Antibiofilm Activity of Combretum micranthum G. Don Catechin-Sugar Phytocomplex on Pseudomonas aeruginosa.

Clinicians often have to face infections caused by microorganisms that are difficult to eradicate due to their resistance and/or tolerance to antimicrobials. Among these pathogens, Pseudomonas aeruginosa causes chronic infections due to its ability to form biofilms on medical devices, skin wounds, ulcers and the lungs of patients with Cystic Fibrosis. In this scenario, the plant world represents an important reservoir of natural compounds with antimicrobial and/or antibiofilm properties. In this study, an extract from the leaves of Combretum micranthum G. Don, named Cm4-p, which was previously investigated for its antimicrobial activities, was assayed for its capacity to inhibit biofilm formation and/or to eradicate formed biofilms. The model strain P. aeruginosa PAO1 and its isogenic biofilm hyperproducer derivative B13 were treated with Cm4-p. Preliminary IR, UV-vis, NMR, and mass spectrometry analyses showed that the extract was mainly composed of catechins bearing different sugar moieties. The phytocomplex (3 g/L) inhibited the biofilm formation of both the PAO1 and B13 strains in a significant manner. In light of the obtained results, Cm4-p deserves deeper investigations of its potential in the antimicrobial field.

Screening Antibacterial Photodynamic Effect of Monascus Red Yeast Rice (Hong-Qu) and Mycelium Extracts.

The fungus Monascus is a well-known source of secondary metabolites with interesting pharmaceutical and nutraceutical applications. In particular, Monascus pigments possess a wide range of biological activities (e.g. antimicrobial, antioxidant, anti-inflammatory or antitumoral). To broaden the scope of their possible application, this study focused on testing Monascus pigment extracts as potential photosensitizing agents efficient in antimicrobial photodynamic therapy (aPDT) against bacteria. For this purpose, eight different extracts of secondary metabolites from the liquid- and solid-state fermentation of Monascus purpureus DBM 4360 and Monascus sp. DBM 4361 were tested against Gram-positive and Gram-negative model bacteria, Bacillus subtilis and Escherichia coli and further screened for ESKAPE pathogens, Staphylococcus aureus and Pseudomonas aeruginosa. To the bacterial culture, increasing concentration of extracts was added and it was found that all extracts showed varying antimicrobial activity against Gram-positive bacteria in dark, which was further increased after irradiation. Gram-negative bacteria were tolerant to the extracts' exposure in the dark but sensitivity to almost all extracts that occurred after irradiation. The Monascus sp. DBM 4361 extracts seemed to be the best potential candidate for aPDT against Gram-positive bacteria, being efficient at low doses, i.e. the lowest total concentration of Monascus pigments exhibiting aPDT effect was 3.92 ± 1.36 mg/L for E. coli. Our results indicate that Monascus spp., forming monascuspiloin as the major yellow pigment and not-forming mycotoxin citrinin, is a promising source of antimicrobials and photoantimicrobials.

Ruthenium(II)-Arene Curcuminoid Complexes as Photosensitizer Agents for Antineoplastic and Antimicrobial Photodynamic Therapy: In Vitro and In Vivo Insights.

Photodynamic therapy (PDT) is an anticancer/antibacterial strategy in which photosensitizers (PSs), light, and molecular oxygen generate reactive oxygen species and induce cell death. PDT presents greater selectivity towards tumor cells than conventional chemotherapy; however, PSs have limitations that have prompted the search for new molecules featuring more favorable chemical-physical characteristics. Curcumin and its derivatives have been used in PDT. However, low water solubility, rapid metabolism, interference with other drugs, and low stability limit curcumin use. Chemical modifications have been proposed to improve curcumin activity, and metal-based PSs, especially ruthenium(II) complexes, have attracted considerable attention. This study aimed to characterize six Ru(II)-arene curcuminoids for anticancer and/or antibacterial PDT. The hydrophilicity, photodegradation rates, and singlet oxygen generation of the compounds were evaluated. The photodynamic effects on human colorectal cancer cell lines were also assessed, along with the ability of the compounds to induce ROS production, apoptotic, necrotic, and/or autophagic cell death. Overall, our encouraging results indicate that the Ru(II)-arene curcuminoid derivatives are worthy of further investigation and could represent an interesting option for cancer PDT. Additionally, the lack of significant in vivo toxicity on the larvae of Galleria mellonella is an important finding. Finally, the photoantimicrobial activity of HCurc I against Gram-positive bacteria is indeed promising.

