A new diatom-based multimetric index to assess lake ecological status.

Eutrophication impairs lake ecosystems at a global scale. In this context, as benthic microalgae are well-established warnings for a large range of stressors, particularly nutrient enrichment, the Water Framework Directive required the development of diatom-based methods to monitor lake eutrophication. Here, we present the diatom-based index we developed for French lakes, named IBDL (Indice Biologique Diatomées en Lacs). Data were collected in 93 lakes from 2015 to 2020. A challenge arose from the discontinuous pressure gradient of our dataset, especially the low number of nutrient-impacted lakes. To analyze the data we opted for the so-called "Threshold Indicator Taxa ANalysis" method, which makes it possible to determine a list of "alert taxa." We obtained a multimetric index based on specific pressure gradients (Kjeldahl nitrogen, suspended matter, biological oxygen demand, and total phosphorous). Considering the European intercalibration process, the very good correlation between IBDL and the common metric (R2 from 0.52 to 0.87 according to the lake alkalinity type) makes us very confident in our ability to match future IBDL quality thresholds with European standards. The IBDL proved at last to be particularly relevant as it has a twofold interest: an excellent relationship with total phosphorus (R2 from 0.63 to 0.83 according to the lake alkalinity type) and a possible application to any lake metatype. Its complementarity with macrophyte-based indices moreover justifies the use of at least two primary producer components for lake ecological status classification.

Dendron based antifouling, MRI and magnetic hyperthermia properties of different shaped iron oxide nanoparticles.

Owing to the great potential of iron oxide nanoparticles (NPs) for nanomedicine, large efforts have been made to better control their magnetic properties, especially their magnetic anisotropy to provide NPs able to combine imaging by MRI and therapy by magnetic hyperthermia. In that context, the design of anisotropic NPs appears as a very promising and efficient strategy. Furthermore, their bioactive coating also remains a challenge as it should provide colloidal stability, biocompatibility, furtivity along with good water diffusion for MRI. By taking advantage of our controlled synthesis method of iron oxide NPs with different shapes (cubic, spherical, octopod and nanoplate), we demonstrate here that the dendron coating, shown previously to be very suitable for 10 nm sized iron oxide, also provided very good colloidal, MRI and antifouling properties to the anisotropic shaped NPs. These antifouling properties, demonstrated through several experiments and characterizations, are very promising to achieve specific targeting of disease tissues without affecting healthy organs. On the other hand, the magnetic hyperthermia properties were shown to depend on the saturation magnetization and the ability of NPs to self-align, confirming the need of a balance between crystalline and dipolar magnetic anisotropies.

VSION as high field MRI T1 contrast agent: evidence of their potential as positive contrast agent for magnetic resonance angiography.

Because of their outstanding magnetic properties, iron oxide nanoparticles have already been the subject of numerous studies in the biomedical field, in particular as a negative contrast agent for T2-weighted nuclear magnetic resonance imaging, or as therapeutic agents in hyperthermia experiments. Recent studies have shown that below a given particle size (i.e. 5 nm), iron oxide may be used to provide a significant positive (brightening) effect on T1-weighted MRI. In such an application, not only the size of the crystal, but also the control of the coating process is essential to ensure optimal properties, especially at a very high field (> 3 T). In this work, we focused on the development of very small iron oxide nanoparticles as a potential platform for high field T1 magnetic resonance angiography (MRA) applications. The feasibility has been evaluated in vivo at 9.4 T, demonstrating the usefulness of the developed system for MRA applications.

Evaluation of the Active Targeting of Melanin Granules after Intravenous Injection of Dendronized Nanoparticles.

The biodistribution of dendronized iron oxides, NPs10@D1_DOTAGA and melanin-targeting NPs10@D1_ICF_DOTAGA, was studied in vivo using magnetic resonance imaging (MRI) and planar scintigraphy through [177Lu]Lu-radiolabeling. MRI experiments showed high contrast power of both dendronized nanoparticles (DPs) and hepatobiliary and urinary excretions. Little tumor uptake could be highlighted after intravenous injection probably as a consequence of the negatively charged DOTAGA-derivatized shell, which reduces the diffusion across the cells' membrane. Planar scintigraphy images demonstrated a moderate specific tumor uptake of melanoma-targeted [177Lu]Lu-NPs10@D1_ICF_DOTAGA at 2 h post-intravenous injection (pi), and the highest tumor uptake of the control probe [177Lu]Lu-NPs10@D1_DOTAGA at 30 min pi, probably due to the enhanced permeability and retention effect. In addition, ex vivo confocal microscopy studies showed a high specific targeting of human melanoma samples impregnated with NPs10@D1_ICF_Alexa647_ DOTAGA.

How a grafting anchor tailors the cellular uptake and in vivo fate of dendronized iron oxide nanoparticles.

