All SNAP25 molecules in the vesicle-plasma membrane contact zone change conformation during vesicle priming.

In neuronal cell types, vesicular exocytosis is governed by the SNARE (soluble NSF attachment receptor) complex consisting of synaptobrevin2, SNAP25, and syntaxin1. These proteins are required for vesicle priming and fusion. We generated an improved SNAP25-based SNARE COmplex Reporter (SCORE2) incorporating mCeruelan3 and Venus and overexpressed it in SNAP25 knockout embryonic mouse chromaffin cells. This construct rescues vesicle fusion with properties indistinguishable from fusion in wild-type cells. Combining electrochemical imaging of individual release events using electrochemical detector arrays with total internal reflection fluorescence resonance energy transfer (TIR-FRET) imaging reveals a rapid FRET increase preceding individual fusion events by 65 ms. The experiments are performed under conditions of a steady-state cycle of docking, priming, and fusion, and the delay suggests that the FRET change reflects tight docking and priming of the vesicle, followed by fusion after ~65 ms. Given the absence of wt SNAP25, SCORE2 allows determination of the number of molecules at fusion sites and the number that changes conformation. The number of SNAP25 molecules changing conformation in the priming step increases with vesicle size and SNAP25 density in the plasma membrane and equals the number of copies present in the vesicle-plasma membrane contact zone. We estimate that in wt cells, 6 to 7 copies of SNAP25 change conformation during the priming step.

Single-cell RNA sequencing reveals the transcriptional heterogeneity of Tbx18-positive cardiac cells during heart development.

The T-box family transcription factor 18 (Tbx18) has been found to play a critical role in regulating the development of the mammalian heart during the primary stages of embryonic development while the cellular heterogeneity and landscape of Tbx18-positive (Tbx18+) cardiac cells remain incompletely characterized. Here, we analyzed prior published single-cell RNA sequencing (scRNA-seq) mouse heart data to explore the heterogeneity of Tbx18+ cardiac cell subpopulations and provide a comprehensive transcriptional landscape of Tbx18+ cardiac cells during their development. Bioinformatic analysis methods were utilized to identify the heterogeneity between cell groups. Based on the gene expression characteristics, Tbx18+ cardiac cells can be classified into a minimum of two distinct cell populations, namely fibroblast-like cells and cardiomyocytes. In terms of temporal heterogeneity, these cells exhibit three developmental stages, namely the MEM stage, ML_P0 stage, and P stage Tbx18+ cardiac cells. Furthermore, Tbx18+ cardiac cells encompass several cell types, including cardiac progenitor-like cells, cardiomyocytes, and epicardial/stromal cells, as determined by specific transcriptional regulatory networks. The scRNA-seq results revealed the involvement of extracellular matrix (ECM) signals and epicardial epithelial-to-mesenchymal transition (EMT) in the development of Tbx18+ cardiac cells. The utilization of a lineage-tracing model served to validate the crucial function of Tbx18 in the differentiation of cardiac cells. Consequently, these findings offer a comprehensive depiction of the cellular heterogeneity within Tbx18+ cardiac cells.

Aberrant Activation of Immune and Non-Immune Cells Contributes to Joint Inflammation and Bone Degradation in Rheumatoid Arthritis.

Abnormal activation of multiple immune and non-immune cells and proinflammatory factors mediate the development of joint inflammation in genetically susceptible individuals. Although specific environmental factors like smoking and infections are associated with disease pathogenesis, until now, we did not know the autoantigens and arthritogenic factors that trigger the initiation of the clinical disease. Autoantibodies recognizing specific post-translationally modified and unmodified antigens are generated and in circulation before the onset of the joint disease, and could serve as diagnostic and prognostic markers. The characteristic features of autoantibodies change regarding sub-class, affinity, glycosylation pattern, and epitope spreading before the disease onset. Some of these antibodies were proven to be pathogenic using animal and cell-culture models. However, not all of them can induce disease in animals. This review discusses the aberrant activation of major immune and non-immune cells contributing to joint inflammation. Recent studies explored the protective effects of extracellular vesicles from mesenchymal stem cells and bacteria on joints by targeting specific cells and pathways. Current therapeutics in clinics target cells and inflammatory pathways to attenuate joint inflammation and protect the cartilage and bones from degradation, but none cure the disease. Hence, more basic research is needed to investigate the triggers and mechanisms involved in initiating the disease and relapses to prevent chronic inflammation from damaging joint architecture.

