Characterization of scorpion alpha-like toxin group using two new toxins from the scorpion Leiurus quinquestriatus hebraeus.

Two novel toxins, Lqh6 and Lqh7, isolated from the venom of the scorpion Leiurus quinquestriatus hebraeus, have in their sequence a molecular signature (8Q/KPE10) associated with a recently defined group of alpha-toxins that target Na channels, namely the alpha-like toxins [reviewed in Gordon, D., Savarin, P., Gurevitz, M. & Zinn-Justin, S. (1998) J. Toxicol. Toxin Rev. 17, 131-159]. Lqh6 and Lqh7 are highly toxic to insects and mice, and inhibit the binding of alpha-toxins to cockroach neuronal membranes. Although they kill rodents by intracerebroventricular injection, they do not inhibit the binding of antimammal alpha-toxins (e.g. Lqh2) to rat brain synaptosomes, not even at high concentrations. Furthermore, in voltage-clamp experiments, rat brain Na channels IIA (rNav1.2A) expressed in Xenopus oocytes are not affected by Lqh6 nor by Lqh7 below 3 micro m. In contrast, muscular Na channels (rNav1.4 and hNav1.5) expressed in the same cells respond to nanomolar concentrations of Lqh6 and Lqh7 by slowing of Na current inactivation and a leftward shift of the peak conductance-voltage curve. The structural and pharmacological properties of the new toxins are compared to those of other scorpion alpha-toxins in order to re-examine the hallmarks previously set for the alpha-like toxin group.     

A realistic in silico model for structure/function studies of molybdenum–copper CO dehydrogenase

CO dehydrogenase (CODH) is an environmentally crucial bacterial enzyme that oxidizes CO to CO₂ at a Mo–Cu active site. Despite the close to atomic resolution structure (1.1 Å), significant uncertainties have remained with regard to the protonation state of the water-derived equatorial ligand coordinated at the Mo-center, as well as the nature of intermediates formed during the catalytic cycle. To address the protonation state of the equatorial ligand, we have developed a realistic in silico QM model (~179 atoms) containing structurally essential residues surrounding the active site. Using our QM model, we examined each plausible combination of redox states (Mo^VI–Cu^I, Mo^V–Cu^II, Mo^V–Cu^I, and Mo^IV–Cu^I) and Mo-coordinated equatorial ligands (O^2?, OH^?, H₂O), as well as the effects of second-sphere residues surrounding the active site. Herein, we present a refined computational model for the Mo(VI) state in which Glu763 acts as an active site base, leading to a MoO₂-like core and a protonated Glu763. Calculated structural and spectroscopic data (hyperfine couplings) are in support of a MoO₂-like core in agreement with XRD data. The calculated two-electron reduction potential (E = ?467 mV vs. SHE) is in reasonable agreement with the experimental value (E = ?558 mV vs. SHE) for the redox couple comprising an equatorial oxo ligand and protonated Glu763 in the Mo^VI–Cu^I state and an equatorial water in the Mo^IV–Cu^I state. We also suggest a potential role of second-sphere residues (e.g., Glu763, Phe390) based on geometric changes observed upon exclusion of these residues in the most plausible oxidized states.     

Condensation of heptaminol hydrochloride for its spectrofluorimetric determination in pure form and tablets: application in human plasma

A sensitive, simple, accurate and less expensive fluorimetric method was designed and validated for analysis of heptaminol HCl in both its pure and dosage forms, as well as in human plasma. The main principle used in the proposed approach was the condensation reaction between heptaminol's primary amino moiety and ethyl acetoacetate/formaldehyde reagents, giving a derivative that was highly fluorescent at 416 nm after excitation at 350 nm. Various experimental parameters that affected either the product's development or its stability were evaluated and optimized. The constructed calibration curve was linear over the range 0.2–2 μg/ml, with a good correlation coefficient (0.9996). Both the calculated limit of detection and limit of quantitation were 0.06 and 0.18 μg/ml, respectively. The presented approach was a success when used to determine Corasore® tablets and was validated according to International Council for Harmonisation guidelines.     

