Biology subjects

Khan, S.

Publications and source records attributed to Khan, S..

At least 199 records · Page 11Linked to original sources

bioRxiv2020

A Modular Microarray Imaging System for Highly Specific COVID-19 Antibody Testing

To detect the presence of antibodies in blood against SARS-CoV-2 in a highly sensitive and specific manner, here we describe a robust, inexpensive ($200), 3D-printable portable imaging platform (TinyArray imager) that can be deployed immediately in areas with minimal infrastructure to read coronavirus antigen microarrays (CoVAMs) that contain a panel of antigens from SARS-CoV-2, SARS-1, MERS, and other respiratory viruses. Application includes basic laboratories and makeshift field clinics where a few drops of blood from a finger prick could be rapidly tested in parallel for the presence of antibodies to SARS-CoV-2 with a test turnaround time of only 2-4 h. To evaluate our imaging device, we probed and imaged coronavirus microarrays with COVID-19-positive and negative sera and achieved a performance on par with a commercial microarray reader 100x more expensive than our imaging device. This work will enable large scale serosurveillance, which can play an important role in the months and years to come to implement efficient containment and mitigation measures, as well as help develop therapeutics and vaccines to treat and prevent the spread of COVID-19.

microbiology↗

bioRxiv2020

Genome Analysis of SARS-CoV-2 Isolate from Bangladesh

Recently the first genome sequence for a Severe acute respiratory syndrome coronavirus 2 or SARS-CoV-2 isolate from Bangladesh became available. The sequencing was carried out by the Child Health Research Foundation and provided the first insight into the genetic details of the viral strain responsible for the SARS-CoV-2 infections in Bangladesh. Here we carried out a comparative study were we explored the phylogenetic relationship between the Bangladeshi isolate with other isolates from different parts of the world. Afterwards we identified single nucleotide variants in the Bangladeshi isolate, using the Wuhan virus reference sequence. We found a total of 9 variants in the Bangladeshi isolate using 2 separate tools. Barring 2, the rest of these variants were also observed in other isolates from different countries. Most of the variants occurred in the ORF1ab gen. Another noteworthy finding was a sequence of three consecutive variants in the N protein gene that were observed in other isolates as well. Lastly the phylogenetic analysis revealed a close relationship between the Bangladeshi isolate and those from Taiwan, Kazakhstan, Greece, California, Spain, Israel, and Sri Lanka.

genomics↗

bioRxiv2020

Analysis of SARS-CoV-2 Antibodies in COVID-19 Convalescent Plasma using a Coronavirus Antigen Microarray

The current practice for diagnosis of COVID-19, based on SARS-CoV-2 PCR testing of pharyngeal or respiratory specimens in a symptomatic patient at high epidemiologic risk, likely underestimates the true prevalence of infection. Serologic methods can more accurately estimate the disease burden by detecting infections missed by the limited testing performed to date. Here, we describe the validation of a coronavirus antigen microarray containing immunologically significant antigens from SARS-CoV-2, in addition to SARS-CoV, MERS-CoV, common human coronavirus strains, and other common respiratory viruses. A comparison of antibody profiles detected on the array from control sera collected prior to the SARS-CoV-2 pandemic versus convalescent blood specimens from virologically confirmed COVID-19 cases demonstrates near complete discrimination of these two groups, with improved performance from use of antigen combinations that include both spike protein and nucleoprotein. This array can be used as a diagnostic tool, as an epidemiologic tool to more accurately estimate the disease burden of COVID-19, and as a research tool to correlate antibody responses with clinical outcomes.

immunology↗

bioRxiv2020

The RecQ DNA helicase Rqh1 promotes Rad3ATR kinase signaling in the DNA replication checkpoint pathway of fission yeast