Molecular and Cellular Pathways Contributing to Joint Damage in Rheumatoid Arthritis.

Rheumatoid arthritis is a chronic autoimmune syndrome associated with several genetic, epigenetic, and environmental factors affecting the articular joints contributing to cartilage and bone damage. Although etiology of this disease is not clear, several immune pathways, involving immune (T cells, B cells, dendritic cells, macrophages, and neutrophils) and nonimmune (fibroblasts and chondrocytes) cells, participate in the secretion of many proinflammatory cytokines, chemokines, proteases (MMPs, ADAMTS), and other matrix lysing enzymes that could disturb the immune balance leading to cartilage and bone damage. The presence of autoantibodies preceding the clinical onset of arthritis and the induction of bone erosion early in the disease course clearly suggest that initiation events damaging the cartilage and bone start very early during the autoimmune phase of the arthritis development. During this process, several signaling molecules (RANKL-RANK, NF-κB, MAPK, NFATc1, and Src kinase) are activated in the osteoclasts, cells responsible for bone resorption. Hence, comprehensive knowledge on pathogenesis is a prerequisite for prevention and development of targeted clinical treatment for RA patients that can restore the immune balance improving clinical therapy.

Precise Time Superresolution by Event Correlation Microscopy.

Fluorescence imaging is often used to monitor dynamic cellular functions under conditions of very low light intensities to avoid photodamage to the cell and rapid photobleaching. Determination of the time of a fluorescence change relative to a rapid high time-resolution event, such as an action potential or pulse stimulation, is challenged by the low photon rate and the need to use imaging frame durations that limit the time resolution. To overcome these limitations, we developed a time superresolution method named event correlation microscopy that aligns repetitive events with respect to the high time-resolution events. We describe the algorithm of the method, its step response function, and a theoretical, computational, and experimental analysis of its precision, providing guidelines for camera exposure time settings depending on imaging signal properties and camera parameters for optimal time resolution. We also demonstrate the utility of the method to recover rapid nonstepwise kinetics by deconvolution fits. The event correlation microscopy method provides time superresolution beyond the photon rate limit and imaging frame duration with well-defined precision.

Autoantibodies as Diagnostic Markers and Mediator of Joint Inflammation in Arthritis.

Rheumatoid arthritis is a systemic, polygenic, and multifactorial syndrome characterized by erosive polyarthritis, damage to joint architecture, and presence of autoantibodies against several self-structures in the serum and synovial fluid. These autoantibodies (anticitrullinated protein/peptide antibodies (ACPAs), rheumatoid factors (RF), anticollagen type II antibodies, antiglucose-6 phosphate isomerase antibodies, anticarbamylated protein antibodies, and antiacetylated protein antibodies) have different characteristics, diagnostic/prognostic value, and pathological significance in RA patients. Some of these antibodies are present in the patients' serum several years before the onset of clinical disease. Various genetic and environmental factors are associated with autoantibody production against different autoantigenic targets. Both the activating and inhibitory FcγRs and the activation of different complement cascades contribute to the downstream effector functions in the antibody-mediated disease pathology. Interplay between several molecules (cytokines, chemokines, proteases, and inflammatory mediators) culminates in causing damage to the articular cartilage and bones. In addition, autoantibodies are proven to be useful disease markers for RA, and different diagnostic tools are being developed for early diagnosis of the clinical disease. Recently, a direct link was proposed between the presence of autoantibodies and bone erosion as well as in the induction of pain. In this review, the diagnostic value of autoantibodies, their synthesis and function as a mediator of joint inflammation, and the significance of IgG-Fc glycosylation are discussed.

Hierarchical Morphology of Polymer Blend Films Induced by Convection-Driven Solvent Evaporation.

