High resolution light-sheet based high-throughput imaging cytometry system enables visualization of intra-cellular organelles

Visualization of intracellular organelles is achieved using a newly developed high throughput imaging cytometry system. This system interrogates the microfluidic channel using a sheet of light rather than the existing point-based scanning techniques. The advantages of the developed system are many, including, single-shot scanning of specimens flowing through the microfluidic channel at flow rate ranging from micro-to nano- lit./min. Moreover, this opens-up in-vivo imaging of sub-cellular structures and simultaneous cell counting in an imaging cytometry system. We recorded a maximum count of 2400 cells/min at a flow-rate of 700 nl/min, and simultaneous visualization of fluorescently-labeled mitochondrial network in HeLa cells during flow. The developed imaging cytometry system may find immediate application in biotechnology, fluorescence microscopy and nano-medicine. (C) 2014 Author(s). All article content, except where otherwise noted, is licensed under a Creative Commons Attribution 3.0 Unported License.

Tailoring viscoelastic response of carbon nanotubes cellular structure using electric field

Cellular structures of carbon nanotubes (CNT) are novel engineering materials, which are finding applications due to their remarkable structural and functional properties. Here, we report the effects of electric field, one of the most frequently used stimulants for harnessing the functional properties of CNT, on the viscoelastic response, an important design consideration for the structural applications of a cellular CNT sample. The application of an electric field results in electrostriction induced large actuation in freestanding CNT samples; however, if the CNT are prohibited to expand, an electric field dependent force is exerted by the sample on the constraining platens. In addition, the above force monotonically decreases with the pre-compressive strain imposed onto the sample. The viscoelastic recovery reveals a decrease in the stress relaxation with an increase in the pre-compressive strain in both the presence and absence of the electric field; however, the stress relaxation was significantly higher in the presence of the electric field. A model, based on a simple linear viscoelastic solid incorporating electric field, is developed to understand the experimental observations.

LANDSCAPE DYNAMICS MODELING THROUGH INTEGRATED MARKOV, FUZZY-AHP AND CELLULAR AUTOMATA

Multi temporal land use information were derived using two decades remote sensing data and simulated for 2012 and 2020 with Cellular Automata (CA) considering scenarios, change probabilities (through Markov chain) and Multi Criteria Evaluation (MCE). Agents and constraints were considered for modeling the urbanization process. Agents were nornmlized through fiizzyfication and priority weights were assigned through Analytical Hierarchical Process (AHP) pairwise comparison for each factor (in MCE) to derive behavior-oriented rules of transition for each land use class. Simulation shows a good agreement with the classified data. Fuzzy and AHP helped in analyzing the effects of agents of growth clearly and CA-Markov proved as a powerful tool in modelling and helped in capturing and visualizing the spatiotemporal patterns of urbanization. This provided rapid land evaluation framework with the essential insights of the urban trajectory for effective sustainable city planning.

Characterization of rice small heat shock proteins targeted to different cellular organelles

Small heat shock proteins (sHSPs) are a family of ATP-independent molecular chaperones which prevent cellular protein aggregation by binding to misfolded proteins. sHSPs form large oligomers that undergo drastic rearrangement/dissociation in order to execute their chaperone activity in protecting substrates from stress. Substrate-binding sites on sHSPs have been predominantly mapped on their intrinsically disordered N-terminal arms. This region is highly variable in sequence and length across species, and has been implicated in both oligomer formation and in mediating chaperone activity. Here, we present our results on the functional and structural characterization of five sHSPs in rice, each differing in their subcellular localisation, viz., cytoplasm, nucleus, chloroplast, mitochondria and peroxisome. We performed activity assays and dynamic light scattering studies to highlight differences in the chaperone activity and quaternary assembly of sHSPs targeted to various organelles. By cloning constructs that differ in the length and sequence of the tag in the N-terminal region, we have probed the sensitivity of sHSP oligomer assembly and chaperone activity to the length and amino acid composition of the N-terminus. In particular, we have shown that the incorporation of an N-terminal tag has significant consequences on sHSP quaternary structure.

