Mikhail Drobizhev - Academia.edu (original) (raw)

Papers by Mikhail Drobizhev

Biophysical Journal, 2014

Smith-Waterman is a dynamic programming algorithm that locally aligns sequences of discrete value... more Smith-Waterman is a dynamic programming algorithm that locally aligns sequences of discrete values by rewarding matched elements and penalizing mismatched elements. We present an adapted algorithm that aligns sequences of real-valued vectors, applied in our case to the output of nanopore DNA sequencing experiments using MspA. We choose each step of the algorithm to correspond to a driving behavior, such as a polymerase's synthesis and proofreading, and each penalty to be the logarithm of the probability that the step occurs. This allows us to interpret the total score of an alignment as its probability of being the true alignment. Given a known sequence but unknown kinetics, optimizing the score with respect to the penalties will set the penalties to the probabilistic values. This is a useful source of information about molecular motor kinetics. The algorithm's modularity and direct relation to physical behavior make it potentially useful for any physical probes of discrete time series. This work was supported by NIH/NHGRI grant R01HG005115 and R01HG006321.

Biophysical Journal, 2014

Fluorescence microscopy is a widely used non-invasive tool for investigating intracellular struct... more Fluorescence microscopy is a widely used non-invasive tool for investigating intracellular structure and processes. Although the palette of fluorescent proteins has revolutionized detection and dynamics in molecular and cellular biology, the limited brightness, low spectral discrimination, and high cellular autofluorescence continue to limit applications. In contrast to photoswitchbased methods, we have developed a spectroscopic method to recover fluorescence from photoaccessible dark states via long-wavelength secondary laser co-illumination. We selectively recover the higher energy fluorescence by modulating this secondary laser without modulating background autofluorescence. Using hypothesized photoreversible isomerizations of the chromophore, we have identified specific fluorescent protein mutants, ranging from blue to red, capable of optical modulation. Employing these methods, we have demonstrated the ability to recover the modulated signal, with >10-fold improvement, from background in live cells. Such modulation schemes enable new imaging modalities for probing intracellular kinetics and equilibria of low-abundance proteins.

Angewandte Chemie (International ed. in English), Jan 8, 2015

We present a new approach for determining the strength of the dipolar solute-induced reaction fie... more We present a new approach for determining the strength of the dipolar solute-induced reaction field, along with the ground- and excited-state electrostatic dipole moments and polarizability of a solvated chromophore, using exclusively one-photon and two-photon absorption measurements. We verify the approach on two benchmark chromophores N,N-dimethyl-6-propionyl-2-naphthylamine (prodan) and coumarin 153 (C153) in a series of toluene/dimethyl sulfoxide (DMSO) mixtures and find that the experimental values show good quantitative agreement with literature and our quantum-chemical calculations. Our results indicate that the reaction field varies in a surprisingly broad range, 0-10(7) V cm(-1) , and that at close proximity, on the order of the chromophore radius, the effective dielectric constant of the solute-solvent system displays a unique functional dependence on the bulk dielectric constant, offering new insight into the close-range molecular interaction.

Reporters, Markers, Dyes, Nanoparticles, and Molecular Probes For Biomedical Applications Vi, 2014

ABSTRACT Two-photon excitation of fluorescent proteins (FPs) is widely used in imaging whole orga... more ABSTRACT Two-photon excitation of fluorescent proteins (FPs) is widely used in imaging whole organisms or living tissues. Many different FPs are now available but these proteins have only been optimized for their one-photon properties. We have developed a technique for screening entire libraries of E. coli colonies expressing FPs that utilizes multiple wavelengths of linear excitation as well as two-photon excitation. Single mutations in a particular protein that affect one or twophoton properties are easily identified, providing new views of structure/function relationships. An amplified femtosecond Ti:sapphire laser and a spectrally filtered lamp source are used to acquire the fluorescence signals of up to ~1000 E. coli colonies on a standard Petri dish. Automation of the analysis and acquisition of the fluorescent signals makes it feasible to rapidly screen tens of thousands of colonies. In a proof of principle experiment with the commonly used EGFP, we used two rounds of error prone PCR and selection to evolve new proteins with shifted absorption and increased two-photon cross sections at 790nm. This method of screening, coupled with careful measurements of photo bleaching dynamics and two-photon cross sections, should make it possible to optimize a wide variety of fluorescent proteins and biosensors for use in two-photon microscopes.

