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Background: During the surge of COVID-19 pandemic, the mechanism of reporting and documentation of deaths was inadequate. It is crucial to establish the mechanism for measuring the actual events of COVID-19 and associated mortalities as well during this pandemic situation. This is the first instance of application of different rapid methods for death estimation during the pandemic situation. If found to be valid, these methods may be useful for other countries with limited resources and less developed death registration system. Objective: To find out the feasibility, acceptability of different rapid method for estimating the cause of deaths among the general population & healthcare workers and also to estimate the number of deaths among allopathic doctors. Methodology: The study was conducted from September 2022 to October 2023. Target population being adult deceased person between 1st April 2020 to 30th November 2022 among general population, health care worker and allopathic doctor. For the general population, the study was conducted in one urban municipal area and two sub-centres of Habra Block I under North 24 Parganas district with WHO Verbal Autopsy (VA) tool for adult. Complete enumeration with snowballing, compartmental and capture-recapture method was adopted for general population, health care worker and allopathic doctor respectively. Results: Overall, 300 VA could be performed (121 urban & 179 rural) among general population. There were challenges related to both feasibility & acceptability in terms of data access, permission and cooperation. Research team employed a combination of persistence, local collaboration, and strategic decision-making to successfully collect data. Acceptability was more in rural area. Total number of deceased doctors was estimated to be 246 from two different lists. Conclusion: Rapid methods may be employed as a part of response systems during pandemic or any future disasters if adequate governmental support and assistance is obtained along with aid from the local people.

Malignant Perivascular Epitheloid Cell Tumour (PEComa) of the lung is very rare, with only six cases reported in literature. This case presented with a large mass originating from right upper lobe of the lung with dilemma in its histopathological diagnosis and management. Postoperative histopathology after a right upper and middle lobectomy describes a tumour with an alveolar/nested pattern of growth and epitheloid morphology with expression of TFE-3 and diagnosed as PEComa. After 6 months the patient had a local recurrence inside the thorax & chest wall.This case qualifies it as a rare type of malignant PEComa with younger age of presentation, aggressive clinical behaviour & malignant histological features along with TFE3 positivity on immunohistochemistry. This case is probably the first of its kind with the largest reported size involving two lobes of the lung.

Coronaviruses express their structural and accessory genes via a set of subgenomic RNAs, whose synthesis is directed by transcription regulatory sequences (TRSs) in the 5' genomic leader and upstream of each body open reading frame. In SARS-CoV-2, the TRS has the consensus AAACGAAC; upon searching for emergence of this motif in the global SARS-CoV-2 sequences, we find that it evolves frequently, especially in the 3' end of the genome. We show well-supported examples upstream of the Spike gene-within the nsp16 coding region of ORF1b-which is expressed during human infection, and upstream of the canonical Envelope gene TRS, both of which have evolved convergently in multiple lineages. The most frequent neo-TRS is within the coding region of the Nucleocapsid gene, and is present in virtually all viruses from the B.1.1 lineage, including the variants of concern Alpha, Gamma, Omicron and descendants thereof. Here, we demonstrate that this TRS leads to the expression of a novel subgenomic mRNA encoding a truncated C-terminal portion of Nucleocapsid, which is an antagonist of type I interferon production and contributes to viral fitness during infection. We observe distinct phenotypes when the Nucleocapsid coding sequence is mutated compared to when the TRS alone is ablated. Our findings demonstrate that SARS-CoV-2 is undergoing evolutionary changes at the functional RNA level in addition to the amino acid level., Competing Interests: I have read the journal’s policy and the authors of this manuscript have the following competing interests: While the authors declare no competing interests directly related to this work, CS receives grants from Bristol Myers Squibb, Ono Pharmaceuticals, Boehringer Ingelheim, Roche-Ventana, Pfizer, and Archer Dx; receives personal fees from Genentech, the Sarah Canon Research Institute, Medicxi, Bicycle Therapeutics, GRAIL, Amgen, AstraZeneca, Bristol Myers Squibb, Illumina, GlaxoSmithKline, MSD, and Roche-Ventana; holds stock options in Apogen Biotech, Epic Biosciences, GRAIL, and Achilles Therapeutics; is a member of a scientific advisory board for Bicycle Therapeutics, GRAIL, Relay Therapeutics, SAGA Diagnostics, and Achilles Therapeutics; is a co-founder of Achilles Therapeutics; receives consulting fees from Genentech, Medicxi, MetaboMed, Novartis, the China Innovation Centre of Roche, and the Sarah Cannon Research Institute; and receives honoraria from Amgen, AstraZeneca, Bristol Myers Squibb, Illumina, and Incyte. DLVB receives grants, paid to their institution, from AstraZeneca and GSK related to COVID-19, and is a member of the UK Genotype-to-Phenotype 2 Consortium. All other authors have declared that no competing interests exist., (Copyright: © 2025 Mears et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

