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Clustering patterns of behavioral and metabolic risk factors for noncommunicable diseases in Iran: findings from a national STEPS survey.

BACKGROUND: Noncommunicable diseases (NCDs) are the leading cause of mortality in Iran, driven by behavioral and metabolic risk factors that frequently co-occur. OBJECTIVE: To identify patterns of co-occurring behavioral and metabolic NCD risk factors among Iranian adults and characterize their demographic and socioeconomic correlates. METHODS: This cross-sectional study analyzed data from 16,618 adults aged ≥25 years who participated in Iran's 2021 nationally representative STEPS survey. Thirteen behavioral and metabolic variables, including physical activity, nutrition score, smoking frequency, alcohol intake, salt intake, body mass index, blood pressure, fasting plasma glucose, and lipid markers, were entered into a K-means clustering analysis. Clusters were characterized by their risk profiles and demographic/socioeconomic attributes. Multinomial logistic regression examined associations between cluster membership and sociodemographic factors. RESULTS: Five distinct behavioral-metabolic clusters emerged. The smokers-drinkers (SD) cluster (3.1%) comprised mostly older, less-educated men with high smoking and alcohol use. The healthy-low-risk (HLR) cluster (40.3%) showed favorable profiles and included younger, more educated individuals. The physically active (PA) cluster (6.6%) was characterized mainly by younger men with markedly high physical activity levels. The dyslipidemic (DLP) cluster (26.0%) exhibited high dyslipidemia and overweight prevalence, while the hypertensive-diabetic (HTD) cluster (24.0%) had the highest obesity, hypertension, and diabetes rates, common among older urban adults. CONCLUSION: Behavioral and metabolic NCD risk factors in Iran formed five distinct co-occurrence patterns. Nearly half of adults belonged to metabolically high-risk clusters, highlighting the need for targeted prevention strategies that combine lifestyle interventions with screening and management of obesity, hypertension, diabetes, and dyslipidemia.

Humans

The potential of clustering methods for pre-test triage in sleep medicine: A systematic review.

Sleep disorders exhibit substantial heterogeneity, and traditional classifications may not fully capture clinically relevant subtypes. Clustering techniques can identify patient subgroups that improve phenotypic characterization and may support personalized management. This systematic review evaluated the application of clustering in sleep medicine, with particular focus on its potential use as a pre-test triage tool prior to formal sleep testing. PubMed/MEDLINE, Embase, Web of Science, and Scopus were searched to February 2025. Eligible studies applied clustering to classify sleep disorders in adults. Two reviewers independently conducted screening, data extraction, and risk-of-bias assessment using QUADAS-2. The protocol was registered on PROSPERO. Fifty-one studies (1983-2025) were included, predominantly focused on obstructive sleep apnea (OSA) (n = 38, 74%). Hierarchical clustering (n = 20) and K-means clustering (n = 14) were the most frequently used techniques. Internal validation was reported in only 18% of studies, and external validation was reported in only 1 study. Seven studies relied exclusively on baseline clinical, demographic, or questionnaire data, representing pre-test scenarios, whereas most incorporated polysomnography-derived variables, limiting their applicability to early clinical stratification. Hierarchical clustering was the most commonly applied method; however, the overall lack of validation limits confidence in the robustness and clinical applicability of identified phenotypes. The potential role of clustering as a pre-test triage strategy remains largely unexplored, as most studies focused on post-diagnostic phenotyping and were affected by incorporation bias. Future research should prioritize pre-test clinical variables, rigorously validate internally and externally, and adopt standardized methodological and reporting practices to facilitate clinical translation.

Humans

Efficient rDNA-mediated multi-copy integration of gene clusters in Aureobasidium melanogenum.

