Cloning and functional characterization of endo-β-1,4-glucanase gene from metagenomic library of vermicompost

In the vermicomposting of paper mill sludge, the activity of earthworms is very dependent on dietetic polysaccharides including cellulose as energy sources. Most of these polymers are degraded by the host microbiota and considered potentially important source for cellulolytic enzymes. In the present study, a metagenomic library was constructed from vermicompost (VC) prepared with paper mill sludge and dairy sludge (fresh sludge, FS) and functionally screened for cellulolytic activities. Eighteen cellulase expressing clones were isolated from about 89,000 fosmid clones libraries. A short fragment library was constructed from the most active positive clone (cMGL504) and one open reading frame (ORF) of 1,092 bp encoding an endo-β-1,4-glucanase was indentified which showed 88% similarity with Cellvibrio mixtus cellulase A gene. The endo-β-1,4-glucanase cmgl504 gene was overexpressed in Escherichia coli. The purified recombinant cmgl504 cellulase displayed activities at a broad range of temperature (25–55°C) and pH (5.5–8.5). The enzyme degraded carboxymethyl cellulose (CMC) with 15.4 U, while having low activity against avicel. No detectable activity was found for xylan and laminarin. The enzyme activity was stimulated by potassium chloride. The deduced protein and three-dimensional structure of metagenome-derived cellulase cmgl504 possessed all features, including general architecture, signature motifs, and N-terminal signal peptide, followed by the catalytic domain of cellulase belonging to glycosyl hydrolase family 5 (GHF5). The cellulases cloned in this work may play important roles in the degradation of celluloses in vermicomposting process and could be exploited for industrial application in future.     

Antifungal and antibacterial activities of Taxus wallichiana Zucc

Current study was undertaken to evaluate the in vitro antifungal and antibacterial potential of methanol extract and subsequent fractions obtained after partitioning in organic solvents with variable polarity of the aerial parts of the tree Taxus wallichiana Zucc. Traditionally, this plant is often used in folk medicines in Pakistan for treating microbial infections. In order to rationalize the traditional use, methanol extracts of leaf, bark, and heartwood of Taxus wallichiana Zucc. were tested against six bacteria and six fungal strains using the Hole diffusion and macro-dilution methods. All extracts and fractions displayed significant antimicrobial effect. Only three fungal strains, Trichophyton longifusus, Microspoum canis, and Fusarium solani were susceptible to the extracts and fractions with MICs ranging from 0.08 to 200 mg/mL. In case of bacterial strains, Staphylococcus aureus, Pseudomonas aeruginosa and Salmonella typhi were susceptible to the extracts and fractions with MICs ranging from 0.08 to 200 mg/mL. Comparison results were carried out using imipinem, miconazole and amphotericin B as standard antibiotics.     

Optimization of enzymatic synthesis of L-ascorbyl palmitate by solvent engineering and statistical experimental designs

Statistical experimental designs combined with solvent engineering for optimization of enzymatic synthesis of L-ascorbyl palmitate were developed. First, the composition of the solvent for co-dissolving polar and apolar substrates was determined. The co-solvent mixture of tert-pentanol: DMSO at a ratio of 9:1 (v/v) and the optimal biocatalyst were obtained. Then, the Plackett-Burman design was implemented to screen the variables that significantly influence the conversion. The method of steepest ascent was used to approach the proximity of optimum. After determining the Plackett-Burman and steepest ascent designs, the optimum values were determined by central composite design under response surface methodology. The statistical analysis showed that the optimum reaction conditions (temperature 50°C, enzyme concentration 5.8 g/L, and substrate molar ratio 11:1, stirring rate 160 rpm, amount of molecular sieve 50 g/L, time 18 h) led to the maximum conversion (66.44%) and production concentration (20.63 g/L). A very satisfactory conversion (64.74%) and production concentration (20.13 g/L) could be achieved in short time (6 h).     

The New Perspectives on Genetic Studies of Type 2 Diabetes and Thyroid Diseases

Recently, genome-wide association studies (GWAS) have led to the discovery of hundreds of susceptibility loci that are associated with complex metabolic diseases, such as type 2 diabetes and hyperthyroidism. The majority of the susceptibility loci are common across different races or populations; while some of them show ethnicity-specific distribution. Though the abundant novel susceptibility loci identified by GWAS have provided insight into biology through the discovery of new genes or pathways that were previously not known, most of them are in introns and the associated variants cumulatively explain only a small fraction of total heritability. Here we reviewed the genetic studies on the metabolic disorders, mainly type 2 diabetes and hyperthyroidism, including candidate genes-based findings and more recently the GWAS discovery; we also included the clinical relevance of these novel loci and the gene-environmental interactions. Finally, we discussed the future direction about the genetic study on the exploring of the pathogenesis of the metabolic diseases.     

