bluuebunny/crossref_metadata_2025 huggingface dataset:
Closed‐head injury increased platform‐mediated avoidance in rats
Authors: Rodriguez‐Rosado Sian Y. , Martínez‐Guzmán Osmarie , Cáceres‐Chacón Mauricio , Rivera‐López Melissa , Haddock‐Martínez Héctor , Sierra‐Mercado Demetrio
Published: May 2022

The most common form of brain injury, concussion, is frequently seen in contact sports and military combat. Converging lines of evidence suggest that concussion may impair fear‐related behaviors, such as platform‐mediated avoidance. In platform‐mediated avoidance, rats are conditioned in an operant chamber to auditory tones co‐terminating with a mild foot shock. An acrylic platform in the opposite corner of the sucrose‐delivering bar allowed rats to avoid the shocks. The effects of concussion on platform‐mediated avoidance remain unclear. Concussion can be modeled in rodents with a closed head injury (CHI). In our model, a guide tube is placed above the head of anesthetized rats, and a weight is dropped through the tube. In the current study, we hypothesize that CHI will impair avoidance behavior. We observed that rats that underwent a CHI spent more time on the platform throughout the test session during the absence of the auditory tone (p=0.0127), even though they had learned that the absence of the tone was a safe period. These results suggest that concussive‐like brain injury results in excess avoidance behavior.
Female‐Specific Features of Metabolic Syndrome in an LH Congenic Rat
Authors: Clark Karen C. , Wagner Valerie A. , Holl Katie L. , Reho John J. , Grobe Justin L. , Kwitek Anne E.
Published: May 2022

Central obesity, high blood pressure, dyslipidemia, and insulin resistance are a collection of cardiovascular and metabolic risk factors that form the basis of the Metabolic Syndrome (MetS) and represent a major public health burden worldwide. The phenotypes that define MetS are all highly heritable, but their genetic complexity necessitates the use of animal models to tease apart novel pathways.

The Lyon Hypertensive (LH) rat is a well‐characterized model of MetS, exhibiting spontaneous and profound differences in features of MetS compared to its metabolically healthy control, the Lyon Normotensive (LN) rat. To understand the genomic causes of MetS in the LH rat, we developed a congenic rat model, where a portion of LN chromosome 17 is introgressed on the LH genomic background. Our hypothesis was that differences in metabolic features between the LH controls and the congenic rats (LH17LNa) would be due to the replaced region of the genome. Male and female LH17LNa rats and LH controls were phenotyped for a variety of MetS characteristics, including weekly body growth until tissue collection at 15 weeks of age, body composition by nuclear magnetic resonance (NMR) at 8, 11, and 14 weeks of age, metabolic rate (Promethion system) between 10 and 12 weeks of age, and adipose tissue collection and histological examination.

We observed a significant decrease in total body growth in female congenics, beginning at 12 weeks of age and persisting throughout the rest of the study. Prior to the divergence in body weight, female congenics showed significant increases in fat mass and significant decreases in fat free mass that remained consistent at all timepoints measured. We also found significant female‐specific decreases in metabolic rate. Tissue collection revealed the source of the increased adiposity in the female LH17LNa rats was specific to abdominal white adipose tissue, and this phenomenon was further explained by significant hypertrophy in those adipocytes, with no evidence of adipocyte hyperplasia.

Genome resequencing of the parental strains identified a nonsense mutation in Ercc6l2, implicating this gene as a strong contender underlying the phenotype differences in the congenics. Ercc6l2 is a helicase involved in transcription‐coupled DNA repair, which is specifically mobilized to repair oxidative stress‐induced DNA damage in post‐mitotic cells. Truncating mutations in Ercc6l2 have previously been implicated in genome instability and premature aging in hematopoetic stem cells in patients with bone marrow failure. The discovery of the significance of Ercc6l2 in the context of female‐specific adipocyte biology could represent a novel role of DNA repair syndromes in MetS pathogenesis.
Peroxiredoxin‐4 Interacts with Dopamine D5 Receptor to Reduce Endoplasmic Reticulum Stress
Authors: Amatya Bibhas , Yang Sufei , Yu Peiying , Armando Ines , Zeng Chunyu , Felder Robin A. , Asico Laureano D. , Jose Pedro A. , Lee Hewang
Published: May 2022

