Lukasz Krzeminski | Cornell University (original) (raw)
Books by Lukasz Krzeminski
This thesis starts from a general idea of a biosensors up to commercially successful blood glucos... more This thesis starts from a general idea of a biosensors up to commercially successful blood glucose sensors that one can buy in almost every pharmacy. In the first chapter we can find what actually a biosensor is, parameters characterizing the analytical device, biosensors’ classification according to the different biological recognition elements as well as the physicochemical transducers and a review of methods used for the biomolecules immobilization on a transducer surface. The second chapter contains the electrochemistry background needed for understanding the working principle of amperometric biosensors, a simple Michaelis–Menten enzyme catalysis theory and finally a kinetic model of a amperometric biosensor considered for two boundary assumptions: membrane/enzyme/electrode and enzymatic biofilm/electrode. In the chapter three there is a description of three generations of amperometric glucose sensors, their comparison, a progress that has been made since the very beginning of a biosensor in late 70s and the a short list of the commercially available electrochemical blood glucose sensors. The fourth chapter was dedicated on an amperometric biosensor applications and gives us a feeling about other electrochemical sensors and research that is being done on them nowadays, in the labs all over the world.
Papers by Lukasz Krzeminski
Proceedings of the National Academy of Sciences, 2020
Metal detoxification is essential for bacteria’s survival in adverse environments and their patho... more Metal detoxification is essential for bacteria’s survival in adverse environments and their pathogenesis in hosts. Understanding the underlying mechanisms is crucial for devising antibacterial treatments. In the Gram-negative bacterium Escherichia coli , membrane-bound sensor CusS and its response regulator CusR together regulate the transcription of the cus operon that plays important roles in cells’ resistance to copper/silver, and they belong to the two-component systems (TCSs) that are ubiquitous across various organisms and regulate diverse cellular functions. In vitro protein reconstitution and associated biochemical/physical studies have provided significant insights into the functions and mechanisms of CusS–CusR and related TCSs. Such studies are challenging regarding multidomain membrane proteins like CusS and also lack the physiological environment, particularly the native spatial context of proteins inside a cell. Here, we use stroboscopic single-molecule imaging and trac...
Physical forces have long been recognized for their effects on the growth, morphology, locomotion... more Physical forces have long been recognized for their effects on the growth, morphology, locomotion, and survival of eukaryotic organisms. Recently, mechanical forces have been shown to regulate processes in bacteria, including cell division, motility, virulence, biofilm initiation, and cell shape, although it remains unclear how mechanical forces in the cell envelope lead to changes in molecular processes. In Gram-negative bacteria, multicomponent protein complexes that form rigid links across the cell envelope directly experience physical forces and mechanical stresses applied to the cell. Here we manipulate tensile and shear mechanical stress in the bacterial cell envelope and use single-molecule tracking to show that shear (but not tensile) stress within the cell envelope promotes disassembly of the tripartite efflux complex CusCBA, a system used by E. coli to resist copper and silver toxicity, thereby making bacteria more susceptible to metal toxicity. These findings provide the ...
Journal of Inflammation Research
Sarcoidosis is a systemic disease of unknown etiology characterized by granuloma formation in the... more Sarcoidosis is a systemic disease of unknown etiology characterized by granuloma formation in the affected tissues. The pathologically activated macrophages are causatively implicated in disease pathogenesis and play important role in granuloma formation. Chitotriosidase (CHIT1), macrophage-derived protein, is upregulated in sarcoidosis and its levels correlate with disease severity implicating CHIT1 in pathology. Methods: CHIT1 was evaluated in serum and bronchial mucosa and mediastinal lymph nodes specimens from sarcoidosis patients. The therapeutic efficacy of OATD-01 was assessed ex vivo on human bronchoalveolar lavage fluid (BALF) macrophages and in vivo in the murine models of granulomatous inflammation. Results: CHIT1 activity was significantly upregulated in serum from sarcoidosis patients. CHIT1 expression was restricted to granulomas and localized in macrophages. Ex vivo OATD-01 inhibited pro-inflammatory mediators' production (CCL4, IL-15) by lung macrophages. In the acute model of granulomatous inflammation in mice, OATD-01 showed anti-inflammatory effects reducing the percentage of neutrophils and CCL4 concentration in BALF. In the chronic model, inhibition of CHIT1 led to a decrease in the number of organized lung granulomas and the expression of sarcoidosis-associated genes. Conclusion: In summary, CHIT1 activity was increased in sarcoidosis patients and OATD-01, a first-in-class CHIT1 inhibitor, demonstrated efficacy in murine models of granulomatous inflammation providing a proof-of-concept for its clinical evaluation in sarcoidosis.
