Abstract: Background: GLP-1 receptor agonists and related incretin-based therapies are effective pharmacological treatments for weight loss in adults with obesity and type 2 diabetes. However, weight regain after treatment discontinuation or attenuation remains a major clinical challenge. Appetite dysregulation may be one mechanism contributing to this regain. Objective: This systematic review aimed to evaluate whether appetite-related changes contribute to weight regain following GLP-1-based weight loss, with particular attention to the distinction between direct post-treatment evidence and indirect evidence from active-treatment studies. Methods: A systematic literature search was conducted in PubMed, Scopus, Embase, and the Cochrane Library for studies published between 2016 and 2026, following PRISMA 2020 guidelines. Eligible studies included adults with obesity, overweight, and/or type 2 diabetes receiving GLP-1 receptor agonists or related incretin-based therapies and reporting appetite-related outcomes, weight outcomes, weight maintenance, or weight regain. Randomized controlled trials, clinical trials, crossover studies, and observational studies were included. Findings were synthesized qualitatively. Results: Eighteen studies published between 2017 and 2026 met the inclusion criteria. Most studies reported that GLP-1-based therapy reduced appetite, hunger, food cravings, energy intake, or dietary intake, while increasing satiety and promoting weight loss during active treatment. Direct appetite measures were commonly reported using visual analogue scales, appetite questionnaires, food craving measures, and energy intake assessments. However, only a small number of studies directly evaluated appetite changes during treatment withdrawal, weight maintenance, or post-treatment weight regain. Therefore, the available evidence supports a plausible mechanistic link between loss of appetite regulation and weight regain, but the evidence remains largely indirect. Conclusion: GLP-1-based therapies consistently improve appetite regulation and promote weight loss during active treatment. However, direct evidence linking post-treatment appetite dysregulation to subsequent weight regain remains limited. Appetite dysregulation should therefore be interpreted as a plausible and clinically relevant contributor, rather than a definitively established causal driver.

Abstract:

Microbial protein production from acetate represents a promising route for sustainable protein supply, yet its efficiency is constrained by limited understanding of carbon–nitrogen metabolic coordination. In this study, nitrogen availability was systematically varied to investigate its role in regulating carbon partitioning and protein biosynthesis in Yarrowia lipolytica. Nitrogen limitation markedly reduced cell growth and protein accumulation (19.56% of dry cell weight) while increasing lipid content (up to 34.16%), indicating a redistribution of carbon flux from protein to lipid synthesis. Transcriptomic analysis revealed a global downregulation of anabolic pathways under nitrogen limitation, accompanied by a shift in nitrogen assimilation from the glutamate dehydrogenase (GDH) pathway to the glutamine synthetase/glutamate synthase (GS–GOGAT) pathway, as well as significant upregulation of genes related to ammonium and amino acid transport. Guided by these findings, metabolic engineering of key nitrogen assimilation pathways was performed. Co-overexpression of GDH and GS increased protein content from 48.52% to 55.77% and improved amino acid composition, whereas GOGAT overexpression impaired growth and protein accumulation. These results demonstrate that nitrogen availability governs carbon allocation through coordinated regulation of nitrogen transport and assimilation, and that balanced enhancement of GDH and GS is an effective strategy to improve protein production from acetate, supporting the development of sustainable fermentation processes using CO₂-derived substrates.

Abstract: The mechanosensitive ion channel Piezo1 acts as a crucial molecule for cellular mechanical signal sensing and transduction. It transforms physical mechanical cues in the microenvironment, including matrix stiffness, fluid shear stress, and tissue tension, into intracellular biochemical signals through Ca2⁺ influx-mediated mechanisms. Consequently, it regulates the activation, proliferation, differentiation, migration, and effector functions of blood cells. Herein, we review the research progress of Piezo1 in various white blood cells, with a particular emphasis on its functional regulatory mechanisms in neutrophils, macrophages, platelets, and T and B lymphocytes. We briefly summarize its current functional status in natural killer cells, dendritic cells, plasma cells, platelets, and other cell types. We analyze the integrated effects and multi-cellular cooperative interactions of Piezo1-mediated blood cell mechanotransduction across physiological and pathological contexts. We discuss the potential value of Piezo1 as a mechano-immunotherapeutic target, therapeutic strategies, and challenges facing clinical translation. Finally, we provide perspectives on future research directions, offering theoretical references for deepening the understanding of the molecular mechanisms by which mechanical microenvironments regulate white blood cell function and disease progression and for developing novel therapeutic strategies.