J. Phys. Chem. B, 2014

Genetically encoded fluorescent proteins (FPs), and biosensors based on them, provide new insight... more Genetically encoded fluorescent proteins (FPs), and biosensors based on them, provide new insights into how living cells and tissues function. Ultimately, the goal of the bioimaging community is to use these probes deep in tissues and even in entire organisms, and this will require two-photon laser scanning microscopy (TPLSM), with its greater tissue penetration, lower autofluorescence background, and minimum photodamage in the out-offocus volume. However, the extremely high instantaneous light intensities of femtosecond pulses in the focal volume dramatically increase the probability of further stepwise resonant photon absorption, leading to highly excited, ionizable and reactive states, often resulting in fast bleaching of fluorescent proteins in TPLSM. Here, we show that the femtosecond multiphoton excitation of red FPs (DsRed2 and mFruits), both in solution and live cells, results in a chain of consecutive, partially reversible reactions, with individual rates driven by a high-order (3−5 photon) absorption. The first step of this process corresponds to a three-(DsRed2) or fourphoton (mFruits) induced fast isomerization of the chromophore, yielding intermediate fluorescent forms, which then subsequently transform into nonfluorescent products. Our experimental data and model calculations are consistent with a mechanism in which ultrafast electron transfer from the chromophore to a neighboring positively charged amino acid residue triggers the first step of multiphoton chromophore transformations in DsRed2 and mFruits, consisting of decarboxylation of a nearby deprotonated glutamic acid residue.

The Journal of Physical Chemistry B, 2012

Chemical Communications, 2013

A π-extended bis-porphyrin bridged via a diketopyrrolopyrrole unit was prepared in 5 steps. This ... more A π-extended bis-porphyrin bridged via a diketopyrrolopyrrole unit was prepared in 5 steps. This fully conjugated π-system displays strongly distorted linear absorption, while its two-photon absorption cross-section reaches 2500-3000 GM at 940 nm. LC-like behaviour, easy orientation and low viscosity are, according to XRD, POM and DSC measurements, due to formation of plastic rather than a liquid crystal.

Scientific Reports, 2015

Directed evolution has been used extensively to improve the properties of a variety of fluorescen... more Directed evolution has been used extensively to improve the properties of a variety of fluorescent proteins (FPs). Evolutionary strategies, however, have not yet been used to improve the two-photon absorption (2PA) properties of a fluorescent protein, properties that are important for two-photon imaging in living tissues, including the brain. Here we demonstrate a technique for quantitatively screening the two-photon excited fluorescence (2PEF) efficiency and 2PA cross section of tens of thousands of mutant FPs expressed in E. coli colonies. We use this procedure to move EGFP through three rounds of two-photon directed evolution leading to new variants showing up to a 50% enhancement in peak 2PA cross section and brightness within the near-IR tissue transparency wavelength range.

Nature chemical biology, 2018

We developed a new way to engineer complex proteins toward multidimensional specifications using ... more We developed a new way to engineer complex proteins toward multidimensional specifications using a simple, yet scalable, directed evolution strategy. By robotically picking mammalian cells that were identified, under a microscope, as expressing proteins that simultaneously exhibit several specific properties, we can screen hundreds of thousands of proteins in a library in just a few hours, evaluating each along multiple performance axes. To demonstrate the power of this approach, we created a genetically encoded fluorescent voltage indicator, simultaneously optimizing its brightness and membrane localization using our microscopy-guided cell-picking strategy. We produced the high-performance opsin-based fluorescent voltage reporter Archon1 and demonstrated its utility by imaging spiking and millivolt-scale subthreshold and synaptic activity in acute mouse brain slices and in larval zebrafish in vivo. We also measured postsynaptic responses downstream of optogenetically controlled neu...

Nature chemical biology, Jan 8, 2018

In the version of this article originally published, the bottom of Figure 4f,g was partially trun... more In the version of this article originally published, the bottom of Figure 4f,g was partially truncated in the PDF. The error has been corrected in the PDF version of this article.

