Culture conditions affect virulence and production of insect toxic proteins in the entomopathogenic fungus Metarhizium anisopliae

Conidial spores are often used as the infectious agent during insect biocontrol applications of entomopathogenic fungi. Here we show differential virulence of conidia derived from Metarhizium anisopliae strain EAMa 01/58-Su depending upon the solid substrata used for cultivation, where LC₅₀ values differed by up to ~10-fold (5.3×10⁶?4.5×10⁵ conidia/ml) and LT₅₀ values by ~40% (9.8?7.1 d). This fungal strain is also known to secrete proteins that are toxic towards adult Mediterranean fruit flies, Ceratitis capitata, and the Greater wax moth, Galleria mellonella, larvae. In vitro production and intrahemoceol injection using G. mellonella as the host was used to test fractions during purification of the protein toxins, demonstrating that they elicited defence-related responses including melanisation and tissue necrosis. Production of these proteins/peptides along with a number of potential cuticle degrading enzymes was confirmed both in vitro and during the infection process (in vivo). Two-dimensional gel electrophoresis, followed by gel elution and bioassay, was used to identify at least three proteins or peptides (molecular mass=11, 15 and 15 kDa) as mediating the observed insect toxicity. These data demonstrate that in vitro screening for insect toxins can mimic in vivo (i.e. during the infection process) secretion and applies the use of proteomics to invertebrate pathology.     

Exome Sequencing of Index Patients with Retinal Dystrophies as a Tool for Molecular Diagnosis

Background

Retinal dystrophies (RD) are a group of hereditary diseases that lead to debilitating visual impairment and are usually transmitted as a Mendelian trait. Pathogenic mutations can occur in any of the 100 or more disease genes identified so far, making molecular diagnosis a rather laborious process. In this work we explored the use of whole exome sequencing (WES) as a tool for identification of RD mutations, with the aim of assessing its applicability in a diagnostic context.

Methodology/Principal Findings

We ascertained 12 Spanish families with seemingly recessive RD. All of the index patients underwent mutational pre-screening by chip-based sequence hybridization and resulted to be negative for known RD mutations. With the exception of one pedigree, to simulate a standard diagnostic scenario we processed by WES only the DNA from the index patient of each family, followed by in silico data analysis. We successfully identified causative mutations in patients from 10 different families, which were later verified by Sanger sequencing and co-segregation analyses. Specifically, we detected pathogenic DNA variants (∼50% novel mutations) in the genes RP1, USH2A, CNGB3, NMNAT1, CHM, and ABCA4, responsible for retinitis pigmentosa, Usher syndrome, achromatopsia, Leber congenital amaurosis, choroideremia, or recessive Stargardt/cone-rod dystrophy cases.

Conclusions/Significance

Despite the absence of genetic information from other family members that could help excluding nonpathogenic DNA variants, we could detect causative mutations in a variety of genes known to represent a wide spectrum of clinical phenotypes in 83% of the patients analyzed. Considering the constant drop in costs for human exome sequencing and the relative simplicity of the analyses made, this technique could represent a valuable tool for molecular diagnostics or genetic research, even in cases for which no genotypes from family members are available.     

Succession of bacterivorous protists on laboratory-made marine snow

Colonization and succession over time by bacterivorous protistson laboratory-made marine snow were analysed in five assaysduring 1994. Marine snow was made hom natural seawater usingrolling tanks. In all experiments, the macroaggregates werestable in size and consistency after the fourth day, and thecolonization and succession processes were similar. Newly formedmacre aggregates became colonized by heterotrophic nanoflagellateson the fourth day, mmt of them kinete plastids (Bodo designisand Rhynchomonns nasuta) and bicosoecids (Pseudobodo tremulansand Bicosoeca sp.). Sarcodines and ciliates appeared 1 day later.Among the former, the most abundant genus was Vannella sp.,while scuticociliates (Uronema marinum) and hypotrichs (Euplotesvannus and Aspidisca steini) were the most abundant ciliates.Most of the species observed in the study were more common tobenthic habitats than to pelagic ones. The planktonic existenceof the genera Bodo, Rhynchomonas, Bicosoeca, Euplotes and Aspidiscadepends on the presence of surfaces because they are poor swimmersor immotile, and Pseudobodo and Vannella need attachment forfeeding. The only pelagic protist observed was Uronema, probablybecause its opportunistic behaviour leads it to exploit enrichedenvironments such as marine snow. Flagellate and ciliate abundancesin laboratory-made macroaggregates were much higher than insurrounding water, which indicates that marine snow representsan enhanced habitat for protist growth.     

