bluuebunny/crossref_metadata_2025 huggingface dataset:
The Effect of Damaging Electric Muscle Contraction on Mitochondrial Health and Function
Authors: Atkinson Erin L. , Murphy Trevor L. , Marchant Erik D. , Pfeifer Brandon S. , Hyldahl Robert D. , Nelson W. B. , Hancock Chad R.
Published: May 2022

High intensity and prolonged exercise have been known to cause at least a temporary reduction in mitochondrial function. However, it is unclear how long mitochondrial function may remain impaired. Electrically stimulated eccentric muscle contractions (EEMC) may offer insights into the mechanisms by which mitochondrial function is restored following damaging muscle contractions like what is seen following high intensity exercise. The effects of EEMC on mitochondrial function have not been investigated. The aim of this study was to determine the impact that EEMC has on mitochondrial function one week following a single bout of the electrical stimulation. Muscle biopsies were taken from the vastus lateralis of female subjects one week prior and one week after EEMC. Muscle fibers were then prepared for High Resolution Respirometry and Fluorometry analysis on an Oroboros O2K Fluo‐Respirometer. This approach allowed for real‐time evaluation of mitochondrial function and production of reactive oxygen species (ROS). We measured mitochondrial function using two different substrate‐uncoupler‐inhibitor‐titrations (SUITs) designed to evaluate carbohydrate‐supported respiration (SUIT‐1) and fatty‐acid supported respiration (SUIT‐2). Under SUIT‐1 conditions, maximum coupled respiration (OXPHOS) was not significantly different between the pre and post EEMC biopsies. Under SUIT‐2 fatty‐acid supported and phosphorylating conditions (with ADP), there was a significant increase in respiratory function in the post EEMC biopsy (p=0.045). Mitochondrial ROS production was not significantly different following recovery than the baseline measures under SUIT‐1 conditions. These data represent the first evaluation of mitochondrial function one week following EEMC. We also show that the previously demonstrated negative effect of high‐intensity exercise on mitochondrial function may be fully recovered one week after damaging contractions and that fatty‐acid supported respiration is improved at the same time point.
Synthesis of A Tetrahydrocannabinol (THC) Analog For Analyzing Cellular Proliferation in Concert With Epigallocatechin Gallate (EGCG) on A Bone‐like Cancer Cell Line (UMR 106‐01 BSP)
Authors: Mamora Kaitlyn , Joseph Eugene
Published: May 2022

Cannabinoids have been extensively studied in the field of cancer research. Tetrahydrocannabinol (THC) has shown promising results in influencing cellular proliferation when in association with other cannabinoids. This traditional entourage effect solely focuses on the study of THC with other cannabinoids. However, not many studies have been done to explore the synergistic effect of THC analogs when used with non‐cannabinoid compounds. THC in its isolate form for experimentation is very strictly regulated. Therefore, this study was conducted in the pursuit of synthesizing and experimenting with analogs of THC to observe a potential entourage effect with epigallocatechin gallate (EGCG), a compound known for its efficacy to reduce proliferation at higher concentrations in UMR cells. It was hypothesized that active analogs of THC can be synthesized and used in concert with EGCG to potentiate decreased proliferation in the bone‐like cancer cell line UMR 106‐01 BSP (UMR cells). Briefly, a Knoevenagel condensation and a Diels‐Alder reaction using 1,3‐cyclohexanediol dissolved in methanol (MeOH) and citronellal with ethylenediamine diacetic acid (EDDA) at a temperature of 60℃ was used to synthesize a novel THC analog, 3,10,10‐Trimethyl‐1,2,3,4,4a,6,7,8,10,10a‐decahydro‐9‐oxa‐5‐phenanthrenone (TDP). UMR cells were routinely passaged, counted, plated in six‐well culture plates at 480,00 cells/mL, then treated with 10‐fold dilutions of TDP. The plates were incubated for 72 hours in a humidified incubator at 37 degrees Celsius with 5% carbon dioxide infusion. At the end of the experiment, the cells were routinely washed with HANKS buffered saline solution (HBSS), then routinely counted using the Luna Automated Cell Counter. In another experiment, designated cells were co‐treated with TDP+EGCG, following the protocol above. F test ANOVA was used to compare variances and all values in the results were expressed as means ± SD. The results from the attempted cannabinoid analog synthesis yielded a novel active THC analog, TDP. Serial dilutions treatment of the UMR cells with TDP alone showed its ability to decrease cell count in a concentration dependent manner. However, when coupled with higher concentrations of EGCG, the co‐treatment increased cell count rather than potentiating the effect of decreasing cellular proliferation. The F Test ANOVA showed robust statistical significance (p values <0.05) with regard to TDP’s effect of decreasing cell proliferation in UMR cells in a concentration‐dependent manner. Overall, the outcomes of this study suggest that active forms of THC analogs can be synthesized and tested in concert with other non‐cannabinoid compounds like EGCG. This study opens the door to explore the entourage effect of TDP with other non‐cannabinoid compounds that may provide another tool in the therapeutic treatment of bone cancer cells.
Characterizing the Mechanism of a Conserved G‐Protein Coupled Receptor in Caenorhabditis elegans
Authors: McCarthy Teagan , Honer Madison , Nelson Matthew D.
Published: May 2022

