Characterization of Two New CTX-M-25-Group Extended-Spectrum β-Lactamase Variants Identified in Escherichia coli Isolates from Israel

Objectives

We characterized two new CTX-M-type extended-spectrum β-lactamase (ESBL) variants in Escherichia coli isolates from stool samples of two elderly patients admitted at the Tel Aviv Sourasky Medical Center, Israel. Both patients underwent treatment with cephalosporins prior to isolation of the E. coli strains.

Methods

ESBLs were detected by the double-disk synergy test and PCR-sequencing of β-lactamase genes. The bla _CTX-M genes were cloned into the pCR-BluntII-TOPO vector in E. coli TOP10. The role of amino-acid substitutions V77A and D240G was analyzed by site-directed mutagenesis of the bla _CTX-M-94 and bla _CTX-M-100 genes and comparative characterization of the resulting E. coli recombinants. MICs of β-lactams were determined by Etest. Plasmid profiling, mating experiments, replicon typing and sequencing of bla _CTX-M flanking regions were performed to identify the genetic background of the new CTX-M variants.

Results

The novel CTX-M β-lactamases, CTX-M-94 and -100, belonged to the CTX-M-25-group. Both variants differed from CTX-M-25 by the substitution V77A, and from CTX-M-39 by D240G. CTX-M-94 differed from all CTX-M-25-group enzymes by the substitution F119L. Glycine-240 was associated with reduced susceptibility to ceftazidime and leucine-119 with increased resistance to ceftriaxone. bla _CTX-M-94 and bla _CTX-M-100 were located within ISEcp1 transposition units inserted into ∼93 kb non-conjugative IncFI and ∼130 kb conjugative IncA/C plasmids, respectively. The plasmids carried also different class 1 integrons.

Conclusions

This is the first report on CTX-M-94 and -100 ESBLs, novel members of the CTX-M-25-group.     

The Drosophila blood brain barrier is maintained by GPCR-dependent dynamic actin structures

The blood brain barrier (BBB) is essential for insulation of the nervous system from the surrounding environment. In Drosophila melanogaster, the BBB is maintained by septate junctions formed between subperineurial glia (SPG) and requires the Moody/G protein-coupled receptor (GPCR) signaling pathway. In this study, we describe novel specialized actin-rich structures (ARSs) that dynamically form along the lateral borders of the SPG cells. ARS formation and association with nonmuscle myosin is regulated by Moody/GPCR signaling and requires myosin activation. Consistently, an overlap between ARS localization, elevated Ca(2+) levels, and myosin light chain phosphorylation is detected. Disruption of the ARS by inhibition of the actin regulator Arp2/3 complex leads to abrogation of the BBB. Our results suggest a mechanism by which the Drosophila BBB is maintained by Moody/GPCR-dependent formation of ARSs, which is supported by myosin activation. The localization of the ARSs close to the septate junctions enables efficient sealing of membrane gaps formed during nerve cord growth.     

The structural flexibility of the human copper chaperone Atox1: Insights from combined pulsed EPR studies and computations

Metallochaperones are responsible for shuttling metal ions to target proteins. Thus, a metallochaperone's structure must be sufficiently flexible both to hold onto its ion while traversing the cytoplasm and to transfer the ion to or from a partner protein. Here, we sought to shed light on the structure of Atox1, a metallochaperone involved in the human copper regulation system. Atox1 shuttles copper ions from the main copper transporter, Ctr1, to the ATP7b transporter in the Golgi apparatus. Conventional biophysical tools such as X‐ray or NMR cannot always target the various conformational states of metallochaperones, owing to a requirement for crystallography or low sensitivity and resolution. Electron paramagnetic resonance (EPR) spectroscopy has recently emerged as a powerful tool for resolving biological reactions and mechanisms in solution. When coupled with computational methods, EPR with site‐directed spin labeling and nanoscale distance measurements can provide structural information on a protein or protein complex in solution. We use these methods to show that Atox1 can accommodate at least four different conformations in the apo state (unbound to copper), and two different conformations in the holo state (bound to copper). We also demonstrate that the structure of Atox1 in the holo form is more compact than in the apo form. Our data provide insight regarding the structural mechanisms through which Atox1 can fulfill its dual role of copper binding and transfer.     

