Characterization of type I interferon pathway during hepatic differentiation of human pluripotent stem cells and hepatitis C virus infection

Pluripotent stem cells are being actively studied as a cell source for regenerating damaged liver. For long-term survival of engrafting cells in the body, not only do the cells have to execute liver-specific function but also withstand the physical strains and invading pathogens. The cellular innate immune system orchestrated by the interferon (IFN) pathway provides the first line of defense against pathogens. The objective of this study is to assess the innate immune function as well as to systematically profile the IFN-induced genes during hepatic differentiation of pluripotent stem cells. To address this objective, we derived endodermal cells (day 5 post-differentiation), hepatoblast (day 15) and hepatocyte-like cells (day 21) from human embryonic stem cells (hESCs). Day 5, 15 and 21 cells were stimulated with IFN-alpha and subjected to IFN pathway analysis. Transcriptome analysis was carried out by RNA sequencing. The results showed that the IFN-alpha treatment activated STAT-JAK pathway in differentiating cells. Transcriptome analysis indicated stage specific expression of classical and non-classical IFN-stimulated genes (ISGs). Subsequent validation confirmed the expression of novel ISGs including RASGRP3, CLMP and TRANK1 by differentiated hepatic cells upon IFN treatment. Hepatitis C virus replication in hESC-derived hepatic cells induced the expression of ISGs - LAMP3, ETV7, RASGRP3, and TRANK1. The hESC-derived hepatic cells contain intact innate system and can recognize invading pathogens. Besides assessing the tissue-specific functions for cell therapy applications, it may also be important to test the innate immune function of engrafting cells to ensure adequate defense against infections and improve graft survival. (C) 2015 The Authors. Published by Elsevier B.V.

Differential proteomic and genomic profiling of mouse striatal cell model of Huntington's disease and control; probable implications to the disease biology

Huntington's disease (HD) is an autosomal dominant disorder of central nervous system caused by expansion of CAG repeats in exon1 of the huntingtin gene (Htt). Among various dysfunctions originated from the mutation in Htt gene, transcriptional deregulation has been considered to be one of the most important abnormalities. Large numbers of investigations identified altered expressions of genes in brains of HD patients and many models of HD. In this study we employed 2D SDS-PAGE/MALDI-MS coupled with 2D-DIGE and real-time PCR experiments of an array of genes focused to HD pathway to determine altered protein and gene expressions in STHdh(Q111)/Hdh(Q111) cells, a cell model of HD and compared with STHdh(Q7)/Hdh(Q7) cells, its wild type counterpart. We annotated 76 proteins from these cells and observed differential expressions of 31 proteins (by 2D-DIGE) involved in processes like unfolded protein binding, negative regulation of neuron apoptosis, response to superoxides etc. Our PCR array experiments identified altered expressions of 47 genes. Altogether significant alteration of 77 genes/proteins could be identified in this HD cell line with potential relevance to HD biology. Biological significance: In this study we intended to find out differential proteomic and genomic profiles in HD condition. We used the STHdh cells, a cellular model for HD and control. These are mouse striatal neuronal cell lines harboring 7 and 111 knock -in CAG repeats in their two alleles. The 111Q containing cell line (STHdh(Q111)/Hdh(Q111)) mimics diseased condition, whereas the 7Q containing ones (STHdh(Q7)/Hdh(Q7)), serves as the proper control cell line. Proteomic experiments were performed earlier to obtain differential expressions of proteins in R6/2 mice models, Hdh(Q) knock -in mice and in plasma and CSF from HD patients. However, no earlier report on proteomic alterations in these two HD cell lines and control was available in literature. It was, therefore, an important objective to find out differential expressions of proteins in these two cell lines. In this study, we annotated 76 proteins from STHdh(Q7)/Hdh(Q7) and STHdh(Q111)/Hdh(Q111) cells using 2D-gel/mass spectrometry. Next, by performing 2D-DIGE, we observed differential expressions of 31 proteins (16 upregulated and 15 downregulated) between these two cell lines. We also performed customized qRT-PCR array focused to HD pathway and found differential expressions of 47 genes (8 gene exptessions increased and 39 genes were decreased significantly). A total of 77 genes/proteins (Htt downregulated in both the studies) were found to be significantly altered from both the experimental paradigms. We validated the differential expressions of Vim, Hypk, Ran, Dstn, Hspa5 and Sod2 either by qRT-PCR or Western blot analysis or both. Out of these 77, similar trends in alteration of 19 out of 31 and 38 out of 47 proteins/genes were reported in earlier studies. Thus our study confirmed earlier observations on differential gene/protein expressions in HD and are really useful. Additionally, we observed differential expression of some novel genes/proteins. One of this was Hypk, a Htt-interacting chaperone protein with the ability to solubilize mHtt aggregated structures in cell lines. We propose that downregulation of Hypk in STHdh-Qm (Q111)/Hdh(Q111) has a causal effect towards HD pathogenesis. Thus the novel findings from our study need further research and might be helpful to understand the molecular mechanism behind HD pathogenesis. (C) 2015 Elsevier B.V. All rights reserved.

