Photoreceptors of Nrl −/− Mice Coexpress Functional S- and M-cone Opsins Having Distinct Inactivation Mechanisms

The retinas of mice null for the neural retina leucine zipper transcription factor (Nrl −/−) contain no rods but are populated instead with photoreceptors that on ultrastructural, histochemical, and molecular criteria appear cone like. To characterize these photoreceptors functionally, responses of single photoreceptors of Nrl −/− mice were recorded with suction pipettes at 35–37°C and compared with the responses of rods of WT mice. Recordings were made either in the conventional manner, with the outer segment (OS) drawn into the pipette (“OS in”), or in a novel configuration with a portion of the inner segment drawn in (“OS out”). Nrl −/− photoreceptor responses recorded in the OS-out configuration were much faster than those of WT rods: for dim-flash responses tpeak = 91 ms vs. 215 ms; for saturating flashes, dominant recovery time constants, τD = 110 ms vs. 240 ms, respectively. Nrl −/− photoreceptors in the OS-in configuration had reduced amplification, sensitivity, and slowed recovery kinetics, but the recording configuration had no effect on rod response properties, suggesting Nrl −/− outer segments to be more susceptible to damage. Functional coexpression of two cone pigments in a single mammalian photoreceptor was established for the first time; the responses of every Nrl −/− cell were driven by both the short-wave (S, λmax ≈ 360 nm) and the mid-wave (M, λmax ≈ 510 nm) mouse cone pigment; the apparent ratio of coexpressed M-pigment varied from 1:1 to 1:3,000 in a manner reflecting a dorso-ventral retinal position gradient. The role of the G-protein receptor kinase Grk1 in cone pigment inactivation was investigated in recordings from Nrl −/−/Grk1−/− photoreceptors. Dim-flash responses of cells driven by either the S- or the M-cone pigment were slowed 2.8-fold and 7.5-fold, respectively, in the absence of Grk1; the inactivation of the M-pigment response was much more seriously retarded. Thus, Grk1 is essential to normal inactivation of both S- and M-mouse cone opsins, but S-opsin has access to a relatively effective, Grk1-independent inactivation pathway

Peroxisome Turnover and Diurnal Modulation of Antioxidant Activity in Retinal Pigment Epithelia Utilizes Microtubule-Associated Protein 1 Light Chain 3B (LC3B)

The retinal pigment epithelium (RPE) supports the outer retina through essential roles in the retinoid cycle, nutrient supply, ion exchange, and waste removal. Each day the RPE removes the oldest ∼10% of photoreceptor outer segment (OS) disk membranes through phagocytic uptake, which peaks following light onset. Impaired degradation of phagocytosed OS material by the RPE can lead to toxic accumulation of lipids, oxidative tissue damage, inflammation, and cell death. OSs are rich in very long chain fatty acids, which are preferentially catabolized in peroxisomes. Despite the importance of lipid degradation in RPE function, the regulation of peroxisome number and activity relative to diurnal OS ingestion is relatively unexplored. Using immunohistochemistry, immunoblot analysis, and catalase activity assays, we investigated peroxisome abundance and activity at 6 AM, 7 AM (light onset), 8 AM, and 3 PM, in wild-type (WT) mice and mice lacking microtubule-associated protein 1 light chain 3B (Lc3b), which have impaired phagosome degradation. We found that catalase activity, but not the amount of catalase protein, is 50% higher in the morning compared with 3 PM, in RPE of WT, but not Lc3b-/-, mice. Surprisingly, we found that peroxisome abundance was stable during the day in RPE of WT mice; however, numbers were elevated overall in Lc3b-/- mice, implicating LC3B in autophagic organelle turnover in RPE. Our data suggest that RPE peroxisome function is regulated in coordination with phagocytosis, possibly through direct enzyme regulation, and may serve to prepare RPE peroxisomes for daily surges in ingested lipid-rich OS. Copyright © 2019 the American Physiological Society

Treatment of effluents containing 2-chlorophenol by adsorption onto chemically and physically activated biochars

