bluuebunny/crossref_metadata_2025 huggingface dataset:
Absence of Erythropoietin and Cardio‐respiratory Responses to Hypobaric Hypoxia in Olfactory Receptor 78 Null mice
Authors: Wang Benjamin , Peng Ying‐Jie , Su Xiaoyu , Zhang Chongxu , Nagati Jason , Garcia Joseph A. , Prabhakar Nanduri R.
Published: May 2022

Hypobaric hypoxia (HH) evokes a series of physiological adaptations, including carotid body (CB)–dependent ventilatory acclimatization to hypoxia (VAH), elevated blood pressure (BP), and stimulation of erythropoietin (Epo) gene in the kidney. Olfactory receptor 78 (Olfr78) is a G‐protein coupled receptor that has been implicated in CB response to acute hypoxia. Given that both CB and kidney express Olfr78, we tested the hypothesis that Olfr78 plays a role in the cardio‐respiratory and renal Epo gene responses to HH. Studies were performed on wild type (WT) and Olfr78 null mice reared in room air (control) or exposed to 18h of HH (0.4 atmospheres). HH treated WT exhibited: 1) VAH manifested as increased baseline breathing, and enhanced hypoxic ventilatory response (HVR; 12% O2); 2) elevated BP and plasma norepinephrine levels; and 3) increased baseline CB sensory nerve activity and augmented CB sensory nerve response to subsequent acute hypoxia‐ all these responses to HH are either markedly attenuated or absent in Olfr78 null mice. WT mice responded to HH with activation of the renal Epo gene expression and elevated plasma Epo levels, and these effects were attenuated or absent in Olfr78 null mice. The attenuated Epo activation by HH was accompanied with markedly reduced hypoxia‐inducible factor (HIF)‐2α protein and reduced activation of HIF‐2 target gene Sod‐1 in Olfr78 null mice, suggesting impaired transcriptional activation of HIF‐2 contributes to attenuated Epo responses to HH. These results suggest a role for Olfr78 in physiological adaptations to HH experienced at high altitude
Loss of Endothelial Cell RIPK3 Leads to Increased Vascular Permeability Following Intestinal Ischemia‐Reperfusion Injury
Authors: Johnson Charmain F. , Griffin Courtney T.
Published: May 2022

The receptor‐interacting serine/threonine‐protein kinase 3 (RIPK3) is primarily known for promoting necroptosis, but recent findings suggest that RIPK3 has non‐necroptotic roles as well, particularly in endothelial cells (ECs). Our lab has reported both beneficial and detrimental roles for endothelial RIPK3 during vascular development, which has prompted us to study RIPK3 in the context of different postnatal vascular pathologies. Our current study focuses on roles for endothelial RIPK3 following intestinal ischemia‐reperfusion (I/R) injury in mice. First‐order branches of the superior mesenteric artery that feeds the ileum were clamped for 1 hr to induce ischemia, and clamps were removed subsequently to allow 24 hr reperfusion in tamoxifen‐induced Ripk3;Cdh5(PAC)‐CreERT2 mice (RIPK3ECKO) compared to wildtype littermate control mice (WT) (males and females, 12 weeks old). Unclamped small intestinal regions were considered as sham control in the same mouse. An Evans blue permeability assay was utilized in assessing small intestinal and distal organ vascular permeability. Small intestinal vascular permeability directly downstream of I/R injury was significantly increased in both RIPK3ECKO and WT mice (n=6 mice/group), however permeability was also significantly increased in sham small intestinal regions and in livers of RIPK3ECKO unlike in WT controls (n=6 mice/group). No difference in vascular permeability was observed between RIPK3ECKO and WT mice (n=5 mice/group) at baseline without I/R injury in all organs assessed. However, in uninjured RIPK3ECKO mice we do observe an increase in the CD68+ proinflammatory macrophage marker in the liver and submucosal layer of the small intestine compared to uninjured WT mice (n=5 mice/group). When clodronate liposomes were administered to deplete macrophages and determine whether this CD68+ macrophage population was contributing to the increased permeability in RIPK3ECKO mice following I/R injury, we observed a permeability rescue in the small intestine but not in the liver (n=4‐5 mice/group). This finding suggests that there are organ‐specific differences in macrophage contribution to vascular permeability following I/R injury. Our current studies are evaluating circulating and tissue specific cytokines and endothelial cell adhesion molecules to understand why RIPK3ECKO vasculature becomes more permeable in response to I/R injury. Discovering cellular mechanisms for vascular endothelial RIPK3 following I/R injury could provide novel therapeutic targets to promote vessel recovery after I/R injury.
Ethanol Inhibits the Metabolism of the Multiple Sclerosis Drug Dimethyl Fumarate to its Active Metabolite and Decreases Brain Exposure
Authors: Yang Bing , Parker Robert B. , Meibohm Bernd , Srivastava Ashish , Temrikar Zaid H. , Laizure S. C.
Published: May 2022

Hypothesis

Dimethyl fumarate is primarily metabolized to the monomethyl fumarate active metabolite by carboxylesterase 1 and is subject to inhibition by ethanol.

Methods

A pharmacokinetic study was conducted to determine the effect of ethanol on dimethyl fumarate disposition in ES1e mice, a strain that is a better model of human carboxylesterase drug metabolism than typical rodent strains. The disposition of dimethyl fumarate and monomethyl fumarate, the purported active metabolite, was determined in a control group (n=30) receiving 100 mg/kg of dimethyl fumarate via oral gavage versus an ethanol group (n=30) receiving 3 g/kg of ethanol by oral gavage 10 minutes prior to dosing with 100 mg/kg of dimethyl fumarate. The area‐under‐the‐curve (AUC) and maximum plasma concentration (Cmax) were determined in plasma and brain tissue from the drug and metabolite concentrations determined by LC‐ mass spectrometry. The AUC and Cmax were compared between the control and ethanol group.

