Single-strand Conformation Polymorphism Research Articles

Leveraging biological nitrogen fixation and soybean yield by planting duration under varying onset of rainy seasons in Uganda

Rainfall distribution and onset of rainy seasons, both of which are key determinants of the decision to plant most annual crops in sub-Saharan Africa (SSA), are increasingly difficult to predict due to climate change. Consequently, planting schedules affect both crops and vital natural systems such as biological nitrogen fixation in legumes. The objective of this study was to establish the optimum planting duration for soybean (Glycine max L.), from the onset of the rainy season, in the context of nodulation and grain yield, and diversity of rhizobia in rhizosphere soil and nodules. A field experiment was set up with treatments, Bradyrhizobia inoculated and non- inoculated soybean variety Maksoy3N planted at four dates; 0, 10, 20 and 30 days from the onset of rain season. Diversity of rhizobia in the rhizosphere soil and nodules were fingerprinted using Single Strand Conformation Polymorphism (SSCP) method. Inoculation significantly (P<0.05) increased the number but not effectiveness of nodules and grain yield; which was attributed to presence of native strains in the soil. Planting after 10 days following the onset of a rainy season significantly (P<0.05) reduced grain yields and total yield loss was realised when seasonal cumulative rainfall was about 500 mm. Planting after 20 days from onset of rainy season significantly (P< 0.05) reduced the total number of nodules. A total of 8 and 12 clusters of bacteria from the soil and nodules at 5% dissimilarity, respectively, were recorded. The Bradyrhizobia species consisted of B. japonicum, B. japonicum (USDA 6), B. elkanii, B. liaoningense, B. canariense, and B. yuanmingense.Key words: Climate change, inoculation, nodulation, rhizobia

Challenges of next-generation sequencing in conservation management: Insights from long-term monitoring of corridor effects on the genetic diversity of mouse lemurs in a fragmented landscape.

Long‐term genetic monitoring of populations is essential for efforts aimed at preserving genetic diversity of endangered species. Here, we employ a framework of long‐term genetic monitoring to evaluate the effects of fragmentation and the effectiveness of the establishment of corridors in restoring population connectivity and genetic diversity of mouse lemurs Microcebus ganzhorni. To this end, we supplement estimates of neutral genetic diversity with the assessment of adaptive genetic variability of the major histocompatibility complex (MHC). In addition, we address the challenges of long‐term genetic monitoring of functional diversity by comparing the genotyping performance and estimates of MHC variability generated by single‐stranded conformation polymorphism (SSCP)/Sanger sequencing with those obtained by high‐throughput sequencing (next‐generation sequencing [NGS], Illumina), an issue that is particularly relevant when previous work serves as a baseline for planning management strategies that aim to ensure the viability of a population. We report that SSCP greatly underestimates individual diversity and that discrepancies in estimates of MHC diversity attributable to the comparisons of traditional and NGS genotyping techniques can influence the conclusions drawn from conservation management scenarios. Evidence of migration among fragments in Mandena suggests that mouse lemurs are robust to the process of fragmentation and that the effect of corridors is masked by ongoing gene flow. Nonetheless, results based on a larger number of shared private alleles at neutral loci between fragment pairs found after the establishment of corridors in Mandena suggest that gene flow is augmented as a result of enhanced connectivity. Our data point out that despite low effective population size, M. ganzhorni maintains high individual heterozygosity at neutral loci and at MHC II DRB gene and that selection plays a predominant role in maintaining MHC diversity. These findings highlight the importance of long‐term genetic monitoring in order to disentangle between the processes of drift and selection maintaining adaptive genetic diversity in small populations.

