Effect of argon implantation on solid-state dewetting: control of size and surface density of silicon nanocrystals.

Thermally induced solid-state dewetting of ultra-thin films on insulators is a process of prime interest, since it is capable of easily forming nanocrystals. If no particular treatment is performed to the film prior to the solid-state dewetting, it is already known that the size, the shape and the density of nanocrystals is governed by the initial film thickness. In this paper, we report a novel approach to control the size and the surface density of silicon nanocrystals based on an argon-implantation preliminary surface treatment. Using 7.5 nm thin layers of silicon, we show that increasing the implantation dose tends to form smaller silicon nanocrystals with diameter and height lower than 50 nm and 30 nm, respectively. Concomitantly, the surface density is increased by a factor greater than 20, going from 5 µm-2 to values over 100 µm-2.

Inappropriate prescribing in elderly people with diabetes admitted to hospital.

AIMS: To assess inappropriate prescribing in older people with diabetes mellitus during the month prior to a hospitalization, using tools on potentially inappropriate medicines (PIMs) and potential prescribing omissions (PPOs) and comparing inappropriate prescribing in patients with without diabetes. METHODS: In an observational, prospective multicentric study, we assessed inappropriate prescribing in 672 patients aged 75 years and older during hospital admission. The Beers, Screening Tool of Older Person's Prescriptions (STOPP) and Screening Tool to Alert Doctors to Right Treatment (START) criteria and Assessing Care of Vulnerable Elders (ACOVE-3) medicine quality indicators were used. We analysed demographic and clinical factors associated with inappropriate prescribing. RESULTS: Of 672 patients, 249 (mean age 82.4 years, 62.9% female) had a diagnosis of diabetes mellitus. The mean number of prescribing drugs per patient with diabetes was 12.6 (4.5) vs. 9.4 (4.3) in patients without diabetes (P < 0.001). Of those patients with diabetes, 74.2% used 10 or more medications; 54.5% of patients with diabetes had at least one Beers-listed PIM, 68.1% had at least one STOPP-listed PIM, 64.6% had at least one START-listed PPO and 62.8% had at least one ACOVE-3-listed PPO. Except for the Beers criteria, these prevalences were significantly higher in patients with diabetes than in those without. After excluding diabetes-related items from these tools, only STOPP-listed PIMs remained significantly higher among patients with diabetes (P = 0.04). CONCLUSIONS: Polypharmacy is common among older patients with diabetes mellitus. Inappropriate prescribing is higher in older patients with diabetes, even when diabetes-related treatment is excluded from the inappropriate prescribing evaluation.

Towards Q-PCR of pathogenic bacteria with improved electrochemical double-tagged genosensing detection.

A very sensitive assay for the rapid detection of pathogenic bacteria based on electrochemical genosensing has been designed. The assay was performed by the PCR specific amplification of the eaeA gene, related with the pathogenic activity of Escherichia coli O157:H7. The efficiency and selectivity of the selected primers were firstly studied by using standard Quantitative PCR (Q-PCR) based on TaqMan fluorescent strategy. The bacteria amplicon was detected by using two different electrochemical genosensing strategies, a highly selective biosensor based on a bulk-modified avidin biocomposite (Av-GEB) and a highly sensitive magneto sensor (m-GEC). The electrochemical detection was achieved in both cases by the enzyme marker HRP. The assay showed to be very sensitive, being able to detect 4.5 ng microl(-1) and 0.45 ng microl(-1) of the original bacterial genome after only 10 cycles of PCR amplification, when the first and the second strategies were used, respectively. Moreover, the electrochemical strategies for the detection of the amplicon showed to be more sensitive compared with Q-PCR strategies based on fluorescent labels such as TaqMan probes.

In situ DNA amplification with magnetic primers for the electrochemical detection of food pathogens.

A sensitive and selective genomagnetic assay for the electrochemical detection of food pathogens based on in situ DNA amplification with magnetic primers has been designed. The performance of the genomagnetic assay was firstly demonstrated for a DNA synthetic target by its double-hybridization with both a digoxigenin probe and a biotinylated capture probe, and further binding to streptavidin-modified magnetic beads. The DNA sandwiched target bound on the magnetic beads is then separated by using a magneto electrode based on graphite-epoxy composite. The electrochemical detection is finally achieved by an enzyme marker, anti-digoxigenin horseradish peroxidase (HRP). The novel strategy was used for the rapid and sensitive detection of polymerase chain reaction (PCR) amplified samples. Promising resultants were also achieved for the DNA amplification directly performed on magnetic beads by using a novel magnetic primer, i.e., the up PCR primer bound to magnetic beads. Moreover, the magneto DNA biosensing assay was able to detect changes at single nucleotide polymorphism (SNP) level, when stringent hybridization conditions were used. The reliability of the assay was tested for Salmonella spp., the most important pathogen affecting food safety.

