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Search Results (514)

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Keywords = real-time fluorescence PCR

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22 pages, 4418 KB  
Article
Mechanistic Investigation of Vitexin in Ameliorating Ovarian Fibrosis in PCOS Mice via the NR4A1/NLRP3 Signaling Pathway
by Haoran Sun, Jiejing Xu, Chengxue Pan, Jia-Le Song and Yanyuan Zhou
Metabolites 2026, 16(5), 332; https://doi.org/10.3390/metabo16050332 - 15 May 2026
Viewed by 165
Abstract
Objective: In this study, Dehydroepiandrosterone (DHEA-induced Polycystic Ovary Syndrome (PCOS) mice were used as models to evaluate the improvement effect of Vitexin (Vit) on ovarian fibrosis and explore the mechanism of action of the NR4A1/NLRP3 signaling pathway. Method: Sixty 4-week-old female ICR mice [...] Read more.
Objective: In this study, Dehydroepiandrosterone (DHEA-induced Polycystic Ovary Syndrome (PCOS) mice were used as models to evaluate the improvement effect of Vitexin (Vit) on ovarian fibrosis and explore the mechanism of action of the NR4A1/NLRP3 signaling pathway. Method: Sixty 4-week-old female ICR mice of the same batch number were selected and their systems were divided into 6 groups (n = 10): normal (Control, Ctrl) group, model (Polycystic Ovary Syndrome, PCOS) group, treatment (Vitexin, The Vit group, normal NR4A1 gene silencing group (Ctrl NR4A1-/-), NR4A1 gene silencing model group (PCOS NR4A1-/-), and NR4A1 gene silencing treatment group (Vit NR4A1-/-). Silencing gene modeling was performed by tail vein injection of adeno-associated virus (serotype AAV-8), and the mouse genotypes were detected by qRT-PCR technology 14 days after injection. After the genotype was determined, the PCOS group and the PCOS NR4A1-/- group were administered dehydroepandrosterone (6 mg/100 g/d) by gavage for 28 consecutive days for modeling, while the Vit group and the Vit NR4A1-/- group were treated with dehydroepandrosterone + vitexin (10 mg/kg/d) by gavage for 28 consecutive days. All mice were raised with pure water and regular maintenance food. After 4 weeks of drug intervention, the mice were euthanized and samples were collected. The pathological changes in ovarian tissue were observed by H&E staining, and the degree of ovarian tissue fibrosis was observed by Masson staining. The levels of superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GSH-Px), malondialdehyde (MDA), total cholesterol (TC), triglycerides (TG), high-density lipoprotein cholesterol (HDL-C), and low-density lipoprotein cholesterol (LDL-C) in mouse serum were detected by biochemical kits. The levels of inflammatory factors (IL-1β, IL-6, IL-18, TNF-α) in mouse serum were determined by enzyme-linked immunosorbent assay. Real-time fluorescence quantitative PCR (qRT-PCR) was used to detect oxidative kinase (Gsta4, Prdx3, Mgst1, Gpx3, Gsr), inflammatory factors (Nlrp3, Caspase-1, Asc, Il-1β, Il-18, Tnf-α) and fibrotic pathway-related genes (Tgf-β1, Smad3, Collagen1, CTGF, α-SMA, Mmp-13, and β-catenin) in ovarian tissues. The levels of inflammatory factors (NLRP3, Caspase-1, ASC, IL-1β, IL-18, TNF-α, IκBα) and fibrosis in mice were determined by Western blot method, and statistical description and analysis were performed using SPSS software. Result: In the wild-type genotype group, compared with the PCOS group, Vit treatment could effectively regulate the metabolic abnormalities of PCOS mice, including inhibiting excessive weight gain, restoring normal glucose tolerance, and reducing body fat content. After Vit treatment, the levels of MDA, TC, TG, LDL, IL-1β, IL-6, IL-18 and TNF-α in the serum of PCOS mice were significantly reduced, while the levels of SOD and HDL in the serum of PCOS mice were increased. The staining results indicated that Vit treatment could significantly inhibit the process of ovarian fibrosis in PCOS mice. The results of WB and PCR demonstrated that after Vit gavage treatment in mice, inflammatory and fibrotic factors such as Nlrp3, Caspase-1, Asc, Il-1β, Il-18, Tgf-β1, Smad3, Collagen1, CTGF, and α-SMA in ovarian tissues could be significantly down-regulated, and the fibrotic level of ovarian tissues could be reduced. Among the same measurement indicators, the silenced NR4A1 group showed a certain degree of increase compared with the wild genotype group, but there was no significant difference. Conclusions: Vit intervention can restore the sex hormone levels and follicular development in ovarian tissues of PCOS mice, regulate reproductive endocrine disorders and abnormal lipid metabolism levels, and regulate the expression of Collagen I, a-SMA and CTGF in the ovaries by inhibiting the NR4A1/NLRP3 signaling pathway, thereby improving the ovarian fibrosis level of PCOS mice. It is suggested that it may play a key role in the treatment of PCOS and the prevention and delay of its long-term complications. Full article
(This article belongs to the Section Plant Metabolism)
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17 pages, 5705 KB  
Article
Identification and Functional Analysis of ZmMAPKKKA-Interacting Proteins Involved in Cold Stress Response in Maize (Zea mays L.)
by Tao Yu, Jianguo Zhang, Xuena Ma, Shiliang Cao, Wenyue Li and Gengbin Yang
Agronomy 2026, 16(10), 978; https://doi.org/10.3390/agronomy16100978 (registering DOI) - 14 May 2026
Viewed by 125
Abstract
Maize (Zea mays L.), a typical thermophilic crop originating from tropical regions, exhibits an inherent sensitivity to low-temperature stress. Cold stress severely restricts maize seed germination, seedling growth, the physiological metabolism, and the final grain yield, which greatly limits its geographical cultivation [...] Read more.
