Restriction Fragment Polymorphism Research Articles

Genetic Variation of Calpastation Gene of Indigenous Bali Cattle (Bos javanicus) in Indonesia

Calpastatin is one of gene markers affecting meat tenderness. The study aimed to evaluate genetic variation of calpastatin (CAST) gene of Bali cattle (Bos javanicus) in lndonesia. A total of 61 samples consisting of 21 Bali cattle, 22 Ongole cattle (Bos indicus), and 18 Friesian Holstein (FH) cattle (Bos taurus) were applied. The Ongole and FH cattle were involved for breed comparison. DNA was extracted from fresh blood using a High Salt method and measured their quality by a Spectrophotometer. A 523 bp of Calpastatin gene fragment was amplified by Polymerase Chain Reaction and Restriction Fragment Polymorphism (PCR-RFLP) technique with RsaI restriction enzyme for genotyping. Result showed that two variants alleles (C and G) and three genotypes (CC, GC, GG) were found in those Bali, Ongole and FH samples. Allele G was dominant allele with the highest G allele was in Bali cattle population (0.88). The higher percentage of allele C was found in Ongole and Friesian Holstein compared to that in Bali cattle. The Ongole breed tends to have a potential source of lean meat quality. This finding identified that genetic variation of CAST gene was exist in Bali cattle and adapted cattle of Ongole and FH in Indonesian.

Effects of Rhizosphere and Long-Term Fertilization Practices on the Activity and Community Structure of Denitrifiers Under Double-Cropping Rice Field

ABSTRACTThe soil physicochemical properties, soil denitrification rates (PDR), denitrifiers via nitrite reductases (nirK and nirS) and nitrous oxide reductase (nosZ), abundance and community composition of denitrifiers in both the rhizosphere and bulk soil from a long-term (32 year) fertilizer field experiment conducted during late rice season were investigated by using the MiSeq sequencing, quantitative PCR, terminal restriction fragment polymorphism (T-RFLP). The experiment including four treatments: without fertilizer input (CK), chemical fertilizer alone (MF), rice straw residue and chemical fertilizer (RF), and organic manure and chemical fertilizer (OM). The results showed that the application of rice straw residue and organic manure increased soil organic carbon (C), total nitrogen (N), and NH4+-N contents. The nirK, nirS, and nosZ copy numbers with OM and RF treatments were significant higher than that of the MF and CK treatments in the rhizosphere and bulk soil (p < 0.05). The principal coordinate analysis (PCoA) analysis showed that the different parts of root zone are the most important factors for the variation of denitrifying bacteria community, and the different fertilization treatments is the second important factors for the variation of denitrifying bacteria community. The MiSeq sequencing result showed that nirK, nirS and nosZ-type denitrifiers communities within bulk soil had lower species diversity compared with rhizosphere soil, and were dominated by Rhizobiales, Rhodobacterales, Burkholderiales, and Pseudomonadales. As a result, the application of fertilization practices had significant effects on soil N and PDR levels, and affected the abundance and community composition of N-functional microbes.

Prevalence and genotyping of Helicobacter pylori in endoscopic biopsy samples from a Chinese population

AbstractBackground:Helicobacter pyloriinhabit the gastric mucosa of humans and are associated with several gastrointestinal diseases which include gastric cancer, peptic ulcer, chronic gastritis and gastric mucosa-associated lymphoid tissue lymphoma.Helicobacter pyloriexhibit a high degree of genetic variability and are associated with its epidemiological, pathological characteristics and dynamics of transmission. The objective of the study was to determine the prevalence and genetic heterogeneity ofH. pyloriisolated from endoscopic biopsy samples from a Chinese population.Methods:Gastric biopsy samples from 86 patients (males, 55; females, 35) who presented to the endoscopic section for various gastrointestinal abnormalities were collected. The samples were subjected to a real-time polymerase chain reaction (PCR) and microbial culture for the isolation ofH. pylori. Further, the isolates were subjected to randomly amplified polymorphic DNA (RAPD) and restriction fragment length polymorphism (RFLP) analysis.Results:Of the 86 gastric biopsy samples, 61 (70.9%) samples were positive for rapid urease test and 37 (43%) samples (28 from male and nine from female) grewH. pylori. Among the biopsy samples subjected to real-time PCR, 39 (45.3%) samples were found to be positive forH. pylori.The RAPD analysis yielded 15 different patterns (four to 17 different sized fragments per strain). The phylogenetic analysis of RAPD yielded 22 clusters at a similarity level ranging from 63% to 100%. RFLP analysis yielded nine different patterns (two to six different sized fragments per strain). Two major restriction patterns were identified, of which 14 (37.8%) strains forms the most common pattern (genotype I) followed by five (13.5%, genotype II) strains with an intra-strain similarity of 100%.Conclusions:The overall prevalence ofH. pyloriwas 45.3%. Despite reports on the declining trend in the prevalence ofH. pyloriinfections, our prevalence rate was still higher than those reported from other developed countries. However, further studies involving a large sample size and covering more regions of China is highly warranted.

