The relationship between the conformational degree of freedom of template-containing threads and slippage in the formation of [2]rotaxane building blocks.

A straightforward slippage strategy has been used for the synthesis of three [2]rotaxane building blocks that all contain an ammonium template for the dibenzo-24-crown-8 macrocycle and an N-hydroxysuccinimide end. The kinetic rate of the slipping-on process proved to be highly dependent on both the length and flexibility of the thread.

The Time-resolved and Extreme-conditions XAS (TEXAS) facility at the European Synchrotron Radiation Facility: the energy-dispersive X-ray absorption spectroscopy beamline ID24.

The European Synchrotron Radiation Facility has recently made available to the user community a facility totally dedicated to Time-resolved and Extreme-conditions X-ray Absorption Spectroscopy--TEXAS. Based on an upgrade of the former energy-dispersive XAS beamline ID24, it provides a unique experimental tool combining unprecedented brilliance (up to 10(14) photons s(-1) on a 4 µm × 4 µm FWHM spot) and detection speed for a full EXAFS spectrum (100 ps per spectrum). The science mission includes studies of processes down to the nanosecond timescale, and investigations of matter at extreme pressure (500 GPa), temperature (10000 K) and magnetic field (30 T). The core activities of the beamline are centered on new experiments dedicated to the investigation of extreme states of matter that can be maintained only for very short periods of time. Here the infrastructure, optical scheme, detection systems and sample environments used to enable the mission-critical performance are described, and examples of first results on the investigation of the electronic and local structure in melts at pressure and temperature conditions relevant to the Earth's interior and in laser-shocked matter are given.

Synthesis of triazolium-based mono- and tris-branched [1]rotaxanes using a molecular transporter of dibenzo-24-crown-8.

We report a diverted route to [1]rotaxane and tris-branched [1]rotaxane that are devoid of any efficient template and which could not be obtained by classical straightforward strategies. The described chemical route relies on the utilization of a "macrocycle transporter", which is able first to bind a macrocycle, second to link temporarily a triazolium-containing molecular axle, and third to deliver the macrocycle around the new docked axle through molecular machinery in a [1]rotaxane structure. The extended encircled thread is eventually cleaved by an amine or a triamine to afford the triazolium-containing [1]rotaxanes, releasing at the same time, the macrocycle transporter as a recyclable species.

Large contribution of human papillomavirus in vaginal neoplastic lesions: a worldwide study in 597 samples.

AIM: This work describes the human papillomavirus (HPV) prevalence and the HPV type distribution in a large series of vaginal intraepithelial neoplasia (VAIN) grades 2/3 and vaginal cancer worldwide. METHODS: We analysed 189 VAIN 2/3 and 408 invasive vaginal cancer cases collected from 31 countries from 1986 to 2011. After histopathological evaluation of sectioned formalin-fixed paraffin-embedded samples, HPV DNA detection and typing was performed using the SPF-10/DNA enzyme immunoassay (DEIA)/LiPA25 system (version 1). A subset of 146 vaginal cancers was tested for p16(INK4a) expression, a cellular surrogate marker for HPV transformation. Prevalence ratios were estimated using multivariate Poisson regression with robust variance. RESULTS: HPV DNA was detected in 74% (95% confidence interval (CI): 70-78%) of invasive cancers and in 96% (95% CI: 92-98%) of VAIN 2/3. Among cancers, the highest detection rates were observed in warty-basaloid subtype of squamous cell carcinomas, and in younger ages. Concerning the type-specific distribution, HPV16 was the most frequently type detected in both precancerous and cancerous lesions (59%). p16(INK4a) overexpression was found in 87% of HPV DNA positive vaginal cancer cases. CONCLUSIONS: HPV was identified in a large proportion of invasive vaginal cancers and in almost all VAIN 2/3. HPV16 was the most common type detected. A large impact in the reduction of the burden of vaginal neoplastic lesions is expected among vaccinated cohorts.

Association of endothelial lipase Thr111Ile polymorphism with proliferative retinopathy in type 2 diabetes patients.