BMC biology, Jan 16, 2018

Genetically encoded calcium ion (Ca2+) indicators (GECIs) are indispensable tools for measuring C... more Genetically encoded calcium ion (Ca2+) indicators (GECIs) are indispensable tools for measuring Ca2+ dynamics and neuronal activities in vitro and in vivo. Red fluorescent protein (RFP)-based GECIs have inherent advantages relative to green fluorescent protein-based GECIs due to the longer wavelength light used for excitation. Longer wavelength light is associated with decreased phototoxicity and deeper penetration through tissue. Red GECI can also enable multicolor visualization with blue- or cyan-excitable fluorophores. Here we report the development, structure, and validation of a new RFP-based GECI, K-GECO1, based on a circularly permutated RFP derived from the sea anemone Entacmaea quadricolor. We have characterized the performance of K-GECO1 in cultured HeLa cells, dissociated neurons, stem-cell-derived cardiomyocytes, organotypic brain slices, zebrafish spinal cord in vivo, and mouse brain in vivo. K-GECO1 is the archetype of a new lineage of GECIs based on the RFP eqFP578 sc...

The journal of physical chemistry letters, Jan 15, 2017

Fluorescent proteins (FPs) are indispensable markers for two-photon imaging of live tissue, espec... more Fluorescent proteins (FPs) are indispensable markers for two-photon imaging of live tissue, especially in the brains of small model organisms. The quantity of physiologically relevant data collected, however, is limited by heat-induced damage of the tissue due to the high intensities of the excitation laser. We seek to minimize this damage by developing FPs with improved brightness. Among FPs with the same chromophore structure, the spectral properties can vary widely due to differences in the local protein environment. Using a physical model that describes the spectra of FPs containing the anionic green FP (GFP) chromophore, we predict that those that are blue-shifted in one-photon absorption will have stronger peak two-photon absorption cross sections. Following this prediction, we present 12 blue-shifted GFP homologues and demonstrate that they are up to 2.5 times brighter than the commonly used enhanced GFP (EGFP).

PloS one, 2017

The goal of this work is to determine how GCaMP6m's fluorescence is altered in response to Ca... more The goal of this work is to determine how GCaMP6m's fluorescence is altered in response to Ca2+-binding. Our detailed spectroscopic study reveals the simplest explanation for how GCaMP6m changes fluorescence in response to Ca2+ is with a four-state model, in which a Ca2+-dependent change of the chromophore protonation state, due to a shift in pKa, is the predominant factor. The pKa shift is quantitatively explained by a change in electrostatic potential around the chromophore due to the conformational changes that occur in the protein when calmodulin binds Ca2+ and interacts with the M13 peptide. The absolute pKa values for the Ca2+-free and Ca2+-saturated states of GCaMP6m are critical to its high signal-to-noise ratio. This mechanism has important implications for further improvements to GCaMP6m and potentially for other similarly designed biosensors.

Biophysical Journal, 2017

classification indicated that fecal samples were enriched in metabolism-related genes involved in... more classification indicated that fecal samples were enriched in metabolism-related genes involved in glycolysis, methionine degradation, maltose utilization, and butyrate production, while cloacal samples were enriched in aromatic metabolism, virulence genes, and membrane transporters. Our analysis was further supported by the recovery of protein sequences for phylogenetic analysis, such as the RNA polymerase of Delftia, a Shigella toxin, a nitrate reductase of Delftia, and beta lactamases from several species. Our work identifies several strategies by which metagenomic sequencing of condor microbiomes may inform us about condor diet, infections, intoxication, and other types of stress.

Biophysical journal, Jan 7, 2017

Several series of near-infrared (NIR) fluorescent proteins (FPs) were recently engineered from ba... more Several series of near-infrared (NIR) fluorescent proteins (FPs) were recently engineered from bacterial phytochromes but were not systematically compared in neurons. To fluoresce, NIR FPs utilize an enzymatic derivative of heme, the linear tetrapyrrole biliverdin, as a chromophore whose level in neurons is poorly studied. Here, we evaluated NIR FPs of the iRFP protein family, which were reported to be the brightest in non-neuronal mammalian cells, in primary neuronal culture, in brain slices of mouse and monkey, and in mouse brain in vivo. We applied several fluorescence imaging modes, such as wide-field and confocal one-photon and two-photon microscopy, to compare photochemical and biophysical properties of various iRFPs. The iRFP682 and iRFP670 proteins exhibited the highest brightness and photostability under one-photon and two-photon excitation modes, respectively. All studied iRFPs exhibited efficient binding of the endogenous biliverdin chromophore in cultured neurons and in ...

