EBioMedicine

Streptococcus pneumoniae is the leading cause of empyema and pneumonia in children, and monitoring of effectiveness of polyvalent pneumococcal vaccines has been essential for controlling invasive pneumococcal disease (IPD) in children and elderly adults. Conventional serotyping of pneumococci has relied on Quellung reaction following laboratory culture, however more recently whole genome sequencing (WGS) has been implemented in many reference laboratories to enhance traditional typing. Pleural fluid samples from cases with empyema are often culture negative, limiting the utility of WGS and requiring polymerase chain reaction (PCR) or 16S rRNA sequencing to detect S. pneumoniae. These molecular methods have limited sensitivity and capacity to characterise pneumococcus in clinical samples, especially in specimens with a low pathogen abundance. This study applied capture-based enrichment (tNGS) to identify and characterise S. pneumoniae directly from pleural fluid samples. A total of 51 pleural fluid samples were subjected to tNGS with a custom probe panel, for 39 known positive fluids collected from IPD cases between 2018-2025 in New South Wales, Australia. tNGS results were benchmarked against molecular-based serotyping. Our tNGS achieved 100% sensitivity and specificity in detecting S. pneumoniae. Serotyping results were concordant with PCR and 95% (37/39) of S. pneumoniae PCR positive pleural fluid cases could be serotyped using tNGS. Standard molecular methods however could only determine serotype in 56% (22/39) of samples. This tNGS enabled 39% improvement in ability to directly identify and serotype IPD-associated serotypes of S. pneumoniae in difficult-to-culture pleural fluids can significantly enhance laboratory surveillance of IPD as well as our understanding of vaccine effectiveness.

Genome-wide association studies of idiopathic pulmonary fibrosis (IPF) have identified 35 common genetic risk loci associated with IPF susceptibility. In this study, we evaluated the effects of the reported variants in clinically curated non-European individuals. Despite limited sample sizes, we observed partial replication, limited transferability of some variants and evidence of ancestry-specific effects. The MUC5B promoter variant rs35705950 emerged as the dominant and most consistent signal across ancestries. Our findings highlight the need for larger, well-characterised studies in understudied populations to support robust discovery and translation.

Background: Host genetics alone explains limited susceptibility to tuberculosis (TB), particularly in people with HIV (PWH). Protein quantitative trait loci (pQTLs), genetic variants that regulate plasma protein levels, may bridge genetic and immunological mechanisms underlying TB progression. Methods: We conducted cis-pQTL mapping in 60 PWH who progressed to active TB and 194 matched controls from the Swiss HIV Cohort Study. Plasma proteomes were quantified via high-resolution mass spectrometry (dia-PASEF), and genotype-protein associations were analyzed separately in TB and control groups. Results: TB progressors harbored 26 cis-pQTLs linked to 12 proteins uniquely enriched in immune pathways (antigen presentation, complement activation, phagocytosis, and T-cell regulation). Controls showed 107 cis-pQTLs linked to 14 targets. Gene Ontology enrichment revealed 46 immune biological processes in TB versus only 1 in controls, with HLA-C, C4B, and CHIT1 as key TB-specific proteins. Conclusions: Integrating proteomics with genomics suggests differential regulation of immune proteins associated with TB progression in PWH. hese genetically anchored protein candidates support follow-up studies and future biomarker evaluation for TB risk prediction.

Equine asthma (EA) is a highly prevalent, chronic, inflammatory disease of the lower airways ranging from mild-to-moderate to severe clinical presentations. Diagnosis currently relies on bronchoalveolar lavage fluid (BALF) cytology, an invasive method associated with interobserver variability, which highlights the need for more reproducible approaches. MicroRNAs (miRNAs) are small noncoding RNAs involved in post-transcriptional gene regulation. They are stable and readily detectable in body fluids and have shown promising results as biomarkers in human asthma. The aim of this study was to characterize miRNA abundance profiles in BALF and serum from horses with distinct EA endotypes to evaluate their biomarker potential and explore their involvement in disease pathogenesis. A total of 43 horses were included and classified as either EA (n=32) or controls (n=11), based on clinical examination and BALF cytology. The EA horses were further divided into three endotypes based on BALF inflammatory cell composition: neutrophilic asthma (n=10), mastocytic asthma (n=15), and mixed asthma (n=7). RNA was isolated from both serum and BALF samples and analyzed by quantitative real-time PCR (qPCR) targeting 103 miRNAs linked to asthma and pulmonary inflammation in humans. Differential miRNA abundance was analyzed across EA endotypes. The most significantly differentially abundant miRNAs were used for in silico target prediction and pathway enrichment analyses. Horses with mixed EA had significantly lower levels of eca-miR-125a-3p and eca-miR-125b-5p in BALF compared to controls. Additionally, eca-miR-146a-5p abundance was significantly increased in BALF from horses with neutrophilic EA compared to mastocytic EA. Target and pathway enrichment analyses for eca-miR-146a-5p identified immune-relevant pathways, such as MAPK and T-cell receptor signaling, supporting its involvement in inflammatory processes associated with asthma. This study identified three promising candidates, eca-miR-125a-3p, eca-miR-125b-5p, and eca-miR-146a-5p, as potential biomarkers associated with different EA endotypes. These miRNAs are interesting candidates for further investigation in an independent cohort.

