Enzymatic degradation of sulphonated azo dye using purified azoreductase from facultative Klebsiella pneumoniae.

Heterologously expressed and purified azoreductase enzyme from facultative Klebsiella pneumoniae was used to degrade sulphonated azo dye. Methyl orange (MO) was used as the model dye to study the azo dye decolorization potential of the purified enzyme at different conditions. The enzyme had maximum activity at 40 °C and pH 8.0. The enzyme was observed to be thermo-stable as some enzyme activity was retained even at 80 °C. The apparent kinetic parameters, i.e., appKm and appVmax, for azoreductase using MO as a substrate were found to be 17.18 µM and 0.08/min, respectively. The purified enzyme was able to decolorize approximately 83% of MO (20 µM) within 10 min in the presence of NADH. Thus, efficient decolorization of MO was observed by the purified enzyme. The recombinant enzyme was purified approximately 18-fold with 46% yield at the end of four steps of the purification process. Enzyme was present in a tetrameric structure as confirmed by the volume at which protein was eluted in gel filtration chromatography, and the monomeric molecular mass of enzyme was found to be 23 kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The dye degradation efficiency of azoreductase cloned from Klebsiella pneumoniae and purified from recombinant Escherichia coli was observed to be much higher as compared with the efficiencies of the reported azoreductases from other bacterial strains. In the present study, we report the purification and characterization of the azoreductase cloned from Klebsiella pneumoniae and expressed in Escherichia coli.

Cyclic enaminone as new chemotype for selective cyclooxygenase-2 inhibitory, anti-inflammatory, and analgesic activities.

The cyclic enaminone moiety has been identified as a new scaffold for selective inhibition of cyclooxygenase-2 with anti-inflammatory and analgesic activities. The designed cyclic enaminones have been synthesized conveniently through the development of a new catalyst-free methodology and evaluated for cyclooxygenase (COX-1 and COX-2) inhibitory activities. Three compounds 7d, 8, and 9 predominantly inhibited COX-2 with selectivity index of 74.09, 19.45 and 108.68, respectively, and were assessed for in vivo anti-inflammatory activity in carrageenan induced rat paw edema assay. The anti-inflammatory activity of 7d was comparable to that of celecoxib at a dose of 12.5 mg/kg. However, the compounds 8 and 9 were more/equally effective as anti-inflammatory agent compared to celecoxib at the doses of 12.5 mg/kg and 25 mg/kg and also exhibited anti-inflammatory activity comparable to that of diclofenac. The therapeutic potential of the most active compound 9 was further assessed by performing in vivo thermal and mechanical hyperalgesia tests using various models that revealed its analgesic activity. The in vivo non-ulcerogenicity of 9 revealed the gastrointestinal safety as compared to the non-selective COX inhibitor indomethacin. The in vitro antioxidant activity and in vivo experiments on heart rate and blood pressure provided the cardiovascular safety profile of 9. The molecular docking studies rationalize the COX-2 selectivity of the newly found anti-inflammatory compounds 7d, 8, and 9.

Design of diffusion-controlled drug delivery devices for controlled release of Paclitaxel.

Controlled drug delivery devices were predicted in a reverse engineering framework for the controlled release of Paclitaxel, an anti-cancer drug, widely used in the treatment of solid tumors. Using quantitative structure-property relationship models for mutual diffusion coefficients of the drug in biocompatible and biodegradable polymers and partition coefficients of the drug between polymers and blood, a framework was developed to predict optimal drug delivery devices for desired dosage regimens. The validation of the predicted mutual diffusion and partition coefficients using experimental data was reported in previous studies. Optimal design parameters along with selection of most appropriate polymers suitable for different dosage regimens, selected based on current clinical practice, were predicted for maximum bioavailability of the drug while maintaining the released drug concentration in blood within the therapeutic range. Reservoir and monolithic type of diffusion-controlled drug delivery devices of different shapes and sizes were predicted with different initial drug loadings and bioavailability for different dosage regimens. The effects of the released Paclitaxel from these devices on the tumor growth were also modeled using a previously reported mathematical pharmacokinetic-pharmacodynamic model. The proposed approach can easily be used to design other diffusion-controlled drug delivery devices.

