First Identification of Novel NDM Carbapenemase,NDM-7, in Escherichia coli in France

Background

The NDM-1 carbapenemase has been identified in 2008 in Enterobacteriaceae. Since then, several reports have emphasized its rapid dissemination throughout the world. The spread of NDM carbapenemases involve several bla _NDM gene variants associated with various plasmids among several Gram negative species.

Methodology

A multidrug-resistant E. coli isolate recovered from urine of a patient who had travelled to Burma has been characterized genetically and biochemically.

Principal Findings

E. coli COU was resistant to all antibiotics tested except amikacin, tigecycline, fosfomycin, and chloramphenicol. Analysis of the antibiotic resistance traits identified a metallo-ß-lactamase, a novel NDM variant, NDM-7. It differs from NDM-4 by a single amino acid substitution sharing an identical extended spectrum profile towards carbapenems. The bla _NDM-7 gene was located on an untypeable conjugative plasmid and associated with a close genetic background similar to those described among the bla _NDM-1 genes. The isolate also harbours bla _CTXM-15 and bla _OXA-1 genes and belonged to ST167.

Significance

This study highlights that spread of NDM producers correspond to spread of multiple bla _NDM genes and clones and therefore will be difficult to control.     

Do Male and Female Cowbirds See Their World Differently? Implications for Sex Differences in the Sensory System of an Avian Brood Parasite

Background

Male and female avian brood parasites are subject to different selection pressures: males compete for mates but do not provide parental care or territories and only females locate hosts to lay eggs. This sex difference may affect brain architecture in some avian brood parasites, but relatively little is known about their sensory systems and behaviors used to obtain sensory information. Our goal was to study the visual resolution and visual information gathering behavior (i.e., scanning) of brown-headed cowbirds.

Methodology/Principal Findings

We measured the density of single cone photoreceptors, associated with chromatic vision, and double cone photoreceptors, associated with motion detection and achromatic vision. We also measured head movement rates, as indicators of visual information gathering behavior, when exposed to an object. We found that females had significantly lower density of single and double cones than males around the fovea and in the periphery of the retina. Additionally, females had significantly higher head-movement rates than males.

Conclusions

Overall, we suggest that female cowbirds have lower chromatic and achromatic visual resolution than males (without sex differences in visual contrast perception). Females might compensate for the lower visual resolution by gazing alternatively with both foveae in quicker succession than males, increasing their head movement rates. However, other physiological factors may have influenced the behavioral differences observed. Our results bring up relevant questions about the sensory basis of sex differences in behavior. One possibility is that female and male cowbirds differentially allocate costly sensory resources, as a recent study found that females actually have greater auditory resolution than males.     

A Novel Pulse-Chase SILAC Strategy Measures Changes in Protein Decay and Synthesis Rates Induced by Perturbation of Proteostasis with an Hsp90 Inhibitor

Standard proteomics methods allow the relative quantitation of levels of thousands of proteins in two or more samples. While such methods are invaluable for defining the variations in protein concentrations which follow the perturbation of a biological system, they do not offer information on the mechanisms underlying such changes. Expanding on previous work [1], we developed a pulse-chase (pc) variant of SILAC (stable isotope labeling by amino acids in cell culture). pcSILAC can quantitate in one experiment and for two conditions the relative levels of proteins newly synthesized in a given time as well as the relative levels of remaining preexisting proteins. We validated the method studying the drug-mediated inhibition of the Hsp90 molecular chaperone, which is known to lead to increased synthesis of stress response proteins as well as the increased decay of Hsp90 “clients”. We showed that pcSILAC can give information on changes in global cellular proteostasis induced by treatment with the inhibitor, which are normally not captured by standard relative quantitation techniques. Furthermore, we have developed a mathematical model and computational framework that uses pcSILAC data to determine degradation constants k_d and synthesis rates V_s for proteins in both control and drug-treated cells. The results show that Hsp90 inhibition induced a generalized slowdown of protein synthesis and an increase in protein decay. Treatment with the inhibitor also resulted in widespread protein-specific changes in relative synthesis rates, together with variations in protein decay rates. The latter were more restricted to individual proteins or protein families than the variations in synthesis. Our results establish pcSILAC as a viable workflow for the mechanistic dissection of changes in the proteome which follow perturbations. Data are available via ProteomeXchange with identifier PXD000538.     

