Publication of descriptions of novel bacterial taxa in <Emphasis Type="Italic">Antonie van Leeuwenhoek</Emphasis>

The species from the order Neisseriales are currently distinguished from other bacteria on the basis of branching in 16S rRNA gene trees. For this order containing a single family, Neisseriaceae, no distinctive molecular, biochemical, or phenotypic characters are presently known. We report here detailed phylogenetic and comparative analyses on the 27 genome sequenced species of the order Neisseriales. Our comparative genomic analyses have identified 54 conserved signature indels (CSIs) in widely distributed proteins that are specific for either all of the sequenced Neisseriales species or a number of clades within this order that are also supported by phylogenetic analyses. Of these CSIs, 11 are specifically present in all of the sequenced species from this order, but are not found in homologous proteins from any other bacteria. These CSIs provide novel molecular markers specific for, and delimiting, this order. Twenty-one CSIs in diverse proteins are specific for a group comprised of the genera Neisseria, Eikenella, Kingella, and Simonsiella (Clade I), which are obligate host-associated organisms, lacking flagella and exhibiting varied morphology. The species from these genera also formed a strongly supported clade in phylogenetic trees based upon concatenated protein sequences; a monophyletic grouping of these genera and other genera displaying similar morphological characteristics was also observed in the 16S rRNA gene tree. A second clade (Clade II), supported by seven of the identified CSIs and phylogenetic trees based upon concatenated protein sequences, grouped together species from the genera Chromobacterium, Laribacter, and Pseudogulbenkiania that are rod-shaped bacteria, which display flagella-based motility and are capable of free living. The remainder of the CSIs were uniquely shared by smaller groups within these two main clades. Our analyses also provide novel insights into the evolutionary history of the Neisseriales and suggest that the CSIs that are specific for the Clade I species may play an important role in the evolution of obligate host-association within this order. On the basis of phylogenetic analysis, the identified CSIs, and conserved phenotypic characteristics of different Neisseriales genera, we propose a division of this order into two families: an emended family Neisseriaceae (corresponding to Clade I) containing the genera Alysiella, Bergeriella, Conchiformibius, Eikenella, Kingella, Neisseria, Simonsiella, Stenoxybacter, Uruburuella and Vitreoscilla and a new family, Chromobacteriaceae fam. nov., harboring the remainder of the genera from this order (viz. Andreprevotia, Aquaspirillum, Aquitalea, Chitinibacter, Chitinilyticum, Chitiniphilus, Chromobacterium, Deefgea, Formivibrio, Gulbenkiania, Iodobacter, Jeongeupia, Laribacter, Leeia, Microvirgula, Paludibacterium, Pseudogulbenkiania, Silvimonas, and Vogesella).     

Kinetic and anion inhibition studies of a β-carbonic anhydrase (FbiCA 1) from the C4 plant Flaveria bidentis

Several β-carbonic anhydrases (CAs, EC 4.2.1.1) are present in all land plants examined thus far. Here we report the first detailed biochemical characterization of one such isoform, FbiCA 1, from the C₄ plant Flaveria bidentis, which was cloned, purified and characterized as recombinant protein. FbiCA 1 has an interesting CO₂ hydrase catalytic activity (k_cat of 1.2 × 10⁵ and k_cat/K_m of 7.5 × 10⁶ M^?1 × s^?1) and was moderately inhibited by most simple/complex inorganic anions. Potent FbiCA 1 inhibitors were also detected, such as trithiocarbonate, diethyldithiocarbamate, sulfamide, sulfamic acid, phenylboronic acid and phenylarsonic acid (K_Is in the range of 4–60 μM). Such inhibitors may be used as tools to better understand the role of various β-CA isoforms in photosynthesis.     

In silico and in vitro studies to elucidate the role of Cu2+ and galanthamine as the limiting step in the amyloid beta (1–42) fibrillation process

