Molecular characterization of plasmids encoding blaCTX-M from faecal Escherichia coli in travellers returning to the UK from South Asia.

BACKGROUND: The global prevalence of extended-spectrum beta-lactamase-producing Escherichia coli is rising and is dominated by blaCTX-M spread by plasmids. Travellers to South Asia from Western Europe have high rates of acquisition of faecal CTX-M-producing E. coli (CTX-M-EC). AIMS: To determine the conjugative ability of CTX-M-EC acquired by healthy volunteers after travel to South Asia, the proportion of travel-acquired CTX-M-EC where blaCTX-M is encoded on a plasmid vs on the bacterial chromosome, and the relatedness of travel-acquired CTX-M-EC plasmids to previously sequenced plasmids. METHODS: Faecal samples were collected pre- and post-travel from 23 volunteers who visited South Asia, and CTX-M-EC were cultured. After short- and long-read sequencing, 10 plasmid sequences were identified and compared with previously sequenced plasmids in GenBank. Conjugation to E. coli K-12 was undertaken using filter mating. FINDINGS: Thirty-five percent of CTX-M-EC isolates tested transferred the blaCTX-M plasmid by conjugation. Travel-acquired CTX-M-EC carried blaCTX-M on a plasmid in 62% of isolates, whereas 38% of isolates had blaCTX-M on the chromosome. CTX-M-EC plasmids acquired after travel to South Asia had close homology to previously described epidemic plasmids which are widely disseminated in humans, animals and the natural environment. CONCLUSION: Globally successful epidemic plasmids are involved in the spread of CTX-M-EC. Targeted strategies may be used to displace such plasmids from the host strain as part of efforts in infection prevention and control in healthcare settings. Bacteria with blaCTX-M plasmids were readily acquired by healthy volunteers, and were carried on return to the UK, providing opportunities for onward dissemination.

A systematic review and meta-analysis of tenderness metrics in control groups used in comparative nutrition experiments.

This review reports the pork quality attributes, Warner-Bratzler Shear Force, Slice Shear Force, Star Probe, pH, marbling, color (Minolta L*/L or Hunter L*/L), and sensory tenderness evaluation, in control groups used in comparative nutrition experiments over the past 20 yr. The original aim of this study was to evaluate if changes in pork quality based on the above metrics occurred over time. To address this question, it was anticipated that data may come from 3 sources with decreasing relevance: representative retail pork surveys, representative post-harvest carcass surveys, and control groups from comparative nutrition experiments. To identify the study population, a review of studies reported in Centre for Agricultural Biosciences International Abstracts (Web of Knowledge; 1994-2014) was conducted. Two national level surveys of retail pork and 146 relevant nutritional experiments studies, with 228 control groups, were identified by the search. It was not possible to conduct a meta-analysis of the retail pork surveys based on only 2 time points. For the comparative studies, a random effects meta-analysis was conducted with year as a covariate to assess the impact of time on the outcome. In the absence of modifiers, there was no evidence of meaningful change in the mean Warner-Bratzler Shear Force, pH, color, marbling, or sensory scores over the study period. There was evidence of substantial between-study heterogeneity in the characteristics of control pigs used over the years for Warner-Bratzler Shear Force and measures of color. The absence of publicly-available representative surveys of pork quality meant the changes in pork quality over time were not clear. If changes in pork quality have occurred, the data suggest that pigs used as controls in experiments may have become less representative of commercial pigs over time and the translatability of study findings from nutrition experiments might be reduced over time. Alternately, if commercial pigs have not changed, then control pigs reflect this. The study does not address if control groups in other experimental intervention studies had similar tenderness patterns as reported here for nutritional interventions. A large amount of potentially available data was excluded from the analysis due to incomplete reporting in the original study reports.

Direct and continuous synthesis of VO2 nanoparticles.

