Bionomics of temperate and tropical Culicoides midges: knowledge gaps and consequences for transmission of Culicoides-borne viruses.

Culicoides midges are abundant hematophagous flies that vector arboviruses of veterinary and medical importance. Dramatic changes in the epidemiology of Culicoides-borne arboviruses have occurred since 1998, including the emergence of exotic viruses in northern temperate regions, increases in global disease incidence, and enhanced virus diversity in tropical zones. Drivers may include changes in climate, land use, trade, and animal husbandry. New Culicoides species and new wild reservoir hosts have been implicated in transmission, highlighting the dynamic nature of pathogen-vector-host interactions. Focusing on potential vector species worldwide and key elements of vectorial capacity, we review the sensitivity of Culicoides life cycles to abiotic and biotic factors. We consider implications for designing control measures and understanding impacts of environmental change in different ecological contexts. Critical geographical, biological, and taxonomic knowledge gaps are prioritized. Recent developments in genomics and mathematical modeling may enhance ecological understanding of these complex arbovirus systems.

The piezoresistive effect in graphene-based polymeric composites.

The strain-dependent electrical resistance of polyvinyl ester-based composites filled with different weight fractions of graphene nanoplatelets (GNPs) has been experimentally investigated. The GNP synthesis and nanocomposite fabrication process have been optimized in order to obtain highly homogeneous filler dispersion and outstanding electrical properties. The produced nanocomposites showed a low percolation threshold of 0.226 wt% and electrical conductivity of nearly 10 S m(-1) at only 4 wt% of GNPs. The piezoresistive response of thin nanocomposite laminae has been assessed by measuring the variation of the electrical resistance as a function of the flexural strain in three-point bending tests under both quasi-static monotonic and dynamic cyclic loading conditions. The obtained results showed higher strain sensitivity than traditional metal foil strain gauges or recently investigated carbon-based nanocomposite films.

Non-classic cystic fibrosis associated with D1152H CFTR mutation.

BACKGROUND: Limited knowledge exists on phenotypes associated with the D1152H cystic fibrosis transmembrane conductance regulator (CFTR) mutation. METHODS: Subjects with a D1152H allele in trans with another CFTR mutation were identified using the French Cystic Fibrosis Registry. Phenotypic characteristics were compared with those of pancreatic insufficient (PI) and pancreatic sufficient (PS) cystic fibrosis (CF) subjects in the Registry (CF cohort). RESULTS: Forty-two subjects with D1152H alleles were identified. Features leading to diagnosis included chronic sinopulmonary disease (n = 25), congenital absence of the vas deferens (n = 11), systematic neonatal screening (n = 4), and genetic counseling (n = 2). Median age at diagnosis was 33 [interquartile range (IQR, 24-41)] years in D1152H subjects. Median sweat chloride concentrations were 43.5 (39-63) mmol/l in D1152H subjects and were markedly lower than in PI and PS CF subjects (p < 0.05). Bronchiectasis was present in 67% of D1152H subjects, but Pseudomonas aeruginosa colonization and pancreatic insufficiency were present in <30% of subjects. Estimated rates of decline in forced expiratory volume in 1 s (FEV(1)) were lower in D1152H subjects vs PI CF subjects (p < 0.05). None of the D1152H subjects identified since 1999 had died or required lung transplantation. CONCLUSIONS: When present in trans with a CF-causing mutation, D1152H causes significant pulmonary disease, but all subjects had prolonged survival.

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.

Disposable plastic microreactors for genomic analyses.

We show the design, development and assessment of disposable, biocompatible, fully plastic microreactors, which are demonstrated to be highly efficient for genomic analyses, such as amplification of DNA, quantitative analyses in real time, multiplex PCR (both in terms of efficiency and selectivity), as compared to conventional laboratory equipment for PCR. The plastic microreactors can easily be coupled to reusable hardware, enabling heating/cooling processes and, in the case of qPCR applications, the real-time detection of the signal from a suitable fluorescent reporter present in the reaction mixture during the analysis. The low cost production of these polymeric microreactors, along with their applicability to a wide range of biochemical targets, may open new perspectives towards practical applications of biochips for point of care diagnostics.

Algorithm for automatic genotype calling of single nucleotide polymorphisms using the full course of TaqMan real-time data.