Peroxiredoxin‐4 (PRDX4), an endoplasmic reticulum (ER)‐localized antioxidant enzyme, plays an essential role in cellular redox homeostasis by reducing hydrogen peroxide from thiol‐containing compounds. The dopamine D5 Receptor (D5R) also plays a protective role against oxidative stress. We hypothesize that D5R interacts with the PRDX4 to reduce oxidative stress in the kidney. In D5R‐HEK 293 cells, fenoldopam (FEN, 25 nM/12 hr, n=4), a D1R and D5R receptor agonist, increased PRDX4 protein expression (1.92±0.12‐fold over basal level, n=4), mainly in non‐lipid raft (LR) fractions (LRs: 24.9±11.4%, non‐LRs: 75.1±11.4%, baseline; LRs: 30.9±13.9%, non‐LRs: 174.1±16.7%, FEN). By contrast, fenoldopam did not affect PRDX4 protein expression in D1R‐HEK 293 cells, indicating a D5R‐specific effect on PRDX4 protein expression. In human renal proximal tubule cells (hRPTCs) and D5R HEK293 cells, fenoldopam increased the co‐immunoprecipitation between PRDX4 and D5R and their co‐localization in the ER. SiRNA‐mediated silencing of PRDX4 increased hydrogen peroxide production in both the vehicle (Veh)‐ and fenoldopam‐treated hRPTCs [(scrambled siRNA: 100 ±15.1% and 55.2± 7.2% with Veh and FEN, respectively; PRDX4 siRNA: 161.8±15.3% and 145.1±14.6 % with Veh and FEN, respectively, n=4)]. The D5R protects against inflammation and siRNA silencing of PRDX4 increased the production of the pro‐inflammatory cytokines, interleukin‐1β [26.88±3.8 and 46.40±4.2 pg/mL (n=3, D5R‐HEK 293); 15.87±1.2 and 37.9±1.4 pg/mL (n=3, hRPTCs), and tumor necrosis factors [131.7±6.5 and 271.2±18.1 pg/mL (n=4, D5R‐HEK 293); 108.8±11.8 and 240.1±13.7 pg/mL (n=4, hRPTCs)]. In D5R‐HEK293 and hRPTCs, the fenoldopam‐mediated decrease in ER‐resident caspase‐12 was also impaired by gene silencing of PRDX4. Furthermore, PRDX4 protein expression was reduced in the kidney cortices of Drd5‐/‐ mice, relative to Drd5 WT mice (WT: 1.00±0.12, n=4; Drd5‐/‐: 0.64±0.13, n=4; P<0.05). We conclude that D5R positively interacts with PRDX4 to reduce ER stress in the kidney.
Na”Nobody” likes COVID‐19!: The Role of Nanobodies in Fighting COVID‐19
Authors: Bah Fatimah , Chandra Aradhna , Krier Luke , Wisniewski Anna , Lane Jim
Published: May 2022

The SARS‐CoV‐2 pandemic has placed increased stress and importance on the development of new and effective treatments for COVID‐19. According to Huo et. al, nanobodies are a new and promising treatment that may be the future of preventing COVID‐19 and other pathogens. Nanobodies are a monomeric protein consisting solely of heavy chain amino acids with only one chain in its Receptor Binding Domain(RBD), in contrast to the antibody that consists of two heavy and two light chains in its RBD. The structural difference affects antibodies’ and nanobodies’ function in reference to the binding to the SARS‐CoV‐2 virus spike, and their structural difference allows for a comprehensive treatment against COVID‐19 by providing unique opportunities in their application. Our research and 3‐D modeling using jMol suggests that while nanobodies are much smaller in size, they have more ridges and bends which creates more overall surface area for hydrogen bonds. These hydrogen bonds take place mainly on alanine and arginine side chains. This means nanobodies satisfy more hydrogen bonds with the spike protein, and therefore have a stronger and more consistent binding affinity to COVID‐19. In comparison, antibodies have a smoother surface, with less bends and folds, that allows for less space for hydrogen bonds, creating, while still effective, a less consistent and weaker binding affinity to the COVID‐19 spike. In addition to nanobodies binding ability and simple structure, they can also be produced synthetically granting immediate care. The Mahtomedi MSOE Center for BioMolecular Modeling MAPS Team used 3‐D modeling and printing technology to compare the structural and functional properties of nanobodies and antibodies in fighting SARS‐CoV‐2 virus.
Vasodilators stimulate SK3 channel anterograde trafficking in endothelial cells to elicit vasodilation
Authors: Peixoto‐Neves Dieniffer , Yadav Shambhu , Mackay Charles , Leo M. Dennis , Mata‐Daboin Alejandro , Mbiakop Ulrich C. , Jaggar Jonathan H.
Published: May 2022

Endothelial cells (ECs) line the lumen of blood vessels and regulate functions, including contractility. Physiological stimuli, such as acetylcholine (ACh) and intraluminal flow, activate small conductance calcium‐activated potassium (SK3) channels in ECs, leading to hyperpolarization and vasodilation. Whether these stimuli modulate SK3 surface abundance in ECs to elicit vasodilation is unclear. Here, we investigated physiological regulation of SK3 channel surface trafficking in mesenteric artery (MA) ECs. Biotinylation experiments demonstrated that ACh or intravascular flow rapidly (2 min) increased surface SK3 protein ~ 2.0‐ and 2.1‐fold, respectively in MAs, with similar results obtained in primary‐cultured ECs. Brefeldin A, an anterograde trafficking inhibitor, removal of extracellular Ca2+, GSK2193874, a selective TRPV4 channel blocker or TRPV4 knockdown using siRNA, each inhibited the ACh‐induced increase in surface SK3 channels in MAs. Super resolution microscopy (SMLM) experiments demonstrated that ACh increased the size of surface SK3 clusters in ECs. ACh activated whole‐cell SK currents and this effect was inhibited by brefeldin A. Brefeldin A also reduced vasodilation induced by ACh and intravascular flow to between ~48 and 57 % of those in control. Brefeldin A blocked the SK channel component of ACh‐ and flow‐mediated vasodilations. In summary, our data indicate that ACh and flow stimulate anterograde trafficking of SK3 channels in ECs via a TRPV4‐dependent Ca2+ signaling mechanism to elicit vasodilation.
Exercise Training Shifts Dependency from NADPH Oxidase 2 to NADPH Oxidase 4 in Endothelium‐Dependent Dilation in Coronary Arterioles Distal to Chronic Occlusion
Authors: Sytha Sharanee P. , Bray Jeff F. , Ramirez Cristina R. , Heaps Cristine L.
Published: May 2022

