Virus Evolution

Japanese encephalitis virus (JEV) is a leading cause of viral encephalitis in humans in Asia. Prior to 2021, only sporadic outbreaks had been detected in Australia's tropical northern extremes. However, during 2021-23, JEV infection was identified in 45 people and in more than 85 piggeries, together with many detections in feral pigs, mosquitoes, and other livestock across Australia, including in the temperate far south. Here we estimated the spatial extent of virus transmission during the outbreak (the realised niche) and the area environmentally suitable for transmission (the potential niche). We collated a national dataset of JEV surveillance and detection in humans, mosquitoes, domestic pigs, feral pigs, and sentinel chickens from 2021-23. We combined these data with (1) previously published predictions of habitat suitability for vertebrate host and vector species, and (2) estimated densities of humans, domestic pigs, and feral pigs, in a multipart assessment of spatial risk. Our model projected broad potential environmental suitability over much of northern and eastern Australia during the outbreak period, but floodplain wetland areas in the north and the inland east of Australia had both the highest estimated environmental suitability for transmission, and the strongest evidence for virus circulation.

Population genomic studies have revealed strong regional structure in Plasmodium knowlesi across Southeast Asia. To facilitate routine genomic surveillance, we developed a targeted amplicon sequencing panel that preserves the large- and small-scale population genetic structure. We screened published genomes for short, highly polymorphic targets and a 30-SNP barcode. We validated multiplexed amplification and sequencing using a portable Oxford Nanopore Technologies MinION sequencer on 28 clinical infections from four southern Thai provinces collected between 2018-2023. Twenty-seven loci yielded sequence data and 23 infections with data from at least eight loci were retained for analysis. Across the 23 Thai infections and 104 published Malaysian genomes, 586 diverse SNPs were observed. The amplicon panel and 30-SNP barcode retained the major population structure observed in genome-wide data, including a clear distinction between Bornean and Peninsular Malaysian infections and two clusters in Borneo. Across newly sequenced Thai infections, 25 loci were diverse and showed substantial diversity (mean expected heterozygosity = 0.69). All infections were effectively monoclonal. Pairwise identity-by-descent revealed substantial recent relatedness, with 46 of 253 pairs at or above 0.5. Two Thai genetic groups (n = 12 and n = 11) were observed, mirroring previously observed structure in peninsular Malaysia. Groups were not significantly associated with province or collection year. In conclusion, targeted sequencing can reveal geographically informative population structure across scales and thus support surveillance.

Background: Noninvasive biomarkers capable of detecting kidney allograft injury and distinguishing rejection from infection remain an important clinical need. Torque teno virus (TTV) reflects host immune status, but its utility in urine has not been well established. We evaluated urinary TTV, total cell-free DNA (cfDNA), cfDNA fragmentation, and BK polyomavirus (BKV) as potential biomarkers of kidney transplant rejection and BK polyomavirus-associated nephropathy (BKPyVAN). Methods: Urine samples from 334 kidney transplant recipients were analyzed in association with kidney biopsies performed within 30 days of sample collection. TTV and cfDNA were measured by multiplex quantitative PCR (qPCR), with a subset analyzed by digital PCR (dPCR), and BKV was quantified by qPCR. Associations with biopsy results were evaluated using group comparisons and nominal logistic regression. Changes associated with outcomes of consecutive biopsies were assessed by matched pairs analysis. Results: Among 544 unique urine samples, the urinary TTV/cfDNA ratio was significantly lower in rejection than in non-rejection samples (P<0.0001). Logistic regression adjusted for demographic factors confirmed an inverse association between the qPCR-derived TTV/cfDNA metric and rejection (adjusted odds ratio per delta Cq, 0.91; 95% CI, 0.88-0.94; P<0.00001). Significant differences were observed for all types of rejection. Association was confirmed by dPCR: higher TTV was associated with lower rejection odds, whereas higher cfDNA was associated with greater rejection odds. A nominal logistic model incorporating dPCR measurements achieved an AUROC of 0.76 (95% CI, 0.69-0.82). Matched pairs analysis showed a significant decrease in TTV/cfDNA when rejection developed and an increase following recovery from rejection, with no significant changes when biopsy status remained unchanged. BKV levels were strongly associated with BKPyVAN. No correlation between TTV and BKV was observed. Nominal logistic regression integrating TTV, cfDNA, and BKV measurements yielded AUROCs of 0.75, 0.97, and 0.73 for rejection, BKPyVAN, and non-rejection, respectively. Conclusions: A reduced urinary TTV/cfDNA ratio is associated with biopsy-defined kidney transplant rejection, and changes in the ratio correlate with transitions in biopsy results. Combined TTV, cfDNA, and BKV measurements showed differential associations with rejection and BKPyVAN, supporting further evaluation as complementary urinary biomarkers.