Prediction of the partition coefficients using QSPR modeling and simulation of paclitaxel release from the diffusion-controlled drug delivery devices.

An in silico approach is proposed to first predict the partition coefficient of the model drug, paclitaxel, in different biocompatible and biodegradable polymer versus the blood plasma using artificial neural networks (ANNs) and semi-empirical quantitative structure property relationships (QSPRs). A simplified molecular-input line-entry system (SMILES) notation is used to represent the structures of the different polymers and the drug. The SMILES notation is then used to calculate the various structure-based descriptors. These descriptors are then used in the ANNs and semi-empirical QSPRs to predict the properties for a given drug-polymer device. A fluid flow model is subsequently solved to simulate the controlled drug release in the blood plasma. The effects of various parameters are also studied on the drug release profiles from these devices. The proposed approach provides a systematic framework to simulate the controlled release of the drug from the diffusion-controlled drug-polymer release systems. The developed models can be used in a reverse engineer framework to design the controlled delivery devices for a target drug release profile in near future.

siRNA delivery using polyelectrolyte-gold nanoassemblies in neuronal cells for BACE1 gene silencing.

Small interfering RNA (siRNA) mediated RNA interference is a versatile therapeutic tool for many intractable genetic disorders. Various nanoassemblies specifically designed to deliver the siRNAs could be utilized for efficient siRNA delivery which is one of the major concern for the success of this therapeutic. Thus, in the present study, polyelectrolyte-gold nanoassemblies (PE-Gold NAs) were selected for siRNA delivery of an in vitro verified siRNA. Three different polyelectrolytes (polyethyleneimine, citraconic anhydride modified poly (allylamine) hydrochloride and poly l-arginine) were used to formulate the PE-Gold NAs using the layer-by-layer technique. Successful physico-chemical characterizations of these PE-Gold NAs were performed using UV-Visible, FTIR, 1H-NMR spectroscopies, XRD, TEM, DLS and Zeta potential measurements. In vitro studies for the cytotoxicity, the uptake of these nanoassemblies and the gene silencing were carried out using these PE-Gold NAs in N2a and NB4 1A3 (murine neuronal) cell lines. The three selected PE-Gold NAs showed significant BACE1 (ß-site APP cleaving enzyme 1) gene silencing (50-60%). This work demonstrates the potential of PE-Gold NAs to deliver siRNA targeting BACE1 in neuronal cells. Finally, it was concluded that different polyelectrolytes used in the PE-Gold NAs achieve different gene silencing due to the variation in their delivery efficiencies.

2-(2-Arylphenyl)benzoxazole As a Novel Anti-Inflammatory Scaffold: Synthesis and Biological Evaluation.

The 2-(2-arylphenyl)benzoxazole moiety has been found to be a new and selective ligand for the enzyme cyclooxygenase-2 (COX-2). The 2-(2-arylphenyl)benzoxazoles 3a-m have been synthesized by Suzuki reaction of 2-(2-bromophenyl)benzoxazole. Further synthetic manipulation of 3f and 3i led to 3o and 3n, respectively. The compounds 3g, 3n, and 3o selectively inhibited COX-2 with selectivity index of 3n much better than that of the COX-2 selective NSAID celecoxib. The in vivo anti-inflammatory potency of 3g and 3n is comparable to that of celecoxib and the nonselective NSAID diclofenac at two different doses, and 3o showed better potency compared to these clinically used NSAIDs.

Application of a novel bacterial consortium for mineralization of sulphonated aromatic amines.

A novel bacterial consortium (TJ-2) for mineralization of aromatic amines resulting from decolorization of azo dyes was developed. Three bacterial strains were identified as Pseudomonas pseudoalcaligenes (TJ-21,EU072476), Pseudomonas citronellolis (TJ-22,EU072477) and Pseudomonas testosterone (TJ-23,EU072477) by 16S rRNA gene sequence analysis. Aromatic amine mineralization under aerobic conditions was observed to be significantly higher with the consortium as compared to pure strains indicating complementary interactions among these strains. It was observed that more than 90% mineralization of aromatic amines was achieved within 18h for different initial aromatic amines concentrations. It was also observed that aromatic amine mineralization depends upon the structure of aromatic amine. Para- and meta-hydroxy substituted aromatic amine were easily mineralized as compared to ortho-substituted which undergoes autoxidation when exposed to oxygen. The consortium was capable of mineralizing other aromatic amines, thus, conferring the possibility of application of TJ-2 for the treatment of industrial wastewaters containing aromatic amines.

