Search Results (1,425)

Objective: Diabetic kidney disease (DKD) is characterized by podocyte injury and impaired autophagy. Bone marrow mesenchymal stem cell-derived exosomes (BMSC-Exos) exhibit therapeutic potential for DKD, yet their mechanisms remain unclear. This study investigated whether BMSC-Exos restore podocyte autophagy via the miR-143-3p/Bcl-2/Beclin1 axis to delay DKD progression. Methods: A high-glucose (HG)-induced podocyte injury model was established using mouse podocytes (MPC5). Autophagy-related proteins (Beclin1, Bcl-2, LC3) and the injury marker desmin were analyzed by Western blot and immunofluorescence (IF). High-throughput sequencing identified BMSC-Exos-enriched miRNAs, with the miR-143-3p/Bcl-2 targeting relationship validated by dual-luciferase reporter assays. BMSCs transfected with miR-143-3p mimic or inhibitor were used to assess exosomes effects on autophagy and podocin expression. In vivo, DKD mice received tail vein injections of modified BMSC-Exos, followed by evaluation of physiological parameters, biochemical indices, and renal histopathology. Results: BMSC-Exos were successfully isolated and characterized. Fluorescence microscopy confirmed exosomes internalization by HG-treated MPC5 cells. BMSC-Exos upregulated Beclin1 and LC3-II while downregulating Bcl-2 and desmin, indicating enhanced autophagy. High-throughput sequencing revealed miR-143-3p enrichment in BMSC-Exos, and Bcl-2 was confirmed as a direct target of miR-143-3p. Exosomes from miR-143-3p mimic-transfected BMSCs further promoted autophagy and podocin expression. In DKD mice, BMSC-Exos reduced blood glucose, urinary albumin-to-creatinine ratio (UACR), and ameliorated renal damage, whereas miR-143-3p inhibition attenuated these effects. Conclusions: BMSC-Exos deliver miR-143-3p to target Bcl-2, thereby activating Beclin1-mediated autophagy and ameliorating DKD. This study elucidates a novel autophagy regulatory mechanism supporting BMSC-Exos as a cell-free therapy for DKD. Full article

Cucumber downy mildew, caused by the obligate parasitic oomycete Pseudoperonospora cubensis [(Berkeley & M. A. Curtis) Rostovzev], is a major threat to global cucumber production. Effective disease management relies on rapid and accurate pathogen detection. However, due to the specialized parasitic nature of P. cubensis, conventional methods are often laborious, low-throughput and inadequate, necessitating the development of a new approach for high-throughput sporangia counting. To address this limitation, we developed a rapid, high-throughput flow cytometry (FCM) assay for the direct quantification of P. cubensis sporangia. The optimal staining protocol involved adding 30 µL of 1000× diluted SYBR Green I to 500 µL of sporangial suspension and incubating at room temperature for 20 min. The flow cytometry parameters were set to a high sample loading speed with a 30-s acquisition time. Instrumental settings included an FL1 (green fluorescence) threshold of 8 × 10⁴ and an SSC (side scatter) threshold of 3 × 10⁵, with low gain. Validation against hemocytometer counts revealed a strong positive correlation (r = 0.8352). The assay demonstrated high reproducibility, with relative standard deviations (RSDs) ranging from 1.96–9.84%, and a detection limit of 1–10 sporangia/µL. Operator-dependent variability ranged from 8.85% to 18.79%. These results confirm that the established flow cytometry assay is a reliable and efficient tool for P. cubensis quantification, offering considerable potential for improving cucumber downy mildew monitoring and control strategies. Full article

