An Intrigrated Novel Approach in MCDM under Fuzziness

Multiple Criteria Decision Making (MCDM) shows promising areas of applications in the field of computational technique of proper project selection. There are four distinct families of methods in MCDM: (a) the outranking,(b) the theory based on value and utility, (c) the multiple objective programming and (d) collaborative decision and negotiation theory based method. An Analytical way to reach the best possible solution of project selection is most desirable. Analytical Hierarchy Process (AHP) is one of the best ways for deciding among the complex criteria structure in different levels. Fuzzy AHP is a synthetic extension of classical AHP method under fuzziness. A fuzzy decision may be viewed as an intersection of the given goals and constraints. A maximizing decision is defined as a point in the space of alternatives at which the membership function of a fuzzy decision attains its maximum value. This paper aims at the integration of fuzzy AHP and Additive Ratio Assessment Method (ARAS). It actually deals with a novel integrated approach of dual synthesis of project selection. At first fuzzy AHP, method is used to find the criteria coefficient with the performance evaluation in a certain environment where triangular fuzzy number describes the subjectivity of vagueness of the criteria. In the second phase, ARAS method is used to determine the rank of the final project selection

Interaction of the intrinsically disordered C?terminal domainof the sesbania mosaic virus RNA?dependent RNA polymerasewith the viral protein P10 in vitro: modulation of the oligomeric stateand polymerase activity

AbstractThe RNA-dependent RNA polymerase (RdRp) of sesbania mosaic virus (SeMV) was previously shown to interact withthe viral protein P10, which led to enhanced polymerase activity. In the present investigation, the equilibrium dissociationconstant for the interaction between the two proteins was determined to be 0.09 ?M using surface plasmon resonance, andthe disordered C-terminal domain of RdRp was shown to be essential for binding to P10. The association with P10 broughtabout a change in the oligomeric state of RdRp, resulting in reduced aggregation and increased polymerase activity. Interestingly,unlike the wild-type RdRp, C-terminal deletion mutants (C del 43 and C del 72) were found to exist predominantlyas monomers and were as active as the RdRp-P10 complex. Thus, either the deletion of the C-terminal disordered domainor its masking by binding to P10 results in the activation of polymerase activity. Further, deletion of the C-terminal 85 residuesof RdRp resulted in complete loss of activity. Mutation of a conserved tyrosine (RdRp Y480) within motif E, locatedbetween 72 and 85 residues from the C-terminus of RdRp, rendered the protein inactive, demonstrating the importance ofmotif E in RNA synthesis in vitro

Functional insights into the role of C-terminal disordered domain of Sesbania mosaic virus RNA-dependent RNA polymerase and the coat protein in viral replication in vivo

The C-terminal disordered domain of sesbania mosaic virus (SeMV) RNA-dependent RNA polymerase (RdRp) interacts with the viral protein P10. The functional significance of this interaction in viral replication was examined by a comparative analysis of genomic and sub-genomic RNA levels (obtained by quantitative real time PCR) in the total RNA extracted from Cyamopsis plants agro-infiltrated with wild-type or mutant forms of SeMV infectious cDNA (icDNA). The sgRNA copy numbers were found to be significantly higher than those of gRNA in the wild-type icDNA transfected plants. Transfection of a mutant icDNA expressing an RdRp lacking the Cterminal disordered domain led to a drastic reduction in the copy numbers of both forms of viral RNA. This could be due to the loss of interaction between the disordered domain of RdRp and P10 and possibly other viral/host proteins that might be required for the assembly of viral replicase. The C-terminal disordered domain also harbours the motif E which is essential for the catalytic function of RdRp. Mutation of the conserved tyrosine within this motif in the full length icDNA resulted in complete inhibition of progeny RNA synthesis in the transfected plants confirming the importance of motif E in the polymerase function in vivo. The role of coat protein (CP) in viral infection was also investigated by agro-infiltration of a CP start codon mutant icDNA which suggested that CP is essential for the encapsidation of viral progeny RNAs at later stages of infection

Comparative Analysis of NanoLuc Luciferase and Alkaline Phosphatase Luminescence Reporter Systems for Phage-Based Detection of Bacteria