Rad3 is the orthologue of ATR and the sensor kinase of the DNA replication checkpoint in Schizosaccharomyces pombe. Under replication stress, it initiates checkpoint signaling at the forks necessary for maintaining genome stability and cell survival. To better understand the checkpoint initiation process, we have carried out a genetic screen in fission yeast by random mutation of the genome looking for mutants with defects in Rad3 kinase signaling. In addition to the previously reported tel2-C307Y mutant (1), this screen has identified six mutations in rqh1 encoding a RecQ DNA helicase. Surprisingly, these rqh1 mutations except a start codon mutation are all in the helicase domain, indicating that the helicase activity of Rqh1 plays an important role in the replication checkpoint. In support of this notion, integration of two helicase-inactive mutations or deletion of rqh1 generated a similar Rad3 signaling defect and heterologous expression of human RECQ1, BLM and RECQ4 restored the Rad3 signaling and partially rescued a rqh1 helicase mutant. Therefore, the replication checkpoint function of Rqh1 is highly conserved and mutations in the helicase domain of these human enzymes may cause the checkpoint defect and contribute to the cancer predisposition syndromes.

cell biology↗

bioRxiv2020

Analysis of Serologic Cross-Reactivity Between Common Human Coronaviruses and SARS-CoV-2 Using Coronavirus Antigen Microarray

The current practice for diagnosis of SARS-CoV-2 infection relies on PCR testing of nasopharyngeal or respiratory specimens in a symptomatic patient at high epidemiologic risk. This testing strategy likely underestimates the true prevalence of infection, creating the need for serologic methods to detect infections missed by the limited testing to date. Here, we describe the development of a coronavirus antigen microarray containing immunologically significant antigens from SARS-CoV-2, in addition to SARS-CoV, MERS-CoV, common human coronavirus strains, and other common respiratory viruses. A preliminary study of human sera collected prior to the SARS-CoV-2 pandemic demonstrates overall high IgG reactivity to common human coronaviruses and low IgG reactivity to epidemic coronaviruses including SARS-CoV-2, with some cross-reactivity of conserved antigenic domains including S2 domain of spike protein and nucleocapsid protein. This array can be used to answer outstanding questions regarding SARS-CoV-2 infection, including whether baseline serology for other coronaviruses impacts disease course, how the antibody response to infection develops over time, and what antigens would be optimal for vaccine development.

immunology↗

bioRxiv2020

Computational vaccinology based development of multi-epitope subunit vaccine for protection against the Norovirus infections

Human Norovirus belong to family Calciviridae, it was identified in the outbreak of gastroenteritis in Norwalk, due to its seasonal prevalence known as "winter vomiting disease". Treatment of Norovirus infection is still mysterious because there is no effective antiviral drugs or vaccine developed to protect against the infection, to eradicate the infection an effective vaccine should be developed. In this study capsid protein (A7YK10), small protein (A7YK11) and polyprotein (A7YK09) were utilized. These proteins were subjected to B and T cell epitopes prediction by using reliable immunoinformatics tools. The antigenic and non-allergenic epitopes were selected for subunit vaccine, which can activate cellular and humoral immune responses. Linkers joined these epitopes together. The vaccine structure was modelled and validated by using Errat, ProSA and rampage servers. The modelled vaccine was docked with TLR-7. Stability of the docked complex was evaluated by MD simulation. In order to apply the concept in a wet lab, the reverse translated vaccine sequence was cloned in pET28a (+). The vaccine developed in this study requires experimental validation to ensure its effectiveness against the disease.

immunology↗

bioRxiv2020

Mutagenic effectiveness and efficiency of gamma rays and sodium azide in M2 generation of Cowpea

Legumes play a pivotal role in combating the chronic hunger and malnutrition in the developing nations and are also ideal crops to achieve global food and nutrition security. In the era of climate change, erratic rainfalls, depleting arable land and water resource, feeding the rapidly growing population is a challenging task. Like most other pulses, cowpea is a self pollinated, nutritious, versatile and widely adapted grain legume, but harbor a little accessible genetic variability. Lack of sufficient genetic variability and small size of flowers, traditional plant breeding methods are not enough to meet the demands of improvement of cowpea. Hence, induced mutagenesis was employed to induce significant genetic variability across a range of agro-economical traits in two cowpea varieties Gomati VU-89 and Pusa- 578 from M1 to M4 generations. The success of induced mutagenesis largely depends on the selection of appropriate mutagen, its dose, effectiveness and efficiency. Hence present study was conduct to assess the effectiveness and efficiency of single and combined doses of sodium azide and gamma rays to set an appropriate protocol for induced mutagenesis experimentation in cowpea.

