Simple and sensitive electrochemical impedimetric approach towards analysis of biophysical interaction.

Study of biophysical interactions have been carried out using specific combination of proteins such as human IgG (as antigen) and anti-human IgG (as complementary antibody; raised in rabbit). Bovine serum albumin (BSA) was used to block any nonspecific interaction between antigen and antibody as BSA has been reported to bind to several sites non-specifically. Optimization of BSA concentration was done in order to make the antigen-antibody interaction relatively more pronounced and specific. We have used gold electrode in order to provide a relatively inert platform for adsorption/immobilization of protein molecules. The interaction between the antigen and antibody caused an increase in the charge transfer resistance (parallel resistance in Randles cell model) for an indicator molecule (hexacyanoferrate) and this was monitored by impedance spectroscopy. Detection limit for the antigen was found to be about 50 ng/mL. The approach presented is general and versatile and can be used for any antigen-antibody pair without any significant modification.

Kinetic modelling of coupled transport across biological membranes.

In this report, we have modelled a secondary active co-transporter (symport and antiport), based on the classical kinetics model. Michaelis-Menten model of enzyme kinetics for a single substrate, single intermediate enzyme catalyzed reaction was proposed more than a hundred years ago. However, no single model for the kinetics of co-transport of molecules across a membrane is available in the literature We have made several simplifying assumptions and have followed the basic Michaelis-Menten approach. The results have been simulated using GNU Octave. The results will be useful in general kinetic simulations and modelling.

Promoters, toll like receptors and microRNAs: a strange association.

Toll-like receptors (TLRs) are proteins that play key role in the innate immune system. In the present study, -1000 base pairs upstream are taken from the transcription start site of the various TLR genes (10 known) in human. About 40 microRNAs have been identified that share 12-19 nucleotide sequence similarity with the promoter regions of 10 TLRs. It is proposed that the microRNA performs potential role in identification of promoter sequence and initiation of transcription.

Signals in the promoter regions of several cancerous genes.

The eukaryotic core promoter regions are complex and fuzzy, usually lacking any conserved regions. However, they contain signals in the form of short stretches of nucleic acid sequences, for transcription start sites (TSS) that are recognized by the transcription factors (TFs). The core promoter region thus plays an important role in biological pathways (gene network and activation). It has been reported that these signals are composed of nucleotide hexamers in the promoter sequence (smaller sequences are likely to have too little information to be useful and longer sequences are too complex to be recognized by proteins) reasonably close to the TSS. The signals (nucleotide hexamers) have been identified by a similarity search on the eukaryotic promoter database (EPD, Homo sapiens). The signals have been classified, depending on their base composition. They have been have clustered using an algorithm, such that there are two and three nucleotide differences between the classes and a single nucleotide difference within a class. We have reclassified the hexamers taking the highest frequent hexamers present in the EPD (Homo sapiens) as the class representatives. Also we have tried to find whether the same composition is reflected on the miRNAs but found that they probably have other functions unrelated to promoter recognition. In this report melanoma carcinoma pathway has been chosen as the reference pathway and the promoters of the driver genes has been searched for the presence of the major classes. A few of these were found and are reported here. Several non-cancerous genes have also been studied as reference and comparison.

Direct electrochemistry of horseradish peroxidase-gold nanoparticles conjugate.

We have studied the direct electrochemistry of horseradish peroxidase (HRP) coupled to gold nanoparticles (AuNP) using electrochemical techniques, which provide some insight in the application of biosensors as tools for diagnostics because HRP is widely used in clinical diagnostics kits. AuNP capped with (i) glutathione and (ii) lipoic acid was covalently linked to HRP. The immobilized HRP/AuNP conjugate showed characteristic redox peaks at a gold electrode. It displayed good electrocatalytic response to the reduction of H(2)O(2), with good sensitivity and without any electron mediator. The covalent linking of HRP and AuNP did not affect the activity of the enzyme significantly. The response of the electrode towards the different concentrations of H(2)O(2) showed the characteristics of Michaelis Menten enzyme kinetics with an optimum pH between 7.0 to 8.0. The preparation of the sensor involves single layer of enzyme, which can be carried out efficiently and is also highly reproducible when compared to other systems involving the layer-by-layer assembly, adsorption or encapsulation of the enzyme. The immobilized AuNP-HRP can be used for immunosensor applications.

Short nucleotide sequences signal spliceosomal binding in nucleic acids.

We have explored the region around the splice sites of the human intron and exons from the exon-intron database (EID) and located a number of short 6-nucleotide and 7-nucleotide sequences that are relatively common in the regions. These short sequences, we expect play an important role in the selection of the appropriate splicing process. We propose that the external signals via short recognition sequences play the deterministic role in the actual splicing process. We have obtained 50 such sequences each from the exon and intron from the beginning and from the ending and noted a number of common features.

