bluuebunny/crossref_metadata_2025 huggingface dataset:
Analysis of Global and Residue‐Specific yB Crystallin Protein Dynamics via NMR Spectroscopy and Dynamic Light Scattering
Authors: Williams Zachary E. , Labbe Natalie , Cirrincione Lucas , Hasselbeck Jimmy , Michel Lea V. , Thurston George M. , Mills Jeffery
Published: May 2022

The proteins that comprise the water‐soluble interior of the optic lens are referred to as crystallin proteins, of which three major categories have been identified: alpha (a), beta (b), and gamma (y). Gamma and beta crystallin proteins have been observed to undergo aggregation and phase‐separation over time, which can lead to the formation of cataracts, while alpha crystallin limits this formation by removing deformed protein aggregates from solution. Here, we describe our studies on bovine yB crystallin, which is the homologue to human yD crystallin. To better understand the intermolecular interactions between yB crystallins, we overexpressed the 15N‐labeled recombinant protein in Escherichia coli, purified it via column chromatography, and studied the effect of solution conditions on protein dynamics using NMR spectroscopy and dynamic light scattering techniques. Prior research from our group employed T1‐T2 NMR experiments to probe the effect of temperature and protein concentration on global protein dynamics, while more recent 1H‐15N HSQC experiments allowed us to visualize the amide proton peaks for yB crystallin protein at various concentrations. We observed small peak shifts for select residues as the protein concentration increased, which we hypothesized were due to increased intermolecular interactions between yB crystallin molecules. Analysis of these NMR spectra, combined with dynamic light scattering data on the same samples, provide further insight into both global dynamics as well as residue‐specific interactions.
Kinetic and stability study on the immobilized enzymatic step of one‐pot dimerization of 2‐[2‐(dimethylamino)ethoxy]ethanol
Authors: Espinosa Kurt , Schmitz Jonathan , Fitzhenry J.J. , Beck Madison , Watkins Linette
Published: May 2022

Tertiary amine catalysts (TACs) are essential organic molecules used in polyurethane manufacturing. Synthesis of these chemicals involves the use of catalytic transition metals at high temperatures and high pressures. Simple amine systems react easily with transition metals; however, amine systems with multiple hydroxyl groups and methylamine complexes produce significant byproducts. In this study, synthesis that forgo transition metals is explored. Enzymes can provide a clean industrial alternative to transition metal use. 2‐[2‐(dimethylamino)ethoxy]ethanol (DMAEE) is the monomer used in the synthesis of a model TAC. When DMAEE is first enzymatically oxidized, the aldehyde can serve as an electrophile for 2‐(methylamino)ethanol (MMEA), forming the DMAEE dimer. Choline oxidase catalysis is the first step of this pathway. Steady state kinetic parameters of choline oxidase with choline and DMAEE as substrates, optimum pH, and temperature stability are reported in this study. The effect of MMEA on the enzymatic reaction is examined as well. In an industrial application, immobilized enzymes can enhance stability over a broad range of temperature and pH. Therefore, choline oxidase was immobilized on CNBr‐activated Sepharose beads, and the effects of immobilization are reported in this study.
Tau modulates mRNA transcription, alternative polyadenylation (APA) profiles of hnRNPs, chromatin remodeling and spliceosome complexes
Authors: Montalbano Mauro , Jaworski Elizabeth , Garcia Stephanie , Ellsworth Anna , McAllen Salome , Routh Andrew , Kayed Rakez
Published: May 2022

Tau protein is a known contributor in several neurodegenerative diseases, including Alzheimer ’s disease (AD) and frontotemporal dementia (FTD). It is well‐established that tau forms pathological aggregates and fibrils in these diseases. Tau has been observed within the nuclei of neurons, but there is a gap in understanding regarding the mechanism by which tau modulates transcription and RNA processing. We are interested in the P301L mutation of tau, which has been associated with FTD and increased tau aggregation. Our study utilized tau‐inducible HEK (iHEK) cells to reveal that WT and P301L tau distinctively alter the transcription and alternative polyadenylation (APA) profiles of numerous nuclear precursors mRNAs, which then translate to form proteins involved in chromatin remodeling and splicing, including several RNA‐Binding Proteins (RBPs). We isolated total mRNA before and after over‐expressing tau and then performed Poly(A)‐ClickSeq (PAC‐Seq) to characterize mRNA expression and APA profiles. We characterized changes in Gene Ontology (GO) pathways using EnrichR and Gene Set Enrichment Analysis (GSEA). We observed that P301L tau up‐regulates genes associated with reactive oxygen species responsiveness as well as genes involved in dendrite, microtubule, and nuclear body/speckle formation. The number of genes regulated by WT tau is greater than the mutant form, which indicates that the P301L mutation causes loss‐of‐function at the transcriptional level. WT tau up‐regulates genes contributing to cytoskeleton‐dependent intracellular transport, microglial activation, microtubule and nuclear chromatin organization, formation of nuclear bodies and speckles. Interestingly, both WT and P301L tau commonly down‐regulate genes responsible for ubiquitin‐proteasome system. In addition, WT tau significantly down‐regulates several genes implicated in chromatin remodeling and nucleosome organization. Although there are limitations inherent to the model systems used, this study will improve understanding regarding the nuclear impact of tau at the transcriptional and post‐transcriptional level. This study also illustrates the potential impact of P301L tau on the human brain genome during early phases of pathogenesis.
The Role of Sex in the Effects of Cigarette Smoke in Vascular Smooth Muscle Cells
Authors: Centner Ann M.
Published: May 2022

Cigarette smoke (CS) damages the vasculature and causes atherosclerosis or arterial plaque build‐up. While mechanistic differences in males and females likely exist, this area is largely unexplored. In fact, our lab observed that male atherosclerotic mice exposed to CS for 4 months had less severe plaque build‐up than their female counterparts. Thus, we hypothesized that male cells had a protective mechanism against the adverse effects of CS.

