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The single-cell gel electrophoresis—the comet assay—has proved to be a sensitive and relatively simple method that is much used in research for the analysis of specific types of DNA damage, and its use in genotoxicity testing is increasing. The efficiency of the comet assay, in terms of number of samples processed per experiment, has been rather poor, and both research and toxicological testing should profit from an increased throughput. We have designed and validated a format involving 96 agarose minigels supported by a hydrophilic polyester film. Using simple technology, hundreds of samples may be processed in one experiment by one person, with less time needed for processing, less use of chemicals and requiring fewer cells per sample. Controlled electrophoresis, including circulation of the electrophoresis solution, improves the homogeneity between replicate samples in the 96-minigel format. The high-throughput method described in this paper should greatly increase the overall capacity, versatility and robustness of the comet assay. [ABSTRACT FROM AUTHOR]

STUDY QUESTION Which human sperm proteins interact with zona pellucida (ZP) glycoproteins, ZPA/2, ZPB/4 and ZPC/3? SUMMARY ANSWER Co-precipitation experiments with recombinant human ZP (rhZP) coated beads demonstrated interactions with various proteins, including glutathione S-transferase M3 (GSTM) with ZPB/4 and voltage-dependent anion channel 2 (VDAC2) with ZPA/2 and ZPC/3. WHAT IS KNOWN ALREADY Regarding sperm–ZP binding, several target spot/proteins have been detected in several species, but not all have been characterized. The limit of these studies was that a mixture of the different ZP glycoproteins was used and did not allow the identification of the specific ZP glycoprotein (ZPA/2, ZPC/3 or ZPB/4) involved in the interaction with the sperm proteins. STUDY DESIGN, SIZE, DURATION To identify the human sperm proteins interacting with the oocyte ZP, we combined two approaches: immunoblot of human spermatozoa targeted by antisperm antibodies (ASAs) from infertile men and far western blot of human sperm proteins overlayd by each of the rhZP proteins. MATERIALS, SETTING, METHODS We used rhZP expressed in Chinese hamster ovary (CHO) cells and ASA eluted from infertile patients undergoing IVF failure. Sperm proteins separated by two-dimensional (2D) electrophoresis recognized by both sperm-eluted ASAs from infertile patients and rhZP were identified by mass spectrometry (MALDI-MS/MS). Some of these proteins were further validated by co-precipitation experiments with rhZP and functional zona binding tests. MAIN RESULTS AND THE ROLE OF CHANCE We identified proteins that are glycolytic enzymes such as pyruvate kinase 3, enolase 1, glyceraldehyde-3-phosphate dehydrogenase, aldolase A, triosephosphate isomerase, detoxification enzymes such as GSTM or phospholipid hydroperoxide glutathione peroxidase, ion channels such as VDAC2 and structural proteins such as outer dense fibre 2. Several of the proteins were localized on the sperm head. However, these proteins have also been described to exert other functions in the flagellum. Co-precipitation experiments with rhZP-coated beads confirmed the direct interaction of GSTM with ZP4 and of VDAC2 with ZP2 and ZP3. LIMITATIONS, REASONS FOR CAUTION We used recombinant ZP in place of native ZP. Thus, the post-translational modifications of the proteins, such as glycosylations, can be different and can influence their function. However, CHO cell-expressed rhZP are functional, e.g. can bind human spermatozoa and induce the acrosome reaction. Moreover, the identification of relevant proteins was limited by the need for sufficient amounts of proteins on the preparative 2D-gel to be subsequently analysed in MALDI-TOF MS/MS. WIDER IMPLICATIONS OF THE FINDINGS Our results bring new insights on the ability of sperm proteins to exert several functions depending on their sub-cellular localization, either the head or flagellum. Their multiple roles suggest that these sperm proteins are multifaceted or moonlighting proteins. STUDY FUNDING/COMPETING INTEREST(S) This work was supported by the grant ReproRio (CNRS, INRA, INSERM and CEA) and the Société d'Andrologie de Langue Française. Trial registration number Not applicable. [ABSTRACT FROM PUBLISHER]

We report the engineering of a DnaE intein able to catalyze rapid C-terminal cleavage in the absence of N-terminal cleavage. A single mutation in DnaE intein from Nostoc punctiforme PCC73102 (NpuDnaE), Asp118Gly, was introduced based on sequence alignment with a previously engineered C-terminal cleaving intein mini-MtuRecA. This mutation was able to both suppress N-terminal cleavage and significantly elevate C-terminal cleavage efficiency. Molecular modeling suggests that in NpuDnaE Asp118 forms a hydrogen bond with the penultimate Asn, preventing its spontaneous cyclization prior to N-terminal cleavage. Mutation of Asp118 to Gly essentially abolishes this restriction leading to subsequent C-terminal cleavage in the absence of N-terminal cleavage. The Gly118 NpuDnaE mutant exhibits rapid thio-dependent C-terminal cleavage kinetics with 80% completion within 3 h at room temperature. We used this newly engineered intein to develop both column-free and chromatography-based protein purification methods utilizing the elastin-like-polypeptide and chitin-binding protein as removable purification tags, respectively. We demonstrate rapid target protein purification to electrophoretic purity at yields up to 84 mg per liter of Escherichia coli culture. [ABSTRACT FROM AUTHOR]

The LYS12 gene from Candida albicans, coding for homoisocitrate dehydrogenase was cloned and expressed as a His-tagged protein in Escherichia coli. The purified gene product catalyzes the Mg2+- and K+-dependent oxidative decarboxylation of homoisocitrate to α-ketoadipate. The recombinant enzyme demonstrates strict specificity for homoisocitrate. SDS- PAGE of Ca HIc DH revealed its molecular mass of 42.6 ± 1 kDa, whereas in size-exclusion chromatography, the enzyme eluted in a single peak corresponding to a molecular mass of 158 ± 3 kDa. Native electrophoresis showed that Ca HIc DH may exist as a monomer and as a tetramer and the latter form is favored by homoisocitrate binding. Ca HIc DH is an hysteretic enzyme. The K M values of the purified His-tagged enzyme for NAD+ and homoisocitrate were 1.09 mM and 73.7 μM, respectively, and kcat was 0.38 s−1. Kinetic parameters determined for the wild-type Ca HIc DH were very similar. The enzyme activity was inhibited by ( 2R,3S)-3-( p-carboxybenzyl)malate ( CBMA), with IC50 = 3.78 mM. CBMA demonstrated some moderate antifungal activity in minimal media that could be enhanced upon conversion of the enzyme inhibitor into its trimethyl ester derivative ( TMCBMA). TMCBMA is the first reported antifungal for which an enzyme of the AAP was identified as a molecular target. [ABSTRACT FROM AUTHOR]

On the basis of anecdotal instances in which atypical laboratory findings suggested the possibility of unsuspected cryoglobulinemia, we applied predetermined criteria to determine how often such findings predict the presence of clinically significant cryoglobulinemia. The laboratory criteria are smeared M-spike (paraprotein) in agarose gel serum protein electrophoresis, precipitated protein at the serum application point of agarose electrophoresis gel, greater than 50% quantitative discrepancy between the densitometrically estimated M-spike and the relevant corresponding serum immunoglobulin isotype concentration from the same specimen, and smeared protein observed on an agarose electrophoresis immunofixation gel. Cases that fulfilled any of these criteria were prospectively collected for 2 years. Brouet types and clinical findings were determined in cases where cryoglobulins were subsequently identified and clinical data available. Among 83 patients in whom any of the above laboratory findings were identified, 52 had subsequent cryoglobulin evaluations. Fourteen of these 52 patients had cryoglobulinemia. Findings indicative of clinically significant cryoglobulinemia were present in 8 of the 10 patients in whom follow-up clinical data were available. [ABSTRACT FROM AUTHOR]

The article discusses a research study on determining bisphenol A (BPA) and triclocan (TCS) in beverage, water and urine samples through the dispersive liquid-liquid microextraction in combination with capillary zone electrophoresis-ultraviolet (UV) detection. Optimized were factors affecting efficiences of microextraction such as dispersive solvent, salt effect and volume of extraction with the use of background electrolyte tetraborate. Conclusions indicated convenient use of the method.

