Treatment of antibiotic-resistant bacteria colonizing diabetic foot ulcers by OLED induced antimicrobial photodynamic therapy.

We evaluate the efficacy of antimicrobial Photodynamic Therapy (APDT) for inactivating a variety of antibiotic-resistant clinical strains from diabetic foot ulcers. Here we are focused on APDT based on organic light-emitting diodes (OLED). The wound swabs from ten patients diagnosed with diabetic foot ulcers were collected and 32 clinical strains comprising 22 bacterial species were obtained. The isolated strains were identified with the use of mass spectrometry coupled with a protein profile database and tested for antibiotic susceptibility. 74% of isolated bacterial strains exhibited adaptive antibiotic resistance to at least one antibiotic. All strains were subjected to the APDT procedure using an OLED as a light source and 16 µM methylene blue as a photosensitizer. APDT using the OLED led to a large reduction in all cases. For pathogenic bacteria, the reduction ranged from 1.1-log to > 8 log (Klebsiella aerogenes, Enterobacter cloaca, Staphylococcus hominis) even for high antibiotic resistance (MRSA 5-log reduction). Opportunistic bacteria showed a range from 0.4-log reduction for Citrobacter koseri to > 8 log reduction for Kocuria rhizophila. These results show that OLED-driven APDT is effective against pathogens and opportunistic bacteria regardless of drug resistance.

Effect of Ovocystatin on Amyloid ß 1-42 Aggregation-In Vitro Studies.

Amyloid ß peptides (Aß) aggregating in the brain have a potential neurotoxic effect and are believed to be a major cause of Alzheimer's disease (AD) development. Thus, inhibiting amyloid polypeptide aggregation seems to be a promising approach to the therapy and prevention of this neurodegenerative disease. The research presented here is directed at the determination of the inhibitory activity of ovocystatin, the cysteine protease inhibitor isolated from egg white, on Aß42 fibril genesis in vitro. Thioflavin-T (ThT) assays, which determine the degree of aggregation of amyloid peptides based on fluorescence measurement, circular dichroism spectroscopy (CD), and transmission electron microscopy (TEM) have been used to assess the inhibition of amyloid fibril formation by ovocystatin. Amyloid beta 42 oligomer toxicity was measured using the MTT test. The results have shown that ovocystatin possesses Aß42 anti-aggregation activity and inhibits Aß42 oligomer toxicity in PC12 cells. The results of this work may help in the development of potential substances able to prevent or delay the process of beta-amyloid aggregation-one of the main reasons for Alzheimer's disease.

The role of the light source in antimicrobial photodynamic therapy.

Antimicrobial photodynamic therapy (APDT) is a promising approach to fight the growing problem of antimicrobial resistance that threatens health care, food security and agriculture. APDT uses light to excite a light-activated chemical (photosensitiser), leading to the generation of reactive oxygen species (ROS). Many APDT studies confirm its efficacy in vitro and in vivo against bacteria, fungi, viruses and parasites. However, the development of the field is focused on exploring potential targets and developing new photosensitisers. The role of light, a crucial element for ROS production, has been neglected. What are the main parameters essential for effective photosensitiser activation? Does an optimal light radiant exposure exist? And finally, which light source is best? Many reports have described the promising antibacterial effects of APDT in vitro, however, its application in vivo, especially in clinical settings remains very limited. The restricted availability may partially be due to a lack of standard conditions or protocols, arising from the diversity of selected photosensitising agents (PS), variable testing conditions including light sources used for PS activation and methods of measuring anti-bacterial activity and their effectiveness in treating bacterial infections. We thus sought to systematically review and examine the evidence from existing studies on APDT associated with the light source used. We show how the reduction of pathogens depends on the light source applied, radiant exposure and irradiance of light used, and type of pathogen, and so critically appraise the current state of development of APDT and areas to be addressed in future studies. We anticipate that further standardisation of the experimental conditions will help the field advance, and suggest key optical and biological parameters that should be reported in all APDT studies. More in vivo and clinical studies are needed and are expected to be facilitated by advances in light sources, leading to APDT becoming a sustainable, alternative therapeutic option for bacterial and other microbial infections in the future.

Non-toxic Polymeric Dots with the Strong Protein-Driven Enhancement of One- and Two-Photon Excited Emission for Sensitive and Non-destructive Albumin Sensing.

