Analysis of Rev1p and Pol zeta in mitochondrial mutagenesis suggests an alternative pathway of damage tolerance

Ultraviolet light is a potent DNA damaging agent that induces bulky lesions in DNA which block the replicative polymerases. In order to ensure continued DNA replication and cell viability, specialized translesion polymerases bypass these lesions at the expense of introducing mutations in the nascent DNA strand. A recent study has shown that the N-terminal sequences of the nuclear translesion polymerases Rev1p and Pol zeta can direct GFP to the mitochondrial compartment of Saccharomyces cerevisiae. We have investigated the role of these polymerases in mitochondrial mutagenesis. Our analysis of mitochondrial DNA point mutations, microsatellite instability, and the spectra of mitochondrial mutations indicate that these translesion polymerases function in a less mutagenic pathway in the mitochondrial compartment than they do in the nucleus. Mitochondrial phenotypes resulting from the loss of Rev1p and Pol zeta suggest that although these polymerases are responsible for the majority of mitochondrial frameshift mutations, they do not greatly contribute to mitochondrial DNA point mutations. Analysis of spontaneous mitochondrial DNA point mutations suggests that Pol zeta may play a role in general mitochondrial DNA maintenance. In addition, we observe a 20-fold increase in UV-induced mitochondrial DNA point mutations in rev deficient strains. Our data provides evidence for an alternative damage tolerance pathway that is specific to the mitochondrial compartment.     

A randomised, double-blind, controlled vaccine efficacy trial of DNA/MVA ME-TRAP against malaria infection in Gambian adults

Background

Many malaria vaccines are currently in development, although very few have been evaluated for efficacy in the field. Plasmodium falciparum multiple epitope (ME)– thrombospondin-related adhesion protein (TRAP) candidate vaccines are designed to potently induce effector T cells and so are a departure from earlier malaria vaccines evaluated in the field in terms of their mechanism of action. ME-TRAP vaccines encode a polyepitope string and the TRAP sporozoite antigen. Two vaccine vectors encoding ME-TRAP, plasmid DNA and modified vaccinia virus Ankara (MVA), when used sequentially in a prime-boost immunisation regime, induce high frequencies of effector T cells and partial protection, manifest as delay in time to parasitaemia, in a clinical challenge model.

Methods and Findings

A total of 372 Gambian men aged 15–45 y were randomised to receive either DNA ME-TRAP followed by MVA ME-TRAP or rabies vaccine (control). Of these men, 296 received three doses of vaccine timed to coincide with the beginning of the transmission season (141 in the DNA/MVA group and 155 in the rabies group) and were followed up. Volunteers were given sulphadoxine/pyrimethamine 2 wk before the final vaccination. Blood smears were collected weekly for 11 wk and whenever a volunteer developed symptoms compatible with malaria during the transmission season. The primary endpoint was time to first infection with asexual P. falciparum. Analysis was per protocol.DNA ME-TRAP and MVA ME-TRAP were safe and well-tolerated. Effector T cell responses to a non-vaccine strain of TRAP were 50-fold higher postvaccination in the malaria vaccine group than in the rabies vaccine group. Vaccine efficacy, adjusted for confounding factors, was 10.3% (95% confidence interval, −22% to +34%; p = 0.49). Incidence of malaria infection decreased with increasing age and was associated with ethnicity.

Conclusions

DNA/MVA heterologous prime-boost vaccination is safe and highly immunogenic for effector T cell induction in a malaria-endemic area. But despite having produced a substantial reduction in liver-stage parasites in challenge studies of non-immune volunteers, this first generation T cell–inducing vaccine was ineffective at reducing the natural infection rate in semi-immune African adults.     

