Polymerase Activity, Protein-Protein Interaction, and Cellular Localization of the Usutu Virus NS5 Protein.

Usutu virus (USUV) has become increasingly relevant in recent years, with large outbreaks that sporadically have affected humans being reported in wildlife. Similarly to the rest of flaviviruses, USUV contains a positive-sense single-stranded RNA genome which is replicated by the activity of nonstructural protein 5 (NS5). USUV NS5 shows high sequence identity with the remaining viruses in this genus. This permitted us to identify the predicted methyltransferase domain and the RNA-dependent RNA polymerase domain (RdRpD). Owing to their high degree of conservation, viral polymerases are considered priority targets for the development of antiviral compounds. In the present study, we cloned and expressed the entire NS5 and the RdRpD in a heterologous system and used purified preparations for protein characterizations. We determined the optimal reaction conditions by investigating how variations in different physicochemical parameters, such as buffer concentration, temperature, and pH, affect RNA polymerization activity. We also found that USUV polymerase, but not the full-length NS5, exhibits cooperative activity in the synthesis of RNA and that the RdRp activity is not inhibited by sofosbuvir. To further examine the characteristics of USUV polymerase in a more specifically biological context, we have expressed NS5 and the RdRpD in eukaryotic cells and analyzed their subcellular location. NS5 is predominantly found in the cytoplasm; a significant proportion is directed to the nucleus, and this translocation involves nuclear location signals (NLS) located at least between the MTase and RdRpD domains.

Hepatitis B virus quasispecies evolution after liver transplantation in patients under long-term lamivudine prophylaxis with or without hepatitis B immune globulin.

AIMS: To investigate an optimal long-term prophylactic strategy for prevention of hepatitis B virus (HBV) recurrence after liver transplantation, we conducted a randomized study of 29 transplant recipients receiving a short course of hepatitis B immune globulin (HBIg) + lamivudine (LAM), followed by randomization to long-term prophylaxis with LAM with or without HBIg. METHODS: The efficacy and safety, and impact on survival and HBV recurrence of these 2 prophylactic regimens were compared over a mean period of 10 years. In patients with viral recurrence, the HBV quasispecies in the surface/polymerase region were studied by ultra-deep pyrosequencing (UDPS). RESULTS: The 10-year survival rate was 76% and was not affected by the type of prophylaxis. Four patients had hepatitis B surface antigen (HBsAg) recurrence within the first 48 months after orthotopic liver transplantation (OLT). HBsAg-positive and -negative patients showed similar mean survival times, with no differences between the 2 regimens. Low HBV DNA levels were transiently detected in 32% of HBsAg-negative patients. UDPS showed major changes after OLT in the HBV quasispecies of patients with viral recurrence, which may be explained by a "bottleneck" effect of OLT together with prophylactic therapy. CONCLUSION: Long-term survival after OLT in end-stage chronic hepatitis B patients was good with both prophylactic strategies. However, low, transient HBV DNA levels were detected even in the absence of HBsAg, showing the importance of continuing HBV prophylaxis.

Bariatric surgery: to whom and when?

Bariatric surgery is the most effective treatment for obesity. Its effects go beyond weight loss, in a high percentage of cases achieving remission of comorbidities associated with obesity and reducing mortality. However, not all patients achieve satisfactory weight loss or resolution of comorbidities and perioperative complications are a constant risk. Correct preoperative evaluation is essential to predict the likelihood of success and choose the most appropriate surgical technique for this purpose. The aim of this review was to ascertain which obese subjects will benefit from bariatric surgery taking into account body mass index, age, comorbidities, risk of complications and the impact of different bariatric surgery techniques.

[Cerebrotendinous xanthomatosis: a multicentric retrospective study of 15 adults, clinical and paraclinical typical and atypical aspects]. / Étude rétrospective multicentrique de 15 cas adultes de xanthomatose cérébrotendineuse : aspects cliniques et paracliniques typiques et atypiques.

