Middle East respiratory syndrome coronavirus vaccine based on a propagation-defective RNA replicon elicited sterilizing immunity in mice.

Self-amplifying RNA replicons are promising platforms for vaccine generation. Their defects in one or more essential functions for viral replication, particle assembly, or dissemination make them highly safe as vaccines. We previously showed that the deletion of the envelope (E) gene from the Middle East respiratory syndrome coronavirus (MERS-CoV) produces a replication-competent propagation-defective RNA replicon (MERS-CoV-ΔE). Evaluation of this replicon in mice expressing human dipeptidyl peptidase 4, the virus receptor, showed that the single deletion of the E gene generated an attenuated mutant. The combined deletion of the E gene with accessory open reading frames (ORFs) 3, 4a, 4b, and 5 resulted in a highly attenuated propagation-defective RNA replicon (MERS-CoV-Δ[3,4a,4b,5,E]). This RNA replicon induced sterilizing immunity in mice after challenge with a lethal dose of a virulent MERS-CoV, as no histopathological damage or infectious virus was detected in the lungs of challenged mice. The four mutants lacking the E gene were genetically stable, did not recombine with the E gene provided in trans during their passage in cell culture, and showed a propagation-defective phenotype in vivo. In addition, immunization with MERS-CoV-Δ[3,4a,4b,5,E] induced significant levels of neutralizing antibodies, indicating that MERS-CoV RNA replicons are highly safe and promising vaccine candidates.

Dataset reporting BCKDK interference in a BCAA-catabolism restricted environment.

This data article contains complementary figures to the research article "Mitochondrial response to the BCKDK-deficiency: some clues to understand the positive dietary response in this form of autism" [1]. Herein we present data relative to the effect of knocking down BCKDK gene on the real time oxygen consumption rate of fibroblasts obtained from a Maple Syrup Urine Disease (MSUD) patient. Interference of BCKDK expression on such cells showing a reduced branched-chain α-ketoacid dehydrogenase (BCKDHc) activity; let us generate a scenario to study the direct effect of BCKDK absence in an environment of high branched-chain amino acids (BCAAs) concentrations. Data relative to the effectiveness of the knockdown together with the potentiality of the BCKDK-knockdown to increase the deficient branched-chain α-ketoacid dehydrogenase activity detected in MSUD patients are also shown.

Volcanic ash leaching as a means of tracing the environmental impact of the 2011 Grímsvötn eruption, Iceland.

The Grímsvötn volcanic eruption, from 21 to 28 May, 2011, was the largest eruption of the Grímsvötn Volcanic System since 1873, with a Volcanic Explosivity Index (VEI) of magnitude 4. The main geochemical features of the potential environmental impact of the volcanic ash-water interaction were determined using two different leaching methods as proxies (batch and vertical flow-through column experiments). Ash consists of glass with minor amounts of plagioclase, clinopyroxene, diopside, olivine and iron sulphide; this latter mineral phase is very rare in juvenile ash. Ash grain morphology and size reflect the intense interaction of magma and water during eruption. Batch and column leaching tests in deionised water indicate that Na, K, Ca, Mg, Si, Cl, S and F had the highest potential geochemical fluxes to the environment. Release of various elements from volcanic ash took place immediately through dissolution of soluble salts from the ash surface. Element solubilities of Grímsvötn ash regarding bulk ash composition were <1 %. Combining the element solubilities and the total estimated mass of tephra (7.29 × 10(14) g), the total inputs of environmentally important elements were estimated to be 8.91 × 10(9) g Ca, 7.02 × 10(9) g S, 1.10 × 10(9) g Cl, 9.91 × 10(8) g Mg, 9.91 × 10(8) g Fe and 1.45 × 10(8) g P The potential environmental problems were mainly associated with the release of F (5.19 × 10(9) g).

Mitochondrial response to the BCKDK-deficiency: Some clues to understand the positive dietary response in this form of autism.

