S1 Nuclease Digestion Research Articles

C5-Propynyl modified 2'-fluoroarabinonucleic acids form stable duplexes with RNA that are RNase H competent.

The clinical success of the antisense approach for the treatment of genetic disorders is indisputably the result of chemical modifications along the oligonucleotide (ON) scaffold, which impart desirable properties including high RNA affinity, nuclease stability and improved drug delivery. While effective, many modifications are not capable of eliciting an RNase H response limiting their application in antisense systems. To contribute to the structural design and inventory of nucleoside analogues with favorable antisense properties, herein we describe the synthesis of C5-propynyl-2'-fluoroarabinonucleic acids (FANAP). Incorporation of individual and multiple uridine (FaraUP) and cytidine (FaraCP) inserts into ONs revealed, both stabilized duplexes formed with RNA. In contrast, these modifications demonstrated a negligible (FaraUP) or reduced (FaraCP) effect on DNA binding. Moreover, modified ONs containing these analogues supported E. coli RNase H cleavage of RNA with an altered cleavage pattern observed relative to controls. Moreover, a 2'-O-methoxyethyl (2'-O-MOE) gapmer with a FANAP core was able to elicit RNA cleavage at an increased rate compared to C5-propynyl-arabinonucleic acids (ANAP). Enzymatic hydrolysis of these gapmers was assessed with nuclease S1 digestion and revealed greater stability of ANAP compared to FANAP. These results suggest C5-propynyl ANA/FANA modifications demonstrate promising potential for the design of therapeutic ONs.

Potentially conjugative plasmids harboring Tn6636, a multidrug-resistant and composite mobile element, in Staphylococcus aureus.

This study aimed to provide detailed genetic characterization of Tn6636, a multidrug-resistant and composite mobile element, in clinical isolates of Staphylococcus aureus. A total of 112 ermB-positive methicillin-susceptible S. aureus (MSSA) and 224 ermB-positive methicillin-resistant S. aureus (MRSA) isolates collected from 2000 to 2015 were tested for the presence of Tn6636. Detection of the plasmids harboring Tn6636 was performed by S1 nuclease digestion pulsed-field gel electrophoresis (PFGE) analysis, conjugation test, and whole genome sequencing (WGS). Prevalence of Tn6636 in MSSA is higher than that in MRSA. Ten MSSA isolates and 10 MRSA isolates carried Tn6636. The 10 MSSA isolates belonged to three sequence types (ST), including ST7 (n=6), ST5 (n=3), and ST59 (n=1). The 10 MRSA isolates belonged to ST188 (n=8) and ST965 (n=2). Analysis of plasmid sequences revealed that Tn6636 was harbored by six different mosaic plasmids. In addition to resistance genes, some plasmids also harbored toxin genes. The presence of multi-resistant Tn6636 in plasmids of both MSSA and MRSA with various STs suggests its broad dissemination. Results indicate that Tn6636 has existed for at least 16 years in Taiwan. The mosaic plasmids harboring Tn6636 can be transferred by conjugation. Ongoing surveillance of Tn6636 is essential to avoid continued spreading of resistant plasmids.

Characterization of Three Porcine Acinetobacter towneri Strains Co-Harboring tet(X3) and blaOXA-58.

Tigecycline is the antibiotic of last resort for the treatment of extensively drug-resistant bacterial infections, mainly those of multidrug-resistant Gram-negative bacteria. The plasmid-mediated tet(X3) gene has recently been described in various pathogens that are resistant to tigecycline. We report three tigecycline-resistant Acinetobacter towneri strains isolated from porcine faeces in China, which all contained the tet(X3)-harboring plasmids. A broth microdilution method was used to examine the antimicrobial susceptibility of the isolates, and S1-Nuclease digestion pulsed-field gel electrophoresis (S1-PFGE) was used to characterize their plasmid profiles. The whole-genome sequences of the isolates were determined with the Nanopore PromethION platform. The sequence analysis indicated that the strains were A. towneri. They showed resistance to multiple antibiotics, and all the resistance genes were located on plasmids. The three tet(X3)-harboring plasmids had a similar backbone structure, and all contained bla OXA-58 with various insertion elements (IS). ISCR2 is considered an important factor in tet(X3) mobilization. In addition to ISCR2, we demonstrate that IS26 generates a circular intermediate containing the tet(X3) gene, which could increase the dissemination risk. To our knowledge, this is the first report of tet(X3)- and bla OXA-58-harboring plasmids in A. towneri. Because the IS26 is frequently found in front of tet(X3), research should be directed toward the action of IS26 in the spread of tet(X3).

