bluuebunny/crossref_metadata_2025 huggingface dataset:
Regulation of activated Protein C/PAR1 endothelial cytoprotective signaling by G protein‐coupled receptor kinases
Authors: Gonzalez Ramirez Monica L. , Lin Ying , Qin Huaping , Birch Cierra , Trejo JoAnn
Published: May 2022

Endothelial dysfunction is a hallmark of inflammation and associated with vascular diseases. Our goal is to define pathways by which the endothelium can resist injury and disruption to facilitate the advancement of new targets for therapeutic development. Activated Protein C (APC) has been shown in preclinical studies to increase endothelial barrier protection and reduce inflammatory responses and signals via protease‐activated receptor‐1 (PAR1). APC enhances endothelial cytoprotection through cleavage of PAR1 at an N‐terminal arginine (R)‐46 site, that is distinct from the canonical thrombin cleavage site at R‐41. This results in the generation of a distinct tethered ligand that promotes PAR1 bias signaling. We discovered that APC/PAR1 signals preferentially via b‐arrestin2 (b‐arr2), rather than heterotrimeric G proteins to promote endothelial barrier protection. The objective of this study is to develop a mechanistic understanding of how APC/PAR1 generates b‐arr2 cytoprotective signaling. G protein‐coupled receptor kinases (GRKs) phosphorylate thrombin‐activated PAR1 and facilitate β‐arrestin‐mediated desensitization. However, contribution of GRKs to β‐arrestin driven APC/PAR1‐induced cytoprotection is unclear. We hypothesize that compartmentalization of APC/PAR1 together with GRK5 is essential to initiate b‐arr2 recruitment and biased signaling. Here we will present studies examining the function and mechanism by which GRKs regulate endothelial cytoprotective responses using GRK‐specific siRNA to assess function as well as confocal immunofluorescence imaging and biochemical sucrose fraction to examine GRK localization and compartmentalization in caveolae. The work is expected to provide important new insight into the role of GRKs in regulating APC/PAR1 cytoprotection in human cultured endothelial cells.
N‐terminal Intrinsic Disorder of G Protein Gamma Subunits Is Important for Their Function as Governors of G Protein Signaling
Authors: Su Xinya , Li Wei , Pang Yui Tik , Gumbart James C. , Torres Matthew
Published: May 2022

Emerging evidence suggests that heterotrimeric G protein gamma subunits (Gγ) are important governors of G protein signaling, a function that is mediated through GPCR‐ and pH‐dependent combinatorial phosphorylation of their intrinsically disordered N‐terminal tails (Gγ‐Nt) that controls Gβγ/effector interactions and signaling. Intrinsic disorder is a universally conserved structural feature of all Gγ subunit N‐termini, which prompted us to hypothesize that, beyond phosphorylation, intrinsic disorder itself is inherently important to the signal‐governing roles of Gγ subunits. To test this hypothesis we devised a strategy in which single amino acid substitutions are sequentially introduced into the Gγ tail, producing a series of isoforms that proceed step‐wise from a fully‐disordered to fully‐ordered (α‐helical) Nt tail structure. As a control for the increasing mutation load, we compare these mutants to those in which the same number of amino acid substitutions are incorporated that do not alter the inherent structural disorder of the tail. These mutant isoforms were then structurally analyzed by circular dichroism (CD) in vitro, by molecular dynamics (MD) simulation in silico, and by functional analysis of Gβγ‐dependent molecular signaling in vivo. Here, we apply this approach to the yeast Gγ subunit, Ste18. CD and MD analyses of Ste18‐Nt tail isoforms indicate that a successful transition from a fully‐disordered to fully‐ordered state is achievable through precise point mutation. Replacing the wild type Gγ subunit with each of the mutant isoforms in yeast, we further show that intrinsic disorder of Gγ‐Nt controls the stability of the Gγ subunit in a manner that is proportional to the loss of intrinsic disorder in vivo. pH‐dependent phosphorylation at Ser3 in the tail is largely unaffected by these changes. However, unexpectedly, we found that the GPCR‐dependent phosphorylation site, Ser7, becomes pH‐sensitive in response to changes in tail structure. Ongoing experiments reveal the effects of Ste18‐Nt tail structure on the interaction of yeast Gbg with its primary effector Ste5, and subsequent effects on activation of MAPKs, which have been shown to be highly sensitive to Gγ‐Nt tail phosphorylation previously. Taken together, these data provide evidence that intrinsic structural disorder plays a direct role in functionality of Gγ subunits as governors of G proteins signaling and substantiates the rationale for exploring similar roles for these tails in mammalian Gβγ‐dependent signaling pathways.
Reviving Microscopy in Paramedical Anatomy Courses Using Web‐based Virtual Microscopy and Interactive Exercises
Authors: Ressetar Holly , Palmer Bruce
Published: May 2022