Background: Filoviruses, specifically Ebola virus (EBOV), Bundibugyo virus (BDBV), and Marburg virus (MARV), have been the etiologic agents of multiple outbreaks of severe disease in the Democratic Republic of the Congo (DRC). Two EBOV-specific vaccine regimens have been approved for use in outbreak scenarios, including the Ad26.ZEBOV/MVA-BN-Filo (Zabdeno/Mvabea) two-dose vaccine, where the MVA-BN-Filo booster dose contains EBOV, MARV, and Sudan (SUDV) virus glycoproteins (GPs) and the nucleoprotein from Tai Forest virus. While long-term immunogenicity of up to two years has been found in clinical trial settings, limited real-world data exists. Methods: Using a multiplexed immunoassay, samples (n=698 at baseline) collected in Mbandaka, Equateur province, DRC, between September 2023 and October 2024 were assessed for seroreactivity to six filovirus antigens (1 BDBV, 2 EBOV, 2 MARV, and 1 SUDV) at three distinct periods: baseline/pre-vaccination, 6-8 weeks post-first dose (Zabdeno), at booster administration (Mvabea), and one-year post-boost dose. Results: Substantial filovirus seroreactivity was found at baseline, with 48.7% (n=340/698) of individuals being seroreactive to at least one virus antigen. High baseline seroreactivity was found against BDBV GP (31.1%, n=217/698), with lower baseline seroreactivity to EBOV GP (15.5%, n=106/698), comparatively. Overall EBOV GP seroreactivity increased to 66.8% (n=376/563) at one year-post vaccine boost and 57.6% (n=250/434) of participants were seroreactive. Despite the booster dose inclusion of SUDV GP and MARV GP, there were no substantial increases in reactivity against either antigen at subsequent timepoints. Overall filovirus seroreactivity increased at all timepoints for the entire cohort, although filovirus reactivity decreased over the study period for those who were seroreactive at baseline. Findings: These data highlight detection of significantly elevated seoreactivity to EBOV GP following first-dose administration of Ad26.ZEBOV, which remained relatively stable one-year post-booster dose administration. Additionally, this study identifies high levels of baseline seroreactivity to BDBV GP despite no previous documented outbreaks in this region of DRC. These data suggest that vaccination with the Ad26.ZEBOV-MVA-BN-Filo vaccine induces stable seroreactivity against EBOV GP, but not to other filovirus antigens.

Brazil experienced its worst dengue epidemic in 2024, with an increase in severe outcomes among elderly individuals, highlighting the need for vaccination strategies informed by population immunity. We reconstructed state- and age-specific immune profiles by fitting an age-structured catalytic model to dengue surveillance data from 2000-2025. This inferred immune profile was incorporated into an SIR-type transmission model to project disease burden and to evaluate the public health impact of vaccine allocation strategies. We observed substantial spatial heterogeneity in accumulated immunity, which was closely associated with projected burden and optimal allocation. Strategies prioritizing states with lower historical incidence and larger susceptible populations yielded greater benefits. Under limited vaccine supply, targeting children resulted in the greatest reductions in DALYs, whereas targeting elderly populations achieved smaller DALY reductions but larger reductions in mortality. We further showed that accelerated vaccine supply markedly increased impact, while allocation choices remained critical when availability was constrained.

Introduction: Acute gastroenteritis (AGE) is a global public health problem and a major cause of mortality in low- and lower-middle-income countries. Human bocavirus (HBoV) is among the viral agents associated with this condition. Therefore, investigating the presence of HBoV in cases of AGE in Salvador, Bahia, as well as its prevalence and circulating strains, is important. Objective: To investigate the occurrence of HBoV infection during an AGE outbreak in Salvador. Methods: A total of 178 samples were analyzed for HBoV using nested polymerase chain reaction (nested PCR), and the amplification products were visualized by agarose gel electrophoresis. HBoV was detected in 5.7% of the samples analyzed. Conclusion: This study contributes to the understanding of HBoV epidemiology as a secondary agent associated with acute gastroenteritis during a norovirus outbreak in Salvador. Continued investigation and identification of the etiological agents involved in outbreaks are essential for evaluating the causes of AGE. These findings also highlight the need for broader diagnostic approaches and the importance of phylogenetic analysis for monitoring and controlling viral infections.

