BioMed Research International

BackgroundThere is a close correlation between neuroendocrine regulation and pulpitis progression. This study aims to identify key neuroendocrine regulation-related genes in pulpitis, providing insights for its treatment. MethodsGSE77459 and GSE92681 datasets were used to validate experimental findings. Key neuroendocrine regulation-related genes were identified via Cytoscape plugin cytoHubba and expression validation. Gene set enrichment analysis, RNA-binding protein regulatory networks, post-translational modifications, molecular regulatory networks, and drug prediction were performed. Key gene expression was experimentally verified in clinical samples. ResultsTop 10 genes were obtained via cytoHubba; 4 (IL6R, OSM, IL1RN, CCL4) with significant differences between pulpitis and control samples and consistent trends in both datasets were identified as key genes. Gene set enrichment analysis showed key genes participate in pathways like cytokine-cytokine receptor interaction. Related RNA-binding proteins were ELAVL1 and HNRNPA1, with phosphorylation as the main post-translational modification. Core regulatory microRNAs included miR-519, miR-765, miR-23, and regulatory factors included FOXC1, PRRX2. Targeted drugs (e.g., sarilumab, haloperidol decanoate, cyclosporine) were predicted, and clinical sample verification confirmed consistent expression trends. Conclusion4 key neuroendocrine regulation-related genes were identified, which may have clinical significance for the diagnosis and treatment of pulpitis.

Background: Previous studies have shown the benefit of dual antiplatelet therapy (DAPT) for acute minor ischemic stroke. Argatroban, is a thrombin inhibitor and is primarily used in patients with acute ischemic stroke experiencing early neurological deterioration. There is no study about the benefit of antiplatelet plus anticoagulant in this population. We aim to study the difference between the combination of argatroban and clopidogrel and DAPT in the outcomes of patients with acute minor ischemic stroke (AMIS, NIHSS <5) presenting within 72 hours after onset. Methods: Argatroban combined with clopidogrel versus aspirin combined with clopidogrel in Stroke (ACAP study) is an investigator-initiated, multicenter, prospective, randomized, open-label trial with blinded endpoint evaluation conducted at four centers in China. This trial will randomize 464 eligible patients with minor ischemic stroke of NIHSS 5 (232 in each arm) within 72 hours of the last known well to receive intravenous argatroban with clopidogrel (treatment group) or aspirin plus clopidogrel (control group). The primary outcome is the proportion of patients achieving excellent outcome, defined as a score of 0-1 on the modified Rankin scale, at 90 days. Conclusions: The ACAP trial will provide important data on the role of intravenous argatroban in patients with acute minor ischemic stroke presenting within 72 hours of last known well.

Mitochondrial potassium channels, located in the inner mitochondrial membrane, play a crucial role in the cells life/death phenomenon. Activation of mitochondrial potassium channels by potassium channel openers may protect cells against ischemia-reperfusion injury. It is known that mitochondrial large conductance calcium-activated potassium channels interact with various mitochondrial proteins, including enzymes of the respiratory chain. Numerous studies indicate that the mitochondria, especially cytochrome c oxidase, play a crucial role as a chromatophore in the cellular response to red and near-infrared light. In this study, we employ the patch-clamp technique and single-channel recordings to investigate the regulation of glioblastoma mitochondrial large conductance calcium-activated potassium channel activity by infrared light. Specifically, we examined the effects of wavelengths 620 nm, 680 nm, 760 nm, and 820 nm in a redox-controlled environment. Our findings suggest that illuminating the inner mitochondrial membrane with these wavelengths may activate mitochondrial large conductance calcium-activated potassium channels. These results offer new insights into the regulation of mitochondrial potassium channels by cytochrome c oxidase, which may lead to the development of non-pharmacological interventions with potential cytoprotective benefits.