As the paramedical programs evolved at West Virginia University, large combined anatomy courses with gross and microanatomy laboratories were replaced with predominantly didactic courses geared towards each individual field. The first year pharmacy anatomy course and the dental hygiene oral histology both place emphasis on histology but the courses did not give students the opportunity to experience student‐directed active learning using a microscope. We created a series of laboratory exercises using a virtual microscopy website developed within our department that features a large collection of annotated normal and pathological histological specimens. The website mimics the actual use of a microscope and facilitates student independent study. Students were provided with laboratory guidelines and faculty video tutorials to facilitate their use of the programs and understanding of histology. In addition, we created identification exercises using a separate website where students could view and label specimens, enter text and share comments with peers and faculty via a chat room. Students readily adapted to using the virtual microscope and the on‐line platform proved to be extremely advantageous when COVID restricted personal contact with students. There was renewed engagement with students taking the lead in interpretation of specimens and group discussion. Students were allowed unlimited attempts in the identification exercises which promoted “educated guessing” and continual faculty feedback. Incorporation of a laboratory component into the courses allowed students to practice their interpretational skills and improved overall academic performance by testing students on other facets of learning. Improvement in academic performance of the dental hygiene students was most pronounced since the laboratory component was integrated throughout the semester. Student feedback was very positive encouraging our further development of clinically‐related exercises.
G protein‐coupled receptor regulation of the alpha‐arrestin ARRDC3
Authors: Wedegaertner Helen , Trejo JoAnn
Published: May 2022

Adaptor proteins are an important class of proteins that help to maintain proper spatiotemporal control of cell signaling. One of the best‐studied families of adaptor proteins are the ubiquitously expressed beta‐arrestins, which are known to be key regulators of G protein‐coupled receptor (GPCR) function. Recently another family of arrestins, termed the alpha‐arrestins, were discovered and shown to be expressed in mammalian cells. While alpha‐ and beta‐arrestins share minimal sequence homology, they are predicted to be structurally similar, and therefore may be subject to common regulatory mechanisms. However, while GPCR regulation of beta‐arrestin function has been well characterized, it remains entirely unknown how GPCRs might regulate alpha‐arrestins, such as ARRDC3. We recently showed that ARRDC3 is a multifunctional adaptor that distinctly regulates protease‐activated receptor‐1 (PAR1) signaling and trafficking, but it remains unknown how these distinct ARRDC3 functions may be regulated. PAR1 is a GPCR for the protease thrombin and is known to regulate a diverse array of physiological functions in health and disease, thus, it is important to better understand the molecular mechanisms that control PAR1 function. The objective of this study is to understand the mechanisms that control ARRDC3 multifaceted functions. We hypothesize that GPCRs regulate ARRDC3 function via distinct processes that enable diverse functions. Using biochemical and microscopy‐based techniques, we show that PAR1 may regulate ARRDC3 through modulation of post‐translational modifications, including ubiquitination. We also show that ubiquitin is critical for regulating distinct ARRDC3 functions, including regulation of GPCR signaling and trafficking, through various mechanisms that may include control of protein stability, subcellular localization, and protein‐protein interactions. Additionally, ARRDC3 has previously been shown to act as a tumor suppressor in highly invasive triple‐negative breast cancers. Thus, in future studies we will assess the role of ubiquitination on ARRDC3 tumor suppressor function in vitro in vivo. The results of these studies will advance our understanding of how alpha‐arrestins may contribute to GPCR regulation and may lead to the identification of novel targets for therapeutic development to treat metastatic breast cancer.
The Promoting Active Learning & Mentoring (PALM) Research Coordination Network Supports the Design and Implementation of Active Learning Strategies, while Increasing Educator Confidence
Authors: Johnson Kathryn M. , Cary Tawnya L. , Wick Sue , Stieben Margaret
Published: May 2022

The Promoting Active Learning & Mentoring (PALM) Research Coordination Network supports current and aspiring biological science educators (PALM Fellows) who seek to incorporate active learning strategies into their undergraduate courses. Through the collaboration of professional scientific societies and educators, PALM Fellows are provided one‐on‐one guidance by PALM Mentors, educators experienced in curricular reform. This 4–12‐month mentorship includes PALM Fellows observing, either virtually or in‐person, PALM Mentors applying active learning strategies in their own classrooms. PALM Fellows work with their PALM Mentors to design, implement, and assess active learning course modules adapted to the needs of the PALM Fellows and their students. Additional mentorship and professional network development are provided to PALM Fellows in the form of regular journal club meetings and group gatherings with other current and former PALM Fellows and Mentors. Over five years, 54 PALM Fellows have received individualized mentoring and assistance in the development and implementation of active learning strategies. At the completion of one year as a PALM Fellow, participants (n=17) were surveyed to determine changes in their teaching approaches that can be attributed to the PALM Network. When asked to rate their confidence since starting their work with the PALM Network, 82% responded that they have more confidence (as indicated by the response of a 7 or 6 on a 7‐point Likert scale) in their understanding of active learning, 88% have more confidence in their ability to incorporate active learning in their teaching, and 71% have more confidence in their understanding of backward design. Additionally, after one year in the PALM Network, all participants (given the option of yes, no, or not sure) responded that they had developed an active learning course module, and 82% had implemented their active learning module in their own classroom. Therefore, the PALM Network successfully supports the design and implementation of active learning strategies in the classrooms of educators seeking to reform their teaching, as well as increasing educator confidence in their ability to do this work.
The Role Lnc‐RHL in Hepatic Cell Lineage Decisions and Proliferation in HepaRG Cells
Authors: Lee SooWan
Published: May 2022

