Uteroplacental insufficiency in rats induces renal apoptosis and delays nephrogenesis completion.

AIM: Uteroplacental insufficiency in rats reduces nephron endowment, leptin concentrations and programmes cardiorenal disease in offspring. Cross-fostering growth-restricted (Restricted) offspring onto a mother with normal lactation restores leptin concentrations and nephron endowment. This study aimed to determine whether the reduced nephron endowment in Restricted offspring is due to delayed glomerular formation and dysregulation of renal genes regulating branching morphogenesis, apoptosis or leptin signalling. Furthermore, we aimed to investigate whether cross-fostering Restricted offspring onto Control mothers could improve glomerular maturation and restore renal gene abundance. METHODS: Uteroplacental insufficiency was induced by bilateral uterine vessel ligation (Restricted) or sham (Control) surgery on gestation day 18 (E18). Kidneys were collected at E20, postnatal day 1 (PN1) and PN7. An additional cohort was cross-fostered onto separate mothers at birth and kidneys collected at PN7. RESULTS: Kidneys were lighter in the Restricted group, but weight was restored with cross-fostering. At E20, abundance of Bax, Flt1 and Vegfa was increased in Restricted offspring, while Ret and Bcl2 transcripts were increased only in Restricted females. At PN7, abundance of Gdnf and Ret was higher in Restricted offspring, as was Casp3. Restricted offspring had a wider nephrogenic zone with more immature glomeruli suggesting a delayed or extended nephrogenic period. Cross-fostering had subtle effects on gene abundance and glomerular maturity. CONCLUSION: Uteroplacental insufficiency induced apoptosis in the developing kidney and delayed and extended nephrogenesis. Cross-fostering Restricted offspring onto Control mothers had beneficial effects on kidney growth and renal maturity, which may contribute to the restoration of nephron endowment.

Rapid Optimization of Engineered Metabolic Pathways with Serine Integrase Recombinational Assembly (SIRA).

Metabolic pathway engineering in microbial hosts for heterologous biosynthesis of commodity compounds and fine chemicals offers a cheaper, greener, and more reliable method of production than does chemical synthesis. However, engineering metabolic pathways within a microbe is a complicated process: levels of gene expression, protein stability, enzyme activity, and metabolic flux must be balanced for high productivity without compromising host cell viability. A major rate-limiting step in engineering microbes for optimum biosynthesis of a target compound is DNA assembly, as current methods can be cumbersome and costly. Serine integrase recombinational assembly (SIRA) is a rapid DNA assembly method that utilizes serine integrases, and is particularly applicable to rapid optimization of engineered metabolic pathways. Using six pairs of orthogonal attP and attB sites with different central dinucleotide sequences that follow SIRA design principles, we have demonstrated that ΦC31 integrase can be used to (1) insert a single piece of DNA into a substrate plasmid; (2) assemble three, four, and five DNA parts encoding the enzymes for functional metabolic pathways in a one-pot reaction; (3) generate combinatorial libraries of metabolic pathway constructs with varied ribosome binding site strengths or gene orders in a one-pot reaction; and (4) replace and add DNA parts within a construct through targeted postassembly modification. We explain the mechanism of SIRA and the principles behind designing a SIRA reaction. We also provide protocols for making SIRA reaction components and practical methods for applying SIRA to rapid optimization of metabolic pathways.

Engineering a novel self-powering electrochemical biosensor.

