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Background Acne is a common skin disorder that involves an infection inside the hair follicle, which is usually treated with antibiotics, resulting in unbalanced skin microbiota and microbial resistance. For this reason, we developed polymeric nanoparticles encapsulating thymol, a natural active compound with antimicrobial and antioxidant properties. In this work, optimization physicochemical characterization, biopharmaceutical behavior and therapeutic efficacy of this novel nanostructured system were assessed. Results Thymol NPs (TH-NP) resulted on suitable average particle size below 200 nm with a surface charge around − 28 mV and high encapsulation efficiency (80%). TH-NP released TH in a sustained manner and provide a slow-rate penetration into the hair follicle, being highly retained inside the skin. TH-NP possess a potent antimicrobial activity against Cutibacterium acnes and minor effect towards Staphylococcus epidermis, the major resident of the healthy skin microbiota. Additionally, the stability and sterility of developed NPs were maintained along storage. Conclusion TH-NP showed a promising and efficient alternative for the treatment of skin acne infection, avoiding antibiotic administration, reducing side effects, and preventing microbial drug resistance, without altering the healthy skin microbiota. Additionally, TH-NP enhanced TH antioxidant activity, constituting a natural, preservative-free, approach for acne treatment. Graphical Abstract

Trefoil factor 3 (TFF3) plays a key role in the maintenance and repair of intestinal mucosa. TFF3 expression is upregulated by the microbiota through TLR2. At the posttranscriptional level, TFF3 is downregulated by miR-7-5p. Reduced TFF3 levels have been detected in the damaged tissue of IBD patients. Here, we investigate the regulation of TFF3 expression by microbiota extracellular vesicles (EVs) in LS174T goblet cells using RT-qPCR and inhibitors of the TLR2 or PI3K pathways. To evaluate the subsequent impact on epithelial barrier function, conditioned media from control and vesicle-stimulated LS174T cells were used to treat Caco-2 monolayers. The barrier-strengthening effects were evaluated by analysing the expression and subcellular distribution of tight junction proteins, and the repairing effects were assessed using wound-healing assays. The results showed a differential regulation of TFF3 in LS174T via EVs from the probiotic EcN and the commensal ECOR12. EcN EVs activated the TFF3 production through TLR2 and downregulated miR7-5-p through PI3K. Consistently, high levels of secreted TFF3 reinforced the tight junctions and stimulated wound healing in the Caco-2 cells. ECOR12 EVs did not cause these effects. TFF3 is a potential therapeutic target in IBD. This study contributes to understanding the molecular players (microbiota EVs) connecting gut microbes to health and may help in designing better nutritional interventions based on microbiota bioactive compounds.

Gut bacteria release extracellular vesicles (BEVs) as an intercellular communication mechanism that primes the host innate immune system. BEVs from E. coli activate dendritic cells (DCs) and subsequent T-cell responses in a strain-specific manner. The specific immunomodulatory effects were, in part, mediated by differential regulation of miRNAs. This study aimed to deepen understanding of the mechanisms of BEVs to drive specific immune responses by analyzing their impact on DC-secreted cytokines and exosomes. DCs were challenged with BEVs from probiotic and commensal E. coli strains. The ability of DC-secreted factors to activate T-cell responses was assessed by cytokine quantification in indirect DCs/naïve CD4+ T-cells co-cultures on Transwell supports. DC-exosomes were characterized in terms of costimulatory molecules and miRNAs cargo. In the absence of direct cellular contacts, DC-secreted factors triggered secretion of effector cytokines by T-cells with the same trend as direct DC/T-cell co-cultures. The main differences between the strains influenced the production of Th1- and Treg-specific cytokines. Exosomes released by BEV-activated DCs were enriched in surface proteins involved in antigen presentation and T-cell activation, but differed in the content of immune-related miRNA, depending on the origin of the BEVs. These differences were consistent with the derived immune responses. Keywords: bacterial membrane vesicles; exosomes; probiotics; intestinal homeostasis; immune regulation; miRNAs

Dry eye disease (DED) is a high prevalent multifactorial disease characterized by a lack of homeostasis of the tear film which causes ocular surface inflammation, soreness, and visual disturbance. Conventional ophthalmic treatments present limitations such as low bioavailability and side effects. Lactoferrin (LF) constitutes a promising therapeutic tool, but its poor aqueous stability and high nasolacrimal duct drainage hinder its potential efficacy. In this study, we incorporate lactoferrin into hyaluronic acid coated liposomes by the lipid film method, followed by high pressure homogenization. Pharmacokinetic and pharmacodynamic profiles were evaluated in vitro and ex vivo. Cytotoxicity and ocular tolerance were assayed both in vitro and in vivo using New Zealand rabbits, as well as dry eye and anti-inflammatory treatments. LF loaded liposomes showed an average size of 90 nm, monomodal population, positive surface charge and a high molecular weight protein encapsulation of 53%. Biopharmaceutical behaviour was enhanced by the nanocarrier, and any cytotoxic effect was studied in human corneal epithelial cells. Developed liposomes revealed the ability to reverse dry eye symptoms and possess anti-inflammatory efficacy, without inducing ocular irritation. Hence, lactoferrin loaded liposomes could offer an innovative nanotechnological tool as suitable approach in the treatment of DED., FCT -Fundação para a Ciência e a Tecnologia(2017SGR1477), info:eu-repo/semantics/publishedVersion

