Biology subjects

Raman, S.

Publications and source records attributed to Raman, S..

At least 37 records · Page 2Linked to original sources

bioRxiv2024

Systematic genome-wide discovery of host factors governing bacteriophage infectivity

Bacterial host factors regulate the infection cycle of bacteriophages. Except for some well-studied host factors (e.g., receptors or restriction-modification systems), the contribution of the rest of the host genome on phage infection remains poorly understood. We developed PHAGEPACK, a pooled assay that systematically and comprehensively measures each host genes impact on phage fitness. PHAGEPACK combines CRISPR interference with phage packaging to link host perturbation to phage fitness during active infection. Using PHAGEPACK, we constructed a genome-wide map of genes impacting T7 phage fitness in permissive E. coli, revealing pathways previously unknown to affect phage packaging. When applied to the non-permissive E. coli O121, PHAGEPACK identified pathways leading to host resistance; their removal increased phage susceptibility up to a billion-fold. Bioinformatic analysis indicates phage genomes carry homologs or truncations of key host factors, potentially for fitness advantage. In summary, PHAGEPACK offers valuable insights into phage-host interactions, phage evolution, and bacterial resistance.

synthetic biology↗

bioRxiv2024

Highly multiplexed design of an allosteric transcription factor to sense novel ligands

Allosteric transcription factors (aTF), widely used as biosensors, have proven challenging to design for detecting novel molecules because mutation of ligand-binding residues often disrupts allostery. We developed Sensor-seq, a high-throughput platform to design and identify aTF biosensors that bind to non-native ligands. We screened a library of 17,737 variants of the aTF TtgR, a regulator of a multidrug exporter, against six non-native ligands of diverse chemical structures - four derivatives of the cancer therapeutic tamoxifen, the antimalarial drug quinine, and the opiate analog naltrexone - as well as two native flavonoid ligands, naringenin and phloretin. Sensor-seq identified novel biosensors for each of these ligands with high dynamic range and diverse specificity profiles. The structure of a naltrexone-bound design showed shape-complementary methionine-aromatic interactions driving ligand specificity. To demonstrate practical utility, we developed cell-free detection systems for naltrexone and quinine. Sensor-seq enables rapid, scalable design of new biosensors, overcoming constraints of natural biosensors.

synthetic biology↗

bioRxiv2023

Serotonin Reduces Belief Stickiness

Serotonin fosters cognitive flexibility, but how, exactly, remains unclear. We show that serotonin reduces belief stickiness: the tendency to get "stuck" in a belief about the state of the world despite incoming contradicting evidence. Participants performed a task assessing belief stickiness in a randomized, double-blind, placebo-controlled study using a single dose of the selective serotonin reuptake inhibitor (SSRI) escitalopram. In the escitalopram group, higher escitalopram plasma levels reduced belief stickiness more, resulting in better inference about the state of the world. Moreover, participants with sufficiently high escitalopram plasma levels had less belief stickiness, and therefore better state inference, than participants on placebo. Exaggerated belief stickiness is exemplified by obsessions: "sticky" thoughts that persist despite contradicting evidence. Indeed, participants with more obsessions had greater belief stickiness, and therefore worse state inference. The opposite relations of escitalopram and obsessions with belief stickiness may explain the therapeutic effect of SSRIs in obsessive-compulsive disorder.

neuroscience↗

bioRxiv2023

Bacteriophage-host interactions in microgravity onboard the International Space Station

Bacteriophage-host interactions play a fundamental role in shaping microbial ecosystems. While extensively studied on Earth, their behavior in microgravity remains largely unexplored. Here, we report the dynamics between T7 bacteriophage and E. coli in microgravity aboard the International Space Station (ISS). Phage activity was initially delayed in microgravity but ultimately successful. We identified de novo mutations in both phage and bacteria that enhanced fitness in microgravity. Deep mutational scanning of the phage receptor binding domain revealed striking differences in the number, position, and mutational preferences between terrestrial and microgravity conditions, reflecting underlying differences in bacterial adaptation. Combinatorial libraries informed by microgravity selections yielded T7 variants capable of productively infecting uropathogenic E. coli resistant to wild-type T7 under terrestrial conditions. These findings help lay the foundation for future research on the impact of microgravity on phage-host interactions and microbial communities and the terrestrial benefits of this research.

synthetic biology↗

bioRxiv2023

Discovering chromatin dysregulation induced by protein-coding perturbations at scale

