Polymerase Chain Reaction Analysis Research Articles

Enhanced anti-tumor efficacy of S3I-201 in breast cancer mouse model through Wharton jelly- exosome

ObjectiveExosomes, membrane-enveloped vesicles found in various cell types, including Wharton’s jelly mesenchymal stem cells, play a crucial role in intercellular communication and regulation. Their use as a cell-free nanotechnology and drug delivery system has attracted attention. Triple-negative breast cancer (TNBC) is a major global health problem and is characterized by a high mortality rate. This study investigates the potential of Wharton’s Jelly mesenchymal stem cell-derived exosomes (WJ-Exo) as carriers of S3I-201 and their effects on STAT3 expression in breast cancer cell lines, and evaluates whether these exosomes can enhance the anti-tumor effect of S3I-201.MethodsThe filtered WJ-Exos were analyzed by Transmission Electron Microscopy (TEM), Scanning electron microscopy (SEM), Dynamic Light Scattering (DLS), flow cytometry, and Western blotting. These exosomes were then used for loading with S3I-201, resulting in the nano-formulation WJ-Exo(S3I-201). The effect of WJ-Exo(S3I-201) on 4T1 cancer cells was investigated in vitro using MTT assay, flow cytometry, wound healing assay, Western blotting and Quantitative Real-Time Polymerase chain reaction (qPCR) analysis. Finally, the therapeutic efficacy of the nano-formulation was investigated in vivo using a tumor-bearing mouse model.ResultsIn vitro experiments showed that co-incubation of 4T1 cells with the nano-formulation resulted in a significant reduction in p-STAT3 levels, induction of apoptosis, modulation of Bcl-2, Bax and caspase-3 protein and gene expression, and inhibition of migration. In vivo, treatment of tumor-bearing mice with WJ-Exo(S3I-201) showed a strong antitumor effect that exceeded the efficacy observed in the S3I-201 group.ConclusionOur results demonstrate that WJ-Exo is an effective carrier for targeting S3I-201 to tumor cells and enhances the therapeutic efficacy of S3I-201 in tumor-bearing mice.

The first detection of SARS-CoV-2 RNA in the wastewater of Bucharest, Romania

Wastewater-based epidemiology (WBE) has been previously used as a tool for pathogen identification within communities. After the SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2) outbreak, in 2020, Daughton proposed the implementation of a wastewater surveillance strategy that could determine the incidence of COVID-19 (coronavirus disease 2019) nationally. Individuals in various stages of SARS-CoV-2 infection, including presymptomatic, asymptomatic and symptomatic patients, can be identified as carriers of the virus in their urine, saliva, stool and other bodily secretions. Studies using this method were conducted to monitor the prevalence of the virus in high-density populations, such as cities but also in smaller communities, such as schools and college campuses. The aim of this pilot study was to assess the feasibility and effectiveness of wastewater surveillance in Bucharest, Romania, and wastewater samples were collected weekly from seven locations between July and September 2023. RNA (ribonucleic acid) extraction, followed by dPCR (digital polymerase chain reaction) analysis, was performed to detect viral genetic material. Additionally, NGS (next generation sequencing) technology was used to identify the circulating variants within the wastewater of Bucharest, Romania. Preliminary results indicate the successful detection of SARS-CoV-2 RNA in wastewater, providing valuable insights into the circulation of the virus within the community.

The ICF2 gene Zbtb24 specifically regulates the differentiation of B1 cells via promoting heme synthesis

BackgroundLoss-of-function mutations of ZBTB24 cause immunodeficiency, centromeric instability, and facial anomalies syndrome 2 (ICF2). ICF2 is a rare autosomal recessive disorder with immunological defects in serum antibodies and circulating memory B cells, resulting in recurrent and sometimes fatal respiratory and gastrointestinal infections. The genotype–phenotype correlation in patients with ICF2 indicates an essential role of ZBTB24 in the terminal differentiation of B cells.MethodsWe used the clustered regularly interspaced short palindromic repeats (CRISPER)/Cas9 technology to generate B cell specific Zbtb24-deficient mice and verified the deletion specificity and efficiency by quantitative polymerase chain reaction (Q-PCR) and western blotting analyses in fluorescence-activated cell sorting (FACS)-sorted cells. The development, phenotype of B cells and in vivo responses to T cell dependent or independent antigens post immunization were analyzed by flow cytometry and enzyme-linked immunosorbent assay (ELISA). Adoptive transfer experiment in combination with in vitro cultures of FACS-purified B cells and RNA-Seq analysis were utilized to specifically determine the impact of Zbtb24 on B cell biology as well as the underlying mechanisms.ResultsZbtb24 is dispensable for B cell development and maintenance in naive mice. Surprisingly, B cell specific deletion of Zbtb24 does not evidently compromise germinal center reactions and the resulting primary and secondary antibody responses induced by T cell dependent antigens (TD-Ags), but significantly inhibits T cell independent antigen-elicited antibody productions in vivo. At the cellular level, Zbtb24-deficiency specifically impedes the plasma cell differentiation of B1 cells without impairing their survival, activation and proliferation in vitro. Mechanistically, Zbtb24-ablation attenuates heme biosynthesis partially through mTORC1 in B1 cells, and addition of exogenous hemin abrogates the differentiation defects of Zbtb24-null B1 cells.ConclusionsZbtb24 seems to regulate antibody responses against TD-Ags B cell extrinsically, but it specifically promotes the plasma cell differentiation of B1 cells via heme synthesis in mice. Our study also suggests that defected B1 functions contribute to recurrent infections in patients with ICF2.

