Caspase-mediated apoptosis induction in zebrafish cerebellar Purkinje neurons.

The zebrafish is a well-established model organism in which to study in vivo mechanisms of cell communication, differentiation and function. Existing cell ablation methods are either invasive or they rely on the cellular expression of prokaryotic enzymes and the use of antibiotic drugs as cell death-inducing compounds. We have recently established a novel inducible genetic cell ablation system based on tamoxifen-inducible Caspase 8 activity, thereby exploiting mechanisms of cell death intrinsic to most cell types. Here, we prove its suitability in vivo by monitoring the ablation of cerebellar Purkinje cells (PCs) in transgenic zebrafish that co-express the inducible caspase and a fluorescent reporter. Incubation of larvae in tamoxifen for 8 h activated endogenous Caspase 3 and cell death, whereas incubation for 16 h led to the near-complete loss of PCs by apoptosis. We observed synchronous cell death autonomous to the PC population and phagocytosing microglia in the cerebellum, reminiscent of developmental apoptosis in the forebrain. Thus, induction of apoptosis through targeted activation of caspase by tamoxifen (ATTACTM) further expands the repertoire of genetic tools for conditional interrogation of cellular functions.

Corallocins A-C, Nerve Growth and Brain-Derived Neurotrophic Factor Inducing Metabolites from the Mushroom Hericium coralloides.

Three new natural products, corallocins A-C (1-3), along with two known compounds were isolated from the mushroom Hericium coralloides. Their benzofuranone and isoindolinone structures were elucidated by spectral methods. All corallocins induced nerve growth factor and/or brain-derived neurotrophic factor expression in human 1321N1 astrocytes. Furthermore, corallocin B showed antiproliferative activity against HUVEC and human cancer cell lines MCF-7 and KB-3-1.

The effects of Provent on moderate to severe obstructive sleep apnoea during continuous positive airway pressure therapy withdrawal: a randomised controlled trial.

OBJECTIVES: The aim of this study was to test the effectiveness of Provent, an expiratory nasal resistance valve, to prevent the recurrence of OSA following CPAP withdrawal. DESIGN: Randomised, partially blinded, parallel, placebo-controlled trial. SETTING: Outpatient sleep clinics in the UK (Oxford) and Switzerland (Zurich). PARTICIPANTS: 67 patients with OSA receiving CPAP were randomised to one of three groups for 2 weeks: continuing CPAP, Provent or placebo Provent. MAIN OUTCOME MEASURES: Primary outcomes included for Provent versus placebo Provent, OSA severity (oxygen desaturation index (ODI), apnoea-hypopnoea index (AHI)) and Epworth Sleepiness Scale (ESS) score. Secondary outcomes for Provent versus placebo Provent included ODI from ambulatory pulse oximetry and blood pressure (BP). For CPAP versus Provent, or CPAP versus placebo Provent, secondary outcomes included ODI/AHI, ESS and BP. RESULTS: 63 patients were included in the per protocol analysis. OSA recurred in the Provent (ODI 35.8, SD 17.4) and placebo Provent (ODI 28.2, SD 18.3) groups, and there was no significant difference in ODI, AHI and ESS between Provent and placebo Provent at 2 weeks (mean difference ODI -1.0, 95% CI -10.0 to +12.0, p=0.85; AHI +3.2, 95% CI -7.7 to +14.1, p=0.52; and ESS -1.4, 95% CI -4.1 to +1.4, p=0.33). ODI from ambulatory pulse-oximetry and BP at 2 weeks were not different in the Provent versus placebo Provent groups. ODI, AHI and BP, but not ESS, were significantly higher in the Provent and placebo Provent groups compared with CPAP. CONCLUSIONS: Provent cannot be recommended as an alternative short-term therapy for patients with moderate to severe OSA already on CPAP. TRIALREGNO: NCT01332175.

Lifelong regeneration of cerebellar Purkinje cells after induced cell ablation in zebrafish.

