Viruthachalam Thiagarajan | Bharathidasan University (original) (raw)
Papers by Viruthachalam Thiagarajan
A rational design of the novel phenanthro[9,10-d]imidazole based pH sensing fluorophore PITP was ... more A rational design of the novel phenanthro[9,10-d]imidazole based pH sensing fluorophore PITP was developed, in which phenanthroimidazole acts as a donor and pyridine acts as an acceptor. Various spectral studies confirm that the protonation occurs at two different nitrogen centres, each of which produces an observable color change depending on the pH of the buffer solution. DFT calculations also support the experimental results. We show that PITP can be used as a ratiometric and colorimetric pH sensor in the acidic region.
Novel triphenylamine-based star-like molecules 11 were synthesized and characterized using FT-IR,... more Novel triphenylamine-based star-like molecules 11 were synthesized and characterized using FT-IR, 1H,
13C, and DEPT-135 NMR spectroscopy, and MALDI-TOF mass spectrometry. The absorption and
emission spectra of star-like molecules were studied in different solvents. The effect of solvent polarity
and aggregation studies on the absorption and emission spectra has also been studied. The new starlike
molecules are found to exhibit broad absorption and emission band along with intramolecular
charge transfer character. The fluorescence spectra of triphenylamine derivatives shift from blue to
green wavelength on increasing the extended conjugation of the molecule. The experimental results
indicate that there is cooperative enhancement originating from the inter-branch coupling and an
increase in the light-harvesting ability upon increasing the conjugated molecule size.
Activation of plasminogen to its active form plasmin is essential for several key mechanisms, inc... more Activation of plasminogen to its active form plasmin is essential for several key mechanisms, including the dissolution of blood clots. Activation occurs naturally via enzymatic proteolysis. We report that activation can be achieved with 280 nm light. A 2.6 fold increase in proteolytic activity was observed after 10 min illumination of human plasminogen. Irradiance levels used are in the same order of magnitude of the UVB solar irradiance. Activation is correlated with light induced disruption of disulphide bridges upon UVB excitation of the aromatic residues and with the formation of photochemical products, e.g. dityrosine and N-formylkynurenine. Most of the protein fold is maintained after 10 min illumination since no major changes are observed in the near-UV CD spectrum. Far-UV CD shows loss of secondary structure after illumination (33.4% signal loss at 206 nm). Thermal unfolding CD studies show that plasminogen retains a native like cooperative transition at ~70 ºC after UV-illumination. We propose that UVB activation of plasminogen occurs upon photo-cleavage of a functional allosteric disulphide bond, Cys737-Cys765, located in the catalytic domain and in van der Waals contact with Trp761 (4.3 Å). Such proximity makes its disruption very likely, which may occur upon electron transfer from excited Trp761. Reduction of Cys737-Cys765 will result in likely conformational changes in the catalytic site. Molecular dynamics simulations reveal that reduction of Cys737-Cys765 in plasminogen leads to an increase of the fluctuations of loop 760–765, the S1-entrance frame located close to the active site. These fluctuations affect the range of solvent exposure of the catalytic triad, particularly of Asp646 and Ser74, which acquire an exposure profile similar to the values in plasmin. The presented photonic mechanism of plasminogen activation has the potential to be used in clinical applications, possibly together with other enzymatic treatments for the elimination of blood clots.
The epidermal growth factor receptor (EGFR) is a member of the ErbB family of receptor tyrosine k... more The epidermal growth factor receptor (EGFR) is a member of the ErbB family of receptor tyrosine kinases. EGFR is activated
upon binding to e.g. epidermal growth factor (EGF), leading to cell survival, proliferation and migration. EGFR overactivation
is associated with tumor progression. We have previously shown that low dose UVB illumination of cancer cells
overexpressing EGFR prior to adding EGF halted the EGFR signaling pathway. We here show that UVB illumination of the
extracellular domain of EGFR (sEGFR) induces protein conformational changes, disulphide bridge breakage and formation of
tryptophan and tyrosine photoproducts such as dityrosine, N-formylkynurenine and kynurenine. Fluorescence
spectroscopy, circular dichroism and thermal studies confirm the occurrence of conformational changes. An immunoassay
has confirmed that UVB light induces structural changes in the EGF binding site. A monoclonal antibody which competes
with EGF for binding sEGFR was used. We report clear evidence that UVB light induces structural changes in EGFR that
impairs the correct binding of an EGFR specific antibody that competes with EGF for binding EGFR, confirming that the 3D
structure of the EGFR binding domain suffered conformational changes upon UV illumination. The irradiance used is in the
same order of magnitude as the integrated intensity in the solar UVB range. The new photonic technology disables a key
receptor and is most likely applicable to the treatment of various types of cancer, alone or in combination with other therapies.