Previously, we have reported that exercise training enhances endothelium‐dependent dilation in coronary arterioles distal to chronic occlusion, through increased contribution of the redox signaling molecule, hydrogen peroxide (H2O2). In the present study, we tested the hypothesis that enhanced endothelium‐dependent dilation in collateral‐dependent arterioles after exercise training would be attributable to an increased contribution of NADPH oxidase (NOX) 4‐derived reactive oxygen species. Yucatan miniature swine were surgically instrumented with an ameroid constrictor around the proximal left circumflex artery, which gradually induced occlusion of the artery and created a collateral‐dependent vascular bed. Eight weeks postoperatively, swine were assigned to either a sedentary protocol consisting of normal pen activity or a progressive exercise‐training regimen comprised of treadmill running 5 days a week, for 14 weeks. Coronary arterioles were isolated from the nonoccluded and collateral‐dependent myocardial regions and bradykinin‐mediated dilation via pressure myography was performed in the presence and absence of ML‐171, gp91ds‐tat and GKT136901, inhibiting NOX1, NOX2, and NOX1/4 isoforms, respectively. Additionally, using the superoxide probe, dihydroethidium, HPLC analysis was performed on isolated microvascular endothelial cells, pretreated with NOX inhibitors, to determine candidate sources of superoxide. Inhibition of NOX2 produced a significant rightward shift in EC50 values of arterioles from sedentary animals, independent of occlusion, as well as the nonoccluded arterioles of exercise‐trained pigs. EC50 values of collateral‐dependent arterioles from exercise‐trained swine were not significantly altered by NOX2 inhibition. Contrastingly, collateral‐dependent arterioles from exercise‐trained swine exhibited a significant shift in EC50 values in the presence NOX1/4 inhibition, which was absent in other arteriole treatment groups. Importantly, NOX1 inhibition did not significantly shift EC50 values in any treatment group, suggesting responses to the NOX1/4 inhibitor, GKT136901, reflect the contribution of NOX4. Interestingly, microvascular endothelial cell superoxide levels were not different across arteriole treatment groups. Our present findings suggest that exercise training produces adaptations in collateral‐dependent arterioles that appear to shift dependence from NOX2‐ to NOX4‐derived reactive oxygen species for endothelium‐dependent dilation. Taken together with our previous findings, our data support the assertion that enhanced dilation of collateral‐dependent arterioles after exercise training is driven by enhanced NOX4‐derived H2O2 levels.
Morphological Trends of the Pinniped Tympanic Bulla, with Emphasis on Phocidae
Authors: Koper Lindsey , Koretsky Irina , Rahmat Sulman J.
Published: May 2022

Research over the past century is extremely limited on the morphology of the auditory apparatuses of pinnipeds, which include the Families Phocidae (true seals), Otariidae (sea lions and fur seals), and Odobenidae (walruses). An extensive literature review revealed in accurate terminology of this region, incorrect information, mislabeled figures, and details that only correspond to terrestrial taxa. Pinnipeds are unique semiaquatic taxa, possessing adaptations that allow them to hear efficiently both in water and on land. The severe lack of information on this region, as well as nonexistent current studies, demonstrates the need for evaluation and comparison of the auditory morphologies of modern carnivorans in relation to hearing. Initial questions addressed in this study include: comparing the tympanic bulla morphologies of Phocidae to other pinnipeds; comparing distinct morphologies of the tympanic bulla found within phocid subfamilies; and demonstrating any discernable trends in regard to ontogeny or sexual dimorphism. This project explored the morphological correlations of the auditory region of the basicrania of the following representatives of the carnivoran families: Phocidae (including looking at individual subfamilies of Cystophorinae, Monachinae, and Phocinae), Otariidae, Odobenidae, Ursidae (bears), and Mustelidae (weasels). Twelve measurements of the skull and nine calculated ratios were included in multiple principal component analyses (PCA) to determine what areas of the auditory apparatus had the most significant variation in morphology. This is the first study using this type of methodology, especially in reference to the hearing adaptations of pinnipeds, and specifically in phocids. The results of the principal component analyses did exhibit distinct trends in phocid bullar morphology in relation to the other pinnipeds. Phocids tend to have larger tympanic bullar ratios in comparison to other carnivorans and otariids present tympanic bullae that are wider apart and medially parallel. Phocid subfamilial trends include: Cystophorinae on average have wider tympanic bullar ratios; Monachinae have greater distances between their tympanic bullae; and Phocinae overall presents with more generalized morphology. There are distinct trends in the auditory morphology of juveniles (their tympanic bullae make up a greater portion of their skull), but only phocid species that have extreme differences between males and females exhibit distinct morphology within the PCA. To establish more significant trends in ontogeny and sexual dimorphism, more taxa need to be included in future studies. Subsequent studies on auditory morphology will be able to incorporate behavioral hearing information so that a clear, accurate picture of pinniped hearing may emerge. Understanding the modern semiaquatic pinniped ear will help clarify the evolutionary process of aquatic adaptations found within fossil carnivoran taxa, a significant and unknown field of study.
Acute And Chronic Learning Impairment Following Seizures In Larval Zebrafish
Authors: Ramon Alfonsina , Belamorich Nicole , Salami Faisha , Singh Harsimran , Lippman‐Bell Jocelyn
Published: May 2022