Although population-scale databases have expanded to millions of protein-coding variants, insight into variant mechanisms has not kept pace. We present PROD-ATAC, a high-throughput method for discovering the effects of protein-coding variants on chromatin. A pooled library of variants is expressed in a disease-agnostic cell line, and single-cell ATAC resolves each variants effect on chromatin. Using PROD-ATAC, we characterized the effects of >100 oncofusions (a class of cancer-causing chimeric proteins) and controls and revealed that pioneer activity is a common feature of fusions spanning an enormous range of fusion frequencies. Further, fusion-induced dysregulation can be context-agnostic as observed mechanisms often overlapped with cancer and cell-type specific prior knowledge. We also showed that gain-of-function pioneering is common among oncofusions. This work provides a global view of fusion-induced chromatin. We uncovered convergent mechanisms among disparate oncofusions and shared modes of dysregulation across different cancers. PROD-ATAC is generalizable to any set of protein-coding variants.

genomics↗

bioRxiv2023

A parametrized two-domain thermodynamic model explains diverse mutational effects on protein allostery

New experimental findings continue to challenge our understanding of protein allostery. Recent deep mutational scanning study showed that allosteric hotspots in the tetracycline repressor (TetR) and its homologous transcriptional factors are broadly distributed rather than spanning well-defined structural pathways as often assumed. Moreover, hotspot mutation-induced allostery loss was rescued by distributed additional mutations in a degenerate fashion. Here, we develop a two-domain thermodynamic model for TetR, which readily rationalizes these intriguing observations. The model accurately captures the in vivo activities of various mutants with changes in physically transparent parameters, allowing the data-based quantification of mutational effects using statistical inference. Our analysis reveals the intrinsic connection of intra- and inter-domain properties for allosteric regulation and illustrate epistatic interactions that are consistent with structural features of the protein. The insights gained from this study into the nature of two-domain allostery are expected to have broader implications for other multidomain allosteric proteins.

biophysics↗

bioRxiv2023

Rhythm generating mechanisms in rat sino-atrial node and ventricle

The major membrane currents responsible for sinoatrial and idioventricular rhythm-generation were studied in isolated rat heart preparations, perfused in Langendorff mode. The rates of whole isolated hearts beating with sinoatrial rhythm decreased with cesium and ivabradine, both blockers of the funny current, and were not affected by nickel, at a dose which blocks T-type calcium current. The sinoatrial rhythm was completely abolished by reduction or removal of sodium from the perfusate (interventions that inhibit calcium-extrusive mode of the sodium/calcium exchanger), or by nifedipine, an L-type calcium channel blocker. Idioventricular rhythm, however, was arrested only by reduction of sodium in the perfusate. Ivabradine reduced the idioventricular rate, nickel did not cause any change, while nifedipine in some cases increased it. The inferences made based on these observations are that INCX and ICaL are obligatory rhythm-generating currents in the sinoatrial node, while INCX is the only obligatory mechanism for an idioventricular rhythm. The funny current is not an obligatory requirement for sinoatrial as well as idioventricular rhythm-generation. However, it enhances the frequency of LCRs. Our results in the isolated whole heart are in corroboration with results from isolated cells.

physiology↗

bioRxiv2023

Deep metagenomic mining reveals bacteriophage sequence motifs driving host specificity

Bacteriophages can adapt to new hosts by altering sequence motifs through recombination or convergent evolution. Where such motifs exist and what fitness advantage they confer remains largely unknown. We report a new method, Metagenomic Sequence Informed Functional Scoring (Meta-SIFT), to discover sequence motifs in metagenomic datasets that can be used to engineer phage activity. Meta-SIFT uses experimental deep mutational scanning data to create sequence profiles to enable deep mining of metagenomes for functional motifs which are otherwise invisible to searches. We experimentally tested over 17,000 Meta-SIFT derived sequence motifs in the receptor-binding protein of the T7 phage. The screen revealed thousands of T7 variants with novel host specificity with functional motifs sourced from distant families. Position, substitution and location preferences dictated specificity across a panel of 20 hosts and conditions. To demonstrate therapeutic utility, we engineered active T7 variants against foodborne pathogen E. coli O121. Meta-SIFT is a powerful tool to unlock the functional potential encoded in phage metagenomes to engineer bacteriophages.

synthetic biology↗

bioRxiv2022

Lectin-Seq: a method to profile lectin-microbe interactions in native communities

