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Immunochemical characterization of antigens of Brucella canis and their use in seroprevalence study of canine brucellosis

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Asian Pacific Journal of Tropical Medicine 2011年第11期4卷 857-861页

作者： Sharma Barkha Sinha Dharmendra Kumar Singh Dhirendra Kumar Department of Epidemiology and Preventive Medicine College of Veterinary SciencePandit Deen Dayal Upadhaya Veterinary UniversityMathuraUttar PradeshPin-281001India Division of Epidemiology Indian Veterinary Research InstituteIzatnagarBareillyUttar Pradesh243122India Division of Veterinary Public Health Indian Veterinary Research InstituteIzatnagarBareillyUttar Pradesh243122 India

Objective:To explore immunochemical characterization of antigens of Brucella canis(B. canis),and the use in seroprevalence study of canine brucellosis.Methods:External hot phosphate buffer saline extract(HPBSE) and internal sonicated(SA) antigens were prepared from B.canis strain MEX 51 and imniunochemically characterized.These antigens were used to test 527 serum samples of dogs by 2-mercaptoethanol-tubc agglutination test(2 ME-TAT), agar gel immunodiffusion test(AGID).dot-ELISA and indirect enzyme-linked immunosorbent assay(I-ELISA) to assess the seroprevalence of canine brucellosis.Results:The protein content of HPBSE and SA antigens was 0.387 mg/ml.and 0.195 mg/mL,respectively,whereas carbohydrate content was 0.174 mg/mL and 0.150 mg/mL,respectively.The sodium dodecyl sulfate-polyacrylamide gel electrophoresis(12.5%) of HPBSE and SA,revealed 6 and 8 visible peptide bands ranging from 18-80 kDa and 12-45 kDa,respectively.Western blot analysis showed immunodominant bands of MW 12.28.39 and 45 kl)a for HPBSE and 20-24 kl)a for SA. The AGII) revealed HPBSE as more specific antigen than SA but both I-ELISA and dot-ELISA indicated SA antigen to be more specific and reliable than HPBSE.The seroprevalence of canine brucellosis was 2.27%by 2ME-TAT.1.5%by AGID.3.03%by dot-ELISA and 16.12%by I-ELISA. Conclusions:On the basis of the results of present study,we concluded that HPBSE is suitable antigen for AGID,which is more specific:whereas SA antigen is suitable for I-ELISA,which is highly sensitive.Therefore,initial screening of serum samples should be carried out by I-ELISA followed by confirmation with AGID.

关键词： Brucella canis SDS-PAGE Western blotting Agar gel immunodiffusion test Dot-ELISA I-ELISA

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Role of beneficial microbial gene pool in mitigating salt/nutrient stress of plants in saline soils through underground phytostimulating signalling molecules

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Pedosphere 2023年第1期33卷 153-171页

作者： Shalini TIWARI barkha sharma Neha BISHT Lakshmi TEWARI Department of Microbiology College of Basic Science&HumanitiesG.B.Pant University of Agriculture&TechnologyPantnagarUttarakhand 263145India

Soil salinity diminishes soil health and reduces crop yield,which is becoming a major global concern.Salinity stress is one of the primary stresses,leading to several other secondary stresses that restrict plant growth and soil fertility.The major secondary stresses induced in plants under saline-alkaline conditions include osmotic stress,nutrient limitation,and ionic stress,all of which negatively impact overall plant growth.Under stressed conditions,certain beneficial soil microflora are known to have evolved phytostimulating mechanisms,such as the synthesis of osmoprotectants,siderophores,1-aminocyclopropane-1-carboxylic acid(ACC)deaminase activity,phosphate solubilization,and hormone production,which enhance plant growth and development while mitigating nutrient stress.Beneficial soil-borne bacterial species such as Bacillus,Pseudomonas,and Klebsiella and fungal strains such as Trichoderma,Aspergillus,Penicillium,Alternaria,and Fusarium also aid in reducing salinity stress.Phosphate-solubilizing microorganisms also assist in nutrient acquisition via both enzymatic and non-enzymatic processes.In the case of enzymatic processes,they produce different enzymes such as alkaline phosphatases and phytases,whereas non-enzymatic processes produce organic acids such as gluconic,citric,malic,and oxalic acids.The native halotolerant/halophilic soil microbial gene pool with multifunctional traits and stress-induced gene expression can be developed as suitable bio-inoculants to enhance stress tolerance and optimize plant growth in saline soils.

