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Chlamydia trachomatis is the most prevalent infection of the genital tract in women worldwide. C. trachomatis has a tendency to cause persistent infection and induce a state of chronic inflammation, which has been reported to play a role in carcinogenesis. We report that persistent C. trachomatis infection increases the expression of inflammatory tumour cytokines and upregulates molecular biomarkers such as podoplanin, Wilms' tumour gene 1 and osteopontin in primary cultures of mesothelial cells (Mes1) and human mesothelioma cells (NCI). Infection experiments showed that Mes1 and NCI supported the growth of C. trachomatisin vitro, and at an m.o.i. of 4, the inclusion-forming units/cell showed many intracellular inclusion bodies after 3 days of infection. However, after 7 days of incubation, increased proliferative and invasive activity was also observed in Mes1 cells, which was more evident after 14 days of incubation. ELISA analysis revealed an increase in vascular endothelial growth factor, IL-6, IL-8, and TNF-α release in Mes1 cells infected for a longer period (14 days). Finally, real-time PCR analysis revealed a strong induction of podoplanin, Wilms' tumour gene 1 and osteopontin gene expression in infected Mes1 cells. The aim of the present study was to investigate the inflammatory response elicited by C. trachomatis persistent infection and the role played by inflammation in cell proliferation, secretion of proinflammatory cytokines and molecular biomarkers of cancer. The results of this study suggest that increased molecular biomarkers of cancer by persistent inflammation from C. trachomatis infection might support cellular transformation, thus increasing the risk of cancer.

Cytochrome P450 CYP1A1 and CYP1B1 enzymes are regulated by the aryl hydrocarbon receptor (AhR), a transcription factor activated by a variety of ligands among which Malassezia metabolites. In this study, we analyzed the modulation of CYP1A1, CYP1B1, and AhR in human keratinocytes infected with different strains of Malassezia pachydermatis, as well as the upregulation of some genes involved in the epidermal homeostasis. We demonstrated that all the strains induced AhR activation and its nuclear translocation in HaCaT cells infected for 24 h, compared to untreated cells. The expression of CYP1A1 and CYP1B1, prototypical markers of the AhR signaling pathway, were upregulated with the level of CYP1A1 mRNA approximately 100-fold greater than that for CYP1B1. Filaggrin, involucrin, and TGaseI, proteins involved in epidermal differentiation, were all modulated by Malassezia pachydermatis strains, with the strongest induction observed for filaggrin. By contrast, quinone oxidoreductase 1 (NQO1), which is part of the antioxidant defense system involved in detoxification, was not modulated in our experimental model. In conclusions, our findings suggest that Malassezia pachydermatis infection of human keratinocytes induces activation of the AhR, and increases the expression of its responsive genes and markers of epidermal differentiation, paving the way for occurrence/exacerbation of pathological skin conditions.

Malassezia pachydermatis is part of the normal cutaneous microbiota of most warm-blooded vertebrates and is associated with otitis externa and seborrhoeic dermatitis in dogs and cats. In this study, we evaluated the growth capacity of nine M. pachydermatis strains on Sabouraud medium in the presence of a high concentration of gentamicin. Strains of M. pachydermatis cultured on Sabouraud medium in the presence of 50 and 100 mg gentamicin ml-1 displayed different growth patterns such as growth or lack of growth. We hypothesized that this difference in growth of M. pachydermatis strains was correlated with the different genotypes of the strains. Random amplification of polymorphic DNA (RAPD) was applied for genetic typing of M. pachydermatis isolates, derived from the external ears of house pet cats suffering from otitis externa. The M. pachydermatis strains were cultured on commercial or home-made Sabouraud medium supplemented or not with gentamicin. RAPD analysis demonstrated a genetic heterogeneity between each strain. In particular, five out of nine strains tested were able to form colonies in the presence of gentamicin. However, a correlation between M. pachydermatis genotype and growth capacity in the presence of gentamicin was not widely demonstrated.