The need for efficient probing, sensing, and control of the bioactivity of biomolecules (e.g., albumins) has led to the engineering of new fluorescent albumins' markers fulfilling very specific chemical, physical, and biological requirements. Here, we explore acetone-derived polymer dots (PDs) as promising candidates for albumin probes, with special attention paid to their cytocompatibility, two-photon absorption properties, and strong ability to non-destructively interact with serum albumins. The PDs show no cytotoxicity and exhibit high photostability. Their pronounced green fluorescence is observed upon both one-photon excitation (OPE) and two-photon excitation (TPE). Our studies show that both OPE and TPE emission responses of PDs are proteinaceous environment-sensitive. The proteins appear to constitute a matrix for the dispersion of fluorescent PDs, limiting both their aggregation and interactions with the aqueous environment. It results in a large enhancement of PD fluorescence. Meanwhile, the PDs do not interfere with the secondary protein structures of albumins, nor do they induce their aggregation, enabling the PD candidates to be good nanomarkers for non-destructive probing and sensing of albumins.

Design and Development of a New Type of Hybrid PLGA/Lipid Nanoparticle as an Ursolic Acid Delivery System against Pancreatic Ductal Adenocarcinoma Cells.

Despite many attempts, trials, and treatment procedures, pancreatic ductal adenocarcinoma (PDAC) still ranks among the most deadly and treatment-resistant types of cancer. Hence, there is still an urgent need to develop new molecules, drugs, and therapeutic methods against PDAC. Naturally derived compounds, such as pentacyclic terpenoids, have gained attention because of their high cytotoxic activity toward pancreatic cancer cells. Ursolic acid (UA), as an example, possesses a wide anticancer activity spectrum and can potentially be a good candidate for anti-PDAC therapy. However, due to its minimal water solubility, it is necessary to prepare an optimal nano-sized vehicle to overcome the low bioavailability issue. Poly(lactic-co-glycolic acid) (PLGA) polymeric nanocarriers seem to be an essential tool for ursolic acid delivery and can overcome the lack of biological activity observed after being incorporated within liposomes. PLGA modification, with the addition of PEGylated phospholipids forming the lipid shell around the polymeric core, can provide additional beneficial properties to the designed nanocarrier. We prepared UA-loaded hybrid PLGA/lipid nanoparticles using a nanoprecipitation method and subsequently performed an MTT cytotoxicity assay for AsPC-1 and BxPC-3 cells and determined the hemolytic effect on human erythrocytes with transmission electron microscopic (TEM) visualization of the nanoparticles and their cellular uptake. Hybrid UA-loaded lipid nanoparticles were also examined in terms of their stability, coating dynamics, and ursolic acid loading. We established innovative and repeatable preparation procedures for novel hybrid nanoparticles and obtained biologically active nanocarriers for ursolic acid with an IC50 below 20 µM, with an appropriate size for intravenous dosage (around 150 nm), high homogeneity of the sample (below 0.2), satisfactory encapsulation efficiency (up to 70%) and excellent stability. The new type of hybrid UA-PLGA nanoparticles represents a further step in the development of potentially effective PDAC therapies based on novel, biologically active, and promising triterpenoids.

Timber-colonizing gram-negative bacteria as potential causative agents of respiratory diseases in woodworkers.

