Biology subjects

Paul, D.

Publications and source records attributed to Paul, D..

52 records · Page 3Linked to original sources

bioRxiv2020

In vitro and in silico analyses of the angiotensin-I converting enzyme inhibitory activity of peptides identified from Bellamya bengalensis protein hydrolysates

The study focuses on identification of ACE-inhibitory peptides from the proteolytic digests of muscle protein of Bellamya bengalensis and its underlying mechanism. 120 min Alcalase-hydrolysates were ultrafiltered to isolate the small peptide fraction (<3kDa) and in vitro ACE-inhibitory activity was analyzed. The IC50 value of the 120 min hydrolysate ultafiltered fraction was found to be 86.74 {+/-} 0.575 {micro}g/mL, while the IC50 of Lisinopril is 0.31 {+/-} 0.07 {micro}g/mL. This fraction was assessed in MALDI-ToF mass-spectrometer and five peptides were sequenced via de novo sequencing. The ACE-inhibitory potential of the peptides have a positive correlation with the hydrophobicity of the amino acids. Synthetic analogue of the peptide (IC50 value 8.52 {+/-} 0.779 {micro}g/mL) was used to understand the thermodynamics of the inhibition by checking the binding affinity of the peptide to ACE by Isothermal titration calorimetry compared with lisinopril, and further substantiated by in silico site specific molecular docking study.

biochemistry↗

bioRxiv2020

Proteins and Ions Compete for Membrane Interaction: the case of Lactadherin

Charged molecular species, such as ions, play a vital role in the life of the cell. In particular, divalent calcium ions (Ca2+) are critical for activating cellular membranes. Interactions between Ca2+ and anionic phosphatidylserine (PS) lipids result in structural changes of the plasma membrane and are vital for many signaling pathways, such as the tightly regulated blood coagulation cascade. Upon cell damage, PS lipids are externalized to the outer leaflet, where they are not only exposed to Ca2+, but also to proteins. Lactadherin is a glycoprotein, important for cell-adhesion, that can act as an anticoagulant. While a number of experimental studies have been performed on lactadherins C2 domains (LactC2) binding affinity for PS molecules, an atomistic description of LactC2 interactions with PS lipids in the plasma membrane is lacking. We performed extensive all-atom molecular dynamics simulations of mixed lipid bilayers and experimental characterization of LactC2-membrane interactions in the presence and absence of Ca2+ and characterized PS-Ca2+ and PS-LactC2 interactions to guide our understanding of how these interactions initiate and impede blood coagulation, respectively. The captured spontaneously formed long-lived PS-Ca2+ and PS-LactC2 complexes revealed that the protein side chains involved in PS-LactC2 interactions appear to be affected by the presence of Ca2+. The degree of LactC2 insertion into the lipid bilayer also appears to be dependent on the presence of Ca2+. Characterizing the interactions between Ca2+ and LactC2 with PS lipids can lead to a greater understanding of the activation and regulation of the blood coagulation cascade and of the basis of charged species interactions with the lipid membrane. STATEMENT OF SIGNIFICANCELactadherin plays an important role in cellular signaling including blood coagulation. Many of these processes involve lactadherin interacting with the lipids of the cell plasma membrane. Lactadherin acts as an anticoagulant and contributes to a number of health issues. Understanding the interactions that drive lactadherins anticoagulant properties can lead to potential new drug targets.

biophysics↗

bioRxiv2020

Impact of DNA sequences in the DNA duplex opening by the Rad4/XPC nucleotide excision repair complex

Rad4/XPC is a key DNA damage sensor for nucleotide excision repair (NER) in eukaryotes. Rad4/XPC recognizes diverse bulky lesions by flipping out two lesion-containing nucleotide pairs and inserting a {beta}-hairpin from the BHD3 domain ({beta}-hairpin3) into the DNA duplex. We have previously observed that Rad4 can form the same open structure when covalently tethered to a normal DNA sequence containing consecutive C/Gs (CCC/GGG) and that a similar open-like structure can be formed even when the {beta}-hairpin3 is lacking. Here, we report a crystal structure of the {Delta}{beta}-hairpin3 mutant tethered to a sequence containing alternating C/Gs (CGC/GCG). In contrast to the previous structures, Rad4 bound to CGC/GCG in a 180{degrees}-reversed manner, capping the end of the duplex without flipping out the nucleotides. MD simulations showed that CGC/GCG was inherently less openable than CCC/GGG and that Rad4 failed to engage with its minor groove, a hallmark of productive binding towards opening. These results reveal that DNA sequences significantly influence the thermodynamic barrier for DNA opening by Rad4, which may render certain DNA structures/sequences resistant to opening despite a long residence time of Rad4. The reverse- mode may indicate unproductive binding for NER whereas the DNA end-binding may hint at Rad4/XPCs functions beyond NER.

