We report a chromosome-level assembly of a male red coachwhip snake (Masticophis flagellum piceus) generated exclusively with nanopore sequencing. Using Hifiasm-ONT for assembly and RagTag for scaffold polishing, we produced a 1.61 Gb nuclear genome comprising 8 macrochromosomes and 10 microchromosomes with a 97.7% BUSCO completeness score. Annotation with LiftOn found 19,832 loci, including 18,025 protein-coding genes. The mitochondrial genome, assembled with MitoHiFi and annotated with MitoFinder, was 17,119 bp with 13 coding genes, 22 tRNAs and 2 rRNAs. All sequencing was performed in a simulated mobile laboratory using a portable sequencer and a laptop with analyses done both locally and remotely. These results highlight the feasibility of decentralized genomics and its potential to accelerate biodiversity research globally.

Background: HER2-low breast cancer is a biologically heterogeneous subgroup in which hormone receptor (HR) expression critically shapes prognosis and treatment, but the underlying regulatory mechanisms remain unclear. MicroRNAs (miRNAs) are key post-transcriptional regulators of gene expression and may contribute to HR heterogeneity. This study aimed to identify deregulated miRNAs and associated gene networks distinguishing HER2-low/HR-positive from HER2-low/HR-negative tumors, elucidating the molecular mechanisms underlying this divergence. Methods: Differential expression analyses of miRNAs and genes were performed using Wilcoxon tests and DESeq2 (|log2FC| > 1; FDR-adjusted p-value < 0.05). Survival analyses were conducted using Cox proportional hazards models to evaluate the individual miRNAs and miRNA signature. Functional enrichment analyses, including GO, KEGG and Reactome pathways, were performed. Correlation analysis and the miRNA target prediction were integrated to identify regulatory interactions. Results: Comparisons between HER2-low/HR-positive and HER2-low/HR-negative tumors identified 165 significantly deregulated miRNAs and 170 strongly deregulated genes. Intersection analysis highlighted miR-9-5p, miR-532-5p and miR-576-5p as specifically associated with HR-negative status. Survival analyses showed non-significant trends for the overall survival and progression-free interval. Functional enrichment analysis revealed hormone-related pathways in HR-positive tumors and immune, inflammatory and proliferative pathways in HR-negative tumors. Integrative correlation and target prediction analyses identified two miRNA–mRNA regulatory axes, miR-576-5p/TGFBI and miR-9-5p/POU2F2. Conclusions: Our study demonstrated that HER2-low breast cancer exhibits distinct miRNA and gene expression profiles, which highlight different transcriptomic profiles according to HR status for the first time. Specific miRNA–gene networks may drive transcriptional heterogeneity, serving as potential biomarkers for stratification and as therapeutic targets. These findings provide insight into the molecular basis of HER2-low tumor diversity and support future development of HR-directed therapeutic strategies.

Background/Objectives: To identify US military unknowns, the Armed Forces Medical Examiner System’s Armed Forces DNA Identification Laboratory has historically relied upon mitochondrial DNA and Y-chromosomal short tandem repeat testing. Where no appropriate family reference sample (FRS) is available or skeletal samples are degraded, autosomal single nucleotide polymorphism (SNP) testing with next-generation sequencing could assist. Methods: A method utilizing hybridization capture enrichment of a 95,000 (95K) SNP panel, amenable to FRS and extremely challenging samples, was validated. The Parabon Fx Forensic Analysis Platform was used for analysis and extended kinship inference. Skeletal samples (n = 65) and associated FRS (n = 64) were selected for a performance evaluation and case-type sample study. Results: Considering FRS with ≥7 ng DNA input into library preparation, 94% yielded ≥66,320 SNPs at ≥5X coverage. SNP recovery for skeletal samples at ≥1X coverage ranged from 5 to 94,197 SNPs, averaging 40,770 SNPs. When skeletal samples resulted in ≥13,000 SNPs, the most likely relationship category was consistent with the expected relationship. A log10 likelihood ratio of ≥4 and a posterior probability of ≥99.99% were established as thresholds for strong statistical support, and 87% of inferences met these thresholds while 13% were considered inconclusive. Pairwise kinship inference between unrelated individuals yielded an unrelated result in 85% of comparisons, 66% with strong statistical support. There were 170 instances of false positive 4th degree relationship inferences with strong statistical support. All false positives involved skeletal samples from individuals of admixed ancestry. Conclusions: With this approach, autosomal SNP testing can result in reliable kinship inferences between related individuals out to 3rd, and in some cases 4th, degree relationships, increasing the scope of eligible FRS to aid in identifications.

