Photoantimicrobial Biohybrids by Supramolecular Immobilization of Cationic Phthalocyanines onto Cellulose Nanocrystals

This is the peer-reviewed version of the following article: Anaya‐Plaza, E., van de Winckel, E., Mikkilä, J., Malho, J. M., Ikkala, O., Gulías, O., ... & Kostiainen, M. A. (2017). Photoantimicrobial biohybrids by supramolecular immobilization of cationic phthalocyanines onto cellulose nanocrystals. Chemistry–A European Journal, 23(18), 4320-4326., which has been published in final form at https://doi.org/10.1002/chem.201605285. This article may be used for non-commercial purposes in accordance with Wiley-VCH Terms and Conditions for Self-ArchivingThe development of photoactive and biocompatible nanostructures is a highly desirable goal to address the current threat of antibiotic resistance. Here, we describe a novel supramolecular biohybrid nanostructure based on the non-covalent immobilization of cationic zinc phthalocyanine (ZnPc) derivatives onto unmodified cellulose nanocrystals (CNC), following an easy and straightforward protocol, in which binding is driven by electrostatic interactions. These non-covalent biohybrids show strong photodynamic activity against S. aureus and E. coli, representative examples of Gram-positive and Gram-negative bacteria, respectively, and C. albicans, a representative opportunistic fungal pathogen, outperforming the free ZnPc counterparts and related nanosystems in which the photosensitizer is covalently linked to the CNC surfaceA.d.l.E. acknowledges a Ramón y Cajal contract from the Spanish Ministry of Economy (MINECO). The work at Madrid was supported by the EU [SO2S (FP7‐PEOPLE‐2012‐ITN, 316975); and CosmoPHOS‐nano (FP7‐NMP‐2012‐6, 310337‐2)], the Spanish MINECO [CTQ‐2014‐52869‐P (T.T.) and CTQ‐2014‐53673‐P (A.d.l.E.)] and Comunidad de Madrid [FOTOCARBON (S2013/MIT‐2841)]. J.M., V.L., and M.A.K. acknowledge support through the Emil Aaltonen Foundation and the Academy of Finland (grants 267497, 273645 and 263504). This work was supported by the Academy of Finland through its Centers of Excellence Programme (2014–2019) and made use of the Aalto University Nanomicroscopy Centre (Aalto NMC). The work in Barcelona was supported by the Spanish MINECO (grant CTQ2013‐48767‐C3‐1‐R). R.B.‐O. thanks the European Social Funds and the SUR del DEC de la Generalitat de Catalunya for his predoctoral fellowship (2016 FI B1 00021)

Ischémie et angiogénèse tumorale : effets des carences en glucose et en acides aminés sur l'expression du VEGF-A par les cellules tumorales et implication de la réponse (« Unfolded Protein Response »).