Circ_0001947 encapsulated by small extracellular vesicles promotes gastric cancer progression and anti-PD-1 resistance by modulating CD8+ T cell exhaustion

BackgroundWhile small extracellular vesicles (sEVs)-derived circular RNAs (circRNAs) have been emerged as significant players in cancer, the function and underlying mechanism of sEVs-derived circRNAs in anti-cancer immunity remain unclear.MethodsGastric cancer (GC)-derived circRNAs were identified using RNA-seq data from GEO datasets and quantitative reverse transcription polymerase chain reaction (qRT-PCR), RNA immunoprecipitation, dual-luciferase assay, and bioinformatics analysis were performed to investigate the regulatory axis. Transwell assay, wound healing assay, cell counting kit-8 (CCK-8) assay, and xenograft models were used to evaluate its role in GC progression in vivo and in vitro. The delivery of specific circRNAs into sEVs were verified through electron microscopy, nanoparticle tracking analysis (NTA) and fuorescence in situ hybridization (FISH). Flow cytometric analysis and immunohistochemical staining were conducted to find out how specific circRNAs mediated CD8+ T cell exhaustion and resistant to anti-programmed cell death 1 (PD-1) therapy.ResultsWe identified that circ_0001947, packaged by GC-derived sEVs, was obviously elevated in GC and was associated with poor clinical outcome. High circ0001947 level augmented the proliferation, migration, and invasion of GC cells. Mechanistically, circ0001947 sponged miR−661 and miR−671−5p to promote the expression of CD39, which further facilitated CD8+ T cell exhaustion and immune resistance. Conversely, blocking circ_0001947 attenuated CD8+ T cell exhaustion and increased the response to anti-PD−1 therapy.ConclusionsOur study manifested the therapeutic potential of targeting sEVs-transmitted circ_0001947 to prohibit CD8+ T cell exhaustion and immune resistance in GC.

Acute nicotine exposure attenuates neurological deficits, ischemic injury and brain inflammatory responses and restores hippocampal long-term potentiation in ischemic stroke followed by lipopolysaccharide-induced sepsis-like state

Ischemic stroke is followed by an increased susceptibility to bacterial infections, which exacerbate histological stroke outcome, neurological deficits and memory impairment due to increased neuroinflammation and neurotransmitter dysfunction. Pharmacological activation of nicotinic acetylcholine receptors was suggested to mitigate brain inflammatory responses in ischemic stroke. The functional responses associated with nicotinic acetylcholine receptor activation were unknown. In this study, male NMRI mice subjected to transient intraluminal middle cerebral artery occlusion (MCAO) were intraperitoneally exposed to vehicle treatment or Escherichia coli lipopolysaccharide (LPS; 4 mg/kg)-induced sepsis-like state 24 h post-MCAO, followed by intraperitoneal administration of vehicle or nicotine (0.5 mg/kg) 30 min later. Over 96 h, rectal temperature, neurological deficits, spontaneous locomotor activity, working memory, ischemic injury, synaptic plasticity, and brain inflammatory responses were evaluated by temperature measurement, behavioral analysis, infarct volumetry, electrophysiological recordings, and polymerase-chain reaction analysis. LPS-induced sepsis induced hypothermia, increased general and focal neurological deficits, reduced spontaneous exploration behavior, reduced working memory, and increased infarct volume post-MCAO. Additional treatment with nicotine attenuated LPS-induced hypothermia, reduced neurological deficits, restored exploration behavior, restored working memory, and reduced infarct volume. Local field potential recordings revealed that LPS-induced sepsis decreased long-term potentiation (LTP) in the dentate gyrus post-MCAO, whereas concomitant nicotine exposure restored LTP in the contralateral dentate gyrus. LPS-induced sepsis increased microglial/ macrophage Iba-1 mRNA and astrocytic GFAP mRNA levels post-MCAO, whereas add-on nicotine treatment reduced astrocytic GFAP mRNA. Taken together, these findings indicate that acute nicotine exposure enhances functional stroke recovery. Future studies will have to evaluate the effects of (1) chronic nicotine exposure, a clinically relevant vascular risk factor, and (2) the cessation of nicotine exposure, which is widely recommended post-stroke, but might have detrimental effects in the early stroke recovery phase.

