibited these effects. Microscopic damage score and MPO levels were reduced in co‐infected animals at 14 days post‐infection. In the colon of C. rodentium‐infected mice, we observed attachment of bacteria to the intestinal epithelium and translocation into the lamina propria. In Giardia co‐infected animals, mucosal adhesion and translocation of C. rodentium was reduced and associated with lower tissue C. rodentium counts. Colonic caspase‐1 and ‐11 protein levels were increased in the co‐infected group compared to animals given C. rodentium alone. In colonic tissues of mice co‐infected with G. muris, mRNA and protein levels of the AMPs β‐Defensin‐2 and TFF3 proteins were significantly increased compared to animals given C. rodentium alone. Immunofluorescence of colonic tissues revealed increased intensity of TFF3 and β‐Defensin‐2 in co‐infected animals, particularly in the lumen and intestinal epithelium.

Conclusion

The data indicate that G. muris infection protects against C. rodentium‐‐induced colitis, in association with upregulation of intestinal AMPs and activation of inflammasome pathways. Our data shed new light on how microbial‐microbial interactions may protect against bacterial enteritis in zones endemic for giardiasis.

Support or Funding Information

This work was supported by Canadian Institutes of Health Research
The Biophysical Determinants of Prion Neuroinvasion
Authors: Sigurdson Christina , Lawrence Jessica , Bett Cyrus , Kurt Tim , Wu Chengbiao , Surewicz Witold , Nilsson K. Peter R.
Published: November 2023

Prions naturally transmit to other individuals as infectious pathogens and have led to large scale epidemics, for example, as occurred during the bovine spongiform encephalopathy (BSE) epidemic. In humans, prions have been transmitted iatrogenically through the transplantation of dura mater grafts and cornea from prion‐infected donors. Most cases of natural prion transmission follow a peripheral exposure and prions spread into the CNS in a process known as neuroinvasion. This ability of most prions to shuttle in and out of the CNS is remarkable and not described for other amyloidogenic proteins. Using mouse models, we have found that certain prion aggregates rarely invade the CNS following intraperitoneal or intra‐tongue inoculation, yet accumulate in extraneural organs, leading to lifelong subclinical carriers of infectious prions. Intriguingly, these poorly neuroinvasive prions are fibrillar, which are uncommon structures in prion disease, as most prions form oligomers. We found that reducing the prion aggregate size markedly impacts the neuroinvasive ability of fibrillar prions, suggesting that size plays an important role in prion neuroinvasion. Additionally, we have found that the intravenous route is surprisingly efficient for fibrillar prion entry into the CNS, which may help explain how human‐to‐human transmission of variant‐Creutzfeldt‐Jakob disease prions occurred following blood transfusion. Taken together, these results indicate that aggregate size and route of exposure are major determinants that impact the ability of a prion to neuroinvade.

Support or Funding Information

NIH R01NS076896
Changes in plasma and urine docosahexaenoic acid levels in Alzheimer's disease
Authors: Fonteh Alfred N. , Castor Katherine , Chung Eun , Applegate Sarah , Harrington Michael
Published: November 2023

Objectives

To test the hypothesis that the major brain omega‐3 fatty acid (docosahexaenoic acid, DHA, 22:6n‐3), is excreted at a faster rate from study participants with Alzheimer's disease (AD) compared with cognitively normal study participants.

Methods

We recruited and collected cerebrospinal fluid (CSF), plasma, and urine from cognitively normal study participants with normal amyloid beta 42 (Aβ42) to total tau (CN‐nAT, n= 36), pathological Aβ42/tau (CN‐pAT, n=34), mild cognitive impairment (MCI) with normal (MCI‐nAT, n= 28) or pathological Aβ42/tau (MCI‐pAT, n=10), and AD (n=29). Lipids were extracted from pooled plasma and urine samples and DHA levels measured using gas chromatography/negative ion chemical ionization/mass spectrometry.

Results

Plasma free DHA and total DHA levels were lower in AD compared with CN and MCI. In contrast, free and total DHA levels in urine were higher in AD compared with CN and MCI. CN and MCI subjects with normal Aβ42/tau had higher plasma DHA than those with pathological Aβ42/tau.

Conclusions

Dysfunctional regulation of DHA metabolism may account for reduced resolution of inflammation in AD study participants. Measurement of plasma and urine DHA levels may be viable approaches for monitoring metabolic pathways associated with dementia in an aging population.

