Mapping of a Leishmania major gene/locus that confers pentamidine resistance by deletion and insertion of transposable element

Pentamidine (PEN) is an alternative compound to treat antimony-resistant leishmaniasis patients, which cellular target remains unclear. One approach to the identification of prospective targets is to identify genes able to mediate PEN resistance following overexpression. Starting from a genomic library of transfected parasites bearing a multicopy episomal cosmid vector containing wild-type Leishmania major DNA, we isolated one locus capable to render PEN resistance to wild type cells after DNA transfection. In order to map this Leishmania locus, cosmid insert was deleted by two successive sets of partial digestion with restriction enzymes, followed by transfection into wild type cells, overexpression, induction and functional tests in the presence of PEN. To determine the Leishmania gene related to PEN resistance, nucleotide sequencing experiments were done through insertion of the transposon Mariner element of Drosophila melanogaster (mosK) into the deleted insert to work as primer island. Using general molecular techniques, we described here this method that permits a quickly identification of a functional gene facilitating nucleotide sequence experiments from large DNA fragments. Followed experiments revealed the presence of a P-Glycoprotein gene in this locus which role in Leishmania metabolism has now been analyzed.A Pentamidina (PEN) é um composto alternativo para o tratamento de pacientes com leishmaniose que apresentam resistência ao antimônio, cujo alvo celular continua incerto. Uma abordagem para se identificar prováveis alvos seria a identificação e super-expressão de genes capazes de mediar resistência a PEN. A partir de uma genoteca construída com o DNA de Leishmania major em um vetor - cosmídio que se desenvolve tanto em bactérias como nas células do parasita, isolamos um locus que após transfecção é capaz de produzir resistência a PEN às células do parasita. Almejando o mapeamento desse locus de leishmania, o inserto clonado nesse cosmídio foi deletado através de duas digestões parciais sucessivas com enzimas de restrição, seguida de transfecção em células selvagens, super-expressão gênica, indução e testes funcionais na presença de PEN. Para determinar o gene de Leishmania relacionado com a resistência a PEN, o seqüenciamento de nucleotídeos foi executado após inserção de elementos transposicionais de Drosophila melanogaster no interior do inserto deletado para atuar como 'ilhas de iniciadores'. Descrevemos aqui o mapeamento desse locus, após a inserção transposicional, que além de facilitar o seqüenciamento de nucleotídeos de grandes fragmentos de DNA, permite uma rápida identificação do gene relacionado com esse fenótipo. Experimentos posteriores revelaram neste locus a presença do gene de uma Glicoproteína-P de membrana, cujo papel no metabolismo na Leishmania está sendo analisado

Produção de concentrados de ácidos graxos por hidrólise de óleos vegetais mediada por lipase vegetal

The aim of this work was to verify the ability of enzymatic crude extract from dormant castor bean seeds to yield concentrated fatty acids by hydrolysis of polyunsaturated vegetable oils such as corn and sunflower. The enzymatic extract exhibited higher activity towards corn oil, which was selected for further studies to determine optimum hydrolysis conditions by factorial design. Maximum hydrolysis percentage (≈84%) was reached at 60% wt. oil:buffer acetate 100 mM pH 4.5, 33 ºC and 5.0% wt. of crude extract after 70 min of reaction. These results suggest that the use of low-cost lipase from castor bean seeds has potential for oil hydrolysis

Ros3 (Lem3p/CDC50) gene dosage is implicated in miltefosine susceptibility in Leishmania (Viannia) braziliensis clinical isolates and in Leishmania (Leishmania) major