BlaCTX-M-1/IncI1-Iγ Plasmids Circulating in Escherichia coli From Norwegian Broiler Production Are Related, but Distinguishable.

Escherichia coli carrying blaCTX–M–1 mediating resistance to extended-spectrum cephalosporins was recently described as a new genotype in Norwegian broiler production. The aim of this study was to characterize these isolates (n = 31) in order to determine whether the emergence of the genotype was caused by clonal expansion or horizontal dissemination of blaCTX–M–1-carrying plasmids. All included isolates were subjected to whole genome sequencing. Plasmid transferability was determined by conjugation, and plasmid replicons in the transconjugants were described using PCR-based replicon typing. Plasmid sizes were determined using S1 nuclease digestion. Plasmids in a subset of strains were reconstructed and compared to plasmids from broiler production in other European countries. The isolates belonged to nine different sequence types (STs), with the largest group being ST57 (n = 12). The vast majority of blaCTX–M–1-carrying plasmids were conjugative. All transconjugants were positive for the IncI1-Iγ replicon, and several also harbored the IncFIB replicon. Highly similar plasmids were present in different E. coli STs. Additionally, high similarity to previously published plasmids was detected. A reconstructed plasmid from an ST57 isolate harbored both IncI1-Iγ and IncFIB replicons and was considered to be co-integrated. The presence of one large plasmid was confirmed by S1 nuclease digestion. Our results show that dissemination of blaCTX–M–1 in Norwegian broiler production is due to both clonal expansion and horizontal transfer of plasmids carrying blaCTX–M–1. The blaCTX–M–1/IncI1-Iγ plasmids grouped into two main lineages, namely clonal complex (CC)-3 and CC-7. The genetic diversity at both strain and plasmid level indicates multiple introductions to Norway. We also show that the blaCTX–M–1 plasmids circulating in Norwegian broiler production are highly similar to plasmids previously described in other countries.

Transmission of mcr-1-Producing Multidrug-resistant Enterobacteriaceae in Public Transportation in Guangzhou, China.

mcr-1-mediated colistin resistance in bacteria is concerning, as colistin is used in treating multidrug-resistant bacterial infections. And mcr-1-producing bacteria have been identified in multiple sources. Up to 248 million people use public transportation daily in China, however; public transportation hasn't been studied as a potential source of community-based transmission of mcr-1. Herein we investigated mcr-1-producing isolates from public transportation and explored the genomic characteristics of them. Surface samples were collected from public transportation in Guangzhou, China, from October 2016 to April 2017. Polymerase chain reaction was performed to detect mcr-1 gene, plasmid replicon type and phylogenetic group. Minimum inhibitory concentrations (MICs) were determined by microdilution method. S1-nuclease digestion and pulsed-field gel electrophoresis (S1-PFGE) and Southern blotting were performed with mcr-1-harboring plasmids. Whole-genome sequencing was performed with mcr-1-producing isolates. Of the 737 samples with bacterial growth, 26 isolates were positive for mcr-1, including 23 Escherichia coli and 3 Klebsiella pneumoniae isolates. The E. coli isolates belonged to phylogroups A and B1. Most mcr-1-producing isolates were resistant to ampicillin (25), cefotaxime (21), fosfomycin (16), and gentamicin (15). S1-PFGE, Southern blotting and replicon typing showed that mcr-1 was mainly located on ~33.3 kb to ~220 kb IncX4, IncI2 and IncHI2 plasmids in E. coli, while located on ~33.3 kb untyped plasmid in K. pneumoniae. Several sequence types (ST), including ST2253, ST101, ST10 complex and ST37, were revealed. Between 53 and 66 (mean = 61.8) resistance genes were identified among mcr-1-producing isolates. Public transportation may serve as a source of mcr-1-producing bacteria.

Prevalence, Serotyping, Molecular Typing, and Antimicrobial Resistance of Salmonella Isolated From Conventional and Organic Retail Ground Poultry.

