DNA sequence profiles of the colorectal cancer critical gene set KRAS-BRAF-PIK3CA-PTEN-TP53 related to age at disease onset

The incidence of colorectal cancer (CRC) increases with age and early onset indicates an increased likelihood for genetic predisposition for this disease. The somatic genetics of tumor development in relation to patient age remains mostly unknown. We have examined the mutation status of five known cancer critical genes in relation to age at diagnosis, and compared the genomic complexity of tumors from young patients without known CRC syndromes with those from elderly patients. Among 181 CRC patients, stratified by microsatellite instability status, DNA sequence changes were identified in KRAS (32%), BRAF (16%), PIK3CA (4%), PTEN (14%) and TP53 (51%). In patients younger than 50 years (n = 45), PIK3CA mutations were not observed and TP53 mutations were more frequent than in the older age groups. The total gene mutation index was lowest in tumors from the youngest patients. In contrast, the genome complexity, assessed as copy number aberrations, was highest in tumors from the youngest patients. A comparable number of tumors from young (<50 years) and old patients (>70 years) was quadruple negative for the four predictive gene markers (KRAS-BRAF-PIK3CA-PTEN); however, 16% of young versus only 1% of the old patients had tumor mutations in PTEN/PIK3CA exclusively. This implies that mutation testing for prediction of EGFR treatment response may be restricted to KRAS and BRAF in elderly (>70 years) patients. Distinct genetic differences found in tumors from young and elderly patients, whom are comparable for known clinical and pathological variables, indicate that young patients have a different genetic risk profile for CRC development than older patients.     

A cardiovascular drug rescues mice from lethal sepsis by selectively attenuating a late-acting proinflammatory mediator, high mobility group box 1

The pathogenesis of sepsis is mediated in part by bacterial endotoxin, which stimulates macrophages/monocytes to sequentially release early (e.g., TNF, IL-1, and IFN-gamma) and late (e.g., high mobility group box 1 (HMGB1) protein) proinflammatory cytokines. The recent discovery of HMGB1 as a late mediator of lethal sepsis has prompted investigation for development of new experimental therapeutics. We found that many steroidal drugs (such as dexamethasone and cortisone) and nonsteroidal anti-inflammatory drugs (such as aspirin, ibuprofen, and indomethacin) failed to influence endotoxin-induced HMGB1 release even at superpharmacological concentrations (up to 10-25 microM). However, several steroid-like pigments (tanshinone I, tanshinone IIA, and cryptotanshinone) of a popular Chinese herb, Danshen (Salvia miltiorrhiza), dose dependently attenuated endotoxin-induced HMGB1 release in macrophage/monocyte cultures. A water-soluble tanshinone IIA sodium sulfonate derivative (TSNIIA-SS), which has been widely used as a Chinese medicine for patients with cardiovascular disorders, selectively abrogated endotoxin-induced HMGB1 cytoplasmic translocation and release in a glucocorticoid receptor-independent manner. Administration of TSNIIA-SS significantly protected mice against lethal endotoxemia and rescued mice from lethal sepsis even when the first dose was given 24 h after the onset of sepsis. The therapeutic effects were partly attributable to attenuation of systemic accumulation of HMGB1 (but not TNF and NO) and improvement of cardiovascular physiologic parameters (e.g., decrease in total peripheral vascular resistance and increase in cardiac stroke volume) in septic animals. Taken together, these data re-enforce the pathogenic role of HMGB1 in lethal sepsis, and support a therapeutic potential for TSNIIA-SS in the treatment of human sepsis.     