The profiling of pre- and post-warming DNA in mouse embryos with microsatellite method

Aims:This research aimed to identify the deoxyribonucleic acid (DNA) profile and changes of post-warming embryo after being frozen with vitrification method using microsatellite method.Materials and Methods:This research examined the mouse embryo blastocysts that were divided into four groups: Post-warming living blastocyst, post-warming living blastocyst with half fragmented cell, post-warming dead blastocyst, and pre-freezing living blastocyst. The isolation sample applied phenol-chloroform method. After obtaining polymerase chain reaction results, all the samples of pre-freezing fresh embryo, post-warming living embryo, dead embryo, and degenerated embryo were then examined by single-strand conformation polymorphism (SSCP).Results:The amplification with D18mit14 primer was 100 bp and 150bp with D18mit87 primer, 150bp with D7mit22, and 300bp with D7mit25. The result of SSCP with D18mit14 primer showed that the blastocysts were fragmented and dead after warming process and formed into two DNA strand fragments, while the fresh embryos which passed freezing process did not form any fragment. D18mit87 primer SSCP indicated different fragments for each treatment. The result of SSCP using D7mit22 formed two different fragments for each treatment. While using D7mit25, the SSCP result formed some different fragments for each sample. Post-warming living embryo had similar ribbon to pre-freezing fresh embryo.Conclusion:D7mit222, D7mit25, and D18mit87 primers could be used as the aneuploidy marker on mouse embryos that were induced by post-warming process. The profile of living blastocyst, dead blastocyst, and post-warming fragmented blastocyst had different DNA tapes.

Detection of Staphylococcal Species Diversity within Human Skin Microbiome Using a Simple PCR-SSCP Technique


 Objective. The aim of this study was to detect the diversity of Staphylococcus species of a healthy human skin using a simple techniquevPCR-SSCP.
 Methods: Blood samples, saliva, and skin swaps samples were collected from 50 persons from Hilla City - Iraq. The genomic DNA was extracted from these samples using the Bacteria Genomic DNA Kit. The concentrations and purity of DNA extract estimated by NanoDrop spectrophotometer. Polymerase chain reaction – single-strand conformational polymorphism (PCR–SSCP) technique was performed to detect the diversity between Staphylococcal species in the human skin microbiome using a specific primer of the 16SrRNA gene.
 Results: The PCR results, indicated that the Staphylococcal species were found within the ski community, but it's not infected blood and mouth of test healthy individuals. SSCP-heteroduplex patterns of PCR products appeared the presence Staphylococcal species diversity within skin microbiome of test healthy individuals.
 Conclusion: In spite of the PCR-SSCP, heteroduplex method was simple and cheap and appeared the diversity betweenvStaphylococcalspeciesvin the human skin microbiome, but it's not diagnosed the bacterial strains. So these results required to confirm by DNAvsequencingvtechnique.

Novel gross deletion at the GHRHR gene locus possibly mediated by Alu specific microhomology identified in a Sri Lankan patient with isolated growth hormone deficiency

ObjectiveCharacterization of a deletion in the exon 1 and 5′ regulatory region of the GHRHR gene in a proband with isolated growth hormone deficiency. MethodsMultiple ligation dependent probe amplification (MLPA) assay was carried out to confirm the homozygous deletion which was suspected during screening of the GHRHR gene by single strand conformation polymorphism. A series of short range PCR amplifications were carried out to map the approximate location of the break points of the deletion. Sanger sequencing was carried out to locate the break points and to identify the length of the deletion. Long range PCR amplification was carried out to confirm the length of the deletion and to screen the parents of the proband for the deletion. ResultsA homozygous deletion was confirmed via MLPA assay. Zones of sequence similarity between upstream intergenic region and intron 1 of the GHRHR gene were identified. Break points of the deletion were identified within perfectly matching 32 bp repeat sequences ie: microhomologies in the specified zones. The novel deletion may have arisen via Alu specific microhomology mediated non-recurrent rearrangement in the maternal lineage of the proband. The deletion being reported in this study include, last 3118 bp from the upstream intergenic region and complete exon 1 and first 2620 bp from intron 1 and one of the 32 bp microhomologies. The total length of the deleted segment was 5875 bp. As the deleted region contained significant elements essential for gene expression, the identified deletion is being reported as likely pathogenic. The same deletion was identified in the mother in heterozygous state. ConclusionWe have characterized a novel deletion that seems to have arisen via Alu specific microhomology mediated non-recurrent rearrangement at GHRHR gene locus. HGVS nomenclature of the deletion is c.-3166_58-2057del. This novel structural variant was identified to be the cause of IGHD of the affected proband.

