Prevalence and Study of the Clonality of Extended-Spectrum Beta-Lactamase-Producing Klebsiella pneumoniae Strains in Neonatology at the University Hospitals of Abidjan by the Pulsed Field Gel Electrophoresis and the Quantitative Antibiogram

Background: ESBL-producing strains of Klebsiella pneumoniae, one of the main causes of nosocomial and hospital-acquired infections, are commonly associated with therapeutic impasses. Surveillance of these multidrug-resistant pathogens is a crucial tool for controlling and preventing infections. This surveillance involves the use of appropriate molecular and phenotypic typing techniques. The choice of techniques is based on criteria such as discriminatory power, intra- and inter-laboratory reproducibility, epidemiological concordance, ease of use and cost. The aim of our study was to identify clusters of Extended-Spectrum Beta-Lactamase-producing Klebsiella pneumoniae (ESBL-K. pneumoniae) strains circulating in neonatology using quantitative antibiogram (QA) and Pulsed Field Gel Electrophoresis (PFGE). Materials and Methods: This cross-sectional study included 55 K. pneumoniae strains isolated from a total of 513 samples. These various samples are taken from newborns, healthcare personnel, and the environment. K. pneumoniae identification followed standard bacteriological procedures and was confirmed using the Vitek® 2 (bioMérieux). The detection of the ESBL phenotype was performed using the synergy test. QA and PFGE were used to identify clonal relationships between the various strains isolated. Concordance between these two methods was assessed by calculating Cohen’s KAPPA coefficient and Simpson’s diversity index. Results: Among the 55 K. pneumoniae strains included in this study, 58.2% (32/55) were found to be Extended-Spectrum Beta-Lactamase (ESBL) producers. Most of these strains were isolated from neonatal samples (blood samples and rectal swabs). The quantitative antibiogram method applied to 28 out of the 32 ESBL-producing strains revealed that the isolates were grouped into 5 clusters. Pulsed Field Gel Electrophoresis performed on a total of 16 ESBL-producing strains showed the existence of four profiles. A perfect concordance was observed between the two methods. Conclusion: The results of this study highlighted the existence of clonal strains of various origins within neonatology units.

Comparison between Four Types of Buffers for DNA Agarose Gel Electrophoresis

To detect the genome of viruses (in environmental and clinical samples), we use electrophoresis running buffer after PCR reaction. Also, electrophoresis buffers were used widely to separate any DNA molecule. In this paper, we used four types of previously known electrophoresis buffers to compare which is easy for preparation, simple in structure, low cost and good performance in agarose gel electrophoresis. For this, we used two agarose concentration (1%, 2%) and two types of DNA ladder (100 bp, 1 kb) represent both smaller and larger sizes of molecule for each type of buffers, from the result we found in first level both supper buffer and TAE buffer with good performance and in second level we found bicarbonate buffer also with good performance also. Finally, we found the tang buffer cannot pose any electrophoretic activity on DNA agarose gel electrophoresis.

Analysis of Resistance-related Proteins in Rice Against Brown Planthopper by Two-dimensional Electrophoresis

A recombinant inbred population (RI) was constructed from a cross between B5, an introgression. line from the wild rice Oryza officinalis Wall. ex Watt, and susceptible cultivar Minghui 63 ( O. sativa L.). The brown planthopper ( BPH) resistances of RI lines were evaluated. Based on bulked segregant analysis (BSA), two protein bulks were made by extracting proteins from equally mixed seedlings of extremely resistant and susceptible plants selected from the RI population, respectively. Two-dimensional electrophoresis was used to detect the changes of polypeptide pattern. Results showed that a protein P40 ( pI 6.3, Mw 40 kD) was significantly reduced or vanished after BPH infestation for 48 h in the susceptible bulk, while it remained uninfluenced in the resistant bulk. In connection with the physiological changes of the resistant and susceptible lines subjected to BPH sucking, we suppose that the protein P40 is related to the interaction responses of lice plants to BPH infestation.