The 3' untranslated region (3'UTR) plays a crucial role in determining mRNA stability, localisation, translation and degradation. Cap analysis of gene expression (CAGE), a method for the detection of capped 5' ends of mRNAs, additionally reveals a large number of apparently 5' capped RNAs derived from locations within the body of the transcript, including 3'UTRs. Here, we provide direct evidence that these 3'UTR-derived RNAs are indeed capped and widespread in mammalian cells. By using a combination of AGO2 enhanced individual nucleotide resolution UV crosslinking and immunoprecipitation (eiCLIP) and CAGE following siRNA treatment, we find that these 3'UTR-derived RNAs likely originate from AGO2-binding sites, and most often occur at locations with G-rich motifs bound by the RNA-binding protein UPF1. High-resolution imaging and long-read sequencing analysis validate several 3'UTR-derived RNAs, showcase their variable abundance and show that they may not co-localise with the parental mRNAs. Taken together, we provide new insights into the origin and prevalence of 3'UTR-derived RNAs, show the utility of CAGE-seq for their genome-wide detection and provide a rich dataset for exploring new biology of a poorly understood new class of RNAs., (© 2024. The Author(s).)

Neuronal differentiation requires building a complex intracellular architecture, and therefore the coordinated regulation of defined sets of genes. RNA-binding proteins (RBPs) play a key role in this regulation. However, while their action on individual mRNAs has been explored in depth, the mechanisms used to coordinate gene expression programs shaping neuronal morphology are poorly understood. To address this, we studied how the paradigmatic RBP IMP1 (IGF2BP1), an essential developmental factor, selects and regulates its RNA targets during the human neuronal differentiation. We perform a combination of system-wide and molecular analyses, revealing that IMP1 developmentally transitions to and directly regulates the expression of mRNAs encoding essential regulators of the microtubule network, a key component of neuronal morphology. Furthermore, we show that m6A methylation drives the selection of specific IMP1 mRNA targets and their protein expression during the developmental transition from neural precursors to neurons, providing a molecular principle for the onset of target selectivity., (© 2024. The Author(s).)

Amyotrophic lateral sclerosis (ALS) is characterized by nucleocytoplasmic mislocalization of the RNA-binding protein (RBP) TDP-43. However, emerging evidence suggests more widespread mRNA and protein mislocalization. Here, we employed nucleocytoplasmic fractionation, RNA sequencing, and mass spectrometry to investigate the localization of mRNA and protein in induced pluripotent stem cell-derived motor neurons (iPSMNs) from ALS patients with TARDBP and VCP mutations. ALS mutant iPSMNs exhibited extensive nucleocytoplasmic mRNA redistribution, RBP mislocalization, and splicing alterations. Mislocalized proteins exhibited a greater affinity for redistributed transcripts, suggesting a link between RBP mislocalization and mRNA redistribution. Notably, treatment with ML240, a VCP ATPase inhibitor, partially restored mRNA and protein localization in ALS mutant iPSMNs. ML240 induced changes in the VCP interactome and lysosomal localization and reduced oxidative stress and DNA damage. These findings emphasize the link between RBP mislocalization and mRNA redistribution in ALS motor neurons and highlight the therapeutic potential of VCP inhibition., Competing Interests: Declaration of interests R.P. and J.H. are named inventors on a patent related to VCP inhibitors (WO2023281030A3)., (Copyright © 2023 The Author(s). Published by Elsevier Inc. All rights reserved.)

