ObjectiveTo establish a highly specific and sensitive triplex TaqMan quantitative PCR method for the detection of mouse hepatitis virus (MHV), mouse parvovirus (MPV), and reovirus type 3 (Reo-3) in laboratory mice. MethodsRecombinant plasmid standards were constructed using conserved genomic regions of the three target viruses. Specific primers and probes were designed, and a triplex TaqMan quantitative PCR (qPCR) system was optimized through preliminary experiments. Sensitivity was evaluated using 10-fold serial dilutions (10¹-10⁷ copies/μL). Intra- and inter-assay repeatability were assessed via reproducibility experiments. ResultsA strong linear correlation (R ²>0.99) was observed between copy number and Ct value at template concentrations of 10²-10⁷ copies/μL. Intra-assay and inter-assay coefficients of variation (CV) were both below 5%. No cross-reactivity was observed with mouse cytomegalovirus (MCMV), vesicular stomatitis virus (VSV), Sendai virus (SeV), or pneumonia virus of mice (PVM). ConclusionThe triplex TaqMan quantitative PCR method established in this study has high sensitivity, good repeatability, and strong specificity. It enables the rapid, specific, and sensitive detection of MHV, MPV, and Reo-3, allows the simultaneous detection of multiple pathogens in a single tube, and can be applied to the detection of clinical samples from laboratory animals and to epidemiological investigations, thereby providing effective scientific and technological support for the prevention and control of viral infection and the interruption of transmission.

Purpose: This study aimed to explore the practices, perceptions, and barriers related to specialty palliative care (SPC) referrals among oncologists in Korea, highlighting the clinical implications of early integration. Materials and Methods: A cross-sectional online survey targeting board-certified hemato-oncology specialists was conducted between August 1-25, 2024. The survey assessed referral practices, attitudes toward early SPC integration, referral criteria, barriers, and institutional characteristics. Results: A total of 227 oncologists participated (response rate, 36.7%). Among them, 68.7% reported frequent SPC referrals, with higher referral rates observed among younger physicians, those in tertiary hospitals, and institutions with in-house SPC teams (p < 0.001). Although 74.9% supported early SPC integration, referrals were often inconsistently timed, frequently occurring after disease progression or at the discontinuation of chemotherapy. For time-based referrals, the most commonly endorsed triggers were disease progression despite palliative second-line treatment and a prognosis of expected mortality within 6-12 months. Need-based referral triggers such as patient or family requests (96.5%), psychological distress (89.9%), or uncontrolled symptoms (83.3%), were also widely endorsed. The major barriers to early SPC integration included patient and family resistance (70.0%) and limited availability of SPC teams (34.4%). Conclusion This study emphasizes the importance of systematic efforts to promote timely SPC integration in Korea, including education to raise patient awareness, improved referral systems, and enhanced infrastructure. The positive attitudes toward early SPC among oncologists reflect a growing recognition of its value, highlighting the need for strategies that align with international standards.

Nonunion of osteoporotic vertebral fractures (OVF), predominantly affecting the elderly, can lead to intractable pain, vertebral collapse, progressive kyphotic deformity, and neurological impairment, significantly compromising patients′ quality of life. There exists considerable debate on diagnosis and management of OVF, encompassing key issues such as clinical diagnosis and staging criteria for nonunion, surgical indications and procedure selection, and postoperative rehabilitation planning. Currently, there lacks standardized clinical guideline and expert consensus on the diagnosis and management of OVF nonunion in China. To address this gap, Minimally Invasive Surgery Group of Chinese Orthopedic Association, Osteoporosis Committee of Chinese Association of Orthopedic Surgeons, Prevention and Rehabilitation Committee for Osteoporosis of Chinese Association of Rehabilitation Medicine and Minimally Invasive Orthopedic Surgery Branch of China Association for Geriatric Care jointly organized domestic experts in spinal surgery, endocrinology, and rehabilitation to formulate the Clinical guideline for the diagnosis and treatment for nonunion of osteoporotic vertebral fractures ( version 2025), based on existing literature and clinical experience and adhering to principles of scientific rigor and practicality. The guideline provided 13 evidence-based recommendations encompassing diagnosis and treatment of OVF nonunion, aiming to standardize its clinical management.

