The Effect of Photographic Light Exposure on Cup to Disc Ratio Grading

Background: Digital optic disc images are integral in remote telehealthcare, yet no quality control standards exist for the exposure setting of the images. This study evaluated the relationship between exposure setting and cup/disc ratio (c/d) grading among glaucoma specialists. Methods: Color disc photos were taken of 50 eyes of pediatric patients under anesthesia at 3 light exposure settings: dark, medium, and bright. 9 glaucoma specialists evaluated the c/d of the imaged discs in random order. The relationships between the exposure levels and the c/d estimates as well as between c/d size and variability were evaluated.Results: A total of 150 photos of 50 optic discs were graded. The c/d estimates were significantly larger among bright when compared to photos taken at the medium exposure setting(0.53 vs 0.48, P < 0.001) as well as the dark exposure setting (0.47, P < 0.001). In addition, images with larger mean c/d did not show more variability amongst readers (P = 0.59). Conclusion: Image exposure affects c/d grading of nonstereoscopic disc images. The brighter exposure is associated with larger c/d grading. Exposure consistency is key in longitudinal care.

Genomic and Spectral Visual Adaptation in Southern Leopard Frogs during the Ontogenetic Transition from Aquatic to Terrestrial Light Environments

Many animals have complex life cycles where larval and adult forms have distinct ecologies and habitats that impose different demands on their sensory systems. While the adaptive decoupling hypothesis predicts reduced genetic correlations between life stages, how sensory systems adapt across life stages at the molecular level is not well understood. Frogs are a compelling system to study this question in because most species rely on vision as both aquatic tadpoles and terrestrial adults, but these habitats present vastly different light environments. Here we used whole eye transcriptome sequencing to investigate differential expression between aquatic tadpoles and terrestrial juveniles of the southern leopard frog (Lithobates sphenocephalus). Because visual physiology changes with light levels, we also tested how constant light or dark exposure affected gene expression. We found 42% of genes were differentially expressed in the eyes of tadpoles versus juveniles, versus 5% for light/dark exposure. Analyses targeting a curated set of visual genes revealed significant differential expression between life stages in genes that control aspects of visual function and development, including spectral sensitivity and lens composition. Light/dark exposure had a significant effect on a smaller set of visual genes. Finally, microspectrophotometry of photoreceptors confirmed shifts in spectral sensitivity predicted by the expression results, consistent with adaptation to distinct light environments. Overall, we identified extensive expression-level differences in the eyes of tadpole and juvenile frogs related to observed morphological and physiological changes through metamorphosis, and corresponding adaptive shifts to optimize vision in aquatic versus terrestrial environments.

Coping with darkness: The adaptive response of marine picocyanobacteria to repeated light energy deprivation

The picocyanobacteria Prochlorococcus and Synechococcus are found throughout the ocean’s euphotic zone, where the daily light:dark cycle drives their physiology. Periodic deep mixing events can, however, move cells below this zone, depriving them of light for extended periods of time. Here we demonstrate that Prochlorococcus and Synechococcus can adapt to tolerate repeated periods of light energy deprivation. Cyanobacterial cultures kept in the dark for 3 days and then returned to the light initially required 18-26 days to resume growth, but after multiple rounds of dark exposure the strains began to regrow after only 1-2 days. This dark-tolerant phenotype was stable and heritable; cultures retained the trait across at least 18-21 generations even when grown in a standard 13:11 light:dark cycle. We found no genetic differences between the dark-tolerant and parental strains of Prochlorococcus NATL2A, indicating that an epigenetic change is responsible for the adaptation. To begin to explore this possibility, we asked whether DNA methylation – an epigenetic mechanism in bacteria – occurs in Prochlorococcus. LC-MS/MS analysis showed that while DNA methylations, including 6mA and 5mC, are found in some other Prochlorococcus strains, no methylations were detected in either the parental or dark-tolerant strain used in our experiments –i.e. the NATL2A strain. These findings suggest that Prochlorococcus utilizes a yet-to-be-determined epigenetic mechanism to adapt to the stress of extended light energy deprivation.