Adenomyosis: genetics of estrogen metabolism.

Background To analyze the allelic variants of genes of enzymes involved in estrogen metabolism: CYP1A1, CYP1A2, CYP19 and SULT1A1 using polymerase chain reaction-restriction fragment length polymorphism-restriction fragment length polymorphism (PCR-RFLP) analysis of women with histologically confirmed adenomyosis and women without proliferative diseases of pelvic organs was performed. We studied the following polymorphisms: CYP1A1 M1, T264 → C transition in the 3'-noncoding region; CYP1A2*1F, C734 → A transversion in CYP1A2 gene; C → T transition (Arg264Cys) in exon 7 of CYP19; SULT1A1*2, G638 → A transition (Arg213His) in the SULT1A1 gene. Materials and methods The study included 804 patients. Group I (experimental group) consisted of 268 women with adenomyosis. Inclusion criteria were: histological verification of adenomyosis, consent of patients to participate in the study. Group II (control group) - 536 women without proliferative diseases of the uterus. Inclusion criteria were: lack of proliferative processes of the uterus histologically confirmed by ultrasound examination, patient's consent to participate in the study. Results We found the significant association of C allele, T/C and C/C genotypes of the CYP1A1 gene (CYP1A1 M1 polymorphism), A allele, C/A and A/A genotypes of the CYP1A2 gene (CYP1A2*1F polymorphism) and the T allele, C/T and C/C genotypes of the CYP19 (Arg264Cys polymorphism) gene with the risk for adenomyosis. Conclusions Patients with adenomyosis had increased frequency of C allele, T/C and C/C genotypes of the CYP1A1 gene, A allele, C/A and A/A genotypes of the CYP1A2 gene and T allele and C/T and C/C genotypes of the CYP19 gene and, on the contrary, decreased frequency of the mutant allele and heterozygous and mutant homozygous genotype of the CYP1A2 gene compared to women without proliferative diseases of the uterus.

Inducible nitric oxide synthase 2 promoter polymorphism and malaria disease severity in children in Southern Ghana.

ObjectiveWe assessed the association of mutant allele frequencies of nitric oxide synthase 2 (NOS2) gene at two SNPs (-954 and -1173) with malaria disease severity in children from a malaria endemic area in Southern Ghana.MethodUsing children recruited at the hospital, assigned into clinical subgroups of uncomplicated and severe malaria and matching with their “healthy control” counterparts, we designed a case control study. Genomic DNA was extracted and genotyping using Restriction Fragment Polymorphism was done.ResultA total of 123 malaria cases (91 uncomplicated, 32 severe) and 100 controls were sampled. Their corresponding mean Hbs were 9.6, 9.3 and 11.2g/dl and geometric mean parasite densities of 32097, 193252 and 0 parasites/ml respectively. Variant allele frequencies varied from 0.09 through 0.03 to 0.12 for G-954C and 0.06 through 0.03 to 0.07 for C-1173T in the uncomplicated, severe and healthy control groups respectively. There was a strong linkage disequilibrium between the two alleles (p<0.001). For the -954 position, the odds of developing severe malaria was found to be 2.5 times lower with the carriage of a C allele compared to those without severe malaria (χ2; p< 0.05) though this isn’t the case with -1173.ConclusionThe carriage of a mutant allele in the -954 NOS2 gene may have a protective effect on malaria among Southern Ghanaian children.