Effect of Human Platelet-Rich Fibrin Lysate on Collagen Type I, Collagen Type III, and Matrix Metalloproteinase 1 : A Protocol Study on Rat Models with Pelvic Organ Prolapse

Background Pelvic organ prolapse (POP) is a prevalent condition caused by weakened pelvic floor support structures. Extracellular matrix alterations, including changes in collagen type I, collagen type III, and matrix metalloproteinase 1 (MMP-1), contribute to the pathogenesis of this condition. Human platelet-rich fibrin lysate (hPRF-L) is a novel regenerative treatment that has shown beneficial results in treating structural weaknesses related to various pelvic floor diseases, including POP. Methods This study protocol aims to investigate the effects of hPRF-L injection on collagen I, III, and MMP-1 in the vaginal mucosa of a rat POP model. POP will be induced in female Sprague-Dawley rats, which will be randomly assigned to control, sham, and hPRF-L treatment groups. The hPRF-L group will receive weekly injections of hPRF-L (25, 50, or 75 μL) into the vaginal mucosa for 4 weeks. Vaginal tissue samples will be collected, and collagen type I, collagen type III, and MMP-1 expression will be evaluated using quantitative reverse transcription polymerase chain reaction and immunohistochemical analyses. Data analysis will be performed with ANOVA and post-hoc tests. Discussion The findings from this study protocol are expected to provide valuable insights into the mechanisms by which hPRF-L impacts the structural integrity of the pelvic floor. By elucidating these mechanisms, this study aims to inform future POP treatment strategies. The anticipated results are an increase in collagen type I and III expression and a reduction in MMP-1 levels in the hPRF-L treatment group compared to the control and sham groups. These outcomes could support the use of hPRF-L as a regenerative therapy for managing POP, offering a potential alternative to more invasive surgical interventions. Conclusion The expected results will contribute to the development of less invasive treatments for POP, improving patient outcomes and quality of life.

Molecular detection of OXA-48 and NDM-1 carbapenemase genes among clinical isolates of Klebsiella pneumoniae recovered from patients attending a private tertiary hospital in Southwestern Nigeria

There have been increasing reports of Klebsiella pneumoniae resistant to β-lactam antibiotics. This study aimed to determine the prevalence of some selected carbapenemase genes among clinical isolates of Klebsiella pneumoniae recovered from patients attending a private tertiary hospital in Southwestern Nigeria. The study was conducted over two months (February-March 2024). A total of 50 clinical isolates of Klebsiella pneumoniae from different clinical specimens were obtained from the Medical Microbiology Department, Babcock University Teaching Hospital (BUTH). The clinical isolates were then characterized using standard microbiological procedures and were tested for susceptibility to meropenem and other classes of antibiotics according to Clinical and Laboratory Standards Institute (CLSI) guidelines. Polymerase Chain Reaction (PCR) detection for OXA-48 and NDM-1 carbapenemase genes was performed on the 50 clinical isolates. PCR analysis showed that 9 (18%) clinical isolates were positive for the OXA-48 gene, 22 (44%) were positive for the NDM-1 gene, 4 (8%) possessed both the OXA-48 and NDM-1 genes, and 23 (46%) possessed neither the OXA-48 nor NDM-1 genes. Antibiotic Susceptibility Testing (AST) revealed that all the clinical isolates were resistant to meropenem. In conclusion, this study demonstrates the presence of OXA-48 and NDM-1 genes in clinical isolates of Klebsiella pneumoniae recovered from patients attending a private tertiary hospital in Southwestern Nigeria, highlighting the role of ESBL (extended-spectrum beta-lactamase) as a major resistance mechanism alongside other mechanisms. Population-based surveillance programs should be implemented to monitor the prevalence and epidemiology of Klebsiella pneumoniae infections at the community level, facilitating early detection of outbreaks and identification of emerging antimicrobial resistance patterns.Core TipThis study highlights the significant prevalence of NDM-1 and OXA-48 carbapenemase genes among Klebsiella pneumoniae clinical isolates in a private tertiary hospital in Southwestern Nigeria, with 44% and 18% of isolates harboring these genes, respectively. Notably, 46% of isolates were resistant to carbapenems despite lacking these genes, suggesting alternative resistance mechanisms. The findings underscore the urgent need for enhanced surveillance, infection control measures, and antibiotic stewardship programs to combat the spread of multidrug-resistant Klebsiella pneumoniae in healthcare settings.

