FGF2, but not EGF, Induces multiciliated ependymal cells to dedifferentiate and adopt radial glial features in vitro

Multiciliated ependymal cells form an epithelium lining most of the ventricular cavities of vertebrates brain. Although considered postmitotic and completely differentiated, ependymal cells maintain some phenotypic characteristics of neural stem cells. Thereby, under specific conditions they behave as neural stem cells, developing radial glia characteristics, and undergoing asymmetric division. Our group is searching for factors that promote dedifferentiation of ependymal cells in vitro. We developed a simple method to obtain pure cultures of non-adherent multiciliated ependymal cells from adult rats. These cultures were used to investigate the effect of FGF2 on the differentiation state and the aggregation of ependymal cells. Thus, FGF2 treated ependymal cells lose cilia and hence mobility, and after 7 days they aggregate to form irregular spheres (diameter ≥ 20-30 μm). Such changes were not observed when EFG was used instead of FGF2. To assess the specificity of FGF2 action on cell aggregation, the FGF receptor inhibitor PD166866 and an anti-FGF2 neutralizing antibody were used. In both conditions the aggregative effect of FGF2 was abolished. No cell proliferation was observed during sphere formation, at least in such experimental conditions. Spheres were analyzed by immunocytochemistry using radial glia markers. They were positive for GFAP, vimentin, BLBP and GLAST. These data suggest that FGF2 promotes the identity loss in multiciliated ependymal cells in vitro, which are transformed into cells with radial glia features.Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech

Neuraminidase-activated microglia compromise the viability of ependymocytes

Neuraminidase (NA) is a sialidase present in the envelope/wall of some virus/bacteria responsible for brain infections, such as flu, mumps or meningitis. The intracerebroventricular injection of NA in the rat brain provokes ependymal detachment and death, and an acute inflammatory process. Although inflammation reverses, ependymal lining is not regenerated. Complement system activation within the CSF contributes to ependymal damage, but is not the only cause (Granados-Duran et al, 2016). Here we aimed to investigate if microglial activation might also play a role. For this purpose we used pure isolated ependymocytes (Grondona et al, 2013) and ventricular wall explants, which were co-cultured with microglial cells, both in basal conditions and with agents that induce microglial activation: NA, LPS, or Pam3CSK4 (synthetic lipopeptide). The viability of the ependymal cells was assessed by trypan blue exclusion. The viability of isolated ependymocytes was reduced when NA or LPS were added to the culture, compared to controls without additives. In the absence of microglia, NA or LPS did not compromise viability significantly, indicating that microglia was involved in ependymocytes death. The addition of NA to cultured explants reduced ependymocytes viability only when microglial cells were present in the culture; a similar reduction was observed when LPS or Pam3CSK4 were added. Conversely, explants cultured in the absence of microglia did not suffer a significant decrease in ependymocytes viability upon NA addition to the medium. We hypothesized that cytokines released by activated microglia, such as IL1β or TNFα, could mediate ependymocytes death. RT-PCR performed in RNA obtained from pure ependymocytes confirmed the presence of IL1β and TNFα receptors in ependymal cells. Nevertheless further experiments are required to confirm this hypothesis. We conclude that microglia activated by NA mediates, at least in part, ependymal cell death, what might be relevant for neuroinflammatory diseases mediated by NA bearing virus/bacteria.Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech

Enhancement of photoacoustic detection of inhomogeneities in polymers

We report a series of experiments on laser pulsed photoacoustic excitationin turbid polymer samples addressed to evaluate the sound speed in the samples and the presence of inhomogeneities in the bulk. We describe a system which allows the direct measurement of the speed of the detected waves by engraving the surface of the piece under study with a fiduciary pattern of black lines. We also describe how this pattern helps to enhance the sensitivity for the detection of an inhomogeneity in the bulk. These two facts are useful for studies in soft matter systems including, perhaps, biological samples. We have performed an experimental analysis on Grilon(R) samples in different situations and we show the limitations of the method.Comment: 8 pages, 7 figure

Electrical studies and plasma characterization of an atmospheric pressure plasma jet operated at low frequency

Low-temperature, high-pressure plasma jets have an extensive use in medical and biological applications. Much work has been devoted to study these applications while comparatively fewer studies appear to be directed to the discharge itself. In this work, in order to better understand the kind of electrical discharge and the plasma states existing in those devices, a study of the electrical characteristics of a typical plasma jet, operated at atmospheric pressure, using either air or argon, is reported. It is found that the experimentally determined electrical characteristics are consistent with the model of a thermal arc discharge, with a highly collisional cathode sheet. The only exception is the case of argon at the smallest electrode separation studied, around 1 mm in which case the discharge is better modeled as either a non-thermal arc or a high-pressure glow. Also, variations of the electrical behavior at different gas flow rates are interpreted, consistently with the arc model, in terms of the development of fluid turbulence in the external jet.Fil: Giuliani, Leandro Estanislao. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Física del Plasma. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Física del Plasma; ArgentinaFil: Xaubet Brea, Magalí Noel. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Física del Plasma. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Física del Plasma; ArgentinaFil: Grondona, Diana Elena. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Física del Plasma. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Física del Plasma; ArgentinaFil: Minotti, Fernando Oscar. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Física del Plasma. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Física del Plasma; ArgentinaFil: Kelly, Hector Juan. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Física del Plasma. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Física del Plasma; Argentin