Single nucleotide polymorphisms (SNPs) are often determined using TaqMan real-time PCR assays (Applied Biosystems) and commercial software that assigns genotypes based on reporter probe signals at the end of amplification. Limitations to the large-scale application of this approach include the need for positive controls or operator intervention to set signal thresholds when one allele is rare. In the interest of optimizing real-time PCR genotyping, we developed an algorithm for automatic genotype calling based on the full course of real-time PCR data. Best cycle genotyping algorithm (BCGA), written in the open source language R, is based on the assumptions that classification depends on the time (cycle) of amplification and that it is possible to identify a best discriminating cycle for each SNP assay. The algorithm is unique in that it classifies samples according to the behavior of blanks (no DNA samples), which cluster with heterozygous samples. This method of classification eliminates the need for positive controls and permits accurate genotyping even in the absence of a genotype class, for example when one allele is rare. Here, we describe the algorithm and test its validity, compared to the standard end-point method and to DNA sequencing.

A 3-year prognostic score for adults with cystic fibrosis.

BACKGROUND: Therapeutic progress in patients with cystic fibrosis (CF) has resulted in improved prognosis over the past decades. We aim to reevaluate prognostic factors of CF and provide a prognostic score to predict the risk of death or lung transplantation (LT) within a 3-year period in adult patients. METHODS: We developed a logistic model using data from the French CF Registry and combined the coefficients into a prognostic score. The discriminative abilities of the model and the prognostic score were assessed by c-statistic. The prognostic score was validated using a 10-fold cross-validation. RESULTS: The risk of death or LT within 3years was related to eight characteristics. The development and the validation provided excellent results for the prognostic score; the c-statistic was 0.91 and 0.90 respectively. CONCLUSION: The score developed to predict 3-year death or LT in adults with CF might be useful for clinicians to identify patients requiring specialized evaluation for LT.

Culicoides Latreille (Diptera: Ceratopogonidae) taxonomy: current challenges and future directions.

Culicoides Latreille biting midges (Diptera: Ceratopogonidae) cause a significant biting nuisance to humans, livestock and equines, and are the biological vectors of a range of internationally important pathogens of both veterinary and medical importance. Despite their economic significance, the delimitation and identification of species and evolutionary relationships between species within this genus remains at best problematic. To date no phylogenetic study has attempted to validate the subgeneric classification of the genus and the monophyly of many of the subgenera remains doubtful. Many informal species groupings are also known to exist but few are adequately described, further complicating accurate identification. Recent contributions to Culicoides taxonomy at the species level have revealed a high correlation between morphological and molecular analyses although molecular analyses are revealing the existence of cryptic species. This review considers the methods for studying the systematics of Culicoides using both morphological and genetic techniques, with a view to understanding the factors limiting our current understanding of Culicoides biology and hence arbovirus epidemiology. In addition, we examine the global status of Culicoides identification, highlighting areas that are poorly addressed, including the potential implementation of emerging technologies.

Gut microbiota trajectory in pediatric patients undergoing hematopoietic SCT.

Acute GvHD (aGvHD) is the main complication of hematopoietic SCT (HSCT) during the treatment of hematological disorders. We carried out the first longitudinal study to follow the gut microbiota trajectory, from both the phylogenetic and functional points of view, in pediatric patients undergoing HSCT. Gut microbiota trajectories and short-chain fatty acid production profiles were followed starting from before HSCT and through the 3-4 months after transplant in children developing and not developing aGvHD. According to our findings, HSCT procedures temporarily cause a structural and functional disruption of the gut microbial ecosystem, describing a trajectory of recovery during the following 100 days. The onset of aGvHD is associated with specific gut microbiota signatures both along the course of gut microbiota reconstruction immediately after transplant and, most interestingly, prior to HSCT. Indeed, in pre-HSCT samples, non-aGvHD patients showed higher abundances of propionate-producing Bacteroidetes, highly adaptable microbiome mutualists that showed to persist during the HSCT-induced ecosystem disruption. Our data indicate that structure and temporal dynamics of the gut microbial ecosystem can be a relevant factor for the success of HSCT and opens the perspective to the manipulation of the pre-HSCT gut microbiota configuration to favor mutualistic persisters with immunomodulatory properties in the gut.

Solid phase purification and SSCP analysis of amplified genomic DNA by capillary electrophoresis.