biochemistry↗

bioRxiv2019

In vivo characterisation of endogenous cardiovascular extracellular vesicles in larval and adult zebrafish

ObjectiveExtracellular vesicles (EVs) facilitate molecular transport across extracellular space, allowing local and systemic signalling during homeostasis and in disease. Extensive studies have described functional roles for EV populations, including during cardiovascular disease, but the in vivo characterisation of endogenously produced EVs is still in its infancy. Due to their genetic tractability and opportunities for live imaging, zebrafish represent an ideal but under-used model to investigate endogenous EVs. The overall aim of this study was to establish a transgenic zebrafish model to allow the in vivo identification, tracking and extraction of endogenous EVs produced by different cell types. Approach and ResultsUsing a membrane-tethered fluorophore reporter system, we show that EVs can be fluorescently labelled in larval and adult zebrafish and demonstrate that multiple cell types including endothelial cells and cardiomyocytes actively produce EVs in vivo. Cell-type specific EVs can be tracked by high spatiotemporal resolution light-sheet live imaging and modified flow cytometry methods allow these EVs to be further evaluated. Importantly, we demonstrate the utility of this model by showing that cardiomyocytes, endothelial cells and macrophages exchange EVs in the adult heart and that ischaemic injury models dynamically alter EV production. ConclusionsWe have developed a powerful in vivo zebrafish model for the investigation of endogenous EVs in all aspects of cardiovascular biology and pathology. A cell membrane fluorophore labelling approach allows cell type-specific tracing of EV origin without bias towards the expression of individual protein markers and will allow detailed future examination of their function.

cell biology↗

bioRxiv2019

A fast microfluidic device to measure the deformability of red blood cells

We report a microfluidic device to determine the shear elastic modulus and the Youngs modulus of red blood cells (RBCs). Our device consists of a single channel opening into a funnel, with a semi-circular obstacle placed at the mouth of the funnel. As a RBC passes the obstacle, it deflects from its original path. Using populations of artificially-stiffened RBCs, we show that the stiffer RBCs deflect more compared to the normal RBCs. We use calibration curves obtained from numerical simulations to map a trajectory of each RBC to its elastic constants. Our estimates of the shear elastic modulus and the Youngs modulus of normal RBCs are within the same range of values reported in the literature using AFM, optical tweezers and micropipette measurements. We also estimate indirectly the elongation index of normal and artificially hardened RBCs from their tracks, without any direct observation of their shapes. Finally, we sort a mixed population of RBCs based on their deformability alone. Our device could potentially be further miniaturized to sort and obtain the elastic constants of nanoscale objects, whose shape change is difficult to monitor by optical microscopy.

biophysics↗

bioRxiv2019

An integrated one-step assay combining thermal lysis and loop-mediated isothermal DNA amplification (LAMP) in 30 min from E. coli and M. smegmatis cells on a paper substrate

Developing sensors in the domains of food safety, soil analysis, water quality monitoring and healthcare often requires distinguishing between different species of bacteria. The most rapid, sensitive and specific method to identify bacteria is by analysing their DNA sequence, which comprises of disinfection and lysis of bacterial cells, amplification of the isolated DNA and detection of the amplified sequence. Seamless integration of these assays on a paper substrate remains a big challenge in paperfluidic nucleic acid analyis. Combining lysis and isothermal amplification in a single reaction step is difficult because the porosity of paper and the presence of cell debris following lysis reduces the efficiency of DNA amplification. On the other hand, extracting and purifying the DNA after lysis to improve the amplification efficiency involves addition of chemical reagents, one or more wash steps and manual intervention. This problem is even more complex for mycobacteria as its thick cell wall structure impedes lysis and the high GC-content of the genome requires careful optimization of enzymatic denaturation during isothermal amplification. Here we successfully combine thermal lysis and loop-mediated isothermal amplification (LAMP) into a single reaction step on paper without the need for any intermediate intervention. We demonstrate our integrated assay by amplifying DNA from 100 CFU/mL of Escherichia coli (MG1655) and Mycobacterium smegmatis (mc2155) cells in 30 min on a paper substrate. We also confirm that E. coli and M. smegmatis can be completely disinfected on paper by heating at 60 {degrees}C for 5 min and 15 min respectively, making this assay safe and suitable for incorporation into diverse paperfluidic sensors for field use.\n\nElectronic Supplementary Information (ESI) is available.