Research on Sex and Tissue Differences in Isozymic Phenotype of Varicorhinus macrolepis through Isozyme Electrophoresis

Polyacrylamide gel electrophoresis (PAGE) and biochemical staining method were used in this study for the analysis on malate dehydrogenase (MDH,E.C. 1.1.1.37) isozymes zymogram in 11 different types of tissues of male and female Varicorhinus macrolepis. It had been found for the first time that the phenotype of malate dehydrogenase (MDH),acid phosphatase (ACP) and superoxide dismutase (SOD) showed difference between male and female V. macrolepis,and there was no difference among different individuals in the same sex. Therefore,the electrophoresis band of malate dehydrogenase,acid phosphatase and superoxide dismutase could be used as an indicator for the identification of gender and tissues of V. macrolepis,which would provide basic data for the developmental genetics,variety improvement and directed breeding of V. macrolepis groups,thus facilitating the development and protection of this valuable fish species.

Study on the Optimization of Two-dimensional Electrophoresis Technology System for Rapeseed Proteome

[Objective] The research aimed to establish the two-dimensional electrophoresis（2-DE）technology which was suitable for the rapeseed proteome research.[Method] Xiangyou 17 was as the material.The sample preparation method,gel concentration and loading amount,etc.in 2-DE technology were optimized.[Result] The best extraction method of total protein of rapeseed was TCA-acetone method,and the protein spots on 2-DE map were the most.When IPG strip（pH 3-10）and 12% gel were used,and the loading amount was 250 μg,the two-dimensional electrophoresis map with the clear background,good repeatability and high protein spot resolution was obtained.[Conclusion] The research laid the foundation for carrying out the rapeseed proteomics research.

Single Cell Gel Electrophoresis Assay of Porcine Leydig Cell DNA Damage Induced by Zearalenone

[Objective] This study aimed to investigate the effect of zearalenone (ZEN) on DNA damage of porcine leydig cells. [Method] Porcine leydig cells cultured in vitro were collected to determine the median lethal dose (LD50) of ZEN with tetrazolium-based colorimetric assay (MTT assay). Comet assay was carried out to detect the DNA damage of porcine leydig cells exposed to at 0 (negative group), 1, 5, 10, 20, 40 μmol/L of ZEN. [Result] The percentage of cell tail was 16.67%, 34.00%, 40.67%, 52.00% and 64.67% under 0, 1, 5, 10 and 20 μmol/L of ZEN, respectively; the differences between the percentages of cell tail in various experimental groups had extremely significant statistical significance compared with the negative group (P<0.01), showing a significant dose-effect relationship; Tail length in various groups was 57.60±4.78, 57.75±6.25, 78.97±5.83, 100.50±6.94 and 146.83±12.31 μm, respectively; Tail DNA % in various groups was 21.29±2.25%, 22.24±2.43%, 31.21±6.27%, 37.45±4.33% and 60.68±9.83%, respectively; Tail length and Tail DNA % in experimental groups with ZEN concentration above 5 μmol/L showed significant differences (P<0.05) compared with the negative group, which showed an upward trend with the increase of ZEN concentration. [Conclusion] ZEN has genotoxic effect on porcine leydig cells, which can cause DNA damage, with a significant dose-effect relationship.

Interactions Between Thrombin and Natural Proudcts of Coreopsis tinctoria Nuttt. and Cistanche deserticola Ma. in Capillary Zone Electrophoresis

Aim A capillary zone electrophoretic method （CZE） was used to determine the interactions between natural products and thrombin. Methods Samples containing natural products and thrombin at various ratios were incubated at 25 ℃ and then were separated by CZE with Tris-acetate buffer at pH 7.2. Each run could be accomplished within 5 min. Results In CZE, the peak width broadened due to the affinity interaction between natural products and thrombin. Compared with positive and negative control, the natural products （CB-1, CB-2） from Coreopsis tinctoria Nuttt. interacted with thrombin; CB-3 from Coreopsis tinctoria Nuttt. and HC-1, HC-2, HC-3 from Cistanche deserticola Ma. did not bind to thrombin. Both qualification and quantification characterizations of the binding were determined. Conclusion The established method is capable of sensitive and fast determination of natural products and thrombin interactions, it can be employed as an alternative method.

