Unusual evolution of a catalytic core element in CCA-adding enzymes

CCA-adding enzymes are polymerases existing in two distinct enzyme classes that both synthesize the C-C-A triplet at tRNA 3′-ends. Class II enzymes (found in bacteria and eukaryotes) carry a flexible loop in their catalytic core required for switching the specificity of the nucleotide binding pocket from CTP- to ATP-recognition. Despite this important function, the loop sequence varies strongly between individual class II CCA-adding enzymes. To investigate whether this loop operates as a discrete functional entity or whether it depends on the sequence context of the enzyme, we introduced reciprocal loop replacements in several enzymes. Surprisingly, many of these replacements are incompatible with enzymatic activity and inhibit ATP-incorporation. A phylogenetic analysis revealed the existence of conserved loop families. Loop replacements within families did not interfere with enzymatic activity, indicating that the loop function depends on a sequence context specific for individual enzyme families. Accordingly, modeling experiments suggest specific interactions of loop positions with important elements of the protein, forming a lever-like structure. Hence, although being part of the enzyme’s catalytic core, the loop region follows an extraordinary evolutionary path, independent of other highly conserved catalytic core elements, but depending on specific sequence features in the context of the individual enzymes.     

Vector Choice Determines Immunogenicity and Potency of Genetic Vaccines against Angola Marburg Virus in Nonhuman Primates

The immunogenicity and durability of genetic vaccines are influenced by the composition of gene inserts and choice of delivery vector. DNA vectors are a promising vaccine approach showing efficacy when combined in prime-boost regimens with recombinant protein or viral vectors, but they have shown limited comparative efficacy as a stand-alone platform in primates, due possibly to suboptimal gene expression or cell targeting. Here, regimens using DNA plasmids modified for optimal antigen expression and recombinant adenovirus (rAd) vectors, all encoding the glycoprotein (GP) gene from Angola Marburg virus (MARV), were compared for their ability to provide immune protection against lethal MARV Angola infection. Heterologous DNA-GP/rAd5-GP prime-boost and single-modality rAd5-GP, as well as the DNA-GP-only vaccine, prevented death in all vaccinated subjects after challenge with a lethal dose of MARV Angola. The DNA/DNA vaccine induced humoral responses comparable to those induced by a single inoculation with rAd5-GP, as well as CD4⁺ and CD8⁺ cellular immune responses, with skewing toward CD4⁺ T-cell activity against MARV GP. Vaccine regimens containing rAd-GP, alone or as a boost, exhibited cellular responses with CD8⁺ T-cell dominance. Across vaccine groups, CD8⁺ T-cell subset dominance comprising cells exhibiting a tumor necrosis factor alpha (TNF-α) and gamma interferon (IFN-γ) double-positive functional phenotype was associated with an absence or low frequency of clinical symptoms, suggesting that both the magnitude and functional phenotype of CD8⁺ T cells may determine vaccine efficacy against infection by MARV Angola.The filoviruses Marburgvirus (MARV) and Ebolavirus (EBOV) are endemic primarily to central Africa and cause a severe form of viral hemorrhagic fever. Of all the filovirus strains or species, the Angola strain of MARV is associated with the highest mortality rate (90%) in humans observed to date (26). An increase in natural filovirus outbreak frequency over the past decade and the potential for use to cause deliberate human mortality have focused attention on the need for therapeutics and vaccines against filoviruses. While regulatory pathways have been proposed to facilitate licensing of a preventive vaccine against potently lethal pathogens such as these, there is as yet no licensed vaccine for use in humans, and efforts remain targeted to the optimization of vaccine performance in nonhuman primates (NHP) since this animal model recapitulates many aspects of disease pathogenesis observed in humans.Genetic vaccines are a promising approach for immunization against pathogens that are rapidly changing due to natural evolution, cross-species transmission, or intentional modification. Gene-based vaccines are produced rapidly and can be delivered by a variety of vectors. DNA vectors are advantageous because they are inherently safe and stable and can be used repeatedly without inducing antivector immune responses. However, while filovirus DNA vaccines have demonstrated efficacy in small animal models, efforts to induce protective immunity by injection of plasmid DNA alone into NHP have yielded less encouraging results. EBOV DNA vectors generate immune protection in mice and guinea pigs, but this has not been demonstrated in NHP unless DNA immunization is boosted with a viral vector vaccine (23). MARV DNA fully protects mice and guinea pigs but provides only partial protection in NHP (17). The discordant results between rodent and primate species may be due to the use of slightly modified infectious challenge viruses in rodent models or may reflect underlying differences in vaccine performance and the mechanisms of immune protection between rodents and NHP.In the current study, we examined whether DNA plasmid-based vaccines could be improved to increase potency in NHP and compared immunogenicity of this vaccine modality with those of viral vector and prime-boost approaches. DNA-vectored vaccines were modified by codon optimizing gene target inserts for enhanced expression in primates. These vectors induced antigen-specific cellular and humoral immune responses similar to immunization using a recombinant adenoviral vector and provided protection after lethal challenge with MARV Angola. However, macaques vaccinated with DNA vectors exhibited clinical symptoms associated with MARV hemorrhagic fever (MHF) that were absent in NHP receiving a single inoculation with recombinant adenovirus (rAd) vectors, suggesting qualitative differences in the immune responses elicited by the different modalities.     

