Biology subjects

Das, R.

Publications and source records attributed to Das, R..

At least 91 records · Page 5Linked to original sources

bioRxiv2022

Genome-first detection of emerging resistance to novel therapeutic agents for SARS-CoV-2

Some COVID-19 patients are unable to clear their infection or are at risk of severe disease, requiring treatment with neutralising monoclonal antibodies (nmAb) and/or antivirals. The rapid roll-out of novel therapeutics means there is limited understanding of the likely genetic barrier to drug resistance. Unprecedented genomic surveillance of SARS-CoV-2 in the UK has enabled a genome-first approach to the detection of emerging drug resistance. Here we report the accrual of mutations in Delta and Omicron cases treated with casirivimab+imdevimab and sotrovimab respectively. Mutations occur within the epitopes of the respective nmAbs. For casirivimab+imdevimab these are present on contiguous raw reads, simultaneously affecting both components. Using surface plasmon resonance and pseudoviral neutralisation assays we demonstrate these mutations reduce or completely abrogate antibody affinity and neutralising activity, suggesting they are driven by immune evasion. In addition, we show that some mutations also reduce the neutralising activity of vaccine-induced serum.

genetics↗

bioRxiv2022

Further antibody escape by Omicron BA.4 and BA.5 from vaccine and BA.1 serum

The Omicron lineage of SARS-CoV-2, first described in November 2021, spread rapidly to become globally dominant and has split into a number of sub-lineages. BA.1 dominated the initial wave but has been replaced by BA.2 in many countries. Recent sequencing from South Africas Gauteng region uncovered two new sub-lineages, BA.4 and BA.5 which are taking over locally, driving a new wave. BA.4 and BA.5 contain identical spike sequences and, although closely related to BA.2, contain further mutations in the receptor binding domain of spike. Here, we study the neutralization of BA.4/5 using a range of vaccine and naturally immune serum and panels of monoclonal antibodies. BA.4/5 shows reduced neutralization by serum from triple AstraZeneca or Pfizer vaccinated individuals compared to BA.1 and BA.2. Furthermore, using serum from BA.1 vaccine breakthrough infections there are likewise, significant reductions in the neutralization of BA.4/5, raising the possibility of repeat Omicron infections.

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↗

bioRxiv2022

Neuroendocrine control of the proteostatic network by HPK-1 delays aging

The nervous system systemically coordinates proteostasis to delay organismal aging. However, the neuronal regulatory mechanisms that coordinate cellular anti-aging programs across tissue and cell-types are relatively unknown. In this work, we identify the homeodomain-interacting protein kinase (HPK-1), a transcriptional cofactor, as a novel neuronal component of the proteostatic network: its overexpression produces a paracrine signal to hyper-induce molecular chaperones and a neuroendocrine signal to induce autophagy in peripheral tissues. Neuronal HPK-1 signaling improves proteostasis in distal tissues through neurotransmitters. These pro-longevity modalities are independently regulated within serotonergic and GABAergic neurons, respectively, through distinct adaptive responses, either of which improve proteostasis in a cell non-autonomous manner. Serotonergic HPK-1 activity amplifies the heat shock response and protects the proteome from acute stress, without altering longevity. Conversely, increased GABAergic HPK-1 activity is sufficient to induce autophagy and extend longevity, without altering acute stress survival. Consistently, GABAergic neurons, but not serotonin, is essential for the cell non-autonomous induction of autophagy by neuronal HPK-1. These findings provide novel insight into how the nervous system partitions and coordinates unique adaptive response pathways to delay organismal aging, and reveals a key role for neuronal HPK-1 in regulating the proteostatic network throughout an intact metazoan animal. Significance StatementAging and the age-associated decline of the proteome is determined in part through neuronal control of evolutionarily conserved transcriptional effectors, which safeguard homeostasis under fluctuating metabolic and stress conditions by regulating an expansive proteostatic network in peripheral tissues. How neuronal signaling mechanisms are primed, relayed through an organism, and specific responses are initiated in receiving cell types remain poorly understood. We have discovered that the Caenorhabditis elegans homeodomain-interacting protein kinase (HPK-1) is a novel transcriptional effector that functions within two distinct neuronal cell-types to non-autonomously regulate divergent components of the proteostatic network to enhance stress resistance, improve proteostasis and delay aging.

