Genome-wide gene expression profile induced by exposure to cadmium acetate in Leishmania infantum promastigotes

Leishmania infantum is the etiological agent of visceral leishmaniasis in Mediterranean areas. The life cycle ofthe protist is dimorphic and heteroxene, as promastigotes develop inside the gut of sand-fly vectors and amastigotes multiply inside mammalian phagocytic cells. In previous studies, we analyzed the expression profiles of these stages and the modulation of gene expression triggered by temperature increase and acidification, both of which are crucial in the differentiation of promastigotes to amastigotes. Differential expression profiles of translation initiation and elongation factors were detected.Here we report that the presence of 1 mM cadmium acetate in the culture medium leads to a shock response consisting ofgrowth arrest, morphological changes, the absence of motility, and the up-regulation of genes that code for: a heavy metaltransporter, trypanothione reductase, a haloacid dehalogenase-like hydrolase, and a metalloexopeptidase from the M20 family, among others. This response is probably controlled by the differential expression of regulatory genes such as those encoding initiation factors 4E, eukaryotic translation initiation factor 3 subunits 8 and 2α, and elongation factor 1β. The initiation factor 2α gene is induced in anomalous environments, i.e., those outside of the protist’s normal life-cycle progression, for example, in response to the presence of cadmium ions, acidification without temperature increase, and vice versa. Our results suggest that the regulation of gene expression is a key component of the shock response. [Int Microbiol 2011; 14(1):1-11

Functional genomics in sand fly-derived Leishmania promastigotes

BACKGROUND: Leishmania development in the sand fly gut leads to highly infective forms called metacyclic promastigotes. This process can be routinely mimicked in culture. Gene expression-profiling studies by transcriptome analysis have been performed with the aim of studying promastigote forms in the sand fly gut, as well as differences between sand fly-and culture-derived promastigotes. FINDINGS: Transcriptome analysis has revealed the crucial role of the microenvironment in parasite development within the sand fly gut because substantial differences and moderate correlation between the transcriptomes of cultured and sand fly-derived promastigotes have been found. Sand fly-derived metacyclics are more infective than metacyclics in culture. Therefore, some caution should be exercised when using cultured promastigotes, depending on the experimental design. The most remarkable examples are the hydrophilic acidic surface protein/small endoplasmic reticulum protein (HASP/SHERP) cluster, the glycoprotein 63 (gp63), and autophagy genes, which are up-regulated in sand fly-derived promastigotes compared with cultured promastigotes. Because HASP/SHERP genes are up-regulated in nectomonad and metacyclic promastigotes in the sand fly, the encoded proteins are not metacyclic specific. Metacyclic promastigotes are distinguished by morphology and high infectivity. Isolating them from the sand fly gut is not exempt from technical difficulty, because other promastigote forms remain in the gut even 15 days after infection. Leishmania major procyclic promastigotes within the sand fly gut up-regulate genes involved in cell cycle regulation and glucose catabolism, whereas metacyclics increase transcript levels of fatty acid biosynthesis and ATP-coupled proton transport genes. Most parasite's signal transduction pathways remain uncharacterized. Future elucidation may improve understanding of parasite development, particularly signaling molecule-encoding genes in sand fly versus culture and between promastigote forms in the sand fly gut. CONCLUSIONS: Transcriptome analysis has been demonstrated to be technically efficacious to study differential gene expression in sand fly gut promastigote forms. Transcript and protein levels are not well correlated in these organisms (approximately 25% quantitative coincidences), especially under stress situations and at differentiation processes. However, transcript and protein levels behave similarly in approximately 60% of cases from a qualitative point of view (increase, decrease, or no variation). Changes in translational efficiency observed in other trypanosomatids strongly suggest that the differences are due to translational regulation and regulation of the steady-state protein levels. The lack of low-input sample strategies does not allow translatome and proteome analysis of sand fly-derived promastigotes so far.The authors thank the Ramón Areces Foundation for a contract. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.S

Leishmania infantum UBC1 in Metacyclic Promastigotes from Phlebotomus perniciosus, a Vaccine Candidate for Zoonotic Visceral Leishmaniasis

