Preinvasion Assessment of Exotic Bark Beetle-Vectored Fungi to Detect Tree-Killing Pathogens.

Exotic diseases and pests of trees have caused continental-scale disturbances in forest ecosystems and industries, and their invasions are considered largely unpredictable. We tested the concept of preinvasion assessment of not yet invasive organisms, which enables empirical risk assessment of potential invasion and impact. Our example assesses fungi associated with Old World bark and ambrosia beetles and their potential to impact North American trees. We selected 55 Asian and European scolytine beetle species using host use, economic, and regulatory criteria. We isolated 111 of their most consistent fungal associates and tested their effect on four important southeastern American pine and oak species. Our test dataset found no highly virulent pathogens that should be classified as an imminent threat. Twenty-two fungal species were minor pathogens, which may require context-dependent response for their vectors at North American borders, while most of the tested fungi displayed no significant impact. Our results are significant in three ways; they ease the concerns over multiple overseas fungus vectors suspected of heightened potential risk, they provide a basis for the focus on the prevention of introduction and establishment of species that may be of consequence, and they demonstrate that preinvasion assessment, if scaled up, can support practical risk assessment of exotic pathogens.

Generation of three-dimensional retinal organoids expressing rhodopsin and S- and M-cone opsins from mouse stem cells.

PURPOSE: Three-dimensional retinal organoids can be differentiated from embryonic stem cells/induced pluripotent stem cells (ES/iPS cells) under defined medium conditions. We modified the serum-free floating culture of embryoid body-like aggregates with quick reaggregation (SFEBq) culture procedure to obtain retinal organoids expressing more rod photoreceptors and S- and M-cone opsins. METHODS: Retinal organoids differentiated from mouse Nrl-eGFP iPS cells were cultured in various mediums during photoreceptor development. To promote rod photoreceptor development, organoids were maintained in media containing 9-cis retinoic acids (9cRA). To obtain retinal organoids with M-opsin expression, we cultured in medium with 1% fetal bovine serum (FBS) supplemented with T3, BMP4, and DAPT. Section immunohistochemistry was performed to visualize the expression of photoreceptor markers. RESULTS: In three-dimensional (3D) retinas exposed to 9cRA, rhodopsin was expressed earlier and S-cone opsins were suppressed. We could maintain 3D retinas up to DD 35 in culture media with 1% FBS. The 3D retinas expressed rhodopsin, S- and M-opsins, but most cone photoreceptors expressed either S- or M-opsins. CONCLUSION: By modifying culture conditions in the SFEBq protocol, we obtained rod-dominated 3D retinas and S- and M-opsin expressing 3D retinas.

Light-dark changes in iris thickness and anterior chamber angle width in eyes with occludable angles.

BACKGROUND: The purpose of this study was to investigate and measure light-dark changes in iris thickness (IT) and anterior chamber angle width in eyes with occludable angles and open angles by using anterior segment optical coherence tomography (AS-OCT). METHODS: We examined 153 eyes of 153 Japanese patients with primary angle closure suspect, primary angle closure, primary angle closure glaucoma, or primary open angle glaucoma. AS-OCT was used to determine pupil diameter, IT, angle opening distance at 500 µm (AOD500), and trabecular-iris space area at 500 µm (TISA500) in each quadrant of the anterior chamber angle (superior, inferior, temporal, and nasal) under light and dark conditions. RESULTS: In the angle closure cases, IT, AOD500 and TISA500 in the dark varied significantly among the four quadrants (P < 0.05, analysis of variance). In contrast, in the open angle cases, IT, AOD500 and TISA500 in the dark did not differ significantly among the four quadrants. In the angle closure cases and the open angle cases, significant negative associations were found between IT difference [IT(light)-IT(dark)] and AOD500 difference [AOD500(light)-AOD500(dark)] (R = -0.411, P < 0.001 and R = -0.501, P = 0.001, respectively) and between IT difference and TISA500 difference [TISA500(light)-TISA500(dark)] (R = -0.475, P < 0.001 and R = -0.462, P = 0.002, respectively). CONCLUSION: Our results suggest that thickening of the iris root under dark conditions is related to the mechanism of primary angle closure. It is important to analyze both the angle structure and peripheral IT in each quadrant.

Enkianthus campanulatus (Ericaceae) is commonly associated with arbuscular mycorrhizal fungi.

