bluuebunny/crossref_metadata_2025 huggingface dataset:
Educational/Professional Partnership in the Development of a Novel Strain of S‐11 Saccharomyces cerevisiae Brewing Yeast
Authors: McGee Taylor J. , Wolyniak Michael J.
Published: May 2022

Craft beers have revolutionized the beer industry. In 2018, the craft beer market made up 20% of the sales of beer in the United States, totaling over 26 billion dollars and experiencing significant growth during a period when many traditional breweries are reporting losses or shrinkage in their market values (Gaille, 2019). Survival in an increasingly competitive market is reliant on the ability to develop novel products that match the ever‐evolving tastes of consumers. I worked with the Three Roads Brewing Company of Farmville and Lynchburg, Virginia to meet this goal using molecular biology. My institution has a preexisting relationship with Three Roads Brewery, as my research advisor has been doing similar but less intensive projects to teach students about the fundamentals of genetic engineering. The Brewmaster uses a strain of baker’s yeast (Saccharomyces cerevisiae) called S‐11 to generate a French Saison beer, a highly carbonated pale ale. My research worked to modify the S‐11 strain to generate a French Saison beer that lacks fusel alcohols while still maintaining the complex flavor profile of the rest of the beer. Because fusel alcohols are “key contributors to the intoxicating effect [of alcohol products],” this request is very common in the brewing community (Xie 2018). By removing these fusel alcohols, the Brewmaster will have a product that he feels will be more enjoyable for his consumers to drink. During the fermentation process, fusel alcohols are produced through a process called the Ehrlich pathway, which is normally responsible for the catabolism of amino acids. There is a key divergence point within the Ehrlich pathway that is especially relevant to brewers during which the fusel aldehyde is either oxidized into a fusel alcohol or reduced into a fusel acid (Hazelwood, 2008). Using molecular genetics to disrupt key genes in this pathway, I eliminated relevant proteins, including ADH1, ADH2, ADH3, ADH4, ADH6, and AAD4 in this process to force reduction as opposed to oxidation, and thereby decrease the production of fusel alcohols. Two strains, S11‐ΔAAD4 and S11‐ΔADH4 produced fewer in fusel alcohols, a 104% and 87% decrease respectively. The Southside region of Virginia in which the brewery is located is one of the most historically disenfranchised regions of the state, and by generating a strain of yeast that better satisfies the needs of local businesses, my lab was able to make a tangible impact in the support of the long‐term high‐paying jobs generated by local businesses such as Three Roads Brewing Company.
Uncovering Pre‐messenger RNA Splicing Mechanisms in Retinitis Pigmentosa
Authors: Wagle Ashna S. , Vargas Marina
Published: May 2022

Disruptions of pre‐messenger RNA splicing is a common mechanism found in human diseases, like autosomal dominant Retinitis Pigmentosa (adRP). adRP affects ~1 in 4,000 people and is associated with the breakdown and loss of cells in the peripheral retina. Genetic analyses of patients harboring adRP have linked eight proteins that are associated with the spliceosome to adRP. Currently, there are two working hypotheses for how mutations in these splicing proteins contribute to adRP. First, splicing defects are occurring ubiquitously in all tissues but are more noticeable in the retinal tissue. Alternatively, that these splicing defects are strictly happening in the retina. This study focuses on understanding how mutations in one of these splicing proteins, PRPF31, impact splicing and cellular growth. In this study, we use a mouse retinal ganglion precursor‐like cell line model to identify various mRNAs effects of adRP mutants splicing function. We are examining six prpf31 mutants adRP mutants selected from patients with adRP. Expression constructs have been prepared for wildtype and our adRP mutants and successfully transfected into the mouse cell lines. We have also established a splicing assay for our mouse photoreceptor cell studies utilizing Pladienolide B, a splicing inhibitor as a model, and will examine the PRPF31 mutants using this assay. Our ongoing work will be described.
A Ternary Antimicrobial System Assaying Bacteriophage Against Human Pathogens using C. elegans Nematodes
Authors: Bennett Evan , Page Shallee
Published: May 2022

Antibiotic resistance is a critical problem in medicine, whereby pathogenic bacteria become able to overcome medicinal treatments One approach to fighting antibiotic resistance is the use of bacteriophages (“phages”), viruses that attack bacteria, a strategy known as “phage therapy”. Phages can target specific bacteria, thereby avoiding the problem of antibiotics that kill a wide spectrum of bacteria, including neutral or beneficial bacteria. Phages have been used as antibiotics for a century, but suffer from two limitations: a) bacteria acquire phage resistance; and b) phage effects in the test tube were observed to differ from their effects in the body. Thus, we have isolated a bacteriophage against a human pathogen Salmonella enterica serovar typhimurium and have fully characterized its growth and annotated its genome. Then, we tested its ability to mitigate Salmonella infection in C. elegans.
Parasitology assays to assess Trichomonas vaginalis nucleoside ribohydrolase inhibitors
Authors: Chimarios Maria , Ventura Carlos , Stockman Brian J.
Published: May 2022

