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The large majority of human immunodeficiency virus type 1 (HIV-1) markers of disease progression/severity previously identified have been associated with alterations in host genetic and immune responses, with few studies focused on viral genetic markers correlate with changes in disease severity. This study presents a cross-sectional/longitudinal study of HIV-1 single nucleotide polymorphisms (SNPs) contained within the viral promoter or long terminal repeat (LTR) in patients within the Drexel Medicine CNS AIDS Research and Eradication Study (CARES) Cohort. HIV-1 LTR SNPs were found to associate with the classical clinical disease parameters CD4+ T-cell count and log viral load. They were found in both defined and undefined transcription factor binding sites of the LTR. A novel SNP identified at position 108 in a known COUP (chicken ovalbumin upstream promoter)/AP1 transcription factor binding site was significantly correlated with binding phenotypes that are potentially the underlying cause of the associated clinical outcome (increase in viral load and decrease in CD4+ T-cell count).

Dendritic cells (DC) present tumour antigens to T cells but this process is defective in the tumour microenvironment. Here the authors find that downregulation of cholesterol 25-hydroxylase underlies defective cross presentation of tumour antigens.

Radiography identifies suspicious lung nodules that are not always easy to diagnose via biopsy. Here, the authors utilize a fluorescent dye that targets the folate receptor and show using needle based endomicroscopy that it can be used to identify cancer cells during biopsy procedures

Summary: Here, we describe a protocol for fluorescence-activated cell sorting (FACS) of human EpCAM+ cells from fresh surgically resected specimens. We then use Q-PCR to identify specific molecular targets associated with the metastatic phenotype. This combined approach enables a qualitative and quantitative gene expression analysis of lung cancer samples. We describe how to use the protocol for Rac GEFs, but it can be applied broadly to other molecular targets.For complete details on the use and execution of this protocol, please refer to Cooke et al. (2021) and Quatromoni et al. (2015).

Viral latency of human immunodeficiency virus type 1 (HIV-1) has become a major hurdle to a cure in the highly effective antiretroviral therapy (ART) era. The clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 system has successfully been demonstrated to excise or inactivate integrated HIV-1 provirus from infected cells by targeting the long terminal repeat (LTR) region. However, the guide RNAs (gRNAs) have classically avoided transcription factor binding sites (TFBSs) that are readily observed and known to be important in human promoters. Although conventionally thought unfavorable due to potential impact on human promoters, our computational pipeline identified gRNA sequences that were predicted to inactivate HIV-1 transcription by targeting the nuclear factor κB (NF-κB) binding sites (gNFKB0, gNFKB1) with a high safety profile (lack of predicted or observed human edits) and broad-spectrum activity (predicted coverage of known viral sequences). Genome-wide, unbiased identification of double strand breaks (DSBs) enabled by sequencing (GUIDE-seq) showed that the gRNAs targeting NF-κB binding sites had no detectable CRISPR-induced off-target edits in HeLa cells. 5′ LTR-driven HIV-1 transcription was significantly reduced in three HIV-1 reporter cell lines. These results demonstrate a working model to specifically target well-known TFBSs in the HIV-1 LTR that are readily observed in human promoters to reduce HIV-1 transcription with a high-level safety profile and broad-spectrum activity.

Summary: Despite the undisputable role of the small GTPase Rac1 in the regulation of actin cytoskeleton reorganization, the Rac guanine-nucleotide exchange factors (Rac-GEFs) involved in Rac1-mediated motility and invasion in human lung adenocarcinoma cells remain largely unknown. Here, we identify FARP1, ARHGEF39, and TIAM2 as essential Rac-GEFs responsible for Rac1-mediated lung cancer cell migration upon EGFR and c-Met activation. Noteworthily, these Rac-GEFs operate in a non-redundant manner by controlling distinctive aspects of ruffle dynamics formation. Mechanistic analysis reveals a leading role of the AXL-Gab1-PI3K axis in conferring pro-motility traits downstream of EGFR. Along with the positive association between the overexpression of Rac-GEFs and poor lung adenocarcinoma patient survival, we show that FARP1 and ARHGEF39 are upregulated in EpCam+ cells sorted from primary human lung adenocarcinomas. Overall, our study reveals fundamental insights into the complex intricacies underlying Rac-GEF-mediated cancer cell motility signaling, hence underscoring promising targets for metastatic lung cancer therapy.

Human immunodeficiency virus type-1 (HIV-1) infection has resulted in the death of upward of 39 million people since being discovered in the early 1980s. A cure strategy for HIV-1 has eluded scientists, but gene editing technologies such as clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) offer a new approach to developing a cure for HIV infection. While the CRISPR/Cas9 system has been used successfully in a number of different types of studies, there remains a concern for off-target effects. This review details the different aspects of the Cas9 system and how they play a role in off-target events. In addition, this review describes the current technologies available for detecting off-target cleavage events and their advantages and disadvantages. While some studies have utilized whole genome sequencing (WGS), this method sacrifices depth of coverage for interrogating the whole genome. A number of different approaches have now been developed to take advantage of next generation sequencing (NGS) without sacrificing depth of coverage. This review highlights four widely used methods for detecting off-target events: (1) genome-wide unbiased identification of double-stranded break events enabled by sequencing (GUIDE-Seq), (2) discovery of in situ Cas off-targets and verification by sequencing (DISCOVER-Seq), (3) circularization for in vitro reporting of cleavage effects by sequencing (CIRCLE-Seq), and (4) breaks labeling in situ and sequencing (BLISS). Each of these technologies has advantages and disadvantages, but all center around capturing double-stranded break (DSB) events catalyzed by the Cas9 endonuclease. Being able to define off-target events is crucial for a gene therapy cure strategy for HIV-1.