Caenorhabditis elegans RAC1/ced-10 mutants as a new animal model to study very early stages of Parkinson's disease.

Patients with Parkinson's disease (PD) display non-motor symptoms arising prior to the appearance of motor signs and before a clear diagnosis. Motor and non-motor symptoms correlate with progressive deposition of the protein alpha-synuclein (Asyn) both within and outside of the central nervous system, and its accumulation parallels neurodegeneration. The genome of Caenorhabditis elegans does not encode a homolog of Asyn, thus rendering this nematode an invaluable system with which to investigate PD-related mechanisms in the absence of interference from endogenous Asyn aggregation. CED-10 is the nematode homolog of human RAC1, a small GTPase needed to maintain the function and survival of dopaminergic neurons against human Asyn-induced toxicity in C. elegans. Here, we introduce C. elegans RAC1/ced-10 mutants as a predictive tool to investigate early PD symptoms before neurodegeneration occurs. Deep phenotyping of these animals reveals that, early in development, they displayed altered defecation cycles, GABAergic abnormalities and an increased oxidation index. Moreover, they exhibited altered lipid metabolism evidenced by the accumulation of lipid droplets. Lipidomic fingerprinting indicates that phosphatidylcholine and sphingomyelin, but not phosphatidylethanolamine or phosphatidylserine, were elevated in RAC1/ced-10 mutant nematodes. These collective characteristics reflect the non-motor dysfunction, GABAergic neurotransmission defects, upregulation of stress response mechanisms, and metabolic changes associated with early-onset PD. Thus, we put forward an easy-to-manipulate preclinical animal model to deepen our understanding of early-stage PD and accelerate the translational path for therapeutic target discovery.

Ocular involvement in sphenopalatin ganglion pulsed radiofrequency.

Pulsed radiofrequency (PRF) treatment of the sphenopalatine ganglion is an important interventional treatment in refractory cases of trigeminal neuralgia (TN) or atypical facial pain, given the easy access to its location. Despite the fact that complications from this technique are rare and it is a fairly safe procedure, ophthalmologists should know about it due to the anatomical relations of this ganglion.

Rapid method for isolation of desiccation-tolerant strains and xeroprotectants.

A novel biotechnological process has been developed for the isolation of desiccation-tolerant microorganisms and their xeroprotectants, i.e., compatible solutes involved in long-term stability of biomolecules in the dry state. Following exposure of soil samples to chloroform, we isolated a collection of desiccation-tolerant microorganisms. This collection was screened for the production of xeroprotectants by a variation of the bacterial milking (osmotic downshock) procedure and by a novel air-drying/rehydration ("dry milking") incubation method. The resultant solutes were shown to protect both proteins and living cells against desiccation damage, thereby validating them as xeroprotectants. Nuclear magnetic resonance (NMR) analytical studies were performed to identify the xeroprotectants; synthetic mixtures of these compounds were shown to perform similarly to natural isolates in drying experiments with proteins and cells. This new approach has biotechnological and environmental implications for the identification of new xeroprotectants of commercial and therapeutic value.

Synergistic effect of Si-hydroxyapatite coating and VEGF adsorption on Ti6Al4V-ELI scaffolds for bone regeneration in an osteoporotic bone environment.

The osteogenic and angiogenic responses to metal macroporous scaffolds coated with silicon substituted hydroxyapatite (SiHA) and decorated with vascular endothelial growth factor (VEGF) have been evaluated in vitro and in vivo. Ti6Al4V-ELI scaffolds were prepared by electron beam melting and subsequently coated with Ca10(PO4)5.6(SiO4)0.4(OH)1.6 following a dip coating method. In vitro studies demonstrated that SiHA stimulates the proliferation of MC3T3-E1 pre-osteoblastic cells, whereas the adsorption of VEGF stimulates the proliferation of EC2 mature endothelial cells. In vivo studies were carried out in an osteoporotic sheep model, evidencing that only the simultaneous presence of both components led to a significant increase of new tissue formation in osteoporotic bone. STATEMENT OF SIGNIFICANCE: Reconstruction of bones after severe trauma or tumors extirpation is one of the most challenging tasks in the field of orthopedic surgery. This scenario is even more complicated in the case of osteoporotic patients, since their bone regeneration capability is decreased. In this work we present a porous implant that promotes bone regeneration even in osteoporotic bone. By coating the implant with osteogenic bioceramics such as silicon substituted hydroxyapatite and subsequent adsorption of vascular endothelial growth factor, these implants stimulate the bone ingrowth when they are implanted in osteoporotic sheep.