Trichomonas vaginalis is a protozoan parasite that causes trichomoniasis, a sexually transmitted disease that affects an estimated 275 million people worldwide. The Centers for Disease Control and Prevention recognizes trichomoniasis as a neglected parasitic infection, with an estimated 2.6 million prevalent infections in the United States in 2018. Clinical manifestations of infections are typically mild, but the immune system can be compromised resulting in higher susceptibility to more serious conditions such as pelvic inflammatory disease, HIV‐1, and other infections. Trichomonal infection has also been associated with prostate cancer and benign prostatic hyperplasia. Traditionally, compounds such as metronidazole or tinidazole have been used to treat trichomoniasis, however, strains resistant to these drugs are becoming more widespread. T. vaginalis requires nucleoside salvage pathways for its survival. Nucleoside ribohydrolase inhibitors thus represent a possible target for the development of new treatments with different mechanisms of action compared to metronidazole. Toward this goal, fragment screening identified nucleoside ribohydrolase inhibitors as starting points for medicinal chemistry efforts. Newly synthesized compounds are routinely evaluated for potency against purified enzymes. However, a better test of potency is antitrichomonal activity. Metronidazole‐sensitive strain B7RC2 (ATCC 50167) of T. vaginalis was cultured anaerobically at 37 ℃ in TYM Diamond’s media supplemented with streptomycin, penicillin and iron solution. Compounds were prepared as 25 mM DMSO stock solutions and tested at 100 µM. DMSO and metronidazole were used as vehicle and positive controls respectively. Parasite cellular viability was measured after 24 hours of incubation with compounds or controls. Cells were counted using a hemocytometer. Active compounds were then tested in a dose‐dependent manner to determine antitrichomonal IC50 values. Compounds with the best combination of enzyme inhibition and T. vaginalis activity were then prioritized for guiding the design of compounds to be synthesized. It is expected that this process will simultaneously optimize enzyme inhibition and antitrichomonal activity resulting in compounds with nM activity in enzyme assays, against metronidazole‐sensitive T. vaginalis, and ultimately against metronidazole‐resistant T. vaginalis.
Inhibition of Escherichia coliATP synthase by dietary pomegranate phenolics
Authors: Lakhani Muhaib , Ahmad Zulfiqar
Published: May 2022

Background

A wide range of natural and synthetic compounds bind and inhibit ATP synthase. Dietary polyphenols found in fruits, vegetables, and spices such as tea, olives, grapes, cantaloupes, garlic, and pomegranate possess antimicrobial activities. Dietary pomegranate polyphenols (DPPs) have been shown to exhibit anticancer, antioxidant, and antimicrobial properties. In the current study we are exploring the connection between antimicrobial properties of DPPs and the inhibition of microbial ATP synthase. ATP synthase is the main source of ATP in almost all organisms from bacteria to man. Selective inhibition of bacterial ATP synthase could help in combating microbial infections.

Methods

Wild type E. coli pBWU13.4 is grown on minimal media to late log phase. Cells are harvested, French Pressed and centrifuged to isolate and purify membrane bound F1FO ATP synthase. Our null control is E. coli pUC118 with deleted ATPase gene. DPPs and its analogs induced inhibition of wild‐type and mutant membrane‐bound ATP synthase is performed at room temperature. Wild‐type, mutant, and null cell growth assays on limiting glucose are evaluated at OD595 using AccuSkan Plate reader over a period of 24 hours.

Results

Variable oxidative phosphorylation and ATPase activity pattern was observed between wild type phytochemical binding site mutant strains. Western blot showed purity and integrity of wild type and mutant enzymes. DPPs and its analogs caused variable inhibition of ATP synthase. The wild‐type enzyme was fully inhibited while mutant enzyme showed variable resistance in presence of DPPs. Variation in the growth pattern was in agreement with the inhibitory profiles of isolated enzymes.

Conclusions

The inhibitory profiles of isolated E. coli membrane‐bound ATP synthase shows that DPPs are potent inhibitors of ATP synthase. Inhibition of E. coli cell growth was also related to DPPs caused ATP synthase inhibition suggesting that ATP synthase is a valuable molecular drug target to combat microbial infection.
Recipe for Success: Correlates of Student Performance in Human Gross Anatomy
Authors: Klein Harrison J. , McCarthy Sarah A. , Allen Erika
Published: May 2022

Anatomy is the first subject incoming medical students must conquer at the Lake Erie College of Osteopathic Medicine (LECOM). As anatomy is so fundamental to clinical medicine, we sought to identify the characteristics and behaviors that most strongly influence success in the Human Gross Anatomy course. First‐year medical students at the Erie, Seton Hill, and Elmira campuses were surveyed about personal wellbeing, academic habits, test taking strategies, and perceived stress. Responses were compared to final course grades via principal component analysis (PCA) and Spearman’s rank correlation test. PCA indicates that 49.66% of the variance in final grades was due to six principal components (PC). PC1 (15.56% of variance) was associated with poor study techniques, such as cramming and lack of self‐assessment, and academic confidence. PC2 (10.20% of variance) was related to student anxiety and help seeking behavior. PC3 (7.21% of variance) emphasized student interaction with lecture material. PC4 (6.14% of variance) and PC5 (5.70% of variance) accounted for group studying and preparedness for lecture, respectively. PC6 (4.86% of variance) was associated with student stress. Spearman’s rank correlation test further identified key characteristics influencing final course grade. Academic confidence (r(133) = .41, p < .0001) and life satisfaction (r(133) = .30, p = .0004) were positively correlated with final course grade whereas negative correlations were found with intra‐test anxiety (r(134) = ‐0.37, p < .0001), cramming (r(135) = ‐.31, p = .0002), and fear of failure (r(135) = ‐.39, p < .0001). These findings suggest that academic counseling for new medical students should focus on establishing effective study techniques early in the semester, properly engaging and preparing for lecture sessions, and encouraging students to utilize mental health resources at LECOM.
Understanding the role of RTL8 proteins in Ubiquilin‐2 biology
Authors: Milaganur Mohan Harihar , Trzeciakiewicz Hanna , Safren Nathaniel , Paulson Henry , Sharkey Lisa
Published: May 2022