Presence of the p.L456V polymorphism in Cuban patients clinically diagnosed with Wilson's disease

Introduction and aimsWilson's disease is characterized by the accumulation of copper in different organs, mainly affecting the liver, brain, and cornea, and is caused by mutations in the ATP7B gene. More than 120 polymorphisms in the ATP7B gene have been reported in the medical literature. The aim of the present study was to identify the conformational changes in the exon 3 region of the ATP7B gene and detect the p.L456V polymorphism in Cuban patients clinically diagnosed with Wilson's disease. Material and methodsA descriptive study was conducted at the Centro Nacional de Genética Médica and the Instituto Nacional de Gastroenterología within the time frame of 2007-2012 and included 105 patients with a clinical diagnosis of Wilson's disease. DNA extraction was performed through the salting-out method and the fragment of interest was amplified using the polymerase chain reaction technique. The conformational shift changes in the exon 3 region and the presence of the p.L456V polymorphism were identified through the Single-Strand Conformation Polymorphism analysis. ResultsThe so-called b and c conformational shift changes, corresponding to the p.L456V polymorphism in the heterozygous and homozygous states, respectively, were identified. The allelic frequency of the p.L456V polymorphism in the 105 Cuban patients that had a clinical diagnosis of Wilson's disease was 41% and liver-related symptoms were the most frequent in the patients with that polymorphism. ConclusionThe p.L456V polymorphism was identified in 64 Cuban patients clinically diagnosed with Wilson's disease, making future molecular study through indirect methods possible.

Sirtuin3 (SIRT3) gene molecular characterization and SNP detection in prolific and low prolific goat breeds

Sirtuin3 (SIRT3) is a member of the Sirtuin family of NAD+-dependent deacetylase. They have evolved as a vital protein in preventing the activation of reactive oxygen species (ROS) in oocytes. A novel study on caprine SIRT3 was conducted, to characterize caprine SIRT3, to detect potential polymorphisms in SIRT3 and to analyze their association with litter size in the two indigenous goat breeds of India viz., the prolific Malabari and low prolific Attappady Black goats. A 1070 bp mRNA sequence of SIRT3 cDNA comprised of an ORF of 1002 bp encoding 333 amino acids, having 96% identity with bovine SIRT3. The genomic DNAs from the goats (n = 222) were subjected to PCR and single strand conformation polymorphism (SSCP) of exon 5 fragment (213 bp) of caprine SIRT3. On analysis, two genotypes viz., DD and DE were observed with frequencies of 0.63 and 0.37 respectively. Further sequencing of the PCR products of the respective genotypes revealed a novel synonymous SNP (MF176159:c.691C > T). Genotypes of this fragment had a significant influence on number of kids born (P < 0.05) with DD genotype being superior to DE genotype. These results highlight the role of SIRT3 in reproduction traits and the detected novel SNP would aid in the Marker Assisted Selection programmes and thus SIRT3 can considered as a potential candidate gene for reproduction traits in goats.

Application of MootralTM Reduces Methane Production by Altering the Archaea Community in the Rumen Simulation Technique.

