Purification, characterization and potential detergent industry application of a thermostable α-amylase from Bacillus licheniformis RA31

331-342α-Amylase is an endoamylase catalysing the degradation of starch into maltose, maltotriose and glucose. The enzyme isolated from microbial sources possess unique properties of thermostability thus making it a useful tool in the detergent industry. Here, we explored a strain of thermophilic bacteria Bacillus licheniformis for its potential application in detergent industry. The B. licheniformis RA31 was isolated from soil samples of hot spring in Rampur district of Himachal Pradesh, India and grown on optimized media to produce thermostable α-amylase. The enzyme was ethanol precipitated, purified (12.93 fold, 55.52% yield and 621.93 U/mg specific activity) to homogeneity with a single band on SDS-PAGE (66 kDa) and native-PAGE (68 kDa). Purified enzyme displayed best activity in pH 8 buffer and 80% activity was retained in pH 7 and 10. It showed temperature optima at 70°C. Its activity was decreased at 70°C (70% after 4 h), 80°C (65% after 4 h) and 90°C (50% after 1 h). The enzyme was stimulated (126%; 5 mM) by barium chloride. It was relatively stable in the presence of commercial detergents (109-125%), SDS (84%), Tween 20 (88%), EDTA (72%) and β-ME (70% at 10 mM). Km and Vmax for the enzymatic hydrolysis of starch were 0.339 mg/mL and 1.450 mg/min, respectively. The enzyme revealed the highest specificity towards wheat starch granule (140% after 1 h) and SEM analysis displayed its biodegradation (2-10 h). Improved cleaning efficiency of potato curry stained fine cotton clothes were observed with enzyme assisted detergent advance treatment (0.02% w/v). The enzyme showed potential applications in detergent industry

Nicotine, IFN-γ and retinoic acid mediated induction of MUC4 in pancreatic cancer requires E2F1 and STAT-1 transcription factors and utilize different signaling cascades

BACKGROUND: The membrane-bound mucins are thought to play an important biological role in cell–cell and cell–matrix interactions, in cell signaling and in modulating biological properties of cancer cell. MUC4, a transmembrane mucin is overexpressed in pancreatic tumors, while remaining undetectable in the normal pancreas, thus indicating a potential role in pancreatic cancer pathogenesis. The molecular mechanisms involved in the regulation of MUC4 gene are not yet fully understood. Smoking is strongly correlated with pancreatic cancer and in the present study; we elucidate the molecular mechanisms by which nicotine as well as agents like retinoic acid (RA) and interferon-γ (IFN-γ) induce the expression of MUC4 in pancreatic cancer cell lines CD18, CAPAN2, AsPC1 and BxPC3. RESULTS: Chromatin immunoprecipitation assays and real-time PCR showed that transcription factors E2F1 and STAT1 can positively regulate MUC4 expression at the transcriptional level. IFN-γ and RA could collaborate with nicotine in elevating the expression of MUC4, utilizing E2F1 and STAT1 transcription factors. Depletion of STAT1 or E2F1 abrogated the induction of MUC4; nicotine-mediated induction of MUC4 appeared to require α7-nicotinic acetylcholine receptor subunit. Further, Src and ERK family kinases also mediated the induction of MUC4, since inhibiting these signaling molecules prevented the induction of MUC4. MUC4 was also found to be necessary for the nicotine-mediated invasion of pancreatic cancer cells, suggesting that induction of MUC4 by nicotine and other agents might contribute to the genesis and progression of pancreatic cancer. CONCLUSIONS: Our studies show that agents that can promote the growth and invasion of pancreatic cancer cells induce the MUC4 gene through multiple pathways and this induction requires the transcriptional activity of E2F1 and STAT1. Further, the Src as well as ERK signaling pathways appear to be involved in the induction of this gene. It appears that targeting these signaling pathways might inhibit the expression of MUC4 and prevent the proliferation and invasion of pancreatic cancer cells

Altered expression of transmembrane mucins, MUC1 and MUC4, in bladder cancer: pathological implications in diagnosis.