BackgroundIt has been shown that n-3 polyunsaturated fatty acids (n-3 PUFA) of marine origin exert significant beneficial effects on myocardial infarction (MI); however, the underlying mechanisms remained unclear. Ceramides play a vital role in the regulation of energy metabolism, mitochondrial function, and apoptosis. Through the integration of clincial studies and animal experiments, this study aimed to determine whether n-3 PUFA improved myocardial function by modulating ceramide metabolism. MethodsIn a case-control study, 100 patients with AMI and 100 healthy pariticipants were enrolled to measure serum ceramide concentrations. Meanwhile, mice were randomly allocated into 4 groups and administrated to a 3-week intervention with n-3 PUFA in triglyceride and phospholipid forms. A mouse model of MI was then established, followed by an additional 4 weeks of continuous intervention. Subsequent comprehensive assessments of cardiac function were performed in the mice. Finally, the mice were euthanized to conduct targeted ceramide lipidomic analysis and other relevant assays. ResultsThe levels of serum C16:0-, C18:0-, C20:0-, C24:1-ceramides and total ceramides in patients with acute myocardial infarction (AMI) were significantly higher compared with the healthy controls. In the murine model of myocardial infarction, pathological analysis via TTC staining demonstrated that interventions with fish oil (triglyceride form) and krill oil (phospholipid form) both significantly reduced myocardial infarct size. Concomitant echocardiographic assessment confirmed that both treatments markedly elevated left ventricular ejection fraction (LVEF), with the magnitude of improvement being significantly superior to that of the model control group. Concurrently, compared with the model group, the concentrations of ceramides in cardiac tissue and serum were significantly lower in the groups with fish oil and krill oil intervention. Western blot analysis further confirmed that n-3 PUFA intervention upregulated adiponectin expression, reduced ceramide accumulation in myocardial tissue, and inhibited mitochondria-mediated cardiomyocyte apoptosis, thereby improving cardiac function and prognosis following myocardial infarction. ConclusionsThis work demonstrates that n-3 PUFA exert cardioprotective effects following MI mediated by adiponectin-ceramide axis. However, there is no significant difference regarding therapeutic efficacy of n-3 PUFA in triglyceride or phospholipid forms.

Flaxseeds have high nutritive value due to the presence of proteins, lignins (SDG), fatty acids, vitamins, dietary fibers, minerals and carbohydrates. This research was conducted to evaluate the effect of distinct doses of flaxseeds on hematological parameters, immunity and lipid profile of male rabbit. In this research, 60 male rabbits were isolated into four groups, three treatment groups T1, T2 and T3 and a control group T0, with 15 rabbits in each group. The treatment groups were given 4%, 6% and 8% of flaxseeds per daily diet for 45 days. On 15th, 30th and 45th day of experiment, blood samples were collected to examine their hematological parameters. Serum was separated from the collected blood sample to perform ELISA and serum lipid profile test to assess antibody titer and lipid profile of the rabbits respectively. The results indicated a significant reduction in TC, TG, and LDL-C levels in contrast to HDL-C which increased significantly in all treatment groups. Whereas, the statistical analysis of hematological parameters showed an HSD (p[&le;]0.05) in flaxseed treated groups. A maximum level of Hb, WBCs, RBCs, MCHC, MCV, HCT, MCH and differential leukocytes count was recorded in high dose group T3 (8% flaxseeds) followed by medium dose group T2 (6% flaxseeds) and low dose group T1 (4% flaxseeds) respectively. There was a significant rise in antibody titer (p[&le;]0.05) against RHDV (Rabbit Hemorrhagic Disease Virus) comparable to non-treated group. The outcomes illustrated that flaxseeds as nutritional supplement are undoubtedly beneficial to health and prevent various diseases. Study contributionThis research specifically explores how dietary supplementation with flaxseeds, a widely recognized source of omega-3 fatty acids, fiber, and antioxidants, can influence metabolic health and immune function. These findings have significant implications for nutritional interventions aimed at improving cardiovascular health, immune support, and overall well-being, making it highly relevant to the journals readership. The aim of this study was to investigates the dose-dependent effect of flaxseeds on hematological parameters, lipid profile and immunity of male rabbits. Using a controlled experimental design, male rabbits were fed a diet supplemented with varying doses of flaxseeds over a period of 45 days. Key parameters such as total cholesterol, triglycerides, LDL-C, HDL-C, antibody titer, red, white blood cell, platelet counts, Hb, HCT, MCV. MCHC, MCH and differential leukocytes levels were measured to assess the impact of flaxseeds. The results demonstrated that flaxseed supplementation significantly restored lipid profiles by reducing total cholesterol and triglycerides, LDL-C and increasing HDL-C while also enhancing immune function by rising antibody titer and maintaining healthy blood profiles in the subjects.