Dans les tumeurs solides, les cellules soumises à des conditions ischémiques induisent des voies de signalisation distinctes contribuant à la commutation angiogénique et au développement tumoral. La composante hypoxique de l'ischémie induit l'expression du VEGF-A par la voie de signalisation dépendante de HIF-1. Par contre, la contribution des carences en glucose ou en glutamine est peu connue. En utilisant un milieu sans sérum, nous montrons que l'activation et la signalisation dépendante d'IRE1 sont des événements liant les réponses dépendantes de l'hypoxie et des carences en glucose à la surexpression du VEGF-A dans divers modèles cellulaires. Des cellules tumorales exprimant un dominant négatif de IRE1 et des cellules MEF IRE1-/- n'induisent pas l'expression du VEGF-A en conditions d'hypoxie ou de carences en glucose. Ces stress mènent à l'induction du VEGF-A par des mécanismes en partie distincts. Ces données sont corrélées avec une réduction de l'angiogenèse et de la croissance tumorale in vivo et démontrent le rôle essentiel joué par IRE1 en réponse à l'ischémie. En absence de glutamine, les ARNm du VEGF-A sont surexprimés indépendamment de l'accumulation des protéines HIF et de la transcription des gènes dépendants de la réponse UPR. De plus, l'expression protéique du VEGF-A est diminuée en corrélation avec la réduction de la synthèse protéique globale. Ces résultats suggèrent que les carences en glutamine n'ont aucun effet sur le processus angiogénique induit par le VEGF-A dans certaines tumeurs. Nous avons également recherché l'implication des carences en glutamine et en glucose dans les réponses métaboliques des cellules tumorales. Ces stress augmentent rapidement le processus d'apoptose, d'une manière plus importante en absence de glutamine qu'en absences de glucose. La spectrométrie FTIR a permis d'évaluer le statut métabolique des cellules tumorales ischémiques et a corrélé leur phénotype hyper-glycolytique avec leur statut prolifératif et agressif.In solid tumors, cells subjected to ischemic conditions trigger distinct signaling pathways which contribute to the angiogenic switch and tumor development. The hypoxic component of ischemia leads to the expression of VEGF-A by the HIF-1-dependent signaling pathways. Alternatively, the contribution of glucose or glutamine deprivation is still not well understood. Using a serum-free medium, we demonstrate that IRE1 activation and signaling is a common molecular determinant linking hypoxia- and hypoglycemia-dependent responses to the upregulation of VEGF-A in various cell models. Indeed, tumor cells expressing a dominant negative IRE1 transgene as well as IRE1-/- MEF were unable to trigger VEGF-A upregulation upon either hypoxic or glucose deprivation conditions. We showed that these various cellular stresses lead to the induction of VEGF-A by partly distinct mechanisms. These data correlated with a reduction of tumor angiogenesis and growth in vivo and demonstrated the essential role played by IRE1 in response to tumor ischemia. Under glutamine deprivation, VEGF-A mRNA was up-regulated and this effect was neither associated to HIF proteins accumulation nor to the transcriptional induction of UPR-dependent genes. Besides, VEGF-A protein expression was down-regulated in correlation to the global decrease of protein synthesis. These results suggested that amino-acids deprivation has no effect on the VEGF-A-driven angiogenic process in a number of tumors. We then questioned the implication of glucose and glutamine deprivations in the metabolic responses of tumor cells. This stress rapidly increase the apoptotic response of the cells, and to a much greater extent in the absence of glutamine than under glucose deprivation. FTIR spectrometry was used to evaluate the metabolic status of tumor cells and correlated the hyper-glycolytic phenotype with their proliferative and aggressive status

Salmonella enterica biofilm-mediated dispersal by nitric oxide donors in association with cellulose nanocrystal hydrogels

Protected by extracellular polymers, microbes within biofilms are significantly more resistant to disinfectants. Current research has been instrumental in identifying nitric oxide donors and hydrogels as potential disinfectant additives. Nitric oxide (NO) donors are considered a very promising molecule as biofilm dispersal agents and hydrogels have recently attracted a lot of interest due to their biocompatible properties and ability to form stable thin films. When the NO donor MAHMA NONOate was dissolved in phosphate saline buffer, it was able to reduce the biomass of well-established biofilms up to 15% for at least 24 h of contact time. Encapsulation of MAHMA NONOate and molsidomine within a hydrogel composed of cellulose nanocrystals (CNC) has shown a synergistic effect in dispersing well-established biofilms: after 2 h of exposure, moderate but significant dispersion was measured. After 6 h of exposure, the number of cells transitioning from the biofilm to the planktonic state was up to 0.6 log higher when compared with non-treated biofilms. To further explore the transport processes of NO donors within hydrogels, we measured the nitric oxide flux from gels, at 25°C for a composite of 0.1 µM MAHMA NONOate–CNC. Nitric oxide diffuses up to 500 µm from the hydrogel surface, with flux decreasing according to Fick’s law. 60% of NO was released from the hydrogel composite during the first 23 min. These data suggest that the combined treatments with nitric oxide donor and hydrogels may allow for new sustainable cleaning strategies

One-Pot Silver Nanoring Synthesis

Silver colloidal nanorings have been synthesized by reducing silver ions with NaBH4 in trisodium citrate buffers. pH increase, by addition of NaOH, was used to speed up reduction reaction. The UV–vis absorption spectra of resulting silver nanorings showed two peaks accounting for transverse and longitudinal surface plasmon resonance, at ≈400 nm, and between 600 and 700 nm, respectively. The shapes of these silver nanoparticles (nanorings) depended on AgNO3/NaBH4 ratio, pH and reaction temperature. Particles were analysed by transmission electron microscopy, scanning electron microscopy and X-ray diffraction. A reaction pathway is proposed to explain silver nanoring formation