Synthesis and biological activity of novel acridinylidene and benzylidene thiazolidinediones.

A novel set of acridinylidene thiazolidinediones and benzylidene thiazolidinediones was synthesized by nucleophilic addition of cyanoacrylates. Some of these compounds were evaluated for their glucose lowering capability and their effects on the triglyceride level in alloxan diabetic mice.

Synthesis and schistosomicidal activity of new substituted thioxo-imidazolidine compounds.

Synthesis and physico-chemical properties of 3-benzyl-5-(4-fluoro-benzylidene)-1-methyl-2-thioxo-imidazolidin-4-ones, 5-benzylidene-3-(4-nitro-benzyl)-2-thioxo-imidazolidin-4-ones and 4-acridin-9-ylmethylene-1-benzyl-5-thioxo-imidazolidin-2-ones compounds are described. These thioxo-imidazolidine derivatives were prepared by alkylation and condensation with 4-fluoro-benzaldehyde or nucleophilic Michael addition with cyanoacrylates. The schistosomicidal activity of 3-benzyl-5-(4-fluoro-benzylidene)-1-methyl-2-thioxo-imidazolidin-4-one compounds was evaluated.

Cloning, sequence and regulation of expression of the lexA gene of Aeromonas hydrophila.

The lexA gene of Aeromonas hydrophila (Ah) has been isolated by using a specific one-step cloning system. The Ah LexA repressor is able to block Escherichia coli (Ec) SOS gene expression and is likely to be cleaved by the activated RecA protein of this bacterial species after DNA damage. Ah lexA would encode a protein of 207 amino acids (aa), which is 75% identical to the LexA repressor of Ec. Two Ec-like SOS boxes have been located upstream from Ah lexA, the distance between them being 4 bp, whereas this same distance in Ec lexA is 5 bp. The structure and sequence of the DNA-binding domain of the LexA repressor of Ec, as well as the region at which its hydrolysis occurs, are highly conserved in Ah LexA. Moreover, a residue of the region implicated in the specific cleavage reaction, and which is present in all known RecA-cleavable repressors, is changed in the Ah LexA. Expression of Ah lexA is DNA-damage inducible in both the Ah and Ec genetic backgrounds to the same extent. In contrast, Ec lexA is poorly induced in DNA-injured Ah cells.

Two different operons for the same function: comparison of the Salmonella typhimurium nrdAB and nrdEF genes.

By using a P22 phage-mediated cloning system, the nrdAB genes of Salmonella typhimurium (St), encoding a ribonucleotide reductase (RR) of class I, have been isolated. The coding regions of the St nrdAB operon show a very high identity with those of the homologous operon of Escherichia coli (Ec). Nevertheless, there are significant differences in their promoter regions since, although the promoters of both operons present two DnaA boxes, these boxes are located downstream from the transcription start point in St, being upstream in Ec. Moreover, the deduced amino-acid sequences of the St nrdAB showed a very limited overall identity (28%) with the products of St nrdEF, which encode a second class-I RR. Expression of St nrdAB and nrdEF is inducible by hydroxyurea, an inhibitor of RR activity. Alignment of the promoter regions of the nrdAB and nrdEF operons of both St and Ec reveals the presence of a consensus sequence. St is the first organism from which two different RR belonging to the same biochemical class are known.

Cloning and sequencing of the gyrA gene from the plant pathogen Erwinia carotovora.

The gyrA gene of Erwinia carotovora subsp. carotovora has been cloned and sequenced. The deduced protein possessed 86% identity with the Escherichia coli GyrA protein. E. carotovora gyrA was also shown to complement an E. coli gyrA43ts mutation.

Inhibition of Trypanosoma cruzi trypanothione reductase by acridines: kinetic studies and structure-activity relationships.