Resistance rates are increasing among several problematic Gram-negative pathogens, a fact that ha... more Resistance rates are increasing among several problematic Gram-negative pathogens, a fact that has encouraged the
development of new antimicrobial agents. This paper characterizes a Salmonella phage endolysin (Lys68) and demonstrates
its potential antimicrobial effectiveness when combined with organic acids towards Gram-negative pathogens. Biochemical
characterization reveals that Lys68 is more active at pH 7.0, maintaining 76.7% of its activity when stored at 4uC for two
months. Thermostability tests showed that Lys68 is only completely inactivated upon exposure to 100uC for 30 min, and
circular dichroism analysis demonstrated the ability to refold into its original conformation upon thermal denaturation. It
was shown that Lys68 is able to lyse a wide panel of Gram-negative bacteria (13 different species) in combination with the
outer membrane permeabilizers EDTA, citric and malic acid. While the EDTA/Lys68 combination only inactivated
Pseudomonas strains, the use of citric or malic acid broadened Lys68 antibacterial effect to other Gram-negative pathogens
(lytic activity against 9 and 11 species, respectively). Particularly against Salmonella Typhimurium LT2, the combinatory
effect of malic or citric acid with Lys68 led to approximately 3 to 5 log reductions in bacterial load/CFUs after 2 hours,
respectively, and was also able to reduce stationary-phase cells and bacterial biofilms by approximately 1 log. The broad
killing capacity of malic/citric acid-Lys68 is explained by the destabilization and major disruptions of the cell outer
membrane integrity due to the acidity caused by the organic acids and a relatively high muralytic activity of Lys68 at low
pH. Lys68 demonstrates good (thermo)stability properties that combined with different outer membrane permeabilizers,
could become useful to combat Gram-negative pathogens in agricultural, food and medical industry.
In the present work we explored the ABP-CM4 peptide properties from Bombyx mori for the creation ... more In the present work we explored the ABP-CM4 peptide properties from Bombyx mori for the creation of biopolymers with broad antimicrobial activity. An antimicrobial recombinant protein-based polymer (rPBP) was designed by cloning the DNA sequence coding for ABP-CM4 in frame with the N-terminus of the elastin-like recombinamer consisting of 200 repetitions of the pentamer VPAVG, here named A200. The new rPBP, named CM4-A200, was purified via a simplified nonchromatographic method, making use of the thermoresponsive behavior of the A200 polymer. ABP-CM4 peptide was also purified through the incorporation of a formic acid cleavage site between the peptide and the A200 sequence. In soluble state the antimicrobial activity of both CM4-A200 polymer and ABP-CM4 peptide was poorly effective. However, when the CM4-A200 polymer was processed into free-standing films high antimicrobial activity against Gram-positive and Gram-negative bacteria, yeasts and filamentous fungi was observed. The antimicrobial activity of CM4-A200 was dependent on the physical contact of cells with the film surface. Furthermore, CM4-A200 films did not reveal a cytotoxic effect against both normal human skin fibroblasts and human keratinocytes. Finally, we have developed an optimized ex vivo assay with pig skin demonstrating the antimicrobial properties of the CM4-A200 cast films for skin applications.
![Research paper thumbnail of 10-[2-(Dimethylamino)ethyl]-9-(4-methoxyphenyl)-3,3,6,6-tetramethyl-3,4,6,7,9,10-hexahydroacridine-1,8(2 H ,5 H )-dione](https://attachments.academia-assets.com/49008356/thumbnails/1.jpg)
](Acta Crystallographica Section E-structure Reports Online, 2009
Biochemistry, 2010
CPD photolyase enzymatically repairs the major UV-induced lesion in DNA, the cyclobutane pyrimidi... more CPD photolyase enzymatically repairs the major UV-induced lesion in DNA, the cyclobutane pyrimidine dimer (CPD), by photoreversion of the damage reaction. An enzyme-bound reduced flavin (FADH -) cofactor functions as photosensitizer. Upon excitation, it transiently transfers an electron to the CPD, triggering scission of the interpyrimidine bonds. After repair completion, the electron returns to the flavin to restore its functional reduced form. A major difficulty for time-resolved spectroscopic monitoring of the enzymatic repair reaction is that absorption changes around 265 nm accompanying pyrimidine restoration are obscured by the strong background absorption of the nondimerized bases in DNA. Here we present a novel substrate for CPD photolyase that absorbs only weakly around 265 nm: a modified thymidine 10-mer with a central CPD and all bases, except the one at the 3 0 end, replaced by 5,6-dihydrothymine which virtually does not absorb around 265 nm. Repair of this substrate by photolyases from Anacystis nidulans and from Escherichia coli was compared with repair of two conventional substrates: a 10-mer of unmodified thymidines containing a central CPD and an acetone-sensitized thymidine 18-mer that contained in average six randomly distributed CPDs per strand. In all cases, the novel substrate was repaired with an efficiency very similar to that of the conventional substrates (quantum yields in the order of 0.5 upon excitation of FADH -). Flashinduced transient absorption changes at 267 nm could be recorded on a millisecond time scale with a single subsaturating flash and yielded very similar signals for all three substrates. Because of its low background absorption around 265 nm and the defined structure, the novel substrate is a promising tool for fast and ultrafast transient absorption studies on pyrimidine dimer splitting by CPD photolyase. † This work was supported by Agence Nationale de la Recherche grant