Ground poultry is marketed as a healthier alternative to ground beef despite the fact that poultry is a major source of foodborne Salmonella. The objectives of this study were to determine the prevalence of Salmonella in Oklahoma retail ground poultry and to characterize representative isolates by serotyping, antimicrobial resistance, PFGE patterns, and large plasmid profiling. A total of 199 retail ground poultry samples (150 ground turkey and 49 ground chicken) were investigated. The overall prevalence of Salmonella in ground poultry was 41% (82/199), and the incidence in conventional samples (47%, 66/141) was higher than in organic samples (27%, 16/58). The prevalence of Salmonella in organic ground chicken and organic ground turkey was 33% (3/9) and 26% (13/49), respectively. Twenty six Salmonella isolates (19 conventional and 7 organic) were chosen for further characterization. The following six serotypes and number of isolates per serotype were identified as follows: Tennessee, 8; Saintpaul, 4; Senftenberg, 4; Anatum, 4 (one was Anatum_var._15+); Ouakam, 3; and Enteritidis, 3. Resistance to 16 tested antimicrobials was as follows: gentamycin, 100% (26/26); ceftiofur, 100% (26/26); amoxicillin/clavulanic acid, 96% (25/26); streptomycin, 92% (24/26); kanamycin, 88% (23/26); ampicillin, 85% (22/26); cephalothin, 81% (21/26); tetracycline, 35% (9/26); sulfisoxazole, 27% (7/26); nalidixic acid, 15% (4/26); and cefoxitin, 15% (4/26). All isolates were susceptible to amikacin, chloramphenicol, ceftriaxone, and trimethoprim/sulfamethoxazole. All screened isolates were multidrug resistant (MDR) and showed resistance to 4–10 antimicrobials; isolates from organic sources showed resistance to 5–7 antimicrobials. PFGE was successful in clustering the Salmonella isolates into distinct clusters that each represented one serotype. PFGE was also used to investigate the presence of large plasmids using S1 nuclease digestion. A total of 8/26 (31%) Salmonella isolates contained a ∼100 Kb plasmid that was present in all Anatum and Ouakam isolates. In conclusion, the presence of multidrug resistant Salmonella with various serotypes, PFGE profiles, and large plasmids in ground poultry stresses the importance of seeking novel interventions to reduce the risk of this foodborne pathogen. Multidrug resistance (MDR) is considered a high additional risk and continued surveillance at the retail level could minimize the risk for the consumer.

Excision of the doubly methylated base N4,5-dimethylcytosine from DNA by Escherichia coli Nei and Fpg proteins.

Cytosine (C) in DNA is often modified to 5-methylcytosine (m5C) to execute important cellular functions. Despite the significance of m5C for epigenetic regulation in mammals, damage to m5C has received little attention. For instance, almost no studies exist on erroneous methylation of m5C by alkylating agents to doubly or triply methylated bases. Owing to chemical evidence, and because many prokaryotes express methyltransferases able to convert m5C into N4,5-dimethylcytosine (m N4,5C) in DNA, m N4,5C is probably present in vivo We screened a series of glycosylases from prokaryotic to human and found significant DNA incision activity of the Escherichia coli Nei and Fpg proteins at m N4,5C residues in vitro The activity of Nei was highest opposite cognate guanine followed by adenine, thymine (T) and C. Fpg-complemented Nei by exhibiting the highest activity opposite C followed by lower activity opposite T. To our knowledge, this is the first description of a repair enzyme activity at a further methylated m5C in DNA, as well as the first alkylated base allocated as a Nei or Fpg substrate. Based on our observed high sensitivity to nuclease S1 digestion, we suggest that m N4,5C occurs as a disturbing lesion in DNA and that Nei may serve as a major DNA glycosylase in E. coli to initiate its repair.This article is part of a discussion meeting issue 'Frontiers in epigenetic chemical biology'.

