Biology subjects

Malla, S.

Publications and source records attributed to Malla, S..

25 records · Page 2Linked to original sources

bioRxiv2022

Biological misinterpretation of transcriptional signatures in tumour samples can unknowingly undermine mechanistic understanding and faithful alignment with preclinical data

Precise mechanism-based gene expression signatures (GESs) have been developed in appropriate in vitro and in vivo model systems, to identify important cancer-related signalling processes. However, some GESs originally developed to represent specific disease processes, primarily with an epithelial cell focus, are being applied to heterogeneous tumour samples where the expression of the genes in the signature may no longer be epithelial-specific. Therefore, unknowingly, even small changes in tumour stroma percentage can directly influence GESs, undermining the intended mechanistic signalling. Using colorectal cancer as an exemplar, we deployed numerous orthogonal profiling methodologies, including laser capture microdissection, flow cytometry, bulk and multiregional biopsy clinical samples, single cell RNAseq and finally spatial transcriptomics, to perform a comprehensive assessment of the potential for the most widely-used GESs to be influenced, or confounded, by stromal content in tumour tissue. To complement this work, we generated a freely-available resource, ConfoundR; https://confoundr.qub.ac.uk/, that enables users to test the extent of stromal influence on an unlimited number of the genes/signatures simultaneously across colorectal, breast, pancreatic, ovarian and prostate cancer datasets. Findings presented here demonstrate the clear potential for misinterpretation of the meaning of GESs, due to widespread stromal influences, which in-turn can undermine faithful alignment between clinical samples and preclinical data/models, particularly cell lines and organoids, or tumour models not fully recapitulating the stromal and immune microenvironment. As such, efforts to faithfully align preclinical models of disease using phenotypically-designed GESs must ensure that the signatures themselves remain representative of the same biology when applied to clinical samples.

cancer biology↗

bioRxiv2021

The Molecular Subtyping Resource (MouSR): a user-friendly tool for rapid biological discovery from human or mouse transcriptional data

Generation of transcriptional data has dramatically increased in the last decade, driving the development of analytical algorithms that enable interrogation of the biology underpinning the profiled samples. However, these resources require users to have expertise in data wrangling and analytics, reducing opportunities for biological discovery by "wet-lab" users with a limited programming skillset. Although commercial solutions exist, costs for software access can be prohibitive for academic research groups. To address these challenges, we have developed an open source and user-friendly data analysis platform for on-the-fly bioinformatic interrogation of transcriptional data derived from human or mouse tissue, called "MouSR". This internet-accessible analytical tool, https://mousr.qub.ac.uk/, enables users to easily interrogate their data using an intuitive "point and click" interface, which includes a suite of molecular characterisation options including QC, differential gene expression, gene set enrichment and microenvironmental cell population analyses from RNA-Seq. Users are provided with adjustable options for analysis parameters to generate results that can be saved as publication-quality images. To highlight its ability to perform high quality data analysis, we utilise the MouSR tool to interrogate our recently published tumour dataset, derived from genetically engineered mouse models and matched organoids, where we rapidly reproduced the key transcriptional findings. The MouSR online tool provides a unique freely-available option for users to perform rapid transcriptomic analyses and comprehensive interrogation of the signalling underpinning transcriptional datasets, which alleviates a major bottleneck for biological discovery.

bioinformatics↗

bioRxiv2021

ZFP207 controls pluripotency by multiple post-transcriptional mechanisms

The pluripotent state is not solely governed by the action of the core transcription factors Oct4, Sox2, and Nanog, but also by a series of co-transcriptional and post-transcriptional events, including alternative splicing (AS) and the interaction of RNA-binding proteins (RBPs) with defined subpopulations of RNAs. Zinc Finger Protein 207 (ZFP207) is an essential transcription factor for mammalian embryonic development. Here, we employ multiple functional analyses to characterize the role of ZFP207 in mouse embryonic stem cells (ESCs). We find that ZFP207 plays a pivotal role in ESC maintenance, and silencing of Zfp207 leads to severe neuroectodermal differentiation defects. In striking contrast to human ESCs, ZFP207 does not transcriptionally regulate stem cell and neuronal-related genes but exerts its effects by control AS networks and acting as an RBP. Our study expands the role of ZFP207 to maintain ESC identity, and underscores ZFP207 functional versatility with key roles in neural fate commitment.