Engaging inexpensive hands-on activities using Chlamydomonas reinhardtii (a green micro-alga) beads to teach the interplay of photosynthesis and cellular respiration to K4–K16 Biology students

Background Photosynthesis and cellular respiration play major roles in energy metabolism and are important Life Science topics for K16 Biology students. Algae beads are used for photosynthesis and cellular respiration labs. Currently there are a few companies that sell biology educational kits for making algae beads using non-motile green micro-algae to introduce students to photosynthesis. These kits are expensive and, do not come with detailed guidelines for trouble shooting and customizations for different grade levels. Chlamydomonas reinhardtii is a motile green micro-alga and is an excellent model system for photosynthesis studies. In this article, we are presenting the work conducted in the student-driven, American Society of Plant Biologists-funded, Plant-BLOOME educational outreach project. This project is a supervised collaborative effort of three undergraduates and one high school student. We have generated a protocol which can be used to make Chlamydomonas beads. We have used these beads to design two simple and inexpensive plant biology hands-on activities. These laboratory activities have been customized to teach the interplay of photosynthesis and cellular respiration to K4–K16 Biology students. Methods Chlamydomonas beads were used for two different laboratory activities that involved monitoring pH changes over time using a pH indicator. Our first activity centers on making and, using light-powered algae bead bracelets to monitor dramatic color/pH changes over time when exposed to darkness or light. Our second activity employs strain-specific algae beads with approximately equal cell numbers to conduct comparative photosynthesis and cellular respiration studies in two Chlamydomonas strains namely, wild type, 4A+ and, a high light-sensitive, photosynthetic mutant, 10E35/lsr1a. Results We optimized our experimental protocol using algae beads in a 5.5 mL screw capped glass vials before performing the same experiment in algae bead bracelets. We found that the algal cell density/bead, water type used in the experiment and, the duration of dark exposure of algal beads can affect successful implementation of the lab activities. Light-powered algae bead bracelets showed dramatic color/pH changes within 3 h upon exposure to light or darkness. These bracelets could be switched back and forth between darkness and light multiple times within 48–72 h to display color/pH changes, provided prior dark exposure time did not exceed 9 h. Our comparative studies of photosynthesis and cellular respiration in 10E35 and in 4A+ showed that relative respiration rate and photosynthetic rate is higher and lower in 10E35, respectively, compared to that in 4A+. Additionally, 10E35 failed to display the expected photosynthesis-induced pH/color changes in the light after prolonged exposure to darkness which indicated that prolonged dark exposure of 10E35, hindered photosynthesis.

Physiological Responses of Tomato and Cucumber Seedlings under Different Light–Dark Cycles

Tomato and cucumber seedlings were grown in a growth chamber to evaluate the effects of different cycles of light–dark exposure conditions (T0 (control treatment) (1 cycle of 24 h distributed in 18 h of light exposure and six hours of dark), T1 (two cycles of 12 h distributed in nine hours of light exposure and three hours of dark) and T2 (three cycles of eight hours distributed in six hours of light exposure and two hours of dark) on growth, nutrient status, pigment concentration and physiological changes. Total dry weight showed different behaviors in both species, since in tomato the total dry weight remained unchanged under varying light–dark cycles, whereas in cucumber seedlings there was a clear decrease compared to the control treatment. In both species, plants grown under T2 showed the best water content. Nitrogen, P and K content—as well as partitioning in the different organs of the plants—displayed different patterns under varying cycles of light–dark conditions in both species. Chlorophyll (b and a + b) concentration decreased significantly in both species in T1 and T2 compared to the control treatment (T0). At physiological level, the concentration of total soluble sugars and proline in leaf showed the highest value in the control treatment with 18 h of light and six hours of dark.

Homeostatic regulation of perisynaptic matrix metalloproteinase 9 (MMP9) activity in the amblyopic visual cortex

Dark exposure (DE) followed by light reintroduction (LRx) reactivates robust synaptic plasticity in adult mouse primary visual cortex (V1), which allows subsequent recovery from amblyopia. Previously we showed that perisynaptic proteolysis by MMP9 mediates the enhancement of plasticity by LRx in binocular adult mice (Murase et al., 2017). However, it was unknown if a visual system compromised by amblyopia could engage this pathway. Here we show that LRx to adult amblyopic mice induces perisynaptic MMP2/9 activity and extracellular matrix (ECM) degradation in deprived and non-deprived V1. Indeed, LRx restricted to the amblyopic eye is sufficient to induce robust MMP2/9 activity at thalamo-cortical synapses and ECM degradation in deprived V1. Two-photon live imaging demonstrates that the history of visual experience regulates MMP2/9 activity in V1, and that DE lowers the threshold for the proteinase activation. The homeostatic reduction of the MMP2/9 activation threshold by DE enables visual input from the amblyopic pathway to trigger robust perisynaptic proteolysis.