INTRODUCTION: Cerebrotendinous xanthomatosis, a metabolic leukodystrophy with an autosomal recessive inheritance, is secondary to deficiency of sterol 27-hydroxylase, an enzyme involved in cholesterol catabolism. Classical symptoms include clinical or infraclinical xanthomas affecting the skin and tendons, early cataracts, neurological signs and diarrhea. Brain imaging reveals involvement of the dentate nuclei and periventricular white matter hyperintensities. The diagnosis is based on an increased cholestanol level in serum, confirmed by the presence of a mutation in the CYP27A1 gene. Treatment is based on chenodeoxycholic acid. METHOD: We report a retrospective multicentric study of 15 cases of cerebrotendinous xanthomatosis diagnosed in French adults. Clinical, molecular and MRI findings were recorded in all patients. RESULTS: The average age at diagnosis was 39years (range 27-65). Disease onset occurred in childhood in 73% of patients and in adulthood in 27%. All patients with a pediatric onset were diagnosed during adulthood (age range 28-65years). Clinical symptoms variably associated cerebellar syndrome, pyramidal syndrome, cognitive decline, epilepsy, neuropathy (sought in 10 of our patients, present in forms in 8), psychiatric disorders, cataract and xanthomas. One patient had an atypical presentation: monoparesis associated with xanthomas. Brain MRI was abnormal in all: findings consisted in T2-weighted hyperintensity of the dentate nuclei (47%), periventricular leuoencephalopathy (73%) which preferentially involved the posterior cerebral part (60%), leucoencephalopathy with a vascular pattern (7%), hyperintensity of the cortico-spinal tracts (53%), globi pallidi, corpus callosum and cerebral atrophy (33%). Serum cholestanol was elevated in 93% of patients. The most frequent mutation was 1183C>T (n=5/15). Under treatment with chenodeoxycholic acid, eight patients improved initially, followed by stabilization in five of them, and worsening in the others. Four patients died. CONCLUSION: Patients with the xanthoma-neurological disorder association should be tested for cerebrotendinous xanthomatosis. The disease often begins in childhood with a diagnostic delay but also in adulthood. Involvement of the dentate nuclei is specific but not sensitive and the supratentorial leucoencephalopathy is not specific but with an antero-posterior gradient. A vascular distribution and involvement of the corpus callosum are possible. Serum cholestanol assay is very reliable: an elevated level provides the diagnosis, which must nevertheless be confirmed by molecular biology.

Leucine-rich repeat-containing G-protein-coupled receptor 5 (Lgr5) as a putative human endometrial stem cell marker.

The endometrium is recognized for its remarkable regenerative and remodeling capacity. Every month this hormonally regulated organ undergoes cycles of growth (from 0.5-2 to 7 mm), regression and shedding of two-third of the tissue, leading to its monthly renewal that occurs ∼400 times in a woman's reproductive lifetime. Several groups have suggested the existence of a human endometrial somatic stem cell (SSC) population located around the spiral arterioles of the basalis. Different groups have isolated, identified and characterized putative endometrial SSC populations in human endometrium based on the general features of undifferentiated cells, such as slow cycling detected using the 5-bromo-2-deoxyuridine technique or identification of a side population using the Hoechst efflux dye technique. Nevertheless, specific markers to isolate these endometrial SSC have not yet been consistently elucidated. Accumulated evidence based on lineage tracing studies indicates that a surface protein named Leucine-rich repeat-containing G-protein-coupled receptor 5 (Lgr5) is a marker that can identify SSC in several tissues such as small intestine mucosa (endodermal origin), hair follicles (ectodermal origin) or mature kidney nephrons (mesodermal origin). This protein plays a crucial role in the Wnt/ß-catenin signaling system by acting on the self-renewal and maintenance of the SSC population. In this work, we present novel data suggestive of Lgr5 as a putative human endometrial SSC marker, and since this is a mesoderm-derived tissue, these findings reinforce the concept that Lgr5 can be considered a universal SSC marker.

