Negative Epistasis between Sickle and Foetal Haemoglobin Suggests a Reduction in Protection against Malaria

BackgroundHaemoglobin variants, Sickle (HbS) and foetal (HbF) have been associated with malaria protection. This study explores epistatic interactions between HbS and HbF on malaria infection.MethodsThe study was conducted between March 2004 and December 2013 within the sickle cell disease (SCD) programme at Muhimbili National Hospital, Tanzania. SCD status was categorized into HbAA, HbAS and HbSS using hemoglobin electrophoresis and High Performance Liquid Chromatography (HPLC). HbF levels were determined by HPLC. Malaria was diagnosed using rapid diagnostic test and/or blood film. Logistic regression and generalized estimating equations models were used to evaluate associations between SCD status, HbF and malaria.Findings2,049 individuals with age range 0-70 years, HbAA 311(15.2%), HbAS 241(11.8%) and HbSS 1,497(73.1%) were analysed. At enrolment, malaria prevalence was significantly higher in HbAA 13.2% compared to HbAS 1.24% and HbSS 1.34% (p<0.001). Mean HbF was lower in those with malaria compared to those without malaria in HbAA (0.43% vs 0.82%) but was the reverse in HbSS (8.10% vs 5.59%). An increase in HbF was associated with a decrease in risk of malaria OR=0.50 (95%CI: 0.28, 0.90; p=0.021) in HbAA, whereas for HbSS the risk of malaria increased OR=2.94 (1.44, 5.98; p=0.003). A similar pattern was seen during multiple visits; HbAA OR=0.52 (0.34, 0.80; p=0.003) vs HbSS OR=2.01 (1.27, 3.23; p=0.003).ConclusionHigher prevalence of malaria in HbAA compared to HbAS and HbSS confirmed the protective effect of HbS. Lower prevalence of malaria in HbAA with high HbF supports a protective effect of HbF. However, in HbSS, the higher prevalence of malaria with high levels of HbF suggests loss of malaria protection. This is the first epidemiological study to suggest a negative epistasis between HbF and HbS on malaria.     

Members of the Chloride Intracellular Ion Channel Protein Family Demonstrate Glutaredoxin-Like Enzymatic Activity

The Chloride Intracellular Ion Channel (CLIC) family consists of six evolutionarily conserved proteins in humans. Members of this family are unusual, existing as both monomeric soluble proteins and as integral membrane proteins where they function as chloride selective ion channels, however no function has previously been assigned to their soluble form. Structural studies have shown that in the soluble form, CLIC proteins adopt a glutathione S-transferase (GST) fold, however, they have an active site with a conserved glutaredoxin monothiol motif, similar to the omega class GSTs. We demonstrate that CLIC proteins have glutaredoxin-like glutathione-dependent oxidoreductase enzymatic activity. CLICs 1, 2 and 4 demonstrate typical glutaredoxin-like activity using 2-hydroxyethyl disulfide as a substrate. Mutagenesis experiments identify cysteine 24 as the catalytic cysteine residue in CLIC1, which is consistent with its structure. CLIC1 was shown to reduce sodium selenite and dehydroascorbate in a glutathione-dependent manner. Previous electrophysiological studies have shown that the drugs IAA-94 and A9C specifically block CLIC channel activity. These same compounds inhibit CLIC1 oxidoreductase activity. This work for the first time assigns a functional activity to the soluble form of the CLIC proteins. Our results demonstrate that the soluble form of the CLIC proteins has an enzymatic activity that is distinct from the channel activity of their integral membrane form. This CLIC enzymatic activity may be important for protecting the intracellular environment against oxidation. It is also likely that this enzymatic activity regulates the CLIC ion channel function.     

Evidence of a Double Burden of Malnutrition in Urban Poor Settings in Nairobi,Kenya

Background

Many low- and middle-income countries are undergoing a nutrition transition associated with rapid social and economic transitions. We explore the coexistence of over and under- nutrition at the neighborhood and household level, in an urban poor setting in Nairobi, Kenya.

Methods

Data were collected in 2010 on a cohort of children aged under five years born between 2006 and 2010. Anthropometric measurements of the children and their mothers were taken. Additionally, dietary intake, physical activity, and anthropometric measurements were collected from a stratified random sample of adults aged 18 years and older through a separate cross-sectional study conducted between 2008 and 2009 in the same setting. Proportions of stunting, underweight, wasting and overweight/obesity were dettermined in children, while proportions of underweight and overweight/obesity were determined in adults.