Sleep is evolutionarily conserved and essential. Yet, it is disadvantageous as it renders animals vulnerable to predation and limits other essential behaviors, such as feeding and reproduction. Thus, animals must balance their sleep drive with behaviors that ensure their survival. How this is regulated at the cellular and molecular level is largely unknown. The genetically tractable nematode, Caenorhabditis elegans, is a powerful sleep model, possessing a compact and completely mapped nervous system of only 302 neurons. Stress‐induced sleep (SIS), analogous to sickness sleep of mammals, is a behavior that allows for a genetic dissection of these key questions. When C. elegans is exposed to noxious stimuli, avoidance responses are initiated, promoting their survival. However, if cellular damage is incurred SIS takes place, promoting recovery. Thus, SIS is an interesting model for identifying mechanisms underlying essential sleep/wake decisions. We conducted a forward genetic screen searching for sleep‐defective mutants, and isolated a strain containing a loss‐of‐function allele of the gene T10E10.3, which encodes a G‐protein coupled receptor (GPCR). Using CRISPR and cell‐specific rescue, we find that T10E10.3 acts to inhibit the sleep‐promoting RIS interneuron to promote arousal, while simultaneously signaling to multiple cells to promote sleep, including the ALA interneuron and a pharyngeal neuron called I6. Over‐expression of T10E10.3 results in a profound early sleep phenotype, suggesting an imbalance in the sleep/wake mechanisms. Our data supports a model in which a single GPCR can regulate the balance between wake/sleep by functioning in an antagonistic manner in unique cell types. We propose that this allows the worm to escape harmful stimuli early and then ensures proper levels of recovery sleep once a safe environment is reached.
Sphingomyelin‐enriched extracellular vesicles from hypertensive diabetic mice regulate calcium mobilization and amiloride‐sensitive transepithelial current in mouse cortical collecting duct cells
Authors: Carson‐Marino Morgan W. , Ramsay Hunter G. , Alli Abdel A.
Published: May 2022

Uncontrolled hypertension (HTN) and diabetes mellitus (DM) are two major public health burdens contributing to increased hospitalization, readmission costs, and adverse clinical outcomes. Moreover, DM is a leading cause of death and long‐term morbidity. Overexpression and activity of epithelial sodium channels (ENaC) in the kidney results in hypertension, but the mechanism for its regulation in the diabetic kidney is not entirely understood. Cholesterol is known to increase ENaC activity in cultured kidney cells in a calcium and PI(4,5)P2‐dependent mechanism. Presumably ENaC is regulated by other lipids in pathophysiology. Extracellular vesicles (EVs) are nano‐sized vesicles that play an important role in intercellular communication and intracellular signaling. Here, we investigated whether EVs isolated from the urine of hypertensive diabetic db/db mice or diabetic db/db mice augment ENaC activity by inhibiting calcium mobilization in mpkCCD cells. Adult diabetic db/db mice were salt loaded (4% NaCl) for 7 days to induce hypertension. Blood pressure was measured by tail‐cuff, and urine from db/db mice with systolic blood pressure of greater than 140mmHg or from normotensive db/db mice were used to isolate EVs. Nanoparticle tracking analysis was performed to determine EV concentration. EVs from hypertensive diabetic db/db mice (N=5) compared to diabetic db/db mice (N=5) suppressed ionomycin‐induced calcium mobilization in mpkCCD cells cultured on glass bottom 35 mm dishes (N=3 experiments, p<0.05) which was measured by confocal microscopy and augmented amiloride‐sensitive transepithelial current in mpKCCD cells cultured in 12mm diameter permeable supports (N=6 permeable supports per group) measured by an epithelial volt ohm meter (EVOM). Sphingomyelin were found to be enriched in urinary EVs from hypertensive diabetic db/db mice compared to diabetic only db/db mice. Exogenous application of sphingomyelin to mpkCCD cells mimicked the suppression of calcium mobilization and increase in amiloride‐sensitive transepithelial current in these cells. Taken together, these data suggest differences in the packaged cargo of urinary EVs between HTN and DM mice that presumably plays a role in the regulation of ENaC activity.
Safety Profile of Short‐Term Creatine Nitrate and Caffeine Co‐Ingested in Resistance Trained Participants
Authors: Mabrey Gina M. , Koozehchian Majid , Newton Andrew , Naderi Alireza , Haddad Monoem , Forbes Scott
Published: May 2022

Caffeine and creatine nitrate are two commonly used ergogenic aids purported to augment resistance and anaerobic performance; however, the safety profile of these supplements co‐ingested is unknown. The purpose was to examine the effects of caffeine and creatine nitrate alone and co‐ingested on several biochemical blood markers (creatine kinase, lactate dehydrogenase, cholesterol, alanine aminotransferase, aspartate transaminase, alkaline phosphatase, white blood cells, lymphocytes, red blood cells, hematocrit, and hemoglobin), resting heart rate and blood pressure, and self‐reported side effects. METHODS: In a double‐blind, randomized, placebo‐controlled cross over study, 12 resistance‐trained participants (22±1 y; 180.0±9.5 cm; 85.9±15.1 kg) completed 4 conditions in random order: creatine nitrate (CN: 5 g/day + 0.675 g/day of maltodextrin), caffeine (CAF: 400 mg/day + 5 g/day of maltodextrin), CN+CAF (5 g/day of creatine nitrate + 400 mg/day of caffeine), and placebo (PL: 5.4 g/day of maltodextrin). Supplements were ingested for 7 days with a 7‐day washout period between conditions. Before and after supplementation, participants provided a blood sample collected from the antecubital vein and measured resting heart rate and blood pressure. A standardized side effect questionnaire was also completed following each condition. Data were analyzed using a two‐way (within‐within) analysis of variance (p≤0.05) with a Tukey’s post hoc when appropriate. For side effects, a chi‐squared non‐parametric test was used. RESULTS: There was no interaction or main effects for all blood parameters (p>0.05). Resting heart rate and blood pressure were also not different between conditions or over time (p>0.05). Lastly, there were no significant differences between conditions for any side effects on the side effect questionnaire (p>0.05). CONCLUSION: Short‐term CAF, CN, and CAF+CN co‐ingestion appears safe for healthy young resistance‐trained participants.
The Effect of Pregnancy Status on Rat Main Uterine Artery and Radial Uterine Artery Structure and Function
Authors: Dunlap Jazmin N. , Wagner Katherine L. , Gusek Brikena , Jones Carleton (. , Eckman Delrae M.
Published: May 2022