There are a large number of remedies in traditional medicine focused on relieving pain and inflammation. Flavanones have been a potential source in the search for leading compounds and biologically active components, and they have been the focus of much research and development in recent years. Eysenhardtia platycarpa is used in traditional medicine for the treatment of kidney diseases, bladder infections, and diabetes mellitus. Many compounds have been isolated from this plant, such as flavones, flavanones, phenolic compounds, triterpenoid acids, chalcones, sugars, and fatty acids, among others. In this paper, natural flavanone 1 (extracted from Eysenhardtia platycarpa) as lead compound and flavanones 1a–1d as its structural analogues were screened for anti-inflammatory activity using Molinspiration® and PASS Online in a computational study. The hydro alcoholic solutions (FS) of flavanones 1, 1a–1d (FS1, FS1a–FS1d) were also assayed to investigate their in vivo anti-inflammatory cutaneous effect using two experimental models, a rat ear edema induced by arachidonic acid (AA) and a mouse ear edema induced by 12-O-tetradecanoylphorbol acetate (TPA). Histological studies and analysis of pro-inflammatory cytokines TNF-α, IL-1β, and IL-6 were also assessed in AA-inflamed rat ear tissue. The results showed that the flavanone hydro alcoholic solutions (FS) caused edema inhibition in both evaluated models. This study suggests that the evaluated flavanones will be effective when used in the future in skin pathologies with inflammation, with the results showing 1b and 1d to be the best.

Communication between cells is crucial to preserve body homeostasis and health. Tightly controlled intercellular dialog is particularly relevant in the gut, where cells of the intestinal mucosa are constantly exposed to millions of microbes that have great impact on intestinal homeostasis by controlling barrier and immune functions. Recent knowledge involves extracellular vesicles (EVs) as mediators of such communication by transferring messenger bioactive molecules including proteins, lipids, and miRNAs between cells and tissues. The specific functions of EVs principally depend on the internal cargo, which upon delivery to target cells trigger signal events that modulate cellular functions. The vesicular cargo is greatly influenced by genetic, pathological, and environmental factors. This finding provides the basis for investigating potential clinical applications of EVs as therapeutic targets or diagnostic biomarkers. Here, we review current knowledge on the biogenesis and cargo composition of EVs in general terms. We then focus the attention to EVs released by cells of the intestinal mucosa and their impact on intestinal homeostasis in health and disease. We specifically highlight their role on epithelial barrier integrity, wound healing of epithelial cells, immunity, and microbiota shaping. Microbiota-derived EVs are not reviewed here.

Ocular inflammation is one of the most common comorbidities associated to ophthalmic surgeries and disorders. Since conventional topical ophthalmic treatments present disadvantages such as low bioavailability and relevant side effects, natural alternatives constitute an unmet medical need. In this sense, lactoferrin, a high molecular weight protein, is a promising alternative against inflammation. However, lactoferrin aqueous instability and high nasolacrimal duct drainage compromises its potential effectiveness. Moreover, nanotechnology has led to an improvement in the administration of active compounds with compromised biopharmaceutical profiles. Here, we incorporate lactoferrin into biodegradable polymeric nanoparticles and optimized the formulation using the design of experiments approach. A monodisperse nanoparticles population was obtained with an average size around 130 nm and positive surface charge. Pharmacokinetic and pharmacodynamic behaviour were improved by the nanoparticles showing a prolonged lactoferrin release profile. Lactoferrin nanoparticles were non-cytotoxic and non-irritant neither in vitro nor in vivo. Moreover, nanoparticles exhibited significantly increased anti-inflammatory efficacy in cell culture and preclinical assays. In conclusion, lactoferrin loaded nanoparticles constitute a safe and novel nanotechnological tool suitable for the treatment of ocular inflammation., This work was supported by Portuguese Science and Technology Foundation (FCT/MCT) and from European Funds (PRODER/ COMPETE) under the project reference UIDB/04469/2020 (strategic found), co-financed by FEDER, under the Partnership Agreement PT2020: UIDB/04469/2020., info:eu-repo/semantics/publishedVersion

The present work is focused on the development of novel surface-functionalized poly(lactic-co-glycolic acid) nanoparticles loaded with thymol (TH-NPs) for topical administration enhancing thymol anti-inflammatory, antioxidant and wound healing activities against acne. TH-NPs were prepared by solvent evaporation method using different surface functionalization strategies and obtaining suitable physicochemical parameters and a good short-term stability at 4 °C. Moreover, TH-NPs skin penetration and antioxidant activity were assessed in ex vivo pig skin models. Skin penetration of TH-NPs followed the follicular route, independently of the surface charge and they were able to enhance antioxidant capacity. Furthermore, antimicrobial activity against Cutibacterium acnes was evaluated in vitro by the suspension test showing improved antibacterial performance. Using human keratinocyte cells (HaCat), cytotoxicity, cellular uptake, antioxidant, anti-inflammatory and wound healing activities were studied. TH-NPs were non-toxic and efficiently internalized inside the cells. In addition, TH-NPs displayed significant anti-inflammatory, antioxidant and wound healing activities, which were highly influenced by TH-NPs surface modifications. Moreover, a synergic activity between TH-NPs and their surface functionalization was demonstrated. To conclude, surface-modified TH-NPs had proven to be suitable to be used as anti-inflammatory, antioxidant and wound healing agents, constituting a promising therapy for treating acne infection and associated inflammation.