Maize (Zea mays L.), a typical thermophilic crop originating from tropical regions, exhibits an inherent sensitivity to low-temperature stress. Cold stress severely restricts maize seed germination, seedling growth, the physiological metabolism, and the final grain yield, which greatly limits its geographical cultivation range and sustainable industrial development. Elucidating the molecular regulatory mechanisms underlying maize cold tolerance and excavating cold-resistant functional genes are essential for the molecular breeding of cold-tolerant maize varieties and expanding maize planting areas in high-latitude and low-temperature-prone regions. In this study, using the strongly cold-tolerant maize inbred line B144 as the experimental material, we cloned the ZmMAPKKKA gene (NCBI accession: LOC103651289) and systematically screened and verified its cold-stress-specific interacting proteins via multiple molecular biological assays. The full-length coding sequence (CDS) of ZmMAPKKKA is 1134 bp, encoding a 377-amino-acid protein with a predicted molecular weight of 40.37 kDa. The quantitative real-time PCR (qRT-PCR) results demonstrated that the ZmMAPKKKA expression was significantly upregulated by 16.56-fold in maize roots after 12 h of low-temperature treatment, indicating a tissue-specific and robust cold response in root tissues. A total of 25 interacting proteins were identified through yeast two-hybrid screening, among which three stress-responsive proteins, including a protein kinase (LOC100286253), a protein phosphatase 2C (PP2C) (LOC542176), and a NAC transcription factor (LOC118474710), were selected for subsequent verification. The Pull-Down, Co-immunoprecipitation (Co-IP), and bimolecular fluorescence complementation (BiFC) assays consistently confirmed that ZmMAPKKKA specifically interacts with these three proteins both in vitro and in vivo under cold stress conditions. This study is the first to construct a ZmMAPKKKA-centered protein interaction module in the maize mitogen-activated protein kinase (MAPK) cascade under cold stress, establishing a novel kinase–phosphatase–transcription factor regulatory cascade that improves the current understanding of cold signal transduction mechanisms in maize. Homologous genes of ZmMAPKKKA in gramineous crops including rice (Oryza sativa) and sorghum (Sorghum bicolor) have been proven to participate in diverse abiotic stress responses, suggesting the conserved functional roles of MAPKKK family genes across gramineous species. Collectively, our findings provide comprehensive insights into the molecular mechanism of the maize MAPK signaling pathway mediating cold stress adaptation and supply valuable functional gene resources for cold-tolerant maize germplasm innovation and molecular breeding. Full article
(This article belongs to the Special Issue Plant Stress Tolerance: From Genetic Mechanism to Cultivation Methods)
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13 pages, 1194 KB  
Article
Early Osteogenic and Stromal Marker Responses of Osteoblast-like and Bone-Marrow Stromal Cell Lines to a Hyaluronic Acid-Coated Xenogeneic Bone Graft: An Exploratory In Vitro Analysis
by Yaniv Mayer, Hia Abu Sada, Hadar Zigdon Giladi, Eran Gabay, Ofri Doppelt-Flikshtain and Ofir Ginesin
Dent. J. 2026, 14(5), 290; https://doi.org/10.3390/dj14050290 - 12 May 2026
Viewed by 239
Abstract
Background: To investigate whether coating xenogeneic bone grafts with hyaluronic acid influences early osteogenic and fibrotic marker expression in vitro. Methods: Three xenograft materials were evaluated, including one hyaluronic acid-coated product and two uncoated deproteinized bovine bone mineral products, all commercially available. Human [...] Read more.