The association between vitamin D receptor gene polymorphisms and hepato-cellular carcinoma in Egyptian patients with chronic liver disease

AimTo evaluate the possible association between vitamin D receptor single nucleotide polymorphisms and hepato cellular carcinoma in Egyptian patients with chronic liver disease. MethodSpecific primer sequences of the vitamin D receptor gene rs1544410, rs7975232, and rs731236 were used to detect vitamin D receptor polymorphisms in blood sample by polymerase chain reaction restriction fragment polymorphism. Allele specific restriction enzymes BsmI, ApaI and TaqI were used to digested PCR amplified products and the bands of specific PCR-RFLP fragments were resolved by 2% Ethidium bromide agrose gel electrophoresis. Genotypes and alleles were identified by their specific banding pattern on the gel electrophoresis via ultraviolet illumination. ResultThe frequencies of the 3 single nucleotide polymorphisms BsmI, ApaI, and TaqI genotype and the haplotype distribution were similar in patients with chronic liver disease either with or without hepato-cellular carcinoma. Neither of vitamin D receptor gene polymorphisms were a risk or protective factor for HCC; nor did these polymorphisms alter the risk for HCC development by coexisting HCC with chronic hepatitis C virus infection. TaqI polymorphism was associated with increased risk of progression of chronic liver disease to liver cell failure and decompensated liver disease. Conclusionvitamin D receptor single nucleotide polymorphisms at BsmI, ApaI, and TaqI did not provide significant association with HCC cancer risk or protection in the overall analysis in Egyptian population with chronic liver disease.

Analysis of the exome aggregation consortium (ExAC) database suggests that the BAP1-tumor predisposition syndrome is underreported in cancer patients.

The BAP1-tumor predisposition syndrome (BAP1-TPDS) has been recently identified to predispose patients to a variety of cancers and preneoplastic lesions. About 130 unrelated probands have been identified worldwide; however, the impact of the syndrome is suspected to be much larger given the diversity of the cancer phenotype. To evaluate the frequency of germline BAP1 mutations in the general and cancer populations, we analyzed the Exome Aggregation Consortium (ExAC), a database that contains 53105 exomes of unrelated individuals unaffected by cancer (general population) and exomes of 7601 unrelated individuals affected by cancer provided by the Cancer Genome Atlas (TCGA, cancer subjects). BAP1 null variants were seen at much higher frequency in the cancer subjects (0.0526%) compared to the general population (0.00188%) with a relative risk of 27.93 and (P = 0.0011, [95% CI: 3.122-249.883], Fisher's exact test). We also studied a reported BAP1 null variant, c.1203T > G, p.T401* (rs200156887), observed commonly in the general population. Sequencing and restriction fragment polymorphism of the RT-4 cell line that contains this variant revealed that it is in fact a 3bp deletion/insertion, c.1201_1203delinsGAG, a likely benign missense alteration p.Y401E explaining the relative high frequency of this variant in the general population. In conclusion, germline null mutations in BAP1 have a significantly higher frequency in cancer patients than the general population. Given the low frequency of reported families with BAP1-TPDS, our results suggest that the syndrome is underreported especially in patients with cancer.

Prevalence and genotyping ofHelicobacter pyloriin endoscopic biopsy samples from a Chinese population

AbstractBackground:Helicobacter pyloriinhabit the gastric mucosa of humans and are associated with several gastrointestinal diseases which include gastric cancer, peptic ulcer, chronic gastritis and gastric mucosa-associated lymphoid tissue lymphoma.Helicobacter pyloriexhibit a high degree of genetic variability and are associated with its epidemiological, pathological characteristics and dynamics of transmission. The objective of the study was to determine the prevalence and genetic heterogeneity ofH. pyloriisolated from endoscopic biopsy samples from a Chinese population.Methods:Gastric biopsy samples from 86 patients (males, 55; females, 35) who presented to the endoscopic section for various gastrointestinal abnormalities were collected. The samples were subjected to a real-time polymerase chain reaction (PCR) and microbial culture for the isolation ofH. pylori. Further, the isolates were subjected to randomly amplified polymorphic DNA (RAPD) and restriction fragment length polymorphism (RFLP) analysis.Results:Of the 86 gastric biopsy samples, 61 (70.9%) samples were positive for rapid urease test and 37 (43%) samples (28 from male and nine from female) grewH. pylori. Among the biopsy samples subjected to real-time PCR, 39 (45.3%) samples were found to be positive forH. pylori.The RAPD analysis yielded 15 different patterns (four to 17 different sized fragments per strain). The phylogenetic analysis of RAPD yielded 22 clusters at a similarity level ranging from 63% to 100%. RFLP analysis yielded nine different patterns (two to six different sized fragments per strain). Two major restriction patterns were identified, of which 14 (37.8%) strains forms the most common pattern (genotype I) followed by five (13.5%, genotype II) strains with an intra-strain similarity of 100%.Conclusions:The overall prevalence ofH. pyloriwas 45.3%. Despite reports on the declining trend in the prevalence ofH. pyloriinfections, our prevalence rate was still higher than those reported from other developed countries. However, further studies involving a large sample size and covering more regions of China is highly warranted.

