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Correlative 3D imaging method for analysing lesion architecture in susceptible mice infected with Mycobacterium tuberculosis.
No full textTuberculosis (TB) is characterized by the formation of heterogeneous, immune-rich granulomas in the lungs. Host and pathogen factors contribute to this heterogeneity, but the molecular and cellular drivers of granuloma diversity remain inadequately understood owing to limitations in experimental techniques. In this study, we developed an approach that combines passive CLARITY (PACT)-based clearing with light-sheet fluorescence microscopy to visualize lesion architecture and lung involvement in Mycobacterium tuberculosis-infected C3HeB/FeJ mice. Three-dimensional rendering of post-mortem lungs revealed critical architectural features in lesion development that traditional thin-section imaging could not detect. Wild-type M. tuberculosis infection resulted in organized granulomas, with median sizes increasing to 3.74×108 µm3 and occupying ∼10% of the total lung volume by day 70 post-infection. In contrast, infection with the avirulent ESX-1 deletion mutant strain resulted in diffuse and sparsely organized CD11b recruitment (median size of 8.22×107 µm3), primarily located in the lung periphery and minimally involving the airways (0.23% of the total lung space). Additionally, we present a method for volumetric correlative light and electron microscopy, enabling tracking of individual immune cell populations within granulomas.</p
DNA methylation cooperates with genomic alterations during non-small cell lung cancer evolution.
No full textAberrant DNA methylation has been described in nearly all human cancers, yet its interplay with genomic alterations during tumor evolution is poorly understood. To explore this, we performed reduced representation bisulfite sequencing on 217 tumor and matched normal regions from 59 patients with non-small cell lung cancer from the TRACERx study to deconvolve tumor methylation. We developed two metrics for integrative evolutionary analysis with DNA and RNA sequencing data. Intratumoral methylation distance quantifies intratumor DNA methylation heterogeneity. MR/MN classifies genes based on the rate of hypermethylation at regulatory (MR) versus nonregulatory (MN) CpGs to identify driver genes exhibiting recurrent functional hypermethylation. We identified DNA methylation-linked dosage compensation of essential genes co-amplified with neighboring oncogenes. We propose two complementary mechanisms that converge for copy number alteration-affected chromatin to undergo the epigenetic equivalent of an allosteric activity transition. Hypermethylated driver genes under positive selection may open avenues for therapeutic stratification of patients
Charles Bonnet syndrome: taking another look at visual hallucinations in sight loss.
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Characterisation of people living with chronic hepatitis B virus infection in England and stratification by HBsAg levels: A cross-sectional study.
No full textCharacterisation of people with hepatitis B (PwHB) remains limited, particularly regarding treatment status, disease severity and biomarker profiles. Quantitative hepatitis B surface antigen (qHBsAg) is a key predictor of response to emerging therapies, but its distribution is poorly described. Using a large, ethnically diverse UK cohort, we assessed demographics, clinical features and HBsAg levels to guide treatment strategies. This cross-sectional analysis of PwHB (N = 2000 [prespecified]) used data from four English hospitals, collected via the National Institute for Health and Care Research Health Informatics Collaborative framework. Individual characteristics were assessed overall, and post hoc by qHBsAg levels (≤ 3000/> 3000 IU/mL; 1000 IU/mL) available from one centre (N = 457). The cohort had a slight male predominance (54%) and a mean age of 44.9 years. White and Asian ethnicity each accounted for 25%, and 23% were on nucleos(t)ide analogue therapy. Centres collecting HBsAg data had more individuals with undetectable HBV DNA or on treatment. Among individuals with non-missing qHBsAg data (263/457), 167/263 (63.5%) had qHBsAg ≤ 3000 IU/mL. These were older (49.6 vs. 43.5 years), more likely to be male (53.9% vs. 35.4%), Asian (40.7% vs. 20.8%) or have undetectable HBV DNA (35.9% vs. 17.7%), and less likely to be Black (13.2% vs. 34.4%) versus those with qHBsAg > 3000 IU/mL. Fifty-six (21.3%) people had qHBsAg 1000 IU/mL. This cohort of PwHB highlights qHBsAg distribution in clinical settings and could identify people more likely to achieve functional cure with emerging therapies
A phase II trial of mTORC1/2 inhibition in STK11 deficient non small cell lung cancer.