OCCURRENCE: Gram-negative bacteria occur commonly in the inner tissues of stored coniferous and deciduous timber, showing a marked variation in numbers. The greatest maximal numbers are found in the sapwood of coniferous timber. The common constituents of the Gram-negative biota are potentially pathogenic species of Enterobacteriaceae family of the genera Rahnella, Pantoea, Enterobacter, and Klebsiella. The air of wood-processing facilities is polluted with the wood-borne Gram-negative bacteria and produced by them endotoxin, as demonstrated worldwide by numerous studies. EFFECTS: There are three potential pathways of the pathogenic impact of wood-borne Gram-negative bacteria on exposed woodworkers: allergic, immunotoxic, and infectious. Allergic impact has been underestimated for a long time with relation to Gram-negative bacteria. Hopefully, the recent demonstration of the first documented case of hypersensitivity pneumonitis (HP) in woodworkers caused by Pantoea agglomerans which developed in extremely large quantities in birch sapwood, would speed up finding of new wood-related cases of HP caused by Gram-negative bacteria. The second pathway is associated with endotoxin, exerting strong immunotoxic (excessively immunostimulative) action. It has been demonstrated that endotoxin is released into wood dust in the form of nano-sized microvesicles, by peeling off the outer membrane of bacteria. Endotoxin microvesicles are easily inhaled by humans together with dust because of small dimensions and aerodynamic shape. Afterwards, they cause a nonspecific activation of lung macrophages, which release numerous inflammatory mediators causing an inflammatory lung reaction, chest tightness, fever, gas exchange disorders, and bronchospasm, without radiographic changes. The resulting disease is known as "Organic Dust Toxic Syndrome" or "toxic pneumonitis." The potential third pathway of pathogenic impact is infection. The suspected species is Klebsiella pneumoniae that may occur commonly in wood dust; however, until now this pathway has not been confirmed. CONCLUSION: Summarizing, Gram-negative bacteria-inhabiting timber should be considered, besides filamentous fungi and actinobacteria, as important risk factors of occupational disease in woodworkers that could be either HP with allergenic background or toxic pneumonitis elicited by endotoxin.

GntR-like SCO3932 Protein Provides a Link between Actinomycete Integrative and Conjugative Elements and Secondary Metabolism.

Streptomyces bacteria produce a plethora of secondary metabolites including the majority of medically important antibiotics. The onset of secondary metabolism is correlated with morphological differentiation and controlled by a complex regulatory network involving numerous regulatory proteins. Control over these pathways at the molecular level has a medical and industrial importance. Here we describe a GntR-like DNA binding transcription factor SCO3932, encoded within an actinomycete integrative and conjugative element, which is involved in the secondary metabolite biosynthesis regulation. Affinity chromatography, electrophoresis mobility shift assay, footprinting and chromatin immunoprecipitation experiments revealed, both in vitro and in vivo, SCO3932 binding capability to its own promoter region shared with the neighboring gene SCO3933, as well as promoters of polyketide metabolite genes, such as cpkD, a coelimycin biosynthetic gene, and actII-orf4-an activator of actinorhodin biosynthesis. Increased activity of SCO3932 target promoters, as a result of SCO3932 overproduction, indicates an activatory role of this protein in Streptomyces coelicolor A3(2) metabolite synthesis pathways.

Coelimycin Synthesis Activatory Proteins Are Key Regulators of Specialized Metabolism and Precursor Flux in Streptomyces coelicolor A3(2).

Many microbial specialized metabolites are industrially relevant agents but also serve as signaling molecules in intra-species and even inter-kingdom interactions. In the antibiotic-producing Streptomyces, members of the SARP (Streptomyces antibiotic regulatory proteins) family of regulators are often encoded within biosynthetic gene clusters and serve as their direct activators. Coelimycin is the earliest, colored specialized metabolite synthesized in the life cycle of the model organism Streptomyces coelicolor A3(2). Deletion of its two SARP activators cpkO and cpkN abolished coelimycin synthesis and resulted in dramatic changes in the production of the later, stationary-phase antibiotics. The underlying mechanisms of these phenotypes were deregulation of precursor flux and quorum sensing, as shown by label-free, bottom-up shotgun proteomics. Detailed profiling of promoter activities demonstrated that CpkO is the upper-level cluster activator that induces CpkN, while CpkN activates type II thioesterase ScoT, necessary for coelimycin synthesis. What is more, we show that cpkN is regulated by quorum sensing gamma-butyrolactone receptor ScbR.

Porphyrin-Loaded Lignin Nanoparticles Against Bacteria: A Photodynamic Antimicrobial Chemotherapy Application.