Soluble human lectins are critical components of innate immunity. Genetic models suggest lectins influence host-resident microbiota, but their specificity for commensal and mutualist species is understudied. Elucidating lectins roles in regulating microbiota requires understanding which microbial species they bind within native communities. To profile human lectin recognition, we developed Lectin-Seq. We apply Lectin-Seq to human fecal microbiota using mannose-binding lectin (MBL) and intelectin-1 (hItln1). The microbial interactomes of MBL and hItln1 differ in composition and diversity. MBL binding is highly selective for a small subset of species commonly associated with humans. In contrast, hItln1s interaction profile encompasses a broad range of lower-abundance species. Thus, human lectins have evolved to recognize distinct species of commensals, suggesting they directly influence microbiome composition. Lectin-Seq offers a new means of annotating microbial communities. One-Sentence SummarySoluble human lectins bind distinct bacterial species in fecal microbiota.

biochemistry↗

bioRxiv2022

Computation-guided redesign of promoter specificity of a bacterial RNA polymerase

The ability to regulate genetic circuits and metabolic pathways is central to cellular control. The existing toolkit is predominantly comprised of local transcription regulators that are unsuitable for exerting control at a global genome-wide scale. Bacterial sigma factors are ideal global regulators as together they direct the RNA polymerase to thousands of transcription sites. Here, we redesigned the promoter specificity of the E. coli housekeeping sigma factor, sigma-70, toward five orthogonal promoter targets not recognized by the native sigma-70. These orthogonal sigma-70 factors were developed by screening a pooled library of computationally designed variants of the -35 DNA recognition helix, each tailored to a specific target promoter. In the redesigned sigma factors new target-specific interactions facilitate new promoter recognition. Activity of the top performing redesigned sigma-70s varied across the promoter targets and ranged from 17% to 77% of native sigma-70 on its canonical active promoter. These orthogonal sigma factors represent a new suite of regulators for global transcriptional control.

synthetic biology↗

bioRxiv2022

The rugged DNA-binding sequence-fitness landscape of the LacI/GalR Family is a product of asymmetry in the operator:repressor complex

How a proteins function influences the shape of its fitness landscape, smooth or rugged, is a fundamental question in evolutionary biochemistry. Smooth landscapes arise when incremental mutational steps lead to a progressive change in function, as commonly seen in enzymes and binding proteins. On the other hand, rugged landscapes are poorly understood because of the inherent unpredictability of how sequence changes affect function. Here, we experimentally characterize the entire sequence phylogeny, comprising 1158 extant and ancestral sequences, of the DNA-binding domain (DBD) of the LacI/GalR transcriptional repressor family. Our analysis revealed an extremely rugged landscape with rapid switching of specificity even between adjacent nodes. Further, the ruggedness arises due to the necessity of the repressor to simultaneously evolve specificity for asymmetric operators and disfavors potentially adverse regulatory crosstalk. Our study provides fundamental insight into evolutionary, molecular, and biophysical rules of genetic regulation through the lens of fitness landscapes.

biochemistry↗

bioRxiv2022

Cell envelope remodeling requires high concentrations of biotin during Mycobacterium abscessus model lung infection

Mycobacterium abscessus is an emerging pathogen resistant to most frontline antibiotics. M. abscessus causes lung infection, predominantly in patients with lung disease or structural abnormalities. To interrogate mechanisms required for M. abscessus survival in the lung, we developed a lung infection model using air-liquid interface culture and performed a screen to identify differentially required genes. In the lung model, synthesis of the cofactor biotin is required due to increased intracellular biotin demand, and pharmacological inhibition of biotin synthesis halts M. abscessus proliferation. Increased quantities of biotin are required to sustain fatty acid remodeling that serves to increase cell envelope fluidity, which in turn promotes M. abscessus survival in the alkaline lung environment. Together, these results indicate that biotin-dependent fatty acid remodeling plays a critical role in pathogenic adaptation to the lung niche and suggests that biotin synthesis and fatty acid metabolism are therapeutic targets for treatment of M. abscessus infection.

microbiology↗

bioRxiv2022

Deep mutational scanning and machine learning reveal structural and molecular rules governing allosteric hotspots in homologous proteins

A fundamental question in protein science is where allosteric hotspots - residues critical for allosteric signaling - are located, and what properties differentiate them. We carried out deep mutational scanning (DMS) of four homologous bacterial allosteric transcription factors (aTF) to identify hotspots and built a machine learning model with this data to glean the structural and molecular properties of allosteric hotspots. We found hotspots to be distributed protein-wide rather than being restricted to "pathways" linking allosteric and active sites as is commonly assumed. Despite structural homology, the location of hotspots was not superimposable across the aTFs. However, common signatures emerged when comparing hotspots coincident with long-range interactions, suggesting that the allosteric mechanism is conserved among the homologs despite differences in molecular details. Machine learning with our large DMS datasets revealed that global structural and dynamic properties to be a strong predictor of whether a residue is a hotspot than local and physicochemical properties. Furthermore, a model trained on one protein can predict hotspots in a homolog. In summary, the overall allosteric mechanism is embedded in the structural fold of the aTF family, but the finer, molecular details are sequence-specific.