Early‐life seizures (ELS) can disrupt brain development, often leading to cognitive impairments in humans and rodents. Here, we investigated whether seizures in larval zebrafish (Danio rerio) lead to acute and long‐term learning deficits. Based on ELS studies in rodents, we hypothesized that seizures in larval zebrafish would impair their performance in declarative memory tasks. To test this, we induced seizures in larval zebrafish using pentylenetetrazole (PTZ), which causes behavioral and electrical seizures in zebrafish. We modified the existing PTZ seizure model, exposing zebrafish to PTZ daily from 5‐7 days post‐fertilization (dpf). We then compared later‐life learning through short‐term memory tasks that lacked external punishment or motivation and focused on conscious recognition and interest in novel stimuli. To test learning and memory acutely after seizures, we used the visual lateralization novel object recognition (VLNOR) test, based on the novel object recognition test frequently used in rodents. Zebrafish show asymmetries in the use of left (LES) and right eye systems (RES), and will initially use LES to observe novel objects and RES to view familiar objects. We found that, when tested for recall of a previously novel object one hour after initially viewing it, 12‐14 dpf unhandled controls (UC group) reached a statistically significant majority of RES approaches by the end of the 8‐minute recall test (p=0.048). The PTZ group did not, and trended towards a significant decrease from 50% in the first 2 minute bin of the recall test (p=0.054). Taken together, these data indicate that the PTZ fish were not able to recall a previously viewed object, and when the object was reintroduced 1 hour later, responded as if the object were novel. We next used a y‐maze task to assess memory 3 months after seizures. In this task, fish had to recall the novel arm of the maze 90 minutes after being removed from the maze. The PTZ group spent significantly less time in the novel arm of the y‐maze during the recall test compared to their UC clutchmates (mean % time in novel arm +/‐ SE = 44.0+/‐3.2 for UC, 29.7+/‐2.1 for PTZ; p=0.002 by t‐test). Furthermore, our preliminary molecular studies show distinct expression profiles of synaptic plasticity genes between PTZ and UC groups by qPCR. Taken together, these results imply that inducing seizures in larval zebrafish leads to both acute and chronic learning and memory impairments. These findings expand the behavioral characterization of the larval zebrafish seizure model, strengthening the power of this model for researching the cognitive consequences of ELS.
Wnt signaling promotes IL‐33 expression in the colonic epithelium
Authors: Schumacher Michael A. , Thai Megan H. , Frey Mark R.
Published: May 2022

Background

Interleukin‐33 (IL‐33) released from the colonic epithelium promotes type 2 innate lymphoid cell activity as part of the colonic injury response. We have previously shown that the intracellular signaling regulator, Sprouty2, restrains colonic IL‐33 via a GSK3β‐dependent mechanism. GSK3β can regulate canonical Wnt signaling, but the role of Wnt signaling and the specific components of this pathway involved in driving IL‐33 are unknown. We tested the hypothesis that activation of discrete arms of Wnt signaling induce epithelial IL‐33 expression.

Methods

Bulk RNA sequencing was performed on colons from mice with an intestinal epithelial‐specific Sprouty2 deletion (Spry2IEKO) or littermate controls to determine the effect of Sprouty2 on Wnt targets. Colonoid and young adult mouse colon (YAMC) epithelial cell cultures were treated with Wnt ligands. Small molecule inhibitors were used to inhibit CBP‐ or p300‐mediated Wnt signaling arms (ICG‐001 or IQ‐1 respectively), in YAMC cells. IL‐33 levels were assayed by qPCR and ELISA.

Results

Spry2IEKO mice have increased colonic transcript levels of the Wnt target genes Mmp7 (p<0.01), Irx3 (p<0.05), Mapk10 (p<0.05), and Jag1 (p<0.05), correlating with the increased expression of IL‐33 that we have previously reported in these mice. Pathway analysis suggests specifically altered CBP/p300‐mediated Wnt signaling in Spry2IEKO colons. Treatment of wild‐type colonoids or YAMC cells with the Wnt ligands Wnt3a and R‐spondin induced IL‐33 expression by 2.1‐fold and 2.3‐fold respectively (p<0.05). Pre‐treatment of cells with ICG‐001 blocked the Wnt‐induced increase in IL‐33 at both the RNA and protein level, whereas IQ‐1 had no effect.

Conclusion

CBP‐mediated Wnt signaling promotes colonic epithelial expression of IL‐33 in vitro. Elevated Wnt activity in Spry2IEKO mice is associated with elevated IL‐33 levels. Dysregulated Wnt signaling may result in impairment of the injury response in inflammatory disease.
Upregulated iRhom2 in the Hypothalamic Paraventricular Nucleus is Associated with TACE‐Mediated Shedding of Transforming Growth Factor‐α and Sympathetic Excitation in Heart Failure Rats
Authors: Yu Yang , Li Hanzeng , Li Mingxuan , Chen Qing , Irfan Nafis M. , Weiss Robert , Wei Shun‐Guang
Published: May 2022

