Recent evidence suggests extracellular vesicles (EVs) as another route of cellular communication. These EVs may be utilized for future therapeutics. In this article, the authors compared ultrafiltration with size-exclusion liquid chromatography (UF-LC) and ultra-centrifugation (UC) for EV recovery.

BackgroundNeurotropic flaviviruses such as tick-borne encephalitis virus (TBEV), Japanese encephalitis virus (JEV), West Nile virus (WNV), and Zika virus (ZIKV) are causative agents of severe brain-related diseases including meningitis, encephalitis, and microcephaly. We have previously shown that local type I interferon response within the central nervous system (CNS) is involved in the protection of mice against tick-borne flavivirus infection. However, the cells responsible for mounting this protective response are not defined.MethodsPrimary astrocytes were isolated from wild-type (WT) and interferon alpha receptor knock out (IFNAR−/−) mice and infected with neurotropic flaviviruses. Viral replication and spread, IFN induction and response, and cellular viability were analyzed. Transcriptional levels in primary astrocytes treated with interferon or supernatant from virus-infected cells were analyzed by RNA sequencing and evaluated by different bioinformatics tools.ResultsHere, we show that astrocytes control viral replication of different TBEV strains, JEV, WNV, and ZIKV. In contrast to fibroblast, astrocytes mount a rapid interferon response and restrict viral spread. Furthermore, basal expression levels of key interferon-stimulated genes are high in astrocytes compared to mouse embryonic fibroblasts. Bioinformatic analysis of RNA-sequencing data reveals that astrocytes have established a basal antiviral state which contributes to the rapid viral recognition and upregulation of interferons. The most highly upregulated pathways in neighboring cells were linked to type I interferon response and innate immunity. The restriction in viral growth was dependent on interferon signaling, since loss of the interferon receptor, or its blockade in wild-type cells, resulted in high viral replication and virus-induced cytopathic effects. Astrocyte supernatant from TBEV-infected cells can restrict TBEV growth in astrocytes already 6 h post infection, the effect on neurons is highly reinforced, and astrocyte supernatant from 3 h post infection is already protective.ConclusionsThese findings suggest that the combination of an intrinsic constitutive antiviral response and the fast induction of type I IFN production by astrocytes play an important role in self-protection of astrocytes and suppression of flavivirus replication in the CNS.Electronic supplementary materialThe online version of this article (doi:10.1186/s12974-016-0748-7) contains supplementary material, which is available to authorized users.

Comparative vertebrate genome sequencing offers a powerful method for detecting conserved regulatory sequences. We propose that the compact genome of the teleost Fugu rubripes is well suited for this purpose. The evolutionary distance of teleosts from other vertebrates offers the maximum stringency for such evolutionary comparisons. To illustrate the comparative genome approach for F. rubripes, we use sequence comparisons between mouse and Fugu Hoxb-4 noncoding regions to identify conserved sequence blocks. We have used two approaches to test the function of these conserved blocks. In the first, homologous sequences were deleted from a mouse enhancer, resulting in a tissue-specific loss of activity when assayed in transgenic mice. In the second approach, Fugu DNA sequences showing homology to mouse sequences were tested for enhancer activity in transgenic mice. This strategy identified a neural element that mediates a subset of Hoxb-4 expression that is conserved between mammals and teleosts. The comparison of noncoding vertebrate sequences with those of Fugu, coupled to a transgenic bioassay, represents a general approach suitable for many genome projects.The major challenges for all genome projects will be deciphering gene function and regulation. In the absence of useful grammars, testing the value of sequences by transgenesis is an important approach to finding regulatory information buried in the noncoding regions that constitute 95% of mammalian genomes; however, transgenic analysis is costly and labor intensive, and means of facilitating this approach are required. We have shown (1) that the Japanese puffer fish Fugu rubripes (Fugu)

Positivity for anti-HSV IgM, a sign of reactivated infection, was found to almost double the risk for AD, whereas the presence of anti-HSV IgG antibodies did not affect the risk.

Objective:In an ongoing, open-label, phase 1b study on the intrathecal administration of rituximab for progressive multiple sclerosis, an intraventricular catheter was inserted for drug delivery. The objective of this study was to characterize the limited white matter axonal injury evoked by catheter insertion by analyzing a panel of markers for tissue damage in CSF and serum.Methods:Lumbar CSF and serum were collected before catheter insertion and at regular intervals during the follow-up period of 1 year. Levels of neurofilament light polypeptide (NF-L), glial fibrillary acidic protein, microtubule-associated protein tau, and S100 calcium binding protein B were measured in the CSF, and NF-L was also quantified in serum at each time point.Results:One month after neurosurgical trauma, there was a distinct peak in NF-L concentration in both CSF and serum. In contrast, the biomarkers S100 calcium binding protein B, glial fibrillary acidic protein, and microtubule-associated protein tau did not show any significant changes. NF-L levels in both CSF and serum peaked at 1 month post surgery, returning to baseline after 6 to 9 months. A strong correlation was observed between the concentrations of NF-L in CSF and serum.Conclusions:The NF-L level, in CSF and serum, appears to be both a sensitive and specific marker for white matter axonal injury. This makes NF-L a valuable tool with which to evaluate acute white matter axonal damage in a clinical setting. Serum analysis of NF-L may become a convenient way to follow white matter axonal damage longitudinally.ClinicalTrials.gov identifier:NCT01719159.