The Ros3 protein is a component of the MT-Ros3 transporter complex, considered as the main route of miltefosine entry in Leishmania. L. braziliensis clinical isolates presenting differences in miltefosine susceptibility and uptake were previously shown to differentially express ros3. In this work, we showed that the ros3 gene copy number was increased in the isolate presenting the highest rates of miltefosine uptake and, thus, the highest susceptibility to this drug. The role of the ros3 gene dosage in miltefosine susceptibility was then investigated through a modulation of the gene copy number using two distinct approaches: through an overexpression of ros3 in a tolerant L. braziliensis clinical isolate and in L. major and by generating mono- and diallelic knockouts of this gene in L. major using clustered regularly interspaced short palindromic repeats (CRISPR) Cas9 (Cas = CRISPR-associated). Although the levels of ros3 mRNA were increased at least 40-fold in overexpressing clones, no significant reduction in the half-maximal effective concentration (EC50) for miltefosine was observed in these parasites. The partial or complete deletion of ros3 in L. major, in turn, resulted in a significant increase of 3 and 20 times, respectively, in the EC50 to miltefosine. We unequivocally showed that the ros3 copy number is one of the factors involved in the differential susceptibility and uptake of miltefosine

Potencial de extração de antocianinas em diferentes genótipos de capim-elefante.

As antocianinas são compostos fenólicos derivados do metabolismo secundário de plantas, conferindo-lhes uma pigmentação que varia do vermelho ao azul, dependendo do pH da planta. Esses fitocompostos apresentam grande potencial como base para o desenvolvimento de novos corantes, fármacos e produtos relacionados à química verde, devido à sua ampla aplicabilidade. O objetivo deste trabalho foi avaliar o potencial extrativo de antocianinas em genótipos de capimelefante, utilizando a metodologia do pH Único. Foram detectadas diferenças significativas para todas as características qualitativas avaliadas. Considerando o potencial de produção de biomassa total e de folhas, infere-se que a extração de antocianinas a partir das folhas do capim-elefante roxo (genótipo T_44.1) é o mais recomendado para a produção de novos compostos bioativos, devido a sua alta produção de biomassa e de antocianinas

Cd doping effects in the heavy-fermion compounds Ce2MIn8 (M = Rh and Ir)

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)Low-temperature magnetic properties of Cd-doped Ce2MIn8 (M = Rh and Ir) single crystals are investigated. Experiments of temperature-dependent magnetic-susceptibility, heat-capacity, and electrical-resistivity measurements revealed that Cd doping enhances the antiferromagnetic (AFM) ordering temperature from T-N = 2.8 K (x=0) to T-N=4.8 K (x=0.21) for Ce2RhIn8-xCdx and induces long-range AFM ordering with T-N = 3.8 K (x=0.21) for Ce2IrIn8-xCdx. Additionally, x-ray and neutron magnetic scattering studies showed that Cd-doped samples present below T-N a commensurate antiferromagnetic structure with a propagation vector (epsilon) over right arrow=(1/2, 1/2, 0). The resolved magnetic structures for both compounds indicate that the Cd doping tends to rotate the direction of the ordered magnetic moments toward the ab plane. This result suggests that the Cd doping affects the Ce3+ ground-state single-ion anisotropy modifying the crystalline electrical field (CEF) parameters at the Ce3+ site. Indications of CEF evolution induced by Cd doping were also found in the electrical-resistivity measurements. Comparisons between our results and the general effects of Cd doping on the related compounds CeMIn5 (M=Co, Rh, and Ir) confirms the claims that the Cd doping induced electronic tuning is the main effect favoring AFM ordering in these compounds.8124Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES

Rarity of monodominance in hyperdiverse Amazonian forests.

Tropical forests are known for their high diversity. Yet, forest patches do occur in the tropics where a single tree species is dominant. Such "monodominant" forests are known from all of the main tropical regions. For Amazonia, we sampled the occurrence of monodominance in a massive, basin-wide database of forest-inventory plots from the Amazon Tree Diversity Network (ATDN). Utilizing a simple defining metric of at least half of the trees ≥ 10 cm diameter belonging to one species, we found only a few occurrences of monodominance in Amazonia, and the phenomenon was not significantly linked to previously hypothesized life history traits such wood density, seed mass, ectomycorrhizal associations, or Rhizobium nodulation. In our analysis, coppicing (the formation of sprouts at the base of the tree or on roots) was the only trait significantly linked to monodominance. While at specific locales coppicing or ectomycorrhizal associations may confer a considerable advantage to a tree species and lead to its monodominance, very few species have these traits. Mining of the ATDN dataset suggests that monodominance is quite rare in Amazonia, and may be linked primarily to edaphic factors

