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The over-accumulation of glycogen appears as a hallmark in various glycogen storage diseases (GSDs), including Pompe, Cori, Andersen and Lafora disease. Accumulating evidence suggests that suppression of glycogen accumulation represents a potential therapeutic approach for treating these GSDs. Using a fluorescence polarization assay designed to screen for inhibitors of the key glycogen synthetic enzyme, glycogen synthase (GS), we identified a substituted imidazole, (rac)-2-methoxy-4-(1-(2-(1-methylpyrrolidin-2-yl)ethyl)-4-phenyl-1H-imidazol-5-yl)phenol (H23), as a first-in-class inhibitor for yeast glycogen synthase 2 (yGsy2p). Data from X-ray crystallography at 2.85 Å, as well as kinetic data, revealed that H23 bound within the UDP-glucose binding pocket of yGsy2p. The high conservation of residues between human and yeast GS in direct contact with H23 informed the development of around 500 H23 analogs. These analogs produced a structure-activity relationship (SAR) profile that led to the identification of a substituted pyrazole, 4-(4-(4-hydroxyphenyl)-3-(trifluoromethyl)-1H-pyrazol-5-yl)pyrogallol, with 300-fold improved potency against human GS. These substituted pyrazoles possess a promising scaffold for drug development efforts targeting GS activity in GSDs associated with excess glycogen accumulation.

APETALA2/ETHYLENE RESPONSIVE FACTOR (AP2/ERF) family transcription factors have well-documented functions in stress responses, but their roles in brassinosteroid (BR)-regulated growth and stress responses have not been established. Here, we show that the Arabidopsis (Arabidopsis thaliana) stress-inducible AP2/ERF transcription factor TINY inhibits BR-regulated growth while promoting drought responses. TINY-overexpressing plants have stunted growth, increased sensitivity to BR biosynthesis inhibitors, and compromised BR-responsive gene expression. By contrast, tiny tiny2 tiny3 triple mutants have increased BR-regulated growth and BR-responsive gene expression. TINY positively regulates drought responses by activating drought-responsive genes and promoting abscisic acid–mediated stomatal closure. Global gene expression studies revealed that TINY and BRs have opposite effects on plant growth and stress response genes. TINY interacts with and antagonizes BRASSINOSTERIOID INSENSITIVE1-ETHYL METHANESULFONATE SUPRESSOR1 (BES1) in the regulation of these genes. Glycogen synthase kinase 3-like protein kinase BR-INSENSITIVE2 (BIN2), a negative regulator in the BR pathway, phosphorylates and stabilizes TINY, providing a mechanism for BR-mediated downregulation of TINY to prevent activation of stress responses under optimal growth conditions. Taken together, our results demonstrate that BR signaling negatively regulates TINY through BIN2 phosphorylation and TINY positively regulates drought responses, as well as inhibiting BR-mediated growth through TINY-BES1 antagonistic interactions. Our results thus provide insight into the coordination of BR-regulated growth and drought responses.

Pipelines that cross complex geological terrains are inevitably threatened by natural hazards, among which landslide attracts extensive attention when pipelines cross mountainous areas. The landslides are typically associated with ground movements that would induce additional stress on the pipeline. Such stress state of pipelines under landslide interference seriously damage structural integrity of the pipeline. Up to the date, limited research has been done on the combined landslide hazard and pipeline stress state analysis. In this paper, a multi-parameter integrated monitoring system was developed for the pipeline stress-strain state and landslide deformation monitoring. Also, data-driven models for the pipeline additional stress prediction was established. The developed predictive models include individual and ensemble-based machine learning approaches. The implementation procedure of the predictive models integrates the field data measured by the monitoring system, with k-fold cross validation used for the generalization performance evaluation. The obtained results indicate that the XGBoost model has the highest performance in the prediction of the additional stress. Besides, the significance of the input variables is determined through sensitivity analyses by using feature importance criteria. Thus, the integrated monitoring system together with the XGBoost prediction method is beneficial to modeling the additional stress in oil and gas pipelines, which will further contribute to pipeline geohazards monitoring management.

Summary Glycosylation defects are a hallmark of many nervous system diseases. However, the molecular and metabolic basis for this pathology is not fully understood. In this study, we found that N-linked protein glycosylation in the brain is metabolically channeled to glucosamine metabolism through glycogenolysis. We discovered that glucosamine is an abundant constituent of brain glycogen, which functions as a glucosamine reservoir for multiple glycoconjugates. We demonstrated the enzymatic incorporation of glucosamine into glycogen by glycogen synthase, and the release by glycogen phosphorylase by biochemical and structural methodologies, in primary astrocytes, and in vivo by isotopic tracing and mass spectrometry. Using two mouse models of glycogen storage diseases, we showed that disruption of brain glycogen metabolism causes global decreases in free pools of UDP-N-acetylglucosamine and N-linked protein glycosylation. These findings revealed fundamental biological roles of brain glycogen in protein glycosylation with direct relevance to multiple human diseases of the central nervous system.

