Optogenetic control of cell function using engineered photoreceptors

Over the past decades, there has been growing recognition that light can provide a powerful stimulus for biological interrogation. Light‐actuated tools allow manipulation of molecular events with ultra‐fine spatial and fast temporal resolution, as light can be rapidly delivered and focused with sub‐micrometre precision within cells. While light‐actuated chemicals such as photolabile ‘caged’ compounds have been in existence for decades, the use of genetically encoded natural photoreceptors for optical control of biological processes has recently emerged as a powerful new approach with several advantages over traditional methods. Here, we review recent advances using light to control basic cellular functions and discuss the engineering challenges that lie ahead for improving and expanding the ever‐growing optogenetic toolkit.     

The translation initiation factor 3 subunit eIF3K interacts with PML and associates with PML nuclear bodies

The promyelocytic leukemia protein (PML) is a tumor suppressor protein that regulates a variety of important cellular processes, including gene expression, DNA repair and cell fate decisions. Integral to its function is the ability of PML to form nuclear bodies (NBs) that serve as hubs for the interaction and modification of over 90 cellular proteins. There are seven canonical isoforms of PML, which encode diverse C-termini generated by alternative pre-mRNA splicing. Recruitment of specific cellular proteins to PML NBs is mediated by protein–protein interactions with individual PML isoforms. Using a yeast two-hybrid screen employing peptide sequences unique to PML isoform I (PML-I), we identified an interaction with the eukaryotic initiation factor 3 subunit K (eIF3K), and in the process identified a novel eIF3K isoform, which we term eIF3K-2. We further demonstrate that eIF3K and PML interact both in vitro via pull-down assays, as well as in vivo within human cells by co-immunoprecipitation and co-immunofluorescence. In addition, eIF3K isoform 2 (eIF3K-2) colocalizes to PML bodies, particularly those enriched in PML-I, while eIF3K isoform 1 associates poorly with PML NBs. Thus, we report eIF3K as the first known subunit of the eIF3 translation pre-initiation complex to interact directly with the PML protein, and provide data implicating alternative splicing of both PML and eIF3K as a possible regulatory mechanism for eIF3K localization at PML NBs.     

Evaluation of dental enamel caries assessment using Quantitative Light Induced Fluorescence and Optical Coherence Tomography

An in vitro study of morphological alterations between sound dental structure and artificially induced white spot lesions in human teeth, was performed through the loss of fluorescence by Quantitative Light‐Induced Fluorescence (QLF) and the alterations of the light attenuation coefficient by Optical Coherence Tomography (OCT). To analyze the OCT images using a commercially available system, a special algorithm was applied, whereas the QLF images were analyzed using the software available in the commercial system employed. When analyzing the sound region against white spot lesions region by QLF, a reduction in the fluorescence intensity was observed, whilst an increase of light attenuation by the OCT system occurred. Comparison of the percentage of alteration between optical properties of sound and artificial enamel caries regions showed that OCT processed images through the attenuation of light enhanced the tooth optical alterations more than fluorescence detected by QLF System.

QLF versus OCT imaging of enamel caries: a photonics assessment     

Dim light at night prior to adolescence increases adult anxiety-like behaviors

Dim light at night (dLAN) disrupts circadian organization and influences adult behavior. We examined early dLAN exposure on adult affective responses. Beginning 3 (juvenile) or 5 weeks (adolescent) of age, mice were maintained in standard light–dark cycles or exposed to nightly dLAN (5 lx) for 5 weeks, then anxiety-like and fear responses were assessed. Hypothalami were collected around the clock to assess core clock genes. Exposure to dLAN at either age increased anxiety-like responses in adults. Clock and Rev-ERB expression were altered by exposure to dLAN. In contrast to adults, dLAN exposure during early life increases anxiety and fear behavior.     

Role of electroencephalogram and oxygen saturation in the induction mechanism of arousal for obstructive sleep apnea-hypopnea syndrome patients

Arousal concomitant with obstructive sleep apnea-hypopnea syndrome (OSAHS) is known to result in sleep fragmentation and excessive daytime sleepiness. The cause of arousal is multifarious, and the mechanism is not yet clear. The aim of this study was to further research the induction mechanism of arousal by investigating the variation of electroencephalogram (EEG) and oxygen saturation (SaO₂). This study enrolled 20 subjects with a clinical diagnosis of OSAHS who underwent overnight polysomnography. Respiratory events and arousals were scored, and individuals with insufficient samples (<30) were excluded. Thus, 13 subjects mostly with severe OSAHS were analyzed in this study. The wavelet coefficients, spectral power of EEG (C4-M1 and C3-M2) before arousal or airway reopening, and the maximum desaturations of SaO₂ during respiratory events were analyzed. For most subjects, EEG (in stages N1 and N2) during respiratory events with arousals exhibited significantly lower values of wavelet coefficients and spectral power (p < 0.05). The maximum desaturations of SaO₂ during respiratory events with arousals are larger than those without among individual. In binary logistic regression analysis, the P values of EEG features and SaO₂ desaturation were both less than 0.001. Our results demonstrate that in light NREM stage, less activity in EEG during respiratory events and larger SaO₂ drop both independently were related to the occurrence of arousal. These significant differences come from major subjects based on the statistical analysis, and help supplement the induction mechanism of arousal.     