Introduction: Acute gastroenteritis (AGE) is a cause of childhood morbidity and may be associated with human adenovirus (HAdV) infection. This study aimed to investigate the occurrence of HAdV in fecal samples from children with AGE in Salvador, Bahia, Brazil, as well as to molecularly characterize the identified strains and evaluate their cytopathic effects in cell culture. Methods: A total of 131 samples were analyzed using the nested polymerase chain reaction (Nested-PCR) technique. Molecular characterization was performed through Sanger sequencing and phylogenetic analysis, while Vero cell cultures were used to investigate the occurrence of cytopathic effects. Results: Of the samples analyzed, 13 (9.9%) tested positive for HAdV, while 118 (90.1%) were negative. The highest detection frequency was observed in children aged three years or younger, particularly those under one year of age. Phylogenetic analysis identified variants related to HAdV-F41 and HAdV-B3, revealing genetic differences and distinct clustering patterns compared with reference sequences. In cell culture, cytopathic effects were observed four days after inoculation, although further analyses are required to confirm productive viral replication. Conclusion: These findings demonstrate the presence of HAdV in the samples studied and contribute to understanding its genetic diversity and circulation in Salvador, highlighting the importance of epidemiological surveillance and further studies on its role in the etiology of childhood AGE.

Retention in antiretroviral therapy (ART) programmes remains a major public health challenge in HIV care, with loss to follow-up (LTFU) and death threatening treatment success. Because these outcomes are competing events, conventional survival methods may yield biased estimates of their incidence and associated risk factors. To estimate the cumulative incidence of LTFU and mortality and identify factors associated with these outcomes among adults receiving ART in Northern Ghana using a competing-risks framework. We conducted a retrospective cohort study of 329 adults living with HIV who initiated ART at Nalerigu Baptist Medical Centre, Ghana, between January 2013 and December 2019. Fine--Gray subdistribution hazard models were used to evaluate predictors of LTFU and death while accounting for competing risks. Cause-specific Cox proportional hazards models were fitted as a sensitivity analysis. The incidence rates were 2.2 per 100 person-months (95% CI: 1.9--2.6) for LTFU and 0.74 per 100 person-months (95% CI: 0.57--0.98) for death. At 36 months, the cumulative incidence was 50.2% for LTFU and 16.0% for death. Patients residing closer to the clinic had a lower cumulative incidence of LTFU than those living farther away (SHR = 0.68, 95% CI: 0.49--0.93), whereas higher baseline weight was associated with increased LTFU (SHR = 1.02 per kg, 95% CI: 1.00--1.04). Mortality was lower among patients receiving efavirenz-based regimens (SHR = 0.27, 95% CI: 0.15--0.50), females (SHR = 0.39, 95% CI: 0.18--0.85), and those with higher baseline weight (SHR = 0.93 per kg, 95% CI: 0.90--0.96). Findings were broadly consistent in cause-specific Cox models. LTFU was substantially more frequent than mortality among adults receiving ART in Northern Ghana. Travel distance to the treatment facility was an important barrier to retention, while ART regimen, sex, and baseline weight were associated with mortality. Expanding access to ART services through decentralized and community-based models may improve retention and support efforts to achieve HIV control targets.

Wolbachia-based interventions were less effective in achieving introgression in Rio de Janeiro than other settings. Reduced introgression occurred in locations where dengue was historically high, raising concerns of inequity. We examined factors affecting Wolbachia introgression in Rio through trajectory modeling, spatiotemporal Bayesian models, and Ae. aegypti abundance estimation. During the intervention (2017-2020), Wolbachia introgression was 26% (median; IQR,14-44%), with four distinct trajectories. Despite receiving more releases, densely populated areas with low socioeconomic status had lower introgression. In spatiotemporally explicit multivariable regressions, higher Ae. aegypti abundance, temperature, population density and lower socioeconomic status were associated with reduced introgression. Improved characterization of Ae. aegypti abundance and other structural/environmental predictors of introgression could be used to calibrate release doses to ensure equitable benefits from Wolbachia interventions.

