A quinonoid is an intermediate of oxidative deamination reaction catalyzed by Dopa decarboxylase

The reactions of Dopa decarboxylase (DDC) with l- and d-enantiomers of tryptophan methyl ester are described. Although both the enantiomers bind to the active site of the enzyme with similar affinity, their binding modes are different. l-enantiomer binds in an unproductive mode, while d-enantiomer acts as an oxidative deamination substrate. For the first time a quinonoid has been detected as intermediate of this reaction. By using rapid-scanning stopped-flow kinetic technique rate constants for formation and decay of this species have been determined. All these data, besides validating the functional DDC active site model, represent an important step toward the elucidation of the catalytic pathway of oxidative deamination.     

TLRs govern neutrophil activity in aspergillosis

Polymorphonuclear neutrophils (PMNs) are essential in initiation and execution of the acute inflammatory response and subsequent resolution of fungal infection. PMNs, however, may act as double-edged swords, as the excessive release of oxidants and proteases may be responsible for injury to organs and fungal sepsis. To identify regulatory mechanisms that may balance PMN-dependent protection and immunopathology in fungal infections, the involvement of different TLR-activation pathways was evaluated on human PMNs exposed to the fungus Aspergillus fumigatus. Recognition of Aspergillus and activation of PMNs occurred through the involvement of distinct members of the TLR family, each likely activating specialized antifungal effector functions. By affecting the balance between fungicidal oxidative and nonoxidative mechanisms, pro- and anti-inflammatory cytokine production, and apoptosis vs necrosis, the different TLRs ultimately impacted on the quality of microbicidal activity and inflammatory pathology. Signaling through TLR2 promoted the fungicidal activity of PMNs through oxidative pathways involving extracellular release of gelatinases and proinflammatory cytokines while TLR4 favored the oxidative pathways through the participation of azurophil, myeloperoxidase-positive, granules and IL-10. This translated in vivo in the occurrence of different patterns of fungal clearance and inflammatory pathology. Both pathways were variably affected by signaling through TLR3, TLR5, TLR6, TLR7, TLR8, and TLR9. The ability of selected individual TLRs to restore antifungal functions in defective PMNs suggests that the coordinated outputs of activation of multiple TLRs may contribute to PMN function in aspergillosis.     

Epidemiology of onychomycosis and paronychia in the area of ANCONA (ITALY) over a period of 5 years

We retrospectively evaluated the epidemiology of onychomycosis and/or paronychia in 172 patients attending the Clinic of Dermatology and Venereology over a 5 year period. Although yeast isolates, belonging to the Candida species, represented the most frequent etiologic agents of these infections, an increasing prevalence of fungal infections due to emerging fungal pathogens (EFP) was noted throughout this time period. In particular, EFP as causative agents of these infections increased from 0 to 28.4% from 1998 to 2002.     

Genotype Analysis of Bacillus anthracis Strains Circulating in Bangladesh

In Bangladesh, anthrax, caused by the bacterium Bacillus anthracis, is considered an endemic disease affecting ruminants with sporadic zoonotic occurrences in humans. Due to the lack of knowledge about risks from an incorrect removal of infected carcasses, the disease is not properly monitored, and because of the socio-economic conditions, the situation is under-reported and under-diagnosed. For sensitive species, anthrax represents a fatal outcome with sudden death and sometimes bleeding from natural orifices. The most common source of infection for ruminants is ingestion of spores during grazing in contaminated pastures or through grass and water contaminated with anthrax spores. Domestic cattle, sheep and goats can also become infected through contaminated bone meal (used as feed) originating from anthrax-infected carcasses. The present investigation was conducted to isolate B. anthracis organisms from 169 samples (73 soil, 1 tissue, 4 bone and 91 bone meal samples) collected from 12 different districts of Bangladesh. The sampling was carried out from 2012 to 2015. Twelve samples resulted positive for B. anthracis. Biomolecular analyses were conducted starting from the Canonical Single Nucleotide Polymorphism (CanSNP) to analyze the phylogenetic origin of strains. The analysis of genotype, obtained through the Multiple Locus Variable Number Tandem Repeat Analysis (MLVA) with the analysis of 15 Variable Number Tandem Repeats (VNTR), demonstrated four different genotypes: two of them were previously identified in the district of Sirajganj. The sub-genotyping, conducted with Single Nucleotide Repeats analysis, revealed the presence of eight subgenotypes. The data of the present study concluded that there was no observed correlation between imported cattle feed and anthrax occurrence in Bangladesh and that the remarkable genetic variations of B. anthracis were found in the soil of numerous outbreaks in this country.     

