Alva Biran | The Hebrew University of Jerusalem (original) (raw)

Papers by Alva Biran

Developmental Cell, 2021

Highlights d Integrative mRNA sequencing of single clones shows enhanced sensitivity d PEGDT-MALD... more Highlights d Integrative mRNA sequencing of single clones shows enhanced sensitivity d PEGDT-MALDEX hydrogel supports stemness in cancer cells and ESCs d CloneSeq identifies cancer stem-like subpopulation d Differentiation decisions of ESCs are maintained during clonal expansion

The Journal of cell biology, Feb 28, 2017

Neuronal stimulation leads to immediate early gene (IEG) expression through calcium-dependent mec... more Neuronal stimulation leads to immediate early gene (IEG) expression through calcium-dependent mechanisms. In recent years, considerable attention has been devoted to the transcriptional responses after neuronal stimulation, but relatively little is known about the changes in chromatin dynamics that follow neuronal activation. Here, we use fluorescence recovery after photobleaching, biochemical fractionations, and chromatin immunoprecipitation to show that KCl-induced depolarization in primary cultured cortical neurons causes a rapid release of the linker histone H1 from chromatin, concomitant with IEG expression. H1 release is repressed by PARP inhibition, PARP1 deletion, a non-PARylatable H1, as well as phosphorylation inhibitions and a nonphosphorylatable H1, leading to hindered IEG expression. Further, H1 is replaced by PARP1 on IEG promoters after neuronal stimulation, and PARP inhibition blocks this reciprocal binding response. Our results demonstrate the relationship between n...

Ever since the introduction of the Salmonella typhimuriummammalian microsome mutagenicity assay (... more Ever since the introduction of the Salmonella typhimuriummammalian microsome mutagenicity assay (the Ames test)over three decades ago, there has been a constant development of additional genotoxicity assays based upon the use of genetically engineered microorganisms. Such assays rely either on reversion principles similar to those of the Ames test or on promoter-reporter fusions that generate a quantifiable dose-dependent signal in the presence of potential DNA damaging compounds and the induction of repair mechanisms. While some of these assays were only briefly described in the scientific literature, others have been developed all the way to commercial products. Out of these, only one, the umu-testhas been fully validated and ISO- and OECD-standardized. Herein we review the main directions undertaken in the construction and testing of bacterial-based genotoxicity bioassays, including the attempts to incorporate at least a partial metabolic activation capacity into the molecular de...

Cell reports, Jan 31, 2015

Embryonic stem cells (ESCs) possess a distinct chromatin conformation maintained by specialized c... more Embryonic stem cells (ESCs) possess a distinct chromatin conformation maintained by specialized chromatin proteins. To identify chromatin regulators in ESCs, we developed a simple biochemical assay named D-CAP (differential chromatin-associated proteins), using brief micrococcal nuclease digestion of chromatin, followed by liquid chromatography tandem mass spectrometry (LC-MS/MS). Using D-CAP, we identified several differentially chromatin-associated proteins between undifferentiated and differentiated ESCs, including the chromatin remodeling protein SMARCD1. SMARCD1 depletion in ESCs led to altered chromatin and enhanced endodermal differentiation. Gene expression and chromatin immunoprecipitation sequencing (ChIP-seq) analyses suggested that SMARCD1 is both an activator and a repressor and is enriched at developmental regulators and that its chromatin binding coincides with H3K27me3. SMARCD1 knockdown caused H3K27me3 redistribution and increased H3K4me3 around the transcription st...

Current Pharmaceutical Biotechnology, 2010

Whole-cell bio-chips for functional sensing integrate living cells on miniaturized platforms made... more Whole-cell bio-chips for functional sensing integrate living cells on miniaturized platforms made by microsystem-technologies (MST). The cells are integrated, deposited or immersed in a media which is in contact with the chip. The cells behavior is monitored via electrical, electrochemical or optical methods. In this paper we describe such wholecell biochips where the signal is generated due to the genetic response of the cells. The solid-state platform hosts the biological component, i.e. the living cells, and integrates all the required micro-system technologies, i.e. the microelectronics, micro-electro optics, micro-electro or magneto mechanics and micro-fluidics. The genetic response of the cells expresses proteins that generate: a. light by photo-luminescence or bioluminescence, b. electrochemical signal by interaction with a substrate, or c. change in the cell impedance. The cell response is detected by a front end unit that converts it to current or voltage amplifies and filters it. The resultant signal is analyzed and stored for further processing. In this paper we describe three examples of whole-cell bio chips, photo-luminescent, bioluminescent and electrochemical, which are based on the genetic response of genetically modified E. coli microbes integrated on a micro-fluidics MEMS platform. We describe the chip outline as well as the basic modeling scheme of such sensors. We discuss the highlights and problems of such system, from the point of view of micro-system-technology.