The reduction of methane emissions by ruminants is a highly desirable goal to mitigate greenhouse gas emissions. Various feed additives have already been tested for their ability to decrease methane production; however, practical use is often limited due to negative effects on rumen fermentation or high costs. Organosulphur compounds from garlic (Allium sativum) and flavonoids have been identified as promising plant-derived compounds which are able to reduce methane production. Here, we evaluated the effects of a combination of garlic powder and bitter orange (Citrus aurantium) extracts, Mootral, on ruminal methane production, ruminal fermentation and the community of methanogenic Archaea by using the rumen simulation technique as ex vivo model. The experiment consisted of an equilibration period of 7 days, an experimental period of 8 days and a withdrawal period of 4 days. During the experimental period three fermenters each were either treated as controls (CON), received a low dose of Mootral (LD), a high dose of Mootral (HD), or monensin (MON) as positive control. Application of Mootral strongly reduced the proportion of methane in the fermentation gas and the production rate of methane. Moreover, the experimental mixture induced a dose-dependent increase in the production rate of short chain fatty acids and in the molar proportion of butyrate. Some effects persisted during the withdrawal period. Both, single strand conformation polymorphism and Illumina MiSeq 16S rRNA amplicon sequencing indicated an archaeal community distinct from CON and MON samples in the LD and HD samples. Among archaeal families the percentage of Methanobacteriaceae was reduced during application of both doses of Mootral. Moreover, several significant differences were observed on OTU level among treatment groups and after withdrawal of the additives for LD and HD group. At day 14, 4 OTUs were positively correlated with methane production. In conclusion this mixture of garlic and citrus compounds appears to effectively reduce methane production by alteration of the archaeal community without exhibiting negative side effects on rumen fermentation.

Detection of Somatic Mutation in Exon 12 of DNA Polymerase β in Ovarian Cancer Tissue Samples

BackgroundDNA polymerase β (pol β) is a key enzyme of base excision repair pathway. It is a 1-kb gene consisting of 14 exons. Its catalytic part lies between exon 8 and exon 14. Exon 12 has a role in deoxyribonucleotide triphosphate selection for nucleotide transferase activity.MethodsGenomic DNA was isolated from ovarian carcinoma samples. Single strand conformation polymorphism method was used to detect mutation in genomic DNA.Results:Twenty-four patients of the 152 pair of tumor samples (15.8%) exhibited a point mutation (C→G) in position 725 in exon 12, which shifts proline to arginine (P242R). Statistical analysis showed a significant (p < 0.001) relationship between pol β mutation and the age of detection.ConclusionThis is a newly reported somatic mutation of pol β in ovarian carcinoma patients from India.

"Mirror" Method to Estimate Mutagenic Activity of DNA Lesions.

We propose an improved method for detecting mutations that arise in DNA due to misincorporations of deoxyadenosine-5′-monophosphate (dAMP) opposite 7,8-dihydro-8-oxoguanine (8-oxoG). The method is based on the synthesis of complementary chains (“mirror” products) using a template containing 8-oxoG. The misincorporation of dAMP in the “mirror” product forms EcoRI sites. The restriction analysis of double-stranded DNAs obtained by PCR of “mirror” product allows quantification of the mutagenesis frequency. In addition, single-strand conformational polymorphism (SSCP) analysis of the single-stranded “mirror” products shows that different DNA polymerases only incorporate dA or dC opposite 8-oxoG. The proposed approach used in developing this technique can be applied in the study of other lesions as well, both single and clustered.

Identification of single nucleotide variations in the Toll-like receptor 9 (TLR9) gene and its association to mastitis susceptibility in dairy cattle

ABSTRACTToll-like receptors (TLRs) play a significant role in the host immune response. The TLR9 recognizes CpG DNA of bacteria and stimulate both the innate and adaptive immune responses. In the present study, functional domain (LRR) of TLR9 gene of mastitis-affected cows in comparison to apparently healthy animals was analyzed for any nucleotide variation as it may influence host susceptibility to disease conditions. LRR domain of TLR9 was amplified and analyzed for single-strand conformational polymorphism (SSCP). Samples shown altered SSCP mobility pattern were sequenced. Three nucleotide variations each in the two regions of functional domain were identified and the one corresponding to G→C variation at position 2318 of reference sequence (Accession No. AJ509824), suggesting the possible association with mastitis. Though the nucleotide variation did not result in amino acid change, it created an additional restriction site for Hae III which can be utilized in developing a new polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) marker.

