Outer Cell Layers Research Articles

A Review on Surface Modification of Bast Fibre as Reinforcement in Polymer Composites

In the past few decades, natural fibres have shown remarkable achievements in the field of reinforced polymer composites because of environmental issues and petroleum crises. Biodegradable fibres (bast, animals hair, stalk, seed etc.) has been used by researchers because of its low cost, low density, abundant in nature, high stiffness etc. over other man-made fibers. The majority of plant fibers which are being considered as reinforcement materials for polymer composites are bast fibers (the outer cell layers of various plants) such as hemp, jute, kenaf, grewia optiva etc. Among these fibers Grewia optiva is recently explored fiber and is presently being assessed as reinforcement in polymeric composites. In this paper, we have discussed and reviewed the extraction process of Grewia optiva fibers and various surface treatments such as graft copolymerization, mercerization, Benzoylation and silane methods for enhancement of the surface adhesion properties of Grewia optiva fibres with their matrice and the effects of these surface treatments on mechanical properties of such fabricated composites.

EXPRESSION OF TUMOR ASSOSIATED AND EPITHELIAL-MESENCHYMAL TRANSITION MARKERS IN 2D AND 3D CELL CULTURES OF MCF-7

The target effects on the expression of epithelial-mesenchymal transition regulation molecules are promising for cancer therapy, including breast cancer. 3D cell culture is a model for studying epithelial-mesenchymal transition in vitro and may become a test system for anticancer therapy. Aim of research. The aim of this research was to evaluate and compare the expression of tumor associated and epithelial-mesenchymal transition markers in tumor cells of breast adenocarcinoma (MCF-7 cell line) in 2D and 3D cell culture. Methods. For realization of the aim MCF-7 cell line (breast adenocarcinoma) was chosen as an experimental model in vitro. The monolayer cell culture was cultured in standard conditions (37 0C, 5 % CO2, humidity 95 %). The initial density of inoculated cells was 2 x 104 cells/cm2. The cells were incubated for two days before their use in the experiment. For the initial generation of spheroids the monolayer cell culture was removed off the substrate after the four days of incubation, using 0,25 % Trypsin-EDTA, and placed in nutrient medium with 5 % carboxymethyl cellulose (Bio-Rad, USA) at concentration of 5 x 105 cells/ml. Then the plates were incubated on an orbital shaker (Orbital shaker, PSU-10i, Biosan, Latvia) at 50 rpm for 3–5 hours. Half of culture medium was replenished every 3 days. A spheroid culture was maintained for 14 days. Detection of markers (ER, p53, EpCAM, vim, AE1/AE3, panCK, EGFR) in 2D and 3D cell culture was performed using immunohistochemistry method with primary monoclonal antibodies. Histological samples of cells were photographed to compare the morphological characteristics and the expression of proteins in monolayer and spheroid culture Results. The results demonstrated that the percentage of tumor marker positive cells (ER+, EGFR+, EpCAM+, panCK+, AE1/AE3+) in monolayer culture is 1.25–2 times than more in spheroid culture. In contrast, tumor spheroids consist of fewer cells with the expression of epithelial markers such as EpCAM and AE1/AE3, but they contain a large number of cells that expressed mesenchymal marker vimentin by 5 % and p53 by 10 %. This may indicate that the cells acquire a mesenchymal phenotype. However, tumor cells of monolayer cell culture were not expressed vimentin. Conclusions. Our results demonstrated the differences of expression of tumor associated and epithelial-mesenchymal transition markers in 2D and 3D breast cancer cell cultures. Thus, the percentage of epithelial markers (Cytokeratines and epithelial cell adhesion molecule) in tumor spheroids is less than in cells of monolayer however spheroids cells begin expressing a mesenchymal marker – vimentin. In 3D cell culture only the outer cell layers expressed tumor associated proteins unlike 2D cell culture in which all of cells showed equally expression. Reduced of manifestation of tumor associated markers in 3D cell culture may indicate an increase of stem properties. These data showed that 3D cell culture more than 2D cell culture characterized processes of epithelial-mesenchymal transition.