Long noncoding RNAs (lncRNAs) are functional transcripts with lengths of over 200 nucleotides that do not encode proteins. LncRNAs have received wide attention as key regulators of stem cell proliferation and differentiation, but knowledge of the specific role of lncRNAs in hepatocyte differentiation is still limited. Recently, our group identified and characterized a novel lncRNA, lnc‐RHL (regulator of hepatic lineage), which plays a critical role in the differentiation of bipotent hepatoblasts to hepatocytes and cholangiocytes. Lnc‐RHL maps to human chromosome 11q23.3 in an apolipoprotein (APO) gene cluster and consists of a 670‐base pair polyadenylated lncRNA with two exons and an intron. We knocked down lnc‐RHL expression with a doxycycline (Dox) inducible shRNA lentiviral vector in HepaRG cells to investigate the role of lnc‐RHL. Interestingly, knockdown of lnc‐RHL inhibited differentiation to hepatocytes but not to cholangiocytes in HepaRG cells. We observed a significant reduction in the number and size of hepatocyte colonies upon Dox induction. Moreover, knockdown of lnc‐RHL downregulated the mRNA levels of hepatocyte markers such as HNF4a and albumin, and many APO genes including APOA1, APOC3, and APOA5 but upregulated mRNA levels of the cholangiocyte marker, cytokeratin 7 (CK7). We also performed RNA‐sequencing analysis and ingenuity pathway analysis (IPA) to further investigate functions of lnc‐RHL and the top impacted canonical pathways due to loss of lncRHL during HepaRG cell differentiation. IPA results showed that several cell cycle‐ and proliferation‐associated canonical pathways were significantly regulated. In addition, a key proliferation‐associated transcription factor, FOXM1 was markedly upregulated after the loss of lnc‐RHL. In summary, this study explores the role and expression of lnc‐RHL in HepaRG cell differentiation and shows that it is required for the proper production of hepatocytes in these cells.
Fenofibrate induced‐hepatomegaly and liver regeneration is PPARα dependent and partially related to the activation of YAP pathway
Authors: Fan Shicheng , Gao Yue , Qu Aijuan , Jiang Yiming , Xie Guomin , Yao Xinpeng , Yang Xiao , Yagai Tomoki , Tian Jianing , Wang Ruimin , Gonzalez Frank J. , Huang Min , Bi Huichang
Published: May 2022

Background and Objective

Fenofibrate, an agonist of peroxisome proliferator activated receptor α (PPARα), has been widely used as a lipid‐regulating agent in clinical practice. Recent studies found that fenofibrate can significantly induce hepatomegaly in mice, but the involved mechanisms remain unclear.

Methods

The effect of fenofibrate on hepatomegaly and liver regeneration was investigated in several strains of genetically‐modified mice including Pparafl/fland PparaΔHep mice. Staining assays were performed to investigate the effect of fenofibrate on hepatocyte size and proliferation during hepatomegaly and liver regeneration. AAV‐Yap shRNA mice model was further used to determine the role of YAP in PPARα‐induced hepatomegaly. Western blot analysis was used to detect the protein expressions of YAP signaling pathway as well as other factors potentially involved in fenofibrate‐induced hepatomegaly.

Results

The study demonstrated that fenofibrate significantly induced liver enlargement in wild‐type and Pparafl/fl mice, while this effect was abolished in hepatocyte‐specific Ppara‐deficient (PparaΔHep) mice. Furthermore, fenofibrate significantly promoted liver regeneration in wild‐type mice following 70% partial hepatectomy (PHx). Staining assays revealed that fenofibrate promoted hepatocyte enlargement around the central vein area and hepatocyte proliferation around the portal vein area in wild‐type, Pparafl/fl and PHx mice. Fenofibrate regulated protein expressions of YAP and its downstream targets (CTGF, CRY61 and ANKRD1) as well as proliferation‐related proteins (CCNA1, CCND1 and CCNE1). Suppression of YAP using AAV‐Yap shRNA repressed fenofibrate‐induced hepatomegaly, as well as hepatocyte enlargement and proliferation. Other factors might also participate in fenofibrate‐induced hepatomegaly, including MYC, KRT23, RHOA and RAS.

Conclusion

These studies revealed a novel role of fenofibrate in promoting liver enlargement and regeneration, which is PPARα dependent and partially related to the activation of YAP signaling pathway. These findings provide clinical relevance of fenofibrate as a potential medication for promoting liver regeneration.
Nucleolar function is regulated by the mitochondrial protein sulfite oxidase (SUOX)
Authors: McFadden Emily J. , Baserga Susan J.
Published: May 2022

Ribosome biogenesis occurs in the nucleolus and relies upon RNA Polymerase I (Pol I) transcription of the rDNA into rRNA. This process is critical for normal cells but is dysregulated in diseases such as Alzheimer’s disease and cancer. To date, however, we have not yet explored the potential relationship between nucleoli and mitochondria. Mitochondria are critical for cellular energy and mitochondrial disorders exhibit dysregulation of nutrient and energy homeostasis. Like nucleoli, mitochondria are also dysregulated in Alzheimer’s disease and cancer, suggesting we may have overlooked a regulatory relationship between these two organelles.