Molecular epidemiology of blaOXA-23-producing carbapenem-resistant Acinetobacter baumannii in a single institution over a 65-month period in north China

BackgroundCarbapenem-resistant Acinetobacter baumannii poses a significant threat to hospitalized patients, as few therapeutic options remain. Thus, we investigated the molecular epidemiology and mechanism of resistance of carbapenem-resistant A.baumannii isolates in Beijing, China.MethodsCarbapenem-resistant A.baumannii isolates (n = 101) obtained between June 2009 and November 2014 were used. Multilocus sequence typing (MLST) and PCR assays for class C and D β-lactamase were performed on all isolates. S1 nuclease pulsed-field gel electrophoresis (PFGE) and Southern blot hybridization were performed to identify the resistance gene location.ResultsAll 101 A.baumannii isolates were highly resistant to frequently used antimicrobials, and were considered multidrug resistant. A total of 12 sequence types (STs) were identified, including 10 reported STs and 2 novel STs. Eighty-seven isolates were classified to clonal complex 92 (CC92), among which ST191 and ST195 were the most common STs. The blaOXA-23 gene was positive in most (n = 95) of the A.baumannii isolates. Using S1-nuclease digestion PFGE and Southern blot hybridization, 3 patterns of plasmids carrying blaOXA-23 were confirmed. ST191 and ST195 (both harboring blaOXA-23) caused outbreaks during the study period, and this is the first report of outbreaks caused by ST191 and ST195 in north China.ConclusionblaOXA-23-producing A.baumannii ST191 and ST 195 isolates can disseminate in a hospital and are potential nosocomial outbreak strains. Surveillance of imipenem-resistant A.baumannii and antimicrobial stewardship should be strengthened.

Characterisation of multidrug-resistant Shiga toxin-producing Escherichia coli cultured from pigs in China: co-occurrence of extended-spectrum β-lactamase- and mcr-1-encoding genes on plasmids

Identification of Enterobacteriaceae harbouring the plasmid-mediated transferable colistin resistance gene mcr-1 presents a new challenge to public health. The aim of this study was to characterise multidrug-resistant Shiga toxin-producing Escherichia coli (STEC) harbouring the mcr-1 gene on plasmids cultured from pigs in China. Using CHROMagar™ ECC plates combined with stx gene detection by PCR, 93 STEC were recovered from 326 faecal, 351 small intestine content and 326 colon content samples taken from healthy pigs in 2011 and 2012 in China. This study, in which ten colistin-resistant isolates with minimum inhibitory concentrations (MICs) of 8–12 mg/L were identified and found to be positive by PCR for the mcr-1 gene, is a follow-up to an earlier investigation. Plasmid profiling by S1-nuclease digestion followed by pulsed-field gel electrophoresis (PFGE) identified several high-molecular-weight plasmids and these were typed by PCR-based replicon typing (PBRT). Two of the ten isolates, namely STEC-CQ09 (O116:H11/CC23/ST88) and CQ10 (O2:H32/ST3628), were selected for further study as described in this report.

Characterization of Extended-Spectrum β-Lactamase-Carrying Plasmids in Clinical Isolates ofKlebsiella pneumoniaefrom Taiwan

We analyzed the replicon types, sizes, and restriction fragment length polymorphism (RFLP) typing of plasmids carrying extended-spectrum β-lactamase (ESBL) genes in Klebsiella pneumoniae isolates from Taiwan. Fifty-one Escherichia coli transconjugant strains with plasmids from ESBL-producing K. pneumoniae from the Taiwan Surveillance of Antimicrobial Resistance III Program in 2002 were included. All the 51 plasmids carried a blaCTX-M gene, the majority of which were blaCTX-M-3 (28/51 [54.9%]). Plasmids ranged in size from 126 to 241 kb by S1 nuclease digestion and subsequent pulsed-field gel electrophoresis, and the most common plasmid size (37.3%) was 161-170 kb. The most common replicon type of plasmids was incompatibility group (Inc)A/C (60.8%). The IncA/C plasmids all carried blaCTX-M (blaCTX-M-3, -14, -15), and some also carried blaSHV (blaSHV-5, -12) genes. All 51 plasmids could be typed with PstI, and 27 (52.9%) belonged to 10 clusters. Thirty-eight of the 51 plasmids were typable with BamHI, and 21 plasmids (55.3%) fell into 7 clusters. Plasmids in the same cluster belonged to the same incompatibility group, with the exception of cluster C6. In conclusion, IncA/C plasmids are the main plasmid type responsible for the dissemination of ESBL genes of K. pneumoniae from Taiwan. RFLP with PstI possessed better discriminatory power than that with BamHI and PCR-based replicon typing for ESBL-carrying plasmids in K. pneumoniae in this study. Greater than 50% of plasmids fell into clusters, and >60% of cluster-classified plasmids were present in clonally unrelated isolates, indicating that horizontal transfer of plasmids plays an important role in the spread of ESBL genes.