Monoclinic VO2 nanoparticles are of interest due to the material's thermochromic properties, however, direct synthesis routes to VO2 nanoparticles are often inaccessible due to the high synthesis temperatures or long reaction times required. Herein, we present a two-step synthesis route for the preparation of monoclinic VO2 nanoparticles using Continuous Hydrothermal Flow Synthesis (CHFS) followed by a short post heat treatment step. A range of particle sizes, dependent on synthesis conditions, were produced from 50 to 200 nm by varying reaction temperatures and the residence times in the process. The nanoparticles were characterised by powder X-ray diffraction, Raman and UV/Vis spectroscopy, transmission electron microscopy (TEM), scanning electron microscopy (SEM) and differential scanning calorimetry (DSC). The nanoparticles were highly crystalline with rod and sphere-like morphologies present in TEM micrographs, with the size of both the rod and spherical particles being highly dependent on both reaction temperature and residence time. SEM micrographs showed the surface of the powders produced from the CHFS process to be highly uniform. The samples were given a short post synthesis heat treatment to ensure that they were phase pure monoclinic VO2, which led to them exhibiting a large and reversible switch in optical properties (at near-IR wavelengths), which suggests that if such materials can be incorporated into coatings or in composites, they could be used for fenestration in architectural applications.

Preparation and testing of diabetes autoantibody controls.

Stocks of the WHO islet cell antibody, GAD(65) antibody, and IA-2 antibody standard (NIBSC 97/550) are now very limited. We have therefore made and tested a series of control preparations in which human monoclonal autoantibodies to IA-2 and to GAD(65) were diluted in antibody-negative human serum to different concentrations. Three different diabetes autoantibody controls (DAC 1-3) were made as was a negative control preparation. Aliquots containing 1 mL of autoantibodies in serum were freeze-dried. After reconstitution (with 1 mL of water) the controls were tested by (125)I-IA-2 immunoprecipitation assay (IPA), (125)I-GAD(65) IPA, GAD(65) Ab ELISA and IA-2 Ab ELISA (kits from RSR Ltd.) and for ICA by immunofluorescence test (IFT). DAC1 is particularly suitable as a control for the (125)I IA-2 IPA; DAC2 is suitable for the (125)I-GAD(65) IPA, GAD(65) Ab ELISA, and IA-2 Ab ELISA; and DAC3 is suitable for the ICA IFT. Freeze-dried preparations showed good stability at 37 degrees C. Reconstituted liquid preparations were stable when stored at 4 degrees C and at 37 degrees C. Availability of an essentially unlimited supply of these reagents should be useful in establishing reproducible and comparable measurements of diabetes autoantibodies in different laboratories using different assays.

Challenges in research with incarcerated parents and importance in violence prevention.

Incarcerated parents present several risk factors for later violence by their children. This study uses comparison groups and repeated measures to evaluate an inmate parenting program. Subjects are inmates at a county detention center, their children, and primary caregivers. Challenges to program implementation and longitudinal research with inmates were identified, along with recommendations to assist future research and programming. Training material should use illustrated, basic language format. Acceptance and participation by inmates and staff require ongoing outreach and communication. Severed relationships are common and future research on inmates with stable family relationships is recommended. Because of inmate transience, integrating parent training into post-release programming is suggested.

IL-10 gene expression is controlled by the transcription factors Sp1 and Sp3.

IL-10 is an 18-kDa cytokine with a key role in homeostatic control of inflammatory and immune responses. We have investigated how transcription of the IL-10 gene is regulated, so as to be able to understand the circumstances of IL-10 expression in both health and disease. In the mouse, IL-10 gene expression is regulated by a TATA-type promoter with a critical cis-acting element containing GGA repeats located at -89 to -77. Its complementary sequence is similar to the cis-acting elements (TCC repeats) in the promoters of genes encoding epidermal growth factor receptor and CD58. All these elements comprise a common CCTCCT sequence with less conserved C + T-rich sequences. Eliminating this CCTCCT sequence results in a marked reduction in promoter activity, suggesting a necessary role in IL-10 gene expression. Despite its dissimilarity to the G + C-rich Sp1 consensus sequence (GC box), Sp1 and Sp3 transcription factors could be shown to bind to this motif. The requirement for Sp1 and Sp3 in transcription of IL-10 was confirmed using Drosophila SL2 cells, which lack endogenous Sp factors. These results suggest that the transcription of IL-10 is positively regulated by both Sp1 and Sp3.

