The Role of Voltage-Gated Calcium Channels in Neurotransmitter Phenotype Specification: Coexpression and Functional Analysis in Xenopus laevis

Calcium activity has been implicated in many neurodevelopmental events, including the specification of neurotransmitter phenotypes. Higher levels of calcium activity lead to an increased number of inhibitory neural phenotypes, whereas lower levels of calcium activity lead to excitatory neural phenotypes. Voltage-gated calcium channels (VGCCs) allow for rapid calcium entry and are expressed during early neural stages, making them likely regulators of activity-dependent neurotransmitter phenotype specification. To test this hypothesis, multiplex fluorescent in situ hybridization was used to characterize the coexpression of eight VGCC alpha 1 subunits with the excitatory and inhibitory neural markers xVGlut1 and xVIAAT in Xenopus laevis embryos. VGCC coexpression was higher with xVGlut1 than xVIAAT, especially in the hindbrain, spinal cord, and cranial nerves. Calcium activity was also analyzed on a single-cell level, and spike frequency was correlated with the expression of VGCC alpha 1 subunits in cell culture. Cells expressing Ca(V)2.1 and Ca(V)2.2 displayed increased calcium spiking compared with cells not expressing this marker. The VGCC antagonist diltiazem and agonist (-)BayK 8644 were used to manipulate calcium activity. Diltiazem exposure increased the number of glutamatergic cells and decreased the number of gamma-aminobutyric acid (GABA)ergic cells, whereas (2) BayK 8644 exposure decreased the number of glutamatergic cells without having an effect on the number of GABAergic cells. Given that the expression and functional manipulation of VGCCs are correlated with neurotransmitter phenotype in some, but not all, experiments, VGCCs likely act in combination with a variety of other signaling factors to determine neuronal phenotype specification. (C) 2014 Wiley Periodicals, Inc

Developmental changes in spinal neuronal properties, motor network configuration, and neuromodulation at free-swimming stages of Xenopus tadpoles

We describe a novel preparation of the isolated brainstem and spinal cord from pro-metamorphic tadpole stages of the South African clawed frog (Xenopus laevis) that permits whole cell patch-clamp recordings from neurons in the ventral spinal cord. Previous research on earlier stages of the same species has provided one of the most detailed understandings of the design and operation of a CPG circuit. Here we have addressed how development sculpts complexity from this more basic circuit. The preparation generates bouts of fictive31 swimming activity either spontaneously or in response to electrical stimulation of the optic tectum, allowing an investigation into how the neuronal properties, activity patterns and neuromodulation of locomotor rhythm generation change during development. We describe an increased repertoire of cellular responses compared to younger larval stages and investigate the cellular level effects of nitrergic neuromodulation as well as the development of a sodium pump-mediated ultra-slow afterhyperpolarisation (usAHP) in these free-swimming larval animals.PostprintPeer reviewe

Effectiveness and cost-effectiveness of a nurse-delivered intervention to improve adherence to treatment for HIV : a pragmatic, multicentre, open-label, randomised clinical trial

This trial was funded from public money by the Netherlands Organisation for Health Research and Development (ZonMW; grant number 171002208). Aardex provided support on the development of the study website. We thank all the HIV nurses and physicians from the seven HIV clinics involved in the AIMS study for their input and collaboration (Academic Medical Centre, Slotervaart hospital, and St. Lucas-Andreas hospital, all in Amsterdam; the Leiden University Medical Centre, Leiden; HAGA hospital, The Hague; Erasmus Medical Centre, Rotterdam; and Isala clinic, Zwolle), the study participants, and the Stichting HIV Monitoring (SHM) for their support in accessing the SHM database for identifying patient inclusion criteria and developing the Markov model. Finally, we thank and remember Herman Schaalma (deceased) for his contribution to the study design and grant application.Peer reviewedPostprin

Takverkenning fruitteelt

Door het Ministerie van Landbouw, Natuurbeheer en Visserij is aan LEI-DLO gevraagd een visie op te stellen van de te verwachten ontwikkelingen in de fruitsector tot 2010. De verkenning vormt de basis voor het door het ministerie te formuleren overheidsbeleid dat gericht is op de ontwikkelingen in de fruitteelt. Deze verkenning start met een beschrijving van de ontwikkelingen in de periode 1985-1997. Aansluitend komen de te verwachten ontwikkelingen aan bod, gegroepeerd naar de centrale thema's markt en keten, milieu en bedrijfsstructuur. Per onderwerp wordt een toekomstbeeld gegeven en zijn de drijvende krachten die deze ontwikkelingsrichting beïnvloeden, aangegeven. Op grond van deze toekomstbeelden wordt een visie gegeven op hoe de primaire fruitsector er in 2010 uitziet

