Mohd. Mohsin

Alpha amylase assisted synthesis of TiO2 nanoparticles: Structural characterization and application as antibacterial agents

The enzyme alpha amylase was used as the sole reducing and capping agent for the synthesis of TiO2 nanoparticles. The biosynthesized nanoparticles were characterized by X-ray diffraction (XRD) and transmission electron microscopic (TEM) methods. The XRD data confirms the monophasic crystalline nature of the nanoparticles formed. TEM data shows that the morphology of nanoparticles depends upon the enzyme concentration used at the time of synthesis. The presence of alpha amylase on TiO2 nanoparticles was confirmed by FTIR. The nanoparticles were investigated for their antibacterial effect on Staphylococcus aureus and Escherichia coli. The minimum inhibitory concentration value of the TiO2 nanoparticles was found to be 62.50 μg/ml for both the bacterial strains. The inhibition was further confirmed using disc diffusion assay. It is evident from the zone of inhibition that TiO2 nanoparticles possess potent bactericidal activity. Further, growth curve study shows effect of inhibitory concentration of TiO2 nanoparticles against S. aureus and E. coli. Confocal microscopy and TEM investigation confirm that nanoparticles were disrupting the bacterial cell wall.

Identification and Comparative Analysis of MicroRNAs Associated with Low-N Tolerance in Rice Genotypes

Background Nitrogen [N] is a critical limiting nutrient for plants and has to be exogenously supplied to many crops, to achieve high yield with significant economic and environmental costs, specifically for rice. Development of low-input nitrogen sustainable crop is necessary for sustainable agriculture. Identification of regulatory elements associated with low-N tolerance is imperative for formulating innovative approaches for developing low-N tolerant crop plants, using gene manipulation. MicroRNAs (miRNAs) are known to play crucial roles in the modulation of gene expression in plants under various environmental conditions. Methodology/Principal Findings MiRNAs associated with low-N tolerance have not been identified so far. In this study, we investigated microarray-based miRNA expression in low-N tolerant and low-N sensitive rice genotypes under low N condition. Expressions of 32 miRNAs differed significantly in the two genotypes. Of these 32 miRNAs, expressions of nine miRNAs were further validated experimentally in leaves as well as in roots. Of these differentially expressed miRNAs, six miRNAs (miR156, miR164, miR528, miR820, miR821 and miR1318) were reported in leaves and four (miR164, miR167, miR168 and miR528) in roots. Target genes of all the 32 miRNAs were predicted, which encode transcription factors, and proteins associated with metabolic processes or stress responses. Expression levels of some of the corresponding miRNA targets were analysed and found to be significantly higher in low N-tolerant genotype than low-N sensitive genotype. These findings suggested that miRNAs played an important role in low-N tolerance in rice. Conclusions/Significance Genome-wide differences in expression of miRNA in low N-tolerant and low N-sensitive rice genotypes were reported. This provides a platform for selection as well as manipulation of genotypes for better N utilization efficiency.

Genetically encoded FRET-based nanosensor for in vivo measurement of leucine

Besides fundamental role in protein synthesis, leucine has metabolic roles as energy substrates, precursors for synthesis of other amino acids and as a modulator of muscle protein synthesis via the insulin-signaling pathway. Leucine concentration in cell and tissue is temporally dynamic as the metabolism of leucine is regulated through multiple enzymes and transporters. Assessment of cell-type specific activities of transporters and enzymes by physical fractionation is extremely challenging. Therefore, a method of reporting leucine dynamics at the cellular level is highly desirable. Given this, we developed a series of genetically encoded nanosensors for real-time in vivo measurement of leucine at cellular level. A leucine binding periplasmic binding protein (LivK) of Escherichia coli K12 was flanked with CFP (cyan fluorescent protein) and YFP (yellow fluorescent protein) at N-terminus and C-terminus, respectively. The constructed nanosensors allowed in vitro determination of fluorescence resonance energy transfer (FRET) changes in a concentration-dependent manner. These sensors were found to be specific to leucine, and stable to pH-changes within a physiological range. Genetically encoded sensors can be targeted to a specific cell type, and allow dynamic measurement of leucine concentration in bacterial and yeast cells.

Genetically-encoded nanosensor for quantitative monitoring of methionine in bacterial and yeast cells

Metabolic engineering of microorganisms for production of biological molecules represent a key goal for industrial biotechnology. The metabolic engineering requires detailed knowledge of the concentrations and flux rates of metabolites and metabolic intermediates in vivo. Genetically-encoded fluorescence resonance energy transfer (FRET) sensors represent a promising technology for measuring metabolite levels and corresponding rate changes in live cells. In the present paper, we report the development of genetically-encoded FRET-based nanosensor for methionine as metabolic engineering of microbial strains for the production of l-methionine is of major interest in industrial biotechnology. In this nanosensor, methionine binding protein (MetN) from Escherichia coli (E. coli) K12 was taken and used as the reporter element of the sensor. The MetN was sandwiched between cyan fluorescent protein (CFP) and yellow fluorescent protein (YFP). Specificity, affinity, pH stability and metal effects was analyzed for the in vitro characterization of this nanosensor, named as FLIPM. The FLIPM is very specific to methionine and found to be stable with the pH within the physiological range. The calculated affinity (Kd) of FLIPM was 203 µM. This nanosensor successfully monitored the intracellular level of methionine in bacterial as well as yeast cell. The data suggest that these nanosensors may be a versatile tool for studying the in vivo dynamics of methionine level non-invasively in living cells.

