Search Results (196)

Exosomes are nanoscale extracellular vesicles that mediate intercellular communication by transporting microRNAs, proteins, and lipids. Generated through Endosomal Sorting Complex Required for Transport (ESCRT)-dependent mechanisms or ESCRT-independent pathways, exosomes are released when multivesicular bodies fuse with the plasma membrane. The ESCRT-dependent pathway involves sequential protein complexes (ESCRT-0, I, II, III) that recognize and sort ubiquitinated cargo, induce membrane budding, and facilitate vesicle scission. In contrast, the ESCRT-independent pathway relies on membrane lipids such as ceramide and proteins like tetraspanins (CD9, CD63, CD81) to promote vesicle formation without ESCRT machinery. Furthermore, post-translational modifications, including ubiquitination, sumoylation, and phosphorylation, further serve as molecular switches, modulating the affinity of ESCRT complexes or cargo proteins for membrane domains and affecting ILV formation rates. In reproductive medicine, exosomes regulate oocyte maturation, embryo–endometrial crosstalk, placental development, and maternal–fetal communication. Altered exosomal signaling contributes to obstetric complications, including preeclampsia, gestational diabetes mellitus, and preterm birth, whereas distinct exosomal miRNA signatures serve as potential diagnostic biomarkers. In gynecology, dysregulated exosomes are implicated in endometriosis, polycystic ovary syndrome, premature ovarian insufficiency, and gynecological malignancies. In contrast, mesenchymal stem cell-derived exosomes show therapeutic promise in restoring ovarian function and enhancing fertility outcomes. The distinctive molecular profiles of circulating exosomes enable minimally invasive diagnosis, while their biocompatibility and ability to cross biological barriers position them as vehicles for targeted drug delivery. Characterization of accessible data provides non-invasive opportunities for disease monitoring. However, clinical translation faces challenges, including standardization of isolation protocols, establishment of reference ranges for biomarkers, and optimization of therapeutic dosing. This review summarizes exosome biogenesis, characterization methods, physiological functions, and clinical applications in obstetrics and gynecology, with an emphasis on their diagnostic and therapeutic potential. Future directions include large-scale biomarker validation studies, engineering approaches to enhance exosome targeting, and integration with precision medicine platforms to advance personalized reproductive healthcare. Full article

Continuous-flow separation of magnetically labeled cells according to surface-marker expression levels is increasingly needed to study phenotypic heterogeneity and support downstream assays. Here, we present a microfluidic platform that uses spatially engineered soft magnetic strips (SMS) to sculpt lateral magnetic deflection fields for quantitative, label-guided cell fractionation. Under a uniform bias field, the SMS generates controllable magnetic gradients within the microchannel, producing distinct lateral velocities among EpCAM-labeled tumor cells that carry different Dynabead loads, which indirectly report membrane protein expression. Multi-outlet collection converts these “race-based” trajectory differences into discrete expression-level-resolved fractions. A COMSOL–MATLAB framework and a force-equivalent metric |(H·∇)H| are used to optimize key structural parameters of the magnetic interface, including strip thickness, width, and vertical spacing from the flow channel. Three journey nodes at 1.5, 3, and 9 mm along the flow path define a three-stage cascade that partitions MDA-MB-231, Caco-2, and A549 cells into four EpCAM-related magnetic subgroups: high (H), medium (M), low (L), and near-negative (N). Experiments show that the sorted fractions follow the expected expression trends reported in the literature, while maintaining high cell recovery (>90%) and viability retention of 98.2 ± 1.3%, indicating compatibility with downstream whole-blood assays and culture. Rather than introducing a new biomarker, this work establishes a quantitative magnetic-field design strategy for continuous microfluidic sorting, in which the spatial configuration of soft magnetic elements is exploited to implement expression-level-dependent fractionation in next-generation magneto-fluidic separation systems. Full article