Genetic & virulence profiling of ESBL-positive E. coli from nosocomial & veterinary sources

CTX-M genes are the most prevalent ESBL globally, infiltrating nosocomial, community and environmental settings. Wild and domesticated animals may act as effective vectors for the dissemination of CTX-producing Enterobacteriaceae. This study aimed to contextualise blaCTX-M-14-positive, cephalosporin-resistant Enterobacteriaceae human infections and compared resistance and pathogenicity markers with veterinary isolates.Epidemiologically related human (n=18) and veterinary (n=4) blaCTX-M-14-positive E. coli were fully characterised. All were typed by XbaI pulsed field gel electrophoresis and ST. Chromosomal/plasmidic locations of blaCTX-M-14 were deduced by S1-nuclease digestion, and association with ISEcp1 was investigated by sequencing. Conjugation experiments assessed transmissibility of plasmids carrying blaCTX-M-14. Presence of virulence determinants was screened by PCR assay and pathogenicity potential was determined by in vitro Galleria mellonella infection models.84% of clinical E. coli originated from community patients. blaCTX-M-14 was found ubiquitously downstream of ISEcp1 upon conjugative plasmids (25–150kb). blaCTX-M-14 was also found upon the chromosome of eight E. coli isolates. CTX-M-14-producing E. coli were found at multiple hospital sites. Clonal commonality between patient, hospitals and livestock microbial populations was found. In vivo model survival rates from clinical isolates (30%) and veterinary isolates (0%) were significantly different (p<0.05). Co-transfer of blaCTX-M-14 and virulence determinants was demonstrated.There is evidence of clonal spread of blaCTX-M-14-positive E. coli involving community patients and farm livestock. blaCTX-M-14 positive human clinical isolates carry a lower intrinsic pathogenic potential than veterinary E. coli highlighting the need for greater veterinary practices in preventing dissemination of MDR E. coli among livestock.

Carbapenem-resistant Klebsiella pneumoniae isolates from Egypt containing blaNDM-1 on IncR plasmids and its association with rmtF.

The aim of this study was to characterize carbapenem-resistant Klebsiella pneumoniae (CRKP) isolates recovered from clinical specimens at a tertiary care hospital in Egypt over a period of 15 months. Eight CRKP isolates were included in this study. The minimum inhibitory concentrations of different antibiotics were determined by broth microdilution and Etest methods. Multilocus sequence typing was performed. Antibiotic resistance genes were assessed by PCR and DNA sequencing. Plasmid analysis was done by S1 nuclease digestion of whole genomic DNA followed by pulsed-field gel electrophoresis (S1-PFGE). Eight carbapenem-resistant NDM-1-producing K. pneumoniae isolates of three different sequence types (ST) were identified (ST147, ST11, and ST17), in which blaNDM-1 was carried by either IncR or untypeable plasmids. Seven out of the eight isolates also contained the rmtF methylase gene. This study describes the occurrence of IncR plasmids carrying blaNDM-1 and rmtF in Egypt, raising concerns regarding this type of replicon and its role in the transmission of these resistance determinants.

Distribution of Plasmids in Distinct Leptospira Pathogenic Species.