Enkianthus is the most basal extant genus in the phylogeny of ericaceous plants. Its members harbor arbuscular mycorrhiza (AM)-like hyphal structures in their roots but, as yet, no study has surveyed the AM fungal species component. Roots from six species of Enkianthus were collected from five distantly located sites in Japan. Intracellular hyphal coils were observed in the root cortical cells of all species. Fungal DNA sequences of the small subunit ribosomal RNA gene were obtained from 73 of 75 segments of Enkianthus campanulatus roots by PCR using either AML2 or NS31/AM1primer pairs. Results indicated that all E. campanulatus trees were extensively associated with Glomus spp. A phylogenetic analysis showed that 71 root segments harbored fungi belonging to Glomus group A. Among eight delineated clades, seven did not nest with any known AM fungal species. One clade was detected in all roots at all sites at relatively high frequencies, but the rest were detected sporadically at each site. The placement of sequences from distantly located sites into a single clade without known AM fungal species suggests the common association of E. campanulatus with particular AM fungal taxa.

Seasonal and environmental changes of mycorrhizal associations and heterotrophy levels in mixotrophic Pyrola japonica (Ericaceae) growing under different light environments.

PREMISE OF THE STUDY: Mixotrophy is a strategy whereby plants acquire carbon both through photosynthesis and heterotrophic exploitation of mycorrhizal fungi. In Euro-American Pyroleae species studied hitherto, heterotrophy levels vary according to species, sites of study, and possibly light conditions. We investigated mycorrhizal association and mixotrophy in the Asiatic forest species Pyrola japonica, and their plasticity under different light conditions. METHODS: Pyrola japonica was sampled bimonthly in sunny and shaded conditions from a deciduous broadleaf forest. We microscopically assessed the rate of fungal colonization and sequenced the ITS to identify the mycorrhizal fungi. We measured (13)C and (15)N isotopic abundances in P. japonica as compared with neighboring autotrophic and mycoheterotrophic plants, to evaluate P. japonica's heterotrophy level. KEY RESULTS: Pyrola japonica formed arbutoid mycorrhizas devoid of fungal mantles, with intracellular hyphal coils and a Hartig net. It tended to be more colonized by mycorrhizal fungi in spring and summer. Most associated fungi belonged to ectomycorrhizal taxa, and 84% of identified fungi were Russula spp. Rate of mycorrhizal colonization and Russula frequency tended to be higher in shaded conditions. Both δ(13)C and δ(15)N values of P. japonica were significantly higher in autotrophic plants, showing that about half of the carbon on average was received from mycorrhizal fungi. Both isotopic values negatively correlated with light availability, suggesting higher heterotrophy levels in shaded conditions. CONCLUSIONS: The mixotrophic P. japonica undergoes changes in mycorrhizal symbionts and carbon nutrition according to light availability. Our results suggest that during Pyroleae evolution, a tendency to increased heterotrophy emerged in the Pyrola/Orthilia clade.

Mycorrhizal fungi associated with Monotropastrum humile (Ericaceae) in central Japan.

We explored the diversity of mycorrhizal fungi associated with Monotropastrum humile in the central part of Japan's main island. We collected 103 M. humile individuals from 12 sites with various forest types. We analyzed the DNA sequences of the internal transcribed spacer region from fungal and plant nuclear ribosomal DNAs to assess the genetic diversity of the fungi associated with M. humile roots and to position the plant with respect to known Monotropoideae groups, respectively. The plants formed a monophyletic clade with other members of M. humile but were separated from M. humile var. glaberrimum and other monotropes (97% bootstrap support). Of the 50 fungal phylotypes, 49 had best matches with the Russulales, and the other had highest similarity with the Thelephoraceae. Our phylogenetic analysis suggests that M. humile roots have a highly specialized association with fungal partners in the Russulaceae. Moreover, a few fungal phylotypes from the M. humile roots had positions neighboring those from Monotropa uniflora roots. These results indicated that the genetic diversity of mycorrhizal fungi of M. humile was highly specific to the Russulaceae, but with high diversity within that family, and that the fungi associated with M. humile differ from those associated with M. uniflora.

Bilateral retinitis after influenza virus infection in a case report.

