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Effects of acute hypoxia followed by reoxygenation on intestinal histomorphology, oxidative stress and hypoxia signaling biomarkers, and microbiota in pikeperch (Sander lucioperca).

In aquatic environments, natural and anthropogenic factors commonly reduce dissolved oxygen (DO) and trigger hypoxia, which threatens the health and survival of aquatic organisms. As an important economic fish species in China, pikeperch (Sander lucioperca) is extremely sensitive to hypoxia. However, there are relatively few reports on how hypoxia and reoxygenation affect its intestinal physiology and microbial community. Three treatment groups were set for pikeperch: normoxia (DO = 8.5 ± 0.5 mg/L), 48 h hypoxia (DO = 2.5 ± 0.1 mg/L), and reoxygenation (48 h hypoxia followed by 6 h reoxygenation at normal DO), to evaluate alterations in intestinal histopathology, tight junction gene expression, oxidative stress, hypoxia signaling molecules and intestinal microbiota composition. The results showed that hypoxia significantly decreased muscularis thickness by approximately 32.5% and reduced the expression of tight junction genes (Occludin, Claudin2, and ZO-2). Moreover, hypoxia significantly increased oxidative stress index levels (GSH-Px, CAT, and MDA), markedly upregulated the expression of Bax, Caspase3, and HIF-1α, while significantly downregulating the expression of Bcl-2, Egln1, and Egln2. Notably, reoxygenation elicited partial compensatory effects against these hypoxia-induced changes. 16S rRNA sequencing analysis revealed that hypoxic stress altered the intestinal microbial community composition of pikeperch and increased its diversity. In the hypoxia group, the abundance of the phylum Bacillota, along with the genera Halomonas and Acinetobacter, was significantly elevated, whereas in the reoxygenation group, the genus Lactobacillus increased approximately 180-fold. The results indicated that hypoxia caused intestinal oxidative damage, cell apoptosis, and intestinal microbiota dysbiosis in pikeperch, while short-term reoxygenation achieved partial recovery from these hypoxia-triggered intestinal injuries. The present research provides valuable references for in-depth exploration of the molecular mechanisms behind the response of pikeperch to acute hypoxia and reoxygenation stress, while also offering a novel perspective to understand the mechanism by which hypoxia impacts intestinal health in fish.

Animals

Comparative analysis of gut microbiota in yaks under different feeding management strategies during cold seasons.

Yaks (Bos grunniens) are crucial for the livelihoods of pastoral communities in cold regions, where feed scarcity during the cold season poses challenges to their health and productivity, underscoring the necessity of understanding how dietary management influences the gut microbiota. In this study, 24 yak steers matched for body weight and health status were randomly allocated to four groups: natural grazing or indoor feeding with roughage-to-concentrate ratios of 50:50, 70:30, or 90:10. Fecal samples were collected for 16S rDNA sequencing and subsequent functional prediction of the microbiota. The results showed that Firmicutes and Bacteroidetes were the dominant phyla across all groups, and UCG-005 and Rikenellaceae_RC9_gut_group were the predominant genera. Concentrate supplementation during the cold season significantly enhanced microbial richness and diversity, with the 70:30 ratio exerting the most pronounced beneficial effects on microbiota structure and key taxa enrichment. These findings highlight the critical role of dietary management in shaping the yak gut microbiota during cold seasons and suggest that the 70:30 ratio optimally improves microbial community structure, thereby promoting yak health and productivity under harsh climatic conditions. Future research should explore the long-term implications of such dietary strategies.

Animals

Molecular characterization of 16 MAPK genes in silver carp (Hypophthalmichthys molitrix) and the differences of their mRNA expression between Qiandao Lake and Taihu Lake.

Mitogen-activated protein kinase (MAPK), a serine-threonine protein kinase, is involved in a variety of stress-induced responses and also plays an important regulatory role in cell metabolism. In the study the open reading frames (ORFs) of 16 MAPK genes in silver carp (Hypophthalmichthys molitrix) were obtained and verified, with the evaluations of their taxonomy, structures, conserved motifs, and evolutionary linkages. And the expression patterns of these genes in the silver carp from Qiandao Lake and Taihu Lake were explored for better understanding the response of MAPK genes to different water environment. MAPK genes of silver carp were divided into three subfamilies, including extracellular signal-regulated kinase (ERK) subfamily, p38 subfamily and C-Jun N-terminal kinase (JNK) subfamily. All these genes possessed similar structures and conserved motifs of MAPK family. Realtime qPCR revealed that the expression patterns of 10 MAPK genes (ScMAPK1, ScMAPK3, ScMAPK4, ScMAPK7, ScMAPK15, ScMAPK8a, ScMAPK8b, ScMAPK9, ScMAPK10 and ScMAPK11) in head kidney, spleen and gill of silver carp in Taihu Lake and Qiandao Lake were different. These findings provide a basis for further research on the function of MAPK in silver carp.