Modelling within-host spatiotemporal dynamics of invasive bacterial disease

Mechanistic determinants of bacterial growth, death, and spread within mammalian hosts cannot be fully resolved studying a single bacterial population. They are also currently poorly understood. Here, we report on the application of sophisticated experimental approaches to map spatiotemporal population dynamics of bacteria during an infection. We analyzed heterogeneous traits of simultaneous infections with tagged Salmonella enterica populations (wild-type isogenic tagged strains [WITS]) in wild-type and gene-targeted mice. WITS are phenotypically identical but can be distinguished and enumerated by quantitative PCR, making it possible, using probabilistic models, to estimate bacterial death rate based on the disappearance of strains through time. This multidisciplinary approach allowed us to establish the timing, relative occurrence, and immune control of key infection parameters in a true host-pathogen combination. Our analyses support a model in which shortly after infection, concomitant death and rapid bacterial replication lead to the establishment of independent bacterial subpopulations in different organs, a process controlled by host antimicrobial mechanisms. Later, decreased microbial mortality leads to an exponential increase in the number of bacteria that spread locally, with subsequent mixing of bacteria between organs via bacteraemia and further stochastic selection. This approach provides us with an unprecedented outlook on the pathogenesis of S. enterica infections, illustrating the complex spatial and stochastic effects that drive an infectious disease. The application of the novel method that we present in appropriate and diverse host-pathogen combinations, together with modelling of the data that result, will facilitate a comprehensive view of the spatial and stochastic nature of within-host dynamics. © 2008 Grant et al.

Apuntes en torno al pensamiento político de Joseph Ratzinger

Un ameno libro de circunstancia sobre la elección de Benedicto XVI, publicado apenas un mes después de esta, me servirá para iniciar mi exposición sobre la persona y labor de Joseph Ratzinger. Allí el autor, Alfredo Urdaci, ensaya una interesante tesis, ¿cómo fue posible tal elección? Porque si bien Ratzinger estaba entre los papables, no era muy probable que realmente fuera elegido. Tenía y –por qué no decirlo– sigue teniendo oposición dentro de la Curia y dentro de algunos sectores de la Iglesia. Y cómo no habría de levantarla un cardenal tan políticamente incorrecto como Ratzinger. No me detengo en sus distintas declaraciones a lo largo de su carrera, pero sí me gustaría presentar un par de frases suyas dichas tan solo días antes de su elección como Papa.

El problema de la razón moderna y el estado de derecho según Joseph Ratzinger

Resumen: El presente texto analiza dos cuestiones centrales en el pensamiento de Joseph Ratzinger: por un lado, el problema de la racionalidad del derecho y, en segundo lugar, el fundamento del Estado de Derecho en el mundo moderno. Para tal fin, previo diagnóstico sobre la situación jurídica contemporánea, se analizan ambos tópicos de acuerdo a los principales discursos realizados por el Cardenal Ratzinger y luego papa Benedicto XVI llegando a la conclusión de que el problema subyacente a ambos es el problema de la verdad tanto metafísica como moral.

Age-dependent decrease in glutamine synthetase expression in the hippocampal astroglia of the triple transgenic Alzheimer's disease mouse model : mechanism for deficient glutamatergic transmission?