PURPOSE: To report 2 years' longitudinal retinal changes using spectral domain optical coherence tomography (SD-OCT) images in a case of retinitis after influenza virus infection. OBSERVATIONS: A 48-year-old female complained of scotoma in the central visual field after influenza virus infection. Her best visual acuity was 20/16, her fundus examination was normal, and fluorescein angiography demonstrated no evident leakage in either the retina or the optic disc. However, SD-OCT images showed a disrupted, blurred inner-segment ellipsoid zone in the macula of both eyes. Two steroid pulse therapy sessions in the first 3 months showed temporary improvement of the central scotoma. However, atrophy of the photoreceptor layer at the juxta fovea gradually progressed in OCT images during the follow-up period. In contrast, the fovea itself was mostly intact and visual acuity was maintained in the 2-year period. CONCLUSIONS AND IMPORTANCE: We experienced a unique case of retinitis after influenza infection, in whom progressive atrophy of the photoreceptor layer was observed in SD-OCT images.

Chemically-induced photoreceptor degeneration and protection in mouse iPSC-derived three-dimensional retinal organoids.

Induced pluripotent stem cells (iPSCs), which can be differentiated into various tissues and cell types, have been used for clinical research and disease modeling. Self-organizing three-dimensional (3D) tissue engineering has been established within the past decade and enables researchers to obtain tissues and cells that almost mimic in vivo development. However, there are no reports of practical experimental procedures that reproduce photoreceptor degeneration. In this study, we induced photoreceptor cell death in mouse iPSC-derived 3D retinal organoids (3D-retinas) by 4-hydroxytamoxifen (4-OHT), which induces photoreceptor degeneration in mouse retinal explants, and then established a live-cell imaging system to measure degeneration-related properties. Furthermore, we quantified the protective effects of representative ophthalmic supplements for treating the photoreceptor degeneration. This drug evaluation system enables us to monitor drug effects in photoreceptor cells and could be useful for drug screening.

iPSC-Derived Retina Transplants Improve Vision in rd1 End-Stage Retinal-Degeneration Mice.

Recent success in functional recovery by photoreceptor precursor transplantation in dysfunctional retina has led to an increased interest in using embryonic stem cell (ESC) or induced pluripotent stem cell (iPSC)-derived retinal progenitors to treat retinal degeneration. However, cell-based therapies for end-stage degenerative retinas that have lost the outer nuclear layer (ONL) are still a big challenge. In the present study, by transplanting mouse iPSC-derived retinal tissue (miPSC retina) in the end-stage retinal-degeneration model (rd1), we visualized the direct contact between host bipolar cell terminals and the presynaptic terminal of graft photoreceptors by gene labeling, showed light-responsive behaviors in transplanted rd1 mice, and recorded responses from the host retina with transplants by ex vivo micro-electroretinography and ganglion cell recordings using a multiple-electrode array system. Our data provides a proof of concept for transplanting ESC/iPSC retinas to restore vision in end-stage retinal degeneration.

Differentiation/Purification Protocol for Retinal Pigment Epithelium from Mouse Induced Pluripotent Stem Cells as a Research Tool.

PURPOSE: To establish a novel protocol for differentiation of retinal pigment epithelium (RPE) with high purity from mouse induced pluripotent stem cells (iPSC). METHODS: Retinal progenitor cells were differentiated from mouse iPSC, and RPE differentiation was then enhanced by activation of the Wnt signaling pathway, inhibition of the fibroblast growth factor signaling pathway, and inhibition of the Rho-associated, coiled-coil containing protein kinase signaling pathway. Expanded pigmented cells were purified by plate adhesion after Accutase® treatment. Enriched cells were cultured until they developed a cobblestone appearance with cuboidal shape. The characteristics of iPS-RPE were confirmed by gene expression, immunocytochemistry, and electron microscopy. Functions and immunologic features of the iPS-RPE were also evaluated. RESULTS: We obtained iPS-RPE at high purity (approximately 98%). The iPS-RPE showed apical-basal polarity and cellular structure characteristic of RPE. Expression levels of several RPE markers were lower than those of freshly isolated mouse RPE but comparable to those of primary cultured RPE. The iPS-RPE could form tight junctions, phagocytose photoreceptor outer segments, express immune antigens, and suppress lymphocyte proliferation. CONCLUSION: We successfully developed a differentiation/purification protocol to obtain mouse iPS-RPE. The mouse iPS-RPE can serve as an attractive tool for functional and morphological studies of RPE.

Spatial distribution and genetic structure of Cenococcum geophilum in coastal pine forests in Japan.