Objective To investigate the role of homocysteine(Hcy)in the pathogenesis of irritable bowel syn-drome(IBS)and its effects on intestinal motility,visceral hypersensitivity,and barrier function.Methods Clinical cohorts and animal models were combined for this study.Clinically,fifteen IBS patients meeting Rome Ⅲcriteria and 15 control individuals were enrolled to detect fecal Hcy levels and their correlation with symptoms.As for animal experiments,water avoidance stress(WAS)and 2,4,6-trinitrobenzene sulfonic acid(TNBS)chemical induction were utilized to establish IBS rat and mouse models,combined with a high-methionine diet(HMD)to simulate hyperhomocysteinemia.IBS symptoms were evaluated through fecal water content,carmine red intestinal transit time,and visceral hypersensitivity scores.Immune-fluorescence and Western blot were used to detect intes-tinal epithelial tight junction proteins.Serum and fecal Hcy concentrations were measured to assess Hcy levels.Statistical analyses included t-tests and One-way Anova.Results Fecal Hcy level in IBS patients were signifi-cantly higher than those in the healthy control group which demonstrated a positive correlation with defecation fre-quency(P＜0.01).In animal models,the combination of TNBS administration and a high-methionine diet markedly elevated serum and fecal Hcy levels in mice,while synergistically exacerbated intestinal motility disor-ders and visceral hypersensitivity.In vitro experiments showed that Hcy treatment down-regulates the expression of tight junction proteins in human colon cancer cell line(Caco-2).Conclusions Hcy plays an important role in the pathogenesis of IBS by impairing intestinal barrier function and enhancing visceral hypersensitivity,and it may serve as a potential new target for the treatment of IBS.

Objective To investigate the effects of peripheral blood neutrophil infiltration on the polarization regulation of cerebral resident microglia under a permanent ischemic stroke model.Methods Fifty-eight C57BL/6 mice were divided into two groups.One group was sham group,and the other group of mice was subjected to permanent middle cerebral artery occlusion surgery.Mice were euthanized 48 hours,7 days,14 days,and 30 days after surgery for tissue collection.Western blotting was used to detect expression levels of M1 microglia markers CD 16,M2 microglia marker arginase 1(Arg1),inflammatory cytokine interleukin-1 β(IL-1β),and neutrophil marker myeloperoxidase(MPO)in brain tissue.Immunofluorescence histochemical staining was used to assess neutrophil infiltration and M2 microglial distribution around the infarct area in brain sections.In vitro,purified neutrophils were co-cultured with BV2 microglial cells.After lipopolysaccharide stimulation,the phagocytosis of neutrophils by BV2 cells was observed,and the expression levels of CD16 and Arg1 proteins in BV2 cells were detected.Results Western blotting showed that the levels of CD16(P＜0.05),IL-1β(P＜0.001),and MPO(P＜0.05)in brain tissue increased significantly 48 hours and 7 days after surgery,then decreased,with MPO expression returning to normal levels 30 days after surgery.Immunofluorescence showed a significant increase of MPO-positive cells around the infarct area of the mouse cerebral cortex 48 hours after surgery(P＜0.001),followed by a decrease(P＜0.05).The number of ionized calcium binding adapter molecule 1(Iba1)and MPO double-positive cells gradually increased after surgery,and reached their peak at 14 days(P＜0.05).Iba1 and Arg1 double-positive cells also increased significantly 7 days(P＜0.05)and 14 days(P＜0.01)after surgery.In vitro,co-culture experiments showed that after BV2 phagocytosing neutrophils,CD 16(P＜0.05)significantly decreased and Arg1 significantly upregulated(P＜0.05).Conclusion In a permanent ischemic stroke model,microglia transition from M1 to M2 type after phagocytosing neutrophils,and the injured brain area changes from pro-inflammatory state to anti-inflammatory state.