bioengineering↗

bioRxiv2019

Francisella novicida Cas9 interrogates genomic DNA with very high specificity and can be used for mammalian genome editing

Genome editing using the CRISPR Cas9 system has been used to manipulate eukaryotic DNA and make precise heritable changes. Although the widely used Streptococcus pyogenes Cas9 (SpCas9) and its engineered variants have been efficiently harnessed for numerous gene-editing applications across different platforms, concerns remain, regarding their putative off targeting at multiple loci across the genome. Here we report that Francisella novicida Cas9 (FnCas9) shows a very high specificity of binding to its intended targets and negligible binding to off-target loci. The specificity is determined by its minimal binding affinity with DNA when mismatches to the target sgRNA are present in the sgRNA:DNA heteroduplex. FnCas9 produces staggered cleavage, higher HDR rates and very low non-specific genome editing compared to SpCas9. We demonstrate FnCas9 mediated correction of the sickle cell mutation in patient derived iPSCs and propose that it can be used for precise therapeutic genome editing for a wide variety of genetic disorders.\n\nSIGNIFICANCE STATEMENTTherapeutic genome editing has been significantly accentuated by the advent of CRISPR based gene correction. However, genomic off-targeting has been a major setback for clinical translation. Although high fidelity versions of Cas9 have been rationally designed, they recognize and bind to off-targets. In this study, we characterize a naturally occurring Cas9 from Francisella novicida (FnCas9) that shows negligible binding affinity to off targets differing by one or more mismatches, rendering it highly specific in target recognition and editing. We show that FnCas9 can direct both HDR and NHEJ mediated DNA repair, generates higher rate of HDR and negligible off-target editing. Finally we show its potential in therapeutic genome editing by correcting the sickle cell anemia mutation in patient derived iPSCs.

molecular biology↗

bioRxiv2019

Probing Ligand and Cation Binding Sites in G-Quadruplex Nucleic Acids by Mass Spectrometry and Electron Photodetachment Dissociation Sequencing

Mass spectrometry provides exquisite detail on ligand and cation binding stoichiometries with a DNA target. The next important step is to develop reliable methods to determine the cation and ligand binding sites in each complex separated by the mass spectrometer. To circumvent the caveat of ligand derivatization for cross-linking, which may alter the ligand binding mode, we explored a tandem mass spectrometry (MS/MS) method that does not require ligand derivatization, and is therefore also applicable to localize metal cations. By obtaining more negative charge states for the complexes using supercharging agents, and by creating radical ions by electron photodetachment, oligonucleotide bonds become weaker than the DNA-cation or DNA-ligand noncovalent bonds upon collision-induced dissociation of the radicals. This electron photodetachment (EPD) method allows to locate the binding regions of cations and ligands by top-down sequencing of the oligonucleotide target. The very potent G-quadruplex ligands 360A and PhenDC3 were found to replace a potassium cation and bind close to the central loop of 4-repeat human telomeric sequences.

biophysics↗

bioRxiv2018

Cell and tissue type independent age-associated DNA methylation changes are not rare but common

Age-associated DNA methylation changes have been widely reported across many different tissue and cell types. Epigenetic clocks that can predict chronological age with a surprisingly high degree of accuracy appear to do so independently of tissue and cell-type, suggesting that a component of epigenetic drift is cell-type independent. However, the relative amount of age-associated DNAm changes that are specific to a cell or tissue type versus the amount that occurs independently of cell or tissue type is unclear and a matter of debate, with a recent study concluding that most epigenetic drift is tissue-specific. Here, we perform a novel comprehensive statistical analysis, including matched multi cell-type and multi-tissue DNA methylation profiles from the same individuals and adjusting for cell-type heterogeneity, demonstrating that a substantial amount of epigenetic drift, possibly over 70%, is shared between significant numbers of different tissue/cell types. We further show that ELOVL2 is not unique and that many other CpG sites, some mapping to genes in the Wnt and glutamate receptor signaling pathways, are altered with age across at least 10 different cell/tissue types. We propose that while most age-associated DNAm changes are shared between cell-types that the putative functional effect is likely to be tissue-specific.

bioinformatics↗

bioRxiv2018

Emergence of Multidrug-Resistant Uropathogens harboring ESBL, Carbapenem, Aminoglycosides and AmpC resistant genes from Northern India