Background/Objectives: The latest National Comprehensive Cancer Network (NCCN) Central Nervous System (CNS) Guidelines recommend utilizing next-generation sequencing (NGS) to enable comprehensive genomic profiling (CGP) as the preferred approach for molecular characterization of central nervous system (CNS) malignancies. CNS malignancies present distinct challenges due to the infeasibility of tissue-based testing for many patients and the restrictive nature of the blood–brain barrier (BBB) making plasma-based liquid biopsy an ineffective alternative. Recent advances in liquid biopsy have extended molecular testing beyond plasma to include cerebrospinal fluid (CSF), which serves as a valuable source for tumor-derived nucleic acids. Methods: The Belay Summit™ 2.0 is a high-throughput CGP assay capable of detecting multiple variant types, including single nucleotide variants (SNVs) and small insertion and deletions (Indels), copy number variations (CNVs), gene fusions, splice variants, and immunotherapy biomarkers such as microsatellite instability (MSI) and tumor mutational burden (TMB). This study details the analytical and clinical validation of Summit™ 2.0 to assess its technical performance and clinical sensitivity. Analytical validation was conducted using 68 specimens, demonstrating robust and reproducible detection of all variant types with 15 ng of CSF-derived total nucleic acid (tNA). Results: The analytical sensitivity of the Belay Summit™ 2.0 assay for SNVs and Indels was determined to be 96.7% with a 100% limit of detection (LoD) at a variant allele frequency of 0.3%. Clinical validity was evaluated across a cohort of 118 CSF specimens, including both primary and metastatic CNS tumors, demonstrating 96% sensitivity and 98% specificity. Conclusions: These findings support the use of the Belay Summit™ 2.0 assay for accurate and reproducible genomic profiling of CNS tumors using tumor-derived nucleic acids from CSF in patients for whom tissue-based testing is considered infeasible, unsafe, or not deemed by the prescribing physician to be clinically appropriate. Full article

Mammarenaviruses (MaAv) cause persistent infection in their natural rodent hosts across the world and, via zoonotic events, can cause severe disease in humans. Thus, the MaAv Lassa virus (LASV) in Western Africa and the Junin virus (JUNV) in the Argentinean Pampas cause hemorrhagic fever diseases with significant case fatality rates in their endemic regions. In addition, the globally distributed MaAv lymphocytic choriomeningitis virus (LCMV) is an underrecognized human pathogen of clinical significance capable of causing devastating infections in neonates and immunocompromised individuals. Despite their impact on human health, there are currently no FDA-approved vaccines or specific antiviral treatments for MaAv infections. Existing anti-MaAv therapies are limited to the off-label use of ribavirin, whose efficacy remains controversial; hence, the development of novel therapeutics to combat human pathogenic MaAv is vital. We employed a high-throughput cell-based infection assay to screen the Pandemic Response Box, a collection of 400 diverse compounds with established antimicrobial activity, for MaAv inhibitors. We identified Ro-24-7429, an antagonist of the HIV-1 Tat protein and RUNX family transcription factor 1 inhibitor; WO 2006118607 A2, a dihydroorotate dehydrogenase inhibitor; and verdinexor, a novel selective inhibitor of nuclear export (SINE) targeting the XPO1/CRM1, as potent anti-MaAv compounds. Consistent with their distinct validated targets, verdinexor and WO 2006118607 A2 exhibited very strong synergistic antiviral activity when used in combination therapy. Our findings pave the way for the development of verdinexor as a potent host-directed antiviral against MaAv, which could be integrated into the development of combination therapy with direct- or host-acting antivirals to combat human pathogenic MaAv. Full article

