Efficient and accurate estimation of relative order tensors from lambda-maps.

The rapid increase in the availability of RDC data from multiple alignment media in recent years has necessitated the development of more sophisticated analyses that extract the RDC data's full information content. This article presents an analysis of the distribution of RDCs from two media (2D-RDC data), using the information obtained from a lambda-map. This article also introduces an efficient algorithm, which leverages these findings to extract the order tensors for each alignment medium using unassigned RDC data in the absence of any structural information. The results of applying this 2D-RDC analysis method to synthetic and experimental data are reported in this article. The relative order tensor estimates obtained from the 2D-RDC analysis are compared to order tensors obtained from the program REDCAT after using assignment and structural information. The final comparisons indicate that the relative order tensors estimated from the unassigned 2D-RDC method very closely match the results from methods that require assignment and structural information. The presented method is successful even in cases with small datasets. The results of analyzing experimental RDC data for the protein 1P7E are presented to demonstrate the potential of the presented work in accurately estimating the principal order parameters from RDC data that incompletely sample the RDC space. In addition to the new algorithm, a discussion of the uniqueness of the solutions is presented; no more than two clusters of distinct solutions have been shown to satisfy each lambda-map.

Estimation of relative order tensors, and reconstruction of vectors in space using unassigned RDC data and its application.

Advances in NMR instrumentation and pulse sequence design have resulted in easier acquisition of Residual Dipolar Coupling (RDC) data. However, computational and theoretical analysis of this type of data has continued to challenge the international community of investigators because of their complexity and rich information content. Contemporary use of RDC data has required a-priori assignment, which significantly increases the overall cost of structural analysis. This article introduces a novel algorithm that utilizes unassigned RDC data acquired from multiple alignment media (nD-RDC, n3) for simultaneous extraction of the relative order tensor matrices and reconstruction of the interacting vectors in space. Estimation of the relative order tensors and reconstruction of the interacting vectors can be invaluable in a number of endeavors. An example application has been presented where the reconstructed vectors have been used to quantify the fitness of a template protein structure to the unknown protein structure. This work has other important direct applications such as verification of the novelty of an unknown protein and validation of the accuracy of an available protein structure model in drug design. More importantly, the presented work has the potential to bridge the gap between experimental and computational methods of structure determination.

Gene expression changes during HPV-mediated carcinogenesis: a comparison between an in vitro cell model and cervical cancer.

We used oligonucleotide microarrays to investigate gene expression changes associated with multi-step human papillomavirus type 16 (HPV16)-mediated carcinogenesis in vitro. Gene expression profiles in 4 early passage HPV16-immortalized human keratinocyte (HKc) lines derived from different donors were compared with their corresponding 4 late-passage, differentiation-resistant cell lines, and to 4 pools of normal HKc, each composed of 3 individual HKc strains, on Agilent 22 k human oligonucleotide microarrays. The resulting data were analyzed using a modified T-test coded in R to obtain lists of differentially expressed genes. Gene expression changes identified in this model system were then compared with gene expression changes described in published studies of cervical intraepithelial neoplasia (CIN) and cervical cancer. Common genes in these lists were further studied by cluster analysis. Genes whose expression changed in the same direction as in CIN or cervical cancer (concordant) at late stages of HPV16-mediated transformation in vitro formed one major cluster, while those that changed in the opposite direction (discordant) formed a second major cluster. Further annotation found that many discordant expression changes involved gene products with an extracellular localization. Two novel genes were selected for further study: overexpression of SIX1 and GDF15, observed during in vitro progression in our model system, was confirmed in tissue arrays of cervical cancer. These microarray-based studies show that our in vitro model system reflects many cellular and molecular alterations characteristic of cervical cancer, and identified SIX1 and GDF15 as 2 novel potential biomarkers of cervical cancer progression.

TALI: local alignment of protein structures using backbone torsion angles.

UNLABELLED: Torsion angle alignment (TALI) is a novel approach to local structural motif alignment, based on backbone torsion angles (phi, psi) rather than the more traditional atomic distance matrices. Representation of a protein structure in the form of a sequence of torsion angles enables easy integration of sequence and structural information, and adopts mature techniques in sequence alignment to improve performance and alignment quality. We show that TALI is able to match local structural motifs as well as identify global structural similarity. TALI is also compared to other structure alignment methods such as DALI, CE, and SSM, as well as sequence alignment based on PSI-BLAST; TALI is shown to be equally successful as, or more successful than, these other methods when applied to challenging structural alignments. The inference of the evolutionary tree of class II aminoacyl-tRNA synthetase shows the potential for TALI in estimating protein structural evolution and in identifying structural divergence among homologous structures. AVAILABILITY: http://redcat.cse.sc.edu/index.php/ PROJECT: TALI/.

