Pitfalls and challenges associated with phenoconversion in forensic toxcicology.

PURPOSE: In recent years, several publications have demonstrated the interest and the usefulness of pharmacogenetics in forensic toxicology. However, this approach remains namely focused on DNA-based phenotype, which may potentially lead to misinterpretation. Other determinants such as co-medication or physiological parameters may also impact the phenotype. This article aims to highlight the importance of considering such determinants in forensic toxicology, through the original case of a heroin-related fatality. METHOD: Ethanol concentration determination and toxicological screening were performed using gas chromatography with flame ionization detection, liquid chromatography with diode array detection and gas chromatography with mass spectrometry detection. CYP2C19 and CYP2D6 genotypes were determined by Taqman® real-time PCR analyses. RESULTS: Femoral blood analyses revealed the presence of ethanol, morphine, codeine, venlafaxine (VEN), O-desmethylvenlafaxine (ODV) and N-desmethylvenlafaxine (NDV), paroxetine, and risperidone. 6-acetylmorphine was also identified in urine. VEN, paroxetine and risperidone were quantified at supra-therapeutic or toxic blood concentrations. NDV was not quantified. The metabolic ratio of VEN (ODV to VEN) was exceptionally low (about 0.7). Pharmacogenetics testing showed that the patient was heterozygous for the CYP2C19*2 loss-of-function allele, which predict an intermediate metabolism for CYP2C19. None of the deficient CYP2D6 alleles investigated were identified. Those results suggest an extensive CYP2D6-metabolism phenotype. CONCLUSION: A discrepancy was seen between the results of the genomic evaluation and the observed metabolic ratio of VEN. This tends to exclude a genetic origin and lead us to formulate other hypotheses, such as phenoconversion that may have been induced by drug interaction involving patients' regular medications. Phenoconversion is as a complex phenomenon that leads to genotype-phenotype mismatch without any genetic abnormality particularly described for cytochromes P450 2D6 and 2C19. Although transient, phenoconversion can have a significant impact on the analysis and interpretation of genotype-focused clinical outcomes correlation and in forensic toxicology conclusions.

Author Correction: Climatic controls of decomposition drive the global biogeography of forest-tree symbioses.

In this Letter, a middle initial and additional affiliation have been added for author G. J. Nabuurs; two statements have been added to the Supplementary Acknowledgements; and a citation to the French National Institute has been added to the Methods; see accompanying Author Correction for further details.

Climatic controls of decomposition drive the global biogeography of forest-tree symbioses.

The identity of the dominant root-associated microbial symbionts in a forest determines the ability of trees to access limiting nutrients from atmospheric or soil pools1,2, sequester carbon3,4 and withstand the effects of climate change5,6. Characterizing the global distribution of these symbioses and identifying the factors that control this distribution are thus integral to understanding the present and future functioning of forest ecosystems. Here we generate a spatially explicit global map of the symbiotic status of forests, using a database of over 1.1 million forest inventory plots that collectively contain over 28,000 tree species. Our analyses indicate that climate variables-in particular, climatically controlled variation in the rate of decomposition-are the primary drivers of the global distribution of major symbioses. We estimate that ectomycorrhizal trees, which represent only 2% of all plant species7, constitute approximately 60% of tree stems on Earth. Ectomycorrhizal symbiosis dominates forests in which seasonally cold and dry climates inhibit decomposition, and is the predominant form of symbiosis at high latitudes and elevation. By contrast, arbuscular mycorrhizal trees dominate in aseasonal, warm tropical forests, and occur with ectomycorrhizal trees in temperate biomes in which seasonally warm-and-wet climates enhance decomposition. Continental transitions between forests dominated by ectomycorrhizal or arbuscular mycorrhizal trees occur relatively abruptly along climate-driven decomposition gradients; these transitions are probably caused by positive feedback effects between plants and microorganisms. Symbiotic nitrogen fixers-which are insensitive to climatic controls on decomposition (compared with mycorrhizal fungi)-are most abundant in arid biomes with alkaline soils and high maximum temperatures. The climatically driven global symbiosis gradient that we document provides a spatially explicit quantitative understanding of microbial symbioses at the global scale, and demonstrates the critical role of microbial mutualisms in shaping the distribution of plant species.

Structural patterns of the human ABCC4/MRP4 exporter in lipid bilayers rationalize clinically observed polymorphisms.

