"Refined mapping of a quantitative trait locus on chromosome 1 responsible for mouse embryonic death"

Recurrent spontaneous abortion (RSA) is defined as the loss of three or more consecutive pregnancies during the first trimester of embryonic intrauterine development. This kind of human infertility is frequent among the general population since it affects 1 to 5% of women. In half of the cases the etiology remains unelucidated. In the present study, we used interspecific recombinant congenic mouse strains (IRCS) in the aim to identify genes responsible for embryonic lethality. Applying a cartographic approach using a genotype/phenotype association, we identified a minimal QTL region, of about 6 Mb on chromosome 1, responsible for a high rate of embryonic death (,30%). Genetic analysis suggests that the observed phenotype is linked to uterine dysfunction. Transcriptomic analysis of the uterine tissue revealed a preferential deregulation of genes of this region compared to the rest of the genome. Some genes from the QTL region are associated with VEGF signaling, mTOR signaling and ubiquitine/proteasome-protein degradation pathways. This work may contribute to elucidate the molecular basis of a multifactorial and complex human disorder as RSA.

Refined mapping of a quantitative trait locus on chromosome 1 responsible for mouse embryonic death

Recurrent spontaneous abortion (RSA) is defined as the loss of three or more consecutive pregnancies during the first trimester of embryonic intrauterine development. This kind of human infertility is frequent among the general population since it affects 1 to 5% of women. In half of the cases the etiology remains unelucidated. In the present study, we used interspecific recombinant congenic mouse strains (IRCS) in the aim to identify genes responsible for embryonic lethality. Applying a cartographic approach using a genotype/phenotype association, we identified a minimal QTL region, of about 6 Mb on chromosome 1, responsible for a high rate of embryonic death (similar to 30%). Genetic analysis suggests that the observed phenotype is linked to uterine dysfunction. Transcriptomic analysis of the uterine tissue revealed a preferential deregulation of genes of this region compared to the rest of the genome. Some genes from the QTL region are associated with VEGF signaling, mTOR signaling and ubiquitine/proteasome-protein degradation pathways. This work may contribute to elucidate the molecular basis of a multifactorial and complex human disorder as RSA.

Uso de la matriz extracelular como material para ingeniería de tejidos

Introducción: La evaluación de injertos vasculares de submucosa de intestino delgado para la regeneración de vasos sanguíneos ha producido una permeabilidad variable (0-100%) que ha sido concurrente con la variabilidad en las técnicas de fabricación. Metodología: Investigamos los efectos de fabricación en permeabilidad y regeneración en un diseño experimental de 22factorial que combino: 1) preservación (P) o remoción (R) de la capa estratum compactum del intestino, y 2) deshidratada (D) o hidratada (H), dentro de cuatro grupos de estudio (PD, RD, PH, RH). Los injertos fueron implantados en las Arterias Carótidas de porcinos (ID 4.5mm, N=4, 7d). Permeabilidad, trombogenicidad, reacción inflamatoria, vascularización, infiltración de fibroblastos, perfil de polarización de macrófagos y fuerza tensil biaxial fueron evaluadas. Resultados: Todos los injertos PD permanecieron permeables (4/4), pero tuvieron escasa vascularización e infiltración de fibroblastos. El grupo RD permaneció permeable (4/4), presentó una extensa vascularización e infiltración de fibroblastos, y el mayor número del fenotipo de macrófagos (M2) asociado a regeneración. El grupo RH presentó menor permeabilidad (3/4), una extensa vascularización e infiltración de fibroblastos, y un perfil dominante de M2. El grupo PH presentó el menor grado de permeabilidad, y a pesar de mayor infiltración celular que PD, exhibió un fenotipo de macrófagos dominante adverso. La elasticidad de los injertos R evolucionó de una manera similar a las Carótidas nativas (particularmente RD, mientras que los injertos P mantuvieron su rigidez inicial. Discusión: Concluimos que los parámetros de fabricación afectan drásticamente los resultados, siendo los injertos RD los que arrojaron mejores resultados.