Droplet microarrays are increasingly used for miniaturized, high-throughput biochemical assays, yet their fabrication commonly relies on complex lithographic processes, custom masks, or specialized coatings. Here we present a simple method for generating hydrophilic arrays on hydrophobic plastic substrates by combining ultrasonic atomization with off-the-shelf perforated masks. A fine mist of poly(vinyl alcohol) (PVA) solution is directed through commercial diamond sieves onto polypropylene (PP) sheets and polystyrene (PS) sheets, forming hydrophilic spots surrounded by the native hydrophobic background. Static contact angle measurements confirm a strong local contrast in wettability (from 100.85 ± 0.91° on untreated PP to 39.96 ± 0.71° on patterned spots, from 95.68 ± 3.61° on untreated PS to 52.00 ± 0.85° on patterned spots), while Image analysis shows droplet CVs of 6–8% in aqueous dye solutions for 1.2–2.0 mm masks; in complex media (LB), droplet uniformity decreases. By mounting the moving mask on a motorized stage, we generate one-dimensional reagent gradients simply by controlling the moving mask motion during atomization. We further demonstrate biological compatibility by culturing Escherichia coli in LB droplets containing resazurin, and by performing localized antibiotic screening using a moving mask-guided streptomycin gradient. The resulting droplet-wise viability data yield an on-chip dose–response curve with an IC₅₀ of 5.1 µg · mL⁻¹ (95% CI: 4.5–5.6 µg·mL⁻¹), obtained from a single array. Covering droplets with Electronic Fluorinated Fluid maintains volumes within 5% of their initial value over 24 h. Compared with conventional droplet microarray fabrication, the proposed method eliminates custom mask production and cleanroom steps, is compatible with standard plastic labware, and intrinsically supports spatial gradients. These attributes make masked ultrasonic atomization a practical platform for high-throughput microfluidic assays, especially in resource-limited settings. Full article

Background: Three-dimensional (3D) cancer models are currently essential tools in high-throughput screening (HTS), serving as a bridge between in vitro and in vivo approaches during drug development. Even though spheroids offer many advantages over 2D cultures, analyzing 3D cultures with heterogeneous morphology remains challenging due to the lack of standardized visualization techniques and multiparameter analysis. Methods: In this work, an optimized CellProfiler pipeline and a Python algorithm for weighting morphological features are used to visualize, extract, and analyze morphological data from spheroids derived from colorectal and pancreatic cancer cell lines with diverse morphologies (HCT116, LoVo, PANC-1, and CFPAC-1). Results: We developed a feature weighting process that combines multiple morphological parameters into a single metric using principal component analysis (PCA). There is a strong correlation between this process and a standard Alamar Blue proliferation assay (r = 0.89, ρ = 0.91, p < 0.001). Using this method, we were able to ascertain the IC₅₀ values of substances that did not produce results in cell lines with heterogeneous morphology (LoVo and CFPAC-1) using a standard proliferation assay. Conclusions: By removing the need for tracer dyes, the resulting methodology may lower costs while accelerating preclinical drug development through informative multiparameter analysis of compound efficacy. Full article

Hepatitis B virus (HBV) persists in infected hepatocytes through covalently closed circular DNA (cccDNA), a stable episomal form that serves as the transcriptional template for viral replication. Accurate and sensitive quantification of intrahepatic cccDNA is crucial for evaluating antiviral therapies, particularly those targeting a functional cure. Here, we report the development of a novel, cccDNA-specific detection system combining recombinase polymerase amplification (RPA) with CRISPR/Cas12a-based fluorescence detection. We designed and validated CRISPR RNAs (crRNAs) targeting HBV cccDNA-specific regions conserved across genotypes A–D. Reaction conditions for both RPA and Cas12a detection were optimized to enhance sensitivity, specificity, and accuracy. The system reliably detected as few as 10 copies of cccDNA-containing plasmid per reaction and showed no cross-reactivity with non-cccDNA forms in serum or plasma, indicating assay specificity. When applied to liver tissue samples from 10 HBV-infected and 6 non-HBV patients, the RPA-CRISPR/Cas12a assay exhibited a high sensitivity (90%) and a strong correlation with qPCR results (R² = 0.9155), confirming its accuracy. In the conclusion, the RPA-CRISPR/Cas12a system provides a robust, cost-effective, and scalable platform for sensitive and specific quantification of intrahepatic HBV cccDNA. This method holds promises for research and high-throughput therapeutic screening applications targeting cccDNA clearance. Full article