CLIP technologies are now widely used to study RNA-protein interactions and many data sets are now publicly available. An important first step in CLIP data exploration is the visual inspection and assessment of processed genomic data on selected genes or regions and performing comparisons: either across conditions within a particular project, or incorporating publicly available data. However, the output files produced by data processing pipelines or preprocessed files available to download from data repositories are often not suitable for direct comparison and usually need further processing. Furthermore, to derive biological insight it is usually necessary to visualize a CLIP signal alongside other data such as annotations, or orthogonal functional genomic data (e.g., RNA-seq). We have developed a simple, but powerful, command-line tool: clipplotr , which facilitates these visual comparative and integrative analyses with normalization and smoothing options for CLIP data and the ability to show these alongside reference annotation tracks and functional genomic data. These data can be supplied as input to clipplotr in a range of file formats, which will output a publication quality figure. It is written in R and can both run on a laptop computer independently or be integrated into computational workflows on a high-performance cluster. Releases, source code, and documentation are freely available at https://github.com/ulelab/clipplotr., (© 2023 Chakrabarti et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society.)

The structure of mRNA molecules plays an important role in its interactions with trans-acting factors, notably RNA binding proteins (RBPs), thus contributing to the functional consequences of this interplay. However, current transcriptome-wide experimental methods to chart these interactions are limited by their poor sensitivity. Here we extend the hiCLIP atlas of duplexes bound by Staufen1 (STAU1) ∼10-fold, through careful consideration of experimental assumptions, and the development of bespoke computational methods which we apply to existing data. We present Tosca, a Nextflow computational pipeline for the processing, analysis and visualisation of proximity ligation sequencing data generally. We use our extended duplex atlas to discover insights into the RNA selectivity of STAU1, revealing the importance of structural symmetry and duplex-span-dependent nucleotide composition. Furthermore, we identify heterogeneity in the relationship between transcripts with STAU1-bound 3' UTR duplexes and metabolism of the associated RNAs that we relate to RNA structure: transcripts with short-range proximal 3' UTR duplexes have high degradation rates, but those with long-range duplexes have low rates. Overall, our work enables the integrative analysis of proximity ligation data delivering insights into specific features and effects of RBP-RNA structure interactions., (© The Author(s) 2023. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Amyotrophic Lateral Sclerosis (ALS) causes motor neuron degeneration, with 97% of cases exhibiting TDP-43 proteinopathy. Elucidating pathomechanisms has been hampered by disease heterogeneity and difficulties accessing motor neurons. Human induced pluripotent stem cell-derived motor neurons (iPSMNs) offer a solution; however, studies have typically been limited to underpowered cohorts. Here, we present a comprehensive compendium of 429 iPSMNs from 15 datasets, and 271 post-mortem spinal cord samples. Using reproducible bioinformatic workflows, we identify robust upregulation of p53 signalling in ALS in both iPSMNs and post-mortem spinal cord. p53 activation is greatest with C9orf72 repeat expansions but is weakest with SOD1 and FUS mutations. TDP-43 depletion potentiates p53 activation in both post-mortem neuronal nuclei and cell culture, thereby functionally linking p53 activation with TDP-43 depletion. ALS iPSMNs and post-mortem tissue display enrichment of splicing alterations, somatic mutations, and gene fusions, possibly contributing to the DNA damage response., (© 2023. The Author(s).)

RNA-binding proteins (RBPs) act as posttranscriptional regulators controlling the fate of target mRNAs. Unraveling how RNAs are recognized by RBPs and in turn are assembled into neuronal RNA granules is therefore key to understanding the underlying mechanism. While RNA sequence elements have been extensively characterized, the functional impact of RNA secondary structures is only recently being explored. Here, we show that Staufen2 binds complex, long-ranged RNA hairpins in the 3'-untranslated region (UTR) of its targets. These structures are involved in the assembly of Staufen2 into RNA granules. Furthermore, we provide direct evidence that a defined Rgs4 RNA duplex regulates Staufen2-dependent RNA localization to distal dendrites. Importantly, disrupting the RNA hairpin impairs the observed effects. Finally, we show that these secondary structures differently affect protein expression in neurons. In conclusion, our data reveal the importance of RNA secondary structure in regulating RNA granule assembly, localization and eventually translation. It is therefore tempting to speculate that secondary structures represent an important code for cells to control the intracellular fate of their mRNAs.

