Dorit Avrahami - Academia.edu (original) (raw)

Papers by Dorit Avrahami

Protein-membrane interactions and proteinprotein recognition within the membrane milieu are of fu... more Protein-membrane interactions and proteinprotein recognition within the membrane milieu are of fundamental importance to fully comprehend a wide range of cellular processes in all organisms. About 40% of all genes in the mammalian genome transcribe for membrane proteins. The paucity in 3D structures for most membrane proteins, the high complexity of the forces involved, and the technical difficulties present challenging obstacles to overcome before fully understanding biological events within membranes. Using a multidisciplinary approach, including cell molecular biology (cells and viruses), biophysics and biochemistry, we study the mode of action of membrane proteins, particularly those involved in infectious diseases. The principle underlying our approach is the dissection of the proteins to small fragments, which are studied for their structure, function, their ability to interfere with the function of the parental intact proteins, and network of peptide-peptide recognition withi...

New Biotechnology, 2018

Highlights  HCV NS3 binding partners were discovered by a microfluidic membrane protein array  ... more Highlights  HCV NS3 binding partners were discovered by a microfluidic membrane protein array  NRG1 was the only binding partner revealed that was cleaved by the NS3/4A protease  NRG1 RNA level increased with NS3/4A, independent of its proteolytic activity  NRG1 EGF-like domain levels increased in HCV infected cells

Advanced Biosystems, 2019

of tumor's DNA, RNA, or proteins, and by integration of tumor cells into chemo sensitivity and re... more of tumor's DNA, RNA, or proteins, and by integration of tumor cells into chemo sensitivity and resistance assays (CSRA). Diagnosis by molecular profiling of DNA, RNA, or proteins is used to identify mole cular biomarkers that are predictive of patient response to a drug. [7,8] Diagnosis by CSRA is used to determine tumor cells ex vivo response to a drug. [9] Although these diagnosis methods improve clinical out come, cancer mortality remains high. [10] Importantly, scientific literature shows that gaps in tumor cellular and mole cular heterogeneity characterization [11] is a major limitation of the personalized medicine approach in cancer. [12] The significant genomic evolution that often occurs during cancer progression creates variability within primary tumors as well as between the primary tumors and metastases. [13-15] Although new high resolution sequencing and bioinformatics methods improved the molecular charac terization of tumors, these technologies remain limited by tissue sampling and analysis methods. [15,16] Recent studies show that during analysis stages, a positive result based on both successful biopsy and molecular charac terization is a reliable indication of the presence of the high risk disease, although a negative result does not reliably exclude the presence of highrisk disease. [17] Thus, new approaches for characterization of tumor heterogeneity and heterogeneity impact on drug resistance are needed. [18] Microfluidic approaches could provide a more detailed pic ture of heterogenous cancer cell population response to drugs than traditional culture methods. [19-23] Therefore, such a device could provide a new direction for CSRA models development. The potential of CSRA models has long been recognized by the scientific community. However, classic tools for CSRA models faced multiple challenges that hindered their success. [24] Some examples of current challenges include; poor and unrepeat able in vitro culture conditions, [24-26] the limited information provided by traditional in vitro techniques to clinicians, [27,28] and tumor heterogeneity. [11] These challenges could potentially explain the observed discordance between in vivo and in vitro therapeutic responses. Microfluidics is already used in multiple molecular biology techniques, such as polymerase chain reaction, electropho resis on a chip, DNA microarrays, and diagnostic devices that Cancer is the second leading cause of death globally. Matching proper treatment and dosage is crucial for a positive outcome. Any given drug may affect patients with similar tumors differently. Personalized medicine aims to address this issue. Unfortunately, most cancer samples cannot be expanded in culture, limiting conventional cell-based testing. Herein, presented is a microfluidic device that combines a drug microarray with cell microscopy. The device can perform 512 experiments to test chemosensitivity and resistance to a drug array. MCF7 and 293T cells are cultured inside the device and their chemosensitivity and resistance to docetaxel, applied at various concentrations, are determined. Cell mortality is determined as a function of drug concentration and exposure time. It is found that both cell types form cluster morphology within the device, not evident in conventional tissue culture under similar conditions. Cells inside the clusters are less sensitive to drugs than dispersed cells. These findings support a heterogenous response of cancer cells to drugs. Then demonstrated is the principle of drug microarrays by testing cell response to four different drugs at four different concentrations. This approach may enable the personalization of treatment to the particular tumor and patient and may eventually improve final patient outcome.