Inhibition of DDR1 enhances in vivo chemosensitivity in KRAS-mutant lung adenocarcinoma

Platinum-based chemotherapy in combination with immune-checkpoint inhibitors is the current standard of care for patients with advanced lung adenocarcinoma (LUAD). However, tumor progression evolves in most cases. Therefore, predictive bioma ricers are needed for better patient stratification and for the identification of new therapeutic strategies, including enhancing the efficacy of chemotoxic agents. Here, we hypothesized that discoidin domain receptor 1 (DDR1) may be both a predictive factor for chemoresistance in patients with LUAD and a potential target positively selected in resistant cells. By using biopsies from patients with LUAD, KRAS-mutant LUAD cell lines, and in vivo genetically engineered KRAS-driven mouse models, we evaluated the role of DDR1 in the context of chemotherapy treatment. We found that DORT is upregulated during chemotherapy both in vitro and in viva. Moreover, analysis of a cohort of patients with LUAD suggested that high DOR1 levels in pretreatment biopsies correlated with poor response to chemotherapy. Additionally, we showed that combining DORI inhibition with chemotherapy prompted a synergistic therapeutic effect and enhanced cell death of KRAS-mutant tumors in vivo. Collectively, this study suggests a potential role for DDR1 as both a predictive and prognostic biomarker, potentially improving the chemotherapy response of patients with LUAD

The cytoprotective drug amifostine modifies both expression and activity of the pro-angiogenic factor VEGF-A

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Efficient mouse transgenesis using Gateway-compatible ROSA26 locus targeting vectors and F1 hybrid ES cells

The ability to rapidly and efficiently generate reliable Cre/loxP conditional transgenic mice would greatly complement global high-throughput gene targeting initiatives aimed at identifying gene function in the mouse. We report here the generation of Cre/loxP conditional ROSA26-targeted ES cells within 3–4 weeks by using Gateway® cloning to build the target vectors. The cDNA of the gene of interest can be expressed either directly by the ROSA26 promoter providing a moderate level of expression or by a CAGG promoter placed in the ROSA26 locus providing higher transgene expression. Utilization of F1 hybrid ES cells with exceptional developmental potential allows the production of germ line transmitting, fully or highly ES cell-derived mice by aggregation of cells with diploid embryos. The presented streamlined procedures accelerate the examination of phenotypical consequences of transgene expression. It also provides a unique tool for comparing the biological activity of polymorphic or splice variants of a gene, or products of different genes functioning in the same or parallel pathways in an overlapping manner

Histone H2B ubiquitylation represses gametogenesis by opposing RSC-dependent chromatin remodeling at the ste11 master regulator locus

In fission yeast, the ste11 gene encodes the master regulator initiating the switch from vegetative growth to gametogenesis. In a previous paper, we showed that the methylation of H3K4 and consequent promoter nucleosome deacetylation repress ste11 induction and cell differentiation (Materne et al., 2015) but the regulatory steps remain poorly understood. Here we report a genetic screen that highlighted H2B deubiquitylation and the RSC remodeling complex as activators of ste11 expression. Mechanistic analyses revealed more complex, opposite roles of H2Bubi at the promoter where it represses expression, and over the transcribed region where it sustains it. By promoting H3K4 methylation at the promoter, H2Bubi initiates the deacetylation process, which decreases chromatin remodeling by RSC. Upon induction, this process is reversed and efficient NDR (nucleosome depleted region) formation leads to high expression. Therefore, H2Bubi represses gametogenesis by opposing the recruitment of RSC at the promoter of the master regulator ste11 gene.This work was supported by grant BFU2014-52143-P from the Spanish Ministerio de Economía y Competitividad to FA and by grants PR T.0012.14, MIS F.4523.11, Ceruna and Marie Curie Action to DH. DH is a FNRS Senior Research Associate.Peer Reviewe