To test our hypothesis and fill the gap in the literature, we treated male and female vascular smooth muscle cells (VSMCs) with cigarette smoke extract (CSE). Using increasing concentrations of CSE (10‐50%), we observed that in female cells mortality increased significantly at 40% and 50% CSE, while in males’ significant mortality only occurred at 50% CSE. Female cells treated with CSE (30%) had higher levels of reactive oxygen species (ROS) and cellular senescence than male cells. Increased resistance of male cells to CSE correlated with a decrease in phosphorylated AKT, which drove an upregulation of endogenous antioxidants (catalase, superoxide dismutase 1 – SOD1) as well as levels of autophagy markers including Sequestosome 1/p62 (SQSTM1) and microtubule‐associated protein light chain 3 II (LC3‐II). This mechanism was not seen in female cells. Sqstm1‐/‐ VSMCs express lower levels of catalase and SOD1, increased ROS levels and showed a higher mortality to CSE compared with wild type cells, suggesting that the upregulation of SQSTM1 is required for male cell’s protective effects. Differences in cellular and secreted ECM‐regulators, metalloproteinases (MMPs), were also observed between sexes, suggesting that plaque stability is different between males and females exposed to CS.

Altogether, these data show that intrinsic sex‐dependent differences regulate the Akt/SQSTM1 pathway in response to CS and that SQSTM1 is a possible target to reduce the effects of CS in the cardiovascular system.
Loop‐Diuretics Increase Pendrin through alpha‐ketoglutarate receptor, Oxgr1
Authors: Tahaei Seyedmohammad E. , Wall Susan , Welling Paul A.
Published: May 2022

Introduction

It is well appreciated that loop diuretic resistance is governed by pendrin and activation of the thiazide‐sensitive sodium chloride cotransporter, NCC. Recently, our lab discovered pendrin increases as the natriuretic response wanes, coincident with the activation of a‐ketoglutarate (AKG) synthesis, secretion of a‐KG into the pro‐urine, and increased gene expression of the AKG GPCR, Oxgr1. Here we test the hypothesis that activation of Oxgr1 is necessary for the development of loop diuretic resistance.

Methods

10 week old male OXGR1 KO and WT (C57B6J) mice were challenged with furosemide (100 mg/kg) for 4 days. Urine was collected over 24 hours, daily, and urinary electrolytes were measured. Mice were euthanized at the end of the experiment and plasma electrolytes and aldosterone levels were measured using standard methods. Cortical protein and RNA abundances were measured using western blot and qRT‐PCR, respectively.

Results

WT and OXGR1 KO displayed distinguishing phenotypes only after furosemide administration. The loop‐diuretic challenge increased pendrin protein in WT mice but not in the OXGR1 KO mice despite identical increases in the chief pendrin stimulators, aldosterone and metabolic alkalosis. Pendrin transcript increased to the same extent in both genotypes, indicating OXGR1 augments pendrin via a post‐translational mechanism. Even with compromised upregulation of pendrin, OXGR1 KO mice exhibited the same diuretic response as WT mice. Upregulation of NCC was not compromised in the OXGR1 mice.

Conclusion

OXGR1 is required for post‐translational upregulation of pendrin protein post diuretic challenge, consistent with activation of a aKG‐OXGR1‐pendrin signaling axis. Upregulation of NCC and residual pendrin activity may be sufficient to brake the loop‐diuretic response.
Eight Weeks of Daily Cannabidiol Ingestion Does Not Alter Peripheral Blood Mononuclear Cell Profile or Natural Killer Cell Cytotoxicity
Authors: Kisiolek Jacob N. , Haughian James M. , Flores Victoria A. , Ramani Arjun H. , Stewart Laura K.
Published: May 2022

Objective

The purpose of this randomized placebo‐controlled trial was to determine the effects of an 8‐week cannabidiol (CBD) intervention on peripheral blood mononuclear cells (PBMCs) profile and natural killer cell (NKC) cytotoxicity.

Hypothesis

Following an 8‐week CBD intervention, participants consuming CBD will have an increased percentage of NKC in peripheral blood, as well as decreased NKC cytotoxicity determined by a measure of K562 cell viability.

Methods

Physically active men and women (18‐45 y), currently meeting the American College of Sports Medicine (ACSM) guidelines for physical activity were randomly assigned to placebo (CN, n=5) or CBD (CB, n=9) groups. Participants consumed capsulized control; coconut derived medium chain triglycerides (MCT; 225mg/day) or CBD (50mg/day with 175mg of MCT) daily for 8 weeks. Participants completed measures of body size, body composition (BodPod) and fitness (VO2max), and peripheral blood was drawn before and after the intervention. Ficoll‐isolated PBMCs were co‐incubated for 4h with K562 human leukemia cells before cytotoxicity analysis by flow cytometry. Before co‐culture, PBMCs were stained with T cell marker CD3, and NKC marker, CD56, for immune profiling. K562 cells were pre‐stained with cell‐specific CD71 (transferrin receptor) and the cell viability dye Calcein AM. Data are presented as mean ± standard deviation with significance set at a=0.05. At the pre intervention time point, data was not normally distributed, so a Welch’s t‐test was preformed to determine group differences. A 2 (group) x 2 (time) analysis of variance (ANOVA) was used to identify any interactions or main effects.