Pubertal Ductal Morphogenesis: Isolation and Transcriptome Analysis of the Terminal End Bud.

The terminal end bud (TEB) is the growing part of the ductal mammary epithelium during puberty, enabling the formation of a primary epithelial network. These highly proliferative bulbous end structures that drive the ductal expansion into the mammary fat pad comprise an outer cap cell layer, containing the progenitor cells of the ductal myoepithelium, and the body cells, which form the luminal epithelium. As TEB make up only a very small part of the whole mammary tissue, TEB-associated factors can be easily missed when whole-tissue sections are being analyzed. Here we describe a method to enzymatically separate TEB and ducts, respectively, from the surrounding stroma of pubertal mice in order to perform transcriptomic or proteomic analysis on the isolated structures and identify potential novel regulators of epithelial outgrowth, or to allow further cell culturing. This approach has previously allowed us to identify novel TEB-associated proteins, including several axonal guidance proteins. We further include protocols for the culturing of isolated TEB, processing of mammary tissue into paraffin and immunohistochemical/fluorescent staining for verification, and localization of protein expression in the mammary tissue at different developmental time points.

Mammary gland stem cells and their application in breast cancer.

The mammary gland is an organ comprising two primary lineages, specifically the inner luminal and the outer myoepithelial cell layers. Mammary gland stem cells (MaSCs) are highly dynamic and self-renewing, and can give rise to these mammary gland lineages. The lineages are responsible for gland generation during puberty as well as expansion during pregnancy. In recent years, researchers have focused on understanding how MaSCs are regulated during mammary gland development and transformation of breast cancer. Here, we summarize the identification of MaSCs, and how they are regulated by the signaling transduction pathways, mammary gland microenvironment, and non-coding RNAs (ncRNAs). Moreover, we debate the evidence for their serving as the origin of breast cancer, and discuss the therapeutic perspectives of targeting breast cancer stem cells (BCSCs). In conclusion, a better understanding of the key regulators of MaSCs is crucial for the clinical treatment of breast cancer.

The HvNramp5 Transporter Mediates Uptake of Cadmium and Manganese, But Not Iron.

The Natural Resistance Associated Macrophage Protein (Nramp) represents a transporter family for metal ions in all organisms. Here, we functionally characterized a member of Nramp family in barley (Hordeum vulgare), HvNramp5. This member showed different expression patterns, transport substrate specificity, and cellular localization from its close homolog in rice (Oryza sativa), OsNramp5, although HvNramp5 was also localized to the plasma membrane. HvNramp5 was mainly expressed in the roots and its expression was not affected by Cd and deficiency of Zn, Cu, and Mn, but slightly up-regulated by Fe deficiency. Spatial expression analysis showed that the expression of HvNramp5 was higher in the root tips than that in the basal root regions. Furthermore, analysis with laser microdissection revealed higher expression of HvNramp5 in the outer root cell layers. HvNramp5 showed transport activity for both Mn2+ and Cd2+, but not for Fe2+ when expressed in yeast. Immunostaining with a HvNramp5 antibody showed that this protein was localized in the root epidermal cells without polarity. Knockdown of HvNramp5 in barley resulted in a significant reduction in the seedling growth at low Mn supply, but this reduction was rescued at high Mn supply. The concentration of Mn and Cd, but not other metals including Cu, Zn, and Fe, was decreased in both the roots and shoots of knockdown lines compared with the wild-type barley. These results indicate that HvNramp5 is a transporter required for uptake of Mn and Cd, but not for Fe, and that barley has a distinct uptake system from rice.

Comparative study of the effect of low-dose versus high-dose aspirin on the retina of aged male albino rats