Background: To investigate whether coating xenogeneic bone grafts with hyaluronic acid influences early osteogenic and fibrotic marker expression in vitro. Methods: Three xenograft materials were evaluated, including one hyaluronic acid-coated product and two uncoated deproteinized bovine bone mineral products, all commercially available. Human osteoblast-like cells (U2OS) and bone marrow stromal cells (HS5) were cultured with material extracts. Proliferation was assessed using XTT assay at 24 and 48 h. Cell adhesion was evaluated through fluorescence microscopy. Osteogenic markers (RUNX2, COL1A1) and fibrotic markers (COL3A1, TGF-β3) were quantified using quantitative real-time PCR. Statistical analysis employed one-way ANOVA with Benjamini–Krieger–Yekutieli (BKY) two-stage FDR correction for datasets that met the normality assumption, and the Kruskal–Wallis test with Dunn’s post hoc test for non-normally distributed data (HS5 XTT assay). Pairwise comparisons were restricted to each xenograft group versus the untreated control; an adjusted p-value < 0.05 was considered statistically significant. Results: At 48 h, the HA-coated xenograft (Xeno1) showed the highest mean metabolic activity in U2OS cells (0.538 ± 0.056) compared with the uncoated Xeno2 (0.450 ± 0.120) and Xeno3 (0.439 ± 0.073); however, after FDR correction no statistically significant differences were observed between groups. The coated material was associated with upregulation of early osteogenic markers, 2.61-fold RUNX2 upregulation (p = 0.01) compared to untreated cells. Both coated and uncoated xenografts demonstrated equivalent suppression of fibrotic markers in HS5 cells, reducing COL3A1 by 92.7% (p = 0.001) and TGF-β3 by 92.1% (p = 0.001). Conclusions: These exploratory in vitro findings suggest that HA coating may enhance early osteogenic marker expression. The observed effects on stromal markers warrant further investigation using primary cells, additional fibrotic endpoints (e.g., TGF-β1, ACTA2), and in vivo models before translational conclusions can be drawn. Full article
(This article belongs to the Special Issue Implant Dentistry—the Surgical Prosthetic Interplay)
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21 pages, 3794 KB  
Article
Stage-Dependent Antibiofilm Effects of UVA Combined with Cinnamaldehyde Against Staphylococcus aureus Biofilms on Titanium Surfaces
by Le Wan, Chan-Young Lee, Woochul Jung, Hongyan Zhou, Youzhen Zheng and Kyung-Soon Park
Antioxidants 2026, 15(5), 574; https://doi.org/10.3390/antiox15050574 - 1 May 2026
Viewed by 263
Abstract
Staphylococcus aureus biofilms formed on titanium surfaces are highly relevant to orthopedic implant-associated infection and remain difficult to control after maturation. This study aimed to evaluate whether ultraviolet A (UVA, 365 nm) combined with cinnamaldehyde (CA) could improve antibiofilm activity against titanium-associated S. [...] Read more.
Staphylococcus aureus biofilms formed on titanium surfaces are highly relevant to orthopedic implant-associated infection and remain difficult to control after maturation. This study aimed to evaluate whether ultraviolet A (UVA, 365 nm) combined with cinnamaldehyde (CA) could improve antibiofilm activity against titanium-associated S. aureus biofilms in a stage-resolved in vitro model and to examine whether the observed responses were associated with reactive oxygen species (ROS). Early stage (8 h) and 24 h biofilm models were established on total hip arthroplasty (THA)-derived titanium discs. After condition screening, 0.5 mM CA combined with 5 min UVA exposure was selected for subsequent experiments. Biofilm biomass was assessed by crystal violet staining, bacterial viability by live/dead staining and colony-forming unit (CFU) enumeration, ROS-associated fluorescence by dihydroethidium (DHE) imaging, and biofilm-associated gene expression by quantitative real-time PCR (qRT-PCR). Chondrocyte viability was also evaluated under the selected antibiofilm-effective conditions. The combined treatment showed stage-dependent antibiofilm effects, with greater biomass reduction in the 8 h biofilm model and marked impairment of bacterial viability and culturability in both models. ROS-associated fluorescence increased under combined exposure and was partially attenuated by N-acetyl-L-cysteine (NAC) in the 24 h biofilm model. In parallel, CA + UVA was associated with lower expression levels of clfA, icaA, and icaD in the 8 h biofilm model and of icaA, icaB, and icaD in the 24 h biofilm model, with partial NAC attenuation in the latter. Chondrocyte viability was lower in all treatment groups than in the untreated control, although the combined treatment did not show an obvious additional decrease compared with the single-treatment groups. These findings indicate that UVA combined with CA exerts stage-dependent antibiofilm effects in an in vitro titanium-associated S. aureus biofilm model. The observed ROS-associated responses were consistent with, but do not establish, mechanistic involvement. The current treatment setting also requires further optimization before translational applicability can be more confidently considered. Full article
(This article belongs to the Section ROS, RNS and RSS)
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18 pages, 34659 KB  
Article
Terahertz Waves Trigger Apoptosis in Cutaneous Squamous Cell Carcinoma via Apoptosis-Inducing Factor Mediated Mitochondrial Pathway
by Liu Sun, Wenxia Wang, Shuocheng She, Lei Wang, Jinwu Zhao, Pandeng Hou and Mingxia He
Cells 2026, 15(9), 810; https://doi.org/10.3390/cells15090810 - 29 Apr 2026
Viewed by 389
Abstract
Background: Terahertz (THz) waves exhibit both photon-like and electron-like properties, showing emerging potential in biomedical applications. Cutaneous squamous cell carcinoma (CSCC) is one of the most common skin tumors. Studies have reported that THz waves can induce apoptosis in cancer cells or ablate [...] Read more.
