A Small Non-Coding RNA Modulates Expression of Pilus-1 Type in Streptococcus pneumoniae.

Streptococcus pneumoniae is a major cause of morbidity and mortality worldwide, and about 30% of the pneumococcal clinical isolates show type I pili-like structures. These long proteinaceous polymers extending from the bacterial surface are encoded by pilus islet 1 and play major roles in adhesion and host colonization. Pili expression is bistable and is controlled by the transcriptional activator RlrA. In this work, we demonstrate that the previously identified small noncoding RNA srn135 also participates in pilus regulation. Our findings show that srn135 is generated upon processing of the 5'-UTR region of rrgA messenger and its deletion prevents the synthesis of RrgA, the main pili adhesin. Moreover, overexpression of srn135 increases the expression of all pili genes and rises the percentage of piliated bacteria within a clonal population. This regulation is mediated by the stabilization of rlrA mRNA since higher levels of srn135 increase its half-life to 165%. Our findings suggest that srn135 has a dual role in pilus expression acting both in cis- (on the RrgA levels) and in trans- (modulating the levels of RlrA) and contributes to the delicate balance between pili expressing and non-expressing bacteria.

Characterization of the canine rostral ventricular-subventricular zone: Morphological, immunohistochemical, ultrastructural, and neurosphere assay studies.

The mammalian ventricular-subventricular zone (V-SVZ) presents the highest neurogenic potential in the brain of the adult individual. In rodents, it is mainly composed of chains of neuroblasts. In humans, it is organized in layers where neuroblasts do not form chains. The aim of this study is to describe the cytoarchitecture of canine V-SVZ (cV-SVZ), to assess its neurogenic potential, and to compare our results with those previously described in other species. We have studied by histology, immunohistochemistry (IHC), electron microscopy and neurosphere assay the morphology, cytoarchitecture and neurogenic potential of cV-SVZ. Age groups of animals were performed. Histological and ultrastructural studies indicated that the cV-SVZ is organized in layers as in humans, but including migratory chains as in rodents. Neural progenitors were organized in niches in the subependymal area and a decline in their number was observed with age. Adult-young dogs contained migratory cells capable to expand and differentiate in vitro according with previous results obtained in rodents, primates, humans, pigs, and dogs. Some adult animals presented perivascular niches outside the V-SVZ. Our observations evidence a great similarity between canine and human V-SVZ indicating that the dog may be better representative of neurogenic events in humans, compared with rodents. Accordingly with our results, we conclude that dogs are a valuable animal model of adult neurogenesis in comparative and preclinical studies.

Helios expression coordinates the development of a subset of striatopallidal medium spiny neurons.

Here, we unravel the mechanism of action of the Ikaros family zinc finger protein Helios (He) during the development of striatal medium spiny neurons (MSNs). He regulates the second wave of striatal neurogenesis involved in the generation of striatopallidal neurons, which express dopamine 2 receptor and enkephalin. To exert this effect, He is expressed in neural progenitor cells (NPCs) keeping them in the G1/G0 phase of the cell cycle. Thus, a lack of He results in an increase of S-phase entry and S-phase length of NPCs, which in turn impairs striatal neurogenesis and produces an accumulation of the number of cycling NPCs in the germinal zone (GZ), which end up dying at postnatal stages. Therefore, He-/- mice show a reduction in the number of dorso-medial striatal MSNs in the adult that produces deficits in motor skills acquisition. In addition, overexpression of He in NPCs induces misexpression of DARPP-32 when transplanted in mouse striatum. These findings demonstrate that He is involved in the correct development of a subset of striatopallidal MSNs and reveal new cellular mechanisms for neuronal development.

Insights in spatio-temporal characterization of human fetal neural stem cells.