Extracellular vesicles (EVs) are bilayer-membrane cellular components that deliver protected cargo to the extracellular environment and can mediate long-distance signaling. We have previously reported that EVs isolated from the virulent fungal pathogen Paracoccidioides brasiliensis Vpb18 can revert the expression, in the attenuated variant Apb18, of stress-related virulence traits. We presently show that the Vev and Aev, respectively, produced by these variants display distinct proteomes, with prevalent functional enrichment in Vev related to oxidative stress response, signal transduction, transport, and localization, in addition to richer protein–protein interaction. Proteome sequences were obtained by nanoflow liquid chromatography coupled with tandem mass spectrometry (nano LC-ESI-MS/MS). The Vev and corresponding Vpb18 proteomes also differed, suggesting a selective bias in vesicle protein cargo. Moreover, sublethal oxidative (VevOxi) and nitrosative (VevNO) stress modulated the Vev proteome and a positive correlation between VevOxi/VevNO-enriched and Vev-enriched (relative to Aev) proteins was observed. Out of 145 fungal virulence factors detected in Vev, 64% were enriched, strongly suggesting that molecules with virulence roles in Paracoccidioides are selectively concentrated in Vev. Our study significantly advanced the field by exploring protein N-terminal acetylation to a dimension rarely investigated in fungal EV proteomics. The proportion of N-terminally acetylated proteins in Vev was higher than in Vpb18 and the presence of Nt-acetylation in Vev-enriched virulence factors varied across the samples, suggesting that it might interfere with protein sorting into EVs and/or protein functionality. Our findings highlight the relevance of our fungal model to unraveling the significance of fungal EVs in pathogenesis and phenotypic transfer. Full article

Protein S-palmitoylation is a reversible lipid modification that regulates various aspects of protein function, including membrane association, subcellular localization, trafficking, stability, and activity. The depalmitoylase ABHD17A removes palmitate from multiple substrates, but its cellular positioning and the role of its own palmitoylation in regulating its function remain unclear. This study identifies a palmitoylation code within the conserved N-terminal cysteine cluster of ABHD17A, which governs its intracellular distribution and plasma membrane (PM) targeting. N-terminal palmitoylation is essential for PM localization. Through the use of code-restricted mutants, we found that modifications in the middle region (C14, C15) are critical for PM targeting and catalytic activity, while modifications at the front (C10, C11) and rear (C18) influence endosomal routing and delivery to the PM. Alanine scanning revealed that adjacent hydrophobic residues, particularly L9 and F13, are crucial for initial engagement with endomembranes. Sequence analysis and mutagenesis identified two tyrosine-based YXXØ motifs within the alpha/beta hydrolase fold; disruption of the proximal motif (L115A) decreased surface abundance and redirected ABHD17A to autophagosomes, indicating a need for YXXØ-dependent endosomal sorting, likely at the trans-Golgi network. Biochemical assays demonstrated a continuum of acylation states influenced by the palmitoylation code. This requirement for the middle region was conserved in ABHD17B and ABHD17C. Overall, our findings suggest a stepwise mechanism for ABHD17A delivery to the PM, enabling its depalmitoylase activity on membrane-bound substrates. Full article

Alzheimer’s disease (AD) is a complex neurodegenerative disorder where age, genetic factors and sleep disturbance significantly influence disease risk. Recent genome-wide association studies identified a C/T missense variant (rs141749679) in the sortilin (SORT1) gene linked to heightened AD risk, revealing SORT1’s role as a key player in the disease’s pathophysiology. This type I membrane glycoprotein is implicated in amyloid β (Aβ) accumulation and associated lipid dysregulation, particularly through its interaction with apolipoprotein E (ApoE). SORT1 facilitates the uptake of ApoE-bound polyunsaturated fatty acids (PUFAs), conversion to endocannabinoids (eCBs), and the regulation of anti-inflammatory pathways via peroxisome proliferator-activated receptors (PPARs). Notably, this neuroprotective signaling is contingent on the APOE allele, exhibiting functionality in presence of ApoE3 but disrupted with ApoE4. Additionally, the brain-type fatty acid binding protein, FABP7, mediates this signaling cascade, emphasizing its role in neuron-glia communication. FABP7 is known to regulate sleep across species and binds PUFAs and eCBs. Therefore, dysfunction of the ApoE-SORT1-FABP7 axis may underlie the neuroprotective loss observed in AD, linking sleep disruption and lipid homeostasis to disease progression. This perspective aims to elucidate the intricate neural-glial mechanisms governing the ApoE-SORT1-FABP7 interaction and their implications for targeting therapeutic interventions in Alzheimer’s disease. Full article

