Source inference of exogenous gamma-hydroxybutyric acid (GHB) administered to humans by means of carbon isotopic ratio analysis: novel perspectives regarding forensic investigation and intelligence issues.

γ-Hydroxybutyric acid (GHB) is an endogenous short-chain fatty acid popular as a recreational drug due to sedative and euphoric effects, but also often implicated in drug-facilitated sexual assaults owing to disinhibition and amnesic properties. Whilst discrimination between endogenous and exogenous GHB as required in intoxication cases may be achieved by the determination of the carbon isotope content, such information has not yet been exploited to answer source inference questions of forensic investigation and intelligence interests. However, potential isotopic fractionation effects occurring through the whole metabolism of GHB may be a major concern in this regard. Thus, urine specimens from six healthy male volunteers who ingested prescription GHB sodium salt, marketed as Xyrem(®), were analysed by means of gas chromatography/combustion/isotope ratio mass spectrometry to assess this particular topic. A very narrow range of δ(13)C values, spreading from -24.810/00 to -25.060/00, was observed, whilst mean δ(13)C value of Xyrem(®) corresponded to -24.990/00. Since urine samples and prescription drug could not be distinguished by means of statistical analysis, carbon isotopic effects and subsequent influence on δ(13)C values through GHB metabolism as a whole could be ruled out. Thus, a link between GHB as a raw matrix and found in a biological fluid may be established, bringing relevant information regarding source inference evaluation. Therefore, this study supports a diversified scope of exploitation for stable isotopes characterized in biological matrices from investigations on intoxication cases to drug intelligence programmes.

Allergy to betalactam antibiotics in children: a prospective follow-up study in retreated children after negative responses in skin and challenge tests.

BACKGROUND: Up to 10% of the patients in whom suspected betalactam hypersensitivity (HS) has been excluded by skin and challenge tests report suspected allergic reactions during subsequent treatments with the same or very similar betalactams. It has been suggested that the reactions may result from a resensitization induced by the challenge performed at the time of the allergological work-up. However, most patients did not undergo a second allergological work-up, to determine if the reactions resulted from betalactam HS or not. OBJECTIVES: We aimed to determine if children diagnosed nonallergic to betalactams have tolerated subsequent treatments with the initially suspected and/or other betalactams, and, in case of a reaction, if the reaction resulted from betalactam HS. Methods: We sent a questionnaire concerning the clinical history of their children to the parents of 256 children previously diagnosed nonallergic to betalactams. A second allergological work-up was performed in the children reporting suspected allergic reactions during subsequent treatments with the same and/or other betalactams. Skin tests were performed with the soluble form of the suspected (or very similar) betalactams and other betalactams from the same and other classes. Skin test responses were assessed at 15-20 min (immediate), 6-8 h (semi-late) and 48-72 h (late). Oral challenge (OC) was performed in children with negative skin tests, either at the hospital (immediate and accelerated reactions), or at home (delayed reactions). RESULTS: A response was obtained from 141 children (55.3%). Forty-eight (34%) of those children had not been treated with the betalactams for whom a diagnosis of allergy had been ruled out previously. Seven (7.5%) of the 93 children who had been treated again reported suspected allergic reactions. Skin tests and OC were performed in six of those children, and gave negative results in five children. In one child previously diagnosed nonallergic to amoxicillin associated with clavulanic acid, we diagnosed a delayed HS to clavulanic acid and a serum sickness-like disease to cefaclor. Thus, the frequency of reactions resulting from betalactam HS in children with negative skin and challenge tests is very low, and does not exceed 2.1% (2/93) if we consider that the child which refused a second allergological work-up is really allergic to betalactams. CONCLUSION: Our results in a very large number of children show that reactions presumed to result from betalactam HS are rare in children in whom the diagnosis of betalactam allergy has been ruled out previously. Moreover, they suggest that, as shown for the initial reactions, most of the reactions during subsequent treatments are rather a consequence of the infectious diseases for whom betalactams have been prescribed than a result of betalactam HS. Finally, they suggest that the risk of resensitization by OC is very low, and do not support the notion that skin testing should be repeated in children diagnosed nonallergic to betalactams.

Zapfen-Stäbchen-Dystrophie durch eine neue reinerbige RPE65-Mutation in Leberscher kongenitaler Amaurose Cone-Rod Dystrophy Caused by a Novel Homozygous RPE65 Mutation in Leber Congenital Amaurosis.