关键词： abiotic stress crop improvement gene expression halotolerant microbial diversity phosphate solubilization plant growth promoting traits soil health

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Silicon and plant nutrition—dynamics,mechanisms of transport and role of silicon solubilizer microbiomes in sustainable agriculture:A review

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Pedosphere 2023年第4期33卷 534-555页

作者： barkha sharma Kailash Chand KUMAWAT Shalini TIWARI Ajay KUMAR Rouf Ahmad DAR Ummed SINGH Massimiliano CARDINALE Department of Microbiology G.B.Pant University of Agriculture&TechnologyPantnagarUttarakhand 263145(India) Department of Industrial Microbiology Jacob Institute of Biotechnology and BioengineeringSam Higginbottom University of AgricultureTechnology and Sciences(SHUATS)PrayagrajUttar Pradesh 211007(India) College of Agriculture Agriculture UniversityJodhpurBaytuBarmerRajasthan 344034(India) Department of Biological and Environmental Sciences and Technologies-DiSTeBA University of SalentoSP6 Lecce-MonteroniLecce I-73100(Italy)

Silicon(Si)is the second most abundant element in the Earth’s crust and has numerous roles in both soils and plants,although it is inaccessible to plants in its native state(insoluble silicate minerals).This inaccessibility can lead to insufficiency,which induces anomalies in plant growth and development.Specifically,Si alleviates various biotic and abiotic stresses in plants by enhancing tolerance mechanisms at different stages of uptake/deposition as a monosilicic acid.Exclusive utilization of conventional Si fertilizers to boost agricultural productivity in an attempt to meet the world’s ever-increasing food demand may result in surface and groundwater pollution,waterway eutrophication,soil fertility depletion,and accumulation of hazardous elements such as arsenic in soil.By addressing the drawbacks of chemical fertilizers,this review emphasizes an environmentally friendly alternative,namely,Si-solubilizing microorganisms(SSMs),as bioinoculants to liberate soluble Si and thus making it available to plants.A deep understanding of recent advances in the functional diversity,colonization patterns,modes of action,role in biogeochemical cycling,and plant stress tolerance of SSMs and their implementation in Si-based agrowaste management through the fabrication of nanoparticles,could help to establish a reliable tool for economically and environmentally sustainable agriculture.From this perspective,in this review,we comprehensively summarize the latest methodologies for the isolation,screening,and characterization of SSMs and Si solubilization mechanisms,including ligands(divalent cations),acids(inorganic and organic),alkali(nucleophilic attack),extracellular polysaccharides,and factors affecting them,as well as Si-mediated regulation of gene expression involved in Si uptake,transportation,and mineralization.We have critically revised the role of SSMs according to the current literature.The contributions of SSMs to biofertilization are still being explored;hence,we also discuss trajectories for future research in rel

关键词： agrowaste management biofertilizer bioinoculant monosilicic acid plant stress silicon-solubilizing microorganism

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Development of a sandwich ELISA for the detection of bovine herpesvirus type 1

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Asian Pacific Journal of Tropical Medicine 2011年第5期4卷 363-366页

作者： Shanaz Bashir Rashmi Singh barkha sharma Sharad K Yadav Department of Epidemiology and Vety.Preventive medicine College of Veterinary SciencesVeterinary UniversityMathura(U.P.)India

Objective:To develop a standard enzyme-linked immunosorbent assay（ELJSA） for the detection of bovine herpesvirus type 1（BHV-1）.Methods:The assay was based on hyperimmune rabbit and guinea pig antisera raised against purified BHV-1.Polyethylene glycol precipitation and sucrose density gradient methods were adopted for viral concentration and purification.Antisera were raised using Freund’s adjuvant followed by extraction of IgG of high purity.Results: Optimum antisera dilutions as determined by titrations were chosen as 14 000,whereas the conjugate was used at 1:2 000 dilution.Using 95 clinical specimens,the ELISA test showed a sensitivity and specificity of 91.90%and 93.10%,respectively when compared to PCR.The cutoff value was fixed at 0.15490) and a P/N ratio of>1.30 indicated a significant positive reaction. Conclusions:The results have demonstrated that this ELISA could efficiently detect BHV-1 and can be used as an important diagnostic tool.

