Ral GTPases have long been proposed as regulators of the metazoan exocyst, a conserved secretory vesicle-tethering complex, but direct evidence for this role has been scarce. In contrast, the well-studied yeast exocyst relies on multiple Rab GTPases to regulate function, but yeast do not encode Ral. Using Caenorhabditis elegans, we demonstrate that endogenous RAL-1 directly engages the exocyst through conserved binding sites in its subunits. Loss of RAL-1 disrupts dendritic arborization of PVD sensory neurons, impairs vesicle trafficking, and causes broad developmental defects, acting both cell autonomously in neurons and non-autonomously through supporting epithelial cells. Structure-guided genome editing of RAL-1-exocyst interfaces produced synthetic phenotypes, underscoring the physiological importance of these contacts. Taken together, our findings establish RAL-1 as a bona fide regulator of the metazoan exocyst in vivo and suggest that Ral-exocyst interactions operate in parallel with other secretory pathways. More broadly, this work positions C. elegans as a powerful system to dissect Ral-exocyst mechanisms across molecular, cellular, and developmental scales.

Lipid oversupply induces insulin resistance in skeletal muscle (SkM), partly by altering mitochondrial function and dynamics. Although electrical pulse stimulation (EPS) improves insulin signaling in myotubes, the mechanisms contributing to this effect under lipotoxic conditions remain unclear. Thus, we investigated whether EPS restores lipid-induced insulin resistance in association with changes in mitochondrial respiration and dynamics, lipotoxic intermediates and evaluated the contribution of contraction-induced secreted factors, including small extracellular vesicles (sEV). Murine C2C12 myotubes were treated with palmitate (PA, 0.3 mmol/l) and subjected to EPS (1 Hz, 11.5 V) for 24 h. Insulin signaling, mitochondrial dynamics, respiratory capacity and lipotoxic intermediates were assessed by Western blotting, high-resolution respirometry and liquid chromatography-tandem mass spectrometry in control (CON) and PA-treated cells before and after EPS. Additionally, CON and PA-treated cells were incubated for 24 h with conditioned media and sEV from unstimulated or EPS-stimulated CON and PA-treated cells to evaluate the effect of the myocellular secretome on AKT signaling. PA reduced insulin-stimulated AKT(Ser473) and (Thr308) phosphorylation, which was partially rescued by EPS. In PA cells, EPS decreased the fusion proteins mitofusin 1 and 2 (MFN1, MFN2) and increased the fission marker dynamin-related protein 1 (DRP1). However, mitochondrial respiration and lipid accumulation were unaffected by EPS. Furthermore, conditioned media and sEV from EPS-stimulated cells enhanced AKT phosphorylation in PA-treated cells. These findings demonstrate that attenuation of lipid-induced insulin resistance is independent of mitochondrial respiratory capacity and lipid accumulation, but it is associated with alterations in proteins involved in mitochondrial dynamics and is likely mediated by exercise-derived sEV.

Stem cells endowed with properties of self-renewal and differentiation have opened new avenues for restoration and regeneration of damaged tissues. The same adult stem cells, when they undergo irreversible mutations, transform into cancer cells and generate a tumor. In this lecture, I present our work on these two aspects of stem cells: one focuses on regeneration of the ocular surface, whereas the other targets cancer stem cells (CSCs) in retinoblastoma (Rb). In regeneration, I present our experience of transplantation of the cultured limbal epithelial cells to restore the ocular surface in severe limbal stem cell deficiency, with 60% to 70% success. The technique proved that transplanted cells survive, network, integrate, contribute to function, and cause no harm to the patient. We also developed a technique wherein both limbal and conjunctival cells could be cultured on a single membrane utilized ring barrier. This approach was effective in reconstructing the conjunctiva and fornices in patients with severe ocular disease. Furthermore, our work led identification of a subpopulation of limbal-derived stromal cells with features of mesenchymal stem cells. We also saw success with culturing human lacrimal gland tissues marked by the presence of epithelial stem cells and mesenchymal stem cells with secretory potential. These cultures we believe could pave the way of lacrimal gland regeneration through cell therapy. We then flip to the "dark side" of stem cells: the isolation and characterization of CSCs in Rb. We worked on both primary tumors as well as Rb y79 cell lines, with a special focus on reversing their chemoresistant properties. Our work demonstrated the possible role of small extracellular vesicles in Rb as a diagnostic and prognostic marker. The reduction of chemoresistance using nano-formulated drugs and targeted delivery of drug-loaded small extracellular vesicles derived from the tumor cells shows promise.