Assessment of poly-L-lysine dendrigrafts for virus concentration in water: use of MS2 bacteriophage as proof of concept.

AIMS: Virus detection has often been difficult due to a low concentration in water. In this study, we developed a new procedure based on concentration of virus particles on an innovative support: poly-L-lysine dendrigrafts (DGL), coupled with directed nucleic acid extraction and real-time PCR quantification. METHODS AND RESULTS: This method was evaluated using the bacteriophage MS2 as a model virus. This virus exhibited the size and structural properties of human pathogenic enteric viruses and has often been used to assess new supports of concentration. Moreover, this bacteriophage is also a faecal contamination indicator. In this study, many water filtration conditions were tested (volume of water, concentration, etc.), and more than 80% of bacteriophage were recovered after filtration on polymer, in most conditions. We demonstrated that the method was linear (slope = 0·99 ± 0·04 and Y intercept when x = -0·02 ± 0·28), valid (as manipulators, tested concentrations, volumes of sample and batch of polymer did not have any influence on concentration) and sensitive (allowing to concentrate up to 16,600-fold 1 l of sample and to detect and quantify down to 750 GC l(-1) and 7500 GC l(-1), respectively). CONCLUSIONS: To conclude, this support exhibits high interest to retain viruses and to allow to detect low concentration of virus in water. SIGNIFICANCE AND IMPACT OF THE STUDY: This study gives valuable advance in the methods of concentration and diagnosis of virus in water.

Role of A1c in the postpartum screening of women with gestational diabetes.

OBJECTIVE: This study aimed to determine whether A1c detects a different prediabetes prevalence in women with a history of gestational diabetes mellitus (GDM) compared to those diagnosed with oral glucose tolerance test (OGTT) and the influence of haemoglobin concentrations on A1c levels. DESIGN AND PATIENTS: We evaluated carbohydrate metabolism status by performing OGTT and A1c tests in 141 postpartum women with prior GDM in the first year post-delivery. RESULTS: The overall prevalence of prediabetes was 41.8%. Prevalence of isolated A1c 5.7-6.4%, impaired glucose tolerance (IGT) and impaired fasting glucose (IFG) was 10.6%, 7.1%, and 9.2%, respectively. Isolated A1c 5.7-6.4% was associated with Caucasian origin (66.7% versus 32.6%, p = 0.02) and with higher LDL cholesterol concentrations (123 ± 28.4 mg/dl versus 101.6 ± 19.2 mg/dl, p = 0.037) compared with patients diagnosed by OGTT (IFG or IGT). Women with postpartum anaemia had similar A1c levels to those with normal haemoglobin concentrations (5.5% ± 0.6% versus 5.4% ± 0.4%, p = 0.237). CONCLUSIONS: Use of A1c in postpartum screening of women with GDM detected an additional 10.6% of patients with prediabetes and a more adverse lipid profile. Haemoglobin concentrations did not influence A1c values.

Acetobacter strains isolated during the acetification of blueberry (Vaccinium corymbosum L.) wine.

Highbush blueberries (Vaccinium corymbosum L.) are known to have positive health benefits. The production of blueberry vinegar is one method to preserve this seasonal fruit and allow extended consumption. In this study, blueberry wine acetification was performed with naturally occurring micro-organisms and with an inoculated Acetobacter cerevisiae strain. Acetifications were carried out in triplicate using the Schützenbach method. The successful spontaneous processes took up to 66% more time than the processes involving inoculation. The isolation of acetic acid bacteria (AAB) and the analysis of these AAB using molecular methods allowed the identification of the main genotypes responsible of the blueberry acetification. Although the Acet. cerevisiae strain was the predominant strain isolated from the inoculated process samples, Acetobacter pasteurianus was isolated from samples for both processes and was the only species present in the spontaneous acetification samples. To the best of our knowledge, this is the first report describing the identification and variability of AAB isolated during blueberry acetification. The isolated Acet. pasteurianus strains could be used for large-scale blueberry vinegar production or as a starter culture in studies of other vinegar production methods.