Endocrine disruptors, including bisphenol A, S, AF, and F, have been demonstrated to exhibit endocrine-disrupting activity. This phenomenon has been associated with a variety of health problems, including (but not limited to) neurological and reproductive disorders. Given the potential hazards, it is essential to have effective tools to assess their toxicity. The nematode Caenorhabditis elegans has become a widely used model organism for studying bisphenols because of its genetic simplicity and the conservation of its fundamental biological processes. This review article summarizes current knowledge of bisphenol toxicity and the use of the model organism C. elegans as a high-throughput system for investigating the toxicological profiles of BPA and its emerging alternatives. Furthermore, we highlight the specific methodologies for assessing the toxic effects of bisphenols in C. elegans. While highlighting its advantages, we critically discuss its limitations, including the absence of specific metabolic organs, which constrain direct extrapolation to mammalian systems. Based on available evidence, we conclude that C. elegans serves as an essential bridge between in vitro assays and mammalian models, offering a powerful platform for the early hazard identification and mechanistic screening of bisphenol analogues. Full article

The JinEr mushroom (“Golden Ear”), a globally rare edible and medicinal macrofungus, comprises a symbiotic complex formed by the symbiotic association of Naematelia aurantialba (Tremellomycetes) and Stereum hirsutum (Agaricomycetes). However, the interactions between these fungi and their associated microbiome remain poorly understood. This study employed high-throughput amplicon sequencing, in situ microbial isolation and culture, and microbial confrontation assays to analyze microbial diversity, community structure, and potential functional roles of the endomycotic bacterial community within JinEr basidiomata and its cultivation substrate. Molecular analysis confirmed the heterogenous composition of the basidiomata, revealing N. aurantialba constitutes less than 20% of the fungal biomass, while S. hirsutum predominates, accounting for approximately 80%. Endomycotic fungi accounted for 0.33% (relative abundance) of the fungal community. Prokaryotic analysis identified Delftia and Sphingomonas as the dominant endomycotic bacterial genera within basidiomata, comprising 85.42% of prokaryotic sequences. Endomycotic bacterial diversity differed significantly (p < 0.05) between basidiomata and substrate, indicating host-specific selection. Cultivation-based approaches yielded 140 culturable bacterial isolates (spanning four families and seven genera) from basidiomata core tissues. In vitro co-culture experiments demonstrated that eight representative bacterial strains exhibited compatible growth with both hosts, while one Enterobacteriaceae strain displayed antagonism towards them. These findings confirm that the heterogeneous JinEr basidiomata harbor a specific prokaryotic assemblage potentially engaged in putative symbiotic or commensal associations with the host fungi. This research advances the understanding of microbial ecology in this unique fungal complex and establishes a culture repository of associated bacteria. This collection facilitates subsequent screening for beneficial bacterial strains to enhance the JinEr cultivation system through the provision of symbiotic microorganisms. Full article

Lung cancer is the leading cause of cancer-related mortality worldwide, with non-small-cell lung cancer (NSCLC) accounting for the majority of cases. Standard tissue biopsies are invasive and unsuitable for repeated monitoring. Liquid biopsy technologies, particularly circulating tumor cell (CTC) analysis, offer a minimally invasive alternative for real-time disease tracking. To address the need for efficient and reproducible CTC isolation, we developed the Cellsway microfluidic CTC enrichment and identification platform, which employs inertial hydrodynamics in a spiral-shaped microfluidic channel comprising hydrofoil-shaped pillars to enable high-throughput, label-free enrichment of CTCs while preserving cell integrity, followed by an optimized CTC identification assay. Analytical performance assessed through spiking experiments using NSCLC cell lines demonstrated recovery rates of 91.9% for H1975 cells and 78.3% for A549 cells. Clinical validation was performed on blood samples from 51 stage IV NSCLC patients. A 7.5 mL volume of peripheral blood was processed with the SwayBox platform, and enriched CTCs were identified through an optimized multiplex immunofluorescence protocol. CTCs were detected in 47% of NSCLC patients, with counts ranging from 0 to 72 cells per 7.5 mL of blood. At a cutoff of 1 CTC per 7.5 mL, the assay achieved a specificity of 95%. Patient-derived CTCs exhibited smaller mean diameters compared to cultured NSCLC cell lines, yet were effectively enriched through hydro-dynamic tuning. These findings demonstrate that the Cellsway platform enables efficient and re-producible CTC isolation with high specificity, supporting its potential utility for clinical monitoring and precision oncology in NSCLC. Full article