AIM: Our previous study demonstrated that the endothelial lipase (EL) C.584C>T polymorphism (rs2000813, p.Thr111Ile) was significantly associated with diabetic retinopathy (DR). The present work was conducted to see if this specific variant of the EL gene was more specifically linked to the severity of DR. METHODS: This retrospective cohort study was based on a review of the institutional charts of 287 type 2 diabetes patients (mean age = 59.7 years; mean BMI = 29.0 kg/m(2); mean HbA1c=8.4%) genotyped for the EL C.584C>T polymorphism (rs2000813, p.Thr111Ile). The stage of DR was also determined for each genotype (CC, CT, TT). RESULTS: On univariate analysis, the minor allele homozygote TT variant was significantly associated with severe DR (OR: 4.3; 95% CI: 1.4, 13.1) compared with the major CC homozygote. No significant result was found for the CT heterozygote. Multivariate analysis revealed an increased risk for TT homozygotes to present with severe non-proliferative DR (OR: 8.09; 95% CI: 1.23, 53.1) or proliferative DR. Other associations were not significant. CONCLUSION: Minor allele homozygosity for this EL variant (c.584C>T) could be a significant risk factor for developing severe, sight-threatening disease due to proliferative DR. Further prospective studies of this EL polymorphism in a larger population sample are needed to confirm these results.

Etravirine concentrations in the cervicovaginal compartment in HIV-1-infected women receiving etravirine-containing antiretroviral therapy: DIVA 02 study.

We studied the penetration of etravirine and HIV shedding in the genital tract among 12 HIV-1-infected women receiving an etravirine-containing regimen who had <40 copies/ml blood plasma (BP) HIV RNA. None of the cervicovaginal fluid (CVF) samples showed detectable HIV RNA. Median etravirine concentrations were 663 ng/ml in BP and 857 ng/ml in CVF, with a CVF/BP etravirine ratio of approximately 1.2. This good penetration of etravirine may contribute to the control of viral replication in the female genital tract.

Association of endothelial lipase Thr111Ile polymorphism with lipid metabolism and microvascular complications in type 2 diabetic patients.

AIM: Endothelial lipase (EL) is a key enzyme in lipid metabolism, and a polymorphism in the EL gene may be a candidate for modulating lipid parameters in type 2 diabetic (T2D) patients. METHODS: In 396 T2D patients (age: 59.5 ± 10.7 years; BMI: 28.9 ± 5.3 kg/m(2); HbA(1c): 8.2 ± 1.9%), the c.584C>T polymorphism (rs2000813, p.Thr111Ile) was studied in 225 men (frequency of c.584T: 0.351) and 171 women (frequency of c.584T: 0.304). Patients' metabolic parameters, and macrovascular and microvascular complications, were assessed at baseline and at follow-up (mean: 4.2 years). RESULTS: Patients who were homozygous for the minor allele displayed modestly decreased low-density lipoprotein (LDL) cholesterol and raised apolipoprotein B at baseline, and raised systolic blood pressure and high-density lipoprotein (HDL) cholesterol on follow-up. Homozygosity for the minor allele was significantly associated with frequency of retinopathy (P=0.025), with TT homozygous patients more likely to have diabetic retinopathy (OR: 3.505; 95% CI: 1.491-8.239) both initially and at follow-up. CONCLUSION: The c.584C>T EL polymorphism is associated with a higher risk of diabetic retinopathy that could be linked to modifications in HDL-cholesterol metabolism and blood pressure levels.

The very low penetrance of cystic fibrosis for the R117H mutation: a reappraisal for genetic counselling and newborn screening.

