Near-Infrared Light-Activated Phototherapy by Gold Nanoclusters for Dispersing Biofilms.

A bacterial biofilm is strongly associated with chronic infections and is difficult to be eradicated, posing serious threats to public health. Development of effective therapeutic strategies to prevent and control hospital-acquired infections via eradication of bacteria shielded by biofilms is challenging. Herein, we developed deoxyribonuclease (DNase)-functionalized gold nanoclusters (AuNCs) (DNase-AuNCs), which are capable of killing Gram-positive and Gram-negative bacteria, especially dispersing the surrounding biofilms. The DNase can break down the extracellular polymeric substance matrix to expose the defenseless bacteria to photothermal therapy (PTT) and photodynamic therapy (PDT) by DNase-AuNCs under 808 nm laser irradiation. The combination of enzymolysis, PDT, and PTT can effectively remove biofilms with a dispersion rate of up to 80% and kill ∼90% of the shielded bacteria. DNase-AuNCs exhibit an outstanding therapeutic effect in treating bacterial biofilm-coated orthodontic devices (Invisalign aligners), suggesting their potential applications in medical devices.

Neutrophil enhancement of Pseudomonas aeruginosa biofilm development: human F-actin and DNA as targets for therapy.

In the cystic fibrosis (CF) airway, chronic infection by Pseudomonas aeruginosa results from biofilm formation in a neutrophil-rich environment. We tested the capacity of human neutrophils to modify early biofilm formation of P. aeruginosa strain PAO1, and an isogenic CF strain isolated early and years later in infection. In a static reactor, P. aeruginosa biofilm density of all strains was enhanced at 24 h in the presence of neutrophils, with the greatest relative increase associated with the lowest inoculum of P. aeruginosa tested. Previously, neutrophil-induced biofilm enhancement was shown to largely result from the incorporation of F-actin and DNA polymers into the bacterial biofilm. This finding was advanced by the comparison of biofilm enhancement from intact unstimulated neutrophils and from lysed or apoptotic neutrophils. Apoptotic neutrophils, with an intact cell membrane, were unable to contribute to biofilm enhancement, while lysed neutrophils evoked a similar response to that of intact cells. Using F-actin and DNA as targets, the capacity of negatively charged poly(amino acids) to disrupt, or prevent, early biofilm formation was tested. Anionic poly(aspartic acid) effectively prevented or disrupted biofilm formation. Combination of poly(aspartic acid) with DNase resulted in a synergistic increase in biofilm disruption. These results demonstrate that the presence of dying neutrophils can facilitate the initial stages of biofilm development by low inocula of P. aeruginosa. Neutrophil F-actin represents a potential new therapeutic target for disruption of pathogenic biofilms.

Nucleases isolated from Chelidonium majus L. milky sap can induce apoptosis in human cervical carcinoma HeLa cells but not in Chinese Hamster Ovary CHO cells.

Milky sap isolated from Chelidonium majus L. (Greater Celandine) serves as a rich source of various biologically active substances such as alkaloids, flavonoids and phenolic acids. Previous research showed that the activity of Ch. majus milky sap may depend also on the presence of biologically active proteins. The goal of this study was to evaluate the biological effect of two nucleases isolated from Ch. majus milk sap, CMN1 of 20 kDa and CMN2 of 36 kDa, on HeLa and CHO tumour cell lines. Both studied nucleases together with other proteins in the sap of the plant are involved in stress and defence reactions against different pathogens. After 48 h incubation of CMN1 and CMN2 only with HeLa cells, the dependence between the number of apoptotic lesions and the concentration of applied nuclease was observed. The highest proapoptotic activity was induced by 13.3 ng/ml concentration of CMN2 collected in May (62 +/- 3% HeLa cells were apoptotic). Moreover, the proportion of necrotic cells in all concentrations of the nucleases and both cell lines was relatively low (1-8 +/- 0.5%). In summary, results of this study show that purified nucleases CMN1 and CMN2 isolated from Ch. majus milky sap exhibit apoptotic activity in HeLa tumour cell line, but not in CHO cells, without inflammatory reaction.

Reusable photonucleases: plasmid scission by a uranyl ion impregnated adenine homopolymer in the presence of visible light and sunlight.

Relaxation of supercoiled plasmid by uranyl ions impregnated on a adenylated polymeric support has been observed in the presence of visible light and sunlight. This insoluble polymer support permits facile reusability of the catalytic system and it failed to cleave lysozyme under the conditions employed for plasmid modification.

The effect of wound ointments on tissue microcirculation and leucocyte behaviour.