IMAGe association: additional clinical features and evidence for recessive autosomal inheritance

Congenital adrenal hypoplasia (CAH) normally occurs in the neonatal period, with patients presenting with more or less severe salt-wasting syndrome. X-linked CAH has been associated with mutations in the DAX-1 gene, and boys have also been shown to have hypogonadotrophic hypogonadism. Recently, in three unrelated boys, CAH was associated with intrauterine growth retardation (IUGR), metaphyseal dysplasia and genital abnormalities, defining a new association called IMAGe. We now report four additional patients with this association, including the first living female. The four patients belong to two unrelated families (one brother and one sister from each family). These patients have the main clinical characteristics of IMAGe association: IUGR, facial dysmorphy (frontal bossing, broad nasal bridge, low-set ears), short limbs due to metaphyseal dysplasia, and adrenal insufficiency. As these patients are older than the initial three patients, we can also describe additional features: short adult height, normal puberty in boys as well as in the living girl. The boys have hypospadias associated with micropenis. The living girl came to clinical attention at the age of 5 years as a result of a familial survey, and careful questioning revealed that she had been suffering from mild adrenal insufficiency since early childhood. At least one boy has congenital hypotonia due to muscular dystrophy. In conclusion, these four new cases display familial transmission, strongly suggesting Mendelian autosomal recessive inheritance. Adrenal insufficiency may be mild. Hypotonia, described in all the patients, might be related to paucisymptomatic muscular dystrophy, as this condition is clearly heterogeneous varying with regard to severity, associated manifestations and outcome. If this symptom is part of the syndrome, which we cannot assume, it could help to localize the candidate gene.     

Free Fatty Acid Effects on the Atrial Myocardium: Membrane Ionic Currents Are Remodeled by the Disruption of T-Tubular Architecture

Background

Epicardial adiposity and plasma levels of free fatty acids (FFAs) are elevated in atrial fibrillation, heart failure and obesity, with potentially detrimental effects on myocardial function. As major components of epicardial fat, FFAs may be abnormally regulated, with a potential to detrimentally modulate electro-mechanical function. The cellular mechanisms underlying such effects of FFAs are unknown.

Objective

To determine the mechanisms underlying electrophysiological effects of palmitic (PA), stearic (SA) and oleic (OA) FFAs on sheep atrial myocytes.

Methods

We used electrophysiological techniques, numerical simulations, biochemistry and optical imaging to examine the effects of acutely (≤ 15 min), short-term (4–6 hour) or 24-hour application of individual FFAs (10 μM) on isolated ovine left atrial myocytes (LAMs).

Results

Acute and short-term incubation in FFAs resulted in no differences in passive or active properties of isolated left atrial myocytes (LAMs). 24-hour application had differential effects depending on the FFA. PA did not affect cellular passive properties but shortened (p<0.05) action potential duration at 30% repolarization (APD₃₀). APD₅₀ and APD₈₀ were unchanged. SA had no effect on resting membrane potential but reduced membrane capacitance by 15% (p<0.05), and abbreviated APD at all values measured (p≤0.001). OA did not significantly affect passive or active properties of LAMs. Measurement of the major voltage-gated ion channels in SA treated LAMs showed a ~60% reduction (p<0.01) of the L-type calcium current (I_Ca-L) and ~30% reduction (p<0.05) in the transient outward potassium current (I_TO). A human atrial cell model recapitulated SA effects on APD. Optical imaging showed that SA incubated for 24 hours altered t-tubular structure in isolated cells (p<0.0001).

Conclusions

SA disrupts t-tubular architecture and remodels properties of membrane ionic currents in sheep atrial myocytes, with potential implications in arrhythmogenesis.     