Characterization of transforming growth factor beta regulator 1-like and association between its expression levels and growth of the giant tiger shrimp Penaeus monodon

Molecular markers that allow selection of juveniles and broodstock with improved growth performances are useful for the shrimp industry. Here, the full-length cDNA of transforming growth factor beta regulator 1-like (PmTbrg1-l) in the giant tiger shrimp Penaeus monodon was determined. It was 1184 bp in length and contained an open reading frame (ORF) of 975 bp corresponding to a deduced polypeptide of 324 amino acids. Successful RNA interference (RNAi) carried out using juveniles injected with PmTbrg1-l dsRNA revealed reduced levels of PmTbrg1-l and myostatin (PmMstm) in hemocytes when compared to shrimp injected with saline solution and GFP dsRNA (P < .05). Associations between single-strand conformational polymorphism (SSCP) patterns or single nucleotide polymorphism (SNP) patterns and growth-related parameters (average body weight and total length) were examined. Juveniles with pattern III (corresponding to A/A918; N = 37) showed a trend for greater average body weight and total length than those with patterns II (G/G918; N = 42) and IV (A/G918; N = 75). The expression level of PmTbrg1-l in the hepatopancreas of females was significantly higher than that in males (P < .05) in two sample sets of three-month-old domesticated juveniles (N = 59 and 50; P < .05). Moreover, its expression level in large-size juveniles was significantly higher than that in medium-size and small-size juveniles in both groups of samples (P < .05). Results indicated that PmTbrg1-l is functionally related with growth of P. monodon.

Non-host larvae negatively impact persistence of the entomopathogen Beauveria bassiana in soil.

A better understanding of the ecology of the insect pathogenic fungus, Beauveria bassiana, in soil is needed to identify reasons behind the variable efficacy often seen after field application. A transformed strain of a candidate commercial strain of B. bassiana (F418 gfp tr3), expressing the green fluorescent protein and the hygromycin B resistance gene, was used to assess the effects of the larvae of a host insect, Tenebrio molitor L. (Coleoptera: Tenebrionidae), a non-host, Costelytra zealandica (Coleoptera: Scarabaeidae) and the absence of larvae on the persistence of F418 gfp tr3 in pasteurised and non-sterile soil over 4 months. In the presence of a T. molitor larvae, F418 gfp tr3 populations increased significantly in pasteurised and non-sterile soil; however, populations increased less in non-sterile soil than in pasteurised soil. Lower populations of F418 gfp tr3 were recovered in pasteurised soil in the presence of C. zealandica larvae than in pasteurised soil without larvae. No difference was observed between F418 gfp tr3 populations in non-sterile soil with a non-host larvae or without larvae. Accompanying studies showed that F418 gfp tr3 conidia germinated and produced appressoria on live and excised cuticle of non-host (C. zealandica) larvae but infection did not occur, leading to a net loss of viable conidia in the soil. Conidia administrated orally to C. zealandica larvae were viable on recovery from faecal samples, suggesting that ingestion of the fungus by the larvae had little impact on the viable fungal population. Soil bacterial and fungal community patterns were analysed using Single-Strand Conformation Polymorphism (SSCP) and showed a correlation between changes in F418 gfp tr3 persistence in pasteurised and non-sterile soil and changes in soil communities in the presence of a host insect, non-host insect or in the absence of insect. In pasteurised soil, non-specific germination of F418 gfp tr3 conidia on the non-host larval cuticle and the presence of antagonistic bacteria introduced with the field-collected larvae are most likely responsible for the differences observed. The more complex microbial community structures in non-sterile soil could lead to fungistasis, preventing potentially antagonistic bacteria degrading conidia or inhibiting attachment and germination on the non-host larval cuticle, resulting in the observed lack of difference between non-host and no larval treatments.