Reporter phage assays are a promising alternative to culture-based assays for rapidly detecting viable bacteria. The reporter systems used in phage-based detection are typically enzymes and their corresponding substrates that provide a signal following infection and expression. While several reporter systems have been developed, comparing reporter systems based on reported bacteria detection limits from literature can be challenging due to factors other than the reporter system that influence detection capabilities. To advance the development of phage-based assays, a systematic comparison and understanding of the components are necessary. The objective of this study was to directly compare two common enzyme-mediated luminescence reporter systems, NanoLuc/Nano-Glo and alkaline phosphatase (ALP*)/DynaLight, for phage-based detection of bacteria. The detection limits of the purified enzymes were determined, as well as the expression levels and bacteria detection capabilities following engineering of the coding genes into T7 phage and infection of E. coli BL21. When comparing the sensitivity of the purified enzymes, NLuc/Nano-Glo enzyme/substrate system demonstrated a lower detection limit than ALP*/DynaLight. In addition, the expression of the NLuc reporter following phage infection of E. coli was greater than ALP*. The lower detection limit combined with the higher expression resulted in a greater than 100-fold increase in sensitivity for the NLuc/Nano-Glo® reporter system compared to ALP*/DynaLight when used for the detection of E. coli in a model system. These findings provide a comparative analysis of two common reporter systems used for phage-based detection of bacteria and a foundational understanding of these systems for engineering future reporter phage assays.This article is published as Wijeratne, S.; Bakshi, A.; Talbert, J. Comparative Analysis of NanoLuc Luciferase and Alkaline Phosphatase Luminescence Reporter Systems for Phage-Based Detection of Bacteria. Bioengineering 2022, 9, 479. https://doi.org/10.3390/bioengineering9090479. Posted with permission. © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/)..<br/

Comparative Analysis of NanoLuc Luciferase and Alkaline Phosphatase Luminescence Reporter Systems for Phage-Based Detection of Bacteria

Reporter phage assays are a promising alternative to culture-based assays for rapidly detecting viable bacteria. The reporter systems used in phage-based detection are typically enzymes and their corresponding substrates that provide a signal following infection and expression. While several reporter systems have been developed, comparing reporter systems based on reported bacteria detection limits from literature can be challenging due to factors other than the reporter system that influence detection capabilities. To advance the development of phage-based assays, a systematic comparison and understanding of the components are necessary. The objective of this study was to directly compare two common enzyme-mediated luminescence reporter systems, NanoLuc/Nano-Glo and alkaline phosphatase (ALP*)/DynaLight, for phage-based detection of bacteria. The detection limits of the purified enzymes were determined, as well as the expression levels and bacteria detection capabilities following engineering of the coding genes into T7 phage and infection of E. coli BL21. When comparing the sensitivity of the purified enzymes, NLuc/Nano-Glo enzyme/substrate system demonstrated a lower detection limit than ALP*/DynaLight. In addition, the expression of the NLuc reporter following phage infection of E. coli was greater than ALP*. The lower detection limit combined with the higher expression resulted in a greater than 100-fold increase in sensitivity for the NLuc/Nano-Glo® reporter system compared to ALP*/DynaLight when used for the detection of E. coli in a model system. These findings provide a comparative analysis of two common reporter systems used for phage-based detection of bacteria and a foundational understanding of these systems for engineering future reporter phage assays

Interaction of Sesbania mosaic virus (SeMV) RNA-dependent RNA polymerase (RdRp) with the p10 domain of polyprotein 2a and its implications in SeMV replication

Identification of viral encoded proteins that interact with RNA-dependent RNA polymerase (RdRp) is an important step towards unraveling the mechanism of replication. Sesbania mosaic virus (SeMV) RdRp was shown to interact strongly with p10 domain of polyprotein 2a and moderately with the protease domain. Mutational analysis suggested that the C-terminal disordered domain of RdRp is involved in the interaction with p10. Coexpression of full length RdRp and p10 resulted in formation of RdRp–p10 complex which showed significantly higher polymerase activity than RdRp alone. Interestingly, CΔ43 RdRp also showed a similar increase in activity. Thus, p10 acts as a positive regulator of RdRp by interacting with the C-terminal disordered domain of RdRp

Interaction of the intrinsically disordered C-terminal domain of the sesbania mosaic virus RNA-dependent RNA polymerase with the viral protein P10 in vitro: modulation of the oligomeric state and polymerase activity