The ABCC4/MRP4 exporter has a clinical impact on membrane transport of a broad range of xenobiotics. It is expressed at key locations for drug disposition or effects such as in the liver, the kidney and blood cells. Several polymorphisms and mutations (e.g., p.Gly187Trp) leading to MRP4 dysfunction are associated with an increased risk of toxicity of some drugs. So far, no human MRP4 structure has been elucidated, precluding rationalization of these dysfunctions at a molecular level. We constructed an atomistic model of the wild type (WT) MRP4 and the p.Gly187Trp mutant embedded in different lipid bilayers and relaxed them for hundreds of nanoseconds by molecular dynamics simulations. The WT MRP4 molecular structure confirmed and ameliorated the general knowledge about the transmembrane helices and the two nucleotide binding domains. Moreover, our model elucidated positions of three generally unresolved domains: L1 (linker between the two halves of the exporter); L0 (N-terminal domain); and the zipper helices (between the two NBDs). Each domain was thoroughly described in view of its function. The p.Gly187Trp mutation induced a huge structural impact on MRP4, mainly affecting NBD 1 structure and flexibility. The structure of transporter enabled rationalization of known dysfunctions associated with polymorphism of MRP4. This model is available to the pharmacology community to decipher the impact of any other clinically observed polymorphism and mutation on drug transport, giving rise to in silico predictive pharmacogenetics.

Response to Comment on "The extent of forest in dryland biomes".

Griffith et al do not question the quality of our analysis, but they question our results with respect to the definition of forest we employed. In our response, we explain why the differences we report result from a difference of technique and not of definition, and how anyone can adapt-as we did-our data set to any forest definition and tree cover threshold of interest.

Response to Comment on "The extent of forest in dryland biomes".

De la Cruz et al question the reliability of our results, claiming that we do not refer to the most appropriate spatial extent of drylands. In our response, we explain why we chose an existing and internationally recognized delineation of drylands among several options, and why our findings are due to a difference of remote sensing technique and not to the definition of drylands we have selected.

Response to Comment on "The extent of forest in dryland biomes".

Schepaschenko et al question our findings, claiming that we did not refer to all existing maps and that we did not account for all sources of uncertainty. In our response, we detail our selection criteria for reference maps, which clarify why the work of Schepaschenko et al was not used, and we explain why our uncertainty assessment is complete and how it was misunderstood by Schepaschenko et al.

Short implants compared to implants in vertically augmented bone: a systematic review.

OBJECTIVES: To assess relevant data comparing short implants or implants associated with vertical ridge augmentation derived from RCT's and CCT's. MATERIAL AND METHODS: A PubMed and hand search was performed to identify all RCT's and CCT's published in English language comparing short implants to implants associated with vertical ridge augmentation. RESULTS: The initial search resulted in 3387 articles. A total of 17 articles were eligible for full-text analysis and four were finally included. This review tends to demonstrate similar implant survival rates between implants placed in vertically augmented bone and short implants (95.09% vs. 96.24%, respectively) with a follow-up ranging from 1 to 5 years. In terms of prosthetic survival rates, there were no differences between the treatments. More surgical complications were reported when using implants placed in vertically augmented bone compared to short implants (56 patients with surgical complications compared to 18 patients, respectively). CONCLUSIONS: This evidence should, however, be interpreted with caution as it is derived from four RCT's with limited sample size (ranging from 15 to 30 per group), limited follow-up and performed by the same research group.

Spatio-temporal variation of biting flies, Stomoxys spp. (Diptera: Muscidae), along a man-made disturbance gradient, from primary forest to the city of Makokou (North-East, Gabon).

Understanding the pattern of abundance of vector populations is important to control the potential of transmission of associated pathogens. The pattern of abundance of Stomoxys Geoffroy, an ubiquitous blood-sucking fly, is poorly known in tropical Africa. In this study, we investigated the spatio-temporal pattern of abundance of the Stomoxys genus along a gradient of man-made disturbance in north-eastern Gabon. Three sites (one in primary forest, one in secondary forest and one in a man-made environment) were monitored during 13 months using Vavoua traps. Seven species and subspecies were found to live in sympatry, but with distinct patterns of abundance with respect to space and time. The most abundant species was Stomoxys transvittatus Villeneuve, whereas the rarest species was S. xanthomelas Roubaud. Stomoxys calcitrans Linné was preferentially found in man-made environments, whereas S. xanthomelas was preferentially found in primary forest. Stomoxys abundance was the greatest in secondary forest, then in man-made environments and finally in primary forest. A seasonal variation in Stomoxys abundance was also found. In conclusion, forest degradation and deforestation are likely both to favour the concentration of populations of Stomoxys, and to change the specific composition of the Stomoxys community.

