Monitoring the natural attenuation of a sewage sludge toxicity using the Allium cepa test

Appropriate final disposal of sewage sludge (SS) generated by wastewater treatment plants (WWTP) has been considered a serious environmental problem, but also a viable alternative to be applied in agriculture, once SS is rich in organic matter and nutrients. However, SS can be a source of contamination of several toxic agents. Therefore, its use in agriculture requires special care to avoid possible damage to the environment and exposed organisms. Detoxification of toxic wastes can be performed using the monitored natural attenuation, which involves biological, physical and chemical processes that frequently occur in the environment. This study aimed to assess the feasibility of decontaminating SS after different periods of monitored natural attenuation. To this end, samples of SS and associations of soil/SS with proportions of 10, 25 and 50% SS were buried for 0, 2, 6 and 12 months in holes prepared in a place free of contamination. Allium cepa was used as an indicator to assess the efficiency of the natural attenuation process. According to chemical analysis, the SS samples presented a high concentration of m- and p-cresol, especially for samples analyzed after 0 or 2 months of natural attenuation. The microorganisms present in the SS belonged to 17 different genera of bacteria, which varied in the microbial composition among samples. Both, raw SS and aqueous SS extracts induced DNA damage in A. cepa, even when associated with soil. However, this effect was observed to decline during the attenuation period, although significant effects were detected for the highest tested concentration (100% SS) even at the end of this process. These results thus indicated the necessity of applying a stabilization process associating SS and soil for a period of at least 12 months and showed that the studied raw SS is not a viable material for use as a soil reconditioner, even after natural attenuation. A. cepa test proved to be a useful tool to assess the efficiency of SS detoxification process. Therefore, we suggest that the application of SS in agriculture should be approached with caution and that the SS must be previously submitted to methodologies that evaluate its toxic potential.     

Comparison of the relative sensitivity of human lymphocytes and mouse splenocytes to two spindle poisons

Aneugenic compounds act on non-DNA targets to exert genotoxicity via an indirect mechanism. In contrast to DNA-binding agents, these compounds are expected to possess threshold levels of activity. Therefore, the risk for adverse effects following human exposure to an aneugen could be minimal, if the threshold of activity has been clearly determined in vivo and in vitro and providing the human exposure level is below this threshold. Thus, the development of a single-cell model to allow comparisons between in vitro and in vivo threshold values for aneugenic compounds is of importance.The in vivo micronucleus test is one of the main assays used in genetic toxicology, and is often performed in the mouse. Thus, an extensive database is available in the literature. However, there are only few data concerning the in vitro micronucleus assay using mouse cells, as the majority of in vitro micronucleus assays have been performed using human lymphocytes. In addition, there is a lack of data concerning thresholds for any compound using this model.First, we evaluated whether the use of mouse splenocytes would be an acceptable alternative to that of human lymphocytes to identify aneugens. To allow valid comparisons, the two protocols were first harmonized. Thus, phytohemagglutinin (PHA) and concanavalin A were used as specific mitogens for human lymphocytes and mouse splenocytes, respectively, in order to achieve similar cell-proliferation rates. To achieve similar and sufficient numbers of binucleated cells, cytochalasin B was added 44 and 56 h after culture initiation of the human and mouse cells, respectively.Second, we compared the sensitivity of the mouse protocol with that of the human protocol by exposing the cells to the aneugens nocodazole and paclitaxel.There was good reproducibility of the cytotoxic/genotoxic responses of the two cell models following exposure to the aneugens. The sensitivity of the mouse splenocytes to paclitaxel was higher than that of the human lymphocytes. The two cell types were equally sensitive to nocodazole.     