Application of cellulases from an alkalothermophilic Thermomonospora sp. in biopolishing of denims

Use of cellulase for denim washing is a standard eco-friendly technique to achieve desirable appearance and softness for cotton fabrics and denims. But enzymatic washing of denim till date involved acid cellulase (Trichoderma reesei) and neutral cellulase (Humicola isolens) the use of which has a drawback of backstaining of the indigo dye on to the fabric. Though it has been suggested that pH is a major factor in controlling backstaining there are no reports on use of cellulase under alkaline conditions for denim washing. In this study for the first time an alkali stable endoglucanase from alkalothermophilic Thermomonospora sp. (T-EG) has been used for denim biofinishing under alkaline conditions. T-EG is effective in removing hairiness with negligible weight loss and imparting softness to the fabric. Higher abrasive activity with lower backstaining was a preferred property for denim biofinishing exhibited by T-EG. The activities were comparable to acid and neutral cellulases that are being regularly used. The enzyme was also effective under non-buffering conditions which is an added advantage for use in textile industry. A probable mechanism of enzymatic finishing of cotton fabric has been represented based on the unique properties of T-EG.     

The combined activation of positive costimulatory signals with modulation of a negative costimulatory signal for the enhancement of vaccine-mediated T-cell responses

Blockade of CTLA-4 by monoclonal antibodies (mAb) can mediate regression of tumors and increase the efficacy of tumor antigen specific vaccines. Blockade of CTLA-4 has also been shown to significantly increase the avidity of antigen-specific T cells after immunization with live recombinant viral vector based vaccine. Here, we demonstrate a biological synergy between CTLA-4 blockade and active vaccine therapy consisting of recombinant vaccinia and avipox viruses expressing carcinoembryonic antigen (CEA) and three T cell costimulatory molecules to enhance antitumor effects. However, this synergy was very much dependent on the temporal relationship of scheduling of the two agents. We evaluated the strategies in both a foreign antigen model using β-galactosidase as immunogen, and in a “self” antigen model using CEA as immunogen. For antitumor activity the model used consisted of mice transgenic for human CEA and a murine carcinoma cell line transfected with CEA. The enhanced antitumor activity after vaccine and CTLA-4 blockade did not result in any signs of autoimmunity. These studies form a rational basis for the use of vector-based vaccines with anti-CTLA-4 and demonstrate that both enhancement of positive costimulatory signals and inhibition of negative costimulatory signals can be simultaneously exploited. These studies also underscore the importance of “drug” scheduling in vaccine combination therapies.     

A dual receptor crosstalk model of G-protein-coupled signal transduction

Macrophage cells that are stimulated by two different ligands that bind to G-protein-coupled receptors (GPCRs) usually respond as if the stimulus effects are additive, but for a minority of ligand combinations the response is synergistic. The G-protein-coupled receptor system integrates signaling cues from the environment to actuate cell morphology, gene expression, ion homeostasis, and other physiological states. We analyze the effects of the two signaling molecules complement factors 5a (C5a) and uridine diphosphate (UDP) on the intracellular second messenger calcium to elucidate the principles that govern the processing of multiple signals by GPCRs. We have developed a formal hypothesis, in the form of a kinetic model, for the mechanism of action of this GPCR signal transduction system using data obtained from RAW264.7 macrophage cells. Bayesian statistical methods are employed to represent uncertainty in both data and model parameters and formally tie the model to experimental data. When the model is also used as a tool in the design of experiments, it predicts a synergistic region in the calcium peak height dose response that results when cells are simultaneously stimulated by C5a and UDP. An analysis of the model reveals a potential mechanism for crosstalk between the Galphai-coupled C5a receptor and the Galphaq-coupled UDP receptor signaling systems that results in synergistic calcium release.     

Impact of exogenous sequences on the characteristics of an epidemic type 2 recombinant vaccine-derived poliovirus

Pathogenic circulating vaccine-derived polioviruses (cVDPVs) have become a major obstacle to the successful completion of the global polio eradication program. Most cVDPVs are recombinant between the oral poliovirus vaccine (OPV) and human enterovirus species C (HEV-C). To study the role of HEV-C sequences in the phenotype of cVDPVs, we generated a series of recombinants between a Madagascar cVDPV isolate and its parental OPV type 2 strain. Results indicated that the HEV-C sequences present in this cVDPV contribute to its characteristics, including pathogenicity, suggesting that interspecific recombination contributes to the phenotypic biodiversity of polioviruses and may favor the emergence of cVDPVs.     

