Zeev Smilansky - Academia.edu (original) (raw)

Papers by Zeev Smilansky

(70) U-c Fingerprint: Glycoprotein Analysis Based on A Lectin Array Rakefet Rosenfeld, Ruth Maya,... more (70) U-c Fingerprint: Glycoprotein Analysis Based on A Lectin Array Rakefet Rosenfeld, Ruth Maya, Zeev Smilansky, Yehudit Amor, Chani Rothmann-Scherz, Yossi Cohen, Mirit Kolog Gulko, Ana Moraga Grosz, Haim Bangio, Revital Rosenberg, Leonid Schvartzer and Ofer Markman Procognia Limited, Unit 4, The Switchback, Gardner Road, Maidenhead, Berks SL6 7RJ, United Kingdom. Glycoproteins are produced by cells as a mixture of glycoforms, in which the same protein scaffold is adorned with variable glycan structures. The specific glycoforms and their relative proportions vary significantly with host cell types and growth conditions. Analysis of the glycan structure of a glycoprotein is challenging and recognition of the importance of glycan structure is rapidly increasing. The number of protein therapeutics under development is also increasing, yet production capacity for recombinant protein drugs has become rate limiting in meeting market need. Consequently there is significant effort to devel...

2013 Proceedings of the ESSCIRC (ESSCIRC), 2013

ABSTRACT The presented vision sensor features low-power pixel-level programmable dynamic backgrou... more ABSTRACT The presented vision sensor features low-power pixel-level programmable dynamic background subtraction as low-level image processing aimed at detecting unusual events occurring in the scene. Each pixel compares its current photogenerated signal with two threshold voltages, defining the boundary conditions outside which the signal is to be considered anomalous, if compared to its past history. In case of anomalous behavior, the pixel one-bit output will be asserted. The 17 transistors square pixel has a pitch of 16μm with a fill-factor of 21%. The 104 × 104 pixel sensor, fabricated in a 0.35 μm CMOS, has a power consumption of 80μW at 30 fps, when the sensor array is powered at 3.3V and the digital part at 1.5V. This turns into a pixel power consumption per frame of 246pW/pixel · frame.

Proceedings of the 2012 ACM/IEEE international symposium on Low power electronics and design - ISLPED '12, 2012

A 64x64 pixel vision sensor performs adaptive background subtraction and event detection at very ... more A 64x64 pixel vision sensor performs adaptive background subtraction and event detection at very low power consumption. The chip is based on a VLSI-oriented vision algorithm, implemented at pixel-level, mimicking the basic process of pre-attentive visual perception. Anomalous pixel behaviors are detected and coded into a 2-bit/pixel. Each pixel integrates two programmable Switched-Capacitors Low-Pass Filters and two clocked comparators, which are fundamental blocks for the execution of the vision algorithm. The 45T square pixel has a pitch of 26μm and a fill factor of 12%. The vision sensor consumes 33μW at 13 fps and 3.3V. This turns into a computing performance of 42 GOPS/W and 4 GOPS/mm2, which are values aligned with the most advanced computational vision sensors.

PROTEOMICS, 2003

Large-scale two-dimensional gel experiments have the potential to identify proteins that play an ... more Large-scale two-dimensional gel experiments have the potential to identify proteins that play an important role in elucidating cell mechanisms and in various stages of drug discovery. Such experiments, typically including hundreds or even thousands of related gels, are notoriously difficult to perform, and analysis of the gel images has until recently been virtually impossible. In this paper we describe a scalable computational model that permits the organization and analysis of a large gel collection. The model is implemented in Compugen's Z4000  system. Gels are organized in a hierarchical, multidimensional data structure that allow the user to view a large-scale experiment as a tree of numerous simpler experiments, and carry out the analysis one step at a time. Analyzed sets of gels form processing units that can be combined into higher level units in an iterative framework. The different conditions at the core of the experiment design, termed the dimensions of the experiment, are transformed from a multidimensional structure to a single hierarchy. The higher level comparison is performed with the aid of a synthetic "adaptor" gel image, called a Raw Master Gel (RMG). The RMG allows the inclusion of data from an entire set of gels to be presented as a gel image, thereby enabling the iterative process. Our model includes a flexible experimental design approach that allows the researcher to choose the condition to be analyzed a posteriori. It also enables data reuse, the performing of several different analysis designs on the same experimental data. The stability and reproducibility of a protein can be analyzed by tracking it up or down the hierarchical dimensions of the experiment.

