Isolation of autochthonous non-white rot fungi with potential for enzymatic upgrading of Venezuelan extra-heavy crude oil

The increasing world demand for fuels makes it necessary to exploit the largest reserve of extra-heavy crude oil (EHCO) of the Orinoco Oil Belt from Venezuela. We propose the use of extracellular oxidative enzymes, in particular, lignin-degrading enzyme systems (LDS) of fungi, for enzymatic improvement of EHCO. Autochthonous non-white rot fungal strains able to use EHCO, and several polycyclic aromatic hydrocarbons (PAHs) as sole carbon source and energy, were isolated from EHCO-polluted soils and identified as belonging to the genera Fusarium, Penicillium, Trichoderma, Aspergillus, Neosartorya, Pseudallescheria, Cladosporium, Pestalotiopsis, Phoma and Paecillomyces. Phenotypic and biochemical assays revealed the ability of these filamentous fungi to synthesize extracellular oxidative enzymes, and suggested a relationship between the LDS and EHCO bioconversion. This work reports, for the first time, the use of o-phenylenediamine dihydrochloride (OPD) as substrate to measure extracellular ligninolytic peroxidases (ELP) in culture broths of filamentous fungi (Fusarium solani HP-1), and constitutes the first formal study of the fungal community associated with the EHCO of the Orinoco Oil Belt.     

First evidence of mineralization of petroleum asphaltenes by a strain of Neosartorya fischeri

A fungal strain isolated from a microbial consortium growing in a natural asphalt lake is able to grow in purified asphaltenes as the only source of carbon and energy. The asphaltenes were rigorously purified in order to avoid contamination from other petroleum fractions. In addition, most of petroporphyrins were removed. The 18S rRNA and β‐tubulin genomic sequences, as well as some morphologic characteristics, indicate that the isolate is Neosartorya fischeri. After 11 weeks of growth, the fungus is able to metabolize 15.5% of the asphaltenic carbon, including 13.2% transformed to CO₂. In a medium containing asphaltenes as the sole source of carbon and energy, the fungal isolate produces extracellular laccase activity, which is not detected when the fungus grow in a rich medium. The results obtained in this work clearly demonstrate that there are microorganisms able to metabolize and mineralize asphaltenes, which is considered the most recalcitrant petroleum fraction.     

Mutation screening of multiple genes in Spanish patients with autosomal recessive retinitis pigmentosa by targeted resequencing

Retinitis Pigmentosa (RP) is a heterogeneous group of inherited retinal dystrophies characterised ultimately by the loss of photoreceptor cells. RP is the leading cause of visual loss in individuals younger than 60 years, with a prevalence of about 1 in 4000. The molecular genetic diagnosis of autosomal recessive RP (arRP) is challenging due to the large genetic and clinical heterogeneity. Traditional methods for sequencing arRP genes are often laborious and not easily available and a screening technique that enables the rapid detection of the genetic cause would be very helpful in the clinical practice. The goal of this study was to develop and apply microarray-based resequencing technology capable of detecting both known and novel mutations on a single high-throughput platform. Hence, the coding regions and exon/intron boundaries of 16 arRP genes were resequenced using microarrays in 102 Spanish patients with clinical diagnosis of arRP. All the detected variations were confirmed by direct sequencing and potential pathogenicity was assessed by functional predictions and frequency in controls. For validation purposes 4 positive controls for variants consisting of previously identified changes were hybridized on the array. As a result of the screening, we detected 44 variants, of which 15 are very likely pathogenic detected in 14 arRP families (14%). Finally, the design of this array can easily be transformed in an equivalent diagnostic system based on targeted enrichment followed by next generation sequencing.     

The synaptic behaviour of the wild forms of Triticum turgidum and T. timopheevii.

Different wild allopolyploid species of Triticeae show extensive bivalent formation at zygotene while a considerable number of multivalents is present in cultivated polyploid wheats. To study the chromosome behaviour at early meiotic stages in wild forms of tetraploid wheats Triticum turgidum and T timopheevii (2n = 4x = 28) we have analysed the synaptic pattern in fully traced spread nuclei at mid- and late zygotene and at pachytene of wild accessions of these species. The mean number of synaptonemal complex (SC) bivalents at mid-zygotene ranged from 12.22 to 13.14 among the accessions studied indicating a strong restriction of synapsis initiation to homologous chromosomes. The mean of bivalents increased at pachytene because of the transformation of multivalents into bivalents. Ring bivalents observed at metaphase I support that SC bivalents were formed by homologous chromosomes. The average values of SC bivalents at mid-zygotene in the wild forms are much higher than the average values observed in the cultivated tetraploid wheats but similar to that of a mutant line of T turgidum with a duplication that includes Ph1, the major homoeologous pairing suppressor locus. These results suggest that the efficiency of the mechanism operating in the homologous recognition for synapsis is higher in wild wheat populations than in cultivated varieties. Apparently, a relatively detrimental modification of the pairing regulating genetic system accompanied the domestication of the wild wheat forms.     