Pioglitazone (PGZ) is a drug used to treat type 2 diabetes mellitus that has been reported to show additional therapeutic activities on diverse inflammatory parameters. The aim of this study was to optimize a topical PGZ-loaded nanoemulsion (PGZ-NE) in order to evaluate its effectiveness for treating atopic dermatitis (AD). The composition of the nanoformulation was established by pseudo-ternary diagram. Parameters such as physical properties, stability, in vitro release profile, and ex vivo permeation were determined. The efficacy study was carried out using oxazolone-induced AD model in hairless mice. PGZ-NE released the drug following a hyperbolic kinetic. Additionally, its properties provided high retention potential of drug inside the skin. Therapeutic benefits of PGZ-NE were confirmed on diverse events of the inflammatory process, such as reduction of lesions, enhancement of skin barrier function, diminished infiltration of inflammatory cells, and expression of pro-inflammatory cytokines. These results were reinforced by atomic force microscope (AFM), which demonstrated the ability of the formulation to revert the rigidification caused by oxazolone and consequently improve the elasticity of the skin. These results suggest that PGZ-NE may be a promising treatment for inflammatory dermatological conditions such as AD., This work was supported by Secretaría de Educación Superior, Ciencia, Tecnología e Innovación (SENESCYT—Ecuador) (grant number 073-CIBAE-2015).

The synthesis and characterization of two hybrid N-methylated carbazole derivatives containing a thiazolyl or a thienyl ring is reported. The thiazolyl derivative has been also characterised by X-ray diffraction analysis. The study of its reactivity in front of [MCl2(dmso)2] (M = Pd or Pt) or Na2[PdCl4] in methanol has allowed us to isolate and characterize its complexes. However, for the thienyl analogue, the formation of any Pd(II) or Pt(II) complex was not detected, indicating that it is less prone to bind to the M(II) ions than its thiazolyl analogue. Density Functional Theory (DFT) and Time-Dependent Density Functional Theory (TD-DFT) calculations have also been carried out in order to rationalize the influence of the nature of the thiazolyl or thienyl group on the electronic delocalization. Molecular mechanics calculations show that the free rotation of the thiazolyl in relation to the carbazole requires a greater energy income than for its thienyl analogue. Studies of the cytotoxic activity of the new compounds on colon (HCT116) and breast (MDA-MB231 and MCF7) cancer cell lines show that the thiazolyl carbazole ligand and its Pt(II) complex are the most active agents of the series and in the MCF7 line their potency is higher than that of cisplatin. In the non-tumoral human skin fibroblast BJ cell line, all the compounds were less toxic than cisplatin. Their potential ability to modify the electrophoretic mobility of pBluescript SK+ plasmid DNA and to act as inhibitors of Topoisomerases I and IIα or cathepsin B has also been investigated.

The overexpression and increased activity of the serine protease Kallikrein 5 (KLK5) is characteristic of inflammatory skin diseases such as Rosacea. The use of inhibitors of this enzyme¿such as 4-(2-aminoethyl)benzenesulfonyl fluoride hydrochloride (AEBSF·HCl) or the anti-human recombinant Kallikrein 5 (anti-KLK5) antibody¿in the treatment of the disease has been limited due to their low bioavailability, for which their immobilization in drug delivery agents can contribute to making serine protease inhibitors clinically useful. In this work, we synthesized gold nanoparticles (GNP) coated with a mixture of hydroxyl- and carboxyl-terminated thiolates (GNP.OH/COOH), whose carboxyl groups were used to further functionalize the nanoparticles with the serine protease inhibitor AEBSF·HCl either electrostatically or covalently (GNP.COOH AEBSF and GNP.AEBSF, respectively), or with the anti-KLK5 antibody (GNP.antiKLK5). The synthesized and functionalized GNP were highly water-soluble, and they were extensively characterized using UV-vis absorption spectroscopy, Transmission Electron Microscopy (TEM), Dynamic Light Scattering (DLS), and Thermogravimetric Analysis (TGA). GNP.OH/COOH and their subsequent functionalizations effectively inhibited KLK5 in vitro. Internalization of fluorophore-coated GNP.OH/COOH in human keratinocytes (HaCaT cells) was proven using confocal fluorescence microscopy. Cell viability assays revealed that the cytotoxicity of free AEBSF is importantly decreased when it is incorporated in the nanoparticles, either ionically (GNP.COOH AEBSF) or, most importantly, covalently (GNP.AEBSF). The functionalized nanoparticles GNP.AEBSF and GNP.antiKLK5 inhibited intracellular KLK5 activity in HaCaT cells and diminished secretion of IL-8 under inflammatory conditions triggered by TLR-2 ligands. This study points to the great potential of these GNP as a new intracellular delivery strategy for both small drugs and antibodies in the treatment of skin diseases such as Rosacea.