Journal of molecular cell biology, 2014

This paper presents a whole-cell bio-chip system where viable, functioning cells are deposited on... more This paper presents a whole-cell bio-chip system where viable, functioning cells are deposited onto solid surfaces that are a part of a micro-machined system. The development of such novel hybrid functional sensors depends on the cell deposition methods; in this work new approach integrating live bacterial cells on a bio-chip using electrophoretic deposition is presented. The bio-material deposition technique was characterized under various driving potential and chamber configurations. The deposited bio-mass included genetically engineered bacterial cells generating electrochemically active byproduct upon exposure to toxic materials in the aqueous solution. In this paper we present the deposition apparatus and methods, as well as the characterization results, e.g. signal vs. time and induction factor, of such chips and discussing the highlight and problems of the new deposition method.

This work presents a novel micro-fluidic whole cell biosensor for water toxicity analysis. The bi... more This work presents a novel micro-fluidic whole cell biosensor for water toxicity analysis. The biosen- sor presented here is based on bacterial cells that are genetically “tailored” to generate a sequence of biochemical reactions that eventually generate an electrical signal in the presence of genotoxicants. The bacterial assay was affected by toxicant contaminated water for an induction time that ranged between 30 min and 120 min. Enzymatic substrate (pAPP) was added to the assay generating the electrochemi- cal active material (pAP) only when toxicants are sensed by the bacteria. The bacteria were integrated onto a micro-chip that was manufactured by MEMS technology and comprises various micro-chambers with volume ranging between 2.5 nl and 157 nl with electrode radius between 37.5 m and 300 m. We describe the biochip operation, its electrochemical response to calibration solutions as well as to the whole cell assays. The potential use of the whole cell biochip for toxicity detection of two different geno- toxicants, nalidixic acid (NA) and 2-amino-3-methylimidazo[4,5-f]quinoline (IQ), is demonstrated. We demonstrate minimal toxicant detection of 10 g/ml for NA using 30 min for induction and 0.31 M for IQ using 120 min for induction, both 3 min after the addition of the substrate material.

Electrochemical signal detection can be readily integrated in biosensors and is thus an attractiv... more Electrochemical signal detection can be readily integrated in biosensors and is thus an attractive alter- native to optical detection methods. In the field of environmental chemistry and ecotoxicology there is a growing demand for lab-independent devices based on whole cell biosensors for the detection of genotoxic compounds. Because of the broad occurrence of pre-genotoxic compounds that need to be bio-activated, the integration of a system for metabolic activation into such a biosensor is important. The present study evaluates a chrono-amperometric detection method in which para-aminophenyl -d- galactopyranoside is used as substrate for a reporter gene assay based on the bacterial SOS-response in comparison to a test system for the determination of genotoxicity in water that is standardized according to the International Organization for Standardization (ISO). The evaluation was done in order to analyze the potential of the electrochemical signal detection to be used as a complementary method for the stan- dard test system and thus to evaluate the usability of electrochemical biosensors for the assessment of genotoxicity of environmental samples. In the present study it is shown that the chrono-amperometric detection of para-aminophenol is specific even in the presence of electro-active species generated by the enzymatic system used for the external bio-activation of contaminants. Under optimized conditions electrochemistry is sufficiently sensitive with a limit of detection that is comparable to the respective ISO-standard.

Over the last few years, the physical dimensions of microchip devices have decreased, enhancing t... more Over the last few years, the physical dimensions of microchip devices have decreased, enhancing the interest in the integration of various devices and complex operations onto a compatible "lab on a chip" system with desirable characteristics and capabilities. This work presents a novel μ-fluidics whole cell biosensor for water toxicity analysis. The biosensor is based on bacterial cells genetically "tailored" to generate an electrochemical bio-signal in the presence of toxic materials. The μ-chip was electrochemically characterized, and demonstrated the potential toxicity analysis with a model toxicant. A novel concept of bacterial biosensors deposition by means of electrophoretic force was examined for the first time. Preliminary results demonstrated the ability to detect electrochemical signal generated by the deposited bacterial cells indicating the potential use for patterning of bacterial cells on solid-state surfaces for the use in bio-sensing.

