Changes in water content and distribution in Quercus ilex leaves during progressive drought assessed by in vivo 1H magnetic resonance imaging

12 páginas, 7 figuras.-- Metodología.[Background]: Drought is a common stressor in many regions of the world and current climatic global circulation models predict further increases in warming and drought in the coming decades in several of these regions, such as the Mediterranean basin. The changes in leaf water content, distribution and dynamics in plant tissues under different soil water availabilities are not well known. In order to fill this gap, in the present report we describe our study withholding the irrigation of the seedlings of Quercus ilex, the dominant tree species in the evergreen forests of many areas of the Mediterranean Basin. We have monitored the gradual changes in water content in the different leaf areas, in vivo and non-invasively, by 1H magnetic resonance imaging (MRI) using proton density weighted (ρw) images and spin-spin relaxation time (T2) maps. [Results]: ρw images showed that the distal leaf area lost water faster than the basal area and that after four weeks of similar losses, the water reduction was greater in leaf veins than in leaf parenchyma areas and also in distal than in basal leaf area. There was a similar tendency in all different areas and tissues, of increasing T2 values during the drought period. This indicates an increase in the dynamics of free water, suggesting a decrease of cell membranes permeability. [Conclusions]: The results indicate a non homogeneous leaf response to stress with a differentiated capacity to mobilize water between its different parts and tissues. This study shows that the MRI technique can be a useful tool to follow non-intrusively the in vivo water content changes in the different parts of the leaves during drought stress. It opens up new possibilities to better characterize the associated physiological changes and provides important information about the different responses of the different leaf areas what should be taken into account when conducting physiological and metabolic drought stress studies in different parts of the leaves during drought stress.This research was supported by the European project NEU NITROEUROPE (GOCE017841), by the Spanish Government grants CGL2006-04025/BOS and Consolider-Ingenio Montes CSD2008-00040 and the Catalan Government grant SGR 2009-1458.Peer reviewe

NMR elastometry of fluid membranes in the mesoscopic regime

In solid-state 2H NMR of fluid lipid bilayers, quasielastic deformations at MHz frequencies are detected as a square-law dependence of the nuclear spin-lattice (R1Z) relaxation rates and order parameters (SCD). The signature square-law slope is found to decrease progressively with the mole fraction of cholesterol and with the acyl chain length, due to a stiffening of the membrane. The correspondence to thermal vesicle fluctuations and molecular dynamics simulations implies that a broad distribution of modes is present, ranging from the membrane size down to the molecular dimensions

Efficacy of adeno-associated virus gene therapy in a MNGIE murine model enhanced by chronic exposure to nucleosides

Preclinical studies have shown that gene therapy is a feasible approach to treat mitochondrial neurogastrointestinal encephalomyopathy (MNGIE). However, the genetic murine model of the disease (Tymp/Upp1 double knockout, dKO) has a limited functional phenotype beyond the metabolic imbalances, and so the studies showing efficacy of gene therapy have relied almost exclusively on demonstrating correction of the biochemical phenotype. Chronic oral administration of thymidine (dThd) and deoxyuridine (dUrd) to dKO mice deteriorates the phenotype of the animals, providing a better model to test therapy approaches. dKO mice were treated with both dThd and dUrd in drinking water from weaning until the end of the study. At 8 - 11 weeks of age, mice were treated with several doses of adeno-associated virus (AAV) serotype 8 vector carrying the human TYMP coding sequence under the control of different liver-specific promoters (TBG, AAT, or HLP). The biochemical profile and functional phenotype were studied over the life of the animals. Nucleoside exposure resulted in 30-fold higher plasma nucleoside levels in dKO mice compared with non-exposed wild type mice. AAV-treatment provided elevated TP activity in liver and lowered systemic nucleoside levels in exposed dKO mice. Exposed dKO mice had enlarged brain ventricles (assessed by magnetic resonance imaging) and motor impairment (rotarod test); both were prevented by AAV treatment. Among all promoters tested, AAT showed the best efficacy. Our results show that AAV-mediated gene therapy restores the biochemical homeostasis in the murine model of MNGIE and, for the first time, demonstrate that this treatment improves the functional phenotype. This work was funded in part by the Spanish Instituto de Salud Carlos III, and the Generalitat de Catalunya. The disclosed funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript

Metabolomics of Therapy Response in Preclinical Glioblastoma : a Multi-Slice MRSI-Based Volumetric Analysis for Noninvasive Assessment of Temozolomide Treatment