Background

Healthy pregnancy is associated with remodeling of the maternal uterine vasculature to meet the metabolic demands of the developing fetus. In contrast to the systemic circulation during pregnancy, it has been suggested that pregnancy decreases radial uterine artery (RUA) distensibility, potentially persisting throughout the postpartum period. The intent of this study is to compare RUA and main uterine artery (MUA) during late pregnancy and the postpartum period to determine whether pregnancy‐related changes in distensibility is similar in both large and small uterine vessels.

Methods

MUAs and RUAs from NP (n=7‐9), LP (gestational age: 21±1 d, n=6‐8), and 30±1 days postpartum (PP30; n=4‐6) Sprague‐Dawley rats were isolated, cleaned of connective tissue, cut into 8mm segments, and cannulated in an arteriograph chamber to assess vascular reactivity to the addition of 60 mM KCl and phenylephrine (PE, 1 µM). In addition, passive mechanical properties such as lumen diameter, wall thickness, distensibility and arterial stress/strain were assessed at intraluminal pressures ranging from 10mm Hg to 140mm Hg. Data were analyzed using one‐way ANOVA or two‐way ANOVA. Data were considered significant at P<0.05.

Results

K+‐ induced constriction was similar in RUAs and MUAs from NP, LP, and PP30 rats. PE‐induced constrictions were similar at all pregnancy states in RUAs. In contrast, PE‐induced constriction in LP MUA was greater than NP MUA (P<0.05) and PP30 MUA (P<0.01). Passive LP RUA lumen diameter was greater than PP30 RUA (P<0.05) and trended toward significance in NP RUA (P<0.07). Passive NP MUA lumen diameters were smaller than MUA diameters from LP (P<0.05) and PP30 (P<0.05) rats. There was no difference in arterial wall thickness between NP, LP and PP30 MUAs (P=NS) and NP, LP and PP30 RUAs (P=NS). Distensibility in NP RUA was greater than PP30 RUAs (P<0.05), but there was no statistical difference in distensibility between LP RUA and PP30 RUAs. There was no difference in distensibility in MUAs at all pregnancy states. Stress/strain relationships unmasked vascular remodeling in both RUAs and MUAs. There was a significantly greater leftward shift in stress/strain values in RUAs from PP30 rats than RUAs from LP (P<0.05) and NP (P<0.05) rats. Furthermore, stress/strain values from LP RUAs were different than RUAs from PP30 (P<0.05) and NP (P<0.05) animals. Similar to RUA, the leftward shift in stress/strain values from PP30 MUA were statistically greater than MUA from LP and NP animals.

Conclusions

These preliminary data suggest that significant vascular remodeling occurs with pregnancy which persists 30 days post parturition. Additional studies need to be completed to determine how multiple pregnancies may alter RUA and MUA structure and function.
Myofibers sarcolemma mechanical properties are affected by the isolation method
Authors: Garcia‐Pelagio Karla , Lovering Richard
Published: May 2022

For understanding the mechanics and function of skeletal muscle in vivo, isolated cell fibers are used. Single isolated myofibers can be obtained from direct dissection or enzymatic dissociation. Isolation via direct dissection allows control of sarcomere length, preserves tendon attachment and, maintains the physiological environment, but is labor‐intensive and time‐consuming. On the contrary, enzymatic dissociation is fast and facile, produces hundreds of myofibers, reduces the number of muscles/animals needed for studies but alters the physiological environment and sarcomere length cannot be controlled. Biomechanical properties of the sarcolemma from mice were studied using the Elastimetry technique applied on myofibers from the extensor digitorum longus dissected directly and from the flexor digitorum brevis obtained by dissociation. Membrane stiffness, Young’s modulus, strain, and viscoelasticity in single isolated fibers were measured. Results showed that the membrane biomechanical properties were up to two times higher using direct isolation compared to the enzymatic dissociation technique. This leads us to conclude that the method used to obtain the myofibers changes the mechanical properties in cells.
A genetic screen for proteins involved in Non‐Stop mRNA decay in C. elegans
Authors: Diamandi Michelle , Brennan Catherine , Youngman Elaine M.
Published: May 2022

Non‐stop mRNAs, which lack a stop codon due to mutation or errors in mRNA transcription or processing, serve as templates for production of aberrant and potentially toxic protein products. Ultimately, translation of non‐stop mRNAs leads to a failure in normal translation termination and ribosome collisions, which in eukaryotes initiates ubiquitin‐dependent degradation of the protein product and Non‐Stop Decay (NSD) of the problematic mRNA. Ribosome collisions in yeast are linked to activation of cellular stress responses and failure to resolve stalled ribosomes leads to neurodegeneration in mice, highlighting the biological importance of mRNA quality control.

