Voriconazole MICs are predictive for the outcome of experimental disseminated scedosporiosis

Scedosporiosis is associated with a mortality rate of up to 90% in patients suffering from disseminated infections. Recommended first-line treatment is voriconazole, but epidemiological cut-off values and clinical breakpoints have not been determined. Objectives: To correlate voriconazole treatment response in mice suffering from disseminated scedosporiosis with MIC values determined using CLSI broth microdilution, Etest (bioMe´rieux) and disc diffusio

The sterol C-24 methyltransferase encoding gene, erg6, is essential for viability of Aspergillus species

Triazoles, the most widely used class of antifungal drugs, inhibit the biosynthesis of ergosterol, a crucial component of the fungal plasma membrane. Inhibition of a separate ergosterol biosynthetic step, catalyzed by the sterol C-24 methyltransferase Erg6, reduces the virulence of pathogenic yeasts, but its effects on filamentous fungal pathogens like Aspergillus fumigatus remain unexplored. Here, we show that the lipid droplet-associated enzyme Erg6 is essential for the viability of A. fumigatus and other Aspergillus species, including A. lentulus, A. terreus, and A. nidulans. Downregulation of erg6 causes loss of sterol-rich membrane domains required for apical extension of hyphae, as well as altered sterol profiles consistent with the Erg6 enzyme functioning upstream of the triazole drug target, Cyp51A/Cyp51B. Unexpectedly, erg6-repressed strains display wild-type susceptibility against the ergosterol-active triazole and polyene antifungals. Finally, we show that erg6 repression results in significant reduction in mortality in a murine model of invasive aspergillosis. Taken together with recent studies, our work supports Erg6 as a potentially pan-fungal drug target

Effect of mistimed eating patterns on breast and prostate cancer risk (MCC-Spain Study)

Altres ajuts: Grant sponsor: Regional Government of the Basque Country; Grant sponsor: Consejerıa de Sanidad de la Region de Murcia; Grant sponsor: European Commission grants FOOD-CT-2006-036224-HIWATE; Grant sponsor: Spanish Association Against Cancer (AECC) Scientific Foundation; Grant sponsor: Fundacion Caja de Ahorros de Asturias and by the University of OviedoModern life involves mistimed sleeping and eating patterns that in experimental studies are associated with adverse health effects. We assessed whether timing of meals is associated with breast and prostate cancer risk taking into account lifestyle and chronotype, a characteristic correlating with preference for morning or evening activity. We conducted a population-based case-control study in Spain, 2008-2013. In this analysis we included 621 cases of prostate and 1,205 of breast cancer and 872 male and 1,321 female population controls who had never worked night shift. Subjects were interviewed on timing of meals, sleep and chronotype and completed a Food Frequency Questionaire. Adherence to the World Cancer Research Fund/American Institute of Cancer Research recommendations for cancer prevention was examined. Compared with subjects sleeping immediately after supper, those sleeping two or more hours after supper had a 20% reduction in cancer risk for breast and prostate cancer combined (adjusted Odds Ratio [OR] = 0.80, 95%CI 0.67-0.96) and in each cancer individually (prostate cancer OR = 0.74, 0.55-0.99; breast cancer OR = 0.84, 0.67-1.06). A similar protection was observed in subjects having supper before 9 pm compared with supper after 10 pm. The effect of longer supper-sleep interval was more pronounced among subjects adhering to cancer prevention recommendations (OR both cancers= 0.65, 0.44-0.97) and in morning types (OR both cancers = 0.66, 0.49-0.90). Adherence to diurnal eating patterns and specifically a long interval between last meal and sleep are associated with a lower cancer risk, stressing the importance of evaluating timing in studies on diet and cancer. What's new? Evidence shows that long-term disruption of endogenous circadian rhythms may be associated with cancer. The effects of mistimed sleeping and eating patterns that come with modern life are however less clear. This large Spanish population-based study examined whether meal timing and sleep patterns are associated with the two most common nightshift-related cancers. Adherence to a more diurnal eating pattern, and specifically an early supper and a long interval between last meal and sleep were associated with a lower breast and prostate cancer risk, stressing the importance of evaluating circadian rhythms in diet and cancer studies and revisiting recommendations for prevention

Comparison of seven prognostic tools to identify low-risk pulmonary embolism in patients aged <50 years

publishersversionPeer reviewe

Experiencias en el aula: tercer encuentro de prácticas pedagógicas innovadoras.