Evolutionary Computation-Based Memetic Algorithm Against Genetic Algorithm to Improve PCR-RFLP Assay Primers of SNP Genotyping

A genetic algorithm (GA) combines the restriction enzyme mining core of single nucleotide polymorphism (SNP) restriction fragment length polymorphism (RFLP) to design polymerase chain reaction (PCR)-RFLP primer pairs for SNP-based genotyping with feasible estimated GA parameters. However, this GA method is easily trapped into local optima. An improved design of PCR-RFLP assay primers for SNP genotyping is needed. A memetic algorithm (MA) was used to design more robust primers for the PCR-RFLP assay to enable SNP genotyping. The novel restriction enzymes hunting (REHUNT) package was embedded into the MA method to provide available restriction enzymes. A formula to calculate more accurate thermodynamic primer melting temperatures was also introduced. Using the criteria of the GA method, in silico simulations for the MA method under different parameter settings were performed with the SNPs of SLC6A4, and results were compared. Appropriate MA parameter settings were superior in providing robust PCR-RFLP primers to achieve SNP genotyping compared with the GA method. Improvements included an accurate thermodynamic SantaLucia’s formula for the calculation of melting temperature, use of the novel REHUNT for restriction enzymes mining, and selection of primers that better conformed to the primer constraints. The appropriate parameter settings for the proposed MA method were identified and carefully evaluated to design robust PCR-RFLP primers for SNP genotyping. Compared with the former GA method, the MA method is more feasible for PCR-RFLP SNP genotyping.

Porcine reproductive and respiratory disease virus: Evolution and recombination yields distinct ORF5 RFLP 1-7-4 viruses with individual pathogenicity

Recent cases of porcine reproductive and respiratory syndrome virus (PRRSV) infection in United States swine-herds have been associated with high mortality in piglets and severe morbidity in sows. Analysis of the ORF5 gene from such clinical cases revealed a unique restriction fragment polymorphism (RFLP) of 1-7-4. The genome diversity of seventeen of these viruses (81.4% to 99.8% identical; collected 2013–2015) and the pathogenicity of 4 representative viruses were compared to that of SDSU73, a known moderately virulent strain. Recombination analyses revealed genomic breakpoints in structural and nonstructural regions of the genomes with evidence for recombination events between lineages. Pathogenicity varied between the isolates and the patterns were not consistent. IA/2014/NADC34, IA/2013/ISU-1 and IN/2014/ISU-5 caused more severe disease, and IA/2014/ISU-2 did not cause pyrexia and had little effect on pig growth. ORF5 RFLP genotyping was ineffectual in providing insight into isolate pathogenicity and that other parameters of virulence remain to be identified.

Polimorfismos del gen ob en bovinos de raza holstein en la Comarca Lagunera, México

