Improved genetic resolution for linkage mapping of resistance to potato wart in monoparental dihaploids with potential diagnostic value in tetraploid potato varieties.

KEY MESSAGE: We achieved improved mapping resolution of the major wart resistance locus Xla-TNL containing also Sen1 in a dihaploid population using SNP data and developed additional markers with diagnostic value in tetraploid varieties. We analyzed a segregating monoparental dihaploid potato population comprising 215 genotypes derived from a tetraploid variety that is highly resistant to Synchytrium endobioticum pathotypes 18 and 6. The clear bimodal segregation for both pathotypes indicated that a major dominant resistance factor in a simplex allele configuration was present in the tetraploid donor genotype. Compared to that in previous analyses of the same tetraploid donor in conventional crosses with susceptible tetraploid genotypes, a segregation pattern with a reduced genetic complexity of resistance in dihaploids was observed here. Using the 12.8 k SolCAP SNP array, we mapped a resistance locus to the Xla-TNL region containing also Sen1 on potato chromosome 11. The improved mapping resolution provided by the monoparental dihaploids allowed for the localization of the genes responsible for the resistance to both pathotypes in an interval spanning less than 800 kbp on the reference genome. Furthermore, we identified eight molecular markers segregating without recombination to pathotype 18 and pathotype 6 resistance. Also, two developed markers display improved diagnostic properties in an independent panel of tetraploid varieties. Overall, our data provide the highest resolution mapping of wart resistance genes at the Xla-TNL locus thus far.

Characteristics of PCR-SSCP and RAPD-HPCE methods for identifying authentication of Penis et testis cervi in Traditional Chinese Medicine based on cytochrome b gene.

The use of Penis et testis cervi, as a kind of precious Traditional Chinese Medicine (TCM), which is derived from dry deer's testis and penis, has been recorded for many years in China. There are abundant species of deer in China, the Penis et testis from species of Cervus Nippon and Cervus elaphusL were authentic, others species were defined as adulterant (different subspecies of deer) or counterfeits (different species). Identification of their origins or authenticity becomes a key in controlling the herbal products. A modified column chromatography was used to extract mitochondrial DNA of dried deer's testis and penis from sika deer (C. Nippon) and red deer (C. elaphusL) in addition to adulterants and counterfeits. Column chromatography requires for a short time to extract mitochondrial DNA of high purity with little damage of DNA molecules, which provides the primary structure of guarantee for the specific PCR; PCR-SSCP method showed a clear intra-specific difference among patterns of single-chain fragments, and completely differentiate Penis et testis origins from C. Nippon and C. elaphusL. RAPD-HPCE was based on the standard electropherograms to compute a control spectrum curve as similarity reference (R) among different samples. The similarity analysis indicated that there were significant inter-species differences among Penis et testis' adulterant or counterfeits. Both techniques provide a fast, simple, and accurate way to directly identify among inter-species or intra-species of Penis et testis.

[Fungal composition in massa medicata fermentata based on culture dependent method and independent PCR-SSCP technique].

OBJECTIVE: To analyze the fungal composition in Massa Medicata Fermentata based on culture dependent method and independent PCR-SSCP technique. METHOD: Fungi were directly isolated from Massa Medicata Fermentata samples. The obtained strains were identified according to morphology and DNA sequence. Meanwhile the total fungal DNA was extracted from Massa Medicata Fermentata samples, the cultural independent PCR-SSCP technique based on ß-tubulin gene were used to identify the mycobiota. RESULT: According to cultural method, Aspergillus flavus and Rhizopus oryzae were present in Massa Medicata Fermentata samples, while A. flavus and A. niger were present in fried Massa Medicata Fermentata samples. In contrast, 5 species were obtained by PCR-SSCP technique, A. flavus was overlapped with fungal taxa derived from culture dependent method; A. ambiguu and A. s ivoriensis were dominant with relative abundance of 57% and 35% respectively, while the relative abundance of A. flavus was as low as 4%. None species was obtained from fried Massa Medicata Fermentata samples. CONCLUSION: PCR-SSCP based on ß-tubulin gene could distinguish fungi into species, culture dependent method combined with culture independent method could better understand the fungal composition associated with Massa Medicata Fermentata fermentation.