A case of familial progressive hyperpigmentation with or without hypopigmentation presenting with hypopigmented striae along the lines of Blaschko.

Familial progressive hyperpigmentation with or without hypopigmentation (FPHH) is an autosomal dominant disorder characterized by widespread skin hyperpigmentation, café-au-lait spots, and hypopigmented circular macules, resulting from KITLG variants. KITLG, expressed by keratinocytes, binds to KIT on melanocytes, stimulating melanogenesis. Disturbances in the KITLG-KIT interaction result in diffuse hyperpigmentation in FPHH. However, the mechanisms behind hypopigmented macule formation remain unclear. This report presents a unique FPHH case in a patient with a novel KITLG mutation (Ser78Leu). Notably, the patient showed multiple hypopigmented macules and striae along the lines of Blaschko. Digital polymerase chain reaction analysis of the DNA from skin and blood tissues indicated a copy-neutral loss of heterozygosity at the KITLG locus, only in the hypopigmented macule. These findings suggest that the hypopigmented macules might result from revertant mosaicism. Conversely, café-au-lait spots do not follow the lines of Blaschko and can superimpose on the hypopigmented striae, indicating a distinct pathogenesis. This case contributes to the understanding of the genetic mechanisms in FPHH.

Comparison In Vitro Study on the Interface between Skin and Bone Cell Cultures and Microporous Titanium Samples Manufactured with 3D Printing Technology Versus Sintered Samples.

Percutaneous implants osseointegrated into the residuum of a person with limb amputation need to provide mechanical stability and protection against infections. Although significant progress has been made in the biointegration of percutaneous implants, the problem of forming a reliable natural barrier at the level of the surface of the implant and the skin and bone tissues remains unresolved. The use of a microporous implant structure incorporated into the Skin and Bone Integrated Pylon (SBIP) should address the issue by allowing soft and bone tissues to grow directly into the implant structure itself, which, in turn, should form a reliable barrier to infections and support strong osseointegration. To evaluate biological interactions between dermal fibroblasts and MC3T3-E1 osteoblasts in vitro, small titanium discs (with varying pore sizes and volume fractions to achieve deep porosity) were fabricated via 3D printing and sintering. The cell viability MTT assay demonstrated low cytotoxicity for cells co-cultured in the pores of the 3D-printed and sintered Ti samples during the 14-day follow-up period. A subsequent Quantitative Real-Time Polymerase Chain Reaction (RT-PCR) analysis of the relative gene expression of biomarkers that are associated with cell adhesion (α2, α5, αV, and β1 integrins) and extracellular matrix components (fibronectin, vitronectin, type I collagen) demonstrated that micropore sizes ranging from 200 to 500 µm of the 3D printed and sintered Ti discs were favorable for dermal fibroblast adhesion. For example, for representative 3D-printed Ti sample S6 at 72 h the values were 4.71 ± 0.08 (α2 integrin), 4.96 ± 0.08 (α5 integrin), 4.71 ± 0.08 (αV integrin), and 1.87 ± 0.12 (β1 integrin). In contrast, Ti discs with pore sizes ranging from 400 to 800 µm demonstrated the best results (in terms of marker expression related to osteogenic differentiation, including osteopontin, osteonectin, osteocalcin, TGF-β1, and SMAD4) for MC3T3-E1 cells. For example, for the representative 3D sample S4 on day 14, the marker levels were 11.19 ± 0.77 (osteopontin), 7.15 ± 0.29 (osteonectin), and 6.08 ± 0.12 (osteocalcin), while for sintered samples the levels of markers constituted 5.85 ± 0.4 (osteopontin), 4.45 ± 0.36 (osteonectin), and 4.46 ± 0.3 (osteocalcin). In conclusion, the data obtained show the high biointegrative properties of porous titanium structures, while the ability to implement several pore options in one structure using 3D printing makes it possible to create personalized implants for the best one-time integration with both skin and bone tissues.