Horizontal gene transfer plays an important role in bacterial evolution. DNA acquired by horizontal gene transfer has to be incorporated into existing regulatory networks. The histone-like nucleoid structuring protein H- NS acts as a silencer of horizontally acquired genes to avoid potential damage. However, specific regulators can overcome H- NS repression, resulting in the integration of newly acquired genes into existing regulatory networks. Here, we analyzed the influence of H- NS on the transcription of the Yersinia enterocolitica hreP gene and its regulators pypA, pypB, and pypC by establishing a dominant-negative H- NS version. Using transcriptional fusions and electrophoretic mobility shift assays, we show that H- NS silences hreP, pypA, pypB, and pypC by direct interactions. While the H- NS antagonist RovA activates pypC, it has no effect on pypA and pypB. Furthermore, H- NS affects biofilm formation in Y. enterocolitica. [ABSTRACT FROM AUTHOR]

Objectives To determine whether pan-protease inhibitor (PI)-resistant virus populations are composed predominantly of viruses with resistance to all PIs or of diverse virus populations with resistance to different subsets of PIs. Methods We performed deep sequencing of plasma virus samples from nine patients with high-level genotypic and/or phenotypic resistance to all licensed PIs. The nine virus samples had a median of 12 PI resistance mutations by direct PCR Sanger sequencing. Results For each of the nine virus samples, deep sequencing showed that each of the individual viruses within a sample contained nearly all of the mutations detected by Sanger sequencing. Indeed, a median of 94.9% of deep sequence reads had each of the PI resistance mutations present as a single chromatographic peak in the Sanger sequence. A median of 5.0% of reads had all but one of the Sanger mutations that were not part of an electrophoretic mixture. Conclusions The collinearity of PI resistance mutations in the nine virus samples demonstrated that pan-PI-resistant viruses are able to replicate in vivo despite their highly mutated protease enzymes. We hypothesize that the marked collinearity of PI resistance mutations in pan-PI-resistant virus populations results from the unique requirements for multi-PI resistance and the extensive cross-resistance conferred by many of the accessory PI resistance mutations. [ABSTRACT FROM PUBLISHER]

Background Recent evidence has suggested that deranged immune responses play a role in the pathogenesis of hidradenitis suppurativa (HS). Objectives To investigate the role of single nucleotide polymorphisms (SNPs) of the tumour necrosis factor ( TNF) and Toll-like receptor 4 ( TLR4) genes in the physical course of HS; these genes encode for proteins implicated in the immune response of the host. Methods DNA was isolated from 190 patients with HS and 84 healthy controls. SNPs at the promoter regions −376G/A, −238G/A and −308G/A of the TNF gene and the Asp299Gly and Thr399Ile SNPs of the TLR4 gene were determined by polymerase chain reaction (PCR) and digestion of the PCR product by restriction enzymes; after electrophoresis on 2·0% agarose gel, products were visualized on under ultraviolet radiation. Results The presence of the −238 TNF gene polymorphism was associated with a predisposition to HS ( P = 0·027). Susceptibility to the disease was strongly correlated with the presence of AGG/GGA/AGA/GAA TNF haplotypes in 32 (17%) patients compared with two (2%) controls ( P < 0·001, odds ratio 8·30, 95% confidence interval 1·94-35·52). The frequency of HS exacerbations and disease severity were greater in patients carrying any of the GAG/AGG/GGA/AGA/GAA haplotypes of the TNF gene. Thirty-two patients were given TNF antagonists. Nineteen of these patients were carriers of the GGG haplotype of the TNF gene, whereas 13 were carriers of other haplotypes; favourable responses as evidenced by the Sartorius score were registered in 15 (79%) and five (38%, P = 0·025), respectively. Carriage of the TLR4 gene alleles was not associated with any disease parameter. Conclusions A significant role of SNPs at the promoter region of the TNF gene is indicated for susceptibility to HS and for response to TNF antagonists. [ABSTRACT FROM AUTHOR]

α1-Antitrypsin (AAT) deficiency leads to deterioration of the lungs that can be prevented with diagnosis and treatment. Isoelectric focusing (IEF) electrophoresis is the current biochemical gold standard for detecting AAT deficiency variants but involves complex interpretation. Variant AAT samples were collected over a 2-year period. Stability of AAT for phenotype determination was assessed in whole blood, dried blood spots, and dried serum spots. A compendium displaying 13 common and 5 rare AAT phenotypes was created, and a detailed methodology describing how to recognize AAT banding patterns and interpret a rare phenotype accompanied these visual data. AAT was stable for IEF phenotype analysis for at least 1 week in whole blood and for 24 hours on dried serum spots. In conclusion, a reference compendium of known AAT phenotypes was established that can serve as a resource for interpreting AAT phenotypes. [ABSTRACT FROM AUTHOR]

Objective: To determine the sensitivities of various testing methods for diagnosing brucellosis. Methods: The 267 patients in our cohort were suspected to be infected with Brucella and had been referred to a hospital in Mianeh City, Iran. The mean (SD) age was 37.0 (11.3) years for female patients and 37.1 (13.7) years for male patients. All serum specimens from these patients were examined by the standard agglutination test (SAT), Coombs Wright test, 2-mercaptoethanol (2ME) test, enzyme-linked immunosorbent assay (ELISA) for determining levels of immunoglobulin G (IgG) and immunoglobulin M (IgM), and multiplex polymerase chain reaction (PCR). DNA was extracted. Extracted DNA was checked with human PCR-targeting CRP gene-encoding 485 base pair (bp) as an internal control to ensure that the proper extraction method had been used. Specific primers targeting IS 711 were used for Brucellosis melitensis, B abortus, and B suis. Results: Brucellosis was confirmed in 110 patients (58.2% male and 41.8% female) based on applied diagnostic methods and clinical features. Results of ELISA, the SAT, and PCR were positive in 92, 42, and 51 patients, respectively. B abortus and B melitensis were detected in 16 and 35 patients, respectively. B suis was not detected in any of the specimens. Conclusions: All methods tested in our study need to be used to ensure accurate diagnosis of brucellosis, although ELISA displayed the highest level of efficiency. Also, B melitensis showed a higher frequency rate than B abortus in our cohort. [ABSTRACT FROM AUTHOR]

The article presents a study which investigated molecular diversity in six viroid species and various molecular variants using single-strand conformation polymorphism (SSCP) and direct sequencing analysis. The study compared and optimized polyacrylamide gel electrophoresis (PAGE) conditions. Results indicated that the differences of some electrophoresis parameters for every species allowed the detection of single nucleotide differences among clones that were clustered into the same group.