Calcite-forming Bacillus licheniformis Thriving on Underwater Speleothems of a Hydrothermal Cave

The underwater environment of Grotta Giusti (Monsummano Terme, Italy) is a suggestive setting with different types of speleothems including “leafy” and “cauliflower” concretions along the walls and roof, and conical pseudo-stalagmites on the floor. Very high calcium and dissolved CO2 levels, and massive calcium carbonate precipitation characterize this cave environment. Yet, life thrives on the leafy concretion surfaces with loads of cultivable heterotrophic microorganisms around 105 colony-forming units per cm2. Bacillus licheniformis appeared to be the prevalent cultivable microorganism on a low-nutrient medium that was used for screening. 16S rRNA gene-based polymerase chain reaction–single strand conformation polymorphism profiling indicated that Group VI Bacillaceae species was well represented in the bacterial community of underwater speleothems. Interpretation of X-ray diffraction spectra and Raman spectroscopy data indicated that the B. licheniformis isolate produced in vitro abundant calcite microcrystals that were also characterized by scanning electron microscopy coupled with energy dispersive X-ray spectroscopy. Production of calcite microcrystals was analyzed in different media (Christensen’s urea agar and B4 calcium carbonate precipitation medium) and incubation conditions, and it was found to be enhanced by nitrate supplement in B4 medium under low-oxygen conditions. B4 and B4-nitrate media also stimulated antibiotic production by the B. licheniformis isolate, which was analyzed by microbiological assays.

A nucleotide substitution in the ovine KAP20-2 gene leads to a premature stop codon that affects wool fibre curvature.

The keratin-associated proteins (KAPs) form a matrix that cross-links the main component of the wool fibre, the keratin intermediate filaments. They are therefore believed to play an important role in defining the physical and mechanical properties of the fibre. Although over 80 functional KAP genes (KRTAPs) have been identified in humans, only 29 have been found in sheep,1-3 All of these are polymorphic, and variation in some of these ovine KRTAPs has been associated with wool traits.1-6 Variation in the gene encoding the high glycine-tyrosine KAP20-2 protein (KRTAP20-2) has been reported to affect cashmere fibre weight traits.7 A blast search in the Ovine Genome Assembly Oar_v4.0 with the caprine KRTAP20-2 coding sequence (MF973462) identified on chromosome 1 an orthologous open reading frame (NC_019458.2:12334029_123134217), which has seven nucleotide substitutions relative to the caprine sequence. The sequence was located in a region that contains 13 other known KRTAPs (Fig. S1). A comparison of the sheep KRTAP20-2 with the human and caprine homologues and with other ovine HGT-KAP genes is shown in Fig. S2. Analysis of a 273-bp PCR fragment containing this open reading frame by single-stranded conformational polymorphism (SSCP) revealed two nucleotide sequence variants (Fig. S3), corresponding to a single nucleotide polymorphism (SNP) c.160A>T. This is the only difference between these sequences and the sheep v4.0 sequence, with the A nucleotide being found in that genome assembly sequence. Both sequences have been submitted to GenBank with accession nos. MH071391 and MH071392, for the A- and T-containing sequences respectively. This introduces a premature stop codon that removes the nine C-terminal amino acid residues. In spite of a high frequency of SNPs and insertion/deletions in KRTAPs,1 a nonsense mutation has not been reported to date. The PCR-SSCP analysis of 250 Merino × Southdown-cross sheep (from three sire lines) for which wool trait data were available, revealed that the A and T variants occur at a frequency of 71.8% and 28.2% respectively. Analyses using general linear models (GLMs) in minitab version 16 indicate that the wool fibre from homozygous AA sheep have a higher mean fibre curvature (MFC) than does the wool fibre from sheep carrying the TT genotype (Table 1). Theoretically, this effect may also reflect linkage between KRTAP20-2 and other genes in the same chromosome region. However, the effect observed for KRTAP20-2 differs from the associations observed for three nearby KRTAPs (KRTAP6-1, KRTAP22-1 and KRTAP6-3), which are at a distance of approximately 73 kb, 80 kb and 87 kb respectively from KRATP20-2 (Fig. S1). Specifically, previous research on Merino-cross sheep has revealed that KRTAP22-1 appears to affect wool yield,3 whereas KRTAP6-1 and KRTAP6-3 appear to affect mean fibre diameter (MFD) associated traits, but none of these genes were associated with the curvature.4, 8 Our data also suggest an association between variation in KRTAP20-2 and MFD (P = 0.091), but this trait in these Merino-cross sheep correlated with MFC (r = 0.490; P < 0.001) (Table S1). This study was financially supported by Ningxia Agricultural Breeding Program and the Lincoln University Gene-Marker Laboratory. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.

