F99 is Critical for Dimerization and Activation of South African HIV-1 Subtype C Protease

HIV-1 protease (PR) is an obligate homodimer which plays a pivotal role in the maturation and hence propagation of HIV. Although successful developments on PR active site inhibitors have been achieved, the major limiting factor has been the emergence of HIV drug-resistant strains. Disruption of the dimer interface serves as an alternative mechanism to inactivate the enzyme. The terminal residue, F99, was mutated to an alanine to investigate its contribution to dimer stability in the South African HIV-1 subtype C (C-SA) PR. The F99A PR and wild-type C-SA PR were overexpressed and purified. The activities of the PRs and their ability to bind an active site inhibitor, acetyl-pepstatin, were determined in vitro. The F99A PR showed no activity and the inability to bind to the inhibitor. Secondary and quaternary structure analysis were performed and revealed that the F99A PR is monomeric with reduced β-sheet content. The mutation of F99 to alanine disrupted the presumed ‘lock-and-key’ motif at the terminal dimer interface, in turn creating a cavity at the N- and C-terminal antiparallel β-sheet. These findings support the design of inhibitors targeting the C-terminus of the C-SA PR, centered on interactions with the bulky F99.     

Challenges of Diagnosing Acute HIV-1 Subtype C Infection in African Women: Performance of a Clinical Algorithm and the Need for Point-of-Care Nucleic-Acid Based Testing

Background

Prompt diagnosis of acute HIV infection (AHI) benefits the individual and provides opportunities for public health intervention. The aim of this study was to describe most common signs and symptoms of AHI, correlate these with early disease progression and develop a clinical algorithm to identify acute HIV cases in resource limited setting.

Methods

245 South African women at high-risk of HIV-1 were assessed for AHI and received monthly HIV-1 antibody and RNA testing. Signs and symptoms at first HIV-positive visit were compared to HIV-negative visits. Logistic regression identified clinical predictors of AHI. A model-based score was assigned to each predictor to create a risk score for every woman.

Results

Twenty-eight women seroconverted after a total of 390 person-years of follow-up with an HIV incidence of 7.2/100 person-years (95%CI 4.5–9.8). Fifty-seven percent reported ≥1 sign or symptom at the AHI visit. Factors predictive of AHI included age <25 years (OR = 3.2; 1.4–7.1), rash (OR = 6.1; 2.4–15.4), sore throat (OR = 2.7; 1.0–7.6), weight loss (OR = 4.4; 1.5–13.4), genital ulcers (OR = 8.0; 1.6–39.5) and vaginal discharge (OR = 5.4; 1.6–18.4). A risk score of 2 correctly predicted AHI in 50.0% of cases. The number of signs and symptoms correlated with higher HIV-1 RNA at diagnosis (r = 0.63; p<0.001).

Conclusions

Accurate recognition of signs and symptoms of AHI is critical for early diagnosis of HIV infection. Our algorithm may assist in risk-stratifying individuals for AHI, especially in resource-limited settings where there is no routine testing for AHI. Independent validation of the algorithm on another cohort is needed to assess its utility further. Point-of-care antigen or viral load technology is required, however, to detect asymptomatic, antibody negative cases enabling early interventions and prevention of transmission.     

Streptococcus pneumoniae Serotypes and Mortality in Adults and Adolescents in South Africa: Analysis of National Surveillance Data, 2003 - 2008

BackgroundAn association between pneumococcal serotypes and mortality has been suggested. We aimed to investigate this among individuals aged ≥15 years with invasive pneumococcal disease (IPD) in South Africa.MethodsIPD cases were identified through national laboratory-based surveillance at 25 sites, pre-pneumococcal conjugate vaccine (PCV) introduction, from 2003–2008. We assessed the association between the 20 commonest serotypes and in-hospital mortality using logistic regression with serotype 4 (the third commonest serotype with intermediate case-fatality ratio (CFR)) as referent.ResultsAmong 3953 IPD cases, CFR was 55% (641/1166) for meningitis and 23% (576/2484) for bacteremia (p<0.001). Serotype 19F had the highest CFR (48%, 100/207), followed by serotype 23F (39%, 99/252) and serotype 1 (38%, 246/651). On multivariable analysis, factors independently associated with mortality included serotype 1 (OR 1.9, 95%CI 1.1–3.5) and 19F (OR 2.9, 95%CI 1.4–6.1) vs. serotype 4; increasing age (25–44 years, OR 1.8, 95%CI 1.0–3.0; 45–64 years, OR 3.6, 95%CI 2.0–6.4; ≥65 years, OR 5.2, 95%CI 1.9–14.1; vs. 15–24 years); meningitis (OR 4.1, 95%CI 3.0–5.6) vs. bacteremic pneumonia; and HIV infection (OR1.7, 95%CI 1.0–2.8). On stratified multivariate analysis, serotype 19F was associated with increased mortality amongst bacteremic pneumococcal pneumonia cases, while no serotype was associated with increased mortality in meningitis cases.ConclusionMortality was increased in HIV-infected individuals, which may be reduced by increased antiretroviral therapy availability. Serotypes associated with increased mortality are included in the 10-and-13-valent PCV and may become less common in adults due to indirect effects following routine infant immunization.     