The brain expressed ubiquilins (UBQLNs) 1, 2 and 4 are a family of ubiquitin adaptor proteins that participate broadly in protein quality control (PQC) pathways, including the ubiquitin proteasome system (UPS). One family member, UBQLN2, plays a role in numerous neurodegenerative diseases including ALS/FTD and Huntington’s disease. UBQLN2 typically resides in the cytoplasm but can translocate to the nucleus under proteotoxic stress and in disease to facilitate nuclear PQC. How UBQLN2 translocates to the nucleus and clears aberrant nuclear proteins, however, is not well understood. In a mass spectrometry screen to discover UBQLN2 interactors, we identified a family of small (13 kDa), highly homologous uncharacterized family of proteins, retrotransposon Gag‐like 8 (RTL8). We then confirmed the interaction between UBQLN2 and a representative RTL8 family member, RTL8A, both in vitro using recombinant proteins and in vivo using mouse brain tissue. We further show that RTL8A promotes nuclear translocation of UBQLN2. The robust effect of RTL8A on nuclear translocation of UBQLN2 does not extend to the other brain‐expressed ubiquilins, UBQLN1 and UBQLN4. Depletion of all RTL8 proteins in human cell lines perturbs nuclear translocation of UBQLN2 following heat shock. Finally, compared to UBQLN1 and UBQLN4, UBQLN2 preferentially stabilizes endogenous RTL8 levels in cells, supporting functional heterogeneity among UBQLNs. As novel UBQLN2 interactors that recruit UBQLN2 to specific subnuclear compartments, RTL8 proteins may regulate the role of UBQLN2 in nuclear PQC.
Microvascular anatomy and physiology laboratory class using the Australian cane toad
Authors: Murphy Timothy V. , Perry Michael A. , Ulman Lesley G. , Marden Nicole A. , Daniel Balu , Hanna Ghada , Housley Gary D.
Published: May 2022

The microvasculature is a key part of the cardiovascular system and teaching microvascular structure and function is a key part of any cardiovascular science course. The Australian cane toad Bufo marinus / Rhinella marina is an economical and widely available species within Australia which can be used to demonstrate basic principles of blood flow and microvascular anatomy and physiology in an in vivo setting.

In the laboratory class, cane toads of either gender (approx. 120 g) are anesthetized by immersion in 0.3 % ethyl 3‐aminobenzoate methanesulfonate (MS‐222) for 15 min and for the duration of the class (about 2 hr) swabs soaked in MS‐222 are placed on the skin. Level of anaesthesia is regularly assessed by the corneal reflex. At the end of the class toads are euthanized by cardiac removal (UNSW ACEC approval #19/67B). The surgical procedure involves a small incision in the upper abdomen, through which a lung, the bladder and the mesentery may be exteriorized and the microvasculature examined under a microscope equipped with a camera for video streaming to high definition monitors.

The class is designed to demonstrate principles delivered in lectures and allow students to integrate knowledge of these principles into microvascular homeostasis and adaptation to interventions. Microvascular features which can be clearly demonstrated include microvascular anatomy and structural adaptations in different tissues, including the lung (pulmonary microcirculation), urinary bladder and mesentery. Observable physiological phenomena include blood flow velocity and its’ effect blood cell behaviour, axial streaming, plasma skimming, leukocyte rolling and adhesion, vasoconstriction and the response of the mesenteric microvasculature to haemorrhage.

Student learning is assessed through pre‐ and post‐class exercises completed on‐line. The pre‐class exercise includes demonstrative videos and a brief quiz. This is intended to ensure students have read and understood the laboratory class notes, along with some basic vascular science, prior to the class, avoiding time‐wasting. The post‐class exercise includes video material recorded during the class accompanied by a quiz aimed at testing the students’ understanding of the observations made; for example, the physiological advantage of unique microvascular anatomy in certain tissues, or the mechanism underlying capillary swelling and hemodilution during haemorrhage. The videos can be edited into a package and used to deliver the class in an on‐line format if required. Thus, the cane toad preparation allows key aspects of microvascular anatomy, physiology, pharmacology and rheology to be demonstrated and taught in a clear and compelling fashion.
Effects of the Mouse Estrous Cycle on Neuropeptide S Receptor‐Mediated Behaviors
Authors: Costa Paula , Runyon Scott , Clark Stewart
Published: May 2022

The neuropeptide S receptor (NPSR) is a G protein‐coupled receptor that has been implicated in various disease states, such as anxiety and post‐traumatic stress disorder, and has shown promise as a target for the treatment of anxiety. Central brain administration of Neuropeptide S (NPS) in mice produces anxiolytic‐like effects, hyperlocomotion, and memory enhancement. Since current anxiety drugs cause undesirable side effects, development of more efficacious drugs is important. However, research into this system has been conducted solely in male rodents, despite females showing a higher prevalence for anxiety disorders. The current study investigated whether NPS‐mediated behavioral phenotypes seen in males translate to females, and whether they are affected by estrous cycle stage. Female C57BL/6 mice were intracerebroventricularly (ICV) cannulated and underwent various behavioral paradigms. Estrous cycle stage was determined through examination of vaginal cytology. Consistent with findings in male mice, NPS‐treated females displayed hyperlocomotion. Additionally, NPS‐treated females displayed anxiolytic‐like behaviors, but preliminary data suggests this effect is contingent on different phases of the estrous cycle. These results indicate that the NPSR could be a viable pharmaceutical target to treat anxiety disorders, as the anxiolytic phenotype is present in both sexes. However, in females, these effects seem to be dependent on the stage of the estrous cycle.
Epigenetic Evidence for Distinct Contributions of Resident and Acquired Myonuclei During Long‐Term Exercise Adaptation Using Timed In Vivo Myonuclear Labeling
Authors: Murach Kevin , Dungan Cory , von Walden Ferdinand , Wen Yuan
Published: May 2022