Background Aspirin is a very popular drug worldwide. Recent studies have suggested that long-term aspirin use might be linked to wet-type acute macular degeneration. Aim This study was undertaken to investigate and compare the effects of chronic daily intake of low-dose versus high-dose aspirin on the structure of the retina in aged male albino rats for the first time. Materials and methods Twelve-month-old male albino rats were assigned to three groups: control, low-dose (2mg/kg) aspirin, and high-dose (10mg/kg) aspirin. The rats were given oral aspirin daily for 1 month. At the end of the experiment, the animals were euthanized and their eyes were enucleated and processed for paraffin blocks. Some of the paraffin sections were stained with H&E, whereas others were immunohistochemically stained with TUNEL, CD31, and glial fibrillary acidic protein markers. Histomorphometric count of ganglion cells, outer nuclear layer cells, CD31-positive cells, and TUNEL- positive cells was carried out using image analysis software. Statistical analysis of cell counts was performed. Results This study showed that chronic aspirin administration induced many degenerative changes in the aged rat retina. Stratification and vascularization of the retinal pigment epithelium was detected in addition to disruption of the photoreceptors. Apoptotic changes were detected in the inner nuclear layer and ganglion cell layer with significant decrease in cell number, which was pronounced with the high dose of aspirin. An increase in glial fibrillary acidic protein immunoreactivity of Müller cells was detected, especially in the high-dose group. Also, retinal neovascularization was clearly detected in the high-dose aspirin group. Conclusion Chronic aspirin intake is likely to produce retinal degenerative changes in aged male rat retina. High-dose aspirin was more detrimental than low dose. Results of the present experiment confirmed some clinical observations that chronic aspirin use is associated with retinal degenerative changes.

Numb and Numbl act to determine mammary myoepithelial cell fate, maintain epithelial identity, and support lactogenesis.

Mammary epithelium is comprised of an inner layer of luminal epithelial cells and an outer layer of contractile myoepithelial cells with mesenchymal properties. These two compartments interact throughout mammary morphogenesis to form branching ducts during puberty and terminate in secretory alveoli during lactation. It is not known how the myoepithelial cell lineage is specified, nor how signals in myoepithelial cells contribute to lactogenesis. Here, we show that Numb and Numbl are enriched in mammary myoepithelial cells, with their expression peaking during pregnancy. We use conditional Numb- and Numbl-knockout mouse models to demonstrate that loss of Numb/Numbl compromised the myoepithelial layer and expanded the luminal layer, led epithelial cells to undergo epithelial-to-mesenchymal transition, and resulted in lactation failure as a result of abnormal alveolar formation during pregnancy. Numb and Numbl function via repression of the Notch signaling pathway and of the p53-p21 axis during mammary gland development. These findings highlight the importance of Numb and Numbl in the control of myoepithelial cell fate determination, epithelial identity, and lactogenesis.-Zhang Y., Li, F., Song, Y., Sheng, X., Ren, F., Xiong, K., Chen, L., Zhang, H., Liu, D., Lengner, C. J., Xue, L., Yu, Z. Numb and Numbl act to determine mammary myoepithelial cell fate, maintain epithelial identity, and support lactogenesis.

Conservation of the role of INNER NO OUTER in development of unitegmic ovules of the Solanaceae despite a divergence in protein function.

BackgroundThe INNER NO OUTER (INO) gene is expressed in the outermost cell layer of the outer integument of bitegmic ovules and is essential for this organ’s growth. The role and cross-species functional conservation of INO orthologs were examined in members of the Solanaceae, which have unitegmic ovules. Unitegmy has evolved several times in disparate angiosperm lineages. INO expression has been observed in the outermost cell layers of all examined unitegmic ovules, but the functional role of INO in unitegmic ovules has not previously been evaluated.ResultsINO orthologs were unambiguously identified in tobacco and tomato by sequence homology. Expression of the tomato INO gene was limited to the outer cell layer of the single integument indicating that this single integument has properties of the outer integument. Expression occurred only after integument initiation, later than observed in ovules of other examined angiosperms. Virus-induced knock-down of expression of the INO ortholog in tobacco inhibited growth of the outer cell layer of the integument leading to a decrease in both integument extension and curvature of the ovule. The altered ovules closely resemble those of the aberrant testa shape (ats) ino mutant combination in Arabidopsis where we see the effect of the ino mutation on a single fused integument produced by the ats mutation. Despite significant sequence identity and similar expression patterns, the tomato INO coding region was not able to complement the Arabidopsis ino mutant.ConclusionsThe similarity of effects of ino mutations on the unitegmic ovules of tobacco and the fused integuments of the Arabidopsis ats mutant show that: 1) INO orthologs play the same role in promoting integument growth in ovules of tobacco and Arabidopsis; and 2) the unitegmic ovules of tobacco (and hence other solanaceous species) are most likely the result of a congenital fusion of two ancestral integuments. Our results further indicate that INO has a conserved role in growth of the outermost cell layer of integuments. The curvature of solanaceous ovules is driven by unequal growth of the outer layers of the single integument that likely correspond to an ancestral outer integument.Electronic supplementary materialThe online version of this article (doi:10.1186/s12870-016-0835-z) contains supplementary material, which is available to authorized users.