Chemokine Expression in Inflamed Adipose Tissue Is Mainly Mediated by NF-κB

Immune cell infiltration of expanding adipose tissue during obesity and its role in insulin resistance has been described and involves chemokines. However, studies so far have focused on a single chemokine or its receptor (especially CCL2 and CCL5) whereas redundant functions of chemokines have been described. The objective of this work was to explore the expression of chemokines in inflamed adipose tissue in obesity. Human and mouse adipocytes were analyzed for expression of chemokines in response to inflammatory signal (TNF-α) using microarrays and gene set enrichment analysis. Gene expression was verified by qRT-PCR. Chemokine protein was determined in culture medium with ELISA. Chemokine expression was investigated in human subcutaneous adipose tissue biopsies and mechanism of chemokine expression was investigated using chemical inhibitors and cellular and animal transgenic models. Chemokine encoding genes were the most responsive genes in TNF-α treated human and mouse adipocytes. mRNA and protein of 34 chemokine genes were induced in a dose-dependent manner in the culture system. Furthermore, expression of those chemokines was elevated in human obese adipose tissue. Finally, chemokine expression was reduced by NF-κB inactivation and elevated by NF-κB activation. Our data indicate that besides CCL2 and CCL5, numerous other chemokines such as CCL19 are expressed by adipocytes under obesity-associated chronic inflammation. Their expression is regulated predominantly by NF-κB. Those chemokines could be involved in the initiation of infiltration of leukocytes into obese adipose tissue.     

Kubergandian (Roadian,Middle Permian) of the Lycian and Aladağ Nappes (Southern Turkey)

The Ni?angahtepe Formation (Teke Dere unit) in the Lycian Nappes, and reworked pebbles and blocks from the Late Triassic Gevne Formation in the Alada? unit (both in Southern Turkey), present a well-developed Kubergandian microfauna and microflora found for the first time in this area. The following cyanobacterial and algal taxa are newly established: Clinortonella nov. gen., Ramovsia lyciana nov. sp., and Kantia? guevenci nov. sp.; some Cancellina and Armenina are revised. These Kubergandian faunas and floras present strong palaeobiogeographical affinities with the Eurasian southern margin (northern Palaeotethyan border). The Teke Dere unit represents an accretionary prism/fore-arc sequence containing different parts of the Palaeotethyan succession. The Late Triassic Gevne Formation marks the Eo-Cimmerian unconformity related to the closure of the Palaeotethys between the Taurus and Anatolian terranes.     

His74 conservation in the bilin reductase PcyA family reflects an important role in protein-substrate structure and dynamics

Phycocyanobilin:ferredoxin oxidoreductase (PcyA) catalyzes the proton-coupled four-electron reduction of biliverdin IXα’s two vinyl groups to produce phycocyanobilin, an essential chromophore for phytochromes, cyanobacteriochromes and phycobiliproteins. Previous site directed mutagenesis studies indicated that the fully conserved residue His74 plays a critical role in the H-bonding network that permits proton transfer. Here, we exploit X-ray crystallography, enzymology and molecular dynamics simulations to understand the functional role of this invariant histidine. The structures of the H74A, H74E and H74Q variants of PcyA reveal that a “conserved” buried water molecule that bridges His74 and catalytically essential His88 is not required for activity. Despite distinct conformations of Glu74 and Gln74 in the H74E and H74Q variants, both retain reasonable activity while the H74A variant is inactive, suggesting smaller residues may generate cavities that increase flexibility, thereby reducing enzymatic activity. Molecular dynamic simulations further reveal that the crucial active site residue Asp105 is more dynamic in H74A compared to wild-type PcyA and the two other His74 variants, supporting the conclusion that the Ala74 mutation has increased the flexibility of the active site.     