The Journal of Physical Chemistry B, 2011
Copper-containing nitrite reductases (NiRs) are enzymes that efficiently reduce nitrite to nitric... more Copper-containing nitrite reductases (NiRs) are enzymes that efficiently reduce nitrite to nitric oxide in potent denitrifying bacteria. There has been an interest in their application in amperometric biosensors for monitoring nitrite levels in natural and waste waters. NiRs have a complex enzyme mechanism and depend on nitrite concentration and pH. Although the mechanism has been intensively studied, it is still controversial. In this thesis, a combined fluorescence and electrochemical method is used to simultaneously monitor the nitrite turn-over rate of a NiR from Alcaligenes faecalis S-6 and the oxidation state of the type-1 copper electron transfer site inside the enzyme. The catalytic activity of NiR is measured electrochemically by exploiting a direct electron transfer to fluorescently labelled enzyme molecules immobilised on modified gold, whereas the redox state of the type-1 copper site is determined from fluorescence intensity changes caused by F6rster resonance energy transfer (FRET) between a fluorophore attached to NiR and its type-1 copper site. Here, a determining role of internal electron transfer is found in NiR's mechanism. Moreover, the heterogeneous interfacial electron transfer to adsorbed NiRs is observed. The electro- activity and binding of labelled and unlabelled NiRs on gold modified with different self-assembled monolayers (SAMs) are studied to understand the effect of NiR labelling on the protein-electrode interactions. Here, electro-active and well-ordered biofilms of NiRs are found on electrodes with SAMs carrying the positive charge (negative NiRs) or when NiRs are modified with fluorophores that help orienting them on SAM-modified gold. Attempts have also been made to implement fluorescently labelled NiRs into the biosensor-like device with a fluorescence output using gold electrodes modified with tethered lipid bilayers (tBLMs) or conducting polymers (CPs). In spite of being unsuccessful, these studies give a better understanding of potential-dependent polymer dynamics and a control over protein immobilisation on functionalised lipid bilayer platforms.EThOS - Electronic Theses Online ServiceGBUnited Kingdo
Journal of the American Chemical Society, 2011
Membrane proteins are key components of the plasma membrane and are responsible for control of ch... more Membrane proteins are key components of the plasma membrane and are responsible for control of chemical ionic gradients, metabolite and nutrient transfer, and signal transduction between the interior of cells and the external environment. Of the genes in the human genome, 30% code for membrane proteins (Krogh et al. J. Mol. Biol. 2001, 305, 567). Furthermore, many FDA-approved drugs target such proteins (Overington et al. Nat. Rev. Drug Discovery 2006, 5, 993). However, the structureÀfunction relationships of these are notably sparse because of difficulties in their purification and handling outside of their membranous environment. Methods that permit the manipulation of membrane components while they are still in the membrane would find widespread application in separation, purification, and eventual structureÀfunction determination of these species (Poo et al. Nature 1977, 265, 602). Here we show that asymmetrically patterned supported lipid bilayers in combination with AC electric fields can lead to efficient manipulation of charged components. We demonstrate the concentration and trapping of such components through the use of a "nested trap" and show that this method is capable of yielding an approximately 30-fold increase in the average protein concentration. Upon removal of the field, the material remains trapped for several hours as a result of topographically restricted diffusion. Our results indicate that this method can be used for concentrating and trapping charged membrane components while they are still within their membranous environment. We anticipate that our approach could find widespread application in the manipulation and study of membrane proteins.