Abstract Background: Acute ischemic stroke (AIS) remains a significant cause of disability worldwide. Current treatments, primarily intravenous thrombolysis (IVT), are limited by narrow time windows and reperfusion injury, leading to suboptimal outcomes for many patients. Chuanzhi Tongluo (CZTL), a traditional Chinese medicine, has been preliminarily recognized as a novel cerebral protection agent in animal models. Objectives: This trial investigates the efficacy and safety of CZTL capsule in patients with AIS who are not eligible for IVT or who experience early neurological deterioration after IVT. Methods and design: The CONCERN trial is an investigator-initiated, prospective, multicenter, double-blind, parallel-control, randomized clinical study in China. An estimated 1,208 eligible participants will be consecutively randomized to receive CZTL capsule therapy or placebo in 1:1 ratio across approximately 70 stroke centers in China. All enrolled patients are orally administered 2 capsules of CZTL or placebo 3 times a day together with antiplatelet agents for 3 months. Outcomes: The primary endpoint is an excellent functional outcome, defined as a score of 0 or 1 on the mRS at 90 days. Lead safety endpoints included 90-day mortality and symptomatic intracranial hemorrhage within 48 hours. Conclusions: Results of CONCERN trial will determine the clinical efficacy and safety of the traditional Chinese medicine CZTL capsule in the treatment of AIS patients. Trial registry number: ChiCTR2300074147 (www.chictr.org.cn).

Objective. To evaluate the diagnostic and prognostic significance of C reactive protein (CRP) level dynamics within the first five days after surgery for the early detection of surgical site infections (SSI) and to identify independent risk factors, taking into account regional specifics of surgical management (types of surgeries, duration of procedures), as well as the local hospital microbial landscape. Materials and Methods. A single-center retrospective cohort analysis of data from 127 patients who underwent surgical procedures between 2022 and 2024 was conducted. CRP levels on postoperative days 1, 3, and 5 were assessed, and delta values were calculated. Descriptive statistics, ROC analysis, and multivariate logistic regression were used to identify predictors of SSI. Results. Patients with SSI lacked the physiological decrease in CRP levels by day 5. The most informative indicator was the CRP level on day 3: a threshold of >106 mg/L was associated with a high risk of SSI (AUC=0.76; sensitivity 85%, specificity 63%). Independent predictors of SSI included surgery duration (OR=1.015 per 1 min; p<0.001) and the increase in CRP between days 3 and 5 (delta CRP3-5: OR=1.027; p=0.023). A combined model (clinical parameters + CRP) demonstrated the highest predictive ability (AUC=0.79). Conclusion. Monitoring CRP dynamics, particularly on days 3 and 5, is a highly informative and accessible method for the early diagnosis of SSI. A CRP threshold of >100 mg/L on day 3 and its subsequent increase should serve as a trigger for in-depth diagnostic investigation and rationalization of antimicrobial therapy. Keywords: C reactive protein, postoperative complications, surgical site infection, antibiotic therapy, predictive factors, diagnosis

BackgroundDental caries and periodontal disease represent the most prevalent global oral health conditions, collectively affecting several billion people. The diagnostic interpretation of dental radiographs, a cornerstone of modern dentistry, is associated with considerable inter-observer variability. In routine clinical practice, clinicians are required to evaluate a high volume of radiographic images daily, a cognitively demanding task in which diagnostic fatigue, time constraints, and the inherent complexity of overlapping anatomical structures can lead to the inadvertent oversight of early-stage pathologies. Artificial intelligence (AI) offers a transformative opportunity to augment clinical decision-making by providing rapid, objective, and consistent radiographic analysis, thereby serving as a tireless adjunct capable of flagging findings that may be missed during routine human inspection. MethodsThis study developed and validated a deep learning system for the automated detection of dental caries and alveolar bone loss using a dataset of 1,063 periapical and bitewing radiographs. Two separate YOLOv8s object detection models were trained and evaluated using a rigorous 5-fold cross-validation methodology. To align with the clinical use-case of a screening tool where high sensitivity is paramount, a custom image-level evaluation criterion was employed: a true positive was recorded if any predicted bounding box had a Jaccard Index (IoU) > 0 with any ground truth annotation. Model performance was systematically evaluated at confidence thresholds of 0.10 and 0.05. ResultsAt a confidence threshold of 0.05, the caries detection model achieved a mean precision of 84.41% ({+/-}0.72%), recall of 85.97% ({+/-}4.72%), and an F1-score of 85.13% ({+/-}2.61%). The alveolar bone loss model demonstrated exceptionally high performance, with a mean precision of 95.47% ({+/-}0.94%), recall of 98.60% ({+/-}0.49%), and an F1-score of 97.00% ({+/-}0.46%). ConclusionThe YOLOv8-based models demonstrated high accuracy and high sensitivity for detecting dental caries and alveolar bone loss on periapical radiographs. The system shows significant potential as a reliable automated assistant for dental practitioners, helping to improve diagnostic consistency, reduce the risk of missed pathology, and ultimately enhance the standard of patient care.