Bacterial pigments: A colorful palette reservoir for biotechnological applications.

Synthetic derivatives are currently used instead of pigments in many applicative fields, from food to feed, from pharmaceutical to diagnostic, from agronomy to industry. Progress in organic chemistry allowed to obtain rather cheap compounds covering the whole color spectrum. However, several concerns arise from this chemical approach, as it is mainly based on nonrenewable resources such as fossil oil, and the toxicity or carcinogenic properties of products and/or precursors may be harmful for personnel involved in the productive processes. In this scenario, microorganisms and their pigments represent a colorful world to discover and reconsider. Each living bacterial strain may be a source of secondary metabolites with peculiar functions. The aim of this review is to link the physiological role of bacterial pigments with their potential use in different biotechnological fields. This enormous potential supports the big challenge for the development of strategies useful to identify, produce, and purify the right pigment for the desired application. At the end of this ideal journey through the world of bacterial pigments, the attention will be focused on melanin compounds, whose production relies upon different techniques ranging from natural producers, heterologous hosts, or isolated enzymes. In a green workflow, the microorganisms represent the starting and final point of pigment production.

Free and Poly-Methyl-Methacrylate-Bounded BODIPYs: Photodynamic and Antimigratory Effects in 2D and 3D Cancer Models.

Several limitations, including dark toxicity, reduced tumor tissue selectivity, low photostability and poor biocompatibility hamper the clinical use of Photodynamic therapy (PDT) in cancer treatment. To overcome these limitations, new PSs have been synthetized, and often combined with drug delivery systems, to improve selectivity and reduce toxicity. In this context, BODIPYs (4,4-difluoro-4-bora-3a,4a-diaza-s-indacene) have recently emerged as promising and easy-to-handle scaffolds for the preparation of effective PDT antitumor agents. In this study, the anticancer photodynamic effect of newly prepared negatively charged polymethyl methacrylate (nPMMA)-bounded BODIPYs (3@nPMMA and 6@nPMMA) was evaluated on a panel of 2D- and 3D-cultured cancer cell lines and compared with free BODIPYs. In particular, the effect on cell viability was evaluated, along with their ability to accumulate into the cells, induce apoptotic and/or necrotic cell death, and inhibit cellular migration. Our results indicated that 3@nPMMA and 6@nPMMA reduce cancer cell viability in 3D models of HC116 and MCF7 cells more effectively than the corresponding free compounds. Importantly, we demonstrated that MDA-MB231 and SKOV3 cell migration ability was significantly impaired by the PDT treatment mediated by 3@nPMMA and 6@nPMMA nanoparticles, likely indicating the capability of this approach to reduce metastatic tumor potential.

Photoinactivation of Pseudomonas aeruginosa Biofilm by Dicationic Diaryl-Porphyrin.

