Evidence for positive selection and recombination hotspots in Deformed wing virus (DWV).

Deformed wing virus (DWV) is considered one of the most damaging pests in honey bees since the spread of its vector, Varroa destructor. In this study, we sequenced the whole genomes of two virus isolates and studied the evolutionary forces that act on DWV genomes. The isolate from a Varroa-tolerant bee colony was characterized by three recombination breakpoints between DWV and the closely related Varroa destructor virus-1 (VDV-1), whereas the variant from the colony using conventional Varroa management was similar to the originally described DWV. From the complete sequence dataset, nine independent DWV-VDV-1 recombination breakpoints were detected, and recombination hotspots were found in the 5' untranslated region (5' UTR) and the conserved region encoding the helicase. Partial sequencing of the 5' UTR and helicase-encoding region in 41 virus isolates suggested that most of the French isolates were recombinants. By applying different methods based on the ratio between non-synonymous (dN) and synonymous (dS) substitution rates, we identified four positions that showed evidence of positive selection. Three of these positions were in the putative leader protein (Lp), and one was in the polymerase. These findings raise the question of the putative role of the Lp in viral evolution.

A New Stratified Sampling Procedure which Decreases Error Estimation of Varroa Mite Number on Sticky Boards.

A new procedure of stratified sampling is proposed in order to establish an accurate estimation of Varroa destructor populations on sticky bottom boards of the hive. It is based on the spatial sampling theory that recommends using regular grid stratification in the case of spatially structured process. The distribution of varroa mites on sticky board being observed as spatially structured, we designed a sampling scheme based on a regular grid with circles centered on each grid element. This new procedure is then compared with a former method using partially random sampling. Relative error improvements are exposed on the basis of a large sample of simulated sticky boards (n=20,000) which provides a complete range of spatial structures, from a random structure to a highly frame driven structure. The improvement of varroa mite number estimation is then measured by the percentage of counts with an error greater than a given level.

Nutritional parameters modify muricidal behavior of male Wistar rats: preventive effects of amino acids and 4' Cl diazepam.

In male Wistar rats fed a diet enriched in polyunsaturated fatty acids and starch (PUFA+S), the percentage of muricidal (Mu) rats increased to 82% within 60 days. Mu rats had higher serum triglyceride levels and lower cholesterol levels than non-Mu rats. Water intake decreased in all rats on the PUFA+S diet concurrently with the increase in the proportion of Mu rats; protracted water restriction in rats fed standard diet also increased the percentage of Mu rats. In the offspring of two Wistar females fed the PUFA+S diet, the proportion of young Mu rats was 67%. When the PUFA+S diet was replaced with standard diet, the induced Mu behavior was not reversed. PK11195 (6 mg/kg i.p.), clonazepam (0.2 mg/kg i.p.), and flumazenil (15 mg/kg i.p.) were ineffective in reversing the induced Mu behavior, whereas 4'-chlorodiazepam (5 mg/kg i.p.) or muscimol (0.5 mg/kg i.p.) caused reversals of 63% or 50%, respectively. A 5-hydroxytryptophan overload (60 mg/kg i.p.) also reversed Mu behavior by 71%. All reversal effects were temporary. Pretreatment with yeast for 7 days before the PUFA+S diet was given prevented induction for more than 90 days on the PUFA+S diet, while similar pretreatment 4'Cl-diazepam resulted in 71% prevention of induction. The results are analyzed in terms of the involvement of endozepin, vasopressin, and serotonin receptors, and of possible genetic parameters.

Dietary lipids affect human ethanol-inducible CYP2E1 gene expression in vivo in mononuclear cells.

We tested the hypothesis that dietary cholesterol modulate human ethanol-inducible CYP2E1 expression in vivo in circulating mononuclear cells. Healthy volunteers (n= 10) were submitted to a low fat low cholesterol diet for 4 days (day 0-day 3, LFLC). Cholesterol (595 +/- 56 mg/day) was then reintroduced for 7 days (day 4-day 10, LFHC). In the same time, controls subjects (n=7) did not change their habitual daily diet. CYP2E1 mRNA levels, evaluated in mononuclear cells, decreased in experimental subjects during both LFLC and LFHC from 100% to 53 +/- 5%, (p<0.001) with a main decrease during LFLC period (100% to 71 +/- 16%, p=0.05). Immunoreactive CYP2E1 showed a similar pattern and decreased from 100 to 62 +/- 12% during the trial (p<0.05). No significant change occured in control subjects. Between day 0 and day 11, changes in CYP2E1 mRNA correlated positively with plasma cholesterol (r2=0.67, p<0.001) and HDL cholesterol concentrations (r2=0.61, p<0.001). In contrast, no correlation was found between plasma fatty acids concentrations and CYP2E1 expression. The present results suggest that lipid factors regulate CYP2E1 expression, in vivo, in human mononuclear cells. In particular, plasma cholesterol concentrations may play an important role in this regulation.