The application of adsorption using biochars for the remediation of effluents containing emerging contaminants, including chlorophenols, is a hotspot and trend development in the literature. This treatment is more interesting when using readily available wastes and at no cost, such as malt bagasse, for example. Here, the biochars were produced from malt bagasse, by physical and chemical activation (with CO2 and ZnCl2, respectively) and employed as adsorbents in the remediation of effluents containing 2-chlorophenol. Results revealed that the activated biochars have mesoporous structures and surface areas of 161 m² g-1 (CO2) and 545 m² g-1 (ZnCl2). For both activated biochars, adsorption of 2-chlorophenol was favored under acid conditions, with the highest adsorption capacities found using ZnCl2-activated biochar. The maximum adsorption capacity using ZnCl2-activated biochar was 150 mg g-1. The process was endothermic and spontaneous. ZnCl2-activated biochar exhibited an efficiency of 98% (using a dosage of 10 g L-1) in the treatment of industrial effluents containing 2-chlorophenol.La aplicación de la adsorción mediante biocarros para la remediación de efluentes que contienen contaminantes emergentes, incluidos los clorofenoles, es un punto crítico y un desarrollo de tendencia en la literatura. Este tratamiento es más interesante cuando se utilizan residuos fácilmente disponibles y sin costo, como el bagazo de malta, por ejemplo. Aquí, los biocarros se produjeron a partir de bagazo de malta, mediante activación física y química (con CO2 y ZnCl2, respectivamente) y se emplearon como adsorbentes en la remediación de efluentes que contienen 2-clorofenol. Los resultados revelaron que los biocarros activados tienen estructuras mesoporosas y áreas superficiales de 161 m² g-1 (CO2) y 545 m² g-1 (ZnCl2). Para ambos biocarros activados, la adsorción de 2-clorofenol se vio favorecida en condiciones ácidas, con las capacidades de adsorción más altas encontradas utilizando biocarbón activado con ZnCl2. La capacidad máxima de adsorción usando biocarbón activado con ZnCl2 fue de 150 mg g-1. El proceso fue endotérmico y espontáneo. El biocarbón activado con ZnCl2 exhibió una eficiencia del 98% (usando una dosis de 10 g L-1) en el tratamiento de efluentes industriales que contienen 2-clorofenol

Native Environment Modulates Leaf Size and Response to Simulated Foliar Shade across Wild Tomato Species

The laminae of leaves optimize photosynthetic rates by serving as a platform for both light capture and gas exchange, while minimizing water losses associated with thermoregulation and transpiration. Many have speculated that plants maximize photosynthetic output and minimize associated costs through leaf size, complexity, and shape, but a unifying theory linking the plethora of observed leaf forms with the environment remains elusive. Additionally, the leaf itself is a plastic structure, responsive to its surroundings, further complicating the relationship. Despite extensive knowledge of the genetic mechanisms underlying angiosperm leaf development, little is known about how phenotypic plasticity and selective pressures converge to create the diversity of leaf shapes and sizes across lineages. Here, we use wild tomato accessions, collected from locales with diverse levels of foliar shade, temperature, and precipitation, as a model to assay the extent of shade avoidance in leaf traits and the degree to which these leaf traits correlate with environmental factors. We find that leaf size is correlated with measures of foliar shade across the wild tomato species sampled and that leaf size and serration correlate in a species-dependent fashion with temperature and precipitation. We use far-red induced changes in leaf length as a proxy measure of the shade avoidance response, and find that shade avoidance in leaves negatively correlates with the level of foliar shade recorded at the point of origin of an accession. The direction and magnitude of these correlations varies across the leaf series, suggesting that heterochronic and/or ontogenic programs are a mechanism by which selective pressures can alter leaf size and form. This study highlights the value of wild tomato accessions for studies of both morphological and light-regulated development of compound leaves, and promises to be useful in the future identification of genes regulating potentially adaptive plastic leaf traits

Microtubule-Associated Protein 1 Light Chain 3B, (LC3B) is Necessary to Maintain Lipid-Mediated Homeostasis in the Retinal Pigment Epithelium