(E)-N-([1,1'-biphenyl]-2-yl)-1-mesitylmethanimine (imine a) and two structural cyclopalladated isomers of imine a, which contained a six-membered metallacycle and an aliphatic and an aromatic metallated carbon of formula [Pd{CH2-3,5-Me2-6-(CH=N-C6H4-2′-C6H5)C6H2}Br(PPh3)] (3a-endo) and [Pd(C6H4-2-C6H4-2′-N=CH-2,4,6-C6H2Me3)Br(PPh3)] (3a-exo), were synthetized by condensation reaction (imine a) and subsequent cyclometallation, metathesis and splitting reactions (compounds 3a-endo and 3a-exo). Imine a was characterized by high resolution ESI-(+) mass spectrometry, IR, and 1H-NMR, and compounds 3a by elemental analysis, high resolution ESI-(+) mass spectrometry, IR, and 1H- and 31P{1H}-NMR. The structural isomer 3a-exo consisted of a mixture of Z and E geometrical isomers in relation to its carbon-nitrogen double bond. In vitro anticancer, antibacterial and antioxidant activities of structural isomers 3a were compared with those of imine a and those of the six membered cyclopalladated primary amine 3b of formula [Pd(C6H4-2-C6H4-2′-NH2)Br(PPh3)]. Compound 3b was prepared as reported. Compounds 3 cytotoxicity towards cancer and normal cells was very dependent on their structure. Compounds 3a-exo and 3b were more cytotoxic than imine a towards the assayed cancer cell lines. Interestingly, compound 3a-exo with an exo imine function to the palladacycle presented a cytotoxicity in the low micromolar range (5–20 μM) towards the assayed cancer cell lines and was at least seven times less cytotoxic than cisplatin against normal BJ human fibroblast cells. Compounds 3 and imine a presented a noticeable antibacterial and antioxidant activity. Notably, imine a produced an antioxidant activity similar to that of ascorbic acid. Finally, compounds 3 were not efficient for altering the DNA tertiary structure or as inhibitors of cathepsin B.

Platinum-based chemotherapy persists to be the only effective therapeutic option against a wide variety of tumours. Nevertheless, the acquisition of platinum resistance is utterly common, ultimately cornering conventional platinum drugs to only palliative in many patients. Thus, encountering alternatives that are both effective and non-cross-resistant is urgent. In this work, we report the synthesis, reduction studies, and luminescent properties of a series of cyclometallated (C,N,N')PtIV compounds derived from amine- imine ligands, and their remarkable efficacy at the high nanomolar range and complete lack of cross57 resistance, as an intrinsic property of the platinacycle, against multiplatinum-resistant colorectal cancer (CRC) and castration-resistant prostate cancer (CRPC) metastatic cell lines generated for this work. We have also determined that the compounds are effective and selective for a broader cancer panel, including breast and lung cancer. Additionally, selected compounds have been further evaluated, finding a shift in their antiproliferative mechanism towards more cytotoxic and less cytostatic than cisplatin against cancer cells, being also able to oxidize cysteine residues and inhibit topoisomerase II, thereby holding great promise as future improved alternatives to conventional platinum drugs.

Gut microbiota plays an essential role in maintaining intestinal homeostasis and human health. Microbiota establishes a complex network of dynamic and reciprocal interactions with the intestinal epithelium and immune system. The mucin layer that covers the epithelium prevents luminal bacteria from accessing host cells. Thus, microbiota–host communication mainly relies on secreted factors and membrane vesicles (MVs), which can cross the inner mucus layer and reach the epithelium. This chapter focuses on the role of microbiota-secreted MVs as key players in signaling processes in the intestinal mucosa. This is an emerging research topic, with the first reports dating from 2012. Microbiota-derived MVs are involved in interspecies communication in the gut, between bacteria and between microbiota and host. Here we present current knowledge on the mechanisms used by microbiota MVs to assist and control the gut microbial community and to modulate host immune and defense responses. Constant stimulation of immune receptors by microbiota MVs results in tightly controlled inflammation that contributes to tolerogenic responses essential to maintaining intestinal homeostasis. Moreover, gut microbiota MVs are emerging as physical vehicles for distribution and delivery of bacterial effectors to distal tissues in human health and disease.

Aim: Development of fluorometholone-loaded PEG-PLGA nanoparticles (NPs) functionalized with cell-penetrating peptides (CPPs) for the treatment of ocular inflammatory disorders. Materials & methods: Synthesized polymers and peptides were used for elaboration of functionalized NPs, which were characterized physicochemically. Cytotoxicity and ability to modulate the expression of proinflammatory cytokines were evaluated in vitro using human corneal epithelial cells (HCE-2). NPs uptake was assayed in both in vitro and in vivo models. Results: NPs showed physicochemical characteristics suitable for ocular administration without evidence of cytotoxicity. TAT-NPs and G2-NPs were internalized and displayed anti-inflammatory activity in both HCE-2 cells and mouse eye. Conclusion: TAT-NPs and G2-NPs could be considered a novel strategy for the treatment of ocular inflammatory diseases of the anterior and posterior segment.

Platinum-based chemotherapy persists to be the only effective therapeutic option against a wide variety of tumours. Nevertheless, the acquisition of platinum resistance is utterly common, ultimately cornering conventional platinum drugs to only palliative in many patients. Thus, encountering alternatives that are both effective and non-cross-resistant is urgent. In this work, we report the synthesis, reduction studies, and luminescent properties of a series of cyclometallated (C,N,N')Pt

Extracellular membrane vesicles (MVs) released by gut microbiota are key players in the communication with the host. The aim of this study was to evaluate the immunomodulatory properties of MVs from the probiotic E. coli Nissle 1917 (EcN) in terms of DC-derived adaptive immune responses and to compare the effects with those elicited by commensal E. coli. The effects of MVs were analysed in monocyte-derived DCs by measuring cytokine expression and the ability of activated-DCs to differentiate CD4+ T cells towards specific effector subsets. EcN MVs derived intricate Th1/Th2/Th17/Th22/Treg responses consistent with the beneficial effects of this probiotic. Th2/Th17/Th22 responses were common to commensal E. coli-derived vesicles but specific differences were observed for Th1 and Treg responses. Since MVs activate DCs in a strain-specific manner, probiotic-derived MVs could be explored as a safe (bacteria-free) strategy to develop new functional food ingredients targeting gut microbiota balance or intestinal inflammation.