Effect of inoculation on strawberry fermentation and acetification processes using native strains of yeast and acetic acid bacteria.

The aim of this work was to analyze the microbiota involved in the traditional vinegar elaboration of strawberry fruit during a spontaneous and inoculated process. In the spontaneous processes, low biodiversity was detected in both alcoholic fermentation (AF) and acetification. Nevertheless, a strain of Saccharomyces cerevisiae and of Acetobacter malorum were selected and tested as starter cultures in the inoculation study. The inoculated processes with these strains were compared with another spontaneous process, yielding a significant reduction in time for AF with a total imposition of the S. cerevisiae strain. The resulting strawberry wine was acetified in different containers (glass and wood) yielding an initial imposition of the A. malorum inoculated strain, although displacement by Gluconacetobacter species was observed in the wood barrels.

Validation of the French version of the MSA health-related Quality of Life scale (MSA-QoL).

INTRODUCTION: Multiple system atrophy (MSA) has considerable impact on health-related quality of life. The MSA health-related Quality of Life scale (MSA-QoL) is a patient-reported questionnaire, which has been recently designed to evaluate the quality of life in MSA. The objective of the present study was to validate the French version of the MSA-QoL questionnaire. METHODS: One hundred and thirty-six consecutive MSA patients were included in the study. Four patients with more than 10% missing responses were excluded from the final analysis. Data quality, scaling assumptions, acceptability, reliability and validity were assessed similar to the original validation of the English version. RESULTS: Missing responses were low, item and subscale scores were evenly distributed and floor and ceiling effects were negligible. Item-total correlations were higher than the recommended greater than 0.30 and internal consistency was high for all subscales. Test-retest reliability was good for all subscales. Validity was supported by moderate interscale correlations between the subscales and the predicted correlations with other scales assessing motor disability, activities of daily living, quality of life and mood. DISCUSSION: The French version of the MSA-QoL displays robust psychometric properties similar to the English version. CONCLUSION: The French version of MSA-QoL seems suitable for assessing quality of life in French speaking MSA patients.

Clinical presentation, diagnostic investigations, treatment protocols and outcomes of dogs diagnosed with tick-borne diseases living in the United Kingdom: 76 cases (2005-2019).

OBJECTIVES: To report the presence of tick-borne diseases in dogs living in the United Kingdom. MATERIALS AND METHODS: Dogs with a final diagnosis of tick-borne diseases made between January 2005 and August 2019 at seven referral institutions in the United Kingdom were included in the study. RESULTS: Seventy-six dogs were included: 25 were diagnosed with ehrlichiosis, 23 with babesiosis, eight with Lyme borreliosis and six with anaplasmosis. Fourteen dogs had co-infections with two or three pathogens. Except for those dogs with anaplasmosis and Lyme borreliosis, most dogs with tick-borne diseases had a history of travel to or from endemic countries. However, three dogs with ehrlichiosis, and one dog each infected with Babesia canis and Babesia vulpes did not have any history of travel. A variety of non-specific clinical signs and laboratory abnormalities were reported. Targeted treatment was successful at achieving clinical remission in 64 (84%) dogs. CLINICAL SIGNIFICANCE: Even in non-endemic areas, veterinary surgeons should consider tick-borne diseases in dogs with compatible clinical presentation and laboratory findings and especially where there is a history of travel. As autochthonous transmission of tick-borne-pathogens does occur, an absence of travel should not rule out tick-borne diseases. Specific diagnostic testing is required to confirm infection, and this enables prompt targeted treatment and often a positive outcome.

Sip18 hydrophilin prevents yeast cell death during desiccation stress.