Experiencias de profesores en su quehacer en los distintos ambientes de aprendizaje presenciales y a distancia.Para el Centro de Excelencia Docente aeiou constituye un honor presentar la publicación del Tercer Encuentro de Prácticas Pedagógicas Innovadoras en el que se destacan cuarenta trabajos de profesores de UNIMINUTO provenientes de diferentes sedes. Con este encuentro son ya tres que bajo la dirección de aeiou los profesores han compartido su quehacer en los distintos ambientes de aprendizaje presenciales y a distancia. Cada año el Centro de Excelencia Docente invita a los profesores a participar en este evento, para el 2108 además de la inscripción voluntaria por parte de cada profesor, se invitó al estudiantado a que postularan a sus profesores que consideraban eran innovadores y creativos en el cumplimiento de su función docente y se obtuvo una respuesta importante por parte de los estudiantes, que para algunos profesores resultó sorpresiva porque quizás no habían considerado que el trabajo que hacían en su ambiente de aprendizaje era diferente, fuera de lo común. Luego de una evaluación de jurados nacionales e internacionales de las prácticas presentadas y de la realización del evento, que tuvo como novedad hacerlo de forma simultánea en cuatro sedes donde UNIMINUTO tiene presencia: Buga, Ibagué, Pereira y Bogotá, se comparte la presente publicación para tener como referencia y evidencia el trabajo que los profesores hacen a diario

Whole-genome sequencing reveals highly specific gene targeting by in vitro assembled Cas9-ribonucleoprotein complexes in Aspergillus fumigatus

Abstract Background CRISPR/Cas9-based genome editing is quickly becoming a powerful tool within the field of fungal genetics. Adaptation of CRISPR/Cas9 systems are allowing for rapid and highly efficient gene targeting within fungi. We recently reported the adaptation of a simple CRISPR/Cas9 system for gene deletion that is effective across multiple genetic backgrounds of Aspergillus fumigatus. This system employs in vitro assembly of Cas9 ribonucleoproteins (RNPs) coupled with micro-homology repair templates for gene deletion. Although highly efficient at gene targeting in wild type genetic backgrounds of A. fumigatus, the potential for our system to produce unwanted off-target mutations has not been addressed. Results Next-generation Illumina sequencing was used to identify genome mutations among transformants isolated from standard (no Cas9) and Cas9-mediated integration of a hygromycin deletion cassette. Two different concentrations of Cas9 were utilized to examine the association of Cas9 concentration with total numbers and types of genomic mutations. For each of the three test groups (zero, low, and high Cas9), three transformants were sequenced and compared to the parent strain. Bioinformatics analyses revealed the average number of total mutations to be similar among all three test groups. A. fumigatus transformation using standard, non-Cas9-mediated methods resulted in an average of 373 ± 28 mutations. In comparison, transformation with in vitro assembled Cas9-RNPs using either high (1 µg/µl) or low (0.5 µg/µl) levels of Cas9 resulted in an average of 326 ± 19 and 395 ± 69 mutations, respectively. In all cases, the vast majority of mutations identified were intergenic. No correlation between the amount of Cas9 utilized for transformation and the overall number of mutations was found. Finally, the specific type of mutation introduced during the transformation process was not Cas9-dependent, as both single-nucleotide polymorphisms and insertion/deletion events were not significantly different between the experimental groups. Conclusions CRISPR/Cas9-based genome editing in A. fumigatus using in vitro assembled RNPs coupled with microhomology templates is a reliable method of gene targeting. This system is highly efficient and is not associated with increased off-target mutations caused by introduction of the Cas9 nuclease

C-terminus Proteolysis and Palmitoylation Cooperate for Optimal Plasma Membrane Localization of RasA in Aspergillus fumigatus

