Browsing by Author "Wang, Hong"

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    Comprehensive off-target analysis of dCas9-SAM-mediated HIV reactivation via long noncoding RNA and mRNA profiling
    Zhang, Yonggang; Arango-Argoty, Gustavo; Li, Fang; Xiao, Xiao; Putatunda, Raj; Yu, Jun; Yang, Xiao-Feng; Wang, Hong; Watson, Layne T.; Zhang, Liqing; Hu, Wenhui (2018-09-10)
    Background CRISPR/CAS9 (epi)genome editing revolutionized the field of gene and cell therapy. Our previous study demonstrated that a rapid and robust reactivation of the HIV latent reservoir by a catalytically-deficient Cas9 (dCas9)-synergistic activation mediator (SAM) via HIV long terminal repeat (LTR)-specific MS2-mediated single guide RNAs (msgRNAs) directly induces cellular suicide without additional immunotherapy. However, potential off-target effect remains a concern for any clinical application of Cas9 genome editing and dCas9 epigenome editing. After dCas9 treatment, potential off-target responses have been analyzed through different strategies such as mRNA sequence analysis, and functional screening. In this study, a comprehensive analysis of the host transcriptome including mRNA, lncRNA, and alternative splicing was performed using human cell lines expressing dCas9-SAM and HIV-targeting msgRNAs. Results The control scrambled msgRNA (LTR_Zero), and two LTR-specific msgRNAs (LTR_L and LTR_O) groups show very similar expression profiles of the whole transcriptome. Among 839 identified lncRNAs, none exhibited significantly different expression in LTR_L vs. LTR_Zero group. In LTR_O group, only TERC and scaRNA2 lncRNAs were significantly decreased. Among 142,791 mRNAs, four genes were differentially expressed in LTR_L vs. LTR_Zero group. There were 21 genes significantly downregulated in LTR_O vs. either LTR_Zero or LTR_L group and one third of them are histone related. The distributions of different types of alternative splicing were very similar either within or between groups. There were no apparent changes in all the lncRNA and mRNA transcripts between the LTR_L and LTR_Zero groups. Conclusion This is an extremely comprehensive study demonstrating the rare off-target effects of the HIV-specific dCas9-SAM system in human cells. This finding is encouraging for the safe application of dCas9-SAM technology to induce target-specific reactivation of latent HIV for an effective “shock-and-kill” strategy.
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    Critical factors controlling regrowth of opportunistic pathogens in premise plumbing
    Wang, Hong (Virginia Tech, 2013-03-28)
    Opportunistic pathogens (e.g., Legionella pneumophila, Mycobacterium avium complex, Acanthamoeba polyphaga, Pseudomonas aeruginosa) residing in human-made water systems, particularly premise plumbing, are now the primary source of water-borne disease in developed countries. The prevention and control of opportunistic pathogens is a new challenge in premise plumbing due to the limited knowledge concerning the factors driving their occurrence and regrowth mechanisms, and also the complexity of premise plumbing conditions. The goal of this study is to identify key factors governing occurrence of opportunistic pathogens in drinking water distribution systems, particularly premise plumbing, via field investigations and lab-scale experiments. A molecular survey of three opportunistic pathogens (L. pneumophila, M. avium, P. aeruginosa), related groups (Legionella and mycobacteria) and two amoeba hosts (Acanthamoeba spp. and Hartmanella vermiformis) was performed in two real-word chloraminated drinking water distribution systems using quantitative polymerase chain reaction (q-PCR). A high occurrence of Legionella (" 69.0%) and mycobacteria (100%), lower occurrence of L. pneumophila (" 20%) and M. avium (" 33.3%), and rare detection of Pseudomonas aeruginosa (" 13.3%) was observed in both systems. Hartmanella vermiformis was more prevalent than Acanthamoeba. Three-minute flushing resulted in reduced gene copies of Legionella, mycobacteria, H. vermiformis and 16S rRNA genes (P<0.05) and distinct microbial community structure in postflushing water, implying strong regrowth potential of opportunistic pathogens in premise pluming. In order to examine the influence of pipe material, disinfectant type, and water age on occurrence and persistence of the target microorganisms, triplicate simulated distribution systems (SDSs) comparing iron, cement and PVC pipe materials were fed either chlorinated or chloraminated tap water, and were sampled at water ages ranging from 1d to 5.7d. q-PCR quantification of target microorganisms in both biofilm and bulk water revealed that Legionella, mycobacteria, P. aeruginosa and both amoebas naturally colonized the six SDSs, but L. pneumophila and M. avium were not detected. Disinfectant type and dose have the strongest influence on the microbiota. Disinfectant decay was noted with water age, particularly in chloraminated SDSs (due to nitrification), generally resulting in increased microbial detection frequencies and densities with water age. Influence of pipe material became apparent at water ages corresponding to low disinfectant residual. Natural colonization of Legionella spp., Mycobacterium spp., Acanthamoeba spp., H. vermiformis and M. avium was also observed in biofilms from five annular reactors, which were used to investigate effects of prior granular activated carbon (GAC) biofiltration and disinfectant type (chlorine, chloramine) on opportunistic pathogens under premise plumbing conditions. GAC pre-treatment effectively reduced total organic carbon (TOC). In most cases, total bacteria and opportunistic pathogens were higher in undisinfected annular reactors, but the levels were not proportional to the level of GAC pre-treatment/TOC. Chlorine was more effective for controlling mycobacteria and Acanthamoeba, whereas chloramine was more effective for controlling Legionella. Both chlorine and chloramine effectively reduced M. avium and H. vermiformis numbers. Pyrosequencing of 16S rRNA genes in biofilms revealed a significant effect of GAC pre-treatment and disinfectant type on the microbial community structure. Overall, the study provides insights to critical factors triggering proliferation of opportunistic pathogens in drinking water systems. Knowledge gained from this study can assist in formulating practical guidance for drinking water utilities and water consumers in terms of opportunistic pathogen prevention and control.