Fungal composition in massa medicata fermentata based on culture dependent method and independent PCR-SSCP technique / 中国中药杂志

<p><b>OBJECTIVE</b>To analyze the fungal composition in Massa Medicata Fermentata based on culture dependent method and independent PCR-SSCP technique.</p><p><b>METHOD</b>Fungi were directly isolated from Massa Medicata Fermentata samples. The obtained strains were identified according to morphology and DNA sequence. Meanwhile the total fungal DNA was extracted from Massa Medicata Fermentata samples, the cultural independent PCR-SSCP technique based on β-tubulin gene were used to identify the mycobiota.</p><p><b>RESULT</b>According to cultural method, Aspergillus flavus and Rhizopus oryzae were present in Massa Medicata Fermentata samples, while A. flavus and A. niger were present in fried Massa Medicata Fermentata samples. In contrast, 5 species were obtained by PCR-SSCP technique, A. flavus was overlapped with fungal taxa derived from culture dependent method; A. ambiguu and A. s ivoriensis were dominant with relative abundance of 57% and 35% respectively, while the relative abundance of A. flavus was as low as 4%. None species was obtained from fried Massa Medicata Fermentata samples.</p><p><b>CONCLUSION</b>PCR-SSCP based on β-tubulin gene could distinguish fungi into species, culture dependent method combined with culture independent method could better understand the fungal composition associated with Massa Medicata Fermentata fermentation.</p>

Development of single-strand conformational polymorphism to screen for mutations in hotspot regions of beta-globin gene of beta-thalassemia patients of Sri Lanka.

Beta-thalassemia is prevalent in Sri Lanka and imposes a heavy economic and social burden in the country due to the patients' life-long need for regular blood transfusion and treatment with iron chelation therapy. Thus, there is a need to develop a rapid, reliable and effective population-based presymptomatic and prenatal screening method for beta-thalassemia. Single-strand conformational polymorphism (SSCP) technique was developed as an adjunct for the previously developed allele-specific PCR (ASP) technique to screen the presence of mutations in beta-globin gene. A hotspot region of beta-globin gene containing 98% of known beta-thalassemia mutations was amplified from 24 clinically diagnosed beta-thalassemia patients and two normal individuals. Two overlapping amplicons of 238 bp and 268 bp were subjected to SSCP analysis. The SSCP banding patterns of these two fragments from beta-thalassemia patients were different from the corresponding regions of normal individuals. Sequence analysis of these regions revealed the presence of 4 mutations in the form of deletion and substitution that have not been reported previously from Sri Lanka. Therefore, the SSCP protocol developed in this study together with ASP should provide an appropriate screening approach for presymptomatic and parental diagnosis of beta-thalassemia in the Sri Lankan population.

Validation of candidate gene markers for marker-assisted selection of potato cultivars with improved tuber quality.

Tuber yield, starch content, starch yield and chip color are complex traits that are important for industrial uses and food processing of potato. Chip color depends on the quantity of reducing sugars glucose and fructose in the tubers, which are generated by starch degradation. Reducing sugars accumulate when tubers are stored at low temperatures. Early and efficient selection of cultivars with superior yield, starch yield and chip color is hampered by the fact that reliable phenotypic selection requires multiple year and location trials. Application of DNA-based markers early in the breeding cycle, which are diagnostic for superior alleles of genes that control natural variation of tuber quality, will reduce the number of clones to be evaluated in field trials. Association mapping using genes functional in carbohydrate metabolism as markers has discovered alleles of invertases and starch phosphorylases that are associated with tuber quality traits. Here, we report on new DNA variants at loci encoding ADP-glucose pyrophosphorylase and the invertase Pain-1, which are associated with positive or negative effect with chip color, tuber starch content and starch yield. Marker-assisted selection (MAS) and marker validation were performed in tetraploid breeding populations, using various combinations of 11 allele-specific markers associated with tuber quality traits. To facilitate MAS, user-friendly PCR assays were developed for specific candidate gene alleles. In a multi-parental population of advanced breeding clones, genotypes were selected for having different combinations of five positive and the corresponding negative marker alleles. Genotypes combining five positive marker alleles performed on average better than genotypes with four negative alleles and one positive allele. When tested individually, seven of eight markers showed an effect on at least one quality trait. The direction of effect was as expected. Combinations of two to three marker alleles were identified that significantly improved average chip quality after cold storage and tuber starch content. In F1 progeny of a single-cross combination, MAS with six markers did not give the expected result. Reasons and implications for MAS in potato are discussed.

