bluuebunny/crossref_metadata_2025 huggingface dataset:
Identifying Cellular and Viral Factor Recruitment to Herpes Simplex Virus Type 1 Replication Forks
Authors: Packard Jessica E. , Dembowski Jill A.
Published: May 2022

Herpes simplex virus type 1 (HSV‐1) is a ubiquitous pathogen that replicates the 152kbp viral genome within the nucleus of host cells. Replication of the HSV‐1 genome is catalyzed by viral replication machinery consisting of a DNA polymerase (UL30), processivity factor (UL42), helicase‐primase complex, origin binding protein (UL9), and a single stranded DNA binding protein. Despite encoding a viral DNA polymerase processivity factor, we found that host Proliferating Cell Nuclear Antigen (PCNA) interacts with HSV‐1 DNA at replication forks and associates with viral DNA in a replication‐dependent manner. PCNA is a homotrimer that provides processivity to cellular DNA polymerases and selectively recruits DNA damage response and DNA repair factors to cellular replication forks. We therefore hypothesized that PCNA associates at viral replication forks to promote HSV‐1 replication while also tethering cellular DNA repair proteins to replicating viral DNA. To test this, we used two models to identify how associated factors change as a function of PCNA: a known PCNA inhibitor, PCNA‐I1, and a cell line that was engineered to inducibly express a shRNA targeting PCNA. We performed a technique adapted from isolation of proteins on nascent DNA (iPOND) to isolate HSV‐1 DNA from infected cells and identified associated viral and cellular proteins. Cells were infected and replicating viral DNA was selectively labeled with EdC. Labeled DNA was specifically and irreversibly tagged via the covalent attachment of biotin azide via click reaction. Biotin‐tagged DNA was purified on streptavidin‐coated beads and associated proteins were eluted and identified by mass spectrometry. As a complimentary approach, we also performed immunofluorescence (IF) to confirm iPOND data. We found that PCNA is associated with viral DNA despite treatment with PCNA‐I1, an observation that is consistent with IF imaging data. Viral replication proteins UL30, UL42, and UL9 decreased in the presence of PCNA‐I1. DNA damage response proteins such as mismatch repair proteins and RECQL both decreased up to 10‐fold. Of note, however, proteins that make up the MRN complex, a class of proteins that functions in DNA break repair, restart of stalled replication forks, and viral DNA infection, increased in response to PCNA‐I1 treatment. Given this data, PCNA may be involved in tethering viral replication proteins, cellular DNA repair proteins, and/or virion assembly proteins to viral replication forks.
Lithium‐associated nephrotoxicity: role of GDF15‐mediated collecting duct cells proliferation
Authors: Tabibzadeh Nahid , Cheval Lydie , Morla Luciana , Chevillard Lucie , Megarbane Bruno , Bienaime Frank , Vidal‐Petiot Emmanuelle , Crambert Gilles
Published: May 2022

Lithium (Li+) salts are the most effective treatment of bipolar disorder (BD), affecting 1 to 4% of the population worldwide. However, this efficacy is counterbalanced by adverse long‐term events, mostly renal effects. Lithium treatment discontinuation is usually not an option as it substantially increases suicidal risk. Understanding the sequence of events in the kidneys might thus improve therapeutic strategies while maintaining this treatment.

Li+‐induced nephrogenic diabetes insipidus (NDI) is the earliest and the most frequent side effect of chronic lithium use, leading to polyuria and polydipsia. Li+ treatment decreases AQP2 expression both in vivo and in vitro. Li+ treatment also induces a microcystic tubulointerstitial nephropathy eventually leading to end‐stage kidney disease. The mechanisms of tubular toxicity and microcyst formation have been related to a proliferative effect of Li+. The time course, the site and the mechanisms of this proliferation are still a matter of debate.

Previous studies from our lab have shown that acidosis‐induced collecting duct cell proliferation depended on the overexpression of GDF15 by principal cells, especially in the early phase of linear proliferation along the tubular axis.

Our objective during this project is to determine the pathways leading to planar and cystic proliferation of collecting duct cells, in particular GDF15 signaling, and their role in lithium nephrotoxicity, and to identify potential biomarkers that could help to predict the course of Li‐induced nephropathy.

We have established a rat and a mouse experimental model treated with therapeutic concentrations of Li+, displaying as expected polyuria and polydipsia, with microcystic tubular dilations and a global proliferation of collecting duct cells (both principal and intercalated) on kidney slices and on microdissected collecting ducts, with an increase in the intercalated/principal cells ratio (figure). We examined the effect of Li+ in GDF15‐/‐ and wild type (WT) mice and found significantly lower polyuria and polydipsia in GDF15‐/‐ mice, along with a higher urine osmolality, and decreased intercalated/principal cell ratio compared to WT mice (figure).

As collecting duct cell proliferation is mediated by GDF15, we studied urinary GDF15 levels (GDF ELISA kit) in Li+‐treated patients with proven diabetes insipidus and found them significantly increased compared to control patients (p<0.01) (figure).

In conclusion, these results suggest a direct relationship between lithium‐induced proliferation signals, collecting duct cell dysfunction and GDF15 expression. Further investigation is needed to determine if GDF15 signaling might represent a therapeutic target in Li+‐induced nephrotoxicity.
Reinforcing Effects of Fentanyl and a Fentanyl Analog Found in Illicit Drug Markets
Authors: McGriff Shelby A. , Thorndike Eric , Chojnacki Michael , Baumann Michael , Schindler Charles
Published: May 2022

HYPOTHESIS

Illicitly manufactured fentanyl and fentanyl analogs are the main drivers of the worsening opioid epidemic. Although the pharmacology of fentanyl is well characterized, there is little information about the reinforcing effects of newly emerging fentanyl analogs that are contributing to opioid overdose. Here, we compare the effects of fentanyl and the clandestine fentanyl analog, cyclopropylfentanyl, on drug self‐administration behavior in male and female rats.

