Remodeling of the Nuclear Envelope and Lamina during Bovine Preimplantation Development and Its Functional Implications.

PubMed

Popken, Jens; Graf, Alexander; Krebs, Stefan; Blum, Helmut; Schmid, Volker J; Strauss, Axel; Guengoer, Tuna; Zakhartchenko, Valeri; Wolf, Eckhard; Cremer, Thomas

2015-01-01

The present study demonstrates a major remodeling of the nuclear envelope and its underlying lamina during bovine preimplantation development. Up to the onset of major embryonic genome activation (MGA) at the 8-cell stage nuclei showed a non-uniform distribution of nuclear pore complexes (NPCs). NPCs were exclusively present at sites where DNA contacted the nuclear lamina. Extended regions of the lamina, which were not contacted by DNA, lacked NPCs. In post-MGA nuclei the whole lamina was contacted rather uniformly by DNA. Accordingly, NPCs became uniformly distributed throughout the entire nuclear envelope. These findings shed new light on the conditions which control the integration of NPCs into the nuclear envelope. The switch from maternal to embryonic production of mRNAs was accompanied by multiple invaginations covered with NPCs, which may serve the increased demands of mRNA export and protein import. Other invaginations, as well as interior nuclear segments and vesicles without contact to the nuclear envelope, were exclusively positive for lamin B. Since the abundance of these invaginations and vesicles increased in concert with a massive nuclear volume reduction, we suggest that they reflect a mechanism for fitting the nuclear envelope and its lamina to a shrinking nuclear size during bovine preimplantation development. In addition, a deposit of extranuclear clusters of NUP153 (a marker for NPCs) without associated lamin B was frequently observed from the zygote stage up to MGA. Corresponding RNA-Seq data revealed deposits of spliced, maternally provided NUP153 mRNA and little unspliced, newly synthesized RNA prior to MGA, which increased strongly at the initiation of embryonic expression of NUP153 at MGA.

Concise Review: Kidney Generation with Human Pluripotent Stem Cells.

PubMed

Morizane, Ryuji; Miyoshi, Tomoya; Bonventre, Joseph V

2017-11-01

Chronic kidney disease (CKD) is a worldwide health care problem, resulting in increased cardiovascular mortality and often leading to end-stage kidney disease, where patients require kidney replacement therapies such as hemodialysis or kidney transplantation. Loss of functional nephrons contributes to the progression of CKD, which can be attenuated but not reversed due to inability to generate new nephrons in human adult kidneys. Human pluripotent stem cells (hPSCs), by virtue of their unlimited self-renewal and ability to differentiate into cells of all three embryonic germ layers, are attractive sources for kidney regenerative therapies. Recent advances in stem cell biology have identified key signals necessary to maintain stemness of human nephron progenitor cells (NPCs) in vitro, and led to establishment of protocols to generate NPCs and nephron epithelial cells from human fetal kidneys and hPSCs. Effective production of large amounts of human NPCs and kidney organoids will facilitate elucidation of developmental and pathobiological pathways, kidney disease modeling and drug screening as well as kidney regenerative therapies. We summarize the recent studies to induce NPCs and kidney cells from hPSCs, studies of NPC expansion from mouse and human embryonic kidneys, and discuss possible approaches in vivo to regenerate kidneys with cell therapies and the development of bioengineered kidneys. Stem Cells 2017;35:2209-2217. © 2017 AlphaMed Press.

Electric double-layer capacitors based on highly graphitized nanoporous carbons derived from ZIF-67.

PubMed

Torad, Nagy L; Salunkhe, Rahul R; Li, Yunqi; Hamoudi, Hicham; Imura, Masataka; Sakka, Yoshio; Hu, Chi-Chang; Yamauchi, Yusuke

2014-06-23

Nanoporous carbons (NPCs) have large specific surface areas, good electrical and thermal conductivity, and both chemical and mechanical stability, which facilitate their use in energy storage device applications. In the present study, highly graphitized NPCs are synthesized by one-step direct carbonization of cobalt-containing zeolitic imidazolate framework-67 (ZIF-67). After chemical etching, the deposited Co content can be completely removed to prepare pure NPCs with high specific surface area, large pore volume, and intrinsic electrical conductivity (high content of sp(2) -bonded carbons). A detailed electrochemical study is performed using cyclic voltammetry and galvanostatic charge-discharge measurements. Our NPC is very promising for efficient electrodes for high-performance supercapacitor applications. A maximum specific capacitance of 238 F g(-1) is observed at a scan rate of 20 mV s(-1) . This value is very high compared to previous works on carbon-based electric double layer capacitors. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Chromatin organization at the nuclear periphery as revealed by image analysis of structured illumination microscopy data.

PubMed

Fišerová, Jindřiška; Efenberková, Michaela; Sieger, Tomáš; Maninová, Miloslava; Uhlířová, Jana; Hozák, Pavel

2017-06-15

The nuclear periphery (NP) plays a substantial role in chromatin organization. Heterochromatin at the NP is interspersed with active chromatin surrounding nuclear pore complexes (NPCs); however, details of the peripheral chromatin organization are missing. To discern the distribution of epigenetic marks at the NP of HeLa nuclei, we used structured illumination microscopy combined with a new MATLAB software tool for automatic NP and NPC detection, measurements of fluorescent intensity and statistical analysis of measured data. Our results show that marks for both active and non-active chromatin associate differentially with NPCs. The incidence of heterochromatin marks, such as H3K27me2 and H3K9me2, was significantly lower around NPCs. In contrast, the presence of marks of active chromatin such as H3K4me2 was only decreased very slightly around the NPCs or not at all (H3K9Ac). Interestingly, the histone demethylases LSD1 (also known as KDM1A) and KDM2A were enriched within the NPCs, suggesting that there was a chromatin-modifying mechanism at the NPCs. Inhibition of transcription resulted in a larger drop in the distribution of H1, H3K9me2 and H3K23me2, which implies that transcription has a role in the organization of heterochromatin at the NP. © 2017. Published by The Company of Biologists Ltd.

Systematic analysis of barrier-forming FG hydrogels from Xenopus nuclear pore complexes

PubMed Central

Labokha, Aksana A; Gradmann, Sabine; Frey, Steffen; Hülsmann, Bastian B; Urlaub, Henning; Baldus, Marc; Görlich, Dirk

2013-01-01

Nuclear pore complexes (NPCs) control the traffic between cell nucleus and cytoplasm. While facilitating translocation of nuclear transport receptors (NTRs) and NTR·cargo complexes, they suppress passive passage of macromolecules ⩾30 kDa. Previously, we reconstituted the NPC barrier as hydrogels comprising S. cerevisiae FG domains. We now studied FG domains from 10 Xenopus nucleoporins and found that all of them form hydrogels. Related domains with low FG motif density also substantially contribute to the NPC's hydrogel mass. We characterized all these hydrogels and observed the strictest sieving effect for the Nup98-derived hydrogel. It fully blocks entry of GFP-sized inert objects, permits facilitated entry of the small NTR NTF2, but arrests importin β-type NTRs at its surface. O-GlcNAc modification of the Nup98 FG domain prevented this arrest and allowed also large NTR·cargo complexes to enter. Solid-state NMR spectroscopy revealed that the O-GlcNAc-modified Nup98 gel lacks amyloid-like β-structures that dominate the rigid regions in the S. cerevisiae Nsp1 FG hydrogel. This suggests that FG hydrogels can assemble through different structural principles and yet acquire the same NPC-like permeability. PMID:23202855

Ran-dependent nuclear export mediators: a structural perspective

PubMed Central

Güttler, Thomas; Görlich, Dirk

2011-01-01

Nuclear export is an essential eukaryotic activity. It proceeds through nuclear pore complexes (NPCs) and is mediated by soluble receptors that shuttle between nucleus and cytoplasm. RanGTPase-dependent export mediators (exportins) constitute the largest class of these carriers and are functionally highly versatile. All of these exportins load their substrates in response to RanGTP binding in the nucleus and traverse NPCs as ternary RanGTP–exportin–cargo complexes to the cytoplasm, where GTP hydrolysis leads to export complex disassembly. The different exportins vary greatly in their substrate range. Recent structural studies of both protein- and RNA-specific exporters have illuminated how exportins bind their cargoes, how Ran triggers cargo loading and how export complexes are disassembled in the cytoplasm. Here, we review the current state of knowledge and highlight emerging principles as well as prevailing questions. PMID:21878989

Regulation of mRNA Trafficking by Nuclear Pore Complexes

PubMed Central

Bonnet, Amandine; Palancade, Benoit

2014-01-01

Over the last two decades, multiple studies have explored the mechanisms governing mRNA export out of the nucleus, a crucial step in eukaryotic gene expression. During transcription and processing, mRNAs are assembled into messenger ribonucleoparticles (mRNPs). mRNPs are then exported through nuclear pore complexes (NPCs), which are large multiprotein assemblies made of several copies of a limited number of nucleoporins. A considerable effort has been put into the dissection of mRNA export through NPCs at both cellular and molecular levels, revealing the conserved contributions of a subset of nucleoporins in this process, from yeast to vertebrates. Several reports have also demonstrated the ability of NPCs to sort out properly-processed mRNPs for entry into the nuclear export pathway. Importantly, changes in mRNA export have been associated with post-translational modifications of nucleoporins or changes in NPC composition, depending on cell cycle progression, development or exposure to stress. How NPC modifications also impact on cellular mRNA export in disease situations, notably upon viral infection, is discussed. PMID:25184662

The Nuclear Transport Factor Kap121 Is Required for Stability of the Dam1 Complex and Mitotic Kinetochore Bi-orientation.

PubMed

Cairo, Lucas V; Wozniak, Richard W

2016-03-15

The karyopherin (Kap) family of nuclear transport factors facilitates macromolecular transport through nuclear pore complexes (NPCs). The binding of Kaps to their cargos can also regulate, both temporally and spatially, the interactions of the cargo protein with interacting partners. Here, we show that the essential yeast Kap, Kap121, binds Dam1 and Duo1, components of the microtubule (MT)-associated Dam1 complex required for linking dynamic MT ends with kinetochores (KTs). Like mutations in the Dam1 complex, loss of Kap121 function compromises the formation of normal KT-MT attachments during mitosis. We show that the stability of the Dam1 complex in vivo is dependent on its association with Kap121. Furthermore, we show that the Kap121/Duo1 complex is maintained in the presence of RanGTP but Kap121 is released by the cooperative actions of RanGTP and tubulin. We propose that Kap121 stabilizes the Dam1 complex and participates in escorting it to spindle MTs. Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.

Sec66-Dependent Regulation of Yeast Spindle-Pole Body Duplication Through Pom152

PubMed Central

Katta, Santharam S.; Chen, Jingjing; Gardner, Jennifer M.; Friederichs, Jennifer M.; Smith, Sarah E.; Gogol, Madelaine; Unruh, Jay R.; Slaughter, Brian D.; Jaspersen, Sue L.

2015-01-01

In closed mitotic systems such as Saccharomyces cerevisiae, the nuclear envelope (NE) does not break down during mitosis, so microtubule-organizing centers such as the spindle-pole body (SPB) must be inserted into the NE to facilitate bipolar spindle formation and chromosome segregation. The mechanism of SPB insertion has been linked to NE insertion of nuclear pore complexes (NPCs) through a series of genetic and physical interactions between NPCs and SPB components. To identify new genes involved in SPB duplication and NE insertion, we carried out genome-wide screens for suppressors of deletion alleles of SPB components, including Mps3 and Mps2. In addition to the nucleoporins POM152 and POM34, we found that elimination of SEC66/SEC71/KAR7 suppressed lethality of cells lacking MPS2 or MPS3. Sec66 is a nonessential subunit of the Sec63 complex that functions together with the Sec61 complex in import of proteins into the endoplasmic reticulum (ER). Cells lacking Sec66 have reduced levels of Pom152 protein but not Pom34 or Ndc1, a shared component of the NPC and SPB. The fact that Sec66 but not other subunits of the ER translocon bypass deletion mutants in SPB genes suggests a specific role for Sec66 in the control of Pom152 levels. Based on the observation that sec66∆ does not affect the distribution of Ndc1 on the NE or Ndc1 binding to the SPB, we propose that Sec66-mediated regulation of Pom152 plays an NPC-independent role in the control of SPB duplication. PMID:26510791

The multifunctional nuclear pore complex: a platform for controlling gene expression

PubMed Central

Ptak, Christopher; Aitchison, John D.; Wozniak, Richard W.

2014-01-01

In addition to their established roles in nucleocytoplasmic transport, the intimate association of nuclear pore complexes (NPCs) with chromatin has long led to speculation that these structures influence peripheral chromatin structure and regulate gene expression. These ideas have their roots in morphological observations, however recent years have seen the identification of physical interactions between NPCs, chromatin, and the transcriptional machinery. Key insights into the molecular functions of specific NPC proteins have uncovered roles for these proteins in transcriptional activation and elongation, mRNA processing, as well as chromatin structure and localization. Here, we review recent studies that provide further molecular detail on the role of specific NPC components as distinct platforms for these chromatin dependent processes. PMID:24657998

Soyasaponin I Improved Neuroprotection and Regeneration in Memory Deficient Model Rats

PubMed Central

Hong, Sung-Woon; Heo, Hwon; Yang, Jeong-hwa; Han, Maeum; Kim, Dong-Hyun; Kwon, Yunhee Kim

2013-01-01

Soy (Glycine Max Merr, family Leguminosae) has been reported to possess anti-cancer, anti-lipidemic, estrogen-like, and memory-enhancing effects. We investigated the memory-enhancing effects and the underlying mechanisms of soyasaponin I (soya-I), a major constituent of soy. Impaired learning and memory were induced by injecting ibotenic acid into the entorhinal cortex of adult rat brains. The effects of soya-I were evaluated by measuring behavioral tasks and neuronal regeneration of memory-deficient rats. Oral administration of soya-I exhibited significant memory-enhancing effects in the passive avoidance, Y-maze, and Morris water maze tests. Soya-Ι also increased BrdU incorporation into the dentate gyrus and the number of cell types (GAD67, ChAT, and VGluT1) in the hippocampal region of memory-deficient rats, whereas the number of reactive microglia (OX42) decreased. The mechanism underlying memory improvement was assessed by detecting the differentiation and proliferation of neural precursor cells (NPCs) prepared from the embryonic hippocampus (E16) of timed-pregnant Sprague-Dawley rats using immunocytochemical staining and immunoblotting analysis. Addition of soya-Ι in the cultured NPCs significantly elevated the markers for cell proliferation (Ki-67) and neuronal differentiation (NeuN, TUJ1, and MAP2). Finally, soya-I increased neurite lengthening and the number of neurites during the differentiation of NPCs. Soya-Ι may improve hippocampal learning and memory impairment by promoting proliferation and differentiation of NPCs in the hippocampus through facilitation of neuronal regeneration and minimization of neuro-inflammation. PMID:24324703

Regulation of adult neural progenitor cell functions by purinergic signaling.

PubMed

Tang, Yong; Illes, Peter

2017-02-01

Extracellular purines are signaling molecules in the neurogenic niches of the brain and spinal cord, where they activate cell surface purinoceptors at embryonic neural stem cells (NSCs) and adult neural progenitor cells (NPCs). Although mRNA and protein are expressed at NSCs/NPCs for almost all subtypes of the nucleotide-sensitive P2X/P2Y, and the nucleoside-sensitive adenosine receptors, only a few of those have acquired functional significance. ATP is sequentially degraded by ecto-nucleotidases to ADP, AMP, and adenosine with agonistic properties for distinct receptor-classes. Nucleotides/nucleosides facilitate or inhibit NSC/NPC proliferation, migration and differentiation. The most ubiquitous effect of all agonists (especially of ATP and ADP) appears to be the facilitation of cell proliferation, usually through P2Y1Rs and sometimes through P2X7Rs. However, usually P2X7R activation causes necrosis/apoptosis of NPCs. Differentiation can be initiated by P2Y2R-activation or P2X7R-blockade. A key element in the transduction mechanism of either receptor is the increase of the intracellular free Ca 2+ concentration, which may arise due to its release from intracellular storage sites (G protein-coupling; P2Y) or due to its passage through the receptor-channel itself from the extracellular space (ATP-gated ion channel; P2X). Further research is needed to clarify how purinergic signaling controls NSC/NPC fate and how the balance between the quiescent and activated states is established with fine and dynamic regulation. GLIA 2017;65:213-230. © 2016 Wiley Periodicals, Inc.

Prolonged cultivation of hippocampal neural precursor cells shifts their differentiation potential and selects for aneuploid cells.

PubMed

Nguyen, The Duy; Widera, Darius; Greiner, Johannes; Müller, Janine; Martin, Ina; Slotta, Carsten; Hauser, Stefan; Kaltschmidt, Christian; Kaltschmidt, Barbara

2013-12-01

Neural precursor cells (NPCs) are lineage-restricted neural stem cells with limited self-renewal, giving rise to a broad range of neural cell types such as neurons, astrocytes, and oligodendrocytes. Despite this developmental potential, the differentiation capacity of NPCs has been controversially discussed concerning the trespassing lineage boundaries, for instance resulting in hematopoietic competence. Assessing their in vitro plasticity, we isolated nestin+/Sox2+, NPCs from the adult murine hippocampus. In vitro-expanded adult NPCs were able to form neurospheres, self-renew, and differentiate into neuronal, astrocytic, and oligodendrocytic cells. Although NPCs cultivated in early passage efficiently gave rise to neuronal cells in a directed differentiation assay, extensively cultivated NPCs revealed reduced potential for ectodermal differentiation. We further observed successful differentiation of long-term cultured NPCs into osteogenic and adipogenic cell types, suggesting that NPCs underwent a fate switch during culture. NPCs cultivated for more than 12 passages were aneuploid (abnormal chromosome numbers such as 70 chromosomes). Furthermore, they showed growth factor-independent proliferation, a hallmark of tumorigenic transformation. In conclusion, our findings substantiate the lineage restriction of NPCs from adult mammalian hippocampus. Prolonged cultivation results, however, in enhanced differentiation potential, which may be attributed to transformation events leading to aneuploid cells.

Wnt signaling maintains the notochord fate for progenitor cells and supports the posterior extension of the notochord.

PubMed

Ukita, Kanako; Hirahara, Shino; Oshima, Naoko; Imuta, Yu; Yoshimoto, Aki; Jang, Chuan-Wei; Oginuma, Masayuki; Saga, Yumiko; Behringer, Richard R; Kondoh, Hisato; Sasaki, Hiroshi

2009-10-01

The notochord develops from notochord progenitor cells (NPCs) and functions as a major signaling center to regulate trunk and tail development. NPCs are initially specified in the node by Wnt and Nodal signals at the gastrula stage. However, the underlying mechanism that maintains the NPCs throughout embryogenesis to contribute to the posterior extension of the notochord remains unclear. Here, we demonstrate that Wnt signaling in the NPCs is essential for posterior extension of the notochord. Genetic labeling revealed that the Noto-expressing cells in the ventral node contribute the NPCs that reside in the tail bud. Robust Wnt signaling in the NPCs was observed during posterior notochord extension. Genetic attenuation of the Wnt signal via notochord-specific beta-catenin gene ablation resulted in posterior truncation of the notochord. In the NPCs of such mutant embryos, the expression of notochord-specific genes was down-regulated, and an endodermal marker, E-cadherin, was observed. No significant alteration of cell proliferation or apoptosis of the NPCs was detected. Taken together, our data indicate that the NPCs are derived from Noto-positive node cells, and are not fully committed to a notochordal fate. Sustained Wnt signaling is required to maintain the NPCs' notochordal fate.

Simple SPION Incubation as an Efficient Intracellular Labeling Method for Tracking Neural Progenitor Cells Using MRI

PubMed Central

D. M., Jayaseema; Lai, Jiann-Shiun; Hueng, Dueng-Yuan; Chang, Chen

2013-01-01

Cellular magnetic resonance imaging (MRI) has been well-established for tracking neural progenitor cells (NPC). Superparamagnetic iron oxide nanoparticles (SPIONs) approved for clinical application are the most common agents used for labeling. Conventionally, transfection agents (TAs) were added with SPIONs to facilitate cell labeling because SPIONs in the native unmodified form were deemed inefficient for intracellular labeling. However, compelling evidence also shows that simple SPION incubation is not invariably ineffective. The labeling efficiency can be improved by prolonged incubation and elevated iron doses. The goal of the present study was to establish simple SPION incubation as an efficient intracellular labeling method. To this end, NPCs derived from the neonatal subventricular zone were incubated with SPIONs (Feridex®) and then evaluated in vitro with regard to the labeling efficiency and biological functions. The results showed that, following 48 hours of incubation at 75 µg/ml, nearly all NPCs exhibited visible SPION intake. Evidence from light microscopy, electron microscopy, chemical analysis, and magnetic resonance imaging confirmed the effectiveness of the labeling. Additionally, biological assays showed that the labeled NPCs exhibited unaffected viability, oxidative stress, apoptosis and differentiation. In the demonstrated in vivo cellular MRI experiment, the hypointensities representing the SPION labeled NPCs remained observable throughout the entire tracking period. The findings indicate that simple SPION incubation without the addition of TAs is an efficient intracellular magnetic labeling method. This simple approach may be considered as an alternative approach to the mainstream labeling method that involves the use of TAs. PMID:23468856

Expression of Pluripotency Markers in Nonpluripotent Human Neural Stem and Progenitor Cells.

PubMed

Vincent, Per Henrik; Benedikz, Eirikur; Uhlén, Per; Hovatta, Outi; Sundström, Erik

2017-06-15

Nonpluripotent neural progenitor cells (NPCs) derived from the human fetal central nervous system were found to express a number of messenger RNA (mRNA) species associated with pluripotency, such as NANOG, REX1, and OCT4. The expression was restricted to small subpopulations of NPCs. In contrast to pluripotent stem cells, there was no coexpression of the pluripotency-associated genes studied. Although the expression of these genes rapidly declined during the in vitro differentiation of NPCs, we found no evidence that the discrete expression was associated with the markers of multipotent neural stem cells (CD133 + /CD24 lo ), the capacity of sphere formation, or high cell proliferation rates. The rate of cell death among NPCs expressing pluripotency-associated genes was also similar to that of other NPCs. Live cell imaging showed that NANOG- and REX1-expressing NPCs continuously changed morphology, as did the nonexpressing cells. Depletion experiments showed that after the complete removal of the subpopulations of NANOG- and REX1-expressing NPCs, the expression of these genes appeared in other NPCs within a few days. The percentage of NANOG- and REX1-expressing cells returned to that observed before depletion. Our results are best explained by a model in which there is stochastic transient expression of pluripotency-associated genes in proliferating NPCs.

Organic Cation Transporter-Mediated Ergothioneine Uptake in Mouse Neural Progenitor Cells Suppresses Proliferation and Promotes Differentiation into Neurons

PubMed Central

Ishimoto, Takahiro; Nakamichi, Noritaka; Hosotani, Hiroshi; Masuo, Yusuke; Sugiura, Tomoko; Kato, Yukio

2014-01-01

The aim of the present study is to clarify the functional expression and physiological role in neural progenitor cells (NPCs) of carnitine/organic cation transporter OCTN1/SLC22A4, which accepts the naturally occurring food-derived antioxidant ergothioneine (ERGO) as a substrate in vivo. Real-time PCR analysis revealed that mRNA expression of OCTN1 was much higher than that of other organic cation transporters in mouse cultured cortical NPCs. Immunocytochemical analysis showed colocalization of OCTN1 with the NPC marker nestin in cultured NPCs and mouse embryonic carcinoma P19 cells differentiated into neural progenitor-like cells (P19-NPCs). These cells exhibited time-dependent [3H]ERGO uptake. These results demonstrate that OCTN1 is functionally expressed in murine NPCs. Cultured NPCs and P19-NPCs formed neurospheres from clusters of proliferating cells in a culture time-dependent manner. Exposure of cultured NPCs to ERGO or other antioxidants (edaravone and ascorbic acid) led to a significant decrease in the area of neurospheres with concomitant elimination of intracellular reactive oxygen species. Transfection of P19-NPCs with small interfering RNA for OCTN1 markedly promoted formation of neurospheres with a concomitant decrease of [3H]ERGO uptake. On the other hand, exposure of cultured NPCs to ERGO markedly increased the number of cells immunoreactive for the neuronal marker βIII-tubulin, but decreased the number immunoreactive for the astroglial marker glial fibrillary acidic protein (GFAP), with concomitant up-regulation of neuronal differentiation activator gene Math1. Interestingly, edaravone and ascorbic acid did not affect such differentiation of NPCs, in contrast to the case of proliferation. Knockdown of OCTN1 increased the number of cells immunoreactive for GFAP, but decreased the number immunoreactive for βIII-tubulin, with concomitant down-regulation of Math1 in P19-NPCs. Thus, OCTN1-mediated uptake of ERGO in NPCs inhibits cellular proliferation via regulation of oxidative stress, and also promotes cellular differentiation by modulating the expression of basic helix-loop-helix transcription factors via an unidentified mechanism different from antioxidant action. PMID:24586778

Simple rules for passive diffusion through the nuclear pore complex

PubMed Central

Mironska, Roxana; Kim, Seung Joong

2016-01-01

Passive macromolecular diffusion through nuclear pore complexes (NPCs) is thought to decrease dramatically beyond a 30–60-kD size threshold. Using thousands of independent time-resolved fluorescence microscopy measurements in vivo, we show that the NPC lacks such a firm size threshold; instead, it forms a soft barrier to passive diffusion that intensifies gradually with increasing molecular mass in both the wild-type and mutant strains with various subsets of phenylalanine-glycine (FG) domains and different levels of baseline passive permeability. Brownian dynamics simulations replicate these findings and indicate that the soft barrier results from the highly dynamic FG repeat domains and the diffusing macromolecules mutually constraining and competing for available volume in the interior of the NPC, setting up entropic repulsion forces. We found that FG domains with exceptionally high net charge and low hydropathy near the cytoplasmic end of the central channel contribute more strongly to obstruction of passive diffusion than to facilitated transport, revealing a compartmentalized functional arrangement within the NPC. PMID:27697925

Exportin Crm1 is repurposed as a docking protein to generate microtubule organizing centers at the nuclear pore.

PubMed

Bao, Xun X; Spanos, Christos; Kojidani, Tomoko; Lynch, Eric M; Rappsilber, Juri; Hiraoka, Yasushi; Haraguchi, Tokuko; Sawin, Kenneth E

2018-05-29

Non-centrosomal microtubule organizing centers (MTOCs) are important for microtubule organization in many cell types. In fission yeast Schizosaccharomyces pombe , the protein Mto1, together with partner protein Mto2 (Mto1/2 complex), recruits the g-tubulin complex to multiple non-centrosomal MTOCs, including the nuclear envelope (NE). Here, we develop a comparative-interactome mass spectrometry approach to determine how Mto1 localizes to the NE. Surprisingly, we find that Mto1, a constitutively cytoplasmic protein, docks at nuclear pore complexes (NPCs), via interaction with exportin Crm1 and cytoplasmic FG-nucleoporin Nup146. Although Mto1 is not a nuclear export cargo, it binds Crm1 via a nuclear export signal-like sequence, and docking requires both Ran in the GTP-bound state and Nup146 FG repeats. In addition to determining the mechanism of MTOC formation at the NE, our results reveal a novel role for Crm1 and the nuclear export machinery in the stable docking of a cytoplasmic protein complex at NPCs. © 2018, Bao et al.

Exportin Crm1 is repurposed as a docking protein to generate microtubule organizing centers at the nuclear pore

PubMed Central

Bao, Xun X; Spanos, Christos; Kojidani, Tomoko; Lynch, Eric M; Rappsilber, Juri; Hiraoka, Yasushi; Haraguchi, Tokuko

2018-01-01

Non-centrosomal microtubule organizing centers (MTOCs) are important for microtubule organization in many cell types. In fission yeast Schizosaccharomyces pombe, the protein Mto1, together with partner protein Mto2 (Mto1/2 complex), recruits the γ-tubulin complex to multiple non-centrosomal MTOCs, including the nuclear envelope (NE). Here, we develop a comparative-interactome mass spectrometry approach to determine how Mto1 localizes to the NE. Surprisingly, we find that Mto1, a constitutively cytoplasmic protein, docks at nuclear pore complexes (NPCs), via interaction with exportin Crm1 and cytoplasmic FG-nucleoporin Nup146. Although Mto1 is not a nuclear export cargo, it binds Crm1 via a nuclear export signal-like sequence, and docking requires both Ran in the GTP-bound state and Nup146 FG repeats. In addition to determining the mechanism of MTOC formation at the NE, our results reveal a novel role for Crm1 and the nuclear export machinery in the stable docking of a cytoplasmic protein complex at NPCs. PMID:29809148

Generation of neural progenitor cells by chemical cocktails and hypoxia

PubMed Central

Cheng, Lin; Hu, Wenxiang; Qiu, Binlong; Zhao, Jian; Yu, Yongchun; Guan, Wuqiang; Wang, Min; Yang, Wuzhou; Pei, Gang

2014-01-01

Neural progenitor cells (NPCs) can be induced from somatic cells by defined factors. Here we report that NPCs can be generated from mouse embryonic fibroblasts by a chemical cocktail, namely VCR (V, VPA, an inhibitor of HDACs; C, CHIR99021, an inhibitor of GSK-3 kinases and R, Repsox, an inhibitor of TGF-β pathways), under a physiological hypoxic condition. These chemical-induced NPCs (ciNPCs) resemble mouse brain-derived NPCs regarding their proliferative and self-renewing abilities, gene expression profiles, and multipotency for different neuroectodermal lineages in vitro and in vivo. Further experiments reveal that alternative cocktails with inhibitors of histone deacetylation, glycogen synthase kinase, and TGF-β pathways show similar efficacies for ciNPC induction. Moreover, ciNPCs can also be induced from mouse tail-tip fibroblasts and human urinary cells with the same chemical cocktail VCR. Thus our study demonstrates that lineage-specific conversion of somatic cells to NPCs could be achieved by chemical cocktails without introducing exogenous factors. PMID:24638034

Generation of Regionally Specific Neural Progenitor Cells (NPCs) and Neurons from Human Pluripotent Stem Cells (hPSCs).

PubMed

Cutts, Josh; Brookhouser, Nicholas; Brafman, David A

2016-01-01

Neural progenitor cells (NPCs) derived from human pluripotent stem cells (hPSCs) are a multipotent cell population capable of long-term expansion and differentiation into a variety of neuronal subtypes. As such, NPCs have tremendous potential for disease modeling, drug screening, and regenerative medicine. Current methods for the generation of NPCs results in cell populations homogenous for pan-neural markers such as SOX1 and SOX2 but heterogeneous with respect to regional identity. In order to use NPCs and their neuronal derivatives to investigate mechanisms of neurological disorders and develop more physiologically relevant disease models, methods for generation of regionally specific NPCs and neurons are needed. Here, we describe a protocol in which exogenous manipulation of WNT signaling, through either activation or inhibition, during neural differentiation of hPSCs, promotes the formation of regionally homogenous NPCs and neuronal cultures. In addition, we provide methods to monitor and characterize the efficiency of hPSC differentiation to these regionally specific cell identities.

In situ architecture of the algal nuclear pore complex.

PubMed

Mosalaganti, Shyamal; Kosinski, Jan; Albert, Sahradha; Schaffer, Miroslava; Strenkert, Daniela; Salomé, Patrice A; Merchant, Sabeeha S; Plitzko, Jürgen M; Baumeister, Wolfgang; Engel, Benjamin D; Beck, Martin

2018-06-18

Nuclear pore complexes (NPCs) span the nuclear envelope and mediate nucleocytoplasmic exchange. They are a hallmark of eukaryotes and deeply rooted in the evolutionary origin of cellular compartmentalization. NPCs have an elaborate architecture that has been well studied in vertebrates. Whether this architecture is unique or varies significantly in other eukaryotic kingdoms remains unknown, predominantly due to missing in situ structural data. Here, we report the architecture of the algal NPC from the early branching eukaryote Chlamydomonas reinhardtii and compare it to the human NPC. We find that the inner ring of the Chlamydomonas NPC has an unexpectedly large diameter, and the outer rings exhibit an asymmetric oligomeric state that has not been observed or predicted previously. Our study provides evidence that the NPC is subject to substantial structural variation between species. The divergent and conserved features of NPC architecture provide insights into the evolution of the nucleocytoplasmic transport machinery.

Nuclear networking.

PubMed

Xie, Wei; Burke, Brian

2017-07-04

Nuclear lamins are intermediate filament proteins that represent important structural components of metazoan nuclear envelopes (NEs). By combining proteomics and superresolution microscopy, we recently reported that both A- and B-type nuclear lamins form spatially distinct filament networks at the nuclear periphery of mouse fibroblasts. In particular, A-type lamins exhibit differential association with nuclear pore complexes (NPCs). Our studies reveal that the nuclear lamina network in mammalian somatic cells is less ordered and more complex than that of amphibian oocytes, the only other system in which the lamina has been visualized at high resolution. In addition, the NPC component Tpr likely links NPCs to the A-type lamin network, an association that appears to be regulated by C-terminal modification of various A-type lamin isoforms. Many questions remain, however, concerning the structure and assembly of lamin filaments, as well as with their mode of association with other nuclear components such as peripheral chromatin.

T cell mediated suppression of neurotropic coronavirus replication in neural precursor cells

PubMed Central

Plaisted, Warren C.; Weinger, Jason G.; Walsh, Craig M.; Lane, Thomas E.

2014-01-01

Neural precursor cells (NPCs) are the subject of intense investigation for their potential to treat neurodegenerative disorders, yet the consequences of neuroinvasive virus infection of NPCs remain unclear. This study demonstrates that NPCs support replication following infection by the neurotropic JHM strain of mouse hepatitis virus (JHMV). JHMV infection leads to increased cell death and dampens IFN-γ-induced MHC class II expression. Importantly, cytokines secreted by CD4+ T cells inhibit JHMV replication in NPCs, and CD8+ T cells specifically target viral peptide-pulsed NPCs for lysis. Furthermore, treatment with IFN-γ inhibits JHMV replication in a dose-dependent manner. Together, these findings suggest that T cells play a critical role in controlling replication of a neurotropic virus in NPCs, a finding which has important implications when considering immune modulation for NPC-based therapies for treatment of human neurologic diseases. PMID:24418558

Differentiated human midbrain-derived neural progenitor cells express excitatory strychnine-sensitive glycine receptors containing α2β subunits.

PubMed

Wegner, Florian; Kraft, Robert; Busse, Kathy; Härtig, Wolfgang; Ahrens, Jörg; Leffler, Andreas; Dengler, Reinhard; Schwarz, Johannes

2012-01-01

Human fetal midbrain-derived neural progenitor cells (NPCs) may deliver a tissue source for drug screening and regenerative cell therapy to treat Parkinson's disease. While glutamate and GABA(A) receptors play an important role in neurogenesis, the involvement of glycine receptors during human neurogenesis and dopaminergic differentiation as well as their molecular and functional characteristics in NPCs are largely unknown. Here we investigated NPCs in respect to their glycine receptor function and subunit expression using electrophysiology, calcium imaging, immunocytochemistry, and quantitative real-time PCR. Whole-cell recordings demonstrate the ability of NPCs to express functional strychnine-sensitive glycine receptors after differentiation for 3 weeks in vitro. Pharmacological and molecular analyses indicate a predominance of glycine receptor heteromers containing α2β subunits. Intracellular calcium measurements of differentiated NPCs suggest that glycine evokes depolarisations mediated by strychnine-sensitive glycine receptors and not by D-serine-sensitive excitatory glycine receptors. Culturing NPCs with additional glycine, the glycine-receptor antagonist strychnine, or the Na(+)-K(+)-Cl(-) co-transporter 1 (NKCC1)-inhibitor bumetanide did not significantly influence cell proliferation and differentiation in vitro. These data indicate that NPCs derived from human fetal midbrain tissue acquire essential glycine receptor properties during neuronal maturation. However, glycine receptors seem to have a limited functional impact on neurogenesis and dopaminergic differentiation of NPCs in vitro.

Differentiated Human Midbrain-Derived Neural Progenitor Cells Express Excitatory Strychnine-Sensitive Glycine Receptors Containing α2β Subunits

PubMed Central

Wegner, Florian; Kraft, Robert; Busse, Kathy; Härtig, Wolfgang; Ahrens, Jörg; Leffler, Andreas; Dengler, Reinhard; Schwarz, Johannes

2012-01-01

Background Human fetal midbrain-derived neural progenitor cells (NPCs) may deliver a tissue source for drug screening and regenerative cell therapy to treat Parkinson’s disease. While glutamate and GABAA receptors play an important role in neurogenesis, the involvement of glycine receptors during human neurogenesis and dopaminergic differentiation as well as their molecular and functional characteristics in NPCs are largely unknown. Methodology/Principal Findings Here we investigated NPCs in respect to their glycine receptor function and subunit expression using electrophysiology, calcium imaging, immunocytochemistry, and quantitative real-time PCR. Whole-cell recordings demonstrate the ability of NPCs to express functional strychnine-sensitive glycine receptors after differentiation for 3 weeks in vitro. Pharmacological and molecular analyses indicate a predominance of glycine receptor heteromers containing α2β subunits. Intracellular calcium measurements of differentiated NPCs suggest that glycine evokes depolarisations mediated by strychnine-sensitive glycine receptors and not by D-serine-sensitive excitatory glycine receptors. Culturing NPCs with additional glycine, the glycine-receptor antagonist strychnine, or the Na+-K+-Cl− co-transporter 1 (NKCC1)-inhibitor bumetanide did not significantly influence cell proliferation and differentiation in vitro. Conclusions/Significance These data indicate that NPCs derived from human fetal midbrain tissue acquire essential glycine receptor properties during neuronal maturation. However, glycine receptors seem to have a limited functional impact on neurogenesis and dopaminergic differentiation of NPCs in vitro. PMID:22606311

Nup133 Is Required for Proper Nuclear Pore Basket Assembly and Dynamics in Embryonic Stem Cells.

PubMed

Souquet, Benoit; Freed, Ellen; Berto, Alessandro; Andric, Vedrana; Audugé, Nicolas; Reina-San-Martin, Bernardo; Lacy, Elizabeth; Doye, Valérie

2018-05-22

Nup133 belongs to the Y-complex, a key component of the nuclear pore complex (NPC) scaffold. Studies on a null mutation in mice previously revealed that Nup133 is essential for embryonic development but not for mouse embryonic stem cell (mESC) proliferation. Using single-pore detection and average NE-fluorescence intensity, we find that Nup133 is dispensable for interphase and postmitotic NPC scaffold assembly in pluripotent mESCs. However, loss of Nup133 specifically perturbs the formation of the nuclear basket as manifested by the absence of Tpr in about half of the NPCs combined with altered dynamics of Nup153. We further demonstrate that its central domain mediates Nup133's role in assembling Tpr and Nup153 into a properly configured nuclear basket. Our findings thus revisit the role of the Y-complex in pore biogenesis and provide insights into the interplay between NPC scaffold architecture, nuclear basket assembly, and the generation of heterogeneity among NPCs. Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.

Programmed Death-Ligand 1 Expression, Microsatellite Instability, Epstein-Barr Virus, and Human Papillomavirus in Nasopharyngeal Carcinomas of Patients from the Philippines.

PubMed

Chang, Ann Margaret V; Chiosea, Simion I; Altman, Alexey; Pagdanganan, Hester A; Ma, Changqing

2017-06-01

Most nasopharyngeal carcinomas (NPCs) in a high-incidence population are driven by Epstein-Barr virus (EBV) infection. EBV-associated malignancies have increased expression of the programmed death-ligand 1 (PD-L1). Immunotherapy agents targeting the PD-1/PD-L1 pathway have achieved durable treatment effects in patients with various cancer types including EBV-associated malignancies. In this study, we sought to investigate PD-L1 expression in a cohort of patients with NPCs from the Philippines. Fifty-six NPCs were studied for PD-L1, p16, and DNA mismatch repair (MMR) deficiency by immunohistochemistry. One case with MMR deficiency was also assessed for microsatellite instability (MSI) by polymerase chain reaction. EBV and human papillomavirus (HPV) status were tested by in situ hybridization. All NPCs were p16 negative. Three of the 56 NPCs (5%) were EBV negative (EBV-) and HPV negative, while one NPC (1/56, 2%) was EBV positive and showed MSI (EBV+/MSI). Positive PD-L1 expression (PD-L1+), defined as membranous staining in ≥1% tumor cells, was seen in 64% (36/56) of NPCs. All three EBV- NPCs were PD-L1+ as was the EBV+/MSI NPC. PD-L1+ was seen significantly more often in NPCs from non-smokers than those from smokers (23/28, 82% vs 9/18, 50%; P = 0.047). PD-L1+ was not associated with pT, pN, distant metastasis, or clinical stage (P > 0.05). PD-L1+ was not associated with overall survival (P = 0.473). In summary, our results show frequent PD-L1 expression in NPCs regardless of EBV status and a preferential PD-L1 expression in non-smokers. MSI and HPV positivity are exceedingly rare in NPCs.

Regulation of Nephron Progenitor Cell Self-Renewal by Intermediary Metabolism.

PubMed

Liu, Jiao; Edgington-Giordano, Francesca; Dugas, Courtney; Abrams, Anna; Katakam, Prasad; Satou, Ryousuke; Saifudeen, Zubaida

2017-11-01

Nephron progenitor cells (NPCs) show an age-dependent capacity to balance self-renewal with differentiation. Older NPCs (postnatal day 0) exit the progenitor niche at a higher rate than younger (embryonic day 13.5) NPCs do. This behavior is reflected in the transcript profiles of young and old NPCs. Bioenergetic pathways have emerged as important regulators of stem cell fate. Here, we investigated the mechanisms underlying this regulation in murine NPCs. Upon isolation and culture in NPC renewal medium, younger NPCs displayed a higher glycolysis rate than older NPCs. Inhibition of glycolysis enhanced nephrogenesis in cultured embryonic kidneys, without increasing ureteric tree branching, and promoted mesenchymal-to-epithelial transition in cultured isolated metanephric mesenchyme. Cotreatment with a canonical Wnt signaling inhibitor attenuated but did not entirely block the increase in nephrogenesis observed after glycolysis inhibition. Furthermore, inhibition of the phosphatidylinositol 3-kinase/Akt self-renewal signaling pathway or stimulation of differentiation pathways in the NPC decreased glycolytic flux. Our findings suggest that glycolysis is a pivotal, cell-intrinsic determinant of NPC fate, with a high glycolytic flux supporting self-renewal and inhibition of glycolysis stimulating differentiation. Copyright © 2017 by the American Society of Nephrology.

Synthesis of ZnO nanopencils using wet chemical method and its investigation as LPG sensor

NASA Astrophysics Data System (ADS)

Shimpi, Navinchandra G.; Jain, Shilpa; Karmakar, Narayan; Shah, Akshara; Kothari, D. C.; Mishra, Satyendra

2016-12-01

ZnO nanopencils (NPCs) were prepared by a novel wet chemical process, using triethanolamine (TEA) as a mild base, which is relatively simple and cost effective method as compared to hydrothermal method. ZnO NPCs were characterized using powder X-ray diffraction (XRD), Fourier Transform Infra-Red (FTIR) spectroscopy in mid-IR and far-IR regions, X-ray Photoelectron Spectroscopy (XPS), UV-vis (UV-vis) absorption spectroscopy, room temperature Photoluminescence (PL) spectroscopy and Field Emission Scanning Electron Microscopy (FESEM). ZnO NPCs obtained, were highly pure, uniform and monodispersed.XRD pattern indicated hexagonal unit cell structure with preferred orientation along the c-axis. Sensing behaviour of ZnO NPCs was studied towards Liquefied Petroleum Gas (LPG) at different operating temperatures. The study shows that ZnO NPCs were most sensitive and promising candidate for detection of LPG at 250 °C with gas sensitivity > 60%. The high response towards LPG is due to high surface area of ZnO NPCs and their parallel alignment.

Dual personality of Mad1: regulation of nuclear import by a spindle assembly checkpoint protein.

PubMed

Cairo, Lucas V; Ptak, Christopher; Wozniak, Richard W

2013-01-01

Nuclear transport is a dynamic process that can be modulated in response to changes in cellular physiology. We recently reported that the transport activity of yeast nuclear pore complexes (NPCs) is altered in response to kinetochore-microtubule (KT-MT) interaction defects. Specifically, KT detachment from MTs activates a signaling pathway that prevents the nuclear import of cargos by the nuclear transport factor Kap121p. This loss of Kap121p-mediated import is thought to influence the nuclear environment, including the phosphorylation state of nuclear proteins. A key regulator of this process is the spindle assembly checkpoint protein Mad1p. In response to unattached KTs, Mad1p dynamically cycles between NPCs and KTs. This cycling appears to induce NPC molecular rearrangements that prevent the nuclear import of Kap121p-cargo complexes. Here, we discuss the underlying mechanisms and the physiological relevance of Mad1p cycling and the inhibition of Kap121p-mediated nuclear import, focusing on outstanding questions within the pathway.

Spatial structure of disordered proteins dictates conductance and selectivity in nuclear pore complex mimics

PubMed Central

Frey, Steffen; Dwarkasing, Arvind; Versloot, Roderick; van der Giessen, Erik

2018-01-01

Nuclear pore complexes (NPCs) lined with intrinsically disordered FG-domains act as selective gatekeepers for molecular transport between the nucleus and the cytoplasm in eukaryotic cells. The underlying physical mechanism of the intriguing selectivity is still under debate. Here, we probe the transport of ions and transport receptors through biomimetic NPCs consisting of Nsp1 domains attached to the inner surface of solid-state nanopores. We examine both wildtype FG-domains and hydrophilic SG-mutants. FG-nanopores showed a clear selectivity as transport receptors can translocate across the pore whereas other proteins cannot. SG mutant pores lack such selectivity. To unravel this striking difference, we present coarse-grained molecular dynamics simulations that reveal that FG-pores exhibit a high-density, nonuniform protein distribution, in contrast to a uniform and significantly less-dense protein distribution in the SG-mutant. We conclude that the sequence-dependent density distribution of disordered proteins inside the NPC plays a key role for its conductivity and selective permeability. PMID:29442997

A role for the nucleoporin Nup170p in chromatin structure and gene silencing

PubMed Central

Van de Vosse, David W.; Wan, Yakun; Lapetina, Diego L.; Chen, Wei-Ming; Chiang, Jung-Hsien; Aitchison, John D.; Wozniak, Richard W.

2013-01-01

Embedded in the nuclear envelope, nuclear pore complexes (NPCs) not only regulate nuclear transport, but also interface with transcriptionally active euchromatin, largely silenced heterochromatin, as well as the boundaries between these regions. It is unclear what functional role NPCs play in establishing or maintaining these distinct chromatin domains. We report that the yeast NPC protein Nup170p interacts with regions of the genome containing ribosomal protein and subtelomeric genes. Here, it functions in nucleosome positioning and as a repressor of transcription. We show that the role of Nup170p in subtelomeric gene silencing is linked to its association with the RSC chromatin-remodeling complex and the silencing factor Sir4p, and that the binding of Nup170p and Sir4p to subtelomeric chromatin is cooperative and necessary for the association of telomeres with the nuclear envelope. Our results establish the NPC as an active participant in silencing and the formation of peripheral heterochromatin. PMID:23452847

The Yeast Nuclear Pore Complex and Transport Through It

PubMed Central

Aitchison, John D.; Rout, Michael P.

2012-01-01

Exchange of macromolecules between the nucleus and cytoplasm is a key regulatory event in the expression of a cell’s genome. This exchange requires a dedicated transport system: (1) nuclear pore complexes (NPCs), embedded in the nuclear envelope and composed of proteins termed nucleoporins (or “Nups”), and (2) nuclear transport factors that recognize the cargoes to be transported and ferry them across the NPCs. This transport is regulated at multiple levels, and the NPC itself also plays a key regulatory role in gene expression by influencing nuclear architecture and acting as a point of control for various nuclear processes. Here we summarize how the yeast Saccharomyces has been used extensively as a model system to understand the fundamental and highly conserved features of this transport system, revealing the structure and function of the NPC; the NPC’s role in the regulation of gene expression; and the interactions of transport factors with their cargoes, regulatory factors, and specific nucleoporins. PMID:22419078

Computational and Organotypic Modeling of Microcephaly ...

EPA Pesticide Factsheets

Microcephaly is associated with reduced cortical surface area and ventricular dilations. Many genetic and environmental factors precipitate this malformation, including prenatal alcohol exposure and maternal Zika infection. This complexity motivates the engineering of computational and experimental models to probe the underlying molecular targets, cellular consequences, and biological processes. We describe an Adverse Outcome Pathway (AOP) framework for microcephaly derived from literature on all gene-, chemical-, or viral- effects and brain development. Overlap with NTDs is likely, although the AOP connections identified here focused on microcephaly as the adverse outcome. A query of the Mammalian Phenotype Browser database for ‘microcephaly’ (MP:0000433) returned 85 gene associations; several function in microtubule assembly and centrosome cycle regulated by (microcephalin, MCPH1), a gene for primary microcephaly in humans. The developing ventricular zone is the likely target. In this zone, neuroprogenitor cells (NPCs) self-replicate during the 1st trimester setting brain size, followed by neural differentiation of the neocortex. Recent studies with human NPCs confirmed infectivity with Zika virions invoking critical cell loss (apoptosis) of precursor NPCs; similar findings have been shown with fetal alcohol or methylmercury exposure in rodent studies, leading to mathematical models of NPC dynamics in size determination of the ventricular zone. A key event

Ex vivo delivery of GDNF maintains motor function and prevents neuronal loss in a transgenic mouse model of Huntington's disease.

PubMed

Ebert, Allison D; Barber, Amelia E; Heins, Brittany M; Svendsen, Clive N

2010-07-01

Huntington's disease (HD) is an autosomal dominant disorder caused by expansion of polyglutamine repeats in the huntingtin gene leading to loss of striatal and cortical neurons followed by deficits in cognition and choreic movements. Growth factor delivery to the brain has shown promise in various models of neurodegenerative diseases, including HD, by reducing neuronal death and thus limiting motor impairment. Here we used mouse neural progenitor cells (mNPCs) as growth factor delivery vehicles in the N171-82Q transgenic mouse model of HD. mNPCs derived from the developing mouse striatum were isolated and infected with lentivirus expressing either glial cell line-derived neurotrophic factor (GDNF) or green fluorescent protein (GFP). Next, mNPCs(GDNF) or mNPCs(GFP) were transplanted bilaterally into the striatum of pre-symptomatic N171-82Q mice. We found that mNPCs(GDNF), but not mNPCs(GFP), maintained rotarod function and increased striatal neuron survival out to 3months post-transplantation. Importantly, histological analysis showed GDNF expression through the duration of the experiment. Our data show that mNPCs(GDNF) can survive transplantation, secrete GDNF for several weeks and are able to maintain motor function in this model of HD. Copyright 2010 Elsevier Inc. All rights reserved.

Switching Plasmons: Gold Nanorod-Copper Chalcogenide Core-Shell Nanoparticle Clusters with Selectable Metal/Semiconductor NIR Plasmon Resonances.

PubMed

Muhammed, Madathumpady Abubaker Habeeb; Döblinger, Markus; Rodríguez-Fernández, Jessica

2015-09-16

Exerting control over the near-infrared (NIR) plasmonic response of nanosized metals and semiconductors can facilitate access to unexplored phenomena and applications. Here we combine electrostatic self-assembly and Cd(2+)/Cu(+) cation exchange to obtain an anisotropic core-shell nanoparticle cluster (NPC) whose optical properties stem from two dissimilar plasmonic materials: a gold nanorod (AuNR) core and a copper selenide (Cu(2-x)Se, x ≥ 0) supraparticle shell. The spectral response of the AuNR@Cu2Se NPCs is governed by the transverse and longitudinal plasmon bands (LPB) of the anisotropic metallic core, since the Cu2Se shell is nonplasmonic. Under aerobic conditions the shell undergoes vacancy doping (x > 0), leading to the plasmon-rich NIR spectrum of the AuNR@Cu(2-x)Se NPCs. For low vacancy doping levels the NIR optical properties of the dually plasmonic NPCs are determined by the LPBs of the semiconductor shell (along its major longitudinal axis) and of the metal core. Conversely, for high vacancy doping levels their NIR optical response is dominated by the two most intense plasmon modes from the shell: the transverse (along the shortest transversal axis) and longitudinal (along the major longitudinal axis) modes. The optical properties of the NPCs can be reversibly switched back to a purely metallic plasmonic character upon reversible conversion of AuNR@Cu(2-x)Se into AuNR@Cu2Se. Such well-defined nanosized colloidal assemblies feature the unique ability of holding an all-metallic, a metallic/semiconductor, or an all-semiconductor plasmonic response in the NIR. Therefore, they can serve as an ideal platform to evaluate the crosstalk between plasmonic metals and plasmonic semiconductors at the nanoscale. Furthermore, their versatility to display plasmon modes in the first, second, or both NIR windows is particularly advantageous for bioapplications, especially considering their strong absorbing and near-field enhancing properties.

Surgical task-shifting in a low-resource setting: outcomes after major surgery performed by nonphysician clinicians in Tanzania.

PubMed

Beard, Jessica H; Oresanya, Lawrence B; Akoko, Larry; Mwanga, Ally; Mkony, Charles A; Dicker, Rochelle A

2014-06-01

Little is known about the breadth and quality of nonobstetric surgical care delivered by nonphysician clinicians (NPCs) in low-resource settings. We aimed to document the scope of NPC surgical practice and characterize outcomes after major surgery performed by nonphysicians in Tanzania. A retrospective records review of major surgical procedures (MSPs) performed in 2012 was conducted at seven hospitals in Pwani Region, Tanzania. Patient and procedure characteristics and level of surgical care provider were documented for each procedure. Rates of postoperative morbidity and mortality after nonobstetric MSPs performed by NPCs and physicians were compared using multivariate logistic regression. There were 6.5 surgical care providers per 100,000 population performing a mean rate of 461 procedures per 100,000 population during the study period. Of these cases, 1,698 (34.7 %) were nonobstetric MSPs. NPCs performed 55.8 % of nonobstetric MSPs followed by surgical specialists (28.7 %) and medical officers (15.5 %). The most common nonobstetric MSPs performed by NPCs were elective groin hernia repair, prostatectomy, exploratory laparotomy, and hydrocelectomy. Postoperative mortality was 1.7 % and 1.5 % in cases done by NPCs and physicians respectively. There was no significant difference in outcomes after procedures performed by NPCs compared with physicians. Surgical output is low and the workforce is limited in Tanzania. NPCs performed the majority of major surgical procedures during the study period. Outcomes after nonobstetric major surgical procedures done by NPCs and physicians were similar. Task-shifting of surgical care to nonphysicians may be a safe and sustainable way to address the global surgical workforce crisis.

Implanted neural progenitor cells regulate glial reaction to brain injury and establish gap junctions with host glial cells.

PubMed

Talaverón, Rocío; Matarredona, Esperanza R; de la Cruz, Rosa R; Macías, David; Gálvez, Victoria; Pastor, Angel M

2014-04-01

Transplantation of neural stem/progenitor cells (NPCs) in the lesioned brain is able to restore morphological and physiological alterations induced by different injuries. The local microenvironment created at the site of grafting and the communication between grafted and host cells are crucial in the beneficial effects attributed to the NPC implants. We have previously described that NPC transplantation in an animal model of central axotomy restores firing properties and synaptic coverage of lesioned neurons and modulates their trophic factor content. In this study, we aim to explore anatomical relationships between implanted NPCs and host glia that might account for the implant-induced neuroprotective effects. Postnatal rat subventricular zone NPCs were isolated and grafted in adult rats after transection of the medial longitudinal fascicle. Brains were removed and analyzed eight weeks later. Immunohistochemistry for different glial markers revealed that NPC-grafted animals displayed significantly greater microglial activation than animals that received only vehicle injections. Implanted NPCs were located in close apposition to activated microglia and reactive astrocytes. The gap junction protein connexin43 was present in NPCs and glial cells at the lesion site and was often found interposed within adjacent implanted and glial cells. Gap junctions were identified between implanted NPCs and host astrocytes and less frequently between NPCs and microglia. Our results show that implanted NPCs modulate the glial reaction to lesion and establish the possibility of communication through gap junctions between grafted and host glial cells which might be involved in the restorative effects of NPC implants. Copyright © 2014 Wiley Periodicals, Inc.

The effect of pulsed electric fields on the electrotactic migration of human neural progenitor cells through the involvement of intracellular calcium signaling.

PubMed

Hayashi, Hisamitsu; Edin, Fredrik; Li, Hao; Liu, Wei; Rask-Andersen, Helge

2016-12-01

Endogenous electric fields (EFs) are required for the physiological control of the central nervous system development. Application of the direct current EFs to neural stem cells has been studied for the possibility of stem cell transplantation as one of the therapies for brain injury. EFs generated within the nervous system are often associated with action potentials and synaptic activity, apparently resulting in a pulsed current in nature. The aim of this study is to investigate the effect of pulsed EF, which can reduce the cytotoxicity, on the migration of human neural progenitor cells (hNPCs). We applied the mono-directional pulsed EF with a strength of 250mV/mm to hNPCs for 6h. The migration distance of the hNPCs exposed to pulsed EF was significantly greater compared with the control not exposed to the EF. Pulsed EFs, however, had less of an effect on the migration of the differentiated hNPCs. There was no significant change in the survival of hNPCs after exposure to the pulsed EF. To investigate the role of Ca 2+ signaling in electrotactic migration of hNPCs, pharmacological inhibition of Ca 2+ channels in the EF-exposed cells revealed that the electrotactic migration of hNPCs exposed to Ca 2+ channel blockers was significantly lower compared to the control group. The findings suggest that the pulsed EF induced migration of hNPCs is partly influenced by intracellular Ca 2+ signaling. Copyright © 2016 Elsevier B.V. All rights reserved.

Optimal route of diphtheria toxin administration to eliminate native nephron progenitor cells in vivo for kidney regeneration.

PubMed

Fukunaga, Shohei; Yamanaka, Shuichiro; Fujimoto, Toshinari; Tajiri, Susumu; Uchiyama, Taketo; Matsumoto, Kei; Ito, Takafumi; Tanabe, Kazuaki; Yokoo, Takashi

2018-02-19

To address the lack of organs for transplantation, we previously developed a method for organ regeneration in which nephron progenitor cell (NPC) replacement is performed via the diphtheria toxin receptor (DTR) system. In transgenic mice with NPC-specific expression of DTR, NPCs were eliminated by DT and replaced with NPCs lacking the DTR with the ability to differentiate into nephrons. However, this method has only been verified in vitro. For applications to natural models, such as animal fetuses, it is necessary to determine the optimal administration route and dose of DT. In this study, two DT administration routes (intra-peritoneal and intra-amniotic injection) were evaluated in fetal mice. The fetus was delivered by caesarean section at E18.5, and the fetal mouse kidney and RNA expression were evaluated. Additionally, the effect of the DT dose (25, 5, 0.5, and 0.05 ng/fetus-body) was studied. Intra-amniotic injection of DT led to a reduction in kidney volume, loss of glomeruli, and decreased differentiation marker expression. The intra-peritoneal route was not sufficient for NPC elimination. By establishing that intra-amniotic injection is the optimal administration route for DT, these results will facilitate studies of kidney regeneration in vivo. In addition, this method might be useful for analysis of kidney development at various time points by deleting NPCs during development. Copyright © 2018 Elsevier Inc. All rights reserved.

Molecular stages of rapid and uniform neuralization of human embryonic stem cells.

PubMed

Bajpai, R; Coppola, G; Kaul, M; Talantova, M; Cimadamore, F; Nilbratt, M; Geschwind, D H; Lipton, S A; Terskikh, A V

2009-06-01

Insights into early human development are fundamental for our understanding of human biology. Efficient differentiation of human embryonic stem cells (hESCs) into neural precursor cells is critical for future cell-based therapies. Here, using defined conditions, we characterized a new method for rapid and uniform differentiation of hESCs into committed neural precursor cells (designated C-NPCs). Dynamic gene expression analysis identified several distinct stages of ESC neuralization and revealed functional modules of coregulated genes and pathways. The first wave of gene expression changes, likely corresponding to the transition through primitive ectoderm, started at day 3, preceding the formation of columnar neuroepithelial rosettes. The second wave started at day 5, coinciding with the formation of rosettes. The majority of C-NPCs were positive for both anterior and posterior markers of developing neuroepithelium. In culture, C-NPCs became electrophysiologically functional neurons; on transplantation into neonatal mouse brains, C-NPCs integrated into the cortex and olfactory bulb, acquiring appropriate neuronal morphologies and markers. Compared to rosette-NPCs,(1) C-NPCs exhibited limited in vitro expansion capacity and did not express potent oncogenes such as PLAG1 or RSPO3. Concordantly, we never detected tumors or excessive neural proliferation after transplantation of C-NPCs into mouse brains. In conclusion, our study provides a framework for future analysis of molecular signaling during ESC neuralization.

Highly efficient methods to obtain homogeneous dorsal neural progenitor cells from human and mouse embryonic stem cells and induced pluripotent stem cells.

PubMed

Zhang, Meixiang; Ngo, Justine; Pirozzi, Filomena; Sun, Ying-Pu; Wynshaw-Boris, Anthony

2018-03-15

Embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) have been widely used to generate cellular models harboring specific disease-related genotypes. Of particular importance are ESC and iPSC applications capable of producing dorsal telencephalic neural progenitor cells (NPCs) that are representative of the cerebral cortex and overcome the challenges of maintaining a homogeneous population of cortical progenitors over several passages in vitro. While previous studies were able to derive NPCs from pluripotent cell types, the fraction of dorsal NPCs in this population is small and decreases over several passages. Here, we present three protocols that are highly efficient in differentiating mouse and human ESCs, as well as human iPSCs, into a homogeneous and stable population of dorsal NPCs. These protocols will be useful for modeling cerebral cortical neurological and neurodegenerative disorders in both mouse and human as well as for high-throughput drug screening for therapeutic development. We optimized three different strategies for generating dorsal telencephalic NPCs from mouse and human pluripotent cell types through single or double inhibition of bone morphogenetic protein (BMP) and/or SMAD pathways. Mouse and human pluripotent cells were aggregated to form embryoid bodies in suspension and were treated with dorsomorphin alone (BMP inhibition) or combined with SB431542 (double BMP/SMAD inhibition) during neural induction. Neural rosettes were then selected from plated embryoid bodies to purify the population of dorsal NPCs. We tested the expression of key dorsal NPC markers as well as nonectodermal markers to confirm the efficiency of our three methods in comparison to published and commercial protocols. Single and double inhibition of BMP and/or SMAD during neural induction led to the efficient differentiation of dorsal NPCs, based on the high percentage of PAX6-positive cells and the NPC gene expression profile. There were no statistically significant differences in the variation of PAX6 and SOX1-positive NPCs between the two human pluripotent cell-derived methods; therefore, both methods are suitable for producing stable dorsal NPCs. When further differentiated into mature neurons, NPCs gave rise to a population of almost exclusively forebrain cortical neurons, confirming the dorsal fate commitment of the progenitors. The methods described in this study show improvements over previously published studies and are highly efficient at differentiating human and mouse pluripotent cell types into dorsal PAX6-positive NPCs and eventually into forebrain cortical neurons.

Conserved and Divergent Features of Mesenchymal Progenitor Cell Types within the Cortical Nephrogenic Niche of the Human and Mouse Kidney.

PubMed

Lindström, Nils O; Guo, Jinjin; Kim, Albert D; Tran, Tracy; Guo, Qiuyu; De Sena Brandine, Guilherme; Ransick, Andrew; Parvez, Riana K; Thornton, Matthew E; Basking, Laurence; Grubbs, Brendan; McMahon, Jill A; Smith, Andrew D; McMahon, Andrew P

2018-03-01

Cellular interactions among nephron, interstitial, and collecting duct progenitors drive mammalian kidney development. In mice, Six2 + nephron progenitor cells (NPCs) and Foxd1 + interstitial progenitor cells (IPCs) form largely distinct lineage compartments at the onset of metanephric kidney development. Here, we used the method for analyzing RNA following intracellular sorting (MARIS) approach, single-cell transcriptional profiling, in situ hybridization, and immunolabeling to characterize the presumptive NPC and IPC compartments of the developing human kidney. As in mice, each progenitor population adopts a stereotypical arrangement in the human nephron-forming niche: NPCs capped outgrowing ureteric branch tips, whereas IPCs were sandwiched between the NPCs and the renal capsule. Unlike mouse NPCs, human NPCs displayed a transcriptional profile that overlapped substantially with the IPC transcriptional profile, and key IPC determinants, including FOXD1 , were readily detected within SIX2 + NPCs. Comparative gene expression profiling in human and mouse Six2/SIX2 + NPCs showed broad agreement between the species but also identified species-biased expression of some genes. Notably, some human NPC-enriched genes, including DAPL1 and COL9A2 , are linked to human renal disease. We further explored the cellular diversity of mesenchymal cell types in the human nephrogenic niche through single-cell transcriptional profiling. Data analysis stratified NPCs into two main subpopulations and identified a third group of differentiating cells. These findings were confirmed by section in situ hybridization with novel human NPC markers predicted through the single-cell studies. This study provides a benchmark for the mesenchymal progenitors in the human nephrogenic niche and highlights species-variability in kidney developmental programs. Copyright © 2018 by the American Society of Nephrology.

GBM secretome induces transient transformation of human neural precursor cells.

PubMed

Venugopal, Chitra; Wang, X Simon; Manoranjan, Branavan; McFarlane, Nicole; Nolte, Sara; Li, Meredith; Murty, Naresh; Siu, K W Michael; Singh, Sheila K

2012-09-01

Glioblastoma (GBM) is the most aggressive primary brain tumor in humans, with a uniformly poor prognosis. The tumor microenvironment is composed of both supportive cellular substrates and exogenous factors. We hypothesize that exogenous factors secreted by brain tumor initiating cells (BTICs) could predispose normal neural precursor cells (NPCs) to transformation. When NPCs are grown in GBM-conditioned media, and designated as "tumor-conditioned NPCs" (tcNPCs), they become highly proliferative and exhibit increased stem cell self-renewal, or the unique ability of stem cells to asymmetrically generate another stem cell and a daughter cell. tcNPCs also show an increased transcript level of stem cell markers such as CD133 and ALDH and growth factor receptors such as VEGFR1, VEGFR2, EGFR and PDGFRα. Media analysis by ELISA of GBM-conditioned media reveals an elevated secretion of growth factors such as EGF, VEGF and PDGF-AA when compared to normal neural stem cell-conditioned media. We also demonstrate that tcNPCs require prolonged or continuous exposure to the GBM secretome in vitro to retain GBM BTIC characteristics. Our in vivo studies reveal that tcNPCs are unable to form tumors, confirming that irreversible transformation events may require sustained or prolonged presence of the GBM secretome. Analysis of GBM-conditioned media by mass spectrometry reveals the presence of secreted proteins Chitinase-3-like 1 (CHI3L1) and H2A histone family member H2AX. Collectively, our data suggest that GBM-secreted factors are capable of transiently altering normal NPCs, although for retention of the transformed phenotype, sustained or prolonged secretome exposure or additional transformation events are likely necessary.

Nup98 FG domains from diverse species spontaneously phase-separate into particles with nuclear pore-like permselectivity

PubMed Central

Schmidt, Hermann Broder; Görlich, Dirk

2015-01-01

Nuclear pore complexes (NPCs) conduct massive transport mediated by shuttling nuclear transport receptors (NTRs), while keeping nuclear and cytoplasmic contents separated. The NPC barrier in Xenopus relies primarily on the intrinsically disordered FG domain of Nup98. We now observed that Nup98 FG domains of mammals, lancelets, insects, nematodes, fungi, plants, amoebas, ciliates, and excavates spontaneously and rapidly phase-separate from dilute (submicromolar) aqueous solutions into characteristic ‘FG particles’. This required neither sophisticated experimental conditions nor auxiliary eukaryotic factors. Instead, it occurred already during FG domain expression in bacteria. All Nup98 FG phases rejected inert macromolecules and yet allowed far larger NTR cargo complexes to rapidly enter. They even recapitulated the observations that large cargo-domains counteract NPC passage of NTR⋅cargo complexes, while cargo shielding and increased NTR⋅cargo surface-ratios override this inhibition. Their exquisite NPC-typical sorting selectivity and strong intrinsic assembly propensity suggest that Nup98 FG phases can form in authentic NPCs and indeed account for the permeability properties of the pore. DOI: http://dx.doi.org/10.7554/eLife.04251.001 PMID:25562883

CCL11 promotes migration and proliferation of mouse neural progenitor cells.

PubMed

Wang, Feifei; Baba, Nobuyasu; Shen, Yuan; Yamashita, Tatsuyuki; Tsuru, Emi; Tsuda, Masayuki; Maeda, Nagamasa; Sagara, Yusuke

2017-02-07

Neonatal hypoxia-ischemia induces massive brain damage during the perinatal period, resulting in long-term consequences to central nervous system structural and functional maturation. Although neural progenitor cells (NPCs) migrate through the parenchyma and home in to injury sites in the rodent brain, the molecular mechanisms are unknown. We examined the role of chemokines in mediating NPC migration after neonatal hypoxic-ischemic brain injury. Nine-day-old mice were exposed to a 120-minute hypoxia following unilateral carotid occlusion. Chemokine levels were quantified in mouse brain extract. Migration and proliferation assays were performed using embryonic and infant mouse NPCs. The neonatal hypoxic-ischemic brain injury resulted in an ipsilateral lesion, which was extended to the cortical and striatal areas. NPCs migrated toward an injured area, where a marked increase of CC chemokines was detected. In vitro studies showed that incubation of NPCs with recombinant mouse CCL11 promoted migration and proliferation. These effects were partly inhibited by a CCR3 antagonist, SB297006. Our data implicate an important effect of CCL11 for mouse NPCs. The effective activation of NPCs may offer a promising strategy for neuroregeneration in neonatal hypoxic-ischemic brain injury.

Extracellular Vesicles from Neural Stem Cells Transfer IFN-γ via Ifngr1 to Activate Stat1 Signaling in Target Cells

PubMed Central

Cossetti, Chiara; Iraci, Nunzio; Mercer, Tim R.; Leonardi, Tommaso; Alpi, Emanuele; Drago, Denise; Alfaro-Cervello, Clara; Saini, Harpreet K.; Davis, Matthew P.; Schaeffer, Julia; Vega, Beatriz; Stefanini, Matilde; Zhao, CongJian; Muller, Werner; Garcia-Verdugo, Jose Manuel; Mathivanan, Suresh; Bachi, Angela; Enright, Anton J.; Mattick, John S.; Pluchino, Stefano

2015-01-01

SUMMARY The idea that stem cell therapies work only via cell replacement is challenged by the observation of consistent intercellular molecule exchange between the graft and the host. Here we defined a mechanism of cellular signaling by which neural stem/precursor cells (NPCs) communicate with the microenvironment via extracellular vesicles (EVs), and we elucidated its molecular signature and function. We observed cytokine-regulated pathways that sort proteins and mRNAs into EVs. We described induction of interferon gamma (IFN-γ) pathway in NPCs exposed to proinflammatory cytokines that is mirrored in EVs. We showed that IFN-γ bound to EVs through Ifngr1 activates Stat1 in target cells. Finally, we demonstrated that endogenous Stat1 and Ifngr1 in target cells are indispensable to sustain the activation of Stat1 signaling by EV-associated IFN-γ/Ifngr1 complexes. Our study identifies a mechanism of cellular signaling regulated by EV-associated IFN-γ/Ifngr1 complexes, which grafted stem cells may use to communicate with the host immune system. PMID:25242146

Plant nuclear pore complex proteins are modified by novel oligosaccharides with terminal N-acetylglucosamine.

PubMed Central

Heese-Peck, A; Cole, R N; Borkhsenious, O N; Hart, G W; Raikhel, N V

1995-01-01

Only a few nuclear pore complex (NPC) proteins, mainly in vertebrates and yeast but none in plants, have been well characterized. As an initial step to identify plant NPC proteins, we examined whether NPC proteins from tobacco are modified by N-acetylglucosamine (GlcNAc). Using wheat germ agglutinin, a lectin that binds specifically to GlcNAc in plants, specific labeling was often found associated with or adjacent to NPCs. Nuclear proteins containing GlcNAc can be partially extracted by 0.5 M salt, as shown by a wheat germ agglutinin blot assay, and at least eight extracted proteins were modified by terminal GlcNAc, as determined by in vitro galactosyltransferase assays. Sugar analysis indicated that the plant glycans with terminal GlcNAc differ from the single O-linked GlcNAc of vertebrate NPC proteins in that they consist of oligosaccharides that are larger in size than five GlcNAc residues. Most of these appear to be bound to proteins via a hydroxyl group. This novel oligosaccharide modification may convey properties to the plant NPC that are different from those of vertebrate NPCs. PMID:8589629

Nuclear pore complex-mediated modulation of TCR signaling is required for naïve CD4+ T cell homeostasis.

PubMed

Borlido, Joana; Sakuma, Stephen; Raices, Marcela; Carrette, Florent; Tinoco, Roberto; Bradley, Linda M; D'Angelo, Maximiliano A

2018-06-01

Nuclear pore complexes (NPCs) are channels connecting the nucleus with the cytoplasm. We report that loss of the tissue-specific NPC component Nup210 causes a severe deficit of naïve CD4 + T cells. Nup210-deficient CD4 + T lymphocytes develop normally but fail to survive in the periphery. The decreased survival results from both an impaired ability to transmit tonic T cell receptor (TCR) signals and increased levels of Fas, which sensitize Nup210 -/- naïve CD4 + T cells to Fas-mediated cell death. Mechanistically, Nup210 regulates these processes by modulating the expression of Cav2 (encoding Caveolin-2) and Jun at the nuclear periphery. Whereas the TCR-dependent and CD4 + T cell-specific upregulation of Cav2 is critical for proximal TCR signaling, cJun expression is required for STAT3-dependent repression of Fas. Our results uncover an unexpected role for Nup210 as a cell-intrinsic regulator of TCR signaling and T cell homeostasis and expose NPCs as key players in the adaptive immune system.

Vesicular glutamate transporters play a role in neuronal differentiation of cultured SVZ-derived neural precursor cells

PubMed Central

Sánchez-Mendoza, Eduardo H.; Bellver-Landete, Victor; Arce, Carmen; Doeppner, Thorsten R.; Hermann, Dirk M.

2017-01-01

The role of glutamate in the regulation of neurogenesis is well-established, but the role of vesicular glutamate transporters (VGLUTs) and excitatory amino acid transporters (EAATs) in controlling adult neurogenesis is unknown. Here we investigated the implication of VGLUTs in the differentiation of subventricular zone (SVZ)-derived neural precursor cells (NPCs). Our results show that NPCs express VGLUT1-3 and EAAT1-3 both at the mRNA and protein level. Their expression increases during differentiation closely associated with the expression of marker genes. In expression analyses we show that VGLUT1 and VGLUT2 are preferentially expressed by cultured SVZ-derived doublecortin+ neuroblasts, while VGLUT3 is found on GFAP+ glial cells. In cultured NPCs, inhibition of VGLUT by Evans Blue increased the mRNA level of neuronal markers doublecortin, B3T and MAP2, elevated the number of NPCs expressing doublecortin protein and promoted the number of cells with morphological appearance of branched neurons, suggesting that VGLUT function prevents neuronal differentiation of NPCs. This survival- and differentiation-promoting effect of Evans blue was corroborated by increased AKT phosphorylation and reduced MAPK phosphorylation. Thus, under physiological conditions, VGLUT1-3 inhibition, and thus decreased glutamate exocytosis, may promote neuronal differentiation of NPCs. PMID:28493916

Layered hydrogels accelerate iPSC-derived neuronal maturation and reveal migration defects caused by MeCP2 dysfunction

NASA Astrophysics Data System (ADS)

Zhang, Zhen-Ning; Freitas, Beatriz C.; Qian, Hao; Lux, Jacques; Acab, Allan; Trujillo, Cleber A.; Herai, Roberto H.; Nguyen Huu, Viet Anh; Wen, Jessica H.; Joshi-Barr, Shivanjali; Karpiak, Jerome V.; Engler, Adam J.; Fu, Xiang-Dong; Muotri, Alysson R.; Almutairi, Adah

2016-03-01

Probing a wide range of cellular phenotypes in neurodevelopmental disorders using patient-derived neural progenitor cells (NPCs) can be facilitated by 3D assays, as 2D systems cannot entirely recapitulate the arrangement of cells in the brain. Here, we developed a previously unidentified 3D migration and differentiation assay in layered hydrogels to examine how these processes are affected in neurodevelopmental disorders, such as Rett syndrome. Our soft 3D system mimics the brain environment and accelerates maturation of neurons from human induced pluripotent stem cell (iPSC)-derived NPCs, yielding electrophysiologically active neurons within just 3 wk. Using this platform, we revealed a genotype-specific effect of methyl-CpG-binding protein-2 (MeCP2) dysfunction on iPSC-derived neuronal migration and maturation (reduced neurite outgrowth and fewer synapses) in 3D layered hydrogels. Thus, this 3D system expands the range of neural phenotypes that can be studied in vitro to include those influenced by physical and mechanical stimuli or requiring specific arrangements of multiple cell types.

Aging Neural Progenitor Cells Have Decreased Mitochondrial Content and Lower Oxidative Metabolism*

PubMed Central

Stoll, Elizabeth A.; Cheung, Willy; Mikheev, Andrei M.; Sweet, Ian R.; Bielas, Jason H.; Zhang, Jing; Rostomily, Robert C.; Horner, Philip J.

2011-01-01

Although neurogenesis occurs in discrete areas of the adult mammalian brain, neural progenitor cells (NPCs) produce fewer new neurons with age. To characterize the molecular changes that occur during aging, we performed a proteomic comparison between primary-cultured NPCs from the young adult and aged mouse forebrain. This analysis yielded changes in proteins necessary for cellular metabolism. Mitochondrial quantity and oxygen consumption rates decrease with aging, although mitochondrial DNA in aged NPCs does not have increased mutation rates. In addition, aged cells are resistant to the mitochondrial inhibitor rotenone and proliferate in response to lowered oxygen conditions. These results demonstrate that aging NPCs display an altered metabolic phenotype, characterized by a coordinated shift in protein expression, subcellular structure, and metabolic physiology. PMID:21900249

Non-parenchymal liver cells support the growth advantage in the first stages of hepatocarcinogenesis.

PubMed

Drucker, Claudia; Parzefall, Wolfram; Teufelhofer, Olga; Grusch, Michael; Ellinger, Adolf; Schulte-Hermann, Rolf; Grasl-Kraupp, Bettina

2006-01-01

Hepatocellular carcinoma almost always arises in chronically inflamed livers. We developed a culture model to study the role of non-parenchymal cells (NPCs) for inflammation-driven hepatocarcinogenesis. Rats were treated with the carcinogen N-nitrosomorpholine, which induced initiated hepatocytes expressing the marker placental glutathione-S-transferase (GSTp). After 21 days two preparations of hepatocytes were made: (i) conventional ones (Hep-conv) containing NPCs and (ii) hepatocytes purified of NPCs (Hep-pur). Initiated hepatocytes, being positive for GSTp (GSTp-pos) were present in both preparations and were cultured along with normal hepatocytes, being negative for GSTp (GSTp-neg). Under any culture condition DNA synthesis was approximately 4-fold higher in GSTp-pos than in GSTp-neg hepatocytes demonstrating the inherent growth advantage of the first stages of hepatocarcinogenesis. Hepatocytes showed approximately 3-fold lower rates of DNA synthesis in Hep-pur than in Hep-conv, which was elevated above Hep-conv levels by addition of NPC or NPC-supernatant. Pretreatment of NPCs with proinflammatory lipopolysaccharide (LPS) further increased DNA synthesis. Thus, NPCs release soluble growth stimulators. Next we investigated the effect of specific cytokines produced by NPCs. Tumour necrosis factor alpha and interleukin 6 barely altered DNA synthesis, whereas hepatocyte growth factor (HGF), keratinocyte growth factor (KGF) and the heparin-binding epidermal growth factor-like growth factor (HB-EGF) were potent inducers of DNA replication in both, GSTp-neg and GSTp-pos cells. In conclusion, DNA synthesis of hepatocytes is increased by factors released from NPCs, an effect augmented by LPS-stimulation. NPC-derived cytokines, such as KGF, HGF and HB-EGF, stimulate DNA synthesis preferentially in initiated hepatocytes, presumably resulting in tumour promotion. Similar mechanisms may contribute to carcinogenesis in human inflammatory liver diseases.

Pluripotent stem cell-derived radial glia-like cells as stable intermediate for efficient generation of human oligodendrocytes.

PubMed

Gorris, Raphaela; Fischer, Julia; Erwes, Kim Lina; Kesavan, Jaideep; Peterson, Daniel A; Alexander, Michael; Nöthen, Markus M; Peitz, Michael; Quandel, Tamara; Karus, Michael; Brüstle, Oliver

2015-12-01

Neural precursor cells (NPCs) derived from human pluripotent stem cells (hPSCs) represent an attractive tool for the in vitro generation of various neural cell types. However, the developmentally early NPCs emerging during hPSC differentiation typically show a strong propensity for neuronal differentiation, with more limited potential for generating astrocytes and, in particular, for generating oligodendrocytes. This phenomenon corresponds well to the consecutive and protracted generation of neurons and GLIA during normal human development. To obtain a more gliogenic NPC type, we combined growth factor-mediated expansion with pre-exposure to the differentiation-inducing agent retinoic acid and subsequent immunoisolation of CD133-positive cells. This protocol yields an adherent and self-renewing population of hindbrain/spinal cord radial glia (RG)-like neural precursor cells (RGL-NPCs) expressing typical neural stem cell markers such as nestin, ASCL1, SOX2, and PAX6 as well as RG markers BLBP, GLAST, vimentin, and GFAP. While RGL-NPCs maintain the ability for tripotential differentiation into neurons, astrocytes, and oligodendrocytes, they exhibit greatly enhanced propensity for oligodendrocyte generation. Under defined differentiation conditions promoting the expression of the major oligodendrocyte fate-determinants OLIG1/2, NKX6.2, NKX2.2, and SOX10, RGL-NPCs efficiently convert into NG2-positive oligodendroglial progenitor cells (OPCs) and are subsequently capable of in vivo myelination. Representing a stable intermediate between PSCs and OPCs, RGL-NPCs expedite the generation of PSC-derived oligodendrocytes with O4-, 4860-, and myelin basic protein (MBP)-positive cells that already appear within 7 weeks following growth factor withdrawal-induced differentiation. Thus, RGL-NPCs may serve as robust tool for time-efficient generation of human oligodendrocytes from embryonic and induced pluripotent stem cells. © 2015 Wiley Periodicals, Inc.

The Effect of Agmatine on Expression of IL-1β and TLX Which Promotes Neuronal Differentiation in Lipopolysaccharide-Treated Neural Progenitors.

PubMed

Song, Juhyun; Kumar, Bokara Kiran; Kang, Somang; Park, Kyung Ah; Lee, Won Taek; Lee, Jong Eun

2013-12-01

Differentiation of neural progenitor cells (NPCs) is important for protecting neural cells and brain tissue during inflammation. Interleukin-1 beta (IL-1β) is the most common pro- inflammatory cytokine in brain inflammation, and increased IL-1β levels can decrease the proliferation of NPCs. We aimed to investigate whether agmatine (Agm), a primary polyamine that protects neural cells, could trigger differentiation of NPCs by activating IL-1β in vitro. The cortex of ICR mouse embryos (E14) was dissociated to culture NPCs. NPCs were stimulated by lipopolysaccharide (LPS). After 6 days, protein expression of stem cell markers and differentiation signal factors was confirmed by using western blot analysis. Also, immunocytochemistry was used to confirm the cell fate. Agm treatment activated NPC differentiation significantly more than in the control group, which was evident by the increased expression of a neuronal marker, MAP2, in the LPS-induced, Agm-treated group. Differentiation of LPS-induced, Agm-treated NPCs was regulated by the MAPK pathway and is thought to be related to IL-1β activation and decreased expression of TLX, a transcription factor that regulates NPC differentiation. Our results reveal that Agm can promote NPC differentiation to neural stem cells by modulating IL-1β expression under inflammatory condition, and they suggest that Agm may be a novel therapeutic strategy for neuroinflammatory diseases.

Prolyl Isomerase Pin1 Regulates Neuronal Differentiation via β-Catenin

PubMed Central

Nakamura, Kazuhiro; Kosugi, Isao; Lee, Daniel Y.; Hafner, Angela; Sinclair, David A.