Background and Aims: Acute myocarditis (AM) is a T cell-mediated myocardial disease with clinical manifestations ranging from mild chest pain to cardiogenic shock. Reliable biomarkers to stratify patients and guide therapy are currently lacking. In particular, the extent of the dysregulation of inflammatory pathways, and the impact on myocardial dysfunction, remain elusive. Methods: Serum analyses were performed in prospectively recruited AM patients (n = 103) from two independent cohorts. Multimodal data integration combining profiling of cytokine and chemokine dysregulation with clinical biomarkers was used to define clinical phenotypes with distinct inflammatory signatures. Machine-learning and regression models were applied to determine biomarkers that indicate clinical severity. Results: Immuno-proteomic profiling revealed conserved inflammatory patterns across AM cohorts, dominated by T cell-related cytokines and chemokines. In addition, AM patients showed dysregulation of fibroblast-derived cytokines, including hepatocyte growth factor (HGF), bone morphogenic protein 4 (BMP4) and the BMP4 inhibitors Gremlin-1 (GREM1) and Gremlin-2 (GREM2). Data integration and unsupervised clustering revealed two immuno-clinical phenotypes, linking T cell activation and fibroblast dysregulation to disease severity. Machine learning-based analysis identified CXCL10, GREM2 and LVEF as critical parameters for stratifying disease severity. Conclusions: These findings highlight a systemic T cell activation signature as diagnostic hallmark of AM. In addition, dysregulation of fibroblast-derived tissue cytokines serves as an indicator for distinct immuno-clinical phenotypes in myocardial inflammatory disease. Thus, the clinically relevant link between T cell-driven immune activation, myocardial inflammation and fibroblast-driven remodelling provides a versatile set of parameters to identify severe manifestations of AM.

BackgroundAfter the global deployment of pneumococcal conjugate vaccines (PCVs), serotype 12F has become the predominant serotype responsible for invasive pneumococcal disease (IPD) worldwide. As PCVs that include serotype 12F are gradually introduced, we aim to characterise the global population structure and genetic diversity of the 12F capsule locus using whole-genome sequencing. Capsule variants with vaccine evasion potential were further investigated by functional experiments. MethodsA global collection of pneumococcal serotype 12F genomes (n=806) from 37 countries across six continents were included in this study. To characterise the serotype 12F population, Global Pneumococcal Sequence Cluster (GPSC), in silico serotype, and antimicrobial resistance profile were inferred from whole-genome data for each isolate. The capsule biosynthesis (cps) locus was analysed for gene content variations that could alter polysaccharide capsule production or structure, thereby influencing recognition by vaccine-induced antibodies. These isolates were further investigated by assessing their capsule production using immunofluorescence assays and its susceptibility to vaccine-elicited antibody killing by opsonophagocytosis assays. FindingsThe global increase in serotype 12F was driven by both distinct pneumococcal lineages across different continents, and a globally-disseminated and multidrug-resistant lineage GPSC26. We identified six capsule variants in nine isolates that had disruptive mutations in cps genes including wze, wcil, wciJ and fnlA. Most (6/9) of the disruptive mutations were a result of strand-slippage mutations. A convergent strand-slippage mutation disrupting the glycosyltransferase gene wciJ was identified in four isolates from distinct lineages and countries. Despite the truncation, three of four isolates with available Quellung typing results still identified them as 12F, indicating the production of the capsule. We then created a genetically engineered lab strain with wciJ knockout and complemented with wciJ containing the strand-slipppage mutation. The knockout strain did not produce any capsule. In contrast, the lab strain with wciJ containing the strand-slippage mutation produced a mixed population of encapsulated and non-encapsulated pneumococci, even within the same chain of pneumococcal cells. This observation indicated encapsulated subpopulation possesses a functional WciJ and rapidly reversible strand-slippage mutation during replication. Opsonophagocytosis assays indicated that the clinical 12F strain with strand-slippage mutation in wciJ exhibited reduced susceptibility to vaccine-elicited serum killing, compared to a genetically closely related 12F clinical strain with an intact wciJ. However, substantial inter-individual antisera variation limits definitive interpretation. InterpretationOur work revealed the global rise of serotype 12F pneumococci has been driven by both regional-specific lineages, and a globally-disseminated and multidrug-resistant lineage GPSC26. We demonstrated that strand-slippage mutation is one of the major drivers of serotype 12F capsule variants and represents a novel mechanism enabling reversible on-off switching of capsule production. The ability to switch off capsule expression in a subpopulation may enable evasion of antibody-mediated killing but increase susceptibility to innate immune clearance. FundingBill & Melinda Gates Foundation, Wellcome Sanger Institute, and the US Centers for Disease Control and Prevention.

