Fnr (EtrA) acts as a fine-tuning regulator of anaerobic metabolism in <Emphasis Type="Italic">Shewanella oneidensis</Emphasis>MR-1

Background  

EtrA in Shewanella oneidensis MR-1, a model organism for study of adaptation to varied redox niches, shares 73.6% and 50.8% amino acid sequence identity with the oxygen-sensing regulators Fnr in E. coli and Anr in Pseudomonas aeruginosa, respectively; however, its regulatory role of anaerobic metabolism in Shewanella spp. is complex and not well understood.     

Sentinel network for monitoring in vitro susceptibility of Plasmodium falciparum to antimalarial drugs in Colombia: a proof of concept

Drug resistance is one of the principal obstacles blocking worldwide malaria control. In Colombia, malaria remains a major public health concern and drug-resistant parasites have been reported. In vitro drug susceptibility assays are a useful tool for monitoring the emergence and spread of drug-resistant Plasmodium falciparum. The present study was conducted as a proof of concept for an antimalarial drug resistance surveillance network based on in vitro susceptibility testing in Colombia. Sentinel laboratories were set up in three malaria endemic areas. The enzyme linked immunosorbent assay-histidine rich protein 2 and schizont maturation methods were used to assess the susceptibility of fresh P. falciparum isolates to six antimalarial drugs. This study demonstrates that an antimalarial drug resistance surveillance network based on in vitro methods is feasible in the field with the participation of a research institute, local health institutions and universities. It could also serve as a model for a regional surveillance network. Preliminary susceptibility results showed widespread chloroquine resistance, which was consistent with previous reports for the Pacific region. However, high susceptibility to dihydroartemisinin and lumefantrine compounds, currently used for treatment in the country, was also reported. The implementation process identified critical points and opportunities for the improvement of network sustainability strategies.     

Susceptibility of Colombian Plasmodium falciparum isolates to 4-aminoquinolines and the definition of amodiaquine resistance in vitro

There are wide variations in the threshold used to define in vitro resistance of Plasmodium falciparum to amodiaquine (AQ), probably due to differences in methodology and interpretation. In vitro susceptibility data of Colombian P. falciparum strains to AQ and N-desethylamodiaquine is used to illustrate the need to standardized methodologies and compare inhibitory concentrations, instead of resistant/susceptible phenotypes, when studying the mechanisms of resistance to AQ and monitoring drug susceptibility trends in the field.     

Respiratory nitrate ammonification by Shewanella oneidensis MR-1

Anaerobic cultures of Shewanella oneidensis MR-1 grown with nitrate as the sole electron acceptor exhibited sequential reduction of nitrate to nitrite and then to ammonium. Little dinitrogen and nitrous oxide were detected, and no growth occurred on nitrous oxide. A mutant with the napA gene encoding periplasmic nitrate reductase deleted could not respire or assimilate nitrate and did not express nitrate reductase activity, confirming that the NapA enzyme is the sole nitrate reductase. Hence, S. oneidensis MR-1 conducts respiratory nitrate ammonification, also termed dissimilatory nitrate reduction to ammonium, but not respiratory denitrification.     

Crystal structure and DNA repair activities of the AP endonuclease from Leishmania major

Apurinic/apyrimidinic endonucleases initiate the repair of abasic sites produced either spontaneously, from attack of bases by reactive oxygen species or as intermediates during base excision repair. The catalytic properties and crystal structure of Leishmania major apurinic/apyrimidinic endonuclease are described and compared with those of human APE1 and bacterial exonuclease III. The purified enzyme is shown to possess apurinic/apyrimidinic endonuclease activity of the same order as eukaryotic and prokaryotic counterparts and an equally robust 3'-phosphodiesterase activity. Consistent with this, expression of the L. major endonuclease confers resistance to both methyl methane sulphonate and H2O2 in Escherichia coli repair-deficient mutants while expression of the human homologue only reverts methyl methane sulphonate sensitivity. Structural analyses and modelling of the enzyme-DNA complex demonstrates a high degree of conservation to previously characterized homologues, although subtle differences in the active site geometry might account for the high 3'-phosphodiesterase activity. Our results confirm that the L. major's enzyme is a key element in mediating repair of apurinic/apyrimidinic sites and 3'-blocked termini and therefore must play an important role in the survival of kinetoplastid parasites after exposure to the highly oxidative environment within the host macrophage.     