Abstract: Ageing remains one of biology’s most fundamental unresolved questions. Existing explanations often attribute ageing to stochastic damage accumulation, adaptive programmes, or interacting hallmarks, yet none fully explains why ageing emerges so broadly in organisms that undergo development. Here, I introduce the Double Code Hypothesis of Ageing, which frames ageing as an inevitable consequence of life’s dual inheritance system: the genome and the epigenome. In this manuscript, “code” is used in a computer-science-like sense, closer to source code than to the specialised meaning used in code biology: an organised set of biological instructions whose effects depend on being read, interpreted, maintained, and executed by cellular machinery.I propose that ageing is not merely the progressive accumulation of epigenetic noise within an individual, but the consequence of an inherent instability in a dual inheritance system whose two informational layers must remain functionally aligned across cellular and organismal generations. The relative stability of the genome allows long-term information preservation, whereas the plasticity of the epigenome enables development, differentiation, and adaptation, but also makes this layer vulnerable to cumulative misalignment.This progressive loss of genome–epigenome coordination acts as a natural, non-programmed limit on lifespan in complex organisms. Rather than an encoded death programme, ageing is described as increasing informational disorder emerging from the unresolved tension between genetic stability and epigenetic plasticity. This falsifiable framework offers experimental predictions in model systems such as Schizosaccharomyces pombe and provides a mechanistic explanation for why ageing exists.

Abstract:

Kelch-like ECH-associated protein 1 (Keap1) acts as a repressor of nuclear factor-erythroid 2-related factor 2 (Nrf2), a major transcription factor regulating cellular antioxidant response. Keap1 is the substrate adaptor subunit of the cullin 3-RING E3 ubiquitin ligase complex that specifically facilitates Nrf2 ubiquitination and its proteasomal degradation. Keap1 is rich in cysteine residues and several of them undergo various modifications, such as sulfhydration, nitrosylation and glutathionylation under cellular stress conditions. Some of these modifications alter the conformation of Keap1, preventing Nrf2 from ubiquitination and subsequent proteasome-mediated degradation. As a result, newly synthesised Nrf2 translocates to the nucleus to induce the expression of diverse genes involved in protecting cells against oxidative stress. Protein CoAlation is a reversible redox-dependent post-translational modification (PTM) in which coenzyme A (CoA) forms disulphide bonds with oxidised cysteine residues under oxidative or metabolic stress. In this study, we demonstrate for the first time that disulphide Keap1 dimer undergoes CoAlation in cellular response to oxidative stress induced by various oxidising compounds. Furthermore, glucose deprivation also induces CoAlation of disulphide Keap1 dimer in HEK293/Pank1β cells. We also demonstrate that Keap1^{11 Cys-less} mutant is not CoAlated in response to diamide treatment or glucose deprivation. In summary, this study uncovers a novel PTM of Keap1 by the key metabolic integrator CoA, which provides new insights into the regulation of the Keap1-Nrf2 antioxidant pathway under oxidative and metabolic stress.