Animals

[Exploring the mechanism underlying the efficacy differences of "Tiaojing Cuyun Acupuncture"for premature ovarian failure based on vaginal microbiota structure].

OBJECTIVE: To observe the efficacy of "Tiaojing Cuyun Acupuncture" combined with hormone replacement therapy in the treatment of premature ovarian failure (POF), and to investigate differences in vaginal microbiota among patients with different therapeutic responses, thereby preliminarily exploring the mechanism underlying efficacy differences. METHODS: Ninety-eight patients with POF were randomly divided into an observation group (49 cases, 1 case was eliminated)and a control group (49 cases, 1 case dropped out). The control group was treated with hormone replacement therapy with Femoston, one tablet each time, once daily. On the basis of the control group, the observation group was additionally treated with "Tiaojing Cuyun Acupuncture". Acupoint group 1 included Baihui (GV20), Zhongwan (CV12), Guanyuan (CV4), and bilateral Tianshu (ST25), Zigong (EX-CA1), Dahe (KI12), Zusanli (ST36), Sanyinjiao (SP6), and Taichong (LR3). Acupoint group 2 included Baihui (GV20), bilateral Shenshu (BL23), Ciliao (BL32), and Taixi (KI3). The two acupoint groups were used alternately, beginning with acupoint group 1. Treatment was administered once every other day, three times per week.Both groups were treated for three menstrual cycles. Serum follicle-stimulating hormone (FSH), luteinizing hormone (LH),estradiol (E2), and anti-M &#xfc; llerian hormone (AMH) levels on days 3-5 of the menstrual cycle were measured before and after treatment in the two groups. Self-rating anxiety scale (SAS) scores were assessed before and after treatment, and clinical efficacy was evaluated in the two groups. Five effective patients (G0 subgroup) and five ineffective patients (G1 subgroup)were randomly selected from each group, and their vaginal microbiota structure was analyzed using 16S rRNA sequencing. RESULTS: After treatment, FSH levels in both groups were lower than those before treatment (P<0.05); SAS scores in both groups were lower than those before treatment (P<0.05), and the observation group had lower SAS score than the control group (P<0.05). The total effective rate was 77.1% (37/48) in the observation group, which was higher than 56.3% (27/48) in the control group (P<0.05). Vaginal microbiota analysis showed that at the phylum level, the relative abundance of Firmicutes in the G0 subgroup of the control group was higher than that in the G1 subgroup, while the relative abundances of Bacteroidetes, Actinobacteria and Proteobacteria were lowed in the G0 subgroup. In contrast, the observation group exhibited an opposite trend: the G0 subgroup had lower Firmicutes abundance but higher abundances of Actinobacteria, Bacteroidetes and Proteobacteria compared with the G1 subgroup. At the family and genus levels, Bifidobacterium was enriched in the G0 subgroups of both groups, whereas Lactobacillus predominated in the G1 subgroups. The G0 subgroup of the control group showed higher abundance of Gardnerella, while both Lactobacillus and Gardnerella were concurrently elevated in the G1 subgroup of the observation group. Additionally, in the observation group indicated that Clostridiales and Rikenellaceae were significantly more abundant in the G0 subgroup than in the G1 subgroup (P<0.05). CONCLUSION: The "Tiaojing Cuyun Acupuncture" combined with hormone replacement therapy is superior to hormone replacement therapy alone in improving anxiety. There are differences in vaginal microbiota structure among patients with different theraputic efficacy, suggesting that the vaginal microecological environment may be involved in the mechanism of treatment response in POF.

Humans

Responding to a protracted tuberculosis outbreak: lessons from multiple rounds of investigation in a Chinese boarding school.