Like other neurons, retinal cells utilize autophagic pathways to maintain cell homeostasis. The mammalian retina relies on heterophagy and selective autophagy to efficiently degrade and metabolize ingested lipids with disruption in autophagy associated degradation contributing to age related retinal disorders. The retinal pigment epithelium (RPE) supports photoreceptor cell renewal by daily phagocytosis of shed photoreceptor outer segments (OS). The daily ingestion of these lipid-rich OS imposes a constant degradative burden on these terminally differentiated cells. These cells rely on Microtubule-Associated Protein 1 Light Chain 3 (LC3) family of proteins for phagocytic clearance of the ingested OS. The LC3 family comprises of three highly homologous members, MAP1LC3A (LC3A), MAP1LC3B (LC3B), and MAP1LC3C (LC3C). The purpose of this study was to determine whether the LC3B isoform plays a specific role in maintaining RPE lipid homeostasis. We examined the RPE and retina of the LC3B-/- mouse as a function of age using in vivo ocular imaging and electroretinography coupled with ex vivo, lipidomic analyses of lipid mediators, assessment of bisretinoids as well as imaging of lipid aggregates. Deletion of LC3B resulted in defects within the RPE including increased phagosome accumulation, decreased fatty acid oxidation and a subsequent increase in RPE and sub-RPE lipid deposits. Age-dependent RPE changes included elevated levels of oxidized cholesterol, deposition of 4-HNE lipid peroxidation products, bisretinoid lipofuscin accumulation, and subretinal migration of microglia, collectively likely contributing to loss of retinal function. These observations are consistent with a critical role for LC3B-dependent processes in the maintenance of normal lipid homeostasis in the aging RPE, and suggest that LC3 isoform specific disruption in autophagic processes contribute to AMD-like pathogenesis. © 2018 Dhingra, Bell, Peachey, Daniele, Reyes-Reveles, Sharp, Jun, Bazan, Sparrow, Kim, Philp and Boesze-Battaglia

Cone-like morphological, molecular, and electrophysiological features of the photoreceptors of the Nrl knockout mouse

PURPOSE. To test the hypothesis that Nrl Ϫ/Ϫ photoreceptors are cones, by comparing them with WT rods and cones using morphological, molecular, histochemical, and electrophysiological criteria. METHODS. The photoreceptor layer of fixed retinal tissue of 4-to 6-week-old mice was examined in plastic sections by electron microscopy, and by confocal microscopy in frozen sections immunolabeled for the mouse UV-cone pigment and colabeled with PNA. Quantitative immunoblot analysis was used to determine the levels of expression of key cone-specific proteins. Single-and paired-flash methods were used to extract the spectral sensitivity, kinetics, and amplification of the awave of the ERG. RESULTS. Outer segments of Nrl Ϫ/Ϫ photoreceptors (ϳ7 m) are shorter than those of wild-type (WT) rods (ϳ25 m) and cones (ϳ15 m); but, like WT cones, they have 25 or more basal discs open to the extracellular space, extracellular matrix sheaths stained by PNA, chromatin "clumping" in their nuclei, and mitochondria two times shorter than rods. Nrl Ϫ/Ϫ photoreceptors express the mouse UV cone pigment, cone transducin, and cone arrestin in amounts expected, given the relative size and density of cones in the two retinas. The ERG a-wave was used to assay the properties of the photocurrent response. The sensitivity of the Nrl -/-a-wave is at its maximum at 360 nm, with a secondary mode at 510 nm having approximately one-tenth the maximum sensitivity. These wavelengths are the max of the two mouse cone pigments. The time to peak of the dim-flash photocurrent response was ϳ50 ms, more than two times faster than that of rods. CONCLUSIONS. Many morphological, molecular, and electrophysiological features of the Nrl Ϫ/Ϫ photoreceptors are cone-like, and strongly distinguish these cells from rods. This retina provides a model for the investigation of cone function and cone-specific genetic disease. (Invest Ophthalmol Vis Sci