Overexpression of Maize ZmC1 and ZmR Transcription Factors in Wheat Regulates Anthocyanin Biosynthesis in a Tissue-Specific Manner

Maize ZmC1 and ZmR transcription factors belong to the MYB-type and bHLH families, respectively, and control anthocyanin biosynthesis. In this study, Agrobacterium-mediated transformation was used to generate transgenic wheat plants that overexpress ZmC1 and ZmR or both, with the objective of developing anthocyanin-enriched wheat germplasm. Three kinds of stable transgenic wheat lines were obtained. The integration of target genes in the transgenic wheat plants was confirmed by fluorescence in situ hybridization (FISH) analysis. We found that single overexpression of ZmC1 regulates pigmentation in the vegetative tissues such as coleoptiles, auricles, and stems. The single overexpression of ZmR controls the coloration in reproductive tissue like spikelets and seeds. The simultaneous overexpression of ZmC1 and ZmR showed the strongest pigmentation in almost all tissues. Furthermore, quantitative real-time PCR (qRT-PCR) analysis revealed that expression of the two transgenes, and of two conserved homologous and six associated structural genes involved in anthocyanin biosynthesis in wheat were greatly up-regulated in the transgenic plants. Similarly, quantitative analysis for anthocyanin amounts based on HPLC-MS also confirmed that the transgenic wheat plants with combined overexpression of ZmC1 and ZmR accumulated the highest quantity of pigment products. Moreover, developing seeds overexpressing ZmR exposed to light conditions showed up-regulated transcript levels of anthocyanin biosynthesis-related genes compared to dark exposure, which suggests an important role of light in regulating anthocyanin biosynthesis. This study provides a foundation for breeding wheat materials with high anthocyanin accumulation and understanding the mechanism of anthocyanin biosynthesis in wheat.

Homeostatic regulation of perisynaptic MMP9 activity in the amblyopic visual cortex

Dark exposure (DE) followed by light reintroduction (LRx) reactivates robust synaptic plasticity in adult mouse V1, which allows recovery from amblyopia. Previously we showed that LRx-induced perisynaptic proteolysis of extracellular substrates by MMP9 mediates the enhanced plasticity in binocular adult mice (Murase et al., 2017). However, it is unknown if a visual system compromised by amblyopia could engage this pathway. Here we show that LRx to adult amblyopic mice induces perisynaptic MMP2/9 activity and degradation of ECM in the deprived and non-deprived V1. LRx restricted to the amblyopic eye induces equally robust MMP2/9 activity at thalamo-cortical synapses and ECM degradation in deprived V1. Two-photon live imaging demonstrates that the history of visual experience regulates MMP2/9 activity in V1, and that DE lowers the threshold for the proteinase activation. This homeostatic reduction of MMP2/9 activation threshold by DE enables the visual input from the amblyopic pathway to trigger robust perisynaptic proteolysis.

Modification of Peak Plasticity Induced by Brief Dark Exposure

The capacity for neural plasticity in the mammalian central visual system adheres to a temporal profile in which plasticity peaks early in postnatal development and then declines to reach enduring negligible levels. Early studies to delineate the critical period in cats employed a fixed duration of monocular deprivation to measure the extent of ocular dominance changes induced at different ages. The largest deprivation effects were observed at about 4 weeks postnatal, with a steady decline in plasticity thereafter so that by about 16 weeks only small changes were measured. The capacity for plasticity is regulated by a changing landscape of molecules in the visual system across the lifespan. Studies in rodents and cats have demonstrated that the critical period can be altered by environmental or pharmacological manipulations that enhance plasticity at ages when it would normally be low. Immersion in complete darkness for long durations (dark rearing) has long been known to alter plasticity capacity by modifying plasticity-related molecules and slowing progress of the critical period. In this study, we investigated the possibility that brief darkness (dark exposure) imposed just prior to the critical period peak can enhance the level of plasticity beyond that observed naturally. We examined the level of plasticity by measuring two sensitive markers of monocular deprivation, namely, soma size of neurons and neurofilament labeling within the dorsal lateral geniculate nucleus. Significantly larger modification of soma size, but not neurofilament labeling, was observed at the critical period peak when dark exposure preceded monocular deprivation. This indicated that the natural plasticity ceiling is modifiable and also that brief darkness does not simply slow progress of the critical period. As an antecedent to traditional amblyopia treatment, darkness may increase treatment efficacy even at ages when plasticity is at its highest.