Electrochemical studies on horseradish peroxidase covalently coupled with redox dyes.

The present study aims at investigating the use of redox dyes as non-diffusional electron mediators in hydrogen peroxide biosensors using horseradish peroxidase (HRP). We observe that the two redox dyes Safranine O and Neutral Red covalently bound to HRP, efficiently mediate electron transfer from the active site of the enzyme to the electrode surface. Dyes bound to the enzyme using a spacer arm diaminohexane further enhance the electron transfer. The enzyme electrodes show a linear response to the concentration of H2O2 up to 500 microM concentration and with a detection limit of around 50 microM. The dyes can be used as coupled mediators to develop a successful electro-optical biosensor.

Protein seeding of gold nanoparticles and mechanism of glycation sensing.

The plasmon resonance of gold nanoparticles (GNPs) synthesized on a protein template senses formation of advanced glycosylated end products (AGEs). A graded alteration of plasmon resonance (both the peak and intensity are affected) is observed as the glycation progresses. Transmission electron microscopy shows significant shift of the size distribution of GNPs in presence of glycation. The higher plasmon resonance is thus caused by increased formation of GNPs, which in turn is attributed to a larger number of smaller particles. To study the binding of the protein with the GNP, infrared (IR) spectroscopy and circular dichroism (CD) studies were undertaken. Whereas the CD studies confirmed the emergence of beta-structure and loss of alpha-helix, the IR data indicated glycation-induced alterations in the amide I region. The proposed sensor for formation of AGEs thus apparently operates by direct or indirect conjugation with amino groups. Incidentally, glycation and AGE formation are responsible for a number of diabetes-related clinical conditions, and the present approach could be adopted for use for a simple colorimetric assay for the AGEs.

Electrochemical studies of glucose oxidase immobilized on glutathione coated gold nanoparticles.

Glutathione (L-gamma-glutamyl-L-cysteinyl-L-glycine; GSH) forms a surface monolayer on gold nanoparticles by tethering via sulfur bonds (Au:GSH). In the present study, glucose oxidase (GOx; EC 1.1.3.4) was immobilized by covalent chemical coupling reactions on to Au:GSH nanoparticles and the enzyme coupled nanoparticles formed a stable colloid (stable for several weeks) in water. The immobilized enzyme was investigated for electrochemical characteristics to monitor the FAD (prosthetic group of the GOx) redox potentials. Various concentrations of substrate (glucose) were added to check the oxidation characteristics. It was observed that with increase in substrate concentrations, the oxidation rate increased proportionally with the current. The present study demonstrated that GOx was effectively coupled to the gold nanoparticle (Au:GSH). The coupled nanoparticle system could be used in a potential biosensor application. Similarly, other enzymes (e.g., horseradish peroxidase) could be immobilized to the Au:GSH nanoparticles via the peptide arm (GSH) to achieve the desired characteristics needed for a specific application in biosensor.

Dynamics of miRNA biogenesis and nuclear transport.

MicroRNAs (miRNAs) are short noncoding RNA sequences ~22 nucleotides in length that play an important role in gene regulation-transcription and translation. The processing of these miRNAs takes place in both the nucleus and the cytoplasm while the final maturation occurs in the cytoplasm. Some mature miRNAs with nuclear localisation signals (NLS) are transported back to the nucleus and some remain in the cytoplasm. The functional roles of these miRNAs are seen in both the nucleus and the cytoplasm. In the nucleus, miRNAs regulate gene expression by binding to the targeted promoter sequences and affect either the transcriptional gene silencing (TGS) or transcriptional gene activation (TGA). In the cytoplasm, targeted mRNAs are translationally repressed or cleaved based on the complementarity between the two sequences at the seed region of miRNA and mRNA. The selective transport of mature miRNAs to the nucleus follows the classical nuclear import mechanism. The classical nuclear import mechanism is a highly regulated process, involving exportins and importins. The nuclear pore complex (NPC) regulates all these transport events like a gate keeper. The half-life of miRNAs is rather low, so within a short time miRNAs perform their function. Temporal studies of miRNA biogenesis are, therefore, useful. We have carried out simulation studies for important miRNA biogenesis steps and also classical nuclear import mechanism using ordinary differential equation (ODE) solver in the Octave software.

Comparative analysis of core promoter region: information content from mono and dinucleotide substitution matrices.

We have studied the core promoter region in five sets of promoter sequences by calculating the average mutual information content H (relative entropy). We have used specially constructed substitution matrices to calculate mono and dinucleotide replacements in a given block of aligned sequences. These substitution matrices use log-odds form of scores, which are in bits of information. Here, we constructed and applied nucleotide substitution matrices for the core promoter region to calculate the information content to study the Transcription Start Site (TSS), TATA-box and downstream regions. As expected, the information content decreases with increasing block size. This clearly implies that the TSS region is likely to be 5-10 bases in size (length). We also notice that both in the case of mouse and humans, both TATA-boxes and TSS regions are likely to play important roles in proper transcriptional initiation.