PURPOSE: This study analysed a multi-semester pulmonary tuberculosis (PTB) cluster outbreak in a Chinese boarding school to provide evidence for future epidemic control. METHODS: Contacts were screened via symptoms, infection tests and chest radiography. Screening expanded progressively from close contacts to same-floor contacts, then all students and staff. Whole-genome sequencing (WGS) with single nucleotide polymorphism (SNP) and bioinformatics analysis was used for lineage classification, transmission clustering (&#x2264;12 SNPs defining a cluster) and drug resistance prediction. RESULTS: From 2020 to 2022, 20 students were diagnosed with PTB, half laboratory-confirmed. Most cases clustered in class 16 and were epidemiologically linked to the primary case (case 0), who had household PTB exposure. Case 0 and case 1 had diagnostic delays exceeding 3 and 6&#xa0;months, respectively. WGS of five isolates (case 1, 3, 4, 9 and 10) collected over three semesters showed all belonged to lineage 2 and differed by &#x2264;12 SNPs, confirming the same transmission chain. The infection rate in class 16 (46.34%) was significantly higher than other case classes (19.05%) and classes without cases (8.27%) (&#x3c7;2&#xa0;=&#xa0;61.169, p&#xa0;<&#xa0;0.001). No new cases were detected during a one-year follow-up of students involved in the outbreak after the final round of screening, nor among household contacts of all cases followed up to the present. CONCLUSIONS: Lack of entry health examinations facilitated the outbreak. Delayed diagnosis, incomplete contact screening and absence of preventive treatment led to cross-semester persistence. The infection rate disparity confirms class 16 as the outbreak epicentre. Improving community case management, extending contact follow-up and enhancing cluster outbreak measures are recommended to prevent future outbreaks.

Humans

Prenatal exome sequencing of fetuses with central nervous system anomalies based on prenatal ultrasound and magnetic resonance imaging diagnosis: A retrospective cohort study with a systematic review and meta-analysis.

INTRODUCTION: Fetal central nervous system (CNS) abnormalities have diverse etiologies, with genetic factors as a major contributor. Prenatal exome sequencing (ES) is a powerful tool for precise molecular diagnosis of CNS anomalies, but its diagnostic yield varies among studies. This study aimed to evaluate the additional diagnostic yield of prenatal ES compared with chromosomal microarray analysis (CMA) in fetuses with CNS anomalies detected by prenatal imaging. MATERIAL AND METHODS: We collected ES results from fetuses diagnosed with CNS anomalies by prenatal imaging (2019-2024) who had negative results. Subgroup analyses assessed phenotype-specific ES diagnostic yield for associated genes and variants. A systematic review and meta-analysis incorporating our data and published studies further explored the association between phenotype and diagnostic yield. RESULTS: In the cohort study of 219 cases, ES identified pathogenic/likely pathogenic single nucleotide variations in 36 cases (16%). The highest diagnostic yield of ES was in cases with multisystem malformations (25%, 14/55), followed by multiple CNS anomalies (15%, 2/13) and isolated CNS anomalies (13%, 20/151). The most commonly identified isolated CNS anomaly was agenesis of the corpus callosum (31%, 5/16). Neural tube defects with urogenital anomalies were associated with a positive ES finding in 57% (4/7) of cases. The meta-analysis of 989 cases from 22 studies showed a pooled diagnostic yield of ES of 27% (95% CI, 21%-34%). The highest diagnostic yield of ES was in cases of corpus callosum anomalies with facial abnormalities (75%, 8/11) and neural tube defects with urogenital malformations (80%, 12/15). The diagnostic yield of ES for three or more CNS abnormalities was 43% (95% CI, 31%-58%), significantly higher than that for only two abnormalities (10%, 95% CI, 4%-18%). No significant difference in diagnostic yield was found between cases identified by prenatal MRI combined with ultrasound (27%, 95% CI, 20%-36%) and those identified by ultrasound alone (25%, 95% CI, 17%-35%). CONCLUSIONS: ES provided a significantly higher diagnostic yield than CMA for fetal CNS abnormalities, with diagnostic yields varying by phenotype. The systematic review and meta-analysis confirmed that the complexity and combination of malformations are key factors associated with differences in ES diagnostic yield.

Humans

Performance comparison of rapid and native barcoding methods for Oxford Nanopore sequencing of Poliovirus Viral Protein 1 (VP1) amplicons.