METHODS

Sprague‐Dawley rats fitted with intravenous (i.v.) jugular catheters were placed in experimental chambers containing two nose poke holes. Active nose poke responses resulted in delivery of a constant volume of drug (0.2 mL) over a period of 2 s on a fixed‐ratio 1 schedule, followed by a 20 s timeout. Acquisition doses for the compounds were 0.01 mg/kg/inj for fentanyl and cyclopropylfentanyl, while 0.1 mg/kg/inj was used for heroin. Following 10 days of acquisition training, dose‐effect testing began with three additional doses, each of which were tested for 3 days. Subjects then returned to the original training dose until responses stabilized and underwent saline extinction.

RESULTS

Acquisition of fentanyl self‐administration was achieved by both male and female rats, with significant differences between active versus inactive nose pokes for both sexes over the last 4 days of training (p<0.05). A similar profile of acquisition was seen for heroin and cyclopropylfentanyl. Both fentanyl and cyclopropylfentanyl showed a typical inverted‐U dose‐effect function during dose‐effect testing. Maximal response rates were similar among all drugs, with fentanyl showing maximum responding at 0.001 mg/kg/inj, cyclopropylfentanyl at 0.003 mg/kg/inj, and heroin at 0.003 mg/kg/inj.

CONCLUSION

Both male and female rats showed comparable rates of fentanyl acquisition. Further comparison between heroin, fentanyl, and cyclopropylfentanyl show that these compounds support self‐administration, indicating that emerging fentanyl analogs display significant abuse liability and may contribute to compulsive use in humans.
Structural studies of an androgen receptor complex reveal modes of allosteric regulation
Authors: Wasmuth Elizabeth V. , Vanden Broeck Arnaud , LaClair Justin , Lawrence Kayla E. , Hoover Elizabeth A. , Pappas Kyrie , Wang Biran , Manova‐Todorova Katia , Weintraub Susan T. , Klinge Sebastian , Sawyers Charles L.
Published: May 2022

The androgen receptor (AR) is the master transcription factor governing gene expression programs required for prostate epithelium development and male phenotype maintenance. AR misregulation is a hallmark of prostate cancer where AR hyperactivation and transcriptome expansion occur in part through AR amplification and interaction with oncoprotein cofactors. Despite its biological importance, how AR’s domains and cofactors cooperate to bind DNA have remained elusive. We isolated three conformations of AR bound to DNA showing AR forms a non‐obligate dimer, with the buried dimer interface utilized by ancestral steroid receptors repurposed to facilitate cooperative DNA binding. We identify novel allosteric surfaces which are compromised in androgen insensitivity syndrome, and reinforced by AR’s oncoprotein cofactor, ERG, and DNA binding motifs. Finally, we present evidence this plastic dimer interface was adopted for transactivation at the expense of DNA binding. Our work highlights how fine‐tuning AR’s cooperative interactions translate to consequences in development and disease.
Investigating the Molecular Mechanisms Driving 7α,25‐dihydroxycholesterol‐GPR183‐Induced Hypersensitivity
Authors: Braden Kathryn , Campolo Michela , Chen Zhoumou , Giancotti Luigi , Doyle Timothy M. , Esposito Emanuela , Zhang Jinsong , Cuzzocrea Salvatore , Arnatt Christopher K. , Salvemini Daniela
Published: May 2022

Neuropathic pain is a debilitating health concern and there is an urgent need for non‐opioid analgesic targets. Our group has identified GPR183 as a novel potential therapeutic target for neuropathic pain. GPR183 is a G‐protein coupled receptor that promotes the migration of immune cells in response to its ligand, 7α,25‐dihydroxycholesterol (7α,25‐OHC). We have shown that GPR183 is upregulated in the dorsal horn spinal cord during neuropathic pain states in rodents and intrathecal injections of 7α,25‐OHC is able to induce allodynia in mice in a GPR183‐dependent manner. However, the mechanism by which GPR183 activation leads to pain is unknown. These studies aim to elucidate the molecular signaling pathways that contribute to 7α,25‐OHC‐induced hypersensitivity. Based on previous literature, we hypothesized that GPR183 activation in the spinal cord would activate mitogen‐activated protein kinases (MAPKs), extracellular signal‐regulated kinase (ERK) and p38, leading to the production of neuroexcitatory cytokines contributing to hypersensitivity.

Methods

To investigate the signaling pathways downstream of GPR183 activation in the spinal cord, we performed unbiased RNA‐Sequencing following intrathecal injections of 7α,25‐OHC with or without the GPR183 antagonist, SAE‐14, in male and female ICR mice. We then performed follow‐up studies giving pharmacological inhibitors of the identified pathways prior to intrathecal 7α,25‐OHC to confirm functional relevance. We also took spinal cord tissue for western blot and enzyme‐linked immunoassay (ELISA) analysis following intrathecal injections.

Results

Through our unbiased RNA‐sequencing we found that many of the genes that were differentially regulated by GPR183 activation contain binding sites for NF‐κB or transcription factors that are regulated by the MAPK’s, ERK or p38. Additionally, we found that small molecule inhibitors of these pathways delayed 7α,25‐OHC‐induced mechanical allodynia. Importantly, the GPR183 antagonist, SAE‐14, blocked the 7α,25‐OHC‐induced upregulation of these pathways, including the upregulation of neuroexcitatory cytokines interleukin‐1β and tumor necrosis factor α.