genetics↗

bioRxiv2022

nPoRe: n-Polymer Realigner for improved pileup variant calling

Despite recent improvements in nanopore basecalling accuracy, germline variant calling of small insertions and deletions (INDELs) remains poor. Although precision and recall for single nucleotide polymorphisms (SNPs) now regularly exceeds 99.5%, INDEL recall at relatively high coverages (85x) remains below 80% for standard R9.4.1 flow cells [22, 23, 31]. Current nanopore variant callers work in two stages: an efficient pileup-based method identifies candidates of interest, and then a more expensive full-alignment model provides the final variant calls. Most false negative INDELs are lost during the first (pileup-based) step, particularly in low-complexity repeated regions. We show that read phasing and realignment can recover a significant portion of INDELs lost during this stage. In particular, we extend Needleman-Wunsch affine gap alignment by introducing new gap penalties for more accurately aligning repeated n-polymer sequences such as homopolymers (n = 1) and tandem repeats (2 [≤] n [≤] 6). On our dataset with 60.6x coverage, haplotype phasing improves INDEL recall in all evaluated high confidence regions from 63.76% to 70.66% and then nPoRe realignment improves it further to 73.04%, with no loss of precision.

bioinformatics↗

bioRxiv2022

Mechanical feedback controls the emergence of dynamical memory in growing tissue monolayers

The growth of a tissue, which depends on cell-cell interactions and biologically relevant process such as cell division and apoptosis, is regulated by a mechanical feedback mechanism. We account for these effects in a minimal two-dimensional model in order to investigate the consequences of mechanical feedback, which is controlled by a critical pressure, pc. A cell can only grow and divide if the pressure it experiences, due to interaction with its neighbors, is less than pc. Because temperature is an irrelevant variable in the model, the cell dynamics is driven by self-generated active forces (SGAFs) that are created by cell division. It is shown that even in the absence of intercellular interactions, cells undergo diffusive behavior. The SGAF-driven diffusion is indistinguishable from the well-known dynamics of a free Brownian particle at a fixed finite temperature. When intercellular interactions are taken into account, we find persistent temporal correlations in the force-force autocorrelation function (FAF) that extends over timescale of several cell division times. The time-dependence of the FAF reveals memory effects, which increases as pc increases. The observed non-Markovian effects emerge due to the interplay of cell division and mechanical feedback, and is inherently a non-equilibrium phenomenon.