Proceedings of the National Academy of Sciences, 2011

During protein synthesis, deacylated transfer RNAs leave the ribosome via an exit (E) site after ... more During protein synthesis, deacylated transfer RNAs leave the ribosome via an exit (E) site after mRNA translocation. How the ribosome regulates tRNA dissociation and whether functional linkages between the aminoacyl (A) and E sites modulate the dynamics of protein synthesis have long been debated. Using single molecule fluorescence resonance energy transfer experiments, we find that, during early cycles of protein elongation, tRNAs are often held in the E site until being allosterically released when the next aminoacyl tRNA binds to the A site. This process is regulated by the length and sequence of the nascent peptide and by the conformational state, detected by tRNA proximity, prior to translocation. In later cycles, E-site tRNA dissociates spontaneously. Our results suggest that the distribution of pretranslocation tRNA states and posttranslocation pathways are correlated within each elongation cycle via communication between distant subdomains in the ribosome, but that this correlation between elongation cycle intermediates does not persist into succeeding cycles.

PLoS ONE, 2012

We present proof-of-concept in vitro results demonstrating the feasibility of using single molecu... more We present proof-of-concept in vitro results demonstrating the feasibility of using single molecule fluorescence resonance energy transfer (smFRET) measurements to distinguish, in real time, between individual ribosomes programmed with several different, short mRNAs. For these measurements we use either the FRET signal generated between two tRNAs labeled with different fluorophores bound simultaneously in adjacent sites to the ribosome (tRNA-tRNA FRET) or the FRET signal generated between a labeled tRNA bound to the ribosome and a fluorescent derivative of ribosomal protein L1 (L1-tRNA FRET). With either technique, criteria were developed to identify the mRNAs, taking into account the relative activity of the mRNAs. These criteria enabled identification of the mRNA being translated by a given ribosome to within 95% confidence intervals based on the number of identified FRET traces. To upgrade the approach for natural mRNAs or more complex mixtures, the stoichiometry of labeling should be enhanced and photobleaching reduced. The potential for porting these methods into living cells is discussed.

Nucleic Acids Research, 2011

We have developed a novel technique of using fluorescent tRNA for translation monitoring (FtTM). ... more We have developed a novel technique of using fluorescent tRNA for translation monitoring (FtTM). FtTM enables the identification and monitoring of active protein synthesis sites within live cells at submicron resolution through quantitative microscopy of transfected bulk uncharged tRNA, fluorescently labeled in the D-loop (fl-tRNA). The localization of fl-tRNA to active translation sites was confirmed through its co-localization with cellular factors and its dynamic alterations upon inhibition of protein synthesis. Moreover, fluorescence resonance energy transfer (FRET) signals, generated when fl-tRNAs, separately labeled as a FRET pair occupy adjacent sites on the ribosome, quantitatively reflect levels of protein synthesis in defined cellular regions. In addition, FRET signals enable detection of intrapopulational variability in protein synthesis activity. We demonstrate that FtTM allows quantitative comparison of protein synthesis between different cell types, monitoring effects of antibiotics and stress agents, and characterization of changes in spatial compartmentalization of protein synthesis upon viral infection.

