The proteasome, a validated anticancer target, participates in an array of biochemical activities, which range from the proteolysis of defective proteins to antigen presentation. We report the preparation of biochemically and photophysically distinct green, red, and far-red real-time sensors designed to simultaneously monitor the proteasome's chymotrypsin-, trypsin-, and caspase-like activities, respectively. These sensors were employed to assess the effect of simultaneous multiple active site catalysis on the kinetic properties of the individual subunits. Furthermore, we have found that the catalytic signature of the proteasome varies depending on the source, cell type, and disease state. Trypsin-like activity is more pronounced in yeast than in mammals, whereas chymotrypsin-like activity is the only activity detectable in B-cells (unlike other mammalian cells). Furthermore, chymotrypsin-like activity is more prominent in transformed B cells relative to their counterparts from healthy donors.

Metastatic progression is a multistep process that involves tumor growth and survival, motility and invasion, and subsequent proliferation in an inappropriate environment. The Src protein tyrosine kinase has been implicated in many of the biochemical pathways that drive these behaviors. Although Src itself is only rarely mutated in human tumors, its aberrant activity has been noted in various cancers and suggested to serve as a barometer of metastatic potential. With these features in mind, we examined Src kinase regulation at the structural, enzymatic, and expression levels as a function of progressively invasive prostate cancer cell lines. Surprisingly, both total Src content and kinase activity decrease with increasing cell line aggressiveness, an observation that appears to be inconsistent with the well-documented role of Src in the signaling pathways that drive growth and invasion. However, we do observe a direct correlation between Src kinase specific activity (total Src kinase activity/total Src content) and metastatic aggressiveness, possibly suggesting that in highly aggressive cell lines, key signaling enzymes are globally recruited to drive the cancerous phenotype. In addition, although the expected enhanced phosphorylation of Src at Tyr-416 (activation site) is present in the most aggressive prostate cancer cell lines, unexpectedly high phosphorylation levels at the Tyr-527 inhibitory site are observed as well. The latter, rather than representative of inhibited enzyme, is more indicative of primed Src responsive to local phosphorylated binding partners.

Single-cell analysis has revealed that transcription is dynamic and stochastic, but tools are lacking that can determine the mechanism operating at a single gene. Here we utilize single-molecule observations of RNA in fixed and living cells to develop a single-cell model of steroid-receptor mediated gene activation. We determine that steroids drive mRNA synthesis by frequency modulation of transcription. This digital behavior in single cells gives rise to the well-known analog dose response across the population. To test this model, we developed a light-activation technology to turn on a single steroid-responsive gene and follow dynamic synthesis of RNA from the activated locus. DOI:http://dx.doi.org/10.7554/eLife.00750.001.

Although the cAMP-dependent protein kinase (PKA) is ubiquitously expressed, it is sequestered at specific subcellular locations throughout the cell, thereby resulting in compartmentalized cellular signaling that triggers site-specific behavioral phenotypes. We developed a three-step engineering strategy to construct an optogenetic PKA (optoPKA) and demonstrated that, upon illumination, optoPKA migrates to specified intracellular sites. Furthermore, we designed intracellular spatially segregated reporters of PKA activity and confirmed that optoPKA phosphorylates these reporters in a light-dependent fashion. Finally, proteomics experiments reveal that light activation of optoPKA results in the phosphorylation of known endogenous PKA substrates as well as potential novel substrates.

There are limited data describing clinical flucytosine pharmacokinetics (PK). The variability of flucytosine partitioning into the CNS is not known. We described the interindividual variability in flucytosine PK in patients with HIV-associated cryptococcal meningoencephalitis. In addition, we quantified the extent and variability of CSF partitioning of flucytosine.