Programa de tr??nsito a la vida adulta en la Comunidad Aut??noma de Canarias

Aulas enclave y centros espec??ficos de educaci??n especial

Cuestionarios TDAH para profesores : un an??lisis desde los criterios del DSM-IV-TR y DSM-V

Resumen basado en el de la publicaci??n

The four major N- and C-terminal splice variants of the excitatory amino acid transporter GLT-1 form cell surface homomeric and heteromeric assemblies

The L-glutamate transporter GLT-1 is an abundant CNS membrane protein of the excitatory amino acid transporter (EAAT) family which controls extracellular L-glutamate levels and is important in limiting excitotoxic neuronal death. Using RT-PCR, we have determined that four mRNAs encoding GLT-1 exist in mouse brain, with the potential to encode four GLT-1 isoforms that differ in their N- and C-termini. We expressed all four isoforms (termed MAST-KREK, MPK-KREK, MAST-DIETCI and MPK-DIETCI according to amino acid sequence) in a range of cell lines and primary astrocytes and show that each isoform can reach the cell surface. In transfected HEK-293 or COS-7 cells, all four isoforms support high-affinity sodium-dependent L-glutamate uptake with identical pharmacological and kinetic properties. Inserting a viral epitope (V5, HA or FLAG) into the second extracellular domain of each isoform allowed co-immunoprecipitation and tr-FRET studies using transfected HEK-293 cells. Here we show for the first time that each of the four isoforms are able to combine to form homomeric and heteromeric assemblies, each of which are expressed at the cell surface of primary astrocytes. After activation of protein kinase C by phorbol ester, V5-tagged GLT-1 is rapidly removed from the cell surface of HEK-293 cells and degraded. This study provides direct biochemical evidence for oligomeric assembly of GLT-1 and reports the development of novel tools to provide insight into the trafficking of GLT-1.

The M1 matrix protein controls the filamentous phenotype of influenza A virus

We show that most isolates of influenza A induce filamentous changes in infected cells in contrast to A/WSN/33 and A/PR8/34 strains which have undergone extensive laboratory passage and are mouse-adapted. Using reverse genetics, we created recombinant viruses in the naturally filamentous genetic background of A/Victoria/3/75 and established that this property is regulated by the M1 protein sequence, but that the phenotype is complex and several residues are involved. The filamentous phenotype was lost when the amino acid at position 41 was switched from A to V, at the same time, this recombinant virus also became insensitive to the antibody 14C2. On the other hand, the filamentous phenotype could be fully transferred to a virus containing RNA segment 7 of the A/WSN/33 virus by a combination of three mutations in both the amino and carboxy regions of the M1 protein. This observation suggests that an interaction among these regions of M1 may occur during assembly. (C) 2004 Elsevier Inc. All rights reserved.

Disease progression in heterosexual patients infected with closely related subtype B strains of HIV type 1 with differing coreceptor usage properties

Previously we described a heterosexual outbreak of HIV-1 subtype B in a town in the north of England (Doncaster) where 11 of 13 infections were shown to be linked by phylogenetic analysis of the env gp120 region. The 11 infections were related to a putative index case, Don1, and further divided into two groups based on the patients' disease status, their viral sequences, and other epidemiological information. Here we describe two further findings. First, we found that viral isolates and gp120 recombinant viruses derived from patients from one group used the CCR5 coreceptor, whereas viruses from the other group could use both the CCR5 and CXCR4 coreceptors. Patients with the X4/R5 dual tropic strains were symptomatic when diagnosed and progressed rapidly, in contrast to the other patient group that has remained asymptomatic, implying a link between the tropism of the strains and disease outcome. Second, we present additional sequence data derived from the index case, demonstrating the presence of sequences from both clades, with an average interclade distance of 9.56%, providing direct evidence of a genetic link between these two groups. This new study shows that Don1 harbored both strains, implying he was either dually infected or that over time intrahost diversification from the R5 to R5/X4 phenotype occurred. These events may account for/have led to the spread of two genetically related strains with different pathogenic properties within the same heterosexual community.