In recent years, antimicrobial photodynamic therapy (aPDT) has received increasing attention as a promising tool aimed at both treating microbial infections and sanitizing environments. Since biofilm formation on biological and inert surfaces makes difficult the eradication of bacterial communities, further studies are needed to investigate such tricky issue. In this work, a panel of 13 diaryl-porphyrins (neutral, mono- and di-cationic) was taken in consideration to photoinactivate Pseudomonas aeruginosa. Among cationic photosensitizers (PSs) able to efficiently bind cells, in this study two dicationic showed to be intrinsically toxic and were ruled out by further investigations. In particular, the dicationic porphyrin (P11) that was not toxic, showed a better photoinactivation rate than monocationic in suspended cells. Furthermore, it was very efficient in inhibiting the biofilms produced by the model microorganism Pseudomonas aeruginosa PAO1 and by clinical strains derived from urinary tract infection and cystic fibrosis patients. Since P. aeruginosa represents a target very difficult to inactivate, this study confirms the potential of dicationic diaryl-porphyrins as photo-activated antimicrobials in different applicative fields, from clinical to environmental ones.

AIF-1 and RNASET2 Play Complementary Roles in the Innate Immune Response of Medicinal Leech.

Recent studies demonstrated that allograft inflammatory factor-1 (AIF-1) and RNASET2 act as chemoattractants for macrophages and modulate the inflammatory processes in both vertebrates and invertebrates. The expression of these proteins significantly increases after bacterial infection; however, the mechanisms by which they regulate the innate immune response are still poorly defined. Here, we evaluate the effect of bacterial lipopolysaccharide injection on the expression pattern of these genes and the interrelation between them during innate immune response in the medicinal leech, an invertebrate model with a simple anatomy and a marked similarity with vertebrates in inflammatory processes. Collectively, prokaryotic-eukaryotic co-cultures and in vivo infection assays suggest that RNASET2 and AIF-1 play a crucial role in orchestrating a functional cross-talk between granulocytes and macrophages in leeches, resulting in the activation of an effective response against pathogen infection. RNASET2, firstly released by granulocytes, likely plays an early antibacterial role. Subsequently, AIF-1+ RNASET2-recruited macrophages further recruit other macrophages to potentiate the antibacterial inflammatory response. These experimental data are in keeping with the notion of RNA-SET2 acting as an alarmin-like molecule whose role is to locally transmit a "danger" signal (such as a bacterial infection) to the innate immune system in order to trigger an appropriate host response.

Catalase A is involved in the response to photooxidative stress in Pseudomonas aeruginosa.

BACKGROUND: Pseudomonas aeruginosa is the etiological agent of systemic and skin infections that are often difficult to treat. Photodynamic therapy (PDT) and, more recently, phototherapy (PT), are emerging among antimicrobial treatments to be combined with antibiotics. Visible light, either alone or combined with a photosensitizer (PS), elicits photooxidative stress that induces microbial death. The response of bacteria to phototherapy seems to involve the antioxidant machinery. This study relies on the effects of detoxifying catalase A (KatA) in response to PDT and PT-induced photooxidative stress. METHODS: The photo- and photodynamic inactivation experiments have been targeted at P. aeruginosa PAO1 and its isogenic derivative katA- mutant. The microorganisms were irradiated by a wide-spectrum halogen-tungsten lamp or light-emitting diodes (LEDs). Two photosensitizers, Tetrakis-(1-methyl-4-pyridyl)-21H, 23porphine, tetra-p-tosylate (TMPyP) porphyrin and Toluidine Blue O (TBO), were applied as part of the photodynamic approach. RESULTS: P. aeruginosa katA- mutant was more sensitive than wild-type strain PAO1 to wide-spectrum light and blue LED (464 nm) treatments. The complementation of KatA, in katA- mutant, restored the light response of wild-type PAO1. Upon TBO treatment and irradiation by visible light (halogen lamp or LED), the sensitivity of katA- mutant was significant higher (p = 0.028 and p = 0.045, respectively) than that of the PAO1 strain. CONCLUSIONS: This study provides the first description of KatA in the response to photooxidative stress induced by photo- and photodynamic therapy.

Pigments influence the tolerance of Pseudomonas aeruginosa PAO1 to photodynamically induced oxidative stress.

