Search Results (1,934)

Mechanical stimulation critically regulates mesenchymal stem cell (MSC) differentiation, yet its effects in three-dimensional (3D) environments remain poorly defined. Here, we developed a custom dynamic stretcher integrating poly(dimethylsiloxane) (PDMS) chambers to apply cyclic strain to human MSCs encapsulated in Fmoc-diphenylalanine (Fmoc-FF) peptide hydrogels—a fully synthetic, tunable extracellular matrix mimic. Finite element modeling verified uniform strain transmission across the hydrogel. Dynamic stretching at 0.5 Hz and 10% strain induced pronounced cytoskeletal alignment, enhanced actin stress fiber formation (coherency index $\approx 0.85$), and significantly increased proliferation compared to static or high-frequency (2.5 Hz, 1%) conditions (coherency index $\approx 0.6$). Quantitative image analysis confirmed strain-dependent increases in coherency index and F-actin intensity, indicating enhanced mechanotransductive remodeling. Biochemical assays and qRT–PCR revealed 2–3-fold upregulation of osteogenic markers—RUNX2, ALP, COL1A1, OSX, BMP, ON, and IBSP—under optimal strain. These results demonstrate that low-frequency, high-strain mechanical loading in 3D peptide hydrogels activates RhoA/ROCK and YAP/TAZ pathways, driving osteogenic differentiation. The integrated experimental–computational approach provides a robust platform for studying mechanobiological regulation and advancing mechanically tunable biomaterials for bone tissue engineering. Full article

Salmonella, a major global foodborne pathogen, is a leading cause of salmonellosis. Quantitative detection of Salmonella provides a scientific basis for establishing microbiological criteria and conducting risk assessments. The plate count method remains the primary approach for bacterial quantification, whereas the most probable number (MPN) method is commonly used for detecting low levels of bacterial contamination. However, both methods are time-consuming and labor-intensive. Validated digital polymerase chain reaction (dPCR) techniques are emerging as promising alternatives because they enable rapid, absolute quantification with high specificity and sensitivity. Herein, we developed a novel droplet dPCR (ddPCR) assay for identifying and quantifying Salmonella using invA as the target. The assay demonstrated high specificity and sensitivity, with a limit of quantification of 1.1 × 10² colony-forming units/mL in meat samples. Furthermore, the log₁₀ values obtained via ddPCR and plate counting exhibited a strong linear relationship (R² > 0.99). Mathematical modeling of growth kinetics further confirmed a high correlation between plate count and ddPCR measurements (Pearson correlation coefficient: 0.996; calculated bias factor: 0.88). Collectively, these results indicate that ddPCR is a viable alternative to the MPN method and represents a powerful tool for the quantitative risk assessment of food safety. Full article

Methicillin-resistant Staphylococcus aureus (MRSA), a major global public health threat due to its broad resistance, urgently requires the development of new antibiotic alternatives. (‒)‒Epigallocatechin-3-gallate (EGCG) is considered a natural bioactive compound with anti-MRSA properties. The L-Lectin module of serine-rich adhesin for platelets (SraP) is considered an important target for blocking MRSA-infected hosts. This study aims to investigate the mechanism of action of EGCG against MRSA. Surface plasmon resonance (SPR), cell adhesion and invasion, biofilm formation, checkerboard assays, RNA sequencing (RNA-seq) and quantitative real-time polymerase chain reaction (qRT-PCR) were performed. The results showed that EGCG bound to SraP L Lectin with high affinity and effectively inhibited MRSA colonization. Additionally, EGCG significantly suppressed pyrimidine metabolism and downregulated related genes, thereby potentially inhibiting bacterial growth. It also markedly reduced the expression of multiple genes associated with β-lactam resistance and inhibited biofilm formation. A strong synergistic effect was observed between EGCG and the bactericidal agent ceftriaxone (CRO). When combined with 10 μg/mL EGCG, CRO required 75% less dosage and exhibited a prolonged antimicrobial effect. In conclusion, EGCG exerts anti-MRSA effects through multiple pathways and represents a promising candidate as an alternative therapeutic agent against MRSA infections. Full article