Coordinate regulation of estrogen receptor ß degradation by Mdm2 and CREB-binding protein in response to growth signals.

The biological actions of estrogen are mediated via estrogen receptors ERα and ERß. Yet, other cellular signaling events that also impact ER functions have an important role in breast carcinogenesis. Here, we show that activation of ErbB2/ErbB3 tyrosine kinase receptors with growth factor heregulin-ß prompts ERß degradation by the 26S proteasome, a mechanism that requires the coactivator cAMP response element-binding (CREB)-binding protein (CBP). We found that CBP promotes ERß ubiquitination and degradation through enhancement of the PI3-K/Akt pathway by heregulin-ß, an effect potentiated by a negatively charged hinge region of ERß. Activated Akt triggered the recruitment of E3 ubiquitin ligase Mdm2 to ERß, which was further stabilized by CBP, resulting in ERß poly-ubiquitination. Mutation of CBP Thr-1872 or Mdm2 Ser-186/188 Akt sites resulted in a dissociation of the ERß-CBP-Mdm2 complex and reduced ERß turnover. We found that the decrease in ERß induced by heregulin-ß was associated with reduced target gene promoter occupancy and enhanced proliferation of breast cancer cells. However, knockdown of Mdm2 restored endogenous ERß levels resulting in reduction of breast cancer cell growth. These studies identify a tripartite Akt-regulated phosphorylation mechanism that functions to hamper normal ERß activity and turnover through the concerted actions of CBP and Mdm2 in response to growth factor signaling pathways in breast cancer cells.

Donor P-gp polymorphisms strongly influence renal function and graft loss in a cohort of renal transplant recipients on cyclosporine therapy in a long-term follow-up.

Cyclosporin A (CsA) is a substrate for cytochrome P450 3A and the efflux transporter P-glycoprotein (P-gp; ABCB1), both abundantly expressed in the kidney. In a long-term follow-up of a cohort of patients who had received kidney transplants between the years 1990 and 2005, we retrospectively investigated the effect of CYP3A4, CYP3A5, and ABCB1 polymorphisms in kidney graft donors on recipients' renal function and risk of subsequent graft loss. DNA samples from 227 donors and clinical data from the 259 respective recipients were analyzed. Graft loss was significantly associated with the presence of the ABCB1 variant haplotype 1236T/2677T/3435T in the donor (1236T/2677T/3435T vs. other haplotypes: hazard ratio = 9.346; 95% confidence interval (CI) (2.278-38.461); P = 0.0019) and with previous episodes of acute organ rejection (hazard ratio = 3.077; 95% CI (1.213-7.812); P = 0.0178). The variant haplotype was also associated with a greater decrease in renal function (homozygotes for TTT -3.047 mlxmin(-1)/year; heterozygotes for TTT -4.435 mlxmin(-1)/year; others -2.186 mlxmin(-1)/year; P = 0.0240). The study showed that the presence of ABCB1 polymorphisms in donors influences long-term graft outcome adversely with decrease in renal function and graft loss in transplant recipients receiving CsA.

[High-risk airway management in pediatric prehospital emergency medicine]. / Une gestion des voies aériennes à haut risque en médecine pédiatrique préhospitalière.

We report the case of high-risk airway management performed in prehospital conditions in a 3-year-old boy suffering from a severe head and maxillofacial trauma. Tracheal intubation was decided because of a comatose status associated with an acute upper airway obstruction resulting in severe hypoxaemia. One minute after a rapid sequence induction, difficult laryngoscopy was encountered. Two tracheal intubation attempts failed. During maintained laryngoscopy, a pediatric angulated Eschmann-like stylet was blindly blocked into the trachea using a rotational maneuver. A tracheal tube was railroaded over the stylet while a hypoxic bradycardia installed. The young child was tracheostomized upon arrival in the hospital, and recovered without neurological complication. In the present case, neither facemask nor laryngeal mask ventilation would have been efficient because of oral cavity jaw and sub-mental pharyngeal open wounds. Since most paediatric emergency medicine physician are not familiar with infraglottic airway techniques, our observation questions the safety of rapid sequence induction in case of severe maxillofacial trauma and reinforces the value of pediatric Eschmann-like stylet. Minimal airway tools equipment for difficult paediatric airway management is discussed.