Loss of activating EGFR mutant gene contributes to acquired resistance to EGFR tyrosine kinase inhibitors in lung cancer cells.

Non-small-cell lung cancer harboring epidermal growth factor receptor (EGFR) mutations attains a meaningful response to EGFR-tyrosine kinase inhibitors (TKIs). However, acquired resistance to EGFR-TKIs could affect long-term outcome in almost all patients. To identify the potential mechanisms of resistance, we established cell lines resistant to EGFR-TKIs from the human lung cancer cell lines PC9 and11-18, which harbored activating EGFR mutations. One erlotinib-resistant cell line from PC9 and two erlotinib-resistant cell lines and two gefitinib-resistant cell lines from 11-18 were independently established. Almost complete loss of mutant delE746-A750 EGFR gene was observed in the erlotinib-resistant cells isolated from PC9, and partial loss of the mutant L858R EGFR gene copy was specifically observed in the erlotinib- and gefitinib-resistant cells from 11-18. However, constitutive activation of EGFR downstream signaling, PI3K/Akt, was observed even after loss of the mutated EGFR gene in all resistant cell lines even in the presence of the drug. In the erlotinib-resistant cells from PC9, constitutive PI3K/Akt activation was effectively inhibited by lapatinib (a dual TKI of EGFR and HER2) or BIBW2992 (pan-TKI of EGFR family proteins). Furthermore, erlotinib with either HER2 or HER3 knockdown by their cognate siRNAs also inhibited PI3K/Akt activation. Transfection of activating mutant EGFR complementary DNA restored drug sensitivity in the erlotinib-resistant cell line. Our study indicates that loss of addiction to mutant EGFR resulted in gain of addiction to both HER2/HER3 and PI3K/Akt signaling to acquire EGFR-TKI resistance.

[PCR-SSCP molecular identification of Panax ginseng and P. quinquefolius based on ITS2 bar coding SNPs].

OBJECTIVE: To establish a method for identifying Panax ginseng and P. quinquefolius with PCR-SSCP, on the basis of specific SNP identification sites of their ITS2 bar codes. METHOD: ITS2 sequences of P. ginseng and P. quinquefolius recorded in GenBank were searched to conduct a comparative analysis and screen out specific SNP identification sites of their ITS2 bar codes. Based on that, the Polymerase Chain Reaction-Single Strand Conformation Polymorphism (PCR-SSCP) method was adopted for analyzing 11 P. ginseng samples and 10 P. quinquefolius samples and verifying sequencing of their PCR products. RESULT: The P. ginseng and P. quinquefolius samples had the same agarose mages of PCR-SSCP with the standard medicines. There were significant differences between the PCR-SSCP agarose mages of P. ginseng and P. quinquefolius, with identifical identification results between PCR-SSCP and sequencing. CONCLUSION: Compared with the sequencing method, PCR-SSCP is so rapid and accurate that it can be used for identifying P. ginseng and P. quinquefolius medicines.

PCR-SSCP molecular identification of Panax ginseng and P. quinquefolius based on ITS2 bar coding SNPs / 中国中药杂志

<p><b>OBJECTIVE</b>To establish a method for identifying Panax ginseng and P. quinquefolius with PCR-SSCP, on the basis of specific SNP identification sites of their ITS2 bar codes.</p><p><b>METHOD</b>ITS2 sequences of P. ginseng and P. quinquefolius recorded in GenBank were searched to conduct a comparative analysis and screen out specific SNP identification sites of their ITS2 bar codes. Based on that, the Polymerase Chain Reaction-Single Strand Conformation Polymorphism (PCR-SSCP) method was adopted for analyzing 11 P. ginseng samples and 10 P. quinquefolius samples and verifying sequencing of their PCR products.</p><p><b>RESULT</b>The P. ginseng and P. quinquefolius samples had the same agarose mages of PCR-SSCP with the standard medicines. There were significant differences between the PCR-SSCP agarose mages of P. ginseng and P. quinquefolius, with identifical identification results between PCR-SSCP and sequencing.</p><p><b>CONCLUSION</b>Compared with the sequencing method, PCR-SSCP is so rapid and accurate that it can be used for identifying P. ginseng and P. quinquefolius medicines.</p>

Asymmetric single-strand conformation polymorphism: an accurate and cost-effective method to amplify and sequence allelic variants.