Distribution and abundance of the ascidian Ecteinascidia turbinata (Ascidiacea: Perophoridae) in Cuba

Permanently submerged mangrove roots (Rhizophora mangle) are the main habitat of the ascidian Ecteinascidia turbinata in Cuba. It was occasionally found on black coral (Antiphates caribeana) between 22 and 38 meters deep. This species exhibits a wide distribution in all the mangrove keys surrounding the Island of Cuba but does not occur in riparian or fringing mangroves. Populations of this species are abundant in Cuba: in 75% of the 58 localities sampled the species was present and in 57% more than 50% of the roots held at least one colony. The highest colony densities were found in the northern coast of Pinar del Rio province with values near one colony per lineal meter of mangrove root. We found the highest density (1.46 col/m) and greatest biomass at Jutías Key, with values between 25 and 660 g/m. The average of wet biomass in the studied mangroves was 73.63 g/m.     

Some isolates of the nematophagous fungus Pochonia chlamydosporia promote root growth and reduce flowering time of tomato

The fungal parasite of nematode eggs Pochonia chlamydosporia is also a root endophyte known to promote growth of some plants. In this study, we analysed the effect of nine P. chlamydosporia isolates from worldwide origin on tomato growth. Experiments were performed at different scales (Petri dish, growth chamber and greenhouse conditions) and developmental stages (seedlings, plantlets and plants). Seven P. chlamydosporia isolates significantly (P < 0.05) increased the number of secondary roots and six of those increased total weight of tomato seedlings. Six P. chlamydosporia isolates also increased root weight of tomato plantlets. Root colonisation varied between different isolates of this fungus. Again P. chlamydosporia significantly increased root growth of tomato plants under greenhouse conditions and reduced flowering and fruiting times (up to 5 and 12 days, respectively) versus uninoculated tomato plants. P. chlamydosporia increased mature fruit weight in tomato plants. The basis of the mechanisms for growth, flowering and yield promotion in tomato by the fungus are unknown. However, we found that P. chlamydosporia can produce Indole‐3‐acetic acid and solubilise mineral phosphate. These results suggest that plant hormones or nutrient ability could play an important role. Our results put forward the agronomic importance of P. chlamydosporia as biocontrol agent of plant parasitic nematodes with tomato growth promoting capabilities.     

K+ Conduction and Mg2+ Blockade in a Shaker Kv-Channel Single Point Mutant with an Unusually High Conductance

Potassium channels exhibit a large diversity of single-channel conductances. Shaker is a low-conductance K-channel in which Pro475→Asp, a single-point mutation near the internal pore entrance, promotes 6- to 8-fold higher unitary current. To assess the mechanism for this higher conductance, we measured Shaker-P475D single-channel current in a wide range of symmetrical K⁺ concentrations and voltages. Below 300 mM K⁺, the current-to-voltage relations (i-V) showed inward rectification that disappeared at 1000 mM K⁺. Single-channel conductance reached a maximum of ～190 pS at saturating [K⁺], a value 4- to 5-fold larger than that estimated for the native channel. Intracellular Mg²⁺ blocked this variant with ～100-fold higher affinity. Near zero voltage, blockade was competitively antagonized by K⁺; however, at voltages >100 mV, it was enhanced by K⁺. This result is consistent with a lock-in effect in a single-file diffusion regime of Mg²⁺ and K⁺ along the pore. Molecular-dynamics simulations revealed higher K⁺ density in the pore, especially near the Asp-475 side chains, as in the high-conductance MthK bacterial channel. The molecular dynamics also showed that K⁺ ions bound distally can coexist with other K⁺ or Mg²⁺ in the cavity, supporting a lock-in mechanism. The maximal K⁺ transport rate and higher occupancy could be due to a decrease in the electrostatic energy profile for K⁺ throughout the pore, reducing the energy wells and barriers differentially by ～0.7 and ～2 kT, respectively.     

Influence of methylmalonyl-CoA mutase alleles on resistance to bovine tuberculosis in the European wild boar (Sus scrofa)

An association study was carried out to examine the influence of methylmalonyl-CoA mutase (MUT) polymorphisms on the susceptibility of a well-studied wild boar population from southern Spain to develop bovine tuberculosis (bTB). To this end, we examined polymorphisms at a closely linked dinucleotide microsatellite flanking exon 2 of the MUT gene in 37 wild boars with bTB and 36 non-infected individuals. The microsatellite showed low polymorphism in the studied population, with only three alleles (MUTm-A, MUTm-B and MUTm-C) found, in contrast to the 11 alleles previously reported for domestic pigs. Our case-control study showed that the MUTm-B allele was associated with disease in a dominant pattern (odds ratio = 3.36; 95% CI = 1.05-10.72; P = 0.04), while the MUTm AA genotype appeared to have a protective effect against bTB infection (odds ratio = 4.33; 95% CI = 1.20-14.96; P = 0.02). Interestingly, infected wild boars heterozygous for MUTm AB are at an advantage (11-fold) to contain the systemic spread of the disease when compared to other genotypes, implying that a balanced polymorphism may be present in the population. These results strengthen previous observations regarding the importance of the MUT gene on bTB resistance in wild boars and indicate that polymorphisms at this locus will influence the risk of acquiring and maintaining bTB in the studied population.