Aureobasidium melanogenum is a promising non-conventional yeast chassis for synthetic biology. However, techniques recombining large genetic fragments, such as gene clusters, are still unavailable, hindering further metabolic reprogramming in this chassis. To achieve multi-copy integration of genes, we employed highly repetitive ribosomal DNA (rDNA) sequences in A. melanogenum as homologous recombination sites for large genetic fragments. First, integration efficiency of three different regions of A. melanogenum rDNA were investigated: RNA polymerase I promoter region (rDNA1, 1.0 kb), partial 26S rDNA region (rDNA2, 1.0 kb), and RNA polymerase I terminator region (rDNA3, 1.0 kb). Our findings revealed that the highest copy numbers and expression stability were observed for the short heterologous green fluorescent protein gene (gfp, 0.7 kb) and the long native polyketide synthase gene (pks, 7.0 kb) after rDNA1-mediated integration. Specifically, the copy numbers reached 7.0 and 8.0 for gfp and pks, respectively, and they remained stably expressed in the genome after 120-h subculturing. Furthermore, an 11.0 kb gene cluster (comprising the native pks, phosphopantetheinyl transferase (npg1), and scytalone dehydratase genes (scd) responsible for melanin biosynthesis) was integrated at the rDNA1 site, resulting in stable recombination with 15.0 copies and an approximately 12-fold increase in melanin production. Overall, the convenience and efficiency of the proposed rDNA-mediated multi-copy insertion strategy will facilitate superior metabolic engineering of A. melanogenum chassis cells.

Multigene Family

Assessing the accuracy and efficiency of an electronic platform for managing childhood illnesses in rural China: A cluster randomized controlled trial.

OBJECTIVES: The Integrated Management of Childhood Illness (IMCI) faces challenges in capacity building and quality control. This trial aims to assess an electronic IMCI (eIMCI) platform in improving the effectiveness and efficiency in disease classification and management by community health workers (CHWs). DESIGN: Cluster randomized controlled trial. SETTING: Rural western China. PARTICIPANTS: 24 CHWs and 72 ill children aged 2 months to 5 years (3 children per CHW). CHWs were randomly assigned to intervention or control groups. INTERVENTIONS: The intervention CHWs received online training and performed disease management using the eIMCI platform featuring integrated training modules and decision-support tools. The control group received traditional face-to-face training and used paper-based IMCI protocols. MAIN OUTCOME MEASURES: Proportion of children correctly diagnosed or classified by CHWs, as determined by a pediatric specialist. Relative risk (RR) between groups was estimated using Poisson Generalized Linear Mixed Models incorporating a random intercept for CHW to account for clustering of children within individual CHWs and adjusting for key covariates at both the CHW and child levels. RESULTS: The intervention group (13 CHWs, 39 children) had a higher rate of correct classification (64.1%) compared to the control group (11 CHWs, 33 children) (39.4%, P&#x2009;=&#x2009;.056). Multivariable regression analysis confirmed this (RR&#x2009;=&#x2009;2.1, 95% CI: 1.5-3.1; P&#x2009;<&#x2009;.001). No significant difference was found in correct treatment rates (38.5% vs. 27.3%, P&#x2009;=&#x2009;.316). Online training reduced time and costs by approximately 80%, though with a slight decrease in post-training evaluation scores. CONCLUSIONS: The eIMCI platform shows potential in enhancing IMCI implementation and significantly reducing the training burden in resource-limited settings. Trial registration: Chinese Clinical Trial Registry: ChiCTR2100042533, https://www.chictr.org.cn/showproj.html?proj=119995.

Humans

Assessment and CommuniCation ExcelLEnce foR sAfe paTient outcomEs (ACCELERATE): A stepped-wedge cluster randomised trial evaluating the effectiveness of a nurse-led assessment and handover communication intervention on patient adverse events.

