Biology subjects

Uppal, S.

Publications and source records attributed to Uppal, S..

9 recordsLinked to original sources

bioRxiv2026

Structure of a secreted lancelet carotenoid cleavage dioxygenase (CCD), from an ancestral CCD clade

Carotenoid cleavage dioxygenases (CCDs) are a superfamily of non-heme iron-containing enzymes ubiquitous across all kingdoms of life. Previously, we identified a new subfamily of CCDs termed beta-carotene oxygenase-like (BCOL) proteins, a chordate expansion of an ancestral sister clade of metazoan CCDs, which are widely distributed in marine invertebrates, including mollusks and lancelets. Here we present the three-dimensional structure of a representative BCOL protein from the lancelet Branchiostoma floridae (BCOLg) at 2.4 [A] resolution and its functional properties. BCOLg symmetrically cleaves {beta}-carotene into all-trans retinal. RNAseq of wild-caught animals detected six of the seven annotated BCOL transcripts in Branchiostoma gut tissue. We expressed and crystallized recombinant BCOLg. BCOLg is heavily glycosylated, with four N-linked glycosylation sites, and adopts the seven-bladed propeller fold characteristic of the superfamily, with one disulfide bond contributing to structural stability. BCOLg and several other BCOL proteins contain Asp substitutions at two of three Glu positions found in other CCDs, stabilizing three of four His residues involved in Fe2+ coordination. Notably, BCOLg lacks the canonical -PDPCK- motif characteristic of other metazoan CCDs.

biochemistry↗

bioRxiv2026

Community-wide adaptive evolution of gut bacteria during dietary transitions

Diet exerts selection on bacterial species within the mammalian gut microbiota1,2,3,4,5, but the extent, modes, genomic bases, and repeatability of gut bacterial adaptive evolution in response to dietary variation within hosts are poorly understood. Here, we show that within-host dietary transitions drive ubiquitous and highly reproducible adaptive evolution of gut bacterial populations. We deeply profiled allele-frequency dynamics in gut microbiota of an experimental cohort of litter-matched wild-derived isogenic mice either transitioned from low-fat/high-carbohydrate diets to high-fat/low-carbohydrate diets or maintained on their ancestral low-fat/high-carbohydrate diet. Dietary transitions induced significantly parallel allele-frequency changes across replicates in 61/62 core gut bacterial species within five months, whereas 0/62 species displayed significant across-replicate parallelism in the control group, indicating deterministic adaptive evolutionary responses across the gut microbiota. Adaptation proceeded through multiple modes, including strain sorting and gene-wise responses. The latter were enriched in a subset of gene families and functions across bacterial species, including invertases. Culturing revealed an invertible DNA switch in Phocaeicola sartorii that, in all replicates transitioned to high-fat/low-carbohydrate diets, localized a promoter sequence upstream of cell-surface glycan foraging genes. These findings elucidate widespread gut bacterial adaptive evolution mediated by within-host dietary variation.

evolutionary biology↗

bioRxiv2024

A granular view of the traits controlling soil bacterial community reconstruction following a prescribed burn

Prescribed burns and wildfires both cause transient reduction of soil microbiome diversity, which has wide-ranging functions in soil and plant health. However, a genetic basis for understanding the mechanisms behind post-fire microbiome recovery is not well-established. Here, we conducted prescribed burns in multiple plots with paired unburned controls, at two prairie locations, sampling each over 5 months. We assembled >300 bacterial genomes that were conserved in both time and space, then identified genomes that were either enriched or depleted post-fire compared to timepoint controls. On a metagenome level, burned and unburned samples were functionally equivalent, but on a species level we determined that post- fire survival is more nuanced than possession of previously hypothesized pyrophilous traits. Our study therefore advances the understanding of how both function and taxonomy contribute to success during the reconstruction of a complex soil microbiome.

microbiology↗

bioRxiv2024

Repeated horizontal acquisition of lagriamide-producing symbionts in Lagriinae beetles