Optimization of SSR-PCR Non-denatured Polyacrylamide Gel Electrophoresis Conditions in Kernelled Apricot

[Objective] The aim was to optimize the SSR-PCR non-denatured polyacrylamide gel electrophoresis conditions in kernelled apricot.[Method]25 accessions of kernelled apricot and three accessions of edible apricot were selected as experimental materials to screen the repeatable SSR loci with high polymorphism by the use of SSR markers combined with non-denatured polyacrylamide gel electrophoresis.And the effect of different factors on electrophoresis conditions was compared to explore the optimal SSR-PCR non-denatured polyacrylamide gel electrophoresis conditions in kernelled apricot.[Result]The optimal non-denatured polyacrylamide gel electrophoresis conditions for SSR-PCR were established as follows：polyacrylamide gel concentration 6%,the ratio of acrylamide to bisacrylamide 29∶1,electrophoresis at 1 000 V for 2-3 h,and staining for 15 min within 0.1% AgNO3.[Conclusion]The optimum electrophoresis system has provided some technical foundations to further study the phylogenetic relationship of kernelled apricots by SSR markers.

The Superiority of Like-rocket Immunoelectrophoresis Using in Single Cell Gel Electrophoresis Assay

[Objective] The like-rocket immunoelectrophoresis was used to explore a new feasible electrophoresis method for single cell gel electrophoresis assay （comet assay）.[Method] The like-rocket immunoelectrophoresis was used for single cell gel electrophoresis assay to detect DNA damage at single cell level,then it was compared with traditional electrophoresis method to analyze its advantage and disadvantages.[Result] Under cell DNA undamaged state,the results of two electrophoresis methods were consistent.When cell DNA was damaged,the comet tail divergence of some cells under traditional electrophoresis method were drifted,however,the comet tail image of like-rocket immunoelectrophoresis was concentrated and not shifted.[Conclusion] The like-rocket immunoelectrophoresis had some advantages.

Enantioseparation of Several PharmaceuticalRacemates with High PerformanceCapillary electrophoresis

With high performance capillary electrophoresis using -cyclodextrin or its deriveatives as the chiral selectors, five pairs of drug enantiomers were separated, The PH of the back- gmund electrolyte and the chiral selector concentrations were optimized; and the effect of organic modifier on separation of chlorpheniramine enantiomers was also inver

Electrophoresis Analysis on the Protein Expression in Heteromorphic Leaves of Populus euphratica Oliv.

[Objective] This study was to elucidate the cellular and molecular mechanism of the development of heteromorphic leaves of Populus euphratica Oliv. [Method] By employing SDS-PAGE and 2-demensional electrophoresis (2-DE) techniques,proteins in various heteromorphic leaves from the same adult tree of P. euphratica were isolated and separated to the electrophoresis technique suitable for the separation and analysis of proteins in leaves of P. euphratica tree. [Results] There were significant differences in the expressions of proteins in various heteromorphic leaves of P. euphratica tree. SDS-PAGE pattern showed that bands of proteins with molecular weight of 57.2,13.2,30.2,23.9 and 33.3 kDa were remarkably different. 2-D electrophoresis pattern presented that proteins in leaves of P. euphratica tree mainly belong to acidic proteins distributed at pH value of 5.0-6.5 and with molecular weight of 20-40 kDa; totally 73 different protein spots were observed,of which 51 were up expressed and other 22 were down expressed in the serrated ovate leaves. [Conclusion] Based on these results,we speculate that regulated gene expression in leaves of P. euphratica tree results in the generation of different shapes of leaves,in order to adapt to the surroundings better.

Electrophoresis of poly(dT)_(20) through α-hemolysin nanopore in high concentration potassium chloride solution

Experiments of poly（dT）20 electrophoresis throughα-hemolysin nanopores were performed to unveil the electrophoretic transport mechanism of DNA through nanopores in high concentration potassium chloride solution. It is found that there are two obvious current blockades induced by poly（dT）20 translocation and collision events. Both blockade currents increase linearly with the applied bias voltage. However, the normalized blockade currents are almost kept the same although variable bias voltages are applied. The collision time of poly（dT）20 in the luminal site of the pore remains constant for different voltages. The translocation speed of poly（dT）20through the nanopore decreases with the increase of bias voltage. It is because as the potential increases, the drag force on the homopolymer helps it to crumple into a cluster much easier due to the poor stacking of thymine residues compared with homopolymers consisting of other nucleotides. Molecular dynamics simulations further confirm the experimental results. Increasing the applied bias voltage can slowdown the translocation velocity of the flexible poly（dT）20, which favors increasing the precision of single molecule detection by using nanopores.

