The blood-brain barrier: cellular basis.

Perfusion experiments with horseradish peroxidase have established that the morphological substrate of the blood-brain barrier is represented by microvascular endothelial cells. They are characterized by complexly arranged tight junctions and a very low rate of transcytotic vesicular transport. They express transport enzymes, carrier systems and brain endothelial cell-specific molecules of unknown function not expressed by any other endothelial cell population. These blood-brain barrier properties are not intrinsic to these cells but are inducible by the surrounding brain tissue. Type I astrocytes injected into the anterior eye chamber of the rat or onto the chick chorioallantoic membrane are able to induce a host-derived angiogenesis and some blood-brain barrier properties in endothelial cells of non-neural origin. Recently we have shown that this cellular interaction is due to the secretion of a soluble astrocyte derived factor(s). Astrocytes are also implicated in the maintenance, functional regulation and the repair of the blood-brain barrier. Complex interactions between other constituents of the microenvironment surrounding the endothelial cells, such as the basement membrane, pericytes, nerve endings, microglial cells and the extracellular fluid, take place and are required for the proper functioning of the blood-brain barrier, which in addition is regionally different as reflected by endothelial cell heterogeneity.

The role of community and population ecology in applying mycorrhizal fungi for improved food security.

The global human population is expected to reach ∼9 billion by 2050. Feeding this many people represents a major challenge requiring global crop yield increases of up to 100%. Microbial symbionts of plants such as arbuscular mycorrhizal fungi (AMF) represent a huge, but unrealized resource for improving yields of globally important crops, especially in the tropics. We argue that the application of AMF in agriculture is too simplistic and ignores basic ecological principals. To achieve this challenge, a community and population ecology approach can contribute greatly. First, ecologists could significantly improve our understanding of the determinants of the survival of introduced AMF, the role of adaptability and intraspecific diversity of AMF and whether inoculation has a direct or indirect effect on plant production. Second, we call for extensive metagenomics as well as population genomics studies that are crucial to assess the environmental impact that introduction of non-local AMF may have on native AMF communities and populations. Finally, we plead for an ecologically sound use of AMF in efforts to increase food security at a global scale in a sustainable manner.

Produção de mudas do tipo rebentão, utilizando coroas de três cultivares de abacaxi inoculadas com fungos micorrízicos

Um dos principais gargalos para o desenvolvimento da abacaxicultura no Brasil tem sido a ausência tanto de mudas em quantidade quanto em qualidade, para propagação. Entre as alternativas, verifica-se a obtenção de mudas a partir da brotação de gemas de coroas dos frutos, que são, normalmente, descartadas pelo consumidor. Além disso, a utilização de fungos micorrízicos arbusculares (FMAs) pode ser uma alternativa para melhorar a produção das mudas, visto que estes fungos podem abreviar o tempo de formação de mudas de diversas frutíferas. O objetivo deste trabalho foi avaliar a produção de mudas do tipo rebentão, através do método de destruição do meristema apical da coroa de cultivares do abacaxizeiro inoculadas com FMAs. Utilizou-se o delineamento em blocos casualizados, num fatorial 3x3, com três cultivares de abacaxi ('Smooth Cayenne', 'Pérola' e 'Jupi') e três tratamentos microbiológicos (Sem inoculação, inoculação com Glomus etunicatum e inoculação com uma mistura dos fungos Glomus clarum e Gigaspora margarita), com quatro repetições. As primeiras emissões foram registradas aos 30; 60 e 90 dias após o plantio para as cultivares 'Smooth Cayenne', 'Pérola' e 'Jupi', respectivamente. O abacaxizeiro 'Smooth Cayenne' produziu 80 e 69% de mudas a mais do que as cultivares 'Pérola' e 'Jupi', respectivamente. Coroas de abacaxi inoculadas com a mistura de fungos micorrízicos apresentaram maior número de emissão de brotações quando comparadas com aquelas inoculadas apenas com G. etunicatum. Nas avaliações nutricionais das coroas, a inoculação com a mistura de dois fungos micorrizicos promoveu, em folhas da coroa do abacaxizeiro, incrementos de 85 e 66% nos teores de P; de 22 e 13% para os de N, e de 6 e 19% para os de K, em relação aos tratamentos G. etunicatum e sem inoculação, respectivamente. Conclui-se que a produção de rebentos oriundos de coroas cuja gema principal foi decapitada é uma alternativa para a produção de mudas de abacaxizeiro, sendo mais eficiente na cultivar 'Smooth Cayenne', que produziu 26 mudas em coroas cultivadas até 420 dias após o plantio.