Development of a software tool and criteria evaluation for efficient design of small interfering RNA.

RNA interference can be used as a tool for gene silencing mediated by small interfering RNAs (siRNA). The critical step in effective and specific RNAi processing is the selection of suitable constructs. Major design criteria, i.e., Reynolds's design rules, thermodynamic stability, internal repeats, immunostimulatory motifs were emphasized and implemented in the siRNA design tool. The tool provides thermodynamic stability score, GC content and a total score based on other design criteria in the output. The viability of the tool was established with different datasets. In general, the siRNA constructs produced by the tool had better thermodynamic score and positional properties. Comparable thermodynamic scores and better total scores were observed with the existing tools. Moreover, the results generated had comparable off-target silencing effect. Criteria evaluations with additional criteria were achieved in WEKA.

Progress in COX-2 inhibitors: a journey so far.

The non-steroidal anti-inflammatory drugs (NSAIDs) are diverse group of compounds used for the treatment of inflammation, since the introduction of acetylsalicylic acid in 1899. Traditional (first generation) NSAIDs exert antiinflammatory, analgesic, and antipyretic effects through the blockade of prostaglandin synthesis via non-selective inhibition of cyclooxygenase (COX-1 and COX-2) isozymes. Their use is associated with side effects such as gastrointestinal and renal toxicity. A number of selective (second generation) COX-2 inhibitors (rofecoxib, celecoxib, valdecoxib etc.) were developed as safer NSAIDs with improved gastric safety profile. Observation of increased cardiovascular risks in APPROVe (Adenomatous Polyp Prevention on Vioxx) study sent tremors and led to voluntary withdrawn of Vioxx (rofecoxib) by Merck from the market in September 2004 followed by Bextra (valdecoxib) in 2005 raising a question on the safety of selective COX-2 inhibitors. This leads to the belief that these effects are mechanism based and may be class effect. However, some studies suggested association of traditional NSAIDs with similar effects requiring a relook into the whole class of NSAIDs rather than simply victimizing the selective COX-2 inhibitors. Recognition of new avenues for selective COX-2 inhibitors such as cancer, Alzheimer's disease, Parkinson's disease, schizophrenia, major depression, ischemic brain injury and diabetic peripheral nephropathy has kindled the interest in these compounds. This review highlights the various structural classes of selective COX-2 inhibitors developed during past seven years (2003-2009) with special emphasis on diaryl-hetero/carbo-cyclic class of compounds. Molecular modeling aspects are also briefly discussed.

Optimization of extrusion conditions for defatted soy-rice blend extrudates.

Processing parameters of feed including moisture content of feed (12, 15, 18, 21 and 24%), soy flour-rice blend ratio (10:90, 14:86, 18:82, 22:78 and 26:74), operational parameters of extruder like barrel temperature (100, 110, 120, 130 and 140 °C), die head temperature (160, 170, 180, 190 and 200 °C) and screw speed (100, 110, 120, 130 and 140 rpm) were optimized for physical and textural properties of soy-rice blend extrudates. The minimum value of longitudinal expansion index lied near 185 °C die head temperature at about 120 rpm. The minimum value of bulk density was observed in between 18:82 blend ratio and 115 °C barrel temperature.

Isolation, identification and application of novel bacterial consortium TJ-1 for the decolourization of structurally different azo dyes.