Astrocytes are fundamental for brain homeostasis and the progression and outcome of many neuropathologies including Alzheimer's disease (AD). In the triple transgenic mouse model of AD (3xTg-AD) generalised hippocampal astroglia atrophy precedes a restricted and specific beta-amyloid (A beta) plaque-related astrogliosis. Astrocytes are critical for CNS glutamatergic transmission being the principal elements of glutamate homeostasis through maintaining its synthesis, uptake and turnover via glutamate-glutamine shuttle. Glutamine synthetase (GS), which is specifically expressed in astrocytes, forms glutamine by an ATP-dependent amination of glutamate. Here, we report changes in GS astrocytic expression in two major cognitive areas of the hippocampus (the dentate gyrus, DG and the CA1) in 3xTg-AD animals aged between 9 and 18 months. We found a significant reduction in Nv (number of cell/mm(3)) of GS immunoreactive (GS-IR) astrocytes starting from 12 months (28.59%) of age in the DG, and sustained at 18 months (31.65%). CA1 decrease of GS-positive astrocytes Nv (33.26%) occurs at 18 months. This Nv reduction of GSIR astrocytes is paralleled by a decrease in overall GS expression (determined by its optical density) that becomes significant at 18 months (21.61% and 19.68% in DG and CA1, respectively). GS-IR Nv changes are directly associated with the presence of A beta deposits showing a decrease of 47.92% as opposed to 23.47% in areas free of A beta. These changes in GS containing astrocytes and GS-immunoreactivity indicate AD-related impairments of glutamate homeostatic system, at the advanced and late stages of the disease, which may affect the efficacy of glutamatergic transmission in the diseased brain that may contribute to the cognitive deficiency.

Alvará com força de Ley, porque Vossa Magestade he servido prohibir, que se possam carregar, nem transportar escravos pretos de hum, e outro sexo dos pórtos da America, Africa, e Asia, para os destes Reinos de Portugal, e dos Algarves ; applicando as penas nelle declaradas a todos os que contravierem a dita Ley, passado o termo de seis mezes, a respeito dos primeiros, e segundos dos referidos pórtos, e hum anno a respeito dos terceiros : tudo na fórma que acima se contém : para V. Magestade ver / Joaquim Joseph Borralho o fez

Ao alto do título: Conde de Oeyras.

Voz sagrada, política, rhetorica, e metrica ou supplemento a's Vozes Saudosas da eloquencia do espirito, do zelo, e eminente sabedoria do Padre Antonio Vieira da Companhia de Jesus, pregador de S. Magestade, e príncipe dos oradores evangélicos : offerecida ao Senhor Doutor Joseph de Lima Pinheiro e Aragam

Padre Antônio Vieira nasceu em Lisboa em 6 de novembro de 1608 e morreu na Bahia em 18 de julho de 1697. Ordenado sacerdote em 1634, logo se destacou como excepcional pregador. Exerceu importante papel politico e diplomático durante o reinado de D. João IV e fez do púlpito uma tribuna a serviço da nação, em guerra com a Espanha. De 1652 a 1661 atuou como missionário no Maranhão. Em Portugal foi denunciado como herético ao Tribunal da Inquisição. Doente e encarcerado defendeu-se com extraordinário vigor, mas foi condenado a internamento numa casa jesuítica e proibido de pregar. Retirou-se em 1681 para o Brasil, onde trabalhou na edição completa dos Sermoens, iniciada em 1679. Voz sagrada... é um suplemento às Vozes saudosas. Foi publicado, após a morte do autor, por Francisco Luiz Ameno em 1748. Compõe-se de cartas, poemas e outros escritos em latim e português e uma oração fúnebre celebrada pelo Conde da Ericeira nas exéquias do Padre Vieira

Historia de Portugal restaurado, offerecida ao Illustmo e Excellentmo Senhor D. Joseph Mascarenhas, do Conselhor de sua Magestade, seu mordomo mór... : escrita por D. Luiz de Menezes, conde da Ericeira, do Conselho de Estado de Sua Magestade.