Mutations on the mitochondrial-expressed Branched Chain α-Keto acid Dehydrogenase Kinase (BCKDK) gene have been recently associated with a novel dietary-treatable form of autism. But, being a mitochondrial metabolism disease, little is known about the impact on mitochondrial performance. Here, we analyze the mitochondrial response to the BCKDK-deficiency in patient's primary fibroblasts by measuring bioenergetics, ultra-structural and dynamic parameters. A two-fold increase in superoxide anion production, together with a reduction in ATP-linked respiration and intracellular ATP levels (down to 60%) detected in mutants fibroblasts point to a general bioenergetics depletion that could affect the mitochondrial dynamics and cell fate. Ultrastructure analysis of BCKDK-deficient fibroblasts shows an increased number of elongated mitochondria, apparently associated with changes in the mediator of inner mitochondria membrane fusion, GTPase OPA1 forms, and in the outer mitochondrial membrane, mitofusin 2/MFN2. Our data support a possible hyperfusion response of BCKDK-deficient mitochondria to stress. Cellular fate also seems to be affected as these fibroblasts show an altered proportion of the cells on G0/G1 and G2/M phases. Knockdown of BCKDK gene in control fibroblasts recapitulates most of these features. Same BCKDK-knockdown in a MSUD patient fibroblasts unmasks the direct involvement of the accelerated BCAAs catabolism in the mitochondrial dysfunction. All these data give us a clue to understand the positive dietary response to an overload of branched-chain amino acids. We hypothesize that a combination of the current therapeutic option with a protocol that considers the oxidative damage and energy expenditure, addressing the patients' individuality, might be useful for the physicians.

Molecular characterization of a new PToV strain. Evolutionary implications.

Toroviruses are emergent viruses, belonging to the Nidovirales order, that remain mostly ignored, despite they are able to infect different species of domestic animals and humans, causing enteric diseases and diarrhea. Thus far, only five variants of porcine torovirus (PToV) have been identified. In this report we describe the identification and partial characterization of a new strain of porcine torovirus (PToV-BRES) that was detected by RT-PCR in a swine faecal specimen from a farm in Brescia (Italy). The complete genes coding for the nucleocapsid (N), hemagglutinin-esterase (HE) and membrane (M) proteins were amplified, and sequence analysis showed that PToV-BRES is a new PToV strain that, based on the HE gene sequence, is phylogenetically related to P4 strain, that was up to now the only member of a distinct PToV lineage. The nucleocapsid protein from PToV-BRES was expressed in insect cells as a his-tagged protein, purified by affinity chromatography and used to develop an ELISA method to detect antibodies against PToV. This assay was evaluated using a serum collection including 45 samples from three commercial farms from Spain. High antibody prevalence against PToV was observed in the three farms, both in adult animals and in piglets, which could suggest that PToV might be endemic in Spanish porcine population. The ELISA method developed in this work could be useful in future epidemiological surveys about toroviruses.

Autophagy in wine making.

Aging that involves contact with dying yeast cells is one of the differential processes between sparkling and still wine production. The release of the products of autolysis during this aging step is fundamental for the quality of sparkling wines made by the traditional method. These cells undergo an autolysis process characterized by self-digestion of yeast intracellular and cell-wall macromolecules, and the release of the degradation products to the wine. Autolysis is the source of several molecules responsible for the quality of sparkling wines, as well as still wines aged on lees (yeast cells). Autolysis is a slow process under sparkling wine production conditions, and there is interest, from the industrial side, in the design of strategies for rapid development of autolysis. Some years ago our research group hypothesized that, during the process of sparkling wine production, autophagy would take place. This had important implications for the design of genetic engineering strategies aimed to accelerate autolysis. The relationships between autolysis and autophagy are not completely elucidated, but in case autophagy preceded autolysis during the aging step of sparkling wine production, there were at least two possibilities for accelerating autolysis by targeting genes involved in autophagy. This chapter discusses methods to demonstrate the development of autophagy under enological conditions. This is accomplished by using either laboratory strains defective in autophagy and/or the Cvt pathway, in conditions that mimic sparkling wine production or industrial wine yeast strains under real sparkling wine production conditions.

Psychosis, migration and social environment: an age--and--gender controlled study.