Revision of the taxonomic position of the xylanolytic Bacillus sp. MIR32 reidentified as Bacillus halodurans and plasmid-mediated transformation of B. halodurans

Bacillus sp. MIR32 has been isolated using xylan as the only carbon source, and one of its xylanolytic enzymes has been extensively studied. Biochemical analysis first related this strain to Bacillus amyloliquefaciens, but further studies based on a comparison of 16S rDNA sequences, G+C content, and DNA-DNA hybridization showed that strain MIR32 should be classified as a member of the species Bacillus halodurans. This change is also supported by the typical phenotype observed and by the results of PCR amplification directed toward spacers in rDNA and tDNA genes, which were assayed and compared with those of B. halodurans DSM 497(T). Although among alkaliphilic bacilli competence development has not been experimentally demonstrated, in this work both B. halodurans MIR32 and DSM 497(T) were transformed according to a simple procedure developed in our laboratory, reaching 10(2)-10(3) stable transformants per microgram of plasmid DNA.     

Smoking Is Associated with More Abdominal Fat in Morbidly Obese Patients

IntroductionWhile the association between cigarette smoking and abdominal fat has been well studied in normal and overweight patients, data regarding the influence of tobacco use in patients with morbid obesity remain scarce. The aim of this study is to evaluate body fat distribution in morbidly obese smokers.MethodsWe employed a cross-sectional study and grouped severely obese patients (body mass index [BMI] >40 kg/m² or >35 kg/m² with comorbidities) according to their smoking habits (smokers or non-smokers). We next compared the anthropometrical measurements and body composition data (measured by electric bioimpedance) of both groups. We analyzed the effect of smoking on body composition variables using univariate and multiple linear regression (MLR); differences are presented as regression coefficients (b) and their respective 95% confidence intervals.ResultsWe included 536 morbidly obese individuals, 453 (84.5%) non-smokers and 83 (15.5%) smokers. Male smokers had a higher BMI (b=3.28 kg/m², p=0.036), larger waist circumference (b=6.07 cm, p=0.041) and higher percentage of body fat (b=2.33%, p=0.050) than non-smokers. These differences remained significant even after controlling for confounding factors. For females, the only significant finding in MLR was a greater muscle mass among smokers (b=1.34kg, p=0.028). No associations were found between tobacco load measured in pack-years and anthropometric measures or body composition.DiscussionPositive associations between smoking and BMI, and waist circumference and percentage of body fat, were found among male morbidly obese patients, but not among females. To the best of our knowledge, this study is the first investigation of these aspects in morbidly obese subjects. We speculate that our findings may indicate that the coexistence of morbid obesity and smoking helps to explain the more serious medical conditions, particularly cardiovascular diseases and neoplasms, seen in these patients.     

Estimating the infectivity of CCR5-tropic simian immunodeficiency virus SIV(mac251) in the gut

CD4+ T-cell depletion during acute human immunodeficiency virus infection occurs predominantly in the gastrointestinal mucosa. Using experimental data on SIV(mac251) viral load in blood and CD4+ T cells in the jejunum, we modeled the kinetics of CD4+ T-cell infection and death and estimated the viral infectivity. The infectivity of SIV(mac251) is higher than previously estimated for SHIV89.6P infection, but this higher infectivity is offset by a lower average peak viral load in SIV(mac251). Thus, the dynamics of target cell infection and death are remarkably similar between a CXCR4- and a CCR5-tropic infection in vivo.     

