Identification of potential drug targets in Salmonella Typhimurium using metabolic modelling and experimental validation

almonella enterica serovar Typhimurium is an established model organism for Gram-negative, intracellular pathogens. Owing to the rapid spread of resistance to antibiotics among this group of pathogens, new approaches to identify suitable target proteins are required. Based on the genome sequence of Salmonella Typhimurium and associated databases, a genome-scale metabolic model was constructed. Output was based on an experimental determination of the biomass of Salmonella when growing in glucose minimal medium. Linear programming was used to simulate variations in energy demand, while growing in glucose minimal medium. By grouping reactions with similar flux responses, a sub-network of 34 reactions responding to this variation was identified (the catabolic core). This network was used to identify sets of one and two reactions, that when removed from the genome-scale model interfered with energy and biomass generation. 11 such sets were found to be essential for the production of biomass precursors. Experimental investigation of 7 of these showed that knock-outs of the associated genes resulted in attenuated growth for 4 pairs of reactions, while 3 single reactions were shown to be essential for growth.

The identification of genes important in pseudomonas syringae pv. phaseolicola plant colonisation using in vitro screening of transposon libraries

The bacterial plant pathogen Pseudomonas syringae pv. phaseolicola (Pph) colonises the surface of common bean plants before moving into the interior of plant tissue, via wounds and stomata. In the intercellular spaces the pathogen proliferates in the apoplastic fluid and forms microcolonies (biofilms) around plant cells. If the pathogen can suppress the plant’s natural resistance response, it will cause halo blight disease. The process of resistance suppression is fairly well understood, but the mechanisms used by the pathogen in colonisation are less clear. We hypothesised that we could apply in vitro genetic screens to look for changes in motility, colony formation, and adhesion, which are proxies for infection, microcolony formation and cell adhesion. We made transposon (Tn) mutant libraries of Pph strains 1448A and 1302A and found 106/1920 mutants exhibited alterations in colony morphology, motility and biofilm formation. Identification of the insertion point of the Tn identified within the genome highlighted, as expected, a number of altered motility mutants bearing mutations in genes encoding various parts of the flagellum. Genes involved in nutrient biosynthesis, membrane associated proteins, and a number of conserved hypothetical protein (CHP) genes were also identified. A mutation of one CHP gene caused a positive increase in in planta bacterial growth. This rapid and inexpensive screening method allows the discovery of genes important for in vitro traits that can be correlated to roles in the plant interaction

Identification of Hammondia heydorni oocysts by a heminested-PCR (hnPCR-AP10) based on the H-heydorni RAPD fragment AP10

Toxoplasma gondii, Hammondia hammondi, Neospora caninum, Neospora hughesi and Hammondia heydorni are members of the Toxoplasmatinae sub-family. They are closely related coccidians with similarly sized oocysts. Molecular diagnostic techniques, especially those based on polymerase chain reaction (PCR), can be successfully applied for the differentiation of Hammondia-like oocysts. In this paper, we describe a rapid and simple method for the identification of H. heydorni oocysts among other members of the Toxoplasmatinae sub-family, using a heminested-PCR (hnPCR-AP10) based on a H. heydorni RAPD fragment available in molecular database. DNA of oocysts of H. heydorni yielded a specific fragment of 289-290 bp in the heminested-PCR assay. No product was yielded when the primers were used for the amplification of DNA extracted from T. gondii, N. caninum, N. hughesi and H. hammondi, thus allowing the differentiation of H. heydorni among other members of the Toxoplasmatinae sub-family. The hnPCR-AP10 was capable of detecting H. heydorni genetic sequences from suspensions with at least 10 oocysts. In conclusion, the hnPCR-AP10 proved to be a reliable method to be used in the identification of H. heydorni oocysts from feces of dogs. (C) 2010 Elsevier B.V. All rights reserved.

