Identifying cancer sub-types from genomic scale data sets using confidence based integration (CBI).

Precision medicine is a method involving refined diagnosis of patients and searching for causes that are unseen in their patient cohorts who otherwise have largely similar health conditions. As the technology evolved to extract features from a wide variety of sources including genetics, a large quantum of data is available to the researchers for conducting micro studies in the field of disease and cures. In cancer research, integrative methods using genomic data sets has become a major area of interest. The petabytes of data that is available at The Cancer Genome Atlas (TCGA), a program jointly under NCI and National Human Genome Research Institute, has made possible more nuanced research in cancer genomics. Our method, Confidence Based Integration (CBI) is an integration method to extract similar as well as complementing information from the genomic data sets. This information will provide insight into the status of patients and their prospects. We used the expression data sets of gene, miRNA and DNA methylation in our fusion experiments on five different cancer types. These data sets, after fusion, are clustered using 'Spectral Clustering' algorithm, which derives clusters that form the disease sub types. Survival properties of each sub type demonstrates the reasons to consider the samples inside them highly similar. The performance of CBI, we report, is better, in terms of P-value in log-rank test, than other methods like similarity network fusion or SNF in forming clusters of significance. Individual features clustered extremely poor compared to CBI in most of the experiments.

Allelic frequency database of 15 polymorphic autosomal STRs in the Malayalam-speaking population of Kerala, India.

In this study, we assessed and established an allelic frequency database of Malayalam-speaking population of south western Indian state Kerala, using 15 polymorphic short tandem repeats (STRs) genetic markers. For this study, 464 unrelated healthy individuals were randomly selected following the ethical standards. The most polymorphic and most discriminating locus was D2S1338, with a value of 0.860 and 0.968, respectively. The range of heterozygosity extended from a minimum of 0.668 (TH01) to a maximum of 0.847 (D2S1338). The combined discrimination power (CPD) and combined exclusion power (CPE) were 1 and 0.999997861, respectively, for all 15 autosomal STR loci under study. The combined probability of match (CPM) and combined paternity index (CPI) for all 15 autosomal STR loci were found to be 9.85 × 10-19 and 4.18 × 105, respectively.

FOXO3 expression during colorectal cancer progression: biomarker potential reflects a tumour suppressor role.

BACKGROUND: In previous studies, the Forkhead/winged-helix-box-class-O3 (FOXO3) transcription factor has displayed both tumour suppressive and metastasis-promoting properties.To clarify its role in human colorectal cancer (CRC) progression, we examined in vivo FOXO3 expression at key points of the metastatic cascade. METHODS: Formalin-fixed paraffin-embedded resection specimens from normal colon, adenomas, primary CRC specimens of different pathological stage and CRC specimens with matched liver metastases were used to generate three separate custom-designed tissue microarray (TMA) representations of metastatic progression. Triplicate cores, immunostained for FOXO3 were scored semiquantitatively by two investigators. RESULTS: The FOXO3 expression is significantly reduced in CRC specimens compared with normal tissue, and progressive FOXO3 downregulation is associated with advancing pathological stage. In addition, recurrent stage I/II primary tumours show a significantly lower FOXO3 expression compared with stage-matched non-recurrent tumours. When stratified according to high and low FOXO3 expression, mean disease-free survival in the low-expressing group was 28 months (95% CI 15.8-50.6) compared with 64 months (95% CI 52.9-75.4) in the high-expressing group. CONCLUSION: We have demonstrated an association between low FOXO3 expression and CRC progression in vivo using purpose-designed TMAs. Forkhead/winged-helix-box-class-O3 may represent a novel biomarker of nodal and distant disease spread with clinical utility in CRC.

Regulation of p73 activity by post-translational modifications.

The transcription factor p73 is a member of the p53 family that can be expressed as at least 24 different isoforms with pro- or anti-apoptotic attributes. The TAp73 isoforms are expressed from an upstream promoter and are regarded as bona fide tumor suppressors; they can induce cell cycle arrest/apoptosis and protect against genomic instability. On the other hand, ΔNp73 isoforms lack the N-terminus transactivation domain; hence, cannot induce the expression of pro-apoptotic genes, but still can oligomerize with TAp73 or p53 to block their transcriptional activities. Therefore, the ratio of TAp73 isoforms to ΔNp73 isoforms is critical for the quality of the response to a genomic insult and needs to be delicately regulated at both transcriptional and post-translational level. In this review, we will summarize the current knowledge on the post-translational regulatory pathways involved to keep p73 protein under control. A comprehensive understanding of p73 post-translational modifications will be extremely useful for the development of new strategies for treating and preventing cancer.

