Adaptive laboratory evolution to hypersaline conditions of lactic acid bacteria isolated from seaweed.

Seaweed biomass has been proposed as a promising alternative carbon source for fermentation processes using microbial factories. However, the high salinity content of seaweed biomass is a limiting factor in large scale fermentation processes. To address this shortcoming, three bacterial species (Pediococcus pentosaceus, Lactobacillus plantarum, and Enterococcus faecium) were isolated from seaweed biomass and evolved to increasing concentrations of NaCl. Following the evolution period, P. pentosaceus reached a plateau at the initial NaCl concentration, whereas L. plantarum, and E. faecium showed a 1.29 and 1.75-fold increase in their salt tolerance, respectively. The impact that salt evolution had on lactic acid production using hypersaline seaweed hydrolysate was investigated. Salinity evolved L. plantarum produced 1.18-fold more lactic acid than the wild type, and salinity evolved E. faecium was able to produce lactic acid, while the wild type could not. No differences in lactic acid production were observed between the P. pentosaceus salinity evolved and wild type strains. Evolved lineages were analyzed for the molecular mechanisms underlying the observed phenotypes. Mutations were observed in genes affecting the ion balance in the cell, the composition of the cell membrane and proteins acting as regulators. This study demonstrates that bacterial isolates from saline niches are promising microbial factories for the fermentation of saline substrates, without the requirement of previous desalination steps, while preserving high final product yields.

Increased Lipophilicity of Halogenated Ruthenium(II) Polypyridyl Complexes Leads to Decreased Phototoxicity in†vitro when Used as Photosensitizers for Photodynamic Therapy.

In the fight against cancer, photodynamic therapy is generating great interest thanks to its ability to selectively kill cancer cells without harming healthy tissues. In this field, ruthenium(II) polypyridyl complexes, and more specifically, complexes with dipyrido[3,2-a:2',3'-c]phenazine (dppz) as a ligand are of particular interest due to their DNA-binding and photocleaving properties. However, ruthenium(II) polypyridyl complexes can sometimes suffer from low lipophilicity, which hampers cellular internalisation through passive diffusion. In this study, four new [Ru(dppz-X2 )3 ]2+ complexes (X=H, F, Cl, Br, I) were synthesized and their lipophilicity (logP), cytotoxicity and phototoxicity on cancerous and noncancerous cell lines were assessed. This study shows that, counterintuitively, the phototoxicity of these complexes decreases as their lipophilicity increases; this could be due solely to the atomic radius of the halogen substituents.

Ruthenium(II) Complex Containing a Redox-Active Semiquinonate Ligand as a Potential Chemotherapeutic Agent: From Synthesis to In Vivo Studies.

Chemotherapy remains one of the dominant treatments to cure cancer. However, due to the many inherent drawbacks, there is a search for new chemotherapeutic drugs. Many classes of compounds have been investigated over the years to discover new targets and synergistic mechanisms of action including multicellular targets. In this work, we designed a new chemotherapeutic drug candidate against cancer, namely, [Ru(DIP)2(sq)](PF6) (Ru-sq) (DIP = 4,7-diphenyl-1,10-phenanthroline; sq = semiquinonate ligand). The aim was to combine the great potential expressed by Ru(II) polypyridyl complexes and the singular redox and biological properties associated with the catecholate moiety. Experimental evidence (e.g., X-ray crystallography, electron paramagnetic resonance, electrochemistry) demonstrates that the semiquinonate is the preferred oxidation state of the dioxo ligand in this complex. The biological activity of Ru-sq was then scrutinized in vitro and in vivo, and the results highlight the promising potential of this complex as a chemotherapeutic agent against cancer.

Synthesis and Characterization of an Epidermal Growth Factor Receptor-Selective RuII Polypyridyl-Nanobody Conjugate as a Photosensitizer for Photodynamic Therapy.