Extended-spectrum {beta}-lactamase (ESBL) producing bacteria acts as a serious threat, and its co-existence with other antibiotic resistant gene makes the clinical scenario worse nowadays. Therefore in this study, we investigated the occurrence of ESBL genes coexisting with carbapenem, AmpC and aminoglycoside resistance gene in uropathogens. Out of 1516 urine samples, 454 showed significant bacteriuria with a prevalence rate of 29.94 %. Escherichia coli (n=340) were found to be the most predominant uropathogen followed by Klebsiella pneumoniae (n=92), Pseudomonas aeruginosa (n=10) and Proteus mirabilis (n=9). Among the total uropathogens, sixty-three ESBL-producers were identified which included blaCTX-M-15 (n=32), followed by blaCTX-M-15 + blaOXA-2 (n=15), blaCTX-M-15 + blaOXA-2 + blaTEM (n=6), blaOXA-2 (n=5), bla OXA-2 + bla SHV-76 (n=1), bla TEM+SHV-76 (n= 1) and bla TEM (n=1). All ESBL genes were found on plasmid incompatibility types: HI1, I1, FIA+FIB, FIA and Y and were horizontally transferable. Among 63 ESBL-producers, 59 isolates harboured carbapenem-resistant genes which included blaNDM-5 (n=48), blaNDM-5 + blaOXA-48 (n=5), blaNDM-5 + blaIMP (n=5) and blaNDM-5 + blaIMP + blaVIM (n=1). The ESBL producing uropathogens also harbored 16S rRNA methylase genes which included rmtB (n=9), rmtA (n=4), rmtC (n=1) and ArmA (n=1) followed by AmpC genes which includes CIT (n=8) and DHA-1 (n=1) genes. Imipenem and gentamicin were found to be more effective. We speculating, this is the first report showing the prevalence of multidrug-resistant uropathogens in this area demanding regular surveillance for such resistance mechanisms which will be useful for health personnel to treat ESBL infection and its co-existence with another antibiotic resistance gene.

microbiology↗

bioRxiv2018

Tensorial Blind Source Separation for Improved Analysis of Multi-Omic Data

There is an increased need for integrative analyses of multi-omic data. Although several algorithms for analysing multi-omic data exist, no study has yet performed a detailed comparison of these methods in biologically relevant contexts. Here we benchmark a novel tensorial independent component analysis (tICA) algorithm against current state-of-the-art methods. Using simulated and real multi-omic data, we find that tICA outperforms established methods in identifying biological sources of data variation at a significantly reduced computational cost. Using two independent multi cell-type EWAS, we further demonstrate how tICA can identify, in the absence of genotype information, mQTLs at a higher sensitivity than competing multi-way algorithms. We validate mQTLs found with tICA in an independent set, and demonstrate that approximately 75% of mQTLs are independent of blood cell subtype. In an application to multi-omic cancer data, tICA identifies many gene modules whose expression variation across tumors is driven by copy number or DNA methylation changes, but whose deregulation relative to the normal state is independent such alterations, an important finding that we confirm by direct analysis of individual data types. In summary, tICA is a powerful novel algorithm for decomposing multi-omic data, which will be of great value to the research community.

bioinformatics↗

bioRxiv2018

The role of stochastic sequestration dynamics for intrinsic noise filtering in signaling network motifs

The relation between design principles of signaling network motifs and their robustness against intrinsic noise still remains illusive. In this work we investigate the role of cascading for coping with intrinsic noise due to stochasticity in molecular reactions. We use stochastic approaches to quantify fluctuations in the terminal kinase of phosphorylation-dephosphorylation cascade motifs and demonstrate that cascading highly affects these fluctuations. We show that this purely stochastic effect can be explained by time-varying sequestration of upstream kinase molecules. In particular, we discuss conditions on time scales and parameter regimes which lead to a reduction of output fluctuations. Our results are put into biological context by adapting rate parameters of our modeling approach to biologically feasible ranges for general binding-unbinding and phosphorylation-dephosphorylation mechanisms. Overall, this study reveals a novel role of stochastic sequestration for dynamic noise filtering in signaling cascade motifs.