PURPOSE: Radical changes in both expression and glycosylation pattern of transmembrane mucins have been observed in various malignancies. We and others have shown that MUC1 and MUC4, two transmembrane mucins, play a sentinel role in cell signaling events that drive several epithelial malignancies. In the present study, we investigated the expression profile of MUC1 and MUC4 in the non-neoplastic bladder urothelium, in various malignant neoplasms of bladder and in bladder carcinoma cell lines. MATERIAL AND METHODS: Immunohistochemistry was performed on tissue sections from the urinary bladder biopsies, resection samples and tissue microarrays (TMAs) with monoclonal antibodies specific for MUC1 and MUC4. We also investigated their expression in bladder carcinoma cell lines by RT-PCR and immunoblotting. RESULTS: MUC1 is expressed on the apical surface or in umbrella cells of the normal non-neoplastic bladder urothelium. Strong expression of MUC1 was also observed in urothelial carcinoma (UC). MUC1 staining increased from normal urothelium (n = 27, 0.35±0.12) to urothelial carcinoma (UC, n = 323, H-score, 2.4±0.22, p≤0.0001). In contrast to MUC1, MUC4 was expressed in all the layers of non-neoplastic bladder urothelium (n = 14, 2.5±0.28), both in the cell membrane and cytoplasm. In comparison to non-neoplastic urothelium, the loss of MUC4 expression was observed during urothelial carcinoma (n = 211, 0.56±0.06). However, re-expression of MUC4 was observed in a subset of metastatic cases of urothelial carcinoma (mean H-score 0.734±0.9). CONCLUSION: The expression of MUC1 is increased while that of MUC4 decreased in UC compared to the normal non-neoplastic urothelium. Expression of both MUC1 and MUC4, however, are significantly higher in urothelial carcinoma metastatic cases compared to localized UC. These results suggest differential expression of MUC1 and MUC4 during development and progression of bladder carcinoma

A blockchain and deep neural networks-based secure framework for enhanced crop protection

The problem faced by one farmer can also be the problem of some other farmer in other regions. Providing information to farmers and connecting them has always been a challenge. Crowdsourcing and community building are considered as useful solutions to these challenges. However, privacy concerns and inactivity of users can make these models inefficient. To tackle these challenges, we present a cost-efficient and blockchain-based secure framework for building a community of farmers and crowdsourcing the data generated by them to help the farmers’ community. Apart from ensuring privacy and security of data, a revenue model is also incorporated to provide incentives to farmers. These incentives would act as a motivating factor for the farmers to willingly participate in the process. Through integration of a deep neural network-based model to our proposed framework, prediction of any abnormalities present within the crops and their predicted possible solutions would be much more coherent. The simulation results demonstrate that the prediction of plant pathology model is highly accurate

Purification, characterization and potential detergent industry application of a thermostable α-amylase from Bacillus licheniformis RA31

α-Amylase is an endoamylase catalysing the degradation of starch into maltose, maltotriose and glucose. The enzyme isolated from microbial sources possess unique properties of thermostability thus making it a useful tool in the detergent industry. Here, we explored a strain of thermophilic bacteria Bacillus licheniformis for its potential application in detergent industry. The B. licheniformis RA31 was isolated from soil samples of hot spring in Rampur district of Himachal Pradesh, India and grown on optimized media to produce thermostable α-amylase. The enzyme was ethanol precipitated, purified (12.93 fold, 55.52% yield and 621.93 U/mg specific activity) to homogeneity with a single band on SDS-PAGE (66 kDa) and native-PAGE (68 kDa). Purified enzyme displayed best activity in pH 8 buffer and 80% activity was retained in pH 7 and 10. It showed temperature optima at 70°C. Its activity was decreased at 70°C (70% after 4 h), 80°C (65% after 4 h) and 90°C (50% after 1 h). The enzyme was stimulated (126%; 5 mM) by barium chloride. It was relatively stable in the presence of commercial detergents (109-125%), SDS (84%), Tween 20 (88%), EDTA (72%) and β-ME (70% at 10 mM). Km and Vmax for the enzymatic hydrolysis of starch were 0.339 mg/mL and 1.450 mg/min, respectively. The enzyme revealed the highest specificity towards wheat starch granule (140% after 1 h) and SEM analysis displayed its biodegradation (2-10 h). Improved cleaning efficiency of potato curry stained fine cotton clothes were observed with enzyme assisted detergent advance treatment (0.02% w/v). The enzyme showed potential applications in detergent industry