Antibiotic resistance profiles of <Emphasis Type="Italic">Escherichia coli</Emphasis> isolates from river sources in Durban,South Africa

Lack of access to potable water has forced many inhabitants of informal settlements in South Africa to rely on surface water sources for their daily water needs, thus exposing these communities to microbial contamination that can result in water-borne diseases. These water sources also serve as natural habitats of pathogenic E. coli strains which harbour virulence factors, which could play a role in the disease process, as well as various multi-drug resistant water-borne pathogens. This study investigated the microbiological quality of two river waters in Durban, South Africa, using total coliform and faecal coliform population as indices. The virulence markers and antibiogram profiles of the E. coli isolates from these rivers were also determined. The results indicated that water from these river sources were of poor microbiological quality and unfit for human consumption. Antibiotic Resistance Profiles of the isolates revealed that 97.1% of the Palmiet River isolates and 71.15% of the Umgeni River isolates were multi-resistant to the antibiotics tested, with all the isolates found to be resistant to novobiocin. Characterization of the virulence markers revealed the presence of stx1, cnf1 and eaeA genes, indicating the possible health risk associated with the ingestion of water from these rivers. The inherent health risks associated with the use of these river water emphasises the need for safe water supply and provision of proper sanitation facilities for the inhabitants of the informal settlements along these river banks.     

Kallikrein and kinin receptor expression in inflammation and cancer

The kallikrein family of serine proteases has been investigated in many inflammatory disorders as molecular mapping, gene characterisation and cloning of kinin receptor genes have unfolded experimentally. In the molecular events of the inflammatory response the kallikrein cascade plays a significant role, since it is considered to initiate and maintain systemic inflammatory responses and immune-modulated disorders. A primary event is the chemotactic attraction of neutrophils which deliver the kallikrein-kinin cascade to sites of cellular injury and carcinogenic transformation of cells. The present study establishes the casual involvement of the kallikrein cascade in infection, inflammatory joint disease, acute transplant rejection, renal glomerular diseases, angiogenesis and carcinoma. We provide strong evidence for new or enhanced expression of kinin B1 receptors in inflammation, and additionally the induction of kallikrein genes in angiogenesis and carcinoma. The results provide insights into possible roles of kallikrein inhibitors and kinin receptor antagonists.     

Diagnostic Accuracy of the HemoCue Hb 301, STAT-Site MHgb and URIT-12 Point-of-Care Hemoglobin Meters in a Central Laboratory and a Community Based Clinic in Durban,South Africa

In South Africa, various point-of-care hemoglobin meters are used. However, the regulatory framework for approval, implementation and oversight of use of point-of-care hemoglobin meters is suboptimal. We assessed the diagnostic accuracy of the HemoCue Hb 301, STAT-Site M^Hgb and URIT-12 point-of-care hemoglobin meters, compared to a central laboratory based reference assay, in a central laboratory and a community based clinic in Durban, South Africa. Differences in performance of the point-of-care assays, compared to the reference assay, were more pronounced in the community based clinic. Results were reasonable for the HemoCue Hb 301, but poor for the STAT-Site M^Hgb and the URIT-12. Poor test performance of point-of-care hemoglobin meters, and inadequate evaluations and oversight in South Africa, leads to suboptimal clinical care and clinical research, and increased costs. There is a need for proper evaluation and quality assurance of point-of-care tests, the results of which should be made widely available to key stakeholders.     

Predictors of long-term prognosis of depression

Background:

Many people with depression experience repeated episodes. Previous research into the predictors of chronic depression has focused primarily on the clinical features of the disease; however, little is known about the broader spectrum of sociodemographic and health factors inherent in its development. Our aim was to identify factors associated with a long-term negative prognosis of depression.

