Obesity-Related Genetic Determinants of Heart Failure Prognosis.

PURPOSE: Recent advances in genomics offer a smart option for predicting future risk of disease and prognosis. The objective of this study was to examine the prognostic value in heart failure (HF) patients, of a series of single nucleotide polymorphisms (SNPs). METHODS: A selection of 192 SNPs found to be related with obesity, body mass index, circulating lipids or cardiovascular diseases were genotyped in 191 patients with HF. Anthropometrical and clinical variables were collected for each patient, and death and readmission by HF were registered as the primary endpoint. RESULTS: A total of 53 events were registered during a follow-up period of 438 (263-1077) days (median (IQR)). Eight SNPs strongly related to obesity and HF prognosis were selected as possible prognostic variables. From these, rs10189761 and rs737337 variants were independently associated with HF prognosis (HR 2.295 (1.287-4.089, 95% CI); p = 0.005), whereas rs10423928, rs1800437, rs737337 and rs9351814 were related with bad prognosis only in obese patients (HR 2.142 (1.438-3.192, 95% CI); p = 0.00018). Combined scores of the genomic variants were highly predictive of poor prognosis. CONCLUSIONS: SNPs rs10189761 and rs737337 were identified, for the first time, as independent predictors of major clinical outcomes in patients with HF. The data suggests an additive predictive value of these SNPs for a HF prognosis. In particular for obese patients, SNPs rs10423928, rs1800437, rs737337 and rs9351814 were related with a bad prognosis. Combined scores weighting the risk of each genomic variant could effect interesting new tools to stratify the prognostic risk of HF patients.

Synthesis of α-aminoboronic acids.

This review describes available methods for the preparation of α-aminoboronic acids in their racemic or in their enantiopure form. Both, highly stereoselective syntheses and asymmetric procedures leading to the stereocontrolled generation of α-aminoboronic acid derivatives are included. The preparation of acyclic, carbocyclic and azacyclic α-aminoboronic acid derivatives is covered. Within each section, the different synthetic approaches have been classified according to the key bond which is formed to complete the α-aminoboronic acid skeleton.

ß-Phenylproline: the high ß-turn forming propensity of proline combined with an aromatic side chain.

The conformational propensities of the proline analogue bearing a phenyl substituent attached to the ß carbon, in either a cis or a trans configuration relative to the carbonyl group, have been investigated. The behaviour of cis- and trans(ßPh)Pro has been compared with that of proline in homochiral and heterochiral dipeptide sequences. NMR and IR studies as well as X-ray diffraction analysis provide evidence that the ß-phenyl substituent does not disrupt the tendency of proline to occupy the i+1 position of a ß-turn. The puckering of the pyrrolidine ring is significantly affected by the presence of the aromatic substituent, which tends to occupy positions that minimize steric repulsions. As a consequence, this substituent adopts specific well-defined orientations, which are more restricted for the cis derivative. Interactions between this aromatic group and that in the adjacent phenylalanine residue may be responsible for some of the conformational differences observed among the different peptides studied.

Access to enantiomerically pure cis- and trans-ß-phenylproline by high-performance liquid chromatography resolution.

The preparation of all four stereoisomers of the proline analog that bears a phenyl group attached to the ß carbon either cis or trans to the carboxylic acid (cis- and trans-ß-phenylproline, respectively) has been addressed. The methodology developed allows access to multigram quantities of the target amino acids in enantiomerically pure form and suitably protected for use in peptide synthesis. Racemic precursors of cis-ß-phenylproline and trans-ß-phenylproline were prepared from easily available starting materials and subjected to high-performance liquid chromatography enantioseparation. Semipreparative columns (250 × 20 mm) containing chiral stationary phases based on amylose (Chiralpak IA) (Daicel-Chiral Technologies Europe, Illkirch, France) or cellulose (Chiralpak IC) were used respectively for the resolution of the cis- and trans-ß-phenylproline precursors.