SPATIAL ANALYSIS OF YIELD TRIALS USING SEPARABLE ARIMA PROCESSES

Spatial analysis procedures based on one-dimensional and two-dimensional (separable) ARIMA (Auto Regressive Integrated Moving Average) processes were used to analyze several yield trials. Two criteria were used to determine the best spatial model: 1) standard error of the treatment difference (SED) and 2) mean squared error (MSE) of prediction based on a cross-validation approach. It is found that spatial models with two-dimensional exponential covariance functions are frequently the best models regarding SED and MSE. Differenced models are frequently the best models regarding SED and the worst with respect to MSE

Characterization of beet necrotic yellow vein furovirus from Spanish sugar beets

Rhizomania is a viral disease, caused by beet necrotic yellow vein furovirus (BNYVV), which was detected in Spanish sugar beets in 1988, it being focused on the Castilla y León region. BNYVV has five RNA fragments with specific functions, and the different composition and proportion of RNA in the virions allow their separation and the characterization of their activities during the development of the disease. Thirty–six samples of sugar beet rootlets and frozen pulps from three different sugar beet zones of Castilla y León were analyzed by DAS-ELISA and Immunocapture-Reverse Transcription-Polymerase Chain Reaction (IC-RT-PCR) using specific primers. The identity of the cDNA products was confirmed by nested- PCR and restriction fragment length polymorphism (RFLP). The uniformity of the patterns obtained by RFLP analyses with nine endonucleases showed the existence of a unique strain of BNYVV in 80,000 Ha of crop surface which could be explained by a recent arrival of the rhizomania disease to this region. The isolates studied were more similar to type A, which has been previously described in BNYVV, but a nonexpected cleavage site for this molecular group was observed with endonuclease Hinc II on the RNA-2 IC-RT-PCR product (nt 2133–3293) in the thirty–six Spanish samples and also in a North American strain taken as reference. The use of frozen pulps obtained as a previous step to the industrial extraction of sugar avoids problems due to erratic distribution of the virus in the roots, provides repetitive results for a particular sample, and facilitates epidemiological and distributional studies on rhizomania disease

Rostral floor plate (flexural organ) secretes glycoproteins immunologically similar to subcommissural organ glycoproteins in dogfish (Scyliorhinus canicula) embryos

The subcommissural organ of vertebrates secretes glycoproteins into the cerebrospinal fluid of the third cerebral ventricle. This material polymerizes in Reissner's fiber. During ontogenetic development, besides the subcommissural organ, the ependyma lining the pontine flexure constitutes an additional Reissner's fiber-secreting gland named flexural organ. We have studied the secretion of the flexural organ and the subcommissural organ in dogfish (Scyliorhinus canicula) embryos using three different antisera and the lectins concanavalin A and wheat germ agglutinin. AFRU is an antiserum against the bovine Reissner's fiber; Ab-600 is an antiserum against 600 kDa dogfish subcommissural organ glycoproteins; and APSO is an antiserum against immunoaffinity purified bovine subcommissural organ secretory glycoproteins. These three antisera immunostained the flexural organ indicating that it contains epitopes similar to those present in bovine and dogfish subcommissural organ glycoproteins. It seems highly probable that the flexural or an and the subcommissural organ of dogfish embryos secrete similar compound(s). Other ependymal regions were also immunostained with Ab-600 and APSO antisera. Then, Reissner's fiber-like glycoproteins were transiently expressed by most embryonary ependymal cells. These glycoproteins might play a role in the development of the central nervous system of vertebrates. (C) 1997 Elsevier Science B.V

Neuraminidase-induced neuroinflammation is largely dependent on microglial TLR4 receptor

The sialidase neuraminidase (NA) cleaves terminal sialic acid from glycoproteins and glycolipids. Among its various locations, it is present in the envelope/membrane of some bacteria/viruses (e.g. influenza virus), where it is involved in infectiveness and dispersion. The injection of NA within the brain lateral ventricle represents a model of acute sterile inflammation. The relevance of the toll-like receptors TLR2 and TLR4 (particularly those in microglial cells) in such process was investigated using mouse strains deficient in these receptors. In septofimbria and hypothalamus, IBA1-positive and IL-1β-positive cell counts increased after NA injection in wild type (WT) mice. In TLR4-/- mice such increases were largely abolished, while only slightly affected in TLR2-/- mice. Similarly, the NA-induced expression of IL-1β, TNFα and IL-6 (evaluated by qPCR) was completely blocked in TLR4-/- mice, and only partially reduced in TLR2-/- mice. Microglia was isolated from the three mouse strains and exposed to NA or to specific TLR2 and TLR4 agonists (Pam3CSK4 and LPS respectively) in vitro. NA induced a cytokine response (IL-1β, TNFα and IL-6) in WT microglia, but was unable to do so in TLR4-/- microglia; TLR2 deficiency partially affected the NA-induced microglia response. To investigate if such response of microglial cells to NA was dependent on the sialidase activity of the enzyme, WT microglia was exposed in vitro to NA previously inactivated with heat, or inhibited with two different sialidase inhibitors (oseltamivir phosphate and N-acetyl-2,3-dehydro-2-deoxyneuraminic acid). In all cases, NA- induced microglia activation was dependent on the intact sialidase activity of NA. Therefore, we conclude that NA is able to directly activate microglial cells, mostly through TLR4 receptor and due to its sialidase activity. Accordingly, the inflammatory reaction induced by NA in vivo is partially dependent on TLR2, while TLR4 plays a crucial role.Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech