Biology subjects

Sareen, D.

Publications and source records attributed to Sareen, D..

9 recordsLinked to original sources

bioRxiv2025

An unusual ring pattern in the Rosβ lanthipeptide of the two-component lantibiotic roseocin

Two-component lantibiotics comprise two post-translationally modified peptides that synergistically exert antimicrobial activity. Most known two-component lantibiotics are made up of a structurally conserved -lanthipeptide that binds to the peptidoglycan precursor lipid II and a {beta}-lanthipeptide that is believed to interact with the -peptide-lipid II complex to form pores in the membranes of susceptible bacteria. A few two-component lantibiotics including roseocin do not follow this general scheme and act by different, currently unresolved mechanisms. An important first step in studying this group of lanthipeptides is determination of their chemical structure. Roseocins {beta}-peptide (Ros{beta}) is formed by the RosM lanthipeptide synthetase from the RosA1 precursor peptide. RosM carries out nine dehydrations of Ser and Thr residues to the corresponding dehydroamino acids followed by six Michael-type additions of the thiols of Cys residues to a subset of the dehydroamino acids forming six thioether crosslinks. Sequence alignment with structurally characterized lanthipeptides does not allow prediction of its ring pattern. In this study, Ros{beta} was produced in Escherichia coli and its ring pattern was established by multi-dimensional NMR spectroscopy. The stereochemistry of the lanthionine and methyllanthionine residues was determined by Marfeys analysis with authentic standards. Ros{beta} is shown to have a unique ring pattern amongst previously characterized lanthipeptides. TOC graphic O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=131 SRC="FIGDIR/small/684949v2_ufig1.gif" ALT="Figure 1"> View larger version (20K): org.highwire.dtl.DTLVardef@125226borg.highwire.dtl.DTLVardef@f6a170org.highwire.dtl.DTLVardef@1e0e57corg.highwire.dtl.DTLVardef@1fa4c8d_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

bioRxiv2024

Identification of Disease-relevant, Sex-based Proteomic Differences in iPSC-derived Vascular Smooth Muscle

The prevalence of cardiovascular disease varies with sex, and the impact of intrinsic sex-based differences on vasculature is not well understood. Animal models can provide important insight into some aspects of human biology, however not all discoveries in animal systems translate well to humans. To explore the impact of chromosomal sex on proteomic phenotypes, we used iPSC-derived vascular smooth muscle cells from healthy donors of both sexes to identify sex-based proteomic differences and their possible effects on cardiovascular pathophysiology. Our analysis confirmed that differentiated cells have a proteomic profile more similar to healthy primary aortic smooth muscle than iPSCs. We also identified sex-based differences in iPSC- derived vascular smooth muscle in pathways related to ATP binding, glycogen metabolic process, and cadherin binding as well as multiple proteins relevant to cardiovascular pathophysiology and disease. Additionally, we explored the role of autosomal and sex chromosomes in protein regulation, identifying that proteins on autosomal chromosomes also show sex-based regulation that may affect the protein expression of proteins from autosomal chromosomes. This work supports the biological relevance of iPSC-derived vascular smooth muscle cells as a model for disease, and further exploration of the pathways identified here can lead to the discovery of sex-specific pharmacological targets for cardiovascular disease. SignificanceIn this work, we have differentiated 4 male and 4 female iPSC lines into vascular smooth muscle cells, giving us the ability to identify statistically-significant sex-specific proteomic markers that are relevant to cardiovascular disease risk (such as PCK2, MTOR, IGFBP2, PTGR2, and SULTE1).

molecular biology↗

bioRxiv2023

Disease related changes in ATAC-Seq of more than 450 iPSC-derived motor neuron lines from ALS patients and controls