Results

At the pre‐intervention time points, there were no significant differences between groups with respect to participant characteristics (age: 27.1 ± 6.1 y; height: 169 ± 9.7 cm; weight: 73.8 ± 15.5 kg; lean body mass: 57.5 ± 12.0 kg; body fat: 21.5 ± 8.3%; VO2Peak: 45.2 ± 7.6 ml/kg/min). There were no significant differences in percentage of NKC (Pre: 8.8 ± 5.8%; Post:7.4 ± 5.7%), natural killer T‐cells (NKT; Pre: 1.7 ±1.1; Post: 1.5 ±1.1%), or T‐cells (Pre: 47.37±13.3; Post: 46.29±10.17%) when CN was compared to CB following the intervention. Furthermore, there were no differences in K562 cell mean or median fluorescence intensity of Calcein AM when CN was compared to CB following the intervention.

Conclusion

Eight weeks of CBD (50mg/day) did not alter NKC, NKT, or T‐cell distribution in PBMCs. In line with these findings, NKC showed similar cytolytic function through no change in mean or median fluorescence intensity of Calcein AM expression of K562 human leukemia cells, indicating no change in cell viability following CBD supplementation. This preliminary study suggests that CBD does not alter the distribution of circulating mononuclear immune cells or NKC cytolytic function.
Detection of Genomic Variations of SARS‐CoV‐2 on the S‐Gene in the Orange County, CA Population
Authors: Gallo Jessie K‐M , Nguyen Jaqueline , Tena Melanie , Calleja‐Macias Itzel
Published: May 2022

Severe Acute Respiratory Syndrome Coronavirus 2 (SARS‐CoV‐2), more commonly known as COVID‐19, is a virus that has spread at a rapid rate beginning in 2019 causing a pandemic. Currently, we remain in this pandemic, and as this virus progresses it is creating mutations of itself, called variants. With these variants, it has become increasingly difficult to control COVID‐19 and keep track of new variants that continue to infect people everywhere. In Orange County, California, COVID‐19 and its variants have enabled county wide shut downs and thousands of infections and deaths. To investigate its devastating effect, five positive COVID‐19 nasal samples were collected between February 2021 to July 2021, and the RNA was extracted. After extraction, a Reverse Transcription Polymerase Chain Reaction (RT‐PCR) was done to amplify 1,727 base pairs of the S‐ Gene within the COVID‐19 genome and was sent to sequencing. These sequences were then aligned using the Basic Local Alignment Search Tool (BLAST) and compared to the original COVID‐19 S‐Gene genome to analyze possible variations. Within the five samples, 30 different mutations were detected and a phylogenetic tree was generated. Awareness of the genetic variation of SARS‐CoV‐2 provides potential future development for more effective targeted research.
Investigating the Molecular and Phenotypic Differences in Pancreatic Neuroendocrine Tumor (PanNET) Cells with Functional Loss of Either ATRX or DAXX
Authors: Nelson Elizabeth B. , Stojanova Marija , Heaphy Christopher M.
Published: May 2022

Telomeres are hexameric DNA repeats at the ends of linear chromosomes that protect genomic integrity. Telomeres shorten after each cell division until a critically shortened length is reached, whereby the cell enters a state of cellular senescence. Cancer cells must overcome this replicative barrier by either upregulating the enzyme telomerase or by using the Alternative Lengthening of Telomeres (ALT) pathway, which uses homology‐directed repair to maintain telomere lengths. ALT is present in ~10% of cancers, yet the exact underlying mechanisms are still being elucidated. However, two known drivers of ALT are loss of function mutations in either ATRX or DAXX, genes that encode a chromatin remodeling complex. Mutations in these genes were initially identified and linked to ALT in pancreatic neuroendocrine tumors (PanNETs), which are the second most common malignancy of the pancreas. This study evaluated the molecular and phenotypic differences between parental PanNET cells (BON1 and QGP1) and isogenic clones with either ATRX or DAXX knocked out via CRISPR‐Cas9 gene editing. The matched parental and isogenic clones were assessed for cell proliferation (MTS assay), clonogenic survival (colony formation assay), and invasion potential (Matrigel invasion assay). Preliminary results for QGP1 cells indicate that the ATRXKO clones show decreased proliferation and colony formation, as well as alterations in invasion potential compared to the parental cell line, thereby suggesting that functional loss of ATRX alters these phenotypic properties. Future studies will help to contribute to the ongoing efforts to understand the underlying mechanisms of ALT and work towards the overall goal of identifying potential biomarkers for early detection, prognosis, and targeted therapies for ALT‐positive cancers.
Insulin is Not Driving Urinary Bladder Enlargement in Rodent Models of Diabetes
Authors: Michel Martin C. , Beltrans‐Ornelas Jesus H. , Silva‐Velasco Diana L. , Castañeda Tamara R. , Elvert Ralf , Kannt Aimo , Xia Ning , Li Huige , Arioglu‐Inan Ebru , Centurion David
Published: May 2022

The urinary bladder is enlarged in all models of type 1 and several of type 2 diabetes (doi 10.1002/nau.23786). We have previously reported that such enlargement is correlated to insulin, but not glucose levels in fructose‐fed rats (doi 10.1096/fasebj.2020.34.s1.00542). Because this was surprising, we have now repeated that study and expanded our analysis to other models of diabetes and obesity.