Series of 9-amino and 9-thioacridines have been synthesized and studied as inhibitors of trypanothione reductase (TR) from Trypanosoma cruzi, the causative agent of Chagas' disease. The compounds are structural analogues of the acridine drug mepacrine (quinacrine), which is a competitive inhibitor of the parasite enzyme, but not of human glutathione reductase, the closest related host enzyme. The 9-aminoacridines yielded apparent K(i) values for competitive inhibition between 5 and 43 microM. The most effective inhibitors were those with the methoxy and chlorine substituents of mepacrine and NH(2) or NHCH(CH(3))(CH(2))(4)N(Et)(2) at C9. Detailed kinetic analyses revealed that in the case of 9-aminoacridines more than one inhibitor molecule can bind to the enzyme. In contrast, the 9-thioacridine derivatives inhibit TR with mixed-type kinetics. The kinetic data are discussed in light of the three-dimensional structure of the TR-mepacrine complex. The conclusion that structurally very similar acridine compounds can give rise to completely different inhibition patterns renders modelling studies and quantitative structure-activity relationships difficult.

New pyridoquinoline derivatives as potential inhibitors of the fluoroquinolone efflux pump in resistant Enterobacter aerogenes strains.

Enterobacter aerogenes, one of the most frequently isolated nosocomial pathogens in France, is exhibiting increasing multidrug resistance mechanisms associated with a change in membrane permeability. For drugs of the quinolone family, mutations in the target and active efflux play a prominent role in the resistance. We report here the effect of several pyridoquinoline derivatives that restore a noticeable fluoroquinolone accumulation to resistant strains that overexpress the MarA activator. Studies of the energy-dependent quinolone efflux indicate that the most efficient derivatives tested probably inhibit the resistance process by acting as substrate competitors on the pump extruding intracellular norfloxacin.

Antimicrobial activity of 9-oxo and 9-thio acridines: correlation with interacalation into DNA and effects on macromolecular biosynthesis.

The antimicrobial activity of several new 9-acridinones and 9-thioalkylacridines towards Escherichia coli, Staphylococcus aureus, Mycobacterium smegmatis and Candida albicans was investigated. Minimal inhibitory, bactericidal and fungicidal concentrations were determined using a microplate assay which enabled inhibitory, bactericidal and fungicidal indices to be calculated. These indices facilitated structure/activity relationship studies. DNA-intercalating capability and DNA supercoiling inhibitory effects as well as inhibitory effects on macromolecular synthesis were determined. Results showed that intercalation into DNA, which is the mechanism of action usually postulated for acridines, cannot be correlated with the properties examined. However, inhibition of RNA synthesis may be involved in the antimicrobial activity of the drugs.

Cyclic AMP stimulates transcription of the structural gene of the outer-membrane protein OmpA of Escherichia coli.

To analyze the effect of cyclic AMP on the expression of the ompA gene of Escherichia coli, encoding the outer-membrane protein OmpA, a fusion between this gene and the lacZ gene was constructed in vitro by using a promoter-probe plasmid. The results obtained indicated that the presence of glucose in the culture medium decreased the transcription of the ompA gene. Likewise, cya and crp mutants exhibited lower levels of ompA gene expression than the wild-type strain. Furthermore, the addition of cyclic AMP increased the expression of the ompA gene in both cya and wild-type strains but not in a crp mutant. All these data show that the cyclic AMP receptor protein-cyclic AMP complex positively modulates ompA transcription in E. coli K-12.

Analysis of the DNA damage-mediated induction of Pseudomonas putida and Pseudomonas aeruginosa lexA genes.

A fusion between the lexA gene of Pseudomonas aeruginosa and Pseudomonas putida and the lacZ gene was constructed in vitro and cloned in a mini-Tn5 transposon derivative to obtain chromosomal insertions which enable to quantitatively examine their transcriptional regulation in both Pseudomonas and E. coli. Analysis of DNA damage-mediated induction of these lexA-lacZ fusions showed that expression of P. putida and P. aeruginosa lexA genes was always higher and earlier than the expression of the lexA gene of E. coli. Furthermore, and in contrast to the lexA gene fusion of E. coli, the rates and extent of the induction of lexA gene fusion of P. putida and P. aeruginosa were largely independent of the UV doses applied. The behaviour of the lexA-lacZ fusions of two Pseudomonas species was the same regardless of whether they were inserted into their own chromosome or into E. coli.

Expression of nrdA and nrdB genes of Escherichia coli is decreased under anaerobiosis.

By using plasmid nrdA-lacZ, nrdAB-lacZ, and nrdB-lacZ gene fusions, the expression of nrdA and nrdB genes of Escherichia coli under anaerobiosis has been studied. The results obtained show that cells of E. coli growing under either fermentative or nitrate respiring conditions present a lower basal level of both nrdA and nrdB genes transcription from the nrdPA promoter. On the other hand, transcription of the nrdB gene from the internal nrdPB promoter was not affected by the absence of oxygen. Moreover, the DNA damage-mediated inducing factor of these nrd genes was the same in both aerobic and anaerobic cultures.