In pursuing novel regulators of nucleoli number and function, we have identified a mitochondrial protein that when individually depleted by siRNA in breast epithelial cells (MCF10As) causes a change in nucleolar number – sulfite oxidase (SUOX). Upon SUOX depletion, the nucleolar number decreases from the typical 2‐3 nucleoli to 1 nucleolus. SiRNA‐mediated SUOX depletion reduces nucleolar rRNA levels in a high‐throughput 5‐EU assay (Bryant et al. 2021) and reduces global protein translation by a puromycin incorporation assay, indicating defective nucleolar function. Furthermore, combined metabolomic and transcriptomic analyses following siRNA‐mediated SUOX depletion reveal disruption in the activated methyl cycle, consistent with aberrant ribosome biogenesis. Experiments are underway to validate defective methylation experimentally at specific steps in ribosome biogenesis. The glutamate cycle is also disrupted, congruent with the presentation of the recessive nervous system disorder Isolated Sulfite Oxidase Deficiency (ISOD). Thus, we have defined a novel mitochondrial regulator of nucleolar number and function, nucleolar rRNA biogenesis, and protein translation with implications in human disease. Our work will open up a new avenue of nucleolus‐mitochondria relationships and regulation, and potentially connect mitochondrial disorders with ribosome biogenesis as well as identify how mitochondrial homeostasis is critical for nucleolar function.
Discovery of Novel Inhibitors for Mycobacterium tuberculosis D‐alanine: D‐alanine Ligase through Virtual Screening
Authors: Vu Haily , Gilari Krithika , Sathiyamoorthy Vaishnavi , Beckham Josh
Published: May 2022

Tuberculosis (TB), although not as common in high‐income countries due to vaccines and antibiotic therapy, remains one of the highest causes of death in low‐income countries. However, overuse of antibiotics has led to the emergence of antibiotic‐resistant strains of the disease. To offset these issues, potential small molecule drug compounds must be identified to combat this bacteria in order to further the drug development process. Tuberculosis can be caused by a variety of other strains of the bacterium, but Mycobacterium tuberculosis is the most common and the most virulent. The D‐alanine:D‐alanine ligase (Ddl) enzyme is an attractive target for drug discovery as it catalyzes a crucial reaction that is necessary for the survival of the bacteria: the formation of the D‐alanyl‐D‐alanine dipeptide from adenosine triphosphate (ATP) and two D‐alanine amino acids. This dipeptide complex then plays a pivotal role in crosslinking peptidoglycan chains during the assembly of the bacterial cell wall. Drugs that target Ddl currently exist on the market, but antibiotic resistance has reduced their efficacy. A crystallography structure of Ddl from M. tuberculosis(PDB ID: 3LWB) was used in conjunction with the virtual screening program GOLD (Genetic Optimization for Ligand Docking by the Cambridge Crystallographic Data Center) and ICM (Internal Coordinate Mechanics by Molsoft) to search for ligands that had a predicted strong binding affinity to the enzyme’s active site. Both the D‐alanine and the ATP sites were targeted in this study. Various ligand libraries were screened and ranked based on binding interactions with the active site. The docking poses of the top‐ranking ligands were further verified through the molecular visualization program PyMol (Schrodinger). The highest‐ranking ligand targeting the ATP site from GOLD, SAM001246657, came from the National Institutes of Health’s Clinical Collection library (NIH) and received an overall fitness score of 104.4. The highest‐ranking compound from the ICM screen targeting the ATP site was SPB06710SC from the HitFinder 9 library and received a score of ‐36.32. For the D‐alanine site, the highest docking ligand from GOLD was ZINC14539627 with a score of 100.77. From ICM, the highest scoring ligand was ZINC11535505 with a score of ‐32.27. Both ligands are from the Zinc ChemBridge library. The inhibitory effects of these top‐scoring ligands may be further validated through wet‐lab research using binding assays and enzyme inhibition assays.
Examination of the Interplay between Membrane Tension and the Tight Junction Protein, ZO‐1
Authors: Wilkinson Nancy J. , Peng Lindsey N. , Doran John E. , Cheng Kwan H. , King Jonathan M.
Published: May 2022

Zonula occluden‐1 (ZO‐1) has been implicated as a major regulator of tight junction formation, functioning as a scaffolding protein and mediator between the junction and the actomyosin cytoskeleton. MDCK II cells that have ZO‐1 knocked‐down (ZO‐1 KD) or knocked‐out (ZO‐1 KO) have observable phenotypic changes, including increased apical actin accumulation, increased cell height, and linearized junctions. The apical actin accumulation, resulting from the dysregulation of myosin II, is suspected to cause an increase in membrane tension seen in the ZO‐1 KO, which may explain the phenotypic changes. The function of ZO‐1 as a regulator of myosin II was confirmed by exposing the ZO‐1 KO to blebbistatin, a myosin II inhibitor, which restored the cell height to that of wild‐type MDCK II cells (WT) and the curvature of the junctions. Flipper‐TR, which is a live cell fluorescent membrane tension probe, along with fluorescence‐lifetime imaging microscopy (FLIM) analysis, are being used to test the membrane tension of the ZO‐1 KO cells compared to WT cells. The Flipper‐TR probe works by inserting itself into a lipid membrane where it undergoes conformational changes induced by membrane tension. The relationship between membrane tension and actin was examined using latrunculin A (LatA), which causes the depolymerization of actin filaments. When both the ZO‐1 KO and WT cells were incubated with LatA, the membrane tension of both decreased to a similar extent. The preliminary results of this testing have shown an increased membrane tension in the ZO‐1 KO cells. To measure the response of ZO‐1 KO cells to increased tension, the expression of tension‐related genes is being measured using quantitative real‐time polymerase chain reaction (qRT‐PCR). These tension‐related genes include the ERM (ezrin, radixin, and moesin) proteins, with a primary interest in ezrin given its known activity as a major regulator of membrane tension in epithelial cells. This study aims to understand the mechanism between ZO‐1 loss and regulation of membrane tension.
The Effect of Mitochondrially Targeted Antioxidants on Wound Healing of Human Dermal Fibroblasts
Authors: Carney Jessica M. , Flateland Megan L. , Hetland Brynn L. , Renquist Kevin J. , Owens Kjerstin M.
Published: May 2022