Detection and identification of point mutations in genomic DNA has proven increasingly important in biomedical research. A variety of methods for the analysis of single base substitutions have been proposed among which Single Strand Conformational Polymorphism (SSCP) quickly gained success due to its simplicity. In this work we present an analytical on-line tool which combines the ease of solid phase purification of amplified genomic DNA, the simplicity of SSCP and the significant potential advantages offered by capillary electrophoresis (CE).

Anion-exchange HPLC analysis of biotinylated oligonucleotides.

Biotinylated oligonucleotides combined with streptavidin-coated magnetic beads are commonly used in current molecular biology. Their quality and the level of incorporated biotin are essential for yielding good results in either solid-phase DNA sequencing or solid-phase purification procedures. This paper presents a very simple analytical test using anion-exchange HPLC and avidin to ascertain the quality of biotinylated oligonucleotides and to predetermine their ability to bind to avidin, which is a prerequisite for functionality in some solid-phase methods.

Fluorescence-based automated DNA sequencing by limited primer labeling.

The applicability of automated DNA sequencing systems to sequencing strategies that require a large number of primers is limited by the necessity for expensive fluorescence-labeled oligonucleotides. Here we present a simple procedure that allows the use of unlabeled oligonucleotides to perform fluorescence-based DNA sequencing. This method is based on a limited primer extension that incorporates three deoxynucleotides, one of which carries a fluorescent moiety. The elongated fluorescent primer is then used in a standard T7 sequencing reaction. This labeling procedure is both economical and straightforward and offers a valid alternative to current fluorescence-labeling protocols. Results of this method with different DNA templates demonstrate the reliability of the protocol.

Dideoxy linear PCR on a commercial fluorescent automated DNA sequencer.

The use of automated fluorescent DNA sequencer systems and PCR-based DNA sequencing methods play an important role in the actual effort to improve the efficiency of large-scale DNA analysis. Here we show the application of the linear PCR using a single fluorescent primer and dideoxynucleotide terminators in four separate sequencing reactions on the EMBL/Pharmacia's fluorescent automated DNA sequencer. We have used dideoxy/deoxynucleoside triphosphate ratios and linear amplification cycle conditions to obtain an accurate sequencing response of up to, and over, 500 bases from just 400 ng of double-stranded DNA template without chemical denaturation. The sequencing protocol described in this paper is effectively suited for enhancement of sensitivity and performance of the automated DNA sequencing system.

Analysis of DNA microarrays by non-destructive fluorescent staining using SYBR green II.

A simple, non-destructive procedure is described to determine the quality of DNA arrays before they are used. It consists of a preliminary staining step of the DNA microarray by using SYBR green II, a fluorophore with specific affinity for ssDNA, followed by a laser scan analysis. The surface quality, integrity and homogeneity of each DNA spot of the array can thus be assessed. After this preliminary control, which may avoid further analytical steps that lead to the waste of precious biological samples, a fully reversible staining procedure is performed that produces an array ready for subsequent use.

Mixed-mode fluorescent DNA sequencing.

Fluorescence-based, automated DNA sequences represent one of the major advances in recent molecular biology. Two main technologies have been developed in this field: the single-label/four-lane system and the four-label/one-lane system. The following present the use of single-label-sequencing chemistry, which resembles traditional radioactive DNA sequencing, using the four-label system ABI 373A that expands its flexibility and obtains data that are immediately interpretable without software manipulation. This method has been named mixed-mode fluorescent DNA sequencing. Here we show one of its possible applications in molecular genetic analysis.

A more stringent choice of primers can improve the performance of fluorescent automated DNA sequencers.

Some primers frequently used in the double-stranded dideoxy DNA sequencing technique with radioactive markers are not suited for fluorescent detection. In fact oligonucleotides have a different annealing efficiency related to their base sequence, and this is reflected in nonequivalent results, particularly in fluorescent automated DNA sequencing. We present a method for the evaluation of primer performance in automated DNA sequencers and show its application to the search for a better set of primers for pBluescript vector.

Metabolism and pharmacokinetics of p-(3,3-dimethyl-1-triazeno) benzoic acid in M5076 sarcoma-bearing mice.