Homogeneous thin films of polymer blends with a desired morphology are necessary because of their applications in the fields such as optoelectronics, sensors, biomedicine, and so on. The frequently employed approach for the thin film preparation, spin coating is only able to achieve a homogeneous film for a small area because of the overwhelming spin-driven solvent evaporation with increased size. Here, a convection-guided morphology formation for polystyrene:poly(methyl methacrylate) blend films is reported. In situ observation shows that the morphology changed from homogeneous deposition with a scale less than 10 µm to a self-organized cellular pattern with a scale of more than 100 µm after the fluid flow is involved. Selective dissolution of the hierarchical films reveals that the cellular morphology is attributed to the flow-field-guided deposition of sequentially generated precipitates. The coupling of phase separation and fluid convection results in the hierarchical morphology that includes Voronoi cellular division as the primary structure and the detailed heterogeneous inner-cell features as the secondary structure. Isolated modulation of either micro- or mesoscale in the hierarchical morphology could be carried out via adjusting phase interaction or the convection disturbance correspondingly, providing a flexible and straightforward strategy to construct designed hierarchical structures for polymer thin films toward desired function or property.

Positively charged amino acids at the SNAP-25 C terminus determine fusion rates, fusion pore properties, and energetics of tight SNARE complex zippering.

SNAP-25 is a Q-SNARE protein mediating exocytosis of neurosecretory vesicles including chromaffin granules. Previous results with a SNAP-25 construct lacking the nine C terminal residues (SNAP-25Δ9) showed changed fusion pore properties (Fang et al., 2008), suggesting a model for fusion pore mechanics that couple C terminal zipping of the SNARE complex to the opening of the fusion pore. The deleted fragment contains the positively charged residues R198 and K201, adjacent to layers 7 and 8 of the SNARE complex. To determine how fusion pore conductance and dynamics depend on these residues, single exocytotic events in bovine chromaffin cells expressing R198Q, R198E, K201Q, or K201E mutants were investigated by carbon fiber amperometry and cell-attached patch capacitance measurements. Coarse grain molecular dynamics simulations revealed spontaneous transitions between a loose and tightly zippered state at the SNARE complex C terminus. The SNAP-25 K201Q mutant showed no changes compared with SNAP-25 wild-type. However, K201E, R198Q, and R198E displayed reduced release frequencies, slower release kinetics, and prolonged fusion pore duration that were correlated with reduced probability to engage in the tightly zippered state. The results show that the positively charged amino acids at the SNAP-25 C terminus promote tight SNARE complex zippering and are required for high release frequency and rapid release in individual fusion events.

Rapid structural change in synaptosomal-associated protein 25 (SNAP25) precedes the fusion of single vesicles with the plasma membrane in live chromaffin cells.

The SNARE complex consists of the three proteins synaptobrevin-2, syntaxin, and synaptosomal-associated protein 25 (SNAP25) and is thought to execute a large conformational change as it drives membrane fusion and exocytosis. The relation between changes in the SNARE complex and fusion pore opening is, however, still unknown. We report here a direct measurement relating a change in the SNARE complex to vesicle fusion on the millisecond time scale. In individual chromaffin cells, we tracked conformational changes in SNAP25 by total internal reflection fluorescence resonance energy transfer (FRET) microscopy while exocytotic catecholamine release from single vesicles was simultaneously recorded using a microfabricated electrochemical detector array. A local rapid and transient FRET change occurred precisely where individual vesicles released catecholamine. To overcome the low time resolution of the imaging frames needed to collect sufficient signal intensity, a method named event correlation microscopy was developed, which revealed that the FRET change was abrupt and preceded the opening of an exocytotic fusion pore by ∼90 ms. The FRET change correlated temporally with the opening of the fusion pore and not with its dilation.

How could SNARE proteins open a fusion pore?

The SNARE (Soluble NSF Attachment protein REceptor) complex, which in mammalian neurosecretory cells is composed of the proteins synaptobrevin 2 (also called VAMP2), syntaxin, and SNAP-25, plays a key role in vesicle fusion. In this review, we discuss the hypothesis that, in neurosecretory cells, fusion pore formation is directly accomplished by a conformational change in the SNARE complex via movement of the transmembrane domains.

Relationship between cathepsins and cardiovascular diseases: a Mendelian randomized study.