PREMISE OF THE STUDY: An efficient alternative strategy to conventional cloning was needed to generate high-quality DNA sequences from a variety of nuclear orthologs for phylogenetic studies. This method would facilitate studies and minimize technical problems typically encountered in cloning methodologies. METHODS: We tested a variety of single-strand conformation polymorphism (SSCP) protocols including purified and unpurified symmetric and asymmetric PCR, loading buffers, and electrophoresis conditions (buffers, matrix, running time, temperature). Results obtained from direct SSCP band sequencing were compared to those obtained from cloning. KEY RESULTS: Our optimized protocol uses asymmetric PCR, with the majority of the samples run in polyacrylamide gel electrophoresis (PAGE). It consistently separated PCR products from 450 to 1200 bp. CONCLUSIONS: Asymmetric PCR single-strand conformation polymorphism is an efficient alternative technique for isolating allelic variants of highly heterozygous individuals, with its greatest applications in sequencing allopolyploids. It eliminates two common problems encountered in cloning: PCR recombination and heteroduplex fixation. In addition, our protocol greatly lowers costs and time associated with procedures.

High diversity of the fungal community structure in naturally-occurring Ophiocordyceps sinensis.

BACKGROUND: Ophiocordyceps sinensis (syn. Cordyceps sinensis), which is a parasite of caterpillars and is endemic to alpine regions on the Tibetan Plateau, is one of the most valuable medicinal fungi in the world. "Natural O. sinensis specimens" harbor various other fungi. Several of these other fungi that have been isolated from natural O. sinensis specimens have similar chemical components and/or pharmaceutical effects as O. sinensis. Nevertheless, the mycobiota of natural O. sinensis specimens has not been investigated in detail. METHODOLOGY/PRINCIPAL FINDINGS: Based on the technique of PCR-single-strand conformation polymorphism (PCR-SSCP), the mycobiota of three different sections (stromata, sclerotia, and mycelial cortices) from natural O. sinensis specimens were investigated using both culture-dependent and -independent methods. For the culture-dependent method, 572 fungal strains were isolated, and 92 putative operational taxonomic units (OTUs) were identified from 226 sequenced strains with the threshold of 97%. For the culture-independent method, 490 fungal clones were identified from about 3000 clones of ITS fragments from the whole-community DNA; based on PCR-SSCP analyses, 266 of these clones were selected to be sequenced, and 118 putative OTUs were detected. The overwhelming majority of isolates/clones and OTUs were detected from mycelial cortices; only a few were detected from stromata and sclerotia. The most common OTUs detected with both methods belonged to Ascomycota; however, only 13 OTUs were detected simultaneously by both methods. Potential novel lineages were detected by each of the two methods. CONCLUSIONS/SIGNIFICANCE: A great number of fungal species present in the mycobiota of naturally-occurring O. sinensis specimens were detected, and many of them may represent undescribed lineages. That only a few of the same OTUs were detected by both methods indicated that different methods should be used. This study increased our understanding about the fungal community structure of this valuable medicinal herb.

Promoter hypermethylation in tumour suppressor genes and response to interleukin-2 treatment in bladder cancer: a pilot study.

PURPOSE: Non-muscle invasive bladder cancer (BC) is a highly recurrent disease, with the first recurrences arising shortly after transurethral resection of the bladder (TURB). Topical administration of interleukin-2 (IL-2) has been shown as an effective adjuvant therapy for BC; however, predictive biomarkers that may identify suitable subgroups of patients are lacking. In this pilot study we sought to determine the prognostic value of epigenetic and genetic inactivation of tumour suppressor genes (TSGs) among BC patients treated with IL-2. METHODS: After complete TURB, patients with multifocal superficial BC were treated with five daily intravesical instillations of IL-2. Promoter hypermethylation in six TSGs and the TP53 gene mutations were prospectively assessed by methylation-specific PCR and automated capillary single-strand conformation polymorphism in 21 primary bladder cancer specimens and ten bladder wall biopsies collected during follow-up. RESULTS: After IL-2 treatment, 9 out of 21 (43%) patients did not develop recurrent tumour within the 1 year of follow-up period. The mean duration of recurrence-free survival in the rest of the study group was 112 days. In the current pilot study, BC with p16 gene hypermethylation had a lower risk of recurrence after treatment with IL-2, as compared to IL-2 treated BC without p16 hypermethylation (p = 0.02). Significant associations were observed between tumour grade and the mean methylation index (p = 0.003), as well as the hypermethylation of the RARbeta gene (p = 0.048). CONCLUSION: Our preliminary data suggest that DNA methylation biomarkers may assist in selection of BC patients for efficient IL-2 therapy.