BACKGROUND: Cystic fibrosis (CF) is caused by compound heterozygosity or homozygosity of CF transmembrane conductance regulator gene (CFTR) mutations. Phenotypic variability associated with certain mutations makes genetic counselling difficult, notably for R117H, whose disease phenotype varies from asymptomatic to classical CF. The high frequency of R117H observed in CF newborn screening has also introduced diagnostic dilemmas. The aim of this study was to evaluate the disease penetrance for R117H in order to improve clinical practice. METHODS: The phenotypes in all individuals identified in France as compound heterozygous for R117H and F508del, the most frequent CF mutation, were described. The allelic prevalences of R117H (p(R117H)), on either intron 8 T5 or T7 background, and F508del (p(F508del)) were determined in the French population, to permit an evaluation of the penetrance of CF for the [R117H]+[F508del] genotype. RESULTS: Clinical details were documented for 184 [R117H]+[F508del] individuals, including 72 newborns. The disease phenotype was predominantly mild; one child had classical CF, and three adults' severe pulmonary symptoms. In 5245 healthy adults, p(F508del) was 1.06%, p(R117H;T7) 0.27% and p(R117H;T5)<0.01%. The theoretical number of [R117H;T7]+[F508del] individuals in the French population was estimated at 3650, whereas only 112 were known with CF related symptoms (3.1%). The penetrance of classical CF for [R117H;T7]+[F508del] was estimated at 0.03% and that of severe CF in adulthood at 0.06%. CONCLUSIONS: These results suggest that R117H should be withdrawn from CF mutation panels used for screening programmes. The real impact of so-called disease mutations should be assessed before including them in newborn or preconceptional carrier screening programmes.

[Expected impact of a quadrivalent HPV vaccine in France]. / Modélisation de l'impact de la vaccination HPV quadrivalente en France.

OBJECTIVE: To assess the expected impact in France of a quadrivalent HPV 6/11/16/18 vaccine on the occurrence of genital HPV-induced lesions in women. METHODS: A Markov model based on a quadrivalent vaccination of 14-year-old girls as recommended in France was performed to assess the number of subjects needed to vaccinate to prevent an HPV-related event during their lifetime and the expected annual number of cases which could be prevented by vaccination. This model was based on prevalence data reported in four large French studies (EDiTH I-IV) reporting an HPV 6/11/16/18 prevalence of 82% (95% CI: 78.5-85.1) in cervical cancer (CC), 64% (95% CI: 59.7-68.1) in CIN2/3, 34% (95% CI: 28.9-38.1) in low-grade squamous intraepithelial lesions (LSIL) and 83% (95% CI 77.6-87.8) in female external acuminata condylomata (EAC) cases. RESULTS: Using a theoretical vaccine efficacy of 100%, 130 young women need to be vaccinated to prevent a case of CC, 17 for a case of CIN2/3 and 13 for a case of EAC. Immunization of 80% of 14-year-old girls could prevent 2495 CC (72%), 17,985 CIN2/3 (54%), 8004 CIN1 (27%), and 22,531 EAC female cases (65%) in France annually. CONCLUSION: A good adhesion to the preferentially recommended HPV quadrivalent vaccination would thus substantially reduce the burden of female genital lesions in France.

[Mild cystic fibrosis: genetics - extending follow-up is necessary]. / Les formes atténuées de la mucoviscidose : génétique - suivi prolongé nécessaire.

The diagnosis of mild cystic fibrosis is first suspected on mild lung disease or absence of pancreatic insufficiency and is assessed by biological analysis. The sweat test is not always conclusive. The nasal potential difference and molecular analysis of CFTR gene allow confirming diagnosis. A regular follow-up in cystic fibrosis clinical centre is essential all life long. The genotype, especially during neonatal period, cannot be used to predict individually the course of the disease. Genetic counselling must be recommended to the parents in order to propose an analysis of CFTR gene to give the appropriate genetic counselling and to consider with them which family members could be concerned, especially in the event of parental project. The research of heterozygote status in related for prenatal diagnosis is not recommended for all mutations.

Human papillomavirus 16 E6, L1, L2 and E2 gene variants in cervical lesion progression.

The human papillomavirus (HPV) 16 E6 genome variant 350G has been found to be more prevalent in women with persistent infection and cervical disease progression than the HPV16 E6 prototype 350T. In this study, we examined whether women who progressed to a high-grade lesion, yet were infected with the prototype 350T, showed variants in other HPV genes such as L1, L2 and E2. Although we detected variants within these genes, they could not explain this phenomenon. Indeed they correlated similarly with variant 350G and prototype 350T. These data indicate that polymorphisms in HPV16 E6 rather than in the other analyzed genes play a role in determining the risk for cervical lesion progression and that additional factors are likely to be required as well.

