Department of Bioscience & Bioengineering

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Non-coding RNAs (ncRNAs) are increasingly being implicated in diverse functional roles. Majority of these ncRNAs have their origin in the repetitive elements of genome. Significantly, increase in genomic complexity has been correlated with increase in repetitive content of the genome. Primate-specific Alu repeats, belonging to SINE class of repeats, is the most abundant repeat class inhabiting the human genome. Of the many possible functional roles of Alu repeats, they have been shown to modulate human transcriptome by virtue of harboring diverse array of functional RNA pol II TFBS, cryptic splice-site-mediated Alu exonization and as probable miRNA targets. Retro-transposition of Alu harboring TFBS has shaped up gene-specific regulatory networks. Alu exonized transcripts are raw material for dsRNA-mediated A^I editing leading to nuclear retention of transcripts and change in miRNA target. miRNA targets within Alu may titrate the effective miRNA or transcript concentration, thus acting as 'miRNA sponge'. Differential levels of Alu RNA during different conditions of stress also await clear functional understanding. These have contributed toward evolution of complex regulatory repertoire leading to the evolution of primate-specific functions. Recent reports of co-localization of pol II and pol III binding sites near the gene and elsewhere in the genome, increase the possibility of dynamic co-ordination between both pol II and pol III determining the ultimate transcriptional outcome. Dynamic and functional Alu repeats seem to be centrally placed to modulate the transcriptional landscape of human genome. © The Author 2011. Published by Oxford University Press. All rights reserved.

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Briefings in Functional Genomics

From 'JUNK' to just unexplored noncoding knowledge: The case of transcribed Alus

Globally only 5-10% of people encountering Mycobacterium tuberculosis have a lifetime risk of active disease indicating a strong host genetic bias towards development of tuberculosis. In the current study we investigated genotype variants pertaining to five cytokine genes namely IFNG, TNFA, IL4, IL10 and IL12 in the north Indian population with active pulmonary tuberculosis (APTB) and correlated the serum cytokine levels with the corresponding genotypes. Twenty five single nucleotide polymorphisms (SNPs) including six loci examined for the first time in tuberculosis were selected for genotyping in 108 patients with APTB from north India and 48 healthy regional controls (HC). Applying exclusion criteria 12 SNPs passed all the filters and were analysed further. The serum cytokine concentrations were measured by ELISA. Compared to HC mean serum IFN-γ, IL-12, IL-4, and IL-10 levels were higher in APTB (p= 0.3661, p= 0.0186, p= 0.003, p= 0.7, respectively). In contrast the mean serum TNF-α level was higher in HC (p= 0.007). Comparison of genotypes and serum levels of the corresponding cytokine genes reveal that though IFN-γ and IL-4 levels were higher in APTB the genotype variants showed no difference between HC and APTB. In contrast the genotypes of the selected rsIDs in the TNFA, IL12 and IL10 genes showed significant association with the varying serum levels of corresponding cytokines. The variant of the TNFA gene at rs3093662, the IL12 gene at rs3213094 and rs3212220 and the IL10 gene at rs3024498 did show a strong indication to be of relevance to the immunity to tuberculosis. To our knowledge this is the first report from this region relating genotypes and serum cytokine levels in north Indian population. © 2011 Elsevier B.V.

Infection, Genetics and Evolution

Differential serum cytokine levels are associated with cytokine gene polymorphisms in north Indians with active pulmonary tuberculosis