BACKGROUND: Cranioplasty is an essential procedure to restore cranial integrity, protect neural structures, and improve cosmetic outcomes. However, commercially available implants are often costly, limiting their accessibility in public healthcare systems. Three dimensional (3D) printing offers a low cost alternative for producing patient-specific solutions. METHODS: A retrospective case series of eight patients undergoing cranioplasty using customized polymethylmethacrylate (PMMA) implants fabricated with 3D printed molds was conducted. Computed tomography (CT) scans were used for segmentation and digital modeling. Patient specific molds were designed and printed preoperatively. Variables analyzed included design time, printing time, intraoperative workflow, and clinical outcomes. RESULTS: Design time ranged from approximately 1 hour for small defects to 3 hours for larger defects. Printing time ranged from 2 3 hours for smaller defects and up to 8 10 hours for larger reconstructions. Satisfactory aesthetic outcomes were achieved in 7 of 8 patients (87.5%). No major implant related complications were observed. CONCLUSION: Low cost 3D printing for PMMA cranioplasty is a feasible, accessible, and effective technique for cranial reconstruction, particularly in resource limited settings. Keywords: Cranioplasty; 3D printing; Cranial defect reconstruction; Low cost surgery; Patient specific implants; Polymethylmethacrylate; Skull reconstruction

Chronic kidney disease of unknown etiology (CKDu) has emerged as an important public health challenge, particularly in agricultural communities across Southern Asia and Central America. Our research aims to explore the role of environmental factors in contributing to CKDu prevalence in these regions. Using an Extreme Gradient Boosting Machine Learning (XGBoost) model, we analyzed an environmental dataset from the CKDu endemic region of Sri Lanka. The XGBoost model achieved 85% accuracy in predicting CKDu prevalence in a total of 100 locales. Significant predictor variables included fluoride concentration in water, electrical conductivity of drinking water (EC), pH, and soil type. Fluoride, a common contaminant in drinking water, was the most influential factor, followed by EC and pH, which influence the solubility and bioavailability of nephrotoxic chemicals in water sources. The study findings highlight the urgent need for targeted water analysis programs and interventions in water quality management, agrochemical usage, and soil treatment in CKDu-endemic regions. These insights also provide a framework for future research to identify causative agents and develop strategies for reducing CKDu prevalence.

The molecular basis of triple-negative breast cancer (TNBC), a highly aggressive and therapy-resistant subtype of breast cancer, is poorly understood. This study aims to identify key genes and pathways involved in TNBC development and progression using a systems biology approach followed by experimental validation. Here, two transcriptome microarray datasets from the GEO database were analysed using the R package LIMMA to detect differentially expressed genes (DEGs) in TNBC tumors. Gene Ontology (GO) and Kyoto Encyclopaedia of Genes and Genomes (KEGG) enrichment analyses using the DAVID database were performed to identify DEGs regulated biological functions and pathways. Further, a protein-protein interaction (PPI) network was constructed using the STRING online database, and the topological properties were determined using MCODE and Cytohubba plug-ins. The expression and the prognostic value of the hub genes were validated using the Cancer Genome Atlas (TCGA) survival analysis. We found 727 DEGs, of which 473 were downregulated and 254 were upregulated in TNBC vs. non-TNBC samples. The GO and KEGG analyses indicated that the DEGs were mainly related to cell adhesion, tumorigenesis, and cellular immunity. The PPI network had shown six hub genes, namely CCND1, CDH1, ESR1, FN1, IL6, and PPARG, as the top key regulators. All these genes were validated by quantitative real-time PCR in the TNBC cell line using non-TNBC cell line as a calibrator, and the obtained results were in accordance with the bioinformatics data. This information may contribute to understanding the various molecular mechanisms that drive the development and progression of TNBC tumors.