Cytogenetic effects of nine <Emphasis Type="Italic">Helichrysum</Emphasis> taxa in human lymphocytes culture

Helichrysum Mill. (Asteraceae) species have been used in folk medicine for thousands of years in the world. The in vitro cytogenetic effects in human lymphocytes of nine Helichrysum taxa used in Turkey folk medicine were investigated. Blood samples were obtained from healthy donors, non-smoking volunteers, which were incubated and exposed to increasing concentrations of methanol extracts of Helichrysum taxa (0.01, 0.05, 0.1, 0.5 and 1 mg/mL). The inhibitory effects of H. stoechas (L.) Moench subsp. barrelieri (Ten.) Nyman, H. armenium DC. subsp. armenium, H. armenium DC. subsp. araxinum (Kirp.) Takht., H. plicatum DC. subsp. plicatum, H. compactum Boiss. and H. artvinense P.H.Davis & Kupicha on the mitotic index and replication index indicate that these taxa can have genotoxic and mutagenic effects. They should therefore not be used freely in alternative medicine although their antiproliferative activity may suggest anticarcinogenic properties. Increase effects of H. stoechas subsp. barrelieri, H. armenium subsp. armenium, H. armenium subsp. araxinum, H. chasmolycicum P.H.Davis, H. plicatum subsp. plicatum, H. compactum and H. artvinense on the micronucleus rates showed that these taxa can have genotoxic and carcinogenic effects.     

SNP identification and marker assay development for high-throughput selection of soybean cyst nematode resistance

Background

Soybean cyst nematode (SCN) is the most economically devastating pathogen of soybean. Two resistance loci, Rhg1 and Rhg4 primarily contribute resistance to SCN race 3 in soybean. Peking and PI 88788 are the two major sources of SCN resistance with Peking requiring both Rhg1 and Rhg4 alleles and PI 88788 only the Rhg1 allele. Although simple sequence repeat (SSR) markers have been reported for both loci, they are linked markers and limited to be applied in breeding programs due to accuracy, throughput and cost of detection methods. The objectives of this study were to develop robust functional marker assays for high-throughput selection of SCN resistance and to differentiate the sources of resistance.

Results

Based on the genomic DNA sequences of 27 soybean lines with known SCN phenotypes, we have developed Kompetitive Allele Specific PCR (KASP) assays for two Single nucleotide polymorphisms (SNPs) from Glyma08g11490 for the selection of the Rhg4 resistance allele. Moreover, the genomic DNA of Glyma18g02590 at the Rhg1 locus from 11 soybean lines and cDNA of Forrest, Essex, Williams 82 and PI 88788 were fully sequenced. Pairwise sequence alignment revealed seven SNPs/insertion/deletions (InDels), five in the 6th exon and two in the last exon. Using the same 27 soybean lines, we identified one SNP that can be used to select the Rhg1 resistance allele and another SNP that can be employed to differentiate Peking and PI 88788-type resistance. These SNP markers have been validated and a strong correlation was observed between the SNP genotypes and reactions to SCN race 3 using a panel of 153 soybean lines, as well as a bi-parental population, F₅–derived recombinant inbred lines (RILs) from G00-3213 x LG04-6000.

Conclusions

Three functional SNP markers (two for Rhg1 locus and one for Rhg4 locus) were identified that could provide genotype information for the selection of SCN resistance and differentiate Peking from PI 88788 source for most germplasm lines. The robust KASP SNP marker assays were developed. In most contexts, use of one or two of these markers is sufficient for high-throughput marker-assisted selection of plants that will exhibit SCN resistance.

Electronic supplementary material

The online version of this article (doi:10.1186/s12864-015-1531-3) contains supplementary material, which is available to authorized users.     

PGC-1α-mediated changes in phospholipid profiles of exercise-trained skeletal muscle

Exercise training influences phospholipid fatty acid composition in skeletal muscle and these changes are associated with physiological phenotypes; however, the molecular mechanism of this influence on compositional changes is poorly understood. Peroxisome proliferator-activated receptor γ coactivator 1α (PGC-1α), a nuclear receptor coactivator, promotes mitochondrial biogenesis, the fiber-type switch to oxidative fibers, and angiogenesis in skeletal muscle. Because exercise training induces these adaptations, together with increased PGC-1α, PGC-1α may contribute to the exercise-mediated change in phospholipid fatty acid composition. To determine the role of PGC-1α, we performed lipidomic analyses of skeletal muscle from genetically modified mice that overexpress PGC-1α in skeletal muscle or that carry KO alleles of PGC-1α. We found that PGC-1α affected lipid profiles in skeletal muscle and increased several phospholipid species in glycolytic muscle, namely phosphatidylcholine (PC) (18:0/22:6) and phosphatidylethanolamine (PE) (18:0/22:6). We also found that exercise training increased PC (18:0/22:6) and PE (18:0/22:6) in glycolytic muscle and that PGC-1α was required for these alterations. Because phospholipid fatty acid composition influences cell permeability and receptor stability at the cell membrane, these phospholipids may contribute to exercise training-mediated functional changes in the skeletal muscle.     