Luis de Menezes, terceiro Conde da Ericeira, nasceu em Lisboa em 1632 e suicidou-se em 1690. Foi chamado de Colbert português por ter estimulado a introdução das artes fabris e industriais em Portugal. Escreveu ‘Historia de Portugal restaurado offerecida ao Illustmo e Excellentmo Senhor D. Joseph Mascarenhas’ em duas partes: a primeira, em 1679 e a segunda, em 1698. A mesma historia obteve depois diversas reimpressões, a saber: Parte I, dividida nos tomos I e II, Lisboa, na Oficina de Domingos Rodrigues, em 1751; Parte II, dividida nos tomos III e IV, Lisboa, na Oficina dos herdeiros de Antonio Pedroso Galram, em 1751. Saiu uma terceira vez em Lisboa em 1759. Segundo Inocêncio, ‘compreende esta historia a narração de todos os sucessos militares e políticos ocorridos em Portugal, desde a restauração de 1640 até o ano de 1668, em que se celebraram as pazes com o reino de Castela’

Raridades da natureza e da arte : divididas pelos quatro elementos, escritas, e dedicadas a Magestade Fidelissima de el Rey Nosso Senhor D. Joseph I : por Pedro Norberto de Aucourt e Padilha

Aucourt Padilha nasceu em Lisboa, em 6 de junho de 1704. Cavalheiro da Ordem de Cristo e Secretário da Mesa do Desembargo do Paço escreveu, além desta obra, ‘Memorias historicas, geográficas e politicas, observadas de Paris a Lisboa’, em 1746, e ‘Memorias da Serenissima Senhora D. Isabel Luisa Josepha, que foi jurada princeza d'estes reinos’, em 1748, entre outras. Inocêncio define ‘Raridades da natureza, e da arte’ como ‘livro de muita curiosidade e recreação, para o tempo em que seu autor o publicou’

GFAP Isoforms in Adult Mouse Brain with a Focus on Neurogenic Astrocytes and Reactive Astrogliosis in Mouse Models of Alzheimer Disease

24 p.

Coral Disease and Health Workshop: Coral Histopathology II

The health and continued existence of coral reef ecosystems are threatened by an increasing array of environmental and anthropogenic impacts. Coral disease is one of the prominent causes of increased mortality among reefs globally, particularly in the Caribbean. Although over 40 different coral diseases and syndromes have been reported worldwide, only a few etiological agents have been confirmed; most pathogens remain unknown and the dynamics of disease transmission, pathogenicity and mortality are not understood. Causal relationships have been documented for only a few of the coral diseases, while new syndromes continue to emerge. Extensive field observations by coral biologists have provided substantial documentation of a plethora of new pathologies, but our understanding, however, has been limited to descriptions of gross lesions with names reflecting these observations (e.g., black band, white band, dark spot). To determine etiology, we must equip coral diseases scientists with basic biomedical knowledge and specialized training in areas such as histology, cell biology and pathology. Only through combining descriptive science with mechanistic science and employing the synthesis epizootiology provides will we be able to gain insight into causation and become equipped to handle the pending crisis. One of the critical challenges faced by coral disease researchers is to establish a framework to systematically study coral pathologies drawing from the field of diagnostic medicine and pathology and using generally accepted nomenclature. This process began in April 2004, with a workshop titled Coral Disease and Health Workshop: Developing Diagnostic Criteria co-convened by the Coral Disease and Health Consortium (CDHC), a working group organized under the auspices of the U.S. Coral Reef Task Force, and the International Registry for Coral Pathology (IRCP). The workshop was hosted by the U.S. Geological Survey, National Wildlife Health Center (NWHC) in Madison, Wisconsin and was focused on gross morphology and disease signs observed in the field. A resounding recommendation from the histopathologists participating in the workshop was the urgent need to develop diagnostic criteria that are suitable to move from gross observations to morphological diagnoses based on evaluation of microscopic anatomy. (PDF contains 92 pages)

Preprocess and data analysis techniques for affymetrix DNA microarrays using bioconductor: a case study in Alzheimer disease

DNA microarray, or DNA chip, is a technology that allows us to obtain the expression level of many genes in a single experiment. The fact that numerical expression values can be easily obtained gives us the possibility to use multiple statistical techniques of data analysis. In this project microarray data is obtained from Gene Expression Omnibus, the repository of National Center for Biotechnology Information (NCBI). Then, the noise is removed and data is normalized, also we use hypothesis tests to find the most relevant genes that may be involved in a disease and use machine learning methods like KNN, Random Forest or Kmeans. For performing the analysis we use Bioconductor, packages in R for the analysis of biological data, and we conduct a case study in Alzheimer disease. The complete code can be found in https://github.com/alberto-poncelas/ bioc-alzheimer