Conclusion

These findings provide novel insight into how spinal GPR183 signaling produces hypersensitivity and can contribute to neuropathic pain states.
Endothelial Regulation of Microvascular Growth and Stability by Ang‐Tie and VEGF Signaling Pathways: A Mechanistic Computational Systems Biology Model
Authors: Zhang Yu , Kontos Christopher D. , Annex Brian H. , Popel Aleksander S.
Published: May 2022

The Angiopoietin‐Tie (Ang‐Tie) pathway is a key signaling pathway regulating vascular stability and permeability, and it significantly intersects and crosstalk with the vascular endothelial growth factor (VEGF) signaling pathway, a major signaling pathway regulating angiogenesis and vascular permeability. Disrupted Ang‐Tie and VEGF signaling is linked to vascular dysfunction related to cancer, systemic inflammation, cardiovascular disease, and diabetic macular edema. Ang‐Tie pathway is regulated by various molecular mechanisms, including ligand‐receptor interactions, receptor multimerization, binding of co‐receptor Tie1, inhibition by vascular endothelial protein tyrosine phosphatase (VE‐PTP), receptor extracellular domain shedding, junctional localization, trafficking, and turnover. VEGF pathway is regulated by ligand binding, homo‐ and heterodimerization of VEGF receptors VEGFR1 and VEGFR2, interaction with neuropilin, thrombospondin‐1/CD47, receptor internalization, recycling, and degradation. Ang‐Tie and VEGF signaling pathways also share crosstalk mechanisms and integrative downstream signaling through phosphoinositide 3‐kinases (PI3K)/Akt, sphingosine‐dependent Erk activation, Src sequestration via RhoA/mDia, and calcium cycling, forming a complex reaction network that requires an integrative model to study the signaling outcomes on a systems level. The present study uses a mechanistic computational model of the Ang‐Tie and VEGF signaling pathways, their regulatory mechanisms, crosstalk, and integrative downstream signaling to quantitatively characterize the molecular control of vascular growth, permeability, and stability by endothelial cells. The model captures and reproduces key experimental observations of the Ang‐Tie/VEGF signaling network with detailed molecular mechanisms. The model significantly expands our previous computational models of the Ang‐Tie signaling pathway [1,2] and integrates our systems biology understanding of the VEGF pathway and their crosstalk. The model serves as a platform for quantitatively predicting the signaling outcome of varying ligand concentrations and receptor expressions in physiological and pathological conditions, identifying molecular mechanisms and targets for therapeutic interventions targeting the signaling network, and simulating the effects of drugs and their combinations on the signaling outcome on a network level.
Activation of satellite glia in stellate ganglia from chronic heart failure rats
Authors: Tu Huiyin , Zhang Dongze , Wadman Michael C. , Li Yu‐Long
Published: May 2022

Cardiac sympathetic overactivation is linked to ventricular arrhythmogenesis and is associated with mortality in patients with chronic heart failure (CHF). However, the sites and mechanisms responsible for increased cardiac sympathetic activity are still poorly understood. Our previous studies showed that CHF‐elevated proinflammatory cytokine levels and macrophage expansion in stellate ganglia are critical factors for cardiac sympathoexcitation. Here we tested if activation of satellite glial cells (SGCs) contributes to macrophage expansion in stellate ganglia from CHF rats.

Rat CHF was induced by surgical ligation of the left anterior descending coronary artery. SGCs and postsynaptic sympathetic neurons were respectively identified by anti‐S100B and anti‐tyrosine hydroxylase (TH) antibodies with immunofluorescence staining. As a marker of SGC activation, glial fibrillary acidic protein (GFAP) was measured by immunofluorescence double staining and Western blot analysis.

Sympathetic neurons in stellate ganglia from both sham and CHF rats were totally surrounded by S100B‐positive SGCs. However, However, a few GFAP‐postive SGCs covered sympathetic neurons in sham stellate ganglia, whereas more GFAP‐positive SGCs surrounded sympathetic neurons in CHF stellate ganglia. Western blot data showed that GFAP was elevated in stellate ganglia from CHF rats, compared to sham rats.

From these results, we conclude that activation of SGCs in stellate ganglia is higher in CHF rats than that in sham rats, which might contribute to the neuroinflammation and sympathoexcitation in stellate ganglia.
Ten Best Practices for Taking Experiential Learning Online
Authors: Gillaspy Glenda E. , Cridland Caitlin , Drape Tiffany , Marine Sasha
Published: May 2022

Like many institutions around the world, the COVID‐19 pandemic prompted us to shift our summer 2020 in‐person undergraduate experiential learning program to a remote, virtual format. Here, we present our observations, summarized in ten best practices, for moving a STEM‐focused research experience for undergraduates, experiential learning program or research‐based course to an online format. We will also discuss how our program was originally designed and implemented, and how we adapted our activities to deliver an at‐home research experience that maintained student engagement, mentorship, and a shared sense of community.
L‐Citrulline Attenuates Inflammation Induced Phenotypic Changes in Neonatal Rat Alveolar Type 1 Cells
Authors: Mohammadi Atefeh , Gauda Estelle B.
Published: May 2022

Introduction

Bronchopulmonary dysplasia (BPD) is a chronic lung disease initiated by inflammation and oxidative stress during the early stages of lung development, disrupting alveolar growth. BPD damages alveolar Type 1 (AT1) epithelial cells, causing decreased alveolarization and impaired gas exchange. Recently, it was shown that AT1 cells undergo cell reprogramming into surfactant producing alveolar Type 2 cells (AT2) in hyperoxic and hypoxic lung injury models, causing significant structural damage in the neonatal lung. While current treatments have improved neonatal care, they cause significant side effects. L‐citrulline, a naturally occurring amino acid, protects the newborn rat lung from injury, preserving alveolar growth in various neonatal BPD models. Due to the significance of inflammation in BPD, we sought to investigate the effect of inflammatory stimuli, lipopolysaccharide (LPS) and tumour necrosis factor α (TNF‐α), and L‐citrulline treatment on AT1 cell programming in vitro. We hypothesize that L‐citrulline treatment will prevent inflammation‐induced AT1 cell reprogramming in vitro.