Isolation and molecular identification of α-amylase producing bacteria from hot water spring by 16S rRNA gene sequencing

937-944Among different types of microbial enzymes, amylases are the most widely used in industries as they are produced in large quantity and in an economic way as compared to plants and animals. Moreover, thermostable amylase has significance as compared to the amylase from mesophiles. Therefore, hot water springs are explored to dig into its bacterial diversity. In the current study, we tried to isolate amylase producing bacteria from the soil and water samples collected from the hot water spring in Rampur, Himachal Pradesh, India. The samples were serially diluted before plating on the Luria Bertani agar plates. A total of 42 bacterial morphotypes were isolated and were screened for amylolytic activity by starch agar plate method. Among the 42 bacterial isolates 25 showed amylolytic activity. Production of amylase was carried out at different temperatures and pH to optimize the temperature and pH conditions for each isolate. All the 25 isolates were characterized based on morphology, biochemical tests and molecular analysis. Sequencing of 16S rRNA gene for the 25 isolates followed by BLAST search revealed a majority of them (19) identified as Bacillus licheniformis. Other isolates were identified as B. subtilis, B. safensis, B. halodurans, B. stratosphericus, Caldimonas hydrothermale and Exiguobacterium mexicanum. An attempt was made to amplify amyN gene which codes for &alpha;-amylase but successful amplification was achieved only from Bacillus licheniformis alone.</span

Radiofrequency ablation of superior hypogastric plexus for the management of pelvic cancer pain

Radiofrequency ablation is a safe and minimally invasive procedure that has been found effective for the treatment of various chronic pain conditions. Hereby, we report a case of severe pelvic pain due to advanced carcinoma cervix refractory to oral medications that was managed successfully by radiofrequency ablation of the superior hypogastric plexus under fluoroscopy. Her pain relieved markedly immediately after the procedure with significant reduction in opioid consumption. The effect was sustained during her follow-up period with improved quality of life

Oral microbiome and health

The oral microbiome is diverse in its composition due to continuous contact of oral cavity with the external environment. Temperatures, diet, pH, feeding habits are important factors that contribute in the establishment of oral microbiome. Both culture dependent and culture independent approaches have been employed in the analysis of oral microbiome. Gene-based methods like PCR amplification techniques, random amplicon cloning, PCR-RELP, T-RELP, DGGE and DNA microarray analysis have been applied to increase oral microbiome related knowledge. Studies revealed that microbes from the phyla Firmicutes, Proteobacteria, Bacteroidetes, Actinobacteria, Fusobacteria, Neisseria, TM7 predominately inhabits the oral cavity. Culture-independent molecular techniques revealed the presence of genera Megasphaera, Parvimonas and Desulfobulbus in periodontal disease. Bacteria, fungi and protozoa colonize themselves on various surfaces in oral cavity. Microbial biofilms are formed on the buccal mucosa, dorsum of the tongue, tooth surfaces and gingival sulcus. Various studies demonstrate relationship between unbalanced microflora and development of diseases like tooth caries, periodontal diseases, type 2 diabetes, circulatory system related diseases etc. Transcriptome-based remodelling of microbial metabolism in health and disease associated states has been well reported. Human diets and habitat can trigger virus activation and influence phage members of oral microbiome. As it is said, “Mouth, is the gateway to the total body wellness, thus oral microbiome influences overall health of an individual”

No Association of PTGDR −441C/T Polymorphism with Asthma in a North Indian Population

Background: Asthma is the most prevalent disease in India according to the national survey conducted by NFHS 2 in 1998–399. Prostaglandin D2 (PGD2) is a bronchoconstriction inducing metabolite of arachidonic acid in the mast cells, which is produced on exposure to allergens and acts as a ligand for the Prostaglandin D2 Receptor (PTGDR). Polymorphisms in the PTGDR gene have been suggested to be involved in the mechanism of asthma