Gene Expression Profiling and Molecular Characterization of Antimony Resistance in Leishmania amazonensis

Leishmania are unicellular microorganisms that can be transmitted to humans by the bite of sandflies. They cause a spectrum of diseases called leishmaniasis, which are classified as neglected tropical diseases by the World Health Organization. The treatment of leishmaniasis is based on the administration of antimony-containing drugs. These drugs have been used since 1947 and still constitute the mainstay for leishmaniasis treatment in several countries. One of the problems with these compounds is the emergence of resistance. Our work seeks to understand how these parasites become resistant to the drug. We studied antimony-resistant Leishmania amazonensis mutants. We analyzed gene expression at the whole genome level in antimony-resistant parasites and identified mechanisms used by Leishmania for resistance. This work could help us in developing new strategies for treatment in endemic countries where people are unresponsive to antimony-based chemotherapy. The identification of common mechanisms among different species of resistant parasites may also contribute to the development of diagnostic kits to identify and monitor the spread of resistance

Long-Lasting Efficacy of Radio Electric Asymmetric Conveyer Neuromodulation Treatment on Functional Dysmetria, an Adaptive Motor Behavior

BackgroundFluctuating asymmetry (FA) is widely defined as the deviation from perfect bilateral symmetry and is considered an epigenetic measure of environmental stress. Rinaldi and Fontani hypothesized that the FA morpho-functional changes originate from an adaptive motor behavior determined by functional alterations in the cerebellum and neural circuits, not caused by a lesion, but induced by environmental stress. They called this phenomenon functional dysmetria (FD). On this premise, they developed the radio electric asymmetric conveyer (REAC) technology, a neuromodulation technology aimed at optimizing the best neuro-psycho-motor strategies in relation to environmental interaction.AimsPrevious studies showed that specific REAC neuro postural optimization (NPO) treatment can induce stable FD recovery. This study aimed to verify the duration of the NPO effect in inducing the stable FD recovery over timeMaterials and methodsData were retrospectively collected from a population of 29,794 subjects who underwent a specific semiological FD assessment and received the NPO treatment, regardless of the pathology referred.ResultsThe analysis of the data collected by the various participants in the study led us to ascertain the disappearance of FD in 100% of the cases treated, with a stability of the result detected up to 18 years after the single administration of the REAC NPO treatment.ConclusionsThe REAC NPO neurobiological modulation treatment consisting of a single administration surprisingly maintains a very long efficacy in the correction of FD. This effect can be explained as the long-lasting capacity of the NPO treatment to induce greater functional efficiency of the brain dynamics as proven in previous studies

Multiple Mutations in Heterogeneous Miltefosine-Resistant Leishmania major Population as Determined by Whole Genome Sequencing

Leishmania spp. are parasitic protozoa responsible for a spectrum of diseases known as leishmaniasis. There are few drugs available for the treatment of these diseases, and miltefosine is the first oral drug used in treatment of visceral leishmaniasis, a form of the disease that can be lethal if not treated. In this study, we seek to understand the mechanism of action and identify targets of the drug by generating promastigote mutants highly resistant to miltefosine. Two independent mutants were submitted to short read whole genome sequencing. Genome analysis of these mutants has permitted us to identify point mutations in three genes (P-type ATPase, pyridoxal kinase and α-adaptin like protein) that were also present in other independent miltefosine resistant mutants. Some of the new genes identified here could be useful as potential markers for miltefosine resistance in Leishmania. Moreover, our approach has permitted us to highlight that resistance can be highly heterogeneous at the population level with individual clones derived from this population differing both in terms of genotypes but also susceptibility phenotypes. This may have practical applications while studying resistance