Background: Cardiovascular diseases (CVDs) are the leading age-related disorders worldwide, with their prevalence increasing annually. Cathepsins are protein-degrading enzymes essential for processes such as intracellular protein breakdown, apoptosis, and immune responses. Recent studies suggest a potential link between cathepsins and CVDs, yet the exact causal relationship remains to be elucidated. To address this, we propose using Mendelian randomization (MR) to explore the causal relationships between cathepsins and CVDs. Methods: We obtained single nucleotide polymorphism (SNP) data for cathepsins from the INTERVAL study, a publicly accessible genome-wide association study (GWAS) dataset. Outcome SNP data were sourced from seven distinct GWAS datasets, ensuring a comprehensive analysis across multiple cardiovascular outcomes. For MR analysis, we primarily employed the inverse variance weighted (IVW) method, known for its efficiency when all SNPs are valid instruments. This was supplemented by the weighted median and MR-Egger methods to provide robustness against potential violations of MR assumptions, such as pleiotropy. The IVW method offers precision and efficiency, the weighted median method adds robustness against invalid instruments, and the MR-Egger method helps identify and correct for pleiotropic biases. Cochran's Q test was utilized to assess heterogeneity, and sensitivity analyses were conducted using MR-PRESSO and the leave-one-out approach. Results: The strength of the associations between exposure and outcome was measured using odds ratios (ORs), and results were presented with 95% confidence intervals (CIs). The cathepsin E increases the risk of myocardial infarction (MI) (OR = 1.053%, 95% CI: 1.007-1.101, p = 0.024) and ischemic stroke (IS) (OR = 1.06%, 95% CI: 1.019-1.103, p = 0.004). Conversely, cathepsin L2 decreases the risk of chronic heart failure (CHF) (OR = 0.922%, 95% CI: 0.859-0.99, p = 0.025) and atrial fibrillation (AF) (OR = 0.956%, 95% CI: 0.918-0.996, p = 0.033). Cathepsin O was associated with an increased risk of IS (OR = 1.054%, 95% CI: 1.008-1.102, p = 0.021) and AF (OR = 1.058%, 95% CI: 1.02-1.098, p = 0.002). Conclusion: Our MR analysis reveals that cathepsin E is a risk factor for MI and IS, cathepsin L2 offers protective effects against CHF and AF, and cathepsin O increases the risk for IS and AF.

Role of the synaptobrevin C terminus in fusion pore formation.

Neurotransmitter release is mediated by the SNARE proteins synaptobrevin II (sybII, also known as VAMP2), syntaxin, and SNAP-25, generating a force transfer to the membranes and inducing fusion pore formation. However, the molecular mechanism by which this force leads to opening of a fusion pore remains elusive. Here we show that the ability of sybII to support exocytosis is inhibited by addition of one or two residues to the sybII C terminus depending on their energy of transfer from water to the membrane interface, following a Boltzmann distribution. These results suggest that following stimulation, the SNARE complex pulls the C terminus of sybII deeper into the vesicle membrane. We propose that this movement disrupts the vesicular membrane continuity leading to fusion pore formation. In contrast to current models, the experiments suggest that fusion pore formation begins with molecular rearrangements at the intravesicular membrane leaflet and not between the apposed cytoplasmic leaflets.

Androgen receptor nucleocytoplasmic trafficking - A one-way journey.

The androgen receptor (AR) is a key regulator of the growth and proliferation of prostate cancer. The majority of lethal castration-resistant prostate cancer (CRPC) growth is still dependent on AR activity. The AR need to be in the nucleus to exert its biological action as a transcription factor. As such, defining the mechanisms that regulate the subcellular localization of AR are important. Previously it was believed that AR was imported into the nucleus in a ligand-dependent manner and subsequently exported out of the nucleus upon ligand withdrawal. Recent evidence has challenged this decades-old paradigm and showed that the AR is degraded, not exported, in the nucleus. This review discusses the current understanding of how AR nucleocytoplasmic localization is regulated by import and through nuclear degradation.

Relationship between Parkinson's disease and cardio-cerebrovascular diseases: a Mendelian randomized study.