Comparative analysis of splice site regions by information content.

We have applied concepts from information theory for a comparative analysis of donor (gt) and acceptor (ag) splice site regions in the genes of five different organisms by calculating their mutual information content (relative entropy) over a selected block of nucleotides. A similar pattern that the information content decreases as the block size increases was observed for both regions in all the organisms studied. This result suggests that the information required for splicing might be contained in the consensus of approximately 6-8 nt at both regions. We assume from our study that even though the nucleotides are showing some degrees of conservation in the flanking regions of the splice sites, certain level of variability is still tolerated, which leads the splicing process to occur normally even if the extent of base pairing is not fully satisfied. We also suggest that this variability can be compensated by recognizing different splice sites with different spliceosomal factors.

Comparative analysis of transcription start sites using mutual information.

The transcription start site (TSS) region shows greater variability compared with other promoter elements. We are interested to search for its variability by using information content as a measure. We note in this study that the variability is significant in the block of 5 nucleotides (nt) surrounding the TSS region compared with the block of 15 nt. This suggests that the actual region that may be involved is in the range of 5-10 nt in size. For Escherichia coli, we note that the information content from dinucleotide substitution matrices clearly shows a better discrimination, suggesting the presence of some correlations. However, for human this effect is much less, and for mouse it is practically absent. We can conclude that the presence of short-range correlations within the TSS region is species-dependent and is not universal. We further observe that there are other variable regions in the mitochondrial control element apart from TSS. It is also noted that effective comparisons can only be made on blocks, while single nucleotide comparisons do not give us any detectable signals.

1/f correlations in viral genomes--a Fast-Fourier Transformation (FFT) Study.

We have studied the presence of long-range correlations in the complete genomes of ten different dsDNA viruses and Saccharomyces cerevisiae (bakers' yeast) chromosome I. We have also studied the correlation between the distribution of the gene length and the domain of "1/f region" of their genomes. Linear regression analysis was done for the power-law region of these organisms and the slope values obtained were approximately -1, which signify the existence of "1/f noise" in the low and medium (intermediate) frequency regions. This suggests the presence of long-range correlations in their genomes. The presence of 1/f noise in a given frequency interval indicates the existence of a fractal (self-similar) structure in the corresponding range of wavelengths. The results of our study suggest that genes have correlations within themselves, and the correlations appear to be related with the scaling exponent alpha.

Kinetic simulation of malate-aspartate and citrate-pyruvate shuttles in association with Krebs cycle.

In the present work, we have kinetically simulated two mitochondrial shuttles, malate-aspartate shuttle (used for transferring reducing equivalents) and citrate-pyruvate shuttle (used for transferring carbon skeletons). However, the functions of these shuttles are not limited to the points mentioned above, and they can be used in different arrangements to meet different cellular requirements. Both the shuttles are intricately associated with Krebs cycle through the metabolites involved. The study of this system of shuttles and Krebs cycle explores the response of the system in different metabolic environments. Here, we have simulated these subsets individually and then combined them to study the interactions among them and to bring out the dynamics of these pathways in focus. Four antiports and a pyruvate pump were modelled along with the metabolic reactions on both sides of the inner mitochondrial membrane. Michaelis-Menten approach was extended for deriving rate equations of every component of the system. Kinetic simulation was carried out using ordinary differential equation solver in GNU Octave. It was observed that all the components attained steady state, sooner or later, depending on the system conditions. Progress curves and phase plots were plotted to understand the steady state behaviour of the metabolites involved. A comparative analysis between experimental and simulated data show fair agreement thus validating the usefulness and applicability of the model.

Modelling the Krebs cycle and oxidative phosphorylation.

The Krebs cycle and oxidative phosphorylation are the two most important sets of reactions in a eukaryotic cell that meet the major part of the total energy demands of a cell. In this paper, we present a computer simulation of the coupled reactions using open source tools for simulation. We also show that it is possible to model the Krebs cycle with a simple black box with a few inputs and outputs. However, the kinetics of the internal processes has been modelled using numerical tools. We also show that the Krebs cycle and oxidative phosphorylation together can be combined in a similar fashion - a black box with a few inputs and outputs. The Octave script is flexible and customisable for any chosen set-up for this model. In several cases, we had no explicit idea of the underlying reaction mechanism and the rate determining steps involved, and we have used the stoichiometric equations that can be easily changed as and when more detailed information is obtained. The script includes the feedback regulation of the various enzymes of the Krebs cycle. For the electron transport chain, the pH gradient across the membrane is an essential regulator of the kinetics and this has been modelled empirically but fully consistent with experimental results. The initial conditions can be very easily changed and the simulation is potentially very useful in a number of cases of clinical importance.