Posttranscriptional regulation of IL-10 gene expression through sequences in the 3'-untranslated region.

IL-10 is an 18-kDa immunoregulatory cytokine the transcription of which is controlled by the ubiquitously expressed transcription factors Sp1 and Sp3. Although many cell types express IL-10 mRNA, not all make detectable amounts of protein, and levels of protein expression vary enormously. We show here that much of this variation can be accounted for by posttranscriptional mechanisms. Multiple copies of potential mRNA destabilizing motifs AUUUA and related sequences can be found to the 3'-untranslated region (UTR) of IL-10 mRNA distributed through three potential regulatory regions. Evidence of RNA-destabilizing activities in all three regions was deduced from luciferase reporter assays. The half-life of RNA containing the 3'-UTR of IL-10 mRNA was quite short in both nonstimulated (t1/2 = 1 h), and PMA-stimulated EL-4 cell (t1/2 = 3 h). In contrast, the half-life of RNA lacking the 3'-UTR was much longer (t1/2 = >12 h) whether cells were stimulated or not. This suggests that many cells are poised to secrete IL-10 and will do so if they receive appropriate posttranscriptional signals.

Naturally occurring dicistronic cricket paralysis virus RNA is regulated by two internal ribosome entry sites.

Cricket paralysis virus is a member of a group of insect picorna-like viruses. Cloning and sequencing of the single plus-strand RNA genome revealed the presence of two nonoverlapping open reading frames, ORF1 and ORF2, that encode the nonstructural and structural proteins, respectively. We show that each ORF is preceded by one internal ribosome entry site (IRES). The intergenic IRES is located 6,024 nucleotides from the 5' end of the viral RNA and is more active than the IRES located at the 5' end of the RNA, providing a mechanistic explanation for the increased abundance of structural proteins relative to nonstructural proteins in infected cells. Mutational analysis of this intergenic-region IRES revealed that ORF2 begins with a noncognate CCU triplet. Complementarity of this CCU triplet with sequences in the IRES is important for IRES function, pointing to an involvement of RNA-RNA interactions in translation initiation. Thus, the cricket paralysis virus genome is an example of a naturally occurring, functionally dicistronic eukaryotic mRNA whose translation is controlled by two IRES elements located at the 5' end and in the middle of the mRNA. This finding argues that eukaryotic mRNAs can express multiple proteins not only by polyprotein processing, reinitiation and frameshifting but also by using multiple IRES elements.

Differential fates of invertase mutants in the yeast endoplasmic reticulum.

A number of proteins have been identified as substrates for endoplasmic reticulum (ER)-associated protein degradation (ERAD) and we describe here a new model substrate with which to study this process. Two secretion-defective forms of yeast invertase that accumulated in the ER to greatly different levels were examined: Suc2-538p levels were low, while Suc2-533p was present in high amounts. Because Suc2-533p and Suc2-538p mRNA levels were comparable, we examined whether Suc2-538p was targeted for degradation. Both mutant polypeptide levels were unaffected in a yeast strain deficient in vacuolar protease activity and, additionally, we showed that Suc2-538p was stabilized in ERAD-deficient strains, demonstrating that Suc2-538p was a substrate for ERAD.

Effect of heat pretreatment on the yield and composition of oil extracted from corn fiber.