Iron(III) Complexes of a Pyridoxal Schiff Base for Enhanced Cellular Uptake with Selectivity and Remarkable Photocytotoxicity

Iron(III) complexes of pyridoxal (vitamin B6, VB6) or salicylaldehyde Schiff bases and modified dipicolylamines, namely, Fe(B)(L)](NO3) (15), where B is phenyl-N,N-bis((pyridin-2-yl)methyl)methanamine (phbpa in 1), (anthracen-9-yl)-N,N-bis((pyridin-2-yl)methyl)methanamine (anbpa in 2, 4) and (pyren-1-yl)-N,N-bis((pyridin-2-yl)methyl)methanamine (pybpa in 3, 5) (H2L1 is 3-hydroxy-5-(hydroxymethyl)-4-(((2-hydroxyphenyl)imino)methyl)-2-methylp yridine (13) and H2L2 is 2-(2-hydroxyphenyl-imino)methyl]phenol), were prepared and their uptake in cancer cells and photocytotoxicity were studied. Complexes 4 and 5, having a non-pyridoxal Schiff base, were prepared to probe the role of the pyridoxal group in tumor targeting and cellular uptake. The PF6 salt (1a) of complex 1 is structurally characterized. The complexes have a distorted six-coordinate FeN4O2 core where the metal is in the +3 oxidation state with five unpaired electrons. The complexes display a ligand to metal charge transfer band near 520 and 420 nm from phenolate to the iron(III) center. The photophysical properties of the complexes are explained from the time dependent density functional theory calculations. The redox active complexes show a quasi-reversible Fe(III)/Fe(II) response near -0.3 V vs saturated calomel electrode. Complexes 2 and 3 exhibit remarkable photocytotoxicity in various cancer cells with IC50 values ranging from 0.4 to 5 mu M with 10-fold lower dark toxicity. The cell death proceeded by the apoptotic pathway due to generation of reactive oxygen species upon light exposure. The nonvitamin complexes 4 and 5 display 3-fold lower photocytotoxicity compared to their VB6 analogues, possibly due to preferential and faster uptake of the vitamin complexes in the cancer cells. Complexes 2 and 3 show significant uptake in the endoplasmic reticulum, while complexes 4 and 5 are distributed throughout the cells without any specific localization pattern.

Reversible induction of translational isoforms of p53 in glucose deprivation

Tumor suppressor protein p53 is a master transcription regulator, indispensable for controlling several cellular pathways. Earlier work in our laboratory led to the identification of dual internal ribosome entry site (IRES) structure of p53 mRNA that regulates translation of full-length p53 and Delta 40p53. IRES-mediated translation of both isoforms is enhanced under different stress conditions that induce DNA damage, ionizing radiation and endoplasmic reticulum stress, oncogene-induced senescence and cancer. In this study, we addressed nutrient-mediated translational regulation of p53 mRNA using glucose depletion. In cell lines, this nutrient-depletion stress relatively induced p53 IRES activities from bicistronic reporter constructs with concomitant increase in levels of p53 isoforms. Surprisingly, we found scaffold/matrix attachment region-binding protein 1 (SMAR1), a predominantly nuclear protein is abundant in the cytoplasm under glucose deprivation. Importantly under these conditions polypyrimidine-tract-binding protein, an established p53 ITAF did not show nuclear-cytoplasmic relocalization highlighting the novelty of SMAR1-mediated control in stress. In vivo studies in mice revealed starvation-induced increase in SMAR1, p53 and Delta 40p53 levels that was reversible on dietary replenishment. SMAR1 associated with p53 IRES sequences ex vivo, with an increase in interaction on glucose starvation. RNAi-mediated-transient SMAR1 knockdown decreased p53 IRES activities in normal conditions and under glucose deprivation, this being reflected in changes in mRNAs in the p53 and Delta 40p53 target genes involved in cell-cycle arrest, metabolism and apoptosis such as p21, TIGAR and Bax. This study provides a new physiological insight into the regulation of this critical tumor suppressor in nutrient starvation, also suggesting important functions of the p53 isoforms in these conditions as evident from the downstream transcriptional target activation.