2012-01-01

The Wnt/β-catenin pathway promotes proliferation of neural progenitor cells (NPCs) at early stages and induces neuronal differentiation from NPCs at late stages, but the molecular mechanisms that control this stage-specific response are unclear. Pin1 is a prolyl isomerase that regulates cell signaling uniquely by controlling protein conformation after phosphorylation, but its role in neuronal differentiation is not known. Here we found that whereas Pin1 depletion suppresses neuronal differentiation, Pin1 overexpression enhances it, without any effects on gliogenesis from NPCs in vitro. Consequently, Pin1-null mice have significantly fewer upper layer neurons in the motor cortex and severely impaired motor activity during the neonatal stage. A proteomic approach identified β-catenin as a major substrate for Pin1 in NPCs, in which Pin1 stabilizes β-catenin. As a result, Pin1 knockout leads to reduced β-catenin during differentiation but not proliferation of NPCs in developing brains. Importantly, defective neuronal differentiation in Pin1 knockout NPCs is fully rescued in vitro by overexpression of β-catenin but not a β-catenin mutant that fails to act as a Pin1 substrate. These results show that Pin1 is a novel regulator of NPC differentiation by acting on β-catenin and provides a new postphosphorylation signaling mechanism to regulate developmental stage-specific functioning of β-catenin signaling in neuronal differentiation. PMID:22645310

Non-physician cataract surgeons in Sub-Saharan Africa: situation analysis.

PubMed

Lewallen, Susan; Etya'ale, Daniel; Kello, Amir Bedri; Courtright, Paul

2012-11-01

Non-physician cataract surgeons (NPCS) provide cataract surgical services in some Sub-Saharan African (SSA) countries. However, their training, placement, legal framework and supervision have not been documented. We sought to do so to inform decision-making regarding future training. Standard questionnaires were sent to national eye coordinators and other ophthalmologic leaders in Africa to collect information. Face-to-face interviews were conducted at training programmes in Ethiopia, Tanzania and Kenya, and email interviews were conducted with directors at training programmes in the Gambia and Malawi. Responses were provided for 31/39 (79%) countries to which questionnaires were sent. These countries represent about 90% of the population of SSA. Overall, 17 countries have one or more NPCS; two-thirds of the total 245 NPCS are found in only three countries. Thirty-six percent of NPCS work alone, but a formal functioning supervision system was reported to be present in only one country. The training centres are similar and face similar challenges. There is considerable variation across SSA in the use and acceptance of NPCS. The placement and support of NPCS after training generally does not follow expectations, and training centres have little role in this. Overall, there was no consensus on whether the cadre, as it is currently viewed, is necessary, desirable or will contribute to addressing cataract surgical needs in SSA. © 2012 Blackwell Publishing Ltd.

Mutations in glycyl-tRNA synthetase impair mitochondrial metabolism in neurons.

PubMed

Boczonadi, Veronika; Meyer, Kathrin; Gonczarowska-Jorge, Humberto; Griffin, Helen; Roos, Andreas; Bartsakoulia, Marina; Bansagi, Boglarka; Ricci, Giulia; Palinkas, Fanni; Zahedi, René P; Bruni, Francesco; Kaspar, Brian; Lochmüller, Hanns; Boycott, Kym M; Müller, Juliane S; Horvath, Rita

2018-06-15

The nuclear-encoded glycyl-tRNA synthetase gene (GARS) is essential for protein translation in both cytoplasm and mitochondria. In contrast, different genes encode the mitochondrial and cytosolic forms of most other tRNA synthetases. Dominant GARS mutations were described in inherited neuropathies, while recessive mutations cause severe childhood-onset disorders affecting skeletal muscle and heart. The downstream events explaining tissue-specific phenotype-genotype relations remained unclear. We investigated the mitochondrial function of GARS in human cell lines and in the GarsC210R mouse model. Human-induced neuronal progenitor cells (iNPCs) carrying dominant and recessive GARS mutations showed alterations of mitochondrial proteins, which were more prominent in iNPCs with dominant, neuropathy-causing mutations. Although comparative proteomic analysis of iNPCs showed significant changes in mitochondrial respiratory chain complex subunits, assembly genes, Krebs cycle enzymes and transport proteins in both recessive and dominant mutations, proteins involved in fatty acid oxidation were only altered by recessive mutations causing mitochondrial cardiomyopathy. In contrast, significant alterations of the vesicle-associated membrane protein-associated protein B (VAPB) and its downstream pathways such as mitochondrial calcium uptake and autophagy were detected in dominant GARS mutations. The role of VAPB has been supported by similar results in the GarsC210R mice. Our data suggest that altered mitochondria-associated endoplasmic reticulum (ER) membranes (MAM) may be important disease mechanisms leading to neuropathy in this condition.

Ultrastructural Characteristics of Rat Hepatic Oval Cells and Their Intercellular Contacts in the Model of Biliary Fibrosis: New Insights into Experimental Liver Fibrogenesis

PubMed Central

Lebensztejn, Dariusz Marek; Daniluk, Urszula; Sobaniec, Piotr; Sendrowski, Krzysztof; Daniluk, Jaroslaw; Debek, Wojciech

2017-01-01

Purpose Recently, it has been emphasized that hepatic progenitor/oval cells (HPCs) are significantly involved in liver fibrogenesis. We evaluated the multipotential population of HPCs by transmission electron microscope (TEM), including relations with adherent hepatic nonparenchymal cells (NPCs) in rats with biliary fibrosis induced by bile duct ligation (BDL). Methods The study used 6-week-old Wistar Crl: WI(Han) rats after BDL for 1, 6, and 8 weeks. Results Current ultrastructural analysis showed considerable proliferation of HPCs in experimental intensive biliary fibrosis. HPCs formed proliferating bile ductules and were scattered in periportal connective tissue. We distinguished 4 main types of HPCs: 0, I, II (bile duct-like cells; most common), and III (hepatocyte-like cells). We observed, very seldom presented in literature, cellular interactions between HPCs and adjacent NPCs, especially commonly found transitional hepatic stellate cells (T-HSCs) and Kupffer cells/macrophages. We showed the phenomenon of penetration of the basement membrane of proliferating bile ductules by cytoplasmic processes sent by T-HSCs and the formation of direct cell-cell contact with ductular epithelial cells related to HPCs. Conclusions HPC proliferation induced by BDL evidently promotes portal fibrogenesis. Better understanding of the complex cellular interactions between HPCs and adjacent NPCs, especially T-HSCs, may help develop antifibrotic therapies in the future. PMID:28769978

Non-physician Clinicians in Sub-Saharan Africa and the Evolving Role of Physicians

PubMed Central

Eyal, Nir; Cancedda, Corrado; Kyamanywa, Patrick; Hurst, Samia A.

2016-01-01

Responding to critical shortages of physicians, most sub-Saharan countries have scaled up training of non-physician clinicians (NPCs), resulting in a gradual but decisive shift to NPCs as the cornerstone of healthcare delivery. This development should unfold in parallel with strategic rethinking about the role of physicians and with innovations in physician education and in-service training. In important ways, a growing number of NPCs only renders physicians more necessary – for example, as specialized healthcare providers and as leaders, managers, mentors, and public health administrators. Physicians in sub-Saharan Africa ought to be trained in all of these capacities. This evolution in the role of physicians may also help address known challenges to the successful integration of NPCs in the health system. PMID:26927585

Testing Brain Overgrowth and Synaptic Models of Autism Using NPCs and Neurons From Patient Derived iPS Cells

DTIC Science & Technology

2015-12-01

AWARD NUMBER: W81XWH-13-1-0414 TITLE: Testing Brain Overgrowth and Synaptic Models of Autism Using NPCs and Neurons From Patient-Derived iPS...3. DATES COVERED 15 Sep 2013 - 14 Sep 2015 4. TITLE AND SUBTITLE Testing Brain Overgrowth and Synaptic Models of Autism Using NPCs and Neurons From...AVAILABILITY STATEMENT Approved for Public Release; Distribution Unlimited 13. SUPPLEMENTARY NOTES 14. ABSTRACT Autism and autism spectrum disorders (ASD

Conditioned Medium Derived from Notochordal Cell-Rich Nucleus Pulposus Tissue Stimulates Matrix Production by Canine Nucleus Pulposus Cells and Bone Marrow-Derived Stromal Cells

PubMed Central

de Vries, Stefan A.H.; Potier, Esther; van Doeselaar, Marina; Meij, Björn P.; Tryfonidou, Marianna A.

2015-01-01

Objectives: Conditioned medium derived from notochordal cell-rich nucleus pulposus tissue (NCCM) was previously shown to have a stimulatory effect on bone marrow stromal cells (BMSCs) and nucleus pulposus cells (NPCs) individually, in mixed species in vitro cell models. The objective of the current study was to assess the stimulatory effect of NCCM on NPCs in a homologous canine in vitro model and to investigate whether combined stimulation with NCCM and addition of BMSCs provides a synergistic stimulatory effect. Methods: BMSCs and NPCs were harvested from chondrodystrophic dogs with confirmed early intervertebral disc (IVD) degeneration. NCCM was produced from NP tissue of nonchondrodystrophic dogs with healthy IVDs. BMSCs or NPCs alone (3×106 cells/mL) and NPCs+BMSCs (6×106 cells/mL; mixed 1:1) were cultured for 4 weeks in 1.2% alginate beads under base medium (BM), NCCM, or with addition of 10 ng/mL transforming growth factor-β1 (TGF-β1) as a positive control. Beads were assessed for glycosaminoglycan (GAG) and DNA contents by biochemical assays, GAG deposition by Alcian blue staining, and gene expression (aggrecan, versican, collagen 1 and 2, SOX9, A disintegrin and metalloproteinase with thrombospondin motifs 5 (ADAMTS5), and matrix metalloproteinase 13 [MMP13]) with real-time quantitative RT-PCR. Results: NCCM increased NPC proliferation, proteoglycan production, and expression of genes associated with a healthy NP-like phenotype. BMSCs also showed increased proteoglycan production under NCCM, but these effects were not observed at the gene level. Combined stimulation of NPCs with NCCM and coculturing with BMSCs did not result in increased proteoglycan content compared to stimulation with NCCM alone. Discussion: NCCM stimulates matrix production by both NPCs and BMSCs and directs NPCs toward a healthier phenotype. NCCM is therefore promising for IVD regeneration and identification of the bioactive components will be helpful to further develop this approach. In the current study, no synergistic effect of adding BMSCs was observed. PMID:25370929

Entry into the nuclear pore complex is controlled by a cytoplasmic exclusion zone containing dynamic GLFG-repeat nucleoporin domains.

PubMed

Fiserova, Jindriska; Spink, Matthew; Richards, Shane A; Saunter, Christopher; Goldberg, Martin W

2014-01-01

Nuclear pore complexes (NPCs) mediate nucleocytoplasmic movement. The central channel contains proteins with phenylalanine-glycine (FG) repeats, or variations (GLFG, glycine-leucine-phenylalanine-glycine). These are 'intrinsically disordered' and often represent weak interaction sites that become ordered upon interaction. We investigated this possibility during nuclear transport. Using electron microscopy of S. cerevisiae, we show that NPC cytoplasmic filaments form a dome-shaped structure enclosing GLFG domains. GLFG domains extend out of this structure and are part of an 'exclusion zone' that might act as a partial barrier to entry of transport-inert proteins. The anchor domain of a GLFG nucleoporin locates exclusively to the central channel. By contrast, the localisation of the GLFG domains varied between NPCs and could be cytoplasmic, central or nucleoplasmic and could stretch up to 80 nm. These results suggest a dynamic exchange between ordered and disordered states. In contrast to diffusion through the NPC, transport cargoes passed through the exclusion zone and accumulated near the central plane. We also show that movement of cargo through the NPC is accompanied by relocation of GLFG domains, suggesting that binding, restructuring and movement of these domains could be part of the translocation mechanism.

Nanofibrous scaffolds for the guidance of stem cell-derived neurons for auditory nerve regeneration.

PubMed

Hackelberg, Sandra; Tuck, Samuel J; He, Long; Rastogi, Arjun; White, Christina; Liu, Liqian; Prieskorn, Diane M; Miller, Ryan J; Chan, Che; Loomis, Benjamin R; Corey, Joseph M; Miller, Josef M; Duncan, R Keith

2017-01-01

Impairment of spiral ganglion neurons (SGNs) of the auditory nerve is a major cause for hearing loss occurring independently or in addition to sensory hair cell damage. Unfortunately, mammalian SGNs lack the potential for autonomous regeneration. Stem cell based therapy is a promising approach for auditory nerve regeneration, but proper integration of exogenous cells into the auditory circuit remains a fundamental challenge. Here, we present novel nanofibrous scaffolds designed to guide the integration of human stem cell-derived neurons in the internal auditory meatus (IAM), the foramen allowing passage of the spiral ganglion to the auditory brainstem. Human embryonic stem cells (hESC) were differentiated into neural precursor cells (NPCs) and seeded onto aligned nanofiber mats. The NPCs terminally differentiated into glutamatergic neurons with high efficiency, and neurite projections aligned with nanofibers in vitro. Scaffolds were assembled by seeding GFP-labeled NPCs on nanofibers integrated in a polymer sheath. Biocompatibility and functionality of the NPC-seeded scaffolds were evaluated in vivo in deafened guinea pigs (Cavia porcellus). To this end, we established an ouabain-based deafening procedure that depleted an average 72% of SGNs from apex to base of the cochleae and caused profound hearing loss. Further, we developed a surgical procedure to implant seeded scaffolds directly into the guinea pig IAM. No evidence of an inflammatory response was observed, but post-surgery tissue repair appeared to be facilitated by infiltrating Schwann cells. While NPC survival was found to be poor, both subjects implanted with NPC-seeded and cell-free control scaffolds showed partial recovery of electrically-evoked auditory brainstem thresholds. Thus, while future studies must address cell survival, nanofibrous scaffolds pose a promising strategy for auditory nerve regeneration.

Non-physician Clinicians in Sub-Saharan Africa and the Evolving Role of Physicians.

PubMed

Eyal, Nir; Cancedda, Corrado; Kyamanywa, Patrick; Hurst, Samia A

2015-12-30

Responding to critical shortages of physicians, most sub-Saharan countries have scaled up training of non-physician clinicians (NPCs), resulting in a gradual but decisive shift to NPCs as the cornerstone of healthcare delivery. This development should unfold in parallel with strategic rethinking about the role of physicians and with innovations in physician education and in-service training. In important ways, a growing number of NPCs only renders physicians more necessary - for example, as specialized healthcare providers and as leaders, managers, mentors, and public health administrators. Physicians in sub-Saharan Africa ought to be trained in all of these capacities. This evolution in the role of physicians may also help address known challenges to the successful integration of NPCs in the health system. © 2016 by Kerman University of Medical Sciences.

A chemically defined substrate for the expansion and neuronal differentiation of human pluripotent stem cell-derived neural progenitor cells.

PubMed

Tsai, Yihuan; Cutts, Josh; Kimura, Azuma; Varun, Divya; Brafman, David A

2015-07-01

Due to the limitation of current pharmacological therapeutic strategies, stem cell therapies have emerged as a viable option for treating many incurable neurological disorders. Specifically, human pluripotent stem cell (hPSC)-derived neural progenitor cells (hNPCs), a multipotent cell population that is capable of near indefinite expansion and subsequent differentiation into the various cell types that comprise the central nervous system (CNS), could provide an unlimited source of cells for such cell-based therapies. However the clinical application of these cells will require (i) defined, xeno-free conditions for their expansion and neuronal differentiation and (ii) scalable culture systems that enable their expansion and neuronal differentiation in numbers sufficient for regenerative medicine and drug screening purposes. Current extracellular matrix protein (ECMP)-based substrates for the culture of hNPCs are expensive, difficult to isolate, subject to batch-to-batch variations, and, therefore, unsuitable for clinical application of hNPCs. Using a high-throughput array-based screening approach, we identified a synthetic polymer, poly(4-vinyl phenol) (P4VP), that supported the long-term proliferation and self-renewal of hNPCs. The hNPCs cultured on P4VP maintained their characteristic morphology, expressed high levels of markers of multipotency, and retained their ability to differentiate into neurons. Such chemically defined substrates will eliminate critical roadblocks for the utilization of hNPCs for human neural regenerative repair, disease modeling, and drug discovery. Copyright © 2015. Published by Elsevier B.V.

Derivation and Expansion Using Only Small Molecules of Human Neural Progenitors for Neurodegenerative Disease Modeling

PubMed Central

Reinhardt, Peter; Glatza, Michael; Hemmer, Kathrin; Tsytsyura, Yaroslav; Thiel, Cora S.; Höing, Susanne; Moritz, Sören; Parga, Juan A.; Wagner, Lydia; Bruder, Jan M.; Wu, Guangming; Schmid, Benjamin; Röpke, Albrecht; Klingauf, Jürgen; Schwamborn, Jens C.; Gasser, Thomas; Schöler, Hans R.; Sterneckert, Jared

2013-01-01

Phenotypic drug discovery requires billions of cells for high-throughput screening (HTS) campaigns. Because up to several million different small molecules will be tested in a single HTS campaign, even small variability within the cell populations for screening could easily invalidate an entire campaign. Neurodegenerative assays are particularly challenging because neurons are post-mitotic and cannot be expanded for implementation in HTS. Therefore, HTS for neuroprotective compounds requires a cell type that is robustly expandable and able to differentiate into all of the neuronal subtypes involved in disease pathogenesis. Here, we report the derivation and propagation using only small molecules of human neural progenitor cells (small molecule neural precursor cells; smNPCs). smNPCs are robust, exhibit immortal expansion, and do not require cumbersome manual culture and selection steps. We demonstrate that smNPCs have the potential to clonally and efficiently differentiate into neural tube lineages, including motor neurons (MNs) and midbrain dopaminergic neurons (mDANs) as well as neural crest lineages, including peripheral neurons and mesenchymal cells. These properties are so far only matched by pluripotent stem cells. Finally, to demonstrate the usefulness of smNPCs we show that mDANs differentiated from smNPCs with LRRK2 G2019S are more susceptible to apoptosis in the presence of oxidative stress compared to wild-type. Therefore, smNPCs are a powerful biological tool with properties that are optimal for large-scale disease modeling, phenotypic screening, and studies of early human development. PMID:23533608

Brownian Dynamics Simulation of Nucleocytoplasmic Transport: A Coarse-Grained Model for the Functional State of the Nuclear Pore Complex

PubMed Central

Moussavi-Baygi, Ruhollah; Jamali, Yousef; Karimi, Reza; Mofrad, Mohammad R. K.

2011-01-01

The nuclear pore complex (NPC) regulates molecular traffic across the nuclear envelope (NE). Selective transport happens on the order of milliseconds and the length scale of tens of nanometers; however, the transport mechanism remains elusive. Central to the transport process is the hydrophobic interactions between karyopherins (kaps) and Phe-Gly (FG) repeat domains. Taking into account the polymeric nature of FG-repeats grafted on the elastic structure of the NPC, and the kap-FG hydrophobic affinity, we have established a coarse-grained model of the NPC structure that mimics nucleocytoplasmic transport. To establish a foundation for future works, the methodology and biophysical rationale behind the model is explained in details. The model predicts that the first-passage time of a 15 nm cargo-complex is about 2.6±0.13 ms with an inverse Gaussian distribution for statistically adequate number of independent Brownian dynamics simulations. Moreover, the cargo-complex is primarily attached to the channel wall where it interacts with the FG-layer as it passes through the central channel. The kap-FG hydrophobic interaction is highly dynamic and fast, which ensures an efficient translocation through the NPC. Further, almost all eight hydrophobic binding spots on kap-β are occupied simultaneously during transport. Finally, as opposed to intact NPCs, cytoplasmic filaments-deficient NPCs show a high degree of permeability to inert cargos, implying the defining role of cytoplasmic filaments in the selectivity barrier. PMID:21673865

Bmp6 Expression in Murine Liver Non Parenchymal Cells: A Mechanism to Control their High Iron Exporter Activity and Protect Hepatocytes from Iron Overload?

PubMed Central

Rausa, Marco; Pagani, Alessia; Nai, Antonella; Campanella, Alessandro; Gilberti, Maria Enrica; Apostoli, Pietro; Camaschella, Clara; Silvestri, Laura

2015-01-01

Bmp6 is the main activator of hepcidin, the liver hormone that negatively regulates plasma iron influx by degrading the sole iron exporter ferroportin in enterocytes and macrophages. Bmp6 expression is modulated by iron but the molecular mechanisms are unknown. Although hepcidin is expressed almost exclusively by hepatocytes (HCs), Bmp6 is produced also by non-parenchymal cells (NPCs), mainly sinusoidal endothelial cells (LSECs). To investigate the regulation of Bmp6 in HCs and NPCs, liver cells were isolated from adult wild type mice whose diet was modified in iron content in acute or chronic manner and in disease models of iron deficiency (Tmprss6 KO mouse) and overload (Hjv KO mouse). With manipulation of dietary iron in wild-type mice, Bmp6 and Tfr1 expression in both HCs and NPCs was inversely related, as expected. When hepcidin expression is abnormal in murine models of iron overload (Hjv KO mice) and deficiency (Tmprss6 KO mice), Bmp6 expression in NPCs was not related to Tfr1. Despite the low Bmp6 in NPCs from Tmprss6 KO mice, Tfr1 mRNA was also low. Conversely, despite body iron overload and high expression of Bmp6 in NPCs from Hjv KO mice, Tfr1 mRNA and protein were increased. However, in the same cells ferritin L was only slightly increased, but the iron content was not, suggesting that Bmp6 in these cells reflects the high intracellular iron import and export. We propose that NPCs, sensing the iron flux, not only increase hepcidin through Bmp6 with a paracrine mechanism to control systemic iron homeostasis but, controlling hepcidin, they regulate their own ferroportin, inducing iron retention or release and further modulating Bmp6 production in an autocrine manner. This mechanism, that contributes to protect HC from iron loading or deficiency, is lost in disease models of hepcidin production. PMID:25860887

Exosomes as potential alternatives to stem cell therapy for intervertebral disc degeneration: in-vitro study on exosomes in interaction of nucleus pulposus cells and bone marrow mesenchymal stem cells.

PubMed

Lu, Kang; Li, Hai-Yin; Yang, Kuang; Wu, Jun-Long; Cai, Xiao-Wei; Zhou, Yue; Li, Chang-Qing

2017-05-10

The stem cell-based therapies for intervertebral disc degeneration have been widely studied. However, the mechanisms of mesenchymal stem cells interacting with intervertebral disc cells, such as nucleus pulposus cells (NPCs), remain unknown. Exosomes as a vital paracrine mechanism in cell-cell communication have been highly focused on. The purpose of this study was to detect the role of exosomes derived from bone marrow mesenchymal stem cells (BM-MSCs) and NPCs in their interaction with corresponding cells. The exosomes secreted by BM-MSCs and NPCs were purified by differential centrifugation and identified by transmission electron microscope and immunoblot analysis of exosomal marker proteins. Fluorescence confocal microscopy was used to examine the uptake of exosomes by recipient cells. The effects of NPC exosomes on the migration and differentiation of BM-MSCs were determined by transwell migration assays and quantitative RT-PCR analysis of NPC phenotypic genes. Western blot analysis was performed to examine proteins such as aggrecan, sox-9, collagen II and hif-1α in the induced BM-MSCs. Proliferation and the gene expression profile of NPCs induced by BM-MSC exosomes were measured by Cell Counting Kit-8 and qRT-PCR analysis, respectively. Both the NPCs and BM-MSCs secreted exosomes, and these exosomes underwent uptake by the corresponding cells. NPC-derived exosomes promoted BM-MSC migration and induced BM-MSC differentiation to a nucleus pulposus-like phenotype. BM-MSC-derived exosomes promoted NPC proliferation and healthier extracellular matrix production in the degenerate NPCs. Our study indicates that the exosomes act as an important vehicle in information exchange between BM-MSCs and NPCs. Given a variety of functions and multiple advantages, exosomes alone or loaded with specific genes and drugs would be an appropriate option in a cell-free therapy strategy for intervertebral disc degeneration.

Actions of Brain-Derived Neurotrophic Factor and Glucocorticoid Stress in Neurogenesis

PubMed Central

Numakawa, Tadahiro; Odaka, Haruki; Adachi, Naoki

2017-01-01

Altered neurogenesis is suggested to be involved in the onset of brain diseases, including mental disorders and neurodegenerative diseases. Neurotrophic factors are well known for their positive effects on the proliferation/differentiation of both embryonic and adult neural stem/progenitor cells (NSCs/NPCs). Especially, brain-derived neurotrophic factor (BDNF) has been extensively investigated because of its roles in the differentiation/maturation of NSCs/NPCs. On the other hand, recent evidence indicates a negative impact of the stress hormone glucocorticoids (GCs) on the cell fate of NSCs/NPCs, which is also related to the pathophysiology of brain diseases, such as depression and autism spectrum disorder. Furthermore, studies including ours have demonstrated functional interactions between neurotrophic factors and GCs in neural events, including neurogenesis. In this review, we show and discuss relationships among the behaviors of NSCs/NPCs, BDNF, and GCs. PMID:29099059

Gene expression changes in the retina following subretinal injection of human neural progenitor cells into a rodent model for retinal degeneration.

PubMed

Jones, Melissa K; Lu, Bin; Saghizadeh, Mehrnoosh; Wang, Shaomei

2016-01-01

Retinal degenerative diseases (RDDs) affect millions of people and are the leading cause of vision loss. Although treatment options for RDDs are limited, stem and progenitor cell-based therapies have great potential to halt or slow the progression of vision loss. Our previous studies have shown that a single subretinal injection of human forebrain derived neural progenitor cells (hNPCs) into the Royal College of Surgeons (RCS) retinal degenerate rat offers long-term preservation of photoreceptors and visual function. Furthermore, neural progenitor cells are currently in clinical trials for treating age-related macular degeneration; however, the molecular mechanisms of stem cell-based therapies are largely unknown. This is the first study to analyze gene expression changes in the retina of RCS rats following subretinal injection of hNPCs using high-throughput sequencing. RNA-seq data of retinas from RCS rats injected with hNPCs (RCS(hNPCs)) were compared to sham surgery in RCS (RCS(sham)) and wild-type Long Evans (LE(sham)) rats. Differential gene expression patterns were determined with in silico analysis and confirmed with qRT-PCR. Function, biologic, cellular component, and pathway analyses were performed on differentially expressed genes and investigated with immunofluorescent staining experiments. Analysis of the gene expression data sets identified 1,215 genes that were differentially expressed between RCS(sham) and LE(sham) samples. Additionally, 283 genes were differentially expressed between the RCS(hNPCs) and RCS(sham) samples. Comparison of these two gene sets identified 68 genes with inverse expression (termed rescue genes), including Pdc, Rp1, and Cdc42ep5. Functional, biologic, and cellular component analyses indicate that the immune response is enhanced in RCS(sham). Pathway analysis of the differential expression gene sets identified three affected pathways in RCS(hNPCs), which all play roles in phagocytosis signaling. Immunofluorescent staining detected the increased presence of macrophages and microglia in RCS(sham) retinas, which decreased in RCS(hNPCs) retinas similar to the patterns detected in LE(sham). The results from this study provide evidence of the gene expression changes that occur following treatment with hNPCs in the degenerating retina. This information can be used in future studies to potentially enhance or predict responses to hNPC and other stem cell therapies for retinal degenerative diseases.

Multiparametric Phenotypic Screening System for Profiling Bioactive Compounds Using Human Fetal Hippocampal Neural Stem/Progenitor Cells.

PubMed

Tabata, Yoshikuni; Murai, Norio; Sasaki, Takeo; Taniguchi, Sachie; Suzuki, Shuichi; Yamazaki, Kazuto; Ito, Masashi

2015-10-01

Stem cell research has been progressing rapidly, contributing to regenerative biology and regenerative medicine. In this field, small-molecule compounds affecting stem cell proliferation/differentiation have been explored to understand stem cell biology and support regenerative medicine. In this study, we established a multiparametric screening system to detect bioactive compounds affecting the cell fate of human neural stem/progenitor cells (NSCs/NPCs), using human fetal hippocampal NSCs/NPCs, HIP-009 cells. We examined effects of 410 compounds, which were collected based on mechanisms of action (MOAs) and chemotypes, on HIP-009's cell fate (self-renewal, neuronal and astrocytic differentiation) and morphology by automated multiparametric assays and profiled induced cellular phenotypes. We found that this screening classified compounds with the same MOAs into subgroups according to additional pharmacological effects (e.g., mammalian target of rapamycin complex 1 [mTORC1] inhibitors and mTORC1/mTORC2 dual inhibitors among mTOR inhibitors). Moreover, it identified compounds that have off-target effects under matrix analyses of MOAs and structure similarities (e.g., neurotropic effects of amitriptyline among tri- and tetracyclic compounds). Therefore, this automated, medium-throughput and multiparametric screening system is useful for finding compounds that affect the cell fate of human NSCs/NPCs for supporting regenerative medicine and to fingerprint compounds based on human stem cells' multipotency, leading to understanding of stem cell biology. © 2015 Society for Laboratory Automation and Screening.

Altered proliferation and networks in neural cells derived from idiopathic autistic individuals.

PubMed

Marchetto, Maria C; Belinson, Haim; Tian, Yuan; Freitas, Beatriz C; Fu, Chen; Vadodaria, Krishna; Beltrao-Braga, Patricia; Trujillo, Cleber A; Mendes, Ana P D; Padmanabhan, Krishnan; Nunez, Yanelli; Ou, Jing; Ghosh, Himanish; Wright, Rebecca; Brennand, Kristen; Pierce, Karen; Eichenfield, Lawrence; Pramparo, Tiziano; Eyler, Lisa; Barnes, Cynthia C; Courchesne, Eric; Geschwind, Daniel H; Gage, Fred H; Wynshaw-Boris, Anthony; Muotri, Alysson R

2017-06-01

Autism spectrum disorders (ASD) are common, complex and heterogeneous neurodevelopmental disorders. Cellular and molecular mechanisms responsible for ASD pathogenesis have been proposed based on genetic studies, brain pathology and imaging, but a major impediment to testing ASD hypotheses is the lack of human cell models. Here, we reprogrammed fibroblasts to generate induced pluripotent stem cells, neural progenitor cells (NPCs) and neurons from ASD individuals with early brain overgrowth and non-ASD controls with normal brain size. ASD-derived NPCs display increased cell proliferation because of dysregulation of a β-catenin/BRN2 transcriptional cascade. ASD-derived neurons display abnormal neurogenesis and reduced synaptogenesis leading to functional defects in neuronal networks. Interestingly, defects in neuronal networks could be rescued by insulin growth factor 1 (IGF-1), a drug that is currently in clinical trials for ASD. This work demonstrates that selection of ASD subjects based on endophenotypes unraveled biologically relevant pathway disruption and revealed a potential cellular mechanism for the therapeutic effect of IGF-1.

One-step pyrolysis route to three dimensional nitrogen-doped porous carbon as anode materials for microbial fuel cells

NASA Astrophysics Data System (ADS)

Bi, Linlin; Ci, Suqin; Cai, Pingwei; Li, Hao; Wen, Zhenhai

2018-01-01

The design and synthesis of low-cost and favourable anode materials is crucial to both power production efficiency and overall performance of microbial fuel cells (MFCs). Herein, we reported the preparation of three dimensional (3D) nitrogen-doped porous carbons (N/PCs) by one-step pyrolysis of solid mixture of sodium citrate and melamine. a variety of techniques, including electron microscopy, X-ray diffraction (XRD), X-ray photoelectron spectroscopy (XPS), etc., were applied to characterize the surface physicochemical properties of the products, featuring macroporous structure with rich nitrogen doping on the as-prepared N/PCs. When applied as anode materials of MFC, the N/PCs exhibits a maximum power density of 2777.7 mW m-2, approximately twice higher than that of the MFCs with the commercial carbon cloth (CC) as anode. The significantly improved performance of the N/PCs was attributed to the unique structure and properties, such as favourable porous structure, good electrical conductivity, and large pore volume (0.7 cm3 g-1) in the present N/PCs. Nitrogen dopant on the surface of porous carbon contributed to an increasing in biocompatibility, resulting in a suitable micro-environment for microbial growth and thus helps to decrease charge transfer resistance at the electrode interface.

Curcumin Stimulates Proliferation of Spinal Cord Neural Progenitor Cells via a Mitogen-Activated Protein Kinase Signaling Pathway

PubMed Central

Son, Sihoon; Cho, Dae-Chul; Kim, Hye-Jeong; Sung, Joo-Kyung; Bae, Jae-Sung

2014-01-01

Objective The aims of our study are to evaluate the effect of curcumin on spinal cord neural progenitor cell (SC-NPC) proliferation and to clarify the mechanisms of mitogen-activated protein (MAP) kinase signaling pathways in SC-NPCs. Methods We established cultures of SC-NPCs, extracted from the spinal cord of Sprague-Dawley rats weighing 250 g to 350 g. We measured proliferation rates of SC-NPCs after curcumin treatment at different dosage. The immuno-blotting method was used to evaluate the MAP kinase signaling protein that contains extracellular signal-regulated kinases (ERKs), p38, c-Jun NH2-terminal kinases (JNKs) and β-actin as the control group. Results Curcumin has a biphasic effect on SC-NPC proliferation. Lower dosage (0.1, 0.5, 1 µM) of curcumin increased SC-NPC proliferation. However, higher dosage decreased SC-NPC proliferation. Also, curcumin stimulates proliferation of SC-NPCs via the MAP kinase signaling pathway, especially involving the p-ERK and p-38 protein. The p-ERK protein and p38 protein levels varied depending on curcumin dosage (0.5 and 1 µM, p<0.05). Conclusion Curcumin can stimulate proliferation of SC-NPCs via ERKs and the p38 signaling pathway in low concentrations. PMID:25289117

Pulsed DC Electric Field–Induced Differentiation of Cortical Neural Precursor Cells

PubMed Central

Chang, Hui-Fang; Lee, Ying-Shan; Tang, Tang K.; Cheng, Ji-Yen

2016-01-01

We report the differentiation of neural stem and progenitor cells solely induced by direct current (DC) pulses stimulation. Neural stem and progenitor cells in the adult mammalian brain are promising candidates for the development of therapeutic neuroregeneration strategies. The differentiation of neural stem and progenitor cells depends on various in vivo environmental factors, such as nerve growth factor and endogenous EF. In this study, we demonstrated that the morphologic and phenotypic changes of mouse neural stem and progenitor cells (mNPCs) could be induced solely by exposure to square-wave DC pulses (magnitude 300 mV/mm at frequency of 100-Hz). The DC pulse stimulation was conducted for 48 h, and the morphologic changes of mNPCs were monitored continuously. The length of primary processes and the amount of branching significantly increased after stimulation by DC pulses for 48 h. After DC pulse treatment, the mNPCs differentiated into neurons, astrocytes, and oligodendrocytes simultaneously in stem cell maintenance medium. Our results suggest that simple DC pulse treatment could control the fate of NPCs. With further studies, DC pulses may be applied to manipulate NPC differentiation and may be used for the development of therapeutic strategies that employ NPCs to treat nervous system disorders. PMID:27352251

Novel pyropheophorbide steryl esters in Black Sea sediments

NASA Astrophysics Data System (ADS)

King, Linda L.; Repeta, Daniel J.

1991-07-01

A series of non-polar chlorophyll degradation products (NPCs) with greater than 10 components has been isolated from Black Sea sediment and identified as pyropheophorbide steryl esters by visible and mass spectrometry. These compounds have been previously observed in seawater and sediment trap samples, and may be formed during grazing of phytoplankton by zooplanktonic herbivores. In Black Sea sediments, NPCs constitute 14% of the total phorbins determined spectroscopically at 660 nm, and 39% of the total chlorophyll degradation products measured by high pressure liquid chromatography. NPCs therefore constitute a significant sedimentary sink for chlorophyll. The distribution of sterols released by hydrolysis of NPCs most closely resembles sterols in suspended particulate matter collected from the euphotic zone and is quite different from the distribution of solvent-extractable sterols in sediments. Sterols extracted from sediments have high concentrations of 4-methylsterols and high stanol/stenol ratios. NPC-derived sterols have very low concentrations of 4-methylsterols and low stanol/stenol ratios. We suggest that these differences reflect an enhanced preservation of NPCs in sediments relative to free sterols and phorbins. As a result, the original production of sterols in the euphotic zone may be more closely approximated by the distribution of NPC-derived sterols than by the distribution of free sterols in sediments.

Adult DRG Stem/Progenitor Cells Generate Pericytes in the Presence of Central Nervous System (CNS) Developmental Cues, and Schwann Cells in Response to CNS Demyelination.

PubMed

Vidal, Marie; Maniglier, Madlyne; Deboux, Cyrille; Bachelin, Corinne; Zujovic, Violetta; Baron-Van Evercooren, Anne

2015-06-01

It has been proposed that the adult dorsal root ganglia (DRG) harbor neural stem/progenitor cells (NPCs) derived from the neural crest. However, the thorough characterization of their stemness and differentiation plasticity was not addressed. In this study, we investigated adult DRG-NPC stem cell properties overtime, and their fate when ectopically grafted in the central nervous system. We compared them in vitro and in vivo to the well-characterized adult spinal cord-NPCs derived from the same donors. Using micro-dissection and neurosphere cultures, we demonstrate that adult DRG-NPCs have quasi unlimited self-expansion capacities without compromising their tissue specific molecular signature. Moreover, they differentiate into multiple peripheral lineages in vitro. After transplantation, adult DRG-NPCs generate pericytes in the developing forebrain but remyelinating Schwann cells in response to spinal cord demyelination. In addition, we show that axonal and endothelial/astrocytic factors as well astrocytes regulate the fate of adult DRG-NPCs in culture. Although the adult DRG-NPC multipotency is restricted to the neural crest lineage, their dual responsiveness to developmental and lesion cues highlights their impressive adaptive and repair potentials making them valuable targets for regenerative medicine. © 2015 AlphaMed Press.

Alternating current electric fields of varying frequencies: effects on proliferation and differentiation of porcine neural progenitor cells.

PubMed

Lim, Ji-Hey; McCullen, Seth D; Piedrahita, Jorge A; Loboa, Elizabeth G; Olby, Natasha J

2013-10-01

Application of sinusoidal electric fields (EFs) has been observed to affect cellular processes, including alignment, proliferation, and differentiation. In the present study, we applied low-frequency alternating current (AC) EFs to porcine neural progenitor cells (pNPCs) and investigated the effects on cell patterning, proliferation, and differentiation. pNPCs were grown directly on interdigitated electrodes (IDEs) localizing the EFs to a region accessible visually for fluorescence-based assays. Cultures of pNPCs were exposed to EFs (1 V/cm) of 1 Hz, 10 Hz, and 50 Hz for 3, 7, and 14 days and compared to control cultures. Immunocytochemistry was performed to evaluate the expression of neural markers. pNPCs grew uniformly with no evidence of alignment to the EFs and no change in cell numbers when compared with controls. Nestin expression was shown in all groups at 3 and 7 days, but not at 14 days. NG2 expression was low in all groups. Co-expression of glial fibrillary acidic protein (GFAP) and TUJ1 was significantly higher in the cultures exposed to 10- and 50-Hz EFs than the controls. In summary, sinusoidal AC EFs via IDEs did not alter the alignment and proliferation of pNPCs, but higher frequency stimulation appeared to delay differentiation into mature astrocytes.

Phenobarbital Induces Alterations in the Proteome of Hepatocytes and Mesenchymal Cells of Rat Livers

PubMed Central

Klepeisz, Philip; Sagmeister, Sandra; Haudek-Prinz, Verena; Pichlbauer, Melanie; Grasl-Kraupp, Bettina; Gerner, Christopher

2013-01-01

Preceding studies on the mode of action of non-genotoxic hepatocarcinogens (NGCs) have concentrated on alterations induced in hepatocytes (HCs). A potential role of non-parenchymal liver cells (NPCs) in NGC-driven hepatocarcinogenesis has been largely neglected so far. The aim of this study is to characterize NGC-induced alterations in the proteome profiles of HCs as well as NPCs. We chose the prototypic NGC phenobarbital (PB) which was applied to male rats for a period of 14 days. The livers of PB-treated rats were perfused by collagenase and the cell suspensions obtained were subjected to density gradient centrifugation to separate HCs from NPCs. In addition, HCs and NPC isolated from untreated animals were treated with PB in vitro. Proteome profiling was done by CHIP-HPLC and ion trap mass spectrometry. Proteome analyses of the in vivo experiments showed many of the PB effects previously described in HCs by other methods, e.g. induction of phase I and phase II drug metabolising enzymes. In NPCs proteins related to inflammation and immune regulation such as PAI-1 and S100-A10, ADP-ribosyl cyclase 1 and to cell migration such as kinesin-1 heavy chain, myosin regulatory light chain RLC-A and dihydropyrimidinase-related protein 1 were found to be induced, indicating major PB effects on these cells. Remarkably, in vitro treatment of HCs and NPCs with PB hardly reproduced the proteome alterations observed in vivo, indicating differences of NGC induced responses of cells at culture conditions compared to the intact organism. To conclude, the present study clearly demonstrated that PB induces proteome alterations not only in HCs but also in NPCs. Thus, any profound molecular understanding on the mode of action of NGCs has to consider effects on cells of the hepatic mesenchyme. PMID:24204595

Signaling through three chemokine receptors triggers the migration of transplanted neural precursor cells in a model of multiple sclerosis.

PubMed

Cohen, Mikhal E; Fainstein, Nina; Lavon, Iris; Ben-Hur, Tamir

2014-09-01

Multiple sclerosis (MS) is a multifocal disease, and precursor cells need to migrate into the multiple lesions in order to exert their therapeutic effects. Therefore, cell migration is a crucial element in regenerative processes in MS, dictating the route of delivery, when cell transplantation is considered. We have previously shown that inflammation triggers migration of multi-potential neural precursor cells (NPCs) into the white matter of experimental autoimmune encephalomyelitis (EAE) rodents, a widely used model of MS. Here we investigated the molecular basis of this attraction. NPCs were grown from E13 embryonic mouse brains and transplanted into the lateral cerebral ventricles of EAE mice. Transplanted NPC migration was directed by three tissue-derived chemokines. Stromal cell-derived factor-1α, monocyte chemo-attractant protein-1 and hepatocyte growth factor were expressed in the EAE brain and specifically in microglia and astrocytes. Their cognate receptors, CXCR4, CCR2 or c-Met were constitutively expressed on NPCs. Selective blockage of CXCR4, CCR2 or c-Met partially inhibited NPC migration in EAE brains. Blocking all three receptors had an additive effect and resulted in profound inhibition of NPC migration, as compared to extensive migration of control NPCs. The inflammation-triggered NPC migration into white matter tracts was dependent on a motile NPC phenotype. Specifically, depriving NPCs from epidermal growth factor (EGF) prevented the induction of glial commitment and a motile phenotype (as indicated by an in vitro motility assay), hampering their response to neuroinflammation. In conclusion, signaling via three chemokine systems accounts for most of the inflammation-induced, tissue-derived attraction of transplanted NPCs into white matter tracts during EAE. Copyright © 2014. Published by Elsevier B.V.

Premature aging of the hippocampal neurogenic niche in adult Bmal1-deficient mice.

PubMed

Ali, Amira A H; Schwarz-Herzke, Beryl; Stahr, Anna; Prozorovski, Timour; Aktas, Orhan; von Gall, Charlotte

2015-06-01

Hippocampal neurogenesis undergoes dramatic age-related changes. Mice with targeted deletion of the clock geneBmal1 (Bmal1(-/-)) show disrupted regulation of reactive oxygen species homeostasis, accelerated aging, neurodegeneration and cognitive deficits. As proliferation of neuronal progenitor/precursor cells (NPCs) is enhanced in young Bmal1(-/-) mice, we tested the hypothesis that this results in premature aging of hippocampal neurogenic niche in adult Bmal1(-/-) mice as compared to wildtype littermates. We found significantly reduced pool of hippocampal NPCs, scattered distribution, enhanced survival of NPCs and an increased differentiation of NPCs into the astroglial lineage at the expense of the neuronal lineage. Immunoreaction of the redox sensitive histone deacetylase Sirtuine 1, peroxisomal membrane protein at 70 kDa and expression of the cell cycle inhibitor p21(Waf1/CIP1) were increased in adult Bmal1(-/-) mice. In conclusion, genetic disruption of the molecular clockwork leads to accelerated age-dependent decline in adult neurogenesis presumably as a consequence of oxidative stress.

Human iPSC-Derived Neural Progenitors Are an Effective Drug Discovery Model for Neurological mtDNA Disorders.

PubMed

Lorenz, Carmen; Lesimple, Pierre; Bukowiecki, Raul; Zink, Annika; Inak, Gizem; Mlody, Barbara; Singh, Manvendra; Semtner, Marcus; Mah, Nancy; Auré, Karine; Leong, Megan; Zabiegalov, Oleksandr; Lyras, Ekaterini-Maria; Pfiffer, Vanessa; Fauler, Beatrix; Eichhorst, Jenny; Wiesner, Burkhard; Huebner, Norbert; Priller, Josef; Mielke, Thorsten; Meierhofer, David; Izsvák, Zsuzsanna; Meier, Jochen C; Bouillaud, Frédéric; Adjaye, James; Schuelke, Markus; Wanker, Erich E; Lombès, Anne; Prigione, Alessandro

2017-05-04

Mitochondrial DNA (mtDNA) mutations frequently cause neurological diseases. Modeling of these defects has been difficult because of the challenges associated with engineering mtDNA. We show here that neural progenitor cells (NPCs) derived from human induced pluripotent stem cells (iPSCs) retain the parental mtDNA profile and exhibit a metabolic switch toward oxidative phosphorylation. NPCs derived in this way from patients carrying a deleterious homoplasmic mutation in the mitochondrial gene MT-ATP6 (m.9185T>C) showed defective ATP production and abnormally high mitochondrial membrane potential (MMP), plus altered calcium homeostasis, which represents a potential cause of neural impairment. High-content screening of FDA-approved drugs using the MMP phenotype highlighted avanafil, which we found was able to partially rescue the calcium defect in patient NPCs and differentiated neurons. Overall, our results show that iPSC-derived NPCs provide an effective model for drug screening to target mtDNA disorders that affect the nervous system. Copyright © 2016 Elsevier Inc. All rights reserved.

Essential Roles for Caenorhabditis elegans Lamin Gene in Nuclear Organization, Cell Cycle Progression, and Spatial Organization of Nuclear Pore Complexes

PubMed Central

Liu, Jun; Ben-Shahar, Tom Rolef; Riemer, Dieter; Treinin, Millet; Spann, Perah; Weber, Klaus; Fire, Andrew; Gruenbaum, Yosef

2000-01-01

Caenorhabditis elegans has a single lamin gene, designated lmn-1 (previously termed CeLam-1). Antibodies raised against the lmn-1 product (Ce-lamin) detected a 64-kDa nuclear envelope protein. Ce-lamin was detected in the nuclear periphery of all cells except sperm and was found in the nuclear interior in embryonic cells and in a fraction of adult cells. Reductions in the amount of Ce-lamin protein produce embryonic lethality. Although the majority of affected embryos survive to produce several hundred nuclei, defects can be detected as early as the first nuclear divisions. Abnormalities include rapid changes in nuclear morphology during interphase, loss of chromosomes, unequal separation of chromosomes into daughter nuclei, abnormal condensation of chromatin, an increase in DNA content, and abnormal distribution of nuclear pore complexes (NPCs). Under conditions of incomplete RNA interference, a fraction of embryos escaped embryonic arrest and continue to develop through larval life. These animals exhibit additional phenotypes including sterility and defective segregation of chromosomes in germ cells. Our observations show that lmn-1 is an essential gene in C. elegans, and that the nuclear lamins are involved in chromatin organization, cell cycle progression, chromosome segregation, and correct spacing of NPCs. PMID:11071918

The N-terminal domain of the mammalian nucleoporin p62 interacts with other nucleoporins of the FXFG family during interphase

DOE Office of Scientific and Technical Information (OSTI.GOV)

Stochaj, Ursula; Banski, Piotr; Kodiha, Mohamed

2006-08-01

Nuclear pore complexes (NPCs) provide the only sites for macromolecular transport between nucleus and cytoplasm. The nucleoporin p62, a component of higher eukaryotic NPCs, is located at the central gated channel and involved in nuclear trafficking of various cargos. p62 is organized into an N-terminal segment that contains FXFG repeats and binds the soluble transport factor NTF2, whereas the C-terminal portion associates with other nucleoporins and importin-{beta}1. We have now identified new components that interact specifically with the p62 N-terminal domain. Using the p62 N-terminal segment as bait, we affinity-purified nucleoporins Nup358, Nup214 and Nup153 from crude cell extracts. Inmore » ligand binding assays, the N-terminal p62 segment associated with Nup358 and p62, suggesting their direct binding to the p62 N-terminal portion. Furthermore, p62 was isolated in complex with Nup358, Nup214 and Nup153 from growing HeLa cells, indicating that the interactions Nup358/p62, Nup214/p62 and p62/Nup153 also occur in vivo. The formation of Nup358/p62 and p62/Nup153 complexes was restricted to interphase cells, whereas Nup214/p62 binding was detected in interphase as well as during mitosis. Our results support a model of complex interactions between FXFG containing nucleoporins, and we propose that some of these interactions may contribute to the movement of cargo across the NPC.« less

Increased spontaneous apoptosis of rat primary neurospheres in vitro after experimental autoimmune encephalomyelitis.

PubMed

Sajad, Mir; Zargan, Jamil; Sharma, Jyoti; Chawla, Raman; Arora, Rajesh; Umar, Sadiq; Khan, Haider A

2011-06-01

Survival of neuronal progenitors (NPCs) is a critical determinant of the regenerative capacity of brain following cellular loss. Herein, we report for the first time, the increased spontaneous apoptosis of the first acute phase of Experimental Autoimmune Encephalomyelitis (EAE) derived neurospheres in vitro. Neuronal as well as oligodendroglial loss occurs during experimental autoimmune encephalomyelitis (EAE). This loss is replenished spontaneously by the concomitant increase in the NPC proliferation evidenced by the presence of thin myelin sheaths in the remodeled lesions. However, remyelination depends upon the survival of NPCs and their lineage specific differentiation. We observed significant increase (P < 0.001) in number of BrdU (+) cells in ependymal subventricular zone (SVZ) in EAE rats. EAE derived NPCs showed remarkable increase in S-phase population which was indeed due to the decrease in G-phase progeny suggesting activation of neuronal progenitor cells (NPCs) from quiescence. However, EAE derived neurospheres showed limited survival in vitro which was mediated by the significantly (P < 0.01) depolarized mitochondria, elevated Caspase-3 (P < 0.001) and fragmentation of nuclear DNA evidenced by single cell gel electrophoresis. Our results suggest EAE induced spontaneous apoptosis of NPCs in vitro which may increase the possibility of early stage cell death in the negative regulation of the proliferative cell number and may explain the failure of regeneration in human multiple sclerosis.

76 FR 33416 - Proposed Information Collection (Nonprofit Research and Education Corporations (NPCs) Data...

Federal Register 2010, 2011, 2012, 2013, 2014

2011-06-08

... Collection (Nonprofit Research and Education Corporations (NPCs) Data Collection) Activity; Comment Request... comment period, comments may be viewed online through FDMS. FOR FURTHER INFORMATION CONTACT: Cynthia... information; (3) ways to enhance the quality, utility, and clarity of the information to be collected; and (4...

Testing Brain Overgrowth and Synaptic Models of Autism Using NPCs and Neurons from Patient-Derived IPS Cells

DTIC Science & Technology

2015-12-01

Award Number: W81XWH-13-1-0415 TITLE: Testing Brain Overgrowth and Synaptic Models of Autism Using NPC’s and Neurons from Patient-Derived IPS...Approved for Public Release; Distribution Unlimited 13. SUPPLEMENTARY NOTES 14. ABSTRACT Autism and autism spectrum disorders (ASD) are complex...impaired social interaction, and limited and repetitive interests and behavior. Recent studies have led to two major hypotheses for autism

Transcriptional activation of NAD+-dependent protein deacetylase SIRT1 by nuclear receptor TLX.

PubMed

Iwahara, Naotoshi; Hisahara, Shin; Hayashi, Takashi; Horio, Yoshiyuki

2009-09-04

An orphan nuclear receptor TLX is a transcriptional repressor that promotes the proliferation and self-renewal of neural precursor cells (NPCs). SIRT1, an NAD(+)-dependent protein deacetylase, is highly expressed in the NPCs and participates in neurogenesis. Here, we found that TLX colocalized with SIRT1 and knockdown of TLX by small interfering RNAs decreased SIRT1 levels in NPCs. TLX increased the SIRT1 expression by binding to the newly identified TLX-activating element in the SIRT1 gene promoter in HEK293 cells. Thus, TLX is an inducer of SIRT1 and may contribute to neurogenesis both as a transactivator and as a repressor.

Transcriptional activation of NAD{sup +}-dependent protein deacetylase SIRT1 by nuclear receptor TLX

DOE Office of Scientific and Technical Information (OSTI.GOV)

Iwahara, Naotoshi; Hisahara, Shin; Hayashi, Takashi

2009-09-04

An orphan nuclear receptor TLX is a transcriptional repressor that promotes the proliferation and self-renewal of neural precursor cells (NPCs). SIRT1, an NAD{sup +}-dependent protein deacetylase, is highly expressed in the NPCs and participates in neurogenesis. Here, we found that TLX colocalized with SIRT1 and knockdown of TLX by small interfering RNAs decreased SIRT1 levels in NPCs. TLX increased the SIRT1 expression by binding to the newly identified TLX-activating element in the SIRT1 gene promoter in HEK293 cells. Thus, TLX is an inducer of SIRT1 and may contribute to neurogenesis both as a transactivator and as a repressor.

Activating Endogenous Neural Precursor Cells Using Metformin Leads to Neural Repair and Functional Recovery in a Model of Childhood Brain Injury.

PubMed

Dadwal, Parvati; Mahmud, Neemat; Sinai, Laleh; Azimi, Ashkan; Fatt, Michael; Wondisford, Fredric E; Miller, Freda D; Morshead, Cindi M

2015-08-11

The development of cell replacement strategies to repair the injured brain has gained considerable attention, with a particular interest in mobilizing endogenous neural stem and progenitor cells (known as neural precursor cells [NPCs]) to promote brain repair. Recent work demonstrated metformin, a drug used to manage type II diabetes, promotes neurogenesis. We sought to determine its role in neural repair following brain injury. We find that metformin administration activates endogenous NPCs, expanding the size of the NPC pool and promoting NPC migration and differentiation in the injured neonatal brain in a hypoxia-ischemia (H/I) injury model. Importantly, metformin treatment following H/I restores sensory-motor function. Lineage tracking reveals that metformin treatment following H/I causes an increase in the absolute number of subependyma-derived NPCs relative to untreated H/I controls in areas associated with sensory-motor function. Hence, activation of endogenous NPCs is a promising target for therapeutic intervention in childhood brain injury models. Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.

MiRNA-128 regulates the proliferation and neurogenesis of neural precursors by targeting PCM1 in the developing cortex

PubMed Central

Zhang, Wei; Kim, Paul Jong; Chen, Zhongcan; Lokman, Hidayat; Qiu, Lifeng; Zhang, Ke; Rozen, Steven George; Tan, Eng King; Je, Hyunsoo Shawn; Zeng, Li

2016-01-01

During the development, tight regulation of the expansion of neural progenitor cells (NPCs) and their differentiation into neurons is crucial for normal cortical formation and function. In this study, we demonstrate that microRNA (miR)-128 regulates the proliferation and differentiation of NPCs by repressing pericentriolar material 1 (PCM1). Specifically, overexpression of miR-128 reduced NPC proliferation but promoted NPC differentiation into neurons both in vivo and in vitro. In contrast, the reduction of endogenous miR-128 elicited the opposite effects. Overexpression of miR-128 suppressed the translation of PCM1, and knockdown of endogenous PCM1 phenocopied the observed effects of miR-128 overexpression. Furthermore, concomitant overexpression of PCM1 and miR-128 in NPCs rescued the phenotype associated with miR-128 overexpression, enhancing neurogenesis but inhibiting proliferation, both in vitro and in utero. Taken together, these results demonstrate a novel mechanism by which miR-128 regulates the proliferation and differentiation of NPCs in the developing neocortex. DOI: http://dx.doi.org/10.7554/eLife.11324.001 PMID:26883496

Establishing a nurse practitioner collaborative: evolution, development, and outcomes.

PubMed

Quinn, Karen; Hudson, Peter

2014-09-01

The first Australian palliative care nurse practitioner (NP) was endorsed in 2003. In 2009 the Victoria Department of Health funded the development of the Victorian Palliative Care Nurse Practitioner Collaborative (VPCNPC). Its aim was to promote the NP role, develop resources, and provide education and mentorship to NPs, nurse practitioner candidates (NPCs), and health service managers. Four key objectives were developed: identify the demographic profile of palliative care NPCs in Victoria; develop an education curriculum and practical resources to support the training and education of palliative care NPCs and NPs; provide mentorship to NPs, NPCs, and service managers; and ensure effective communication with all key stakeholders. An NPC survey was also conducted to explore NPC demographics, models of care, the hours of study required for the role, the mentoring process, and education needs. This paper reports on the establishment of the VPCNPC, the steps taken to achieve its objectives, and the results of the survey. The NP role in palliative care in Australia continues to evolve, and the VPCNPC provides a structure and resources to clearly articulate the benefits of the role to nursing and clinical services.

A syngeneic glioma model to assess the impact of neural progenitor target cell age on tumor malignancy

PubMed Central

Mikheev, Andrei M; Stoll, Elizabeth A; Mikheeva, Svetlana A; Maxwell, John-Patrick; Jankowski, Pawel P; Ray, Sutapa; Uo, Takuma; Morrison, Richard S; Horner, Philip J; Rostomily, Robert C

2010-01-01

Summary Human glioma incidence, malignancy and treatment resistance are directly proportional to patient age. Cell intrinsic factors are reported to contribute to human age-dependent glioma malignancy but suitable animal models to examine the role of aging are lacking. Here we developed an orthotopic syngeneic glioma model to test the hypothesis that the age of neural progenitor cells (NPCs), presumed cells of glioma origin, influences glioma malignancy. Gliomas generated from transformed donor 3-, 12-, and 18-month-old NPCs in same-aged adult hosts all formed highly invasive glial tumors that phenocopied the human disease. Survival analysis indicated increased malignancy of gliomas generated from older 12- and 18-month-old transformed NPCs compared with their 3-month counterparts (median survival of 38.5 and 42.5 vs. 77 days, respectively). This study showed for the first time that age of target cells at the time of transformation can affect malignancy and demonstrated the feasibility of a syngeneic model using transformed NPCs for future examination of the relative impacts of age-related cell intrinsic and cell-extrinsic factors in glioma malignancy. PMID:19489742

In Vivo Analysis of the Neurovascular Niche in the Developing Xenopus Brain

PubMed Central

Li, Jianli

2017-01-01

Abstract The neurovascular niche is a specialized microenvironment formed by the interactions between neural progenitor cells (NPCs) and the vasculature. While it is thought to regulate adult neurogenesis by signaling through vascular-derived soluble cues or contacted-mediated cues, less is known about the neurovascular niche during development. In Xenopus laevis tadpole brain, NPCs line the ventricle and extend radial processes tipped with endfeet to the vascularized pial surface. Using in vivo labeling and time-lapse imaging in tadpoles, we find that intracardial injection of fluorescent tracers rapidly labels Sox2/3-expressing NPCs and that vascular-circulating molecules are endocytosed by NPC endfeet. Confocal imaging indicates that about half of the endfeet appear to appose the vasculature, and time-lapse analysis of NPC proliferation and endfeet-vascular interactions suggest that proliferative activity does not correlate with stable vascular apposition. Together, these findings characterize the neurovascular niche in the developing brain and suggest that, while signaling to NPCs may occur through vascular-derived soluble cues, stable contact between NPC endfeet and the vasculature is not required for developmental neurogenesis. PMID:28795134

Integrative analysis of genes and miRNA alterations in human embryonic stem cells-derived neural cells after exposure to silver nanoparticles.

PubMed

Oh, Jung-Hwa; Son, Mi-Young; Choi, Mi-Sun; Kim, Soojin; Choi, A-Young; Lee, Hyang-Ae; Kim, Ki-Suk; Kim, Janghwan; Song, Chang Woo; Yoon, Seokjoo

2016-05-15

Given the rapid growth of engineered and customer products made of silver nanoparticles (Ag NPs), understanding their biological and toxicological effects on humans is critically important. The molecular developmental neurotoxic effects associated with exposure to Ag NPs were analyzed at the physiological and molecular levels, using an alternative cell model: human embryonic stem cell (hESC)-derived neural stem/progenitor cells (NPCs). In this study, the cytotoxic effects of Ag NPs (10-200μg/ml) were examined in these hESC-derived NPCs, which have a capacity for neurogenesis in vitro, at 6 and 24h. The results showed that Ag NPs evoked significant toxicity in hESC-derived NPCs at 24h in a dose-dependent manner. In addition, Ag NPs induced cell cycle arrest and apoptosis following a significant increase in oxidative stress in these cells. To further clarify the molecular mechanisms of the toxicological effects of Ag NPs at the transcriptional and post-transcriptional levels, the global expression profiles of genes and miRNAs were analyzed in hESC-derived NPCs after Ag NP exposure. The results showed that Ag NPs induced oxidative stress and dysfunctional neurogenesis at the molecular level in hESC-derived NPCs. Based on this hESC-derived neural cell model, these findings have increased our understanding of the molecular events underlying developmental neurotoxicity induced by Ag NPs in humans. Copyright © 2015 Elsevier Inc. All rights reserved.

Intracellularly monitoring/imaging the release of doxorubicin from pH-responsive nanoparticles using Förster resonance energy transfer.

PubMed

Chen, Ko-Jie; Chiu, Ya-Ling; Chen, Yu-Ming; Ho, Yi-Cheng; Sung, Hsing-Wen

2011-04-01

Stimuli-responsive nanoparticles (NPs) have been receiving much attention as a drug-delivery vehicle for therapeutic applications; once internalized into cells, the intracellular fate of NPs and their drug release behavior in response to local stimuli must be understood for efficient delivery of therapeutics. In this study, we prepared pH-responsive doxorubicin (DOX)-loaded NPs, made of N-palmitoyl chitosan bearing a Cy5 moiety (Cy5-NPCS), as an anticancer delivery device. The results of our molecular dynamic simulations showed that the ability of Cy5-NPCS to self-associate offered the close proximity between the donor (DOX) and the acceptor (Cy5) required for Förster resonance energy transfer (FRET), while the pH-driven structure transition prescribed the on-to-off switch of the energy transfer. The caveolae-mediated pathway played a major role in the internalization of NPCS NPs. Using the concept of FRET, we found that the DOX fluorescence in the cytosol was first seen when NPCS NPs were present in the slightly acidic early endosomes. Following NPCS NPs trafficking into a more acidic organelle (late endosomes/lysosomes), a more evident release of DOX into the cytosol was observed; the released DOX was then gradually accumulated in the cell nuclei, leading to a significant cytotoxicity. Understanding the fate of NPs with respect to their intracellular localization and drug release behavior is crucial for the rational design of drug carriers. Copyright © 2010 Elsevier Ltd. All rights reserved.

The effects of hyaluronic acid hydrogels with tunable mechanical properties on neural progenitor cell differentiation.

PubMed

Seidlits, Stephanie K; Khaing, Zin Z; Petersen, Rebecca R; Nickels, Jonathan D; Vanscoy, Jennifer E; Shear, Jason B; Schmidt, Christine E

2010-05-01

We report the ability to direct the differentiation pathway of neural progenitor cells (NPCs) within hydrogels having tunable mechanical properties. By modifying the polymeric sugar hyaluronic acid (HA), a major extracellular matrix component in the fetal mammalian brain, with varying numbers of photocrosslinkable methacrylate groups, hydrogels could be prepared with bulk compressive moduli spanning the threefold range measured for neonatal brain and adult spinal cord. Ventral midbrain-derived NPCs were photoencapsulated into HA hydrogels and remained viable after encapsulation. After three weeks, the majority of NPCs cultured in hydrogels with mechanical properties comparable to those of neonatal brain had differentiated into neurons (ss-III tubulin-positive), many of which had extended long, branched processes, indicative of a relatively mature phenotype. In contrast, NPCs within stiffer hydrogels, with mechanical properties comparable to those of adult brain, had differentiated into mostly astrocytes (glial fibrillary acidic protein (GFAP)-positive). Primary spinal astrocytes cultured in the hydrogel variants for two weeks acquired a spread and elongated morphology only in the stiffest hydrogels evaluated, with mechanical properties similar to adult tissue. Results demonstrate that the mechanical properties of these scaffolds can assert a defining influence on the differentiation of ventral midbrain-derived NPCs, which have strong clinical relevance because of their ability to mature into dopaminergic neurons of the substantia nigra, cells that idiopathically degenerate in individuals suffering from Parkinson's disease. Copyright 2010 Elsevier Ltd. All rights reserved.

Non-physician communities in Japan: are they still disadvantaged?

PubMed

Kashima, S; Inoue, K; Matsumoto, M; Takeuchi, K

2014-01-01

Non-physician community' (NPC) is a policy term that indicates a medically underserved area in Japan. Designated NPCs are politically targeted as the foci of medical resource allocation. NPC is defined as a specified district where 50 or more persons dwell within a geographic diameter of 4 km and medical care is not easily accessible. The definition of NPC was first introduced in 1960 and has been unchanged for more than half a century despite radical social changes in rural Japan. This study examines whether designated NPCs are still more disadvantaged in terms of geographical access to healthcare in comparison to other communities. Hiroshima prefecture, which has the largest number of NPCs in terms of tertiary healthcare areas of Japan, was used as the study area. Targeted communities were all the NPCs in the prefecture, and, as controls, two community groups were selected: non-NPC adjacent to NPC, and municipal center. We measured driving time from NPCs and control communities to the nearest healthcare facilities, which were classified into the following two types: primary or secondary care facilities (n=2636) and tertiary care facilities (equal to tertiary emergency care centers; n=6). We further calculated the driving time to the nearest facilities for secondary emergency care (n=246) extracted from the 2636 primary or secondary care facilities. The median driving times to the nearest primary or secondary healthcare facility for NPC, non-NPC, and municipal center were 11 minutes, 11 minutes, and 1 minute, respectively; the times to a tertiary healthcare facility (equal to an accident and emergency care center) were 80 minutes, 84 minutes, and 68 minutes, respectively; and the times to a secondary emergency care facility were 24 minutes, 18 minutes, and 15 minutes, respectively. Although a municipal center was significantly more advantageous in driving time compared to a primary or secondary care facility, the disadvantage of a NPC in access was no more obvious than an adjacent non-NPC for any type of healthcare facility. NPCs had a disadvantage in access time to primary, secondary and tertiary medical care compared with a municipal center. NPCs, however, did not have a greater access disadvantage in comparison to adjacent rural communities for any type of medical facility. As such, future resource allocation policies in Japan need to redefine medically underserved communities.

Mutant Ataxin-1 Inhibits Neural Progenitor Cell Proliferation in SCA1

PubMed Central

Cvetanovic, Marija; Hu, Yuan-Shih; Opal, Puneet

2017-01-01

Spinocerebellar ataxia type 1 (SCA1) is a dominantly inherited neurodegenerative disease caused by the expansion of a polyglutamine (Q) repeat tract in the protein ataxin-1 (ATXN1). Beginning as a cerebellar ataxic disorder, SCA1 progresses to involve the cerebral cortex, hippocampus, and brainstem. Using SCA1 knock-in mice that mirror the complexity of the human disease, we report a significant decrease in the capacity of adult neuronal progenitor cells (NPCs) to proliferate. Remarkably, a decrease in NPCs proliferation can be observed in vitro, outside the degenerative milieu of surrounding neurons or glia, demonstrating that mutant ATXN1 acting cell autonomously within progenitor cells interferes with their ability to proliferate. Our findings suggest that compromised adult neurogenesis contributes to the progressive pathology of the disease particularly in areas such as the hippocampus and cerebral cortex where stem cells provide neurotropic factors and participate in adult neurogenesis. These findings not only shed light on the biology of the disease but also have therapeutic implications in any future stem cell- based clinical trials. PMID:27306906

Mutant Ataxin-1 Inhibits Neural Progenitor Cell Proliferation in SCA1.

PubMed

Cvetanovic, Marija; Hu, Yuan-Shih; Opal, Puneet

2017-04-01

Spinocerebellar ataxia type 1 (SCA1) is a dominantly inherited neurodegenerative disease caused by the expansion of a polyglutamine (Q) repeat tract in the protein ataxin-1 (ATXN1). Beginning as a cerebellar ataxic disorder, SCA1 progresses to involve the cerebral cortex, hippocampus, and brainstem. Using SCA1 knock-in mice that mirror the complexity of the human disease, we report a significant decrease in the capacity of adult neuronal progenitor cells (NPCs) to proliferate. Remarkably, a decrease in NPCs proliferation can be observed in vitro, outside the degenerative milieu of surrounding neurons or glia, demonstrating that mutant ATXN1 acting cell autonomously within progenitor cells interferes with their ability to proliferate. Our findings suggest that compromised adult neurogenesis contributes to the progressive pathology of the disease particularly in areas such as the hippocampus and cerebral cortex where stem cells provide neurotropic factors and participate in adult neurogenesis. These findings not only shed light on the biology of the disease but also have therapeutic implications in any future stem cell-based clinical trials.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zhu Shanshan; Zhang Hong; Matunis, Michael J.

SUMOs (small ubiquitin-related modifiers) are eukaryotic proteins that are covalently conjugated to other proteins and thereby regulate a wide range of important cellular processes. The molecular mechanisms by which SUMO modification influences the functions of most target proteins and cellular processes, however, remain poorly defined. A major obstacle to investigating the effects of SUMO modification is the availability of a system for selectively inducing the modification or demodification of an individual protein. To address this problem, we have developed a procedure using the rapamycin heterodimerizer system. This procedure involves co-expression of rapamycin-binding domain fusion proteins of SUMO and candidate SUMOmore » substrates in living cells. Treating cells with rapamycin induces a tight association between SUMO and a single SUMO substrate, thereby allowing specific downstream effects to be analyzed. Using RanGAP1 as a model SUMO substrate, the heterodimerizer system was used to investigate the molecular mechanism by which SUMO modification targets RanGAP1 from the cytoplasm to nuclear pore complexes (NPCs). Our results revealed a dual role for Ubc9 in targeting RanGAP1 to NPCs: In addition to conjugating SUMO-1 to RanGAP1, Ubc9 is also required to form a stable ternary complex with SUMO-1 modified RanGAP1 and Nup358. As illustrated by our studies, the rapamycin heterodimerizer system represents a novel tool for studying the molecular effects of SUMO modification.« less

Brief Report: Robo1 Regulates the Migration of Human Subventricular Zone Neural Progenitor Cells During Development.

PubMed

Guerrero-Cazares, Hugo; Lavell, Emily; Chen, Linda; Schiapparelli, Paula; Lara-Velazquez, Montserrat; Capilla-Gonzalez, Vivian; Clements, Anna Christina; Drummond, Gabrielle; Noiman, Liron; Thaler, Katrina; Burke, Anne; Quiñones-Hinojosa, Alfredo

2017-07-01

Human neural progenitor cell (NPC) migration within the subventricular zone (SVZ) of the lateral ganglionic eminence is an active process throughout early brain development. The migration of human NPCs from the SVZ to the olfactory bulb during fetal stages resembles what occurs in adult rodents. As the human brain develops during infancy, this migratory stream is drastically reduced in cell number and becomes barely evident in adults. The mechanisms regulating human NPC migration are unknown. The Slit-Robo signaling pathway has been defined as a chemorepulsive cue involved in axon guidance and neuroblast migration in rodents. Slit and Robo proteins expressed in the rodent brain help guide neuroblast migration from the SVZ through the rostral migratory stream to the olfactory bulb. Here, we present the first study on the role that Slit and Robo proteins play in human-derived fetal neural progenitor cell migration (hfNPC). We describe that Robo1 and Robo2 isoforms are expressed in the human fetal SVZ. Furthermore, we demonstrate that Slit2 is able to induce a chemorepellent effect on the migration of hfNPCs derived from the human fetal SVZ. In addition, when Robo1 expression is inhibited, hfNPCs are unable to migrate to the olfactory bulb of mice when injected in the anterior SVZ. Our findings indicate that the migration of human NPCs from the SVZ is partially regulated by the Slit-Robo axis. This pathway could be regulated to direct the migration of NPCs in human endogenous neural cell therapy. Stem Cells 2017;35:1860-1865. © 2017 AlphaMed Press.

BRAIN REGENERATION IN PHYSIOLOGY AND PATHOLOGY: THE IMMUNE SIGNATURE DRIVING THERAPEUTIC PLASTICITY OF NEURAL STEM CELLS

PubMed Central

Martino, Gianvito; Pluchino, Stefano; Bonfanti, Luca; Schwartz, Michal

2013-01-01

Regenerative processes occurring under physiological (maintenance) and pathological (reparative) conditions are a fundamental part of life and vary greatly among different species, individuals, and tissues. Physiological regeneration occurs naturally as a consequence of normal cell erosion, or as an inevitable outcome of any biological process aiming at the restoration of homeostasis. Reparative regeneration occurs as a consequence of tissue damage. Although the central nervous system (CNS) has been considered for years as a “perennial” tissue, it has recently become clear that both physiological and reparative regeneration occur also within the CNS to sustain tissue homeostasis and repair. Proliferation and differentiation of neural stem/progenitor cells (NPCs) residing within the healthy CNS, or surviving injury, are considered crucial in sustaining these processes. Thus a large number of experimental stem cell-based transplantation systems for CNS repair have recently been established. The results suggest that transplanted NPCs promote tissue repair not only via cell replacement but also through their local contribution to changes in the diseased tissue milieu. This review focuses on the remarkable plasticity of endogenous and exogenous (transplanted) NPCs in promoting repair. Special attention will be given to the cross-talk existing between NPCs and CNS-resident microglia as well as CNS-infiltrating immune cells from the circulation, as a crucial event sustaining NPC-mediated neuroprotection. Finally, we will propose the concept of the context-dependent potency of transplanted NPCs (therapeutic plasticity) to exert multiple therapeutic actions, such as cell replacement, neurotrophic support, and immunomodulation, in CNS repair. PMID:22013212

Biomimetic hydrogels direct spinal progenitor cell differentiation and promote functional recovery after spinal cord injury.

PubMed

Geissler, Sydney A; Sabin, Alexandra L; Besser, Rachel R; Gooden, Olivia M; Shirk, Bryce D; Nguyen, Quan M; Khaing, Zin Z; Schmidt, Christine E

2018-04-01

Demyelination that results from disease or traumatic injury, such as spinal cord injury (SCI), can have a devastating effect on neural function and recovery. Many researchers are examining treatments to minimize demyelination by improving oligodendrocyte availability in vivo. Transplantation of stem and oligodendrocyte progenitor cells is a promising option, however, trials are plagued by undirected differentiation. Here we introduce a biomaterial that has been optimized to direct the differentiation of neural progenitor cells (NPCs) toward oligodendrocytes as a cell delivery vehicle after SCI. A collagen-based hydrogel was modified to mimic the mechanical properties of the neonatal spinal cord, and components present in the developing extracellular matrix were included to provide appropriate chemical cues to the NPCs to direct their differentiation toward oligodendrocytes. The hydrogel with cells was then transplanted into a unilateral cervical contusion model of SCI to examine the functional recovery with this treatment. Six behavioral tests and histological assessment were performed to examine the in vivo response to this treatment. Our results demonstrate that we can achieve a significant increase in oligodendrocyte differentiation of NPCs compared to standard culture conditions using a three-component biomaterial composed of collagen, hyaluronic acid, and laminin that has mechanical properties matched to those of neonatal neural tissue. Additionally, SCI rats with hydrogel transplants, with and without NPCs, showed functional recovery. Animals transplanted with hydrogels with NPCs showed significantly increased functional recovery over six weeks compared to the media control group. The three-component hydrogel presented here has the potential to provide cues to direct differentiation in vivo to encourage regeneration of the central nervous system.

Generation of Oligodendrogenic Spinal Neural Progenitor Cells From Human Induced Pluripotent Stem Cells.

PubMed

Khazaei, Mohamad; Ahuja, Christopher S; Fehlings, Michael G

2017-08-14

This unit describes protocols for the efficient generation of oligodendrogenic neural progenitor cells (o-NPCs) from human induced pluripotent stem cells (hiPSCs). Specifically, detailed methods are provided for the maintenance and differentiation of hiPSCs, human induced pluripotent stem cell-derived neural progenitor cells (hiPS-NPCs), and human induced pluripotent stem cell-oligodendrogenic neural progenitor cells (hiPSC-o-NPCs) with the final products being suitable for in vitro experimentation or in vivo transplantation. Throughout, cell exposure to growth factors and patterning morphogens has been optimized for both concentration and timing, based on the literature and empirical experience, resulting in a robust and highly efficient protocol. Using this derivation procedure, it is possible to obtain millions of oligodendrogenic-NPCs within 40 days of initial cell plating which is substantially shorter than other protocols for similar cell types. This protocol has also been optimized to use translationally relevant human iPSCs as the parent cell line. The resultant cells have been extensively characterized both in vitro and in vivo and express key markers of an oligodendrogenic lineage. © 2017 by John Wiley & Sons, Inc. Copyright © 2017 John Wiley and Sons, Inc.

A novel human model of the neurodegenerative disease GM1 gangliosidosis using induced pluripotent stem cells demonstrates inflammasome activation.

PubMed

Son, Mi-Young; Kwak, Jae Eun; Seol, Binna; Lee, Da Yong; Jeon, Hyejin; Cho, Yee Sook

2015-09-01

GM1 gangliosidosis (GM1) is an inherited neurodegenerative disorder caused by mutations in the lysosomal β-galactosidase (β-gal) gene. Insufficient β-gal activity leads to abnormal accumulation of GM1 gangliosides in tissues, particularly in the central nervous system, resulting in progressive neurodegeneration. Here, we report an in vitro human GM1 model, based on induced pluripotent stem cell (iPSC) technology. Neural progenitor cells differentiated from GM1 patient-derived iPSCs (GM1-NPCs) recapitulated the biochemical and molecular phenotypes of GM1, including defective β-gal activity and increased lysosomes. Importantly, the characterization of GM1-NPCs established that GM1 is significantly associated with the activation of inflammasomes, which play a critical role in the pathogenesis of various neurodegenerative diseases. Specific inflammasome inhibitors potently alleviated the disease-related phenotypes of GM1-NPCs in vitro and in vivo. Our data demonstrate that GM1-NPCs are a valuable in vitro human GM1 model and suggest that inflammasome activation is a novel target pathway for GM1 drug development. Copyright © 2015 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Molecular Insights into the Complex Relationship between Capacitance and Pore Morphology in Nanoporous Carbon-based Supercapacitors.

PubMed

Pak, Alexander J; Hwang, Gyeong S

2016-12-21

Electrochemical double layer capacitors, or supercapacitors, are high-power energy storage devices that consist of large surface area electrodes (filled with electrolyte) to accommodate ion packing in accordance with classical electric double layer (EDL) theory. Nanoporous carbons (NPCs) have recently emerged as a class of electrode materials with the potential to dramatically improve the capacitance of these devices by leveraging ion confinement. However, the molecular mechanisms underlying such enhancements are a clear departure from EDL theory and remain an open question. In this paper, we present the concept of ion reorganization kinetics during charge/discharge cycles, especially within highly confining subnanometer pores, which necessarily dictates the capacitance. Our molecular dynamics voltammetric simulations of ionic liquid immersed in NPC electrodes (of varying pore size distributions) demonstrate that the most efficient ion migration, and thereby largest capacitance, is facilitated by nonuniformity of shape (e.g., from cylindrical to slitlike) along nanopore channels. On the basis of this understanding, we propose that a new structural descriptor, coined as the pore shape factor, can provide a new avenue for materials optimization. These findings also present a framework to understand and evaluate ion migration kinetics within charged nanoporous materials.

Novel Fe-based nanocrystalline powder cores with excellent magnetic properties produced using gas-atomized powder

NASA Astrophysics Data System (ADS)

Chang, Liang; Xie, Lei; Liu, Min; Li, Qiang; Dong, Yaqiang; Chang, Chuntao; Wang, Xin-Min; Inoue, Akihisa

2018-04-01

FeSiBPNbCu nanocrystalline powder cores (NPCs) with excellent magnetic properties were fabricated by cold-compaction of the gas-atomized amorphous powder. Upon annealing at the optimum temperature, the NPCs showed excellent magnetic properties, including high initial permeability of 88, high frequency stability up to 1 MHz with a constant value of 85, low core loss of 265 mW/cm3 at 100 kHz for Bm = 0.05 T, and superior DC-bias permeability of 60% at a bias field of 100 Oe. The excellent magnetic properties of the present NPCs could be attributed to the ultrafine α-Fe(Si) phase precipitated in the amorphous matrix and the use of gas-atomized powder coated with a uniform insulation layer.

Nuclear translocation of proteins and the effect of phosphatidic acid.

PubMed

Yao, Hongyan; Wang, Geliang; Wang, Xuemin

2014-01-01

Transport of proteins containing a nuclear localization signal (NLS) into the nucleus is mediated by nuclear transport receptors called importins, typically dimmers of a cargo-binding α-subunit and a β-subunit that mediates translocation through the nuclear pore complexes (NPCs). However, how proteins without canonical NLS move into the nucleus is not well understood. Recent results indicate that phospholipids, such as phosphatidic acid, play important roles in the intracellular translocation of proteins between the nucleus and cytoplasm.

Nuclear translocation of proteins and the effect of phosphatidic acid

PubMed Central

Yao, Hongyan; Wang, Geliang; Wang, Xuemin

2014-01-01

Transport of proteins containing a nuclear localization signal (NLS) into the nucleus is mediated by nuclear transport receptors called importins, typically dimmers of a cargo-binding α-subunit and a β-subunit that mediates translocation through the nuclear pore complexes (NPCs). However, how proteins without canonical NLS move into the nucleus is not well understood. Recent results indicate that phospholipids, such as phosphatidic acid, play important roles in the intracellular translocation of proteins between the nucleus and cytoplasm. PMID:25482760

Improving Prescripted Agent Behavior with Neuroevolution

DTIC Science & Technology

2005-01-01

learned behaviors might be undesirable, violating the designer’s intentions. This problem could be avoided if game designers could specify an...compatible throughout evolution, and the problem of matching different topologies (Radcliffe 1993) is essentially avoided . Second, NEAT speciates the...interaction with NPCs, such as a Massive Multiplayer Online Role Playing Game ( MMORPG ), the ability for NPCs to slowly adapt to humans would keep the game

Nuclear NF-κB contributes to chlorpyrifos-induced apoptosis through p53 signaling in human neural precursor cells.

PubMed

Lee, Jeong Eun; Lim, Mi Sun; Park, Jae Hyeon; Park, Chang Hwan; Koh, Hyun Chul

2014-05-01

Chlorpyrifos (CPF) is one of the most widely used organophosphate insecticides with several harmful effects, including neurotoxicity. Although many studies have addressed the neurotoxicity induced by CPF, most data on neurodevelopmental damage was obtained from animal models. We are the first group to use human neural precursor cells (hNPCs) derived from human embryonic stem cells (hESCs) as a developing neuron model to evaluate the mechanisms involved in CPF-induced neurotoxicity. CPF was cytotoxic to these cells in a concentration-dependent manner, as shown by decreased cell viability and increased lactate dehydrogenase release. Furthermore, CPF reduced the expression of AKT and ERK proteins which are involved in intracellular survival pathways. Exposure of hNPCs to CPF led to the production of reactive oxygen species (ROS), and the antioxidant N-acetyl-cystein (NAC) attenuated ROS production induced by CPF. In addition, CPF increased cytochrome c release into the cytosol and activated caspase-9 and -3, indicating that cell death induced by CPF was due to apoptosis in hNPCs. Consistent with these findings, CPF treatment reduced the level of Bcl-2 protein and increased the level of Bax protein. Especially, CPF increased the translocation of BAX into the mitochondria. CPF also induced nuclear accumulation of NF-κB and p53 proteins in a concentration-dependent manner, and their inhibitors attenuated CPF-induced cytotoxicity. In addition, an inhibitor of NF-κB nuclear translocation blocked the increase of p53 in CPF-treated hNPCs. These findings show that CPF induced hNPCs death in part through NF-κB activation via ROS generation, enabling the interaction of p53 with Bcl-2 and Bax and subsequent release of cytochrome c. Collectively, these results represent a unique molecular characterization of CPF-induced cytotoxicity in hNPCs. These data suggest that CPF may affect neurodevelopment in a manner similar to that of several known and suspected neurotoxicants. Crown Copyright © 2014. Published by Elsevier B.V. All rights reserved.