Using Isolated Mitochondria from Minimal Quantities of Mouse Skeletal Muscle for High throughput Microplate Respiratory Measurements

Skeletal muscle mitochondria play a specific role in many disease pathologies. As such, the measurement of oxygen consumption as an indicator of mitochondrial function in this tissue has become more prevalent. Although many technologies and assays exist that measure mitochondrial respiratory pathways in a variety of cells, tissue and species, there is currently a void in the literature in regards to the compilation of these assays using isolated mitochondria from mouse skeletal muscle for use in microplate based technologies. Importantly, the use of microplate based respirometric assays is growing among mitochondrial biologists as it allows for high throughput measurements using minimal quantities of isolated mitochondria. Therefore, a collection of microplate based respirometric assays were developed that are able to assess mechanistic changes/adaptations in oxygen consumption in a commonly used animal model. The methods presented herein provide step-by-step instructions to perform these assays with an optimal amount of mitochondrial protein and reagents, and high precision as evidenced by the minimal variance across the dynamic range of each assay.     

Binding of colchicine and lumicolchicine to components in plant extracts

Irradiation of dilute, aqueous solutions of colchicine and of colcemid by long wavelength UV resulted, in each case, in a mixture of three major lumi-products whose relative concentrations depended upon the degree of UV exposure. Components in plant extracts, after concentration by ammonium sulphate precipitation, bound the drugs and their lumi-derivatives. Consideration of the different ratios of colchicine- to lumicolchicine-binding activity in different fractions of a plant extract, and after various enzymatic and temperature treatments of the plant preparations, suggest the involvement of different binding sites for the various forms of the alkaloid.     

猪链球菌2型唾液酸合成酶neu B基因敲除突变株的构建及其生物学特性

利用同源重组基因敲除方法构建猪链球菌2型强毒株05ZYH33唾液酸合成酶neuB基因敲除突变株。PCR和Southern杂交结果均显示neuB基因在1株转化重组体中完全被壮观霉素抗性基因替代, 表明neuB基因敲除突变体构建成功。生物学特性鉴定显示, 突变体与强毒株在菌落形态、溶血活性以及染色特性方面均无明显差异; 电镜检查发现突变体表面结构组分与强毒株有显著差异, 荚膜明显变薄, 质地更加紧密; 小鼠致病性试验结果显示, 突变体毒力显著减弱。研究结果提示菌体荚膜中的唾液酸对于猪链球菌2型侵袭和致病具有重要作用。     

中红侧沟茧蜂化学感受蛋白MmedCSP1的结合特征

化学感受蛋白是一类存在于昆虫化学感受器中的可溶性蛋白, 被认为与昆虫识别外界化学信息有关。本研究使用pGEX-4T-1表达载体在BL21 (DE3)异源表达系统中表达中红侧沟茧蜂Microplitis mediator化学感受蛋白MmedCSP1, 并通过亲和层析法纯化得到去表达标签的MmedCSP1;使用bis-ANS作为荧光配基, 在荧光分光光度计上研究它与50种气味标样的结合特征, 从而得到此类化学感受蛋白在中红侧沟茧蜂嗅觉识别中识别气味的种类。结果表明: MmedCSP1只能与水杨酸甲酯、戊烷、罗勒烯、β-紫罗兰酮、3, 4-二甲基苯甲醛、2-己酮和叶醇结合。但只有脂类化合物β-紫罗兰酮能在浓度为1 mmol/L下将bis-ANS从MmedCSP1中替换50%, β-紫罗兰酮与MmedCSP1的结合常数为16.89 μmol/L。这些结果提示MmedCSP1参与中红侧沟茧蜂对水杨酸甲酯、戊烷、罗勒烯、β-紫罗兰酮、3, 4-二甲基苯甲醛、2-己酮和叶醇等气味的识别过程, 且在不同气味中的识别过程中对于气味的运输能力有差异。