Ecological immunogenetics of life-history traits in a model amphibian

Major histocompatibility complex (MHC) genes determine immune repertoires and social preferences of vertebrates. Immunological regulation of microbial assemblages associated with individuals influences their sociality, and should also affect their life-history traits. We exposed Xenopus laevis tadpoles to water conditioned by adult conspecifics. Then, we analysed tadpole growth, development and survivorship as a function of MHC class I and class II peptide-binding region amino acid sequence similarities between tadpoles and frogs that conditioned the water to which they were exposed. Tadpoles approached metamorphosis earlier and suffered greater mortality when exposed to immunogenetically dissimilar frogs. The results suggest that developmental regulatory cues, microbial assemblages or both are specific to MHC genotypes. Tadpoles may associate with conspecifics with which they share microbiota to which their genotypes are well adapted

Elektriciteitsverbruik op glastuinbouwbedrijven

Het aandeel van het elektriciteitsverbruik in de totale energiebehoefte van de glastuinbouw is beperkt maar neemt jaarlijks toe. Op glastuinbouwbedrijven blijkt een grote diversiteit in het verbruik van elektriciteit. Een klein aantal bedrijven (10%) neemt het grootste deel van het elektriciteitsverbruik voor zijn rekening (66%). Het hoogste elektriciteitsverbruik valt waar te nemen op bloemenbedrijven, gevolgd door potplanten- en groentebedrijven. De verschillen worden voor het grootste deel bepaald door de mate waarin assimilatiebelichting wordt toegepast en door de hoogte van de brandstofintensiteit. De groei in het verbruik is veroorzaakt door een toenemend areaal met assimilatiebelichting

4-Octyl itaconate reduces influenza A replication by targeting the nuclear export protein CRM1

In recent years, especially since the outbreak of the severe acute respiratory syndrome coronavirus 2 pandemic, the cell-permeable itaconate derivative 4-octyl itaconate (4-OI) has gained traction as a potential antiviral agent. Here, we demonstrate that 4-OI inhibits replication of multiple influenza A viruses (IAV) by restricting nuclear export of viral ribonucleoproteins, a key step in the IAV replication cycle. This nuclear retention is achieved by deactivation and subsequent degradation of chromosomal maintenance 1 protein (CRM1), also known as exportin 1 (XPO1), a host cell protein exploited by IAV during replication. 4-OI-mediated deactivation of CRM1 resulted in the accumulation of the IAV nucleoprotein, the Rev protein of feline immunodeficiency virus, as well as the natural CRM1 cargos p53 and p65, in the nucleus of treated cells. Further mechanism of action studies revealed that, similar to known CRM1 inhibitors, 4-OI modifies a key cysteine in the cargo binding pocket of CRM1 at position 528 through an alkylation reaction called 2,3-dicarboxypropylation. Subsequent studies in a cell line in which the cysteine at position 528 in CRM1 protein was substituted by a serine confirmed that modification of this residue was indeed the cause for the observed inhibitory effect induced by 4-OI on CRM1 function. Overall, this study demonstrated a mechanism through which 4-OI directly interferes with the replication cycle of CRM1-dependent viruses, which contributes to the understanding of the antiviral and anti-inflammatory properties of this multifaceted immuno-metabolite. IMPORTANCE Itaconate derivates, as well as the naturally produced metabolite, have been proposed as antivirals against influenza virus. Here, the mechanism behind the antiviral effects of exogenous 4-octyl itaconate (4-OI), a derivative of itaconate, against the influenza A virus replication is demonstrated. The data indicate that 4-OI targets the cysteine at position 528 of the CRM1 protein, resulting in inhibition of the nuclear export of viral ribonucleoprotein complexes in a similar manner as previously described for other selective inhibitors of nuclear export. These results postulate a mechanism not observed before for this immuno-metabolite derivative. This knowledge is helpful for the development of derivatives of 4-OI as potential antiviral and anti-inflammatory therapeutics.</p

Combining cytotoxicity assessment and Xenopus laevis phenotypic abnormality assay as a predictor of nanomaterial safety

The African clawed frog, Xenopus laevis, has been used as an efficient pre-clinical screening tool to predict drug safety during the early stages of the drug discovery process. X. laevis is a relatively inexpensive model that can be used in whole organism high-throughput assays whilst maintaining a high degree of homology to the higher vertebrate models often used in scientific research. Despite an ever-increasing volume of biomedical nanoparticles (NPs) in development, their unique physico-chemical properties challenge the use of standard toxicology assays. Here, we present a protocol that directly compares the sensitivity of X. laevis development as a tool to assess potential NP toxicity by observation of embryo phenotypic abnormalities/lethality after NP exposure to in vitro cytotoxicity obtained using mammalian cell lines. In combination with conventional cytotoxicity assays, the X. laevis phenotypic assay provides accurate data to efficiently assess the safety of a novel biomedical NP