Background: The aim of this study was to describe an unexpected phenotype in a family with Leber congenital amaurosis (LCA) due to a retinal pigment epithelium-specific protein 65 kDa (RPE65) homozygous mutation. History and Signs: We analyzed a family from Yemen in which 3 individuals were affected with LCA. Linkage analysis using markers flanking the known LCA genes was done, followed by direct sequencing of RPE65. Therapy and Outcome: Severe visual impairment and night blindness were observed during infancy. We observed photophobia only in the 8-year-old patient. The youngest affected had bilateral hyperopia of + 3.50 and visual acuity of 1/60. The oldest two had visual acuity limited to hand movements in the right eye (OD) and counting fingers in the left eye (OS) for the oldest and of 5/60 OD, 6/60 OS for the other. They showed disc pallor, attenuated vessels, white flecks in the retina mid-periphery and bull's eye maculopathy. ERGs of the oldest child were completely unresponsive. Genomic sequencing identified a novel homozygous missense mutation, IVS2-3C > G, in the second RPE65 intron. Conclusions: We identified a novel LCA-related homozygous RPE65 mutation associated with a severe clinical presentation including an early and severe cone dysfunction. This is in contrast with the presentation associated with other RPE65 mutations predominantly causing rod-cone dystrophy with residual visual function.

Genomics of secondary metabolite production by Pseudomonas fluorescens Pf-5

The complete sequence of the 7.07 Mb genome of the biological control agent Pseudomonas fluorescens Pf-5 is now available, providing a new opportunity to advance knowledge of biological control through genomics. P. fluorescens Pf-5 is a rhizosphere bacterium that suppresses seedling emergence diseases and produces a spectrum of antibiotics toxic to plant-pathogenic fungi and oomycetes. In addition to six known secondary metabolites produced by Pf-5, three novel secondary metabolite biosynthesis gene clusters identified in the genome could also contribute to biological control. The genomic sequence provides numerous clues as to mechanisms used by the bacterium to survive in the spermosphere and rhizosphere. These features include broad catabolic and transport capabilities for utilizing seed and root exudates, an expanded collection of efflux systems for defense against environmental stress and microbial competition, and the presence of 45 outer membrane receptors that should allow for the uptake of iron from a wide array of siderophores produced by soil microorganisms. As expected for a bacterium with a large genome that lives in a rapidly changing environment, Pf-5 has an extensive collection of regulatory genes, only some of which have been characterized for their roles in regulation of secondary metabolite production or biological control. Consistent with its commensal lifestyle, Pf-5 appears to lack a number of virulence and pathogenicity factors found in plant pathogen.

Siglec-1 is a novel dendritic cell receptor that mediates HIV-1 trans-infection through recognition of viral membrane gangliosides.

Dendritic cells (DCs) are essential antigen-presenting cells for the induction of immunity against pathogens. However, HIV-1 spread is strongly enhanced in clusters of DCs and CD4(+) T cells. Uninfected DCs capture HIV-1 and mediate viral transfer to bystander CD4(+) T cells through a process termed trans-infection. Initial studies identified the C-type lectin DC-SIGN as the HIV-1 binding factor on DCs, which interacts with the viral envelope glycoproteins. Upon DC maturation, however, DC-SIGN is down-regulated, while HIV-1 capture and trans-infection is strongly enhanced via a glycoprotein-independent capture pathway that recognizes sialyllactose-containing membrane gangliosides. Here we show that the sialic acid-binding Ig-like lectin 1 (Siglec-1, CD169), which is highly expressed on mature DCs, specifically binds HIV-1 and vesicles carrying sialyllactose. Furthermore, Siglec-1 is essential for trans-infection by mature DCs. These findings identify Siglec-1 as a key factor for HIV-1 spread via infectious DC/T-cell synapses, highlighting a novel mechanism that mediates HIV-1 dissemination in activated tissues.