The pharmacokinetics of the anticancer agent p-(3,3-dimethyl-1-triazeno) benzoic acid (pCOOH-DMT), a drug now in phase I clinical trial in Europe, was investigated in C57Bl female mice with M5076 reticulum-cell sarcoma that were treated i.v. with 200 mg/kg pCOOH-DMT. The drug disappeared from plasma with a terminal half-life of about 2.5 h. Plasma clearance was approximately 6 ml/min per kg. Distribution studies showed some differences in drug levels in different tissues. The highest levels were found in the tumor, liver, kidney and lung; lower levels were found in the spleen and gut, and the lowest, in the brain. The N-desmethyl derivative of pCOOH-DMT was not detectable in plasma or tissues of mice treated with the drug. Therefore, the previous evidence of low N-demethylation of pCOOH-DMT was confirmed. pCOOH-DMT glucuronide was identified by mass spectrometry and quantified by high-performance liquid chromatography (HPLC) in plasma, tissues and urine samples. pCOOH-DMT glucuronide appears to be the major urinary metabolite of pCOOH-DMT in mice. Another metabolite identified by mass spectrometry and quantified by HPLC in some tissues and urine was pCOOH-DMT glycinate.

Determinant factors for diagnostic delay in operable breast cancer patients.

Randomized trials of mammographic screening have provided strong evidence that early diagnosis and treatment of breast cancer can reduce the specific mortality. Moreover, in a recent systematic review of published studies, delays of 3-6 months between symptom onset and treatment have been clearly found to be associated with lower survival rates for breast cancer patients. The aim of this study was to examine delays registered among breast cancer patients in southern Italy, in order to recognize their determining factors so as to provide women with a better opportunity for survival. The variables examined were age (< 50, 50-64, > or = 65 years), education (< or = 5, > 5 school years); symptom status at first presentation (symptomatic or asymptomatic); date of first symptom presentation; date of first consultation with a health provider; the type of health provider consulted; tumour size and nodal status according to the pTNM system. Time intervals were categorized into: < 1 month, 1-3 months and > 3 months for patient and medical delay; 1-3 months, 3-6 months, > 6 months for overall delay. Patient delay was associated with age and education: a higher risk was found for women of over 65 years age (odds ratio (OR) 2.1, 95% confidence interval (CI) 1.2-3.5) and with < or = 5 years school attendance (OR 3.3, 95% CI 2.0-5.6). Medical delay was seen to be associated with the professional figure: significant differences were found between senologists (oncologists exclusively dedicated to breast cancer operation) and other specialists (OR 3.5, 95% CI 1.5-8.4). Young age and symptomatic presentation were found to be high risk factors. Concerning tumour size in overall delay, in cases where the tumour was > 2 cm the OR was 2.4 (95% CI 1.5-3.7). Our study suggests that diagnostic delay can be reduced by providing more efficient training programmes for members of the medical profession and by producing educational training programmes targeted specifically at each age category (i.e. in older women more attention to education in prevention; in younger women correct information about mammography and specialized structures).

Electrospray ionization mass spectrometry of synthetic oligonucleotides using 2-propanol and spermidine

Oligonucleotides have become widely used tools in molecular biology and molecular diagnostics. Their parallel synthesis in large numbers and the increasing interest in microarray technology has raised the requirement for fast and informative analytical tools for their quality control. A direct injection electrospray ionization mass spectrometry (ESI-MS) technique based on the use of aqueous 2-propanol as running eluent, and spermidine (or triethylamine) as DNA modifiers, has been applied to analyze a large set of samples (about 200 synthetic oligonucleotides) ranging from 5 to 15 kDa (17-51mers) with good results in terms of sensitivity, suppression of sodium adduct formation, and speed of analysis. Copyright 2000 John Wiley & Sons, Ltd.

Application of capillary electrophoresis at low pH to oligonucleotides quality control.

A method for oligonucleotides analysis by using capillary electrophoresis at low pH in free solution is described. It may be considered an alternative to classical analytical techniques which use basic buffers and require the presence of sieving media to separate oligonucleotides as a function of their length. On the contrary, at low pH oligo nucleotides can be separated only depending on their base composition. A large set of samples consisting of 72 synthetic oligonucleotides bearing a 5'-alkylamine moiety and designed for HLA genotyping were analysed. The quality of these synthetic oligos was easily assessed, and a single base difference in oligonucleotides of equal sequence was detected. The results suggest the application of this method to the emerging field of mutation detection and single nucleotide polymorfism analysis.