Background: Terahertz (THz) waves exhibit both photon-like and electron-like properties, showing emerging potential in biomedical applications. Cutaneous squamous cell carcinoma (CSCC) is one of the most common skin tumors. Studies have reported that THz waves can induce apoptosis in cancer cells or ablate tumor tissues. Our previous studies also confirmed that 0.1 THz radiation could significantly promote apoptosis in cutaneous melanoma cells, while it had no apparent effect on fibroblast viability, proliferation, migration, and apoptosis. However, the effects of 0.1 THz radiation on CSCC cells have not yet been explored. Furthermore, there remains a lack of investigation into the structural and functional effects on fibroblasts. Therefore, it is necessary to conduct a systematic study to evaluate the influence of 0.1 THz radiation on both CSCC cells and fibroblasts in order to better understand its potential therapeutic applications in the treatment of skin cancer. Purpose: This study aims to explore the biological effects of 0.1 THz radiation on SCC-7 cells and to uncover the molecular mechanisms underlying THz-induced apoptosis, as well as its potential effect on L-929 cells. Methods: Cell viability was evaluated through the CCK-8 assay, while cell cycle distribution was analyzed with the DNA content detection kit. Wound healing assays were performed to assess cell migration, and Annexin V-FITC staining was used to detect apoptosis. Caspase-3 activity was measured using the caspase-3 activity assay kit. Cell morphology was observed using the Atomic Force Microscope (AFM) and the Transmission Electron Microscopy (TEM). Alterations in membrane potential were detected with the M09 membrane potential probe kit, and intracellular Ca2+ levels were quantified using the Fluo-8 AM fluorescent probe. Mitochondrial permeability transition pore (mPTP) opening was assessed with the MPTP detection kit, mitochondrial membrane potential changes were measured using the JC-1 probe kit, and cellular ATP levels were measured with the enhanced ATP assay kit. Subsequently, proteomic analysis was performed. Intracellular reactive oxygen species (ROS) levels were quantified with the ROS detection kit, and cytochrome c (Cyt c) release was quantified using the mouse Cyt c ELISA kit. Apoptosis-inducing factor (AIF) expression was analyzed at both mRNA and protein levels by quantitative real-time PCR (qPCR) and Western blot. AIF expression in CSCC tissues was further evaluated based on the GSE42677 and GSE45164 databases. Finally, cyclosporin A (CsA) was used to inhibit mPTP, and in combination with the iMAC inhibitor, the Aifm1 expression and Cyt c release were examined. Results: Our results showed that THz waves significantly disrupted the membrane integrity of SCC-7 cells and induced mitochondrial structural and functional damage. This resulted in a significant increase in ROS levels and the activation of mPTP and the mitochondrial apoptosis channel (MAC). THz radiation promoted the release of Cyt c and AIF from mitochondria, triggering a noncanonical caspase-3-dependent apoptosis pathway. Notably, L-929 cells did not show significant phenotypic or apoptotic changes under the same irradiation conditions. Bioinformatics analysis of the Gene Expression Omnibus (GEO) database revealed that AIF expression was significantly altered in CSCC tissues compared to normal skin tissues. Conclusions: These findings indicated that 0.1 THz radiation effectively induced apoptosis in SCC-7 cells by triggering mitochondrial dysfunction and ROS generation, which led to the release of AIF. Furthermore, the dysregulation of AIF in CSCC tissues suggested its potential as a promising biomarker. These results provided important molecular insights into the therapeutic potential of THz radiation, particularly for the treatment of cutaneous squamous cell carcinoma. Full article
(This article belongs to the Section Cellular Biophysics)
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16 pages, 1194 KB  
Article
A Multiplex One-Tube Nested Real-Time PCR Assay for the Point-of-Care Testing of Infectious Meningitis
by Duoxiao Zhang, Jie Wang, Zijin Zhao, Yanqing Tie, Jianing Wu, Shihao Jiao, Xingyu Liu, Yuxin Wang, Shijue Gao, Mengchuan Zhao, Pei Zhao, Zhiqiang Han, Xiaona Lyu, Xinxin Shen, Xuejun Ma and Zhishan Feng
Pathogens 2026, 15(5), 456; https://doi.org/10.3390/pathogens15050456 - 22 Apr 2026
Viewed by 336
Abstract
In this study, we developed a multiplex one-tube nested real-time fluorescent quantitative PCR (mONRT-PCR) assay integrated with a portable, fully automated nucleic acid point-of-care testing (POCT) platform for the detection of Haemophilus influenzae (H. influenzae), Listeria monocytogenes (L. monocytogenes), [...] Read more.
In this study, we developed a multiplex one-tube nested real-time fluorescent quantitative PCR (mONRT-PCR) assay integrated with a portable, fully automated nucleic acid point-of-care testing (POCT) platform for the detection of Haemophilus influenzae (H. influenzae), Listeria monocytogenes (L. monocytogenes), and Cryptococcus neoformans (C. neoformans) in cerebrospinal fluid (CSF). The assay enables nested amplification within a closed system using conventional primers and probes, thereby reducing operational complexity and minimizing contamination risk. Analytical evaluation demonstrated limits of detection of 100 copies/μL for H. influenzae and L. monocytogenes, and 101 copies/μL for C. neoformans using recombinant plasmids, as well as 10−7 to 10−6 ng/μL using genomic DNA. No cross-reactivity was observed when tested against a panel of 17 common non-target microorganisms encountered in clinical microbiology laboratories. In simulated CSF samples, the assay maintained detectable amplification at low pathogen concentrations. When implemented on the POCT platform, detection limits reached 5, 10, and 50 CFU/mL for the three pathogens, respectively. Clinical evaluation using 43 CSF samples showed almost perfect agreement with conventional qPCR (κ = 0.861, p < 0.001). Notably, additional C. neoformans detections were observed by mONRT-PCR-POCT compared with qPCR, suggesting improved sensitivity under clinical conditions. The assay yielded results within approximately 1 h and 47 min. These findings indicate that the proposed assay provides a rapid, sensitive, and integrated approach for meningitis pathogen detection, while maintaining a practical balance between analytical performance and operational simplicity. Further validation in larger cohorts is warranted. Full article
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10 pages, 2411 KB  
Article
Diagnostic and Phylogenetic Insights into a Human Rabies Virus Isolate from Romania
by Vlad Vuta, Maria Gradinaru, Mihnea Hurmuzache, Florica Bărbuceanu, Lenuta Zamfir, Răzvan Moțiu, Laura Schmid, Dirk Höper, Sten Calvelage, Thomas Müller and Conrad M. Freuling
Viruses 2026, 18(4), 475; https://doi.org/10.3390/v18040475 - 17 Apr 2026
Viewed by 493
Abstract
Rabies is a fatal zoonotic disease once clinical symptoms develop. In Europe, sustained animal rabies control programs have led to a marked decline in animal rabies and subsequently human rabies cases; however, sporadic infections continue to occur. In July 2025, a fatal case [...] Read more.