Langmuir, 2019

Interest in PDMS microfluidic devices has grown dramatically in recent years, particularly in the... more Interest in PDMS microfluidic devices has grown dramatically in recent years, particularly in the context of improved performance lab-on-a-chip devices with decreasing channel size enabling more devices on ever smaller chips. As channels become smaller, the resistance to flow increases and the device structure must be able to withstand higher internal pressures. We report herein the fabrication of microstructured surfaces that promote water mobility independent of surface static wetting properties. The key tool in this approach is the growth of ZnO nanorods on the bottom face of the microfluidic device. We show that water flow in these devices is similar whether the textured nanorod-bearing surface is hydrophilic or

Lab on a Chip, 2017

A computerized semiautomatic aligner aligns multilayer devices comprising intricate microfeatures... more A computerized semiautomatic aligner aligns multilayer devices comprising intricate microfeatures several-fold more accurately than manual alignment.

ACS nano, Apr 25, 2017

Realization of bioinspired molecular machines that can perform many and diverse operations in res... more Realization of bioinspired molecular machines that can perform many and diverse operations in response to external chemical commands is a major goal in nanotechnology, but current molecular machines respond to only a few sequential commands. Lack of effective methods for introduction and removal of command compounds and low efficiencies of the reactions involved are major reasons for the limited performance. We introduce here a user interface based on a microfluidics device and single-molecule fluorescence spectroscopy that allows efficient introduction and removal of chemical commands and enables detailed study of the reaction mechanisms involved in the operation of synthetic molecular machines. The microfluidics provided 64 consecutive DNA strand commands to a DNA-based motor system immobilized inside the microfluidics, driving a bipedal walker to perform 32 steps on a DNA origami track. The microfluidics enabled removal of redundant strands, resulting in a 6-fold increase in proc...

Genes & Development, 2014

Transcription of protein-coding genes is highly dependent on the RNA polymerase II core promoter.... more Transcription of protein-coding genes is highly dependent on the RNA polymerase II core promoter. Core promoters, generally defined as the regions that direct transcription initiation, consist of functional core promoter motifs (such as the TATA-box, initiator [Inr], and downstream core promoter element [DPE]) that confer specific properties to the core promoter. The known basal transcription factors that support TATA-dependent transcription are insufficient for in vitro transcription of DPE-dependent promoters. In search of a transcription factor that supports DPE-dependent transcription, we used a biochemical complementation approach and identified the Drosophila TBP (TATA-box-binding protein)-related factor 2 (TRF2) as an enriched factor in the fractions that support DPE-dependent transcription. We demonstrate that the short TRF2 isoform preferentially activates DPE-dependent promoters. DNA microarray analysis reveals the enrichment of DPE promoters among short TRF2 up-regulated ...

It is important for robots to model other robots' unob- served actions, plans, goals and beh... more It is important for robots to model other robots' unob- served actions, plans, goals and behaviors. However, clas- sic plan recognition is ill-suited to modeling robotic sys- tems, as (i) it assumes that actions are discrete, instanta- neous and cannot take place in parallel; and (ii) it uses a planning operator-based representation, which differs sig- nificantly from the behavior-based controllers often used with robots—thus making it difficult to represent the re- active components of robots interactions with their envi- ronment. We present a behavior-based approach to plan- recognition, in which hierarchical behaviors are used to model the observed robots. We show that our new model allows efficient, practical inference based on observations of parallel and continuous actions, and knowledge of the observed robots. We present highly efficient symbolic algo- rithms for answering key queries about observed robots, un- der conditions of lossy and lossless observations.