Leptospirosis, caused by pathogenic Leptospira, is a worldwide zoonotic infection. The genus Leptospira includes at least 21 species clustered into three groups—pathogens, non-pathogens, and intermediates—based on 16S rRNA phylogeny. Research on Leptospira is difficult due to slow growth and poor transformability of the pathogens. Recent identification of extrachromosomal elements besides the two chromosomes in L. interrogans has provided new insight into genome complexity of the genus Leptospira. The large size, low copy number, and high similarity of the sequence of these extrachromosomal elements with the chromosomes present challenges in isolating and detecting them without careful genome assembly. In this study, two extrachromosomal elements were identified in L. borgpetersenii serovar Ballum strain 56604 through whole genome assembly combined with S1 nuclease digestion following pulsed-field gel electrophoresis (S1-PFGE) analysis. Further, extrachromosomal elements in additional 15 Chinese epidemic strains of Leptospira, comprising L. borgpetersenii, L. weilii, and L. interrogans, were successfully separated and identified, independent of genome sequence data. Southern blot hybridization with extrachromosomal element-specific probes, designated as lcp1, lcp2 and lcp3-rep, further confirmed their occurrences as extrachromosomal elements. In total, 24 plasmids were detected in 13 out of 15 tested strains, among which 11 can hybridize with the lcp1-rep probe and 11 with the lcp2-rep probe, whereas two can hybridize with the lcp3-rep probe. None of them are likely to be species-specific. Blastp search of the lcp1, lcp2, and lcp3-rep genes with a nonredundant protein database of Leptospira species genomes showed that their homologous sequences are widely distributed among clades of pathogens but not non-pathogens or intermediates. These results suggest that the plasmids are widely distributed in Leptospira species, and further elucidation of their biological significance might contribute to our understanding of biology and infectivity of pathogenic spirochetes.

Electrochemical biosensor for microRNA detection based on hybridization protection against nuclease S1 digestion

Nuclease S1 can catalyze the nonspecific endo- and exonucleolytic cleavage of single-stranded DNA and RNA to yield nucleoside 5′-phosphates and 5′-phosphooligonucleotides. However, it cannot hydrolyze double-stranded DNA, double-stranded RNA, or DNA-RNA hybrid. Inspired by this specific property, a simple electrochemical method was developed for microRNA detection based on hybridization protection against nuclease S1 digestion. In the absence of hybridization process, the assembled probe DNA on the electrode surface can be easily digested by nuclease S1 and a strong electrochemical signal can be generated due to the decreased repulsive force towards the redox probe. However, after hybridization with target microRNA, the digestion activity of nuclease S1 is inhibited, which can lead to a weak electrochemical signal. Based on the change of the electrochemical signal, the detection of target microRNA-319a can be achieved. Under optimal experiment conditions, the electrochemical signal was proportional to microRNA-319a concentration from 1000 to 5 pM and the detection limit was 1.8 pM (S/N = 3). The developed method also showed high detection selectivity and reproducibility. Furthermore, the proposed method was successfully applied to assay the expression level of microRNA-319a in the leaves of rice seedlings after being incubated with different concentrations of 6-benzylaminopurine.

Label-free selective sensing of Pb2+ lead(II) sensors based on the aggregation of a pyrene fluorescent probe

In the present work, we report a label-free fluorescence turn-on approach for the sensitive and selective sensing of Pb2+. Pyrene with one positive charge was used as the fluorescent probe, and thrombin aptamer (TBA), which was a G-rich oligonucleotide, was employed to form G-quadruplex with lead(II). When TBA and Pb2+ were mixed with lead(II) in an aqueous solution, it was folded into a stable G-quadruplex. Subsequently, a single-stranded nucleic acid-specific nuclease S1 was added. The G-quadruplex stabilized by Pb2+ lead(II) had markedly a significant resistant ability to nuclease S1 digestion. However, in the absence of Pb2+ lead(II), no quadruplex or less stable quadruplex was formed and TBA was digested by nuclease S1 in 3 min under the optimized experimental conditions. Finally, pyrene probe was mixed with oligonucleotide in Pb2+ lead(II). Electrostatic interactions between oligonucleotide (a polyanion) and the probe induced the aggregation of the probe, which in turn produced strong emission of the strong pyrene excimer emission. The intensity of the induced excimer emission was directly proportional to the amount of Pb2+ added. Our approach shows good selectivity and sensitivity for the detection of Pb2+ with a limit of detection limit as low as 800 nmol/L.

Hubungan Metilasi DNA dengan Ekspresi Gen MADS-box pada Buah Mantel Tanaman Kelapa Sawit (Elaeis guineensis Jacq.)