plant biology↗

bioRxiv2020

Long-read genome sequencing and assembly of Leptopilina boulardi: a specialist Drosophila parasitoid

Leptopilina boulardi (Hymenoptera: Figitidae) is a specialist parasitoid of Drosophila. The Drosophila-Leptopilina system has emerged as a suitable model for understanding several aspects of host-parasitoid biology. However, a good quality genome of the wasp counterpart was lacking. Here, we report a whole-genome assembly of L. boulardi to bring it in the scope of the applied and fundamental research on Drosophila parasitoids with access to epigenomics and genome editing tools. The 375Mb draft genome has an N50 of 275Kb with 6315 scaffolds >500bp and encompasses >95% complete BUSCOs. Using a combination of ab-initio and RNA-Seq based methods, 25259 protein-coding genes were predicted and 90% (22729) of them could be annotated with at least one function. We demonstrate the quality of the assembled genome by recapitulating the phylogenetic relationship of L. boulardi with other Hymenopterans. The key developmental regulators like Hox genes and sex determination genes are well conserved in L. boulardi, and so is the basic toolkit for epigenetic regulation. The search for epigenetic regulators has also revealed that L. boulardi genome possesses DNMT1 (maintenance DNA methyltransferase), DNMT2 (tRNA methyltransferase) but lacks the de novo DNA methyltransferase (DNMT3). Also, the heterochromatin protein 1 family appears to have expanded as compared to other hymenopterans. The draft genome of L. boulardi (Lb17) will expedite the research on Drosophila parasitoids. This genome resource and early indication of epigenetic aspects in its specialization make it an interesting system to address a variety of questions on host-parasitoid biology.

genomics↗

bioRxiv2020

Identification of a novel HIF-1α-αMβ2 Integrin-NETosis axis in fibrotic interstitial lung disease

Neutrophilic inflammation correlates with mortality in fibrotic interstitial lung disease (ILD) however, the underlying mechanisms remain unclear. We aimed to determine whether aberrant neutrophil activation is a feature of ILD and the relative role of hypoxia. We used lung biopsies and bronchoalveolar lavage (BAL) from ILD patients to investigate the extent of hypoxia and neutrophil activation in lungs of patients with ILD. We complemented these findings with ex vivo functional studies of neutrophils from healthy volunteers to determine the effects of hypoxia. We demonstrate for the first time HIF-1 staining in neutrophils and endothelial cells in ILD lung biopsies. Hypoxia enhanced both spontaneous and phorbol 12-myristate 13-acetate (PMA)-induced neutrophil extracellular trap (NET) release (NETosis), neutrophil adhesion, and trans-endothelial migration. Hypoxia also increased neutrophil expression of the M and X integrin subunits. Interestingly, NETosis was induced by M{beta}2 integrin activation and prevented by cation chelation. Finally, NETs were demonstrated in the BAL from ILD patients, and quantification showed significantly increased cell-free DNA content and MPO-citrullinated histone H3 complexes in ILD patients compared to non-ILD controls. Our work indicates that HIF-1 upregulation may augment neutrophil recruitment and activation within the lung interstitium through activation of {beta}2 integrins. Our results identify a novel HIF-1-Integrin-NETosis axis for future exploration in therapeutic approaches to fibrotic ILD.

immunology↗

bioRxiv2019

Factors Affecting Detection Of GMO Traces In Food And Crop Samples

Rice is one of the highly consumable and demanded food crops throughout the world. To meet this requirement, genetically modified (GM) crops were introduced, which were met with sturdy and frequently angry opposition by the user. To handle the situation labeling of GM food / crop was advised, thus demand for traceability and detection of unknown modified genes increased. Modified genes are detected by various methods; these methods are affected by several factors as reported in international standards. Those factors include presence / absence of shells, husk, and dust, method of DNA extraction and different PCR primers. These factors ultimately pose hindrance in detection of modified genes. This study was conducted to analyze the effects of the above mentioned factors on detection of GM rice samples. Furthermore two different sets of primers were used with same samples and their impact on the PCR detection was observed. The results showed a significant difference in DNA concentration between the rice samples with husk, without husk and after seed germination. Furthermore, the change of PCR primer set also affected the detection of genetic modifications. This allows analysis of the potential factors that may have impact on the final results.

