Physiological level of norepinephrine increases adenine nucleotides hydrolysis in rat blood serum

Extracellular adenosine 5′-triphosphate (ATP) and its breakdown products, adenosine 5′-diphosphate (ADP) and adenosine, have significant effects on a variety of biological processes. NTPDase enzymes, responsible for adenine nucleotides hydrolysis, are considered the major regulators of purinergic signaling in the blood. Previous work by our group demonstrated that ATP and ADP hydrolysis in rat blood serum are higher during the dark (activity) phase compared to the light (rest) phase. In nocturnal animals (e.g., rats), important physiological changes occur during the dark phase, such as increased circulating levels of melatonin, corticosterone, and norepinephrine (NE). This study investigated the physiological effects, in vivo and in vitro, of melatonin, dexamethasone, and NE upon nucleotides hydrolysis in rat blood serum. For in vivo experiments, the animals received a single injection of saline (control), melatonin (0.05 mg/kg), dexamethasone (0.1 mg/kg), or NE (0.03 mg/kg). For in vitro experiments, melatonin (1.0 nM), dexamethasone (1.0 μM), or NE (1.0 nM) was added directly to the reaction medium with blood serum before starting the enzyme assay. The results demonstrated that ATP and ADP hydrolysis in both in vitro and in vivo experiments were significantly higher with NE treatment compared to control (in vitro: ATP = 36.63%, ADP = 22.43%, P < 0.05; in vivo: ATP = 44.1%, ADP = 37.28%, P < 0.001). No significant differences in adenine nucleotides hydrolysis were observed with melatonin and dexamethasone treatments. This study suggests a modulatory role of NE in the nucleotidases pathway, decreasing extracellular ATP and ADP, and suggests that NE might modulate its own release by increasing the activities of soluble nucleotidases.     

Mapping of quantitative trait loci controlling broomrape (Orobanche crenata Forsk.) resistance in faba bean (Vicia faba L.).

Orobanche crenata Forsk. is a root parasite that produces devastating effects on many crop legumes and has become a limiting factor for faba bean production in the Mediterranean region. The efficacy of available control methods is minimal and breeding for broomrape resistance remains the most promising method of control. Resistance seems to be scarce and complex in nature, being a quantitative characteristic difficult to manage in breeding programmes. To identify and map the QTLs (quantitative trait loci) controlling the trait, 196 F2 plants derived from the cross between a susceptible and a resistant parent were analysed using isozymes, RAPD, seed protein genes, and microsatellites. F2-derived F3 lines were studied for broomrape resistance under field conditions. Of the 130 marker loci segregating in the F2 population, 121 could be mapped into 16 linkage groups. Simple interval mapping (SIM) and composite interval mapping (CIM) were performed using QTL Cartographer. Composite interval mapping using the maximum number of markers as cofactors was clearly the most efficient way to locate putative QTLs. Three QTLs for broomrape resistance were detected. One of the three QTLs explained more than 35% of the phenotypic variance, whereas the others accounted for 11.2 and 25.5%, respectively. This result suggests that broomrape resistance in faba bean can be considered a polygenic trait with major effects of a few single genes.     

Evolutionary History of the PER3 Variable Number of Tandem Repeats (VNTR): Idiosyncratic Aspect of Primate Molecular Circadian Clock

The PER3 gene is one of the clock genes, which function in the core mammalian molecular circadian system. A variable number of tandem repeats (VNTR) locus in the 18th exon of this gene has been strongly associated to circadian rhythm phenotypes and sleep organization in humans, but it has not been identified in other mammals except primates. To better understand the evolution and the placement of the PER3 VNTR in a phylogenetical context, the present study enlarges the investigation about the presence and the structure of this variable region in a large sample of primate species and other mammals. The analysis of the results has revealed that the PER3 VNTR occurs exclusively in simiiforme primates and that the number of copies of the primitive unit ranges from 2 to 11 across different primate species. Two transposable elements surrounding the 18th exon of PER3 were found in primates with published genome sequences, including the tarsiiforme Tarsius syrichta, which lacks the VNTR. These results suggest that this VNTR may have evolved in a common ancestor of the simiiforme branch and that the evolutionary copy number differentiation of this VNTR may be associated with primate simiiformes sleep and circadian phenotype patterns.     