PURPOSE: Adverse social experiences are frequently invoked to explain the higher rate of psychosis among migrant groups. The aim of the present study was to establish the socio-environmental factors distinguishing migrant psychotic patients from autochthonous patients. SUBJECTS AND METHOD: We conducted a cross-sectional survey involving 341 migrant psychotic patients matched for age and gender with 341 autochthonous psychotic patients. RESULTS: Migrant patients lived more often with their parental family, were less often enrolled with a referral psychiatrist, presented a lower rate of employment, a lower percentage of alcohol misuse and of suicide attempts. DISCUSSION: Our findings add to the growing body of results showing that more attention needs to be focused on socio-environmental variables in psychosis research. However, several limitations have to be taken into account, particularly with regard to selection biases and age of onset of the psychotic illness. CONCLUSION: Our results are compatible with the hypothesis that unemployment is a contributing factor in the risk for psychosis among migrant groups. Migrants' families are an important keystone in the mental health care process of their sick relatives. Our service models need to be adapted with the aim to make the treatment easier for migrant patients.

Immunolocalization of a novel cholesteryl ester hydrolase in the endoplasmic reticulum of murine and human hepatocytes.

We have recently purified a cholesteryl ester hydrolase (CEH) from rat liver microsomes. Antibodies raised against the purified protein specifically reacted with a 106-kd protein and neutralized 90% of the CEH activity of rat liver microsomes (J Lipid Res 1999;40:715-725). In this work we have used the anti-CEH antibody to study both the subcellular distribution of the protein in hepatocytes as well as its tissue-specific expression in rat. Western blotting of subcellular fractions obtained from isolated rat hepatocytes revealed that the immunoreactive 106-kd CEH was exclusively localized in microsomes. The antibody also recognized a 106-kd protein in microsomes from mouse and human liver but not from rat nonparenchymal liver cells. Confocal microscopy of HepG2 cells revealed that CEH immunoreactive material colocalized with calnexin, a marker of the endoplasmic reticulum. Furthermore, high-resolution immunoelectron microscopy of rat liver thin sections exclusively localized the CEH immunoreactivity to the endoplasmic reticulum of the hepatocyte. No CEH immunoreactivity was observed in microsomes derived from adrenal glands, ovaries, testis, pancreas, intestine, white adipose tissue, mammary gland, lung, spleen, brain, aorta, and macrophages. We report a CEH localized to the endoplasmic reticulum, erCEH, in the mammalian hepatocyte. The subcellular localization and tissue-restricted pattern of expression of erCEH suggests that it might have unique functions in liver cholesterol metabolism.

[Molecular characterization of thalassemias in the Valencia community and its relationship with the hematological phenotype]. / Caracterización molecular de las talasemias en la Comunidad Valenciana y su relación con el fenotipo hematológico.

PURPOSE: The aim of the study is to present the first results of molecular characterization of thalassaemias in Valencian Community and their relationship with the haematological parameters. PATIENTS AND METHODS: The study includes 87 thalassemic patients: 30 alpha-thalassaemias, 40 beta-thalassaemias and 17 delta beta-thalassaemias. The molecular alterations were studied in white cell blood DNA, either following different PCR methods or by testing the digestion of the amplified PCR products with selective restriction enzymes. RESULTS: The molecular characterization of beta-thalassaemias was achieved in 94% of the subjects, being the transition C-->T in CD-39 the most frequent (44%) of the mutations studied. 94% of the delta beta-thalassaemias studied corresponded to the delta beta-Spanish type. All the alpha thalassaemias characterized (64%) corresponded to the -alpha 3.7 deletion. The reamining 36% were negative for the alpha 0 deletions --MED, --20.5, or the non deletional mutations Hph I and NocI. DISCUSSION: In the Valencian Community, like what has been described for the beta-thalassaemias in other Mediterranean regions of Spain (Barcelona, Granada and Mallorca), a high incidence in C-->T transition in CD-39 was observed, in contrast with central and south-western regions of Spain, where the G-->A IVS-I-1 is the most frequent mutation. Our study supports that the IVS-I-6 mutations is the one with lower repercussions on the haematological parameters. Our study confirms the Spanish type of delta beta-thalassaemia as the most frequent in the Valencian Community, and that the 3.7 kb alpha deletion is the most frequent mutation for the alpha-thalassaemia, although alpha thalassaemia is also the poorly characterized form of thalassaemia.