Integrating the intrinsic conformational preferences of noncoded α-amino acids modified at the peptide bond into the noncoded amino acids database.

Recently, we reported a database (Noncoded Amino acids Database; http://recerca.upc.edu/imem/index.htm) that was built to compile information about the intrinsic conformational preferences of nonproteinogenic residues determined by quantum mechanical calculations, as well as bibliographic information about their synthesis, physical and spectroscopic characterization, the experimentally established conformational propensities, and applications (Revilla-López et al., J Phys Chem B 2010;114:7413-7422). The database initially contained the information available for α-tetrasubstituted α-amino acids. In this work, we extend NCAD to three families of compounds, which can be used to engineer peptides and proteins incorporating modifications at the--NHCO--peptide bond. Such families are: N-substituted α-amino acids, thio-α-amino acids, and diamines and diacids used to build retropeptides. The conformational preferences of these compounds have been analyzed and described based on the information captured in the database. In addition, we provide an example of the utility of the database and of the compounds it compiles in protein and peptide engineering. Specifically, the symmetry of a sequence engineered to stabilize the 3(10)-helix with respect to the α-helix has been broken without perturbing significantly the secondary structure through targeted replacements using the information contained in the database.

Deep sequencing of GDF5 reveals the absence of rare variants at this important osteoarthritis susceptibility locus.

OBJECTIVE: The common single nucleotide polymorphism (SNP) rs143383 in the 5' untranslated region (5'UTR) of growth and differentiation factor 5 (GDF5) is strongly associated with osteoarthritis (OA) and influences GDF5 allelic expression in vitro and in the joint tissues of OA patients. This effect is modulated in cis by another common SNP, also located within the 5'UTR, whilst a common SNP in the 3'UTR influences allelic expression independent of rs143383. DNA variants can be common, rare or extremely rare/unique. To therefore enhance our understanding of the allelic architecture of this very important OA susceptibility locus we sequenced the gene for potentially functional and novel rare variants. METHOD: Using the Sanger method we sequenced GDF5 in 992 OA patients and 944 controls, with DNA changes identified by sequencing software. We encompassed the protein-coding region of the two GDF5 exons, both untranslated regions and approximately 100 bp of the proximal promoter of the gene. RESULTS: We detected 13 variants. Six were extremely rare with minor allele frequencies (MAFs) of ≤ 0.0006. One is in a predicted transcription factor binding site in the GDF5 promoter whilst two substitute conserved amino acids. The remaining seven variants were common and are previously known variants, with MAFs ranging from 0.025 to 0.39. There was a complete absence of variants with frequencies in-between the extremely rare (n=6) and the common (n=7). CONCLUSIONS: This is the first report of the deep sequencing of an OA susceptibility locus. The absence of rare variants informs us that within the regions of the gene that we have sequenced GDF5 does not harbour any novel variants that are able to contribute, at a population level, to the OA association signal mediated by rs143383 nor does it harbour, at a population level, any novel variants that can influence OA susceptibility independent of rs143383.

Practical access to the proline analogs (S,S,S)- and (R,R,R)-2-methyloctahydroindole-2-carboxylic acids by HPLC enantioseparation.

An efficient methodology for the preparation of the α-tetrasubstituted proline analog (S,S,S)-2-methyloctahydroindole-2-carboxylic acid, (S,S,S)-(αMe)Oic, and its enantiomer, (R,R,R)-(αMe)Oic, has been developed. Starting from easily available substrates and through simple transformations, a racemic precursor has been synthesized in excellent yield and further subjected to HPLC resolution using a cellulose-derived chiral stationary phase. Specifically, a semipreparative (250 mm × 20 mm ID) Chiralpak® IC column has allowed the efficient resolution of more than 4 g of racemate using a mixture of n-hexane/tert-butyl methyl ether/2-propanol as the eluent. Multigram quantities of the target amino acids have been isolated in enantiomerically pure form and suitably protected for incorporation into peptides.