BACKGROUND: Patients continue to experience harm from undetected deterioration, falls and pressure injuries. We aimed to implement and evaluate an organisational, ward-level nurse-led assessment and communication intervention to proactively reduce patient adverse events. METHODS: A stepped-wedge cluster randomised Trial over 12-months was conducted at three metropolitan hospitals. Our intervention comprised a comprehensive, systematic patient assessment at shift commencement; a structured patient-centred bedside nurse-to-nurse clinical handover; and multidisciplinary communication consisting of nurse participation in medical ward rounds. Evidence-based implementation strategies informed intervention delivery to nine clusters (20-35 bed-wards with &#x2265;70% permanent nurses) over three sequential 14-week steps. Routinely collected patient-level data were used to measure intervention effect. The primary outcome was a composite measure of medical emergency team calls, unplanned intensive care unit admissions, in-hospital falls; and stage 2-4 pressure injuries. Secondary outcomes were: individual measures of the primary outcome; nurse-reported perceptions of safety culture; organisational readiness to change; barriers to physical assessment; staff engagement; and patient-reported experience measures of safety and overall hospital experience. Analyses were adjusted for age, sex, hospital, pre/post intervention, and Trial step (fortnight), with random effects for ward and patient. RESULTS: There were 13,753 eligible admissions. No change was observed in the primary composite outcome measure (odds ratio (OR) [95% confidence interval (CI)]: 0.99 [0.77, 1.28]; p&#xa0;=&#xa0;0.95). There was no significant difference in medical emergency team calls (OR [95% CI]: 1.02 [0.75, 1.39]; p&#xa0;=&#xa0;0.91); unplanned intensive care unit admissions (OR [95% CI]: 1.35 [0.57, 3.20]; p&#xa0;=&#xa0;0.50) and falls (OR [95% CI]: 1.53 [0.96, 2.45]; p&#xa0;=&#xa0;0.07). However, stage 2-4 pressure injuries significantly decreased by 41% (OR [95% CI]: 0.59 [0.38, 0.93]; p&#xa0;=&#xa0;0.02); a significant absolute effect improvement of 0.8% ([95% CI: 0.3%-1.3%], p&#xa0;<&#xa0;0.01). There were statistically significant improvements in nurses' overall perceptions of Safety Attitudes (Pre: 74.6, Post: 79.7; p&#xa0;=&#xa0;0.02), and the Organisational Readiness to Change subscales of, leader culture (Pre: 3.73, Post 3.91; p&#xa0;=&#xa0;0.02), leadership behaviour (Pre: 3.85, Post: 4.11; p&#xa0;=&#xa0;0.03), and general resources (Pre: 3.06, Post: 3.30; p&#xa0;=&#xa0;0.03). A statistically significant decrease in Barriers to Physical Assessment (Pre: 2.48, Post: 2.24; p&#xa0;<0.001) and in six of seven sub-scales was observed. Patients' overall Measure of Safety remained high, but unchanged (Pre: 3.94 Post: 3.92; p&#xa0;=&#xa0;0.07). CONCLUSION: The ACCELERATE Trial demonstrated that nurse-driven initiatives, emphasising structured physical assessments by nurses, patient-centred clinical handovers, and multidisciplinary communication, significantly: reduced pressure injuries; decreased nurses' perceived barriers to performing physical assessments; and improved leadership behaviour, communication, and ward safety culture perceptions. Results highlight the transformative potential of this approach, which now warrants testing at scale for broader implementation. TRIAL REGISTRATION: Australian New Zealand Clinical Trials Registry ID: ACTRN12621000265875.

Humans

Open Dialogue versus treatment as usual for adults presenting in crisis to mental health services in England (the ODDESSI Trial): a multisite cluster-randomised trial.

BACKGROUND: Open Dialogue is a person-centred, transdiagnostic model of mental health care that emphasises continuity, therapeutic relationships, and collaboration with the service user's social network. Open Dialogue is a service-wide approach to care involving network meetings with the service user, members of their social network, and usually two practitioners who support the network throughout the duration of care. In this cluster-randomised trial, we aimed to evaluate the clinical effectiveness of Open Dialogue versus treatment as usual for adults presenting in crisis to community mental health services in England. METHODS: This multicentre, parallel two-arm, cluster-randomised, controlled superiority trial was conducted in mental health services in five National Health Service trusts in London and the South of England. Clusters were defined at the level of primary care practices within service catchment areas. Participants were adults aged 18 years or older presenting in crisis to mental health services and registered with a practice within trial clusters. Randomisation was done at the cluster level (1:1), stratified by catchment area, and balanced on average general practice (GP) list size and Index of Multiple Deprivation (2015). The chief investigator, senior statistician, and assessors of the primary outcome were masked in the study. Participants either received Open Dialogue or treatment as usual, which refers to the functional team model currently implemented throughout English mental health services. The primary outcome was time (days) to first relapse following initial recovery from the index crisis censored at the end of the 2-year follow-up period. Participant-reported secondary outcomes were EuroQol Visual Analogue Scale, Social Provisions Scale, Lubben Social Network Scale, Questionnaire about the Process of Recovery, and the Client Satisfaction Questionnaire, measured at five timepoints over 2 years, and clinical measures were extracted from electronic health records. People with relevant lived experience were involved in the design and execution of the study. Fidelity to the model of care in Open Dialogue and treatment as usual, and adherence to the delivery of Open Dialogue, were measured prior to each site starting participant recruitment, then every 6 months thereafter until the final participant follow-up in that site. The trial was retrospectively registered (ISRCTN52653325) and is complete. FINDINGS: 185 general practices associated with six mental health Trusts across England were identified for screening. 105 practices were excluded, and 80 were included in cluster formation, forming 32 clusters that were randomly assigned (16 to treatment as usual and 16 to the Open Dialogue intervention). One mental health trust (two clusters) withdrew, resulting in five mental health trusts (30 clusters) participating in the trial. Between June 25, 2019, and Dec 9, 2021, 494 participants (266 [54%] female gender, 221 [45%] male gender, 341 [69%] White British) with a mean age of 38&#xb7;1 years (SD 13&#xb7;4) provided consent for study inclusion (223 in the treatment as usual group and 271 in the Open Dialogue group). Of these, 174 (78%) in the treatment as usual group and 225 (83%) in the Open Dialogue group recovered and had data enabling relapse determination; there was no significant difference between groups on the primary outcome of time to relapse following initial recovery (marginal hazard ratio 0&#xb7;95 [95% CI 0&#xb7;67-1&#xb7;32]). For secondary outcomes, Open Dialogue was associated with significantly lower probabilities of psychiatric inpatient admission and re-referral to crisis care or secondary mental health services, and with improvements in self-rated recovery, health-related quality of life, and satisfaction with services. There were no significant differences in social network quality or size. There were 386 serious adverse events (281 in the treatment as usual group and 105 in the Open Dialogue group); 376 (97%) were deemed to be unrelated to the intervention. INTERPRETATION: Open Dialogue did not reduce time to first relapse compared with treatment as usual, the primary outcome, but it reduced acute inpatient bed use, improved service user reported outcomes and experience, and there were no significant safety concerns. Further investigation is required to determine whether Open Dialogue can enhance the effectiveness and acceptability of crisis care and continuing care in community mental health services. FUNDING: National Institute for Health Research.