Biodiversity is increasingly exploited worldwide for the finding of new pharmaceuticals. In relation to a competitive aptitude developed in many and diverse environments, microorganisms are able to produce secondary metabolites with cytotoxic and antiproliferative properties that are valuable in the perspective of antitumor drug discovery. Particularly, fungal species in the genus Penicillium represent a prolific source of biologically active extrolites that in some cases have already disclosed possible relevance for an application in cancer chemotherapy. Antiproliferative, pro-apoptotic, anti-angiogenic, anti-metastatic, DNA synthesis and cell cycle inhibitory properties of these compounds are reviewed in the present paper. _____________________________________________________________________________________________________________

Seminal vesicle protein IV (SV-IV) is a secretory anti-inflammatory, procoagulant, and immunomodulatory protein produced in large amounts by the seminal vesicle epithelium of the rat under the strict transcriptional control of androgen. In particular, this protein was shown to possess the ability to markedly inhibit in vivo the humoral and cell-mediated immune responses of mice to nonbacterial cellular antigens (sheep erythrocytes and spermatozoa). We report data that demonstrate that in mice treated with SV-IV and infected with Salmonella enterica serovar Typhimurium, SV-IV is able to downregulate some important immunological and biochemical parameters that serovar Typhimurium normally upregulates in these animals. This event did not correlate with a lower bacterial burden but was associated with a markedly increased one (300%). Furthermore, the treatment of mice with SV-IV alone also produced a significant increase in the rate of mortality among serovar Typhimurium-infected animals. The mechanism underlying these phenomena was investigated, and the strong immunosuppression produced by SV-IV in serovar Typhimurium-infected mice was suggested to be the basis for the increased rate of mortality. The SV-IV-mediated immunosuppression was characterized by a decrease in the humoral and cell-mediated immune responses, altered lymphocyte-macrophage interaction, downregulation of cytokine and inducible nitric oxide synthase gene expression, inhibition of macrophage phagocytosis and intracellular killing activities, and absence of apoptosis in the splenocyte population of SV-IV- and serovar Typhimurium-treated mice. The immunosuppressive activity of SV-IV was specific and was not due to aspecific cytotoxic effects. SV-IV-specific receptors (K(d) = 10(-8) M) occurring on the macrophage and lymphocyte plasma membranes may be involved in the molecular mechanism underlying the SV-IV-mediated immunosuppression. Some results obtained by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay also revealed a functional impairment of mitochondria (a decrease in mitochondrial dehydrogenase activity), thus indicating the possible implication of these organelles in the immunosuppressive process.

Antibiotic resistance and biofilm tolerance are among the principal factors involved in the persistence of chronic infections. The need for new antimicrobials is an ever-increasing challenge in clinical environments and in the control of global health. Arylfurans form a set of structures that have been identified in many natural products, e.g. lignans. Lignans are a sub-group of non-flavonoid polyphenols that play an active role in plants’ defense against bacteria and fungi infections. The aim of this study was to identify novel synthetic arylfurans and lignan-like arylbenzylfurans exhibiting antimicrobial properties. The molecules synthetized were tested against Pseudomonas aeruginosa, Escherichia coli, Staphylococcus aureus and S. epidermidis. We found that among tested compounds, arylbenzylfuran 11 was active against S. aureus and S. epidermidis with an MIC of 4 μg ml-1. Compound 11 was also active on methicillin-resistant S. aureus and S. epidermidis. By confocal laser scanning microscopy, we showed that 32 μg ml-1 of compound 11 was able to induce a significant reduction in S. aureus and S. epidermidis biofilms viability. Finally, we demonstrated that compound 11 was not cytotoxic on HaCat cells up to 128 μg ml-1. This work shows the antimicrobial and anti-biofilm potential of a synthetic lignan-like furan.

The generation of peptidomimetic substructures for medicinal chemistry purposes requires effective and divergent synthetic methods. We present in this work an efficient flow process that allows quick modulation of reagents for Joullié-Ugi multicomponent reaction, using spiroindolenines as core motifs. This sterically hindered imine equivalent could successfully be diversified using various isocyanides and amino acids in generally good space-time yields. A telescoped flow process combining interrupted Fischer reaction for spiroindolenine synthesis and subsequent Joullié-Ugi-type modification resulted in product formation in very good overall yield in less than 2 hours compared to 48 hours required in batch mode. The developed protocol can be seen as a general tool for rapid and facile generation of peptidomimetic compounds. We also showcase preliminary biological assessments for the prepared compounds.

