Synthesis and in vivo evaluation of [11C]SN003 as a PET ligand for CRF1 receptors

Synthesis and evaluation of [O-methyl-11C](4-methoxy-2-methylphenyl)[1-(1-methoxymethylpropyl)-6-methyl-1H-[1,2,3]triazolo[4,5-c]pyridin-4-yl]amine or [11C]SN003 ([11C]6), as a PET imaging agent for CRF1 receptors, in baboons is described. 4-[1-(1-Methoxymethylpropyl)-6-methyl-1H-[1,2,3]triazolo[4,5-c]pyridin-4-ylamino]-3-methylphenol (5), the precursor molecule for the radiolabeling, was synthesized from 2,4-dichloro-6-methyl-3-nitropyridine in seven steps with 20% overall yield. The total time required for the synthesis of [11C]SN003 is 30 min from EOB using [11C]methyl triflate in the presence of NaOH in acetone. The yield of the synthesis is 22% (EOS) with >99% chemical and radiochemical purities and a specific activity of >2000 Ci/mmol. PET studies in baboon show that [11C]6 penetrates the BBB and accumulates in brain. No detectable specific binding was observed, likely due to the rapid metabolism or low density of CRF1 receptors in primate brain.     

Gamma irradiation as a useful tool for the isolation of astaxanthin-overproducing mutant strains of Phaffia rhodozyma

Astaxanthin is a carotenoid pigment responsible for the red color of the flesh of many marine animals. There is an increasing interest in the use of astaxanthin in aquaculture, chemical, pharmaceutical, and alimentary industries. Phaffia rhodozyma has been identified as the best biological source of astaxanthin. Mutagenesis was carried out using different doses of gamma irradiation (1.0, 2.0, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, and 7.0 kGy), and 10 mutant colonies (Gam1-Gam10) were obtained. Highly pigmented mutant strains produced astaxanthin at approximately 15?887.5?μg/L dry mass of yeast, whereas the parental strain produced it at 1061.64?μg/g dry mass of yeast. In the thin-layer chromatography analysis, P. rhodozyma JH-82 and Gam1 mutant strain produced the same retention factor (R(f)) values, but Gam1 showed a higher astaxanthin content than JH-82.     

Identification of genes expressed in the angiosperm female gametophyte

Until recently, identification of gene regulatory networks controlling the development of the angiosperm female gametophyte has presented a significant challenge to the plant biology community. The angiosperm female gametophyte is fairly inaccessible because it is a highly reduced structure relative to the sporophyte and is embedded within multiple layers of the sporophytic tissue of the ovule. Moreover, although mutations affecting the female gametophyte can be readily isolated, their analysis can be difficult because most affect genes involved in basic cellular processes that are also required in the diploid sporophyte. In recent years, expression-based approaches in multiple species have begun to uncover gene sets expressed in specific female gametophyte cells as a means of identifying regulatory networks controlling cell differentiation in the female gametophyte. Here, recent efforts to identify and analyse gene expression programmes in the Arabidopsis female gametophyte are reviewed.     

Diminished transmission of drug resistant HIV-1 variants with reduced replication capacity in a human transmission model

Background

Different patterns of drug resistance are observed in treated and therapy naïve HIV-1 infected populations. Especially the NRTI-related M184I/V variants, which are among the most frequently encountered mutations in treated patients, are underrepresented in the antiretroviral naïve population. M184I/V mutations are known to have a profound effect on viral replication and tend to revert over time in the new host. However it is debated whether a diminished transmission efficacy of HIV variants with a reduced replication capacity can also contribute to the observed discrepancy in genotypic patterns.As dendritic cells (DCs) play a pivotal role in HIV-1 transmission, we used a model containing primary human Langerhans cells (LCs) and DCs to compare the transmission efficacy M184 variants (HIV-M184V/I/T) to HIV wild type (HIV-WT). As control, we used HIV harboring the NNRTI mutation K103N (HIV-K103N) which has a minor effect on replication and is found at a similar prevalence in treated and untreated individuals.

Results

In comparison to HIV-WT, the HIV-M184 variants were less efficiently transmitted to CCR5⁺ Jurkat T cells by both LCs and DCs. The transmission rate of HIV-K103N was slightly reduced to HIV-WT in LCs and even higher than HIV-WT in DCs. Replication experiments in CCR5⁺ Jurkat T cells revealed no apparent differences in replication capacity between the mutant viruses and HIV-WT. However, viral replication in LCs and DCs was in concordance with the transmission results; replication by the HIV-M184 variants was lower than replication by HIV-WT, and the level of replication of HIV-K103N was intermediate for LCs and higher than HIV-WT for DCs.

Conclusions

Our data demonstrate that drug resistant M184-variants display a reduced replication capacity in LCs and DCs which directly impairs their transmission efficacy. As such, diminished transmission efficacy may contribute to the lower prevalence of drug resistant variants in therapy naive individuals.

     

Hypermethylated RASSF1 and SLC5A8 promoters alongside BRAFV600E mutation as biomarkers for papillary thyroid carcinoma

Circulating cell-free DNA (cfDNA) has been considered as a diagnostic source to track genetic and epigenetic alterations in cancer. We aimed to study mutation in addition to the methylation status in the promoter regions of RASSF1 and SLC5A8 genes in tissues and circulating free DNA samples of patients affected with papillary thyroid carcinoma (PTC) and thyroid nodules as controls. BRAF^V600E mutation was studied by ARMS-scorpion real-time polymerase chain reaction method in 57 PTC and 45 thyroid nodule cases. Methylation status of RASSF1 and SLC5A8 promoter regions was analyzed by methylation-specific high-resolution melting curve analysis. BRAF^V600E mutation was found in 39 (68.4%) out of 57 PTC tissue samples, while in 33 (49.1%) cases of cfDNA, this mutation was detected. The frequency of BRAF^V600E mutation in cfDNA was significantly different between metastatic and nonmetastatic PTC cases (22 of 33 PTC cases vs. 5 of 34 thyroid nodule samples). Methylation levels of three promoter regions of SLC5A8 and proximal promoter region of RASSF1 was significantly different between PTC and thyroid nodule cases in both cfDNA and tissue DNA. In addition, the methylation status of these two genes in tissue DNA was reflected in methylation status observed in cfDNA. This study confirmed that BRAF^V600E mutation is better for discrimination between papillary thyroid carcinoma and thyroid nodules. On the other hand, hypermethylation in the more proximal promoter regions to RASSF1 and SLC5A8 genes showed higher sensitivity and more acceptable specificity for this discrimination.