Thiopurine S-methyltransferase gene polymorphism is predictive of azathioprine-induced myelosuppression in heart transplant recipients.

Azathioprine (AZA) is metabolized via the cytosolic enzyme thiopurine S-methyltransferase (TPMT). TPMT activity exhibits genetic polymorphism with four prevalent (75%) mutant alleles TPMT*2 (G238C) and TPMT*3 (A719G and/or G460A) and a wild-type allele TPMT*1. To test the hypothesis that presence of these mutations is associated with greater toxicity of AZA in heart transplant recipients, 30 consecutive patients treated with AZA were followed up for the first month after heart transplant. Mutation of TPMT gene (mutation-specific polymerase chain reaction-based methods) was observed in four patients (A719G: n = 2; A719G plus G460: n = 2). Agranulocytosis did not occur in patients with the wild genotype. It occurred in the two patients with mutation A719G and there was a 40% drop in neutrophils in the two other patients. Discontinuation of AZA in the four mutant patients corrected for the drop. Presence of TPMT mutations is associated with a greater likelihood of agranulocytosis. Determination of these mutations could reduce the risk for hematological side-effects.

Cholesterol and triglyceride levels in the serum of muricidal male Wistar rats: indices of mitochondrial benzodiazepine receptors?

Cholesterol and triglyceride levels were studied in the serum of aggressive muricidal and non-muricidal male Wistar rats. The muricidal behavior was either spontaneous or induced by a long-term isolation or by adrenalectomy. Cholesterol levels were slightly higher in the whole population of muricidal rats; this was mainly observed in spontaneously and in adrenalectomized muricidal rats, as compared to non-muricidal rats of the same series. As regards triglyceride levels, they were significantly higher in the whole population of muricidal rats, mainly in isolation- and adrenalectomy-induced muricidal rats; the ratio of triglycerides to body weight was higher in the serum of muricidal rats of all series. The possible significance of these results is discussed in light of the data of the literature and related to the functional role of either mitochondrial benzodiazepine receptors or serotonin.

Arsenic speciation in humans and food products: a review.

Although acute intoxication has become rare, arsenic (As) is still a dangerous pollution agent for industrial workers and people living in the vicinity of emission sources. In humans, only inorganic As is toxic; organic forms present in large amounts in the environment are nontoxic. It is therefore important to be able to differentiate one group from the other using appropriate speciation methods. The authors review the present knowledge of the distribution of As in humans and food products. The three steps of the speciation methods (sample preparation, species separation, and detection) are described. For liquid samples, a clean-up step (C18 cartridge extraction, dilution, or freezing) is necessary to eliminate proteins and salts from the matrix. For solid organic samples, the first step consists of the digestion of tissues followed by solvent extraction sometimes coupled with a C18 extraction. The separation of As species is accomplished by different high-performance liquid chromatography (HPLC) methods (ion-exchange, ion-pairing, and micellar liquid chromatography). The detection methods are compatible with HPLC and are able to detect As species in the microgram-per-liter range. Inductively coupled plasma (ICP) atomic emission spectrometry is more frequently used, but suffers from interference by organic solvents in the mobile phases. Atomic absorption spectrometry methods give sensitivities of the same order. ICP-mass spectrometry has the advantage of specificity and can be 100- to 1000-fold more sensitive than previous methods.

Arsenic speciation in human organs following fatal arsenic trioxide poisoning--a case report.