A multiplex diagnostic test is evaluated for self-reported long COVID associated persistent symptoms and a poor recovery from a SARS-CoV-2 infection. A mass-standardised concentration of total antibodies (AC), high-quality (HQ) antibodies and percentage of HQ antibodies (HQ%) is assessed against a spectrum of spike proteins to the SARS-CoV-2 variants: Wuhan, , {delta}, and the Omicron variants BA.1, BA.2, BA.2.12.1, BA.2.75, BA.5, CH.1.1, BQ.1.1 and XBB.1.5 in three cohorts. A cohort of control patients (n = 46) recovered (CC) and a cohort of self-declared long COVID patients (n = 113) (LCC). A nested Receiver Operating Characteristic (ROC) analysis, performed for the variant with lowest HQ concentration in the spectrum, produced an area under the curve and AUC = 0.61 (0.53-0.70) for the CC vs LCC cohorts. For the LCC cohort, the cut-off thresholds for AC = 0.8 mg/L, HQ = 1.5 mg/L and HQ% of 34% were determined, leading to a 71% sensitivity and 66% specificity derived by the Youden metric. The cohorts may be fully classified based on ROC and outlier analysis to give an incidence of persistent virus 62% (95% CI 52% - 71%), hyperimmune 12% (95% CI 7% - 20%) and unclassified, 26% (95% CI 18% - 35%). The overall diagnostic accuracy for both the hyper and hypo immune is 69%. All clinical interventions can now be tailored for the heterogenous long COVID patient cohort.

Lung fibroblasts are key regulators of tissue homeostasis and extracellular matrix (ECM) remodeling, and their aberrant activation drives the progressive parenchymal scarring characteristic of idiopathic pulmonary fibrosis (IPF), a fatal disease with limited therapeutic options. Despite their central pathogenic role, lung fibroblasts are difficult to isolate due to their embedded position within the ECM, and standard in vitro culture conditions may lead to the loss of their native functional and transcriptional characteristics, hampering the study of fibroblast behavior in disease. The transcriptional heterogeneity of lung fibroblast subtypes and the extent to which culture-induced alterations diverge from native tissue signatures remain poorly understood. Here, we integrated single-cell RNA sequencing (scRNA-seq) and spatial transcriptomics of lung tissue from IPF patients and age-matched healthy donors with transcriptomic profiling of cultured fibroblasts collected at passages 1 and 6 after isolation using three optimized protocols: whole lung cell suspension (WLCS), negative fraction enrichment, and outgrowth. Tissue-based analysis identified six transcriptionally distinct mesenchymal subtypes: alveolar, adventitial, inflammatory, peribronchial, CTHRC1+ and smooth muscle cell (SMC). The fibroblast subtype CTHRC1+ represented the most transcriptionally activated pro-fibrotic subtype, showing the greatest upregulation of ECM biosynthesis genes, a prominent role in intercellular communication, and preferential enrichment within fibroblastic foci in IPF lung tissue. Pseudotime trajectory analysis supported a directional transcriptional continuum from alveolar and inflammatory fibroblasts toward the CTHRC1+ state, driven by coordinated activation of pro-fibrotic transcription factors, including RUNX2, CREB3L1, and SCX. In vitro culture progressively reshaped fibroblast transcriptional identity relative to native tissue, with increased collagen and matrix metalloproteinase (MMP) expression during passaging, loss of distinct CTHRC1+ fibroblasts, and gain of alveolar fibroblasts displaying pro-fibrotic activation across all isolation protocols. These findings provide a high-resolution transcriptional map of lung fibroblast heterogeneity in IPF and highlight critical limitations of standard in vitro culture systems for recapitulating native fibroblast diversity, with important implications for the development and evaluation of fibroblast-targeted therapeutic strategies in IPF.