J-aggregates of tiacarbocyanine derivative are investigated in frozen solution at low temperature... more J-aggregates of tiacarbocyanine derivative are investigated in frozen solution at low temperatures by optical spectroscopy and spectral hole burning. It is found that absorption and luminescence bands have inhomogeneous widths of 220 and 130 cm-1 at 5 K respectively used in contrast to pseudoisocyanine (PIC) J-bands are separated by Stokes shift of 100 cm-1. Moreover, J-aggregate fluorescence spectrum depends drastically

Proceedings of Spie the International Society For Optical Engineering, Feb 1, 2009

We report simultaneous two-photon absorption (2PA) spectra in a series of substituted corroles an... more We report simultaneous two-photon absorption (2PA) spectra in a series of substituted corroles and related porphyrins in 800-1400 nm laser wavelength range. Compared to the porphyrins, the 2PA spectrum of corroles contains a distinct and relatively high intensity peak, sigma(2) = 60-130 GM, close to twice the wavelength of Soret maximum (800-850 nm). The increase of 2PA peak cross section is explained in terms of decreased symmetry of the contracted macro-cycle, and is most likely related to relaxing of the parity selection rules that restrict 2PA in the Soret band for more symmetrical porphyrins. We also observe that the strength of the 2PA peak in Soret region decreases with the electron-withdrawing ability (increasing Hammett constant) of the side substituents, which can be explained by assuming that the corrole core itself possesses electron-accepting ability. The peak 2PA cross sections in the Q-region are much less than in Soret-region, and can be quantitatively described within the two-level approximation taking into account permanent dipole moments in the ground and excited states.

Biophysical Journal, 2014

Smith-Waterman is a dynamic programming algorithm that locally aligns sequences of discrete value... more Smith-Waterman is a dynamic programming algorithm that locally aligns sequences of discrete values by rewarding matched elements and penalizing mismatched elements. We present an adapted algorithm that aligns sequences of real-valued vectors, applied in our case to the output of nanopore DNA sequencing experiments using MspA. We choose each step of the algorithm to correspond to a driving behavior, such as a polymerase's synthesis and proofreading, and each penalty to be the logarithm of the probability that the step occurs. This allows us to interpret the total score of an alignment as its probability of being the true alignment. Given a known sequence but unknown kinetics, optimizing the score with respect to the penalties will set the penalties to the probabilistic values. This is a useful source of information about molecular motor kinetics. The algorithm's modularity and direct relation to physical behavior make it potentially useful for any physical probes of discrete time series. This work was supported by NIH/NHGRI grant R01HG005115 and R01HG006321.

Biophysical Journal, 2014

Fluorescence microscopy is a widely used non-invasive tool for investigating intracellular struct... more Fluorescence microscopy is a widely used non-invasive tool for investigating intracellular structure and processes. Although the palette of fluorescent proteins has revolutionized detection and dynamics in molecular and cellular biology, the limited brightness, low spectral discrimination, and high cellular autofluorescence continue to limit applications. In contrast to photoswitchbased methods, we have developed a spectroscopic method to recover fluorescence from photoaccessible dark states via long-wavelength secondary laser co-illumination. We selectively recover the higher energy fluorescence by modulating this secondary laser without modulating background autofluorescence. Using hypothesized photoreversible isomerizations of the chromophore, we have identified specific fluorescent protein mutants, ranging from blue to red, capable of optical modulation. Employing these methods, we have demonstrated the ability to recover the modulated signal, with >10-fold improvement, from background in live cells. Such modulation schemes enable new imaging modalities for probing intracellular kinetics and equilibria of low-abundance proteins.