Kinetic modelling of mitochondrial translation.

Mitochondrial genome contains 13 protein coding genes, all being part of the oxidative phosphorylation complexes. The process of translation of these protein coding mRNAs in mitochondrial matrix is a good miniature model of translation in cytoplasm. In this work, we have simulated three phases of mitochondrial translation viz. initiation, elongation and termination (including ribosome recycling). The kinetic equations for these phases have been deduced based on the information available in literature. Various factors involved in the process have been included explicitly. Kinetic simulation was done using Octave, open source software. Scripts were written individually for each phase. Initiation begins with mitoribosome, mRNA, fMet-tRNA and initiation factors. The final product of the initiation script, the initiation complex, was introduced as the start point in the successive step, i.e. elongation. Elongation is a particular extensive process where the various aminoacyl-tRNAs already present in the matrix check for matching with the triplet codon in A-site of mitoribosome. This script consists of two parts: one with the time behaviour of the factors involved in the molecular process (using ordinary differential equation solver) and the other including the reading of triplet codon on the mRNA and incorporating the corresponding aminoacyl-tRNA, and then at each step elongating the peptide chain (using loops and conditions). The peptide chain thus formed in the elongation step (in the loops and conditions segment) was released in the termination step. This was followed by mitoribosome recycling where the mitoribosome reached the native state and was ready for the next cycle of translation.

Functional, structural, and sequence studies of microRNA.

In this review, current knowledge and ideas regarding several important functional-, structural-, and sequence-related aspects of microRNAs (miRNAs) are summarized. The current research on structural and functional aspects of miRNAs is rapidly growing, and new information appears regularly. Well-established information from literature useful for researchers working in this area has been compiled in this work. Although details of the methodology have not been elaborated, the outline should be useful for a broad and general understanding. The current information is highly interdisciplinary including molecular biology and bioinformatics; we have tried to bring both sides together. Little is known about the 3-D structure of miRNAs and its significance. miRNAs are usually active in conjunction with some proteins, and the complex is responsible for the intended task. Little information is available in the literature about the protein-miRNA interactions and their nature and properties. Nonetheless, we believe that the review will be useful to both bioinformaticians and molecular biologists.

Conserved short sequences in promoter regions of human genome.

Recognition of promoter elements by the transcription factors is one of the early initial and crucial steps in gene expression and regulation. In prokaryotes, there are clear signals to identify the promoter regions like TATAAT at around -10 and TTGACA at -35 positions from transcription start site (TSS). In eukaryotes the promoter regions are structurally more complex and there are no conserved or consensus sequences similar to the ones found in prokaryotic promoters. We have located a set of GC rich short sequences (< 8 nt) that are relatively common in human promoter sequences around the TSS (+/- 100 relative to TSS). These sequences were sorted based on their frequency of occurrence in the database and the most common 50 sequences were used for further studies. Sigmoidal behavior of the high end of the frequency distribution of these sequences suggests presence of some internal co-operativity. These short sequences are distributed on both sides of TSS, suggesting that probably the transcription factors recognize these sequences on both upstream and downstream of TSS. As eukaryotic promoters lack any conserved sequences, we expect that these short sequences may help in recognition of promoter regions by relevant transcription factors prior to the initiation of transcription process. We postulate that a cluster of genes with common short sequences in the promoter region can be recognized by a particular transcription factor. We also found that most of these short sequences are fairly common within miRNA (both mature and stem-loop sequences). Our studies indicate that eukaryotic transcription is more complex than currently believed.

Gold nanoparticles based sandwich electrochemical immunosensor.

In this report we have used gold nanoparticles (AuNPs) to covalently attach an antibody (Ab(1)) using a spacer arm. The AuNPs/Ab(1) modified gold electrode was used for a sandwich electrochemical immunoassay. The detection was done using cyclic voltammetry and impedance measurements using Horse Radish Peroxidase (HRP) as enzyme label on secondary antibody (Ab(2)) and 3,3', 5,5'-tertramethyl benzidine (TMB) as an electroactive dye. The cyclic voltammetric experiments showed three clear peaks at potentials 154 mV, -33 mV and -156 mV. There was an increase in the both anodic and cathodic current values for the peak at potential -33 mV, when H2O2 was added and the other peaks observed at potential 154 mV and -156 mV resulted due to the oxidation and reduction of TMB. The detection limit of this electrode was 2 ng/mL or 10 pg/5 microL of the analyte. The electrochemical impedance spectroscopy studies demonstrate that the formation of antigen-antibody complexes increases the series resistance and thus confirms the assembly on the electrode. This study showed that AuNPs was efficient in preserving the activity and orientation of the antibody and it can form a major platform in many clinical immunoassays.