Virulence and mutation rates of Salmonella typhimurium strains with increased mutagenic strength in a mouse model.

Two strains of Salmonella typhimurium presenting increased mutation rates, either spontaneous or mediated by DNA damage, have been constructed. One of the strains carries a null mutS mutation, while the other harbors plasmid pRW30, which contains the Escherichia coli umuDC operon. The virulence of these strains has been determined by inoculating BALB/c or Swiss mice. The 50% lethal dose of both strains is identical to that obtained for the wild-type. Likewise, the two strains and the wild-type contribute equally to animal death in mixed infections. The frequency of Nal(R) mutants recovered from animals inoculated with either wild-type or MutS(-) cells was not affected by the presence of pRW30. These results indicate that the DNA damage which S. typhimurium cells can suffer during the infectious process by host cell metabolites does not cause induction of the SOS response at levels able to trigger the error-prone DNA repair pathway.

Non-reciprocal regulation of Rhodobacter capsulatus and Rhodobacter sphaeroides recA genes expression.

The Rhodobacter capsulatus recA gene has been isolated and sequenced. Its deduced amino acid sequence showed the closest identity with the Rhodobacter sphaeroides RecA protein (91% identity). However, the promoter regions of both R. capsulatus and R. sphaeroides recA genes are only 64% similar. An Escherichia coli-like LexA binding site was not present in the upstream region of the R. capsulatus recA gene. Nevertheless, the R. capsulatus recA gene is inducible by DNA damage in both hetero- and phototrophically growing conditions. The R. capsulatus recA gene is poorly induced when inserted into the chromosome of R. sphaeroides, indicating that the recA gene of both bacteria possess different control sequences despite their phylogenetically close relationship.

Nucleotide sequence of the methoxyneurosporene dehydrogenase gene from Rhodobacter sphaeroides: comparison with other bacterial carotenoid dehydrogenases.

The nucleotide sequence of the 1794-bp fragment containing the crtD gene from Rhodobacter sphaeroides 2.4.1 encoding for methoxyneurosporene dehydrogenase has been determined. A 63% sequence identity was found when compared with the nucleotide sequence of the crtD gene from Rhodobacter capsulatus. A putative regulatory palindromic motif present in the crtD gene from R. capsulatus also exists in this gene from R. sphaeroides. The translated open reading frame of the crtD gene of R. sphaeroides has identified a polypeptide of 495 amino acids which shares a 56% sequence identity with the same CrtD protein of R. capsulatus. The N- and C-termini of these CrtD proteins present a high degree of similarity with the N- and C-termini of other carotenoid dehydrogenases including those encoded by crtI genes. This is in good agreement with the previously hypothesized homology between CrtI and CrtD proteins.

A consensus sequence for the Rhodospirillaceae SOS operators.

The sequences controlling the expression of the Rhodobacter capsulatus recA and uvrA genes belonging to the SOS DNA repair system have been identified by PCR mutagenesis. Data obtained demonstrated that the GTTCN7GTAC and GAACN7GAAC motifs present upstream of the recA gene and the GTTCN7GTTC motif found upstream of the uvrA gene are required for their respective DNA damage-mediated induction. Alignment of recA promoters of R. capsulatus, Rhodobacter sphaeroides and Rhodopseudomonas viridis with the uvrA promoters of R. capsulatus and R. sphaeroides has identified the consensus sequence GTTCVYVYTWTGTTC as the SOS operator site of the Rhodospirillaceae family.

Cloning and characterization of the recA of Paracoccus denitrificans and construction of a recA-deficient mutant.

The recA gene of Paracoccus denitrificans has been isolated from a genomic library by hybridization with the Rhodobacter sphaeroides recA gene. Its complete nucleotide sequence consists of 1071 bp encoding a polypeptide of 356 amino acids. Nucleotide sequence analysis of the P. denitrificans recA gene revealed the closest identities with the R. sphaeroides and the Rhodobacter capsulatus recA genes. Nevertheless, and surprisingly, recA genes of these two phototrophic bacteria are not DNA damage-inducible when introduced into P. denitrificans cells, whereas recA genes of both P. denitrificans and Rhizobium etli are. These results suggest that the promoters of P. denitrificans and R. etli recA genes have a similar regulatory sequence. A recA-defective mutant of P. denitrificans has also been constructed by replacement of the active recA gene by an in vitro inactivated gene copy.