Background/Objectives: Chlamydia trachomatis (CT) infection is one of the most prevalent sexually transmitted infections (STIs) worldwide and has been consistently associated with adverse reproductive outcomes, including female infertility. However, the molecular mechanisms underlying this association remain incompletely understood. This study aimed to investigate whether genes previously associated with female infertility display altered expression patterns in response to CT infection by reanalyzing publicly available transcriptomic data derived from a human in vitro infection model. Methods: An integrative in silico approach was employed. A curated list of 106 genes associated with female infertility was compiled from publicly available databases and integrated with transcriptomic data from the Gene Expression Omnibus (GEO) dataset GSE109428, which profiles primary human fallopian tube mesenchymal cells infected in vitro with CT serovar L2. Gene expression changes were evaluated at two time points (24 and 48 h post-infection) by comparing infected cells with uninfected control samples, followed by functional and phenotype enrichment analyses. Results: One female infertility-associated gene (AKAP12) was consistently dysregulated at both 24 and 48 h post-infection. In addition, fourteen genes (ANAPC4, BMP1, BNC2, BTG4, EFHD1, FBXO43, INHBB, PATL2, SCARB1, SND1, SYNE1, TRIP13, TTC28, and TUBA1C) became significantly dysregulated exclusively at 48 h post-infection, indicating a time-dependent host transcriptional response to CT infection. Functional and phenotype enrichment analyses revealed associations with biological processes related to embryonic development and meiosis, as well as phenotypes linked to female infertility. These enriched terms were supported by a small subset of genes and were therefore interpreted cautiously. Conclusions: Overall, these findings suggest that CT infection modulates the expression of several infertility-associated genes and may influence biological pathways critical for female reproductive function. While exploratory, this study provides a molecular context that aligns with previously reported associations between CT infection and female infertility.

Background/Objectives: The Subo Merino sheep is a high-quality fine-wool breed developed through progressive hybridization, characterized by high wool yield and excellent wool quality. This study is designed to investigate the effects of two gene polymorphisms in Subo Merino sheep on wool traits, thereby providing critical theoretical and technical support for the breeding of high-quality fine-wool sheep. Methods: In this study, 944 one-year-old Subo Merino sheep were genotyped for coding regions of the BLTP1 and KIF27 genes using the Fluidigm BioMark™ HD system. Association between SNP loci and wool traits was analyzed via the least squares means method in SAS 9.4. Protein–protein interaction networks were constructed using the STRING database, and protein structures before and after mutation were predicted with SOPMA and SWISS-MODEL. Results: The results revealed that BLTP1 gene identified a missense mutation site SNP1, which resulted in a nucleotide change c.812 (C > T) and an amino acid change p.Pro271Leu. KIF27 gene identified a missense mutation site SNP2, which resulted in a nucleotide change c.3896 (T > C) and an amino acid change p.Met1299Thr. Association analysis showed that SNP1 had a significant effect on wool crimp number (CN) and staple length (SL) (p < 0.05), while SNP2 significantly affected live weight after shearing (LWAS) (p < 0.05). Protein structure prediction showed that mutations at SNP1 and SNP2 primarily led to changes in α-helix, extended chain, and random coil structures. Conclusions: These results suggest that SNP1 in BLTP1 and SNP2 in KIF27 could serve as potential molecular markers for wool traits in Subo Merino sheep. This study provides theoretical support and candidate gene targets for molecular marker-assisted breeding, contributing to genetic improvement and efficient breeding of this fine-wool breed.