Update on the management of vasoproliferative tumour. / Actualización sobre el manejo del tumor vasoproliferativo.

CASE REPORT: Here we report a 19-year-old female patient who presented a vasoproliferative tumour. It caused complications, such as epiretinal membrane, macular oedema, vitreous haemorrhage, and exudative retinal detachment. The patient was treated with 3 injections of intravitreal bevacizumab, an intravitreal dexamethasone implant, tocilizumab, and double freeze-thaw cryotherapy. DISCUSSION: Therapeutic options are: observation, if it is small, if it is a peripheral lesion, and if there seems to be no threat to vision. If it requires treatment, laser photocoagulation, intravitreal bevacizumab, trans-conjunctival cryotherapy, transpupillary thermotherapy, photodynamic therapy, brachytherapy plaques and surgery are the different options available. Recently, tocilizumab and intravitreal dexamethasone implants have been reported to be beneficial.

Crystallochemistry, textural properties, and in vitro biocompatibility of different silicon-doped calcium phosphates.

Three silicon-doped calcium phosphates (Si-CaPs) were synthesized by heating precipitated silicon-doped apatite via different thermal treatments. Temperatures of 700 degrees C, 900 degrees C, and 1100 degrees C led to an apatite-glass biphasic material, nanocrystalline Si-doped apatite (SiHA), and Si-doped apatite-alpha tricalcium phosphate biphasic material, respectively. Structure, microstructure, textural properties, and chemical differences were determined for the three bioceramics. Biocompatibility tests were carried out by seeding osteblast-like cells onto the three substrates. Si-CaP treated at 700 degrees C and 900 degrees C led to Ca decrease in the culture media, partially impeding the cell proliferation over them. However, the proliferation capability is restored when additional culture medium is added. Finally, cytotoxicity results indicated that cell damage is much lower in osteblast-like cells seeded onto SiHA and SiHA-alpha tricalcium phosphate samples than in plastic culture control.

Vitreous SiO2-CaO coatings on Ti6Al4V alloys: reactivity in simulated body fluid versus osteoblast cell culture.

Vitreous coatings of the SiO(2)-CaO system have been prepared on Ti6Al4V substrates by the sol-gel method. The textural parameters (porosity and roughness) and thickness of the films obtained increase when the concentration of the precursor solutions is raised. In vitro studies of these coatings have been performed using two approaches: soaking in simulated body fluid, and by growing osteoblasts on these materials. The results of both studies show differences in terms of chemical reactivity. While in simulated body fluid the coatings were dissolved without forming a bioactive surface, when osteoblast-like cells grew on the coatings they were more stable. Furthermore, cell culture assays show biocompatible behavior of these coatings making them of potential interest for clinical applications. The effect of the textural parameters of the obtained coatings on the cell functions (attachment, spreading, proliferation and differentiation) has also been studied. The results show an increase in these cell parameters as the roughness and porosity of the coatings increase.

The relationship between nuclear magnetic resonance-visible lipids, lipid droplets, and cell proliferation in cultured C6 cells.