Lattice Monte Carlo simulation of semiconductor nanocrystal synthesis in microemulsion droplets.

A lattice Monte Carlo model has been developed to describe the formation of a single semiconductor nanocrystal (quantum dot) inside a droplet of a microemulsion. The motivation stems from the need to understand the kinetics of quantum dot formation in microemulsion templates with minimal droplet-droplet coalescence. In these systems, a fixed amount of a reactant is dissolved in each droplet, and another reactant is supplied by diffusion through the interface. Nucleation is facilitated by a spontaneous reaction between the precursors at the droplet interface, and the coalescence of nuclei and clusters ultimately leads to the formation of a single particle. The size of the final particle is controlled by the concentration of the first reactant. A hard-sphere potential is used to describe cluster-cluster interactions. The overall particle formation time initially increases with final particle size, quickly passes through a maximum, and subsequently decreases due to the formation of large intermediate clusters apparently acting as effective collision partners to smaller ones. Studies of the evolution of intermediate cluster sizes provided mechanistic details of the final particle formation through cluster-cluster coalescence. A generalized dimensionless equation is obtained that relates the formation time of the final particle to its size for various droplet sizes and diffusivities of the first reactant and clusters. A parametric study reveals that the final particle formation time is more sensitive to changes in the cluster-cluster coalescence probability than in the probability of nucleation.

Maternal high-fat diet: effects on offspring bone structure.

UNLABELLED: Peak bone mass is believed to partly be programmed in utero. Mouse dams and offspring were given a high-fat diet and offspring studied as adults. Female offspring from high-fat dams exhibited altered trabecular structure indicative of in utero programming. In utero nutrition has consequences in later life. INTRODUCTION: Epidemiological studies suggest that skeletal growth is programmed during intrauterine and early postnatal life. We hypothesise that development of optimal peak bone mass has, in part, a foetal origin and investigated this using a mouse model of maternal dietary fat excess. METHODS: Offspring from mouse dams fed either standard chow (C) or lifetime high-fat diet (HF) were maintained on a HF diet to adulthood. Femur samples were taken at 30 weeks of age and bone structure, adiposity and strength analysed. Sample sizes were four to six for each sex and each diet group. RESULTS: Offspring from HF-fed dams showed increased adiposity in the femur in comparison to offspring from C-fed dams. Female offspring from HF dams exhibited altered trabecular structure indicative of in utero programming. CONCLUSIONS: A maternal HF diet during pregnancy increases bone marrow adiposity and alters bone structure in their offspring.

Patellar button dissociation in a mobile-bearing LCS patellofemoral joint arthroplasty.

Patellofemoral arthroplasty is an effective treatment for isolated patellofemoral arthritis. Midterm results reveal a success rate of approximately 80% to 90% with modern designs. The reported failure mechanisms associated with patellofemoral arthroplasty include progressive tibiofemoral arthritis, patellar pain, catching or subluxation caused by soft-tissue imbalance, component malposition, and problematic designs. We present a previously unreported new complication of patellar button dissociation in a mobile-bearing LCS Patellofemoral Joint Replacement Prosthesis.

Volatile sulfur compounds produced by probiotic bacteria in the presence of cysteine or methionine.

AIMS: To investigate the abilities of various probiotic bacteria to produce volatile sulfur compounds (VSCs) relevant to food flavour and aroma. METHODS AND RESULTS: Probiotic strains (Lactobacillus acidophilus NCFM, Lactobacillus plantarum 299v, Lactobacillus rhamnosus GG, Lactobacillus reuteri ATCC55730 and L. reuteri BR11), Lactobacillus delbrueckii ATCC4797, L. plantarum ATCC14917 and Lactococcus lactis MG1363 were incubated with either cysteine or methionine. Volatile compounds were captured, identified and quantified using a sensitive solid-phase microextraction (SPME) technique combined with gas chromatography coupled to a pulsed flame photometric detector (SPME/GC/PFPD). Several VSCs were identified including H(2)S, methanethiol, dimethyldisulfide and dimethyltrisulfide. The VSC profiles varied substantially for different strains of L. plantarum and L. reuteri and it was found that L. reuteri ATCC55730 and L. lactis MG1363 produced the lowest levels of VSCs (P < 0.05). Levels of VSCs generated by bacteria were found to be equivalent to, or higher than, that found in commercial cheeses. CONCLUSIONS: Several probiotic strains are able to generate considerable levels of VSCs and substantial variations in VSC generating potential exists between different strains from the same species. SIGNIFICANCE AND IMPORTANCE OF THE STUDY: This study demonstrates that probiotic bacteria are able to efficiently generate important flavour and aroma compounds and therefore has implications for the development of probiotic containing foods.