Plant Growth Regulation, 2007
Ascorbate levels and redox states, as well as the activities of the enzymes of ascorbate metaboli... more Ascorbate levels and redox states, as well as the activities of the enzymes of ascorbate metabolism, were analyzed in roots of tomato seedlings during the culture on a medium supplemented with auxin and compared to the control cultured on an auxin-free medium. Biochemical parameters were determined separately in the distal part of the root where the inhibitory effect of auxin
Journal of the American Chemical Society, 2011
Particle and fibre toxicology, Jan 23, 2012
Mechanisms for cellular uptake of nanoparticles have important implications for nanoparticulate d... more Mechanisms for cellular uptake of nanoparticles have important implications for nanoparticulate drug delivery and toxicity. We have explored the mechanism of uptake of amorphous silica nanoparticles of 14 nm diameter, which agglomerate in culture medium to hydrodynamic diameters around 500 nm. In HT29, HaCat and A549 cells, cytotoxicity was observed at nanoparticle concentrations ≥ 1 μg/ml, but DNA damage was evident at 0.1 μg/ml and above. Transmission electron microscopy (TEM) combined with energy-dispersive X-ray spectroscopy confirmed entry of the silica particles into A549 cells exposed to 10 μg/ml of nanoparticles. The particles were observed in the cytoplasm but not within membrane bound vesicles or in the nucleus. TEM of cells exposed to nanoparticles at 4°C for 30 minutes showed particles enter cells when activity is low, suggesting a passive mode of entry. Plasma lipid membrane models identified physical interactions between the membrane and the silica NPs. Quartz crystal ...
Molecular microbiology, Jan 14, 2016
In bacteria, trigger factor (TF) is the molecular chaperone that interacts with the ribosome to a... more In bacteria, trigger factor (TF) is the molecular chaperone that interacts with the ribosome to assist the folding of nascent polypeptides. Studies in vitro have provided insights into the function and mechanism of TF. Much is to be elucidated, however, about how TF functions in vivo. Here we use single-molecule tracking, in combination with genetic manipulations, to study the dynamics and function of TF in living E. coli cells. We find that TF, besides interacting with the 70S ribosome, may also bind to ribosomal subunits and form TF-polypeptide complexes that may include DnaK/DnaJ proteins. The TF-70S ribosome interactions are highly dynamic inside cells, with an average residence time of ∼0.2 s. Our results confirm that the signal recognition particle weakens TF's interaction with the 70S ribosome, and further identify that this weakening mainly results from a change in TF's binding to the 70S ribosome, rather than its unbinding. Moreover, using photoconvertible bimolecul...
Chemical Research in Toxicology, Feb 27, 2014
ZnO nanoparticles (NPs) are prone to dissolution, and uncertainty remains whether biological/cell... more ZnO nanoparticles (NPs) are prone to dissolution, and uncertainty remains whether biological/cellular responses to ZnO NPs are solely due to the release of Zn(2+) or whether the NPs themselves have additional toxic effects. We address this by establishing ZnO NP solubility in dispersion media (Dulbecco's modified Eagle's medium, DMEM) held under conditions identical to those employed for cell culture (37 °C, 5% CO2, and pH 7.68) and by systematic comparison of cell-NP interaction for three different ZnO NP preparations. For NPs at concentrations up to 5.5 μg ZnO/mL, dissolution is complete (with the majority of the soluble zinc complexed to dissolved ligands in the medium), taking ca. 1 h for uncoated and ca. 6 h for polymer coated ones. Above 5.5 μg/mL, the results are consistent with the formation of zinc carbonate, keeping the solubilized zinc fixed to 67 μM of which only 0.45 μM is as free Zn(2+), i.e., not complexed to dissolved ligands. At these relatively high concentrations, NPs with an aliphatic polyether-coating show slower dissolution (i.e., slower free Zn(2+) release) and reprecipitation kinetics compared to those of uncoated NPs, requiring more than 48 h to reach thermodynamic equilibrium. Cytotoxicity (MTT) and DNA damage (Comet) assay dose-response curves for three epithelial cell lines suggest that dissolution and reprecipitation dominate for uncoated ZnO NPs. Transmission electron microscopy combined with the monitoring of intracellular Zn(2+) concentrations and ZnO-NP interactions with model lipid membranes indicate that an aliphatic polyether coat on ZnO NPs increases cellular uptake, enhancing toxicity by enabling intracellular dissolution and release of Zn(2+). Similarly, we demonstrate that needle-like NP morphologies enhance toxicity by apparently frustrating cellular uptake. To limit toxicity, ZnO NPs with nonacicular morphologies and coatings that only weakly interact with cellular membranes are recommended.
