Rapid whole-plant bioassay for phosphorus phytoavailability in soils

Chemical P extraction from soils is an indirect and frequently questionable index for P availability. To monitor the dynamics of P availability in soils more directly following the application of P fertilizer, manure or sludge, a rapid, whole-plant bioassay was developed using tomato (Lycopersicon esculentum Mill.), Chinese cabbage (Brassica rapa L. var.pekinensis) and wheat (Triticum aestivum L.). Plant P extracted in 0.1 M H₂SO₄ (P_i) and total P (P_t) concentration or content in stem, leaves or whole shoots were highly correlated (P < 0.01) with P fertilizer rates or water-soluble (WSP) or Olsen P in various soils, over wide ranges of soil P status. The whole-plant P_i content was found to be as informative as the more complicated indices of P_t or P_iconcentration. The assay was used to compare availability of fertilizer-P and sewage-sludge-P after incorporation into alluvial soil during 1–100 days of incubation. While both soil and plant indices had shown that fertilizer-P was more highly available than sewage-sludge-P in each period, the bioassay was much more sensitive than the Olsen-P or WSP soil indices in showing P fixation and decrease of availability during incubation time. The bioassay is sufficiently rapid (5–12 days) to allow a study of short-term changes in soil-P availability following incorporation of various P additives, and it is applicable to a very wide range of P availability values (6–535 mg Olsen-P kg^–1), extending from lower than desired for crop production to higher than permitted from an environmental standpoint.     

Evaluating Hemorrhage in Renal Cell Carcinoma Using Susceptibility Weighted Imaging

Background

Intratumoral hemorrhage is a frequent occurrence in renal cell carcinoma and is an indicator of tumor subtype. We hypothesize that susceptibility weighted imaging (SWI) is sensitive to hemorrhage in renal cell carcinoma and can give a more diagnostic image when compared to conventional imaging techniques.

Materials and Methods

A retrospective review of 32 patients with clear cell renal cell carcinoma was evaluated. All patients underwent magnetic resonance imaging (MRI) and 22 out of 32 patients also underwent a computed tomography (CT) scan. Hemorrhage was classified into 3 different categories according to shape and distribution. Histopathology was obtained from all masses by radical nephrectomy. The ability to detect the presence of hemorrhage using CT, non-contrast conventional MRI and SWI was evaluated, and the patterns of hemorrhage were compared.

Results

Using pathologic results as the gold standard, the sensitivities of non-contrast conventional MRI, SWI and CT in detecting hemorrhage in clear cell renal cell carcinoma were 65.6%, 100% and 22.7%, respectively. Accuracy of non-contrast conventional MRI and SWI in evaluating hemorrhagic patterns were 31.3% and 100%, respectively.

Conclusion

These results demonstrate that SWI can better reveal hemorrhage and characterize the pattern more accurately than either non-contrast conventional MRI or CT. This suggests that SWI is the technique of choice for detecting hemorrhagic lesions in patients with renal cancer.     

miR-192 Directly Binds and Regulates Dicer1 Expression in Neuroblastoma

Neuroblastoma (NB) arises from the embryonic neural crest and is the most common extracranial solid tumor in children under 5 years of age. Reduced expression of Dicer1 has recently been shown to be in correlation with poor prognosis in NB patients. This study aimed to investigate the mechanisms that could lead to the down-regulation of Dicer1 in neuroblastoma. We used computational prediction to identify potential miRs down-regulating Dicer1 in neuroblastoma. One of the miRs that were predicted to target Dicer1 was miR-192. We measured the levels of miR-192 in 43 primary tumors using real time PCR. Following the silencing of miR-192, the levels of dicer1 cell viability, cell proliferation and migration capability were analyzed. Multivariate analysis identified miR-192 as an independent prognostic marker for relapse in neuroblastoma patients (p=0.04). We were able to show through a dual luciferase assay and side-directed mutational analysis that miR-192 directly binds the 3'' UTR of Dicer1 on positions 1232-1238 and 2282-2288. An increase in cell viability, proliferation and migration rates were evident in NB cells transfected with miR-192-mimic. Yet, there was a significant decrease in proliferation when NB cells were transfected with an miR-192-inhibitor We suggest that miR-192 might be a key player in NB by regulating Dicer1 expression.     