Rabies is a fatal zoonotic disease once clinical symptoms develop. In Europe, sustained animal rabies control programs have led to a marked decline in animal rabies and subsequently human rabies cases; however, sporadic infections continue to occur. In July 2025, a fatal case of autochthonous (locally acquired) human rabies was confirmed in Romania following a stray dog bite in a patient who did not receive post-exposure prophylaxis (PEP). Here, we report the first molecular characterization of a human rabies virus (RABV) strain isolated in Romania and place it in the context of contemporaneously circulating animal-derived RABV strains. Rabies virus infection was confirmed intra vitam by fluorescent antibody testing and both conventional and real-time RT-PCR on cerebrospinal fluid and saliva, with postmortem confirmation on skin and brain tissue. Whole-genome sequencing was performed on the human isolate and on 22 animal-derived RABV strains collected in northern Romania in 2025. Phylogenetic analyses revealed that all recent Romanian sequences clustered within the North-East European (NEE) rabies virus phylogenetic group and segregated into two geographically distinct genetic clusters: a north-western cluster, closely related to strains from Slovakia and Poland, and a larger north-eastern cluster, linked to viruses circulating in eastern Romania and the Republic of Moldova. The human-derived RABV genome was grouped within the north-eastern cluster and showed the highest genetic similarity to animal viral strains from the same geographical area, supporting a local transmission event. This demonstrates the importance of integrating human viral genomic data into the national rabies surveillance framework. Full article
(This article belongs to the Section Animal Viruses)
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14 pages, 751 KB  
Article
Transient miR-92a Induction in Intermediate Monocytes (CD14++CD16+) in Acute Coronary Syndrome (ACS)
by Lukas Harbaum, Julian Kreutz, Carina Weibler, Gerhild Euler, Michael Malysa, Hartmann Raifer, Bernhard Schieffer, Karsten Grote and Mariana Parahuleva
Int. J. Mol. Sci. 2026, 27(7), 3281; https://doi.org/10.3390/ijms27073281 - 4 Apr 2026
Viewed by 403
Abstract
Intermediate monocytes (CD14++CD16+), a highly pro-inflammatory subset, are linked to endothelial activation, thrombus formation, and poor outcomes in acute coronary syndrome (ACS), suggesting a role in the transition to plaque vulnerability. MicroRNA-92a (miR-92a) promotes vascular inflammation by repressing the [...] Read more.
Intermediate monocytes (CD14++CD16+), a highly pro-inflammatory subset, are linked to endothelial activation, thrombus formation, and poor outcomes in acute coronary syndrome (ACS), suggesting a role in the transition to plaque vulnerability. MicroRNA-92a (miR-92a) promotes vascular inflammation by repressing the transcription factors Kruppel-like factors (KLFs) 2/4, thereby inducing endothelial dysfunction and increasing leukocyte adhesion. Because both intermediate monocytes and miR-92a contribute to plaque instability, their expression profiles appear relevant in acute ischemia. We investigated whether miR-92a is differentially regulated in monocyte subpopulations in ACS compared to chronic coronary syndrome (CCS). Patients with ACS (STEMI/NSTEMI) undergoing urgent coronary angiography and patients with CCS were enrolled. Blood samples were collected peripherally (T0P) and from the culprit coronary artery (T0C) during catheterization. Additional peripheral samples were collected 48 h after intervention (T1) and at the 3-month follow-up (T2). Peripheral blood mononuclear cells (PBMCs) were isolated by Ficoll density-gradient centrifugation. Monocytes were sorted by fluorescence-activated cell sorting (FACS) into classical (CD14++CD16), intermediate (CD14++CD16+), and non-classical (CD14+CD16++) subsets. MiR-92a expression was measured using real-time PCR and analyzed across predefined time points. In classical and non-classical monocytes, miR-92a levels remained stable throughout the observation period and did not differ between ACS and CCS patients. No spatial expression gradient was observed between intracoronary and peripheral samples at baseline. In contrast, intermediate monocytes in the ACS cohort showed a transient increase in miR-92a expression at T1 compared with baseline (T0p) and the 3-month follow-up (T2). No comparable temporal changes were observed in CCS patients. These findings indicate a temporary alteration of miR-92a expression in intermediate monocytes during the early post-interventional phase following ACS. However, given the exploratory nature of this study and the limited sample size, the biological significance of this observation requires confirmation in larger cohorts. Full article
(This article belongs to the Section Molecular Pathology, Diagnostics, and Therapeutics)
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13 pages, 3076 KB  
Article
A Rapid Visual Detection Method for Fasciola hepatica Based on RAA-CRISPR/Cas12b
by Jiangying Li, Tao Zhang, Jingkai Ai, Zijuan Zhao, Zhi Li, Yong Fu, Dan Jia, Hong Duo, Xiuying Shen, Ru Meng, Yingna Jian and Xueyong Zhang
Animals 2026, 16(7), 1093; https://doi.org/10.3390/ani16071093 - 2 Apr 2026
Viewed by 476
Abstract
Fascioliasis, a globally prevalent zoonosis, severely threatens public health and livestock security. Current diagnostic approaches, hindered by the need for sophisticated instrumentation and specialized expertise, are inadequate for on-site surveillance in resource-constrained settings. This study developed a rapid, visual detection assay for Fasciola [...] Read more.