Short lifetime structures appearing in RNA and DNA.

The structure and function of unstable single-stranded DNA (ssDNA) have not been widely examined. While numerous studies have investigated DNA as an information molecule, the different potentials of DNA, particularly those of ssDNA, remain unclear. For polypeptides, the significance of denatured structures has been established in the past two decades. Polynucleotides have chemically distinct properties from polypeptides, but their behaviours have not been thoroughly studied. In this review, three different phenomena related to unstable ssDNA are discussed: i) ssDNA cleavage of restriction enzymes; ii) single-stranded conformation polymorphism, which can be theoretically explained by single-stranded conformation dynamics; and iii) random PCR (Polymerase Chain Reaction). These features can be utilized for scientific or technical applications. Previous studies showed that the phenomena exhibited by ssDNA were correctly understood only when unstable and transient structures were taken into account. Transient structures of ssDNA may have undiscovered functions governed by very rapid processes and/or multi-diversity states because of their intrinsic natures.

Effect of CNT/PDMS Nanocomposites on the Dynamics of Pioneer Bacterial Communities in the Natural Biofilms of Seawater.

In this study, the antifouling (AF) performance of different carbon nanotubes (CNTs)-modified polydimethylsiloxane (PDMS) nanocomposites (PCs) was examined directly in the natural seawater, and further analyzed using the Multidimensional Scale Analyses (MDS) method. The early-adherent bacterial communities in the natural biofilms adhering to different PC surfaces were investigated using the single-stranded conformation polymorphism (SSCP) technique. The PCs demonstrated differences and reinforced AF properties in the field, and they were prone to clustering according to the discrepancies within different CNT fillers. Furthermore, most PC surfaces only demonstrated weak modulating effects on the biological colonization and successional process of the early bacterial communities in natural biofilms, indicating that the presence of the early colonized prokaryotic microbes would be one of the primary causes of colonization and deterioration of the PCs. C6 coating seems to be promising for marine AF applications, since it has a strong perturbation effect on pioneer prokaryotic colonization.

Prion protein testis specific (PRNT) gene polymorphisms and transcript level in ovine spermatozoa: Implications in freezability, fertilization and embryo production

An essential role of prion protein testis specific (PRNT) and prion protein 2 dublet (PRND) genes in the male reproductive function has been highlighted, although a deeper knowledge for the mechanisms involved is still lacking. Our goal was to determine the importance of the PRNT haplotypic variants and mRNA expression levels in ovine spermatozoa freezability and ability for fertilization and embryo developmental processes. Their association with the PRND gene polymorphisms was also analyzed. DNA from rams belonging to three Portuguese sheep breeds (n = 28) was screened by single-strand conformation polymorphism (SSCP) analysis to identify the PRNT and PRND polymorphisms. Semen collected from these rams was cryopreserved and fertility traits evaluated. The SSCP analyses revealed polymorphisms in the codons 6, 38, 43 and 48 of the PRNT coding region - respectively c.17C > T (p.Ser6Phe, which disrupts a consensus arginine-X-X serine/threonine motif); c.112G > C (p.Gly38 > Arg); and synonymous c.129T > C and c.144A > G. The polymorphisms in codons 6, 38 and 48 occur simultaneously while the one in codon 43 occurs independently. Six haplotypes were identified in the PRNT coding region, resulting in three different amino acid polymorphic variants (6S-38G-43C-48V, S6F-G38R-43C-48V and 6F-38R-43C-48V). The PRNT gene mRNA transcript level in spermatozoa was related to the identified haplotypic variants, either considering the codons 6-38-48 (P ≤ 0.0001) or the codon 43 alone (P ≤ 0.0001) or altogether (P ≤ 0.0001). An interaction between PRNT haplotypes and PRND genotypes on PRNT transcript level was also identified (P = 0.0003). Rams carrying the 17C-112G-144A PRNT haplotype had sperm with the highest post-thawed individual motility (P ≤ 0.03). Combined PRNT and PRND polymorphic variation influenced the post-thawed individual motility (P = 0.01). The male PRNT haplotypic, either considering the codons 6-38-48 and 43 altogether or the codon 43 alone, interfered (P ≤ 0.04) in embryo production rates. In conclusion, our data confirm that the PRNT gene is highly polymorphic in sheep and that the PRNT and PRND genotypes are associated. The identified polymorphisms of PRNT coding region seems to interfere on the ram spermatozoa mRNA transcript level and on male fertility, specifically in sperm freezability and ability for embryo development.