Background: Alu RNAs are present at elevated levels in stress conditions and, consequently, Alu repeats are increasingly being associated with the physiological stress response. Alu repeats are known to harbor transcription factor binding sites that modulate RNA pol II transcription and Alu RNAs act as transcriptional co-repressors through pol II binding in the promoter regions of heat shock responsive genes. An observation of a putative heat shock factor (HSF) binding site in Alu led us to explore whether, through HSF binding, these elements could further contribute to the heat shock response repertoire.Results: Alu density was significantly enriched in transcripts that are down-regulated following heat shock recovery in HeLa cells. ChIP analysis confirmed HSF binding to a consensus motif exhibiting positional conservation across various Alu subfamilies, and reporter constructs demonstrated a sequence-specific two-fold induction of these sites in response to heat shock. These motifs were over-represented in the genic regions of down-regulated transcripts in antisense oriented Alus. Affymetrix Exon arrays detected antisense signals in a significant fraction of the down-regulated transcripts, 50% of which harbored HSF sites within 5 kb. siRNA knockdown of the selected antisense transcripts led to the over-expression, following heat shock, of their corresponding down-regulated transcripts. The antisense transcripts were significantly enriched in processes related to RNA pol III transcription and the TFIIIC complex.Conclusions: We demonstrate a non-random presence of Alu repeats harboring HSF sites in heat shock responsive transcripts. This presence underlies an antisense-mediated mechanism that represents a novel component of Alu and HSF involvement in the heat shock response. © 2011 Pandey et al.; licensee BioMed Central Ltd.

Genome Biology

Heat shock factor binding in Alu repeats expands its involvement in stress through an antisense mechanism

A highly facile and feasible strategy on the fabrication of advanced intrinsic peroxidase mimetics based on Mn2+ doped mixed ferrite (MnIIxFeII1-xFeIII 2O4) nanoparticles was demonstrated for the quantitative and sensitive detection of mouse IgG (as a model analyte). Mn2+ doped Fe1-xMnxFe2O4 nanoparticles were synthesized using varying ratios of Mn2+:Fe2+ ions and characterized by the well known complementary techniques. The increase of Mn2+ proportion had remarkably enhanced the peroxidase activity and magnetism. The catalytic activity of mixed ferrites was found to follow Michaelis-Menten kinetics and was noticeably higher than native Fe 3O4. The calculated Km and Kcat exhibited strong affinity with substrates which were remarkably higher than similar sized native magnetite nanoparticles and horseradish peroxidase (HRP). These findings stimulated us to develop carboxyl modified Fe 1-xMnxFe2O4 nanoparticles using phosphonomethyl immunodiacetic acid (PMIDA) to engineer PMIDA-Fe 1-xMnxFe2O4 fabricated enzyme linked immunosorbent assay (ELISA). Results of both PMIDA-Fe1-xMn xFe2O4 linked ELISA revealed that the enhancements in absorbance during the catalysis of enzyme substrate were linearly proportional to the concentration of mouse IgG within the range between 0.1 μg/ml and 2.5 μg/ml. Further, this detection was ten times lower than previous reports and the detection limit of mouse IgG was 0.1 μg/ml. The advantages of our fabricated artificial peroxidase mimetics are combined of low cost, easy to prepare, better stability and tunable catalytic activity. Moreover, this method provides a new horizon for the development of promising analytical tools in the application of biocatalysis, bioassays, and bioseparation. © 2011 Elsevier B.V. All rights reserved.

Talanta

Development of phosphonate modified Fe(1-x)MnxFe 2O4 mixed ferrite nanoparticles: Novel peroxidase mimetics in enzyme linked immunosorbent assay

Pdcd4 is a novel tumor suppressor protein that functions in the nucleus and the cytoplasm, and appears to be involved in the regulation of transcription and translation. In the cytoplasm, Pdcd4 has been implicated in the suppression of translation of mRNAs containing structured 5′-untranslated regions; however, the mechanisms that recruit Pdcd4 to specific target mRNAs and the identities of these mRNAs are mostly unknown. In this study, we have identified c-myb mRNA as the first natural translational target mRNA of Pdcd4. We have found that translational suppression of c-myb mRNA by Pdcd4 is dependent on sequences located within the c-myb-coding region. Furthermore, we have found that the N-terminal domain of Pdcd4 has an important role in targeting Pdcd4 to c-myb RNA by mediating preferential RNA binding to the Pdcd4-responsive region of c-myb mRNA. Overall, our work demonstrates for the first time that Pdcd4 is directly involved in translational suppression of a natural mRNA and provides the first evidence for a key role of the RNA-binding domain in targeting Pdcd4 to a specific mRNA. © 2011 Macmillan Publishers Limited All rights reserved.