Background/Objective: Pancreatic and duodenal homeobox 1 (PDX1) is a key transcription factor required for pancreatic development and maintenance of β-cell function. Genetic variants in PDX1 have been associated with monogenic forms of diabetes, including maturity-onset diabetes of the young type 4 (MODY4). However, the func-tional consequences of many reported non-synonymous single-nucleotide polymorphisms (nsSNPs) in PDX1 remain unclear. In this study, an integrated in silico approach was applied to systematically identify and characterize po-tentially deleterious nsSNPs in the PDX1 gene. Methods: Missense variants were retrieved from public databases and evaluated using multiple sequence- and structure-based prediction tools to assess functional impact, disease association, protein stability, and structural consequences. Variants considered deleterious were further examined through three-dimensional structural modeling and molecular dynamics simulation. Results: Several nsSNPs were identified with consistent predictions of pathogenicity, reduced protein stability, and pronounced structural and dynamic perturbations. Variants including R197G, Y170N, and T151K in the PDX1 Protein were considered the highest deleterious mutants. Conclusion: These findings will provide insight into the molecular mechanisms by which PDX1 mutations may contribute to β-cell dysfunction and diabetes development and offer a rational framework for prior-itizing variants for experimental validation and clinical interpretation.

Background/Objectives: Recombinant adeno-associated virus (AAV) vectors have revolutionized gene therapy for monogenic diseases such as Duchenne muscular dystrophy (DMD). However, high systemic doses required for muscle transduction cause a spectrum of toxicities ranging from transient hepatic inflammation to fatal multi-organ failure leading to death. These adverse events have reshaped the risk–benefit considerations for gene therapy in DMD. Methods: We conducted a narrative review describing complications associated with AAV-mediated gene therapies in the DMD field. PubMed and Clinicaltrials databases were used to search for peer-reviewed manuscripts published between 1987 and 2025. Publicly available abstracts and press releases were also used to describe AAV-mediated adverse events that have been discovered. Priority was given to large prospective cohorts, meta-analyses, and high-impact publications. Results: We outlined the mechanistic basis of AAV toxicity—spanning innate and adaptive immune activation, vector–host interactions, transgene overexpression, and host vulnerability—and discussed their therapeutic implications for DMD. We also highlighted ongoing strategies for vector re-design, immune modulation, patient selection, and regulatory adaptation, aiming to improve efficacy with safety in the next generation of muscular dystrophy gene therapies. Conclusions: Patient safety remains the number one priority in the AAV-mediated gene therapies field. Achieving long-term benefits requires continued optimization of existing vectors, implementation of strict criteria for patient selection, and regulation of immune responses, with close collaboration and transparent dialog among scientists, clinicians, and regulatory agencies, informed by both successful cases as well as tragic deaths reported in the fields of neuromuscular diseases.

Background/Objectives: Salvia miltiorrhiza (Danshen) is a well-known medicinal herb in traditional Chinese medicine. It produces tanshinones and salvianolic acids as key bioactive constituents in its roots, yet the molecular basis for their tissue-specific accumulation is still poorly understood. This study aims to identify candidate functional genes involved in the biosynthesis of tanshinones and salvianolic acids, and to reveal the molecular basis underlying their tissue-specific accumulation in S. miltiorrhiza. Methods: For this purpose, we compared transcriptomic and metabolomic differences between the roots and leaves, and further measured a set of physiological parameters, including the POD, SOD, CAT and PAL activities, as well as the total phenols and flavones contents. Results: Metabolomic analysis identified 6805 metabolites, of which 241 were differentially accumulated between roots and leaves, with 172 upregulated in roots. The elevated metabolites included gibberellin, cryptotanshinone, decursinol angelate, chalcone, and psoralenol. Transcriptome analysis identified 32,700 annotated genes, with 9895 showing differential expression between roots and leaves, including 4199 upregulated in roots. Roots exhibited higher levels of phenols and flavones, as well as significantly greater POD, SOD, CAT, and PAL activities. Conclusions: Integrated omics analysis identified putative candidate genes including CPS, KS, and P450s as potential contributors for tanshinone and salvianolic acid biosynthesis. The identified genes provide valuable resources for molecular breeding, offering opportunities to improve the medicinal quality of S. miltiorrhiza.