Journal of the American Chemical Society, Jan 17, 2015
Heme-copper oxidases (HCOs) are key enzymes in prokaryotes and eukaryotes for energy production d... more Heme-copper oxidases (HCOs) are key enzymes in prokaryotes and eukaryotes for energy production during aerobic respiration. They catalyze the reduction of the terminal electron acceptor, oxygen, and utilize the Gibbs free energy to transport protons across a membrane to generate a proton (ΔpH) and electrochemical gradient termed proton motive force (PMF), which provides the driving force for the adenosine triphosphate (ATP) synthesis. Excessive PMF is known to limit the turnover of HCOs, but the molecular mechanism of this regulatory feedback remains relatively unexplored. Here we present a single-enzyme study that reveals that cytochrome bo3 from Escherichia coli, an HCO closely homologous to Complex IV in human mitochondria, can enter a rare, long-lifetime leak state during which proton flow is reversed. The probability of entering the leak state is increased at higher ΔpH. By rapidly dissipating the PMF, we propose that this leak state may enable cytochrome bo3, and possibly othe...
The journal of physical chemistry. B, Jan 22, 2015
Single-molecule tracking (SMT) of fluorescently-tagged cytoplasmic proteins can provide valuable ... more Single-molecule tracking (SMT) of fluorescently-tagged cytoplasmic proteins can provide valuable information on the underlying biological processes in living cells via subsequent analysis of the displacement distributions. However, the confinement effect originated from the small size of a bacterial cell skews the protein's displacement distribution and complicates the quantification of the intrinsic diffusive behaviors. Using the inverse transformation method, we convert the skewed displacement distribution (for both 2D and 3D imaging conditions) back to that in free space for systems containing one or multiple (non)interconverting Brownian diffusion states, from which we can reliably extract the number of diffusion states as well as their intrinsic diffusion coefficients and respective fractional populations. We further demonstrate a successful application to experimental SMT data of a transcription factor in living E. coli cells. This work allows a direct quantitative connect...
Nature communications, Jan 6, 2015
Binding and unbinding of transcription regulators at operator sites constitute a primary mechanis... more Binding and unbinding of transcription regulators at operator sites constitute a primary mechanism for gene regulation. While many cellular factors are known to regulate their binding, little is known on how cells can modulate their unbinding for regulation. Using nanometer-precision single-molecule tracking, we study the unbinding kinetics from DNA of two metal-sensing transcription regulators in living Escherichia coli cells. We find that they show unusual concentration-dependent unbinding kinetics from chromosomal recognition sites in both their apo and holo forms. Unexpectedly, their unbinding kinetics further varies with the extent of chromosome condensation, and more surprisingly, varies in opposite ways for their apo-repressor versus holo-activator forms. These findings suggest likely broadly relevant mechanisms for facile switching between transcription activation and deactivation in vivo and in coordinating transcription regulation of resistance genes with the cell cycle.
Chemical Research in Toxicology, 2014
ZnO nanoparticles (NPs) are prone to dissolution, and uncertainty remains whether biological/cell... more ZnO nanoparticles (NPs) are prone to dissolution, and uncertainty remains whether biological/cellular responses to ZnO NPs are solely due to the release of Zn(2+) or whether the NPs themselves have additional toxic effects. We address this by establishing ZnO NP solubility in dispersion media (Dulbecco's modified Eagle's medium, DMEM) held under conditions identical to those employed for cell culture (37 °C, 5% CO2, and pH 7.68) and by systematic comparison of cell-NP interaction for three different ZnO NP preparations. For NPs at concentrations up to 5.5 μg ZnO/mL, dissolution is complete (with the majority of the soluble zinc complexed to dissolved ligands in the medium), taking ca. 1 h for uncoated and ca. 6 h for polymer coated ones. Above 5.5 μg/mL, the results are consistent with the formation of zinc carbonate, keeping the solubilized zinc fixed to 67 μM of which only 0.45 μM is as free Zn(2+), i.e., not complexed to dissolved ligands. At these relatively high concentrations, NPs with an aliphatic polyether-coating show slower dissolution (i.e., slower free Zn(2+) release) and reprecipitation kinetics compared to those of uncoated NPs, requiring more than 48 h to reach thermodynamic equilibrium. Cytotoxicity (MTT) and DNA damage (Comet) assay dose-response curves for three epithelial cell lines suggest that dissolution and reprecipitation dominate for uncoated ZnO NPs. Transmission electron microscopy combined with the monitoring of intracellular Zn(2+) concentrations and ZnO-NP interactions with model lipid membranes indicate that an aliphatic polyether coat on ZnO NPs increases cellular uptake, enhancing toxicity by enabling intracellular dissolution and release of Zn(2+). Similarly, we demonstrate that needle-like NP morphologies enhance toxicity by apparently frustrating cellular uptake. To limit toxicity, ZnO NPs with nonacicular morphologies and coatings that only weakly interact with cellular membranes are recommended.