Pseudomonas aeruginosa is an opportunistic pathogen known to be resistant to different classes of antibiotics and disinfectants. P. aeruginosa also displays a certain degree of tolerance to photodynamic therapy (PDT), an alternative antimicrobial approach exploiting a photo-oxidative stress induced by exogenous photosensitizers and visible light. To evaluate whether P. aeruginosa pigments can contribute to its relative tolerance to PDT, we analysed the response to this treatment of isogenic transposon mutants of P. aeruginosa PAO1 with altered pigmentation. In general, in the presence of pigments a higher tolerance to PDT-induced photo-oxidative stress was observed. Hyperproduction of pyomelanin makes the cells much more tolerant to stress caused by either radicals or singlet oxygen generated by different photosensitizers upon photoactivation. Phenazines, pyocyanin and phenazine-1-carboxylic acid, produced in different amounts depending on the cultural conditions, are able to counteract both types of PDT-elicited reactive oxygen species. Hyperproduction of pyoverdine, caused by a mutation in a quorum-sensing gene, rendered P. aeruginosa more tolerant to a photosensitizer that generates mainly singlet oxygen, although in this case the observed tolerance to photo-oxidative stress cannot be exclusively attributed to the presence of the pigment.

Characterization of the human EDF-1 minimal promoter: involvement of NFY and Sp1 in the regulation of basal transcription.

Endothelial Differentiation Factor (EDF)-1 is a calmodulin binding protein involved in the repression of endothelial cell differentiation, a crucial, late step in angiogenesis. Its expression is cell cycle regulated, although its transcriptional regulation is yet to be determined. To map the promoter region and to understand its regulation, we cloned and fused 2300 bp upstream of EDF-1 translational start site to a luciferase reporter gene. After transient transfection in HeLa cells, this fragment was shown to possess a promoter activity. Deletion constructs of the 5' flanking region of EDF-1 lead to the identification of the minimal promoter region which was highly homologous to the mouse sequence. No TATA box was detected, whereas three consensus sequences--two GC boxes and a CAAT box--were identified. EMSA supershift and chromatin immunoprecipitation demonstrated that these sequences were binding sites for Sp1/Sp3 and NFY, respectively. Deletion of Sp1/Sp3 and NF-Y consensus sequences resulted in the total loss of EDF-1 promoter activity. Our studies indicate that Sp1 and NFY binding is essential for EDF-1 promoter activity.

Complex transcriptional effects of p63 isoforms: identification of novel activation and repression domains.

p63 is a transcription factor structurally related to the p53 tumor suppressor. The C-terminal region differs from p53's in that it contains a sterile alpha motif (SAM) domain and is subject to multiple alternative splicings. The N-terminal region is present in the transactivation (TA) and DeltaN configurations, with the latter lacking the transcriptional activation domain 1. Single amino acid substitutions and frameshift mutations of p63 cause the human ankyloblepharon ectodermal dysplasia clefting (AEC) or ectrodactyly ectodermal dysplasia and facial clefting (EEC) syndromes. We have systematically compared the activities of the wild-type p63 isoforms and of the natural mutants in activation and repression assays on three promoters modulated by p53. We found that p63 proteins with an altered SAM domain or no SAM domain-the beta isoforms, the EEC frameshift mutant, and the missense AEC mutations-all showed a distinctly higher level of activation of the MDM2 promoter and decreased repression on the HSP70 promoter. Fusion of SAM to the GAL4 DNA-binding domain repressed a heterologous promoter. A second activation domain, TA2, corresponding to exons 11 to 12, was uncovered by comparing the activation of DeltaN isoforms on natural promoters and in GAL4 fusion systems. In colony formation assays, the AEC mutants, but not the EEC frameshift, were consistently less efficient in suppressing growth, in both the TA version and the DeltaN version, with respect to their p63alpha counterparts. These data highlight the modularity of p63, identifying the SAM domain as a dominant transcriptional repression module and indicating that the AEC and EEC frameshift mutants are characterized by a subversion of the p63 transcriptional potential.