This paper records the efforts of a multi-disciplinary team of undergraduate students from Glasgow University to collectively design and carry out a 10 week project in Synthetic Biology as part of the international Genetic Engineered Machine competition (iGEM). The aim of the project was to design and build a self-powering electrochemical biosensor called 'ElectrEcoBlu'. The novelty of this engineered machine lies in coupling a biosensor with a microbial fuel cell to transduce a pollution input into an easily measurable electrical output signal. The device consists of two components; the sensor element which is modular, allowing for customisation to detect a range of input signals as required, and the universal reporter element which is responsible for generating an electrical signal as an output. The genetic components produce pyocyanin, a competitive electron mediator for microbial fuel cells, thus enabling the generation of an electrical current in the presence of target chemical pollutants. The pollutants tested in our implementation were toluene and salicylate. ElectrEcoBlu is expected to drive forward the development of a new generation of biosensors. Our approach exploited a range of state-of-the-art modelling techniques in a unified framework of qualitative, stochastic and continuous approaches to support the design and guide the construction of this novel biological machine. This work shows that integrating engineering techniques with scientific methodologies can provide new insights into genetic regulation and can be considered as a reference framework for the development of biochemical systems in synthetic biology.

The diagnostic value of light reflection rheology for suspected deep vein thrombosis: a five-and-a-half year single centre experience.

OBJECTIVES: Previous studies evaluating light reflection rheology (LRR) in the preliminary diagnosis of suspected deep vein thrombosis (DVT) have been based on limited patient numbers. The objective of this study was to demonstrate the diagnostic value of LRR in this setting for a large patient series. METHODS: A retrospective study was carried out using prospectively collected data over a five-and-a-half year period. During the study period, 3342 limbs were assessed with LLR in 3105 patients referred with suspected DVT (both inpatients and outpatients). RESULTS: In total, 2396 (71.7%) of all the LRR tests carried out yielded an abnormal result. Of the remaining 946 (28.3%) with a normal result, 204 went on to have a duplex scan due to a high clinical index of suspicion, and 14 DVTs were identified on duplex scanning. Of the remaining 742 patients with a normal LRR result who were not scanned, six re-presented within three months with a confirmed DVT, and one with a confirmed pulmonary embolus (PE). These results yield a negative predictive value of 97.8%, a sensitivity of 96.4% with a three-month post-test thromboembolism incidence of 0.9%. Use of LRR allowed duplex scanning to be avoided for 22.2% of referrals. CONCLUSIONS: Despite the increasing use of D-dimers and clinical probability scoring in the preliminary investigation of thromboembolic events, LRR remains a viable alternative in safely risk-stratifying patients with suspected DVT.

Phytodetoxification of TNT by transgenic plants expressing a bacterial nitroreductase.

There is major international concern over the wide-scale contamination of soil and associated ground water by persistent explosives residues. 2,4,6-Trinitrotoluene (TNT) is one of the most recalcitrant and toxic of all the military explosives. The lack of affordable and effective cleanup technologies for explosives contamination requires the development of better processes. Significant effort has recently been directed toward the use of plants to extract and detoxify TNT. To explore the possibility of overcoming the high phytotoxic effects of TNT, we expressed bacterial nitroreductase in tobacco plants. Nitroreductase catalyzes the reduction of TNT to hydroxyaminodinitrotoluene (HADNT), which is subsequently reduced to aminodinitrotoluene derivatives (ADNTs). Transgenic plants expressing nitroreductase show a striking increase in ability to tolerate, take up, and detoxify TNT. Our work suggests that expression of nitroreductase (NR) in plants suitable for phytoremediation could facilitate the effective cleanup of sites contaminated with high levels of explosives.

Gene cloning and nucleotide sequencing and properties of a cocaine esterase from Rhodococcus sp. strain MB1.

A strain of Rhodococcus designated MB1, which was capable of utilizing cocaine as a sole source of carbon and nitrogen for growth, was isolated from rhizosphere soil of the tropane alkaloid-producing plant Erythroxylum coca. A cocaine esterase was found to initiate degradation of cocaine, which was hydrolyzed to ecgonine methyl ester and benzoate; both of these esterolytic products were further metabolized by Rhodococcus sp. strain MB1. The structural gene encoding a cocaine esterase, designated cocE, was cloned from Rhodococcus sp. strain MB1 genomic libraries by screening recombinant strains of Rhodococcus erythropolis CW25 for growth on cocaine. The nucleotide sequence of cocE corresponded to an open reading frame of 1,724 bp that codes for a protein of 574 amino acids. The amino acid sequence of cocaine esterase has a region of similarity with the active serine consensus of X-prolyl dipeptidyl aminopeptidases, suggesting that the cocaine esterase is a serine esterase. The cocE coding sequence was subcloned into the pCFX1 expression plasmid and expressed in Escherichia coli. The recombinant cocaine esterase was purified to apparent homogeneity and was found to be monomeric, with an M(r) of approximately 65,000. The apparent K(m) of the enzyme (mean +/- standard deviation) for cocaine was measured as 1.33 +/- 0.085 mM. These findings are of potential use in the development of a linked assay for the detection of illicit cocaine.