Results

Like dimethyl fumarate disposition in humans, only monomethyl fumarate concentrations were quantifiable in the plasma in the control group. In the ethanol group, dimethyl fumarate was quantifiable in both the plasma and brain tissue.

Control
Ethanol

DMF
MMF
DMF
MMF

AUCPLASMA(ng/ml*min)
ND
6072
4629
3948

CmaxPLASMA(ng/ml)
ND
210
64
65

AUCBRAIN (ng/ml*min)
ND
980
20
787

DMF=dimethyl fumarate; MMF=monomethyl fumarate (active metabolite); ND=not detected

The terminal elimination half‐life of monomethyl fumarate in the control and ethanol groups was 14.4 and 44.3 minutes, respectively.

Conclusions

Ethanol inhibits the hydrolysis of dimethyl fumarate to active monomethyl fumarate by carboxylesterase 1 and has a drastic effect on the disposition of dimethyl fumarate and monomethyl fumarate. The therapeutic effect of dimethyl fumarate is likely to be significantly altered by the consumption of ethanol or other drug that inhibits carboxylesterase 1 potentially altering the beneficial effect of chronic dimethyl fumarate therapy on Relapsing‐Remitting Multiple Sclerosis.
How Lipids Regulate the Cell Surface Localization of HSPA1A, a Stress‐inducible 70‐kDa Heat Shock Protein
Authors: Altman Rachel T. , Nikolaidis Nikolas
Published: May 2022

HSPA1A is a molecular chaperone that regulates survival stressed and cancer cells. In addition to its cytosolic pro‐survival functions, HSPA1A also localizes at the plasma membrane (PM) of stressed and tumor cells. In cancer cells, the presence of HSPA1A at the cell surface allows the latter cells to develop resistance to radiation therapy, show increased invasiveness, and develop distant metastasis. Therefore, abolishing HSPA1A from the surface of tumor cells is a promising therapeutic. However, HSPA1A’s pathway to the cell surface remains enigmatic because this protein lacks membrane localization signals. Considering that HSPA1A binds with high selectivity to negatively charged lipids, like phosphatidylserine (PS) and mono‐phosphorylated phosphoinositides (PIPs), we hypothesized that the interaction of HSPA1A with these lipids allows the chaperone to translocate and anchor to the PM. To test this hypothesis, we subjected human cell lines to heat shock, depleted specific lipid targets, and quantified HSPA1A’s PM localization using confocal microscopy and cell surface biotinylation. These experiments revealed that depletion of PS, PI(4)P, and PI(3)P by co‐transfecting HSPA1A with known lipid‐biosensors, significantly reduced HSPA1A’s surface presentation. In contrast, reducing PI(4,5)P2 availability by overexpression of the PLCδ‐PH biosensor had minimal effects on HSPA1A’s PM‐localization. Next, we manipulated the cellular lipid content using ionomycin (a PLC activator), phenylarsine oxide (PAO) and GSK‐A1 (general and a selective PI4‐Kinase inhibitors, respectively), wortmannin (a PI3‐Kinase inhibitor), and fendiline (a repurposed FDA approved drug that reduces PS) using lipid‐biosensors as positive and negative controls. These experiments revealed that HSPA1A’s PM localization was unaffected by ionomycin but was greatly reduced in the presence of PAO, GSK‐A1, wortmannin, and fendiline, corroborating the findings obtained by the co‐transfection experiments. We further verified these results by selectively depleting PI(4,5)P2 and PI(4)P using a well‐established rapamycin‐induced phosphatase system. Our findings strongly support the notion that HSPA1A’s surface presentation is a multifaceted lipid‐driven phenomenon controlled by the binding of the chaperone to specific endosomal and plasma membrane lipids. These findings provide the basis for future interventions to render tumor cells sensitive to radiation therapy.
Insights Into the Na+‐Dependent Anion Exchange in the SLC4 Family From the Cryo‐EM Structure of the Sodium‐Driven Chloride/Bicarbonate Exchanger SLC4A8
Authors: Zhekova Hristina R. , Wang Weiguang , Tsirulnikov Kirill , Kayık Gülru , Khan Hanif M. , Azimov Rustam , Abuladze Natalia , Kao Liyo , Newman Debbie , Noskov Sergei , Tieleman D. P. , Zhou Z. H. , Pushkin Alexander , Kurtz Ira
Published: May 2022

