Development of efficient one-pot three-component assembly of trityl olmesartan medoxomil

We have elaborated a one-pot three-component assembly of trityl olmesartan medoxomil starting from commercially available ethyl 4-(2-hydroxypropan-2-yl)-2-propyl-1H-imidazole-5-carboxylate, 5-(4′-(bromomethyl)-[1,1′-biphenyl]-2-yl)-1-trityl-1H-tetrazole and 4-(chloromethyl)-5-methyl-1,3-dioxol-2-one intermediates. The developed and optimized one-pot process provides 72–75% yield of trityl olmesartan medoxomil over three steps, which represents in average ca. 90% yield per synthetic step, on a 300?g scale. The process is conducted in simple fashion and provides highly pure trityl olmesartan medoxomil (up to 97.5% by HPLC), which can be easily converted to olmesartan medoxomil that fully complies with all ICH requirements. Furthermore, the described process significantly improves the primary process to trityl olmesartan medoxomil by drastic reduction of required unit operations and application of single reaction solvent through the reaction sequence. Moreover, the amount of used organic solvents was notably reduced. The developed process has provided solid bases for industrial production of trityl olmesartan medoxomil.     

Hepatocellular Carcinoma Displays Distinct DNA Methylation Signatures with Potential as Clinical Predictors

Background

Hepatocellular carcinoma (HCC) is characterized by late detection and fast progression, and it is believed that epigenetic disruption may be the cause of its molecular and clinicopathological heterogeneity. A better understanding of the global deregulation of methylation states and how they correlate with disease progression will aid in the design of strategies for earlier detection and better therapeutic decisions.

Methods and Findings

We characterized the changes in promoter methylation in a series of 30 HCC tumors and their respective surrounding tissue and identified methylation signatures associated with major risk factors and clinical correlates. A wide panel of cancer-related gene promoters was analyzed using Illumina bead array technology, and CpG sites were then selected according to their ability to classify clinicopathological parameters. An independent series of HCC tumors and matched surrounding tissue was used for validation of the signatures. We were able to develop and validate a signature of methylation in HCC. This signature distinguished HCC from surrounding tissue and from other tumor types, and was independent of risk factors. However, aberrant methylation of an independent subset of promoters was associated with tumor progression and etiological risk factors (HBV or HCV infection and alcohol consumption). Interestingly, distinct methylation of an independent panel of gene promoters was strongly correlated with survival after cancer therapy.

Conclusion

Our study shows that HCC tumors exhibit specific DNA methylation signatures associated with major risk factors and tumor progression stage, with potential clinical applications in diagnosis and prognosis.     

The reverse cholesterol transport pathway improves understanding of genetic networks for fat deposition and muscle growth in beef cattle

In the present study, thirteen genes involved in the reverse cholesterol transport (RCT) pathway were investigated for their associations with three fat depositions, eight fatty acid compositions and two growth-related phenotypes in a Wagyu x Limousin reference population, including 6 F(1) bulls, 113 F(1) dams, and 246 F(2) progeny. A total of 37 amplicons were used to screen single nucleotide polymorphisms (SNPs) on 6 F(1) bulls. Among 36 SNPs detected in 11 of these 13 genes, 19 were selected for genotyping by the Sequenom assay design on all F(2) progeny. Single-marker analysis revealed seven SNPs in ATP binding cassette A1, apolipoproteins A1, B and E, phospholipid transfer protein and paraoxinase 1 genes significantly associated with nine phenotypes (P<0.05). Previously, we reported genetic networks associated with 19 complex phenotypes based on a total of 138 genetic polymorphisms derived from 71 known functional genes. Therefore, after Bonferroni correction, these significant (adjusted P<0.05) and suggestive (adjusted P<0.10) associations were then used to identify genetic networks related to the RCT pathway. Multiple-marker analysis suggested possible genetic networks involving the RCT pathway for kidney-pelvic-heart fat percentage, rib-eye area, and subcutaneous fat depth phenotypes with markers derived from paraoxinase 1, apolipoproteins A1 and E, respectively. The present study confirmed that genes involved in cholesterol homeostasis are useful targets for investigating obesity in humans as well as for improving meat quality phenotypes in a livestock production.     

Supramolecular organization of photosynthetic membrane proteins in the chlorosome-containing bacterium Chloroflexus aurantiacus

The arrangement of core antenna complexes (B808-866-RC) in the cytoplasmic membrane of filamentous phototrophic bacterium Chloroflexus aurantiacus was studied by electron microscopy in cultures from different light conditions. A typical nearest-neighbor center-to-center distance of ~18 nm was found, implying less protein crowding compared to membranes of purple bacteria. A mean RC:chlorosome ratio of 11 was estimated for the occupancy of the membrane directly underneath each chlorosome, based on analysis of chlorosome dimensions and core complex distribution. Also presented are results of single-particle analysis of core complexes embedded in the native membrane.     