Zinc Piracy as a Mechanism of Neisseria meningitidis for Evasion of Nutritional Immunity

The outer membrane of Gram-negative bacteria functions as a permeability barrier that protects these bacteria against harmful compounds in the environment. Most nutrients pass the outer membrane by passive diffusion via pore-forming proteins known as porins. However, diffusion can only satisfy the growth requirements if the extracellular concentration of the nutrients is high. In the vertebrate host, the sequestration of essential nutrient metals is an important defense mechanism that limits the growth of invading pathogens, a process known as “nutritional immunity.” The acquisition of scarce nutrients from the environment is mediated by receptors in the outer membrane in an energy-requiring process. Most characterized receptors are involved in the acquisition of iron. In this study, we characterized a hitherto unknown receptor from Neisseria meningitidis, a causative agent of sepsis and meningitis. Expression of this receptor, designated CbpA, is induced when the bacteria are grown under zinc limitation. We demonstrate that CbpA functions as a receptor for calprotectin, a protein that is massively produced by neutrophils and other cells and that has been shown to limit bacterial growth by chelating Zn²⁺ and Mn²⁺ ions. Expression of CbpA enables N. meningitidis to survive and propagate in the presence of calprotectin and to use calprotectin as a zinc source. Besides CbpA, also the TonB protein, which couples energy of the proton gradient across the inner membrane to receptor-mediated transport across the outer membrane, is required for the process. CbpA was found to be expressed in all N. meningitidis strains examined, consistent with a vital role for the protein when the bacteria reside in the host. Together, our results demonstrate that N. meningitidis is able to subvert an important defense mechanism of the human host and to utilize calprotectin to promote its growth.     

Power consumption of three impeller combinations in mixing Xanthan fermentation broths

Xanthan gum fermentation represents a good model for the study of the mixing of rheologically complex culture broths. Most of the previous work on power consumption dealt with ‘standard’, single impellers and used model fluids to simulate xanthan broths. This work describes the characterization of three dual-impeller combinations (D/T = 0·53) for the mixing of dehydrated—reconstituted fermentation broths of Xanthomonas campestris that had matched rheology to the actual broths. The bottom impeller was a Rushton turbine (RT) and the top impeller was another RT, a 45° pitched blade turbine (PT) or an A-310 Lightnin mixer (A310). The experiments were carried out in a tank of 0·0094 m³ working volume equipped with an air bearing dynamometer. The power was measured in a wide range of xanthan concentrations (5–40 kg m⁻³) in aerated (0·25, 0·5 and 1·0 vvm) and unaerated conditions. Unaerated power number (Po) vs. Reynolds number (Re) curves showed similar trends for the three combinations. Exponents close to −1 were obtained in the laminar region. A minimum in Po (Po_min) occurred at Re = 30–40, then increasing to a plateau value which was evident at Re> 200. In the transition region Po_min values were 4·3 (RT and RT), 3·6 (RT and PT) and 2·4 (RT and A310). The aerated power data for (RT and PT) and (RT and A-310) showed higher torque instabilities than the dual RT combinations at higher xanthan concentrations. The higher the xanthan concentrations, the higher the drop in power and the less important the effect of the aeration rate. Among the combinations tested, when using Rushton turbines, the well-mixed ‘cavern’ reached the tank wall (i.e., fluid motion was observed) at the lowest volumetric power input. High     

Identification of Bioactive Compounds against Aedes aegypti (Diptera: Culicidae) by Bioassays and in Silico Assays