An intact microcirculation is essential for normal healing to occur. Wound repair may be impaired by various endogenous and exogenous factors, such as reduced microvascular perfusion, infection and debris. In the nonhealing wound, radical surgical debridement is critical. To supplement healing, various ointments are used in clinical practice. Little is known about their effects on tissue perfusion. We have therefore selected two substances widely used, the antiseptic Betadine and the enzyme combination Elase and investigated their impact on the microcirculation and on leucocyte activity, using the cremaster muscle as a model. We found that functional capillary density and arteriolar diameters were significantly reduced by Betadine, whereas leucocyte activity was not affected. In the Elase group, capillary flow and arteriolar diameters were significantly increased, and again leucocyte activity was not changed. The mechanism by which Betadine reduces microvascular flow is believed to be the same as in reperfusion injury. The positive effect of Elase on the microcirculation might be attributed to plasmin, which has been shown to dilate blood vessels.

[Silastic gel and elastomer in the cicatrization of wounds in the rabbit]. / Le gel et l'élastomère de Silastic dans la cicatrisation des plaies chez le lapin.

The aim of this experimental study was to examine two new wound healing adjuvants. Four experimental wounds were created on the back of rabbits to compare the kinetics of wound healing after applying the standard treatment, Silastic gel, and elastomer with and without compounds known to act on wound healing. We observed a tendency towards more rapid healing in the gel groups (13.7 days), the elastomer group (17.3 days) and the elastomer with active compound group (15.3 days) compared with the control group (17.3 days) and the standard treatment group (17.8 days). The differences were not however statistically different. Elastomer is the better carrier for active wound healing compounds because it has better stability over time. Silicon dressings are interesting for wound healing because they give satisfactory results in terms of healing time and in terms of redressing and patient comfort.

Decreased stability of DNA in cells treated with alkylating agents.

A modified highly sensitive procedure for the evaluation of DNA damage in individual cells treated with alkylating agents is reported. The new methodology is based on the amplification of single-strandedness in alkylated DNA by heating in the presence of Mg2+. Human ovarian carcinoma cells A2780 were treated with nitrogen mustard (HN2), fixed in methanol, and stained with monoclonal antibody (MOAB) F7-26 generated against HN2-treated DNA. Binding of MOAB was measured by flow cytometry with indirect immunofluorescence. The maximal difference in fluorescence between untreated and HN2-treated cells was observed after heating at 100 degrees C for 5 min in PBS containing 1.25 mM MgCl2. Higher concentrations of MgCl2 inhibited MOAB binding to HN2-treated cells and heating at lower concentrations induced binding to control cells. Intensive binding of MOAB to control and drug-treated cells was observed after heating in Tris buffer supplemented with MgCl2. Thus, the presence of phosphates and MgCl2 during heating was necessary for the detection of HN2-induced changes in DNA stability. Fluorescence of HN2-treated cells decreased to background levels after treatment with single-strand-specific S1 nuclease. MOAB F7-26 interacted with single-stranded regions in DNA and did not bind to dsDNA or other cellular antigens. Specific reactivity of MOAB F7-26 with deoxycytidine was established by avidin-biotin ELISA. Single-stranded conformation was necessary for the binding of MOAB to deoxycytidine on the DNA molecule. It is suggested that alkylation of guanines decreased the stability of the DNA molecule and increased the access of MOAB F7-26 to deoxycytidines on the opposite DNA strand.

DNA organization in nucleosomes.

A new technique known as electron spectroscopic imaging has allowed the direct visualization of DNA within the nucleosomes of chromatin. The results presented here confirm the model which suggests that approximately two supercoil turns of DNA are wound about the nucleosome core. The structure of nucleosomes from putative transcriptionally active genes, fractionated by preferential sensitivity to DNAase II and solubility in 2 mM MgCl2, has been examined using both dark field electron microscopy and electron spectroscopic imaging. Oligomeric strands of nucleosomes in this fraction have a less distinct beaded appearance than those of bulk chromatin. The phosphorus distribution in this chromatin suggests that the DNA has a less recognizable organization, lacking a two-turn supercoil per subunit. The unique appearance of this fraction in 30 mM NaCl is reversibly changed to the classical beaded appearance when dialyzed into 0.4 M NaCl.

Entrapment of plasmid DNA by liposomes and their interactions with plant protoplasts.

Lecithin and lecithin/cholesterol liposomes formed in aqueous solutions of DNA entrap covalently closed circular, open circular and linear DNA molecules of size up to at least 13 kilobases. The sequestered DNA molecules are efficiently protected against exogenous deoxyribonuclease action although nicking and linearization of circular DNA can be observed. The size of these liposomes ranges from approximately 0.5 to 7.5 mu with an average of 2.5--4 mu. DNA filled liposomes strongly interact with plant protoplasts under conditions inducing protoplast fusion. Results suggest that sequestered plasmid DNA can be transferred to protoplast nuclei.

Growth cycle of predacious Bdellovibrios in a host-free extract system and some properties of the host extract.