Methods

AT1 cells were isolated from newborn rats on postnatal day 4 using fluorescence activated cell sorting. AT1 cells were pre‐treated with L‐citrulline (4mM/mL or 10mM/mL) for 1hr prior to LPS (10µg/mL), TNF‐α (5ng/mL), or a combination of LPS and TNF‐α for 24hrs in vitro. Whole cell lysates were prepared for immunoblotting. AT1 cells were also assayed for the level of injury‐induced cell viability using the MTT assay.

Results

AT1 cell lysates from the LPS+TNF‐α group had significantly decreased RTI‐40 protein expression compared to the control group (p<0.04 LPS+TNF‐α vs. control, n=6). In the presence of L‐citrulline, RTI‐40 protein expression of cells exposed to LPS+ TNF‐α was maintained (p<0.02 L‐citrulline+LPS+TNF‐α vs. LPS+TNF‐α, n=4) (Figure 1). AT1 cell viability in LPS, TNF‐α, and the combined exposure group was comparable to the control group (n=8) (Figure 2).

Conclusion

Our preliminary data strongly suggests that L‐citrulline treatment mitigates inflammation‐induced cell reprogramming in neonatal AT1 cells. Our findings show that the injury‐induced decrease in RTI‐40 expression is not due to reduced AT1 cell viability, rather due to phenotypic changes. Given the importance of AT1 cells in alveolarization, gas exchange, and BPD pathogenesis, characterizing the cell specific effects of L‐citrulline on AT1 cell reprograming is considerably critical and highly relevant to identifying a safe and effective treatment for premature infants that can mitigate the effects of inflammation on the developing lung.
Resuscitating Learner Satisfaction: Enhancing ACLS Pharmacology Education Through a Case‐Based Escape Room Simulation
Authors: Conway Nicholas B. , Daniele Alexander L. , Uchiyama Emiri , Saunders John M. , Athauda Gagani , Toonkel Rebecca L.
Published: May 2022

Background

Training in Advanced Cardiac Life Support (ACLS) is a vital component of medical education. However, data suggests that learners are not satisfied with traditional ACLS learning formats and quickly lose mastery of the pharmacologic knowledge obtained. Prior studies have shown high learner satisfaction and knowledge mastery related to the ‘gamification’ of learning sessions. We hypothesize that the utilization of an online pre‐session learning module followed by a case‐based escape room simulation will lead to high levels of learner knowledge, satisfaction, and confidence in ACLS pharmacology.

Methods

All fourth‐year medical students (n=112) in the Class of 2021 participated in an online pre‐session module and escape room activity. Initially, all students completed a short (16‐minute) online learning module designed to teach the basics of ACLS pharmacology. After this module, students completed a 28‐item pre‐session MCQ quiz to assess baseline knowledge and to ensure preparation for the escape room activity. Students then completed a high‐fidelity case‐based escape room activity consisting of six ten‐minute stations incorporating simulators, puzzles, and clues related to ACLS relevant pharmacologic concepts and algorithms. After the activity, students completed the same 28‐items as a post‐session MCQ quiz and a 14‐item satisfaction/confidence survey. Pre‐ and post‐session results were compared via paired t‐tests.

Results

All 112 participants (RR=100%) completed both the pre‐ and post‐session knowledge assessments. Overall mean performance on pre‐ and post‐session assessments was 87.6% (SD 8.4) vs. 95.7% (SD 5.0), respectively (Table 1, p<0.0001). Mean pre‐ and post‐session performance on pharmacology specific items was 77.7% (SD 11%) and 92.8% (SD 9.5%), respectively (Table 1, p<0.0001). 111 participants (RR=99%) completed the post‐session confidence survey. On a 7‐point Likert scale (1‐“Strongly Disagree” through 7‐“Strongly Agree, ”Figure 1), mean agreement with the statement “The ACLS module improved my knowledge of medical decision making for patients in cardiac arrest” was 6.6 (SD 0.8). On a 10‐point Likert scale, students rated their pre‐ and post‐session confidence in “Addressing a patient in cardiac arrest” at 7.7 (SD 2.1) and 8.4 (SD 1.6), respectively (p=0.0019).

Conclusion

Participation in the online pre‐session module and escape room activity led to significant knowledge gains in ACLS pharmacology along with high levels of learner confidence and satisfaction. This innovative approach may be used to teach ACLS pharmacology to fourth‐year medical students and to supplement traditional ACLS certification courses.
Effect of Acute Heat Exposure on the Pressor Response to a Voluntary Hypoxic Apnea: A Cross‐Tolerance Study
Authors: Cope Heidi L. , Hemingway Holden W. , Saul Benjamin M. , Richey Rauchelle E. , Moore Amy M. , Shokraeifard Austin M. , Olivencia‐Yurvati Albert H. , Smith Michael L. , Romero Steven A.
Published: May 2022

