Structural properties of the linkers connecting the n- and c- terminal domains in the mocr bacterial transcriptional regulators

Peptide inter-domain linkers are peptide segments covalently linking two adjacent domains within a protein. Linkers play a variety of structural and functional roles in naturally occurring proteins. In this work we analyze the sequence properties of the predicted linker regions of the bacterial transcriptional regulators belonging to the recently discovered MocR subfamily of the GntR regulators. Analyses were carried out on the MocR sequences taken from the phyla Actinobacteria, Firmicutes, Alpha-, Beta- and Gammaproteobacteria. The results suggest that MocR linkers display phylum-specific characteristics and unique features different from those already described for other classes of inter-domain linkers. They show an average length significantly higher: 31.8 ± 14.3 residues reaching a maximum of about 150 residues. Compositional propensities displayed general and phylum-specific trends. Pro is dominating in all linkers. Dyad propensity analysis indicate Pro–Pro as the most frequent amino acid pair in all linkers. Physicochemical properties of the linker regions were assessed using amino acid indices relative to different features: in general, MocR linkers are flexible, hydrophilic and display propensity for β-turn or coil conformations. Linker sequences are hypervariable: only similarities between MocR linkers from organisms related at the level of species or genus could be found with sequence searches. The results shed light on the properties of the linker regions of the new MocR subfamily of bacterial regulators and may provide knowledge-based rules for designing artificial linkers with desired properties. © 2016 The Author(s

Biochemical data from the characterization of a new pathogenic mutation of human pyridoxine-5'-phosphate oxidase (PNPO)

PNPO deficiency is responsible of severe neonatal encephalopathy, responsive to pyridoxal-5’-phosphate (PLP) or pyridoxine. Recent studies widened the phenotype of this condition and detected new genetic variants on PNPO gene, whose pathogenetic role and clinical expression remain to be established. One of these mutations, Arg116Gln, is of particular interest because of its later onset of symptoms (beyond the first months of life) and its peculiar epileptic manifestations in patients. This protein variant was expressed as recombinant protein in E coli, purified to homogeneity, and characterized with respect to structural and kinetic properties, stability, binding constants of cofactor flavin mononucleotide (FMN) and product (PLP) in order to define the molecular and structural bases of its pathogenicity. For interpretation and discussion of reported data, together with the description of clinical studies, refer to the article [7][1] (doi: 10.1016/j.ymgme.2017.08.003)

Role of housing system and season on the carcass and meat quality traits of growing rabbits reared in Italian commercial farms

The aim of this study was to compare the carcass traits and meat quality of growing rabbits reared in four types of commercially available housing systems (i.e. bicellular cages, dual-purpose cages, enriched cages and elevated pens) across three production cycles covering different seasons (i.e. autumn, winter and summer) under field conditions. The rabbits originated from 12 commercial farms (three farms per housing system) located in the Northeast of Italy and were slaughtered in five commercial slaughterhouses at a slaughter age from 71 to 90 d. Twenty carcasses per farm and cycle were randomly selected at the slaughterhouse and carcass and meat quality traits were evaluated 24 h post-mortem. The heaviest carcass weights were found in rabbits reared in enriched cages, followed by those in parks, while the lightest carcasses were found in rabbits from bicellular cages. Carcass fat and longissimus lumborum proportions were higher and hind legs proportion lower in carcasses of rabbits kept in enriched cages compared to those from the other systems. Based on significant differences for meat quality traits (i.e. pH, L*, thawing losses and shear force) among housing systems, ante-mortem stress was likely lower in rabbits from enriched cages and parks than in those from bicellular and dual-purpose cages. Nevertheless, these differences were not translated into noticeable changes in meat quality because of the small variations among housing systems. Overall, the observed differences could not be strictly and exclusively related to the enclosure in which the animals were housed, but also to other production and ante-mortem factors. The rearing season significantly affected slaughter traits, with lower slaughter and carcass weights in summer compared to winter and autumn. Despite the limit in the sample size of investigated farms, this study highlighted that under field conditions the final product quality of rabbits is not clearly distinguishable based on the housing system per se

Isolation of a Complex Formed Between Acinetobacter baumannii HemA and HemL, Key Enzymes of Tetrapyrroles Biosynthesis

Plants, algae and most bacteria synthesize 5-aminolevulinic acid (ALA), the universal precursor of tetrapyrroles such as heme, chlorophyll and coenzyme B12, by a two-step transformation involving the NADPH-dependent glutamyl-tRNA reductase (HemA), which reduces tRNA-bound glutamate to glutamate-1-semialdehyde (GSA), and the pyridoxamine 5′-phosphate-dependent glutamate-1-semialdehyde-2,1-aminomutase (HemL), responsible for the isomerization of GSA into ALA. Since GSA is a very unstable compound at pH values around neutrality, the formation of a HemA-HemL complex has been proposed to occur, allowing for direct channeling of this intermediate from HemA to HemL. Experimental evidence of the formation of this complex has been obtained with the enzymes from Escherichia coli and Chlamydomonas reinhardtii. However, its isolation has never been attained, probably because HemA is degraded when intracellular heme accumulates. In this work, we devised a co-expression and co-purification strategy of HemA and HemL from Acinetobacter baumannii, which allowed the isolation of the HemA-HemL complex. Our results indicate that HemA is stabilized when co-expressed with HemL. The addition of citrate throughout the expression and purification procedure further promotes the formation of the HemA-HemL complex, which can be isolated in fair amount for functional and structural studies. This work lays the bases for a rational design of HemA-HemA inhibitors to be developed as antibacterial agents against A. baumannii, a multidrug resistant opportunistic pathogen responsible for a broad range of severe nosocomial infections