Effectiveness of high-throughput miniaturized sorbent- and solid phase microextraction techniques combined with gas chromatography–mass spectrometry analysis for a rapid screening of volatile and semi-volatile composition of wines: a comparative study

In this study the feasibility of different extraction procedures was evaluated in order to test their potential for the extraction of the volatile (VOCs) and semi-volatile constituents (SVOCs) from wines. In this sense, and before they could be analysed by gas chromatography–quadrupole first stage masss spectrometry (GC–qMS), three different high-throughput miniaturized (ad)sorptive extraction techniques, based on solid phase extraction (SPE), microextraction by packed sorbents (MEPS) and solid phase microextraction (SPME), were studied for the first time together, for the extraction step. To achieve the most complete volatile and semi-volatile signature, distinct SPE (LiChrolut EN, Poropak Q, Styrene-Divinylbenzene and Amberlite XAD-2) and MEPS (C2, C8, C18, Silica and M1 (mixed C8-SCX)) sorbent materials, and different SPME fibre coatings (PA, PDMS, PEG, DVB/CAR/PDMS, PDMS/DVB, and CAR/PDMS), were tested and compared. All the extraction techniques were followed by GC–qMS analysis, which allowed the identification of up to 103 VOCs and SVOCs, distributed by distinct chemical families: higher alcohols, esters, fatty acids, carbonyl compounds and furan compounds. Mass spectra, standard compounds and retention index were used for identification purposes. SPE technique, using LiChrolut EN as sorbent (SPELiChrolut EN), was the most efficient method allowing for the identification of 78 VOCs and SVOCs, 63 and 19 more than MEPS and SPME techniques, respectively. In MEPS technique the best results in terms of number of extractable/identified compounds and total peak areas of volatile and semi-volatile fraction, were obtained by using C8 resin whereas DVB/CAR/PDMS was revealed the most efficient SPME coating to extract VOCs and SVOCs from Bual wine. Diethyl malate (18.8 ± 3.2%) was the main component found in wine SPELiChrolut EN extracts followed by ethyl succinate (13.5 ± 5.3%), 3-methyl-1-butanol (13.2 ± 1.7%), and 2-phenylethanol (11.2 ± 9.9%), while in SPMEDVB/CAR/PDMS technique 3-methyl-1-butanol (43.3 ± 0.6%) followed by diethyl succinate (18.9 ± 1.6%), and 2-furfural (10.4 ± 0.4%), are the major compounds. The major VOCs and SVOCs isolated by MEPSC8 were 3-methyl-1-butanol (26.8 ± 0.6%, from wine total volatile fraction), diethyl succinate (24.9 ± 0.8%), and diethyl malate (16.3 ± 0.9%). Regardless of the extraction technique, the highest extraction efficiency corresponds to esters and higher alcohols and the lowest to fatty acids. Despite some drawbacks associated with the SPE procedure such as the use of organic solvents, the time-consuming and tedious sampling procedure, it was observed that SPELiChrolut EN, revealed to be the most effective technique allowing the extraction of a higher number of compounds (78) rather than the other extraction techniques studied.

Fractal analysis of leaf-texture properties as a tool for taxonomic and identification purposes: a case study with species from Neotropical Melastomataceae (Miconieae tribe)

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Identification of Staphylococcus saprophyticus isolated from patients with urinary tract infection using a simple set of biochemical tests correlating with 16S-23S interspace region molecular weight patterns

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

A Streamlined DNA Tool for Global Identification of Heavily Exploited Coastal Shark Species (Genus Rhizoprionodon)

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Genetic identification of the sharks Rhizoprionodon porosus and R. lalandii by PCR-RFLP and nucleotide sequence analyses of 5S rDNA

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Identification of triterpenes and sterols from pterogyne nitens (fabaceae-caesalpinioideae) using high-resolution gas chromatography

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Rapid spot test analysis for the detection of urotropine in pharmaceutical preparations

The widespread falsification and/or adulteration of commercially available pharmaceutical preparations call for reliable methods of drug identification, preferably through selective and rapid sorting color tests that could be undertaken with minimum equipment remote from laboratory facilities. The present work deals with a convenient adaptation and refinement of a spot test devised by Feigl (1966) for urotropine, based on the hydrolytic cleavage of that substance in the presence of sulfuric acid, splitting out formaldehyde which is identified by its color reaction with chromotropic acid. A simple emergency kit was developed for the quick, efficient, inexpensive and easy performance of urotropine tests by semiskilled personnel even in the drugstore laboratory (or office) as well as in a mobile screening operation. It is shown that when the reagents are added according to the recommended sequence a self-heating system is generated, increasing substantially the reactions' rates and the test sensitivity as well. The identification limit found was 25 mug of urotropine, for both solid and liquid samples. The possible interference of 84 substances/materials was investigated. Interference was noted only for methylene blue, acriflavine, Ponceau Red, Bordeaux Red (these dyes are often included in urotropine dosage forms), pyramidone, dipyrone, quinine and tetracycline. A simple procedure for removing most of the interferences is described. Data for 8 commercial dosage forms and results obtained from their analysis are presented.