β-glucosidase from <Emphasis Type="Italic">Sclerotinia sclerotiorum</Emphasis>: a new and efficient purification procedure and use as a suitable marker in immuno-enzymatic assay

The β-glucosidase enzyme β-glu2 isolated from Sclerotinia sclerotiorum was purified and used as tracer in enzyme linked immunosorbent assay. A novel purification procedure of the protein was developed that consists of ammonium sulphate precipitation, gel filtration on Sephacryl S-200-HR column, ion exchange chromatography on DEAE-Toyopearl and polybuffer exchanger PBE 94 TM chromatofocusing. The pI value was 4.45. K _m and V _max values for the enzyme towards p-nitrophenyl-β-D-glucopyranoside were respectively 0.45 mM and 0.2 U/mL. Thermal stability showed that β-glu2 has a half-life of 85 min at 55 °C and of 25 min at 65 °C. β-glu2 was conjugated to goat anti-rabbit antibodies with glutaraldehyde as cross linking agent according to the one-step method. Conjugates were purified by HPLC gel filtration on TSK 2000. Enzymatic and immunological activities of the β-glucosidase conjugate component were tested by the ELISA method.     

Mapping the DNA- and zinc-binding domains of ASR1 (abscisic acid stress ripening), an abiotic-stress regulated plant specific protein

Abscisic acid stress ripening (ASR1) is a highly charged low molecular weight plant specific protein that is regulated by salt- and water-stresses. The protein possesses a zinc-dependent DNA-binding activity (Kalifa et al., Biochem. J. 381 (2004) 373) and overexpression in transgenic plants results in an increased salt-tolerance (Kalifa et al., Plant Cell Environ. 27 (2004) 1459). There are no structure homologs of ASR1, thus the structural and functional domains of the protein cannot be predicted. Here, we map the protein domains involved in the binding of Zn(2+) and DNA. Using mild acid hydrolysis, and a series of ASR1 carboxy-terminal truncations we show that the zinc-dependent DNA-binding could be mapped to the central/carboxy-terminal domain. In addition, using MALDI-TOF-MS with a non-acidic matrix, we show that two zinc ions are bound to the amino-terminal domain. Other zinc ion(s) bind the DNA-binding domain. Binding of zinc to ASR1 induces conformational changes resulting in a decreased sensitivity to proteases.     

A Non-Inferiority,Individually Randomized Trial of Intermittent Screening and Treatment versus Intermittent Preventive Treatment in the Control of Malaria in Pregnancy

Background

The efficacy of intermittent preventive treatment for malaria with sulfadoxine-pyrimethamine (IPTp-SP) in pregnancy is threatened in parts of Africa by the emergence and spread of resistance to SP. Intermittent screening with a rapid diagnostic test (RDT) and treatment of positive women (ISTp) is an alternative approach.

Methods and Findings

An open, individually randomized, non-inferiority trial of IPTp-SP versus ISTp was conducted in 5,354 primi- or secundigravidae in four West African countries with a low prevalence of resistance to SP (The Gambia, Mali, Burkina Faso and Ghana). Women in the IPTp-SP group received SP on two or three occasions whilst women in the ISTp group were screened two or three times with a RDT and treated if positive for malaria with artemether-lumefantrine (AL). ISTp-AL was non-inferior to IPTp-SP in preventing low birth weight (LBW), anemia and placental malaria, the primary trial endpoints. The prevalence of LBW was 15.1% and 15.6% in the IPTp-SP and ISTp-AL groups respectively (OR = 1.03 [95% CI: 0.88, 1.22]). The mean hemoglobin concentration at the last clinic attendance before delivery was 10.97g/dL and 10.94g/dL in the IPTp-SP and ISTp-AL groups respectively (mean difference: -0.03 g/dL [95% CI: -0.13, +0.06]). Active malaria infection of the placenta was found in 24.5% and in 24.2% of women in the IPTp-SP and ISTp-AL groups respectively (OR = 0.95 [95% CI 0.81, 1.12]). More women in the ISTp-AL than in the IPTp-SP group presented with malaria parasitemia between routine antenatal clinics (310 vs 182 episodes, rate difference: 49.4 per 1,000 pregnancies [95% CI 30.5, 68.3], but the number of hospital admissions for malaria was similar in the two groups.

Conclusions

Despite low levels of resistance to SP in the study areas, ISTp-AL performed as well as IPTp-SP. In the absence of an effective alternative medication to SP for IPTp, ISTp-AL is a potential alternative to IPTp in areas where SP resistance is high. It may also have a role in areas where malaria transmission is low and for the prevention of malaria in HIV positive women receiving cotrimoxazole prophylaxis in whom SP is contraindicated.