Humans

The HOXA gene cluster: a critical regulator in bone-related disorders.

BACKGROUND: Skeletal homeostasis relies on the dynamic balance between bone formation and bone resorption. The disruption of this balance acts as the central pathological mechanism of multiple metabolic bone diseases including osteoporosis, and is closely correlated with the progression of various other bone-related disorders. As pivotal transcription factors regulating embryonic development and cell fate, the homeobox A (HOXA) gene family plays an essential role in skeletal physiological and pathological processes. METHODS: This review systematically summarizes recent research advances of the HOXA gene family in bone-related diseases, concludes the evolutionarily conserved regulatory patterns of HOXA members, and clarifies the molecular mechanisms by which HOXA genes mediate bone metabolic disorders and the occurrence as well as development of bone diseases. RESULTS: Accumulating evidence demonstrates that HOXA family members present complex functions and strong heterogeneity in bone-related diseases. They participate in the pathogenesis of bone diseases via three evolutionarily conserved regulatory manners: determining regional patterning, modulating signaling pathways, and integrating epigenetic and non-coding RNA (ncRNA) regulatory networks. CONCLUSION: Further exploring the underlying mechanisms of the HOXA family in bone-related diseases provides novel insights into the pathogenesis of bone disorders. Meanwhile, it also supplies solid theoretical basis and potential therapeutic targets for the development of novel HOXA-targeted therapeutic strategies against bone diseases.

Humans

Enhancement of secondary organic aerosol formation from isoprene photooxidation by ammonia.

Ammonia (NH3) can participate in atmospheric secondary organic aerosol (SOA) formation by reacting with organic acids and carbonyl compounds in particle phase, but its influence on the gas phase chemistry remains unclear. This study performed a series of smog chamber experiments to investigate the influence of NH3 on the formation of SOA from isoprene photooxidation by OH radicals. Both gas and particle phase products were measured with a series of state-of- art instruments including a nitrate ion chemical ionization mass spectrometer (nitrate-CIMS) and high-resolution time-of-flight aerosol mass spectrometer (HR-TOF-AMS). Our results showed that in the presence of NH3 SOA in the chamber significantly increased, along with an enhanced oxidation of isoprene. CIMS analysis further showed that NH3 in the chamber homogeneously reacts not only with gas-phase organic acids but also with gaseous low volatility oxygenated organic molecules (OOMs) to generate extremely low volatility and ultralow volatility NH3-OOMs clusters. Quantum chemical calculation showed that NH3 can spontaneously interact with OOMs to form NH3-OOMs clusters by forming hydrogen bonds with RCOOH, R-OOH, and R-OH. These clusters can promote new particles formation and particle growth through nucleation and condensation, directly enhancing the isoprene SOA production with a contribution of 78% to the enhanced SOA. Moreover, the formation of NH3-OOMs clusters also results in more isoprene consumed by OH radicals, indirectly increasing the SOA production with a contribution of 22 % to the enhanced SOA. Our work for the first time clarified a synergetic effect of NH3 on isoprene SOA formation, which should be accounted for by models.