Biomimetic hydrogels direct spinal progenitor cell differentiation and promote functional recovery after spinal cord injury

NASA Astrophysics Data System (ADS)

Geissler, Sydney A.; Sabin, Alexandra L.; Besser, Rachel R.; Gooden, Olivia M.; Shirk, Bryce D.; Nguyen, Quan M.; Khaing, Zin Z.; Schmidt, Christine E.

2018-04-01

Objective. Demyelination that results from disease or traumatic injury, such as spinal cord injury (SCI), can have a devastating effect on neural function and recovery. Many researchers are examining treatments to minimize demyelination by improving oligodendrocyte availability in vivo. Transplantation of stem and oligodendrocyte progenitor cells is a promising option, however, trials are plagued by undirected differentiation. Here we introduce a biomaterial that has been optimized to direct the differentiation of neural progenitor cells (NPCs) toward oligodendrocytes as a cell delivery vehicle after SCI. Approach. A collagen-based hydrogel was modified to mimic the mechanical properties of the neonatal spinal cord, and components present in the developing extracellular matrix were included to provide appropriate chemical cues to the NPCs to direct their differentiation toward oligodendrocytes. The hydrogel with cells was then transplanted into a unilateral cervical contusion model of SCI to examine the functional recovery with this treatment. Six behavioral tests and histological assessment were performed to examine the in vivo response to this treatment. Main results. Our results demonstrate that we can achieve a significant increase in oligodendrocyte differentiation of NPCs compared to standard culture conditions using a three-component biomaterial composed of collagen, hyaluronic acid, and laminin that has mechanical properties matched to those of neonatal neural tissue. Additionally, SCI rats with hydrogel transplants, with and without NPCs, showed functional recovery. Animals transplanted with hydrogels with NPCs showed significantly increased functional recovery over six weeks compared to the media control group. Significance. The three-component hydrogel presented here has the potential to provide cues to direct differentiation in vivo to encourage regeneration of the central nervous system.

Abnormal neural precursor cell regulation in the early postnatal Fragile X mouse hippocampus.

PubMed

Sourial, Mary; Doering, Laurie C

2017-07-01

The regulation of neural precursor cells (NPCs) is indispensable for a properly functioning brain. Abnormalities in NPC proliferation, differentiation, survival, or integration have been linked to various neurological diseases including Fragile X syndrome. Yet, no studies have examined NPCs from the early postnatal Fragile X mouse hippocampus despite the importance of this developmental time point, which marks the highest expression level of FMRP, the protein missing in Fragile X, in the rodent hippocampus and is when hippocampal NPCs have migrated to the dentate gyrus (DG) to give rise to lifelong neurogenesis. In this study, we examined NPCs from the early postnatal hippocampus and DG of Fragile X mice (Fmr1-KO). Immunocytochemistry on neurospheres showed increased Nestin expression and decreased Ki67 expression, which collectively indicated aberrant NPC biology. Intriguingly, flow cytometric analysis of the expression of the antigens CD15, CD24, CD133, GLAST, and PSA-NCAM showed a decreased proportion of neural stem cells (GLAST + CD15 + CD133 + ) and an increased proportion of neuroblasts (PSA-NCAM + CD15 + ) in the DG of P7 Fmr1-KO mice. This was mirrored by lower expression levels of Nestin and the mitotic marker phospho-histone H3 in vivo in the P9 hippocampus, as well as a decreased proportion of cells in the G 2 /M phases of the P7 DG. Thus, the absence of FMRP leads to fewer actively cycling NPCs, coinciding with a decrease in neural stem cells and an increase in neuroblasts. Together, these results show the importance of FMRP in the developing hippocampal formation and suggest abnormalities in cell cycle regulation in Fragile X. Crown Copyright © 2017. Published by Elsevier B.V. All rights reserved.

Rejuvenation of MPTP-induced human neural precursor cell senescence by activating autophagy

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zhu, Liang; Dong, Chuanming; Department of Anatomy and Neurobiology, The Jiangsu Key Laboratory of Neuroregeneration, Nantong University, Nantong

Aging of neural stem cell, which can affect brain homeostasis, may be caused by many cellular mechanisms. Autophagy dysfunction was found in aged and neurodegenerative brains. However, little is known about the relationship between autophagy and human neural stem cell (hNSC) aging. The present study used 1-methyl-4-phenyl-1, 2, 3, 6-tetrahydropyridine (MPTP) to treat neural precursor cells (NPCs) derived from human embryonic stem cell (hESC) line H9 and investigate related molecular mechanisms involved in this process. MPTP-treated NPCs were found to undergo premature senescence [determined by increased senescence-associated-β-galactosidase (SA-β-gal) activity, elevated intracellular reactive oxygen species level, and decreased proliferation] and weremore » associated with impaired autophagy. Additionally, the cellular senescence phenotypes were manifested at the molecular level by a significant increase in p21 and p53 expression, a decrease in SOD2 expression, and a decrease in expression of some key autophagy-related genes such as Atg5, Atg7, Atg12, and Beclin 1. Furthermore, we found that the senescence-like phenotype of MPTP-treated hNPCs was rejuvenated through treatment with a well-known autophagy enhancer rapamycin, which was blocked by suppression of essential autophagy gene Beclin 1. Taken together, these findings reveal the critical role of autophagy in the process of hNSC aging, and this process can be reversed by activating autophagy. - Highlights: • We successfully establish hESC-derived neural precursor cells. • MPTP treatment induced senescence-like state in hESC-derived NPCs. • MPTP treatment induced impaired autophagy of hESC-derived NPCs. • MPTP-induced hESC-derived NPC senescence was rejuvenated by activating autophagy.« less

Integrative analysis of genes and miRNA alterations in human embryonic stem cells-derived neural cells after exposure to silver nanoparticles

DOE Office of Scientific and Technical Information (OSTI.GOV)

Oh, Jung-Hwa; Department of human and environmental toxicology, University of Science & Technology, Daejeon 34113; Son, Mi-Young

Given the rapid growth of engineered and customer products made of silver nanoparticles (Ag NPs), understanding their biological and toxicological effects on humans is critically important. The molecular developmental neurotoxic effects associated with exposure to Ag NPs were analyzed at the physiological and molecular levels, using an alternative cell model: human embryonic stem cell (hESC)-derived neural stem/progenitor cells (NPCs). In this study, the cytotoxic effects of Ag NPs (10–200 μg/ml) were examined in these hESC-derived NPCs, which have a capacity for neurogenesis in vitro, at 6 and 24 h. The results showed that Ag NPs evoked significant toxicity in hESC-derivedmore » NPCs at 24 h in a dose-dependent manner. In addition, Ag NPs induced cell cycle arrest and apoptosis following a significant increase in oxidative stress in these cells. To further clarify the molecular mechanisms of the toxicological effects of Ag NPs at the transcriptional and post-transcriptional levels, the global expression profiles of genes and miRNAs were analyzed in hESC-derived NPCs after Ag NP exposure. The results showed that Ag NPs induced oxidative stress and dysfunctional neurogenesis at the molecular level in hESC-derived NPCs. Based on this hESC-derived neural cell model, these findings have increased our understanding of the molecular events underlying developmental neurotoxicity induced by Ag NPs in humans. - Highlights: • This system served as a suitable model for developmental neurotoxicity testing. • Ag NPs induce the apoptosis in human neural cells by ROS generation. • Genes for development of neurons were dysregulated in response to Ag NPs. • Molecular events during early developmental neurotoxicity were proposed.« less

Large-scale generation of human iPSC-derived neural stem cells/early neural progenitor cells and their neuronal differentiation.

PubMed

D'Aiuto, Leonardo; Zhi, Yun; Kumar Das, Dhanjit; Wilcox, Madeleine R; Johnson, Jon W; McClain, Lora; MacDonald, Matthew L; Di Maio, Roberto; Schurdak, Mark E; Piazza, Paolo; Viggiano, Luigi; Sweet, Robert; Kinchington, Paul R; Bhattacharjee, Ayantika G; Yolken, Robert; Nimgaonka, Vishwajit L; Nimgaonkar, Vishwajit L

2014-01-01

Induced pluripotent stem cell (iPSC)-based technologies offer an unprecedented opportunity to perform high-throughput screening of novel drugs for neurological and neurodegenerative diseases. Such screenings require a robust and scalable method for generating large numbers of mature, differentiated neuronal cells. Currently available methods based on differentiation of embryoid bodies (EBs) or directed differentiation of adherent culture systems are either expensive or are not scalable. We developed a protocol for large-scale generation of neuronal stem cells (NSCs)/early neural progenitor cells (eNPCs) and their differentiation into neurons. Our scalable protocol allows robust and cost-effective generation of NSCs/eNPCs from iPSCs. Following culture in neurobasal medium supplemented with B27 and BDNF, NSCs/eNPCs differentiate predominantly into vesicular glutamate transporter 1 (VGLUT1) positive neurons. Targeted mass spectrometry analysis demonstrates that iPSC-derived neurons express ligand-gated channels and other synaptic proteins and whole-cell patch-clamp experiments indicate that these channels are functional. The robust and cost-effective differentiation protocol described here for large-scale generation of NSCs/eNPCs and their differentiation into neurons paves the way for automated high-throughput screening of drugs for neurological and neurodegenerative diseases.

Novel pyropheophorbide steryl esters in Black Sea sediments

DOE Office of Scientific and Technical Information (OSTI.GOV)

King, L.L.; Repeta, D.J.

1991-07-01

A series of non-polar chlorophyll degradation products (NPCs) with greater than 10 components has been isolated from Black Sea sediment and identified as pyropheophorbide steryl esters by visible and mass spectrometry. These compounds have been previously observed in seawater and sediment trap samples, and may be formed during grazing of phytoplankton by zooplanktonic herbivores. In Black Sea sediments, NPCs constitute 14% of the total phorbins determined spectroscopically at 660 nm, and 39% of the total chlorophyll degradation products measured by high pressure liquid chromatography. NPCs therefore constitute a significant sedimentary sink for chlorophyll. The distribution of sterols released by hydrolysismore » of NPCs most closely resembles sterols in suspended particulate matter collected from the euphotic zone and is quite different from the distribution of solvent-extractable sterols in sediments. Sterols extracted from sediemtns have high concentrations of 4-methylsterols and high stanol/stenol ratios. BNPC-derived sterols have very low concentrations of 4-methylsterols and low stanol/stenol ratios. The authors suggest that these differences reflect an enhanced preservation of HPCs in sediments relative to free sterols and phorbins. As a result, the original production of sterols in the euphotic zone may be more closely approximated by the distribution of NPC-derived sterols than by the distribution of free sterols in sediments.« less

Preparation of isolated nuclei from K 562 haemopoietic cell line for high resolution scanning electron microscopy.

PubMed

Reipert, S; Reipert, B M; Allen, T D

1994-09-01

The aim of the work is to visualise nuclear pore complexes (NPCs) in mammalian cells by high resolution scanning electron microscopy. A detergent-free isolation protocol was employed to obtain clean nuclei from the haemopoietic cell line K 562. Nuclear isolation was performed by mechanical homogenisation under hypotonic conditions followed by purification of the nuclear fraction. The isolated nuclei were attached to silicon chips, fixed, critical point dried, and sputter coated with a thin film (3-4 nm) of tantalum. Analysis of the nuclear surface by scanning electron microscopy (SEM) revealed a strong sensitivity of the outer nuclear membrane (ONM) to disruption during the isolation procedure. A significant reduction of the characteristic pattern of damage to the ONM was achieved by means of an isopicnic centrifugation on an isoosmolar balanced Percoll gradient. Analysis of the population of isolated nuclei by flow cytometry showed no signs of cell cycle specific losses of nuclei during isolation. The SEM investigations of the morphology of the nuclear envelope (NE) and of substructural details of NPCs and polyribosomes were performed using an in-lens field emission scanning electron microscope.

High resolution microscopy reveals the nuclear shape of budding yeast during cell cycle and in various biological states

PubMed Central

Kamgoue, Alain; Normand, Christophe; Léger-Silvestre, Isabelle; Mangeat, Thomas

2016-01-01

ABSTRACT How spatial organization of the genome depends on nuclear shape is unknown, mostly because accurate nuclear size and shape measurement is technically challenging. In large cell populations of the yeast Saccharomyces cerevisiae, we assessed the geometry (size and shape) of nuclei in three dimensions with a resolution of 30 nm. We improved an automated fluorescence localization method by implementing a post-acquisition correction of the spherical microscopic aberration along the z-axis, to detect the three dimensional (3D) positions of nuclear pore complexes (NPCs) in the nuclear envelope. Here, we used a method called NucQuant to accurately estimate the geometry of nuclei in 3D throughout the cell cycle. To increase the robustness of the statistics, we aggregated thousands of detected NPCs from a cell population in a single representation using the nucleolus or the spindle pole body (SPB) as references to align nuclei along the same axis. We could detect asymmetric changes of the nucleus associated with modification of nucleolar size. Stereotypical modification of the nucleus toward the nucleolus further confirmed the asymmetric properties of the nuclear envelope. PMID:27831493

Biomechanics of the transport barrier in the nuclear pore complex.

PubMed

Stanley, George J; Fassati, Ariberto; Hoogenboom, Bart W

2017-08-01

The nuclear pore complex (NPC) is the selective gateway through which all molecules must pass when entering or exiting the nucleus. It is a cog in the gene expression pathway, an entrance to the nucleus exploited by viruses, and a highly-tuned nanoscale filter. The NPC is a large proteinaceous assembly with a central lumen occluded by natively disordered proteins, known as FG-nucleoporins (or FG-nups). These FG-nups, along with a family of soluble proteins known as nuclear transport receptors (NTRs), form the selective transport barrier. Although much is known about the transport cycle and the necessity of NTRs for chaperoning cargo molecules through the NPC, the mechanism by which NTRs and NTR•cargo complexes translocate the selective transport barrier is not well understood. How can disordered FG-nups and soluble NTRs form a transport barrier that is selective, ATP-free, and fast? In this work, we review various mechanical approaches - both experimental and theoretical/computational - employed to better understand the morphology of the FG-nups, and their role in nucleocytoplasmic transport. Recent experiments on FG-nups tethered to planar surfaces, coupled with quantitative modelling work suggests that FG-nup morphologies are the result of a finely balanced system with significant contributions from FG-nup cohesiveness and entropic repulsion, and from NTR•FG-nup binding avidity; whilst AFM experiments on intact NPCs suggest that the FG-nups are sufficiently cohesive to form condensates in the centre of the NPC lumen, which may transiently dissolve to facilitate the transport of larger cargoes. Copyright © 2017 Elsevier Ltd. All rights reserved.

Neurodevelopmental origin and adult neurogenesis of the neuroendocrine hypothalamus

PubMed Central

Maggi, Roberto; Zasso, Jacopo; Conti, Luciano

2015-01-01

The adult hypothalamus regulates many physiological functions and homeostatic loops, including growth, feeding and reproduction. In mammals, the hypothalamus derives from the ventral diencephalon where two distinct ventricular proliferative zones have been described. Although a set of transcription factors regulating the hypothalamic development has been identified, the exact molecular mechanisms that drive the differentiation of hypothalamic neural precursor cells (NPCs) toward specific neuroendocrine neuronal subtypes is yet not fully disclosed. Neurogenesis has been also reported in the adult hypothalamus at the level of specific niches located in the ventrolateral region of ventricle wall, where NPCs have been identified as radial glia-like tanycytes. Here we review the molecular and cellular systems proposed to support the neurogenic potential of developing and adult hypothalamic NPCs. We also report new insights on the mechanisms by which adult hypothalamic neurogenesis modulates key functions of this brain region. Finally, we discuss how environmental factors may modulate the adult hypothalamic neurogenic cascade. PMID:25610370

Neurofibromin Modulates Adult Hippocampal Neurogenesis and Behavioral Effects of Antidepressants

PubMed Central

Li, Yun; Li, Yanjiao; McKay, Renée M.; Riethmacher, Dieter; Parada, Luis F.

2012-01-01

Neurogenesis persists in the rodent dentate gyrus (DG) throughout adulthood but declines with age and stress. Neural progenitor cells (NPCs) residing in the subgranular zone of the DG are regulated by an array of growth factors and respond to the microenvironment, adjusting their proliferation level to determine the rate of neurogenesis. Here we report that genetic deletion of neurofibromin (Nf1), a tumor suppressor with RAS-GAP activity,in adult NPCs enhanced DG proliferation and increased generation of new neurons in mice. Nf1 loss-associated neurogenesis had the functional effect of enhancing behavioral responses to subchronic antidepressants and, over time, led to spontaneous antidepressive-like behaviors. Thus, our findings establish an important role for the Nf1-Ras pathway in regulating adult hippocampal neurogenesis, and demonstrate that activation of adult NPCs is sufficient to modulate depression- and anxiety-like behaviors. PMID:22399775

Testing Brain Overgrowth and Synaptic Models of Autism Using NPCs and Neurons from Patient-Derived iPS Cells

DTIC Science & Technology

2014-10-01

AD_________________ Award Number: W81XWH-13-1-0415 TITLE: Testing Brain Overgrowth and Synaptic Models of Autism Using NPCs and Neurons from...REPORT TYPE Annual 3. DATES COVERED 15 Sept 2013 – 14 Sept 2014 4. TITLE AND SUBTITLE Testing Brain Overgrowth and Synaptic Models of Autism Using...STATEMENT Approved for Public Release; Distribution Unlimited 13. SUPPLEMENTARY NOTES 14. ABSTRACT Autism and autism spectrum disorders (ASD

A comparison of methods to assess the antimicrobial activity of nanoparticle combinations on bacterial cells.

PubMed

Bankier, Claire; Cheong, Yuen; Mahalingam, Suntharavathanan; Edirisinghe, Mohan; Ren, Guogang; Cloutman-Green, Elaine; Ciric, Lena

2018-01-01

Bacterial cell quantification after exposure to antimicrobial compounds varies widely throughout industry and healthcare. Numerous methods are employed to quantify these antimicrobial effects. With increasing demand for new preventative methods for disease control, we aimed to compare and assess common analytical methods used to determine antimicrobial effects of novel nanoparticle combinations on two different pathogens. Plate counts of total viable cells, flow cytometry (LIVE/DEAD BacLight viability assay) and qPCR (viability qPCR) were used to assess the antimicrobial activity of engineered nanoparticle combinations (NPCs) on Gram-positive (Staphylococcus aureus) and Gram-negative (Pseudomonas aeruginosa) bacteria at different concentrations (0.05, 0.10 and 0.25 w/v%). Results were analysed using linear models to assess the effectiveness of different treatments. Strong antimicrobial effects of the three NPCs (AMNP0-2) on both pathogens could be quantified using the plate count method and flow cytometry. The plate count method showed a high log reduction (>8-log) for bacteria exposed to high NPC concentrations. We found similar antimicrobial results using the flow cytometry live/dead assay. Viability qPCR analysis of antimicrobial activity could not be quantified due to interference of NPCs with qPCR amplification. Flow cytometry was determined to be the best method to measure antimicrobial activity of the novel NPCs due to high-throughput, rapid and quantifiable results.

Histone deacetylases 1 and 2 regulate the transcriptional programs of nephron progenitors and renal vesicles.

PubMed

Liu, Hongbing; Chen, Shaowei; Yao, Xiao; Li, Yuwen; Chen, Chao-Hui; Liu, Jiao; Saifudeen, Zubaida; El-Dahr, Samir S

2018-05-18

Nephron progenitor cells (NPCs) are Six2-positive metanephric mesenchyme cells, which undergo self-renewal and differentiation to give rise to nephrons until the end of nephrogenesis. Histone deacetylases (HDACs) are a group of epigenetic regulators that control cell fate, but their role in balancing NPC renewal and differentiation is unknown. Here, we report that NPC-specific deletion of Hdac1 and Hdac2 genes in mice results in early postnatal lethality owing to renal hypodysplasia and loss of NPCs. HDAC1/2 interact with the NPC renewal regulators Six2, Osr1 and Sall1, and are co-bound along with Six2 on the Six2 enhancer. Although the mutant NPCs differentiate into renal vesicles (RVs), Hdac1/2 mutant kidneys lack nascent nephrons or mature glomeruli, a phenocopy of Lhx1 mutants. Transcriptional profiling and network analysis identified disrupted expression of Lhx1 and its downstream genes, Dll1 and Hnf1a/4a , as key mediators of the renal phenotype. Finally, although HDAC1/2-deficient NPCs and RVs overexpress hyperacetylated p53, Trp53 deletion failed to rescue the renal dysgenesis. We conclude that the epigenetic regulators HDAC1 and HDAC2 control nephrogenesis via interactions with the transcriptional programs of nephron progenitors and renal vesicles. © 2018. Published by The Company of Biologists Ltd.

Human neural progenitors differentiate into astrocytes and protect motor neurons in aging rats.

PubMed

Das, Melanie M; Avalos, Pablo; Suezaki, Patrick; Godoy, Marlesa; Garcia, Leslie; Chang, Christine D; Vit, Jean-Philippe; Shelley, Brandon; Gowing, Genevieve; Svendsen, Clive N

2016-06-01

Age-associated health decline presents a significant challenge to healthcare, although there are few animal models that can be used to test potential treatments. Here, we show that there is a significant reduction in both spinal cord motor neurons and motor function over time in the aging rat. One explanation for this motor neuron loss could be reduced support from surrounding aging astrocytes. Indeed, we have previously shown using in vitro models that aging rat astrocytes are less supportive to rat motor neuron function and survival over time. Here, we test whether rejuvenating the astrocyte niche can improve the survival of motor neurons in an aging spinal cord. We transplanted fetal-derived human neural progenitor cells (hNPCs) into the aging rat spinal cord and found that the cells survive and differentiate into astrocytes with a much higher efficiency than when transplanted into younger animals, suggesting that the aging environment stimulates astrocyte maturation. Importantly, the engrafted astrocytes were able to protect against motor neuron loss associated with aging, although this did not result in an increase in motor function based on behavioral assays. We also transplanted hNPCs genetically modified to secrete glial cell line-derived neurotrophic factor (GDNF) into the aging rat spinal cord, as this combination of cell and protein delivery can protect motor neurons in animal models of ALS. During aging, GDNF-expressing hNPCs protected motor neurons, though to the same extent as hNPCs alone, and again had no effect on motor function. We conclude that hNPCs can survive well in the aging spinal cord, protect motor neurons and mature faster into astrocytes when compared to transplantation into the young spinal cord. While there was no functional improvement, there were no functional deficits either, further supporting a good safety profile of hNPC transplantation even into the older patient population. Copyright © 2016 Elsevier Inc. All rights reserved.

Route of steroid-activated macromolecules through nuclear pores imaged with atomic force microscopy.

PubMed

Oberleithner, H; Schäfer, C; Shahin, V; Albermann, L

2003-02-01

In eukaryotic cells, two concentric membranes, the nuclear envelope (NE), separate the nucleus from the cytoplasm. The NE is punctured by nuclear pore complexes (NPCs; molecular mass 120 MDa) that serve as regulated pathways for macromolecules entering and leaving the nuclear compartment. Transport across NPCs occurs through central channels. Such import and export of macromolecules through individual NPCs can be elicited in the Xenopus laevis oocyte by injecting the mineralocorticoid aldosterone and can be visualized with atomic force microscopy. The electrical NE resistance in intact cell nuclei can be measured in parallel. Resistance increases when macromolecules are engaged with the NPC. This article describe six observations made from these experiments and the conclusions that can be drawn from them. (i) A homogeneous population of macromolecules (approx. 100 kDa) attaches to the cytoplasmic face of the NPC 2 min after aldosterone injection. They are most likely to be aldosterone receptors. After a few minutes, they have disappeared. (ii) Large plugs (approx. molecular mass 1 MDa) appear in the central channels 20 min after hormone injection. They are most likely to be ribonucleoproteins exiting the nucleus. (iii) Electrical resistance measurements in isolated nuclei reveal transient electrical NE resistance peaks: an early (2 min) peak and a late (20 min) peak. Electrical peaks reflect macromolecule interaction with the NPC. (iv) Spironolactone blocks both the early and late peaks. This indicates that classic aldosterone receptors are involved in the pregenomic (early) and post-genomic (late) responses. (v) Actinomycin D and, independently, RNase A block the late electrical peak, confirming that plugs are genomic in nature. (vi) Intracellular calcium chelation blocks both early and late electrical peaks. Thus, the release of calcium from internal stores, which is known to be the first intracellular signal in response to aldosterone, is a prerequisite for the late genomic response.

Contrasting impact of organic and inorganic nanoparticles and colloids on the behavior of particle-reactive elements in tropical estuaries: An experimental study

NASA Astrophysics Data System (ADS)

Merschel, Gila; Bau, Michael; Dantas, Elton Luiz

2017-01-01

Estuarine processes may affect the flux of dissolved organic carbon (DOC), iron and other particle-reactive elements such as the rare earth elements and yttrium (REY), into the ocean via salt-induced coagulation and subsequent removal of river-borne (nano-)particles and colloids. We experimentally assessed the impact of the admixture of seawater on DOC, Fe and REY associated with inorganic and organic nanoparticles and colloids (NPCs) present in tropical rivers, using Rio Solimões and Rio Negro, which are particularly rich in inorganic and organic NPCs, respectively, as river water endmembers. Similar to the conservative elements Sr, Rb and U, DOC behaves conservatively in all mixing experiments, whereas strong removal of Fe and REY (and preferential removal of light over heavy REY and of Ce relative to La and Pr) is confined to experiments with inorganic NPC-rich Rio Solimões water. This removal already occurs at very low salinity and is due to the aggregation of the inorganic NPCs. However, REY removal efficiency increases gradually with increasing salinity, which is in marked contrast to DOC-poor Arctic river waters from which REY removal at lowest salinity is significantly stronger. This suggests that the DOC concentrations in the water have a profound impact on the estuarine mixing behavior of particle-reactive elements. In marked contrast to the Rio Solimões mixing experiment, Fe and the REY in experiments with Rio Negro water behave similarly to DOC and mix conservatively with seawater, indicating that the organic NPCs, most of which are humic and fulvic acids, and their associated trace elements are much less susceptible to coagulation and estuarine removal than inorganic ones. Even at higher salinities, estuarine REY removal from inorganic NPC-rich Rio Solimões water significantly exceeds REY removal from organic NPC-rich Rio Negro water. Hence, the combination of higher element concentrations in and of less estuarine removal from organic NPC-rich rivers compared to inorganic NPC-rich rivers indicates that the former are a more important source of particle-reactive elements to the oceans than previously thought. This suggests that chemical complexation with organic ligands, such as humic and fulvic acids, may have a strong impact on the riverine flux and on the marine inventory of particle-reactive elements, and hence may play an important role for the isotopic composition of such elements in seawater.

Epigenome profiling and editing of neocortical progenitor cells during development.

PubMed

Albert, Mareike; Kalebic, Nereo; Florio, Marta; Lakshmanaperumal, Naharajan; Haffner, Christiane; Brandl, Holger; Henry, Ian; Huttner, Wieland B

2017-09-01

The generation of neocortical neurons from neural progenitor cells (NPCs) is primarily controlled by transcription factors binding to DNA in the context of chromatin. To understand the complex layer of regulation that orchestrates different NPC types from the same DNA sequence, epigenome maps with cell type resolution are required. Here, we present genomewide histone methylation maps for distinct neural cell populations in the developing mouse neocortex. Using different chromatin features, we identify potential novel regulators of cortical NPCs. Moreover, we identify extensive H3K27me3 changes between NPC subtypes coinciding with major developmental and cell biological transitions. Interestingly, we detect dynamic H3K27me3 changes on promoters of several crucial transcription factors, including the basal progenitor regulator Eomes We use catalytically inactive Cas9 fused with the histone methyltransferase Ezh2 to edit H3K27me3 at the Eomes locus in vivo , which results in reduced Tbr2 expression and lower basal progenitor abundance, underscoring the relevance of dynamic H3K27me3 changes during neocortex development. Taken together, we provide a rich resource of neocortical histone methylation data and outline an approach to investigate its contribution to the regulation of selected genes during neocortical development. © 2017 The Authors.

Pushing the envelope: microinjection of Minute virus of mice into Xenopus oocytes causes damage to the nuclear envelope.

PubMed

Cohen, Sarah; Panté, Nelly

2005-12-01

Parvoviruses are small DNA viruses that replicate in the nucleus of their host cells. It has been largely assumed that parvoviruses enter the nucleus through the nuclear pore complex (NPC). However, the details of this mechanism remain undefined. To study this problem, the parvovirus Minute virus of mice (MVM) was microinjected into the cytoplasm of Xenopus oocytes and a transmission electron microscope was used to visualize the effect of the virus on the host cell. It was found that MVM caused damage to the nuclear envelope (NE) in a time- and concentration-dependent manner. Damage was predominantly to the outer nuclear membrane and was often near the NPCs. However, microinjection experiments in which the NPCs were blocked showed that NE damage induced by MVM was independent of the NPC. To address the question of whether this effect of MVM is specific to the NE, purified organelles were incubated with MVM. Visualization by electron microscopy revealed that MVM did not affect all intracellular membranes. These data represent a novel form of virus-induced damage to host cell nuclear structure and suggest that MVM is imported into the nucleus using a unique mechanism that is independent of the NPC, and involves disruption of the NE and import through the resulting breaks.

Multiphoton photochemical crosslinking-based fabrication of protein micropatterns with controllable mechanical properties for single cell traction force measurements

NASA Astrophysics Data System (ADS)

Tong, Ming Hui; Huang, Nan; Zhang, Wei; Zhou, Zhuo Long; Ngan, Alfonso Hing Wan; Du, Yanan; Chan, Barbara Pui

2016-01-01

Engineering 3D microstructures with predetermined properties is critical for stem cell niche studies. We have developed a multiphoton femtosecond laser-based 3D printing platform, which generates complex protein microstructures in minutes. Here, we used the platform to test a series of fabrication and reagent parameters in precisely controlling the mechanical properties of protein micropillars. Atomic force microscopy was utilized to measure the reduced elastic modulus of the micropillars, and transmission electron microscopy was used to visualize the porosity of the structures. The reduced elastic modulus of the micropillars associated positively and linearly with the scanning power. On the other hand, the porosity and pore size of the micropillars associated inversely and linearly with the scanning power and reagent concentrations. While keeping the elastic modulus constant, the stiffness of the micropillars was controlled by varying their height. Subsequently, the single cell traction forces of rabbit chondrocytes, human dermal fibroblasts, human mesenchymal stem cells, and bovine nucleus pulposus cells (bNPCs) were successfully measured by culturing the cells on micropillar arrays of different stiffness. Our results showed that the traction forces of all groups showed positive relationship with stiffness, and that the chondrocytes and bNPCs generated the highest and lowest traction forces, respectively.

Nanoparticle Clusters: Assembly and Control Over Internal Order, Current Capabilities, and Future Potential.

PubMed

Stolarczyk, Jacek K; Deak, Andras; Brougham, Dermot F

2016-07-01

The current state of the art in the use of colloidal methods to form nanoparticle assemblies, or clusters (NPCs) is reviewed. The focus is on the two-step approach, which exploits the advantages of bottom-up wet chemical NP synthesis procedures, with subsequent colloidal destabilization to trigger assembly in a controlled manner. Recent successes in the application of functional NPCs with enhanced emergent collective properties for a wide range of applications, including in biomedical detection, surface enhanced Raman scattering (SERS) enhancement, photocatalysis, and light harvesting, are highlighted. The role of the NP-NP interactions in the formation of monodisperse ordered clusters is described and the different assembly processes from a wide range of literature sources are classified according to the nature of the perturbation from the initial equilibrium state (dispersed NPs). Finally, the future for the field and the anticipated role of computational approaches in developing next-generation functional NPCs are briefly discussed. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Excitation-neurogenesis coupling in adult neural stem/progenitor cells.

PubMed

Deisseroth, Karl; Singla, Sheela; Toda, Hiroki; Monje, Michelle; Palmer, Theo D; Malenka, Robert C

2004-05-27

A wide variety of in vivo manipulations influence neurogenesis in the adult hippocampus. It is not known, however, if adult neural stem/progenitor cells (NPCs) can intrinsically sense excitatory neural activity and thereby implement a direct coupling between excitation and neurogenesis. Moreover, the theoretical significance of activity-dependent neurogenesis in hippocampal-type memory processing networks has not been explored. Here we demonstrate that excitatory stimuli act directly on adult hippocampal NPCs to favor neuron production. The excitation is sensed via Ca(v)1.2/1.3 (L-type) Ca(2+) channels and NMDA receptors on the proliferating precursors. Excitation through this pathway acts to inhibit expression of the glial fate genes Hes1 and Id2 and increase expression of NeuroD, a positive regulator of neuronal differentiation. These activity-sensing properties of the adult NPCs, when applied as an "excitation-neurogenesis coupling rule" within a Hebbian neural network, predict significant advantages for both the temporary storage and the clearance of memories.

PSA-NCAM-Negative Neural Crest Cells Emerging during Neural Induction of Pluripotent Stem Cells Cause Mesodermal Tumors and Unwanted Grafts

PubMed Central

Lee, Dongjin R.; Yoo, Jeong-Eun; Lee, Jae Souk; Park, Sanghyun; Lee, Junwon; Park, Chul-Yong; Ji, Eunhyun; Kim, Han-Soo; Hwang, Dong-Youn; Kim, Dae-Sung; Kim, Dong-Wook

2015-01-01

Summary Tumorigenic potential of human pluripotent stem cells (hPSCs) is an important issue in clinical applications. Despite many efforts, PSC-derived neural precursor cells (NPCs) have repeatedly induced tumors in animal models even though pluripotent cells were not detected. We found that polysialic acid-neural cell adhesion molecule (PSA-NCAM)− cells among the early NPCs caused tumors, whereas PSA-NCAM+ cells were nontumorigenic. Molecular profiling, global gene analysis, and multilineage differentiation of PSA-NCAM− cells confirm that they are multipotent neural crest stem cells (NCSCs) that could differentiate into both ectodermal and mesodermal lineages. Transplantation of PSA-NCAM− cells in a gradient manner mixed with PSA-NCAM+ cells proportionally increased mesodermal tumor formation and unwanted grafts such as PERIPHERIN+ cells or pigmented cells in the rat brain. Therefore, we suggest that NCSCs are a critical target for tumor prevention in hPSC-derived NPCs, and removal of PSA-NCAM− cells eliminates the tumorigenic potential originating from NCSCs after transplantation. PMID:25937368

Modulation of the Innate Immune Response by Human Neural Precursors Prevails over Oligodendrocyte Progenitor Remyelination to Rescue a Severe Model of Pelizaeus-Merzbacher Disease.

PubMed

Marteyn, Antoine; Sarrazin, Nadège; Yan, Jun; Bachelin, Corinne; Deboux, Cyrille; Santin, Mathieu D; Gressens, Pierre; Zujovic, Violetta; Baron-Van Evercooren, Anne

2016-04-01

Pelizaeus-Merzbacher disease (PMD) results from an X-linked misexpression of proteolipid protein 1 (PLP1). This leukodystrophy causes severe hypomyelination with progressive inflammation, leading to neurological dysfunctions and shortened life expectancy. While no cure exists for PMD, experimental cell-based therapy in the dysmyelinated shiverer model suggested that human oligodendrocyte progenitor cells (hOPCs) or human neural precursor cells (hNPCs) are promising candidates to treat myelinopathies. However, the fate and restorative advantages of human NPCs/OPCs in a relevant model of PMD has not yet been addressed. Using a model of Plp1 overexpression, resulting in demyelination with progressive inflammation, we compared side-by-side the therapeutic benefits of intracerebrally grafted hNPCs and hOPCs. Our findings reveal equal integration of the donor cells within presumptive white matter tracks. While the onset of exogenous remyelination was earlier in hOPCs-grafted mice than in hNPC-grafted mice, extended lifespan occurred only in hNPCs-grafted animals. This improved survival was correlated with reduced neuroinflammation (microglial and astrocytosis loads) and microglia polarization toward M2-like phenotype followed by remyelination. Thus modulation of neuroinflammation combined with myelin restoration is crucial to prevent PMD pathology progression and ensure successful rescue of PMD mice. These findings should help to design novel therapeutic strategies combining immunomodulation and stem/progenitor cell-based therapy for disorders associating hypomyelination with inflammation as observed in PMD. © 2015 AlphaMed Press.

Melatonin resists oxidative stress-induced apoptosis in nucleus pulposus cells.

PubMed

He, Ruijun; Cui, Min; Lin, Hui; Zhao, Lei; Wang, Jiayu; Chen, Songfeng; Shao, Zengwu

2018-04-15

Intervertebral disc degeneration (IVDD) is thought to be the major cause of low back pain (LBP), which is still in lack of effective etiological treatment. Oxidative stress has been demonstrated to participate in the impairment of nucleus pulposus cells (NPCs). As the most important neuroendocrine hormone in biological clock regulation, melatonin (MLT) is also featured by good antioxidant effect. In this study, we investigated the effect and mechanisms of melatonin on oxidative stress-induced damage in rat NPCs. Cytotoxicity of H 2 O 2 and protecting effect of melatonin were analyzed with Cell Counting kit-8 (CCK-8). Cell apoptosis rate was detected by Annexin V-FITC/PI staining. DCFH-DA probe was used for the reactive oxygen species (ROS) detection. The mitochondrial membrane potential (MMP) changes were analyzed with JC-1 probe. Intracellular oxidation product and reductants were measured through enzymatic reactions. Extracellular matrix (ECM) and apoptosis associated proteins were analyzed with Western blot assays. Melatonin preserved cell viability of NPCs under oxidative stress. The apoptosis rate, ROS level and malonaldehyde (MDA) declined with melatonin. MLT/H 2 O 2 group showed higher activities of GSH and SOD. The fall of MMP receded and the expression of ECM protein increased with treatment of melatonin. The mitochondrial pathway of apoptosis was inhibited by melatonin. Melatonin alleviated the oxidative stress-induced apoptosis of NPCs. Melatonin could be a promising alternative in treatment of IVDD. Copyright © 2018 Elsevier Inc. All rights reserved.

Gap junction-mediated calcium waves define communication networks among murine postnatal neural progenitor cells.

PubMed

Lacar, Benjamin; Young, Stephanie Z; Platel, Jean-Claude; Bordey, Angélique

2011-12-01

In the postnatal neurogenic niche, two populations of astrocyte-like cells (B cells) persist, one acting as neural progenitor cells (NPCs, B1 cells) and one forming a structural boundary between the neurogenic niche and the striatum (B2 cells, niche astrocytes). Despite being viewed as two distinct entities, we found that B1 and B2 cells express the gap junction protein connexin 43 and display functional coupling involving 50-60 cells. Using neonatal electroporation to label slowly cycling radial glia-derived B1 cells, which send a basal process onto blood vessels, we further confirmed dye coupling between NPCs. To assess the functionality of the coupling, we used calcium imaging in a preparation preserving the three-dimensional architecture of the subventricular zone. Intercellular calcium waves were observed among B cells. These waves travelled bidirectionally between B1 and B2 cells and propagated on blood vessels. Inter-B-cell calcium waves were absent in the presence of a gap junction blocker but persisted with purinergic receptor blockers. These findings show that privileged microdomains of communication networks exist among NPCs and niche astrocytes. Such functional coupling between these two cell types suggests that niche astrocytes do not merely have a structural role, but may play an active role in shaping the behavior of NPCs. © 2011 The Authors. European Journal of Neuroscience © 2011 Federation of European Neuroscience Societies and Blackwell Publishing Ltd.

BDE-99 impairs differentiation of human and mouse NPCs into the oligodendroglial lineage by species-specific modes of action

PubMed Central

Dach, Katharina; Bendt, Farina; Huebenthal, Ulrike; Giersiefer, Susanne; Lein, Pamela J.; Heuer, Heike; Fritsche, Ellen

2017-01-01

Polybrominated diphenyl ethers (PBDEs) are bioaccumulating flame retardants causing developmental neurotoxicity (DNT) in humans and rodents. Their DNT effects are suspected to involve thyroid hormone (TH) signaling disruption. Here, we tested the hypothesis whether disturbance of neural progenitor cell (NPC) differentiation into the oligodendrocyte lineage (O4+ cells) by BDE-99 involves disruption of TH action in human and mouse (h,m)NPCs. Therefore, we quantified differentiation of NPCs into O4+ cells and measured their maturation via expression of myelin-associated genes (hMBP, mMog) in presence and absence of TH and/or BDE-99. T3 promoted O4+ cell differentiation in mouse, but not hNPCs, and induced hMBP/mMog gene expression in both species. BDE-99 reduced generation of human and mouse O4+ cells, but there is no indication for BDE-99 interfering with cellular TH signaling during O4+ cell formation. BDE-99 reduced hMBP expression due to oligodendrocyte reduction, but concentrations that did not affect the number of mouse O4+ cells inhibited TH-induced mMog transcription by a yet unknown mechanism. In addition, ascorbic acid antagonized only the BDE-99-dependent loss of human, not mouse, O4+ cells by a mechanism probably independent of reactive oxygen species. These data point to species-specific modes of action of BDE-99 on h/mNPC development into the oligodendrocyte lineage. PMID:28317842

BDE-99 impairs differentiation of human and mouse NPCs into the oligodendroglial lineage by species-specific modes of action.

PubMed

Dach, Katharina; Bendt, Farina; Huebenthal, Ulrike; Giersiefer, Susanne; Lein, Pamela J; Heuer, Heike; Fritsche, Ellen

2017-03-20

Polybrominated diphenyl ethers (PBDEs) are bioaccumulating flame retardants causing developmental neurotoxicity (DNT) in humans and rodents. Their DNT effects are suspected to involve thyroid hormone (TH) signaling disruption. Here, we tested the hypothesis whether disturbance of neural progenitor cell (NPC) differentiation into the oligodendrocyte lineage (O4 + cells) by BDE-99 involves disruption of TH action in human and mouse (h,m)NPCs. Therefore, we quantified differentiation of NPCs into O4 + cells and measured their maturation via expression of myelin-associated genes (hMBP, mMog) in presence and absence of TH and/or BDE-99. T3 promoted O4 + cell differentiation in mouse, but not hNPCs, and induced hMBP/mMog gene expression in both species. BDE-99 reduced generation of human and mouse O4 + cells, but there is no indication for BDE-99 interfering with cellular TH signaling during O4 + cell formation. BDE-99 reduced hMBP expression due to oligodendrocyte reduction, but concentrations that did not affect the number of mouse O4 + cells inhibited TH-induced mMog transcription by a yet unknown mechanism. In addition, ascorbic acid antagonized only the BDE-99-dependent loss of human, not mouse, O4 + cells by a mechanism probably independent of reactive oxygen species. These data point to species-specific modes of action of BDE-99 on h/mNPC development into the oligodendrocyte lineage.

Mcl1 regulates the terminal mitosis of neural precursor cells in the mammalian brain through p27Kip1.

PubMed

Hasan, S M Mahmudul; Sheen, Ashley D; Power, Angela M; Langevin, Lisa Marie; Xiong, Jieying; Furlong, Michael; Day, Kristine; Schuurmans, Carol; Opferman, Joseph T; Vanderluit, Jacqueline L

2013-08-01

Cortical development requires the precise timing of neural precursor cell (NPC) terminal mitosis. Although cell cycle proteins regulate terminal mitosis, the factors that influence the cell cycle machinery are incompletely understood. Here we show in mice that myeloid cell leukemia 1 (Mcl1), an anti-apoptotic Bcl-2 protein required for the survival of NPCs, also regulates their terminal differentiation through the cell cycle regulator p27(Kip1). A BrdU-Ki67 cell profiling assay revealed that in utero electroporation of Mcl1 into NPCs in the embryonic neocortex increased NPC cell cycle exit (the leaving fraction). This was further supported by a decrease in proliferating NPCs (Pax6(+) radial glial cells and Tbr2(+) neural progenitors) and an increase in differentiating cells (Dcx(+) neuroblasts and Tbr1(+) neurons). Similarly, BrdU birth dating demonstrated that Mcl1 promotes premature NPC terminal mitosis giving rise to neurons of the deeper cortical layers, confirming their earlier birthdate. Changes in Mcl1 expression within NPCs caused concomitant changes in the levels of p27(Kip1) protein, a key regulator of NPC differentiation. Furthermore, in the absence of p27(Kip1), Mcl1 failed to induce NPC cell cycle exit, demonstrating that p27(Kip1) is required for Mcl1-mediated NPC terminal mitosis. In summary, we have identified a novel physiological role for anti-apoptotic Mcl1 in regulating NPC terminal differentiation.

Interdisciplinary Discrepancies Between Parenteral Nutrition Macronutrient Prescribing and Recommendations: Is Body Mass Index a Factor?

PubMed

Elliott, Katie L; Kandiah, Jay; Walroth, Todd A

2017-07-01

Formal nutrition training in medical schools and residencies is lacking and needed. Registered dietitians (RDs) are formally trained in nutrition support and considered experts in the nutrition field. Our purpose was to examine prescribing and recommending discrepancies of parenteral nutrition macronutrients between medical residents (MRs) and RDs and compare results with the ASPEN clinical care guidelines. We also looked at discrepancies among obese patients, due to their increased risk of mortality. The primary end point of this retrospective review was discrepancies in nonprotein calories (NPCs) and grams of protein (PRO) between MRs and RDs. The secondary end point was discrepancies in NPCs and PRO between MRs and RDs among patients stratified by body mass index category. MRs prescribed 300 NPCs more versus RDs ( P < .001). When compared with RDs, MRs prescribed fewer NPCs for underweight patients and more for obese patients ( P < .001). The same analysis found that the PRO discrepancies significantly varied by body mass index classification as well ( P = .022). When these results were compared with the ASPEN clinical care guidelines, RDs adhered closer to the guidelines than did MRs in terms of permissive underfeeding of obese patients. It is widely accepted that MRs are in need of increased formal training, and the results of our study confirm this need and suggest a short-term solution of increasing order-writing privileges for the RD. RDs with this privilege may adhere more closely to clinical care guidelines and therefore increase patient safety.

Mesothelioma incidence and asbestos exposure in Italian national priority contaminated sites.

PubMed

Binazzi, Alessandra; Marinaccio, Alessandro; Corfiati, Marisa; Bruno, Caterina; Fazzo, Lucia; Pasetto, Roberto; Pirastu, Roberta; Biggeri, Annibale; Catelan, Dolores; Comba, Pietro; Zona, Amerigo

2017-11-01

Objectives This study aimed to (i) describe mesothelioma incidence in the Italian national priority contaminated sites (NPCS) on the basis of data available from the Italian National Mesothelioma Registry (ReNaM) and (ii) profile NPCS using Bayesian rank analysis. Methods Incident cases of mesothelioma and standardized incidence ratios (SIR) were estimated for both genders in each of the 39 selected NPCS in the period 2000-2011. Age-standardized rates of Italian geographical macro areas were used to estimate expected cases. Rankings of areas were produced by a hierarchical Bayesian model. Asbestos exposure modalities were discussed for each site. Results In the study period, 2683 incident cases of mesothelioma (1998 men, 685 women) were recorded. An excess of mesothelioma incidence was confirmed in sites with a known past history of direct use of asbestos (among men) such as Balangero (SIR 197.1, 95% CI 82.0-473.6), Casale Monferrato (SIR 910.7, 95% CI 816.5-1012.8), and Broni (SIR 1288.5, 95% CI 981.9-1691.0), in sites with shipyards and harbors (eg, Trieste, La Spezia, Venice, and Leghorn), and in settings without documented direct use of asbestos. The analysis ranked the sites of Broni and Casale Monferrato (both genders) and Biancavilla (only for women) the highest. Conclusions The present study confirms that asbestos pollution is a risk for people living in polluted areas, due to not only occupational exposure in industrial settings with direct use of asbestos but also the presence of asbestos in the environment. Epidemiological surveillance of asbestos-related diseases is a fundamental tool for monitoring the health profile in NPCS.

Impairment of adult hippocampal neural progenitor proliferation by methamphetamine: role for nitrotyrosination

PubMed Central

2011-01-01

Methamphetamine (METH) abuse has reached epidemic proportions, and it has become increasingly recognized that abusers suffer from a wide range of neurocognitive deficits. Much previous work has focused on the deleterious effects of METH on mature neurons, but little is known about the effects of METH on neural progenitor cells (NPCs). It is now well established that new neurons are continuously generated from NPCs in the adult hippocampus, and accumulating evidence suggests important roles for these neurons in hippocampal-dependent cognitive functions. In a rat hippocampal NPC culture system, we find that METH results in a dose-dependent reduction of NPC proliferation, and higher concentrations of METH impair NPC survival. NPC differentiation, however, is not affected by METH, suggesting cell-stage specificity of the effects of METH. We demonstrate that the effects of METH on NPCs are, in part, mediated through oxidative and nitrosative stress. Further, we identify seventeen NPC proteins that are post-translationally modified via 3-nitrotyrosination in response to METH, using mass spectrometric approaches. One such protein was pyruvate kinase isoform M2 (PKM2), an important mediator of cellular energetics and proliferation. We identify sites of PKM2 that undergo nitrotyrosination, and demonstrate that nitration of the protein impairs its activity. Thus, METH abuse may result in impaired adult hippocampal neurogenesis, and effects on NPCs may be mediated by protein nitration. Our study has implications for the development of novel therapeutic approaches for METH-abusing individuals with neurologic dysfunction and may be applicable to other neurodegenerative diseases in which hippocampal neurogenesis is impaired. PMID:21708025

Pilocarpine-Induced Status Epilepticus Increases the Sensitivity of P2X7 and P2Y1 Receptors to Nucleotides at Neural Progenitor Cells of the Juvenile Rodent Hippocampus.

PubMed

Rozmer, Katalin; Gao, Po; Araújo, Michelle G L; Khan, Muhammad Tahir; Liu, Juan; Rong, Weifang; Tang, Yong; Franke, Heike; Krügel, Ute; Fernandes, Maria José S; Illes, Peter

2017-07-01

Patch-clamp recordings indicated the presence of P2X7 receptors at neural progenitor cells (NPCs) in the subgranular zone of the dentate gyrus in hippocampal brain slices prepared from transgenic nestin reporter mice. The activation of these receptors caused inward current near the resting membrane potential of the NPCs, while P2Y1 receptor activation initiated outward current near the reversal potential of the P2X7 receptor current. Both receptors were identified by biophysical/pharmacological methods. When the brain slices were prepared from mice which underwent a pilocarpine-induced status epilepticus or when brain slices were incubated in pilocarpine-containing external medium, the sensitivity of P2X7 and P2Y1 receptors was invariably increased. Confocal microscopy confirmed the localization of P2X7 and P2Y1 receptor-immunopositivity at nestin-positive NPCs. A one-time status epilepticus in rats caused after a latency of about 5 days recurrent epileptic fits. The blockade of central P2X7 receptors increased the number of seizures and their severity. It is hypothesized that P2Y1 receptors after a status epilepticus may increase the ATP-induced proliferation/ectopic migration of NPCs; the P2X7 receptor-mediated necrosis/apoptosis might counteract these effects, which would otherwise lead to a chronic manifestation of recurrent epileptic fits. © The Author 2016. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

Three-Dimensional Reconstruction of Nuclear Envelope Architecture Using Dual-Color Metal-Induced Energy Transfer Imaging.

PubMed

Chizhik, Anna M; Ruhlandt, Daja; Pfaff, Janine; Karedla, Narain; Chizhik, Alexey I; Gregor, Ingo; Kehlenbach, Ralph H; Enderlein, Jörg

2017-12-26

The nuclear envelope, comprising the inner and the outer nuclear membrane, separates the nucleus from the cytoplasm and plays a key role in cellular functions. Nuclear pore complexes (NPCs), which are embedded in the nuclear envelope, control transport of macromolecules between the two compartments. Here, using dual-color metal-induced energy transfer (MIET), we determine the axial distance between Lap2β and Nup358 as markers for the inner nuclear membrane and the cytoplasmic side of the NPC, respectively. Using MIET imaging, we reconstruct the 3D profile of the nuclear envelope over the whole basal area, with an axial resolution of a few nanometers. This result demonstrates that optical microscopy can achieve nanometer axial resolution in biological samples and without recourse to complex interferometric approaches.

Integrity and Function of the Saccharomyces cerevisiae Spindle Pole Body Depends on Connections Between the Membrane Proteins Ndc1, Rtn1, and Yop1

PubMed Central

Casey, Amanda K.; Dawson, T. Renee; Chen, Jingjing; Friederichs, Jennifer M.; Jaspersen, Sue L.; Wente, Susan R.

2012-01-01

The nuclear envelope in Saccharomyces cerevisiae harbors two essential macromolecular protein assemblies: the nuclear pore complexes (NPCs) that enable nucleocytoplasmic transport, and the spindle pole bodies (SPBs) that mediate chromosome segregation. Previously, based on metazoan and budding yeast studies, we reported that reticulons and Yop1/DP1 play a role in the early steps of de novo NPC assembly. Here, we examined if Rtn1 and Yop1 are required for SPB function in S. cerevisiae. Electron microscopy of rtn1Δ yop1Δ cells revealed lobular abnormalities in SPB structure. Using an assay that monitors lateral expansion of the SPB central layer, we found that rtn1Δ yop1Δ SPBs had decreased connections to the NE compared to wild type, suggesting that SPBs are less stable in the NE. Furthermore, large budded rtn1Δ yop1Δ cells exhibited a high incidence of short mitotic spindles, which were frequently misoriented with respect to the mother–daughter axis. This correlated with cytoplasmic microtubule defects. We found that overexpression of the SPB insertion factors NDC1, MPS2, or BBP1 rescued the SPB defects observed in rtn1Δ yop1Δ cells. However, only overexpression of NDC1, which is also required for NPC biogenesis, rescued both the SPB and NPC associated defects. Rtn1 and Yop1 also physically interacted with Ndc1 and other NPC membrane proteins. We propose that NPC and SPB biogenesis are altered in cells lacking Rtn1 and Yop1 due to competition between these complexes for Ndc1, an essential common component of both NPCs and SPBs. PMID:22798490

A-type Lamins Form Distinct Filamentous Networks with Differential Nuclear Pore Complex Associations.

PubMed

Xie, Wei; Chojnowski, Alexandre; Boudier, Thomas; Lim, John S Y; Ahmed, Sohail; Ser, Zheng; Stewart, Colin; Burke, Brian

2016-10-10

The nuclear lamina is a universal feature of metazoan nuclear envelopes (NEs) [1]. In mammalian cells, it appears as a 10-30 nm filamentous layer at the nuclear face of the inner nuclear membrane (INM) and is composed primarily of A- and B-type lamins, members of the intermediate filament family [2]. While providing structural integrity to the NE, the lamina also represents an important signaling and regulatory platform [3]. Two A-type lamin isoforms, lamins A and C (LaA and LaC), are expressed in most adult human cells. Encoded by a single gene, these proteins are largely identical, diverging only in their C-terminal tail domains. By contrast with that of LaC, the unique LaA tail undergoes extensive processing, including farnesylation and endo-proteolysis [4, 5]. However, functional differences between LaA and LaC are still unclear. Compounding this uncertainty, the structure of the lamina remains ill defined. In this study, we used BioID, an in vivo proximity-labeling method to identify differential interactors of A-type lamins [6]. One of these, Tpr, a nuclear pore complex (NPC) protein, is highlighted by its selective association with LaC. By employing superresolution microscopy, we demonstrate that this Tpr association is mirrored in enhanced interaction of LaC with NPCs. Further superresolution studies visualizing both endogenous A- and B-type lamins have allowed us to construct a nanometer-scale model of the mammalian nuclear lamina. Our data indicate that different A- and B-type lamin species assemble into separate filament networks that together form an extended composite structure at the nuclear periphery providing attachment sites for NPCs, thereby regulating their distribution. Copyright © 2016 Elsevier Ltd. All rights reserved.

Attenuation of Mouse Melanoma by A/C Magnetic Field after Delivery of Bi-Magnetic Nanoparticles by Neural Progenitor Cells

PubMed Central

Rachakatla, Raja Shekar; Balivada, Sivasai; Seo, Gwi-Moon; Myers, Carl B; Wang, Hongwang; Samarakoon, Thilani N.; Dani, Raj; Pyle, Marla; Kroh, Franklin O.; Walker, Brandon; Leaym, Xiaoxuan; Koper, Olga B.; Chikan, Viktor; Bossmann, Stefan H.; Tamura, Masaaki; Troyer, Deryl L.

2010-01-01

Localized magnetic hyperthermia as a treatment modality for cancer has generated renewed interest, particularly if it can be targeted to the tumor site. We examined whether tumor-tropic neural progenitor cells (NPCs) could be utilized as cell delivery vehicles for achieving preferential accumulation of core/shell iron/iron oxide magnetic nanoparticles (MNPs) within a mouse model of melanoma. We developed aminosiloxane-porphyrin functionalized MNPs, evaluated cell viability and loading efficiency, and transplanted neural progenitor cells loaded with this cargo into mice with melanoma. NPCs were efficiently loaded with core/shell Fe/Fe3O4 MNPs with minimal cytotoxicity; the MNPs accumulated as aggregates in the cytosol. The NPCs loaded with MNPs could travel to subcutaneous melanomas, and after A/C (alternating current) magnetic field (AMF) exposure, the targeted delivery of MNPs by the cells resulted in a measurable regression of the tumors. The tumor attenuation was significant (p<0.05) a short time (24 hours) after the last of three AMF exposures. PMID:21058696

Mitochondrial Superoxide Production Negatively Regulates Neural Progenitor Proliferation and Cerebral Cortical Development

PubMed Central

Hou, Yan; Ouyang, Xin; Wan, Ruiqian; Cheng, Heping; Mattson, Mark P.; Cheng, Aiwu

2012-01-01

Although high amounts of reactive oxygen species (ROS) can damage cells, ROS can also play roles as second messengers, regulating diverse cellular processes. Here we report that embryonic mouse cerebral cortical neural progenitor cells (NPCs) exhibit intermittent spontaneous bursts of mitochondrial superoxide (SO) generation (mitochondrial SO flashes) that require transient opening of membrane permeability transition pores (mPTP). This quantal SO production negatively regulates NPC self-renewal. Mitochondrial SO scavengers and mPTP inhibitors reduce SO flash frequency and enhance NPC proliferation, whereas prolonged mPTP opening and SO generation increase SO flash incidence and decrease NPC proliferation. The inhibition of NPC proliferation by mitochondrial SO involves suppression of extracellular signal-regulated kinases. Moreover, mice lacking SOD2 (SOD2−/− mice) exhibit significantly fewer proliferative NPCs and differentiated neurons in the embryonic cerebral cortex at mid-gestation compared with wild type littermates. Cultured SOD2−/− NPCs exhibit a significant increase in SO flash frequency and reduced NPC proliferation. Taken together, our findings suggest that mitochondrial SO flashes negatively regulate NPC self-renewal in the developing cerebral cortex. PMID:22949407

High-Content Screening in hPSC-Neural Progenitors Identifies Drug Candidates that Inhibit Zika Virus Infection in Fetal-like Organoids and Adult Brain.

PubMed

Zhou, Ting; Tan, Lei; Cederquist, Gustav Y; Fan, Yujie; Hartley, Brigham J; Mukherjee, Suranjit; Tomishima, Mark; Brennand, Kristen J; Zhang, Qisheng; Schwartz, Robert E; Evans, Todd; Studer, Lorenz; Chen, Shuibing

2017-08-03

Zika virus (ZIKV) infects fetal and adult human brain and is associated with serious neurological complications. To date, no therapeutic treatment is available to treat ZIKV-infected patients. We performed a high-content chemical screen using human pluripotent stem cell-derived cortical neural progenitor cells (hNPCs) and found that hippeastrine hydrobromide (HH) and amodiaquine dihydrochloride dihydrate (AQ) can inhibit ZIKV infection in hNPCs. Further validation showed that HH also rescues ZIKV-induced growth and differentiation defects in hNPCs and human fetal-like forebrain organoids. Finally, HH and AQ inhibit ZIKV infection in adult mouse brain in vivo. Strikingly, HH suppresses viral propagation when administered to adult mice with active ZIKV infection, highlighting its therapeutic potential. Our approach highlights the power of stem cell-based screens and validation in human forebrain organoids and mouse models in identifying drug candidates for treating ZIKV infection and related neurological complications in fetal and adult patients. Copyright © 2017 Elsevier Inc. All rights reserved.

Supercapacitor electrode materials with hierarchically structured pores from carbonization of MWCNTs and ZIF-8 composites.

PubMed

Li, Xueqin; Hao, Changlong; Tang, Bochong; Wang, Yue; Liu, Mei; Wang, Yuanwei; Zhu, Yihua; Lu, Chenguang; Tang, Zhiyong

2017-02-09

Due to their high specific surface area and good electric conductivity, nitrogen-doped porous carbons (NPCs) and carbon nanotubes (CNTs) have attracted much attention for electrochemical energy storage applications. In the present work, we firstly prepared MWCNT/ZIF-8 composites by decoration of zeolitic imidazolate frameworks (ZIF-8) onto the surface of multi-walled CNTs (MWCNTs), then obtained MWCNT/NPCs by the direct carbonization of MWCNT/ZIF-8. By controlling the reaction conditions, MWCNT/ZIF-8 with three different particle sizes were synthesized. The effect of NPCs size on capacitance performance has been evaluated in detail. The MWCNT/NPC with large-sized NPC (MWCNT/NPC-L) displayed the highest specific capacitance of 293.4 F g -1 at the scan rate of 5 mV s -1 and only lost 4.2% of capacitance after 10 000 cyclic voltammetry cycles, which was attributed to the hierarchically structured pores, N-doping and high electrical conductivity. The studies of symmetric two-electrode supercapacitor cells also confirmed MWCNT/NPC-L as efficient electrode materials that have good electrochemical performance, especially for high-rate applications.

Ascorbic acid alters cell fate commitment of human neural progenitors in a WNT/β-catenin/ROS signaling dependent manner.

PubMed

Rharass, Tareck; Lantow, Margareta; Gbankoto, Adam; Weiss, Dieter G; Panáková, Daniela; Lucas, Stéphanie

2017-10-16

Improving the neuronal yield from in vitro cultivated neural progenitor cells (NPCs) is an essential challenge in transplantation therapy in neurological disorders. In this regard, Ascorbic acid (AA) is widely used to expand neurogenesis from NPCs in cultures although the mechanisms of its action remain unclear. Neurogenesis from NPCs is regulated by the redox-sensitive WNT/β-catenin signaling pathway. We therefore aimed to investigate how AA interacts with this pathway and potentiates neurogenesis. Effects of 200 μM AA were compared with the pro-neurogenic reagent and WNT/β-catenin signaling agonist lithium chloride (LiCl), and molecules with antioxidant activities i.e. N-acetyl-L-cysteine (NAC) and ruthenium red (RuR), in differentiating neural progenitor ReNcell VM cells. Cells were supplemented with reagents for two periods of treatment: a full period encompassing the whole differentiation process versus an early short period that is restricted to the cell fate commitment stage. Intracellular redox balance and reactive oxygen species (ROS) metabolism were examined by flow cytometry using redox and ROS sensors. Confocal microscopy was performed to assess cell viability, neuronal yield, and levels of two proteins: Nucleoredoxin (NXN) and the WNT/β-catenin signaling component Dishevelled 2 (DVL2). TUBB3 and MYC gene responses were evaluated by quantitative real-time PCR. DVL2-NXN complex dissociation was measured by fluorescence resonance energy transfer (FRET). In contrast to NAC which predictably exhibited an antioxidant effect, AA treatment enhanced ROS metabolism with no cytotoxic induction. Both drugs altered ROS levels only at the early stage of the differentiation as no changes were held beyond the neuronal fate commitment stage. FRET studies showed that AA treatment accelerated the redox-dependent release of the initial pool of DVL2 from its sequestration by NXN, while RuR treatment hampered the dissociation of the two proteins. Accordingly, AA increased WNT/β-catenin signaling output i.e. MYC mRNA level, whereas RuR attenuated it. Moreover, AA improved neurogenesis as much as LiCl as both TUBB3-positive cell yield and TUBB3 mRNA level increased, while NAC or RuR attenuated neurogenesis. Markedly, the neurogenesis outputs between the short and the full treatment with either NAC or AA were found unchanged, supporting our model that neuronal yield is altered by events taking place at the early phase of differentiation. Our findings demonstrate that AA treatment elevates ROS metabolism in a non-lethal manner prior to the NPCs commitment to their neuronal fate. Such effect stimulates the redox-sensitive DVL2 activation and WNT/β-catenin signaling response that would enhance the ensuing neuronal cell differentiation.

Acid-Sensing Ion Channel 1a Regulates Fate of Rat Nucleus Pulposus Cells in Acid Stimulus Through Endoplasmic Reticulum Stress.

PubMed

Xie, Zhi-Yang; Chen, Lu; Zhang, Cong; Liu, Lei; Wang, Feng; Cai, Feng; Wang, Xiao-Hu; Shi, Rui; Sinkemani, Arjun; Yu, Hao-Min; Hong, Xin; Wu, Xiao-Tao

2018-01-01

Acid-sensing ion channel 1a (ASIC1a) participates in human intervertebral disc degeneration (IVDD) and regulates the destiny of nucleus pulposus cells (NPCs) in acid stimulus. However, the mechanism of ASIC1a activation and its downstream pathway remain unclear. Endoplasmic reticulum (ER) stress also participates in the acid-induced apoptosis of NPCs. The main purpose of this study was to investigate whether there is any connection between ASIC1a and ER stress in an acid-induced nucleus pulposus degeneration model. The IVDs of Sprague-Dawley rats were stained by immunohistochemical staining to evaluate the expression of ASIC1a in normal and degenerated rat nucleus pulposus. ASIC1a expression was also quantified by quantitative real-time-polymerase chain reaction and Western blotting analysis. NPCs were exposed to the culture media with acidity at pH 7.2 and 6.5 for 24 h, with or without 4-phenylbutyrate (4-PBA, a blocker of the ER stress pathway). Cell apoptosis was examined by Annexin V/Propidium Iodide (PI) staining and was quantified using flow cytometry analysis. ASIC1a-mediated intracellular calcium was determined by Ca 2+ imaging using Fura-2-AM. Acidity-induced changes in ER stress markers were studied using Western blotting analysis. In vivo , ASIC1a expression was upregulated in natural degeneration. In vitro , acid stimulus increased intracellular calcium levels, but this effect was blocked by knockdown of ASIC1a, and this reversal was partly inhibited by 4-PBA. In addition, blockade of ASIC1a reduced expression of ER stress markers, especially the proapoptotic markers. ASIC1a partly regulates ER stress and promotes apoptosis of NPCs under acid stimulus and may be a novel therapeutic target in IVDD.

Mesencephalic human neural progenitor cells transplanted into the neonatal hemiparkinsonian rat striatum differentiate into neurons and improve motor behaviour

PubMed Central

Hovakimyan, Marine; Haas, Stefan Jean-Pierre; Schmitt, Oliver; Gerber, Bernd; Wree, Andreas; Andressen, Christian

2006-01-01

Neural stem cell transplantation is a promising strategy for the treatment of neurodegenerative diseases. To evaluate the differentiation potential of human neural progenitor cells (hNPCs) as a prerequisite for clinical trials, we intracerebrally transplanted in vitro expanded fetal mesencephalic hNPCs into hemiparkinsonian rats. On postnatal day one (P1), 17 animals underwent a unilateral intraventricular 6-hydroxydopamine injection into the right lateral ventricle. At P3, animals (n = 10) received about 100 000 hNPCs (1 µL) in the right striatum. Five weeks after birth, animals underwent behaviour tests prior to fixation, followed by immunohistochemistry on brain slices for human nuclei, glial fibrillary acidic protein, S100β, neuronal nuclei antigen, neuron-specific enolase and tyrosine hydroxylase. Compared with the apomorphine-induced rotations in the lesioned-only group (7.4 ± 0.5 min−1), lesioned and successfully transplanted animals (0.3 ± 0.1 min−1) showed a significant therapeutic improvement. Additionally, in the cylinder test, the lesioned-only animals preferred to use the ipsilateral forepaw. Conversely, the lesioned and transplanted animals showed no significant side bias similar to untreated control animals. Transplanted human nuclei-immunoreactive cells were found to survive and migrate up to 2000 µm into the host parenchyma, many containing the pan-neuronal markers neuronal nuclei antigen and neuron-specific enolase. In the striatum, tyrosine hydroxylase-immunoreactive somata were also found, indicating a dopaminergic differentiation capacity of transplanted hNPCs in vivo. However, the relative number of tyrosine hydroxylase-immunoreactive neurons in vivo seemed to be lower than in corresponding in vitro differentiation. To minimize donor tissue necessary for transplantation, further investigations will aim to enhance dopaminergic differentiation of transplanted cells in vivo. PMID:17118060

p63 and p73 coordinate p53 function to determine the balance between survival, cell death, and senescence in adult neural precursor cells

PubMed Central

Fatt, M P; Cancino, G I; Miller, F D; Kaplan, D R

2014-01-01

The p53 family members p73 and p63 have been implicated in various aspects of stem cell regulation. Here, we have asked whether they work together to regulate stem cell biology, focusing upon neural precursor cells (NPCs) in the adult murine brain. By studying mice that are haploinsufficient for p63 and/or p73, we show that these two proteins cooperate to ensure appropriate NPC self-renewal and long-term maintenance in the hippocampus and forebrain, and that when both are haploinsufficient, the NPC deficits are significantly greater than haploinsufficiency for either alone. We show that, in the case of p63+/− mice, this decrease in adult NPCs is caused by enhanced apoptosis. However, when p73 is coincidently haploinsufficient, this rescues the enhanced apoptosis of p63+/− NPCs under both basal conditions and following genotoxic stress, instead causing increased cellular senescence. This increase in cellular senescence is likely due, at least in part, to increased levels of basal DNA damage and p53 activation, as genetic ablation of p53 completely rescues the senescence phenotype observed in p63+/−; p73+/− mice. Thus, the presence of p73 determines whether p63+/− NPCs exhibit increased p53-dependent apoptosis or senescence. Together, these studies demonstrate that p63 and p73 cooperate to maintain adult NPC pools through regulation of p53 function; p63 antagonizes p53 to promote cellular survival, whereas p73 regulates self-renewal and p53-mediated apoptosis versus senescence. PMID:24809925

FOXOs modulate proteasome activity in human-induced pluripotent stem cells of Huntington's disease and their derived neural cells.

PubMed

Liu, Yanying; Qiao, Fangfang; Leiferman, Patricia C; Ross, Alan; Schlenker, Evelyn H; Wang, Hongmin

2017-11-15

Although it has been speculated that proteasome dysfunction may contribute to the pathogenesis of Huntington's disease (HD), a devastating neurodegenerative disorder, how proteasome activity is regulated in HD affected stem cells and somatic cells remains largely unclear. To better understand the pathogenesis of HD, we analyzed proteasome activity and the expression of FOXO transcription factors in three wild-type (WT) and three HD induced-pluripotent stem cell (iPSC) lines. HD iPSCs exhibited elevated proteasome activity and higher levels of FOXO1 and FOXO4 proteins. Knockdown of FOXO4 but not FOXO1 expression decreased proteasome activity. Following neural differentiation, the HD-iPSC-derived neural progenitor cells (NPCs) demonstrated lower levels of proteasome activity and FOXO expressions than their WT counterparts. More importantly, overexpression of FOXO4 but not FOXO1 in HD NPCs dramatically enhanced proteasome activity. When HD NPCs were further differentiated into DARPP32-positive neurons, these HD neurons were more susceptible to death than WT neurons and formed Htt aggregates under the condition of oxidative stress. Similar to HD NPCs, HD-iPSC-derived neurons showed reduced proteasome activity and diminished FOXO4 expression compared to WT-iPSC-derived neurons. Furthermore, HD iPSCs had lower AKT activities than WT iPSCs, whereas the neurons derived from HD iPSC had higher AKT activities than their WT counterparts. Inhibiting AKT activity increased both FOXO4 level and proteasome activity, indicating a potential role of AKT in regulating FOXO levels. These data suggest that FOXOs modulate proteasome activity, and thus represents a potentially valuable therapeutic target for HD. © The Author 2017. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

Programmed hyperphagia secondary to increased hypothalamic SIRT1.

PubMed

Desai, Mina; Li, Tie; Han, Guang; Ross, Michael G

2014-11-17

Small for gestational age (SGA) offspring exhibit reduced hypothalamic neural satiety pathways leading to programmed hyperphagia and adult obesity. Appetite regulatory site, the hypothalamic arcuate nucleus (ARC) contains appetite (NPY/AgRP) and satiety (POMC) neurons. Using in vitro culture of hypothalamic neuroprogenitor cells (NPC) which form the ARC, we demonstrated that SGA offspring exhibit reduced NPC proliferation and neuronal differentiation. bHLH protein Hes1 promotes NPC self-renewal and inhibits differentiation by repressing neuronal differentiation genes (Mash1, neurogenin3). We hypothesized that Hes1/Mash1 and ultimately ARC neuronal differentiation and expression of NPY/POMC neurons are influenced by SIRT1 which is a nutrient sensor and a histone deacetylase. Control dams received ad libitum food, whereas study dams were 50% food-restricted from pregnancy day 10 to 21 (SGA). In vivo studies showed that SGA newborns and adult offspring had increased protein expression of hypothalamic/ARC SIRT1 and AgRP with decreased POMC. Additionally, SGA newborns had decreased expression of hypothalamic neurogenic factors with reduced in vivo NPC proliferation. In vitro culture of hypothalamic NPCs showed similar changes with elevated SIRT1 binding to Hes1 in SGA newborn. Silencing SIRT1 increased NPC proliferation and Hes1 and Tuj1expression in both Control and SGA NPCs. Although SGA NPC proliferation remained below that of Controls, it was higher than Control NPCs in the absence of SIRT1 siRNA. The direct impact of SIRT1 on NPC proliferation and differentiation were further confirmed with pharmacologic SIRT1 inhibitor and activator. Thus, in SGA newborns elevated SIRT1 induces premature differentiation of NPCs, reducing the NPC pool and cell proliferation. Copyright © 2014 Elsevier B.V. All rights reserved.

Programmed Hyperphagia secondary to Increased Hypothalamic SIRT1

PubMed Central

Desai, Mina; Li, Tie; Han, Guang; Ross, Michael G.

2014-01-01

Small for gestational age (SGA) offspring exhibit reduced hypothalamic neural satiety pathways leading to programmed hyperphagia and adult obesity. Appetite regulatory site, the hypothalamic arcuate nucleus (ARC) contains appetite (NPY/AgRP) and satiety (POMC) neurons. Using in vitro culture of hypothalamic neuroprogenitor cells (NPC) which form the ARC, we demonstrated that SGA offspring exhibit reduced NPC proliferation and neuronal differentiation. bHLH protein Hes1 promotes NPC self-renewal and inhibits differentiation by repressing neuronal differentiation genes (Mash1, neurogenin3). We hypothesized that Hes1/Mash1 and ultimately ARC neuronal differentiation and expression of NPY/POMC neurons are influenced by SIRT1 which is a nutrient sensor and a histone deacetylase. Control dams received ad libitum food, whereas study dams were 50% food-restricted from pregnancy day 10 to 21 (SGA). In vivo studies showed that SGA newborns and adult offspring had increased protein expression of hypothalamic/ARC SIRT1 and AgRP with decreased POMC. Additionally, SGA newborns had decreased expression of hypothalamic neurogenic factors with reduced in vivo NPC proliferation. In vitro culture of hypothalamic NPCs showed similar changes with elevated SIRT1 binding to Hes1 in SGA newborn. Silencing SIRT1 increased NPC proliferation and Hes1 and Tuj1expression in both Control and SGA NPCs. Although SGA NPC proliferation remained below that of Controls, it was higher than Control NPCs in the absence of SIRT1 siRNA. The direct impact of SIRT1 on NPC proliferation and differentiation were further confirmed with pharmacologic SIRT1 inhibitor and activator. Thus, in SGA newborns elevated SIRT1 induces premature differentiation of NPCs, reducing the NPC pool and cell proliferation. PMID:25245521

Protein phosphatase 2ACα gene knock-out results in cortical atrophy through activating hippo cascade in neuronal progenitor cells.

PubMed

Liu, Bo; Sun, Li-Hua; Huang, Yan-Fei; Guo, Li-Jun; Luo, Li-Shu

2018-02-01

Protein phosphatase 2ACα (PP2ACα), a vital member of the protein phosphatase family, has been studied primarily as a regulator for the development, growth and protein synthesis of a lot of cell types. Dysfunction of PP2ACα protein results in neurodegenerative disease; however, this finding has not been directly confirmed in the mouse model with PP2ACα gene knock-out. Therefore, in this study presented here, we generated the PP2ACα gene knock-out mouse model by the Cre-loxP targeting gene system, with the purpose to directly observe the regulatory role of PP2ACα gene in the development of mouse's cerebral cortex. We observe that knocking-out PP2ACα gene in the central nervous system (CNS) results in cortical neuronal shrinkage, synaptic plasticity impairments, and learning/memory deficits. Further study reveals that PP2ACα gene knock-out initiates Hippo cascade in cortical neuroprogenitor cells (NPCs), which blocks YAP translocation into the nuclei of NPCs. Notably, p73, directly targeted by Hippo cascade, can bind to the promoter of glutaminase2 (GLS2) that plays a dominant role in the enzymatic regulation of glutamate/glutamine cycle. Finally, we find that PP2ACα gene knock-out inhibits the glutamine synthesis through up-regulating the activity of phosphorylated-p73 in cortical NPCs. Taken together, it concludes that PP2ACα critically supports cortical neuronal growth and cognitive function via regulating the signaling transduction of Hippo-p73 cascade. And PP2ACα indirectly modulates the glutamine synthesis of cortical NPCs through targeting p73 that plays a direct transcriptional regulatory role in the gene expression of GLS2. Copyright © 2017 Elsevier Ltd. All rights reserved.

DNA methylation and copy number variation analyses of human embryonic stem cell-derived neuroprogenitors after low-dose decabromodiphenyl ether and/or bisphenol A exposure.

PubMed

Du, L; Sun, W; Li, X M; Li, X Y; Liu, W; Chen, D

2018-05-01

The polybrominated diphenyl ether flame retardants decabromodiphenyl ether (BDE-209) and bisphenol A (BPA) are environmental contaminants that can cross the placenta and exert toxicity in the developing fetal nervous system. Copy number variants (CNVs) play a role in a number of genetic disorders and may be implicated in BDE-209/BPA teratogenicity. In this study, we found that BDE-209 and/or BPA exposure decreased neural differentiation efficiency of human embryonic stem cells (hESCs), although there was a >90% induction of neuronal progenitor cells (NPCs) from exposed hESCs. However, the mean of CNV numbers in the NPCs with BDE-209 + BPA treatment was significantly higher compared to the other groups, whereas DNA methylation was lower and DNA methyltransferase(DNMT1 and DNMT3A) expression were significantly decreased in all of the BDE-209 and/or BPA treatment groups compared with the control groups. The number of CNVs in chromosomes 3, 4, 11, 22, and X in NPCs with BDE-209 and/or BPA exposure was higher compared to the control group. In addition, CNVs in chromosomes 7, 8, 14, and 16 were stable in hESCs and hESCs-derived NPCs irrespective of BDE-209/BPA exposure, and CNVs in chromosomes 20 q11.21 and 16 p13.11 might be induced by neural differentiation. Thus, BDE-209/BPA exposure emerges as a potential source of CNVs distinct from neural differentiation by itself. BDE-209 and/or BPA exposure may cause genomic instability in cultured stem cells via reduced activity of DNA methyltransferase, suggesting a new mechanism of human embryonic neurodevelopmental toxicity caused by this class of environmental toxins.

Engineering quadratic nonlinear photonic crystals for frequency conversion of lasers

NASA Astrophysics Data System (ADS)

Chen, Baoqin; Hong, Lihong; Hu, Chenyang; Zhang, Chao; Liu, Rongjuan; Li, Zhiyuan

2018-03-01

Nonlinear frequency conversion offers an effective way to extend the laser wavelength range. Quadratic nonlinear photonic crystals (NPCs) are artificial materials composed of domain-inversion structures whose sign of nonlinear coefficients are modulated with desire to implement quasi-phase matching (QPM) required for nonlinear frequency conversion. These structures can offer various reciprocal lattice vectors (RLVs) to compensate the phase-mismatching during the quadratic nonlinear optical processes, including second-harmonic generation (SHG), sum-frequency generation and the cascaded third-harmonic generation (THG). The modulation pattern of the nonlinear coefficients is flexible, which can be one-dimensional or two-dimensional (2D), be periodic, quasi-periodic, aperiodic, chirped, or super-periodic. As a result, these NPCs offer very flexible QPM scheme to satisfy various nonlinear optics and laser frequency conversion problems via design of the modulation patterns and RLV spectra. In particular, we introduce the electric poling technique for fabricating QPM structures, a simple effective nonlinear coefficient model for efficiently and precisely evaluating the performance of QPM structures, the concept of super-QPM and super-periodically poled lithium niobate for finely tuning nonlinear optical interactions, the design of 2D ellipse QPM NPC structures enabling continuous tunability of SHG in a broad bandwidth by simply changing the transport direction of pump light, and chirped QPM structures that exhibit broadband RLVs and allow for simultaneous radiation of broadband SHG, THG, HHG and thus coherent white laser from a single crystal. All these technical, theoretical, and physical studies on QPM NPCs can help to gain a deeper insight on the mechanisms, approaches, and routes for flexibly controlling the interaction of lasers with various QPM NPCs for high-efficiency frequency conversion and creation of novel lasers.

A comparative transcriptomic analysis of astrocytes differentiation from human neural progenitor cells.

PubMed

Magistri, Marco; Khoury, Nathalie; Mazza, Emilia Maria Cristina; Velmeshev, Dmitry; Lee, Jae K; Bicciato, Silvio; Tsoulfas, Pantelis; Faghihi, Mohammad Ali

2016-11-01

Astrocytes are a morphologically and functionally heterogeneous population of cells that play critical roles in neurodevelopment and in the regulation of central nervous system homeostasis. Studies of human astrocytes have been hampered by the lack of specific molecular markers and by the difficulties associated with purifying and culturing astrocytes from adult human brains. Human neural progenitor cells (NPCs) with self-renewal and multipotent properties represent an appealing model system to gain insight into the developmental genetics and function of human astrocytes, but a comprehensive molecular characterization that confirms the validity of this cellular system is still missing. Here we used an unbiased transcriptomic analysis to characterize in vitro culture of human NPCs and to define the gene expression programs activated during the differentiation of these cells into astrocytes using FBS or the combination of CNTF and BMP4. Our results demonstrate that in vitro cultures of human NPCs isolated during the gliogenic phase of neurodevelopment mainly consist of radial glial cells (RGCs) and glia-restricted progenitor cells. In these cells the combination of CNTF and BMP4 activates the JAK/STAT and SMAD signaling cascades, leading to the inhibition of oligodendrocytes lineage commitment and activation of astrocytes differentiation. On the other hand, FBS-derived astrocytes have properties of reactive astrocytes. Our work suggests that in vitro culture of human NPCs represents a valuable cellular system to study human disorders characterized by impairment of astrocytes development and function. Our datasets represent an important resource for researchers studying human astrocytes development and might set the basis for the discovery of novel human-specific astrocyte markers. © 2016 The Authors. European Journal of Neuroscience published by Federation of European Neuroscience Societies and John Wiley & Sons Ltd.

The Nuclear Pore-Associated TREX-2 Complex Employs Mediator to Regulate Gene Expression

PubMed Central

Schneider, Maren; Hellerschmied, Doris; Schubert, Tobias; Amlacher, Stefan; Vinayachandran, Vinesh; Reja, Rohit; Pugh, B. Franklin; Clausen, Tim; Köhler, Alwin

2015-01-01

Summary Nuclear pore complexes (NPCs) influence gene expression besides their established function in nuclear transport. The TREX-2 complex localizes to the NPC basket and affects gene-NPC interactions, transcription, and mRNA export. How TREX-2 regulates the gene expression machinery is unknown. Here, we show that TREX-2 interacts with the Mediator complex, an essential regulator of RNA Polymerase (Pol) II. Structural and biochemical studies identify a conserved region on TREX-2, which directly binds the Mediator Med31/Med7N submodule. TREX-2 regulates assembly of Mediator with the Cdk8 kinase and is required for recruitment and site-specific phosphorylation of Pol II. Transcriptome and phenotypic profiling confirm that TREX-2 and Med31 are functionally interdependent at specific genes. TREX-2 additionally uses its Mediator-interacting surface to regulate mRNA export suggesting a mechanism for coupling transcription initiation and early steps of mRNA processing. Our data provide mechanistic insight into how an NPC-associated adaptor complex accesses the core transcription machinery. PMID:26317468

Apoptosis leads to a degradation of vital components of active nuclear transport and a dissociation of the nuclear lamina.