Oncogene

Pdcd4 directly binds the coding region of c-myb mRNA and suppresses its translation

Aggregation in polymer composites is one of the major obstacles in the carbon nanotubes (CNTs) applications. Authentic CNTs are known to have very good electrical conductivity and mechanical strengths. Surface functionalization can avoid aggregation and help dispersion of CNTs, but reduces CNT's electrical conductivities and mechanical strengths dramatically. It needs a good way to resolve the above dilemma situation; i.e., poor dispersion-good conductivity vs. good dispersion-poor conductivity. Herein, we demonstrate that in-situ polymerized polyaniline (PANI)-coated CNTs have good polymer matrix compatibility, and are superior electrically conductive fillers to nylon 6 composites. In this report, multi-walled CNTs (MWCNTs) were surface-modified with poly(acrylic acids) (PAA), followed by further coating with PANI. The electrical conductivity of (PANI-MWCNTs)-nylon 6 composite thin film was increased from 10-12 to 7.3 × 10-5 S/cm in the presence of 1 wt% PANI-coated MWCNTs prepared by physical mixing of PANI and PAA-grafted MWCNTs. When in-situ polymerized PANI-coated MWCNTs were added, the electrical conductivity of MWCNTs-nylon 6 composite was further enhanced by 3 orders to be 3.4 × 10-2 S/cm at the same 1 wt% loading of MWCNTs. Both Fourier-transformed infrared and uv-visible absorption spectra indicate that there exist very strong site-specific charge transfer interactions between the quinoid rings of PANI and MWCNTs, which results in the superior electrical conductivity of MWCNT-nylon 6 composite. © 2011 Elsevier Ltd. All rights reserved.

Polymer

Enhanced electrical conductivity of nylon 6 composite using polyaniline-coated multi-walled carbon nanotubes as additives

The study of protein conformational changes in the presence of surfactants and lipids is important in the context of protein folding and misfolding. In the present study, we have investigated the mechanism of the protein conformational change coupled with aggregation leading to size growth of Hen Egg White Lysozyme (HEWL) in the presence of an anionic detergent such as sodium dodecyl sulphate (SDS) in alkaline pH. We have utilized intrinsic protein fluorescence (tryptophan) and extrinsic fluorescent reporters such as 8-anilinonaphthalene-1- sulfonic acid (ANS), dansyl and fluorescein to follow the protein conformational change in realtime. By analyzing the kinetics of fluorescence intensity and anisotropy of multiple fluorescent reporters, we have been able to delineate the mechanism of surfactant-induced aggregation of lysozyme. The kinetic parameters reveal that aggregation proceeds with an initial fast-phase (conformational change) followed by a slow-phase (self-assembly). Our results indicate that SDS, below critical micelle concentration, induces conformational expansion that triggers the aggregation process at a micromolar protein concentration range. © 2010 Springer Science+Business Media, LLC.

Journal of Fluorescence

Kinetics of surfactant-induced aggregation of lysozyme studied by fluorescence spectroscopy

We have investigated the fibrillation propensity of different conformational isomers of an archetypal, all α-helical protein, namely, bovine serum albumin (BSA), under different pH conditions and ionic strengths using fluorescence and circular dichroism (CD) spectroscopy. At low pH and higher protein concentration, the partially folded conformers associate to form oligomers that are converted into ordered amyloid-like fibrils when incubated at elevated temperature. We have elucidated the mechanism of fibril formation, especially the early steps, by monitoring the kinetics of structural changes during the aggregation process. Various structural probes in tandem were utilized to decipher the temporal evolution of both conformational and size changes by measuring the time dependence of fluorescence intensity and anisotropy of intrinsic tryptophans and several extrinsic fluorophores during the aggregation. Additionally, CD spectroscopy was utilized to monitor the changes in protein secondary structural content during fibrillation. Our findings suggest that the conformational conversion occurs in the oligomers that serve as precursors to amyloid fibrils and precedes the overall fibrillar growth. © 2011 American Chemical Society.