biophysics↗

bioRxiv2021

The Biomechanics of the Locust Ovipositor Valves: a Unique Digging Apparatus

The female locust has a unique mechanism for digging in order to deposit its eggs deep in the ground. It utilizes two pairs of sclerotized valves to displace the granular matter, while extending its abdomen as it propagates underground. This ensures optimal conditions for the eggs to incubate, and provides them with protection from predators. Here, two major axes of operation of the digging valves are identified, one in parallel to the propagation direction of the ovipositor, and one perpendicular to it. The direction-dependent biomechanics of the locust major, dorsal digging valves are quantified and analyzed, under forces in the physiological range and beyond, considering hydration level, as well as the females age, or sexual maturation state. Our findings reveal that the responses of the valves to compression forces in the specific directions change upon sexual maturation to follow their function, and depend on environmental conditions. Namely, in the physiological force range, the valves are resistant to mechanical failure. In addition, mature females, which lay eggs, have stiffer valves, up to roughly nineteen times the stiffness of the pre-mature locusts. The valves are stiffer in the major working direction, corresponding to soil shuffling and compression, compared to the direction of propagation. Hydration of the valves reduces their stiffness but increases their resilience against failure. These findings provide mechanical and materials guidelines for the design of novel non-drilling excavating tools, including 3D-printed anisotropic materials based on composites. Statement of significanceThe female locust lay its eggs underground in order to protect them from predators and to provide them with optimal conditions for hatching. In order to dig into the ground, it uses two pairs of valves: The ventral pair is plugged as a wedge, while the dorsal pair performs the digging of the oviposition tunnel. We study the mechanical response of the digging valves, depending on age, hydration level and direction of operation. Our findings show that during the course of roughly two weeks in the life of the adult female, the digging valves become up to nineteen-fold stiffer against failure, in order to fulfill their function as diggers. While hydration reduces the stiffness, it also increases the resilience against failure and renders the valves unbreakable within the estimated physiological force range and beyond. The digging valves are consistently stiffer in the digging direction than in the perpendicular direction, implying on their form-follows-function design. Graphical Abstract O_FIG_DISPLAY_L [Figure 1] M_FIG_DISPLAY C_FIG_DISPLAY

bioengineering↗

bioRxiv2021

An evolutionary divergent thermodynamic brake in ZAP-70 finetunes the kinetic proofreading in T cell

T cell signaling starts with assembling several tyrosine kinases and adaptor proteins to the T cell receptor (TCR), following the antigen binding. The lifetime of the TCR: antigen complex and the time delay between the recruitment and activation of each kinase determines the T cell response. The mechanism by which the time delays are implemented in TCR signaling is not fully understood. Combining experiments and kinetic modeling, we here report a thermodynamic-brake in the regulatory module of ZAP-70, which determines the ligand selectivity, and may delay the ZAP-70 activation in TCR. Phylogenetic analysis revealed that the evolution of the thermodynamic-brake coincides with the divergence of the adaptive immune system to the cell-mediated and humoral responses. Paralogous kinase Syk expressed in B cells, does not possess such a functional thermodynamic brake, which may explain higher basal activation and lack of ligand selectivity by Syk.

biochemistry↗

bioRxiv2021

Community science designed ribosomes with beneficial phenotypes

Functional design of ribosomes with mutant ribosomal RNA (rRNA) could expand opportunities for understanding molecular translation, building cells from the bottom-up, and engineering ribosomes with altered capabilities. However, such efforts have been hampered by cell viability constraints, an enormous combinatorial sequence space, and limitations on large-scale, 3D design of RNA structures and functions. To address these challenges, we developed an integrated community science and experimental screening approach for rational design of ribosomes. This approach couples Eterna, an online video game that crowdsources RNA sequence design to community scientists in the form of puzzles, with in vitro ribosome synthesis, assembly, and translation in multiple design-build-test-learn cycles. We applied our framework to discover mutant rRNA sequences that improve protein synthesis in vitro and cell growth in vivo, relative to wild type ribosomes, under diverse environmental conditions. This work provides new insights into ribosome rRNA sequence-function relationships, with implications for synthetic biology.