The Gulf killifish, Fundulus grandis, is a small teleost fish that inhabits marshes of the Gulf of Mexico and demonstrates high tolerance of environmental variation, making it an excellent subject for the study of physiological and molecular adaptations to environmental stress. In the present study, two-dimensional (2D) gel electrophoresis and matrix-assisted laser desorption/ionization time-of-flight tandem mass spectrometry were used to resolve and identify proteins from five tissues: skeletal muscle, liver, brain, heart, and gill. Of 864 protein features excised from 2D gels, 424 proteins were identified, corresponding to a 49% identification rate. For any given tissue, several protein features were identified as the same protein, resulting in a total of 254 nonredundant proteins. These nonredundant proteins were categorized into a total of 11 molecular functions, including catalytic activity, structural molecule, binding, and transport. In all tissues, catalytic activity and binding were the most highly represented molecular functions. Comparing across the tissues, proteome coverage was lowest in skeletal muscle, due to a combination of a low number of gel spots excised for analysis and a high redundancy of identifications among these spots. Nevertheless, the identification of a substantial number of proteins with high statistical confidence from other tissues suggests that F. grandis may serve as a model fish for future studies of environmental proteomics and ultimately help to elucidate proteomic responses of fish and other vertebrates to environmental stress. [ABSTRACT FROM AUTHOR]

A novel technique of insect ecdysone analysis was established by capillary zone electrophoresis using 20-hydroxy ecdysone (purity ≥93%) as a standard sample. It showed that 20-hydroxy ecdysone and other trace impurities were completely separated within 5 min with an electrolyte containing 10 mmol/L borate, at pH 9.18, 20 kV applied voltage and 0.5 psi X 5 s injected volume. Under this optimal condition, effects of azadirachtin on ecdysone in the hemolymph of Ostrinia furnacalis (Guenee) were investigated. The results showed that the peak values of 20-hydroxy ecdysone were strongly suppressed by azadirachtin and the titers remained at a lower level than those in the control samples. When compared with high-performance liquid chromatography, the primary advantages of capillary zone electrophoresis include a much shorter analysis time and detection limit and the elimination of complex pretreatment of samples. [ABSTRACT FROM AUTHOR]

The purpose of this study was to isolate the coding sequence of the p24 protein (encoded by the gag gene) from sera collected from Iranian patients, to clone and express this sequence in Escherichia coli (E coli), and to assess the immunoreactivity of the expressed recombinant protein. The complete coding sequence for the Gag protein was isolated by nested real-time polymerase chain reaction {RT-PCR) from the serum of an infected individual and then cloned into a PTZ57R (T) vector. The resulting vector, PTZ-gag53-IR, was sequenced. Subsequently, the coding sequence of p24 protein was isolated by PCR from the PTZ-gag53-IR vector and inserted into a pET102/D-TOPO vector using TOPO directional cloning and then expressed in E coli in fusion with His-Patch (HP)-thioredoxin. The immunoreactivity of the expressed protein was tested by immunoblotting and enzyme-linked immunosorbent assay (ELISA). RT-PCR revealed a sequence belonging to the CRF35_AD subtype of the HIV-1 virus, which is highly prevalent in Iran and Afghanistan. Recombinant p24 protein from this isolate showed 100% sensitivity and 100% specificity in an antibody assay. [ABSTRACT FROM AUTHOR]

The article presents abstracts on topics related to Chemistry in the November 2012 issue of the periodical which include monoclonal gammopathy using Capillary Electrophoreses, Detection of Hemoglobin A, and Acute Splenic Sequestration Crisis in patient suffering from Thalassemia.

Temperature can affect the performance of insects through its influence on enzyme function. We report a series of laboratory and field experiments investigating the putatively adaptive temperature-dependent effects of phosphoglucomutase ( pgm) genotype on development time, a central life-history fitness component, as mediated by the microhabitat choice of ovipositing female yellow dung flies ( Scathophaga stercoraria). The allozyme corresponding to the most common pgm allele 3 (approximate 90% natural frequency) had the highest Q10 and showed higher in vitro activity at warmer temperatures (17-27 ° C), whereas the allozyme corresponding to allele 4 (4.5%) showed relatively higher activity at the lowest temperature (7 ° C), and the allozyme corresponding to allele 2 (2.5%) showed lower activity throughout. A laboratory experiment revealed that egg development time for allele 3 was shortest (i.e. best) at all temperatures tested, although egg-to-adult development time was longest for offspring derived from field females bearing allele 3. Importantly, over 3 years in the field, allele 4 did not increase in frequency late in the season as the temperatures dropped. Although females augmented their proportion of eggs laid on the warmer south slope of a dung pat (adaptive response), the development times of their offspring consistently increased towards the end of the season (maladaptive response). Regardless, females did not systematically bias the pgm composition of eggs laid on the north versus south slopes, as was expected from previous work hypothesizing that females exert microhabitat choice based on the pgm locus. We conclude that, although PGM allozymes differ in activity and the pgm genotype does differentially affect development time, these effects are inconsistent, and not predictable from in vitro allozyme assays, and therefore difficult to reconcile with an adaptive framework of cryptic female choice. © 2012 The Linnean Society of London, Biological Journal of the Linnean Society, 2012, ••, ••-••. [ABSTRACT FROM AUTHOR]

A particular fungal population is present in the main stages of the manufacturing process of cork discs. Its diversity was studied using both dependent (isolation) and independent culture methods (denaturing gel gradient electrophoresis and cloning of the ITS1-5.8 S- ITS2 region). The mycobiota in the samples taken in the stages before and after the first boiling seems to be distinct from the population in the subsequent manufacturing stages. Most isolated fungi belong to the genera Penicillium, Eurotium and Cladosporium. The presence of uncultivable fungi, Ascomycota and endophytes in raw cork was confirmed by sequencing. The samples taken after the first boiling contained uncultivable fungi, but in a few samples some isolated fungi were also detected. The main taxa present in the following stages were Chrysonilia sitophila, Penicillium glabrum and Penicillium spp. All applied techniques had complementary outcomes. The main factors driving the shift in cork fungal colonization seem to be the high levels of humidity and temperature to which the slabs are subjected during the boiling process. [ABSTRACT FROM AUTHOR]

Serum protein electrophoresis (SPE) and immunofixation electrophoresis (IFE) are standard methods for detection and monitoring of monoclonal (M) proteins. However, these tests are rarely available in the remote areas, especially in developing countries. Transportation of fresh serum (FS) samples is also usually inconvenient. This study investigated M-protein identification using serum blot on filter paper (FP). SPE and IFE were performed on FS and FP specimens using the Sebia Hydrasys automated electrophoresis system. Statistical analyses were conducted to assess sample stability and agreement of FS vs FP. The FP method showed good agreement with the FS method. The r values for correlation of albumin levels--α1, α2, β, and γ (%)--between FP and FS samples in SPE were all more than 0.95 (P < .01). IFE displayed no significant difference between those 2 methods in the identification of M protein. The FP method demonstrated an accurate and reproducible alternative to FS for identification of M protein using SPE and IFE. [ABSTRACT FROM AUTHOR]

Monoclonal gammopathy of undetermined significance (MGUS) that presents with no quantifiable M spike on immunofixation electrophoresis (IFE) can be termed IFE MGUS. We retrospectively identified patients with IFE MGUS who were monitored with at least 1 subsequent assessment that included an IFE, and evaluated the persistence of the monoclonal protein and the progression of disease. Although the monoclonal proteins persisted in the majority of patients, 16% did not experience this persistence, and had no documented immunomodulatory therapy. After a median follow-up of 3.9 years, the disease clinically progressed in 14 patients (3.2%). Eight of these 14 patients with clinical progression had an immunoglobulin (Ig) A IFE M protein and 6 had an IgG M protein. This study demonstrates that in some patients with IFE MGUS, the M proteins are transient and that IgA IFE MGUS is more likely to persist and progress to myeloma. [ABSTRACT FROM AUTHOR]