SLC4 transporters are involved in the transport of HCO3‾, CO32‾, Na+, Cl‾ and H+ required for regulation of pH and ion homeostasis in several important physiological processes. Dysfunction of these transporters can lead to acid‐base abnormalities, pH dysregulation, and various disease processes. Understanding of their transport mechanism is therefore critical for development of pharmacological strategies for SLC4‐related diseases. The studies of the SLC4 transport mechanisms have been hindered by lack of information on the specific substrate binding sites within the protein matrix. No binding sites have been resolved in the two previously published structures of SLC4 proteins, the chloride/bicarbonate anion exchanger 1 (AE1, SLC4A1) and the electrogenic sodium carbonate cotransporter 1 (NBCe1, SLC4A4), although putative binding sites have been identified on the basis of these structures from computational modelling and functional mutagenesis experiments. Here we present a new cryo‐EM structure at 3.4Å of the outward facing open (OF) conformation of the sodium dependent chloride/bicarbonate exchanger (NDCBE, SLC4A8) [1], which exchanges Na+‐CO32‾ for Cl‾, combining the feature functionalities of both AE1 and NBCe1. The structure resolves for the first time a Na+ and a CO32‾ ion bound at the center of the protein in the area of the previously identified putative binding site S1 in NBCe1 and AE1. Mutation of the residues involved in ion binding leads to significant impairment of NDCBE transport. Computational modelling with the new NDCBE structure outlines two separate ion pathways for cations and anions in the OF cavity and demonstrates that a Na+ ion bound at D800 is necessary for anion binding in site S1 and that a Na+‐CO32‾ ion pair in this site is the most likely ion load. A second anion binding site, S2, is also identified in the permeation cavity in proximity to site S1. We conclude that Na+‐dependent transport in the SLC4 family is influenced both by thermodynamic effects at the S1 binding site and by permeation effects governed by the residues lining the cation and anion permeation pathways in the OF cavity and site S2.

[1] Wang W, Tsirulnikov K, Zhekova HR, et al. Cryo‐EM structure of the sodium‐driven chloride/bicarbonate exchanger NDCBE. Nat Commun. 2020;12: 5690
Anatomical Structures Object Library of the Human Reference Atlas
Authors: Börner Katy , Quardokus Ellen M. , Browne Kristen
Published: May 2022

The Human BioMolecular Atlas Program (HuBMAP) aims to create a computable, open Human Reference Atlas (HRA) [1]. This HRA will redefine our understanding of the human body by recovering multi‐scale tissue organization from the human body to the single cell resolution. In the initial three years of the project, we created an authoritative ontology of anatomical structures, cell types, and biomarkers; this anatomical structure terminology was then linked to three‐dimensional reference objects in the size and shape of human organs, functional tissue units, and anatomical micro‐structures [2]. Many of the current 52 reference organs were derived from the Visible Human Project data. An initial subset can be explored at https://hubmapconsortium.github.io/ccf/pages/ccf‐3d‐reference‐library.html. The growing collection of reference objects form the basis of tissue data registration via the Registration User interface (RUI, https://hubmapconsortium.github.io/ccf‐ui/rui); registered tissue data can be explored in the context of the atlas via the Exploration User Interface (EUI, https://portal.hubmapconsortium.org/ccf‐eui).

We are in the process of developing standard operating procedures and design style guides for the HuBMAP Reference Object Library and would like to present first drafts of both at the AAA meeting to benefit from feedback by the larger expert community.

References

Snyder MP, Greenleaf WJ, Ahadi S, Nevins SA, Lee H, Esplin E, et al. The human body at cellular resolution: the NIH Human Biomolecular Atlas Program. Nature 2019; 574:187–92.

Börner K, Teichmann SA, Quardokus EM, Gee JC, Browne K, Osumi‐Sutherland D, et al. Anatomical structures, cell types and biomarkers of the Human Reference Atlas. Nat Cell Biol 2021; 23:1117–28. https://doi.org/10.1038/s41556‐021‐00788‐6.
Digital Dissection with CT Scans Enhances Anatomy Education: First‐year Medical Students’ Perception of Radiology Teaching During the Covid‐19 Pandemic
Authors: Spouge Rebecca , Forster Bruce B. , Tsai Olivia , Gil‐Mohapel Joana , Darras Kathryn E.
Published: May 2022

OBJECTIVE

The aim of this study was to evaluate students’ perceptions of the educational value of online, interactive anatomy radiology laboratories.

HYPOTHESIS

Digital dissection is performed with computed tomography (CT) scans on near life‐size anatomy visualization tables and has been used to teach radiology anatomy at our institution. These sessions were pivoted to online delivery in response to COVID‐19. With the ongoing movement towards technology integration in medical education, if home access to these online laboratories is made available then we hypothesize the perceived learning experience should be preserved.

METHODS

290 first‐year medical students participated in 10 weekly digital radiology anatomy laboratories, which employed 3D CT scans to emphasize the clinical applications of anatomy. Students reviewed the cases at home prior to the lab on a mobile platform and labs were delivered synchronously via the Zoom platform in a facilitated large‐group format. After the course, a voluntary anonymous online survey was distributed assessing learner satisfaction, task value, emotional achievement, and cognitive load.

RESULTS

Survey response rate was 44%. Most participants reported the labs improved their knowledge of anatomy (82%), disease (81%) and clinical decision making (65%), including surgical knowledge (76%). The large group, synchronous learning format was found to be the most effective (68%). Positive emotional achievement was reported (mean 2.98). The extraneous cognitive load of the at‐home mobile technology and lack of time were perceived as the greatest programmatic challenges.

CONCLUSION

Synchronous digital dissection labs were perceived as a valuable addition to the first‐year medical curriculum, enhancing learners’ clinical decision making and preparation for medical sub‐specialties. This format of radiology anatomy education can be integrated into blended learning environments to provide students with additional learning opportunities.
Variants and Cellular Functional Verification of CITED2 Gene Promoter Region in Patients with Atrial Septal Defect
Authors: Chen Zhuo , Chen Huan‐Xin , Yin Xiu‐Yun , Han Jun , Yang Qin , He Guo‐Wei
Published: May 2022

Objectives

Congenital heart disease (CHD) is a common disease of human birth defects, and atrial septal defect (ASD) is the most common form of ASD. Genetic variants in the coding region of the CITED2 gene is known to be significantly correlated with cardiac malformations, but variants of the CITED2 promoter region and its relationship with the formation of ASD are still unclear. We hypothesize that the variants of the CITED2 promoter may be related to pathogenesis of ASD. The purpose of this study was to screen variants in the promoter region of the CITED2 gene and to verify the effect of the variants on gene expression at the cellular level.

