Antibiotic resistance genes in the bacteriophage DNA fraction of environmental samples

Antibiotic resistance is an increasing global problem resulting from the pressure of antibiotic usage, greater mobility of the population, and industrialization. Many antibiotic resistance genes are believed to have originated in microorganisms in the environment, and to have been transferred to other bacteria through mobile genetic elements. Among others, ß-lactam antibiotics show clinical efficacy and low toxicity, and they are thus widely used as antimicrobials. Resistance to ß-lactam antibiotics is conferred by ß-lactamase genes and penicillin-binding proteins, which are chromosomal- or plasmid-encoded, although there is little information available on the contribution of other mobile genetic elements, such as phages. This study is focused on three genes that confer resistance to ß-lactam antibiotics, namely two ß-lactamase genes (blaTEM and blaCTX-M9) and one encoding a penicillin-binding protein (mecA) in bacteriophage DNA isolated from environmental water samples. The three genes were quantified in the DNA isolated from bacteriophages collected from 30 urban sewage and river water samples, using quantitative PCR amplification. All three genes were detected in the DNA of phages from all the samples tested, in some cases reaching 104 gene copies (GC) of blaTEM or 102 GC of blaCTX-M and mecA. These values are consistent with the amount of fecal pollution in the sample, except for mecA, which showed a higher number of copies in river water samples than in urban sewage. The bla genes from phage DNA were transferred by electroporation to sensitive host bacteria, which became resistant to ampicillin. blaTEM and blaCTX were detected in the DNA of the resistant clones after transfection. This study indicates that phages are reservoirs of resistance genes in the environment.

Proof of concept for microarray-based detection of DNA-binding oncogenes in cell extracts.

The function of DNA-binding proteins is controlled not just by their abundance, but mainly at the level of their activity in terms of their interactions with DNA and protein targets. Moreover, the affinity of such transcription factors to their target sequences is often controlled by co-factors and/or modifications that are not easily assessed from biological samples. Here, we describe a scalable method for monitoring protein-DNA interactions on a microarray surface. This approach was designed to determine the DNA-binding activity of proteins in crude cell extracts, complementing conventional expression profiling arrays. Enzymatic labeling of DNA enables direct normalization of the protein binding to the microarray, allowing the estimation of relative binding affinities. Using DNA sequences covering a range of affinities, we show that the new microarray-based method yields binding strength estimates similar to low-throughput gel mobility-shift assays. The microarray is also of high sensitivity, as it allows the detection of a rare DNA-binding protein from breast cancer cells, the human tumor suppressor AP-2. This approach thus mediates precise and robust assessment of the activity of DNA-binding proteins and takes present DNA-binding assays to a high throughput level.

Oligomerization and DNA binding of Ler, a master regulator of pathogenicity of enterohemorrhagic and enteropathogenic Escherichia coli

Ler is a DNA-binding, oligomerizable protein that regulates pathogenicity islands in enterohemorrhagic and enteropathogenic Escherichia coli strains. Ler counteracts the transcriptional silencing effect of H-NS, another oligomerizable nucleoid-associated protein. We studied the oligomerization of Ler in the absence and presence of DNA by atomic force microscopy. Ler forms compact particles with a multimodal size distribution corresponding to multiples of 35 units of Ler. DNA wraps around Ler particles that contain more than 1516 Ler monomers. The resulting shortening of the DNA contour length is in agreement with previous measurements of the length of DNA protected by Ler in footprinting assays. We propose that the repetition unit corresponds to the number of monomers per turn of a tight helical Ler oligomer. While the repressor (H-NS) and anti-repressor (Ler) have similar DNA-binding domains, their oligomerization domains are unrelated. We suggest that the different oligomerization behavior of the two proteins explains the opposite results of their interaction with the same or proximal regions of DNA.

Solid-phase synthesis and DNA binding studies of dichloroplatinum(II) conjugates of dicarba analogues of octreotide as a new anticancer drugs

The first dichloroplatinum(II) conjugates of dicarba analogues of octreotide , which is expected to act as a"tumour-targeting device", have been efficiently synthesized following a stepwise solid-phase approach; these compounds emulate the mechanism of cisplatin since they form a 1,2-intrastrand cross-link with two consecutive guanines of an oligonucleotide.