Journal of molecular cell biology, 2011

Embryonic stem cells (ESCs) exhibit unique chromatin features, including a permissive transcripti... more Embryonic stem cells (ESCs) exhibit unique chromatin features, including a permissive transcriptional program and an open, decondensed chromatin state. Induced pluripotent stem cells (iPSCs), which are very similar to ESCs, hold great promise for therapy and basic research. However, the mechanisms by which reprogramming occurs and the chromatin organization that underlies the reprogramming process are largely unknown. Here we characterize and compare the epigenetic landscapes of partially and fully reprogrammed iPSCs to mouse embryonic fibroblasts (MEFs) and ESCs, which serves as a standard for pluripotency. Using immunofluorescence and biochemical fractionations, we analyzed the levels and distribution of a battery of histone modifications (H3ac, H4ac, H4K5ac, H3K9ac, H3K27ac, H3K4me3, H3K36me2, H3K9me3, H3K27me3, and γH2AX), as well as HP1α and lamin A. We find that fully reprogrammed iPSCs are epigenetically identical to ESCs, and that partially reprogrammed iPSCs are closer to M...

Current Pharmaceutical Biotechnology, 2010

Whole-cell bio-chips for functional sensing integrate living cells on miniaturized platforms made... more Whole-cell bio-chips for functional sensing integrate living cells on miniaturized platforms made by microsystem-technologies (MST). The cells are integrated, deposited or immersed in a media which is in contact with the chip. The cells behavior is monitored via electrical, electrochemical or optical methods. In this paper we describe such wholecell biochips where the signal is generated due to the genetic response of the cells. The solid-state platform hosts the biological component, i.e. the living cells, and integrates all the required micro-system technologies, i.e. the microelectronics, micro-electro optics, micro-electro or magneto mechanics and micro-fluidics. The genetic response of the cells expresses proteins that generate: a. light by photo-luminescence or bioluminescence, b. electrochemical signal by interaction with a substrate, or c. change in the cell impedance. The cell response is detected by a front end unit that converts it to current or voltage amplifies and filters it. The resultant signal is analyzed and stored for further processing. In this paper we describe three examples of whole-cell bio chips, photo-luminescent, bioluminescent and electrochemical, which are based on the genetic response of genetically modified E. coli microbes integrated on a micro-fluidics MEMS platform. We describe the chip outline as well as the basic modeling scheme of such sensors. We discuss the highlights and problems of such system, from the point of view of micro-system-technology.

Cell Reports, Mar 31, 2015

Alajem et al. develop an assay that indicates differential association of SMARCD1 with chromatin ... more Alajem et al. develop an assay that indicates differential association of SMARCD1 with chromatin in embryonic stem cells (ESCs) and early differentiating cells. SMARCD1 is associated with bivalent genes in ESCs, regulates H3K4me3/H3K27me3 distribution, and binds and regulates the pluripotency factor Klf4.

Cell reports, Jan 31, 2015

Embryonic stem cells (ESCs) possess a distinct chromatin conformation maintained by specialized c... more Embryonic stem cells (ESCs) possess a distinct chromatin conformation maintained by specialized chromatin proteins. To identify chromatin regulators in ESCs, we developed a simple biochemical assay named D-CAP (differential chromatin-associated proteins), using brief micrococcal nuclease digestion of chromatin, followed by liquid chromatography tandem mass spectrometry (LC-MS/MS). Using D-CAP, we identified several differentially chromatin-associated proteins between undifferentiated and differentiated ESCs, including the chromatin remodeling protein SMARCD1. SMARCD1 depletion in ESCs led to altered chromatin and enhanced endodermal differentiation. Gene expression and chromatin immunoprecipitation sequencing (ChIP-seq) analyses suggested that SMARCD1 is both an activator and a repressor and is enriched at developmental regulators and that its chromatin binding coincides with H3K27me3. SMARCD1 knockdown caused H3K27me3 redistribution and increased H3K4me3 around the transcription st...