Mutations causing amyotrophic lateral sclerosis (ALS) often affect the condensation properties of RNA-binding proteins (RBPs). However, the role of RBP condensation in the specificity and function of protein-RNA complexes remains unclear. We created a series of TDP-43 C-terminal domain (CTD) variants that exhibited a gradient of low to high condensation propensity, as observed in vitro and by nuclear mobility and foci formation. Notably, a capacity for condensation was required for efficient TDP-43 assembly on subsets of RNA-binding regions, which contain unusually long clusters of motifs of characteristic types and density. These "binding-region condensates" are promoted by homomeric CTD-driven interactions and required for efficient regulation of a subset of bound transcripts, including autoregulation of TDP-43 mRNA. We establish that RBP condensation can occur in a binding-region-specific manner to selectively modulate transcriptome-wide RNA regulation, which has implications for remodeling RNA networks in the context of signaling, disease, and evolution., Competing Interests: Declaration of interests J.S. is a consultant for Dewpoint Therapeutics, Maze Therapeutics, and Vivid Sciences. B.P. is an employee of Dewpoint Therapeutics., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Reactive astrocytes are implicated in amyotrophic lateral sclerosis (ALS), although the mechanisms controlling reactive transformation are unknown. We show that decreased intron retention (IR) is common to human-induced pluripotent stem cell (hiPSC)-derived astrocytes carrying ALS-causing mutations in VCP, SOD1 and C9orf72. Notably, transcripts with decreased IR and increased expression are overrepresented in reactivity processes including cell adhesion, stress response and immune activation. This was recapitulated in public-datasets for (i) hiPSC-derived astrocytes stimulated with cytokines to undergo reactive transformation and (ii) in vivo astrocytes following selective deletion of TDP-43. We also re-examined public translatome sequencing (TRAP-seq) of astrocytes from a SOD1 mouse model, which revealed that transcripts upregulated in translation significantly overlap with transcripts exhibiting decreased IR. Using nucleocytoplasmic fractionation of VCP mutant astrocytes coupled with mRNA sequencing and proteomics, we identify that decreased IR in nuclear transcripts is associated with enhanced nonsense mediated decay and increased cytoplasmic expression of transcripts and proteins regulating reactive transformation. These findings are consistent with a molecular model for reactive transformation in astrocytes whereby poised nuclear reactivity-related IR transcripts are spliced, undergo nuclear-to-cytoplasmic translocation and translation. Our study therefore provides new insights into the molecular regulation of reactive transformation in astrocytes., (© The Author(s) 2021. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Studies of spliceosomal interactions are challenging due to their dynamic nature. Here we used spliceosome iCLIP, which immunoprecipitates SmB along with small nuclear ribonucleoprotein particles and auxiliary RNA binding proteins, to map spliceosome engagement with pre-messenger RNAs in human cell lines. This revealed seven peaks of spliceosomal crosslinking around branchpoints (BPs) and splice sites. We identified RNA binding proteins that crosslink to each peak, including known and candidate splicing factors. Moreover, we detected the use of over 40,000 BPs with strong sequence consensus and structural accessibility, which align well to nearby crosslinking peaks. We show how the position and strength of BPs affect the crosslinking patterns of spliceosomal factors, which bind more efficiently upstream of strong or proximally located BPs and downstream of weak or distally located BPs. These insights exemplify spliceosome iCLIP as a broadly applicable method for transcriptomic studies of splicing mechanisms.

The CRISPR-Cas9 system has successfully been adapted to edit the genome of various organisms. However, our ability to predict the editing outcome at specific sites is limited. Here, we examined indel profiles at over 1,000 genomic sites in human cells and uncovered general principles guiding CRISPR-mediated DNA editing. We find that precision of DNA editing (i.e., recurrence of a specific indel) varies considerably among sites, with some targets showing one highly preferred indel and others displaying numerous infrequent indels. Editing precision correlates with editing efficiency and a preference for single-nucleotide homologous insertions. Precise targets and editing outcome can be predicted based on simple rules that mainly depend on the fourth nucleotide upstream of the protospacer adjacent motif (PAM). Indel profiles are robust, but they can be influenced by chromatin features. Our findings have important implications for clinical applications of CRISPR technology and reveal general patterns of broken end joining that can provide insights into DNA repair mechanisms., (Copyright © 2018 The Author(s). Published by Elsevier Inc. All rights reserved.)