Rapid degradation of propanil in rice crop fields

Propanil and its major degradation product, 3,4-dichloroaniline (DCA), were monitored in surface water and soil samples from two rice fields of the Ebre Delta area (Tarragona, Spain) following agricultural application. On-line solid-phase extraction (SPE) (water) and Soxhlet extraction (soil) followed by liquid chromatography/diode array detection (LC/DAD) were used for the trace determination of both compounds. Unequivocal confirmation/identification was conducted by using liquid chromatography/atmospheric pressure chemical ionization-mass spectrometry, LC/APCI/MS (using negative and positive ionization modes). Concentrations of the herbicide propanil in water samples varied from 1.9 to 55.9 mu g/L. Propanil degraded very rapidly to DCA, and high concentrations of this product were found, varying from 16.5 to 470 mu g/L in water and 119 +/- 22 mu g/kg in soil samples. No detectable DCA (<0.001%) was found in the applied pesticide formulation, indicating that DCA formation took place after propanil application. These field results compared favorably with laboratory experiments showing that humic interactions had a strong influence on the pesticide degradation. The half-lifes under real conditions for propanil and DCA, calculated using a first-order decay, were 1.2 and 1.6 days, respectively.

Use of on-line liquid chromatography-nuclear magnetic resonance spectroscopy for the rapid investigation of flavonoids from Sorocea bomplandii

This paper reports the identification of di- and triglycosylated flavonoids from Sorocea bomplandii (Moraceae) by liquid chromatography coupled on-line to nuclear magnetic resonance (LC-NMR). These glycosylated flavonoids may be used as a taxonomic marker in future work. (C) 2002 Elsevier B.V. B.V All rights reserved.

Rapid molecular diagnosis of tuberculosis and other mycobacteriosis in smear-negative clinical specimens

Delay in diagnosis of pulmonary and other forms of tuberculosis (TB) can be fatal, particularly in HIV-infected patients. Hence, techniques based on nucleic acid amplification, which are both rapid and of high specificity and sensitivity, are now widely used and recommended for laboratories that diagnose TB. In the present study, diagnostic methods based on mycobacterial DNA amplification were evaluated in comparative trials alongside tradicional bacterial methods, using negative smear samples from patients with clinically-suspected TB (sputum samples from 25 patients with suspected pulmonary TB, urine samples from two patients with suspected renal TB and cerebrospinal fluid samples from one patient with suspected meningeal TB). A specificity of 100% was achieved with DNA amplification methods and tradicional culture/identification methods, in relation to clinical findings and treatment results. For the smear-negative sputa, conventional PCR for M. tuberculosis was positive in 62% of suspected lung TB case, showing the same sensitivity as bacterial identification. Both techniques failed in the detection of extra-pulmonary samples. Nested PCR showed, after species-specific amplification, a sensitivity of 100% for M. avium and 85% for M. tuberculosis. For extra-pulmonary smear-negative samples, only Nested PCR detected M. tuberculosis and all cases were confirmed clinically. Nested PCR, in which two-step amplification reactions are performed, can identify the two most important mycobacteria in human pathology quickly and directly from clinical spicimens.

Multiplex PCR use for Staphylococcus aureus identification and oxacillin and mupirocin resistance evaluation

