The Early Intestinal Microbiota of Healthy Korean Newborns

Background: The microflora hypothesis may be the underlying explanation for the growth of inflammatory disease. In addition to many known affecting factors, knowing the gut microbiota of healthy newborns can help to understand the gut immunity and modulate it. Objectives: This study examined the microbiota of healthy newborns from urban regions. Patients and Methods: We enrolled 128 full-term newborns, born at Seoul St. Mary and St. Paul hospital from January 2009 to February 2010. All 143 samples of feces were cultivated in six culture plates to determine the amounts of total bacteria, anaerobes, gram-positive bacteria, coliforms, lactobacilli, and bifidobacteria. The samples were evaluated with a bivariate correlation between coliforms and lactobacilli. Terminal restriction fragment length polymorphism (T-RFLP) analysis with HhaI and MspI and a clustering analysis were performed for determination of diversity. Results: Bacteria were cultured in 61.5% of feces in the following order: anaerobes, gram-positive bacteria, lactobacilli, coliform, and bifidobacteria. The growth of total bacteria and lactobacilli increased in feces defecated after 24 hours of birth (P < 0.001, P = 0.008) and anaerobes decreased (P = 0.003). A negative correlation between the growth of lactobacilli and coliforms was found (r = -463, P < 0.001). Conclusions: This study confirms that bacterial colonization of healthy newborns born in cities is non-sterile, but has early diversification and inter-individuality.

Gender-specific association of MSA2756G with hypertension in patients attending a health facility in Ningxia Province, China

Purpose: To investigate the distribution of methionine synthase A2756G (MSA2756G) in the hypertensive patients in northwest Chinese population. Methods: A total of 378 unrelated hypertensive patients attending Ningxia Peoples Hospital, Ningxia Province, China, were recruited for this study. We analyzed genotype by amplication - created restriction sites (ACRS) and polymerase chain reaction - restrict fragment length polymorphism (PCR - RFLP) in hypertensive patients, and inspected the relation of the genotype with hypertension by χ2 and t test. Results: The frequency of G allele was 10.25 % in the control group and 14.04 % in hypertension group; it was not statistically different (p > 0.05). In the male group, the frequency of allele G was 11.50 % in control group, and 8.79 % in hypertension group. There was no significant difference between control and hypertension groups (p > 0.05). In the female group, the frequency of allele G was 9.00 %, in control and 19.54 % in hypertension group (p < 0.05), while in the hypertension group, allele G was 8.79 % in males which is significantly lower (p < 0.05) than in females (19.54 %) . Conclusion: Allele G of MSA2756G is a risk factor for hypertension in female in this Chinese population of this study.

Oral chondroitin sulfate and prebiotics for the treatment of canine Inflammatory Bowel Disease: a randomized, controlled clinical trial

BACKGROUND Canine inflammatory bowel disease (IBD) is a chronic enteropathy of unknown etiology, although microbiome dysbiosis, genetic susceptibility, and dietary and/or environmental factors are hypothesized to be involved in its pathogenesis. Since some of the current therapies are associated with severe side effects, novel therapeutic modalities are needed. A new oral supplement for long-term management of canine IBD containing chondroitin sulfate (CS) and prebiotics (resistant starch, β-glucans and mannaoligosaccharides) was developed to target intestinal inflammation and oxidative stress, and restore normobiosis, without exhibiting any side effects. This double-blinded, randomized, placebo-controlled trial in dogs with IBD aims to evaluate the effects of 180 days administration of this supplement together with a hydrolyzed diet on clinical signs, intestinal histology, gut microbiota, and serum biomarkers of inflammation and oxidative stress. RESULTS Twenty-seven client-owned biopsy-confirmed IBD dogs were included in the study, switched to the same hydrolyzed diet and classified into one of two groups: supplement and placebo. Initially, there were no significant differences between groups (p > 0.05) for any of the studied parameters. Final data analysis (supplement: n = 9; placebo: n = 10) showed a significant decrease in canine IBD activity index (CIBDAI) score in both groups after treatment (p < 0.001). After treatment, a significant decrease (1.53-fold; p < 0.01) in histologic score was seen only in the supplement group. When groups were compared, the supplement group showed significantly higher serum cholesterol (p < 0.05) and paraoxonase-1 (PON1) levels after 60 days of treatment (p < 0.01), and the placebo group showed significantly reduced serum total antioxidant capacity (TAC) levels after 120 days (p < 0.05). No significant differences were found between groups at any time point for CIBDAI, WSAVA histologic score and fecal microbiota evaluated by PCR-restriction fragment length polymorphism (PCR-RFLP). No side effects were reported in any group. CONCLUSIONS The combined administration of the supplement with hydrolyzed diet over 180 days was safe and induced improvements in selected serum biomarkers, possibly suggesting a reduction in disease activity. This study was likely underpowered, therefore larger studies are warranted in order to demonstrate a supplemental effect to dietary treatment of this supplement on intestinal histology and CIBDAI.