Induction of aquaporin 1 but not aquaporin 4 messenger RNA in rat primary brain microvessel endothelial cells in culture

Aquaporins (AQPs) are a family of proteins that mediate water transport across cells, but the extent to which they are involved in water transport across endothelial cells of the blood-brain barrier is not clear. Expression of AQP1 and AQP4 in rat brain microvessel endothelial cells was investigated in order to determine whether these isoforms were present and, in particular, to examine the hypothesis that brain endothelial expression of AQPs is dynamic and regulated by astrocytic influences. Reverse-transcriptase-polymerase chain reaction (RT-PCR) and immunocytochemistry showed that AQP1 mRNA and protein are present at very low levels in primary rat brain microvessel endothelial cells, and are up-regulated in passaged cells. Upon passage, endothelial cell expression of mdr1a mRNA is decreased, indicating loss of blood-brain barrier phenotype. In passage 4 endothelial cells, AQP1 mRNA levels are reduced by coculture above rat astrocytes, demonstrating that astrocytic influences are important in maintaining the low levels of AQP1 characteristic of the blood-brain barrier endothelium. Reverse-transcriptase-PCR revealed very low levels of AQP1 mRNA present in the RBE4 rat brain microvessel endothelial cell line, with no expression detected in primary cultures of rat astrocytes or in the C6 rat glioma cell line. In contrast, AQP4 mRNA is strongly expressed in astrocytes, but no expression is found in primary or passaged brain microvessel endothelial cells, or in RBE4 or C6 cells. Our results support the concept that expression of AQP1, which is seen in many non-brain endothelia, is suppressed in the specialized endothelium of the blood-brain barrier.

Regulation of the cardiomyocyte transcriptome vs translatome by endothelin-1 and insulin: translational regulation of 5' terminal oligopyrimidine tract (TOP) mRNAs by insulin

Background: Changes in cellular phenotype result from underlying changes in mRNA transcription and translation. Endothelin-1 stimulates cardiomyocyte hypertrophy with associated changes in mRNA/protein expression and an increase in the rate of protein synthesis. Insulin also increases the rate of translation but does not promote overt cardiomyocyte hypertrophy. One mechanism of translational regulation is through 5' terminal oligopyrimidine tracts (TOPs) that, in response to growth stimuli, promote mRNA recruitment to polysomes for increased translation. TOP mRNAs include those encoding ribosomal proteins, but the full panoply remains to be established. Here, we used microarrays to compare the effects of endothelin-1 and insulin on the global transcriptome of neonatal rat cardiomyocytes, and on mRNA recruitment to polysomes (i.e. the translatome). Results: Globally, endothelin-1 and insulin (1 h) promoted >1.5-fold significant (false discovery rate < 0.05) changes in expression of 341 and 38 RNAs, respectively. For these transcripts with this level of change there was little evidence of translational regulation. However, 1336 and 712 RNAs had >1.25-fold significant changes in expression in total and/or polysomal RNA induced by endothelin-1 or insulin, respectively, of which ~35% of endothelin-1-responsive and ~56% of insulin-responsive transcripts were translationally regulated. Of mRNAs for established proteins recruited to polysomes in response to insulin, 49 were known TOP mRNAs with a further 15 probable/possible TOP mRNAs, but 49 had no identifiable TOP sequences or other consistent features in the 5' untranslated region. Conclusions: Endothelin-1, rather than insulin, substantially affects global transcript expression to promote cardiomyocyte hypertrophy. Effects on RNA recruitment to polysomes are subtle, with differential effects of endothelin-1 and insulin on specific transcripts. Furthermore, although insulin promotes recruitment of TOP mRNAs to cardiomyocyte polysomes, not all recruited mRNAs are TOP mRNAs.

Lack of association between lipaemia and central adiposity in subjects with an atherogenic lipoprotein phenotype (ALP)