The formation of fibrils and oligomers of amyloid beta (Aβ) with 42 amino acid residues (Aβ_1–42) is the most important pathophysiological event associated with Alzheimer''s disease (AD). The formation of Aβ fibrils and oligomers requires a conformational change from an α-helix to a β-sheet conformation, which is encouraged by the formation of a salt bridge between Asp 23 or Glu 22 and Lys 28. Recently, Cu²⁺ and various drugs used for AD treatment, such as galanthamine (Reminyl®), have been reported to inhibit the formation of Aβ fibrils. However, the mechanism of this inhibition remains unclear. Therefore, the aim of this work was to explore how Cu²⁺ and galanthamine prevent the formation of Aβ_1–42 fibrils using molecular dynamics (MD) simulations (20 ns) and in vitro studies using fluorescence and circular dichroism (CD) spectroscopies. The MD simulations revealed that Aβ_1–42 acquires a characteristic U-shape before the α-helix to β-sheet conformational change. The formation of a salt bridge between Asp 23 and Lys 28 was also observed beginning at 5 ns. However, the MD simulations of Aβ₁₋₄₂ in the presence of Cu²⁺ or galanthamine demonstrated that both ligands prevent the formation of the salt bridge by either binding to Glu 22 and Asp 23 (Cu²⁺) or to Lys 28 (galanthamine), which prevents Aβ₁₋₄₂ from adopting the U-characteristic conformation that allows the amino acids to transition to a β-sheet conformation. The docking results revealed that the conformation obtained by the MD simulation of a monomer from the 1Z0Q structure can form similar interactions to those obtained from the 2BGE structure in the oligomers. The in vitro studies demonstrated that Aβ remains in an unfolded conformation when Cu²⁺ and galanthamine are used. Then, ligands that bind Asp 23 or Glu 22 and Lys 28 could therefore be used to prevent β turn formation and, consequently, the formation of Aβ fibrils.     

Household Transmission of Rotavirus in a Community with Rotavirus Vaccination in Quininde,Ecuador

Background

We studied the transmission of rotavirus infection in households in peri-urban Ecuador in the vaccination era.

Methods

Stool samples were collected from household contacts of child rotavirus cases, diarrhea controls and healthy controls following presentation of the index child to health facilities. Rotavirus infection status of contacts was determined by RT-qPCR. We examined factors associated with transmissibility (index-case characteristics) and susceptibility (household-contact characteristics).

Results

Amongst cases, diarrhea controls and healthy control household contacts, infection attack rates (iAR) were 55%, 8% and 2%, (n = 137, 130, 137) respectively. iARs were higher from index cases with vomiting, and amongst siblings. Disease ARs were higher when the index child was <18 months and had vomiting, with household contact <10 years and those sharing a room with the index case being more susceptible. We found no evidence of asymptomatic infections leading to disease transmission.

Conclusion

Transmission rates of rotavirus are high in households with an infected child, while background infections are rare. We have identified factors associated with transmission (vomiting/young age of index case) and susceptibility (young age/sharing a room/being a sibling of the index case). Vaccination may lead to indirect benefits by averting episodes or reducing symptoms in vaccinees.     

Criterion-Related Validity of the Distance- and Time-Based Walk/Run Field Tests for Estimating Cardiorespiratory Fitness: A Systematic Review and Meta-Analysis

Objectives

The main purpose of the present meta-analysis was to examine the criterion-related validity of the distance- and time-based walk/run tests for estimating cardiorespiratory fitness among apparently healthy children and adults.

Materials and Methods

Relevant studies were searched from seven electronic bibliographic databases up to August 2015 and through other sources. The Hunter-Schmidt’s psychometric meta-analysis approach was conducted to estimate the population criterion-related validity of the following walk/run tests: 5,000 m, 3 miles, 2 miles, 3,000 m, 1.5 miles, 1 mile, 1,000 m, ½ mile, 600 m, 600 yd, ¼ mile, 15 min, 12 min, 9 min, and 6 min.

Results

From the 123 included studies, a total of 200 correlation values were analyzed. The overall results showed that the criterion-related validity of the walk/run tests for estimating maximum oxygen uptake ranged from low to moderate (r_p = 0.42–0.79), with the 1.5 mile (r_p = 0.79, 0.73–0.85) and 12 min walk/run tests (r_p = 0.78, 0.72–0.83) having the higher criterion-related validity for distance- and time-based field tests, respectively. The present meta-analysis also showed that sex, age and maximum oxygen uptake level do not seem to affect the criterion-related validity of the walk/run tests.

Conclusions

When the evaluation of an individual’s maximum oxygen uptake attained during a laboratory test is not feasible, the 1.5 mile and 12 min walk/run tests represent useful alternatives for estimating cardiorespiratory fitness. As in the assessment with any physical fitness field test, evaluators must be aware that the performance score of the walk/run field tests is simply an estimation and not a direct measure of cardiorespiratory fitness.     