The SBT6.1 subtilase processes the GOLVEN1 peptide controlling cell elongation.

The GOLVEN (GLV) gene family encode small secreted peptides involved in important plant developmental programs. Little is known about the factors required for the production of the mature bioactive GLV peptides. Through a genetic suppressor screen in Arabidopsis thaliana, two related subtilase genes, AtSBT6.1 and AtSBT6.2, were identified that are necessary for GLV1 activity. Root and hypocotyl GLV1 overexpression phenotypes were suppressed by mutations in either of the subtilase genes. Synthetic GLV-derived peptides were cleaved in vitro by the affinity-purified SBT6.1 catalytic enzyme, confirming that the GLV1 precursor is a direct subtilase substrate, and the elimination of the in vitro subtilase recognition sites through alanine substitution suppressed the GLV1 gain-of-function phenotype in vivo Furthermore, the protease inhibitor Serpin1 bound to SBT6.1 and inhibited the cleavage of GLV1 precursors by the protease. GLV1 and its homolog GLV2 were expressed in the outer cell layers of the hypocotyl, preferentially in regions of rapid cell elongation. In agreement with the SBT6 role in GLV precursor processing, both null mutants for sbt6.1 and sbt6.2 and the Serpin1 overexpression plants had shorter hypocotyls. The biosynthesis of the GLV signaling peptides required subtilase activity and might be regulated by specific protease inhibitors. The data fit with a model in which the GLV1 signaling pathway participates in the regulation of hypocotyl cell elongation, is controlled by SBT6 subtilases, and is modulated locally by the Serpin1 protease inhibitor.

NADPH Oxidase Contributes to Photoreceptor Degeneration in Constitutively Active RAC1 Mice.

PurposeThe active form of small GTPase RAC1 is required for activation of NADPH oxidase (NOX), which in turn generates reactive oxygen species (ROS) in nonphagocytic cells. We explored whether NOX-induced oxidative stress contributes to rod degeneration in retinas expressing constitutively active (CA) RAC1.MethodsTransgenic (Tg)–CA-RAC1 mice were given apocynin (10 mg/kg, intraperitoneal), a NOX inhibitor, or vehicle daily for up to 13 weeks. Superoxide production and oxidative damage were assessed by dihydroethidium staining and by protein carbonyls and malondialdehyde levels, respectively. Outer nuclear layer (ONL) cells were counted and electroretinogram (ERG) amplitudes measured in Tg-CA-RAC1 mice. Outer nuclear layer cells were counted in wild-type (WT) mice after transfer of CA-Rac1 gene by subretinal injection of AAV8-pOpsin-CA Rac1-GFP.ResultsTransgenic-CA-RAC1 retinas had significantly fewer photoreceptor cells and more apoptotic ONL cells than WT controls from postnatal week (Pw) 3 to Pw13. Superoxide accumulation and protein and lipid oxidation were increased in Tg-CA-RAC1 retinas and were reduced in mice treated with apocynin. Apocynin reduced the loss of photoreceptors and increased the rod ERG a- and b-wave amplitudes when compared with vehicle-injected transgenic controls. Photoreceptor loss was also observed in regions of adult WT retina transduced with AAV8-pOpsin-CA Rac1-GFP but not in neighboring regions that were not transduced or in AAV8-pOpsin-GFP–transduced retinas.ConclusionsConstitutively active RAC1 promotes photoreceptor cell death by oxidative damage that occurs, at least partially, through NOX-induced ROS. Reactive oxygen species are likely involved in multiple forms of retinal degenerations, and our results support investigating RAC1 inhibition as a therapeutic approach that targets this disease pathway.