Background: Tuberculosis recurrence accounts for a substantial proportion of incident tuberculosis in many settings. Distinguishing between its mechanisms can inform public health interventions for prevention. Methods: We conducted a retrospective study of individuals with multiple culture-confirmed TB episodes and available sequential isolates from 2012 to 2023 in Dourados and Campo Grande, Mato Grosso do Sul state, Brazil. Patients were classified as having recurrent TB after treatment completion or retreatment following non-curative outcomes. Whole-genome sequencing was used to assess pairwise genetic distances between isolates, classifying relapse or persistent infection ([≤]12 single-nucleotide polymorphisms [SNPs]) versus reinfection or retreatment with reinfection (>12 SNPs). Results: Among 9,293 individuals with TB, 772 recurrent or retreatment episodes were identified. Paired isolates from 82 individuals were available for comparisons. Among individuals who completed treatment, reinfection accounted for 74.1% (40/54) of recurrent episodes, while 25.9% (14/54) were classified as relapse. Among individuals with non-curative outcomes, persistent infection (53.6%, 15/28) and retreatment with reinfection (46.4%, 13/28) occurred at similar frequencies. Persistent infection and relapse occurred earlier after the initial episode, whereas reinfection and retreatment with reinfection predominated after two years. Incarceration history was strongly associated with reinfection after treatment completion (92.5%, p=0.012) and after non-curative outcomes (76.9%, p=0.016). Conclusions: In this high-burden setting, reinfection drives TB recurrence among individuals who complete treatment, particularly at longer intervals after initial disease, reflecting sustained exposure risk. Relapse and persistent infection remain clinically important, especially following non-curative outcomes. These findings underscore the need for integrated strategies combining adherence support to prevent treatment-related recurrence with interventions to reduce transmission, particularly in high-risk settings.

Background: Renal impairment is associated with increased risk of Parkinson's disease (PD) in general populations; however, the renal-PD link within cardiovascular disease (CVD) patients remains unclear through the high comorbidity of renal dysfunction and elevated PD risk among this special population. Objectives: To assess renal function's association, longitudinal trajectories and predictive value for PD specifically within a cardiovascular disease cohort. Methods: Among 29,266 UK Biobank CVD patients, we assessed baseline renal function via creatinine-based (eGFRcr) and cystatin C-based (eGFRcys) estimated glomerular filtration. Multivariable Cox regression analyzed associations with incident PD and all-cause mortality, with wide sensitivity analyses addressing reverse causation/confounding. Nested case-control analysis characterized pre-PD eGFR trajectories over 14 years. We finally evaluated whether renal function improved the PREDICT-PD's predictive ability. Results: Over a median 13.1-year follow-up, 489 incident PD cases and 5,919 deaths occurred. Lower eGFR levels exhibited dose-dependent associations with increased PD risk (eGFRcr: HR=0.87 [0.80~0.95]; eGFRcys: HR=0.90 [0.82~0.99]) and all-cause mortality (eGFRcr: HR=0.77 [0.75~0.79]; eGFRcys: HR=0.64 [0.63~0.66]). Pre-PD eGFR trajectories diverged significantly from controls starting over 14 years before diagnosis. eGFR-defined chronic kidney disease (<60 ml/min/1.73m2) conferred 38~60% higher PD risk and 159~234% elevated mortality risk, and could significantly enhance PREDICT-PD's discrimination, with a 1.18~1.34% increase in prediction accuracy. Conclusions: Impaired renal function is an independent PD and all-cause mortality risk factor of CVD patients, preceded by a slow, progressive eGFR decline starting >14 years before diagnosis. Incorporating renal function substantially improves PD risk prediction in this population.

BackgroundMutations in the E3 ubiquitin ligase Parkin (encoded by PRKN) are the most frequently known cause of recessively inherited Parkinsons disease. In addition to the loss of dopaminergic neurons, microglial activation is another pathological feature observed in Parkinsons disease. While postmortem brain samples show the end stage of the disease, neurons and glia derived from patients induced pluripotent stem cells (iPSCs) provide a model for detecting early pre-degenerative disease trajectories. However, mixed cell populations often confound these cultures, leading to heterogeneous disease phenotypes. MethodsHere, we tease apart the cell type-specific phenotypes underlying Parkin-linked Parkinsons disease by performing single-nucleus RNA sequencing in iPSC-derived co-cultures of dopaminergic neurons and microglia from PRKN mutation carriers and healthy controls. We validated our transcriptomic key findings through inflammatory cytokine profiling and live-cell calcium imaging. ResultsSingle-nucleus RNA sequencing identified seven major cell types composed of neuronal, glial, and precursor cells, with dopaminergic neurons accounting for the largest cell population. Pathway analysis revealed cell type-specific dysregulated biological processes in Parkin-deficient cells, including gene expression differences in dopaminergic neurons that control mitophagy and dopamine homeostasis, whereas microglia showed changes in calcium homeostasis and inflammatory signaling. Functional analysis verified elevated secretion of monocyte chemotactic protein 1 in PRKN-mutant co-cultures compared with controls, linking Parkin deficiency to increased microglial chemotactic signaling. Furthermore, lower intracellular calcium levels and diminished calcium release following treatment confirmed impaired calcium homeostasis in PRKN-mutant microglia. ConclusionsProfiling at the single-cell level resolved distinct cell subpopulations, enabling us to identify cell type-specific pathway disturbances underlying Parkin deficiency. This unique dataset provides a basis for understanding the impairment of individual cell types and the impact of cellular crosstalk in Parkinsons disease pathology.