Muscle fibers are syncytial post‐mitotic cells that can acquire exogenous nuclei from resident muscle stem cells, called satellite cells. Myonuclei are added to muscle fibers by satellite cells during conditions such as load‐induced hypertrophy. It is difficult to dissect the molecular contributions of resident versus satellite cell‐derived myonuclei during adaptation due to the complexity of labeling distinct nuclear populations in multinuclear cells without label transference between nuclei. To sidestep this barrier, we utilized a genetic mouse model where myonuclear DNA can be specifically and stably labeled via non‐constitutive H2B‐GFP at any point in the lifespan. Resident myonuclei (Mn) were GFP‐tagged in vivo before eight weeks of progressive weighted wheel running (PoWeR) in adult mice (>4‐month‐old). Resident+satellite cell‐derived myonuclei (Mn+SC Mn) were labeled at the end of PoWeR in a separate cohort. Following myonuclear isolation, promoter DNA methylation profiles acquired with low‐input RRBS were compared to deduce epigenetic contributions of satellite cell‐derived myonuclei during adaptation. Resident myonuclear DNA has hypomethylated promoters in genes related to protein turnover, while the addition of satellite cell‐derived myonuclei shifts myonuclear methylation profiles to favor transcription factor regulation and cell‐cell signaling. By comparing myonucleus‐specific methylation profiling to previously published single‐nucleus transcriptional analysis in the absence (Mn) versus presence of satellite cells (Mn+SC Mn) with PoWeR, we provide evidence that satellite cell‐derived myonuclei may preferentially supply ribosomal proteins to growing myofibers and retain an epigenetic “memory” of prior stem cell identity. These data offer insights on distinct epigenetic myonuclear characteristics and contributions during adult muscle growth.
The long noncoding RNA uc.230/CUG‐binding protein 1 axis sustains intestinal epithelial homeostasis and response to tissue injury
Authors: Yu Tingxi , Kalakonda Sudhakar , Liu Xiangzheng , Han Naomi , Chung Hee K. , Xiao Lan , Jaladanki Rao N. , He Tong‐Chuan , Raufman Jean‐Pierre , Wang Jian‐Ying
Published: May 2022

BACKGROUND & AIMS

The mammalian intestinal epithelium is colonized by complex microbiota and exposed to a wide variety of luminal noxious substances. Intestinal epithelial integrity is commonly disrupted in patients with critical disorders, but the exact underlying mechanisms are unclear. Long noncoding RNAs transcribed from ultraconserved regions (T‐UCRs) control different cell functions and are involved in gut pathologies. Here, we investigated the role of T‐UCRs in intestinal epithelial homeostasis and identified T‐UCR uc.230 as a major regulator of epithelial renewal, apoptosis, and barrier function.

METHODS

Intestinal mucosal tissues were collected from mice following dextran sodium sulfate (DSS) treatment or fasting, and from patients with ulcerative colitis and Crohn’s disease. We isolated primary enterocytes from the small intestine of mice and developed intestinal organoids. Levels of uc.230 were silenced in intestinal epithelial cells (IECs) and organoids by transfection with small interfering RNAs or elevated using a plasmid vector that overexpressed uc.230. Association of uc.230 with miR‐503 was determined by biotinylated RNA pulldown assays.

RESULTS

Compared with controls, intestinal mucosal tissues from mice with DSS‐induced colitis or fasted for 48 h and from patients with ulcerative colitis had increased levels of uc.230. The uc.230 content in Crohn’s disease tissues was the same as in control tissue. Silencing uc.230 inhibited the growth of IECs and intestinal organoids and resulted in epithelial barrier dysfunction. Silencing uc.230 also increased IEC vulnerability to apoptotic cell death, whereas increasing uc.230 levels protected IECs against apoptosis. Mechanistic studies revealed that uc.230 increased CUG‐binding protein 1 (CUGBP1) by acting as a natural decoy RNA for miR‐503, which interacts with Cugbp1 mRNA and represses its translation.

CONCLUSIONS

Our results indicate that uc.230 sustains intestinal mucosal homeostasis by promoting epithelial renewal and barrier function and protects IECs against apoptosis at least in part by serving as a natural sponge for miR‐503, thereby preserving CUGBP1 expression.
Conditional Disruption of the BBSome in POMC Neurons Alter Energy and Glucose Homeostasis
Authors: Guo Deng F. , Williams Paul A. , Rahmouni Kamal
Published: May 2022