synthetic biology↗

bioRxiv2021

Redesigning the Eterna100 for the Vienna 2 folding engine

The rational design of RNA is becoming important for rapidly developing technologies in medicine and biochemistry, spurring development of numerous RNA secondary structure design algorithms and benchmarks to evaluate their performance. However, the problem of RNA design is dependent upon the reverse problem of RNA structure prediction through "folding engines" that predict structure from sequence. We hypothesized that differences in RNA folding engines could impact design algorithms, and recruited an online community of RNA design experts to modify the widely-used RNA secondary structure design benchmark, Eterna100, to address unsolvability of some cases when changing the folding engine used (Vienna 1.8 updated to Vienna 2.6). We tested this new Eterna100-V2 benchmark with five RNA design algorithms, and found that while overall rankings remained similar, the performance of RNA design algorithms that depended on folding engines in their training did indeed depend on which underlying parameter set was used in training. This work demonstrates that the design "difficulty" of RNA structures is intrinsically linked to thermodynamic models, and suggests that future RNA design algorithms that are agnostic to thermodynamic models will result in optimal performance and development. Eterna100-V1 and Eterna100-V2 benchmarks and example solutions are freely available at https://github.com/eternagame/eterna100-benchmarking. Author SummaryDesigning RNA molecules that fold to a desired target structure is an algorithmic problem gathering increasing attention due to the emergence of RNA-based therapies and the need for rational design of RNAs. The Eterna100 dataset, a collection of target structures with increasing design difficulty, designed and selected by players of the crowdsourced RNA game Eterna, has been widely used to benchmark RNA design algorithms. However, these puzzles were originally developed using the now-deprecated version 1 of the ViennaRNA folding engine. In this manuscript, we introduce an updated benchmark, called the Eterna100-V2. We found that nineteen puzzles using Vienna 1 were unsolvable in Vienna 2, but that Eterna participants were able to re-design the puzzles with minimal modifications to make them solvable in Vienna 2. We confirmed that the rankings of 5 RNA design algorithms remained consistent between Eterna100-V1 and -V2. However, discrepancies in performance from algorithms that relied on thermodynamic models in their training suggest that algorithms will benefit from being agnostic to thermodynamic models as these models continue to improve.

bioinformatics↗

bioRxiv2021

Identification and targeting of a pan-genotypic influenza A virus RNA structure that mediates packaging and disease.

Currently approved anti-influenza drugs target viral proteins, are subtype limited, and are challenged by rising antiviral resistance. To overcome these limitations, we sought to identify a conserved essential RNA secondary structure within the genomic RNA predicted to have greater constraints on mutation in response to therapeutics targeting this structure. Here, we identified and genetically validated an RNA stemloop structure we termed PSL2, which serves as a packaging signal for genome segment PB2 and is highly conserved across influenza A virus (IAV) isolates. RNA structural modeling rationalized known packaging-defective mutations and allowed for predictive mutagenesis tests. Disrupting and compensating mutations of PSL2s structure give striking attenuation and restoration, respectively, of in vitro virus packaging and mortality in mice. Antisense Locked Nucleic Acid oligonucleotides (LNAs) designed against PSL2 dramatically inhibit IAV in vitro against viruses of different strains and subtypes, possess a high barrier to the development of antiviral resistance, and are equally effective against oseltamivir carboxylate-resistant virus. A single dose of LNA administered 3 days after, or 14 days before, a lethal IAV inoculum provides 100% survival. Moreover, such treatment led to the development of strong immunity to rechallenge with a ten-fold lethal inoculum. Together, these results have exciting implications for the development of a versatile novel class of antiviral therapeutics capable of prophylaxis, post-exposure treatment, and "just-in-time" universal vaccination against all IAV strains, including drug-resistant pandemics. One Sentence SummaryTargeting a newly identified conserved RNA structure in the packaging signal region of influenza segment PB2 abrogates virus production in vitro and dramatically attenuates disease in vivo.

microbiology↗

bioRxiv2021

Microbial byproducts determine reproductive fitness of free-living and parasitic nematodes

A distinguishing feature of Trichuris nematodes is that these parasitic worms reproduce within the digestive tracts of humans and other mammalian hosts shedding thousands of eggs daily, facilitating their sustained presence in the environment and hampering eradication efforts. Although this aspect of the lifecycle places Trichuris in a microbiota-rich environment, metabolic byproducts of bacteria that facilitate the reproductive development of parasites are unknown. Here, we employ a pipeline using the well-characterized free-living nematode C. elegans to identify microbial factors with conserved roles in the reproduction of nematodes. A screen for E. coli mutants that impair C. elegans fertility identified genes in fatty acid biosynthesis and ethanolamine utilization pathways, including fabH and eutN. Trichuris muris eggs displayed defective hatching in the presence of E. coli deficient in fabH or eutN due to reduction in arginine or elevated levels of aldehydes, respectively. Remarkably, T. muris reared in gnotobiotic mice colonized with these E. coli mutants displayed profound abnormalities including morphological defects and a failure to lay viable eggs. These findings indicate that microbial byproducts mediate evolutionarily conserved transkingdom interactions that impact the reproductive fitness of distantly-related nematodes.