Improving heat tolerance in betel palm (Areca catechu) by characterisation and expression analyses of heat shock proteins under thermal stress

Context Heat shock proteins play a vital role in cellular homeostasis by protecting proteins against various environmental stresses, which facilitates the survival of plants under unfavourable conditions. Aims We aimed to provide the first comprehensive genomic and expression analysis of the HSP70 gene family in betel palm (Areca catechu) to elucidate its role in heat stress response. Methods Genomic analysis revealed 34 putative HSP70 genes distributed across 13 chromosomes. These were renamed AcatHSP70 and classified into five subfamilies (A–E) based on phylogenetic analysis. These genes are mostly localised in the chloroplast, cytoplasm, and nucleus. Gene ontology revealed that these genes are mostly involved in heat stress. The gene duplication events of HSP70 genes involved only segmental duplications. We subjected betel palm seedlings (2 years old) to heat stress under controlled conditions for 30 days at high, low, and room temperatures for expression analyses of HSP70 genes. Key results Expression analysis revealed eight putative candidate genes (AcatHSP70-3, AcatHSP70-13, AcatHSP70-22, AcatHSP70-19, AcatHSP70-21, AcatHSP70-24, AcatHSP70-25, and AcatHSP70-26) that showed significantly higher expression under high-temperature stress. AcatHSP70-5 showed higher expression under low-temperature treatment, and AcatHSP70-16 was responsive at room temperature treatment. Conclusion We conclude that the majority of AcatHSP70 genes play a crucial role under thermal stress conditions, and respond to high-temperature stress as shown by the quantitative reverse transcription polymerase chain reaction analysis. Implications This comprehensive characterisation of the HSP70 gene family provides novel insights into the thermal protection mechanisms of betel palms in changing climates.

Simulated Gastrointestinal Digestion and Fecal Fermentation Characteristics of Exopolysaccharides Synthesized by Schleiferilactobacillus harbinensis Z171.

Exopolysaccharides (EPSs) produced by Lactobacillus have important physiological activities and are commonly used as novel prebiotics. A strain of Lactobacillus with high EPS yield was identified as Schleiferilactobacillus harbinensis (S. harbinensis Z171), which was isolated from Chinese sauerkraut. The objective of this study was to investigate the in vitro simulated digestion and fecal fermentation behavior of the purified exopolysaccharide fraction F-EPS1A from S. harbinensis Z171 and its influence on the human intestinal flora composition. The in vitro digestion results showed that the primary structural characteristics of F-EPS1A, such as morphology, molecular weight, and monosaccharide composition remained stable after saliva and gastrointestinal digestion. Compared with the blank group, the fermentation of F-SPS1A by fecal microbiota decreased the diversity of the bacterial communities, significantly promoted the relative abundance of Bifidobacterium and Faecalibacterium, and decreased the relative abundance of Lachnospiraceae_Clostridium, Fusobacterium, and Oscillospira. Reverse transcription polymerase chain reaction (RT-PCR) analysis also showed that the population of Bifidobacterium markedly increased. Furthermore, the total short-chain fatty acid levels increased significantly, especially for butyric acid. Gas chromatography-mass spectrometry (GC-MS) results showed that F-EPS1A could be fermented by the human gut microbiota to synthesize organic acids and derivative metabolites that are beneficial to gut health. Therefore, these findings suggest that F-EPS1A could be exploited as a potential prebiotic.

OSGIN1 regulates PM2.5-induced fibrosis via mediating autophagy in an in vitro model of COPD

Fine particulate matter (PM2.5) has been identified as a significant contributing factor to the exacerbation of chronic obstructive pulmonary disease (COPD). It has been observed that PM2.5 may induce lung fibrosis in COPD, although the precise molecular mechanism behind this remains unclear. In a previous study, we demonstrated that PM2.5 upregulates oxidative stress induced growth inhibitor 1 (OSGIN1), which in turn leads to injury in airway epithelial cells, thereby, suggesting a potential link between PM2.5 exposure and COPD. Based on this, we hypothesized that OSGIN1 plays a role in PM2.5-induced fibrosis in COPD. Human bronchial epithelial cells (HBEs) were treated with cigarette smoke extract (CSE) to construct an in vitro model of COPD. Our findings revealed that PM2.5 increased fibrosis indicators and upregulated OSGIN1 in CSE-stimulated HBEs (CSE-HBEs), and knockdown of OSGIN1 reduced the expression of fibrosis indicators. Through the use of microRNA target prediction software and the Gene Expression Omnibus database, we predicted miRNAs that targeted OSGIN1 in COPD. Subsequently, real-time polymerase chain reaction and western blot analysis confirmed that PM2.5 modulated miR-654–5p to regulate OSGIN1 in CSE-HBEs. Western blot demonstrated that OSGIN1 induced autophagy, thereby exacerbating fibrosis in CSE-HBEs. In summary, our results suggest that PM2.5 upregulates OSGIN1 through inhibiting miR-654–5p, leading to increased autophagy and fibrosis in CSE-HBEs.

Polydatin ameliorates early brain injury after subarachnoid hemorrhage through up-regulating SIRT1 to suppress endoplasmic reticulum stress.