biochemistry↗

bioRxiv2022

CXCR2 inhibition enables NASH-HCC immunotherapy

ObjectiveHepatocellular carcinoma (HCC) is increasingly associated with non-alcoholic steatohepatitis (NASH). HCC immunotherapy offers great promise; however, recent data suggests NASH-HCC may be less sensitive to conventional immune checkpoint inhibition (ICI). We hypothesised that targeting neutrophils using a CXCR2 small molecule inhibitor may sensitise NASH-HCC to ICI therapy. DesignNeutrophil infiltration was characterised in human HCC and mouse models of HCC. Late-stage intervention with anti-PD1 and/or a CXCR2 inhibitor was performed in murine models of NASH-HCC. The tumour immune microenvironment was characterised by imaging mass cytometry, RNA-seq and flow cytometry. ResultsNeutrophils expressing CXCR2, a receptor crucial to neutrophil recruitment in acute-injury, are highly represented in human NASH-HCC. In models of NASH-HCC lacking response to ICI, the combination of a CXCR2 antagonist with anti-PD1 suppressed tumour burden and extended survival. Combination therapy increased intratumoral XCR1+ dendritic cell activation and CD8+ T cell numbers which are associated with anti-tumoral immunity, this was confirmed by loss of therapeutic effect upon genetic impairment of myeloid cell recruitment, neutralisation of the XCR1-ligand XCL1 or depletion of CD8+ T cells. Therapeutic benefit was accompanied by an unexpected increase in tumour-associated neutrophils (TANs) which switched from a pro-tumor to anti-tumour progenitor-like neutrophil phenotype. Reprogrammed TANs were found in direct contact with CD8+ T cells in clusters that were enriched for the cytotoxic anti-tumoural protease granzyme B. Neutrophil reprogramming was not observed in the circulation indicative of the combination therapy selectively influencing TANs. ConclusionCXCR2-inhibition induces reprogramming of the tumour immune microenvironment that promotes ICI in NASH-HCC. Significance of this studyWhat is already known on this subject? O_LIImmune checkpoint inhibition therapy is emerging as a promising new therapy for the treatment of advanced hepatocellular carcinoma (HCC). C_LIO_LIOnly a minority of HCC patients will respond to immune checkpoint inhibition (ICI) therapy and recent data suggest that HCC on the background of NASH may have reduced sensitivity to this treatment strategy. C_LIO_LINeutrophils are a typical myeloid component of the liver in NASH and are found either within the HCC tumour microenvironment or in a peritumoural location. C_LIO_LINeutrophils have considerable phenotypic plasticity and can exist in both tumour promoting and tumour suppressing states. C_LIO_LINeutrophils may have the ability to influence ICI therapy. C_LI What are the new findings? O_LICXCR2+ neutrophils are found in human NASH and within the tumour of both human and mouse models of NASH-HCC. C_LIO_LIThe resistance of NASH-HCC to anti-PD1 therapy is overcome by co-treatment with a CXCR2 small molecule inhibitor, with evidence of reduced tumour burden and extended survival. C_LIO_LIAnti-PD1 and CXCR2 inhibitor combine to selectively reprogramme tumour-associated neutrophils (TANs) from a pro- to an anti-tumour phenotype. C_LIO_LIReprogrammed TANs proliferate locally within Granzyme B+ immune clusters that contain physically associating CD8+ T cells and antigen presenting cells. C_LIO_LIConventional XCR1+ dendritic cells (cDC1s) are found to be elevated in anti-PD1 and CXCR2 inhibitor treated HCCs and together with CD8+ T cells are required for therapeutic benefit. C_LI How might it impact on clinical practice in the foreseeable future? O_LITANs can be selectively manipulated to adopt an anti-tumour phenotype which unlocks their potential for cancer therapy. The ability of CXCR2 antagonism to combine with ICI therapy to bring about enhanced therapeutic benefit in NASH-HCC (and potentially in HCC of other aetiologies) warrents clinical investigation. C_LI

cancer biology↗

bioRxiv2022

Antimicrobial resistance in dairy slurry tanks: a critical point for measurement and control