Journal of molecular cell biology, 2014

Cell-based toxicity bioassays harbor the potential for efficient detection and monitoring of haza... more Cell-based toxicity bioassays harbor the potential for efficient detection and monitoring of hazardous materials. However, their use in the field has been limited by harsh and unstable environmental condi- tions that shorten shelf-life, introduce significant noise, and reduce the signal and signal-to-noise ratio; such conditions may thus decrease the probability of correct decisions, increasing both false positive and false negative outcomes. Therefore, there is a need for a stable cell-on-chip integration that offers long- term storage and resilience to environmental factors. The use of intact microbial biofilms as biological elements in a whole-cell biosensor, and their integration into specialized biochips, holds promise for enhancing sensor stability as well as providing an innovative platform for biofilm research. We report here for the first time on the integration of a bacterial biofilm as the sensing element of a whole-cell biosensor, as a means to stabilize and preserve reproducibility, viability and functionality of the bacte- rial sensor cells. We have employed a genetically engineered Escherichia coli sensor strain, tailored to respond to the presence of genotoxic (DNA damaging) agents by the induction of a reporter enzyme, alkaline phosphatase, and tested its functionality in colorimetric and electrochemical assays. Three dif- ferent bacterial integration forms were examined: planktonic cells, electronically deposited sessile cells, and biofilms. For all integration forms, a clear dose-dependent positive response to the presence of the model toxicant nalidixic acid was observed, with biofilms displaying higher current density and detec- tion sensitivity than planktonic and sessile cells. We present the electrode apparatus and methods and biochip characterization of such chips, e.g. signal vs. time and induction factor, and discuss the advantage and potential problems of the new biofilm-biochip technology.

Ever since the introduction of the Salmonella typhi- murium mammalian microsome mutagenicity assa... more Ever since the introduction of the Salmonella typhi- murium mammalian microsome mutagenicity assay (the ‘Ames test’) over three decades ago, there has been a constant development of additional genotox- icity assays based upon the use of genetically engi- neered microorganisms. Such assays rely either on reversion principles similar to those of the Ames test, or on promoter–reporter fusions that generate a quantifiable dose-dependent signal in the presence of potential DNA damaging compounds and the induc- tion of repair mechanisms; the latter group is the subject of the present review. Some of these assays were only briefly described in the scientific literature, whereas others have been developed all the way to commercial products. Out of these, only one, the umu-test, has been fully validated and ISO- and OECD standardized. Here we review the main directions undertaken in the construction and testing of bacterial-based genotoxicity bioassays, including the attempts to incorporate at least a partial metabolic activation capacity into the molecular design. We list the genetic modifications introduced into the tester strains, compare the performance of the different assays, and briefly describe the first attempts to incorporate such bacterial reporters into actual geno- toxicity testing devices.

Electrochimica Acta, 2009

This work presents a novel micro-fluidic whole cell biosensor for water toxicity analysis. The bi... more This work presents a novel micro-fluidic whole cell biosensor for water toxicity analysis. The biosensor presented here is based on bacterial cells that are genetically "tailored" to generate a sequence of biochemical reactions that eventually generate an electrical signal in the presence of genotoxicants. The bacterial assay was affected by toxicant contaminated water for an induction time that ranged between 30 min and 120 min. Enzymatic substrate (pAPP) was added to the assay generating the electrochemical active material (pAP) only when toxicants are sensed by the bacteria. The bacteria were integrated onto a micro-chip that was manufactured by MEMS technology and comprises various micro-chambers with volume ranging between 2.5 nl and 157 nl with electrode radius between 37.5 m and 300 m. We describe the biochip operation, its electrochemical response to calibration solutions as well as to the whole cell assays. The potential use of the whole cell biochip for toxicity detection of two different genotoxicants, nalidixic acid (NA) and 2-amino-3-methylimidazo[4,5-f]quinoline (IQ), is demonstrated. We demonstrate minimal toxicant detection of 10 g/ml for NA using 30 min for induction and 0.31 M for IQ using 120 min for induction, both 3 min after the addition of the substrate material.

A bacterial genotoxicity reporter strain was con- structed in which the tightly controlled strong... more A bacterial genotoxicity reporter strain was con- structed in which the tightly controlled strong promoter of the Escherichia coli SOS response gene sulA was fused to the alkaline phosphatase-coding phoA reporter gene. The bio- reporter responded in a dose-dependent manner to three model DNA-damaging agents—hydrogen peroxide, nalidixic acid (NA), and mitomycin C (MMC)—detected 30–60 min after exposure. Detection thresholds were 0.15 μM for MMC, 7.5 μM for nalidixic acid, and approximately 50 μM for hydrogen peroxide. A similar response to NA was observed when the bioreporter was integrated into a specially designed, portable electrochemical detection platform. Reporter sensi- tivity was further enhanced by single and double knockout mutations that enhanced cell membrane permeability (rfaE) and inhibited DNA damage repair mechanisms (umuD, uvrA). The rfaE mutants displayed a five- and tenfold increase in sensitivity to MMC and NA, respectively, while the uvrA mutation was advantageous in the detection of hydrogen peroxide. A similar sensitivity was displayed by the double rfaE/uvrA mutant when challenged with the pre-genotoxic agents 2-amino-3-methylimidazo[4,5-f]quinoline and 2- aminoanthracene following metabolic activation with an S9 mammalian liver fraction.