Electron transport pathway for a Streptomyces cytochrome P450: cytochrome P450 105D5-catalyzed fatty acid hydroxylation in Streptomyces coelicolor A3(2)

Streptomyces and other bacterial actinomycete species produce many important natural products, including the majority of known antibiotics, and cytochrome P450 (P450) enzymes catalyze important biosynthetic steps. Relatively few electron transport pathways to P450s have been characterized in bacteria, particularly streptomycete species. One of the 18 P450s in Streptomyces coelicolor A3(2), P450 105D5, was found to bind fatty acids tightly and form hydroxylated products when electrons were delivered from heterologous systems. The six ferredoxin (Fdx) and four flavoprotein Fdx reductase (FDR) proteins coded by genes in S. coelicolor were expressed in Escherichia coli, purified, and used to characterize the electron transfer pathway. Of the many possibilities, the primary pathway was NADH --> FDR1 --> Fdx4 --> P450 105D5. The genes coding for FDR1, Fdx4, and P450 105D5 are located close together in the S. coelicolor genome. Several fatty acids examined were substrates, including those found in S. coelicolor extracts, and all yielded several products. Mass spectra of the products of lauric acid imply the 8-, 9-, 10-, and 11-hydroxy derivatives. Hydroxylated fatty acids were also detected in vivo in S. coelicolor. Rates of electron transfer between the proteins were measured; all steps were faster than overall hydroxylation and consistent with rates of NADH oxidation. Substrate binding, product release, and oxygen binding were relatively fast in the catalytic cycle; high kinetic deuterium isotope effects for all four lauric acid hydroxylations indicated that the rate of C-H bond breaking is rate-limiting in every case. Thus, an electron transfer pathway to a functional Streptomyces P450 has been established.     

Dynamics of arrestin-rhodopsin interactions: loop movement is involved in arrestin activation and receptor binding

In this study we investigate conformational changes in Loop V-VI of visual arrestin during binding to light-activated, phosphorylated rhodopsin (Rho*-P) using a combination of site-specific cysteine mutagenesis and intramolecular fluorescence quenching. Introduction of cysteines at positions in the N-domain at residues predicted to be in close proximity to Ile-72 in Loop V-VI of arrestin (i.e. Glu-148 and Lys-298) appear to form an intramolecular disulfide bond with I72C, significantly diminishing the binding of arrestin to Rho*-P. Using a fluorescence approach, we show that the steady-state emission from a monobromobimane fluorophore in Loop V-VI is quenched by tryptophan residues placed at 148 or 298. This quenching is relieved upon binding of arrestin to Rho*-P. These results suggest that arrestin Loop V-VI moves during binding to Rho*-P and that conformational flexibility of this loop is essential for arrestin to adopt a high affinity binding state.     

Identification of novel bacterial plasminogen-binding proteins in the human pathogen Mycobacterium tuberculosis

Binding and activation of human plasminogen (Plg) to generate the proteolytic enzyme plasmin (Plm) have been associated with the invasive potential of certain bacteria. In this work, proteomic analysis together with ligand blotting assays identified several major Plg-binding spots in Mycobacterium tuberculosis soluble extracts (SEs) and culture filtrate proteins. The identity of 15 different proteins was deduced by N-terminal and/or MS and corresponded to DnaK, GroES, GlnA1, Ag85 complex, Mpt51, Mpt64, PrcB, MetK, SahH, Lpd, Icl, Fba, and EF-Tu. Binding of Plg to recombinant M. tuberculosis DnaK, GlnA1, and Ag85B was further confirmed by ELISA and ligand blotting assays. The binding was inhibited by epsilon-aminocaproic acid, indicating that the interaction involved lysine residues. Plg bound to recombinant mycobacterial proteins was activated to Plm by tissue-type Plg activator. In contrast with recombinant proteins, M. tuberculosis SE enhanced several times the Plg activation mediated by the activator. Interestingly, GlnA1 was able to bind the extracellular matrix (ECM) protein fibronectin. Together these results show that M. tuberculosis posses several Plg receptors suggesting that bound Plg to bacteria surface, can be activated to Plm, endowing bacteria with the ability to break down ECM and basal membranes proteins contributing to tissue injury in tuberculosis.     

Palmitic Acid-Induced NAD+ Depletion is Associated with the Reduced Function of SIRT1 and Increased Expression of BACE1 in Hippocampal Neurons

Increased levels of circulating fatty acids, such as palmitic acid (PA), are associated with the development of obesity, insulin resistance, type-2 diabetes and metabolic syndrome. Furthermore, these diseases are linked to an increased risk of cancer, cardiovascular diseases, mild cognitive impairment and even Alzheimer’s disease (AD). However, the precise actions of elevated PA levels on neurons and their association with neuronal metabolic disruption that leads to the expression of pathological markers of AD, such as the overproduction and accumulation of the amyloid-β peptide, represent an area of intense investigation. A possible molecular mechanism involved in the effects of PA may be through dysfunction of the NAD⁺ sensor enzyme, SIRT1. Therefore, the aim of the present study was to analyze the relationship between the effects of PA metabolism on the function of SIRT1 and the upregulation of BACE1 in cultured hippocampal neurons. PA reduced the total amount of NAD⁺ in neurons that caused an increase in p65 K310 acetylation due to inhibition of SIRT1 activity and low protein content. Furthermore, BACE1 protein and its activity were increased, and BACE1 was relocated in neurites after PA exposure.