Monitoring of malaria parasite resistance to chloroquine and sulphadoxine-pyrimethamine in the Solomon Islands by DNA microarray technology.

BACKGROUND: Little information is available on resistance to anti-malarial drugs in the Solomon Islands (SI). The analysis of single nucleotide polymorphisms (SNPs) in drug resistance associated parasite genes is a potential alternative to classical time- and resource-consuming in vivo studies to monitor drug resistance. Mutations in pfmdr1 and pfcrt were shown to indicate chloroquine (CQ) resistance, mutations in pfdhfr and pfdhps indicate sulphadoxine-pyrimethamine (SP) resistance, and mutations in pfATPase6 indicate resistance to artemisinin derivatives. METHODS: The relationship between the rate of treatment failure among 25 symptomatic Plasmodium falciparum-infected patients presenting at the clinic and the pattern of resistance-associated SNPs in P. falciparum infecting 76 asymptomatic individuals from the surrounding population was investigated. The study was conducted in the SI in 2004. Patients presenting at a local clinic with microscopically confirmed P. falciparum malaria were recruited and treated with CQ+SP. Rates of treatment failure were estimated during a 28-day follow-up period. In parallel, a DNA microarray technology was used to analyse mutations associated with CQ, SP, and artemisinin derivative resistance among samples from the asymptomatic community. Mutation and haplotype frequencies were determined, as well as the multiplicity of infection. RESULTS: The in vivo study showed an efficacy of 88% for CQ+SP to treat P. falciparum infections. DNA microarray analyses indicated a low diversity in the parasite population with one major haplotype present in 98.7% of the cases. It was composed of fixed mutations at position 86 in pfmdr1, positions 72, 75, 76, 220, 326 and 356 in pfcrt, and positions 59 and 108 in pfdhfr. No mutation was observed in pfdhps or in pfATPase6. The mean multiplicity of infection was 1.39. CONCLUSION: This work provides the first insight into drug resistance markers of P. falciparum in the SI. The obtained results indicated the presence of a very homogenous P. falciparum population circulating in the community. Although CQ+SP could still clear most infections, seven fixed mutations associated with CQ resistance and two fixed mutations related to SP resistance were observed. Whether the absence of mutations in pfATPase6 indicates the efficacy of artemisinin derivatives remains to be proven.

Inhibitory effect of DNA supercoiling on DNA knotting

Using numerical simulations we investigated the effect of DNA supercoiling on the topological equilibrium of DNA molecules. We showed that under the steady state conditions that maintain the same effective deficit of the linking number in unknotted and knotted DNA molecules the topological equilibrium results in a much smaller fraction of knots than in the case of torsionally relaxed DNA molecules. Based on these results we propose that one of the important functions of DNA supercoiling is to reduce formation of DNA knots.

Mutations in the DNA-binding domain of NR2E3 affect in vivo dimerization and interaction with CRX.

BACKGROUND: NR2E3 (PNR) is an orphan nuclear receptor essential for proper photoreceptor determination and differentiation. In humans, mutations in NR2E3 have been associated with the recessively inherited enhanced short wavelength sensitive (S-) cone syndrome (ESCS) and, more recently, with autosomal dominant retinitis pigmentosa (adRP). NR2E3 acts as a suppressor of the cone generation program in late mitotic retinal progenitor cells. In adult rod photoreceptors, NR2E3 represses cone-specific gene expression and acts in concert with the transcription factors CRX and NRL to activate rod-specific genes. NR2E3 and CRX have been shown to physically interact in vitro through their respective DNA-binding domains (DBD). The DBD also contributes to homo- and heterodimerization of nuclear receptors. METHODOLOGY/PRINCIPAL FINDINGS: We analyzed NR2E3 homodimerization and NR2E3/CRX complex formation in an in vivo situation by Bioluminescence Resonance Energy Transfer (BRET(2)). NR2E3 wild-type protein formed homodimers in transiently transfected HEK293T cells. NR2E3 homodimerization was impaired in presence of disease-causing mutations in the DBD, except for the p.R76Q and p.R104W mutant proteins. Strikingly, the adRP-linked p.G56R mutant protein interacted with CRX with a similar efficiency to that of NR2E3 wild-type and p.R311Q proteins. In contrast, all other NR2E3 DBD-mutant proteins did not interact with CRX. The p.G56R mutant protein was also more effective in abolishing the potentiation of rhodospin gene transactivation by the NR2E3 wild-type protein. In addition, the p.G56R mutant enhanced the transrepression of the M- and S-opsin promoter, while all other NR2E3 DBD-mutants did not. CONCLUSIONS/SIGNIFICANCE: These results suggest different disease mechanisms in adRP- and ESCS-patients carrying NR2E3 mutations. Titration of CRX by the p.G56R mutant protein acting as a repressor in trans may account for the severe clinical phenotype in adRP patients.

