Calcium/calmodulin kinase1 and its relation to thermotolerance and HSP90 in Sporothrix schenckii: an RNAi and yeast two-hybrid study

<p>Abstract</p> <p>Background</p> <p><it>Sporothrix schenckii </it>is a pathogenic dimorphic fungus of worldwide distribution. It grows in the saprophytic form with hyaline, regularly septated hyphae and pyriform conidia at 25°C and as the yeast or parasitic form at 35°C. Previously, we characterized a calcium/calmodulin kinase in this fungus. Inhibitors of this kinase were observed to inhibit the yeast cell cycle in <it>S. schenckii</it>.</p> <p>Results</p> <p>The presence of RNA interference (RNAi) mechanism in this fungus was confirmed by the identification of a Dicer-1 homologue in <it>S. schenckii </it>DNA. RNAi technology was used to corroborate the role of calcium/calmodulin kinase I in <it>S. schenckii </it>dimorphism. Yeast cells were transformed with the pSilent-Dual2G (pSD2G) plasmid w/wo inserts of the coding region of the calcium/calmodulin kinase I (<it>sscmk1</it>) gene. Transformants were selected at 35°C using resistance to geneticin. Following transfer to liquid medium at 35°C, RNAi transformants developed as abnormal mycelium clumps and not as yeast cells as would be expected. The level of <it>sscmk1 </it>gene expression in RNAi transformants at 35°C was less than that of cells transformed with the empty pSD2G at this same temperature. Yeast two-hybrid analysis of proteins that interact with SSCMK1 identified a homologue of heat shock protein 90 (HSP90) as interacting with this kinase. Growth of the fungus similar to that of the RNAi transformants was observed in medium with geldanamycin (GdA, 10 μM), an inhibitor of HSP90.</p> <p>Conclusions</p> <p>Using the RNAi technology we silenced the expression of <it>sscmk1 </it>gene in this fungus. RNAi transformants were unable to grow as yeast cells at 35°C showing decreased tolerance to this temperature. The interaction of SSCMK1 with HSP90, observed using the yeast two-hybrid assay suggests that this kinase is involved in thermotolerance through its interaction with HSP90. SSCMK1 interacted with the C terminal domain of HSP90 where effector proteins and co-chaperones interact. These results confirmed SSCMK1 as an important enzyme involved in the dimorphism of <it>S. schenckii</it>, necessary for the development of the yeast phase of this fungus. Also this study constitutes the first report of the transformation of <it>S. schenckii </it>and the use of RNAi to study gene function in this fungus.</p

Antiviral evaluation of new synthetic bioconjugates based on GA-Hecate: A new class of antivirals targeting different steps of Zika Virus replication

Re-emerging arboviruses represent a serious health problem due to their rapid vector-mediated spread, mainly in urban tropical areas. The 2013–2015 Zika virus (ZIKV) outbreak in South and Central America has been associated with cases of microcephaly in newborns and Guillain–Barret syndrome. We previously showed that the conjugate gallic acid—Hecate (GA-FALALKALKKALKKLKKALKKAL-CONH2)—is an efficient inhibitor of the hepatitis C virus. Here, we show that the Hecate peptide is degraded in human blood serum into three major metabolites. These metabolites conjugated with gallic acid were synthesized and their effect on ZIKV replication in cultured cells was evaluated. The GA-metabolite 5 (GA-FALALKALKKALKKL-COOH) was the most efficient in inhibiting two ZIKV strains of African and Asian lineage at the stage of both virus entry (virucidal and protective) and replication (post-entry). We also demonstrate that GA-metabolite 5 does not affect cell growth after 7 days of continuous treatment. Thus, this study identifies a new synthetic antiviral compound targeting different steps of ZIKV replication in vitro and with the potential for broad reactivity against other flaviviruses. Our work highlights a promising strategy for the development of new antivirals based on peptide metabolism and bioconjugation

Virus-like particles of louping ill virus elicit potent neutralizing antibodies targeting multimers of viral envelope protein