Ric-8B is a GTP-dependent G protein alphas guanine nucleotide exchange factor

ric-8 (resistance to inhibitors of cholinesterase 8) genes have positive roles in variegated G protein signaling pathways, including Gα(q) and Gα(s) regulation of neurotransmission, Gα(i)-dependent mitotic spindle positioning during (asymmetric) cell division, and Gα(olf)-dependent odorant receptor signaling. Mammalian Ric-8 activities are partitioned between two genes, ric-8A and ric-8B. Ric-8A is a guanine nucleotide exchange factor (GEF) for Gα(i)/α(q)/α(12/13) subunits. Ric-8B potentiated G(s) signaling presumably as a Gα(s)-class GEF activator, but no demonstration has shown Ric-8B GEF activity. Here, two Ric-8B isoforms were purified and found to be Gα subunit GDP release factor/GEFs. In HeLa cells, full-length Ric-8B (Ric-8BFL) bound endogenously expressed Gα(s) and lesser amounts of Gα(q) and Gα(13). Ric-8BFL stimulated guanosine 5'-3-O-(thio)triphosphate (GTPγS) binding to these subunits and Gα(olf), whereas the Ric-8BΔ9 isoform stimulated Gα(s short) GTPγS binding only. Michaelis-Menten experiments showed that Ric-8BFL elevated the V(max) of Gα(s) steady state GTP hydrolysis and the apparent K(m) values of GTP binding to Gα(s) from ～385 nm to an estimated value of ～42 μM. Directionality of the Ric-8BFL-catalyzed Gα(s) exchange reaction was GTP-dependent. At sub-K(m) GTP, Ric-BFL was inhibitory to exchange despite being a rapid GDP release accelerator. Ric-8BFL binds nucleotide-free Gα(s) tightly, and near-K(m) GTP levels were required to dissociate the Ric-8B·Gα nucleotide-free intermediate to release free Ric-8B and Gα-GTP. Ric-8BFL-catalyzed nucleotide exchange probably proceeds in the forward direction to produce Gα-GTP in cells.     

Association of Genes,Pathways, and Haplogroups of the Mitochondrial Genome with the Risk of Colorectal Cancer: The Multiethnic Cohort

The mitochondrial genome encodes for the synthesis of 13 proteins that are essential for the oxidative phosphorylation (OXPHOS) system. Inherited variation in mitochondrial genes may influence cancer development through changes in mitochondrial proteins, altering the OXPHOS process, and promoting the production of reactive oxidative species. To investigate the role of the OXPHOS pathway and mitochondrial genes in colorectal cancer (CRC) risk, we tested 185 mitochondrial SNPs (mtSNPs), located in 13 genes that comprise four complexes of the OXPHOS pathway and mtSNP groupings for rRNA and tRNA, in 2,453 colorectal cancer cases and 11,930 controls from the Multiethnic Cohort Study. Using the sequence kernel association test, we examined the collective set of 185 mtSNPs, as well as subsets of mtSNPs grouped by mitochondrial pathways, complexes, and genes, adjusting for age, sex, principal components of global ancestry, and self-reported maternal race/ethnicity. We also tested for haplogroup associations using unconditional logistic regression, adjusting for the same covariates. Stratified analyses were conducted by self-reported maternal race/ethnicity. In European Americans, a global test of all genetic variants of the mitochondrial genome identified an association with CRC risk (P = 0.04). In mtSNP-subset analysis, the NADH dehydrogenase 2 (MT-ND2) gene in Complex I was associated with CRC risk at a P-value of 0.001 (q = 0.015). In addition, haplogroup T was associated with CRC risk (OR = 1.66, 95% CI: 1.19–2.33, P = 0.003). No significant mitochondrial pathway and gene associations were observed in the remaining four racial/ethnic groups—African Americans, Asian Americans, Latinos, and Native Hawaiians. In summary, our findings suggest that variations in the mitochondrial genome and particularly in the MT-ND2 gene may play a role in CRC risk among European Americans, but not in other maternal racial/ethnic groups. Further replication is warranted and future studies should evaluate the contribution of mitochondrial proteins encoded by both the nuclear and mitochondrial genomes to CRC risk.     

HSP60 is transported through the secretory pathway of 3-MCA-induced fibrosarcoma tumour cells and undergoes N-glycosylation

There is increasing evidence localizes the mitochondrial chaperone heat shock protein (HSP)60, outside the cell, where it mediates interactions between immune cells and other body tissues. However, the mechanisms by which HSP60 is secreted into the extracellular environment are not fully understood. Recent studies have shown that HSP60 is actively released by a nonconventional secretion mechanism, the lipid raft-exosome pathway. In the present study, we show for the first time that HSP60, produced by 3-methylcholantrene-induced fibrosarcoma tumour cells, is secreted through the conventional endoplasmic reticulum-Golgi secretory pathway. Confocal microscopy using anti-TGN38 and anti-HSP60 antibodies together with monensin, a Golgi transport inhibitor, demonstrated the relocation of HSP60 to the Golgi of malignant cells but not primary fibroblast cells subjected to heat shock or fibroblast cell lines. Transmission electron microscopy, flow cytometry and cell fractionation of cell treated with brefeldin A, an inhibitor of endoplasmic reticulum to Golgi protein transport, further indicated that HSP60 is present both in the endoplasmic reticulum and the Golgi complex of malignant cells. We found a single mRNA with a mitochondrial targeting sequence encoding for HSP60 in the malignant cells but two HSP60 translation products, namely the native unmodified protein and a protein post-translationally modified by N-glycosylation. The N-glycans observed were composed of high-mannose structures and bi-, tri- and tetra-antennary complex type structures occupying sites of the three potential glycosylation sites present on HSP60. Accordingly, we propose that HSP60 in malignant cells is transported through the endoplasmic reticulum-Golgi secretion pathway, where it acquires N-glycans, and thus can affect the immunological properties of the proteins in the tumour microenvironment.     