Imaging cellular signalling: many `moving tales' in MAP kinase odyssey

Cellular signalling events are at the core of every adaptive response. Signalling events link environmental changes to physiological responses, consequently allowing cellular and organismal sustenance and survival. Classical approaches to study cellular signalling have relied on a variety of cell disruptive techniques which yield limited kinetic information, while the underlying events are much more complex. In this article, we discuss how modern live cell imaging microscopy has found increasing utilization in revealing spatio temporal dynamics of various signalling pathways. Utilizing the well studied mitogen-activated protein kinase (MAPK) signalling cascade as a template, the design, construction and utilization of `mobile' (translocation proficient) biosensors, suitable for studying MAPK signalling in living cells are described in detail. Experimental setup and results obtained from these biosensors, based on different proteins involved in the MAPK signalling cascade, have been described along with the setup of a microscope optimal for live cell imaging applications. Utilizing the ability to activate or deactivate signalling pathways using defined activators and specific pharmacological inhibitors, we also show how these sensors can yield unique spatial and temporal kinetic information of signalling in living cells.

Metal Complexes of Curcumin for Cellular Imaging, Targeting, and Photoinduced Anticancer Activity

CONSPECTUS: Curcumin is a polyphenolic species. As an active ingredient of turmeric, it is well-known for its traditional medicinal properties. The therapeutic values include antioxidant, anti-inflammatory, antiseptic, and anticancer activity with the last being primarily due to inhibition of the transcription factor NF-kappa B besides affecting several biological pathways to arrest tumor growth and its progression. Curcumin with all these positive qualities has only remained a potential candidate for cancer treatment over the years without seeing any proper usage because of its hydrolytic instability involving the diketo moiety in a cellular medium and its poor bioavailability. The situation has changed considerably in recent years with the observation that curcumin in monoanionic form could be stabilized on binding to a metal ion. The reports from our group and other groups have shown that curcumin in the metal-bound form retains its therapeutic potential. This has opened up new avenues to develop curcumin-based metal complexes as anticancer agents. Zinc(II) complexes of curcumin are shown to be stable in a cellular medium. They display moderate cytotoxicity against prostate cancer and neuroblastoma cell lines. A similar stabilization and cytotoxic effect is reported for (arene)ruthenium(II) complexes of curcumin against a variety of cell lines. The half-sandwich 1,3,5-triaza-7-phosphatricyclo-3.3.1.1]decane (RAPTA)-type ruthenium(II) complexes of curcumin are shown to be promising cytotoxic agents with low micromolar concentrations for a series of cancer cell lines. In a different approach, cobalt(III) complexes of curcumin are used for its cellular delivery in hypoxic tumor cells using intracellular agents that reduce the metal and release curcumin as a cytotoxin. Utilizing the photophysical and photochemical properties of the curcumin dye, we have designed and synthesized photoactive curcumin metal complexes that are used for cellular imaging by fluorescence microscopy and damaging the cancer cells on photoactivation in visible light while being minimally toxic in darkness. In this Account, we have made an attempt to review the current status of the chemistry of metal curcumin complexes and present results from our recent studies on curcumin complexes showing remarkable in vitro photocytotoxicity. The undesirable dark toxicity of the complexes can be reduced with suitable choice of the metal and the ancillary ligands in a ternary structure. The complexes can be directed to specific subcellular organelles. Selectivity by targeting cancer cells over normal cells can be achieved with suitable ligand design. We expect that this methodology is likely to provide an impetus toward developing curcumin-based photochemotherapeutics for anticancer treatment and cure.

Is the cellular initiation of translation an exclusive property of the initiator tRNAs?

Translation of mRNAs is the primary function of the ribosomal machinery. Although cells allow for a certain level of translational errors/mistranslation (which may well be a strategic need), maintenance of the fidelity of translation is vital for the cellular function and fitness. The P-site bound initiator tRNA selects the start codon in an mRNA and specifies the reading frame. A direct P-site binding of the initiator tRNA is a function of its special structural features, ribosomal elements, and the initiation factors. A highly conserved feature of the 3 consecutive G:C base pairs (3GC pairs) in the anticodon stem of the initiator tRNAs is vital in directing it to the P-site. Mutations in the 3GC pairs diminish/abolish initiation under normal physiological conditions. Using molecular genetics approaches, we have identified conditions that allow initiation with the mutant tRNAs in Escherichia coli. During our studies, we have uncovered a novel phenomenon of in vivo initiation by elongator tRNAs. Here, we recapitulate how the cellular abundance of the initiator tRNA, and nucleoside modifications in rRNA are connected with the tRNA selection in the P-site. We then discuss our recent finding of how a conserved feature in the mRNA, the Shine-Dalgarno sequence, influences tRNA selection in the P-site.