Genetic diversity of Ophiocordyceps sinensis, a medicinal fungus endemic to the Tibetan Plateau: implications for its evolution and conservation.

BACKGROUND: Ophiocordyceps sinensis (syn. Cordyceps sinensis), endemic to alpine regions on the Tibetan plateau, is one of the most valuable medicinal fungi in the world. Huge commercial demand has led to excessive harvest and a dramatic decline in its numbers. The diversity of terrains and climates on the Tibetan Plateau and the broad insect host range (more than 50 species in the family Hepialidae) may have resulted in substantial intraspecific genetic diversity for this fungus. The objective of this study was to evaluate the population distribution of O. sinensis from geographically diverse regions of the Tibetan Plateau based on nrDNA ITS and MAT1-2-1 gene sequences. Understanding of the genetic diversity and genesis of O. sinensis will provide important information for the evolution and conservation of this fungus. RESULTS: Significant sequence variations in the ITS and MAT1-2-1 genes (27 and 23 informative sites, eight and seven haplotypes, respectively) were observed. Phylogenetic analysis based on ITS sequences, MAT1-2-1 sequences, or their combined data set, clustered isolates from northern regions in one clade (clade I), whereas isolates from southern regions were dispersed in all four clades (clade I-IV). Single-strand conformation polymorphism (SSCP) analyses of 2639 ITS clones from seven samples revealed 91 different SSCP patterns that were subsequently sequenced. ITS heterogeneity was found in XZ-LZ07-H1 (Nyingchi population), and 17 informative sites and five haplotypes were detected from 15 clones. The five haplotypes clustered into three clades (clade I, II, and IV). CONCLUSIONS: Significant genetic divergence in O. sinensis was observed and the genetic diversification was greater among southern isolates than that among northern isolates. The polymorphism of nrDNA ITS sequences suggested that O. sinensis spread from a center of origin (the Nyingchi District) to southern regions and subsequently to northern areas. These results suggest that southern populations are important reservoirs of genetic diversity and should be taken into account in conservation programs.

UGT1A1 gene polymorphisms in North Indian neonates presenting with unconjugated hyperbilirubinemia.

Genetic factors are implicated in pathogenesis of neonatal hyperbilirubinemia. In this nested case-control study, we determined 1) frequency of thymine-adenine (TA)n promoter polymorphism and Gly71Arg mutation in uridine diphosphoglucuronate-glucuronosyltransferase 1A1 (UGT1A1) gene in neonates > or =35-wk gestation presenting with bilirubin levels > or =18 mg/dL and controls, 2) interaction among (TA)n promoter polymorphism, glucose-6-phosphate dehydrogenase (G6PD) gene mutations, and peak bilirubin. The number of TA repeats was assessed by PCR-single-strand conformation polymorphism (SSCP) analysis and Gly71Arg mutation by PCR-RFLP. Fifty samples of both mutations were verified with DNA sequencing. One hundred twenty-seven neonates were enrolled (77 hyperbilirubinemics, 50 controls). The incidence of (TA)n polymorphism was higher in babies with hyperbilirubinemia [89.6% vs. 50%, OR 8.63 (95% CI, 3.2-24.1)]. Gly71Arg mutation was not found either in hyperbilirubinemics or controls. A novel polymorphism (Ala72Pro) at codon position 72 of exon 1 was detected in all 50 samples (21 hyperbilirubinemics, 29 controls), which were sequenced. Presence of variant (TA)n promoter (adjusted OR, 10.6; 95% CI, 3.3-34.2), G6PD deficiency (adjusted OR, 20.6; 95% CI, 3.6-117.3), and history of jaundice in sibling requiring phototherapy (adjusted OR, 12.6; 95% CI, 1.1-141.6) were independent risk factors for bilirubin levels > or =18 mg/dL.

Petrochemical wastewater odor treatment by biofiltration.