PubMed

Kramer, A; Liashkovich, I; Oberleithner, H; Ludwig, S; Mazur, I; Shahin, V

2008-08-12

Apoptosis, a physiologically critical process, is characterized by a destruction of the cell after sequential degradation of key cellular components. Here, we set out to explore the fate of the physiologically indispensable nuclear envelope (NE) in this process. The NE mediates the critical nucleocytoplasmic transport through nuclear pore complexes (NPCs). In addition, the NE is involved in gene expression and contributes significantly to the overall structure and mechanical stability of the cell nucleus through the nuclear lamina, which underlies the entire nucleoplasmic face of the NE and thereby interconnects the NPCs, the NE, and the genomic material. Using the nano-imaging and mechanical probing approach atomic force microscopy (AFM) and biochemical methods, we unveiled the fate of the NE during apoptosis. The doomed NE sustains a degradation of both the mediators of the critical selective nucleocytoplasmic transport, namely NPC cytoplasmic filaments and basket, and the nuclear lamina. These observations are paralleled by marked softening and destabilization of the NE and the detection of vesicle-like nuclear fragments. We conclude that destruction of the cell nucleus during apoptosis proceeds in a strategic fashion. Degradation of NPC cytoplasmic filaments and basket shuts down the critical selective nucleocytoplasmic cross-talk. Degradation of the nuclear lamina disrupts the pivotal connection between the NE and the chromatin, breaks up the overall nuclear architecture, and softens the NE, thereby enabling the formation of nuclear fragments at later stages of apoptosis.

Caulimoviridae Tubule-Guided Transport Is Dictated by Movement Protein Properties ▿

PubMed Central

Sánchez-Navarro, Jesús; Fajardo, Thor; Zicca, Stefania; Pallás, Vicente; Stavolone, Livia

2010-01-01

Plant viruses move through plasmodesmata (PD) either as nucleoprotein complexes (NPCs) or as tubule-guided encapsidated particles with the help of movement proteins (MPs). To explore how and why MPs specialize in one mechanism or the other, we tested the exchangeability of MPs encoded by DNA and RNA virus genomes by means of an engineered alfalfa mosaic virus (AMV) system. We show that Caulimoviridae (DNA genome virus) MPs are competent for RNA virus particle transport but are unable to mediate NPC movement, and we discuss this restriction in terms of the evolution of DNA virus MPs as a means of mediating DNA viral genome entry into the RNA-trafficking PD pathway. PMID:20130061

Bayes classification of terrain cover using normalized polarimetric data

NASA Technical Reports Server (NTRS)

Yueh, H. A.; Swartz, A. A.; Kong, J. A.; Shin, R. T.; Novak, L. M.

1988-01-01

The normalized polarimetric classifier (NPC) which uses only the relative magnitudes and phases of the polarimetric data is proposed for discrimination of terrain elements. The probability density functions (PDFs) of polarimetric data are assumed to have a complex Gaussian distribution, and the marginal PDF of the normalized polarimetric data is derived by adopting the Euclidean norm as the normalization function. The general form of the distance measure for the NPC is also obtained. It is demonstrated that for polarimetric data with an arbitrary PDF, the distance measure of NPC will be independent of the normalization function selected even when the classifier is mistrained. A complex Gaussian distribution is assumed for the polarimetric data consisting of grass and tree regions. The probability of error for the NPC is compared with those of several other single-feature classifiers. The classification error of NPCs is shown to be independent of the normalization function.

Super-resolution mapping of scaffold nucleoporins in the nuclear pore complex.

PubMed

Ma, Jiong; Kelich, Joseph M; Junod, Samuel L; Yang, Weidong

2017-04-01

The nuclear pore complex (NPC), composed of ∼30 different nucleoporins (Nups), is one of the largest supramolecular structures in eukaryotic cells. Its octagonal ring scaffold perforates the nuclear envelope and features a unique molecular machinery that regulates nucleocytoplasmic transport. However, the precise copy number and the spatial location of each Nup in the native NPC remain obscure due to the inherent difficulty of counting and localizing proteins inside of the sub-micrometer supramolecular complex. Here, we combined super-resolution single-point edge-excitation subdiffraction (SPEED) microscopy and nanobody-specific labeling to reveal the spatial distribution of scaffold Nups within three separate layers in the native NPC with a precision of ∼3 nm. Our data reveal both the radial and axial spatial distributions for Pom121, Nup37 and Nup35 and provide evidence for their copy numbers of 8, 32 and 16, respectively, per NPC. This approach can help pave the path for mapping the entirety of Nups in native NPCs and also other structural components of macromolecular complexes. © 2017. Published by The Company of Biologists Ltd.

Super-resolution mapping of scaffold nucleoporins in the nuclear pore complex

PubMed Central

Ma, Jiong; Kelich, Joseph M.; Junod, Samuel L.

2017-01-01

ABSTRACT The nuclear pore complex (NPC), composed of ∼30 different nucleoporins (Nups), is one of the largest supramolecular structures in eukaryotic cells. Its octagonal ring scaffold perforates the nuclear envelope and features a unique molecular machinery that regulates nucleocytoplasmic transport. However, the precise copy number and the spatial location of each Nup in the native NPC remain obscure due to the inherent difficulty of counting and localizing proteins inside of the sub-micrometer supramolecular complex. Here, we combined super-resolution single-point edge-excitation subdiffraction (SPEED) microscopy and nanobody-specific labeling to reveal the spatial distribution of scaffold Nups within three separate layers in the native NPC with a precision of ∼3 nm. Our data reveal both the radial and axial spatial distributions for Pom121, Nup37 and Nup35 and provide evidence for their copy numbers of 8, 32 and 16, respectively, per NPC. This approach can help pave the path for mapping the entirety of Nups in native NPCs and also other structural components of macromolecular complexes. PMID:28202688

Yeast silencing factor Sir4 and a subset of nucleoporins form a complex distinct from nuclear pore complexes

PubMed Central

Ptak, Christopher; Roesner, Ulyss K.

2017-01-01

Interactions occurring at the nuclear envelope (NE)–chromatin interface influence both NE structure and chromatin organization. Insights into the functions of NE–chromatin interactions have come from the study of yeast subtelomeric chromatin and its association with the NE, including the identification of various proteins necessary for tethering subtelomeric chromatin to the NE and the silencing of resident genes. Here we show that four of these proteins—the silencing factor Sir4, NE-associated Esc1, the SUMO E3 ligase Siz2, and the nuclear pore complex (NPC) protein Nup170—physically and functionally interact with one another and a subset of NPC components (nucleoporins or Nups). Importantly, this group of Nups is largely restricted to members of the inner and outer NPC rings, but it lacks numerous others including cytoplasmically and nucleoplasmically positioned Nups. We propose that this Sir4-associated Nup complex is distinct from holo-NPCs and that it plays a role in subtelomeric chromatin organization and NE tethering. PMID:28883038

Yeast silencing factor Sir4 and a subset of nucleoporins form a complex distinct from nuclear pore complexes.

PubMed

Lapetina, Diego L; Ptak, Christopher; Roesner, Ulyss K; Wozniak, Richard W

2017-10-02

Interactions occurring at the nuclear envelope (NE)-chromatin interface influence both NE structure and chromatin organization. Insights into the functions of NE-chromatin interactions have come from the study of yeast subtelomeric chromatin and its association with the NE, including the identification of various proteins necessary for tethering subtelomeric chromatin to the NE and the silencing of resident genes. Here we show that four of these proteins-the silencing factor Sir4, NE-associated Esc1, the SUMO E3 ligase Siz2, and the nuclear pore complex (NPC) protein Nup170-physically and functionally interact with one another and a subset of NPC components (nucleoporins or Nups). Importantly, this group of Nups is largely restricted to members of the inner and outer NPC rings, but it lacks numerous others including cytoplasmically and nucleoplasmically positioned Nups. We propose that this Sir4-associated Nup complex is distinct from holo-NPCs and that it plays a role in subtelomeric chromatin organization and NE tethering. © 2017 Lapetina et al.

Lead decreases cell survival, proliferation, and neuronal differentiation of primary cultured adult neural precursor cells through activation of the JNK and p38 MAP kinases

PubMed Central

Engstrom, Anna; Wang, Hao; Xia, Zhengui

2015-01-01

Adult hippocampal neurogenesis is the process whereby adult neural precursor cells (aNPCs) in the subgranular zone (SGZ) of the dentate gyrus (DG) generate adult-born, functional neurons in the hippocampus. This process is modulated by various extracellular and intracellular stimuli, and the adult-born neurons have been implicated in hippocampus-dependent learning and memory. However, studies on how neurotoxic agents affect this process and the underlying mechanisms are limited. The goal of this study was to determine whether lead, a heavy metal, directly impairs critical processes in adult neurogenesis and to characterize the underlying signaling pathways using primary cultured SGZ-aNPCs isolated from adult mice. We report here that lead significantly increases apoptosis and inhibits proliferation in SGZ-aNPCs. In addition, lead significantly impairs spontaneous neuronal differentiation and maturation. Furthermore, we found that activation of the c-Jun NH2-terminal kinase (JNK) and p38 mitogen activated protein (MAP) kinase signaling pathways are important for lead cytotoxicity. Our data suggest that lead can directly act on adult neural stem cells and impair critical processes in adult hippocampal neurogenesis, which may contribute to its neurotoxicity and adverse effects on cognition in adults. PMID:25967738

Lead decreases cell survival, proliferation, and neuronal differentiation of primary cultured adult neural precursor cells through activation of the JNK and p38 MAP kinases.

PubMed

Engstrom, Anna; Wang, Hao; Xia, Zhengui

2015-08-01

Adult hippocampal neurogenesis is the process whereby adult neural precursor cells (aNPCs) in the subgranular zone (SGZ) of the dentate gyrus (DG) generate adult-born, functional neurons in the hippocampus. This process is modulated by various extracellular and intracellular stimuli, and the adult-born neurons have been implicated in hippocampus-dependent learning and memory. However, studies on how neurotoxic agents affect this process and the underlying mechanisms are limited. The goal of this study was to determine whether lead, a heavy metal, directly impairs critical processes in adult neurogenesis and to characterize the underlying signaling pathways using primary cultured SGZ-aNPCs isolated from adult mice. We report here that lead significantly increases apoptosis and inhibits proliferation in SGZ-aNPCs. In addition, lead significantly impairs spontaneous neuronal differentiation and maturation. Furthermore, we found that activation of the c-Jun NH2-terminal kinase (JNK) and p38 mitogen activated protein (MAP) kinase signaling pathways are important for lead cytotoxicity. Our data suggest that lead can directly act on adult neural stem cells and impair critical processes in adult hippocampal neurogenesis, which may contribute to its neurotoxicity and adverse effects on cognition in adults. Copyright © 2015 Elsevier Ltd. All rights reserved.

A human neurodevelopmental model for Williams syndrome

PubMed Central

Chailangkarn, Thanathom; Trujillo, Cleber A.; Freitas, Beatriz C.; Hrvoj-Mihic, Branka; Herai, Roberto H.; Yu, Diana X.; Brown, Timothy T.; Marchetto, Maria C. N.; Bardy, Cedric; McHenry, Lauren; Stefanacci, Lisa; Järvinen, Anna; Searcy, Yvonne M.; DeWitt, Michelle; Wong, Wenny; Lai, Philip; Ard, M. Colin; Hanson, Kari L.; Romero, Sarah; Jacobs, Bob; Dale, Anders M.; Dai, Li; Korenberg, Julie R.; Gage, Fred H.; Bellugi, Ursula; Halgren, Eric; Semendeferi, Katerina; Muotri, Alysson R.

2016-01-01

Summary Williams syndrome (WS) is a genetic neurodevelopmental disorder characterized by an uncommon hypersociability and a mosaic of retained and compromised linguistic and cognitive abilities. Nearly all clinically diagnosed individuals with WS lack precisely the same set of genes, with breakpoints in chromosome band 7q11.231–5. The contribution of specific genes to the neuroanatomical and functional alterations, leading to behavioral pathologies in humans, remains largely unexplored. Here, we investigate neural progenitor cells (NPCs) and cortical neurons derived from WS and typically developing (TD) induced pluripotent stem cells (iPSCs). WS NPCs have an increased doubling time and apoptosis compared to TD NPCs. Using an atypical WS subject6, 7, we narrowed this cellular phenotype to a single gene candidate, FZD9. At the neuronal stage, WS-derived layers V/VI cortical neurons were characterized by longer total dendrites, increased numbers of spines and synapses, aberrant calcium oscillation and altered network connectivity. Morphometric alterations observed in WS neurons were validated after Golgi staining of postmortem layers V/VI cortical neurons. This human iPSC model8 fills in the current knowledge gap in WS cellular biology and could lead to further insights into the molecular mechanism underlying the disorder and the human social brain. PMID:27509850

Protein-based human iPS cells efficiently generate functional dopamine neurons and can treat a rat model of Parkinson disease.

PubMed

Rhee, Yong-Hee; Ko, Ji-Yun; Chang, Mi-Yoon; Yi, Sang-Hoon; Kim, Dohoon; Kim, Chun-Hyung; Shim, Jae-Won; Jo, A-Young; Kim, Byung-Woo; Lee, Hyunsu; Lee, Suk-Ho; Suh, Wonhee; Park, Chang-Hwan; Koh, Hyun-Chul; Lee, Yong-Sung; Lanza, Robert; Kim, Kwang-Soo; Lee, Sang-Hun

2011-06-01

Parkinson disease (PD) involves the selective loss of midbrain dopamine (mDA) neurons and is a possible target disease for stem cell-based therapy. Human induced pluripotent stem cells (hiPSCs) are a potentially unlimited source of patient-specific cells for transplantation. However, it is critical to evaluate the safety of hiPSCs generated by different reprogramming methods. Here, we compared multiple hiPSC lines derived by virus- and protein-based reprogramming to human ES cells (hESCs). Neuronal precursor cells (NPCs) and dopamine (DA) neurons delivered from lentivirus-based hiPSCs exhibited residual expression of exogenous reprogramming genes, but those cells derived from retrovirus- and protein-based hiPSCs did not. Furthermore, NPCs derived from virus-based hiPSCs exhibited early senescence and apoptotic cell death during passaging, which was preceded by abrupt induction of p53. In contrast, NPCs derived from hESCs and protein-based hiPSCs were highly expandable without senescence. DA neurons derived from protein-based hiPSCs exhibited gene expression, physiological, and electrophysiological properties similar to those of mDA neurons. Transplantation of these cells into rats with striatal lesions, a model of PD, significantly rescued motor deficits. These data support the clinical potential of protein-based hiPSCs for personalized cell therapy of PD.

Conditioned Medium Derived from Neural Progenitor Cells Induces Long-term Post-ischemic Neuroprotection, Sustained Neurological Recovery, Neurogenesis, and Angiogenesis.

PubMed

Doeppner, Thorsten R; Traut, Viktorija; Heidenreich, Alexander; Kaltwasser, Britta; Bosche, Bert; Bähr, Mathias; Hermann, Dirk M

2017-03-01

Adult neural progenitor cells (NPCs) induce post-ischemic long-term neuroprotection and brain remodeling by releasing of survival- and plasticity-promoting mediators. To evaluate whether secreted factors may mimic neuroprotective and restorative effects of NPCs, we exposed male C57BL6 mice to focal cerebral ischemia and intravenously applied conditioned medium (CM) derived from subventricular zone NPCs. CM dose-dependently reduced infarct volume and brain leukocyte infiltration after 48 h when delivered up to 12 h after focal cerebral ischemia. Neuroprotection persisted in the post-acute stroke phase yielding enhanced neurological recovery that lasted throughout the 28-day observation period. Increased Bcl-2, phosphorylated Akt and phosphorylated STAT-3 abundance, and reduced caspase-3 activity and Bax abundance were noted in ischemic brains of CM-treated mice at 48 h post-stroke, indicative of enhanced cell survival signaling. Long-term neuroprotection was associated with increased brain glial cell line-derived neurotrophic factor (GDNF) and vascular endothelial growth factor (VEGF) concentrations at 28 days resulting in increased neurogenesis and angiogenesis. The observation that NPC-derived CM induces sustained neuroprotection and neurological recovery suggests that cell transplantation may be dispensable when secreted factors are instead administered.

miR-196a Ameliorates Cytotoxicity and Cellular Phenotype in Transgenic Huntington’s Disease Monkey Neural Cells

PubMed Central

Carter, Richard L.; Prucha, Melinda S.; Yang, Jinjing; Parnpai, Rangsun; Chan, Anthony W. S.

2016-01-01

Huntington’s disease (HD) is an inherited neurodegenerative disorder caused by the expansion of polyglutamine (polyQ) tract that leads to motor, cognitive and psychiatric impairment. Currently there is no cure for HD. A transgenic HD nonhuman primate (HD-NHP) model was developed with progressive development of clinical and pathological features similar to human HD, which suggested the potential preclinical application of the HD-NHP model. Elevated expression of miR-196a was observed in both HD-NHP and human HD brains. Cytotoxicity and apoptosis were ameliorated by the overexpression of miR-196a in HD-NHP neural progenitor cells (HD-NPCs) and differentiated neural cells (HD-NCs). The expression of apoptosis related gene was also down regulated. Mitochondrial morphology and activity were improved as indicated by mitotracker staining and the upregulation of CBP and PGC-1α in HD-NPCs overexpressing miR-196a. Here we demonstrated the amelioration of HD cellular phenotypes in HD-NPCs and HD-NCs overexpressing miR-196a. Our results also suggested the regulatory role of miR-196a in HD pathogenesis that may hold the key for understanding molecular regulation in HD and developing novel therapeutics. PMID:27631085

Co-evolution of breast-to-brain metastasis and neural progenitor cells.

PubMed

Neman, Josh; Choy, Cecilia; Kowolik, Claudia M; Anderson, Athena; Duenas, Vincent J; Waliany, Sarah; Chen, Bihong T; Chen, Mike Y; Jandial, Rahul

2013-08-01

Brain colonization by metastatic tumor cells offers a unique opportunity to investigate microenvironmental influences on the neoplastic process. The bi-directional interplay of breast cancer cells (mesodermal origin) and brain cells (neuroectodermal origin) is poorly understood and rarely investigated. In our patients undergoing neurosurgical resection of breast-to-brain metastases, specimens from the tumor/brain interface exhibited increased active gliosis as previously described. In addition, our histological characterization revealed infiltration of neural progenitor cells (NPCs) both outside and inside the tumor margin, leading us to investigate the cellular and molecular interactions between NPCs and metastases. Since signaling by the TGF-β superfamily is involved in both developmental neurobiology and breast cancer pathogenesis, we examined the role of these proteins in the context of brain metastases. The brain-metastatic breast cancer cell line MDA-MB-231Br (231Br) expressed BMP-2 at significantly higher levels compared to its matched primary breast cancer cell line MDA-MB-231 (231). Co-culturing was used to examine bi-directional cellular effects and the relevance of BMP-2 overexpression. When co-cultured with NPCs, 231 (primary) tumor cells failed to proliferate over 15 days. However, 231Br (brain metastatic) tumor cells co-cultured with NPCs escaped growth inhibition after day 5 and proliferated, occurring in parallel with NPC differentiation into astrocytes. Using shRNA and gene knock-in, we then demonstrated BMP-2 secreted by 231Br cells mediated NPC differentiation into astrocytes and concomitant tumor cell proliferation in vitro. In xenografts, overexpression of BMP-2 in primary breast cancer cells significantly enhanced their ability to engraft and colonize the brain, thereby creating a metastatic phenotype. Conversely, BMP-2 knockdown in metastatic breast cancer cells significantly diminished engraftment and colonization. The results suggest metastatic tumor cells create a permissive neural niche by steering NPC differentiation toward astrocytes through paracrine BMP-2 signaling.

Co-evolution of breast-to-brain metastasis and neural progenitor cells

PubMed Central

Neman, Josh; Choy, Cecilia; Kowolik, Claudia M.; Anderson, Athena; Duenas, Vincent J.; Waliany, Sarah; Chen, Bihong T.; Chen, Mike Y.

2013-01-01

Brain colonization by metastatic tumor cells offers a unique opportunity to investigate microenvironmental influences on the neoplastic process. The bi-directional interplay of breast cancer cells (mesodermal origin) and brain cells (neuroectodermal origin) is poorly understood and rarely investigated. In our patients undergoing neurosurgical resection of breast-to-brain metastases, specimens from the tumor/brain interface exhibited increased active gliosis as previously described. In addition, our histological characterization revealed infiltration of neural progenitor cells (NPCs) both outside and inside the tumor margin, leading us to investigate the cellular and molecular interactions between NPCs and metastases. Since signaling by the TGF-β superfamily is involved in both developmental neurobiology and breast cancer pathogenesis, we examined the role of these proteins in the context of brain metastases. The brain-metastatic breast cancer cell line MDA-MB-231Br (231Br) expressed BMP-2 at significantly higher levels compared to its matched primary breast cancer cell line MDA-MB-231 (231). Co-culturing was used to examine bi-directional cellular effects and the relevance of BMP-2 overexpression. When co-cultured with NPCs, 231 (primary) tumor cells failed to proliferate over 15 days. However, 231Br (brain meta-static) tumor cells co-cultured with NPCs escaped growth inhibition after day 5 and proliferated, occurring in parallel with NPC differentiation into astrocytes. Using shRNA and gene knock-in, we then demonstrated BMP-2 secreted by 231Br cells mediated NPC differentiation into astrocytes and concomitant tumor cell proliferation in vitro. In xenografts, overexpression of BMP-2 in primary breast cancer cells significantly enhanced their ability to engraft and colonize the brain, thereby creating a metastatic phenotype. Conversely, BMP-2 knockdown in metastatic breast cancer cells significantly diminished engraftment and colonization. The results suggest metastatic tumor cells create a permissive neural niche by steering NPC differentiation toward astrocytes through paracrine BMP-2 signaling. PMID:23456474

Upregulated expression of Nogo-A and NgR in an experimental model of focal microgyria regulates the migration, proliferation and self-renewal of subventricular zone neural progenitors

DOE Office of Scientific and Technical Information (OSTI.GOV)

Yu, Sixun; Shu, Haifeng; Yang, Tao

Nogo-A and its receptor (NgR) were first described as myelin-associated inhibitors of neuronal regeneration in response to injury. In recent years, knowledge about the important role of the Nogo-A protein in several neuronal pathologies has grown considerably. Here, we employed a neonatal cortex freeze-lesion (NFL) model in neonatal rats and measured the expression of Nogo-A and NgR in the resulting cerebrocortical microdysgenesis 5–75 days after freezing injury. We observed marked upregulation of Nogo-A and NgR in protein levels. Furthermore, the migration of neural precursor cells (NPCs) derived from the subventricular zone (SVZ) toward the sits of injury was perturbed bymore » treatment of NgR antagonist peptide NEP1-40. In vitro analysis showed that the knockdown of NgR by lentivirus-delivered siRNA promoted in axonal regeneration and SVZ-derived neural stem cell/progenitor cell (SVZ-NPCs) adhesion and migration, findings which were similar to the effects of NEP1-40. Taken together, our results indicate an important role for NgR in regulating the physiological processes of SVZ-NPCs. The observation of upregulated Nogo-A/NgR in lesion sites in the NFL model suggest that the effects of the perturbed Nogo-A are a key feature during the development and/or the progression of cortical malformation. - Highlights: • NFL model is an accurate experimental reproduction of focal microgyria of FCD. • The increase of the Nogo-A Levels occurs in response to freeze-induced focal lesioning. • Nogo-A/NgR may play a critical role for in the pathologic progression of FCD. • Nogo-A is associated with the migration, proliferation and self-renewal of SVZ-NPCs.« less

Magnetic Enhancement of Stem Cell-Targeted Delivery into the Brain Following MR-Guided Focused Ultrasound for Opening the Blood-Brain Barrier.

PubMed

Shen, Wei-Bin; Anastasiadis, Pavlos; Nguyen, Ben; Yarnell, Deborah; Yarowsky, Paul J; Frenkel, Victor; Fishman, Paul S

2017-07-01

Focused ultrasound (FUS)-mediated blood-brain barrier disruption (BBBD) can enable even large therapeutics such as stem cells to enter the brain from the bloodstream. However, the efficiency is relatively low. Our previous study showed that human neural progenitor cells (hNPCs) loaded with superparamagnetic iron oxide nanoparticles (SPIONs) in culture were attracted by an external magnetic field. In vivo, enhanced brain retention was observed near a magnet mounted on the skull in a rat model of traumatic brain injury, where BBBD also occurs. The goal of the current study was to determine whether magnetic attraction of SPION-loaded hNPCs would also enhance their retention in the brain after FUS-mediated BBBD. A small animal magnetic resonance imaging (MRI)-guided FUS system operating at 1.5 MHz was used to treat rats (∼120 g) without tissue damage or hemorrhage. Evidence of successful BBBD was validated with both radiologic enhancement of gadolinium on postsonication TI MRI and whole brain section visualization of Evans blue dye. The procedure was then combined with the application of a powerful magnet to the head directly after intravenous injection of the hNPCs. Validation of cells within the brain was performed by staining with Perls' Prussian blue for iron and by immunohistochemistry with a human-specific antigen. By injecting equal numbers of iron oxide (SPIONs) and noniron oxide nanoparticles-loaded hNPCs, each labeled with a different fluorophore, we found significantly greater numbers of SPIONs-loaded cells retained in the brain at the site of BBBD as compared to noniron loaded cells. This result was most pronounced in regions of the brain closest to the skull (dorsal cortex) in proximity to the magnet surface. A more powerful magnet and a Halbach magnetic array resulted in more effective retention of SPION-labeled cells in even deeper brain regions such as the striatum and ventral cortex. There, up to 90% of hNPCs observed contained SPIONs compared to 60% to 70% with the less powerful magnet. Fewer cells were observed at 24 h posttreatment compared to 2 h (primarily in the dorsal cortex). These results demonstrate that magnetic attraction can substantially enhance the retention of stem cells after FUS-mediated BBBD. This procedure could provide a safer and less invasive approach for delivering stem cells to the brain, compared to direct intracranial injections, substantially reducing the risk of bleeding and infection.

Bioinspired nanovalves with selective permeability and pH sensitivity

NASA Astrophysics Data System (ADS)

Zheng, Z.; Huang, X.; Schenderlein, M.; Moehwald, H.; Xu, G.-K.; Shchukin, D. G.

2015-01-01

Biological systems with controlled permeability and release functionality, which are among the successful examples of living beings to survive in evolution, have attracted intensive investigation and have been mimicked due to their broad spectrum of applications. We present in this work, for the first time, an example of nuclear pore complexes (NPCs)-inspired controlled release system that exhibits on-demand release of angstrom-sized molecules. We do so in a cost-effective way by stabilizing porous cobalt basic carbonates as nanovalves and realizing pH-sensitive release of entrapped subnano cargo. The proof-of-concept work also consists of the establishment of two mathematical models to explain the selective permeability of the nanovalves. Finally, gram-sized (or larger) quantities of the bio-inspired controlled release system can be synthesized through a scaling-up strategy, which opens up opportunities for controlled release of functional molecules in wider practical applications.Biological systems with controlled permeability and release functionality, which are among the successful examples of living beings to survive in evolution, have attracted intensive investigation and have been mimicked due to their broad spectrum of applications. We present in this work, for the first time, an example of nuclear pore complexes (NPCs)-inspired controlled release system that exhibits on-demand release of angstrom-sized molecules. We do so in a cost-effective way by stabilizing porous cobalt basic carbonates as nanovalves and realizing pH-sensitive release of entrapped subnano cargo. The proof-of-concept work also consists of the establishment of two mathematical models to explain the selective permeability of the nanovalves. Finally, gram-sized (or larger) quantities of the bio-inspired controlled release system can be synthesized through a scaling-up strategy, which opens up opportunities for controlled release of functional molecules in wider practical applications. Electronic supplementary information (ESI) available. See DOI: 10.1039/c4nr06378c

In vivo and ex vivo magnetic resonance spectroscopy of the infarct and the subventricular zone in experimental stroke

PubMed Central

Jiménez-Xarrié, Elena; Davila, Myriam; Gil-Perotín, Sara; Jurado-Rodríguez, Andrés; Candiota, Ana Paula; Delgado-Mederos, Raquel; Lope-Piedrafita, Silvia; García-Verdugo, José Manuel; Arús, Carles; Martí-Fàbregas, Joan

2015-01-01

Ex vivo high-resolution magic-angle spinning (HRMAS) provides metabolic information with higher sensitivity and spectral resolution than in vivo magnetic resonance spectroscopy (MRS). Therefore, we used both techniques to better characterize the metabolic pattern of the infarct and the neural progenitor cells (NPCs) in the ipsilateral subventricular zone (SVZi). Ischemic stroke rats were divided into three groups: G0 (non-stroke controls, n=6), G1 (day 1 after stroke, n=6), and G7 (days 6 to 8 after stroke, n=12). All the rats underwent MRS. Three rats per group were analyzed by HRMAS. The remaining rats were used for immunohistochemical studies. In the infarct, both techniques detected significant metabolic changes. The most relevant change was in mobile lipids (2.80 ppm) in the G7 group (a 5.53- and a 3.95-fold increase by MRS and HRMAS, respectively). In the SVZi, MRS did not detect any significant metabolic change. However, HRMAS detected a 2.70-fold increase in lactate and a 0.68-fold decrease in N-acetylaspartate in the G1 group. None of the metabolites correlated with the 1.37-fold increase in NPCs detected by immunohistochemistry in the G7 group. In conclusion, HRMAS improves the metabolic characterization of the brain in experimental ischemic stroke. However, none of the metabolites qualifies as a surrogate biomarker of NPCs. PMID:25605287

Quantitative Analyses of Synergistic Responses between Cannabidiol and DNA-Damaging Agents on the Proliferation and Viability of Glioblastoma and Neural Progenitor Cells in Culture

PubMed Central

Deng, Liting; Ng, Lindsay; Ozawa, Tatsuya

2017-01-01

Evidence suggests that the nonpsychotropic cannabis-derived compound, cannabidiol (CBD), has antineoplastic activity in multiple types of cancers, including glioblastoma multiforme (GBM). DNA-damaging agents remain the main standard of care treatment available for patients diagnosed with GBM. Here we studied the antiproliferative and cell-killing activity of CBD alone and in combination with DNA-damaging agents (temozolomide, carmustine, or cisplatin) in several human GBM cell lines and in mouse primary GBM cells in cultures. This activity was also studied in mouse neural progenitor cells (NPCs) in culture to assess for potential central nervous system toxicity. We found that CBD induced a dose-dependent reduction of both proliferation and viability of all cells with similar potencies, suggesting no preferential activity for cancer cells. Hill plot analysis indicates an allosteric mechanism of action triggered by CBD in all cells. Cotreatment regimens combining CBD and DNA-damaging agents produced synergistic antiproliferating and cell-killing responses over a limited range of concentrations in all human GBM cell lines and mouse GBM cells as well as in mouse NPCs. Remarkably, antagonistic responses occurred at low concentrations in select human GBM cell lines and in mouse GBM cells. Our study suggests limited synergistic activity when combining CBD and DNA-damaging agents in treating GBM cells, along with little to no therapeutic window when considering NPCs. PMID:27821713

Contrasted frequencies of p53 accumulation in the two age groups of North African nasopharyngeal carcinomas.

PubMed

Khabir, A; Sellami, A; Sakka, M; Ghorbel, A M; Daoud, J; Frikha, M; Drira, M M; Busson, P; Jlidi, R

2000-10-01

EBV-associated nasopharyngeal carcinomas (NPCs) from Southeast Asia and North Africa have many common clinical and biological characteristics. However, they differ with regard to their age distribution. In Asia, NPC mainly affects patients in the 4th or 5th decade of their life, whereas in North Africa an additional peak of incidence is found between the ages of 10 and 20. The p53 gene is rarely mutated in NPC. However, several groups have reported a consistent accumulation of p53 in Asian NPCs. To determine whether p53 was also accumulated in North African NPCs, we investigated its expression, by immunohistochemistry, in a series of 90 Tunisian biopsies. Bc12 and CD95, two proteins involved in the regulation of cell survival and apoptosis, were investigated in the same study. We found accumulation of p53 in 81% of the cases for patients over 30 years of age, but in only 38% of specimens for younger patients (P = 0.00013). There was a trend toward a higher frequency of Bc12 detection in patients over 30, but it was not statistically significant. CD95 expression was detected in all biopsies, generally at a high level, even at advanced stages of the disease. The changing frequency of p53 accumulation, below and over 30, suggests that NPC cells often achieve malignant transformation through different pathways in both age groups.

MicroRNA-130b targets Fmr1 and regulates embryonic neural progenitor cell proliferation and differentiation

DOE Office of Scientific and Technical Information (OSTI.GOV)

Gong, Xi; Zhang, Kunshan; Wang, Yanlu

2013-10-04

Highlights: •We found that the 3′ UTR of the Fmr1 mRNA is a target of miR-130b. •MiR-130b suppresses the expression of Fmr1 in mouse embryonic stem cell. •MiR-130b alters the proliferation of mouse embryonic stem cell. •MiR-130b alters fate specification of mouse embryonic stem cell. -- Abstract: Fragile X syndrome, one of the most common forms of inherited mental retardation, is caused by expansion of the CGG repeat in the 5′-untranslated region of the X-linked Fmr1 gene, which results in transcriptional silencing and loss of expression of its encoded protein FMRP. The loss of FMRP increases proliferation and alters fatemore » specification in adult neural progenitor cells (aNPCs). However, little is known about Fmr1 mRNA regulation at the transcriptional and post-transcriptional levels. In the present study, we report that miR-130b regulated Fmr1 expression by directly targeting its 3′-untranslated region (3′ UTR). Up-regulation of miR-130b in mouse embryonic neural progenitor cells (eNPCs) decreased Fmr1 expression, markedly increased eNPC proliferation and altered the differentiation tendency of eNPCs, suggesting that antagonizing miR-130b may be a new therapeutic entry point for treating Fragile X syndrome.« less

Oxytocin alters cell fate selection of rat neural progenitor cells in vitro

PubMed Central

Kannappan, Ramaswamy; Xu, Zhiqiang; Martino, Audrey; Friese, Matthew B.; Boyd, Justin D.; Crosby, Gregory; Culley, Deborah J.

2018-01-01

Synthetic oxytocin (sOT) is widely used during labor, yet little is known about its effects on fetal brain development despite evidence that it reaches the fetal circulation. Here, we tested the hypothesis that sOT would affect early neurodevelopment by investigating its effects on neural progenitor cells (NPC) from embryonic day 14 rat pups. NPCs expressed the oxytocin receptor (OXTR), which was downregulated by 45% upon prolonged treatment with sOT. Next, we examined the effects of sOT on NPC death, apoptosis, proliferation, and differentiation using antibodies to NeuN (neurons), Olig2 (oligodendrocytes), and GFAP (astrocytes). Treated NPCs were analysed with unbiased high-throughput immunocytochemistry. Neither 6 nor 24 h exposure to 100 pM or 100 nM sOT had an effect on viability as assessed by PI or CC-3 immunocytochemistry. Similarly, sOT had negligible effect on NPC proliferation, except that the overall rate of NPC proliferation was higher in the 24 h compared to the 6 h group regardless of sOT exposure. The most significant finding was that sOT exposure caused NPCs to select a predominantly neuronal lineage, along with a concomitant decrease in glial cells. Collectively, our data suggest that perinatal exposure to sOT can have neurodevelopmental consequences for the fetus, and support the need for in vivo anatomical and behavioral studies in offspring exposed to sOT in utero. PMID:29346405

Oxytocin alters cell fate selection of rat neural progenitor cells in vitro.

PubMed

Palanisamy, Arvind; Kannappan, Ramaswamy; Xu, Zhiqiang; Martino, Audrey; Friese, Matthew B; Boyd, Justin D; Crosby, Gregory; Culley, Deborah J

2018-01-01

Synthetic oxytocin (sOT) is widely used during labor, yet little is known about its effects on fetal brain development despite evidence that it reaches the fetal circulation. Here, we tested the hypothesis that sOT would affect early neurodevelopment by investigating its effects on neural progenitor cells (NPC) from embryonic day 14 rat pups. NPCs expressed the oxytocin receptor (OXTR), which was downregulated by 45% upon prolonged treatment with sOT. Next, we examined the effects of sOT on NPC death, apoptosis, proliferation, and differentiation using antibodies to NeuN (neurons), Olig2 (oligodendrocytes), and GFAP (astrocytes). Treated NPCs were analysed with unbiased high-throughput immunocytochemistry. Neither 6 nor 24 h exposure to 100 pM or 100 nM sOT had an effect on viability as assessed by PI or CC-3 immunocytochemistry. Similarly, sOT had negligible effect on NPC proliferation, except that the overall rate of NPC proliferation was higher in the 24 h compared to the 6 h group regardless of sOT exposure. The most significant finding was that sOT exposure caused NPCs to select a predominantly neuronal lineage, along with a concomitant decrease in glial cells. Collectively, our data suggest that perinatal exposure to sOT can have neurodevelopmental consequences for the fetus, and support the need for in vivo anatomical and behavioral studies in offspring exposed to sOT in utero.

Regulation of differentiation flux by Notch signalling influences the number of dopaminergic neurons in the adult brain

PubMed Central

Trujillo-Paredes, Niurka; Valencia, Concepción; Guerrero-Flores, Gilda; Arzate, Dulce-María; Baizabal, José-Manuel; Guerra-Crespo, Magdalena; Fuentes-Hernández, Ayari; Zea-Armenta, Iván; Covarrubias, Luis

2016-01-01

ABSTRACT Notch signalling is a well-established pathway that regulates neurogenesis. However, little is known about the role of Notch signalling in specific neuronal differentiation. Using Dll1 null mice, we found that Notch signalling has no function in the specification of mesencephalic dopaminergic neural precursor cells (NPCs), but plays an important role in regulating their expansion and differentiation into neurons. Premature neuronal differentiation was observed in mesencephalons of Dll1-deficient mice or after treatment with a Notch signalling inhibitor. Coupling between neurogenesis and dopaminergic differentiation was indicated from the coincident emergence of neuronal and dopaminergic markers. Early in differentiation, decreasing Notch signalling caused a reduction in NPCs and an increase in dopaminergic neurons in association with dynamic changes in the proportion of sequentially-linked dopaminergic NPCs (Msx1/2+, Ngn2+, Nurr1+). These effects in differentiation caused a significant reduction in the number of dopaminergic neurons produced. Accordingly, Dll1 haploinsufficient adult mice, in comparison with their wild-type littermates, have a consistent reduction in neuronal density that was particularly evident in the substantia nigra pars compacta. Our results are in agreement with a mathematical model based on a Dll1-mediated regulatory feedback loop between early progenitors and their dividing precursors that controls the emergence and number of dopaminergic neurons. PMID:26912775

Regulation of differentiation flux by Notch signalling influences the number of dopaminergic neurons in the adult brain.

PubMed

Trujillo-Paredes, Niurka; Valencia, Concepción; Guerrero-Flores, Gilda; Arzate, Dulce-María; Baizabal, José-Manuel; Guerra-Crespo, Magdalena; Fuentes-Hernández, Ayari; Zea-Armenta, Iván; Covarrubias, Luis

2016-02-24

Notch signalling is a well-established pathway that regulates neurogenesis. However, little is known about the role of Notch signalling in specific neuronal differentiation. Using Dll1 null mice, we found that Notch signalling has no function in the specification of mesencephalic dopaminergic neural precursor cells (NPCs), but plays an important role in regulating their expansion and differentiation into neurons. Premature neuronal differentiation was observed in mesencephalons of Dll1-deficient mice or after treatment with a Notch signalling inhibitor. Coupling between neurogenesis and dopaminergic differentiation was indicated from the coincident emergence of neuronal and dopaminergic markers. Early in differentiation, decreasing Notch signalling caused a reduction in NPCs and an increase in dopaminergic neurons in association with dynamic changes in the proportion of sequentially-linked dopaminergic NPCs (Msx1/2+, Ngn2+, Nurr1+). These effects in differentiation caused a significant reduction in the number of dopaminergic neurons produced. Accordingly, Dll1 haploinsufficient adult mice, in comparison with their wild-type littermates, have a consistent reduction in neuronal density that was particularly evident in the substantia nigra pars compacta. Our results are in agreement with a mathematical model based on a Dll1-mediated regulatory feedback loop between early progenitors and their dividing precursors that controls the emergence and number of dopaminergic neurons. © 2016. Published by The Company of Biologists Ltd.

Biochemical profiling of rat embryonic stem cells grown on electrospun polyester fibers using synchrotron infrared microspectroscopy.

PubMed

Doncel-Pérez, Ernesto; Ellis, Gary; Sandt, Christophe; Shuttleworth, Peter S; Bastida, Agatha; Revuelta, Julia; García-Junceda, Eduardo; Fernández-Mayoralas, Alfonso; Garrido, Leoncio

2018-06-01

Therapeutic options for spinal cord injuries are severely limited; current treatments only offer symptomatic relief and rehabilitation focused on educating the individual on how to adapt to their new situation to make best possible use of their remaining function. Thus, new approaches are needed, and interest in the development of effective strategies to promote the repair of neural tracts in the central nervous system inspired us to prepare functional and highly anisotropic polymer scaffolds. In this work, an initial assessment of the behavior of rat neural progenitor cells (NPCs) seeded on poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) fiber scaffolds using synchrotron-based infrared microspectroscopy (SIRMS) is described. Combined with a modified touch imprint cytology sample preparation method, this application of SIRMS enabled the biochemical profiles of NPCs on the coated polymer fibers to be determined. The results showed that changes in the lipid and amide I-II spectral regions are modulated by the type and coating of the substrate used and the culture time. SIRMS studies can provide valuable insight into the early-stage response of NPCs to the morphology and surface chemistry of a biomaterial, and could therefore be a useful tool in the preparation and optimization of cellular scaffolds. Graphical abstract Synchrotron IR microspectroscopy can provide insight into the response of neural progenitor cells to synthetic scaffolds.

Block voter model: Phase diagram and critical behavior

NASA Astrophysics Data System (ADS)

Sampaio-Filho, C. I. N.; Moreira, F. G. B.

2011-11-01

We introduce and study the block voter model with noise on two-dimensional square lattices using Monte Carlo simulations and finite-size scaling techniques. The model is defined by an outflow dynamics where a central set of NPCS spins, here denoted by persuasive cluster spins (PCS), tries to influence the opinion of their neighboring counterparts. We consider the collective behavior of the entire system with varying PCS size. When NPCS>2, the system exhibits an order-disorder phase transition at a critical noise parameter qc which is a monotonically increasing function of the size of the persuasive cluster. We conclude that a larger PCS has more power of persuasion, when compared to a smaller one. It also seems that the resulting critical behavior is Ising-like independent of the range of interaction.

Nup53 Is Required for Nuclear Envelope and Nuclear Pore Complex Assembly

PubMed Central

Hawryluk-Gara, Lisa A.; Platani, Melpomeni; Santarella, Rachel

2008-01-01

Transport across the nuclear envelope (NE) is mediated by nuclear pore complexes (NPCs). These structures are composed of various subcomplexes of proteins that are each present in multiple copies and together establish the eightfold symmetry of the NPC. One evolutionarily conserved subcomplex of the NPC contains the nucleoporins Nup53 and Nup155. Using truncation analysis, we have defined regions of Nup53 that bind to neighboring nucleoporins as well as those domains that target Nup53 to the NPC in vivo. Using this information, we investigated the role of Nup53 in NE and NPC assembly using Xenopus egg extracts. We show that both events require Nup53. Importantly, the analysis of Nup53 fragments revealed that the assembly activity of Nup53 depleted extracts could be reconstituted using a region of Nup53 that binds specifically to its interacting partner Nup155. On the basis of these results, we propose that the formation of a Nup53–Nup155 complex plays a critical role in the processes of NPC and NE assembly. PMID:18256286

Mechanisms of nuclear pore complex assembly - two different ways of building one molecular machine.

PubMed

Otsuka, Shotaro; Ellenberg, Jan

2018-02-01

The nuclear pore complex (NPC) mediates all macromolecular transport across the nuclear envelope. In higher eukaryotes that have an open mitosis, NPCs assemble at two points in the cell cycle: during nuclear assembly in late mitosis and during nuclear growth in interphase. How the NPC, the largest nonpolymeric protein complex in eukaryotic cells, self-assembles inside cells remained unclear. Recent studies have started to uncover the assembly process, and evidence has been accumulating that postmitotic and interphase NPC assembly use fundamentally different mechanisms; the duration, structural intermediates, and regulation by molecular players are different and different types of membrane deformation are involved. In this Review, we summarize the current understanding of these two modes of NPC assembly and discuss the structural and regulatory steps that might drive the assembly processes. We furthermore integrate understanding of NPC assembly with the mechanisms for rapid nuclear growth in embryos and, finally, speculate on the evolutionary origin of the NPC implied by the presence of two distinct assembly mechanisms. © 2017 The Authors. FEBS Letters published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.

Non-FG mediated transport of the large pre-ribosomal subunit through the nuclear pore complex by the mRNA export factor Gle2

PubMed Central

Occhipinti, Laura; Chang, Yiming; Altvater, Martin; Menet, Anna M.; Kemmler, Stefan; Panse, Vikram G.

2013-01-01

Multiple export receptors passage bound pre-ribosomes through nuclear pore complexes (NPCs) by transiently interacting with the Phe-Gly (FG) meshwork of their transport channels. Here, we reveal how the non-FG interacting yeast mRNA export factor Gly-Leu-FG lethal 2 (Gle2) functions in the export of the large pre-ribosomal subunit (pre-60S). Structure-guided studies uncovered conserved platforms used by Gle2 to export pre-60S: an uncharacterized basic patch required to bind pre-60S, and a second surface that makes non-FG contacts with the nucleoporin Nup116. A basic patch mutant of Gle2 is able to function in mRNA export, but not pre-60S export. Thus, Gle2 provides a distinct interaction platform to transport pre-60S to the cytoplasm. Notably, Gle2’s interaction platforms become crucial for pre-60S export when FG-interacting receptors are either not recruited to pre-60S or are impaired. We propose that large complex cargos rely on non-FG as well as FG-interactions for their efficient translocation through the nuclear pore complex channel. PMID:23907389

Sr-doped nanowire modification of Ca-Si-based coatings for improved osteogenic activities and reduced inflammatory reactions

NASA Astrophysics Data System (ADS)

Li, Kai; Hu, Dandan; Xie, Youtao; Huang, Liping; Zheng, Xuebin

2018-02-01

Biomedical coatings for orthopedic implants should facilitate osseointegration and mitigate implant-induced inflammatory reactions. In our study, Ca-Si coatings with Sr-containing nanowire-like structures (NW-Sr-CS) were achieved via hydrothermal treatment. In order to identify the effect of nanowire-like topography and Sr dopant on the biological properties of Ca-Si-based coatings, the original Ca-Si coating, Ca-Si coatings modified with nanoplate (NP-CS) and similar nanowire-like structure (NW-CS) were fabricated as the control. Surface morphology, phase composition, surface area, zeta potential and ion release of these coatings were characterized. The in vitro osteogenic activities and immunomodulatory properties were evaluated with bone marrow stromal cells (BMSCs) and RAW 264.7 cells, a mouse macrophage cell line. Compared with the CS and NP-CS coatings, the NW-CS coating possessed a larger surface area and pore volume, beneficial protein adsorption, up-regulated the expression levels of integrin β1, Vinculin and focal adhesion kinase and promoted cell spreading. Furthermore, the NW-CS coating significantly enhanced the osteogenic differentiation and mineralization as indicated by the up-regulation of ALP activity, mineralized nodule formation and osteoblastogenesis-related gene expression. With the introduction of Sr, the NW-Sr-CS coatings exerted a greater effect on the BMSC proliferation rate, calcium sensitive receptor gene expression as well as PKC and ERK1/2 phosphorylation. In addition, the Sr-doped coatings significantly up-regulated the ratio of OPG/RANKL in the BMSCs. The NW-Sr-CS coatings could modulate the polarization of macrophages towards the wound-healing M2 phenotype, reduce the mRNA expression levels of pro-inflammatory cytokines (TNF-α, IL-1β, IL-6) and enhance anti-inflammatory cytokines (IL-1ra, IL-10). The Sr-doped nanowire modification may be a valuable approach to enhance osteogenic activities and reduce inflammatory reactions.

Identification of genetic loci that modulate cell proliferation in the adult rostral migratory stream using the expanded panel of BXD mice.

PubMed

Poon, Anna; Goldowitz, Daniel

2014-03-19

Adult neurogenesis, which is the continual production of new neurons in the mature brain, demonstrates the strikingly plastic nature of the nervous system. Adult neural stem cells and their neural precursors, collectively referred to as neural progenitor cells (NPCs), are present in the subgranular zone (SGZ) of the dentate gyrus, the subventricular zone (SVZ), and rostral migratory stream (RMS). In order to harness the potential of NPCs to treat neurodegenerative diseases and brain injuries, it will be important to understand the molecules that regulate NPCs in the adult brain. The genetic basis underlying NPC proliferation is still not fully understood. From our previous quantitative trait locus (QTL) analysis, we had success in using a relatively small reference population of recombinant inbred strains of mice (AXBXA) to identify a genetic region that is significantly correlated with NPC proliferation in the RMS. In this study, we expanded our initial QTL mapping of RMS proliferation to a far richer genetic resource, the BXD RI mouse strains. A 3-fold difference in the number of proliferative, bromodeoxyuridine (BrdU)-labeled cells was quantified in the adult RMS of 61 BXD RI strains. RMS cell proliferation is highly dependent on the genetic background of the mice with an estimated heritability of 0.58. Genome-wide mapping revealed a significant QTL on chromosome (Chr) 6 and a suggestive QTL on Chr 11 regulating the number of NPCs in the RMS. Composite interval analysis further revealed secondary QTLs on Chr 14 and Chr 18. The loci regulating RMS cell proliferation did not overlap with the suggestive loci modulating cell proliferation in the SGZ. These mapped loci serve as starting points to identify genes important for this process. A subset of candidate genes in this region is associated with cell proliferation and neurogenesis. Interconnectivity of these candidate genes was demonstrated using pathway and transcriptional covariance analyses. Differences in RMS cell proliferation across the BXD RI strains identifies genetic loci that serve to provide insights into the interplay of underlying genes that may be important for regulating NPC proliferation in the adult mouse brain.

Natively Unfolded FG Repeats Stabilize the Structure of the Nuclear Pore Complex.

PubMed

Onischenko, Evgeny; Tang, Jeffrey H; Andersen, Kasper R; Knockenhauer, Kevin E; Vallotton, Pascal; Derrer, Carina P; Kralt, Annemarie; Mugler, Christopher F; Chan, Leon Y; Schwartz, Thomas U; Weis, Karsten

2017-11-02

Nuclear pore complexes (NPCs) are ∼100 MDa transport channels assembled from multiple copies of ∼30 nucleoporins (Nups). One-third of these Nups contain phenylalanine-glycine (FG)-rich repeats, forming a diffusion barrier, which is selectively permeable for nuclear transport receptors that interact with these repeats. Here, we identify an additional function of FG repeats in the structure and biogenesis of the yeast NPC. We demonstrate that GLFG-containing FG repeats directly bind to multiple scaffold Nups in vitro and act as NPC-targeting determinants in vivo. Furthermore, we show that the GLFG repeats of Nup116 function in a redundant manner with Nup188, a nonessential scaffold Nup, to stabilize critical interactions within the NPC scaffold needed for late steps of NPC assembly. Our results reveal a previously unanticipated structural role for natively unfolded GLFG repeats as Velcro to link NPC subcomplexes and thus add a new layer of connections to current models of the NPC architecture. Copyright © 2017 Elsevier Inc. All rights reserved.

Liability of physicians supervising nonphysician clinicians.

PubMed

Paterick, Barbara B; Waterhouse, Blake E; Paterick, Timothy E; Sanbar, Sandy S

2014-01-01

Physicians confront a variety of liability issues when supervising nonphysician clinicians (NPC) including: (1) direct liability resulting from a failure to meet the state-defined standards of supervision/collaboration with NPCs; (2) vicarious liability, arising from agency law, where physicians are held accountable for NPC clinical care that does not meet the national standard of care; and (3) responsibility for medical errors when the NPC and physician are co-employees of the corporate enterprise. Physician-NPC co-employee relationships are highlighted because they are new and becoming predominant in existing healthcare models. Because of their novelty, there is a paucity of judicial decisions determining liability for NPC errors in this setting. Knowledge of the existence of these risks will allow physicians to make informed decisions on what relationships they will enter with NPCs and how these relationships will be structured and monitored.

In vivo maternal and in vitro BPA exposure effects on hypothalamic neurogenesis and appetite regulators.

PubMed

Desai, Mina; Ferrini, Monica G; Han, Guang; Jellyman, Juanita K; Ross, Michael G

2018-07-01

In utero exposure to the ubiquitous plasticizer, bisphenol A (BPA) is associated with offspring obesity. As food intake/appetite is one of the critical elements contributing to obesity, we determined the effects of in vivo maternal BPA and in vitro BPA exposure on newborn hypothalamic stem cells which form the arcuate nucleus appetite center. For in vivo studies, female rats received BPA prior to and during pregnancy via drinking water, and newborn offspring primary hypothalamic neuroprogenitor (NPCs) were obtained and cultured. For in vitro BPA exposure, primary hypothalamic NPCs from healthy newborns were utilized. In both cases, we studied the effects of BPA on NPC proliferation and differentiation, including putative signal and appetite factors. Maternal BPA increased hypothalamic NPC proliferation and differentiation in newborns, in conjunction with increased neuroproliferative (Hes1) and proneurogenic (Ngn3) protein expression. With NPC differentiation, BPA exposure increased appetite peptide and reduced satiety peptide expression. In vitro BPA-treated control NPCs showed results that were consistent with in vivo data (increase appetite vs satiety peptide expression) and further showed a shift towards neuronal versus glial fate as well as an increase in the epigenetic regulator lysine-specific histone demethylase1 (LSD1). These findings emphasize the vulnerability of stem-cell populations that are involved in life-long regulation of metabolic homeostasis to epigenetically-mediated endocrine disruption by BPA during early life. Copyright © 2018. Published by Elsevier Inc.

Quantitative Analyses of Synergistic Responses between Cannabidiol and DNA-Damaging Agents on the Proliferation and Viability of Glioblastoma and Neural Progenitor Cells in Culture.

PubMed

Deng, Liting; Ng, Lindsay; Ozawa, Tatsuya; Stella, Nephi

2017-01-01

Evidence suggests that the nonpsychotropic cannabis-derived compound, cannabidiol (CBD), has antineoplastic activity in multiple types of cancers, including glioblastoma multiforme (GBM). DNA-damaging agents remain the main standard of care treatment available for patients diagnosed with GBM. Here we studied the antiproliferative and cell-killing activity of CBD alone and in combination with DNA-damaging agents (temozolomide, carmustine, or cisplatin) in several human GBM cell lines and in mouse primary GBM cells in cultures. This activity was also studied in mouse neural progenitor cells (NPCs) in culture to assess for potential central nervous system toxicity. We found that CBD induced a dose-dependent reduction of both proliferation and viability of all cells with similar potencies, suggesting no preferential activity for cancer cells. Hill plot analysis indicates an allosteric mechanism of action triggered by CBD in all cells. Cotreatment regimens combining CBD and DNA-damaging agents produced synergistic antiproliferating and cell-killing responses over a limited range of concentrations in all human GBM cell lines and mouse GBM cells as well as in mouse NPCs. Remarkably, antagonistic responses occurred at low concentrations in select human GBM cell lines and in mouse GBM cells. Our study suggests limited synergistic activity when combining CBD and DNA-damaging agents in treating GBM cells, along with little to no therapeutic window when considering NPCs. Copyright © 2016 by The American Society for Pharmacology and Experimental Therapeutics.

Conventional and array-based comparative genomic hybridization analysis of nasopharyngeal carcinomas from the Mediterranean area.

PubMed

Rodriguez, S; Khabir, A; Keryer, C; Perrot, C; Drira, M; Ghorbel, A; Jlidi, R; Bernheim, A; Valent, A; Busson, P

2005-03-01

Nasopharyngeal carcinoma (NPC) occurs with a high incidence in Southeast Asia and to a lesser extent in the Mediterranean area, especially in Tunisia, Algeria, and Morocco. Cellular gene alterations combined with latent Epstein-Barr virus infection are thought to be essential for NPC oncogenesis. To date, chromosome analysis with comparative genomic hybridization (CGH) has been reported exclusively for NPCs from Southeast Asia. Although NPCs from the Mediterranean area have several distinct clinical and epidemiological features, CGH investigations have been lacking. Chromosome analysis was therefore undertaken on a series of NPC xenografts and biopsies derived from patients of Mediterranean origin. Four xenografts were investigated with a combination of conventional CGH, array-based CGH, and comparative expressed sequence hybridization. In addition, 23 fresh NPC biopsies were analyzed with conventional CGH. Data obtained from xenografts and fresh biopsies were consistent, except that amplification of genes at 18p was observed only in xenografts derived from metastatic tissues. Frequent gains associated with gene overexpression were detected at 1q25 approximately qter (64%) and 12p13 (50%). Losses were noticed mainly at 11q14 approximately q23 (50%), 13q12 approximately q31 (50%), 14q24 approximately q31 (43%), and 3p13 approximately p23 (43%). Comparison with previous reports suggests that Mediterranean NPCs have higher frequencies of gains at 1q and losses at 13q than their Asian counterparts.

Combination of the clustered regularly interspaced short palindromic repeats (CRISPR)-associated 9 technique with the piggybac transposon system for mouse in utero electroporation to study cortical development.

PubMed

Cheng, Man; Jin, Xubin; Mu, Lili; Wang, Fangyu; Li, Wei; Zhong, Xiaoling; Liu, Xuan; Shen, Wenchen; Liu, Ying; Zhou, Yan

2016-09-01

In utero electroporation (IUE) is commonly used to study cortical development of cerebrum by downregulating or overexpressing genes of interest in neural progenitor cells (NPCs) of small mammals. However, exogenous plasmids are lost or diluted over time. Furthermore, gene knockdown based on short-hairpin RNAs may exert nonspecific effects that lead to aberrant neuronal migration. Genomic engineering by the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated 9 (Cas9) system has great research and therapeutic potentials. Here we integrate the CRISPR/Cas9 components into the piggyBac (PB) transposon system (the CRISPR/Cas9-PB toolkit) for cortical IUEs. The mouse Sry-related HMG box-2 (Sox2) gene was selected as the target for its application. Most transduced cortical NPCs were depleted of SOX2 protein as early as 3 days post-IUE, whereas expressions of SOX1 and PAX6 remained intact. Furthermore, both the WT Cas9 and the D10A nickase mutant Cas9n showed comparable knockout efficiency. Transduced cortical cells were purified with fluorescence-activated cell sorting, and effective gene editing at the Sox2 loci was confirmed. Thus, application of the CRISPR/Cas9-PB toolkit in IUE is a promising strategy to study gene functions in cortical NPCs and their progeny. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

Inducible and Conditional Deletion of Extracellular Signal-regulated Kinase 5 Disrupts Adult Hippocampal Neurogenesis*

PubMed Central

Pan, Yung-Wei; Zou, Junhui; Wang, Wenbin; Sakagami, Hiroyuki; Garelick, Michael G.; Abel, Glen; Kuo, Chay T.; Storm, Daniel R.; Xia, Zhengui

2012-01-01

Recent studies have led to the exciting idea that adult-born neurons in the dentate gyrus of the hippocampus may play a role in hippocampus-dependent memory formation. However, signaling mechanisms that regulate adult hippocampal neurogenesis are not well defined. Here we report that extracellular signal-regulated kinase 5 (ERK5), a member of the mitogen-activated protein kinase family, is selectively expressed in the neurogenic regions of the adult mouse brain. We present evidence that shRNA suppression of ERK5 in adult hippocampal neural stem/progenitor cells (aNPCs) reduces the number of neurons while increasing the number of cells expressing markers for stem/progenitor cells or proliferation. Furthermore, shERK5 attenuates both transcription and neuronal differentiation mediated by Neurogenin 2, a transcription factor expressed in adult hippocampal neural progenitor cells. By contrast, ectopic activation of endogenous ERK5 signaling via expression of constitutive active MEK5, an upstream activating kinase for ERK5, promotes neurogenesis in cultured aNPCs and in the dentate gyrus of the mouse brain. Moreover, neurotrophins including NT3 activate ERK5 and stimulate neuronal differentiation in aNPCs in an ERK5-dependent manner. Finally, inducible and conditional deletion of ERK5 specifically in the neurogenic regions of the adult mouse brain delays the normal progression of neuronal differentiation and attenuates adult neurogenesis in vivo. These data suggest ERK5 signaling as a critical regulator of adult hippocampal neurogenesis. PMID:22645146

Postage for the messenger: Designating routes for Nuclear mRNA Export

PubMed Central

Natalizio, Barbara J.; Wente, Susan R.

2013-01-01

Transcription of messenger(m) RNA occurs in the nucleus, making the translocation of mRNA across the nuclear envelope (NE) boundary a critical determinant of proper gene expression and cell survival. A major mRNA export route occurs via the NXF1-dependent pathway through the nuclear pore complexes (NPCs) embedded in the NE. However, recent findings have discovered new evidence supporting the existence of multiple mechanisms for crossing the NE, including both NPC-mediated and NE budding-mediated pathways. An analysis of the trans-acting factors and cis components that define these pathways reveals shared elements as well as mechanistic differences. We review here the current understanding of the mechanisms that characterize each pathway and highlight the determinants that influence mRNA transport fate. PMID:23583578

Valproic Acid Induces Telomerase Reverse Transcriptase Expression during Cortical Development.

PubMed

Kim, Ki Chan; Choi, Chang Soon; Gonzales, Edson Luck T; Mabunga, Darine Froy N; Lee, Sung Hoon; Jeon, Se Jin; Hwangbo, Ram; Hong, Minha; Ryu, Jong Hoon; Han, Seol-Heui; Bahn, Geon Ho; Shin, Chan Young

2017-10-01

The valproic acid (VPA)-induced animal model is one of the most widely utilized environmental risk factor models of autism. Autism spectrum disorder (ASD) remains an insurmountable challenge among neurodevelopmental disorders due to its heterogeneity, unresolved pathological pathways and lack of treatment. We previously reported that VPA-exposed rats and cultured rat primary neurons have increased Pax6 expression during post-midterm embryonic development which led to the sequential upregulation of glutamatergic neuronal markers. In this study, we provide experimental evidence that telomerase reverse transcriptase (TERT), a protein component of ribonucleoproteins complex of telomerase, is involved in the abnormal components caused by VPA in addition to Pax6 and its downstream signals. In embryonic rat brains and cultured rat primary neural progenitor cells (NPCs), VPA induced the increased expression of TERT as revealed by Western blot, RT-PCR, and immunostainings. The HDAC inhibitor property of VPA is responsible for the TERT upregulation. Chromatin immunoprecipitation revealed that VPA increased the histone acetylation but blocked the HDAC1 binding to both Pax6 and Tert genes. Interestingly, the VPA-induced TERT overexpression resulted to sequential upregulations of glutamatergic markers such as Ngn2 and NeuroD1, and inter-synaptic markers such as PSD-95, α-CaMKII, vGluT1 and synaptophysin. Transfection of Tert siRNA reversed the effects of VPA in cultured NPCs confirming the direct involvement of TERT in the expression of those markers. This study suggests the involvement of TERT in the VPA-induced autistic phenotypes and has important implications for the role of TERT as a modulator of balanced neuronal development and transmission in the brain.

Upregulation of neurovascular communication through filamin abrogation promotes ectopic periventricular neurogenesis.

PubMed

Houlihan, Shauna L; Lanctot, Alison A; Guo, Yan; Feng, Yuanyi

2016-09-24

Neuronal fate-restricted intermediate progenitors (IPs) are derived from the multipotent radial glia (RGs) and serve as the direct precursors for cerebral cortical neurons, but factors that control their neurogenic plasticity remain elusive. Here we report that IPs' neuron production is enhanced by abrogating filamin function, leading to the generation of periventricular neurons independent of normal neocortical neurogenesis and neuronal migration. Loss of Flna in neural progenitor cells (NPCs) led RGs to undergo changes resembling epithelial-mesenchymal transition (EMT) along with exuberant angiogenesis that together changed the microenvironment and increased neurogenesis of IPs. We show that by collaborating with β-arrestin, Flna maintains the homeostatic signaling between the vasculature and NPCs, and loss of this function results in escalated Vegfa and Igf2 signaling, which exacerbates both EMT and angiogenesis to further potentiate IPs' neurogenesis. These results suggest that the neurogenic potential of IPs may be boosted in vivo by manipulating Flna-mediated neurovascular communication.

Resveratrol Inhibits the Proliferation of Neural Progenitor Cells and Hippocampal Neurogenesis*

PubMed Central

Park, Hee Ra; Kong, Kyoung Hye; Yu, Byung Pal; Mattson, Mark P.; Lee, Jaewon

2012-01-01

Resveratrol is a phytoalexin and natural phenol that is present at relatively high concentrations in peanuts and red grapes and wine. Based upon studies of yeast and invertebrate models, it has been proposed that ingestion of resveratrol may also have anti-aging actions in mammals including humans. It has been suggested that resveratrol exerts its beneficial effects on health by activating the same cellular signaling pathways that are activated by dietary energy restriction (DR). Some studies have reported therapeutic actions of resveratrol in animal models of metabolic and neurodegenerative disorders. However, the effects of resveratrol on cell, tissue and organ function in healthy subjects are largely unknown. In the present study, we evaluated the potential effects of resveratrol on the proliferation and survival of neural progenitor cells (NPCs) in culture, and in the hippocampus of healthy young adult mice. Resveratrol reduced the proliferation of cultured mouse multi-potent NPCs, and activated AMP-activated protein kinase (AMPK), in a concentration-dependent manner. Administration of resveratrol to mice (1–10 mg/kg) resulted in activation of AMPK, and reduced the proliferation and survival of NPCs in the dentate gyrus of the hippocampus. Resveratrol down-regulated the levels of the phosphorylated form of cyclic AMP response element-binding protein (pCREB) and brain-derived neurotrophic factor (BDNF) in the hippocampus. Finally, resveratrol-treated mice exhibited deficits in hippocampus-dependent spatial learning and memory. Our findings suggest that resveratrol, unlike DR, adversely affects hippocampal neurogenesis and cognitive function by a mechanism involving activation of AMPK and suppression of CREB and BDNF signaling. PMID:23105098

Ablation of cdk4 and cdk6 affects proliferation of basal progenitor cells in the developing dorsal and ventral forebrain.

PubMed

Grison, Alice; Gaiser, Carine; Bieder, Andrea; Baranek, Constanze; Atanasoski, Suzana

2018-03-23

Little is known about the molecular players driving proliferation of neural progenitor cells (NPCs) during embryonic mouse development. Here, we demonstrate that proliferation of NPCs in the developing forebrain depends on a particular combination of cell cycle regulators. We have analyzed the requirements for members of the cyclin-dependent kinase (cdk) family using cdk-deficient mice. In the absence of either cdk4 or cdk6, which are both regulators of the G1 phase of the cell cycle, we found no significant effects on the proliferation rate of cortical progenitor cells. However, concomitant loss of cdk4 and cdk6 led to a drastic decrease in the proliferation rate of NPCs, specifically the basal progenitor cells of both the dorsal and ventral forebrain at embryonic day 13.5 (E13.5). Moreover, basal progenitors in the forebrain of Cdk4;Cdk6 double mutant mice exhibited altered cell cycle characteristics. Cdk4;cdk6 deficiency led to an increase in cell cycle length and cell cycle exit of mutant basal progenitor cells in comparison to controls. In contrast, concomitant ablation of cdk2 and cdk6 had no effect on the proliferation of NCPs. Together, our data demonstrate that the expansion of the basal progenitor pool in the developing telencephalon is dependent on the presence of distinct combinations of cdk molecules. Our results provide further evidence for differences in the regulation of proliferation between apical and basal progenitors during cortical development. © 2018 Wiley Periodicals, Inc. Develop Neurobiol, 2018. © 2018 Wiley Periodicals, Inc.

Nanoarchitectures for Metal-Organic Framework-Derived Nanoporous Carbons toward Supercapacitor Applications.

PubMed

Salunkhe, Rahul R; Kaneti, Yusuf Valentino; Kim, Jeonghun; Kim, Jung Ho; Yamauchi, Yusuke

2016-12-20

The future advances of supercapacitors depend on the development of novel carbon materials with optimized porous structures, high surface area, high conductivity, and high electrochemical stability. Traditionally, nanoporous carbons (NPCs) have been prepared by a variety of methods, such as templated synthesis, carbonization of polymer precursors, physical and chemical activation, etc. Inorganic solid materials such as mesoporous silica and zeolites have been successfully utilized as templates to prepare NPCs. However, the hard-templating methods typically involve several synthetic steps, such as preparation of the original templates, formation of carbon frameworks, and removal of the original templates. Therefore, these methods are not favorable for large-scale production. Metal-organic frameworks (MOFs) with high surface areas and large pore volumes have been studied over the years, and recently, enormous efforts have been made to utilize MOFs for electrochemical applications. However, their low conductivity and poor stability still present major challenges toward their practical applications in supercapacitors. MOFs can be used as precursors for the preparation of NPCs with high porosity. Their parent MOFs can be prepared with endless combinations of organic and inorganic constituents by simple coordination chemistry, and it is possible to control their porous architectures, pore volumes, surface areas, etc. These unique properties of MOF-derived NPCs make them highly attractive for many technological applications. Compared with carbonaceous materials prepared using conventional precursors, MOF-derived carbons have significant advantages in terms of a simple synthesis with inherent diversity affording precise control over porous architectures, pore volumes, and surface areas. In this Account, we will summarize our recent research developments on the preparation of three-dimensional (3-D) MOF-derived carbons for supercapacitor applications. This Account will be divided into three main sections: (1) useful background on carbon materials for supercapacitor applications, (2) the importance of MOF-derived carbons, and (3) potential future developments of MOF-derived carbons for supercapacitors. This Account focuses mostly on carbons derived from two types of MOFs, namely, zeolite imidazolate framework-8 (ZIF-8) and ZIF-67. By using examples from our previous works, we will show the uniqueness of these carbons for achieving high performance by control of the chemical reactions/conditions as well proper utilization in asymmetric/symmetric supercapacitor configurations. This Account will promote further developments of MOF-derived multifunctional carbon materials with controlled porous architectures for optimization of their electrochemical performance toward supercapacitor applications.

Pom121 links two essential subcomplexes of the nuclear pore complex core to the membrane

PubMed Central

Mitchell, Jana M.; Mansfeld, Jörg; Capitanio, Juliana; Kutay, Ulrike

2010-01-01

Nuclear pore complexes (NPCs) control the movement of molecules across the nuclear envelope (NE). We investigated the molecular interactions that exist at the interface between the NPC scaffold and the pore membrane. We show that key players mediating these interactions in mammalian cells are the nucleoporins Nup155 and Nup160. Nup155 depletion massively alters NE structure, causing a dramatic decrease in NPC numbers and the improper targeting of membrane proteins to the inner nuclear membrane. The role of Nup155 in assembly is likely closely linked to events at the membrane as we show that Nup155 interacts with pore membrane proteins Pom121 and NDC1. Furthermore, we demonstrate that the N terminus of Pom121 directly binds the β-propeller regions of Nup155 and Nup160. We propose a model in which the interactions of Pom121 with Nup155 and Nup160 are predicted to assist in the formation of the nuclear pore and the anchoring of the NPC to the pore membrane. PMID:20974814

Synthesis of polybenzoxazine based nitrogen-rich porous carbons for carbon dioxide capture

NASA Astrophysics Data System (ADS)

Wan, Liu; Wang, Jianlong; Feng, Chong; Sun, Yahui; Li, Kaixi

2015-04-01

Nitrogen-rich porous carbons (NPCs) were synthesized from 1,5-dihydroxynaphthalene, urea, and formaldehyde based on benzoxazine chemistry by a soft-templating method with KOH chemical activation. They possess high surface areas of 856.8-1257.8 m2 g-1, a large pore volume of 0.15-0.65 cm3 g-1, tunable pore structure, high nitrogen content (5.21-5.32 wt%), and high char yields. The amount of the soft-templating agent F127 has multiple influences on the textural and chemical properties of the carbons, affecting the surface area and pore structure, impacting the compositions of nitrogen species and resulting in an improvement of the CO2 capture performance. At 1 bar, high CO2 uptake of 4.02 and 6.35 mmol g-1 at 25 and 0 °C was achieved for the sample NPC-2 with a molar ratio of F127 : urea = 0.010 : 1. This can be attributed to its well-developed micropore structure and abundant pyridinic nitrogen, pyrrolic nitrogen and pyridonic nitrogen functionalities. The sample NPC-2 also exhibits a remarkable selectivity for CO2/N2 separation and a fast adsorption/desorption rate and can be easily regenerated. This suggests that the polybenzoxazine-based NPCs are desirable for CO2 capture because of possessing a high micropore surface area, a large micropore volume, appropriate pore size distribution, and a large number of basic nitrogen functionalities.Nitrogen-rich porous carbons (NPCs) were synthesized from 1,5-dihydroxynaphthalene, urea, and formaldehyde based on benzoxazine chemistry by a soft-templating method with KOH chemical activation. They possess high surface areas of 856.8-1257.8 m2 g-1, a large pore volume of 0.15-0.65 cm3 g-1, tunable pore structure, high nitrogen content (5.21-5.32 wt%), and high char yields. The amount of the soft-templating agent F127 has multiple influences on the textural and chemical properties of the carbons, affecting the surface area and pore structure, impacting the compositions of nitrogen species and resulting in an improvement of the CO2 capture performance. At 1 bar, high CO2 uptake of 4.02 and 6.35 mmol g-1 at 25 and 0 °C was achieved for the sample NPC-2 with a molar ratio of F127 : urea = 0.010 : 1. This can be attributed to its well-developed micropore structure and abundant pyridinic nitrogen, pyrrolic nitrogen and pyridonic nitrogen functionalities. The sample NPC-2 also exhibits a remarkable selectivity for CO2/N2 separation and a fast adsorption/desorption rate and can be easily regenerated. This suggests that the polybenzoxazine-based NPCs are desirable for CO2 capture because of possessing a high micropore surface area, a large micropore volume, appropriate pore size distribution, and a large number of basic nitrogen functionalities. Electronic supplementary information (ESI) available: Elemental and XPS analyses and XPS peak positions and relative content of N species in the NPCs. See DOI: 10.1039/c4nr07409b

Wnt/B-Catenin Signaling is Required to Rescue Midbrain Dopaminergic Progenitors and Promote Neurorepair in Ageing Mouse Model of Parkinson’s Disease

PubMed Central

L’Episcopo, Francesca; Tirolo, Cataldo; Testa, Nunzio; Caniglia, Salvatore; Morale, Maria Concetta; Serapide, Maria Francesca; Pluchino, Stefano; Marchetti, Bianca

2014-01-01

SUMMARY Wnt/β-catenin signaling is required for specification and neurogenesis of midbrain dopaminergic (mDA) neurons, the pivotal neuronal population that degenerates in Parkinson’s disease (PD) and in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) mouse model of PD. Wnt/β-catenin signaling plays a vital role in adult neurogenesis but whether it might engage DA neurogenesis/neurorepair in the affected PD brain is yet unresolved. Recently, the adult midbrain aqueduct periventricular regions (Aq-PVRs) were shown to harbor neural stem/progenitor cells (mNPCs) with DA potential in vitro, but restrictive mechanisms in vivo are believed to limit their DA regenerative capacity. Using in vitro mNPC culture systems we herein demonstrate that aging is one most critical factor restricting mNPC neurogenic potential via dysregulation of Wnt/β-catenin signaling. Cococulture paradigms between young/aged (Y/A) mNPCs and Y/A astrocytes identified glial age and a decline of glial-derived factors including Wnts as key determinants of impaired neurogenic potential, whereas Wnt activation regimens efficiently reversed the diminished proliferative, neuronal and DA differentiation potential of A-mNPCs. Next, in vivo studies in wild (Wt) and transgenic β-catenin reporter mice uncovered Wnt/β-catenin signaling activation and remarkable astrocyte remodeling of Aq-PVR in response to MPTP-induced DA neuron death. Spatio-temporal analyses unveiled β-catenin signaling in predopaminergic (Nurr1+/TH−) and imperiled or rescuing DAT+ neurons during MPTP-induced DA neuron injury and self-repair. Aging inhibited Wnt signaling, whereas β-catenin activation in situ with a specific GSK-3β antagonist promoted a significant degree of DA neurorestoration associated with reversal of motor deficit, with implications for neurorestorative approaches in PD. PMID:24648001

SIRB, sans iron oxide rhodamine B, a novel cross-linked dextran nanoparticle, labels human neuroprogenitor and SH-SY5Y neuroblastoma cells and serves as a USPIO cell labeling control.

PubMed

Shen, Wei-Bin; Vaccaro, Dennis E; Fishman, Paul S; Groman, Ernest V; Yarowsky, Paul

2016-05-01

This is the first report of the synthesis of a new nanoparticle, sans iron oxide rhodamine B (SIRB), an example of a new class of nanoparticles. SIRB is designed to provide all of the cell labeling properties of the ultrasmall superparamagnetic iron oxide (USPIO) nanoparticle Molday ION Rhodamine B (MIRB) without containing the iron oxide core. MIRB was developed to label cells and allow them to be tracked by MRI or to be manipulated by magnetic gradients. SIRB possesses a similar size, charge and cross-linked dextran coating as MIRB. Of great interest is understanding the biological and physiological changes in cells after they are labeled with a USPIO. Whether these effects are due to the iron oxide buried within the nanoparticle or to the surface coating surrounding the iron oxide core has not been considered previously. MIRB and SIRB represent an ideal pairing of nanoparticles to identify nanoparticle anatomy responsible for post-labeling cytotoxicity. Here we report the effects of SIRB labeling on the SH-SY5Y neuroblastoma cell line and primary human neuroprogenitor cells (hNPCs). These effects are contrasted with the effects of labeling SH-SY5Y cells and hNPCs with MIRB. We find that SIRB labeling, like MIRB labeling, (i) occurs without the use of transfection reagents, (ii) is packaged within lysosomes distributed within cell cytoplasm, (iii) is retained within cells with no loss of label after cell storage, and (iv) does not alter cellular viability or proliferation, and (v) SIRB labeled hNPCs differentiate normally into neurons or astrocytes. Copyright © 2016 John Wiley & Sons, Ltd. Copyright © 2016 John Wiley & Sons, Ltd.

A prenatal interruption of DISC1 function in the brain exhibits a lasting impact on adult behaviors, brain metabolism, and interneuron development.

PubMed

Deng, Dazhi; Jian, Chongdong; Lei, Ling; Zhou, Yijing; McSweeney, Colleen; Dong, Fengping; Shen, Yilun; Zou, Donghua; Wang, Yonggang; Wu, Yuan; Zhang, Limin; Mao, Yingwei

2017-10-17

Mental illnesses like schizophrenia (SCZ) and major depression disorder (MDD) are devastating brain disorders. The SCZ risk gene, disrupted in schizophrenia 1 ( DISC1 ), has been associated with neuropsychiatric conditions. However, little is known regarding the long-lasting impacts on brain metabolism and behavioral outcomes from genetic insults on fetal NPCs during early life. We have established a new mouse model that specifically interrupts DISC1 functions in NPCs in vivo by a dominant-negative DISC1 (DN-DISC1) with a precise temporal and spatial regulation. Interestingly, prenatal interruption of mouse Disc1 function in NPCs leads to abnormal depression-like deficit in adult mice. Here we took a novel unbiased metabonomics approach to identify brain-specific metabolites that are significantly changed in DN-DISC1 mice. Surprisingly, the inhibitory neurotransmitter, GABA, is augmented. Consistently, parvalbumin (PV) interneurons are increased in the cingulate cortex, retrosplenial granular cortex, and motor cortex. Interestingly, somatostatin (SST) positive and neuropeptide Y (NPY) interneurons are decreased in some brain regions, suggesting that DN-DISC1 expression affects the localization of interneuron subtypes. To further explore the cellular mechanisms that cause this change, DN-DISC1 suppresses proliferation and promotes the cell cycle exit of progenitors in the medial ganglionic eminence (MGE), whereas it stimulates ectopic proliferation of neighboring cells through cell non-autonomous effect. Mechanistically, it modulates GSK3 activity and interrupts Dlx2 activity in the Wnt activation. In sum, our results provide evidence that specific genetic insults on NSCs at a short period of time could lead to prolonged changes of brain metabolism and development, eventually behavioral defects.

Transient enhancement of proliferation of neural progenitors and impairment of their long-term survival in p25 transgenic mice

PubMed Central

Dong, Fengping; Shu, Tianzhi; Zhou, Ying; Tsai, Li-Huei; Mao, Yingwei

2016-01-01

Cyclin-dependent kinase 5 (CDK5) regulates important neuronal functions via p35. p35 undergoes cleavage in response to neuronal activity and neurotoxic conditions to release its subunit p25. Although p25 has been implicated in various neurodegenerative diseases, the mechanisms by which p25 mediates neurodegenerative impairment have not been fully elucidated. We aimed to determine the role of p25-mediated neurodegeneration on neurogenesis in an inducible transgenic mouse line overexpressing p25 (p25 TG) in the forebrain. Adult neuronal progenitor cells (NPCs) were labeled with BrdU in vivo, which were significantly increased in numbers in the subventricular zone, the hippocampus, and the cortex of p25 TG mice. Consistently, more mitotic cells were observed in p25 TG mice than in controls, even in the cortex and the CA1, which are not neurogenic regions. BrdU-positive cells were negative for GFAP or γ-H2AX, suggesting that they are not astrocytes or dying cells. Neurospheres derived from the dentate gyrus and the cortex were significantly increased in p25 TG mice and can be differentiated into astrocytes and neurons. However, p25 TG decreased the long-term survival of proliferating NPCs and severely impaired adult neurogenesis. A Transwell co-culture system was used to assess the influence of p25-expressing primary neurons on adult NPCs. Co-culture with p25-expressing neurons downregulated Ki67 expression and upregulated cleaved caspase-3, indicating that the paracrine signaling in cell-cell communication is essential for NPC survival and proliferation. Moreover, increased CDK5 activity impairs Wnt activation. This study demonstrates that hyperactivation of p25 may temporarily enhance NPC proliferation, but impair their long-term survival. PMID:27283769

Transient enhancement of proliferation of neural progenitors and impairment of their long-term survival in p25 transgenic mice.

PubMed

Zou, Donghua; Zhou, Yijing; Liu, Long; Dong, Fengping; Shu, Tianzhi; Zhou, Ying; Tsai, Li-Huei; Mao, Yingwei

2016-06-28

Cyclin-dependent kinase 5 (CDK5) regulates important neuronal functions via p35. p35 undergoes cleavage in response to neuronal activity and neurotoxic conditions to release its subunit p25. Although p25 has been implicated in various neurodegenerative diseases, the mechanisms by which p25 mediates neurodegenerative impairment have not been fully elucidated. We aimed to determine the role of p25-mediated neurodegeneration on neurogenesis in an inducible transgenic mouse line overexpressing p25 (p25 TG) in the forebrain. Adult neuronal progenitor cells (NPCs) were labeled with BrdU in vivo, which were significantly increased in numbers in the subventricular zone, the hippocampus, and the cortex of p25 TG mice. Consistently, more mitotic cells were observed in p25 TG mice than in controls, even in the cortex and the CA1, which are not neurogenic regions. BrdU-positive cells were negative for GFAP or γ-H2AX, suggesting that they are not astrocytes or dying cells. Neurospheres derived from the dentate gyrus and the cortex were significantly increased in p25 TG mice and can be differentiated into astrocytes and neurons. However, p25 TG decreased the long-term survival of proliferating NPCs and severely impaired adult neurogenesis. A Transwell co-culture system was used to assess the influence of p25-expressing primary neurons on adult NPCs. Co-culture with p25-expressing neurons downregulated Ki67 expression and upregulated cleaved caspase-3, indicating that the paracrine signaling in cell-cell communication is essential for NPC survival and proliferation. Moreover, increased CDK5 activity impairs Wnt activation. This study demonstrates that hyperactivation of p25 may temporarily enhance NPC proliferation, but impair their long-term survival.

Quercetin prevents protein nitration and glycolytic block of proliferation in hydrogen peroxide insulted cultured neuronal precursor cells (NPCs): Implications on CNS regeneration.

