Coexpression of factor VIII heavy and light chain adeno-associated viral vectors produces biologically active protein

We are interested in using recombinant adeno-associated viral vectors in the treatment of hemophilia A. Because of the size constraints of recombinant adeno-associated viral vectors, we delivered the heavy and light chains of the human factor 8 (hFVIII) cDNA independently by using two separate vectors. Recombinant AAV vectors were constructed that utilized the human elongation factor 1α promoter, a human growth factor polyadenylation signal, and the cDNA sequences encoding either the heavy or light chain of hFVIII. Portal vein injections of each vector alone, a combination of both vectors, or a hFIX control vector were performed in C57BL/6 mice. An ELISA specific for the light chain of hFVIII demonstrated very high levels (2–10 μg/ml) of protein expression in animals injected with the light chain vector alone or with both vectors. We utilized a chromogenic assay in combination with an antibody specific to hFVIII to determine the amount of biologically active hFVIII in mouse plasma. In animals injected with both the heavy and light chain vectors, greater than physiological levels (200–400 ng/ml) of biologically active hFVIII were produced. This suggests that coexpression of the heavy and light chains of hFVIII may be a feasible approach for treatment of hemophilia A.

Parkin functions as an E2-dependent ubiquitin– protein ligase and promotes the degradation of the synaptic vesicle-associated protein, CDCrel-1

Parkinson's disease is a common neurodegenerative disorder in which familial-linked genes have provided novel insights into the pathogenesis of this disorder. Mutations in Parkin, a ring-finger-containing protein of unknown function, are implicated in the pathogenesis of autosomal recessive familial Parkinson's disease. Here, we show that Parkin binds to the E2 ubiquitin-conjugating human enzyme 8 (UbcH8) through its C-terminal ring-finger. Parkin has ubiquitin–protein ligase activity in the presence of UbcH8. Parkin also ubiquitinates itself and promotes its own degradation. We also identify and show that the synaptic vesicle-associated protein, CDCrel-1, interacts with Parkin through its ring-finger domains. Furthermore, Parkin ubiquitinates and promotes the degradation of CDCrel-1. Familial-linked mutations disrupt the ubiquitin–protein ligase function of Parkin and impair Parkin and CDCrel-1 degradation. These results suggest that Parkin functions as an E3 ubiquitin–protein ligase through its ring domains and that it may control protein levels via ubiquitination. The loss of Parkin's ubiquitin–protein ligase function in familial-linked mutations suggests that this may be the cause of familial autosomal recessive Parkinson's disease.

Regulation of the stem cell leukemia (SCL) gene: A tale of two fishes

The stem cell leukemia (SCL) gene encodes a tissue-specific basic helix–loop–helix (bHLH) protein with a pivotal role in hemopoiesis and vasculogenesis. Several enhancers have been identified within the murine SCL locus that direct reporter gene expression to subdomains of the normal SCL expression pattern, and long-range sequence comparisons of the human and murine SCL loci have identified additional candidate enhancers. To facilitate the characterization of regulatory elements, we have sequenced and analyzed 33 kb of the SCL genomic locus from the pufferfish Fugu rubripes, a species with a highly compact genome. Although the pattern of SCL expression is highly conserved from mammals to teleost fish, the genes flanking pufferfish SCL were unrelated to those known to flank both avian and mammalian SCL genes. These data suggest that SCL regulatory elements are confined to the region between the upstream and downstream flanking genes, a region of 65 kb in human and 8.5 kb in pufferfish. Consistent with this hypothesis, the entire 33-kb pufferfish SCL locus directed appropriate expression to hemopoietic and neural tissue in transgenic zebrafish embryos, as did a 10.4-kb fragment containing the SCL gene and extending to the 5′ and 3′ flanking genes. These results demonstrate the power of combining the compact genome of the pufferfish with the advantages that zebrafish provide for studies of gene regulation during development. Furthermore, the pufferfish SCL locus provides a powerful tool for the manipulation of hemopoiesis and vasculogenesis in vivo.

Epstein-Barr viral latency is disrupted by the immediate-early BRLF1 protein through a cell-specific mechanism.

Epstein-Barr virus (EBV), the causative agent of infectious mononucleosis, is a human herpesvirus associated with epithelial cell malignancies (nasopharyngeal carcinoma) as well as B-cell malignancies. Understanding how viral latency is disrupted is a central issue in herpesvirus biology. Epithelial cells are the major site of lytic EBV replication within the human host, and viral reactivation occurs in EBV-associated nasopharyngeal carcinomas. It is known that expression of a single viral immediate-early protein, BZLF1, is sufficient to initiate the switch from latent to lytic infection in B cells. Cellular regulation of BZLF1 transcription is therefore thought to play a key role in regulating the stringency of viral latency. Here we show that, unexpectedly, expression of another viral immediate-early protein, BRLF1, can disrupt viral latency in an epithelial cell-specific fashion. Therefore, the mechanisms leading to disruption of EBV latency appear to be cell-type specific.