systems biology↗

bioRxiv2018

DNA methylation oscillation defines classes of enhancers

Understanding the regulatory landscape of human cells requires the integration of genomic and epigenomic maps, capturing combinatorial levels of cell type-specific and invariant activity states.\n\nHere, we segmented whole-genome bisulfite sequencing-derived methylomes into consecutive blocks of co-methylation (COMETs) to obtain spatial variation patterns of DNA methylation (DNAm oscillations) integrated with histone modifications and promoter-enhancer interactions derived from promoter capture Hi-C (PCHi-C) sequencing of the same purified blood cells.\n\nMapping DNAm oscillations onto regulatory genome annotation revealed that enhancers are enriched for DNAm hyper-oscillations (>30-fold), where multiple machine learning models support DNAm as predictive of enhancer location. Based on this analysis, we report overall predictive power of 99% for DNAm oscillations, 77.3% for DNaseI, 41% for CGIs, 20% for UMRs and 0% for LMRs, demonstrating the power of DNAm oscillations over other methods for enhancer prediction. Methylomes of activated and non-activated CD4+ T cells indicate that DNAm oscillations exist in both states irrespective of activation; hence they can be used to determine the location of latent enhancers.\n\nOur approach advances the identification of tissue-specific regulatory elements and outperforms previous approaches defining enhancer classes based on DNA methylation.

genomics↗

bioRxiv2017

ProGeM: A framework for the prioritisation of candidate causal genes at molecular quantitative trait loci

Quantitative trait locus (QTL) mapping of molecular phenotypes such as metabolites, lipids, and proteins through genome-wide association studies (GWAS) represents a powerful means of highlighting molecular mechanisms relevant to human diseases. However, a major challenge of this approach is to identify the causal gene(s) at the observed QTLs. Here we present a framework for the \"Prioritisation of candidate causal Genes at Molecular QTLs\" (ProGeM), which incorporates biological domain-specific annotation data alongside genome annotation data from multiple repositories. We assessed the performance of ProGeM using a reference set of 227 previously reported and extensively curated metabolite QTLs. For 98% of these loci, the expert-curated gene was one of the candidate causal genes prioritised by ProGeM. Benchmarking analyses revealed that 69% of the causal candidates were nearest to the sentinel variant at the investigated molecular QTLs, indicating that genomic proximity is the most reliable indicator of \"true positive\" causal genes. In contrast, cis-gene expression QTL data led to three false positive candidate causal gene assignments for every one true positive assignment. We provide evidence that these conclusions also apply to other molecular phenotypes, suggesting that ProGeM is a powerful and versatile tool for annotating molecular QTLs. ProGeM is freely available via GitHub.

bioinformatics↗

bioRxiv2017

Platelet enrichment from whole blood in a clog-free microfluidic radial pillar device (RAPID)

Pillar-based passive microfluidic devices combine the advantages of simple designs, low device footprint, and high selectivity for size-based separation of blood cells. Most of these device designs have been validated with dilute blood samples. Handling whole blood in pillar-based devices is extremely challenging due to clogging. The high proportion of cells (particularly red blood cells) in blood, the varying sizes and stiffness of the different blood cells, and the tendency of the cells to aggregate lead to clogging of the pillars within a short period. We recently reported a radial pillar device (RAPID) design for contin-uous and high throughput separation of multi-sized rigid polystyrene particles in a single experiment. In this manuscript, we have given detailed guidelines to modify the design of RAPID for any application with deformable objects (e.g. cells). We have adapted RAPID to work with blood samples directly without any pre-processing steps. We were successful in operating the device with whole blood for almost 6 hours, which is difficult to achieve with most pillar-based devices. Finally, we demonstrated up to ~ 60-fold enrichment of platelets as an illustration of the improved device design. Whole blood pillar-based platelet clog-free RAPID

physiology↗

bioRxiv2017

A radial pillar device (RAPID) for continuous and high-throughput separation of multi-sized particles

Pillar-based microfluidic sorting devices are preferred for isolation of rare cells due to their simple designs and passive operation. Dead-end pillar filters can efficiently capture large rare cells, such as, circulating tumor cells (CTCs), nucleated red blood cells (NRBCs), etc., but they get clogged easily. Cross flow filters are preferred for smaller rare particles (e.g. separating bacteria from blood), but they need additional buffer inlets and a large device footprint for efficient operation. We have designed a new microparticle separation device i.e. Radial Pillar Device (RAPID) that combines the advantages of dead-end and cross flow filters. RAPID can simultaneously isolate both large and small rare particles from a mixed population, while functioning for several hours without clogging. We have achieved simultaneous separation of 10{micro}m and 2{micro}m polystyrene particles from a mixture of 2 {micro}m, 7 {micro}m and 10 {micro}m beads. RAPID achieved average separation purity and recovery in excess of 90%. The throughput of our device ( 3ml/min) is 10 and 100 times higher compared to cross flow and dead-end filters respectively, thereby justifying the name RAPID.

bioengineering↗