La Comarca Lagunera es la cuenca lechera más importante de México. En la actualidad se están utilizando diversas técnicas que permiten evaluar genéticamente el animal a una edad temprana, permitiendo seleccionar futuros reproductores con características deseables. Entre los genes relacionados con la producción de leche, se encuentran el gen Ob también llamado gen Leptina el cual actúa sobre el sistema nervioso central y tejidos periféricos jugando un papel muy importante en la modulación regulación del apetito, ganancia de peso vivo, incremento del metabolismo energético y el anabolismo muscular. Este trabajo se realizó para determinar el polimorfismo de longitud del fragmento de restricción ACI I de gen leptina en el exón 2 y correlacionarlo con los parámetros de producción y calidad de leche. Se recolectaron 100 muestra de sangre de vacas en producción del establo “Lácteos Florida” de Francisco I. Madero municipio de Coahuila, México con tres estatus de producción: altas, medias y bajas La extracción de ADN se realizó por el método modificado de Salting-Out. Se realizó PCR del gen leptina originando un fragmento de 272 bp de longitud y se realizó PCR-RFLP con la enzima de restricción ACI I y secuenciación, correlacionando los genotipos TT, CT Y CC con tres estatus de producción de leche: altas, medias, bajas. El análisis estadístico indicó que las vacas portadoras del genotipo homocigoto (TT) tienen un efecto significativo (P&lt;0.01) con respecto a las características de producción y calidad de leche ya que tuvieron un mayor consumo de alimento, ganancia de peso, además de una elevada producción de leche en comparación a los genotipos heterocigoto (CT) y homocigoto (CC). Los resultados obtenidos muestran que la identificación molecular de polimorfismos del gen Ob puede usarse como herramienta de selección genética en bovinos de raza Holstein.

Genetic variation within species and haplotypes of the Wiseana (Lepidoptera: Hepialidae) complex and development of non-sequenced based identification tools to aid field studies.

Porina is the common name used to describe moths and caterpillars of the seven endemic species and three haplotypes of Wiseana in New Zealand. Several species have adapted to eating introduced pasture plants; however, a paucity of defining morphological characteristics has meant that porina are grouped as indistinguishable species within a complex. This study aimed to develop non-sequencing identification methods for porina species. We sequenced 1718 bp of the mitochondrial cytochrome oxidase genes for all Wiseana species and haplotypes using 75 specimens collected from 42 sites throughout New Zealand. Two identification methods were developed; the high-resolution melt method uses 106 bp of the cytochrome oxidase I gene, and the restriction fragment polymorphism method uses a larger 700 bp region from the same gene. Validation was performed on a further 275 specimens. Melt curve profiles varied more with population heterogeneity than digest results. In both methods, W. mimica and W. jocosa were inseparable. W. fuliginea grouped with W. mimica and W. jocosa in the restriction method, but the melt curve profile differed. Until now, ecological studies of porina at the species level have been implausible. Our non-sequencing based methods allow rapid identification of moths and caterpillars to species and haplotype level, paving the way for ecological studies of pasture pest species and the development of species-specific mitigation strategies. © 2017 Society of Chemical Industry.

Effects of different fertilizer application methods on the community of nitrifiers and denitrifiers in a paddy soil

Nitrifiers and denitrifiers are the key drivers of N cycling in paddy soil. Little is known about the effects of different fertilization methods, especially side bar fertilization, on the community of nitrifiers and denitrifiers in paddy soils. Here we assess the relationships between soil physicochemical properties, denitrification and nitrification activities, and the underlying microbial communities in a surface layer of paddy field soil treated with different fertilization methods. Soil was unfertilized (control), treated with conventional chemical fertilizer (CF), CF plus pig manure (MC), or slow-release fertilizer (SR), or by slow-release side bar fertilization (SB). Soil was sampled after one season of early and late rice growth. We determined soil physicochemical properties, potential nitrification rates (PNR), and denitrification enzyme activities (DEA). Ammonia-oxidizing archaeal (AOA) and bacterial (AOB) communities were assessed via their ammonia monooxygenase (amoA) genes, and denitrifiers via nitrite reductases (nirK and nirS) and nitrous oxide reductase (nosZ). Quantitative PCR was used to assess gene abundance, terminal restriction fragment polymorphism (T-RFLP) to investigate fertilization effects on microbial communities, and clone library sequencing and phylogenetic analysis to explore the taxonomic diversity of the nitrifiers and denitrifiers. Fertilization significantly increased the amount of NH4 +-N in the soil of SB and MC treatments, whereas MC lowered the NO3 −-N level. DEA was higher for MC and CF than the other treatments. The PNR in MC-treated soil was significantly lower than that in CF-treated soil. There were no significant differences in AOA and nirS copy numbers; however, nirK and nosZ copy numbers were higher for MC compared with CF. The number of AOA terminal restriction fragments (TRFs) increased significantly with N addition and reached the highest level for SB, whereas the number of AOB TRFs did not change significantly between treatments. Similarly, the number of nirK TRFs increased under fertilization, with the highest number obtained for SR; however, no significant change was observed for nirS and nosZ TRFs across different treatments, except for their relative abundance. All AOA amoA genotypes were in archaeal group 1.1b, whereas 95% AOB were in Nitrosospira cluster 3a. More than 40% of nirS OTUs were affiliated with Herbaspirillum, a key N-cycle player in this paddy soil. The SB and MC treatments had significant effects on soil N, DEA, and PNR levels, and affected the community of N-functional microbes. SB in combination with pig manure would contribute to the improvement of paddy soil fertility.