ABSTRACT The presence of mantled fruit on large-scale clonal production of oil palm had trully decreased the oil productivity. Mantled phenotype is likely to be that of an epigenetic change involving DNA methylation and the MADS-box transcription factor gene which encoded floral organ homeotic transformation. The objectives of this research were to quantify the degree of methylation which determined fruit abnormality through Ultra Performance Liquid Chromatography (UPLC) and to study its correlation with the MADS-box EgAGL6, EgAG2, and EgAGA genes expressed on mantled fruit derived from oil palm clonal plants which have been quantified using Quantitative Real-Time PCR (qPCR). This research was arranged in two replications for each gene and cDNA. The expression of the target genes were compared to EF1-α1 as the reference gene. Through the Least Significant Difference (LSD) Test at 95% confidence level of qPCR result, EgAGL6 expression was significantly lower in mantled fruit which decreased from 1.88 fold in Abn m to 0.46 fold in Abn. EgAG2 expression was increased non-significantly from 0.91 fold in Abn m to 1.13 fold in Abn, while EgAGA expression was higher in mantled which increased significantly from 1.48 fold in Abn m to 1.71 fold in Abn. Nuclease S1 digestion and UPLC revealed the genome-wide increase in DNA methylation on mantled fruit (18.33-19.55%) compared to its normal counterparts (5.67%). This increased in global DNA methylation was showed by the significant decreased in EgAGL6 transcript level of mantled fruit. This gene assumed to be involved in the development of mantled fruit. Keywords: DNA-methylation, MADS-box genes, Mantled fruit, Quantitative Real-Time PCR

Acquired carbapenemases in Enterobactericeae in Singapore, 1996-2012

To characterise carbapenemase-producing Enterobacteriaceae (CRE) isolated in Singapore. Carbapenemase genes and their flanking regions were amplified and sequenced by PCR. Isolates were typed by pulsed-field gel electrophoresis and multi-locus sequence typing. Plasmids bearing carbapenemase genes were sized by S1 nuclease digestion, Southern blotting, and DNA hybridisation with appropriate probes. Transfer of these plasmids was attempted by conjugation and transformation. Successfully transferred plasmids were characterised by polymerase chain reaction (PCR) replicon typing and restriction digestion. Isolates of Klebsiella pneumoniae, Escherichia coli, Enterobacter cloacae and Citrobacter species carried a variety of carbapenemase genes including blaIMP-1, blaIMP-4, blaNDM-1, blaNDM-7, blaOXA-181, blaOXA-48 and blaKPC-2. Apart from K. pneumoniae with blaOXA-181, and some K. pneumoniae with blaNDM-1, the other isolates were not clonal. However there appears to be some spread of plasmids with blaIMP-1, blaNDM-1,blaKPC-2, and blaOXA-48. The number of isolates of CRE has increased in Singapore, especially since 2010. There is a diversity of carbapenemase types that reflects the geographical proximity of other countries with CRE.

Physicochemical and immunological studies on mitochondrial DNA modified by peroxynitrite: implications of neo-epitopes of mitochondrial DNA in the etiopathogenesis of systemic lupus erythematosus

Recent evidence has demonstrated that mitochondria possess their own nitric oxide synthase (mtNOS) and can produce endogenous reactive-nitrogen-species (RNS) including peroxynitrite (ONOO(-)). This study was undertaken to investigate the role of mitochondrial DNA (mtDNA) damage by ONOO(-) in systemic lupus erythematosus (SLE) autoimmunity. MtDNA was isolated from fresh goat liver and modified by ONOO(-), generated by synergistic action of nitric oxide (NO) and superoxide (O (-) 2) donors. Modifications occurring in mtDNA were characterized by physicochemical techniques. SLE patients (n = 50) with varying disease activity according to the SLE Disease Activity Index (SLEDAI) and healthy controls (n = 34) were evaluated for antibodies to native and ONOO(-)-modified mtDNA by immunoassays. Gel retardation assays were performed to cross-examine the immunoassay results using affinity-purified SLE immunoglobulin G (IgG). Nitrosative stress in SLE patients was studied by measuring nitrotyrosine and inducible NO synthase (iNOS). The ONOO(-) caused extensive damage to mtDNA as evident by ultraviolet (UV) hyperchromicity and loss of florescence intensity. Thermal melting studies, agarose gel electrophoresis and nuclease S1 digestibility clearly indicate structural perturbation in mtDNA by ONOO(-). Quenching studies with specific NO or O (-) 2 quenchers confirmed that the damaging agent was ONOO(-). SLE autoantibodies exhibited enhanced binding with ONOO(-)-mtDNA as compared to their native analog. Interestingly, not only was there an increased number of subjects positive for ONOO(-) -mtDNA, but also the levels of anti-ONOO(-) -mtDNA antibodies were statistically significantly higher among SLE patients whose SLEDAI scores were ≥ 20 as compared with SLE patients with lower SLEDAI scores (SLEDAI < 20). Normal healthy controls showed negligible binding with either antigen. Furthermore, SLE patients had higher levels of nitrotyrosine and iNOS compared with their respective healthy controls. Our novel results provide an important insight into the immunological basis of anti-DNA autoantibody generation in SLE. Our data conclude that modification of mtDNA by ONOO(-) causes structural perturbations, resulting in the generation of neo-epitopes, and making it a potential immunogen in SLE. The mtDNA modified by ONOO(-) may be useful in evaluating the progression of SLE and in elucidating the mechanisms of disease pathogenesis.