Cryptococcal meningitis (CM) is a major cause of mortality in HIV programmes in Africa despite increasing access to antiretroviral therapy (ART). Mortality is driven in part by limited availability of amphotericin-based treatment, drug-induced toxicities of amphotericin B deoxycholate and prolonged hospital admissions. A single, high-dose of liposomal amphotericin (L-AmB, Ambisome) on a fluconazole backbone has been reported as non-inferior to 14 days of standard dose L-AmB in reducing fungal burden. This trial examines whether single, high-dose L-AmB given with high-dose fluconazole and flucytosine is non-inferior to a seven-day course of amphotericin B deoxycholate plus flucytosine (the current World Health Organization [WHO] recommended treatment regimen).

It is essential that clinical trial participants are representative of the population under investigation. Using HIV-associated cryptococcal meningitis (CM) as a case study, we conducted a systematic review of clinical trials to determine how inclusive and representative they were both in terms of the affected population and the involvement of local investigators.

Protein therapeutics are a powerful class of drugs known for their selectivity and potency. However, the potential efficacy of these therapeutics is commonly offset by short circulatory half-lives and undesired action at otherwise healthy tissue. We describe herein a targeted protein delivery system that employs engineered red blood cells (RBCs) as carriers and light as the external trigger that promotes hemolysis and drug release. RBCs internally loaded with therapeutic proteins are readily surface modified with a dormant hemolytic peptide. The latter is activated via easily assigned wavelengths that extend into the optical window of tissue. We have demonstrated that photorelease transpires with spatiotemporal control and that the liberated proteins display the anticipated biological effects in vitro. Furthermore, we have confirmed targeted delivery of a clot-inducing enzyme in a mouse model. Finally, we anticipate that this strategy is not limited to RBC carriers but also should be applicable to nano- and microtransporters comprised of bilayer lipid membranes.

Advanced HIV disease (AHD; CD4 counts <200 cells/µL) remains common in many low- and middle-income settings. An instrument-free point-of-care test to rapidly identify patients with AHD would facilitate implementation of the World Health Organization (WHO) recommended package of care. We performed a laboratory-based validation study to evaluate the performance of the VISITECT CD4 Advanced Disease assay in Botswana.

An optimized peptide substrate was used to measure protein kinase B (PKB) activity in single cells. The peptide substrate was introduced into single cells, and capillary electrophoresis was used to separate and quantify nonphosphorylated and phosphorylated peptide. The system was validated in three model pancreatic cancer cell lines before being applied to primary cells from human pancreatic adenocarcinomas propagated in nude mice. As measured by phosphorylation of peptide substrate, each tumor cell line exhibited statistically different median levels of PKB activity (65%, 21%, and 4% phosphorylation in PANC-1 (human pancreatic carcinoma), CFPAC-1 (human metastatic ductal pancreatic adenocarcinoma), and HPAF-II cells (human pancreatic adenocarcinoma), respectively) with CFPAC-1 cells demonstrating two populations of cells or bimodal behavior in PKB activation levels. The primary cells exhibited highly variable PKB activity at the single cell level, with some cells displaying little to no activity and others possessing very high levels of activity. This system also enabled simultaneous characterization of peptidase action in single cells by measuring the amount of cleaved peptide substrate in each cell. The tumor cell lines displayed degradation rates statistically similar to one another (0.02, 0.06, and 0.1 zmol pg(-1) s(-1), for PANC-1, CFPAC-1, and HPAF-II cells, respectively) while the degradation rate in primary cells was 10-fold slower. The peptide cleavage sites also varied between tissue-cultured and primary cells, with 5- and 8-residue fragments formed in tumor cell lines and only the 8-residue fragment formed in primary cells. These results demonstrate the ability of chemical cytometry to identify important differences in enzymatic behavior between primary cells and tissue-cultured cell lines.