Accurate and timely sequencing of poliovirus is critical for global eradication efforts, particularly for molecular epidemiology based on the typing region of the genome, viral protein 1 (VP1). While Oxford Nanopore Technologies (ONT) sequencing has expanded capabilities for poliovirus surveillance, the relative performance of different ONT library preparation methods, including ligation-based (Native Barcoding) and transposase-based (Rapid Barcoding) approaches, has not been systematically evaluated. In this study, we compared rapid barcoding and native barcoding workflows for sequencing VP1 amplicons from 17 type 2 poliovirus-positive samples, each processed in triplicate. Native barcoding generated significantly more sequencing output, producing approximately 2.3-fold greater total read yield than rapid barcoding, and demonstrated higher run-to-run reproducibility (R2 = 0.979-0.998 vs. 0.847-0.929, respectively; p&#x202f;<&#x202f;0.001). In addition, native barcoding generated 80% of the total yield achieved by rapid barcoding within approximately 7&#x202f;h, whereas rapid barcoding required approximately 40&#x202f;h to reach the same output. Despite these differences, both methods produced identical VP1 consensus sequences across all samples, with comparable read quality (median per-base Q-scores of approximately Q17-Q18). Rapid barcoding provided substantial practical advantages, reducing hands-on library preparation time (55 vs. 200&#x202f;min) and per-sample cost ($12.82 vs. $16.54), while simplifying workflow and reducing technical complexity. These findings indicate that sequencing yield may not be a determinant of downstream analytical outcomes for poliovirus VP1 ONT sequencing. Rapid barcoding therefore represents a cost-effective and efficient approach for routine poliovirus surveillance, whereas native barcoding remains advantageous in applications requiring rapid data generation or maximal sequencing depth.

Poliovirus

Diagnostic and clinical utility of exome sequencing and chromosomal microarray in children with GDD/iD: a meta-analysis.

BACKGROUND: Global developmental delay/intellectual disability (GDD/ID) is among the most common neurodevelopmental disorders, with up to half of cases are attributed to genetic factors. Chromosome microarray (CMA) has traditionally been the primary genetic test for idiopathic GDD/ID. However, whole exome sequencing (WES) and whole genome sequencing (WGS) have recently emerged, substantially increasing diagnostic yields in these populations. METHODS: We conducted a comprehensive literature search of PubMed, Scopus, EMBASE, and the Cochrane Library from inception to April 29, 2025. Studies reporting the diagnostic utility of these tests in children with GDD/ID were included and analyzed. RESULTS: A total of 102 studies, comprising 55,752 children, were reviewed. The pooled diagnostic yield of WES was 0.37 (95% CI: 0.33-0.41; I2 = 93%), significantly higher than that of CMA at 0.19 (95% CI: 0.16-0.21; I2 = 95%). Subgroup analyses showed that WES yielded significantly higher diagnostic rates than CMA in both same-sample comparisons (OR = 2.27, 95% CI: 1.08-4.78) and different-sample comparisons (OR = 1.65, 95% CI: 1.15-2.37). Only one study evaluated WGS, reporting a diagnostic yield of 0.27. Meta-regression revealed a significant association between CMA diagnostic yield and the proportion of male participants (p&#x2009;<&#x2009;0.01), but not with WES. No significant difference in diagnostic utility was observed between isolated GDD/ID and GDD/ID with comorbidities. CONCLUSION: In children with unexplained GDD/ID, WES demonstrates superior diagnostic and clinical utility compared to CMA. Incorporating WES as a first-line investigation in the diagnostic evaluation of GDD/ID may be warranted.

Humans

Metatranscriptomic analysis of viral sequences associated with Culex nigripalpus at an Alabama aquaculture site.

Mosquitoes associated with aquaculture habitats can harbor diverse viruses, yet the viromes of many locally abundant species remain poorly characterized. At an aquaculture-associated site in Auburn, Alabama, we surveyed mosquito populations and found Culex nigripalpus to be the dominant species collected. To characterize viruses associated with this mosquito, we performed RNA-seq on pooled female Cx. nigripalpus and compared complementary bioinformatic workflows for viral detection and genome recovery. One workflow removed host-associated reads by mapping to the closest available mosquito reference genome prior to assembly, whereas a second workflow used fully de novo assembly and viral database annotation. Additional protein-level filtering, cross-workflow comparison, and comparison of Trinity and rnaSPAdes assemblies were used to prioritize well-supported viral candidates. Across the original analyses, 16 submitted accessions corresponding to 12 collapsed virus/name groups were recovered, including Merida virus, Hubei mosquito virus 5, Zhejiang mosquito virus, Hubei virga-like virus 3, Rinkaby virus, Elemess virus, Qingnian mosquito virus, Serbia narna-like virus 2, XiangYun narna-levi-like virus 8, Ecclesville picorna-like virus, and baculovirus-like fragments. Several candidates were supported across multiple workflows, while others were recovered only under specific analytical conditions, indicating that candidate recovery was influenced by assembly and filtering choices. Selected viral contigs were independently supported by RT-PCR amplification. Overall, these results provide a first characterization of viral sequences associated with Cx. nigripalpus from an Alabama aquaculture-associated site and show that comparison across assembly and filtering strategies helped prioritize the most consistently supported viral candidates.