Aerosols

Community-tailored One Health educational intervention to enhance knowledge and practices for zoonotic disease prevention in rural Thailand: A protocol for a prospective cluster randomised controlled Trial in Chanthaburi, Thailand (Saan Suk trial).

BACKGROUND: Zoonotic infectious disease risk arises at human-animal-environment interfaces where pathogen spillover can occur. Rural communities living in biodiverse settings may experience frequent contact with wildlife and shared environments through livelihoods, food practices, and economic activities. Reducing spillover risk and strengthening pandemic prevention requires both structural and individual-level change. Community-based interventions that promote awareness, risk perception, self-efficacy, pro-environmental behaviour, and safe coexistence with wildlife may support prevention by shifting behavioural determinants of zoonotic disease risk. The Saan Suk intervention was co-developed with rural communities in Thailand using a Human-Centred Design approach and is grounded in the Health Belief Model and One Health principles. The intervention is intended to be feasible, acceptable, and deliverable through Thailand's established Village Health Volunteer (VHV) system. METHODS: This protocol describes a parallel-arm, cluster-randomised controlled superiority trial that will be conducted during July - October 2026, in Chanthaburi Province, Thailand. 24 villages will be equally randomised to the Saan Suk intervention or the current practice (control). In intervention villages, trained VHVs will deliver, once a week over four weeks, a multimodal One Health educational intervention designed to improve knowledge of zoonotic spillover, promote protective behaviours, reduce risky wildlife-related contacts, and support respectful coexistence with wildlife. Trained outcome assessment teams will conduct structured interviews with 42 adult participants per village, yielding a total sample size of 1,008 participants. The sample size was calculated for the primary outcome, accounting for clustering, with 90% power to detect a medium effect size (6 points on the 0-100 knowledge scale) at a significance level of 0.05, accounting for a design effect with an ICC of 0.028. The primary outcome is knowledge of zoonotic spillover, transmission pathways, risk factors, protective and risky behaviours, and safe coexistence with wildlife. Secondary outcomes include attitudes, self-efficacy, preventive and risky behaviours, and reported contacts with major local reservoir hosts. A structured questionnaire was developed, expert-reviewed, and piloted for the outcome assessment. Outcomes will be analysed using mixed-effects regression models with random effects for village and adjustment for relevant pre-specified confounders. Primary analyses will follow the intention-to-treat principle. DISCUSSION: This trial will evaluate whether a co-designed, VHV-delivered One Health educational programme can improve knowledge of zoonotic disease prevention and behavioural determinants in rural communities living in close contact with wildlife and shared ecosystems. If effective and feasible, Saan Suk could inform integration into routine VHV training and community-based zoonotic disease and pandemic prevention strategies. TRIAL REGISTRATION: The Saan Suk trial is registered with the German Clinical Trials Register (DRKS). Registration ID: DRKS00038582; date of registration: 11 May 2026.

Zoonoses

Ethiopia missed opportunities for vaccination study: Cluster-randomized evaluation of 5-dose measles vaccine vials and a flexible open-vial policy, 2021-2022.