Methods

The blood samples of 332 ASD patients and 293 unrelated healthy children used as controls were studied. The total DNA of all subjects was extracted, and the CITED2 promoter variants were screened by PCR combined with Sanger sequencing. The luciferase activity of the CITED2 promoter was measured by a dual luciferase reporter at the cellular level. Electrophoretic mobility change assay (EMSA) and bioinformatics analysis were used to test the effect of CITED2 promoter changes on transcription factor binding sites.

Results

Four variants in the promoter region of the CITED2 gene were found only in the 332 ASD patients with zero occurrence in the 293 control subjects. These included one novel heterozygous variant (g.4933C>A) and three SNPs [1 case of g.4078 A>C(rs1165649373), 4 cases of g.4935C>T(rs111470468), 2 cases of g.5027C>T (rs112831934)]. The cellular functional analysis showed that these four variants significantly changed the transcriptional activity of the CITED2 gene promoter in HEK‐293 cells (P<0.05). In addition, EMSA results and database analysis showed that these variants created or destroyed a series of possible transcription factor binding sites, resulting in changes of the expression of CITED2 protein.

Conclusion

The variants of CITED2 promoter sequence in ASD patients affect transcriptional activity and are likely involved in the occurrence and development of ASD. These findings may provide new perspectives on the molecular pathogenesis and potential therapeutic insights of ASD patients.
Plasma Concentrations of Trace Elements Selenium and Cobalt During and After Coronary Artery Bypass Grafting Surgery
Authors: Zhou Jia‐Yi , Hou Hai‐Tao , Wang Shi‐Fu , Yang Qin , He Guo‐Wei
Published: May 2022

Objectives

Trace elements selenium (Se) and cobalt (Co) have been known to be essential in the human body and the correlation between Se and cardiac surgery has been suggested. The present study was designed to investigate the plasma concentration of Se and Co during and after coronary artery bypass grafting (CABG) surgery under cardiopulmonary bypass (CPB).

Methods

From December 2019 to January 2020, preoperative plasma samples from isolated first‐time CABG patients (N=20; 10 males and 10 females) were prospectively collected at five points: after anesthesia and before CPB (T1), 45 min after CPB started (T2), 90 min after CPB started (T3), postoperative day1 (T4), and day 4 (T5), respectively. The concentration of Se and Co was measured.

Results

The concentration significantly decrease for Se at T2 (105.24 ± 4.08 vs. 68.56 ± 2.42μg/L, P < 0.001) and T3 (105.24 ± 4.08 vs. 80.41± 3.40μg/L, P < 0.001) and for Co at T4 (0.35 ± 0.19 vs. 0.26 ± 0.13μg/L, P < 0.01) and T5 (0.35 ± 0.19 vs. 0.23 ± 0.11μg/L, P < 0.001). There was no operative mortality or major morbidity except that five patients developed atrial fibrillation (AF).

Conclusions

This study for the first time identified the alteration of plasma concentration of Se and Co during and after CABG surgery. The findings may suggest that supplementation of Se before or during CABG and that of Co after CABG may become necessary for patients undergoing CABG. Further studies are warranted.
Role of GBP5 in NLRP3 inflammasome mediated intestinal inflammation in Crohn’s disease
Authors: Li Yichen , Lin Xutao , Cheng Sijing , Wang Wenxia , Huang Yibo , Ke Jia , Zhu Lixin
Published: May 2022

Background

NLRP3 inflammasome is implicated in the pathogenesis of inflammatory bowel diseases (IBD). Since guanylate binding protein 5 (GBP5) induces the NLRP3 inflammasome activity, we aim to investigate the potential role of GBP5 in IBD pathogenesis.

Methods

The expression of GBP5, NLRP3 inflammasome and related cytokines and chemokines were examined in two cohorts of IBD patients and healthy controls, by microarray transcriptome analysis, quantitative real time PCR, Western blot and Luminex Multi‐Analyte Profiling technology. Cellular and subcellular localization of GBP5 in colonic biopsies were examined by immunohistochemistry and immunofluorescence with confocal microscopy. For functional studies, GBP5 expression was induced by IFNγ or silenced by siRNA or CRISPR/CAS9 technique.

Results

Expression of GBP5 was elevated in colonic mucosa in two geographically and culturally distinct IBD cohorts. In colonic tissues of IBD patients, the expression of GBP5 was mainly confined to immune cells and the levels of GBP5 expression were correlated with those of the inflammatory cytokines and chemokines. In cultured T and macrophage cells, expression of proinflammatory cytokines and chemokines were increased when GBP5 was induced, while GBP5 deficiency leads to decreased expression of the proinflammatory mediators including gasdermin D, caspase 1 and pro‐IL1β.