Phylogeographical research has revealed several paradigm patterns of postglacial range expansion from the Mediterranean peninsulas to more northern parts of Europe. These range expansions have consequences for the genetic constitution of populations. Although many studies have been performed in mainland Europe, the colonization history of the British Isles is relatively poorly studied; the genetic consequences of the last glacial readvances and the climate optimum conditions, as well as the implications of the recent climatic conditions on the population genetic structures, are little understood. Therefore, we selected the common blue butterfly Polyommatus icarus as a model species for understanding more generally the colonization patterns of the British Isles and the genetic dynamics on these islands. Allozyme analyses of this butterfly show a rather high genetic diversity over continental Europe without major genetic differentiation. The situation on the British Isles is completely different. Here, populations show a much lower genetic diversity compared to mainland Europe. The genetic constitution is well differentiated from that observed on the European mainland, and the genetic differentiation among populations in Britain is stronger than at the European scale. These results support the hypothesis that a relatively cold-tolerant species such as the common blue could have colonized the British Isles early during the late glacial period and survived the last glacial readvances in small refugia in the South. The retraction of this species in small isolated populations could have caused the genetic impoverishment found. The subsequent forest climax during the climate optimum possibly restricted further expansion of this early succession species to small pockets all over the British Isles, resulting in the genetic patchwork that is still observed. Additionally, the relatively cool and rainy conditions one these islands might have caused bottlenecks, possibly enforcing these genetic patterns. © 2012 The Linnean Society of London, Biological Journal of the Linnean Society, 2012, ••, ••-••. [ABSTRACT FROM AUTHOR]

This study used the general applicability of 2,6-didi-o-methyl-β-cyclodextrin (DM-β-CD) as the chiral selector in capillary electrophoresis for fast and efficient chiral separation of repaglinide enantiomers. A systematic study of the parameters affecting separation was performed with UV detection at 243 nm. The optimum conditions were determined to be 1.25% (w/v) DM-β-CD in 20 mM sodium phosphate (pH 2.5) as the running buffer and separation voltage at 20 kV. DM-β-CD had the best enantiomer resolution properties under the tested conditions, whereas other β-cyclodextrins showed inferior performances or no performance. The proposed method had a linear calibration curve in the concentration range of 12.5–400 µg/mL. The limit of detection was 100 ng/mL. The intra-day and inter-day precisions were 2.8 and 3.2%, respectively. Recoveries of 97.9–100.9% were obtained. The proposed method was fast and convenient, and was determined to be efficient for separating enantiomers and applicable for analyzing repaglinide enantiomers in quality control of pharmaceutical production. [ABSTRACT FROM PUBLISHER]

The Biofilm ( BF) building capacity of different serotypes of Salmonella enterica derived from the poultry farm environment was investigated. Starting point for the investigation was the question if farm-isolated Salmonella serotypes with high importance for poultry meat and egg production are capable of forming a BF under defined laboratory conditions. Several isolates from different stages of the production cycle were chosen and compared to laboratory grown strains of the same serotype. BF building capacity was analyzed in a 96-well format during a time period of 2 days. Pulse field gel electrophoresis was used to establish a relationship between different isolates. The BF building capacity of a monospecies BF was strongly dependent on the temperature used for incubation. Results indicated further that certain farm isolates were capable of forming BF under laboratory conditions, whereas laboratory grown strains were not. Considerable differences between different field serovars and within one serovar exist. In conclusion, the BF building capacity of poultry-derived isolates is a function of adaptation to their host environment. Thus, the control of BF as a reservoir for Salmonella in the farm environment is of crucial importance for the overall improvement of food safety. Mechanical and substance-based approaches for this control exist in several variations, but overall decontamination success is difficult to achieve and needs to be especially adapted to the farm environment. [ABSTRACT FROM AUTHOR]

The article presents a study which investigates on the proteomic identification of peroxidase stabilized with acetylated polymannan polysaccharide (ApmP) of Aloe barbadensis Miller. The study performed an electrophoretic band test and proteomic analysis which detected seven tryptic peptides. The study reveals that all the tested characteristics of the peroxidase indicate that activities stabilized with ApmP pertain to the basic secretory peroxidase family.

We identified a highly expressed protein ( BUNA2) by two-dimensional gel electrophoresis from the hyper lignin-degrading fungus Phanerochaete sordida YK-624 under wood-rotting conditions. Partial amino acid sequences of BUNA2 were determined by LC- MS/ MS analysis, and BUNA2 gene ( bee2) and promoter region were PCR-cloned and sequenced. The bee2 promoter was used to drive the expression of the manganese peroxidase gene ( mnp4) in P. sordida YK-624. Eighteen mnp4-expressing clones were obtained, with most showing higher ligninolytic activity and selectivity than wild-type YK-624. Examination of the ligninolytic properties of the most effective lignin-degrading transformant, BM-65, cultured on wood meal revealed that this strain exhibited higher lignin degradation and MnP activities than those of wild type. Transcriptional analysis confirmed the increased expression of recombinant mnp4 in the transformant. These results indicate that use of the bee2 promoter to drive the expression of ligninolytic enzymes may be an effective approach for improving the lignin-degrading properties of white-rot fungi. [ABSTRACT FROM AUTHOR]

A bacterial strain, designated as TSB-6, was isolated from the sediments of a Tantloi ( India) hot spring at 65 ° C. The strain showed 98% 16 S rRNA gene sequence similarity with Anoxybacillus kualawohkensis strain KW12 and was found to grow optimally at 37 ° C. However, growing cells, cell suspensions, and cell-free extracts from 65 ° C cultures showed higher Cr(VI) reduction activities when assayed at either 37 or 65 ° C than those obtained from 37 ° C cultures. On fractionation of extracts from cells grown at 65 ° C, the chromate reductase activity assayed at 65 ° C was found mostly in the soluble fraction. When log-phase cells growing at 37 ° C were shifted to 65 ° C, the stressed cells produced larger quantities of reactive oxygen species. Consequently, growth of the cells was retarded, but specific Cr(VI) reduction activity increased. 2 D gel electrophoresis followed by MALDI- TOF MS/ MS identified the proteins whose expression level changed as a result of heat stress. The upregulated set included proteins involved in cellular metabolism of sugar, nucleotide, amino acids, lipids and vitamins, oxidoreductase activity, and protein folding. The downregulated proteins are also involved in cellular metabolism, DNA binding, and environmental signal processing. [ABSTRACT FROM AUTHOR]

Ochratoxin A (OTA) is a toxic isocoumarin derivative produced by various species of mould which mainly grow on grain, coffee, and nuts. Recent studies have suggested that OTA induces cell death in plants. To investigate possible mechanisms of OTA phytotoxicity, both digital gene expression (DGE) transcriptomic and two-dimensional electrophoresis proteomic analyses were used, through which 3118 genes and 23 proteins were identified as being up- or down-regulated at least 2-fold in Arabidopsis leaf in response to OTA treatment. First, exposure of excised Arabidopsis thaliana leaves to OTA rapidly causes the hypersensitive reponse, significantly accelerates the increase of reactive oxygen species and malondialdehyde, and enhances antioxidant enzyme defence responses and xenobiotic detoxification. Secondly, OTA stimulation causes dynamic changes in transcription factors and activates the membrane transport system dramatically. Thirdly, a concomitant persistence of compromised photosynthesis and photorespiration is indicative of a metabolic shift from a highly active to a weak state. Finally, the data revealed that ethylene, salicylic acid, jasmonic acid, and mitogen-activated protein kinase signalling molecules mediate the process of toxicity caused by OTA. Profiling analyses on Arabidopsis in response to OTA will provide new insights into signalling transduction that modulates the OTA phytotoxicity mechanism, facilitate mapping of regulatory networks, and extend the ability to improve OTA tolerance in Arabidopsis. [ABSTRACT FROM PUBLISHER]