Effects of Short and Long-Term Hydrological Regimes on the Composition and Function of Denitrifying Bacteria in Experimental Wetlands

We investigated effects of short- and long-term hydrologic fluctuations on denitrification in constructed wetland basins. N2 and N2O emission rates, their ratio and a number of physicochemical variables were compared between pulsed (with annual drawdown-flooding cycles) and static (constantly flooded) wetland basins. The abundance and structure of general and denitrifying bacterial communities were compared based on quantitative PCR and terminal restriction fragment polymorphism analyses of 16S rRNA and nosZ genes, respectively. These variables significantly differed among sampling months, but not between the two long-term hydrologic treatments. The same set of variables were measured and compared among samples from different sites of one pulsed basin. The single basin experiment revealed temporal shifts in measured variables, which coincided with short-term hydrological changes. Bank sites, which had larger changes in abiotic conditions, showed greater variability in denitrifying activities than basin sites. Significant difference in N2 and N2O emission rates, 16S rRNA and nosZ gene abundance and structural composition was also identified between basin and bank sites. Our results suggest that activity and community structure of denitrifying bacteria in wetland soils are insensitive to moderate hydrology change, but can be significantly impacted by large hydrological fluctuation at both short-term and long-term scales.

GENETIC VARIANTS RELATED TO LIPID METABOLISM AS A RISK FACTOR TO LATE-ONSET ALZHEIMER’S DISEASE

Background: Genetic polymorphisms in genes regulating cholesterol metabolism have been suggested to risk factor of developing Alzheimer’s disease (AD). Objective: to analyze the frequency of polymorphisms apolipoprotein E (APOE-HhaI) and adenosine triphosphate binding cassette transporter 1 (ABCA1-StyI) in patients with late-onset AD. Design: case-control study. Participants: We studied 166 subjects (≥65 years old): Study Group (SG)- 88 patients and Control Group (CG)- 88 without dementia. Setting: The polymorphisms were determined using the polymorphism chain reaction and restriction fragment length polymorphism (PCR-RFLP) methods. It was applied Fisher's exact/chi-square tests (P&lt;0.05). Results: Genotypes with APOE*4 prevailed in SG. The genotypic combination between APOE-HhaI and ABCA1-StyI polymorphisms showed a prevalence of heterozygous genotypes of risk for AD. Conclusion: Although genetic variants for ABCA1-StyI alone does not differentiate patients and controls, the G allele in synergy with APOE*4 allele is highlighted in patients suggesting the influence of ABCA1 in the disease.

Tomato second cycle hybrids as a source of genetic variability for fruit quality traits

The objective of this study was to investigate the phenotypic and molecular variability in a F2 generation derived from a SCH (Second Cycle Hybrid) in order to detect QTLs for some fruit traits of tomato. Genome coverage at different levels was achieved by three types of molecular markers (polypeptides, sequence-related amplified polymorphism-SRAP and amplified restriction fragment polymorphism - AFLP). Different degrees of polymorphism were detected by SRAP and AFLP at the DNA structure level and also by polypeptides at the DNA expression level. The first two markers, associated with phenotypic variation, detected QTLs involved in important agronomic traits such as fruit shelf life, soluble solids content, pH, and titratable acidity. New gene blocks originated by recombination during the first cycle of crossing were detected. This study confirmed that the observed phenotypic differences represent a new gene rearrangement and that these new gene blocks are responsible for the presence of the genetic variability detected for these traits.

Gastric microbiota in the functional dyspepsia patients treated with probiotic yogurt

ObjectiveTo investigate the structure of the gastric microbiota in functional dyspepsia (FD) and its role in the pathophysiology.DesignWe compared the basic physiological properties of the gastric fluid (GF) and the...