Decreasing negative impacts of harvesting over insect communities using variable retention in southern Patagonian forests

Variable retention is an alternative silvicultural approach to timber forest management, which consist in a regeneration treatment with different degrees and patterns of stand retention. It has been proposed to mitigate harmful effects of harvesting, but effectiveness in insect conservation remains unknown in southern Patagonian Nothofagus pumilio forests. Here, the objectives were to: (1) define a baseline of insect diversity in old-growth forests along a site quality gradient (high, medium and low, associated to the forest productivity of each site); (2) evaluate stands with different retention treatments [aggregated (AR) surrounded by dispersed (DR) retention, and aggregated retention surrounded by clear-cut (CC)] and to compare with old-growth unmanaged forests (OGF); and (3) assess temporal changes during the first 4 years after harvesting (YAH). In a long term forest research plot, mobile epigean insect richness and relative abundance were characterized and classified in seven response type groups, using a wide spectrum sampling set. Data analyses included parametric and permutational ANOVAs, multivariate classification and ordinations. There were found 79 species before harvesting, and that richness was not related to site quality. After harvesting, 84 new species were added considering all treatments along the first four sampled YAH, of which 65 % were added to OGF, while in harvested sites richness and abundance directly diminished with retention degree (OGF > AR > DR > CC) due to incoming species cannot compensate the lost of them. However, fluctuations in diversity were observed along the YAH. Therefore, harvesting reduces insect richness in N. pumilio forests independently of the treatment, but the original insect assemblage significantly changes due to loss of sensitive species and introduction of others from surrounding environments. Despite this, inclusion of aggregates greatly diminished harvesting impacts because insect assemblage is favoured when structural complexity is preserved, conserving richness and abundance at similar levels than in old-growth forests. However, more studies are necessary to evaluate effects of different aggregate size, shape and distribution into harvested forests, as well as their fragmentation and connectivity at landscape level.     

Gly161 mutations associated with Primary Hyperoxaluria Type I induce the cytosolic aggregation and the intracellular degradation of the apo-form of alanine:glyoxylate aminotransferase

Primary Hyperoxaluria Type I (PH1) is a severe rare disorder of metabolism due to inherited mutations on liver peroxisomal alanine:glyoxylate aminotransferase (AGT), a pyridoxal 5′-phosphate (PLP)-dependent enzyme whose deficiency causes the deposition of calcium oxalate crystals in the kidneys and urinary tract. PH1 is an extremely heterogeneous disease and there are more than 150 disease-causing mutations currently known, most of which are missense mutations. Moreover, the molecular mechanisms by which missense mutations lead to AGT deficiency span from structural, functional to subcellular localization defects. Gly161 is a highly conserved residue whose mutation to Arg, Cys or Ser is associated with PH1. Here we investigated the molecular bases of the AGT deficit caused by Gly161 mutations with expression studies in a mammalian cellular system paired with biochemical analyses on the purified recombinant proteins. Our results show that the mutations of Gly161 (i) strongly reduce the expression levels and the intracellular half-life of AGT, and (ii) make the protein in the apo-form prone to an electrostatically-driven aggregation in the cell cytosol. The coenzyme PLP, by shifting the equilibrium from the apo- to the holo-form, is able to reduce the aggregation propensity of the variants, thus partly decreasing the effect of the mutations. Altogether, these results shed light on the mechanistic details underlying the pathogenicity of Gly161 variants, thus expanding our knowledge of the enzymatic phenotypes leading to AGT deficiency.     

The Hydrophobic TraM Protein of pKM101 Is Required for Conjugal Transfer and Sensitivity to Donor-Specific Bacteriophage

pKM101 is a self-transmissible plasmid of the IncN incompatibility group. Analysis of the DNA sequences of the genes required for conjugal transfer suggested the existence of a previously uncharacterized open reading frame, designatedtraM,that might be required for conjugation. Merodiploid strains containing transposon insertion mutations either intraMor in neighboringtragenes were used to demonstrate thattraMconstitutes a new complementation group essential for conjugation and donor phage sensitivity. The hydrophobicity profile of TraM suggests that it contains a signal sequence. The remainder of TraM is also composed predominantly of hydrophobic amino acids but contains one possible surface exposed loop. TraM–alkaline phosphatase and TraM–β-galactosidase fusion proteins supported the hypothesis that TraM has a small cytoplasmic loop. We were unable to detect heterologous complementation between anytramutation and its homolog from thevirBoperon ofAgrobacterium tumefaciens.