Biology subjects

Bajaj, P.

Publications and source records attributed to Bajaj, P..

9 recordsLinked to original sources

bioRxiv2025

Knowledge-informed multimodal cfDNA analysis improves sensitivity and generalization in cancer detection

Liquid biopsy offers a minimally invasive opportunity to detect and monitor cancers through analysis of cell-free DNA (cfDNA). However, current approaches face challenges of limited sensitivity at low tumor fractions, technical variability, and poor generalization across cohorts. Tumor-informed targeted methods offer high specificity but suffer from low sensitivity due to random sampling, tumor evolution and adaptation (including resistance mechanisms), and other sources of heterogeneity. Conversely, tumor-naive genome-wide methods can increase sensitivity but often sacrifice specificity, particularly at low tumor fractions. We developed Fragmentomics Analysis for Tumor Evaluation with AI (Fate-AI), a multimodal framework that integrates fragmentomic and methylation-derived features from low-pass whole-genome sequencing (LPWGS) and cell-free methylated DNA immunoprecipitation and high-throughput sequencing (cfMeDIP-seq). It employs a knowledge-informed strategy to select recurrently altered genomic regions and tissue-specific methylation loci to combine the advantages of tumor-naive approaches with the specificity of tumor-informed approaches. This approach derives robust per-sample normalized features that mitigate batch effects and enhance cross-cohort reproducibility. We evaluated Fate-AI on a total of 1,219 plasma samples spanning ten cancer types and healthy controls from multiple laboratories and sequencing centers, including 432 newly profiled cases (280 with both cfMeDIP-seq and LPWGS) together with 787 samples from four independent public datasets. Fate-AI achieved superior sensitivity and specificity compared to state-of-the-art methods, detecting tumor-derived signals at fractions as low as 10-5 in experimental dilutions. Fate-AI scores correlated with disease stage and tracked longitudinal progression, anticipating relapse months before clinical progression. Furthermore, Fate-AI enabled tissue-of-origin classification, with AUCs ranging from 0.84 to 0.97 across six cancer types. Collectively, our results demonstrate that Fate-AI provides a sensitive, generalizable, and clinically actionable platform for early detection, minimal residual disease monitoring, and tissue-of-origin classification, supporting its potential as a liquid biopsy framework in precision oncology.

cancer biology↗

bioRxiv2025

Product-stabilized filamentation by human glutamine synthetase allosterically tunes metabolic activity

To maintain metabolic homeostasis, enzymes must adapt to fluctuating nutrient levels through mechanisms beyond gene expression. Here, we demonstrate that human glutamine synthetase (GS) can reversibly polymerize into filaments aided by a composite binding site formed at the filament interface by the product, glutamine. Time-resolved cryo-electron microscopy (cryo-EM) confirms that glutamine binding stabilizes these filaments, which in turn exhibit reduced catalytic specificity for ammonia at physiological concentrations. This inhibition appears induced by a conformational change that remodulates the active site loop ensemble gating substrate entry. Metadynamics ensemble refinement revealed >10 [A] conformational range for the active site loop and that the loop is stabilized by transient contacts. This disorder is significant, as we show that the transient contacts which stabilize this loop in a closed conformation are essential for catalysis both in vitro and in cells. We propose that GS filament formation constitutes a negative-feedback mechanism, directly linking product concentration to the structural and functional remodeling of the enzyme.

bioinformatics↗

bioRxiv2024

CcdA chaperones CcdB against irreversible misfolding and aggregation via a cotranslational folding mechanism