CYP2B6rs2279343 Is Associated with Improved Survival of Pediatric Rhabdomyosarcoma Treated with Cyclophosphamide.

BackgroundRhabdomyosarcoma (RMS) is a small round blue cell malignant tumor, representing 7% of childhood malignancies, and over 50% of all soft tissue sarcomas. Cyclophosphamide (CPA) is a prodrug and is the mainstay of RMS treatment. CYP2B6 is a highly polymorphic drug metabolizing enzyme involved in CPA bioactivation. The influence of CYP2B6 single nucleotide polymorphisms (SNPs) on the survival of RMS is still unknown.MethodsWe genotyped CYP2B6SNPs rs2279343, rs3745274, and rs3211371 by restriction fragment polymorphism (RFLP) after PCR amplification in a cohort of 73 pediatric RMS patients treated with CPA-based first line treatment. We then analyzed the association between those genotypes and survival outcome of RMS.ResultsThe frequencies of CYP2B6 rs2279343, rs3745274, and rs3211371 were 63%, 45.2%, and 5.5%, respectively. There was no association between rs3745274, rs3211371 genotypes and survival outcomes of RMS. However, the carriers of at least one mutant allele CYP2B6rs2279343 had significantly longer event-free survival (p-value = 0.03).ConclusionOur results demonstrated that CYP2B6 rs2279343 may predict EFS in RMS patients and warrants future studies to clarify the pharmacogenetics of CPA in pediatrics. If validated, integration of genetic factors with clinical and molecular characteristics could be used for a composite algorithm to better stratify risk prior to treatment.

Change of methane production pathway with sediment depth in a lake on the Tibetan plateau

Methane production is the terminal step in the degradation of organic matter in most anoxic lake sediments. It has been suggested that CH4 production in lake sediments is dominated by hydrogenotrophic methanogenesis, especially in deeper layers. We investigated the vertical sediment profile from the surface to 50cm depth in the sediment of Bangong Co, an oligotrophic high altitude lake on the Tibetan plateau. We measured CH4 production, stable carbon isotopefractionation and the archaeal community structure. We found that the methane production rates, the acetate concentrations and the numbers of bacteria and archaea strongly decreased with sediment depth. The enrichment factor (ε) for hydrogenotrophic methanogenesis also decreased with depth, while δ13C of acetate stayed fairly constant. The contribution of hydrogenotrophic methanogenesis to total CH4 production increased with depth from ~36% to 100%. Analysis of terminal restriction fragment polymorphism (T-RFLP) of archaeal 16S rRNA genes showed that the relative abundance of aceticlastic (Methanosaetaceae) methanogens also decreased with depth disappearing completely at 50cm depth. Our study firstly showed that the methanogenic pathway and the methanogenic archaeal community systematically changed with sediment depth in a high altitude lake, probably controlled by the availability of easily degradable organic matter.

Population Dynamics and Community Composition of Ammonia Oxidizers in Salt Marshes after the Deepwater Horizon Oil Spill.

The recent oil spill in the Gulf of Mexico had significant effects on microbial communities in the Gulf, but impacts on nitrifying communities in adjacent salt marshes have not been investigated. We studied persistent effects of oil on ammonia-oxidizing archaeal (AOA) and bacterial (AOB) communities and their relationship to nitrification rates and soil properties in Louisiana marshes impacted by the Deepwater Horizon oil spill. Soils were collected at oiled and unoiled sites from Louisiana coastal marshes in July 2012, 2 years after the spill, and analyzed for community differences based on ammonia monooxygenase genes (amoA). Terminal Restriction Fragment Polymorphism and DNA sequence analyses revealed significantly different AOA and AOB communities between the three regions, but few differences were found between oiled and unoiled sites. Community composition of nitrifiers was best explained by differences in soil moisture and nitrogen content. Despite the lack of significant oil effects on overall community composition, we identified differences in correlations of individual populations with potential nitrification rates between oiled and unoiled sites that help explain previously published correlation patterns. Our results suggest that exposure to oil, even 2 years post-spill, led to subtle changes in population dynamics. How, or if, these changes may impact ecosystem function in the marshes, however, remains uncertain.