Serum apolipoproteins C-I and C-III are reduced in stomach cancer patients: results from MALDI-based peptidome and immuno-based clinical assays

Finding new peptide biomarkers for stomach cancer in human sera that can be implemented into a clinically practicable prediction method for monitoring of stomach cancer. We studied the serum peptidome from two different biorepositories. We first employed a C8-reverse phase liquid chromatography approach for sample purification, followed by mass-spectrometry analysis. These were applied onto serum samples from cancer-free controls and stomach cancer patients at various clinical stages. We then created a bioinformatics analysis pipeline and identified peptide signature discriminating stomach adenocarcinoma patients from cancer-free controls. Matrix Assisted Laser Desorption/Ionization-Time of Flight (MALDI-TOF) results from 103 samples revealed 9 signature peptides; with prediction accuracy of 89% in the training set and 88% in the validation set. Three of the discriminating peptides discovered were fragments of Apolipoproteins C-I and C-III (apoC-I and C-III); we further quantified their serum levels, as well as CA19-9 and CRP, employing quantitative commercial-clinical assays in 142 samples. ApoC-I and apoC-III quantitative results correlated with the MS results. We then employed apoB-100-normalized apoC-I and apoC-III, CA19-9 and CRP levels to generate rules set for stomach cancer prediction. For training, we used sera from one repository, and for validation, we used sera from the second repository. Prediction accuracies of 88.4% and 74.4% were obtained in the training and validation sets, respectively. Serum levels of apoC-I and apoC-III combined with other clinical parameters can serve as a basis for the formulation of a diagnostic score for stomach cancer patients.     

Designed aromatic homo-dipeptides: formation of ordered nanostructures and potential nanotechnological applications

Molecular self-assembly offers new routes for the fabrication of novel materials at the nano-scale. Peptide-based nanostructures represent nano-objects of particular interest, as they are biocompatible, can be easily synthesized in large amounts, can be decorated with functional elements and can be used in various biological and non-biological applications. We had previously revealed the formation of highly ordered tubular structures by the diphenylalanine peptide, the core recognition motif of Alzheimer's beta-amyloid polypeptide, due to specific aromatic interactions. We further confirmed this model and demonstrated that a non-charged peptide analogue, Ac-Phe-Phe-NH2, self-assembled into similar tubular structures. We later explored other amine and carboxyl modified diphenylalanine peptide analogues and revealed that these dipeptides can form ordered tubular structures at the nanometric scale. Moreover, a very similar peptide, the diphenylglycine, self-assembled into ordered nano-spherical assemblies. Here we extend our research and explore the self-assembly of other homo-aromatic dipeptides in which their phenyl side-chains are modified with halogen atoms (di-para-fluoro-Phe, di-pentafluoro-Phe, di-para-iodo-Phe), additional phenyl groups (di-4-phenyl-Phe), or with nitro substitutions (di-para-nitro-Phe). We also probed the effect of the alteration of the phenyl groups with naphtyl groups (di-D-1-Nal and di-D-2-Nal). In all cases, well-ordered nanostructures were obtained and studied by scanning electron microscopy, transmission electron microscopy and vibrational spectroscopy. Taken together, the current work and previous ones define the homo-aromatic dipeptide as a central motif for the formation of ordered self-assembled tubular, spherical and two-dimensional structures at the nano-scale.     

Effect of desiccation on mosquito oviposition site selection in Mediterranean temporary habitats

1. Hydroperiod duration has been identified as the main factor determining the faunal composition and structure of aquatic communities in temporary habitats. It is hypothesised that desiccation will positively affect mosquito oviposition habitat selection during the post-drought period due to the lack of antagonists. 2. An experiment was carried out in outdoor mesocosms to assess whether desiccation events have post-drought effects on community richness and mosquito oviposition. Three different treatments were randomly assigned to the mesocosms: (i) eight mesocosms were left dried for a week and then reflooded; (ii) eight mesocosms stayed dry for 4 weeks before they were reflooded; (iii) eight mesocosms were maintained at a constant water volume of 30 litres during the entire experiment as controls. Mosquito oviposition and invertebrate community richness were monitored in every mesocosm, along with environmental parameters [water temperature, pH, conductivity, dissolved oxygen, total suspended solids (TSS) and chlorophyll a concentration]. 3. Post-drought mosquito oviposition and larval abundance were higher in the short-drought and long-drought pools than in the control. Desiccation negatively affected the biomass of the filter feeder invertebrates in both desiccation treatments. Chlorophyll a concentrations were higher in the long-drought pools than in controls. The negative impact of desiccation on zooplankton led to a post-drought increase in algae, associated with an increase in mosquito oviposition. 4. Despite immediate negative effect on mosquitoes, pulsed disturbances can benefit mosquitoes as they favour oviposition during the post-disturbance recovery period due to a lower abundance of mosquito antagonists and higher food resources for their offspring.