Abstract: Background: Genitourinary (GU) cancers — encompassing prostate, urothelial, and renal cell carcinomas — collectively represent a leading source of oncologic morbidity and mortality worldwide. Tumor heterogeneity and the dynamic evolution of resistance mechanisms limit the clinical utility of static tissue biopsies, creating an urgent need for real-time, non-invasive biomarkers. Circulating tumor DNA (ctDNA), isolated from peripheral blood or urine, captures the somatic mutational landscape of the tumor in a temporally resolved manner and has emerged as a compelling candidate predictive biomarker for treatment selection and resistance monitoring across GU malignancies. Methods: An integrative review was conducted following the Whittemore and Knafl (2005) framework. A systematic literature search was performed across PubMed/MEDLINE, Cochrane Central Register of Controlled Trials, Embase, and Web of Science, covering the period January 2020 to April 2026. Search strategies combined MeSH and free-text terms pertaining to ctDNA, liquid biopsy, and the three principal GU cancer types. After duplicate removal and staged screening, 23 studies met inclusion criteria and formed the basis of the qualitative synthesis. Results: The integrated evidence demonstrates that ctDNA functions as a dynamic predictive biomarker across three GU cancer domains. In castration-resistant prostate cancer (CRPC), ctDNA tumor fraction detected at cycle 3, day 1 of enzalutamide therapy independently predicts radiographic progression-free survival and overall survival (mOS 16.0 vs. 22.1 months for ctDNA-positive vs. ctDNA-negative patients). In muscle-invasive bladder cancer (MIBC), post-cystectomy ctDNA identifies metastatic relapse with 94% sensitivity and 98% specificity and stratifies benefit from adjuvant atezolizumab in IMvigor010. In renal cell carcinoma (RCC), emerging data support ctDNA for VHL and PBRM1 mutation tracking and treatment response assessment, though prospective validation remains limited. ctDNA-guided detection of androgen receptor splice variant 7 (AR-V7) and FGFR3 alterations enables biologically informed treatment sequencing in CRPC and urothelial carcinoma, respectively. Conclusion: ctDNA constitutes a molecularly rigorous, clinically actionable biomarker platform with demonstrated predictive and prognostic value across the GU cancer spectrum. Integration into prospective clinical trials and regulatory frameworks represents the critical next step toward embedding ctDNA-guided decision-making into standard oncologic practice.

Abstract: Polymerase chain reaction (PCR) is a widely used molecular biology technique; however, it remains highly susceptible to non-specific primer binding, particularly in genomic regions with extensive sequence similarity. Such off-target amplification can generate false amplicons that are difficult to detect using conventional quality control methods and may lead to erroneous downstream interpretation. Here, we present FalseAmpHunter, a pipeline designed to detect, assemble, and characterize false amplicons from paired-end next-generation sequencing (NGS) data generated from PCR amplification. FalseAmpHunter reconstructs candidate amplicons, maps them genome-wide, evaluates primer-binding orientation, and distinguishes true target amplification from paralog-driven off-target products and sequencing artifacts. We validated FalseAmpHunter using a synthetic dataset derived from in silico PCR of paralogous olfactory receptor (OR) genes. The pipeline successfully identified both the intended target amplicon and false amplicons originating from paralogous loci, while excluding a random decoy control. FalseAmpHunter provides a systematic and transparent solution for investigating false PCR amplification events and is applicable to assay development, primer validation, and troubleshooting of targeted sequencing experiments. By transforming raw sequencing data into interpretable genomic evidence, it enhances confidence in PCR-based analyses, particularly in paralog-rich genomic contexts. The pipeline is accessible online at: https://github.com/xoaib4/FalseAmpHunter.

Abstract: Background: Recent work has revealed that protein-coding sequences encode regulatory information influencing mRNA stability and translation through a nascent peptide code. However, the evolutionary origin of this regulatory layer remains unclear. This study aims to determine when peptide-mediated translational control emerged during the evolution of the proteome and genetic code. Methods: Dipeptide-specific effects on mRNA stability and translation were integrated with a phylogenetic timeline of dipeptide emergence derived from dipeptide sequences across proteomes. Each of the 400 canonical dipeptides was assigned an evolutionary age, and experimentally derived regulatory effects were mapped onto this timeline, with associations assessed using rank-based correlation and regression analyses. Results: A weak but statistically significant negative association was observed between dipeptide age and mRNA stability, indicating that more recently evolved dipeptides tend to destabilize transcripts. This trend was stronger at the amino acid level, where later-emerging residues showed greater contributions to reduced mRNA levels. Destabilizing effects were associated with physicochemical properties such as positive charge, side-chain bulkiness, and β-strand propensity. Mapping these effects onto codon space revealed a non-random distribution aligned with the evolutionary and structural organization of the genetic code. Destabilizing effects were also enriched within specific codon exchange groups, indicating that regulatory signals are structured within the degeneracy and mutational neighborhoods of the code. Conclusions: These findings indicate that the nascent peptide code is a late evolutionary innovation linked to amino acid expansion and proteomic complexity, with regulation embedded within both peptide sequences and the degeneracy structure of the standard genetic code.