IL-22R, IL-10R2, and IL-22BP binding sites are topologically juxtaposed on adjacent and overlapping surfaces of IL-22.

Interleukin (IL) 22 is a type II cytokine that is produced by immune cells and acts on nonimmune cells to regulate local tissue inflammation. As a product of the recently identified T helper 17 lineage of CD4(+) effector lymphocytes, IL-22 plays a critical role in mucosal immunity as well as in dysregulated inflammation observed in autoimmune diseases. We used comprehensive mutagenesis combined with mammalian cell expression, ELISA cell-based, and structural methods to evaluate how IL-22 interacts with its cell surface receptor, IL-22R/IL-10R2, and with secreted IL-22 binding protein. This study identifies those amino acid side chains of IL-22 that are individually important for optimal binding to IL-22R, considerably expands the definition of IL-22 surface required for binding to IL-10R2, and demonstrates how IL-22 binding protein prevents IL-22R from binding to IL-22. The IL-22R and IL-10R2 binding sites are juxtaposed on adjacent IL-22 surfaces contributed mostly by helices A, D, and F and loop AB. Our results also provide a model for how IL-19, IL-20, IL-24, and IL-26 which are other IL-10-like cytokines, interact with their respective cell surface receptors.

Identification of critical amino acid residues for human iNOS functional activity.

Nitric oxide (NO) is a short-lived signaling molecule that mediates a variety of biological functions, including vascular homeostasis, neurotransmission, antimicrobial defense and antitumor activities. Three known NOS isoforms (eNOS, nNOS and iNOS) have been cloned and sequenced. Here, we show that upon expression in Escherichia coli using a novel expression vector, an iNOS sequence containing three mutations (A805D, F831S and L832P) within the iNOS reductase domain produced very little functionally active iNOS protein compared to the wild type (wt) iNOS. Each of these point mutations also was individually constructed into the wt iNOS sequence. The activity of the iNOS protein containing the A805D mutation was comparable to wt, while a drastic reduction in iNOS activity was observed for the F831S and L832P mutants. A comparison of the molecular models of the reductase domain of the wt and mutant iNOS revealed a reduced core packing density for the F831S and L832P mutations compared to wt. In addition, the modeling also suggests altered hydrogen bonding, van der Waals and hydrophobic interactions of these mutants.

Microarray technologies for gene transcript analysis in pancreatic cancer.

Pancreatic cancer is a devastating condition that is most often characterized by a poor prognosis. Microarray technologies are promising screening methods for the identification of potential markers for early diagnosis and chemotherapeutic intervention. In this article, we review the current state of pancreatic cancer research as it relates to the measurement of gene transcript levels by DNA microarray analysis.

Antioxidant enzymes and peroxidation in galactogenic cataract.

The antioxidant enzymes like catalase and superoxide dismutase (SOD) were studied in erythrocytes and lens at various stages of cataractogenesis in albino rats. The rate of peroxidation was measured by assessing the malondiadehyde (MDA) in lens and plasma. The insoluble and soluble protein fractions were measured in lens to study the protein crosslinkings in relation to the above said parameters. Cataract was induced in albino rats by feeding it with 30% galactose as part of the normal diet (w/w) for 30 days. The results show a decrease of SOD and catalase with concomitant increase of MDA and insoluble protein with the advancement of cataract.

Effects of caloric restriction on mitochondrial function and gene transcripts in rat muscle.

Rodent skeletal muscle mitochondrial DNA has been shown to be a potential site of oxidative damage during aging. Caloric restriction (CR) is reported to reduce oxidative stress and prolong life expectancy in rodents. Gene expression profiling and measurement of mitochondrial ATP production capacity were performed in skeletal muscle of male rats after feeding them either a control diet or calorie-restricted diet (60% of control diet) for 36 wk to determine the potential mechanism of the beneficial effects of CR. CR enhanced the transcripts of genes involved in reactive oxygen free radical scavenging function, tissue development, and energy metabolism while decreasing expression of those genes involved in signal transduction, stress response, and structural and contractile proteins. Real-time PCR measurements confirmed the changes in transcript levels of cytochrome-c oxidase III, superoxide dismutase (SOD)1, and SOD2 that were noted by the microarray approach. Mitochondrial ATP production and citrate synthase were unaltered by the dietary changes. We conclude that CR alters transcript levels of several genes in skeletal muscle and that mitochondrial function in skeletal muscle remains unaltered by the dietary intervention. Alterations in transcripts of many genes involved in reactive oxygen scavenging function may contribute to the increase in longevity reported with CR.