The pressor response induced by a voluntary hypoxic apnea is exaggerated in individuals with obstructive sleep apnea and is strongly correlated to sympathetic overactivity. Acute heat exposure alters neural control of blood pressure, but its effect on the pressor response to a voluntary hypoxic apnea has never been explored. Therefore, we tested the hypothesis that acute heat exposure would attenuate the pressor response to a voluntary hypoxic apnea, and thereby manifest as a form of physiological cross‐tolerance. Six young adults (2 females, 25 ± 1 yrs) were exposed to passive heat stress (water‐perfused suit) sufficient to increase body core temperature by 1.2 °C. Voluntary hypoxic apneas were performed in duplicate before acute heat exposure (pre‐heat) and in recovery when body core temperature returned to ≤ 0.3 °C of baseline. Participants breathed gas mixtures of varying FiO2 (21%, 16%, and 12%; randomized) for 1 min followed immediately by a 15 s end‐expiratory apnea. Beat‐by‐beat arterial blood pressure (Finometer) and arterial oxygen saturation (finger pulse oximetry) were measured throughout. The pressor response was calculated as the difference between baseline mean arterial pressure and the peak response following each apnea. The change in arterial oxygen saturation during each apnea did not differ from pre‐heat to recovery (FiO2 21%: pre‐heat, Δ0 ± 1 % vs. recovery, Δ1 ± 2 %; FiO2 16%: pre‐heat, Δ‐4 ± 1 % vs. recovery, Δ‐3 ± 1 %; FiO2 12%: pre‐heat, Δ‐9 ± 3 % vs. recovery, Δ‐10 ± 4 %; P = 0.6 for interaction). The pressor response to a voluntary apnea was attenuated in recovery from acute heat exposure across all gas mixtures (FiO2 21%: pre‐heat, Δ14 ± 11 mmHg vs. recovery, Δ10 ± 7 mmHg; FiO2 16%: pre‐heat, Δ22 ± 15 mmHg vs. recovery, Δ15 ± 11 mmHg; FiO2 12%: pre‐heat, Δ28 ± 15 mmHg vs. recovery, Δ21 ± 16 mmHg; P < 0.01 for main effect of time). These data suggest that acute heat exposure induces a cross‐tolerance effect such that the pressor response to a voluntary hypoxic apnea is reduced. Acute heat exposure could improve hypertension in adults with obstructive sleep apnea, secondary to altered chemoreflex function and sympathetic neural control, and provide additional therapeutic options for this population to improve cardiovascular health.
Chemical crosslinking and peptide mapping reveal structural characteristics of interdomain 1 and 2 of the placental malaria adherent protein VAR2CSA
Authors: M. G. Jagadeeshaprasad , Bewley Maria C. , Lovely Gautam , Flanagan John M. , Gowda Channe D.
Published: May 2022

The sequestration of Plasmodium falciparum in the placenta contributes to multiple placental malaria pathologies. VAR2CSA, a member of P. falciparum erythrocyte membrane protein 1 family of variant proteins, mediates the sequestration via the binding of parasite‐infected red blood cells to chondroitin 4‐sulfate in the placenta. This selective binding makes VAR2CSA a specific target for developing placental malaria treatment strategies. VAR2CSA contains ~310‐kDa C4S‐binding ectodomain, which thought to consist of an N‐terminal segment (NTS), six canonical Duffy‐binding‐like domains (DBL1‐DBL6), and interdomain (ID1 and ID2) segments. The ectodomain contains 116 cysteine residues. Up to eight intradomain disulfide bonds may be observed in canonical DBL domains; however, not all cysteine residues in a DBL domain conform to this pattern and additional ones are located outside the DBL domains. Recent cryo‐electron microscopy reconstructions revealed the functional core structure containing a third interdomain component (ID3) and substantiated the carbohydrate‐binding channel identified earlier. However, up to 46 % of the core, including the paths of ID1, regions of ID2 remain untraced in these structures. Here, by chemical cross‐linking/mass spectrometry of VAR2CSA, we identify additional interdomain interactions in the unmodeled regions of VAR2CSA. Disulfide bond mapping identifies roles for non‐canonical disulfide bonds and reveals previously un‐modelled interdomain disulfide bonds that link ID2 to DBL4ε. In addition, we resolve a disagreement in the interpretation of ID3 by unambiguously identifying the cysteine residues involved in the disulfide bond between DBL4ε and ID3.
Dantrolene Significantly Improves Cerebral Blood Flow In Diabetic Rats With Cerebral Vasospasms
Authors: Roman Marie , Martins Antonio H. , Ferrer Yancy , Torres Hector , Crespo Maria J.
Published: May 2022

Cerebral vasospasm (CVSP) is a potent vasoconstriction of the cerebral vasculature, and the primary cause of morbidity and mortality following a hemorrhagic stroke. Diabetics have a greater predisposition for developing CVSPs than nondiabetics, even when under glycemic control. The middle cerebral artery (MCA) is one of the most common vessels affected by CVSPs. Experimental evidence shows that concomitant administration of dantrolene (a ryanodine receptor blocker) and nimodipine (a Ca+2 channel blocker) synergistically reduces vasospasms in aortic rings from streptozotocin (STZ)‐induced diabetic rats. To determine if the effects observed in the systemic vasculature extend to the cerebral circulation, we investigated the effects of intravenous administration of dantrolene (2.5 mg/kg) and nimodipine (1 mg/kg and 2 mg/kg) on MCA blood flow velocity (BFV) seven days after the induction of CVSP in diabetic rats. Vasospasms were induced by bathing the external surface of the left common carotid artery with autologous whole blood. BFV was measured with a PeriFlux 5000 Laser Doppler System before, and sixty minutes after administering the drugs. Morphometric evaluations of the common carotid arteries were also performed to assess vascular alterations secondary to the induction of vasospasms. BFV was reduced by 30% with dantrolene alone (from 495.90 ±21.38 to 345.50±31.46 perfusion units, n=6, p≤0.05), however, nimodipine alone (1mg/kg or 2 mg/kg) did not affect this variable. The combination of dantrolene with 1 mg/kg nimodipine decreased BFV by 22% (from 518.90±31.49 to 405.50±23.84 perfusion units, n=7, p≤0.05), while a 32% reduction was obtained with the combination of dantrolene and 2 mg/kg nimodipine (from 504.60±15.38 to 344.90±36.98 perfusion units, n=6, p≤0.05). Furthermore, seven days after the induction of vasospasms, the lumen area of the left common carotid artery decreased, whereas media thickness and the wall‐to‐lumen ratio increased when compared to contralateral controls. The latter findings suggest that a period of seven days after the induction of vasospasms, was sufficient to cause vascular remodeling. Thus, our results indicate that, in diabetic rats, 2.5 mg/kg dantrolene alone was as effective as the combination of dantrolene and the highest dose of nimodipine in reducing BFV in the MCA. Therefore, dantrolene may provide a promising alternative therapy for the treatment of CVSP in the diabetic population.
Oxidative Stress Contributes to Microvascular Endothelial Dysfunction in Women With a History of Gestational Diabetes
Authors: Stanhewicz Anna E. , Schlarmann Rowan L. , Brustkern Kaila M. , Jalal Diana I.
Published: May 2022