Protein tyrosine phosphatases (PTPs) are important signaling enzymes that control such fundamental processes as proliferation, differentiation, survival/apoptosis, as well as adhesion and motility. Potent and selective PTP inhibitors serve not only as powerful research tools, but also as potential therapeutics against a variety illness including cancer and diabetes. PTP activity-based assays are widely used in high throughput screening (HTS) campaigns for PTP inhibitor discovery. These assays suffer from a major weakness, in that the reactivity of the active site Cys can cause serious problems as highly reactive oxidizing and alkylating agents may surface as hits. We describe the development of a fluorescence polarization (FP)-based displacement assay that makes the use of an active site Cys to Ser mutant PTP (e.g., PTP1B/C215S) that retains the wild-type binding affinity. The potency of library compounds is assessed by their ability to compete with the fluorescently labeled active site ligand for binding to the Cys to Ser PTP mutant. Finally, the substitution of the active site Cys by a Ser renders the mutant PTP insensitive to oxidation and alkylation and thus will likely eliminate "false" positives due to modification of the active site Cys that destroy the phosphatase activity.

HIV-associated cryptococcal, TB and pneumococcal meningitis are the leading causes of adult meningitis in sub-Saharan Africa (SSA). We performed a systematic review and meta-analysis with the primary aim of estimating mortality from major causes of adult meningitis in routine care settings, and to contrast this with outcomes from clinical trial settings.

To determine effects of cryptococcal meningitis (CM) on human immunodeficiency virus (HIV)-1C cerebrospinal fluid (CSF) viral escape, CSF/plasma viral discordance, and drug resistance mutation (DRM) discordance between CSF and plasma compartments, we compared CSF and plasma viral load (VL) and DRMs in individuals with HIV-associated CM in Botswana.This cross-sectional study utilized 45 paired CSF/plasma samples from participants in a CM treatment trial (2014-2016). HIV-1 VL was determined and HIV-1 protease and reverse transcriptase genotyping performed. DRMs were determined using the Stanford HIV database. CSF viral escape was defined as HIV-1 ribonucleic acid ≥0.5 log10 higher in CSF than plasma and VL discordance as CSF VL > plasma VL.HIV-1 VL was successfully measured in 39/45 pairs, with insufficient sample volume in 6; 34/39 (87.2%) participants had detectable HIV-1 in plasma and CSF, median 5.1 (interquartile range: 4.7-5.7) and 4.6 (interquartile range:3.7-4.9) log10 copies/mL, respectively (P≤.001). CSF viral escape was present in 1/34 (2.9%) and VL discordance in 6/34 (17.6%). Discordance was not associated with CD4 count, antiretroviral status, fungal burden, CSF lymphocyte percentage nor mental status. Twenty-six of 45 (57.8%) CSF/plasma pairs were successfully sequenced. HIV-1 DRM discordance was found in 3/26 (11.5%); 1 had I84IT and another had M46MI in CSF only. The third had K101E in plasma and V106 M in CSF.Our findings suggest that HIV-1 escape and DRM discordance may occur at lower rates in participants with advanced HIV-disease and CM compared to those with HIV associated neurocognitive impairment.

HIV-1 compartmentalization in reservoir sites remains a barrier to complete HIV eradication. It is unclear whether there is variation in HIV-1 env and gag between cerebrospinal fluid (CSF) and plasma of individuals with HIV-associated cryptococcal meningitis (CM). We compared HIV-1 env characteristics and the gag cytotoxic T-lymphocyte (CTL) escape mutations from CSF and plasma samples. Employing population-based Sanger sequencing, we sequenced HIV-1 env from CSF of 25 patients and plasma of 26 patients. For gag, 15 CSF and 21 plasma samples were successfully sequenced. Of these, 18 and 9 were paired env and gag CSF/plasma samples, respectively. There was no statistically significant difference in the proportion of CCR5-using strains in the CSF and plasma, (p = 0.50). Discordant CSF/plasma virus co-receptor use was found in 2/18 pairs (11.1%). The polymorphisms in the HIV-1 V3 loop were concordant between the two compartments. From the HIV-1 gag sequences, three pairs had discordant CTL escape mutations in three different epitopes of the nine analyzed. These findings suggest little variation in the HIV-1 env between plasma and CSF and that the CCR5-using strains predominate in both compartments. HIV-1 gag CTL escape mutations also displayed little variation in CSF and plasma suggesting similar CTL selective pressure.