The role of organic anion-transporting polypeptides and their common genetic variants in mycophenolic acid pharmacokinetics.

The goal of this study was to determine the roles of the organic anion-transporting polypeptides (OATPs) OATP1A2, OATP1B1, and OATP1B3 and their genetic variants in the pharmacokinetics of the immunosuppressive drug mycophenolate mofetil (MMF). Using OATP-transfected human embryonic kidney (HEK) cells, we measured the uptake of mycophenolic acid (MPA) and its glucuronide (MPAG). MPAG, but not MPA, significantly accumulated in cells expressing OATP1B3 or OATP1B1 (P < 0.05). The pharmacokinetics of both MPA and MPAG were significantly influenced by the OATP1B3 polymorphism 334T>G/699G>A in 70 renal transplant patients receiving combination treatment of MMF with either tacrolimus or sirolimus, but not in 115 patients receiving MMF and cyclosporine. The decrease in dose-normalized (dn) MPA exposure and the concomitant increase in the MPAG/MPA metabolic ratio are consistent with reduced enterohepatic cycling in patients carrying the OATP1B3 334G-699A haplotype. Further studies demonstrated that this variant of OATP1B3 exhibited a reduced maximal velocity (V(max)) in transfected HEK cells, thereby providing functional evidence to support our clinical findings.

Maternal caffeine ingestion during gestation and lactation influences respiratory adaptation to acute alveolar hypoxia in newborn rats and adenosine A2A and GABA A receptor mRNA transcription.

Caffeine is a widely used psychostimulant freely crossing the placental barrier. At the doses usually absorbed, it acts as an antagonist of both A1 and A2A adenosine receptors. Pregnant women are generally not advised to limit their caffeine consumption and thus expose their progeny to the drug during the whole of gestation and lactation. The possibility that such caffeine exposure may have long-term consequences on brain development has led to several behavioral investigations on animal models. Despite the crucial role played by adenosine receptor systems in neonatal breathing control, few studies in vitro have been concerned with the consequences of maternal caffeine absorption on breathing, and none in the unrestrained intact animal. The present investigation analyzed the influence of caffeine exposure via placental and milk transfer on resting ventilation and on the response to moderate alveolar hypoxia of 0 to 2-day-old newborn rat (P0-P2) together with the possible underlying mechanisms. Dams absorbed caffeine (46+/-3 mg/kg/day) via drinking fluid (0.2 g/L) throughout gestation, in conditions mimicking moderate human consumption. Caffeine exposure did not significantly affect basal respiratory parameters. In contrast, it attenuated both the early increase and the secondary decrease in ventilation triggered by moderate alveolar hypoxia (11% O2 inhaled). The abolition of Fos protein expression evoked by hypoxia suggested that caffeine exposure may decrease the activity of O2-sensing peripheral chemoreceptor pathway. From real-time PCR data, those functional alterations were associated to increases in A2A adenosine receptor and alpha2 GABA(A) receptor subunit mRNAs in the medulla. This indicates that, even at moderate doses, maternal caffeine consumption may induce a series of subtle developmental alterations that may affect modulation of breathing control in the neonate in pathological situations such hypoxia.

[Molecular pharmacogenetics in hospital laboratories in France: current data and future prospects]. / La pharmacogénétique moléculaire hospitalière en France: données actuelles et perspectives.

Molecular pharmacogenetic units have recently been established in several hospital laboratories in France. The clinical impact of these units is still limited and numerous problems of organizational, ethical, legal, technical, social and economical nature remain to be resolved. However, an increasing number of these units, a rise in their activities and an enlargement of their scope of application are foreseeable in the future. Ultimately, these units would significantly contribute to limit the public health problem caused by interindividual variabilities in drug effects. In view of these prospects, it seems essential that such hospital activity should be quickly recognised by the authorities and the various health sectors in France. It is also essential that the problems that arise from such pharmacogenetic activities should be considered by the authorities and would profit from the organization of a national network and from financial guarantees.

[Pharmacogenetics and immunosuppressor drugs: impact and clinical interest in transplantation]. / Pharmacogénétique des médicaments immunosuppresseurs. Impact et intérêt clinique en transplantation.