Zebrafish have an impressive capacity to regenerate neurons in the central nervous system. However, regeneration of the principal neuron of the evolutionary conserved cerebellum, the Purkinje cell (PC), is believed to be limited to developmental stages based on invasive lesions. In contrast, non-invasive cell type-specific ablation by induced apoptosis closely represents a process of neurodegeneration. We demonstrate that the ablated larval PC population entirely recovers in number, quickly reestablishes electrophysiological properties, and properly integrates into circuits to regulate cerebellum-controlled behavior. PC progenitors are present in larvae and adults, and PC ablation in adult cerebelli results in an impressive PC regeneration of different PC subtypes able to restore behavioral impairments. Interestingly, caudal PCs are more resistant to ablation and regenerate more efficiently, suggesting a rostro-caudal pattern of de- and regeneration properties. These findings demonstrate that the zebrafish cerebellum is able to regenerate functional PCs during all stages of the animal's life.

Visual improvement following continuous positive airway pressure therapy in diabetic subjects with clinically significant macular oedema and obstructive sleep apnoea: proof of principle study.

BACKGROUND: Diabetic retinopathy and diabetic macular oedema are more prevalent in patients with coexistent obstructive sleep apnoea (OSA). OBJECTIVES: We assessed if treatment of OSA with continuous positive airway pressure (CPAP) might improve visual acuity (VA). METHODS: A total of 35 patients with clinically significant macular oedema (CSMO) and OSA [oxygen desaturation index (ODI) ≥10 or apnoea-hypopnoea index (AHI) ≥15] were identified and agreed to be studied. VA (expressed as the logarithm of the minimum angle of resolution, logMAR), macular thickness, fundal photographs, glycosylated haemoglobin (HbA1c) and rhodopsin mRNA were measured twice at baseline and at 3 and 6 months post-CPAP. Fluorescein angiography and the Epworth Sleepiness Scale (ESS) were obtained once at baseline and at 6 months. RESULTS: Three patients withdrew before the first trial visit. Thus, a total of 32 patients (17 males) entered the study, and 4 subsequently withdrew; thus 28 completed 6 months of follow-up. Baseline characteristics of the subjects were as follows [mean (SD or inter-quartile range)]: age 66.2 (7.1) years, body mass index 31.7 (6.3), HbA1c 7.4% (1.44) [57.1 (15.7) mmol/mol], AHI 16.5 (11-25), ODI 16.0 (12-25), ESS 6.5 (4.0-12.0) and duration of diabetes 9.5 years (5.0-16.5). Participants were divided into 13 high and 15 low CPAP compliers (≥ and <2.5 h/night over the 6 months, respectively). At 6 months, the adjusted treatment effect on VA of high compliance versus low compliance was 0.11 (95% confidence interval 0.21 to -0.002; p = 0.047), equivalent to a one-line improvement on the logMAR chart. There was no significant improvement in macular oedema or fundal photographs. CONCLUSIONS: This hypothesis-generating, uncontrolled study suggests that ≥2.5 h/night CPAP usage over 6 months in individuals with CSMO and OSA may be associated with improvement in VA. This provides justification for a randomised controlled trial of CPAP therapy in such patients.

Erinacine C Activates Transcription from a Consensus ETS DNA Binding Site in Astrocytic Cells in Addition to NGF Induction.

Medicinal mushrooms of the genus Hericium are known to produce secondary metabolites with homeostatic properties for the central nervous system. We and others have recently demonstrated that among these metabolites cyathane diterpenoids and in particular erinacine C possess potent neurotrophin inducing properties in astrocytic cells. Yet, the signaling events downstream of erinacine C induced neurotrophin acitivity in neural-like adrenal phaeochromocytoma cells (PC12) cells have remained elusive. Similar, signaling events activated by erinacine C in astrocytic cells are unknown. Using a combination of genetic and pharmacological inhibitors we show that erinacine C induced neurotrophic activity mediates PC12 cell differentiation via the TrkA receptor and likely its associated PLCγ-, PI3K-, and MAPK/ERK pathways. Furthermore, a small library of transcriptional activation reporters revealed that erinacine C induces transcriptional activation mediated by DNA consensus binding sites of selected conserved transcription factor families. Among these, transcription is activated from an ETS consensus in a concentration dependent manner. Interestingly, induced ETS-consensus transcription occurs in parallel and independent of neurotrophin induction. This finding helps to explain the many pleiotropic functions of cyathane diterpenoids. Moreover, our studies provide genetic access to cyathane diterpenoid functions in astrocytic cells and help to mechanistically understand the action of cyathanes in glial cells.