AS101 Prevents Diabetic Nephropathy Progression and Mesangial Cell Dysfunction: Regulation of the AKT Downstream Pathway

Diabetic nephropathy (DN) is characterized by proliferation of mesangial cells, mesangial expansion, hypertrophy and extracellular matrix accumulation. Previous data have cross-linked PKB (AKT) to TGFβ induced matrix modulation. The non-toxic compound AS101 has been previously shown to favorably affect renal pathology in various animal models and inhibits AKT activity in leukemic cells. Here, we studied the pharmacological properties of AS101 against the progression of rat DN and high glucose-induced mesangial dysfunction. In-vivo administration of AS101 to Streptozotocin injected rats didn’t decreased blood glucose levels but ameliorated kidney hypotrophy, proteinuria and albuminuria and downregulated cortical kidney phosphorylation of AKT, GSK3β and SMAD3. AS101 treatment of primary rat glomerular mesangial cells treated with high glucose significantly reduced their elevated proliferative ability, as assessed by XTT assay and cell cycle analysis. This reduction was associated with decreased levels of p-AKT, increased levels of PTEN and decreased p-GSK3β and p-FoxO3a expression. Pharmacological inhibition of PI3K, mTORC1 and SMAD3 decreased HG-induced collagen accumulation, while inhibition of GSK3β did not affect its elevated levels. AS101 also prevented HG-induced cell growth correlated to mTOR and (rp)S6 de-phosphorylation. Thus, pharmacological inhibition of the AKT downstream pathway by AS101 has clinical potential in alleviating the progression of diabetic nephropathy.     

An international comparability study on quantification of total methyl cytosine content

Various methods have been developed for quantitative analysis of DNA methylation. However, there is currently no reference analysis system regarding DNA methylation with which other analytical approaches can be compared and evaluated. A standard measurement system that includes reference methods and reference materials may improve comparability and credibility of data obtained from different analytical environments. In an effort to establish a standard system for measurement of DNA methylation, the Korea Research Institute of Standards and Science (KRISS) coordinated an international comparison study among different national metrology institutes. An initial stage of the study involved an intercomparison regarding quantitative measurement of total methyl cytosine contents in artificially constructed DNA samples. The measurement principle involved measurement of dNMP contents following enzymatic hydrolysis of DNA samples. Results of the study showed good comparability among four of five participants and close agreement with reference values assigned by the coordinating laboratory. Conflicting data from one participant may have resulted from incomplete hydrolysis of samples due to use of insufficient amounts of enzymes. These results indicate that comparable and accurate results can be obtained from different measurement environments if digestion conditions are controlled appropriately and valid calibration systems are employed.     

Regulation of Mating and Meiosis in Yeast by the Mating-Type Region

A supposed sporulation-deficient mutation of Saccharomyces cerevisiae is found to affect mating in haploids and in diploids, and to be inseparable from the mating-type locus by recombination. The mutation is regarded as a defective a allele and is designated a*. This is confirmed by its dominance relations in diploids, triploids, and tetraploids. Tetrad analysis of tetraploids and of their sporulating diploid progeny suggests the existence of an additional locus, RME, which regulates sporulation in yeast strains that can mate. Thus the recessive homozygous constitution rme/rme enables the diploids a*/α, a/a*, and α/α to go through meiosis. Haploids carrying rme show apparent premeiotic DNA replication in sporulation conditions. This new regulatory locus is linked to the centromere of the mating-type chromosome, and its two alleles, rme and RME, are found among standard laboratory strains.     