Angewandte Chemie (International ed. in English), Jan 8, 2015

We present a new approach for determining the strength of the dipolar solute-induced reaction fie... more We present a new approach for determining the strength of the dipolar solute-induced reaction field, along with the ground- and excited-state electrostatic dipole moments and polarizability of a solvated chromophore, using exclusively one-photon and two-photon absorption measurements. We verify the approach on two benchmark chromophores N,N-dimethyl-6-propionyl-2-naphthylamine (prodan) and coumarin 153 (C153) in a series of toluene/dimethyl sulfoxide (DMSO) mixtures and find that the experimental values show good quantitative agreement with literature and our quantum-chemical calculations. Our results indicate that the reaction field varies in a surprisingly broad range, 0-10(7) V cm(-1) , and that at close proximity, on the order of the chromophore radius, the effective dielectric constant of the solute-solvent system displays a unique functional dependence on the bulk dielectric constant, offering new insight into the close-range molecular interaction.

Reporters, Markers, Dyes, Nanoparticles, and Molecular Probes For Biomedical Applications Vi, 2014

ABSTRACT Two-photon excitation of fluorescent proteins (FPs) is widely used in imaging whole orga... more ABSTRACT Two-photon excitation of fluorescent proteins (FPs) is widely used in imaging whole organisms or living tissues. Many different FPs are now available but these proteins have only been optimized for their one-photon properties. We have developed a technique for screening entire libraries of E. coli colonies expressing FPs that utilizes multiple wavelengths of linear excitation as well as two-photon excitation. Single mutations in a particular protein that affect one or twophoton properties are easily identified, providing new views of structure/function relationships. An amplified femtosecond Ti:sapphire laser and a spectrally filtered lamp source are used to acquire the fluorescence signals of up to ~1000 E. coli colonies on a standard Petri dish. Automation of the analysis and acquisition of the fluorescent signals makes it feasible to rapidly screen tens of thousands of colonies. In a proof of principle experiment with the commonly used EGFP, we used two rounds of error prone PCR and selection to evolve new proteins with shifted absorption and increased two-photon cross sections at 790nm. This method of screening, coupled with careful measurements of photo bleaching dynamics and two-photon cross sections, should make it possible to optimize a wide variety of fluorescent proteins and biosensors for use in two-photon microscopes.

J. Phys. Chem. B, 2014

Genetically encoded fluorescent proteins (FPs), and biosensors based on them, provide new insight... more Genetically encoded fluorescent proteins (FPs), and biosensors based on them, provide new insights into how living cells and tissues function. Ultimately, the goal of the bioimaging community is to use these probes deep in tissues and even in entire organisms, and this will require two-photon laser scanning microscopy (TPLSM), with its greater tissue penetration, lower autofluorescence background, and minimum photodamage in the out-offocus volume. However, the extremely high instantaneous light intensities of femtosecond pulses in the focal volume dramatically increase the probability of further stepwise resonant photon absorption, leading to highly excited, ionizable and reactive states, often resulting in fast bleaching of fluorescent proteins in TPLSM. Here, we show that the femtosecond multiphoton excitation of red FPs (DsRed2 and mFruits), both in solution and live cells, results in a chain of consecutive, partially reversible reactions, with individual rates driven by a high-order (3−5 photon) absorption. The first step of this process corresponds to a three-(DsRed2) or fourphoton (mFruits) induced fast isomerization of the chromophore, yielding intermediate fluorescent forms, which then subsequently transform into nonfluorescent products. Our experimental data and model calculations are consistent with a mechanism in which ultrafast electron transfer from the chromophore to a neighboring positively charged amino acid residue triggers the first step of multiphoton chromophore transformations in DsRed2 and mFruits, consisting of decarboxylation of a nearby deprotonated glutamic acid residue.

The Journal of Physical Chemistry B, 2012

Chemical Communications, 2013

A π-extended bis-porphyrin bridged via a diketopyrrolopyrrole unit was prepared in 5 steps. This ... more A π-extended bis-porphyrin bridged via a diketopyrrolopyrrole unit was prepared in 5 steps. This fully conjugated π-system displays strongly distorted linear absorption, while its two-photon absorption cross-section reaches 2500-3000 GM at 940 nm. LC-like behaviour, easy orientation and low viscosity are, according to XRD, POM and DSC measurements, due to formation of plastic rather than a liquid crystal.