This study aims to investigate the inhibitory effect of Polydatin (PD) on endoplasmic reticulum (ER) stress following subarachnoid hemorrhage (SAH) and to elucidate the underlying mechanisms. A standard intravascular puncture model was established to mimic SAH in mice. Neurological functions were assessed using neurological scoring, Grip test, and Morris water maze. Brain edema and Evans blue extravasation were measured to evaluate blood-brain barrier permeability. Western blot and quantitative real-time polymerase chain reaction (PCR) analyses were performed to examine protein and mRNA expressions related to ER stress. Terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) staining was used to detect cell apoptosis, and transmission electron microscopy was used to observe the ultrastructure of the endoplasmic reticulum. The results indicated that PD significantly reduced brain edema and Evans blue extravasation after SAH, improving neurological function. Compared to the SAH group, the expression levels of ER stress-related proteins including glucose-regulated protein 78 (GRP78), phosphorylated protein kinase R-like endoplasmic reticulum kinase (p-PERK), phosphorylated eukaryotic initiation factor 2α (p-eIF2α), activating transcription factor 4 (ATF4), and C/EBP homologous protein (CHOP), were significantly lower in the PD-treated group. Moreover, PD significantly enhances the protein expression of Sirtuin 1 (SIRT1). Validation with sh-SIRT1 confirmed the critical role of SIRT1 in ER stress, with PD's inhibitory effect on ER stress being dependent on SIRT1 expression. Additionally, PD attenuated ER stress-mediated neuronal apoptosis and SAH-induced ferroptosis through upregulation of SIRT1. PD alleviates ER stress following SAH by upregulating SIRT1 expression, thereby mitigating early brain injury. The protective effects of PD are mediated through SIRT1, which inhibits ER stress and reduces neuronal apoptosis and ferroptosis.

Melatonin Mitigates Cisplatin-Induced Submandibular Gland Damage by Inhibiting Oxidative Stress, Inflammation, Apoptosis, and Fibrosis.

The study aims to examine the possible effect of melatonin against cisplatin-induced submandibular degeneration in experimental rats exploring its ameliorative mechanisms. Rats were classified into four experimental groups; control group; melatonin group; cisplatin group; and cisplatin+melatonin group. Submandibular tissues were collected. Biochemical, histopathological, and immunohistopathological examination and quantitative reverse transcription polymerase chain reaction (qRT-PCR) analysis were performed. The results indicate that intraperitoneal administration of melatonin (30 mg/kg body weight) alongsidecisplatin significantly elevated submandibular glands (SMG) and reduced glutathione (GSH) and superoxide dismutase (SOD) levels (p < 0.001), while it reducedmalondialdehyde (MDA) levels, NF-κB gene expression, the protein level of tumor necrosis factor-alpha (TNF-α), interleukin-6 (IL-6), interleukin-1 beta (IL-1β), immunoexpression of low-dose cyclooxygenase-2 (Cox-2), and CD68. Moreover, melatonin reduced immune and gene expression of alpha-smooth muscle actin (α-SMA), immunoexpression of caspase-3, and gene expression of Bax in comparison to the cisplatin group. Melatonin attenuated cisplatin-induced submandibular destruction alleviating SMG oxidative stress, inflammation, and fibrosis in addition to halting cellular apoptosis, sheds light on its usage in clinical application.

Pirfenidone targeted mechanisms for alleviating methotrexate-induced testiculopathy in Wistar rats.

Testicular injury and affected spermatogenesis are major complications of methotrexate (MTX) use. Oxidative stress is one contributing process leading to inflammation and apoptosis induction. Pirfenidone (PFD) is a well-known anti-fibrotic drug prescribed for interstitial lung fibrosis, in addition to anti-inflammatory, antioxidative, and antiapoptotic capabilities. The study aimed to explore the potential protection afforded by PFD in a rat model of MTX-induced testiculopathy. The experimental design included four groups, each containing seven adult Wistar rats: control, PFD (500mg/kg/day, orally)-, MTX (0.5mg/kg, intraperitoneal, twice weekly)-, and PFD/MTX-treated groups. Treatment continued for 4weeks. Blood and testicular samples were harvested for biochemical, histological, immunohistochemical, and polymerase chain reaction (PCR) analyses. Also, the testicular damage and spermatogenic activity were graded by the testicular injury and Johnsen scoring system, respectively. PFD positively affected the serum testosterone (TST) level, reduced the testicular inflammatory mediators [tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β)], reduced the testicular oxidative burden, increased superoxide dismutase (SOD), and protected the testicular histological structure. In addition, antifibrotic effects, anti-caspase-3, and PCNA enhancement activity were recorded. PFD exhibited a protective potential and mitigated the MTX-induced testiculopathy via suppression of testicular oxidative stress, inflammation, fibrosis, and apoptosis and retaining the testicular proliferative efficacy as confirmed by histological, immunohistochemical, and biochemical methods.