Primary human fetal cells have been used in clinical trials of cell replacement therapy for the treatment of neurodegenerative disorders such as Huntington's disease (HD). However, human fetal primary cells are scarce and difficult to work with and so a renewable source of cells is sought. Human fetal neural stem cells (hfNSCs) can be generated from human fetal tissue, but little is known about the differences between hfNSCs obtained from different developmental stages and brain areas. In the present work we characterized hfNSCs, grown as neurospheres, obtained from three developmental stages: 4-5, 6-7 and 8-9weeks post conception (wpc) and four brain areas: forebrain, cortex, whole ganglionic eminence (WGE) and cerebellum. We observed that, as fetal brain development proceeds, the number of neural precursors is diminished and post-mitotic cells are increased. In turn, primary cells obtained from older embryos are more sensitive to the dissociation process, their viability is diminished and they present lower proliferation ratios compared to younger embryos. However, independently of the developmental stage of derivation proliferation ratios were very low in all cases. Improvements in the expansion rates were achieved by mechanical, instead of enzymatic, dissociation of neurospheres but not by changes in the seeding densities. Regardless of the developmental stage, neurosphere cultures presented large variability in the viability and proliferation rates during the initial 3-4 passages, but stabilized achieving significant expansion rates at passage 5 to 6. This was true also for all brain regions except cerebellar derived cultures that did not expand. Interestingly, the brain region of hfNSC derivation influences the expansion potential, being forebrain, cortex and WGE derived cells the most expandable compared to cerebellar. Short term expansion partially compromised the regional identity of cortical but not WGE cultures. Nevertheless, both expanded cultures were multipotent and kept the ability to differentiate to region specific mature neuronal phenotypes.

Development of a transplantable glioma tumour model from genetically engineered mice: MRI/MRS/MRSI characterisation.

The initial aim of this study was to generate a transplantable glial tumour model of low-intermediate grade by disaggregation of a spontaneous tumour mass from genetically engineered models (GEM). This should result in an increased tumour incidence in comparison to GEM animals. An anaplastic oligoastrocytoma (OA) tumour of World Health Organization (WHO) grade III was obtained from a female GEM mouse with the S100ß-v-erbB/inK4a-Arf (+/-) genotype maintained in the C57BL/6 background. The tumour tissue was disaggregated; tumour cells from it were grown in aggregates and stereotactically injected into C57BL/6 mice. Tumour development was followed using Magnetic Resonance Imaging (MRI), while changes in the metabolomics pattern of the masses were evaluated by Magnetic Resonance Spectroscopy/Spectroscopic Imaging (MRS/MRSI). Final tumour grade was evaluated by histopathological analysis. The total number of tumours generated from GEM cells from disaggregated tumour (CDT) was 67 with up to 100 % penetrance, as compared to 16 % in the local GEM model, with an average survival time of 66 ± 55 days, up to 4.3-fold significantly higher than the standard GL261 glioblastoma (GBM) tumour model. Tumours produced by transplantation of cells freshly obtained from disaggregated GEM tumour were diagnosed as WHO grade III anaplastic oligodendroglioma (ODG) and OA, while tumours produced from a previously frozen sample were diagnosed as WHO grade IV GBM. We successfully grew CDT and generated tumours from a grade III GEM glial tumour. Freezing and cell culture protocols produced progression to grade IV GBM, which makes the developed transplantable model qualify as potential secondary GBM model in mice.

Small regulatory RNAs in Streptococcus pneumoniae: discovery and biological functions.

Streptococcus pneumoniae is a prominent human pathogen responsible for many severe diseases and the leading cause of childhood mortality worldwide. The pneumococcus is remarkably adept at colonizing and infecting different niches in the human body, and its adaptation to dynamic host environment is a central aspect of its pathogenesis. In the last decade, increasing findings have evidenced small RNAs (sRNAs) as vital regulators in a number of important processes in bacteria. In S. pneumoniae, a small antisense RNA was first discovered in the pMV158 plasmid as a copy number regulator. More recently, genome-wide screens revealed that the pneumococcal genome also encodes multiple sRNAs, many of which have important roles in virulence while some are implicated in competence control. The knowledge of the sRNA-mediated regulation in pneumococcus remains very limited, and future research is needed for better understanding of functions and mechanisms. Here, we provide a comprehensive summary of the current knowledge on sRNAs from S. pneumoniae, focusing mainly on the trans-encoded sRNAs.