Atherosclerosis(AS)is the major pathologic basis of cardiovascular disease,and its complications,such as myocardial infarction and stroke,are primarily triggered by rupture of atherosclerotic vulnerable plaques(VASPs).Conventional imaging techniques rely on anatomical changes,making it difficult to assess plaque vulnerability at an early stage.In recent years,molecular imaging combined with nanoprobe technology has enabled precise visualization at the cellular and molecular levels by targeting pathological features of plaques(e.g.,lipid core,inflammation,thin fibrous cap,etc.).This article reviews the pathological features of VASPs,the limitations of existing imaging techniques,and the design principles and application progress of molecular imaging probes and discusses the challenges and future directions for their clinical translation.

This study was aimed at identifying the pathogenic bacteria responsible for the death of a young tiger at the Fujian Meihua Mountain South China Tiger Breeding Research Institute.Tissue samples from the lungs,liver,and intestines of the deceased tiger were collected,and the bacteria were cultured inasterile environment.The bacterial strains were characterized according to their morphological and molecular biological properties,including assessment of virulence genes and antibiotic resistance genes,mouse lethality tests,and antibiotic susceptibility evaluations.A predominant bacterial strain isolated from the liver of the deceased tiger was identified as enterotoxigenic Escherichia coli(ETEC)strain Tiger22513F.Phylogenetic analysis of the 16S rRNA gene revealed that the Tiger22513F strain exhibited close genetic similarity to the reference strain ETEC(MF919609.1),with 99.9%nucleotide similarity,and resided on the same evolutionary branch.The Tiger22513F strain contained 11 antibiotic resistance genes(tetA,sul1,sul3,cmlA,floR,blaTEM,blaSHV,blaCMY-2,qnrA,qnrS,and qnrD)along with five virulence genes(VT1,fyuA,tsh,iucD,and ST).Mouse lethality tests indicated significant pathogenicity toward mice,affecting primarily the lungs,liver,and intestines.Antibiotic susceptibility testing demonstrated that this strain exhibited resistance to various classes of beta-lactam antibiotics,as well as quinolones and aminoglycosides.This investigation successfully isolated a multi-drug resistant enterotoxigenic Escherichia coli strain with pronounced pathogenicity from the liver of a deceased tiger;thus providing valuable scientific insights for clinical diagnosis,as well as prevention and control measures,against ETEC infections in South China tigers.

Objective:To evaluate the safety of neoadjuvant therapy with pembrolizumab combined with 5-fluorouracil (5-FU) and cisplatin in patients with advanced temporal bone squamous cell carcinoma (TBSCC), and its impact on tumor response rate and disease-free survival (DFS).Methods:This prospective, single-arm, open-label clinical study enrolled patients with advanced (Stage Ⅲ/Ⅳ) TBSCC from Sun Yat-sen Memorial Hospital. Patients received 2-3 cycles of neoadjuvant therapy with pembrolizumab, 5-FU, and cisplatin, followed by definitive surgery. Postoperatively, patients received 6 cycles of pembrolizumab combined with radiotherapy. The primary endpoint was the 2-year disease-free survival (DFS) rate. Secondary endpoints included objective response rate (ORR) and safety indicators. Survival analysis was performed using the Kaplan-Meier method. Adverse events (AE) were assessed using the National Cancer Institute′s Common Terminology Criteria for Adverse Events (CTCAE), version 5.0. Statistical analyses were conducted using SPSS software, version 22.0.Results:From August 2021 to April 2024, 16 patients with advanced TBSCC were enrolled (13 males and 3 females), with a median age of 54 years and a median follow-up time of 2.32 years. Following neoadjuvant therapy, the objective response rate (ORR) was 64.3% (9/14), and the disease control rate (DCR) was 92.9% (13/14). The 2-year DFS rate was 86.6%. Common treatment-related adverse events (TRAE) included leukopenia (56.3%, 9/16), nausea and vomiting (50.0%, 8/16), diarrhea, oral mucositis, and elevated liver function tests (25.0%, 4/16). One patient (6.25%) experienced a grade 3 adverse event.Conclusion:Neoadjuvant pembrolizumab-chemotherapy significantly enhances objective response rate and disease-free survival in advanced TBSCC.