Herein we report the preparation of 3,4-dibenzylfurans and some oxidized derivatives with lignan backbone. The compounds were prepared using the Friedel-Crafts reaction with BF3 etherate as catalyst, demethylation with iodocyclohexane, acetylation and oxidation reactions. The antimicrobial activity was evaluated through their capacity to inhibit the growth of Gram positive and Gram negative bacteria, and of the yeast Candida albicans. Among ten products assayed four furans displayed a good antimicrobial activity against Staphylococcus aureus, S. epidermidis and C. albicans; on the contrary, none of the compounds were active against Pseudomonas aeruginosa. One of them inhibited the growth of S. aureus, S. epidermidis (biofilm producer strain) and C. albicans at 16 μg/mL, showing a bactericidal activity already after one hour of treatment. In summary, the results suggest a possible use of these derivatives for general disinfection practices or antimicrobial agents in cosmesis skin-care.

The increasing resistance of fungi to conventional antifungal drugs has prompted worldwide the search for new compounds. In this work, we investigated the antifungal properties of acylated Temporin L derivatives, Pent-1B and Dec-1B, against Candida albicans, including the multidrug-resistant strains. Acylated peptides resulted to be active both on reference and clinical strains with MIC values ranging from 6.5 to 26 µM, and they did not show cytotoxicity on human keratinocytes. In addition, we also observed a synergistic or additive effect with voriconazole for peptides Dec-1B and Pent-1B through the checkerboard assay on voriconazole-resistant Candida strains. Moreover, fluorescence-based assays, NMR spectroscopy, and confocal microscopy elucidated a potential membrane-active mechanism, consisting of an initial electrostatic interaction of acylated peptides with fungal membrane, followed by aggregation and insertion into the lipid bilayer and causing membrane perturbation probably through a carpeting effect.

The pharmacodynamic and pharmacokinetic properties of bioactive peptides can be modulated by introducing conformational constraints such as intramolecular macrocyclizations, which can involve either the backbone and/or side chains. Herein, we aimed at increasing the α-helicity content of temporin L, an isoform of an intriguing class of linear antimicrobial peptides (AMPs), endowed with a wide antimicrobial spectrum, by the employment of diverse side-chain tethering strategies, including lactam, 1,4-substituted [1,2,3]-triazole, hydrocarbon, and disulfide linkers. Our approach resulted in a library of cyclic temporin L analogues that were biologically assessed for their antimicrobial, cytotoxic, and antibiofilm activities, leading to the development of the first-in-class cyclic peptide related to this AMP family. Our results allowed us to expand the knowledge regarding the relationship between the α-helical character of temporin derivatives and their biological activity, paving the way for the development of improved antibiotic cyclic AMP analogues.

WHEN CELLS are damaged, as often occurs during trauma and metabolic stress, the organism has to choose whether to repair the damage by promoting cell survival or remove irreparably injured cells. Cell injury occurs when an adverse stimulus reversibly disrupts the normal, complex homeostatic balance of the cellular metabolism. In this case, after injury the cells attempt to seal breaks in their membranes, chaperone the removal or refolding of altered proteins, and repair damaged DNA. On the contrary, when cell injury is too extensive to permit reparative responses, the cell reaches a “point of no return” and the irreversible injury culminates in programmed cell death (PCD). Specific properties or features of cells make them more or less vulnerable to external stimuli, thus determining the kind of cellular response. In addition, the characteristic of the injury (type of injury, exposure time, or severity) will also affect the extent of the damage. We present a short overview of the best-known PCD pathways. We emphasize the apoptotic pathway, considered for years the hallmark of PCD, and the different stimuli that produce cell injury. CELL INJURY The survival of multicellular organisms depends on the function of a diverse set of differentiated cell types. After development is complete, the viability of the organism depends on the maintenance and renewal of these diverse lineages. Within each lineage homeostasis is maintained through a delicate balance between cell proliferation and cell death.