Additional data for nuclear DNA give new insights into the phylogenetic position of Sorex granarius within the Sorex araneus group.

Many species contain genetic lineages that are phylogenetically intermixed with those of other species. In the Sorex araneus group, previous results based on mtDNA and Y chromosome sequence data showed an incongruent position of Sorex granarius within this group. In this study, we explored the relationship between species within the S. araneus group, aiming to resolve the particular position of S. granarius. In this context, we sequenced a total of 2447 base pairs (bp) of X-linked and nuclear genes from 47 individuals of the S. araneus group. The same taxa were also analyzed within a Bayesian framework with nine autosomal microsatellites. These analyses revealed that all markers apart from mtDNA showed similar patterns, suggesting that the problematic position of S. granarius is best explained by an incongruent behavior by mtDNA. Given their close phylogenetic relationship and their close geographic distribution, the most likely explanation for this pattern is past mtDNA introgression from S. araneus race Carlit to S. granarius.

Bacillus subtilis bacteriophage SPP1 hexameric DNA helicase, G40P, interacts with forked DNA.

SPP1-encoded replicative DNA helicase gene 40 product (G40P) is an essential product for phage replication. Hexameric G40P, in the presence of AMP-PNP, preferentially binds unstructured single-stranded (ss)DNA in a sequence-independent manner. The efficiency of ssDNA binding, nucleotide hydrolysis and the unwinding activity of G40P are affected in a different manner by different nucleotide cofactors. Nuclease protection studies suggest that G40P protects the 5' tail of a forked molecule, and the duplex region at the junction against exonuclease attack. G40P does not protect the 3' tail of a forked molecule from exonuclease attack. By using electron microscopy we confirm that the ssDNA transverses the centre of the hexameric ring. Our results show that hexameric G40P DNA helicase encircles the 5' tail, interacts with the duplex DNA at the ss-double-stranded DNA junction and excludes the 3' tail of the forked DNA.

Quality Control/Quality Assurance Testing for Joint Density and Segregation of Asphalt Mixtures, TR-623, 2013

Longitudinal joint quality control/assurance is essential to the successful performance of asphalt pavements and it has received considerable amount of attention in recent years. The purpose of the study is to evaluate the level of compaction at the longitudinal joint and determine the effect of segregation on the longitudinal joint performance. Five paving projects with the use of traditional butt joint, infrared joint heater, edge restraint by milling and modified butt joint with the hot pinch longitudinal joint construction techniques were selected in this study. For each project, field density and permeability tests were made and cores from the pavement were obtained for in-lab permeability, air void and indirect tensile strength. Asphalt content and gradations were also obtained to determine the joint segregation. In general, this study finds that the minimum required joint density should be around 90.0% of the theoretical maximum density based on the AASHTO T166 method. The restrained-edge by milling and butt joint with the infrared heat treatment construction methods both create the joint density higher than this 90.0% limit. Traditional butt joint exhibits lower density and higher permeability than the criterion. In addition, all of the projects appear to have segregation at the longitudinal joint except for the edge-restraint by milling method.

Deicer Scaling Resistance of Concrete Mixtures Containing Slag Cement Phase 2: Evaluation of Different Laboratory Scaling Test Methods, TPF-5(100), 2012