Long-acting injectable human immunodeficiency virus (HIV) medicines expand treatment and prevention choices, but willingness to use them does not establish readiness to begin. We examined readiness to start these options, associated factors and conditions for acceptance among people living with HIV and pre-exposure prophylaxis (PrEP)-service clients in Uganda. This cross-sectional study analysed 225 participants at Mildmay Hospital and Kajjansi Health Centre IV in central Uganda. Readiness meant preferring initiation immediately or within three months. Modified Poisson regression estimated adjusted prevalence ratios (aPRs) and 95% confidence intervals (CIs), including age-by-service-group interaction. Separate models examined informational, perceptual and financial factors, adjusting for service group, age and sex. Logistic marginal standardisation checked demographic and expanded age comparisons. Overall, 108 participants (48.0%) were ready to start injectable HIV treatment or prevention, 111 (49.3%) wanted more information about these options, and six (2.7%) preferred initiation in six to twelve months. Readiness was 80.8% among people living with HIV aged 15-24 years and 34.4% among those aged 25 years or older (aPR 2.35, 95% CI 1.63-3.38). Corresponding proportions among PrEP-service clients were 39.2% and 41.4% (aPR 0.95, 95% CI 0.60-1.50; interaction p=0.002). In separate adjusted models, clinic-staff information (aPR 1.94, 95% CI 1.42-2.66), belief in greater effectiveness (2.00, 1.43-2.80) and a one-point increase in benefit score (3.27, 2.36-4.55) were associated with higher readiness. A one-point increase in concern score (0.70, 0.60-0.80) and financial difficulty sometimes or more often (0.68, 0.48-0.96) were associated with lower readiness. Fewer side effects was the leading condition for accepting injectable HIV treatment or prevention across service and readiness groups. Nearly half of participants were ready to start, while a similar proportion wanted further information about injectable options. Programmes should assess intended initiation timing alongside willingness and address safety, appointment flexibility, privacy and affordability through counselling and service planning.

The accrual of somatic copy number alterations (CNAs) in the premalignant condition Barrett's oesophagus (BO) predicts transformation to oesophageal adenocarcinoma (OAC). The dynamics of CNA evolution, namely when CNAs first appear and their rate of clonal expansion, are unknown. Here we show that CNAs typically begin to accrue in BO many decades prior to first clinical detection of BO or cancer occurrence, and in some cases during childhood. We constructed a hierarchical Bayesian methodology called CARBINE to calculate the calendar year occurrence time of CNAs from single-timepoint contemporary genetic data. We analysed data from 80 individuals who had received longitudinal endoscopic surveillance of their BO and had known cancer progression status. Loss of heterozygosity (LOH) on chromosome 9p tended to be the first CNA event and often occurred when patients were between 10-30 years old. Genome doubling (GD), known to be a strong risk factor for cancer development, also typically originated multiple decades prior to cancer develop and the GD clone expanded very slowly. Patients who progressed to OAC had an enrichment of CNAs late in life. CNA accrual dynamics were more predictive of cancer risk than CNA burden measurements alone. Analysis of 97 OAC whole genomes, of which 38 had sufficient purity for CNA timing analysis, confirmed the early-life origins of CNAs found in the cancer and indicated that the seeds of OAC exist within an increasingly cancerised BO field for many years. CNA timing shows that the malignant transformation potential of BO may be recorded within the somatic genome in the first decades of life, giving a strong rationale for the effectiveness of risk stratification in early life for Barrett's patients.

Brazil has one of the highest dengue burdens globally, with transmission influenced by climate events. Yet, the effects of compound climate extremes on dengue risk and prediction have not been quantified to date. Using a Bayesian spatiotemporal model of monthly dengue cases across 435 health regions from 2010 to 2025, we estimated relative risk associated with interacting long-lag and short-lag drought indicators and temperature anomalies across nine Koppen-Geiger climatic zones, adjusting for absolute temperature and El Nino-Southern Oscillation. Long-lag dry, mid-lag warm and short-lag wet conditions were associated with the highest relative risk in five climatic zones, with temperature anomalies modifying hydrometeorological dynamics more strongly in temperate than tropical areas. Climate covariates improved the prediction of case counts and outbreaks compared to baseline models at lead times of 1, 3, 6 and 12 months, and for the upcoming season 5 months ahead. Improvements were greatest in the Northeast, Southeast, and Midwest. Recent dengue incidence provided the greatest improvement at the 1-month lead, whereas combining incidence with climate information performed best at 3- and 6-month leads. We extended our framework to weekly state-level predictions of the season ahead to align with national public health planning. We proposed a multi-staged dengue forecasting approach to enhance early warning and response across Brazil.