Vitamin B6 rescues insulin resistance and glucose-induced DNA damage caused by reduced activity of Drosophila PI3K

: The insulin signaling pathway controls cell growth and metabolism, thus its deregulation is associated with both cancer and diabetes. Phosphatidylinositol 3-kinase (PI3K) contributes to the cascade of phosphorylation events occurring in the insulin pathway by activating the protein kinase B (PKB/AKT), which phosphorylates several substrates, including those involved in glucose uptake and storage. PI3K inactivating mutations are associated with insulin resistance while activating mutations are identified in human cancers. Here we show that RNAi-induced depletion of the Drosophila PI3K catalytic subunit (Dp110) results in diabetic phenotypes such as hyperglycemia, body size reduction, and decreased glycogen content. Interestingly, we found that hyperglycemia produces chromosome aberrations (CABs) triggered by the accumulation of advanced glycation end-products and reactive oxygen species. Rearing PI3KRNAi flies in a medium supplemented with pyridoxal 5'-phosphate (PLP; the catalytically active form of vitamin B6) rescues DNA damage while, in contrast, treating PI3KRNAi larvae with the PLP inhibitor 4-deoxypyridoxine strongly enhances CAB frequency. Interestingly, PLP supplementation rescues also diabetic phenotypes. Taken together, our results provide a strong link between impaired PI3K activity and genomic instability, a crucial relationship that needs to be monitored not only in diabetes due to impaired insulin signaling but also in cancer therapies based on PI3K inhibitors. In addition, our findings confirm the notion that vitamin B6 is a good natural remedy to counteract insulin resistance and its complications

A pilot study about on-farm assessment of health and welfare in rabbits kept in different housing systems

This pilot study tested an on-farm protocol based on resource, management, and animal-based measures to evaluate the on-farm health and welfare of rabbits kept in four different housing systems. In detail, the four housing systems were (1) standard breeding cages for reproducing does (3,300 cm2) with their litters associated with bicellular cages for growing rabbits (1,200 cm2); (2) dual-purpose cages for both reproducing does and growing rabbits (3,655 cm2); (3) enriched cages (4,739 cm2) for both reproducing does and growing rabbits equipped with a wire-mesh elevated platform (1,015 cm2); (4) parks (30,977 cm2) made up of four modules (7,744 cm2 each) joined by removing the wire net walls between them with growing rabbits kept in collective parks and reproducing does individually in the single modules. A total of 12 commercial farms (three farms/four housing systems) were visited during three seasons (summer, autumn, and winter) on two occasions each: (1) a pre-weaning visit for recordings on reproducing does and litters and (2) a pre-slaughtering visit for recordings on growing rabbits. At the pre-weaning visit, the prevalence of health concerns did not differ among does and litters kept in the different housing systems. At the pre-slaughtering visit, a higher prevalence of dermatomycosis was found in farms with dual-purpose cages and parks. Overall, taking into account the limitations due to the small sample size per housing system and the field conditions, the on-farm assessment tested in the present pilot study did not highlight major differences in the welfare and health of reproducing does and their kits as well as of growing rabbits in farms using different housing systems, which need to be confirmed on a large number of farms. The study also outlined the role of several management and environmental factors changing from one farm to another, which stresses the troubles of accounting for on-farm rabbit welfare and health exclusively to the housing system.info:eu-repo/semantics/publishedVersio

Curcumin-1,2,3-Triazole Conjugation for Targeting the Cancer Apoptosis Machinery

The burden of neoplastic diseases is widely recognized as a severe cause of mortality. The clinical inadequacy of most anticancer therapeutics urgently prompted intense drug discovery efforts toward the identification of new chemical entities endowed with a potent and safe antitumor profile. In this scenario, targeting cancer cells apoptosis machinery has emerged as a relevant strategy, useful for tackling the emergence of drug resistance. On this basis, a small library of naturally inspired hybrid molecules was obtained by combining, through a click chemistry approach, "privileged" synthons such as curcumin scaffold and 1,2,3-triazole building block. Compound1, bearing apara-fluoro phenyl moiety, showed low-micromolar potency against T acute lymphoblastic leukemia cell growth. More in-depth biologic studies demonstrated, for this analog, cell death-inducing properties associated with its capability to simultaneously activate both the receptor and the mitochondrial apoptosis cascades. This peculiar behavior offers promises for achieving an expanded anticancer effect, namely intense cytotoxic response coupled with reduced predisposition of chemoresistance insurgence. Altogether, this study allowed the identification of compound1as a lead compound worth to be progressed as an anticancer drug candidate

Structural insights into the DNA recognition mechanism by the bacterial transcription factor PdxR

Specificity in protein-DNA recognition arises from the synergy of several factors that stem from the structural and chemical signatures encoded within the targeted DNA molecule. Here, we deciphered the nature of the interactions driving DNA recognition and binding by the bacterial transcription factor PdxR, a member of the MocR family responsible for the regulation of pyridoxal 5 & PRIME;-phosphate (PLP) biosynthesis. Single particle cryo-EM performed on the PLP-PdxR bound to its target DNA enabled the isolation of three conformers of the complex, which may be considered as snapshots of the binding process. Moreover, the resolution of an apo-PdxR crystallographic structure provided a detailed description of the transition of the effector domain to the holo-PdxR form triggered by the binding of the PLP effector molecule. Binding analyses of mutated DNA sequences using both wild type and PdxR variants revealed a central role of electrostatic interactions and of the intrinsic asymmetric bending of the DNA in allosterically guiding the holo-PdxR-DNA recognition process, from the first encounter through the fully bound state. Our results detail the structure and dynamics of the PdxR-DNA complex, clarifying the mechanism governing the DNA-binding mode of the holo-PdxR and the regulation features of the MocR family of transcription factors