The aim of this investigation was to study the distribution of arsenic species in human organs following fatal acute intoxication by arsenic trioxide. The collected autopsy samples of most organs were ground and dried, and the total arsenic was measured by electrothermal atomic absorption spectrometry (ETAAS). The arsenic species--inorganic arsenic, in the form of arsenite [As(III)] and arsenate [As(V)], and its metabolites [monomethylarsonic acid (MMA) and dimethylarsinic acid (DMA)]--were quantified by ETAAS after extraction with methanol/water (1:1, by volume) and separation by HPLC. The results indicate that after acute intoxication, the liver and kidneys show the highest concentrations of total arsenic and that the total concentration in blood is 7- to 350-fold less concentrated than in organs. In all organs, As(III) is the predominant species, and MMA is more concentrated than DMA. MMA and DMA are more prevalent in lipidic organs (49% of total arsenic) compared with other organs (25% of total arsenic). As(V) was found in small quantities in the liver, kidneys, and blood.

Contribution to chromium and nickel enrichment during cooking of foods in stainless steel utensils.

Nickel ingestion can cause exacerbation of dermatitis in patients who are already nickel-sensitive; Chromium (Cr VI) is the 2nd allergen, after nickel. However, stainless steel is widely used in home cookware. In this study, we determined nickel and chromium levels by atomic absorption spectrometry in 11 habitual menus cooked in different grades of stainless steel utensils. We noted a great difference in nickel and chromium intake depending on the menu, and a significant difference between the glass and stainless steel saucepans, but this was very low compared with the levels of nickel and chromium contained in the menus; mean intakes of these elements were under the tolerable daily intake (TDI) recommended by the World Health Organization. Hence, there is no advantage for nickel-sensitive patients in switching to materials other than stainless steel, provided that this is of good quality.

Effect of dietary cholesterol on low density lipoprotein-receptor, 3-hydroxy-3-methylglutaryl-CoA reductase, and low density lipoprotein receptor-related protein mRNA expression in healthy humans.

We investigated the possibility that dietary cholesterol downregulates the expression of low density lipoprotein (LDL) receptor and 3-hydroxy-3-methylglutaryl (HMG)-CoA reductase genes of circulating mononuclear cells in vivo in healthy humans. We also studied the variations of the LDL receptor-related protein (LRP) gene in the same conditions. Dieters (n = 5) were submitted to a 4-d fat restriction (mean cholesterol intake: 6+/-4 mg/d), followed by a 7-d cholesterol (a mean of 791+/-150 mg/d) supplementation. Controls (n = 3) did not change their diet. During fat restriction, serum total and LDL cholesterol decreased significantly (P < 0.05), and LDL receptor and HMG-CoA reductase mRNA copy numbers in mononuclear cells increased by 57 and 147%, respectively (P < 0.05). After reintroducing cholesterol, serum cholesterol was stable whereas LDL receptor and HMG-CoA reductase mRNA decreased by 46 and 72% (P < 0.05) and LRP mRNA increased by 59% (P < 0.005). The changes in LDL receptor and HMG-CoA reductase mRNA abundance were correlated (r = +0.79, P = 0.02) during cholesterol reintroduction as were LDL receptor and LRP mRNA levels, but negatively (r = -0.70, P = 0.05). Also, 70% of the variability in LRP mRNA (P < 0.005) was explained by dietary cholesterol. Thus, the basic mechanisms regulating cellular cholesterol content, the coordinate feedback repression of genes governing the synthesis and uptake of cholesterol, are operating in vivo in humans. However, serum cholesterol did not increase in response to dietary cholesterol, suggesting that these mechanisms may not play as predominant a role as previously believed in the short-term control of serum cholesterol in vivo in humans. A new finding is that LRP gene is also sensitive to dietary cholesterol, suggesting that it may participate in the control of serum cholesterol. Further in vivo studies in humans are warranted to explore the molecular mechanisms of the physiological response to dietary cholesterol in humans.

Validated determination of total arsenic species of toxicological interest (arsenite, arsenate and their metabolites) by atomic absorption spectrometry after separation from dietary arsenic by liquid extraction: toxicological applications.

A validated method for the selective extraction of total As species of toxicological interest (arsenite, arsenate and mono- and dimethylated arsenic species) from urine, followed by atomic absorption spectrometric determination, is described. The mechanisms involved in extraction were studied and the extraction method was optimized. The urine sample was acidified with concentrated HCl and KI and sodium hypophosphite were added. Under these conditions, As species were reduced to their corresponding iodide arsines, extracted with toluene and back-extracted with 1 mmol l-1 NaOH solution. Only inorganic arsenic and its metabolites in humans (monomethylarsonic and dimethylarsinic acid) were extracted. Arsenobetaine of dietary origin was not extracted. This method can detect if any As increase in urine originates from inorganic As intoxication or only from dietary non-toxic As species such as arsenobetaine.