Five studies were performed in models of type 2 diabetes and obesity, one in fructose‐fed rats (n = 16), one each in ZSF1 rats at age 20 and 28 weeks (a cross between a female Zucker Diabetic Fatty and a male spontaneously hypertensive SHHF rat; n = 30 and 45, respectively), and two in mice on a high‐fat diet (n = 23 and 24, respectively). Bladder weight at study end and blood insulin concentrations were subjected to linear correlation analysis based on individual animal data, and data expressed a squared Pearsson correlation coefficient (r2).

Animals in the diabetic/obese groups were hyperinsulinemic, whereas bladder enlargement was observed in both studies with ZSF1 rats, the old but not the new in fructose‐fed rats, and in neither study with mice on a high‐fat diet. Bladder weight and insulin levels were not correlated in the new study in fructose‐fed rats or any of the other four studies; the numerical correlation coefficient was negative in two of these studies. When data from all six studies were pooled, a very weak inverse correlation was observed (r2 0.0682, descriptive p‐value 0.0094; Figure 1).

We conclude that, contrary to our previous observation, bladder weight is not positively correlated with insulin levels in rodent models of type 2 diabetes and obesity.
The Bile Acid Receptor Tgr5 and High Fat, High Sugar‐Induced Liver Injury
Authors: Ferrell Jessica M. , Dilts Matthew , Stahl Zachary , Pokhrel Sabita , Boehme Shannon , Chiang John Y. L.
Published: May 2022

Background

Progression of fatty liver disease to NASH/fibrosis is a major health concern. Bile acids regulate metabolic homeostasis and inflammation in the liver and gut via the activation of nuclear farnesoid‐X‐receptor (Fxr) and the membrane G protein‐coupled bile acid receptor 1 (Gpbar1, aka Tgr5). Tgr5 is expressed in the gut and skeletal muscle, and in cholangiocytes and Kupffer cells of the liver. Activation of Tgr5 reduces inflammation through suppression of NFκB. Tgr5 also induces GLP‐1 secretion from enterocytes to improve insulin sensitivity. Here, we fed Tgr5‐/‐ mice a high fat, high fructose, and high sucrose diet to test the hypothesis that lack of Tgr5 would exacerbate the progression from fatty liver to NASH/fibrosis.

Methods

Female C57Bl/6J control wild type mice (WT) and Tgr5 knockout mice (Tgr5‐/‐) were fed a high fat (40% kcal), high fructose diet + 20% sucrose water (HFS) for 20 weeks. Metabolic phenotypes were characterized through examination of lipid and cholesterol metabolism pathways, and fibrosis and inflammation pathways.

Results

Tgr5‐/‐ mice fed HFS were more glucose intolerant compared to WT mice, despite gaining the same amount of weight. Tgr5‐/‐ mice fed HFS accumulated significantly more hepatic cholesterol and triglycerides compared to WT mice on the same diet, and gene expression of Acc and Fasn were significantly upregulated. Surprisingly, hepatic expression of genes involved in inflammation and fibrosis were significantly reduced in Tgr5‐/‐ mice fed HFS diet. Tgr‐/‐mice had significantly reduced expression of Col1a1, Col1a2, Tgfb, and Sma. Tgr5‐/‐ also had significantly reduced liver hydroxyproline, a measure of collagen production. Liver fibrosis is partly mediated through serotonin signaling. Tgr5‐/‐mice had significantly reduced expression of Maoa and the serotonin receptors Htr2a and Htr2b, which may have protected against the development of liver fibrosis in these mice.

Conclusion

Altered serotonin metabolism may protect Tgr5‐/‐ mice from the progression of steatosis to NASH/fibrosis induced by a high fat, high sugar diet.
Effect of a chromium‐fructooligosaccharide complex on glycemic parameters in obese and obese‐NIDDM rats
Authors: Tulp Orien L. , Krasner Susan P. , Einstein George P. , Awan Aftab R.
Published: May 2022