Conclusions

GBP5 is required in the expression of many inflammatory cytokines and chemokines in intestinal immune cells. In addition, GBP5 may up‐regulate inflammatory reactions through an inflammasome mediated mechanism. Highly elevated GBP5 expression at the inflamed colonic mucosa in two geographically and culturally distinct IBD cohorts indicates that GBP5 plays a common and important role in IBD pathogenesis, and therefore, is a potential therapeutic target for the management of IBD patients of various genetic and environmental backgrounds.
Interaction of epithelium and myofibroblasts through ERBB3‐PDGF growth pathways crosstalk drive the maturation of distal lung epithelium in late stage lung development
Authors: Zhang Xin , Heydarian Motaharehsadat , Voss Carola , Sucre Jennifer , Hilgendorff Anne
Published: May 2022

Rationale

The last stage of lung development is realized by coordinated growth of septae from the alveolar walls to subdivide the distal saccules into alveoli, resulting in the expansion of surface area for gas exchange. The process is accompanied by microvascular maturation and underlying matrix formation of the interstitial scaffold. Secondary septation is driven by the orchestration of the distal epithelium, endothelium, and fibroblasts through the spatial and temporal distribution of various growth pathways and their crosstalk.

This study investigated the role of the receptor tyrosine kinases 3 (ERBB3) pathway in the fibroblast‐epithelial crosstalk during septation using both in vitro co‐cultures and ex vivo organoid model as well as transgenic mice.

Methods

Primary myofibroblasts were extracted from neonatal C57BL/6wildtype (WT) mice at postnatal day 7 (PND7) and co‐cultured with a murine epithelial cell line (MLE12) in transwell inserts for 24h before sample collection. Gene expression was analyzed by quantitative PCR. Neonatal lung organoids were generated from primary lung epithelial cells (CD45‐/CD31‐) from neonatal C57BL/6 WT mice (PND7) and cultured in matrigel together with primary myofibroblasts from neonatal WT mice (PND7) as well as age matched mice heterozygot for the Platelet‐derived growth factor receptors (PDGF‐Rα+/‐). After organoid formation, cultures were treated with Neuregulin (NRG), an activating ligand of the ERBB3 receptor. We measured organoid number and size and Erbb3 signaling was assessed by immunofluorescence and quantitative gene expression analyses.

Results

Proximal culture with myofibroblasts upregulated the expression level of ERBB3 receptor in epithelial cells, as well as homeodomain‐only protein homeobox (HOPX), GATA‐binding factor 6 (GATA6), both of which are essential in the differentiation of distal epithelial cells and surfactant production during late stage lung development. In neonatal lung organoids, myofibroblasts with reduced PDGFRα expression led to reduction in organoid size and number. Treatment with the Erbb3 ligand NRG significantly increased both the expression and phosphorylation of Erbb3 in epithelial cells together with an upregulation of surfactant C (SPC) expression, and an increase in myofibroblasts PDGF‐R α expression.

Conclusion

Activated Erbb3 signaling results from the interaction with fibroblasts involving PDGF‐Rα signaling and drives distal epithelium maturation.
In‐Silico Investigation of Castration on Vas Deferens Smooth Muscle Electrophysiology
Authors: Mahapatra Chitaranjan , Manchanda Rohit
Published: May 2022

OBJECTIVE

The vas deferens smooth muscle (VDSM) generates spontaneous contractions for the sperm transportation. The underlying electrical activities of the VDSM cell are prominently correlated with these contractions. According to a recent experimental study, the castration has down regulated the A‐type K+channel activities in the VDSM cell. However, the modulating properties of castration into VDSM electrophysiology are not investigated yet. In the present time, the in‐silico study plays a key role in understanding various complex physiological systems. To explore the quantitative contribution of castration to the VDSM membrane action potential (AP), a biophysically detailed single VDSM in‐silico cell model is simulated.

METHODS

The in‐silico guinea‐pig VDSM cell model is constructed after incorporating a voltage‐gated Na+ ion channel, two voltage‐gated Ca2+ ion channels, a hyperpolarization‐activated ion channel, two voltage‐gated K+ ion channels, a Ca2+‐activated K+ ion channel, and a nonspecific background leak ion channel. All ion channel models are validated by comparing the simulated currents and current‐voltage relationship with those reported in the experimental studies. The modulating properties of the castration are simulated by mimicking the A‐type K+ channel on VDSM cell excitability.

RESULTS

The ion channel conductnaces are set to maintain the resting membrane potential (RMP) at ̶ 50 mV as the physiological range of RMP in VDSM cell varies from ̶ 45 mV to ̶ 70 mV (Figure 1). The AP and membrane depolarization are generated in the whole cell model by applying an external current stimulus (10‐30 pA), as a brief square pulse of 10 ms duration. The A‐type K+ current is elicited by a 1000 ms depolarizing‐voltage‐step between ̶ 70 and +40 mV from a holding potential (̶ 80 mV) with 10 mV increments. The maximum conductance of A‐type K+ channel is altered to observe the changes in VDSM AP. The RMP, AP width, and AP peak are elevated by 2 mV, 15 ms, and 1 mV respectively after the castration (Figure 1). It reveals that the VDSM cell is more electrically excited due to the castration.

CONCLUSIONS

The present in‐silico model, constrained heavily by physiological data, provides a powerful tool to investigate the ionic mechanisms underlying the genesis of VDSM electrical activity. In the guinea‐pig, following castration, the VDSM cell is excited by membrane depolarization, which causes to evoke more spontaneous contractions. The new biological insight from this investigation shows the agonist of the A‐type K+ channel might control the much‐diminished sex drive experiences for men after the castration.
Benefit of Circadian Clocks in Adaptive Immunity And Vaccination Responses
Authors: Barnoud Coline , Ince Louise M. , Lutes Lydia K. , Pick Robert , Wang Chen , Jemelin Stéphane , Scheiermann Christoph
Published: May 2022