Upon sensing Yersinia pseudotuberculosis (Yptb), receptor-mediated pathways are stimulated to trigger polymorphonuclear (PMN) antimicrobial responses. Yptb injects multiple type 3 secreted effector proteins, Yops (Yersinia outer proteins), that possess distinct biochemical functions, into PMNs to inhibit PMN responses. We show that several Yops, YopE, YopH, and YopO, each partially interfered with CD63 plasma membrane localization, a marker for primary degranulation. The host pathways involved in CD63 mobilization are complex, and it is not completely understood how Yops collaborate to inactivate this process. Here, CRISPR/Cas9 technology was used in an immortalized system of myeloid progenitor cells (Cas9-ER-HoxB8) to generate knockout PMN cell lines. To probe the impact of different Yops on the neutrophil pathways activated upon encountering Yptb, we interrogated the panel of genetically modified neutrophils with genetically modified bacteria. This approach of targeted gene deletion to inactivate specific pathways/proteins uncovered host pathways that synergize to induce CD63 mobilization that are distinctly targeted by YopE and YopH. YopE inhibited CD63 mobilization in the absence of SKAP2, a YopH target, whereas YopH inhibited CD63 mobilization in the absence of RhoG, a YopE target, indicating that these Yops inactivate distinct signaling pathways. Furthermore, the SKAP2-independent pathway inactivated by YopE is involved in primary granule release and ROS production. Overall, this work highlights multiple signaling pathways involved in mounting degranulation and ROS production and the diverse Yop-mediated mechanisms that WT-Yptb employs to effectively disarm them. Importantly, this work provides an avenue to untangle neutrophil signaling pathways targeted by pathogens using Cas9-ER-HoxB8 cells.IMPORTANCEWhen sensing invading bacteria, neutrophils become activated through multiple receptors that trigger signal-transduction cascades resulting in the generation of antimicrobial responses. The enteric pathogen, Yersinia pseudotuberculosis (Yptb), is equipped to effectively inhibit these responses using its collection of effector proteins (Yops). Here, we developed a system to overcome the limitations of performing genetic manipulations in neutrophils by implementing CRISPR/Cas9 technology in an engineered system of myeloid progenitor cells (Cas9-ER-HoxB8), which can be induced to differentiate into neutrophils. By infecting genetically modified neutrophils with Yptb strains expressing individual Yops, we identified distinct host signaling pathways that synergize to induce neutrophil antimicrobial responses. Our findings provide insight into several signaling events triggered by Yptb infection and show how YopE and YopH target distinct pathways to block vesicle trafficking and extracellular ROS production. This powerful genetic system can be applied to other pathogens to dissect the intricacies of neutrophil-pathogen interactions.

Ivacaftor, a CFTR potentiator, is clinically effective in treating cystic fibrosis (CF), but it has poor lung targeting, variable oral bioavailability, and poor aqueous solubility. The objective of the study was to develop and optimize an inhalable proliposomal dry powder inhaler (DPI) of ivacaftor for pulmonary delivery. The optimized ivacaftor-loaded proliposomes were optimized using a face-centered central composite design (α = 1) by varying soy phosphatidylcholine (SPC) concentration and drug: cholesterol ratio, with particle size, zeta potential, and entrapment efficiency. The optimized formulation was spray dried with mannitol and leucine to obtain inhalable microparticles. The optimized proliposomal formulation had nanosized vesicles (∼198 nm), high entrapment efficiency (∼73%), and good colloidal stability. The solid-state characterization confirmed amorphization and molecular dispersion of ivacaftor without chemical incompatibility. The spray-dried formulation exhibited high emitted dose (96.45%), fine particle fraction (52.38%), and MMAD (1.66 µm), and sustained drug release, good cytocompatibility, and satisfactory accelerated stability supported its suitability. Conclusively, proliposomal inhalation delivery is a promising approach to enhance pulmonary targeting of ivacaftor in cystic fibrosis.

Giardiasis, caused by Giardia intestinalis, is a prevalent intestinal protozoan infection worldwide. Clinical manifestations are variable, and many infections remain asymptomatic. Disease pathogenesis is multifactorial, involving brush border enzyme deficiency, apoptosis, dysbiosis and increased intestinal permeability. Advances in genomics, genetic tools, and model systems have substantially improved our understanding of the parasite's virulence mechanisms. Giardia exhibits considerable genetic diversity, with distinct assemblages associated with differences in parasite biology. Attachment is mediated by the ventral disk, while the parasite's eight flagella contribute to motility and intestinal persistence. Antigenic variation of cysteine-rich surface proteins enables immune evasion and promotes chronic infection but the level of inflammation is low. The parasite releases a variety of proteins and extracellular vesicles containing proteins and small RNAs that play important roles in host-cell interactions and modulation of the gut microbiome. This review describes areas that need further research to connect pathogenesis to specific virulence mechanisms.