The incidence and odds ratio of obesity and Type 2 diabetes, a common sequela of obesity, are increasing in incidence to the point of becoming epidemic in Western society. To determine the potential beneficial effects of a chromium‐fructooligosaccharide complex (FCC) on glycemic responses of male obese and obese NIDDM rats, groups of 3 ‐ 4 month old lean and obese LA/Ntul//‐cp and SHR/Ntul//‐cp rats (NIDDM‐rats, n= 6 rats/group) reared normally on Purina #5012 chow from the time of weaning, were subjected to an oral glucose tolerance (OGT) after a brief 4‐hour fast. A second group of NIDDM rats (n=5) aged one year were also administered the OGT and FCC challenges. NIDDM rats develop IGT by adolescence and renal disease, cataracts, and impaired nerve conduction velocity typical of DM by adulthood. Animals were administered a glucose solution equivalent to 2.5 grams/kg BW via intragastric gavage, or the same glucose plus the FCC (2.5 g/kg BW, containing 200µg Cr and 2.25 mg/serving) or the FCC alone over a brief (1‐2 minute) duration. Measures of blood glucose (BG) were determined at 30‐minute intervals from zero to +120 minutes post gavage via tail bleeding, and the area under the curve (AUC) of each group determined. Final body weights (BW) of obese were greater than lean (p=0.05), and obese‐NIDDM > Obese rats (p=<0.05) and increased in NIDDM rats with older age. OGT of lean rats remained normal throughout, and FCC resulted in minor decreases in AUC and in BG concentrations at all time points (p= n.s.) The OGT of obese rats demonstrated significant increases in BG at +60, +90‐, and +120‐minutes post gavage but remained non‐diabetic, and FCC decreased the glycemic increase and AUC by ~50%, similar to the glycemic response off lean littermates. The OGT of young NIDDM was significantly impaired and FCC resulted in a 32% reduction in the AUC. The OGT and AUC of OLD NIDDM rats was greater than in young rats and was decreased by 25% with FCC. OGT and OGG+FCC were similar at both ages in NIDDM rats. These results are consistent with an FCC‐mediated improvement in glycemic control in congenic animal models for obesity and obesity+NIDDM and suggest that the FCC complex may be a useful nutraceutical adjunct in the dietary treatment of obesity and other glucose intolerant states as they occur in man and animals.
Improved student performance in cardiovascular physiology despite the pandemic, class size increase and switching to a pass/fail grading system
Authors: Johnson Fruzsina K. , Johnson Robert A.
Published: May 2022

Physiology has been a foundational course in medical curriculums for several decades. At William Carey University College of Osteopathic Medicine (WCUCOM), Medical Physiology is taught in both semesters of the first‐year curriculum. Cardiovascular physiology is the first major organ system we teach after the foundational lectures and takes up most of the Fall semester. Going from the 2019‐2020 to the 2020‐2021 academic year we faced multiple challenges: 1) the world‐wide COVID pandemic has forced us to switch to a hybrid curriculum, which for the physiology course meant switching from live lectures to lecture recordings with once weekly online Q&A sessions, 2) also due to the pandemic we switched from the A,B,C,F grading system to pass/fail for all courses, 3) we were undergoing the first stage of a planned class size increase from 100 students to 150 students. Any of these factors could have negatively affected student performance. In this study, we compared overall student performance on the traditionally challenging cardiovascular physiology section between the Fall 2019 and Fall 2020 student cohorts. In 2019, 105 students completed the Fall semester at WCUCOM. Their average exam score on the cardiovascular questions was 79.91±9.68%, and the average standard deviation between the three exams was 8.59±5.18%. In 2020, 156 students completed the Fall semester at WCUCOM. Their average exam score on the cardiovascular questions was 83.11±9.51%, and the average standard deviation between the three exams was 9.37±5.68%. The increase in the mean exam score between cohorts is statistically significant (P=0.013, Mann‐Whitney rank sum test), while the average standard deviation between exams is not significantly different between the two groups (P=0.397, Mann‐Whitney rank sum test). These findings show that student performance on the cardiovascular section of the Medical Physiology course at WCUCOM improved from the Fall 2019 to Fall 2020 semester. Our results suggest that factors traditionally considered to adversely affect student performance, such as changes in curriculum delivery during the pandemic, changes in the grading system, as well as a significant class size increase, are not necessarily negative influences.
A Peep into The Insulin Signaling Pathway of Palmatine: In Vitro, In Vivo & Ex‐Vivo
Authors: Okechukwu Patrick N. , Sharma Mridula N. , Selvaraja Malarvili N. , Nalweyiso Jacinta N. , Balachandran Abbirami N. , Chan Hor Kuan N. , Gaurav Anand N.
Published: May 2022

Background

Insulin resistance, a characteristic of Diabetes Mellitus (DM), is caused by the inactivation of the insulin signaling pathway: IRS‐ PI3K ‐PKC ‐AKT2, and GLUT4. Conditions such as oxidative stress, glycated proteins, hyperglycemia, endoplasmic stress, fatty acid accumulation are major factors that cause inactivation of the insulin signaling pathway. Palmatine, an alkaloid, has been previously reported to possess antidiabetic, antioxidant, antiglycation properties and inhibition of endoplasmic stress.

Objective

The current study evaluated the rejuvenation of insulin signaling pathway by Palmatine in insulin‐resistant L6 skeletal muscle cells, extracted soleus muscle tissue and streptozotocin (STZ)‐induced diabetic rats.

Method

Differentiated L6 skeletal muscle cells and extracted soleus muscle tissue was induced insulin resistance using high insulin and glucose levels media. While STZ was used to induce type 2 DM in Sprague Dawley rats. The cells/tissue/animal was treated with palmatine, while glimepiride and metformin were used as standard drug. The differential gene expression of IRS‐1, PI3K, PKC‐α, AKT2, and GLUT4 was evaluated using qPCR. In silico docking of palmatine, tyrosine phosphatase SHP‐2 (SHP‐2) domains protein and Protein tyrosine phosphatase‐1B (PTP1B) was done using CDOCKER program available in DS V2.0 software.

Result

The results showed that IRS‐1, PI3K, AKT2, and GLUT4 were down‐regulated, while PKC‐α was upregulated in insulin resistant cells, muscle tissue and STZ‐induced diabetic rats. In contrast to the insulin‐resistant cells of the afore mentioned genes were up‐regulated in Palmatine treated cells/tissue/animals, while PKC‐α was down‐regulated. In silico docking of Palmatine showed a good affinity for tyrosine phosphatase SHP‐2 (SHP‐2) domains protein and Protein tyrosine phosphatase‐1B (PTP1B) proteins suggesting the modulation of these proteins.