PubMed

Sajad, Mir; Zargan, Jamil; Zargar, Mohammad Afzal; Sharma, Jyoti; Umar, Sadiq; Arora, Rajesh; Khan, Haider A

2013-05-01

Survival along with optimal proliferation of neuronal precursors determines the outcomes of the endogenous cellular repair in CNS. Cellular-oxidation based cell death has been described in several neurodegenerative disorders. Therefore, this study was aimed at the identification of the potent targets of oxidative damage to the neuronal precursors and its effective prevention by a natural flavonoid, Quercetin. Neuronal precursor cells (NPCs), Nestin+ and GFAP (Glial fibrillary acidic protein)+ were isolated and cultured from adult rat SVZ (subventricular zone). These cells were challenged with a single dose of H2O2 (50μM) and/or pre-treated with different concentrations of Quercetin. H2O2 severely limited the cellular viability and expansion of the neurospheres. Cellular-oxidation studies revealed reduction in glutathione dependent redox buffering along with depletion of enzymatic cellular antioxidants that might potentiate the nitrite (NO2(-)) and superoxide anion (O2(-)) mediated peroxynitrite (ONOO(-)) formation and irreversible protein nitration. We identified depleted PK-M2 (M2 isoform of pyruvate kinase) activity and apoptosis of NPCs revealed by the genomic DNA fragmentation and elevated PARP (poly ADP ribose polymerase) activity along with increased Caspase activity initiated by severely depolarised mitochondrial membranes. However, the pre-treatment of Quercetin in a dose-response manner prevented these changes and restored the expansion of neurospheres preferably by neutralizing the oxidative conditions and thereby reducing peroxynitrite formation, protein nitration and PK-M2 depletion. Our results unravel the potential interactions of oxidative environment and respiration in the survival and activation of precursors and offer a promise shown by a natural flavonoid in the protective strategy for neuronal precursors of adult brain. Copyright © 2013 Elsevier Inc. All rights reserved.

Is the Role of Physicians Really Evolving Due to Non-physician Clinicians Predominance in Staff Makeup in Sub-Saharan African Health Systems? Comment on "Non-physician Clinicians in Sub-Saharan Africa and the Evolving Role of Physicians".

PubMed

Sidat, Mohsin M

2016-07-02

Health workforce shortages in Sub-Saharan Africa are widely recognized, particularly of physicians, leading the training and deployment of Non-physician clinicians (NPCs). The paper by Eyal et al provides interesting and legitimate viewpoints on evolving role of physicians in context of decisive increase of NPCss in Sub-Saharan Africa. Certainly, in short or mid-term, NPCs will continue to be a proxy solution and a valuable alternative to overcome physicians' shortages in sub-Saharan Africa. Indeed, NPCs have an important role at primary healthcare (PHC) level. Physicians at PHC level can certainly have all different roles that were suggested by Eyal et al, including those not directly related to healthcare provision. However, at secondary and higher levels of healthcare, physicians would assume other roles that are mainly related to patient clinical care. Thus, attempting to generalize the role of physicians without taking into account the context where they will work would be not entirely appropriate. It is true that often physicians start the professional carriers at PHC level and progress to other levels of healthcare particularly after clinical post-graduation training. Nevertheless, the training programs offered by medical institutions in sub-Saharan Africa need to be periodically reviewed and take into account professional and occupational roles physicians would take in context of evolving health systems in sub-Saharan Africa. © 2016 The Author(s); Published by Kerman University of Medical Sciences. This is an open-access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Direct exposure to mild heat promotes proliferation and neuronal differentiation of neural stem/progenitor cells in vitro

PubMed Central

Hossain, Md Emon; Katakura, Masanori; Sugimoto, Naotoshi; Mamun, Abdullah Al; Islam, Rafiad; Hashimoto, Michio; Shido, Osamu

2017-01-01

Heat acclimation in rats is associated with enhanced neurogenesis in thermoregulatory centers of the hypothalamus. To elucidate the mechanisms for heat acclimation, we investigated the effects of direct mild heat exposure on the proliferation and differentiation of neural stem/progenitor cells (NSCs/NPCs). The NSCs/NPCs isolated from forebrain cortices of 14.5-day-old rat fetuses were propagated as neurospheres at either 37.0°C (control) or 38.5°C (mild heat exposure) for four days, and the effects on proliferation were investigated by MTS cell viability assay, measurement of neurosphere diameter, and counting the total number of cells. The mRNA expressions of heat shock proteins (HSPs) and brain-derived neurotrophic factor (BDNF), cAMP response element-binding (CREB) protein and Akt phosphorylation levels, and intracellular reactive oxygen species (ROS) levels were analyzed using real time PCR, Western blotting and CM-H2DCFDA assay respectively. Heat exposure under proliferation condition increased NSC/NPC viability, neurosphere diameter, and cell count. BDNF mRNA expression, CREB phosphorylation, and ROS level were also increased by heat exposure. Heat exposure increased HSP27 mRNA expression concomitant with enhanced p-Akt level. Moreover, treatment with LY294002 (a PI3K inhibitor) abolished the effects of heat exposure on NSC/NPC proliferation. Furthermore, heat exposure under differentiation conditions increased the proportion of cells positive for Tuj1 (a neuronal marker). These findings suggest that mild heat exposure increases NSC/NPC proliferation, possibly through activation of the Akt pathway, and also enhances neuronal differentiation. Direct effects of temperature on NSCs/NPCs may be one of the mechanisms involved in hypothalamic neurogenesis in heat-acclimated rats. Such heat-induced neurogenesis could also be an effective therapeutic strategy for neurodegenerative diseases. PMID:29287093

Vector-Free and Transgene-Free Human iPS Cells Differentiate into Functional Neurons and Enhance Functional Recovery after Ischemic Stroke in Mice

PubMed Central

Mohamad, Osama; Faulkner, Ben; Chen, Dongdong; Yu, Shan Ping; Wei, Ling

2013-01-01

Stroke is a leading cause of human death and disability in the adult population in the United States and around the world. While stroke treatment is limited, stem cell transplantation has emerged as a promising regenerative therapy to replace or repair damaged tissues and enhance functional recovery after stroke. Recently, the creation of induced pluripotent stem (iPS) cells through reprogramming of somatic cells has revolutionized cell therapy by providing an unlimited source of autologous cells for transplantation. In addition, the creation of vector-free and transgene-free human iPS (hiPS) cells provides a new generation of stem cells with a reduced risk of tumor formation that was associated with the random integration of viral vectors seen with previous techniques. However, the potential use of these cells in the treatment of ischemic stroke has not been explored. In the present investigation, we examined the neuronal differentiation of vector-free and transgene-free hiPS cells and the transplantation of hiPS cell-derived neural progenitor cells (hiPS-NPCs) in an ischemic stroke model in mice. Vector-free hiPS cells were maintained in feeder-free and serum-free conditions and differentiated into functional neurons in vitro using a newly developed differentiation protocol. Twenty eight days after transplantation in stroke mice, hiPS-NPCs showed mature neuronal markers in vivo. No tumor formation was seen up to 12 months after transplantation. Transplantation of hiPS-NPCs restored neurovascular coupling, increased trophic support and promoted behavioral recovery after stroke. These data suggest that using vector-free and transgene-free hiPS cells in stem cell therapy are safe and efficacious in enhancing recovery after focal ischemic stroke in mice. PMID:23717557

Comparison of 2D and 3D neural induction methods for the generation of neural progenitor cells from human induced pluripotent stem cells.

PubMed

Chandrasekaran, Abinaya; Avci, Hasan X; Ochalek, Anna; Rösingh, Lone N; Molnár, Kinga; László, Lajos; Bellák, Tamás; Téglási, Annamária; Pesti, Krisztina; Mike, Arpad; Phanthong, Phetcharat; Bíró, Orsolya; Hall, Vanessa; Kitiyanant, Narisorn; Krause, Karl-Heinz; Kobolák, Julianna; Dinnyés, András

2017-12-01

Neural progenitor cells (NPCs) from human induced pluripotent stem cells (hiPSCs) are frequently induced using 3D culture methodologies however, it is unknown whether spheroid-based (3D) neural induction is actually superior to monolayer (2D) neural induction. Our aim was to compare the efficiency of 2D induction with 3D induction method in their ability to generate NPCs, and subsequently neurons and astrocytes. Neural differentiation was analysed at the protein level qualitatively by immunocytochemistry and quantitatively by flow cytometry for NPC (SOX1, PAX6, NESTIN), neuronal (MAP2, TUBB3), cortical layer (TBR1, CUX1) and glial markers (SOX9, GFAP, AQP4). Electron microscopy demonstrated that both methods resulted in morphologically similar neural rosettes. However, quantification of NPCs derived from 3D neural induction exhibited an increase in the number of PAX6/NESTIN double positive cells and the derived neurons exhibited longer neurites. In contrast, 2D neural induction resulted in more SOX1 positive cells. While 2D monolayer induction resulted in slightly less mature neurons, at an early stage of differentiation, the patch clamp analysis failed to reveal any significant differences between the electrophysiological properties between the two induction methods. In conclusion, 3D neural induction increases the yield of PAX6 + /NESTIN + cells and gives rise to neurons with longer neurites, which might be an advantage for the production of forebrain cortical neurons, highlighting the potential of 3D neural induction, independent of iPSCs' genetic background. Copyright © 2017 The Authors. Published by Elsevier B.V. All rights reserved.

Topological defects control collective dynamics in neural progenitor cell cultures

NASA Astrophysics Data System (ADS)

Kawaguchi, Kyogo; Kageyama, Ryoichiro; Sano, Masaki

2017-04-01

Cultured stem cells have become a standard platform not only for regenerative medicine and developmental biology but also for biophysical studies. Yet, the characterization of cultured stem cells at the level of morphology and of the macroscopic patterns resulting from cell-to-cell interactions remains largely qualitative. Here we report on the collective dynamics of cultured murine neural progenitor cells (NPCs), which are multipotent stem cells that give rise to cells in the central nervous system. At low densities, NPCs moved randomly in an amoeba-like fashion. However, NPCs at high density elongated and aligned their shapes with one another, gliding at relatively high velocities. Although the direction of motion of individual cells reversed stochastically along the axes of alignment, the cells were capable of forming an aligned pattern up to length scales similar to that of the migratory stream observed in the adult brain. The two-dimensional order of alignment within the culture showed a liquid-crystalline pattern containing interspersed topological defects with winding numbers of +1/2 and -1/2 (half-integer due to the nematic feature that arises from the head-tail symmetry of cell-to-cell interaction). We identified rapid cell accumulation at +1/2 defects and the formation of three-dimensional mounds. Imaging at the single-cell level around the defects allowed us to quantify the velocity field and the evolving cell density; cells not only concentrate at +1/2 defects, but also escape from -1/2 defects. We propose a generic mechanism for the instability in cell density around the defects that arises from the interplay between the anisotropic friction and the active force field.

Similar BCL-X but different BCL-2 levels in the two age groups of north African nasopharyngeal carcinomas.

PubMed

Khabir, Abdelmajid; Ghorbel, Abdelmoneem; Daoud, Jamel; Frikha, Mounir; Drira, Mohamed Mokhtar; Laplanche, Agnès; Busson, Pierre; Jlidi, Rachid

2003-01-01

Nasopharyngeal carcinomas (NPCs) are consistently associated with the Epstein-Barr virus (EBV). As Bcl-2 and Bcl-X are co-expressed in EBV-transformed B-lymphocytes, we attempted to determine their status in malignant NPC cells. A retrospective series of 100 NPC specimens from untreated Tunisian patients was investigated by immuno-histochemistry. Twenty seven of the patients were below 30 years old and therefore classified in the "juvenile" form of north African NPCs. Bcl-2 and Bcl-X expression was assessed semi-quantitatively using a score based on the percentage of positive cells and staining intensity. Intense Bcl-X expression was detected in malignant cells of 100% biopsy samples with similar scores for patients below 30 years or those aged 30 or over. Bcl-2 was detected in 89% biopsies but its expression differed considerably between the samples. The average Bcl-2 score was much lower for patients under 30 years (4.4+/-1.5 compared to 6.5+/-2 for older patients; P<10(-6)). Multivariate analysis demonstrated that no other clinical parameter, except the primary tumor size, was correlated to the Bcl-2 score. Bcl-X and Bcl-2 are co-expressed in 89% of NPCs whereas their expression is mutually exclusive in other head and neck carcinomas (particularly squamous cell carcinomas, SCC). The constantly high expression of Bcl-X is consistent with it being induced by the EBV protein Epstein-Barr nuclear antigen 1 (EBNA1), as recently reported in a murine model. The contrasted levels of Bcl-2 expression in the two age groups strengthen the hypothesis that these clinical forms result from distinct oncogenic mechanisms.

Estradiol-17β-Induced Human Neural Progenitor Cell Proliferation Is Mediated by an Estrogen Receptor β-Phosphorylated Extracellularly Regulated Kinase Pathway

PubMed Central

Wang, Jun Ming; Liu, Lifei; Brinton, Roberta Diaz

2008-01-01

Estradiol-17β (E2) induces rodent hippocampal neural progenitor cell (NPC) proliferation in vitro, in vivo, and after brain injury. The purpose of the present investigation was to determine whether E2-induced proliferation observed in rodent model systems generalized to cells of human neural origin and the signaling pathway by which E2 promotes mitosis of human NPCs (hNPCs). Results of these analyses indicate that E2 induced a significant increase in hNPC proliferation in a time- and dose-dependent manner. E2-induced hNPC DNA replication was paralleled by elevated cell cycle protein expression and centrosome amplification, which was associated with augmentation of total cell number. To determine whether estrogen receptor (ER) and which ER subtype were required for E2-induced hNPC proliferation, ER expression was first determined by real-time RT-PCR, followed by Western blot analysis, and subsequently verified pharmacologically using ERα or β-selective ligands. Results of these analyses indicated that ERβ expression was predominant relative to ERα, which was barely detectable in hNPCs. Activation of ERβ by the ERβ-selective ligand, diarylpropionitrile, led to an increase in phosphorylated extracellular signal-regulated kinase, and subsequent centrosome amplification and hNPC proliferation, which were blocked by the MEKK antagonist, UO126, but not its inactive analog, UO124. These findings, for the first time, demonstrate the molecular cascade and related cell biology events involved in E2-induced hNPC proliferation in vitro. Therapeutic implications of these findings relevant to hormone therapy and prevention of neurodegenerative disease are discussed. PMID:17962344

Transplantation of embryonic stem cells improves nerve repair and functional recovery after severe sciatic nerve axotomy in rats.

PubMed

Cui, Lin; Jiang, Jun; Wei, Ling; Zhou, Xin; Fraser, Jamie L; Snider, B Joy; Yu, Shan Ping

2008-05-01

Extensive research has focused on transplantation of pluripotent stem cells for the treatment of central nervous system disorders, the therapeutic potential of stem cell therapy for injured peripheral nerves is largely unknown. We used a rat sciatic nerve transection model to test the ability of implanted embryonic stem (ES) cell-derived neural progenitor cells (ES-NPCs) in promoting repair of a severely injured peripheral nerve. Mouse ES cells were neurally induced in vitro; enhanced expression and/or secretion of growth factors were detected in differentiating ES cells. One hour after removal of a 1-cm segment of the left sciatic nerve, ES-NPCs were implanted into the gap between the nerve stumps with the surrounding epineurium as a natural conduit. The transplantation resulted in substantial axonal regrowth and nerve repair, which were not seen in culture medium controls. One to 3 months after axotomy, co-immunostaining with the mouse neural cell membrane specific antibody M2/M6 and the Schwann cell marker S100 suggested that transplanted ES-NPCs had survived and differentiated into myelinating cells. Regenerated axons were myelinated and showed a uniform connection between proximal and distal stumps. Nerve stumps had near normal diameter with longitudinally oriented, densely packed Schwann cell-like phenotype. Fluoro-Gold retrogradely labeled neurons were found in the spinal cord (T12-13) and DRG (L4-L6), suggesting reconnection of axons across the transection. Electrophysiological recordings showed functional activity recovered across the injury gap. These data suggest that transplanted neurally induced ES cells differentiate into myelin-forming cells and provide a potential therapy for severely injured peripheral nerves.

Neural stem cell transplantation in central nervous system disorders: from cell replacement to neuroprotection.

PubMed

De Feo, Donatella; Merlini, Arianna; Laterza, Cecilia; Martino, Gianvito

2012-06-01

Transplantation of neural stem/precursor cells (NPCs) has been proposed as a promising therapeutic strategy in almost all neurological disorders characterized by the failure of central nervous system (CNS) endogenous repair mechanisms in restoring the tissue damage and rescuing the lost function. Nevertheless, recent evidence consistently challenges the limited view that transplantation of these cells is solely aimed at protecting the CNS from inflammatory and neurodegenerative damage through cell replacement. Recent preclinical data confirmed that transplanted NPCs may also exert a 'bystander' neuroprotective effect and identified a series of molecules - for example, immunomodulatory substances, neurotrophic growth factors, stem cell regulators as well as guidance molecules - whose in-situ secretion by NPCs is temporally and spatially orchestrated by environmental needs. A better understanding of the molecular and cellular mechanisms sustaining this 'therapeutic plasticity' is of pivotal importance for defining crucial aspects of the bench-to-beside translation of neural stem cell therapy, that is route and timing of administration as well as the best cellular source. Further insight into those latter issues is eagerly expected from the ongoing phase I/II clinical trials, while, on the other hand, new cellular sources are being developed, mainly by exploiting the new possibilities offered by cellular reprogramming. Nowadays, the research on NPC transplantation in neurological disorders is advancing on two different fronts: on one hand, recent preclinical data are uncovering the molecular basis of NPC therapeutic plasticity, offering a more solid rational framework for the design of clinical studies. On the other hand, pilot trials are highlighting the safety and feasibility issues of neural stem cell transplantation that need to be addressed before efficacy could be properly evaluated.

Transgenic analysis of a SoxB gene reveals neural progenitor cells in the cnidarian Nematostella vectensis.

PubMed

Richards, Gemma Sian; Rentzsch, Fabian

2014-12-01

Bilaterian neurogenesis is characterized by the generation of diverse neural cell types from dedicated neural stem/progenitor cells (NPCs). However, the evolutionary origin of NPCs is unclear, as neurogenesis in representatives of the bilaterian sister group, the Cnidaria, occurs via interstitial stem cells that also possess broader, non-neural, developmental potential. We address this question by analysing neurogenesis in an anthozoan cnidarian, Nematostella vectensis. Using a transgenic reporter line, we show that NvSoxB(2) - an orthologue of bilaterian SoxB genes that have conserved roles in neurogenesis - is expressed in a cell population that gives rise to sensory neurons, ganglion neurons and nematocytes: the three primary neural cell types of cnidarians. EdU labelling together with in situ hybridization, and within the NvSoxB(2)::mOrange transgenic line, demonstrates that cells express NvSoxB(2) before mitosis and identifies asymmetric behaviours of sibling cells within NvSoxB(2)(+) lineages. Morpholino-mediated gene knockdown of NvSoxB(2) blocks the formation of all three neural cell types, thereby identifying NvSoxB(2) as an essential positive regulator of nervous system development. Our results demonstrate that diverse neural cell types derive from an NvSoxB(2)-expressing population of mitotic cells in Nematostella and that SoxB genes are ancient components of a neurogenic program. To our knowledge this is the first description of a lineage-restricted, multipotent cell population outside the Bilateria and we propose that neurogenesis via dedicated, SoxB-expressing NPCs predates the split between cnidarians and bilaterians. © 2014. Published by The Company of Biologists Ltd.

The Amyloid Precursor Protein (APP) Triplicated Gene Impairs Neuronal Precursor Differentiation and Neurite Development through Two Different Domains in the Ts65Dn Mouse Model for Down Syndrome*

PubMed Central

Trazzi, Stefania; Fuchs, Claudia; Valli, Emanuele; Perini, Giovanni; Bartesaghi, Renata; Ciani, Elisabetta

2013-01-01

Intellectual disability in Down syndrome (DS) appears to be related to severe proliferation impairment during brain development. Recent evidence shows that it is not only cellular proliferation that is heavily compromised in DS, but also cell fate specification and dendritic maturation. The amyloid precursor protein (APP), a gene that is triplicated in DS, plays a key role in normal brain development by influencing neural precursor cell proliferation, cell fate specification, and neuronal maturation. APP influences these processes via two separate domains, the APP intracellular domain (AICD) and the soluble secreted APP. We recently found that the proliferation impairment of neuronal precursors (NPCs) from the Ts65Dn mouse model for DS was caused by derangement of the Shh pathway due to overexpression of patched1(Ptch1), its inhibitory regulator. Ptch1 overexpression was related to increased levels within the APP/AICD system. The overall goal of this study was to determine whether APP contributes to neurogenesis impairment in DS by influencing in addition to proliferation, cell fate specification, and neurite development. We found that normalization of APP expression restored the reduced neuronogenesis, the increased astrogliogenesis, and the reduced neurite length of trisomic NPCs, indicating that APP overexpression underpins all aspects of neurogenesis impairment. Moreover, we found that two different domains of APP impair neuronal differentiation and maturation in trisomic NPCs. The APP/AICD system regulates neuronogenesis and neurite length through the Shh pathway, whereas the APP/secreted AP system promotes astrogliogenesis through an IL-6-associated signaling cascade. These results provide novel insight into the mechanisms underlying brain development alterations in DS. PMID:23740250

A galvanotaxis assay for analysis of neural precursor cell migration kinetics in an externally applied direct current electric field.

PubMed

Babona-Pilipos, Robart; Popovic, Milos R; Morshead, Cindi M

2012-10-13

The discovery of neural stem and progenitor cells (collectively termed neural precursor cells) (NPCs) in the adult mammalian brain has led to a body of research aimed at utilizing the multipotent and proliferative properties of these cells for the development of neuroregenerative strategies. A critical step for the success of such strategies is the mobilization of NPCs toward a lesion site following exogenous transplantation or to enhance the response of the endogenous precursors that are found in the periventricular region of the CNS. Accordingly, it is essential to understand the mechanisms that promote, guide, and enhance NPC migration. Our work focuses on the utilization of direct current electric fields (dcEFs) to promote and direct NPC migration - a phenomenon known as galvanotaxis. Endogenous physiological electric fields function as critical cues for cell migration during normal development and wound repair. Pharmacological disruption of the trans-neural tube potential in axolotl embryos causes severe developmental malformations(1). In the context of wound healing, the rate of repair of wounded cornea is directly correlated with the magnitude of the epithelial wound potential that arises after injury, as shown by pharmacological enhancement or disruption of this dcEF(2-3). We have demonstrated that adult subependymal NPCs undergo rapid and directed cathodal migration in vitro when exposed to an externally applied dcEF. In this protocol we describe our lab's techniques for creating a simple and effective galvanotaxis assay for high-resolution, long-term observation of directed cell body translocation (migration) on a single-cell level. This assay would be suitable for investigating the mechanisms that regulate dcEF transduction into cellular motility through the use of transgenic or knockout mice, short interfering RNA, or specific receptor agonists/antagonists.

Generation of kisspeptin-responsive GnRH neurons from human pluripotent stem cells.

PubMed

Poliandri, Ariel; Miller, Duncan; Howard, Sasha; Nobles, Muriel; Ruiz-Babot, Gerard; Harmer, Stephen; Tinker, Andrew; McKay, Tristan; Guasti, Leonardo; Dunkel, Leo

2017-05-15

GnRH neurons are fundamental for reproduction in all vertebrates, integrating all reproductive inputs. The inaccessibility of human GnRH-neurons has been a major impediment to studying the central control of reproduction and its disorders. Here, we report the efficient generation of kisspeptin responsive GnRH-secreting neurons by directed differentiation of human Embryonic Stem Cells and induced-Pluripotent Stem Cells derived from a Kallman Syndrome patient and a healthy family member. The protocol involves the generation of intermediate Neural Progenitor Cells (NPCs) through long-term Bone morphogenetic protein 4 inhibition, followed by terminal specification of these NPCs in media containing Fibroblast Growth Factor 8 and a NOTCH inhibitor. The resulting GnRH-expressing and -secreting neurons display a neuroendocrine gene expression pattern and present spontaneous calcium transients that can be stimulated by kisspeptin. These in vitro generated GnRH expressing cells provide a new resource for studying the molecular mechanisms underlying the development and function of GnRH neurons. Copyright © 2017 Elsevier B.V. All rights reserved.

Development of Gonadotropin-Releasing Hormone-Secreting Neurons from Human Pluripotent Stem Cells.

PubMed

Lund, Carina; Pulli, Kristiina; Yellapragada, Venkatram; Giacobini, Paolo; Lundin, Karolina; Vuoristo, Sanna; Tuuri, Timo; Noisa, Parinya; Raivio, Taneli

2016-08-09

Gonadotropin-releasing hormone (GnRH) neurons regulate human puberty and reproduction. Modeling their development and function in vitro would be of interest for both basic research and clinical translation. Here, we report a three-step protocol to differentiate human pluripotent stem cells (hPSCs) into GnRH-secreting neurons. Firstly, hPSCs were differentiated to FOXG1, EMX2, and PAX6 expressing anterior neural progenitor cells (NPCs) by dual SMAD inhibition. Secondly, NPCs were treated for 10 days with FGF8, which is a key ligand implicated in GnRH neuron ontogeny, and finally, the cells were matured with Notch inhibitor to bipolar TUJ1-positive neurons that robustly expressed GNRH1 and secreted GnRH decapeptide into the culture medium. The protocol was reproducible both in human embryonic stem cells and induced pluripotent stem cells, and thus provides a translational tool for investigating the mechanisms of human puberty and its disorders. Copyright © 2016 The Author(s). Published by Elsevier Inc. All rights reserved.

A quantitative framework to evaluate modeling of cortical development by neural stem cells

PubMed Central

Stein, Jason L.; de la Torre-Ubieta, Luis; Tian, Yuan; Parikshak, Neelroop N.; Hernandez, Israel A.; Marchetto, Maria C.; Baker, Dylan K.; Lu, Daning; Hinman, Cassidy R.; Lowe, Jennifer K.; Wexler, Eric M.; Muotri, Alysson R.; Gage, Fred H.; Kosik, Kenneth S.; Geschwind, Daniel H.

2014-01-01

Summary Neural stem cells have been adopted to model a wide range of neuropsychiatric conditions in vitro. However, how well such models correspond to in vivo brain has not been evaluated in an unbiased, comprehensive manner. We used transcriptomic analyses to compare in vitro systems to developing human fetal brain and observed strong conservation of in vivo gene expression and network architecture in differentiating primary human neural progenitor cells (phNPCs). Conserved modules are enriched in genes associated with ASD, supporting the utility of phNPCs for studying neuropsychiatric disease. We also developed and validated a machine learning approach called CoNTExT that identifies the developmental maturity and regional identity of in vitro models. We observed strong differences between in vitro models, including hiPSC-derived neural progenitors from multiple laboratories. This work provides a systems biology framework for evaluating in vitro systems and supports their value in studying the molecular mechanisms of human neurodevelopmental disease. PMID:24991955

Efficient protein targeting to the inner nuclear membrane requires Atlastin-dependent maintenance of ER topology

PubMed Central

Pawar, Sumit; Ungricht, Rosemarie; Tiefenboeck, Peter; Leroux, Jean-Christophe

2017-01-01

Newly synthesized membrane proteins are targeted to the inner nuclear membrane (INM) by diffusion within the membrane system of the endoplasmic reticulum (ER), translocation through nuclear pore complexes (NPCs) and retention on nuclear partners. Using a visual in vitro assay we previously showed that efficient protein targeting to the INM depends on nucleotide hydrolysis. We now reveal that INM targeting is GTP-dependent. Exploiting in vitro reconstitution and in vivo analysis of INM targeting, we establish that Atlastins, membrane-bound GTPases of the ER, sustain the efficient targeting of proteins to the INM by their continued activity in preserving ER topology. When ER topology is altered, the long-range diffusional exchange of proteins in the ER network and targeting efficiency to the INM are diminished. Highlighting the general importance of proper ER topology, we show that Atlastins also influence NPC biogenesis and timely exit of secretory cargo from the ER. PMID:28826471

A physical model describing the interaction of nuclear transport receptors with FG nucleoporin domain assemblies.

PubMed

Zahn, Raphael; Osmanović, Dino; Ehret, Severin; Araya Callis, Carolina; Frey, Steffen; Stewart, Murray; You, Changjiang; Görlich, Dirk; Hoogenboom, Bart W; Richter, Ralf P

2016-04-08

The permeability barrier of nuclear pore complexes (NPCs) controls bulk nucleocytoplasmic exchange. It consists of nucleoporin domains rich in phenylalanine-glycine motifs (FG domains). As a bottom-up nanoscale model for the permeability barrier, we have used planar films produced with three different end-grafted FG domains, and quantitatively analyzed the binding of two different nuclear transport receptors (NTRs), NTF2 and Importin β, together with the concomitant film thickness changes. NTR binding caused only moderate changes in film thickness; the binding isotherms showed negative cooperativity and could all be mapped onto a single master curve. This universal NTR binding behavior - a key element for the transport selectivity of the NPC - was quantitatively reproduced by a physical model that treats FG domains as regular, flexible polymers, and NTRs as spherical colloids with a homogeneous surface, ignoring the detailed arrangement of interaction sites along FG domains and on the NTR surface.

Enrollment Management's Sleeping Giant: The Net Price Calculator Mandate

ERIC Educational Resources Information Center

Fallon, Mary A. C.

2011-01-01

Enrollment managers will be watching to see how recruitment strategies change when higher education's sleeping giant--net price calculators (NPCs)--wakes in the fall of 2011. Some predict yield projections may be more difficult and reputations will be challenged as prospective students, their families, high school counselors, and independent…

20 CFR 655.1309 - Labor certification determinations.

Code of Federal Regulations, 2010 CFR

2010-04-01

... Administrator, OFLC is the Department's National CO. The Administrator, OFLC, and the CO(s) in the NPC(s) (by... otherwise meeting certification criteria by that date, the CO will make a determination either to grant or deny the Application for Temporary Employment Certification. The CO will grant the application if and...

20 CFR 655.1309 - Labor certification determinations.

Code of Federal Regulations, 2011 CFR

2011-04-01

... Administrator, OFLC is the Department's National CO. The Administrator, OFLC, and the CO(s) in the NPC(s) (by... otherwise meeting certification criteria by that date, the CO will make a determination either to grant or deny the Application for Temporary Employment Certification. The CO will grant the application if and...

A statistical image analysis framework for pore-free islands derived from heterogeneity distribution of nuclear pore complexes.

PubMed

Mimura, Yasuhiro; Takemoto, Satoko; Tachibana, Taro; Ogawa, Yutaka; Nishimura, Masaomi; Yokota, Hideo; Imamoto, Naoko

2017-11-24

Nuclear pore complexes (NPCs) maintain cellular homeostasis by mediating nucleocytoplasmic transport. Although cyclin-dependent kinases (CDKs) regulate NPC assembly in interphase, the location of NPC assembly on the nuclear envelope is not clear. CDKs also regulate the disappearance of pore-free islands, which are nuclear envelope subdomains; this subdomain gradually disappears with increase in homogeneity of the NPC in response to CDK activity. However, a causal relationship between pore-free islands and NPC assembly remains unclear. Here, we elucidated mechanisms underlying NPC assembly from a new perspective by focusing on pore-free islands. We proposed a novel framework for image-based analysis to automatically determine the detailed 'landscape' of pore-free islands from a large quantity of images, leading to the identification of NPC intermediates that appear in pore-free islands with increased frequency in response to CDK activity. Comparison of the spatial distribution between simulated and the observed NPC intermediates within pore-free islands showed that their distribution was spatially biased. These results suggested that the disappearance of pore-free islands is highly related to de novo NPC assembly and indicated the existence of specific regulatory mechanisms for the spatial arrangement of NPC assembly on nuclear envelopes.

In the Right Ballpark? Assessing the Accuracy of Net Price Calculators

ERIC Educational Resources Information Center

Anthony, Aaron M.; Page, Lindsay C.; Seldin, Abigail

2016-01-01

Large differences often exist between a college's sticker price and net price after accounting for financial aid. Net price calculators (NPCs) were designed to help students more accurately estimate their actual costs to attend a given college. This study assesses the accuracy of information provided by net price calculators. Specifically, we…

Designer Self-Assembling Peptide Nanofiber Scaffolds Containing Link Protein N-Terminal Peptide Induce Chondrogenesis of Rabbit Bone Marrow Stem Cells

PubMed Central

Wang, Baichuan; Sun, Caixia; Shao, Zengwu; Yang, Shuhua; Che, Biao; Wu, Qiang; Liu, Jianxiang

2014-01-01

Designer self-assembling peptide nanofiber hydrogel scaffolds have been considered as promising biomaterials for tissue engineering because of their excellent biocompatibility and biofunctionality. Our previous studies have shown that a novel designer functionalized self-assembling peptide nanofiber hydrogel scaffold (RLN/RADA16, LN-NS) containing N-terminal peptide sequence of link protein (link N) can promote nucleus pulposus cells (NPCs) adhesion and three-dimensional (3D) migration and stimulate biosynthesis of type II collagen and aggrecan by NPCs in vitro. The present study has extended these investigations to determine the effects of this functionalized LN-NS on bone marrow stem cells (BMSCs), a potential cell source for NP regeneration. Although the functionalized LN-NS cannot promote BMSCs proliferation, it significantly promotes BMSCs adhesion compared with that of the pure RADA16 hydrogel scaffold. Moreover, the functionalized LN-NS remarkably stimulates biosynthesis and deposition of type II collagen and aggrecan. These data demonstrate that the functionalized peptide nanofiber hydrogel scaffold containing link N peptide as a potential matrix substrate will be very useful in the NP tissue regeneration. PMID:25243141

Ferritin nanoparticles for improved self-renewal and differentiation of human neural stem cells.

PubMed

Lee, Jung Seung; Yang, Kisuk; Cho, Ann-Na; Cho, Seung-Woo

2018-01-01

Biomaterials that promote the self-renewal ability and differentiation capacity of neural stem cells (NSCs) are desirable for improving stem cell therapy to treat neurodegenerative diseases. Incorporation of micro- and nanoparticles into stem cell culture has gained great attention for the control of stem cell behaviors, including proliferation and differentiation. In this study, ferritin, an iron-containing natural protein nanoparticle, was applied as a biomaterial to improve the self-renewal and differentiation of NSCs and neural progenitor cells (NPCs). Ferritin nanoparticles were added to NSC or NPC culture during cell growth, allowing for incorporation of ferritin nanoparticles during neurosphere formation. Compared to neurospheres without ferritin treatment, neurospheres with ferritin nanoparticles showed significantly promoted self-renewal and cell-cell interactions. When spontaneous differentiation of neurospheres was induced during culture without mitogenic factors, neuronal differentiation was enhanced in the ferritin-treated neurospheres. In conclusion, we found that natural nanoparticles can be used to improve the self-renewal ability and differentiation potential of NSCs and NPCs, which can be applied in neural tissue engineering and cell therapy for neurodegenerative diseases.

Acute stress enhances adult rat hippocampal neurogenesis and activation of newborn neurons via secreted astrocytic FGF2

PubMed Central

Kirby, Elizabeth D; Muroy, Sandra E; Sun, Wayne G; Covarrubias, David; Leong, Megan J; Barchas, Laurel A; Kaufer, Daniela

2013-01-01

Stress is a potent modulator of the mammalian brain. The highly conserved stress hormone response influences many brain regions, particularly the hippocampus, a region important for memory function. The effect of acute stress on the unique population of adult neural stem/progenitor cells (NPCs) that resides in the adult hippocampus is unclear. We found that acute stress increased hippocampal cell proliferation and astrocytic fibroblast growth factor 2 (FGF2) expression. The effect of acute stress occurred independent of basolateral amygdala neural input and was mimicked by treating isolated NPCs with conditioned media from corticosterone-treated primary astrocytes. Neutralization of FGF2 revealed that astrocyte-secreted FGF2 mediated stress-hormone-induced NPC proliferation. 2 weeks, but not 2 days, after acute stress, rats also showed enhanced fear extinction memory coincident with enhanced activation of newborn neurons. Our findings suggest a beneficial role for brief stress on the hippocampus and improve understanding of the adaptive capacity of the brain. DOI: http://dx.doi.org/10.7554/eLife.00362.001 PMID:23599891

Tumour necrosis factor-alpha impairs neuronal differentiation but not proliferation of hippocampal neural precursor cells: Role of Hes1.

PubMed

Keohane, Aoife; Ryan, Sinead; Maloney, Eimer; Sullivan, Aideen M; Nolan, Yvonne M

2010-01-01

Tumour necrosis factor-alpha (TNFalpha) is a pro-inflammatory cytokine, which influences neuronal survival and function yet there is limited information available on its effects on hippocampal neural precursor cells (NPCs). We show that TNFalpha treatment during proliferation had no effect on the percentage of proliferating cells prepared from embryonic rat hippocampal neurosphere cultures, nor did it affect cell fate towards either an astrocytic or neuronal lineage when cells were then allowed to differentiate. However, when cells were differentiated in the presence of TNFalpha, significantly reduced percentages of newly born and post-mitotic neurons, significantly increased percentages of astrocytes and increased expression of TNFalpha receptors, TNF-R1 and TNF-R2, as well as expression of the anti-neurogenic Hes1 gene, were observed. These data indicate that exposure of hippocampal NPCs to TNFalpha when they are undergoing differentiation but not proliferation has a detrimental effect on their neuronal lineage fate, which may be mediated through increased expression of Hes1. Copyright 2009 Elsevier Inc. All rights reserved.

Functional Convergence of Neurons Generated in the Developing and Adult Hippocampus

PubMed Central

Piatti, Verónica C; Morgenstern, Nicolás A; Zhao, Chunmei; van Praag, Henriette; Gage, Fred H; Schinder, Alejandro F

2006-01-01

The dentate gyrus of the hippocampus contains neural progenitor cells (NPCs) that generate neurons throughout life. Developing neurons of the adult hippocampus have been described in depth. However, little is known about their functional properties as they become fully mature dentate granule cells (DGCs). To compare mature DGCs generated during development and adulthood, NPCs were labeled at both time points using retroviruses expressing different fluorescent proteins. Sequential electrophysiological recordings from neighboring neurons of different ages were carried out to quantitatively study their major synaptic inputs: excitatory projections from the entorhinal cortex and inhibitory afferents from local interneurons. Our results show that DGCs generated in the developing and adult hippocampus display a remarkably similar afferent connectivity with regard to both glutamate and GABA, the major neurotransmitters. We also demonstrate that adult-born neurons can fire action potentials in response to an excitatory drive, exhibiting a firing behavior comparable to that of neurons generated during development. We propose that neurons born in the developing and adult hippocampus constitute a functionally homogeneous neuronal population. These observations are critical to understanding the role of adult neurogenesis in hippocampal function. PMID:17121455

Conditional ablation of the prorenin receptor in nephron progenitor cells results in developmental programming of hypertension.

PubMed

Song, Renfang; Kidd, Laura; Janssen, Adam; Yosypiv, Ihor V

2018-04-01

Nephron induction during kidney development is driven by reciprocal interactions between progenitor cells (NPCs) of the cap mesenchyme (CM) and the ureteric bud (UB). The prorenin receptor (PRR) is a receptor for renin and prorenin, and an accessory subunit of the vacuolar proton pump V-ATPase. Previously, we demonstrated that conditional ablation of the PRR in Six2 + NPCs in mice (Six2 PRR -/- ) causes early neonatal death. Here, we identified genes that are regulated by PRR in Six2 + NPCs FACS-isolated from Six2 PRR -/- and control kidneys on embryonic day E15.5 using whole-genome expression analysis. Seven genes with expression in CM cells previously shown to direct kidney development, including Notch1, β-catenin, Lef1, Lhx1, Jag1, and p53, were downregulated. The functional groups within the downregulated gene set included genes involved in embryonic and cellular development, renal regeneration, cellular assembly and organization, cell morphology, death and survival. Double-transgenic Six2 PRR -/- /BatGal + mice, a reporter strain for β-catenin transcriptional activity, showed decreased β-catenin activity in the UB in vivo. Reduced PRR gene dosage in heterozygous Six2 PRR +/- mice was associated with decreased glomerular number, segmental thickening of the glomerular basement membrane with focal podocyte foot process effacement, development of hypertension and increased soluble PRR (sPRR) levels in the urine at 2 months of age. Together, these data demonstrate that NPC PRR performs essential functions during nephrogenesis via control of hierarchy of genes that regulate critical cellular processes. Both reduced nephron endowment and augmented urine sPRR likely contribute to programming of hypertension in Six2 PRR +/- mice. © 2018 The Authors. Physiological Reports published by Wiley Periodicals, Inc. on behalf of The Physiological Society and the American Physiological Society.

Effects of gintonin-enriched fraction on hippocampal cell proliferation in wild-type mice and an APPswe/PSEN-1 double Tg mouse model of Alzheimer's disease.

PubMed

Kim, Hyeon-Joong; Kim, Dae-Joong; Shin, Eun-Ju; Lee, Byung-Hwan; Choi, Sun-Hye; Hwang, Sung-Hee; Rhim, Hyewhon; Cho, Ik-Hyun; Kim, Hyoung-Chun; Nah, Seung-Yeol

2016-12-01

We previously showed that gintonin, an exogenous lysophosphatidic acid (LPA) receptor ligand, attenuated β-amyloid plaque formation in the cortex and hippocampus, and restored β-amyloid-induced memory dysfunction. Both endogenous LPA and LPA receptors play a key role in embryonic brain development. However, little is known about whether gintonin can induce hippocampal cell proliferation in adult wild-type mice and an APPswe/PSEN-1 double Tg mouse model of Alzheimer's disease (AD). In the present study, we examined the effects of gintonin on the proliferation of hippocampal neural progenitor cells (NPCs) in vitro and its effects on the hippocampal cell proliferation in wild-type mice and a transgenic AD mouse model. Gintonin treatment increased 5-bromo-2'-deoxyuridine (BrdU) incorporation in hippocampal NPCs in a dose- and time-dependent manner. Gintonin (0.3 μg/ml) increased the immunostaining of glial fibrillary acidic protein, NeuN, and LPA1 receptor in hippocampal NPCs. However, the gintonin-induced increase in BrdU incorporation and immunostaining of biomarkers was blocked by an LPA1/3 receptor antagonist and Ca 2+ chelator. Oral administration of the gintonin-enriched fraction (50 and 100 mg/kg) increased hippocampal BrdU incorporation and LPA1/3 receptor expression in adult wild-type and transgenic AD mice. The present study showed that gintonin could increase the number of hippocampal neurons in adult wild-type mice and a transgenic AD mouse model. Our results indicate that gintonin-mediated hippocampal cell proliferation contributes to the gintonin-mediated restorative effect against β-amyloid-induced hippocampal dysfunction. These results support the use of gintonin for the prevention or treatment of neurodegenerative diseases such as AD via promotion of hippocampal neurogenesis. Copyright © 2016 Elsevier Ltd. All rights reserved.

Sequence‑dependent effect of sorafenib in combination with natural phenolic compounds on hepatic cancer cells and the possible mechanism of action.

PubMed

Bahman, Abdulmajeed A; Abaza, Mohamed Salah I; Khoushiash, Sarah I; Al-Attiyah, Rajaa J

2018-06-08

Sorafenib (Nexavar, BAY43‑9006 or Sora) is the first molecular targeted agent that has exhibited significant therapeutic benefits in advanced hepatocellular carcinoma (HCC). However, not all HCC patients respond well to Sora and novel therapeutic strategies to optimize the efficacy of Sora are urgently required. Plant‑based drugs have received increasing attention owing to their excellent chemotherapeutic and chemopreventive activities; they are also well tolerated, non‑toxic, easily available and inexpensive. It is well known that certain biologically active natural products act synergistically with synthetic drugs used in clinical applications. The present study aimed to investigate whether a combination therapy with natural phenolic compounds (NPCs), including curcumin (Cur), quercetin (Que), kaempherol (Kmf) and resveratrol (Rsv), would allow a dose reduction of Sora without concomitant loss of its effectiveness. Furthermore, the possible molecular mechanisms of this synergy were assessed. The hepatic cancer cell lines Hep3b and HepG2 were treated with Sora alone or in combination with NPCs in concomitant, sequential, and inverted sequential regimens. Cell proliferation, cell cycle, apoptosis and expression of proteins associated with the cell cycle and apoptosis were investigated. NPCs markedly potentiated the therapeutic efficacy of Sora in a sequence‑, type‑, NPC dose‑ and cell line‑dependent manner. Concomitant treatment with Sora and Cur [sensitization ratio (SR)=28], Kmf (SR=18) or Que (SR=8) was associated with the highest SRs in Hep3b cells. Rsv markedly potentiated the effect of Sora (SR=17) on Hep3b cells when administered in a reverse sequential manner. By contrast, Rsv and Que did not improve the efficacy of Sora against HepG2 cells, while concomitant treatment with Cur (SR=10) or Kmf (SR=4.01) potentiated the cytotoxicity of Sora. Concomitant treatment with Sora and Cur or Kmf caused S‑phase and G2/M phase arrest of liver cancer cells and markedly induced apoptosis compared with mono‑treatment with Sora, Cur or Kmf. Concomitant treatment with Sora and Cur reduced the protein levels of cyclins A, B2 and D1, phosphorylated retinoblastoma and B‑cell lymphoma (Bcl) extra‑large protein. By contrast, Sora and Cur co‑treatment increased the protein levels of Bcl‑2‑associated X protein, cleaved caspase‑3 and cleaved caspase‑9 in a dose‑dependent manner. In conclusion, concomitant treatment with Sora and Cur or Kmf appears to be a potent and promising therapeutic approach that may control hepatic cancer by triggering cell cycle arrest and apoptosis. Additional studies are required to examine the potential of combined treatment with Sora and NPCs in human hepatic cancer and other solid tumor types in vivo.

Analysis of the initiation of nuclear pore assembly by ectopically targeting nucleoporins to chromatin

PubMed Central

Schwartz, Michal; Travesa, Anna; Martell, Steven W; Forbes, Douglass J

2015-01-01

Nuclear pore complexes (NPCs) form the gateway to the nucleus, mediating virtually all nucleocytoplasmic trafficking. Assembly of a nuclear pore complex requires the organization of many soluble sub-complexes into a final massive structure embedded in the nuclear envelope. By use of a LacI/LacO reporter system, we were able to assess nucleoporin (Nup) interactions, show that they occur with a high level of specificity, and identify nucleoporins sufficient for initiation of the complex process of NPC assembly in vivo. Eleven nucleoporins from different sub-complexes were fused to LacI-CFP and transfected separately into a human cell line containing a stably integrated LacO DNA array. The LacI-Nup fusion proteins, which bound to the array, were examined for their ability to recruit endogenous nucleoporins to the intranuclear LacO site. Many could recruit nucleoporins of the same sub-complex and a number could also recruit other sub-complexes. Strikingly, Nup133 and Nup107 of the Nup107/160 subcomplex and Nup153 and Nup50 of the nuclear pore basket recruited a near full complement of nucleoporins to the LacO array. Furthermore, Nup133 and Nup153 efficiently targeted the LacO array to the nuclear periphery. Our data support a hierarchical, seeded assembly pathway and identify Nup133 and Nup153 as effective “seeds” for NPC assembly. In addition, we show that this system can be applied to functional studies of individual nucleoporin domains as well as to specific nucleoporin disease mutations. We find that the R391H cardiac arrhythmia/sudden death mutation of Nup155 prevents both its subcomplex assembly and nuclear rim targeting of the LacO array. PMID:25602437

Inner/Outer nuclear membrane fusion in nuclear pore assembly: biochemical demonstration and molecular analysis.

PubMed

Fichtman, Boris; Ramos, Corinne; Rasala, Beth; Harel, Amnon; Forbes, Douglass J

2010-12-01

Nuclear pore complexes (NPCs) are large proteinaceous channels embedded in double nuclear membranes, which carry out nucleocytoplasmic exchange. The mechanism of nuclear pore assembly involves a unique challenge, as it requires creation of a long-lived membrane-lined channel connecting the inner and outer nuclear membranes. This stabilized membrane channel has little evolutionary precedent. Here we mapped inner/outer nuclear membrane fusion in NPC assembly biochemically by using novel assembly intermediates and membrane fusion inhibitors. Incubation of a Xenopus in vitro nuclear assembly system at 14°C revealed an early pore intermediate where nucleoporin subunits POM121 and the Nup107-160 complex were organized in a punctate pattern on the inner nuclear membrane. With time, this intermediate progressed to diffusion channel formation and finally to complete nuclear pore assembly. Correct channel formation was blocked by the hemifusion inhibitor lysophosphatidylcholine (LPC), but not if a complementary-shaped lipid, oleic acid (OA), was simultaneously added, as determined with a novel fluorescent dextran-quenching assay. Importantly, recruitment of the bulk of FG nucleoporins, characteristic of mature nuclear pores, was not observed before diffusion channel formation and was prevented by LPC or OA, but not by LPC+OA. These results map the crucial inner/outer nuclear membrane fusion event of NPC assembly downstream of POM121/Nup107-160 complex interaction and upstream or at the time of FG nucleoporin recruitment.

EVALUATION OF HUMAN NEURAL PROGENITOR CELLS FOR DEVELOPMENTAL NEUROTOXICITY SCREENING: TIME COURSE OF EFFECTS ON CELL PROLIFERATION AND VIABILITY.

EPA Science Inventory

Current testing methods for developmental neurotoxicity (DNT) make evaluation of the effects of large numbers of chemicals impractical and prohibitively expensive. As such, we are evaluating human neural progenitor cells (NPCs) as a screen for DNT. ReNcell CX (ReN CX) cells are a...

Plasticity of an ultrafast interaction between nucleoporins and nuclear transport receptors.

PubMed

Milles, Sigrid; Mercadante, Davide; Aramburu, Iker Valle; Jensen, Malene Ringkjøbing; Banterle, Niccolò; Koehler, Christine; Tyagi, Swati; Clarke, Jane; Shammas, Sarah L; Blackledge, Martin; Gräter, Frauke; Lemke, Edward A

2015-10-22

The mechanisms by which intrinsically disordered proteins engage in rapid and highly selective binding is a subject of considerable interest and represents a central paradigm to nuclear pore complex (NPC) function, where nuclear transport receptors (NTRs) move through the NPC by binding disordered phenylalanine-glycine-rich nucleoporins (FG-Nups). Combining single-molecule fluorescence, molecular simulations, and nuclear magnetic resonance, we show that a rapidly fluctuating FG-Nup populates an ensemble of conformations that are prone to bind NTRs with near diffusion-limited on rates, as shown by stopped-flow kinetic measurements. This is achieved using multiple, minimalistic, low-affinity binding motifs that are in rapid exchange when engaging with the NTR, allowing the FG-Nup to maintain an unexpectedly high plasticity in its bound state. We propose that these exceptional physical characteristics enable a rapid and specific transport mechanism in the physiological context, a notion supported by single molecule in-cell assays on intact NPCs. Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.

Plasticity of an Ultrafast Interaction between Nucleoporins and Nuclear Transport Receptors

PubMed Central

Milles, Sigrid; Mercadante, Davide; Aramburu, Iker Valle; Jensen, Malene Ringkjøbing; Banterle, Niccolò; Koehler, Christine; Tyagi, Swati; Clarke, Jane; Shammas, Sarah L.; Blackledge, Martin; Gräter, Frauke; Lemke, Edward A.

2015-01-01

Summary The mechanisms by which intrinsically disordered proteins engage in rapid and highly selective binding is a subject of considerable interest and represents a central paradigm to nuclear pore complex (NPC) function, where nuclear transport receptors (NTRs) move through the NPC by binding disordered phenylalanine-glycine-rich nucleoporins (FG-Nups). Combining single-molecule fluorescence, molecular simulations, and nuclear magnetic resonance, we show that a rapidly fluctuating FG-Nup populates an ensemble of conformations that are prone to bind NTRs with near diffusion-limited on rates, as shown by stopped-flow kinetic measurements. This is achieved using multiple, minimalistic, low-affinity binding motifs that are in rapid exchange when engaging with the NTR, allowing the FG-Nup to maintain an unexpectedly high plasticity in its bound state. We propose that these exceptional physical characteristics enable a rapid and specific transport mechanism in the physiological context, a notion supported by single molecule in-cell assays on intact NPCs. PMID:26456112

Nucleoporin MOS7/Nup88 contributes to plant immunity and nuclear accumulation of defense regulators.

PubMed

Wiermer, Marcel; Germain, Hugo; Cheng, Yu Ti; García, Ana V; Parker, Jane E; Li, Xin

2010-01-01

Controlled nucleocytoplasmic trafficking is an important feature for fine-tuning signaling pathways in eukaryotic organisms. Nuclear pore complexes (NPCs) composed of nucleoporin proteins (Nups) are essential for the exchange of macromolecules across the nuclear envelope. A recent genetic screen in our laboratory identified a partial loss-of-function mutation in Arabidopsis MOS7/Nup88 that causes defects in basal immunity, Resistance (R) protein-mediated defense and systemic acquired resistance. In Drosophila and mammalian cells, exportin-mediated nuclear export of activated Rel/NFκB transcription factors is enhanced in nup88 mutants resulting in immune response failure. Consistent with Nup88 promoting nuclear retention of NFκB, our functional analyses revealed that MOS7/Nup88 is required for appropriate nuclear accumulation of the autoactivated R protein snc1, as well as the key immune regulators EDS1 and NPR1. These results suggest that controlling the nuclear concentrations of specific immune regulators is fundamental for defining defense outputs.

Investigating molecular crowding within nuclear pores using polarization-PALM

PubMed Central

Fu, Guo; Tu, Li-Chun; Zilman, Anton

2017-01-01

The key component of the nuclear pore complex (NPC) controlling permeability, selectivity, and the speed of nucleocytoplasmic transport is an assembly of natively unfolded polypeptides, which contain phenylalanine-glycine (FG) binding sites for nuclear transport receptors. The architecture and dynamics of the FG-network have been refractory to characterization due to the paucity of experimental methods able to probe the mobility and density of the FG-polypeptides and embedded macromolecules within intact NPCs. Combining fluorescence polarization, super-resolution microscopy, and mathematical analyses, we examined the rotational mobility of fluorescent probes at various locations within the FG-network under different conditions. We demonstrate that polarization PALM (p-PALM) provides a rich source of information about low rotational mobilities that are inaccessible with bulk fluorescence anisotropy approaches, and anticipate that p-PALM is well-suited to explore numerous crowded cellular environments. In total, our findings indicate that the NPC’s internal organization consists of multiple dynamic environments with different local properties. PMID:28949296

The molecular mechanism of nuclear transport revealed by atomic-scale measurements

PubMed Central

Hough, Loren E; Dutta, Kaushik; Sparks, Samuel; Temel, Deniz B; Kamal, Alia; Tetenbaum-Novatt, Jaclyn; Rout, Michael P; Cowburn, David

2015-01-01

Nuclear pore complexes (NPCs) form a selective filter that allows the rapid passage of transport factors (TFs) and their cargoes across the nuclear envelope, while blocking the passage of other macromolecules. Intrinsically disordered proteins (IDPs) containing phenylalanyl-glycyl (FG)-rich repeats line the pore and interact with TFs. However, the reason that transport can be both fast and specific remains undetermined, through lack of atomic-scale information on the behavior of FGs and their interaction with TFs. We used nuclear magnetic resonance spectroscopy to address these issues. We show that FG repeats are highly dynamic IDPs, stabilized by the cellular environment. Fast transport of TFs is supported because the rapid motion of FG motifs allows them to exchange on and off TFs extremely quickly through transient interactions. Because TFs uniquely carry multiple pockets for FG repeats, only they can form the many frequent interactions needed for specific passage between FG repeats to cross the NPC. DOI: http://dx.doi.org/10.7554/eLife.10027.001 PMID:26371551

A physical model describing the interaction of nuclear transport receptors with FG nucleoporin domain assemblies

PubMed Central

Zahn, Raphael; Osmanović, Dino; Ehret, Severin; Araya Callis, Carolina; Frey, Steffen; Stewart, Murray; You, Changjiang; Görlich, Dirk; Hoogenboom, Bart W; Richter, Ralf P

2016-01-01

The permeability barrier of nuclear pore complexes (NPCs) controls bulk nucleocytoplasmic exchange. It consists of nucleoporin domains rich in phenylalanine-glycine motifs (FG domains). As a bottom-up nanoscale model for the permeability barrier, we have used planar films produced with three different end-grafted FG domains, and quantitatively analyzed the binding of two different nuclear transport receptors (NTRs), NTF2 and Importin β, together with the concomitant film thickness changes. NTR binding caused only moderate changes in film thickness; the binding isotherms showed negative cooperativity and could all be mapped onto a single master curve. This universal NTR binding behavior – a key element for the transport selectivity of the NPC – was quantitatively reproduced by a physical model that treats FG domains as regular, flexible polymers, and NTRs as spherical colloids with a homogeneous surface, ignoring the detailed arrangement of interaction sites along FG domains and on the NTR surface. DOI: http://dx.doi.org/10.7554/eLife.14119.001 PMID:27058170

Generation of Neural Progenitor Spheres from Human Pluripotent Stem Cells in a Suspension Bioreactor.

PubMed

Yan, Yuanwei; Song, Liqing; Tsai, Ang-Chen; Ma, Teng; Li, Yan

2016-01-01

Conventional two-dimensional (2-D) culture systems cannot provide large numbers of human pluripotent stem cells (hPSCs) and their derivatives that are demanded for commercial and clinical applications in in vitro drug screening, disease modeling, and potentially cell therapy. The technologies that support three-dimensional (3-D) suspension culture, such as a stirred bioreactor, are generally considered as promising approaches to produce the required cells. Recently, suspension bioreactors have also been used to generate mini-brain-like structure from hPSCs for disease modeling, showing the important role of bioreactor in stem cell culture. This chapter describes a detailed culture protocol for neural commitment of hPSCs into neural progenitor cell (NPC) spheres using a spinner bioreactor. The basic steps to prepare hPSCs for bioreactor inoculation are illustrated from cell thawing to cell propagation. The method for generating NPCs from hPSCs in the spinner bioreactor along with the static control is then described. The protocol in this study can be applied to the generation of NPCs from hPSCs for further neural subtype specification, 3-D neural tissue development, or potential preclinical studies or clinical applications in neurological diseases.

Hyperbranched-polymer dispersed nanocomposite volume gratings for holography and diffractive optics

NASA Astrophysics Data System (ADS)

Tomita, Yasuo; Takeuchi, Shinsuke; Oyaizu, Satoko; Urano, Hiroshi; Fukamizu, Taka-aki; Nishimura, Naoya; Odoi, Keisuke

2016-10-01

We review our experimental investigations of photopolymerizable nanoparticle-polymer composites (NPCs) for holography and diffractive optics. Various types of hyperbranched polymer (HBP) were systhesized and used as transporting organic nanoparticles. These HBPs include hyperbranched poly(ethyl methacrylate) (HPEMA), hyperbranched polystyrene (HPS) and hyperbranched triazine/aromatic polymer units (HTA) whose refractive indices are 1.51, 1.61 and 1.82, respectively. Each HBP was dispersed in (meth)acrylate monomer whose refractive index was so chosen that a refractive index difference between HBP and the formed polymer was large. Such monomer-HBP syrup was mixed with a titanocene photoinitiator for volume holographic recording in the green. We used a two-beam interference setup to write an unslanted transmission volume grating at grating spacing of 1 μm and at a wavelength of 532 nm. It is shown that NPC volume gratings with the saturated refractive index modulation amplitudes as large as 0.008, 0.004 and 0.02 can be recorded in NPCs incorporated with HPEMA, HPS and HTA at their optimum concentrations of 34, 34 and 25 vol.%, respectively. We show the usefulness of HBP-dispersed NPC volume gratings for holographic applications such as holographic data storage and diffractive optical devices.

Comparative transcriptome analysis in induced neural stem cells reveals defined neural cell identities in vitro and after transplantation into the adult rodent brain.

PubMed

Hallmann, Anna-Lena; Araúzo-Bravo, Marcos J; Zerfass, Christina; Senner, Volker; Ehrlich, Marc; Psathaki, Olympia E; Han, Dong Wook; Tapia, Natalia; Zaehres, Holm; Schöler, Hans R; Kuhlmann, Tanja; Hargus, Gunnar

2016-05-01

Reprogramming technology enables the production of neural progenitor cells (NPCs) from somatic cells by direct transdifferentiation. However, little is known on how neural programs in these induced neural stem cells (iNSCs) differ from those of alternative stem cell populations in vitro and in vivo. Here, we performed transcriptome analyses on murine iNSCs in comparison to brain-derived neural stem cells (NSCs) and pluripotent stem cell-derived NPCs, which revealed distinct global, neural, metabolic and cell cycle-associated marks in these populations. iNSCs carried a hindbrain/posterior cell identity, which could be shifted towards caudal, partially to rostral but not towards ventral fates in vitro. iNSCs survived after transplantation into the rodent brain and exhibited in vivo-characteristics, neural and metabolic programs similar to transplanted NSCs. However, iNSCs vastly retained caudal identities demonstrating cell-autonomy of regional programs in vivo. These data could have significant implications for a variety of in vitro- and in vivo-applications using iNSCs. Copyright © 2016 Roslin Cells Ltd. Published by Elsevier B.V. All rights reserved.

Single-cell mechanical phenotype is an intrinsic marker of reprogramming and differentiation along the mouse neural lineage.

PubMed

Urbanska, Marta; Winzi, Maria; Neumann, Katrin; Abuhattum, Shada; Rosendahl, Philipp; Müller, Paul; Taubenberger, Anna; Anastassiadis, Konstantinos; Guck, Jochen

2017-12-01

Cellular reprogramming is a dedifferentiation process during which cells continuously undergo phenotypical remodeling. Although the genetic and biochemical details of this remodeling are fairly well understood, little is known about the change in cell mechanical properties during the process. In this study, we investigated changes in the mechanical phenotype of murine fetal neural progenitor cells (fNPCs) during reprogramming to induced pluripotent stem cells (iPSCs). We find that fNPCs become progressively stiffer en route to pluripotency, and that this stiffening is mirrored by iPSCs becoming more compliant during differentiation towards the neural lineage. Furthermore, we show that the mechanical phenotype of iPSCs is comparable with that of embryonic stem cells. These results suggest that mechanical properties of cells are inherent to their developmental stage. They also reveal that pluripotent cells can differentiate towards a more compliant phenotype, which challenges the view that pluripotent stem cells are less stiff than any cells more advanced developmentally. Finally, our study indicates that the cell mechanical phenotype might be utilized as an inherent biophysical marker of pluripotent stem cells. © 2017. Published by The Company of Biologists Ltd.

Epigallocatechin gallate (EGCG) inhibits adhesion and migration of neural progenitor cells in vitro.

PubMed

Barenys, Marta; Gassmann, Kathrin; Baksmeier, Christine; Heinz, Sabrina; Reverte, Ingrid; Schmuck, Martin; Temme, Thomas; Bendt, Farina; Zschauer, Tim-Christian; Rockel, Thomas Dino; Unfried, Klaus; Wätjen, Wim; Sundaram, Sivaraj Mohana; Heuer, Heike; Colomina, Maria Teresa; Fritsche, Ellen

2017-02-01

Food supplements based on herbal products are widely used during pregnancy as part of a self-care approach. The idea that such supplements are safe and healthy is deeply seated in the general population, although they do not underlie the same strict safety regulations than medical drugs. We aimed to characterize the neurodevelopmental effects of the green tea catechin epigallocatechin gallate (EGCG), which is now commercialized as high-dose food supplement. We used the "Neurosphere Assay" to study the effects and unravel underlying molecular mechanisms of EGCG treatment on human and rat neural progenitor cells (NPCs) development in vitro. EGCG alters human and rat NPC development in vitro. It disturbs migration distance, migration pattern, and nuclear density of NPCs growing as neurospheres. These functional impairments are initiated by EGCG binding to the extracellular matrix glycoprotein laminin, preventing its binding to β1-integrin subunits, thereby prohibiting cell adhesion and resulting in altered glia alignment and decreased number of migrating young neurons. Our data raise a concern on the intake of high-dose EGCG food supplements during pregnancy and highlight the need of an in vivo characterization of the effects of high-dose EGCG exposure during neurodevelopment.

Nasopharyngeal carcinoma heterogeneity of DNA content identified on cytologic preparations.

PubMed

Maohuai, C; Chang, A R; Lo, D

2001-06-01

To evaluate tumor heterogeneity of DNA content in nasopharyngeal carcinoma (NPC) performed on cytologic specimens. Image cytometric analysis of DNA ploidy status of 40 NPCs was performed on nasopharyngeal brushing smears stained with the Feulgen method after hematoxylin eosin staining. If the DNA distribution pattern from the same tumor exhibited diploid, aneuploid or/and tetraploid peaks or some combination of these patterns, the presence of tumor heterogeneity of DNA content was identified. Thirty-four cases (85%) had a nondiploid DNA pattern among the 40 NPCs. Twenty-eight cases exhibited tumor heterogeneity of DNA content (70%). Of the 28 tumors, 13 (46%) had a combination of diploid and tetraploid patterns, 10 (37%) had a combination of diploid and aneuploid patterns, 3 cases (11%) had a combination of tetraploid and aneuploid patterns, and 2 cases had two aneuploid stem lines. The relationship between DNA ploidy pattern and tumor histologic and cytologic morphology was also examined. There is a high incidence of DNA content heterogeneity in NPC. The relevance of tumor heterogeneity to the biologic behavior of NPC awaits further study. DNA quantification with image cytometry on destained cytologic preparations is feasible and reliable.

The Immune Adaptor SLP-76 Binds to SUMO-RANGAP1 at Nuclear Pore Complex Filaments to Regulate Nuclear Import of Transcription Factors in T Cells

PubMed Central

Liu, Hebin; Schneider, Helga; Recino, Asha; Richardson, Christine; Goldberg, Martin W.; Rudd, Christopher E.

2015-01-01

Summary While immune cell adaptors regulate proximal T cell signaling, direct regulation of the nuclear pore complex (NPC) has not been reported. NPC has cytoplasmic filaments composed of RanGAP1 and RanBP2 with the potential to interact with cytoplasmic mediators. Here, we show that the immune cell adaptor SLP-76 binds directly to SUMO-RanGAP1 of cytoplasmic fibrils of the NPC, and that this interaction is needed for optimal NFATc1 and NF-κB p65 nuclear entry in T cells. Transmission electron microscopy showed anti-SLP-76 cytoplasmic labeling of the majority of NPCs in anti-CD3 activated T cells. Further, SUMO-RanGAP1 bound to the N-terminal lysine 56 of SLP-76 where the interaction was needed for optimal RanGAP1-NPC localization and GAP exchange activity. While the SLP-76-RanGAP1 (K56E) mutant had no effect on proximal signaling, it impaired NF-ATc1 and p65/RelA nuclear entry and in vivo responses to OVA peptide. Overall, we have identified SLP-76 as a direct regulator of nuclear pore function in T cells. PMID:26321253

The Immune Adaptor SLP-76 Binds to SUMO-RANGAP1 at Nuclear Pore Complex Filaments to Regulate Nuclear Import of Transcription Factors in T Cells.

PubMed

Liu, Hebin; Schneider, Helga; Recino, Asha; Richardson, Christine; Goldberg, Martin W; Rudd, Christopher E

2015-09-03

While immune cell adaptors regulate proximal T cell signaling, direct regulation of the nuclear pore complex (NPC) has not been reported. NPC has cytoplasmic filaments composed of RanGAP1 and RanBP2 with the potential to interact with cytoplasmic mediators. Here, we show that the immune cell adaptor SLP-76 binds directly to SUMO-RanGAP1 of cytoplasmic fibrils of the NPC, and that this interaction is needed for optimal NFATc1 and NF-κB p65 nuclear entry in T cells. Transmission electron microscopy showed anti-SLP-76 cytoplasmic labeling of the majority of NPCs in anti-CD3 activated T cells. Further, SUMO-RanGAP1 bound to the N-terminal lysine 56 of SLP-76 where the interaction was needed for optimal RanGAP1-NPC localization and GAP exchange activity. While the SLP-76-RanGAP1 (K56E) mutant had no effect on proximal signaling, it impaired NF-ATc1 and p65/RelA nuclear entry and in vivo responses to OVA peptide. Overall, we have identified SLP-76 as a direct regulator of nuclear pore function in T cells. Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.

Aspects of nuclear envelope dynamics in mitotic cells.

PubMed

Burke, Brian; Shanahan, Catherine; Salina, Davide; Crisp, Melissa

2005-01-01

Major features of the nuclear envelope (NE) are a pair of inner and outer nuclear membranes (INM, ONM) spanned by nuclear pore complexes. While the composition of the ONM resembles that of the endoplasmic reticulum, the INM contains a unique spectrum of proteins. Localization of INM proteins involves a mechanism of selective retention whereby integral proteins are immobilized and concentrated by virtue of interactions with nuclear components. In the case of emerin, INM localization involves interaction with A-type lamins. Interactions between membrane proteins may also play a significant role in INM localization. This conclusion stems from studies on nesprins, a family of membrane proteins that feature a large cytoplasmic domain, a single C-terminal membrane-spanning domain and a small lumenal domain. The nesprin membrane anchor and lumenal (KASH) domains are related to the Drosophila Klarsicht protein. Evidence is emerging that this KASH region interacts with other NE proteins and may influence their distributions. Overexpression of GFP-KASH causes loss of emerin and LAP2 from the NE. This is not due to global reorganization of the NE since LAP1 as well as lamins and NPCs remain unaffected. Our results suggest that interactions between NE membrane components are far more extensive and complex than current models suggest.

Neuroinflammatory Dynamics Underlie Memory Impairments after Repeated Social Defeat

PubMed Central

McKim, Daniel B.; Niraula, Anzela; Tarr, Andrew J.; Wohleb, Eric S.

2016-01-01

Repeated social defeat (RSD) is a murine stressor that recapitulates key physiological, immunological, and behavioral alterations observed in humans exposed to chronic psychosocial stress. Psychosocial stress promotes prolonged behavioral adaptations that are associated with neuroinflammatory signaling and impaired neuroplasticity. Here, we show that RSD promoted hippocampal neuroinflammatory activation that was characterized by proinflammatory gene expression and by microglia activation and monocyte trafficking that was particularly pronounced within the caudal extent of the hippocampus. Because the hippocampus is a key area involved in neuroplasticity, behavior, and cognition, we hypothesize that stress-induced neuroinflammation impairs hippocampal neurogenesis and promotes cognitive and affective behavioral deficits. We show here that RSD caused transient impairments in spatial memory recall that resolved within 28 d. In assessment of neurogenesis, the number of proliferating neural progenitor cells (NPCs) and the number of young, developing neurons were not affected initially after RSD. Nonetheless, the neuronal differentiation of NPCs that proliferated during RSD was significantly impaired when examined 10 and 28 d later. In addition, social avoidance, a measure of depressive-like behavior associated with caudal hippocampal circuitry, persisted 28 d after RSD. Treatment with minocycline during RSD prevented both microglia activation and monocyte recruitment. Inhibition of this neuroinflammatory activation in turn prevented impairments in spatial memory after RSD but did not prevent deficits in neurogenesis nor did it prevent the persistence of social avoidance behavior. These findings show that neuroinflammatory activation after psychosocial stress impairs spatial memory performance independent of deficits in neurogenesis and social avoidance. SIGNIFICANCE STATEMENT Repeated exposure to stress alters the homeostatic environment of the brain, giving rise to various cognitive and mood disorders that impair everyday functioning and overall quality of life. The brain, previously thought of as an immune-privileged organ, is now known to communicate extensively with the peripheral immune system. This brain–body communication plays a significant role in various stress-induced inflammatory conditions, also characterized by psychological impairments. Findings from this study implicate neuroimmune activation rather than impaired neurogenesis in stress-induced cognitive deficits. This idea opens up possibilities for novel immune interventions in the treatment of cognitive and mood disturbances, while also adding to the complexity surrounding the functional implications of adult neurogenesis. PMID:26937001

Putative members of the Arabidopsis Nup107-160 nuclear pore sub-complex contribute to pathogen defense.

PubMed

Wiermer, Marcel; Cheng, Yu Ti; Imkampe, Julia; Li, Meilan; Wang, Dongmei; Lipka, Volker; Li, Xin

2012-06-01

In eukaryotic cells, transduction of external stimuli into the nucleus to induce transcription and export of mRNAs for translation in the cytoplasm is mediated by nuclear pore complexes (NPCs) composed of nucleoporin proteins (Nups). We previously reported that Arabidopsis MOS3, encoding the homolog of vertebrate Nup96, is required for plant immunity and constitutive resistance mediated by the de-regulated Toll interleukin 1 receptor/nucleotide-binding/leucine-rich repeat (TNL)-type R gene snc1. In vertebrates, Nup96 is a component of the conserved Nup107-160 nuclear pore sub-complex, and implicated in immunity-related mRNA export. Here, we used a reverse genetics approach to examine the requirement for additional subunits of the predicted Arabidopsis Nup107-160 complex in plant immunity. We show that, among eight putative complex members, beside MOS3, only plants with defects in Nup160 or Seh1 are impaired in basal resistance. Constitutive resistance in the snc1 mutant and immunity mediated by TNL-type R genes also depend on functional Nup160 and have a partial requirement for Seh1. Conversely, resistance conferred by coiled coil-type immune receptors operates largely independently of both genes, demonstrating specific contributions to plant defense signaling. Our functional analysis further revealed that defects in nup160 and seh1 result in nuclear accumulation of poly(A) mRNA, and, in the case of nup160, considerable depletion of EDS1, a key positive regulator of basal and TNL-triggered resistance. These findings suggest that Nup160 is required for nuclear mRNA export and full expression of EDS1-conditioned resistance pathways in Arabidopsis. © 2012 The Authors. The Plant Journal © 2012 Blackwell Publishing Ltd.

Before We're Up to Our Necks in Aggregators, Let's Get out Our Net "Value" Calculators!

ERIC Educational Resources Information Center

Butler, Lawrence

2012-01-01

No sooner has the Net Price Calculator (NPC) wave crashed ashore, the next wave of college-choice transparency in the form of third-party data aggregators is threatening to engulf American colleges and universities. Since last October, NPCs have become a fact of life for American colleges and universities. Some are doing the bare minimum to comply…

Functional self-assembled peptide scaffold inhibits tumor necrosis factor-alpha-induced inflammation and apoptosis in nucleus pulposus cells by suppressing nuclear factor-κB signaling.

PubMed

Li, Xiaochuan; Cheng, Shi; Wu, Yaohong; Ying, Jingwei; Wang, Chaofeng; Wen, Tianyong; Bai, Xuedong; Ji, Wei; Wang, Deli; Ruan, Dike

2018-04-01

Although nucleus pulposus (NP) tissue engineering has achieved tremendous success, researches still face the huge obstacles in maintaining cell survival and function. A novel functional self-assembled peptide RADA-KPSS was constructed by conjugating BMP-7 short active fragment (KPSS) to the C-terminus of RADA16-I that displays anti-inflammatory and anti-apoptosis effects. However, whether this functional self-assembled RADA-KPSS peptide can alleviate inflammation and NPC apoptosis induced by tumor necrosis factor-alpha (TNF-α) has not been studied. Therefore, we cultured NPCs treated with TNF-α for 48 h with the RADA-KPSS peptide, and compared the results to those with RADA16-I peptide. The cell apoptosis rate, inflammatory mediator secretion, expression of matrix-degrading enzymes, and extracellular matrix (ECM) protein levels were evaluated. The expression of nuclear factor-κB-p65 (NF-κB-p65) protein was also tested. TNF-α-treated NPCs cultured with the RADA16-I peptide showed up-regulated gene expression for matrix-degrading enzymes, such as matrix metalloproteinases-3 (MMP-3), MMP-9, and a disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS-4), and down-regulated gene expression for ECM proteins such as aggrecan, collagen II, and Sox-9. The RADA-KPSS peptide could attenuate the expression of MMP-3, MMP-9, and ADAMTS-4, promote accumulation of ECM proteins, and increase secretion of glycosaminoglycan as compared with the RADA16-I peptide. Moreover, the TNF-α-damaged NPCs was further demonstrated to inhibit NF-κB-p65, IL-1, IL-6, and prostaglandin E-2 proteins and decrease cell apoptosis in RADA-KPSS peptide. In conclusion, the functional self-assembled RADA-KPSS peptides have anti-inflammatory and anti-apoptotic effects by promoting anabolic processes and inhibiting catabolic processes in intervertebral disk degeneration. These peptides may be feasible for clinical applications in NP tissue engineering. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 106A: 1082-1091, 2018. © 2017 Wiley Periodicals, Inc.

Overcoming the Practical Barriers to Spinal Cord Cell Transplantation for ALS

DTIC Science & Technology

2013-10-01

not be neglected. Moreover, escalating numbers and volumes of injections seem to be associated with lack of accuracy and reflux . Histological...with intact segments. Histological analysis will also determine whether reflux occurs with volume escalation as well as with fast (hand-held...analysis of reflux and transient morbidity with number and volume of injection of hNPCs (Boulis). Create a cell bank of astrocyte restricted

Co-Administration of an Excipient Oligonucleotide Helps Delineate Pathways of Productive and Nonproductive Uptake of Phosphorothioate Antisense Oligonucleotides in the Liver.

PubMed

Donner, Aaron J; Wancewicz, Edward V; Murray, Heather M; Greenlee, Sarah; Post, Noah; Bell, Melanie; Lima, Walt F; Swayze, Eric E; Seth, Punit P

2017-08-01

Phosphorothioate (PS) modified antisense oligonucleotides (ASOs) have progressed rapidly in the clinic for treating a variety of disease indications. We previously demonstrated that the activity of PS ASOs in the liver can be enhanced by co-infusion of an excipient oligonucleotide (EON). It was posited that the EON saturates a nonproductive uptake pathway(s) thereby permitting accumulation of the PS ASO in a productive tissue compartment. In this report, we measured PS ASO activity following administration by bolus, infusion or co-fusion with EON within hepatocytes and nonparenchymal cells (NPCs), of the liver. This revealed that while ASOs accumulate preferentially in NPCs, they are intrinsically more active in hepatocytes. Furthermore, we show that the EON enhances ASO potency when infused up to 72 h before or after administration of the active ASO suggesting that the EON can saturate and displace the ASO from nonproductive to productive compartments. Physical presence of the EON in tissues was required for optimal potentiation suggesting that there is a dynamic distribution of the ASO and EON between the compartments. Lastly, using a candidate approach, we confirmed Stabilin-2 as a molecular pathway for ASO uptake in sinusoidal endothelial cells and the ASGR as a pathway for ASO uptake into hepatocytes in the liver.

A Host-Configured Lithium-Sulfur Cell Built on 3D Nickel Photonic Crystal with Superior Electrochemical Performances.

PubMed

Lin, Shengxuan; Yan, Yang; Cai, Zihe; Liu, Lin; Hu, Xiaobin

2018-04-18

The insulator of the sulfur cathode and the easy dendrites growth of the lithium anode are the main barriers for lithium-sulfur cells in commercial application. Here, a 3D NPC@S/3D NPC@Li full cell is reported based on 3D hierarchical and continuously porous nickel photonic crystal (NPC) to solve the problems of sulfur cathode and lithium anode at the same time. In this case, the 3D NPC@S cathode can not only offer a fast transfer of electron and lithium ion, but also effectively prevent the dissolution of polysulfides and the tremendous volume change during cycling, and the 3D NPC@Li anode can efficiently inhibit the growth of lithium dendrites and volume expansion, too. As a result, the cell exhibits a high reversible capacity of 1383 mAh g -1 at 0.5 C (the current density of 837 mA g -1 ), superior rate ability (the reversible capacity of 735 mAh g -1 at the extremely high current density of 16 750 mA g -1 ) with excellent coulombic efficiency of about 100% and an excellent cycle life over 500 cycles with only about 0.026% capacity loss per cycle. © 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Electro-acupuncture exerts beneficial effects against cerebral ischemia and promotes the proliferation of neural progenitor cells in the cortical peri-infarct area through the Wnt/β-catenin signaling pathway

PubMed Central

CHEN, BIN; TAO, JING; LIN, YUKUN; LIN, RUHUI; LIU, WEILIN; CHEN, LIDIAN

2015-01-01

Electro-acupuncture (EA) is a novel therapy based on combining traditional acupuncture with modern electrotherapy, and it is currently being investigated as a treatment for ischemic stroke. In the present study, we aimed to investigate the mechanisms through which EA regulates the proliferation of neural progenitor cells (NPCs) in the cortical peri-infarct area after stroke. The neuroprotective effects of EA on ischemic rats were evaluated by determining the neurological deficit scores and cerebral infarct volumes. The proliferation of the NPCs and the activation of the Wnt/β-catenin signaling pathway in the cortical peri-infarct area were examined. Our results revealed that EA significantly alleviated neurological deficits, reduced the infarct volume and enhanced NPC proliferation [nestin/glial fibrillary acidic protein (GFAP)-double positive] in the cortex of rats subjected to middle cerebral artery occlusion (MCAO). Moreover, the Wnt1 and β-catenin mRNA and protein levels were increased, while glycogen synthase kinase-3 (GSK3) transcription was suppressed by EA. These results suggest that the upregulatory effects of EA on the Wnt/β-catenin signaling pathway may promote NPC proliferation in the cortical peri-infarct area after stroke, consequently providing a therapeutic effect against cerebral ischemia. PMID:26329606

Effects of Chronic Low-Dose Radiation on Human Neural Progenitor Cells

NASA Astrophysics Data System (ADS)

Katsura, Mari; Cyou-Nakamine, Hiromasa; Zen, Qin; Zen, Yang; Nansai, Hiroko; Amagasa, Shota; Kanki, Yasuharu; Inoue, Tsuyoshi; Kaneki, Kiyomi; Taguchi, Akashi; Kobayashi, Mika; Kaji, Toshiyuki; Kodama, Tatsuhiko; Miyagawa, Kiyoshi; Wada, Youichiro; Akimitsu, Nobuyoshi; Sone, Hideko

2016-01-01

The effects of chronic low-dose radiation on human health have not been well established. Recent studies have revealed that neural progenitor cells are present not only in the fetal brain but also in the adult brain. Since immature cells are generally more radiosensitive, here we investigated the effects of chronic low-dose radiation on cultured human neural progenitor cells (hNPCs) derived from embryonic stem cells. Radiation at low doses of 31, 124 and 496 mGy per 72 h was administered to hNPCs. The effects were estimated by gene expression profiling with microarray analysis as well as morphological analysis. Gene expression was dose-dependently changed by radiation. By thirty-one mGy of radiation, inflammatory pathways involving interferon signaling and cell junctions were altered. DNA repair and cell adhesion molecules were affected by 124 mGy of radiation while DNA synthesis, apoptosis, metabolism, and neural differentiation were all affected by 496 mGy of radiation. These in vitro results suggest that 496 mGy radiation affects the development of neuronal progenitor cells while altered gene expression was observed at a radiation dose lower than 100 mGy. This study would contribute to the elucidation of the clinical and subclinical phenotypes of impaired neuronal development induced by chronic low-dose radiation.

Debt, demographics, and dual degrees: American medicine at the crossroads: part 3: A paradigm shift or a return to the basics?

PubMed

Ellerin, Bruce E

2007-08-01

An alternative solution to the predicted physician workforce shortage would be the incorporation of nonphysician caregivers (NPCs) into the primary care workforce under the supervision of a limited number of internists, pediatricians, and family practitioners, thus freeing medical students and residents who currently enter primary care medicine for work in medical and surgical specialties in which there are current shortages that require specific medical training beyond the scope of NPCs' competencies. At the same time, the profession should follow the lead of the multidisciplinary ethic of contemporary natural science, in which collaboration among disciplines has become increasingly crucial for high-level research, by creating a training pathway, on the model of the Medical Science Training Program, for dual-degree physicians who seek to combine their medical expertise with training in the social sciences and the humanities. In addition to recognizing and rewarding an existing and growing trend within medical education, the creation of a cadre of dual-degree experts with access to centers of power and influence in law, business, government, and the media could create the nucleus of a medically trained intellectual elite that would be in a better position in the future to advocate for physician interests in crucial centers of power.

Engineered LINE-1 retrotransposition in nondividing human neurons

PubMed Central

Macia, Angela; Widmann, Thomas J.; Heras, Sara R.; Ayllon, Veronica; Sanchez, Laura; Benkaddour-Boumzaouad, Meriem; Muñoz-Lopez, Martin; Rubio, Alejandro; Amador-Cubero, Suyapa; Blanco-Jimenez, Eva; Garcia-Castro, Javier; Menendez, Pablo; Ng, Philip; Muotri, Alysson R.; Goodier, John L.; Garcia-Perez, Jose L.