The asexual ectomycorrhizal fungus Cenococcum geophilum has a wide geographic range in diverse forest ecosystems. Although its genetic diversity has been documented at a stand or regional scale, knowledge of spatial genetic structure is limited. We studied the genetic diversity and spatial structure of C. geophilum in eight Japanese coastal pine forests with a maximum geographic range of 1364 km. A total of 225 samples were subjected to phylogenetic analysis based on the glyceraldehyde 3-phosphate dehydrogenase gene (GAPDH) followed by microsatellite analysis with five loci. The phylogenetic analysis based on GAPDH resolved three groups with most isolates falling into one dominant lineage. Microsatellite analyses generated 104 multilocus genotypes in the overall populations. We detected significant genetic variation within populations and genetic clusters indicating that high genetic diversity may be maintained by possible recombination processes at a stand scale. Although no spatial autocorrelation was detected at a stand scale, the relationship between genetic and geographic distances among the populations was significant, suggesting a pattern of isolation by distance. These results indicate that cryptic recombination events at a local scale and unknown migration events at both stand and regional scales influence spatial distribution and genetic structure of C. geophilum in coastal pine forests of Japan.

Influence of anterior segment biometric parameters on the anterior chamber angle width in eyes with angle closure.

PURPOSE: To predict angle narrowing in eyes with angle closure in a Japanese population using anterior segment optical coherence tomography (AS-OCT) quantitative parameters. PATIENTS AND METHODS: AS-OCT was used to examine 118 eyes of 118 patients with angle closure and 40 eyes of 40 patients with open angle under dark conditions. After measuring the angle opening distance 500 (AOD500), anterior chamber depth, iris thickness (IT), iris convexity (IC), pupil diameter, anterior chamber width, and crystalline lens rise, multivariate regression analyses were performed for the AOD500 in each group. RESULTS: With the exception of IT, significant differences were observed between the AS-OCT parameters for the angle closure and open-angle groups. Anterior chamber depth, IT, and IC were the explanatory variables associated with AOD500 for each group (P≤0.001). A significant negative association was found between IT and IC only in the angle-closure group (P<0.001). CONCLUSIONS: This study quantitatively confirmed that shallow anterior chamber depth was a major mechanism of angle narrowing, and that both IT and IC had a strong impact on angle narrowing. Moreover, the negative association found between IT and IC in only the angle closure group indicated the existence of the stretch force placed on the iris by relative pupillary block.

The Effect of Raffaelea quercus-mongolicae Inoculations on the Formation of Non-conductive Sapwood of Quercus mongolica.

In Korea, mass mortality of Quercus mongolica trees has become obvious since 2004. Raffaelea quercus-mongolicae is believed to be a causal fungus contributing the mortality. To evaluate the pathogenicity of the fungus to the trees, the fungus was multiple- and single-inoculated to the seedlings and twigs of the mature trees, respectively. In both the inoculations, the fungus was reisolated from more than 50% of inoculated twigs and seedlings. In the single inoculations, proportions of the transverse area of non-conductive sapwood at inoculation points and vertical lengths of discoloration expanded from the points were significantly different between the inoculation treatment and the control. In the multiple inoculations, no mortality was confirmed among the seedlings examined. These results showed that R. quercus-mongolicae can colonize sapwood, contribute to sapwood discoloration and disrupt sap flows around inoculation sites of Q. mongolica, although the pathogenicity of the fungus was not proven.

Association between external limiting membrane status and visual acuity in diabetic macular oedema.

AIM: To evaluate the association between the foveal external limiting membrane (ELM) status and visual acuity (VA) in diabetic macular oedema (DMO). METHODS: We retrospectively reviewed the spectral domain optical coherence tomography images of 127 eyes from 127 patients with DMO and evaluated the correlation between the logarithm of the minimal angle of resolution (logMAR) VA and the statuses of the foveal ELM, inner segment/outer segment (IS/OS) and cone outer segment tips (COST); foveal macular thickness (FMT); and presence or absence of hard exudates (HE), serous retinal detachment (SRD) and vitreous adhesion. The integrity of the ELM, IS/OS, and COST was classified into three categories (absent, disrupted and complete). RESULTS: There was a strong correlation between VA and the statuses of the ELM (r=0.699, p<0.001), IS/OS (r=0.716, p<0.001) and COST (r=0.471, p<0.001). There was no correlation between FMT and logMAR VA (r=-0.036, p=0.687). However, when we analysed the correlation between FMT and VA by dividing patients into those with FMT ≤250 µm and those with FMT >250 µm, there was a positive correlation between FMT and VA in eyes with FMT ≤250 µm (r=-0.601, p<0.0001) and a negative correlation in eyes with FMT>250 µm (r=0.290, p<0.01). Other factors HE, SRD and vitreous adhesion did not correlate with VA. CONCLUSIONS: In DMO, the ELM status may be as closely related to VA as the IS/OS status.