Developmental Cell, 2021

Highlights d Integrative mRNA sequencing of single clones shows enhanced sensitivity d PEGDT-MALD... more Highlights d Integrative mRNA sequencing of single clones shows enhanced sensitivity d PEGDT-MALDEX hydrogel supports stemness in cancer cells and ESCs d CloneSeq identifies cancer stem-like subpopulation d Differentiation decisions of ESCs are maintained during clonal expansion

The Journal of cell biology, Feb 28, 2017

Neuronal stimulation leads to immediate early gene (IEG) expression through calcium-dependent mec... more Neuronal stimulation leads to immediate early gene (IEG) expression through calcium-dependent mechanisms. In recent years, considerable attention has been devoted to the transcriptional responses after neuronal stimulation, but relatively little is known about the changes in chromatin dynamics that follow neuronal activation. Here, we use fluorescence recovery after photobleaching, biochemical fractionations, and chromatin immunoprecipitation to show that KCl-induced depolarization in primary cultured cortical neurons causes a rapid release of the linker histone H1 from chromatin, concomitant with IEG expression. H1 release is repressed by PARP inhibition, PARP1 deletion, a non-PARylatable H1, as well as phosphorylation inhibitions and a nonphosphorylatable H1, leading to hindered IEG expression. Further, H1 is replaced by PARP1 on IEG promoters after neuronal stimulation, and PARP inhibition blocks this reciprocal binding response. Our results demonstrate the relationship between n...

Ever since the introduction of the Salmonella typhimuriummammalian microsome mutagenicity assay (... more Ever since the introduction of the Salmonella typhimuriummammalian microsome mutagenicity assay (the Ames test)over three decades ago, there has been a constant development of additional genotoxicity assays based upon the use of genetically engineered microorganisms. Such assays rely either on reversion principles similar to those of the Ames test or on promoter-reporter fusions that generate a quantifiable dose-dependent signal in the presence of potential DNA damaging compounds and the induction of repair mechanisms. While some of these assays were only briefly described in the scientific literature, others have been developed all the way to commercial products. Out of these, only one, the umu-testhas been fully validated and ISO- and OECD-standardized. Herein we review the main directions undertaken in the construction and testing of bacterial-based genotoxicity bioassays, including the attempts to incorporate at least a partial metabolic activation capacity into the molecular de...

Cell reports, Jan 31, 2015

Embryonic stem cells (ESCs) possess a distinct chromatin conformation maintained by specialized c... more Embryonic stem cells (ESCs) possess a distinct chromatin conformation maintained by specialized chromatin proteins. To identify chromatin regulators in ESCs, we developed a simple biochemical assay named D-CAP (differential chromatin-associated proteins), using brief micrococcal nuclease digestion of chromatin, followed by liquid chromatography tandem mass spectrometry (LC-MS/MS). Using D-CAP, we identified several differentially chromatin-associated proteins between undifferentiated and differentiated ESCs, including the chromatin remodeling protein SMARCD1. SMARCD1 depletion in ESCs led to altered chromatin and enhanced endodermal differentiation. Gene expression and chromatin immunoprecipitation sequencing (ChIP-seq) analyses suggested that SMARCD1 is both an activator and a repressor and is enriched at developmental regulators and that its chromatin binding coincides with H3K27me3. SMARCD1 knockdown caused H3K27me3 redistribution and increased H3K4me3 around the transcription st...

Current Pharmaceutical Biotechnology, 2010

Whole-cell bio-chips for functional sensing integrate living cells on miniaturized platforms made... more Whole-cell bio-chips for functional sensing integrate living cells on miniaturized platforms made by microsystem-technologies (MST). The cells are integrated, deposited or immersed in a media which is in contact with the chip. The cells behavior is monitored via electrical, electrochemical or optical methods. In this paper we describe such wholecell biochips where the signal is generated due to the genetic response of the cells. The solid-state platform hosts the biological component, i.e. the living cells, and integrates all the required micro-system technologies, i.e. the microelectronics, micro-electro optics, micro-electro or magneto mechanics and micro-fluidics. The genetic response of the cells expresses proteins that generate: a. light by photo-luminescence or bioluminescence, b. electrochemical signal by interaction with a substrate, or c. change in the cell impedance. The cell response is detected by a front end unit that converts it to current or voltage amplifies and filters it. The resultant signal is analyzed and stored for further processing. In this paper we describe three examples of whole-cell bio chips, photo-luminescent, bioluminescent and electrochemical, which are based on the genetic response of genetically modified E. coli microbes integrated on a micro-fluidics MEMS platform. We describe the chip outline as well as the basic modeling scheme of such sensors. We discuss the highlights and problems of such system, from the point of view of micro-system-technology.