Louping ill virus (LIV) is a tick-borne flavivirus that predominantly causes disease in livestock, especially sheep in the British Isles. A preventive vaccine, previously approved for veterinary use but now discontinued, was based on an inactivated whole virion that likely provided protection by induction of neutralizing antibodies recognizing the viral envelope (E) protein. A major disadvantage of the inactivated vaccine was the need for high containment facilities for the propagation of infectious virus, as mandated by the hazard group 3 status of the virus. This study aimed to develop high-efficacy non-infectious protein-based vaccine candidates. Specifically, soluble envelope protein (sE), and virus-like particles (VLPs), comprised of the precursor of membrane and envelope proteins, were generated, characterized, and studied for their immunogenicity in mice. Results showed that the VLPs induced more potent virus neutralizing response compared to sE, even though the total anti-envelope IgG content induced by the two antigens was similar. Depletion of anti-monomeric E protein antibodies from mouse immune sera suggested that the neutralizing antibodies elicited by the VLPs targeted epitopes spanning the highly organized structure of multimer of the E protein, whereas the antibody response induced by sE focused on E monomers. Thus, our results indicate that VLPs represent a promising LIV vaccine candidate

Zika virus-like particles bearing covalent dimer of envelope protein protect mice from lethal challenge

Zika virus (ZIKV) envelope (E) protein is the major target of neutralizing antibodies in infected host, and thus represents a candidate of interest for vaccine design. However, a major concern in the development of vaccines against ZIKV and the related dengue virus is the induction of cross-reactive poorly neutralizing antibodies that can cause antibody-dependent enhancement (ADE) of infection. This risk necessitates particular care in vaccine design. Specifically, the engineered immunogens should have their cross-reactive epitopes masked, and they should be optimized for eliciting virus-specific strongly neutralizing antibodies upon vaccination. Here, we developed ZIKV subunit- and virus-like particle (VLP)-based vaccines displaying E in its wild type form, or E locked in a covalently linked dimeric (cvD) conformation to enhance the exposure of E dimers to the immune system. Compared with their wild-type derivatives, cvD immunogens elicited antibody with higher capacity of neutralizing virus infection of cultured cells. More importantly, these immunogens protected animals from lethal challenge with both the African and Asian lineages of ZIKV, impairing virus dissemination to brain and sexual organs. Moreover, the locked conformation of E reduced the exposure of epitopes recognized by cross-reactive antibodies and therefore showed a lower potential to induce ADE in vitro. Our data demonstrated a higher efficacy of the VLPs in comparison with the soluble dimer and support VLP-cvD as a promising ZIKV vaccine

Rational Zika vaccine design via the modulation of antigen membrane anchors in chimpanzee adenoviral vectors

Zika virus (ZIKV) emerged on a global scale and no licensed vaccine ensures long-lasting anti-ZIKV immunity. Here we report the design and comparative evaluation of four replication-deficient chimpanzee adenoviral (ChAdOx1) ZIKV vaccine candidates comprising the addition or deletion of precursor membrane (prM) and envelope, with or without its transmembrane domain (TM). A single, non-adjuvanted vaccination of ChAdOx1 ZIKV vaccines elicits suitable levels of protective responses in mice challenged with ZIKV. ChAdOx1 prME ∆TM encoding prM and envelope without TM provides 100% protection, as well as long-lasting anti-envelope immune responses and no evidence of in vitro antibody-dependent enhancement to dengue virus. Deletion of prM and addition of TM reduces protective efficacy and yields lower anti-envelope responses. Our finding that immunity against ZIKV can be enhanced by modulating antigen membrane anchoring highlights important parameters in the design of viral vectored ZIKV vaccines to support further clinical assessments