Mutation screening of the BRCA1 gene in sporadic breast cancer in the Central of Iran

The purpose of the work was to study mutation screening of BRCA1 in Iranian patients with sporadic breast cancer. We carried out a mutational analysis of BRCA1 gene in 101 breast cancer patients from a population in Central of Iran. The comparison of DNA of paraffin-embedded breast cancer tissue from patients was studied, and breast tissue from 30 unrelated normal women without cancer was selected as controls. The entire BRCA1 coding sequence was amplified by PCR with primers especially designed for comprehensive mutation screening by single-strand conformation polymorphism (SSCP) analysis. The PCR products revealing abnormal SSCP migration pattern were sequenced. Then, in silico investigation was performed to identify the effects of new mutations on stability and function of BRCA 1.A total of ten nucleotide alterations were observed in the breast cancer tissue DNA. Six cases of single nucleotide changes in BRCA1 were detected in the study without records in the BIC database consisted four polymorphism in exon 11 (1543 Del G, 1597C>T, 2246 Del T, 2612C>G), one missense mutation in exon 7 (490C>T), and one deletion mutation in exon 10 (743 Del C). No nucleotide alterations were detected in the controls. In addition, the results of in silico analysis indicated that three mutations including 2612C>G, 1543 Del G and 743 Del C were not recorded in the especial database. Furthermore, the results of molecular docking studies confirmed that 2612C>G could change physicochemical properties of BRCA1.The findings of the present study suggest that screened mutations may have an important role on the incidence of breast cancer in women.

MicroRNA-326 and microRNA-200c: Two novel biomarkers for diagnosis and prognosis of pediatric acute lymphoblastic leukemia.

Multidrug resistance (MDR) is considered as the major obstacle for treating pediatric acute lymphoblastic leukemia (ALL). MicroRNAs (miRNAs) are small non coding RNAs which may potentially regulate response to chemotherapy. In this study, total RNA was isolated from bone marrow samples of 46 children with de novo ALL and 16 controls. Quantitative reverse transcriptase polymerase chain reaction was used to investigate the expression profile of the predicted miRNAs; miR-326 and miR-200c, and their predicted targets ABCA2, and ABCA3 transporters. The presence of minimal residual disease was studied using PCR-SSCP (single-strand conformation polymorphism) 1 year after treatment. The association between the miRNA expression and drug resistance was analyzed statistically. Results showed a significant down-regulation of both miR-326 and miR-200c expressions in ALL patients compared with non-cancer controls (P = 0.0002, AUC = 0.813 and P = 0.035, AUC = 0.79, respectively). A considerable negative association between miR-326 expression and MDR was identified which could raise the risk of chemoresistance by 4.8- fold. The expression profiles of miR-326 and ABCA2 transporter were inversely correlated. Data revealed, a novel diagnostic role for miR-326 and miR-200c as potential biomarkers of pediatric ALL. Down-regulation of miR-326 was introduced, for the first time, as a prognostic factor for drug resistance in childhood ALL. To the best of our knowledge, this is the first time that ABCA2 transporter is proposed as a target gene for miR-326, through which it can exert its impact on drug resistance. These data may provide novel approaches to new therapeutics and diagnostics.