Biology subjects

Nguyen, T. H.

Publications and source records attributed to Nguyen, T. H..

66 records · Page 4Linked to original sources

bioRxiv2020

iBRAB: in silico based-designed Broad-spectrum Fab against H1N1 Influenza A Virus

Influenza virus A is a significant agent involved in the outbreak of worldwide epidemics, causing millions of fatalities around the world by respiratory diseases and seasonal illness. Many projects had been conducting to investigate recovered infected patients for therapeutic vaccines that have broad-spectrum activity. With the aid of the computational approach in biology, the designation for a vaccine model is more accessible. We developed an in silico protocol called iBRAB to design a broad-reactive Fab on a wide range of influenza A virus. The Fab model was constructed based on sequences and structure of available broad-spectrum Abs or Fabs against a wide range of H1N1 influenza A virus. As a result, the proposed Fab model followed iBRAB has good binding affinity over 27 selected HA of different strains of H1 influenza A virus, including wild-type and mutated ones. The examination also took by computational tools to fasten the procedure. This protocol could be applied for a fast designed therapeutic vaccine against different types of threats.

bioinformatics↗

bioRxiv2020

High-throughput cultivation of stable, diverse, fecal-derived microbial communities to model the intestinal microbiota

SummaryMechanistic understanding of the impacts of the gut microbiota on human health has been hampered by limited throughput in animal models. To enable systematic interrogation of gut-relevant microbial communities, here we generated hundreds of in vitro communities cultured from diverse stool samples in various media. Species composition revealed stool-derived communities that are phylogenetically complex, diverse, stable, and highly reproducible. Community membership depended on both medium and initial inoculum, with certain media preserving inoculum compositions. Different inocula yielded different community compositions, indicating their potential for personalized therapeutics. Communities were robust to freezing and large-volume culturing, enabling future translational applications. Defined communities were generated from isolates and reconstituted growth and composition similar to those of communities derived from stool inocula. Finally, in vitro experiments probing the response to ciprofloxacin successfully predicted many changes observed in vivo, including the resilience and sensitivity of each Bacteroides species. Thus, stool-derived in vitro communities constitute a powerful resource for microbiota research.Competing Interest StatementThe authors have declared no competing interest.View Full Text

microbiology↗

bioRxiv2020

WHOLE-GENOME SEQUENCING AND DE NOVO ASSEMBLY OF A 2019 NOVEL CORONAVIRUS (SARS-COV-2) STRAIN ISOLATED IN VIETNAM

The pandemic COVID-19 caused by the zoonotic virus SARS-CoV-2 has devastated countries worldwide, infecting more than 4.5 million people and leading to more than 300,000 deaths. Whole genome sequencing (WGS) is an effective tool to monitor emerging strains and provide information for intervention, thus help to inform outbreak control decisions. Here, we reported the first effort to sequence and de novo assemble the whole genome of SARS-CoV-2 using PacBios SMRT sequencing technology in Vietnam. We also presented the annotation results and a brief analysis of the variants found in our SARS-CoV-2 strain, which was isolated from a Vietnamese patient. The sequencing was successfully completed and de novo assembled in less than 30 hours, resulting in one contig with no gap and a length of 29,766 bp. All detected variants as compared to the NCBI reference were highly accurate as confirmed by Sanger sequencing. The results have shown the potential of long read sequencing to provide high quality WGS data to support public health responses, and advance understanding of this and future pandemics.

genomics↗

bioRxiv2020

A comparative study of isothermal nucleic acid amplification methods for SARS-CoV-2 detection at point of care