In the nematode Caenorhabditis elegans, loss of the core NSD components pelo‐1 and skih‐2 leads to temperature‐sensitive sterility, as does the loss of endogenous siRNAs that are triggered by NSD at some loci. To further probe the link between NSD and the biology of germ cells, we have used an RNAi approach to screen for temperature‐sensitive reduction in brood size for the C. elegans orthologs of several proteins known to be part of the NSD machinery in other organisms. Preliminary results have identified 2 NSD‐associated genes with conditionally reduced brood sizes. We will present progress on examining the requirement of these genes for Non‐Stop decay in C. elegans, as well as investigation of their specific contributions to the biology of the germ line.
Label free DIA and DDA nano‐LC/MS/MS improved quantitative profiling of redox stress mediated proteomic changes in mouse dendritic cells
Authors: Clement Cristina C. , Soni Rajesh K.
Published: May 2022

A diet rich in saturated fat and carbohydrates causes a low‐grade chronic inflammation in several organs. To investigate the impact of oxidative stress and metabolic syndrome on the whole cellular proteome of professional antigen presenting cells (APCs), we isolated six biological replicates consisted of total proteomic extracts of dendritic cells (DCs) from B6 mice on normal and high fructose/high fat (HFHF) diet and performed quantitative label‐free data independent (DIA) and data dependent (DDA) nano LC‐MS/MS analysis of protein expression profiles. Our optimized primary DCs processing for label free proteomics and DIA quantitative analysis retrieved 1451 proteins (with at least 1 unique peptide), identified at 2% attained FDR (0.5% FDR for peptides) out of which 744 proteins were eligible for quantitative analysis and exhibited statistically significant changes in mouse DCs proteomes harvested from mice on HFHF vs normal diet (ANOVA, p<0.05). Bioinformatics and GO annotations mapped quantitative changes in energy and stress related proteins associated with several metabolic pathways including glycolysis, lipolysis and mitochondrial oxidative phosphorylation, reflecting the possible onset of insulin‐resistance phenotype. The (LFQ) DIA analysis highlighted qualitative and quantitative changes in the proteomes from mouse DCs, spanning a wide spectrum of up‐regulated inflammatory pathways, which in turn, could wire the reshaping of immunopeptidome landscape presented by MHC‐II under redox metabolic stress.
Introns in yeast regulate translation to generate protein and cellular function
Authors: Hossain Munshi Azad , Bolech Emma , Codallos Nicholas , Johnson Tracy L.
Published: May 2022

While RNA splicing has been recognized as an important mode of eukaryotic gene regulation, the role that introns themselves play in gene expression and proteome diversity has been less clear. Here, we describe how the intron of GCR1, a transcription factor that regulates glycolytic gene expression, contains intronic elements that control translation initiated from an in‐frame, intronic‐AUG. Moreover, the intronic elements regulate translation under stress conditions. To understand how the translation start site within the intron is recognized, we analyzed the structures and functions of sequences around the start site. These studies reveal an internal ribosome entry site (IRES) that regulates translation initiation from within the intron. In addition to this sequence, we find that a ribosomal protein, Rps25, regulates utilization of the intronic AUG. These data suggest that splicing regulation and intron retention set up a novel mode of translational control. When we analyzed all intron‐containing genes in yeast, we discovered that this architecture—an intronic AUG that is in frame with the annotated stop codon—is found in other intron‐containing genes. Moreover, the proteins generated appear to be functional, particularly under stress conditions. Together these studies illustrate a role for introns in translational control and tuning the proteome in response to environmental conditions.
Unfolding RFX1 Mediated Regulation of Cancer Stem Cells
Authors: Raveendran Pooja S. , Issaac Joby , Kunnummal Midhunaraj , Das Ani V.
Published: May 2022

Regulatory factor X1(RFX1) is a unique transcription factor that can act both as an activator and repressor in a context‐dependent manner. In most of the cases, it has been shown to exert an inhibitory action on its targets. It belongs to the evolutionarily conserved RFX (Regulatory Factor binding to the X‐box) family of transcription factors consisting of eight homologs (RFX1‐8) in humans. RFX1 is seen down‐regulated in many cancers and is speculated to have a critical role in regulating stem cell properties. Cancer stem cells are major hurdles in cancer therapy as they are capable of repopulating a tumor after treatment. We aim to understand the regulation of cancer stemness by RFX1 in human teratocarcinomal (NTERA‐2) cancer stem like cells.

We have chosen NTERA2 cl.D1 (NT2) embryonic carcinoma cells since they are the malignant counterparts of embryonic stem cells and are pluripotent in nature, making them an ideal candidate to be used as a cancer stem cell model. The cells were differentiated using DISC (Days in suspension culture) method to obtain differentiated populations (NT2D). Using real time analysis, the expression of RFX1 was evaluated in proliferating (NT2P) and differentiating (NT2D) cell populations. A significant increase in RFX1 expression was observed in NT2D cells compared to NT2P cells. For further validation, the same was done in stem cell population enriched using Abcg2 promoter where the stem (GFP+ve) and non‐stem (GFP‐ve) cell populations were sorted out by FACS. Further RFX1 expression was perturbed in NTERA cells, and the assessment of cardinal properties of CSCs, such as clonogenicity/self‐renewal properties, proliferation potential, and migration/invasive properties in vitro,revealed that RFX1 plays a significant role in cancer stemness. Therefore, understanding the regulation of cancer stemness by RFX1 will allow us to formulate better diagnostic and therapeutic approaches to address chemoresistant cancers.
Conformational Dynamics Linking Kinase and Endonuclease Domains Explain the Divergent Function of IRE1 Paralogues
Authors: Grey Michael J. , Jainchill Dylan , Simpson Mariska S. , Lencer Wayne I.
Published: May 2022

