Single-molecule RNA-FISH analysis reveals stochasticity in reactivation of latent HIV-1 regulated by Nuclear Orphan Receptors NR4A and cMYC.

HIV-1 eradication strategies require complete reactivation of HIV-1 latent cells by Latency Reversing Agents (LRA). Current methods lack effectiveness due to incomplete proviral reactivation. We employed a single-molecule RNA-FISH (smRNA-FISH) and FISH-Quant analysis and found that proviral reactivation is highly variable from cell-to-cell, stochastic, and occurs in bursts and waves, with different kinetics in response to diverse LRAs.

Generic Multiplex Digital PCR for Accurate Quantification of T Cells in Copy Number Stable and Unstable DNA Samples.

An accurate T cell quantification is prognostically and therapeutically relevant in various clinical applications, including oncology care and research. In this chapter, we describe how T cell quantifications can be obtained from bulk DNA samples with a multiplex digital PCR experiment. The experimental setup includes the concurrent quantification of three different DNA targets within one reaction: a unique T cell DNA marker, a regional corrector, and a reference DNA marker.

Quantification of Unmethylated Insulin DNA Using Methylation Sensitive Restriction Enzyme Digital Polymerase Chain Reaction.

Assessment of specific β-cell death can be used to determine the quality and viability of pancreatic islets prior to transplantation and hence predict the suitability of the pancreas for isolation. Recently, several groups have demonstrated that unmethylated insulin ()-DNA is correlated to β-cell death in type 1 diabetes patients and during clinical islet isolation and subsequent transplantation. Here, we present a step-by-step protocol of our novel developed method for quantification of the relative amount of unmethylated -DNA using methylation sensitive restriction enzyme digital polymerase chain reaction This method provides a novel and sensitive way to quantify the relative amount of β-cell derived unmethylated -DNA in cellular lysate.

A novel digital PCR-based method to quantify (switched) B cells reveals the extent of allelic involvement in different recombination processes in the IGH locus.

B cells fulfill an important role in the adaptive immunity. Upon activation and immunoglobulin (IG) class switching, these cells function in the humoral immunity compartment as plasma cells. For clinical applications, it can be important to quantify (switched) B cells accurately in a variety of body fluids and tissues of benign, inflammatory and malignant origin.

Scientific and clinical implications of genetic and cellular heterogeneity in uveal melanoma.

Here, we discuss the presence and roles of heterogeneity in the development of uveal melanoma. Both genetic and cellular heterogeneity are considered, as their presence became undeniable due to single cell approaches that have recently been used in uveal melanoma analysis. However, the presence of precursor clones and immune infiltrate in uveal melanoma have been described as being part of the tumour already decades ago.

Histone deacetylase inhibition reduces deleterious cytokine release induced by ingenol stimulation.

Latency reversing agents (LRAs), such as protein kinase C (PKC) agonists, constitute a promising strategy for exposing and eliminating the HIV-1 latent reservoir. PKC agonists activate NF-κB and induce deleterious pro-inflammatory cytokine production. Adjuvant pharmacological agents, such as ruxolitinib, a JAK inhibitor, have previously been combined with LRAs to reduce deleterious pro-inflammatory cytokine secretion without inhibiting HIV-1 reactivation in vitro.

Accurate Quantification of T Cells in Copy Number Stable and Unstable DNA Samples Using Multiplex Digital PCR.

An accurate T-cell quantification is prognostically and therapeutically relevant in various malignancies. We previously developed a digital PCR-based approach offering a precise T-cell enumeration in small amounts of DNA. However, it may be challenging to apply this method in malignant specimens, as genetic instability can disturb the underlying mathematical model.

In Uveal Melanoma, Angiopoietin-2 but Not Angiopoietin-1 Is Increased in High-Risk Tumors, Providing a Potential Druggable Target.

Uveal melanoma (UM) metastasize haematogeneously, and tumor blood vessel density is an important prognostic factor. We hypothesized that proangiogenic factors such as angiopoietin-1 (ANG-1) and angiopoietin-2 (ANG-2), two targetable cytokines, might play a role in tumor development and metastatic behavior. mRNA levels of ANG-1 and ANG-2 were determined in 64 tumors using an Illumina HT-12 v4 mRNA chip and compared to clinical, pathologic, and genetic tumor parameters.

Activation of the Anti-Oxidative Stress Response Reactivates Latent HIV-1 Through the Mitochondrial Antiviral Signaling Protein Isoform MiniMAVS.

The mitochondrial antiviral signaling protein (MAVS) is part of the cell's innate immune mechanism of defense. MAVS mRNA is bicistronic and can give rise to a full length-MAVS and a shorter isoform termed miniMAVS. In response to viral infections, viral RNA can be sensed by the cytosolic RNA sensors retinoic acid-inducible gene I (RIG-I) and/or melanoma differentiation-associated protein 5 (MDA5) and activate NF-κB through interaction with MAVS.

Diverse Mucosal-Associated Invariant TCR Usage in HIV Infection.

Mucosal-associated invariant T (MAIT) cells are innate-like T cells that specifically target bacterial metabolites but are also identified as innate-like sensors of viral infection. Individuals with chronic HIV-1 infection have lower numbers of circulating MAIT cells compared with healthy individuals, yet the features of the MAIT TCR repertoire are not well known. We isolated and stimulated human PBMCs from healthy non-HIV-infected donors (HD), HIV-infected progressors on antiretroviral therapy, and HIV-infected elite controllers (EC).