Leishmania parasites cause outstanding levels of morbidity and mortality in many developing countries in tropical and subtropical regions. Numerous gene expression profiling studies have been performed comparing different Leishmania species' life-cycles and stage forms in regard to their distinct infective ability. Based on expression patterns, homology to human orthologues, in silico HLA-binding predictions, and annotated functions, we were able to select several vaccine candidates which are currently under study. One of these candidates is the Leishmania infantum ubiquitin-conjugating enzyme E2 (LiUBC1), whose relative levels, subcellular location, in vitro infectivity in the U937 myeloid human cell model, and protection levels in Syrian hamsters against L. infantum infection were studied herein. LiUBC1 displays a low level of similarity with the mammalian orthologs and relevant structure differences, such as the C-terminal domain, which is absent in the human ortholog. LiUBC1 is present in highly infective promastigotes. Knock-in parasites overexpressing the enzyme increased their infectivity, according to in vitro experiments. Syrian hamsters immunized with the recombinant LiUBC1 protein did not show any parasite burden in the spleen, unlike the infection control group. The IFN-γ transcript levels in splenocytes were significantly higher in the LiUBC1 immunized group. Therefore, LiUBC1 induced partial protection against L. infantum in the Syrian hamster model.This work was financed by a contract with CZ Vaccines, Porriño, Spain, and partially defrayed by a grant from the Fundación Ramón Areces. JL thanks CZ Vaccines for the fellowship.S

Temperature increase prevails over acidification in gene expression modulation of amastigote differentiation in Leishmania infantum

<p>Abstract</p> <p>Background</p> <p>The extracellular promastigote and the intracellular amastigote stages alternate in the digenetic life cycle of the trypanosomatid parasite <it>Leishmania</it>. Amastigotes develop inside parasitophorous vacuoles of mammalian phagocytes, where they tolerate extreme environmental conditions. Temperature increase and pH decrease are crucial factors in the multifactorial differentiation process of promastigotes to amastigotes. Although expression profiling approaches for axenic, cell culture- and lesion-derived amastigotes have already been reported, the specific influence of temperature increase and acidification of the environment on developmental regulation of genes has not been previously studied. For the first time, we have used custom <it>L. infantum </it>genomic DNA microarrays to compare the isolated and the combined effects of both factors on the transcriptome.</p> <p>Results</p> <p>Immunofluorescence analysis of promastigote-specific glycoprotein gp46 and expression modulation analysis of the amastigote-specific A2 gene have revealed that concomitant exposure to temperature increase and acidification leads to amastigote-like forms. The temperature-induced gene expression profile in the absence of pH variation resembles the profile obtained under combined exposure to both factors unlike that obtained for exposure to acidification alone. In fact, the subsequent fold change-based global iterative hierarchical clustering analysis supports these findings.</p> <p>Conclusions</p> <p>The specific influence of temperature and pH on the differential regulation of genes described in this study and the evidence provided by clustering analysis is consistent with the predominant role of temperature increase over extracellular pH decrease in the amastigote differentiation process, which provides new insights into <it>Leishmania </it>physiology.</p

A putative Leishmania DNA polymerase theta protects the parasite against oxidative damage

Leishmania infantum is a protozoan parasite that is phagocytized by human macrophages. The host macrophages kill the parasite by generating oxidative compounds that induce DNA damage. We have identified, purified and biochemically characterized a DNA polymerase θ from L. infantum (LiPolθ), demonstrating that it is a DNA-dependent DNA polymerase involved in translesion synthesis of 8oxoG, abasic sites and thymine glycol lesions. Stably transfected L. infantum parasites expressing LiPolθ were significantly more resistant to oxidative and interstrand cross-linking agents, e.g. hydrogen peroxide, cisplatin and mitomycin C. Moreover, LiPolθ-overexpressing parasites showed an increased infectivity toward its natural macrophage host. Therefore, we propose that LiPolθ is a translesion synthesis polymerase involved in parasite DNA damage tolerance, to confer resistance against macrophage aggression.Spanish Ministry of Science and Innovation [AGL2010-21806-C02-01 to V.L., BFU2012-37969 to L.B.]; Comunidad deMadrid [S2010/BMD-2361 to L.B.]; Ramón Areces Foundation [050204100014 to V.L.]Peer Reviewe