RasA is a major regulator of fungal morphogenesis and virulence in Aspergillus fumigatus. The proper localization of RasA to the plasma membrane is essential for the formation of invasive hyphae during infection. In yeast, the localization of Ras2p to the plasma membrane is orchestrated by several post-translational modifications (PTM) at the C-terminal CAAX box that are thought to occur in sequential order. These PTMs include: (1) CAAX motif farnesylation by the farnesyltransferase complex composed of Ram1p and Ram2p; (2) proteolysis of the -AAX residues by Rce1p or Ste24p; (3) methylation of the remaining prenylated cysteine residue by Ste14p, and; (4) palmitoylation at a single conserved cysteine residue mediated by the Erf2p/Erf4p palmitoyltransferase. We previously reported that homologs of each RasA PTM enzyme are conserved in A. fumigatus. Additionally, we delineated a major role for protein farnesylation in A. fumigatus growth and virulence. In this work, we characterize the post-prenylation processing enzymes of RasA in A. fumigatus. The genes encoding the RasA post-prenylation enzymes were first deleted and examined for their roles in growth and regulation of RasA. Only when strains lacked cppB, the A. fumigatus homologue of yeast RCE1, there was a significant reduction in fungal growth and conidial germination. In addition, cppB-deletion mutants displayed hypersensitivity to the cell wall-perturbing agents Calcofluor White and Congo Red and the cell wall biosynthesis inhibitor Caspofungin. In contrast to the previously published data in yeast, the deletion of post-prenylation modifying enzymes did not alter the plasma membrane localization or activation of RasA. To delineate the molecular mechanisms underlying these differences, we investigated the interplay between dual-palmitoylation of the RasA hypervariable region and CAAX proteolysis for stabilization of RasA at the plasma membrane. Our data indicate that, in the absence of proper CAAX proteolysis, RasA accumulation at the plasma membrane is stabilized by dual palmitoyl groups on the dual cysteine residues. Therefore, we conclude CAAX proteolysis and dual-palmitoylation of the hypervariable region is important for maintaining a stable attachment association of RasA with the plasma membrane to support optimal fungal growth and development

Image_4.TIF

<p>RasA is a major regulator of fungal morphogenesis and virulence in Aspergillus fumigatus. The proper localization of RasA to the plasma membrane is essential for the formation of invasive hyphae during infection. In yeast, the localization of Ras2p to the plasma membrane is orchestrated by several post-translational modifications (PTM) at the C-terminal CAAX box that are thought to occur in sequential order. These PTMs include: (1) CAAX motif farnesylation by the farnesyltransferase complex composed of Ram1p and Ram2p; (2) proteolysis of the -AAX residues by Rce1p or Ste24p; (3) methylation of the remaining prenylated cysteine residue by Ste14p, and; (4) palmitoylation at a single conserved cysteine residue mediated by the Erf2p/Erf4p palmitoyltransferase. We previously reported that homologs of each RasA PTM enzyme are conserved in A. fumigatus. Additionally, we delineated a major role for protein farnesylation in A. fumigatus growth and virulence. In this work, we characterize the post-prenylation processing enzymes of RasA in A. fumigatus. The genes encoding the RasA post-prenylation enzymes were first deleted and examined for their roles in growth and regulation of RasA. Only when strains lacked cppB, the A. fumigatus homologue of yeast RCE1, there was a significant reduction in fungal growth and conidial germination. In addition, cppB-deletion mutants displayed hypersensitivity to the cell wall-perturbing agents Calcofluor White and Congo Red and the cell wall biosynthesis inhibitor Caspofungin. In contrast to the previously published data in yeast, the deletion of post-prenylation modifying enzymes did not alter the plasma membrane localization or activation of RasA. To delineate the molecular mechanisms underlying these differences, we investigated the interplay between dual-palmitoylation of the RasA hypervariable region and CAAX proteolysis for stabilization of RasA at the plasma membrane. Our data indicate that, in the absence of proper CAAX proteolysis, RasA accumulation at the plasma membrane is stabilized by dual palmitoyl groups on the dual cysteine residues. Therefore, we conclude CAAX proteolysis and dual-palmitoylation of the hypervariable region is important for maintaining a stable attachment association of RasA with the plasma membrane to support optimal fungal growth and development.</p