Structure, tissue distribution, and chromosomal localization of the prepronociceptin gene.

Nociceptin (orphanin FQ), the newly discovered natural agonist of opioid receptor-like (ORL1) receptor, is a neuropeptide that is endowed with pronociceptive activity in vivo. Nociceptin is derived from a larger precursor, prepronociceptin (PPNOC), whose human, mouse, and rat genes we have now isolated. The PPNOC gene is highly conserved in the three species and displays organizational features that are strikingly similar to those of the genes of preproenkephalin, preprodynorphin, and preproopiomelanocortin, the precursors to endogenous opioid peptides, suggesting the four genes belong to the same family-i.e., have a common evolutionary origin. The PPNOC gene encodes a single copy of nociceptin as well as of other peptides whose sequence is strictly conserved across murine and human species; hence it is likely to be neurophysiologically significant. Northern blot analysis shows that the PPNOC gene is predominantly transcribed in the central nervous system (brain and spinal cord) and, albeit weakly, in the ovary, the sole peripheral organ expressing the gene. By using a radiation hybrid cell line panel, the PPNOC gene was mapped to the short arm of human chromosome 8 (8p21), between sequence-tagged site markers WI-5833 and WI-1172, in close proximity of the locus encoding the neurofilament light chain NEFL. Analysis of yeast artificial chromosome clones belonging to the WC8.4 contig covering the 8p21 region did not allow to detect the presence of the gene on these yeast artificial chromosomes, suggesting a gap in the coverage within this contig.

kappa-Opioid receptor in humans: cDNA and genomic cloning, chromosomal assignment, functional expression, pharmacology, and expression pattern in the central nervous system.

Using the mouse delta-opioid receptor cDNA as a probe, we have isolated genomic clones encoding the human mu- and kappa-opioid receptor genes. Their organization appears similar to that of the human delta receptor gene, with exon-intron boundaries located after putative transmembrane domains 1 and 4. The kappa gene was mapped at position q11-12 in human chromosome 8. A full-length cDNA encoding the human kappa-opioid receptor has been isolated. The cloned receptor expressed in COS cells presents a typical kappa 1 pharmacological profile and is negatively coupled to adenylate cyclase. The expression of kappa-opioid receptor mRNA in human brain, as estimated by reverse transcription-polymerase chain reaction, is consistent with the involvement of kappa-opioid receptors in pain perception, neuroendocrine physiology, affective behavior, and cognition. In situ hybridization studies performed on human fetal spinal cord demonstrate the presence of the transcript specifically in lamina II of the dorsal horn. Some divergences in structural, pharmacological, and anatomical properties are noted between the cloned human and rodent receptors.

Loss of the catalytic subunit of the DNA-dependent protein kinase in DNA double-strand-break-repair mutant mammalian cells.

The DNA-dependent protein kinase (DNA-PK) consists of three polypeptide components: Ku-70, Ku-80, and an approximately 350-kDa catalytic subunit (p350). The gene encoding the Ku-80 subunit is identical to the x-ray-sensitive group 5 complementing gene XRCC5. Expression of the Ku-80 cDNA rescues both DNA double-strand break (DSB) repair and V(D)J recombination in group 5 mutant cells. The involvement of Ku-80 in these processes suggests that the underlying defect in these mutant cells may be disruption of the DNA-PK holoenzyme. In this report we show that the p350 kinase subunit is deleted in cells derived from the severe combined immunodeficiency mouse and in the Chinese hamster ovary cell line V-3, both of which are defective in DSB repair and V(D)J recombination. A centromeric fragment of human chromosome 8 that complements the scid defect also restores p350 protein expression and rescues in vitro DNA-PK activity. These data suggest the scid gene may encode the p350 protein or regulate its expression and are consistent with a model whereby DNA-PK is a critical component of the DSB-repair pathway.