The need for alternative strategies to fight bacteria is evident from the emergence of antimicrobial resistance. To that respect, photodynamic antimicrobial chemotherapy steadily rises in bacterial eradication by using light, a photosensitizer and oxygen, which generates reactive oxygen species that may kill bacteria. Herein, we report the encapsulation of 5,10,15,20-tetrakis(4-hydroxyphenyl)-21H,23H-porphyrin into acetylated lignin water-dispersible nanoparticles (THPP@AcLi), with characterization of those systems by standard spectroscopic and microscopic techniques. We observed that THPP@AcLi retained porphyrin's photophysical/photochemical properties, including singlet oxygen generation and fluorescence. Besides, the nanoparticles demonstrated enhanced stability on storage and light bleaching. THPP@AcLi were evaluated as photosensitizers against two Gram-negative bacteria, Escherichia coli and Pseudomonas aeruginosa, and against three Gram-positive bacteria, Staphylococcus aureus, Staphylococcus epidermidis, and Enterococcus faecalis. THPP@AcLi were able to diminish Gram-positive bacterial survival to 0.1% when exposed to low white LED light doses (4.16 J/cm2), requiring concentrations below 5 µM. Nevertheless, the obtained nanoparticles were unable to diminish the survival of Gram-negative bacteria. Through transmission electron microscopy observations, we could demonstrate that nanoparticles did not penetrate inside the bacterial cell, exerting their destructive effect on the bacterial wall; also, a high affinity between acetylated lignin nanoparticles and bacteria was observed, leading to bacterial flocculation. Altogether, these findings allow to establish a photodynamic antimicrobial chemotherapy alternative that can be used effectively against Gram-positive topic infections using the widely available natural polymeric lignin as a drug carrier. Further research, aimed to inhibit the growth and survival of Gram-negative bacteria, is likely to enhance the wideness of acetylated lignin nanoparticle applications.

Acute hypersensitivity pneumonitis in woodworkers caused by inhalation of birch dust contaminated with Pantoea agglomerans and Microbacterium barkeri.

CASE DESCRIPTION: Five workers (2 males and 3 females) employed in a furniture factory located in eastern Poland developed hypersensitivity pneumonitis (HP) after the pine wood used for furniture production was replaced by birch wood. All of them reported onset of respiratory and general symptoms (cough, shortness of breath, general malaise) after inhalation exposure to birch dust, showed crackles at auscultation, ground-glass attenuations in HRCT examination, and lymphocytosis in the BAL examination. The diagnosis of acute HP was set in 4 persons and the diagnosis of subacute HP in one. IDENTIFICATION OF SPECIFIC ALLERGEN: Samples of birch wood associated with evoking disease symptoms were subjected to microbiological analysis with the conventional and molecular methods. Two bacterial isolates were found to occur in large quantities (of the order 108 CFU/g) in examined samples: Gram-negative bacterium of the species Pantoea agglomerans and a non-filamentous Gram-positive actinobacterium of the species Microbacterium barkeri. In the test for inhibition of leukocyte migration, 4 out of 5 examined patients showed a positive reaction in the presence of P. agglomerans and 2 in the presence of M. barkeri. Only one person showed the presence of precipitins to P. agglomerans and none to M. barkeri. In the inhalation challenge, which is the most relevant allergological test in the HP diagnostics, all patients reacted positively to P. agglomerans and only one to M. barkeri. The results indicate that P. agglomerans developing in birch wood was the main agent causing HP in the workers exposed to the inhalation of dust from this wood, while the etiologic role of M. barkeri is probably secondary. CONCLUSION: The results demonstrate that apart from fungi and filamentous actinobacteria, regarded until recently as causative agents of HP in woodworkers, Gram-negative bacteria and non-filamentous actinobacteria may also elicit disease symptoms in the workers processing wood infected with large amounts of these microorganisms. The results obtained also seem to indicate that cellular-mediated reactions are more significant for causing disease symptoms compared to those that are precipitin-mediated.

A GntR-Like Transcription Factor HypR Regulates Expression of Genes Associated With L-Hydroxyproline Utilization in Streptomyces coelicolor A3(2).