Responses of plant 15N natural abundance and isotopic fractionation to N addition reflect the N status of a temperate steppe in China

Aims Elevated anthropogenic nitrogen (N) deposition could alter N status in temperate steppe. However, threshold observations of N status change from N limit to N saturation by far are not conclusive in these ecosystems. Research on the natural abundance of¹⁵N (δ¹⁵N) could greatly help provide integrated information about ecosystem N status. The goal of this study was to investigate the suitability of measurements of δ¹⁵N of major ecosystem N pools and several key species, plant¹⁵N fractionation, together with key vegetation and soil indicators in response to N fertilization as a tool to identify the N status in a temperate steppe in Inner Mongolia.     

Influence of mycorrhizal fungi on seed germination and growth in terrestrial and epiphytic orchids

Epiphytes constitute over 70% of orchid diversity, but little is known about the functioning of their mycorrhizal associations. Terrestrial orchid seeds germinate symbiotically in soil and leaf litter, whereas epiphytic orchids may be exposed to relatively high light levels from an early stage of development and often produce green seeds. This suggests that seedlings of the two groups of orchids may differ in their responses to light and requirements for mycorrhiza-supplied carbon. The interactive effects of light, exogenous carbon and mycorrhizal status on germination and growth were investigated in vitro using axenic agar microcosms for one tropical epiphyte and three geophytic orchid species. The geophytic species strongly depended on their mycorrhiza for growth and this could not be substituted by exogenous sucrose, whereas the epiphytic species achieved 95% of the mycorrhizal seedling volume when supplied with exogenous sucrose in the dark. Mycorrhiza status strongly interacted with light exposure, enabling germination. Light inhibited or severely reduced growth, especially for the terrestrial orchids in the absence of mycorrhiza. For the first time, this study showed the parallel ecological importance of mycorrhizal fungi in overcoming light inhibition of seed germination and growth in both terrestrial and epiphytic orchids.     

Carbanilation of cereal beta-glucans for molecular weight determination and conformational studies

Cereal beta-glucans can form aggregates in aqueous solution. The presence of aggregates in cereal beta-glucan solutions led to inaccurate determination of molecular weights and it was believed that intermolecular hydrogen bonding caused the aggregation. To eliminate aggregates, a carbanilation method for molecular weight determination of cereal beta-glucans was developed. Wheat beta-glucan samples were selected for investigation. The carbanilation method can prevent intermolecular hydrogen bonding by blocking hydroxyl groups with phenyl carbamate groups. The carbanilates of cereal beta-glucans were prepared by the reaction of cereal beta-glucans with phenylisocyanate catalyzed by DMSO and pyridine. To avoid degradation during the carbanilation reaction, relatively mild conditions were used, which led to incomplete substitution (DS: approximately 2). However, after the carbanilation reaction, the carbanilates dissolved completely in 1,4-dioxane solution without any detectable aggregates, which allowed accurate molecular weight determination. The degree of substitution (DS) of carbanilates was determined by both a nitrogen content method and an FT-IR method. The FT-IR method proved to be the more effective for DS estimation. Using this method, the converted molecular weights of cereal beta-glucans were in good agreement with the results measured in 0.5M NaOH solution, which previously was shown to be a good solvent for cereal beta-glucans. After the carbanilation reaction, conformational changes of carbanilates were studied by static and dynamic light scattering techniques. The fractal dimension (d(f)=2.27) and the structure sensitive parameters (rho >2) suggested a porous globular structure for partially carbanilated beta-glucans.     

Wood decay under the microscope

Many aspects of the interactions between host wood structure and fungal activity can be revealed by high resolution light microscopy, and this technique has provided much of the information discussed here. A wide range of different types of decay can result from permutations of host species, fungal species and conditions within wood. Within this spectrum, three main types are commonly recognised: brown rot, white rot and soft rot. The present review explores parts of the range of variation that each of these encompasses and emphasizes that degradation modes appear to reflect a co-evolutionary adaptation of decay fungi to different wood species or the lignin composition within more primitive and advanced wood cell types. One objective of this review is to provide evidence that the terms brown rot, white rot and soft rot may not be obsolete, but rigid definitions for fungi that are placed into these categories may be less appropriate than thought previously. Detailed knowledge of decomposition processes does not only aid prognosis of decay development in living trees for hazard assessment but also allows the identification of wood decay fungi that can be used for biotechnology processes in the wood industry. In contrast to bacteria or commercial enzymes, hyphae can completely ramify through solid wood. In this review evidence is provided that wood decay fungi can effectively induce permeability changes in gymnospermous heartwood or can be applied to facilitate the identification of tree rings in diffuse porous wood of angiosperms. The specificity of their enzymes and the mild conditions under which degradation proceeds is partly detrimental for trees, but also make wood decay fungi potentially efficient biotechnological tools.