[Usage analysis of the Nursing Activities Score in two Spanish ICUS]. / Análisis de la utilización de la escala Nursing Activities Score en dos UCIS Españolas.

The aim of this study was to analyze the discourse of health managers on aspects related to delay in tuberculosis diagnosis. This was a qualitative research study, conducted with 16 Family Health Unit managers. The empirical data were obtained through semi-structured interviews. The analysis was based on the theoretical framework of the French school of discourse analysis. According to the managers' statements, the delay in tuberculosis diagnosis is related to patient and health service aspects. As for patient aspects, managers report fear, prejudice and lack of information as factors that may promote a delayed diagnosis. Regarding health service aspects, structural problems and lack of professional skills were reported. The discourse of managers should be considered to qualify tuberculosis control actions and to prevent delays in diagnosis.

Usage analysis of the Nursing Activities Score in two Spanish ICUS / Análise da utilização da escala Nursing Activities Score em duas UCIS Espanholas / Análisis de la utilización de la escala Nursing Activities Score en dos UCIS Españolas

The aim of this study was to analyze the discourse of health managers on aspects related to delay in tuberculosis diagnosis. This was a qualitative research study, conducted with 16 Family Health Unit managers. The empirical data were obtained through semi-structured interviews. The analysis was based on the theoretical framework of the French school of discourse analysis. According to the managers’ statements, the delay in tuberculosis diagnosis is related to patient and health service aspects. As for patient aspects, managers report fear, prejudice and lack of information as factors that may promote a delayed diagnosis. Regarding health service aspects, structural problems and lack of professional skills were reported. The discourse of managers should be considered to qualify tuberculosis control actions and to prevent delays in diagnosis. .

Estudo prospetivo cujo objetivo foi analisar as diferenças no preenchimento da escala Nursing Activities Score (NAS) em duas UTI polivalentes de dois hospitais espanhóis. Dados relativos internados nas unidades durante o período de outubro a novembro de 2011. Os dados recolhidos de 103 pacientes produziram 941 medições na escala NAS. Diferenças significativas foram encontradas nos itens: monitoramento, procedimentos de higiene, mobilização e posicionamento, atividades administrativas e monitoramento auricular à esquerda (p < 0,001). Conclui-se que o uso de instrumentos padronizados é essencial quando se compara a carga de trabalho em unidades diferentes. A escala apresenta itens com uma componente de avaliação subjetiva, sendo por isso importante a unificação de critérios para a comparação de resultados entre diferentes unidades.

Estudio prospectivo cuyo objetivo fue analizar las diferencias en el llenado de la escala Nursing Activities Score (NAS) en dos UCIs polivalentes de dos hospitales españoles. Datos relacionados a la carga de trabajo se recogieron diariamente, mediante la escala para los pacientes internados en las unidades durante el periodo de octubre a noviembre del 2011. Se recogieron datos de 103 pacientes obteniéndose un total de 941 medidas de la escala NAS. Diferencias significativas se encontraron en los ítems: monitorización, procedimientos de higiene, movilización y posición, tareas administrativas y monitorización de la aurícula izquierda (p < 0.001). Se concluyó que el empleo de instrumentos estandarizados es fundamental para poder comparar la carga de trabajo en diferentes unidades. La escala presenta ítems con un componente de valoración subjetiva, siendo importante la unificación de criterios para poder comparar los resultados entre las distintas unidades.

Evaluation of the nursing workload through the Nine Equivalents for Nursing Manpower Use Scale and the Nursing Activities Score: a prospective correlation study.