Background/Objectives: We investigated whether palmitic acid (PA) induced the expression of inflammatory cytokines and was involved in pyroptosis in a human dental pulp cell line. Methods: Human dental pulp cells cultured in Minimum Essential Medium Alpha (αMEM) were treated with 1 µg/mL LPS and/or PA (100, 300 and 500 µM). As a control, αMEM was added in the culture medium. The WST-1 assay was performed to assess cell proliferation, and morphological changes in cells were examined. RNA expression of IL-1β, IL-6, TNF-α, caspase-4 and gasdermin d were detected by quantitative RT-PCR (qPCR). Results: The WST-1 assay showed that cell viability decreased by 36% at 300 µM and 47% at 500 µM PA compared to the control (p < 0.05). Cell morphology revealed slight shrinkage in 100, 300 and 500 µM PA groups. RNA expression of IL-1β and IL-6 in the PA groups was significantly higher than that in the control groups (p < 0.05), while RNA expression of TNF-α in the PA group was the same as that of control group. The mRNA expression of caspase-4 and gasdermin d in PA groups was significantly higher than that in control group (p < 0.05). Likewise, the concentration of IL-1β and IL-6 was significantly higher in both LPS and PA groups than that in the LPS or PA groups (p < 0.05). Conclusions: The results of this study suggest that PA induces the expression of inflammatory cytokines and is involved in pyroptosis in a human dental pulp cell line. Full article

Background and Objectives: Kidney transplant recipients (KTRs) face increased susceptibility to persistent viral infections due to prolonged immunosuppression. While high-risk human papillomavirus (HR-HPV) infection is known to be more prevalent in this population, little is known about the co-occurrence of HPV with human herpesviruses (HHVs) infection in the female genital tract. This study aimed to investigate the presence, dynamics, and potential interactions between HR-HPV and HHVs infections—including HSV-1, HSV-2, EBV, CMV, HHV-6, and HHV-7—in female KTRs during the first year after transplantation. Materials and Methods: A total of 39 female KTRs and 79 age-matched healthy controls were enrolled in the study. Cervicovaginal swabs from recipients were obtained at three time points: two weeks, six months, and one year post-transplantation. HPV DNA was screened using PCR, followed by high-risk HPV genotyping and quantitative viral load assessment using two commercial PCR kits. HHVs were detected using a multiplex PCR assay. Results: HPV DNA was detected in 98% of the KTRs at least once during follow-up, which was significantly greater than in the controls (38%). HR-HPV was identified in 46% of the recipients over the study period, with the highest viral load at one year post-transplantation. HHVs were detected in 72% of the KTRs but not in 43% of the controls (p < 0.01), with EBV and CMV being the most common. Coinfection with HR-HPV and HHVs occurred in 46% of the recipients but not in the controls. Samples containing both EBV and CMV had significantly higher HR-HPV viral loads than samples with no HHVs or with single/other HHV combinations (p < 0.01). All cervical intraepithelial neoplasia patients were found to have combined HPV and HHV infection. Conclusions: Female KTRs present a high burden of both HR-HPV and herpesviruses infections, with increased HPV viral loads. These findings suggest a potential synergistic interaction between HR-HPV and herpesviruses in the immunosuppressed setting. Full article

The primary objective of this study was to develop and optimize a colorimetric reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay for porcine abortion-associated pestivirus (PAAPeV)—an emerging pathogen that causes severe reproductive disorders in swine, for which no effective treatments or vaccines are currently available. In this study, four sets of LAMP primers were designed and screened for the colorimetric RT-LAMP assay, targeting the highly conserved 5′ untranslated region (5′UTR) of PAAPeV. Three reaction parameters, including reaction temperature, reaction duration, and inner-to-outer primer ratio, were then optimized based on cycle threshold (Ct) values, fluorescence intensity, and color changes of the endpoint products. Subsequently, the specificity and sensitivity of the optimized colordetect RT-LAMP assay were systematically validated, and its diagnostic performance was compared with that of the gold-standard reverse transcription quantitative polymerase chain reaction (RT-qPCR). The results demonstrated that the optimized assay achieved a detection limit of 2 copies/μL under the conditions of 65 °C incubation for 25 min and an inner-to-outer primer ratio of 8:1, with results amenable to naked-eye interpretation. Furthermore, this assay exhibited high specificity, showing no cross-reactivity with other known pestiviruses or prevalent swine pathogens. Clinical sample testing results showed 100% concordance between colordetect RT-LAMP and RT-qPCR. Collectively, this colordetect RT-LAMP assay represents a rapid, sensitive, and specific tool for PAAPeV RNA detection in both clinical laboratories and field settings. Full article