Mitochondrial disease arises from variants in either the mitochondrial or nuclear genome that impair mitochondrial oxidative phosphorylation. Complex III (ubiquinol:cytochrome c oxidoreductase, CIII2) is a key enzyme of the mitochondrial respiratory chain, catalyzing electron transfer from reduced coenzyme Q10 (ubiquinol) to complex IV, via cytochrome c. CIII2 has one subunit encoded by mitochondrial DNA, the remainder being nuclear encoded. Individuals with CIII2 deficiency present with significant clinical heterogeneity, on account of pathogenic variants in genes encoding structural CIII2 subunits or assembly factors. UQCC1 (GenBank: NM_018244.5) encodes the mitochondrial complex III assembly factor Ubiquinol-cytochrome c reductase complex assembly factor 1, which is currently not associated with human disease. Here we describe nine subjects from eight unrelated families harboring biallelic UQCC1 variants with heterogeneous, early-onset mitochondrial disease presentations and lactic acidosis. Subject-derived fibroblasts demonstrated a decrease in the steady-state levels of UQCC1 and its interacting partner UQCC2, with structural modelling supportive of variant pathogenicity. BN-PAGE and quantitative proteomics demonstrated decreased CIII2 abundance whilst mass spectrometry (MS)-complexome profiling corroborated these findings, identifying impaired respiratory supercomplex formation and a delay in the incorporation of newly synthesized CIII2 subunits into native assemblies. Lentiviral transduction experiments in subject-derived fibroblasts restored the levels of both UQCC1 protein and CIII2 activity. Collectively, these findings provide convergent genetic, biochemical, proteomic and functional evidence that bi-allelic UQCC1 variants disrupt CIII2 assembly, establishing UQCC1 deficiency as a cause of mitochondrial CIII2 deficiency.

Objective To examine the association between patient-reported shared decision-making (SDM) and documented advance care planning (ACP) among adults with Alzheimer disease and related dementias (ADRD), and to assess whether this association differed between adults with and without ADRD. Design Retrospective observational study using linked survey and electronic health record (EHR) data. Setting and Participants Participants aged [&ge;]50 years in the All of Us Research Program who completed a patient-reported SDM measure and had linked EHR records. The primary analytic cohort included 3,635 adults with ADRD; 123,681 adults without ADRD comprised a secondary comparison cohort. Methods SDM was measured by how often clinicians asked participants about their opinions or preferences regarding medical care or treatment and was categorized as high (always/most of the time) or low (some of the time/ None of the time). Documented ACP was identified within 3 years before or after the SDM assessment. Modified Poisson regression with robust variance estimated prevalence ratios (PRs) and adjusted PRs (aPRs). Results Among adults with ADRD, 60.6% reported high SDM. Documented ACP prevalence was 5.1% among participants with high SDM and 3.6% among those with low SDM (P=0.027). High SDM was associated with greater ACP prevalence in the unadjusted model (PR, 1.44; 95% CI, 1.04-1.99); the association remained positive but was attenuated after full adjustment (aPR, 1.28; 95% CI, 0.93-1.77). Among adults without ADRD, high SDM was associated with documented ACP (aPR, 1.17; 95% CI, 1.03-1.33). Conclusions and Implications Among adults with ADRD, high SDM was associated with greater ACP prevalence in unadjusted analyses, although the fully adjusted association was not statistically significant. In contrast, higher SDM remained significantly associated with ACP among those without ADRD. The low prevalence of ACP highlights an important opportunity to expand discussions of current and future care preferences as a routine component of patient-centered care.