Abstract: Protein conformational inheritance is documented across bacteria, fungi, and neurons, and while several authors have argued for its conceptual inclusion in molecular inheritance frameworks, a concise, widely adopted formal vocabulary that unifies these literatures remains lacking. Naming by pathological outcome rather than mechanism has partitioned prion disease research, adaptive conformational biology, and protein engineering into separate silos despite a shared mechanistic basis. This review formalises the shared mechanism with three terms. Alt-F protein (alternatively folded protein) names the mechanistic class irrespective of outcome. Conformoreplication names the protein-to-protein templated propagation of fold geometry — a third conceptual extension of the Central Dogma. The conformotype is the third molecular inheritance tier, transmitted through cell division independently of DNA sequence or epigenetic marks, initiated by post-translational modifications acting as environmental transducers, and regulated by the chaperone network. The framework identifies blind spots in three research fields that the current sequence-centric paradigm structurally excludes: AMR surveillance that cannot detect conformationally-encoded resistance in genotypically susceptible isolates; industrial biotechnology that screens enzyme variants by sequence but not conformational state; and neurodegeneration therapeutics that targets downstream aggregates rather than the upstream chaperone regulatory failure. To move beyond conceptual identification of these blind spots, the review operationalises each through a concrete analytical pipeline — integrating conformational proteomics, MSA-subsampled AlphaFold screening, and chaperone modulation assays — demonstrating that the conformotype framework is not merely taxonomic but immediately actionable across all three fields.

Abstract: This study supports the use of human skin explants as a versatile and translational model for evaluating pharmacologic skin responses and topical bioactivity. The approach allows mechanistic insight beyond single biomarkers and may serve as a scalable pharmacologic platform for efficacy testing, particularly when expanded to include multiple donors and broader readouts.

Abstract: Background: Autism spectrum disorder (ASD) affects approximately 1–2% of children worldwide, yet its etiology remains incompletely understood. Emerging evidence suggests that offspring of parents with autoimmune diseases show elevated autism prevalence. Notably, children of parents with psoriasis (OR 1.59), type 1 diabetes (OR 1.49–2.36), and rheumatoid arthritis (OR 1.51) demonstrate particularly strong associations. Hypothesis: I propose that autism may be conceptualized as an immune-metabolic disorder in which TNF-α-mediated mitochondrial dysfunction contributes to cerebral energy deficiency. This energy deficit may impair three critical processes: (1) synaptic pruning during neurodevelopment, (2) real-time social cognition including gaze processing and emotion recognition, and (3) protein synthesis of critical synaptic scaffolding molecules. The proposed mechanism is TNF-α pathway dysregulation arising from inherited inflammatory susceptibility and/or direct fetal exposure to elevated maternal TNF-α during pregnancy. I further propose that the well-documented “firstborn effect” in autism reflects maternal immune maladaptation during primigravid pregnancies. Additionally, for cases without parental autoimmune history, a speculative secondary mechanism is proposed: mitonuclear immune conflict, in which paternal immune genes may partially recognize maternal mitochondria as non-self, generating endogenous TNF-α. A newly expanded component of the model is the decidua basalis–placenta–fetal brain transmission pathway, in which disruption of the decidual tolerogenic circuit, placental amplification of inflammatory signals, and fetal microglial reprogramming may together contribute to altered neurodevelopment during sensitive gestational windows. The model further proposes that this process may occur even in the setting of a clinically silent, chronic low-to-intermediate pro-inflammatory cytokine state that is insufficient to endanger maternal or fetal survival, yet sufficient to disrupt fetal brain development. Implications: This framework may provide an integrative account of disparate observations about autism pathophysiology and suggests that TNF-α-related pathways merit further investigation for potential risk modification, particularly in pregnancies identified as high-risk through parental autoimmune or inflammatory disease.