A lectin was purified from the mushroom Hygrophorus russula by affinity chromatography on a Sephadex G-50 column and BioAssist S cation exchange chromatography and designated H. russula lectin (HRL). The results of sodium dodecyl sulfate–polyaclylamidegel electrophoresis, gel filtration and matrix-assisted laser desorption ionization time-of-flight mass spectrometry of HRL indicated that it was composed of four identical 18.5 kDa subunits with no S-S linkage. Isoelectric focusing of the lectin showed bands near pI 6.40. The complete sequence of 175 amino acid residues was determined by amino acid sequencing of intact or enzyme-digested HRL. The sequence showed homology with Grifola frondosa lectin. The cDNA of HRL was cloned from RNA extracted from the mushroom. The open reading frame of the cDNA consisted of 528 bp encoding 176 amino acids. In hemagglutination inhibition assay, α1-6 mannobiose was the strongest inhibitor and isomaltose, Glcα1-6Glc, was the second strongest one, among mono- and oligosaccharides tested. Frontal affinity chromatography indicated that HRL had the highest affinity for Manα1-6(Manα1-3)Manβ1-4GlcNAcβ1-4GlcNAc, and non-reducing terminal Manα1-6 was essential for the binding of HRL to carbohydrate chains. The sugar-binding specificity of HRL was also analyzed by using BIAcore. The result from the analysis exhibited positive correlations with that of the hemagglutination inhibition assay. All the results suggested that HRL recognized the α1-6 linkage of mannose and glucose, especially the Manα1-6 bond. HRL showed a mitogenic activity against spleen lymph cells of an F344 rat. Furthermore, an enzyme-linked immunosorbent assay showed strong binding of HRL to human immunodeficiency virus type-1 gp120. [ABSTRACT FROM PUBLISHER]

A simple and accurate capillary electrophoresis (CE) method was developed to simultaneously separate and quantify heparin, chondroitin sulfate and hyaluronic acid. The relative standard deviations (intra-day) of migration time, peak height and peak area for heparin, chondroitin sulfate and hyaluronic acid were lower than 1.11, 5.45 and 2.82%, respectively. The limits of detection of heparin, chondroitin sulfate and hyaluronic acid were 0.91, 0.12 and 9.04 × 10−3 mg/mL, respectively. The developed electrophoretic method was successfully applied to the analysis of commercial drug products and biological samples containing chondroitin sulfate and/or hyaluronic acid. The recoveries for chondroitin sulfate and hyaluronic acid were in the range of 95.9 ∼107.0%. This was the first time the content of hyaluronic acid in the synovial fluids from osteoarthritic rabbits was investigated by CE. The results suggested that hyaluronic acid in the synovial fluids from osteoarthritic rabbits may be further metabolized and the administration of chondroitin sulfate or hyaluronic acid could affect the content and metabolism of hyaluronic acid in the synovial fluids. The developed CE method was simple to implement without sample pretreatment such as depolymerisation, very repeatable and easily transferred from lab to lab. [ABSTRACT FROM PUBLISHER]

The chromosomal passenger complex (CPC) senses tension defects at the kinetochore to activate the spindle assembly checkpoint, and helps to position the cleavage furrow. The CPC, consisting of INCENP, Survivin, Borealin and Aurora B localizes to the inner centromere at metaphase and re-localizes to the spindle midzone at anaphase; several CPC functions are regulated by post-translational modification. Borealin is phosphorylated at multiple sites and phosphorylation at S219 causes Borealin to migrate more slowly upon electrophoresis. Here we find that Cdk1 can induce a mobility shift of Borealin, suggesting that S219 phosphorylation is under Cdk1 control. However, Cdk1 is inefficient at phosphorylating purified Borealin in vitro. A yeast orthologue of Borealin, Npl1, is dephosphorylated by the phosphatase Cdc14. We find no difference in the mobility shift of Borealin in human cells lacking either Cdc14A or Cdc14B. In contrast, the phosphatase inhibitor okadaic acid does delay the dephosphorylation of Borealin as cells exit mitosis. The proteasome inhibitor MG132 reduces Borealin phosphorylation in mitosis and increases the steady-state level of Borealin, especially in mutants lacking the C-terminus. However, a second, structurally unrelated proteasome inhibitor, lactacystin did not up-regulate Borealin. These results suggest that the effect of MG132 on Borealin is due to the inhibition of an intracellular protease other than the proteasome. [ABSTRACT FROM PUBLISHER]

The study of protein–protein interactions (PPIs) is essential to uncover unknown functions of proteins at the molecular level and to gain insight into complex cellular networks. Affinity purification and mass spectrometry (AP-MS), yeast two-hybrid, imaging approaches and numerous diverse databases have been developed as strategies to analyze PPIs. The past decade has seen an increase in the number of identified proteins with the development of MS and large-scale proteome analyses. Consequently, the false-positive protein identification rate has also increased. Therefore, the general consensus is to confirm PPI data using one or more independent approaches for an accurate evaluation. Furthermore, identifying minor PPIs is fundamental for understanding the functions of transient interactions and low-abundance proteins. Besides establishing PPI methodologies, we are now seeing the development of new methods and/or improvements in existing methods, which involve identifying minor proteins by MS, multidimensional protein identification technology or OFFGEL electrophoresis analyses, one-shot analysis with a long column or filter-aided sample preparation methods. These advanced techniques should allow thousands of proteins to be identified, whereas in-depth proteomic methods should permit the identification of transient binding or PPIs with weak affinity. Here, the current status of PPI analysis is reviewed and some advanced techniques are discussed briefly along with future challenges for plant proteomics. [ABSTRACT FROM AUTHOR]

We report the precision of the mean migration position of hemoglobin (Hb)S, HbC, HbG (Philadelphia), and HbD (Los Angeles) in 193 samples of whole blood assayed by capillary electrophoresis (CE) and high-performance liquid chromatography (HPLC). By expressing the migration of Hb variants by CE relative to that of HbA2 in the same sample, there was a significant improvement in the coefficient of variation for each variant studied. The potential usefulness of expressing Hb variants relative to that of HbA2 was evaluated by comparing the separation of 2 closely migrating Hbs. When expressed by their initial migrations on CE, 25 of the 43 cases of HbG and HbD overlapped. However, when the migrations of these variants were expressed relative to the HbA2 in the same sample, the 24 cases of HbG separated completely from the 19 cases of HbD. These findings suggest that expressing Hb variants relative to an internal standard, such as HbA2, may be of value for establishing a library of variant Hbs evaluated by CE. [ABSTRACT FROM AUTHOR]

The function of whcB, one of the four whiB homologues of Corynebacterium glutamicum, was assessed. Cells carrying the P180- whcB clone, and thus overexpressing the whcB gene, showed retarded growth, probably due to increased sensitivity to oxidants, whereas cells lacking whcB ( Δ whcB) did not. However, growth retardation was not observed in cells with additionally whcE deleted. Furthermore, the Δ whcE phenotype, characterized by slow growth and sensitivity to oxidants, was reversed in cells carrying P180- whcB. Like the whcE gene, which is also known as a whiB homologue, the whcB gene was preferentially expressed in stationary phase. Determination of the genes under regulation of whcB using two-dimensional polyacrylamide gel electrophoresis identified several genes involved in electron transfer reactions that were regulated in cells carrying P180- whcB. Collectively, these findings indicate that whcB function requires whcE. Furthermore, whcB and whcE are paralogues but perform distinct regulatory roles during growth under oxidative stress. [ABSTRACT FROM AUTHOR]