Cotranslational subunit assembly is thought to be a prominent feature throughout the proteome, but in bacteria, there are only a limited number of experimentally confirmed examples and most involve addition of extraneous tag sequences for experimental convenience. CcdA and CcdB are the antitoxin and toxin components, respectively, of the ccdAB operon. They assemble in a hetero-multimeric complex in vivo. Mutant phenotypes in a saturation mutagenesis library of CcdB were inferred from deep sequencing in two contexts, one when CcdB is expressed alone, and the other when CcdB is present in an operonic context downstream of its cognate antitoxin, CcdA. When expressed in the absence of CcdA, charged and polar mutations in the CcdB core cause the protein to misfold. However, many such deleterious mutations are rescued when expressed along with CcdA in the native operon. CcdA thus acts as a chaperone and relieves the folding defect in CcdBcotranslationally. Assembly efficiency and efficacy decreases when CcdA and CcdB are expressed from separate mRNAs relative to when they are expressed from the same polycistronic mRNA. Gene organisation in operons in bacteria may thus reflect a fundamental cotranslational mechanism that is important for effective assembly of protein complexes and can potentially buffer substantial genetic variation. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=161 SRC="FIGDIR/small/618527v1_ufig1.gif" ALT="Figure 1"> View larger version (20K): org.highwire.dtl.DTLVardef@26e1ceorg.highwire.dtl.DTLVardef@b2a4b1org.highwire.dtl.DTLVardef@5a7c85org.highwire.dtl.DTLVardef@a50512_HPS_FORMAT_FIGEXP M_FIG C_FIG In BriefUsing the CcdAB Type II Toxin-Antitoxin system, the authors show that the antitoxin is able to suppress folding defects of the toxin, likely by processes involving cotranslational assembly. HighlightsO_LIDeep mutational scanning is used to assess and compare the foldability of buried-site CcdB mutants in the absence and presence of CcdA. C_LIO_LIFolding defects of CcdB mutants are rescued in the presence of CcdA when expressed in an operonic context. C_LIO_LICcdAB complex formation is enhanced when both the toxin and antitoxin components are synthesized from the same polycistronic mRNA, relative to synthesis from separate mRNAs. C_LIO_LICcdA acts as chaperone both to assist correct folding, and to buffer mutations in CcdB, likely via cotranslational assembly mechanisms. C_LI

biophysics↗

bioRxiv2023

Molecular bases for strong phenotypic effects of single-site synonymous substitutions in the E.coli ccdB toxin gene

Single-site synonymous mutations typically have only minor or no effects on gene function. Here, we estimate the effects on cell growth of ~200 single-site synonymous mutations in an operonic context by mutating each position of ccdB, the 101-residue long cytotoxin of the ccdAB Toxin-Antitoxin (TA) operon to all possible degenerate codons. Phenotypes were assayed by transforming the mutant library into CcdB sensitive and resistant E. coli strains, isolating plasmid pools, and subjecting them to deep sequencing. Since autoregulation is a hallmark of TA operons, phenotypes obtained for ccdB synonymous mutants after transformation in a RelE toxin reporter strain followed by deep sequencing provided information on the amount of CcdAB complex formed. Synonymous mutations in the N-terminal region involved in translation initiation showed the strongest non-neutral phenotypic effects. We observe an interplay of numerous factors, namely, location of the codon, codon usage, t-RNA abundance, formation of anti-Shine Dalgarno sequences, transcript secondary structure, and evolutionary conservation in determining phenotypic effects of ccdB synonymous mutations. Incorporation of an N-terminal, hyperactive synonymous mutation, in the background of the single-site synonymous mutant library sufficiently increased translation initiation, such that mutational effects on either folding or termination of translation became more apparent. Introduction of putative pause sites not only affects the translational rate but might also alter the folding kinetics of the protein in vivo. This information is useful in optimizing heterologous gene expression in E. coli and understanding the molecular bases for alteration in gene expression that arise due to synonymous mutations. SIGNIFICANCE STATEMENTSynonymous mutations which do not change amino-acid identity, typically have only minor or no effects on gene function. Using sensitive genetic screens in the context of the ccdAB bacterial toxin-antitoxin operon, we demonstrate that many single-site synonymous mutations of the ccdB toxin gene display significant phenotypic effects in an operonic context. The largest effects were seen for synonymous mutations in the N-terminal region involved in translation initiation. Synonymous mutations that affected either folding or translation termination were also identified. Lack of translational pausing due to synonymous mutations in hydrophobic residue stretches, was found to decrease the amount of properly folded CcdB protein. In summary the study provides novel insights into diverse mechanisms by which synonymous mutations modulate gene function.