The treatment of odorous pollutants by microorganisms on packed waste straw and cortex was investigated at the wastewater treatment plant of the Shanghai petrochemical factory. The removal efficiency of H(2)S, NH(3) and VOCs (volatile organic compounds) reached 98%, 91% and 90%, respectively after operation for one month at an empty bed retention time (EBRT) of 120s. The heterotrophic bacteria were found to be the dominant microorganism in the biofilter, while fungi and actinomycetes were also present. The bacteria were mostly identified as the members of the genus Bacillus (62.5% of cultured bacteria). The single strand conformation polymorphism (SSCP) results revealed that the genus Bacillus and Pseudomonas were the predominant bacteria. The microbial diversity gradually increased as the treatment progressed, which indicated that the microbial community in the biofilter became more stable upon pollutant removal. The scanning electron microscopy (SEM) was performed to evaluate the microorganism growth on the media. It was found that the waste straw and cortex were suitable for microorganism attachment and growth, and may have potential application in odor treatment.

Pyruvate dehydrogenase deficiency: identification of a novel mutation in the PDHA1 gene which responds to amino acid supplementation.

UNLABELLED: The pyruvate dehydrogenase complex (PDHc) is an intramitochondrial multienzyme system, which plays a key role in aerobic glucose metabolism by catalysing the oxidative decarboxylation of pyruvate to acetyl-CoA. Genetic defects in the PDHc lead to lactic acidemia and neurological abnormalities. In the majority of the cases, the defect appears to reside in the E(1)alpha subunit, the first catalytic component of the complex. The report is on a 6-year-old Portuguese boy with mild neurological involvement and low PDHc activity with absence of E1alpha on immunoblotting analysis. Molecular studies showed a novel and "de novo" mutation in the PDHA1 gene, R253G. Treatment with arginine aspartate showed complete clinical and biochemical recovery. We hypothesise that arginine aspartate acts as a chemical or pharmacological chaperone, and suggest amino acid supplementation as a possible therapy in PDHA1 mutations with mild phenotypes. CONCLUSION: our results encourage the use of amino acid supplementation to overcome the metabolic/biochemical changes induced by PDHA1 gene specific mutations associated with mild PDHc phenotypes.

Clinical and molecular characteristics of isolated colonic Crohn's disease.

BACKGROUND: Clinical, serological, and molecular data support the existence of discrete subsets of Crohn's disease (CD) defined by location of disease. Little is known about the epidemiology and natural history of isolated CD of the colon (Montreal Classification L2) because most studies have not accurately distinguished it from ileocolonic disease. Our objectives were to describe the clinical features and natural history of isolated colonic CD in a rigorously characterized patient cohort and to investigate the association of polymorphisms in a number of genes with colonic location of disease and disease behavior. METHODS: Patients with L2 disease were identified from a database of 675 CD patients. Only patients with a normal small bowel enema (70%), ileoscopy alone (30%), or both (20%) were included. Genotyping was performed using PCR-SSP or the iPLEX platform. RESULTS: In all, 135 patients were classified with L2 disease. L2 disease was more common in women (74.0% versus 58.0%; P = 0.0004; odds ratio [OR] = 2.11, 95% confidence interval [CI] 1.36-3.26) and in never smokers (48.9% versus 36.9%; P = 0.008; OR = 1.64, 95% CI 1.09-2.45); 20.7% underwent colonic resection for severe disease. We confirmed that carriage of the HLA-DRB1*0103 allele is strongly associated with isolated colonic CD (14.9% versus 4.0%; P = 0.000016; OR 4.6, 95% CI 2.25-9.47) and report the novel association of this allele with time to first surgical event (log rank P = 0.001). There was no association with any of the known CD susceptibility loci (NOD2, IBD5, NOD1, IL23R, ATG16L1) and isolated colonic CD. A nonsynonymous polymorphism in MEKK1 (rs832582) was associated with CD susceptibility overall (15% versus 19%; P = 0.0083; OR = 1.28, 95% CI 1.07-1.54). The association was strongest in those patients not carrying a NOD2 mutation and had no effect on disease location. CONCLUSIONS: This study describes the clinical features of isolated colonic CD and demonstrates the importance of the HLA region in determining the molecular basis of colonic inflammation.

Polymorphisms and methylation of the reduced folate carrier in osteosarcoma.