Crescimento de mudas de maracujazeiro-doce inoculadas com fungos micorrízicos arbusculares e bactérias diazotróficas sob diferentes doses de fósforo

Conduziu-se o experimento em casa de vegetação com o objetivo de avaliar os efeitos da inoculação de fungos micorrízicos arbusculares (FMAs) e de bactérias diazotróficas endofíticas no crescimento de mudas de maracujazeiro-doce sob diferentes doses de fósforo. O delineamento experimental foi em blocos casualizados, em arranjo fatorial 3x2x4, sendo três tratamentos com fungos: Gigaspora margarita, Glomus clarum e controle (sem fungo); dois tratamentos com bactérias: Burkholderia sp. + Burkholderia silvatlantica e controle (sem bactéria); e quatro doses de fósforo: 0; 15; 30 e 60 mg dm-3, com quatro repetições. A unidade experimental foi composta por um vaso de 3,5 dm³ com três plantas. As mudas foram produzidas a partir de sementes e transplantadas para vasos, contendo mistura de solo e areia (1:2 v:v) esterilizada. Aos noventa dias após o transplante das mudas, foram avaliados: altura, diâmetro do caule, área foliar, massa seca da parte aérea, porcentagem de raízes colonizadas por fungos e enumeração de bactérias nas raízes. Independentemente da presença de bactérias diazotróficas, a inoculação com FMAs proporcionou incrementos na altura, massa seca de parte aérea, área foliar e diâmetro do caule das mudas de maracujazeiro-doce, nas doses de 0 e 15 mg dm-3 de P. As bactérias promoveram incrementos na altura e área foliar das mudas quando associadas ao FMA G. margarita, entretanto, sem a presença dos FMAs, estas reduziram a massa seca da parte aérea, independentemente das doses de P no substrato.

Cell wall maturation of Arabidopsis trichomes is dependent on exocyst subunit EXO70H4 and involves callose deposition.

Arabidopsis (Arabidopsis thaliana) leaf trichomes are single-cell structures with a well-studied development, but little is understood about their function. Developmental studies focused mainly on the early shaping stages, and little attention has been paid to the maturation stage. We focused on the EXO70H4 exocyst subunit, one of the most up-regulated genes in the mature trichome. We uncovered EXO70H4-dependent development of the secondary cell wall layer, highly autofluorescent and callose rich, deposited only in the upper part of the trichome. The boundary is formed between the apical and the basal parts of mature trichome by a callose ring that is also deposited in an EXO70H4-dependent manner. We call this structure the Ortmannian ring (OR). Both the secondary cell wall layer and the OR are absent in the exo70H4 mutants. Ecophysiological aspects of the trichome cell wall thickening include interference with antiherbivore defense and heavy metal accumulation. Ultraviolet B light induces EXO70H4 transcription in a CONSTITUTIVE PHOTOMORPHOGENIC1-dependent way, resulting in stimulation of trichome cell wall thickening and the OR biogenesis. EXO70H4-dependent trichome cell wall hardening is a unique phenomenon, which may be conserved among a variety of the land plants. Our analyses support a concept that Arabidopsis trichome is an excellent model to study molecular mechanisms of secondary cell wall deposition.

TLR3-Mediated CD8+ Dendritic Cell Activation Is Coupled with Establishment of a Cell-Intrinsic Antiviral State.

Because of their unique capacity to cross-present Ags to CD8(+) T cells, mouse lymphoid tissue-resident CD8(+) dendritic cells (DCs) and their migratory counterparts are critical for priming antiviral T cell responses. High expression of the dsRNA sensor TLR3 is a distinctive feature of these cross-presenting DC subsets. TLR3 engagement in CD8(+) DCs promotes cross-presentation and the acquisition of effector functions required for driving antiviral T cell responses. In this study, we performed a comprehensive analysis of the TLR3-induced antiviral program and cell-autonomous immunity in CD8(+) DC lines and primary CD8(+) DCs. We found that TLR3-ligand polyinosinic-polycytidylic acid and human rhinovirus infection induced a potent antiviral protection against Sendai and vesicular stomatitis virus in a TLR3 and type I IFN receptor-dependent manner. Polyinosinic-polycytidylic acid-induced antiviral genes were identified by mass spectrometry-based proteomics and transcriptomics in the CD8(+) DC line. Nanostring nCounter experiments confirmed that these antiviral genes were induced by TLR3 engagement in primary CD8(+) DCs, and indicated that many are secondary TLR3-response genes requiring autocrine IFN-β stimulation. TLR3-activation thus establishes a type I IFN-dependent antiviral program in a DC subtype playing crucial roles in priming adaptive antiviral immune responses. This mechanism is likely to shield the priming of antiviral responses against inhibition or abrogation by the viral infection. It could be particularly relevant for viruses detected mainly by TLR3, which may not trigger type I IFN production by DCs that lack TLR3, such as plasmacytoid DCs or CD8(-) DCs.