Cold stress limits the growth and productivity of Hevea brasiliensis, the primary source of natural rubber. This study investigated early transcriptomic responses in Reyan ‘7-33-97’ seedlings exposed to 4 °C, 10 °C, and 15 °C for 1, 2, and 4 h with room temperature (25 °C) as the control. RNA sequencing identified 9894 differentially expressed genes (DEGs), with the most significant transcriptional changes observed at 10 °C, indicating that genes to resist cold stress could not be mobilized at 4 °C, resulting in poor cold resistance of the rubber tree. KEGG enrichment analysis of DEGs between 10 °C (2 h) and 4 °C (2 h) revealed that genes involved in tryptophan metabolism (HbKynL.x1, HbKynL, HbCLP1, HbCLP2) and carbon metabolism (TCH4, XTH23), which contribute to cell wall modification, exhibited higher expression at 10 °C. Gene Ontology enrichment analysis highlighted significant involvement of “thylakoid,” “photosystems,” and “photosynthetic membrane,” alongside molecular functions such as “xyloglucan transferase activity” and “transcriptional regulator activity.” The interacting network of key pathways, including carbon metabolism (ko01200) and carbon fixation (ko00710) pathways, was sorted out, highlighting their integration with plant hormone signal transduction. Complex signaling networks, including MAPK, and kynurenine pathways coordinate the expression of cold-responsive genes and protective proteins, and it was confirmed and speculated that there is crosstalk response in cold defense mechanisms. Furthermore, 61 DEGs were associated with antioxidant processes, including major catalase and peroxidase enzymes. Our study shows that rubber trees physiological activities that respond to low-temperature signals cannot be carried out normally at 4 °C. The newly discovered metabolic pathway and the reason for abnormal cold signal transduction at low temperatures are the focus of future research on cold resistance. Full article

SNX11, a sorting nexin protein localized on the endosomal membrane, is an important protein closely related to protein sorting and endosomal trafficking. Previously, through a genome-wide CRISPR screening, we identified SNX11 as a critical protein for the entry of Dabie bandavirus. SNX11 deletion significantly inhibits the replication of Dabie bandavirus. We further discovered that the loss of SNX11 alters endosomal pH, potentially affecting the release process of Dabie bandavirus from endosomes to the cytoplasm. However, the mechanism by which SNX11 modulates endosomal pH and whether SNX11 deletion similarly inhibits other viruses remain to be elucidated. This study reveals that SNX11 can interact with the V1 subunit of the endosomal proton pump V-ATPase, affecting the expression level of this subunit on the endosomal membrane and thereby disrupting the assembly of V-ATPase. Additionally, we found that SNX11 deletion significantly inhibits the replication of dengue virus, hantavirus, and influenza virus. These findings suggest that SNX11 may be a key protein in the process of viral infection and could serve as a broad-spectrum antiviral target. Full article

Background: Cell-penetrating peptides cross cell membrane barriers while carrying cargoes in a functional form. Our work identified two novel lung-targeting peptides, S7A and R11A. Here, we present studies on biodistribution, the cell types targeted, and an in vitro proof of application. Methods: Studies were performed in human bronchial epithelial cells (HBECs) with and without various endocytic inhibitors, and coincubation with fluorescently labeled transferrin or endocytic markers. Cyclic R11A (cR11A) was conjugated to siRNA duplexes and anti-viral activity against SARS-CoV-2 was tested. Biodistribution studies were performed by injecting wild-type mice with fluorescently labeled peptides, and various circulation times were allowed for, as well as cross-staining of lung sections or isolated single cells with various cellular markers, followed by fluorescence-activated cell sorting or confocal microscopy. Results: cR11A showed peak uptake in 15 min, with the highest uptake in airway epithelial type II (ATII) cells, followed by p63+ basal cells and ionocytes. Cyclization increased transduction efficiencies ~100-fold. Endocytosis studies showed a decrease in peptide uptake by pre-treatment with Pitstop2 but not Amiloride or Nystatin. Endocytic marker Lamp1 showed colocalization at the earliest time point, with the escape of the peptide from endocytic vesicles later. cR11A conjugated to ant-spike and anti-envelop proteins showed anti-viral effects with an EC₉₀ of 0.6 μM and 1.0 µM, respectively. Conclusions: We have identified a novel peptide, cR11A, that targets ATII, basal cells, and ionocytes, the cyclization of which increased transduction efficiency in vitro and in vivo. The uptake mechanism appears to be via clathrin-mediated endocytosis with escape from endocytic vesicles. cR11A can act as a vector to deliver anti-viral siRNA to epithelial cells. Full article

Endosomal dysfunction is one of the earliest cellular signs in Alzheimer’s disease. Tumor susceptibility gene 101 protein (TSG101) is a component of the endosomal sorting complex required for transport (ESCRT)-I, which plays a key role in sorting ubiquitinated cell surface proteins and lipids onto intraluminal vesicles of multivesicular bodies for trafficking to lysosomes or autophagosomes for degradation, or to the plasma membrane for exosomal secretion. TSG101-dependent trafficking has been implicated in the propagation and spread of misfolded proteins associated with neurodegenerative diseases. We used transgenesis mice to study the in vivo consequences of disrupting TSG101-dependent trafficking in adult neurons. Mice lacking Tsg101 in forebrain neurons (Tsg101^ck2-null) showed rapid loss of hippocampal neurons and progressive forebrain atrophy. Astrogliosis was apparent in the dentate gyrus within 1 week of deleting Tsg101, followed by apoptosis of hippocampal CA3 neurons and accumulation of the autophagy adapter P62/SQSTM1 and ubiquitinated proteins. Failure to detect lipidated LC3 indicated autophagy was impaired rather than upregulated. Endosomal markers (RAB5 and RAB7) and amyloid protein also accumulated in hippocampal neurons of Tsg101^ck2-null mice. Our data establish a critical role for TSG101 in neuronal survival and demonstrate the importance of the in vivo assessment of gene and protein functions. Full article

Cardiovascular complications are a hallmark of Post-Acute Sequelae of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) infection (PASC), yet the mechanisms driving persistent cardiac dysfunction remain poorly understood. Emerging evidence implicates mitochondrial dysfunction in immune cells as a key contributor. This study investigated whether CD14⁺⁺ monocytes from long COVID patients exhibit bioenergetic impairment, mitochondrial DNA (mtDNA) damage, and defective oxidative stress adaptation, which may underlie cardiovascular symptoms in PASC. CD14⁺⁺ monocytes were isolated from 14 long COVID patients with cardiovascular symptoms (e.g., dyspnea, angina) and 10 age-matched controls with similar cardiovascular risk profiles. Mitochondrial function was assessed using a Seahorse Agilent Analyzer under basal conditions and after oxidative stress induction with buthionine sulfoximine (BSO). Mitochondrial membrane potential was measured via Tetramethylrhodamine Ethyl Ester (TMRE) assay, mtDNA integrity via qPCR, and reactive oxygen species (ROS) dynamics via Fluorescence-Activated Cell Sorting (FACS). Parallel experiments exposed healthy monocytes to SARS-CoV-2 spike protein to evaluate direct viral effects. CD14⁺⁺ monocytes from long COVID patients with cardiovascular symptoms (n = 14) exhibited profound mitochondrial dysfunction compared to age-matched controls (n = 10). Under oxidative stress induced by buthionine sulfoximine (BSO), long COVID monocytes failed to upregulate basal respiration (9.5 vs. 30.4 pmol/min in controls, p = 0.0043), showed a 65% reduction in maximal respiration (p = 0.4035, ns) and demonstrated a 70% loss of spare respiratory capacity (p = 0.4143, ns) with significantly impaired adaptation to BSO challenge (long COVID + BSO: 9.9 vs. control + BSO: 54 pmol/min, p = 0.0091). Proton leak, a protective mechanism against ROS overproduction, was blunted in long COVID monocytes (3-fold vs. 13-fold elevation in controls, p = 0.0294). Paradoxically, long COVID monocytes showed reduced ROS accumulation after BSO treatment (6% decrease vs. 1.2-fold increase in controls, p = 0.0015) and elevated mitochondrial membrane potential (157 vs. 113.7 TMRE fluorescence, p = 0.0179), which remained stable under oxidative stress. mtDNA analysis revealed severe depletion (80% reduction, p < 0.001) and region-specific damage, with 75% and 70% reductions in amplification efficiency for regions C and D (p < 0.05), respectively. In contrast, exposure of healthy monocytes to SARS-CoV-2 spike protein did not recapitulate these defects, with preserved basal respiration, ATP production, and spare respiratory capacity, though coupling efficiency under oxidative stress was reduced (p < 0.05). These findings suggest that mitochondrial dysfunction in long COVID syndrome arises from maladaptive host responses rather than direct viral toxicity, characterized by bioenergetic failure, impaired stress adaptation, and mitochondrial genomic instability. This study identifies persistent mitochondrial dysfunction in long COVID monocytes as a critical driver of cardiovascular complications in PASC. Key defects—bioenergetic failure, impaired stress adaptation and mtDNA damage—correlate with clinical symptoms like heart failure and exercise intolerance. The stable elevation of mitochondrial membrane potential and resistance to ROS induction suggest maladaptive remodeling of mitochondrial physiology. These findings position mitochondrial resilience as a therapeutic target, with potential strategies including antioxidants, mtDNA repair agents or metabolic modulators. The dissociation between spike protein exposure and mitochondrial dysfunction highlights the need to explore host-directed mechanisms in PASC pathophysiology. This work advances our understanding of long COVID cardiovascular sequelae and provides a foundation for biomarker development and targeted interventions to mitigate long-term morbidity. Full article