Novel Homozygous Rax Gene Mutation in Patients With Bilateral Anophthalmia

Purpose: To report the clinical and genetic study of one family and one isolated case of Egyptian origin with clinical anophthalmia. To further determine the role of RAX in anophthalmia and associated cerebral malformations. Methods: Three patients with clinical anophthalmia and first-degree relatives from 2 consanguineous families of Egyptian origin underwent full ophthalmologic, general and neurological examination, and blood drawing. Cerebral MRI was performed in the index case of the family and in the isolated case. Genomic DNA was prepared from venous leukocytes and direct sequencing of all the exons and intron-exon junctions of the RAX gene was performed after PCR amplification Results: Clinical bilateral anophthalmia was observed in all three patients. General and neurological examination was free in the family; obesity and psychomotor developmental delay was noticed in the isolated case. Orbital MRI showed the presence of cystic remnants and reduced optic nerves. Thin optic chiasm was the only observed cerebral malformation on MRI in the index case while the isolated case harboured diffuse cerebral atrophy and absence of the pituitary gland in addition. The three patients carried a novel homozygous mutation (IVS2-3G>A) in the RAX gene, while their parents were heterozygous healthy carriers. Conclusions: To our knowledge, only two isolated cases of anophthalmia have been found to be caused by compound heterozygote RAX mutations, three null and one missense, affecting nuclear localization or DNA-binding homeodomain. We identified a novel homozygous RAX mutation in three patients with bilateral anophthalmia from Northern Egypt. The mutation potentially affects splicing of the last exon and, if not submitted to non-stop decay, could result in a protein that has an aberrant homeodomain and no paired-tail domain. Functional consequences of this change still need to be characterized. This is the first report of homozygous RAX mutation associated with autosomal recessive bilateral anophthalmia

Molecular and functional characterization of a novel cardiac-specific human tropomyosin isoform.

BACKGROUND: Tropomyosin (TM), an essential actin-binding protein, is central to the control of calcium-regulated striated muscle contraction. Although TPM1alpha (also called alpha-TM) is the predominant TM isoform in human hearts, the precise TM isoform composition remains unclear. METHODS AND RESULTS: In this study, we quantified for the first time the levels of striated muscle TM isoforms in human heart, including a novel isoform called TPM1kappa. By developing a TPM1kappa-specific antibody, we found that the TPM1kappa protein is expressed and incorporated into organized myofibrils in hearts and that its level is increased in human dilated cardiomyopathy and heart failure. To investigate the role of TPM1kappa in sarcomeric function, we generated transgenic mice overexpressing cardiac-specific TPM1kappa. Incorporation of increased levels of TPM1kappa protein in myofilaments leads to dilated cardiomyopathy. Physiological alterations include decreased fractional shortening, systolic and diastolic dysfunction, and decreased myofilament calcium sensitivity with no change in maximum developed tension. Additional biophysical studies demonstrate less structural stability and weaker actin-binding affinity of TPM1kappa compared with TPM1alpha. CONCLUSIONS: This functional analysis of TPM1kappa provides a possible mechanism for the consequences of the TM isoform switch observed in dilated cardiomyopathy and heart failure patients.

Diplostigmaty in plants: a novel mechanism that provides reproductive assurance.

Differentiation of female sexual organs in flowering plants is rare and contrasts with the wide range of male reproductive strategies. An unusual example involves diplostigmaty, the possession of spatially and temporally distinct stigmas in Sebaea (Gentianaceae). Here, the single pistil within a flower has an apical stigma, as occurs in most flowering plants, but also a secondary stigma that occurs midway down the style, which is physically discrete and receptive several days after the apical stigma. We examined the function of diplostigmaty in Sebaea aurea, an insect-pollinated species of the Western Cape of South Africa. Floral manipulations and measurements of fertility and mating patterns provided evidence that basal stigmas function to enable autonomous delayed self-pollination, without limiting opportunities for outcrossing and thus avoiding the costs of seed discounting. We suggest that delayed selfing serves as a mechanism of reproductive assurance in populations with low plant density. The possession of dimorphic stigma function provides a novel example of a flexible mixed-mating strategy in plants that is responsive to changing demographic conditions.

Effect of a novel beta-adrenoceptor agonist (Ro 40-2148) on resting energy expenditure in obese women.

The aim of this single-blind, placebo-controlled study was to investigate the effects of the new beta-adrenergic compound Ro 40-2148 on resting energy expenditure (REE) at rest and after an oral glucose load in non-diabetic obese women before and after two weeks of treatment. After one week of placebo administration and after an overnight fast and one hour rest, REE and glucose and lipid oxidation rates were measured by indirect calorimetry (hood system) before and for 6 h after a single dose of placebo solution. A 75 g oral glucose tolerance test (OGTT) was performed during this period starting 90 min after the placebo administration. During the following two weeks, using a randomization design, six patients received Ro 40-2148 at a dose of 400 mg diluted in 100 ml water twice a day (i.e. 800 mg per day), while six others continued with the placebo administration. The same tests and measurements were repeated after two weeks, except for the treatment group which received the drug instead of the placebo. The 14-day period of drug administration did not increase REE measured in post-absorptive conditions. Similarly, there was no acute effect on REE of a 400 mg dose of Ro 40-2148. In contrast, glucose-induced thermogenesis was significantly increased after two weeks in the treatment group (means +/- s.e.m.: 3.7 +/- 1.3%, P = 0.047), while no change was observed in the placebo group (-0.8 +/- 0.7%, not significant). Since there was no significant change in the respiratory quotient, the increase in energy expenditure observed in the treatment group was due to stimulation of both lipid and glucose oxidation. The drug induced no variations in heart rate, blood pressure, axillary temperature or in plasma glucose, insulin and free fatty acid levels. In conclusion, this study shows that Ro 40-2148 activates glucose-induced thermogenesis in obese non-diabetic patients.