The RNA-dependent RNA polymerase (RdRp) of sesbania mosaic virus (SeMV) was previously shown to interact with the viral protein P10, which led to enhanced polymerase activity. In the present investigation, the equilibrium dissociation constant for the interaction between the two proteins was determined to be 0.09 mu M using surface plasmon resonance, and the disordered C-terminal domain of RdRp was shown to be essential for binding to P10. The association with P10 brought about a change in the oligomeric state of RdRp, resulting in reduced aggregation and increased polymerase activity. Interestingly, unlike the wild-type RdRp, C-terminal deletion mutants (C del 43 and C del 72) were found to exist predominantly as monomers and were as active as the RdRp-P10 complex. Thus, either the deletion of the C-terminal disordered domain or its masking by binding to P10 results in the activation of polymerase activity. Further, deletion of the C-terminal 85 residues of RdRp resulted in complete loss of activity. Mutation of a conserved tyrosine (RdRp Y480) within motif E, located between 72 and 85 residues from the C-terminus of RdRp, rendered the protein inactive, demonstrating the importance of motif E in RNA synthesis in vitro

Effect of tamarind seed husk supplementation on ruminal methanogenesis, methanogen diversity and fermentation characteristics

Tamarind seed husk, an agricultural waste, was evaluated both in vitro & in vivo as a methane-ameliorating agent. Basal diets for in vitro study were formulated using finger millet straw and concentrate in 70:30 proportions, and tamarind seed husk was included as part of the concentrate with equal (w/w) replacement of wheat bran to achieve 2.5 (T1), 5.0 (T2) and 10.0% (T3) levels. Based on the in vitro results, two levels (2.5%, T1 and 5.0%, T2) of tamarind seed husk were selected for the in vivo study in male cattle to investigate the effect on enteric methane emission. Fifteen crossbred cattle divided into three groups were assigned to the following diets: control (T0, no husk), test group 1 (T1, 2.5% husk) and test group 2 (T2, 5.0% husk). The in vivo study showed a decrease of about 17% in enteric methane emission with a higher level (5%) of tamarind seed husk supplementation. The in vivo study demonstrated a non-adverse impact of supplementation on dry matter intake and digestibility, while rumen methanogens notably decreased at 5% inclusion level. It may be concluded that tamarind seed husk supplementation achieved significant methane reduction via direct inhibition of rumen methanogens without affecting fibre degradability

A FACS-based novel isolation technique identifies heterogeneous CTCs in oral squamous cell carcinoma

PurposeIsolating circulating tumour cells (CTCs) from the blood is challenging due to their low abundance and heterogeneity. Limitations of conventional CTC detection methods highlight the need for improved strategies to detect and isolate CTCs. Currently, the Food and Drug Administration (FDA)-approved CellSearch™ and other RUO techniques are not available in India. Therefore, we wanted to develop a flexible CTC detection/isolation technique that addresses the limitation(s) of currently available techniques and is suitable for various downstream applications.MethodsWe developed a novel, efficient, user-friendly CTC isolation strategy combining density gradient centrifugation and immuno-magnetic hematogenous cell depletion with fluorescence-activated cell sorting (FACS)-based positive selection using multiple CTC-specific cell-surface markers. For FACS, a stringent gating strategy was optimised to exclude debris and doublets by side scatter/forward scatter (SSC/FSC) discriminator, remove dead cells by 4′,6-diamidino-2-phenylindole (DAPI) staining, and eliminate non-specific fluorescence using a “dump” channel. APC-labelled anti-CD45mAB was used to gate remaining hematogenous cells, while multiple epithelial markers (EpCAM, EGFR, and Pan-Cytokeratin) and an epithelial–mesenchymal transition (EMT) marker (Vimentin) labelled with fluorescein isothiocyanate (FITC) were used to sort cancer cells. The technique was initially developed by spiking Cal 27 cancer cells into the blood of healthy donors and then validated in 95 biopsy-proven oral squamous cell carcinoma (OSCC) patients. CTCs isolated from patients were reconfirmed by Giemsa staining, immuno-staining, and whole transcriptome amplification (WTA), followed by qRT-PCR. In vitro culture and RNA sequencing (RNA-Seq) were also performed to confirm their suitability for various downstream applications.ResultsThe mean detection efficiency for the Cal 27 tongue cancer cells spiked in the whole blood of healthy donors was 32.82% ± 12.71%. While ~75% of our patients (71/95) had detectable CTCs, the CTC positivity was independent of the TNM staging. The isolated potential cancer cells from OSCC patients were heterogeneous in size. They expressed different CTC-specific markers in various combinations as identified by qRT-PCR after WTA in different patients. Isolated CTCs were also found to be suitable for downstream applications like short-term CTC culture and RNA-Seq.ConclusionWe developed a sensitive, specific, flexible, and affordable CTC detection/isolation technique, which is scalable to larger patient cohorts, provides a snapshot of CTC heterogeneity, isolates live CTCs ready for downstream molecular analysis, and, most importantly, is suitable for developing countries