EphA4 blockers promote axonal regeneration and functional recovery following spinal cord injury in mice

Upregulation and activation of developmental axon guidance molecules, such as semaphorins and members of the Eph receptor tyrosine kinase family and their ligands, the ephrins, play a role in the inhibition of axonal regeneration following injury to the central nervous system. Previously we have demonstrated in a knockout model that axonal regeneration following spinal cord injury is promoted in the absence of the axon guidance protein EphA4. Antagonism of EphA4 was therefore proposed as a potential therapy to promote recovery from spinal cord injury. To further assess this potential, two soluble recombinant blockers of EphA4, unclustered ephrin-A5-Fc and EphA4-Fc, were examined for their ability to promote axonal regeneration and to improve functional outcome following spinal cord hemisection in wildtype mice. A 2-week administration of either of these blockers following spinal cord injury was sufficient to promote substantial axonal regeneration and functional recovery by 5 weeks following injury. Both inhibitors produced a moderate reduction in astrocytic gliosis, indicating that much of the effect of the blockers may be due to promotion of axon growth. These studies provide definitive evidence that soluble inhibitors of EphA4 function offer considerable therapeutic potential for the treatment of spinal cord injury and may have broader potential for the treatment of other central nervous system injuries.     

Activation of myeloid cell-specific adhesion class G protein-coupled receptor EMR2 via ligation-induced translocation and interaction of receptor subunits in lipid raft microdomains

The adhesion class G protein-coupled receptors (adhesion-GPCRs) play important roles in diverse biological processes ranging from immunoregulation to tissue polarity, angiogenesis, and brain development. These receptors are uniquely modified by self-catalytic cleavage at a highly conserved GPCR proteolysis site (GPS) dissecting the receptor into an extracellular subunit (α) and a seven-pass transmembrane subunit (β) with cellular adhesion and signaling functions, respectively. Using the myeloid cell-restricted EMR2 receptor as a paradigm, we exam the mechanistic relevance of the subunit interaction and demonstrate a critical role for GPS autoproteolysis in mediating receptor signaling and cell activation. Interestingly, two distinct receptor complexes are identified as a result of GPS proteolysis: one consisting of a noncovalent α-β heterodimer and the other comprising two completely independent receptor subunits which distribute differentially in membrane raft microdomains. Finally, we show that receptor ligation induces subunit translocation and colocalization within lipid rafts, leading to receptor signaling and inflammatory cytokine production by macrophages. Our present data resolve earlier conflicting results and provide a new mechanism of receptor signaling, as well as providing a paradigm for signal transduction within the adhesion-GPCR family.     

Anti-proliferative effects of a novel isoflavone derivative in medullary thyroid carcinoma: An in vitro study

Currently available treatments for patients with medullary thyroid carcinoma (MTC) with residual or recurrent disease after primary surgery have low efficacy rates. In view of the possible role of estrogen in the development of thyroid neoplasia, we explored whether proliferation of the human MTC TT cell line, might be curbed by carboxy-daidzein-tBoc (cD-tBoc), a novel isoflavone derivative. Estrogen receptor (ER) α mRNA expression in TT cells was more abundant than ERβ, with a ratio of 48:1. Estradiol-17β (E2) increased DNA synthesis in a dose dependent manner. [(3)H]-thymidine incorporation was also stimulated by the ERβ agonist DPN and the ERα agonist PPT. cD-tBoc inhibited TT cell growth as assessed by thymidine incorporation, XTT assay, and microscopic analysis of culture wells. Creatine kinase specific activity, a marker of the modulatory effects of estrogen on cell energy metabolism, was likewise inhibited. The inhibitory effect of cD-tBoc on [(3)H]-thymidine incorporation could be blocked by the ERβ antagonist PTHPP but not by the ERα antagonist MPP, suggesting that the antiproliferative effect of cD-tBoc on these cells is mediated through ERβ. Furthermore, cD-tBoc potently increased apoptosis and cell necrosis. Co-incubation with the antiapoptotic agent Z-VAD-FMK reversed the growth inhibitory effect elicited by cD-tBoc. These results support the hypothesis that estrogens are involved in the proliferation of MTC. The potent anti-proliferative effects mediated by isoflavone derivatives in the human MTC cell line TT suggest and that this property may be utilized to design effective anti-neoplastic agents.