Conjugation of cytostatic drugs to nanomaterials seeks to improve their low bioavailability and selectivity to overcome the important associated side effects. In this work, we aimed to synthesize water-soluble gold nanoparticles as transporters for synthetic cyclic peptides with a potential anticancer activity but with a limited bioavailability. The highly water-soluble nanoparticles (2.5 nm diameter gold core) are coated with a mixture of polyethylene glycol linkers, one bearing a terminal hydroxyl group for increasing dispersibility in water, and the second bearing a carboxylic acid group for peptide conjugation through amide bond formation. Peptide-functionalized particles have a 9.7 ± 1.8 nm hydrodynamic diameter and are highly water-soluble and stable in solution for at least one year. The morphology of the gold cores as well as their organic coating was studied using Transmission Electron Microscopy, showing that the attachment of a limited number of peptides per nanoparticle leads to a uneven organic coating of two different thicknesses, one of 2.0 ± 0.6 nm formed by polyethylene glycol linkers, and a second of 3.6 ± 0.5 nm which includes the peptide. GNP significantly enhance the internalization of the cyclic peptide BPC734 in cells as compared to peptide in solution, with improved uptake in cancerous HT29 cells. Cytotoxicity studies show that peptide BPC734 in solution is toxic in the micromolar range, whereas peptide-functionalized particles are toxic at nanomolar peptide concentrations and with a significantly higher toxicity for cancerous cells. All these results, besides the stability and expected passive tumor targeting, make these particles a promising option for improving the bioavailability, efficacy, and selectivity in cancer therapy.

The synthesis of three new cyclometallated platinum(II) compounds containing a para-tolyl ligand and a tridentate [C,N,N'] (cm1) or a bidentate [C,N] ligand and an additional ligand such as SEt2 (cm2) or Ph3 (cm3) is reported. The X-ray molecular structure of platinum(II) compound cm3 is also presented. Intermolecular oxidative addition of methyl iodide or iodine upon cm1, cm2 and cm3 produced six novel cyclometallated platinum(IV) compounds. The cytotoxic activity against a panel of human adenocarcinoma cell lines (A-549 lung, MDA-MB-231 and MCF-7 breast, and HCT-116 colon), DNA interaction, topoisomerase I, IIα, and cathepsin B inhibition, and cell cycle arrest, apoptosis and ROS generation of the investigated complexes are presented. The best results for antiproliferative activity were obtained for platinum (IV) compounds cm1MeI and cm1I2 arising from oxidative addition of methyl iodide and iodine, respectively, to cm1. Cyclometallated platinum(IV) compounds cm1MeI and cm3MeI induce significant changes in the mobility of DNA and, in addition, cm1MeI, cm3MeI and cm1I2, showed considerable topoisomerase IIα inhibitory activity. Moreover, the compounds exhibiting the higher antiproliferative activity (cm1MeI and cm1I2) were found to generate ROS and to supress HCT-116 colon cancer cell growth by a mixture of cell cycle arrest and apoptosis induction. 1H NMR experiments carried out in a buffered aqueous medium (pH 7.40) indicate that compound cm1MeI is not reduced by common biologically relevant reducing agents such as ascorbic acid, glutathione or cysteine.

Cisplatin is an efficient anticancer drug, but its effects are often lost after several chemotherapy cycles, showing important secondary effects. For these reasons, new anticancer agents, with different coordination properties and mechanisms of action, are needed. Here we describe the reaction of 2-phenylaniline with cis-[PtCl2(dmso)(2)] and sodium acetate to afford a cycloplatinated compound 2 and the synthesis and some biological studies of 3-6 (two neutral and two ionic compounds): [PtCl(C-N)(L)], C-N cycloplatinated 2-phenylaniline with L = PPh3(3) or P(4-FC6H4)(3) (4) and [Pt(C-N)(L-L)]Cl with L-L = Ph2PCH2CH2Ph2(5) or (C6F5)(2)PCH2-CH2(C6F5)(2) (6). Ionic platinacycles 5 and 6 show a greater antiproliferative activity than that of cisplatin in human lung, breast, and colon cancer cell lines (A-549, MDA-MB-231 and MCF-7, and HCT-116), a remarkable result given the fact that they do not show covalent interaction with DNA. 5 and 6 have also been found able to oxidize NADH by a catalytic process prod- oducing H2O2 as ROS. The activity of these complexes to generate ROS seems to be the key factor to explain their potent anticancer activity; it should be noted that platinum(II) complexes showing biocatalytic activity for hydride transfer from NADH have not been described so far. Ionic complex 6 shows low affinity to some target proteins; the presence of perfluoroaromatic rings seems to hinder its interaction with some biomolecules.