AIMS: For this study, we performed a genetic screen of S. cerevisiae's deletion library for mutants sensitive to dehydration stress, with which we aimed to discover cell dehydration-tolerant genes. METHODS AND RESULTS: We used a yeast gene deletion set (YGDS) of 4850 viable mutant haploid strains to perform a genome-wide screen for the identification of desiccation stress modifiers. SIP18 is among the genes identified as essential for cells surviving to drying/rehydration process. Deletion of SIP18 promotes the accumulation of reactive oxygen species and enhances apoptotic and necrotic cell death in response to dehydration/rehydration process. CONCLUSIONS: SIP18p acts as an inhibitor of apoptosis in yeast under dehydration stress, as suggested by its antioxidative capacity through the ROS accumulation reduction after an H(2) O(2) attack. SIGNIFICANCE AND IMPACT OF THE STUDY: To our knowledge, this is the first systematic screen for the identification of putative genes essential to overcoming cell dehydration process. A broad range of identified genes could help to understand why some strains of high biotechnological interest cannot cope with the drying and rehydration treatments. Dehydration sensitivity makes these strains not suitable to be commercialized by yeast manufactures.

Identification of yeast and acetic acid bacteria isolated from the fermentation and acetification of persimmon (Diospyros kaki).

Persimmon (Diospyros kaki) is a seasonal fruit with important health benefits. In this study, persimmon use in wine and condiment production was investigated using molecular methods to identify the yeast and acetic acid bacteria (AAB) isolated from the alcoholic fermentation and acetification of the fruit. Alcoholic fermentation was allowed to occur either spontaneously, or by inoculation with a commercial Saccharomyces cerevisiae wine strain, while acetification was always spontaneous; all these processes were performed in triplicates. Non-Saccharomyces yeast species were particularly abundant during the initial and mid-alcoholic fermentation stages, but S. cerevisiae became dominant toward the end of these processes. During spontaneous fermentation, S. cerevisiae Sc1 was the predominant strain isolated throughout, while the commercial strain of S. cerevisiae was the most common strain isolated from the inoculated fermentations. The main non-Saccharomyces strains isolated included Pichia guilliermondii, Hanseniaspora uvarum, Zygosaccharomyces florentinus and Cryptococcus sp. A distinct succession of AAB was observed during the acetification process. Acetobacter malorun was abundant during the initial and mid-stages, while Gluconacetobacter saccharivorans was the main species during the final stages of these acetifications. Four additional AAB species, Acetobacter pasteurianus, Acetobacter syzygii, Gluconacetobacter intermedius and Gluconacetobacter europaeus, were also detected. We observed 28 different AAB genotypes, though only 6 of these were present in high numbers (between 25%-60%), resulting in a high biodiversity index.

Crystal structure refinement of magnesium zinc divanadate, MgZnV2O7, from powder X-ray diffraction data.

The crystal structure of magnesium zinc divanadate, MgZnV2O7, was determined and refined from laboratory X-ray powder diffraction data. The title compound was synthesized by a solid-state reaction at 1023 K in air. The crystal structure is isotypic with Mn0.6Zn1.4V2O7 (C2/m; Z = 6) and is related to the crystal structure of thortveitite. The asymmetric unit contains two metal sites with statistically distributed magnesium and zinc atoms with the atomic ratio close to 1:1. One (Mg/Zn) metal site (M1) is located on Wyckoff position 8j and the other (M2) on 4h. Three V sites (all on 4i), and eight O (three 8j, four 4i, and one 2b) sites complete the asymmetric unit. The structure is an alternate stacking of V2O7 layers and (Mg/Zn) atom layers along [20]. It is distinct from other related structures in that each V2O7 layer consists of two groups: a V2O7 dimer and a V4O14 tetra-mer. Mixed-occupied M1 and M2 are coordinated by oxygen atoms in distorted trigonal bipyramidal and octa-hedral sites, respectively.

[Corneal wound healing-Pathophysiology and principles]. / Wundheilung der Kornea ­ Pathophysiologie und Grundlagen.