There is an ongoing controversy about the subcellular origin of the fatty acyl chains that give rise to the NMR visible mobile lipids (MLs) resonance at approximately 1.24 ppm in the 1H spectra of cells and solid tumors. Some groups have been supporting the hypothesis that triglycerides originating MLs are isotropically tumbling in small membrane microdomains, whereas other authors back the proposal that they are inside cytosolic or extracellular (necrotic areas) lipid droplets. Furthermore, MLs are frequently present in in vivo spectra recorded from human brain tumors, but the meaning of this detection is not fully clear. We have addressed the possible contribution of intracellular droplets to the ML pattern recorded from human brain tumors in vivo by studying cultured C6 rat glioma cells as a model system for astrocytic tumors. We show here that cultured C6 cells display ML resonances in high field (9.4 T) 1H NMR spectra recorded at 136 ms echo time when grown at saturation density conditions, but no MLs are visible for log-phase cells. Fluorescence microscopy analysis of cells stained with the lipophylic dye Nile red shows intracellular spherical yellow-gold droplets containing neutral lipids; cells at saturation density present lipid droplets of diameters about 1.6 microm in most cells (85%), whereas they are almost absent in log-phase cells (only 6% of the cells contain them). Furthermore, log-phase cells can be induced to display MLs and accumulate Nile red-positive droplets by culturing them for 24 h at pH 6.2. This acid pH effect can be fully reversed by 24 h of standard media incubation. Lipid droplet volume calculated from fluorescence microscopy preparations in an average cell is different for both culture conditions (2.2 times higher volume for saturation density than for pH-stressed cells). This difference in lipid droplet volume is reflected by a different ML peak height at 1.24 ppm (about 2 times higher for saturation density than for pH-stressed cells). Flow cytometry analysis shows that both culture conditions result in a slowing down of the proliferation rate of the cells. The fact that MLs are found to originate in lipid droplets inside cells that are growth compromised but still viable suggests that changes in the proliferative state of tumor cells, in the absence of necrosis, may be detected non invasively by in vivo NMR spectroscopy.

Evidence that mobile lipids detected in rat brain glioma by 1H nuclear magnetic resonance correspond to lipid droplets.

Mobile lipids have been detected by proton nuclear magnetic resonance (NMR) in animal and human tumors (cultured cells, biopsies, and in vivo), but their origin and subcellular location are still unclear. They have been associated with malignancy, metastatic ability, drug resistance, and necrosis. We wanted to determine whether these lipids are located within plasma membrane microdomains or in lipid droplets for a C6 cell-induced rat glioma. NMR-visible mobile lipids were found in all subcellular fractions isolated from the rat tumor, except in the cytosolic supernatants. Transmission electron microscopy showed that lipid droplets were present in all subcellular fractions containing NMR-visible lipids and in the necrotic and perinecrotic areas of the tumor. The mean diameter of droplets isolated by flotation in the subcellular fractionation protocol was 0.97 microm (n = 682; droplet profile diameter range between 0.2 and 5.0 microm). The apparent diffusion coefficient for these lipids (46 +/- 17 microm2 s(-1) measured in vivo by proton spectroscopy was four orders of magnitude higher than would be expected if mobile lipids were inside plasma membrane microdomains. The combined results demonstrated that mobile lipids detected in vivo by proton NMR in the C6 rat glioma are located in large lipid droplets, associated with the necrotic process.

Cognitive and hedonic responses to meal ingestion correlate with changes in circulating metabolites.

BACKGROUND: We have previously shown that meal ingestion induces cognitive perception (sensations) with a hedonic dimension (well-being) that depends on the characteristics of the meal and the appropriateness of the digestive response. The aim of the present study is to identify metabolomic biomarkers of the cognitive response to meal ingestion. METHODS: In 18 healthy subjects, the response to a test meal (Edanec, 1 kcal/mL) ingested until maximum satiation (50 mL/min) was assessed. Perception measurements and blood samples were taken before, at the end of the meal, and 20 min after ingestion. The cognitive response and the hedonic dimension were measured on 10 cm scales. Metabolomic analysis was performed using nuclear magnetic resonance (NMR) spectroscopy and values of triglycerides, insulin, peptide YY (PYY), and glucagon-like peptide-1 (GLP-1) were determined using conventional laboratory techniques. KEY RESULTS: Ingestion up to maximum satiation induced sensation of fullness and decreased digestive well-being. The total amount ingested by each subject correlated with the basal sensation of hunger, but not with other sensations or blood metabolite levels. Immediately after ingestion, satiation correlated with an increase in glucose (R = 0.49; p = 0.038) and valine levels (R = 0.48; p = 0.043). Twenty-minutes after finalizing ingestion, triglyceride levels had significantly increased which correlated with the recovery in well-being (R = 0.48; p = 0.046) and the decrease in desire to eat a food of choice (R = -0.56; p = 0.016). The increase in lipids inversely correlated with abdominal discomfort (R = -0.51; p = 0.032). CONCLUSIONS & INFERENCES: Cognitive and hedonic responses to meal ingestion correlate with changes in circulating metabolites, which may serve as objective biomarkers of perception.