Conclusion

Palmatine seem to have rejuvenate the impaired insulin signaling pathway through upregulation of the gene expression of IRS‐1, PI3K, AKT2, and GLUT4 and downregulation of PKC‐α expression. In silico docking shows a reduced modulation of PTP1B and increase affinity to SHP‐2. Reduced expression of PKC‐α and modulation of PTP1B maybe the reason for the observed increased expression of IRS‐1, PI3K, AKT2, and GLUT4.
The Neural basis of Complex Spatial, Social and Acoustic Behaviors – in Freely Behaving and Flying Bats
Authors: Yartsev Michael M.
Published: May 2022

Research in our lab focused on the neural mechanisms of three main behavioral functions: (i) complex spatial navigation, (2) acoustic communication and (3) group social behaviors. To do so, we utilize an unusual model system, the bat, that offers several advantages.

For the study of complex spatial navigation, we utilize bats because they are one of the most exquisite navigators on the planet and the only mammal capable of self‐propelled flight. We developed a human‐free, fully automated experimental setup where we discovered a temporally dissociated code for the representation of space and time in the hippocampus of flying bats. Furthermore, we developed a host of novel technologies, such as wireless calcium imaging in freely flying bats, which enables studying the stability of hippocampal codes in bats for the very first time.

For the study of acoustic communication, we utilize bats because they are vocal experts and are one of the only mammals that are believed to acquire their vocalizations through an active learning process. If correct, this would be incredibly important because vocal learning has yet to be rigorously studied in the mammalian brain. Using advanced anatomical mapping methods and in combination with wireless neurophysiological approaches and behavioral monitoring systems we able to study this complex behavior in a detailed manner.

Finally, for the study of group social behavior, we build on the fact bats are highly social, long‐lived, mammals that spend their entire life in group social living. We established technologies for multi‐animal wireless neurophysiological approaches that provided us a unique opportunity to study the neural computations underlying group social behavior. Through a series of studies, we uncovered an incredibly rich repertoire of neural representations for group social interactions in bats. These included the representation of social information both within and across the brains of socially interacting individuals as well as a unique representation of individual identity and social relationships between group members during communication.
Journey through a Phenomic Universe: Frontiers in Analysis of Morphological Data
Authors: Watanabe Akinobu
Published: May 2022

Advancements in imaging modalities now allow for high‐throughput acquisition of high‐fidelity phenotypic data. Analytical pipelines that harness these rich sources of anatomical information, from data collection to results, are vital for the emergence and maturation of phenome‐level research that aims to analyze phenotypes at organism‐wide scale. Towards this goal, my colleagues and I have employed existing and newly developed high‐dimensional geometric morphometric techniques––an approach that permits a more holistic investigation of not only how anatomical structures change forms across treatments or through time, but also how their subunits interact within and between each other through analysis of integration and modularity. Here, I highlight the utility of this high‐dimensional approach with studies on skull shape evolution in squamate reptiles and neuroanatomical evolution in modern birds from their dinosaurian ancestors. Collectively, these studies show that evolutionary dynamics differ across taxa, time, and anatomical regions; and that diverse phenotypes could spring from conserved patterns of integration, as well as from more strongly integrated structures contrary to historical notion. As we collect and analyze high‐dimensional data, however, we must also consider whether these datasets are, in fact, “better” at characterizing morphology. To address this issue, I present analytical explorations that demonstrate how and when a high‐dimensional approach is superior to standard morphometric methods, including greater stability and reliability in morphological characterization and statistical analysis of variation and covariation. Combined with modern imaging technologies, a high‐dimensional approach to shape analysis empowers morphologists to capture and understand phenotypic variation at unprecedented resolution and scale.
Engaging biochemistry students virtually utilizing problem‐based learning and at home lab activities
Authors: Wodzanowski Kimberly A. , Anonick Madison V. , Genova Lauren A. , Kloxin April M. , Grimes Catherine L.
Published: May 2022

The unprecedented 2020 COVID‐19 pandemic prompted the rapid shift to online learning in higher education. For courses particularly in the natural sciences, this shift posed a challenge to adapt complex course material that relied on in‐person demonstrations and hands‐on laboratory experiences to successfully be mastered. Online learning continues grow in these fields where it was once limited. In this piece, we describe the rejuvenation of our problem‐based learning Introduction to Biochemistry for students in a virtual environment. We utilized Zoom for engaging discussions between student‐led discussion groups focused on their reading of primary literature, adapted labs with course content to enable them to be performed at home, and utilized the online format to connect with a scientist whose work we studied. In particular, our course centered around the mechanisms of antibiotics, beginning with Pasteur’s discovery of fermentation and ending with present day papers deconvoluting the synthesis of bacterial peptidoglycan and how different antibiotics inhibit this pathway. Throughout the course, we were able to utilize the online format and at home labs to connect science to everyday life in different ways than traditional classroom learning. Students overall were engaged and enjoyed how this course was different than other online courses. Our observations provide guidance for how to improve future online‐based science courses, particularly problem‐based learning ones, to make them most effective for successful student learning.
The Role of Endothelial PARs in Modulating Insulin Signaling
Authors: Rajala Rahul , Griffin Courtney T.
Published: May 2022

Endothelial cell (EC) dysfunction occurs in diabetes. Thrombin, a circulating serine protease with increased activity in diabetics, signals through the protease‐activated receptors 1 and 4 (PAR1/PAR4). To determine EC‐specific roles of PARs in insulin‐dependent diabetes mellitus (IDDM), we generated Par1fl//fl;Par4fl/fl;iCdh5(PAC)‐CreERT2 (Par1/4ECko) mice, which we induced with tamoxifen to delete Par1/Par4 from ECs and subsequently challenged with streptozotocin (STZ) to induce diabetes.