Animals

Target Capture of Ancient Shell DNA Enables Phylogenetic Reconstruction of Deep-Sea Molluscs.

Target capture is widely used to enrich endogenous DNA from calcium phosphate skeletal material in vertebrates, but its performance on calcium carbonate hard parts widely produced by invertebrates remains poorly understood. Here, we compared DNA recovery from four fresh and 12 ancient (eight radiocarbon-dated to 1671-1135&#x2009;years old before present) deep-sea vesicomyid clam shells, including species Archivesica marissinica, A. nanshaensis and A. okutanii, using whole-genome sequencing (WGS) or target capture of ultraconserved elements (UCEs). WGS achieved 16.65% on-target read recovery of UCEs from fresh soft tissue, but <&#x2009;1% from shell specimens. By contrast, UCE capture in the same specimen increased on-target reads by up to 155-fold, reaching 29.84% in fresh shells and up to 72-fold, reaching 19.89% in ancient shells. Target capture of UCEs recovered 142-1001 loci per sample compared to 0-230 with WGS alone. Ancient shells of A. marissinica and A. okutanii, based on reads mapped with bwa-mem2 and bbmap, exhibited characteristic post-mortem DNA damage signals, with average 5'-end C-to-T misincorporation rates of 3.46% and 15.97%, respectively, exceeding the levels observed in fresh A. marissinica shells (maximum 1.24%). UCE-based phylogenetic reconstructions incorporating shell ancient DNA recovered two major clades within Pliocardiinae, consistent with published phylogenomic trees. Together, these findings demonstrate that target-capture enrichment enables effective recovery of highly degraded DNA from ancient mollusc shells and supports robust phylogenetic inference at the intrageneric scale, expanding the utility of shells-one of the most abundant invertebrate remains-for evolutionary, biogeographic and conservation studies.

Animals

Real-world clinical utility of exome sequencing in pediatric drug-resistant epilepsy: Experience from a tertiary center in Thailand.

BACKGROUND: Genomic testing has increasingly contributed to the diagnosis and management of pediatric drug-resistant epilepsy (DRE), particularly in patients with suspected genetic etiologies. This study evaluated the diagnostic yield and real- world clinical utility of whole-exome sequencing (WES) in children with DRE. METHODS: Children with DRE and seizure onset before 15&#xa0;years of age were enrolled between January 2020 and December 2023. Clinical data, including demographics, seizure characteristics, developmental history, electroencephalography (EEG), brain magnetic resonance imaging (MRI), and prior investigations, were reviewed. WES was performed in all probands and, when available, their parents. Variants were interpreted according to standard guidelines. Clinical utility and 1-year seizure and developmental outcomes were assessed from follow-up records. RESULTS: Fifty-six patients (23 males, 33 females) were included. The median age at seizure onset was 1&#xa0;year (interquartile range [IQR] 0.3-4&#xa0;years), and 96.4% had developmental comorbidities. Pathogenic or likely pathogenic variants were identified in 39% (22/56), with the highest diagnostic yield in children with seizure onset before 3&#xa0;years of age. Channelopathies accounted for most genetically solved cases (68%), predominantly involving sodium channel genes. Genetic diagnoses provided clinical utility in 73% (16/22) of solved cases by guiding treatment and precision management. At 1-year follow-up, genetically solved patients showed more favorable seizure and developmental outcomes than those with genetically unsolved patients. CONCLUSION: WES achieved a 39% diagnostic yield and substantial clinical utility in pediatric DRE, particularly in early-onset and channelopathy-related disorders. These findings support early molecular diagnosis to facilitate genotype-informed management in appropriately selected children. However, the more favorable developmental and seizure outcomes observed in genetically solved patients should be interpreted with caution, as they may have been influenced by multiple factors beyond genetic diagnosis. In resource-limited settings, careful clinical phenotyping remains essential for treatment decisions and for prioritizing children for genomic testing.