Doxorubicin modulates telomerase activity in Ewing's sarcoma in vitro and in vivo.

Since most tumours escape replicative senescence by re-activation of the enzyme telomerase, telomerase is a promising target in the treatment of cancer and a promising marker for diagnosis and therapeutic response. We evaluated the effects of doxorubicin, one of the most active drugs in the treatment of Ewing's sarcoma, on telomerase in the human Ewing's sarcoma cell line STA-ET-1 in vitro and in STA-ET-1 xenografts in vivo. Telomerase activity (TA) was examined by TRAP-assay and real-time PCR. Real-time PCR was also used to quantify the mRNA expression of the catalytic subunit of telomerase (hTERT). In vitro growth inhibition was determined by the MTT-assay. Tumour xenografts were analyzed for tumour volume, apoptosis, necrosis, and proliferation. Doxorubicin concentrations that inhibited in vitro growth of STA-ET-1 by 50% compared to untreated controls ranged between 0.14 microM after 24 h and 0.01 microM after 72 h. Compared to untreated controls doxorubicin reduced TA in STA-ET-1 at toxic concentrations, but increased TA at non-toxic concentrations. In comparison with untreated xenografts, TA was reduced to 65% and hTERT expression dropped to 25% within 72 h in xenografts treated with 17.5 mg/kg doxorubicin i.p.; both recovered to initial values after 264 h. The rate of proliferating cells dropped to 70% within 96 h and increased thereafter. The highest rates of necrosis and apoptosis were seen after 96 h. hTERT expression co-varied significantly with proliferation but not with TA, apoptosis, and necrosis. No correlation was observed between TA, proliferation, apoptosis and necrosis. The results suggest doxorubicin induces down-regulation of hTERT gene expression that at least in part modulates TA in these tumours.

La tuberculosis en el cárcel: un retrato de las adversidades del sistema prisional brasileño / Tuberculosis in prison: a portrait of the adversities of the Brazilian prison system

Resumo:Tuberculose (TB) é uma doença infectocontagiosa que tem na forma pulmonar a maior importância clínica e epidemiológica.O Brasil apresenta alta incidência de TB, ocupando o 20º lugar no mundo e a situação entre as pessoas privadas de liberdade (PPL) é ainda mais grave. Por meio de pesquisa exploratória quali-quantitativao, buscou-se analisar o atual panorama da tuberculose no Brasil, com ênfase no sistema prisional. Na prisão, o desenvolvimento da TB é favorecido pela superlotação, nutrição precária, más condições higiênico-sanitárias, além de altas taxas de comorbidades. Assim, a incidência de TB entre PPL é cerca de 31 vezes superior ao notificado extramuros, colocando-as entre o grupos mais vulneráveis, equiparadas às pessoas vivendo com HIV. Este fato torna-se ainda mais preocupante quando se reconhece que o Brasil é o quarto país com a maior população prisional do mundo e com taxas crescentes de aprisionamento. Deve-se ter em mente que a população intramuros não está completamente isolada, tornando-se fonte de transmissão da tuberculose para o restante da população, através de visitas, transferências e contato com os profissionais do presídio. Diversos programas tiveram relativo êxito em reduzir o índice de incidência da tuberculose nos últimos anos, contudo, para as PPL as mazelas ainda se mantêm, tornando o sistema prisional um verdadeiro reservatório de tuberculose.

AbstractTuberculosis (TB) is an infectious disease that has the greatest clinical and epidemiological importance in pulmonary form. Brazil has a high incidence of TB, occupying the 20th place in the world and the situation among people deprived of liberty (PPL) is even more serious. Through exploratory qualitative-quantitative research, we sought to analyze the current panorama of tuberculosis in Brazil, with emphasis on the prison system. In prison, the development of TB is favored by overcrowding, poor nutrition, poor hygienic-sanitary conditions, and high rates of comorbidities. Thus, the incidence of TB among PLWA is about 31 times higher than reported outside the city walls, placing them among the most vulnerable groups, equal to those living with HIV. This fact becomes even more worrying when one recognizes that Brazil is the fourth country with the largest prison population in the world and with increasing rates of imprisonment. It should be borne in mind that the intramural population is not completely isolated, becoming a source of transmission of tuberculosis to the rest of the population through visits, transfers and contact with prison staff. Several programs have been relatively successful in reducing the incidence of tuberculosis in recent years; however, for PPLs the adversities still remain, making the prison system a true reservoir of tuberculosis.