INTRODUCTION: In 2024, an estimated 95,000 people died from measles globally, largely from suboptimal coverage with the measles-containing vaccine (MCV). Health workers may defer vaccinating eligible children to avoid wasting doses from 10-dose MCV vials, which must be discarded six hours after opening. These missed opportunities for vaccination (MOV) reduce coverage and timeliness. Ethiopia, which provides MCV at 9 and 15&#xa0;months, considered switching to 5-dose vials. METHODS: We conducted a 15-month, randomized controlled trial with a nested cross-sectional design in Ethiopia. Sixty woredas were randomized to: (1) policy-only, instructing health workers to open 10-dose vials for any number of eligible children, with additional stock for increased wastage; (2) 5-dose switch, combining this policy with replacement of 10-dose by 5-dose vials; or (3) control (routine 10-dose practice). Household and health-facility surveys at baseline and endline assessed effects on first-dose (MCV1) and second-dose (MCV2) coverage, MCV1 timeliness, and wastage. Generalized estimating equation models estimated net intervention effects versus control. We also estimated the government cost of a nationwide 5-dose switch. RESULTS: MCV1 coverage was similar between policy-only and control (adjusted risk difference [ARD]&#xa0;=&#xa0;-1%, 95%CI: -17%, 15%), with no significant differences in MCV1 timeliness or MCV2 coverage. In the 5-dose switch group, MCV1 coverage changed little (ARD&#xa0;=&#xa0;1%, 95%CI: -17%, 18%), but timely MCV1 at 9&#xa0;months (ARD&#xa0;=&#xa0;18%, 95%CI: 7%, 28%) and MCV2 coverage (ARD&#xa0;=&#xa0;17%, 95%CI: 1%, 34%) rose significantly, and wastage fell (ARD&#xa0;=&#xa0;-7%, 95%CI: -14%, -1%). By endline, 29% of 5-dose health workers opened vials &#x2265;10 times monthly (none in control); caregivers in both intervention groups reported 11% fewer measles-related MOVs. A nationwide 5-dose switch was estimated to save 17% in procurement cost per fully vaccinated (two-dose) child. CONCLUSIONS: The combined 5-dose intervention improved MCV1 timeliness and MCV2 coverage and reduced wastage, addressing a key operational barrier and potentially supporting measles elimination in Ethiopia.

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

Molecular Landscape and Advanced Diagnostic Technologies for BRAF Mutations in Cancer: From Quantitative PCR and ddPCR to CRISPR-Based Platforms.

BRAF mutations are key oncogenic alterations across multiple malignancies, including melanoma, thyroid carcinoma, colorectal cancer, non-small cell lung cancer, glioma, and hairy cell leukemia. The most prevalent variant, BRAF-V600E, induces constitutive activation of the MAPK signaling pathway, promoting tumor progression and influencing therapeutic responsiveness. Accurate detection of BRAF alterations is therefore essential for molecular classification, prognostic assessment, treatment selection, and resistance surveillance. This review summarizes the molecular heterogeneity of BRAF mutations and critically evaluates current diagnostic methodologies. Conventional approaches such as allele-specific PCR and Sanger sequencing are compared with advanced quantitative platforms, including high-resolution melting analysis, droplet digital PCR, and next-generation sequencing, with emphasis on analytical sensitivity, mutation coverage, and clinical applicability. Emerging technologies such as CRISPR-based assays, rolling circle amplification systems, and nanoparticle-based biosensors and point-of-care diagnostic platforms are also discussed for their potential to enhance ultra-sensitive detection, particularly in liquid biopsy settings. These emerging tools are highlighted for their potential to enable ultra-sensitive, rapid, and decentralized mutation detection, particularly in liquid biopsy settings. Key challenges, including intratumoral heterogeneity, low allele-frequency variants, FFPE-associated artifacts, and clonal evolution under therapeutic pressure, are examined within a translational framework. In addition, we examine critical barriers to clinical implementation, including standardization, cost, and global accessibility of molecular diagnostics, and outline potential solutions through scalable technologies and decentralized testing strategies. We propose that optimal BRAF testing requires a mutation subclass-informed and clinically integrated strategy combining comprehensive baseline profiling with longitudinal molecular monitoring. Future diagnostic paradigms will likely integrate multi-omics data and artificial intelligence (AI)-assisted interpretation to refine precision oncology implementation. Looking forward, we propose that optimal BRAF testing will require integration of multi-omics profiling with AI-assisted interpretation, enabling automated variant classification, real-time clinical decision support, and improved prediction of therapeutic response and resistance.

Humans

Single-cell transcriptomics reveals heterogeneous stress responses and Mg2+-mediated survival mechanisms in Lactobacillus delbrueckii subsp. bulgaricus during freeze-drying and storage.