Estradiol promotes cell shape changes and glial fibrillary acidic protein redistribution in hypothalamic astrocytes in vitro: a neuronal-mediated effect.

We have previously shown that in hypothalamic mixed neuronal-glial cultures both astrocytic shape and distribution of glial fibrillary acidic protein (GFAP) are modified by estradiol. In the present study, we have investigated whether or not the presence of neurons is necessary for these hormonal effects. In mixed neuronal-glial hypothalamic cultures the proportion of process-bearing GFAP-immunoreactive cells was significantly increased after treatment for 30 min with 10(-12) M 17 beta estradiol. This effect was present for at least 1 day and was reverted by incubating the cells in estradiol-free medium. Estradiol incubation resulted in a progressive differentiation of GFAP-immunoreactive cells from a flattened epithelioid morphology to bipolar, radial, and stellate shapes. This effect was not observed in pure hypothalamic glial cultures. Furthermore, incubation of hypothalamic glial cells with medium conditioned by estradiol-treated mixed hypothalamic cultures did not affect the shape of GFAP-immunoreactive astrocytes. In contrast, addition of hypothalamic neurons, but not cerebellar neurons or fibroblasts, to established hypothalamic glial cultures affected the development of estradiol sensitivity in astrocytes. These results indicate that estradiol induction of shape changes in hypothalamic astrocytes is not only dependent on the presence of hypothalamic neurons, but that physical contact between astrocytes and neurons is necessary for the manifestation of the effect of this hormone.

Ontogeny of insulin-like growth factor I, its receptor, and its binding proteins in the rat hypothalamus.

A role for the insulin-like growth factors (IGFs) in brain growth and differentiation has recently been suggested. In previous studies on fetal hypothalamic cells we found a trophic influence of IGF-I on in vitro survival and differentiation of both neurons and glia. We have now investigated the expression of IGF-I, its receptor and its binding proteins in the rat hypothalamus to determine whether endogenous IGF-I might serve as a trophic factor during development of this brain area. Both IGF-I receptors and IGF-I binding proteins showed marked developmental stage-dependent variations. Thus, IGF-I receptors as measured by both binding and cross-linking techniques, were highest during fetal life and steadily decreased thereafter to reach low adult levels. Changes in receptor numbers rather than in its affinity constant accounted for the differences seen in binding activity during development. In addition, we found 3 different IGF-I binding proteins (IGFBPs) of apparent Mr of 24, 29 and 32 kDa respectively, whose levels also showed a specific developmental pattern. Highest levels of the 29 and 32 kDA IGFBPs were found in fetal and early postnatal life, whereas levels of the 24 kDa form were highest in young adults. Changes in the concentration of IGFBPs rather than in their affinities for IGF-I accounted for the different binding capacities found. Using a specific IGF-I radioimmunoassay we found that IGF-I-like immunoreactivity (IGF-I-li) levels had no direct correlation with developmental stage. IGF-I-li levels oscillated with no apparent trend throughout development of the hypothalamus.(ABSTRACT TRUNCATED AT 250 WORDS)

Localization of insulin-like growth factor I (IGF-I)-like immunoreactivity in the developing and adult rat brain.