Background: Diabetic nephropathy (DN) is largely driven by transforming growth factor-β1 (TGF-β1)-mediated fibrosis. Clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein 9 (Cas9) ribonucleoprotein (RNP) complexes offer precise gene disruption, yet effective non-viral delivery remains a challenge. This study developed cationic lipid-based hybrid nanostructured lipid carriers (NLCs) for intracellular delivery of TGFB1-targeting RNP as an early-stage platform for DN gene modulation. Methods: A single-guide RNA (sgRNA) targeting human TGFB1 was assembled with Cas9 protein (1:1 and 1:2 molar ratios). Hybrid NLCs comprising squalene, glyceryl trimyristate, and the cationic lipid 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP) were formulated via optimized emulsification–sonication to achieve sub-100 nm particles. Physicochemical properties, including polydispersity index (PDI), were assessed via dynamic light scattering (DLS), while silencing efficacy in HEK293T cells was quantified using quantitative reverse transcription PCR (RT-qPCR) and enzyme-linked immunosorbent assay (ELISA). Results: Optimized NLCs achieved hydrodynamic diameters of 65–99 nm (PDI < 0.5) with successful RNP complexation. The 1:2 Cas9:sgRNA formulation produced the strongest gene-editing response, reducing TGFB1 mRNA by 67% (p < 0.01) compared with 39% for the 1:1 ratio. This translated to a significant reduction in TGF-β1 protein (p < 0.05) within 24 h. Conclusions: DOTAP-based hybrid NLCs enable efficient delivery of CRISPR–Cas9 RNP and achieve significant suppression of TGFB1 expression at both transcriptional and protein levels. These findings establish a promising non-viral platform for upstream modulation of profibrotic signaling in DN and support further evaluation in kidney-derived cells and in vivo renal models. Full article

The Rhodomyrtus tomentosa (Aiton.) Hassk. berry is rich in structurally diverse polysaccharides with potential biological activity. However, its immunomodulatory properties remain understudied, limiting our current understanding of its functional significance. Two structurally distinct polysaccharides from Rhodomyrtus tomentosa (RTP-1 and RTP-2) were evaluated for immunostimulatory activity in RAW264.7 macrophages. Phagocytic function was assessed by neutral red assay, nitric oxide (NO) and reactive oxygen species were measured using the Griess assay and fluorescent probes, and cytokines (TNF-α, IL-6 and IL-1β) were quantified by enzyme-linked immunosorbent assay. Analysis of RNA-seq data using weighted gene co-expression network analysis revealed co-expression modules. The selected transcripts were independently validated by quantitative real-time PCR (RT-qPCR). The results showed that both polysaccharides enhanced phagocytosis, increased NO/ROS levels, and promoted cytokine secretion. Transcriptome results indicated that RTP-2 activated the MEturquoise co-expression module containing 222 hub genes, whereas RTP-1 was mainly associated with the MECyan module containing 49 hub genes. Module enrichment for RTP-2 revealed links with mitophagy–immune regulation, proteostasis/stress, and innate immune signaling. RT-qPCR further confirmed that in the RTP-2 group, Dram1 expression was upregulated approximately 121 times, Bmf1 expression was upregulated approximately 18 times, and Bnip3 was significantly downregulated, whereas Bnip3l expression remained unchanged. Overall, RTP-2 exhibited a more pronounced and coherent macrophage-stimulating profile in vitro, supporting its potential as a macrophage-targeted immunostimulatory ingredient. Full article