FRET-based genetically-encoded sensors for quantitative monitoring of metabolites

Neighboring cells in the same tissue can exist in different states of dynamic activities. After genomics, proteomics and metabolomics, fluxomics is now equally important for generating accurate quantitative information on the cellular and sub-cellular dynamics of ions and metabolite, which is critical for functional understanding of organisms. Various spectrometry techniques are used for monitoring ions and metabolites, although their temporal and spatial resolutions are limited. Discovery of the fluorescent proteins and their variants has revolutionized cell biology. Therefore, novel tools and methods targeting sub-cellular compartments need to be deployed in specific cells and targeted to sub-cellular compartments in order to quantify the target-molecule dynamics directly. We require tools that can measure cellular activities and protein dynamics with sub-cellular resolution. Biosensors based on fluorescence resonance energy transfer (FRET) are genetically encoded and hence can specifically target sub-cellular organelles by fusion to proteins or targetted sequences. Since last decade, FRET-based genetically encoded sensors for molecules involved in energy production, reactive oxygen species and secondary messengers have helped to unravel key aspects of cellular physiology. This review, describing the design and principles of sensors, presents a database of sensors for different analytes/processes, and illustrate examples of application in quantitative live cell imaging.

Gum ghatti mediated, one pot green synthesis of optimized gold nanoparticles: Investigation of process-variables impact using Box-Behnken based statistical design

Due to unique inherent catalytic characteristics of different size, shape and surface functionalized gold nanoparticles, their potential applications, are being explored in various fields such as drug delivery, biosensor, diagnosis and theranostics. However conventional process for synthesis of these metallic nanoparticles utilizes toxic reagents as reducing agents, additional capping agent for stability as well as surface functionalization for drug delivery purposes. Hence, in this work suitability of gum Ghatti for reducing, capping and surface functionalization during the synthesis of stable Gold nanoparticles were duly explored. Role and impact of key process variables i.e. volume of chloroauric acid solution, gum solution and temperature at their respective three different levels, as well as mechanism of formation of optimized gold nanoparticles were also investigated using Box- Behnken design. These novel synthesized optimized Gold nanoparticles were further characterized by UV spectrophotometer for its surface plasmon resonance (SPR) at around ∼530nm, dynamic light scattering (DLS) for its hydrodynamic size (112.5nm), PDI (0.222) and zeta potential (-21.3mV) while, transmission electron microscopy (TEM) further revealed surface geometry of these nanoparticles being spherical in shape.

Role of green fluorescent proteins and their variants in development of FRET-based sensors

Since the last decade, a lot of advancement has been made to understand biological processes involving complex intracellular pathways. The major challenge faced was monitoring and trafficking of metabolites in real time. Although a range of quantitative and imaging techniques have been developed so far, the discovery of green fluorescent proteins (GFPs) has revolutionized the advancement in the field of metabolomics. GFPs and their variants have enabled researchers to ‘paint’ a wide range of biological molecules. Fluorescence resonance energy transfer (FRET)-based genetically encoded sensors is a promising technology to decipher the real-time monitoring of the cellular events inside living cells. GFPs and their variants, due to their intrinsic fluorescence properties, are extensively being used nowadays in cell-based assays. This review focuses on structure and function of GFP and its derivatives, mechanism emission and their use in the development of FRET-based sensors for metabolites.

Nanoscale gizmos – the novel fluorescent probes for monitoring protein activity

Abstract Nanobiotechnology has emerged inherently as an interdisciplinary field, with collaborations from researchers belonging to diverse backgrounds like molecular biology, materials science and organic chemistry. Till the current times, researchers have been able to design numerous types of nanoscale fluorescent tool kits for monitoring protein–protein interactions through real time cellular imagery in a fluorescence microscope. It is apparent that supplementing any protein of interest with a fluorescence habit traces its function and regulation within a cell. Our review therefore highlights the application of several fluorescent probes such as molecular organic dyes, quantum dots (QD) and fluorescent proteins (FPs) to determine activity state, expression and localization of proteins in live and fixed cells. The focus is on Fluorescence Resonance Energy Transfer (FRET) based nanosensors that have been developed by researchers to visualize and monitor protein dynamics and quantify metabolites of diverse nature. FRET based toolkits permit the resolution of ambiguities that arise due to the rotation of sensor molecules and flexibility of the probe. Achievements of live cell imaging and efficient spatiotemporal resolution however have been possible only with the advent of fluorescence microscopic technology, equipped with precisely sensitive automated softwares.