There is a current surge of interest in the development of novel photosensitizers (PSs) for photodynamic therapy (PDT), as those currently approved are not completely ideal. Among the tested compounds, we have previously investigated the use of RuII polypyridyl complexes with a [Ru(bipy)2 (dppz)]2+ and [Ru(phen)2 (dppz)]2+ scaffold (bipy=2,2'-bipyridine; dppz=dipyrido[3,2-a:2',3'-c]phenazine; phen=1,10-phenanthroline). These complexes selectively target DNA. However, because DNA is ubiquitous, it would be of great interest to increase the selectivity of our PDT PSs by linking them to a targeting vector in view of targeted PDT. Herein, we present the synthesis, characterization, and in-depth photophysical evaluation of a nanobody-containing RuII polypyridyl conjugate selective for the epidermal growth factor receptor (EGFR) in view of targeted PDT. Using ICP-MS and confocal microscopy, we could demonstrate that our conjugate has high selectivity for the EGFR receptor, which is a crucial oncological target because it is overexpressed and/or deregulated in a variety of solid tumors. However, in contrast to expectations, this conjugate was found to not produce reactive oxygen species (ROS) in cancer cells and is therefore not phototoxic.

Mesoporous silica nanoparticles functionalised with a photoactive ruthenium(ii) complex: exploring the formulation of a metal-based photodynamic therapy photosensitiser.

A series of nanomaterials based on mesoporous silica have been synthesised and functionalised with a photoactive polypyridyl ruthenium(ii) complex, namely [Ru(bipy)2-dppz-7-hydroxymethyl][PF6]2 (bipy = 2,2'-bipyridine, dppz = dipyrido[3,2-a:2',3'-c]phenazine), by various methods. The functionalisation reactions were based on the covalent binding to different ligands attached to the pores of the mesoporous nanoparticles and a simple physisorption using polyamino-functionalised mesoporous silica nanoparticles. The resulting nanostructured systems have been characterised by XRD, XRF, BET, SEM and TEM, observing the incorporation of the metallodrug onto the nanostructured silica in a different way depending on the synthetic method used in the loading reactions. In our studies, we have also observed that functionalisation with the metallodrug causes changes in the structural and textural features of the materials. The phototherapeutic activity of the ruthenium-functionalised materials in HeLa cervical cancer cells has been tested and the preliminary results are presented herein.

Comparison of the octadentate bifunctional chelator DFO*-pPhe-NCS and the clinically used hexadentate bifunctional chelator DFO-pPhe-NCS for 89Zr-immuno-PET.

PURPOSE: All clinical 89Zr-immuno-PET studies are currently performed with the chelator desferrioxamine (DFO). This chelator provides hexadentate coordination to zirconium, leaving two coordination sites available for coordination with, e.g., water molecules, which are relatively labile ligands. The unsaturated coordination of DFO to zirconium has been suggested to result in impaired stability of the complex in vivo and consequently in unwanted bone uptake of 89Zr. Aiming at clinical improvements, we report here on a bifunctional isothiocyanate variant of the octadentate chelator DFO* and the in vitro and in vivo comparison of its 89Zr-DFO*-mAb complex with 89Zr-DFO-mAb. METHODS: The bifunctional chelator DFO*-pPhe-NCS was prepared from previously reported DFO* and p-phenylenediisothiocyanate. Subsequently, trastuzumab was conjugated with either DFO*-pPhe-NCS or commercial DFO-pPhe-NCS and radiolabeled with Zr-89 according to published procedures. In vitro stability experiments were carried out in saline, a histidine/sucrose buffer, and blood serum. The in vivo performance of the chelators was compared in N87 tumor-bearing mice by biodistribution studies and PET imaging. RESULTS: In 0.9 % NaCl 89Zr-DFO*-trastuzumab was more stable than 89Zr-DFO-trastuzumab; after 72 h incubation at 2-8 °C 95 % and 58 % intact tracer were left, respectively, while in a histidine-sucrose buffer no difference was observed, both products were ≥ 92 % intact. In vivo uptake at 144 h post injection (p.i.) in tumors, blood, and most normal organs was similar for both conjugates, except for skin, liver, spleen, ileum, and bone. Tumor uptake was 32.59 ± 11.95 and 29.06 ± 8.66 % ID/g for 89Zr-DFO*-trastuzumab and 89Zr-DFO-trastuzumab, respectively. The bone uptake was significantly lower for 89Zr-DFO*-trastuzumab compared to 89Zr-DFO-trastuzumab. At 144 h p.i. for 89Zr-DFO*-trastuzumab and 89Zr-DFO-trastuzumab, the uptake in sternum was 0.92 ± 0.16 and 3.33 ± 0.32 % ID/g, in femur 0.78 ± 0.11 and 3.85, ± 0.80 and in knee 1.38 ± 0.23 and 8.20 ± 2.94 % ID/g, respectively. The uptake in bone decreased from 24 h to 144 h p.i. about two fold for the DFO* conjugate, while it increased about two fold for the DFO conjugate. CONCLUSIONS: Zr-DFO*-trastuzumab showed superior in vitro stability and in vivo performance when compared to 89Zr-DFO-trastuzumab. This makes the new octadentate DFO* chelator a candidate successor of DFO for future clinical 89Zr-immuno-PET.

