Bin Ma

This study aimed to investigate the impact of outer membrane proteins 35 (OmpK35) and 36 on the virulence of Klebsiella pneumoniae and the corresponding mechanisms. Serotype K1 strain NTUH-K2044 was utilized to create single-deletion mutants (ΔompK35 and ΔompK36) and a double-deletion mutant (ΔompK35/36), alongside their complementation variants. Multiple analyses were performed, including capsule staining, transmission electron microscopy, serum resistance assays, phagocytosis tests, and infection models using mice and Galleria mellonella. The mutants retained hypercapsules similar to NTUH-K2044, but looser capsule structures revealed by transmission electron microscopy. All mutants exhibited decreased virulence, with median lethal doses under 5 × 10⁴ colony forming units in mice. Specifically, only ΔompK35/36 showed reduced serum resistance compared to the wild strain, similar for the anti- phagocytosis capabilities. Additionally, ΔompK35 induced liver inflammation, whereas ΔompK36 and ΔompK35/36 exerted milder effects. Despite these differences, all mutants displayed normal growth rates and upregulated expression of gnd and wcaJ, crucial for capsular polysaccharide (CPS) biosynthesis. The hypercapsule, composed mainly of CPS, plays a crucial role in the hypervirulence of K. pneumoniae. OmpK35/36 may modulate the virulence of K. pneumoniae by regulating CPS export. The online version contains supplementary material available at 10.1186/s12866-026-05212-3.

Digitoxin, a cardiac glycoside from Digitalis spp., is clinically employed for heart failure and atrial fibrillation, and yet its reproductive toxicity is

Background: The investigation of ovarian development, dysfunction, and aging is essential for female reproductive health. Despite extensive research on the cellular functions of Brefeldin A (BFA) as an intracellular transport inhibitor, its specific effects and mechanisms on ovarian development/aging remain inadequately understood. Methods: Mice and porcine oocytes/granulosa cells (GCs) were treated with BFA. Morphological and omics analyses (including Western blot, real‐time polymerase chain reaction (RT‐PCR), transcriptomics, and metabolomics) were conducted. Results: In 3‐week‐old female mice, BFA treatment significantly suppressed oocyte maturation, induced apoptosis, and increased estradiol and LH levels. This treatment upregulated apoptosis‐related genes while downregulating proliferation‐associated genes. Additionally, BFA elevated senescence markers (p21 and p26) and decreased the activity of the longevity gene SIRT6. In porcine oocytes, BFA reduced the maturation rate and lowered mRNA levels of key maturation‐related genes, LHX8 and GDF9. In porcine GCs, BFA increased apoptosis and upregulated genes such as Caspase‐3, BAX, and P21, while downregulating genes associated with proliferation and longevity. Similar effects were observed in 12‐month‐old female mice, indicating consistency across age groups. Metabolomic analysis in these mice revealed that BFA primarily impacted pathways related to steroid biosynthesis, ovarian steroidogenesis, and estrogen signaling. Transcriptomic analysis in 12‐month‐old female mice further demonstrated that BFA disrupted ovarian function through multiple mechanisms, including modulation of the GnRH signaling pathway, activation of the FOXO pathway, and interference with meiosis‐related gene expression. Conclusion: Our findings are pivotal for advancing the understanding of ovarian aging, dysfunctions, and diseases, and ultimately facilitate addressing BFA's potential adverse effects on reproductive health/aging.

Long non-coding RNAs (lncRNAs) are essential regulatory molecules involved in various biological processes in mammals. However, their expression patterns across multiple porcine tissues have not been systematically characterized. We analyzed 607 RNA-seq datasets derived from 14 porcine tissues, including backfat, gallbladder, heart, ileum, jejunum, kidney, longissimus dorsi, liver, lung, skeletal muscle, ovary, pituitary, skeletal muscle, and spleen. Additionally, we examined 63 single-cell RNA-seq datasets from porcine primary oocytes at five developmental stages. For comparative analysis, we included 20 human and 17 mouse oocyte RNA-seq datasets. We identified 52,798 porcine lncRNAs, with tissue-specific expression patterns most prominent in oocytes and least in skeletal muscle. Among them, 2169 were classified as housekeeping and 14,469 as tissue-specific lncRNAs. Cross-species analysis revealed that a small subset of oocyte-expressed lncRNAs is conserved in humans and mice, associated with catalytic activity and circadian regulation. Additionally, 44 lncRNAs were differentially expressed during oocyte development, implicating them in neurogenesis, vesicle transport, and protein modification. Our findings not only contribute to the growing body of knowledge regarding lncRNAs in porcine biology but also pave the way for future research aimed at elucidating their functional roles in reproductive biology and other physiological processes.