OBJECTIVE: To investigate the associations between indices of adiposity and cardiovascular risk factors in individuals with an atherogenic lipoprotein phenotype (ALP). SUBJECTS: Fifty-five men, aged 34-69 y, body mass index (BMI) 22-35 kg/m2, with an ALP lipid profile (triglycerides (TG) 1.5-4.0 mmol/l, HDL<1.1 mmol/l; %LDL-3>40% total LDL). DESIGN: Each participant provided a fasting blood sample and underwent an 8 h postprandial assessment and had anthropometric measurements taken. OUTCOME MEASURES: BMI, waist circumference (W), waist-to-hip ratio (W/H), sum of skinfolds (SSK), fasting and postprandial concentrations of glucose, insulin and plasma lipids, post-heparin lipase activity, and apoE genotype. RESULTS: The expected positive associations between BMI, W and SSK and fasting and postprandial insulin were observed (r=0.42-0.65). Little association between glucose responses and any measures of adiposity was evident. Unexpectedly, there were no positive associations between measures of central adiposity (W and W/H) and fasting and postprandial TG responses, with a trend towards negative associations in this study group (TG AUC vs W, r=-0.23, P=0.097; TG IAUC vs W/H, r=-0.26, P=0.068). Subgroup analysis indicated that lack of a positive association between central adiposity and postprandial TG values was more evident in those with one E4 allele (r=-0.42, P=0.077) relative to non-E4 carriers (r=-0.16, P=0.430). The expected positive associations between insulin and TG responses were not observed (r=-0.03 to -0.36). CONCLUSION: In this ALP group the expected positive association between TG responses and a centralized distribution of body fat was not observed, particularly in individuals with an apoE4 genotype. Our findings are not in line with the view that there is a clear causal relationship between insulin resistance and the lipid abnormalities associated with ALP.

ApoE polymorphism and fish oil supplementation in subjects with an antherogenic lipoprotein phenotype

The study assessed the efficacy of fish oil supplementation in counteracting the classic dyslipidemia of the atherogenic lipoprotein phenotype (ALP). In addition, the impact of the common apolipoprotein E (apoE) polymorphism on the fasting and postprandial lipid profile and on responsiveness to the dietary intervention was established. Fifty-five ALP males (aged 34 to 69 years, body mass index 22 to 35 kg/m2, triglyceride [TG] levels 1.5 to 4.0 mmol/L, high density lipoprotein cholesterol [HDL-C] <1.1 mmol/l, and percent low density lipoprotein [LDL]-3 >40% total LDL) completed a randomized placebo-controlled crossover trial of fish oil (3.0 g eicosapentaenoic acid/docosahexaenoic acid per day) and placebo (olive oil) capsules with the 6-week treatment arms separated by a 12-week washout period. In addition to fasting blood samples, at the end of each intervention arm, a postprandial assessment of lipid metabolism was carried out. Fish oil supplementation resulted in a reduction in fasting TG level of 35% (P<0.001), in postprandial TG response of 26% (TG area under the curve, P<0.001), and in percent LDL-3 of 26% (P<0.05). However, no change in HDL-C levels was evident (P=0.752). ANCOVA showed that baseline HDL-C levels were significantly lower in apoE4 carriers (P=0.035). The apoE genotype also had a striking impact on lipid responses to fish oil intervention. Individuals with an apoE2 allele displayed a marked reduction in postprandial incremental TG response (TG incremental area under the curve, P=0.023) and a trend toward an increase in lipoprotein lipase activity relative to non-E2 carriers. In apoE4 individuals, a significant increase in total cholesterol and a trend toward a reduction in HDL-C relative to the common homozygous E3/E3 profile was evident. Our data demonstrate the efficacy of fish oil fatty acids in counteracting the proatherogenic lipid profile of the ALP but also that the apoE genotype influences responsiveness to this dietary treatment.

Inter-relationships between small, dense low-density lipoprotein (LDL), plasma triacylglycerol and LDL apoprotein B in an atherogenic lipoprotein phenotype in free-living subjects