2017-01-01

Half the human genome is made of transposable elements (TEs), whose ongoing activity continues to impact our genome. LINE-1 (or L1) is an autonomous non-LTR retrotransposon in the human genome, comprising 17% of its genomic mass and containing an average of 80–100 active L1s per average genome that provide a source of inter-individual variation. New LINE-1 insertions are thought to accumulate mostly during human embryogenesis. Surprisingly, the activity of L1s can further impact the somatic human brain genome. However, it is currently unknown whether L1 can retrotranspose in other somatic healthy tissues or if L1 mobilization is restricted to neuronal precursor cells (NPCs) in the human brain. Here, we took advantage of an engineered L1 retrotransposition assay to analyze L1 mobilization rates in human mesenchymal (MSCs) and hematopoietic (HSCs) somatic stem cells. Notably, we have observed that L1 expression and engineered retrotransposition is much lower in both MSCs and HSCs when compared to NPCs. Remarkably, we have further demonstrated for the first time that engineered L1s can retrotranspose efficiently in mature nondividing neuronal cells. Thus, these findings suggest that the degree of somatic mosaicism and the impact of L1 retrotransposition in the human brain is likely much higher than previously thought. PMID:27965292

The role of cannabinoids in adult neurogenesis

PubMed Central

Prenderville, Jack A; Kelly, Áine M; Downer, Eric J

2015-01-01

The processes underpinning post-developmental neurogenesis in the mammalian brain continue to be defined. Such processes involve the proliferation of neural stem cells and neural progenitor cells (NPCs), neuronal migration, differentiation and integration into a network of functional synapses within the brain. Both intrinsic (cell signalling cascades) and extrinsic (neurotrophins, neurotransmitters, cytokines, hormones) signalling molecules are intimately associated with adult neurogenesis and largely dictate the proliferative activity and differentiation capacity of neural cells. Cannabinoids are a unique class of chemical compounds incorporating plant-derived cannabinoids (the active components of Cannabis sativa), the endogenous cannabinoids and synthetic cannabinoid ligands, and these compounds are becoming increasingly recognized for their roles in neural developmental processes. Indeed, cannabinoids have clear modulatory roles in adult neurogenesis, probably through activation of both CB1 and CB2 receptors. In recent years, a large body of literature has deciphered the signalling networks involved in cannabinoid-mediated regulation of neurogenesis. This timely review summarizes the evidence that the cannabinoid system is intricately associated with neuronal differentiation and maturation of NPCs and highlights intrinsic/extrinsic signalling mechanisms that are cannabinoid targets. Overall, these findings identify the central role of the cannabinoid system in adult neurogenesis in the hippocampus and the lateral ventricles and hence provide insight into the processes underlying post-developmental neurogenesis in the mammalian brain. PMID:25951750

The role of neural precursor cells and self assembling peptides in nerve regeneration

PubMed Central

2013-01-01

Objective Cranial nerve injury involves loss of central neural cells in the brain stem and surrounding support matrix, leading to severe functional impairment. Therapeutically targeting cellular replacement and enhancing structural support may promote neural regeneration. We examined the combinatorial effect of neural precursor cells (NPC) and self assembling peptide (SAP) administration on nerve regeneration. Methods Nerve injury was induced by clip compression of the rodent spinal cord. SAPs were injected immediately into the injured cord and NPCs at 2 weeks post-injury. Behavioral analysis was done weekly and rats were sacrificed at 11 weeks post injury. LFB-H&E staining was done on cord tissue to assess cavitation volume. Motor evoked potentials (MEP) were measured at week 11 to assess nerve conduction and Kaplan meier curves were created to compare survival estimates. Results NPCs and SAPs were distributed both caudal and rostral to the injury site. Behavioral analysis showed that SAP + NPC transplantation significantly improved locomotor score p <0.03) and enhanced survival (log rank test, p = 0.008) compared to control. SAP + NPC treatment also improved nerve conduction velocity (p = 0.008) but did not affect cavitation volume (p = 0.73). Conclusion Combinatorial NPC and SAP injection into injured nerve tissue may enhance neural repair and regeneration. PMID:24351041

The role of neural precursor cells and self assembling peptides in nerve regeneration.

PubMed

Zhao, Xiao; Yao, Gordon S; Liu, Yang; Wang, Jian; Satkunendrarajah, Kajana; Fehlings, Michael

2013-12-19

Cranial nerve injury involves loss of central neural cells in the brain stem and surrounding support matrix, leading to severe functional impairment. Therapeutically targeting cellular replacement and enhancing structural support may promote neural regeneration. We examined the combinatorial effect of neural precursor cells (NPC) and self assembling peptide (SAP) administration on nerve regeneration. Nerve injury was induced by clip compression of the rodent spinal cord. SAPs were injected immediately into the injured cord and NPCs at 2 weeks post-injury. Behavioral analysis was done weekly and rats were sacrificed at 11 weeks post injury. LFB-H&E staining was done on cord tissue to assess cavitation volume. Motor evoked potentials (MEP) were measured at week 11 to assess nerve conduction and Kaplan Meier curves were created to compare survival estimates. NPCs and SAPs were distributed both caudal and rostral to the injury site. Behavioral analysis showed that SAP + NPC transplantation significantly improved locomotor score p <0.03) and enhanced survival (log rank test, p = 0.008) compared to control. SAP + NPC treatment also improved nerve conduction velocity (p = 0.008) but did not affect cavitation volume (p = 0.73). Combinatorial NPC and SAP injection into injured nerve tissue may enhance neural repair and regeneration.

A microfluidically perfused three dimensional human liver model.

PubMed

Rennert, Knut; Steinborn, Sandra; Gröger, Marko; Ungerböck, Birgit; Jank, Anne-Marie; Ehgartner, Josef; Nietzsche, Sandor; Dinger, Julia; Kiehntopf, Michael; Funke, Harald; Peters, Frank T; Lupp, Amelie; Gärtner, Claudia; Mayr, Torsten; Bauer, Michael; Huber, Otmar; Mosig, Alexander S

2015-12-01

Within the liver, non-parenchymal cells (NPCs) are critically involved in the regulation of hepatocyte polarization and maintenance of metabolic function. We here report the establishment of a liver organoid that integrates NPCs in a vascular layer composed of endothelial cells and tissue macrophages and a hepatic layer comprising stellate cells co-cultured with hepatocytes. The three-dimensional liver organoid is embedded in a microfluidically perfused biochip that enables sufficient nutrition supply and resembles morphological aspects of the human liver sinusoid. It utilizes a suspended membrane as a cell substrate mimicking the space of Disse. Luminescence-based sensor spots were integrated into the chip to allow online measurement of cellular oxygen consumption. Application of microfluidic flow induces defined expression of ZO-1, transferrin, ASGPR-1 along with an increased expression of MRP-2 transporter protein within the liver organoids. Moreover, perfusion was accompanied by an increased hepatobiliary secretion of 5(6)-carboxy-2',7'-dichlorofluorescein and an enhanced formation of hepatocyte microvilli. From this we conclude that the perfused liver organoid shares relevant morphological and functional characteristics with the human liver and represents a new in vitro research tool to study human hepatocellular physiology at the cellular level under conditions close to the physiological situation. Copyright © 2015 Elsevier Ltd. All rights reserved.

A PML/Slit Axis Controls Physiological Cell Migration and Cancer Invasion in the CNS.

PubMed

Amodeo, Valeria; A, Deli; Betts, Joanne; Bartesaghi, Stefano; Zhang, Ying; Richard-Londt, Angela; Ellis, Matthew; Roshani, Rozita; Vouri, Mikaella; Galavotti, Sara; Oberndorfer, Sarah; Leite, Ana Paula; Mackay, Alan; Lampada, Aikaterini; Stratford, Eva Wessel; Li, Ningning; Dinsdale, David; Grimwade, David; Jones, Chris; Nicotera, Pierluigi; Michod, David; Brandner, Sebastian; Salomoni, Paolo

2017-07-11

Cell migration through the brain parenchyma underpins neurogenesis and glioblastoma (GBM) development. Since GBM cells and neuroblasts use the same migratory routes, mechanisms underlying migration during neurogenesis and brain cancer pathogenesis may be similar. Here, we identify a common pathway controlling cell migration in normal and neoplastic cells in the CNS. The nuclear scaffold protein promyelocytic leukemia (PML), a regulator of forebrain development, promotes neural progenitor/stem cell (NPC) and neuroblast migration in the adult mouse brain. The PML pro-migratory role is active also in transformed mouse NPCs and in human primary GBM cells. In both normal and neoplastic settings, PML controls cell migration via Polycomb repressive complex 2 (PRC2)-mediated repression of Slits, key regulators of axon guidance. Finally, a PML/SLIT1 axis regulates sensitivity to the PML-targeting drug arsenic trioxide in primary GBM cells. Taken together, these findings uncover a drug-targetable molecular axis controlling cell migration in both normal and neoplastic cells. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

Diffusion and retention are major determinants of protein targeting to the inner nuclear membrane

PubMed Central

Ungricht, Rosemarie; Klann, Michael; Horvath, Peter

2015-01-01

Newly synthesized membrane proteins are constantly sorted from the endoplasmic reticulum (ER) to various membranous compartments. How proteins specifically enrich at the inner nuclear membrane (INM) is not well understood. We have established a visual in vitro assay to measure kinetics and investigate requirements of protein targeting to the INM. Using human LBR, SUN2, and LAP2β as model substrates, we show that INM targeting is energy-dependent but distinct from import of soluble cargo. Accumulation of proteins at the INM relies on both a highly interconnected ER network, which is affected by energy depletion, and an efficient immobilization step at the INM. Nucleoporin depletions suggest that translocation through nuclear pore complexes (NPCs) is rate-limiting and restricted by the central NPC scaffold. Our experimental data combined with mathematical modeling support a diffusion-retention–based mechanism of INM targeting. We experimentally confirmed the sufficiency of diffusion and retention using an artificial reporter lacking natural sorting signals that recapitulates the energy dependence of the process in vivo. PMID:26056139

Actin-Cytoskeleton- and Rock-Mediated INM Are Required for Photoreceptor Regeneration in the Adult Zebrafish Retina

PubMed Central

Lahne, Manuela; Li, Jingling; Marton, Rebecca M.

2015-01-01

Loss of retinal neurons in adult zebrafish (Danio rerio) induces a robust regenerative response mediated by the reentry of the resident Müller glia into the cell cycle. Upon initiating Müller glia proliferation, their nuclei migrate along the apicobasal axis of the retina in phase with the cell cycle in a process termed interkinetic nuclear migration (INM). We examined the mechanisms governing this cellular process and explored its function in regenerating the adult zebrafish retina. Live-cell imaging revealed that the majority of Müller glia nuclei migrated to the outer nuclear layer (ONL) to divide. These Müller glia formed prominent actin filaments at the rear of nuclei that had migrated to the ONL. Inhibiting actin filament formation or Rho-associated coiled-coil kinase (Rock) activity, which is necessary for phosphorylation of myosin light chain and actin myosin-mediated contraction, disrupted INM with increased numbers of mitotic nuclei remaining in the basal inner nuclear layer, the region where Müller glia typically reside. Double knockdown of Rho-associated coiled-coil kinase 2a (Rock2a) and Rho-associated coiled-coil kinase 2b (Rock2b) similarly disrupted INM and reduced Müller glial cell cycle reentry. In contrast, Rock inhibition immediately before the onset of INM did not affect Müller glia proliferation, but subsequently reduced neuronal progenitor cell proliferation due to early cell cycle exit. Long-term, Rock inhibition increased the generation of mislocalized ganglion/amacrine cells at the expense of rod and cone photoreceptors. In summary, INM is driven by an actin-myosin-mediated process controlled by Rock2a and Rock2b activity, which is required for sufficient proliferation and regeneration of photoreceptors after light damage. SIGNIFICANCE STATEMENT The human retina does not replace lost or damaged neurons, ultimately causing vision impairment. In contrast, zebrafish are capable of regenerating lost neurons. Understanding the mechanisms that regulate retinal regeneration in these organisms will help to elucidate approaches to stimulate a similar response in humans. In the damaged zebrafish retina, Müller glia dedifferentiate and proliferate to generate neuronal progenitor cells (NPCs) that differentiate into the lost neurons. We show that the nuclei of Müller glia and NPCs migrate apically and basally in phase with the cell cycle. This migration is facilitated by the actin cytoskeleton and Rho-associated coiled-coil kinases (Rocks). We demonstrate that Rock function is required for sufficient proliferation and the regeneration of photoreceptors, likely via regulating nuclear migration. PMID:26609156

Actin-Cytoskeleton- and Rock-Mediated INM Are Required for Photoreceptor Regeneration in the Adult Zebrafish Retina.

PubMed

Lahne, Manuela; Li, Jingling; Marton, Rebecca M; Hyde, David R

2015-11-25

Loss of retinal neurons in adult zebrafish (Danio rerio) induces a robust regenerative response mediated by the reentry of the resident Müller glia into the cell cycle. Upon initiating Müller glia proliferation, their nuclei migrate along the apicobasal axis of the retina in phase with the cell cycle in a process termed interkinetic nuclear migration (INM). We examined the mechanisms governing this cellular process and explored its function in regenerating the adult zebrafish retina. Live-cell imaging revealed that the majority of Müller glia nuclei migrated to the outer nuclear layer (ONL) to divide. These Müller glia formed prominent actin filaments at the rear of nuclei that had migrated to the ONL. Inhibiting actin filament formation or Rho-associated coiled-coil kinase (Rock) activity, which is necessary for phosphorylation of myosin light chain and actin myosin-mediated contraction, disrupted INM with increased numbers of mitotic nuclei remaining in the basal inner nuclear layer, the region where Müller glia typically reside. Double knockdown of Rho-associated coiled-coil kinase 2a (Rock2a) and Rho-associated coiled-coil kinase 2b (Rock2b) similarly disrupted INM and reduced Müller glial cell cycle reentry. In contrast, Rock inhibition immediately before the onset of INM did not affect Müller glia proliferation, but subsequently reduced neuronal progenitor cell proliferation due to early cell cycle exit. Long-term, Rock inhibition increased the generation of mislocalized ganglion/amacrine cells at the expense of rod and cone photoreceptors. In summary, INM is driven by an actin-myosin-mediated process controlled by Rock2a and Rock2b activity, which is required for sufficient proliferation and regeneration of photoreceptors after light damage. The human retina does not replace lost or damaged neurons, ultimately causing vision impairment. In contrast, zebrafish are capable of regenerating lost neurons. Understanding the mechanisms that regulate retinal regeneration in these organisms will help to elucidate approaches to stimulate a similar response in humans. In the damaged zebrafish retina, Müller glia dedifferentiate and proliferate to generate neuronal progenitor cells (NPCs) that differentiate into the lost neurons. We show that the nuclei of Müller glia and NPCs migrate apically and basally in phase with the cell cycle. This migration is facilitated by the actin cytoskeleton and Rho-associated coiled-coil kinases (Rocks). We demonstrate that Rock function is required for sufficient proliferation and the regeneration of photoreceptors, likely via regulating nuclear migration. Copyright © 2015 the authors 0270-6474/15/3515612-23$15.00/0.

Potential Role of Activated Nonparenchymal Cells in Acetaminophen-Induced Potentiation of Hepatotoxicity

DTIC Science & Technology

1991-06-14

ALT is either being degraded or the activity is inhibited by something in the 133 media. AST activity in cocultures of NPCs and hepatocytes was... Paracetamol Hepatotoxicity: IN VITRO Studies in Isolated Mouse Hepatocytes. Toxicology Letters. 2229: 37-48. Casini, A. M., P. A. Ferrali and M...Acute Liver Necrosis Following Overdose of Paracetamol . British Medical Journal. 2: 497-499. Decker, T., M. L. Lohmann-Matthes, U. Karck, T. Peters

Task shifting to non-physician clinicians for integrated management of hypertension and diabetes in rural Cameroon: a programme assessment at two years

PubMed Central

2010-01-01

Background The burden of non-communicable chronic diseases, such as hypertension and diabetes, increases in sub-Saharan Africa. However, the majority of the rural population does still not have access to adequate care. The objective of this study is to examine the effectiveness of integrating care for hypertension and type 2 diabetes by task shifting to non-physician clinician (NPC) facilities in eight rural health districts in Cameroon. Methods Of the 75 NPC facilities in the area, 69 (87%) received basic equipment and training in hypertension and diabetes care. Effectiveness was assessed after two years on status of equipment, knowledge among trained NPCs, number of newly detected patients, retention of patients under care, treatment cost to patients and changes in blood pressure (BP) and fasting plasma glucose (FPG) among treated patients. Results Two years into the programme, of 54 facilities (78%) available for re-assessment, all possessed a functional sphygmomanometer and stethoscope (65% at baseline); 96% stocked antihypertensive drugs (27% at baseline); 70% possessed a functional glucose meter and 72% stocked oral anti-diabetics (15% and 12% at baseline). NPCs' performance on multiple-choice questions of the knowledge-test was significantly improved. During a period of two years, trained NPCs initiated treatment for 796 patients with hypertension and/or diabetes. The retention of treated patients at one year was 18.1%. Hypertensive and diabetic patients paid a median monthly amount of 1.4 and 0.7 Euro respectively for their medication. Among hypertensive patients with ≥ 2 documented visits (n = 493), systolic BP decreased by 22.8 mmHg (95% CI: -20.6 to -24.9; p < 0.0001) and diastolic BP by 12.4 mmHg (-10.9 to -13.9; p < 0.0001). Among diabetic patients (n = 79) FPG decreased by 3.4 mmol/l (-2.3 to -4.5; p < 0.001). Conclusions The integration of hypertension and diabetes into primary health care of NPC facilities in rural Cameroon was feasible in terms of equipment and training, accessible in terms of treatment cost and showed promising BP- and FPG-trends. However, low case-detection rates per NPC and a very high attrition among patients enrolled into care, limited the effectiveness of the programme. PMID:21144064

Task shifting to non-physician clinicians for integrated management of hypertension and diabetes in rural Cameroon: a programme assessment at two years.

PubMed

Labhardt, Niklaus D; Balo, Jean-Richard; Ndam, Mama; Grimm, Jean-Jacques; Manga, Engelbert

2010-12-14

The burden of non-communicable chronic diseases, such as hypertension and diabetes, increases in sub-Saharan Africa. However, the majority of the rural population does still not have access to adequate care. The objective of this study is to examine the effectiveness of integrating care for hypertension and type 2 diabetes by task shifting to non-physician clinician (NPC) facilities in eight rural health districts in Cameroon. Of the 75 NPC facilities in the area, 69 (87%) received basic equipment and training in hypertension and diabetes care. Effectiveness was assessed after two years on status of equipment, knowledge among trained NPCs, number of newly detected patients, retention of patients under care, treatment cost to patients and changes in blood pressure (BP) and fasting plasma glucose (FPG) among treated patients. Two years into the programme, of 54 facilities (78%) available for re-assessment, all possessed a functional sphygmomanometer and stethoscope (65% at baseline); 96% stocked antihypertensive drugs (27% at baseline); 70% possessed a functional glucose meter and 72% stocked oral anti-diabetics (15% and 12% at baseline). NPCs' performance on multiple-choice questions of the knowledge-test was significantly improved. During a period of two years, trained NPCs initiated treatment for 796 patients with hypertension and/or diabetes. The retention of treated patients at one year was 18.1%. Hypertensive and diabetic patients paid a median monthly amount of 1.4 and 0.7 Euro respectively for their medication. Among hypertensive patients with ≥ 2 documented visits (n = 493), systolic BP decreased by 22.8 mmHg (95% CI: -20.6 to -24.9; p < 0.0001) and diastolic BP by 12.4 mmHg (-10.9 to -13.9; p < 0.0001). Among diabetic patients (n = 79) FPG decreased by 3.4 mmol/l (-2.3 to -4.5; p < 0.001). The integration of hypertension and diabetes into primary health care of NPC facilities in rural Cameroon was feasible in terms of equipment and training, accessible in terms of treatment cost and showed promising BP- and FPG-trends. However, low case-detection rates per NPC and a very high attrition among patients enrolled into care, limited the effectiveness of the programme.

Neuroinflammatory Dynamics Underlie Memory Impairments after Repeated Social Defeat.

PubMed

McKim, Daniel B; Niraula, Anzela; Tarr, Andrew J; Wohleb, Eric S; Sheridan, John F; Godbout, Jonathan P

2016-03-02

Repeated social defeat (RSD) is a murine stressor that recapitulates key physiological, immunological, and behavioral alterations observed in humans exposed to chronic psychosocial stress. Psychosocial stress promotes prolonged behavioral adaptations that are associated with neuroinflammatory signaling and impaired neuroplasticity. Here, we show that RSD promoted hippocampal neuroinflammatory activation that was characterized by proinflammatory gene expression and by microglia activation and monocyte trafficking that was particularly pronounced within the caudal extent of the hippocampus. Because the hippocampus is a key area involved in neuroplasticity, behavior, and cognition, we hypothesize that stress-induced neuroinflammation impairs hippocampal neurogenesis and promotes cognitive and affective behavioral deficits. We show here that RSD caused transient impairments in spatial memory recall that resolved within 28 d. In assessment of neurogenesis, the number of proliferating neural progenitor cells (NPCs) and the number of young, developing neurons were not affected initially after RSD. Nonetheless, the neuronal differentiation of NPCs that proliferated during RSD was significantly impaired when examined 10 and 28 d later. In addition, social avoidance, a measure of depressive-like behavior associated with caudal hippocampal circuitry, persisted 28 d after RSD. Treatment with minocycline during RSD prevented both microglia activation and monocyte recruitment. Inhibition of this neuroinflammatory activation in turn prevented impairments in spatial memory after RSD but did not prevent deficits in neurogenesis nor did it prevent the persistence of social avoidance behavior. These findings show that neuroinflammatory activation after psychosocial stress impairs spatial memory performance independent of deficits in neurogenesis and social avoidance. Repeated exposure to stress alters the homeostatic environment of the brain, giving rise to various cognitive and mood disorders that impair everyday functioning and overall quality of life. The brain, previously thought of as an immune-privileged organ, is now known to communicate extensively with the peripheral immune system. This brain-body communication plays a significant role in various stress-induced inflammatory conditions, also characterized by psychological impairments. Findings from this study implicate neuroimmune activation rather than impaired neurogenesis in stress-induced cognitive deficits. This idea opens up possibilities for novel immune interventions in the treatment of cognitive and mood disturbances, while also adding to the complexity surrounding the functional implications of adult neurogenesis. Copyright © 2016 the authors 0270-6474/16/362590-15$15.00/0.

Cell of Origin and Cancer Stem Cell Phenotype in Medulloblastomas

DTIC Science & Technology

2017-09-01

AWARD NUMBER: W81XWH-14-1-0115 TITLE: Cell of Origin and Cancer Stem Cell Phenotype in Medulloblastomas PRINCIPAL INVESTIGATOR: Kyuson Yun...CA130273 - Cell of Origin and Cancer Stem Cell Phenotype in Medulloblastomas 5a. CONTRACT NUMBER 5b. GRANT NUMBER W81XWH-14-1-0115 5c. PROGRAM...hypothesis, we originally proposed to transform neural stem cells (NSCs) and neural progenitor cells (NPCs) in vivo by expressing an activated form

Engineered LINE-1 retrotransposition in nondividing human neurons.

PubMed

Macia, Angela; Widmann, Thomas J; Heras, Sara R; Ayllon, Veronica; Sanchez, Laura; Benkaddour-Boumzaouad, Meriem; Muñoz-Lopez, Martin; Rubio, Alejandro; Amador-Cubero, Suyapa; Blanco-Jimenez, Eva; Garcia-Castro, Javier; Menendez, Pablo; Ng, Philip; Muotri, Alysson R; Goodier, John L; Garcia-Perez, Jose L

2017-03-01

Half the human genome is made of transposable elements (TEs), whose ongoing activity continues to impact our genome. LINE-1 (or L1) is an autonomous non-LTR retrotransposon in the human genome, comprising 17% of its genomic mass and containing an average of 80-100 active L1s per average genome that provide a source of inter-individual variation. New LINE-1 insertions are thought to accumulate mostly during human embryogenesis. Surprisingly, the activity of L1s can further impact the somatic human brain genome. However, it is currently unknown whether L1 can retrotranspose in other somatic healthy tissues or if L1 mobilization is restricted to neuronal precursor cells (NPCs) in the human brain. Here, we took advantage of an engineered L1 retrotransposition assay to analyze L1 mobilization rates in human mesenchymal (MSCs) and hematopoietic (HSCs) somatic stem cells. Notably, we have observed that L1 expression and engineered retrotransposition is much lower in both MSCs and HSCs when compared to NPCs. Remarkably, we have further demonstrated for the first time that engineered L1s can retrotranspose efficiently in mature nondividing neuronal cells. Thus, these findings suggest that the degree of somatic mosaicism and the impact of L1 retrotransposition in the human brain is likely much higher than previously thought. © 2017 Macia et al.; Published by Cold Spring Harbor Laboratory Press.

Maternal dietary intake of choline in mice regulates development of the cerebral cortex in the offspring

PubMed Central

Wang, Yanyan; Surzenko, Natalia; Friday, Walter B.; Zeisel, Steven H.

2015-01-01

Maternal diets low in choline, an essential nutrient, increase the risk of neural tube defects and lead to low performance on cognitive tests in children. However, the consequences of maternal dietary choline deficiency for the development and structural organization of the cerebral cortex remain unknown. In this study, we fed mouse dams either control (CT) or low-choline (LC) diets and investigated the effects of choline on cortical development in the offspring. As a result of a low choline supply between embryonic day (E)11 and E17 of gestation, the number of 2 types of cortical neural progenitor cells (NPCs)—radial glial cells and intermediate progenitor cells—was reduced in fetal brains (P < 0.01). Furthermore, the number of upper layer cortical neurons was decreased in the offspring of dams fed an LC diet at both E17 (P < 0.001) and 4 mo of age (P < 0.001). These effects of LC maternal diet were mediated by a decrease in epidermal growth factor receptor (EGFR) signaling in NPCs related to the disruption of EGFR posttranscriptional regulation. Our findings describe a novel mechanism whereby low maternal dietary intake of choline alters brain development.—Wang, Y., Surzenko, N., Friday, W. B., Zeisel, S. H. Maternal dietary intake of choline in mice regulates development of the cerebral cortex in the offspring. PMID:26700730

Maternal dietary intake of choline in mice regulates development of the cerebral cortex in the offspring.

PubMed

Wang, Yanyan; Surzenko, Natalia; Friday, Walter B; Zeisel, Steven H

2016-04-01

Maternal diets low in choline, an essential nutrient, increase the risk of neural tube defects and lead to low performance on cognitive tests in children. However, the consequences of maternal dietary choline deficiency for the development and structural organization of the cerebral cortex remain unknown. In this study, we fed mouse dams either control (CT) or low-choline (LC) diets and investigated the effects of choline on cortical development in the offspring. As a result of a low choline supply between embryonic day (E)11 and E17 of gestation, the number of 2 types of cortical neural progenitor cells (NPCs)-radial glial cells and intermediate progenitor cells-was reduced in fetal brains (P< 0.01). Furthermore, the number of upper layer cortical neurons was decreased in the offspring of dams fed an LC diet at both E17 (P< 0.001) and 4 mo of age (P< 0.001). These effects of LC maternal diet were mediated by a decrease in epidermal growth factor receptor (EGFR) signaling in NPCs related to the disruption of EGFR posttranscriptional regulation. Our findings describe a novel mechanism whereby low maternal dietary intake of choline alters brain development.-Wang, Y., Surzenko, N., Friday, W. B., Zeisel, S. H. Maternal dietary intake of choline in mice regulates development of the cerebral cortex in the offspring. © FASEB.

Generation of H1 PAX6WT/EGFP reporter cells to purify PAX6 positive neural stem/progenitor cells.

PubMed

Wu, Wei; Liu, Juli; Su, Zhenghui; Li, Zhonghao; Ma, Ning; Huang, Ke; Zhou, Tiancheng; Wang, Linli

2018-08-25

Neural conversion from human pluripotent cells (hPSCs) is a potential therapy to neurological disease in the future. However, this is still limited by efficiency and stability of existed protocols used for neural induction from hPSCs. To overcome this obstacle, we developed a reporter system to screen PAX6 + neural progenitor/stem cells using transcription activator like effector nuclease (TALEN). We found that knock-in 2 A-EGFP cassette into PAX6 exon of human embryonic stem cells H1 with TALEN-based homology recombination could establish PAX6 WT/EGFP H1 reporter cell line fast and efficiently. This reporter cell line could differentiate into PAX6 and EGFP double positive neural progenitor/stem cells (NPCs/NSCs) after neural induction. Those PAX6 WT/EGFP NPCs could be purified, expanded and specified to post-mitotic neurons in vitro efficiently. With this reporter cell line, we also screened out 1 NPC-specific microRNA, hsa-miR-99a-5p, and 3 ESCs-enriched miRNAs, hsa-miR-302c-5p, hsa-miR-512-3p and hsa-miR-518 b. In conclusion, the TALEN-based neural stem cell screening system is safe and efficient and could help researcher to acquire adequate and pure neural progenitor cells for further application. Copyright © 2018 Elsevier Inc. All rights reserved.

Elucidating the impact of neurofibromatosis-1 germline mutations on neurofibromin function and dopamine-based learning.

PubMed

Anastasaki, Corina; Woo, Albert S; Messiaen, Ludwine M; Gutmann, David H

2015-06-15

Neurofibromatosis type 1 (NF1) is a common autosomal dominant neurologic condition characterized by significant clinical heterogeneity, ranging from malignant cancers to cognitive deficits. Recent studies have begun to reveal rare genotype-phenotype correlations, suggesting that the specific germline NF1 gene mutation may be one factor underlying disease heterogeneity. The purpose of this study was to define the impact of the germline NF1 gene mutation on brain neurofibromin function relevant to learning. Herein, we employ human NF1-patient primary skin fibroblasts, induced pluripotent stem cells and derivative neural progenitor cells (NPCs) to demonstrate that NF1 germline mutations have dramatic effects on neurofibromin expression. Moreover, while all NF1-patient NPCs exhibit increased RAS activation and reduced cyclic AMP generation, there was a neurofibromin dose-dependent reduction in dopamine (DA) levels. Additionally, we leveraged two complementary Nf1 genetically-engineered mouse strains in which hippocampal-based learning and memory is DA-dependent to establish that neuronal DA levels and signaling as well as mouse spatial learning are controlled in an Nf1 gene dose-dependent manner. Collectively, this is the first demonstration that different germline NF1 gene mutations differentially dictate neurofibromin function in the brain. © The Author 2015. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

Targeted Knockdown of Bone Morphogenetic Protein Signaling within Neural Progenitors Protects the Brain and Improves Motor Function following Postnatal Hypoxia-Ischemia

PubMed Central

Dettman, Robert W.; Birch, Derin; Fernando, Augusta; Kessler, John A.; Dizon, Maria L.V.

2018-01-01

Hypoxic-ischemic injury (HI) to the neonatal human brain results in myelin loss that, in some children, can manifest as cerebral palsy. Previously, we had found that neuronal overexpression of the bone morphogenic protein (BMP) inhibitor noggin during development increased oligodendroglia and improved motor function in an experimental model of HI utilizing unilateral common carotid artery ligation followed by hypoxia. As BMPs are known to negatively regulate oligodendroglial fate specification of neural stem cells and alter differentiation of committed oligodendroglia, BMP signaling is likely an important mechanism leading to myelin loss. Here, we showed that BMP signaling is upregulated within oligodendroglia of the neonatal brain. We tested the hypothesis that inhibition of BMP signaling specifically within neural progenitor cells (NPCs) is sufficient to protect oligodendroglia. We conditionally deleted the BMP receptor 2 subtype (BMPR2) in NG2-expressing cells after HI. We found that BMPR2 deletion globally protects the brain as assessed by MRI and protects motor function as assessed by digital gait analysis, and that conditional deletion of BMPR2 maintains oligodendrocyte marker expression by immunofluorescence and Western blot and prevents loss of oligodendroglia. Finally, BMPR2 deletion after HI results in an increase in noncompacted myelin. Thus, our data indicate that inhibition of BMP signaling specifically in NPCs may be a tractable strategy to protect the newborn brain from HI. PMID:29324456

Active integrated filters for RF-photonic channelizers.

PubMed

El Nagdi, Amr; Liu, Ke; LaFave, Tim P; Hunt, Louis R; Ramakrishna, Viswanath; Dabkowski, Mieczyslaw; MacFarlane, Duncan L; Christensen, Marc P

2011-01-01

A theoretical study of RF-photonic channelizers using four architectures formed by active integrated filters with tunable gains is presented. The integrated filters are enabled by two- and four-port nano-photonic couplers (NPCs). Lossless and three individual manufacturing cases with high transmission, high reflection, and symmetric couplers are assumed in the work. NPCs behavior is dependent upon the phenomenon of frustrated total internal reflection. Experimentally, photonic channelizers are fabricated in one single semiconductor chip on multi-quantum well epitaxial InP wafers using conventional microelectronics processing techniques. A state space modeling approach is used to derive the transfer functions and analyze the stability of these filters. The ability of adapting using the gains is demonstrated. Our simulation results indicate that the characteristic bandpass and notch filter responses of each structure are the basis of channelizer architectures, and optical gain may be used to adjust filter parameters to obtain a desired frequency magnitude response, especially in the range of 1-5 GHz for the chip with a coupler separation of ∼9 mm. Preliminarily, the measurement of spectral response shows enhancement of quality factor by using higher optical gains. The present compact active filters on an InP-based integrated photonic circuit hold the potential for a variety of channelizer applications. Compared to a pure RF channelizer, photonic channelizers may perform both channelization and down-conversion in an optical domain.

The Role of NG2 Glial Cells in ALS Pathogenesis

DTIC Science & Technology

2014-12-01

NG2+ glial cells ( OPCs ), NPCs were further differentiated to pre- OPCs and OPCs , outlined in Figure 2, which showed reliable cell morphology and cell...marker expression. Pre- OPCs were determined by their expression of Olig2 and NKX2.2 (Figure 1). The majority of colonies showed Olig2+, NKX2.2...and Olig2+/NKX2.2+ cells, however the percentage of each marker expression varied among colonies. To further differentiate pre- OPCs to OPCs , the

DOE Office of Scientific and Technical Information (OSTI.GOV)

Busch, Albert; Kiel, Tilman; Heupel, Wolfgang-M.

Lamins, which form the nuclear lamina, not only constitute an important determinant of nuclear architecture, but additionally play essential roles in many nuclear functions. Mutations in A-type lamins cause a wide range of human genetic disorders (laminopathies). The importance of lamin A (LaA) in the spatial arrangement of nuclear pore complexes (NPCs) prompted us to study the role of LaA mutants in nuclear protein transport. Two mutants, causing prenatal skin disease restrictive dermopathy (RD) and the premature aging disease Hutchinson Gilford progeria syndrome, were used for expression in HeLa cells to investigate their impact on the subcellular localization of NPC-associatedmore » proteins and nuclear protein import. Furthermore, dynamics of the LaA mutants within the nuclear lamina were studied. We observed affected localization of NPC-associated proteins, diminished lamina dynamics for both LaA mutants and reduced nuclear import of representative cargo molecules. Intriguingly, both LaA mutants displayed similar effects on nuclear morphology and functions, despite their differences in disease severity. Reduced nuclear protein import was also seen in RD fibroblasts and impaired lamina dynamics for the nucleoporin Nup153. Our data thus represent the first study of a direct link between LaA mutant expression and reduced nuclear protein import.« less

A coarse-grained computational model of the nuclear pore complex predicts Phe-Gly nucleoporin dynamics

DOE Office of Scientific and Technical Information (OSTI.GOV)

Pulupa, Joan; Rachh, Manas; Tomasini, Michael D.

The phenylalanine-glycine–repeat nucleoporins (FG-Nups), which occupy the lumen of the nuclear pore complex (NPC), are critical for transport between the nucleus and cytosol. Although NPCs differ in composition across species, they are largely conserved in organization and function. Transport through the pore is on the millisecond timescale. Here, to explore the dynamics of nucleoporins on this timescale, we use coarse-grained computational simulations. These simulations generate predictions that can be experimentally tested to distinguish between proposed mechanisms of transport. Our model reflects the conserved structure of the NPC, in which FG-Nup filaments extend into the lumen and anchor along the interiormore » of the channel. The lengths of the filaments in our model are based on the known characteristics of yeast FG-Nups. The FG-repeat sites also bind to each other, and we vary this association over several orders of magnitude and run 100-ms simulations for each value. The autocorrelation functions of the orientation of the simulated FG-Nups are compared with in vivo anisotropy data. We observe that FG-Nups reptate back and forth through the NPC at timescales commensurate with experimental measurements of the speed of cargo transport through the NPC. Our results are consistent with models of transport where FG-Nup filaments are free to move across the central channel of the NPC, possibly informing how cargo might transverse the NPC.« less

A coarse-grained computational model of the nuclear pore complex predicts Phe-Gly nucleoporin dynamics

DOE PAGES

Pulupa, Joan; Rachh, Manas; Tomasini, Michael D.; ...

2017-09-08

The phenylalanine-glycine–repeat nucleoporins (FG-Nups), which occupy the lumen of the nuclear pore complex (NPC), are critical for transport between the nucleus and cytosol. Although NPCs differ in composition across species, they are largely conserved in organization and function. Transport through the pore is on the millisecond timescale. Here, to explore the dynamics of nucleoporins on this timescale, we use coarse-grained computational simulations. These simulations generate predictions that can be experimentally tested to distinguish between proposed mechanisms of transport. Our model reflects the conserved structure of the NPC, in which FG-Nup filaments extend into the lumen and anchor along the interiormore » of the channel. The lengths of the filaments in our model are based on the known characteristics of yeast FG-Nups. The FG-repeat sites also bind to each other, and we vary this association over several orders of magnitude and run 100-ms simulations for each value. The autocorrelation functions of the orientation of the simulated FG-Nups are compared with in vivo anisotropy data. We observe that FG-Nups reptate back and forth through the NPC at timescales commensurate with experimental measurements of the speed of cargo transport through the NPC. Our results are consistent with models of transport where FG-Nup filaments are free to move across the central channel of the NPC, possibly informing how cargo might transverse the NPC.« less

The nuclear pore complex protein ALADIN is anchored via NDC1 but not via POM121 and GP210 in the nuclear envelope

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kind, Barbara, E-mail: barbara.kind@uniklinikum-dresden.de; Koehler, Katrin, E-mail: katrin.koehler@uniklinikum-dresden.de; Lorenz, Mike, E-mail: mlorenz@mpi-cbg.de

2009-12-11

The nuclear pore complex (NPC) consists of {approx}30 different proteins and provides the only sites for macromolecular transport between cytoplasm and nucleus. ALADIN was discovered as a new member of the NPC. Mutations in ALADIN are known to cause triple A syndrome, a rare autosomal recessive disorder characterized by adrenal insufficiency, alacrima, and achalasia. The function and exact location of the nucleoporin ALADIN within the NPC multiprotein complex is still unclear. Using a siRNA-based approach we downregulated the three known membrane integrated nucleoporins NDC1, GP210, and POM121 in stably expressing GFP-ALADIN HeLa cells. We identified NDC1 but not GP210 andmore » POM121 as the main anchor of ALADIN within the NPC. Solely the depletion of NDC1 caused mislocalization of ALADIN. Vice versa, the depletion of ALADIN led also to disappearance of NDC1 at the NPC. However, the downregulation of two further membrane-integral nucleoporins GP210 and POM121 had no effect on ALADIN localization. Furthermore, we could show a direct association of NDC1 and ALADIN in NPCs by fluorescence resonance energy transfer (FRET) measurements. Based on our findings we conclude that ALADIN is anchored in the nuclear envelope via NDC1 and that this interaction gets lost, if ALADIN is mutated. The loss of integration of ALADIN in the NPC is a main pathogenetic aspect for the development of the triple A syndrome and suggests that the interaction between ALADIN and NDC1 may be involved in the pathogenesis of the disease.« less

Human olfactory bulb neural stem cells expressing hNGF restore cognitive deficit in Alzheimer's disease rat model.

PubMed

Marei, Hany E S; Farag, Amany; Althani, Asma; Afifi, Nahla; Abd-Elmaksoud, Ahmed; Lashen, Samah; Rezk, Shaymaa; Pallini, Roberto; Casalbore, Patrizia; Cenciarelli, Carlo

2015-01-01

In this study, we aim to demonstrate the fate of allogenic adult human olfactory bulb neural stem/progenitor cells (OBNSC/NPCs) transplanted into the rat hippocampus treated with ibotenic acid (IBO), a neurotoxicant specific to hippocampal cholinergic neurons that are lost in Alzheimer's disease. We assessed their possible ability to survive, integrate, proliferate, and differentiate into different neuronal and glial elements: we also evaluate their possible therapeutic potential, and the mechanism(s) relevant to neuroprotection following their engraftment into the CNS milieu. OBNSC/NPCs were isolated from adult human olfactory bulb patients, genetically engineered to express GFP and human nerve growth factor (hNGF) by lentivirus-mediated infection, and stereotaxically transplanted into the hippocampus of IBO-treated animals and controls. Stereological analysis of engrafted OBNSCs eight weeks post transplantation revealed a 1.89 fold increase with respect to the initial cell population, indicating a marked ability for survival and proliferation. In addition, 54.71 ± 11.38%, 30.18 ± 6.00%, and 15.09 ± 5.38% of engrafted OBNSCs were identified by morphological criteria suggestive of mature neurons, oligodendrocytes and astrocytes respectively. Taken together, this work demonstrated that human OBNSCs expressing NGF ameliorate the cognitive deficiencies associated with IBO-induced lesions in AD model rats, and the improvement can probably be attributed primarily to neuronal and glial cell replacement as well as the trophic influence exerted by the secreted NGF. © 2014 Wiley Periodicals, Inc.

KCa3.1 Modulates Neuroblast Migration Along the Rostral Migratory Stream (RMS) In Vivo

PubMed Central

Turner, Kathryn L.; Sontheimer, Harald

2014-01-01

From the subventricular zone (SVZ), neuronal precursor cells (NPCs), called neuroblasts, migrate through the rostral migratory stream (RMS) to become interneurons in the olfactory bulb (OB). Ion channels regulate neuronal migration during development, yet their role in migration through the adult RMS is unknown. To address this question, we utilized Nestin-CreERT2/R26R-YFP mice to fluorescently label neuroblasts in the adult. Patch-clamp recordings from neuroblasts reveal K+ currents that are sensitive to intracellular Ca2+ levels and blocked by clotrimazole and TRAM-34, inhibitors of intermediate conductance Ca2+-activated K+ (KCa3.1) channels. Immunolabeling and electrophysiology show KCa3.1 expression restricted to neuroblasts in the SVZ and RMS, but absent in OB neurons. Time-lapse confocal microscopy in situ showed inhibiting KCa3.1 prolonged the stationary phase of neuroblasts' saltatory migration, reducing migration speed by over 50%. Both migration and KCa3.1 currents could also be inhibited by blocking Ca2+ influx via transient receptor potential (TRP) channels, which, together with positive immunostaining for transient receptor potential canonical 1 (TRPC1), suggest that TRP channels are an important Ca2+ source modulating KCa3.1 activity. Finally, injecting TRAM-34 into Nestin-CreERT2/R26R-YFP mice significantly reduced the number of neuroblasts that reached the OB, suggesting an important role for KCa3.1 in vivo. These studies describe a previously unrecognized protein in migration of adult NPCs. PMID:23585521

Nutraceutical intervention reverses the negative effects of blood from aged rats on stem cells.

PubMed

Bickford, Paula C; Kaneko, Yuji; Grimmig, Bethany; Pappas, Colleen; Small, Brent; Sanberg, Cyndy D; Sanberg, Paul R; Tan, Jun; Douglas Shytle, R

2015-10-01

Aging is associated with a decline in function in many of the stem cell niches of the body. An emerging body of literature suggests that one of the reasons for this decline in function is due to cell non-autonomous influences on the niche from the body. For example, studies using the technique of parabiosis have demonstrated a negative influence of blood from aged mice on muscle satellite cells and neurogenesis in young mice. We examined if we could reverse this effect of aged serum on stem cell proliferation by treating aged rats with NT-020, a dietary supplement containing blueberry, green tea, vitamin D3, and carnosine that has been shown to increase neurogenesis in aged rats. Young and aged rats were administered either control NIH-31 diet or one supplemented with NT-020 for 28 days, and serum was collected upon euthanasia. The serum was used in cultures of both rat hippocampal neural progenitor cells (NPCs) and rat bone marrow-derived mesenchymal stem cells (MSCs). Serum from aged rats significantly reduced cell proliferation as measured by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and 5-bromo-2'-deoxyuridine (BrdU) assays in both NPCs and MSCs. Serum from aged rats treated with NT-020 was not different from serum from young rats. Therefore, NT-020 rescued the effect of serum from aged rats to reduce stem cell proliferation.

Proteomic Analysis of Zika Virus Infected Primary Human Fetal Neural Progenitors Suggests a Role for Doublecortin in the Pathological Consequences of Infection in the Cortex.

PubMed

Jiang, Xuan; Dong, Xiao; Li, Shi-Hua; Zhou, Yue-Peng; Rayner, Simon; Xia, Hui-Min; Gao, George F; Yuan, Hui; Tang, Ya-Ping; Luo, Min-Hua

2018-01-01

Zika virus (ZIKV) infection is associated with severe neurological defects in fetuses and newborns, such as microcephaly. However, the underlying mechanisms remain to be elucidated. In this study, proteomic analysis on ZIKV-infected primary human fetal neural progenitor cells (NPCs) revealed that virus infection altered levels of cellular proteins involved in NPC proliferation, differentiation and migration. The transcriptional levels of some of the altered targets were also confirmed by qRT-PCR. Among the altered proteins, doublecortin (DCX) plays an important role in NPC differentiation and migration. Results showed that ZIKV infection downregulated DCX, at both mRNA and protein levels, as early as 1 day post infection (1 dpi), and lasted throughout the virus replication cycle (4 days). The downregulation of DCX was also observed in a ZIKV-infected fetal mouse brain model, which displayed decreased body weight, brain size and weight, as well as defective cortex structure. By screening the ten viral proteins of ZIKV, we found that both the expression of NS4A and NS5 were correlated with the downregulation of both mRNA and protein levels of DCX in NPCs. These data suggest that DCX is modulated following infection of the brain by ZIKV. How these observed changes of DCX expression translate in the pathological consequences of ZIKV infection and if other cellular proteins are equally involved remains to be investigated.

Environment and health in contaminated sites: the case of Taranto, Italy.

PubMed

Pirastu, Roberta; Comba, Pietro; Iavarone, Ivano; Zona, Amerigo; Conti, Susanna; Minelli, Giada; Manno, Valerio; Mincuzzi, Antonia; Minerba, Sante; Forastiere, Francesco; Mataloni, Francesca; Biggeri, Annibale

2013-01-01

The National Environmental Remediation programme in Italy includes sites with documented contamination and associated potential health impacts (National Priority Contaminated Sites-NPCSs). SENTIERI Project, an extensive investigation of mortality in 44 NPCSs, considered the area of Taranto, a NPCS where a number of polluting sources are present. Health indicators available at municipality level were analyzed, that is, mortality (2003-2009), mortality time trend (1980-2008), and cancer incidence (2006-2007). In addition, the cohort of individuals living in the area was followed up to evaluate mortality (1998-2008) and morbidity (1998-2010) by district of residence. The results of the study consistently showed excess risks for a number of causes of death in both genders, among them: all causes, all cancers, lung cancer, and cardiovascular and respiratory diseases, both acute and chronic. An increased infant mortality was also observed from the time trends analysis. Mortality/morbidity excesses were detected in residents living in districts near the industrial area, for several disorders including cancer, cardiovascular, and respiratory diseases. These coherent findings from different epidemiological approaches corroborate the need to promptly proceed with environmental cleanup interventions. Most diseases showing an increase in Taranto NPCS have a multifactorial etiology, and preventive measures of proven efficacy (e.g., smoking cessation and cardiovascular risk reduction programs, breast cancer screening) should be planned. The study results and public health actions are to be communicated objectively and transparently so that a climate of confidence and trust between citizens and public institutions is maintained.

Increased Re-Entry into Cell Cycle Mitigates Age-Related Neurogenic Decline in the Murine Subventricular Zone

PubMed Central

Stoll, Elizabeth A.; Habibi, Behnum A.; Mikheev, Andrei M.; Lasiene, Jurate; Massey, Susan C.; Swanson, Kristin R.; Rostomily, Robert C.; Horner, Philip J.

2012-01-01

Although new neurons are produced in the subventricular zone (SVZ) of the adult mammalian brain, fewer functional neurons are produced with increasing age. The age-related decline in neurogenesis has been attributed to a decreased pool of neural progenitor cells (NPCs), an increased rate of cell death, and an inability to undergo neuronal differentiation and develop functional synapses. The time between mitotic events has also been hypothesized to increase with age, but this has not been directly investigated. Studying primary-cultured NPCs from the young adult and aged mouse forebrain, we observe that fewer aged cells are dividing at a given time; however, the mitotic cells in aged cultures divide more frequently than mitotic cells in young cultures during a 48-hour period of live-cell time-lapse imaging. Double-thymidine-analog labeling also demonstrates that fewer aged cells are dividing at a given time, but those that do divide are significantly more likely to re-enter the cell cycle within a day, both in vitro and in vivo. Meanwhile, we observed that cellular survival is impaired in aged cultures. Using our live-cell imaging data, we developed a mathematical model describing cell cycle kinetics to predict the growth curves of cells over time in vitro and the labeling index over time in vivo. Together, these data surprisingly suggest that progenitor cells remaining in the aged SVZ are highly proliferative. PMID:21948688

Enhanced in vivo targeting of murine nonparenchymal liver cells with monophosphoryl lipid A functionalized microcapsules.

PubMed

Pietrzak-Nguyen, Anette; Fichter, Michael; Dedters, Marvin; Pretsch, Leah; Gregory, Stephen H; Meyer, Claudius; Doganci, Aysefa; Diken, Mustafa; Landfester, Katharina; Baier, Grit; Gehring, Stephan

2014-07-14

A broad spectrum of infectious liver diseases emphasizes the need of microparticles for targeted delivery of immunomodulatory substances to the liver. Microcapsules (MCs) are particularly attractive for innovative drug and vaccine formulations, enabling the combination of antigen, drugs, and adjuvants. The present study aimed to develop microcapsules characterized by an enhanced liver deposition and accelerated uptake by nonparenchymal liver cells (NPCs). Initially, two formulations of biodegradable microcapsules were synthesized from either hydroxyethyl starch (HES) or mannose. Notably, HES-MCs accumulated primarily in the liver, while mannose particles displayed a lung preference. Functionalization of HES-MCs with anti-CD40, anti-DEC205, and/or monophosphoryl lipid A (MPLA) enhanced uptake of MCs by nonparenchymal liver cells in vitro. In contrast, only MPLA-coated HES-MCs promoted significantly the in vivo uptake by NPCs. Finally, HES-MCs equipped with MPLA, anti-CD40, and anti-DEC205 induced the secretion of TNF-α, IL-6 by Kupffer cells (KCs), and IFN-γ and IL-12p70 by liver dendritic cells (DCs). The enhanced uptake and activation of KCs by MPLA-HES-MCs is a promising approach to prevent or treat infection, since KCs are exploited as an entry gate in various infectious diseases, such as malaria. In parallel, loading and activating liver DCs, usually prone to tolerance, bears the potential to induce antigen specific, intrahepatic immune responses necessary to prevent and treat infections affecting the liver.

Fluoxetine Regulates Neurogenesis In Vitro Through Modulation of GSK-3β/β-Catenin Signaling

PubMed Central

Hui, Jiaojie; Zhang, Jianping; Kim, Hoon; Tong, Chang; Ying, Qilong; Li, Zaiwang; Mao, Xuqiang; Shi, Guofeng; Yan, Jie; Zhang, Zhijun

2015-01-01

Background: It is generally accepted that chronic treatment with antidepressants increases hippocampal neurogenesis, but the molecular mechanisms underlying their effects are unknown. Recently, glycogen synthase kinase-3 beta (GSK-3β)/β-catenin signaling was shown to be involved in the mechanism of how antidepressants might influence hippocampal neurogenesis. Methods: The aim of this study was to determine whether GSK-3β/β-catenin signaling is involved in the alteration of neurogenesis as a result of treatment with fluoxetine, a selective serotonin reuptake inhibitor. The mechanisms involved in fluoxetine’s regulation of GSK-3β/β-catenin signaling pathway were also examined. Results: Our results demonstrated that fluoxetine increased the proliferation of embryonic neural precursor cells (NPCs) by up-regulating the phosphorylation of Ser9 on GSK-3β and increasing the level of nuclear β-catenin. The overexpression of a stabilized β-catenin protein (ΔN89 β-catenin) significantly increased NPC proliferation, while inhibition of β-catenin expression in NPCs led to a significant decrease in the proliferation and reduced the proliferative effects induced by fluoxetine. The effects of fluoxetine-induced up-regulation of both phosphorylation of Ser9 on GSK-3β and nuclear β-catenin were significantly prevented by the 5-hydroxytryptamine-1A (5-HT1A) receptor antagonist WAY-100635. Conclusions: The results demonstrate that fluoxetine may increase neurogenesis via the GSK-3β/β-catenin signaling pathway that links postsynaptic 5-HT1A receptor activation. PMID:25522429

Cell mediated therapeutics for cancer treatment: Tumor homing cells as therapeutic delivery vehicles

NASA Astrophysics Data System (ADS)

Balivada, Sivasai

Many cell types were known to have migratory properties towards tumors and different research groups have shown reliable results regarding cells as delivery vehicles of therapeutics for targeted cancer treatment. Present report discusses proof of concept for 1. Cell mediated delivery of Magnetic nanoparticles (MNPs) and targeted Magnetic hyperthermia (MHT) as a cancer treatment by using in vivo mouse cancer models, 2. Cells surface engineering with chimeric proteins for targeted cancer treatment by using in vitro models. 1. Tumor homing cells can carry MNPs specifically to the tumor site and tumor burden will decrease after alternating magnetic field (AMF) exposure. To test this hypothesis, first we loaded Fe/Fe3O4 bi-magnetic NPs into neural progenitor cells (NPCs), which were previously shown to migrate towards melanoma tumors. We observed that NPCs loaded with MNPs travel to subcutaneous melanoma tumors. After alternating magnetic field (AMF) exposure, the targeted delivery of MNPs by the NPCs resulted in a mild decrease in tumor size (Chapter-2). Monocytes/macrophages (Mo/Ma) are known to infiltrate tumor sites, and also have phagocytic activity which can increase their uptake of MNPs. To test Mo/Ma-mediated MHT we transplanted Mo/Ma loaded with MNPs into a mouse model of pancreatic peritoneal carcinomatosis. We observed that MNP-loaded Mo/Ma infiltrated pancreatic tumors and, after AMF treatment, significantly prolonged the lives of mice bearing disseminated intraperitoneal pancreatic tumors (Chapter-3). 2. Targeted cancer treatment could be achieved by engineering tumor homing cell surfaces with tumor proteases cleavable, cancer cell specific recombinant therapeutic proteins. To test this, Urokinase and Calpain (tumor specific proteases) cleavable; prostate cancer cell (CaP) specific (CaP1 targeting peptide); apoptosis inducible (Caspase3 V266ED3)- rCasp3V266ED3 chimeric protein was designed in silico. Hypothesized membrane anchored chimeric protein (rCasp3V266ED3, rMcherry red) plasmids were constructed. Membrane anchoring and activity of designed proteins were analyzed in RAW264.7 Mo/Ma and HEK293 cells in vitro. Further, Urokinase (uPA) mediated cleavage and release of rCasp3V266ED3 from engineered cells was tested (Chapter-4). Animal models for cancer therapy are invaluable for preclinical testing of potential cancer treatments. Final chapter of present report shows evidence for immune-deficient line of pigs as a model for human cancers (Chapter-5)

Tests of Rock Cores Michigamme Study Area, Michigan

DTIC Science & Technology

1970-06-01

cca;c-rraincd tona- l ".1 ., reck with dis - ’ pte, --’gmatite bands. MG-CR-26 SAMcO-9, DC-2 2 17 Black and white, medium- grtlned gn.insic ,onalite. I...4 . :t:::*.::: l N . . ......... 20,000 W:., ..... ... ... ... ...... ....... ... . .1. 1:: t: :1:1:1 N.: .111:::: Fiji. milIA : -mit: 15.00C :N...PCS13.60/MF$3.00 SutrdPormigSre.Vl l siae V Bilogcal ervcesProgam.Fisal Yar 975Software Project Resource Requirement* PADA251 738/PAT 52 p PC$4.50

Facilitating the Evaluation of Complexity in the Public Sector: Learning from the NHS in Scotland

ERIC Educational Resources Information Center

Connolly, John; Reid, Garth; Mooney, Allan

2015-01-01

It is necessary for public managers to be able to evaluate programmes in the context of complexity. This article offers key learning and reflections based on the experience of facilitating the evaluation of complexity with a range of public sector partners in Scotland. There have been several articles that consider evaluating complexity and…

Nucleoporin 62 and Ca(2+)/calmodulin dependent kinase kinase 2 regulate androgen receptor activity in castrate resistant prostate cancer cells.

PubMed

Karacosta, Loukia G; Kuroski, Laura A; Hofmann, Wilma A; Azabdaftari, Gissou; Mastri, Michalis; Gocher, Angela M; Dai, Shuhang; Hoste, Allen J; Edelman, Arthur M

2016-02-15

Re-activation of the transcriptional activity of the androgen receptor (AR) is an important factor mediating progression from androgen-responsive to castrate-resistant prostate cancer (CRPC). However, the mechanisms regulating AR activity in CRPC remain incompletely understood. Ca(2+) /calmodulin-dependent kinase kinase (CaMKK) 2 was previously shown to regulate AR activity in androgen-responsive prostate cancer cells. Our objective was to further explore the basis of this regulation in CRPC cells. The abundance of CaMKK2 in nuclear fractions of androgen-responsive prostate cancer and CRPC, cells were determined by subcellular fractionation and Western blotting. CaMKK2 association with nuclear pore complexes (NPCs) and nucleoporins (Nups) including Nup62, were imaged by structured illumination and super-resolution fluorescence microscopy and co-immunoprecipitation, respectively. The abundance and subcellular localization of CaMKK2 and Nup62 in human clinical specimens of prostate cancer was visualized by immunohistochemistry. The role of Nups in the growth and viability of CRPC cells was assessed by RNA interference and cell counting. The involvement of CaMKK2 and Nup62 in regulating AR transcriptional activity was addressed by RNA interference, chromatin immunoprecipitation, androgen response element reporter assay, and Western blotting. CaMKK2 was expressed at higher levels in the nuclear fraction of CPRC C4-2 cells, than in that of androgen-responsive LNCaP cells. In C4-2 cells, CaMKK2 associated with NPCs of the nuclear envelope and physically interacted with Nup62. CaMKK2 and Nup62 demonstrated pronounced, and similar increases in both expression and perinuclear/nuclear localization in human clinical specimens of advanced prostate cancer relative to normal prostate. Knockdown of Nup62, but not of Nups, 98 or 88, reduced growth and viability of C4-2 cells. Knockdown of Nup62 produced a greater reduction of the growth and viability of C4-2 cells than of non-neoplastic RWPE-1 prostatic cells. Nup62, CaMKK2, and the AR were recruited to androgen response elements of the AR target genes, prostate specific antigen, and transmembrane protease, serine 2. Knockdown of CaMKK2 and Nup62 reduced prostate specific antigen expression and AR transcriptional activity driven by androgen response elements from the prostate-specific probasin gene promoter. Nup62 and CaMKK2 are required for optimal AR transcriptional activity and a potential mechanism for AR re-activation in CRPC. © 2015 Wiley Periodicals, Inc.

Working alone or in the presence of others: exploring social facilitation in baggage X-ray security screening tasks.

PubMed

Yu, Rui-feng; Wu, Xin

2015-01-01

This study investigated whether the mere presence of a human audience would evoke a social facilitation effect in baggage X-ray security screening tasks. A 2 (target presence: present vs. absent) ×  2 (task complexity: simple vs. complex) ×  2 (social presence: alone vs. human audience) within-subject experiment simulating a real baggage screening task was conducted. This experiment included 20 male participants. The participants' search performance in this task was recorded. The results showed that the presence of a human audience speeded up responses in simple tasks and slowed down responses in complex tasks. However, the social facilitation effect produced by the presence of a human audience had no effect on response accuracy. These findings suggested that the complexity of screening tasks should be considered when designing work organisation modes for security screening tasks. Practitioner summary: This study investigated whether the presence of a human audience could evoke a social facilitation effect in baggage X-ray security screening tasks. An experimental simulation was conducted. The results showed that the presence of a human audience facilitated the search performance of simple tasks and inhibited the performance of complex tasks.

Negative regulation of TLX by IL-1β correlates with an inhibition of adult hippocampal neural precursor cell proliferation.

PubMed

Ryan, Sinead M; O'Keeffe, Gerard W; O'Connor, Caitriona; Keeshan, Karen; Nolan, Yvonne M

2013-10-01

Adult hippocampal neurogenesis is modulated by a number of intrinsic and extrinsic factors including local signalling molecules, exercise, aging and inflammation. Inflammation is also a major contributor to several hippocampal-associated disorders. Interleukin-1beta (IL-1β) is the most predominant pro-inflammatory cytokine in the brain, and an increase in its concentration is known to decrease the proliferation of both embryonic and adult hippocampal neural precursor cells (NPCs). Recent research has focused on the role of nuclear receptors as intrinsic regulators of neurogenesis, and it is now established that the orphan nuclear receptor TLX is crucial in maintaining the NPC pool in neurogenic brain regions. To better understand the involvement of TLX in IL-1β-mediated effects on hippocampal NPC proliferation, we examined hippocampal NPC proliferation and TLX expression in response to IL-1β treatment in an adult rat hippocampal neurosphere culture system. We demonstrate that IL-1β reduced the proliferation of hippocampal NPCs and TLX expression in a dose and time-dependent manner and that co-treatment with IL-1β receptor antagonist or IL-1 receptor siRNA prevented these effects. We also report a dose-dependent effect of IL-1β on the composition of cell phenotypes in the culture and on expression of TLX in these cells. This study thus provides evidence of an involvement of TLX in IL-1β-induced changes in adult hippocampal neurogenesis, and offers mechanistic insight into disorders in which neuroinflammation and alterations in neurogenesis are characteristic features. Copyright © 2013 Elsevier Inc. All rights reserved.

How Stem Cells Speak with Host Immune Cells in Inflammatory Brain Diseases

PubMed Central

Pluchino, Stefano; Cossetti, Chiara

2014-01-01

Advances in stem cell biology have raised great expectations that diseases and injuries of the central nervous system (CNS) may be ameliorated by the development of non-hematopoietic stem cell medicines. Yet, the application of adult stem cells as CNS therapeutics is challenging and the interpretation of some of the outcomes ambiguous. In fact, the initial idea that stem cell transplants work only via structural cell replacement has been challenged by the observation of consistent cellular signaling between the graft and the host. Cellular signaling is the foundation of coordinated actions and flexible responses, and arises via networks of exchanging and interacting molecules that transmit patterns of information between cells. Sustained stem cell graft-to-host communication leads to remarkable trophic effects on endogenous brain cells and beneficial modulatory actions on innate and adaptive immune responses in vivo, ultimately promoting the healing of the injured CNS. Among a number of adult stem cell types, mesenchymal stem cells (MSCs) and neural stem/precursor cells (NPCs) are being extensively investigated for their ability to signal to the immune system upon transplantation in experimental CNS diseases. Here, we focus on the main cellular signaling pathways that grafted MSCs and NPCs use to establish a therapeutically relevant cross talk with host immune cells, while examining the role of inflammation in regulating some of the bidirectionality of these communications. We propose that the identification of the players involved in stem cell signaling might contribute to the development of innovative, high clinical impact therapeutics for inflammatory CNS diseases. PMID:23633288

Competitive adsorption of a binary CO2-CH4 mixture in nanoporous carbons: effects of edge-functionalization.

PubMed

Lu, Xiaoqing; Jin, Dongliang; Wei, Shuxian; Zhang, Mingmin; Zhu, Qing; Shi, Xiaofan; Deng, Zhigang; Guo, Wenyue; Shen, Wenzhong

2015-01-21

The effect of edge-functionalization on the competitive adsorption of a binary CO2-CH4 mixture in nanoporous carbons (NPCs) has been investigated for the first time by combining density functional theory (DFT) and grand canonical Monte Carlo (GCMC) simulation. Our results show that edge-functionalization has a more positive effect on the single-component adsorption of CO2 than CH4, therefore significantly enhancing the selectivity of CO2 over CH4, in the order of NH2-NPC > COOH-NPC > OH-NPC > H-NPC > NPC at low pressure. The enhanced adsorption originates essentially from the effects of (1) the conducive environment with a large pore size and an effective accessible surface area, (2) the high electronegativity/electropositivity, (3) the strong adsorption energy, and (4) the large electrostatic contribution, due to the inductive effect/direct interaction of the embedded edge-functionalized groups. The larger difference from these effects results in the higher competitive adsorption advantage of CO2 in the binary CO2-CH4 mixture. Temperature has a negative effect on the gas adsorption, but no obvious influence on the electrostatic contribution on selectivity. With the increase of pressure, the selectivity of CO2 over CH4 first decreases sharply and subsequently flattens out to a constant value. This work highlights the potential of edge-functionalized NPCs in competitive adsorption, capture, and separation for the binary CO2-CH4 mixture, and provides an effective and superior alternative strategy in the design and screening of adsorbent materials for carbon capture and storage.

"One-for-All" Strategy in Fast Energy Storage: Production of Pillared MOF Nanorod-Templated Positive/Negative Electrodes for the Application of High-Performance Hybrid Supercapacitor.

PubMed

Qu, Chong; Liang, Zibin; Jiao, Yang; Zhao, Bote; Zhu, Bingjun; Dang, Dai; Dai, Shuge; Chen, Yu; Zou, Ruqiang; Liu, Meilin

2018-06-01

Currently, metal-organic frameworks (MOFs) are intensively studied as active materials for electrochemical energy storage applications due to their tunable structure and exceptional porosities. Among them, water stable pillared MOFs with dual ligands have been reported to exhibit high supercapacitor (SC) performance. Herein, the "One-for-All" strategy is applied to synthesize both positive and negative electrodes of a hybrid SC (HSC) from a single pillared MOF. Specifically, Ni-DMOF-TM ([Ni(TMBDC)(DABCO) 0.5 ], TMBDC: 2,3,5,6-tetramethyl-1,4-benzenedicarboxylic acid, DABCO: 1,4-diazabicyclo[2.2.2]-octane) nanorods are directly grown on carbon fiber paper (CFP) (denoted as CFP@TM-nanorods) with the help of triethylamine and function as the positive electrode of HSC under alkaline electrolyte. Meanwhile, calcinated N-doped hierarchical porous carbon nanorods (CFP@TM-NPCs) are produced and utilized as the negative counter-electrode from a one-step heat treatment of CFP@TM-nanorods. After assembling these two electrodes together to make a hybrid device, the TM-nanorods//TM-NPCs exhibit a wide voltage window of 1.5 V with a high sloping discharge plateau between 1-1.2 V, indicating its great potential for practical applications. This as-described "One-for-All" strategy is widely applicable and highly reproducible in producing MOF-based electrode materials for HSC applications, which shortens the gap between experimental synthesis and practical application of MOFs in fast energy storage. © 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Fluoxetine regulates neurogenesis in vitro through modulation of GSK-3β/β-catenin signaling.

PubMed

Hui, Jiaojie; Zhang, Jianping; Kim, Hoon; Tong, Chang; Ying, Qilong; Li, Zaiwang; Mao, Xuqiang; Shi, Guofeng; Yan, Jie; Zhang, Zhijun; Xi, Guangjun

2014-12-07

It is generally accepted that chronic treatment with antidepressants increases hippocampal neurogenesis, but the molecular mechanisms underlying their effects are unknown. Recently, glycogen synthase kinase-3 beta (GSK-3β)/β-catenin signaling was shown to be involved in the mechanism of how antidepressants might influence hippocampal neurogenesis. The aim of this study was to determine whether GSK-3β/β-catenin signaling is involved in the alteration of neurogenesis as a result of treatment with fluoxetine, a selective serotonin reuptake inhibitor. The mechanisms involved in fluoxetine's regulation of GSK-3β/β-catenin signaling pathway were also examined. Our results demonstrated that fluoxetine increased the proliferation of embryonic neural precursor cells (NPCs) by up-regulating the phosphorylation of Ser9 on GSK-3β and increasing the level of nuclear β-catenin. The overexpression of a stabilized β-catenin protein (ΔN89 β-catenin) significantly increased NPC proliferation, while inhibition of β-catenin expression in NPCs led to a significant decrease in the proliferation and reduced the proliferative effects induced by fluoxetine. The effects of fluoxetine-induced up-regulation of both phosphorylation of Ser9 on GSK-3β and nuclear β-catenin were significantly prevented by the 5-hydroxytryptamine-1A (5-HT1A) receptor antagonist WAY-100635. The results demonstrate that fluoxetine may increase neurogenesis via the GSK-3β/β-catenin signaling pathway that links postsynaptic 5-HT1A receptor activation. © The Author 2015. Published by Oxford University Press on behalf of CINP.

Comparative interactomics provides evidence for functional specialization of the nuclear pore complex.

PubMed

Obado, Samson O; Field, Mark C; Rout, Michael P

2017-07-04

The core architecture of the eukaryotic cell was established well over one billion years ago, and is largely retained in all extant lineages. However, eukaryotic cells also possess lineage-specific features, frequently keyed to specific functional requirements. One quintessential core eukaryotic structure is the nuclear pore complex (NPC), responsible for regulating exchange of macromolecules between the nucleus and cytoplasm as well as acting as a nuclear organizational hub. NPC architecture has been best documented in one eukaryotic supergroup, the Opisthokonts (e.g. Saccharomyces cerevisiae and Homo sapiens), which although compositionally similar, have significant variations in certain NPC subcomplex structures. The variation of NPC structure across other taxa in the eukaryotic kingdom however, remains poorly understood. We explored trypanosomes, highly divergent organisms, and mapped and assigned their NPC proteins to specific substructures to reveal their NPC architecture. We showed that the NPC central structural scaffold is conserved, likely across all eukaryotes, but more peripheral elements can exhibit very significant lineage-specific losses, duplications or other alterations in their components. Amazingly, trypanosomes lack the major components of the mRNA export platform that are asymmetrically localized within yeast and vertebrate NPCs. Concomitant with this, the trypanosome NPC is ALMOST completely symmetric with the nuclear basket being the only major source of asymmetry. We suggest these features point toward a stepwise evolution of the NPC in which a coating scaffold first stabilized the pore after which selective gating emerged and expanded, leading to the addition of peripheral remodeling machineries on the nucleoplasmic and cytoplasmic sides of the pore.

Sensitive Immunofluorescent Staining of Cells via Generation of Fluorescent Nanoscale Polymer Films in Response to Biorecognition

PubMed Central

Avens, Heather J.; Berron, Brad J.; May, Allison M.; Voigt, Katerina R.; Seedorf, Gregory J.; Balasubramaniam, Vivek; Bowman, Christopher N.

2011-01-01

Immunofluorescent staining is central to nearly all cell-based research, yet only a few fluorescent signal amplification approaches for cell staining exist, each with distinct limitations. Here, the authors present a novel, fluorescent polymerization-based amplification (FPBA) method that is shown to enable similar signal intensities as the highly sensitive, enzyme-based tyramide signal amplification (TSA) approach. Being non-enzymatic, FPBA is not expected to suffer from nonspecific staining of endogenous enzymes, as occurs with enzyme-based approaches. FPBA employs probes labeled with photopolymerization initiators, which lead to the controlled formation of fluorescent polymer films only at targeted biorecognition sites. Nuclear pore complex proteins (NPCs; in membranes), vimentin (in filaments), and von Willebrand factor (in granules) were all successfully immunostained by FPBA. Also, FPBA was demonstrated to be capable of multicolor immunostaining of multiple antigens. To assess relative sensitivity, decreasing concentrations of anti-NPC antibody were used, indicating that both FPBA and TSA stained NPC down to a 1:100,000 dilution. Nonspecific, cytoplasmic signal resulting from NPC staining was found to be reduced up to 5.5-fold in FPBA as compared to TSA, demonstrating better signal localization with FPBA. FPBA’s unique approach affords a combination of preferred attributes, including high sensitivity and specificity not otherwise available with current techniques. PMID:21339175

Nucleoporins redistribute inside the nucleus after cell cycle arrest induced by histone deacetylases inhibition.

PubMed

Pérez-Garrastachu, Miguel; Arluzea, Jon; Andrade, Ricardo; Díez-Torre, Alejandro; Urtizberea, Marta; Silió, Margarita; Aréchaga, Juan

2017-09-03

Nucleoporins are the main components of the nuclear-pore complex (NPC) and were initially considered as mere structural elements embedded in the nuclear envelope, being responsible for nucleocytoplasmic transport. Nevertheless, several recent scientific reports have revealed that some nucleoporins participate in nuclear processes such as transcription, replication, DNA repair and chromosome segregation. Thus, the interaction of NPCs with chromatin could modulate the distribution of chromosome territories relying on the epigenetic state of DNA. In particular, the nuclear basket proteins Tpr and Nup153, and the FG-nucleoporin Nup98 seem to play key roles in all these novel functions. In this work, histone deacetylase inhibitors (HDACi) were used to induce a hyperacetylated state of chromatin and the behavior of the mentioned nucleoporins was studied. Our results show that, after HDACi treatment, Tpr, Nup153 and Nup98 are translocated from the nuclear pore toward the interior of the cell nucleus, accumulating as intranuclear nucleoporin clusters. These transitory structures are highly dynamic, and are mainly present in the population of cells arrested at the G0/G1 phase of the cell cycle. Our results indicate that the redistribution of these nucleoporins from the nuclear envelope to the nuclear interior may be implicated in the early events of cell cycle initialization, particularly during the G1 phase transition.

Surface-plasmon enhanced photoemission of a silver nano-patterned photocathode

NASA Astrophysics Data System (ADS)

Zhang, Z.; Li, R.; To, H.; Andonian, G.; Pirez, E.; Meade, D.; Maxson, J.; Musumeci, P.

2017-09-01

Nano-patterned photocathodes (NPC) take advantage of plasmonic effects to resonantly increase absorption of light and localize electromagnetic field intensity on metal surfaces leading to surface-plasmon enhanced photoemission. In this paper, we report the status of NPC research at UCLA including in particular the optimization of the dimensions of a nanohole array on a silver wafer to enhance plasmonic response at 800 nm light, the development of a spectrally-resolved reflectivity measurement setup for quick nanopattern validation, and of a novel cathode plug to enable high power tests of NPCs on single crystal substrates in a high gradient radiofrequency gun.

Decision-making capacity for research participation among addicted people: a cross-sectional study.

PubMed

Morán-Sánchez, Inés; Luna, Aurelio; Sánchez-Muñoz, Maria; Aguilera-Alcaraz, Beatriz; Pérez-Cárceles, Maria D

2016-01-13

Informed consent is a key element of ethical clinical research. Addicted population may be at risk for impaired consent capacity. However, very little research has focused on their comprehension of consent forms. The aim of this study is to assess the capacity of addicted individuals to provide consent to research. 53 subjects with DSM-5 diagnoses of a Substance Use Disorder (SUD) and 50 non psychiatric comparison subjects (NPCs) participated in the survey from December 2014 to March 2015. This cross-sectional study was carried out at a community-based Outpatient Treatment Center and at an urban-located Health Centre in Spain. A binary judgment of capacity/incapacity was made guided by the MacArthur Competence Assessment Tool for Clinical Research (MacCAT-CR) and a clinical interview. Demographics and clinical characteristics were assessed by cases notes and the Mini-Mental State Examination, the Global Assessment Functional Scale and the Clinical Global Impression Scale. NPCs performed the best on the MacCAT-CR, and patients with SUD had the worst performance, particularly on the Understanding and Appreciation subscales. 32.7% SUD people lacked research-related decisional capacity. There were no statistically significant differences between the groups in terms of capacity to consent to research. The findings of our study provide evidence that a large proportion of individuals with SUD had decisional capacity for consent to research. It is therefore inappropriate to draw conclusions about capacity to make research decisions on the basis of a SUD diagnosis. In the absence of advanced cognitive impairment, acute withdrawal or intoxication, we should assume that addicted persons possess decision-making capacity. Thus, the view that people with SUD would ipso facto lose decision-making power for research consent is flawed and stigmatizing.

[North African and Southeast Asian nasopharyngeal carcinomas: between the resemblance and the dissemblance].

PubMed

Ayadi, W; Khabir, A; Hadhri-Guiga, B; Fki, L; Toumi, N; Siala, W; Charfi, S; Fendri, A; Makni, H; Boudawara, T; Ghorbel, A; Gargouri, A; Jlidi, R; Gargouri, R; Busson, P; Drira, M; Daoud, J; Frikha, M; Hammami, A; Karray-Hakim, H

2010-04-01

Nasopharyngeal carcinoma (NPC) is an unusual head and neck cancer because of its unequal geographical distribution and its consistent association with the Epstein-Barr virus (EBV). This malignant tumor poses a serious public health problem in many countries, especially in Southeast Asia and North Africa where the recorded incidence are highest. During the past decade, a growing number of studies were undertaken to define the molecular basis of NPC. However, the analysis of several clinical and biological parameters of North African and Southeast Asian NPCs has shown notable differences, suggesting that they could result from a distinct combination of etiological factors. One intriguing characteristic of North African NPC, concerns its bimodal age distribution with a secondary peak of incidence in the range of 15-25 years, not observed in Asian NPC. In this juvenile form of NPC, immuno-histochemistry assay has shown that the two key proteins controlling the apoptotic-survival balance p53 and Bcl-2 are less frequently expressed whereas the transmembrane tyrosine-kinase receptor c-kit and the main EBV oncoprotein LMP1 were more abundant. In addition, the EBV serological alterations are less informative for the diagnosis of the juvenile compared to the adult form. In addition, most North African NPCs contain EBV strains with genetic polymorphisms distinct from those described in the Southeast Asia series (predominance of F, D, H1-H2, XhoI+ and f, C, H, XhoI- respectively). In contrast, studies relating on tumor chromosomal alterations or aberrant promoter methylation result in data very similar to those obtained from the Southeast Asia series, supporting the concept of a common molecular basis for all NPC regardless of patient geographic origin.

Dimethyl Fumarate Protects Neural Stem/Progenitor Cells and Neurons from Oxidative Damage through Nrf2-ERK1/2 MAPK Pathway.

PubMed

Wang, Qin; Chuikov, Sergei; Taitano, Sophina; Wu, Qi; Rastogi, Arjun; Tuck, Samuel J; Corey, Joseph M; Lundy, Steven K; Mao-Draayer, Yang

2015-06-17

Multiple sclerosis (MS) is the most common multifocal inflammatory demyelinating disease of the central nervous system (CNS). Due to the progressive neurodegenerative nature of MS, developing treatments that exhibit direct neuroprotective effects are needed. Tecfidera™ (BG-12) is an oral formulation of the fumaric acid esters (FAE), containing the active metabolite dimethyl fumarate (DMF). Although BG-12 showed remarkable efficacy in lowering relapse rates in clinical trials, its mechanism of action in MS is not yet well understood. In this study, we reported the potential neuroprotective effects of dimethyl fumarate (DMF) on mouse and rat neural stem/progenitor cells (NPCs) and neurons. We found that DMF increased the frequency of the multipotent neurospheres and the survival of NPCs following oxidative stress with hydrogen peroxide (H2O2) treatment. In addition, utilizing the reactive oxygen species (ROS) assay, we showed that DMF reduced ROS production induced by H2O2. DMF also decreased oxidative stress-induced apoptosis. Using motor neuron survival assay, DMF significantly promoted survival of motor neurons under oxidative stress. We further analyzed the expression of oxidative stress-induced genes in the NPC cultures and showed that DMF increased the expression of transcription factor nuclear factor-erythroid 2-related factor 2 (Nrf2) at both levels of RNA and protein. Furthermore, we demonstrated the involvement of Nrf2-ERK1/2 MAPK pathway in DMF-mediated neuroprotection. Finally, we utilized SuperArray gene screen technology to identify additional anti-oxidative stress genes (Gstp1, Sod2, Nqo1, Srxn1, Fth1). Our data suggests that analysis of anti-oxidative stress mechanisms may yield further insights into new targets for treatment of multiple sclerosis (MS).

Effects of neural progenitor cells on post-stroke neurological impairment—a detailed and comprehensive analysis of behavioral tests

PubMed Central

Doeppner, Thorsten R.; Kaltwasser, Britta; Bähr, Mathias; Hermann, Dirk M.

2014-01-01

Systemic transplantation of neural progenitor cells (NPCs) in rodents reduces functional impairment after cerebral ischemia. In light of upcoming stroke trials regarding safety and feasibility of NPC transplantation, experimental studies have to successfully analyze the extent of NPC-induced neurorestoration on the functional level. However, appropriate behavioral tests for analysis of post-stroke motor coordination deficits and cognitive impairment after NPC grafting are not fully established. We therefore exposed male C57BL6 mice to either 45 min (mild) or 90 min (severe) of cerebral ischemia, using the thread occlusion model followed by intravenous injection of PBS or NPCs 6 h post-stroke with an observation period of three months. Post-stroke motor coordination was assessed by means of the rota rod, tight rope, corner turn, inclined plane, grip strength, foot fault, adhesive removal, pole test and balance beam test, whereas cognitive impairment was analyzed using the water maze, the open field and the passive avoidance test. Significant motor coordination differences after both mild and severe cerebral ischemia in favor of NPC-treated mice were observed for each motor coordination test except for the inclined plane and the grip strength test, which only showed significant differences after severe cerebral ischemia. Cognitive impairment after mild cerebral ischemia was successfully assessed using the water maze test, the open field and the passive avoidance test. On the contrary, the water maze test was not suitable in the severe cerebral ischemia paradigm, as it too much depends on motor coordination capabilities of test mice. In terms of both reliability and cost-effectiveness considerations, we thus recommend the corner turn, foot fault, balance beam, and open field test, which do not depend on durations of cerebral ischemia. PMID:25374509

Dimethyl Fumarate Protects Neural Stem/Progenitor Cells and Neurons from Oxidative Damage through Nrf2-ERK1/2 MAPK Pathway

PubMed Central

Wang, Qin; Chuikov, Sergei; Taitano, Sophina; Wu, Qi; Rastogi, Arjun; Tuck, Samuel J.; Corey, Joseph M.; Lundy, Steven K.; Mao-Draayer, Yang

2015-01-01

Multiple sclerosis (MS) is the most common multifocal inflammatory demyelinating disease of the central nervous system (CNS). Due to the progressive neurodegenerative nature of MS, developing treatments that exhibit direct neuroprotective effects are needed. Tecfidera™ (BG-12) is an oral formulation of the fumaric acid esters (FAE), containing the active metabolite dimethyl fumarate (DMF). Although BG-12 showed remarkable efficacy in lowering relapse rates in clinical trials, its mechanism of action in MS is not yet well understood. In this study, we reported the potential neuroprotective effects of dimethyl fumarate (DMF) on mouse and rat neural stem/progenitor cells (NPCs) and neurons. We found that DMF increased the frequency of the multipotent neurospheres and the survival of NPCs following oxidative stress with hydrogen peroxide (H2O2) treatment. In addition, utilizing the reactive oxygen species (ROS) assay, we showed that DMF reduced ROS production induced by H2O2. DMF also decreased oxidative stress-induced apoptosis. Using motor neuron survival assay, DMF significantly promoted survival of motor neurons under oxidative stress. We further analyzed the expression of oxidative stress-induced genes in the NPC cultures and showed that DMF increased the expression of transcription factor nuclear factor-erythroid 2-related factor 2 (Nrf2) at both levels of RNA and protein. Furthermore, we demonstrated the involvement of Nrf2-ERK1/2 MAPK pathway in DMF-mediated neuroprotection. Finally, we utilized SuperArray gene screen technology to identify additional anti-oxidative stress genes (Gstp1, Sod2, Nqo1, Srxn1, Fth1). Our data suggests that analysis of anti-oxidative stress mechanisms may yield further insights into new targets for treatment of multiple sclerosis (MS). PMID:26090715

Aging-Induced Nrf2-ARE Pathway Disruption in the Subventricular Zone Drives Neurogenic Impairment in Parkinsonian Mice via PI3K-Wnt/β-Catenin Dysregulation

PubMed Central

L’Episcopo, Francesca; Tirolo, Cataldo; Testa, Nunzio; Caniglia, Salvatore; Morale, Maria C.; Impagnatiello, Francesco; Pluchino, Stefano; Marchetti, Bianca

2013-01-01

Aging and exposure to environmental toxins including MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine) are strong risk factors for developing Parkinson’s disease (PD), a common neurologic disorder characterized by selective degeneration of midbrain dopaminergic (DAergic) neurons and astrogliosis. Aging and PD impair the subventricular zone (SVZ), one of the most important brain regions for adult neurogenesis. Because inflammation and oxidative stress are the hallmarks of aging and PD, we investigated the nature, timing, and signaling mechanisms contributing to aging-induced SVZ stem/neuroprogenitor cell (NPC) inhibition in aging male mice and attempted to determine to what extent manipulation of these pathways produces a functional response in the outcome of MPTP-induced DAergic toxicity. We herein reveal an imbalance of Nrf2-driven antioxidant/anti-inflammatory genes, such as Heme oxygenase1 in the SVZ niche, starting by middle age, amplified upon neurotoxin treatment and associated with an exacerbated proinflammatory SVZ microenvironment converging to dysregulate the Wingless-type MMTV integration site (Wnt)/β-catenin signaling, a key regulatory pathway for adult NPCs. In vitro experiments using coculture paradigms uncovered aged microglial proinflammatory mediators as critical inhibitors of NPC proliferative potential. We also found that interruption of PI3K (phosphatidylinositol 3-kinase)/Akt and the Wnt/Fzd/β-catenin signaling cascades, which switch glycogen synthase kinase 3β (GSK-3β) activation on and off, were causally related to the impairment of SVZ-NPCs. Moreover, a synergy between dysfunctional microglia of aging mice and MPTP exposure further inhibited astrocyte proneurogenic properties, including the expression of key Wnts components. Last, pharmacological activation/antagonism studies in vivo and in vitro suggest the potential that aged SVZ manipulation is associated with DAergic functional recovery. PMID:23345222

Developing Collective Training for Small Unmanned Aerial Systems Employment

NASA Technical Reports Server (NTRS)

Durlach, Paula J.; Priest, Heather; Martin, Glenn A.; Saffold, Jay

2010-01-01

The projected use of small unmanned aerial systems (SUAS) in military operations will produce training requirements which go beyond current capabilities. The paper describes the development of prototype training procedures and accompanying research simulations to address this need. We initially constructed a testbed to develop simulation-based training for an SUAS operator equipped with a simulated vertical-lift and land SUAS. However, the required training will go beyond merely training an operator how to pilot an SUAS. In addition to tactics, techniques, and procedures for employment of SUASs, collective training methods must be trained. Moreover, the leader of a unit equipped with SUAS will need to learn how to plan missions which incorporate the SUAS, and take into account air space and frequency management considerations. The demands of the task require the leader to allocate personnel to the SUAS mission, communicate and coordinate with those personnel during the mission, and make use of the information provided. To help address these training issues, we expanded our research testbed to include a command and control node (C2 node), to enable communications between a leader and the SUAS operator. In addition, we added a virtual environment in which dismounted infantry missions can be conducted. This virtual environment provides the opportunity for interactions among human-controlled avatars and non-player characters (NPCs), plus authoring tools to construct scenarios. Using these NPCs, a collective exercise involving friendly, enemy, and civilian personnel can be conducted without the need for a human role-player for every entity. We will describe the results of our first experiment, which examined the ability of players to negotiate use of the C2 node and the virtual environment at the same time, in order to see if this is a feasible combination of tools for training development.