The cellular distribution of insulin-like growth factor I (IGF-I) immunoreactivity was examined in the rat brain from embryonic day 15 to maturity. IGF-I immunoreactivity was found in the perikarya of neurons distributed along the entire extension of the neuronal tube in all the embryonic ages studied (E15, E17, E19 and E21). In E21 animals, the majority of immunoreactive neurons was located in the olfactory bulb, cerebral cortex, hippocampus, striatum, diencephalon, mesencephalic colliculi, trigeminal nuclei, trigeminal ganglion and in motoneurons of the brainstem. In 10- and 20-day-old rats, in addition to the above areas, IGF-I immunoreactivity was also observed in capillary walls, ependymal cells, choroid plexus, glial cells and most fiber paths. In postnatal ages, immunoreactivity in neuronal somas was mainly restricted to the cell nuclei. However, IGF-I immunoreactivity in the neuronal cytoplasm was observed in 20-day-old rats treated with colchicine while fiber paths and neuronal cell nuclei were negative in these animals. In the telencephalon of 20-day-old rats injected with colchicine, the most intense immunoreactive neurons were observed in the olfactory bulb, cerebral cortex, tenia tecta, hippocampus, islands of Calleja, septal nuclei, striatum, endopyriform nucleus and amygdala. Most diencephalic nuclei, the substantia nigra, the mesencephalic colliculi, Purkinje cells in the cerebellar cortex and several nuclei in mesencephalon, pons and medulla oblongata were also immunoreactive. In adult rats injected with colchicine, IGF-I immunoreactivity was located in the same areas as in 20-day-old rats. The number of immunoreactive cells and the intensity of the staining was reduced in adult rats as compared to that found in young postnatal animals.(ABSTRACT TRUNCATED AT 250 WORDS)

Sodium diethyldithiocarbamate restores T lymphocyte proliferation, interleukin-2 production and NK activity in cyclophosphamide-immunosuppressed animals.

The in vivo effect of sodium diethyldithiocarbamate (DTC) on IL-2 production, mitogen-induced proliferation and natural killer (NK) activity of lymphocytes from normal as well as cyclophosphamide (CY)-treated mice has been investigated. DTC was given in a single subcutaneous injection (25 mg/kg) to normal or cyclophosphamide-treated mice (250 mg/kg i.p. simultaneously to DTC). An enhancement of T lymphocyte proliferation in CY-treated animals and an increase of IL-2 production in both normal and immunosuppressed mice was observed. When DTC (10 mg/kg) was administered daily for two weeks an increase in concanavalin A-induced mitogenesis, IL-2 production and NK activity in CY-treated animals was observed. These immune parameters were reduced 12 days after CY treatment by a factor of 2 to 3 times, while DTC treatment restored these responses to normal levels. LPS-induced mitogenesis was not significantly enhanced. The effect of DTC could be partially mediated by changes in IL-2 activity. According to these results some functional parameters of the immune system of the suppressed host can be partially or completely restored by means of an appropriate immunomodulator treatment. DTC could be of interest in the treatment of diseases where immune functions are impaired or in combined treatments with immunosuppressants.

Corticosterone-releasing activity of immune mediators.

Products derived from the activated immune system have been reported to modulate neuroendocrine function. In addition, a direct connection between neuroendocrine and immune responses to stress has recently been proposed. We now provide evidence that heterogeneous lymphokine-containing supernatants from mitogen-stimulated rat spleen cells can stimulate both basal and corticotropin-induced corticosterone secretion from rat adrenal cells in an in vitro perifusion system. Moreover, thymosin alpha 1, a 28-amino acid residue peptide found both in thymus and lymphocyte-derived supernatants was also able to synergistically stimulate corticotropin-stimulated corticosterone release, without affecting basal corticosterone output in this same in vitro adrenal cell perifusion system. These results reinforce the suggestion about the existence of bidirectional interactions between the immune and neuroendocrine systems. They also indicate that this communication may occur directly at the adrenal gland level, a major effector site of the body's response to stress.

Isoprinosine restores in vitro T lymphocyte functions of cyclophosphamide immunosuppressed mice.

The effect and the mechanism of action of isoprinosine has been investigated in several models of in vitro activation of lymphocytes. Isoprinosine added to spleen cell cultures enhanced lymphocyte proliferation induced by concanavalin A or allogeneic stimulation as well as the generation of allospecific cytotoxic T cells. The effect of isoprinosine on T lymphocyte proliferation in vitro was specially marked when mice were treated with cyclophosphamide (75-200 mg/kg) 16-24 h before the onset of cultures. No effect was observed on B cell proliferation to LPS. Addition of inosine or adenosine also enhanced proliferation of cells from both normal and cyclophosphamide treated mice. Isoprinosine and inosine and, more markedly, adenosine, augmented interleukin-2 activity in concanavalin A supernatants of spleen cells from the same animals.