The cornea forms the anterior border of the eye and significantly contributes to a sharp optical image quality on the retina through its transparency, avascular nature and curvature. Because of its anatomical structure and as a barrier to the environment, the cornea is particularly exposed to various external factors, such as injuries and pathogens. A correct wound healing without the formation of light diverging scarring is therefore essential to preserve the integrity and function of the cornea. Misguided wound healing is of outstanding clinical relevance and can lead to corneal fibrogenesis. Corneal fibrosis results in scarring with a loss of optical transparency, which significantly reduces eyesight and can lead to blindness. Understanding the pathophysiological mechanisms of wound healing and fibrogenesis is of great importance for the diagnostics, treatment and evaluation of the subsequent healing process in order to prevent permanent damage as far as possible.

Summary of recommendations and key points of the consensus of Spanish scientific societies (SEPAR, SEMICYUC, SEMES; SECIP, SENEO, SEDAR, SENP) on the use of non-invasive ventilation and high-flow oxygen therapy with nasal cannulas in adult, pediatric, and neonatal patients with severe acute respiratory failure. / Sumario de las recomendaciones y puntos clave del Consenso de las Sociedades Científicas Españolas (SEPAR, SEMICYUC, SEMES; SECIP, SENeo, SEDAR, SENP) para la utilización de la ventilación no invasiva y terapia de alto flujo con cánulas nasales en el paciente adulto, pediátrico y neonatal con insuficiencia respiratoria aguda grave.

Non-invasive respiratory support (NIRS) in adult, pediatric, and neonatal patients with acute respiratory failure (ARF) comprises two treatment modalities, non-invasive mechanical ventilation (NIMV) and high-flow nasal cannula (HFNC) therapy. However, experts from different specialties disagree on the benefit of these techniques in different clinical settings. The objective of this consensus was to develop a series of good clinical practice recommendations for the application of non-invasive support in patients with ARF, endorsed by all scientific societies involved in the management of adult and pediatric/neonatal patients with ARF. To this end, the different societies involved were contacted, and they in turn appointed a group of 26 professionals with sufficient experience in the use of these techniques. Three face-to-face meetings were held to agree on recommendations (up to a total of 71) based on a literature review and the latest evidence associated with 3 categories: indications, monitoring and follow-up of NIRS. Finally, the experts from each scientific society involved voted telematically on each of the recommendations. To classify the degree of agreement, an analogue classification system was chosen that was easy and intuitive to use and that clearly stated whether the each NIRS intervention should be applied, could be applied, or should not be applied.

Summary of recommendations and key points of the consensus of Spanish scientific societies (SEPAR, SEMICYUC, SEMES; SECIP, SENEO, SEDAR, SENP) on the use of non-invasive ventilation and high-flow oxygen therapy with nasal cannulas in adult, pediatric, and neonatal patients with severe acute respiratory failure.

Non-invasive respiratory support (NIRS) in adult, pediatric, and neonatal patients with acute respiratory failure (ARF) comprises two treatment modalities, non-invasive mechanical ventilation (NIMV) and high-flow nasal cannula (HFNC) therapy. However, experts from different specialties disagree on the benefit of these techniques in different clinical settings. The objective of this consensus was to develop a series of good clinical practice recommendations for the application of non-invasive support in patients with ARF, endorsed by all scientific societies involved in the management of adult and pediatric/neonatal patients with ARF. To this end, the different societies involved were contacted, and they in turn appointed a group of 26 professionals with sufficient experience in the use of these techniques. Three face-to-face meetings were held to agree on recommendations (up to a total of 71) based on a literature review and the latest evidence associated with 3 categories: indications, monitoring and follow-up of NIRS. Finally, the experts from each scientific society involved voted telematically on each of the recommendations. To classify the degree of agreement, an analogue classification system was chosen that was easy and intuitive to use and that clearly stated whether the each NIRS intervention should be applied, could be applied, or should not be applied.

Response to creatine analogs in fibroblasts and patients with creatine transporter deficiency.