Previously, hexane extraction of corn fiber was reported to produce a unique and potentially valuable oil that contained high levels of several phytosterols (which have been noted for their cholesterol-lowering properties). Current studies revealed that heat treatment (over the range of 100-175 degrees C) of corn fiber in either a convection oven or a vacuum oven caused only a modest reduction in the levels of the phytosterol components. However, these same heat pretreatments caused a considerable increase (up to 10-fold) in the levels (increasing from 0.34 wt % to a maximum of 3.64 wt % gamma-tocopherol in the oil) and yields (increasing from 5.4 mg of gamma-tocopherol/100 g of corn fiber to a maximum of 52.1 mg of gamma-tocopherol/100 g of corn fiber) of gamma-tocopherol in corn fiber oil. The main differences between the convection oven and vacuum oven pretreatments were associated with the disappearance of free fatty acids and free phytosterols at the higher temperature pretreatments in the vacuum oven, probably due to the lower boiling points of these lipids. Microwave pretreatment was also effective but caused a much smaller increase in the levels of gamma-tocopherol.

VH4-34 encoded antibodies in systemic lupus erythematosus: a specific diagnostic marker that correlates with clinical disease characteristics.

OBJECTIVE: To determine the clinical significance of elevated serum levels of VH4-34 encoded antibodies (VH4-34 Ab) with respect to the diagnosis and clinical characteristics of systemic lupus erythematosus (SLE). METHODS: Ninety-five patients with SLE and 344 controls were studied. The controls included 34 healthy individuals, 282 patients with nonautoimmune diseases, and 28 patients with autoimmune diseases other than SLE. VH4-34 Ab levels were measured by inhibition ELISA using anti-idiotope monoclonal antibody (9G4). SLE disease activity, severity, and damage were assessed by visual analog scales, Systemic Lupus Activity Measure, Lupus Severity of Disease Index, and Systemic Lupus International Collaborating Clinics/American College of Rheumatology Damage Index. RESULTS: Fifty-two of 95 patients with SLE had elevated levels of VH4-34 Ab compared to 18 of 344 controls (5%), giving a sensitivity of 55% and a specificity of 95% for elevated VH4-34 Ab as a serologic test for SLE. The positive predictive value of elevated VH4-34 under these conditions was 74-85%. In this study, anti-dsDNA was not VH4-34 encoded. Significant correlations between VH4-34 and disease activity and severity indices were observed (r = 0.29-0.50). The relative risk for severe disease in SLE patients with VH4-34 antibody level in the highest tertile compared to the lowest tertile was 5.25. Twenty-five of 29 patients with lupus nephritis and 6 of 6 patients with central nervous system (CNS) lupus had elevated VH4-34 Ab. CONCLUSION: With a specificity of 94-95%, the VH4-34 antibody assay may prove valuable as a confirmatory diagnostic test for SLE. In patients with known SLE, serum VH4-34 Ab levels correlate with overall disease severity and activity, but not damage, and with nephritis and CNS lupus.

Mechanism of the alpha-complementation reaction of E. coli beta-galactosidase deduced from fluorescence correlation spectroscopy measurements.

The kinetics of the alpha-complementation reaction of two protein fragments yielding active E. coli beta-galactosidase was measured using fluorescence correlation spectroscopy (FCS). The association reaction was extremely slow with an apparent association rate kapp of 207 M-1 s-1. This low association rate can be explained by a fast pre-equilibrium and slow subsequent steps involving at least two dimeric complexes. The subsequent formation of a tetrameric complex is probable and consistent with the experimental data. The complexes comprise two or four subunits, respectively, of the large fragment (EA)2 and in all cases only one small fragment, ED which has been labeled with Cy5. These kinetics have been compared to the association kinetics of ED to inactivated (EA)2. The kinetics were similar to the association with native (EA)2. The data support the observation that lyophilization of (EA)2 in a reducing environment which causes complete loss of enzymatic activity does not interfere with binding.

Detection of the hereditary hemochromatosis gene mutation by real-time fluorescence polymerase chain reaction and peptide nucleic acid clamping.