Journal of molecular cell biology, 2014

This paper presents a whole-cell bio-chip system where viable, functioning cells are deposited on... more This paper presents a whole-cell bio-chip system where viable, functioning cells are deposited onto solid surfaces that are a part of a micro-machined system. The development of such novel hybrid functional sensors depends on the cell deposition methods; in this work new approach integrating live bacterial cells on a bio-chip using electrophoretic deposition is presented. The bio-material deposition technique was characterized under various driving potential and chamber configurations. The deposited bio-mass included genetically engineered bacterial cells generating electrochemically active byproduct upon exposure to toxic materials in the aqueous solution. In this paper we present the deposition apparatus and methods, as well as the characterization results, e.g. signal vs. time and induction factor, of such chips and discussing the highlight and problems of the new deposition method.

This work presents a novel micro-fluidic whole cell biosensor for water toxicity analysis. The bi... more This work presents a novel micro-fluidic whole cell biosensor for water toxicity analysis. The biosen- sor presented here is based on bacterial cells that are genetically “tailored” to generate a sequence of biochemical reactions that eventually generate an electrical signal in the presence of genotoxicants. The bacterial assay was affected by toxicant contaminated water for an induction time that ranged between 30 min and 120 min. Enzymatic substrate (pAPP) was added to the assay generating the electrochemi- cal active material (pAP) only when toxicants are sensed by the bacteria. The bacteria were integrated onto a micro-chip that was manufactured by MEMS technology and comprises various micro-chambers with volume ranging between 2.5 nl and 157 nl with electrode radius between 37.5 m and 300 m. We describe the biochip operation, its electrochemical response to calibration solutions as well as to the whole cell assays. The potential use of the whole cell biochip for toxicity detection of two different geno- toxicants, nalidixic acid (NA) and 2-amino-3-methylimidazo[4,5-f]quinoline (IQ), is demonstrated. We demonstrate minimal toxicant detection of 10 g/ml for NA using 30 min for induction and 0.31 M for IQ using 120 min for induction, both 3 min after the addition of the substrate material.

Electrochemical signal detection can be readily integrated in biosensors and is thus an attractiv... more Electrochemical signal detection can be readily integrated in biosensors and is thus an attractive alter- native to optical detection methods. In the field of environmental chemistry and ecotoxicology there is a growing demand for lab-independent devices based on whole cell biosensors for the detection of genotoxic compounds. Because of the broad occurrence of pre-genotoxic compounds that need to be bio-activated, the integration of a system for metabolic activation into such a biosensor is important. The present study evaluates a chrono-amperometric detection method in which para-aminophenyl -d- galactopyranoside is used as substrate for a reporter gene assay based on the bacterial SOS-response in comparison to a test system for the determination of genotoxicity in water that is standardized according to the International Organization for Standardization (ISO). The evaluation was done in order to analyze the potential of the electrochemical signal detection to be used as a complementary method for the stan- dard test system and thus to evaluate the usability of electrochemical biosensors for the assessment of genotoxicity of environmental samples. In the present study it is shown that the chrono-amperometric detection of para-aminophenol is specific even in the presence of electro-active species generated by the enzymatic system used for the external bio-activation of contaminants. Under optimized conditions electrochemistry is sufficiently sensitive with a limit of detection that is comparable to the respective ISO-standard.

Over the last few years, the physical dimensions of microchip devices have decreased, enhancing t... more Over the last few years, the physical dimensions of microchip devices have decreased, enhancing the interest in the integration of various devices and complex operations onto a compatible "lab on a chip" system with desirable characteristics and capabilities. This work presents a novel μ-fluidics whole cell biosensor for water toxicity analysis. The biosensor is based on bacterial cells genetically "tailored" to generate an electrochemical bio-signal in the presence of toxic materials. The μ-chip was electrochemically characterized, and demonstrated the potential toxicity analysis with a model toxicant. A novel concept of bacterial biosensors deposition by means of electrophoretic force was examined for the first time. Preliminary results demonstrated the ability to detect electrochemical signal generated by the deposited bacterial cells indicating the potential use for patterning of bacterial cells on solid-state surfaces for the use in bio-sensing.