Inhibition of Sirt1 promotes neural progenitors toward motoneuron differentiation from human embryonic stem cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zhang, Yun; Wang, Jing; Clinical Stem Cell Center, Peking University Third Hospital, 49 North Garden Road, Haidian District, Beijing 100191

2011-01-14

Research highlights: {yields} Nicotinamide inhibit Sirt1. {yields} MASH1 and Ngn2 activation. {yields} Increase the expression of HB9. {yields} Motoneurons formation increases significantly. -- Abstract: Several protocols direct human embryonic stem cells (hESCs) toward differentiation into functional motoneurons, but the efficiency of motoneuron generation varies based on the human ESC line used. We aimed to develop a novel protocol to increase the formation of motoneurons from human ESCs. In this study, we tested a nuclear histone deacetylase protein, Sirt1, to promote neural precursor cell (NPC) development during differentiation of human ESCs into motoneurons. A specific inhibitor of Sirt1, nicotinamide, dramatically increasedmore » motoneuron formation. We found that about 60% of the cells from the total NPCs expressed HB9 and {beta}III-tubulin, commonly used motoneuronal markers found in neurons derived from ESCs following nicotinamide treatment. Motoneurons derived from ESC expressed choline acetyltransferase (ChAT), a positive marker of mature motoneuron. Moreover, we also examined the transcript levels of Mash1, Ngn2, and HB9 mRNA in the differentiated NPCs treated with the Sirt1 activator resveratrol (50 {mu}M) or inhibitor nicotinamide (100 {mu}M). The levels of Mash1, Ngn2, and HB9 mRNA were significantly increased after nicotinamide treatment compared with control groups, which used the traditional protocol. These results suggested that increasing Mash1 and Ngn2 levels by inhibiting Sirt1 could elevate HB9 expression, which promotes motoneuron differentiation. This study provides an alternative method for the production of transplantable motoneurons, a key requirement in the development of hESC-based cell therapy in motoneuron disease.« less

Nucleoporins as components of the nuclear pore complex core structure and Tpr as the architectural element of the nuclear basket.

PubMed

Krull, Sandra; Thyberg, Johan; Björkroth, Birgitta; Rackwitz, Hans-Richard; Cordes, Volker C

2004-09-01

The vertebrate nuclear pore complex (NPC) is a macromolecular assembly of protein subcomplexes forming a structure of eightfold radial symmetry. The NPC core consists of globular subunits sandwiched between two coaxial ring-like structures of which the ring facing the nuclear interior is capped by a fibrous structure called the nuclear basket. By postembedding immunoelectron microscopy, we have mapped the positions of several human NPC proteins relative to the NPC core and its associated basket, including Nup93, Nup96, Nup98, Nup107, Nup153, Nup205, and the coiled coil-dominated 267-kDa protein Tpr. To further assess their contributions to NPC and basket architecture, the genes encoding Nup93, Nup96, Nup107, and Nup205 were posttranscriptionally silenced by RNA interference (RNAi) in HeLa cells, complementing recent RNAi experiments on Nup153 and Tpr. We show that Nup96 and Nup107 are core elements of the NPC proper that are essential for NPC assembly and docking of Nup153 and Tpr to the NPC. Nup93 and Nup205 are other NPC core elements that are important for long-term maintenance of NPCs but initially dispensable for the anchoring of Nup153 and Tpr. Immunogold-labeling for Nup98 also results in preferential labeling of NPC core regions, whereas Nup153 is shown to bind via its amino-terminal domain to the nuclear coaxial ring linking the NPC core structures and Tpr. The position of Tpr in turn is shown to coincide with that of the nuclear basket, with different Tpr protein domains corresponding to distinct basket segments. We propose a model in which Tpr constitutes the central architectural element that forms the scaffold of the nuclear basket.

Surface-plasmon enhanced photoemission of a silver nano-patterned photocathode

DOE PAGES

Zhang, Z.; Li, R.; To, H.; ...

2016-11-22

Here, nano-patterned photocathodes (NPC) take advantage of plasmonic effects to resonantly increase absorption of light and localize electromagnetic field intensity on metal surfaces leading to surface-plasmon enhanced photoemission. In this paper, we report the status of NPC research at UCLA including in particular the optimization of the dimensions of a nanohole array on a silver wafer to enhance plasmonic response at 800 nm light, the development of a spectrally-resolved reflectivity measurement setup for quick nanopattern validation, and of a novel cathode plug to enable high power tests of NPCs on single crystal substrates in a high gradient radiofrequency gun.

Surface-plasmon enhanced photoemission of a silver nano-patterned photocathode

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zhang, Z.; Li, R.; To, H.

Here, nano-patterned photocathodes (NPC) take advantage of plasmonic effects to resonantly increase absorption of light and localize electromagnetic field intensity on metal surfaces leading to surface-plasmon enhanced photoemission. In this paper, we report the status of NPC research at UCLA including in particular the optimization of the dimensions of a nanohole array on a silver wafer to enhance plasmonic response at 800 nm light, the development of a spectrally-resolved reflectivity measurement setup for quick nanopattern validation, and of a novel cathode plug to enable high power tests of NPCs on single crystal substrates in a high gradient radiofrequency gun.

Structural dynamics of the cell nucleus

PubMed Central

Wiegert, Simon; Bading, Hilmar

2011-01-01

Neuronal morphology plays an essential role in signal processing in the brain. Individual neurons can undergo use-dependent changes in their shape and connectivity, which affects how intracellular processes are regulated and how signals are transferred from one cell to another in a neuronal network. Calcium is one of the most important intracellular second messengers regulating cellular morphologies and functions. In neurons, intracellular calcium levels are controlled by ion channels in the plasma membrane such as NMDA receptors (NMDARs), voltage-gated calcium channels (VGCCs) and certain α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors (AMPARs) as well as by calcium exchange pathways between the cytosol and internal calcium stores including the endoplasmic reticulum and mitochondria. Synaptic activity and the subsequent opening of ligand and/or voltage-gated calcium channels can initiate cytosolic calcium transients which propagate towards the cell soma and enter the nucleus via its nuclear pore complexes (NPCs) embedded in the nuclear envelope. We recently described the discovery that in hippocampal neurons the morphology of the nucleus affects the calcium dynamics within the nucleus. Here we propose that nuclear infoldings determine whether a nucleus functions as an integrator or detector of oscillating calcium signals. We outline possible ties between nuclear mophology and transcriptional activity and discuss the importance of extending the approach to whole cell calcium signal modeling in order to understand synapse-to-nucleus communication in healthy and dysfunctional neurons. PMID:21738832

Oligonucleotide facilitators may inhibit or activate a hammerhead ribozyme.

PubMed Central

Jankowsky, E; Schwenzer, B

1996-01-01

Facilitators are oligonucleotides capable of affecting hammerhead ribozyme activity by interacting with the substrate at the termini of the ribozyme. Facilitator effects were determined in vitro using a system consisting of a ribozyme with 7 nucleotides in every stem sequence and two substrates with inverted facilitator binding sequences. The effects of 9mer and 12mer RNA as well as DNA facilitators which bind either adjacent to the 3'- or 5'-end of the ribozyme were investigated. A kinetic model was developed which allows determination of the apparent dissociation constant of the ribozyme-substrate complex from single turnover reactions. We observed a decreased dissociation constant of the ribozyme-substrate complex due to facilitator addition corresponding to an additional stabilization energy of delta delta G=-1.7 kcal/mol with 3'-end facilitators. The cleavage rate constant was increased by 3'-end facilitators and decreased by 5'-end facilitators. Values for Km were slightly lowered by all facilitators and kcat was increased by 3'-end facilitators and decreased by 5'-end facilitators in our system. Generally the facilitator effects increased with the length of the facilitators and RNA provided greater effects than DNA of the same sequence. Results suggest facilitator influences on several steps of the hammerhead reaction, substrate association, cleavage and dissociation of products. Moreover, these effects are dependent in different manners on ribozyme and substrate concentration. This leads to the conclusion that there is a concentration dependence whether activation or inhibition is caused by facilitators. Conclusions are drawn with regard to the design of hammerhead ribozyme facilitator systems. PMID:8602353

Facilitating complex shape drawing in Williams syndrome and typical development.

PubMed

Hudson, Kerry D; Farran, Emily K

2013-07-01

Individuals with Williams syndrome (WS) produce drawings that are disorganised, likely due to an inability to replicate numerous spatial relations between parts. This study attempted to circumvent these drawing deficits in WS when copying complex combinations of one, two and three shapes. Drawing decisions were reduced by introducing a number of facilitators, for example, by using distinct colours and including facilitatory cues on the response sheet. Overall, facilitation improved drawing in the WS group to a comparable level of accuracy as typically developing participants (matched for non-verbal ability). Drawing accuracy was greatest in both groups when planning demands (e.g. starting location, line lengths and changes in direction) were reduced by use of coloured figures and providing easily distinguished and clearly grouped facilitatory cues to form each shape. This study provides the first encouraging evidence to suggest that drawing of complex shapes in WS can be facilitated; individuals with WS might be receptive to remediation programmes for drawing and handwriting. Copyright © 2013 Elsevier Ltd. All rights reserved.

Enhanced expression of FNDC5 in human embryonic stem cell-derived neural cells along with relevant embryonic neural tissues.

PubMed

Ghahrizjani, Fatemeh Ahmadi; Ghaedi, Kamran; Salamian, Ahmad; Tanhaei, Somayeh; Nejati, Alireza Shoaraye; Salehi, Hossein; Nabiuni, Mohammad; Baharvand, Hossein; Nasr-Esfahani, Mohammad Hossein

2015-02-25

Availability of human embryonic stem cells (hESCs) has enhanced the capability of basic and clinical research in the context of human neural differentiation. Derivation of neural progenitor (NP) cells from hESCs facilitates the process of human embryonic development through the generation of neuronal subtypes. We have recently indicated that fibronectin type III domain containing 5 protein (FNDC5) expression is required for appropriate neural differentiation of mouse embryonic stem cells (mESCs). Bioinformatics analyses have shown the presence of three isoforms for human FNDC5 mRNA. To differentiate which isoform of FNDC5 is involved in the process of human neural differentiation, we have used hESCs as an in vitro model for neural differentiation by retinoic acid (RA) induction. The hESC line, Royan H5, was differentiated into a neural lineage in defined adherent culture treated by RA and basic fibroblast growth factor (bFGF). We collected all cell types that included hESCs, rosette structures, and neural cells in an attempt to assess the expression of FNDC5 isoforms. There was a contiguous increase in all three FNDC5 isoforms during the neural differentiation process. Furthermore, the highest level of expression of the isoforms was significantly observed in neural cells compared to hESCs and the rosette structures known as neural precursor cells (NPCs). High expression levels of FNDC5 in human fetal brain and spinal cord tissues have suggested the involvement of this gene in neural tube development. Additional research is necessary to determine the major function of FDNC5 in this process. Copyright © 2014 Elsevier B.V. All rights reserved.

Facilitated recycling protects human RNA polymerase III from repression by Maf1 in vitro.

PubMed

Cabart, Pavel; Lee, JaeHoon; Willis, Ian M

2008-12-26

Yeast cells synthesize approximately 3-6 million molecules of tRNA every cell cycle at a rate of approximately 2-4 transcripts/gene/s. This high rate of transcription is achieved through many rounds of reinitiation by RNA polymerase (pol) III on stable DNA-bound complexes of the initiation factor TFIIIB. Studies in yeast have shown that the rate of reinitiation is increased by facilitated recycling, a process that involves the repeated reloading of the polymerase on the same transcription unit. However, when nutrients become limiting or stress conditions are encountered, RNA pol III transcription is rapidly repressed through the action of the conserved Maf1 protein. Here we examine the relationship between Maf1-mediated repression and facilitated recycling in a human RNA pol III in vitro system. Using an immobilized template transcription assay, we demonstrate that facilitated recycling is conserved from yeast to humans. We assessed the ability of recombinant human Maf1 to inhibit different steps in transcription before and after preinitiation complex assembly. We show that recombinant Maf1 can inhibit the recruitment of TFIIIB and RNA pol III to immobilized templates. However, RNA pol III bound to preinitiation complexes or in elongation complexes is protected from repression by Maf1 and can undergo several rounds of initiation. This indicates that recombinant Maf1 is unable to inhibit facilitated recycling. The data suggest that additional biochemical steps may be necessary for rapid Maf1-dependent repression of RNA pol III transcription.

Does Adaptive Scaffolding Facilitate Students' Ability to Regulate their Learning with Hypermedia?

ERIC Educational Resources Information Center

Azevedo, Roger; Cromley, Jennifer G.; Seibert, Diane

2004-01-01

Is adaptive scaffolding effective in facilitating students' ability to regulate their learning of complex science topics with hypermedia? We examined the role of different scaffolding instructional interventions in facilitating students' shift to more sophisticated mental models as indicated by both performance and process data. Undergraduate…

Plasticity of Subventricular Zone Neuroprogenitors in MPTP (1-Methyl-4-Phenyl-1,2,3,6-Tetrahydropyridine) Mouse Model of Parkinson’s Disease Involves Cross Talk between Inflammatory and Wnt/β-Catenin Signaling Pathways: Functional Consequences for Neuroprotection and Repair

PubMed Central

L’Episcopo, Francesca; Tirolo, Cataldo; Testa, Nunzio; Caniglia, Salvatore; Morale, Maria C.; Deleidi, Michela; Serapide, Maria F.; Pluchino, Stefano; Marchetti, Bianca

2013-01-01

In Parkinson’s disease (PD), neurogenesis is impaired in the subventricular zone (SVZ) of postmortem human PD brains, in primate nonhuman and rodent models of PD. The vital role of Wingless-type MMTV integration site (Wnt)/β-catenin signaling in the modulation of neurogenesis, neuroprotection, and synaptic plasticity coupled to our recent findings uncovering an active role for inflammation and Wnt/β-catenin signaling in MPTP-induced loss and repair of nigrostriatal dopaminergic (DAergic) neurons prompted us to study the impact of neuroinflammation and the Wnt/β-catenin pathway in the response of SVZ neuroprogenitors (NPCs) in MPTP-treated mice. In vivo experiments, using bromodeoxyuridine and cell-specific markers, and ex vivo time course analyses documented an inverse correlation between the reduced proliferation of NPCs and the generation of new neuroblasts with the phase of maximal exacerbation of microglia reaction, whereas a shift in the microglia proinflammatory phenotype correlated with a progressive NPC recovery. Ex vivo and in vitro experiments using microglia–NPC coculture paradigms pointed to NADPH-oxidase (gpPHOX91), a major source of microglial ROS, and reactive nitrogen species as candidate inhibitors of NPC neurogenic potential via the activation of glycogen synthase 3 (pGSK-3βTyr216), leading to loss of β-catenin, a chief downstream transcriptional effector. Accordingly, MPTP/MPP+ (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine) caused β-catenin downregulation and pGSK-3βTyr216 overexpression, whereas manipulation of Wnt/β-catenin signaling with RNA interference-mediated GSK-3β knockdown or GSK-3β antagonism reversed MPTP-induced neurogenic impairment ex vivo/in vitro or in vivo. Reciprocally, pharmacological modulation of inflammation prevented β-catenin downregulation and restored neurogenesis, suggesting the possibility to modulate this endogenous system with potential consequences for DAergic neuroprotection and self-repair. PMID:22323720

Systemic Injection of Neural Stem/Progenitor Cells in Mice with Chronic EAE

PubMed Central

Donegà, Matteo; Giusto, Elena; Cossetti, Chiara; Schaeffer, Julia; Pluchino, Stefano

2014-01-01

Neural stem/precursor cells (NPCs) are a promising stem cell source for transplantation approaches aiming at brain repair or restoration in regenerative neurology. This directive has arisen from the extensive evidence that brain repair is achieved after focal or systemic NPC transplantation in several preclinical models of neurological diseases. These experimental data have identified the cell delivery route as one of the main hurdles of restorative stem cell therapies for brain diseases that requires urgent assessment. Intraparenchymal stem cell grafting represents a logical approach to those pathologies characterized by isolated and accessible brain lesions such as spinal cord injuries and Parkinson's disease. Unfortunately, this principle is poorly applicable to conditions characterized by a multifocal, inflammatory and disseminated (both in time and space) nature, including multiple sclerosis (MS). As such, brain targeting by systemic NPC delivery has become a low invasive and therapeutically efficacious protocol to deliver cells to the brain and spinal cord of rodents and nonhuman primates affected by experimental chronic inflammatory damage of the central nervous system (CNS). This alternative method of cell delivery relies on the NPC pathotropism, specifically their innate capacity to (i) sense the environment via functional cell adhesion molecules and inflammatory cytokine and chemokine receptors; (ii) cross the leaking anatomical barriers after intravenous (i.v.) or intracerebroventricular (i.c.v.) injection; (iii) accumulate at the level of multiple perivascular site(s) of inflammatory brain and spinal cord damage; and (i.v.) exert remarkable tissue trophic and immune regulatory effects onto different host target cells in vivo. Here we describe the methods that we have developed for the i.v. and i.c.v. delivery of syngeneic NPCs in mice with experimental autoimmune encephalomyelitis (EAE), as model of chronic CNS inflammatory demyelination, and envisage the systemic stem cell delivery as a valuable technique for the selective targeting of the inflamed brain in regenerative neurology. PMID:24798882

Abstinence following Alcohol Drinking Produces Depression-Like Behavior and Reduced Hippocampal Neurogenesis in Mice

PubMed Central

Stevenson, Jennie R; Schroeder, Jason P; Nixon, Kimberly; Besheer, Joyce; Crews, Fulton T; Hodge, Clyde W

2010-01-01

Alcoholism and depression show high degrees of comorbidity. Clinical evidence also indicates that depression that emerges during abstinence from chronic alcohol use has a greater negative impact on relapse than pre-existing depression. Although no single neurobiological mechanism can account for the behavioral pathologies associated with these devastating disorders, converging evidence suggests that aspects of both alcoholism and depression are linked to reductions in hippocampal neurogenesis. Here, we report results from a novel preclinical behavioral model showing that abstinence from voluntary alcohol drinking leads to the emergence of depression-like behavior and reductions in neurogenesis. C57BL/6J mice were allowed to self-administer ethanol (10% v/v) vs H2O in the home cage for 28 days. Alcohol was then removed for 1 or 14 days, and mice were tested in the forced swim test to measure depression-like behavior. After 14 days, but not 1 day of abstinence from alcohol drinking, mice showed a significant increase in depression-like behavior. The significant increase in depression-like behavior during abstinence was associated with a reduction in proliferating cell nuclear antigen (PCNA) and doublecortin (DCX) immunoreactivity in the dentate gyrus of the hippocampus indicating that both the number of proliferating neural progenitor cells (NPC) and immature neurons were reduced, respectively. The number of NPCs that were labeled with bromo-deoxyuridine (BrdU) at the beginning of alcohol exposure was not altered indicating that survival of NPCs is not linked to abstinence-induced depression. Chronic treatment (14 days) with the antidepressant desipramine during abstinence prevented both the emergence of depression-like behavior and the reduction in hippocampal neurogenesis indicating that abstinence-induced depression is associated with structural plasticity in the hippocampus. Overall, the results of this study support the conclusion that profound functional (ie behavioral) and structural changes occur during abstinence from alcohol use and suggest that antidepressant treatment may alleviate some of these pathological neurobehavioral adaptations. PMID:18563059

Comparing the performance and preference of students experiencing a Reading Aloud Accommodation to those who do not on a virtual science assessment

NASA Astrophysics Data System (ADS)

Shelton, Angela

Many United States secondary students perform poorly on standardized summative science assessments. Situated Assessments using Virtual Environments (SAVE) Science is an innovative assessment project that seeks to capture students' science knowledge and understanding by contextualizing problems in a game-based virtual environment called Scientopolis. Within Scientopolis, students use an "avatar" to interact with non-player characters (NPCs), artifacts, embedded clues and "sci-tools" in order to help solve the problems of the townspeople. In an attempt to increase students' success on assessments, SAVE science places students in an environment where they can use their inquiry skills to solve problems instead of reading long passages which attempt to contextualize questions but ultimately cause construct-irrelevant variance. However, within these assessments reading is still required to access the test questions and character interactions. This dissertation explores how students' in-world performances differ when exposed to a Reading Aloud Accommodation (RAA) treatment in comparison to a control group. Student perceptions of the treatment are also evaluated. While a RAA is typically available for students with learning disabilities or English language learners, within this study, all students were randomly assigned to either the treatment or control, regardless of any demographic factors or learning barriers. The theories of Universal design for learning and brain-based learning advocate for multiple ways for students to engage, comprehend, and illustrate their content knowledge. Further, through providing more ways for students to interact with content, all students should benefit, not just those with learning disabilities. Students in the experimental group listened to the NPCs speak the dialogue that provides them with the problem, clues, and assessment questions, instead of relying on reading skills to gather the information. Overall, students in the treatment group statistically outperformed those in the control. Student perceptions of using the reading aloud accommodation were generally positive. Ideas for future research are presented to investigate the accommodation further.

Neural progenitor cell implants modulate vascular endothelial growth factor and brain-derived neurotrophic factor expression in rat axotomized neurons.

PubMed

Talaverón, Rocío; Matarredona, Esperanza R; de la Cruz, Rosa R; Pastor, Angel M

2013-01-01

Axotomy of central neurons leads to functional and structural alterations which largely revert when neural progenitor cells (NPCs) are implanted in the lesion site. The new microenvironment created by NPCs in the host tissue might modulate in the damaged neurons the expression of a high variety of molecules with relevant roles in the repair mechanisms, including neurotrophic factors. In the present work, we aimed to analyze changes in neurotrophic factor expression in axotomized neurons induced by NPC implants. For this purpose, we performed immunofluorescence followed by confocal microscopy analysis for the detection of vascular endothelial growth factor (VEGF), brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3) and nerve growth factor (NGF) on brainstem sections from rats with axotomy of abducens internuclear neurons that received NPC implants (implanted group) or vehicle injections (axotomized group) in the lesion site. Control abducens internuclear neurons were strongly immunoreactive to VEGF and BDNF but showed a weak staining for NT-3 and NGF. Comparisons between groups revealed that lesioned neurons from animals that received NPC implants showed a significant increase in VEGF content with respect to animals receiving vehicle injections. However, the immunoreactivity for BDNF, which was increased in the axotomized group as compared to control, was not modified in the implanted group. The modifications induced by NPC implants on VEGF and BDNF content were specific for the population of axotomized abducens internuclear neurons since the neighboring abducens motoneurons were not affected. Similar levels of NT-3 and NGF immunolabeling were obtained in injured neurons from axotomized and implanted animals. Among all the analyzed neurotrophic factors, only VEGF was expressed by the implanted cells in the lesion site. Our results point to a role of NPC implants in the modulation of neurotrophic factor expression by lesioned central neurons, which might contribute to the restorative effects of these implants.

Effects of cell type and configuration on anabolic and catabolic activity in 3D co-culture of mesenchymal stem cells and nucleus pulposus cells.

PubMed

Ouyang, Ann; Cerchiari, Alec E; Tang, Xinyan; Liebenberg, Ellen; Alliston, Tamara; Gartner, Zev J; Lotz, Jeffrey C

2017-01-01

Tissue engineering constructs to treat intervertebral disc degeneration must adapt to the hypoxic and inflammatory degenerative disc microenvironment. The objective of this study was to determine the effects of two key design factors, cell type and cell configuration, on the regenerative potential of nucleus pulposus cell (NPC) and mesenchymal stem cell (MSC) constructs. Anabolic and catabolic activity was quantified in constructs of varying cell type (NPCs, MSCs, and a 50:50 co-culture) and varying configuration (individual cells and micropellets). Anabolic and catabolic outcomes were both dependent on cell type. Gene expression of Agg and Col2A1, glycosaminoglycan (GAG) content, and aggrecan immunohistochemistry (IHC), were significantly higher in NPC-only and co-culture groups than in MSC-only groups, with NPC-only groups exhibiting the highest anabolic gene expression levels. However, NPC-only constructs also responded to inflammation and hypoxia with significant upregulation of catabolic genes (MMP-1, MMP-9, MMP-13, and ADAMTS-5). MSC-only groups were unaffected by degenerative media conditions, and co-culture with MSCs modulated catabolic induction of the NPCs. Culturing cells in a micropellet configuration dramatically reduced catabolic induction in co-culture and NPC-only groups. Co-culture micropellets, which take advantage of both cell type and configuration effects, had the most immunomodulatory response, with a significant decrease in MMP-13 and ADAMTS-5 expression in hypoxic and inflammatory media conditions. Co-culture micropellets were also found to self-organize into bilaminar formations with an MSC core and NPC outer layer. Further understanding of these cell type and configuration effects can improve tissue engineering designs. © 2016 The Authors. Journal of Orthopaedic Research published by Wiley Periodicals, Inc. on behalf of the Orthopaedic Research Society. J Orthop Res 35:61-73, 2017. © 2016 The Authors. Journal of Orthopaedic Research published by Wiley Periodicals, Inc. on behalf of the Orthopaedic Research Society.

Can training in advanced clinical skills in obstetrics, neonatal care and leadership, of non-physician clinicians in Malawi impact on clinical services improvements (the ETATMBA project): a process evaluation

PubMed Central

Ellard, David R; Chimwaza, Wanangwa; Davies, David; O'Hare, Joseph Paul; Kamwendo, Francis; Quenby, Siobhan; Griffiths, Frances

2014-01-01

Objectives The ‘enhancing human resources and the use of appropriate technologies for maternal and perinatal survival in sub-Saharan Africa’ (ETATMBA) project is training emergency obstetric and new-born care (EmONC) non-physician clinicians (NPCs) as advanced clinical leaders. Our objectives were to evaluate the implementation and changes to practice. Design A mixed methods process evaluation with the predominate methodology being qualitative. Setting Rural and urban hospitals in 8 of the 14 districts of northern and central Malawi. Participants 54 EmONC NPCs with 3 years’ plus experience. Intervention Training designed and delivered by clinicians from the UK and Malawi; it is a 2-year plus package of training (classroom, mentorship and assignments). Results We conducted 79 trainee interviews over three time points during the training, as well as a convenience sample of 10 colleagues, 7 district officers and 2 UK obstetricians. Trainees worked in a context of substantial variation in the rates of maternal and neonatal deaths between districts. Training reached trainees working across the target regions. For 46 trainees (8 dropped out of the course), dose delivered in terms of attendance was high and all 46 spent time working alongside an obstetrician. In early interviews trainees recalled course content unprompted indicating training had been received. Colleagues and district officers reported cascading of knowledge and initial changes in practice indicating early implementation. By asking trainees to describe actual cases we found they had implemented new knowledge and skills. These included life-saving interventions for postpartum haemorrhage and eclampsia. Trainees identified the leadership training as enabling them to confidently change their own practice and initiate change in their health facility. Conclusions This process evaluation suggests that trainees have made positive changes in their practice. Clear impacts on maternal and perinatal mortality are yet to be elucidated. PMID:25116455

Complementarity of stability patches at the interfaces of protein complexes: Implication for the structural organization of energetic hot spots.

PubMed

Kuttner, Yosef Y; Engel, Stanislav

2018-02-01

A rational design of protein complexes with defined functionalities and of drugs aimed at disrupting protein-protein interactions requires fundamental understanding of the mechanisms underlying the formation of specific protein complexes. Efforts to develop efficient small-molecule or protein-based binders often exploit energetic hot spots on protein surfaces, namely, the interfacial residues that provide most of the binding free energy in the complex. The molecular basis underlying the unusually high energy contribution of the hot spots remains obscure, and its elucidation would facilitate the design of interface-targeted drugs. To study the nature of the energetic hot spots, we analyzed the backbone dynamic properties of contact surfaces in several protein complexes. We demonstrate that, in most complexes, the backbone dynamic landscapes of interacting surfaces form complementary "stability patches," in which static areas from the opposing surfaces superimpose, and that these areas are predominantly located near the geometric center of the interface. We propose that a diminished enthalpy-entropy compensation effect augments the degree to which residues positioned within the complementary stability patches contribute to complex affinity, thereby giving rise to the energetic hot spots. These findings offer new insights into the nature of energetic hot spots and the role that backbone dynamics play in facilitating intermolecular recognition. Mapping the interfacial stability patches may provide guidance for protein engineering approaches aimed at improving the stability of protein complexes and could facilitate the design of ligands that target complex interfaces. © 2017 Wiley Periodicals, Inc.

Methods to Succeed in Effective Knowledge Translation in Clinical Practice.

PubMed

Kitson, Alison L; Harvey, Gillian

2016-05-01

To explore the evidence around facilitation as an intervention for the successful implementation of new knowledge into clinical practice. The revised version of the Promoting Action on Research Implementation in Health Services (PARIHS) framework, called the integrated or i-PARIHS framework, is used as the explanatory framework. This framework posits that evidence is a multidimensional construct embedded within innovation and operationalized by clinicians (individuals and within teams), working across multiple layers of context. Facilitation is the active ingredient that promotes successful implementation. An emerging body of evidence supports facilitation as a mechanism to getting new knowledge into clinical practice. Facilitation roles are divided into beginner, experienced, and expert facilitators. Facilitators can be internal or external to the organization they work in, and their skills and attributes complement other knowledge translation (KT) roles. Complex KT projects require facilitators who are experienced in implementation methods. Facilitation is positioned as the active ingredient to effectively introduce new knowledge into a clinical setting. Levels of facilitation experience are assessed in relation to the complexity of the KT task. Three core facilitation roles are identified, and structured interventions are established taking into account the nature and novelty of the evidence, the receptiveness of the clinicians, and the context or setting where the new evidence is to be introduced. Roles such as novice, experienced, and expert facilitators have important and complementary parts to play in enabling the successful translation of evidence into everyday practice in order to provide effective care for patients. © 2016 Sigma Theta Tau International.

The potential of sarcospan in adhesion complex replacement therapeutics for the treatment of muscular dystrophy

PubMed Central

Marshall, Jamie L.; Kwok, Yukwah; McMorran, Brian; Baum, Linda G.; Crosbie-Watson, Rachelle H.

2013-01-01

Three adhesion complexes span the sarcolemma and facilitate critical connections between the extracellular matrix and the actin cytoskeleton: the dystrophin- and utrophin-glycoprotein complexes and α7β1 integrin. Loss of individual protein components results in a loss of the entire protein complex and muscular dystrophy. Muscular dystrophy is a progressive, lethal wasting disease characterized by repetitive cycles of myofiber degeneration and regeneration. Protein replacement therapy offers a promising approach for the treatment of muscular dystrophy. Recently, we demonstrated that sarcospan facilitates protein-protein interactions amongst the adhesion complexes and is an important therapeutic target. Here, we review current protein replacement strategies, discuss the potential benefits of sarcospan expression, and identify important experiments that must be addressed for sarcospan to move to the clinic. PMID:23601082

The Race that Precedes Coactivation: Development of Multisensory Facilitation in Children

ERIC Educational Resources Information Center

Barutchu, Ayla; Crewther, David P.; Crewther, Sheila G.

2009-01-01

Rationale: The facilitating effect of multisensory integration on motor responses in adults is much larger than predicted by race-models and is in accordance with the idea of coactivation. However, the development of multisensory facilitation of endogenously driven motor processes and its relationship to the development of complex cognitive skills…

Teaching Note--Constructing Critical Conversations: A Model for Facilitating Classroom Dialogue for Critical Learning

ERIC Educational Resources Information Center

Kang, Hye-Kyung; O'Neill, Peggy

2018-01-01

Discussions of power and privilege, oppression, and structural inequities in classrooms can produce complex understanding and critical analysis when facilitated effectively. In this article we present the critical conversations model for facilitating conversations that open up space for discussing such issues and encourage the development of…

Paper SERS chromatography for detection of trace analytes in complex samples

NASA Astrophysics Data System (ADS)

Yu, Wei W.; White, Ian M.

2013-05-01

We report the application of paper SERS substrates for the detection of trace quantities of multiple analytes in a complex sample in the form of paper chromatography. Paper chromatography facilitates the separation of different analytes from a complex sample into distinct sections in the chromatogram, which can then be uniquely identified using SERS. As an example, the separation and quantitative detection of heroin in a highly fluorescent mixture is demonstrated. Paper SERS chromatography has obvious applications, including law enforcement, food safety, and border protection, and facilitates the rapid detection of chemical and biological threats at the point of sample.

Co-dependence between trypanosome nuclear lamina components in nuclear stability and control of gene expression.

PubMed

Maishman, Luke; Obado, Samson O; Alsford, Sam; Bart, Jean-Mathieu; Chen, Wei-Ming; Ratushny, Alexander V; Navarro, Miguel; Horn, David; Aitchison, John D; Chait, Brian T; Rout, Michael P; Field, Mark C

2016-12-15

The nuclear lamina is a filamentous structure subtending the nuclear envelope and required for chromatin organization, transcriptional regulation and maintaining nuclear structure. The trypanosomatid coiled-coil NUP-1 protein is a lamina component functionally analogous to lamins, the major lamina proteins of metazoa. There is little evidence for shared ancestry, suggesting the presence of a distinct lamina system in trypanosomes. To find additional trypanosomatid lamina components we identified NUP-1 interacting proteins by affinity capture and mass-spectrometry. Multiple components of the nuclear pore complex (NPC) and a second coiled-coil protein, which we termed NUP-2, were found. NUP-2 has a punctate distribution at the nuclear periphery throughout the cell cycle and is in close proximity to NUP-1, the NPCs and telomeric chromosomal regions. RNAi-mediated silencing of NUP-2 leads to severe proliferation defects, gross alterations to nuclear structure, chromosomal organization and nuclear envelope architecture. Further, transcription is altered at telomere-proximal variant surface glycoprotein (VSG) expression sites (ESs), suggesting a role in controlling ES expression, although NUP-2 silencing does not increase VSG switching. Transcriptome analysis suggests specific alterations to Pol I-dependent transcription. NUP-1 is mislocalized in NUP-2 knockdown cells and vice versa, implying that NUP-1 and NUP-2 form a co-dependent network and identifying NUP-2 as a second trypanosomatid nuclear lamina component. © The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.

Nup100 regulates Saccharomyces cerevisiae replicative life span by mediating the nuclear export of specific tRNAs

PubMed Central

Lord, Christopher L.; Ospovat, Ophir; Wente, Susan R.

2017-01-01

Nuclear pore complexes (NPCs), which are composed of nucleoporins (Nups) and regulate transport between the nucleus and cytoplasm, significantly impact the replicative life span (RLS) of Saccharomyces cerevisiae. We previously reported that deletion of the nonessential gene NUP100 increases RLS, although the molecular basis for this effect was unknown. In this study, we find that nuclear tRNA accumulation contributes to increased longevity in nup100Δ cells. Fluorescence in situ hybridization (FISH) experiments demonstrate that several specific tRNAs accumulate in the nuclei of nup100Δ mutants. Protein levels of the transcription factor Gcn4 are increased when NUP100 is deleted, and GCN4 is required for the elevated life spans of nup100Δ mutants, similar to other previously described tRNA export and ribosomal mutants. Northern blots indicate that tRNA splicing and aminoacylation are not significantly affected in nup100Δ cells, suggesting that Nup100 is largely required for nuclear export of mature, processed tRNAs. Distinct tRNAs accumulate in the nuclei of nup100Δ and msn5Δ mutants, while Los1-GFP nucleocytoplasmic shuttling is unaffected by Nup100. Thus, we conclude that Nup100 regulates tRNA export in a manner distinct from Los1 or Msn5. Together, these experiments reveal a novel Nup100 role in the tRNA life cycle that impacts the S. cerevisiae life span. PMID:27932586

Structural dynamics of the cell nucleus: basis for morphology modulation of nuclear calcium signaling and gene transcription.

PubMed

Queisser, Gillian; Wiegert, Simon; Bading, Hilmar

2011-01-01

Neuronal morphology plays an essential role in signal processing in the brain. Individual neurons can undergo use-dependent changes in their shape and connectivity, which affects how intracellular processes are regulated and how signals are transferred from one cell to another in a neuronal network. Calcium is one of the most important intracellular second messengers regulating cellular morphologies and functions. In neurons, intracellular calcium levels are controlled by ion channels in the plasma membrane such as NMDA receptors (NMDARs), voltage-gated calcium channels (VGCCs) and certain α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors (AMPARs) as well as by calcium exchange pathways between the cytosol and internal calcium stores including the endoplasmic reticulum and mitochondria. Synaptic activity and the subsequent opening of ligand and/or voltage-gated calcium channels can initiate cytosolic calcium transients which propagate towards the cell soma and enter the nucleus via its nuclear pore complexes (NPCs) embedded in the nuclear envelope. We recently described the discovery that in hippocampal neurons the morphology of the nucleus affects the calcium dynamics within the nucleus. Here we propose that nuclear infoldings determine whether a nucleus functions as an integrator or detector of oscillating calcium signals. We outline possible ties between nuclear mophology and transcriptional activity and discuss the importance of extending the approach to whole cell calcium signal modeling in order to understand synapse-to-nucleus communication in healthy and dysfunctional neurons.

Complexation facilitated reduction of aromatic N-oxides by aqueous Fe(II)-tiron complex: reaction kinetics and mechanisms.

PubMed

Chen, Yiling; Zhang, Huichun

2013-10-01

Rapid reduction of carbadox (CDX), olaquindox and several other aromatic N-oxides were investigated in aqueous solution containing Fe(II) and tiron. Consistent with previous work, the 1:2 Fe(II)-tiron complex, FeL2(6-), is the dominant reactive species as its concentration linearly correlates with the observed rate constant kobs under various conditions. The N-oxides without any side chains were much less reactive, suggesting direct reduction of the N-oxides is slow. UV-vis spectra suggest FeL2(6-) likely forms 5- or 7-membered rings with CDX and olaquindox through the N and O atoms on the side chain. The formed inner-sphere complexes significantly facilitated electron transfer from FeL2(6-) to the N-oxides. Reduction products of the N-oxides were identified by HPLC/QToF-MS to be the deoxygenated analogs. QSAR analysis indicated neither the first electron transfer nor N-O bond cleavage is the rate-limiting step. Calculations of the atomic spin densities of the anionic N-oxides confirmed the extensive delocalization between the aromatic ring and the side chain, suggesting complex formation can significantly affect the reduction kinetics. Our results suggest the complexation facilitated N-oxide reduction by Fe(II)-tiron involves a free radical mechanism, and the subsequent deoxygenation might also benefit from the weak complexation of Fe(II) with the N-oxide O atom.

SWI/SNF interacts with cleavage and polyadenylation factors and facilitates pre-mRNA 3' end processing.

PubMed

Yu, Simei; Jordán-Pla, Antonio; Gañez-Zapater, Antoni; Jain, Shruti; Rolicka, Anna; Östlund Farrants, Ann-Kristin; Visa, Neus

2018-05-31

SWI/SNF complexes associate with genes and regulate transcription by altering the chromatin at the promoter. It has recently been shown that these complexes play a role in pre-mRNA processing by associating at alternative splice sites. Here, we show that SWI/SNF complexes are involved also in pre-mRNA 3' end maturation by facilitating 3' end cleavage of specific pre-mRNAs. Comparative proteomics show that SWI/SNF ATPases interact physically with subunits of the cleavage and polyadenylation complexes in fly and human cells. In Drosophila melanogaster, the SWI/SNF ATPase Brahma (dBRM) interacts with the CPSF6 subunit of cleavage factor I. We have investigated the function of dBRM in 3' end formation in S2 cells by RNA interference, single-gene analysis and RNA sequencing. Our data show that dBRM facilitates pre-mRNA cleavage in two different ways: by promoting the association of CPSF6 to the cleavage region and by stabilizing positioned nucleosomes downstream of the cleavage site. These findings show that SWI/SNF complexes play a role also in the cleavage of specific pre-mRNAs in animal cells.

The DNA damage checkpoint pathway promotes extensive resection and nucleotide synthesis to facilitate homologous recombination repair and genome stability in fission yeast.

PubMed

Blaikley, Elizabeth J; Tinline-Purvis, Helen; Kasparek, Torben R; Marguerat, Samuel; Sarkar, Sovan; Hulme, Lydia; Hussey, Sharon; Wee, Boon-Yu; Deegan, Rachel S; Walker, Carol A; Pai, Chen-Chun; Bähler, Jürg; Nakagawa, Takuro; Humphrey, Timothy C

2014-05-01

DNA double-strand breaks (DSBs) can cause chromosomal rearrangements and extensive loss of heterozygosity (LOH), hallmarks of cancer cells. Yet, how such events are normally suppressed is unclear. Here we identify roles for the DNA damage checkpoint pathway in facilitating homologous recombination (HR) repair and suppressing extensive LOH and chromosomal rearrangements in response to a DSB. Accordingly, deletion of Rad3(ATR), Rad26ATRIP, Crb2(53BP1) or Cdc25 overexpression leads to reduced HR and increased break-induced chromosome loss and rearrangements. We find the DNA damage checkpoint pathway facilitates HR, in part, by promoting break-induced Cdt2-dependent nucleotide synthesis. We also identify additional roles for Rad17, the 9-1-1 complex and Chk1 activation in facilitating break-induced extensive resection and chromosome loss, thereby suppressing extensive LOH. Loss of Rad17 or the 9-1-1 complex results in a striking increase in break-induced isochromosome formation and very low levels of chromosome loss, suggesting the 9-1-1 complex acts as a nuclease processivity factor to facilitate extensive resection. Further, our data suggest redundant roles for Rad3ATR and Exo1 in facilitating extensive resection. We propose that the DNA damage checkpoint pathway coordinates resection and nucleotide synthesis, thereby promoting efficient HR repair and genome stability. © The Author(s) 2014. Published by Oxford University Press.

The potential of sarcospan in adhesion complex replacement therapeutics for the treatment of muscular dystrophy.

PubMed

Marshall, Jamie L; Kwok, Yukwah; McMorran, Brian J; Baum, Linda G; Crosbie-Watson, Rachelle H

2013-09-01

Three adhesion complexes span the sarcolemma and facilitate critical connections between the extracellular matrix and the actin cytoskeleton: the dystrophin- and utrophin-glycoprotein complexes and α7β1 integrin. Loss of individual protein components results in a loss of the entire protein complex and muscular dystrophy. Muscular dystrophy is a progressive, lethal wasting disease characterized by repetitive cycles of myofiber degeneration and regeneration. Protein-replacement therapy offers a promising approach for the treatment of muscular dystrophy. Recently, we demonstrated that sarcospan facilitates protein-protein interactions amongst the adhesion complexes and is an important potential therapeutic target. Here, we review current protein-replacement strategies, discuss the potential benefits of sarcospan expression, and identify important experiments that must be addressed for sarcospan to move to the clinic. © 2013 FEBS.

Cyclophilins facilitate dissociation of the human papillomavirus type 16 capsid protein L1 from the L2/DNA complex following virus entry.

PubMed

Bienkowska-Haba, Malgorzata; Williams, Carlyn; Kim, Seong Man; Garcea, Robert L; Sapp, Martin

2012-09-01

Human papillomaviruses (HPV) are composed of the major and minor capsid proteins, L1 and L2, that encapsidate a chromatinized, circular double-stranded DNA genome. At the outset of infection, the interaction of HPV type 16 (HPV16) (pseudo)virions with heparan sulfate proteoglycans triggers a conformational change in L2 that is facilitated by the host cell chaperone cyclophilin B (CyPB). This conformational change results in exposure of the L2 N terminus, which is required for infectious internalization. Following internalization, L2 facilitates egress of the viral genome from acidified endosomes, and the L2/DNA complex accumulates at PML nuclear bodies. We recently described a mutant virus that bypasses the requirement for cell surface CyPB but remains sensitive to cyclosporine for infection, indicating an additional role for CyP following endocytic uptake of virions. We now report that the L1 protein dissociates from the L2/DNA complex following infectious internalization. Inhibition and small interfering RNA (siRNA)-mediated knockdown of CyPs blocked dissociation of L1 from the L2/DNA complex. In vitro, purified CyPs facilitated the dissociation of L1 pentamers from recombinant HPV11 L1/L2 complexes in a pH-dependent manner. Furthermore, CyPs released L1 capsomeres from partially disassembled HPV16 pseudovirions at slightly acidic pH. Taken together, these data suggest that CyPs mediate the dissociation of HPV L1 and L2 capsid proteins following acidification of endocytic vesicles.

Cyclophilins Facilitate Dissociation of the Human Papillomavirus Type 16 Capsid Protein L1 from the L2/DNA Complex following Virus Entry

PubMed Central

Bienkowska-Haba, Malgorzata; Williams, Carlyn; Kim, Seong Man; Garcea, Robert L.

2012-01-01

Human papillomaviruses (HPV) are composed of the major and minor capsid proteins, L1 and L2, that encapsidate a chromatinized, circular double-stranded DNA genome. At the outset of infection, the interaction of HPV type 16 (HPV16) (pseudo)virions with heparan sulfate proteoglycans triggers a conformational change in L2 that is facilitated by the host cell chaperone cyclophilin B (CyPB). This conformational change results in exposure of the L2 N terminus, which is required for infectious internalization. Following internalization, L2 facilitates egress of the viral genome from acidified endosomes, and the L2/DNA complex accumulates at PML nuclear bodies. We recently described a mutant virus that bypasses the requirement for cell surface CyPB but remains sensitive to cyclosporine for infection, indicating an additional role for CyP following endocytic uptake of virions. We now report that the L1 protein dissociates from the L2/DNA complex following infectious internalization. Inhibition and small interfering RNA (siRNA)-mediated knockdown of CyPs blocked dissociation of L1 from the L2/DNA complex. In vitro, purified CyPs facilitated the dissociation of L1 pentamers from recombinant HPV11 L1/L2 complexes in a pH-dependent manner. Furthermore, CyPs released L1 capsomeres from partially disassembled HPV16 pseudovirions at slightly acidic pH. Taken together, these data suggest that CyPs mediate the dissociation of HPV L1 and L2 capsid proteins following acidification of endocytic vesicles. PMID:22761365

Argumentation: A Methodology to Facilitate Critical Thinking.

PubMed

Makhene, Agnes

2017-06-20

Caring is a difficult nursing activity that involves a complex nature of a human being in need of complex decision-making and problem solving through the critical thinking process. It is mandatory that critical thinking is facilitated in general and in nursing education particularly in order to render care in diverse multicultural patient care settings. This paper aims to describe how argumentation can be used to facilitate critical thinking in learners. A qualitative, exploratory and descriptive design that is contextual was used. Purposive sampling method was used to draw a sample and Miles and Huberman methodology of qualitative analysis was used to analyse data. Lincoln and Guba's strategies were employed to ensure trustworthiness, while Dhai and McQuoid-Mason's principles of ethical consideration were used. Following data analysis the findings were integrated within literature which culminated into the formulation of guidelines that can be followed when using argumentation as a methodology to facilitate critical thinking.

75 FR 2172 - Self-Regulatory Organizations; Chicago Board Options Exchange, Incorporated; Notice of Filing and...

Federal Register 2010, 2011, 2012, 2013, 2014

2010-01-14

... complex order auction (COA) and book (COB) rule. The COA system facilitates the handling and execution of complex orders by allowing for complex orders to rest in the system and allowing for inbound complex... order), and the stock component of a stock-option complex order handled by the system is executed on...

Body shape helps legged robots climb and turn in complex 3-D terrains

NASA Astrophysics Data System (ADS)

Han, Yuanfeng; Wang, Zheliang; Li, Chen

Analogous to streamlined shapes that reduce drag in fluids, insects' ellipsoid-like rounded body shapes were recently discovered to be ``terradynamically streamlined'' and enhance locomotion in cluttered terrain by facilitating body rolling. Here, we hypothesize that there exist more terradynamic shapes that facilitate other modes of locomotion like climbing and turning in complex 3-D terrains by facilitating body pitching and yawing. To test our hypothesis, we modified the body shape of a legged robot by adding an elliptical and a rectangular shell and tested how it negotiated with circular and square vertical pillars. With a rectangular shell the robot always pitched against square pillars in an attempt to climb, whereas with an elliptical shell it always yawed and turned away from circular pillars given a small initial lateral displacement. Square / circular pillars facilitated pitching / yawing, respectively. To begin to reveal the contact physics, we developed a locomotion energy landscape model. Our model revealed that potential energy barriers to transition from pitching to yawing are high for angular locomotor and obstacle shapes (rectangular / square) but vanish for rounded shapes (elliptical / circular). Our study supports the plausibility of locomotion energy landscapes for understanding the rich locomotor transitions in complex 3-D terrains.

Mediation: a response to aid-in-dying and the Supreme Court decision.

PubMed

Saulo, M; Wagener, R J; Rothschild, I S

1998-01-01

The recent U.S. Supreme Court decision concerning aid-in-dying has drawn attention to the complexity of end-of-life care. The authors summarize the recent Supreme Court's decision and the problems surrounding this complex issue. A case study is provided to demonstrate how mediation facilitates collaborative problem solving. Finally, the authors demonstrate how nurse leaders can apply this three-stage process and its attendant principles to facilitate ethical decision making in end-of-life care.

Evaluation of the tratment of metal-EDTA complexes using Ti0{sub 2} photocatalysis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Madden, T.; Datyte, A.K.; Prairie, M.R.

1996-03-01

This study has demonstrated the feasibility of TiO{sub 2} photocatalysis to treat EDTA and several metal-EDTA complexes that can be found in industrial wastewaters. For the EDTA complexes of metals capable of photodeposition, such as Cu and Pb, certain reaction conditions were shown to facilitate the simultaneous complex degradation and photodeposition of these metals onto the catalyst. With metals that do not easily photodeposit, such as Ni and Cd, it is shown that the complex degradation is still facilitated, and can enhance other metals removal processes after photocatalytic treatment. Because the treatment of these metal-EDTA complexes typically requires special measures,more » there may exist situations where TiO{sub 2} photocatalysis could actually be the preferred method of treatment. However, its use should be compared economically to other more established advanced oxidation technologies. This necessity is demonstrated in the economic comparison to ozone treatment for EDTA degradation alone, where ozone treatment appears to be the clear choice in this application.« less

BLOC-1 Interacts with BLOC-2 and the AP-3 Complex to Facilitate Protein Trafficking on Endosomes

PubMed Central

Di Pietro, Santiago M.; Falcón-Pérez, Juan M.; Tenza, Danièle; Setty, Subba R.G.; Marks, Michael S.; Raposo, Graça

2006-01-01

The adaptor protein (AP)-3 complex is a component of the cellular machinery that controls protein sorting from endosomes to lysosomes and specialized related organelles such as melanosomes. Mutations in an AP-3 subunit underlie a form of Hermansky-Pudlak syndrome (HPS), a disorder characterized by abnormalities in lysosome-related organelles. HPS in humans can also be caused by mutations in genes encoding subunits of three complexes of unclear function, named biogenesis of lysosome-related organelles complex (BLOC)-1, -2, and -3. Here, we report that BLOC-1 interacts physically and functionally with AP-3 to facilitate the trafficking of a known AP-3 cargo, CD63, and of tyrosinase-related protein 1 (Tyrp1), a melanosomal membrane protein previously thought to traffic only independently of AP-3. BLOC-1 also interacts with BLOC-2 to facilitate Tyrp1 trafficking by a mechanism apparently independent of AP-3 function. Both BLOC-1 and -2 localize mainly to early endosome-associated tubules as determined by immunoelectron microscopy. These findings support the idea that BLOC-1 and -2 represent hitherto unknown components of the endosomal protein trafficking machinery. PMID:16837549

Primary brain tumors, neural stem cell, and brain tumor cancer cells: where is the link?

PubMed Central

Germano, Isabelle; Swiss, Victoria; Casaccia, Patrizia

2010-01-01

The discovery of brain tumor-derived cells (BTSC) with the properties of stem cells has led to the formulation of the hypothesis that neural stem cells could be the cell of origin of primary brain tumors (PBT). In this review we present the most common molecular changes in PBT, define the criteria of identification of BTSC and discuss the similarities between the characteristics of these cells and those of the endogenous population of neural stem cells (NPCs) residing in germinal areas of the adult brain. Finally, we propose possible mechanisms of cancer initiation and progression and suggest a model of tumor initiation that includes intrinsic changes of resident NSC and potential changes in the microenvironment defining the niche where the NSC reside. PMID:20045420

[Algorithms for treatment of complex hand injuries].

PubMed

Pillukat, T; Prommersberger, K-J

2011-07-01

The primary treatment strongly influences the course and prognosis of hand injuries. Complex injuries which compromise functional recovery are especially challenging. Despite an apparently unlimited number of injury patterns it is possible to develop strategies which facilitate a standardized approach to operative treatment. In this situation algorithms can be important guidelines for a rational approach. The following algorithms have been proven in the treatment of complex injuries of the hand by our own experience. They were modified according to the current literature and refer to prehospital care, emergency room management, basic strategy in general and reconstruction of bone and joints, vessels, nerves, tendons and soft tissue coverage in detail. Algorithms facilitate the treatment of severe hand injuries. Applying simple yes/no decisions complex injury patterns are split into distinct partial problems which can be managed step by step.

Implementation of a web-based national child health-care programme in a local context: A complex facilitator role.

PubMed

Tell, Johanna; Olander, Ewy; Anderberg, Peter; Berglund, Johan Sanmartin

2018-02-01

The aim of this study was to investigate child health-care coordinators' experiences of being a facilitator for the implementation of a new national child health-care programme in the form of a web-based national guide. The study was based on eight remote, online focus groups, using Skype for Business. A qualitative content analysis was performed. The analysis generated three categories: adapt to a local context, transition challenges and led by strong incentives. There were eight subcategories. In the latent analysis, the theme 'Being a facilitator: a complex role' was formed to express the child health-care coordinators' experiences. Facilitating a national guideline or decision support in a local context is a complex task that requires an advocating and mediating role. For successful implementation, guidelines and decision support, such as a web-based guide and the new child health-care programme, must match professional consensus and needs and be seen as relevant by all. Participation in the development and a strong bottom-up approach was important, making the web-based guide and the programme relevant to whom it is intended to serve, and for successful implementation. The study contributes valuable knowledge when planning to implement a national web-based decision support and policy programme in a local health-care context.

Structure of UreG/UreF/UreH Complex Reveals How Urease Accessory Proteins Facilitate Maturation of Helicobacter pylori Urease

PubMed Central

Fong, Yu Hang; Wong, Ho Chun; Yuen, Man Hon; Lau, Pak Ho; Chen, Yu Wai; Wong, Kam-Bo

2013-01-01

Urease is a metalloenzyme essential for the survival of Helicobacter pylori in acidic gastric environment. Maturation of urease involves carbamylation of Lys219 and insertion of two nickel ions at its active site. This process requires GTP hydrolysis and the formation of a preactivation complex consisting of apo-urease and urease accessory proteins UreF, UreH, and UreG. UreF and UreH form a complex to recruit UreG, which is a SIMIBI class GTPase, to the preactivation complex. We report here the crystal structure of the UreG/UreF/UreH complex, which illustrates how UreF and UreH facilitate dimerization of UreG, and assembles its metal binding site by juxtaposing two invariant Cys66-Pro67-His68 metal binding motif at the interface to form the (UreG/UreF/UreH)2 complex. Interaction studies revealed that addition of nickel and GTP to the UreG/UreF/UreH complex releases a UreG dimer that binds a nickel ion at the dimeric interface. Substitution of Cys66 and His68 with alanine abolishes the formation of the nickel-charged UreG dimer. This nickel-charged UreG dimer can activate urease in vitro in the presence of the UreF/UreH complex. Static light scattering and atomic absorption spectroscopy measurements demonstrated that the nickel-charged UreG dimer, upon GTP hydrolysis, reverts to its monomeric form and releases nickel to urease. Based on our results, we propose a mechanism on how urease accessory proteins facilitate maturation of urease. PMID:24115911

Inhibition of oxidative metabolism leads to p53 genetic inactivation and transformation in neural stem cells

PubMed Central

Bartesaghi, Stefano; Graziano, Vincenzo; Galavotti, Sara; Henriquez, Nick V.; Betts, Joanne; Saxena, Jayeta; Minieri, Valentina; A, Deli; Karlsson, Anna; Martins, L. Miguel; Capasso, Melania; Nicotera, Pierluigi; Brandner, Sebastian; De Laurenzi, Vincenzo; Salomoni, Paolo

2015-01-01

Alterations of mitochondrial metabolism and genomic instability have been implicated in tumorigenesis in multiple tissues. High-grade glioma (HGG), one of the most lethal human neoplasms, displays genetic modifications of Krebs cycle components as well as electron transport chain (ETC) alterations. Furthermore, the p53 tumor suppressor, which has emerged as a key regulator of mitochondrial respiration at the expense of glycolysis, is genetically inactivated in a large proportion of HGG cases. Therefore, it is becoming evident that genetic modifications can affect cell metabolism in HGG; however, it is currently unclear whether mitochondrial metabolism alterations could vice versa promote genomic instability as a mechanism for neoplastic transformation. Here, we show that, in neural progenitor/stem cells (NPCs), which can act as HGG cell of origin, inhibition of mitochondrial metabolism leads to p53 genetic inactivation. Impairment of respiration via inhibition of complex I or decreased mitochondrial DNA copy number leads to p53 genetic loss and a glycolytic switch. p53 genetic inactivation in ETC-impaired neural stem cells is caused by increased reactive oxygen species and associated oxidative DNA damage. ETC-impaired cells display a marked growth advantage in the presence or absence of oncogenic RAS, and form undifferentiated tumors when transplanted into the mouse brain. Finally, p53 mutations correlated with alterations in ETC subunit composition and activity in primary glioma-initiating neural stem cells. Together, these findings provide previously unidentified insights into the relationship between mitochondria, genomic stability, and tumor suppressive control, with implications for our understanding of brain cancer pathogenesis. PMID:25583481

A Programmable DNA Origami Platform for Organizing Intrinsically Disordered Nucleoporins within Nanopore Confinement.

PubMed

Fisher, Patrick D Ellis; Shen, Qi; Akpinar, Bernice; Davis, Luke K; Chung, Kenny Kwok Hin; Baddeley, David; Šarić, Anđela; Melia, Thomas J; Hoogenboom, Bart W; Lin, Chenxiang; Lusk, C Patrick

2018-02-27

Nuclear pore complexes (NPCs) form gateways that control molecular exchange between the nucleus and the cytoplasm. They impose a diffusion barrier to macromolecules and enable the selective transport of nuclear transport receptors with bound cargo. The underlying mechanisms that establish these permeability properties remain to be fully elucidated but require unstructured nuclear pore proteins rich in Phe-Gly (FG)-repeat domains of different types, such as FxFG and GLFG. While physical modeling and in vitro approaches have provided a framework for explaining how the FG network contributes to the barrier and transport properties of the NPC, it remains unknown whether the number and/or the spatial positioning of different FG-domains along a cylindrical, ∼40 nm diameter transport channel contributes to their collective properties and function. To begin to answer these questions, we have used DNA origami to build a cylinder that mimics the dimensions of the central transport channel and can house a specified number of FG-domains at specific positions with easily tunable design parameters, such as grafting density and topology. We find the overall morphology of the FG-domain assemblies to be dependent on their chemical composition, determined by the type and density of FG-repeat, and on their architectural confinement provided by the DNA cylinder, largely consistent with here presented molecular dynamics simulations based on a coarse-grained polymer model. In addition, high-speed atomic force microscopy reveals local and reversible FG-domain condensation that transiently occludes the lumen of the DNA central channel mimics, suggestive of how the NPC might establish its permeability properties.

Nup100 regulates Saccharomyces cerevisiae replicative life span by mediating the nuclear export of specific tRNAs.

PubMed

Lord, Christopher L; Ospovat, Ophir; Wente, Susan R

2017-03-01

Nuclear pore complexes (NPCs), which are composed of nucleoporins (Nups) and regulate transport between the nucleus and cytoplasm, significantly impact the replicative life span (RLS) of Saccharomyces cerevisiae We previously reported that deletion of the nonessential gene NUP100 increases RLS, although the molecular basis for this effect was unknown. In this study, we find that nuclear tRNA accumulation contributes to increased longevity in nup100 Δ cells. Fluorescence in situ hybridization (FISH) experiments demonstrate that several specific tRNAs accumulate in the nuclei of nup100 Δ mutants. Protein levels of the transcription factor Gcn4 are increased when NUP100 is deleted, and GCN4 is required for the elevated life spans of nup100 Δ mutants, similar to other previously described tRNA export and ribosomal mutants. Northern blots indicate that tRNA splicing and aminoacylation are not significantly affected in nup100 Δ cells, suggesting that Nup100 is largely required for nuclear export of mature, processed tRNAs. Distinct tRNAs accumulate in the nuclei of nup100 Δ and msn5 Δ mutants, while Los1-GFP nucleocytoplasmic shuttling is unaffected by Nup100. Thus, we conclude that Nup100 regulates tRNA export in a manner distinct from Los1 or Msn5. Together, these experiments reveal a novel Nup100 role in the tRNA life cycle that impacts the S. cerevisiae life span. © 2017 Lord et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society.

Netrin1 establishes multiple boundaries for axon growth in the developing spinal cord.

PubMed

Varadarajan, Supraja G; Butler, Samantha J

2017-10-01

The canonical model for netrin1 function proposed that it acted as a long-range chemotropic axon guidance cue. In the developing spinal cord, floor-plate (FP)-derived netrin1 was thought to act as a diffusible attractant to draw commissural axons to the ventral midline. However, our recent studies have shown that netrin1 is dispensable in the FP for axon guidance. We have rather found that netrin1 acts locally: netrin1 is produced by neural progenitor cells (NPCs) in the ventricular zone (VZ), and deposited on the pial surface as a haptotactic adhesive substrate that guides Dcc + axon growth. Here, we further demonstrate that this netrin1 pial-substrate has an early role orienting pioneering spinal axons, directing them to extend ventrally. However, as development proceeds, commissural axons choose to grow around a boundary of netrin1 expressing cells in VZ, instead of continuing to extend alongside the netrin1 pial-substrate in the ventral spinal cord. This observation suggests netrin1 may supply a more complex activity than pure adhesion, with netrin1-expressing cells also supplying a growth boundary for axons. Supporting this possibility, we have observed that additional domains of netrin1 expression arise adjacent to the dorsal root entry zone (DREZ) in E12.5 mice that are also required to sculpt axonal growth. Together, our studies suggest that netrin1 provides "hederal" boundaries: a local growth substrate that promotes axon extension, while also preventing local innervation of netrin1-expressing domains. Copyright © 2017 Elsevier Inc. All rights reserved.

Nestin is essential for zebrafish brain and eye development through control of progenitor cell apoptosis.

PubMed

Chen, Hua-Ling; Yuh, Chiou-Hwa; Wu, Kenneth K

2010-02-19

Nestin is expressed in neural progenitor cells (NPC) of developing brain. Despite its wide use as an NPC marker, the function of nestin in embryo development is unclear. As nestin is conserved in zebrafish and its predicted sequence is clustered with the mammalian nestin orthologue, we used zebrafish as a model to investigate its role in embryogenesis. Injection of nestin morpholino (MO) into fertilized eggs induced time- and dose-dependent brain and eye developmental defects. Nestin morphants exhibited characteristic morphological changes including small head, small eyes and hydrocephalus. Histological examinations show reduced hind- and mid-brain size, dilated ventricle, poorly organized retina and underdeveloped lens. Injection of control nestin MO did not induce brain or eye changes. Nestin MO injection reduced expression of ascl1b (achaete-scute complex-like 1b), a marker of NPCs, without affecting its distribution. Nestin MO did not influence Elavl3/4 (Embryonic lethal, abnormal vision, Drosophila-like 3/4) (a neuronal marker), or otx2 (a midbrain neuronal marker), but severely perturbed cranial motor nerve development and axon distribution. To determine whether the developmental defects are due to excessive NPC apoptosis and/or reduced NPC proliferation, we analyzed apoptosis by TUNEL assay and acridine orange staining and proliferation by BrdU incorporation, pcna and mcm5 expressions. Excessive apoptosis was noted in hindbrain and midbrain cells. Apoptotic signals were colocalized with ascl1b. Proliferation markers were not significantly altered by nestin MO. These results suggest that nestin is essential for zebrafish brain and eye development probably through control of progenitor cell apoptosis.

Facilitating Argumentative Knowledge Construction through a Transactive Discussion Script in CSCL

ERIC Educational Resources Information Center

Noroozi, Omid; Weinberger, Armin; Biemans, Harm J. A.; Mulder, Martin; Chizari, Mohammad

2013-01-01

Learning to argue is prerequisite to solving complex problems in groups, especially when they are multidisciplinary and collaborate online. Environments for Computer-Supported Collaborative Learning (CSCL) can be designed to facilitate argumentative knowledge construction. This study investigates how argumentative knowledge construction in…

Addressing Complex Challenges through Adaptive Leadership: A Promising Approach to Collaborative Problem Solving

ERIC Educational Resources Information Center

Nelson, Tenneisha; Squires, Vicki

2017-01-01

Organizations are faced with solving increasingly complex problems. Addressing these issues requires effective leadership that can facilitate a collaborative problem solving approach where multiple perspectives are leveraged. In this conceptual paper, we critique the effectiveness of earlier leadership models in tackling complex organizational…

The role of the equatorial ligands for the redox behavior, mode of cellular accumulation and cytotoxicity of platinum(IV) prodrugs.

PubMed

Göschl, Simone; Varbanov, Hristo P; Theiner, Sarah; Jakupec, Michael A; Galanski, Markus; Keppler, Bernhard K

2016-07-01

The current study aims to elucidate the possible reasons for the significantly different pharmacological behavior of platinum(IV) complexes with cisplatin-, carboplatin- or nedaplatin-like cores and how this difference can be related to their main physicochemical properties. Chlorido-containing complexes are reduced fast (within hours) by ascorbate and are able to unwind plasmid DNA in the presence of ascorbate, while their tri- and tetracarboxylato analogs are generally inert under the same conditions. Comparison of the lipophilicity, cellular accumulation and cytotoxicity of the investigated platinum compounds revealed the necessity to define new structure-property/activity relationships (SPRs and SARs). The higher activity and improved accumulation of platinum(IV) complexes bearing Cl(-) in equatorial position cannot only be attributed to passive diffusion facilitated by their lipophilicity. Therefore, further platinum accumulation experiments under conditions where active/facilitated transport mechanisms are suppressed were performed. Under hypothermic conditions (4°C), accumulation of dichloridoplatinum(IV) complexes is reduced down to 10% of the amount determined at 37°C. These findings suggest the involvement of active and/or facilitated transport in cellular uptake of platinum(IV) complexes with a cisplatin-like core. Studies with ATP depletion mediated by oligomycin and low glucose partially confirmed these observations, but their feasibility was severely limited in the adherent cell culture setting. Copyright © 2016 Elsevier Inc. All rights reserved.

Can training in advanced clinical skills in obstetrics, neonatal care and leadership, of non-physician clinicians in Malawi impact on clinical services improvements (the ETATMBA project): a process evaluation.

PubMed

Ellard, David R; Chimwaza, Wanangwa; Davies, David; O'Hare, Joseph Paul; Kamwendo, Francis; Quenby, Siobhan; Griffiths, Frances

2014-08-12

The 'enhancing human resources and the use of appropriate technologies for maternal and perinatal survival in sub-Saharan Africa' (ETATMBA) project is training emergency obstetric and new-born care (EmONC) non-physician clinicians (NPCs) as advanced clinical leaders. Our objectives were to evaluate the implementation and changes to practice. A mixed methods process evaluation with the predominate methodology being qualitative. Rural and urban hospitals in 8 of the 14 districts of northern and central Malawi. 54 EmONC NPCs with 3 years' plus experience. Training designed and delivered by clinicians from the UK and Malawi; it is a 2-year plus package of training (classroom, mentorship and assignments). We conducted 79 trainee interviews over three time points during the training, as well as a convenience sample of 10 colleagues, 7 district officers and 2 UK obstetricians. Trainees worked in a context of substantial variation in the rates of maternal and neonatal deaths between districts. Training reached trainees working across the target regions. For 46 trainees (8 dropped out of the course), dose delivered in terms of attendance was high and all 46 spent time working alongside an obstetrician. In early interviews trainees recalled course content unprompted indicating training had been received. Colleagues and district officers reported cascading of knowledge and initial changes in practice indicating early implementation. By asking trainees to describe actual cases we found they had implemented new knowledge and skills. These included life-saving interventions for postpartum haemorrhage and eclampsia. Trainees identified the leadership training as enabling them to confidently change their own practice and initiate change in their health facility. This process evaluation suggests that trainees have made positive changes in their practice. Clear impacts on maternal and perinatal mortality are yet to be elucidated. Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://group.bmj.com/group/rights-licensing/permissions.

Cataract surgery in Southern Ethiopia: distribution, rates and determinants of service provision.

PubMed

Habtamu, Esmael; Eshete, Zebiba; Burton, Matthew J

2013-11-19

Cataract is the leading cause of blindness worldwide, with the greatest burden found in low-income countries. Cataract surgery is a curative and cost-effective intervention. Despite major non-governmental organization (NGO) support, the cataract surgery performed in Southern Region, Ethiopia is currently insufficient to address the need. We analyzed the distribution, productivity, cost and determinants of cataract surgery services. Confidential interviews were conducted with all eye surgeons (Ophthalmologists & Non-Physician Cataract Surgeons [NPCS]) in Southern Region using semi-structured questionnaires. Eye care project managers were interviewed using open-ended qualitative questionnaires. All eye units were visited. Information on resources, costs, and the rates and determinants of surgical output were collected. Cataract surgery provision is uneven across Southern Region: 66% of the units are within 200 km of the regional capital. Surgeon to population ratios varied widely from 1:70,000 in the capital to no service provision in areas containing 7 million people. The Cataract Surgical Rate (CSR) in 2010 was 406 operations/million/year with zonal CSRs ranging between 204 and 1349. Average number of surgeries performed was 374 operations/surgeon/year. Ophthalmologists and NPCS performed a mean of 682 and 280 cataract operations/surgeon/year, respectively (p = 0.03). Resources are underutilized, at 56% of capacity. Community awareness programs were associated with increased activity (p = 0.009). Several factors were associated with increased surgeon productivity (p < 0.05): working for >2 years, working in a NGO/private clinic, working in an urban unit, having a unit manger, conducting outreach programs and a satisfactory work environment. The average cost of cataract surgery in 2010 was US$141.6 (Range: US$37.6-312.6). Units received >70% of their consumables from NGOs. Mangers identified poor staff motivation, community awareness and limited government support as major challenges. The uneven distribution of infrastructure and personnel, underutilization by the community and inadequate attention and support from the government are limiting cataract surgery service delivery in Southern Ethiopia. Improved human resource management and implementing community-oriented strategies may help increase surgical output and achieve the "Vision 2020: The Right to Sight" targets for treating avoidable blindness.

Structural organization of the inactive X chromosome in the mouse

PubMed Central

Giorgetti, Luca; Lajoie, Bryan R.; Carter, Ava C.; Attia, Mikael; Zhan, Ye; Xu, Jin; Chen, Chong Jian; Kaplan, Noam; Chang, Howard Y.; Heard, Edith; Dekker, Job

2017-01-01

X-chromosome inactivation (XCI) involves major reorganization of the X chromosome as it becomes silent and heterochromatic. During female mammalian development, XCI is triggered by upregulation of the non-coding Xist RNA from one of the two X chromosomes. Xist coats the chromosome in cis and induces silencing of almost all genes via its A-repeat region1,2, although some genes (constitutive escapees) avoid silencing in most cell types, and others (facultative escapees) escape XCI only in specific contexts3. A role for Xist in organizing the inactive X (Xi) chromosome has been proposed4–6. Recent chromosome conformation capture approaches have revealed global loss of local structure on the Xi chromosome and formation of large mega-domains, separated by a region containing the DXZ4 macrosatellite7–10. However, the molecular architecture of the Xi chromosome, in both the silent and expressed regions, remains unclear. Here we investigate the structure, chromatin accessibility and expression status of the mouse Xi chromosome in highly polymorphic clonal neural progenitors (NPCs) and embryonic stem cells. We demonstrate a crucial role for Xist and the DXZ4-containing boundary in shaping Xi chromosome structure using allele-specific genome-wide chromosome conformation capture (Hi-C) analysis, an assay for transposase-accessible chromatin with high throughput sequencing (ATAC–seq) and RNA sequencing. Deletion of the boundary disrupts mega-domain formation, and induction of Xist RNA initiates formation of the boundary and the loss of DNA accessibility. We also show that in NPCs, the Xi chromosome lacks active/inactive compartments and topologically associating domains (TADs), except around genes that escape XCI. Escapee gene clusters display TAD-like structures and retain DNA accessibility at promoter-proximal and CTCF-binding sites. Furthermore, altered patterns of facultative escape genes in different neural progenitor clones are associated with the presence of different TAD-like structures after XCI. These findings suggest a key role for transcription and CTCF in the formation of TADs in the context of the Xi chromosome in neural progenitors. PMID:27437574

Distinct pattern of Th17/Treg cells in pregnant women with a history of unexplained recurrent spontaneous abortion.

PubMed

Qian, Jinfeng; Zhang, Na; Lin, Jing; Wang, Caiyan; Pan, Xinyao; Chen, Lanting; Li, Dajin; Wang, Ling

2018-05-13

The aim of the current study was to determine the pattern of immune cells and related functional molecules in peripheral blood and at the maternal-fetal interface in women with unexplained recurrent spontaneous abortion (URSA). In part I, 155 women were included and divided into four groups: non-pregnant controls with no history of URSA (NPCs), pregnant controls with no history of URSA (PCs), non-pregnant women with a history of URSA (NPUs), and pregnant women with a history of URSA (PUs). Venous blood samples were collected and analyzed. In part II, 35 subjects with URSA and 40 subjects in the early stage of normal pregnancy who chose to undergo an abortion were recruited. Samples of the decidua were collected, and the proportion of immune cells and the expression of related molecules were evaluated. Peripheral regulatory T cells (Treg cells) increased in PCs compared to NPCs, but in women with URSA the flux of Treg cells disappeared when pregnancy occurred. Levels of interleukin-10 (IL-10), cytotoxic T lymphocyte-associated antigen 4 (CTLA-4), and IL-17 and the ratio of Th17/Treg cells in peripheral blood remained stable among the four groups. At the maternal-fetal interface, the percentage of Treg cells, the level of CTLA-4 of CD4 + CD25 + CD127 lo cells and CD4 + Foxp3 + cells were significantly lower in women with URSA compared to controls, respectively. Levels of transforming growth factor-β1 (TGF-β1) mRNA and protein in the decidua significantly decreased in URSA while levels of IL-6 and tumor necrosis factor-ɑ (TNF-ɑ) and the Th17/Treg ratio significantly increased. In conclusion, peripheral Treg cells did not increase in pregnant women with URSA. The decrease in Treg cells and levels of CTLA-4 and TGF-β1 and as well as the increase in levels of IL-6 and TNF-ɑ, and the Th17/Treg ratio at the maternal-fetal interface might contribute to inappropriate maternal-fetal immune tolerance in URSA.

Managing Postsocialist Transitions: Politicized Sense Making as a Facilitator of Organizational Change

ERIC Educational Resources Information Center

Tiplic, Dijana

2011-01-01

This study explores what organizational strategies are employed to initiate and facilitate organizational change in higher education institutions in the increasingly complex and competitive postsocialist environment of Bosnia-Herzegovina. By studying organizations trapped between their inert socialist-era legacies and desired organizational…

Modeling complex tone perception: grouping harmonics with combination-sensitive neurons.

PubMed

Medvedev, Andrei V; Chiao, Faye; Kanwal, Jagmeet S

2002-06-01

Perception of complex communication sounds is a major function of the auditory system. To create a coherent precept of these sounds the auditory system may instantaneously group or bind multiple harmonics within complex sounds. This perception strategy simplifies further processing of complex sounds and facilitates their meaningful integration with other sensory inputs. Based on experimental data and a realistic model, we propose that associative learning of combinations of harmonic frequencies and nonlinear facilitation of responses to those combinations, also referred to as "combination-sensitivity," are important for spectral grouping. For our model, we simulated combination sensitivity using Hebbian and associative types of synaptic plasticity in auditory neurons. We also provided a parallel tonotopic input that converges and diverges within the network. Neurons in higher-order layers of the network exhibited an emergent property of multifrequency tuning that is consistent with experimental findings. Furthermore, this network had the capacity to "recognize" the pitch or fundamental frequency of a harmonic tone complex even when the fundamental frequency itself was missing.

Cross-Linking and Mass Spectrometry Methodologies to Facilitate Structural Biology: Finding a Path through the Maze

DOE Office of Scientific and Technical Information (OSTI.GOV)

Merkley, Eric D.; Cort, John R.; Adkins, Joshua N.

2013-09-01

Multiprotein complexes, rather than individual proteins, make up a large part of the biological macromolecular machinery of a cell. Understanding the structure and organization of these complexes is critical to understanding cellular function. Chemical cross-linking coupled with mass spectrometry is emerging as a complementary technique to traditional structural biology methods and can provide low-resolution structural information for a multitude of purposes, such as distance constraints in computational modeling of protein complexes. In this review, we discuss the experimental considerations for successful application of chemical cross-linking-mass spectrometry in biological studies and highlight three examples of such studies from the recent literature.more » These examples (as well as many others) illustrate the utility of a chemical cross-linking-mass spectrometry approach in facilitating structural analysis of large and challenging complexes.« less

Visual Complexity and Pictorial Memory: A Fifteen Year Research Perspective.

ERIC Educational Resources Information Center

Berry, Louis H.

For 15 years an ongoing research project at the University of Pittsburgh has focused on the effects of variations in visual complexity and color on the storage and retrieval of visual information by learners. Research has shown that visual materials facilitate instruction, but has not fully delineated the interactions of visual complexity and…

Multi-enzyme complexes on DNA scaffolds capable of substrate channelling with an artificial swinging arm

NASA Astrophysics Data System (ADS)

Fu, Jinglin; Yang, Yuhe Renee; Johnson-Buck, Alexander; Liu, Minghui; Liu, Yan; Walter, Nils G.; Woodbury, Neal W.; Yan, Hao

2014-07-01

Swinging arms are a key functional component of multistep catalytic transformations in many naturally occurring multi-enzyme complexes. This arm is typically a prosthetic chemical group that is covalently attached to the enzyme complex via a flexible linker, allowing the direct transfer of substrate molecules between multiple active sites within the complex. Mimicking this method of substrate channelling outside the cellular environment requires precise control over the spatial parameters of the individual components within the assembled complex. DNA nanostructures can be used to organize functional molecules with nanoscale precision and can also provide nanomechanical control. Until now, protein-DNA assemblies have been used to organize cascades of enzymatic reactions by controlling the relative distance and orientation of enzymatic components or by facilitating the interface between enzymes/cofactors and electrode surfaces. Here, we show that a DNA nanostructure can be used to create a multi-enzyme complex in which an artificial swinging arm facilitates hydride transfer between two coupled dehydrogenases. By exploiting the programmability of DNA nanostructures, key parameters including position, stoichiometry and inter-enzyme distance can be manipulated for optimal activity.

Multi-enzyme complexes on DNA scaffolds capable of substrate channelling with an artificial swinging arm.

PubMed

Fu, Jinglin; Yang, Yuhe Renee; Johnson-Buck, Alexander; Liu, Minghui; Liu, Yan; Walter, Nils G; Woodbury, Neal W; Yan, Hao

2014-07-01

Swinging arms are a key functional component of multistep catalytic transformations in many naturally occurring multi-enzyme complexes. This arm is typically a prosthetic chemical group that is covalently attached to the enzyme complex via a flexible linker, allowing the direct transfer of substrate molecules between multiple active sites within the complex. Mimicking this method of substrate channelling outside the cellular environment requires precise control over the spatial parameters of the individual components within the assembled complex. DNA nanostructures can be used to organize functional molecules with nanoscale precision and can also provide nanomechanical control. Until now, protein-DNA assemblies have been used to organize cascades of enzymatic reactions by controlling the relative distance and orientation of enzymatic components or by facilitating the interface between enzymes/cofactors and electrode surfaces. Here, we show that a DNA nanostructure can be used to create a multi-enzyme complex in which an artificial swinging arm facilitates hydride transfer between two coupled dehydrogenases. By exploiting the programmability of DNA nanostructures, key parameters including position, stoichiometry and inter-enzyme distance can be manipulated for optimal activity.

DigiMemo: Facilitating the Note Taking Process

ERIC Educational Resources Information Center

Kurt, Serhat

2009-01-01

Everyone takes notes daily for various reasons. Note taking is very popular in school settings and generally recognized as an effective learning strategy. Further, note taking is a complex process because it requires understanding, selection of information and writing. Some new technological tools may facilitate the note taking process. Among such…

Writing To Facilitate Learning in Microbiology.

ERIC Educational Resources Information Center

Fisher, Linda E.

This paper describes a microbiology course that utilizes writing to facilitate learning of complex concepts, for communicating experimental results, and as a diagnostic tool for the instructor in monitoring the students' understanding of material on an on-going basis. In-class writing assignments that summarize subject units are accompanied by a…

Design Research as a Mechanism for Consultants to Facilitate and Evaluate Educational Innovations

ERIC Educational Resources Information Center

Castillo, Jose M.; Dorman, Clark; Gaunt, Brian; Hardcastle, Beth; Justice, Kelly; March, Amanda L.

2016-01-01

Schools across the nation are implementing innovative practices; however, questions remain regarding how to facilitate quality implementation. Research designs that emphasize high degrees of control over independent variables result in findings with internal validity, but that may not generalize to complex, dynamic educational systems. The purpose…

Myogenin Recruits the Histone Chaperone Facilitates Chromatin Transcription (FACT) to Promote Nucleosome Disassembly at Muscle-specific Genes*

PubMed Central

Lolis, Alexandra A.; Londhe, Priya; Beggs, Benjamin C.; Byrum, Stephanie D.; Tackett, Alan J.; Davie, Judith K.