Biophysical Journal, 2013
This thesis starts from a general idea of a biosensors up to commercially successful blood glucos... more This thesis starts from a general idea of a biosensors up to commercially successful blood glucose sensors that one can buy in almost every pharmacy. In the first chapter we can find what actually a biosensor is, parameters characterizing the analytical device, biosensors’ classification according to the different biological recognition elements as well as the physicochemical transducers and a review of methods used for the biomolecules immobilization on a transducer surface. The second chapter contains the electrochemistry background needed for understanding the working principle of amperometric biosensors, a simple Michaelis–Menten enzyme catalysis theory and finally a kinetic model of a amperometric biosensor considered for two boundary assumptions: membrane/enzyme/electrode and enzymatic biofilm/electrode. In the chapter three there is a description of three generations of amperometric glucose sensors, their comparison, a progress that has been made since the very beginning of a biosensor in late 70s and the a short list of the commercially available electrochemical blood glucose sensors. The fourth chapter was dedicated on an amperometric biosensor applications and gives us a feeling about other electrochemical sensors and research that is being done on them nowadays, in the labs all over the world.
Proceedings of the National Academy of Sciences, 2020
Metal detoxification is essential for bacteria’s survival in adverse environments and their patho... more Metal detoxification is essential for bacteria’s survival in adverse environments and their pathogenesis in hosts. Understanding the underlying mechanisms is crucial for devising antibacterial treatments. In the Gram-negative bacterium Escherichia coli , membrane-bound sensor CusS and its response regulator CusR together regulate the transcription of the cus operon that plays important roles in cells’ resistance to copper/silver, and they belong to the two-component systems (TCSs) that are ubiquitous across various organisms and regulate diverse cellular functions. In vitro protein reconstitution and associated biochemical/physical studies have provided significant insights into the functions and mechanisms of CusS–CusR and related TCSs. Such studies are challenging regarding multidomain membrane proteins like CusS and also lack the physiological environment, particularly the native spatial context of proteins inside a cell. Here, we use stroboscopic single-molecule imaging and trac...
Physical forces have long been recognized for their effects on the growth, morphology, locomotion... more Physical forces have long been recognized for their effects on the growth, morphology, locomotion, and survival of eukaryotic organisms. Recently, mechanical forces have been shown to regulate processes in bacteria, including cell division, motility, virulence, biofilm initiation, and cell shape, although it remains unclear how mechanical forces in the cell envelope lead to changes in molecular processes. In Gram-negative bacteria, multicomponent protein complexes that form rigid links across the cell envelope directly experience physical forces and mechanical stresses applied to the cell. Here we manipulate tensile and shear mechanical stress in the bacterial cell envelope and use single-molecule tracking to show that shear (but not tensile) stress within the cell envelope promotes disassembly of the tripartite efflux complex CusCBA, a system used by E. coli to resist copper and silver toxicity, thereby making bacteria more susceptible to metal toxicity. These findings provide the ...
Journal of Inflammation Research
Sarcoidosis is a systemic disease of unknown etiology characterized by granuloma formation in the... more Sarcoidosis is a systemic disease of unknown etiology characterized by granuloma formation in the affected tissues. The pathologically activated macrophages are causatively implicated in disease pathogenesis and play important role in granuloma formation. Chitotriosidase (CHIT1), macrophage-derived protein, is upregulated in sarcoidosis and its levels correlate with disease severity implicating CHIT1 in pathology. Methods: CHIT1 was evaluated in serum and bronchial mucosa and mediastinal lymph nodes specimens from sarcoidosis patients. The therapeutic efficacy of OATD-01 was assessed ex vivo on human bronchoalveolar lavage fluid (BALF) macrophages and in vivo in the murine models of granulomatous inflammation. Results: CHIT1 activity was significantly upregulated in serum from sarcoidosis patients. CHIT1 expression was restricted to granulomas and localized in macrophages. Ex vivo OATD-01 inhibited pro-inflammatory mediators' production (CCL4, IL-15) by lung macrophages. In the acute model of granulomatous inflammation in mice, OATD-01 showed anti-inflammatory effects reducing the percentage of neutrophils and CCL4 concentration in BALF. In the chronic model, inhibition of CHIT1 led to a decrease in the number of organized lung granulomas and the expression of sarcoidosis-associated genes. Conclusion: In summary, CHIT1 activity was increased in sarcoidosis patients and OATD-01, a first-in-class CHIT1 inhibitor, demonstrated efficacy in murine models of granulomatous inflammation providing a proof-of-concept for its clinical evaluation in sarcoidosis.