Abstract: (Background) Aiming to reduce synthetic fertilizer dependence and enhance soil fertility, this study isolated and characterized nitrogen-fixing bacteria from the maize rhizosphere. (Methods) Nitrogen-free selective media were used for bacterial isolation, followed by detection of the nifH gene and nitrogenase activity. Phylogenetic identification was conducted via 16S rRNA sequencing. Growth-promoting traits, stress tolerance, and pot-based plant inoculation effects were assessed. Genetic modification of strain GN8811 was performed to improve nitrogen fixation and growth promotion. (Results) Seven isolates that carried the nifH gene and exhibited nitrogenase activity were closely related to four genera. Several isolates showed phosphate solubilization, iron chelation, IAA production, or potassium solubilization, with GN2003 and GN8811 tolerating high salinity and variable pH. Inoculation with GN8811 promoted maize growth comparable to nitrogen fertilization, and its genetically modified derivative (ΔnifL::PrpoD) showed further improvement even under high nitrogen conditions. (Conclusions) These findings highlight the potential of combining microbial screening with genetic engineering to develop efficient bioinoculants for sustainable maize cultivation.

Abstract:

Objectives: This study aimed to investigate the phytochemical content of the endemic plant Thymus fedtschenkoi var. handelii (Ronniger) Jalas and, for the first time, to examine its anticancer potential on various cancer cell lines. Methods: The plant was collected from natural habitat and the essential oils (EOs) composition was analyzed using GC-MS. The anticancer efficacy and cytotoxicity of plant extracts and the EOs were evaluated using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and lactate dehydrogenase (LDH) methods on lung (A549, Calu1, H1650), bone (SW1353, MG63, Saos2), prostate (PC3, DU145, LNCaP) and brain (A172, B35, C6) cancer cell lines, as well as normal cell lines (Beas2B, FL, HC). Results: The main components of the EOs were determined to be high amounts of carvacrol (51.12%), γ-gamma-terpinene (16.87%), and p-cymene (14.76%). Both the extract (GI50: 1.10–3.28 µg/mL) and the EOs (GI50: 1.05–2.03 µg/mL) exhibited strong antiproliferative activity. However, EOs demonstrated markedly superior growth suppression, with TGI values of 1.97–9.19 µg/mL, whereas the extract required substantially higher concentrations (110.6–261.5 µg/mL). The LC50 values of all samples exceeded 500 µg/mL in all cell lines tested, indicating that the natural compounds predominantly had a cytostatic effect. Normal cells showed comparable reduced sensitivity, supporting selectivity. Morphological analyses further confirmed treatment-induced cellular alterations consistent with antiproliferative and apoptotic processes. Overall, EOs emerged as the most potent fraction, combining low TGI values with moderate cytotoxicity, indicating strong therapeutic potential. Conclusions: The potent and selective antiproliferative activity of T. fedtschenkoi var. handelii may hold significant therapeutic potential in the pharmaceutical industry.

Abstract: Current variant interpretation frameworks, including those proposed by the American College of Medical Genetics and Genomics and the Association for Molecular Pathology (ACMG/AMP) and ClinGen, rely on implicit assumptions regarding allele frequencies, linkage disequilibrium (LD), and independence of variables. These assumptions are largely derived from European populations and are not valid in highly admixed populations. Here, we argue that the Brazilian population constitutes a natural stress test for these frameworks, not merely due to the extent of admixture, but due to its qualitative structure, characterized by recent tri-hybrid admixture (European, African, Indigenous American) and extensive recombination generating novel haplotypic configurations. We further highlight the instability of borderline variants of uncertain significance (VUS) under Bayesian classification and propose a reformulation explicitly incorporating haplotype structure and local ancestry.