molecular biology↗

bioRxiv2022

Structural and functional determinants inferred from deep mutational scans

Mutations that affect protein binding to a cognate partner primarily occur either at buried residues or at exposed residues directly involved in partner binding. Distinguishing between these two categories based solely on mutational phenotypes is challenging. The bacterial toxin CcdB kills cells by binding to DNA Gyrase. Cell death is prevented by binding to its cognate antitoxin CcdA, at an extended interface that partially overlaps with the GyrA binding site. Using the CcdAB toxin-antitoxin (TA) system as a model, a comprehensive site-saturation mutagenesis library of CcdB was generated in its native operonic context. The mutational sensitivity of each mutant was estimated by evaluating the relative abundance of each mutant in two strains, one resistant and the other sensitive to the toxic activity of the CcdB toxin, through deep sequencing. The ability to bind CcdA was inferred through a RelE reporter gene assay, since the CcdAB complex binds to its own promoter, repressing transcription. By analysing mutant phenotypes in the CcdB sensitive, CcdB resistant and RelE reporter strains, it was possible to assign residues to buried, CcdA interacting or GyrA interacting sites. A few mutants were individually constructed, expressed, and biophysically characterised to validate molecular mechanisms responsible for the observed phenotypes. Residues inferred to be important for antitoxin binding, are also likely to be important for rejuvenating CcdB from the CcdB-Gyrase complex. Therefore, even in the absence of structural information, when coupled to appropriate genetic screens, such high-throughput strategies can be deployed for predicting structural and functional determinants of proteins. Broader Impact StatementPartial loss-of-function mutations predominantly occur either at buried-site or exposed, active-site residues. We report a facile method to identify multiple binding sites for different interacting partners for a protein, and distinguish them from buried site and exposed non active-site residues, solely from mutational data.

biophysics↗

bioRxiv2022

Signaling modality within gp130 receptor enhances tissue regeneration

Adult mammals are incapable of multi-tissue regeneration and augmentation of this potential may drastically shift current therapeutic paradigms. Here, we found that a common co-receptor of IL-6 cytokines, glycoprotein 130 (gp130), serves as a major nexus integrating various context-specific signaling inputs to either promote regenerative outcomes or aggravate disease progression. Via genetic and pharmacological experiments in vitro and in vivo, we demonstrated that a signaling tyrosine 814 (Y814) within gp130 serves as a major cellular stress sensor. Mice with constitutively inactivated Y814 (F814) exhibit regenerative, not reparative, responses after wounding in skin and anti-degenerative responses in the synovial joint. In addition, pharmacological inhibition of gp130 Y814 results in regeneration of multiple tissues in several species as well as disease modification in animal models of osteoarthritis. Our study characterizes a novel molecular mechanism that, if selectively manipulated, enhances the intrinsic regenerative capacity while preventing pathological outcomes in injury and disease. SummaryGp130 Y814 signaling module serves as a cellular stress sensor responsible for hindering tissue regeneration while triggering pathological outcomes after injury.

molecular biology↗

bioRxiv2020

A novel high-accuracy genome assembly method utilizing a high-throughput workflow

Across domains of biological research using genome sequence data, high-quality reference genome sequences are essential for characterizing genetic variation and understanding the genetic basis of phenotypes. However, the construction of genome assemblies for various species is often hampered by complexities of genome organization, especially repetitive and complex sequences, leading to mis-assembly and missing regions. Here, we describe a high-throughput gold standard genome assembly workflow using a large-scale bacterial artificial chromosome (BAC) library with a refined two-step pooling strategy and the Lamp assembler algorithm. This strategy minimizes the laborious processes of physical map construction and clone-by-clone sequencing, enabling inexpensive sequencing of several thousand BAC clones. By applying this strategy with a minimum tiling path BAC clone library for the short arm of chromosome 2D (2DS) of bread wheat, 98% of BAC sequences, covering 92.7% of the 2DS chromosome, were assembled correctly for this species with a highly complex and repetitive genome. We also identified 48 large mis-assemblies in the reference wheat genome assembly (IWGSC RefSeq v1.0) and corrected these large mis-assemblies in addition to filling 92.2% of the gaps in RefSeq v1.0. Our 2DS assembly represents a new benchmark for the assembly of complex genomes with both high accuracy and efficiency.