The endosomal sorting complexes required for transport (ESCRT) comprise a fundamental cellular machinery with remarkable versatility in membrane remodeling. It is multifunctional in the multivesicular body (MVB) biogenesis, exosome formation and secretion, virus budding, cytokinesis, plasma membrane repair, neuron pruning, and autophagy. ESCRT’s involvement in cellular mechanisms extends beyond basic membrane trafficking. By directly interacting with autophagy-related (ATG) proteins and facilitating autophagosome-lysosome fusion, ESCRT ensures cellular homeostasis. Dysregulation in ESCRT function has been implicated in cancer, neurodegenerative disorders, and infectious diseases, underscoring its critical role in numerous pathologies. Hepatitis B virus (HBV) is an enveloped virus that exploits ESCRT and autophagy pathways for viral replication, assembly, and secretion. This review synthesizes recent mechanistic insights into ESCRT’s multifaceted roles, particularly focusing on its interactions with autophagy formation and the HBV lifecycle. Full article

Background/Objectives: Cytomegalovirus (CMV) infection expands early endosomes (EEs) into tubular extensions that may contribute to the control of virus replication and virion assembly. Sequential recruitment of protein coats and sorting nexins (SNXs) creates membrane zones at the EEs that serve as scaffolds for membrane tubulation and retrieval of cargo proteins, including host cell signaling proteins and viral glycoproteins. This study aims to investigate whether the SNX3-dependent zone of EEs contributes to CMV replication and assembly. Methods: Protein localization was analyzed by confocal imaging and expression by Western blot. The contribution of SNX3 to murine CMV (MCMV) replication, assembly compartment (AC) formation, and virion release was analyzed by siRNA and shRNA depletion. The impact of other downstream SNXs that act in EE tubulation was investigated by combined siRNA knockdowns of SNX1, SNX2, SNX4, SNX17, and SNX27 on cell lines expressing shRNA for SNX3. Results: The SNX3-162 isoform acting at EEs was efficiently knocked down by siRNA and shRNA. The SNX3-dependent EE zone recruited SNX27 and contributed to Rab10-dependent tubulation within the pre-AC. SNX3 was not essential for MCMV replication but contributed to the SNX27-, SNX17- and SNX4-dependent release of virions. Silencing SNX3 further reduced the release of virions after silencing SNX27, SNX4, and SNX17, three SNXs that control recycling to the plasma membrane. Conclusions: SNX3 contributes to the formation of pre-AC and MCMV assembly. It acts sequentially with SNX27, SNX4, and SNX17 along the recycling pathway in the process of the production and release of infection virions, suggesting that multiple membrane sources may contribute to the secondary envelopment of MCMV virions. Full article