Interplay of Substrate Conductivity, Cellular Microenvironment, and Pulsatile Electrical Stimulation toward Osteogenesis of Human Mesenchymal Stem Cells in Vitro

The influences of physical stimuli such as surface elasticity, topography, and chemistry over mesenchymal stem cell proliferation and differentiation are well investigated. In this context, a fundamentally different approach was adopted, and we have demonstrated the interplay of inherent substrate conductivity, defined chemical composition of cellular microenvironment, and intermittent delivery of electric pulses to drive mesenchymal stem cell differentiation toward osteogenesis. For this, conducting polyaniline (PANI) substrates were coated with collagen type 1 (Coll) alone or in association with sulfated hyaluronan (sHya) to form artificial extracellular matrix (aECM), which mimics the native microenvironment of bone tissue. Further, bone marrow derived human mesenchymal stem cells (hMSCs) were cultured on these moderately conductive (10(-4)10(-3) S/cm) aECM coated PANI substrates and exposed intermittently to pulsed electric field (PEF) generated through transformer-like coupling (TLC) approach over 28 days. On the basis of critical analysis over an array of end points, it was inferred that Coll/sHya coated PANI (PANI/Coll/sHya) substrates had enhanced proliferative capacity of hMSCs up to 28 days in culture, even in the absence of PEF stimulation. On the contrary, the adopted PEF stimulation protocol (7 ms rectangular pulses, 3.6 mV/cm, 10 Hz) is shown to enhance osteogenic differentiation potential of hMSCs. Additionally, PEF stimulated hMSCs had also displayed different morphological characteristics as their nonstimulated counterparts. Concomitantly, earlier onset of ALP activity was also observed on PANI/Coll/sHya substrates and resulted in more calcium deposition. Moreover, real-time polymerase chain reaction results indicated higher mRNA levels of alkaline phosphatase and osteocalcin, whereas the expression of other osteogenic markers such as Runt-related transcription factor 2, Col1A, and osteopontin exhibited a dynamic pattern similar to control cells that are cultured in osteogenic medium. Taken together, our experimental results illustrate the interplay of multiple parameters such as substrate conductivity, electric field stimulation, and aECM coating on the modulation of hMSC proliferation and differentiation in vitro.

Temperature dependent compressive behavior of graphene mediated three-dimensional cellular assembly

Mechanical behavior of three-dimensional cellular assembly of graphene foam (GF) presented temperature dependent characteristics evaluated at both low temperature and room temperature conditions. Cellular structure of GF comprised of polydimethyl siloxane polymer as a flexible supporting material demonstrated 94% enhancement in the storage modulus as compared to polymer foam alone. Evaluation of frequency dependence revealed an increase in both storage modulus and tan delta with the increase in frequency. Moreover, strain rate independent highly reversible behavior is measured up to several compression cycles at larger strains. It is elucidated that the interaction between graphene and polymer plays a crucial role in thermo-mechanical stability of the cellular structure. (C) 2015 Elsevier Ltd. All rights reserved.