Journal of Physical Chemistry B

Insights into the mechanism of aggregation and fibril formation from bovine serum albumin

This article delineates the design and synthesis of a novel, bio-functionalized, magneto-fluorescent multifunctional nanoparticles suitable for cancer-specific targeting, detection and imaging. Biocompatible, hydrophilic, magneto-fluorescent nanoparticles with surface-pendant amine, carboxyl and aldehyde groups were designed using o-carboxymethyl chitosan (OCMC). The free amine groups of OCMC stabilized magnetite nanoparticles on the surface allow for the covalent attachment of a fluorescent dye such as rhodamine isothiocyanate (RITC) with the aim to develop a magneto-fluorescent nanoprobe for optical imaging. In order to impart specific cancer cell targeting properties, folic acid and its aminated derivative was conjugated onto these magneto-fluorescent nanoparticles using different pendant groups (-NH 2, -COOH, -CHO). These newly synthesized iron-oxide folate nanoconjugates (FA-RITC-OCMC-SPIONs) showed excellent dispersibility, biocompatibility and good hydrodynamic sizes under physiological conditions which were extensively studied by a variety of complementary techniques. The cellular internalization efficacy of these folate-targeted and its non-targeted counterparts were studied using a folate-overexpressed (HeLa) and a normal (L929 fibroblast) cells by fluorescence microscopy and magnetically activated cell sorting (MACS). Cell-uptake behaviors of nanoparticles clearly demonstrate that cancer cells over-expressing the human folate receptor internalized a higher level of these nanoparticle-folate conjugates than normal cells. These folate targeted nanoparticles possess specific magnetic properties in the presence of an external magnetic field and the potential of these nanoconjugates as T 2-weighted negative contrast MR imaging agent were evaluated in folate-overexpressed HeLa and normal L929 fibroblast cells. © 2011 The Royal Society of Chemistry.

Nanoscale

Folate receptor targeted, carboxymethyl chitosan functionalized iron oxide nanoparticles: A novel ultradispersed nanoconjugates for bimodal imaging

In this article, we report the design and synthesis of a series of well-dispersed superparamagnetic iron oxide nanoparticles (SPIONs) using chitosan as a surface modifying agent to develop a potential T2 contrast probe for magnetic resonance imaging (MRI). The amine, carboxyl, hydroxyl, and thiol functionalities were introduced on chitosancoated magnetic probe via simple reactions with small reactive organic molecules to afford a series of biofunctionalized nanoparticles. Physico-chemical characterizations of these functionalized nanoparticles were performed by TEM, XRD, DLS, FTIR, and VSM. The colloidal stability of these functionalized iron oxide nanoparticles was investigated in presence of phosphate buffer saline, high salt concentrations and different cell media for 1 week. MRI analysis of human cervical carcinoma (HeLa) cell lines treated with nanoparticles elucidated that the amine-functionalized nanoparticles exhibited higher amount of signal darkening and lower T2 relaxation in comparison to the others. The cellular internalization efficacy of these functionalized SPIONs was also investigated with HeLa cancer cell line by magnetically activated cell sorting (MACS) and fluorescence microscopy and results established selectively higher internalization efficacy of amine-functionalized nanoparticles to cancer cells. These positive attributes demonstrated that these nanoconjugates can be used as a promising platform for further in vitro and in vivo biological evaluations. © Springer Science+Business Media B.V. 2011.