Image_3.TIF

<p>RasA is a major regulator of fungal morphogenesis and virulence in Aspergillus fumigatus. The proper localization of RasA to the plasma membrane is essential for the formation of invasive hyphae during infection. In yeast, the localization of Ras2p to the plasma membrane is orchestrated by several post-translational modifications (PTM) at the C-terminal CAAX box that are thought to occur in sequential order. These PTMs include: (1) CAAX motif farnesylation by the farnesyltransferase complex composed of Ram1p and Ram2p; (2) proteolysis of the -AAX residues by Rce1p or Ste24p; (3) methylation of the remaining prenylated cysteine residue by Ste14p, and; (4) palmitoylation at a single conserved cysteine residue mediated by the Erf2p/Erf4p palmitoyltransferase. We previously reported that homologs of each RasA PTM enzyme are conserved in A. fumigatus. Additionally, we delineated a major role for protein farnesylation in A. fumigatus growth and virulence. In this work, we characterize the post-prenylation processing enzymes of RasA in A. fumigatus. The genes encoding the RasA post-prenylation enzymes were first deleted and examined for their roles in growth and regulation of RasA. Only when strains lacked cppB, the A. fumigatus homologue of yeast RCE1, there was a significant reduction in fungal growth and conidial germination. In addition, cppB-deletion mutants displayed hypersensitivity to the cell wall-perturbing agents Calcofluor White and Congo Red and the cell wall biosynthesis inhibitor Caspofungin. In contrast to the previously published data in yeast, the deletion of post-prenylation modifying enzymes did not alter the plasma membrane localization or activation of RasA. To delineate the molecular mechanisms underlying these differences, we investigated the interplay between dual-palmitoylation of the RasA hypervariable region and CAAX proteolysis for stabilization of RasA at the plasma membrane. Our data indicate that, in the absence of proper CAAX proteolysis, RasA accumulation at the plasma membrane is stabilized by dual palmitoyl groups on the dual cysteine residues. Therefore, we conclude CAAX proteolysis and dual-palmitoylation of the hypervariable region is important for maintaining a stable attachment association of RasA with the plasma membrane to support optimal fungal growth and development.</p

Image_2.TIF

<p>RasA is a major regulator of fungal morphogenesis and virulence in Aspergillus fumigatus. The proper localization of RasA to the plasma membrane is essential for the formation of invasive hyphae during infection. In yeast, the localization of Ras2p to the plasma membrane is orchestrated by several post-translational modifications (PTM) at the C-terminal CAAX box that are thought to occur in sequential order. These PTMs include: (1) CAAX motif farnesylation by the farnesyltransferase complex composed of Ram1p and Ram2p; (2) proteolysis of the -AAX residues by Rce1p or Ste24p; (3) methylation of the remaining prenylated cysteine residue by Ste14p, and; (4) palmitoylation at a single conserved cysteine residue mediated by the Erf2p/Erf4p palmitoyltransferase. We previously reported that homologs of each RasA PTM enzyme are conserved in A. fumigatus. Additionally, we delineated a major role for protein farnesylation in A. fumigatus growth and virulence. In this work, we characterize the post-prenylation processing enzymes of RasA in A. fumigatus. The genes encoding the RasA post-prenylation enzymes were first deleted and examined for their roles in growth and regulation of RasA. Only when strains lacked cppB, the A. fumigatus homologue of yeast RCE1, there was a significant reduction in fungal growth and conidial germination. In addition, cppB-deletion mutants displayed hypersensitivity to the cell wall-perturbing agents Calcofluor White and Congo Red and the cell wall biosynthesis inhibitor Caspofungin. In contrast to the previously published data in yeast, the deletion of post-prenylation modifying enzymes did not alter the plasma membrane localization or activation of RasA. To delineate the molecular mechanisms underlying these differences, we investigated the interplay between dual-palmitoylation of the RasA hypervariable region and CAAX proteolysis for stabilization of RasA at the plasma membrane. Our data indicate that, in the absence of proper CAAX proteolysis, RasA accumulation at the plasma membrane is stabilized by dual palmitoyl groups on the dual cysteine residues. Therefore, we conclude CAAX proteolysis and dual-palmitoylation of the hypervariable region is important for maintaining a stable attachment association of RasA with the plasma membrane to support optimal fungal growth and development.</p