Dual mode of cell death upon the photo-irradiation of a RuII polypyridyl complex in interphase or mitosis.

Photodynamic therapy (PDT) is an attractive, complementary medical technique to chemotherapy. Among the different photosensitizers (PSs) employed, Ru(ii) polypyridyl complexes were found to be valid substitutes to porphyrin-based or phthalocyanine-based PSs. Here, we confirm that one such complex, namely [Ru(bipy)2-dppz-7-methoxy][PF6]2 (Ru65), which localizes in the nucleus of various cancer and normal cells, displays cytotoxicity only upon UV-A irradiation. Importantly, we disclose the molecular mechanism of the UV-A mediated cytotoxic action of Ru65. We demonstrate that Ru65 intercalates in DNA and, upon light irradiation, promotes guanine oxidation, resulting in nicks in the double helix. We confirm this mechanism of action in living cells, showing that the UV-A irradiation of cells loaded with Ru65 results in a transient DNA damage response and cell death. Strikingly, the photo-irradiation of Ru65 triggered distinct mechanisms of cell death in interphase or mitotic cells. The former underwent cell cycle arrest at the G2/M phase and massive cytoplasmic vacuolation, which was paralleled by an unfolded-protein stress response, resulting in a reduction of viability and cell death through a paraptosis-like mechanism. On the other hand, the UV-A irradiation of Ru65 in cells synchronized by G2/M block-release with a selective CDK1 inhibitor led to blocking mitotic entry and rapid cell death through classic apoptotic pathways. Importantly, targeting mitotic cells with Ru65 allowed increasing its photo-toxicity by a factor of 3.6. Overall, our findings show that the use of a combination of a cell cycle inhibitor and a PS targeting the nucleus could open up new avenues in PDT.

Transcriptional interactions suggest niche segregation among microorganisms in the human gut.

The human gastrointestinal (GI) tract is the habitat for hundreds of microbial species, of which many cannot be cultivated readily, presumably because of the dependencies between species1. Studies of microbial co-occurrence in the gut have indicated community substructures that may reflect functional and metabolic interactions between cohabiting species2,3. To move beyond species co-occurrence networks, we systematically identified transcriptional interactions between pairs of coexisting gut microbes using metagenomics and microarray-based metatranscriptomics data from 233 stool samples from Europeans. In 102 significantly interacting species pairs, the transcriptional changes led to a reduced expression of orthologous functions between the coexisting species. Specific species-species transcriptional interactions were enriched for functions important for H2 and CO2 homeostasis, butyrate biosynthesis, ATP-binding cassette (ABC) transporters, flagella assembly and bacterial chemotaxis, as well as for the metabolism of carbohydrates, amino acids and cofactors. The analysis gives the first insight into the microbial community-wide transcriptional interactions, and suggests that the regulation of gene expression plays an important role in species adaptation to coexistence and that niche segregation takes place at the transcriptional level.