The effect of neutral nonadditive gene action on the quantitative index of population divergence

For neutral additive genes, the quantitative index of population divergence (Q(ST)) is equivalent to Wright's fixation index (F(ST)). Thus, divergent or convergent selection is usually invoked, respectively, as a cause of the observed increase (Q(ST) > F(ST)) or decrease (Q(ST) < F(ST)) of Q(ST) from its neutral expectation (Q(ST) = F(ST)). However, neutral nonadditive gene action can mimic the additive expectations under selection. We have studied theoretically the effect of consecutive population bottlenecks on the difference F(ST) - Q(ST) for two neutral biallelic epistatic loci, covering all types of marginal gene action. With simple dominance, Q(ST) < F(ST) for only low to moderate frequencies of the recessive alleles; otherwise, Q(ST) > F(ST). Additional epistasis extends the condition Q(ST) < F(ST) to a broader range of frequencies. Irrespective of the type of nonadditive action, Q(ST) < F(ST) generally implies an increase of both the within-line additive variance after bottlenecks over its ancestral value (V(A)) and the between-line variance over its additive expectation (2F(ST)V(A)). Thus, both the redistribution of the genetic variance after bottlenecks and the F(ST) - Q(ST) value are governed largely by the marginal properties of single loci. The results indicate that the use of the F(ST) - Q(ST) criterion to investigate the relative importance of drift and selection in population differentiation should be restricted to pure additive traits.     

High-level production and secretion of recombinant proteins by the dimorphic yeast Arxula adeninivorans

The non-conventional dimorphic thermo- and salt-resistant yeast Arxula adeninivorans has been developed as a host for heterologous gene expression. For assessment of the system two model genes have been selected: the GFP gene encoding the intracellular green fluorescent protein, and the HSA gene encoding the secreted human serum albumin. The expression system includes two host strains, namely A. adeninivorans LS3, which forms budding cells at 30 degrees C and mycelia at >42 degrees C, and the strain A. adeninivorans 135, which forms mycelia at temperatures as low as 30 degrees C. For expression control the constitutive A. adeninivorans-derived TEF1-promoter and S. cerevisiae-derived PHO5-terminator were selected. The basic A. adeninivorans transformation/expression vector pAL-HPH1 is further equipped with the Escherichia coli-derived hph gene conferring hygromycin B resistance and the 25S rDNA from A. adeninivorans for rDNA targeting. Transformants were obtained for both budding cells and mycelia. In both cell types similar expression levels were achieved and the GFP was localised in the cytoplasm while more than 95% of the HSA accumulated in the culture medium. In initial fermentation trials on a 200-ml shake flask scale maximal HSA product levels were observed after 96 h of cultivation.     

Pharmacological Blockade of Cannabinoid CB1 Receptors in Diet-Induced Obesity Regulates Mitochondrial Dihydrolipoamide Dehydrogenase in Muscle

Cannabinoid CB₁ receptors peripherally modulate energy metabolism. Here, we investigated the role of CB₁ receptors in the expression of glucose/pyruvate/tricarboxylic acid (TCA) metabolism in rat abdominal muscle. Dihydrolipoamide dehydrogenase (DLD), a flavoprotein component (E3) of α-ketoacid dehydrogenase complexes with diaphorase activity in mitochondria, was specifically analyzed. After assessing the effectiveness of the CB₁ receptor antagonist AM251 (3 mg kg^-1, 14 days) on food intake and body weight, we could identified seven key enzymes from either glycolytic pathway or TCA cycle—regulated by both diet and CB₁ receptor activity—through comprehensive proteomic approaches involving two-dimensional electrophoresis and MALDI-TOF/LC-ESI trap mass spectrometry. These enzymes were glucose 6-phosphate isomerase (GPI), triosephosphate isomerase (TPI), enolase (Eno3), lactate dehydrogenase (LDHa), glyoxalase-1 (Glo1) and the mitochondrial DLD, whose expressions were modified by AM251 in hypercaloric diet-induced obesity. Specifically, AM251 blocked high-carbohydrate diet (HCD)-induced expression of GPI, TPI, Eno3 and LDHa, suggesting a down-regulation of glucose/pyruvate/lactate pathways under glucose availability. AM251 reversed the HCD-inhibited expression of Glo1 and DLD in the muscle, and the DLD and CB₁ receptor expression in the mitochondrial fraction. Interestingly, we identified the presence of CB₁ receptors at the membrane of striate muscle mitochondria. DLD over-expression was confirmed in muscle of CB ₁ ^-/- mice. AM251 increased the pyruvate dehydrogenase and glutathione reductase activity in C₂C₁₂ myotubes, and the diaphorase/oxidative activity in the mitochondria fraction. These results indicated an up-regulation of methylglyoxal and TCA cycle activity. Findings suggest that CB₁ receptors in muscle modulate glucose/pyruvate/lactate pathways and mitochondrial oxidative activity by targeting DLD.