Point-of-care testing (POCT) has emerged as a vital diagnostic approach in emergency medicine, primary care, and resource-limited environments because of its convenience, affordability, and capacity to provide immediate results. Here, we present a multifunctional portable nucleic acid detection platform integrating reverse transcription polymerase chain reaction (RT-qPCR) and reverse transcription loop-mediated isothermal amplification (RT-LAMP) within a unified microfluidic device. The system leverages Tesla-valve-based passive flow control to enhance reaction efficiency and operational simplicity. A four-channel optical detection unit allows for multiplex fluorescence quantification (CY5, FAM, VIC, ROX) and has high sensitivity and reproducibility for RT-LAMP. The compact design reduces the overall size by approximately 90% compared with conventional qPCR instruments. For RT-PCR, the system achieves a detection limit of 2.0 copies μL⁻¹ and improves analytical efficiency by 27%. For RT-LAMP, the detection limit reaches 2.95 copies μL⁻¹ with a 14% enhancement in analytical efficiency. Compared with commercial qPCR instruments, the device maintains equivalent quantitative accuracy despite significant miniaturization, ensuring reliable performance in decentralized testing. Furthermore, the total RT-LAMP assay time is reduced from more than two hours to 42 min, enabling truly rapid molecular diagnostics. This dual-mode platform offers a flexible, scalable strategy for bridging laboratory-grade molecular assays with real-time POCT applications, supporting early disease detection and epidemic surveillance. Full article

Background: Type 2 diabetes mellitus (T2DM) is typically characterized by the dysregulation of metabolic remodeling. As a systemic metabolic disease, T2DM can affect the mass and function of skeletal muscle by inducing impaired energy metabolism, mitochondrial dysfunction, and chronic low-grade inflammation. β-Hydroxybutyrate dehydrogenase 1 (BDH1) is a rate-limiting enzyme involved in ketone body metabolism, and its activity is down-regulated in various models of diabetic complications. Aerobic exercise (AE) is recognized as an effective intervention to promote energy homeostasis and alleviate metabolic stress. Whether its protective effect on skeletal muscle in T2DM involves the regulatory control of BDH1 expression remains unclear. Methods: Wild-type (WT) and systemic BDH1 knockout (BDH1^−/−) male C57BL/6J mice were used to establish the sedentary control (SED) and AE models of T2DM by providing a high-fat diet combined with streptozotocin injection. The indicators related to metabolic remodeling were detected by hematoxylin and eosin staining, immunofluorescence staining, quantitative real-time PCR, and Western blot assays. Results: After 8 weeks of AE, we found that AE improved glycolipid metabolic disorders and mitochondrial quality control in the gastrocnemius muscle of T2DM mice by up-regulating BDH1, thereby alleviating oxidative stress, inflammation, and fibrosis. Compared with the WT mice, the BDH1^−/− T2DM mice in the SED group exhibited more severe phenotypic impairment. The metabolic improvement effect of AE was attenuated in the BDH1^−/− mice. Conclusions: BDH1 is a key effector enzyme that may mediate the AE-induced improvement in metabolic remodeling in the gastrocnemius muscle of mice with T2DM. Full article

Background/Objectives: Cigarette smoke (CS) drives pathogenesis across the spectrum of chronic respiratory disorders, exerting its detrimental effects primarily through oxidative stress and programmed cell death. Scutellarein (Scu), a botanical-origin flavonoid enriched in respiratory therapeutics-oriented Chinese medicinal herbs, demonstrates established anti-inflammatory applications. This study systematically evaluated the protective roles of Scu against CS-induced lung injury and explored the underlying mechanisms. Methods: Subacute CS-exposed mice were used to evaluate the therapeutic effects of Scu on lung injury. Immunofluorescence and quantitative PCR were used to examine the expression levels of junctional proteins and proinflammatory mediators. Apoptotic cell death was quantified using Annexin V-FITC/7-AAD staining. Transepithelial electrical resistance and dextran permeability assay were used to access the barrier integrity in alveolar epithelial MLE-12 cells. Western blotting was used to detect the changes in the signal pathway. Results: In CS-exposed mice, Scu administration dose-dependently reduced histopathological scores, pulmonary edema, changes in the alveolar structure, and inflammatory cell infiltration. In MLE-12 cells, Scu significantly suppressed cigarette smoke condensate (CSC)-induced inflammatory mediators, oxidative stress, caspase-3 activation, and apoptosis and preserved CSC-suppressed tight junction protein expression and barrier disruption. Scu also rescued CSC-altered expression levels of Hrk, Ecscr, and Myo5b and mitigated the CSC-suppressed PI3K/AKT/mTOR pathway. Conclusions: Scu alleviates CS-induced subacute lung injury through its antioxidant, anti-apoptotic effects to maintain epithelial barrier integrity likely via the mitigation of the CSC-suppressed PI3K/AKT/mTOR pathway. Full article