Fluorescence microscopy is characterized by low background noise, thus a fluorescent object appea... more Fluorescence microscopy is characterized by low background noise, thus a fluorescent object appears as an area of high signal/noise. Thermal gradients may result in apparent motion of the object, leading to a blurred image. Here, we have developed an image processing methodology that may remove/reduce blur significantly for any type of microscopy. A total of ~100 images were acquired with a pixel size of 30 nm. The acquisition time for each image was approximately 1 second. We can quantity the drift in X and Y using the sub pixel accuracy computed centroid location of an image object in each frame. We can measure drifts down to approximately 10 nm in size and a drift-compensated image can therefore be reconstructed on a grid of the same size using the "Shift and Add" approach leading to an image of identical size as the individual image. We have also reconstructed the image using a 3 fold larger grid with a pixel size of 10 nm. The resulting images reveal details at the diffraction limit. In principle we can only compensate for inter-image driftthus the drift that takes place during the acquisition time for the individual image is not corrected. We believe that our results are of general applicability in microscopy and other types of imaging. A prerequisite for our method is the presence of a trackable object in the image such as a cell nucleus. Downloaded From: http://proceedings.spiedigitallibrary.org/ on 03/07/2013 Terms of Use: http://spiedl.org/terms Proc. of SPIE Vol. 8587 85871H-4 Downloaded From: http://proceedings.spiedigitallibrary.org/ on 03/07/2013 Terms of Use: http://spiedl.org/terms Proc. of SPIE Vol. 8587 85871H-5 Downloaded From: http://proceedings.spiedigitallibrary.org/ on 03/07/2013 Terms of Use: http://spiedl.org/terms
Transient absorption spectroscopy is a powerful tool for the study of photoreactions on time scal... more Transient absorption spectroscopy is a powerful tool for the study of photoreactions on time scales from femtoseconds to seconds. Typically, reactions slower than ϳ1 ns are recorded by the "classical" technique; the reaction is triggered by an excitation flash, and absorption changes accompanying the reaction are recorded in real time using a continuous monitoring light beam and a detection system with sufficiently fast response. The pico-and femtosecond region can be accessed by the more recent "pump-probe" technique, which circumvents the difficulties of real time detection on a subnanosecond time scale. This is paid for by accumulation of an excessively large number of shots to sample the reaction kinetics. Hence, it is of interest to extend the classical real time technique as far as possible to the subnanosecond range. In order to identify and minimize detection artifacts common on a subnanosecond scale, like overshoot, ringing, and signal reflections, rigorous testing is required of how the detection system responds to fast changes of the monitoring light intensity. Here, we introduce a novel method to create standard signals for detector fidelity testing on a time scale from a few picoseconds to tens of nanoseconds. The signals result from polarized measurements of absorption changes upon excitation of ruthenium complexes ͕͓Ru͑bpy͒ 3 ͔ 2+ and a less symmetric derivative͖ by a short laser flash. Two types of signals can be created depending on the polarization of the monitoring light with respect to that of the excitation flash: a fast steplike bleaching at magic angle and a monoexponentially decaying bleaching for parallel polarizations. The lifetime of the decay can be easily varied via temperature and viscosity of the solvent. The method is applied to test the performance of a newly developed real time transient absorption setup with 300 ps time resolution and high sensitivity.
The light-dependent DNA repair enzyme photolyase contains a unique evolutionary conserved triple ... more The light-dependent DNA repair enzyme photolyase contains a unique evolutionary conserved triple tryptophan electron transfer chain (W382-W359-W306 in photolyase from E. coli) that bridges the ∼15 Å distance between the buried flavin adenine dinucleotide (FAD) cofactor and the surface of the protein. Upon excitation of the semireduced flavin (FADH°), electron transfer through the chain leads to formation of fully reduced flavin (FADH -; required for DNA repair) and oxidation of the most remote tryptophan residue W306, followed by its deprotonation. The thus-formed tryptophanyl radical W306°+ is reduced either by an extrinsic reductant or by reverse electron transfer from FADH -. Altogether the kinetics of these charge transfer reactions span 10 orders of magnitude, from a few picoseconds to tens of milliseconds. We investigated electron transfer processes in the picosecond-nanosecond time window bridging the time domains covered by ultrafast pump-probe and "classical" continuous probe techniques. Using a recent dedicated setup, we directly show that virtually no absorption change between 300 ps and 10 ns occurs in wild-type photolyase, implying that no charge recombination takes place in this time window. In contrast, W306F mutant photolyase showed a partial absorption recovery with a time constant of 0.85 ns. In wild-type photolyase, the quantum yield of FADH -W306°+ was found at 19 ( 4%, in reference to the established quantum yield of the long-lived excited state of [Ru(bpy) 3 ] 2+ . With this yield, the optical spectrum of the excited state of FADH°can be constructed from ultrafast spectroscopic data; this spectrum is dominated by excited state absorption extending from below 450 to 850 nm. The new experimental results, taken together with previous data, allow us to propose a detailed kinetic and energetic scheme of the electron transfer chain. † Part of the "Benoît Soep Festschrift".