COVID-19, caused by the novel coronavirus SARS-CoV-2, has spread worldwide and put most of the world under lockdown. Despite that there have been emergently approved vaccines for SARS-CoV-2, COVID-19 cases, hospitalizations, and deaths have remained rising. Thus, rapid diagnosis and necessary public health measures are still key parts to contain the pandemic. In this study, the colorimetric isothermal nucleic acid amplification tests (iNAATs) for SARS-CoV-2 detection based on loop-mediated isothermal amplification (LAMP), cross-priming amplification (CPA), and polymerase spiral reaction (PSR) were designed and evaluated. The three methods showed the same limit of detection (LOD) value of 1 copy of the targeted gene per reaction. However, for the direct detection of SARS-CoV-2 genomic-RNA, LAMP outperformed both CPA and PSR, exhibiting the LOD value of roughly 43.14 genome copies/reaction. The results can be read with the naked eye within 45 minutes, without cross-reactivity to closely related coronaviruses. Moreover, the direct detection of SARS-CoV-2 RNA in simulated patient specimens by iNAATs was also successful. Finally, the ready-to-use lyophilized reagents for LAMP reactions were shown to maintain the sensitivity and LOD value of the liquid assays. The results indicate that the colorimetric lyophilized LAMP kit developed herein is highly suitable for detecting SARS-CoV-2 nucleic acids at point-of-care.

molecular biology↗

bioRxiv2020

Weekend light shifts evoke persistent Drosophila circadian neural network desynchrony

Billions of people subject themselves to phase-shifting light signals on a weekly basis by remaining active later at night and sleeping in later on weekends relative to weekday for up to a 3hr weekend light shift (WLS). Unnatural light signals disrupt circadian rhythms and physiology and behavior. Real-time light responses of mammalian suprachiasmatic nucleus are unmeasurable at single cell resolution. We compared Drosophila whole-circadian circuit responses between unshifted daytime/nighttime schedule and a 3hr WLS schedule at the single-cell resolution in cultured adult Drosophila brains using real-time bioluminescence imaging of the PERIOD protein for 11 days to determine how light shifts alter biological clock entrainment and stability. We find that circadian circuits show highly synchronous oscillations across all major circadian neuronal subgroups in unshifted light schedules. In contrast, circadian circuits exposed to a WLS schedule show significantly dampened oscillator synchrony and rhythmicity in most circadian neurons during, and after exposure. The WLS schedule first desynchronizes lateral ventral neuron (LNv) oscillations and the LNv are the last to resynchronize upon returning to a simulated weekday schedule. Surprisingly, one circadian subgroup, the dorsal neuron group-3 (DN3s), robustly increase their within-group synchrony in response to WLS exposure. Intact adult flies exposed to the WLS schedule show post-WLS transient defects in sleep stability, learning, and memory. Our findings suggest that WLS schedules disrupt circuit-wide circadian neuronal oscillator synchrony for much of the week, thus leading to observed behavioral defects in sleep, learning, and memory. Significance StatementThe circadian clock controls numerous aspects of daily animal physiology, metabolism and behavior. Shift work in humans is harmful. Our understanding of circadian circuit-level oscillations stem from ex vivo imaging of mammalian suprachiasmatic nucleus (SCN) brain slices. However, our knowledge is limited to investigations without direct interrogation of phase-shifting light signals. We measured circuit-level circadian responses to a WLS protocol in light sensitive ex vivo Drosophila whole-brain preparation and find robust sub-circuit-specific oscillator desynchrony/resynchrony responses to light. These circuit-level behaviors correspond to our observed functional defects in learning and memory, and sleep pattern disruption in vivo. Our results reflect that WLS cause circadian-circuit desynchronization and correlate with disrupted cognitive and sleep performance.

neuroscience↗

bioRxiv2019

Selective disruption of synaptic BMP signaling by a Smad mutation adjacent to the highly conserved H2 helix