Tumor necrosis factor (TNF)‐α converting enzyme (TACE), also known as a disintegrin and metalloprotease (ADAM)17, is a key mediator of cell signaling by proteolytically cleaving extracellular domains of various cytokines and growth factors. TACE‐mediated shedding of transforming growth factor (TGF)‐α, has been shown to transactivate epidermal growth factor receptor (EGFR) to activate the mitogen‐activated protein kinase signaling pathway in the pathophysiological conditions. We previously reported that both TACE and TGF‐α are upregulated in the hypothalamic paraventricular nucleus (PVN, a critical cardiovascular and autonomic center) and contribute to the sympathetic excitation in heart failure (HF). However, the mechanisms by which TACE and TGF‐α are upregulated in the PVN in HF remain unclear. Recent evidence reveals that inactive Rhomboid proteins (iRhoms) including iRhom1 and iRhom2 are essential upstream regulators of TACE/ADAM17. The present study sought to determine the expression of iRhoms and its relationship with TACE‐mediated shedding of TGF‐α in the PVN in HF. Male rats underwent coronary artery ligation to induce HF or sham surgery (Sham). The left ventricular function and infarction size were assessed by echocardiography. Four weeks later, these animals were euthanized to collect cerebrospinal fluid (CSF), PVN tissues and blood for molecular and biochemical measurements. All values are expressed as the mean ± SD. Real‐time PCR showed abundant mRNA expression of both iRhom1 and iRhom2 in the PVN and brain cortex in Sham or HF rats. Compared with Sham rats, HF rats had significantly (*P<0.05) increased mRNA expression of iRhom2 (2.00 ± 0.57* vs 1.03 ± 0.28, HF vs Sham) but not iRhom1 (1.32 ± 0.41 vs 1.02 ± 0.21) in the PVN. Additionally, HF rats exhibited marked increases in mRNA expression of TACE (2.25 ± 1.15* vs 1.04 ± 0.36) in the PVN and the plasma level of norepinephrine (NE, a marker of sympathetic excitation, 12.39 ± 4.96* vs 5.82 ± 2.76 ng/mL), along with elevated levels of TGF‐α (23.54 ± 6.25* vs 10.08 ± 4.38 pg/mL) in CSF. The levels of epidermal growth factor (9.88 ± 3.59 vs 8.15 ± 4.24 pg/mL) in the CSF did not alter significantly in HF compared with Sham rats. Moreover, the linear regression analysis indicated that the mRNA expression of iRhom2 in the PVN was positively correlated with TACE level in the PVN (R2 = 0.62, P <0.01), TGF‐α level in the CSF (R2 = 0.64, P <0.01) and NE level in the plasma (R2 = 0.63, P <0.01). These results suggest that the upregulated expression of iRhom2 in the PVN may promote ectodomain shedding of TGF‐α by enhancing TACE expression to drive sympathetic activation in HF. Brain iRhom2 is a potential target for therapeutic intervention in HF.
Tacrolimus induces ligand independent TGFβ receptor signaling to promote renal fibrosis
Authors: Ume Adaku , Yesomi‐Wenegieme Tara , Williams Clintoria
Published: May 2022

Background

Although calcineurin inhibitors (CNIs) such as tacrolimus and cyclosporin have dramatically improved the quality of patient care, long‐term therapy causes irreversible damage to the kidneys in the form of renal fibrosis. These morphologic changes ultimately lead to a decline in renal function and can progress to end‐stage renal failure. These detrimental outcomes present a critical need to identify the driving mechanisms by which CNIs cause renal damage.

Objective

It is well established that TGFb is a major contributor to CNI‐induced renal fibrosis. However, the underlying mechanisms remain unknown. The objectives of this study are to 1) investigate whether TGFb secretion is required to stimulate TGFb receptor signaling in a model of CNI‐induced renal fibrosis and 2) investigate whether calcineurin plays a critical role in regulating TGFb receptor activity.

Experimental Design

To examine the role of calcineurin inhibition in altered TGFb receptor signaling, wild type mice were treated with either vehicle (0.01% ethanol) or 10 mg/kg tacrolimus for 7 days. To confirm in vivo findings, wild‐type mouse renal cortical fibroblasts were treated with either vehicle (0.01% ethanol) or 1nM tacrolimus for 24 hours in the presence and absence of anti‐TGFb neutralizing antibodies. TGFb receptor expression and activation, TGFb receptor downstream signaling mediators, profibrotic markers and calcineurin activity were analyzed.

Results

Findings demonstrate that tacrolimus‐induced loss of calcineurin activity is accompanied with enhanced TGFb receptor activation and signaling. Notably, increasing concentrations of anti‐TGFb neutralizing antibodies failed to abolish aberrant TGFb signaling and increased expression of profibrotic markers.

Conclusions

Together, these results demonstrate that 1) CNIs promote ligand‐independent TGFb signaling and 2) calcineurin plays a functional role in regulating TGFb receptor activity.

Significance

This study elucidates a direct pharmacological mechanism by which CNIs promote renal fibrosis and provides a physiological role of calcineurin in regulating TGFb receptor activity. Further insight into the role of calcineurin in regulating TGFb receptor activity could improve the long‐term outcomes of patients on chronic CNI therapy.
Inhibition of Branched‐Chain Ketoacid Dehydrogenase Kinase (BDK) Attenuates BCKA Induced Suppression of Insulin‐Stimulated Glucose Uptake in Muscle Cells
Authors: Mann Gagandeep
Published: May 2022

Objective

Plasma levels of branched‐chain amino acids (BCAAs) and their metabolites, branched‐chain ketoacids (BCKAs) are increased in insulin resistance, a condition that can lead to type 2 diabetes mellitus (T2DM). BCAA catabolic enzymes are downregulated in diabetes and obesity in skeletal muscle. We previously showed that leucine and KIC suppressed insulin‐stimulated glucose uptake in L6 myotubes. We have also shown that knocking down branched‐chain ketoacid dehydrogenase (BCKD), an enzyme that decarboxylates BCKAs, suppressed insulin‐stimulated glucose uptake. The objective of this study was to analyze the effect of increasing BCAA catabolism on measures of insulin sensitivity. We hypothesized that upregulating BCAA catabolism increases insulin‐stimulated glucose transport and attenuates BCKA induced insulin resistance.

Methods

L6 myotubes were starved of serum‐ and amino acids with vehicle (DMSO) or BDK inhibitor BT2 supplementation for 3 hours. Then myotubes were supplemented with or without BCKAs (200 uM) for 30 minutes. After, cells were incubated with or without insulin (100 nM) for 20 minutes. They were then harvested for immunoblotting or used for glucose transport assay.

Results

BDK inhibition had no effect on insulin‐stimulated glucose uptake. BCKAs suppressed insulin‐stimulated glucose uptake by 31% in control cells (p>0.05; n=3); this suppression was attenuated in cells in which BDK was inhibited. BDK inhibition also reduced KIC‐induced IRS‐1Ser612 and S6K1Thr389 phosphorylation by 28% and 60% respectively but had no effect on AktSer473 phosphorylation.