The ER stress sensors IRE1α and IRE1β have dual kinase and endonuclease activities that mediate cellular proteostasis by splicing Xbp1 mRNA and degrading other ER‐targeted transcripts. Although the two paralogues share a high degree of sequence homology, the epithelial cell‐specific paralogue IRE1β, which is expressed at mucosal surfaces, has impaired kinase activity and phosphorylation that restrict stress‐induced activation of the endonuclease domain. To uncover structural features that explain these differences, we used normal mode analysis to build dynamic correlation networks describing active and inactive conformations of IRE1 kinase‐endonuclease domains. For human IRE1α, we identified a fingerprint of intra‐ and interdomain dynamic couplings that distinguish between the two conformations. Mutations that disrupted IRE1α endonuclease activity perturbed the dynamic networks of the active conformation and enhanced features of an inactive conformation, thus validating our approach. Strikingly, the dynamic networks of human IRE1β when modeled in an active conformation resembled the inactive IRE1α network. A set of non‐conserved amino acids were found to distinguish between the dynamic networks linking kinase‐endonuclease domains for the two IRE1 paralogues. In most cases, however, these divergent network‐determining amino acids were found only in IRE1β sequences from mammals. In lower vertebrates, the key amino acids and the dynamic couplings of IRE1α active state were conserved in IRE1β. Thus, IRE1β evolved distinct conformational dynamics and enzymatic activity for a role in mucus barrier function that is unique to higher vertebrates.
Crotalus atrox Induced Cellular Injury Triggers an Oxidative Stress Gene Response in HEK‐293T Cells
Authors: Khoshneviszadeh Shawyun , Albrecht Eric A.
Published: May 2022

Wound and regeneration involves a series of complex processes that can induce oxidative stress by elevating intracellular reactive oxygen species (ROS) levels. In this study, we used RNA‐seq and qPCR to examine the molecular signature of Human Embryonic Kidney cells (HEK 293T) stimulated with Crotalus atrox venom. NGS data was assembled and annotated using BLASTx and UNIPROT. Results showed 243 genes were differentially expressed (106 upregulated, 137 downregulated) between Crotalus atrox stimulated and non‐stimulated cells (p<0.05). HEK 293T stimulated with Crotalus atrox venom for 4 hours showed an increase in the expression of oxidative stress response genes (e.g., TOMM6, EGR (1,2,3), HSP70 (1A, 1B), UPK3BL (1,2)), and protein synthesis (ETV5, EMP1) (fc>2.0). Significantly downregulated (log2(fc)<‐1.20) genes were associated with protein coding (e.g., ABCG1, ABCA1, PPARA, HMGA2, TLCD5). Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses identified 8 significant KEGG pathways, which included an amphetamine addiction pathway (p<0.03). Genes identified in this pathway such as FosB proto‐oncogene (FosB) and activity regulated cytoskeleton protein (ARC) are connected to oxidative stress responses. Quantitative PCR (qPCR) was used to evaluate several genes identified from RNA‐seq data. Cells stimulated with Crotalus atrox venom for 4 hours induced an up‐regulation of HSPA1A (2.49±0.49), HSPA1B (2.04±0.12), and cytochrome P450 1A1 (CYP1A1) (3.24±0.33), matching NGS data trends. Pre‐incubation with the zinc chelator tris(2‐pyridylmethyl) amine (TPA) prior to venom stimulation induced a significant up‐regulation in HSPA1B (13.2±0.04). Extending Crotalus atrox venom stimulation to 8 hours increased the gene expression of metallothionein 1X (MT1X, 4.68±0.26) and peroxiredoxin‐4 (PRDX4, 3.34±0.11) compared to non‐stimulated cells. Interestingly, pre‐incubating cells with polyethylene glycol (PEG) catalase then stimulating cells with Crotalus atrox venom for 8 hours produced PRDX4 and MT1X gene expression values that were not significantly different from the non‐stimulated control. In contrast, glutathione peroxidase‐1 (GPx‐1) was significantly downregulated (0.27±0.16) after an 8 hour stimulation with Crotalus atrox venom, compared to non‐stimulated cells. Our data suggests Crotalus atrox induced cellular injury involves several gene families connected to oxidative stress responses.
Testing a multi‐factorial model of the role of Rhipicephalus sanguineus in the spread of Rocky Mountain Spotted Fever in the Arizona region
Authors: Allwardt Kayla , Roe Nicolette , Logue Mary G. , Mutterperl Jeffery , Oliva Alec , Schaefer Charles P. , Hernandez Jose , Kreisler Rachael , Lee Jung Keun , Quinlan Michael , VandenBrooks John
Published: May 2022

Rocky Mountain Spotted Fever (RMSF) is the deadliest tick‐borne disease in the United States. RMSF is spread from a canine host to humans through incidental contact with a tick vector. Historically, transmission of R. rickettsii, the causative agent, has been associated with the American dog tick (Dermacentor variabilis) and the Rocky Mountain wood tick (Dermacentor andersoni). However, in 2003, an outbreak of RMSF in Arizona was associated with a novel vector, the brown dog tick (Rhipicephalus sanguineus). Since then, the vectoring capacity of the brown dog tick has spread across the American Southwest and into parts of Mexico. Currently, the brown dog tick is not known to vector the bacteria outside of this area despite brown dog ticks being present in every state in the U.S. If this vectoring capacity continues to expand, many parts of the country will be at risk due to the brown dog tick’s ubiquitous nature. This is especially alarming as failure to treat RMSF in humans within the first five days of infection can lead to mortality rates as high as 80%. Therefore, this research was aimed at determining the factors that are currently limiting the vectoring capacity of the brown dog tick to Arizona and the surrounding region in order to prevent further spread of RMSF.