The BBSome, a protein complex composed of 8 Bardet‐Biedl syndrome (BBS) proteins including BBS1 has emerged as critical regulator of energy and glucose homeostasis. Humans and animals bearing disruption of the BBSome through loss of various BBS genes display numerous features including obesity and diabetes, but the underlying mechanisms are not well understood. We previously demonstrated that BBSome deficiency in the anorexigenic proopiomelanocortin (POMC) neurons through deletion of the Bbs1 gene lead to the development of obesity. To determine the effects BBSome deficiency in adulthood, we bred mice expressing an inducible POMCERCre with Bbs1fl/fl mice. tdTomato reporter mice were further breed with these mice to validate Cre recombinase via red tdTomato fluorescence protein expression. To induce Bbs1gene deletion we treated the POMCERCre/Bbs1fl/fl at age of 6 weeks with tamoxifen (75 mg/kg, IP, for 5 days). Importantly, both male and female POMCERCre/Bbs1fl/flmice displayed an obesity phenotype as indicated by the increased (P<0.05) body weight (41.0±1.3 vs 36.0±1.9 g in males and 29.0±1.1 vs 26.3±0.9 g in females at age of 20 weeks) and fat mass (10.8±1.2 vs. 7.8.±1.7g in males and 8.2±1.4 vs. 4.8±0.9 g in females). This was associated with increased (P<0.05) food intake (3.3.±0.3 vs. 2.9±0.2 g in males and 2.8±0.4g vs 2.5±0.2 g in females). These results indicate that hyperphagia contributes to the development of obesity in POMCERCre/Bbs1fl/fl mice. Moreover, using glucose tolerance and insulin tolerance tests we found that both male and female POMCERCre/Bbs1fl/flmice display glucose intolerance and insulin resistance. These findings demonstrate the importance of the BBSome in POMC neurons for the regulation of energy and glucose homeostasis.
Mechanism of Activation of SgrAI via Enzyme Filamentation and Mechanism of DNA Sequence Specificity Expansion
Authors: Horton Nancy , Ghadirian Niloofar , Shan Zelin , Lyumkis Dmitry
Published: May 2022

We describe a new 2.7 Å CryoEM structure of a filament forming enzyme, SgrAI, in a pre‐transition state captured just prior to nucleophilc attack. Filament forming enzymes are enzymes which form linear, helical, and even tubular structures that may act to regulate enzyme activity, play a role in signaling, nucleate phase separated membrane‐less cellular compartments, or other possible functions. SgrAI is a sequence specific DNA endonuclease which plays a role in bacterial innate immunity. The phage‐host arms race is arguably the oldest co‐evolutionary system wherein bacteria evolve systems to defend against invading DNA, and phage evolve systems to outsmart bacterial defenses. SgrAI forms filaments with a left‐handed helical symmetry of four copies per turn, which in principle could extend indefinitely. The kinetics of association and dissociation of the filament and concentrations of DNA bound SgrAI limit filament size under laboratory conditions to 2‐20 enzyme copies, and the number of recognition sites on a phage genome limits filament size in vivo. Filamentation activates the SgrAI rate constant for DNA cleavage 200‐1000 fold, and also expands the DNA sequence specificity of SgrAI to include fourteen additional recognition sequences. Our prior work has shown the details of the filamentous architecture, conformational changes in the bound DNA and protein, as well as the full enzymatic kinetic pathway including microscopic rate constants for each step. These studies suggest that the unusual regulatory mechanism involving filamentation evolved in SgrAI to maximize its anti‐phage activity while protecting the unusually long genome of its host from damaging DNA breaks. Our new structure connects the conformational change seen in SgrAI in the filamentous form to the binding of a second divalent cation in the active site thereby resulting in the observed activated DNA cleavage rate. In combination with a newly reported x‐ray crystal structure of apo SgrAI, we propose mechanisms for DNA sequence specificity expansion upon filamentation involving a disorder‐to‐order transition, energetics of DNA structure, and protein‐DNA interactions.
An organism‐wide atlas of tissue crosstalk in physical activity
Authors: Wei Wei
Published: May 2022

Physical activity is a powerful physiologic stimulus that evokes tissue crosstalk and provides benefits for multiple organ systems. However, our understanding of how exercise remodels endocrine communication pathways remains largely incomplete due to a lack of tools. We have previously developed a chemical biology technology to label the collection of secreted proteins (e.g., secretome) from blood plasma in a cell type‐selective manner in mice1. Here we apply our approach to generating a 10‐organ, 21‐cell type plasma proteome atlas of secreted proteins in response to treadmill running in mice. 3% of detected protein‐cell type pairs exhibit selective bidirectional changes in response to exercise. Our secretome atlas validates known exercise‐regulated cell type–protein pairs, identifies new cellular sources for exercise‐regulated plasma proteins and discovers undescribed protein hormones that recapitulate the potent anti‐obesity effect of physical activity. We further show that the change in protein secretion from multiple cell types is induced in part by the elevation of extracellular lactate. On the cell type level, surprisingly, PDGFRa+ cells exhibit the most dynamic exercise‐regulated secretome, exceeding that of other classically exercise‐responsive cell types including myocytes, adipocytes, and hepatocytes. Further functional analysis reveals dramatic downregulation of several complement proteins from PDGFRa+ cells and may explain the anti‐fibrotic effect of physical activity across multiple organs. Taken together, these data provide a molecular and cell type‐specific map and uncover fundamental regulation of the dynamic changes to secreted polypeptides that mediate tissue crosstalk in physical activity.