Oxidative stress is known to be a destructive result of wounded tissues and is thought to be a source of delayed wound healing; however, the relationship between reactive oxygen species (ROS) and wound repair is not completely known. We hypothesized that the use of mitochondrially targeted antioxidants will increase the rate of wound healing of normal skin fibroblasts by decreasing steady‐state levels of reactive oxygen species at the wound site. Scratch assays showed that ROS accumulation was prominent at the wound site and increased overtime several hours. Cells were treated with mitochondrially targeted antioxidants and their nontargeted counterparts. Treatment with antioxidants decreased ROS levels in wounded and unwounded cells and increased the rate of wound healing. Finding a correlation between ROS and wound healing and how they are affected by antioxidants will offer an increased understanding of the biological process of wound healing and may propose therapeutic options to promote healing efficacy.
Fast Foods, and Processed Foods May Worsen Perceived Stress and Mental Health
Authors: Rosenberg Lexis , Welch Megan , Dempsey Gerard , Colabelli Michael , Kumarasivam Tharsana , Moser Lindsey , Begdache Lina
Published: May 2022

The connection between dietary factors and mental health has been addressed in the literature. However, the association between dietary factors and perceived stress combined with mental distress is still in need of further research. In particular, fast food and processed food are typically devoid of high‐quality nutrients and tend to be energy‐dense containing high levels of simple sugars, saturated fats, and trans fats. These dietary factors promote inflammation, alter brain chemicals, and initiate a stress response. Therefore, the aim of this study was to assess the impact of fast food and processed food consumption on perceived stress and mental distress. It was hypothesized that high amounts of fast foods will have a positive correlation with symptoms of mental distress and perceived stress. An anonymous survey was administered through social media platforms targeting adults 18 years or older. The survey consisted of validated scales: the Food Mood Questionnaire, Kessler‐6 and Perceived Stress Scale, A total of 1591 individuals completed the survey. Data was analyzed using Spearman’s Rho correlation and Principal Component Analysis in IBM SPSS Version 25.0. The results reveal that there is a positive correlation between consumption of high amounts of fast food and processed foods and feelings of anxiety (p <0.01), nervousness (p <0.01), restlessness (p <0.01), lack of motivation (p <0.01), and depressive symptoms (p <0.01). Our results suggest that a diet high in fast food and processed foods is positively correlated with higher perceived stress and mental distress levels; hence, a decreased mental health status.
Diurnal Differences in Mitochondrial Bioenergetics is Lost in Bmal1 Knockout Rats
Authors: Ramsey Anne , Bailey Shannon , Pollock David
Published: May 2022

In the kidney, 95% of O2 consumed through mitochondrial respiration is used for Na+ transport. The circadian clock regulates Na+ transport with a necessary diurnal variation for optimal health. The circadian clock transcription factor BMAL1 is proposed to regulate mitochondrial bioenergetic function; however, this has not been extensively investigated in the kidney. Therefore, we tested the hypothesis that renal mitochondrial respiration may be impaired or altered in a novel Bmal1 clock gene knock‐out rat model and is associated with loss of diurnal control of Na+ excretion. Male and female global Bmal1+/+ and Bmal1‐/‐ rats at 12‐14 weeks of age maintained in a regular 12‐hour light/dark cycle was used to measure renal mitochondrial O2consumption. Outer renal medullary tissue was dissected and prepared for respiration measurements at either day (ZT2‐4) or night (ZT14‐16) periods to correspond with minimum and maximum whole‐body energy consumption as well as peak and trough BMAL1 protein expression, respectively (n = 10, Bmal1+/+; n = 22, Bmal1‐/‐). Respiration was assessed using permeabilized tissue in the Oroboros Oxygraph and analyzed using Data Lab 2 software (O2K, Oroboros Instruments GmbH). Mitochondrial gene expression analysis was assessed using digital drop PCR. Data from both sexes were combined for each genotype at the two time points and analyzed by two‐way ANOVA. Mitochondrial state 3 O2 consumption was significantly higher (main effect of genotype p = 0.0310) in Bmal1‐/‐ (118 ± 156 pmol/s*mg) compared to BMAL1+/+ rats (75 ± 15 pmol/s*mg; Tukey post‐hoc p = 0.0303). We also observed a significant increase in complex IV activity in Bmal1‐/‐ rats during the dark vs. light period (164 ± 40 vs. 306 ± 40 pmol/s*mg; p=0.0031, t‐test). Mitochondrial‐associated genes, optic atrophy 1 (Opal1) and mitofusion 1 (Mfn1) show as significant day‐night difference in mRNA expression in kidneys from BMAL1+/+ control rats that is lost in Bmal1‐/‐ rats consistent with disrupted mitochondrial fusion and fission processes. Our lab previously reported that male Bmal1‐/‐ rats do not have the typical night‐day difference in Na+ excretion, which is consistent with our new data showing an alteration in mitochondrial function in kidneys from Bmal1‐/‐ rats. Therefore, our findings demonstrate that BMAL1 may play a role in maintaining the coupling of mitochondrial respiration and ATP generation in the kidney and support the hypothesis that diurnal Na+handling is regulated in part by BMAL1‐dependent mitochondrial function.
Specialized Feeding Morphology in the Small Crocodyliform Scolomastaxand Implications for Woodbine Paleoecology
Authors: Noto Christopher R.
Published: May 2022