Fascioliasis, a globally prevalent zoonosis, severely threatens public health and livestock security. Current diagnostic approaches, hindered by the need for sophisticated instrumentation and specialized expertise, are inadequate for on-site surveillance in resource-constrained settings. This study developed a rapid, visual detection assay for Fasciola hepatica via recombinase-aided amplification (RAA) integrated with CRISPR/Cas12b, addressing critical equipment and operational constraints. Targeting a specific mitochondrial DNA fragment of F. hepatica, recombinant plasmid standards were constructed, RAA primers and sgRNA optimized, and three detection modalities (real-time fluorescence, UV lamp, test strip) integrated. Clinical validation against PCR demonstrated 45 min turnaround time, F. hepatica-specific positivity, and real-time fluorescence sensitivity of 2.6 copies/μL. Results showed high concordance with PCR and qPCR, with substantially reduced assay duration and streamlined workflow. This highly sensitive, specific, multi-visualized method overcomes limitations of conventional techniques, offering an efficient, field-deployable tool for fascioliasis surveillance and control in grassroots and pastoral regions. Full article
(This article belongs to the Section Veterinary Clinical Studies)
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15 pages, 6578 KB  
Article
Glaesserella parasuis Infection Modulates the Transcriptome of Porcine Peritoneal Mesothelial Primary Cells: Implications for Understanding Peritoneal Invasion Mechanisms
by Pu Guo, Jialong Fan, Yangfan Dong, Jiacheng Zhang, Qirong Lu, Chun Ye, Shulin Fu, Zhongyuan Wu, Yu Liu and Yinsheng Qiu
Biology 2026, 15(7), 565; https://doi.org/10.3390/biology15070565 - 1 Apr 2026
Viewed by 524
Abstract
Polyserositis is an important clinical feature of Glaesserella parasuis (G. parasuis, GPS) infection in pigs, typically presenting as peritonitis, pleuritis, pericarditis, meningitis, and arthritis, resulting in heavy economic losses in the swine industry. However, the current research on the pathogenesis of [...] Read more.
Polyserositis is an important clinical feature of Glaesserella parasuis (G. parasuis, GPS) infection in pigs, typically presenting as peritonitis, pleuritis, pericarditis, meningitis, and arthritis, resulting in heavy economic losses in the swine industry. However, the current research on the pathogenesis of infectious peritonitis, particularly that caused by GPS, remains limited, and this condition has long been poorly reported in both clinical practice and research. In this study, we investigated the overall changes in gene expression in porcine peritoneal mesothelial primary cells (PPMCs) following a GPS infection using transcriptomics analysis. A total of 779 differentially expressed genes (DEGs) were identified after 12 h of infecting the PPMCs with GPS, resulting in 253 and 526 genes being upregulated and downregulated, respectively. Additionally, 220 DEGs, mainly involved in the NOD-like receptor signaling pathway, the TNF signaling pathway, and the metabolic pathway, were enriched in the KEGG analysis. These pathways were associated with the main DEGs (IL-1β, IL6, CCL5, CCL2 and NFKBIA), and their gene expression levels were verified through quantitative real-time fluorescence PCR (qRT-PCR). Moreover, oxidative phosphorylation, Salmonella infection, rheumatoid arthritis, and other regulating pathways were clustered together. Our results provide insights into the molecular mechanism underlying GPS-induced peritonitis in swine, identify novel therapeutic targets, and provide research direction for the control and prevention of GPS infections. These insights provide a foundational basis for advancing intervention and prevention approaches for this overlooked yet clinically significant manifestation of polyserositis. Full article
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14 pages, 1334 KB  
Article
Transcriptome Sequencing and Identification of APOE Gene Polymorphisms, Their Expression and Their Relationship with Body Size Traits in Guizhou White Goats (Capra hircus)
by Wen-Ying Wang, Lin-Guang Dai, Jun-You Huang, Xing-Chao Song, Jin-Zhu Meng, Yuan-Yuan Zhao, Zhen-Yang Wu and Qing-Ming An
Animals 2026, 16(7), 1031; https://doi.org/10.3390/ani16071031 - 27 Mar 2026
Viewed by 445
Abstract
Carcass growth and development are crucial evaluation indicators influencing the economic efficiency of goats (Capra hircus). This study aimed to screen the nucleotide variation sites (SNPs) of the APOE gene in Guizhou white goats and explore the correlation between APOE gene [...] Read more.