Diabetic Par1/4ECko mice had reduced blood glucose levels (347.6 mg/dl, p<0.001) compared to their diabetic control littermates (538.4 mg/dl). At 12 weeks after STZ treatment, Par1/4ECkomice also weighed significantly more than their diabetic control littermates (~6.5%, p<0.024). Par1/4ECko mice likewise had visible abdominal fat pads, which are normally lost during the progression of diabetes. In addition, serum albumin (Reference Range: 2.5‐4.8 g/dl), which can be an indicator of hyperglycemia‐induced dehydration, was significantly elevated in diabetic control mice (5.2 g/dl, p<0.0007) but attenuated in Par1/4ECko mice (4.46 g/dl). Importantly, diabetic Par1/4ECko mice did not have significantly different (p<0.4518) serum insulin levels than their diabetic control littermates. Therefore, since diabetic Par1/4ECkomice showed better glucose uptake parameters than their control littermates, despite comparably reduced insulin levels, the mutant mice have whole‐body phenotypes that are consistent with heightened insulin sensitivity. Moreover, diabetic Par4ECkomice did not phenocopy the reduced blood glucose levels and weight loss seen Par1/4ECko mutants, indicating that endothelial Par1 deletion likely drives the heightened insulin sensitivity phenotypes.

PAR1‐siRNA‐treated primary Human Umbilical Vein Endothelial Cells (HUVECs) showed increased basal insulin receptor (IR) activity (independent of insulin treatment) as measured by the uptake of a fluorescent glucose‐analog. PAR1‐siRNA‐treated HUVECs also showed increased expression of the IR Type A (IR‐A) spliceform, which has high insulin‐independent basal activity. Conversely, treatment of HUVECs with a PAR1 ligand resulted in increased levels of the insulin‐dependent IR Type B (IR‐B)spliceform. These data suggest that endothelial PAR1 can act as a modulator of IR splicing/activity.

IR activity in ECs is implicated in insulin transport from the bloodstream to parenchymal tissues. Increased endothelial IR signaling results in increased whole‐body insulin sensitivity. We propose that endothelial PAR1 acts as a modulator of IR splicing/activity and subsequent insulin transcytosis. Therefore, loss of endothelial PAR1 potentially increases whole‐body insulin sensitivity by driving insulin transport to parenchymal tissues and subsequent glucose uptake.
Purification of the human cone‐rod homeobox protein
Authors: Hayes Jonathan E. , Enke Raymond A. , Berndsen Christopher
Published: May 2022

A “lock and key” is the most common depiction of protein binding, protein has a specific structure, and its binding molecule of interest can fit into it. What happens when a protein domain needs to bind to multiple biomolecules? The transcription factor CRX is one of those with an activation domain that can bind to various biomolecules. CRX regulates the gene expression of cone and rod photoreceptors. To effectively regulate gene expression, CRX’s activation domain interacts with multiple transcriptional co‐activators. Unlike the “lock and key” concept with defined structures, we propose that the activation domain of CRX is intrinsically disordered, allowing for interactions of multiple proteins in the same domain. In support of a disordered activation domain, we observe that proline and serine amino acids are abundant in the CRX activation domain. We also note an abundance of pathogenic frameshift mutations and a lack of point mutants in the activation domain. To characterize the structure of CRX, we attempted to purify full‐length CRX and CRX coexpressed with known transcriptional coactivator NRL. We hypothesize that this latter experiment will enhance the stability and expression of full‐length CRX. We have purified CRX using nickel chromatography and are currently optimizing the next steps to enhance the purity of CRX and NRL. The information from biochemical and structural experiments on purified CRX will provide insight into the transcriptional regulation of CRX, leading to the development of photoreceptor neurons.
Characterization of Buprenorphine Antagonism at Human Mu Opioid Receptors
Authors: Wedemeyer Michael J. , Berg Kelly A. , Clarke William P.
Published: May 2022

In 2017 the US government declared a public health emergency to address the national opioid crisis. Despite this focused effort and increased awareness, opioid overdose deaths have since increased by over 50%. Individuals with opioid use disorder have increased risk of opioid related death, and medication assistance can facilitate recovery by reducing opioid cravings and withdrawal effects. One such medication is buprenorphine, an FDA approved drug for the treatment of opioid use disorder sold under the brand Suboxone®, Subutex®, and Zubsolv®. While buprenorphine is clinically described as a partial agonist of the mu opioid receptor (MOR), the literature demonstrates that its pharmacological characteristics are complex. In ongoing studies we are rigorously characterizing the pharmacological actions of buprenorphine at the human MOR.