Lightening up Ruthenium Complexes to Fight Cancer?

In medicine, light is used in a medical treatment called photodynamic therapy (PDT) to treat some types of cancer and skin diseases. This technique generally allows for reduced side effects compared to traditional chemotherapy. However, PDT is not fully effective on hypoxic tumors (i.e. lacking oxygen). To overcome this important drawback, photoactivated chemotherapy (PACT) agents have been designed to obtain light-mediated cancer cell death via an oxygen-independent mechanism. Ruthenium complexes have already been and are currently deeply explored as traditional anticancer agents. However, as reported in this short review article, such compounds can also bring novel opportunities in the field of light-mediated cancer treatment. Herein, we report on our findings in the optimization of Ru(II) polypyridyl complexes as PDT and PACT agents for the potential treatment of cancer and, interestingly, also of bacterial infections.

Induction of Cytotoxicity through Photorelease of Aminoferrocene.

Reactive oxygen species (ROS)-activated aminoferrocene-based anticancer prodrug candidates successfully take advantage of intrinsically high amounts of ROS in tumor tissues. Interestingly, the ROS-initiated activation of these prodrug candidates leads to formation of unstable aminoferrocene (Fc-NH2) derivatives, which decay to iron ions. The latter catalytically increases ROS concentration to a lethal level. In this work, we prepared light-controlled aminoferrocene prodrug candidates by derivatizing Fc-NH2 with an o-nitrophenyl and an o-nitrobiphenyl photolabile protecting group (PLPG), respectively, and by further conjugation to a mitochondria localization signal (MLS) peptide (Cys-D-Arg-Phe-Lys-NH2). The resulting bioconjugates were found to be more stable and less cytotoxic, in the dark, toward human promyelocytic leukemia cells (HL-60) compared to Fc-NH2. Upon light irradiation at 355 nm, both conjugates released Fc-NH2, albeit with very different photolysis quantum yields. The o-nitrobiphenyl photocage was in fact several orders of magnitude more efficient than the o-nitrophenyl photocage in releasing Fc-NH2. This difference was reflected by the light irradiation experiments on the HL-60 cell line, in which aminoferrocene conjugated with the o-nitrobiphenyl cage and the MLS displayed the highest phototoxicity index (2.5 ± 0.4) of all the compounds tested. The iron release assays confirmed the rise in iron ion concentrations upon light irradiation of both caged aminoferrocene derivatives. Together with the absence of phototoxicity on the nonmalignant hTERT-immortalized retinal pigment epithelial (hTERT RPE-1) cell line, these results indicate catalytic generation of ROS as possible mode of action.

Combination of Ru(ii) complexes and light: new frontiers in cancer therapy.

The synergistic action of light, oxygen and a photosensitizer (PS) has found applications for decades in medicine under the name of photodynamic therapy (PDT) for the treatment of skin diseases and, more recently, for the treatment of cancer. However, of the thirteen PSs currently approved for the treatment of cancer over more than 10 countries, only two contain a metal ion. This fact is rather surprising considering that nowadays around 50% of conventional chemotherapies involve the use of cisplatin and other platinum-containing drugs. In this perspective article, we review the opportunities brought by the use of Ru(ii) complexes as PSs in PDT. In addition, we also present the recent achievements in the application of Ru(ii) complexes in photoactivated chemotherapy (PACT). In this strategy, the presence of oxygen is not required to achieve cell toxicity. This is of significance since tumors are generally hypoxic. Importantly, this perspective article focuses particularly on the Ru(ii) complexes for which an in vitro biological evaluation has been performed and the mechanism of action (partially) unveiled.