Amyotrophic Lateral Sclerosis (ALS), like many other neurodegenerative diseases, is highly heritable, but with only a small fraction of cases explained by monogenic disease alleles. To better understand sporadic ALS, we report epigenomic profiles, as measured by ATAC-seq, of motor neuron cultures derived from a diverse group of 380 ALS patients and 80 healthy controls. We find that chromatin accessibility is heavily influenced by sex, the iPSC cell type of origin, ancestry, and the inherent variance arising from sequencing. Once these covariates are corrected for, we are able to identify robust ALS-specific signals in the data. Additionally, we find that the ATAC-seq data is able to predict ALS disease progression rates with similar accuracy to methods based on biomarkers and clinical status. These results suggest that iPSC-derived motor neurons recapitulate important disease-relevant epigenomic changes.

neuroscience↗

bioRxiv2023

Non-antibiotic pharmaceuticals exhibit toxicity against Escherichia coli at environmentallyrelevant concentrations with no evolution of cross-resistance to antibiotics

Antimicrobial resistance can arise in the natural environment via prolonged exposure to the effluent surrounding manufacturing facilities. These facilities also produce non-antibiotic pharmaceuticals, and the effect of these on the surrounding microbial communities is less clear; whether they have inherent toxicity, or whether long-term exposure might select for cross-resistance to antibiotics. To this end, we screened four non-antibiotic pharmaceuticals (acetaminophen, ibuprofen, propranolol, met formin) and titanium dioxide for toxicity against Escherichia coli K-12 MG1655 and conducted a 30 day selection experiment to assess the effect of long-term exposure. All compounds reduced the maximum optical density reached by E. coli at a range of concentrations including one of environmental relevance, with transcriptome analysis identifying upregulated genes related to stress response and multidrug efflux in response ibuprofen treatment. The non-antibiotic pharmaceuticals did not select for significant genetic changes following a 30 day exposure, and no evidence of selection for cross-resistance to antibiotics was observed for population evolved in the presence of ibuprofen in spite of the differential gene expression after exposure to this compound. This work suggests that these non-antibiotic pharmaceuticals, at environmental concentrations, do not select for cross-resistance to antibiotics in E. coli.

microbiology↗

bioRxiv2021

Generation of iPSC lines with high cytogenetic stability from peripheral blood mononuclear cells (PBMCs)

The utility of human induced pluripotent stem cells (hiPSCs) is contingent upon genomic integrity and stability. Recurrent genomic aberrations have been observed in human iPSC lines upon long-term culture, [~]10-25% demonstrate karyotype abnormalities. We describe a new and reliable non-integrating episomal plasmid reprogramming method for fresh (unexpanded) peripheral blood mononuclear cells (PBMC) into iPSCs (PBMC-iPSCs). PBMC-iPSCs produced using this method have a superior chromosome-level karyotype stability rate ([~]5% abnormality rate for all chromosomes; 2.8% for autosomes). After extended culture PBMC-iPSCs maintain a low rate of abnormalities (2% for autosomes). Deep coverage whole genome sequencing in a subset of PBMC-iPSC lines showed no shared single nucleotide polymorphisms (SNPs) or structural variants are introduced during reprogramming and maintenance of PBMC-iPSCs. iPSCs reprogrammed from unexpanded PBMCs have consistently high cytogenetic stability and minimal genomic aberrations, suggesting this method is highly suited for iPSCs in research and therapeutic clinical applications.

cell biology↗

bioRxiv2020

G4C2 repeat RNA mediates the disassembly of the nuclear pore complex in C9orf72 ALS/FTD

Nucleocytoplasmic transport, controlled by the nuclear pore complex, has recently emerged as a pathomechanism underlying neurodegenerative diseases including C9orf72 ALS/FTD. However, little is known about the underlying molecular events and the underlying biology in human neurons. Using super resolution structured illumination microscopy of twenty three nucleoporins in nuclei from C9orf72 iPSC derived neurons and postmortem human tissue we identify a unique subset of eight nucleoporins lost from human neuronal nuclei. POM121, an integral transmembrane nucleoporin, appears to coordinate the composition of the nucleoporins within human neuronal nuclei ultimately impacting nucleocytoplasmic transport, and subsequent cellular toxicity in C9orf72 iPSNs. These data suggest that POM121 is a critical nucleoporin in the maintenance of the nuclear localization of specific nucleoporins in human neurons. Moreover, loss of nuclear POM121, as a result of expanded C9orf72 ALS/FTD repeat RNA, initiates a pathological cascade affecting nucleoporin composition within neuronal nuclei, nuclear pore complex function, and overall downstream neuronal survival.