A predominance of small, dense low-density lipoprotein (LDL) is a major component of an atherogenic lipoprotein phenotype, and a common, but modifiable, source of increased risk for coronary heart disease in the free-living population. While much of the atherogenicity of small, dense LDL is known to arise from its structural properties, the extent to which an increase in the number of small, dense LDL particles (hyper-apoprotein B) contributes to this risk of coronary heart disease is currently unknown. This study reports a method for the recruitment of free-living individuals with an atherogenic lipoprotein phenotype for a fish-oil intervention trial, and critically evaluates the relationship between LDL particle number and the predominance of small, dense LDL. In this group, volunteers were selected through local general practices on the basis of a moderately raised plasma triacylglycerol (triglyceride) level (>1.5 mmol/l) and a low concentration of high-density-lipoprotein cholesterol (<1.1 mmol/l). The screening of LDL subclasses revealed a predominance of small, dense LDL (LDL subclass pattern B) in 62% of the cohort. As expected, subjects with LDL subclass pattern B were characterized by higher plasma triacylglycerol and lower high-density lipoprotein cholesterol (<1.1 mmol/l) levels and, less predictably, by lower LDL cholesterol and apoprotein B levels (P<0.05; LDL subclass A compared with subclass B). While hyper-apoprotein B was detected in only five subjects, the relative percentage of small, dense LDL-III in subjects with subclass B showed an inverse relationship with LDL apoprotein B (r=-0.57; P<0.001), identifying a subset of individuals with plasma triacylglycerol above 2.5 mmol/l and a low concentration of LDL almost exclusively in a small and dense form. These findings indicate that a predominance of small, dense LDL and hyper-apoprotein B do not always co-exist in free-living groups. Moreover, if coronary risk increases with increasing LDL particle number, these results imply that the risk arising from a predominance of small, dense LDL may actually be reduced in certain cases when plasma triacylglycerol exceeds 2.5 mmol/l.

Phenotype MicroArray (TM) in the metabolic characterisation of Salmonella serotypes Agona, Enteritidis, Give, Hvittingfoss, Infantis, Newport and Typhimurium

The Phenotype MicroArray (TM) (PM) technology was used to study the metabolic characteristics of 29 Salmonella strains belonging to seven serotypes of S. enterica spp. enterica. Strains of serotypes Typhimurium (six strains among definite phage types DTs 1, 40 and 104) and Agona (two strains) were tested for 949 substrates, Enteritidis (six strains of phage type PT1), Give, Hvittingfoss, Infantis and Newport strains (two of each) were tested for 190 substrates and seven other Agona strains for 95 substrates. The strains represented 18 genotypes in pulsed-field gel electrophoresis (PFGE). Among 949 substrates, 18 were identified that could be used to differentiate between the strains of those seven serotypes or within a single serotype. Unique metabolic differences between the Finnish endemic Typhimurium DT1 and Agona strains were detected, for example, in the metabolism of d-tagatose, d-galactonic acid gamma-lactone and l-proline as a carbon source. Thus, the PM technique is a useful tool for identifying potential differential markers on a metabolic basis that could be used for epidemiological surveillance.

Weaning diet induces sustained metabolic phenotype shift in the pig and influences host response to Bifidobacterium lactis NCC2818

Background The process of weaning causes a major shift in intestinal microbiota and is a critical period for developing appropriate immune responses in young mammals.Objective To use a new systems approach to provide an overview of host metabolism and the developing immune system in response to nutritional intervention around the weaning period.Design Piglets (n=14) were weaned onto either an egg-based or soya-based diet at 3 weeks until 7 weeks, when all piglets were switched onto a fish-based diet. Half the animals on each weaning diet received Bifidobacterium lactis NCC2818 supplementation from weaning onwards. Immunoglobulin production from immunologically relevant intestinal sites was quantified and the urinary (1)H NMR metabolic profile was obtained from each animal at post mortem (11 weeks).Results Different weaning diets induced divergent and sustained shifts in the metabolic phenotype, which resulted in the alteration of urinary gut microbial co-metabolites, even after 4 weeks of dietary standardisation. B lactis NCC2818 supplementation affected the systemic metabolism of the different weaning diet groups over and above the effects of diet. Additionally, production of gut mucosa-associated IgA and IgM was found to depend upon the weaning diet and on B lactis NCC2818 supplementation.ConclusionThe correlation of urinary (1)H NMR metabolic profile with mucosal immunoglobulin production was demonstrated, thus confirming the value of this multi-platform approach in uncovering non-invasive biomarkers of immunity. This has clear potential for translation into human healthcare with the development of urine testing as a means of assessing mucosal immune status. This might lead to early diagnosis of intestinal dysbiosis and with subsequent intervention, arrest disease development. This system enhances our overall understanding of pathologies under supra-organismal control.