Bioactivation and clearance of anticancer agents are crucial factors in the efficacy and toxicity of anticancer drugs. Exosomes are membrane-bound vesicles secreted by tumor cells and the tumor microenvironment. These vesicles contain drug-metabolizing enzymes, transporters, and RNA. Exosomes can be non-invasively collected from the blood. This article has been prepared by combining the evidence of the role of exosomal cytochrome P450 isoforms, such as cytochrome P450 family 2 subfamily C member 19, cytochrome P450 family 2 subfamily D member 6, and cytochrome P450 family 3 subfamily A member 5, in the oxidation of anticancer drugs in the first phase of drug metabolism. Exosomal UDP-glucuronosyltransferase 1A1 has been found to help in the conjugation of anticancer drugs in the second phase of drug metabolism. Exosomal ATP-binding cassette transporters, P-glycoprotein, multidrug resistance-associated protein 1, and breast cancer resistance protein have been found to act as intercellular conveyors of drug efflux capacity. These transporters can modulate the efficacy of chemotherapy. Exosomal microRNAs, such as microRNA-21, microRNA-155, and microRNA-1246, have been found to modulate apoptosis, DNA repair, metabolic adaptation, and chemotherapy resistance through the PI3K/AKT pathway, EZH2/STAT3 pathway, and PDCD4 pathway. Long non-coding RNAs, such as UCA1 and HOXA antisense RNA, can reprogram the chromatin structure. These long non-coding RNAs can suppress tumor suppressor genes, thus leading to chemotherapy resistance. Bioinformatics pipelines have been designed to bridge the gap between exosomal RNA and protein levels in the profile of dynamic metabolic states. The challenges in the field of exosomal therapy drug monitoring have been discussed in the article. The article has recommended the use of artificial intelligence in the integration of omics with microfluidic technology for the improvement of exosomal therapy drug monitoring.

Metabolomics provides functional molecular readouts of disease phenotypes, therapeutic responses, and tissue microenvironments, but its extension to low-volume, single-cell, and spatially resolved samples remains analytically challenging. Laser desorption/ionization mass spectrometry (LDI-MS) offers a rapid, direct, and high-throughput route for small-molecule analysis. However, its performance in biomedical metabolomics is largely governed by the matrix-analyte interface. Nanomaterials have emerged as attractive LDI-MS matrices because of their low background in the low-mass region, large surface area, tunable photothermal and electronic properties, and designable surface chemistry. In this review, we summarize recent advances in nanomatrix-assisted LDI-MS for biomedical metabolomics, emphasizing design principles. We discuss three key design dimensions of nanomatrix and interface engineering: structural engineering for energy absorption and desorption efficiency, surface functionalization for metabolite enrichment and low-interference ionization, and interface engineering for reproducibility, throughput, and low-volume sample compatibility. Representative biomedical applications are highlighted, including biofluid metabolic fingerprinting, extracellular vesicle and single-cell metabolomics, and spatial metabolic imaging. Finally, we discuss current challenges in matrix reproducibility, metabolite coverage, workflow standardization, and clinical validation, and outline future opportunities for integrated nanomatrix platforms in multiscale biomedical metabolomics.

The concept of protein corona formation around extracellular vesicles (EVs) has given birth to new insights into how cells may recognize EVs by proteins presented at the EV surface. Here we present spatially resolved proteomics using the biotin ligase TurboID to map proteins interacting at the EV surface, without a need for physical isolation of the EV-corona complexes. TurboID promiscuously biotinylates nearby proteins within a few nm distance from the fused 'bait' protein. We genetically engineered EVs by modifying CD63 as the bait protein to which TurboID was fused facing outward to map EV corona proteins. Biotinylated proteins were then analysed by Western blotting and liquid chromatography-mass spectrometry. Western blots revealed protein patterns that are distinct depending on the localization of the fused TurboID. The mass spectrometry analysis identified many of the serum proteins commonly known to form a corona around a synthetic solid nanoparticle, also supporting those previously reported through the physical isolation approaches. Rather striking is, however, the EV corona footprint of endogenous proteins that tells us about the EV biogenesis and what the intrinsic endogenous corona might look like. This approach, which we coined EV-SPEC (Spatial Proteomics of Endogenous Corona), has thus the potential to revolutionize our understanding of EV biology by shifting the focus from the EVs themselves to the proteins that make up the corona, or how the cell 'sees' them, in analogy with the biomolecular corona extensively characterized for synthetic nanoparticles.