The adaptive immune response is under circadian control, yet, the benefit of this rhythmicity for the organism is unknown. Furthermore, it is not understood why adaptive immune reactions continue to exhibit circadian changes over long periods of time. Using a combination of experimental and mathematical modelling approaches, we show here that dendritic cells (DCs) migrate from the skin to the draining lymph node (LN) in a time‐of‐day‐dependent manner, which provides an enhanced likelihood for functional interactions with T cells. Greater numbers of infiltrating DCs induce rhythmic expression of TNF‐α in the draining LN, which enhances ICAM‐1 expression in high endothelial venules (HEVs), resulting in lymphocyte infiltration and LN expansion. Icam1 is controlled by binding of the core circadian clock transcription factor BMAL1 to its promoter region, and rhythmic Icam1 expression is lost in mice lacking endothelial cell BMAL1. LN cellularity continues to be different for weeks after the initial time‐of‐day‐dependent challenge, which determines the immune response to vaccinations directed against Hepatitis A virus as well as SARS‐CoV‐2, as evidenced by rhythmic T cell reactions, germinal center formation and antibody production. Our results provide the mechanistic understanding of the time‐of‐day dependent development and maintenance of an adaptive immune response, demonstrating its dependency upon the timing of the initial challenge and the interactions of rhythmicity in multiple parameters. This provides a strategy for using time‐of‐day to optimize vaccination regimes.
Metformin Exposure in uteroProgrammes Hypertension in a Sex‐Specific Manner in Adult Offspring of Obese Mice
Authors: Wilsmore Phoebe , Schoonejans Josca , Ashmore Thomas , Blackmore Heather , Fernandez‐Twinn Denise , Giussani Dino , Ozanne Susan
Published: May 2022

Introduction

Metformin is the first‐line pharmacological treatment for gestational diabetes in many countries. Although effective at improving maternal glycaemia, metformin readily crosses the placenta and could directly affect the fetus. Indeed, metformin exposure in utero increases postnatal adiposity. However, other long‐term effects on offspring exposed to metformin in utero have not been investigated. Here we explored the impact of maternal metformin treatment during obese, glucose‐intolerant pregnancy on offspring cardiovascular function at one year of age using an established mouse model of maternal diet‐induced obesity.

Methods

Female C57Bl/6J mice were fed either a standard chow diet [Control dams (Con)] or an obesogenic diet [Obese (Ob) and Obese‐Metformin (Ob‐Met) dams] from weaning up to and including pregnancy and lactation. Ob‐Met dams received a clinically relevant dose of metformin orally from one week prior to mating until day 19 of gestation. All offspring were weaned onto standard chow fed ad libitum at three weeks of age. At one year of age, systolic blood pressure (SBP) was measured by tail cuff plethysmography in the conscious animal and echocardiography was performed under anaesthesia. Post‐mortem, femoral arteries were isolated and vasomotor reactivity tested using in vitro wire myography. Myography dose‐response curves were analysed using mixed effects models, all other data were analysed by One‐Way ANOVA. Post‐hoc multiple comparisons were performed where appropriate. Significance was accepted when P<0.05.

Results

Relative to controls, female offspring of Ob‐Met dams had increased SBP. However, no differences in SBP were observed in male offspring (Fig.1A). Female offspring of both Ob and Ob‐Met dams had enhanced femoral vasoconstrictor reactivity to K+ (Fig. 1B) but normal cardiac function (Fig. 1C). In contrast, male offspring of both Ob and Ob‐Met dams showed indices of diastolic dysfunction (Fig. 1C) and male offspring of Ob‐Met dams had enhanced femoral vasoconstrictor reactivity to phenylephrine (PE) (Fig.1B).

Conclusions

Maternal obesity and metformin exposure in utero caused sex‐specific changes in cardiovascular function in 12‐month‐old offspring. In female offspring, exposure to maternal obesity and metformin programmed hypertension with enhanced peripheral vasoconstrictor reactivity. In male offspring, maternal obesity alone programmed echocardiographic indices of left ventricular restrictive filling which were not corrected by metformin treatment, while combined exposure to maternal obesity and metformin also programmed enhanced peripheral vasoconstrictor reactivity. These findings provide novel insight into the long‐term effects of in utero metformin exposure on the cardiovascular health of the next generation in obese pregnancy, and highlight the importance of considering offspring sex in developmental programming studies.
Parental Obesity Alters Offspring Blood Pressure Regulation and Cardiovascular Responses to Stress: Role of P2X7R and Sex Differences
Authors: do Carmo Jussara M. , Moak Sydney P. , Dai Xuemei , Borges Gisele C. , Omoto Ana C. M. , Wang Zhen , Li Xuan , Mouton Alan J. , Hall John E. , da Silva Alexandre A.
Published: May 2022

