C-C chemokine receptor type 5 (CCR5) is the main co-receptor for HIV entry into the target CD4+ cells, and homozygous CCR5Δ32/Δ32 cells are resistant to CCR5-tropic HIV infection. However, the CCR5Δ32/Δ32 homozygous donors in populations are rare. Here we developed a simple approach to induce CCR5Δ32/Δ32 homozygotes through CRISPR-Cas9 genome-editing technology. Designing a pair of single-guide RNA targeting the flank region of the CCR5Δ32 mutation locus, we applied the CRISPR-Cas9 and lentiviral packaging system to successfully convert wild-type CCR5 into CCR5Δ32/Δ32 homozygotes in the human Jurkat CD4+ cell line and primary CD4+ cells, exactly the same as the naturally occurring CCR5Δ32/Δ32 mutation. The successful rate is up to 20% in Jurkat cells but less in primary CD4+ cells. The modified CCR5Δ32/Δ32 CD4+ cells are resistant to CCR5-tropic HIV infection. Whole-genome sequencing revealed no apparent off-target sites. This approach has the promise to promote HIV/AIDS therapy from the only cured unique Berlin patient to a routine autologous cell-based therapy.
Normally, HIV-1 enters into CD4+ cells through membrane fusion, and newly synthesized HIV-1 viral proteins assemble on the plasma membrane to form viral particles and bud out. In the previous study, we found host factor coiled-coil domain containing protein 8 (CCDC8) can strongly inhibit HIV-1 production, but the underline mechanism is not clear. Here we show that overexpression of CCDC8 reverses the normal HIV-1 production process, and causes newly assembled HIV-1 Gag particles to be endocytosed on the plasma membrane, rather than budding out. Live-cell imaging system captured the moment of CCDC8-mediated Gag internalization on the plasma membrane, and the speed of Gag turnover is up to 1.53 μm/s, much faster than Gag assembly on the plasma membrane. After Gag internalization, it accumulates in the cellular organelle-lysosome for degradation, but not proteasome, autophagosome, endoplasmic reticulum, clathrin or recycling endosome. In addition, CCDC8 is a membrane-associated protein, and N-terminal of CCDC8 is very important for membrane binding, and also important for inhibition of Gag assembly. C-terminal deletion of CCDC8 has a little effect on anti-HIV-1 effect. Moreover, CCDC8 is phosphorylated at amino acid threonine T87 and serine S261, and mono-methylated at lysine K491. Alanine mutations of T87A, S261A and K491A singly or in combination do not affect CCDC8 anti-HIV activity. In conclusion, overexpression of CCDC8 can cause newly assembled HIV-1 Gag particles on the plasma membrane to be endocytosed and degraded in lysosome. Human Immunodeficiency Virus type 1 (HIV-1), the etiologic agent of AIDS, belongs to the retrovirus family 1, 2. In its life cycle, HIV-1 first recognizes and then binds to the CD4+ receptor 2. Under the aid of co-receptors, most importantly CCR5 or CXCR4, HIV-1 enters the target cells through membrane fusion 2. HIV-1 uncoats its capsid and undergoes reverse transcription from genome RNA into double stranded DNA. Viral integrase then inserts the viral double stranded DNA into the human genome. The integrated viral DNA can be silent or be activated by viral accessary protein Tat 2. Tat protein starts viral mRNA transcription in the nucleus and viral mRNAs export to the cytoplasm for translation. The translated viral proteins and transcribed viral genome RNA assemble on the plasma membrane, and then viral particles bud out and release 2. The released mature viral particles infect new target cells and start a new life cycle. Human cells also encode proteins to facilitate or block the normal viral replication cycle. In the previous study, we reported that coiled-coil domain containing protein 8 (CCDC8) can inhibit HIV-1 Gag production 3. We continue our study of CCDC8 against HIV-1 in this study. Some important proteins include coiled-coil domains in the coding regions, for example anti-retrovirus factor Trim5α 4, 5 , Tetherin 6 , and etc. There are other coiled-coil domain containing proteins, temporarily as coiled-coil domain containing (CCDC) family proteins. However, functions of most...