The Journal of Physical Chemistry B, 2011
Copper-containing nitrite reductases (NiRs) are enzymes that efficiently reduce nitrite to nitric... more Copper-containing nitrite reductases (NiRs) are enzymes that efficiently reduce nitrite to nitric oxide in potent denitrifying bacteria. There has been an interest in their application in amperometric biosensors for monitoring nitrite levels in natural and waste waters. NiRs have a complex enzyme mechanism and depend on nitrite concentration and pH. Although the mechanism has been intensively studied, it is still controversial. In this thesis, a combined fluorescence and electrochemical method is used to simultaneously monitor the nitrite turn-over rate of a NiR from Alcaligenes faecalis S-6 and the oxidation state of the type-1 copper electron transfer site inside the enzyme. The catalytic activity of NiR is measured electrochemically by exploiting a direct electron transfer to fluorescently labelled enzyme molecules immobilised on modified gold, whereas the redox state of the type-1 copper site is determined from fluorescence intensity changes caused by F6rster resonance energy transfer (FRET) between a fluorophore attached to NiR and its type-1 copper site. Here, a determining role of internal electron transfer is found in NiR's mechanism. Moreover, the heterogeneous interfacial electron transfer to adsorbed NiRs is observed. The electro- activity and binding of labelled and unlabelled NiRs on gold modified with different self-assembled monolayers (SAMs) are studied to understand the effect of NiR labelling on the protein-electrode interactions. Here, electro-active and well-ordered biofilms of NiRs are found on electrodes with SAMs carrying the positive charge (negative NiRs) or when NiRs are modified with fluorophores that help orienting them on SAM-modified gold. Attempts have also been made to implement fluorescently labelled NiRs into the biosensor-like device with a fluorescence output using gold electrodes modified with tethered lipid bilayers (tBLMs) or conducting polymers (CPs). In spite of being unsuccessful, these studies give a better understanding of potential-dependent polymer dynamics and a control over protein immobilisation on functionalised lipid bilayer platforms.EThOS - Electronic Theses Online ServiceGBUnited Kingdo
Journal of the American Chemical Society, 2011
Membrane proteins are key components of the plasma membrane and are responsible for control of ch... more Membrane proteins are key components of the plasma membrane and are responsible for control of chemical ionic gradients, metabolite and nutrient transfer, and signal transduction between the interior of cells and the external environment. Of the genes in the human genome, 30% code for membrane proteins (Krogh et al. J. Mol. Biol. 2001, 305, 567). Furthermore, many FDA-approved drugs target such proteins (Overington et al. Nat. Rev. Drug Discovery 2006, 5, 993). However, the structureÀfunction relationships of these are notably sparse because of difficulties in their purification and handling outside of their membranous environment. Methods that permit the manipulation of membrane components while they are still in the membrane would find widespread application in separation, purification, and eventual structureÀfunction determination of these species (Poo et al. Nature 1977, 265, 602). Here we show that asymmetrically patterned supported lipid bilayers in combination with AC electric fields can lead to efficient manipulation of charged components. We demonstrate the concentration and trapping of such components through the use of a "nested trap" and show that this method is capable of yielding an approximately 30-fold increase in the average protein concentration. Upon removal of the field, the material remains trapped for several hours as a result of topographically restricted diffusion. Our results indicate that this method can be used for concentrating and trapping charged membrane components while they are still within their membranous environment. We anticipate that our approach could find widespread application in the manipulation and study of membrane proteins.