During vertebrate brain development, the onset of neuronal differentiation is under strict temporal control. In the mammalian thalamus and other brain regions, neurogenesis is regulated also in a spatially progressive manner referred to as a neurogenetic gradient, the underlying mechanism of which is unknown. Here we describe the existence of a neurogenetic gradient in the zebrafish thalamus and show that the progression of neurogenesis is controlled by dynamic expression of the bHLH repressor her6. Members of the Hes/Her family are known to regulate proneural genes, such as Neurogenin and Ascl. Here we find that Her6 determines not only the onset of neurogenesis but also the identity of thalamic neurons, marked by proneural and neurotransmitter gene expression: loss of Her6 leads to premature Neurogenin1-mediated genesis of glutamatergic (excitatory) neurons, whereas maintenance of Her6 leads to Ascl1-mediated production of GABAergic (inhibitory) neurons. Thus, the presence or absence of a single upstream regulator of proneural gene expression, Her6, leads to the establishment of discrete neuronal domains in the thalamus.diencephalon ͉ Hes1 ͉ mash1 ͉ ngn1 ͉ zona limitans intrathalamica N eurogenesis in the developing vertebrate CNS is regulated with a high degree of temporal and spatial precision, with stereotypic patterns of neuronal differentiation and extensive neuronal migration (1, 2). Dynamic patterns of mitotically active neuronal precursors, known as 'neurogenetic gradients' (3) have been described in several brain regions, including the neocortex (4, 5), the dorsal midbrain colliculi (6), and the dopaminergic region of the ventral midbrain (7). In the mammalian diencephalon, in particular the thalamus (formerly known as dorsal thalamus), two main neurogenetic gradients have been described: from posterior to anterior and from lateral to medial (8,9). In rodents, all thalamic neurons are generated in about 6 days, and the orthogonal gradients of glutamatergic neurogenesis sweep across the boundaries of future nuclei. The underlying molecular mechanisms responsible for generating the neurogenetic gradients of the thalamus are unknown.The major constituent of the thalamus is a population of excitatory neurons generated in the caudal thalamus (cTh), whereas a minor population of inhibitory neurons is generated in the rostral thalamus (rTh). The latter is thought to give rise to the reticular nucleus and the ventral lateral geniculate nucleus, including the intergeniculate leaflet (10). During development, this rostro-caudal partitioning is seen in the expression domains of proneural basic helix-loop-helix (bHLH) genes: the achaete-scute-like complex genes (Ascl formerly known as Mash in mouse and Zash in zebrafish) mark the GABAergic rTh and the prethalamus (PTh) and the Neurogenin genes (Neurog, formerly known as Ngn) mark the glutamatergic cTh (2,11,12). Several lines of evidence show that these genes function as determinants of transmitter phenotype: in the mouse telencephalon, Neurog1/2 are required to ...

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SUMMARYThe roof plate is a signalling centre positioned at the dorsal midline of the central nervous system and generates dorsalising morphogenic signals along the length of the neuraxis. Within cranial ventricles, the roof plate gives rise to choroid plexus, which regulates the internal environment of the developing and adult brain and spinal cord via the secretion of cerebrospinal fluid. Using the fourth ventricle as our model, we show that the organiser properties of the roof plate are determined by its boundaries with the adjacent neuroepithelium. Through a combination of in ovo transplantation, co-culture and electroporation techniques in chick embryos between embryonic days 3 and 6, we demonstrate that organiser properties are maintained by interactions between the non-neural roof plate and the neural rhombic lip. At the molecular level, this interaction is mediated by Delta-Notch signalling and upregulation of the chick homologue of Hes1: chairy2. Gain-and loss-of-function approaches reveal that cdelta1 is both necessary and sufficient for organiser function. Our results also demonstrate that while chairy2 is specifically required for the maintenance of the organiser, its ectopic expression is not sufficient to recapitulate organiser properties. Expression of atonal1 in the rhombic lip adjacent at the roof plate boundary is acutely dependent on both boundary cell interactions and Delta-Notch signalling. Correspondingly, the roof plate boundary organiser also signals to the roof plate itself to specify the expression of early choroid plexus markers. Thus, the roof plate boundary organiser signals bi-directionally to acutely coordinate the development of adjacent neural and non-neural tissues.