Journal of Biological Chemistry, 2004

We report on the synthesis, biological function, and a plausible mode of action of a new group of... more We report on the synthesis, biological function, and a plausible mode of action of a new group of lipopeptides with potent antifungal and antibacterial activities. These lipopeptides are derived from positively charged peptides containing D-and L-amino acids (diastereomers) that are palmitoylated (PA) at their N terminus. The peptides investigated have the sequence K 4 X 7 W, where X designates Gly, Ala, Val, or Leu (designated D-X peptides). The data revealed that PAD -G and PAD A gained potent antibacterial and antifungal activity despite the fact that both parental peptides were completely devoid of any activity toward microorganisms and model phospholipid membranes. In contrast, PAD -L lost the potent antibacterial activity of the parental peptide but gained and preserved partial antifungal activity. Interestingly, both D-V and its palmitoylated analog were inactive toward bacteria, and only the palmitoylated peptide was highly potent toward yeast. Both PAD -L and PAD -V lipopeptides were also endowed with hemolytic activity. Mode of action studies were performed by using tryptophan fluorescence and attenuated total reflectance Fourier transform infrared and circular dichroism spectroscopy as well as transmembrane depolarization assays with bacteria and fungi. The data suggest that the lipopeptides act by increasing the permeability of the cell membrane and that differences in their potency and target specificity are the result of differences in their oligomeric state and ability to dissociate and insert into the cytoplasmic membrane. These results provide insight regarding a new approach of modulating hydrophobicity and the self-assembly of non-membrane interacting peptides in order to endow them with both antibacterial and antifungal activities urgently needed to combat bacterial and fungal infections.

Biochemistry, 2002

Our basic understanding of how to combat fungal infections has not kept pace with the recent shar... more Our basic understanding of how to combat fungal infections has not kept pace with the recent sharp rise in life-threatening cases found particularly among immuno-compromised individuals. Current investigations for new potential antifungal agents have focused on antimicrobial peptides, which are used as a cell-free defense mechanism in all organisms. Unfortunately, despite their high antibacterial activity, most of them are not active toward fungi, the reason of which is not clear. Here, we present a new approach to modify an antibacterial peptide, a magainin analogue, to display antifungal activity by its conjugation with lipophilic acids. This approach has the advantage of producing well-defined changes in hydrophobicity, secondary structure, and self-association. These modifications were characterized in solution at physiological concentrations using CD spectroscopy, tryptophan fluorescence, and analytical ultracentrifugation. In order of increasing hydrophobicity, the attachment to the magainin-2 analogue of (i) heptanoic acid results in a monomeric, unordered structure, (ii) undecanoic acid yields concentrationdependent oligomers of R helices, and (iii) palmitic acid yields concentration-independent R-helical monomers, a novel lipopeptide structure, which is resistant to proteolytic digestion. Membrane-lipopeptide interactions and the membrane-bound structures were studied using fluorescence and ATR-FTIR in PC/ PE/PI/ergosterol (5/2.5/2.5/1, w/w) SUV, which constitute the major components of Candida albicans bilayers. A direct correlation was found between oligomerization of the lipopeptides in solution and potent antifungal activity. These results provide insight to a new approach of modulating hydrophobicity and self-assembly of antimicrobial peptides in solution, without altering the sequence of the peptidic chain. These studies also provide a general means of developing a new group of lipopeptide candidates as therapeutic agents against fungal infections.

Biochemistry, 2003

The dramatically increased frequency of opportunistic fungal infections has prompted research to ... more The dramatically increased frequency of opportunistic fungal infections has prompted research to diversify the arsenal of antifungal agents. Antimicrobial peptides constitute a promising family for future antibiotics with a new mode of action. However, only a few are effective against fungal pathogens because of their ability to self-assemble. Recently, we showed that the conjugation of fatty acids to the potent antibacterial peptide magainin endowed it with antifungal activity concomitant with an increase in its oligomeric state in solution. To investigate whether a high potency of the parental peptide is prerequisite for antifungal activity, we conjugated undecanoic acid (UA) and palmitic acid (PA) to inactive diastereomers of magainin containing four d-amino acids ([D]-4-magainin), as well as to a weakly active diastereomeric lytic peptide containing Lys and Leu ([D]-K(5)L(7)). All lipopeptides gained potent activity toward Cryptococcus neoformans. Most importantly, [D]-K(5)L(7)-UA was highly potent against all microorganisms tested, including bacteria, yeast, and opportunistic fungi. All lipopeptides increased the permeability of Escherichia coli spheroplasts and intact C. neoformans, as well as their corresponding membranes, phosphatidylethanol (PE)/phosphatidylglycerol (PG) and phosphatidylcholine (PC)/PE/phosphatidylinositol (PI)/ergosterol, respectively. The extent of membrane-permeating activity correlated with their biological function, suggesting that the plasma membrane was one of their major targets. Circular dichroism (CD) and attenuated total reflectance Fourier transform infrared (ATR-FTIR) spectroscopy revealed that their mode of oligomerization in solution, structure, and organization in membranes have important roles regarding their antibacterial and antifungal activities. Together with the advantage of using diastereomers versus all l-amino acid peptides, this study paves the way to the design of a new group of potent antifungal peptides urgently needed to combat opportunistic fungal infection.

