Biology subjects

Nguyen, H.

Publications and source records attributed to Nguyen, H..

6 recordsLinked to original sources

bioRxiv2018

An open-source software analysis package for Microspheres with Ratiometric Barcode Lanthanide Encoding (MRBLEs)

Multiplexed bioassays, in which multiple analytes of interest are probed in parallel within a single small volume, have greatly accelerated the pace of biological discovery. Bead-based multiplexed bioassays have many technical advantages, including near solution-phase kinetics, small sample volume requirements, many within-assay replicates to reduce measurement error, and, for some bead materials, the ability to synthesize analytes directly on beads via solid-phase synthesis. To allow bead-based multiplexing, analytes can be synthesized on spectrally encoded beads with a 1:1 linkage between analyte identity and embedded codes. Bead-bound analyte libraries can then be pooled and incubated with a fluorescently-labeled macromolecule of interest, allowing downstream quantification of interactions between the macromolecule and all analytes simultaneously via imaging alone. Extracting quantitative binding data from these images poses several computational image processing challenges, requiring the ability to identify all beads in each image, quantify bound fluorescent material associated with each bead, and determine their embedded spectral code to reveal analyte identities. Here, we present a novel open-source Python software package (the mrbles analysis package) that provides the necessary tools to: (1) find encoded beads in a bright-field microscopy image; (2) quantify bound fluorescent material associated with bead perimeters; (3) identify embedded ratiometric spectral codes within beads; and (4) return data aggregated by embedded code and for each individual bead. We demonstrate the utility of this package by applying it towards analyzing data generated via multiplexed measurement of calcineurin protein binding to MRBLEs (Microspheres with Ratiometric Barcode Lanthanide Encoding) containing known and mutant binding peptide motifs. We anticipate that this flexible package should be applicable to a wide variety of assays, including simple bead or droplet finding analysis, quantification of binding to non-encoded beads, and analysis of multiplexed assays that use ratiometric, spectrally encoded beads.

bioengineering↗

bioRxiv2018

Widespread separation of the polypyrimidine tract from 3’ AG by G tracts in association with alternative exons in metazoa and plants

At the end of introns, the polypyrimidine tract (Py) is often close to the 3 AG in a consensus (Y)20NCAGgt in humans. Interestingly, we have found that they could also be separated by purine-rich elements including G tracts in thousands of human genes. These regulatory elements between the Py and 3AG (REPA) mainly regulate alternative 3 splice sites (3SS) and intron retention. Here we show their widespread distribution and special properties across kingdoms. The purine-rich 3SS are found in up to about 60% of the introns among more than 1000 species/lineages by whole genome analysis, and up to 18% of these introns contain the REPA G tracts in about 2.4 millions of 3SS in total. In particular, they are significantly enriched over their 3SS and genome backgrounds in metazoa and plants, and highly associated with alternative splicing of genes in diverse functional clusters. They are also highly enriched (3-6 folds) in the canonical as well as aberrantly used 3 splice sites in cancer patients carrying mutations of the branch point factor SF3B1 or the 3AG binding factor U2AF35. Moreover, the REPA G tract-harbouring 3SS have significantly reduced occurrences of branch point (BP) motifs between the -24 and -4 positions, in particular absent from the -7 - -5 positions in several model organisms examined. The more distant branch points are associated with increased occurrences of alternative splicing in human and zebrafish. The branch points, REPA G tracts and associated 3SS motifs appear to have emerged differentially in a phylum- or species-specific way during evolution. Thus, there is widespread separation of the Py and 3AG by REPA G tracts, likely evolved among different species or branches of life. This special 3SS arrangement contributes to the generation of diverse transcript or protein isoforms in biological functions or diseases through alternative or aberrant splicing.

molecular biology↗

bioRxiv2018

Using a robotic fish to investigate individual differences in social responsiveness in the guppy

Responding towards the actions of others is one of the most important behavioral traits whenever animals of the same species interact. Mutual influences among interacting individuals may modulate the social responsiveness seen and thus makes it often difficult to study the level and variation of individuality in responsiveness. Here, biomimetic robots (BRs) that are accepted as conspecifics but controlled by the experimenter can be a useful tool. Studying the interactions of live animals with BRs allows pinpointing the live animals level of responsiveness by removing confounding mutuality. In this paper, we show that live guppies (Poecilia reticulata) exhibit consistent differences among each other in their responsiveness when interacting with a biomimetic fish robot - Robofish - and a live companion. It has been repeatedly suggested that social responsiveness correlates with other individual behavioral traits like risk-taking behavior ( boldness) or activity level. We tested this assumption in a second experiment. Interestingly, our detailed analysis of individual differences in social responsiveness using the Robofish, suggests that responsiveness is an independent trait, not part of a larger behavioral syndrome formed by boldness and activity.

animal behavior and cognition↗

bioRxiv2018

Increased autophagy in ephrinB2 deficient osteocytes is associated with hypermineralized, brittle bones

Mineralized bone forms when collagen-containing osteoid accrues hydroxyapatite crystals. This process has two phases: a rapid initiation (primary mineralization), followed by slower accrual of mineral (secondary mineralization) that continues until that portion of bone is renewed by remodelling. Within the bone matrix is an interconnected network of cells termed osteocytes. These cells are derived from bone-forming osteoblasts. Osteoblast differentiation requires expression of ephrinB2, and we were intrigued about why ephrinB2 continues to be expressed in mature osteocytes. To determine its function in osteocytes, we developed an osteocyte-specific ephrinB2 null mouse and found they exhibited a brittle bone phenotype. This was not caused by a change in bone mass, but by an intrinsic defect in the strength of the bone material. Although the initiation of osteoid mineralization occurred at a normal rate, the process of secondary mineralization was accelerated in these mice. The maturing mineralized bone matrix incorporated mineral and carbonate more rapidly than controls, indicating that osteocytic ephrinB2 suppresses mineral accumulation in bone. No known regulators of mineralization were modified in the bone of these mice. However, RNA sequencing showed differential expression of a group of autophagy-associated genes, and increased autophagic flux was confirmed in ephrinB2 knockdown osteocytes. This indicates that the process of secondary mineralization in bone makes use of autophagic machinery in a manner that is limited by ephrinB2 in osteocytes, and that this process may be disrupted in conditions of bone fragility.

cell biology↗

bioRxiv2017

Visual evidence accumulation behavior in unrestrained mice

A major challenge to studying the neural circuits underlying perceptual decision making has been the limited availability of tools for manipulation of neural activity. In recent years, rodents have emerged as a desirable model for overcoming the technical hurdle. However, mice, which offer abundant genetic tools for circuit manipulation, are underrepresented in perceptual evidence accumulation studies. Here we describe the behavior of mice performing a visual evidence accumulation task similar to one previously used in rats and humans. We found that although mice were capable of achieving similar accuracy levels as rats, differences in accumulation strategy were apparent. To test the engagement of cortex in the visual evidence accumulation task, we optogenetically inhibited activity in the anteromedial (AM) visual area using JAWS. Importantly, light activation biased choices in both injected and uninjected animals. Fortunately, by varying the stimulus-response contingency while holding constant the stimulated hemisphere, we surmounted this obstacle and demonstrated a role for AM in contralateral choices. Taken together, our results argue that mice accumulate visual evidence to guide decisions, an ability that is supported in part by area AM.

neuroscience↗

bioRxiv2017

Multisite Generalizability Of Schizophrenia Diagnosis Classification Based On Functional Brain Connectivity

Our objective was to assess the generalizability, across sites and cognitive contexts, of schizophrenia classification based on functional brain connectivity. We tested different training-test scenarios combining fMRI data from 191 schizophrenia patients and 191 matched healthy controls obtained at 6 scanning sites and under different task conditions. Diagnosis classification accuracy generalized well to a novel site and cognitive context provided data from multiple sites were used for classifier training. By contrast, lower classification accuracy was achieved when data from a single distinct site was used for training. These findings indicate that it is beneficial to use multisite data to train fMRI-based classifiers intended for large-scale use in the clinical realm.

neuroscience↗