Abstract: The axonal membrane is not the seat of nerve conduction: it is the boundary between two osmotic reservoirs whose asymmetry is the thermodynamic engine of the action potential. Voltage-gated ion channels are not the generators of the nerve signal: they are its osmotic amplifiers, and their spatial distribution along the axon is a geometric necessity, not an arbitrary anatomical feature. The Ionic-Mechano-Hydraulic (IMH) model formalises this principle: intracellular K⁺ adsorbed on the cytoplasmic polyelectrolyte gel triggers an ionic phase transition; extracellular Na⁺ amplifies the resulting hydraulic wave through Nav channels; Kv channels close the osmotic cycle and enforce the refractory period. The conduction velocity is predicted from the elastic modulus of myelin, not from the density of the sodium channel. The model resolves a 75-year-old anomaly that Huxley and Stämpfli themselves described as impossible in a purely electrical system: a positive current enters a node before the membrane potential at the preceding node has reached its maximum. Ten falsifi-able predictions are presented that cover myelin mechanics, mechanoreceptor adaptation, terminal arborisation geometry, velocity-diameter scaling, and axon diameter limits derived from first physical principles. The Hodgkin-Huxley model is not discarded: it is explained.

Abstract: The Apennine wolf (Canis lupus italicus) is a distinct subspecies whose ongoing population recovery in Italy has progressively increased the demand for live capture protocols validated for scientific monitoring and conservation management. Despite the widespread use of mechanical and chemical immobilization in European wolf management, no study has to date systematically evaluated the integrated combina-tion of a humane mechanical restraint system and a structured chemical immobiliza-tion protocol — and specifically the association of the Fremont™ humane foot snare with a medetomidine-ketamine-acepromazine (MKA) protocol, in terms of their joint physiological effects and welfare implications for this subspecies under operational field conditions. Between June 2010 and July 2017, thirteen free-ranging Apennine wolves were captured in Maiella National Park (central Apennines, Italy) using the Fremont™ snare and immobilized with a standardized MKA protocol. Cardiorespira-tory parameters, body temperature, peripheral oxygen saturation, venous blood gas values, and a comprehensive hematological and serum biochemical panel were rec-orded during immobilization. Mean heart rate was 100 ± 15 bpm, respiratory rate 24 ± 13 breaths/min, and body temperature 38.1 ± 1.0°C. No clinically significant hyper-thermia was recorded in the cohort as a whole. Hematological and biochemical values were broadly consistent with published reference ranges for the species, with condi-tion-specific deviations identified in two individuals (one pregnant female and one ju-venile presenting signs of transient capture-related myopathy), both of which resolved without clinical sequelae. No capture-related mortality occurred. All thirteen individ-uals survived the minimum post-capture monitoring period, and preliminary GPS da-ta suggest a transient reduction in movement activity in the immediate post-release period. These findings support the welfare adequacy and operational feasibility of the combined Fremont™ snare–MKA protocol for the Apennine wolf, and provide base-line physiological and hematobiochemical reference data for Canis lupus italicus rele-vant to future capture and conservation management programmes.

Abstract: Psoriasis and psoriatic arthritis (PsA) are systemic immune-mediated diseases, but the features that distinguish cutaneous-dominant psoriasis from musculoskeletal involvement remain unclear. We analyzed four core public cross-sectional datasets spanning whole-blood methylation, PBMC single-cell RNA sequencing summarized at the subject level, skin RNA sequencing, and purified CD4+ T-cell methylation, and used two additional public skin cohorts for external contextual checks to define an inflammatory disease axis (DIR) and a contrast-resolved systemic-state coordinate (CRS) representing additional systemic immune-state variation associated with PsA. In whole-blood methylation, DIR primarily separated healthy controls from psoriasis, whereas CRS separated psoriasis from PsA with minimal correlation to DIR. In PBMC single-cell data, CRS was higher in PsA and in the source-defined PSX subgroup (joint pain without CASPAR-classified PsA) than in PsO. Cell-type-resolved analyses localized CRS-related shifts to CD8 naive T cells, NK cells, CD14 monocytes, and regulatory T cells and identified multicompartment pathway-state remodeling along the CRS continuum. In contrast, skin RNA sequencing mainly captured lesional inflammatory burden and showed only limited additional PsA-related separation within the same tissue state. These findings support a model in which PsA is distinguished from psoriasis by an additional systemic immune-state axis rather than by skin inflammatory burden alone.