The prevalence of obesity has increased dramatically in the United States during the past three decades. This increase has occurred in children, adolescents and adults of both sexes and all ethnic groups. In addition, the proportion of women and men of reproductive age who are overweight and obese has been increasing considerably, and maternal obesity and excessive gestational weight gain may predispose their offspring to health‐related consequences during childhood and adulthood, and increase cardiovascular responses to stress. Therefore, we examined the impact of parental (maternal + paternal) obesity on offspring blood pressure (BP) regulation and cardiovascular responses to stress. Offspring from normal (N) diet‐fed C57BL/6J parents were fed either N (NN, n=5 per sex) or a high fat (H) diet (NH, n=5‐7 per sex) from weaning until adulthood. Offspring from obese H diet‐fed parents were also fed N (HN, n=5‐6 per sex) or H diet (HH, n=5‐8 per sex). Body weight, calorie intake and fat mass were measured at 22 weeks of age when cardiovascular phenotyping was performed. Male and female HH offspring were 15% heavier than NH and 70% heavier than NN offspring. Male HH and HN offspring had elevated BP (121±2 and 115±1 mmHg, by telemetry) compared to male NH and NN offspring (108±6 and 107±3 mmHg, respectively) and augmented BP response to angiotensin II, losartan and hexamethonium. Male HH and HN offspring also showed increased BP response to acute air‐jet stress (37±2 and 38±2 mmHg) compared to only 24±3 and 25±3 mmHg in NH and NN offspring. Baseline heart rate (HR) and HR responses to air‐jet stress were similar among groups. In females, BP and cardiovascular responses to stress were similar among all offspring. Male H diet‐fed offspring from obese H diet‐fed purinoreceptor 7 deficient (HH‐P2X7R‐KO) parents had normal BP that was similar to control NN‐P2X7R‐KO offspring from lean parents. These results indicate that parental obesity leads to increased BP and augmented BP response to stress in their offspring in a sex‐dependent manner and that the impact of parental obesity on male offspring BP regulation is markedly attenuated in P2X7R‐KO mice.
P2Y2 Receptor Upregulation and Signaling during Adipogenesis and Inflammation: a New Mechanism in Insulin Resistance
Authors: Qian Shenqi , Shi Yi , Peng Qianman , Senfeld Jared , Shen Jianzhong
Published: May 2022

It has been known that obesity is associated with inflammation and insulin resistance and that adipose tissue contributes to insulin sensitivity and glucose homeostasis; however, the exact mechanism(s) of insulin resistance in adipose tissue remains incompletely understood. We hypothesized that inflammation promotes adipocyte P2Y2 receptor (P2Y2R) induction, leading to insulin resistance by inhibiting the insulin‐AKT signaling pathway. Real‐time RT‐PCR revealed that the P2Y2R mRNA was significantly upregulated during human visceral pre‐adipocytes differentiation into mature adipocytes in vitro. Stimulation of the mature adipocytes by TNFα further increased P2Y2R mRNA expression. Functional P2Y2R upregulation was confirmed by intracellular Ca2+ mobilization assay in response to ATP or UTP stimulation in mature human visceral adipocytes. In addition, stimulation of the P2Y2R by ATP or UTP suppressed basal and insulin‐induced phosphorylation of AKT in a dose‐dependent manner in mature adipocytes. These effects were significantly diminished by AR‐C118925, a selective P2Y2R antagonist, suggesting a negative role of P2Y2R in insulin receptor signaling. Furthermore, we found that activation of the P2Y2R inhibited insulin‐induced glucose uptake in mature adipocytes. We concluded that the P2Y2R is upregulated during adipocyte differentiation and inflammation and that P2Y2R activation leads to insulin resistance by suppressing the AKT signaling pathway, highlighting P2Y2R being a potential new drug target to combat obesity and type‐II diabetes.
Dysregulation of Skeletal Muscle Mitochondrial Turnover and Dynamics Occurs During Late Symptomatic Stages of Spinal Muscular Atrophy
Authors: Mikhail Andrew I. , Ng Sean Y. , Osborne Aislin K. , Canteenwala Abbas K. , Ljubicic Vladimir
Published: May 2022

Spinal muscular atrophy (SMA) is a debilitating neuromuscular disorder caused by a mutation in the survival motor neuron 1 (SMN1) gene and it is the leading genetic cause of infant mortality. Recently approved genetic therapies designed to augment full length SMN protein in the central nervous system fail to ameliorate abnormal skeletal muscle features, which strongly suggests an important role for SMN protein in skeletal muscle homeostasis. This study aimed to characterize key modifiers of skeletal muscle mitochondrial turnover and dynamics during disease progression in a pre‐clinical murine model of SMA in vivo. Additionally, we investigated the effects of a single dose of exercise on these mitochondrial biology‐regulating pathways in the skeletal muscle of SMA mice. Muscle samples were collected from wild‐type (WT) and Smn2B/‐ (SMA) mice at postnatal day 9 (P9), P13, and P21 to examine skeletal muscle‐specific disease progression. Mitochondrial content did not differ between genotypes at all timepoints as evident by similar levels of mitochondrial oxidative phosphorylation proteins, succinate dehydrogenase staining, and citrate synthase content. However, mRNA content of key genes responsible for regulating mitochondrial quality such as nuclear respiratory factor 2, mitochondrial transcription factor A, and p53 were significantly higher in SMA mice versus WT at P21. Additionally, we observed elevated mitochondrial fission activity as indicated by a 2‐fold increase (p < 0.05) in dynamin related protein 1 (DRP1) activation. Concomitantly, the expression of several mitophagy proteins including BLC2 interacting protein 3, parkin, and PTEN‐induced kinase 1 were significantly increased by 2.9‐, 2.7‐, and 2‐fold, respectively, compared to WT animals at P21. Interestingly, we observed blunted (p < 0.05) inclusion of optic atrophy 1 exon 4b, a key exon in mitochondrial biogenesis, between WT and SMA mice at P21. Acute exercise significantly increased the inhibition of DRP1‐mediated mitochondrial fission activity in the muscles of SMA mice relative to sedentary SMA mice. However, a single exercise dose failed to alter the expression of mitophagy proteins up to three hours after running. Our data demonstrate that skeletal muscle mitochondrial health is compromised in SMA mice due to elevated fission and mitophagy processes. Furthermore, SMA mice display aberrant alternative splicing of Opa1 transcripts that may in turn hinder mitochondrial biogenesis in later disease stages. We also highlight that an acute bout of treadmill running elicits pro‐fusion signaling to potentially improve the mitochondrial reticulum. Collectively, this study is the first to reveal mitochondrial dysfunction in SMA skeletal muscle and outlines signalling associated with mitochondrial plasticity following a single dose of exercise.
Acute Intermittent Hypoxia Improves Orthostatic Tolerance in Chronic but not Acute Spinal Cord Injured Rats
Authors: Wainman Liisa , Ahmadian Mehdi , Foster Glen , Ainslie Philip , Rodrigues Perim Raphael , Mitchell Gordon , West Christopher
Published: May 2022

