This article evaluates the effect of shear stress on the composition of biofilm bacterial communities. For the first time, a Conical Couette–Taylor Reactor (CCTR) was used to develop biofilms at ...varying shear stresses (from 0.055 to 0.27
Pa) and provided a useful model for studying the effect of hydrodynamics on biofilms. The composition, diversity and dynamics of biofilm bacterial communities were analysed using the PCR–SSCP fingerprint method. Results clearly demonstrate a link between shear stress and composition of the microbial communities. High shear stresses decrease biofilm diversity and the analysis of biofilm community dynamics suggests that shear stress would slow down biofilm maturation and tend to maintain a young biofilm.
Purpose To critically evaluate our experience in molecular testing of Leber congenital amaurosis (LCA) and to use this information to devise a general approach to heterogeneous recessive disorders. ...Careful clinical and molecular characterization of large cohorts of patients affected with inherited eye diseases will be an essential step in the development of effective therapy for these diseases, especially when the therapy involves gene replacement. Design A molecular genetic case-control study. Methods Six hundred forty-two unrelated individuals with the clinical diagnosis of LCA and 200 unrelated control individuals were screened for disease-causing sequence variations in eight genes using various combinations of single-strand conformational polymorphism analysis (SSCP), automated DNA sequencing, multiplex allele-specific ligation analysis (SNPlex), and high-density solid-phase single nucleotide polymorphism genotyping. Results Four hundred forty instances of 189 different disease-causing sequence variations were observed in this study, 98 of which have not been previously reported. One hundred forty-six of the 189 variations (77%) were observed in only a single individual. The observed variations were not evenly distributed among the LCA patients or among the eight genes. Empirical analysis of this uneven distribution was used to devise a multi-platform mutation detection strategy that is four times more efficient than a more conventional strategy of completely sequencing all of the coding regions of all LCA genes in all subjects. Hardy-Weinberg analysis of the observed mutations suggests that these eight genes are collectively responsible for about 70% of the cases of LCA in North America. The carrier frequency of the most common LCA allele (an intron 26 variation in CEP290) was found to be 2/3,248, which suggests that the overall prevalence of LCA in this population is about 1/81,000. An allele-specific ligation assay (SNPlex) was designed to detect 68 of the most common LCA-causing alleles, and semi-quantitative analysis of the data from this assay also revealed examples of gene deletion and isodisomy in the cohort. Conclusions The data demonstrates that a tiered screening strategy combining allele-specific detection with automated DNA sequencing can increase the efficiency of autosomal recessive mutation detection four-fold when compared with DNA sequencing alone. However, the very high rate of unique mutations observed in this study (77%) suggests that DNA sequencing will remain an important part of the overall strategy if high sensitivity is to be achieved.
The aim of the present study was to detect the FOXP3 gene polymorphisms in Barki sheep at a variable region covering exon 13, intron 13 and the coding sequence in exon 14 and to test the association ...of these polymorphisms with growth traits. 122 Barki lambs were phenotyped for various growth traits, viz., birth weight (BW), weaning weight (WW), pre-weaning daily gain in weight (ADG1), post-weaning daily gain in weight (ADG2) and marketing bodyweight (MW). The polymerase chain reaction - single-strand conformational polymorphisms (PCR-SSCP) and DNA sequencing methods were used to identify the genetic variants in the FOXP3 gene. The associations between the variation in FOXP3 gene and growth traits were tested using a general linear model. Two variants (F1 and F2 with gene frequencies of 0.64 and 0.36, respectively), and three genotypes (F1F1, F1F2 and F2F2 with frequencies of 0.37, 0.53 and 0.10, respectively) were detected. The association of FOXP3 genotype was significant (p
< 0.05) with ADG2 and MW. It is concluded that FOXP3 genotype might be helpful for sheep breeders to produce fast-growing lambs. However, further studies are needed in a large population to confirm the association we found.