Autophagy plays a WASHing game

EMBO J (2013) 32: 2685–2696 doi:10.1038/emboj.2013.189; published online August232013Beclin 1 is a crucial regulator of autophagy. It forms a complex with ATG14L, VPS34 or the class III phosphatidylinositol 3-kinase AMBRA1 to control autophagosome formation. A study in this issue of The EMBO Journal (Xia et al, 2013) reports that WASH, a protein known to regulate endosomal sorting, hampers autophagy by inhibiting the AMBRA1-dependent polyubiquitylation of Beclin 1. This modification is required to promote VPS34 activity and to initiate autophagy during starvation.Macroautophagy (hereafter called autophagy) is a lysosomal degradation pathway for cytoplasmic components. Autophagy is initiated by the formation of a double-membrane bound vacuole, the autophagosome that sequesters the autophagic cargo and eventually fuses with the lysosomal compartment, leading to cargo degradation. Autophagy plays a major role in cytoplasm homeostasis by removing protein aggregates and controlling organelle quality, and it is stimulated to promote cell survival during stressful situations, such as nutrient starvation and microbial infection. Autophagosome formation is dependent on evolutionarily conserved Atg (Autophagy-related) proteins initially identified in yeast (Mizushima et al, 2011). They function in complexes or functional modules on a membrane known as the phagophore that matures into the autophagosome via several stages (initiation, elongation and sealing). Phosphatidylinositol 3-phosphate kinase complex I (PI3K complex I) plays a key role in the initiation step. In this complex, Beclin 1 (the mammalian homologue of yeast Atg6) interacts with ATG14L, AMBRA1, and class III PI3K or VPS34 (Cecconi and Levine, 2008; Figure 1). The activity of this complex, which produces the lipid phosphatidylinositol 3-phosphate (PI3P) to recruit Atg18 homologues (WIPIs), has to be tightly regulated to keep autophagy under control.Open in a separate windowFigure 1Role of WASH in autophagosome formation. WASH interacts with Beclin 1 via a sequence (121–221) that is not involved in endosomal sorting. In the absence of WASH, when autophagy is stimulated by nutrient deprivation, Beclin 1 interacts with VPS34 (and its adaptor VPS15), ATG14L and AMBRA1. In this complex, Beclin 1 is polyubiquitylated by AMBRA1, which acts as an E3 ligase. The activation of VPS34 produces PI3P at the phagophore to recruit WIPI proteins, and then triggers the machinery to elongate and seal the membrane, thus forming an autophagosome. The VAC domain of WASH involved in endosomal sorting is not required for autophagy regulation.In this issue of The EMBO Journal, Xia et al (2013) demonstrate that WASH (Wiskott-Aldrich syndrome protein (WASP) and SCAR homologue) regulates autophagosome formation in response to nutrient starvation by influencing the ubiquitylation of Beclin 1. WASH forms a complex with four other proteins and plays an essential role in endosomal sorting by promoting actin polymerization to facilitate segregation of endosomal proteins (Seaman et al, 2013). Using WASH−/− mice, Xia et al (2013) observe that WASH deficiency causes embryonic lethality (E7.5−E9.5) with massive apoptosis-independent cell death and the accumulation of autophagic structures. However, whether autophagy is instrumental in the cell death observed in WASH−/− embryos remains to be investigated. From a series of classical readouts, the authors conclude that WASH downregulates starvation-induced autophagy. Interestingly, WASH is detected on the autophagosomal membrane before and after closure of the autophagosome, but not on the autolysosomes (organelles formed when the autophagosome fuses with the lysosome). The authors first show that WASH has distinct roles in autophagy and in endosomal sorting, because deletion of the VCA domain of WASH that is required for its endosomal function and the knockdown of FAM21 (a protein that directs WASH to the endosomal membrane) do not influence autophagy. They then show that WASH interacts with the coil-coiled domain of Beclin 1. WASH depletion induces more VPS34 to interact with Beclin 1, leading to augmented formation of PtdIns3P and increased recruitment of WIPI-1 to the autophagosomal membrane. However, WASH does not influence the stability of Beclin 1, although it does regulate its K63-linked polyubiquitylation at position K437 during starvation (K63-linked polyubiquitylation suggests a regulatory role, whereas K48-linked polyubiquitylation is frequently associated with protein degradation). Overexpression of WASH reduces the ubiquitylation of Beclin 1, and the K437R Beclin 1 mutant that cannot be ubiquitylated has a low level of interaction with VPS34, which prevents the stimulation of autophagy by starvation (Figure 1). Recently, Beclin 1 has been shown to be ubiquitylated at position K117 during TLR4-induced autophagy (Shi and Kehrl, 2010). Interestingly, Xia et al (2013) show that the K117R Beclin 1 mutant is still ubiquitylated in response to nutrient starvation. Finally, the authors identify the substrate-specific E3 ubiquitin ligase for Beclin 1 in this context. They exclude TRAF6 and NEDD4, two E3 ligases reported to ubiquitylate Beclin 1 (Kuang et al, 2013). AMBRA1, which regulates autophagy in the PI3K complex I, is also known as DCAF3 and interacts with the Cullin 4–DDB1 E3 ligase complex (Jin et al, 2006). Following in vitro E3 ligase assays, Xia et al (2013) conclude that AMBRA1 is the substrate receptor for Beclin 1 ubiquitylation at position K437, adding Cullin4/DDB1 and AMBRA1 to the list of E3 ligases involved in autophagy (Kuang et al, 2013). The finding that WASH and AMBRA1 compete to regulate starvation-induced autophagy raises several questions: how do WASH and AMBRA1 influence one another''s binding to Beclin 1? WASH is evolutionarily conserved, although it is not present in yeast (Linardopoulou et al, 2007)—does it also reduce starvation-induced autophagy in non-mammalian cells? WASH has recently been reported to be required for the lysosomal digestion of autophagy cargo in Dictyostelium during starvation (King et al, 2013). Does this mean that the function of WASH in autophagy has evolved from controlling cargo degradation to forming autophagosomes? Xia et al (2013) do not report any association between WASH and the lysosomal compartment during autophagy; the role of WASH in autophagosome maturation calls for further investigation. Finally, a recent report demonstrates that AMBRA1 interacts with the E3 ligase TRAF6 to ubiquitylate ULK1 (the mammalian orthologue of the yeast Atg1) and to ensure its subsequent stabilization and function (Nazio et al, 2013). ULK1 is part of a complex that acts with PI3K complex 1 to regulate the initiation of autophagy (Mizushima et al, 2011). How are these two AMBRA1-dependent ubiquitylation processes coordinated to control autophagy? In conclusion, both the study reported here (Xia et al, 2013) and the recent study of Nazio et al (2013) clearly demonstrate that AMBRA1 plays a central role in regulating the initiation of autophagy.     