Clinical utility

Emergence of a Novel, Phenotypically Difficult-to-Detect Vancomycin-Resistant Enterococcus faecium Clone (ST117/CT7799).

A significant increase of vancomycin-resistant Enterococcus faecium (VREfm) infections was observed in South-Eastern Austria since 2024. The prolonged outbreak is caused by a novel vanB-VREfm clone (ST117/CT7799, "VREfmstyr"). This study characterizes the atypical difficult-to-detect resistance phenotype and assesses the genomic relatedness of the isolates. Patient and outbreak characteristics were investigated including whole genome sequencing of the isolates. Sensitivity of broth microdilution (BMD), gradient tests (GT), disk diffusion (DD), and automated susceptibility testing (VITEK2) was compared. The performance of commercial screening media was evaluated. From sporadic detections in early 2024 case numbers began to rise during the year. In 30/31 (97%) of all cases, intra-hospital transmission was considered likely and an association with invasive procedures was identified in most cases. Core genome multilocus sequence typing revealed only six allelic differences between VREfmstyr isolates collected in a 12-month period, all belonging to the E. faecium ST117/CT7799 lineage. BMD detected vancomycin resistance (MIC&#x2009;>&#x2009;4&#x2009;mg/L) in no more than 16/31 (52%) of isolates after 24&#x2009;h incubation, while GT and DD misclassified all isolates. Only prolonged incubation improved the performance of these assays. VITEK2 analysis, however, correctly classified all 31 isolates. Of four commercially available VRE-screening agars, only one was capable of detecting VREfmstyr after 24&#x2009;h incubation. The emergence and clonal dissemination of VREfm ST117/CT7799 reveals a serious diagnostic gap as commonly used diagnostic algorithms fail to reliably detect this resistance phenotype. Our findings should help to further evaluate the true geographical distribution and clinical significance of this novel VREfm clone.

Enterococcus faecium

Transcriptomic and RNAi analyses reveal chloride channel 3-associated osmoregulation in Litopenaeus vannamei under low-salinity stress.

Chloride channels and transporters are important for cellular volume regulation and salinity adaptation in euryhaline crustaceans, yet the intestinal transcriptional relationship between plasma-membrane and intracellular chloride pathways remains unclear in Litopenaeus vannamei. In this study, RNA interference of anoctamin 1 (ANO1) was combined with intestinal transcriptome sequencing under the production-relevant low-salinity condition of salinity 3. ANO1 silencing produced a focused transcriptional response, with 16 differentially expressed genes (DEGs) identified (11 upregulated and 5 downregulated). Functional enrichment indicated that these genes were associated with transporter activity, cytoskeletal organization, extracellular matrix-receptor interaction, membrane lipid metabolism, and vesicular processes. Notably, a transcript encoding chloride channel protein 3 (CLC-3) was significantly upregulated following ANO1 knockdown, suggesting a potential transcriptional relationship between ANO1 and CLC-3 in chloride homeostasis. Based on this finding, CLC-3 was selected for full-length cDNA cloning, sequence characterization, salinity-gradient expression analysis, and RNAi-based functional assessment. The cloned CLC-3 cDNA was 2883&#xa0;bp in length and encoded an 850 amino acid protein containing a conserved voltage-gated chloride channel (Voltage-CLC) domain and two cystathionine &#x3b2;-synthase domains. Phylogenetic analysis placed LvCLC-3 within the intracellular CLC-c clade, and tissue distribution analysis showed the highest CLC-3 expression in the intestine. Intestinal CLC-3 expression responded nonlinearly to salinity variation, peaking at salinity 20. Under salinity 3, CLC-3 knockdown reduced ANO1, Na+/K+-ATPase alpha subunit, and Na+-K+-2Cl- cotransporter transcript levels, whereas glutamate-gated chloride channel expression increased. Mild hepatopancreatic structural alterations were also observed after CLC-3 knockdown. These findings suggest that CLC-3 is a salinity-responsive intracellular chloride-transporter candidate associated with intestinal ion-transport-related transcriptional responses after ANO1 suppression in L. vannamei, although the underlying physiological mechanism requires further validation.

Animals

Feasibility of routine clinical liquid-based cytology for lung cancer compact panel testing.