OBJECTIVE: To determine the relationship between nursing workload measured through the nine equivalents of nursing manpower use (NEMS) scale and that measured through the nursing activities score (NAS) scale and to analyse staff needs as determined through each of the scales. METHODS: The study used a descriptive prospective correlational design to collect data between October 2007 and July 2009. Nursing workload data for 730 ICU patients were collected daily using the NAS and NEMS scales. Both scales were then correlated and used to estimate staff needs. FINDINGS: 6815 score pairs were collected, which reflected the nursing workload for each patient as calculated daily using both scales. Pearson's correlation coefficient for individual measurements obtained through the NAS and the NEMS corresponded to .672, and to .932 for the daily total workload in the unit. The staffing requirements based on the NAS scale scores were significantly higher than those based on the NEMS scale. A high correlation existed for individual measurements using both scales and for the total workload measurement in the unit. The main difference was found when analysing staffing requirements, with higher staff numbers needed for the NAS scale. CONCLUSION: Both NAS and NEMS can be used to measure the nursing workload in the ICU. Staffing requirements using NAS were higher than those using NEMS.

[Assessment of nursing workload in three groups of patients in a Spanish ICU using the Nursing Activities Score Scale]. / Carga de trabajo en tres grupos de pacientes de UCI Española según el Nursing Activities Score.

The purpose of this study was to assess the nursing workload at admission to and discharge from intensive care of three groups of patients (i.e., acute coronary syndrome, acute respiratory failure, and sepsis). A prospective, descriptive study was performed over a 27-month period and included 563 patients. The workload was assessed using the Nursing Activities Score scale. Significant differences in the workload were determined on the days of admission and discharge: the workload was higher in both cases for patients with acute respiratory failure and sepsis compared with patients diagnosed with acute coronary syndrome. This difference was maintained over the first seven days of their hospital stay. From day 8 on, the difference disappeared, and a workload balance was achieved in the three groups. Good staffing requires adequate tools for measuring care needs and understanding the workload required in the groups of patients who are most frequently admitted to intensive care.

Carga de trabajo en tres grupos de pacientes de UCI Española según el Nursing Activities Score / Carga de trabalho em três grupos de pacientes em uma UTI espanhola segundo Nursing Activites Score / Assessment of nursing workload in three groups of patients in a Spanish ICU using the Nursing Activities Score Scale

Se objetivó valorizar la carga de trabajo al ingreso y al alta en tres grupos de pacientes (síndrome coronario agudo, insuficiencia respiratoria aguda y sepsis) en terapia intensiva. Estudio descriptivo, prospectivo, de 27 meses, incluyéndose 563 pacientes, valorando carga de trabajo según Nursing Activities Score. Existieron diferencias significativas en la carga de trabajo al ingreso y en el alta entre los grupos de pacientes, siendo superior en ambos momentos la de pacientes con insuficiencia respiratoria aguda y sepsis frente a pacientes coronarios. Durante los siete primeros días de estancia se mantuvo esta diferencia, desapareciendo a partir del octavo día, equilibrándose la carga de trabajo para los tres grupos. Para conseguir una adecuada dotación de personal es fundamental contar con instrumentos para medir las necesidades de cuidados y conocer la carga de trabajo de los distintos grupos de enfermos que ingresan con mayor frecuencia en las unidades de terapia intensiva.

O objetivo deste estudo foi avaliar a carga de trabalho na admissão e alta dos pacientes de três grupos (síndrome coronária aguda, insuficiência respiratória aguda e sepsis) em cuidados intensivos. Trata-se de estudo prospectivo, descritivo, que decorreu durante 27 meses, incluindo 563 pacientes. Para a avaliação da carga de trabalho utilizou-se a escala Nursing Activities Score. A partir dos resultados do estudo parecem existir diferenças significativas na carga de trabalho no dia da admissão e alta entre os grupos de pacientes, sendo a carga maior em ambos os tempos a dos pacientes com insuficiência respiratória aguda e sepsis. Durante os primeiros sete dias de internamento essa diferença manteve-se, desaparecendo no oitavo dia, o que equilibrou a carga de trabalho para os três grupos. Conclui-se que para se conseguir os recursos adequados é essencial dispor de instrumentos para medir as necessidades de cuidados e conhecer a carga de trabalho dos diferentes grupos de pacientes que passam com mais frequência pelas unidades de cuidados intensivos.