High-dose methotrexate is a standard component in the treatment of osteogenic sarcoma. Impaired methotrexate uptake associated with decreased reduced folate carrier expression is a common mechanism of methotrexate resistance in osteogenic sarcoma samples. We investigated whether promoter methylation and polymorphisms in the 3' untranslated region are involved in regulating reduced folate carrier expression. In a cohort of 66 osteogenic sarcoma specimens, quantitative methylation-specific polymerase chain reaction and single-strand conformation polymorphism were performed. We found detectable levels of promoter methylation in 84.3% of samples. When related to the reduced folate carrier mRNA levels, a trend was observed that reduced folate carrier expression is lower in samples (median, 0.7) with greater than 10% DNA methylation as compared with those (median, 2.3) with less than 10% DNA methylation. The heterozygous polymorphisms of 2582 T/G and 2617C/T in the 3' untranslated region showed reduced folate carrier expression (median, 0.9) as compared with the wild-type 2582T and 2617C (median, 4.2). The data suggest promoter methylation and polymorphisms in the 3' untranslated region of the reduced folate carrier may be involved in its transcriptional regulation in osteogenic sarcoma. Further study is required to confirm this finding.

Protective versus promotional effects of white tea and caffeine on PhIP-induced tumorigenesis and beta-catenin expression in the rat.

A 1 year carcinogenicity bioassay was conducted in rats treated with three short cycles of 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP)/high-fat (HF) diet, followed by 2% white tea (wt/vol), 0.05% epigallocatechin-3-gallate (EGCG) or 0.065% caffeine as sole source of fluid intake. Thirty-two percent of the PhIP/HF controls survived to 1 year, compared with 50, 48.7 and 18.2% in groups given white tea, EGCG and caffeine, respectively. After 1 year, PhIP/HF controls had tumors in the colon, skin, small intestine, Zymbal's gland, salivary gland and pancreas. For all sites combined, excluding the colon, tumor incidence data were as follows: PhIP/HF 69.5%, PhIP/HF + EGCG 48.7%, PhIP/HF + white tea 46.9% and PhIP/HF + caffeine 13.3%. Unexpectedly, a higher incidence of colon tumors was detected in rats post-treated with white tea (69%) and caffeine (73%) compared with the 42% incidence in PhIP/HF controls. In the colon tumors, beta-catenin mutations were detected at a higher frequency after caffeine posttreatment, and there was a shift toward more tumors harboring substitutions of Gly34 with correspondingly high protein and messenger RNA expression seen for both beta-catenin and c-Myc. c-Myc expression exhibited concordance with tumor promotion, and there was a concomitant increase in cell proliferation versus apoptosis in colonic crypts. A prior report described suppression of PhIP-induced colonic aberrant crypts by the same test agents, but did not incorporate a HF diet. These findings are discussed in the context of epidemiological data which do not support an adverse effect of tea and coffee on colon tumor outcome-indeed, some such studies suggest a protective role for caffeinated beverages.

Sugar beet-associated bacterial and fungal communities show a high indigenous antagonistic potential against plant pathogens.

The aim of this study was to analyze microbial communities in/on sugar beet with special focus on antagonists toward plant pathogens. For this purpose, the composition of microorganisms isolated from the rhizosphere, phyllosphere, endorhiza, and endosphere of field-grown sugar beet plants was analyzed by a multiphasic approach at three different plant development stages at six locations in Europe. The analysis of microbial communities by Single Strand Conformation Polymorphism (SSCP) of 16S/18S rRNA clearly revealed the existence of discrete microenvironment- and site-specific patterns. A total of 1952 bacterial and 1344 fungal isolates screened by dual testing for antagonism toward the pathogens Aphanomyces cochlioides, Phoma betae, Pythium ultimum, and Rhizoctonia solani resulted in 885 bacterial (=45%) and 437 fungal (=33%) antagonists. In general, the indigenous antagonistic potential was very high and influenced by (a) the location, (b) the plant developmental stage, and (3) the microenvironment. Furthermore, we showed for the first time that the antagonistic potential was highly specific for each target pathogen. The majority of antagonistic microorganisms suppressed only one pathogen (bacteria: 664 = 75%; fungi: 256 = 59%), whereas the minority showed a broad host range (bacteria: 4 = 0.5%; fungi: 7 = 1.6%). The bacterial communities harbored the highest antagonistic potential against P. ultimum, whereas the fungal communities contained more antagonists against A. cochlioides and R. solani. In contrast to their high proportion, only a low diversity of antagonists at genotypic and species level was found. Novel antagonistic species, e.g., Subtercola pratensis or Microbacterium testaceum were found in the internal part of the sugar beet body.