Support or Funding Information

L.K. Whittier and the Helen Posthuma Foundations
Pharmacokinetics in an introductory physics lab: experimental monitoring and computer modeling of drug metabolism
Authors: Rodriguez Juan , Weigal Justin , Spitznagel Benjamin , Wilmes Anthony
Published: November 2023

Vision and Change [AAAS, 2011] outlines a blueprint for modernizing biology education that includes a listing of key concepts and competencies students need to succeed in 21st century Biology. Here we describe an introductory physics lab that advances the reports’ recommendations on the concept of systems, and on the abilities to tap into the interdisciplinary nature of science and to use modeling/simulation. These are achieved through an in vitro pharmacokinetics experiment on the metabolism of coumarin by liver microsomes. The apparatus revolves around two fluid compartments separated by a semipermeable membrane. One compartment is a shallow, 3D printed well, that contains a liver microsome suspension. This arrangement mimics a tissue compartment. The second compartment mimics the circulation. It uses a mini‐pump that circulates a fluid continuously across the semipermeable membrane. Coumarin is injected into the circulating fluid, from where it diffuses across the semipermeable membrane and into the microsome‐containing compartment where it is gradually metabolized. The fluid also flows continuously through a small UV/VIS spectrophometer that monitors drug concentration in realtime. Experimental results are contrasted with those obtained with agent‐based modeling that mimics flow conditions in the circulation, diffusion into and out of the tissue compartment, and metabolism.
Improvement in student data analysis skills after out‐of‐class assignments
Authors: Walton Kristen LW
Published: November 2023

The ability to understand and interpret data is a core aspect of scientific thinking. However, although data analysis is an increasing area of focus in many biology majors classes, many textbooks and lab manuals for allied health majors classes do not include significant amounts of graphs and figures showing experimental data. In two allied health majors courses, Medical and Public Health Microbiology or Pathophysiology, students were given assignments from primary journal articles and their data analysis skills were assessed in a pre/post‐test format. The pre/post‐test for the two classes included similar questions placed in a context appropriate to each class. Students were given 3–4 assignments that included a significant data analysis component. These ranged from case studies that included a figure from a journal article, to reading a short journal article and answering questions about multiple figures. Typical data formats were line or bar graphs, gel photographs, and flow charts. The pre‐ and post‐tests incorporated the same types of figures to assess whether the assignments resulted in a change in data analysis skills. The mean class score in Medical ad Public Health Microbiology showed a small but significant improvement from pre‐test to post‐test (50.4% versus 60.0%, p<0.001 by paired t‐test). Scores on individual questions requiring predictions based on presented data or on analysis of a flow diagram improved the most. This supports the conclusion that a relatively small number of out‐of‐class assignments resulted in a significant improvement in data analysis abilities in this population of students.
Clinical Scenario Videos Improve D.M.D Students’ Perception of the Basic Sciences and Ability to Apply Content Knowledge
Authors: Metz Cynthia Jayne , Metz Michael James
Published: November 2023

Students enrolled in D.M.D. programs often have difficulty understanding the importance of basic science classes, such as physiology, for their future careers as practicing dentists. In order to help alleviate this issue, a series of video modules were created using simulated patients and custom‐designed animations that showcase medical emergencies in the dental practice. Students formatively assessed their knowledge using embedded questions. These responses indicated that while students could initially recognize the cause of the medical emergency, they had difficulty in applying their knowledge of physiology to the scenario. In two of the three videos, students drastically improved their ability to answer high‐level clinical questions at the conclusion of the video. Additionally, when compared to the previous year of the course, there was a significant improvement in unit exam scores on clinically‐related questions (6.2% increase), while no change was found for the basic science questions. Surveys were administered to freshmen and senior dental students to compare their perception of these changes. Freshmen students indicated a more positive perception of the physiology course and its importance for success on board examinations and their dental career. The most positive reported aspects of the clinical scenario modules were the clear applications of physiology to real‐life dental situations, the interactive nature of the videos, and the improved student comprehension of course concepts. These results suggest that online modules may successfully be used to improve student perceptions of the basic sciences, and enhance the ability of students to apply basic science content to clinically‐important scenarios.
Clinical Simulations: Engaging Medical Students in Problem‐Based Learning
Authors: Li Andrew , Carvalho Helena
Published: November 2023

Introduction

Problem‐Based Learning (PBL) curricula allow for medical students to learn preclinical material by teaching each other through presentations based on real patient cases. A benefit to PBL is the development of individual student teaching styles. Traditional, lecture‐format presentations are effective for a certain portion of medical students. It is important to develop novel teaching methods that engage the student audience and facilitate learning and retention.