Spinal cord injury (SCI) causes hypotension and orthostatic intolerance due to disrupted sympathetic input to the heart and vasculature following injury1. A neuro‐therapeutic strategy shown to increase somatic motor function below the injury is acute intermittent hypoxia (AIH). While AIH is known to improve control over respiratory and non‐respiratory motor systems with incomplete cervical SCI2,3, cardio‐autonomic responses to AIH after SCI are unknown. In able‐bodied individuals, AIH increases blood pressure (BP), improves orthostatic tolerance (OT), and heightens sympathetic activity4. In incomplete SCI, intact neural pathways regulating sympathetic nerve activity suggest the potential for AIH to increase sub‐lesional sympathetic activity, thus improving cardiovascular function. Previous work has demonstrated that the response to AIH is greatest in rats at least 4 weeks post‐injury due to reestablishment of serotonergic pathways2, therefore the purpose of this project is to investigate whether AIH improves OT in rats following acute (2 weeks) and/or chronic (4 weeks) high‐thoracic SCI. We believe AIH will improve OT more in chronically versus acutely injured rats.

12 Wistar rats underwent T3 300 kdyn SCI. At 14 (n=6) or 28 (n=6) days post‐SCI rats were tracheotomized, ventilated and arterial catheterization was performed to assess BP. Rats were subjected to lower body negative pressure (LBNP; recorded with manometer) to decrease mean arterial pressure (MAP) by 5 and 10 mmHg for 1 min. Rats were exposed to isocapnic AIH (10 x 1‐min FIO2=0.10 separated by 2 min of FIO2=1.00; targeting a PaCO2 of 40 mmHg). LBNP was repeated 90 minutes post‐AIH, the expected peak response. Blood gases were measured pre‐ and post‐AIH. A mixed‐design ANOVA was performed to examine effects of time post‐SCI (group) and AIH (treatment) on change in box pressure required to decrease MAP by ‐5 and ‐10 mmHg.

There was a significant group*treatment interaction for the ‐5 mmHg (p=0.011) and ‐10 mmHg LBNP stage (p=0.017). Post hoc comparisons demonstrate that in chronic SCI the box pressure required to drop MAP by ‐5 and ‐10mmHg was increased by 41±16% (p=0.009) and 46±22% (p=0.079) versus pre‐AIH, respectively, implying improved OT (Fig 1).

We demonstrate that AIH has the capacity to improve OT in a rodent model of SCI in a chronic, but not acute setting.

References

1. DeVeau et al. Autonomic Neuroscience(2018)

2. Golder & Mitchell Journal of Neuroscience (2005)

3. Prosser‐Loose et al. Journal of Neurotrauma (2015)

4. Stuckless et al. Journal of Physiology (2020)
Sleep Disruption Exacerbates Cognitive Dysfunction by Promoting Lipid‐Immune Dysregulation in the Corticohippocampal System of Pentylenetetrazol‐Kindled Rats
Authors: Edem Edem E. , Nebo Kate E. , Rimamchatin Peculiar , Bello Aisha , Akinluyi Elizabeth , Fafure Adedamola , Adeoluwa Olusegun , Adekeye Adesina , Oremosu Ademola
Published: May 2022

Sleep deprivation has been identified as one of the triggers for seizures in patients with generalised epilepsy. Therefore, we tested whether rapid eye movement (REM) sleep deprivation would increase susceptibility to developing generalised seizures established by pentylenetetrazol (PTZ) kindling; and whether REM sleep deprivation would affect treatment response and cognitive outcomes in epileptic rats. Furthermore, possible mechanistic underpinnings such as lipid‐immune responses in the prefrontal cortex and hippocampus were evaluated. Male rats were either sleep‐deprived followed by PTZ kindling, or kindled before being sleep‐deprived, or kindled before sleep deprivation and treated with a known anticonvulsant, diazepam. Thereafter, cognitive behaviours were evaluated. Following euthanasia, the prefrontal cortex and hippocampus were extracted and processed for biochemical and histological/histochemical/immunohistochemical assessments. Our results showed increased susceptibility to epileptogenesis in the group exposed to sleep deprivation before kindling compared to the PTZ‐kindling group without sleep deprivation. Also, sleep deprivation aggravated epilepsy features in the sleep‐deprived PTZ‐kindled rats and negatively impacted drug (diazepam) response in these animals. These were all linked to a disruption in lipid profile, and interleukin‐17 (IL‐17) activity; exacerbation in demyelination, microglial activation, and low‐density lipoprotein receptor‐related protein 1 (LRP1) expression in the prefrontal cortex and hippocampus following sleep deprivation and PTZ kindling. Furthermore, we observed a substantial deterioration in spatial working memory especially in the sleep‐deprived PTZ‐kindled group compared to the kindled group. Overall, our findings suggest that impairment of cognitive functions as seen in the sleep deprivation before kindling and sleep deprivation after kindling groups is attributable to a perturbation in the interaction between lipoproteins, their receptors, immune cells and their inflammatory/phagocytic responses impairing myelination/remyelination, thus aggravating cognitive deficits.