neuroscience↗

bioRxiv2020

Generation of twenty four induced pluripotent stem cell lines from twenty four members of the Lothian Birth Cohort 1936

Cognitive decline is among the most feared aspects of ageing. We have generated induced pluripotent stem cells (iPSCs) from 24 people from the Lothian Birth Cohort 1936, whose cognitive ability was tested in childhood and in older age. Peripheral blood mononuclear cells (PBMCs) were reprogrammed using non-integrating oriP/EBNA1 backbone plasmids expressing six iPSC reprogramming factors (OCT3/4 (POU5F1), SOX2, KLF4, L-Myc, shp53, Lin28, SV40LT). All lines demonstrated STR matched karyotype and pluripotency was validated by multiple methods. These iPSC lines are a valuable resource to study molecular mechanisms underlying individual differences in cognitive ageing and resilience to age-related neurodegenerative diseases.

cell biology↗

bioRxiv2018

Semi-in vitro Reconstitution of Roseocin, a Two-Component Lantibiotic from an Actinomycete

Lantibiotics are lanthionine containing peptide natural products that belong to the class of ribosomally synthesized and post-translationally modified peptides (RiPPs). Recent expansion in the availability of microbial genomic data and in silico analysis tools have accelerated the discovery of these promising alternatives to antibiotics. Following the genome-mining approach, a biosynthetic gene cluster for a putative two-component lantibiotic roseocin was identified in the genome of an Actinomycete, Streptomyces roseosporus NRRL 11379. Post-translationally modified lanthipeptides of this cluster were obtained by heterologous expression of the genes in E. coli, and were in vitro reconstituted to their bioactive form. The two lanthipeptides displayed antimicrobial activity against Gram-positive bacteria only synergistically, a property reminiscent of two-component lantibiotics. Structural analysis of the -component identified a disulfide bridge flanking two of its four thioether bridges and the {beta}-component having six thioether bridges with its C-terminus extended than the previously known two-component lantibiotics.

biochemistry↗

bioRxiv2017

A Comparison of mRNA Sequencing with Random Primed and 3’-Directed Libraries

Deep mRNA sequencing (mRNAseq) is the state-of-the-art for whole transcriptome measurements. A key step is creating a library of cDNA sequencing fragments from RNA. This is generally done by random priming, creating multiple sequencing fragments along the length of each transcript. A 3 end-focused library approach cannot detect differential splicing, but has potentially higher throughput at lower cost (~10-fold lower), along with the ability to improve quantification by using transcript molecule counting with unique molecular identifiers (UMI) to correct for PCR bias. Here, we compare implementation of such a 3-digital gene expression (3-DGE) approach with \"conventional\" random primed mRNAseq, which has not yet been done. We find that while conventional mRNAseq detects ~15% more genes, the resulting lists of differentially expressed genes and therefore biological conclusions and gene signatures are highly concordant between the two techniques. We also find good quantitative agreement on the level of individual genes between the two techniques in terms of both read counts and fold change between two conditions. We conclude that for high-throughput applications, the potential cost savings associated with the 3-DGE approach are a very reasonable tradeoff for modest reduction in sensitivity and inability to observe alternative splicing, and should enable much larger scale studies focused on not only differential expression analysis, but also quantitative transcriptome profiling. The computational scripts and programs, along with experimental standard operating procedures used in our pipeline presented here, are freely available on our website (www.dtoxs.org).

genomics↗