A Peptides Migration Model in capillary Zone Electrophoresis

A peptides migration model based on the principle of mechanics is presented in capillary zone electrophoresis (CZE). It is shown that the migration that the (tr) is a function of electric (Q), relative molecular mass (Mr), conformation parameter (Rc) of peptides and electrophoretic condition parameter(A). The conformation parameter is introduced to characterize multifarious shapes owing to the complex conlormation and the various kinds of macromolecules, where Rc≥1/3. The parameters A and Rc can be obtained from experimental data. The times of migration of the nine standard peptides were measured in pH 2.5buffer on different electrophoretic conditions in CZE. The experimental results agreed well with the theoretical prediction.

Separation of Nucleotides in Swine Tissues Using Capillary Zone Electrophoresis

Capillary zone electrophoresis has been applied to the analysis of nucleotides. The effects of buffer concentration. pH and other operating conditions on the separation were investigated and optimized. By using the method, separation and identification of nuclotides in swine tissues were completed.

AN ALLOZYME ELECTROPHORESIS STUDY ON ELEVEN SPECIES OF MEGOPHRYINAE IN CHINA

Allozymes of eleven species of Megophryinae in China were examined electrophoretically to investigate genetic diversity and phylogenetic relationships. Fourteen enzymes, presumptively coded by 24 loci were detected to be variable. Gene frequencies of each population at each locus were presented. The commonly used measure of genetic diversity, the average heterozygosity (H) were calculated based on gene frequencies. The results indicated that Megophryinae had a high level of genetic diversity in amphibians, an average H of 0.18, ranging from 0.058 to 0.28. Nei's (1978) genetic distances(Nei's D) were calculated for all possible population pairs. A dendrogram of 13 populations representing 11 species, 3 genera of Megophryinae were derived and presented by using UPGMA, based on Nei' s D. The assignment of Ophryophryne as a distinct genus were supported by an average Nei's D of 1.4067 which separated O. microstoma from all other populations.Subdivision of Brachytarsophrys from Megophrys was not supported by this study. Within Megophrys, three groups were recognized: (1)M. lateralis, M. giganticus and M. longipes; (2)M. palpebralespineosa, M. boettgeri and M. parva;(3) M. minor and M. kuatunensis. Three populations of M. omeimontis were closely related and share a clade independent from all other Megophrys, and B. feae as well.

Simultaneous Determination of Clonidine Hydrochloride,Hydrochlorothiazide, and Rutin in Zhenju Jiangya Tablet by Capillary Electrophoresis

Aim To develop a method for the determination of three drug components： clonidine hydrochloride, hydrochlorothiazide and rutin in Zhenju Jiangya tablet. Methods An uncoated capillary tube was used to analyze under 20 kV voltage at 20 ℃. The samples were introduced into the capillary tube by hydrodynamic mode applying 50 kPa for 5 s and detected at 210 nm. Results The linear ranges of clonidine hydrochlofide, hydrochlorothiazide, and rutin were 10 μg· mL^-1 - 100μg· mL^-1, 30μg· mL^-1 - 300 μg· mL^- 1, and 30μg · mL^-1 - 300μg · mL^-1, respectively. Inter-day and intra-day RSD were all below 10.5%. The recoveries were 94.96% for clonidine hydrochloride, 84.45% for hydroehlorothiazide, and 89.88 % for rutin. Conclusion Clonidine hydrochloride, hydrochlorothiazide, and rutin are baseline separated. The method is simple and rapid for simultaneous determination of the three drug components in Zhenju Jiangya tablet.