Characterization of some bacterial strains isolated from animal clinical materials and identified as Corynebacterium xerosis by molecular biological techniques

Eighteen Corynebacterium xerosis strains isolated from different animal clinical specimens were subjected to phenotypic and molecular genetic studies. On the basis of the results of the biochemical characterization, the strains were tentatively identified as C. xerosis. Phylogenetic analysis based on comparative analysis of the sequences of 16S rRNA and rpoB genes revealed that the 18 strains were highly related to C. xerosis, C. amycolatum, C. freneyi, and C. hansenii. There was a good concordance between 16S rRNA and partial rpoB gene sequencing results, although partial rpoB gene sequencing allowed better differentiation of C. xerosis. Alternatively, C. xerosis was also differentiated from C. freneyi and C. amycolatum by restriction fragment length polymorphism analysis of the 16S-23S rRNA gene intergenic spacer region. Phenotypic characterization indicated that besides acid production from D-turanose and 5-ketogluconate, 90% of the strains were able to reduce nitrate. The absence of the fatty acids C(14:0), C(15:0), C(16:1)omega 7c, and C(17:1)omega 8c can also facilitate the differentiation of C. xerosis from closely related species. The results of the present investigation demonstrated that for reliable identification of C. xerosis strains from clinical samples, a combination of phenotypic and molecular-biology-based identification techniques is necessary.

Development of a novel molecular tool for the rapid assessment of the biodiversity changes of benthic nematodes assemblages. Mares Conference Marine Ecosystems Helath and conservation

The molecular profiling system was developed using directed terminal-restriction fragment length polymorphism (dT-RFLP) to characterize soil nematode assemblages by relative abundance of feeding guilds and validation by comparison to traditional morphological method. The good performance of these molecular tools applied to soil nematodes assemblages create an opportunity to develop a novel approach for rapid assessment of the biodiversity changes of benthic nematodes assemblages of marine and estuarine sediments. The main aim of this research is to combine morphological and molecular analysis of estuarine nematodes assemblages, to establish a tool for fast assessment of the biodiversity changes within habitat recovery of Zostera noltii seagrass beds; and validate the dT-RFLP as a high-throughput tool to assess the system recovery. It was also proposed to develop a database of sequences related to individuals identified at species level to develop a new taxonomic reference system. A molecular phylogenetic analysis of the estuarine nematodes has being performed. After morphological identification, barcoding of 18S rDNA are being determined for each nematode species and the results have shown a good degree of concordance between traditional morphology-based identification and DNA sequences. The digest strategy developed for soil nematodes is not suitable for marine nematodes. Then five samples were cloned and sequenced and the sequence data was used to design a new dT-RFLP strategy to adapt this tool to marine assemblages. Several solutions were presented by DRAT and tested empirically to select the solution that cuts most efficiently, separating the different clusters. The results of quantitative PCR showed differences in nematode density between two sampling stations according the abundance of the nematode density obtained by the traditional methods. These results suggest that qPCR could be a robust tool for enumeration of nematode abundance, saving time.