1. Wei, W*., Riley, NM* et al. Cell type‐selective secretome profiling in vivo. Nat Chem Biol (2021).
Knockout of the a4 isoform of the V‐ATPase subunit a reduces tumor growth and metastasis in vivo
Authors: Alshagawi Mohammed , Su Kevin , Collins Michael , McGuire Christina , Forgac Michael
Published: May 2022

The vacuolar ATPase (V‐ATPase) is an ATP‐dependent proton pump that transports protons across intracellular membranes and, in certain cell types including cancer cells, across the plasma membrane, where it promotes tumor survival and metastasis. V‐ATPase localization is regulated by different isoforms of subunit a, and it has been shown that the a3 and a4 isoforms both target V‐ATPases to the plasma membrane and are critical for migration and invasion of different cancer cell lines in vitro. By using CRISPR mediated gene editing to selectively disrupt each of the 4 isoforms (a1‐a4), we previously reported that the a4 isoform appears to be important for plasma membrane targeting of the V‐ATPase and migration and invasion of 4T1‐12B breast cancer cells in vitro. Using BALB/C mice as an animal model, we have investigated the effect of disruption of different isoforms of subunit a on tumor growth and metastasis in vivo. The results suggest that targeting plasma membrane V‐ATPases represents a novel therapeutic approach to inhibition of tumor growth and metastasis in breast cancer patients.
Endothelial Cell Heterogeneity Between Conduit and Resistance Mesenteric Arteries
Authors: Morin Emily E. , Gonzalez Bosc Laura , Kanagy Nancy L. , Naik Jay S.
Published: May 2022

The vascular endothelium contains morphologically similar cells throughout, but individual cells along the length of a single vascular tree or in different regional circulations function quite differently. When observations made in large arteries are extrapolated to explain the function of endothelial cells (EC) in the resistance vasculature/microcirculation, only a fraction of these observations are consistent between artery sizes. To what extent EC from different arteriolar segments of the same tissue are phenotypically different at the single‐cell level remains unknown. Therefore, single‐cell RNA‐seq was performed using a 10X Genomics Chromium system. Cells were enzymatically digested from large (>300 µm) and small (<150 µm) mesenteric arteries to generate single‐cell suspensions from 6 adult male Sprague‐Dawley rats which were pooled to create two samples (3 rats/sample). Cell Ranger software was used for initial data processing, including alignment, filtering, and unique molecular identifier counting (10x Genomics). After initial data processing, Seurat, an R package for single‐cell sequencing analysis, combined datasets and conducted a comparative analysis across the phenotypes. Seurat’s data integration workflow transforms individual datasets into a shared matrix while controlling batch effects and technical differences. After normalized integration, the dataset was integrated and scaled before unsupervised cell clustering and cluster visualization using uniform manifold approximation and projection (UMAP) plots. Differential gene expression (DEG) allowed us to infer the biological identity of individual cells. EC clusters from large and small arteries were further subclustered. Our analysis revealed 601 DEG genes between conduit and resistance arteries. Gene ontology analysis of scRNA‐seq data discovered 449 that were different between large and small arteries. We identified six unique EC subpopulations, with DEG genes and pathways identified for each cluster. The proportion and absolute numbers of EC in each cluster differed between conduit and resistance arteries. These results and this dataset allow the discovery and support novel hypotheses needed to determine the mechanisms that determine the phenotypic heterogeneity between conduit and resistance arteries.
Activation of select airway afferent subpopulations evokes cardiopulmonary reflexes
Authors: Darcey Teresa , Taylor‐Clark Thomas
Published: May 2022

Activation of airway sensory nerves causes respiratory and autonomic reflexes. The majority of the sensory nerves are only sensitive to noxious stimuli, such as inflammation, infection, irritants and pollutants. Activation of these nociceptive sensory nerves evokes protective mechanisms such as apnea, cough and bradycardia that can contribute to disease morbidity. Airway nociceptive sensory nerves are heterogeneous with respect to gene expression and neuroanatomy, and our objective is to characterize the specific reflexes evoked by activation of specific afferent subsets. To selectively activate these vagal afferent subpopulations in vivo, mice were exposed to nebulized selective stimuli such as capsaicin (transient receptor potential (TRP) vanillin 1 (V1) agonist), allyl isothiocyanate (AITC, TRP ankyrin 1 (A1) agonist) and clozapine‐N‐oxide (CNO, selective agonist for the designer receptors exclusively activated by designer drugs (DREADD) stimulatory receptor hM3Dq). hM3Dq expression was selectively expressed in sensory subpopulations under the control of cre recombinase in TRPV1‐cre (all nociceptors) and Tac1‐cre (peptidergic nerves). ECGs were recorded via radiotelemetry following implantation of biopotential sensing modules, and respiration was measured via whole body plethysmography. Our data indicate that stimulation of nociceptive subpopulations selectively evokes bradypnea and bradycardia in freely moving wild type mice.
Cardiac baroreflex sensitivity and heart rate variability are lower in humans with high affinity hemoglobin compared to typical affinity hemoglobin
Authors: Pruter Wyatt W. , Klassen Stephen A. , Dominelli Paolo B. , Wiggins Chad C. , Senefeld Jonathon W. , Roy Tuhin K. , Joyner Michael J. , Baker Sarah E.
Published: May 2022

Background

Humans with high O2 affinity hemoglobin (HAH) have a markedly lower partial pressure of O2at which 50% of hemoglobin is saturated (P50) and a leftward shift of the oxy‐hemoglobin dissociation curve. Humans with HAH have attenuated heart rate responses during both acute and chronic hypoxic exposure relative to humans with typical P50. Despite the attenuated heart rate responses to hypoxia, there are typically no blood pressure differences found in humans with HAH relative to humans with typical P50. Due to the blunted heart rate and normal blood pressure responses to hypoxia in humans with HAH, this study aimed to investigate the impact of HAH on cardiac autonomic function during normoxia and hypoxia. We hypothesized that humans with HAH would have lower cardiac baroreflex sensitivity (cBRS) and heart rate variability (HRV) during normoxia and hypoxia relative to individuals with typical P50.

