A novel lid-covering peptide inhibitor of nicotinic acetylcholine receptors derived from αd-conotoxin GeXXA

Nicotinic acetylcholine receptors (nAChRs) play a fundamental role in nervous signal transmission, therefore various antagonists and agonists are highly desired to explore the structure and function of nAChRs. Recently, a novel dimeric αD-conotoxin GeXXA was identified to inhibit nAChRs by binding at the top surface of the receptors, and the monomeric C-terminal domain (CTD) of αD-GeXXA retains some inhibitory activity. In this study, the internal dimeric N-terminal domain (NTD) of this conopeptide was further investigated. We first developed a regio-selective protection strategy to chemically prepare the anti-parallel dimeric NTD, and found that the isolated NTD part of GeXXA possesses the nAChR-inhibitory activity, the subtype-dependence of which implies a preferred binding of NTD to the β subunits of nAChR. Deletion of the NTD N-terminal residues did not affect the activity of NTD, indicating that the N-terminus is not involved in the interaction with nAChRs. By optimizing the sequence of NTD, we obtained a fully active single-chain cyclic NTD, based on which 4 Arg residues were found to interact with nAChRs. These results demonstrate that the NTD part of αD-GeXXA is a lid-covering nAChR inhibitor, displaying a novel inhibitory mechanism distinct from other allosteric ligands of nAChRs

Aberrantly hydroxymethylated differentially expressed genes and the associated protein pathways in osteoarthritis

Background The elderly population is at risk of osteoarthritis (OA), a common, multifactorial, degenerative joint disease. Environmental, genetic, and epigenetic (such as DNA hydroxymethylation) factors may be involved in the etiology, development, and pathogenesis of OA. Here, comprehensive bioinformatic analyses were used to identify aberrantly hydroxymethylated differentially expressed genes and pathways in osteoarthritis to determine the underlying molecular mechanisms of osteoarthritis and susceptibility-related genes for osteoarthritis inheritance. Methods Gene expression microarray data, mRNA expression profile data, and a whole genome 5hmC dataset were obtained from the Gene Expression Omnibus repository. Differentially expressed genes with abnormal hydroxymethylation were identified by MATCH function. Gene ontology and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses of the genes differentially expressed in OA were performed using Metascape and the KOBAS online tool, respectively. The protein–protein interaction network was built using STRING and visualized in Cytoscape, and the modular analysis of the network was performed using the Molecular Complex Detection app. Results In total, 104 hyperhydroxymethylated highly expressed genes and 14 hypohydroxymethylated genes with low expression were identified. Gene ontology analyses indicated that the biological functions of hyperhydroxymethylated highly expressed genes included skeletal system development, ossification, and bone development; KEGG pathway analysis showed enrichment in protein digestion and absorption, extracellular matrix–receptor interaction, and focal adhesion. The top 10 hub genes in the protein–protein interaction network were COL1A1, COL1A2, COL2A1, COL3A1, COL5A1, COL5A2, COL6A1, COL8A1, COL11A1, and COL24A1. All the aforementioned results are consistent with changes observed in OA. Conclusion After comprehensive bioinformatics analysis, we found aberrantly hydroxymethylated differentially expressed genes and pathways in OA. The top 10 hub genes may be useful hydroxymethylation analysis biomarkers to provide more accurate OA diagnoses and target genes for treatment of OA

Filamentation of asparagine synthetase in Saccharomyces cerevisiae.

Asparagine synthetase (ASNS) and CTP synthase (CTPS) are two metabolic enzymes crucial for glutamine homeostasis. A genome-wide screening in Saccharomyces cerevisiae reveal that both ASNS and CTPS form filamentous structures termed cytoophidia. Although CTPS cytoophidia were well documented in recent years, the filamentation of ASNS is less studied. Using the budding yeast as a model system, here we confirm that two ASNS proteins, Asn1 and Asn2, are capable of forming cytoophidia in diauxic and stationary phases. We find that glucose deprivation induces ASNS filament formation. Although ASNS and CTPS form distinct cytoophidia with different lengths, both structures locate adjacently to each other in most cells. Moreover, we demonstrate that the Asn1 cytoophidia colocalize with the Asn2 cytoophidia, while Asn2 filament assembly is largely dependent on Asn1. In addition, we are able to alter Asn1 filamentation by mutagenizing key sites on the dimer interface. Finally, we show that ASN1D330V promotes filamentation. The ASN1D330V mutation impedes cell growth in an ASN2 knockout background, while growing normally in an ASN2 wild-type background. Together, this study reveals a connection between ASNS and CTPS cytoophidia and the differential filament-forming capability between two ASNS paralogs

Association between combination COVID-19-influenza vaccination and long COVID in middle-aged and older Europeans: A cross-sectional study