Nucleic Acids Research, 2013

The current report represents a further advancement of our previously reported technology termed ... more The current report represents a further advancement of our previously reported technology termed Fluorescent transfer RNA (tRNA) for Translation Monitoring (FtTM), for monitoring of active global protein synthesis sites in single live cells. FtTM measures Fö rster resonance energy transfer (FRET) signals, generated when fluorescent tRNAs (fl-tRNAs), separately labeled as a FRET pair, occupy adjacent sites on the ribosome. The current technology, termed DiCodon Monitoring of Protein Synthesis (DiCoMPS), was developed for monitoring active synthesis of a specific protein. In DiCoMPS, specific fl-tRNA pair combinations are selected for transfection, based on the degree of enrichment of a dicodon sequence to which they bind in the mRNA of interest, relative to the background transcriptome of the cell in which the assay is performed. In this study, we used cells infected with the Epizootic Hemorrhagic Disease Virus 2-Ibaraki and measured, through DiCoMPS, the synthesis of the viral non-structural protein 3 (NS3), which is enriched in the AUA:AUA dicodon. fl-tRNA Ile UAU -generated FRET signals were specifically enhanced in infected cells, increased in the course of infection and were diminished on siRNAmediated knockdown of NS3. Our results establish an experimental approach for the single-cell measurement of the levels of synthesis of a specific viral protein.

Molecular Cell, 2011

We employ single-molecule fluorescence resonance energy transfer (smFRET) to study structural dyn... more We employ single-molecule fluorescence resonance energy transfer (smFRET) to study structural dynamics over the first two elongation cycles of protein synthesis, using ribosomes containing either Cy3-labeled ribosomal protein L11 and A-or P-site Cy5-labeled tRNA or Cy3 and Cy5 labeled tRNAs. Pre-translocation (PRE) complexes demonstrate fluctuations between classical and hybrid forms, with concerted motions of tRNAs away from L11 and from each other when classical complex converts to hybrid complex. EF-G·GTP binding to both hybrid and classical PRE complexes halts these fluctuations prior to catalyzing translocation to form the posttranslocation (POST) complex. EF-G dependent translocation from the classical PRE complex proceeds via transient formation of a short-lived hybrid intermediate. A-site binding of either EF-G to the PRE complex or of aminoacyl-tRNA·EF-Tu ternary complex to the POST complex markedly suppresses ribosome conformational lability.

Biophysical Journal, 2013

at the A-site, and iii) deacylated tRNA and ribosomal protein L1 at the E-site, to determine the ... more at the A-site, and iii) deacylated tRNA and ribosomal protein L1 at the E-site, to determine the rates of steps 1) -3) during elongation cycles of ribosomes programmed with mRNAs either containing or lacking a PK. We find that, whereas the presence of a PK has little or no effect on the rates of steps 1) and 2), it strongly decreases (~2.5-fold) the rate of step 3. Thus, somewhat surprisingly, step 3) appears to be more strongly coupled to the unfolding of PK structure than step 2). In contrast, preliminary results indicate that stem-loop structures can decrease the rates of both steps 2) and 3). Proteins are synthesized by the ribosome and must generally fold to become functionally active. Although it is generally assumed that the ribosome and the process of translation affect folding, this idea has been extremely difficult to demonstrate. We have developed an experimental system to investigate the folding of single ribosome-bound stalled nascent polypeptides with optical tweezers. In T4 lysozyme, the ribosome slows the formation of stable tertiary interactions and the attainment of the native state relative to the free protein. Incomplete T4 lysozyme polypeptides misfold and aggregate when free in solution, but remain folding-competent near the ribosomal surface. Altogether, our results suggest that the ribosome not only decodes the genetic information and synthesizes polypeptides, but also promotes efficient de novo attainment of the native state and thus acts as a molecular chaperone for newly synthesized proteins. We are currently extending our approach to monitor folding on actively elongating ribosomes in the optical tweezers, which allows us to study how elongation dynamics affect nascent protein folding.

Biophysical Journal, 2010

... Allosteric Dissociation of E-Site tRNA During Polypeptide Elongation. Authors: Chen, Chunlai;... more ... Allosteric Dissociation of E-Site tRNA During Polypeptide Elongation. Authors: Chen, Chunlai; Stevens, Benjamin; Kaur, Jaskiran; Cabral, Diana; Smilansky, Zeev; Cooperman, Barry S.; Goldman, Yale E. Publication: Biophysical Journal, vol. 98, issue 3, pp. 262a-262a. ...