Brain Insulin-Like Growth Factor-I Directs the Transition from Stem Cells to Mature Neurons During Postnatal/Adult Hippocampal Neurogenesis.

The specific actions of insulin-like growth factor-I (IGF-I) and the role of brain-derived IGF-I during hippocampal neurogenesis have not been fully defined. To address the influence of IGF-I on the stages of hippocampal neurogenesis, we studied a postnatal/adult global Igf-I knockout (KO) mice (Igf-I(-/-) ) and a nervous system Igf-I conditional KO (Igf-I(Δ/Δ) ). In both KO mice we found an accumulation of Tbr2(+) -intermediate neuronal progenitors, some of which were displaced in the outer granule cell layer (GCL) and the molecular layer (ML) of the dentate gyrus (DG). Similarly, more ectopic Ki67(+) - cycling cells were detected. Thus, the GCL was disorganized with significant numbers of Prox1(+) -granule neurons outside this layer and altered morphology of radial glial cells (RGCs). Dividing progenitors were also generated in greater numbers in clonal hippocampal stem cell (HPSC) cultures from the KO mice. Indeed, higher levels of Hes5 and Ngn2, transcription factors that maintain the stem and progenitor cell state, were expressed in both HPSCs and the GCL-ML from the Igf-I(Δ/Δ) mice. To determine the impact of Igf-I deletion on neuronal generation in vivo, progenitors in Igf-I(-/-) and Igf-I(+/+) mice were labeled with a GFP-expressing vector. This revealed that in the Igf-I(-/-) mice more GFP(+) -immature neurons were formed and they had less complex dendritic trees. These findings indicate that local IGF-I plays critical roles during postnatal/adult hippocampal neurogenesis, regulating the transition from HPSCs and progenitors to mature granule neurons in a cell stage-dependent manner. Stem Cells 2016;34:2194-2209.

Survival and Functionality of hESC-Derived Retinal Pigment Epithelium Cells Cultured as a Monolayer on Polymer Substrates Transplanted in RCS Rats.

To determine the safety, survival, and functionality of human embryonic stem cell-derived RPE (hESC-RPE) cells seeded on a polymeric substrate (rCPCB-RPE1 implant) and implanted into the subretinal (SR) space of Royal College of Surgeons (RCS) rats. Monolayers of hESC-RPE cells cultured on parylene membrane were transplanted into the SR space of 4-week-old RCS rats. Group 1 (n = 46) received vitronectin-coated parylene membrane without cells (rMSPM+VN), group 2 (n = 59) received rCPCB-RPE1 implants, and group 3 (n = 13) served as the control group. Animals that are selected based on optical coherence tomography screening were subjected to visual function assays using optokinetic (OKN) testing and superior colliculus (SC) electrophysiology. At approximately 25 weeks of age (21 weeks after surgery), the eyes were examined histologically for cell survival, phagocytosis, and local toxicity. Eighty-seven percent of the rCPCB-RPE1-implanted animals showed hESC-RPE survivability. Significant numbers of outer nuclear layer cells were rescued in both group 1 (rMSPM+VN) and group 2 (rCPCB-RPE1) animals. A significantly higher ratio of rod photoreceptor cells to cone photoreceptor cells was found in the rCPCB-RPE1-implanted group. Animals with rCPCB-RPE1 implant showed hESC-RPE cells containing rhodopsin-positive particles in immunohistochemistry, suggesting phagocytic function. Superior colliculus mapping data demonstrated that a significantly higher number of SC sites responded to light stimulus at a lower luminance threshold level in the rCPCB-RPE1-implanted group. Optokinetic data suggested both implantation groups showed improved visual acuity. These results demonstrate the safety, survival, and functionality of the hESC-RPE monolayer transplantation in an RPE dysfunction rat model.

Identification and localization of bioactive naphthoquinones in the roots and rhizosphere of Paterson's curse (Echium plantagineum), a noxious invader.