Conclusions

BDK inhibition attenuated BCKA‐induced suppression of insulin‐stimulated glucose uptake, suggesting that BCAA metabolism may be implicated in the pathogenesis of insulin resistance, and is worth further investigation.
On the Flame: The Fab Fragment of Rheumatoid Arthritis
Authors: Chen Kaelyn , Akintola Toluwalase , Lawrence Katelyn , Lane Jim
Published: May 2022

With more than 200,000 cases per year, Rheumatoid Arthritis (RA) is one of the most common autoimmune disorders in the United States. Across the globe, up to 14 million people are affected by its symptoms of joint pain, stiffness, swelling, fatigue, and even physical deformity. Rheumatoid factors (RF) are autoantibodies that correlate with RA severity and recognize epitopes in the Fc region of its antigen immunoglobulin (Ig) G, yet it is paradoxically found circulating in the blood alongside the antigen without binding to it. According to Thomsen, et. al, a 2018 study that contested the idea of IgG aggravation as the prerequisite to RF binding, the native state of IgG is present in a closed form, where the Fab fragment shields the Fc region, only exposing the Fc effector sites when a conformational change is induced. This closed form is a result of a missing galactose between the C□2 domains of the heavy chains, thus causing the IgG to self associate, forming immune complexes to fix the complement system. The subsequent heightened inflammatory response exacerbates the progression of joint damage in RA and other severe symptoms. In this study, we examined RF in relation to IgG forming immune complexes to achieve a better understanding of the immunological progression or pathogenesis of RA. The Mahtomedi MSOE Center for BioMolecular Modeling MAPS Team used 3‐D modeling and printing technology to examine structure‐function relationships of Fab and Fc fragments in IgG. The visual model will be a valuable tool in developing our story.
Understanding the Molecular Mechanism of PLCε Regulation and G Protein‐Mediated Activation
Authors: Muralidharan Kaushik , Tat Rachel , Outlaw Kennedy , Ohri Vaani , Flaherty Hannah , Garland‐Kuntz Elisabeth , Lyon Angeline
Published: May 2022

Cardiovascular disease is the leading cause of death in the world. The phospholipase C (PLC) family of enzymes, in particular the PLCε subfamily, are essential for normal cardiovascular function. PLCε hydrolyzes phosphatidylinositol phosphates at cellular membranes, producing inositol phosphates (IPx) and diacylglycerol (DAG). These crucial secondary messengers activate multiple downstream pathways, including cardiac contractility and the expression of hypertrophic genes. In the cardiovascular system, PLCε is regulated through direct interactions with the RhoA and Rap1A small GTPases, which in turn are activated downstream of G protein‐coupled receptors (GPCRs). RhoA is reported to activate PLCε at the plasma membrane, whereas Rap1A translocates and activates PLCε at the perinuclear membrane. However, the elements within PLCε that regulate basal activity and membrane association have not been fully identified. Similarly, the domains involved in Rap1A versus RhoA binding, activation and translocation to different subcellular membranes have not been mapped. In this work, we use a structure‐guided approach, together with cell‐based activity assays, epifluorescence, and confocal microscopy to identify the roles of PLCε regulatory elements and domains in basal activity, subcellular localization, and regulation by RhoA and Rap1A GTPases. Functional studies show N‐ and C‐terminal regulatory domains of PLCε dictate its location within the cell, and contribute differently to basal and G protein‐dependent activity. We also show that regulatory insertions within the catalytic TIM barrel, including the X–Y linker and Y‐box, aid in interfacial activation and membrane association. These studies provide much needed insights into the molecular determinants of PLCε that regulate its localization and activity in cells, which is critical for elucidating its roles in cardiovascular function.
Endothelial Senescence Mediates Hypoxia‐induced Vascular Remodeling in the Lung through TWIST1 Signaling
Authors: Kyi Priscilla , Hunyenyiwa Tendai , Hendee Kathryn , Matus Kienna , Mammoto Tadanori , Mammoto Akiko
Published: May 2022

Pulmonary hypertension (PH) is a fatal pulmonary vascular disease characterized by a sustained elevation of pulmonary arterial pressure. One of the major characteristics of PH is uncontrolled accumulation of pulmonary artery smooth muscle cells (PASMCs) to normally non‐muscularized distal pulmonary arteries (PAs). Cellular senescence contributes to aging and lung diseases associated with PH. The aim of this study is to understand the mechanism by which cellular senescence controls vascular remodeling in PH. We have reported that a basic helix‐loop‐helix transcription factor, TWIST1, in endothelial cells (ECs) mediates hypoxia‐induced accumulation of PASMCs to PAs by increasing platelet‐derived growth factor (PDGFB) expression. Here we have demonstrated that the levels of senescence markers, p16INK4A and senescence‐associated β‐galactosidase (SA‐β‐gal) are higher in ECs isolated from PH patients compared to those from healthy individuals. Analysis of publicly available RNAseq data of PH patient lungs reveals the interaction between TWIST1 and senescence‐related genes. The levels of PDGFB upregulated in PH patient‐derived ECs are inhibited by knocking down p16INK4A expression or treatment with senolytic reagent ABT‐263. Hypoxia‐induced accumulation of a‐smooth muscle actin (aSMA)‐positive cells to the PAs and TWIST1 expression are attenuated in p16INK4Afl/fl‐Cdh5(PAC)‐CreERT2 mice after tamoxifen induction. These results suggest that EC senescence mediates vascular remodeling in PH through TWIST1 signaling.
Stress‐Induced Gut Dysmotility is Associated with Altered Intestinal BDNF‐TrkB Signaling
Authors: Singh Alpana , Zhang Chengxiu , Kulkarni Subhash
Published: May 2022

Introduction

While stress is associated with incidence of gut dysmotility disorders, mechanisms by which stress causes dysmotility are not well understood. In the central nervous system (CNS), stress causes behavioral deficits by increasing the levels of Glucocorticoid hormone to suppress molecular signaling between brain‐derived neurotrophic factor (BDNF) and its receptor tropomyosin receptor kinase B (TrkB). We hypothesize that stress causes a Glucocorticoid‐mediated reduction in BDNF‐TrkB signaling in the enteric nervous system (ENS), whose neurons, which modulate diverse gut functions, reside entirely within the gut wall.