Exploring the biological role of postzygotic and germinal de novo mutations in ASD.

De novo mutations (DNMs), including germinal and postzygotic mutations (PZMs), are a strong source of causality for Autism Spectrum Disorder (ASD). However, the biological processes involved behind them remain unexplored. Our aim was to detect DNMs (germinal and PZMs) in a Spanish ASD cohort (360 trios) and to explore their role across different biological hierarchies (gene, biological pathway, cell and brain areas) using bioinformatic approaches. For the majority of the analysis, a combined ASD cohort (N = 2171 trios) was created using previously published data by the Autism Sequencing Consortium (ASC). New plausible candidate genes for ASD such as FMR1 and NFIA were found. In addition, genes harboring PZMs were significantly enriched for miR-137 targets in comparison with germinal DNMs that were enriched in GO terms related to synaptic transmission. The expression pattern of genes with PZMs was restricted to early mid-fetal cortex. In contrast, the analysis of genes with germinal DNMs revealed a spatio-temporal window from early to mid-fetal development stages, with expression in the amygdala, cerebellum, cortex and striatum. These results provide evidence of the pathogenic role of PZMs and suggest the existence of distinct mechanisms between PZMs and germinal DNMs that are influencing ASD risk.

Differential upregulation of the three transforming growth factor beta isoforms in human osteoarthritic cartilage.

OBJECTIVES: Decreased levels of transforming growth factor beta (TGFbeta) have been related to the failure of cartilage repair in experimental models of osteoarthritis. This study aimed to examine this aspect of osteoarthritis in human cartilage. METHODS: Cartilage samples were obtained from 11 patients with hip osteoarthritis and 11 patients with femoral neck fracture who were undergoing total hip replacement. Gene expression of the three TGFbeta isoforms, collagen type II (COL2A1) and aggrecan (AGC1) was analysed by reverse transcription quantitative PCR and immunohistochemistry. RESULTS: Expression of the three TGFbeta isoforms was increased in osteoarthritis cartilage. The upregulation was more marked for the TGFbeta3 isoform (2.3-fold) than for TGFbeta1 (1.6-fold) or TGFbeta2 (1.7-fold). The messenger RNA levels of TGFbeta1 and TGFbeta2 were strongly correlated in osteoarthritis cartilage (r(s) = 0.83, p = 0.002), but levels of TGFbeta3 were uncorrelated with any of the two other TGFbeta isoforms. Immunohistochemistry showed an extension of immunoreactivity for the three TGFbeta isoforms to more chondrocytes and to deeper cartilage layers in the more severe osteoarthritis lesions. No correlation of TGFbeta isoforms with COL2A1 or AGC1 expression levels was found. CONCLUSIONS: The three isoforms of TGFbeta were differentially upregulated in late osteoarthritis in relation to an increased percentage of TGFbeta-positive chondrocytes. These results indicate that cartilage damage progresses in spite of the TGFbeta stimulus for cartilage anabolism and that other causes of the failure to cope with the increased cartilage catabolism of osteoarthritis should be investigated.

Testing the druggable endothelial differentiation gene 2 knee osteoarthritis genetic factor for replication in a wide range of sample collections.

OBJECTIVES: To replicate a previously reported association with osteoarthritis (OA) of the promoter single nucleotide polymorphism (SNP) rs10980705 in the endothelial differentiation gene 2 (EDG2). METHODS: Five collections of samples, four from Europe and one from China, were studied. They included patients with 3 OA phenotypes: 1501 with knee OA, 1497 with hip OA and 376 with generalised OA. A total of 2521 controls were also studied. Allele and genotype frequencies of the rs10980705 SNP were analysed in each individual sample collection and in pooled data. In addition, a meta-analysis to incorporate results from the original Japanese report was performed. RESULTS: The association of the rs10980705 SNP with knee OA was not replicated in any of the five sample collections studied or in their combined analysis (odds ratio (OR) 1.10, 95% CI 0.98 to 1.22; p = 0.10). Meta-analysis of all data, including the original Japanese study, did show association with knee OA (OR 1.15, 95% CI 1.06 to 1.26; p = 0.002) but the effect was accounted for by the Japanese data and was less significant than the original report. No association was found with hip OA or with generalised OA. CONCLUSIONS: The original report of a promising genetic association between a druggable G-protein coupled receptor, EDG2, and knee OA has not been replicated. This lack of replication could be due to a modest effect of the promoter polymorphism that will require even larger studies (the winners curse) although a more pronounced effect in the Asian population vs Europeans cannot be excluded.