The Arlington Archosaur Site (AAS) of Texas is a productive fossil locality found in the upper Lewisville Formation of the Woodbine Group (Cenomanian; Late Cretaceous). The AAS preserves a diverse flora and fauna from a lower delta plain, including at least 4‐5 taxa of crocodyliforms: Scolomastax, Deltasuchus, Terminonaris, Woodbinesuchus, and an undescribed eusuchian. In present‐day ecosystems, no more than two crocodyliform taxa are sympatric due to overlapping niche requirements. The greater diversity of sympatric crocodyliform taxa observed at the AAS and similar fossil localities is due to niche partitioning, which is achieved through increased morphological disparities in the feeding apparatus of sympatric taxa. Scolomastax is characterized by a shortened jaw, heterodont dentition, and enlarged coronoid‐like process, which is distinct from the generalist Deltasuchus and small‐prey specialists Terminonaris and Woodbinesuchus. Here, further work reconstructs the jaw musculature of Scolomastax and interprets its feeding ecology. Compared to other herbivorous and durophagus reptiles, Scolomastax possesses similar enlargement of the adductor musculature (particularly M. adductor mandibulae posterior and the M. pterygoideus muscles) and attachments that enhance their mechanical advantage, including some unique features not seen in any other taxa. These features confirm a specialized feeding morphology and further support an omnivorous diet reconstruction for Scolomastax. Such specialized feeding adaptations among crocodyliforms were not uncommon in the Cretaceous, where warmer global temperatures and equable conditions allowed Scolomastaxand similar small crocodyliforms to evolve specialized feeding morphologies and fill niches otherwise occupied by mammals in present‐day ecosystems. These conditions supported greater crocodyliform diversity at the AAS and similar ancient communities.
Activating P2Y1receptors improves function in arteries with repressed autophagy
Authors: Cho Jae Min , Park Seul‐Ki , Kwon Oh Sung , La Salle D. T. , Cerbie James , Fermoyle Caitlin C. , Morgan David , Nelson Ashley , Bledsoe Amber , Bharath Leena P. , Tandar Megan , Kunapuli Satya P. , Richardson Russell S. , Babu Pon Velayutham Anandh , Mookherjee Sohom , Kishore Bellamkonda K. , Wang Fei , Yang Tianxin , Boudina Sihem , Trinity Joel D. , Symons J. D.
Published: May 2022

Objective and hypothesis

The importance of endothelial cell (EC) autophagy to vascular homeostasis in the context of health and disease is evolving. Earlier we reported that intact EC autophagy is requisite to maintain shear‐stress‐induced nitric oxide (NO) generation via glycolysis‐dependent purinergic signaling to eNOS. Here we illustrate the translational and functional significance of these findings.

Methods and Results

First, we assessed translational relevance using older male humans and mice that exhibit blunted EC autophagy and impaired arterial function vs. adult controls. Active hyperemia evoked by rhythmic handgrip exercise elevated radial artery shear rate similarly from baseline in adult (23±1 y) and older (68±2 y) subjects for 60‐min. Compared to baseline, indexes of autophagy initiation, p‐eNOSS1177 activation, and NO generation, occurred in radial artery ECs obtained from adult but not older volunteers. Regarding mice, indexes of autophagy and p‐eNOSS1177 activation were robust in ECs from adult (7 ± 1 months) but not older (23 ± 1 months) animals that completed 60‐min treadmill‐running. Further, results concerning the extracellular acidification rate (ECAR; Seahorse Bioanalyzer) indicate glycolysis and glycolytic capacity were elevated in response to 20 dyn/cm2 laminar shear stress x 45‐min in primary arterial ECs obtained from adult but not older mice. We next questioned whether the inability to initiate EC autophagy, glycolysis, and p‐eNOSS1177 precipitates dysfunction in arteries from older vs. adult mice. Compromised intraluminal flow‐mediated vasodilation displayed by arteries from older vs. adult mice was recapitulated in vessels from adult mice by : (i) NO synthase inhibition; (ii) acute autophagy impairment using 3‐methyladenine (3‐MA); (iii) EC Atg3 depletion (Atg3EC‐/‐ mice); (iv) purinergic 2Y1‐receptor (P2Y1‐R) blockade; and (v) germline depletion of P2Y1‐Rs. Importantly, P2Y1‐R activation using 2‐methylthio‐ADP (2‐Me‐ADP) improved vasodilatory capacity in arteries from : (i) adult mice treated with 3‐MA; (ii) adult Atg3EC‐/‐ mice; and (iii) older animals with repressed EC autophagy.