CPD photolyase enzymatically repairs the major UV-induced lesion in DNA, the cyclobutane pyrimidi... more CPD photolyase enzymatically repairs the major UV-induced lesion in DNA, the cyclobutane pyrimidine dimer (CPD), by photoreversion of the damage reaction. An enzyme-bound reduced flavin (FADH -) cofactor functions as photosensitizer. Upon excitation, it transiently transfers an electron to the CPD, triggering scission of the interpyrimidine bonds. After repair completion, the electron returns to the flavin to restore its functional reduced form. A major difficulty for time-resolved spectroscopic monitoring of the enzymatic repair reaction is that absorption changes around 265 nm accompanying pyrimidine restoration are obscured by the strong background absorption of the nondimerized bases in DNA. Here we present a novel substrate for CPD photolyase that absorbs only weakly around 265 nm: a modified thymidine 10-mer with a central CPD and all bases, except the one at the 3 0 end, replaced by 5,6-dihydrothymine which virtually does not absorb around 265 nm. Repair of this substrate by photolyases from Anacystis nidulans and from Escherichia coli was compared with repair of two conventional substrates: a 10-mer of unmodified thymidines containing a central CPD and an acetone-sensitized thymidine 18-mer that contained in average six randomly distributed CPDs per strand. In all cases, the novel substrate was repaired with an efficiency very similar to that of the conventional substrates (quantum yields in the order of 0.5 upon excitation of FADH -). Flashinduced transient absorption changes at 267 nm could be recorded on a millisecond time scale with a single subsaturating flash and yielded very similar signals for all three substrates. Because of its low background absorption around 265 nm and the defined structure, the novel substrate is a promising tool for fast and ultrafast transient absorption studies on pyrimidine dimer splitting by CPD photolyase. † This work was supported by Agence Nationale de la Recherche grant
This article contains supporting information online at www.pnas.org/lookup/suppl/
Journal of Photochemistry and Photobiology A-chemistry, 2003
Photophysics and photochemistry of acridinedione (ADD) dyes doped in PMMA matrix have been studie... more Photophysics and photochemistry of acridinedione (ADD) dyes doped in PMMA matrix have been studied using fluorescence and flash photolysis technique. The absorption and emission spectra of acridinedione dyes in PMMA matrix are broad. A pronounced influence of λexc on the red shift of ADD fluorescence spectra was found which is independent of the various substituents in the ADD dyes. This observed shift was explained by the inhomogeneity in the PMMA matrix site results in the migration of the electronic excitation energy from the blue centres to red ones. The inhomogeneous distribution of ADD dyes in PMMA matrix causes the non-exponential fluorescence decay. The triplet lifetimes of these dyes in polymer matrix is found to be in millisecond time domain, whereas in solution the triplet lifetime is in microseconds. Laser flash photolysis shows that the photoionisation of ADD dyes in PMMA matrix results in the formation of an electron and a cation radical. Steady state photolysis of ADD dyes in PMMA matrix was carried out. ADD dyes have higher photostability in PMMA matrix than in solution.
Spectrochimica Acta Part A-molecular and Biomolecular Spectroscopy, 2007
Newly synthesised fluorescent chemosensor ADDTU contains the thiourea receptor connected to the a... more Newly synthesised fluorescent chemosensor ADDTU contains the thiourea receptor connected to the acridinedione (ADD) fluorophore via a covalent bond, giving rise to a fluorophore-receptor motif. In this fluorescent chemosensor, the anion recognition takes place at the receptor site which result in the concomitant changes in the photophysical properties of a ADD fluorophore by modulation of photoinduced electron transfer (PET) process. The binding ability of these sensor with the anions F−, Cl−, Br−, I−, HSO4−, ClO4−, AcO−, H2PO4− and BF4− (as their tetrabutylammounium salts) in acetonitrile were investigated using UV–vis, steady state and time-resolved emission techniques. ADDTU system allows for the selective fluorescent sensing of AcO−, H2PO4− and F− over other anions in acetonitrile.
Chemical Physics Letters, 2003
The hydrogen-bonding interaction between acridinedione dye (ADD) and urea dimer in methanol is re... more The hydrogen-bonding interaction between acridinedione dye (ADD) and urea dimer in methanol is reported by using steady state and time resolved fluorescence techniques.
Journal of Luminescence, 2007
We present a simple but highly specific acridinedione fluorophore (ADD-1) that acts both as a flu... more We present a simple but highly specific acridinedione fluorophore (ADD-1) that acts both as a fluorescent and colorimetric sensor for anions in acetonitrile. The specific optical signalling of ADD-1 is due to the formation of new distinct intramolecular charge transfer (ICT) emitting states in the presence of AcO− (490 nm), H2PO4− (440 nm), and F− (510 nm) over other anions. Presence of F− shows a colour change that is perceptible to the naked eye, from colourless to an intense fluorescent green due to the deprotonation of acridinedione ring amino hydrogen.
