Comparison of Retinal Metabolic Activity and Structural Development between rd10 Mice and Normal Mice Using Multiphoton Fluorescence Lifetime Imaging Microscopy.

Fluorescence lifetime imaging microscopy (FLIM) is a technique that analyzes the metabolic state of tissues based on the spatial distribution of fluorescence lifetimes of certain interacting molecules. We used multiphoton FLIM to study the metabolic state of developing C57BL6/J and rd10 retinas based on the fluorescence lifetimes of free versus bound nicotinamide adenine dinucleotide and nicotinamide adenine dinucleotide phosphate (NAD(P)H), with free NAD(P)H percentages suggesting increased glycolysis and bound NAD(P)H percentages indicating oxidative phosphorylation. The mice were sacrificed and enucleated at various time points throughout their first 3 months of life. The isolated eyecups were fixed, sectioned using a polyacrylamide gel embedding technique, and then analyzed with FLIM. The results suggested that in both C57BL6/J mice and rd10 mice, oxidative phosphorylation initially decreased and then increased, plateauing over time. This trend, however, was accelerated in rd10 mice, with its turning point occurring at p10 versus the p30 turning point in C57BL6/J mice. There was also a noticeable difference in oxidative phosphorylation rates between the outer and inner retinas in both strains, with greater oxidative phosphorylation present in the latter. A greater understanding of rd10 and WT metabolic changes during retinal development may provide deeper insights into retinal degeneration and facilitate the development of future treatments.

Gene Expression of Clusterin, Tissue Inhibitor of Metalloproteinase-1, and Their Receptors in Retinal Pigment Epithelial Cells and Müller Glial Cells Is Modulated by Inflammatory Stresses.

Balanced activities of matrix metalloproteinases (MMPs) and their inhibitors are essential for photoreceptor (PR) cell survival. PR rod cell survival in rodent models of inherited retinitis pigmentosa (RP) is prolonged by recombinant tissue inhibitor of metalloproteinase (TIMP)-1 or clusterin (CLU) proteins. Retinal pigment epithelial cells (RPE) and Müller glia (MG) cells support PR cells. In human RPE and MG cell lines, we measured their mRNA levels of the two genes with quantitative real-time PCR (qRT-PCR) with interleukin (IL)-1ß treatment, a key pathological component in retinal degeneration. Endogenous CLU gene expression was significantly downregulated by IL-1ß in both cell types, whereas TIMP-1 expression was upregulated in MG cells, suggesting the transcriptional control of CLU is potentially more sensitive to inflammatory conditions. The expression levels of CLU endocytic receptors revealed that the low-density lipoprotein receptor-related protein 2 (LRP2) was upregulated only in MG cells by the treatment with no detectable change in RPE cells. Like LRP2, IL-1ß upregulated TIMP-1 receptor LRP1 expression in MG cells; however, it was decreased in the expression of RPE cells. These data suggest that the gene expression of CLU and TIMP-1 and their receptors may be dynamically modulated in inflammatory conditions.

MSI2 promotes translation of multiple IRES-containing oncogenes and virus to induce self-renewal of tumor initiating stem-like cells.

RNA-binding protein Musashi 2 (MSI2) is elevated in several cancers and is linked to poor prognosis. Here, we tested if MSI2 promotes MYC and viral mRNA translation to induce self-renewal via an internal ribosome entry sequence (IRES). We performed RIP-seq using anti-MSI2 antibody in tumor-initiating stem-like cells (TICs). MSI2 binds the internal ribosome entry site (IRES)-containing oncogene mRNAs including MYC, JUN and VEGFA as well as HCV IRES to increase their synthesis and promote self-renewal and tumor-initiation at the post-transcriptional level. MSI2 binds a lncRNA to interfere with processing of a miRNA that reduced MYC translation in basal conditions. Deregulation of this integrated MSI2-lncRNA-MYC regulatory loop drives self-renewal and tumorigenesis through increased IRES-dependent translation of MYC mRNA. Overexpression of MSI2 in TICs promoted their self-renewal and tumor-initiation properties. Inhibition of MSI2-RNA binding reduced HCV IRES activity, viral replication and liver hyperplasia in humanized mice predisposed by virus infection and alcohol high-cholesterol high-fat diet. Together MSI2, integrating the MYC oncogenic pathway, can be employed as a therapeutic target in the treatment of HCC patients. A hypothetical model shows that MSI2 binds and activates cap-independent translation of MYC, c-JUN mRNA and HCV through MSI2-binding to Internal Ribosome Entry Sites (IRES) resulting in upregulated MYC, c-JUN and viral protein synthesis and subsequent liver oncogenesis. Inhibitor of the interaction between MYC IRES and MSI2 reduces liver hyperplasia, viral mRNA translation and tumor formation.