Fibroblasts play a fundamental role in maintaining tissue architecture, regulating repair processes, and adapting to metabolic and inflammatory stress. Increasing evidence indicates that endogenous bioelectrical states contribute to gene expression regulation and cellular homeostasis. In this study, we investigated the effects of Radio Electric Asymmetric Conveyer (REAC) Metabolic Optimization–Inside Blue Zone (MO-IBZ) treatment on key regulators of stress response and metabolic control in human foreskin fibroblasts (HFF-1). Cells were exposed to nine standardized REAC MO-IBZ sessions, and changes in gene and protein expression were evaluated. Quantitative RT-PCR revealed a significant downregulation of SIRT1 and an upregulation of PPAR-γ expression in treated cells compared with untreated controls. These findings indicate molecular changes involving stress-responsive and metabolic regulatory pathways; however, they should be interpreted primarily as transcriptional signatures, as no direct functional stress-response or metabolic assays were performed. Immunofluorescence analysis showed visually increased expression of mTOR, IGF-1 receptor, and cytochrome c in REAC-treated fibroblasts, supporting a qualitative indication of activation of pathways associated with anabolic signaling, mitochondrial function, and metabolic efficiency. Taken together, these findings indicate that REAC MO-IBZ induces a coordinated molecular profile compatible with changes in cellular metabolic regulatory capacity. Within the framework of current bioelectrical literature, these changes may plausibly reflect broader regulatory adaptations; however, the present work does not provide direct measurements of bioelectrical parameters, functional metabolic activity, or epigenetic regulation, and therefore such interpretations remain speculative. These results provide descriptive mechanistic evidence supporting further investigation of REAC-based bioelectrical modulation as a potential strategy to influence cellular pathways involved in metabolic balance and tissue repair, encouraging future studies incorporating direct bioelectrical, epigenetic, and functional analyses. Full article

Carpione rhabdovirus (CAPRV) is an emerging virus within the family Rhabdoviridae, posing potential threats to aquaculture species such as golden pompano (Trachinotus anak). However, since the 21st century, and for CAPRV strains isolated from marine fish, only a single CAPRV2023 sequence has previously been available in public databases, with no additional sequences reported. Because the virus undergoes genetic variation, relying on this single sequence likely introduced mismatches or off-target risks in earlier detection assay designs. Notably, the previously developed two-step N-targeting detection assay was designed based solely on that single CAPRV2023 sequence. Consequently, this study involved determining and analyzing the N gene sequences from CAPRV isolates gathered from 2023 to 2025, with the aim of pinpointing conserved regions for assay development, and sequence comparisons subsequently verified the existence of mismatches in the primer–probe binding sites of the previous assay. Since quantitative assays in aquatic virology often define copy numbers utilizing either plasmid DNA templates or RNA templates produced via in vitro transcription, which may lead to variations in amplification kinetics and sensitivity, this study compared both standards to ensure reliable quantification across different nucleic acid types. Based on these findings, a one-step TaqMan quantitative PCR (qPCR) assay was developed and validated using dual nucleic acid standards, namely plasmid DNA and in vitro–transcribed RNA. Compared with conventional two-step qPCR, the one-step format combines cDNA synthesis and subsequent DNA amplification in a single sealed tube, thereby effectively preventing cross-contamination, simplifying the workflow, and improving detection efficiency. The assay exhibited strong linearity (R² > 0.99) and consistent amplification efficiencies between 90% and 110%, demonstrating excellent quantitative performance. The detection limits were 2 copies per reaction for plasmid DNA and 20 copies for in vitro–transcribed RNA templates. No cross-reactivity was observed with other aquatic pathogens, and the assay showed strong repeatability and reproducibility (coefficients of variation below 2.0%), providing a sensitive and reliable tool for epidemiological surveillance and the analysis of CAPRV distribution in marine aquaculture systems of southern China. Full article