Scientific Reports, 2015

Directed evolution has been used extensively to improve the properties of a variety of fluorescen... more Directed evolution has been used extensively to improve the properties of a variety of fluorescent proteins (FPs). Evolutionary strategies, however, have not yet been used to improve the two-photon absorption (2PA) properties of a fluorescent protein, properties that are important for two-photon imaging in living tissues, including the brain. Here we demonstrate a technique for quantitatively screening the two-photon excited fluorescence (2PEF) efficiency and 2PA cross section of tens of thousands of mutant FPs expressed in E. coli colonies. We use this procedure to move EGFP through three rounds of two-photon directed evolution leading to new variants showing up to a 50% enhancement in peak 2PA cross section and brightness within the near-IR tissue transparency wavelength range.

Nature chemical biology, 2018

We developed a new way to engineer complex proteins toward multidimensional specifications using ... more We developed a new way to engineer complex proteins toward multidimensional specifications using a simple, yet scalable, directed evolution strategy. By robotically picking mammalian cells that were identified, under a microscope, as expressing proteins that simultaneously exhibit several specific properties, we can screen hundreds of thousands of proteins in a library in just a few hours, evaluating each along multiple performance axes. To demonstrate the power of this approach, we created a genetically encoded fluorescent voltage indicator, simultaneously optimizing its brightness and membrane localization using our microscopy-guided cell-picking strategy. We produced the high-performance opsin-based fluorescent voltage reporter Archon1 and demonstrated its utility by imaging spiking and millivolt-scale subthreshold and synaptic activity in acute mouse brain slices and in larval zebrafish in vivo. We also measured postsynaptic responses downstream of optogenetically controlled neu...

Nature chemical biology, Jan 8, 2018

In the version of this article originally published, the bottom of Figure 4f,g was partially trun... more In the version of this article originally published, the bottom of Figure 4f,g was partially truncated in the PDF. The error has been corrected in the PDF version of this article.

BMC biology, Jan 16, 2018

Genetically encoded calcium ion (Ca2+) indicators (GECIs) are indispensable tools for measuring C... more Genetically encoded calcium ion (Ca2+) indicators (GECIs) are indispensable tools for measuring Ca2+ dynamics and neuronal activities in vitro and in vivo. Red fluorescent protein (RFP)-based GECIs have inherent advantages relative to green fluorescent protein-based GECIs due to the longer wavelength light used for excitation. Longer wavelength light is associated with decreased phototoxicity and deeper penetration through tissue. Red GECI can also enable multicolor visualization with blue- or cyan-excitable fluorophores. Here we report the development, structure, and validation of a new RFP-based GECI, K-GECO1, based on a circularly permutated RFP derived from the sea anemone Entacmaea quadricolor. We have characterized the performance of K-GECO1 in cultured HeLa cells, dissociated neurons, stem-cell-derived cardiomyocytes, organotypic brain slices, zebrafish spinal cord in vivo, and mouse brain in vivo. K-GECO1 is the archetype of a new lineage of GECIs based on the RFP eqFP578 sc...

The journal of physical chemistry letters, Jan 15, 2017

Fluorescent proteins (FPs) are indispensable markers for two-photon imaging of live tissue, espec... more Fluorescent proteins (FPs) are indispensable markers for two-photon imaging of live tissue, especially in the brains of small model organisms. The quantity of physiologically relevant data collected, however, is limited by heat-induced damage of the tissue due to the high intensities of the excitation laser. We seek to minimize this damage by developing FPs with improved brightness. Among FPs with the same chromophore structure, the spectral properties can vary widely due to differences in the local protein environment. Using a physical model that describes the spectra of FPs containing the anionic green FP (GFP) chromophore, we predict that those that are blue-shifted in one-photon absorption will have stronger peak two-photon absorption cross sections. Following this prediction, we present 12 blue-shifted GFP homologues and demonstrate that they are up to 2.5 times brighter than the commonly used enhanced GFP (EGFP).