ABSTRACTThe potential impact of combined COVID-19 and influenza vaccination on long COVID remains uncertain. In the present cross-sectional study, we aimed to investigate the plausible association between them in middle-aged and older Europeans based on the Survey of Health, Ageing, and Retirement in Europe (SHARE). A total of 1910 participants were recruited in the analyses. The study outcome was long COVID. Participants were divided into 4 groups through the self-reported status of COVID-19 and influenza vaccination. Odds ratios (ORs) and 95% confidence intervals (CIs) were estimated. 1397 participants experienced long COVID. After multivariable adjustment, those vaccinated with neither COVID-19 nor influenza vaccine had higher risk of long COVID (OR, 1.72; 95% CI, 1.26–2.35) compared to those vaccinated with both vaccines. Furthermore, adding the 4 statuses of COVID-19 vaccination/influenza vaccination to conventional risk model improved risk reclassification for long COVID (continuous net reclassification improvement was 16.26% [p = .003], and integrated discrimination improvement was 0.51% [p = .005]). No heterogeneity was found in the subgroup analyses (all p-interaction ≥0.05). Our study might provide a strategy for people aged 50 and over to reduce the occurrence of long COVID, that is, to combine the use of the COVID-19 vaccine and influenza vaccines

Orai–vascular endothelial-cadherin signaling complex regulates high-glucose exposure-induced increased permeability of mouse aortic endothelial cells

Introduction Diabetes-associated endothelial barrier function impairment might be linked to disturbances in Ca2+ homeostasis. To study the role and molecular mechanism of Orais–vascular endothelial (VE)-cadherin signaling complex and its downstream signaling pathway in diabetic endothelial injury using mouse aortic endothelial cells (MAECs).Research design and methods The activity of store-operated Ca2+ entry (SOCE) was detected by calcium imaging after 7 days of high-glucose (HG) or normal-glucose (NG) exposure, the expression levels of Orais after HG treatment was detected by western blot analysis. The effect of HG exposure on the expression of phosphorylated (p)-VE-cadherin and VE-cadherin on cell membrane was observed by immunofluorescence assay. HG-induced transendothelial electrical resistance was examined in vitro after MAECs were cultured in HG medium. FD-20 permeability was tested in monolayer aortic endothelial cells through transwell permeability assay. The interactions between Orais and VE-cadherin were detected by co-immunoprecipitation and immunofluorescence technologies. Immunohistochemical experiment was used to detect the expression changes of Orais, VE-cadherin and p-VE-cadherin in aortic endothelium of mice with diabetes.Results (1) The expression levels of Orais and activity of SOCE were significantly increased in MAECs cultured in HG for 7 days. (2) In MAECs cultured in HG for 7 days, the ratio of p-VE-cadherin to VE-cadherin expressed on the cell membrane and the FD-20 permeability in monolayer endothelial cells increased, indicating that intercellular permeability increased. (3) Orais and VE-cadherin can interact and enhance the interaction ratio through HG stimulation. (4) In MAECs cultured with HG, the SOCE activator ATP enhanced the expression level of p-VE-cadherin, and the SOCE inhibitor BTP2 decreased the expression level of p-VE-cadherin. (5) Significantly increased expression of p-VE-cadherin and Orais in the aortic endothelium of mice with diabetes.Conclusion HG exposure stimulated increased expression of Orais in endothelial cells, and increased VE-cadherin phosphorylation through Orais–VE-cadherin complex and a series of downstream signaling pathways, resulting in disruption of endothelial cell junctions and initiation of atherosclerosis

Physical interaction of tropomyosin 3 and STIM1 regulates vascular smooth muscle contractility and contributes to hypertension

Scope: Tropomyosin (TPM), an actin-binding protein widely expressed across different cell types, is primarily involved in cellular contractile processes. We investigated whether TPM3 physically and functionally interacts with stromal interaction molecule 1 (STIM1) to contribute to vascular smooth muscle cell (VSMC) contraction, store-operated calcium entry (SOCE), and high-salt intake–induced hypertension in rats. Methods and results: Analysis of a rat RNA-seq data set of 80 samples showed that the STIM1 and Tpm3 transcriptome expression pattern is highly correlated, and co-immunoprecipitation results indicated that TPM3 and STIM1 proteins physically interacted in rat VSMCs. Immunohistochemical data displayed obvious co-localization of TPM3 and STIM1 in rat VSMCs. Knockdown of TPM3 or STIM1 in VSMCs with specific small interfering RNA significantly suppressed contractions in tension measurement assays and decreased SOCE in calcium assays. Rats fed a high-salt diet for 4 weeks had significantly higher systolic blood pressure than controls, with significantly increased contractility and markedly increased TPM3 and STIM1 expression levels in the mesenteric resistance artery (shown by tension measurements and immunoblotting, respectively). Additionally, high salt environment in vitro induced significant enhancement of TPM3 and STIM1 expression levels in VSMCs. Conclusions: We showed for the first time that TPM3 and STIM1 physically and functionally interact to contribute to VSMC contraction, SOCE, and high-salt intake–induced hypertension. Our findings provide mechanistic insights and offer a potential therapeutic target for high-salt intake–induced hypertension