Conclusions

Arterial dysfunction concurrent with pharmacological, genetic, and age‐associated EC autophagy disruption is improved by activating P2Y1‐Rs.
Determining The mRNA Which microRNA‐379‐5p Inhibitor Is Targeting in Human Lung A549 Cells
Authors: Schroder Araya K. , Yoo Lina I.
Published: May 2022

MicroRNAs are single‐stranded, non‐coding RNA that are complimentary to messenger RNA and play a role in post‐transcription gene expression and RNA silencing. Understanding the specific mRNA targets of miRNA that are elevated in tumors can provide insight into how cancer develops. The purpose of this experiment was to utilize miR‐379‐5p inhibitor to determine its target genes in human lung cells. Ten target genes for miRNA‐379‐5p were selected from Targetscan and miRDB databases, of which nine were novel and one, Mdm2, was a known target gene used as a positive control. QRT‐PCR primers for target genes were designed. A549 cells were transfected with the miR‐379‐5p inhibitor or a negative control, and then putative target mRNA levels were measured by qRT‐PCR. The Mdm2 positive control gene demonstrated increased levels in the presence of the miR‐379‐5p inhibitor, suggesting the assay was working properly. The putative target gene INSR also showed an increase in mRNA levels in the presence of miR‐379‐5p inhibitor, but a western blot did not show increased expression at the protein level. Other putative target genes examined did not show an increase in mRNA levels in the presence of the miR‐379‐5p inhibitor.
Neuronal Activity Differentially Regulates KCC2 Protein Expression at Distinct Neuronal Maturation Stages
Authors: Pethe Abhishek , Heimrich Bernd , Roussa Eleni
Published: May 2022

The K+/Cl‐ co‐transporter 2 (KCC2), a protein exclusively expressed in neurons, facilitates the co‐transport of a K+ and a Cl‐ ion across the cell membrane. The developmental shift of GABAA receptor mediated responses from depolarizing to hyperpolarizing is mainly due to a developmental upregulation of KCC2. Moreover, in mature neurons, neuronal activity leads to a decrease in surface expression and phosphorylation of Ser940 residue of KCC2, thereby reducing its activity. KCC2 is additionally phosphorylated at Thr906 and Thr1007, which reduces KCC2 surface expression and thus activity.

Given the important role of neuronal activity in regulating KCC2 expression, we sought to investigate the effect of pilocarpine or 4‐aminopyridine (4AP) treatment on KCC2 expression across different maturation stages of neurons using quantitative RT‐PCR, immunocytochemistry (ICC) and immunoblotting. We also investigated the three phosphorylation sites – Ser940, Thr906 and Thr1007 – of KCC2. Immature neurons (day in vitro 4) from embryonic day 18.5 mouse hippocampus were treated with pilocarpine or 4AP (100µM, 60 minutes). The results show that 4AP or pilocarpine treatment has no effect on KCC2 transcript or protein expression, compared to controls. Similarly, ICC and immunoblotting results showed that there was no change in the phosphorylation status of Ser940. ICC results also showed that the phosphorylation status of Thr906 and Thr1007 remained unaltered in immature neurons after pilocarpine treatment.

In day in vitro 17 organotypic hippocampal slices cultured from postnatal day 2 mouse pups, immunoblotting showed that treatment with 4AP did not affect KCC2 protein expression. However, in day in vitro 35 organotypic hippocampal slice cultures, 4AP treatment reduced KCC2 whole protein expression.

These results demonstrate that neuronal activity has a distinctive effect on KCC2 protein as the neurons mature, suggesting a differential regulation of KCC2 protein at different maturation stages.
Hemoglobin Glycation and Oxidative Stress Induce Functional Modification of Band 3 Protein in D‐Galactose‐Treated Erythrocytes: Double Effect of Quercetin
Authors: Spinelli Sara , Remigante Alessia G. D. , Dossena Silvia , Marino Angela , Morabito Rossana
Published: May 2022

D‐Galactose (D‐Gal) is a reducing sugar whose concentration in the blood is very low (between 0.00008 and 0.00018 mmol/L). An abnormal accumulation of D‐Gal, which occurs in pathological conditions such as galactosemia or to reproduce experimental models such as aging, leads to an increase in oxidative events and the formation of advanced glycation end products (AGEs), known to alter erythrocyte homeostasis. Specifically, increased levels of glycated hemoglobin (A1c) and oxidative markers have a negative impact on the exchange capability of Band 3 protein (B3p), responsible for the chloride‐bicarbonate exchange, essential for erythrocyte function. Among the natural compounds that can counteract these glycation and oxidation phenomena, quercetin (Q), a flavonoid found in many fruits and vegetables, shows strong anti‐glycation and antioxidant power at low doses, both in vivo and in vitro.

Based on this, the hypothesis of the present work is that Q counteracts A1c formation and increased oxidative stress induced by exposure to different doses of D‐Gal in two in vitro experimental models: galactosemia (low doses) and aging (high doses).

Therefore, the aim of this study is to verify whether the anti‐glycation and antioxidant action of Q results in a restoration of B3p functionality, measured by the rate constant for SO4²⁻ uptake, slower and better detectable than Cl‐or HCO3‐.

In this regard, rate constant of SO4²⁻ uptake, A1c levels, oxidative stress assessment (TBARS levels, ‐SH groups levels, GSH/GSSG ratio), were determined after 1h incubation of erythrocytes with or without Q (10 µmol / L) at 37°C, followed by 24 h exposure to low (0.1‐10 mmol/L) or high (50‐100 mmol/L) doses of D‐Gal at 25°C.

Our results show that low doses of D‐Gal reduce the rate constant of SO4²⁻ uptake and increase A1c levels, but do not induce oxidative stress. In contrast, high D‐Gal doses accelerate the rate constant, increase A1c levels and oxidative stress. Furthermore, pre‐treatment with Q reduces A1c levels and oxidative stress, bringing the rate constant back to control values.

In conclusion, we can confirm that: i) anion exchange through B3p is a useful tool to monitor the homeostasis of erythrocytes exposed to different concentrations of D‐Gal; ii) all selected doses of D‐Gal affect B3p function via different mechanisms, including glycation events (low doses) and oxidative events (high doses); iii) Q attenuates the D‐Gal effect, impairing the rate constant alterations by preventing A1c formation and reducing oxidative stress.