Bone morphogenetic proteins (BMPs) shape normal development and function via canonical and non-canonical signaling pathways. When activating the canonical pathway, BMPs initiate signaling by binding to transmembrane receptors that phosphorylate pathway effectors, the Smad proteins, inducing their translocation into the nucleus and thus regulation of target genes. Phosphorylated Smads also accumulate at cellular junctions, but this non-canonical signaling modality remains less defined. We have recently reported that phosphorylated Smad (pMad in Drosophila) accumulates at synaptic junctions in complexes with genetically distinct composition and regulation. Here we examined a wide collection of Drosophila Mad alleles and searched for molecular features relevant to pMad accumulation at synaptic junctions. We found that strong Mad alleles generally disrupt both synaptic and nuclear pMad accumulation, whereas moderate Mad alleles have a wider range of phenotypes and could selectively impact different BMP signaling modalities. Interestingly, synaptic pMad appeared more sensitive to net reduction in Mad levels than nuclear pMad. Importantly, a previously uncharacterized allele, Mad8, showed markedly reduced synaptic pMad levels but only moderately diminished nuclear pMad signals. The postsynaptic composition and electrophysiological properties of Mad8 NMJs were similarly altered. Using biochemical approaches, we examined how single point mutations such as S359L, present in Mad8, could influence the Mad-receptor interface and we identified a key molecular determinant, the H2 helix. Our study highlights the biological relevance of the Smad-dependent, non-canonical BMP signaling and uncovers a highly conserved structural feature of Smads, critical for normal development and function.

genetics↗

bioRxiv2019

Metabolic Profiling of Aortic Stenosis and Hypertrophic Cardiomyopathy Identifies Mechanistic Contrasts in Substrate Utilisation

BackgroundAortic stenosis (AS) and hypertrophic cardiomyopathy (HCM) are highly distinct disorders leading to left ventricular hypertrophy (LVH), but whether cardiac metabolism substantially differs between these in humans remains to be elucidated.\n\nMethodWe undertook a detailed invasive (aortic root and coronary sinus) metabolic profiling in patients with severe AS and HCM in comparison to non-LVH controls, to investigate cardiac fuel selection and metabolic remodelling. These patients were assessed under different physiological states (at rest and during stress induced by pacing). The identified changes in the metabolome were further validated by metabolomic and orthogonal transcriptomic analysis, in separately recruited patient cohorts.\n\nResultsWe identified a highly discriminant metabolomic signature in severe AS characterised by striking accumulation of long-chain acylcarnitines, intermediates of long-chain transport and fatty acid metabolism, and validated this in a separate cohort. Mechanistically, we identify a down-regulation in the PPAR- transcriptional network, including expression of genes regulating FAO.\n\nConclusionsWe present a comprehensive analysis of changes in the metabolic pathways (transcriptome to metabolome) in severe AS, and its comparison to HCM. Our results demonstrate fundamental distinctions in substrate preference between AS and HCM, highlighting insufficient long-chain FAO, and the PPAR- signalling network as a specific metabolic therapeutic target in AS.

systems biology↗

bioRxiv2019

An Interscholastic Network to Generate LexA Enhancer Trap Lines in Drosophila

Binary expression systems like the LexA-LexAop system provide a powerful experimental tool kit to study gene and tissue function in developmental biology, neurobiology and physiology. However, the number of well-defined LexA enhancer trap insertions remains limited. In this study, we present the molecular characterization and initial tissue expression analysis of nearly 100 novel StanEx LexA enhancer traps, derived from the StonEx1 index line. This includes 76 insertions into novel, distinct gene loci not previously associated with enhancer traps or targeted LexA constructs. Additionally, our studies revealed evidence for selective transposase-dependent replacement of a previously-undetected KP element on chromosome III within the StanEx1 genetic background during hybrid dysgenesis, suggesting a molecular basis for the over-representation of LexA insertions at the NK7.1 locus in our screen. Production and characterization of novel fly lines were performed by students and teachers in experiment-based genetics classes within a geographically diverse network of public and independent high schools. Thus, unique partnerships between secondary schools and university-based programs have produced and characterized novel genetic and molecular resources in Drosophila for open-source distribution, and provide paradigms for development of science education through experience-based pedagogy.

genetics↗

bioRxiv2019

Activity-by-Contact model of enhancer specificity from thousands of CRISPR perturbations

Mammalian genomes harbor millions of noncoding elements called enhancers that quantitatively regulate gene expression, but it remains unclear which enhancers regulate which genes. Here we describe an experimental approach, based on CRISPR interference, RNA FISH, and flow cytometry (CRISPRi-FlowFISH), to perturb enhancers in the genome, and apply it to test >3,000 potential regulatory enhancer-gene connections across multiple genomic loci. A simple equation based on a mechanistic model for enhancer function performed remarkably well at predicting the complex patterns of regulatory connections we observe in our CRISPR dataset. This Activity-by-Contact (ABC) model involves multiplying measures of enhancer activity and enhancer-promoter 3D contacts, and can predict enhancer-gene connections in a given cell type based on chromatin state maps. Together, CRISPRi-FlowFISH and the ABC model provide a systematic approach to map and predict which enhancers regulate which genes, and will help to interpret the functions of the thousands of disease risk variants in the noncoding genome.