Involvement of mutant and wild-type CYSLTR2 in the development and progression of uveal nevi and melanoma.

Background: Activating Gα signalling mutations are considered an early event in the development of uveal melanoma. Whereas most tumours harbour a mutation in GNAQ or GNA11, CYSLTR2 (encoding G-protein coupled receptor CysLTR) forms a rare alternative. The role of wild-type CysLTR in uveal melanoma remains unknown.

Quantification of DNA methylation independent of sodium bisulfite conversion using methylation-sensitive restriction enzymes and digital PCR.

Epigenetic regulation is important in human health and disease, but the exact mechanisms remain largely enigmatic. DNA methylation represents one epigenetic aspect but is challenging to quantify. In this study, we introduce a digital approach for the quantification of the amount and density of DNA methylation.

Structure-Activity Relationship Analysis of Benzotriazine Analogues as HIV-1 Latency-Reversing Agents.

"Shock and kill" therapeutic strategies toward HIV eradication are based on the transcriptional activation of latent HIV with a latency-reversing agent (LRA) and the consequent killing of the reactivated cell by either the cytopathic effect of HIV or an arm of the immune system. We have recently found several benzotriazole and benzotriazine analogues that have the ability to reactivate latent HIV by inhibiting signal transducer and activator of transcription 5 (STAT5) SUMOylation and promoting STAT5 binding to the HIV long terminal repeat and increasing its transcriptional activity. To understand the essential structural groups required for biological activity of these molecules, we performed a systematic analysis of >40 analogues.

Interplay between TERT promoter mutations and methylation culminates in chromatin accessibility and TERT expression.

The telomerase reverse transcriptase (TERT) gene is responsible for telomere maintenance in germline and stem cells, and is re-expressed in 90% of human cancers. CpG methylation in the TERT promoter (TERTp) was correlated with TERT mRNA expression. Furthermore, two hotspot mutations in TERTp, dubbed C228T and C250T, have been revealed to facilitate binding of transcription factor ETS/TCF and subsequent TERT expression.

Usage of Droplet Digital PCR (ddPCR) Assays for T Cell Quantification in Cancer.

T cells fulfill a central role in cell-mediated immunity and can be found in the circulation and lymphoid organs upon maturation. For clinical applications, it can be important to quantify (infiltrated) T cells accurately in a variety of body fluids and tissues of benign, inflammatory, or malignant origin. For decades, flow cytometry and immunohistochemistry have been the accustomed methods to quantify T cells.

Synthetic Ingenols Maximize Protein Kinase C-Induced HIV-1 Latency Reversal.

Antiretroviral therapy (ART) does not cure HIV-1 infection due to the persistence of proviruses in long-lived resting T cells. Strategies targeting these latently infected cells will be necessary to eradicate HIV-1 in infected individuals. Protein kinase C (PKC) activation is an effective mechanism to reactivate latent proviruses and allows for recognition and clearance of infected cells by the immune system.

Digital PCR-Based T-cell Quantification-Assisted Deconvolution of the Microenvironment Reveals that Activated Macrophages Drive Tumor Inflammation in Uveal Melanoma.

Uveal melanoma progression can be predicted by gene expression profiles enabling a clear subdivision between tumors with a good (class I) and a poor (class II) prognosis. Poor prognosis uveal melanoma can be subdivided by expression of immune-related genes; however, it is unclear whether this subclassification is justified; therefore, T cells in uveal melanoma specimens were quantified using a digital PCR approach. Absolute T-cell quantification revealed that T-cell influx is present in all uveal melanomas associated with a poor prognosis.

CD32 is expressed on cells with transcriptionally active HIV but does not enrich for HIV DNA in resting T cells.

The persistence of HIV reservoirs, including latently infected, resting CD4 T cells, is the major obstacle to cure HIV infection. CD32a expression was recently reported to mark CD4 T cells harboring a replication-competent HIV reservoir during antiretroviral therapy (ART) suppression. We aimed to determine whether CD32 expression marks HIV latently or transcriptionally active infected CD4 T cells.

Accurate Quantification of T Cells by Measuring Loss of Germline T-Cell Receptor Loci with Generic Single Duplex Droplet Digital PCR Assays.

Quantifying T cells accurately in a variety of tissues of benign, inflammatory, or malignant origin can be of great importance in a variety of clinical applications. Flow cytometry and immunohistochemistry are considered to be gold-standard methods for T-cell quantification. However, these methods require fresh, frozen, or fixated cells and tissue of a certain quality.

World Federation of Pediatric Imaging (WFPI) volunteer outreach through tele-reading: the pilot project in South Africa.

Background: Shortages in radiology services are estimated to affect 3.5-4.7 billion people worldwide.

Antibiotic susceptibility patterns of Neisseria gonorrhoeae isolates in Port Elizabeth.

Objective: To survey the antibiotic susceptibility of Neisseria gonorrhoeae isolates.

Design: This was a cohort analytical study.

Setting: Three clinics serving different areas in Port Elizabeth.

Musculoskeletal Symptoms among Computer-assisted Design (CAD) Operators and Evaluation of a Self-assessment Questionnaire.

A self-administered questionnaire with questions related to the physical workload at video display units (VDUs) was tested for intra- and intermethod reliability among 36 men and 64 women working with research and documentation. The results showed an acceptable test-retest agreement. The answers to three questions were validated by direct measurements: the locations of keyboard and mouse on the table, and the distance between elbow and keyboard heights when working.