plant biology↗

bioRxiv2019

The contribution of PIP2-type aquaporins to photosynthesis in response to increased vapour pressure deficit

Roles of three different plasma membrane aquaporins (PIPs) in leaf-level gas exchange of Arabidopsis thaliana were examined using single, double and triple knockout mutants and compared to the Columbia-0 wild type (WT) plants. Since multiple Arabidopsis PIPs are implicated in conducting carbon dioxide across membranes, we focused on identifying whether the examined isoforms affect photosynthesis, either mediated through the control of stomatal conductance to water vapour (gs) or mesophyll conductance of CO2 (gm) or a combination of both. In two separate studies, we grew Arabidopsis plants in a low humidity environment and under high humidity conditions. We found that the contribution of functional PIPs to gs was larger under conditions of low air humidity when the evaporative demand was high, whereas any effect of lacking PIP function was minimal under higher humidity conditions. The pip2;4 knockout mutants had 44% higher gs than the WT under low humidity conditions, which in turn resulted in an increased photosynthetic rate (Anet). AtPIP2;4 is thus likely to be involved in maintaining a positive water balance and high water use efficiency through mediation of transmembrane water flow. The lack of functional AtPIP2;5 on the other hand did not affect gs, but reduced gm indicating a possible role in regulating CO2 membrane permeability. This potential regulatory function was indeed confirmed by subsequent stopped flow measurements of yeast expressing AtPIP2;5.

plant biology↗

bioRxiv2019

Prolonged tonic pain in healthy humans disrupts intrinsic brain networks implicated in pain modulation

Neural mechanisms of ongoing nociceptive processing in the human brain remain largely obscured by the dual challenge of accessing neural dynamics and safely applying sustained painful stimuli. Recently, pain-related neural processing has been measured using fMRI resting state functional connectivity (FC) in chronic pain patients. However, ongoing pain-related processing in normally pain-free humans remains incompletely understood. Therefore, differences between chronic pain patients and controls may be due to comorbidities with chronic pain. Decreased FC among regions of the descending pain modulation network (DPMN) are associated with presence and severity of chronic pain disorders. We aimed to determine if the presence of prolonged tonic pain would lead to disruption of the DPMN. High (10%) concentration topical capsaicin was combined with a warm thermode applied to the leg to create a flexible, prolonged tonic pain model to study the FC of brain networks in otherwise healthy, pain-free subjects in two separate cohorts (n=18; n=32). We contrasted seed-based FC during prolonged tonic pain with a pain-free passive task. In seed-based FC analysis prolonged tonic pain led to enhanced FC between the anterior middle cingulate cortex (aMCC) and the somatosensory leg representation. Additionally, FC was enhanced between the pregenual anterior cingulate cortex (pACC), right mediodorsal thalamus and the posterior parietal cortex bilaterally. Further, in the seed-driven PAG network, positive FC with the left DLPFC became negative FC during prolonged tonic pain. These data suggest that some altered DPMN FC findings in chronic pain could partially be explained by the presence of ongoing pain.

neuroscience↗

bioRxiv2019

Multiclass Disease Classification from Microbial Whole-Community Metagenomes using Graph Convolutional Neural Networks.

There is a wealth of information contained within ones microbiome regarding their physiology and environment, and this is a promising avenue for developing non-invasive diagnostic tools. Here, we utilize 5643 aggregated, annotated whole-community metagenomes from 19 different diseases to implement the first multiclass microbiome disease classifier of this scale. We compared three different machine learning models: random forests, deep neural nets, and a novel graph convolutional architecture which exploits the graph structure of phylogenetic trees as its input. We show that the graph convolutional model outperforms deep neural nets in terms of accuracy (achieving 75% average test-set accuracy), receiver-operator-characteristics (92.1% average AUC), and precision-recall (50% average AUPR). Additionally, the convolutional nets performance complements that of the random forest, achieving similar accuracy but better receiver-operator-characteristics and lower area under precision-recall. Lastly, we are able to achieve over 90% average top-3 accuracy across all of our models. Together, these results indicate that there are predictive, disease specific signatures across microbiomes which could potentially be used for diagnostic purposes.