Objective A large number of studies have highlighted the important role of the gut microbiota in the pathophysiology of neurological disorders, suggesting that its manipulation might serve as a treatment strategy. We hypothesized that the gut microbiota participates in absence seizure development and maintenance in the WAG/Rij rat model and tested this hypothesis by evaluating potential gut microbiota and intestinal alterations in the model, as well as measuring the impact of microbiota manipulation using fecal microbiota transplantation (FMT). Methods Initially, gut microbiota composition and intestinal histology of WAG/Rij rats (a well-recognized genetic model of absence epilepsy) were studied at 1, 4, and 8 months of age in comparison to nonepileptic Wistar rats. Subsequently, in a second set of experiments, at 6 months of age, untreated Wistar or WAG/Rij rats treated with ethosuximide (ETH) were used as gut microbiota donors for FMT in WAG/Rij rats, and electroencephalographic (EEG) recordings were obtained over 4 weeks. At the end of FMT, stool and gut samples were collected, absence seizures were measured on EEG recordings, and microbiota analysis and histopathological examinations were performed. Results Gut microbiota analysis showed differences in beta diversity and specific phylotypes at all ages considered and significant variances in the Bacteroidetes/Firmicutes ratio between Wistar and WAG/Rij rats. FMT, from both Wistar and ETH-treated WAG/Rij donors to WAG/Rij rats, significantly decreased the number and duration of seizures. Histological results indicated that WAG/Rij rats were characterized by intestinal villi disruption and inflammatory infiltrates already at 1 month of age, before seizure occurrence; FMT partially restored intestinal morphology while also significantly modifying gut microbiota and concomitantly reducing absence seizures. Significance Our results demonstrate for the first time that the gut microbiota is modified and contributes to seizure occurrence in a genetic animal model of absence epilepsy and that its manipulation may be a suitable therapeutic target for absence seizure management.

The rapid development of antimicrobial resistance is pushing the search in the discovering of novel antimicrobial molecules to prevent and treat bacterial infections. Self-assembling antimicrobial peptides, as the lipidated peptides, are a novel and promising class of molecules capable of meeting this need. Based on previous work on Temporin L analogs, several new molecules lipidated at the N- or and the C-terminus were synthesised. Our goal is to improve membrane interactions through finely tuning self-assembly to reduce oligomerisation in aqueous solution and enhance self-assembly in bacterial membranes while reducing toxicity against human cells. The results here reported show that the length of the aliphatic moiety is a key factor to control target cell specificity and the oligomeric state of peptides either in aqueous solution or in a membrane-mimicking environment. The results of this study pave the way for the design of novel molecules with enhanced activities.

Previously, we identified a potent antimicrobial analogue of temporin L (TL), [Pro3 ]TL, in which glutamine at position 3 was substituted with proline. In this study, a series of analogues in which position 3 is substituted with non-natural proline derivatives, was investigated for correlations between the conformational properties of the compounds and their antibacterial, cytotoxic, and hemolytic activities. Non-natural proline analogues with substituents at position 4 of the pyrrolidine ring were considered. Structure-activity relationship (SAR) studies of these analogues were performed by means of antimicrobial and cytotoxicity assays along with circular dichroism (CD) and NMR spectroscopic analyses for selected compounds. The most promising peptides were additionally evaluated for their activity against some representative veterinary microbial strains to compare with those from human strains. We identified novel analogues with interesting properties that make them attractive lead compounds.

Resin acids are valued in traditional medicine for their antiseptic properties. Among these, abietic acid has been reported to be active against methicillin-resistant Staphylococcus aureus (MRSA) strains. In veterinary healthcare, the methicillin-resistant Staphylococcus pseudintermedius (MRSP) strain is an important reservoir of antibiotic resistance genes including mecA. The incidence of MRSP has been increasing, and treatment options in veterinary medicine are partial. Here, we investigated the antimicrobial and antibiofilm properties of abietic acid against three MRSP and two methicillin-susceptible Staphylococcus pseudintermedius (MSSP) strains, isolated from diseased pet animals and human wound samples. Abietic acid showed a significant minimal inhibitory concentration (MIC) value ranging from 32 to 64 &mu, g/mL (MRSPs) and 8 &mu, g/mL (MSSP). By checkerboard method we demonstrated that abietic acid increased oxacillin susceptibility of MRSP strains, thus showing a synergistic interaction with oxacillin. Abietic acid was also able to contrast the vitality of treated MSSP and MRSP1 biofilms at 20 &mu, g/mL and 40 &mu, g/mL, respectively. Finally, the compound moderately reduced mecA, mecR1 and mec1 gene expression. In conclusion, the results here reported demonstrate the antimicrobial activity of abietic acid against MRSP and support the use of this compound as a potential therapeutic agent to be used in combinatorial antibiotic therapy.