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 4°C for two months. Thermostability tests showed that Lys68 is only completely inactivated upon exposure to 100°C 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.

CPD photolyase uses light to repair cyclobutane pyrimidine dimers (CPDs) formed between adjacent pyrimidines in UV-irradiated DNA. The enzyme harbors an FAD cofactor in fully reduced state (FADH − ). The CPD repair mechanism involves electron transfer from photoexcited FADH − to the CPD, splitting of its intradimer bonds, and electron return to restore catalytically active FADH − . The two electron transfer processes occur on time scales of 10 −10 and 10 −9 s, respectively. Until now, CPD splitting itself has only been poorly characterized by experiments. Using a previously unreported transient absorption setup, we succeeded in monitoring cyclobutane thymine dimer repair in the main UV absorption band of intact thymine at 266 nm. Flavin transitions that overlay DNA-based absorption changes at 266 nm were monitored independently in the visible and subtracted to obtain the true repair kinetics. Restoration of intact thymine showed a short lag and a biexponential rise with time constants of 0.2 and 1.5 ns. We assign these two time constants to splitting of the intradimer bonds (creating one intact thymine and one thymine anion radical T ∘− ) and electron return from T ∘− to the FAD cofactor with recovery of the second thymine, respectively. Previous model studies and computer simulations yielded various CPD splitting times between <1 ps and <100 ns. Our experimental results should serve as a benchmark for future efforts to model enzymatic photorepair. The technique and methods developed here may be applied to monitor other photoreactions involving DNA.DNA repair | flavin adenine dinucleotide | transient absorption spectroscopy | UV damage E xposure of living organisms to UV light from the sun induces harmful lesions in DNA. To overcome this threat, specific repair enzymes have evolved, probably the most ancient and widespread one being CPD photolyase (1, 2). It uses light to repair cis-syn cyclobutane pyrimidine dimer (CPD) lesions, formed by a UV-induced [2 þ 2] cycloaddition of two adjacent pyrimidines (mostly thymines) in the same strand (Fig. 1). CPD photolyase has been found in organisms from all kingdoms of life, except placental mammals (including humans) that rely on nucleotide excision repair for CPD lesions. Among the various DNA repair mechanisms known, that of CPD photolyase is considered the most "cost-efficient" and least error-prone (3, 4). Note, however, that for CPDs containing cytosine, deamination of the cytosine may occur prior to repair. Uracil containing CPD cleavage by photolyase would then result in harmful cytosine-to-uracil mutations (5).Photolyase is a globular single chain protein of approximately 60 kDa that harbors two buried cofactors: flavin adenine dinucleotide (FAD) in its fully reduced state (FADH − ), which is the essential catalytic cofactor, and an antenna pigment, either a folate or a flavin derivative that absorbs blue or near UV light much stronger than FADH − does and efficiently transfers the excitation energy to FADH − .It is widely accepted (1, 2) that CPD repair by phot...

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 approximately 15 A distance between the buried flavin adenine dinucleotide (FAD) cofactor and the surface of the protein. Upon excitation of the semireduced flavin (FADH(o)), 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(o)(+) 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(o)(+) 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(o) 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.

A novel colorimetric and fluorescent chemosensor ADDTU-1 bearing dual receptor sites, which shows specific optical signaling for AcO-, H2PO4-, and F- over other anions and dual response toward AcO- and F- via PET and ICT mechanisms, is described. [structure: see text]

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.

In the field of environmental remediation and sustainability, the built-in electric field of ferroelectrics has been regarded as a promising strategy to enhance photocatalytic (PC) dye degradation and photoelectrochemical (PEC) water splitting. Here, we report on Ce-doped BaTiO 3 (BT) nanoassemblies prepared by a hydrothermal route. X-ray diffraction reveals the phase transformation from tetragonal to cubic on the sintering temperature and Ce doping. From X-ray photoelectron spectroscopy (XPS), the oxygen vacancies are found to be maximum for 4 mol % of Ce concentration. The ferroelectric and piezoelectric measurements disclose a higher remnant polarization (1.76 μC cm −2 ) and d 33 coefficient (15 pCN −1 ) at 4 mol % due to the built-in electric field. Thus, we observed a significantly improved PC dye degradation with the rate constant (k) of 0.0139 m −1 (methylene blue), 0.0147 m −1 (methyl violet) at 4 mol %, and 0.0117 m −1 (congo red) at 6 mol %. PEC water splitting showed that the photoanode fabricated at 4 mol % of Ce exhibits enriched photocurrent density (1.45 mA cm −2 ), impressive early onset of water oxidation (−0.504 V), and hydrogen gas evolution (22.50 μmol h −1 cm −2 ). Poling studies display a significant enhancement in both PC and PEC properties indicating the built-in electric field assisted activities of Ce-doped BT nanoassemblies. The underlying mechanisms behind the degradation efficiency and improved photocurrent density are established via the built-in electric field facilitating charge carrier detachment and transport as evidenced by the photoluminescence decay and XPS valence band spectra.

Magnetic iron oxide nanoparticles (MIONPs) play a major role in the emerging fields of nanotechnology to facilitate rapid advancements in biomedical and industrial platforms. The superparamagnetic properties of MIONPs and their environment friendly synthetic methods with well-defined particle size have become indispensable to obtain their full potential in a variety of applications ranging from cellular to diverse areas of biomedical science. Thus, the broadened scope and need for MIONPs in their demanding fields of applications required to be highlighted for a comprehensive understanding of their state-of-the-art. Many synthetic methods, however, do not entirely abolish their undesired cytotoxic effects caused by free radical production and high iron dosage. In addition, the agglomeration of MIONPs has also been a major problem. To alleviate these issues, suitable surface modification strategies adaptive to MIONPs has been suggested not only for the effective cytotoxicity control but also to minimize their agglomeration. The surface modification using inorganic and organic polymeric materials would represent an efficient strategy to utilize the diagnostic and therapeutic potentials of MIONPs in various human diseases including cancer. This review article elaborates the structural and magnetic properties of MIONPs, specifically magnetite, maghemite and hematite, followed by the important synthetic methods that can be exploited for biomedical approaches. The in vivo cytotoxic effects and the possible surface modifications employed to eliminate the cytotoxicity thereby enhancing the nanoparticle efficacy are also critically discussed. The roles and applications of surface modified MIONPs in medical and industrial platforms have been described for the benefits of global well-being.

Absorption and emission spectra of 9-N,N-dimethylaniline decahydroacridinedione (DMAADD) have been studied in different solvents. The fluorescence spectra of DMAADD are found to exhibit dual emission in aprotic solvents and single emission in protic solvents. The effect of solvent polarity and viscosity on the absorption and emission spectra has also been studied. The fluorescence excitation spectra of DMAADD monitored at both the emission bands are different. The presence of two different conformation of the same molecule in the ground state has lead to two close lying excited states, local excited (LE) and charge transfer (CT), and thereby results in the dual fluorescence of the dye. A CTstate involving the N,N-dimethylaniline group and the decahy droacridinedione chromophore as donor and acceptor, respectively, has been identified as the source of the long wavelength anomalous fluorescence. The experimental studies were supported by ab initio time dependent-density functional theory (TDDFT) calculations performed at the B3LYP/6-31G* level. The molecule possesses photoinduced electron transfer (PET) quenching in the LE state, which is confirmed by the fluorescence lifetime and fluorescent intensity enhancement in the presence of transition metal ions.