Bioactive plant secondary products are frequently the drivers of complex rhizosphere interactions, including those with other plants, herbivores and microbiota. These chemically diverse molecules typically accumulate in a highly regulated manner in specialized plant tissues and organelles. We studied the production and localization of bioactive naphthoquinones (NQs) in the roots of Echium plantagineum, an invasive endemic weed in Australia. Roots of E. plantagineum produced red-coloured NQs in the periderm of primary and secondary roots, while seedling root hairs exuded NQs in copious quantities. Confocal imaging and microspectrofluorimetry confirmed that bioactive NQs were deposited in the outer layer of periderm cells in mature roots, resulting in red colouration. Intracellular examination revealed that periderm cells contained numerous small red vesicles for storage and intracellular transport of shikonins, followed by subsequent extracellular deposition. Periderm and root hair extracts of field- and phytotron-grown plants were analysed by UHPLC/Q-ToF MS (ultra high pressure liquid chromatography coupled to quadrupole time of flight mass spectrometry) and contained more than nine individual NQs, with dimethylacrylshikonin, and phytotoxic shikonin, deoxyshikonin and acetylshikonin predominating. In seedlings, shikonins were first found 48h following germination in the root-hypocotyl junction, as well as in root hair exudates. In contrast, the root cortices of both seedling and mature root tissues were devoid of NQs. SPRE (solid phase root zone extraction) microprobes strategically placed in soil surrounding living E. plantagineum plants successfully extracted significant levels of bioactive shikonins from living roots, rhizosphere and bulk soil surrounding roots. These findings suggest important roles for accumulation of shikonins in the root periderm and subsequent rhizodeposition in plant defence, interference, and invasion success.

Cell Death Control by Matrix Metalloproteinases.

In contrast to mammalian matrix metalloproteinases (MMPs) that play important roles in the remodeling of the extracellular matrix in animals, the proteases responsible for dynamic modifications of the plant cell wall are largely unknown. A possible involvement of MMPs was addressed by cloning and functional characterization of Sl2-MMP and Sl3-MMP from tomato (Solanum lycopersicum). The two tomato MMPs were found to resemble mammalian homologs with respect to gelatinolytic activity, substrate preference for hydrophobic amino acids on both sides of the scissile bond, and catalytic properties. In transgenic tomato seedlings silenced for Sl2/3-MMP expression, necrotic lesions were observed at the base of the hypocotyl. Cell death initiated in the epidermis and proceeded to include outer cortical cell layers. In later developmental stages, necrosis spread, covering the entire stem and extending into the leaves of MMP-silenced plants. The subtilisin-like protease P69B was identified as a substrate of Sl2- and Sl3-MMP. P69B was shown to colocalize with Sl-MMPs in the apoplast of the tomato hypocotyl, it exhibited increased stability in transgenic plants silenced for Sl-MMP activity, and it was cleaved and inactivated by Sl-MMPs in vitro. The induction of cell death in Sl2/3-MMP-silenced plants depended on P69B, indicating that Sl2- and Sl3-MMP act upstream of P69B in an extracellular proteolytic cascade that contributes to the regulation of cell death in tomato.

96. An Approach to Compare Multiple Adeno-Associated Virus (AAV) Capsids Side-by-Side in a Single Eye Following Intravitreal Injection