Goal

Design and implement PBL learning activities for “Chest Pain Differentials” and “Cardiac Rehabilitation” that involve students in interactive scenarios to capture their attention and encourage learning through participation.

Methods

Chest Pain Differentials: Six systems were addressed (Musculoskeletal, Cardiovascular, Pulmonary, Gastrointestinal, Endocrine/Neurological, and Psychiatric) with six clinical vignettes. Pairs of students were instructed to act as a physician or patient: the “physician” student was tasked with diagnosing underlying etiology of chest pain; the “patient” student was tasked with simulating the clinical vignette containing patient identifier, HPI, and key buzzwords. The scenarios were debriefed with slides containing images of the vignette along with basic information on other differentials in that system. Cardiac Rehabilitation: A simulation of a cardiac rehabilitation orientation was done where students were the incoming admissions. It introduced patient demographics, indications, health assessments, physical activity regimen, diet plan, reducing risk factors, emotional/sexual counseling, risks and benefits.

Results

Voluntary and anonymous survey to students on presentation effectiveness scored 4.17 ± 0.75 (mean ± SD) for Chest Pain Differentials and 3.83 ± 0.98 (mean ± SD) for Cardiac Rehabilitation (with 1 being the least effective and 5 being the most effective).

Discussion

Feedback was generally positive for both presentations. It is interesting that Chest Pain Differentials, which included student interaction in scenarios, scored higher than Cardiac Rehabilitation, which was presenter‐driven. Students favored the engaging component of both presentations, which helped with their learning. Limitations include the short time available (15 minutes) that prevented further discussions on differentials. Opportunities for improvement are to expand the differential scope and give better direction for each scenario.
Race Influences Student Experience in STEM Courses
Authors: Johnson Kathryn M.S. , Mantina Namoonga
Published: November 2023

The rapid diversification of student populations necessitates assessment of teaching practices to ensure success and equity for all students. At a small residential liberal arts college in the Midwest, persistence in science, technology, engineering, and mathematics (STEM) courses is >90%, with no difference in all‐college STEM persistence and persistence in STEM of students from traditionally underrepresented (UR) groups. However, although there is no significant difference in student persistence through STEM courses, it is unclear if students belonging to UR groups and White students are having similar and equitable experiences of inclusion and success inside and beyond the STEM classroom. To assess the effect of race on student experience in STEM courses, current students (n=20, 10 UR, 10 White) were grouped by race (UR or White) and number of STEM courses completed at the institution (1 or 4+), and participated in one of four student‐facilitated student focus groups (UR1, UR4+, White1, White4+). During the focus groups, trained and experienced student facilitators asked a series of questions relating to students’ strategies for success when struggling in STEM courses, perception of support from STEM faculty and other students, how aspects of a student's social identity have affected their experiences, and ideas for an inclusive and equitable STEM environment. Upon analysis of the responses, clear differences emerged between White and UR students. While White students who had completed only one STEM course indicated that they had never contemplated dropping the course, students belonging to UR groups reported having strongly considered dropping their STEM course. When students who had completed at least four STEM courses were asked, “What do you do when you are struggling in a STEM course?”, White students had a variety of responses, but most replied that they would form study groups. Students from UR groups indicated that if they do ask for help, they are most likely to go to the professor for assistance. However, when the same group of UR students were asked if they ever felt unwelcome or unsupported in a STEM course, their responses focused on difficulties communicating with professors. Although students belonging to UR groups are persisting at a similar rate as all students, the results of these focus groups provide evidence of important differences in those students’ STEM experiences.

Support or Funding Information

This research was supported by the Beloit College Office of the Provost and Dean of the College.
Trichloroethylene Produced Defects in the Developing Heart Involve the Transcription Factor, HNF4a
Authors: Chen Sheri , Nunez Martha , Ismail Kareem , Selmin Ornella , Runyan Raymond Bruce
Published: November 2023