O EFEITO DO CONTROLE RESPIRATÓRIO EM VARIÁVEIS ELETROFISIOLÓGICAS DA ATENÇÃO

A prática do ioga tem se tornado cada vez mais popular, não apenas pelos benefícios físicos, mas principalmente pelo bem-estar psicológico trazido pela sua prática. Um dos componentes do ioga é o Prãnãyama, ou controle da respiração. A atenção e a respiração são dois mecanismos fisiológicos e involuntários requeridos para a execução do Prãnãyama. O principal objetivo desse estudo foi verificar se variáveis contínuas do EEG (potência de diferentes faixas que o compõem) seriam moduladas pelo controle respiratório, comparando-se separadamente as duas fases do ciclo respiratório (inspiração e expiração), na situação de respiração espontânea e controlada. Fizeram parte do estudo 19 sujeitos (7 homens/12 mulheres, idade média de 36,89 e DP = ± 14,46) que foram convidados a participar da pesquisa nas dependências da Faculdade de Saúde da Universidade Metodista de São Paulo. Para o registro do eletroencefalograma foi utilizado um sistema de posicionamento de cinco eletrodos Ag AgCl (FPz, Fz, Cz, Pz e Oz) fixados a uma touca de posicionamento rápido (Quick-Cap, Neuromedical Supplies®), em sistema 10-20. Foram obtidos valores de máxima amplitude de potência (espectro de potência no domínio da frequência) nas frequências teta, alfa e beta e delta e calculada a razão teta/beta nas diferentes fases do ciclo respiratório (inspiração e expiração), separadamente, nas condições de respiração espontânea e de controle respiratório. Para o registro do ciclo respiratório, foi utilizada uma cinta de esforço respiratório M01 (Pletismógrafo). Os resultados mostram diferenças significativas entre as condições de respiração espontânea e de controle com valores das médias da razão teta/beta menores na respiração controlada do que na respiração espontânea e valores de média da potência alfa sempre maiores no controle respiratório. Diferenças significativas foram encontradas na comparação entre inspiração e expiração da respiração controlada com diminuição dos valores das médias da razão teta/beta na inspiração e aumento nos valores das médias da potência alfa, sobretudo na expiração. Os achados deste estudo trazem evidências de que o controle respiratório modula variáveis eletrofisiológicas relativas à atenção refletindo um estado de alerta, porém mais relaxado do que na situação de respiração espontânea.

Moderate relationships between NAA and cognitive ability in healthy adults:implications for cognitive spectroscopy

Background: Proton Magnetic Resonance Spectroscopy (H-MRS) is a non-invasive imaging technique that enables quantification of neurochemistry in vivo and thereby facilitates investigation of the biochemical underpinnings of human cognitive variability. Studies in the field of cognitive spectroscopy have commonly focused on relationships between measures of N-acetyl aspartate (NAA), a surrogate marker of neuronal health and function, and broad measures of cognitive performance, such as IQ. Methodology/Principal Findings: In this study, we used H-MRS to interrogate single-voxels in occipitoparietal and frontal cortex, in parallel with assessments of psychometric intelligence, in a sample of 40 healthy adult participants. We found correlations between NAA and IQ that were within the range reported in previous studies. However, the magnitude of these effects was significantly modulated by the stringency of data screening and the extent to which outlying values contributed to statistical analyses. Conclusions/Significance: H-MRS offers a sensitive tool for assessing neurochemistry non-invasively, yet the relationships between brain metabolites and broad aspects of human behavior such as IQ are subtle. We highlight the need to develop an increasingly rigorous analytical and interpretive framework for collecting and reporting data obtained from cognitive spectroscopy studies of this kind. © 2014 Patel, Blyth, Griffiths, Kelly and Talcott.

Hypnotic induction is followed by state-like changes in the organization of EEG functional connectivity in the theta and beta frequency bands in high-hypnotically susceptible individuals

Altered state theories of hypnosis posit that a qualitatively distinct state of mental processing, which emerges in those with high hypnotic susceptibility following a hypnotic induction, enables the generation of anomalous experiences in response to specific hypnotic suggestions. If so then such a state should be observable as a discrete pattern of changes to functional connectivity (shared information) between brain regions following a hypnotic induction in high but not low hypnotically susceptible participants. Twenty-eight channel EEG was recorded from 12 high susceptible (highs) and 11 low susceptible (lows) participants with their eyes closed prior to and following a standard hypnotic induction. The EEG was used to provide a measure of functional connectivity using both coherence (COH) and the imaginary component of coherence (iCOH), which is insensitive to the effects of volume conduction. COH and iCOH were calculated between all electrode pairs for the frequency bands: delta (0.1-3.9 Hz), theta (4-7.9 Hz) alpha (8-12.9 Hz), beta1 (13-19.9 Hz), beta2 (20-29.9 Hz) and gamma (30-45 Hz). The results showed that there was an increase in theta iCOH from the pre-hypnosis to hypnosis condition in highs but not lows with a large proportion of significant links being focused on a central-parietal hub. There was also a decrease in beta1 iCOH from the pre-hypnosis to hypnosis condition with a focus on a fronto-central and an occipital hub that was greater in high compared to low susceptibles. There were no significant differences for COH or for spectral band amplitude in any frequency band. The results are interpreted as indicating that the hypnotic induction elicited a qualitative change in the organization of specific control systems within the brain for high as compared to low susceptible participants. This change in the functional organization of neural networks is a plausible indicator of the much theorized "hypnotic-state". © 2014 Jamieson and Burgess.