Twelve cyclometallated palladium(II) complexes containing primary aromatic amines [benzylamine (a), (R)-1-(1-naphthyl)ethylamine (b) and 2-phenylaniline (c)] as anionic bidentate (C,N)(-) ligands have been evaluated against a panel of human adenocarcinoma cell lines (A549 lung, MDA-MB231 and MCF7 breast, and the cisplatin resistant HCT116 colon). The results revealed a remarkable antiproliferative activity of the triphenylphosphane mononuclear compounds 3-4 (series a, b, c) and the best inhibition was provided for 3c and 4c with the 2-phenylaniline ligand and a six membered chelate ring. Interestingly, 3c and 4c were 14 and 19 times more potent than cisplatin for the inhibition of the cisplatin resistant HCT116 human adenocarcinoma cell line, respectively. Cyclopalladated complexes 3c and 4c exercise their antiproliferative activity over A549 cells mainly through the induction of apoptosis (38 and 31-fold increase in early apoptotic cells, respectively).

The cyclopalladation of a series of symmetric diimines with the formula (RC6H4CHNZ)2, where Z = CH2 or (CH2)2OCH2 and R = p-Cl, p-OMe, p-NO2, and o-Cl, is described. Optimal conditions to obtain the dimetalated compounds were found to be palladium(II) acetate, in toluene, at 60 °C and with a reaction time of 2−4 h. The reactivity of the dimetalated compounds with monodentate, bidentate, and bis(monodentate) Lewis bases was also studied. The cytotoxic activity of some selected compounds was evaluated against a panel of adenocarcinoma cell lines (colon HCT116 and breast MCF7 and MDA-MB231). Compounds containing the fragment NCH2CH2OCH2CH2OCH2CH2N exhibited a remarkable cytotoxic activity in the three cancer cells assayed, but complexes containing the NCH2CH2N fragment showed no activity. It seems that the length and flexibility of the central saturated chain in the imine molecule, as well as its lipophilicity and hydrophilicity, explain the different cytotoxicity of the two series of coordination compounds here reported.

The synthesis and characterization of the new 2-phenylindole derivative: C 8 H 3 N-2-C 6 H 5 -3NOMe-5OMe ( 3c ) and the trans- and cis- isomers of [Pt( 3c )Cl 2 (DMSO)] complexes ( 4c and 5c , respectively) are described. The crystal structures of 4c ·CH 2 Cl 2 and 5c confirm: (a) the existence of a Pt-N indole bond, (b) the relative arrangement of the Cl − ligands [ trans- (in 4c ) or cis- (in 5c )] and (c) the anti- ( E ) configuration of the oxime. The cytotoxic assessment of C 8 H 3 N-2-(C 6 H 4 -4′R 1 )-3NOMe-5R 2 [with R 1 = R 2 = H ( 3a ); R 1 = Cl, R 2 = H ( 3b ) and R 1 = H, R 2 = OMe ( 3c )] and the geometrical isomers of [Pt(L)Cl 2 (DMSO)] with L = 3a – 3c [ trans- ( 4a – 4c ) and cis- ( 5a – 5c ), respectively] against human breast adenocarcinoma cell lines (MDA-MB231 and MCF-7) is also reported and reveals that all the platinum(II) complexes (except 4a ) are more cytotoxic than cisplatin in front of the MCF7 cell line. Electrophoretic DNA migration studies of the synthesized compounds in the absence and in the presence of topoisomerase-I have been performed, in order to get further insights into their mechanism of action.

The cytotoxic activity of two series of platinum(II) complexes containing the polyfunctional imines R1–CH N–R2 [R1 = phenyl or ferrocenyl unit and R2 = (CH2)n–CH2–NMe2 where n = 1 or 2) (1 and 2) or C6H4-2-SMe (3)] acting as a bidentate (N,N′) (4–7) or terdentate [C(phenyl or ferrocenyl),N,N′]− (8–10) or [C(ferrocenyl),N,S]− ligand (11) in front of A549 lung, MDA-MB231 breast and HCT116 colon human adenocarcinoma cell lines is reported. The results reveal that most of the platinum(II) complexes are active against the three assayed lines and compounds 6, 7 and the platinacycles 10 and 11 exhibit a remarkable antiproliferative activity, even greater than cisplatin itself, in the cisplatin resistant HCT116 human cancer cell line. Electrophoretic DNA migration studies showed that most of them modify the DNA tertiary structure in a similar way as the reference cisplatin. Solution studies of a selection of the most relevant complexes have also been performed in order to test: (a) their stability in the aqueous biological medium and/or the formation of biologically active species and (b) their proclivity to react with 9-methylguanine (9-MeG), as a model nucleobase. Computational studies at DFT level have also been performed in order to explain the different solution behaviour of the complexes and their proclivity to react with the nucleobase.

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is considered a multifunctional protein with defined functions in numerous mammalian cellular processes. GAPDH functional diversity depends on various factors such as covalent modifications, subcellular localization, oligomeric state and intracellular concentration of substrates or ligands, as well as protein–protein interactions. In bacteria, alternative GAPDH functions have been associated with its extracellular location in pathogens or probiotics. In this study, new intracellular functions of Escherichia coli GAPDH were investigated following a proteomic approach aimed at identifying interacting partners using in vivo formaldehyde cross-linking followed by mass spectrometry. The identified proteins were involved in metabolic processes, protein synthesis and folding or DNA repair. Some interacting proteins were also identified in immunopurification experiments in the absence of cross-linking. Pull-down experiments and overlay immunoblotting were performed to further characterize the interaction with phosphoglycolate phosphatase (Gph). This enzyme is involved in the metabolism of 2-phosphoglycolate formed in the DNA repair of 3′-phosphoglycolate ends generated by bleomycin damage. We show that interaction between Gph and GAPDH increases in cells challenged with bleomycin, suggesting involvement of GAPDH in cellular processes linked to DNA repair mechanisms.