Monte-Carlo Analysis of the Influence of the Electrical Component Tolerances on the Behavior of Series-Series- and LCC-Compensated IPT Systems

The use of compensation networks increases the power transfer capability of inductive power transfer (IPT) systems in the battery charging process of electric vehicles (EVs). Among the proposed topologies, the Series-Series (SS) and the LCC networks are currently in widespread use in wireless battery chargers based on IPT systems. This paper focuses on the study of the behavior of both compensation topologies when they are detuned due to the tolerances of their components. To compare their performances, a Monte-Carlo analysis was carried out using Simulink and MATLAB. The tolerance values, assigned independently to each component, fall within a [−20, 20] % range according to a normal distribution. Histograms and scatter plots were used for comparison purposes. The analysis reveals that the LCC network allows a tighter control over the currents that flow through the magnetic coupler coils. Moreover, it was found that the increments in those currents can be limited to some extent by selecting capacitors featuring low tolerance values in the LCC compensation. Nevertheless, the SS network remains an appropriate choice if size and cost are essential constraints in a given design

Control Scheme of a Bidirectional Inductive Power Transfer System for Electric Vehicles Integrated into the Grid

Inductive power transfer (IPT) systems have become a very effective technology when charging the batteries of electric vehicles (EVs), with numerous research works devoted to this field in recent years. In the battery charging process, the EV consumes energy from the grid, and this concept is called Grid-to-Vehicle (G2V). Nevertheless, the EV can also be used to inject part of the energy stored in the battery into the grid, according to the so-called Vehicle-to-Grid (V2G) scheme. This bidirectional feature can be applied to a better development of distributed generation systems, thus improving the integration of EVs into the grid (including IPT-powered EVs). Over the past few years, some works have begun to pay attention to bidirectional IPT systems applied to EVs, focusing on aspects such as the compensation topology, the design of the magnetic coupler or the power electronic configuration. Nevertheless, the design of the control system has not been extensively studied. This paper is focused on the design of a control system applied to a bidirectional IPT charger, which can operate in both the G2V and V2G modes. The procedure design of the control system is thoroughly explained and classical control techniques are applied to tailor the control scheme. One of the advantages of the proposed control scheme is the robustness when there is a mismatch between the coupling factor used in the model and the real value. Moreover, the control system can be used to limit the peak value of the primary side current when this value increases, thus protecting the IPT system. Simulation results obtained with PSCADTM/EMTDCTM show the good performance of the overall system when working in both G2V and V2G modes, while experimental results validate the control system behavior in the G2V mode.Inductive power transfer (IPT) systems have become a very effective technology when charging the batteries of electric vehicles (EVs), with numerous research works devoted to this field in recent years. In the battery charging process, the EV consumes energy from the grid, and this concept is called Grid-to-Vehicle (G2V). Nevertheless, the EV can also be used to inject part of the energy stored in the battery into the grid, according to the so-called Vehicle-to-Grid (V2G) scheme. This bidirectional feature can be applied to a better development of distributed generation systems, thus improving the integration of EVs into the grid (including IPT-powered EVs). Over the past few years, some works have begun to pay attention to bidirectional IPT systems applied to EVs, focusing on aspects such as the compensation topology, the design of the magnetic coupler or the power electronic configuration. Nevertheless, the design of the control system has not been extensively studied. This paper is focused on the design of a control system applied to a bidirectional IPT charger, which can operate in both the G2V and V2G modes. The procedure design of the control system is thoroughly explained and classical control techniques are applied to tailor the control scheme. One of the advantages of the proposed control scheme is the robustness when there is a mismatch between the coupling factor used in the model and the real value. Moreover, the control system can be used to limit the peak value of the primary side current when this value increases, thus protecting the IPT system. Simulation results obtained with PSCADTM/EMTDCTM show the good performance of the overall system when working in both G2V and V2G modes, while experimental results validate the control system behavior in the G2V mode