The results of our research show that the current spread of RMSF in the Arizona region is due to a combination of four factors: (1) the presence of a distinct population of brown dog ticks, (2) variation in the genetic strain of the causative agent R. rickettsii, (3) distribution of canine seroprevalence across the region, and (4) geographic and climatic factors. In support of this, we have identified three genetically distinct clades of brown dog tick across the region that vary in the R. rickettsii infection rate. In sequencing intergenic regions of the R. rickettsii isolated from these tick populations, we have identified variation in the rickettsial strain across the region. Using immunofluorescence assays, we determined that canine seroprevalence rates are rising and seropositive dogs were found in regions that have previously not reported any human cases including regions as far north as Petaluma, CA. Even more concerning, 14 of 16 counties surveyed were at medium to high risk for human outbreak. These data were then analyzed at a county level and the relative role of each of the factors varied by geographic location. Together, this multi‐factorial model is a first step towards understanding the epidemiology of RMSF in the Arizona region and provides insights into ways to improve human and canine health by stopping future spread of RMSF.
Student Attitudes Surrounding Active Learning in an Online Anatomy Class
Authors: Mitchell Lauren , Bradley Libby , Handler Emma A.
Published: May 2022

Anatomy classes can be challenging for students due in part to the nature of anatomy course material, in that students are required to learn a large amount of information in a short amount of time. Students who have not identified their study style or have yet to take classes that are materially‐intensive may perform poorly. Additionally, when anatomy courses are presented online, students’ learning can be impacted further due to the lack of access to typical in‐class learning experiences such as peer interaction and dissection. To address these challenges, this study introduces an active learning technique called Jigsaw to supplement peer‐to‐peer learning in an online, graduate anatomy course. Jigsaw was implemented into an online anatomy course in the Spring of 2021. During Jigsaw, students were split up into expert groups in which they were expected to master a specific topic assigned to them. Students from the expert groups were then put into teaching groups, where each student would teach the other students about the material mastered in their expert group material. A previous study had evaluated Jigsaw within an in‐person classroom environment with positive outcomes. The present study included 72 students who were split into 3 expert groups with 24 students per group. Two students from each expert group were assigned to one of 12 teaching groups, 6 students per group with 2 from each expert group A, B, and C. Students’ attitudes surrounding Jigsaw were then evaluated using post‐unit surveys and a final post‐course survey. Results from the post‐course survey indicated that 77.3% of students were either somewhat satisfied or very satisfied when asked about their satisfaction with Jigsaw. The remaining percent were neutral or negative. The satisfaction score took into account student’s likelihood of using Jigsaw again, as well as how they felt Jigsaw improved their understanding of the material. Jigsaw can serve to provide an outlet for peer interaction and learning that is absent is online classrooms, where students do not meet face‐to‐face and do not have hands‐on dissection. Future studies should also take into account grades as well as student attitudes in an effort to evaluate the usefulness of Jigsaw as a supplemental learning resource. Understanding students’ attitudes surrounding active learning techniques in the classroom can inform future anatomy educators as well as other instructors on assisting students in their learning process, especially in material intensive courses such as anatomy and as online learning becomes more commonplace.
Incidence and clinical implications of anatomical variations of the pancreas. A systematic review
Authors: Martínez Daniela V. , Valenzuela Juan J. , Cariseo Carolina V. , Orellana Mathias V. , Iwanaga Joe , Gold Marjorie , Santana Andrés
Published: May 2022

Introduction

The pancreas is retroperitoneal gland located the posterior wall of the abdomen, and plays a central role in digestion and absorption, as well as in the use and storage of energy substrates. Due to its location, as an external organ deep in the retroperitoneum, its irregular surface, its smoothness, its close and varied relationships to adjacent structures, and its vague boundaries, its surgical intervention presents a challenge to the surgeon, hence knowing its anatomical variations is important information previous to consider.

Methods

A systematized search of scientific articles and human anatomy books, written in Spanish or English, published between 1970 and 2020, was carried out in the following databases; MEDLINE, SCIELO, WOS, CINHAL, SCOPUS and GOOGLE SCHOLAR, to describe the characteristics of the anatomical region of the pancreas and clinical complications.

Results

After applying the eligibility criteria 32 studies were included in this review, of which 2 described an annular pancreas, 3 described an ansa pancreatica, 3 mentioned a bifid pancreas, 5 mentioned a circumportal pancreas, 12 mentioned a pancreas divisum, 5 described ductal variations such as the course of the pancreatic duct or anomalies of the ductal system, 2 mentioned variations of the pancreaticobiliary Union and 2 described vascular variations of nearby vessels.