Ocular neovascularization underlies major blinding eye diseases such as “wet” age‐related macular degeneration (AMD). Despite the successes of treatments targeting the vascular endothelial growth factor (VEGF) pathway, resistant and refractory patient populations necessitate discovery of new therapeutic targets. Using a forward chemical genetic approach, we identified the heme synthesis enzyme ferrochelatase (FECH) as necessary for angiogenesis in vitro and in vivo . FECH is overexpressed in wet AMD eyes and murine choroidal neovascularization; siRNA knockdown of Fech or partial loss of enzymatic function in the Fech m1Pas mouse model reduces choroidal neovascularization. FECH depletion modulates endothelial nitric oxide synthase function and VEGF receptor 2 levels. FECH is inhibited by the oral antifungal drug griseofulvin, and this compound ameliorates choroidal neovascularization in mice when delivered intravitreally or orally. Thus, FECH inhibition could be used therapeutically to block ocular neovascularization.

Plant steroid hormones brassinosteroids (BRs) regulate plant growth and development at many different levels. Recent research has revealed that stress-responsive NAC (petunia NAM and Arabidopsis ATAF1, ATAF2, and CUC2) transcription factor RD26 is regulated by BR signaling and antagonizes BES1 in the interaction between growth and drought stress signaling. However, the upstream signaling transduction components that activate RD26 during drought are still unknown. Here, we demonstrate that the function of RD26 is modulated by GSK3-like kinase BIN2 and protein phosphatase 2C ABI1. We show that ABI1, a negative regulator in abscisic acid (ABA) signaling, dephosphorylates and destabilizes BIN2 to inhibit BIN2 kinase activity. RD26 protein is stabilized by ABA and dehydration in a BIN2-dependent manner. BIN2 directly interacts and phosphorylates RD26 in vitro and in vivo. BIN2 phosphorylation of RD26 is required for RD26 transcriptional activation on drought-responsive genes. RD26 overexpression suppressed the brassinazole (BRZ) insensitivity of BIN2 triple mutant bin2 bil1 bil2, and BIN2 function is required for the drought tolerance of RD26 overexpression plants. Taken together, our data suggest a drought signaling mechanism in which drought stress relieves ABI1 inhibition of BIN2, allowing BIN2 activation. Sequentially, BIN2 phosphorylates and stabilizes RD26 to promote drought stress response.

Brassinosteroids (BRs) regulate plant growth and stress responses via the BES1/BZR1 family of transcription factors, which regulate the expression of thousands of downstream genes. BRs are involved in the response to drought, however the mechanistic understanding of interactions between BR signalling and drought response remains to be established. Here we show that transcription factor RD26 mediates crosstalk between drought and BR signalling. When overexpressed, BES1 target gene RD26 can inhibit BR-regulated growth. Global gene expression studies suggest that RD26 can act antagonistically to BR to regulate the expression of a subset of BES1-regulated genes, thereby inhibiting BR function. We show that RD26 can interact with BES1 protein and antagonize BES1 transcriptional activity on BR-regulated genes and that BR signalling can also repress expression of RD26 and its homologues and inhibit drought responses. Our results thus reveal a mechanism coordinating plant growth and drought tolerance., Brassinosteroid (BR) signalling regulates plant development via the BES1/BZR1 family of transcription factors. Here the authors show that BES1 activity can be modified by the drought-responsive RD26 transcription factor providing a molecular basis for the interaction between drought and BR signalling.

The allocation of carbon and nitrogen resources to the synthesis of plant proteins, carbohydrates, and lipids is complex and under the control of many genes; much remains to be understood about this process. QQS (Qua-Quine Starch; At3g30720), an orphan gene unique to Arabidopsis thaliana, regulates metabolic processes affecting carbon and nitrogen partitioning among proteins and carbohydrates, modulating leaf and seed composition in Arabidopsis and soybean. Here the universality of QQS function in modulating carbon and nitrogen allocation is exemplified by a series of transgenic experiments. We show that ectopic expression of QQS increases soybean protein independent of the genetic background and original protein content of the cultivar. Furthermore, transgenic QQS expression increases the protein content of maize, a C4 species (a species that uses 4-carbon photosynthesis), and rice, a protein-poor agronomic crop, both highly divergent from Arabidopsis. We determine that QQS protein binds to the transcriptional regulator AtNF-YC4 (Arabidopsis nuclear factor Y, subunit C4). Overexpression of AtNF-YC4 in Arabidopsis mimics the QQS-overexpression phenotype, increasing protein and decreasing starch levels. NF-YC, a component of the NF-Y complex, is conserved across eukaryotes. The NF-YC4 homologs of soybean, rice, and maize also bind to QQS, which provides an explanation of how QQS can act in species where it does not occur endogenously. These findings are, to our knowledge, the first insight into the mechanism of action of QQS in modulating carbon and nitrogen allocation across species. They have major implications for the emergence and function of orphan genes, and identify a nontransgenic strategy for modulating protein levels in crop species, a trait of great agronomic significance.