C-C chemokine receptor 5 (CCR5) is a receptor for chemokines and a co-receptor for HIV-1 entry into the target CD4+ cells. CCR5 delta 32 deletion is a loss-of-function mutation, resistant to HIV-1 infection. We tried to induce the CCR5 delta 32 mutation harnessing the genome editing technique, CRISPR-Cas9 (Clustered Regularly Interspaced Short Palindromic Repeats, CRISPR and CRISPR associated protein 9, Cas9) in the commonly used cell line human embryonic kidney HEK 293T cells. Surprisingly, we found that HEK293T cells are heterozygous for CCR5 delta 32 mutation, in contrast to the wild type CCR5 cells, human acute T cell leukemia cell line Jurkat and human breast adenocarcinoma cell line MDA-MB-231 cells. This finding indicates that at least one human cell line is heterozygous for the CCR5 delta 32 mutation. We also found that in PCR amplification, wild type CCR5 DNA and mutant delta 32 DNA can form mismatched heteroduplex and move slowly in gel electrophoresis.
Background Various antibiotic resistant bacteria are known to induce repeated pulmonary infections and increase morbidity and mortality. A thorough knowledge of the spectrum of bacteria with antibiotic resistance genes (ARGs) can improve the antibiotic treatment efficiency. In this study, we induced metagenomic next-generation sequencing (mNGS) alignment and assembly methods in the bioinformatics analysis pipeline to reveal the profile of bacteria with ARGs (ARB) in samples from patients with pulmonary infections. Methods A retrospective analysis of 151 clinical samples from 144 patients with pulmonary infections was undertaken by mNGS and conventional microbiological detection methods. Positive ARB were determined according to the analysis results detected both by the alignment and assembly methods. Co-occurrence analysis of ARG-ARB network was conducted to investigate the attributions between ARGs and microbial taxa. We also evaluated the potential application conditions to predict ARGs using those two approaches. Results Compared to that using conventional detection methods, the false-positive detection rate of ARB was significantly higher using mNGS alignment method. The assembly method could assist the determining of the detected pathogens by the alignment method as true ARB and improve the predictive capabilities (46% > 13%). ARG-ARB network revealed the main ARGs in predominant ARB. A total of 361 ARGs were detected, which mostly belonged to the multidrug class and β-lactam antibiotic classes. Specifically, 101 ARGs (existing in two approaches) and 34 ARGs (detected only by assembly method) achieved a clear ARG-bacteria attribution and potentially could optimize the reference antibiotic resistance database. The most prevalent ARB and its corresponding ARG and drug classes were as follows in this study: Acinetobacter baumannii (ADE, multidrug), Pseudomonas aeruginosa (MEX, multidrug), Klebsiella pneumoniae (MDT, aminocoumarin; EMR, fluoroquinolone), Stenotrophomonas maltophilia (SME, multidrug) and Corynebacterium striatum (carA, MLSB). Conclusion Collectively, our findings demonstrated the applicability of mNGS alignment and assembly as antibiotic resistant diagnostic methods and uncovered pulmonary infection-associated ARB and ARGs, potentially, as antibiotic treatment targets for the pulmonary infection.
COVID-19 has spread globally, causing a pandemic and medical interruptions. As more countries control the epidemic, the resumption of work is imperative. However, asymptomatic carriers become the main source of infection. After several months of recovery, Wuhan had much experience with facing the challenge of work resumption. The purpose of this study was to investigate the safety of the resumption strategies, as well as the outcome of the resumption efforts, in the early post-epidemic period. A retrospective study was conducted in patients admitted between April 8 and June 30 to the neurosurgery department of Tongji Hospital, Wuhan. The medical information, past medical history, COVID-19 tests, laboratory parameters, CT results, and management were reviewed and recorded. 768 patients were admitted to the neurosurgery department at Tongji Hospital, and none of them became new infections. Our department recovered to 70% efficiency one month after the resumption of work. Two patients were found to have asymptomatic infections in the outpatient department. Two patients who recovered from COVID-19 underwent the surgery without recurrence of COVID-19. Tumor patients accounted for more than 50% of the surgery patients in the early period. It is feasible and helpful to follow our strict admission algorithm in the early post-epidemic period, even though the challenges of asymptomatic infectors exist. Two COVID-19 tests in 3 days are suggested within the early period. Protective downgrades should be based on the testing of asymptomatic patients in the area. Recovered COVID-19 patients can undergo surgery without recurrence.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.