Bacteria from the genus Streptomyces have been long exploited as the most prolific producers of antibiotics, other secondary metabolites and enzymes. They are important members of soil microbial communities that can adapt to changing conditions thank to the fine regulation of gene expression in response to environmental signals. Streptomyces coelicolor A3(2) is a model organism for molecular studies with the most deeply recognized interactions within the complex metabolic and regulatory network. However, details about molecular signals recognized by specialized regulatory proteins as well as their direct targets are often missing. We describe here a zinc-binding protein HypR (SCO6294) which belongs to FadR subfamily of GntR-like regulators. The DNA sequence 5'-TACAATGTCAC-3' recognized by the HypR protein in its own promoter region was identified by DNase I footprinting. Binding of six DNA fragments containing similar sequences located in other promoter regions were confirmed by the electrophoretic mobility shift assay (EMSA). The sequences of 7 in vitro-determined binding sites were assembled to generate a logo of the HypR binding motif, 5'-CTNTGC(A/C)ATGTCAC-3'. Comparison of luciferase reporter genes expression under the control of cloned promoter regions in S. coelicolor A3(2) wild type and deletion mutant strains revealed, that the HypR protein acts as a repressor of its target genes. Genes belonging to the regulon of HypR code for enzymes putatively involved in collagen degradation and utilization of L-hydroxyproline (L-Hyp) as concluded from predicted structure and conserved domains. Their transcription is induced in the wild type strain by the addition of L-Hyp to the culture medium. Moreover, knockout of one of the genes from the predicted L-Hyp utilization operon abolished the ability of the strain to grow on L-Hyp as a sole source of carbon. To our knowledge, this work is the first indication of the existence of the pathway of L-hydroxyproline catabolism in Streptomycetes.

Multi-level regulation of coelimycin synthesis in Streptomyces coelicolor A3(2).

Despite being a yellow pigment visible to the human eye, coelimycin (CPK) remained to be an undiscovered secondary metabolite for over 50 years of Streptomyces research. Although the function of this polyketide is still unclear, we now know that its "cryptic" nature is attributed to a very complex and precise mechanism of cpk gene cluster regulation in the model actinomycete S. coelicolor A3(2). It responds to the stringent culture density and timing of the transition phase by the quorum-sensing butanolide system and to the specific nutrient availability/uptake signals mediated by the global (pleiotropic) regulators; many of which are two-component signal transduction systems. The final effectors of this regulation cascade are predicted to be two cluster-situated Streptomyces antibiotic regulatory proteins (SARPs) putatively activating the expression of type I polyketide synthase (PKS I) genes. After its synthesis, unstable, colorless antibiotic coelimycin A reacts with specific compounds in the medium losing its antibacterial properties and giving rise to yellow coelimycins P1 and P2. Here we review the current knowledge on coelimycin synthesis regulation in Streptomyces coelicolor A3(2). We focus on the regulatory feedback loop which interconnects the butanolide system with other cpk cluster-situated regulators. We also present the effects exerted on cpk genes expression by the global, pleiotropic regulators, and the regulatory connections between cpk and other biosynthetic gene clusters.

Effect of amyloid curli fibrils and curli CsgA monomers from Escherichia coli on in vitro model of intestinal epithelial barrier stimulated with cytokines.

Amyloid curli fibrils produced by Escherichia coli are well-known virulence factor influencing E. coli adhesion and biofilm formation. However, the impact of curli on intestinal epithelial barrier stimulated with proinflammatory cytokines is unknown. In the study, we examined the effect of curli produced by nonpathogenic E. coli K-12 and wild-type E. coli EC32 strains, and purified CsgA proteins on differentiated Caco-2 cell monolayers stimulated with a mixture of IL-1ß, TNF-α, and INFγ cytokines as a model of 'inflamed intestinal epithelial barrier' in vitro. The results of the study indicated that curliated E. coli adhered better to polarized Caco-2 cells than their curli-deficient mutants and the adherence was further augmented by stimulation of epithelial cells with proinflammatory cytokines. Interestingly, curli reduced internalization but enhanced intracellular survival of the wild-type E. coli strain EC32 within intestinal epithelial cells. Curli-expressing E. coli, as well as purified CsgA proteins, attenuated IL-8 secretion by unstimulated Caco-2 cells, although the effect was barely observed on cytokine-stimulated cells. The findings of the study revealed that curli fibrils are an important virulence factor enabling curliated E. coli to effectively colonize intestinal epithelium especially in individuals with inflammatory intestinal disorders.

Reversible Photocontrol of DNA Melting by Visible-Light-Responsive F4-Coordinated Azobenzene Compounds.

