Biology subjects

Shao, A.

Publications and source records attributed to Shao, A..

3 recordsLinked to original sources

bioRxiv2021

Arap1 Loss Causes RPE Phagocytic Dysfunction and Subsequent Photoreceptor Death

PurposeArap1 is an Arf-directed GTPase-activating protein (GAP) shown to modulate actin cytoskeletal dynamics by regulating Arf and Rho family members. We have previously shown that Arap1-/- mice develop photoreceptor degeneration similar to the human condition retinitis pigmentosa (RP), corroborated by fundus examination, histopathology, and ERG analysis. However, Arap1 expression was not detected in photoreceptors, but in Muller Glia and retinal pigment epithelium (RPE), suggesting a non-cell-autonomous mechanism for degeneration. The aim of this study was to elucidate the role of retinal Arap1 in photoreceptor maintenance. MethodsAlbino Arap1-/- mice were generated via breeding pigmented Arap1-/- mice onto a Tyr-/- C57BL/6J background. Conditional knockout (cKO) mice were generated for Muller Glia/RPE, Muller Glia, and RPE via targeting Cralbp, Glast, and Vmd2 promoters, respectively, to drive Cre recombinase expression to knock out Arap1. Mice were analyzed by fundus photography, optical coherence tomography (OCT), histology, and immunohistochemistry. Arap1 binding partners were assayed by affinity purification mass spectrometry. ResultsVmd2-Cre Arap1tm1c/tm1c and Cralbp-Cre Arap1tm1c/tm1c mice, but not Glast-Cre Arap1tm1c/tm1c mice, recapitulated the photoreceptor degeneration phenotype originally observed in germline Arap1-/- mice. These findings were corroborated by fundus exam, OCT, and histological analysis. Mass spectrometry analysis of ARAP1 co-immunoprecipitation identified putative binding partners of ARAP1, revealing numerous interactants involved in phagocytosis, cytoskeletal composition, intracellular trafficking, and endocytosis. Quantification of rod outer segment (OS) phagocytosis in vivo demonstrated a clear phagocytic defect in Arap1-/- mice compared to Arap1+/+ littermate controls while cone phagocytosis was preserved. ConclusionsArap1 expression, specifically in RPE, is necessary for photoreceptor survival due to its indispensable function in RPE phagocytosis. We propose a model in which Arap1 regulates G-protein function for nonmuscle myosin II targeting during phagocytosis. This novel role of Arap1 is important for further understanding of both the diversity of its functions and the complex molecular regulation of RPE phagocytosis.

molecular biology↗

bioRxiv2020

Intein-assisted bisection mapping systematically splits proteins for Boolean logic and inducibility engineering

Split inteins are powerful tools for seamless ligation of synthetic split proteins. Yet, their use remains limited because the already intricate split site identification problem is often complicated by the requirement of extein junction sequences. To address this, we augmented a mini-Mu transposon-based screening approach and devised the intein-assisted bisection mapping (IBM) method. IBM robustly revealed clusters of split sites on five proteins, converting them into AND or NAND logic gates. We further showed that the use of inteins expands functional sequence space for splitting a protein. We also demonstrated the utility of our approach over rational inference of split sites from secondary structure alignment of homologous proteins. Furthermore, the intein inserted at an identified site could be engineered by the transposon again to become partially chemically inducible, and to some extent enabled post-translational tuning on host protein function. Our work offers a generalizable and systematic route towards creating split protein-intein fusions and conditional inteins for protein activity control.

synthetic biology↗

bioRxiv2019

Facilitators and Barriers to Linkage to HIV Care and Treatment among Female Sex Workers in a Community-based HIV Prevention Intervention in Tanzania: a qualitative study

BackgroundHIV-infected female sex workers (FSWs) have poor linkage to HIV care in sub-Sahara Africa.\n\nMethodsWe conducted 21 focus group discussions (FGDs) to explore factors influencing linkage to HIV care among FSWs tested for HIV through a comprehensive community-based HIV prevention project in Tanzania.\n\nResultsInfluences on linkage to care were present at the system, societal and individual levels. System-level factors included unfriendly service delivery environment, including lengthy pre-enrolment sessions, concerns about confidentiality, stigmatising attitudes of health providers. Societal-level factors included myths and misconceptions about ART and stigma. On the individual level, most notable was fear of not being able to continue to have a livelihood if ones status were to be known. Facilitators were noted, including the availability of transport to services, friendly health care providers and peer-support referral and networks.\n\nConclusionFindings of this study underscore the importance of peer-supported linkages to HIV care and the need for respectful, high-quality care.

epidemiology↗