Psoriasis is a common inflammatory skin disease with chronic manifestation in which the role of neutrophil extracellular traps (NETs) and alarmins are increasingly recognized as contributors to systemic and cutaneous inflammation. However, the interaction between alarmins and NET-driven immune responses remains poorly defined. The main aim of this study is to define the role of target alarmins (i.e., IL-33 and TSLP) in NETs induction and its subsequent impact on oxidative stress and inflammation in the peripheral blood. In the present study, we recruited active psoriasis patients (n = 56) and control (n = 56) subjects. The frequency of circulating neutrophils, the levels of NET-associated markers (MPO (myeloperoxidase)–DNA complex, CitH3 (citrullinated histone H3), PAD4 (peptidyl arginine deiminase4), NADPH oxidase, and NE (neutrophil elastase)), and alarmin transcripts (IL (interleukin)-33, TSLP (thymic stromal lymphopoietin), S100A7, S100B, HSP (heat shock protein) 60/70 were quantified using flow cytometry, ELISA (Enzyme-linked immunosorbent assay), and qPCR (quantitative polymerase chain reaction), respectively, in each group. The NET formation potential of isolated neutrophils was assessed in the presence or absence of rhIL-33 and rhTSLP by immunocytofluorescence. The effect of rhIL-33- and rhTSLP-primed NETs in augmenting oxidative stress and inflammation was evaluated on peripheral blood mononuclear cells (PBMCs) by ELISA. Significantly higher circulating neutrophils (p < 0.001) and levels of NET-associated markers (i.e., MPO–DNA complex, CitH3, PAD4, NADPH oxidase, and NE) were observed in active psoriasis patients compared to controls. Lesional skin exhibited strong expression of MPO (p < 0.001) compared to normal skin. The alarmins, IL-33 and TSLP, were markedly upregulated in the blood and skin (p < 0.05). The rhIL-33 and rhTSLP treated neutrophils demonstrated enhanced NETosis in patients (p < 0.001). Increased expression of inflammatory cytokines and oxidative stress markers were reported in PBMCs when incubated with rhIL-33- and rhTSLP-primed NETs. Taken together, our investigation demonstrated the novel mechanism wherein the alarmins IL-33 and TSLP exacerbate NET formation that may drive enhanced inflammation and oxidative stress in psoriasis. Full article

Heterocapsa bohaiensis is an emerging harmful dinoflagellate increasingly reported from coastal regions of the Pacific. However, an available molecular assay offering rapid and sensitive detection is still lacking. This study developed a SYBR Green real-time quantitative PCR (qPCR) assay for the identification and quantification of H. bohaiensis. Species-specific primers (F: 5′-CCATCGAACCAGAACTCCGT-3′; R: 5′-AGTGTAGTGCACCGCATGTC-3′) were designed and the assay was optimized and evaluated using laboratory cultures for specificity, sensitivity, and quantitative performance. Primer screening and melt-curve analysis confirmed that the selected primer pair produced a single, specific amplification peak for H. bohaiensis, with no cross-reactivity observed in non-target species (Chlorella pyrenoidosa, Phaeocystis globosa, Skeletonema costatum, Alexandrium tamarense) or mixed algal communities. The standard curve displayed strong linearity (R² = 0.9868) and a high amplification efficiency (102.5%). The limit of detection (LOD) was approximately 2–3 cells per reaction, as determined from 24 replicates of 5-cell equivalents and verified at ~2.7-cell equivalents. This sensitivity was comparable to or exceeded that reported for assays targeting other HABs forming dinoflagellates. Quantitative results derived from the qPCR assay closely matched microscopic cell counts, with a relative error of 10.79%, falling within the acceptable threshold for phytoplankton surveys. In summary, this study established and validates a species-specific qPCR assay for H. bohaiensis under controlled laboratory conditions. The method shows strong potential for incorporation into HAB monitoring programs, early-warning systems, and future ecological investigations of this emerging species. Full article