Journal of molecular cell biology, 2011

Embryonic stem cells (ESCs) exhibit unique chromatin features, including a permissive transcripti... more Embryonic stem cells (ESCs) exhibit unique chromatin features, including a permissive transcriptional program and an open, decondensed chromatin state. Induced pluripotent stem cells (iPSCs), which are very similar to ESCs, hold great promise for therapy and basic research. However, the mechanisms by which reprogramming occurs and the chromatin organization that underlies the reprogramming process are largely unknown. Here we characterize and compare the epigenetic landscapes of partially and fully reprogrammed iPSCs to mouse embryonic fibroblasts (MEFs) and ESCs, which serves as a standard for pluripotency. Using immunofluorescence and biochemical fractionations, we analyzed the levels and distribution of a battery of histone modifications (H3ac, H4ac, H4K5ac, H3K9ac, H3K27ac, H3K4me3, H3K36me2, H3K9me3, H3K27me3, and γH2AX), as well as HP1α and lamin A. We find that fully reprogrammed iPSCs are epigenetically identical to ESCs, and that partially reprogrammed iPSCs are closer to M...

Current Pharmaceutical Biotechnology, 2010

Whole-cell bio-chips for functional sensing integrate living cells on miniaturized platforms made... more Whole-cell bio-chips for functional sensing integrate living cells on miniaturized platforms made by microsystem-technologies (MST). The cells are integrated, deposited or immersed in a media which is in contact with the chip. The cells behavior is monitored via electrical, electrochemical or optical methods. In this paper we describe such wholecell biochips where the signal is generated due to the genetic response of the cells. The solid-state platform hosts the biological component, i.e. the living cells, and integrates all the required micro-system technologies, i.e. the microelectronics, micro-electro optics, micro-electro or magneto mechanics and micro-fluidics. The genetic response of the cells expresses proteins that generate: a. light by photo-luminescence or bioluminescence, b. electrochemical signal by interaction with a substrate, or c. change in the cell impedance. The cell response is detected by a front end unit that converts it to current or voltage amplifies and filters it. The resultant signal is analyzed and stored for further processing. In this paper we describe three examples of whole-cell bio chips, photo-luminescent, bioluminescent and electrochemical, which are based on the genetic response of genetically modified E. coli microbes integrated on a micro-fluidics MEMS platform. We describe the chip outline as well as the basic modeling scheme of such sensors. We discuss the highlights and problems of such system, from the point of view of micro-system-technology.

Cell Reports, Mar 31, 2015

Alajem et al. develop an assay that indicates differential association of SMARCD1 with chromatin ... more Alajem et al. develop an assay that indicates differential association of SMARCD1 with chromatin in embryonic stem cells (ESCs) and early differentiating cells. SMARCD1 is associated with bivalent genes in ESCs, regulates H3K4me3/H3K27me3 distribution, and binds and regulates the pluripotency factor Klf4.

Cell reports, Jan 31, 2015

Embryonic stem cells (ESCs) possess a distinct chromatin conformation maintained by specialized c... more Embryonic stem cells (ESCs) possess a distinct chromatin conformation maintained by specialized chromatin proteins. To identify chromatin regulators in ESCs, we developed a simple biochemical assay named D-CAP (differential chromatin-associated proteins), using brief micrococcal nuclease digestion of chromatin, followed by liquid chromatography tandem mass spectrometry (LC-MS/MS). Using D-CAP, we identified several differentially chromatin-associated proteins between undifferentiated and differentiated ESCs, including the chromatin remodeling protein SMARCD1. SMARCD1 depletion in ESCs led to altered chromatin and enhanced endodermal differentiation. Gene expression and chromatin immunoprecipitation sequencing (ChIP-seq) analyses suggested that SMARCD1 is both an activator and a repressor and is enriched at developmental regulators and that its chromatin binding coincides with H3K27me3. SMARCD1 knockdown caused H3K27me3 redistribution and increased H3K4me3 around the transcription st...