Women with a history of gestational diabetes mellitus (GDM) are twice as likely to develop cardiovascular disease as their age‐matched counterparts. GDM is associated with endothelial dysfunction and elevated oxidative stress during pregnancy, and vascular dysfunction postpartum. However, the mechanism(s) mediating this postpartum dysfunction are unknown. Using the cutaneous circulation as a model, we examined if oxidative stress contributes to microvascular dysfunction in healthy women with a history of GDM (GDM). We hypothesized that: 1) endothelium‐ and nitric oxide (NO)‐dependent dilation would be attenuated in GDM compared to women with a history of normal pregnancy (NP), and 2) local antioxidant (L‐ascorbate) administration would augment endothelium‐ and NO‐dependent dilation in GDM. Ten NP (35±1yrs) and 10 GDM (34±1yrs) underwent a standard local heating protocol (42°C; 0.1°C·s‐1). Two intradermal microdialysis fibers were placed in the ventral forearm for local delivery of lactated Ringer’s (control), or 5mM ascorbate. After full expression of the local heating response, 15mM NG‐nitro‐L‐arginine methyl ester (L‐NAME; NO synthase‐inhibition) was perfused. Red cell flux was measured continuously by laser‐Doppler flowmetry and cutaneous vascular conductance (CVC=flux/MAP) was standardized to maximum (%CVCmax; 28mM SNP + 43°C). Urine albumin to creatinine ratio (ACR) and plasma concentrations of oxidized LDL (oxLDL) and thiobarbituric acid reactive substances (TBARS) were measured. Women with history of GDM had attenuated endothelium‐dependent (GDM: 67±7 vs. NP: 90±4%CVCmax; p<0.001) and NO‐dependent (GDM: 54±7 vs. NP: 71±3%; p=0.001) dilation at the control site and tended to have higher urine ACR (p=0.06). Both endothelium‐dependent (r2=0.53, p=0.02) and NO‐dependent (r2=0.56, p=0.01) dilation were related to urine ACR in GDM. Local ascorbate perfusion improved endothelium‐dependent (82±5%CVCmax; p=0.03 vs. control) and NO‐dependent (68±5%; p=0.02 vs. control) dilation in those with history of GDM but had no effect in HC (p>0.05). There were no differences in plasma oxLDL (hxGDM:60.5 vs hxNP:50.9 U/L; p=0.18) or TBARS (hxGDM: 0.3 vs hxNP: 0.2 μM; p=0.26) concentrations between groups. These data suggest that otherwise healthy women with a history of GDM have attenuated microvascular endothelial function and that this dysfunction is mediated, in part, by oxidative stress at the level of the vasculature.
The effective use of the Anatomage table as a teaching tool for learning the Infratemporal Fossa
Authors: Mosaheb Sydney , Afifi Linda , Wisco Jonathan J. , Zumwalt Ann C.
Published: May 2022

The objective of this Vesalius Master’s capstone project was to develop a lesson for teaching the Infratemporal fossa (ITF) using an Anatomage table, to first year students in the Medical Gross Anatomy course. The ITF is an important space within the head and neck, through which many structures pass or are housed but is difficult to visualize both in the cadaver and in 2D images. In contrast, the Anatomage table provides opportunities for rotatable three‐dimensional views. The lesson was designed to enable students to simultaneously visualize structures within the fossa and the relationships of these structures to deeper and more superficial structures in the area. A virtual dissection of the ITF from superficial to deep was created as 4 separate preset screens on the Anatomage table. Preset one showed the structures of the head and neck superficial to the ITF. The second preset showed a deeper view into the ITF in which the zygomatic arch and mandible were removed to see the muscles within in the ITF. Preset three was a view of the cranial nerves (CN), specifically CN V3 and its branches within the ITF. Preset four was a view that showed the vasculature within the ITF, specifically the maxillary artery and its branches. The lesson was designed to use the Anatomage table features to highlight the 3D relationships of these structures, supplemented with a worksheet that followed the virtual dissection and provided students with the high yield information for the course practical and written examinations. The lesson was run 5 times for a total of 35 students. After the session, students completed an evaluation of the effectiveness of this lesson, the lesson resources, and the instructor. Overall, the students felt that this method of teaching the ITF was highly effective for teaching the structures and function of the ITF contents. The students expressed that using the Anatomage table allowed them to orient themselves to the regions in contrast to what other resources allow. The supplemental worksheet was an important complementary resource to the lesson as it functioned as a study guide for the exam. This study demonstrates that the Anatomage table is a useful tool for teaching a region of the body that is difficult to demonstrate in the cadaver. This lesson allowed students to incorporate other relationships within the head and neck and provided context to these relationships. The method of teaching allowed the students to work together and actively engage in the lesson, which was effective for understanding the content. This teaching method can be used both to supplement cadaver teaching and in a context in which cadavers are not accessible.
The TM2.50 Aspartate of the TM1‐2‐7 Hydrogen‐Bond Network Modulates Signaling Bias Effects in G Protein‐Coupled Receptor 19 (GPR19)
Authors: Walter Deborah , Leysen Hanne , Christiaenssen Bregje , Maudsley Stuart
Published: May 2022