Hereditary hemochromatosis (HH), an iron overload disease, is the most common known inheritable disease. The most prevalent form of HH is believed to be the result of a single base-pair mutation. We describe a rapid homogeneous mutation analysis method that does not require post-polymerase chain reaction (PCR) manipulations. This method is a marriage of three emerging technologies: rapid cycling PCR thermal cyclers, peptide nucleic acid (PNA) probes, and a new double-stranded DNA-selective fluorescent dye, Sybr Green I. The LightCycler is a rapid thermal cycler that fluorometrically monitors real-time formation of amplicon with Sybr Green I. PNAs are DNA mimics that are more sensitive to mismatches than DNA probes, and will not serve as primers for DNA polymerases. PNA probes were designed to compete with PCR primers hybridizing to the HH mutation site. Fully complemented PNA probes at an 18:1 ratio over DNA primers with a mismatch result in suppression of amplicon formation. Conversely, PNA probes with a mismatch will not impair the binding of a complementary primer, culminating in amplicon formation. A LightCycler-based rapid genetic assay has been developed to distinguish HH patients from HH carriers and normal individuals using PNA clamping technology.

Identification of ceramide-phosphorylethanolamine in oomycete plant pathogens: Pythium ultimum, Phytophthora infestans, and Phytophthora capsici.

Cellular lipids were extracted from three species of Oomycete plant pathogens (Pythium ultimum, Phytophthora infestans, and Ph. capsici) and analyzed via normal-phase high-performance liquid chromatography with flame-ionization detection. The most abundant polar lipids in each of the three species were the polar membrane lipids, phosphatidylethanolamine (PE), phosphatidylcholine, and a phosphosphingolipid that eluted soon after PE. Structural analysis via mass spectrometry and nuclear magnetic resonance spectrometry revealed that the phosphosphingolipid was ceramide phosphorylethanolamine (Cer-PE). The most abundant molecular species of Cer-PE in P. ultimum had a molecular weight of 670.5, contained an unusual 19-carbon branched triunsaturated sphingoid (C19-delta 4, 8, 10, 9-methyl long-chain base) and palmitic acid as the amide-linked fatty acid. The most abundant molecular species of Cer-PE in Ph. infestans had a molecular weight of 714.5, contained a common 16-carbon 1,3 di-OH sphingoid, and erucic (cis 13-docosenoic, C22-delta 13) acid as the amide-linked fatty acid. The Cer-PE in Ph. capsici comprised a mixture of each of the two molecular species found in P. ultimum and Ph. infestans.

Seamless tiling of amorphous silicon photodiode-TFT arrays for very large area X-ray image sensors.

We propose a method for producing a truly seamless tiled image sensor from four identical subarrays, such that the resulting tiled image sensor has no missing pixels. Four standard amorphous silicon photodiode-TFT (thin film transistor) arrays are cut parallel to the bus-bars, in the space between the bus-bar and the preceding pixel electrode. The cut is done in such a way that it is adjacent to the pixel electrode on the active plate. The four subarrays are then rotated by 90 degrees, with respect to their neighbors, and butted together. In this way, the seam between each tile has no adjacent bus-bar, and it is possible to reproduce the pixel pitch between neighboring tiles, with an acceptable alignment tolerance of the tiles. We have demonstrated the feasibility of the method, with the aid of a small prototype, based on a 192 x 192 pixel array, with a 200-microm pitch. Some image processing is necessary to rotate the images back for display on a conventional display monitor. This can cause artefacts, in some fast moving scenes, in which case an alternative scheme, which uses two mirror image arrays, each rotated by 180 degrees, can be used. However, for static X-ray images and most images in dynamic medical X-ray applications this is not necessary and we can obtain good quality seamless images, free from any significant artefacts.

High prevalence of GAD65 (and IA-2) antibodies in Japanese IDDM patients by a new immunoprecipitation assay based on recombinant human GAD65.