MYB46 and MYB83 are two functionally redundant Arabidopsis thaliana MYB transcription factors that act as master switches regulating secondary wall biosynthesis. Here, we report the identification of the transcriptional responsive elements and global analysis of the direct targets of MYB46 and MYB83. Using the estrogen-inducible direct activation system, we found that a number of previously identified MYB46 downstream transcription factors, including MYB43, MYB52, MYB54, MYB58, MYB63 and KNAT7, are direct targets of MYB46. Promoter deletion coupled with transactivation analysis of the MYB63 promoter led to the identification of a 7 bp sequence that is sufficient to be responsive to MYB46 activation, and therefore this sequence is designated as the secondary wall MYB-responsive element (SMRE). Further single nucleotide mutation together with electrophoretic mobility shift assay mapped the SMRE consensus sequence as ACC(A/T)A(A/C)(T/C). Genome-wide analysis of direct targets of MYB46 demonstrated that it directly regulates the expression of not only a number of downstream transcription factors, but also a suite of secondary wall biosynthetic genes, some of which are also directly activated by secondary wall NAC (SWN) master switches or by MYB46 direct targets. Furthermore, MYB83 was found to bind to the same SMRE consensus sequence and activate the same set of direct targets as MYB46. Our study has revealed that the transcription program regulating secondary wall biosynthesis involves a multileveled feed-forward loop regulatory structure in which MYB46/MYB83 together with their regulators SWNs and their direct targets regulate an array of downstream genes thereby activating the secondary wall biosynthetic program. [ABSTRACT FROM AUTHOR]

Objectives One of the thrust areas in drug delivery research is transdermal drug delivery systems (TDDS) due to their characteristic advantages over oral and parenteral drug delivery systems. Researchers have focused their attention on the use of microneedles to overcome the barrier of the stratum corneum. Microneedles deliver the drug into the epidermis without disruption of nerve endings. Recent advances in the development of microneedles are discussed in this review for the benefit of young scientists and to promote research in the area. Key findings Microneedles are fabricated using a microelectromechanical system employing silicon, metals, polymers or polysaccharides. Solid coated microneedles can be used to pierce the superficial skin layer followed by delivery of the drug. Advances in microneedle research led to development of dissolvable/degradable and hollow microneedles to deliver drugs at a higher dose and to engineer drug release. Iontophoresis, sonophoresis and electrophoresis can be used to modify drug delivery when used in concern with hollow microneedles. Microneedles can be used to deliver macromolecules such as insulin, growth hormones, immunobiologicals, proteins and peptides. Microneedles containing 'cosmeceuticals' are currently available to treat acne, pigmentation, scars and wrinkles, as well as for skin tone improvement. Summary Literature survey and patents filled revealed that microneedle-based drug delivery system can be explored as a potential tool for the delivery of a variety of macromolecules that are not effectively delivered by conventional transdermal techniques. [ABSTRACT FROM AUTHOR]

Hyaluronan (HA) is a linear non-sulfated polysaccharide mainly found in the extracellular matrix. The size of HA can vary from a few disaccharides up to at least 25,000 units, reaching molecular weights of 10 × 103 kDa. HA has many biological functions, and both its size and tissue concentration play an important role in many physiological and pathological processes. It is relatively easy to determine the HA concentration using enzyme-linked binding protein assays, but the molecular weight of HA has so far been shown to be a more challenging task to measure. Here, we present a method for size determination of HA using gas-phase electrophoretic mobility molecular analysis (GEMMA), which utilizes the electrophoretic mobility of molecules in air to estimate the molecular weight of the analyte. We show that this method gives reliable molecular weight estimations of HA in the range of 30–2400 kDa, which covers almost its whole biological range. The average measuring time for one GEMMA spectrum is between 5 and 10 min using only 6 pg of HA. In addition, the peak area in a GEMMA spectrum can be used to estimate the HA concentration in the sample. The high sensitivity and small sample volumes make GEMMA an excellent tool for both size determinations and estimation of concentration of samples with low HA concentration, as is the case for HA extracted from small tissue samples. [ABSTRACT FROM PUBLISHER]

Aims: To investigate the distribution of staphylococcal enterotoxin genes ( se) and the molecular features of community-associated methicillin-sensitive/resistant Staphylococcus aureus (CA-MSSA/MRSA) isolates in the nostrils of healthy pets and their owners. Methods and Results: A total of 114 Staph. aureus isolates were identified from 1563 nasal swab samples, and CA-MRSA accounted for 20·2% ( n = 23) of the total identified isolates. CA-MRSA isolates (91·3%, 21/23) harboured higher percentage of se than did CA-MSSA isolates (58·2%, 53/91) ( P < 0·01), and the two highest se profiles of CA-MRSA were seb- sek- seq (42·9%, 9/21) and seb- sek- seq- sep (28·6%, 6/21). Of the MSSAs, 42·8% (39/91) were resistant to at least one antimicrobial drug and 8·8% (8/91) were multidrug resistant (MDR). We identified nine staphylocoagulase (SC) types (I-VIII and X) and three multilocus sequence types (ST59-MRSA-IV/V, ST-239-MRSA-V and ST241-MRSA-V). SC VII (23·4%, 22/94), a staphylococcal food poisoning isolate found mainly in Japan, and ST-59-MRSA-IV/V (85%, 17/20), a widespread CA-MRSA clone found mainly in Taiwan, both were the most predominant types. Phylogenetic analysis together with se and molecular characteristics obtained using pulsed-field gel electrophoresis showed that high levels of antimicrobial resistance and the se-carrying clone ST59-MRSA-IV/V-SC VII were all clustered in genogroup 5. Conclusions: The CA-MRSA clone of se-carrying-MDR-ST-59-IV/V-SC VII was identified predominantly in this study, and this clone might play a significant role in staphylococcal food poisoning in community settings. Significance and Impact of the Study: To our knowledge, this is the first study focussing on enterotoxin-carrying CA-MRSA/MSSA in pets and their owners, and the results support the future warnings in animal-human bond caused by CA-staphylococci in the commonwealth and the need to take cautions worldwide. [ABSTRACT FROM AUTHOR]

Catharanthus roseus produces a large array of terpenoid indole alkaloids (TIAs) that are an important source of natural or semisynthetic anticancer drugs. The biosynthesis of TIAs is tissue specific and induced by certain phytohormones and fungal elicitors, indicating the involvement of a complex transcriptional control network. However, the transcriptional regulation of the TIA pathway is poorly understood. Here, we describe a C. roseus WRKY transcription factor, CrWRKY1, that is preferentially expressed in roots and induced by the phytohormones jasmonate, gibberellic acid, and ethylene. The overexpression of CrWRKY1 in C. roseus hairy roots up-regulated several key TIA pathway genes, especially Tryptophan Decarboxylase (TDC), as well as the transcriptional repressors ZCT1 (for zinc-finger C. roseus transcription factor 1), ZCT2, and ZCT3. However, CrWRKY1 overexpression repressed the transcriptional activators ORCA2, ORCA3, and CrMYC2. Overexpression of a dominant-repressive form of CrWRKY1, created by fusing the SRDX repressor domain to CrWRKY1, resulted in the down-regulation of TDC and ZCTs but the up-regulation of ORCA3 and CrMYC2. CrWRKY1 bound to the W box elements of the TDC promoter in electrophoretic mobility shift, yeast one-hybrid, and C. roseus protoplast assays. Up-regulation of TDC increased TDC activity, tryptamine concentration, and resistance to 4-methyl tryptophan inhibition of CrWRKY1 hairy roots. Cornpared with control roots, CrWRKY1 hairy roots accumulated up to 3-fold higher levels of serpentine. The preferential expression of CrWRKY1 in roots and its interaction with transcription factors including ORCA3, CrMYC2, and ZCTs may play a key role in determining the root-specific accumulation of serpentine in C. roseus plants. [ABSTRACT FROM AUTHOR]