Conclusions

We found that the annular pancreas, PD, ansa pancreatica and bifid pancreas are variations that play a predisposing role in developing pancreatitis, the annular pancreas could also give operative complications in a pancreatic head resection surgery and postoperative complications such as pancreatic fistula, and biliary duct system variations appear to be predisposing factors to gallbladder stones formation. Hence, incorporating the study of its anatomical variations in the training of health professionals is very important for the process of diagnosis, monitoring and treatment of pancreatic diseases.
Alternative GTPase‐deficient mutants of Gα12/13 show differences in signaling potency and effector binding
Authors: Durall R. T. , Meigs Thomas E.
Published: May 2022

Heterotrimeric G proteins are implicated in tumorigenic signaling in a variety of cancer types. Although G protein α subunits of the Gs, Gi, and Gq subfamilies have been found in tumors as mutationally activated, GTPase‐deficient variants, the G12/13 subfamily has the unusual property of participating in cancer progression as an overexpressed, wildtype form. Recent work by Maziarz et al. (2020) identified two “hotspot” mutations at the Arg200 position of Gα13 in bladder tumor samples, demonstrating that mutational activation of G12/13 subfamily proteins may also play a role in cancer progression. Although corresponding mutations at this Arg residue in Gα12 have not been located in tumors, the recent findings for Gα13 suggest that such Gα12 mutants may be identified in future work. Most in vitro signaling studies employing constitutively active Gα12 and/or Gα13 have utilized a Gln‐to‐Leu (QL) substitution in the switch II region to eliminate GTPase activity. Therefore, we sought to compare these QL variants of Gα12 and Gα13 to the recently reported Arg mutants in several G12/13‐specific signaling readouts. We engineered the previously identified hotspot Gα13 mutants (R200K and R200G) along with corresponding mutants of Gα12 (R203K and R203G). Our preliminary findings from reporter gene assays in HEK293 cells suggest that Gα12.R203K and Gα12.R203G are more potent than the Gα12.QL variant in driving two pathways implicated in Gα12‐mediated tumor progression: serum response factor (SRF) and YAP/TAZ. Gα13.R200K and Gα13.R200G appear to stimulate SRF and TEAD reporters at a similar strength to Gα13.QL, consistent with the findings of Maziarz et al. In addition, we investigated binding of these mutants to the RGS‐homology RhoGEFs LARG, p115RhoGEF, and PDZ‐RhoGEF. Unexpectedly, Gα13.R200G showed a RhoGEF binding profile that was different from Gα13.QL, suggesting these GTPase‐deficient mutants differ in their affinity for specific effector proteins. We currently are examining the comparative binding of these mutants to other targets such as epithelial cadherin (E‐cadherin) and radixin. Further, we found these activated Gα12/13 mutants to cause a decrease in cellular levels of E‐cadherin. Interestingly, these mutants appear to have differential effects on E‐cadherin levels compared to QL variants of Gα12/13. We have found this difference to be most pronounced between Gα12 Arg and QL mutants, in comparison to the Gα13 mutants. Due to our recent finding that Gα12 and Gα13 cause an increase in levels of calreticulin, a protein reported to down‐regulate E‐cadherin gene expression, we are using RNA interference to test the hypothesis that Gα12 and/or Gα13 modulate E‐cadherin levels in a calreticulin‐dependent manner.
Irreversible and noncompetitive mu opioid receptor agonists
Authors: Stahl Edward , Schmid Cullen L. , Acevedo‐Canabal Agnes , Read Cai , Grim Travis , Kennedy Nicole M. , Bannister Thomas D. , Bohn Laura M.
Published: May 2022

The ability of a ligand to preferentially promote engagement of one signaling pathway over another downstream of GPCR activation has been referred to as signaling bias, functional selectivity or biased agonism. The presentation of ligand bias reflects selectivity between active states of the receptor which may result in the display of preferential engagement with one signaling pathway over another. In this study, we provide evidence that the G protein‐biased MOR agonists, SR‐17018 and SR‐14968 stabilize the mu opioid receptor in a wash‐resistant, yet antagonist‐reversible, G protein signaling state. Furthermore, we demonstrate that these structurally related biased agonists, are noncompetitive for radiolabeled MOR antagonist binding and while they stimulate G protein signaling in mouse brain, partial agonists of this class do not compete with full agonist activation. Importantly, opioid antagonists can readily reverse their effects in vivo. Given that chronic treatment with SR‐17018 does not lead to tolerance in several mouse pain models, this feature may be desirable for the development of long lasting opioid analgesics that remain sensitive to antagonist reversal of respiratory suppression.
Effects of Fungus‐Derived Compound N9 on Airway Mucus Secretion: In Vitro and In Vivo Studies
Authors: Yimnual Chantapol , Worakajit Nichakorn , Kaewin Suchada , Rukachaisirikul Vatcharin , Muanprasat Chatchai
Published: May 2022

Asthma is a heterogeneous chronic airway inflammatory disease affecting more than 250 million people worldwide. It is characterized by airway inflammation, smooth muscle contraction, and mucus hypersecretion. The current treatment of asthma includes inhibition of the first two characteristics with little attention being paid for modulation of mucus secretion. In fact, mucus dysregulation is a main contributor to airway obstruction in fatal asthma. Since N9 inhibited TMEM16A chloride channel required for airway mucus secretion, we hypothesized that N9 might be a negative modulator for mucus secretion in asthma. Interestingly, we found that N9 at 10 µM impaired Ca2+‐induced mucin depletion in cytokine‐treated airway epithelial (Calu‐3) cell monolayers as analyzed by immunofluorescence staining of intracellular MUC5AC, a pathologic mucin protein, suggesting that N9 inhibited mucin secretion. Intraperitoneal administration of N9 at 20 mg/kg inhibited OVA‐induced mucus secretion as analyzed by ELISA for detection of MUC5AC in bronchoalveolar lavage of BALB/c mice. Conversely, N9 did not reduce inflammatory score in H&E‐stained lung slides or inflammatory cells in bronchoalveolar lavage with dexamethasone (5 mg/kg) being a positive control. These results suggest that N9 specifically targets mucin secretion without compromising immune function. Taken together, N9 represents the first class of fungus‐derived inhibitor of mucus secretion which might be useful for the treatment of asthma and other airway diseases with mucus hypersecretion being the main pathogenesis.
Host lipids required for Membrane protein matrix formation in new severe acute respiratory syndrome‐related coronavirus (SARS‐CoV‐2), ribonucleocapsid packaging and viral assembly
Authors: Amiar Souad , Stahelin Robert V.
Published: May 2022