Optimization of Multiplex PCR and Multiplex Gel Electrophoresis in Sunflower SSR Analysis Using Infrared Fluorescence and Tailed Primers

In an effort to simplify the procedure and to reduce the cost of fluorescence SSR analysis, the conditions of the multiplex PCR and the multiplex gel electrophoresis were optimized in the genetic analysis of sunflower (Helianthus annuus L.) inbred lines. Results indicated that factors for a successful multiplex PCR assay were related to the cycling touchdown annealing temperature, the balance of primer concentration at the various loci, the concentration of PCR buffer and the Taq DNA polymerase. Based on the optimization, a tailed primer strategy was outlined, and the effective ways were proposed to overcome the troubleshootings commonly encountered in the multiplex PCR and the multiplex gel electrophoresis.

A New Capillary Electrophoresis Apparatus with Piezoelectric Ceramics High Voltage Source and Amperometric Detector

A new capillary electrophoresis apparatus was designed. Piezoelectric ceramics transformer technology was first applied in capillary electrophoresis, a high voltage and stable source was made. Amperometric detector was used in which the working electrode was closely opposite to the end of capillary. The apparatus was characterized in good reproducibility, safety and very low cost.

Optimization of Two-Dimensional Gel Electrophoresis for Kenaf Leaf Proteins

To establish a suitable and effective protocol of protein extraction for two-dimensional gel electrophoresis （2-DE） analysis in kenaf leaf tissues, three extraction methods （trichloroacetic acid/acetone, urea/thiourea, and phenol extraction methods） were applied to the extraction of kenaf leaf protein. The results were compared in regard to protein extraction efficiency, sodiumdodecyl sulfate-polyacrylamide gel electrophoresis （SDS-PAGE）, and 2-DE gels. Furthermore, the 2-DE system was optimized for four aspects： the pH range of IPG （immobilized pH gradient） stripes, sampling methods, sample volumes, and concentration of polyacrylamide gels. The data presented showed that the phenol extraction method is the best method to perform 2-DE analysis of kenaf leaf protein. The protein extracted from phenol extraction method reached the purity of （26.40±0.859）%, showed （25.67±1.53） protein bands in one dimension SDS-PAGE gels, and （1 374±54.44） protein spots on 2-DE gels. The research also indicates that kenaf leaf protein spots were distributed mainly within the pH range of 4-8. More clear background with a better distribution effect and many protein spots could be obtained on 2-DE gels under the conditions of active rehydration loading, 24 cm IPG strips （linear pH gradient of 4-7）, 1.4 mg samples, and 12% SDS-PAGE gels.

Simultaneous Detection of 13 Key Bacterial Respiratory Pathogens by Combination of Multiplex PCR and Capillary Electrophoresis

Objective Lower respiratory tract infections continue to pose a significant threat to human health. It is important to accurately and rapidly detect respiratory bacteria. To compensate for the limits of current respiratory bacteria detection methods, we developed a combination of multiplex polymerase chain reaction （PCR） and capillary electrophoresis （MPCE） assay to detect thirteen bacterial pathogens responsible for lower respiratory tract infections, including Streptococcus pneumoniae, Haemophilus influenzae, Moraxella catorrholis, Pseudomonas aeruginosa, Klebsiella pneumoniae, Escherichia coli, Staphylococcus aureus, Mycoplasma pneumoniae, Legionella spp., Bordetella pertussis, Mycobacterium tuberculosis complex, Corynebactefium diphthefiae, and Streptococcus pyogenes. Methods Three multiplex PCR reactions were built, and the products were analyzed by capillary electrophoresis using the high-throughput DNA analyzer. The specificity of the MPCE assay was examined and the detection limit was evaluated using DNA samples from each bacterial strain and the simulative samples of each strain. This assay was further evaluated using 152 clinical specimens and compared with real-time PCR reactions. For this assay, three nested-multiplex-PCRs were used to detect these clinical specimens. Results The detection limits of the MPCE assay for the 13 pathogens were very low and ranged from 10-7 to 10-2 ng/μL. Furthermore, analysis of the 252 clinical specimens yielded a specificity ranging from 96.5%-100.0%, and a sensitivity of 100.0% for the 13 pathogens. Conclusion This study revealed that the MPCE with high specificity and sensitivity. This assay survey of respiratory pathogens. assay is a rapid, reliable, and high-throughput method has great potential in the molecular epidemiological.