Antiplasmodial activity of targeted zinc(II)-dipicolylamine complexes

This study measured the antiplasmodial activity of nine zinc-dipicolylamine (ZnDPA) complexes against three strains of Plasmodium falciparum, the causative parasite of malaria. Growth inhibition assays showed significant activity against all tested strains, with 50% inhibitory concentrations between 5 and 600 nM and almost no toxic effect against host cells including healthy red blood cells. Fluorescence microscopy studies with a green-fluorescent ZnDPA probe showed selective targeting of infected red blood cells. The results suggest that ZnDPA coordination complexes are promising antiplasmodial agents with potential for targeted malaria treatment.     

Asymmetric synthesis of quaternary α-amino acid derivatives and their fluorinated analogues

In this work, we describe the asymmetric synthesis of a series of fluorinated and non-fluorinated quaternary α-amino acid derivatives. This methodology involves the diastereoselective addition of chiral 2-p-tolylsulfinyl benzylcarbanions to either imines containing a 2-furyl moiety or trifluoromethyl α-imino esters. Synthetic practicality of this method is demonstrated by short (two-steps) and convenient preparation of 2-(trifluoromethyl)indoline-2-carboxylates.     

Targeting SR Proteins Improves SMN Expression in Spinal Muscular Atrophy Cells

Spinal muscular atrophy (SMA) is one of the most common inherited causes of pediatric mortality. SMA is caused by deletions or mutations in the survival of motor neuron 1 (SMN1) gene, which results in SMN protein deficiency. Humans have a centromeric copy of the survival of motor neuron gene, SMN2, which is nearly identical to SMN1. However, SMN2 cannot compensate for the loss of SMN1 because SMN2 has a single-nucleotide difference in exon 7, which negatively affects splicing of the exon. As a result, most mRNA produced from SMN2 lacks exon 7. SMN2 mRNA lacking exon 7 encodes a truncated protein with reduced functionality. Improving SMN2 exon 7 inclusion is a goal of many SMA therapeutic strategies. The identification of regulators of exon 7 inclusion may provide additional therapeutic targets or improve the design of existing strategies. Although a number of regulators of exon 7 inclusion have been identified, the function of most splicing proteins in exon 7 inclusion is unknown. Here, we test the role of SR proteins and hnRNP proteins in SMN2 exon 7 inclusion. Knockdown and overexpression studies reveal that SRSF1, SRSF2, SRSF3, SRSF4, SRSF5, SRSF6, SRSF7, SRSF11, hnRNPA1/B1 and hnRNP U can inhibit exon 7 inclusion. Depletion of two of the most potent inhibitors of exon 7 inclusion, SRSF2 or SRSF3, in cell lines derived from SMA patients, increased SMN2 exon 7 inclusion and SMN protein. Our results identify novel regulators of SMN2 exon 7 inclusion, revealing potential targets for SMA therapeutics.     

Intestinal parasites and genotyping of Giardia duodenalis in children: first report of genotype B in isolates from human clinical samples in Mexico

Giardia duodenalis is one of the most prevalent enteroparasites in children. This parasite produces several clinical manifestations. The aim of this study was to determine the prevalence of genotypes of G. duodenalis causing infection in a region of southeastern Mexico. G. duodenalis cysts were isolated (33/429) from stool samples of children and molecular genotyping was performed by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis, targeting the triosephosphate isomerase ( tpi ) and glutamate dehydrogenase ( gdh ) genes. The tpi gene was amplified in all of the cyst samples, either for assemblage A (27 samples) or assemblage B (6 samples). RFLP analysis classified the 27 tpi -A amplicons in assemblage A, subgenotype I. Samples classified as assemblage B were further analysed using PCR-RFLP of the gdh gene and identified as assemblage B, subgenotype III. To our knowledge, this is the first report of assemblage B of G. duodenalis in human clinical samples from Mexico.