bioinformatics↗

bioRxiv2019

Competition among Gardnerella subgroups from the human vaginal microbiome

Gardnerella spp. are hallmarks of bacterial vaginosis, a clinically significant dysbiosis of the vaginal microbiome. Gardnerella has four subgroups (A, B, C and D) based on cpn60 sequences. Multiple subgroups are often detected in individual women, and interactions between these subgroups are expected to influence their population dynamics and associated signs and symptoms of bacterial vaginosis. In the present study, contact-independent and contact-dependent interactions between the four Gardnerella subgroups were investigated in vitro. The cell free supernatants of mono- and co-cultures had no effect on growth rates of the Gardnerella subgroups suggesting that there are no contact-independent interactions (and no contest competition). For contact-dependent interactions, mixed communities of 2, 3 or 4 subgroups were created and the initial (0 h) and final population sizes (48 h) were quantified using subgroup-specific PCR. Compared to the null hypothesis of additive interactions, most (69.3%) of the mixed communities exhibited competition (p < 0.0001). Competition reduced the growth rates of subgroups A, B and C. In contrast, the growth rate of subgroup D increased in the presence of the other subgroups (p < 0.0001). All subgroups were able to form biofilm alone and in mixed communities. Our study suggests that there is scramble competition among Gardnerella subgroups, which likely contributes to the observed distributions of Gardnerella spp. in vaginal microbiomes and the formation of the multispecies biofilms characteristic of bacterial vaginosis.

microbiology↗

bioRxiv2019

Bioactivity-guided isolation of rosmarinic acid as a principle bioactive compound from the butanol extract of Isodon rugosus against pea aphid, Acyrthosiphon pisum

Aphids are agricultural pest insects that transmit viruses and cause feeding damage on a global scale. Current pest control involving the excessive use of synthetic insecticides over decades has led to multiple forms of aphid resistance to most classes of insecticides. In nature, plants produce secondary metabolites during their interaction with insects and these metabolites can act as toxicants, antifeedants, anti-oviposition agents and deterrents towards the insects. In a previous study, we demonstrated that the butanol fraction from a crude methanolic extract of an important plant species, Isodon rugosus showed strong insecticidal activity against the pea aphid, Acyrthosiphon pisum. It was however not known as which compound was responsible for such activity. To further explore this finding, current study aimed to exploit a bioactivity-guided strategy to isolate and identify the active compound in the butanol fraction of I. rugosus. As such, reversed-phase flash chromatography, acidic extraction and different spectroscopic techniques were used to isolate and identify the new compound, rosmarinic acid as the bioactive compound in I. rugosus. Insecticidal activity of rosmarinic acid was carried out using standard protocols on A. pisum. The data was analyzed using qualitative and quantitative statistical approaches. Considering that a very low concentration of this compound (LC90 = 5.4 ppm) causes significant mortality in A. pisum within 24 h, rosmarinic acid could be exploited as a potent insecticide against this important pest insect. Furthermore, I. rugosus is already used for medicinal purposes and rosmarinic acid is known to reduce genotoxic effects induced by chemicals, hence it is expected to be safer compared to the current conventional pesticides. While this study highlights the potential of I. rugosus as a possible biopesticide source against A. pisum, it also provides the basis for further exploration and development of formulations for effective field application.

plant biology↗

bioRxiv2019

Conformational coupling by trans-phosphorylation in calcium calmodulin dependent kinase II