A genetically encoded biosensor was used to monitor cAMP levels in real time in HEK293 cells expressing a low density of the human MOR. In these cells, buprenorphine acted as an antagonist, shifting the concentration response curve of the MOR agonist, DAMGO, to the right (reduced potency) in a concentration‐dependent manner, similar to the action of the MOR antagonist naloxone. Unlike the effect of naloxone, buprenorphine also decreased the maximal response of DAMGO, an effect that saturated with increasing concentrations of buprenorphine. Similar antagonism by buprenorphine was observed when fentanyl was used as the MOR agonist. The effect of buprenorphine to decrease the potency of DAMGO was reduced by washout of buprenorphine. In contrast, the reduction in the maximal response to DAMGO was resistant to washout of buprenorphine. Both effects of buprenorphine (on DAMGO potency and efficacy) were prevented by pre‐treatment with naloxone, followed by rigorous washout. This suggested that buprenorphine’s effects were mediated by a naloxone‐sensitive binding site, presumably the orthosteric site. Given the slow rate of dissociation of buprenorphine binding to MOR in several published studies and the rapid time‐course involved in the cAMP response, we considered the possibility that the observed effects of buprenorphine were due to hemi‐equilibrium conditions. Computations conducted using hemi‐equilibrium equations support the hypothesis that the antagonist effects of buprenorphine in this system are due to hemi‐equilibrium. These kinetics were further supported in high‐expressing hMOR CHO cells, as buprenorphine agonism was resistant to reversal by naloxone or washing. As a frontline treatment for opioid use disorder, an understanding of buprenorphine’s pseudo‐irreversible binding may elucidate buprenorphine’s unique properties and facilitate future drug discovery.
Assessment of Metabolic Response During High‐Intensity Interval Exercise and Resting Vascular and Mitochondrial Function in Recreational CrossFit Participants
Authors: Pearson Regis C. , Olenick Alyssa A. , Jenkins Nathan T.
Published: May 2022

Background

High‐intensity functional training (HIFT) can play a major role in physiological adaptations to prevent cardiometabolic disease. A majority of HIFT interventions incorporate a CrossFit (CF) training methodology, in which the goal is to increase work capacity over time while using mono‐structural, body weight, and weightlifting movements.

Methods

We measured aerobic capacity, metabolic response during high‐intensity interval exercise (HIIE), resting mitochondrial oxidative capacity, and resting large vessel function and microvascular reactivity in adults who have participated in a CF exercise for greater than one year vs. a sedentary group completing <2 h·wk‐1 of structure exercise for at least one year (SED).

Results

21 healthy, non‐smoking, participants were recruited (CF n = 13 vs. SED n = 8). CF participants had 40.4% greater absolute and 33.0% relative VO2 peak (P < 0.001) and lower body composition (CF = 18.6 (3.8) vs. SED = 30.3 (8.4); P < 0.001). CF participants had lower ratings of perceived exertion (P = 0.022) with higher respiratory responses (P < 0.05) and a lower heart rate response during HIIE (P = 0.024). CF participants had higher fat and carbohydrate oxidation when expressed as absolute and body weight relative values (P < 0.013), but not when expressed relative to lean mass (P > 0.200), indicating that skeletal muscle mass is a primary driver in substrate oxidation. CF participants had greater mitochondrial oxidative capacity (CF n = 12; P = 0.014). There were no differences in large artery function, but CF participants had greater baseline arterial diameter (CF n = 12; P = 0.004) and faster reperfusion following arterial occlusion (CF n = 12; P < 0.05), suggesting some influence of exercise habits on microvascular function.

Conclusion

Overall, these data support the effectiveness of HIFT programs, like CF exercise, to improve fitness and weight status and metabolic, mitochondrial, and vascular function.
Eating a high fat or ketogenic diet increases sensitivity of rats to methamphetamine‐induced locomotion
Authors: Elsey Madeline , Beltran Nina M. , Serafine Katherine M.
Published: May 2022

Eating a high fat diet leads to negative health consequences such as obesity and type 2 diabetes. Recent evidence also suggests that eating a high fat diet can impact drug sensitivity. For example, eating a high fat laboratory chow enhances the sensitivity of rats to the behavioral effects of drugs of abuse, including methamphetamine. Previous research has focused on diets that are high in fat and carbohydrates but low in protein. In contrast, a ketogenic diet is high in fat, low in carbohydrates, and low in protein. Ketogenic diets have been used in humans for the treatment of epilepsy and have been investigated for weight loss. Preclinical studies suggest that rats eating a ketogenic diet are less sensitive to the locomotor stimulating effects of cocaine (e.g., stereotypy). It was hypothesized that eating high fat chow would enhance sensitivity of rats to methamphetamine‐induced locomotion and sensitization; however, rats eating ketogenic chow would not differ from rats eating standard chow. To test this hypothesis, rats eating standard chow (17% kcal from fat, 58% kcal from carbohydrate, 25% kcal from protein), high fat chow (60% kcal from fat, 21% kcal from carbohydrate, 18% kcal from protein), or ketogenic chow (90.5% kcal from fat, 0.3% kcal from carbohydrate, 9.2% kcal from protein) were tested once weekly with cumulative doses of methamphetamine (0.1‐3.2 mg/kg; i.p.). After 4 weeks, rats eating high fat chow were more sensitive to the locomotor‐stimulating effects of smaller and intermediate doses of methamphetamine (e.g., 0.32, 1.0 mg/kg) than rats eating standard chow. Rats eating ketogenic chow were also more sensitive than rats eating standard chow to the locomotor‐stimulating effects of methamphetamine, but only at the largest dose tested (3.2 mg/kg). Future research will investigate the mechanisms underlying these diet‐induced changes in methamphetamine sensitivity.