Leukocyte cell-derived chemotaxin 2 (LECT2) regulates liver ischemia–reperfusion injury

Background and aimHepatic ischemia–reperfusion injury (IRI) is a significant challenge in liver transplantation, trauma, hypovolemic shock, and hepatectomy, with limited effective interventions available. This study aimed to investigate the role of leukocyte cell-derived chemotaxin 2 (LECT2) in hepatic IRI and assess the therapeutic potential of Lect2-short hairpin RNA (shRNA) delivered through adeno-associated virus (AAV) vectors. Materials and methodsThis study analyzed human liver and serum samples from five patients undergoing the Pringle maneuver. Lect2-knockout and C57BL/6J mice were used. Hepatic IRI was induced by clamping the hepatic pedicle. Treatments included recombinant human LECT2 (rLECT2) and AAV-Lect2-shRNA. LECT2 expression levels and serum biomarkers including alanine aminotransferase (ALT), aspartate aminotransferase (AST), creatinine, and blood urea nitrogen (BUN) were measured. Histological analysis of liver necrosis and quantitative reverse-transcription polymerase chain reaction were performed. ResultsSerum and liver LECT2 levels were elevated during hepatic IRI. Serum LECT2 protein and mRNA levels increased post reperfusion. Lect2-knockout mice had reduced weight loss; hepatic necrosis; and serum ALT, AST, creatinine, and BUN levels. rLECT2 treatment exacerbated weight loss, hepatic necrosis, and serum biomarkers (ALT, AST, creatinine, and BUN). AAV-Lect2-shRNA treatment significantly reduced weight loss, hepatic necrosis, and serum biomarkers (ALT, AST, creatinine, and BUN), indicating therapeutic potential. ConclusionsElevated LECT2 levels during hepatic IRI increased liver damage. Genetic knockout or shRNA-mediated knockdown of Lect2 reduced liver damage, indicating its therapeutic potential. AAV-mediated Lect2-shRNA delivery mitigated hepatic IRI, offering a potential new treatment strategy to enhance clinical outcomes for patients undergoing liver-related surgeries or trauma.

Prevalence and molecular characteristics of carbapenem-resistant Escherichia coli isolated from dogs in South Korea.

Carbapenem-resistant Enterobacteriaceae are emerging as a global public health risk. Therefore, assessing the prevalence of carbapenem-resistant Escherichia coli (CRE) in both humans and animals is important. We aimed to ascertain the occurrence and characteristics of CRE isolated from companion animals, dogs and cats. E. coli strains were tested for antimicrobial susceptibility using the broth microdilution technique. Antimicrobial resistance genes were detected by polymerase chain reaction and sequencing analysis. The molecular characteristics of CRE were determined using multi-locus sequence typing, replicon typing, and pulsed-field gel electrophoresis (PFGE). In total, 13 CRE isolates (0.13%) were identified from dogs possessing blaNDM-5 along with β-lactamase genes, mostly blaCMY-2 (92.2%) and blaTEM-1 (53.8%). The commonly observed mutations were S83L and D87N in gyrA, S80I in parC, and S458A in parE. CRE carried non-beta-lactam resistance genes, with the majority being tet(B) (100%), sul (84.6%), and aac(3)-II (53.8%). Nine different PFGE patterns (P1-P9), IncX3-type plasmids (69.2%), and ST410 (84.6%) were predominantly detected. This investigation provides significant insight into the prevalence and molecular characteristics of blaNDM-5-carrying E. coli in dogs. The co-existence of blaNDM-5 and other antimicrobial resistance genes in E. coli potentially poses severe health hazards to humans.

Oligomeric procyanidins combined with Parabacteroides distasonis ameliorate high-fat diet-induced atherosclerosis by regulating lipid metabolism, inflammation reaction and bile acid metabolism in ApoE−/− mice

Atherosclerosis (AS) is the main pathological basis of cardiovascular diseases. Hence, the prevention and treatment strategies of AS have attracted great research attention. As a potential probiotic, Pararabacteroides distasonis has a positive regulatory effect on lipid metabolism and bile acids (BAs) profile. Oligomeric procyanidins have been confirmed to be conducive to the prevention and treatment of AS, whose anti-atherosclerotic effect may be associated with the promotion of gut probiotics. However, it remains unclear whether and how oligomeric procyanidins and P. distasonis combined (PPC) treatment can effectively alleviate high-fat diet (HFD)-induced AS. In this study, PPC treatment was found to significantly decrease atherosclerotic lesion, as well as alleviate the lipid metabolism disorder, inflammation and oxidative stress injury in ApoE−/− mice. Surprisingly, targeted metabolomics demonstrated that PPC intervention altered the BA profile in mice by regulating the ratio of secondary BAs to primary BAs, and increased fecal BAs excretion. Further, quantitative polymerase chain reaction (qPCR) analysis showed that PPC intervention facilitated reverse cholesterol transport by upregulating Srb1 expression; In addition, PPC intervention promoted BA synthesis from cholesterol in liver by upregulating Cyp7a1 expression via suppression of the farnesoid X receptor (FXR) pathway, thus exhibiting a significant serum cholesterol-lowering effect. In summary, PPC attenuated HFD-induced AS in ApoE−/− mice, which provides new insights into the design of novel and efficient anti-atherosclerotic strategies to prevent AS based on probiotics and prebiotics.