Parkinson's disease (PD) and cardio-cerebrovascular diseases are related, according to earlier studies, but these studies have some controversy. Our aim was to assess the impact of PD on cardiocerebrovascular diseases using a Mendelian randomization (MR) method. The data for PD were single nucleotide polymorphisms (SNPs) from a publicly available genome-wide association study (GWAS) dataset containing data on 482,730 individuals. And the outcome SNPs data is were derived from five different GWAS datasets. The basic method for MR analysis was the inverse variance weighted (IVW) approach. We use the weighted median method and the MR-Egger method to supplement the MR analysis conclusion. Finally, We used Cochran's Q test to test heterogeneity, MR-PRESSO method and leave-one-out analysis method to perform sensitivity analysis. We used ratio ratios (OR) to assess the strength of the association between exposure and outcome, and 95% confidence intervals (CI) to show the reliability of the results. Our findings imply that PD is linked to a higher occurrence of coronary artery disease (CAD) (OR = 1.055, 95% CI 1.020-1.091, P = 0.001), stroke (OR = 1.039, 95% CI 1.007-1.072, P = 0.014). IVW analyses for stroke's subgroups of ischemic stroke (IS) and 95% CI 1.007-1.072, P = 0.014). IVW analyses for stroke's subgroups of ischemic stroke (IS) and cardioembolic stroke (CES) also yielded positive results, respectively (OR = 1.043, 95% CI 1.008-1.079, P = 0.013), (OR = 1.076, 95% CI 1.008-1.149, P = 0.026). There is no evidence of a relationship between PD and other cardio-cerebrovascular diseases. Additionally, sensitivity analysis revealed reliable outcomes. Our MR study analysis that PD is related with an elevated risk of CAD, stroke, IS, and CES.

The Impact of Bariatric Surgery on Pancreatic Cancer Risk: a Systematic Review and Meta-Analysis.

A growing body of evidence suggests that bariatric surgery is associated with a reduced risk of some cancers. This meta-analysis aims to determine whether bariatric surgery affects pancreatic cancer risk. We conducted a comprehensive literature search of PubMed, Embase, and Web of Science databases. Fixed-effect models were used to estimate pooled data and presented as odds ratio (OR) and 95% confidence interval (CI). Heterogeneity was assessed using the Cochran Q test and I2 test. A total of 9 cohort studies involving 1,147,473 patients were included in the analysis. The pooled OR was 0.76 (95% CI = 0.64-0.90). The Cochran Q test and I2 test indicated only mild heterogeneity (P = 0.12, I2 = 38%). In the subgroup analyses, the pooled OR was 0.67 (95% CI = 0.54-0.82) for North America. In the subgroup analyses by mean follow-up time, the pooled OR was 0.46 (95% CI = 0.28-0.74) for less than 5 years. In conclusion, bariatric surgery has a positive effect on pancreatic cancer reduction, especially in North America. This effect may diminish or disappear with time.

Probing the role of the C2F domain of otoferlin.

Afferent synapses of cochlear inner hair cells (IHCs) employ a unique molecular machinery. Otoferlin is a key player in this machinery, and its genetic defects cause human auditory synaptopathy. We employed site-directed mutagenesis in mice to investigate the role of Ca2+ binding to the C2F domain of otoferlin. Substituting two aspartate residues of the C2F top loops, which are thought to coordinate Ca2+-ions, by alanines (OtofD1841/1842A) abolished Ca2+-influx-triggered IHC exocytosis and synchronous signaling in the auditory pathway despite substantial expression (~60%) of the mutant otoferlin in the basolateral IHC pole. Ca2+ influx of IHCs and their resting membrane capacitance, reflecting IHC size, as well as the number of IHC synapses were maintained. The mutant otoferlin showed a strong apex-to-base abundance gradient in IHCs, suggesting impaired protein targeting. Our results indicate a role of the C2F domain in otoferlin targeting and of Ca2+ binding by the C2F domain for IHC exocytosis and hearing.

RNA-Seq transcriptome analysis of renal tissue from spontaneously hypertensive rats revealed renal protective effects of dapagliflozin, an inhibitor of sodium-glucose cotransporter 2.

Hypertensive nephropathy (HTN) is a common complication of hypertension. Although various agents for treatment of hypertension exert significant effects, there is currently no effective treatment for hypertensive nephropathy. Sodium-glucose cotransporter 2 (SGLT2) inhibitors, such as dapagliflozin (DAPA), are a new class of hypoglycemic agents shown to improve the prognosis of patients with chronic kidney disease and diabetes mellitus. However, the mechanisms underlying the protective effects of DAPA remain unclear. RNA-sequencing (RNA-Seq)-based computational analysis was conducted to explore the transcriptomic changes to spontaneously hypertensive rats (SHRs) treated with DAPA for 8 weeks. Differentially expressed genes in SHRs were related to dysregulation of lipid metabolism, oxidation-reduction reaction, immunity and inflammation in HTN. Transcriptome analysis showed that 8 weeks of DAPA therapy exerted protective effects on the renal tissues of SHRs through the lysosomal, phagosomal, and autophagic pathways. VENN diagram analysis identified Zinc finger and BTB domain-containing 20 (Zbtb20) as the potential target of DAPA therapy. Consistent with the RNA-Seq findings, real-time quantitative PCR and immunohistochemical analyses revealed increased expression of Zbtb20 in the renal tissues of SHRs, whereas expression was decreased following 8 weeks of DAPA administration. The results of this study clarified the transcriptome signature of HTN and the beneficial effects of DAPA on renal tissues by alleviating dysregulation of metabolic processes and reducing inflammation.