BackgroundThe most prevalent kind of oral cancer is oral squamous cell carcinoma (OSCC), which has a poor prognosis because of delayed detection and a lack of molecular indicators. MethodsTranscriptomic data from TCGA were analyzed to identify differentially expressed genes between OSCC and normal samples. Functional enrichment analysis was performed to determine biological pathways. A protein-protein interaction network was constructed using STRING and visualized in Cytoscape to identify hub genes. ResultsA total of 5732 differentially expressed genes were identified, including 2459 upregulated and 3273 downregulated genes. Network analysis revealed several highly connected hub genes such as CDK1, CCNB1, TOP2A, BUB1, and MMP9. Functional enrichment indicated significant involvement of cell cycle regulation and cancer-associated pathways. ConclusionThis integrative analysis identified key regulatory hub genes that may be involved in OSCC progression. These genes may serve as promising biomarkers and therapeutic targets for future studies.

Primarily inhabiting the harsh, high-altitude environment of the Qiangtang National Nature Reserve exceeding 5,000 meters above the sea (m.a.s.l.), the golden wild yak is critically endangered, with fewer than 300 individuals remaining in the world, a situation exacerbated by the significant challenges of conducting research and conservation of their genetic resources. Somatic cell nuclear transfer (SCNT) can be an effective method for their preservation, but facing several obstacles in this context, including the hypoxic stress at high altitude that impairs embryonic development due to in vitro manipulation, and constraints of long-distance embryo transport. In the present study, the ear tissue was collected from a childhood male golden wild yak at Xizang Geye Wildlife Rescue Station (4800 m.a.s.l.) and send to Institute of Animal Science at Beijing to derive fibroblast cells. Using fibroblast cells of the golden wild yak as nuclear donors, and bovine oocytes from a local slaughterhouse at Beijing as recipients, the interspecific SCNT (iSCNT) embryos were generated and in vitro developed to blastocysts. To maintain the embryonic viability after long-distance transportation from Beijing to Xizang, iSCNT blastocysts were subjected to cryopreservation by vitrification method. Thawing of vitrified iSCNT blastocysts were completed at Xizang Dangxiong Yak Breeding Innovation Base (4200 m.a.s.l.), and transferred into the uterine horn of domestic yaks. 257 days after blastocyst transfer, a cloned golden wild yak was successfully harvested on January 10, 2026. This work demonstrates, for the first time, that interspecies somatic cell nuclear transfer can successfully produce a cloned offspring under extreme conditions, spanning 4800 m.a.s.l. donor origin, long-distance vitrified embryo transportation, and high-altitude blastocyst transfer at 4200 m.a.s.l., establishing a viable strategy for conserving critically endangered high-altitude species.

PurposeDifferentially expressed genes (DEGs) between colorectal cancer liver metastasis (CRLM) epithelium and primary colorectal cancer (CRC) epithelium (LMR DEGs) identified based on single-cell RNA sequencing (scRNA-seq) data may become new biomarkers for CRC prognosis. MethodsAn scRNA-seq dataset was used to describe the cellular landscape of primary CRC and CRLM and identify LMR DEGs. Prognostic LMR DEGs were identified in the bulk RNA-seq dataset. Based on the prognostic LMR DEGs, multiple machine learning algorithm combinations were compared in terms of their C-index, and the best model was selected for the construction of the LMR score. ResultsAmong the 2070 LMR DEGs, 426 prognostic LMR DEGs were ultimately obtained. The combination of the randomized survival forest (RSF) model and ridge regression had the highest C-index and was therefore used to construct a 15-gene scoring system (LMR score). In the external validation set, the 1- and 5-year AUCs of the LMR score were greater than those of the AJCC stage and other scoring systems constructed with a similar dataset. In addition, the LMR score was closely associated with factors that influence CRC outcomes, such as immune infiltration. ConclusionThe LMR score may be a reliable new biomarker for predicting the prognosis of patients with CRC.

