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.

Draft Genome Sequence of the Host-Restricted Salmonella enterica Serovar Abortusovis Strain SS44.

Salmonella enterica serovar Abortusovis is a pathogen strictly adapted to ovines, in which it causes abortion. To enhance our understanding of this pathogen, we assembled the first draft sequence of an S. Abortusovis genome (strain SS44). The obtained genomic data might facilitate the study of S. enterica evolution and host adaptation.

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.

Expression profiling of genes involved in the formation of aroma in two peach genotypes.

The expression profile of flavour-related genes during ripening was investigated in two peach genotypes, Bolero and OroA, which have been selected for their contrasting aroma/ripening behaviour. A new peach microarray containing 4776 oligonucleotide probes corresponding to a set of ESTs specifically enriched in secondary metabolism (µPEACH2.0) was designed to investigate transcriptome changes during three fruit ripening stages, revealing 1807 transcripts differentially expressed within and between the two genotypes. Differences in the expression of genes involved in the biosynthesis of aroma compounds were detected during the ripening process within and between the two genotypes. In particular, a subset of 12 transcripts involved in metabolism of esters, norisoprenoids, phenylpropanoids and lactones, varied in expression during ripening and between Bolero and OroA.

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.

HCV and cancer: a case-control study in a high-endemic area.

BACKGROUND/AIMS: HCV is a RNA virus that cannot be integrated with the host genome; it can, however, exert its oncogenic potential indirectly by contributing to the modulatory effects of the host immune system, probably through a capacity to elude the immune system. We have carried out a case-controlled study on the different oncological pathologies which have, to date, been shown to have a relationship with HCV. METHODS: We screened 495 patients with different types of cancer: 114 cases of liver cancer, 41 of multiple myeloma, 111 non-Hodgkin's lymphomas, 130 thyroid cancers, 63 cases of Hodgkin's disease. The controls were 226 patients with no history of cancer. The relationship between each cancer and HCV infection was assessed by means of odds ratios (OR) and corresponding 95% confidence intervals. RESULTS: Risks were greater for liver cancer (OR=32.9 95% CI 16.5-65.4, p<0.0001), multiple myeloma (OR=4.5 95% CI 1.9-10.7, p=0.0004) and B-cell non-Hodgkin's lymphoma (OR=3.7 95% CI 1.9-7.4, p=0.0001). For Hodgkin's disease there was no significant association (p=0.3). An association between HCV and thyroid cancer was noted (OR=2.8 95% CI 1.2-6.3, p=0.01). CONCLUSION: Our study is particularly important for public health since the high prevalence of HCV in the South of Italy gives reason to expect increases in not only liver cancer, but also tumors associated with the immune system and thyroid cancer in years to come.

An assessment of delays in obtaining definitive breast cancer treatment in Southern Italy.

Female population is medically underserved in Southern Italy (in comparison with other Italian regions). In a recent systematic review of published studies, delays of 3-6 months between symptom onset and treatment have been clearly associated with lower survival rates for breast cancer patients. The aim of this study was to examine breast cancer delays in medically underserved patients in Southern Italy, in order to recognize their determinating factors so as to provide women with a better opportunity for survival. The variables examined were age, education, symptom status at first presentation: symptomatic and asymptomatic, date of first symptom presentation, date of first consultation with a health provider, consulted provider, tumor 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 education: a higher risk was found for women 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 (oncologist exclusively dedicated to breast cancer) and other specialists (OR 3.5, 95%, CI 1.5-8.4). Age and symptomatic presentation were found to be high risk factors. Concerning tumor size in overall delay in cases > 2 cm had OR values were of 2.4 (95%, CI 1.5-3.7). In conclusion our study suggests that diagnostic delay is associated with medically underserved status and can be reduced by educating younger and less educated women, as suggested in other studies and by providing training programs for members in the medical profession.

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).

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.

Association of early-onset Alzheimer's disease with an interleukin-1alpha gene polymorphism.