Objective. Primary OA and Kashin–Beck disease (KBD) show similar pathological changes in articular cartilage. The objective was to screen differentially expressed genes between OA and normal cartilage, confirm the candidate gene expression among OA, KBD and normal cartilage, and then clarify its role in vitro.Methods. Differentially expressed genes in OA cartilage were screened by suppression subtractive hybridization (SSH) and verified by real-time quantitative PCR (Q-PCR) analysis. Heparan sulphate 6-O-sulphotransferase 2 (HS6ST2) expression was identified by Q-PCR and immunohistochemistry. After suppressing HS6ST2 by RNA interference in C28/I2 human chondrocyte line, the effects were analysed through determining the cell viability by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), the aggrecan contents by toluidine blue staining and the mRNA expression levels of SRY-type high mobility group box 9 (SOX9), AGC1, MMP3, a disintegrin and metalloproteinase domain with thrombospondin motifs 4 (ADAMTS4) and ADAMTS5 by Q-PCR.Results. HS6ST2 in the reverse subtraction library was identified as a down-regulated gene in OA and KBD at both mRNA and protein levels. The percentage of safranion O staining area was correlated positively with the percentage of HS6ST2-positive chondrocytes in OA and KBD cartilage. After HS6ST2-specific short interfering RNA (siRNA) transfection to C28/I2 cells, the cell viability was inhibited significantly, and the mRNA expression levels of SOX9 and AGC1 were reduced markedly, while MMP3 expression was increased significantly.Conclusion. HS6ST2 down-regulation was identified in both OA and KBD cartilage. The findings first suggest that HS6ST2 may participate in the pathogenesis of OA and KBD by influencing aggrecan metabolism. [ABSTRACT FROM PUBLISHER]

The article presents a study which determined the localization of gene product At5g19500p from Arabidopsis thaliana using in vitro chloroplastic import assay and translation to a cell-free wheat germ system. The study was investigated by mRNA transcription and translation, subfractionation of chloroplasts and electrophoresis. Result showed that At5g19500p was successfully imported into the chloroplasts and its mature form was found inside the envelope membrane.

Soluble sulfotransferases (SULTs) generate electrophilically reactive metabolites from numerous food-borne compounds, environmental contaminants and drugs, often resulting in mutagenicity and carcinogenicity. Substrate specificity, regulation and tissue distribution of SULTs show large interspecies differences. In humans, therefore, SULTs may be involved in the induction of cancer in different tissues than in standard animal models. To construct a rodent model taking some species differences into account, we transferred a 68.5 kb human (h) genomic sequence that comprised the transcribed and long flanking regions of SULT1A1 and 1A2 into murine oocytes. This approach resulted in several mouse lines expressing these human genes in a copy number-dependent manner with a tissue distribution similar to that in humans. In previous in vitro studies, we had demonstrated that human SULT1A1 and 1A2 efficiently catalyze the terminal activation of 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) to a mutagen. The transgenic mice were used to study the hSULT1A1/1A2-mediated activation. Tissue distribution and levels of DNA adducts were determined in hSULT1A1/1A2 transgenic and wild-type mice after an oral dosage of PhIP. Transgenic mice exhibited significantly elevated PhIP–DNA adduct levels compared with the wild-type in liver (13-fold), lung (3.8-fold), colon (2-fold), kidney (1.6-fold) and cecum (1.5-fold). Moreover, among the eight tissues examined, liver was the one with the lowest and highest adduct levels in wild-type and transgenic mice, respectively. Hence, expression of hSULT1A1/1A2 not only enhanced the genotoxicity but also substantially changed the organotropism of PhIP. [ABSTRACT FROM AUTHOR]

Background: protein glycosylation varies with the physiological and pathological status of the cell. Consequently, analysis of protein-linked glycans has growing importance both in basic glycobiological research and as a potential tool for monitoring the physiological state in humans.Design, setting and participants: a total of 265 healthy northern Chinese men and women were grouped by age and gender. The mean age in males and females was similar.Objective: the study is aimed to evaluate the effects of the age and gender on the human serum N-glycans profiles in the clinical diagnose of ageing and disease.Methods: the 265 human serum N-glycan profiles were obtained by DNA sequencer-assisted fluorophore-assisted carbohydrate electrophoresis. Comparison of N-glycan profiles was carried out among the different genders and age groups and the data were analysed with the GeneMapper software.Results: age-related changes in the three N-glycan structures (NGA2F, NGA2FB and NA2F) were observed. Interestingly, fucosylation of N-glycans was significantly different (P < 0.0001) between men and women: more core-α-1,6-fucosylated glycans were detected in women, whereas more branching-α-1,3-fucosylated N-glycans were seen in men.Conclusions: the N-glycome profile in serum is gender and age dependent. This should be taken into consideration in the development of serum glycome markers. [ABSTRACT FROM AUTHOR]

Multilocus sequence typing reveals that many bacterial species have a clonal structure and that some clones are widespread. This underlying phylogeny was not revealed by pulsed-field gel electrophoresis, a method better suited to short-term outbreak investigation. Some global clones are multiresistant and it is easy to assume that these have disseminated from single foci. Such conclusions need caution, however, unless there is a clear epidemiological trail, as with KPC carbapenemase-positive Klebsiella pneumoniae ST258 from Greece to northwest Europe. Elsewhere, established clones may have repeatedly and independently acquired resistance. Thus, the global ST131 Escherichia coli clone most often has CTX-M-15 extended-spectrum β-lactamase (ESBL), but also occurs without ESBLs and as a host of many other ESBL types. We explore this interaction of clone and resistance for E. coli, K. pneumoniae, Acinetobacter baumannii- a species where three global lineages dominate - and Pseudomonas aeruginosa, which shows clonal diversity, but includes the relatively 'tight' serotype O12/Burst Group 4 cluster that has proved adept at acquiring resistances - from PSE-1 to VIM-1 β-lactamases - for over 20 years. In summary, 'high-risk clones' play a major role in the spread of resistance, with the risk lying in their tenacity - deriving from poorly understood survival traits - and a flexible ability to accumulate and switch resistance, rather than to constant resistance batteries. [ABSTRACT FROM AUTHOR]

Aims: This study evaluated a typing method of O26:H11 enterohaemorrhagic and enteropathogenic Escherichia coli (EHEC and EPEC) based on the variation in genomic location and copy numbers of IS 621. Methods and Results: Two multiplex PCRs, targeting either the left (5′) or right (3′) IS/chromosome junction of 12 IS 621 insertion sites and one PCR specific of another truncated copy, were developed. Thirty-eight amplification profiles were observed amongst a collection of 69 human and bovine O26:H11 EHEC and EPEC. Seventy-one per cent of the 45 EHEC and EPEC with identical IS 621 fingerprints within groups of two, three or four isolates had >85% pulsed field gel electrophoresis (PFGE) profile similarity, including four groups of epidemiologically related EHEC or EPEC, while most of the groups had <85% similarity between each others. Epidemiologically related EHEC from each of three independent outbreaks in Japan and Belgium also exhibited identical IS 621 fingerprints and PFGE profiles. Conclusions: The IS 621 fingerprinting and the PFGE are complementary typing assays of EHEC and EPEC; though, the former is less discriminatory. Significance and Impact of the Study: The IS 621 printing method represents a rapid (24 h) first-line surveillance and typing assay, to compare and trace back O26:H11 EHEC and EPEC during surveys in farms, multiple human cases and outbreaks. [ABSTRACT FROM AUTHOR]