BACKGROUNDRapid, interoperable whole-genome tools for Mycobacterium tuberculosis complex (MTBC) surveillance remain limited for harmonised lineage assignment across recognised lineages and simultaneous resistance-associated variant detection. AIMTo develop and validate Pathotypr, an alignment-free tool for harmonised MTBC lineage assignment and resistance genotyping from assemblies and raw reads. METHODSWe reconstructed an MTBC phylogeny from 26,813 genomes using 609,003 polymorphic sites, derived an updated lineage marker backbone, and implemented a k-mer/Random Forest framework with marker-based lineage and WHO catalogue-based resistance calling. Performance was evaluated on 498 RefSeq assemblies, 88,071 UShER-TB typed sequencing samples, 162 clinical read sets for closest-reference matching, and 7,148 CRyPTIC isolates with phenotypic drug susceptibility data. RESULTSPathotypr supported all 14 currently recognised MTBC lineages (L1-L10, A1-A4). On 498 complete genomes, marker-based and alignment-free lineage calls were 100% concordant, and prediction accuracy remained 100% on 254 independent assemblies. In 88,071 non-ambiguous UShER-TB samples, root-lineage concordance with TB-Profiler was 100%, while Pathotypr additionally identified lineage 10, A1 and A2. Resistance predictions showed 85.0% genotype-phenotype concordance overall, with high performance for rifampicin (95.8% sensitivity, 95.0% specificity) and isoniazid (93.0%, 97.9%). Runtime was about 1 second per sample, enabling analysis of 88,071 samples in approximately 24 hours on four threads. In the MDR-enriched CRyPTIC collection, Pathotypr supported reconstruction of 135 probable introduction events into Germany, Italy and Ukraine; 33.7% of introduction-associated isolates carried MDR/pre-XDR genotypes. CONCLUSIONPathotypr enables rapid, harmonised MTBC lineage assignment and high-confidence resistance screening, supporting near real-time and cross-border tuberculosis surveillance.

Background & AimsAntiviral therapies targeting hepatitis B virus (HBV) suppress viral replication, but rarely achieve functional cure. Understanding HBV-host cell interaction is crucial for developing novel therapeutic approaches. Here, we report host cell proteins associated with HBV virions and filamentous subviral particles (fSVPs) and characterize one of them, apolipoprotein C1 (ApoC1), mechanistically. MethodsHighly purified HBV virions and fSVPs were obtained by sequential use of several biophysical methods. Particles were analyzed by mass spectrometry and associated proteins were evaluated phenotypically using an HBV infection model. The top hit, ApoC1 was characterized in detail. ResultsAssociated with virions and fSVPs, we identified in addition to known chaperones such as HSP90AB1 and HSC70, several apolipoprotein-related factors. RNAi-based phenotypic validation identified strongest effects for ApoC1, likely due to two complementary effects. First, ApoC1 depletion reduced intracellular cholesterol level impairing HBV infection and SVP production, which was compensated by exogenous cholesterol substitution. Second, ApoC1 that is mainly enriched in high-density lipoprotein (HDL), associates with HBV virions and fSVPs and increases HBV infectivity. The same was found for hepatitis D virus (HDV), a satellite virus utilizing HBV envelopes. Supplementation of exogenous HDL enhanced infection most likely via scavenger receptor class B type 1 (SR-B1), the natural HDL receptor. Consistently, inhibition of SR-B1 suppressed HBV and HDV infection. ConclusionsWe established a method for obtaining highly purified HBV virions and fSVPs and identified the HDL component ApoC1 to associate with both particle types. ApoC1 promotes HBV and HDV infection most likely via SR-B1 facilitating viral entry.