Background: Broodiness is a major limiting factor for reproductive efficiency in indigenous avian breeds, a phenomenon underpinned physiologically by granulosa cell (GC) apoptosis and subsequent follicular atresia. While Serine Protease 23 (PRSS23) has been implicated in mammalian ovarian remodeling, its specific regulatory function in avian follicular dynamics remains elusive. Methods: Utilizing the Wuding chicken—an indigenous breed distinguished by robust environmental adaptability but compromised by high broodiness frequency—as a biological model, this study dissected the molecular mechanism of PRSS23-mediated follicular regression. We cloned the complete coding sequence of the Wuding chicken PRSS23 gene, characterized its spatiotemporal expression profile, and interrogated its function in primary GCs via gain- and loss-of-function assays. Results: RT-qPCR analysis revealed that PRSS23 is differentially expressed across the hypothalamic–pituitary–ovarian (HPO) axis, with ovarian expression being significantly upregulated during the broody period compared to the laying period. Mechanistically, PRSS23 overexpression significantly downregulated the expression of follicle-stimulating hormone receptor (FSHR) and key steroidogenic enzymes (STAR, CYP19A1, HSD3β1), thereby suppressing the expression of genes governing the biosynthesis potential of progesterone and estradiol. Concurrently, PRSS23 overexpression was associated with transcriptional repression of components of the PI3K/AKT/mTOR signaling cascade; this transcriptional regulation further induced cell cycle arrest at the G0/G1 phase, and activated the mitochondrial apoptotic pathway characterized by BAX upregulation and BCL2 downregulation. Conversely, siRNA-mediated knockdown of PRSS23 alleviated these inhibitory effects, promoting GC proliferation and survival. Conclusions: These findings establish PRSS23 as a pivotal pro-atretic factor in Wuding chickens, driving ovarian atrophy through the dual transcriptional-level inhibition of steroidogenesis and survival signaling pathways. This study identifies a potential molecular target for marker-assisted selection programs aimed at attenuating broodiness while preserving the superior meat quality traits of indigenous poultry.

Background: Niemann–Pick type C is a lysosomal storage disorder that results from pathogenic variants in the NPC1 gene or in some cases from NPC2 pathogenic alterations. The disease presents a remarkable clinical variability that in some cases resembles common diseases, often resulting in a diagnostic odyssey or at least delaying proper diagnosis. In addition, the NPC1 gene is highly polymorphic, and consequently, when missense variants are identified after gene sequencing, accurate classification of their pathogenicity is essential to ensure appropriate access to available therapies and to provide reliable genetic counseling. Objectives: To get insights into the pathogenicity of a novel variant in NPC1, p.Cys800Ser, we created stable cell lines expressing this variant, in parallel with cell lines expressing the NPC1 wild-type and NPC1 pathogenic variants. Methods: We leveraged an isogenic cell line in which the NPC1 gene was knocked down and subsequently infected it with retroviruses carrying NPC1-WT and NPC1 variants C-terminally fused with an mNeonGreen tag. Three different NPC1 variants were included in this study: two known pathogenic variants, p.Ala1035Val and p.Pro1007Ala, and the novel p.Cys800Ser, whose significance was unknown. Results: We observed in the stable cell line expressing NPC1 p.Cys800Ser that the mutated NPC1 protein is transported to the lysosome similarly to the p.Pro1007Ala variant and affects lysosomal distribution. Conclusions: Using this approach, we could analyze the pathogenicity of each variant separately and these cell lines could be used for personalized medicine-based approaches and multi-omic studies.