With the use of supplementary cementing materials (SCMs) in concrete mixtures, salt scaling tests such as ASTM C672 have been found to be overly aggressive and do correlate well with field scaling performance. The reasons for this are thought to be because at high replacement levels, SCM mixtures can take longer to set and to develop their properties: neither of these factors is taken into account in the standard laboratory finishing and curing procedures. As a result, these variables were studied as well as a modified scaling test, based on the Quebec BNQ scaling test that had shown promise in other research. The experimental research focused on the evaluation of three scaling resistance tests, including the ASTM C672 test with normal curing as well as an accelerated curing regime used by VDOT for ASTM C1202 rapid chloride permeability tests and now included as an option in ASTM C1202. As well, several variations on the proposed draft ASTM WK9367 deicer scaling resistance test, based on the Quebec Ministry of Transportation BNQ test method, were evaluated for concretes containing varying amounts of slag cement. A total of 16 concrete mixtures were studied using both high alkali cement and low alkali cement, Grade 100 slag and Grade 120 slag with 0, 20, 35 and 50 percent slag replacement by mass of total cementing materials. Vinsol resin was used as the primary air entrainer and Micro Air® was used in two replicate mixes for comparison. Based on the results of this study, a draft alternative test method to ASTM C762 is proposed.

Development of Performance Properties of Ternary Mixtures and Concrete Pavement Mixture Design and Analysis (MDA): Effect of Paste Quality on Fresh and Hardened Properties of Ternary Mixtures, TPF-5(117), 2012

The purpose of this study was to investigate the effect of cement paste quality on the concrete performance, particularly fresh properties, by changing the water-to-cementitious materials ratio (w/cm), type and dosage of supplementary cementitious materials (SCM), and airvoid system in binary and ternary mixtures. In this experimental program, a total matrix of 54 mixtures with w/cm of 0.40 and 0.45; target air content of 2%, 4%, and 8%; a fixed cementitious content of 600 pounds per cubic yard (pcy), and the incorporation of three types of SCMs at different dosages was prepared. The fine aggregate-to- total aggregate ratio was fixed at 0.42. Workability, rheology, air-void system, setting time, strength, Wenner Probe surface resistivity, and shrinkage were determined. The effects of paste variables on workability are more marked at the higher w/cm. The compressive strength is strongly influenced by the paste quality, dominated by w/cm and air content. Surface resistivity is improved by inclusion of Class F fly ash and slag cement, especially at later ages. Ternary mixtures performed in accordance with their ingredients. The data collected will be used to develop models that will be part of an innovative mix proportioning procedure.

Development of Performance Properties of Ternary Mixtures: Field Demonstrations and Project Summary, TPF-5(117), 2012

Supplementary cementitious materials (SCM) have become common parts of modern concrete practice. The blending of two or three cementitious materials to optimize durability, strength, or economics provides owners, engineers, materials suppliers, and contractors with substantial advantages over mixtures containing only portland cement. However, these advances in concrete technology and engineering have not always been adequately captured in specifications for concrete. Users need specific guidance to assist them in defining the performance requirements for a concrete application and the selection of optimal proportions of the cementitious materials needed to produce the required durable concrete. The fact that blended cements are currently available in many regions increases options for mixtures and thus can complicate the selection process. Both Portland and blended cements have already been optimized by the manufacturer to provide specific properties (such as setting time, shrinkage, and strength gain). The addition of SCMs (as binary, ternary, or even more complex mixtures) can alter these properties, and therefore has the potential to impact the overall performance and applications of concrete. This report is the final of a series of publications describing a project aimed at addressing effective use of ternary systems. The work was conducted in several stages and individual reports have been published at the end of each stage.

Identification and potential origin of invasive clawed frogs Xenopus (Anura: Pipidae) in Sicily based on mitochondrial and nuclear DNA

African clawed frogs of the widespread polytypic species Xenopus laevis Daudin, 1802 (ranging large parts of sub-Saharan Africa) have been spreading since the 1940s, and have established reproductive populations in Europe, Asia and the Americas, where they can have negative impact as competitors of native amphibians and as disease vectors for chytridomycosis or ranaviruses. Here we use two mitochondrial (cytochrome b, 16S rDNA) and one nuclear (RAG 1: Recombination Associated Gene 1) DNA markers to infer the potential origin of invasive clawed frogs from Sicily that represent the largest invasive population in Europe. Identical mtDNA haplotypes match with those of Xenopus laevis, and Sicilian clawed frogs very probably belong to a lineage from the Cape Region of South Africa, most likely originating from a laboratory stock. Nuclear data support this conclusion. Identical mtDNA sequences (cyt b, 16S) of frogs sampled across their range in Sicily suggest the occurrence of a single source population and a potential bottleneck at their release, but faster evolving multilocus nuclear data (microsatellites, SNPs) on the population genetics would be important in the future to better support this hypothesis