Molecular screening of interleukin-6 gene promoter and influence of −174G/C polymorphism on breast cancer

Interleukin-6 (IL-6) is a cytokine involved in different physiologic and pathophysiologic processes including carcinogenesis. In 2003, a single nucleotide polymorphism (−174G/C) of the IL-6 gene promoter has been linked to breast cancer prognosis in node-positive (N+) breast cancer patients. Since, different studies have led to conflicting conclusions about its role as a prognostic and/or diagnostic marker. The primary aim of our study was to investigate the link between −174G/C polymorphism and breast cancer risk on the one hand, and −174G/C polymorphism and prognosis in different groups of patients: sporadic N+ breast cancers (n = 138), sporadic N− breast cancers (n = 95) and familial breast cancer (n = 60) on the other hand. The variables of interest were disease-free survival and overall survival. The secondary aim of the study was to screen IL-6 gene promoter using direct sequencing to identify new polymorphisms in our French Caucasian breast cancer population. No association or trend of association between −174G/C polymorphism of IL-6 gene promoter gene and breast cancer diagnosis or prognosis was shown, even in meta-analyses. Furthermore, we have identified four novel polymorphic sites in the IL-6 gene promoter region: −764G → A, −757C → T, −233T → A, 15C → A.     

Protein multiple sequence alignment by hybrid bio-inspired algorithms

This article presents an immune inspired algorithm to tackle the Multiple Sequence Alignment (MSA) problem. MSA is one of the most important tasks in biological sequence analysis. Although this paper focuses on protein alignments, most of the discussion and methodology may also be applied to DNA alignments. The problem of finding the multiple alignment was investigated in the study by Bonizzoni and Vedova and Wang and Jiang, and proved to be a NP-hard (non-deterministic polynomial-time hard) problem. The presented algorithm, called Immunological Multiple Sequence Alignment Algorithm (IMSA), incorporates two new strategies to create the initial population and specific ad hoc mutation operators. It is based on the 'weighted sum of pairs' as objective function, to evaluate a given candidate alignment. IMSA was tested using both classical benchmarks of BAliBASE (versions 1.0, 2.0 and 3.0), and experimental results indicate that it is comparable with state-of-the-art multiple alignment algorithms, in terms of quality of alignments, weighted Sums-of-Pairs (SP) and Column Score (CS) values. The main novelty of IMSA is its ability to generate more than a single suboptimal alignment, for every MSA instance; this behaviour is due to the stochastic nature of the algorithm and of the populations evolved during the convergence process. This feature will help the decision maker to assess and select a biologically relevant multiple sequence alignment. Finally, the designed algorithm can be used as a local search procedure to properly explore promising alignments of the search space.     

A three-dimensional analysis of finger and bow string movements during the release in archery

The aim of this paper was to examine finger and bow string movements during archery by investigating a top Austrian athlete (FITA score = 1233) under laboratory conditions. Maximum lateral bow string deflection and angular displacements for index, third, and ring fingers between the full draw position and the end of the release were quantified using a motion tracking system. Stepwise multiple regression analyses were used to determine whether bow string deflection and finger movements are predictive for scoring. Joint ranges of motion during the shot itself were large in the proximal and distal interphalangeal joints, and much smaller in the metacarpophalangeal joints. Contrary to our expectations, greater deflection leads to higher scores (R2 = .18, p < .001) and the distal interphalangeal joint of the third finger weakly predicts the deflection (R2 = .11, p < .014). More variability in the joint angles of the third finger was found in bad shots than in good shots. Findings in this study let presume that maximum lateral bow string deflection does not adversely affect the archer's performance.     

The structure of a native l-amino acid oxidase, the major component of the Vipera ammodytes ammodytes venomic, reveals dynamic active site and quaternary structure stabilization by divalent ions

The crystal structure of the major component of the Vipera ammodytes ammodytes venomic, a flavotoxin, member of the l-amino acid oxidase (LAAO) family, has been determined and refined at 2.6 ? resolution. The asymmetric unit consists of four molecules, each bound to oxidized FAD, representing a dimer of dimers. The binding of four Zn(2+) ions stabilizes the enzymatically active quaternary structure and is considered important for the biological activity of LAAO and other flavoproteins. Each monomer consists of three domains with a cofactor bound between the FAD and substrate binding domains, and a solvent exposed glycosylation site which is considered crucial for the toxicity. Comparison of LAAO structures in the absence and presence of a substrate indicates conformational changes in the dynamic active site. The active site H-bond network involving the triad Lys326-Water-N5 of FAD is formed only upon substrate binding, and results in the increased mobility of the isoalloxazine system. Details of the catalytic transformation of amino acid substrates are discussed.