Results

Of the 3335 children included in the analyses with a total of 6750 visits, 46% (51% boys, 40% girls) were stunted, 11% (13% boys, 9% girls) were underweight, 2.5% (3% boys, 2% girls) were wasted, while 9% of boys and girls were overweight/obese respectively. Among their mothers, 7.5% were underweight while 32% were overweight/obese. A large proportion (43% and 37%%) of overweight and obese mothers respectively had stunted children. Among the 5190 adults included in the analyses, 9% (6% female, 11% male) were underweight, and 22% (35% female, 13% male) were overweight/obese.

Conclusion

The findings confirm an existing double burden of malnutrition in this setting, characterized by a high prevalence of undernutrition particularly stunting early in life, with high levels of overweight/obesity in adulthood, particularly among women. In the context of a rapid increase in urban population, particularly in urban poor settings, this calls for urgent action. Multisectoral action may work best given the complex nature of prevailing circumstances in urban poor settings. Further research is needed to understand the pathways to this coexistence, and to test feasibility and effectiveness of context-specific interventions to curb associated health risks.     

Loss of STK11 expression is an early event in prostate carcinogenesis and predicts therapeutic response to targeted therapy against MAPK/p38

Prostate cancer (PCa) is the second leading cause of cancer-related death in men; however, the molecular mechanisms leading to its development and progression are not yet fully elucidated. Of note, it has been recently shown that conditional stk11 knockout mice develop atypical hyperplasia and prostate intraepithelial neoplasia (PIN). We recently reported an inverse correlation between the activity of the STK11/AMPK pathway and the MAPK/p38 cascade in HIF1A-dependent malignancies. Furthermore, MAPK/p38 overactivation was detected in benign prostate hyperplasia, PIN and PCa in mice and humans. Here we report that STK11 expression is significantly decreased in PCa compared to normal tissues. Moreover, STK11 protein levels decreased throughout prostate carcinogenesis. To gain insight into the role of STK11-MAPK/p38 activity balance in PCa, we treated PCa cell lines and primary biopsies with a well-established MAPK14-MAPK11 inhibitor (SB202190), which has been extensively used in vitro and in vivo. Our results indicate that inhibition of MAPK/p38 significantly affects PCa cell survival in an STK11-dependent manner. Indeed, we found that pharmacologic inactivation of MAPK/p38 does not affect viability of STK11-proficient PCa cells due to the triggering of the AMPK-dependent autophagic pathway, while it induces apoptosis in STK11-deficient cells irrespective of androgen receptor (AR) status. Of note, AMPK inactivation or autophagy inhibition in STK11-proficient cells sensitize SB202190-treated PCa cells to apoptosis. On the other end, reconstitution of functional STK11 in STK11-deficient PCa cells abrogates apoptosis. Collectively, our data show that STK11 is a key factor involved in the early phases of prostate carcinogenesis, and suggest that it might be used as a predictive marker of therapeutic response to MAPK/p38 inhibitors in PCa patients.     

A Multi-Site Knowledge Attitude and Practice Survey of Ebola Virus Disease in Nigeria

Background

The 2014 Ebola Virus Disease (EVD) outbreak was characterised by fear, misconceptions and irrational behaviours. We conducted a knowledge attitude and practice survey of EVD in Nigeria to inform implementation of effective control measures.

Methods

Between July 30^th and September 30^th 2014, we undertook a cross sectional study on knowledge, attitude and practice (KAP) of Ebola Virus Disease (EVD) among adults of the general population and healthcare workers (HCW) in three states of Nigeria, namely Bayelsa, Cross River and Kano states. Demographic information and data on KAP were obtained using a self-administered standardized questionnaire. The percentage KAP scores were categorised as good and poor. Independent predictors of good knowledge of EVD were ascertained using a binary logistic regression model.