genomics↗

bioRxiv2020

Climatic-niche evolution of SARS CoV-2

COVID-19 pandemic has been extensively studied by many researchers. However, it is still unclear why it was restricted to higher latitudes during the initial days and later cascaded in the tropics. Here, we analyzed 176 SARS-CoV-2 genomes across different latitudes and climate (Koppens climate) that provided insights about within species virus evolution and its relation to abiotic factors. Two genetically variant groups, named as G1 and G2 were identified, well defined by four mutations. The G1 group (ancestor), is mainly restricted to warm and moist, temperate climate (Koppens C climate) while its descendent G2 group surpasses the climatic restrictions of G1, initially cascading into neighboring cold climate (D) of higher latitudes and later into hot climate of the tropics (A). It appears that the gradation of temperate climate (Cfa-Cfb) to cold climate (Dfa-Dfb) drives the evolution of G1 into G2 variant group which later adapted to tropical climate (A) as well. It seems this virus followed inverse latitudinal gradient in the beginning due to its preference towards temperate (C) and cold climate (D). Our work elucidates virus evolutionary studies combined with climatic studies can provide crucial information about the pathogenesis and natural spreading pathways in such outbreaks which is hard to achieve through individual studies. Mutational insights gained may help design an efficacious vaccine. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=107 SRC="FIGDIR/small/147074v3_ufig1.gif" ALT="Figure 1"> View larger version (28K): org.highwire.dtl.DTLVardef@f39a3org.highwire.dtl.DTLVardef@1910598org.highwire.dtl.DTLVardef@6516corg.highwire.dtl.DTLVardef@cbb48c_HPS_FORMAT_FIGEXP M_FIG C_FIG In BriefThe authors elucidate adaptation of SARS-CoV-2 to different climates by studying phylogenetics and the distribution of strains on Koppens climate map. HighlightsO_LIPhylogenetic network divides SARS-CoV-2 strains into two variant groups, G1 and G2. C_LIO_LIG1 strains is restricted to Koppens "temperate" climate (mainly Cfa-Cfb). C_LIO_LIG2 strains has evolved from G1 to sustain in other climates mainly "humid-continental" (Dfa-Dfb) and "tropical-savannah" (Aw) climate. C_LI

evolutionary biology↗

bioRxiv2019

Carbohydrate, glutathione, and polyamine metabolism are central to Aspergillus flavus oxidative stress responses over time

The primary and secondary metabolites of fungi are critical for adaptation to environmental stresses, host pathogenicity, competition with other microbes, and reproductive fitness. Drought-derived reactive oxygen species (ROS) have been shown to stimulate aflatoxin production and regulate development in Aspergillus flavus, and may function in signaling with host plants. Here, we have performed global, untargeted metabolomics to better understand the role of aflatoxin production in oxidative stress responses, and also explore isolate-specific oxidative stress responses over time. Two field isolates of A. flavus, AF13 and NRRL3357, possessing high and moderate aflatoxin production, respectively, were cultured in medium with and without supplementation with 15mM H2O2, and mycelia were collected following 4 and 7 days in culture for global metabolomics. Overall, 389 compounds were described in the analysis which were examined for differential accumulation. Significant differences were observed in both isolates in response to oxidative stress and when comparing sampling time points. The moderate aflatoxin-producing isolate, NRRL3357, showed extensive stimulation of antioxidant mechanisms and pathways including polyamines metabolism, glutathione metabolism, TCA cycle, and lipid metabolism while the highly aflatoxigenic isolate, AF13, showed a less vigorous response to stress. Carbohydrate pathway levels also imply that carbohydrate repression and starvation may influence metabolite accumulation at the later timepoint. Higher conidial oxidative stress tolerance and antioxidant capacity in AF13 compared to NRRL3357, inferred from their metabolomic profiles and growth curves over time, may be connected to aflatoxin production capability and aflatoxin-related antioxidant accumulation. The coincidence of several of the detected metabolites in H2O2-stressed A. flavus and drought-stressed hosts suggests their potential role in the interaction between these organisms and their use as markers/targets to enhance host resistance through biomarker selection or genetic engineering.\n\nAuthor SummaryAspergillus flavus is a fungal pathogen of several important crops including maize and peanut. This pathogen produces carcinogenic mycotoxins known as aflatoxins during infection of plant materials, and is particularly severe under drought stress conditions. This results in significant losses in crop value and poses a threat to food safety and security globally. To combat this, understanding how this fungus responds to environmental stresses related to drought can allow us to identify novel methods of mitigating aflatoxin contamination. Here, we analyzed the accumulation of a broad series of metabolites over time in two isolates of A. flavus with differing stress tolerance and aflatoxin production capabilities in response to drought-related oxidative stress. We identified several metabolites and mechanisms in A. flavus which allow it to cope with environmental oxidative stress and may influence aflatoxin production and fungal growth. These may serve as potential targets for selection in breeding programs for the development of new cultivars, or for alteration using genetic engineering approaches to mitigate excessive aflatoxin contamination under drought stress.

microbiology↗