The domain of solid organ transplantation is characterized by the use of variable drug combinations with drug-drug interactions, and the presence of two genomes, that of the transplanted organ and that of the receiver, which can be involved in the pharmacogenetics of these drugs. This paper is a literature review of the impact of the genetic polymorphisms of the metabolic enzymes, efflux transporters and therapeutic targets of the main immunosuppressive drugs (cyclosporine, tacrolimus, sirolimus and mycophenolate) on the dose-concentration and concentration-effect relationships of these drugs. The polymorphisms of metabolic enzymes have significant effects on the pharmacokinetics of all these drugs, but the clinical trials for validating treatment individualization based on these genetic differences are still lacking. It should be noted that the influence of the donor's genome has seldom been studied and has been found to be significant in liver transplant recipients. The influence of efflux transporter genes polymorphisms, in particular of P-glycoprotein and MPR2, is controversial. As for the polymorphisms of the drug targets genes, either they have not been reported (calcineurin, mTOR), or their influence has only been the subject of a few preliminary studies (IMPDH2). The pharmacogenetics of immunosuppressants is thus still an open field for investigations and potential therapeutic progress.

Asymptotic distribution of stage-grouped population models.

Matrix models are often used to predict the dynamics of size-structured or age-structured populations. The asymptotic behaviour of such models is defined by their malthusian growth rate lambda, and by their stationary distribution w that gives the asymptotic proportion of individuals in each stage. As the coefficients of the transition matrix are estimated from a sample of observations, lambda and w can be considered as random variables whose law depends on the distribution of the observations. The goal of this study is to specify the asymptotic law of lambda and w when using the maximum likelihood estimators of the coefficients of the transition matrix. We prove that lambda and w are asymptotically normal, and the expressions of the asymptotic variance of lambda and of the asymptotic covariance matrix of w are given. The convergence speed of lambda and w towards their asymptotic law is studied using simulations. The results are applied to a real case study that consists of a Usher model for a tropical rain forest in French Guiana. They permit to assess the number of trees to measure to get a given precision on the estimated asymptotic diameter distribution, which is an important information on tropical forest management.

Effects of the atrial antiarrhythmic drug AVE0118 on cardiac ion channels.

Previous studies in pigs and goats have demonstrated that AVE0118 prolongs atrial refractoriness without any effect on the QT-interval. The purpose of the present study was to investigate the effect of the compound on various cardiac ion channels. AVE0118 blocked the pig Kv1.5 and the human Kv1.5 expressed in Xenopus oocytes with IC(50) values of 5.4+/-0.7 microM and 6.2+/-0.4 microM respectively. In Chinese hamster ovary (CHO) cells, AVE0118 decreased the steady-state hKv1.5 current with an IC(50) of 1.1+/-0.2 microM. The hKv4.3/KChIP2.2 current in CHO cells was blocked by AVE0118 by accelerating the apparent time-constant of inactivation ( tau(inact)), and the integral current was inhibited with an IC(50) of 3.4+/-0.5 microM. At 10 microM AVE0118 tau(inact) decreased from 9.3+/-0.6 ms ( n=8, control) to 3.0+/-0.3 ms ( n=8). The K(ACh) current was investigated in isolated pig atrial myocytes by application of 10 microM carbachol. At a clamp potential of -100 mV the I(KACh) was half-maximally blocked by 4.5+/-1.6 microM AVE0118. In the absence of carbachol, AVE0118 had no effect on the inward current recorded at -100 mV. Effects on the I(Kr) current were investigated on HERG channels expressed in CHO cells. AVE0118 blocked this current half-maximally at approximately 10 microM. Comparable results were obtained in isolated guinea pig ventricular myocytes, where half-maximal inhibition of the I(Kr) tail current occurred at a similar concentration of AVE0118. Other ionic currents, like the I(Ks), I(KATP) (recorded in guinea pig ventricular myocytes), and L-type Ca(2+) (recorded in pig atrial myocytes) were blocked by 10 microM AVE0118 by 10+/-3% ( n=6), 28+/-7% ( n=4), and 22+/-13% ( n=5) respectively. In summary, AVE0118 preferentially inhibits the atrial K(+) channels I(Kur), I(to) and I(KACH). This profile may explain the selective prolongation of atrial refractoriness described previously in pigs and goats.

Imaging the premotor areas.

Recent imaging studies of motor function provide new insights into the organization of the premotor areas of the frontal lobe. The pre-supplementary motor area and the rostral portion of the dorsal premotor cortex, the 'pre-PMd', are, in many respects, more like prefrontal areas than motor areas. Recent data also suggest the existence of separate functional divisions in the rostral cingulate zone.