Rapid classification of protein structure models using unassigned backbone RDCs and probability density profile analysis (PDPA).

A method of identifying the best structural model for a protein of unknown structure from a list of structural candidates using unassigned 15N1H residual dipolar coupling (RDC) data and probability density profile analysis (PDPA) is described. Ten candidate structures have been obtained for the structural genomics target protein PF2048.1 using ROBETTA. 15N1H residual dipolar couplings have been measured from NMR spectra of the protein in two alignment media and these data have been analyzed using PDPA to rank the models in terms of their ability to represent the actual structure. A number of advantages in using this method to characterize a protein structure become apparent. RDCs can easily and rapidly be acquired, and without the need for assignment, the cost and duration of data acquisition is greatly reduced. The approach is quite robust with respect to imprecise and missing data. In the case of PF2048.1, a 79 residue protein, only 58 and 55 of the total RDC data were observed. The method can accelerate structure determination at higher resolution using traditional NMR spectroscopy by providing a starting point for the addition of NOEs and other NMR structural data.

Vitamin D receptor gene Tru9I polymorphism and risk for incidental sporadic colorectal adenomas.

AIM: Recent laboratory and epidemiological studies suggest that vitamin D is a potential agent for colorectal cancer prevention. Its function is partially mediated by the vitamin D receptor (VDR). The aim of this study was to investigate whether a novel G (allele "U") >A (allele "u") polymorphism (Tru9I) in the VDR intron 8 region is associated with risk for colorectal adenoma in a colonoscopy-based case-control study. METHODS: Genotyping for a total of 391 subjects was carried out through PCR and restriction fragment length polymorphism. RESULTS: The frequencies of "U" and "u" alleles were 89.3% and 10.7%, respectively. The "Uu" and "uu" genotypes were associated with decreased risk for adenoma (OR, 0.71; 95%CI, 0.40-1.25). The inverse association was more pronounced for multiple adenomas and adenomas that were larger had moderate or greater dysplasia, or were sessile: the odds ratios (ORs) were, 0.51 (95%CI, 0.21-1.24), 0.37 (95%CI, 0.11-1.28), 0.68 (95%CI, 0.33-1.41), and 0.36 (95%CI, 0.13-0.97) respectively. In joint/combined analyses, inverse associations were more obvious among those who had at least one "u" allele and also were younger (OR, 0.60; 95%CI, 0.26-1.37), women (OR, 0.38; 95%CI, 0.17-0.88), did not smoke (OR, 0.39; 95%CI, 0.13-1.23), or took NSAID (OR, 0.38; 95%CI, 0.12-1.25), but no evidence existed for interactions with calcium or vitamin D intake. CONCLUSION: Our findings suggest that the VDR Tru9I polymorphism may be associated with lower risk for colorectal adenoma, particularly in interaction with various risk factors, but not with calcium or vitamin D.

Novel genetic variations of the p53R2 gene in patients with colorectal adenoma and controls.

AIM: p53-inducible ribonucleotide reductase small subunit 2 (p53R2) encodes a 351-amino-acid peptide, which catalyzes conversion of ribonucleoside diphosphates to the corresponding deoxyribonucleotides required for DNA replication and repair. A recent study reported that a point mutation (G/T) in the p53 binding sequence in a colon cancer cell line completely impaired p53R2 protein activity. METHODS: We screened the p53R2 gene coding regions and a regulatory region which contains a p53 binding sequence in 100 patients with colorectal adenoma and 100 control subjects using PCR, cold SSCP, and direct DNA sequencing. RESULTS: Although we did not identify genetic variation in all nine exons, four regulatory-region variants were found, of which three were single nucleotide polymorphisms (SNPs) (nt 1 789 C/G, nt 1 928 A/G, 1 933 T/C), and one was 20 bp insertion which replaced a ATTTT between nt 1831 and 1835. Additionally, we determined the frequency of these p53R2 variants in a recently concluded case-control study of incident sporadic colorectal adenomas (163 cases and 210 controls). CONCLUSION: Although more detailed functional characterizations of these polymorphisms remain to be undertaken, these polymorphic sites may be useful for identifying alleles associated with mis-splicing, additional transcript factors and, more generally, in cancer-susceptibility association studies.