Pineapple (Ananas comosus) cv. pérola ex vitro growth and mycorrhizal colonization affected by in vitro sucrose concentration

The aim of this study was to evaluate the effect of sucrose concentration in the culture medium on growth and on the establishment of mycorrhizas during the acclimatization of pineapple cv. Pérola. The plantlets were micropropagated in MS culture medium with 0, 10, 20 and 30 g L-1 of sucrose and then they were acclimatized during 12 weeks under greenhouse conditions, in a sandy soil - compost mixture, uninoculated or inoculated with a Rhizophagus clarus isolate. Plantlets from the culture medium with 20 g and 30 g of sucrose L-1 showed higher shoot and root biomass than those from sugar-free medium. Mycorrhizal colonization was lower in plantlets micropropagated in sucrose-free medium, but the intensity of arbuscules did not differ among treatments. In the 12-week period of acclimatization, mycorrhizal colonization had no effect on plant biomass.

EFEITO DE FUNGOS MICORRÍZICOS ARBUSCULARES E DA ADUBAÇÃO NO CRESCIMENTO DE MUDAS DE Eugenia uniflora L., PRODUZIDAS EM DIFERENTES SUBSTRATOS

RESUMO O objetivo deste estudo foi avaliar o efeito de fungos micorrízicos arbusculares (FMAs), da adubação e da composição do substrato no crescimento de mudas de Eugenia uniflora. As sementes foram germinadas em vermiculita média e repicadas para tubetes (100 cm3) contendo substratos à base de vermicomposto e casca de arroz carbonizada e, como controle, utilizou-se do substrato comercial à base de casca de pínus. Estes substratos foram testados com e sem inoculação micorrízica, adicionada ao substrato, como também se testaram a presença e a ausência de adubação de cobertura. Foram analisadas as propriedades físico-químicas dos substratos formulados. Avaliaram-se a altura, o diâmetro do colo, a agregação das raízes ao substrato, a biomassa seca aérea, a biomassa seca radicial e foram determinados a relação entre altura e diâmetro do colo e o índice de qualidade de Dickson. A inoculação com FMAs não influenciou no crescimento das mudas, enquanto a interação entre substratos e adubação foi significativa para a maioria das variáveis. A ausência de resposta aos FMAs foi, provavelmente, devido às altas concentrações de fósforo nestes substratos. Concluiu-se que o substrato à base de vermicomposto e casca de arroz carbonizada, na proporção de 20/80, pode ser utilizado na produção de mudas desta espécie.

The absence of VGLUT3 predisposes to cocaine abuse by increasing dopamine and glutamate signaling in the nucleus accumbens.

Tonically active cholinergic interneurons (TANs) from the nucleus accumbens (NAc) are centrally involved in reward behavior. TANs express a vesicular glutamate transporter referred to as VGLUT3 and thus use both acetylcholine and glutamate as neurotransmitters. The respective roles of each transmitter in the regulation of reward and addiction are still unknown. In this study, we showed that disruption of the gene that encodes VGLUT3 (Slc17a8) markedly increased cocaine self-administration in mice. Concomitantly, the amount of dopamine (DA) release was strongly augmented in the NAc of VGLUT3(-/-) mice because of a lack of signaling by metabotropic glutamate receptors. Furthermore, dendritic spines and glutamatergic synaptic transmission on medium spiny neurons were increased in the NAc of VGLUT3(-/-) mice. Increased DA and glutamate signaling in the NAc are hallmarks of addiction. Our study shows that TANs use glutamate to reduce DA release and decrease reinforcing properties of cocaine in mice. Interestingly, we also observed an increased frequency of rare variations in SLC17A8 in a cohort of severe drug abusers compared with controls. Our findings identify VGLUT3 as an unexpected regulator of drug abuse.