Matrix metalloproteinase-9 (MMP-9) and tissue inhibitor of metalloproteinases 1 (TIMP-1) are localized in the nucleus of retinal Müller glial cells and modulated by cytokines and oxidative stress.

Matrix metalloproteinases (MMPs) are involved in the pathology of numerous inflammatory retinal degenerations, including retinitis pigmentosa (RP). Our previous work revealed that intravitreal injections with tissue inhibitor of metalloproteinases 1 (TIMP-1) reduce the progression of rod cell death and inhibit cone cell remodeling that involves reactive gliosis in retinal Müller glial cells (MGCs) in rodent models. The underlying cellular and molecular mechanisms of how TIMP-1 functions in the retina remain to be resolved; however, MGCs are involved in structural homeostasis, neuronal cell survival and death. In the present study, MMP-9 and TIMP-1 expression patterns were investigated in a human MGC line (MIO-M1) under inflammatory cytokine (IL-1ß and TNF-α) and oxidative stress (H2O2) conditions. First, both IL-1ß and TNF-α, but not H2O2, have a mild in vitro pro-survival effect on MIO-M1 cells. Treatment with either cytokine results in the imbalanced secretion of MMP-9 and TIMP-1. H2O2 treatment has little effect on their secretion. The investigation of their intracellular expression led to interesting observations. MMP-9 and TIMP-1 are both expressed, not only in the cytoplasm, but also inside the nucleus. None of the treatments alters the MMP-9 intracellular distribution pattern. In contrast to MMP-9, TIMP-1 is detected as speckles. Intracellular TIMP-1 aggregation forms in the cytoplasmic area with IL-1ß treatment. With H2O2 treatments, the cell morphology changes from cobbles to spindle shapes and the nuclei become larger with increases in TIMP-1 speckles in an H2O2 dose-dependent manner. Two TIMP-1 cell surface receptors, low density lipoprotein receptor-related protein-1 (LRP-1) and cluster of differentiation 82 (CD82), are expressed within the nucleus of MIO-M1 cells. Overall, these observations suggest that intracellular TIMP-1 is a target of proinflammatory and oxidative insults in the MGCs. Given the importance of the roles for MGCs in the retina, the functional implication of nuclear TIMP-1 and MMP-9 in MGCs is discussed.

A global database of plant production and carbon exchange from global change manipulative experiments.

Numerous ecosystem manipulative experiments have been conducted since 1970/80 s to elucidate responses of terrestrial carbon cycling to the changing atmospheric composition (CO2 enrichment and nitrogen deposition) and climate (warming and changing precipitation regimes), which is crucial for model projection and mitigation of future global change effects. Here, we extract data from 2,242 publications that report global change manipulative experiments and build a comprehensive global database with 5,213 pairs of samples for plant production (productivity, biomass, and litter mass) and ecosystem carbon exchange (gross and net ecosystem productivity as well as ecosystem and soil respiration). Information on climate characteristics and vegetation types of experimental sites as well as experimental facilities and manipulation magnitudes subjected to manipulative experiments are also included in this database. This global database can facilitate the estimation of response and sensitivity of key terrestrial carbon-cycling variables under future global change scenarios, and improve the robust projection of global change‒terrestrial carbon feedbacks imposed by Earth System Models.

p53 destabilizing protein skews asymmetric division and enhances NOTCH activation to direct self-renewal of TICs.

Tumor-initiating stem-like cells (TICs) are defective in maintaining asymmetric cell division and responsible for tumor recurrence. Cell-fate-determinant molecule NUMB-interacting protein (TBC1D15) is overexpressed and contributes to p53 degradation in TICs. Here we identify TBC1D15-mediated oncogenic mechanisms and tested the tumorigenic roles of TBC1D15 in vivo. We examined hepatocellular carcinoma (HCC) development in alcohol Western diet-fed hepatitis C virus NS5A Tg mice with hepatocyte-specific TBC1D15 deficiency or expression of non-phosphorylatable NUMB mutations. Liver-specific TBC1D15 deficiency or non-p-NUMB expression reduced TIC numbers and HCC development. TBC1D15-NuMA1 association impaired asymmetric division machinery by hijacking NuMA from LGN binding, thereby favoring TIC self-renewal. TBC1D15-NOTCH1 interaction activated and stabilized NOTCH1 which upregulated transcription of NANOG essential for TIC expansion. TBC1D15 activated three novel oncogenic pathways to promote self-renewal, p53 loss, and Nanog transcription in TICs. Thus, this central regulator could serve as a potential therapeutic target for treatment of HCC.

