Biology subjects

Bhadra, S.

Publications and source records attributed to Bhadra, S..

11 recordsLinked to original sources

bioRxiv2026

Agnostic material classification using differential de Bruijn graphs of DNA imprints

The wide variety of physical and chemical properties in materials makes the study of unknown substances challenging. We have previously proposed a theoretical framework for agnostic material characterization based on using nucleic acid imprints of the materials and then analyzing material-specific patterns of derived sequences. Here we demonstrate an experimental and computational pipeline that can agnostically identify and distinguish varied materials based on DNA k-mer imprints and validate the ability of these imprints to distinguish closely related materials. This work lays the foundation for expansion of purely agnostic sensing technologies for the unbiased characterization and categorization of a much wider variety of biotic and abiotic materials.

bioinformatics↗

bioRxiv2025

Functional traits drive speciation in tropical palms through complex interactions between genome size, adaptation and allometry

O_LIThe importance of functional trait evolution and genome size on plant speciation are well established, but their interactive effects remain untested in a single comparative macroevolutionary framework. C_LIO_LIWe integrated phylogenetic, trait and genome size data for palms (Arecaceae) - a large pantropical family (>2600 species) with 167-fold variation in trait and 60-fold variation in genome size. We used structural equation modelling to test three key hypotheses: trait evolution promotes speciation (H1: trait flexibility hypothesis), and, speciation and trait evolution rates are constrained by allometry (H2: allometric constraint hypothesis) and genome size (H3: large genome constraint hypothesis). C_LIO_LIWe detected seven major speciation rate shifts during the ca. 110-million-year history of palms. Tip-derived speciation rates increased with faster evolution in leaves and plant height, supporting H1, whereas correlated evolution between trait evolution rates indirectly influenced speciation, supporting H2. Large genomes decreased plant height and stem diameter evolutionary rates, but increased leaf size evolution and speciation rates, thus partly supporting H3. C_LIO_LIOur findings illustrate how the complex interplay between genome size, allometry and trait evolvability affect speciation, emphasizing the importance of holistic approaches in macroevolution. Furthermore, our results point to potential general mechanisms driving speciation rates throughout the plant Tree of Life. C_LI

evolutionary biology↗

bioRxiv2024

Short lifespan is one's fate, long lifespan is one's achievement: lessons from Daphnia

Studies of longevity and senescence rely on baseline life expectancy of reference genotypes measured in standardized conditions such as food level and group size. Variation in baseline lifespan data across labs and protocols and among genotypes can make longevity intervention studies difficult to compare, particularly when GxE interactions exist. Furthermore, extending the lifespan of a short-lived genotype or of any genotype under suboptimal conditions may be of a lesser theoretical and translational value than extending the maximal possible lifespan. Daphnia is rapidly becoming a model organism of choice for longevity research complementing data obtained on traditional models. In this study we report baseline longevity of several genotypes (parthenogenetic clones) of a long-lived species D. magna under a variety of laboratory protocols, aiming to document the highest possible lifespan, factors reducing it, and physiological parameters that change with age and correlate with longevity. Combining data from 25 different experiments across two labs we report strong differences among clones of different geographic origin, moderate effects of group size and medium composition on longevity, and strong GxE with respect to food level. Specifically, short-lived clones that tend to originate from small intermittent habitats show little or no caloric restriction (CR) longevity extension, while long-lived ones expand their lifespan even further when maintained at 25% of the ad libitum food. We find no evidence of any trade-offs between longevity and fecundity across clones or correlations with age-specific feeding rate. We find that in the short-lived, CR non-responsive clones show little correlation between longevity and two measures of lipid peroxidation (LPO: lipid hydroperoxides and MDA abundance). In contrast, the long-lived, CR-responsive clones show a positive longevity correlation with lipid hydroperoxide abundance at any age, and a negative correlation with MDA concentration measured at about median lifespan. This indicates differences among genotypes in longevity-related accumulation of LPO targets, efficiency of detoxification of LPO products, and/or their effects on longevity. Our observations support the hypothesis that a long lifespan can be affected by food availability and levels of oxidative damage, while genetically determined short lifespan remains short regardless. We suggest a set of condition and genotypes to be used as a reference for longevity studies in Daphnia.

ecology↗

bioRxiv2023

Comprehensive mutational analysis of the checkpoint signaling functions of Rpa1/Ssb1 in fission yeast