2013-01-01

Facilitates chromatin transcription (FACT) functions to reorganize nucleosomes by acting as a histone chaperone that destabilizes and restores nucleosomal structure. The FACT complex is composed of two subunits: SSRP1 and SPT16. We have discovered that myogenin interacts with the FACT complex. Transfection of FACT subunits with myogenin is highly stimulatory for endogenous muscle gene expression in 10T1/2 cells. We have also found that FACT subunits do not associate with differentiation-specific genes while C2C12 cells are proliferating but are recruited to muscle-specific genes as differentiation initiates and then dissociate as differentiation proceeds. The recruitment is dependent on myogenin, as knockdowns of myogenin show no recruitment of the FACT complex. These data suggest that FACT is involved in the early steps of gene activation through its histone chaperone activities that serve to open the chromatin structure and facilitate transcription. Consistent with this hypothesis, we find that nucleosomes are depleted at muscle-specific promoters upon differentiation and that this activity is dependent on the presence of FACT. Our results show that the FACT complex promotes myogenin-dependent transcription and suggest that FACT plays an important role in the establishment of the appropriate transcription profile in a differentiated muscle cell. PMID:23364797

Stalled replication forks within heterochromatin require ATRX for protection

PubMed Central

Huh, M S; Ivanochko, D; Hashem, L E; Curtin, M; Delorme, M; Goodall, E; Yan, K; Picketts, D J

2016-01-01

Expansive growth of neural progenitor cells (NPCs) is a prerequisite to the temporal waves of neuronal differentiation that generate the six-layered neocortex, while also placing a heavy burden on proteins that regulate chromatin packaging and genome integrity. This problem is further reflected by the growing number of developmental disorders caused by mutations in chromatin regulators. ATRX gene mutations cause a severe intellectual disability disorder (α-thalassemia mental retardation X-linked (ATRX) syndrome; OMIM no. 301040), characterized by microcephaly, urogenital abnormalities and α-thalassemia. Although the ATRX protein is required for the maintenance of repetitive DNA within heterochromatin, how this translates to disease pathogenesis remain poorly understood and was a focus of this study. We demonstrate that AtrxFoxG1Cre forebrain-specific conditional knockout mice display poly(ADP-ribose) polymerase-1 (Parp-1) hyperactivation during neurogenesis and generate fewer late-born Cux1- and Brn2-positive neurons that accounts for the reduced cortical size. Moreover, DNA damage, induced Parp-1 and Atm activation is elevated in progenitor cells and contributes to their increased level of cell death. ATRX-null HeLa cells are similarly sensitive to hydroxyurea-induced replication stress, accumulate DNA damage and proliferate poorly. Impaired BRCA1-RAD51 colocalization and PARP-1 hyperactivation indicated that stalled replication forks are not efficiently protected. DNA fiber assays confirmed that MRE11 degradation of stalled replication forks was rampant in the absence of ATRX or DAXX. Indeed, fork degradation in ATRX-null cells could be attenuated by treatment with the MRE11 inhibitor mirin, or exacerbated by inhibiting PARP-1 activity. Taken together, these results suggest that ATRX is required to limit replication stress during cellular proliferation, whereas upregulation of PARP-1 activity functions as a compensatory mechanism to protect stalled forks, limiting genomic damage, and facilitating late-born neuron production. PMID:27171262

Endoglucanase Peripheral Loops Facilitate Complexation of Glucan Chains on Cellulose via Adaptive Coupling to the Emergent Substrate Structures

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lin, Yuchun; Beckham, Gregg T.; Himmel, Michael E.

We examine how the catalytic domain of a glycoside hydrolase family 7 endoglucanase catalytic domain (Cel7B CD) facilitates complexation of cellulose chains from a crystal surface. With direct relevance to the science of biofuel production, this problem also represents a model system of biopolymer processing by proteins in Nature. Interactions of Cel7B CD with a cellulose microfibril along different paths of complexation are characterized by mapping the atomistic fluctuations recorded in free-energy simulations onto the parameters of a coarse-grain model. The resulting patterns of protein-biopolymer couplings also uncover the sequence signatures of the enzyme in peeling off glucan chains frommore » the microfibril substrate. We show that the semiopen active site of Cel7B CD exhibits similar barriers and free energies of complexation over two distinct routes; namely, scooping of a chain into the active-site cleft and threading from the chain end into the channel. On the other hand, the complexation energetics strongly depends on the surface packing of the targeted chain and the resulting interaction sites with the enzyme. A revealed principle is that Cel7B CD facilitates cellulose deconstruction via adaptive coupling to the emergent substrate. The flexible, peripheral segments of the protein outside of the active-site cleft are able to accommodate the varying features of cellulose along the simulated paths of complexation. The general strategy of linking physics-based molecular interactions to protein sequence could also be helpful in elucidating how other protein machines process biopolymers.« less

Design and Implementation of a Mobile Exergaming Platform

NASA Astrophysics Data System (ADS)

Prévost, Laurent; Liechti, Olivier; Lyons, Michael J.

This paper describes the design, implementation, and initial testing of a reusable platform for the creation of pervasive games with geo-localization services. We concentrate on role-playing games built by combining several types of simpler mini-games having three major components: Quests; Collectables; and Non-player characters (NPC). Quests encourage players to be active in their physical environment and take part in collaborative play; Collectables provide motivation; and NPCs enable player-friendly interaction with the platform. Each of these elements poses different technical requirements, which were met by implementing the gaming platform using the inTrack pervasive middle-ware being developed by our group. Several sample games were implemented and tested within the urban environment of Kyoto, Japan, using gaming clients running on mobile phones from NTT DoCoMo, Japan’s largest mobile provider.

Histone modifications controlling native and induced neural stem cell identity.

PubMed

Broccoli, Vania; Colasante, Gaia; Sessa, Alessandro; Rubio, Alicia

2015-10-01

During development, neural progenitor cells (NPCs) that are capable of self-renewing maintain a proliferative cellular pool while generating all differentiated neural cell components. Although the genetic network of transcription factors (TFs) required for neural specification has been well characterized, the unique set of histone modifications that accompanies this process has only recently started to be investigated. In vitro neural differentiation of pluripotent stem cells is emerging as a powerful system to examine epigenetic programs. Deciphering the histone code and how it shapes the chromatin environment will reveal the intimate link between epigenetic changes and mechanisms for neural fate determination in the developing nervous system. Furthermore, it will offer a molecular framework for a stringent comparison between native and induced neural stem cells (iNSCs) generated by direct neural cell conversion. Copyright © 2015 Elsevier Ltd. All rights reserved.

Preventable hospitalizations: does rurality or non-physician clinician supply matter?

PubMed

Nayar, Preethy; Nguyen, Anh T; Apenteng, Bettye; Yu, Fang

2012-04-01

This study examines the relationship between rurality as well as the proportion of non-physician clinicians and county rates of ambulatory care sensitive hospitalizations (ACSHs) for pediatric, adult and elderly populations in Nebraska. The study design was a cross-sectional observational study of county level factors that affect the county level rates of ACSHs using Poisson regression models. Rural (non-metro) counties have significantly higher ACSHs for both pediatric and adult population, but not for the elderly. Frontier counties have significantly higher adult ACSHs. The proportion of primary care providers who are non-physician clinicians does not have a significant association with ACSHs for any of the age groups. The results indicate that rurality may have a greater impact on pediatric and adult ACSHs and the proportion of NPCs in the primary care provider workforce does not significantly impact ACSH rates.

Barriers and Facilitators to Implementing a Change Initiative in Long-Term Care Using the INTERACT® Quality Improvement Program.

PubMed

Tappen, Ruth M; Wolf, David G; Rahemi, Zahra; Engstrom, Gabriella; Rojido, Carolina; Shutes, Jill M; Ouslander, Joseph G

Implementation of major organizational change initiatives presents a challenge for long-term care leadership. Implementation of the INTERACT® (Interventions to Reduce Acute Care Transfers) quality improvement program, designed to improve the management of acute changes in condition and reduce unnecessary emergency department visits and hospitalizations of nursing home residents, serves as an example to illustrate the facilitators and barriers to major change in long-term care. As part of a larger study of the impact of INTERACT® on rates of emergency department visits and hospitalizations, staff of 71 nursing homes were called monthly to follow-up on their progress and discuss successful facilitating strategies and any challenges and barriers they encountered during the yearlong implementation period. Themes related to barriers and facilitators were identified. Six major barriers to implementation were identified: the magnitude and complexity of the change (35%), instability of facility leadership (27%), competing demands (40%), stakeholder resistance (49%), scarce resources (86%), and technical problems (31%). Six facilitating strategies were also reported: organization-wide involvement (68%), leadership support (41%), use of administrative authority (14%), adequate training (66%), persistence and oversight on the part of the champion (73%), and unfolding positive results (14%). Successful introduction of a complex change such as the INTERACT® quality improvement program in a long-term care facility requires attention to the facilitators and barriers identified in this report from those at the frontline.

Navigating Complex Trade-Offs in Conservation and Development: An Integrative Framework

ERIC Educational Resources Information Center

Hirsch, Paul D.; Brosius, J. Peter

2013-01-01

We present a framework that makes space for multiple perspectives and ways of thinking about complex trade-off problems in conservation and development. At the core of the framework are three "integrative lenses" designed to facilitate lines of inquiry according to three unique ways of perceiving complexity. The aim of the framework is…

Unpacking the Complexity of Planning with Persons with Cognitive Disability and Complex Support Needs

ERIC Educational Resources Information Center

Collings, Susan; Dew, Angela; Dowse, Leanne

2018-01-01

Background: Planners will engage with people with cognitive disability and complex support needs in the Australian National Disability Insurance Scheme, but the specific skills needed to build sustainable plans with this group are not yet known. Method: A qualitative study was conducted to explore the barriers and facilitators to planning with…

The informativity of sound modulates crossmodal facilitation of visual discrimination: a fMRI study.

PubMed

Li, Qi; Yu, Hongtao; Li, Xiujun; Sun, Hongzan; Yang, Jingjing; Li, Chunlin

2017-01-18

Many studies have investigated behavioral crossmodal facilitation when a visual stimulus is accompanied by a concurrent task-irrelevant sound. Lippert and colleagues reported that a concurrent task-irrelevant sound reduced the uncertainty of the timing of the visual display and improved perceptional responses (informative sound). However, the neural mechanism by which the informativity of sound affected crossmodal facilitation of visual discrimination remained unclear. In this study, we used event-related functional MRI to investigate the neural mechanisms underlying the role of informativity of sound in crossmodal facilitation of visual discrimination. Significantly faster reaction times were observed when there was an informative relationship between auditory and visual stimuli. The functional MRI results showed sound informativity-induced activation enhancement including the left fusiform gyrus and the right lateral occipital complex. Further correlation analysis showed that the right lateral occipital complex was significantly correlated with the behavioral benefit in reaction times. This suggests that this region was modulated by the informative relationship within audiovisual stimuli that was learnt during the experiment, resulting in late-stage multisensory integration and enhanced behavioral responses.

Cynefin as Reference Framework to Facilitate Insight and Decision-Making in Complex Contexts of Biomedical Research.

PubMed

Kempermann, Gerd

2017-01-01

The Cynefin scheme is a concept of knowledge management, originally devised to support decision making in management, but more generally applicable to situations, in which complexity challenges the quality of insight, prediction, and decision. Despite the fact that life itself, and especially the brain and its diseases, are complex to the extent that complexity could be considered their cardinal feature, complex problems in biomedicine are often treated as if they were actually not more than the complicated sum of solvable sub-problems. Because of the emergent properties of complex contexts this is not correct. With a set of clear criteria Cynefin helps to set apart complex problems from "simple/obvious," "complicated," "chaotic," and "disordered" contexts in order to avoid misinterpreting the relevant causality structures. The distinction comes with the insight, which specific kind of knowledge is possible in each of these categories and what are the consequences for resulting decisions and actions. From student's theses over the publication and grant writing process to research politics, misinterpretation of complexity can have problematic or even dangerous consequences, especially in clinical contexts. Conceptualization of problems within a straightforward reference language like Cynefin improves clarity and stringency within projects and facilitates communication and decision-making about them.

Facilitators & Barriers to the Adoption of Ergonomic Solutions in Construction

PubMed Central

Dale, Ann Marie; Jaegers, Lisa; Welch, Laura; Barnidge, Ellen; Weaver, Nancy; Evanoff, Bradley A.

2017-01-01

Background Rates of musculoskeletal disorders in construction remain high. Few studies have described barriers and facilitators to the use of available ergonomic solutions. This paper describes these barriers and facilitators and their relationship to the level of adoption. Methods Three analysts rated 16 proposed ergonomic solutions from a participatory ergonomics study and assessed the level of adoption, six adoption characteristics, and identified the category of adoption from a theoretical model. Results Twelve solutions were always or intermittently used and were rated positively for characteristics of relative advantage, compatibility with existing work processes, and trialability. Locus of control (worker vs. contractor) was not related to adoption. Simple solutions faced fewer barriers to adoption than those rated as complex. Conclusions Specific adoption characteristics can help predict the use of new ergonomic solutions in construction. Adoption of complex solutions must involve multiple stakeholders, more time, and shifts in culture or work systems. PMID:28195660

Competition and facilitation between a native and a domestic herbivore: trade-offs between forage quantity and quality.

PubMed

Augustine, David J; Springer, Tim L

2013-06-01

Potential competition between native and domestic herbivores is a major consideration influencing the management and conservation of native herbivores in rangeland ecosystems. In grasslands of the North American Great Plains, black-tailed prairie dogs (Cynomys ludovicianus) are widely viewed as competitors with cattle but are also important for biodiversity conservation due to their role in creating habitat for other native species. We examined spatiotemporal variation in prairie dog effects on growing-season forage quality and quantity using measurements from three colony complexes in Colorado and South Dakota and from a previous study of a fourth complex in Montana. At two complexes experiencing below-average precipitation, forage availability both on and off colonies was so low (12-54 g/m2) that daily forage intake rates of cattle were likely constrained by instantaneous intake rates and daily foraging time. Under these dry conditions, prairie dogs (1) substantially reduced forage availability, thus further limiting cattle daily intake rates, and (2) had either no or a small positive effect on forage digestibility. Under such conditions, prairie dogs are likely to compete with cattle in direct proportion to their abundance. For two complexes experiencing above-average precipitation, forage quantity on and off colonies (77-208 g/m2) was sufficient for daily forage intake of cattle to be limited by digestion rather than instantaneous forage intake. At one complex where prairie dogs enhanced forage digestibility and [N] while having no effect on forage quantity, prairie dogs are predicted to facilitate cattle mass gains regardless of prairie dog abundance. At the second complex where prairie dogs enhanced digestibility and [N] but reduced forage quantity, effects on cattle can vary from competition to facilitation depending on prairie dog abundance. Our findings show that the high spatiotemporal variation in vegetation dynamics characteristic of semiarid grasslands is paralleled by variability in the magnitude of competition between native and domestic grazers. Competitive interactions evident during dry periods may be partially or wholly offset by facilitation during periods when forage digestibility is enhanced and forage quantity does not limit the daily intake rate of cattle.

Dynamic Modeling as a Cognitive Regulation Scaffold for Developing Complex Problem-Solving Skills in an Educational Massively Multiplayer Online Game Environment

ERIC Educational Resources Information Center

Eseryel, Deniz; Ge, Xun; Ifenthaler, Dirk; Law, Victor

2011-01-01

Following a design-based research framework, this article reports two empirical studies with an educational MMOG, called "McLarin's Adventures," on facilitating 9th-grade students' complex problem-solving skill acquisition in interdisciplinary STEM education. The article discusses the nature of complex and ill-structured problem solving…

Facilitating and Learning at the Edge of Chaos: Expanding the Context of Experiential Education.

ERIC Educational Resources Information Center

Oekerman, Carl

Significant recent discoveries within a number of scientific disciplines, collectively referred to as the science of complexity, are creating a major shift in how human beings understand the complex, adaptive systems that make up the world. A complex adaptive system consists of networks of large numbers of agents that interact with each other and…

Photodissociation of Non-Covalent Peptide-Crown Ether Complexes

PubMed Central

Wilson, Jeffrey J.; Kirkovits, Gregory J.; Sessler, Jonathan L.; Brodbelt, Jennifer S.

2008-01-01

Highly chromogenic 18-crown-6-dipyrrolylquinoxaline coordinates primary amines of peptides, forming non-covalent complexes that can be transferred to the gas phase by electrospray ionization. The appended chromogenic crown ether facilitates efficient energy transfer to the peptide upon ultraviolet irradiation in the gas phase, resulting in diagnostic peptide fragmentation. Collisional activated dissociation (CAD) and infrared multiphoton dissociation (IRMPD) of these non-covalent complexes results only in their disassembly with the charge retained on either the peptide or crown ether, yielding no sequence ions. Upon UV photon absorption the intermolecular energy transfer is facilitated by the fast activation time scale of UVPD (< 10 ns) and by the collectively strong hydrogen bonding between the crown ether and peptide, thus allowing effective transfer of energy to the peptide moiety prior to disruption of the intermolecular hydrogen bonds. PMID:18077179

Mutual Alignment Comparison Facilitates Abstraction and Transfer of a Complex Scientific Concept

ERIC Educational Resources Information Center

Orton, Judy M.; Anggoro, Florencia K.; Jee, Benjamin D.

2012-01-01

Learning about a scientific concept often occurs in the context of unfamiliar examples. Mutual alignment analogy--a type of analogical comparison in which the analogues are only partially understood--has been shown to facilitate learning from unfamiliar examples . In the present study, we examined the role of mutual alignment analogy in the…

Planning for MacArthur: Harnessing the Complexity of Postwar Japan

DTIC Science & Technology

2014-05-22

change in Japan’s education system, facilitated religious tolerance and strictly controlled information through censorship and targeted information...system, facilitated religious tolerance and strictly controlled information through censorship and targeted information operations. Although many...Supreme Commander for the Allied Powers, it is clear that operational planners on MacArthur’s staff used the principles of understanding the

From Vesalius to Virtual Reality: How Embodied Cognition Facilitates the Visualization of Anatomy

ERIC Educational Resources Information Center

Jang, Susan

2010-01-01

This study examines the facilitative effects of embodiment of a complex internal anatomical structure through three-dimensional ("3-D") interactivity in a virtual reality ("VR") program. Since Shepard and Metzler's influential 1971 study, it has been known that 3-D objects (e.g., multiple-armed cube or external body parts) are visually and…

Identifying Facilitating Factors and Barriers to Improving Student Retention Rates in Tertiary Teaching Courses: A Systematic Review

ERIC Educational Resources Information Center

Bowles, Terence V.; Brindle, Kimberley A.

2017-01-01

Factors which impact student retention in tertiary level teaching courses are complex. This study examined facilitating factors and barriers to student retention for students studying education. Due to a limited number of studies, the search was extended to factors impacting students undertaking tertiary education. A systematic review was…

Does a Discussion by Any Other Name Sound the Same? Teaching Discussion in Three ELA Methods Courses

ERIC Educational Resources Information Center

Alston, Chandra L.; Danielson, Katie A.; Dutro, Elizabeth; Cartun, Ashley

2018-01-01

Facilitating discussions in English Language Arts can develop students' skills as speakers and listeners and their ability to engage with diverse perspectives. However, classroom observations often demonstrate a lack of student talk, raising questions about the complexity of facilitating discussion and teachers' opportunities to learn and hone the…

Immunoprecipitation and Characterization of Membrane Protein Complexes from Yeast

ERIC Educational Resources Information Center

Parra-Belky, Karlett; McCulloch, Kathryn; Wick, Nicole; Shircliff, Rebecca; Croft, Nicolas; Margalef, Katrina; Brown, Jamie; Crabill, Todd; Jankord, Ryan; Waldo, Eric

2005-01-01

In this undergraduate biochemistry laboratory experiment, the vacuolar ATPase protein complex is purified from yeast cell extracts by doing immunoprecipitations under nondenaturing conditions. Immunoprecipitations are performed using monoclonal antibodies to facilitate data interpretation, and subunits are separated on the basis of their molecular…

Graduate Education to Facilitate Interdisciplinary Research Collaboration: Identifying Individual Competencies and Developmental Activities

ERIC Educational Resources Information Center

Holt, Valerie Ciocca

2013-01-01

Interdisciplinary research collaborations (IDRC) are considered essential for addressing the most complex global community problems concerning science, health, education, energy, the environment, and society. In spite of technological advances, supportive funding, and even researcher proclivity to collaborate, these complex interdisciplinary…

The truncated NLR protein TIR-NBS13 is a MOS6/IMPORTIN-α3 interaction partner required for plant immunity.

PubMed

Roth, Charlotte; Lüdke, Daniel; Klenke, Melanie; Quathamer, Annalena; Valerius, Oliver; Braus, Gerhard H; Wiermer, Marcel

2017-12-01

Importin-α proteins mediate the translocation of nuclear localization signal (NLS)-containing proteins from the cytoplasm into the nucleus through nuclear pore complexes (NPCs). Genetically, Arabidopsis IMPORTIN-α3/MOS6 (MODIFIER OF SNC1, 6) is required for basal plant immunity and constitutive disease resistance activated in autoimmune mutant snc1 (suppressor of npr1-1, constitutive 1), suggesting that MOS6 plays a role in the nuclear import of proteins involved in plant defense signaling. Here, we sought to identify and characterize defense-regulatory cargo proteins and interaction partners of MOS6. We conducted both in silico database analyses and affinity purification of functional epitope-tagged MOS6 from pathogen-challenged stable transgenic plants coupled with mass spectrometry. We show that among the 13 candidate MOS6 interactors we selected for further functional characterization, the TIR-NBS-type protein TN13 is required for resistance against Pseudomonas syringae pv. tomato (Pst) DC3000 lacking the type-III effector proteins AvrPto and AvrPtoB. When expressed transiently in N. benthamiana leaves, TN13 co-immunoprecipitates with MOS6, but not with its closest homolog IMPORTIN-α6, and localizes to the endoplasmic reticulum (ER), consistent with a predicted N-terminal transmembrane domain in TN13. Our work uncovered the truncated NLR protein TN13 as a component of plant innate immunity that selectively binds to MOS6/IMPORTIN-α3 in planta. We speculate that the release of TN13 from the ER membrane in response to pathogen stimulus, and its subsequent nuclear translocation, is important for plant defense signal transduction. © 2017 The Authors The Plant Journal © 2017 John Wiley & Sons Ltd.

Evolution of the Karyopherin-β Family of Nucleocytoplasmic Transport Factors; Ancient Origins and Continued Specialization

PubMed Central

O'Reilly, Amanda J.; Dacks, Joel B.; Field, Mark C.

2011-01-01

Background Macromolecular transport across the nuclear envelope (NE) is achieved through nuclear pore complexes (NPCs) and requires karyopherin-βs (KAP-βs), a family of soluble receptors, for recognition of embedded transport signals within cargo. We recently demonstrated, through proteomic analysis of trypanosomes, that NPC architecture is likely highly conserved across the Eukaryota, which in turn suggests conservation of the transport mechanisms. To determine if KAP-β diversity was similarly established early in eukaryotic evolution or if it was subsequently layered onto a conserved NPC, we chose to identify KAP-β sequences in a diverse range of eukaryotes and to investigate their evolutionary history. Results Thirty six predicted proteomes were scanned for candidate KAP-β family members. These resulting sequences were resolved into fifteen KAP-β subfamilies which, due to broad supergroup representation, were most likely represented in the last eukaryotic common ancestor (LECA). Candidate members of each KAP-β subfamily were found in all eukaryotic supergroups, except XPO6, which is absent from Archaeplastida. Phylogenetic reconstruction revealed the likely evolutionary relationships between these different subfamilies. Many species contain more than one representative of each KAP-β subfamily; many duplications are apparently taxon-specific but others result from duplications occurring earlier in eukaryotic history. Conclusions At least fifteen KAP-β subfamilies were established early in eukaryote evolution and likely before the LECA. In addition we identified expansions at multiple stages within eukaryote evolution, including a multicellular plant-specific KAP-β, together with frequent secondary losses. Taken with evidence for early establishment of NPC architecture, these data demonstrate that multiple pathways for nucleocytoplasmic transport were established prior to the radiation of modern eukaryotes but that selective pressure continues to sculpt the KAP-β family. PMID:21556326

mda-9/Syntenin protein positively regulates the activation of Akt protein by facilitating integrin-linked kinase adaptor function during adhesion to type I collagen.

PubMed

Hwangbo, Cheol; Park, Juhee; Lee, Jeong-Hyung

2011-09-23

The integrin-linked kinase (ILK)-PINCH1-α-parvin (IPP) complex functions as a signaling platform for integrins that modulates various cellular processes. ILK functions as a central adaptor for the assembly of IPP complex. We report here that mda-9/syntenin, a positive regulator of cancer metastasis, regulates the activation of Akt (also known as protein kinase B) by facilitating ILK adaptor function during adhesion to type I collagen (COL-I) in human breast cancer cells. COL-I stimulation induced the phosphorylation and plasma membrane translocation of Akt. Inhibition of mda-9/syntenin or expression of mutant ILK (E359K) significantly blocked the translocation of both ILK and Akt to the plasma membrane. mda-9/syntenin associated with ILK, and this association was increased at the plasma membrane by COL-I stimulation. Knockdown of mda-9/syntenin impaired COL-I-induced association of ILK with Akt and plasma membrane targeting of ILK-Akt complex. These results demonstrated that mda-9/syntenin regulates the activation of Akt by controlling the plasma membrane targeting of Akt via a mechanism that facilitates the association of Akt with ILK at the plasma membrane during adhesion to COL-I. On a striking note, inhibition of mda-9/syntenin impaired COL-I-induced plasma membrane translocation of the IPP complex and assembly of integrin β1-IPP signaling complexes. Thus, our study defines the role of mda-9/syntenin in ILK adaptor function and describes a new mechanism of mda-9/syntenin for regulation of cell migration.

Origin of the cell nucleus, mitosis and sex: roles of intracellular coevolution.

PubMed

Cavalier-Smith, Thomas

2010-02-04

The transition from prokaryotes to eukaryotes was the most radical change in cell organisation since life began, with the largest ever burst of gene duplication and novelty. According to the coevolutionary theory of eukaryote origins, the fundamental innovations were the concerted origins of the endomembrane system and cytoskeleton, subsequently recruited to form the cell nucleus and coevolving mitotic apparatus, with numerous genetic eukaryotic novelties inevitable consequences of this compartmentation and novel DNA segregation mechanism. Physical and mutational mechanisms of origin of the nucleus are seldom considered beyond the long-standing assumption that it involved wrapping pre-existing endomembranes around chromatin. Discussions on the origin of sex typically overlook its association with protozoan entry into dormant walled cysts and the likely simultaneous coevolutionary, not sequential, origin of mitosis and meiosis. I elucidate nuclear and mitotic coevolution, explaining the origins of dicer and small centromeric RNAs for positionally controlling centromeric heterochromatin, and how 27 major features of the cell nucleus evolved in four logical stages, making both mechanisms and selective advantages explicit: two initial stages (origin of 30 nm chromatin fibres, enabling DNA compaction; and firmer attachment of endomembranes to heterochromatin) protected DNA and nascent RNA from shearing by novel molecular motors mediating vesicle transport, division, and cytoplasmic motility. Then octagonal nuclear pore complexes (NPCs) arguably evolved from COPII coated vesicle proteins trapped in clumps by Ran GTPase-mediated cisternal fusion that generated the fenestrated nuclear envelope, preventing lethal complete cisternal fusion, and allowing passive protein and RNA exchange. Finally, plugging NPC lumens by an FG-nucleoporin meshwork and adopting karyopherins for nucleocytoplasmic exchange conferred compartmentation advantages. These successive changes took place in naked growing cells, probably as indirect consequences of the origin of phagotrophy. The first eukaryote had 1-2 cilia and also walled resting cysts; I outline how encystation may have promoted the origin of meiotic sex. I also explain why many alternative ideas are inadequate. Nuclear pore complexes are evolutionary chimaeras of endomembrane- and mitosis-related chromatin-associated proteins. The keys to understanding eukaryogenesis are a proper phylogenetic context and understanding organelle coevolution: how innovations in one cell component caused repercussions on others.

Diffusion, capture and recycling of SCAR/WAVE and Arp2/3 complexes observed in cells by single-molecule imaging.

PubMed

Millius, Arthur; Watanabe, Naoki; Weiner, Orion D

2012-03-01

The SCAR/WAVE complex drives lamellipodium formation by enhancing actin nucleation by the Arp2/3 complex. Phosphoinositides and Rac activate the SCAR/WAVE complex, but how SCAR/WAVE and Arp2/3 complexes converge at sites of nucleation is unknown. We analyzed the single-molecule dynamics of WAVE2 and p40 (subunits of the SCAR/WAVE and Arp2/3 complexes, respectively) in XTC cells. We observed lateral diffusion of both proteins and captured the transition of p40 from diffusion to network incorporation. These results suggest that a diffusive 2D search facilitates binding of the Arp2/3 complex to actin filaments necessary for nucleation. After nucleation, the Arp2/3 complex integrates into the actin network and undergoes retrograde flow, which results in its broad distribution throughout the lamellipodium. By contrast, the SCAR/WAVE complex is more restricted to the cell periphery. However, with single-molecule imaging, we also observed WAVE2 molecules undergoing retrograde motion. WAVE2 and p40 have nearly identical speeds, lifetimes and sites of network incorporation. Inhibition of actin retrograde flow does not prevent WAVE2 association and disassociation with the membrane but does inhibit WAVE2 removal from the actin cortex. Our results suggest that membrane binding and diffusion expedites the recruitment of nucleation factors to a nucleation site independent of actin assembly, but after network incorporation, ongoing actin polymerization facilitates recycling of SCAR/WAVE and Arp2/3 complexes.

Diffusion, capture and recycling of SCAR/WAVE and Arp2/3 complexes observed in cells by single-molecule imaging

PubMed Central

Millius, Arthur; Watanabe, Naoki; Weiner, Orion D.

2012-01-01

The SCAR/WAVE complex drives lamellipodium formation by enhancing actin nucleation by the Arp2/3 complex. Phosphoinositides and Rac activate the SCAR/WAVE complex, but how SCAR/WAVE and Arp2/3 complexes converge at sites of nucleation is unknown. We analyzed the single-molecule dynamics of WAVE2 and p40 (subunits of the SCAR/WAVE and Arp2/3 complexes, respectively) in XTC cells. We observed lateral diffusion of both proteins and captured the transition of p40 from diffusion to network incorporation. These results suggest that a diffusive 2D search facilitates binding of the Arp2/3 complex to actin filaments necessary for nucleation. After nucleation, the Arp2/3 complex integrates into the actin network and undergoes retrograde flow, which results in its broad distribution throughout the lamellipodium. By contrast, the SCAR/WAVE complex is more restricted to the cell periphery. However, with single-molecule imaging, we also observed WAVE2 molecules undergoing retrograde motion. WAVE2 and p40 have nearly identical speeds, lifetimes and sites of network incorporation. Inhibition of actin retrograde flow does not prevent WAVE2 association and disassociation with the membrane but does inhibit WAVE2 removal from the actin cortex. Our results suggest that membrane binding and diffusion expedites the recruitment of nucleation factors to a nucleation site independent of actin assembly, but after network incorporation, ongoing actin polymerization facilitates recycling of SCAR/WAVE and Arp2/3 complexes. PMID:22349699

Expanding Evolutionary Theory beyond Darwinism with Elaborating, Self-Organizing, and Fractionating Complex Evolutionary Systems

ERIC Educational Resources Information Center

Fichter, Lynn S.; Pyle, E. J.; Whitmeyer, S. J.

2010-01-01

Earth systems increase in complexity, diversity, and interconnectedness with time, driven by tectonic/solar energy that keeps the systems far from equilibrium. The evolution of Earth systems is facilitated by three evolutionary mechanisms: "elaboration," "fractionation," and "self-organization," that share…

Elongation factor Ts directly facilitates the formation and disassembly of the Escherichia coli elongation factor Tu·GTP·aminoacyl-tRNA ternary complex.

PubMed

Burnett, Benjamin J; Altman, Roger B; Ferrao, Ryan; Alejo, Jose L; Kaur, Navdep; Kanji, Joshua; Blanchard, Scott C

2013-05-10

Aminoacyl-tRNA (aa-tRNA) enters the ribosome in a ternary complex with the G-protein elongation factor Tu (EF-Tu) and GTP. EF-Tu·GTP·aa-tRNA ternary complex formation and decay rates are accelerated in the presence of the nucleotide exchange factor elongation factor Ts (EF-Ts). EF-Ts directly facilitates the formation and disassociation of ternary complex. This system demonstrates a novel function of EF-Ts. Aminoacyl-tRNA enters the translating ribosome in a ternary complex with elongation factor Tu (EF-Tu) and GTP. Here, we describe bulk steady state and pre-steady state fluorescence methods that enabled us to quantitatively explore the kinetic features of Escherichia coli ternary complex formation and decay. The data obtained suggest that both processes are controlled by a nucleotide-dependent, rate-determining conformational change in EF-Tu. Unexpectedly, we found that this conformational change is accelerated by elongation factor Ts (EF-Ts), the guanosine nucleotide exchange factor for EF-Tu. Notably, EF-Ts attenuates the affinity of EF-Tu for GTP and destabilizes ternary complex in the presence of non-hydrolyzable GTP analogs. These results suggest that EF-Ts serves an unanticipated role in the cell of actively regulating the abundance and stability of ternary complex in a manner that contributes to rapid and faithful protein synthesis.

An Investigation of the Relationship between Cohesion and Syntactic Complexity of Take-Away Writing and Advanced Content Complexity and Depth

ERIC Educational Resources Information Center

Gardner, Matthew Thomas

2017-01-01

In secondary and post-secondary content courses, the use of writing to facilitate complex learning in advanced content areas, which is called writing to learn content, can help students to evaluate their understanding, higher order cognition, and thinking about the content to learned (Carifio, 2005; Hayes, 2006; Carifio, 2015). The primary focus…

Student Facilitators' Habits of Mind and Their Influences on Higher-Level Knowledge Construction Occurrences in Online Discussions: A Case Study

ERIC Educational Resources Information Center

Hew, Khe Foon; Cheung, Wing Sum

2011-01-01

Previous research studies on factors influencing student higher-level knowledge construction in asynchronous online discussions have largely focused on the instructors' role, student learning style, and the complexity of the discussion task. This study explores the issue from a different angle--that of student facilitators' habits of mind.…

Constructing Videocases to Help Novices Learn to Facilitate Discussions in Science and English: How Does Subject Matter Matter?

ERIC Educational Resources Information Center

Rosaen, Cheryl L.; Lundeberg, Mary; Terpstra, Marjorie; Cooper, Marjorie; Niu, Rui; Fu, Jing

2010-01-01

Learning to conduct interactive classroom discussions is a high priority for becoming an effective teacher, and most teachers view conducting productive classroom discussions as a complex undertaking. Because the dynamics of facilitating classroom discussions are multifaceted and hard to analyze in real time, there is a growing interest in how…

A Path Analysis of Pre-Service Teachers' Attitudes to Computer Use: Applying and Extending the Technology Acceptance Model in an Educational Context

ERIC Educational Resources Information Center

Teo, Timothy

2010-01-01

The purpose of this study is to examine pre-service teachers' attitudes to computers. This study extends the technology acceptance model (TAM) framework by adding subjective norm, facilitating conditions, and technological complexity as external variables. Results show that the TAM and subjective norm, facilitating conditions, and technological…

Professor as Facilitator: Shaping an Emerging, Living System of Shared Leadership in the Classroom

ERIC Educational Resources Information Center

Bright, David S.; Turesky, Elizabeth Fisher; Putzel, Roger; Stang, Thomas

2012-01-01

From the perspective of emergence, professors can facilitate and shape a class as a complex, adaptive, and living system. A case study illustrates phases of emergence in the classroom by tracing how a professor may use this perspective to empower students to share in the leadership of the classroom. Instead of presenting lessons, the professor…

Citizen social science: a methodology to facilitate and evaluate workplace learning in continuing interprofessional education.

PubMed

Dadich, Ann

2014-05-01

Workplace learning in continuing interprofessional education (CIPE) can be difficult to facilitate and evaluate, which can create a number of challenges for this type of learning. This article presents an innovative method to foster and investigate workplace learning in CIPE - citizen social science. Citizen social science involves clinicians as co-researchers in the systematic examination of social phenomena. When facilitated by an open-source online social networking platform, clinicians can participate via computer, smartphone, or tablet in ways that suit their needs and preferences. Furthermore, as co-researchers they can help to reveal the dynamic interplay that facilitates workplace learning in CIPE. Although yet to be tested, citizen social science offers four potential benefits: it recognises and accommodates the complexity of workplace learning in CIPE; it has the capacity to both foster and evaluate the phenomena; it can be used in situ, capturing and having direct relevance to the complexity of the workplace; and by advancing both theoretical and methodological debates on CIPE, it may reveal opportunities to improve and sustain workplace learning. By describing an example situated in the youth health sector, this article demonstrates how these benefits might be realised.

Experiences of practice facilitators working on the Improved Delivery of Cardiovascular Care project: Retrospective case study.

PubMed

Liddy, Clare; Rowan, Margo; Valiquette-Tessier, Sophie-Claire; Drosinis, Paul; Crowe, Lois; Hogg, William

2018-01-01

To examine the barriers to and facilitators of practice facilitation experienced by participants in the Improving Delivery of Cardiovascular Care (IDOCC) project. Case studies of practice facilitators' narrative reports. Eastern Ontario. Primary care practices that participated in the IDOCC project. Cases were identified by calculating sum scores in order to determine practices' performance relative to their peers. Two case exemplars were selected that scored within ± 1 SD of the total mean score, and a qualitative analysis of practice facilitators' narrative reports was conducted using a 5-factor implementation framework to identify barriers and facilitators. Narratives were divided into 3 phases: planning, implementation, and sustainability. Barriers and facilitators fluctuated over the intervention's 3 phases. Site A reported more barriers (n = 47) than facilitators (n = 38), while site B reported a roughly equal number of barriers (n = 144) and facilitators (n = 136). In both sites, the most common barriers involved organizational and provider factors and the most common facilitators were associated with innovation and structural factors. Both practices encountered various barriers and facilitators throughout the IDOCC's 3 phases. The case studies reveal the complex interactions of these factors over time, and provide insight into the implementation of practice facilitation programs. Copyright© the College of Family Physicians of Canada.

Knockdown Brm and Baf170, components of chromatin remodeling complex, facilitates reprogramming of somatic cells

USDA-ARS?s Scientific Manuscript database

The SWI/SNF (SWItch/Sucrose NonFermentable or BAF, Brg/Brahma-associated factors) complexes are epigenetic modifiers of chromatin structure and undergo progressive changes in subunit composition during cellular differentiation. For example, in embryonic stem cells (ESCs) esBAF contains Brg1 and Baf...

Stimulus Equalization: Temporary Reduction of Stimulus Complexity to Facilitate Discrimination Learning.

ERIC Educational Resources Information Center

Hoko, J. Aaron; LeBlanc, Judith M.

1988-01-01

Because disabled learners may profit from procedures using gradual stimulus change, this study utilized a microcomputer to investigate the effectiveness of stimulus equalization, an error reduction procedure involving an abrupt but temporary reduction of dimensional complexity. The procedure was found to be generally effective and implications for…

Developing and Modeling Complex Social Interventions: Introducing the Connecting People Intervention

ERIC Educational Resources Information Center

Webber, Martin; Reidy, Hannah; Ansari, David; Stevens, Martin; Morris, David

2016-01-01

Objectives: Modeling the processes involved in complex social interventions is important in social work practice, as it facilitates their implementation and translation into different contexts. This article reports the process of developing and modeling the connecting people intervention (CPI), a model of practice that supports people with mental…

Task Complexity, Learning Opportunities, and Korean EFL Learners' Question Development

ERIC Educational Resources Information Center

Kim, YouJin

2012-01-01

Building on the cognitive and interactive perspectives of task research, the cognition hypothesis states that increasing task complexity promotes greater interaction and feedback and thus facilitates second language (L2) development (Robinson, 2001b, 2007a). To date, very little research has explored this claim during learner-learner interactions…

Preparing Teachers to Lead Mathematics Discussions

ERIC Educational Resources Information Center

Boerst, Timothy A.; Sleep, Laurie; Ball, Deborah Loewenberg; Bass, Hyman

2011-01-01

Background/Context: Discussion is central to mathematics teaching and learning, as well as to mathematics as an academic discipline. Studies have shown that facilitating discussions is complex work that is not easily done or learned. To make such complex aspects of the work of teaching learnable by beginners, recent research has focused on…

Leucine facilitates the insulin-stimulated glucose uptake and insulin signaling in skeletal muscle cells: involving mTORC1 and mTORC2.

PubMed

Liu, Hui; Liu, Rui; Xiong, Yufang; Li, Xiang; Wang, Xiaolei; Ma, Yan; Guo, Huailan; Hao, Liping; Yao, Ping; Liu, Liegang; Wang, Di; Yang, Xuefeng

2014-08-01

Leucine, a branched-chain amino acid, has been shown to promote glucose uptake and increase insulin sensitivity in skeletal muscle, but the exact mechanism remains unestablished. We addressed this issue in cultured skeletal muscle cells in this study. Our results showed that leucine alone did not have an effect on glucose uptake or phosphorylation of protein kinase B (AKT), but facilitated the insulin-induced glucose uptake and AKT phosphorylation. The insulin-stimulated glucose uptake and AKT phosphorylation were inhibited by the phosphatidylinositol 3-kinase inhibitor, wortmannin, but the inhibition was partially reversed by leucine. The inhibitor of mammalian target of rapamycin complex 1 (mTORC1), rapamycin, had no effect on the insulin-stimulated glucose uptake, but eliminated the facilitating effect of leucine in the insulin-stimulated glucose uptake and AKT phosphorylation. In addition, leucine facilitation of the insulin-induced AKT phosphorylation was neutralized by knocking down the core component of the mammalian target of rapamycin complex 2 (mTORC2) with specific siRNA. Together, these findings show that leucine can facilitate the insulin-induced insulin signaling and glucose uptake in skeletal muscle cells through both mTORC1 and mTORC2, implicating the potential importance of this amino acid in glucose homeostasis and providing new mechanistic insights.

Interactions between Depression and Facilitation within Neural Networks: Updating the Dual-Process Theory of Plasticity

PubMed Central

Prescott, Steven A.

1998-01-01

Repetitive stimulation often results in habituation of the elicited response. However, if the stimulus is sufficiently strong, habituation may be preceded by transient sensitization or even replaced by enduring sensitization. In 1970, Groves and Thompson formulated the dual-process theory of plasticity to explain these characteristic behavioral changes on the basis of competition between decremental plasticity (depression) and incremental plasticity (facilitation) occurring within the neural network. Data from both vertebrate and invertebrate systems are reviewed and indicate that the effects of depression and facilitation are not exclusively additive but, rather, that those processes interact in a complex manner. Serial ordering of induction of learning, in which a depressing locus precedes the modulatory system responsible for inducing facilitation, causes the facilitation to wane. The parallel and/or serial expression of depression and waning facilitation within the stimulus–response pathway culminates in the behavioral changes that characterize dual-process learning. A mathematical model is presented to formally express and extend understanding of the interactions between depression and facilitation. PMID:10489261

Effects of spectral complexity and sound duration on automatic complex-sound pitch processing in humans - a mismatch negativity study.

PubMed

Tervaniemi, M; Schröger, E; Saher, M; Näätänen, R

2000-08-18

The pitch of a spectrally rich sound is known to be more easily perceived than that of a sinusoidal tone. The present study compared the importance of spectral complexity and sound duration in facilitated pitch discrimination. The mismatch negativity (MMN), which reflects automatic neural discrimination, was recorded to a 2. 5% pitch change in pure tones with only one sinusoidal frequency component (500 Hz) and in spectrally rich tones with three (500-1500 Hz) and five (500-2500 Hz) harmonic partials. During the recordings, subjects concentrated on watching a silent movie. In separate blocks, stimuli were of 100 and 250 ms in duration. The MMN amplitude was enhanced with both spectrally rich sounds when compared with pure tones. The prolonged sound duration did not significantly enhance the MMN. This suggests that increased spectral rather than temporal information facilitates pitch processing of spectrally rich sounds.

Working with lay people in health service research: a model of co-evolution based on complexity theory.

PubMed

Kernick, David; Mitchell, Annie

2010-01-01

Involving lay researchers is an important part of Government policy in the United Kingdom within the context of the National Health Service. Here we draw upon insights from complexity theory to suggest a model that we call consensual qualitative research where lay researchers and professionals are co-producers of knowledge. The focus of attention is on understanding and facilitating the patterns that emerge from non-linear interaction at a local level. We describe some core principles that can facilitate the development of such a model and conclude that the resources in terms of time and effort that such an approach requires, should not be underestimated.

Chronic stress may facilitate the recruitment of habit- and addiction-related neurocircuitries through neuronal restructuring of the striatum

PubMed Central

Taylor, S.B.; Anglin, J.M.; Paode, P.R.; Riggert, A.G.; Olive, M.F.; Conrad, C.D.

2014-01-01

Chronic stress is an established risk factor in the development of addiction. Addiction is characterized by a progressive transition from casual drug use to habitual and compulsive drug use. The ability of chronic stress to facilitate the transition to addiction may be mediated by increased engagement of the neurocircuitries underlying habitual behavior and addiction. In the present study, striatal morphology was evaluated after two weeks of chronic variable stress in male Sprague-Dawley rats. Dendritic complexity of medium spiny neurons was visualized and quantified with Golgi staining in the dorsolateral and dorsomedial striatum, as well as in the nucleus accumbens core and shell. In separate cohorts, the effects of chronic stress on habitual behavior and the acute locomotor response to methamphetamine were also assessed. Chronic stress resulted in increased dendritic complexity in the dorsolateral striatum and nucleus accumbens core, regions implicated in habitual behavior and addiction, while decreased complexity was found in the nucleus accumbens shell, a region critical for the initial rewarding effects of drugs of abuse. Chronic stress did not affect dendritic complexity in the dorsomedial striatum. A parallel shift toward habitual learning strategies following chronic stress was also identified. There was an initial reduction in acute locomotor response to methamphetamine, but no lasting effect as a result of chronic stress exposure. These findings suggest that chronic stress may facilitate the recruitment of habit- and addiction-related neurocircuitries through neuronal restructuring in the striatum. PMID:25242641

Induced pluripotent stem cell models of Zellweger spectrum disorder show impaired peroxisome assembly and cell type-specific lipid abnormalities.

PubMed

Wang, Xiao-Ming; Yik, Wing Yan; Zhang, Peilin; Lu, Wange; Huang, Ning; Kim, Bo Ram; Shibata, Darryl; Zitting, Madison; Chow, Robert H; Moser, Ann B; Steinberg, Steven J; Hacia, Joseph G

2015-08-29

Zellweger spectrum disorder (PBD-ZSD) is a disease continuum caused by mutations in a subset of PEX genes required for normal peroxisome assembly and function. They highlight the importance of peroxisomes in the development and functions of the central nervous system, liver, and other organs. To date, the underlying bases for the cell-type specificity of disease are not fully elucidated. Primary skin fibroblasts from seven PBD-ZSD patients with biallelic PEX1, PEX10, PEX12, or PEX26 mutations and three healthy donors were transduced with retroviral vectors expressing Yamanaka reprogramming factors. Candidate induced pluripotent stem cells (iPSCs) were subject to global gene expression, DNA methylation, copy number variation, genotyping, in vitro differentiation and teratoma formation assays. Confirmed iPSCs were differentiated into neural progenitor cells (NPCs), neurons, oligodendrocyte precursor cells (OPCs), and hepatocyte-like cell cultures with peroxisome assembly evaluated by microscopy. Saturated very long chain fatty acid (sVLCFA) and plasmalogen levels were determined in primary fibroblasts and their derivatives. iPSCs were derived from seven PBD-ZSD patient-derived fibroblasts with mild to severe peroxisome assembly defects. Although patient and control skin fibroblasts had similar gene expression profiles, genes related to mitochondrial functions and organelle cross-talk were differentially expressed among corresponding iPSCs. Mitochondrial DNA levels were consistent among patient and control fibroblasts, but varied among all iPSCs. Relative to matching controls, sVLCFA levels were elevated in patient-derived fibroblasts, reduced in patient-derived iPSCs, and not significantly different in patient-derived NPCs. All cell types derived from donors with biallelic null mutations in a PEX gene showed plasmalogen deficiencies. Reporter gene assays compatible with high content screening (HCS) indicated patient-derived OPC and hepatocyte-like cell cultures had impaired peroxisome assembly. Normal peroxisome activity levels are not required for cellular reprogramming of skin fibroblasts. Patient iPSC gene expression profiles were consistent with hypotheses highlighting the role of altered mitochondrial activities and organelle cross-talk in PBD-ZSD pathogenesis. sVLCFA abnormalities dramatically differed among patient cell types, similar to observations made in iPSC models of X-linked adrenoleukodystrophy. We propose that iPSCs could assist investigations into the cell type-specificity of peroxisomal activities, toxicology studies, and in HCS for targeted therapies for peroxisome-related disorders.

Secondary foundation species as drivers of trophic and functional diversity: evidence from a tree-epiphyte system.

PubMed

Angelini, Christine; Silliman, Brian R

2014-01-01

Facilitation cascades arise where primary foundation species facilitate secondary (dependent) foundation species, and collectively, they increase habitat complexity and quality to enhance biodiversity. Whether such phenomena occur in nonmarine systems and if secondary foundation species enhance food web structure (e.g., support novel feeding guilds) and ecosystem function (e.g., provide nursery for juveniles) remain unclear. Here we report on field experiments designed to test whether trees improve epiphyte survival and epiphytes secondarily increase the number and diversity of adult and juvenile invertebrates in a potential live oak-Tillandsia usneoides (Spanish moss) facilitation cascade. Our results reveal that trees reduce physical stress to facilitate Tillandsia, which, in turn, reduces desiccation and predation stress to facilitate invertebrates. In experimental removals, invertebrate total density, juvenile density, species richness and H' diversity were 16, 60, 1.7, and 1.5 times higher, and feeding guild richness and H' were 5 and 11 times greater in Tillandsia-colonized relative to Tillandsia-removal limb plots. Tillandsia enhanced communities similarly in a survey across the southeastern United States. These findings reveal that a facilitation cascade organizes this widespread terrestrial assemblage and expand the role of secondary foundation species as drivers of trophic structure and ecosystem function. We conceptualize the relationship between foundation species' structural attributes and associated species abundance and composition in a Foundation Species-Biodiversity (FSB) model. Importantly, the FSB predicts that, where secondary foundation species form expansive and functionally distinct structures that increase habitat availability and complexity within primary foundation species, they generate and maintain hot spots of biodiversity and trophic interactions.

The inhibitory spillover effect: Controlling the bladder makes better liars *

PubMed Central

Fenn, Elise; Blandón-Gitlin, Iris; Coons, Jennifer; Pineda, Catherine; Echon, Reinalyn

2015-01-01

The Inhibitory-Spillover-Effect (ISE) on a deception task was investigated. The ISE occurs when performance in one self-control task facilitates performance in another (simultaneously conducted) self-control task. Deceiving requires increased access to inhibitory control. We hypothesized that inducing liars to control urination urgency (physical inhibition) would facilitate control during deceptive interviews (cognitive inhibition). Participants drank small (low-control) or large (high-control) amounts of water. Next, they lied or told the truth to an interviewer. Third-party observers assessed the presence of behavioral cues and made true/lie judgments. In the high-control, but not the low-control condition, liars displayed significantly fewer behavioral cues to deception, more behavioral cues signaling truth, and provided longer and more complex accounts than truth-tellers. Accuracy detecting liars in the high-control condition was significantly impaired; observers revealed bias toward perceiving liars as truth-tellers. The ISE can operate in complex behaviors. Acts of deception can be facilitated by covert manipulations of self-control. PMID:26366466

Control System Architectures, Technologies and Concepts for Near Term and Future Human Exploration of Space

NASA Technical Reports Server (NTRS)

Boulanger, Richard; Overland, David

2004-01-01

Technologies that facilitate the design and control of complex, hybrid, and resource-constrained systems are examined. This paper focuses on design methodologies, and system architectures, not on specific control methods that may be applied to life support subsystems. Honeywell and Boeing have estimated that 60-80Y0 of the effort in developing complex control systems is software development, and only 20-40% is control system development. It has also been shown that large software projects have failure rates of as high as 50-65%. Concepts discussed include the Unified Modeling Language (UML) and design patterns with the goal of creating a self-improving, self-documenting system design process. Successful architectures for control must not only facilitate hardware to software integration, but must also reconcile continuously changing software with much less frequently changing hardware. These architectures rely on software modules or components to facilitate change. Architecting such systems for change leverages the interfaces between these modules or components.

Molecular Speciation of Trace Metal Organic Complexes in the Pacific Ocean

NASA Astrophysics Data System (ADS)

Repeta, D.; Boiteau, R. M.; Bundy, R. M.; Babcock-Adams, L.

2017-12-01

Microbial production across approximately one third of the surface ocean is limited by extraordinarily low (picomolar) concentrations of dissolved iron, essentially all of which is complexed to strong organic ligands of unknown composition. Other biologically important trace metals (cobalt, copper, zinc, nickel) are also complexed to strong organic ligands, which again have not been extensively characterized. Nevertheless, organic ligands exert a strong influence on metal bioavailability and toxicity. For example, amendment experiments using commercially available siderophores, organic compounds synthesized by microbes to facilitate iron uptake, show these ligands can both facilitate or impede iron uptake depending on the siderophore composition and available uptake pathways. Over the past few years we have developed analytical techniques using high pressure liquid chromatography interfaced with inductively coupled plasma and electrospray ionization mass spectrometry to identify and quantify trace metal organic complexes in laboratory cultures of marine microbes and in seawater. We found siderophores to be widely distributed in the ocean, particularly in regions characterized by low iron concentrations. We also find chemically distinct complexes of copper, zinc, colbalt and nickel that we have yet to fully characterize. We will discuss some of our recent work on trace metal organic speciation in seawater and laboratory cultures, and outline future efforts to better understand the microbial cycling of trace metal organic complexes in the sea.

H3K9me3 demethylase Kdm4d facilitates the formation of pre-initiative complex and regulates DNA replication

PubMed Central

Wu, Rentian; Wang, Zhiquan; Zhang, Honglian; Gan, Haiyun; Zhang, Zhiguo

2017-01-01

DNA replication is tightly regulated to occur once and only once per cell cycle. How chromatin, the physiological substrate of DNA replication machinery, regulates DNA replication remains largely unknown. Here we show that histone H3 lysine 9 demethylase Kdm4d regulates DNA replication in eukaryotic cells. Depletion of Kdm4d results in defects in DNA replication, which can be rescued by the expression of H3K9M, a histone H3 mutant transgene that reverses the effect of Kdm4d on H3K9 methylation. Kdm4d interacts with replication proteins, and its recruitment to DNA replication origins depends on the two pre-replicative complex components (origin recognition complex [ORC] and minichromosome maintenance [MCM] complex). Depletion of Kdm4d impairs the recruitment of Cdc45, proliferating cell nuclear antigen (PCNA), and polymerase δ, but not ORC and MCM proteins. These results demonstrate a novel mechanism by which Kdm4d regulates DNA replication by reducing the H3K9me3 level to facilitate formation of pre-initiative complex. PMID:27679476

A STRIPAK complex mediates axonal transport of autophagosomes and dense core vesicles through PP2A regulation

PubMed Central

Neufeld, Thomas P.

2017-01-01

Autophagy plays an essential role in the cellular homeostasis of neurons, facilitating the clearance of cellular debris. This clearance process is orchestrated through the assembly, transport, and fusion of autophagosomes with lysosomes for degradation. The motor protein dynein drives autophagosome motility from distal sites of assembly to sites of lysosomal fusion. In this study, we identify the scaffold protein CKA (connector of kinase to AP-1) as essential for autophagosome transport in neurons. Together with other core components of the striatin-interacting phosphatase and kinase (STRIPAK) complex, we show that CKA associates with dynein and directly binds Atg8a, an autophagosomal protein. CKA is a regulatory subunit of PP2A, a component of the STRIPAK complex. We propose that the STRIPAK complex modulates dynein activity. Consistent with this hypothesis, we provide evidence that CKA facilitates axonal transport of dense core vesicles and autophagosomes in a PP2A-dependent fashion. In addition, CKA-deficient flies exhibit PP2A-dependent motor coordination defects. CKA function within the STRIPAK complex is crucial to prevent transport defects that may contribute to neurodegeneration. PMID:28100687

Poly(ADP-ribose) polymerase 1 escorts XPC to UV-induced DNA lesions during nucleotide excision repair

PubMed Central

Robu, Mihaela; Shah, Rashmi G.; Purohit, Nupur K.; Zhou, Pengbo; Naegeli, Hanspeter

2017-01-01

Xeroderma pigmentosum C (XPC) protein initiates the global genomic subpathway of nucleotide excision repair (GG-NER) for removal of UV-induced direct photolesions from genomic DNA. The XPC has an inherent capacity to identify and stabilize at the DNA lesion sites, and this function is facilitated in the genomic context by UV-damaged DNA-binding protein 2 (DDB2), which is part of a multiprotein UV–DDB ubiquitin ligase complex. The nuclear enzyme poly(ADP-ribose) polymerase 1 (PARP1) has been shown to facilitate the lesion recognition step of GG-NER via its interaction with DDB2 at the lesion site. Here, we show that PARP1 plays an additional DDB2-independent direct role in recruitment and stabilization of XPC at the UV-induced DNA lesions to promote GG-NER. It forms a stable complex with XPC in the nucleoplasm under steady-state conditions before irradiation and rapidly escorts it to the damaged DNA after UV irradiation in a DDB2-independent manner. The catalytic activity of PARP1 is not required for the initial complex formation with XPC in the nucleoplasm but it enhances the recruitment of XPC to the DNA lesion site after irradiation. Using purified proteins, we also show that the PARP1–XPC complex facilitates the handover of XPC to the UV-lesion site in the presence of the UV–DDB ligase complex. Thus, the lesion search function of XPC in the genomic context is controlled by XPC itself, DDB2, and PARP1. Our results reveal a paradigm that the known interaction of many proteins with PARP1 under steady-state conditions could have functional significance for these proteins. PMID:28760956

Poly(ADP-ribose) polymerase 1 escorts XPC to UV-induced DNA lesions during nucleotide excision repair.

PubMed

Robu, Mihaela; Shah, Rashmi G; Purohit, Nupur K; Zhou, Pengbo; Naegeli, Hanspeter; Shah, Girish M

2017-08-15

Xeroderma pigmentosum C (XPC) protein initiates the global genomic subpathway of nucleotide excision repair (GG-NER) for removal of UV-induced direct photolesions from genomic DNA. The XPC has an inherent capacity to identify and stabilize at the DNA lesion sites, and this function is facilitated in the genomic context by UV-damaged DNA-binding protein 2 (DDB2), which is part of a multiprotein UV-DDB ubiquitin ligase complex. The nuclear enzyme poly(ADP-ribose) polymerase 1 (PARP1) has been shown to facilitate the lesion recognition step of GG-NER via its interaction with DDB2 at the lesion site. Here, we show that PARP1 plays an additional DDB2-independent direct role in recruitment and stabilization of XPC at the UV-induced DNA lesions to promote GG-NER. It forms a stable complex with XPC in the nucleoplasm under steady-state conditions before irradiation and rapidly escorts it to the damaged DNA after UV irradiation in a DDB2-independent manner. The catalytic activity of PARP1 is not required for the initial complex formation with XPC in the nucleoplasm but it enhances the recruitment of XPC to the DNA lesion site after irradiation. Using purified proteins, we also show that the PARP1-XPC complex facilitates the handover of XPC to the UV-lesion site in the presence of the UV-DDB ligase complex. Thus, the lesion search function of XPC in the genomic context is controlled by XPC itself, DDB2, and PARP1. Our results reveal a paradigm that the known interaction of many proteins with PARP1 under steady-state conditions could have functional significance for these proteins.

Using a meta-ethnographic approach to explore the nature of facilitation and teaching approaches employed in interprofessional education.

PubMed

Reeves, Scott; Pelone, Ferruccio; Hendry, Julie; Lock, Nicholas; Marshall, Jayne; Pillay, Leontia; Wood, Ruth

2016-12-01

Interprofessional facilitators and teachers are regarded as central to the effective delivery of interprofessional education (IPE). As the IPE literature continues to expand, most studies have focused on reporting learner outcomes, with little attention paid to IPE facilitation. However, a number of studies have recently emerged reporting on this phenomenon. To present a synthesis of qualitative evidence on the facilitation of IPE, using a meta-ethnographic approach. Electronic databases and journals were searched for the past 10 years. Of the 2164 abstracts initially found, 94 full papers were reviewed and subsequently 12 papers were included. Teams of two reviewers independently completed each step in the review process. The quality of these papers was assessed using a modified critical appraisal checklist. Seven key concepts embedded in the included studies were synthesized into three main factors which provided an insight into the nature of IPE facilitation. Specifically, the synthesis found that IPE facilitation is influenced by "contextual characteristics"; "facilitator experiences"; and the "use of different facilitation strategies". IPE facilitation is a complex activity affected by contextual, experiential and pedagogical factors. Further research is needed to explore the effects of these factors on the delivery of IPE.

A geologic and mineral exploration spatial database for the Stillwater Complex, Montana

USGS Publications Warehouse

Zientek, Michael L.; Parks, Heather L.

2014-01-01

This report provides essential spatially referenced datasets based on geologic mapping and mineral exploration activities conducted from the 1920s to the 1990s. This information will facilitate research on the complex and provide background material needed to explore for mineral resources and to develop sound land-management policy.

Achieving Complex Learning Outcomes through Adoption of a Pedagogical Perspective: A Model for Computer Technology Delivered Instruction

ERIC Educational Resources Information Center

Bellard, Breshanica

2018-01-01

Professionals responsible for the delivery of education and training using technology systems and platforms can facilitate complex learning through application of relevant strategies, principles and theories that support how learners learn and that support how curriculum should be designed in a technology based learning environment. Technological…

Education: A Complex and Empowering Social Work Intervention at the End of Life

ERIC Educational Resources Information Center

Cagle, John G.; Kovacs, Pamela J.

2009-01-01

Education is a frequently used social work intervention. Yet it seems to be an underappreciated and a deceptively complex intervention that social workers may not be adequately prepared to use. Reliable, accessible information is essential as it helps prevent unnecessary crises, facilitates coping, and promotes self-determination. This article…

Catalytic Activity of Thiolate-Bridged Diruthenium Complexes Bearing Pendent Ether Moieties in the Oxidation of Molecular Dihydrogen.

PubMed

Yuki, Masahiro; Sakata, Ken; Kikuchi, Shoma; Kawai, Hiroyuki; Takahashi, Tsuyoshi; Ando, Masaki; Nakajima, Kazunari; Nishibayashi, Yoshiaki

2017-01-23

Thiolate-bridged diruthenium complexes bearing pendent ethers have been found to work as effective catalysts toward the oxidation of molecular dihydrogen into protons and electrons in water. The pendent ether moiety in the complex plays an important role to facilitate the proton transfer between the metal center and the external proton acceptor. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

An exploration of teaching presence in online interprofessional education facilitation.

PubMed

Evans, Sherryn Maree; Ward, Catherine; Reeves, Scott

2017-07-01

Although the prevalence of online asynchronous interprofessional education (IPE) has increased in the last decade, little is known about the processes of facilitation in this environment. The teaching presence element of the Community of Inquiry Framework offers an approach to analyze the contributions of online facilitators, however, to date it has only been used on a limited basis in health professions education literature. Using an exploratory case study design, we explored the types of contributions made by IPE facilitators to asynchronous interprofessional team discussions by applying the notion of teaching presence. Using a purposeful sampling approach, we analyzed 14 facilitators' contributions to asynchronous team discussion boards in an online IPE course. We analyzed data using directed content analysis based on the key indicators of teaching presence. The online IPE facilitators undertook the three critical pedagogical functions identified in teaching presence: facilitating discourse, direct instruction, and instructional design and organization. While our data fitted well with a number of key activities embedded in these three functions, further modification of the teaching presence concept was needed to describe our facilitators' teaching presence. This study provides an initial insight into the key elements of online asynchronous IPE facilitation. Further research is required to continue to illuminate the complexity of online asynchronous IPE facilitation.

Building dialogue on complex conservation issues in a conference setting.

PubMed

Rock, Jenny; Sparrow, Andrew; Wass, Rob; Moller, Henrik

2014-10-01

Dialogue about complex science and society issues is important for contemporary conservation agendas. Conferences provide an appropriate space for such dialogue, but despite its recognized worth, best practices for facilitating active dialogue are still being explored. Face-to-face (FTF) and computer-mediated communication (CMC) are two approaches to facilitating dialogue that have different strengths. We assessed the use of these approaches to create dialogue on cultural perspectives of conservation and biodiversity at a national ecology conference. In particular, we aimed to evaluate their potential to enhance dialogue through their integrated application. We used an interactive blog to generate CMC on participant-sourced issues and to prime subsequent discussion in an FTF conference workshop. The quantity and quality of both CMC and FTF discussion indicated that both approaches were effective in building dialogue. Prior to the conference the blog averaged 126 views per day, and 44 different authors contributed a total of 127 comments. Twenty-five participants subsequently participated in active FTF discussion during a 3-h workshop. Postconference surveys confirmed that CMC had developed participants' thinking and deepened FTF dialogue; 88% indicated specifically that CMC helped facilitate the FTF discussion. A further 83% of respondents concluded that preliminary blog discussion would be useful for facilitating dialogue at future conferences. © 2014 Society for Conservation Biology.

Factory Skills Training at Honeywell.

ERIC Educational Resources Information Center

Fey, Carol L.

1981-01-01

Because of increased training needs, caused by complex products and employee turnover, Honeywell provides trainers with training packages which facilitate more effective teaching of new production workers. (JOW)

Chronic stress may facilitate the recruitment of habit- and addiction-related neurocircuitries through neuronal restructuring of the striatum.

PubMed

Taylor, S B; Anglin, J M; Paode, P R; Riggert, A G; Olive, M F; Conrad, C D

2014-11-07

Chronic stress is an established risk factor in the development of addiction. Addiction is characterized by a progressive transition from casual drug use to habitual and compulsive drug use. The ability of chronic stress to facilitate the transition to addiction may be mediated by increased engagement of the neurocircuitries underlying habitual behavior and addiction. In the present study, striatal morphology was evaluated after 2 weeks of chronic variable stress in male Sprague-Dawley rats. Dendritic complexity of medium spiny neurons was visualized and quantified with Golgi staining in the dorsolateral and dorsomedial striatum, as well as in the nucleus accumbens core and shell. In separate cohorts, the effects of chronic stress on habitual behavior and the acute locomotor response to methamphetamine were also assessed. Chronic stress resulted in increased dendritic complexity in the dorsolateral striatum and nucleus accumbens core, regions implicated in habitual behavior and addiction, while decreased complexity was found in the nucleus accumbens shell, a region critical for the initial rewarding effects of drugs of abuse. Chronic stress did not affect dendritic complexity in the dorsomedial striatum. A parallel shift toward habitual learning strategies following chronic stress was also identified. There was an initial reduction in acute locomotor response to methamphetamine, but no lasting effect as a result of chronic stress exposure. These findings suggest that chronic stress may facilitate the recruitment of habit- and addiction-related neurocircuitries through neuronal restructuring in the striatum. Copyright © 2014 IBRO. Published by Elsevier Ltd. All rights reserved.

The Effects of Semantic Transparency and Base Frequency on the Recognition of English Complex Words

ERIC Educational Resources Information Center

Xu, Joe; Taft, Marcus

2015-01-01

A visual lexical decision task was used to examine the interaction between base frequency (i.e., the cumulative frequencies of morphologically related forms) and semantic transparency for a list of derived words. Linear mixed effects models revealed that high base frequency facilitates the recognition of the complex word (i.e., a "base…

An Exploration of the Calcium-Binding Mode of Egg White Peptide, Asp-His-Thr-Lys-Glu, and In Vitro Calcium Absorption Studies of Peptide-Calcium Complex.

PubMed

Sun, Na; Jin, Ziqi; Li, Dongmei; Yin, Hongjie; Lin, Songyi

2017-11-08

The binding mode between the pentapeptide (DHTKE) from egg white hydrolysates and calcium ions was elucidated upon its structural and thermodynamics characteristics. The present study demonstrated that the DHTKE peptide could spontaneously bind calcium with a 1:1 stoichiometry, and that the calcium-binding site corresponded to the carboxyl oxygen, amino nitrogen, and imidazole nitrogen atoms of the DHTKE peptide. Moreover, the effect of the DHTKE-calcium complex on improving the calcium absorption was investigated in vitro using Caco-2 cells. Results showed that the DHTKE-calcium complex could facilitate the calcium influx into the cytosol and further improve calcium absorption across Caco-2 cell monolayers by more than 7 times when compared to calcium-free control. This study facilitates the understanding about the binding mechanism between peptides and calcium ions as well as suggests a potential application of egg white peptides as nutraceuticals to improve calcium absorption.

[Documenting a rehabilitation program using a logic model: an advantage to the assessment process].

PubMed

Poncet, Frédérique; Swaine, Bonnie; Pradat-Diehl, Pascale

2017-03-06

The cognitive and behavioral disorders after brain injury can result in severe limitations of activities and restrictions of participation. An interdisciplinary rehabilitation program was developed in physical medicine and rehabilitation at the Pitié-Salpêtriere Hospital, Paris, France. Clinicians believe this program decreases activity limitations and improves participation in patients. However, the program’s effectiveness had never been assessed. To do this, we had to define/describe this program. However rehabilitation programs are holistic and thus complex making them difficult to describe. Therefore, to facilitate the evaluation of complex programs, including those for rehabilitation, we illustrate the use of a theoretical logic model, as proposed by Champagne, through the process of documentation of a specific complex and interdisciplinary rehabilitation program. Through participatory/collaborative research, the rehabilitation program was analyzed using three “submodels” of the logic model of intervention: causal model, intervention model and program theory model. This should facilitate the evaluation of programs, including those for rehabilitation.

Exploring the Complexities of Children's Inquiries in Science: Knowledge Production Through Participatory Practices

NASA Astrophysics Data System (ADS)

Siry, Christina

2013-12-01

Beginning with the assumption that young children are capable of producing unprecedented knowledges about science phenomena, this paper explores the complexities of children's inquiries within open-ended investigations. I ask two central questions: (1) how can we (teachers, researchers, and children themselves) use and build upon children's explorations in science in practice? and (2) what pedagogical approaches can position children as experts on their experiences to facilitate children's sense of ownership in the process of learning science? Six vignettes from a Kindergarten classroom are analyzed to elaborate the central claim of this work, which is that when children are engaged in collaborative open-ended activities, science emerges from their interactions. Open-ended structures allowed for teachers and children to facilitate further investigations collaboratively, and participatory structures mediated children's representations and explanations of their investigations. Evidence of children's interactions is used to illustrate the complexities of children's explorations, and pedagogical approaches that create the spaces for children to create knowledge are highlighted.

An inducible ER–Golgi tether facilitates ceramide transport to alleviate lipotoxicity

PubMed Central

Choudhary, Vineet

2017-01-01

Ceramides are key intermediates in sphingolipid biosynthesis and potent signaling molecules. However, excess ceramide is toxic, causing growth arrest and apoptosis. In this study, we identify a novel mechanism by which cells prevent the toxic accumulation of ceramides; they facilitate nonvesicular ceramide transfer from the endoplasmic reticulum (ER) to the Golgi complex, where ceramides are converted to complex sphingolipids. We find that the yeast protein Nvj2p promotes the nonvesicular transfer of ceramides from the ER to the Golgi complex. The protein is a tether that generates close contacts between these compartments and may directly transport ceramide. Nvj2p normally resides at contacts between the ER and other organelles, but during ER stress, it relocalizes to and increases ER–Golgi contacts. ER–Golgi contacts fail to form during ER stress in cells lacking Nvj2p. Our findings demonstrate that cells regulate ER–Golgi contacts in response to stress and reveal that nonvesicular ceramide transfer out of the ER prevents the buildup of toxic amounts of ceramides. PMID:28011845

Effect of Binding Components in Complex Sample Matrices on Recovery in Direct Immersion Solid-Phase Microextraction: Friends or Foe?

PubMed

Alam, Md Nazmul; Pawliszyn, Janusz

2018-02-20

The development of matrix compatible coatings for solid-phase microextraction (SPME) has enabled direct extraction of analytes from complex sample matrices. The direct immersion (DI) mode of SPME when utilized in conjunction with such extraction phases facilitates extraction of a wide range of analytes from complex matrices without the incurrence of fouling or coating saturation. In this work, mathematical models and computational simulations were employed to investigate the effect of binding components present in complex samples on the recovery of small molecules varying in logP for extractions carried out using the direct immersion approach. The presented findings corroborate that the studied approach indeed enables the extraction of both polar and nonpolar analytes from complex matrices, provided a suitable sorbent is employed. Further results indicated that, in certain cases, the kinetics of extraction of a given analyte in its free form might be dependent on the desorption kinetics of their bound form from matrix components, which might lower total recoveries of analytes with high affinity for the matrix. However, the binding of analytes to matrix components also enables SPME to extract a balanced quantity of different logP analytes, facilitated by multiphase equilibria, with a single extraction device.

H3K9me3 demethylase Kdm4d facilitates the formation of pre-initiative complex and regulates DNA replication.

PubMed

Wu, Rentian; Wang, Zhiquan; Zhang, Honglian; Gan, Haiyun; Zhang, Zhiguo

2017-01-09

DNA replication is tightly regulated to occur once and only once per cell cycle. How chromatin, the physiological substrate of DNA replication machinery, regulates DNA replication remains largely unknown. Here we show that histone H3 lysine 9 demethylase Kdm4d regulates DNA replication in eukaryotic cells. Depletion of Kdm4d results in defects in DNA replication, which can be rescued by the expression of H3K9M, a histone H3 mutant transgene that reverses the effect of Kdm4d on H3K9 methylation. Kdm4d interacts with replication proteins, and its recruitment to DNA replication origins depends on the two pre-replicative complex components (origin recognition complex [ORC] and minichromosome maintenance [MCM] complex). Depletion of Kdm4d impairs the recruitment of Cdc45, proliferating cell nuclear antigen (PCNA), and polymerase δ, but not ORC and MCM proteins. These results demonstrate a novel mechanism by which Kdm4d regulates DNA replication by reducing the H3K9me3 level to facilitate formation of pre-initiative complex. © The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.

Perceptions of family-centred services in a paediatric rehabilitation programme: strengths and complexities from multiple stakeholders.

PubMed

Arcuri, G G; McMullan, A E; Murray, A E; Silver, L K; Bergthorson, M; Dahan-Oliel, N; Coutinho, F

2016-03-01

Family-centred services (FCS) are best practice in paediatric rehabilitation and describe philosophies and approaches to medical care that emphasize the partnership and involvement of parents. While evidence supports FCS, there are complexities to its successful implementation. This mixed-methods study aimed to measure the extent to which parents and the healthcare provider (HCP) perceive service provision as being family centred, and to describe barriers and facilitators to the delivery of FCS. Parents of children participating in a rehabilitation programme and HCPs providing services participated in this study. Parents completed the measure of processes of care-20 and participated in interviews, while HCPs completed the measure of processes of care-service providers and participated in a focus group. Quantitative analysis revealed that parents were mostly satisfied with features of FCS, which included communication and support between parents and HCPs, respect of diversity and parental collaboration and participation. Parents identified communication methods and psychosocial needs as areas that facilitated but sometimes detracted from FCS. Institutional barriers led to the identification of areas for improvement identified by multiple stakeholders. HCPs identified more areas for improvement than parents. When considering these barriers, it is evident that implementation is a complex process, impacted by institutional barriers. FCS needs to be investigated further, and systemic interventions should be used to facilitate its implementation. © 2015 John Wiley & Sons Ltd.

Kinetic Model Facilitates Analysis of Fibrin Generation and Its Modulation by Clotting Factors: Implications for Hemostasis-Enhancing Therapies

DTIC Science & Technology

2014-01-01

facilitates analysis of fibrin generation and its modulation by clotting factors : implications for hemostasis-enhancing therapies† Alexander Y...investigate the ability of fibrinogen and a recently proposed prothrombin complex concentrate composition, PCC-AT (a combination of the clotting factors II...kinetics. Moreover, the model qualitatively predicted the impact of tissue factor and tPA/tenecteplase level variations on the fibrin output. In the

Mobile health solutions for the aging population: A systematic narrative analysis.

PubMed

Kruse, Clemens Scott; Mileski, Michael; Moreno, Joshua

2017-05-01

Introduction The ubiquitous nature of mobile technology coupled with the acceptance of mobile health (mHealth) among the elderly offers an opportunity to augment the existing medical workforce in long-term care. The objective of this review and narrative analysis is to identify and analyse facilitators and barriers to adoption of mHealth for the elderly. Methods Studies over the last year were identified in multiple database indices, and three reviewers examined abstracts ( k = 0.82) and analysed articles for themes which were tallied in affinity diagrams to identify frequency of occurrence in the literature (n = 36). Results The three facilitators mentioned most often were independence (18%), understanding (13%), and visibility (13%). The three barriers mentioned most often were complexity (21%), limited by users (12%) and ineffective (12%). Discussion and conclusions The reviewers concluded that the work done so far illustrates that mHealth enables a perception of independence. Future research should focus on the barriers of complexity of technology and improving existing medical literacy in order to facilitate further adoption.

Application of Intervention Mapping to the Development of a Complex Physical Therapist Intervention.

PubMed

Jones, Taryn M; Dear, Blake F; Hush, Julia M; Titov, Nickolai; Dean, Catherine M

2016-12-01

Physical therapist interventions, such as those designed to change physical activity behavior, are often complex and multifaceted. In order to facilitate rigorous evaluation and implementation of these complex interventions into clinical practice, the development process must be comprehensive, systematic, and transparent, with a sound theoretical basis. Intervention Mapping is designed to guide an iterative and problem-focused approach to the development of complex interventions. The purpose of this case report is to demonstrate the application of an Intervention Mapping approach to the development of a complex physical therapist intervention, a remote self-management program aimed at increasing physical activity after acquired brain injury. Intervention Mapping consists of 6 steps to guide the development of complex interventions: (1) needs assessment; (2) identification of outcomes, performance objectives, and change objectives; (3) selection of theory-based intervention methods and practical applications; (4) organization of methods and applications into an intervention program; (5) creation of an implementation plan; and (6) generation of an evaluation plan. The rationale and detailed description of this process are presented using an example of the development of a novel and complex physical therapist intervention, myMoves-a program designed to help individuals with an acquired brain injury to change their physical activity behavior. The Intervention Mapping framework may be useful in the development of complex physical therapist interventions, ensuring the development is comprehensive, systematic, and thorough, with a sound theoretical basis. This process facilitates translation into clinical practice and allows for greater confidence and transparency when the program efficacy is investigated. © 2016 American Physical Therapy Association.

Spatio-temporal Model of Endogenous ROS and Raft-Dependent WNT/Beta-Catenin Signaling Driving Cell Fate Commitment in Human Neural Progenitor Cells

PubMed Central

Haack, Fiete; Lemcke, Heiko; Ewald, Roland; Rharass, Tareck; Uhrmacher, Adelinde M.

2015-01-01

Canonical WNT/β-catenin signaling is a central pathway in embryonic development, but it is also connected to a number of cancers and developmental disorders. Here we apply a combined in-vitro and in-silico approach to investigate the spatio-temporal regulation of WNT/β-catenin signaling during the early neural differentiation process of human neural progenitors cells (hNPCs), which form a new prospect for replacement therapies in the context of neurodegenerative diseases. Experimental measurements indicate a second signal mechanism, in addition to canonical WNT signaling, being involved in the regulation of nuclear β-catenin levels during the cell fate commitment phase of neural differentiation. We find that the biphasic activation of β-catenin signaling observed experimentally can only be explained through a model that combines Reactive Oxygen Species (ROS) and raft dependent WNT/β-catenin signaling. Accordingly after initiation of differentiation endogenous ROS activates DVL in a redox-dependent manner leading to a transient activation of down-stream β-catenin signaling, followed by continuous auto/paracrine WNT signaling, which crucially depends on lipid rafts. Our simulation studies further illustrate the elaborate spatio-temporal regulation of DVL, which, depending on its concentration and localization, may either act as direct inducer of the transient ROS/β-catenin signal or as amplifier during continuous auto-/parcrine WNT/β-catenin signaling. In addition we provide the first stochastic computational model of WNT/β-catenin signaling that combines membrane-related and intracellular processes, including lipid rafts/receptor dynamics as well as WNT- and ROS-dependent β-catenin activation. The model’s predictive ability is demonstrated under a wide range of varying conditions for in-vitro and in-silico reference data sets. Our in-silico approach is realized in a multi-level rule-based language, that facilitates the extension and modification of the model. Thus, our results provide both new insights and means to further our understanding of canonical WNT/β-catenin signaling and the role of ROS as intracellular signaling mediator. PMID:25793621

Localizing Protein in 3D Neural Stem Cell Culture: a Hybrid Visualization Methodology

PubMed Central

Fai, Stephen; Bennett, Steffany A.L.

2010-01-01

The importance of 3-dimensional (3D) topography in influencing neural stem and progenitor cell (NPC) phenotype is widely acknowledged yet challenging to study. When dissociated from embryonic or post-natal brain, single NPCs will proliferate in suspension to form neurospheres. Daughter cells within these cultures spontaneously adopt distinct developmental lineages (neurons, oligodendrocytes, and astrocytes) over the course of expansion despite being exposed to the same extracellular milieu. This progression recapitulates many of the stages observed over the course of neurogenesis and gliogenesis in post-natal brain and is often used to study basic NPC biology within a controlled environment. Assessing the full impact of 3D topography and cellular positioning within these cultures on NPC fate is, however, difficult. To localize target proteins and identify NPC lineages by immunocytochemistry, free-floating neurospheres must be plated on a substrate or serially sectioned. This processing is required to ensure equivalent cell permeabilization and antibody access throughout the sphere. As a result, 2D epifluorescent images of cryosections or confocal reconstructions of 3D Z-stacks can only provide spatial information about cell position within discrete physical or digital 3D slices and do not visualize cellular position in the intact sphere. Here, to reiterate the topography of the neurosphere culture and permit spatial analysis of protein expression throughout the entire culture, we present a protocol for isolation, expansion, and serial sectioning of post-natal hippocampal neurospheres suitable for epifluorescent or confocal immunodetection of target proteins. Connexin29 (Cx29) is analyzed as an example. Next, using a hybrid of graphic editing and 3D modelling softwares rigorously applied to maintain biological detail, we describe how to re-assemble the 3D structural positioning of these images and digitally map labelled cells within the complete neurosphere. This methodology enables visualization and analysis of the cellular position of target proteins and cells throughout the entire 3D culture topography and will facilitate a more detailed analysis of the spatial relationships between cells over the course of neurogenesis and gliogenesis in vitro. Both Imbeault and Valenzuela contributed equally and should be considered joint first authors. PMID:21258319

An Examination of Cultural Influences on Team Cognition and Information Sharing in Emergency Crisis Management Domains: A Mixed Methodological Approach

ERIC Educational Resources Information Center

Endsley, Tristan Caroline

2016-01-01

The consequence for better understanding and facilitating teamwork during crisis and disaster response in the coming years is significant. It becomes clear that environmental and situational complexity requires a reliance on teams to carry out response efforts. Crises, regardless of the type of event, require complex decision-making and planning…

Developing a Service Management Strategy Facilitated by Action Learning: An Empirical Study from the UK Health & Fitness Industry

ERIC Educational Resources Information Center

Oliver, John

2006-01-01

One of the principle tenets of action learning is that it provides the potential to explore and solve complex organisational problems. The question of how best to develop a future business strategy is such a problem. Existing literature on strategy making presents a multi-faceted debate, suggesting that the complexity of competitive environments…

Design Patterns for Learning and Assessment: Facilitating the Introduction of a Complex Simulation-Based Learning Environment into a Community of Instructors

ERIC Educational Resources Information Center

Frezzo, Dennis C.; Behrens, John T.; Mislevy, Robert J.

2010-01-01

Simulation environments make it possible for science and engineering students to learn to interact with complex systems. Putting these capabilities to effective use for learning, and assessing learning, requires more than a simulation environment alone. It requires a conceptual framework for the knowledge, skills, and ways of thinking that are…