Spatiotemporal control over the regulation of intra- and intermolecular motions in naturally occurring systems is systematically studied to expand the toolbox of mechanical operations in multicomponent nanoarchitectures. DNA is ideally suited for programming light-powered processes that are based on a minimalist molecular design. Here, the noncovalent incorporation of bistable photoswitches into B-like DNA moieties is shown to trigger the thermal transition midpoint of the duplexes by converting visible light into directed mechanical work by orchestrating the collective actions of the photoresponsive chromophores and the host DNA nanostructures. Besides its practical applications, the resulting hybrid nanosystem bears unique features of modulability, biocompatibility, reversibility, and addressability, which are key components for developing molecular photon-controlled programmed materials.

Evaluation of NMP22 in bladder cancer patients sensitive to environmental toxins.

BACKGROUND: Bladder cancer (BC) is recognized as environmentally related. The interaction of environmental exposure to chemicals and genetic susceptibility seem to play important roles in BC development. In order to improve diagnosis and the recognition of BC risk, a group of markers which combine genetic susceptibility with detoxification and nuclear matrix protein (NMP22) is proposed. OBJECTIVES: The aim of the study was to examine the utility of nuclear matrix protein (NMP22) as a diagnostic marker in BC in genetic susceptibility (NAT2 slow acetylators) combined with detoxification abilities (glutathione S-transferase GST and isoenzyme GST-π). MATERIAL AND METHODS: The NMP22 level in urine, N-acetyltransferase 2 (NAT2) genotype and GST activity in hemolysate blood, as well as isoenzyme GST-π level, were determined in the urine and serum of 43 patients with BC and from 25 non-cancer controls. NMP22 and isoenzyme GST-π levels were measured by ELISA. The NAT2 genotype was examined in DNA isolated from whole blood using the PCR (Polymerase Chain Reaction) technique, while the activity of GST was determined with the spectrophotometric method. RESULTS: In the BC group, NMP22 (p = 0.005) concentration, GST-π (p = 0.003) in urine and GST (p = 0.009) activity in blood were statistically significantly higher than in the healthy controls. The majority of BC patients were slow acetylators (NAT2 genotype). A correlation between the level of nuclear matrix protein NMP22 and GST was found in all BC group (p = 0.007) and also slow acetylators (p = 0.0147). CONCLUSIONS: The results support the utility of a marker combination, which covers the genetic susceptibility to chemicals with the level of detoxification and nuclear matrix protein in BC patients. A relationship between NMP22 level in urine, GST level in blood and NAT2 genotype was observed. Also the isoenzyme GST-π in urine seems useful as a marker of BC.

Hydrolytic activity determination of Tail Tubular Protein A of Klebsiella pneumoniae bacteriophages towards saccharide substrates.

In this paper, the enzymatic activity, substrate specificity and antibiofilm feature of bacteriophage dual-function tail proteins are presented. So far, tail tubular proteins A-TTPAgp31 and TTPAgp44-have been considered as structural proteins of Klebsiella pneumoniae bacteriophages KP32 and KP34, respectively. Our results show that TTPAgp31 is able to hydrolyze maltose as well as Red-starch. The activity of 1 µM of the protein was calculated as 47.6 milli-Units/assay relating to the α-amylase activity. It degrades capsular polysaccharides (cPS), slime polysaccharides (sPS) and lipopolysaccharide (LPS) of K. pneumoniae PCM 2713 and shows antibiofilm reactivity towards S. aureus PCM 519 and E. faecalis PCM 2673. TTPAgp44 hydrolyses trehalose and cPS of E. faecium PCM 1859. TTPAgp44's activity was also observed in the antibiofilm test against P. aeruginosa PCM 2710 and B. subtilis PCM 2021. TTPAgp31 has been identified as α-1,4-glucosidase whereas, TTPAgp44 exhibits trehalase-like activity. Both proteins contain aspartate and glutamate residues in the ß-stranded region which are essential for catalytic activity of glycoside hydrolases. The significant novelty of our results is that for the first time the bacteriophage tubular proteins are described as the unique enzymes displaying no similarity to any known phage hydrolases. They can be used as antibacterial agents directed against bacterial strains producing exopolysaccharides and forming a biofilm.

Specific Recognition of G-Quadruplexes Over Duplex-DNA by a Macromolecular NIR Two-Photon Fluorescent Probe.