Recommendations for sex/gender neuroimaging research:key principles and implications for research design, analysis and interpretation

Neuroimaging (NI) technologies are having increasing impact in the study of complex cognitive and social processes. In this emerging field of social cognitive neuroscience, a central goal should be to increase the understanding of the interaction between the neurobiology of the individual and the environment in which humans develop and function. The study of sex/gender is often a focus for NI research, and may be motivated by a desire to better understand general developmental principles, mental health problems that show female-male disparities, and gendered differences in society. In order to ensure the maximum possible contribution of NI research to these goals, we draw attention to four key principles—overlap, mosaicism, contingency and entanglement—that have emerged from sex/gender research and that should inform NI research design, analysis and interpretation. We discuss the implications of these principles in the form of constructive guidelines and suggestions for researchers, editors, reviewers and science communicators.

Redefining neuromarketing as an integrated science of influence

Multiple transformative forces target marketing, many of which derive from new technologies that allow us to sample thinking in real time (i.e., brain imaging), or to look at large aggregations of decisions (i.e., big data). There has been an inclination to refer to the intersection of these technologies with the general topic of marketing as “neuromarketing”. There has not been a serious effort to frame neuromarketing, which is the goal of this paper. Neuromarketing can be compared to neuroeconomics, wherein neuroeconomics is generally focused on how individuals make “choices”, and represent distributions of choices. Neuromarketing, in contrast, focuses on how a distribution of choices can be shifted or “influenced”, which can occur at multiple “scales” of behavior (e.g., individual, group, or market/society). Given influence can affect choice through many cognitive modalities, and not just that of valuation of choice options, a science of influence also implies a need to develop a model of cognitive function integrating attention, memory, and reward/aversion function. The paper concludes with a brief description of three domains of neuromarketing application for studying influence, and their caveats.

Preferencias, dedicación y problemáticas generadas por los videojuegos: Una perspectiva de género

Entre las actividades de ocio de los/as jóvenes, cabe mencionar las asociadas con los videojuegos. Por ello, la finalidad central de este estudio es analizar las preferencias del colectivo de adolescentes con los videojuegos, así como conocer su dedicación y problemáticas que generan, teniendo en cuenta principalmente la variable de género. El presente trabajo se encuadra en un enfoque metodológico cuantitativo-cualitativo, a partir de la recogida de datos con un cuestionario y la técnica del grupo de discusión. En el estudio han participado un total de 151 adolescentes. 124 a partir de la aplicación de un cuestionario cerrado (62 hombres y 62 mujeres) y 27 a través de la realización de 6 grupos de discusión (17 hombres y 10 mujeres). Como resultados y conclusiones, cabe destacar la existencia diferenciada en el uso de los videojuegos en función de la edad y por razón de género. A su vez, las mujeres muestran una responsabilidad mayor en esta actividad, tanto en el tiempo de dedicación como en la elección. Los chicos destinan una gran cantidad de tiempo a los videojuegos de contenido violento, mientras las chicas prefieren los de estrategia. En general, el juego con videojuegos no genera problemáticas sustanciales y controversias, aunque algunos participantes reflejan manifestaciones de mal humor. Por último, cabe indicar que las prácticas con los videojuegos en la adolescencia se ciñen al mero hecho de divertirse, desperdiciando las posibilidades educativas que esta actividad ofrece.

Cytomegalovirus and Langerhans Cell Histiocytosis: Is There a Link?

Background: Langerhans cell histiocytosis is a rare proliferative histiocytic disease of unknown etiology. Histologically, it is characterized by granuloma-like proliferation of Langerhans-type dendritic cells derived from bone marrow. Many investigators have suggested the possible role of viruses such as Epstein-Barr virus, human herpesvirus-6 (HHV-6), herpes simplex virus (HSV) types 1 and 2, and Cytomegalovirus in the pathogenesis of Langerhans cell histiocytosis. Objectives: In this study, we have investigated the presence of Cytomegalovirus in Langerhans cell histiocytosis in Iranian children. Patients and Methods: In this retrospective study, we have investigated the presence of Cytomegalovirus DNA expression, using paraffin-embedded tissue samples of 30 patients with Langerhans cell histiocytosis and 30 age and site-matched controls by qualitative Polymerase Chain Reaction (PCR) method. Results: No significant difference in prevalence of Cytomegalovirus presence between patients and controls was found. Cytomegalovirus was found by qualitative PCR in only 2 (6.66%) out of 30 patients and in 1 (3.3%) of 30 control samples with a P value of 1 (1.00 > 0.05) using chi-square test with OR: 2.07; 95% CI of OR: 0.18 - 24.15. Conclusions: Our findings do not support the hypothesis of a possible role for Cytomegalovirus in the pathogenesis of Langerhans cell histiocytosis.