Host-free growth and reproduction of a host-dependent strain of Bdellovibrio bacteriovorus incubated with an extract from host cells were studied. The morphological changes occurring in the cells were correlated with deoxyribonucleic acid (DNA) synthesis as measured by labeled nucleotide or orthophosphate incorporation. The host-free developmental cycle of Bdellovibrio is similar to that of the two-membered system; the early loss of flagella, the elongation into filaments, and multiple fission into flagellated progeny are typical for both host-free and intraperiplasmic development of bdellovibrios. Filament length and time of division appear to depend on the concentration of the host extract. Host extract was found to be heat stable and DNase stable, and Pronase sensitive and RNase sensitive. Addition of ribonucleic acid to the extract medium at various times during the Bdellovibrio growth cycle demonstrated that host extract is required continuously during the cycle for growth. The observations reported give a unified picture of Bdellovibrio development and allow for the suggestion that wild-type bdellovibrios depend upon the presence of some host factor for induction of DNA synthesis, whereas depletion of host factor triggers division. The ecological implications of such host dependence are discussed.

Inhibition of macrophage migration by nucleotide-containing streptococcal preparations.

Certain extracts of streptococcal cell walls are known to inhibit macrophage migration in vitro. In this study, we attempted to identify the streptococcal components responsible for this phenomenon. Trypsinized cell walls and cytoplasm from groups A and B streptococci were extracted with hot formamide followed by acetone precipitation. Subsequent gel filtration in aqueous solutions yielded a fraction devoid of C-carbohydrate and containing mostly oligonucleotides, apparently derived from streptococcal cytoplasm. This fraction significantly inhibited the migration of peritoneal exudate cells from rats sensitized to groups A and B streptococci. It was noteworthy that no inhibition of migration was observed with cells from nonsensitized animals or control rats injected with BCG or complete Freund adjuvant. Similarly, no inhibition was obtained with formamide extracts of calf thymus RNA. Although the inhibition does not show specificity for streptococcal groups, it seems to have immunological specificity since prior sensitization with streptococci is required for migration inhibition.

Immunological and chemical studies of the streptococcal group O protein type antigens.

Two antigens present in group O streptococci have been described and are designated as type antigens. No other antigens are present in extracts of group O cells in significant quantity. Antigen I is a cell wall protein, and II is a nucleoprotein of undetermined cell location. The immunological specificity of the latter resides only in the protein. Both antigens have been extracted from the cell with water or 0.2 n HCl (pH 2) at 100 C, and antigen I can be removed by trypsin. After purification, pH 2 was found to destroy the serological activity of each antigen. Antgens I and II and also the O polysaccharide antigen can be distinguished on a single agar-gel diffusion plate with group O whole-cell antiserum. N- and C-terminal amino acid analyses and total amino acid composition prove that I and II are separate molecular species. Each possesses properties which separate them from the streptococcal M, R, and T proteins. Antigen I separated into three fractions, each possessing serological specificity, on a diethylaminoethyl-Sephadex column at pH 8.6 during an increase in buffer molarity. The protein is considered to possess a multiple unit structure. The results indicate that the O streptococci are limited to two serological types. Group O whole-cell antisera, after adsorption with a strain not containing the O polysaccharide, can now be prepared for the positive identification of the types of group O streptococci.

Ribonucleic acid synthesis of vesicular stomatitis virus, II. An RNA polymerase in the virion.

The virions of vesicular stomatitis virus contain an enzyme that catalyzes the incorporation of ribonucleotides into RNA. The product of the reaction is mainly RNA complementary in base sequence to that of vesicular stomatitis virus RNA.

Isolation and partial characterization of an immunogenic moiety obtained from Salmonella typhimurium.

Ribosomal preparations obtained from Salmonella typhimurium by differential centrifugation and sodium dodecyl sulfate (SDS) treatment of the bacillary lysate were found to be immunogenic in F(1) hybrid (C(3)H/HeJ x DBA/2J) and albino Swiss mice, as determined by progressive host survival. The immunity obtained was independent of the need for adjuvant and dependent on the dosage of immunogen given. Immunizations with the ribosomal preparations induced an immune response comparable to that obtained by vaccination with living organisms and significantly greater than that obtained by immunization with heat-killed salmonellae, purified lipopolysaccharide, or crude and SDS-treated endotoxin preparations. No effect on the immunogenicity of the ribosomal fraction was observed by enzymatic treatment with trypsin, Pronase, deoxyribonuclease, and pancreatic ribonuclease. Linear sucrose density gradient resolution of the preparations showed that the immunogenicity of the ribosomal fraction was not unique to any one of its subcomponents. Ethyl alcohol-precipitated, crude ribonucleic acid preparations obtained from the ribosomal and sucrose density-resolved ribosomal preparations were found to induce an immune response comparable to that obtained by immunization with the entire ribosomal fraction. Dialysis in doubly distilled demineralized water slightly reduced the immunogenicity of the preparation; however, comparable dialysis in 10(-4)m MgCl(2)-phosphate buffer did not. Chemical assays of the preparations found to be immunogenic were performed.