Methods

Gut motility was assayed by analyzing the whole gut transit time (WGTT) of an orally gavaged carmine red dye in adult mice. Effect of stress was tested by comparing WGTT of Stress‐Induced Dysmotility (SID) mice, wherein age‐matched adult male mice were restrained for one hour, with that of unrestrained control mice. The effect of Glucocorticoids was tested by comparing WGTT of age‐matched adult C57/BL6 male mice dosed with Dexamethasone (DEX, a Glucocorticoid receptor agonist, intra‐peritoneal 5mg/kg), with that of saline‐dosed control mice. Effect of DEX on enteric BDNF levels (using R&D ELISA kit) was tested by using total proteins isolated from preparations of adult murine longitudinal muscle‐myenteric plexus (LM‐MP) tissues cultured with and without DEX (1 µM) for 4 hours. TrkB exists in full‐length and truncated (TrkB.T1) isoforms, and the presence of TrkB.T1 in ENS neurons was observed by immunofluorescence analyses of ENS tissue of TrkB.T1‐V5 transgenic mice. Involvement of TrkB.T1 isoform in SID was tested by comparing WGTT of male transgenic mice containing partial (T1‐Het) or complete loss (T1 KO) of TrkB.T1. Differences were compared for statistical significance using Student’s t‐test and Mann‐Whitney test.

Results

5 consecutive days of 1 hour restraint caused a significant delay in WGTT of adult C57/BL6 SID mice, when compared to controls (Table 1). DEX caused a significant delay in WGTT, when compared to control mice (Table 1). DEX treatment caused a significant reduction in BDNF levels (Table 1). TrkB.T1‐V5 mice show presence of TrkB.T1 on adult enteric neurons (Fig 1). A single hour of restraint caused a significant delay in WGTT in adult T1 KO mice, when compared with littermate T1 Het mice (Table 1).

Discussion

Restraint stress and DEX caused similar delay in gut transit in wildtype mice, and since DEX decreased gut BDNF levels, we infer that stress‐induced increase in glucocorticoid signaling delayed gut transit by suppressing enteric BDNF levels. Increased susceptibility of T1 KO mice to SID highlights the important role of the truncated TrkB.T1 receptor in maintenance of normal gut motility during acute and moderate stress. Thus, our data implicates altered BDNF‐TrkB.T1 signaling in the etiology of Stress Induced Dysmotility.
Oxidative Stress Suppresses Nrf2 Protein Synthesis through Global Protein Synthesis Inhibition
Authors: LaMorte Joseph , Pensabene Kaitlin , Allender Amanda , Eggler Aimee
Published: May 2022

Oxidative and electrophilic stresses are common aggressors in chronic diseases ranging from arthritis to cancer to neurodegenerative disease. The transcription factor Nrf2 is an essential mediator of the cell’s cytoprotective response to these stressors. Nrf2 is constantly synthesized, ubiquitinated, and degraded with a short half‐life of 20 minutes that maintains low basal Nrf2 protein levels. Electrophiles and oxidative stress cause Nrf2 to accumulate and subsequently to activate cytoprotective genes by modifying cysteines of the Keap1 protein, the repressor that targets Nrf2 for ubiquitination. We previously found that paradoxically, reactive oxygen species (ROS) stimulate the production of cytoprotective proteins, but inhibit Nrf2 protein synthesis, revealing a “Dr. Jekyll and Mr. Hyde” effect of ROS on Nrf2 accumulation. We tested the hypothesis that oxidative conditions that activate the Nrf2 pathway simultaneously inhibit global protein synthesis, thereby suppressing Nrf2 protein synthesis. We investigated both ROS generated by redox cycling of the oxidizable phenol, di‐tert‐butylhydroquinone (dtBHQ), and direct addition of H2O2 to HaCaT keratinocyte cells. We found that 100 μM to 400 μM H2O2 and 50 μM dtBHQ inhibit both Nrf2 protein synthesis and global protein synthesis. Addition of antioxidants, such as the manganese porphyrin MnTMPyP, rescued both Nrf2‐specific and global protein synthesis. Therefore, antioxidants such as MnTMPyP that mitigate the suppressive effects of ROS on global protein synthesis during Nrf2 activation could enhance the Nrf2/cytoprotective response under conditions of electrophilic and oxidative stress.
Investigating the structure of UHRF2 TTD‐PHD with H3K9me3
Authors: Konopka Sean , Trievel Raymond , Albaugh Brittany
Published: May 2022

Epigenetics is the study of how genes are regulated within cells without altering the DNA sequence of the genome. UHRF1 and UHRF2 are two epigenetic proteins that regulate histone modification and DNA methylation. The tandem Tudor domain (TTD) and the plant homeo domain (PHD) of these proteins are histone binding domains that interact with the H3 tail trimethylated at lysine 9 (H3K9me3). While the structure of UHRF1 in complex with an H3K9me3 peptide has been solved, there remains a lack of structural data that provides the exact requirements for this histone interaction in UHRF2. Here we seek to further investigate the histone binding function by elucidating the UHRF2 TTD‐PHD and H3K9me3 structural complex through x‐ray crystallography.
Selection of GDP‐fucose by active site residues in Arabidopsis thaliana SPY
Authors: Rolli Jenelle , Desrochers Kate , Taylor Jack , NewRingeisen Erin , Mohammadzadh Hadia , Joiner Cassandra
Published: May 2022