Trial Registration

ClinicalTrials.gov NCT01084213 Pan African Clinical trials Registry PACT201202000272122     

Health centre surveys as a potential tool for monitoring malaria epidemiology by area and over time

Background

Presently, many malaria control programmes use health facility data to evaluate the impact of their interventions. Facility-based malaria data, although useful, have problems with completeness, validity and representativeness and reliance on routinely collected health facility data might undermine demonstration of the magnitude of the impact of the recent scaleups of malaria interventions. To determine whether carefully conducted health centre surveys can be reliable means of monitoring area specific malaria epidemiology, we have compared malaria specific indices obtained from surveys in health centres with indices obtained from cross-sectional surveys conducted in their catchment communities.

Methods

A series of age stratified, seasonal, cross-sectional surveys were conducted during the peak malaria transmission season in 2008 and during the following dry season in 2009 in six ecologically diverse areas in The Gambia. Participants were patients who attended the health centres plus a representative sample from the catchment villages of these health facilities. Parasitaemia, anaemia, attributable proportion of fever and anti-MSP1-₁₉ antibody seroprevalence were compared in the health facility attendees and community participants.

Results

A total of 16,230 subjects completed the study; approximately half participated in the health centre surveys and half in the wet season surveys. Data from both the health centre and community surveys showed that malaria endemicity in The Gambia is now low, heterogeneous and seasonal. In the wet season, parasitaemia, seroprevalence and fever prevalence were higher in subjects seen in the health centres than in the community surveys. Age patterns of parasitaemia, attributable proportions of fever and seroprevalence rates were similar in subjects who participated in the community and health centre surveys.

Conclusion

Health centre surveys have potential as a surveillance tool for evaluating area specific malaria control activities and for monitoring changes in local malaria epidemiology over time.     

Simple synthetic toll-like receptor 2 ligands

Stimulation of toll-like receptor 2 (TLR2) by bacterial lipoproteins induces fast non-specific immune responses against pathogens followed by slow but specific adaptive immune responses. Development of synthetic TLR2 agonists/antagonists would be useful in the prevention of different infectious and immunologic disorders. The current study reports synthesis and TLR2 activity of two simple TLR2 ligands, which feature minimal structural requirement for TLR2 activity (two long lipid chains) and stimulate agonistic activity at nanomolar concentration.     

Mitochondrial genome maintenance: roles for nuclear nonhomologous end-joining proteins in Saccharomyces cerevisiae

Mitochondrial DNA (mtDNA) deletions are associated with sporadic and inherited diseases and age-associated neurodegenerative disorders. Approximately 85% of mtDNA deletions identified in humans are flanked by short directly repeated sequences; however, mechanisms by which these deletions arise are unknown. A limitation in deciphering these mechanisms is the essential nature of the mitochondrial genome in most living cells. One exception is budding yeast, which are facultative anaerobes and one of the few organisms for which directed mtDNA manipulation is possible. Using this model system, we have developed a system to simultaneously monitor spontaneous direct-repeat-mediated deletions (DRMDs) in the nuclear and mitochondrial genomes. In addition, the mitochondrial DRMD reporter contains a unique KpnI restriction endonuclease recognition site that is not present in otherwise wild-type (WT) mtDNA. We have expressed KpnI fused to a mitochondrial localization signal to induce a specific mitochondrial double-strand break (mtDSB). Here we report that loss of the MRX (Mre11p, Rad50p, Xrs2p) and Ku70/80 (Ku70p, Ku80p) complexes significantly impacts the rate of spontaneous deletion events in mtDNA, and these proteins contribute to the repair of induced mtDSBs. Furthermore, our data support homologous recombination (HR) as the predominant pathway by which mtDNA deletions arise in yeast, and suggest that the MRX and Ku70/80 complexes are partially redundant in mitochondria.