Superparamagnetic spherical iron oxide nanoparticles of 10 nm diameter have been synthesized by thermal decomposition and grafted through a direct ligand exchange protocol with two dendrons bearing respectively a monophosphonic anchor (D2) or a biphosphonic tweezer (D2-2P) at their focal point. Physico-chemical characterization techniques such as dynamic light scattering (DLS), zeta potential, Fourier transform infrared spectroscopy (FTIR), transmission electron microscopy (TEM) and superconducting quantum interference device (SQUID) magnetometry were used to assess their composition, colloidal stability and magnetic properties. High-resolution magic angle spinning (HR-MAS) nuclear magnetic resonance (NMR) spectroscopy studies have been conducted to understand the organic shell composition and to determine both the grafting rate of the dendrons onto the nanoparticle surface and the influence of the remaining oleic acid originating from the synthesis protocol on the cellular uptake. Both dendronized IONPs showed moderate in vitro toxicity (MTT and LDH tests) in human cancer and primary cell lines. Furthermore, in vivo MRI studies showed high contrast enhancement as well as renal and hepatobiliary excretions and highlighted the influence of the grafting anchor (mono- versus bi-phosphonate) on the in vivo fate of dendronized magnetic iron oxides.

Myoferlin regulates cellular lipid metabolism and promotes metastases in triple-negative breast cancer.

Myoferlin is a multiple C2-domain-containing protein that regulates membrane repair, tyrosine kinase receptor function and endocytosis in myoblasts and endothelial cells. Recently it has been reported as overexpressed in several cancers and shown to contribute to proliferation, migration and invasion of cancer cells. We have previously demonstrated that myoferlin regulates epidermal growth factor receptor activity in breast cancer. In the current study, we report a consistent overexpression of myoferlin in triple-negative breast cancer cells (TNBC) over cells originating from other breast cancer subtypes. Using a combination of proteomics, metabolomics and electron microscopy, we demonstrate that myoferlin depletion results in marked alteration of endosomal system and metabolism. Mechanistically, myoferlin depletion caused impaired vesicle traffic that led to a misbalance of saturated/unsaturated fatty acids. This provoked mitochondrial dysfunction in TNBC cells. As a consequence of the major metabolic stress, TNBC cells rapidly triggered AMP activated protein kinase-mediated metabolic reprogramming to glycolysis. This reduced their ability to balance between oxidative phosphorylation and glycolysis, rendering TNBC cells metabolically inflexible, and more sensitive to metabolic drug targeting in vitro. In line with this, our in vivo findings demonstrated a significantly reduced capacity of myoferlin-deficient TNBC cells to metastasise to lungs. The significance of this observation was further supported by clinical data, showing that TNBC patients whose tumors overexpress myoferlin have worst distant metastasis-free and overall survivals. This novel insight into myoferlin function establishes an important link between vesicle traffic, cancer metabolism and progression, offering new diagnostic and therapeutic concepts to develop treatments for TNBC patients.

HDAC7 inhibition resets STAT3 tumorigenic activity in human glioblastoma independently of EGFR and PTEN: new opportunities for selected targeted therapies.

To date, the mutational status of EGFR and PTEN has been shown as relevant for favoring pro- or anti-tumor functions of STAT3 in human glioblastoma multiforme (GBM). We have screened genomic data from 154 patients and have identified a strong positive correlation between STAT3 and HDAC7 expression. In the current work we show the existence of a subpopulation of patients overexpressing HDAC7 and STAT3 that has particularly poor clinical outcome. Surprisingly, the somatic mutation rate of both STAT3 and HDAC7 was insignificant in GBM comparing with EGFR, PTEN or TP53. Depletion of HDAC7 in a range of GBM cells induced the expression of tyrosine kinase JAK1 and the tumor suppressor AKAP12. Both proteins synergistically sustained the activity of STAT3 by inducing its phosphorylation (JAK1) and protein expression (AKAP12). In absence of HDAC7, activated STAT3 was responsible for significant imbalance of secreted pro-/anti-angiogenic factors. This inhibited the migration and sprouting of endothelial cells in paracrine fashion in vitro as well as angiogenesis in vivo. In a murine model of GBM, induced HDAC7-silencing decreased the tumor burden by threefold. The current data show for the first time that silencing HDAC7 can reset the tumor suppressor activity of STAT3, independently of the EGFR/PTEN/TP53 background of the GBM. This effect could be exploited to overcome tumor heterogeneity and provide a new rationale behind the development of specific HDAC7 inhibitors for clinical use.

Electron paramagnetic resonance as a sensitive tool to assess the iron oxide content in cells for MRI cell labeling studies.