Asymmetric responses of plant community structure and composition to precipitation variabilities in a semi-arid steppe.

Changing precipitation regimes can profoundly affect plant growth in terrestrial ecosystems, especially in arid and semi-arid regions. However, how changing precipitation, especially extreme precipitation events, alters plant diversity and community composition is still poorly understood. A 3-year field manipulative experiment with seven precipitation treatments, including - 60%, - 40%, - 20%, 0% (as a control), + 20%, + 40%, and + 60% of ambient growing-season precipitation, was conducted in a semi-arid steppe in the Mongolian Plateau. Results showed total plant community cover and forb cover were enhanced with increased precipitation and reduced under decreased precipitation, whereas grass cover was suppressed under the - 60% treatment only. Plant community and grass species richness were reduced by the - 60% treatment only. Moreover, our results demonstrated that total plant community cover was more sensitive to decreased than increased precipitation under normal and extreme precipitation change, and species richness was more sensitive to decreased than increased precipitation under extreme precipitation change. The community composition and low field water holding capacity may drive this asymmetric response. Accumulated changes in community cover may eventually lead to changes in species richness. However, compared to control, Shannon-Weiner index (H) did not respond to any precipitation treatment, and Pielou's evenness index (E) was reduced under the + 60% treatment across the 3 year, but not in each year. Thus, the findings suggest that plant biodiversity in the semi-arid steppe may have a strong resistance to precipitation pattern changes through adjusting its composition in a short term.

Microbial functional traits are sensitive indicators of mild disturbance by lamb grazing.

Mild disturbances are prevalent in the environment, which may not be easily notable but could have considerable ecological consequences over prolonged periods. To evaluate this, a field study was designed to examine the effects of very light-intensity lamb grazing on grassland soil microbiomes with different soil backgrounds. No significant change (P > 0.05) was observed in any vegetation and soil variables. Nonetheless, hundreds of microbial functional gene families, but not bacterial taxonomy, were significantly (P < 0.05) shifted. The relative abundances of both taxonomic markers and functional genes related to nitrifying bacteria were also changed. The observation highlighted herein, showing a high level of sensitivity with respect to functional traits (functionally categorized taxa or genes) in differentiating mild environmental disturbance, suggests that the key level at which to address microbial responses may not be "species" (by means of rRNA taxonomy), but rather at the functional gene level.

Elevated CO2 does not stimulate carbon sink in a semi-arid grassland.

Elevated CO2 is widely accepted to enhance terrestrial carbon sink, especially in arid and semi-arid regions. However, great uncertainties exist for the CO2 fertilisation effects, particularly when its interactions with other global change factors are considered. A four-factor (CO2 , temperature, precipitation and nitrogen) experiment revealed that elevated CO2 did not affect either gross ecosystem productivity or ecosystem respiration, and consequently resulted in no changes of net ecosystem productivity in a semi-arid grassland despite whether temperature, precipitation and nitrogen were elevated or not. The observations could be primarily attributable to the offset of ecosystem carbon uptake by enhanced soil carbon release under CO2 enrichment. Our findings indicate that arid and semi-arid ecosystems may not be sensitive to CO2 enrichment as previously expected and highlight the urgent need to incorporate this mechanism into most IPCC carbon-cycle models for convincing projection of terrestrial carbon sink and its feedback to climate change.

Shifts of growing-season precipitation peaks decrease soil respiration in a semiarid grassland.

Changing precipitation regimes could have profound influences on carbon (C) cycle in the biosphere. However, how soil C release from terrestrial ecosystems responds to changing seasonal distribution of precipitation remains unclear. A field experiment was conducted for 4 years (2013-2016) to examine the effects of altered precipitation distributions in the growing season on soil respiration in a temperate steppe in the Mongolian Plateau. Over the 4 years, both advanced and delayed precipitation peaks suppressed soil respiration, and the reductions mainly occurred in August. The decreased soil respiration could be primarily attributable to water stress and subsequently limited plant growth (community cover and belowground net primary productivity) and soil microbial activities in the middle growing season, suggesting that precipitation amount in the middle growing season is more important than that in the early, late, or whole growing seasons in regulating soil C release in grasslands. The observations of the additive effects of advanced and delayed precipitation peaks indicate semiarid grasslands will release less C through soil respiratory processes under the projected seasonal redistribution of precipitation in the future. Our findings highlight the potential role of intra-annual redistribution of precipitation in regulating ecosystem C cycling in arid and semiarid regions.