BackgroundPancreatic adenocarcinoma (PDAC) is an abysmal disease, with a poor clinical outcome, largely due to limited life-extending treatments for patients. Notoriously, PDAC displays a T cell-suppressive tumor microenvironment where underlying molecular mechanisms that lead to this phenotype remain poorly understood. To unravel specific mechanisms, we utilized bioinformatic analyses with functional studies and revealed the cytolinker protein plectin (PLEC) as a novel player in regulating the T cell-suppressive tumor microenvironment of PDAC. MethodsUtilizing the TCGA-PAAD dataset, tumor samples were separated by PLEC expression to evaluate patient survival, and pathway analyses associated with increased tumorigenesis. Evaluation of immune infiltration and subsequent immune deconvolution was performed using tidyestimate and CIBERSORTx R packages. Single-cell RNA-seq (scRNA-seq) analysis from 229 PDAC patients was analyzed to investigate signaling dynamics and immune cell infiltration in PLECHigh patients. Functional validation was provided using a monoclonal antibody (mAb) against cell surface plectin (CSP) in two murine PDAC models to examine changes in tumor growth and immune cell subset abundance. ResultsOur studies revealed that high plectin expression results in an overall worse survival associated with activation of pro-tumorigenic pathways and decreased anti-tumor immune signature in PDAC patients. Analysis via GSEA indicates PLECHigh patients display an aggressive phenotype and suppressed pro-inflammatory signaling pathways. Immune ESTIMATE scores were significantly decreased in PLECHigh patients, and scRNA-seq analysis revealed that PLECHigh tumors display a decrease in anti-tumor CD8+ T cells. In vivo analyses using an anti-CSP mAb revealed a reduction in tumor growth kinetics compared to IgG control corresponding with a significant increase in proliferating and activated cytotoxic CD8+ T cells. Anti-CSP-mediated tumor suppression was inhibited when CD8+ T cells were depleted, indicating that anti-CSP treatment is contingent on cytotoxic T cell functionality. ConclusionOur findings identify plectin as a biomarker of aggressive disease in PDAC, with high plectin expression associated with decreased T cell infiltration, and that treatment with anti-CSP mAb reinstates anti-tumor immunity and decreases tumor volume in vivo. These findings position plectin as a high-priority therapeutic target, with the potential to fundamentally reshape immune responses in PDAC and improve outcomes for patients with few remaining options.

BackgroundDrug responses in pancreatic ductal adenocarcinoma (PDAC) vary sharply across in vitro culture formats, but most 2D-3D comparisons conflate microenvironmental cues with time-dependent cellular adaptation. As a result, conventional assays frequently overestimate drug efficacy and poorly reflect clinical pharmacology. Main findingsWe profiled MiaPaCa-2, PANC-1, and CFPAC-1 grown in an extracellular-matrix (ECM) hydrogel for 1-12 days, defining extended 3D cultures ([&ge;]10 days) as mature tumoroids, and quantified 72 h drug responses to a multi-class oncology panel using growth-rate (GR) metrics to normalize for proliferation across formats and durations. Prolonged 3D pre-culture induced broad tolerance, with typical 10-100x reductions in sensitivity to standards of care (5-fluorouracil, SN38, oxaliplatin, gemcitabine, paclitaxel), following a reproducible susceptibility hierarchy (MiaPaCa-2 > PANC-1 > CFPAC-1) after GR correction. In mature tumoroids, GR values closely approximated clinically observed plasma exposures (e.g., within <4x for 5-FU and <0.5x for gemcitabine), whereas 2D and short-term organoid assays markedly underestimated resistance, often by >100x, thereby overstating drug activity. Notably, CFPAC-1 exhibited increased sensitivity to SN38 and trametinib under mature-organoid conditions, demonstrating that microenvironmental conditioning can invert responses for selected mechanisms. Transcriptomic profiling revealed coordinated up-regulation of multiple ABC transporters with extended 3D residence, tracking resistance phenotypes across lines and implicating transporter-linked tolerance programs. SignificanceTogether, these data identify time-in-3D and the emergence of mature tumoroids as dominant, previously under-controlled determinants of PDAC pharmacology that both induce tolerance and unmask context-dependent vulnerabilities. We propose incorporating both short-term and mature-tumoroid screening arms into preclinical workflows, reporting pre-culture duration alongside GR-normalized effect sizes, and leveraging transporter-informed biomarkers to guide regimen prioritization and sequencing. This framework enhances physiological relevance, reproducibility, and translational fidelity in PDAC drug discovery.