Current gene therapy approaches for ocular diseases rely largely on the use of subretinal injection for treatment of outer nuclear layer cells of interest. While subretinal injection allows for efficient transduction of retinal cells close to the site of the injection, there is a significant risk that the injection can further damage retinas with compromised integrity. Several studies have identified new adeno-associated virus (AAV) capsid mutants that are potentially able to transduce outer nuclear layer cells following intravitreal injections. To further explore AAV vector-mediated intravitreal injection approaches for clinical translation, it is imperative to directly compare retinal transduction efficiencies between newly identified and previously published AAV mutants. Using clinically relevant large animal models, these mutant capsids, as well as other commonly used AAV serotypes, can be tested in a comprehensive manner. To this end, we propose to utilize a viral DNA/RNA barcoding approach established by our lab. In this approach, AAV capsids derived from different serotypes and mutants can be identified by DNA and RNA barcodes unique to each AAV capsid. These barcodes are contained in viral genomes and also expressed as RNA barcodes following cellular transduction. As each AAV capsid strain has a unique viral genome, a mass transduction of a number of different AAV strains can be conducted in order to directly compare transduction levels side-by-side in a single eye. To test feasibility of the barcode approach and establish proof-of-principle in the ocular gene therapy research, we produced a DNA/RNA-barcoded AAV library that contained 20 serotypes including AAV serotypes 1-11 and AAV mutants including DJ, LK03, 2i8 and 2G9. Each AAV strain expresses a pair of 12 nucleotide-long RNA barcodes unique to each strain under the control of the human U6 snRNA promoter. Seven C57BL/6J mice have been injected into vitreous humor bilaterally with the DNA/RNA-barcoded AAV library vector at doses ranging from 2 to 9 x109 vg. To date, eyes were harvested from one mouse 3 weeks post injection. Total DNA and RNA were extracted from the retina as a whole from each eye, and were subjected to DNA-PCR or reverse transcriptase (RT)-PCR to amplify viral DNA and RNA barcodes, followed by Illumina barcode sequencing. A preliminary data analysis revealed that AAV2, AAV-DJ and AAV2G9 transduced the murine retina better than other AAV strains including AAV9, the reference control AAV strain contained in the library. Although further optimization of the approach may be required to move this project forward, we plan to apply this method to assess transduction efficiencies of various AAV strains side by side in specific subsets of sorted retinal cells and in various animal species. The safer approach of intravitreal injections with the most optimal AAV vectors would significantly increase the ability to treat over 200 genetic retinal diseases and therefore the data that will be obtained from these studies will considerably benefit the field of retinal gene therapy.

Brain-Region-Specific Organoids Using Mini-bioreactors for Modeling ZIKV Exposure

Cerebral organoids, three-dimensional cultures that model organogenesis, provide a new platform to investigate human brain development. High cost, variability, and tissue heterogeneity limit their broad applications. Here, we developed a miniaturized spinning bioreactor (SpinΩ) to generate forebrain-specific organoids from human iPSCs. These organoids recapitulate key features of human cortical development, including progenitor zone organization, neurogenesis, gene expression, and, notably, a distinct human-specific outer radial glia cell layer. We also developed protocols for midbrain and hypothalamic organoids. Finally, we employed the forebrain organoid platform to model Zika virus (ZIKV) exposure. Quantitative analyses revealed preferential, productive infection of neural progenitors with either African or Asian ZIKV strains. ZIKV infection leads to increased cell death and reduced proliferation, resulting in decreased neuronal cell-layer volume resembling microcephaly. Together, our brain-region-specific organoids and SpinΩ provide an accessible and versatile platform for modeling human brain development and disease and for compound testing, including potential ZIKV antiviral drugs.

Prenatal alcohol exposure alters synaptic activity of adult hippocampal dentate granule cells under conditions of enriched environment.

Prenatal alcohol exposure (PAE) results in fetal alcohol spectrum disorder (FASD), which is characterized by a wide range of cognitive and behavioral deficits that may be linked to impaired hippocampal function and adult neurogenesis. Preclinical studies in mouse models of FASD indicate that PAE markedly attenuates enrichment-mediated increases in the number of adult-generated hippocampal dentate granule cells (aDGCs), but whether synaptic activity is also affected has not been studied. Here, we utilized retroviral birth-dating coupled with whole cell patch electrophysiological recordings to assess the effects of PAE on enrichment-mediated changes in excitatory and inhibitory synaptic activity as a function of DGC age. We found that exposure to an enriched environment (EE) had no effect on baseline synaptic activity of 4- or 8-week-old aDGCs from control mice, but significantly enhanced the excitatory/inhibitory ratio of synaptic activity in 8-week-old aDGCs from PAE mice. In contrast, exposure to EE significantly enhanced the excitatory/inhibitory ratio of synaptic activity in older pre-existing DGCs situated in the outer dentate granule cell layer (i.e., those generated during embryonic development; dDGCs) in control mice, an effect that was blunted in PAE mice. These findings indicate distinct electrophysiological responses of hippocampal DGCs to behavioral challenge based on cellular ontogenetic age, and suggest that PAE disrupts EE-mediated changes in overall hippocampal network activity. These findings may have implications for future therapeutic targeting of hippocampal dentate circuitry in clinical FASD. © 2016 Wiley Periodicals, Inc.