A novel bacterial consortium (TJ-1), which could decolorize Acid Orange 7 (AO7) and manyother azo dyes, was developed. In TJ-1 three bacterial strains were identified as Aeromonas caviae, Proteus mirabilis and Rhodococcus globerulus by 16S rRNA gene sequence analysis. AO7 decolorization was significantly higher with the use of consortium as compared to the use of individual strains, indicating complementary interactions among these strains. AO7 decolorization was observed under microaerophilic condition in the presence of organic carbon source. Either yeast extract (YE) alone or a combination of YE and glucose resulted in much higher decolorization of AO7 as compared to glucose alone, peptone or starch. Kinetic studies with different initial AO7 concentrations showed that more than 90% decolorization could be achieved even at 200mg/l within 16h. Fed-batch studies showed that AO7 decolorization required 10h during the first cycle and 5h in the second and third cycles, showing that bacterial cells could be used for multiple cycles. The consortium also decolorized fifteen other azo dyes individually as well as a simulated wastewater containing a mixture of all the sixteen azo dyes, thus, conferring the possibility of application of TJ-1 for the treatment of industrial wastewaters.

Mononeuritis multiplex secondary to sarcoidosis.

Peripheral nerve involvement is a rare, yet treatable neurological manifestation of sarcoidosis. Most patients respond well to corticosteroids, but relapses are common and the long-term prognosis remains unpredictable. We present a patient with an asymmetrical neurological presentation of previously undiagnosed sarcoidosis. She presented with paresthesias and predominantly distal extremity weakness. Other possible causes of neuropathy were ruled out and she was found to have an elevated serum ACE level. A nerve/lip biopsy demonstrated non-caseating granulomas consistent with sarcoid. Her clinical outcome was favorable after initiating treatment with high dose oral prednisone.

An adaptive strategy for single- and multi-cluster gene assignment.

Strict assignment of genes to one class, dimensionality reduction, a priori specification of the number of classes, the need for a training set, nonunique solution, and complex learning mechanisms are some of the inadequacies of current clustering algorithms. Existing algorithms cluster genes on the basis of high positive correlations between their expression patterns. However, genes with strong negative correlations can also have similar functions and are most likely to have a role in the same pathways. To address some of these issues, we propose the adaptive centroid algorithm (ACA), which employs an analysis of variance (ANOVA)-based performance criterion. The ACA also uses Euclidian distances, the center-of-mass principle for heterogeneously distributed mass elements, and the given data set to give unique solutions. The proposed approach involves three stages. In the first stage a two-way ANOVA of the gene expression matrix is performed. The two factors in the ANOVA are gene expression and experimental condition. The residual mean squared error (MSE) from the ANOVA is used as a performance criterion in the ACA. Finally, correlated clusters are found based on the Pearson correlation coefficients. To validate the proposed approach, a two-way ANOVA is again performed on the discovered clusters. The results from this last step indicate that MSEs of the clusters are significantly lower compared to that of the fibroblast-serum gene expression matrix. The ACA is employed in this study for single- as well as multi-cluster gene assignments.

Multistage gene expression profiling in a differentially susceptible mouse colon cancer model.

The DNA alkylating agent, azoxymethane (AOM), induces tumor formation in the distal colon of susceptible mice. Differential susceptibility to this colonotropic carcinogen has been well characterized in A/J (sensitive) and AKR/J (resistant) mice. However, the reasons underlying the differential response to AOM and the molecular mechanisms involved in colon tumor progression remain unclear. To address these issues, we used a cDNA microarray approach to determine time-related changes in gene expression patterns in A/J and AKR/J colons following carcinogen treatment. In the present study, mice were injected intraperitoneally with either AOM (10mg/kg body weight once a week for 6 weeks) or 0.9% NaCl solution (vehicle controls). Total RNA was isolated from the distal colons at 1, 4, and 24 weeks post-AOM exposure. RNA was reverse transcribed and cDNA samples labeled with Cy3 and Cy5 were hybridized to a glass chip containing 4608 mouse cDNA duplicate clones. The resulting mRNA expression levels were analyzed using GLEAMS 3.0, a Unix/Linux-based software program. Genes with more than twofold variations in expression levels were considered significant. Further clustering analysis was performed based on gene expression patterns at different time points using a novel adaptive centroid algorithm (ACA). Of the 4608 genes, 118 clustered into 11 significant groups that displayed similar and distinct expression patterns between the strains following carcinogen treatment. Nine clusters were selected for further analysis based on their divergence in response between A/J and AKR/J and their potential role in tumorigenesis. Overall, our data indicate time- and strain-specific genetic alterations during different stages of colon tumorigenesis following AOM treatment.