Journal of molecular cell biology, 2014

Cell-based toxicity bioassays harbor the potential for efficient detection and monitoring of haza... more Cell-based toxicity bioassays harbor the potential for efficient detection and monitoring of hazardous materials. However, their use in the field has been limited by harsh and unstable environmental condi- tions that shorten shelf-life, introduce significant noise, and reduce the signal and signal-to-noise ratio; such conditions may thus decrease the probability of correct decisions, increasing both false positive and false negative outcomes. Therefore, there is a need for a stable cell-on-chip integration that offers long- term storage and resilience to environmental factors. The use of intact microbial biofilms as biological elements in a whole-cell biosensor, and their integration into specialized biochips, holds promise for enhancing sensor stability as well as providing an innovative platform for biofilm research. We report here for the first time on the integration of a bacterial biofilm as the sensing element of a whole-cell biosensor, as a means to stabilize and preserve reproducibility, viability and functionality of the bacte- rial sensor cells. We have employed a genetically engineered Escherichia coli sensor strain, tailored to respond to the presence of genotoxic (DNA damaging) agents by the induction of a reporter enzyme, alkaline phosphatase, and tested its functionality in colorimetric and electrochemical assays. Three dif- ferent bacterial integration forms were examined: planktonic cells, electronically deposited sessile cells, and biofilms. For all integration forms, a clear dose-dependent positive response to the presence of the model toxicant nalidixic acid was observed, with biofilms displaying higher current density and detec- tion sensitivity than planktonic and sessile cells. We present the electrode apparatus and methods and biochip characterization of such chips, e.g. signal vs. time and induction factor, and discuss the advantage and potential problems of the new biofilm-biochip technology.

Ever since the introduction of the Salmonella typhi- murium mammalian microsome mutagenicity assa... more Ever since the introduction of the Salmonella typhi- murium mammalian microsome mutagenicity assay (the ‘Ames test’) over three decades ago, there has been a constant development of additional genotox- icity assays based upon the use of genetically engi- neered microorganisms. Such assays rely either on reversion principles similar to those of the Ames test, or on promoter–reporter fusions that generate a quantifiable dose-dependent signal in the presence of potential DNA damaging compounds and the induc- tion of repair mechanisms; the latter group is the subject of the present review. Some of these assays were only briefly described in the scientific literature, whereas others have been developed all the way to commercial products. Out of these, only one, the umu-test, has been fully validated and ISO- and OECD standardized. Here we review the main directions undertaken in the construction and testing of bacterial-based genotoxicity bioassays, including the attempts to incorporate at least a partial metabolic activation capacity into the molecular design. We list the genetic modifications introduced into the tester strains, compare the performance of the different assays, and briefly describe the first attempts to incorporate such bacterial reporters into actual geno- toxicity testing devices.

Electrochimica Acta, 2009

This work presents a novel micro-fluidic whole cell biosensor for water toxicity analysis. The bi... more This work presents a novel micro-fluidic whole cell biosensor for water toxicity analysis. The biosensor presented here is based on bacterial cells that are genetically "tailored" to generate a sequence of biochemical reactions that eventually generate an electrical signal in the presence of genotoxicants. The bacterial assay was affected by toxicant contaminated water for an induction time that ranged between 30 min and 120 min. Enzymatic substrate (pAPP) was added to the assay generating the electrochemical active material (pAP) only when toxicants are sensed by the bacteria. The bacteria were integrated onto a micro-chip that was manufactured by MEMS technology and comprises various micro-chambers with volume ranging between 2.5 nl and 157 nl with electrode radius between 37.5 m and 300 m. We describe the biochip operation, its electrochemical response to calibration solutions as well as to the whole cell assays. The potential use of the whole cell biochip for toxicity detection of two different genotoxicants, nalidixic acid (NA) and 2-amino-3-methylimidazo[4,5-f]quinoline (IQ), is demonstrated. We demonstrate minimal toxicant detection of 10 g/ml for NA using 30 min for induction and 0.31 M for IQ using 120 min for induction, both 3 min after the addition of the substrate material.

A bacterial genotoxicity reporter strain was con- structed in which the tightly controlled strong... more A bacterial genotoxicity reporter strain was con- structed in which the tightly controlled strong promoter of the Escherichia coli SOS response gene sulA was fused to the alkaline phosphatase-coding phoA reporter gene. The bio- reporter responded in a dose-dependent manner to three model DNA-damaging agents—hydrogen peroxide, nalidixic acid (NA), and mitomycin C (MMC)—detected 30–60 min after exposure. Detection thresholds were 0.15 μM for MMC, 7.5 μM for nalidixic acid, and approximately 50 μM for hydrogen peroxide. A similar response to NA was observed when the bioreporter was integrated into a specially designed, portable electrochemical detection platform. Reporter sensi- tivity was further enhanced by single and double knockout mutations that enhanced cell membrane permeability (rfaE) and inhibited DNA damage repair mechanisms (umuD, uvrA). The rfaE mutants displayed a five- and tenfold increase in sensitivity to MMC and NA, respectively, while the uvrA mutation was advantageous in the detection of hydrogen peroxide. A similar sensitivity was displayed by the double rfaE/uvrA mutant when challenged with the pre-genotoxic agents 2-amino-3-methylimidazo[4,5-f]quinoline and 2- aminoanthracene following metabolic activation with an S9 mammalian liver fraction.