An Aluminum-Inducible IREG Gene is Required for Internal Detoxification of Aluminum in Buckwheat.

Buckwheat (Fagopyrum esculentum Moench) is able to detoxify aluminum (Al) both externally and internally, but the molecular mechanisms underlying its high Al tolerance are not understood. We functionally characterized a gene (FeIREG1) belonging to IRON REGULATED/ferroportin in buckwheat, which showed high expression in our previous genome-wide transcriptome analysis. FeIREG1 was mainly expressed in the roots, and its expression was up-regulated by Al, but not by other metals and low pH. Furthermore, in contrast to AtIREG1 and AtIREG2 in Arabidopsis, the expression of FeIREG1 was not induced by Fe deficiency. Spatial expression analysis showed that the Al-induced expression of FeIREG1 was found in the root tips and higher expression was detected in the outer layers of this part. Immunostaining also showed that FeIREG1 was localized at the outer cell layers in the root tip. A FeIREG1-green fluorescent protein (GFP) fusion protein was localized to the tonoplast when transiently expressed in onion epidermal cells. Overexpression of FeIREG1 in Arabidopsis resulted in increased Al tolerance, but did not alter the tolerance to Cd, Co and Fe. The tolerance to Ni was slightly enhanced in the overexpression lines. Mineral analysis showed that the accumulation of total root Al and other essential mineral elements was hardly altered in the overexpression lines. Taken together, our results suggest that FeIREG1 localized at the tonoplast plays an important role in internal Al detoxification by sequestering Al into the root vacuoles in buckwheat.

A Brief Discussion of Association of Radiation and Metabolisms

Diversity of effects of radiation on distinct cell types suggest that mitochondria could be the most important organelle in metabolism of radiation. Mitochondria is known as metabolic machine of cells providing energy. According to the amount of mitochondria content, cells work with different functions. For example, skin cells present fewer amount of mitochondria than heart muscle cells. Inner (basal) layer of skin cells reproduce self and grow to outer layers slowly. Outer layers of skin cells’ mitochondria disappear, gradually die and keratinize to protect inner tissue. Light, sometimes only sun radiation exposure of skin cells can cause inflammation, keratinized layers peel off, basal reproductive skin cells over grow, even genetic mutate to form skin tumor. However, not higher risk rate of skin tumor in children, it maybe because the earlier and severer inflammation alert and avoid the young people expose in long high dose of radioactivity.

Changes of myoid and endothelial cells in the peritubular wall during contraction of the seminiferous tubule.

The wall of the seminiferous tubule in rodents consists of an inner layer of myoid cells covered by an outer layer of endothelial cells. Myoid cells are a type of smooth muscle cell containing α-actin filaments arranged in two independent layers that contract when stimulated by endothelin-1. The irregular surface relief of the tubular wall is often considered a hallmark of contraction induced by a variety of stimuli. We examine morphological changes of the rat seminiferous tubule wall during contraction by a combination of light, confocal, transmission and scanning electron microscopy. During ET-1-induced contraction, myoid cells changed from a flat to a conical shape, but their actin filaments remained in independent layers. As a consequence of myoid cell contraction, the basement membrane became wavy, orientation of collagen fibers in the extracellular matrix was altered and the endothelial cell layer became folded. To observe the basement of the myoid cell cone, the endothelial cell monolayer was removed by collagenase digestion prior to SEM study. In contracted tubules, it is possible to distinguish cell relief: myoid cells have large folds on the external surface oriented parallel to the tubular axis, whereas endothelial cells have numerous cytoplasmic projections facing the interstitium. The myoid cell cytoskeleton is unusual in that the actin filaments are arranged in two orthogonal layers, which adopt differing shapes during contraction with myoid cells becoming cone-shaped. This arrangement impacts on other components of the seminiferous tubule wall and affects the propulsion of the tubular contents to the rete testis.