Ciencias de la Biología y Agronomía

Este volumen I contiene 17 capítulos arbitrados que se ocupan de estos asuntos en Tópicos Selectos de Ciencias de la Biología y Agronomía, elegidos de entre las contribuciones, reunimos algunos investigadores y estudiantes. Se presenta un Estudio Comparativo de los Recursos Hidrológico-Forestales de la Microcuenca de la Laguna de Epatlan, Pue. (1993 a 2014); la Situación Actual de la Mancha de Asfalto en Maíz (Zea mays L.) en los Municipios de Jiquipilas y Ocozocoautla, Chiapas, México; las poblaciones sobresalientes de maíz de la raza Zapalote Chico, en la Región Istmeña de Oaxaca; Se indica el índice de área foliar de cultivo de Chile Poblano mediante dos métodos en condiciones protegidas; Esquivel, Urzúa y Ramírez exploran el efecto de la biofertilización con Azospirillum en el crecimiento y producción de Jitomate; esbozan su artículo sobre la determinación del nivel de Heterosis en híbridos de Maíz para la Comarca Lagunera; una investigación sobre la estabilización de semilla de Solanum lycopersicum durante el almacenamiento y estimulación de la germinación; acotan sobre el CTAB como una nueva opción para la detección de Huanglongbing en cítricos, plantean su evaluación sobre el aluminio y cómo afecta la vida de florero de Heliconia psittacorum; indican sobre el impacto del H-564C, como un híbrido de maíz con alta calidad de proteina para el trópico húmedo de México; presetan su investigación sobre la producción de Piña Cayena Lisa y MD2 (Ananas comosus L.) en condiciones de Loma Bonita, en Oaxaca; acotan sobre el efecto de coberteras como control biológico por conservación contra áfidos en Nogal Pecanero; esbozan sobre la caracterización de cuatro genotipos de Frijol Negro en Martínez de la Torre, Veracruz, México; presentan una caracterización hidroecológica de la microcuenca de Arroyo Prieto, Yuriría, Gto., y alternativas para su restauración ambiental; presentan su investigación sobre el efecto del hongo Beauveria bassiana sobre solubilización de fosfatos y la disponibilidad de fósforo en el suelo; plantean su investigación sobre la Germinación y regeneración in vitro de Epidendrum falcatum LINDL; esbozan su artículo sobre genotipos de frijol negro y su tolerancia a sequía terminal en Veracruz, México

Studies of the mass composition of cosmic rays and proton-proton interaction cross-sections at ultra-high energies with the Pierre Auger Observatory

In this work, we present an estimate of the cosmic-ray mass composition from the distributions of the depth of the shower maximum (Xmax) measured by the fluorescence detector of the Pierre Auger Observatory. We discuss the sensitivity of the mass composition measurements to the uncertainties in the properties of the hadronic interactions, particularly in the predictions of the particle interaction cross-sections. For this purpose, we adjust the fractions of cosmic-ray mass groups to fit the data with Xmax distributions from air shower simulations. We modify the proton-proton cross-sections at ultra-high energies, and the corresponding air shower simulations with rescaled nucleus-air cross-sections are obtained via Glauber theory. We compare the energy-dependent composition of ultra-high-energy cosmic rays obtained for the different extrapolations of the proton-proton cross-sections from low-energy accelerator data

Study of downward Terrestrial Gamma-ray Flashes with the surface detector of the Pierre Auger Observatory

The surface detector (SD) of the Pierre Auger Observatory, consisting of 1660 water-Cherenkov detectors (WCDs), covers 3000 km2 in the Argentinian pampa. Thanks to the high efficiency of WCDs in detecting gamma rays, it represents a unique instrument for studying downward Terrestrial Gamma-ray Flashes (TGFs) over a large area. Peculiar events, likely related to downward TGFs, were detected at the Auger Observatory. Their experimental signature and time evolution are very different from those of a shower produced by an ultrahigh-energy cosmic ray. They happen in coincidence with low thunderclouds and lightning, and their large deposited energy at the ground is compatible with that of a standard downward TGF with the source a few kilometers above the ground. A new trigger algorithm to increase the TGF-like event statistics was installed in the whole array. The study of the performance of the new trigger system during the lightning season is ongoing and will provide a handle to develop improved algorithms to implement in the Auger upgraded electronic boards. The available data sample, even if small, can give important clues about the TGF production models, in particular, the shape of WCD signals. Moreover, the SD allows us to observe more than one point in the TGF beam, providing information on the emission angle

Combined fit to the spectrum and composition data measured by the Pierre Auger Observatory including magnetic horizon effects

The measurements by the Pierre Auger Observatory of the energy spectrum and mass composition of cosmic rays can be interpreted assuming the presence of two extragalactic source populations, one dominating the flux at energies above a few EeV and the other below. To fit the data ignoring magnetic field effects, the high-energy population needs to accelerate a mixture of nuclei with very hard spectra, at odds with the approximate E$^{-2}$ shape expected from diffusive shock acceleration. The presence of turbulent extragalactic magnetic fields in the region between the closest sources and the Earth can significantly modify the observed CR spectrum with respect to that emitted by the sources, reducing the flux of low-rigidity particles that reach the Earth. We here take into account this magnetic horizon effect in the combined fit of the spectrum and shower depth distributions, exploring the possibility that a spectrum for the high-energy population sources with a shape closer to E$^{-2}$ be able to explain the observations