Validation of DNA methylation profiling in formalin-fixed paraffin-embedded samples using the Infinium HumanMethylation450 Microarray

A formalin-fixed paraffin-embedded (FFPE) sample usually yields highly degraded DNA, which limits the use of techniques requiring high-quality DNA, such as Infinium Methylation microarrays. To overcome this restriction, we have applied an FFPE restoration procedure consisting of DNA repair and ligation processes in a set of paired fresh-frozen (FF) and FFPE samples. We validated the FFPE results in comparison with matched FF samples, enabling us to use FFPE samples on the Infinium HumanMethylation450 Methylation array.     

Cross-pathogenicity of Rhizoctonia solani strains on pasture legumes in pasture-crop rotations

In Australia, in the past, pasture legumes were rotated mainly with cereals, but increasingly these rotations now involve pasture legumes with a wider range of crops, including legumes. This increasing frequency of the leguminous host in the rotation system may be associated with increased root rots in legumes in the current pasture-crop rotations. The primary aim of this study was to see whether the pathogenicity on pasture legumes of strains of Rhizoctonia solani sourced from lupins and cereals (common crops in rotation with pastures) is associated with increased incidence of root rots in pasture legumes in the disease conducive sandy soils of the Mediterranean regions of southern Australia. The second aim was to determine sources of resistance among newly introduced pasture legumes to R. solani strains originating from rotational crops as this would reduce the impact of disease in the pasture phase. Fifteen pasture legume genotypes were assessed for their resistance/susceptibility to five different zymogram groups (ZG) of the root rot pathogen R. solani under glasshouse conditions. Of the R. solani groups tested, ZG1–5 and ZG1–4 (both known to be pathogenic on cereals and legumes) overall, caused the most severe root disease across the genotypes tested, significantly more than ZG6 (known to be pathogenic on legumes), in turn significantly >ZG4 (known to be pathogenic on legumes) which in turn was >ZG11 (known to be pathogenic on legumes including tropical species). Overall, Ornithopus sativus Brot. cvs Cadiz and Margurita, Trifolium michelianum Savi. cvs Paradana and Frontier and T. purpureum Loisel. cv. Electro showed a significant level of resistance to root rot caused by R. solani ZG11 (root disease scores ≤1.2 on a 1–3 scale where 3 = maximum disease severity) while O. sativus cvs Cadiz and Erica showed a significant level of resistance to root rot caused by R. solani ZG4 (scores ≤1.2). O. compressus L. cvs Charano and Frontier, O. sativus cv. Erica, and T. purpureum cv. Electro showed some useful resistance to root rot caused by R. solani ZG6 (scores ≤1.8). This is the first time that cvs Cadiz, Electro, Frontier, Margurita and Paradana have been recognised for their levels of resistance to root rot caused by R. solani ZG11; and similarly for cvs Cadiz and Erica against ZG4; and for cvs Charano, Erica, and Electro against ZG6. These genotypes with resistance may also serve as useful sources of resistance in pasture legume breeding programs and also could potentially be exploited directly into areas where other rotation crops are affected by these R. solani strains. None of the tested genotypes showed useful resistance to R. solani ZG1–4 (scores ≥2.0) or ZG1–5 (scores ≥2.5). This study demonstrates the relative potential of the various R. solani ZG strains, and particularly ZG1–4, ZG1–5, ZG4 and ZG6 to attack legume pastures and pose a significant threat to non-pasture crop species susceptible to these strains grown in rotation with these pasture legumes. Significantly, the cross-pathogenicity of these strains could result in the continuous build-up of inoculum of these strains that may seriously affect the productivity eventually of legumes in all rotations. In particular, when choosing pasture legumes as rotation crops, caution needs to be exercised so that the cultivars deployed are those with the best resistance to the R. solani ZGs most likely to be prevalent at the location.     