ResumenLa tuberculosis (TB) es una enfermedad infectocontagiosa que tiene en forma pulmonar la mayor importancia clínica y epidemiológica. Brasil presenta alta incidencia de TB, ocupando el 20º lugar en el mundo y la situación entre las personas privadas de libertad (PPL) es aún más grave. Por medio de una investigación exploratoria cuali-cuantitativa, se buscó analizar el actual panorama de la tuberculosis en Brasil, con énfasis en el sistema penitenciario. En la prisión, el desarrollo de la TB es favorecido por el hacinamiento, la nutrición precaria, las malas condiciones higiénico-sanitarias, además de altas tasas de comorbilidad. Así, la incidencia de TB entre PPL es aproximadamente 31 veces superior al notificado extramuros, colocándolos entre los grupos más vulnerables, equiparados a las personas que viven con el VIH. Este hecho se vuelve aún más preocupante cuando se reconoce que Brasil es el cuarto país con la mayor población prisional del mundo y con tasas crecientes de encarcelamiento.Se debe tener en cuenta que la población intramurosa no está completamente aislada, convirtiéndose en fuente de transmisión de la tuberculosis para el resto de la población, a través de visitas, transferencias y contacto con los profesionales de la cárcel. Diversos programas tuvieron éxito en reducir el índice de incidencia de la tuberculosis en los últimos años, sin embargo, para las PPL las adversidades aún se mantienen, haciendo que el sistema penitenciario un verdadero reservorio de tuberculosis.

Targeted disruption of the homeobox gene Nkx2.9 reveals a role in development of the spinal accessory nerve.

The homeodomain-containing transcription factor Nkx2.9 is expressed in the ventralmost neural progenitor domain of the neural tube together with the related protein Nkx2.2 during early mouse embryogenesis. Cells within this region give rise to V3 interneurons and visceral motoneurons in spinal cord and hindbrain, respectively. To investigate the role of the Nkx2.9 gene, we generated a mutant mouse by targeted gene disruption. Homozygous mutant animals lacking Nkx2.9 were viable and fertile with no apparent morphological or behavioral phenotype. The distribution of neuronal progenitor cells and differentiated neurons in spinal cord was unaffected in Nkx2.9-deficient animals. This finding is in contrast to Nkx2.2-null mutants, which have been shown to exhibit ventral to dorsal transformation of neuronal cell fates in spinal cord. Our results suggest that specification of V3 interneurons in the posterior CNS does not require Nkx2.9, most probably because of functional redundancy with the co-expressed Nkx2.2 protein. In hindbrain, however, absence of Nkx2.9 resulted in a significantly altered morphology of the spinal accessory nerve (XIth), which appeared considerably shorter and thinner than in wild-type animals. Consistent with this phenotype, immature branchial motoneurons of the spinal accessory nerve, which normally migrate from a ventromedial to a dorsolateral position within the neural tube, were markedly reduced in Nkx2.9-deficient embryos at E10.5, while ventromedial motor column cells were increased in numbers. In addition, the vagal and glossopharyngeal nerves appeared abnormal in approximately 50% of mutant embryos, which may be related to the observed reduction of Phox2b expression in the nucleus ambiguus of adult mutant mice. From these observations, we conclude that Nkx2.9 has a specific function in the hindbrain as determinant of the branchial motoneuron precursor cells for the spinal accessory nerve and possibly other nerves of the branchial-motor column. Like other Nkx genes expressed in the CNS, Nkx2.9 seems to be involved in converting positional information into cell fate decisions.

Epitaxial growth of sexiphenyl on Al(111): from monolayer to crystalline films.

A combination of in situ surface sensitive-techniques, UV photoemission and low energy electron diffraction, with ex situ bulk sensitive X-ray diffraction reveals the formation of epitaxial thin films of sexiphenyl on Al(111) starting from the first monolayer. For room temperature growth, highly ordered films are formed with a unique alignment of the sexiphenyl molecules with the long axes of all molecules aligned parallel to both the surface and the <10> azimuthal directions of Al(111). This is related to a densely packed highly commensurate first monolayer, which acts as a template for the unique (21) crystallite orientation observed.