Glioblastoma (GBM) is the most common aggressive primary brain tumor in adults, with a short survival time even after aggressive therapy. Non-invasive surrogate biomarkers of therapy response may be relevant for improving patient survival. Previous work produced such biomarkers in preclinical GBM using semi-supervised source extraction and single-slice Magnetic Resonance Spectroscopic Imaging (MRSI). Nevertheless, GBMs are heterogeneous and single-slice studies could prevent obtaining relevant information. The purpose of this work was to evaluate whether a multi-slice MRSI approach, acquiring consecutive grids across the tumor, is feasible for preclinical models and may produce additional insight into therapy response. Nosological images were analyzed pixel-by-pixel and a relative responding volume, the Tumor Responding Index (TRI), was defined to quantify response. Heterogeneous response levels were observed and treated animals were ascribed to three arbitrary predefined groups: high response (HR, n = 2), TRI = 68.2 ± 2.8%, intermediate response (IR, n = 6), TRI = 41.1 ± 4.2% and low response (LR, n = 2), TRI = 13.4 ± 14.3%, producing therapy response categorization which had not been fully registered in single-slice studies. Results agreed with the multi-slice approach being feasible and producing an inverse correlation between TRI and Ki67 immunostaining. Additionally, ca. 7-day oscillations of TRI were observed, suggesting that host immune system activation in response to treatment could contribute to the responding patterns detected

A new ex vivo method to evaluate the performance of candidate MRI contrast agents: a proof-of-concept study

BACKGROUND: Magnetic resonance imaging (MRI) plays an important role in tumor detection/diagnosis. The use of exogenous contrast agents (CAs) helps to improve the discrimination between lesion and neighbouring tissue, but most of the currently available CAs are non-specific. Assessing the performance of new, selective CAs requires exhaustive assays and large amounts of material. Accordingly, in a preliminary screening of new CAs, it is important to choose candidate compounds with good potential for in vivo efficiency. This screening method should reproduce as close as possible the in vivo environment. In this sense, a fast and reliable method to select the best candidate CAs for in vivo studies would minimize time and investment cost, and would benefit the development of better CAs. RESULTS: The post-mortem ex vivo relative contrast enhancement (RCE) was evaluated as a method to screen different types of CAs, including paramagnetic and superparamagnetic agents. In detail, sugar/gadolinium-loaded gold nanoparticles (Gd-GNPs) and iron nanoparticles (SPIONs) were tested. Our results indicate that the post-mortem ex vivo RCE of evaluated CAs, did not correlate well with their respective in vitro relaxivities. The results obtained with different Gd-GNPs suggest that the linker length of the sugar conjugate could modulate the interactions with cellular receptors and therefore the relaxivity value. A paramagnetic CA (GNP (E_2)), which performed best among a series of Gd-GNPs, was evaluated both ex vivo and in vivo. The ex vivo RCE was slightly worst than gadoterate meglumine (201.9 ± 9.3% versus 237 ± 14%, respectively), while the in vivo RCE, measured at the time-to-maximum enhancement for both compounds, pointed to GNP E_2 being a better CA in vivo than gadoterate meglumine. This is suggested to be related to the nanoparticule characteristics of the evaluated GNP. CONCLUSION: We have developed a simple, cost-effective relatively high-throughput method for selecting CAs for in vivo experiments. This method requires approximately 800 times less quantity of material than the amount used for in vivo administrations

Mutation of the 3-phosphoinositide-dependent protein kinase-1 (PDK1) substrate-docking site in the developing brain causes microcephaly with abnormal brain morphogenesis independently of Akt, leading to impaired cognition and disruptive behaviors

The phosphoinositide 3-kinase (PI 3-kinase)/Akt signaling pathway plays essential roles during neuronal development. The 3-phosphoinositide-dependent protein kinase 1 (PDK1) coordinates the PI 3-kinase signals by activating twenty three kinases of the AGC family including Akt. Phosphorylation of a conserved docking site in the substrate is a requisite for PDK1 to recognize, phosphorylate and activate most of these kinases, with the exception of Akt. We exploited this differential mechanism of regulation by generating neuronal-specific conditional knock-in mice expressing the mutant form of PDK1 L155E in which the substrate-docking site binding motif, termed the PIF-pocket, was disrupted. As a consequence, activation of all the PDK1 substrates tested excluding Akt was abolished. Mice exhibited microcephaly, altered cortical layering and reduced circuitry, leading to cognitive deficits and exacerbated disruptive behavior combined with diminished motivation. The abnormal patterning of the adult brain arise from the reduced ability of the embryonic neurons to polarize and extend their axons, therefore highlighting the essential roles that the PDK1 signaling beyond Akt plays in mediating the neuronal responses that are instructive for brain development

Magnetically amplified photothermal therapies and multimodal imaging with magneto-plasmonic nanodomes