C9orf72 repeat expansions in Wakayama: One potential cause of amyotrophic lateral sclerosis in the Kii Peninsula, Japan

A cluster of cases of amyotrophic lateral sclerosis (ALS) exists in the southern part of the Kii Peninsula in Japan. Although both genetic and environmental factors are thought to be causative, the critical cause of this cluster has not been identified. C9orf72 is the most common genetic factor in both familial and sporadic C9orf72-related ALS in people of European ancestry, but it is rare among Japanese populations. However, a previous report revealed that the frequency of C9orf72-related ALS was significantly higher in the cluster area. We evaluated the proportion of C9orf72 hexanucleotide repeat expansions in 99 cases of ALS diagnosed in Wakayama Prefecture, including the cluster area, by using repeat-primed polymerase chain reaction and fluorescence fragment length analysis. We found that 2 of the 99 patients (0 % of those with familial ALS and 2.4 % of those with sporadic ALS) had hexanucleotide repeat expansions in C9orf72, and long-read sequencing revealed that these expansions were causative. No expansions were observed among 90 patients with Parkinson's disease or among 90 healthy controls. Haplotype analysis with long-read sequencing data revealed that the two patients with repeat expansions shared the common haplotype with that previously reported in Finnish patients with C9orf72-related ALS, which suggests a founder effect. C9orf72 was thought to be a rare causative gene in Japan, but this study revealed that it may be relatively common in Wakayama Prefecture.

Cisplatin induces acute liver injury by triggering caspase-3/GSDME-mediated cell pyroptosis

BackgroundCisplatin triggers Gasdermin E (GSDME) cleavage, causing membrane bubble formation, content release, and inflammation. Caspase-3 activation initiates GSDME cleavage, and thus inhibiting this pathway mitigates cisplatin-induced pyroptosis in hepatocytes. This study aimed to delve into how cisplatin induces liver injury via pyroptosis. MethodsFor animal experiments, C57BL/6J mice were divided into three groups: control, liver injury model group, and Ac-DMLD-CMK (caspase-3 inhibitor) intervention group. The liver histology was evaluated by hematoxylin and eosin staining, immunohistochemistry, immunofluorescence and TUNEL staining. The mRNA and protein levels were detected by real-time polymerase chain reaction (PCR) and Western blot analysis. For in vitro experiments, HL-7702 cells were treated with cisplatin or GSDME siRNA. Cell pyroptosis was determined via cellular morphology, cytotoxicity and viability detection, flow cytometric assay, and Western blot detection for the expression of pyroptosis-related proteins. ResultsCisplatin-induced distinct liver morphological changes, hepatocellular injury, and inflammation in mice, along with elevated serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels and increased pro-inflammatory cytokine expression. Heightened macrophage infiltration and hepatocellular death indicated cisplatin-induced hepatotoxicity. Cisplatin upregulated GSDME activation, along with Bax-mediated caspase-3 cleavage both in vivo and in vitro, implicating caspase-3/GSDME-dependent pyroptosis in liver injury. Treatment with Ac-DMLD-CMK ameliorated cisplatin-induced liver injury, reducing hepatocellular lesions, serum ALT and AST levels, cytokine expression, macrophage infiltration, and hepatocyte death. Ac-DMLD-CMK also attenuated GSDME-dependent pyroptosis post-cisplatin induction, as evidenced by decreased GSDME expression, Bax upregulation, and cleaved caspase-3 activation. For HL-7702 cells, GSDME siRNA transfection reduced GSDME expression, attenuated typical signs of cisplatin-induced pyroptosis, partially restored cell viability, and significantly inhibited cytotoxicity and a decrease in the proportion of propidium iodide-positive cells, indicating protection against cisplatin-induced hepatocyte pyroptosis. ConclusionOur study underscores the role of the caspase-3/GSDME signaling pathway in mediating cisplatin-induced hepatotoxicity, particularly in cases of excessive or cumulative cisplatin exposure. These findings suggest that targeting GSDME could represent a promising therapeutic approach to mitigate cisplatin-induced liver damage.