Low prevalence of VRE gastrointestinal colonization of hospitalized patients in Manitoba tertiary care and community hospitals.

OBJECTIVE: To determine the prevalence of vancomycin-resistant enterococci (VRE) bowel colonization in hospitalized patients in Manitoba who had stool specimens collected for Clostridium difficile toxin and/or culture testing. DESIGN: Two tertiary care and five community hospitals in Winnipeg and three rural Manitoba community hospitals participated in this study. From January 1 to December 31, 1997 stool specimens, one per patient, submitted to hospital microbiology laboratories for C difficile toxin and/or culture testing were screened for VRE on colistin-nalidixic acid-vancomycin (6 microg/mL) (CNAV) agar plates. The study was divided into six, eight-week intervals. Stool specimens received in the first two weeks of each eight week interval were screened for VRE. MAIN RESULTS: A total of 1408 stool specimens were submitted over the 48-week study period. Sixty-seven (4.8%) patients with VRE colonization of their lower gastrointestinal tract were identified. Three of the 67 (4.5%) VRE isolates were Enterococcus faecium, with the remaining 64 (95.5%) were Enterococcus gallinarum. The three vancomycin-resistant E faecium -VREF- (from two different Winnipeg hospitals) demonstrated the vanA genotype, and were resistant to vancomycin, teicoplanin and ampicillin. All three VREF isolates also demonstrated high level resistance to both gentamicin and streptomycin but were susceptible to quinuprisitin/dalfopristin and LY333328. CONCLUSION: VRE colonization in hospitalized patients in Manitoba is infrequent and most commonly due to E gallinarum. The prevalence of VREF colonization in the patients studied was 0.2% (three of 1408).

The effect of pregnancy on the lower-limb venous system of women with varicose veins.

OBJECTIVES: to assess the effect of pregnancy on the lower-limb venous system of women with varicose veins. Design a longitudinal prospective study of 11 pregnant women, with varicose vein disease. METHODS: eleven pregnant women with varicose veins were recruited as part of a larger study. Veins were assessed in both lower limbs using colour-flow duplex scanning at a 75 degrees head-up tilt. The diameter and velocity and duration of reflux were measured in each vein at 12, 20, 26, 34, 38 weeks gestation and 6 weeks postpartum. RESULTS: eleven women had reflux and varicose veins demonstrated at first scan. All veins dilated with increasing gestation. This was maximal in the superficial system, reaching significance (p

Identification and characterization of class 1 integrons in bacteria from an aquatic environment.

In a survey of 3000 Gram-negative bacteria isolated from an estuarine environment over a 2 month period, the incidence of class 1 integrons was determined to be 3.6%. Of 85 integrons studied further, 11 lacked both the qacEdelta1 and sull genes usually present in the 3' conserved segment of the integron. The qacEdelta1 and sull genes were identified in the 3' conserved segment of 36 integrons. The remaining 38 integrons lacked a sull gene but contained a qacE gene. The variable region of 74 integrons was characterized by PCR and sequence analysis. Forty of the integrons were found to lack integrated gene cassettes, although 21 of these 'empty' integrons were shown to contain inserted DNA which has been tentatively identified as a novel insertion sequence (IS) element. Of the 34 integrons which contained inserted gene cassettes, the aadA1a gene was found to be the most prevalent (74%). Nineteen integrons contained additional or other gene cassettes in their variable region, including those encoding resistance to trimethoprim (dfr1a, dfrIIc, dfrV, dfrVII, dfrXII), chloramphenicol (catB3, catB5), aminoglycosides (aadA2, aacA4, aacC1), beta-lactamases (oxa2) and erythromycin (ereA). This study confirms the occurrence of integrons in bacteria from a natural habitat and suggests that in the absence of continued antibiotic selective pressures, integrons which persist appear to preferentially exist without integrated antibiotic resistance gene cassettes.