Synoviocyte infection with adeno-associated virus (AAV) is neutralized by human synovial fluid from arthritis patients and depends on AAV serotype.

Intraarticular gene transfer with adeno-associated viral (AAV) vectors may allow efficient therapeutic transgene expression within the joint in diseases such as rheumatoid arthritis (RA), allowing high expression of the protein within the joint, preventing both systemic diffusion and side effects. However, humans demonstrate antibodies against AAV, which can influence gene transfer. To better understand critical obstacles to intraarticular gene therapy with AAV, we have previously shown that synovial fluid (SF) contains IgG to AAV that neutralizes chondrocyte infection in vitro. Our objective was therefore to compare neutralization exerted by SF from RA patients for four different AAV serotypes (AAV serotypes 1, 2, 5, and 8) on human primary synoviocytes. Serotype 2 infected synoviocytes most efficiently followed, in decreasing order, by serotypes 1, 5, and 8. SF from all patients partially inhibited infection of synoviocytes by at least one of the four serotypes. Infection with serotypes 1 and 2 was the most inhibited by SF, whereas inhibition was weak for serotypes 5 and 8. Last, we have shown that inhibition of AAV1/interleukin (IL)-4 infection of synoviocytes by SF could be reversed by increasing the number of AAV1/IL-4 particles, with a dose-dependent effect. We conclude that the most infectious AAV serotypes (1 and 2) in synoviocytes are also the serotypes most neutralized by SF. Thus, serotype 5 seems to demonstrate the best infection efficiency:immunogenicity ratio for local use in articular diseases. These data may be useful for tailoring intraarticular AAV-mediated gene therapy to individual patients.

Envenenamientos e intoxicaciones de fauna silvestre y doméstica en la región de murcia durante el bienio 2005-2006 / Intentional and unintentional poisonings of wild and domestic fauna in the Region of Murcia during biennium 2005-2006

El presente estudio afronta la revisión y análisis de los datos obtenidos a partir de los casos sospechososde envenenamiento o intoxicación en animales silvestres (y domésticos en menor medida), recibidos duranteel bienio 2005-2006 en el Servicio de Toxicología del Centro de Recuperación de Fauna Silvestre “El Valle”(Murcia). Dicha revisión permite ofrecer información epidemiológica acerca de los principales agentes tóxicosrelacionados con envenenamientos de animales de vida silvestre en el Sureste de España, así como de las causasque conducen a provocarlos. De los 94 casos recibidos y analizados, 41 fueron positivos, con un balance totalde 122 cadáveres. Plaguicidas, sobre todo insecticidas y rodenticidas, fueron las sustancias más frecuentementeinvolucradas. En concreto, aldicarb (29,54%), fentión (25,00%), carbofurano (15,90%) y monocrotofos(9,09%) son los compuestos más utilizados en la preparación de cebos para envenenamientos. La primavera yel invierno son las épocas del año en las que los envenenamientos son más numerosos, y mayoritariamente seproducen en zonas rurales y de tradición cinegética

Data of chemical toxicological analyses carried out over a two year period (2005-2006) by the WildlifeRecovery Center “El Valle” of Murcia (Spain). These results allow to perform an epidemiologic information conclusioaboutthe main toxic agents of poisoning wildlife (and domestic animals in some cases) of Southeastern Spain.From 94 cases received and analyzed, 41 were positive to intentional poisoning (a total of 122 dead animals).Pesticides, especially insecticides and rodenticides, were frequently involved. Aldicarb (29,54%), fenthion(25,00%), carbofuran (15,90%) and monocrotophos (9,09%) were the most usual toxic agents in baits preparedfor intentional poisonings. Spring and winter are the seasons of the year in which the poisonings are morecommon, and mainly they take place in rural zones and those of hunting areas

In the rat, citrullinated autologous fibrinogen is immunogenic but the induced autoimmune response is not arthritogenic.