Peripheral Blood transcriptome analysis evaluated the bulk transcript abundance (TA) covering all leukocyte cell populations. However, there are 2 main problems in using bulk expression as biomarkers: (1) A long list of differential expression genes (DEGs) was found, and (2) DEGs cannot be attributed to a host response of any specific cell-type. TA assays after conventional cell sorting, as the gold-standard method, is too tedious for routine use. Recently, we showed that by using a ratio-based biomarker, RBB (ratio of two stringently selected genes), it is feasible to interrogate the gene expression of a single cell-type (monocyte and B lymphocyte) in peripheral whole blood (WB) directly. Here, we apply this in-silico cell sorting algorithm (DIRECT LS-TA, Direct Leukocyte Single cell-type Transcript Abundance) to granulocytes in WB samples to reveal RBBs specific to granulocytes. This DIRECT LS-TA approach without the need for cell-sorting was applied to public datasets to differentiate the 2 types of infection (bacterial vs viral infection). The following RBBs measured in WB correlate with the expression of target (numerator) genes in purified granulocytes, thus cell-sorting can be avoided by using these RBBs: ARG1/SRGN, ANXA3/SRGN, RSAD2/SRGN. Together with monocyte DIRECT LS-TA biomarkers, IFI27/PSAP, direct quantification of 4 genes provided optimal differentiation of viral from bacterial infection. Meta-analysis and unsupervised machine learning classification confirmed the superior performance of DIRECT LS-TA biomarkers. These RBBs found by prior In-silico cell-sorting identified pairs of genes that are used to formulate as ratio-based biomarkers (RBBs) to represent gene expression of granulocytes inside whole blood cell-mixture samples which was useful to triage febrile patients into two major categories of febrile diseases between viral and bacterial infection with high degree of sensitivity and specificity.

Stroke-associated pneumonia (SAP) is a common, severe complication in acute ischemic stroke (AIS) patients receiving bridging therapy (intravenous thrombolysis + mechanical thrombectomy), worsening prognosis and increasing mortality. Current SAP prediction models rely heavily on subjective clinical factors, limiting accuracy. This study developed an interpretable machine learning (ML) model combining inflammatory biomarkers to stratify SAP risk in AIS patients undergoing bridging therapy. We retrospectively enrolled AIS patients who received bridging therapy, collected baseline clinical data and inflammatory biomarkers, and constructed ML models (including XGBoost, random forest) with SHAP analysis for interpretability. The model integrating inflammatory biomarkers achieved excellent predictive performance (AUC=0.XX, 95%CI: XX-XX), outperforming traditional clinical models. SHAP analysis identified key biomarkers driving SAP risk, enhancing model transparency. This interpretable ML model provides an objective, accurate tool for SAP risk stratification in AIS patients, helping clinicians identify high-risk individuals early and implement targeted interventions to improve outcomes.

Parathyroid hormone-related peptide (PTHrP), encoded by PTHLH, has been implicated in tumor progression through its involvement in epithelial-mesenchymal transition (EMT), angiogenesis, and tumor cell migration. Previous experimental studies suggest that PTHrP may promote these processes in colorectal cancer (CRC), partly through the modulation of factors such as secreted protein acidic and rich in cysteine (SPARC) and vascular endothelial growth factor (VEGFA). These events play a key role in the acquisition of an aggressive phenotype in our experimental models. In this study, we performed an integrative in silico analysis of multiple transcriptomic datasets to investigate the potential role of PTHLH in CRC. Differential expression analysis identified a set of consistently dysregulated genes across independent datasets. Functional enrichment and network analyses revealed that PTHLH expression is associated with biological processes related to extracellular matrix remodeling, EMT, and angiogenesis. Correlation analyses showed a positive association between PTHLH and SPARC expression, while network-based approaches suggested a potential functional connection with VEGFA. To assess the clinical relevance of these findings, survival analysis was performed using publicly available datasets. High expression levels of PTHLH, SPARC, and VEGFA were significantly associated with reduced overall survival in patients. Notably, a combined gene signature based on these three factors demonstrated a stronger prognostic effect than individual genes, indicating enhanced predictive value. These findings suggest that PTHrP is associated with molecular pathways involved in tumor progression and, together with SPARC and VEGF, may contribute to a coordinated regulatory axis with prognostic relevance in CRC, warranting further experimental validation.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has continuously evolved since its emergence in the human population in 2019. As of 1st August 2025, more than 1,700 Omicron subvariants have been designated by the Pango nomenclature system. The Pango nomenclature system designates a new lineage based on genetic and epidemiological information of SARS-CoV-2 strains. However, there is a possibility that strains that have similar genetic backgrounds and the same phenotype are given different Pango lineage names. In this paper, we propose a new algorithm, called FindPart-w, which can identify groups of viral lineages that share the same relative effective reproduction numbers. We introduced a new lineage replacement model, called the constrained RelRe model, which constrains groups of lineages to have the same relative effective reproduction numbers. The FindPart-w algorithm searches the equality constraints that minimise the Akaike Information Criterion of constrained RelRe models. Using hypothetical observation count data created by simulation, we found that the FindPart-w algorithm can identify groups of lineages having the same relative effective reproduction number in a practical computational time. Applying FindPart-w to actual real-world data of time-stamped lineage counts from the United States, we found that the Pango lineage nomenclature system may have given different lineage names to SARS-CoV-2 strains even if they have the same relative effective reproduction number and similar genetic backgrounds. In conclusion, this study showed that viruses that had the same relative effective reproduction number were identifiable from temporal count data of viral sequences. These findings will contribute to the future development of lineage designation systems that consider both genetic backgrounds and transmissibilities of lineages.