Fixing, storage time and DNA extraction applied to dT-RFLP for ecological and molecular studies of marine nematodes assemblages

The application of molecular methods offers an alternative faster than traditional methods based on morphology It is nearly impossible to process all the samples in short period using traditional methods, and the deterioration of marine sediments rapidly occurs The dT-RFLP (directed Terminal-Restriction Fragment Length Polymorphism) allows a rapid assessment of biodiversity changes of nematodes assemblages The use of a not suitable fixing, storage time and DNA extraction could be a limitation in molecular analysis like dT-RFLP and real time PCR.Objetives: the best fixative •the level of DNA degradation over the time •the best DNA extraction method for marine nematodes and suitable for dT-RFLP analysis

Development of molecular approaches to assess the diversity and abundance of marine nematodes assemblages for assessment of Good Environmental Status of the estuarine and marine ecosystems

In Europe, the concerns with the status of marine ecosystems have increased, and the Marine Directive has as main goal the achievement of Good Environmental Status (GES) of EU marine waters by 2020. Molecular tools are seen as promising and emerging approaches to improve ecosystem monitoring, and have led ecology into a new era, representing perhaps the most source of innovation in marine monitoring techniques. Benthic nematodes are considered ideal organisms to be used as biological indicator of natural and anthropogenic disturbances in aquatic ecosystems underpinning monitoring programmes on the ecological quality of marine ecosystems, very useful to assess the GES of the marine environment. dT-RFLP (directed Terminal-Restriction Fragment Length Polymorphism) allows to assess the diversity of nematode communities, but also allows studying the functioning of the ecosystem, and combined with relative real-time PCR (qPCR), provides a high-throughput semi-quantitative characterization of nematode communities. These characteristics make the two molecular tools good descriptors for the good environmental status assessment. The main aim of this study is to develop and optimize the dT-RFLP and qPCR in Mira estuary (SW coast, Portugal). A molecular phylogenetic analysis of marine and estuarine nematodes is being performed combining morphological and molecular analysis to evaluate the diversity of free-living marine nematodes in Mira estuary. After morphological identification, barcoding of 18S rDNA and COI genes are being determined for each nematode species morphologically identified. So far we generated 40 new sequences belonging to 32 different genus and 17 families, and the study has shown a good degree of concordance between traditional morphology-based identification and DNA sequences. These results will improve the assessment of marine nematode diversity and contribute to a more robust nematode taxonomy. The DNA sequences are being used to develop the dT-RFLP with the ability to easily process large sample numbers (hundreds and thousands), rather than typical of classical taxonomic or low throughput molecular analyses. A preliminary study showed that the digest enzymes used in dT-RFLP for terrestrial assemblages separated poorly the marine nematodes at taxonomic level for functional group analysis. A new digest combination was designed using the software tool DRAT (Directed Terminal Restriction Analysis Tool) to distinguished marine nematode taxa. Several solutions were provided by DRAT and tested empirically to select the solution that cuts most efficiently. A combination of three enzymes and a single digest showed to be the best solution to separate the different clusters. Parallel to this, another tool is being developed to estimate the population size (qPCR). An improvement in qPCR estimation of gene copy number using an artificial reference is being performed for marine nematodes communities to quantify the abundance. Once developed, it is proposed to validate both methodologies by determining the spatial and temporal variability of benthic nematodes assemblages across different environments. The application of these high-throughput molecular approaches for benthic nematodes will improve sample throughput and their implementation more efficient and faster as indicator of ecological status of marine ecosystems.

A genetic map of macadamia based on randomly amplified DNA fingerprinting (RAF) markers

The first genetic linkage map of macadamia (Macadamia integrifolia and M. tetraphylla) is presented. The map is based on 56 F-1 progeny of cultivars 'Keauhou' and 'A16'. Eighty-four percent of the 382 markers analysed segregated as Mendelian loci. The two-way pseudo-testcross mapping strategy allowed construction of separate parental cultivar maps. Ninety bridging loci enabled merging of these maps to produce a detailed genetic map of macadamia, 1100 cM in length and spanning 70-80% of the genome. The combined map comprised 24 linkage groups with 265 framework markers: 259 markers from randomly amplified DNA fingerprinting (RAF), five random amplified polymorphic DNA (RAPD), and one sequence-tagged microsatellite site (STMS). The RAF marker system unexpectedly revealed 16 codominant markers, one of them a putative microsatellite locus and exhibiting four distinct alleles in the cross. This molecular study is the most comprehensive examination to date of genetic loci of macadamia, and is a major step towards developing marker-assisted selection for this crop.