The synthesis of six novel cyclometallated platinum(IV) iodido complexes is accomplished by intermolecular oxidative addition of methyl iodide (compounds 2a-2c) or iodine (compounds 3a-3c) upon cyclometallated platinum(II) compounds [PtX{(CH3)(2)N(CH2)(3)NCH(4-ClC6H3)}] (1a-1c: X = Cl, CH3 or I). The X-ray molecular structures of platinum(II) compound 1c and platinum(IV) compounds 3b and 3a' (an isomer of 3a) are reported. The cytotoxic activity against a panel of human adenocarcinoma cell lines (A-549 lung, MDA-MB-231 and MCF-7 breast, and HCT-116 colon), DNA interaction, topoisomerase I, II alpha, and cathepsin B inhibition, and cell cycle arrest, apoptosis and ROS generation of the investigated complexes are presented. Remarkable antiproliferative activity was observed for most of the synthesized cycloplatinated compounds (series 1-3) in all the selected carcinoma cell lines. The best inhibition was provided for the octahedral platinum(IV) compounds 2a-2c exhibiting a methyl and an iodido axial ligand. Preliminary biological results point to a different mechanism of action for the investigated compounds. Cyclometallated platinum(II) compounds 1a-1c modify the DNA migration as cisplatin. In contrast, cyclometallated platinum(IV) compounds 2a-2c and 3a-3c did not modify the DNA tertiary structure neither in the absence nor in the presence of ascorbic acid, which made them incapable of reducing platinum(IV) compounds 2b and 2c in a buffered aqueous medium (pH 7.40) according to H-1 NMR experiments. Remarkable topoisomerase II alpha inhibitory activity is reported for platinum(IV) complexes 2b and 3a and in addition, for the last one, a moderate cathepsin B inhibition is reported. Cell cycle arrest (decrease in G0/G1 and G2 phases and arrest in the S phase), induction of apoptosis and ROS generation are related to the antiproliferative activity of some representative octahedral cyclometallated platinum(IV) compounds (2b and 2c).

The study of the reactivity of (1S,2R) [(η(5)-C(5)H(5))Fe{[(η(5)-C(5)H(4)) CHNCH(Me)CH(OH)C(6)H(5)}] (1a) with cis-[PtCl(2)(DMSO)(2)] under different experimental conditions has allowed to isolate and characterize three pairs of isomeric and diastereomerically pure platinum(II) complexes. Two of the pairs are the trans- and cis- isomers of (1S,2R)[Pt{(η(5)-C(5)H(5))Fe[(η(5)-C(5)H(4))CHNCH(Me)CH(OH)C(6)H(5)]}Cl(2)(DMSO)] [trans-(2a) and cis-(3a), respectively], and of (1S,2R) [Pt{(κ(2)-N,O)(η(5)-C(5)H(5))Fe[(η(5)-C(5)H(4))CHNCH(Me)CH(O)C(6)H(5)]}Cl(DMSO)], {trans-(Cl, N) in (4a)} or a cis-(Cl, N) {in (5a)}; while the third one is formed by platinacycles: [Pt{(κ(2)-C,N[(η(5)-C(5)H(3))]CHN-CH(Me)CH(OH)C(6)H(5)]Fe(η(5)-C(5)H(5))}Cl(DMSO)] with different planar chirality [S(p) (in 6a) or R(p) (in 7a)]. The crystal structures of compounds 2a, 3a, 5a and 6a are also reported. The cytotoxic assessment of 1a-7a on lung (A549), breast (MDA-MB-231) and colon (HCT-116) cancer cell lines is also reported and reveals that the potency of the complexes is strongly dependent on the mode of binding of the iminoalcohol {(N) in 2a and 3a, (N,O)(-) in 4a and 5a or (C,N)(-) in 6a and 7a}, the relative arrangement of the monodentate ligands (in 2a-5a), and the planar chirality of the 1,2-ferrocenylunit in (6a and 7a). Among the new products (2a-7a), compounds 4a and 5a exhibit the highest potency with IC(50) values smaller than cisplatin in the three cancer cell lines assayed. Electrophoretic DNA migration studies in the presence of 2a-7a have been performed in order to get further insights into their mechanism of action. A comparative study of the solution behaviour of all the complexes in DMSO-d(6) or in DMSO-d(6):D(2)O (1:1) mixtures at 298 K is also reported.

Treatment of benzophenone imine with the stoichiometric amount of Pd(OAc) 2 in acetic acid at 60 °C produced the corresponding acetato-bridged five-membered ortho -cyclopalladated dimer [Pd{C 6 H 4 CPh NH}(μ-OAc)] 2 ( 1 ), which was isolated in pure form in a 79% yield. Reaction of 1 with an excess of LiCl in acetone gave rise to the corresponding chlorido-bridged cyclopalladated dimer [Pd{C 6 H 4 CPh NH}(μ-Cl)] 2 ( 2 ) in a 78% yield. Compounds 1 – 2 reacted with an excess of py- d 5 or the stoichiometric amount of PPh 3 to give the mononuclear compounds trans - N , L -[Pd{C 6 H 4 CPh NH}(X)(L)] [ 3 (X = OAc, L = py- d 5 ); 4 (X = Cl, L = py- d 5 ); 5 (X = OAc, L = PPh 3 ) and 6 (X = Cl, L = PPh 3 )]. Compounds 2 – 3 were prepared in CDCl 3 /py- d 5 solution and were studied by 1 H NMR, but were not isolated. In contrast, compounds 5 – 6 were prepared in acetone and were isolated in pure form in 43 and 79% yields, respectively. Compounds 1 , 2 , 5 and 6 were characterized by elemental analyses, mass spectrometry, IR, NMR and electronic spectroscopy. Compounds 1 , 2 , 5 and 6 showed high antiproliferative activity against MDA-MB231 and MCF7 human breast cancer cell lines, especially, compounds 5 – 6 . These two latter compounds presented greater antiproliferative activity than cisplatin and produced IC 50 values in the range 1–5 μM. The interaction of compounds 1 , 2 , 5 and 6 with DNA was also studied by the DNA electrophoretic migration, DNA-ethidium bromide fluorescence quenching and viscometry techniques.