Microbial symbionts associate with multicellular organisms on a continuum from facultative associations to mutual codependency. In some of the oldest intracellular symbioses there is exclusive vertical symbiont transmission, and co-diversification of symbiotic partners over millions of years. Such symbionts often undergo genome reduction due to low effective population sizes, frequent population bottlenecks, and reduced purifying selection. Here, we describe multiple independent acquisition events of closely related defensive symbionts followed by genome erosion in a group of Lagriinae beetles. Previous work in Lagria villosa revealed the dominant genome-eroded symbiont of the genus Burkholderia produces the antifungal compound lagriamide and protects the beetles eggs and larvae from antagonistic fungi. Here, we use metagenomics to assemble 11 additional genomes of lagriamide-producing symbionts from seven different host species within Lagriinae from five countries, to unravel the evolutionary history of this symbiotic relationship. In each host species, we detected one dominant genome-eroded Burkholderia symbiont encoding the lagriamide biosynthetic gene cluster (BGC). Surprisingly, however, we did not find evidence for host-symbiont co-diversification, or for a monophyly of the lagriamide-producing symbionts. Instead, our analyses support at least four independent acquisition events of lagriamide-encoding symbionts and subsequent genome erosion in each of these lineages. By contrast, a clade of plant-associated relatives retained large genomes but secondarily lost the lagriamide BGC. In conclusion, our results reveal a dynamic evolutionary history with multiple independent symbiont acquisitions characterized by high degree of specificity. They highlight the importance of the specialized metabolite lagriamide for the establishment and maintenance of this defensive symbiosis.

genomics↗

bioRxiv2023

Autometa 2: A versatile tool for recovering genomes from highly-complex metagenomic communities

In 2019, we developed Autometa, an automated binning pipeline that is able to effectively recover metagenome-assembled genomes from complex environmental and non-model host-associated microbial communities. Autometa has gained widespread use in a variety of environments and has been applied in multiple research projects. However, the genome-binning workflow was at times overly complex and computationally demanding. As a consequence of Autometas diverse application, non-technical and technical researchers alike have noted its burdensome installation and inefficient as well as error-prone processes. Moreover its taxon-binning and genome-binning behaviors have remained obscure. For these reasons we set out to improve its accessibility, efficiency and efficacy to further enable the research community during their exploration of Earths environments. The highly augmented Autometa 2 release, which we present here, has vastly simplified installation, a graphical user interface and a refactored workflow for transparency and reproducibility. Furthermore, we conducted a parameter sweep on standardized community datasets to show that it is possible for Autometa to achieve better performance than any other binning pipeline, as judged by Adjusted Rand Index. Improvements in Autometa 2 enhance its accessibility for non-bioinformatic oriented researchers, scalability for large-scale and highly-complex samples and interpretation of recovered microbial communities. Graphical abstractAutometa: An automated taxon binning and genome binning workflow for single sample resolution of metagenomic communities.

bioinformatics↗

bioRxiv2022

Uncovering Lasonolide A biosynthesis using genome-resolved metagenomics

Invertebrates, in particular sponges, have been a dominant source of new marine natural products. For example, lasonolide A (LSA) is a potential anti-cancer molecule isolated from the marine sponge Forcepia sp., with nanomolar growth inhibitory activity and a unique cytotoxicity profile against the National Cancer Institute 60 cell line screen. Here, we identified the putative biosynthetic pathway for LSA. Genomic binning of the Forcepia sponge metagenome revealed a gram-negative bacterium belonging to the phylum Verrucomicrobia as the candidate producer of LSA. Phylogenetic analysis showed this bacterium, herein named Candidatus Thermopylae lasonolidus, only has 88.78% 16S rRNA identity with the closest relative Pedosphaera parvula Ellin514, indicating it represents a new genus. The lasonolide A (las) biosynthetic gene cluster (BGC) was identified as a trans-AT polyketide synthase (PKS) pathway. When compared with its host genome, the las BGC exhibits a significantly different GC content and penta-nucleotide frequency, suggesting a potential horizontal acquisition of the gene cluster. Furthermore, three copies of the putative las pathway were identified in the candidate producer genome. Differences between the three las repeats were observed including the presence of three insertions, two single-nucleotide polymorphisms and the absence of a stand-alone acyl carrier protein in one of the repeats. Even though the Verrucomicrobial producer shows signs of genome-reduction, its genome size is still fairly large (about 5Mbp) and when compared to its closest free-living relative contains most of the primary metabolic pathways, suggesting that it is in the early stages of reduction. ImportanceWhile sponges are valuable sources of bioactive natural products, a majority of these compounds are produced in small amounts by uncultured symbionts, hampering the study and clinical development of these unique compounds. Lasonolide A (LSA), isolated from marine sponge Forcepia sp., is a cytotoxic molecule active at nanomolar concentrations and causes premature chromosome condensation, blebbing, cell contraction and loss of cell adhesion, indicating a novel mechanism of action and making it a potential anti-cancer drug lead. However, its limited supply hampers progression to clinical trials. We investigated the microbiome of Forcepia sp. using culture-independent DNA sequencing to uncover how an uncultured bacterium produces LSA. This provides future opportunities for heterologous expression and cultivation efforts that may minimize LSAs supply problem.