microbiology↗

bioRxiv2021

A Combined Spectroscopic and Molecular Modeling Study on Structure-Function-Dynamics under Chemical Modification: Alpha-Chymotrypsin with Formalin Preservative

Enzyme conformations can be altered via modification of its amino acid residues, side chains and large-scale domain modifications, which are closely linked to its function. Herein, we have addressed the role of residue modification in catalytic activity and molecular recognition of an enzyme alpha-chymotrypsin (CHT) in presence of covalent cross-linker formalin. Optical spectroscopy studies exhibit reduced catalytic activity of the enzyme with increased formalin concentration. Polarization gated anisotropy studies of a fluorophore 8-anilino-1- napthelenesulfonic acid (ANS) in CHT show a dip rise pattern in presence of formalin which is consistent with the generation of multiple ANS binding sites in the enzyme owing to modifications of its local amino acid residues. Molecular docking study on minimal local residue modifications in CHT reveals formation of a stable enzyme-substrate complex even with the serine-histidine cross-linked enzyme which prohibits product formation giving rise to reduced catalytic activity.

biophysics↗

bioRxiv2021

G-Protein coupled Purinergic P2Y12 receptor interacts and internalizes TauRD-mediated by membrane-associated actin cytoskeleton remodelling in microglia

In Alzheimers disease, the microtubule-associated protein, Tau misfolds to form aggregates and filaments in the intra- and extracellular region of neuronal cells. Microglial cells are the resident brain macrophage cells that are involved in constant surveillance and are activated by the extracellular deposits. Purinergic receptors are involved in chemotactic migration of microglial cells towards the site of inflammation. In our recent study, we found that microglial P2Y12 receptor has been involved in phagocytosis of full-length Tau species such as monomers, oligomers and aggregates by actin-driven chemotaxis. In this study, we have showed the interaction of repeat-domain of Tau (TauRD) with microglial P2Y12 receptor and analysed the corresponding residues for interaction by various in-silico approaches. In cellular studies, TauRD was found to interact with microglial P2Y12R and induces its cellular expression as confirmed by coimmunoprecipitation and western blot analysis respectively. Similarly, immunofluorescence microscopic studies emphasized that TauRD were phagocytosed by microglial P2Y12R via the membrane-associated actin remodelling as filopodia extension. Furthermore, the P2Yl2R-mediated TauRD internalization has activated the microglia with an increase in the Iba1 level and TauRD become accumulated at peri-nuclear region as localized with Iba1. Altogether, microglial P2Y12R interacts with TauRD and mediates directed migration and activation for its internalization.

neuroscience↗

bioRxiv2021

Ultra-rapid somatic variant detection via real-time threshold sequencing

Molecular markers are becoming increasingly important for cancer diagnosis, proper clinical trial enrollment, and even surgical decision making, motivating ultra-rapid, intraoperative variant detection. Sequencing-based detection is considered the gold standard approach, but typically takes hours to perform. In this work, we present Threshold Sequencing, a methodology for designing protocols for targeted variant detection on real-time sequencers with a minimal time to result. Threshold Sequencing analytically identifies a time-optimal threshold to stop target amplification and begin sequencing. To further reduce diagnostic time, we explore targeted Loop-mediated Isothermal Amplification (LAMP) and design a LAMP-specific bioinformatics tool--LAMPrey--to process sequenced LAMP product. LAMPreys concatemer aware alignment algorithm is designed to maximize recovery of diagnostically relevant information leading to a more rapid detection versus standard read alignment approaches. Coupled with time-optimized DNA extraction and library preparation, we demonstrate confirmation of a hot-spot mutation (250x support) from tumor tissue in less than 30 minutes.