The central histaminergic neurons in vitro, and their neuroprotective role in excitotoxic cell death in the postnatal rat hippocampus.

Histamine acts as a neurotransmitter in the central nervous system. Brain histamine in synthesized in neurons located to the posterior hypothalamus, from where these neurons send their projections to different parts of the brain. Released histamine participates in the regulation of several physiological functions such as arousal, attention and body homeostasis. Disturbances in the histaminergic system have been detected in diseases such as epilepsy, sleep disorders, anxiety, depression, Alzheimer’s disease, and schizophrenia. The purpose of this thesis was to develop optimal culture conditions for the histaminergic neurons, to study their detailed morphology, and to find out their significance in the kainic acid (KA)-induced neuronal death in the immature rat hippocampus. The morphology of the histaminergic neurons in vitro was comparable with the earlier findings. Histamine-containing vesicles were found in the axon but also in the cell body and dendrites suggesting a possibility for the somatodendritic release. Moreover, histamine was shown to be colocalized with the vesicular monoamine transporter 2 (VMAT2) suggesting that VMAT2 transports histamine to the subcellular storage vesicles. Furthermore, histamine was localized with γ-aminobutyric acid (GABA) in distinct storage vesicles and with neuropeptide galanin partly in the same storage vesicles suggesting different corelease mechanisms for GABA and galanin with histamine. In the organotypic hippocampal slice cultures, KA-induced neuronal death was first detected 12 h after the treatment being restricted mainly to the CA3 subregion. Moreover, cell death was irreversible, since the 48 h recovery period did not save the cells, but instead increased the damage. Finally, neuronal death was suggested to be necrotic, since intracellular apoptotic pathways were not activated, and the morphological changes detected with the electron microscopy were characteristic for necrosis. In the coculture system of the hippocampal and posterior hypothalamic slices, histaminergic neurons significantly decreased epileptiform burst activity and neuronal death in the hippocampal slices, this effect being mediated by histamine 1 (H1) and 3 (H3) receptors. In conclusion, the histaminergic neurons were maintained succesfully in the in vitro conditions exhibiting comparable morphological characteristics as detected earlier in vivo. Moreover, they developed functional innervations within the hippocampal slices in the coculture system. Finally, the KA-induced regionspecific, irreversible and necrotic hippocampal pyramidal cell damage was significantly decreased by the histaminergic neurons through H1 and H3 receptors.

Blood microvesicles: from proteomics to physiology

Phospholipid vesicles of less than 1 μm are present in blood in physiological state and their concentration may vary under pathological conditions. Various names such as exosomes (EXS) and microparticles (MPS) have been used to designate these extracellular vesicles (EVS). Although EXs and MPS possibly arise from separate mechanisms, they share numerous similarities representing a challenge for their purification and characterization. These vesicles generally originate from various types of cells such as red blood cells, platelets, leukocytes or endothelial cells but also from tumor cells. They participate in numerous biological processes including hemostasis. It is therefore of major scientific interest to characterize the protein content of these different types of EVS and that of their membranes in order to elucidate the essential functions of these dynamic vesicular compartments. Proteomics has been shown to be a particularly adequate tool in this study field. This review attempts to link proteomic data with physiological roles and functions of blood EVS.

Synaptic Integration of Adult-Born Hippocampal Neurons Is Locally Controlled by Astrocytes.

Adult neurogenesis is regulated by the neurogenic niche, through mechanisms that remain poorly defined. Here, we investigated whether niche-constituting astrocytes influence the maturation of adult-born hippocampal neurons using two independent transgenic approaches to block vesicular release from astrocytes. In these models, adult-born neurons but not mature neurons showed reduced glutamatergic synaptic input and dendritic spine density that was accompanied with lower functional integration and cell survival. By taking advantage of the mosaic expression of transgenes in astrocytes, we found that spine density was reduced exclusively in segments intersecting blocked astrocytes, revealing an extrinsic, local control of spine formation. Defects in NMDA receptor (NMDAR)-mediated synaptic transmission and dendrite maturation were partially restored by exogenous D-serine, whose extracellular level was decreased in transgenic models. Together, these results reveal a critical role for adult astrocytes in local dendritic spine maturation, which is necessary for the NMDAR-dependent functional integration of newborn neurons.