Skeletal degeneration is a major cause of pain, disability, fragility fracture, and loss of independence in aging populations. Although osteoporosis, osteoarthritis, and intervertebral disc degeneration are usually managed as anatomically separate disorders, they share a common regenerative failure: endogenous stem and progenitor cells lose renewal capacity, acquire inflammatory secretory programs, and become less able to rebuild damaged matrix. This narrative review synthesizes evidence on senescence in bone marrow mesenchymal stem/stromal cells, skeletal stem cells, hematopoietic stem cells, cartilage progenitor cells, synovial mesenchymal stromal cells, and disc-derived progenitor populations. Priority is given to primary mechanistic, omics, translational, and early clinical studies that connect senescence markers with functional impairment or intervention response. We discuss telomere-associated DNA damage, mitochondrial and oxidative stress, nutrient- and mechanosensing pathways, epigenetic remodeling, dysregulated autophagy and mitophagy, and the senescence-associated secretory phenotype as interlocking mechanisms that reshape skeletal niches. Disease-specific sections then examine how senescent progenitor compartments drive osteoporosis, osteoarthritis, and intervertebral disc degeneration through impaired osteogenesis or chondrogenesis, matrix catabolism, inflammatory vesicle signaling, and maladaptive mechanical feedback. Finally, we compare senolytics, senomorphics, metabolic and mitochondrial regulators, cell rejuvenation, extracellular vesicle engineering, biomaterials, and gene-based delivery platforms from a regenerative medicine perspective. Stem/progenitor cell senescence should be treated not simply as a biomarker of aging, but as a compartment-specific therapeutic bottleneck. Translation will require rigorous cell identity, multi-parameter senescence assays, disease-stage stratification, and therapies that restore regenerative competence without suppressing physiological repair.

Colorectal cancer (CRC) is one of the most common malignant tumors. Exosomes are key modulators of intercellular communication and important participants in CRC tumorigenesis. However, the influence and underlying mechanism of cancer-secreted exosomes in CRC are not fully understood. In the present study, exosome-enriched extracellular vesicles (EVs) were isolated from paired CRC and adjacent normal control tissue (NC) from patients with CRC using ultracentrifugation, size exclusion chromatography and ultrafiltration. Bowtie software was used to identify mRNA expression patterns. Further analyses included functional enrichment analysis using Gene Ontology and Kyoto Encyclopedia of Genes and Genomes for differentially expressed genes (DEGs) and Gene Set Enrichment Analysis for functional enrichment of the entire transcriptome. Gene Set Variation Analysis was used to identify differentially enriched functions and pathways for each sample. Weighted gene correlation network analysis was used to select candidate key genes. The diagnostic performance of the candidate key genes was validated using the models of multiple logistic regression and a support vector machine with the external CRC exosomal RNA database exoRBase. A total of 814 DEGs were identified in the exosome-enriched EVs from patients with CRC compared with those from NC group. The enriched functions were potentially associated with key steps and pathways involved in CRC tumorigenesis, including cell cycle, DNA replication, RNA activity, protein metabolism and the PI3K/Akt', Wnt, MAPK and KRAS pathways, as well as the regulation of mitochondria, the pentose phosphate pathway and intestinal immunity. A total 14 candidate key genes, namely, p53 effector related to PMP-22, MYC, dyskerin pseudouridine synthase 1), VDAC1 (voltage dependent anion channel 1), adenosylhomocysteinase), TMEM123 (transmembrane protein 123), RPL36A (ribosomal protein L36a), CCT5 (chaperonin containing TCP1 subunit 5), CHCHD2 (coiled-coil-helix-coiled-coil-helix domain containing 2), RPS21 (ribosomal protein S21), KRT18 (keratin 18), TGFBI (transforming growth factor beta induced), ETS2 (ETS proto-oncogene 2) and AXIN2 (axin 2), were identified. The present exploratory study revealed pronounced transcriptomic changes in exosome-enriched EV derived from CRC compared with those derived from paracancerous tissue, and may provide a novel platform for understanding tumor microenvironment interactions.

Reliable processing of biospecimens such as plasma in liquid biopsy is crucial to achieving robust analytical results; however, manual procedures involved in sample preparation remain a major source of variability. Here, we present MECHA (Magnetic-Enrichment and Centrifugal-Homogenization Assembly), a fully automated system that standardizes magnetic bead (MB)-based sample handling. MECHA integrates coordinated magnetic actuation, controlled vibration, and orbital mixing to perform target capture, washing, and labeling steps with minimal operator input. The prototype was designed to process eight samples in parallel for electrochemiluminescence (ECL) detection. The signal variation was <10% across replicates. In a pilot analytical study, the MECHA-ECL pipeline enabled quantitative profiling of extracellular vesicles (EVs) from clinical plasma samples of colorectal cancer patients. The automated EV assay achieved a detection limit of 4 × 104 EVs/mL, a dynamic range spanning 4 orders of magnitude, and identified EGFR and CD133 as predictive biomarkers for chemotherapy response.