Methods

Nine humans with a HAH mutation (6 female, Age: 40±12yrs, P50: 16±1 mmHg) and 12 controls with typical affinity hemoglobin (6 female, Age: 40±10yrs, P50: 26±1 mmHg) laid supine for 10‐minutes prior to 20‐minutes of isocapnic hypoxia, titrated to reduce arterial O2 pressure to ~50 mmHg (Avg. FiO2 = 11.9±0.0%). Beat‐to‐beat arterial pressures (arterial catheter) and heart rate (electrocardiogram) were continuously recorded. Data were averaged in five‐minute segments starting with the last five minutes of supine normoxic rest. Spontaneous cBRS was quantified using the sequence method. HRV was measured in time and frequency domains. Standard deviation of the RR interval (SDRR) was calculated as the standard deviation of all RR intervals in a data segment. Low frequency (LF) HRV was calculated using Fast Fourier Transformation and assessed as absolute spectral intensity in the LF band (0.04‐0.15Hz).

Results

Arterial oxygen (PaO2) and carbon dioxide (PaCO2) pressures were not different between humans with HAH and controls during normoxia or isocapnic hypoxia [PaO2: (HAH vs. Control): 48±3 vs. 47±2 mmHg, p=0.612; PaCO2: (HAH vs. Control): 40±3 vs. 39±1 mmHg, p=0.328] exposure. cBRS was lower in humans with HAH compared to controls in normoxia and throughout isocapnic hypoxia [normoxia (HAH vs. Control): 7±4 vs. 16±10 ms/mmHg; hypoxia minutes 15‐20 (HAH vs. Control): 4±3 vs. 14±11 ms/mmHg; Hb type, p=0.024]. Several metrics of HRV were lower in humans with HAH relative to control (SDRR: Hb type, p≤0.001; LF: Hb type, p=0.030) and HRV was blunted to a similar extent in both groups with hypoxia (SDRR: time effect, p=0.009).

Conclusion

The findings of this study are twofold. Compared to controls, humans with HAH have: 1) attenuated cardiac baroreflex sensitivity and 2) lower heart rate variability in both normoxia and isocapnic hypoxia.
ZO‐1 Knockdown in MDCK Type II Cells Decreases Leak Pathway Opening Size and Increases Opening Number
Authors: Monaco Ashley , Axis Josephine , Amsler Kurt
Published: May 2022

Paracellular solute movement across epithelial cell sheets is divided into at least two pathways, the Pore Pathway (high capacity, small ions and solutes, charge selective) and the Leak Pathway (low capacity, large molecules, charge insensitive). Improved understanding of the Leak Pathway is essential to evaluate its role in epithelial physiology and pathophysiology, as well as, its potential use as a pathway for the delivery of macromolecule drugs. Depletion of the tight junction‐associated cytoplasmic protein Zonula Occludens‐1 (ZO‐1) in the dog kidney epithelial cell line, MDCK Type II, increases Leak Pathway apparent permeability (Papp). There are multiple parameters that could affect Leak Pathway Papp, including the density of the pores/openings (number/unit membrane area), their length, their tortuosity, and their size (radius). A theoretical computational analysis revealed marked differences in the effect of pore size versus pore density on Papp­ as a function of solute size. We, therefore, compared the dependence of Papp on solute size in populations of wild type MDCK Type II cells, ZO‐1 knockdown MDCK Type II (ZO‐1 KD) cells, and ZO‐2 knockdown MDCK Type II (ZO‐2 KD) cells. The flux of fluorescein‐dextrans of different sizes across monolayers of wild type MDCK Type II, ZO‐1 KD, and ZO‐2 KD cell populations grown on permeable supports was measured as a function of time. Flux was linear as a function of time for all solutes across all cell lines. As expected, flux rate decreased with increasing fluorescein‐dextran Stokes radius (3 kDa ‐ ~12 Å to 70 kDa ‐ ~60 Å) in all three cell lines. For the smaller fluorescein‐dextrans, ZO‐1 KD cell populations exhibited several‐fold higher Papp values than wild type MDCK Type II cells. As solute size increased, however, the extent of the increase over the wild type MDCK Type II cells diminished progressively. In contrast, the Papp values for the wild type MDCK Type II cell populations and the ZO‐2 KD cell populations were similar throughout the fluorescein‐dextran size range. These results revealed that Papp values decreased more rapidly as a function of solute size in ZO‐1 KD cell populations than in wild type MDCK Type II cell populations or ZO‐2 KD cell populations. While we cannot determine Leak Pathway opening radius using this approach, our results argue that the Leak Pathway openings in ZO‐1 KD cell populations are smaller than those in either wild type MDCK Type II cell populations or ZO‐2 KD cell populations. Since the ZO‐1 KD cell populations exhibit an increased flux rate for smaller fluorescein‐dextrans, this suggests that the ZO‐1 KD cell populations have an increased number of Leak Pathway openings compared to wild type MDCK Type II cell populations and ZO‐2 KD cell populations. Differences in opening length and/or tortuosity may also play a role in the observed differences in solute flux rates.
The Effect of Tauroursodeoxycholic Acid (TUDCA) Treatment on Pregnancy Outcomes and Uterine Artery Function in a Rat Model of Advanced Maternal Age
Authors: Pasha Mazhar , Kirschenman Raven , Wooldridge Amy , Spaans Floor , Cooke Christy‐ Lynn , Davidge Sandra T.
Published: May 2022

Introduction

Advanced maternal age (AMA; ≥35 years) increases the risk of pregnancy complications and adverse pregnancy outcomes such as fetal growth restriction. Endoplasmic reticulum (ER) and oxidative stress have been linked to vascular dysfunction (i.e. reduced nitric oxide [NO] & increased endothelin‐1 [ET‐1] levels) in aging. Tauroursodeoxycholic acid (TUDCA) is an ER and oxidative stress inhibitor and has been shown to improve embryo development in vitro. However, whether treatment with TUDCA can help improve uterine vascular function and pregnancy outcomes in AMA has not been investigated. We hypothesize that TUDCA treatment will improve pregnancy outcomes and vascular function in a rat model of AMA.