No full textThere are no current stratified medicine options for STK11-deficient NSCLC. STK11 loss mediates mTORC activation, GLUT1 up-regulation and increased glycolysis. This metabolic reprogramming might represent a therapeutic vulnerability targetable with mTORC1/2 inhibition. In arm B2 of the National Lung Matrix Trial 54 patients with NSCLC received vistusertib, of which 49 were STK11-deficient (30 with KRAS mutation (B2D), 19 without (B2S)). Objective response (OR) and durable clinical benefit (DCB) rates with 95% credible intervals (CrI) were estimated from posterior probability distributions generated using Bayesian beta-binomial conjugate analysis. In B2D, 2 per-protocol patients obtained OR (estimated true OR rate (95%CrI) 9.8% (2.4-24.3). Estimates of true DCB rate (95%CrI): B2D 24.4% (11.1-42.3), B2S 14.6% (3.6-34.7). Overall, vistusertib cannot be recommended in this context. Longitudinal ctDNA analysis demonstrates enrichment of SMARCA4 mutations post-treatment. In vitro studies show adaptive resistance to mTORC1/2 inhibition via AKT reactivation. (NCT02664935, ISRCTN38344105, EudraCT 2014-000814-73, 10 June 2015).</p
A systematic benchmark of Nanopore long-read RNA sequencing for transcript-level analysis in human cell lines.
No full textThe human genome contains instructions to transcribe more than 200,000 RNAs. However, many RNA transcripts are generated from the same gene, resulting in alternative isoforms that are highly similar and that remain difficult to quantify. To evaluate the ability to study RNA transcript expression, we profiled seven human cell lines with five different RNA-sequencing protocols, including short-read cDNA, Nanopore long-read direct RNA, amplification-free direct cDNA and PCR-amplified cDNA sequencing, and PacBio IsoSeq, with multiple spike-in controls, and additional transcriptome-wide N6-methyladenosine profiling data. We describe differences in read length, coverage, throughput and transcript expression, reporting that long-read RNA sequencing more robustly identifies major isoforms. We illustrate the value of the SG-NEx data to identify alternative isoforms, novel transcripts, fusion transcripts and N6-methyladenosine RNA modifications. Together, the SG-NEx data provide a comprehensive resource enabling the development and benchmarking of computational methods for profiling complex transcriptional events at isoform-level resolution
Identifying research priorities for pituitary adenoma surgery: an international Delphi consensus statement.
No full textPURPOSE: Pituitary surgery is the mainstay treatment for most pituitary adenomas, but many questions remain about perioperative and long-term management and outcomes. This study aimed to identify the most pressing research priorities in pituitary surgery with input from patients, caregivers, and healthcare professionals. METHODS: An initial survey of patients, caregivers, and healthcare professionals assembled priorities related to preoperative care, surgical techniques, and postoperative management in pituitary surgery. Priorities were thematically grouped into summary priorities, and those answered by existing evidence were omitted following a literature review. An interim survey asked patients, caregivers, and healthcare professionals to select their top 10 priorities from the remaining list. The highest-ranked priorities advanced to a consensus meeting, where the top 10 questions were prioritized. RESULTS: In the initial survey, 147 participants-60.5% of whom were patients, caregivers, or patient support group representatives-submitted 785 priorities, which were then condensed into 52 summary priorities. After a literature review, 33 unanswered priorities were included in the interim survey, completed by 155 respondents, of whom 54.2% were patients, caregivers, or patient support group representatives. The top-ranked priorities were discussed by 14 participants (7 patients and 7 healthcare professionals) during a consensus meeting. The top 10 priorities covered a variety of themes including enhancing diagnosis and management of pituitary adenomas, advancing surgical techniques and technologies, optimizing the prediction of outcomes and complications, and improving patient support and follow-up. CONCLUSIONS: The top 10 research priorities in pituitary surgery aim to align researchers and direct funding in order to maximize impact and champion patient representation
Cryo-electron tomography reveals how COPII assembles on cargo-containing membranes
No full textProteins traverse the eukaryotic secretory pathway through membrane trafficking between organelles. The coat protein complex II (COPII) mediates the anterograde transport of newly synthesized proteins from the endoplasmic reticulum, engaging cargoes with a wide range of size and biophysical properties. The native architecture of the COPII coat and how cargo might influence COPII carrier morphology remain poorly understood. Here we reconstituted COPII-coated membrane carriers using purified Saccharomyces cerevisiae proteins and cell-derived microsomes as a native membrane source. Using cryo-electron tomography with subtomogram averaging, we demonstrate that the COPII coat binds cargo and forms largely spherical vesicles from native membranes. We reveal the architecture of the inner and outer coat layers and shed light on how spherical carriers are formed. Our results provide insights into the architecture and regulation of the COPII coat and advance our current understanding of how membrane curvature is generated
Whole-genome ancestry of an Old Kingdom Egyptian.