Neuropsychiatric symptoms (NPS) are highly prevalent in Mild Cognitive Impairment (MCI), conceptualized as an intermediate clinical state between normal aging and dementia. Although neuroimaging studies have reported associations between NPS and brain alterations in MCI, longitudinal evidence on NPS and their relationship with multimodal MRI measures remains limited. We aimed to investigate (1) longitudinal changes in NPS, volumetric assessment and measurement of regional cerebral blood flow (CBF) within the medial temporal lobe (MTL); (2) associations between longitudinal changes in MRI measures and NPS; (3) whether baseline MRI measures predicted longitudinal changes in NPS. Thirty individuals with MCI aged over 65 years were recruited, of whom eighteen followed up for a mean period of 29 {+/-} 8.9 months. There were significant reductions in total grey matter and white matter volumes, together with volume loss in the bilateral hippocampus, bilateral parahippocampal gyrus and right entorhinal cortex (pFDR[&le;]0.05) over the follow up period. Volume loss in the right parahippocampal gyrus was associated with increase in aggression (pFDR<0.01), and CBF increase in the right hippocampus and parahippocampal gyrus was associated with worsening in agitation (pFDR<0.01). Furthermore, lower baseline volume of the right entorhinal cortex was associated with increase in aggression, and lower baseline CBF in the right hippocampus and parahippocampal gyrus was associated with worsening of agitation (pFDR<0.05). Our findings highlight the importance of identifying NPS in individuals with MCI and the role of structural and perfusion MRI measures as promising biomarkers for monitoring disease progression and improving risk prediction of conversion to dementia.

People experiencing homelessness (PEH) have substantially lower quality-adjusted life expectancy and substantially higher healthcare spending compared to housed populations. California has the largest population of PEH in the United States, and an estimated ten percent of PEH have co-occurring opioid use disorder (OUD). We developed a lifetime, individual-level microsimulation of the 18,928 PEH with OUD in California to evaluate the health and economic effects of expanding access to housing through a Housing First approach, which does not require OUD treatment or abstinence. Increases in permanent housing and shelter access were based on Los Angeles County's Measure A goals, and we accounted for both housing and healthcare costs. Expanding housing access increased mean age at death by 0.34 years, reduced fatal overdoses for the cohort by 39, and produced an incremental cost-effectiveness ratio of $31,083 per quality-adjusted life-year gained, well below conventional willingness-to-pay thresholds. We provide evidence that housing is a high-value health intervention for PEH with OUD in California. Policymakers should support Housing First programs as part of integrated responses to the intertwined crises of homelessness and opioid use.

Background: The relationship between recent viral infection and subsequent susceptibility to, or protection against, other viruses remains unclear, particularly across ages and symptom status. Methods: A prospective cohort study enrolled participants aged 6 months to 49 years in Washington and Oregon, USA, 2022-2024. Weekly nasal swabs were tested for SARS-CoV-2, respiratory syncytial virus (RSV), and influenza A/B; additional swabs were collected during symptomatic illness and were also tested for parainfluenza (PIV) 1-4, endemic human coronaviruses (hCOV), adenovirus, human metapneumovirus, human rhinovirus (hRV), and enteroviruses. Proportional hazards regression models were used to characterize associations between incident symptomatic infection (illness) and history of recent illness due to other viruses (within 1-7, 8-14, 15-30, 31-60, 61-90, 91-180 days), adjusted for geographic region, age, sex, daycare/school/work attendance, household size, and number of children in the household. For SARS-CoV-2, influenza, and RSV, these analyses were repeated to study incidence of infection (including asymptomatic cases). Findings: Of 3,560 participants, 2,969 (83%) had [&ge;]1 illness over 4,299 person-years. Adjusted hazard ratios (aHRs) in the short term ([&le;]2 weeks) after an illness were predominantly below 1, indicating that recent viral illness was often associated with lower risk of subsequent illness with other viruses. Across age groups, recent hRV illness was associated with lower risk of hCOV, PIV, and RSV illness (aHRs 0.07-0.27). SARS-CoV-2 illness was associated with a higher short-term risk of adenovirus and PIV illness (aHRs 3.20-3.33). In children aged 6 months-4 years, recent influenza illness was associated with higher short-term risk of SARS-CoV-2 illness. Including asymptomatic infections generally attenuated these associations. Interpretation: Some viral illnesses may alter short-term susceptibility to subsequent viral illnesses. Interactions may differ by age and symptom status. Funding: US Centers for Disease Control and Prevention; Washington State Department of Health.