BACKGROUND: The Lung Cancer Compact Panel (cPANEL) is a recently approved highly sensitive multiplex gene panel in Japan that supports both DNA- and RNA-based next-generation sequencing. Although cytological specimens are acceptable for cPANEL, unfixed cell pellets or dedicated preservation tubes are typically recommended. However, evidence remains limited regarding whether residual liquid-based cytology (LBC) cell suspensions prepared for routine cytological diagnosis can be used directly for cPANEL testing without dedicated molecular preservation or additional preanalytical processing. In this study, we evaluated the feasibility of applying LBC specimens that are widely used in contemporary clinical practice to cPANEL. METHODS: We analyzed DNA and RNA quality in 69 clinical LBC specimens. Among these, 51 specimens containing non-small cell lung cancer cells with previously determined driver alteration status were subjected to cPANEL testing to evaluate assay concordance with clinical companion diagnostic results. RESULTS: DNA integrity was generally well preserved (DNA Integrity Number [DIN]: 6.2&#xa0;&#xb1;&#xa0;1.5). In contrast, RNA integrity showed greater variability (DV200: 16.4&#xa0;&#xb1;&#xa0;12.1%). ThinPrep-fixed specimens demonstrated lower DIN and DV200 values compared with CytoRich Red-fixed specimens. Although all samples successfully passed the DNA-based cPANEL assay, six cases (11.8%) failed the RNA-based assay, with RNA yield being a major contributing factor. Among the 46 evaluable specimens, concordance was 95.7% and sensitivity was 92.3%, or 88.9% including RNA module failures as cPANEL-negative. CONCLUSIONS: With appropriate fixative selection and adequate cellularity, cPANEL using clinical LBC specimens may serve as a practical diagnostic platform. We demonstrated that routine LBC specimens can be directly applied to cPANEL without special preanalytical processing.

Humans

Dynamic evolution of chaperone-mediated autophagy is associated with tumor microenvironment remodeling and prognostic stratification in lung adenocarcinoma: insights from single-cell transcriptomics, ensemble machine learning, and experimental validation.

BACKGROUND: Lung adenocarcinoma (LUAD) shows prognostic heterogeneity, and tumor-node-metastasis (TNM) staging is limited for individualized management. Chaperone-mediated autophagy (CMA) maintains proteostasis, but its role during adenocarcinoma in situ (AIS)-minimally invasive adenocarcinoma (MIA)-invasive adenocarcinoma (IAC) progression remains unclear. METHODS: Single-cell RNA sequencing (scRNA-seq) data from GSE189357 and bulk transcriptomes from The Cancer Genome Atlas (TCGA)-LUAD and Gene Expression Omnibus (GEO) cohorts were integrated. CMA activity, cell-cell communication, weighted gene co-expression network analysis (WGCNA), tumor-normal differential expression, machine-learning survival modeling, tumor microenvironment (TME) features, drug sensitivity, and EPC1 function were analyzed. RESULTS: CMA-high tumor epithelial cells increased from AIS (58.1%) to MIA (65.7%) but declined in IAC (44.4%; p < 0.001). CMA-low cells preferentially received fibroblast-derived extracellular matrix cues. A CMA-negatively correlated module identified 69 core genes. Random survival forest (RSF) performed best among 117 machine-learning combinations (mean concordance index > 0.873). High-risk patients had worse survival across cohorts, and the risk score was independently associated with overall survival (hazard ratio = 16.013, 95% confidence interval: 9.579-26.768, p < 0.001). High-risk tumors showed proliferative activation and M0 macrophage enrichment, whereas low-risk tumors showed stronger immune-related signaling. EPC1 overexpression suppressed malignant phenotypes in A549 cells. CONCLUSION: CMA dynamics are associated with stromal and immune remodeling during LUAD progression. A CMA-based model provides robust prognostic stratification and may offer a basis for future TME-guided studies.

Chaperone-mediated autophagy

Construction of circRNA-miRNA-mRNA regulatory networks in the intestine of turbot (Scophthalmus maximus) following Vibrio anguillarum infection.