Large-scale material extrusion-based additive manufacturing of short carbon fibre-reinforced silicon carbide ceramic matrix composite preforms

Large-scale short carbon fibre-reinforced silicon carbide (Csf/SiC) ceramic matrix composites (CMCs) have important applications in the field of aerospace engineering. This study proposed the use of material extrusion based additive manufacturing to fabricate large-scale Csf/SiC CMC preforms. In this paper, we determined how the key material extrusion parameters, including solid loading, nozzle diameter and layer height impact the stability of the additively manufactured Csf/SiC CMCs. The solid loading significantly influenced the stability of the Csf/SiC CMCs, and the slurry with 50 vol.% solid loading was better for additive manufacturing. The layer height played a significant role in the void formation in CMCs. It was appropriate for structure retention to set the layer height as 60–75% of the nozzle diameter. The effect of angle from vertical on the stability of out-of-plane structure was also investigated. When the angle was over 40o, the out-of-plane structure additively manufactured without supports tended to collapse. Large-scale Csf/SiC CMC preforms with out-of-plane structures were finally successfully fabricated. This study is believed to provide some fundamental understanding for the fabrication of large-scale fibre-reinforced ceramic matrix composites

Plasma Membrane Mechanical Stress Activates TRPC5 Channels

<div><p>Mechanical forces exerted on cells impose stress on the plasma membrane. Cells sense this stress and elicit a mechanoelectric transduction cascade that initiates compensatory mechanisms. Mechanosensitive ion channels in the plasma membrane are responsible for transducing the mechanical signals to electrical signals. However, the mechanisms underlying channel activation in response to mechanical stress remain incompletely understood. Transient Receptor Potential (TRP) channels serve essential functions in several sensory modalities. These channels can also participate in mechanotransduction by either being autonomously sensitive to mechanical perturbation or by coupling to other mechanosensory components of the cell. Here, we investigated the response of a TRP family member, TRPC5, to mechanical stress. Hypoosmolarity triggers Ca²⁺ influx and cationic conductance through TRPC5. Importantly, for the first time we were able to record the stretch-activated TRPC5 current at single-channel level. The activation threshold for TRPC5 was found to be 240 mOsm for hypoosmotic stress and between −20 and −40 mmHg for pressure applied to membrane patch. In addition, we found that disruption of actin filaments suppresses TRPC5 response to hypoosmotic stress and patch pipette pressure, but does not prevent the activation of TRPC5 by stretch-independent mechanisms, indicating that actin cytoskeleton is an essential transduction component that confers mechanosensitivity to TRPC5. In summary, our findings establish that TRPC5 can be activated at the single-channel level when mechanical stress on the cell reaches a certain threshold.</p></div

Pipette pressure activates TRPC5 on single-channel membrane patch.

<p>(A) schematic diagram depicting single-channel current measurement in TRPC5-expressing CHO-K1 cells. Membrane stretch was elicited by applying suction through the patch pipette, as indicated by <i>red arrow</i>. (B) a representative cell-attached recording (n = 11) of TRPC5-expressing CHO-K1 cell showing changes in channel activity when -40 mmHg pipette pressure was applied (<i>suction</i>) and subsequently released (<i>release</i>). The pipette holding potential was -60 mV. (C) representative traces showing single-channel activities in vector transfected (<i>Vector</i>) and TRPC5-expressing (<i>TRPC5</i>) CHO-K1 cells at 0 mmHg and -40 mmHg pipette pressure with cell-attached configuration. Channel activities were also recorded in TRPC5-expressing cells pretreated with 10 μM BAPTA-AM to buffer cytosolic Ca²⁺ fluctuation (<i>TRPC5+BAPTA-AM</i>). The pipette holding potentials were -60 mV. (D) channel open probability (NPo) values over time for the stretch-activated channel under <i>0 mmHg</i> and <i>-40 mmHg</i>. Shown are analyses from the same cell-attached patch applied with the indicated pipette pressures for 90 seconds. (E) quantification of the single-channel open probabilities (NPo) of the channel activities recorded on TRPC5-expressing CHO-K1 cells as in (C). (F) single-channel <i>I-V</i> relationships of the stretch-activated channels in cell-attached configuration. The bath solution was 130 mM K⁺ solution (<i>High K</i>^<i>+</i>, <i>red circle</i>) for dissipating the membrane potential. The slope conductance is 39 ± 2 pS. (G) schematic diagram showing the two-step backfilling protocol. Patch pipettes were backfilled with T5E3 (15 μg/ml) using a two-step protocol. T5E3 eventually diffused to pipette tip to inhibit TRPC5. Only the patched membrane is depicted for simplicity. Recordings were performed with cell-attached configuration. (H) representative traces showing the stretch (-40 mmHg)-activated channel in the presence of preimmune IgG (15 μg/ml) or T5E3 (15 μg/ml) immediately (<i>0 min</i>) and 10 minutes (<i>10 min</i>) after gigaseal formation. The pipette holding potentials were -60 mV. (I) summary of single-channel open probabilities (NPo) as in (H).</p