Surface zwitterionization of customized 3D Ti6Al4V scaffolds: a promising alternative to eradicate bone infection.

Large and critical bone defect reconstruction is still a hard challenge in tumor resections, non-unions, some traumatisms and articular prosthesis revisions. The application of electron beam melting technology (EBM) to implant manufacturing constitutes a promising alternative, which provides better biomechanics and customized solutions. Implant infections are one of the most serious complications associated with surgical treatments, which require immediate solutions for achieving better clinical results. Herein, to confer antimicrobial properties, a simple and cost-effective approach based on a bifunctionalization process to create a zwitterionic surface on Ti6Al4V EBM implants is proposed. The obtained results show a notable reduction of bacterial adhesion (more than 97%) and total inhibition of biofilm formation, combined with demonstrated biocompatibility and bioactivity, permitting cell adhesion on the entire surface of these 3D zwitterionic scaffolds. This surface zwitterionization provides new perspectives for custom-made Ti6Al4V EBM implants for bone tissue regeneration with antimicrobial properties.

In vitro colonization of stratified bioactive scaffolds by pre-osteoblast cells.

UNLABELLED: Mesoporous bioactive glass-polycaprolactone (MBG-PCL) scaffolds have been prepared by robocasting, a layer by layer rapid prototyping method, by stacking of individual strati. Each stratus was independently analyzed during the cell culture tests with MC3T3-E1 preosteblast-like cells. The presence of MBG stimulates the colonization of the scaffolds by increasing the cell proliferation and differentiation. MBG-PCL composites not only enhanced pre-osteoblast functions but also allowed cell movement along its surface, reaching the upper stratus faster than in pure PCL scaffolds. The cells behavior on each individual stratus revealed that the scaffolds colonization depends on the chemical stimuli supplied by the MBG dissolution and surface changes associated to the apatite-like formation during the bioactive process. Finally, scanning electron and fluorescence microscopy revealed that the kinetic of cell migration strongly depends on the architectural features of the scaffolds, in such a way that layers interconnections are used as migration routes to reach the farther scaffolds locations from the initial cells source. STATEMENT OF SIGNIFICANCE: This manuscript provides new insights on cell behavior in bioceramic/polymer macroporous scaffolds prepared by rapid prototyping methods. The experiments proposed in this work have allowed the evaluation of cell behavior within the different levels of the scaffolds, i.e. from the initials source of cells towards the farther scaffold locations. We could demonstrate that the in vitro cell colonization is encouraged by the presence of a highly bioactive mesoporous glass (MBG). This bioceramic enhances the cell migration towards upper strati through the dissolution of chemical signals and the changes occurred on the scaffolds surface during the bioactive process. In addition the MBG promotes preosteblastic proliferation and differentiation respect to scaffolds made of pure polycaprolactone. Finally, this study reveals the significance of the architectural design to accelerate the cell colonization. These experiments put light on the factors that should be taken into account to accelerate the regeneration processes under in vivo conditions.

Tailoring the biological response of mesoporous bioactive materials.