A rational design of the novel phenanthro[9,10-d]imidazole based pH sensing fluorophore PITP was ... more A rational design of the novel phenanthro[9,10-d]imidazole based pH sensing fluorophore PITP was developed, in which phenanthroimidazole acts as a donor and pyridine acts as an acceptor. Various spectral studies confirm that the protonation occurs at two different nitrogen centres, each of which produces an observable color change depending on the pH of the buffer solution. DFT calculations also support the experimental results. We show that PITP can be used as a ratiometric and colorimetric pH sensor in the acidic region.
Novel triphenylamine-based star-like molecules 11 were synthesized and characterized using FT-IR,... more Novel triphenylamine-based star-like molecules 11 were synthesized and characterized using FT-IR, 1H,
13C, and DEPT-135 NMR spectroscopy, and MALDI-TOF mass spectrometry. The absorption and
emission spectra of star-like molecules were studied in different solvents. The effect of solvent polarity
and aggregation studies on the absorption and emission spectra has also been studied. The new starlike
molecules are found to exhibit broad absorption and emission band along with intramolecular
charge transfer character. The fluorescence spectra of triphenylamine derivatives shift from blue to
green wavelength on increasing the extended conjugation of the molecule. The experimental results
indicate that there is cooperative enhancement originating from the inter-branch coupling and an
increase in the light-harvesting ability upon increasing the conjugated molecule size.
Activation of plasminogen to its active form plasmin is essential for several key mechanisms, inc... more Activation of plasminogen to its active form plasmin is essential for several key mechanisms, including the dissolution of blood clots. Activation occurs naturally via enzymatic proteolysis. We report that activation can be achieved with 280 nm light. A 2.6 fold increase in proteolytic activity was observed after 10 min illumination of human plasminogen. Irradiance levels used are in the same order of magnitude of the UVB solar irradiance. Activation is correlated with light induced disruption of disulphide bridges upon UVB excitation of the aromatic residues and with the formation of photochemical products, e.g. dityrosine and N-formylkynurenine. Most of the protein fold is maintained after 10 min illumination since no major changes are observed in the near-UV CD spectrum. Far-UV CD shows loss of secondary structure after illumination (33.4% signal loss at 206 nm). Thermal unfolding CD studies show that plasminogen retains a native like cooperative transition at ~70 ºC after UV-illumination. We propose that UVB activation of plasminogen occurs upon photo-cleavage of a functional allosteric disulphide bond, Cys737-Cys765, located in the catalytic domain and in van der Waals contact with Trp761 (4.3 Å). Such proximity makes its disruption very likely, which may occur upon electron transfer from excited Trp761. Reduction of Cys737-Cys765 will result in likely conformational changes in the catalytic site. Molecular dynamics simulations reveal that reduction of Cys737-Cys765 in plasminogen leads to an increase of the fluctuations of loop 760–765, the S1-entrance frame located close to the active site. These fluctuations affect the range of solvent exposure of the catalytic triad, particularly of Asp646 and Ser74, which acquire an exposure profile similar to the values in plasmin. The presented photonic mechanism of plasminogen activation has the potential to be used in clinical applications, possibly together with other enzymatic treatments for the elimination of blood clots.
The epidermal growth factor receptor (EGFR) is a member of the ErbB family of receptor tyrosine k... more The epidermal growth factor receptor (EGFR) is a member of the ErbB family of receptor tyrosine kinases. EGFR is activated
upon binding to e.g. epidermal growth factor (EGF), leading to cell survival, proliferation and migration. EGFR overactivation
is associated with tumor progression. We have previously shown that low dose UVB illumination of cancer cells
overexpressing EGFR prior to adding EGF halted the EGFR signaling pathway. We here show that UVB illumination of the
extracellular domain of EGFR (sEGFR) induces protein conformational changes, disulphide bridge breakage and formation of
tryptophan and tyrosine photoproducts such as dityrosine, N-formylkynurenine and kynurenine. Fluorescence
spectroscopy, circular dichroism and thermal studies confirm the occurrence of conformational changes. An immunoassay
has confirmed that UVB light induces structural changes in the EGF binding site. A monoclonal antibody which competes
with EGF for binding sEGFR was used. We report clear evidence that UVB light induces structural changes in EGFR that
impairs the correct binding of an EGFR specific antibody that competes with EGF for binding EGFR, confirming that the 3D
structure of the EGFR binding domain suffered conformational changes upon UV illumination. The irradiance used is in the
same order of magnitude as the integrated intensity in the solar UVB range. The new photonic technology disables a key
receptor and is most likely applicable to the treatment of various types of cancer, alone or in combination with other therapies.