Encapsulation of endoglucanase using a biopolymer Gum Arabic for its controlled release

Gum Arabic, a biodegradable natural polymer was used as a matrix to encapsulate endoglucanase from Thermomonospora sp. The modified enzyme retained complete biocatalytic activity and exhibited a shift in the optimum temperature [50-55 degrees C] and considerable increase in the pH and temperature stabilities as compared to the free enzyme. Encapsulation of the enzyme also protected the activity in presence of detergents and enhanced the shelf life. A 3-fold decrease in the initial rate of reaction indicated a controlled release of the enzyme conferring properties preferred for its potential application in the manufacture of detergents.     

Comparative genomic hybridization detects secondary chromosomal deletions in Escherichia coli K-12 MG1655 mutants and highlights instability in the flhDC region

The use of whole-genome microarrays for monitoring mutagenized or otherwise engineered genetic derivatives is a potentially powerful tool for checking genomic integrity. Using comparative genomic hybridization of a number of unrelated, directed deletion mutants in Escherichia coli K-12 MG1655, we identified unintended secondary genomic deletions in the flhDC region in Δfnr, Δcrp, and ΔcreB mutants. These deletions were confirmed by PCR and phenotypic tests. Our findings show that nonmotile progeny are found in some MG1655 directed deletion mutants, and studies on the effects of gene knockouts should be viewed with caution when the mutants have not been screened for the presence of secondary deletions or confirmed by other methods.     

Mixed substrate solid state fermentation for production and extraction of lipase from Aspergillus niger MTCC 2594

A novel mixed substrate solid-state fermentation (SSF) process has been developed for Aspergillus niger MTCC 2594 using wheat bran (WB) and gingelly oil cake (GOC) and the results showed that addition of GOC to WB (WB : GOC, 3 : 1, w/w) increased the lipase activity by 36.0% and the activity was 384.3+/-4.5 U/g dry substrate at 30 degrees C and 72 h. Scale up of lipase production to 100 g and 1 kg tray-level batch fermentation resulted in 95.0% and 84.0% of enzyme activities respectively at 72 h. A three-stage multiple contact counter-current extraction yielded 97% enzyme recovery with a contact time of 60 min. However, extraction by simple percolation and plug-flow methods resulted in decreased enzyme recoveries. The mixed substrate SSF process has resulted in a significant increase in specific activity (58.9%) when compared to a submerged fermentation (SmF) system. Furthermore, an efficient process of extraction has been standardized with this process. Use of GOC along with WB as potential raw materials for enzyme production could be of great commercial significance. This is the first report on the production and extraction of lipase from Aspergillus niger using mixed solid substrates, WB and GOC, which are potential raw materials for the production of enzymes and other value-added products.     

Licensing virus-specific T cells to secrete the neutrophil attracting chemokine CXCL-8 during hepatitis B virus infection

T cell functional plasticity helps tailor antiviral immunity during different phases of infections. We tested whether, during different phases of HBV infection, virus-specific T cells can acquire specific proinflammatory functions that could drive granulocyte/mononuclear cell liver infiltration. Multifunctional analysis of HBV-specific T cells during acute and chronic HBV infection revealed that HBV-specific T cells had the capacity to produce the neutrophil chemokine CXCL-8 but not IL-17. CXCL-8 producing T cells were detectable in the liver of chronic HBV patients with active hepatitis; while in acute HBV patients CXCL-8 production by T cells was temporally limited to the acute phase of disease, concomitant with the peak of liver inflammation. Characterization of the conditions necessary for the development of CXCL-8 producing T cells showed a requirement for IL-7 and IL-15 during T cell expansion. These data show that functional plasticity of virus-specific T cells spontaneously occurs during HBV infection and that an environment rich IL-7 and IL-15 can license T cells with the ability to produce CXCL-8 and potentially influence liver pathology.