MRI cell tracking is a promising technique to track various cell types (stem cells, tumor cells, etc.) in living animals. Usually, cells are incubated with iron oxides (T(2) contrast agent) in order to take up the particles before being injected in vivo. Iron oxide quantification is important in such studies for validating the labeling protocols and assessing the dilution of the particles with cell proliferation. We here propose to implement electron paramagnetic resonance (EPR) as a very sensitive method to quantify iron oxide concentration in cells. Iron oxide particles exhibit a unique EPR spectrum, which directly reflects the number of particles in a sample. In order to compare EPR with existing methods (Perls's Prussian blue reaction, ICP-MS and fluorimetry), we labeled tumor cells (melanoma and renal adenocarcinoma cell lines) and fibroblasts with fluorescent iron oxide particles, and determined the limits of detection of the different techniques. We show that EPR is a very sensitive technique and is specific for iron oxide quantification as measurements are not affected by endogenous iron. As a consequence, EPR is well adapted to perform ex vivo analysis of tissues after cell tracking experiments in order to confirm MRI results.

Multimodal assessment of early tumor response to chemotherapy: comparison between diffusion-weighted MRI, 1H-MR spectroscopy of choline and USPIO particles targeted at cell death.

The aim of this study was to determine the value of different magnetic resonance (MR) protocols to assess early tumor response to chemotherapy. We used a murine tumor model (TLT) presenting different degrees of response to three different cytotoxic agents. As shown in survival curves, cyclophosphamide (CP) was the most efficient drug followed by 5-fluorouracil (5-FU), whereas the etoposide treatment had little impact on TLT tumors. Three different MR protocols were used at 9.4 Tesla 24 h post-treatment: diffusion-weighted (DW)-MRI, choline measurement by (1) H MRS, and contrast-enhanced MRI using ultrasmall iron oxide nanoparticles (USPIO) targeted at phosphatidylserine. Accumulation of contrast agent in apoptotic tumors was monitored by T(2) -weighted images and quantified by EPR spectroscopy. Necrosis and apoptosis were assessed by histology. Large variations were observed in the measurement of choline peak areas and could not be directly correlated to tumor response. Although the targeted USPIO particles were able to significantly differentiate between the efficiency of each cytotoxic agent and best correlated with survival endpoint, they present the main disadvantage of non-specific tumor accumulation, which could be problematic when transferring the method to the clinic. DW-MRI presents a better compromise by combining longitudinal studies with a high dynamic range; however, DW-MRI was unable to show any significant effect for 5-FU. This study illustrates the need for multimodal imaging in assessing tumor response to treatment to compensate for individual limitations.

Iron oxide particles covered with hexapeptides targeted at phosphatidylserine as MR biomarkers of tumor cell death.

The aim of the study was to evaluate the ability of a new MR contrast agent to detect cell death as a biomarker of the efficacy of anti-cancer treatment. The phosphatidylserine-targeted hexapeptide (E3) was coupled to pegylated ultrasmall iron oxide nanoparticles (USPIO) that can be detected by magnetic resonance imaging (MRI) and by electron paramagnetic resonance (EPR). USPIO binding to staurosporine-treated TLT (transplantable liver tumor) cells, evaluated by X-Band EPR, indicated twice as much binding of USPIO grafted with the E3 peptide, compared with USPIO grafted with a scrambled peptide or ungrafted USPIO. In vivo experiments were carried out using TLT cells implanted intramuscularly into NMRI mice, and tumor cell death was induced by irradiation. After intravenous injection of the different types of USPIO, the accumulation of contrast agent was evaluated ex vivo by X-band EPR, in vivo by L-band EPR and by T(2)-weighted MRI. In irradiated tumors there was greater accumulation of the targeted USPIO particles compared with control particles or compared with the targeted particles in untreated tissues. In conclusion, phosphatidylserine-targeting of USPIO particles can detect dying tissues. This molecular targeted system should be evaluated further as a potential biomarker of tumor response to treatment.

Iron oxide based MR contrast agents: from chemistry to cell labeling.

Superparamagnetic iron oxide nanoparticles can be used for numerous applications such as MRI contrast enhancement, hyperthermia, detoxification of biological fluids, drug delivery, or cell separation. In this work, we will summarize the chemical routes for synthesis of iron oxide nanoparticles, the fluid stabilization, and the surface modification of superparamagnetic iron oxide nanoparticles. Some examples of the numerous applications of these particles in the biomedical field mainly as MRI negative contrast agents for tissue-specific imaging, cellular labeling, and molecular imaging will be given. Larger particles or particles displaying a non-neutral surface (thanks to their coating or to a cell transfection agent with which they are mixed) are very useful tools, although the cells to be labeled have no professional phagocytic function. Labeled cells can then be transplanted and monitored by MRI in a broad spectrum of applications. Direct in vivo magnetic labeling of cells is mainly performed by intravenous injection of long-circulating iron oxide-based MRI contrast agents, which can extravasate and/or undergo a cellular uptake in an amount sufficient to allow an MRI visualization of areas of interest such as inflamed regions or tumors. Particles with long circulation times, or able to induce a strong negative effect individually have been also modified by conjugation to a ligand, so that their cellular uptake, or at least their binding to the cell surface, could occur through a specific ligand-receptor interaction, in vivo as well as in vitro. Thus, experimentally as well as in a few trials on humans, iron oxide particles currently find promising applications.