Resistance rates are increasing among several problematic Gram-negative pathogens, a fact that ha... more Resistance rates are increasing among several problematic Gram-negative pathogens, a fact that has encouraged the
development of new antimicrobial agents. This paper characterizes a Salmonella phage endolysin (Lys68) and demonstrates
its potential antimicrobial effectiveness when combined with organic acids towards Gram-negative pathogens. Biochemical
characterization reveals that Lys68 is more active at pH 7.0, maintaining 76.7% of its activity when stored at 4uC for two
months. Thermostability tests showed that Lys68 is only completely inactivated upon exposure to 100uC for 30 min, and
circular dichroism analysis demonstrated the ability to refold into its original conformation upon thermal denaturation. It
was shown that Lys68 is able to lyse a wide panel of Gram-negative bacteria (13 different species) in combination with the
outer membrane permeabilizers EDTA, citric and malic acid. While the EDTA/Lys68 combination only inactivated
Pseudomonas strains, the use of citric or malic acid broadened Lys68 antibacterial effect to other Gram-negative pathogens
(lytic activity against 9 and 11 species, respectively). Particularly against Salmonella Typhimurium LT2, the combinatory
effect of malic or citric acid with Lys68 led to approximately 3 to 5 log reductions in bacterial load/CFUs after 2 hours,
respectively, and was also able to reduce stationary-phase cells and bacterial biofilms by approximately 1 log. The broad
killing capacity of malic/citric acid-Lys68 is explained by the destabilization and major disruptions of the cell outer
membrane integrity due to the acidity caused by the organic acids and a relatively high muralytic activity of Lys68 at low
pH. Lys68 demonstrates good (thermo)stability properties that combined with different outer membrane permeabilizers,
could become useful to combat Gram-negative pathogens in agricultural, food and medical industry.
In the present work we explored the ABP-CM4 peptide properties from Bombyx mori for the creation ... more In the present work we explored the ABP-CM4 peptide properties from Bombyx mori for the creation of biopolymers with broad antimicrobial activity. An antimicrobial recombinant protein-based polymer (rPBP) was designed by cloning the DNA sequence coding for ABP-CM4 in frame with the N-terminus of the elastin-like recombinamer consisting of 200 repetitions of the pentamer VPAVG, here named A200. The new rPBP, named CM4-A200, was purified via a simplified nonchromatographic method, making use of the thermoresponsive behavior of the A200 polymer. ABP-CM4 peptide was also purified through the incorporation of a formic acid cleavage site between the peptide and the A200 sequence. In soluble state the antimicrobial activity of both CM4-A200 polymer and ABP-CM4 peptide was poorly effective. However, when the CM4-A200 polymer was processed into free-standing films high antimicrobial activity against Gram-positive and Gram-negative bacteria, yeasts and filamentous fungi was observed. The antimicrobial activity of CM4-A200 was dependent on the physical contact of cells with the film surface. Furthermore, CM4-A200 films did not reveal a cytotoxic effect against both normal human skin fibroblasts and human keratinocytes. Finally, we have developed an optimized ex vivo assay with pig skin demonstrating the antimicrobial properties of the CM4-A200 cast films for skin applications.
Acta Crystallographica Section E-structure Reports Online, 2009
Biochemistry, 2010
CPD photolyase enzymatically repairs the major UV-induced lesion in DNA, the cyclobutane pyrimidi... more CPD photolyase enzymatically repairs the major UV-induced lesion in DNA, the cyclobutane pyrimidine dimer (CPD), by photoreversion of the damage reaction. An enzyme-bound reduced flavin (FADH -) cofactor functions as photosensitizer. Upon excitation, it transiently transfers an electron to the CPD, triggering scission of the interpyrimidine bonds. After repair completion, the electron returns to the flavin to restore its functional reduced form. A major difficulty for time-resolved spectroscopic monitoring of the enzymatic repair reaction is that absorption changes around 265 nm accompanying pyrimidine restoration are obscured by the strong background absorption of the nondimerized bases in DNA. Here we present a novel substrate for CPD photolyase that absorbs only weakly around 265 nm: a modified thymidine 10-mer with a central CPD and all bases, except the one at the 3 0 end, replaced by 5,6-dihydrothymine which virtually does not absorb around 265 nm. Repair of this substrate by photolyases from Anacystis nidulans and from Escherichia coli was compared with repair of two conventional substrates: a 10-mer of unmodified thymidines containing a central CPD and an acetone-sensitized thymidine 18-mer that contained in average six randomly distributed CPDs per strand. In all cases, the novel substrate was repaired with an efficiency very similar to that of the conventional substrates (quantum yields in the order of 0.5 upon excitation of FADH -). Flashinduced transient absorption changes at 267 nm could be recorded on a millisecond time scale with a single subsaturating flash and yielded very similar signals for all three substrates. Because of its low background absorption around 265 nm and the defined structure, the novel substrate is a promising tool for fast and ultrafast transient absorption studies on pyrimidine dimer splitting by CPD photolyase. † This work was supported by Agence Nationale de la Recherche grant