The calcium calmodulin dependent protein kinase II (CaMKII) is a dodecameric holoenzyme important for encoding memory. Its activation, triggered by binding of calcium calmodulin, persists autonomously after calmodulin dissociation. One (receiver) kinase captures and subsequently phosphorylates the regulatory domain peptide of a donor kinase forming a chained dimer as a first stage of autonomous activation. Protein dynamics simulations examined the conformational changes triggered by dimer formation and phosphorylation, aimed to provide a molecular rationale for human mutations that result in learning disabilities. Ensembles generated from X-ray crystal structures were characterized by network centrality and community analysis. Mutual information related collective motions to local fragment dynamics encoded with a structural alphabet. Implicit solvent tCONCOORD conformational ensembles revealed the dynamic architecture of Inactive kinase do-mains was co-opted in the activated dimer but the network hub shifted from the nucleotide binding cleft to the captured peptide. Explicit solvent molecular dynamics (MD) showed nucleotide and substrate binding determinants formed coupled nodes in long-range signal relays between regulatory peptides in the dimer. Strain in the extended captured peptide was balanced by reduced flexibility of the receiver kinase C-lobe core. The relays were organized around a hydrophobic patch between the captured peptide and a key binding helix. The human mutations aligned along the relays. Thus, these mutations could disrupt the allosteric network alternatively, or in addition, to altered binding affinities. Non-binding protein sectors distant from the binding sites mediated the allosteric signalling; providing possible targets for inhibitor design. Phosphorylation of the peptide modulated the dielectric of its binding pocket to strengthen the patch, non-binding sectors, domain interface and temporal correlations between parallel relays. These results provide the molecular details underlying the reported positive kinase cooperativity to enrich discussion on how autonomous activation by phosphorylation leads to long-term behavioural effects.

neuroscience↗

bioRxiv2018

A simple cloning-free method to efficiently induce gene expression using CRISPR/Cas9

Gain-of-function studies often require the tedious cloning of transgene cDNA into vectors for overexpression beyond the physiological expression levels. The rapid development of CRISPR/Cas technology presents promising opportunities to address these issues. Here we report a simple, cloning-free method to induce gene expression at endogenous locus using CRISPR/Cas9 activators. Our strategy utilises synthesized sgRNA expression cassettes to direct a nuclease-null Cas9 complex fused with transcriptional activators (VP64, p65 and Rta) for site-specific induction of endogenous genes. This strategy allows rapid initiation of gain-of-function studies in the same day. Using this cloning-free approach, we tested two CRISPR activation systems, dSpCas9VPR and dSaCas9VPR, for induction of multiple genes in human and rat cells. Our results showed that both CRISPR activators allow efficient induction of six different neural development genes (CRX, RORB, RAX, OTX2, ASCL1 and NEUROD1) in human cells, whereas the rat cells exhibit a more variable and less efficient levels of gene induction, as observed in three different genes (Ascl1, Neurod1, Nrl). Altogether, this study provides a simple method to efficiently activate endogenous gene expression using CRISPR/Cas9 activators, which can be applies as a rapid workflow to initiate gain-of-function studies for a range of molecular and cell biology disciplines.

molecular biology↗

bioRxiv2018

Metabolically activated macrophages in mammary adipose tissue link obesity to triple-negative breast cancer

Obesity is associated with increased incidence and severity of triple-negative breast cancer (TNBC); however, mechanisms underlying this relationship are incompletely understood. Macrophages, which accumulate in adipose tissue and are activated during obesity, are an attractive mechanistic link. Here, we show that, during obesity, murine and human mammary adipose tissue macrophages adopt a pro-inflammatory, metabolically- activated (MMe) macrophage phenotype that promotes TNBC stem-like markers and functions, including increased tumorsphere growth in vitro and tumor-initiating potential in vivo. We demonstrate that MMe macrophages release cytokines in an NADPH oxidase 2 (NOX2)-dependent manner that signal through glycoprotein 130 (GP130) on TNBC cells to promote their stem-like properties. Accordingly, deleting Nox2 in myeloid cells or depleting GP130 in TNBC cells attenuates the ability of obesity to drive TNBC tumor formation. Our studies implicate MMe macrophage accumulation in mammary adipose tissue during obesity as a mechanism for promoting TNBC stemness and tumorigenesis.\n\nHIGHLIGHTS Obesity promotes TNBC tumor formation and stemness.\nMammary adipose tissue macrophages are metabolically activated (MMe) in obese mice and humans.\nMMe macrophages in mammary adipose tissue contribute to obesity-induced stemness.\nMMe macrophages promote TNBC stemness through GP130 signaling.

cancer biology↗