Plant Growth Regulation, 2007
Ascorbate levels and redox states, as well as the activities of the enzymes of ascorbate metaboli... more Ascorbate levels and redox states, as well as the activities of the enzymes of ascorbate metabolism, were analyzed in roots of tomato seedlings during the culture on a medium supplemented with auxin and compared to the control cultured on an auxin-free medium. Biochemical parameters were determined separately in the distal part of the root where the inhibitory effect of auxin
Journal of the American Chemical Society, 2011
Particle and fibre toxicology, Jan 23, 2012
Mechanisms for cellular uptake of nanoparticles have important implications for nanoparticulate d... more Mechanisms for cellular uptake of nanoparticles have important implications for nanoparticulate drug delivery and toxicity. We have explored the mechanism of uptake of amorphous silica nanoparticles of 14 nm diameter, which agglomerate in culture medium to hydrodynamic diameters around 500 nm. In HT29, HaCat and A549 cells, cytotoxicity was observed at nanoparticle concentrations ≥ 1 μg/ml, but DNA damage was evident at 0.1 μg/ml and above. Transmission electron microscopy (TEM) combined with energy-dispersive X-ray spectroscopy confirmed entry of the silica particles into A549 cells exposed to 10 μg/ml of nanoparticles. The particles were observed in the cytoplasm but not within membrane bound vesicles or in the nucleus. TEM of cells exposed to nanoparticles at 4°C for 30 minutes showed particles enter cells when activity is low, suggesting a passive mode of entry. Plasma lipid membrane models identified physical interactions between the membrane and the silica NPs. Quartz crystal ...
Molecular microbiology, Jan 14, 2016
In bacteria, trigger factor (TF) is the molecular chaperone that interacts with the ribosome to a... more In bacteria, trigger factor (TF) is the molecular chaperone that interacts with the ribosome to assist the folding of nascent polypeptides. Studies in vitro have provided insights into the function and mechanism of TF. Much is to be elucidated, however, about how TF functions in vivo. Here we use single-molecule tracking, in combination with genetic manipulations, to study the dynamics and function of TF in living E. coli cells. We find that TF, besides interacting with the 70S ribosome, may also bind to ribosomal subunits and form TF-polypeptide complexes that may include DnaK/DnaJ proteins. The TF-70S ribosome interactions are highly dynamic inside cells, with an average residence time of ∼0.2 s. Our results confirm that the signal recognition particle weakens TF's interaction with the 70S ribosome, and further identify that this weakening mainly results from a change in TF's binding to the 70S ribosome, rather than its unbinding. Moreover, using photoconvertible bimolecul...
Chemical Research in Toxicology, Feb 27, 2014
ZnO nanoparticles (NPs) are prone to dissolution, and uncertainty remains whether biological/cell... more ZnO nanoparticles (NPs) are prone to dissolution, and uncertainty remains whether biological/cellular responses to ZnO NPs are solely due to the release of Zn(2+) or whether the NPs themselves have additional toxic effects. We address this by establishing ZnO NP solubility in dispersion media (Dulbecco's modified Eagle's medium, DMEM) held under conditions identical to those employed for cell culture (37 °C, 5% CO2, and pH 7.68) and by systematic comparison of cell-NP interaction for three different ZnO NP preparations. For NPs at concentrations up to 5.5 μg ZnO/mL, dissolution is complete (with the majority of the soluble zinc complexed to dissolved ligands in the medium), taking ca. 1 h for uncoated and ca. 6 h for polymer coated ones. Above 5.5 μg/mL, the results are consistent with the formation of zinc carbonate, keeping the solubilized zinc fixed to 67 μM of which only 0.45 μM is as free Zn(2+), i.e., not complexed to dissolved ligands. At these relatively high concentrations, NPs with an aliphatic polyether-coating show slower dissolution (i.e., slower free Zn(2+) release) and reprecipitation kinetics compared to those of uncoated NPs, requiring more than 48 h to reach thermodynamic equilibrium. Cytotoxicity (MTT) and DNA damage (Comet) assay dose-response curves for three epithelial cell lines suggest that dissolution and reprecipitation dominate for uncoated ZnO NPs. Transmission electron microscopy combined with the monitoring of intracellular Zn(2+) concentrations and ZnO-NP interactions with model lipid membranes indicate that an aliphatic polyether coat on ZnO NPs increases cellular uptake, enhancing toxicity by enabling intracellular dissolution and release of Zn(2+). Similarly, we demonstrate that needle-like NP morphologies enhance toxicity by apparently frustrating cellular uptake. To limit toxicity, ZnO NPs with nonacicular morphologies and coatings that only weakly interact with cellular membranes are recommended.
Journal of the American Chemical Society, Jan 17, 2015
Heme-copper oxidases (HCOs) are key enzymes in prokaryotes and eukaryotes for energy production d... more Heme-copper oxidases (HCOs) are key enzymes in prokaryotes and eukaryotes for energy production during aerobic respiration. They catalyze the reduction of the terminal electron acceptor, oxygen, and utilize the Gibbs free energy to transport protons across a membrane to generate a proton (ΔpH) and electrochemical gradient termed proton motive force (PMF), which provides the driving force for the adenosine triphosphate (ATP) synthesis. Excessive PMF is known to limit the turnover of HCOs, but the molecular mechanism of this regulatory feedback remains relatively unexplored. Here we present a single-enzyme study that reveals that cytochrome bo3 from Escherichia coli, an HCO closely homologous to Complex IV in human mitochondria, can enter a rare, long-lifetime leak state during which proton flow is reversed. The probability of entering the leak state is increased at higher ΔpH. By rapidly dissipating the PMF, we propose that this leak state may enable cytochrome bo3, and possibly othe...