Anticancer profile of a series of gold(III) (2-phenyl)pyridine complexes.

Six phosphorescent (2-phenyl)pyridine (ppy) gold(III) 2,4,6-tris(trifluoromethyl)phenyl (FMes) complexes were synthesized and investigated for their anticancer potential. The compounds demonstrated strong antiproliferative activity, with EC50 values in the low micromolar range, along with significant accumulation in HeLa cancer cells after treatment for only 6 h (up to 119 ng gold per milligram of protein as measured by high-resolution continuum source atomic spectroscopy). Enzyme inhibition studies showed interaction of the gold(III) complexes with thioredoxin reductase (TrxR), a key homeostasis-regulation flavoprotein. TrxR was inhibited with IC50 values in the micromolar range. Furthermore, five of the complexes displayed selectivity toward TrxR against glutathione reductase (GR, a disulfide reductase structurally related to TrxR) by up to >49-fold. Because no major differences in bioactivity were observed across the series, [(ppy)Au(FMes)(PPh3 )OTf] (complex 4) was chosen for further in-depth biological characterization. Complex 4 was also found to interact with guanosine monophosphate in (1) H NMR studies under long incubation times. Interestingly, 4 induced a significant increase in intracellular levels of reactive oxygen species, which led to late apoptotic events and cytocidal effects.

DNA intercalating Ru(II) polypyridyl complexes as effective photosensitizers in photodynamic therapy.

Six substitutionally inert [Ru(II) (bipy)2 dppz](2+) derivatives (bipy=2,2'-bipyridine, dppz=dipyrido[3,2-a:2',3'-c]phenazine) bearing different functional groups on the dppz ligand [NH2 (1), OMe (2), OAc (3), OH (4), CH2 OH (5), CH2 Cl (6)] were synthesized and studied as potential photosensitizers (PSs) in photodynamic therapy (PDT). As also confirmed by DFT calculations, all complexes showed promising (1) O2 production quantum yields, well comparable with PSs available on the market. They can also efficiently intercalate into the DNA double helix, which is of high interest in view of DNA targeting. The cellular localization and uptake quantification of 1-6 were assessed by confocal microscopy and high-resolution continuum source atomic absorption spectrometry. Compound 1, and especially 2, showed very good uptake in cervical cancer cells (HeLa) with preferential nuclear accumulation. None of the compounds studied was found to be cytotoxic in the dark on both HeLa cells and, interestingly, on noncancerous MRC-5 cells (IC50 >100 µM). However, 1 and 2 showed very promising behavior with an increment of about 150 and 42 times, respectively, in their cytotoxicities upon light illumination at 420 nm in addition to a very good human plasma stability. As anticipated, the preferential nuclear accumulation of 1 and 2 and their very high DNA binding affinity resulted in very efficient DNA photocleavage, suggesting a DNA-based mode of phototoxic action.

An octadentate bifunctional chelating agent for the development of stable zirconium-89 based molecular imaging probes.

(89)Zr-based imaging agents hold great promise as novel radio-tracers in nuclear medicine. However, insufficient stability of currently used radiometal complexes in vivo is a safety concern for clinical applications. We herein report the first octadentate bifunctional chelating agent for the development of (89)Zr-labelled (bio)conjugates with improved stability.

A bis(dipyridophenazine)(2-(2-pyridyl)pyrimidine-4-carboxylic acid)ruthenium(II) complex with anticancer action upon photodeprotection.

Improving the selectivity of anticancer drugs towards cancer cells is one of the main goals of drug optimization; the prodrug strategy has been one of the most promising. A light-triggered prodrug strategy is presented as an efficient approach for controlling cytotoxicity of the substitutionally inert cytotoxic complex [Ru(dppz)2(CppH)](PF6)2(C1; CppH=2-(2-pyridyl)pyrimidine-4-carboxylic acid; dppz=dipyrido[3,2-a:2',3'-c]phenazine). Attachment of a photolabile 3-(4,5-dimethoxy-2-nitrophenyl)-2-butyl (DMNPB) ester ("photocaging") makes the otherwise active complex C1 innocuous to both cancerous (HeLa and U2OS) and non-cancerous (MRC-5) cells. The cytotoxic action can be successfully unleashed in living cells upon light illumination (350 nm), reaching similar level of activity as the parent cytotoxic compound C1. This is the first substitutionally inert cytotoxic metal complex to be used as a light-triggered prodrug candidate.