Ovarian aging significantly contributes to the decline of the female reproductive system, adversely affecting fertility and endocrine homeostasis. To address the challenges posed by reproductive aging, natural products have shown promising preventive and therapeutic effects. Here, we investigated the beneficial effects of natural compound celastrol on ovarian development and aging, together with its underlying mechanisms. We found that celastrol administration at a concentration of 3 mg/kg promoted follicle development in young mice and enhanced porcine oocyte maturation, while regulating granulosa cell proliferation and apoptosis. In 12-month-old mice (equivalent to middle-aged adults), celastrol exhibited similar beneficial effects. Transcriptomic analysis revealed that differentially expressed genes post-celastrol treatment were associated with steroid biosynthesis, estrogen signaling pathways, type 2 diabetes, insulin secretion, meiosis, and apoptosis. Additionally, insulin receptor substrate 1 (IRS1), an adapter protein in insulin signaling, was shown to advance puberty in young mice and to facilitate oocyte maturation. Overexpression of IRS1 in oocytes promoted follicular development and oocyte maturation, resulting in enhanced steroid hormone levels, whereas IRS1 knockdown inhibited these processes. Our findings indicate that celastrol may regulate ovarian development and aging by modulating IRS1 expression and its related pathways, suggesting celastrol as a novel small-molecule compound targeting IRS1, and offering new perspectives for potential therapeutic strategies against reproductive aging and infertility.

Bayesian networks (BNs) are an excellent machine learning algorithm for extensively exploring the influencing factors associated with many diseases. However, few researchers have used BNs to examine the influencing factors associated with arthritis in older adults in the Chinese community. Our aim has been to use BNs to construct a complex network of relationships between arthritis and its related influencing factors and to predict arthritis through Bayesian inference, thereby providing scientific references for its control and prevention. Data were downloaded from the 2015 China Health and Retirement Longitudinal Study (CHARLS) online database, a longitudinal survey of the middle-aged and older adults in China. Twenty-two variables such as smoking, depressive symptoms, age, and joint pain were included in this study. First, Elastic networks (ENs) were used to screen for features closely associated with arthritis, and we subsequently incorporated these features into the construction of the BNs model. We performed structural learning of the BNs based on the taboo algorithm and used the maximum likelihood method for parameter learning of the BNs. In total, 15,764 participants were enrolled in this study, which included 5,076 patients with arthritis. ENs identified 13 factors strongly associated with arthritis. The BNs consisted of 14 nodes and 24 directed edges. Among them, depressive symptoms and age were direct influences on arthritis, whereas gender was an indirect influence on the diseases. BNs graphically visualized the complex network of relationships between arthritis and its influences and predicted the development of arthritis through Bayesian inference. These results were in line with clinical practice. BNs thus have a wide range of application prospects.

Maturation of follicles is the primary condition for the initiation of puberty, and excessive apoptosis of granulosa cells (GCs) will hinder the normal development of follicles in pigs. Signal Transducer and Activator of Transcription 4 (STAT4) plays an important role in cell proliferation and apoptosis. However, the mechanism of DNA methylation regulating STAT4 transcription and affecting follicle development in pigs remains unclear. To resolve this problem, we constructed a STAT4 overexpression vector and interference fragment to explore the effects of STAT4 on GC function and investigate the effects of changes in methylation status of the STAT4 promoter region on cell function and kisspeptin-1 (KISS1) expression, as well as the STAT4 effects on the development of the follicles of pigs and mice in vitro. We found that the expression of STAT4 decreased, while DNA methylation of the STAT4 promoter region increased with the growth of the follicles. After overexpression of STAT4, the apoptosis of GCs was increased but the proliferation, cell cycle and estrogen secretion of GCs were inhibited. When GCs were treated with DNA methyltransferase inhibitor (5-Aza-CdR), the methylation of the STAT4 promoter region decreased, resulting in a significant increase in the expression of STAT4. Consequently, the expression of KISS1 was inhibited. At the same time, the expressions of genes related to cell proliferation, cell cycle and estrogen secretion signaling pathways decreased, while the expressions of genes related to the apoptosis signaling pathway increased. After infection with the STAT4 lentiviral vector (LV-STAT4) in follicles of mice, the expression of STAT4 in ovaries of mice significantly increased, and the expression of KISS1 was significantly decreased. The capillaries on the surface of follicles were constricted, the age of puberty onset in mice was delayed while the levels of GnRH, LH, FSH and E2 in serum were decreased. In conclusion, we found that reduced methylation status of the STAT4 promoter region promoted the transcription of STAT4 and then inhibited the expression of KISS1, as well as promoted the apoptosis of GCs and ultimately inhibited the normal development of follicles in mammals.

Early, rapid, and accurate diagnostic tests play critical roles not only in the identification/management of individuals infected by SARS-CoV-2, but also in fast and effective public health surveillance, containment, and response. Our aim has been to develop a fast and robust fluorescence in situ hybridization (FISH) detection method for detecting SARS-CoV-2 RNAs by using an HEK 293 T cell culture model. At various times after being transfected with SARS-CoV-2 E and N plasmids, HEK 293 T cells were fixed and then hybridized with ATTO-labeled short DNA probes (about 20 nt). At 4 h, 12 h, and 24 h after transfection, SARS-CoV-2 E and N mRNAs were clearly revealed as solid granular staining inside HEK 293 T cells at all time points. Hybridization time was also reduced to 1 h for faster detection, and the test was completed within 3 h with excellent results. In addition, we have successfully detected 3 mRNAs (E mRNA, N mRNA, and ORF1a (−) RNA) simultaneously inside the buccal cells of COVID-19 patients. Our high-resolution RNA FISH might significantly increase the accuracy and efficiency of SARS-CoV-2 detection, while significantly reducing test time. The method can be conducted on smears containing cells (e.g., from nasopharyngeal, oropharyngeal, or buccal swabs) or smears without cells (e.g., from sputum, saliva, or drinking water/wastewater) for detecting various types of RNA viruses and even DNA viruses at different timepoints of infection.