Background: Idiopathic pulmonary fibrosis (IPF) is a rare disease with a poor prognosis. Disease risk involves rare and common genetic variants. However, an inverse association have been described between them. Accordingly, IPF patients with a higher polygenic risk score (PRS) for IPF are less likely to carry rare deleterious variants and vice versa. Here, we evaluate weather PRS of IPF could serve as an additional criterion to patient prioritisation for rare variant discovery. Methods: We identified carriers based on the presence of rare qualifying variants (QVs) in genes linked to monogenic forms of pulmonary fibrosis in 888 IPF patients from the Pulmonary Fibrosis Foundation Patient Registry (PFF-PR). Genome-wide association study (GWAS) summary statistics from independent cohorts were used to construct a whole-genome PRS (WG-PRS) using a clumping and thresholding method (C+T) and a Bayesian method (SBayesRC). PRS were also derived from 19 known common sentinel IPF variants (Sentinel-PRS). Logistic regression models were used to evaluate associations between PRS and carrier status. Discriminatory performance was evaluated using area under the curve (AUC) analysis, and comparisons were made with DeLong test. Validation was performed in 472 IPF individuals from the UK PROFILE cohort. Results: IPF-PRS were strongly associated with the QVs carrier status: Odds Ratio [OR] 0.65 (95% Confidence Interval [CI] 0.53-0.79) for WG-PRSC+T, OR 0.71 (95% CI 0.59-0.86) for WG-PRSSBayesRC, and OR 0.77 (95% CI 0.63-0.94) for Sentinel-PRS. Adding WG-PRS to the patient personal clinical history improved the prediction of QVs carriers: AUC=0.62 for the clinical model, AUC=0.68 for WG-PRSC+T (DeLong test, p=9.54x10-4) and AUC=0.66 for WG-PRSSBayesRC (DeLong test, p=0.02). Adding of IPF-PRS to clinical variables correctly reclassified 22.8% of carriers when using WG-PRSC+T, 20.8% when using Sentinel-PRS, and 16.7% for WG-PRSSBayesRC. WG-PRSSBayesRC and the Sentinel-PRS also demonstrated improved prediction of QVs carriers in telomere-related genes in PROFILE. Conclusions: Incorporating IPF-PRS into a model based on the patient clinical history improves the identification of QVs carriers. Although the overall discriminatory power was moderate, these findings raise de the possibility of using WG-PRS as useful criterion for rare variant discovery in patients with IPF and enhance decision-making.

BackgroundThis study investigates the role of the pioneer transcription factor FOXA1 as a master gene in sustaining epithelial cell polarization in early-stage lung adenocarcinoma. The partial loss of FOXA1 is explored to determine if it will affect plasticity and progression of lung adenocarcinoma. The study also addresses the transcriptional circuitry that links polarity defects to lysosome homeostasis. MethodsA multiomics approach was used to define the status of the chromatin in epithelial and mesenchymal states of A549 adenocarcinoma cells obtained with a newly synthetized TGF-{beta} receptor inhibitor or TGF-{beta} respectively. The study leveraged ATAC-seq, RNA sequencing, Cut&Tag sequencing of FOXA1 and histone marks profiling. The functional impact of FOXA1 was examined by partial silencing in vitro and by heterozygous FOXA1 deletion in a KrasG12D mouse model. Three-dimensional organoid culture, high-resolution electron microscopy, spatial transcriptomics and multiplex immunohistochemistry assessed carcinoma cell polarity, proliferation, the tumor microenvironment and organelle content. Group differences were evaluated with two-tailed t tests or one-way analysis of variance. ResultsFOXA1 binding and expression were highest in cells harboring an epithelial phenotype. In mouse KrasG12D LUAD tumors FOXA1 marked polarized, CDH1-positive cells; heterozygous loss diminished CDH1, disrupted apical-basal architecture, lowered organoid-forming efficiency and remodeled the immune microenvironment. Spatial transcriptomics and ultrastructural analyses showed that FOXA1-deficient carcinoma cells accumulated lysosomes, down-regulated vesicle fusion genes of the SNARE family and activated the lysosomal CLEAR gene network. FOXA1 occupied enhancers of lysosome-associated genes and competed with the transcription factor TFE3, thereby suppressing transcription of cathepsin B and cathepsin C and restricting lysosome biogenesis. ConclusionsFOXA1 is a central regulator that preserves epithelial cell polarity and limits lysosome formation in lung adenocarcinoma. Targeting the FOXA1-TFE3-lysosome axis may affect tumor plasticity and provide new therapeutic opportunities.