Impact of high-fat diet and antioxidant supplement on mitochondrial functions and gene transcripts in rat muscle.

High-fat diets are reported to increase oxidative stress in a variety of tissues, whereas antioxidant supplementation prevents many diseases attributed to high-fat diet. Rodent skeletal muscle mitochondrial DNA has been shown to be a potential site of oxidative damage. We hypothesized that the effects of a high-fat diet on skeletal muscle DNA functions would be attenuated or partially reversed by antioxidant supplementation. Gene expression profiling and measurement of mitochondrial ATP production capacity were performed in skeletal muscle from male rats after feeding one of three diets (control, high-fat diet with or without antioxidants) for 36 wk. The high-fat diet altered transcript levels of 18 genes of 800 surveyed compared with the control-fed rats. Alterations included reduced expression of genes involved in free-radical scavenging and tissue development and increased expression of stress response and signal transduction genes. The magnitude of these alterations due to high-fat diet was reduced by antioxidant supplementation. Real-time PCR measurements confirmed the changes in transcript levels of cytochrome c oxidase subunit III and superoxide dismutase-1 and -2 noted by microarray approach. Mitochondrial ATP production was unaltered by dietary changes or antioxidant supplementation. It is concluded that the high-fat diet increases the transcription of genes involved in stress response but reduces those of free-radical scavenger enzymes, resulting in reduced DNA repair/metabolism (increased DNA damage). Antioxidants partially prevent these changes. Mitochondrial functions in skeletal muscle remain unaltered by the dietary intervention due to many adaptive changes in gene transcription.

Early identification of recipients with progressive histologic recurrence of hepatitis C after liver transplantation.

Approximately half of patients undergoing liver transplantation (LT) for hepatitis C virus (HCV) develop histologic evidence of recurrence within the first postoperative year. Early identification of recipients at risk for more severe recurrence of HCV may be useful in selecting patients for antiviral therapy. We determined whether recipients at greatest risk for more severe recurrence of HCV can be identified by pre- and/or early post-LT HCV-RNA levels in serum or tissue. Serum and tissue samples were prospectively collected pre-LT and at 7 days, 4 months, 1 year, and at 3 years posttransplantation from patients undergoing LT for HCV. Hepatitis activity index (HAI) and fibrosis stage (FS) were assessed in all liver biopsies. Forty-seven patients (32 men) were studied. Higher HCV-RNA levels at 4 months post-LT (>/=10(9) copies/mL, n = 29) were associated with higher HAI at 1 year and at 3 years post-LT. The HAI seen on protocol biopsies at 4 months correlated significantly with fibrosis stage (FS) at 1 year (r =.56, P

Progesterone release from glutaraldehyde cross-linked casein microspheres: in vitro studies and in vivo response in rabbits.

Microspheres of bovine milk protein casein loaded with progesterone were fabricated by glutaraldehyde cross-linking of an aqueous alkaline solution of the protein dispersed in a hexane and dichloromethane non-aqueous dispersion medium with an aliphatic polyurethane as the stabilizer. Microspheres were characterized for their surface morphology and internal structure using scanning electron microscopy. In vitro release studies in phosphate buffer at 37 degrees C demonstrated that the rate of release of the steroid from the microsphere matrix was a function of cross-linking density, particle size, and drug payload. Microsphere formulations released 50% to 60% of the incorporated steroid in about 30 days and, thereafter, attained a steady state. In the presence of a protein-digesting enzyme such as protease, complete release of the steroid was observed in about 4 days in vitro into phosphate buffer. Intramuscular injection of progesterone-loaded microspheres into rabbits showed a plasma concentration of 1 to 2 ng/mL up to 5 months without any significant burst effect, whereas the powdered steroid administered in saline demonstrated a large burst effect peaking over 20 ng/mL, and the plasma concentration was not sustained beyond 4 days. Data obtained suggest that casein microspheres would be promising as a biodegradable drug carrier for sustained delivery of steroids.

Activation of ryanodine receptors by imperatoxin A and a peptide segment of the II-III loop of the dihydropyridine receptor.