Methods:

We included 585 people aged 16 years and older who participated in the 2000/01 cycle of the National Population Health Survey and who reported experiencing a major depressive episode in 2000/01. The primary outcome was the course of depression until 2006/07. We grouped individuals into trajectories of depression using growth trajectory models. We included demographic, mental and physical health factors as predictors in the multivariable regression model to compare people with different trajectories.

Results:

Participants fell into two main depression trajectories: those whose depression resolved and did not recur (44.7%) and those who experienced repeated episodes (55.3%). In the multivariable model, daily smoking (OR 2.68, 95% CI 1.54–4.67), low mastery (i.e., feeling that life circumstances are beyond one’s control) (OR 1.10, 95% CI 1.03–1.18) and history of depression (OR 3.5, 95% CI 1.95–6.27) were significant predictors (p < 0.05) of repeated episodes of depression.

Interpretation:

People with major depression who were current smokers or had low levels of mastery were at an increased risk of repeated episodes of depression. Future studies are needed to confirm the predictive value of these variables and to evaluate their accuracy for diagnosis and as a guide to treatment.Depression is a common and often recurrent disorder that compromises daily functioning and is associated with a decrease in quality of life.^1–3 Guidelines for the treatment of depression, such as those published by the Canadian Network for Mood and Anxiety Treatments (CANMAT)⁵ and the National Institute for Health and Clinical Excellence (NICE) in the United Kingdom,⁴ often recommend antidepressant treatment in patients with severe symptoms and outline specific risk factors supporting long-term treatment maintenance.^4,5 However, for patients who do not meet the criteria for treatment of depression, the damaging sequelae of depression are frequently compounded without treatment.⁵ In such cases, early treatment for depression may result in an improved long-term prognosis.^6–8A small but growing number of studies have begun to characterize the long-term course of depression in terms of severity,⁹ life-time prevalence¹⁰ and patterns of recurrence.¹¹ However, a recent systematic review of the risk factors of chronic depression highlighted a need for longitudinal studies to better identify prognostic factors.¹² The capacity to distinguish long-term patterns of recurrence of depression in relation to the wide range of established clinical and nonclinical factors for depression could be highly beneficial. Our objective was to use a population-based cohort to identify and understand the baseline factors associated with a long-term negative prognosis of depression.     

Colon cancer: genomics and apoptotic events

Colon cancer is the third most common cancer globally. The risk of developing colon cancer is influenced by a number of factors that include age and diet, but is primarily a genetic disease, resulting from oncogene over-expression and tumour suppressor gene inactivation. The induction and progression of the disease is briefly outlined, as are the cellular changes that occur in its progression. While colon cancer is uniformly amenable to surgery if detected at the early stages, advanced carcinomas are usually lethal, with metastases to the liver being the most common cause of death. Oncogenes and genetic mutations that occur in colon cancer are featured. The molecules and signals that act to eradicate or initiate the apoptosis cascade in cancer cells, are elucidated, and these include caspases, Fas, Bax, Bid, APC, antisense hTERT, PUMA, 15-LOX-1, ceramide, butyrate, tributyrin and PPARgamma, whereas the molecules which promote colon cancer cell survival are p53 mutants, Bcl-2, Neu3 and COX-2. Cancer therapies aimed at controlling colon cancer are reviewed briefly.     

Post heroin dose tissue distribution of 6-monoacetylmorphine (6-MAM) with MALDI imaging

Heroin is an illicit opioid drug which is commonly abused and leads to dependence and addiction. Heroin is considered a pro-drug and is rapidly converted to its major active metabolite 6-monoacetylmorphine (6-MAM) which mediates euphoria and reward through the stimulation of opioid receptors in the brain. The aim of this study was to investigate the distribution and localization of 6-MAM in the healthy Sprague Dawley rat brain following intraperitoneal (i.p) administration of heroin (10 mg/kg), using matrix-assisted laser desorption/ionization mass spectrometric imaging (MALDI-MSI), in combination with quantification via liquid chromatography mass spectrometry (LC–MS/MS). These findings revealed that 6-MAM is present both in plasma and brain tissue with a T_max of 5 min (2.8 µg/mL) and 15 min (1.1 µg/mL), respectively. MSI analysis of the brain showed high intensities of 6-MAM in the thalamus-hypothalamus and mesocorticolimbic system including areas of the cortex, caudate putamen, and ventral pallidum regions. This finding correlates with the distribution of opioid receptors in the brain, according to literature. In addition, we report a time-dependent distribution in the levels of 6-MAM, from 1 min with the highest intensity of the drug observed at 15 min, with sparse distribution at 45 min before decreasing at 60 min. This is the first study to use MSI as a brain imaging technique to detect a morphine’s distribution over time in the brain.