The yiaK–S operon is required for aerobic growth on l-ascorbate in several Enterobacteriaceae. Here we present evidence that the yiaX2 gene belonging to the yiaK–S operon of Klebsiella pneumoniae 13882, which encodes a protein similar to the putative transporters classified as the major facilitator superfamily, is involved in the uptake of l-ascorbate. Concentration kinetic analysis yielded an apparent Km of YiaX2 for l-ascorbate of 161.38 ±8.28 µmol·L–1 and a Vmax of 3.81 ± 0.60 nmol·mg–1·min–1. This carrier uses the energy from electrochemical gradients, since it was inhibited by carbonyl cyanide m-chlorophenylhydrazone, a hydrophobic proton conductor that dissipates proton motive force.

The capacity to both ferment and oxidize l -ascorbate has been widely documented for a number of enteric bacteria. Here we present evidence that all the strains of Klebsiella pneumoniae tested in this study ferment l -ascorbate using the ula regulon-encoded proteins. Under aerobic conditions, several phenotypes were observed for the strains. Our results showed that the yiaK-S system is required for this aerobic metabolic process. Gel shift experiments performed with UlaR and YiaJ and probes corresponding to the specific promoters indicated that l -ascorbate-6-phosphate is the effector molecule recognized by both regulators, since binding of the repressors to their recognition sites was impaired by the presence of this compound. We demonstrated that in K. pneumoniae cells l -ascorbate-6-phosphate is formed only by the action of the UlaABC phosphotransferase system. This finding explains why strains that lack the ula genetic system and therefore are unable to form the inducer intracellularly cannot efficiently use this vitamin as a carbon source under either anaerobic or aerobic conditions. Thus, efficient aerobic metabolism of l -ascorbate in K. pneumoniae is dependent on the presence of both the yiaK-S and ula systems. The expression of the yiaK-S operon, but not the expression of the ula regulon, is controlled by oxygen availability. Both systems are regulated by the cyclic AMP (cAMP)-cAMP receptor protein (CRP) complex and by IHF.

Escherichia coli NADH dehydrogenase-2 (NDH-2) is a primary dehydrogenase in aerobic respiration that shows cupric-reductase activity. The enzyme is encoded by ndh, which is highly regulated by global transcription factors. It was described that the gene is expressed in the exponential growth phase and repressed in late stationary phase. We report the maintenance of NDH-2 activity and ndh expression in the stationary phase when cells were grown in media containing at least 37 mM phosphate. Gene regulation was independent of RpoS and other transcription factors described to interact with the ndh promoter. At this critical phosphate concentration, cell viability, oxygen consumption rate, and NADH/NAD+ ratio were maintained in the stationary phase. These physiological parameters gradually changed, but NDH-2 activity remained high for up to 94 h. Phosphate seems to trigger an internal signal in the stationary phase mediated by systems not yet described.

A series of cyclometallated platinum(IV) compounds (3a, 3a' and 3b') with a meridional [C,N,N'] terdentate ligand, featuring an halido and an aryl group in the axial positions has been evaluated for electrochemical reduction and preliminary biological behavior against a panel of human adenocarcinoma (A-549 lung, HCT-116 colon, and MCF-7 breast) cell lines and the normal bronquial epithelial BEAS-2B cells. Cathodic reduction potentials (shifting from -1.463 to -1.570 V) reveal that the platinum(IV) compounds under study would be highly reluctant to be reduced in a biological environment. Actually ascorbic acid was not able to reduce complex 3a', the most prone to be reduced according its reduction potential, over a period of one week. These results suggest an intrinsic activity for the investigated platinum(IV) complexes (3a, 3a' and 3b'), which exhibit a remarkable cytotoxicity effectiveness (with IC50 values in the low micromolar range), even greater than that of cisplatin. The IC50 for A-549 lung cells and clog P values were found to follow the same trend: 3b' > 3a' > 3a. However, no correlation was observed between reduction potential and in vitro activity. As a representative example, cyclometallated platinum(IV) compound 3a', exercise its antiproliferative activity directly over non-microcytic A-549 lung cancer cells through a mixture of cell cycle arrest (13% arrest at G1 phase and 46% arrest at G2 phase) and apoptosis induction (increase of early apoptosis by 30 times with regard to control). To gain further insights into the mode of action of the investigated platinum(IV) complexes, drug uptake, cathepsin B inhibition and ROS generation were also evaluated. Interestingly an increased ROS generation could be related with the antiproliferative activity of the cyclometallated platinum(IV) series under study in the cisplatin-resistant A-549 lung and HCT-116 cancer cell lines.