Waste from dairy production is one of the worlds largest sources of contamination from antimicrobial resistant bacteria (ARB) and genes (ARGs). However, studies to date do not provide necessary evidence to inform antimicrobial resistance (AMR) countermeasures. We undertook a detailed, interdisciplinary, longitudinal analysis of dairy slurry waste. The slurry contained a population of ARB and ARGs, with resistances to current, historical and never-used on-farm antibiotics; resistances were associated with Gram-negative and Gram-positive bacteria and mobile elements (ISEcp1, Tn916, Tn21-family transposons). Modelling and experimental work suggested that these populations are in dynamic equilibrium, with microbial death balanced by fresh input. Consequently, storing slurry without further waste input for at least 60 days was predicted to reduce ARB spread onto land, with >99% reduction in cephalosporin resistant Escherichia coli. The model also indicated that for farms with low antibiotic use, further reductions are unlikely to reduce AMR further. We conclude that the slurry tank is a critical point for prevalence and control of AMR, and that measures to limit the spread of AMR from dairy waste should combine responsible antibiotic use, including low total quantity, avoidance of human critical antibiotics, and choosing antibiotics with shorter half-lives, coupled with appropriate slurry storage.

microbiology↗

bioRxiv2021

Engineering a dynamic, controllable infectivity switch in bacteriophage T7

Transcriptional repressors play an important role in regulating phage genomes. Here, we examined how synthetic regulation based on repressors can be to create a dynamic, controllable infectivity switch in bacteriophage T7. We engineered T7 by replacing a large region of the early phage genome with combinations of ligand-responsive promoters and ribosome binding sites (RBS) designed to control the phage RNA polymerase. Phages with the engineered switch showed virulence comparable to wildtype when not repressed, indicating the phage can be engineered without a loss of fitness. When repressed, the most effective switch used a TetR promoter and a weak RBS, resulting in a two-fold increase in latent period (time to lyse host) and change in phage titer. Further, phage activity could be tuned by varying inducer concentrations. Our study provides a proof of concept for a simple circuit for user control over phage infectivity.

synthetic biology↗

bioRxiv2021

TAPAS: an open-source software package for Translational Neuromodeling and Computational Psychiatry

Psychiatry faces fundamental challenges with regard to mechanistically guided differential diagnosis, as well as prediction of clinical trajectories and treatment response of individual patients. This has motivated the genesis of two closely intertwined fields: (i) Translational Neuromodeling (TN), which develops "computational assays" for inferring patient-specific disease processes from neuroimaging, electrophysiological, and behavioral data; and (ii) Computational Psychiatry (CP), with the goal of incorporating computational assays into clinical decision making in everyday practice. In order to serve as objective and reliable tools for clinical routine, computational assays require end-to-end pipelines from raw data (input) to clinically useful information (output). While these are yet to be established in clinical practice, individual components of this general end-to-end pipeline are being developed and made openly available for community use. In this paper, we present the Translational Algorithms for Psychiatry-Advancing Science (TAPAS) software package, an open-source collection of building blocks for computational assays in psychiatry. Collectively, the tools in TAPAS presently cover several important aspects of the desired end-to-end pipeline, including: (i) tailored experimental designs and optimization of measurement strategy prior to data acquisition, (ii) quality control during data acquisition, and (iii) artifact correction, statistical inference, and clinical application after data acquisition. Here, we review the different tools within TAPAS and illustrate how these may help provide a deeper understanding of neural and cognitive mechanisms of disease, with the ultimate goal of establishing automatized pipelines for predictions about individual patients. We hope that the openly available tools in TAPAS will contribute to the further development of TN/CP and facilitate the translation of advances in computational neuroscience into clinically relevant computational assays.

neuroscience↗

bioRxiv2021

Virus-associated organosulfur metabolism in human and environmental systems

Viruses influence the fate of nutrients and human health by killing microorganisms and altering metabolic processes. Organosulfur metabolism and biologically-derived hydrogen sulfide play dynamic roles in manifestation of diseases, infrastructure degradation, and essential biological processes. While microbial organosulfur metabolism is well-studied, the role of viruses in organosulfur metabolism is unknown. Here we report the discovery of 39 gene families involved in organosulfur metabolism encoded by 3,749 viruses from diverse ecosystems, including human microbiomes. The viruses infect organisms from all three domains of life. Six gene families encode for enzymes that degrade organosulfur compounds into sulfide, while others manipulate organosulfur compounds and may influence sulfide production. We show that viral metabolic genes encode key enzymatic domains, are translated into protein, are maintained after recombination, and that sulfide provides a fitness advantage to viruses. Our results reveal viruses as drivers of organosulfur metabolism with important implications for human and environmental health.

microbiology↗