Serial colour flow duplex scanning of the veins of the lower limb throughout pregnancy.

OBJECTIVE: To examine changes in diameter and reflux in normal veins of the lower limb throughout pregnancy. METHODS: Fifty-seven women were recruited into the study and 43 completed the full assessment to six weeks postpartum. Thirteen had pre-existing venous disease and are reported elsewhere. The veins were assessed in both lower limbs using colour flow duplex scanning. This was performed at a 75 degree tilt measuring vein diameter and the presence or absence of reflux. Measurements were made at 12, 20, 26, 34, 38 weeks of gestation and 6 weeks postpartum. RESULTS: No new reflux developed in any of the veins studied. In the superficial system the maximum change was seen in the long saphenous vein at 34 weeks, on the left side the vein diameter failed to return to baseline size. Significant change also occurred in the superficial femoral vein. Dilatation of the deep veins of the calf was observed being greater in the left posterior tibial and the left peroneal at the mid-calf point. CONCLUSION: Colour flow duplex scanning is an acceptable method of assessing the lower limb veins in pregnancy. Maximum diameter changes were seen in the long saphenous vein and in the deep veins at mid-calf. No new reflux developed during the gestation period in veins which were previously normal.

Biodegradation of explosives by transgenic plants expressing pentaerythritol tetranitrate reductase.

Plants offer many advantages over bacteria as agents for bioremediation; however, they typically lack the degradative capabilities of specially selected bacterial strains. Transgenic plants expressing microbial degradative enzymes could combine the advantages of both systems. To investigate this possibility in the context of bioremediation of explosive residues, we generated transgenic tobacco plants expressing pentaerythritol tetranitrate reductase, an enzyme derived from an explosive-degrading bacterium that enables degradation of nitrate ester and nitroaromatic explosives. Seeds from transgenic plants were able to germinate and grow in the presence of 1 mM glycerol trinitrate (GTN) or 0.05 mM trinitrotoluene, at concentrations that inhibited germination and growth of wild-type seeds. Transgenic seedlings grown in liquid medium with 1 mM GTN showed more rapid and complete denitration of GTN than wild-type seedlings. This example suggests that transgenic plants expressing microbial degradative genes may provide a generally applicable strategy for bioremediation of organic pollutants in soil.

E test versus agar dilution for antimicrobial susceptibility testing of viridans group streptococci.

Viridans group streptococci (VGS) are commonly isolated from the blood of hospitalized patients. The E test represents a convenient method for determining the MICs for VGS, but for this purpose it has not been well validated against reference methods. In this study, 180 unselected VGS isolates were identified to a species level, and the MICs of penicillin, cefuroxime, cefotaxime, and vancomycin were determined by both agar dilution and the E test. Available data regarding demographic and laboratory variables for each VGS bacteremic episode were collected, the significance of each VGS isolate was assessed, and the associations between and among laboratory and clinical variables were investigated. Among all VGS isolates, 68.3% (median of three runs) were found to be fully susceptible to penicillin by agar dilution. The E test and agar dilution showed average agreements (within +/-1 dilution) of 92.2% for penicillin, 95.7% for cefuroxime 91.3% for cefotaxime, and 86.7% for vancomycin. Agreements over serial E tests and serial agar dilutions were excellent for beta-lactam agents (intraclass correlation coefficients, >0.9) but less impressive for vancomycin. Very major error rates for the E test were

The Stresst'er ergometer as an alternative to treadmill testing in patients with claudication.