No full textAncient Egyptian society flourished for millennia, reaching its peak during the Dynastic Period (approximately 3150-30 BCE). However, owing to poor DNA preservation, questions about regional interconnectivity over time have not been addressed because whole-genome sequencing has not yet been possible. Here we sequenced a 2× coverage whole genome from an adult male Egyptian excavated at Nuwayrat (Nuerat, نويرات). Radiocarbon dated to 2855-2570 cal. BCE, he lived a few centuries after Egyptian unification, bridging the Early Dynastic and Old Kingdom periods. The body was interred in a ceramic pot within a rock-cut tomb1, potentially contributing to the DNA preservation. Most of his genome is best represented by North African Neolithic ancestry, among available sources at present. Yet approximately 20% of his genetic ancestry can be traced to genomes representing the eastern Fertile Crescent, including Mesopotamia and surrounding regions. This genetic affinity is similar to the ancestry appearing in Anatolia and the Levant during the Neolithic and Bronze Age2-5. Although more genomes are needed to fully understand the genomic diversity of early Egyptians, our results indicate that contacts between Egypt and the eastern Fertile Crescent were not limited to objects and imagery (such as domesticated animals and plants, as well as writing systems)6-9 but also encompassed human migration
Population pharmacokinetics of pyrazinamide and isoniazid in plasma and cerebrospinal fluid from South African adults with tuberculous meningitis.
No full textPyrazinamide and isoniazid are first-line drugs for tuberculous meningitis (TBM), but limited information is available on their plasma pharmacokinetics, and particularly cerebrospinal fluid (CSF) penetration, in patients with TBM. Any potential effect of co-administration with high-dose rifampicin, also being evaluated in trials for TBM, is unknown. Understanding this is important for dose optimisation. We characterized pyrazinamide and isoniazid plasma and CSF pharmacokinetics among adults enrolled in a phase 2 clinical trial of intensified antibiotic therapy for HIV-associated TBM. Participants were randomized to receive either standard TBM treatment (including rifampicin 10 mg/kg) or high-dose rifampicin (35 mg/kg) plus linezolid, with or without aspirin. Plasma and lumbar CSF samples were collected on days 3 and 28 after study enrollment, and drug concentrations were measured using liquid chromatography-tandem mass spectrometry. Data were analysed using nonlinear mixed-effects modeling. Forty-nine participants provided 414 plasma and 44 CSF concentrations. Pyrazinamide CSF concentrations equilibrated with plasma with a half-life of 0.66 h and a pseudo-partition coefficient of 1.05. Isoniazid concentrations equilibrated with a half-life of 3.87 h and a pseudo-partition coefficient of 1.04. Pyrazinamide clearance increased by 30% from day 3 to day 28. NAT2 phenotype determined multi-modal isoniazid clearance. High-dose rifampicin did not affect pyrazinamide or isoniazid plasma pharmacokinetics or CSF penetration. Both drugs achieved exposure in CSF similar to plasma, supporting their crucial role in TBM treatment. Plasma pharmacokinetics of pyrazinamide and isoniazid in TBM were consistent with previously reported values in pulmonary tuberculosis, even when co-administered with high-dose rifampicin