Maintaining the viability of lactic acid bacteria during dehydration and subsequent storage remains a significant challenge. Here, we employed single-cell RNA sequencing to reveal the heterogeneous stress responses of Lactobacillus delbrueckii subsp. bulgaricus, identifying seven distinct transcriptional clusters across the liquid culture, freeze-drying, and storage phases. The dominant clusters in the freeze-drying and storage were not completely consistent, showing significant functional differentiation. Genomic stability may be important for survival during freeze-drying and storage, while intracellular energy homeostasis appears important for viability during storage. The magnesium transporter mgtB was highly expressed in clusters tolerant to freeze-drying and storage, suggesting a critical role for Mg2+ homeostasis. Further experimental validation confirmed that Mg2+ treatment significantly bolstered stress resistance, increasing immediate post-freeze-drying survival by over 2-fold (up to 92.90%) and post-storage survival by over 5-fold (up to 5.98%). Proteomic data indicated that Mg2+ supplementation correlated with the maintenance of several biological functions potentially relevant to bacterial survival during freeze-drying and storage, including DNA repair, translation, and central carbon metabolism. These findings provide a map of microbial stress resistance through population heterogeneity and offer a potential strategy that may be adapted for enhancing the stability of other industrial lactic acid bacteria products.

Freeze Drying

Comparative analyses of olfactory receptor repertoires in Schizothorax fish based on the chromosome-level genomes: Implications for regulatory roles of dietary differentiation and ploidy variation.

The olfactory receptor (OR) genes constitute the molecular basis of fish olfaction, mediating survival behaviors and environmental adaptation while coevolving with habitat-driven evolution. Schizothorax, a cyprinid genus endemic to the Qinghai-Tibetan Plateau, exhibits remarkable dietary divergence and ploidy variation in response to plateau environmental changes, which presumably facilitates the adaptive evolution of OR genes. However, the evolutionary patterns of OR genes associated with trophic divergence and ploidy variation in this genus remain unclear. In this study, three species were selected: the herbivorous diploid S. macropogon, the carnivorous diploid S. lantsangensis, and the herbivorous tetraploid S. curvilabiatus. S. macropogon possessed 142 OR genes (92.25% functional), primarily located on chromosomes 14 and 24, with the fewest sequence clusters. Such compact gene repertoire and highly overlapping chromosomal clusters indicated specialization for a herbivorous olfactory niche. S. lantsangensis contained 127 OR genes (93.70% functional), concentrated on chromosomes 4 and 5, with fewer sequence clusters and a scattered distribution, reflecting evolution of OR genes under carnivorous feeding habits. The herbivorous tetraploid S. curvilabiatus exhibited striking features: 316 OR genes (94.30% functional), the most subfamilies, unique &#x3b5; and &#x3ba; OR subfamilies, and species-specific motifs. These characteristics revealed that ploidy, rather than herbivory, dominated OR gene evolution. In conclusion, dietary differentiation and ploidy variation together drove olfactory adaptive evolution in Schizothorax, providing new insights into vertebrate OR gene ecological adaptation.

Animals

Functional neuroimaging subtypes of obsessive-compulsive disorder: A systematic review and meta-analysis.

Obsessive-compulsive disorder (OCD) exhibits substantial clinical heterogeneity that may reflect underlying neurobiological diversity. Neuroimaging-based subtyping may advance precision psychiatry by identifying biologically distinct subgroups with differential treatment responses. This study systematically synthesized evidence from functional neuroimaging subtyping studies in OCD to identify reproducible neurobiological subtypes, characterize their clinical profiles, and establish a consensus-based classification framework. We reviewed 40 original studies employing machine learning, clustering, normative modeling, or classification approaches, encompassing approximately 8,150 patients. Consensus clustering identified three reproducible neurobiological subtypes. The Limbic-Hyperactive subtype, comprising approximately 40% of patients, exhibited amygdala and insula hyperconnectivity, elevated anxiety levels, predominant contamination and washing symptoms, and favorable response to cognitive-behavioral therapy. The Fronto-Striatal-Hypoconnected subtype, comprising approximately 35% of patients, demonstrated reduced orbitofrontal-striatal connectivity, cognitive inflexibility, predominant checking and ordering symptoms, and a favorable response to selective serotonin reuptake inhibitors. The Global-Disrupted subtype, comprising approximately 25% of patients, exhibited widespread connectivity disruption, greater symptom severity, and poor treatment response. Support vector machine classification achieved 81.5% accuracy for subtype assignment, though classification of OCD versus healthy controls showed limited generalizability in multisite settings (AUC 0.567-0.673). These findings support a neuroimaging-based framework for personalized treatment selection but require prospective validation.

Humans

Single-cell RNA sequencing provides further insights into the immunostimulatory action of freeze-dried Lactiplantibacillus plantarum on Penaeus vannamei shrimp.