Acinetobacter baumannii as a community foodborne pathogen: Peptide mass fingerprinting analysis,genotypic of biofilm formation and phenotypic pattern of antimicrobial resistance

Acinetobacter baumannii (A. baumannii) is one of the most common Gram-negative pathogens that represent a major threat to human life. Because the prevalence of Multidrug-resistant biofilm-forming A. baumannii is increasing all over the world, this may lead to outbreaks of hospital infections. Nonetheless, the role of raw meat as a reservoir for A. baumannii remains unclear. Here our research was aimed to exhibit the frequency, precise identification, and genotyping of biofilm-related genes as well as antimicrobial resistance of A. baumannii isolates of raw meat specimens. Fifty-five A. baumannii strains were recovered from 220 specimens of different animal meat and then identified by Peptide Mass Fingerprinting Technique (PMFT). All identified isolates were genotyped by the qPCR method for the existence of biofilm-related genes (ompA, bap, blaPER-1, csuE, csgA, and fimH). In addition, the antimicrobial resistance against A. baumannii was detected by the Kirby-Bauer method. Based on our findings, the frequency rate of 55 A. baumannii isolates was 46.55%, 32.50%, 15.00%, and 9.68% of sheep, chicken, cow, and camel raw meat samples, respectively. The PMFT was able to identify all strains by 100%. the percentages of csuE, ompA, bla_PER-1, bap, and csgA genes in biofilm and non-biofilm producer A. baumannii were 72.73%, 60%, 58.2%, 52.74%, and 25.45%, respectively. In contrast, the fimH was not detected in all non-biofilm and biofilm producer strains. The ompA, bap, blaPER-1, csgA were detected only in biofilm-producing A. baumannii isolates. The maximum degree of resistance was observed against amoxicillin/clavulanic acid (89.10%), gentamicin (74.55%), tetracycline (72.73%), ampicillin (65.45%), and tobramycin (52.73%). In conclusion, our investigation demonstrated the high incidence of multi-drug resistant A. baumannii in raw meat samples, with a high existence of biofilm-related virulence genes of ompA, bap, blaPER-1, csgA. Therefore, it has become necessary to take the control measures to limit the development of A. baumannii.     

Uncovering genomic causes of co-morbidity in epilepsy: gene-driven phenotypic characterization of rare microdeletions

Background

Patients with epilepsy often suffer from other important conditions. The existence of such co-morbidities is frequently not recognized and their relationship with epilepsy usually remains unexplained.

Methodology/Principal Findings

We describe three patients with common, sporadic, non-syndromic epilepsies in whom large genomic microdeletions were found during a study of genetic susceptibility to epilepsy. We performed detailed gene-driven clinical investigations in each patient. Disruption of the function of genes in the deleted regions can explain co-morbidities in these patients.

Conclusions/Significance

Co-morbidities in patients with epilepsy can be part of a genomic abnormality even in the absence of (known) congenital malformations or intellectual disabilities. Gene-driven phenotype examination can also reveal clinically significant unsuspected condition.     

Detection of two CSN1S1 variants in Egyptian buffalo

The present study investigates CSN1S1 casein gene polymorphism in Egyptian buffalo. CSN1S1 was analyzed in 17 unrelated Egyptian lactating buffalo. The amplified segment includes the last 43 amino acids of Exon 17 and part of Intron 17. In the present study we report for the first time the presence of 2 variants 178^Ser (TCA) and 178^Leu (TTA) in Egyptian buffalo CSN1S1 gene. The genotypic frequencies in the investigated Egyptian buffalo sample were 0.47, 0.058 and 0.47 for homozygous 178^Ser, for homozygous 178^Leu and heterozygous 178^Leu/Ser, respectively. The 178^Ser and 178^Leu variant frequencies are 0.64 and 0.36, respectively which indicates the superiority of variant 178^Ser in Egyptian buffalo. The allelic frequency in Egyptian buffalo is not much different from the corresponding allelic frequency in Italian buffalo (0.69 and 0.31 for 178^Ser and 178^Leu, respectively) as reported by Chianese et al. [3]. This is not surprising since they both belong to Mediterranean type.     

Diazoxide and Pinacidil Uncouple Pyruvate–Malate-Induced Mitochondrial Respiration

We investigated the effects of K_ATP channel openers diazoxide and pinacidil on the respiration rate and membrane potential () of rat heart mitochondria, oxidizing pyruvate and malate. Diazoxide and pinacidil (58.8–1348.3 M) increased the V ₂ (-ADP) respiration rate accordingly by 13–208% and 30–273% and decreased the by 2–17% and 6–55%. These effects were also similar in the respiration medium without K⁺. Moreover, carboxyatractyloside completely abolished diazoxide- and pinacidil-induced uncoupling, indicating a role for the mitochondrial adenine nucleotide translocase in this process.     