Genetic variation in the nuclear factor kappaB pathway in relation to susceptibility to rheumatoid arthritis.

OBJECTIVE: To examine genetic association between rheumatoid arthritis (RA) and known polymorphisms in core genes of the nuclear factor (NF)kappaB pathway, the major intracellular pathway in RA pathogenesis. METHODS: Discovery and replication sample sets of Spanish patients with RA and controls were studied. A total of 181 single nucleotide polymorphisms (SNPs) uniformly spaced along the genomic sequences of 17 core genes of the NFkappaB pathway (REL, RELA, RELB, NFKB1, NFKB2, NFKBIA, NFKBIB, NFKBIE, IKBKA, IKBKB, IKBKE, IKBKAP, KBRAS1, KBRAS2, MAP3K1, MAP3K14, TAX1BP1) were studied by mass spectrometry analysis complemented with 5'-nuclease fluorescence assays in the discovery set, 458 patients with RA and 657 controls. SNPs showing nominal significant differences were further investigated in the replication set of 1189 patients with RA and 1092 controls. RESULTS: No clear reproducible association was found, although 12 SNPs in IKBKB, IKBKE and REL genes showed significant association in the discovery set. Interestingly, two of the SNPs in the IKBKE gene, weakly associated in the discovery phase, showed a trend to significant association in the replication phase. Pooling both sample sets together, the association with these two SNPs was significant. CONCLUSION: We did not find any major effect among the explored members of the NFkappaB pathway in RA susceptibility. However, it is possible that variation in the IKBKE gene could have a small effect that requires replication in additional studies.

In silico molecular engineering for a targeted replacement in a tumor-homing peptide.

A new amino acid has been designed as a replacement for arginine (Arg, R) to protect the tumor-homing pentapeptide CREKA (Cys-Arg-Glu-Lys-Ala) from proteases. This amino acid, denoted (Pro)hArg, is characterized by a proline skeleton bearing a specifically oriented guanidinium side chain. This residue combines the ability of Pro to induce turn-like conformations with the Arg side-chain functionality. The conformational profile of the CREKA analogue incorporating this Arg substitute has been investigated by a combination of simulated annealing and molecular dynamics. Comparison of the results with those previously obtained for the natural CREKA shows that (Pro)hArg significantly reduces the conformational flexibility of the peptide. Although some changes are observed in the backbone...backbone and side-chain...side-chain interactions, the modified peptide exhibits a strong tendency to accommodate turn conformations centered at the (Pro)hArg residue and the overall shape of the molecule in the lowest energy conformations characterized for the natural and the modified peptides exhibit a high degree of similarity. In particular, the turn orients the backbone such that the Arg, Glu, and Lys side chains face the same side of the molecule, which is considered important for bioactivity. These results suggest that replacement of Arg by (Pro)hArg in CREKA may be useful in providing resistance against proteolytic enzymes while retaining conformational features which are essential for tumor-homing activity.

A straightforward route to enantiopure alpha-substituted derivatives of (2S,3aS,7aS)-octahydroindole-2-carboxylic acid.