Creatine transporter (CRTR) deficiency is one of the most frequent causes of X-linked mental retardation. The lack of an effective treatment for this disease, in contrast to creatine (Cr) biosynthesis disorders that respond to Cr monohydrate (CM), led us to analyze the efficacy of a lipophilic molecule derived from Cr, creatine ethyl ester (CEE), in fibroblasts and patients with CRTR deficiency. CM and CEE uptake studies were performed in six controls and four fibroblast cell lines from patients. We found a significant increase in Cr uptake after 72 h of incubation with CEE (500 micromol/L) in patients and control fibroblasts compared to incubation with CM. Subsequently, we assayed the clinical effect of CEE administration in four patients with CRTR deficiency. After 1 year of treatment, a lack of significant improvement in neuropsychological assessment or changes in Cr level in brain (1)H MRS was observed, and CEE was discontinued. In conclusion, this 12-month trial with CEE did not increase the brain concentration of Cr. Our in vitro data lend support to the idea of a certain passive transport of CEE in both pathological and control cells, although more lipophilic molecules or other cell systems that mimic the BBB should be used for a better approach to the in vivo system.

MHC2TA rs4774C and HHV-6A active replication in multiple sclerosis patients.

BACKGROUND AND PURPOSE: In a previous report, a strong gene-environment interaction between human herpesvirus 6A (HHV6A) active replication and MHC2TA rs4774C was demonstrated. The objectives of this study were: (i) to reappraise the association that was found in the previous study; (ii) to evaluate if MS patients with minor allele C and HHV-6A active infection had different clinical behavior; and (iii) to analyze the possible association of MHC2TA rs4774C with Epstein-Barr virus (EBV). METHODS: A total of 149 MS patients were analyzed both at the MHC2TA locus and by HHV-6A status in serum. We studied a G/C polymorphism (rs4774) by a TaqMan Assay-on-Demand. HHV-6A genomes in serum were evaluated by quantitative PCR. A control group of 562 healthy Spanish individuals was included for comparative purposes in the genetic analyses. A battery of clinical data was collected for all the MS patients included in the study. RESULTS: (i) MHC2TA/HHV-6A interaction: we found the same strong association of the rs4774C allele with HHV-6A active replication than in the previous study (P = 0.0001). (ii) CLINICAL DATA: the two main statistical significant differences for MS patients with HHV-6A active infection and minor allele C were: (a) a significant number of them were not free of progression (EDSS = 0) 2 years after the diagnosis (P = 0.01); (b) only a third of them responded to interferon beta treatment (P = 0.05). CONCLUSIONS: This study has verified previous results about the strong gene-environment interaction between HHV6A active replication and MHC2TA rs4774C. Furthermore, a different clinical behavior for MS patients with HHV-6A active infection and minor allele C was found.

Identification and quantification of acetic acid bacteria in wine and vinegar by TaqMan-MGB probes.

A Real-Time PCR (RT-PCR) assay was developed using TaqMan minor groove binder (MGB) probes for the specific detection and quantification of five acetic acid bacteria (AAB) species (Acetobacter pasteurianus, Acetobacter aceti, Gluconacetobacter hansenii, Gluconacetobacter europaeus and Gluconobacter oxydans) in wine and vinegar. The primers and probes, designed from the 16S rRNA gene, showed good specificity with the target AAB species. The technique was tested on AAB grown in glucose medium (GY) and inoculated samples of red wine and wine vinegar. Standard curves were constructed with the five target species in all these matrices. Quantification was linear over at least 5 log units using both serial dilution of purified DNA and cells. When this technique was tested in GY medium and inoculated matrices, at least 10(2)-10(3) cells/ml were detected. To quantify low populations of AAB in microbiologically complex samples, a PCR enrichment including part of the 16S-23S rRNA gene ITS region was needed to increase the amount of target DNA compared to non-target DNA. The RT-PCR assay used in this study is a reliable, specific and fast method for quantifying these five AAB species in wine and vinegar.