Trichloroethylene, TCE, is an organic solvent known to cause congenital heart disease in animals. Prior studies showed that TCE alters calcium homeostasis and myocyte contraction and transcription of more than 4000 genes. Bioinformatic analysis of altered gene transcription suggested that Hepatocyte nuclear factor 4, alpha (HNF4α) may be a central target for TCE. HNF4a was linked to 75% of the nodes in a TCE interactome. HNF4α is transcription factor that plays a significant role in developing liver, kidney, and intestines. Though not previously reported, it can be found in the embryonic heart by PCR and western blot. To explore the connection between TCE and HNF4a, we exposed pregnant mice to TCE (11μM) or the HNF4a agonists/anatagonists Bi6015 and Benfluorex (10 μM) in maternal drinking water. Embryos were collected on day 10.5 and hearts were dissected and pooled for each litter. RNA was isolated and synthesized into cDNA for quantitative PCR analysis of marker gene expression. Fourteen markers were selected from previous microarray studies in mouse or chick embryo models as being significantly altered by TCE exposure during development. TCE inhibited expression of each the selected marker genes while Benfluorex and Bi6015 were agonists of their expression. The overlap between HNF4a and TCE regulation of the selected markers was complete. Bi6015 activity varied as an agonist or antagonist in culture depending on concentration. The data support the hypothesis that HNF4a is a direct target of TCE exposure during heart development. Further studies will explore the direct binding of TCE and Benfluorex to HNF4a.

Support or Funding Information

NIEHS P30 ES006694
Morphometric parameters to evaluate zebrafish heart: a model to study cardiovascular function.
Authors: Vargas Rafael A , Vásquez Isabel Cristina
Published: November 2023

Cardiovascular diseases are a worldwide public health problem. To date, extensive research has been conducted to elucidate the pathophysiological mechanisms that trigger cardiovascular diseases and to evaluate therapeutic options. Animal models are widely used to achieve these goals, and zebrafish have emerged as a low‐cost model that produces rapid results. Currently, a large body of research is devoted to the cardiovascular development and diverse cardiovascular disorders of zebrafish embryos and larvae. However, less research has been conducted on adult zebrafish specimens and, there is insufficient information about cardiosomatic parameters in healthy adult zebrafish. Although various somatic and cardiac morphometric parameters are widely used to assess the developmental conditions of commercial and wild fish species, to evaluate growth, maturation, nutrition, reproduction and overall health status for individuals and populations, we found that surprisingly, these parameters have not previously been fully examined in adult zebrafish. In this study, we describe a method to obtain and to evaluate morphometric parameters (of both the entire animal and the heart) of adult zebrafish. We used these data to calculate additional parameters, such as body mass index, condition factor and cardiac somatic index. This method and its results can be used as reference for researchers who are interested in future studies that aim to evaluate the pathophysiological aspects of the zebrafish cardiovascular system.

Support or Funding Information This work was supported by Research Grant from the Pontificia Universidad Javeriana, Bogotá, Colombia. Project: 5605, budget code 120112Z0401200.
Cross‐talk Among Kupffer Cells and Hepatic Stellate Cells is Critical for Kupffer Cell Activation during Liver Injury
Authors: Roth Katherine , Copple Bryan , Albee Ryan
Published: November 2023

The mechanisms that regulate Kupffer cell activation in the liver after injury are not fully understood. Our recent studies demonstrated that activation of hypoxia‐inducible factor‐1a (HIF‐1α) in hepatic stellate cells (HSCs) is critical for Kupffer cell activation after liver injury. This indicates that cross‐talk among HSCs and Kupffer cells during liver injury is critically important for Kupffer cell activation. In our current studies, we tested the hypothesis that necrotic hepatocytes activate HIF‐1a in HSCs which stimulates release of a mediator that activates Kupffer cells. Our results demonstrate that treatment of primary, mouse HSCs with necrotic hepatocytes activates HIF‐1a. Necrotic hepatocytes failed to activate HIF‐1a in HSCs isolated from MyD88 knockout mice or in HSCs pretreated with the NF‐kB inhibitor MG132. This indicated a key role for toll‐like receptor and/or interleukin receptor signaling in activation of HIF‐1a in HSCs by necrotic hepatocytes. Treatment of Kupffer cells with necrotic hepatocytes did not increase expression of inflammatory cytokines, however, treatment of Kupffer cells with conditioned medium from HSCs treated with necrotic hepatocytes upregulated Cxcl2 and other inflammatory cytokines in Kupffer cells. Pretreatment of HSCs with the phospholipase A2 inhibitor, methyl arachidonyl fluorophosphonate, prevented HSC‐mediated Kupffer cell activation, suggesting that HSCs release an arachidonic acid metabolite after exposure to necrotic hepatocytes that activates Kupffer cells. In conclusion, these in vitro studies support our in vivo findings, and expand upon these studies by demonstrating that necrotic cells activate HIF‐1α in HSCs in a Myd88‐ and NF‐kB‐dependent manner, leading to the release of an arachidonic acid metabolite that activates Kupffer cells. Further characterization of this pathway could lead to the development of therapies aimed at reducing Kupffer cell‐mediated liver injury or increasing Kupffer cell‐mediated liver repair.