Carcass growth and development are crucial evaluation indicators influencing the economic efficiency of goats (Capra hircus). This study aimed to screen the nucleotide variation sites (SNPs) of the APOE gene in Guizhou white goats and explore the correlation between APOE gene variations and body size traits, as APOE had been identified as a key candidate gene regulating growth and development in this breed through transcriptome sequencing screening. A total of 324 Guizhou white goats were used in this study for SNP detection, population genetic analysis, real-time fluorescence quantitative PCR (RT-qPCR) and association analysis. The results showed that one nucleotide mutation site (g.353 A > G) was detected in the APOE gene, which yielded two alleles (A and G) and three genotypes (AA, AG and GG). The site exhibited moderate polymorphism and conformed to Hardy–Weinberg equilibrium. The mRNA expression level of APOE in longissimus dorsi muscle was significantly higher in males than in females. Association analysis revealed a sex-specific effect of this locus on body size traits. The A allele and AA genotype were significantly associated with increased body weight and heart girth in females, whereas no significant effect was detected in males. Therefore, the identified APOE gene mutation site can serve as a candidate molecular marker for the early selection of growth traits in Guizhou white goats. Full article
(This article belongs to the Special Issue Genetics and Breeding for Enhancing Production Traits in Ruminants)
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19 pages, 2736 KB  
Article
Rationally Engineered D-Amino Acid Peptide DT7-3 Combats Multidrug-Resistant Helicobacter pylori via a Novel “Triple-Hit” Mechanism
by Shiying Yan, Xin Yan, Jiarui Zhao, Yue Zhou, Changyi Huang, Yiping Chen, Jia Wang, Jian Zhang, Chaoyi Han, Yu Gao, Tianlan Jiang, Hansheng Zhu, Hao Shi, Fosheng Li, Jian Zhao and Mei Cao
Microorganisms 2026, 14(4), 744; https://doi.org/10.3390/microorganisms14040744 - 26 Mar 2026
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Abstract
Helicobacter pylori (H. pylori) is the primary etiological agent for chronic gastritis, peptic ulcers, and gastric adenocarcinoma. The alarming rise in multidrug-resistant (MDR) strains, particularly against clarithromycin (CLR), metronidazole (MNZ), and levofloxacin (LVX), has severely compromised standard therapies. Thus, there is [...] Read more.
Helicobacter pylori (H. pylori) is the primary etiological agent for chronic gastritis, peptic ulcers, and gastric adenocarcinoma. The alarming rise in multidrug-resistant (MDR) strains, particularly against clarithromycin (CLR), metronidazole (MNZ), and levofloxacin (LVX), has severely compromised standard therapies. Thus, there is an urgent clinical need for novel antimicrobial agents that operate through distinct mechanisms to bypass resistance pathways and mitigate gastric cancer risk. We designed and synthesized a series of antimicrobial peptides, focusing on the proteolytically stable all-D-amino acid enantiomer, DT7-3, derived from a probiotic-sourced template. Minimum inhibitory concentrations (MICs) were determined against standard strains and 11 clinical MDR isolates via the broth microdilution method. Antimicrobial mechanisms were elucidated using scanning electron microscopy (SEM) for morphology, fluorescence-based assays for anti-adhesion activity, and real-time qPCR to quantify virulence gene expression (babA, ureA, and vacA). Biocompatibility was assessed using defibrinated sheep erythrocytes, gastric epithelial cells (GES-1), and representative beneficial gut microbiota. Analysis of the clinical isolates revealed resistance rates of 63.6% for CLR/LVX and 81.8% for MNZ, with 54.5% identified as MDR. DT7-3 exhibited superior potency (MIC 1–32 µg/mL) against all strains, significantly outperforming its L-enantiomer counterparts. Mechanistic studies unveiled a “triple-hit” mechanism: (1) rapid membrane disruption; (2) potent inhibition of bacterial adhesion to host cells (~60% reduction at 0.5 × MIC); (3) significant downregulation of critical virulence factors (babA, ureA, and vacA). Furthermore, DT7-3 showed an excellent safety profile, with negligible hemolysis (<5% at 32 µg/mL) and minimal cytotoxicity toward GES-1 cells, yielding a high selectivity index (SI, MHC/MIC) > 32 relative to mammalian cells. Crucially, DT7-3 showed high selectivity for the pathogen over beneficial gut microbiota (MIC > 128 µg/mL, SI > 16). Crucially, DT7-3 maintained potent bactericidal activity (MIC ≤ 16 µg/mL) even under cholesterol-enriched conditions. The engineered D-peptide DT7-3 is a potent candidate for combating MDR H. pylori. Its multifaceted mechanism, targeting bacterial viability while suppressing core virulence factors, positions it as a robust lead compound for next-generation eradication therapies aimed at reducing the burden of H. pylori-associated diseases. Full article
(This article belongs to the Section Antimicrobial Agents and Resistance)
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22 pages, 6221 KB  
Article
RNA-Seq and RT-qPCR Analysis of the Formation Process from Potato Stolons to Tubers and Functional Study of StLSH10 in Tuberization
by Rong Li, Yihan Zhao, Yifan Zhou, Cheng Sun, Chunna Lv, Jian Wang and Fang Wang
Horticulturae 2026, 12(4), 409; https://doi.org/10.3390/horticulturae12040409 - 25 Mar 2026
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Abstract
The potato tuber is a metamorphic organ formed by the expansion of the underground stolon tip. It is an economically important organ and an excellent material for studying the occurrence and development of modified plant organs. However, genetic studies have lagged due to [...] Read more.