Droplet microfluidics enables high-throughput, compartmentalized reactions using minimal reagent volumes, but most implementations rely on precision-fabricated chips and external pumping systems that limit portability and accessibility. Here, we present a handheld vibrational droplet generator that repurposes a consumer electric toothbrush and a modified disposable pipette tip to produce nearly monodisperse water-in-oil droplets without microfluidic channels or syringe pumps. The device is powered by the toothbrush’s built-in motor and controlled by a simple 3D-printed adapter and adjustable counterweight that tune the vibration amplitude transmitted to the pipette tip. By varying the aperture of the pipette tip, droplets with diameters from ~100–300 µm were generated at rates of ~100 droplets s⁻¹. Image analysis revealed narrow size distributions with coefficients of variation below 5% in typical operating conditions. We further demonstrate proof-of-concept applications in digital loop-mediated isothermal amplification (LAMP) and microbiological colony-forming unit (CFU) assays. A commercial feline parvovirus (FPV) kit manufactured by Beyotime Biotechnology Co., Ltd. (Shanghai, China), three template concentrations yielded emulsified reaction droplets that remained stable at 65 °C for 45 min and produced distinct fractions of fluorescent-positive droplets, allowing estimation of template concentration via a Poisson model. In a second set of experiments, the device was used as a droplet-based spreader to dispense diluted Escherichia coli suspensions onto LB agar plates, achieving uniform colony distributions across the plate at different dilution factors. The proposed handheld vibrational generator is inexpensive, easy to assemble from off-the-shelf components, and minimizes dead volume and cross-contamination because only the pipette tip contacts the sample. Although the current prototype still exhibits device-to-device variability and moving droplets in open containers complicate real-time imaging, these results indicate that toothbrush-based vibrational actuation can provide a practical and scalable route toward “lab-in-hand” droplet assays in resource-limited or educational settings. Full article

Background: Enterococcus faecalis is an opportunistic pathogen that is capable of causing bacterial encephalitis under specific pathological conditions. MicroRNAs (miRNAs) are a class of small, single-stranded non-coding RNAs, typically approximately 21 nucleotides in length. As master regulators of gene expression, they orchestrate critical pathways across diverse organisms and a broad spectrum of diseases; however, their role during E. faecalis neuro-invasion remains unexplored. Methods: A lamb model of E. faecalis-induced encephalitis was established. Integrated analysis of high-throughput sequencing data identified oar-miR-29b as a key differentially expressed miRNA during infection. To first verify its association with inflammation, primary SBMECs were stimulated with lipoteichoic acid (LTA), confirming that oar-miR-29b expression was significantly upregulated under inflammatory conditions. Subsequently, independent gain- and loss-of-function experiments in SBMECs were performed, with inflammatory cytokine expression assessed by qPCR and tight-junction protein levels evaluated by Western blotting. Results: Functional studies demonstrated that oar-miR-29b acts as a pro-inflammatory mediator, significantly upregulating IL-1β, IL-6, and TNF-α while degrading tight-junction proteins (ZO-1, occludin, and claudin-5), thereby compromising endothelial barrier integrity. Mechanistically, bioinformatic prediction and dual-luciferase reporter assays confirmed C1QTNF6 as a direct target of oar-miR-29b. The oar-miR-29b/C1QTNF6 axis is thus defined as a novel regulatory pathway contributing to neuro-inflammation and blood-brain barrier disruption. Conclusions: Collectively, our findings identify the oar-miR-29b/C1QTNF6 axis as a novel pathogenic mechanism that exacerbates E. faecalis-induced neuroinflammation and blood-brain barrier disruption. Full article