Nanotherapies require new ways for controlling and improving the delivery of the therapeutic agents to the site of action to maximize their efficacy and minimize the side effects. This control is particularly relevant in photothermal treatments to reduce the required light intensity and amount of injected nanoparticles, and to minimize necrotic cell deaths. Here we present a novel concept for multifunctional nanobiomedical agents: magneto-plasmonic (MP) nanodomes for magnetically guided and amplified photothermal therapies and as contrast agents for multimodal imaging. The MP nanodomes are composed of a Fe/Au bilayer semi-shell deposited on a 100 nm diameter fluorescent polystyrene nanosphere, which gather a unique combination of straightforward functionalization, high colloidal stability, very strong ferromagnetic behavior and intense optical absorption efficiency in the near infrared. We show that the photothermal conversion efficiency of the Fe/Au nanodomes with high Fe ratios is substantially larger than pure plasmonic Au nanodomes and the state-of-art plasmonic nanoheaters, i.e. Au nanorods and nanoshells, by merging strong optical absorption, minimized scattering and low optical anisotropy. Remarkably, the effective magnetophoretic concentration of the Fe/Au nanodomes at the illumination region enables large local increase of the optically induced temperature rise. The Fe semishell also provides very intense T contrast in nuclear magnetic resonance, which is at least 15-fold larger per particle than commercial iron oxide contrast agents. Moreover, the fluorescent polystyrene nanosphere and the Au semishell integrate valuable fluorescent and X-ray contrasts, respectively, which we have used to assess the nanodomes internalization by cancer cells. The MP nanodomes are nontoxic to cells even in the case of magnetophoretic local enrichment with initially high particle concentration (100 μg/mL). Remarkably, we demonstrate amplified local photothermal treatments by the magnetic enrichment of the nanodomes at the illumination region, which enables reaching nearly 100% reduction of cell viability with low particle concentration (10 μg/mL) and mild NIR laser intensity (5 W/cm). These results highlight the high potential of MP nanodomes for magnetically guided and amplified photothermal therapies

Changes in water content and distribution in Quercus ilex leaves during progressive drought assessed by in vivo 1 H magnetic resonance imaging

Drought is a common stressor in many regions of the world and current climatic global circulation models predict further increases in warming and drought in the coming decades in several of these regions, such as the Mediterranean basin. The changes in leaf water content, distribution and dynamics in plant tissues under different soil water availabilities are not well known. In order to fill this gap, in the present report we describe our study withholding the irrigation of the seedlings of Quercus ilex, the dominant tree species in the evergreen forests of many areas of the Mediterranean Basin. We have monitored the gradual changes in water content in the different leaf areas, in vivo and non-invasively, by 1 H magnetic resonance imaging (MRI) using proton density weighted (ρ) images and spin-spin relaxation time (T) maps. ρimages showed that the distal leaf area lost water faster than the basal area and that after four weeks of similar losses, the water reduction was greater in leaf veins than in leaf parenchyma areas and also in distal than in basal leaf area. There was a similar tendency in all different areas and tissues, of increasing Tvalues during the drought period. This indicates an increase in the dynamics of free water, suggesting a decrease of cell membranes permeability. The results indicate a non homogeneous leaf response to stress with a differentiated capacity to mobilize water between its different parts and tissues. This study shows that the MRI technique can be a useful tool to follow non-intrusively the in vivo water content changes in the different parts of the leaves during drought stress. It opens up new possibilities to better characterize the associated physiological changes and provides important information about the different responses of the different leaf areas what should be taken into account when conducting physiological and metabolic drought stress studies in different parts of the leaves during drought stress

NMR elastometry of fluid membranes in the mesoscopic regime

In solid-state 2H NMR of fluid lipid bilayers, quasielastic deformations at MHz frequencies are detected as a square-law dependence of the nuclear spin-lattice (R1Z) relaxation rates and order parameters (SCD). The signature square-law slope is found to decrease progressively with the mole fraction of cholesterol and with the acyl chain length, due to a stiffening of the membrane. The correspondence to thermal vesicle fluctuations and molecular dynamics simulations implies that a broad distribution of modes is present, ranging from the membrane size down to the molecular dimensions

Microwave-assisted synthesis of SPION-reduced graphene oxide hybrids for magnetic resonance imaging (MRI)

Magnetic resonance imaging (MRI) is a useful tool for disease diagnosis and treatment monitoring. Superparamagnetic iron oxide nanoparticles (SPION) show good performance as transverse relaxation (T) contrast agents, thus facilitating the interpretation of the acquired images. Attachment of SPION onto nanocarriers prevents their agglomeration, improving the circulation time and efficiency. Graphene derivatives, such as graphene oxide (GO) and reduced graphene oxide (RGO), are appealing nanocarriers since they have both high surface area and functional moieties that make them ideal substrates for the attachment of nanoparticles. We have employed a fast, simple and environmentally friendly microwave-assisted approach for the synthesis of SPION-RGO hybrids. Different iron precursor/GO ratios were used leading to SPION, with a median diameter of 7.1 nm, homogeneously distributed along the RGO surface. Good relaxivity (r*) values were obtained in MRI studies and no significant toxicity was detected within in vitro tests following GL261 glioma and J774 macrophage-like cells for 24 h with SPION-RGO, demonstrating the applicability of the hybrids as T-weighted MRI contrast agents