Profiling calcium signals of in vitro polarized human effector CD4+ T cells

Differentiation of naïve CD4⁺ T cells into effector subtypes with distinct cytokine profiles and physiological roles is a tightly regulated process, the imbalance of which can lead to an inadequate immune response or autoimmune disease. The crucial role of Ca²⁺ signals, mainly mediated by the store operated Ca²⁺ entry (SOCE) in shaping the immune response is well described. However, it is unclear if human effector CD4⁺ T cell subsets show differential Ca²⁺ signatures in response to different stimulation methods. Herein, we provide optimized in vitro culture conditions for polarization of human CD4⁺ effector T cells and characterize their SOCE following both pharmacological store depletion and direct T-cell receptor (TCR) activation. Moreover, we measured whole cell Ca²⁺ release activated Ca²⁺ currents (I_CRAC) and investigated whether the observed differences correlate to the expression of CRAC genes. Our results show that Ca²⁺ profiles of helper CD4⁺ Th1, Th2 and Th17 are distinct and in part shaped by the intensity of stimulation. Regulatory T cells (Treg) are unique being the subtype with the most prominent SOCE response. Analysis of in vivo differentiated Treg unraveled the role of differential expression of ORAI2 in fine-tuning signals in Treg vs. conventional CD4⁺ T cells.     

Phylogeny in Echinocereus (Cactaceae) based on combined morphological and molecular evidence: taxonomic implications

Echinocereus is a morphologically diverse genus that includes 64 species grouped into eight taxonomic sections based on morphological traits. In previous molecular phylogenetic analyses, the relationships amongst Echinocereus species were not entirely revealed and useful characters to recognize clades were not provided. The inclusion of several sources of evidence in a phylogenetic analysis is likely to produce more supported hypotheses. Therefore, we performed a combined phylogenetic analysis with a set of 44 morphological characters and six chloroplast DNA sequences. Topologies from parsimony and Bayesian analyses were mostly congruent. However, the relationships of E. poselgeri were not consistent between analyses. A second Bayesian analysis using a long-branch extraction test resulted in a topology with the morphological position of E. poselgeri congruent with that in parsimony analysis. Parsimony and Bayesian analyses corroborated the monophyly of Echinocereus, which included eight monophyletic groups. The combined phylogeny integrated into different clades those taxa that were not determined in previous analyses and changed the relationships of some recognized clades. The clades did not recover the recent infrageneric classification. In the present study, a new sectional classification for Echinocereus is proposed based on the eight recovered clades, which is supported by a combination of morphological and molecular characters. An identification key for sections in the genus is included.     

Nitrogen-induced variations in leaf gas exchange of spring triticale under field conditions

Nitrogen (N) is the key factor limiting photosynthetic processes and crop yield. Little is known about the response of leaf gas exchange of spring triticale (Triticosecale Wittm.) to N supply. The effect of N fertilizers on different gas exchange variables, i.e., photosynthetic rate (A), transpiration rate (E), stomatal conductance (g _s), instantaneous water use efficiency (WUE) and maximum quantum yield of photosystem II (PSII) (F _v/F _m), chlorophyll index (SPAD, soil–plant analysis development), and the relationship of these variables with yield were studied in spring triticale grown under field conditions. Six treatments of N—0, 90, 180, 90 + 30, 90 + 30 + 30 kg ha^?1 (applied as ammonium nitrate, AN) and one treatment of N 90 + 30 + 30 kg ha^?1 (applied as urea ammonium nitrate solution, UAN) were compared. The analysis of variance showed that throughout the triticale growing season, N fertilization had significant effects on A, WUE, g _s and SPAD. On average, N fertilizer application increased A values by 14–70%. E and F _v/F _m values were not influenced by N fertilization levels. The effect of growth stage and year on gas exchange variables and F _v/F _m and SPAD was found to be significant. At different growth stages, A values varied and maximum ones were reached at BBCH 31–33 (decimal code system of growth stages) and BBCH 59. With aging, values of A decreased independently of N fertilization level. The gas exchange variables were equally affected by both fertilizer forms. The interplay among grain yield, leaf gas exchange variables, F _v/F _m and SPAD of spring triticale was estimated. The statistical analysis showed that grain yield positively and significantly correlated with A and SPAD values throughout the growing season.