BackgroundART effectively suppresses HIV replication and restores CD4+ T cells; however, long-lived HIV latent reservoirs enable viral persistence. Tuberculosis (TB) co-infection further impacts HIV latency and enhances viral replication. Given the high prevalence of latent TB infection (LTBI) in TB-endemic settings, understanding its impact on HIV biology is critical. Our study aims to investigate the influence of TB co-infection on HIV reservoir dynamics, viral diversity, and drug resistance mutations in ART-naive individuals. MethodologySamples from 90 ART-naive HIV-1C individuals, stratified based on IGRA and TB diagnosis, were used in this study. Plasma and PBMCs were isolated for viral RNA and DNA extraction respectively. Total proviral DNA was quantified using gag PCR. Full-length env and pol genes were amplified, purified and sequenced using ONT and Illumina platforms. Pol sequences were subjected to Drug Resistance Mutation (DRM) analysis via Stanford HIVdb with a minimum threshold mutation frequency of [&ge;]10%. Full length env sequences were used for phylogenetic analysis by aligning with Indian Subtype C reference sequence and phylogenetic tree was generated using ggplot2. ResultProviral load analysis showed no significant differences across HIV+LTBI-, HIV+LTBI+, and HIV+TB+ groups, although a trend toward higher levels was observed in HIV+TB+ individuals. Correlation analysis revealed distinct immune associations, with HIV+LTBI+ individuals showing positive correlations with activation and PD-1 expression. Longitudinal analysis of proviral loads demonstrated a modest decline in proviral load post-ART but remained persistent for up to 18-20 months following initiation of ART accompanied by low level ongoing viral replication. DRM analysis revealed a 33% prevalence in ART-naive individuals, with higher occurrence in HIV+LTBI+ group. Of the identified DRMs, 38% (5/13) and 71% (5/7) in sequences obtained from PBMC and plasma respectively were attributed to polymorphic mutations associated with Integrase strand transfer inhibitors (INSTIs). DRMs within plasma and PBMC derived viruses showed high concordance. Phylogenetic analysis of env sequences indicated overlapping viral populations between the 3 groups, with greater diversity in PBMCs compared to plasma. ConclusionThe study highlights that HIV reservoir dynamics, drug resistance, and viral diversity are significantly influenced by TB co-infection. While proviral loads were comparable, LTBI-associated immune activation and granuloma niches may have driven viral diversification and DRM emergence. High concordance between compartments and presence of transmitted resistance underscore the need for baseline screening, multi-compartment analysis, and sustained surveillance.

Cardiac transthyretin amyloidosis (ATTR-CA) is caused by myocardial deposition of misfolded transthyretin, leading to progressive heart failure. Disease pathology, however, extends beyond passive amyloid deposition and also involves active processes such as extracellular matrix (ECM) remodeling and immune activation. Mass spectrometry (MS) is the gold standard for amyloid typing in diagnostics. Here, we applied quantitative MS-driven proteomics on formalin-fixed paraffin-embedded whole cardiac tissue sections from six ATTR-CA cases, ten unaffected controls and four AL-CA controls to investigate protein expression changes. In addition to transthyretin, over 500 proteins were upregulated in ATTR-CA biopsies, including complement and coagulation factors as well as extracellular matrix (ECM) remodeling proteins. Among these, members of the A Disintegrin and Metalloproteinase with Thrombospondin Motifs (ADAMTS) family, metalloproteinases (MMPs), and Tissue Inhibitor of Metalloproteinases (TIMP3) showed significant upregulation. These proteins are key regulators of ECM turnover and structural integrity. Immunohistochemistry confirmed ADAMTS4 enrichment in amyloid deposits, while TIMP3 showed strong expression in cardiomyocytes and weaker staining within amyloid deposits. Together, these findings indicate that ECM remodeling, alongside complement and coagulation activation, represents a reproducible feature of cardiac ATTR amyloidosis. Whole-tissue proteomics provides biological insights that extend beyond amyloid typing, with potential implications for biomarker discovery and therapeutic targeting in ATTR-CA.

Tuberculosis (TB) caused by Mycobacterium tuberculosis (M.tb) remains the leading infectious cause of death worldwide, with multidrug-resistant (MDR) and extensively drug-resistant (XDR) strains presenting an escalating therapeutic crisis. All-trans retinoic acid (ATRA), an active vitamin A metabolite with established immunomodulatory properties, has emerged as a candidate host-directed therapy, yet its genome-wide transcriptional effects on macrophage across strains of differing drug-resistance profiles remain uncharacterized. We performed RNA sequencing of murine peritoneal macrophages infected with drug-susceptible H37Rv, MDR-2261, or XDR-MCY M.tb strains, with and without ATRA treatment, validated by RT-PCR and flow cytometry. All three strains activated a conserved pro-inflammatory program dominated by Nos2, Il1b, and Acod1 induction with progressive suppression of host translational and mitochondrial machinery. XDR-MCY uniquely induced Ifnb1/IFN-{beta}, suppressed Prdx1 and Gpx4, the latter shared with MDR-2261 and showed selective downregulation of MHC-II processing genes, suggesting a multilayered immune evasion strategy. ATRA activated canonical retinoid signaling across all infection states and consistently induced Arg1-mediated resolution signaling, with Nos2 suppression observed in H37Rv- and MDR-infected macrophages. ATRA selectively restored Epas1/HIF-2 in H37Rv- and MDR-infected macrophages without disrupting the HIF-1-driven antimicrobial program or itaconate biosynthesis. ATRA responsiveness progressively attenuated with drug resistance, with XDR-MCY-infected macrophages largely refractory to transcriptional reprogramming. These findings provide a transcriptional rationale for evaluating ATRA as a host-directed adjunct in drug-resistant TB and identify Gpx4 suppression in drug-resistant strains and XDR-specific Ifnb1 induction as vulnerabilities necessitating further investigation.