Abstract: Oxidative stress is a major contributor to neuronal apoptosis and subsequent neurofunctional deficits. This study investigates the dual role of the mitochondrial membrane-anchored protein NIX in PC12 cells, a model for mature neurons. We demonstrate that both overexpression and knockdown of NIX attenuate apoptosis under oxidative stress, albeit through distinct mechanisms. Overexpression of NIX promotes cell survival by activating NIX-mediated mitophagy, which clears damaged mitochondria and intracellular reactive oxygen species (ROS), thereby maintaining redox homeostasis. Conversely, knockdown of NIX reduces apoptosis primarily by diminishing the intrinsic pro-apoptotic function of the protein. Collectively, these findings reveal that NIX expression levels critically gate PC12 cell fate under oxidative stress by differentially activating pro-survival or anti-apoptotic pathways.

Abstract: Background: Persistent high-risk human papillomavirus (hrHPV) infection causes over 99% of cervical precancers and cancers worldwide, with HPV genotype 16 (HPV16) responsible for 50% of the cases. Latvia ranks among the top EU countries for cervical cancer incidence and mortality. In the general Latvian population, 4.2% women are hrHPV-infected, mostly with HPV16. However, information on the circulating HPV16 isolates is missing. Objectives: To study the genomic variability of the Latvian HPV16 isolates, compare them with HPV16 in Europe and across the globe, reveal features associated with the severity of cervical disease and uncover eventual sequence changes due the national HPV vaccination. Methods: DNA was extracted from the formalin-fixed paraffin-embedded cervical tissues of women diagnosed with cervical intraepithelial neoplasia (CIN) stages I-III and squamous cell carcinoma (SCC) grades 1-3 collected between 2012 and 2024. Samples positive for HPV16 were subjected to whole genome sequencing (WGS) on the Illumina platform (n=16) or Sanger sequencing of the E6/E7 coding region (n=31). Consensus HPV16 sequence was generated, and single nucleotide polymorphisms (SNP) and eventual amino acid substitutions (AAS) were analysed. Results: Complete genomes of 16 HPV16 variants were reconstructed, 13 related to the European sublineage A1, and 3, to the sublineage A2 references. Sequences showed high conservation, still 93 non-redundant variants were identified. The highest variability was observed for the capsid protein L2, the lowest, for oncoprotein E7. Prevalence of SNPs and AAS in the Latvian HPV16 variants, specifically in capsid protein L1, did not increase with time, showing no effect of HPV vaccination. Associations between HPV16 sequence features and severity of cervical disease were limited to AAS E6:L90V which was significantly more common in SCC grade 2/3 than in CINII/III cases (p=0.015). Conclusions: Highly conserved HPV16 genomes circulating in Latvia harbour series of unique, as well as common nonsynonymous SNPs with respective AAS, with one, AAS E6:L90V, associating with disease severity. No HPV vaccine escape variants were detected. Deciphering complete genomes of HPV16 from CIN and SCC cases in Latvia informs public authorities performing HPV vaccination and is useful for management of HPV-associated cervical diseases.

Abstract: The expansion of captive breeding of the forest musk deer (Moschus berezovskii)—a first-class protected species in China with significant economic and medicinal value—has led to escalating disease threats, underscoring the need for intelligent disease management based on knowledge graphs. However, the unstructured nature of veterinary texts, characterized by nested entities, ambiguous boundaries, and sparse relations, poses substantial challenges to accurate joint entity-relation extraction. To address these issues, this study proposes BRW-GPLinker, an enhanced joint extraction model built upon the GPLinker framework. This model integrates a Boundary-Aware Module (BAM) for precise entity boundary detection, a Relative Distance Bias Module (RDBM) to minimize pairing errors in dense contexts, and a Weighted Sparse Multi-label Cross-Entropy (WSMCE) loss function to improve recall for infrequent relations. Experimental results on the constructed MS-Data dataset demonstrate that BRW-GPLinker achieves an F1 score of 0.887, outperforming the baseline GPLinker by 2.0 percentage points. It also exhibits strong generalization, with an F1 score of 0.590 on the general-domain CMeIE-V2 dataset. These findings confirm that the proposed model provides reliable support for constructing disease knowledge graphs in forest musk deer farming.