Mesoporous bioactive glasses (MBGs) in the SiO2-CaO-P2O5 system have been prepared using different non-ionic structure directing agents (SDA): Brij58, F68, P123 and F127. For the first time, the bioactive response of MBGs can be tailored with the kind of SDA incorporated. This is because, in addition to the textural properties, we can use the SDA to tailor the local atomic environment within the MBG struts. These features lead to differences in the in vitro bioactive behaviour of MBGs. Among the different SDAs used in this work, the triblock copolymer F68 leads to MBGs that exhibit the fastest bioactivity and the fastest differentiation induction from a pre-osteoblast to an osteoblast phenotype. These results are explained in terms of a highly ordered mesoporous structure, more free calcium cations acting as silica network modifiers and small mesopores that avoid the formation of CaP nuclei within pores, which could obstruct the ionic exchange with the surrounding fluids.

Mesoporous SBA-15 HPLC evaluation for controlled gentamicin drug delivery.

Mesoporous silica SBA-15 was prepared to evaluate its application as gentamicin drug delivery system. Two procedures were used to evaluate the delivery: calcined powder and disk conformed. The samples were charged with gentamicin sulphate and the experiments were carried out in vitro. No significant difference between powder and disk was observed in the tests. The release profiles exhibited a pronounced initial burst release effect of 60%, followed by a very slow release pattern. A new HPLC method was employed for calculated gentamicin amount in the delivery test. This method requires a small amount of sample, very advisable in these kinds of assays.

Magnetic resonance spectroscopy of brain hemangiopericytomas: high myoinositol concentrations and discrimination from meningiomas.

OBJECT: Hemangiopericytomas are a rare type of brain tumor that are very similar to meningiomas in appearance and symptoms but require different treatment. It is not normally possible to distinguish between them by using magnetic resonance (MR) imaging and computerized tomography studies. However, discrimination may be possible by using in vivo MR spectroscopy (MRS) because the biochemical composition of these two lesions is different. The goal of this study was to describe the use of MRS in discriminating between these similar tumor types. METHODS: In vivo MRS spectra were acquired in 27 patients (three with hemangiopericytomas and 24 with meningiomas) by using a single-voxel proton brain examination system at 1.5 teslas with short- (20-msec) and long- (135-msec) echo times. In addition, brain biopsy specimens obtained by open craniotomy were frozen within 5 minutes of resection and stored in liquid nitrogen until they were used. The specimens were powdered, extracted with perchloric acid, redissolved in 2H2O2 and high-resolution in vitro MRS was used at 9.4 teslas to record their spectra. CONCLUSIONS: In this study the authors show that hemangiopericytomas could be clearly distinguished from meningiomas because they have a larger peak at 3.56 ppm. Measurements of extracts of the tumors and comparison of spectra acquired with MRS at long- (135-msec) and short- (20-msec) echo times established that this was due to the much higher levels of myoinositol in the hemangiopericytomas.

Localized corrosion of 316L stainless steel with SiO2-CaO films obtained by means of sol-gel treatment.

Sol-gel films on austenitic stainless steel (AISI 316L) polished wafer were prepared from sono-sols obtained from tetraethylorthosilane and hydrated calcium nitrate. However, pitting was observed in different places on the stainless steel surfaces. The corrosion resistance was evaluated by the polarization resistance in simulated body fluid environment at 37 degrees C. The critical current density, the passive current density, the corrosion potential, and the critical pitting potential were studied. The austenitic stainless steel 316L treated presents important electrochemical corrosion and consequently its application as endosseous implants is not possible.

Defective sarcoplasmic reticulum-mitochondria calcium exchange in aged mouse myocardium.