The open reading frame 3a (ORF3a) is a protein important to the pathogenicity of SARS-CoV-2. The cytoplasmic domain of ORF3a has three canonical tyrosine-based sorting signals (160YNSV163, 211YYQL213, and 233YNKI236), and a previous study has indicated that mutation of the 160YNSV163 motif abrogated plasma membrane expression and inhibited ORF3a-induced apoptosis. Here, we have systematically removed all three tyrosine-based motifs and assessed the importance of each motif or combination of motifs in trafficking to the cell surface. Our results indicate that the 160YNSV163 motif alone was insufficient for ORF3a cell-surface trafficking, while the 211YYQL213 motif was the most important. Additionally, an ORF3a with all three YxxΦ motifs disrupted (ORF3a-[ΔYxxΦ]) was not transported to the cell surface, and LysoIP studies indicate that ORF3a but not ORF3a-[ΔYxxΦ] was present in late endosome/lysosome fractions. A growth-curve analysis of different SARS-CoV-2 viruses expressing the different mutant ORF3a proteins revealed no significant differences in virus replication. Finally, the inoculation of K18hACE-2 mice indicated that the SARS-CoV-2 lacking the three YxxΦ motifs was less pathogenic than the unmodified SARS-CoV-2. These results indicate that the tyrosine motifs of ORF3a contribute to cell-surface expression and SARS-CoV-2 pathogenesis Full article

Fuchs’ endothelial corneal dystrophy (FECD) is a progressive eye disease characterized by accelerated loss of endothelial cells and the development of focal excrescence (guttae) on Descemet’s membrane, resulting in cornea opacity and vision deterioration. The development of FECD is assumed to be due to the interplay between genetic and environmental factor risks, causing abnormal extracellular-matrix organization, increased oxidative stress, apoptosis and unfolded protein response. However, the molecular knowledge of FECD is limited. The development of genome-wide platforms and bioinformatics approaches has enabled us to identify numerous genetic loci that are associated with FECD. In this review, we gathered genome-wide studies (n = 31) and sorted them according to genomics (n = 9), epigenomics (n = 3), transcriptomics (n = 15), proteomics (n = 3) and metabolomics (n = 1) levels to characterize progress in understanding FECD. We also extracted validated differentially expressed/spliced genes and proteins identified through comparisons of FECD case and control groups. In addition, highlighted loci from each omics layer were combined according to a comparison with similar study groups from original studies for downstream gene-set enrichment analysis, which provided the most significant biological pathways related to extracellular-matrix organization. In the future, multiomics study approaches are needed to increase the sample size and statistical power to identify strong candidate genes for functional studies on animal models and cell lines for better understanding FECD. Full article

Background: Sorting nexin 19 (SNX19) is important in the localization and trafficking of the dopamine D₁ receptor (D₁R) to lipid raft microdomains. However, the interaction between SNX19 and the lipid raft components caveolin-1 or flotillin-1 and, in particular, their roles in the cellular endocytosis and cell membrane trafficking of the D₁R have not been determined. Methods: Caveolin-1 and flotillin-1 motifs were analyzed by in silico analysis; colocalization was observed by confocal immunofluorescence microscopy; protein-protein interaction was determined by co-immunoprecipitation. Results: In silico analysis revealed the presence of putative caveolin-1 and flotillin-1 binding motifs within SNX19. In mouse and human renal proximal tubule cells (RPTCs), SNX19 was localized mainly in lipid rafts. In mouse RPTCs transfected with wild-type (WT) Snx19, fenoldopam (FEN), a D₁-like receptor agonist, increased the colocalization of SNX19 with caveolin-1 and flotillin-1. FEN also increased the co-immunoprecipitation of SNX19 with caveolin-1 and flotillin-1, effects that were prevented by SCH39166, a D₁-like receptor antagonist. The FEN-mediated increase in the residence of SNX19 in lipid rafts and the colocalization of the D₁R with caveolin-1 and flotilin-1 were attenuated by the deletion of a caveolin-1 (YHTVNRRYREF) (ΔCav1) or a flotillin-1 (EEGPGTETETGLPVS) (ΔFlot1) binding motif. The FEN-mediated increase in intracellular cAMP production was also impaired by the deletion of either the flotillin-1 or caveolin-1 binding motif. Nocodazole, a microtubule depolymerization inhibitor, interfered with the FEN-mediated increase in the colocalization between SNX19 and D₁R. Conclusion: SNX19 contains caveolin-1 and flotillin-1 binding motifs, which play an important role in D₁R endocytosis and signaling. Full article