High yielding and remarkably selective alkylations of a suitably protected derivative of (2S,3aS,7aS)-octahydroindole-2-carboxylic acid are described. The fused bicyclic structure of this proline analogue greatly influences the stereochemical outcome of direct alkylation reactions taking place at the alpha-carbon and provides access to alpha-substituted analogues with retention of the configuration. The overall procedure allows the preparation of enantiopure alpha-substituted derivatives of this Oic isomer, suitably protected for their incorporation into peptides, in a straightforward manner.

Versatile methodology for the synthesis and α-functionalization of (2R,3aS,7aS)-octahydroindole-2-carboxylic acid.

An improved strategy for the effective synthesis of enantiomerically pure (2R,3aS,7aS)-octahydroindole-2-carboxylic acid (Oic), based on the formation of a trichloromethyloxazolidinone derivative, has been developed. Additionally, the completely diastereoselective α-alkylation of such oxazolidinone provides a very convenient and concise route to enantiopure α-tetrasubstituted derivatives of this Oic stereoisomer.

Stereoselective Synthesis of Quaternary Proline Analogues.

This review describes available methods for the diastereoselective and asymmetric synthesis of quaternary prolines. The focus is on the preparation of alpha-functionalized prolines with the pyrrolidine moiety not embedded in a polycyclic frame. The diverse synthetic approaches are classified according to the bond which is formed to complete the quaternary skeleton.

Characterization of EST-derived microsatellites for gene mapping and evolutionary genomics in turbot.

The detection of microsatellite sequences within expressed sequence tags (ESTs) connects potential markers with specific genes, generating type I markers. We have developed and mapped by linkage analysis a set of EST-derived microsatellites in the turbot, Scophthalmus maximus. One hundred and ninety-one microsatellites were identified from 9256 turbot ESTs. Primer design was possible with 98 microsatellites. After genotyping 25 wild turbot and the parents of two reference families for linkage analysis, 43 EST-derived microsatellites were selected because they met technical and polymorphism criteria. A final set of 31 EST-derived microsatellites could be mapped to 17 linkage groups of the turbot consensus map based on 242 anonymous microsatellites. Twenty-four microsatellite-containing ESTs were functionally annotated, confirming them as type I markers. Nineteen were mapped in the turbot consensus map. These EST-derived microsatellites constitute useful tools for genome scanning of turbot populations, marker-assisted selection programmes and comparative mapping.

Highly anti-selective SN2' substitutions of chiral cyclic 2-iodo-allylic alcohol derivatives with mixed zinc-copper reagents.

[reaction: see text] Functionalized allylic electrophilic reagents such as chiral 2-iodo-1-cyclohexenyl and -cyclopentenyl phosphates undergo highly stereoselective anti-S(N)2'-allylic substitution reactions with a wide range of organozinc reagents (R(2)Zn and RZnI) leading to chiral products with a transfer of the chiral information >95%. The use of functionalized organozinc iodides allows preparation of the bicyclic enones 8 and 9 in >or=93% ee.

Stereoselective S N 2-substitutions using polyfunctional lithium arylcuprates prepared by an iodine-copper exchange.

[reaction: see text] Polyfunctional mixed lithium arylcuprates of the type (FG-Ar)(PhMe(2)CCH(2))CuLi obtained via iodine- or bromine-copper exchange with (PhMe(2)CCH(2))(2)CuLi react with cyclic 2-iodoallylic acetates with high S(N)2 selectivity. The addition of ZnBr(2) changes this selectivity and allows the performance of highly regioselective and enantioselective anti S(N)2' substitutions using open-chain allylic pentafluorobenzoates.

Stanna-Brook rearrangement of carboxylic acid derivatives. Synthetic utility and mechanistic studies.

[reaction: see text] The reaction of R(3)SnLi with carboxylic acid derivatives proceeds through a novel, very fast stanna-Brook rearrangement that generates alpha-alkoxyorganolithium compounds as intermediates. The outcome of these reactions depends on the nature of the carboxyl derivatives. Reaction of R(3)SnLi with ester derivatives gives rise to coupled products through a novel C-C bond formation reaction. Experimental evidence of the detailed reaction mechanism is provided.