Communications Biology, 2019

Autophosphorylation of receptor and non-receptor tyrosine kinases is a common molecular switch wi... more Autophosphorylation of receptor and non-receptor tyrosine kinases is a common molecular switch with broad implications for pathogeneses and therapy of cancer and other human diseases. Technologies for large-scale discovery and analysis of autophosphorylation are limited by the inherent difficulty to distinguish between phosphorylation and autophosphorylation in vivo and by the complexity associated with functional assays of receptors kinases in vitro. Here, we report a method for the direct detection and analysis of tyrosine autophosphorylation using integrated microfluidics and freshly synthesized protein arrays. We demonstrate the efficacy of our platform in detecting autophosphorylation activity of soluble and transmembrane tyrosine kinases, and the dependency of in vitro autophosphorylation assays on membranes. Our method, Integrated Microfluidics for Autophosphorylation Discovery (IMAD), is high-throughput, requires low reaction volumes and can be applied in basic and translational research settings. To our knowledge, it is the first demonstration of posttranslational modification analysis of membrane protein arrays.

Methods in Molecular Biology, 2016

Scientific Reports, 2016

Transcription factors (TFs) alter gene expression in response to changes in the environment throu... more Transcription factors (TFs) alter gene expression in response to changes in the environment through sequence-specific interactions with the DNA. These interactions are best portrayed as a landscape of TF binding affinities. Current methods to study sequence-specific binding preferences suffer from limited dynamic range, sequence bias, lack of specificity and limited throughput. We have developed a microfluidic-based device for SELEX Affinity Landscape MAPping (SELMAP) of TF binding, which allows high-throughput measurement of 16 proteins in parallel. We used it to measure the relative affinities of Pho4, AtERF2 and Btd full-length proteins to millions of different DNA binding sites, and detected both high and low-affinity interactions in equilibrium conditions, generating a comprehensive landscape of the relative TF affinities to all possible DNA 6-mers, and even DNA10-mers with increased sequencing depth. Low quantities of both the TFs and DNA oligomers were sufficient for obtaining high-quality results, significantly reducing experimental costs. SELMAP allows in-depth screening of hundreds of TFs, and provides a means for better understanding of the regulatory processes that govern gene expression. Transcription factors (TFs) are important components of gene regulatory networks. They alter gene expression in response to changes in the cellular environment 1. Gene expression is controlled by TFs and co-factors, through their sequence-specific interactions with DNA. The analysis of transcription factor binding to DNA is best portrayed as a landscape of both high-and low-affinity binding sites 2. Recently, technological advances have greatly increased our knowledge of the locations of TF binding sites within genomes and sequence-specific binding preferences for many TFs. These advances include both in vivo and in vitro experimental methods and the development of new methods of computational analysis 3–6. The most commonly used in vivo method for measuring TF-DNA interaction is chromatin immunoprecip-itation (ChIP) (ChIP-chip and ChIP-seq). These methods are used to study the interactions between specific proteins and genomic DNA sequences by identifying occupied genomic regions 7. In a ChIP experiment, the DNA-binding protein is crosslinked to DNA by treating cells with formaldehyde and shredding the chromatin by sonication into small fragments, generally in the 200–600 bp range. An antibody specific to the protein of interest is then used to immunoprecipitate (IP) the DNA-protein complex. Finally, the crosslinks are reversed and the released DNA is assayed to determine its sequences 8. In ChIP-chip the chromatin IP is combined with a DNA microarray, while in ChIP-seq the resulting DNA fragments are sequenced 3. Despite the tremendous value of ChIP methods, they have technical limitations. The analysis requires the genomic DNA to be sheared into sized fragments that enable sequencing or loading into a microarray chip. In addition, a substantial amount of unbound DNA is trapped in the precipitate and generates a nonspecific signal. In many of these experiments, a bias in selection toward GC-rich fragments is observed, both in library preparation and in amplification prior to sequencing. Moreover, the potential of TFs to cross-react with other DNA-binding proteins present in the system may lead to imprecision in specific sequence determination 7–9 .