The potato tuber is a metamorphic organ formed by the expansion of the underground stolon tip. It is an economically important organ and an excellent material for studying the occurrence and development of modified plant organs. However, genetic studies have lagged due to the potato’s complex genetic background. In this study, we used stolons and tubers of the potato ‘Qingshu 9’ at different stages of the tuberization process as samples for transcriptome sequencing and systematically analyzed the transcriptome characteristics of tuberization. Through RT-qPCR analysis, 16 candidate genes related to tuberization were identified. Overexpression verification was performed on one candidate gene, StLSH10, and the results indicated that it might be involved in regulating tuberization. This research provides a theoretical basis for elucidating the molecular mechanism of tuberization and offers a new target to improve potato yield and quality through molecular breeding strategies. Full article
(This article belongs to the Section Genetics, Genomics, Breeding, and Biotechnology (G2B2))
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21 pages, 4808 KB  
Article
Hungatella hathewayi: A Tumor-Derived Bacterium Enriched in Colorectal Cancer Tissues and a Potential Diagnostic Biomarker
by Wenzhe Zhang, Jin Liu, Shanshan Sha, Qiulong Yan and Yufang Ma
Microorganisms 2026, 14(3), 707; https://doi.org/10.3390/microorganisms14030707 - 21 Mar 2026
Cited by 1 | Viewed by 757
Abstract
Colorectal cancer (CRC) is the third most common cancer globally and the second leading cause of cancer-related deaths. While intestinal microbiota dysbiosis is linked to CRC, the direct role of intratumoral bacteria in metastasis remains poorly understood. In this study, we isolated pathogenic [...] Read more.
Colorectal cancer (CRC) is the third most common cancer globally and the second leading cause of cancer-related deaths. While intestinal microbiota dysbiosis is linked to CRC, the direct role of intratumoral bacteria in metastasis remains poorly understood. In this study, we isolated pathogenic bacteria from CRC tumor tissues, identified as Hungatella hathewayi (H. hathewayi), through the 16S rRNA gene and whole-genome sequencing. We developed specific primers (P48/P52) and polyclonal antibodies for detecting H. hathewayi in samples. Using quantitative real-time PCR (qPCR), we found significant enrichment of H. hathewayi in fecal samples from CRC patients compared to healthy controls, with mean fold changes of 137-fold and 142-fold for primers P48 and P52, respectively. Analysis of tissue samples revealed that H. hathewayi abundance was higher in CRC tumor tissues compared to normal tissues, with mean fold changes of 2.90 for P48 and 3.97 for P52. Fluorescence in situ hybridization (FISH), immunofluorescence (IF), and immunohistochemistry (IHC) confirmed its spatial distribution within tumor tissues. In vitro assays using CRC cell lines demonstrated that H. hathewayi-derived succinate upregulates HIF-1α and SUCNR1 expression and promotes cell metastasis by inducing epithelial–mesenchymal transition (EMT). Collectively, these findings identify H. hathewayi as a novel pro-metastatic bacterium and a potential non-invasive biomarker for CRC diagnosis, providing direct evidence for the role of intratumoral bacteria in CRC progression. Full article
(This article belongs to the Section Gut Microbiota)
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12 pages, 263 KB  
Article
Balancing Speed and Cost: Economic Insights from Rapid Diagnostic Testing in Bloodstream Infections
by Gergana Lengerova, Ralitsa Raycheva, Michael M. Petrov and Todor Kantardjiev
Antibiotics 2026, 15(3), 320; https://doi.org/10.3390/antibiotics15030320 - 20 Mar 2026
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Abstract
Background: Rapid diagnostic tests (RDTs) for bloodstream infections (BSIs) reduce time to pathogen identification, yet evidence on their real-world economic and clinical value remains inconsistent. This study aimed to compare clinical outcomes, antibiotic utilization, and hospital costs associated with different rapid microbiological identification [...] Read more.
Background: Rapid diagnostic tests (RDTs) for bloodstream infections (BSIs) reduce time to pathogen identification, yet evidence on their real-world economic and clinical value remains inconsistent. This study aimed to compare clinical outcomes, antibiotic utilization, and hospital costs associated with different rapid microbiological identification methods versus standard culture. Methods: A retrospective observational study was conducted in a tertiary university hospital including 115 hospitalized patients with suspected or confirmed BSIs. Multiplex PCR (mPCR), fluorescence in situ hybridization (FISH), and MALDI-TOF MS were compared with conventional culture. Outcomes included mortality, length of stay, antibiotic-days, and direct and indirect hospital costs. Nonparametric and exploratory adjusted analyses were performed. Results: No significant differences were observed across diagnostic groups for age, sex, mortality, or length of stay. Patients tested with mPCR showed higher empirical and total antibiotic-days and increased antibiotic-related costs (p < 0.05). Median direct and indirect hospital costs were numerically lower with FISH and mPCR but did not reach statistical significance. Adjusted analyses confirmed that diagnostic modality was not independently associated with mortality or costs. Conclusions: Rapid diagnostics accelerate identification but demonstrate heterogeneous downstream clinical and economic effects. Their value appears to depend more on local implementation and antimicrobial stewardship integration than on diagnostic speed alone. Full article
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