Introduction: This study aimed to characterize the neurochemical profile of Alzheimer s disease (AD) continuum stages using proton magnetic resonance spectroscopy (1H-MRS) data for brain metabolites and macromolecules. We used Orthogonal Projection to Latent Structures Discriminant Analysis (OPLS-DA) to classify diagnostic groups and assess feature importance. Methods: Our dataset consisted of 7 T short-echo time 1H-MRS spectra of the posterior cingulate cortex/precuneus of individuals diagnosed with AD (n = 27) or Mild Cognitive Impairment (MCI, n = 27) and Healthy Controls (HC, n = 31). Classification used 100-round Monte Carlo cross-validation based on metabolite concentrations and macromolecular peak intensities. Variable importance in projection (VIP) was assessed across classification models to identify the most relevant neurochemical features. The resulting metabolite rankings were compared with previous literature to evaluate their biological plausibility. Results: Classification was strongest for AD versus HC (AUC=0.88) and lower for comparisons involving MCI. Metabolites contributing most strongly to classification included myo-inositol, ascorbate, and choline-containing compounds, consistent with previous studies on AD-related neurochemical alterations. In addition, specific macromolecular components were identified as relevant features, suggesting a potential contribution of MM signals to the characterization of AD-related neurochemistry. Discussion: These findings support the use of short-echo-time 1H-MRS as a complementary tool for characterizing neurochemical changes in the AD continuum, with potential applications in disease characterization and monitoring, warranting validation in larger cohorts.

BACKGROUND: The chromosome 9p21.3 locus is among the strongest genetic risk loci for cardiovascular disease (CVD), yet the regulatory mechanisms underlying this association remain poorly understood. Previous vascular disease studies focused predominantly on the role of this locus in smooth muscle cell (SMC) dysfunction, whereas its potential role in endothelial cells (ECs) remains less well characterized. Here, we sought to identify candidate regulatory variants at 9p21.3, their distal target genes, and effector transcripts that contribute to CVD-related phenotypes in ECs. METHODS: Promoter capture Hi-C was used to map chromatin interactions at 9p21.3 in primary human ascending aortic SMCs (n=6), supplementing the previously obtained ECs (n=16) dataset. To support the regulatory relevance of these interactions, expression quantitative trait locus analysis was performed in 136 vascular biopsies. Variant effects were prioritized in silico using AlphaGenome. The regulatory activity of the prioritized region was validated using CRISPR activation. RESULTS: Chromatin interaction mapping identified a complex regulatory architecture at the 9p21.3 locus involving 19 putative regulatory elements and 15 genes across ECs and SMCs. MIR31HG showed the most recurrent distal promoter connectivity with the 9p21.3 region. Consistent with the chromatin interaction map, expression of MIR31HG transcripts was associated with genetic variation at the 9p21.3 locus. In ECs, the lncRNA MIR31HG, but not miR-31, was responsive to hemodynamic flow. Depletion of MIR31HG in ECs was associated with endothelial activation. Epigenetic tracks and deep learning predictions highlighted an active enhancer element at 9p21.3, which was subsequently validated by CRISPR activation and increased MIR31HG expression, confirming its capacity to regulate MIR31HG over a large genomic distance of approximately 538 kb. CONCLUSIONS: Altogether, these data implicate lncRNA MIR31HG as a likely effector of the 9p21.3 CVD locus, linking the cis-regulatory element and heritable genetic variation in the respective region to endothelial activation through long-range enhancer regulation.

Estimating the correlation between subject-specific rates of change is a common objective in longitudinal studies. A natural approach is to fit ordinary least squares regressions per subject and correlate the resulting slope estimates directly. Under complete data, this slopes-first estimator is algebraically equivalent to a contrast-based plug-in estimator derived from the joint variance-covariance matrix of the two signals across time. Under independent dropout, however, the slopes-first approach implicitly applies heterogeneous weights across missing-data patterns, introducing bias whenever the cross-covariance between the two signals is not constant over time. We formalise this result and propose an unbiased alternative that estimates the joint covariance matrix before applying the contrast. We further extend our results to include covariates as well. Simulations confirm that the slopes-first estimator is biased under non-constant cross-covariance while the proposed alternative remains unbiased. Further, we show in a real-world example that the two estimators can lead to different scientific conclusions and recommend that researchers working with longitudinal data inspect the missing data pattern to make an informed decision about the estimator of choice.