Salmonella entericaserovarTyphimurium Virulence-Resistance Plasmids Derived from the pSLT Carrying Nonconventional Class 1 Integrons withdfrA12Gene in Their Variable Region andsul3in the 3′ Conserved Segment

Drug-resistant derivatives of serovar-specific virulence plasmids, such as pSLT, in clinically-relevant Salmonella enterica serovar Typhimurium strains, represent a threat for human health. We have analysed 14 S. Typhimurium isolates recovered in Italy and the United Kingdom from swine and from cases of human infection for the presence of virulence-resistance (VR) plasmids. They were negative for the multidrug resistance (MDR) region of the Salmonella genomic island 1 (SGI1), but expressed resistance to ampicillin, chloramphenicol, streptomycin/spectinomycin, sulfamethoxazole, and tetracyclines. The isolates were characterised by XbaI pulsed-field gel electrophoresis, multilocus sequence typing, and detection of resistance and virulence determinants (PCR/sequencing). Identification of VR plasmids was accomplished by PCR detection of bla genes (encoding ampicillin resistance), class 1 integrons and the pSLT virulence gene spvC. Plasmid analyses were performed by alkaline lysis, S1-nuclease digestion, replicon typing, conjugation, restriction analyses, and Southern blot/hybridization. Two blaOXA-1 positive isolates contained pSLT-derived plasmids related to pUO-StVR2. In nine isolates, eight from swine and one from a patient, MDR-conferring-IncFII-VR plasmids were detected. They contained the blaTEM-1 gene as well as a nonconventional class 1 integron with dfrA12-aadA2 gene cassettes in its variable region, and a sul3 gene in the 3' conserved segment. Restriction analysis suggested a novel pSLT variant. The results obtained underline the role of swine as a potential reservoir for the blaTEM-1-IncFII-plasmids. The occurrence and spread of virulence- and MDR-conferring plasmids should be considered as a potential public health problem.

63 EMSA detection of p53 sequence non-specific binding in PCR produced human DNA analogues

p53 is a tumor suppressor that induces cell cycle arrest and apoptosis in response to DNA damage and cancerogenesis. Its ability to bind DNA, and thus play its biological role, is possible in two manners: sequence-specific binding to its consensus sequence (p53CON) and sequence non-specific binding, which occurs preferably in higher DNA structures. Recently, it has been proven that DNA quadruplexes occur in regulation areas of most cancer genes. In our study, we have tested human DNA cloned into plasmid vectors. The DNAs were obtained by chromatin immunoprecipitation of regions which were bound by p53 with high affinity, although they do not contain p53CON. The sequence studied in this work is located in a noncoding region of human chromosome 7. We suggest that structure-specific binding is responsible for higher affinity of p53 binding in these areas. It has been previously found that some single-stranded regions appeared in these areas, suggesting the presence of higher DNA structures by S1 nuclease digestion (unpublished results). Because we were unable to detect the exact location of p53 binding with sufficient resolution by standard methods, we have amplified different parts of immunoprecipitated DNAs by PCR and found, using EMSA, to what part of the insert p53 binds with the highest affinity. This area is represented by cca 150 nucleotides. The strongest preference of p53 was found for the region which contained repeated short tracts of 3–4 Ts and a short polyPu.polyPy sequence. It is known that dAn:dTn blocks can cause DNA curvature, and the polyPu.polyPy sequence is able to form an intramolecular triplex.