Enhancement of Flotation Selectivity of Sphalerite Using Mineral-Stressed Paenibacillus polymyxa and Its Cellular Components

The selective flotation of sphalerite from a sphalerite-galena mineral mixture was achieved using cellular components of Paenibacillus polymyxa after adaptation to the above minerals. The soluble and insoluble fractions of the thermolysed bacterial cells adapted to sphalerite yielded higher flotation recoveries of sphalerite with selectivity indices ranging between 22 and 29. The protein profile for the unadapted and mineral-stressed cells was found to differ distinctly, attesting to variation in the yield and nature of extra-cellular polymeric substances. The changes induced in the bacterial cell wall components after adaptation to sphalerite or galena with respect to the contents of phosphate, uronic acid and acetylated sugars of P. polymyxa were quantified. In keeping with these changes, a marginal morphological transition of P. polymyxa from rods to spheres was observed. The role of the dissolved metal ions from the minerals as well as that of the constituents of extracellular secretions in modulating the surface potential of the mineral-stressed cells were demonstrated. These studies highlighted that, mineral stress led to qualitative and quantitative changes in the cellular components, which facilitated the enhancement of flotation selectivity of sphalerite.

An efficient design of serial and parallel memory using Quantum dot cellular automata

Quantum cellular automata (QCA) is a new technology in the nanometer scale and has been considered as one of the alternative to CMOS technology. In this paper, we describe the design and layout of a serial memory and parallel memory, showing the layout of individual memory cells. Assuming that we can fabricate cells which are separated by 10nm, memory capacities of over 1.6 Gbit/cm2 can be achieved. Simulations on the proposed memories were carried out using QCADesigner, a layout and simulation tool for QCA. During the design, we have tried to reduce the number of cells as well as to reduce the area which is found to be 86.16sq mm and 0.12 nm2 area with the QCA based memory cell. We have also achieved an increase in efficiency by 40%.These circuits are the building block of nano processors and provide us to understand the nano devices of the future.

Thermal co-reduction approach to vary size of silver nanoparticle: its microbial and cellular toxicology

In recent years, silver nanoparticles (AgNPs) have attracted considerable interest in the field of food, agriculture and pharmaceuticals mainly due to its antibacterial activity. AgNPs have also been reported to possess toxic behavior. The toxicological behavior of nanomaterials largely depends on its size and shape which ultimately depend on synthetic protocol. A systematic and detailed analysis for size variation of AgNP by thermal co-reduction approach and its efficacy toward microbial and cellular toxicological behavior is presented here. With the focus to explore the size-dependent toxicological variation, two different-sized NPs have been synthesized, i.e., 60 nm (Ag60) and 85 nm (Ag85). A detailed microbial toxicological evaluation has been performed by analyzing minimum inhibitory concentration (MIC), minimum bactericidal concentration (MBC), diameter of inhibition zone (DIZ), growth kinetics (GrK), and death kinetics (DeK). Comparative cytotoxicological behavior was analyzed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. It has been concluded by this study that the size of AgNPs can be varied, by varying the concentration of reactants and temperature called as ``thermal co-reduction'' approach, which is one of the suitable approaches to meet the same. Also, the smaller AgNP has shown more microbial and cellular toxicity.

Stimuli-responsive colorimetric and NIR fluorescence combination probe for selective reporting of cellular hydrogen peroxide

Hydrogen peroxide (H2O2) is a key reactive oxygen species and a messenger in cellular signal transduction apart from playing a vital role in many biological processes in living organisms. In this article, we present phenyl boronic acid-functionalized quinone-cyanine (QCy-BA) in combination with AT-rich DNA (exogenous or endogenous cellular DNA), i.e., QCy-BA subset of DNA as a stimuli-responsive NIR fluorescence probe for measuring in vitro levels of H2O2. In response to cellular H2O2 stimulus, QCy-BA converts into QCy-DT, a one-donor-two-acceptor (D2A) system that exhibits switch-on NIR fluorescence upon binding to the DNA minor groove. Fluorescence studies on the combination probe QCy-BA subset of DNA showed strong NIR fluorescence selectively in the presence of H2O2. Furthermore, glucose oxidase (GOx) assay confirmed the high efficiency of the combination probe QCy-BA subset of DNA for probing H2O2 generated in situ through GOx-mediated glucose oxidation. Quantitative analysis through fluorescence plate reader, flow cytometry and live imaging approaches showed that QCy-BA is a promising probe to detect the normal as well as elevated levels of H2O2 produced by EGF/Nox pathways and post-genotoxic stress in both primary and senescent cells. Overall, QCy-BA, in combination with exogenous or cellular DNA, is a versatile probe to quantify and image H2O2 in normal and disease-associated cells.