Immunostimulation through dietary interventions opened new avenues in developing disease control and prevention tools for shrimp aquaculture. We have previously shown that feeding with freeze-dried Lactiplantibacillus plantarum (LAB) increased disease resistance of Penaeus vannamei against both Vibrio parahaemolyticus and white spot syndrome virus (WSSV) based on bulk RNA sequencing of shrimp gills. This tissue participates in ion transport and serves as a first line of defense against environmental stressors and pathogenic infections. However, characterization of their cell composition and functions remains limited. Here, we implemented a single-cell RNA sequencing approach to further gather insights into how feeding with freeze-dried LAB modulates host immunity which may not be evident with bulk RNA sequencing approach. A total of five clusters with unique transcriptional signatures were identified, corresponding to pillar cells, septal cells, and sessile hemocytes. Pseudo-bulk analyses at global- and cluster-levels showed differential expression of genes related to host immunity and metabolism. We further revealed how overall transcriptomic changes are not exclusively caused by gene expression changes but may also be driven by cell population dynamics. This study highlighted how single-cell RNA sequencing approach may shed light on the mechanisms of action of immunostimulants which may be masked in bulk transcriptome analyses.

Animals

Genome-wide identification and expression profiling of CSP and OBP genes in Stictocephala bisonia reveals candidate genes potentially associated with insecticide response.

Stictocephala bisonia is an important invasive agricultural pest. Due to the frequent application of insecticides in its habitat, this species is under intense selection pressure. Chemosensory proteins (CSPs) and odorant-binding proteins (OBPs) are known to play key roles in insecticide resistance, but their specific functions in S. bisonia remain unclear. In this study, we identified a total of 22 SbisCSPs and 16 SbisOBPs based on the S. bisonia genome. To screen for candidate genes potentially linked to insecticide resistance, we adopted a multi-criteria screening strategy that integrated phylogenetic analysis, molecular docking with three insecticides, and tissue-specific expression profiling. Phylogenetic analysis identified several SbisCSPs and SbisOBPs clustering with genes known to be involved in insecticide resistance, serving as an initial evolutionary filter. Molecular docking results indicated that &#x3bb;-Cyhalothrin exhibited the strong predicted binding affinity with most of SbisCSPs and SbisOBPs. Subsequent qPCR validation of seven prioritized candidates revealed distinct expression patterns: SbisCSP22 was highly expressed in adults and demonstrated strong binding affinity to all three insecticides tested, suggesting a potential role in mediating multi-insecticide response. Conversely, SbisCSP17 was significantly upregulated in larvae, clustered with genes known to mediate imidacloprid resistance, and exhibited strong binding affinity to imidacloprid. Given its larval-specific expression and the soil-dwelling behavior of larvae, we hypothesize that SbisCSP17 is a key candidate gene for larvae coping with soil-treated insecticides.

Animals

Depression and amyloid-&#x3b2; across CSF, PET, and plasma biomarkers: a systematic review and meta-analysis.

Alzheimer's disease is increasingly defined by biomarker evidence of amyloid-&#x3b2; and tau pathology, sharpening questions about whether late-life depression contributes to, or instead reflects, this pathology. We conducted a systematic review and meta-analysis of studies published between 2000 and 2025 that compared amyloid-&#x3b2; biomarkers in adults with and without depression, with depression defined by validated clinical diagnoses or symptom rating scales. Twenty-four studies were included, spanning three biomarker sources: cerebrospinal fluid, positron emission tomography imaging, and plasma. Across all sources, the pooled difference in amyloid-&#x3b2; burden between depressed and non-depressed individuals was small and clustered near zero, indicating only a weak, statistically non-significant tendency toward higher amyloid in depression. When the three sources were examined separately, each yielded a similar near-null result, although between-study heterogeneity was considerable for cerebrospinal fluid and plasma and moderate for imaging. Importantly, a prespecified subgroup analysis showed that imaging results diverged by quantification method: studies using the simpler standardized uptake value ratio clustered around zero, whereas the smaller group of studies using kinetic distribution volume ratio modelling showed a significant positive association, suggesting that methodological choices critically influence the observed relationship. Taken together, these findings indicate that depression is not consistently accompanied by greater amyloid-&#x3b2; burden across widely used biomarker platforms. The distribution volume ratio signal nonetheless raises the possibility of subtle associations that cruder methods may obscure, and suggests that depression may shape Alzheimer's disease trajectories more by modifying the clinical impact of amyloid than by altering its amount.

Humans