OBJECTIVES: To compare a new pedal ergometer (the Stresst'er) with conventional treadmill examination in patients with leg pain on exercise. DESIGN: Patients presenting with claudication were assessed by standard treadmill test and by the new device in two District General Hospitals. METHODS: Ninety-four subjects were studied. Symptoms induced by both types of exercise were compared. Distance walked on the treadmill was compared to the number of pumps using the Stresst'er. The percentage change in ankle systolic pressure produced by the two tests was compared. RESULTS: Subjective symptoms induced by the tests were similar. Distance walked did correlate with pumps on the ergometer, as did change in systolic pressure. Analysis of a subgroup of 41 cases with receiver operator curve (ROC) tests showed that the new device is sensitive and specific. CONCLUSION: This new device is comparable with treadmill testing, but being easier to use, may have a place in the vascular clinic.

In-utero cocaine exposure and neonatal intestinal perforation: a case report.

This article describes a case of spontaneous neonatal intestinal perforation in a preterm infant with evidence of intrauterine exposure to cocaine. Clinical, laboratory, radiological, surgical, and pathological finds were inconsistent with necrotizing enterocolitis. Three previous case reports had made the association between spontaneous focal neonatal intestinal perforation and prior intrauterine cocaine exposure; however, in each case, there were extenuating circumstances that may weaken the etiological link. The case described here appears to strengthen the association/etiological link. After all other causes of focal neonatal intestinal perforation have been excluded, the possibility of intrauterine exposure to cocaine should be considered.

Recent development of rigors during infusion of urokinase: is it related to an endotoxin?

PURPOSE: This study was undertaken to determine the prevalence of rigors associated with the use of urokinase (UK) and to assay for the presence of an endotoxin in the UK solution. PATIENTS AND METHODS: Records of 75 patients who underwent 86 UK infusions between January 1988 and July 1992 were reviewed to evaluate for the development of UK-associated rigors. A modified chromogenic limulus amebocyte lysate (LAL) test was performed to determine the presence of endotoxin in four samples of UK from lots associated with rigors, one sample of UK not associated with rigors, sterile water, nonionic contrast medium, and ionic contrast medium. RESULTS: Between January 1, 1988, and July 10, 1990, 43 patients underwent 46 UK treatments (group 1) with no documented rigors (0% prevalence). In 45 of these 46 treatments, a standard, non-pulse-spray bolus of 75,000-500,000 IU of UK (mean dose, 182,222 IU) was used. Between July 11, 1990, and July 6, 1992, 38 patients underwent 40 UK treatments (group 2). In 33 of these 40 treatments, a standard bolus was given. Five patients received a pulse-spray bolus. The mean bolus was 213,768 IU (range, 100,000-500,000 IU). Eleven group 2 patients developed rigors (28% prevalence; P = .0005 vs group 1). The chromogenic LAL tests demonstrated no endotoxin in sterile water, nonionic contrast media, or ionic contrast media. Endotoxin was detected in small concentrations in the four samples of UK associated with rigors and in the UK sample not associated with rigors. CONCLUSION: The increase in the prevalence of rigors associated with the use of UK does not appear to be related to an endotoxin in UK, since the concentration of endotoxin detected is well below the threshold pyrogenic dose in humans.

Is there androcentric bias in psychiatric diagnosis?

Flaws, biases, and ethical problems surrounding research and diagnosis may lead to inappropriate or inequitable treatments that exacerbate or fail to improve the misery that some individuals face due to their psychiatric conditions. Possible androcentric biases in the choice and definition of categories for diagnosis available in DSM-III-R may in turn influence the approaches of therapists to clients, particularly male therapists towards female clients. Androcentric bias in diagnosis, which may also be reflected in the values of the psychiatrist, may lead to treatment regimens designed to make clients fit into roles, positions, and norms prescribed by a culture reflecting patriarchal values. Some acceptance of attempts by feminists to correct androcentrism are beginning to emerge in psychiatric diagnosis.