A Discrete-Time Control Method for Fast Transient Voltage-Sag Compensation in DVR

This paper presents a discrete-time domain control scheme for balanced voltage sag compensation using a Dynamic Voltage Restorer (DVR), which is recognized to be an appropriate and economical power electronic device with which to ameliorate these disturbances. The proposed control method is implemented in the synchronous reference frame (SRF), with two nested regulators, one of which includes an integral action. This algorithm has some advantages with respect to other control algorithms, such as the fact that the proposed methodology permits all the closed-loop poles of the DVR system to be placed in the desired locations in order to de ne the dynamical behavior with a reduction in the number of the electrical magnitudes to be measured and without the need for state observers, as occurs in traditional control methods. What is more, the well-known inner current loop implemented in other control schemes, which is employed to attenuate the resonance of the plant, is unnecessary. Furthermore, the unbalanced voltage sag compensation can be achieved by adding a ``plug-in'' controller and following the same methodology presented for balanced voltage sags to design the controller. The good performance of the proposed control scheme is validated by means of simulation and experimental results carried out with a 5 kW DVR laboratory prototype. The discrete-time control method is also compared with two control schemes previously proposed in literature

Supplementary Materials for U-Th dating of carbonate crusts reveals Neanderthal origin of Iberian cave art

Materiales suplementarios al artículo de Science 2018 sobre las dataciones de arte rupestre en la Península Ibérica mediante U-TH

The antibiotic resistance-free vaccine based on the non-replicative pPAL vector is fully protective against SARS-CoV-2 in the murine model

1 p.Background. The main objective of this work is the development of a DNA vaccine against the SARS-CoV-2 virus based on the non-replicative antibiotic resistance marker gene-free the plasmid vector pPAL.Methods. We designed pPAL-Sfs and pPAL-structural protein constructs. A PCR cloning procedure was carried out to obtain the pPAL-based recombinant vaccine and laboratory-scale batches of pPAL-based SARS-CoV-2 vaccine constructs were produced. Transfection was performed on the human HEK293 cell line with the pPAL-based recombinant vaccine. Expression was evaluated by Western blot. Evaluation of protection experiments against a lethal dose of 105 pfu of SARS-CoV-2 (Wuhan-Hu-1 and Delta strains) in K18-hACE2 female mice vaccinated intramuscularly with a prime/boost regimen was carried out by assessing both humoral and cellular immune responses. ELISA was used to evaluate humoral immunity, namely total IgG, as well as IgG1 and IgG2c subclasses. The cellular immune response was evaluated by quantifying the rate of IFN-γ producing splenocyte clones used ELISpot. In addition, characterization of the cellular response was carried out by intracellular staining (ICS) to identify of the rate of IFN-γ and TNF-α producing TCD4+ lymphocytes, as well as the proportion of TCD8+ lymphocytes. Determination of viral load in the main target organs was done by RT-PCR (lungs, heart, and brain). Virus replication capacity was also evaluated in target organs tissues. In vitro assays were performed out to determine the levels of neutralizing antibodies against SARS-CoV-2 virus.Results. The results show 100% protection of vaccinated animals in terms of symptomatology, animal weight, level of neutralizing antibodies against the virus and the rate of IFN-γ and TNF-α producing splenocyte clones. The analysis of IgG subclasses shows a predominance of IgG2c over IgG1, indicating the activation of a specific and cytotoxic Th1 protective cellular immune response and immunological memory. Finally, a reduction of viral load has been observed in vaccinated animals, with a clear reduction of virus replication in the main target organs. Furthermore, there is a synergistic effect increasing protection using the two plasmids p-PALSfs + pPAL-structural protein (under patent).Conclusions. The DNA vaccine pPAL-Sfs + pPAL-structural protein is fully protective in the mouse model in terms of maintenance of body weight, absence of significant clinical signs, viral load clearance in target organs and immune response. The immune response included neutralizing antibodies, predominance of IgG2c over IgG1 ratio, a Th1 response, and a multifunctional cytotoxic cellular response.Peer reviewe