Conversion of arginyl to citrullyl residues (citrullination) is essential for the formation of the epitopes recognized by rheumatoid arthritis (RA)-associated autoantibodies to citrullinated proteins (ACPA). ACPA are secreted by plasma cells of the rheumatoid synovial tissue where their major target, citrullinated fibrin, is abundant. Although numerous arguments suggest that ACPA play an important role in RA, their pathological relevance remains to be established. In the present study, we assessed the immunogenicity and arthritogenicity of complete Freund's adjuvant-emulsified autologous citrullinated (C-rFBG) or non-citrullinated (NC-rFBG) fibrinogen in Lewis (LEW) and Brown-Norway rats, which exhibit drastic differences in their susceptibility to induced autoimmune diseases. NC-rFBG induced no antibody response. In contrast, a single injection of C-rFBG induced an IgG response directed mainly to citrullinated determinants of rFBG. However, all rat strains remained devoid of clinical and histological signs of arthritis up to 3 months after C-rFBG inoculation. Next, in LEW rats, we tested whether autoimmunity to C-rFBG could aggravate acute ankle arthritis triggered by intra-articular injection of incomplete Freund's adjuvant (IFA). However, such arthritis evolved identically in the presence or absence of anti-C-rFBG autoantibodies. However, IFA-injected joints were devoid of citrullinated fibrin deposits. Therefore, citrullination allows breakdown of immunological tolerance but the autoimmune response developed is not spontaneously arthritogenic. Whether or not it can aggravate arthritis with citrullinated fibrin deposits remains to be evaluated.

. Association of HindIII and PvuII genetic polymorphisms of lipoprotein lipase with lipid metabolism and macrovascular events in type 2 diabetic patients.

AIM: Lipoprotein lipase (LPL) is a key enzyme of lipid metabolism, and its genetic polymorphism may be a candidate for modulating lipid parameters in type 2 diabetic subjects (D2). METHODS: In a group of 404 type 2 diabetic patients, aged 59.5+/-10.8y, BMI=28.9+/-5.3 kg/m2, HbA1c=8.2+/-1.9%, we studied the H and P polymorphisms at the LPL locus detectable with the restriction enzymes HindIII and PvuII. Patients were separated into 229 males (17H1H1, 84H1H2, 128H2H2 and 51P1P1, 110P1P2, 68P2P2) and 175 females (16H1H1, 69H1H2, 90H2H2 and 51P1P1, 85P1P2, 39P2P2), and compared on the basis of their lipid parameters and their macrovascular complications. RESULTS: Triglyceride (TG) and HDL-cholesterol(c) concentrations differed between patients with and without coronary heart disease (CHD) (3.44+/-2.09 and 1.96+/-1.40 mmol/l for TGs and 1.05+/-0.24 and 1.34+/-0.40 mmol/l for HDL-c, P<0.001). HDL-c concentrations were lower in male H2H2 and P2P2 subjects (P<0.001), and TG levels were higher in male H2H2 and P2P2 subjects (P<0.0001 for Hind III and P<0.05 for PvuII). Allele frequency of the HindIII and PvuII restriction site was similar to those reported in other Caucasian populations and the presence of the H2/P2 variants was significantly higher in CHD patients. The prevalence of CHD in this population was 18% but was 29% in H2H2 and 38% in P2P2 subjects (P<0.02). CONCLUSION: Thus, HindIII and PvuII polymorphisms seem to exert a modulating role on lipid profile particularly in male D2, contributing to increase the risk of macrovascular events.

Identification of type-specific and cross-reactive neutralizing conformational epitopes on the major capsid protein of human papillomavirus type 31.

The majority of the neutralizing epitopes of papillomaviruses (PV) are conformation-specific and have not been fully characterised. Studies have, to date, been limited to a few HPV types only. We analysed the epitopes on the major capsid protein (L1) of Human papillomavirus (HPV) type 31 using monoclonal antibodies (MAbs) generated against HPV-31 virus-like particles (VLPs). The type-specific MAbs against HPV-31 were all found to be neutralizing and recognized conformation-dependent epitopes. Two other MAbs directed against a conformational epitope were found to be cross-reactive with other HPV types, and one of them was found to be cross-neutralizing. Cross-reactive antibodies were further investigated using wild-type HPV-16 L1 VLPs and two mutants. The results obtained suggested the existence of a cross-neutralizing conformational epitope at the N-terminal part of the FG loop of the major capsid protein, and the other four cross-reactive MAbs recognized epitopes also located at the N-terminal part of the FG loop.