Support or Funding Information

This study was supported by NIH grant 2 R01 DK073566 to Bryan Copple. Katherine Roth was supported by NIH Training Grant T32 ES007255.
A Balancing Act: Role of HNF4α and β ‐catenin in Hepatobiliary Development and Cholangiocarcinoma Pathogenesis
Authors: Walesky Chad , Goessling Wolfram
Published: November 2023

Cholangiocarcinoma (CC) is among the deadliest cancers worldwide, with a five‐year survival rate of ~15% in intrahepatic cholangiocarcinoma (ICC) and ~30% in extrahepatic cholangiocarcinoma (ECC). It is a rare cancer, affecting ~8,500 patients in the U.S. each year. The incidence of CC has increased, and there are few effective strategies for prevention, diagnosis, or treatment, indicating a significant unmet medical need. Often, altered developmental signaling can shed light on pathways that may also be altered during cancer pathogenesis. Therefore, our strategy is to understand mechanisms that are important in hepatobiliary development and examine these pathways in the context of CC pathogenesis in hopes of discovering novel therapeutic targets.

We have recently shown that loss of hepatocyte nuclear factor 4 alpha (HNF4α) contributes to the pathogenesis of CC. Further, Wnt/β‐catenin has recently been shown to be upregulated in patients with CC. Therefore, we hypothesized that an HNF4α/β‐catenin interaction may be important in the context of CC pathogenesis.

Hepatocytes and cholangiocytes are thought to originate from a bipotent progenitor cell population called hepatoblasts (Prox1‐positive). In the current study we have demonstrated the importance of proper HNF4α/β‐catenin signaling in the development of both hepatocytes and cholangiocytes within the zebrafish liver. HNF4α knockdown results in a loss of the hepatoblast marker Prox1. Interestingly, zebrafish maintain development of cholangiocytes following a loss of the Prox1‐positive hepatoblast population. Further, genetic or chemical increase in β‐catenin (APC‐/‐ ; Wnt agonist) results in a loss of HNF4α, Prox1, and a maintenance of cholangiocyte differentiation. Therefore, we hypothesize that over‐activation of β‐catenin results in a loss of HNF4α, causing hepatoblasts to preferentially differentiate into cholangiocytes.

These studies demonstrate that HNF4α/β‐catenin signaling are directly linked to regulate hepatobiliary development. Further investigation into the mechanisms by which these pathways interact can provide valuable knowledge in understanding hepatobiliary development and how alterations in these pathways may contribute to cholangiocarcinoma pathogenesis.

Support or Funding Information

Supported by a research fellowship from the Cholangiocarcinoma Foundation
Cells lining the lateral ventricle show dynamic protein expression following tail loss in the leopard gecko
Authors: McDonald Rebecca Paula , Gilbert Emily Anne Beatrice , Macdonald Alaina , Vickaryous Matthew Kenneth
Published: November 2023

Neural stem progenitor cells (NSPCs) have been identified in the adult mammalian central nervous system, specifically in the dentate gyrus of the hippocampus and in the subventricular zone of the lateral ventricle. Despite this, mammals are incapable of central nervous system regeneration following injury. In contrast, many lizard species can regenerate their spinal cord (following tail loss) and some can even repair the optic nerve and parts of the brain. However, the source of cells contributing to brain regeneration remains unclear. Here we characterize cells lining the lateral ventricle of the brain in the leopard gecko, Eublepharis macularius. We show that these ventricular lining, or periventricular cells are a potential progenitor population capable of responding to central nervous system injury. Using a bromodeoxyuridine (BrdU) pulse‐chase experiment we determined that, prior to injury, periventricular cells are slow‐cycling. Moreover, periventricular cells show widespread expression of several common NSPC markers, including the transcription factor Sox2 and the intermediate filament glial fibrillary acidic protein (GFAP). In addition, a subset of periventricular cells express the neural progenitor cell marker Sox9. Following injury to the spinal cord (as a result of tail loss) populations of periventricular cells lining the lateral ventricle continue to express NSPC markers but also begin to proliferate, as evidenced by expression of both proliferating cell nuclear antigen (PCNA) and phosphohistone H3. Intriguingly, some of these cells newly express HuC/D, a marker of neuronal differentiation. Dynamic protein expression by periventricular cells indicates that this population is activated in response to injury, and points towards their potential to participate in neural regeneration.

Support or Funding Information

Natural Sciences and Engineering Research Council (NSERC) Discovery Grant 400358