G protein‐coupled receptors (GPCRs) are successful drug targets due to their functional involvement in nearly every biological process. GPCRs can generate functional signals via both G proteins and β‐arrestins. Our research has demonstrated that an additional non‐G protein signaling function may exist for GPCRs, i.e. the GIT2 (GRK interacting transcript 2) signaling pathway. The structural bases of differential signaling paradigm control in GPCRs are still relatively unclear. Through structural modification of conserved structural motifs in GPCRs, we have sought to unravel this conundrum. The hydrogen‐bond network, including transmembrane helix 1 (TM1) Asparagine (N1.50), TM2 Aspartate (D2.50), and TM7 Asparagine (N7.49), is conserved across the majority of Rhodopsin‐like GPCRs and is known to affect GPCR signal initiation. Human GPR19 however possesses a natural variation in this network, i.e. a Lysine substitution for the TM7 Asparagine N7.49. Here we have introduced selective point mutations to several of these conserved sites and have investigated how these changes affect G protein, β‐arrestin, and GIT2 signaling bias effects. We have investigated both the proteomic cellular response to expression level variation of wild type (WT) and mutant (MT) GPR19 forms, as well as the physical interactome of WT and MT forms by using quantitative mass spectrometry. With these data we have performed targeted bioinformatic analyses to investigate signaling bias effects at GPR19. Our results have demonstrated that the TM2 MT D2.50A was able to indeed shift signaling bias from β‐arrestin paradigms to GIT2 signaling paradigms. Our research, as well as others, have shown that β‐arrestin and GIT2 signaling can act in an opposite manner regarding stress resistance and DNA damage (both drivers of aging and age‐related disease), with the former facilitating damage and the latter attenuating damage. Controlling the ability of a GPCR to selectively regulate these pathways could show beneficial effects concerning DNA damage in the context of aging and age‐related disease.
Hyperbaric Oxygen Therapy Following Cervical Spinal Cord Injury Increases BDNF/TrkB Signaling in Diaphragm Muscle
Authors: Boeno Franccesco P. , Sunshine Michael D. , Bindi Victoria E. , Fuller David D. , Smuder Ashley J.
Published: May 2022

Respiratory insufficiency is the leading cause of morbidity and mortality following cervical spinal cord injury (SCI). Diaphragm atrophy following SCI occurs as a result of damage to respiratory neural circuitry, which leads to impaired muscle contractility. Disruption of redox balance occurs after SCI and increased mitochondrial reactive oxygen species production is an upstream trigger for diaphragm muscle atrophy and weakness. Recently, we demonstrated that increased antioxidant capacity in response to hyperbaric oxygen (HBO) therapy may play a role in preserving mitochondrial redox homeostasis following SCI. However, a precise understanding of the mechanisms by which HBO therapy can prevent diaphragm dysfunction is lacking. In this regard, evidence indicates that the secretion of neurotrophic factors, such as brain derived neurotrophic factor (BDNF), may also contribute to enhanced functional recovery when HBO therapy is initiated acutely following cervical SCI. Through activation of the 5′ adenosine monophosphate‐activated protein kinase (AMPK) and serine‐threonine protein kinase (AKT) signaling via the phosphorylation of its receptor tropomyosin receptor kinase B (TrKb), BDNF expression has been shown to modulate mitochondrial biogenesis and skeletal muscle proteolysis. Therefore, we tested the hypothesis that 10 days of HBO therapy initiated in the acute phase following cervical SCI can increase diaphragm mitochondrial biogenesis and prevent diaphragm proteolytic signaling by activating BDNF/TrkB signaling. To test this, adult male Sprague‐Dawley rats were separated into three groups (n=8/group): (1) non‐injured, room air exposure (CON); (2) lateral‐cervical spinal cord contusion, room air exposure (SCI); and (3) lateral‐cervical spinal cord contusion, HBO therapy (SCI+HBO). Animals in the SCI and SCI+HBO groups were anesthetized, and following laminectomy at the C3–C4 level, a lateral‐cervical spinal contusion was made using a mechanical impactor. HBO therapy consisted of 10 days of exposure starting on the day of SCI, using a 40 L chamber flushed with 100% O2 and pressurized to 3 ATA. Our results indicate that HBO therapy delivered in the acute phase of SCI positively affected BDNF/TrkB signaling. Specifically, diaphragm protein expression of BDNF, p‐AKT/AKT and p‐AMPK/AMPK were increased after 10 days of HBO therapy compared to CON animals (p<0.05). Additionally, HBO therapy prevented the SCI‐induced reduction in diaphragm peroxisome proliferator‐activated receptor‐gamma coactivator (PGC1‐α) protein expression (p<0.05), as well as the caspase‐3‐specific cleavage of α‐II‐spectrin observed in the SCI group (p<0.05). In summary, these findings demonstrate that HBO therapy can induce BDNF signaling and highlight an additional mechanism for the protective effects of HBO therapy to preserve diaphragm muscle function following SCI.
Chronic Hypoxia Blunts the Hypoxic Ventilatory Response in Neonatal and Adult Rats
Authors: Lee Darya , Murphy Erin J. , West Hannah , Reynoso Williams Maya , Bavis Ryan W.
Published: May 2022