PloS one, 2017

The goal of this work is to determine how GCaMP6m's fluorescence is altered in response to Ca... more The goal of this work is to determine how GCaMP6m's fluorescence is altered in response to Ca2+-binding. Our detailed spectroscopic study reveals the simplest explanation for how GCaMP6m changes fluorescence in response to Ca2+ is with a four-state model, in which a Ca2+-dependent change of the chromophore protonation state, due to a shift in pKa, is the predominant factor. The pKa shift is quantitatively explained by a change in electrostatic potential around the chromophore due to the conformational changes that occur in the protein when calmodulin binds Ca2+ and interacts with the M13 peptide. The absolute pKa values for the Ca2+-free and Ca2+-saturated states of GCaMP6m are critical to its high signal-to-noise ratio. This mechanism has important implications for further improvements to GCaMP6m and potentially for other similarly designed biosensors.

Biophysical Journal, 2017

classification indicated that fecal samples were enriched in metabolism-related genes involved in... more classification indicated that fecal samples were enriched in metabolism-related genes involved in glycolysis, methionine degradation, maltose utilization, and butyrate production, while cloacal samples were enriched in aromatic metabolism, virulence genes, and membrane transporters. Our analysis was further supported by the recovery of protein sequences for phylogenetic analysis, such as the RNA polymerase of Delftia, a Shigella toxin, a nitrate reductase of Delftia, and beta lactamases from several species. Our work identifies several strategies by which metagenomic sequencing of condor microbiomes may inform us about condor diet, infections, intoxication, and other types of stress.

Biophysical journal, Jan 7, 2017

Several series of near-infrared (NIR) fluorescent proteins (FPs) were recently engineered from ba... more Several series of near-infrared (NIR) fluorescent proteins (FPs) were recently engineered from bacterial phytochromes but were not systematically compared in neurons. To fluoresce, NIR FPs utilize an enzymatic derivative of heme, the linear tetrapyrrole biliverdin, as a chromophore whose level in neurons is poorly studied. Here, we evaluated NIR FPs of the iRFP protein family, which were reported to be the brightest in non-neuronal mammalian cells, in primary neuronal culture, in brain slices of mouse and monkey, and in mouse brain in vivo. We applied several fluorescence imaging modes, such as wide-field and confocal one-photon and two-photon microscopy, to compare photochemical and biophysical properties of various iRFPs. The iRFP682 and iRFP670 proteins exhibited the highest brightness and photostability under one-photon and two-photon excitation modes, respectively. All studied iRFPs exhibited efficient binding of the endogenous biliverdin chromophore in cultured neurons and in ...

J-aggregates of tiacarbocyanine derivative are investigated in frozen solution at low temperature... more J-aggregates of tiacarbocyanine derivative are investigated in frozen solution at low temperatures by optical spectroscopy and spectral hole burning. It is found that absorption and luminescence bands have inhomogeneous widths of 220 and 130 cm-1 at 5 K respectively used in contrast to pseudoisocyanine (PIC) J-bands are separated by Stokes shift of 100 cm-1. Moreover, J-aggregate fluorescence spectrum depends drastically

Proceedings of Spie the International Society For Optical Engineering, Feb 1, 2009

We report simultaneous two-photon absorption (2PA) spectra in a series of substituted corroles an... more We report simultaneous two-photon absorption (2PA) spectra in a series of substituted corroles and related porphyrins in 800-1400 nm laser wavelength range. Compared to the porphyrins, the 2PA spectrum of corroles contains a distinct and relatively high intensity peak, sigma(2) = 60-130 GM, close to twice the wavelength of Soret maximum (800-850 nm). The increase of 2PA peak cross section is explained in terms of decreased symmetry of the contracted macro-cycle, and is most likely related to relaxing of the parity selection rules that restrict 2PA in the Soret band for more symmetrical porphyrins. We also observe that the strength of the 2PA peak in Soret region decreases with the electron-withdrawing ability (increasing Hammett constant) of the side substituents, which can be explained by assuming that the corrole core itself possesses electron-accepting ability. The peak 2PA cross sections in the Q-region are much less than in Soret-region, and can be quantitatively described within the two-level approximation taking into account permanent dipole moments in the ground and excited states.