Replication protein A (RPA) is a heterotrimeric complex and the major single-strand DNA (ssDNA) binding protein in eukaryotes. It plays important roles in DNA replication, repair, recombination, telomere maintenance, and checkpoint signaling. Because RPA is essential for cell survival, understanding its checkpoint signaling function in cells has been challenging. Several RPA mutants have been reported previously in fission yeast. None of them, however, has a defined checkpoint defect. A separation-of-function mutant of RPA, if identified, would provide significant insights into the checkpoint initiation mechanisms. We have explored this possibility and carried out an extensive genetic screening for Rpa1/Ssb1, the large subunit of RPA in fission yeast, looking for mutants with defects in checkpoint signaling. This screen has identified twenty-five primary mutants that are sensitive to genotoxins. Among these mutants, two have been confirmed partially defective in checkpoint signaling primarily at the replication fork, not the DNA damage site. The remaining mutants are likely defective in other functions such as DNA repair or telomere maintenance. Our screened mutants, therefore, provide a valuable tool for future dissection of the multiple functions of RPA in fission yeast. AUTHOR SUMMARYOriginally discovered as a protein required for replication of simian virus SV40 DNA, replication protein A is now known to function in DNA replication, repair, recombination, telomere maintenance, and checkpoint signaling in all eukaryotes. The protein is a complex of three subunits and the two larger ones are essential for cell growth. This essential function however complicates the studies in living cells, and for this reason, its checkpoint function remains to be fully understood. We have carried out an genetic screening of the largest subunit of this protein in fission yeast, aiming to find a non-lethal mutant that lacks the checkpoint function. This extensive screen has uncovered two mutants with a partial defect in checkpoint signaling when DNA replication is arrested. Surprisingly, although the two mutants also have a defect in DNA repair, their checkpoint signaling remains largely functional in the presence of DNA damage. We have also uncovered twenty-three mutants with defects in DNA repair or telomere maintenance, but not checkpoint signaling. Therefore, the non-lethal mutants uncovered by this study provide a valuable tool for dissecting the multiple functions of this biologically important protein in fission yeast.

genetics↗

bioRxiv2021

Multi-modal engineering of Bst DNA polymerase for thermostability in ultra-fast LAMP reactions

DNA polymerase from Geobacillus stearothermophilus, Bst DNA polymerase (Bst DNAP), is a versatile enzyme with robust strand-displacing activity that enables loop-mediated isothermal amplification (LAMP). Despite its exclusive usage in LAMP assay, its properties remain open to improvement. Here, we describe logical redesign of Bst DNAP by using multimodal application of several independent and orthogonal rational engineering methods such as domain addition, supercharging, and machine learning predictions of amino acid substitutions. The resulting Br512g3 enzyme is not only thermostable and extremely robust but it also displays improved reverse transcription activity and the ability to carry out ultrafast LAMP at 74 {degrees}C. Our study illustrates a new enzyme engineering strategy as well as contributes a novel engineered strand displacing DNA polymerase of high value to diagnostics and other fields.

bioengineering↗

bioRxiv2020

High-surety isothermal amplification and detection of SARS-CoV-2, including with crude enzymes

ABSTRACTIsothermal nucleic acid amplification tests (iNAT), such as loop-mediated isothermal amplification (LAMP), are good alternatives to polymerase chain reaction (PCR)-based amplification assays, especially for point-of-care and low resource use, in part because they can be carried out with relatively simple instrumentation. However, iNATs can generate spurious amplicons, especially in the absence of target sequences, resulting in false positive results. This is especially true if signals are based on non-sequence-specific probes, such as intercalating dyes or pH changes. In addition, pathogens often prove to be moving, evolving targets, and can accumulate mutations that will lead to inefficient primer binding and thus false negative results. Internally redundant assays targeting different regions of the target sequence can help to reduce such false negatives. Here we describe rapid conversion of three previously described SARS-CoV-2 LAMP assays that relied on non-sequence-specific readout into assays that can be visually read using sequence-specific fluorogenic oligonucleotide strand exchange (OSD) probes. We evaluate one-pot operation of both individual and multiplex LAMP-OSD assays and demonstrate detection of SARS-CoV-2 virions in crude human saliva.Competing Interest StatementThe authors have declared no competing interest.View Full Text