Mechanisms and targeting of proteosome-dependent androgen receptor degradation in prostate cancer.

The androgen receptor (AR) remains to be a key target for the treatment of prostate cancer, including the majority of castration-resistant prostate cancer (CRPC). AR is stabilized in CRPC and the ubiquitin-proteasome system (UPS) plays a major role in AR degradation. Targeting AR for degradation provides a potential approach to overcome the resistance of CRPC to current AR antagonists, including the next generation AR signaling inhibitors. Different types of AR degraders have been developed, including the proteolysis-targeting chimeras (PROTACs), selective AR degraders (SARDs), and novel AR degraders, with several AR PROTACs currently in clinical trials. The present mini-review discusses the regulation of AR degradation by the UPS, the potential role of a novel nuclear degradation signal in AR, and different types of AR degraders.

Proteus mirabilis Vesicles Induce Mitochondrial Apoptosis by Regulating miR96-5p/Abca1 to Inhibit Osteoclastogenesis and Bone Loss.

Bone loss due to an increased osteoclast activity is common in osteoporosis and rheumatoid arthritis. For the first time, we observed an inhibition of osteoclast formation and bone resorption by outer-membrane vesicles (OMVs) from a Gram-negative, pathogenic bacterium, Proteus mirabilis (P.M). Gene ontogeny and KEGG enrichment analyses of miRNA and mRNA sequencing data demonstrated a significant effect of P.M OMVs on mitochondrial functions and apoptotic pathways. OMVs induced mitochondrial dysfunction through an increased level of intracellular ROS, collapse of mitochondrial membrane potential (ΔΨm), and modulation of Bax, Bcl-2, caspase-3, and cytochrome c expression. In addition, P.M OMVs strongly inhibited miR-96-5p expression, which caused an upregulation of ATP binding cassette subfamily A member 1 (Abca1) in osteoclasts leading to an increased level of mitochondria-dependent apoptosis. Moreover, treatment with P.M but not Escherichia coli OMVs attenuated bone loss in experimental osteoporosis and collagen-induced arthritis. Collectively, we demonstrated osteoprotective functions of OMVs from Proteus mirabilis, which downregulated miR-96-5p causing an increased Abca1 expression and mitochondria-dependent apoptosis.

Transcriptomic profile analysis of the left atrium in spontaneously hypertensive rats in the early stage.

Left atrial remodeling, characterized by enlargement and hypertrophy of the left atrium and increased fibrosis, was accompanied by an increased incidence of atrial fibrillation. While before morphological changes at the early stage of hypertension, how overloaded hypertension influences the transcriptomic profile of the left atrium remains unclear. Therefore, RNA-sequencing was performed to define the RNA expressing profiles of left atrium in spontaneously hypertensive rats (SHRs) and normotensive Wistar-Kyoto (WKY) rats as a control group. We also compared the changes in the RNA expression profiles in SHRs treated with an angiotensin receptor blocker (ARB) and angiotensin receptor-neprilysin inhibitor (ARNI) to assess the distinct effects on the left atrium. In total, 1,558 differentially expressed genes were found in the left atrium between WKY rats and SHRs. Bioinformatics analysis showed that these mRNAs could regulate upstream pathways in atrial remodeling through atrial fibrosis, inflammation, electrical remodeling, and cardiac metabolism. The regulated transcripts detected in the left atrial tissue in both the ARB-treated and ARNI-treated groups were related to metabolism. In contrast to the ARB-treated rates, the transcripts in ARNI-treated rats were mapped to the cyclic guanosine monophosphate-protein kinase G signaling pathway.