Oxacillin-resistant Staphylococcus aureus represents a serious problem in hospitals worldwide, increasing infected patients' mortality and morbidity and raising treatment costs and internment time. In this study, the results of using the Multiplex PCR technique to amplify fragments of the genes femA (specific-species), mecA (oxacillin resistance) and ileS-2 (mupirocin resistance) were compared with those of tests conventionally used to identify S. aureus isolates and ascertain their resistance to drugs. Fifty S. aureus strains were isolated from patients receiving treatment at UNOESTE University Hospital in Presidente Prudente, SP, Brazil. The 686 bp fragment corresponding to the gene femA was amplified and detected in all the isolates. On the other hand, the 310 bp fragment corresponding to the mecA gene was amplified in 29 (58%) of the isolates. All of the isolates showed sensitivity to mupirocin in the agar diffusion test, which was corroborated by the lack of any amplicon of the 456 bp fragment corresponding to the ileS-2 gene, in the PCR bands. The conventional tests to identify S. aureus and detect resistance to oxacillin and mupirocin showed 100% agreement with the PCR Multiplex results. The use of techniques for rapid and accurate identification of bacteria and assessment of their resistance may be valuable in the control of infection by resistant strains, allowing the rapid isolation and treatment of an infected patient. However, the results demonstrate that traditional phenotypic tests are also reliable, though they take more time.

Air versus saline in the loss of resistance technique for identification of the epidural space

BackgroundThe success of epidural anaesthesia depends on correct identification of the epidural space. For several decades, the decision of whether to use air or physiological saline during the loss of resistance technique for identification of the epidural space has been governed by the personal experience of the anaesthesiologist. Epidural block remains one of the main regional anaesthesia techniques. It is used for surgical anaesthesia, obstetrical analgesia, postoperative analgesia and treatment of chronic pain and as a complement to general anaesthesia. The sensation felt by the anaesthesiologist from the syringe plunger with loss of resistance is different when air is compared with saline (fluid). Frequently fluid allows a rapid change from resistance to non-resistance and increased movement of the plunger. However, the ideal technique for identification of the epidural space remains unclear.ObjectivesTo evaluate the efficacy and safety of both air and saline in the loss of resistance technique for identification of the epidural space.To evaluate complications related to the air or saline injected.Search methodsWe searched the Cochrane Central Register of Controlled Trials (CENTRAL) (2013, Issue 9), MEDLINE, EMBASE and the Latin American and Caribbean Health Science Information Database (LILACS) (from inception to September 2013). We applied no language restrictions. The date of the most recent search was 7 September 2013.Selection criteriaWe included randomized controlled trials (RCTs) and quasi-randomized controlled trials (quasi-RCTs) on air and saline in the loss of resistance technique for identification of the epidural space.Data collection and analysisTwo review authors independently assessed trial quality and extracted data.Main resultsWe included in the review seven studies with a total of 852 participants. The methodological quality of the included studies was generally ranked as showing low risk of bias inmost domains, with the exception of one study, which did not mask participants. We were able to include data from 838 participants in the meta-analysis. We found no statistically significant differences between participants receiving air and those given saline in any of the outcomes evaluated: inability to locate the epidural space (three trials, 619 participants) (risk ratio (RR) 0.88, 95% confidence interval (CI) 0.33 to 2.31, low-quality evidence); accidental intravascular catheter placement (two trials, 223 participants) (RR 0.90, 95% CI 0.33 to 2.45, low-quality evidence); accidental subarachnoid catheter placement (four trials, 682 participants) (RR 2.95, 95% CI 0.12 to 71.90, low-quality evidence); combined spinal epidural failure (two trials, 400 participants) (RR 0.98, 95% CI 0.44 to 2.18, low-quality evidence); unblocked segments (five studies, 423 participants) (RR 1.66, 95% CI 0.72 to 3.85); and pain measured by VAS (two studies, 395 participants) (mean difference (MD) -0.09, 95% CI -0.37 to 0.18). With regard to adverse effects, we found no statistically significant differences between participants receiving air and those given saline in the occurrence of paraesthesias (three trials, 572 participants) (RR 0.89, 95% CI 0.69 to 1.15); difficulty in advancing the catheter (two trials, 227 participants) (RR 0.91, 95% CI 0.32 to 2.56); catheter replacement (two trials, 501 participants) (RR 0.69, 95% CI 0.26 to 1.83); and postdural puncture headache (one trial, 110 participants) (RR 0.83, 95% CI 0.12 to 5.71).Authors' conclusionsLow-quality evidence shows that results do not differ between air and saline in terms of the loss of resistance technique for identification of the epidural space and reduction of complications. Applicability might be compromised, as most of the results described in this review were obtained from parturient patients. This review underlines the need to conduct well-designed trials in this field.