molecular biology↗

bioRxiv2020

A one-enzyme RT-qPCR assay for SARS-CoV-2, and procedures for reagent production

ABSTRACTGiven the scale of the ongoing COVID-19 pandemic, the need for reliable, scalable testing, and the likelihood of reagent shortages, especially in resource-poor settings, we have developed a RT-qPCR assay that relies on an alternative to conventional viral reverse transcriptases, a thermostable reverse transcriptase / DNA polymerase (RTX)1. Here we show that RTX performs comparably to the other assays sanctioned by the CDC and validated in kit format. We demonstrate two modes of RTX use – (i) dye-based RT-qPCR assays that require only RTX polymerase, and (ii) TaqMan RT-qPCR assays that use a combination of RTX and Taq DNA polymerases (as the RTX exonuclease does not degrade a TaqMan probe). We also provide straightforward recipes for the purification of this alternative reagent. We anticipate that in low resource or point-of-need settings researchers could obtain the available constructs from Addgene or our lab and begin to develop their own assays, within whatever regulatory framework exists for them.We lay out protocols for dye-based and TaqMan probe-based assays, in order to best compare with ‘gold standard’ reagents. These protocols should form the basis of further modifications that can simplify the assay to the use of overexpressing cells themselves as reagents.Developing dye-based and TaqMan probe-based RT-qPCR assays with RTXCompeting Interest StatementThe authors have declared no competing interest.View Full Text

molecular biology↗

bioRxiv2018

From Space to Sequence and Back Again: Iterative DNA Proximity Ligation and its Applications to DNA-Based Imaging

We extend the concept of DNA proximity ligation from a single readout per oligonucleotide pair to multiple reversible, iterative ligations re-using the same oligonucleotide molecules. Using iterative proximity ligation (IPL), we can in principle capture multiple ligation events between each oligonucleotide and its various neighbors and thus recover a far richer knowledge about their relative positions than single, irreversible ligation events. IPL would thus act to sample and record local molecular neighborhoods. By integrating a unique DNA barcode into each participating oligonucleotide, we can catalog the individual ligation events and thus capture the positional information contained therein in a high throughput manner using next-generation DNA sequencing. We propose that by interpreting IPL sequencing results in the context of graph theory and by applying spring layout algorithms, we can recover geometric patterns of objects labeled by DNA. Using simulations, we demonstrate that we can in principle recover letter patterns photolithographed onto slide surfaces using only IPL sequencing data, illustrating how our technique maps complex spatial configurations into DNA sequences and then - using only this sequence information - recovers them. We complement our theoretical work with an experimental proof-of-concept of iterative proximity ligation on an oligonucleotide population.

bioinformatics↗

bioRxiv2018

Multiplex logic processing isothermal diagnostic assays for an evolving virus

We have developed a generalizable smart molecular diagnostic capable of accurate point-of-care (POC) detection of variable nucleic acid targets. Our one-pot isothermal assay relies on multiplex execution of four loop-mediated isothermal amplification reactions, with primers that are degenerate and redundant, thereby increasing the breadth of targets while reducing the probability of amplification failure. An easy-to-read visual answer is computed directly by a multi-input Boolean OR gate signal transducer that uses degenerate strand exchange probes to assess any combination of amplicons. We demonstrate our platform by using the same assay to detect divergent Asian and African lineages of the evolving Zika virus (ZIKV), while maintaining selectivity against non-target viruses. Direct analysis of biological specimens proved possible, with 20 virions / {micro}l being directly detected in human saliva within 90 minutes, and crudely macerated ZIKV-infected Aedes aegypti mosquitoes being identified with 100% specificity and sensitivity. The ease-of-use with minimal instrumentation, broad programmability, and built-in fail-safe reliability make our smart molecular diagnostic attractive for POC use.

molecular biology↗

bioRxiv2018

Direct nucleic acid analysis of mosquitoes for high fidelity species identification and detection of Wolbachia using a cellphone