A horizon scan of priorities for coastal marine microbiome research

Research into the microbiomes of natural environments is changing the way ecologists and evolutionary biologists view the importance of microbes in ecosystem function. This is particularly relevant in ocean environments, where microbes constitute the majority of biomass and control most of the major biogeochemical cycles, including those that regulate the Earth's climate. Coastal marine environments provide goods and services that are imperative to human survival and well-being (e.g. fisheries, water purification), and emerging evidence indicates that these ecosystem services often depend on complex relationships between communities of microorganisms (the ‘microbiome’) and their hosts or environment – termed the ‘holobiont’. Understanding of coastal ecosystem function must therefore be framed under the holobiont concept, whereby macroorganisms and their associated microbiomes are considered as a synergistic ecological unit. Here we evaluated the current state of knowledge on coastal marine microbiome research and identified key questions within this growing research area. Although the list of questions is broad and ambitious, progress in the field is increasing exponentially, and the emergence of large, international collaborative networks and well-executed manipulative experiments are rapidly advancing the field of coastal marine microbiome research

Loss of the Metalloprotease ADAM9 Leads to Cone-Rod Dystrophy in Humans and Retinal Degeneration in Mice

Cone-rod dystrophy (CRD) is an inherited progressive retinal dystrophy affecting the function of cone and rod photoreceptors. By autozygosity mapping, we identified null mutations in the ADAM metallopeptidase domain 9 (ADAM9) gene in four consanguineous families with recessively inherited early-onset CRD. We also found reduced photoreceptor responses in Adam9 knockout mice, previously reported to be asymptomatic. In 12-month-old knockout mice, photoreceptors appear normal, but the apical processes of the retinal pigment epithelium (RPE) cells are disorganized and contact between photoreceptor outer segments (POSs) and the RPE apical surface is compromised. In 20-month-old mice, there is clear evidence of progressive retinal degeneration with disorganized POS and thinning of the outer nuclear layer (ONL) in addition to the anomaly at the POS-RPE junction. RPE basal deposits and macrophages were also apparent in older mice. These findings therefore not only identify ADAM9 as a CRD gene but also identify a form of pathology wherein retinal disease first manifests at the POS-RPE junction

Microtubule-Associated Protein 1 Light Chain 3B, (LC3B) Is Necessary to Maintain Lipid-Mediated Homeostasis in the Retinal Pigment Epithelium

Like other neurons, retinal cells utilize autophagic pathways to maintain cell homeostasis. The mammalian retina relies on heterophagy and selective autophagy to efficiently degrade and metabolize ingested lipids with disruption in autophagy associated degradation contributing to age related retinal disorders. The retinal pigment epithelium (RPE) supports photoreceptor cell renewal by daily phagocytosis of shed photoreceptor outer segments (OS). The daily ingestion of these lipid-rich OS imposes a constant degradative burden on these terminally differentiated cells. These cells rely on Microtubule-Associated Protein 1 Light Chain 3 (LC3) family of proteins for phagocytic clearance of the ingested OS. The LC3 family comprises of three highly homologous members, MAP1LC3A (LC3A), MAP1LC3B (LC3B), and MAP1LC3C (LC3C). The purpose of this study was to determine whether the LC3B isoform plays a specific role in maintaining RPE lipid homeostasis. We examined the RPE and retina of the LC3B-/- mouse as a function of age using in vivo ocular imaging and electroretinography coupled with ex vivo, lipidomic analyses of lipid mediators, assessment of bisretinoids as well as imaging of lipid aggregates. Deletion of LC3B resulted in defects within the RPE including increased phagosome accumulation, decreased fatty acid oxidation and a subsequent increase in RPE and sub-RPE lipid deposits. Age-dependent RPE changes included elevated levels of oxidized cholesterol, deposition of 4-HNE lipid peroxidation products, bisretinoid lipofuscin accumulation, and subretinal migration of microglia, collectively likely contributing to loss of retinal function. These observations are consistent with a critical role for LC3B-dependent processes in the maintenance of normal lipid homeostasis in the aging RPE, and suggest that LC3 isoform specific disruption in autophagic processes contribute to AMD-like pathogenesis