Celotno besedilo
Dostopno za:
BFBNIB, DOBA, GIS, IJS, IZUM, KILJ, KISLJ, NUK, PILJ, PNG, SAZU, UILJ, UKNU, UL, UM, UPUK
Microbial ecology has undergone a profound change in the last two decades with regard to methods employed for the analysis of natural communities. Emphasis has shifted from culturing to the analysis ...of signature molecules including molecular DNA-based approaches that rely either on direct cloning and sequencing of DNA fragments (shotgun cloning) or often rely on prior amplification of target sequences by use of the polymerase chain reaction (PCR). The pool of PCR products can again be either cloned and sequenced or can be subjected to an increasing variety of genetic profiling methods, including amplified ribosomal DNA restriction analysis, automated ribosomal intergenic spacer analysis, terminal restriction fragment length polymorphism, denaturing gradient gel electrophoresis, temperature gradient gel electrophoresis, single strand conformation polymorphism, and denaturing high-performance liquid chromatography. In this document, we present and critically compare these methods commonly used for the study of microbial diversity.
Purpose: In breast cancer, the PIK3CA gene is frequently mutated at “hotspots” in exons 9 and 20, corresponding to the helical and kinase domains, respectively.
We decided to investigate the ...association of PIK3CA mutations with pathologic features and clinical outcome in a large series of patients with breast cancer.
Experimental Design: Frozen samples from 163 consecutive patients were analyzed for PIK3CA mutations using PCR single-strand conformation polymorphism and sequence analyses.
Results: We identified 46 missense mutations, 24 (53%) in exon 9, and 21 (47%) in exon 20. Twelve (50%) of the 24 mutations in exon
9 were of the E542K type and 11 (46%) were of the E545K type. Twenty (95%) of the 21 mutations in exon 20 were H1047R substitutions.
Mutations in exon 9 were more frequent in lobular carcinomas (42% of cases) than in ductal carcinoma (11% of cases; P = 0.002). At univariate survival analysis, PIK3CA exon 20 mutations were associated with prolonged overall and disease-free survival, whereas mutations in exon 9 were associated
with significantly worse prognosis. At multivariate analysis, exon 9 PIK3CA mutations were the strongest independent factor to predict poor prognosis for disease-free survival ( P = 0.0003) and overall survival ( P = 0.001).
Conclusion: Our data show that exon 9 PIK3CA mutations are typical of infiltrating lobular carcinomas. In addition, they indicate that PIK3CA mutations in different exons are of different prognostic value: exon 9 mutations are independently associated with early
recurrence and death, whereas exon 20 PIK3CA mutations are associated with optimal prognosis.
1. Ovalbumin (SERPINB14) is the most abundant protein present in egg white contributing about 54% of the total egg protein. In this study, the objectives were to clone and characterise the coding ...sequence of the SERPINB14 gene, to explore its expression profile, identify polymorphisms in the promoter of the gene and explore any association with egg quality traits in White Leghorn chickens.
2. SNPs and mRNA expression of SERPINB14 in White Leghorn chicken lines were detected by PCR-single strand conformation polymorphism (SSCP) along with sequencing and qPCR. The open reading frame (ORF) was cloned in an expression plasmid vector and sequenced.
3. The ORF of this gene was 1161 bp encoding a peptide of 386 amino acids. There were three SNPs observed in the coding region of the gene, one of which was of the mis-sense type, having c562G>A transition which resulted in substitution of alanine to threonine at position 188 in the protein sequence. In both the lines, an increase in expression of the gene was observed after onset of egg production with peak expression at the 40
th
week of age compared to before onset of lay. The SERPINB14 gene was expressed in the magnum, but not in ovary and infundibulum, tissues of each White Leghorn line. The promoter region of the gene showed SNPs with three haplotypes; H1, H2, and H3. The haplo groups were associated with the egg weight and age at sexual maturity in the IWI line and Haugh unit and albumin index in the IWK line.
4. It was concluded that the ORF of SERPINB14 gene in White Leghorn chicken lines is polymorphic. The promoter region of the gene is also polymorphic and has significant (P < 0.05) association with Haugh unit and egg weight in IWK and IWI chicken lines, respectively.
A fatal transmissible tumor spread between individuals by biting has emerged in the Tasmanian devil (Sarcophilus harrisii), a carnivorous marsupial. Here we provide genetic evidence establishing that ...the tumor is clonal and therefore foreign to host devils. Thus, the disease is highly unusual because it is not just a tumor but also a tissue graft, passed between individuals without invoking an immune response. The MHC plays a key role in immune responses to both tumors and grafts. The most common mechanism of immune evasion by tumors is down-regulation of classical cell surface MHC molecules. Here we show that this mode of immune escape does not occur. However, because the tumor is a graft, it should still be recognized and rejected by the host's immune system due to foreign cell surface antigens. Mixed lymphocyte responses showed a lack of alloreactivity between lymphocytes of different individuals in the affected population, indicating a paucity of MHC diversity. This result was verified by genotyping, providing a conclusive link between a loss of MHC diversity and spread of a disease through a wild population. This novel disease arose as a direct result of loss of genetic diversity and the aggressive behavior of the host species. The neoplastic clone continues to spread although the population, and, without active disease control by removal of affected animals and the isolation of disease-free animals, the Tasmanian devil faces extinction.