Cell Cycle activation and ouabain-sensitive ion movements of 3T3 and C3H-10T½ fibroblasts

The introduction of either PGF_2α (10^?7 M) or TPA (10^?7 M) stimulated, ouabain-sensitive ⁸⁶Rb⁺ influx at 30 min in postconfluent 3T3-4 mouse fibroblast cultures by 117% and 124%, respectively. Both TPA and PGF_2α at these concentrations stimulated the incorporation of ³H-TdR into DNA. TPA had the greatest stimulatory effect, which was similar to that obtained with 10% fetal calf serum. In accord with the idea that modulation of membrane processes such as Na⁺/K⁺ pump activity in fibroblasts may reflect important events related to the initiation of DNA synthesis, it was observed that in both 3T3-4 and C3H-1 0T½ cells there were parallel increases in ³H-TdR incorporation and ouabain-sensitive ⁸⁶Rb⁺ influxes with 10^?7 M TPA, whereas PGF_2α stimulated a significant increase in ³H-TdR incorporation in 3T3-4 but not C3H-10T½ cells and only marginal increases in ouabain-sensitive ⁸⁶Rb⁺ influx in both. Therefore, although there appears to be a close correlation between Na⁺/K⁺ pump activation and subsequent S-phase entry following TPA stimulation, a similar correlation for PGF_2α cannot be confirmed.     

Origin and phylogeny of Oryza species with the CD genome based on multiple-gene sequence data

The CD genome species in the genus Oryza are endemic to Latin America, including O. alta, O. grandiglumis and O. latifolia. Origins and phylogenetic relationship of these species have long been in dispute and are still ambiguous due to their homogeneous genome type, similar morphological characteristics and overlapping distribution. In the present study, we sequenced two chloroplast fragments (matK and trnL-trnF) and portions of three nuclear genes (Adh1, Adh2 and GPA1) from sixteen accessions representing seven species with the C, CD, and E genomes, as well as one G genome species as the outgroup. Phylogenetic analyses using parsimony and distance methods strongly supported that the CD genome originated from a single hybridization event, and that the C genome species (O. officinalis or O. rhizomatis instead of O. eichingeri) served as the maternal parent while the E genome species (O. australiensis) was the paternal donor during the formation of CD genome. In addition, the consistent phylogenetic relationships among the CCDD species indicated that significant divergence existed between O. latifolia and the other two (O. alta and O. grandiglumis), which corroborated the suggestion of treating the latter two as a single species or as taxa within species.We thank Tao Sang of Michigan State University (East Lansing, USA) and Bao-rong Lu of Fudan University (Shanghai, China) for their encouragement and assistance. We are also grateful to the International Rice Research Institute (Manila, Philippines) for providing plant material for this study. This research was supported by the Chinese Academy of Sciences (kscxz-sw-101A), the National Natural Science Foundation of China (30025005) and the Program for Key International S & T Cooperation Project of P. R. China (2001CB711103).     

Local synthesis of the phosphatidylinositol-3,4-bisphosphate lipid drives focal adhesion turnover

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Upregulation of miR-183 represses neuropathic pain through inhibiton of MAP3K4 in CCI rat models

Many studies have verified that microRNAs contribute a lot to neuropathic pain progression. Furthermore, nerve-related inflammatory cytokines play vital roles in neuropathic pain progression. miR-183 has been identified to have a common relationship with multiple pathological diseases. However, the potential effects of miR-183 in the process of neuropathic pain remain undetermined. Therefore, we performed the current study with the purpose of finding the functions of miR-183 in neuropathic pain progression using a chronic sciatic nerve injury (CCI) rat model. We demonstrated that miR-183 expression levels were evidently reduced in CCI rats in contrast with the control group. Overexpression of miR-183 produced significant relief of mechanical hyperalgesia, as well as thermal hyperalgesia in CCI rats. Furthermore, neuropathic pain-correlated inflammatory cytokine expression levels containing interleukin-6 (IL-6) and interleukin-1β (IL-1β), cyclooxygenase-2 (COX-2) were obviously inhibited by upregulation of miR-183. Meanwhile, dual-luciferase reporter assays showed MAP3K4 was a direct downstream gene of miR-183. The expression levels of MAP3K4 were modulated by the increased miR-183 negatively, which lead to the downregulation of IL-6, IL-1β, and COX-2, and then reduced neuropathic pain progression, respectively. Overall, our study pointed out that miR-183 was a part of the negative regulator which could relieve neuropathic pain by targeting MAP3K4. Thus it may provide a new clinical treatment for neuropathic pain patients clinical therapy.