Methods

Young (4 months) and aged (9.5 months of age; ~35 years in humans) pregnant control or TUDCA‐treated rats were studied on gestational day 20 (term=22 days). Following euthanasia, pregnancy outcomes (litter size, resorption sites, fetal weight, and placental weight) were recorded, and main uterine arteries were isolated. Endothelium‐dependent vasorelaxation to methacholine (MCh) was assessed by wire myography in the presence/absence of pan NO synthase inhibitor (L‐NAME). In addition, big‐endothelin‐1 (bET‐1) vasoconstriction responses were evaluated, in the presence/absence of endothelin converting enzyme (ECE) inhibitor (CGS). Data summarized as maximum vasodilation response (Emax) and analyzed by two‐way ANOVA with Sidak’s post‐test, p<0.05 was considered significant.

Results

Fetal body weights were reduced in AMA control rats compared to young dams (p<0.017), while TUDCA treatment tended to increase fetal body weight in AMA rats (p=0.067); TUDCA did not affect fetal body weight in the young dams. Placental weights, litter size and number of resorptions were similar in all the groups. The maximum relaxation (Emax) to MCh was impaired in the AMA dams compared to young dam (p=0.047), and this reduced relaxation was not evident in the TUDCA treated AMA dams. Ex vivo treatment with L‐ NAME showed an increased contribution of NO in young TUDCA treated rats (ΔEmax [differences in Emax]; p=0.043) compared to young control rats but not in AMA rats. Ex vivo treatment with CGS demonstrated reduced ECE conversion in young TUDCA treated rats (Emax; p<0.001), while CGS had no effect in the young control dams or both AMA groups.

Conclusion

Our study assessed TUDCA as an intervention to improve the pregnancy outcomes in a rat model of AMA. An increased fetal body weight suggests a beneficial effect of TUDCA in AMA pregnancies. However, contrary to our hypothesis, in young dams, TUDCA increased NO contribution and reduced ECE effect, but did not affect uterine artery function in AMA dams. Nevertheless, TUDCA has the potential to improve pregnancy outcomes in complicated pregnancies, but further studies are warranted.
Reversal chemotherapeutic resistance of triple‐negative breast cancer via functionally inhibiting RNA‐binding protein HuR
Authors: Wei Lanjing , Zhang Qi , Zhong Cuncong , Aubé Jeffrey , Welch Danny R. , Wu Xiaoqing , Xu Liang
Published: May 2022

Chemotherapy remains the primary option for the systemic treatment of triple‐negative breast cancer (TNBC). However, chemoresistance frequently occurs with the conventional use of chemotherapeutic drugs, resulting in a poorer prognosis and a higher recurrence rate of TNBC than other subtypes of breast cancer. Therefore, overcoming chemoresistance is a critical challenge to conquer for the successful treatment of TNBC. The RNA‐binding protein Hu antigen R (HuR) is a posttranscriptional regulator. It can stabilize mRNA by binding to U‐ or AU‐rich elements (ARE) mainly in 3’ untranslated region (UTR) of mRNA and therefore upregulate the translation most of the time. The encoded proteins of HuR target mRNAs are implicated in multiple cancer hallmarks, including therapeutic resistance. The cytoplasmic accumulation of HuR is reported to contribute to chemoresistance in several types of cancer cells, and HuR inhibition sensitizes cells to chemodrugs. We hypothesize that inhibition of HuR function by disrupting its interaction with mRNA can accelerate the decay of target mRNA and thus reduce the translation level of proteins responsible for chemoresistance.

Recently, our lab reported a small molecule HuR inhibitor, KH‐3, which potently inhibits HuR function by disrupting HuR‐mRNA interactions. In this study, we investigate the roles HuR played in the chemoresistance of TNBC and evaluate whether HuR inhibition by KH‐3 can enhances the chemotherapy efficacy. To determine whether HuR inhibition overcomes acquired chemoresistance of TNBC, two MDA‐MB‐231 cell sub‐lines resistant to docetaxel (231‐TR) or doxorubicin (231‐DR) were generated. Compared to the parental cell line, two sub‐lines exhibit similar sensitivity to KH‐3, and KH‐3 re‐sensitizes chemoresistant cells to docetaxel or doxorubicin in the MTT‐based cytotoxicity assay and the colony formation assay, indicating that HuR inhibition can overcome the acquired chemoresistance. The combination index suggests that the combination of KH‐3 with docetaxel or doxorubicin has a synergistic effect. The in vivo efficacy studies in both MDA‐MB‐231 and 231‐TR orthotopic xenograft mouse models confirm that KH‐3 synergizes docetaxel treatment. Mechanistically, several HuR direct target mRNAs implicated in chemoresistance were found upregulated in the resistant cells, which were reversed by KH‐3 treatment. The detailed molecular mechanisms of how KH‐3 sensitized TNBC cells to chemodrugs are being investigated. This research suggests that HuR inhibition is a promising strategy for overcoming chemoresistance in TNBC.