The Evolution and Application of a Novel DNA Aptamer Targeting Bone Morphogenetic Protein 2 for Bone Regeneration.

Recombinant human bone morphogenetic protein 2 (rhBMP-2) is an FDA-approved growth factor for bone regeneration and repair in medical practice. The therapeutic effects of rhBMP-2 may be enhanced through specific binding to extracellular matrix (ECM)-like scaffolds. Here, we report the selection of a novel rhBMP-2-specific DNA aptamer, functionalization of the aptamer in an ECM-like scaffold, and its application in a cellular context. A DNA aptamer BA1 was evolved and shown to have high affinity and specificity to rhBMP-2. A molecular docking model demonstrated that BA1 was probably bound to rhBMP-2 at its heparin-binding domain, as verified with experimental competitive binding assays. The BA1 aptamer was used to functionalize a type I collagen scaffold, and fraction ratios were optimized to mimic the natural ECM. Studies in the myoblast cell model C2C12 showed that the aptamer-enhanced scaffold could specifically augment the osteo-inductive function of rhBMP-2 in vitro. This aptamer-functionalized scaffold may have value in enhancing rhBMP-2-mediated bone regeneration.

Directed Evolution of a G-Quadruplex Peroxidase DNAzyme and Application in Proteomic DNAzyme-Aptamer Proximity Labeling.

DNAzymes have been limited in application by their low catalytic rates. Here, we evolved a new peroxidase DNAzyme mSBDZ-X-3 through a directed evolution method based on the capture of self-biotinylated DNA catalyzed by its intrinsic peroxidase activity. The mSBDX-X-3 DNAzyme has a parallel G-quadruplex structure and has more favorable catalytic properties than all previously reported peroxidase DNAzyme variants. We applied mSBDZ-X-3 in an aptamer-coupled proximity-based labeling proteomic assay to determine the proteins that bind to cell surface cancer biomarkers EpCAM and nucleolin. Confocal microscopy, western blot analysis, and LC-MS/MS showed that the hybrid DNAzyme aptamer-coupled proximity assay-labeled proteins associated with EpCAM and nucleolin within 6-12 min in fixed cancer cells. The labeled proteins were identified by mass spectrometry. This study provides a highly efficient peroxidase DNAzyme, a methodology for selection of such variants, and a method for its application in spatial proteomics using entirely nucleic acid-based tooling.

Microfluidic Technology for Nucleic Acid Aptamer Evolution and Application.

The intersection of microfluidics and aptamer technologies holds particular promise for rapid progress in a plethora of applications across biomedical science and other areas. Here, the influence of microfluidics on the field of aptamers, from traditional capillary electrophoresis approaches through innovative modern-day approaches using micromagnetic beads and emulsion droplets, is reviewed. Miniaturizing aptamer-based bioassays through microfluidics has the potential to transform diagnostics and embedded biosensing in the coming years.

Translationally controlled tumor protein (TCTP) is required for TGF-ß1 induced epithelial to mesenchymal transition and influences cytoskeletal reorganization.

Epithelial-mesenchymal transition (EMT) is a programed course of developmental changes resulting in the acquisition of invasiveness and mobility in cells. In cancer, this course is used by epithelial cells to attain movability. Translationally controlled tumor protein (TCTP) has been extensively characterized following the observation on tumor reversion ensuing its depletion. However, the role of TCTP in cancer progression is still elusive. Here, we demonstrate for the first time that TCTP is a target of transforming growth factor-ß1 (TGF-ß1), a key regulator of EMT in A549 cells. We here present changes in expression patterns of intermediate filament markers (vimentin and cytokeratin 18a) of EMT following TCTP knockdown or over expression. The TCTP over-expression in cancer cells is associated with mesenchymal characters, while downregulation promotes the epithelial markers in the cells. Interaction of TCTP with ß-catenin seems to stabilize ß-catenin, preparative to its nuclear localization highlighting a role for ß-catenin signaling in EMT. Moreover, the induction of urokinase plasminogen activator (uPA) following ectopic expression of TCTP leads to destabilization of ECM. The cells knocked down for TCTP show diminished invasiveness and migration under TGF-ß1 treatment. The present results for the first time demonstrate that TGF-ß1 dependent TCTP expression is required for EMT in cells.