Individuals recruited into clinical trials for life-threatening illnesses are particularly vulnerable. This is especially true in low-income settings. The decision to enrol may be influenced by existing inequalities, poor healthcare infrastructure and fear of death. Where patients are confused or unconscious the responsibility for this decision falls to relatives. This qualitative study is nested in the ongoing AMBIsome Therapy Induction OptimisatioN (AMBITION) Trial. AMBITION is recruiting participants from five countries in sub-Saharan Africa and is trialling a novel treatment approach for HIV-associated cryptococcal meningitis, an infection known to affect brain function. We aim to learn from the experiences of participants, relatives and researchers involved in AMBITION.

Single, high-dose liposomal amphotericin B (LAmB; AmBisome, Gilead Sciences) has demonstrated non-inferiority to amphotericin B deoxycholate in combination with other antifungals for averting all-cause mortality from HIV-associated cryptococcal meningitis. There are limited data on the pharmacokinetics (PK) of AmBisome. The aim of this study was to describe population PK of AmBisome and conduct a meta-analysis of the available studies to suggest the optimal dosing for cryptococcal meningoencephalitis.

Syphilis data from low- and middle-income countries are lacking due to limited testing. Point-of-care tests (POCTs) have been promoted to expand testing but previously only included treponemal tests, which cannot distinguish active from past infection. We aimed to assess the feasibility of using a combined treponemal and non-treponemal POCT in HIV clinic patients in Gaborone, Botswana, and estimate syphilis prevalence in our clinic sample using this approach. We recruited 390 non-pregnant patients. Participants underwent a combined treponemal and non-treponemal POCT (Dual Path Platform (DPP®) Syphilis Screen and Confirm Assay (Chembio Diagnostic Systems)) on finger-prick blood sample and a questionnaire. Median age 45 years, 30% men, median CD4 count 565 cells/μL, and 91% had an HIV viral load <400 copies/mL. Five participants had active syphilis (1.3%, 95% CI 0.5-3.0%) and 64 had previous syphilis (16.4%, 95% CI 13.0-20.4%) using the DPP POCT. There was a reasonable level of agreement between digital and visual reading of the POCT (kappa statistic of 0.81); however, visual reading missed three active infections (60%). The level of active syphilis was similar to local antenatal data. The DPP POCT led to five participants with active syphilis being diagnosed and starting same-day treatment. The digital reader should be used.

Because small-molecule activators of adenylyl cyclases (AC) affect ACs cell-wide, it is challenging to explore the signaling consequences of AC activity emanating from specific intracellular compartments. We explored this issue using a series of engineered, optogenetic, spatially restricted, photoactivable adenylyl cyclases (PACs) positioned at the plasma membrane (PM), the outer mitochondrial membrane (OMM), and the nucleus (Nu). The biochemical consequences of brief photostimulation of PAC is primarily limited to the intracellular site occupied by the PAC. By contrast, sustained photostimulation results in distal cAMP signaling. Prolonged cAMP generation at the OMM profoundly stimulates nuclear protein kinase (PKA) activity. We have found that phosphodiesterases 3 (OMM and PM) and 4 (PM) modulate proximal (local) cAMP-triggered activity, whereas phosphodiesterase 4 regulates distal cAMP activity as well as the migration of PKA's catalytic subunit into the nucleus.

Research into life-threatening illnesses which require emergency hospitalisation is essential. This group of patients is unique in that they are experiencing an unfolding emergency when they are approached, enrolled, and followed up in a research study. We aimed to synthesise qualitative data from trial participants and surrogate decision-makers to deepen our understanding and inform the design and conduct of future clinical trials for life-threatening illnesses.