Excitation-contraction coupling in skeletal muscle is believed to be triggered by direct protein-protein interactions between the sarcolemmal dihydropyridine-sensitive Ca2+ channel and the Ca2+ release channel/ryanodine receptor (RyR) of sarcoplasmic reticulum. A 138-amino acid cytoplasmic loop between repeats II and III of the alpha1 subunit of the skeletal dihydropyridine receptor (the II-III loop) interacts with a region of the RyR to elicit Ca2+ release. In addition, small segments (10-20 amino acid residues) of the II-III loop retain the capacity to activate Ca2+ release. Imperatoxin A, a 33-amino acid peptide from the scorpion Pandinus imperator, binds directly to the RyR and displays structural and functional homology with an activating segment of the II-III loop (Glu666-Leu690). Mutations in a structural motif composed of a cluster of basic amino acids followed by Ser or Thr dramatically reduce or completely abolish the capacity of the peptides to activate RyRs. Thus, the Imperatoxin A-RyR interaction mimics critical molecular characteristics of the II-III loop-RyR interaction and may be a useful tool to elucidate the molecular mechanism that couples membrane depolarization to sarcoplasmic reticulum Ca2+ release in vivo.

Biologically active peptides caged on tyrosine.

We have demonstrated the feasibility of preparing caged peptides by derivatizing a single amino acid side chain in peptides up to 20 amino acids long. Two peptides are illustrated whose activities are reduced by nearly 2 orders of magnitude using this caging approach. The specific strategy described here of derivatizing tyrosine side chains with a charged caging moiety should be generally applicable in the preparation of caged peptides that have a critical tyrosine residue (e.g., LSM1) or that have critical hydrophobic patches (e.g., RS-20). Other amino acid side chains are also accessible via this caging strategy. Derivatives of threonine, serine, lysine, cysteine, glutamate, aspartate, glutamine, and asparagine can be prepared and site specifically inserted into peptides in an analogous manner. The caged peptides synthesized and purified by the methods described here are compatible with biological samples, including living cells, and have been used to demonstrate the central importance of calmodulin, MLCK, and, by inference, myosin II in ameboid locomotion in polarized eosinophil cells. Photoactivation of peptides within cells should provide a wealth of new information in future investigations by allowing specific protein activities to be knocked out in an acute and spatially defined way.

Comparative complement activation study of polypropylene hollow fibres of two different makes in static condition.

The complement activation is one of the major problems encountered in the use of extracorporeal devices. The complement-activating potential of two polypropylene hollow fibres (used in membrane oxygenator) of different make and designated as F1 and F2 was tested with time (10, 30, and 180 minutes). The fibres were brought in contact with human blood under in vitro static condition for the comparison. A direct measurement of unadsorbed concentration of the complement protein, C3, present in the liquid phase of human blood before and after the contact with polymer was made using human C3 antisera. This gave a measure of C3 adsorption on the fibres with time and probably also gave an indirect measure of C3a in the blood. IgG was also estimated using antisera of human IgG. The total protein and albumin concentration were measured to obtain an overall adsorption profile of these protein on the fibre surfaces with respect to time. The results showed that C3 adsorption was taking place mainly through the alternative pathway over and above the classical one, being more in the case of F2 than F1. SEM studies revealed poor adhesion of platelets on both fibres, though some activated platelets were also seen with slight deformation at 10 minutes and a few with prominent pseudopodia formations at a later time period on the surface of both fibres. The total protein adsorption was faster, and the surface pores of the F1 were found masked at 10 minutes observation. Later, desorption occurred making the pores visible at 180 minutes. The F2 surface examination showed a continuous deposition of protein layers with time, thereby masking the pores at 180 minutes. The present experimental finding and assessment favoured the F1 as a marginally better candidate to be considered for oxygenator development.

Signaling pathways underlying eosinophil cell motility revealed by using caged peptides.

Insights into structure-function relations of many proteins opens the possibility of engineering peptides to selectively interfere with a protein's activity. To facilitate the use of peptides as probes of cellular processes, we have developed caged peptides whose influence on specific proteins can be suddenly and uniformly changed by near-UV light. Two peptides are described which, on photolysis of a caging moiety, block the action of calcium-calmodulin or myosin light chain kinase (MLCK). The efficacy of theses peptides is demonstrated in vitro and in vivo by determining their effect before and after photolysis on activities of isolated enzymes and cellular functions known to depend on calcium-calmodulin and MLCK. These caged peptides each were injected into motile, polarized eosinophils, and when exposed to light promptly blocked cell locomotion in a similar manner. The results indicate that the action of calcium-calmodulin and MLCK, and by inference myosin II, are required for the ameboid locomotion of these cells. This methodology provides a powerful means for assessing the role of these and other proteins in a wide range of spatio-temporally complex functions in intact living cells.