Long mammalian introns make it challenging for the RNA processing machinery to identify exons accurately. We find that LINE-derived sequences (LINEs) contribute to this selection by recruiting dozens of RNA-binding proteins (RBPs) to introns. This includes MATR3, which promotes binding of PTBP1 to multivalent binding sites within LINEs. Both RBPs repress splicing and 3' end processing within and around LINEs. Notably, repressive RBPs preferentially bind to evolutionarily young LINEs, which are located far from exons. These RBPs insulate the LINEs and the surrounding intronic regions from RNA processing. Upon evolutionary divergence, changes in RNA motifs within LINEs lead to gradual loss of their insulation. Hence, older LINEs are located closer to exons, are a common source of tissue-specific exons, and increasingly bind to RBPs that enhance RNA processing. Thus, LINEs are hubs for the assembly of repressive RBPs and also contribute to the evolution of new, lineage-specific transcripts in mammals. VIDEO ABSTRACT., (Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.)

An interplay of experimental and computational methods is required to achieve a comprehensive understanding of protein-RNA interactions. UV crosslinking and immunoprecipitation (CLIP) identifies endogenous interactions by sequencing RNA fragments that copurify with a selected RNA-binding protein under stringent conditions. Here we focus on approaches for the analysis of the resulting data and appraise the methods for peak calling, visualization, analysis, and computational modeling of protein-RNA binding sites. We advocate that the sensitivity and specificity of data be assessed in combination for computational quality control. Moreover, we demonstrate the value of analyzing sequence motif enrichment in peaks assigned from CLIP data and of visualizing RNA maps, which examine the positional distribution of peaks around regulated landmarks in transcripts. We use these to assess how variations in CLIP data quality and in different peak calling methods affect the insights into regulatory mechanisms. We conclude by discussing future opportunities for the computational analysis of protein-RNA interaction experiments.

The structure of RNA molecules has a critical role in regulating gene expression, largely through influencing their interactions with RNA-binding proteins (RBPs). RNA hybrid and individual-nucleotide resolution UV cross-linking and immunoprecipitation (hiCLIP) is a transcriptome-wide method of monitoring these interactions by identifying RNA duplexes bound by a specific RBP. The hiCLIP protocol consists of the following steps: in vivo cross-linking of RBPs to their bound RNAs; partial RNA digestion and purification of RNA duplexes interacting with the specific RBP using immunoprecipitation; ligation of the two arms of RNA duplexes via a linker; reverse transcription; cDNA library amplification; and finally high-throughput DNA sequencing. Mapping of the sequenced arms to a reference transcriptome identifies the exact locations of duplexes. hiCLIP data can directly identify all types of RNA duplexes bound by RBPs, including those that are challenging to predict computationally, such as intermolecular and long-range intramolecular duplexes. Moreover, the use of an adaptor that links the two arms of the RNA duplex permits hiCLIP to unambiguously identify the duplexes. Here we describe in detail the procedure for a hiCLIP experiment and the subsequent streamlined data analysis with an R package, 'hiclipr' (https://github.com/luslab/hiclipr/). Preparation of the library for high-throughput DNA sequencing takes ∼7 d and the basic bioinformatic pipeline takes 1 d.

There have been a number of developments in the management of venous thromboembolism over the past few years. Old questions, such as thrombolysis, have been revisited in recent trials. New initiatives, such as ambulatory care pathways, are being established across the country. This conference brought together doctors from the UK, USA, Spain and Australia to review the up-to-date management of venous thromboembolism., (© Royal College of Physicians 2015. All rights reserved.)

We describe a young man who presented with a short history of painless haematuria. Subsequent investigations and biopsy of lesions found in his bladder at cystoscopy confirmed the diagnosis of primary lymphoma of the bladder. Computed tomography studies confirmed the disease was localised to his bladder. Unfortunately, the tumour was not eradicated by radiotherapy. However, it was successfully treated with intravesical mitozantrone given in a novel gel formation. Three years after diagnosis the patient remains well with no evidence of recurrence.