Results

Out of 1035 study participants with median age of 32 years, 648 (62.6%) were males, 846 (81.7%) had tertiary education and 441 (42.6%) were HCW. There were 218, 239 and 578 respondents from Bayelsa, Cross River and Kano states respectively. The overall median percentage KAP scores and interquartile ranges (IQR) were 79.46% (15.07%), 95.0% (33.33%) and 49.95% (37.50%) respectively. Out of the 1035 respondents, 470 (45.4%), 544(52.56%) and 252 (24.35%) had good KAP of EVD defined using 80%, 90% and 70% score cut-offs respectively. Independent predictors of good knowledge of EVD were being a HCW (Odds Ratio-OR-2.89, 95% Confidence interval-CI of 1.41–5.90), reporting ‘moderate to high fear of EVD’ (OR-2.15, 95% CI-(1.47–3.13) and ‘willingness to modify habit’ (OR-1.68, 95% CI-1.23–2.30).

Conclusion

Our results reveal suboptimal EVD-related knowledge, attitude and practice among adults in Nigeria. To effectively control future outbreaks of EVD in Nigeria, there is a need to implement public sensitization programmes that improve understanding of EVD and address EVD-related myths and misconceptions, especially among the general population.     

Direct Observation (DO) for Drug-Resistant Tuberculosis: Do We Really DO?

Introduction

Directly-observed therapy (DOT) is recommended for drug-resistant tuberculosis (DR-TB) patients during their entire treatment duration. However, there is limited published evidence on implementation of direct observation (DO) in the field. This study aims to detail whether DO was followed with DR-TB patients in a Médecins Sans Frontières (MSF) tuberculosis program in Mumbai, India.

Methods

This was a cross-sectional, mixed-methods study. Existing qualitative data from a purposively-selected subset of 12 patients, 5 DOT-providers and 5 family members, were assessed in order to determine how DO was implemented. A questionnaire-based survey of DR-TB patients, their DOT-providers and MSF staff was completed between June and August 2014. Patients were defined as”following Strict DO” and “following DO” if a DOT-provider had seen the patient swallow his/her medications “every day” or “most of the days” respectively. If DO was not followed, reasons were also recorded. The qualitative data were analysed for theme and content and used to supplement the questionnaire-based data.

Results

A total of 70 DR-TB patients, 65 DOT-providers and 21 MSF health staff were included. Fifty-five per cent of the patients were HIV-co-infected and 41% had multidrug-resistant-TB plus additional resistance to a fluoroquinolone. Among all patients, only 14% (10/70) and 20% (14/70) self-reported “following Strict DO” and “following DO” respectively. Among DOT-providers, 46% (30/65) reported that their patients “followed DO”. MSF health staff reported none of the patients “followed DO”. Reasons for not implementing DO included the unavailability of DOT-provider, time spent, stigma and treatment adverse events. The qualitative data also revealed that “Strict DO” was rarely followed and noted the same reasons for lack of implementation.

Conclusion

This mixed-methods study has found that a majority of patients with DR-TB in Mumbai did not follow DO, and this was reported by patients and care-providers. These data likely reflect the reality of DO implementation in many high-burden settings, since this relatively small cohort was supported and closely monitored by a skilled team with access to multiple resources. The findings raise important concerns about the necessity of DO as a “pillar” of DR-TB treatment which need further validation in other settings. They also suggest that patient-centred adherence strategies might be better approaches for supporting patients on treatment.     

Hydroxychloroquine Protects against Cardiac Ischaemia/Reperfusion Injury In Vivo via Enhancement of ERK1/2 Phosphorylation

An increasing number of investigations including human studies demonstrate that pharmacological ischaemic preconditioning is a viable way to protect the heart from myocardial ischaemia/reperfusion (I/R) injury. This study investigated the role of hydroxychloroquine (HCQ) in the heart during I/R injury. In vitro and in vivo models of myocardial I/R injury were used to assess the effects of HCQ. It was found that HCQ was protective in neonatal rat cardiomyocytes through inhibition of apoptosis, measured by TUNEL and cleaved caspase-3. This protection in vitro was mediated through enhancement of ERK1/2 phosphorylation mediated by HCQ in a dose-dependent fashion. A decrease in infarct size was observed in an in vivo model of myocardial I/R injury in HCQ treated animals and furthermore this protection was blocked in the presence of the ERK1/2 inhibitor U0126. For the first time, we have shown that HCQ promotes a preconditioning like protection in an in vivo simulated rat myocardial I/R injury model. Moreover, it was shown that HCQ is protective via enhanced phosphorylation of the pro-survival kinase ERK1/2.     