The journal of physical chemistry. B, Jan 22, 2015
Single-molecule tracking (SMT) of fluorescently-tagged cytoplasmic proteins can provide valuable ... more Single-molecule tracking (SMT) of fluorescently-tagged cytoplasmic proteins can provide valuable information on the underlying biological processes in living cells via subsequent analysis of the displacement distributions. However, the confinement effect originated from the small size of a bacterial cell skews the protein's displacement distribution and complicates the quantification of the intrinsic diffusive behaviors. Using the inverse transformation method, we convert the skewed displacement distribution (for both 2D and 3D imaging conditions) back to that in free space for systems containing one or multiple (non)interconverting Brownian diffusion states, from which we can reliably extract the number of diffusion states as well as their intrinsic diffusion coefficients and respective fractional populations. We further demonstrate a successful application to experimental SMT data of a transcription factor in living E. coli cells. This work allows a direct quantitative connect...
Nature communications, Jan 6, 2015
Binding and unbinding of transcription regulators at operator sites constitute a primary mechanis... more Binding and unbinding of transcription regulators at operator sites constitute a primary mechanism for gene regulation. While many cellular factors are known to regulate their binding, little is known on how cells can modulate their unbinding for regulation. Using nanometer-precision single-molecule tracking, we study the unbinding kinetics from DNA of two metal-sensing transcription regulators in living Escherichia coli cells. We find that they show unusual concentration-dependent unbinding kinetics from chromosomal recognition sites in both their apo and holo forms. Unexpectedly, their unbinding kinetics further varies with the extent of chromosome condensation, and more surprisingly, varies in opposite ways for their apo-repressor versus holo-activator forms. These findings suggest likely broadly relevant mechanisms for facile switching between transcription activation and deactivation in vivo and in coordinating transcription regulation of resistance genes with the cell cycle.
Chemical Research in Toxicology, 2014
ZnO nanoparticles (NPs) are prone to dissolution, and uncertainty remains whether biological/cell... more ZnO nanoparticles (NPs) are prone to dissolution, and uncertainty remains whether biological/cellular responses to ZnO NPs are solely due to the release of Zn(2+) or whether the NPs themselves have additional toxic effects. We address this by establishing ZnO NP solubility in dispersion media (Dulbecco's modified Eagle's medium, DMEM) held under conditions identical to those employed for cell culture (37 °C, 5% CO2, and pH 7.68) and by systematic comparison of cell-NP interaction for three different ZnO NP preparations. For NPs at concentrations up to 5.5 μg ZnO/mL, dissolution is complete (with the majority of the soluble zinc complexed to dissolved ligands in the medium), taking ca. 1 h for uncoated and ca. 6 h for polymer coated ones. Above 5.5 μg/mL, the results are consistent with the formation of zinc carbonate, keeping the solubilized zinc fixed to 67 μM of which only 0.45 μM is as free Zn(2+), i.e., not complexed to dissolved ligands. At these relatively high concentrations, NPs with an aliphatic polyether-coating show slower dissolution (i.e., slower free Zn(2+) release) and reprecipitation kinetics compared to those of uncoated NPs, requiring more than 48 h to reach thermodynamic equilibrium. Cytotoxicity (MTT) and DNA damage (Comet) assay dose-response curves for three epithelial cell lines suggest that dissolution and reprecipitation dominate for uncoated ZnO NPs. Transmission electron microscopy combined with the monitoring of intracellular Zn(2+) concentrations and ZnO-NP interactions with model lipid membranes indicate that an aliphatic polyether coat on ZnO NPs increases cellular uptake, enhancing toxicity by enabling intracellular dissolution and release of Zn(2+). Similarly, we demonstrate that needle-like NP morphologies enhance toxicity by apparently frustrating cellular uptake. To limit toxicity, ZnO NPs with nonacicular morphologies and coatings that only weakly interact with cellular membranes are recommended.
Biophysical Journal, 2013