Abstract
The development of characteristic thallus structures in lichen-forming fungi requires the association with suitable photoautotrophic partners. Previous work suggests that fungi have a ...specific range of compatible photobionts and that selected algal strains are also correlated with the habitat conditions. We selected the rock-inhabiting crust lichen Protoparmeliopsis muralis, which exhibits high flexibility in algal associations. We present a geographically extended and detailed analysis of algal association patterns including thalli which host superficial algal colonies. We sampled 17 localities in Europe, and investigated the photobiont genotypic diversity within and between thalli and compared the diversity of intrathalline photobionts and externally associate algal communities between washed and unwashed thalli by single-strand conformation polymorphism analyses and ITS sequence data. The results show that (1) photobiont population within the lichen thalli is homogeneous; (2) multiple photobiont genotypes occur within single areoles and lobes of individual lichens; and (3) algal communities which superficially colonize the lichen thalli host taxa known as photobionts in unrelated lichens. Photobiont association patterns are extremely flexible in this ecologically versatile crust-forming lichen. We suggest that lichen surfaces represent a potential temporary niche for free-living stages of lichen photobionts, which could facilitate the establishment of further lichens in the proximal area.
Genetic variation in coding regions is of strong interest for biologists as it represents an important factor that drives evolution. To analyse polymorphic loci, researchers usually rely on commonly ...used typing techniques such as cloning, SSCP, DGGE or RSCA. However, there are potential pitfalls in screening multi-allelic templates, which are mainly the formation of sequence chimeras during PCR amplification, and mosaic sequences during cloning. One of the most challenging genomic regions to explore is the Major Histocompatibility Complex (
MHC), which codes for peptide-binding proteins of the vertebrate's adaptive immune system and is well known for its exceptional polymorphism. We compared the effect of two different PCR amplification approaches in a study of the
MHC class IIB genes of the three-spined stickleback (
Gasterosteus aculeatus). One approach used standard PCR conditions and the other a combination of several measures to eliminate PCR artefacts. In both approaches, the amplicons obtained were cloned and sequenced. In the first, established approach, 24% of the clones represented artefacts, while in the second approach the number of artefacts were reduced ten-fold. Furthermore, it enabled easy differentiation between real alleles and artificial sequences. We also analysed the potential effects of such artefacts in genetic analysis and evolutionary interpretation, and found a slight reduction in the signature of positive selection and an increase in recombination events. Consequently, we strongly recommend to apply the new PCR approach described in this study when genotyping
MHC or other polymorphic genes.
•The anaerobic digestion of olive mill and abattoir wastewaters was investigated.•Effects of temperature and loading on the microbial structure were examined.•PCR-SSCP showed significant dynamic of ...both bacteria and archaea.•The high COD removal and biogas yield were achieved at 55°C.•Methanobacteriales and Thermoplasmales are predominant archaea.
The effect of increasing the organic loading rates (OLRs) on the performance of the anaerobic codigestion of olive mill (OMW) and abattoir wastewaters (AW) was investigated under mesophilic and thermophilic conditions. The structure of the microbial community was also monitored. Increasing OLR to 9g of chemical oxygen demand (COD) L−1d−1 affected significantly the biogas yield and microbial diversity at 35°C. However, at 55°C digester remained stable until OLR of 12g of CODL−1d−1 with higher COD removal (80%) and biogas yield (0.52Lg−1 COD removed). Significant differences in the bacterial communities were detected between mesophilic and thermophilic conditions. The dominant phyla detected in the digester at both phases were the Firmicutes, Actinobacteria, Bacteroidetes, Synergistetes and Spirochaete. However, Verrucomicrobia, Proteobacteria and the candidate division BRC1 were only detected at thermophilic conditions. The Methanobacteriales and the Thermoplasmales were found as a high predominant archaeal member in the anaerobic sludge.
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