Aim: To comprehensively characterize the salivary proteome in periodontitis using Orbitrap Astral data-independent acquisition mass spectrometry (DIA-MS), identify an atlas of differentially expressed proteins (DEPs), and develop a machine learning-derived multi-protein biomarker panel for non-invasive diagnosis of stage III/IV periodontitis. Materials and Methods: Unstimulated saliva samples from 199 participants (periodontal health/gingivitis, n=120; stage III/IV periodontitis, n=79) were analyzed by Orbitrap Astral DIA-MS. DEPs were identified, and pathway enrichment analysis was performed. A two-tier machine learning pipeline, integrating pathway-based feature selection with cross-validated evaluation, was applied to identify the optimal diagnostic panel. Results: Orbitrap Astral DIA-MS quantified 5,597 salivary proteins and 1,966 DEPs (|log2FC|>0.5, FDR<0.05). Pathway analysis identified 14 periodontitis-relevant KEGG pathways, including Th17 cell differentiation, IL-17 signaling, neutrophil extracellular trap formation, and complement and coagulation cascades. A four-protein panel (TEC, RAC1, MAPK14, KRT17) achieved an area under the curve (AUC) of 0.985 plus-or-minus sign 0.010, with 83% sensitivity and 100% specificity. The panel was corroborated using public datasets. Conclusions: To our knowledge, this study represents the first application of Orbitrap Astral DIA mass spectrometry in periodontitis research, establishing a disease-specific DEPs atlas and a salivary biomarker panel with high diagnostic accuracy for stage III/IV periodontitis, providing a foundation for future external validation studies.

BackgroundMultidrug-resistant (MDR) Gram-negative bacteria have triggered a critical global health crisis. Polymyxin lipopeptide antibiotics are used as a last-line therapy against these problematic pathogens, but their clinical use is largely limited by severe nephrotoxicity. Human oligopeptide transporter 2 (hPepT2) is a membrane transporter mediating the reabsorption of polymyxins in renal proximal tubular cells, substantially contributing to their nephrotoxicity. However, it remains unclear how polymyxins interact with hPepT2. MethodsIn this study, we investigated the structure-interaction relationship (SIR) of polymyxins with hPepT2 by integrating computational, chemical and cell biology approaches. Bioinformatic modelling predicted the residues essential for the binding of polymyxins with hPepT2. Transporter mutagenesis and molecular analysis were employed to explore the role of each residue in the interaction of hPepT2 and polymyxins. Moreover, we synthesised a series of polymyxin-like analogues with altering the moieties that are critical for binding with hPepT2. The antibacterial activity and nephrotoxicity of these analogues were subsequently assessed. ResultsOur bioinformatic modelling proposed an outward-facing structure of hPepT2 with a possible transport pathway that polymyxins bind to the lateral opening site of hPepT2 (e.g. E214, D215, D317, D342, E622). Molecular assays for transporter function and expression confirmed that D215 residue of hPepT2 is critical for polymyxin binding, while several other residues significantly impact on transporter turnover rate and/or protein expression. Our experimental validations showed that the lipopeptide analogues with altering the Dab1, Dab3, Dab5 and Dab9 moieties of polymyxins demonstrated decreased interactions with hPepT2. Among these synthetic analogues, alanine substitution at Dab3 showed reduced nephrotoxicity in mice while reserved antibacterial activity against a range of bacterial strains. ConclusionsOverall, this proof-of-concept study demonstrated that the computationally predicted and experimentally validated polymyxin-hPepT2 SIR model provides a viable approach for the discovery of novel, safer lipopeptide antibiotics.