New karyotype for Mesomys stimulax (Rodentia,Echimyidae) from the Brazilian Amazon: A case for species complex?

Mesomys Wagner, 1845 (Rodentia, Echimyidae, Eumysopinae) currently has four recognized species, three of which occur in Brazil: Mesomys hispidus (probably a species complex), M. occultus, and M. stimulax. Mesomys leniceps is found in montane forests of northern Peru. Mesomys stimulax, the focus of the present study, has a distribution that is restricted to the central and eastern Amazonia south of the Amazon River, extending from the left bank of the Tapajós River to the right bank of the Tocantins River, and south to the southeast portion of Pará State. The genus presents karyotypes with diploid number 2n = 60 and Fundamental Number (FN) = 116 for M. hispidus and M. stimulax, and 2n = 42, FN = 54 for M. occultus. We studied the karyotype of a female specimen of M. stimulax collected from the Tapirapé‐Aquiri National Forest, Marabá, Pará, Brazil, in the Xingu/Tocantins interfluvium. The obtained karyotype (2n = 60 and FN = 110) differs from that described in the literature for both M. stimulax and M. hispidus by exhibiting more biarmed chromosomes, probably due to pericentric inversions and/or centromeric repositioning, and exhibiting differences in the amount and distribution of constitutive heterochromatin (CH). These results suggest that, similar to what has already been proposed for M. hispidus, M. stimulax may represent a species complex and/or cryptic species. The mechanisms of chromosomal diversification in Mesomys and the biogeographic implications are discussed reinforcing the need for broad systematic review for Mesomys.     

The Proteasome-associated Protein Ecm29 Inhibits Proteasomal ATPase Activity and in Vivo Protein Degradation by the Proteasome

Several proteasome-associated proteins regulate degradation by the 26 S proteasome using the ubiquitin chains that mark most substrates for degradation. The proteasome-associated protein Ecm29, however, has no ubiquitin-binding or modifying activity, and its direct effect on substrate degradation is unclear. Here, we show that Ecm29 acts as a proteasome inhibitor. Besides inhibiting the proteolytic cleavage of peptide substrates in vitro, it inhibits the degradation of ubiquitin-dependent and -independent substrates in vivo. Binding of Ecm29 to the proteasome induces a closed conformation of the substrate entry channel of the core particle. Furthermore, Ecm29 inhibits proteasomal ATPase activity, suggesting that the mechanism of inhibition and gate regulation by Ecm29 is through regulation of the proteasomal ATPases. Consistent with this, we identified through chemical cross-linking that Ecm29 binds to, or in close proximity to, the proteasomal ATPase subunit Rpt5. Additionally, we show that Ecm29 preferentially associates with both mutant and nucleotide depleted proteasomes. We propose that the inhibitory ability of Ecm29 is important for its function as a proteasome quality control factor by ensuring that aberrant proteasomes recognized by Ecm29 are inactive.     

Zinc ion coordination as a modulating factor of the ZnuA histidine-rich loop flexibility: A molecular modeling and fluorescence spectroscopy study

ZnuA is the soluble component of the high-affinity ZnuABC zinc transporter belonging to the ATP-binding cassette-type periplasmic Zn-binding proteins. The zinc transporter ZnuABC is composed by three proteins: ZnuB, the membrane permease, ZnuC, the ATPase component and ZnuA, the soluble periplasmic metal-binding protein which captures Zn and delivers it to ZnuB.The ZnuA protein contains a charged flexible loop, rich in histidines and acidic residues, showing significant species-specific differences. Various studies have established that this loop contributes to the formation of a secondary zinc binding site, which has been proposed to be important in the acquisition of periplasmic Zn for its delivery to ZnuB or for regulation of zinc uptake. Due to its high mobility the structure of the histidine-rich loop has never been solved by X-ray diffraction studies. In this paper, through a combined use of molecular modeling, mutagenesis and fluorescence spectroscopy, we confirm the presence of two zinc binding sites characterized by different affinities for the metal ion and show that the flexibility of the loop is modulated by the binding of the zinc ions to the protein. The data obtained by fluorescence spectroscopy have then be used to validate a 3D model including the unsolved histidine-rich loop.