Fluorescence microscopy is characterized by low background noise, thus a fluorescent object appea... more Fluorescence microscopy is characterized by low background noise, thus a fluorescent object appears as an area of high signal/noise. Thermal gradients may result in apparent motion of the object, leading to a blurred image. Here, we have developed an image processing methodology that may remove/reduce blur significantly for any type of microscopy. A total of ~100 images were acquired with a pixel size of 30 nm. The acquisition time for each image was approximately 1 second. We can quantity the drift in X and Y using the sub pixel accuracy computed centroid location of an image object in each frame. We can measure drifts down to approximately 10 nm in size and a drift-compensated image can therefore be reconstructed on a grid of the same size using the "Shift and Add" approach leading to an image of identical size as the individual image. We have also reconstructed the image using a 3 fold larger grid with a pixel size of 10 nm. The resulting images reveal details at the diffraction limit. In principle we can only compensate for inter-image driftthus the drift that takes place during the acquisition time for the individual image is not corrected. We believe that our results are of general applicability in microscopy and other types of imaging. A prerequisite for our method is the presence of a trackable object in the image such as a cell nucleus. Downloaded From: http://proceedings.spiedigitallibrary.org/ on 03/07/2013 Terms of Use: http://spiedl.org/terms Proc. of SPIE Vol. 8587 85871H-4 Downloaded From: http://proceedings.spiedigitallibrary.org/ on 03/07/2013 Terms of Use: http://spiedl.org/terms Proc. of SPIE Vol. 8587 85871H-5 Downloaded From: http://proceedings.spiedigitallibrary.org/ on 03/07/2013 Terms of Use: http://spiedl.org/terms
Transient absorption spectroscopy is a powerful tool for the study of photoreactions on time scal... more Transient absorption spectroscopy is a powerful tool for the study of photoreactions on time scales from femtoseconds to seconds. Typically, reactions slower than ϳ1 ns are recorded by the "classical" technique; the reaction is triggered by an excitation flash, and absorption changes accompanying the reaction are recorded in real time using a continuous monitoring light beam and a detection system with sufficiently fast response. The pico-and femtosecond region can be accessed by the more recent "pump-probe" technique, which circumvents the difficulties of real time detection on a subnanosecond time scale. This is paid for by accumulation of an excessively large number of shots to sample the reaction kinetics. Hence, it is of interest to extend the classical real time technique as far as possible to the subnanosecond range. In order to identify and minimize detection artifacts common on a subnanosecond scale, like overshoot, ringing, and signal reflections, rigorous testing is required of how the detection system responds to fast changes of the monitoring light intensity. Here, we introduce a novel method to create standard signals for detector fidelity testing on a time scale from a few picoseconds to tens of nanoseconds. The signals result from polarized measurements of absorption changes upon excitation of ruthenium complexes ͕͓Ru͑bpy͒ 3 ͔ 2+ and a less symmetric derivative͖ by a short laser flash. Two types of signals can be created depending on the polarization of the monitoring light with respect to that of the excitation flash: a fast steplike bleaching at magic angle and a monoexponentially decaying bleaching for parallel polarizations. The lifetime of the decay can be easily varied via temperature and viscosity of the solvent. The method is applied to test the performance of a newly developed real time transient absorption setup with 300 ps time resolution and high sensitivity.
The light-dependent DNA repair enzyme photolyase contains a unique evolutionary conserved triple ... more The light-dependent DNA repair enzyme photolyase contains a unique evolutionary conserved triple tryptophan electron transfer chain (W382-W359-W306 in photolyase from E. coli) that bridges the ∼15 Å distance between the buried flavin adenine dinucleotide (FAD) cofactor and the surface of the protein. Upon excitation of the semireduced flavin (FADH°), electron transfer through the chain leads to formation of fully reduced flavin (FADH -; required for DNA repair) and oxidation of the most remote tryptophan residue W306, followed by its deprotonation. The thus-formed tryptophanyl radical W306°+ is reduced either by an extrinsic reductant or by reverse electron transfer from FADH -. Altogether the kinetics of these charge transfer reactions span 10 orders of magnitude, from a few picoseconds to tens of milliseconds. We investigated electron transfer processes in the picosecond-nanosecond time window bridging the time domains covered by ultrafast pump-probe and "classical" continuous probe techniques. Using a recent dedicated setup, we directly show that virtually no absorption change between 300 ps and 10 ns occurs in wild-type photolyase, implying that no charge recombination takes place in this time window. In contrast, W306F mutant photolyase showed a partial absorption recovery with a time constant of 0.85 ns. In wild-type photolyase, the quantum yield of FADH -W306°+ was found at 19 ( 4%, in reference to the established quantum yield of the long-lived excited state of [Ru(bpy) 3 ] 2+ . With this yield, the optical spectrum of the excited state of FADH°can be constructed from ultrafast spectroscopic data; this spectrum is dominated by excited state absorption extending from below 450 to 850 nm. The new experimental results, taken together with previous data, allow us to propose a detailed kinetic and energetic scheme of the electron transfer chain. † Part of the "Benoît Soep Festschrift".