Differential Endosomal Sorting of a Novel P2Y12 Purinoreceptor Mutant

P2Y₁₂ receptor internalization and recycling play an essential role in ADP‐induced platelet activation. Recently, we identified a patient with a mild bleeding disorder carrying a heterozygous mutation of P2Y₁₂ (P341A) whose P2Y₁₂ receptor recycling was significantly compromised. Using human cell line models, we identified key proteins regulating wild‐type (WT) P2Y₁₂ recycling and investigated P2Y₁₂‐P341A receptor traffic. Treatment with ADP resulted in delayed Rab5‐dependent internalization of P341A when compared with WT P2Y₁₂. While WT P2Y₁₂ rapidly recycled back to the membrane via Rab4 and Rab11 recycling pathways, limited P341A recycling was observed, which relied upon Rab11 activity. Although minimal receptor degradation was evident, P341A was localized in Rab7‐positive endosomes with considerable agonist‐dependent accumulation in the trans‐Golgi network (TGN). Rab7 activity is known to facilitate recruitment of retromer complex proteins to endosomes to transport cargo to the TGN. Here, we identified that P341A colocalized with Vps26; depletion of which blocked limited recycling and promoted receptor degradation. This study has identified key points of divergence in the endocytic traffic of P341A versus WT‐P2Y₁₂. Given that these pathways are retained in human platelets, this research helps define the molecular mechanisms regulating P2Y₁₂ receptor traffic and explain the compromised receptor function in the platelets of the P2Y₁₂‐P341A‐expressing patient.     

Cell Budding from Normal Appearing Epithelia: A Predictor of Colorectal Cancer Metastasis?

Background: Colorectal carcinogenesis is believed to be a multi-stage process that originates with a localized adenoma, which linearly progresses to an intra-mucosal carcinoma, to an invasive lesion, and finally to metastatic cancer. This progression model is supported by tissue culture and animal model studies, but it is difficult to reconcile with several well-established observations, principally among these are that up to 25% of early stage (Stage I/II), node-negative colorectal cancer (CRC) develop distant metastasis, and that circulating CRC cells are undetectable in peripheral blood samples of up to 50% of patients with confirmed metastasis, but more than 30% of patients with no detectable metastasis exhibit such cells. The mechanism responsible for this diverse behavior is unknown, and there are no effective means to identify patients with pending, or who are at high risk for, developing metastatic CRC.Novel findings: Our previous studies of human breast and prostate cancer have shown that cancer invasion arises from the convergence of a tissue injury, the innate immune response to that injury, and the presence of tumor stem cells within tumor capsules at the site of the injury. Focal degeneration of a capsule due to age or disease attracts lymphocyte infiltration that degrades the degenerating capsules resulting in the formation of a focal disruption in the capsule, which selectively favors proliferating or “budding” of the underlying tumor stem cells. Our recent studies suggest that lymphocyte infiltration also triggers metastasis by disrupting the intercellular junctions and surface adhesion molecules within the proliferating cell buds causing their dissociation. Then, lymphocytes and tumor cells are conjoined through membrane fusion to form tumor-lymphocyte chimeras (TLCs) that allows the tumor stem cell to avail itself of the lymphocyte''s natural ability to migrate and breach cell barriers in order to intravasate and to travel to distant organs. Our most recent studies of human CRC have detected nearly identical focal capsule disruptions, lymphocyte infiltration, budding cells, and the formation of TLCs. Our studies have further shown that age- and type-matched node-positive and -negative CRC have a significantly different morphological and immunohistochemical profile and that the majority of lymphatic ducts with disseminated cells are located within the mucosa adjacent to morphologically normal appearing epithelial structures that express a stem cell-related marker.New hypothesis: Based on these findings and the growth patterns of budding cells revealed by double immunohistochemistry, we further hypothesize that metastatic spread is an early event of carcinogenesis and that budding cells overlying focal capsule disruptions represent invasion- and metastasis-initiating cells that follow one of four pathways to progress: (1) to undergo extensive in situ proliferation leading to the formation of tumor nests that subsequently invade the submucosa, (2) to migrate with associated lymphocytes functioning as “seeds” to grow in new sites, (3) to migrate and intravasate into pre-existing vascular structures by forming TLCs, or (4) to intravasate into vascular structures that are generated by the budding cells themselves. We also propose that only node-positive cases harbor stem cells with the potential for multi-lineage differentiation and unique surface markers that permit intravasation.