Circular RNAs (circRNAs) play pivotal roles in post-transcriptional regulation by acting as molecular sponges for microRNAs (miRNAs) within the competitive endogenous RNA (ceRNA) network. However, the regulatory mechanisms in teleost immune responses remain poorly understood. In this study, circRNA-miRNA-mRNA networks were investigated in turbot (Scophthalmus maximus) following Vibrio anguillarum infection to elucidate host-pathogen interactions. Through high-throughput sequencing of intestinal tissues, a total of 50 differentially expressed circRNAs (DE-circRNAs) (18 at 2 hpi, 16 at 12 hpi, 16 at 48 hpi), 212 DE-miRNAs (11 at 2 hpi, 70 at 12 hpi, 15 at 48 hpi), and 1774 DE-mRNAs were identified. Functional enrichment analyses (GO/KEGG) revealed significant associations with immune pathways, including the MAPK signaling pathway and gap junction. An integrated circRNA-miRNA-mRNA regulatory network was constructed, highlighting key interactions including novel_circ_0002573/DE-miR-27a-3p/FGB and novel_circ_0002423/novel_347/GNE, which may regulate inflammatory and antibacterial responses. The expression patterns of selected circRNAs, miRNAs and mRNAs were validated using qRT-PCR, confirming the reliability of the sequencing results. Importantly, fibrinogen beta chain (FGB) and CXCR4/CXCL12 signaling were identified as critical immune modulators. These findings provide insights of the ceRNA regulatory networks involved in teleost intestinal immunity and provide potential molecular targets for selective breeding of disease resistance in this species.

Animals

Diversity and population connectivity of members of the family Eunicidae inhabiting deep-water corals in the North Atlantic.

Eunicid polychaetes are often found in association with Cold Water Corals (CWCs), even establishing symbiotic relationships, such as those described between Desmophyllum pertusum and Eunice norvegica. While genetic connectivity of CWCs across the North Atlantic has been widely studied, little is known about their associated fauna in this regard. Here, we present a study combining a focused analysis of the genetic and genomic connectivity of E. norvegica with a regional assessment of the distribution and evolutionary relationships of three CWC-associated eunicid species from the Cantabrian Sea and the North of the United Kingdom (190-1,230&#xa0;m depth). An integrative approach using genetic (16S, COI and 18S), morphological and ecological data allowed the identification of the eunicids studied, with new records of Eunice cf. nicidioformis and Leodice cf. antarctica in the Cantabrian Sea, as well as previously undocumented associations with CWC species. In addition, RADseq data contributed to the delimitation of the closely related species E. norvegica and Eunice philocorallia. Moreover, the genetic connectivity of E. norvegica was studied trough a RADseq (1,067 neutral SNPs) approach. Our results indicate a single panmictic population across approximately 2,000&#xa0;km, suggesting that oceanographic currents facilitate passive dispersal of E. norvegica lecithotrophic larvae, aided by coral host stepping-stones. The connectivity patterns observed for E. norvegica mirror those of D. pertusum, on which the worm is ecologically dependent. Our study highlights the importance of using integrated genetic, morphological and ecological data to characterise and delineate understudied CWC-associated species and improve our understanding of their dispersal capabilities and genetic connectivity to inform future conservation recommendations.

Animals

Genetic Screening of Colombian Patients With Early-Onset Parkinson Disease.

BACKGROUND AND OBJECTIVES: Early-onset Parkinson disease (EOPD), defined as symptom onset before 50 years of age, accounts for approximately 10% of patients and is suggested to have a greater genetic component than typical late-onset forms of the disease. Recessive variants in PRKN, PINK1, and DJ-1, are the most common genetic cause of EOPD, however, most studies are in patients of white ancestry. This study aims to analyze genetic variants in PRKN, PINK1, and DJ-1 in Colombian patients to help address the gap in EOPD genetic research of South American populations. METHODS: We analyzed 43 unrelated patients with EOPD using Sanger sequencing for the PRKN, PINK1, and DJ-1 genes and employed multiplex ligation-dependent probe amplification to detect copy number variants. Additionally, long-read whole-genome sequencing was conducted on 3 unresolved patients with age at onset before 30 years of age (long-read sequencing [LRS] patient A-C). RESULTS: We identified known pathogenic single-nucleotide variants and copy number variants in the PRKN gene accounting for 2 patients' disease (4.6% of patients). We observed 2 pathogenic variants in PRKN (c.155delA; p.N52Mfs*29 and c.1083+1G>A) in patient 1, who reported an age at onset of 16 years. We further detected a homozygous duplication of PRKN exons 5-6 in an additional patient, age at onset of 18 years. DISCUSSION: Our study helps characterize genetic contributors to EOPD in Colombian patients, demonstrating genetic forms (PRKN, PINK1, and DJ-1) are rare. Our results highlight a need to include diverse populations in research to improve genetic understanding of disease.

Journal Article