This evidence suggests that Q could be a useful dietary supplement to prevent oxidative events and AGE‐mediated processes linked to disease.
Biophysical Models of Minipig Right Atrial Ganglionic Plexus Principal Neurons Identified from Transcriptomics Data
Authors: Gupta Suranjana , Feldman Jessica M. , Newton Adam J. H. , Kuttippurathu Lakshmi , Moss Alison , Schwaber James S. , Vadigepalli Rajanikanth , Lytton William W.
Published: May 2022

The Right Atrial Ganglionic Plexus (RAGP) is a component of the intrinsic cardiac nervous system. Its neurons are critical to the cardiac pacemaker response to vagal stimulation, regulating sino‐atrial node activity. We developed single‐compartment computational models of RAGP principal neurons by incorporating ion channels identified from transcriptomics data from HT‐qPCR (High Throughput quantitative Polymerase Chain Reaction) and RNA‐sequencing of 405 principal neurons in female Yucatan minipig RAGP. Out of the ~200 genes in the transcriptomics map, we identified 13 genes that encode ion channel proteins, namely: Naf (Scn1a, Nav 1.1), KDR (Kcnc1, Kv 3.1; Kcnab1, Kv 1.1), P‐, N‐, L‐, T‐type Ca2+ (Cacna1a, Cav 2.1; Cacna1b, Cav 2.2; Cacna1c, Cav 1.2; Cacna1g, Cav 3.1; Cacna1i, Cav 3.3), KA (Kcnd2) and HCN1, HCN2, HCN3, HCN4. The corresponding Hodgkin‐Huxley‐based kinetic models of these ion channel isoforms were mined from databases: Ion Channel Genealogy, Channelpedia, and ModelDB. Since each ion channel gene had several kinetic models associated with it, we ran multiple simulations on the NetPyNE software to identify a single model per gene that would best represent the channel's dynamic behaviour in the assembled electrophysiological model. A binary transcriptomic map of the 13 genes showed 101 distinct ion channel combinations. Our 505 electrophysiological models (5 sets of the 101 cells) elicited two primary firing profiles. 65% elicited a single‐spike phasic response, ~32% elicited a tonic firing in response to a current clamp stimulus, and ~3% produced tonic firing that continued after the stimulus. The firing frequencies increased by ~13 Hz for every 0.1 nA increase in the current clamp stimulus and decreased by ~11 Hz for every 0.01 S/cm2 increase in KA channel conductance. Our electrophysiological models are available to the SPARC community in NetPyNE and NIH’s O2S2PARC platform. The models exhibit phasic and tonic excitability profiles with phasic being more prevalent. We next aim to incorporate neuromodulatory inputs and simulate a network of these neuronal models.
The Composition of Bile Acids under Fatty Liver Model in Chicken
Authors: Yang Wen Yuan , Lin Yuan Yu
Published: May 2022

Bile acids is synthesized by cholesterol in the liver and exert by two different pathways, classical and alternative pathway. Two major enzymes, CYP7A1 and CYP27A1 play regulatory roles in two pathways. CA (cholic acid) and CDCA (chenodeoxycholic acid) as known as the primary bile acids which means they are major products in these two pathways and then they can be modified by gut microbiome. After modifying, the metabolites are called secondary bile acids such as lithocholic acid (LCA)、deoxycholic acid (DCA). Some researchers found that total bile acids including primary and secondary bile acids elevate in serum from non‐alcoholic fatty liver (NAFL). However, the composition of bile acids in chickens upon fatty liver model is unclear.

In current study, seven‐week‐old ISA chicken were assigned to two group, there are control group (C) (n=10) and cholesterol with low choline (CLC) group (n=10). The control group was fed with extra 0.05% choline and without cholesterol, the other group was fed with extra 2% cholesterol and without choline for six weeks to simulate as NAFL. After sacrificing, we collected serum and cecum contents to quantitation of bile acids by mass spectrometer and analyze gut microbiome by 16S amplicon sequencing. The gene expression pattern of bile acid synthetic enzymes and related transcription factors was also determined by real‐time PCR.

The bile acids in serum, such as TCDCA (C:CLC=6080.15nM:14555.75nM)、TLCA (C:CLC=122.31nM:2122.62nM)、5β‐Cholenic Acid‐7α‐ol‐3‐one (C:CLC=1.04nM:3.87nM)、CDCA (C:CLC=3.93nM:38.13nM) are significantly elevated in CLC group and the bile acids in cecum contents such as, TLCA (C:CLC=109.8nM:8897.27nM)、TUDCA (C:CLC=15.82nM:985.0nM)、THDCA(C:CLC=718.72nM:3084.36nM)、LCA(C:CLC=4.32nM:22.88nM)、TDCA(C:CLC=10.12nM:172.28nM) are significantly increased in CLC group. The results of gene expression showed that CYP7A1 and CYP8B1 were increased in CLC group compared than control. For gut microbiome, the proportion of bile acid tolerance bacteria Alistipes in the CLC group is 9.13% which is 1.69% higher than the control group.

The pleiotropic roles of bile acid have gradually been valued. In this study, we analyzed the composition of bile acids under the fatty liver of chickens. We also found that the concentration of taurine conjugated bile acid in chickens is much higher than that of glycine.