Mitochondrial alterations are critically involved in increased vulnerability to disease during aging. We investigated the contribution of mitochondria-sarcoplasmic reticulum (SR) communication in cardiomyocyte functional alterations during aging. Heart function (echocardiography) and ATP/phosphocreatine (NMR spectroscopy) were preserved in hearts from old mice (>20 months) with respect to young mice (5-6 months). Mitochondrial membrane potential and resting O2 consumption were similar in mitochondria from young and old hearts. However, maximal ADP-stimulated O2 consumption was specifically reduced in interfibrillar mitochondria from aged hearts. Second generation proteomics disclosed an increased mitochondrial protein oxidation in advanced age. Because energy production and oxidative status are regulated by mitochondrial Ca2+, we investigated the effect of age on mitochondrial Ca2+ uptake. Although no age-dependent differences were found in Ca2+ uptake kinetics in isolated mitochondria, mitochondrial Ca2+ uptake secondary to SR Ca2+ release was significantly reduced in cardiomyocytes from old hearts, and this effect was associated with decreased NAD(P)H regeneration and increased mitochondrial ROS upon increased contractile activity. Immunofluorescence and proximity ligation assay identified the defective communication between mitochondrial voltage-dependent anion channel and SR ryanodine receptor (RyR) in cardiomyocytes from aged hearts associated with altered Ca2+ handling. Age-dependent alterations in SR Ca2+ transfer to mitochondria and in Ca2+ handling could be reproduced in cardiomyoctes from young hearts after interorganelle disruption with colchicine, at concentrations that had no effect in aged cardiomyocytes or isolated mitochondria. Thus, defective SR-mitochondria communication underlies inefficient interorganelle Ca2+ exchange that contributes to energy demand/supply mistmach and oxidative stress in the aged heart.

RNase1 prevents the damaging interplay between extracellular RNA and tumour necrosis factor-α in cardiac ischaemia/reperfusion injury.

Despite optimal therapy, the morbidity and mortality of patients presenting with an acute myocardial infarction (MI) remain significant, and the initial mechanistic trigger of myocardial "ischaemia/reperfusion (I/R) injury" remains greatly unexplained. Here we show that factors released from the damaged cardiac tissue itself, in particular extracellular RNA (eRNA) and tumour-necrosis-factor α (TNF-α), may dictate I/R injury. In an experimental in vivo mouse model of myocardial I/R as well as in the isolated I/R Langendorff-perfused rat heart, cardiomyocyte death was induced by eRNA and TNF-α. Moreover, TNF-α promoted further eRNA release especially under hypoxia, feeding a vicious cell damaging cycle during I/R with the massive production of oxygen radicals, mitochondrial obstruction, decrease in antioxidant enzymes and decline of cardiomyocyte functions. The administration of RNase1 significantly decreased myocardial infarction in both experimental models. This regimen allowed the reduction in cytokine release, normalisation of antioxidant enzymes as well as preservation of cardiac tissue. Thus, RNase1 administration provides a novel therapeutic regimen to interfere with the adverse eRNA-TNF-α interplay and significantly reduces or prevents the pathological outcome of ischaemic heart disease.

Sustained high prevalence of pneumococcal serotype 1 in paediatric parapneumonic empyema in southern Spain from 2005 to 2009.

The epidemiology and microbiological characteristics of paediatric parapneumonic empyema (PPE) before the introduction of the new generation of conjugate pneumococcal vaccines (10-valent and 13-valent) are described. All patients <14 years old admitted to a tertiary paediatric hospital with a diagnosis of PPE were prospectively enrolled from January 2005 to December 2009. Pneumococcal serotyping of culture-negative pleural fluid samples was performed using a multiplex real-time PCR assay. Overall, 219 patients had PPE. Incidence rates for PPE remained stable during the study period with a not significant increase in 2009 compared with 2005 (p 0.13), and were temporally associated with higher circulation of pandemic influenza A H1N1 during the last quarter in our population (p 0.001). Pneumococci were detected in 72% of culture-positive and 79% of culture-negative samples. Serotypes were determined in 104 PPE cases. Serotype 1 was the most prevalent serotype identified (42%) followed by serotypes 7F (20%), 3 (16%), 19A (8%) and 5 (7%). Serotype distribution remained similar during all time periods. Pneumococcal serotype 1 remained the most common cause of PPE during the 5-year study. The new generation of pneumococcal conjugate vaccines offers potential serotype coverage of 73% (10-valent) and 99% (13-valent) in the population studied suffering from PPE. Continuous epidemiological and molecular studies are paramount to monitor the impact of pneumococcal vaccines on the epidemiology of PPE.