O‐linked β‐N‐acetylglucosamine (O‐GlcNAc) transferase (OGT) transfers O‐GlcNAc from the nucleotide‐sugar UDP‐GlcNAc to thousands of nuclear and cytoplasmic proteins. OGT is an essential enzyme, and misregulation of O‐GlcNAcylation has been linked to cancers or neurodegeneration. Most eukaryotic organisms possess a single structural homolog of OGT. However, Arabidopsis thaliana has two: Spindly (SPY) and Secret Agent (SEC). Whilst being structurally similar, SPY and SEC select different nucleotide sugars, GDP‐fucose and UDP‐GlcNAc, respectively. To determine which of SPY's active site amino acids govern GDP‐fucose selection, residues equivalent to those necessary for OGT's function were mutated and expressed in E. coli. Using the in vitro malachite green glycosyltransferase assay, the mutants’ activity was analyzed and compared to wildtype. One mutant displayed wild‐type levels of glycosylation, while another exhibited partial activity. The remaining three mutants lost glycosylation activity, suggesting essentiality for GDP‐fucose selection.
Elucidating the Quality Control Pathway of KCC2, a Critical Synchronizer of Neuronal Development
Authors: Kok Morgan , Singh Ishika , Hartnett‐Scott Karen , Aizenman Elias , Brodsky Jeffrey L.
Published: May 2022

Neurodevelopment is a complex process orchestrated by a multitude of factors, including neuronal ion transporters. The neuron‐specific K+/Cl‐ co‐transporter 2 (KCC2) regulates γ‐aminobutyric acid (GABA) inhibitory neurotransmission. Mutations in KCC2 have been linked to multiple neurodevelopmental disorders, including epilepsy, schizophrenia, and autism. KCC2 is a complex multimeric membrane protein exhibiting reduced cell surface expression and/or activity when disease‐associated mutations are present. This observation is indicative of an underlying instability in the KCC2 protein, potentially making it susceptible to cellular quality control pathways that encourage proper folding and/or target misfolded proteins for degradation. Here, we sought to elucidate the quality control mechanisms that regulate KCC2 in three different model systems: yeast, HEK293 cells, and primary rat neurons. Assessment of protein stability in yeast revealed that KCC2 is targeted for endoplasmic‐reticulum‐associated degradation (ERAD). In contrast, experiments in HEK293 cells supported a more subtle role for ERAD in maintaining steady‐state levels of KCC2, but this system allowed for an analysis of KCC2 glycosylation and transport through the secretory system. In turn, KCC2 was remarkably stable in primary rat neurons. By capitalizing on the advantages of the HEK293 cell system, we have also obtained preliminary data that select disease associated KCC2 mutations are unstable relative to the wildtype protein. Together, this study represents a critical new analysis of the quality control mechanisms regulating KCC2 and suggests that KCC2‐associated diseases might be linked to the ERAD pathway.
Characterization of Hepatic Injury During the Azoxymethane Model of Acute Liver Failure
Authors: McMillin Matthew , Jhawer Ashwin , Frampton Gabriel , Bhattarai Shadikchhya M. , Troyanovskaya Eleonora
Published: May 2022

Introduction

Acute liver failure (ALF) results from a significant loss of liver function and has few effective treatments and poor clinical outcomes. ALF is associated with neurological dysfunction, called type A hepatic encephalopathy (HE), which greatly increases mortality. One of the commonly used models of type A HE is the azoxymethane (AOM) mouse model, which is associated with hepatocyte necrosis, neuroinflammation and oxidative stress. That being said, there have been no extensive analyses into the hepatic pathology associated with AOM‐induced liver injury. Therefore, the aim of this study was to characterize the hepatic injury during the AOM model of type A HE.

Methods

C57Bl/6 mice were administered 100 mg/kg of AOM and were euthanized at various stages of neurological decline and tissue was collected. These stages include normal (vehicle‐treated), prior to neurological decline (pre), minor neurological decline (minor), significant neurological decline (major), and coma. Liver injury was assessed by measuring serum ALT and AST levels as well as liver H&E staining. Serum albumin, bile acids, glutathione (GSH) and ammonia were measured to determine liver function. Various aspects of oxidative stress, inflammation and cell death were assessed by measuring CYP2E1, Nrf2, SOD1, CCL2, IL‐1β, IL‐6, LC3‐II, phosphorylated MLKL, cleaved caspase 3, p21 and TGFβ1 expression using real‐time PCR, immunoblotting, ELISA or immunohistochemistry. All data were analyzed using GraphPad Prism software with results presented as mean +/‐ SEM. Differences between two groups were compared using the Student’s t‐test and differences between three groups were determined using ANOVA.

Results

AOM‐treated mice had significant elevations of serum ALT and AST and hepatic necrosis as neurological decline progressed. CYP2E1 expression decreased throughout the stages of neurological decline, with the greatest suppression in mice at coma. Measures of oxidative stress and signaling had the largest increase when mice were at major neurological decline, which was also associated with the largest increase of inflammatory chemokines and cytokines. Measures of necroptosis, apoptosis and cellular senescence were also dysregulated, with coma mice showing the highest degrees of cell death and increased cellular senescence.

Conclusions

These data demonstrate that AOM‐treated mice have increased oxidative stress and inflammation resulting from the metabolic dysregulation from AOM toxicity. This leads to cell death through multiple different mechanisms and an impairment of regeneration due to increased cellular senescence.