The current model of SARS‐CoV‐2 assembly proposes a multistage process: (i) the viral RNA coated with the nucleoprotein (N) forms the ribonucleoprotein (RNP), (ii) traffics to the endoplasmic reticulum‐Golgi apparatus intermediate compartment (ERGIC)‐derived vesicles to (iii) interact with the membrane (M) assembled along with spike (S) and envelope (E) proteins. Coronaviruses’ M has been described as required for new virus particle assembly. To date, biochemical and structural information on M are unavailable and key host factors required for M lattice formation, trafficking and interaction with the RNP are poorly characterized. The objective of this research project is to progress our understanding of SARS‐CoV‐2 assembly. Our central hypothesis is that M through its interaction with host lipids coordinates viral protein interactions and RNP packaging as key processes for SARS‐CoV‐2 assembly and spread. The role of lipids as molecules regulating the viral life cycle is poorly investigated.

Our current data demonstrates a clear binding of cell expressed M to a Golgi resident sphingolipid. Furthermore, mutations at the sphingolipid binding motif resulted in deficient pull down of M protein by sphingolipid coated beads. In cells, the M sphingolipid‐binding mutant failed to retain S protein at viral assembly membranes resulting in a reduced entry capacity of newly formed virus‐like particles. Our current study is providing a first insight on the direct role of host lipids during SARS‐CoV‐2 assembly, orchestrated by the M protein.
The Effects of Site Specific Histone Modifications on Nucleosomal DNA Accessibility
Authors: Rowlett Chesley C. , Liu Wenshe M.
Published: May 2022

Chromatin accessibility is the ability of nucleosomal DNA to be physically accessed by cellular biomachinery. Accessibility of chromatin is controlled by a slew of post translational modifications to histone proteins, DNA, and chromatin‐binding factors. This growing field of research has become increasingly important as chromatin accessibility has a large effect on transcription and gene regulation. Gene and cell cycle regulation must be understood to properly study their misregulation which commonly occurs in most, if not all, cancers. Thus, understanding the organization and regulation of the chromatin state by post translational modifications plays an important role in understanding the dysfunction of cancerous cells. The most basic unit of chromatin is the nucleosome core particle. The nucleosome core particle is made up of an octamer that has two copies each of the 4 histone proteins H2A, H2B, H3, and H4 with DNA wrapping around the octamer itself. It is on the N‐terminal tails of these histone proteins that many post‐translational modifications take place the regulate the chromatin state. In this study, the effect of histone lysine acetylation on the DNA‐histone core interaction will be assessed by its thermal stability and accessibility of nucleosomal DNA to Pst1 Digestion. We propose to assay singly acetylated mononucleosomes at the following sites: H2A: K5; H2B: K5, K12, K15, and K20; H3: K9, K14, K18, K23, K27, K36, K37, K64, K56, K79, K115, and K122; H4: K5, K8, K12, and K16. We predict that, as previously shown with nu′‐H3K18ac and nu′‐H3K36ac acetylation at lysine sites will facilitate unwrapping of nucleosomal DNA.
Analysis of Domain Structure of the Bacterial Mating Protein ConB of Bacillus subtilis
Authors: Nguyen Kathy X. , Berkmen Melanie B.
Published: May 2022

Conjugation, or mating, is the transfer of DNA from a bacterial donor cell to a recipient cell. Conjugation contributes to the movement of genes involved in antibiotic resistance and pathogenesis. ICEBs1 is an integrative and conjugative element of Bacillus subtilis. ICEBs1 encodes for conjugation machinery, classified as a Type IV Secretion System (T4SS), that consists of multiple interacting proteins including ConB, ConC, ConD, ConE, ConG, and CwlT. Our focus is on the bitopic transmembrane mating protein ConB. ConB has four domains: a cytoplasmic N‐terminal domain (NTD), a transmembrane segment (TMS), and extracellular central and C‐terminal NTF2 domains. Previous research showed that deletion of any of these four domains results in significantly lower mating frequencies, suggesting that each domain is critical for function. Here, we analyze how deletions of ConB’s domains affect ConB protein levels in B. subtilisusing quantitative infrared fluorescence western blotting. conB domain deletion mutants were fused to the mCherry gene to allow for detection by an anti‐mCherry antibody. We found that the mCherry antibody quantitatively detects ConB‐mCherry in B. subtilis. Site‐directed mutagenesis was used to separately delete each of the four domains encoded by conB. So far, we have discovered that separate deletion of three of the four ConB’s domains does not alter ConB proteins levels greatly. We conclude that the defects for at least three of the four mutants in mating were due to defects in protein function, and not due to low protein levels. Our results provide a new understanding on the domain structure of ConB in B. subtilis.