Near full-length genome analysis of low prevalent human immunodeficiency virus type 1 subclade F1 in Sao Paulo, Brazil

Background: The genetic diversity of the human immunodeficiency virus type 1 (HIV-1) is critical to lay the groundwork for the design of successful drugs or vaccine. In this study we aimed to characterize and define the molecular prevalence of HIV-1 subclade F1 currently circulating in Sao Paulo, Brazil. Methods: A total of 36 samples were selected from 888 adult patients residing in Sao Paulo who had previously been diagnosed in two independent studies in our laboratory as being infected with subclade F1 based on pol subgenomic fragment sequencing. Proviral DNA was amplified from the purified genomic DNA of all 36 blood samples by 5 fragments overlapping PCR followed by direct sequencing. Sequence data were obtained from the 5 fragments of pure subclade F1 and phylogenetic trees were constructed and compared with previously published sequences. Subclades F1 that exhibited mosaic structure with other subtypes were omitted from any further analysis Results: Our methods of fragment amplification and sequencing confirmed that only 5 sequences inferred from pol region as subclade F1 also holds true for the genome as a whole and, thus, estimated the true prevalence at 0.56%. The results also showed a single phylogenetic cluster of the Brazilian subclade F1 along with non-Brazilian South American isolates in both subgenomic and the full-length genomes analysis with an overall intrasubtype nucleotide divergence of 6.9%. The nucleotide differences within the South American and Central African F1 strains, in the C2-C3 env, were 8.5% and 12.3%, respectively. Conclusion: All together, our findings showed a surprisingly low prevalence rate of subclade F1 in Brazil and suggest that these isolates originated in Central Africa and subsequently introduced to South America.

Characterization of mannose-binding lectin plasma levels and genetic polymorphisms in HIV-1-infected individuals

INTRODUCTION: The present study investigated the association between mannose-binding lectin (MBL) gene polymorphism and serum levels with infection by HIV-1. METHODS: Blood samples (5mL) were collected from 97 HIV-1-infected individuals resident in Belém, State of Pará, Brazil, who attended the Special Outpatient Unit for Infections and Parasitic Diseases (URE-DIPE). CD4+ T-lymphocyte count and plasma viral load were quantified. A 349bp fragment of exon 1 of the MBL was amplified via PCR, using genomic DNA extracted from controls and HIV-1-infected individuals, following established protocols. MBL plasma levels of the patients were quantified using an enzyme immunoassay kit. RESULTS: Two alleles were observed: MBL*O, with a frequency of 26.3% in HIV-1-infected individuals; and the wild allele MBL*A (73.7%). Similar frequencies were observed in the control group (p > 0.05). Genotype frequencies were distributed according to the Hardy-Weinberg equilibrium in both groups. Mean MBL plasma levels varied by genotype, with statistically significant differences between the AA and AO (p < 0.0001), and AA and OO (p < 0.001) genotypes, but not AO and OO (p = 0.17). Additionally, CD4+ T-lymphocytes and plasma viral load levels did not differ significantly by genotype (p > 0.05). CONCLUSIONS: The results of this study do not support the hypothesis that MBL gene polymorphism or low plasma MBL concentrations might have a direct influence on HIV-1 infection, although a broader study involving a large number of patients is needed.

Risk of multiple myeloma is associated with polymorphisms within telomerase genes and telomere length

Compelling biological and epidemiological evidences point to a key role of genetic variants of the TERT and TERC genes in cancer development. We analyzed the genetic variability of these two gene regions using samples of 2,267 multiple myeloma (MM) cases and 2,796 healthy controls. We found that a TERT variant, rs2242652, is associated with reduced MM susceptibility (OR?=?0.81; 95% CI: 0.72-0.92; p?=?0.001). In addition we measured the leukocyte telomere length (LTL) in a subgroup of 140 cases who were chemotherapy-free at the time of blood donation and 468 controls, and found that MM patients had longer telomeres compared to controls (OR?=?1.19; 95% CI: 0.63-2.24; ptrend ?=?0.01 comparing the quartile with the longest LTL versus the shortest LTL). Our data suggest the hypothesis of decreased disease risk by genetic variants that reduce the efficiency of the telomerase complex. This reduced efficiency leads to shorter telomere ends, which in turn may also be a marker of decreased MM risk.

