Biology subjects

Chang, A.

Publications and source records attributed to Chang, A..

4 recordsLinked to original sources

bioRxiv2018

CRISPR/Cas9 Targeted Capture Of Mammalian Genomic Regions For Characterization By NGS

The robust detection of structural variants in mammalian genomes remains a challenge. It is particularly difficult in the case of genetically unstable Chinese hamster ovary (CHO) cell lines with only draft genome assemblies available. We explore the potential of the CRISPR/Cas9 system for the targeted capture of genomic loci containing integrated vectors in CHO-K1-based cell lines, and compare it to popular target-enrichment methods and to whole genome sequencing (WGS). The CRISPR/Cas9-based techniques allow for amplification-free capture of genomic regions, which reduces the possibility of sequencing artifacts. Other advantages of these methods are the ease of bioinformatics analysis, potential for multiplexing, and the production of longer sequencing templates for real-time sequencing. The utility of these protocols has been proven by identification of transgene integration sites and flanking sequences in a number of CHO cell lines. However, data produced by these and other targeted capture methods are not always sufficient to analyze complex genomic rearrangements (CGRs) or unexpected sequences introduced into genome by vector integration events. In contrast, WGS provides complete information about vector integration sites, vector copy number, CGRs, and foreign DNA-but despite these benefits, WGS is not easily implemented due to the cost and complexity of the analysis.

genomics↗

bioRxiv2018

MeCP2 isoform e1 mutant mice recapitulate motor and metabolic phenotypes of Rett syndrome

Mutations in the X-linked gene MECP2 cause the majority of Rett syndrome (RTT) cases. Two differentially spliced isoforms of exons 1 and 2 (MeCP2-e1 and MeCP2-e2) contribute to the diverse functions of MeCP2, but only mutations in exon 1, not exon 2, are observed in RTT. We previously described an isoform-specific MeCP2-e1 deficient male mouse model of a human RTT mutation that lacks MeCP2-e1 while preserving expression of MeCP2-e2. However, RTT patients are heterozygous females that exhibit delayed and progressive symptom onset beginning in late infancy, including neurologic as well as metabolic, immune, respiratory, and gastrointestinal phenotypes. Consequently, we conducted a longitudinal assessment of symptom development in MeCP2-e1 mutant females and males. A delayed and progressive onset of motor impairments was observed in both female and male MeCP2-e1 mutant mice, including hind limb clasping and motor deficits in gait and balance. Because these motor impairments were significantly impacted by age-dependent increases in body weight, we also investigated metabolic phenotypes at an early stage of disease progression. Both male and female MeCP2-e1 mutants exhibited significantly increased body fat compared to sex-matched wild-type littermates prior to weight differences. Mecp2e1-/y males exhibited significant metabolic phenotypes of hypoactivity, decreased energy expenditure, increased respiratory exchange ratio (RER), but decreased food intake compared to wildtype. Untargeted analysis of lipid metabolites demonstrated a distinguishable profile in MeCP2-e1 female mutant liver characterized by increased triglycerides. Together these results demonstrate that MeCP2-e1 mutation in mice of both sexes recapitulate early and progressive metabolic and motor phenotypes of human RTT.

neuroscience↗

bioRxiv2018

Piezo1 calcium flickers localize to hotspots of cellular traction forces

Piezo channels transduce mechanical stimuli into electrical and chemical signals, and in doing so, powerfully influence development, tissue homeostasis, and regeneration. While much is known about how Piezo1 responds to external forces, its response to internal, cell-generated forces remains poorly understood. Here, using measurements of endogenous Piezo1 activity and traction forces in native cellular conditions, we show that actomyosin-based cellular traction forces generate spatially-restricted Ca2+ flickers in the absence of externally-applied mechanical forces. Although Piezo1 channels diffuse readily in the plasma membrane and are widely distributed across the cell, their flicker activity is enriched in regions proximal to force-producing adhesions. The mechanical force that activates Piezo1 arises from Myosin II phosphorylation by Myosin Light Chain Kinase. We propose that Piezo1 Ca2+ flickers allow spatial segregation of mechanotransduction events, and that diffusion allows channel molecules to efficiently respond to transient, local mechanical stimuli.

biophysics↗

bioRxiv2017

Analysis of the immunological biomarker profile during acute Zika virus infection reveals the overexpression of CXCL10, a chemokine already linked to neuronal damage

Infection with Zika virus (ZIKV) manifests in a broad spectrum of disease ranging from mild illness to severe neurological complications. To define immunologic correlates of ZIKV infection, we characterized the levels of circulating cytokines, chemokines and growth factors in 54 infected patients of both genders, at five different time-points after symptoms onset using microbeads multiplex immunoassay; statistical analysis and data mining compared to 100 age-matched controls. ZIKV-infected patients present a striking systemic inflammatory response with high levels of pro-inflammatory mediators. Despite the strong inflammatory pattern, IL-1Ra and IL-4 are also induced during acute infection. Interestingly, the inflammatory cytokines, IL-1{beta}, IL-13, IL-17, TNF-, IFN-{gamma}; chemokines, CXCL8, CCL2, CCL5; and the growth factor G-CSF display a bimodal distribution accompanying viremia. While this is the first manuscript to document bimodal distributions of viremia in ZIKV infection, bimodal viremia has been documented in other viral infections with primary viremia peaks during mild systemic disease and a secondary viremia with distribution of the virus to organs and tissues. Moreover, biomarker network analysis demonstrated distinct dynamics in consonance with the bimodal viremia profiles at different time-points during ZIKV infection. Such robust cytokine and chemokine response has been associated with blood-brain barrier permeability and neuroinvasiveness in other flaviviral infections. High-dimensional data analysis further established CXCL10, a chemokine involved in fetal neuron apoptosis and Guillain-Barre syndrome, as the most promising biomarker of acute ZIKV infection for a potential clinical application.\n\nAuthor SummaryInfection with Zika virus manifests in a broad spectrum of disease ranging from mild illness to severe neurological complications. This study characterized the levels of circulating cytokines, chemokines and growth factors in Zika-infected patients showing an inflammatory immune response. Specifically, this study identified a chemokine, CXCL10, known to be involved in fetal neuron apoptosis and Guillain-Barre syndrome, as the most promising biomarker to characterize acute Zika virus infection.

immunology↗