microbiology↗

bioRxiv2022

RNase H1 exists as phase-separated assemblies in association with elongating RNA Polymerase II during transcription

R-loops are three stranded nucleic acid structures consisting of a RNA/DNA hybrid and a single stranded displaced DNA. RNase H1 is an endonuclease which specifically degrades the RNA moiety in RNA-DNA hybrids. Here, we report that RNase H1 interacts with transcription elongation machinery during active transcription in a liquid-liquid phase separation (LLPS) dependent manner. We show that RNase H1 interacts with nascent RNA, and transcription elongation machinery in Hela cells, using in-situ nascent RNA labelling and Proximity ligation assay. Further, RNase H1 was found to exhibit properties of liquid-like condensates both in vitro and in vivo. Interestingly, RNase H1 interaction with elongating RNA Polymerase II can be disrupted by chemicals that perturb LLPS. Importantly, we show that LLPS is important for regulation of R-loop levels in the cell. Based on our results, we propose that RNase H1 exists as phase-separated assemblies in association with elongating RNA Polymerase II during active transcription.

cell biology↗

bioRxiv2021

Synthesis, Physiochemical and Biological evaluation of Inclusion Complex of Benzyl Isothiocyanate encapsulated in cyclodextrins for triple negative breast cancer

Benzyl isothiocyanate (BITC), an organic dietary compound, is allied with a major role in the potential chemopreventive effects. BITC has acknowledged rising attention as a therapeutic compound to be used in medicine because of its high potency and characteristic biopharmaceutical properties, like high permeability with marginal aqueous solubility. The highly volatile and hydrophobic nature brought a need to provide a suitable delivery-matrix to BITC to exploit its pharmacological potential to the fullest. It has been successfully incorporated in {beta}-CD and HP-{beta}-CD using acoustic forces and thoroughly characterized using UV-vis spectroscopy, FTIR, DSC, TEM, and SAXS. The complexation helped in masking the acute odour, achieving a controlled release of BITC, and made its use viable by prolonging the retention time and thereby sustaining the biological effects. Different models like Higuchi, first-order kinetic decay, Korsmeyer-Peppas model were applied, suggesting a diffusion-controlled mechanism of release. Also, the bioaccessibility and stability of BITC in an in vitro digestion model was evaluated. The main objective of the present work was to systemically study the credibility of BITC-CD complexes in well-established tumor mimicking 2D cell culture models and produce a conclusive report on its chemotherapeutic activity. The in vitro anti-cancer activity of BITC and the formed sonochemical complexes was confirmed by MTT assay and further evaluated using apoptosis assay and production of ROS like moieties. Cell cycle analysis was done to evaluate the growth inhibitory mechanism of BITC. Strikingly, BITC and its complexes showcased ROS generation and lysosome-mediated cell death. Effect on cell migration was assessed using wound healing assay. The results promptly suggest the functional efficacy of the CDs in releasing BITC and attest the ability of the complexes to provide alternate to otherwise remedially sparse triple-negative breast cancer.

cancer biology↗

bioRxiv2018

Bromodomain Protein, BRD4, Contributes to the Regulation of Alternative Splicing

Bromodomain protein 4 (BRD4) is an atypical kinase and a histone acetyl transferase (HAT) which plays an important role in chromatin remodeling and early transcriptional elongation. During transcription elongation, BRD4 travels with the elongation complex. Since most of the alternative splicing events take place co-transcriptionally, we asked if BRD4 plays a role in regulation of alternative splicing. We find that distinct patterns of alternative splicing are associated with conditional deletion of BRD4 during thymocyte differentiation in vivo. Similarly, depletion of BRD4 in T-ALL cells alters patterns of splicing. Most of the alternatively spliced events affected by BRD4 are usage of exon skipping. In an established insulin receptor minigene model of splicing, BRD4 over expression modulates alternative splicing. Importantly, as assessed by both immunoprecipitation (IP) and proximity ligation (PLA) assays, BRD4 interacts with components of the splicing machinery. BRD4 also co-localizes on chromatin with one of the splicing regulators. We propose that BRD4 contributes to patterns of alternative splicing through its interaction with the splicing machinery during transcription elongation.\n\nSignificance StatementThe bromodomain protein, BRD4, is a transcriptional and epigenetic regulator that plays a critical role in both cancer and inflammation. It has pleiotropic activities, including chromatin organization, transcriptional pause release and initiation. We now report that it also contributes to the regulation of alternative splicing. Taken together, these findings indicate that BRD4 functions to coordinate the various steps in gene expression.

molecular biology↗