DNA Methylation Characteristics of Primary Melanomas with Distinct Biological Behaviour

In melanoma, the presence of promoter related hypermethylation has previously been reported, however, no methylation-based distinction has been drawn among the diverse melanoma subtypes. Here, we investigated DNA methylation changes associated with melanoma progression and links between methylation patterns and other types of somatic alterations, including the most frequent mutations and DNA copy number changes. Our results revealed that the methylome, presenting in early stage samples and associated with the BRAF^V600E mutation, gradually decreased in the medium and late stages of the disease. An inverse relationship among the other predefined groups and promoter methylation was also revealed except for histologic subtype, whereas the more aggressive, nodular subtype melanomas exhibited hypermethylation as well. The Breslow thickness, which is a continuous variable, allowed for the most precise insight into how promoter methylation decreases from stage to stage. Integrating our methylation results with a high-throughput copy number alteration dataset, local correlations were detected in the MYB and EYA4 genes. With regard to the effects of DNA hypermethylation on melanoma patients'' survival, correcting for clinical cofounders, only the KIT gene was associated with a lower overall survival rate. In this study, we demonstrate the strong influence of promoter localized DNA methylation changes on melanoma initiation and show how hypermethylation decreases in melanomas associated with less favourable clinical outcomes. Furthermore, we establish the methylation pattern as part of an integrated apparatus of somatic DNA alterations.     

The analogs of oxytocin and D-homoarginine vasopressin with bulky substituted phenylalanine in position 2

Solid phase technique on p-methylbenzhydrylamine resin wasused for the synthesis of eight analogs of oxytocin and 8-D-homoarginine vasopressin with the non-coded amino acids L- or D-2,3,4,5,6-pentamethylphenylalanine and L- or D-4-phenylphenylalanine in position 2. The preparation of theabove mentioned non-coded amino acids is described as well.All eight analogs were found to be potent inhibitors ofoxytocin activity in the uterotonic in vitro test in theabsence of Mg²⁺ ions. In the uterotonic test invitro in the presence of Mg²⁺ and in the test invivo, their potency is strongly decreased or completelyabolished. The substances are also weak pressor inhibitors.The L or D configuration does not seem to influence theactivity significantly.     

Chickpea facilitates phosphorus uptake by intercropped wheat from an organic phosphorus source

Pot experiments were conducted to investigate interspecific complementation in utilization of phytate and FePO₄ by plants in the wheat (Triticum aestivum L.)/chickpea (Cicer arietinum L.) intercropping under sterile and non-sterile conditions. The pots were separated into two compartments by either a solid root barrier to eliminate root contact and solute movement, by a nylon mesh (30 M) to prevent root contact but permit solute exchange, or not separated between the compartments. Wheat plants were grown in one compartment and chickpea in the other. Two P sources were tested at 60 mg P kg^–1 soil (sodium phytate or FePO₄). Under non-sterile conditions, the biomass of wheat was significantly greater when the roots were intermingled with chickpea than when the roots were separated from chickpea roots by a solid root barrier or nylon mesh. When phytate–P was applied, P concentrations in wheat (2.9 g kg^–1 in shoots and 1.4 g kg^–1 in roots) without root barrier between the two species were higher than those in the treatments with nylon mesh or with the solid root barrier separation (1.9 g kg^–1 in shoots and 1.0 g kg^–1 in roots). In contrast, P concentrations in wheat supplied with FePO₄ were similar between the root separation treatments. There was no significant difference in P uptake by chickpea between the P sources or between the root separation treatments, except that P uptake was greater in the phytate treatment with the root barrier. Total P uptake from phytate was increased by 25% without root separation compared to the root separation treatments. Under sterile conditions and supply of phytate–P, the biomass of wheat was doubled when the roots were intermingled with chickpea and increased by a third with the nylon mesh separation compared to that with the solid root barrier. Biomass production in wheat at various treatments correlated with P concentration in shoot. Biomass production and P concentration in chickpea were unaffected by root separation. Total P uptake by plants was 68% greater with root intermingling and 37% greater with nylon mesh separation than that with the solid root barrier. The results suggest that chickpea roots facilitate P utilization from the organic P by wheat.     

Pigment configuration in the light-harvesting protein of the xanthophyte alga <Emphasis Type="Italic">Xanthonema debile</Emphasis>

Proper biogenesis and maintenance of photosynthetic thylakoid membrane complexes are essential for the photosynthetic light reactions. A thylakoid lumenal protein, Psb27, plays a vital role in assembly or/and maintenance of photosystem II (PSII). In cyanobacteria, it is a small lipoprotein docked to the lumenal side of PSII, and functions in the assembly of the Mn₄Ca cluster and in the PSII repair cycle. However, Psb27 from Arabidopsis thaliana is not a lipoprotein, and it is involved in PSII repair and acclimation to fluctuating light stress, suggesting a functional divergence between Arabidopsis Psb27 and cyanobacterial Psb27s. To gain a better understanding of Psb27 from higher plants, we determined the crystal structure of Arabidopsis Psb27 by X-ray crystallography at a resolution of 1.85 Å. The structure of Arabidopsis Psb27 is a four-helix bundle, similar to its orthologues from cyanobacteria. However, there are several structural differences between Arabidopsis Psb27 and cyanobacterial Psb27s concerning the overall molecular shape, the N- and C-terminal structures, and the surface charge. These differences suggest that Psb27 from higher plants and cyanobacteria may function differently.