Manipulation of natural mosquito populations using the endosymbiotic bacteria Wolbachia is being investigated as a novel strategy to reduce the burden of mosquito-borne viruses. To evaluate the efficacy of these interventions, it will be critical to determine Wolbachia infection frequencies in Aedes aegypti mosquito populations. However, current diagnostic tools are not well-suited to fit this need. Morphological methods cannot identify Wolbachia, immunoassays often suffer from low sensitivity and poor throughput, while PCR and spectroscopy require complex instruments and technical expertise, which restrict their use to centralized laboratories. To address this unmet need, we have used loop-mediated isothermal amplification (LAMP) and oligonucleotide strand displacement (OSD) probes to create a one-pot sample-to-answer nucleic acid diagnostic platform for vector and symbiont surveillance. LAMP-OSD assays can directly amplify target nucleic acids from macerated mosquitoes without requiring nucleic acid purification and yield specific single endpoint yes/no fluorescence signals that are observable to eye or by cellphone camera. We demonstrate cellphone-imaged LAMP-OSD tests for two targets, the Aedes aegypti cytochrome oxidase I (coi) gene and the Wolbachia surface protein (wsp) gene, and show a limit of detection of 4 and 40 target DNA copies, respectively. In a blinded test of 90 field-caught mosquitoes, the coi LAMP-OSD assay demonstrated 98% specificity and 97% sensitivity in identifying Ae. aegypti mosquitoes even after 3 weeks of storage without desiccant at 37 {degrees}C. Similarly, the wsp LAMP-OSD assay readily identified the wAlbB Wolbachia strain in field-collected Aedes albopictus mosquitoes without generating any false positive signals. Modest technology requirements, minimal execution steps, simple binary readout, and robust accuracy make the LAMP-OSD-to-cellphone assay platform well suited for field vector surveillance in austere or resource-limited conditions.\n\nAuthor summaryMosquitoes spread many human pathogens and novel approaches are required to reduce the burden of mosquito-borne disease. One promising approach is transferring Wolbachia into Aedes aegypti mosquitoes where it blocks transmission of arboviruses like dengue, Zika and Yellow fever viruses and spreads through mosquito populations. For effective evaluation of this approach, regular surveillance of Wolbachia infections in Ae. aegypti is required, but current diagnostic tools are not well suited to support these critical surveillance needs. To fill this need we developed a simple, robust and inexpensive assay to identify Ae. aegypti mosquitoes and Wolbachia using our unique one-pot assay platform, LAMP-OSD, which uses loop-mediated isothermal amplification to amplify nucleic acid targets at a single temperature. Unlike other LAMP-based tests, our assays assure accuracy by coupling amplification with novel nucleic acid strand displacement (OSD) probes that hybridize to specific sequences in LAMP amplification products and thereby generate simple yes/no readout of fluorescence readable by human eye and by off-the-shelf cellphones. To facilitate field use, we developed our assays so they are compatible with crushed mosquito homogenate as the template, meaning no nucleic acid extraction is required. In blinded tests using field collected mosquitoes, LAMP-OSD-cellphone tests performed robustly to identify 29 of 30 Ae. aegypti even after 3 weeks of storage at 37 {degrees}C while producing only one false positive out of 60 non-specific mosquitoes. Similarly, our assay could identify Wolbachia in field-caught Aedes albopictus without producing any false positives. Our easy to use and easy to interpret assays should facilitate widespread field mosquito surveillance with minimal instrumentation and high accuracy.

molecular biology↗

bioRxiv2017

Principal Metabolic Flux Mode Analysis

MotivationIn the analysis of metabolism using omics data, two distinct and complementary approaches are frequently used: Principal component analysis (PCA) and Stoichiometric flux analysis. PCA is able to capture the main modes of variability in a set of experiments and does not make many prior assumptions about the data, but does not inherently take into account the flux mode structure of metabolism. Stoichiometric flux analysis methods, such as Flux Balance Analysis (FBA) and Elementary Mode Analysis, on the other hand, produce results that are readily interpretable in terms of metabolic flux modes, however, they are not best suited for exploratory analysis on a large set of samples.\n\nResultsWe propose a new methodology for the analysis of metabolism, called Principal Metabolic Flux Mode Analysis (PMFA), which marries the PCA and Stoichiometric flux analysis approaches in an elegant regularized optimization framework. In short, the method incorporates a variance maximization objective form PCA coupled with a Stoichiometric regularizer, which penalizes projections that are far from any flux modes of the network. For interpretability, we also introduce a sparse variant of PMFA that favours flux modes that contain a small number of reactions. Our experiments demonstrate the versatility and capabilities of our methodology.\n\nAvailabilityMatlab software for PMFA and SPMFA is available in https://github.com/ aalto-ics-kepaco/PMFA.\n\nContactsahely@iitpkd.ac.in, juho.rousu@aalto.fi, Peter.Blomberg@vtt.fi, Sandra.Castillo@vtt.fi\n\nSupplementary informationDetailed results are in Supplementary files. Supplementary data are available at https://github.com/aalto-ics-kepaco/PMFA/blob/master/Results.zip.

bioinformatics↗