关键词： Bovine herpesvirus type 1 Antisera Enzyme-linked immunosorbent assay (ELISA)

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Bacterial Surface Layer Proteins: A Promising Nano-Technological Tool for Bio-Sensing Applications

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Advances in Bioscience and Biotechnology 2019年第3期10卷 42-58页

作者： Anjali sharma barkha Singhal School of Biotechnology Gautam Buddha University Greater Noida (U.P.) India

The phenomenal rise in the demand of biosensors accelerated their rapid development and immersive applications in the myriads of fields. The essential requirement of developing efficient bio-sensing platform is to find stable well organized interfacial architecture that can serve as an excellent matrix for binding and recognizing biomolecules. In this context, the enormous potential has been envisaged in surface layer proteins that represented themselves as most primitive and simplest self-assembled system with repetitive physicochemical properties for the molecular functionalization of surfaces and various interfaces. The prominence of S-layer proteins has been broadened by integrating genetic engineering approaches for the fine tuning of functional groups and protein domains in geometrically well-defined manner. The efficient and stable binding of various nanomaterials with S-layers in regular arrays has led to paradigmatic shift in their nano-biotechnological sensing applications. More recently, functional S-layer supported lipid membranes have been generated through covalent binding of lipid molecules either with native or recombinant S-layer proteins at nano-scale dimensions serving as “proof of concept” for the development of bio-sensing platform. Thus, in the light of benefits conferred by surface layer proteins for the development of highly efficient biosensors, an exciting path has been opened for broadening their translational applications in drug delivery, disease diagnosis, vaccines development, lab-on-chip devices etc. Therefore, this review intends to describe about the importance of surface layer proteins in the development of biosensors.

关键词： Surface Layer Proteins Nano-Patterning Self-Assembly Liposome Biosensors

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Bacterial Surface Layer Proteins: From Moonlighting to Biomimetics: A New Horizonto Lead

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Advances in Bioscience and Biotechnology 2018年第8期9卷 352-372页

作者： Nimisha Gaur Ankit sharma barkha Singhal School of Biotechnology Gautam Buddha University Greater Noida (U.P.) India

The landmark discovery of moonlighting proteins embarks the significant progress in understanding the biological complexity and their closed-circuit analysis. The growing continuum in the variety of moonlighting functions paved the way for further elucidation of structural-functional aspects of protein evolution and design of proteins with novel functions. Currently, the moonlighting functions in various adhesive properties of surface layer proteins, an essential component of cell surface architecture of archaea and all phylogenetic groups of eubacteria become more prominently recognized. The remarkable credentials of surface layer proteins to self-assemble into supramolecular structures at nano-scale dimension have been exploited for the production of smart biomaterials in the form of biomimetics has been thrust area of research. The finely tuned topological features in terms of shape, size, geometry and surface chemistry of surface layer proteins are crucial for the production of biomimetics. The current developments of biomimetic lipid bilayers and composite membranes find applicability in understanding the functional dynamism of evolutionary relationship of bacterial cell envelopes and vaccine development, drug development and drug delivery. Though the development of biomimetics embraces fascination but faces with technological challenges. The plethora of literature has been available for the moonlighting aspects and nano-technological applications separately but none of the review describes towards the rhythmic transition from moonlighting functions of surface layer proteins of bacteria to biomimetics development and applications. Therefore, this review describes certain basic aspects of moonlighting functions and their mechanism of action, surface layer proteins and their moonlighting functions of commensal bacteria and their transition towards biomimetics. The recent developments of biomimetics based on surface layer proteins have been summarized and also posited different challenges and future prospects.

关键词： Moonlighting Biomimetic Surface Layer Proteins Self-Assembly Nano-Biotechnology

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