Proceedings of the National Academy of Sciences, 2016

The discovery of how a pathogen invades a cell requires one to determine which host cell receptor... more The discovery of how a pathogen invades a cell requires one to determine which host cell receptors are exploited. This determination is a challenging problem because the receptor is invariably a membrane protein, which represents an Achilles heel in proteomics. We have developed a universal platform for high-throughput expression and interaction studies of membrane proteins by creating a microfluidic-based comprehensive human membrane protein array (MPA). The MPA is, to our knowledge, the first of its kind and offers a powerful alternative to conventional proteomics by enabling the simultaneous study of 2,100 membrane proteins. We characterized direct interactions of a whole nonenveloped virus (simian virus 40), as well as those of the hepatitis delta enveloped virus large form antigen, with candidate host receptors expressed on the MPA. Selected newly discovered membrane protein-pathogen interactions were validated by conventional methods, demonstrating that the MPA is an important tool for cellular receptor discovery and for understanding pathogen tropism.

Physiologia plantarum, Jan 29, 2016

To appropriately acclimate to environmental stresses, plants have to rapidly activate a specific ... more To appropriately acclimate to environmental stresses, plants have to rapidly activate a specific transcriptional program. Yet, the identity and function of many of the transcriptional regulators that mediate early responses to abiotic stress stimuli is still unknown. In this work we employed the promoter of the multi-stress-responsive zinc finger protein Zat12 in yeast one-hybrid (Y1H) screens to identify early abiotic stress-responsive transcriptional regulators. Analysis of Zat12 promoter fragments fused to luciferase underlined a ~200 bp fragment responsive to NaCl and to reactive oxygen species (ROS). Using these segments and others as baits against Y1H control or stress Arabidopsis prey libraries, we identified 15 potential Zat12 transcriptional regulators. Among the prominent proteins identified were known transcription factors including bZIP29 and ANAC91 as well as unknown function proteins such as a homolog of the human USB1, a U6 snRNA processing protein, and dormancy/auxin...

Nucleic Acids Research, 2015

Protein binding to DNA is a fundamental process in gene regulation. Methodologies such as ChIP-Se... more Protein binding to DNA is a fundamental process in gene regulation. Methodologies such as ChIP-Seq and mapping of DNase I hypersensitive sites provide global information on this regulation in vivo. In vitro methodologies provide valuable complementary information on protein-DNA specificities. However, current methods still do not measure absolute binding affinities. There is a real need for large-scale quantitative protein-DNA affinity measurements. We developed QPID, a microfluidic application for measuring protein-DNA affinities. A single run is equivalent to 4096 gel-shift experiments. Using QPID, we characterized the different affinities of ATF1, c-Jun, c-Fos and AP-1 to the CRE consensus motif and CRE half-site in two different genomic sequences on a single device. We discovered that binding of ATF1, but not of AP-1, to the CRE half-site is highly affected by its genomic context. This effect was highly correlated with ATF1 ChIP-seq and PBM experiments. Next, we characterized the affinities of ATF1 and ATF3 to 128 genomic CRE and CRE half-site sequences. Our affinity measurements explained that in vivo binding differences between ATF1 and ATF3 to CRE and CRE half-sites are partially mediated by differences in the minor groove width. We believe that QPID would become a central tool for quantitative characterization of biophysical aspects affecting protein-DNA binding.

Molecular & Cellular Proteomics, 2015

Protein post-translational modifications mediate dynamic cellular processes with broad implicatio... more Protein post-translational modifications mediate dynamic cellular processes with broad implications in human disease pathogenesis. There is a large demand for high-throughput technologies supporting post-translational modifications research, and both mass spectrometry and protein arrays have been successfully utilized for this purpose. Protein arrays override the major limitation of target protein abundance inherently associated with MS analysis. This technology, however, is typically restricted to pre-purified proteins spotted in a fixed composition on chips with limited life-time and functionality. In addition, the chips are expensive and designed for a single use, making complex experiments cost-prohibitive. Combining microfluidics with in situ protein expression from a cDNA microarray addressed these limitations. Based on this approach, we introduce a modular integrated microfluidic platform for multiple post-translational modifications analysis of freshly synthesized protein arrays (IMPA). The system&amp;amp;amp;amp;amp;amp;amp;amp;amp;#39;s potency, specificity and flexibility are demonstrated for tyrosine phosphorylation and ubiquitination in quasicellular environments. Unlimited by design and protein composition, and relying on minute amounts of biological material and cost-effective technology, this unique approach is applicable for a broad range of basic, biomedical and biomarker research.