Most of the hematophagous insects act as disease vectors, including Aedes aegypti, responsible for transmitting some of the most critical arboviruses globally, such as Dengue. The use of repellents based on natural products is a promising alternative for personal protection compared to industrial chemical repellents. In this study, the repellent effect of essential oils extracted from Lippia thymoides, Lippia alba, Cymbopogon winterianus, and Eucalyptus globulus leaves was evaluated. Essential oils used showed repellent activity against Ae. aegypti in laboratory bioassays, obtaining protection rates above 70 % from 3.75 mg/mL and higher concentration for all analyzed oils. GC/MS identified 57 constituents, which were used in the ligand-based pharmacophore model to expose compounds with requirements for repellents that modulate mosquitoes behavior through odorant-binding protein 1 Ae. aegypti. Ligand-based pharmacophore model approach results suggested that repellent activity from C. winterianus, L. alba, and L. thymoides essential oils’ metabolites is related to Citronelal (QFIT=26.77), Citronelol (QFIT=11.29), Citronelol acetate (QFIT=52.22) and Geranil acetate (QFIT=10.28) with synergistic or individual activity. E. globulus essential oil's repellent activity is associated with Ledol (0.94 %; QFIT=41.95). Molecular docking was applied to understand the binding mode and affinity of the essential oils’ data set at the protein binding site. According to molecular docking, Citronelol (ChemPLP=60.98) and geranyl acetate (ChemPLP=60.55) were the best-classified compounds compared to the others and they can be explored to develop new repellents.     

Mimicking a p53‐MDM2 interaction based on a stable immunoglobulin‐like domain scaffold

Antibodies recognize protein targets with great affinity and specificity. However, posttranslational modifications and the presence of intrinsic disulfide‐bonds pose difficulties for their industrial use. The immunoglobulin fold is one of the most ubiquitous folds in nature and it is found in many proteins besides antibodies. An example of a protein family with an immunoglobulin‐like fold is the Cysteine Protease Inhibitors (ICP) family I42 of the MEROPs database for protease and protease inhibitors. Members of this protein family are thermostable and do not present internal disulfide bonds. Crystal structures of several ICPs indicate that they resemble the Ig‐like domain of the human T cell co‐receptor CD8α As ICPs present 2 flexible recognition loops that vary accordingly to their targeted protease, we hypothesize that members of this protein family would be ideal to design peptide aptamers that mimic protein‐protein interactions. Herein, we use an ICP variant from Entamoeba histolytica (EhICP1) to mimic the interaction between p53 and MDM2. We found that a 13 amino‐acid peptide derived from p53 can be introduced in 2 variable loops (DE, FG) but not the third (BC). Chimeric EhICP1‐p53 form a stable complex with MDM2 at a micromolar range. Crystal structure of the EhICP1‐p53(FG)‐loop variant in complex with MDM2 reveals a swapping subdomain between 2 chimeric molecules, however, the p53 peptide interacts with MDM2 as in previous crystal structures. The structural details of the EhICP1‐p53(FG) interaction with MDM2 resemble the interaction between an antibody and MDM2.     

Determination and diagnostic value of CA9 mRNA in peripheral blood of patients with oral leukoplakia

Background: Oral leukoplakia is one of the most common oral premalignant disorder. The classical evaluation through tissue biopsy is not always valid to evaluate the risk of malignization.

Material and methods: RT-qPCR was performed on 47 blood samples (21 patients with leukoplakia, 2 with oral squamous cell carcinoma (OSCC), and 24 healthy patients) and on 11 tissue samples (3 leukoplakia, 4 OSCC, and 4 samples of healthy tissue).

Results: There are significant differences in expression between the different groups (F?=?4.057, p?=?.006). The Duncan post hoc test shows that the only group that differentiates is the tumour tissue. Using Wilcoxon test, different covariables of patients with leukoplakia were analysed with respect to the group of healthy patients and no significant differences were observed.

Conclusions: The diagnostic route through liquid biopsy has not been conclusive in this study, but there are significant differences in the levels analysed in the different tissue samples.