bioinformatics↗

bioRxiv2021

Ensuring scientific reproducibility in bio-macromolecular modeling via extensive, automated benchmarks

Each year vast international resources are wasted on irreproducible research. The scientific community has been slow to adopt standard software engineering practices, despite the increases in high-dimensional data, complexities of workflows, and computational environments. Here we show how scientific software applications can be created in a reproducible manner when simple design goals for reproducibility are met. We describe the implementation of a test server framework and 40 scientific benchmarks, covering numerous applications in Rosetta bio-macromolecular modeling. High performance computing cluster integration allows these benchmarks to run continuously and automatically. Detailed protocol captures are useful for developers and users of Rosetta and other macromolecular modeling tools. The framework and design concepts presented here are valuable for developers and users of any type of scientific software and for the scientific community to create reproducible methods. Specific examples highlight the utility of this framework and the comprehensive documentation illustrates the ease of adding new tests in a matter of hours.

bioinformatics↗

bioRxiv2021

Combinatorial optimization of mRNA structure, stability, and translation for RNA-based therapeutics

Therapeutic mRNAs and vaccines are being developed for a broad range of human diseases, including COVID-19. However, their optimization is hindered by mRNA instability and inefficient protein expression. Here, we describe design principles that overcome these barriers. We develop a new RNA sequencing-based platform called PERSIST-seq to systematically delineate in-cell mRNA stability, ribosome load, as well as in-solution stability of a library of diverse mRNAs. We find that, surprisingly, in-cell stability is a greater driver of protein output than high ribosome load. We further introduce a method called In-line-seq, applied to thousands of diverse RNAs, that reveals sequence and structure-based rules for mitigating hydrolytic degradation. Our findings show that "superfolder" mRNAs can be designed to improve both stability and expression that are further enhanced through pseudouridine nucleoside modification. Together, our study demonstrates simultaneous improvement of mRNA stability and protein expression and provides a computational-experimental platform for the enhancement of mRNA medicines.

molecular biology↗

bioRxiv2021

Comparative in vitro antioxidant and antibacterial activities of leaf extract fractions of Crimson bottlebrush, Callistemon citrinus (Curtis.) Skeels

ObjectivesThe present study aimed to analyze a comparative in vitro free radical scavenging and antibacterial potentials of leaf aqueous and the successive extract fractions of Callistemon citrinus. MethodsFor in vitro antioxidant activity assessments, 1,1-diphenyl-2-picrylhydrazyl (DPPH) free radical scavenging activity, Fe3+ ion reducing antioxidant power (FRAP) assay, and total antioxidant capacity of the extracts were tested. Antibacterial potentials were tested through Agar well diffusion method using both gram-positive and gram-negative bacterial strains. ResultsData indicate the polar successive aqueous fraction (AQF) possesses the highest free radicals scavenging capacity, with lowest EC50 (the required extract concentration to scavenge half of the free radicals) for DPPH and FRAP assays, and contains the highest total phenolics (308.2{+/-}5.9 {micro}g tannic acid equivalent/mg dry extract, DE), flavonoids (516.7{+/-}3.5 {micro}g quercetin equivalent/mg DE), total antioxidant capacity (441.48{+/-}12.8 {micro}g ascorbic acid equivalent/mg DE). Phenolics and flavonoids contents were positively correlated with the in vitro antioxidant activities. The antibacterial study indicates the petroleum ether and chloroform are suitable solvents for extracting antibacterial phytochemicals from C. citrinus leaves that are effective against both gram-positive and negative bacterial strains. ConclusionThe most polar fraction i.e. the successive aqueous extract fraction of C. citrinus leaves exhibited the highest antioxidant activities while the most non-polar petroleum ether extract fraction showed the highest antibacterial potentials thus these extract fractions might have therapeutic importance.

pharmacology and toxicology↗