Chemiluminescent measurement of oxalate in serum by detection of hydrogen peroxide generated through oxalate oxidase.

Today, chemiluminescence detection reactions have become popular in analytical biochemistry essentially due to their high sensitivity. A chemiluminescent synthetic system (luminol/porphyrin) was successfully used to measure serum oxalate by determination of hydrogen peroxide generated through oxalate oxidase (EC 1.2.3.4.). This new method is efficient and simple, highly sensitive and the results obtained in normal adult subjects are in good agreement with those of approved methods. This original application of such a chemiluminescent system allowed us to achieve a sensitive serum oxalate assay (detection limit of 0.2 mumol/L) characterized by a low serum volume (200 microL) required for analysis.

Serum oxalate microassay using chemiluminescence detection.

Determination of serum oxalate concentration is important for the diagnosis and monitoring of hyperoxalurias, and extends to patients with all types of renal disease. Approximately 5 to 10 ml of blood is required for each test by conventional methods, and the test is not adapted for use in children. We developed a highly sensitive method that limits the volume of blood required for the test. This new and sensitive tool to detect H2O2 can be successfully substituted for the conventional, and expensive, colorimetric reaction to accurately analyze oxalate concentration.

Cyclodextrins and enantiomeric separations of drugs by liquid chromatography and capillary electrophoresis: basic principles and new developments.

Investigation of individual drug enantiomers is required in pharmacokinetic and pharmacodynamic studies of drugs with a chiral centre. Cyclodextrins (CDs) are extensively used in high-performance liquid chromatography as stationary phases bonded to a solid support or as mobile phase additives in HPLC and capillary electrophoresis (CE) for the separation of chiral compounds. We describe here the basis for the liquid chromatographic and capillary electrophoretic resolution of drug enantiomers and the factors affecting their enantiomeric separation. This review covers the use of CDs and some of their derivatives in studies of compounds of pharmacological interest.

Simple high-performance liquid chromatographic separation of oxazepam and its diastereoisomeric glucuronides in serum. Applications in a pharmacokinetic study in sheep.

This paper describes a highly specific and sensitive method for quantifying oxazepam and its diastereoisomeric glucuronides in serum. The method involves sample clean-up by solid-phase extraction on C18 cartridge followed by quantitation on a reversed-phase HPLC column. Diazepam is used as internal standard. Extraction recovery from serum proved to be more than 86%. Precision, expressed as C.V., was in the range 1.2-9.5%. The limits of quantification were 40, 400, and 200 nmol/l for oxazepam, S-(+)- and R-(-)-glucuronides, respectively. This method was applied to the determination of oxazepam and its diastereoisomeric glucuronides in serum collected during a pharmacokinetic study performed in sheep after oral administration of racemic oxazepam. S-(+)/R-(-) ratios were measured all along the sampling time collection and the pharmacokinetic parameters were determined.

Automated Cobas Mira kinetic enzymatic assay for ethylene glycol applied to emergency situations.

An automated kinetic method for assaying ethylene glycol in serum using glycerol dehydrogenase with the multiparametric analyzer Cobas Mira is described. Initially, 5 microL of sample is mixed with tris-NAD buffer; after enzyme addition, the variation of the absorbance at 340 nm is automatically measured, and the instrument calculates the ethylene glycol concentration of the specimen. The method has good precision and specificity and is suitable for emergency screening. Some applications developed in our laboratory are also described.

Quantitation by gas chromatography with selected-ion monitoring mass spectrometry of "natural" diazepam, N-desmethyldiazepam and oxazepam in normal human serum.

During the past five years, the literature has tended to prove the occurrence of "natural benzodiazepines" in tissues and biological fluids of non-medicated humans. Several have been identified but very few papers deal with their quantitation in biological material. We present here a method for the specific and sensitive measurement of serum levels of diazepam, N-desmethyldiazepam and oxazepam by gas chromatography with selected-ion monitoring mass spectrometry in twenty human volunteers without medication. Diazepam was found over the whole population, in the range 7.3-32.0 pg/ml, identical in males and females. The other two were present in only some individuals (1.0-7.6 pg/ml for N-desmethyldiazepam and 2.0-13.0 pg/ml for oxazepam). The origin (endogenous, dietary or microbial) of these substances is still to be elucidated.