The implication of guanine-rich DNA sequences in biologically important roles such as telomerase dysfunction and the regulation of gene expression has prompted the search for structure-specific G-quadruplex agents for targeted diagnostic and therapeutic applications. Herein, we report on a near-infrared (NIR) two-photon poly(cationic) anthracene-based macromolecule able to selectively target G-quadruplexes (G4s) over genomic double-stranded DNA. In particular, the striking changes in its linear and third-order nonlinear optical properties, combined with the emergence of a strong induced electronic circular dichroism (ECD) signal upon binding to canonical and noncanonical DNA secondary structures allowed for a highly specific detection of several different G4s. Furthermore, through a detailed computational analysis we bring compelling evidence that our probe intercalation within G4s is a thermodynamically favored event, and we fully rationalize the spectroscopic evolution resulting from this complexation event by providing a reasonable explanation regarding the origin of the peculiar ECD effect that accompanies it.

Tail tubular protein A: a dual-function tail protein of Klebsiella pneumoniae bacteriophage KP32.

Tail tubular protein A (TTPA) is a structural tail protein of Klebsiella pneumoniae bacteriophage KP32, and is responsible for adhering the bacteriophage to host cells. For the first time, we found that TTPA also exhibits lytic activity towards capsular exopolysaccharide (EPS) of the multiresistant clinical strain of Klebsiella pneumoniae, PCM2713, and thus should be regarded as a dual-function macromolecule that exhibits both structural and enzymatic actions. Here, we present our crystallographic and enzymatic studies of TTPA. TTPA was crystallized and X-ray diffraction data were collected to a resolution of 1.9 Å. In the crystal, TTPA molecules were found to adopt a tetrameric structure with α-helical domains on one side and ß-strands and loops on the other. The novel crystal structure of TTPA resembles those of the bacteriophage T7 tail protein gp11 and gp4 of bacteriophage P22, but TTPA contains an additional antiparallel ß-sheet carrying a lectin-like domain that could be responsible for EPS binding. The enzymatic activity of TTPA may reflect the presence of a peptidoglycan hydrolase domain in the α-helical region (amino acid residues 126 to 173). These novel results provide new insights into the enzymatic mechanism through which TTPA acts on polysaccharides.

SYNTHESIS AND ANTIBACTERIAL ACTIVITY OF NEW SULFONAMIDE ISOXAZOLO[5,4-b]PYRIDINE DERIVATIVES.

A series of novel sulfonamide isoxazolo[5,4-b]pyridines were synthesized. The substrates for their synthesis were 3-aminoisoxazolo[5,4-b]pyridine and selected aryl sulfonic chlorides, chlorosulfonic acid and selected amines. Reactions were carried out using the classical and microwave methods. Selected compounds were tested towards antibacterial and antiproliferative activity. The structure of the obtained new derivatives was determined by elemental analysis and acquired IR and 1H NMR spectra. Among the tested compounds: N- isoxazolo[5,4-b]pyridine-3-yl-benzenesulfonamide (2) and N-isoxazolo[5,4-b]pyridine-3-yl-4-methylbenzene-sulfonamide (5) showed antimicrobial activity towards Pseudomonas aeruginosa (ATCC 27853) and Escherichia coli (ATCC 25922) at doses: 125, 250 and 500 µg. Both compounds showed a 50% inhibition of proliferation of breast carcinoma cell line MCF7 at concentrations of 152.56 µg/mL and 160 161.08 µg/mL, respectively.

Bio-mediated synthesis, characterization and cytotoxicity of gold nanoparticles.

We report here a "green" approach for the synthesis of gold nanoparticles (GNPs) in which the Mentha piperita extract was applied for the bioreduction of chloroauric acid and the stabilization of the formed nanostructures. The obtained GNPs were characterized by UV-Vis absorption spectroscopy and transmission electron microscopy (TEM). The reduction of gold ions with the plant extract leads to the production of nanoparticles with various shapes (spherical, triangular and hexagonal) and sizes (from 10 to 300 nm). The kinetics of the reaction was monitored and various conditions of the synthesis were investigated. As a result, we established protocols optimized towards the synthesis of nanospheres and nanoprisms of gold. The cytotoxic effect of the obtained gold nanoparticles was studied by performing MTT assay, which showed lower cytotoxicity of the biosynthesized GNPs compared to gold nanorods synthesized using the usual seed-mediated growth. The results suggest that the synthesis using plant extracts may be a useful method to produce gold nanostructures for various biological and medical applications.