CPD photolyase enzymatically repairs the major UV-induced lesion in DNA, the cyclobutane pyrimidi... more CPD photolyase enzymatically repairs the major UV-induced lesion in DNA, the cyclobutane pyrimidine dimer (CPD), by photoreversion of the damage reaction. An enzyme-bound reduced flavin (FADH -) cofactor functions as photosensitizer. Upon excitation, it transiently transfers an electron to the CPD, triggering scission of the interpyrimidine bonds. After repair completion, the electron returns to the flavin to restore its functional reduced form. A major difficulty for time-resolved spectroscopic monitoring of the enzymatic repair reaction is that absorption changes around 265 nm accompanying pyrimidine restoration are obscured by the strong background absorption of the nondimerized bases in DNA. Here we present a novel substrate for CPD photolyase that absorbs only weakly around 265 nm: a modified thymidine 10-mer with a central CPD and all bases, except the one at the 3 0 end, replaced by 5,6-dihydrothymine which virtually does not absorb around 265 nm. Repair of this substrate by photolyases from Anacystis nidulans and from Escherichia coli was compared with repair of two conventional substrates: a 10-mer of unmodified thymidines containing a central CPD and an acetone-sensitized thymidine 18-mer that contained in average six randomly distributed CPDs per strand. In all cases, the novel substrate was repaired with an efficiency very similar to that of the conventional substrates (quantum yields in the order of 0.5 upon excitation of FADH -). Flashinduced transient absorption changes at 267 nm could be recorded on a millisecond time scale with a single subsaturating flash and yielded very similar signals for all three substrates. Because of its low background absorption around 265 nm and the defined structure, the novel substrate is a promising tool for fast and ultrafast transient absorption studies on pyrimidine dimer splitting by CPD photolyase. † This work was supported by Agence Nationale de la Recherche grant
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Journal of Photochemistry and Photobiology A-chemistry, 2003
Photophysics and photochemistry of acridinedione (ADD) dyes doped in PMMA matrix have been studie... more Photophysics and photochemistry of acridinedione (ADD) dyes doped in PMMA matrix have been studied using fluorescence and flash photolysis technique. The absorption and emission spectra of acridinedione dyes in PMMA matrix are broad. A pronounced influence of λexc on the red shift of ADD fluorescence spectra was found which is independent of the various substituents in the ADD dyes. This observed shift was explained by the inhomogeneity in the PMMA matrix site results in the migration of the electronic excitation energy from the blue centres to red ones. The inhomogeneous distribution of ADD dyes in PMMA matrix causes the non-exponential fluorescence decay. The triplet lifetimes of these dyes in polymer matrix is found to be in millisecond time domain, whereas in solution the triplet lifetime is in microseconds. Laser flash photolysis shows that the photoionisation of ADD dyes in PMMA matrix results in the formation of an electron and a cation radical. Steady state photolysis of ADD dyes in PMMA matrix was carried out. ADD dyes have higher photostability in PMMA matrix than in solution.
Spectrochimica Acta Part A-molecular and Biomolecular Spectroscopy, 2007
Newly synthesised fluorescent chemosensor ADDTU contains the thiourea receptor connected to the a... more Newly synthesised fluorescent chemosensor ADDTU contains the thiourea receptor connected to the acridinedione (ADD) fluorophore via a covalent bond, giving rise to a fluorophore-receptor motif. In this fluorescent chemosensor, the anion recognition takes place at the receptor site which result in the concomitant changes in the photophysical properties of a ADD fluorophore by modulation of photoinduced electron transfer (PET) process. The binding ability of these sensor with the anions F−, Cl−, Br−, I−, HSO4−, ClO4−, AcO−, H2PO4− and BF4− (as their tetrabutylammounium salts) in acetonitrile were investigated using UV–vis, steady state and time-resolved emission techniques. ADDTU system allows for the selective fluorescent sensing of AcO−, H2PO4− and F− over other anions in acetonitrile.
Chemical Physics Letters, 2003
The hydrogen-bonding interaction between acridinedione dye (ADD) and urea dimer in methanol is re... more The hydrogen-bonding interaction between acridinedione dye (ADD) and urea dimer in methanol is reported by using steady state and time resolved fluorescence techniques.
Journal of Luminescence, 2007
We present a simple but highly specific acridinedione fluorophore (ADD-1) that acts both as a flu... more We present a simple but highly specific acridinedione fluorophore (ADD-1) that acts both as a fluorescent and colorimetric sensor for anions in acetonitrile. The specific optical signalling of ADD-1 is due to the formation of new distinct intramolecular charge transfer (ICT) emitting states in the presence of AcO− (490 nm), H2PO4− (440 nm), and F− (510 nm) over other anions. Presence of F− shows a colour change that is perceptible to the naked eye, from colourless to an intense fluorescent green due to the deprotonation of acridinedione ring amino hydrogen.