Objective:To investigate the clinical application effect of Zishen Yutai Pill in patients with repeated implantation failure (RIF) undergoing frozen-thawed embryo transfer (FET) cycles.Methods:A retrospective case-control study was conducted, selecting 744 cycles of patients with RIF at the Department of Female Reproductive Medicine,State Key Laboratory of Reproductive Medicine and Offspring Health, Center for Reproductive Medicine, Institute of Women, Children and Reproductive Health, Shandong University from October 2017 to April 2023. The patients were divided into experimental group (treated with Zishen Yutai Pill, n=279) and control group (treated without Zishen Yutai Pill, n=465) based on whether Zishen Yutai Pill was added to luteal support. The pregnancy outcomes between the two groups were compared. Based on the different endometrial preparation protocols in the FET cycles, the patients were used down-regulation protocol ( n=271) or non-down-regulation protocol ( n=473). The pregnancy outcomes of the two groups in each protocol were compared. Results:There were no statistically significant differences between the two groups in terms of biochemical pregnancy rate, clinical pregnancy rate, embryo implantation rate, early miscarriage rate, late miscarriage rate, and live birth rate (all P>0.05). However, the biochemical pregnancy rate [69.0% (58/84)], clinical pregnancy rate [59.5% (50/84)], and embryo implantation rate [59.4% (57/96)] in the experimental group of the down-regulated protocol were significantly higher than those in control group [56.1% (105/187), P=0.045; 46.5% (87/187), P=0.048; 44.9% (92/205), P=0.019], with statistically significant differences. In the non-down-regulated protocol, there were no statistically significant differences in pregnancy outcomes between the two groups (all P>0.05). Conclusion:In the FET down-regulated protocol, Zishen Yutai Pill can significantly improve the clinical pregnancy rate and the embryo implantation rate in patients with RIF, thereby improving pregnancy outcomes.

Background: s/Aims: Sarcomatoid hepatocellular carcinoma (HCC) is a rare histological subtype of HCC characterized by extremely poor prognosis; however, its molecular characterization has not been elucidated. Methods: In this study, we conducted an integrated multiomics study of whole-exome sequencing, RNA-seq, spatial transcriptome, and immunohistochemical analyses of 28 paired sarcomatoid tumor components and conventional HCC components from 10 patients with sarcomatoid HCC, in order to identify frequently altered genes, infer the tumor subclonal architectures, track the genomic evolution, and delineate the transcriptional characteristics of sarcomatoid HCCs. Results: Our results showed that the sarcomatoid HCCs had poor prognosis. The sarcomatoid tumor components and the conventional HCC components were derived from common ancestors, mostly accessing similar mutational processes. Clonal phylogenies demonstrated branched tumor evolution during sarcomatoid HCC development and progression. TP53 mutation commonly occurred at tumor initiation, whereas ARID2 mutation often occurred later. Transcriptome analyses revealed the epithelial–mesenchymal transition (EMT) and hypoxic phenotype in sarcomatoid tumor components, which were confirmed by immunohistochemical staining. Moreover, we identified ARID2 mutations in 70% (7/10) of patients with sarcomatoid HCC but only 1–5% of patients with non-sarcomatoid HCC. Biofunctional investigations revealed that inactivating mutation of ARID2 contributes to HCC growth and metastasis and induces EMT in a hypoxic microenvironment. Conclusions We offer a comprehensive description of the molecular basis for sarcomatoid HCC, and identify genomic alteration (ARID2 mutation) together with the tumor microenvironment (hypoxic microenvironment), that may contribute to the formation of the sarcomatoid tumor component through EMT, leading to sarcomatoid HCC development and progression.