genetics↗

bioRxiv2018

Phylotastic: improving access to tree-of-life knowledge with flexible, on-the-fly delivery of trees

(1) A comprehensive phylogeny of species, i.e., a tree of life, has potential uses in a variety of contexts in research and education. This potential is limited if accessing the tree of life requires special knowledge, complex software, or long periods of training.\n\n(2) The Phylotastic project aims to use web-services technologies to lower the barrier for accessing phylogenetic knowledge, making it as easy to get a phylogeny of species as it is to get online driving directions. In prior work, we designed an open system of web services to validate and manage species names, find phylogeny resources, extract subtrees matching a users species list, calibrate them, and mash them up with images and information from online resources.\n\n(3) Here we report a publicly accessible system for on-the-fly delivery of phylogenetic knowledge, developed with user feedback on what types of functionality are considered useful by researchers and educators. The system currently consists of a web portal that implements 3 types of workflows to obtain species phylogenies (scaled by geologic time and decorated with thumbnail images); 19 underlying web services accessible via a common registry; and toolbox code in R and Python so that others can create applications that leverage these services. These resources cover most of the use-cases identified in our analysis of user needs.\n\n(4) The Phylotastic system, accessible via http://www.phylotastic.org, provides a unique resource to access the current state of phylogenetic knowledge, useful for a variety of cases in which a tree extracted quickly from online resources (as distinct from a tree custom-made from character data) is sufficient, as it is for many casual uses of trees identified here.

bioinformatics↗

bioRxiv2018

Bayesian analysis of isothermal titration calorimetry for binding thermodynamics

Isothermal titration calorimetry (ITC) is the only technique able to determine both the enthalpy and entropy of noncovalent association in a single experiment. The standard data analysis method based on nonlinear regression, however, provides unrealistically small uncertainty estimates due to its neglect of dominant sources of error. Here, we present a Bayesian framework for sampling from the posterior distribution of all thermodynamic parameters and other quantities of interest from one or more ITC experiments, allowing uncertainties and correlations to be quantitatively assessed. For a series of ITC measurements on metal:chelator and protein:ligand systems, the Bayesian approach yields uncertainties which represent the variability from experiment to experiment more accurately than the standard data analysis. In some datasets, the median enthalpy of binding is shifted by as much as 1.5 kcal/mol. A Python implementation suitable for analysis of data generated by MicroCal instruments (and adaptable to other calorimeters) is freely available online.

biophysics↗

bioRxiv2017

Label-free quantitative screening of breast tissue using Spatial Light Interference Microscopy (SLIM)

Breast cancer is the most common type of cancer among women worldwide. The standard histopathology of breast tissue, the primary means of disease diagnosis, involves manual microscopic examination of stained tissue by a pathologist. Because this method relies on qualitative information, it can result in inter-observer variation. Furthermore, for difficult cases the pathologist often needs additional markers of malignancy to help in making a diagnosis. We present a quantitative method for label-free tissue screening using Spatial Light Interference Microscopy (SLIM). By extracting tissue markers of malignancy based on the nanostructure revealed by the optical path-length, our method provides an objective and potentially automatable method for rapidly flagging suspicious tissue. We demonstrated our method by imaging a tissue microarray comprising 68 different subjects - 34 with malignant and 34 with benign tissues. Three-fold cross validation results showed a sensitivity of 94% and specificity of 85% for detecting cancer. The quantitative biomarkers we extract provide a repeatable and objective basis for determining malignancy. Thus, these disease signatures can be automatically classified through machine learning packages, since our images do not vary from scan to scan or instrument to instrument, i.e., they represent intrinsic physical attributes of the sample, independent of staining quality.

pathology↗