The purpose of this study was to assess the nursing workload at admission to and discharge from intensive care of three groups of patients (i.e., acute coronary syndrome, acute respiratory failure, and sepsis). A prospective, descriptive study was performed over a 27-month period and included 563 patients. The workload was assessed using the Nursing Activities Score scale. Significant differences in the workload were determined on the days of admission and discharge: the workload was higher in both cases for patients with acute respiratory failure and sepsis compared with patients diagnosed with acute coronary syndrome. This difference was maintained over the first seven days of their hospital stay. From day 8 on, the difference disappeared, and a workload balance was achieved in the three groups. Good staffing requires adequate tools for measuring care needs and understanding the workload required in the groups of patients who are most frequently admitted to intensive care.

Reduced innate immune response, apoptosis, and virus release in cells cured of respiratory syncytial virus persistent infection.

It has been reported that cell clones isolated at different passages from a culture of HEp-2 cells infected persistently with human respiratory syncytial virus (HRSV) were cured of the virus. Further studies on one of these clones (31C1) are reported here, showing that 31C1 cells can still be infected by HRSV but release low amounts of virus to the culture supernatant, develop smaller and less numerous syncytia than the original HEp-2 cells, and display only a weak innate immune response to the infection. Accordingly, uninfected 31C1 cells, but not clones derived from uninfected HEp-2 cells, express low levels of TLR3 and RIG-I. In addition, 31C1 cells are partly resistant to apoptosis. These results indicate that persistent infection of HEp-2 cells by HRSV has selected cell variants, with changes affecting cell survival, virus growth and the innate immune response that may be valuable for studies of virus-cell interaction.

Nolz1 promotes striatal neurogenesis through the regulation of retinoic acid signaling.

BACKGROUND: Nolz1 is a zinc finger transcription factor whose expression is enriched in the lateral ganglionic eminence (LGE), although its function is still unknown. RESULTS: Here we analyze the role of Nolz1 during LGE development. We show that Nolz1 expression is high in proliferating neural progenitor cells (NPCs) of the LGE subventricular zone. In addition, low levels of Nolz1 are detected in the mantle zone, as well as in the adult striatum. Similarly, Nolz1 is highly expressed in proliferating LGE-derived NPC cultures, but its levels rapidly decrease upon cell differentiation, pointing to a role of Nolz1 in the control of NPC proliferation and/or differentiation. In agreement with this hypothesis, we find that Nolz1 over-expression promotes cell cycle exit of NPCs in neurosphere cultures and negatively regulates proliferation in telencephalic organotypic cultures. Within LGE primary cultures, Nolz1 over-expression promotes the acquisition of a neuronal phenotype, since it increases the number of ß-III tubulin (Tuj1)- and microtubule-associated protein (MAP)2-positive neurons, and inhibits astrocyte generation and/or differentiation. Retinoic acid (RA) is one of the most important morphogens involved in striatal neurogenesis, and regulates Nolz1 expression in different systems. Here we show that Nolz1 also responds to this morphogen in E12.5 LGE-derived cell cultures. However, Nolz1 expression is not regulated by RA in E14.5 LGE-derived cell cultures, nor is it affected during LGE development in mouse models that present decreased RA levels. Interestingly, we find that Gsx2, which is necessary for normal RA signaling during LGE development, is also required for Nolz1 expression, which is lost in Gsx2 knockout mice. These findings suggest that Nolz1 might act downstream of Gsx2 to regulate RA-induced neurogenesis. Keeping with this hypothesis, we show that Nolz1 induces the selective expression of the RA receptor (RAR)ß without altering RARα or RARγ. In addition, Nozl1 over-expression increases RA signaling since it stimulates the RA response element. This RA signaling is essential for Nolz1-induced neurogenesis, which is impaired in a RA-free environment or in the presence of a RAR inverse agonist. It has been proposed that Drosophila Gsx2 and Nolz1 homologues could cooperate with the transcriptional co-repressors Groucho-TLE to regulate cell proliferation. In agreement with this view, we show that Nolz1 could act in collaboration with TLE-4, as they are expressed at the same time in NPC cultures and during mouse development. CONCLUSIONS: Nolz1 promotes RA signaling in the LGE, contributing to the striatal neurogenesis during development.