Overexpression of the pluripotent cytokine interleukin-1 (IL-1) by microglial cells correlates with formation of neuritic beta-amyloid plaques in Alzheimer's disease (AD). We evaluated polymorphisms in the genes coding for the IL-1alpha, IL-1beta, and IL-1 receptor antagonist cytokines, and tested their association with the occurrence and age at onset of sporadic AD. We found a strong association between the IL-1A T/T genotype and AD onset before 65 years of age (odds ratio, 4.86), with carriers of this genotype showing an onset of disease 9 years earlier than IL-1A C/C carriers. A weaker association with the age at onset was also shown for the IL-1B and IL-1RN genes. These data suggest either a direct effect of the IL-1 gene family, mainly IL-1A, on the clinical onset of AD, or a linkage dysequilibrium with an unknown locus relevant to AD on chromosome 2.

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.

Time-resolved DNA-microarray reading by an intensified CCD for ultimate sensitivity.

We describe a novel technique for DNA-microarray reading based on time-resolved fluorescence measurements. We used an intensified CCD camera with picosecond resolution to acquire a set of time-delayed fluorescence images from a mutation DNA microarray marked with cyanine 3. We measured the fluorescence lifetimes of the marker and the background separately, and we used this information to calculate the amplitude map of the marker, starting from the time-delayed images. This procedure allowed us to identify hybridized spots that are not visible in fluorescence images acquired with continuous-wave detection.

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.

Apolipoprotein E genotyping by capillary electrophoretic analysis of restriction fragments.

We present the genotyping of apolipoprotein (apo) E by means of restriction fragment analysis of amplified genomic DNA by high-performance capillary electrophoresis and a replaceable non-gel-sieving matrix. This procedure streamlines the genotyping of apo E in large-scale population studies because of the automation and speed of capillary electrophoresis.

Sequence and gene content in 35 kb genomic clone mapping in the human Xq27.1 region.

This paper presents detailed analysis of the entire sequence of a cosmid clone, 26H7, containing 35 kb of human DNA. This cosmid resides on the q27.1 region of the human X chromosome between, DXS1232 and DXS119 loci. Novel potential small exons were detected for which conventional gene identification strategies (Northern blot analysis and extensive cDNA library screening) proved to be inefficient. Of the standard repetitive elements we found: 8 Alu's making up 6.2% of the sequence; 10 MIR segments (4.1%); 5 LINE1 elements (4.8%), 3 MIR2 (1.0%); 2 MLT (2.9%), and 1 MSTA (0.7%) representing about 20% of the total sequence. The overall GC content was rather low, only 42% and no CpG island was detected using rare restriction enzymes. However, a CpG-rich region was identified. Computer aided analysis of the sequence inferred the presence of three possible genes: one of them was found to be homologous to the U7 RNA family elements; a second is reported in this paper, however at the moment no significant homology has been found in the data bank. The third predicted gene has not as yet been found to be detectable by RT-PCR. We also report in this paper the identification of X-chromosome specific repeated sequences.

Multiple G6PD mutations are associated with a clinical and biochemical phenotype similar to that of G6PD Mediterranean.

Glucose-6-phosphate dehydrogenase (G6PD) deficiency, one of the most common red cell abnormalities, is characterized by a wide clinical, biochemical, and molecular heterogeneity. In this study we have determined the molecular basis of G6PD deficiency in a sample of 70 male subjects, originating from different parts of Italy, who all shared a clinical and biochemical phenotype identical or very similar to that of G6PD Mediterranean, the most common variant in Italy. In 59 cases (84%) we found the mutation 563 C --> T, previously known to be underlying the G6PD Mediterranean and the two polymorphic variants G6PD Cagliari and G6PD Sassari. From the remaining 11 we amplified the entire coding region of G6PD in 8 different fragments and subjected them to nonradioactive single-strand conformation analysis. Direct sequencing was then performed on abnormal fragments. By this approach we found six cases (8.5%) with 1360 G --> A mutation (G6PD Union) and two cases (2.8%) with 1376 G --> C (G6PD Cosenza). In the remaining three samples we found two other mutations: 1342 A --> G (two cases, 2.8%) and 1052 G --> T (one case, 1.4%). These two molecular defects have never been described before and were designated by us as G6PD S. Antioco and G6PD Partenope, respectively. Haplotype analysis suggested that all the non-Mediterranean mutations occurred independently on a normal G6PD allele. This study shows that the G6PD Union mutation has a high polymorphic frequency in the Italian population and that the genetic heterogeneity of G6PD Mediterranean-like variants is higher at the molecular level than expected from biochemical characterization.

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.