Biofilms on tracheoesophageal voice prostheses: a confocal laser scanning microscopy demonstration of mixed bacterial and yeast biofilms

The aim of this study was to demonstrate the presence of yeast and bacterial biofilms on the surface of tracheoesophageal voice prostheses (TVPs) by a double-staining technique with confocal laser scanning microscopy (CLSM). Biofilms of 12 removed TVPs were visualized by scanning electron microscopy, then stained with ConA-FITC and propidium iodide for CLSM. Microbial identification was by partial 16S rRNA gene analysis and ITS-2 sequence analysis. Microbial biofilms on the TVPs consisted of bacteria and filamentous cells. Bacterial cells were attached to the filamentous and unicellular yeast cells, thus forming a network. Sequence analyses of six voice prostheses identified the presence of a variety of bacterial and yeast species. In vivo studies showed that Klebsiella oxytoca and Micrococcus luteus efficiently attached to Candida albicans. CLSM with double fluorescence staining can be used to demonstrate biofilm formations composed of a mixture of yeast and bacterial cells on the surface of TVPs.     

The functional vanGCd cluster of Clostridium difficile does not confer vancomycin resistance

vanG_Cd, a cryptic gene cluster highly homologous to the vanG gene cluster of Enterococcus faecalis is largely spread in Clostridium difficile. Since emergence of vancomycin resistance would have dramatic clinical consequences, we have evaluated the capacity of the vanG_Cd cluster to confer resistance. We showed that expression of vanG_Cd is inducible by vancomycin and that VanG_Cd, VanXY_Cd and VanT_Cd are functional, exhibiting D‐Ala : D‐Ser ligase, D,D‐dipeptidase and D‐Ser racemase activities respectively. In other bacteria, these enzymes are sufficient to promote vancomycin resistance. Trans‐complementation of C. difficile with the vanC resistance operon of Enterococcus gallinarum faintly impacted the MIC of vancomycin, but did not promote vancomycin resistance in C. difficile. Sublethal concentration of vancomycin led to production of UDP‐MurNAc‐pentapeptide[D‐Ser], suggesting that the vanG_Cd gene cluster is able to modify the peptidoglycan precursors. Our results indicated amidation of UDP‐MurNAc‐tetrapeptide, UDP‐MurNAc‐pentapeptide[D‐Ala] and UDP‐MurNAc‐pentapeptide[D‐Ser]. This modification is passed on the mature peptidoglycan where a muropeptide Tetra‐Tetra is amidated on the meso‐diaminopimelic acid. Taken together, our results suggest that the vanG_Cd gene cluster is functional and is prevented from promoting vancomycin resistance in C. difficile.     

Analysis of humeral head displacements from sequences of biplanar X-rays: repeatability study and preliminary results in healthy subjects

This work presents an accurate method to measure gleno-humeral translations in a controlled pseudo-kinematic environment. Low-dose biplanar X-rays were acquired from nine healthy subjects at three elevations of the arm in the scapular plane. On each set of images, shoulder bony landmarks were manually located in 3D using a dedicated software. Intra-observer and inter-observer repeatability of landmark identification, as well as humeral head center (GH) translations, were studied. Repeatability for the identification of GH in the global coordinate system (CS) was good with 95% confidence intervals (CIs) ranging from 0.57 to 2.25 mm. Scapular landmark CIs ranged from 0.80 to 12 mm. Gleno-humeral translations of small amplitude ( < 6 mm) were detected in seven out of nine subjects. The results obtained here confirm that calibrated low-dose stereo-radiography is a promising tool for the functional analysis of the shoulder.