Behavioral individuality, often termed animal personality, reflects consistent patterns of behavioral variability across individuals. In fruit flies (Drosophila melanogaster), pharmacological and dietary manipulations affecting neuromodulatory systems have been shown to alter behavior, but their effects on behavioral predictability remain incompletely understood. Here, we investigated whether developmental dietary exposure to tryptophan (a serotonin precursor) or escitalopram (a selective serotonin reuptake inhibitor, SSRI) is associated with changes in lateralized turning behavior. Flies were reared from larval stages on supplemented media and tested in a Y-maze assay to assess movement predictability. Flies exposed to escitalopram displayed significantly reduced behavioral variability compared to controls, indicated by a lower median absolute deviation (MAD) of turning behavior, whereas tryptophan supplementation did not significantly affect variability. Because both compounds were tested at a single dietary dose and serotonergic activity was not directly measured, these findings should be interpreted as dose-specific behavioral effects rather than evidence of altered serotonergic tone or mechanism. Our results demonstrate that chronic developmental exposure to escitalopram is associated with increased behavioral predictability in fruit flies, highlighting the utility of high-throughput behavioral assays for detecting subtle pharmacologically induced changes in individual variability. Future studies incorporating dose–response designs and physiological validation will be required to establish underlying mechanisms. Full article

The continuing spread of SARS-CoV-2 and the associated morbidity and mortality, especially in vulnerable populations, highlight the need for the development of antiviral therapeutics. Reverse genetics systems and reporter viruses are valuable for antiviral screening by simplifying methods to detect and quantify virus infections. This study aimed to generate wild-type and Nluc reporter full-length SARS-CoV-2 molecular clones and viruses as tools for high-throughput antiviral assays. The large SARS-CoV-2 genome (~30 kb) makes cDNA cloning and virus rescue technically challenging, so we opted to use cDNA chemical synthesis services to generate full-length wild-type and reporter Delta and Omicron clones. Clone-derived Delta and Omicron wild-type and reporter viruses were successfully rescued and showed replication kinetics comparable to patient-derived isolates. Nluc reporter viruses displayed stable luciferase expression that correlated with viral titres, supporting their reliability as replication substitutes. Antiviral assays measuring replication inhibition by Remdesivir, Molnupiravir, and Nirmatrelvir, based on Nluc expression, yielded IC₅₀ values and selectivity indices consistent with published ranges. Finally, Delta Nluc viruses replicated in primary human bronchial epithelial cells, demonstrating the application of clone-derived viruses in physiologically relevant models. The SARS-CoV-2 cDNA clones and Nluc reporter viruses derived from DNA synthesis services provide a rapid, scalable reverse genetics platform for generating new viruses and developing assays to rapidly assess antiviral compounds against current and emerging SARS-CoV-2 variants or coronaviruses that may emerge in the future. Full article

Early-onset colorectal cancer (EOCRC) in people < 50 years of age has been rising globally, yet its causes remain unknown. Emerging evidence suggests that environmental factors, including exposure to micro-and nanoplastics (MNPs), may contribute to colorectal carcinogenesis. MNPs can enter the gastrointestinal tract through ingestion, translocate across the epithelial barrier via endocytosis or paracellular pathways, and interact directly with epithelial and immune cells. Once internalized, they may generate events associated with tumor initiation including oxidative stress, disruption of membrane integrity, pro-inflammatory signaling, and disruption of genomic and epigenomic stability. Patient-derived colorectal organoids offer a physiologically relevant and scalable 3D model that closely mimics the cellular architecture and genetic landscape of primary tumors. We highlight how organoid models can be leveraged to study the impact of MNPs on the key processes of inflammation, DNA damage, senescence, and epigenetic modifications. Furthermore, we discuss the application of organoid-based systems to model EOCRC driven by environmental exposures, including the integration of organoid platforms with high-throughput assays, omics profiling, and microfluidics to better capture MNP-induced pathogenic mechanisms. Altogether, colorectal organoids provide a powerful bridge between environmental plastic exposure and EOCRC etiology, offering a tractable platform to identify mechanistic pathways and potential biomarkers of early disease. Full article