Detection of somaclonal variation during cocoa somatic embryogenesis characterised using cleaved amplified polymorphic sequence and the new freeware Artbio

The scarcity and stochastic nature of genetic mutations presents a significant challenge for scientists seeking to characterise de novo mutation frequency at specific loci. Such mutations can be particularly numerous during regeneration of plants from in vitro culture and can undermine the value of germplasm conservation efforts. We used cleaved amplified polymorphic sequence (CAPS) analysis to characterise new mutations amongst a clonal population of cocoa plants regenerated via a somatic embryogenesis protocol used previously for cocoa cryopreservation. Efficacy of the CAPS system for mutation detection was greatly improved after an ‘a priori’ in silico screen of reference target sequences for actual and potential restriction enzyme recognition sites using a new freely available software called Artbio. Artbio surveys known sequences for existing restriction enzyme recognition sites but also identifies all single nucleotide polymorphism (SNP) deviations from such motifs. Using this software, we performed an in silico screen of seven loci for restriction sites and their potential mutant SNP variants that were possible from 21 restriction enzymes. The four most informative locus-enzyme combinations were then used to survey the regenerant populations for de novo mutants. We characterised the pattern of point mutations and, using the outputs of Artbio, calculated the ratio of base substitution in 114 somatic embryo-derived cocoa regenerants originating from two explant genotypes. We found 49 polymorphisms, comprising 26.3% of the samples screened, with an inferred rate of 2.8 × 10−3 substitutions/screened base. This elevated rate is of a similar order of magnitude to previous reports of de novo microsatellite length mutations arising in the crop and suggests caution should be exercised when applying somatic embryogenesis for the conservation of plant germplasm.

Detection of the single nucleotide polymorphism (rs2227307) in the human interleukin 8 gene using a pcr-rflp assay

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Epstein-barr virus infection and single nucleotide Polymorphisms in the promoter region of interleukin 10 gene in patients with Hodgkin lymphoma

Context. - Hodgkin lymphoma is a neoplastic disease in which the immune system plays a major role in its pathogenesis. Interleukin 10 ( IL-10), an immunosuppressive cytokine actively produced in patients with Hodgkin lymphomas, favors the survival of the Hodgkin/Reed-Sternberg cells. Individual variations in IL-10 levels may be due, in part, to the presence of single nucleotide polymorphisms in the IL10 gene promoter.Objective. - To evaluate whether particular single nucleotide polymorphisms in the IL10 gene are found more frequently in Hodgkin lymphoma cases associated with Epstein-Barr virus infection.Design. - the identification of single nucleotide polymorphisms at positions -1082 and -819/-592 in the IL10 gene was performed by polymerase chain reaction and restriction length fragment polymorphisms analysis in 65 cases of Hodgkin lymphoma and 50 cases of reactive benign follicular lymphoid hyperplasia ( non-Hodgkin lymphoma control group).Results. - the frequency of the genotype GG at position -1082 was found to be significantly higher in patients with Epstein-Barr virus-positive Hodgkin lymphoma compared with Epstein-Barr virus-negative cases.Conclusions. - the results suggest that the presence of specific single nucleotide polymorphisms in the IL10 gene, notably those associated with high IL-10 production, may play a role in the susceptibility to Epstein-Barr virus -positive Hodgkin lymphoma development.

Evaluation of DNA polymorphisms amplified by arbitrary primers (RAPD) as genetically associated elements to differentiate virulent and non-virulent Paracoccidioides brasiliensis isolates

Randomly amplified polymorphic DNA (RAPD) analysis of 35 Paracoccidioides brasiliensis isolates was carried out to evaluate the correlation of RAPD profiles with the virulence degree or the type of the clinical manifestations of human paracoccidioidomycosis. The dendrogram presented two main groups sharing 64% genetic similarity. Group A included two isolates from patients with chronic paracoccidioidomycosis; group B comprised the following isolates showing 65% similarity: two non-virulent, six attenuated, five virulent, eight from patients with chronic paracoccidioidomycosis and two from patients with acute paracoccidioidomycosis. The virulent Pb18 isolate and six attenuated or non-virulent samples derived from it were genetically indistinguishable (100% of similarity). Thus, in our study, RAPD patterns could not discriminate among 35 P. brasiliensis isolates according to their differences either in the degree of virulence or in the type of the clinical manifestation of this fungal infection. © 2002 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved.