Cytoskeletal Tropomyosin Tm5NM1 Is Required for Normal Excitation–Contraction Coupling in Skeletal Muscle

The functional diversity of the actin microfilaments relies in part on the actin binding protein tropomyosin (Tm). The muscle-specific Tms regulate actin-myosin interactions and hence contraction. However, there is less known about the roles of the numerous cytoskeletal isoforms. We have shown previously that a cytoskeletal Tm, Tm5NM1, defines a Z-line adjacent cytoskeleton in skeletal muscle. Recently, we identified a second cytoskeletal Tm in this region, Tm4. Here we show that Tm4 and Tm5NM1 define separate actin filaments; the former associated with the terminal sarcoplasmic reticulum (SR) and other tubulovesicular structures. In skeletal muscles of Tm5NM1 knockout (KO) mice, Tm4 localization was unchanged, demonstrating the specificity of the membrane association. Tm5NM1 KO muscles exhibit potentiation of T-system depolarization and decreased force rundown with repeated T-tubule depolarizations consistent with altered T-tubule function. These results indicate that a Tm5NM1-defined actin cytoskeleton is required for the normal excitation–contraction coupling in skeletal muscle.     

Electrochemical checking of aerobic isolates from electrochemically active biofilms formed in compost

Aims:  To design a cyclic voltammetry (CV) procedure to check the electrochemical activity of bacterial isolates that may explain the electrochemical properties of biofilms formed in compost.
Methods and Results:  Bacteria catalysing acetate oxidation in garden compost were able to form electrochemically active biofilms by transferring electrons to an electrode under chronoamperometry. They were recovered from the electrode surface and identification of the isolates using 16S rRNA sequencing showed that most of them were Gammaproteobacteria, mainly related to Enterobacter and Pseudomonas spp. A CV procedure was designed to check the electrochemical activity of both groups of isolates. Preliminary CVs suggested that the bacteria were not responsible for the catalysis of acetate oxidation. In contrast, both groups of isolates were found to catalyse the electrochemical reduction of oxygen under experimental conditions that favoured adsorption of the microbial cells on the electrode surface.
Conclusions:  Members of the genera Enterobacter and Pseudomonas were found to be able to catalyse the electrochemical reduction of oxygen.
Significance and Impact of the Study:  This study has shown the unexpected efficiency of Enterobacter and Pseudomonas spp. in catalysing the reduction of oxygen, suggesting a possible involvement of these species in biocorrosion, or possible application of these strains in designing bio-cathode for microbial fuel cells.     

One-year Weight Losses in the Look AHEAD Study: Factors Associated With Success

This report provides a further analysis of the first year weight losses in the Look AHEAD (Action for Health in Diabetes) study and identifies factors associated with success. Participants were a total of 5,145 men and women with type 2 diabetes who were recruited at 16 sites and randomly assigned to an intensive lifestyle intervention (ILI) or a control condition, Diabetes Support and Education (DSE). During year 1, participants in ILI received comprehensive diet and physical activity counseling in a total of 42 group and individual sessions, compared with three educational sessions for DSE participants. As reported previously, at the end of the year, ILI participants lost 8.6% of initial weight, compared to 0.7% for DSE (P < 0.001). Within the ILI group, all racial/ethnic groups achieved clinically significant weight losses (>5.5%), although there were significant differences among groups. For the year, ILI participants attended an average of 35.4 treatment sessions and reported exercising a mean of 136.6 min/week and consuming a total of 360.9 meal replacement products. Greater self-reported physical activity was the strongest correlate of weight loss, followed by treatment attendance and consumption of meal replacements. The use of orlistat, during the second half of the year, increased weight loss only marginally in those ILI participants who had lost <5% of initial weight during the first 6 months and chose to take the medication thereafter as a toolbox option. The lifestyle intervention was clinically effective in all subsets of an ethnically and demographically diverse population.     

The importance of electrostatic potential in the interaction of sweet proteins with the sweet taste receptor

In addition to many small molecular mass sweeteners there are in nature a few sweet proteins. The molecular volume of sweet proteins is so different from that of common sweeteners that it was difficult to understand how molecules as large as proteins can activate a receptor designed to host small molecules. We have recently shown that sweet proteins can activate the sweet receptor by a mechanism of interaction, called 'wedge model", in which proteins fit a large cavity of the receptor with wedge-shaped surfaces of their structures. In order to substantiate this model we have designed, expressed and characterized seven mutants of MNEI, a single chain monellin. Three uncharged residues of the interaction surface, Met42, Tyr63 and Tyr65, were changed either into acidic or basic residues whereas Asp68, a key acidic residue, was changed into a basic one. As a general trend, we observe that an increase of the negative charge is much more detrimental for sweetness than an increase of positive charge. In addition we show that by a careful choice of a residue at the center of the interface between MNEI and receptor, it is possible even to increase the sweetness of MNEI. These results are fully consistent with the wedge model.     

Fluorescent indicators distributed throughout the pharmacophore of cholecystokinin provide insights into distinct modes of binding and activation of type A and B cholecystokinin receptors

Ligand probes with fluorescent indicators positioned throughout the pharmacophoric domain can provide important insights into the molecular basis of receptor binding and activation as reflected in the microenvironment of each indicator while docked at a receptor. We developed three cholecystokinin-like probes with Aladan situated at the N terminus, in the mid-region, and at the C terminus (positions 24, 29, and 33, respectively). These were studied in solution and docked at type A and B cholecystokinin receptors. This study demonstrated clear differences in mechanisms of cholecystokinin binding and activation of these structurally related receptors with distinct agonist structure-activity relationships. The fluorescence characteristics of Aladan are highly sensitive to the polarity of its microenvironment. The mid-region probe was least accessible to the aqueous milieu as determined by fluorescence emission spectra and iodide quenching, which was not altered by changes in conformation from active to inactive. Accessibility of the N- and C-terminal probes was affected by receptor conformation. The position 24 probe was more easily quenched in the active than in the G protein-uncoupled conformation for both receptors. However, the position 33 probe docked at the type A cholecystokinin receptor was more easily quenched in the active conformation, whereas the same probe docked at the type B cholecystokinin receptor was more easily quenched in the inactive conformation. Fluorescence anisotropy and red edge excitation shift determinations confirmed these observations and supported the proposed movements. Although both type A and B cholecystokinin receptors bind cholecystokinin with high affinity, resulting in fully efficacious biological responses, these receptors utilize distinct molecular modes of binding.     

Identification of key genes responsible for cytokine-induced erythroid and myeloid differentiation and switching of hematopoietic stem cells by RAGE

We utilized a unique culture system to analyze the expression patterns of gene, protein, and cell surface antigen, and the biological process of the related genes in erythroid and myeloid differentiation and switching of hematopoietic stem cells （HSCs） in response to cytokine alterations. Gene-specific fragments （266） identified from five populations of cytokine-stimulated HSCs were categorized into three groups： （1） expressed specifically in a single cell population; （2） expressed in two cell populations, and （3） expressed in three or more populations. Of 145 defined cDNAs, three （2%） were novel genes. Protein two-dimensional gel electrophoresis and flow cytometry analyses showed overlapped and distinguished protein expression profiles in the cell populations studied. Biological process mapping of mRNAs expressed in erythroid and myeloid lineages indicated that mRNAs shared by both lineages attended ＇core processes,＇ whereas genes specifically expressed in either lineage alone were related to specific processes or cellular maturation. Data from this study support the hypothesis that committed HSCs （El4 or G14） cells can still be redirected to develop into myeloid or erythroid cells when erythropoietin （EPO） is replaced with granulocyte-colony stimulating factor （G-CSF） under erythroid-cultured condition or G-CSF with EPO in myeloid-cultured environment, respectively. Our results suggest that genes or proteins co-expressed in erythroid and myeloid lineages may be essential for the lineage maintenance and switching in hematopoiesis.     

Patterns of parental care in Neotropical glassfrogs: fieldwork alters hypotheses of sex‐role evolution

Many animals provide parental care to offspring. Parental sex‐roles vary extensively across taxa, and such patterns are considered well documented. However, information on amphibians is lacking relative to other vertebrate groups. We combine natural history observations with functional and historical analyses to examine the evolution of egg care in glassfrogs (Centrolenidae). Parental care was considered rare and predominately provided by males. Our field observations of 40 species revealed that care occurs throughout the family, and the caregiving sex changes across lineages. We discovered that a brief period of maternal care is widespread and occurs in species previously thought to lack care. Using a combination of female‐removal experiments, prey‐choice tests with egg‐eating katydids, and parental disturbance‐tolerance assays, we confirm the adaptive benefits of short‐term maternal care in wild Cochranella granulosa and Teratohyla pulverata. To examine historical transitions between caregiving sexes, we assembled a molecular phylogeny and estimated ancestral care states using our data and the literature. We assessed patterns indicative of sex‐specific constraints by testing whether transitions between the sexes are associated with changes in care levels. Our analyses support that male‐only care evolved 2–3 times from female‐only care, and this change is associated with substantial increases in care levels – a pattern supporting the hypothesis that male‐only care evolved via constraints on maternal expenditure. Many groups of amphibians remain poorly studied, with emerging evidence indicating that care patterns are more diverse than currently appreciated. Natural history remains fundamental to uncovering this diversity and generating testable hypotheses of sex‐role evolution.     

Intensive care antibiotic consumption and resistance patterns: a cross-correlation analysis

Background

Over recent decades, a dramatic increase in infections caused by multidrug-resistant pathogens has been observed worldwide. The aim of the present study was to investigate the relationship between local resistance bacterial patterns and antibiotic consumption in an intensive care unit in a Romanian university hospital.

Methods

A prospective study was conducted between 1st January 2012 and 31st December 2013. Data covering the consumption of antibacterial drugs and the incidence density for the main resistance phenotypes was collected on a monthly basis, and this data was aggregated quarterly. The relationship between the antibiotic consumption and resistance was investigated using cross-correlation, and four regression models were constructed, using the SPSS version 20.0 (IBM, Chicago, IL) and the R version 3.2.3 packages.

Results

During the period studied, the incidence of combined-resistant and carbapenem-resistant P. aeruginosa strains increased significantly [(gradient = 0.78, R² = 0.707, p = 0.009) (gradient = 0.74, R² = 0.666, p = 0.013) respectively], mirroring the increase in consumption of β-lactam antibiotics with β-lactamase inhibitors (piperacillin/tazobactam) and carbapenems (meropenem) [(gradient = 10.91, R² = 0.698, p = 0.010) and (gradient = 14.63, R² = 0.753, p = 0.005) respectively]. The highest cross-correlation coefficients for zero time lags were found between combined-resistant vs. penicillins consumption and carbapenem-resistant P. aeruginosa strains vs. carbapenems consumption (0.876 and 0.928, respectively). The best model describing the relation between combined-resistant P. aeruginosa strains and penicillins consumption during a given quarter incorporates both the consumption and the incidence of combined-resistant strains in the hospital department during the previous quarter (multiple R² = 0.953, p = 0.017). The best model for explaining the carbapenem resistance of P. aeruginosa strains based on meropenem consumption during a given quarter proved to be the adjusted model which takes into consideration both previous consumption and incidence density of strains during the previous quarter (Multiple R² = 0.921, p = 0.037).

Conclusions

The cross-correlation coefficients and the fitted regression models provide additional evidence that resistance during the a given quarter depends not only on the consumption of antibacterial chemotherapeutic drugs in both that quarter and the previous one, but also on the incidence of resistant strains circulating during the previous quarter.

     

Lactam constraints provide insights into the receptor-bound conformation of secretin and stabilize a receptor antagonist

The natural ligands for family B G protein-coupled receptors are moderate-length linear peptides having diffuse pharmacophores. The amino-terminal regions of these ligands are critical for biological activity, with their amino-terminal truncation leading to production of orthosteric antagonists. The carboxyl-terminal regions of these peptides are thought to occupy a ligand-binding cleft within the disulfide-bonded amino-terminal domains of these receptors, with the peptides in amphipathic helical conformations. In this work, we have characterized the binding and activity of a series of 11 truncated and lactam-constrained secretin(5-27) analogues at the prototypic member of this family, the secretin receptor. One peptide in this series with lactam connecting residues 16 and 20 [c[E(16),K(20)][Y(10)]sec(5-27)] improved the binding affinity of its unconstrained parental peptide 22-fold while retaining the absence of endogenous biological activity and competitive antagonist characteristics. Homology modeling with molecular mechanics and molecular dynamics simulations established that this constrained peptide occupies the ligand-binding cleft in an orientation similar to that of natural full-length secretin and provided insights into why this peptide was more effective than other truncated conformationally constrained peptides in the series. This lactam bridge is believed to stabilize an extended α-helical conformation of this peptide while in solution and not to interfere with critical residue-residue approximations while docked to the receptor.     

Glycosylation genes expressed in seam cells determine complex surface properties and bacterial adhesion to the cuticle of Caenorhabditis elegans

The surface of the nematode Caenorhabditis elegans is poorly understood but critical for its interactions with the environment and with pathogens. We show here that six genes (bus-2, bus-4, and bus-12, together with the previously cloned srf-3, bus-8, and bus-17) encode proteins predicted to act in surface glycosylation, thereby affecting disease susceptibility, locomotory competence, and sexual recognition. Mutations in all six genes cause resistance to the bacterial pathogen Microbacterium nematophilum, and most of these mutations also affect bacterial adhesion and biofilm formation by Yersinia species, demonstrating that both infection and biofilm formation depend on interaction with complex surface carbohydrates. A new bacterial interaction, involving locomotory inhibition by a strain of Bacillus pumilus, reveals diversity in the surface properties of these mutants. Another biological property--contact recognition of hermaphrodites by males during mating--was also found to be impaired in mutants of all six genes. An important common feature is that all are expressed most strongly in seam cells, rather than in the main hypodermal syncytium, indicating that seam cells play the major role in secreting surface coat and consequently in determining environmental interactions. To test for possible redundancies in gene action, the 15 double mutants for this set of genes were constructed and examined, but no synthetic phenotypes were observed. Comparison of the six genes shows that each has distinctive properties, suggesting that they do not act in a linear pathway.