Molecular and cellular characterization of the biological effects of ruthenium(II) complexes incorporating 2-pyridyl-2-pyrimidine-4-carboxylic acid.

A great majority of the Ru complexes currently studied in anticancer research exert their antiproliferative activity, at least partially, through ligand exchange. In recent years, however, coordinatively saturated and substitutionally inert polypyridyl Ru(II) compounds have emerged as potential anticancer drug candidates. In this work, we present the synthesis and detailed characterization of two novel inert Ru(II) complexes, namely, [Ru(bipy)(2)(Cpp-NH-Hex-COOH)](2+) (2) and [Ru(dppz)(2)(CppH)](2+) (3) (bipy = 2,2'-bipyridine; CppH = 2-(2'-pyridyl)pyrimidine-4-carboxylic acid; Cpp-NH-Hex-COOH = 6-(2-(pyridin-2-yl)pyrimidine-4-carboxamido)hexanoic acid; dppz = dipyrido[3,2-a:2',3'-c]phenazine). 3 is of particular interest as it was found to have IC(50) values comparable to cisplatin, a benchmark standard in the field, on three cancer cell lines and a better activity on one cisplatin-resistant cell line than cisplatin itself. The mechanism of action of 3 was then investigated in detail and it could be demonstrated that, although 3 binds to calf-thymus DNA by intercalation, the biological effects that it induces did not involve a nuclear DNA related mode of action. On the contrary, confocal microscopy colocalization studies in HeLa cells showed that 3 specifically targeted mitochondria. This was further correlated by ruthenium quantification using High-resolution atomic absorption spectrometry. Furthermore, as determined by two independent assays, 3 induced apoptosis at a relatively late stage of treatment. The generation of reactive oxygen species could be excluded as the cause of the observed cytotoxicity. It was demonstrated that the mitochondrial membrane potential in HeLa was impaired by 3 as early as 2 h after its introduction and even more with increasing time.

Yin Yang of immunoregulation in organ transplantation and cancer.

The 'Yin Yang of Immunoregulation: Cancer and Organ Transplantation' meeting took place in Nantes, France on 2-3 December 2010 and was dedicated to the biology of myeloid and lymphoid immune cells in the context of cancer and transplantation. This meeting was organized by the Immunotherapy Research group of the Western France Cancer Network Cancéropole Grand-Ouest and the Immunomonitorage et Biothérapies network (IMBIO) research program, which is supported by the Région Pays de la Loire.

The rare G93D mutation causes a slowly progressing lower motor neuron disease.

We describe an ALS family with the rare SOD1 G93D mutation. Three members of the family developed ALS at an age ranging from 45 to 71 years. In all cases pyramidal signs were not evident. Two members of the family were obligate gene carriers, and died at 56 and 81years, respectively, without developing ALS signs or symptoms. The mutation was found in the DNA extracted from the hair bulbs in the two deceased obligate carriers and in another family member who died at 80 years of age without any sign of the disease. This study shows that SOD1 G93D mutation causes a slowly developing lower motor neuron disease with a reduced penetrance.

A morte de idosos asilados: percepção dos cuidadores de enfermagem / The death of elderly people living in retirement homes: perceptions of nursing caregivers

O segmento populacional de idosos cresceu nas últimas décadas. Constatou-se isso em um asilo em Itajaí/SC, despertando assim o desejo de verificar a percepção dos cuidadores de enfermagem sobre a morte dos idosos...