Emergence of plant and rhizospheric microbiota as stable interactomes.

The growing human population and depletion of resources have necessitated development of sustainable agriculture. Beneficial plant-microbe associations have been known for quite some time now. To maintain sustainability, one could show better reliance upon beneficial attributes of the rhizosphere microbiome. To harness the best agronomic traits, understanding the entire process of recruitment, establishment, and maintenance of microbiota as stable interactome within the rhizosphere is important. In this article, we highlight the process of recruitment and establishment of microbiota within rhizosphere. Further, we have discussed the interlinkages and the ability of multiple (microbial and plant) partners to interact with one another forming a stable plant holobiont system. Lastly, we address the possibility of exploring the knowledge gained from the holobiont system to tailor the rhizosphere microbiome for better productivity and maintenance of agroecosystems. The article provide new insights into the broad principles of stable plant-microbe interactions which could be useful for sustaining agriculture and food security.

Molecular epidemiology of Vibrio cholerae associated with flood in Brahamputra River valley, Assam, India.

Cholera is often caused when drinking water is contaminated through environmental sources. In recent years, the drastic cholera epidemics in Odisha (2007) and Haiti (2010) were associated with natural disasters (flood and Earthquake). Almost every year the state of Assam India witnesses flood in Brahamputra River valley during reversal of wind system (monsoon). This is often followed by outbreak of diarrheal diseases including cholera. Beside the incidence of cholera outbreaks, there is lack of experimental evidence for prevalence of the bacterium in aquatic environment and its association with cholera during/after flood in the state. A molecular surveillance during 2012-14 was carried out to study prevalence, strain differentiation, and clonality of Vibrio cholerae in inland aquatic reservoirs flooded by Brahamputra River in Assam. Water samples were collected, filtered, enriched in alkaline peptone water followed by selective culturing on thiosulfate bile salt sucrose agar. Environmental isolates were identified as V. cholerae, based on biochemical assays followed by sero-grouping and detailed molecular characterization. The incidence of the presence of the bacterium in potable water sources was higher after flood. Except one O1 isolate, all of the strains were broadly grouped under non-O1/non-O139 whereas some of them did have cholera toxin (CT). Surprisingly, we have noticed Haitian ctxB in two non-O1/non-O139 strains. MLST analyses based on pyrH, recA and rpoA genes revealed clonality in the environmental strains. The isolates showed varying degree of antimicrobial resistance including tetracycline and ciprofloxacin. The strains harbored the genetic elements SXT constins and integrons responsible for multidrug resistance. Genetic characterization is useful as phenotypic characters alone have proven to be unsatisfactory for strain discrimination. An assurance to safe drinking water, sanitation and monitoring of the aquatic reservoirs is of utmost importance for combating the impending epidemic threat in the flood affected areas. Further, the management of flood through multi-prong approaches and sustainable utilization of environmental resources would be effective in disease management.

Interaction of Piriformospora indica with Azotobacter chroococcum.

Microbial communities in rhizosphere interact with each other and form a basis of a cumulative impact on plant growth. Rhizospheric microorganisms like Piriformospora indica and Azotobacter chroococcum are well known for their beneficial interaction with plants. These features make P. indica/A. chroococcum co-inoculation of crops most promising with respect to sustainable agriculture and to understanding the transitions in the evolution of rhizospheric microbiome. Here, we investigated interactions of P. indica with A. chroococcum in culture. Out of five Azotobacter strains tested, WR5 exhibited growth-promoting while strain M4 exerted growth-inhibitory effect on the fungus in axenic culture. Electron microscopy of co-culture indicated an intimate association of the bacterium with the fungus. 2-D gel electrophoresis followed by mass spectrometry of P. indica cellular proteins grown with or without WR5 and M4 showed differential expression of many metabolic proteins like enolase-I, ureaseD, the GTP binding protein YPT1 and the transmembrane protein RTM1. Fungal growth as influenced by bacterial crude metabolites was also monitored. Taken together, the results conform to a model where WR5 and M4 influence the overall growth and physiology of P. indica which may have a bearing on its symbiotic relationship with plants.