Usefulness of HPV testing in the follow-up of untreated cervical low grade lesions.

The aim of the present work was to evaluate the usefulness of high-risk human papillomavirus (HR-HPV) testing for the follow-up of women with untreated low grade cervical squamous cell lesions (LSIL). For that, 412 women with a cytological diagnosis of LSIL at entry were monitored by cytology, HR-HPV testing with the Hybrid Capture II assay (HC-II) and colposcopy. Our primary endpoint was clinical progression defined by the presence of a high grade cervical intraepithelial neoplasia (CIN2 and CIN3) at the biopsy. At baseline, histological control revealed 10 CIN2 and 11 CIN3 only in the cohort of women HR-HPV+. In the follow-up, 4 CIN2 and 8 CIN3 were detected, always in the women initially HR-HPV+. Thus, the recurrence of a HR-HPV+ infection clearly selects a population at high-risk for CIN2-3. The semi-quantitative appreciation of the viral load with HC-II could not be used as a good prognostic factor for the follow-up of women with LSIL. HR-HPV testing reduces the number of cytology and colposcopy examinations in the follow-up of women aged >35 years when HPV testing is initially negative. Thus HR-HPV testing should be reserved for the follow-up of this population of women initially HR-HPV+ and proposed 6 to 12 months after the cytological diagnosis of LSIL.

IgG subclass distribution of the rheumatoid arthritis-specific autoantibodies to citrullinated fibrin.

In the rheumatoid synovium, deiminated ('citrullinated') forms of fibrin are the major targets of IgG autoantibodies to citrullinated proteins (ACPA), the most specific serological markers of rheumatoid arthritis (RA). To further the characterization of ACPA, we determined their subclass distribution. From a previously validated highly sensitive and specific enzyme-linked immunosorbent assay (ELISA) onto in vitro deiminated human fibrinogen - antihuman fibrin(ogen) autoantibodies (AhFibA)-ELISA - we derived and calibrated four ELISAs, using monoclonal antibodies to each of the four IgG subclasses, to determine the proportions of AhFibA subclasses in the sera. A series of 186 serum samples from RA patients was analysed. All AhFibA-positive sera contained IgG1-AhFibA, which reached the highest titres and accounted for more than 80% of AhFibA in three-quarters of the sera. One or two other subclasses were associated with IgG1 in 39% of the sera, IgG4-AhFibA being observed much more frequently and at higher titres than IgG3- or IgG2-AhFibA. IgG1 alone or IgG(1 + 4)-AhFibA were the AhFibA subclass profiles found in more than 80% of patients. AhFibA are mainly IgG1 and, to a lesser extent, IgG4. Such IgG subclass profiles may influence the effector phases of the immunological conflict between ACPA and deiminated fibrin that takes place specifically in the rheumatoid synovium and therefore may play a critical role in the self-maintenance of rheumatoid inflammation.

Negative human papillomavirus testing in normal smears selects a population at low risk for developing high-grade cervical lesions.

High-risk human papillomaviruses (HR-HPV) are the necessary cause of cervical carcinomas and there is an increasing interest in using HR-HPV DNA detection in adjunction to cytological examination for primary cervical screening. To determine whether women with a normal smear negative for HR-HPV DNA detection with the Hybrid Capture II assay might represent a low-risk population for developing a high-grade squamous intraepithelial lesion (HSIL), 4401 women have been followed in a period of 12-72 months (median=34 months). During this follow-up, four HSIL and one microinvasive carcinoma have been detected in this cohort (three in the cohort of 3526 women >29 years). The global negative predictive value (NPV) of double-negative tests is thus of 99.9% (ninety-five percent confidence interval (95% CI): 99.8-100%), whereas cytology alone gives an NPV of 99.2% (95% CI: 98.9-99.5%). If we obtain a second negative HR-HPV test 1-2 years after the initial test, the NPV is 100%. The NPV is also of 100% in the cohort of women >49 years. We conclude that all these women could be safely screened at longer intervals between 3 and 5 years. This policy will offset the increased costs induced by an additional HR-HPV testing in primary screening.