Ikaros-1 couples cell cycle arrest of late striatal precursors with neurogenesis of enkephalinergic neurons.

During central nervous system development, several transcription factors regulate the differentiation of progenitor cells to postmitotic neurons. Here we describe a novel role for Ikaros-1 in the generation of late-born striatal neurons. Our results show that Ikaros-1 is expressed in the boundary of the striatal germinal zone (GZ)/mantle zone (MZ), where it induces cell cycle arrest of neural progenitors by up-regulation of the cyclin-dependent kinase inhibitor (CDKi) p21(Cip1/Waf1). This effect is coupled with the neuronal differentiation of late precursors, which in turn is critical for the second wave of striatal neurogenesis that gives rise to matrix neurons. Consistently, Ikaros(-/-) mice had fewer striatal projecting neurons and, in particular, enkephalin (ENK)-positive neurons. In addition, overexpression of Ikaros-1 in primary striatal cultures increases the number of calbindin- and ENK-positive neurons. Our results also show that Ikaros-1 acts downstream of the Dlx family of transcription factors, insofar as its expression is lost in Dlx1/2 double knockout mice. However, we demonstrate that Ikaros-1 and Ebf-1 independently regulate the final determination of the two populations of striatal projection neurons of the matrix compartment, ENK- and substance P-positive neurons. In conclusion, our findings identify Ikaros-1 as a modulator of cell cycle exit of neural progenitors that gives rise to the neurogenesis of ENK-positive striatal neurons.

Cultures of HEp-2 cells persistently infected by human respiratory syncytial virus differ in chemokine expression and resistance to apoptosis as compared to lytic infections of the same cell type.

HEp-2 cells that survived a lytic infection with Human Respiratory Syncytial Virus (HRSV) were grown to obtain a persistently infected culture that produced relatively high amounts of virus (10(6)-10(7) pfu/ml) for more than twenty passages. The cells in this culture were heterogeneous with regard to the expression of viral antigens, ranging from high to undetectable levels. However, all cell clones derived from the persistent culture did not produce infectious virus or viral antigens and grew more slowly than the original uninfected HEp-2 cells. When these "cured" cell clones were infected with wild-type HRSV, delayed virus production and reduction in the number and size of syncytia were observed compared to lytically infected HEp-2 cells. Most significantly, differences in gene expression between persistently and lytically infected cultures were also observed, including genes that encode for cytokines, chemokines and other gene products that either promote cell survival or inhibit apoptosis. These results highlight the significantly different responses of the same cell type to HRSV infection depending on the outcome of such infection, i.e., lytic versus persistent.

Germ cell restricted expression of chick Nanog.

Nanog is a pluripotency-associated factor expressed in embryonic stem cells and in the epiblast and primordial germ cells of the mouse embryo. We have identified the chick orthologue of Nanog and found that its expression is limited to primordial germ cells in the early embryo and is not found throughout the epiblast. Genomic analysis has shown that Nanog is an amniote-specific gene and is absent from anamniotes and invertebrates. Furthermore, other pluripotency associated genes that are located in close proximity to Nanog in human and mouse are absent from the chick genome. Such observations lead to a scenario of sequential addition of novel genes to a genomic region associated to pluripotency. These results have profound implications for the study of the evolution of pluripotent lineages in the embryo and of vertebrate stem cells.