Rhamnosidases are enzymes that catalyze the hydrolysis of terminal nonreducing L-rhamnose for the bioconversion of natural or synthetic rhamnosides. They are of great significance in the current biotechnological area, with applications in food and pharmaceutical industrial processes. In this study we isolated and characterized a novel alkaline rhamnosidase from Acrostalagmus luteo albus, an alkali-tolerant soil fungus from Argentina. We also present an efficient, simple, and inexpensive method for purifying the A. luteo albus rhamnosidase and describe the characteristics of the purified enzyme. In the presence of rhamnose as the sole carbon source, this fungus produces a rhamnosidase with a molecular weight of 109 kDa and a pI value of 4.6, as determined by SDS-PAGE and analytical isoelectric focusing, respectively. This enzyme was purified to homogeneity by chromatographic and electrophoretic techniques. Using p-nitrofenil-α- L-rhamnopiranoside as substrate, the enzyme activity showed pH and temperature optima of 8.0 and 55°C, respectively. The enzyme exhibited Michaelis-Menten kinetics, with K and V values of 3.38 mmol l and 68.5 mmol l min, respectively. Neither divalent cations such as Ca, Mg, Mn, and Co nor reducing agents such as β-mercaptoethanol and dithiothreitol showed any effect on enzyme activity, whereas this activity was completely inhibited by Zn at a concentration of 0.2 mM. This enzyme showed the capacity to hydrolyze some natural rhamnoglucosides such as hesperidin, naringin and quercitrin under alkaline conditions. Based on these results, and mainly due to the high activity of the A. luteo albus rhamnosidase under alkaline conditions, this enzyme should be considered a potential new biocatalyst for industrial applications. [ABSTRACT FROM AUTHOR]

BACKGROUND The purpose of this study was to optimize the electrophoretic conditions that should be used for the effective isolation of functional human spermatozoa and to determine whether this method of isolating cells was associated with oxidative stress and DNA damage. METHODS Human spermatozoa were prepared by repeated centrifugation, discontinuous density gradient centrifugation and electrophoresis followed by assessments of sperm quality. RESULTS Systematic analysis of optimal electrophoresis conditions demonstrated that field strength was positively correlated with sperm recovery rates but negatively correlated with sperm movement, irrespective of whether the current or the voltage was held constant. This loss of functionality observed at high power settings was not associated with a major increase in superoxide generation or the induction of oxidative DNA damage. In contrast, discontinuous Percoll gradient centrifugation was shown to produce a significant rise in oxidative DNA base adduct expression in live cells (P < 0.05). As a result of these analyses, optimized electrophoretic conditions were defined that permitted sperm recovery rates of around 20%. These electrophoretically isolated cells were not only free of oxidative stress but exhibited significantly enhanced motility (P < 0.01) and vitality (P < 0.001) compared with the original samples. CONCLUSIONS We conclude that while field strength is positively correlated with sperm recovery rates; it is negatively associated with sperm motility. Optimized conditions are described that represent a balance between these opposing forces and permit the isolation of highly motile, vital sperm populations, free from the oxidative DNA damage associated with conventional density gradient centrifugation technologies. [ABSTRACT FROM PUBLISHER]

The article discusses a study which investigated the production of reactive oxygen species (ROS), as well as its apoptosis induction through Gts1p-mediated protein aggregate formation. The study used agarose gel electrophoresis to examine the degrees of Gts1p-mediated protein aggregation. Results suggested that protein aggregates mediated by Gts1p influence the mitochondrial regulation of ROS apoptosis and production.

Aims: This study applied culture-dependent and molecular approaches to examine the bacterial communities at corrosion sites at Granite Mountain Record Vault (GMRV) in Utah, USA, with the goal of understanding the role of microbes in these unexpected corrosion events. Methods and Results: Samples from corroded steel chunks, rock particles and waters around the corrosion pits were collected for bacterial isolation and molecular analyses. Bacteria cultivated from these sites were identified as members of Alphaproteobacteria, Gammaproteobacteria, Firmicutes and Actinobacteria. In addition, molecular genetic characterization of the communities via nested-polymerase chain reaction-denaturing gradient gel electrophoresis (DGGE) indicated the presence of a broad spectrum of bacterial groups, including Alphaproteobacteria, Betaproteobacteria, Deltaproteobacteria, Actinobacteria, Firmicutes and Bacteroidetes. However, neither cultivation nor molecular approaches identified sulfate-reducing bacteria (SRB), the bacteria commonly implicated as causative organisms were found associated with corrosive lesions in a process referred to as microbially influenced corrosion (MIC). The high diversity of bacterial groups at the corrosion sites in comparison with that seen in the source waters suggested to us a role for the microbes in corrosion, perhaps being an expression of a redox-active group of microbes transferring electrons, harvesting energy and producing biomass. Conclusions: The corrosion sites contained highly diverse microbial communities, consistent with the involvement of microbial activities along the redox gradient at corrosion interface. We hypothesize an electron transport model for MIC, involving diverse bacterial groups such as acid-producing bacteria (APB), SRB, sulfur-oxidizing bacteria (SOB), metal-reducing bacteria (MRB) and metal-oxidizing bacteria (MOB). Significance and Impact of the Study: The characterization of micro-organisms that influence metal-concrete corrosion at GMRV has significant implications for corrosion control in high-altitude freshwater environments. MIC provides a potential opportunity to further our understandings of extracellular electron transfer and interspecies communications. [ABSTRACT FROM AUTHOR]

Background. In the past decade, new methicillin-resistant Staphylococcus aureus (MRSA) strains have emerged as a predominant cause of community-associated skin and soft-tissue infections (SSTIs). Little information exists regarding trends in MRSA prevalence and molecular characteristics or regarding antimicrobial susceptibility profiles of S. aureus isolates. Methods. We enrolled adults with acute, purulent SSTIs presenting to a US network of 12 emergency departments during August 2008. Cultures and clinical information were collected. S. aureus isolates were characterized by antimicrobial susceptibility testing, pulsed-field gel electrophoresis, and toxin genes detection. The prevalence of S. aureus and MRSA and isolate genetic characteristics and susceptibilities were compared with those from a similar study conducted in August 2004. Results. The prevalence of MRSA was 59% among all SSTIs during both study periods; however, the prevalence by site varied less in 2008 (38%-84%), compared with 2004 (15%-74%). Pulsed-field type USA300 continued to account for almost all MRSA isolates (98%). Susceptibility to trimethoprim-sulfamethoxazole, clindamycin, and tetracycline among MRSA isolates remained greater than 90% in 2008. A higher proportion of MRSA infections were treated with an agent to which the infecting isolate was susceptible in vitro in 2008 (97%), compared with 2004 (57%). Conclusions. Similar to 2004, MRSA remained the most common identifiable cause of purulent SSTIs among patients presenting to a network of US emergency departments in 2008. The infecting MRSA isolates continued to be predominantly pulsed-field type USA300 and susceptible to recommended non-β-lactam oral agents. Clinician prescribing practices have shifted from MRSA-inactive to MRSA-active empirical antimicrobial regimens. [ABSTRACT FROM AUTHOR]

Extracellular polymeric substances (EPS) are an important component of microbial biofilms, and it is becoming increasingly apparent that extracellular DNA (eDNA) has a functional role in EPS. This study characterizes the eDNA extracted from the novel activated sludge biofilm process of aerobic granules. Exposing the sludge to cation exchange resin (CER) was used for the extraction of eDNA and intracellular DNA (iDNA) from aerobic granules. This was optimized for eDNA yield while causing minimal cell lysis. We then compared the DNA composition of these extractions using randomly amplified polymorphic DNA (RAPD) fingerprinting and PCR-based denaturing gradient-gel electrophoresis (DGGE). Upon the analysis of the genomic DNA and the 16S rRNA genes, differences were detected between the sludge biofilm eDNA and iDNA. Different bacteria within the biofilm disproportionally release DNA into the EPS matrix of the biofilm. The findings further the idea that eDNA has a functional role in the biofilm state, which is an important conceptual information for industrial application of biofilms. [ABSTRACT FROM AUTHOR]