Chronic hypoxia (CH) may affect the control of breathing differently in neonatal versus adult mammals. It is widely accepted that CH blunts the hypoxic ventilatory response (HVR) in neonatal rats but augments hypoxic ventilation in adult rats. However, there is also evidence that longer durations of CH can progressively attenuate the HVR in adult rats too. Therefore, we predicted that short durations of CH (3 days) would blunt the HVR in neonatal rats and augment the HVR in adult rats, but that longer durations of CH (7 days) would blunt the HVR in adult rats. To test these predictions, rats were exposed to 12% O2 (CH) or room air (Control) for 3 days as neonates (from P0 to P3, or from P7 to P10) or as young adults. Baseline ventilation (21% O2) and the acute HVR (12% O2) were measured by plethysmography immediately following the exposure. The adult rats were then returned to the CH and Control chambers for 4 more days before repeating the breathing measurements. Although Sprague‐Dawley rats were used in all experiments, adults from two different commercial suppliers (Charles River Laboratories and Envigo) were used to determine whether genetic background might influence the results. Baseline ventilation was greater in all CH groups regardless of their age, consistent with ventilatory acclimatization. With the exception of CH rats at P10 in which peak hypoxic ventilation (i.e., minute ventilation during the first minute of 12% O2) was also greater than in Controls, hypoxic ventilation was generally similar between CH and Control groups or reduced in the CH group. Accordingly, the change in ventilation from baseline (i.e., HVR) was blunted by CH regardless of the age of the rats, the duration of the CH exposure (adults only), or the substrain of Sprague‐Dawley rat (adults only). The order in which test gases were presented during breathing measurements (i.e., 21% O2 – 12% O2 vs. 12% O2 – 21% O2) did not influence the calculated HVR in adult rats. We conclude that the respiratory plasticity elicited by normobaric CH is similar across ages in Sprague‐Dawley rats.
Soluble Guanylyl Cyclase Stimulator Improves Contracting Skeletal Muscle Oxygen Pressures in Heart Failure Rats
Authors: Weber Ramona E. , Schulze Kiana , Hageman K. S. , Sandner Peter , Musch Timothy , Poole David
Published: May 2022

Introduction

Impaired convective and diffusive oxygen (O2) transport is a hallmark of heart failure with reduced ejection fraction (HFrEF), largely attributed to nitric oxide‐soluble guanylyl cyclase (sGC)‐cyclic guanosine monophosphate pathway dysfunction. sGC stimulators were synthesized to independently and synergistically (with endothelial derived‐NO) promote vasodilation, thereby demonstrating the potential to improve skeletal muscle oxygenation in HFrEF. We tested the hypotheses that 2 weeks of administration of sGC stimulator BAY 41‐2272 would increase the O2 delivery (Q̇O2)‐to‐O2uptake (V̇O2) ratio in the skeletal muscle interstitial space (PO2is) of HFrEF rats at rest and during twitch contractions.

Methods

HFrEF was induced in adult male Sprague‐Dawley (3‐4 mo. old) rats via myocardial infarction (MI). Following 5 weeks of HFrEF progression, rats were treated with 1.0 mg/kg of BAY 41‐2272 via oral gavage twice per day (HFrEF + BAY; n=6) for 2 weeks prior to the contraction protocol. The control heart failure group (HFrEF; n=5) received vehicle only. Phosphorescence quenching protocols determined the partial pressure of O2 in the spinotrapezius muscle interstitial space at rest and during twitch contractions, reflecting the dynamic relationship between Q̇O2 and V̇O2.

Results

The degree of HFrEF was not different between groups as evidenced by left ventricular end‐diastolic pressure (16 ± 1 mmHg, both groups) and MI size (27 ± 1 vs. 29 ± 1%) (all P>0.05). There were no differences in blood gases following contractions (P>0.05). Kinetic analyses revealed no difference in PO2is at rest, however immediately following the onset of contractions and throughout the contraction steady‐state, HFrEF+BAY rats maintained a ~5‐7 mmHg higher PO2is (32‐180s of contractions, P<0.05). Furthermore, HFrEF+BAY rats had an attenuated undershoot, decreased tau (time constant), and increased end PO2is (all P<0.05) in comparison to HFrEF rats.

Conclusions

During the rest‐contraction transient and throughout the contractions steady‐state BAY 41‐2272 increases PO2isin rats with moderate HFrEF. By improving O2delivery and relieving the HF‐induced O2‐dependence of V̇O2 kinetics these findings support the therapeutic potential for sGC stimulators to reduce vasomotor dysfunction and improve exercise tolerance in HFrEF.
Valproic acid regulates sodium‐dependent vitamin C transporter‐2 (SVCT‐2) functional expression in neuronal cells
Authors: Teafatiller Trevor , Subramanian Veedamali S.
Published: May 2022

Neuronal uptake of ascorbic acid (AA) in humans occurs via the human sodium‐dependent vitamin C transporter‐2 (hSVCT2; the product of the SLC23A2gene). Studies have shown that a significantly lower level of vitamin C is present in the blood plasma of patients with chronic neurological conditions including epilepsy. Therefore, studies focused on determining the molecular mechanisms involved in regulating hSVCT2 functional expression are vital for identifying potential therapeutic strategies to optimize vitamin C body homeostasis. Currently, little is known about the role of valproic acid (VPA), a drug used to treat epilepsy that also acts as a class I histone deacetylase inhibitor (HDACi), on AA uptake in neuronal systems. Thus, this study aims to investigate the effect of VPA on SVCT2 functional expression using human neuroblastoma SH‐SY5Y cells (in vitro) and mouse (in vivo) models. VPA (1mM) treatment up‐regulated the AA uptake and this increased uptake was accompanied by a marked increase in hSVCT2 protein and mRNA expression levels as well as SLC23A2 promoter activity in SH‐SY5Y cells. In addition, the protein and mRNA expression levels of specific protein‐1 (Sp1), a transcription factor that drives the basal activity of SLC23A2 promoter, were also markedly up‐regulated in VPA‐treated SH‐SY5Y cells. Further, knocking down the HDAC2, the predominant isoform in the neuronal system, significantly increased AA uptake and up‐regulated the hSVCT2 protein and mRNA expression levels in SH‐SY5Y cells. VPA administration (150 mg/kg body weight) to wild‐type (WT) mice displayed increased mouse (m)SVCT2 protein, mRNA and hnRNA expression in the brain. Together, our findings suggests that VPA up‐regulates the functional expression of SVCT2 and HDAC2 plays an essential role in neuronal AA uptake.