Marked differences have been reported in the prevalence of glutamic acid decarboxylase (GAD) antibodies between Caucasian (63-84%) and Japanese (30-50%) or Asian (5-50%) IDDM patients. Using a new immunoprecipitation assay based on 125I-labelled recombinant human GAD65 we have reassessed prevalence of GAD65 antibodies in Japanese patients. We also assessed prevalence of IA-2 antibodies. GAD65 antibodies were detected in 83.3% of sera taken within 1 year of onset, comparable to the prevalence reported in Caucasian patients. Positivity decreased to 66.7% after 2 to 3 years and to 54.3% after 3 years from onset, still higher than previously reported Asian prevalence. Except in one patient, high antibody levels persisted chronically, up to 12 years. There was no difference in the prevalence of GAD65 antibodies between Japanese IDDM patients with and without autoimmune thyroid disease (AITD). IA-2 antibodies were detected in 64.7% of sera taken within 1 year of onset. Prevalence of IA-2 antibodies was lower than that of GAD65 antibodies. The difference in positivity in Asian IDDM patients between present and previous reports arose from the sensitivity of our assay for GAD65 antibodies. Additionally, the patients we studied had classic IDDM with a well-defined onset. We conclude that prevalence of GAD65 antibodies in Japanese IDDM patients is comparable to that in Western studies. There was no relationship of GAD65 antibody positivity to coexistence of AITD. Our results suggest that autoimmunity is the most significant cause of Japanese IDDM.

The effect of ethanol and oxygen on the growth of Zymomonas mobilis and the levels of hopanoids and other membrane lipids.

Zymomonas mobilis (ATCC 29191) was grown either aerobically or anaerobically in the presence of 2% (wt/vol) glucose and 0, 3, or 6% (vol/vol) ethanol. The rates of growth and the composition of hopanoids, cellular fatty acids, and other lipids in the bacterial membranes were quantitatively analyzed. The bacterium grew in the presence of 3% and 6% ethanol and was more ethanol tolerant when grown anaerobically. In the absence of ethanol, hopanoids comprised about 30% (by mass) of the total cellular lipids. Addition of ethanol to the media caused complex changes in the levels of hopanoids and other lipids. However, there was not a significant increase in any of the hopanoid lipid classes as ethanol concentration was increased. As previously reported, vaccenic acid was the most abundant fatty acid in the lipids of Z. mobilis, and its high constitutive levels were unaffected by the variations in ethanol and oxygen concentrations. A cyclopropane fatty acid accounted for 2.6-6.4 wt % of the total fatty acids in all treatments.

Modulation of lipoxygenase activity by bacterial hopanoids.

Tetrahydroxybacteriohopane (1), a bacterial hopanoid, inhibited soybean 15-lipoxygenase with an IC50 of about 10 microM. After per-O-acetylation of 1 no inhibition of the 15-lipoxygenase was observed. Two other bacterial hopanoids, tetrahydroxybacteriohopane glucosamine (2) and tetrahydroxybacteriohopane ether (3), stimulated the activity of soybean 15-lipoxygenase. The activities of two other arachidonic acid-metabolizing enzymes, human 5-lipoxygenase and prostaglandin H synthase, were unaffected by 1.

Expression of rat neuronal nitric oxide synthase in Saccharomyces cerevisiae.

The rat neuronal nitric oxide synthase (nNOS) cDNA was expressed in Saccharomyces cerevisiae under the control of a hybrid PGK/GAL promoter, PAL. Galactose induction resulted in the production of a soluble 160 kDa protein that was recognised by anti-neuronal NOS antibody. NOS activity was detected by the conversion of [3H]arginine to [3H]citrulline and required the addition of the cofactors, calmodulin and tetrahydrobiopterin. The activity was inhibited by the arginine analogues NG-nitro-L-arginine and NG-nitro-L-arginine methyl ester.