Methanosarcina acetivorans C2A Topoisomerase IIIα, an Archaeal Enzyme with Promiscuity in Divalent Cation Dependence

Topoisomerases play a fundamental role in genome stability, DNA replication and repair. As a result, topoisomerases have served as therapeutic targets of interest in Eukarya and Bacteria, two of the three domains of life. Since members of Archaea, the third domain of life, have not been implicated in any diseased state to-date, there is a paucity of data on archaeal topoisomerases. Here we report Methanosarcina acetivorans TopoIIIα (MacTopoIIIα) as the first biochemically characterized mesophilic archaeal topoisomerase. Maximal activity for MacTopoIIIα was elicited at 30–35°C and 100 mM NaCl. As little as 10 fmol of the enzyme initiated DNA relaxation, and NaCl concentrations above 250 mM inhibited this activity. The present study also provides the first evidence that a type IA Topoisomerase has activity in the presence of all divalent cations tested (Mg2+, Ca2+, Sr2+, Ba2+, Mn2+, Fe2+, Co2+, Ni2+, Cu2+, Zn2+ and Cd2+). Activity profiles were, however, specific to each metal. Known type I (ssDNA and camptothecin) and type II (etoposide, novobiocin and nalidixic acid) inhibitors with different mechanisms of action were used to demonstrate that MacTopoIIIα is a type IA topoisomerase. Alignment of MacTopoIIIα with characterized topoisomerases identified Y317 as the putative catalytic residue, and a Y317F mutation ablated DNA relaxation activity, demonstrating that Y317 is essential for catalysis. As the role of Domain V (C-terminal domain) is unclear, MacTopoIIIα was aligned with the canonical E. coli TopoI 67 kDa fragment in order to construct an N-terminal (1–586) and a C-terminal (587–752) fragment for analysis. Activity could neither be elicited from the fragments individually nor reconstituted from a mixture of the fragments, suggesting that native folding is impaired when the two fragments are expressed separately. Evidence that each of the split domains plays a role in Zn2+ binding of the enzyme is also provided

Barriers Analysis on Consumer Usage Intention of Freelance Marketplace in Indonesia

Past studies have shown that the informal sector, including freelancers, contributes significantly towards Indonesia's rate of employment by providing economic opportunities to those who cannot be absorbed by the formal sectors. Seizing this opportunity, many businesses are tapping into providing freelancers a place to sell and market their services online, commonly known as freelance marketplace. Yet the penetration of these platforms is still low in Indonesia. While the supply is high, Indonesian consumers still have doubts to use these marketplaces as their go-to platform when searching for freelancers. Thus, this study aims to investigate factors that impede consumers intention to use freelance marketplace in Indonesia, based on the framework of Innovation Resistance Theory (IRT). The data was collected from 370 respondents through online questionnaires, analyzed using Smart PLS software, and interviews, deciphered using selective coding. The results suggest that usage barrier, value barrier, tradition barrier, and image barrier negatively influence consumers' usage intention of freelance marketplace, all except for risk barrier. Additionally, social influence also has a significant effect on value barrier and image barrier. These findings can be useful to construct business strategies for online freelance service providers, by focusing to eliminate the critical barriers mentioned in this study

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Not AvailableThe devastating sheath blight disease caused by Rhizoctonia solani Kuhn (teleomorph: Thanatephorus cucumeris) causes major yield loss in most rice growing regions of the world. In this study, two moderately tolerant and four susceptible genotypes of rice were selected for R. solani induced proteome analysis using two-dimensional polyacrylamide gel electrophoresis. Forty five differentially expressed proteins (DEPs) were identified and analyzed by Mass Spectrometry. Based on their functions, these proteins were classified into different groups, viz., photosynthesis, resistance and pathogenesis, stress, cell wall metabolism and cytoskeleton development associated proteins, and hypothetical or uncharacterized proteins. Expression of 14 genes encoding DEPs was analyzed by quantitative PCR which showed consistency in transcripts and genes expression pattern. Furthermore, the expression of 16 other genes involved in diverse biological functions was analyzed. Up-regulation of these genes in the tolerant genotype Pankaj during sheath blight disease suggested efficient genetic regulation of this cultivar under stress. Also, expression analysis of conserved microRNAs (miRNAs) and their target genes revealed important role of miRNAs in post-transcriptional gene regulation during development of rice sheath blight disease. Genome-wide discovery of miRNAs and further characterization of DEPs and genes will help in better understanding of the molecular events during sheath blight disease development in rice.Department of Biotechnology, Government of India grant no.BT/PR6466/COE/34/16/201