cell biology↗

bioRxiv2020

A Novel Stress Response Pathway Regulates Ribosome Biogenesis

Production of ribosomes is an energy-intensive process owing to the intricacy of these massive macromolecular machines. Each human ribosome contains 80 ribosomal proteins and four non-coding RNAs. Accurate assembly requires precise regulation of protein and RNA subunits. In response to stress, the integrated stress response (ISR) rapidly inhibits global translation. How rRNA is coordinately regulated with the rapid inhibition of ribosomal protein synthesis is not known. Here we show that stress specifically inhibits the first step of rRNA processing. Unprocessed rRNA is stored within the nucleolus, and, when stress resolves, it re-enters the ribosome biogenesis pathway. Retention of unprocessed rRNA within the nucleolus aids in the maintenance of this organelle. This response is independent of the ISR or inhibition of cellular translation but represents an independent stress-response pathway that we term Ribosome Biogenesis Stress Response (RiBiSR). Failure to coordinately regulate ribosomal protein translation and rRNA production results in nucleolar fragmentation. Our study unveils a novel stress response pathway that aims at conserving energy, preserving the nucleolus, and prevents further stress by regulation of rRNA processing.

biochemistry↗

bioRxiv2020

A Novel Efficient L-Lysine Exporter Identified by Functional Metagenomics

Lack of active export system often limits the industrial bio-based production processes accumulating the intracellular product and hence complexing the purification steps. L-lysine, an essential amino acid, is produced biologically in quantities exceeding two million tons per year; yet, L-lysine production is challenged by efficient export system at high titres during fermentation. To address this issue, new exporter candidates for efficient efflux of L-lysine are needed. Using metagenomic functional selection, we identified 58 genes encoded on 28 unique metagenomic fragments from cow gut microbiome library that improved L-lysine tolerance. These genes include a novel putative L-lysine transporter, belonging to a previously uncharacterized EamA superfamily. Characterization using Xenopus oocyte expression system as well as an Escherichia coli host demonstrates activity as a L-lysine transporter. This novel exporter improved L-lysine tolerance in E. coli by 40% and enhanced the specific productivity of L-lysine in an industrial Corynebacterium glutamicum strain by 12%. Our approach allows the sequence-independent discovery of novel exporters and can be deployed to increase titres and productivity of toxicity-limited bioprocesses.

molecular biology↗

bioRxiv2019

Adaptive laboratory evolution reveals general and specific chemical tolerance mechanisms and enhances biochemical production

Tolerance to high product concentrations is a major barrier to achieving economically viable processes for bio-based chemical production. Chemical tolerance mechanisms are often unknown, thus their rational design is not achievable. To reveal unknown tolerance mechanisms we used an automated platform to evolve Escherichia coli to grow in previously toxic concentrations of 11 chemicals that have applications as polymer precursors, chemical intermediates, or biofuels. Re-sequencing of isolates from 88 independently evolved populations, reconstruction of mutations, and cross-compound tolerance profiling was employed to uncover general and specific tolerance mechanisms. We found that: 1) the broad tolerance of strains towards chemicals varied significantly depending on the chemical stress condition under which the strain was evolved; 2) the strains that acquired high levels of NaCl tolerance also became broadly tolerant to most chemicals; 3) genetic tolerance mechanisms included alterations in regulatory, cell wall, transcriptional and translational functions, as well as more chemical-specific mechanisms related to transport and metabolism; 4) using pre-tolerized starting strains can significantly enhance subsequent production of chemicals when a production pathway is inserted; and 5) only a subset of the evolved isolates showed improved production indicating that this approach is especially useful when a large number of independently evolved isolates are screened for production. We provide a comprehensive genotype-phenotype map based on identified mutations and growth phenotypes for 224 chemical tolerant strains.

microbiology↗

bioRxiv2017

Nanopore sequencing and assembly of a human genome with ultra-long reads

Nanopore sequencing is a promising technique for genome sequencing due to its portability, ability to sequence long reads from single molecules, and to simultaneously assay DNA methylation. However until recently nanopore sequencing has been mainly applied to small genomes, due to the limited output attainable. We present nanopore sequencing and assembly of the GM12878 Utah/Ceph human reference genome generated using the Oxford Nanopore MinION and R9.4 version chemistry. We generated 91.2 Gb of sequence data ([~]30x theoretical coverage) from 39 flowcells. De novo assembly yielded a highly complete and contiguous assembly (NG50 [~]3Mb). We observed considerable variability in homopolymeric tract resolution between different basecallers. The data permitted sensitive detection of both large structural variants and epigenetic modifications. Further we developed a new approach exploiting the long-read capability of this system and found that adding an additional 5x-coverage of ultra-long reads (read N50 of 99.7kb) more than doubled the assembly contiguity. Modelling the repeat structure of the human genome predicts extraordinarily contiguous assemblies may be possible using nanopore reads alone. Portable de novo sequencing of human genomes may be important for rapid point-of-care diagnosis of rare genetic diseases and cancer, and monitoring of cancer progression. The complete dataset including raw signal is available as an Amazon Web Services Open Dataset at: https://github.com/nanopore-wgs-consortium/NA12878.

genomics↗