Genomewide association study using a high-density single nucleotide polymorphism array and case-control design identifies a novel essential hypertension susceptibility locus in the promoter region of endothelial NO synthase.

Essential hypertension is a multifactorial disorder and is the main risk factor for renal and cardiovascular complications. The research on the genetics of hypertension has been frustrated by the small predictive value of the discovered genetic variants. The HYPERGENES Project investigated associations between genetic variants and essential hypertension pursuing a 2-stage study by recruiting cases and controls from extensively characterized cohorts recruited over many years in different European regions. The discovery phase consisted of 1865 cases and 1750 controls genotyped with 1M Illumina array. Best hits were followed up in a validation panel of 1385 cases and 1246 controls that were genotyped with a custom array of 14 055 markers. We identified a new hypertension susceptibility locus (rs3918226) in the promoter region of the endothelial NO synthase gene (odds ratio: 1.54 [95% CI: 1.37-1.73]; combined P=2.58 · 10(-13)). A meta-analysis, using other in silico/de novo genotyping data for a total of 21 714 subjects, resulted in an overall odds ratio of 1.34 (95% CI: 1.25-1.44; P=1.032 · 10(-14)). The quantitative analysis on a population-based sample revealed an effect size of 1.91 (95% CI: 0.16-3.66) for systolic and 1.40 (95% CI: 0.25-2.55) for diastolic blood pressure. We identified in silico a potential binding site for ETS transcription factors directly next to rs3918226, suggesting a potential modulation of endothelial NO synthase expression. Biological evidence links endothelial NO synthase with hypertension, because it is a critical mediator of cardiovascular homeostasis and blood pressure control via vascular tone regulation. This finding supports the hypothesis that there may be a causal genetic variation at this locus.

Improved HIV-1 RNA quantitation by COBAS AmpliPrep/COBAS TaqMan HIV-1 Test, v2.0 using a novel dual-target approach.

BACKGROUND: HIV-1 RNA viral load is a key parameter for reliable treatment monitoring of HIV-1 infection. Accurate HIV-1 RNA quantitation can be impaired by primer and probe sequence polymorphisms as a result of tremendous genetic diversity and ongoing evolution of HIV-1. A novel dual HIV-1 target amplification approach was realized in the quantitative COBAS AmpliPrep/COBAS TaqMan HIV-1 Test, v2.0 (HIV-1 TaqMan test v2.0) to cope with the high genetic diversity of the virus. OBJECTIVES AND STUDY DESIGN: The performance of the new assay was evaluated for sensitivity, dynamic range, precision, subtype inclusivity, diagnostic and analytical specificity, interfering substances, and correlation with the COBAS AmpliPrep/COBAS TaqMan HIV-1 (HIV-1 TaqMan test v1.0) predecessor test in patients specimens. RESULTS: The new assay demonstrated a sensitivity of 20 copies/mL, a linear measuring range of 20-10,000,000 copies/mL, with a lower limit of quantitation of 20 copies/mL. HIV-1 Group M subtypes and HIV-1 Group O were quantified within +/-0.3 log(10) of the assigned titers. Specificity was 100% in 660 tested specimens, no cross reactivity was found for 15 pathogens nor any interference for endogenous substances or 29 drugs. Good comparability with the predecessor assay was demonstrated in 82 positive patient samples. In selected clinical samples 35/66 specimens were found underquantitated in the predecessor assay; all were quantitated correctly in the new assay. CONCLUSIONS: The dual-target approach for the HIV-1 TaqMan test v2.0 enables superior HIV-1 Group M subtype coverage including HIV-1 Group O detection. Correct quantitation of specimens underquantitated in the HIV-1 TaqMan test v1.0 test was demonstrated.