Retenção de metais pesados em micélio de fungos micorrízicos arbusculares

This work evaluated the kinetics as well as the retention capacity of Cu, Zn, Cd, and Pb by arbuscular mycorrhizal fungi (AMF) mycelium. The metal retention is a fast process with Cu being retained 3, 30, and 60 times faster than Zn, Cd, and Pb, respectively. Metal retention capacity varied amongst the different tested AMF species and decreased in the following order: Cu>Zn>>Cd>Pb. The Glomus clarum mycelium showed the highest retention capacity for Cu, Cd and Pb, whereas Zn was mostly retained by Gigaspora gigantea mycelium. The simultaneous application of all tested metals in solution decreased Cu and Zn retention by AMF mycelium. The high retention capacity of Cu and Zn by mycelium of G. clarum and G. gigantea suggests a promising use of these isolates in phytoremediation.

Bisphosphonate Inhibition of Prostate Cancer Cell Invasion, Migration and Cytoskeletal Organization

Metastatic bone lesions are commonly associated with prostate cancer affecting approximately 60-80% of the patients. The progression of prostate cancer into an advanced stage is a complex process and its molecular mechanisms are poorly understood. So far, no curative treatment is available for advanced stages of prostate cancer. Bisphosphonates (BPs) are synthetic pyrophosphate analogues, which are used as therapeutics for various metabolic bone diseases because of their ability to inhibit osteoclastic bone resorption. Nitrogen-containing bisphosphonates block the function of osteoclasts by disturbing the vesicular traffic and the mevalonate pathway -related enzymes, for example farnesyl diphosphate synthase, which is involved in post-translational isoprenylation of small GTPases. In addition, the anti-proliferative, anti-invasive and pro-apoptotic effects of nitrogen-containing bisphosphonates on various cancer cell lines have been reported. The aim of this thesis work was to clarify the effects of bisphosphonates on prostate cancer cells, focusing on the mechanisms of adhesion, invasion and migration. Furthermore, the role of the mevalonate pathway and prenylation reactions in invasion and regulation of the cytoskeleton of prostate cancer cells were examined. Finally, the effects of alendronate on cytoskeleton- and actin-related proteins in prostate cancer cells were studied in vitro and in vivo. The results showed that the nitrogen-containing bisphosphonate alendronate inhibited the adhesion of prostate cancer cells to various extracellular matrix proteins and migration and invasion in vitro. Inhibition of invasion and migration was reversed by mevalonate pathway intermediates. The blockage of the prenylation transferases GGTase I and FTase inhibited the invasion, migration and actin organization of prostate cancer cells. The marked decrease of cofilin was observed by the prenylation inhibitors used. Inhibition of GGTase I also disrupted the regulation of focal adhesion kinase and paxillin. In addition, alendronate disrupted the cytoskeletal organization and decreased the level of cofilin in vitro and in vivo. The decrease of the cofilin level by alendronate could be one of the key mechanisms behind the observed inhibition of migration and invasion. Based on the effects of nitrogen-containing bisphosphonates on tumor cell invasion and cytoskeletal organization, they can be suggested to be developed as therapeutics for inhibiting prostate cancer metastasis.

PLANT GROWTH-PROMOTING MICROBIAL INOCULANT FOR Schizolobium parahyba pv. parahyba

ABSTRACTSchizolobium parahyba pv. amazonicum (Huber ex Ducke) Barneby (paricá) occurs naturally in the Amazon and is significant commercial importance due to its rapid growth and excellent performance on cropping systems. The aim of this paper was to evaluate a microbial inoculants such as arbuscular mycorrhiza fungi (AMF) and Rhizobium sp. that promote plant growth. The inocula was 10 g of root colonized and spores of Glomus clarum and/or 1 mL of cell suspension (107 CFU/mL) of Rhizobium sp. and/or 100 g of chemical fertilizer NPK 20-05-20 per planting hole. The experimental design was complete randomized blocks with five replications and eight treatments (n = 800). Plant height, stem diameter and plant survival were measured. The results were tested for normality and homogeneity of variances and analyzed by ANOVA and Tukey test (p < 0.05). Rhizobium sp and AM fungi showed no effect on plant growth. Environmental factors probably influenced the effectiveness of symbiosis of both microorganisms and plant growth. The chemical fertilizer increased S. parahyba growth. During the first 120 days plants suffered with drought and frost, and at 180 days plants inoculated with microorganism plus chemical fertilizer showed higher survival when compared with control. The results showed that the microbial inoculants used showed an important role on plant survival after high stress conditions, but not in plant growth. Also was concluded that the planting time should be between November to December to avoid the presence of young plants during winter time that is dry and cold.