MODIFIED VACUOLE PHENOTYPE1 Is an Arabidopsis Myrosinase-Associated Protein Involved in Endomembrane Protein Trafficking

We identified an Arabidopsis (Arabidopsis thaliana) ethyl methanesulfonate mutant, modified vacuole phenotype1-1 (mvp1-1), in a fluorescent confocal microscopy screen for plants with mislocalization of a green fluorescent protein-δ tonoplast intrinsic protein fusion. The mvp1-1 mutant displayed static perinuclear aggregates of the reporter protein. mvp1 mutants also exhibited a number of vacuole-related phenotypes, as demonstrated by defects in growth, utilization of stored carbon, gravitropic response, salt sensitivity, and specific susceptibility to the fungal necrotroph Alternaria brassicicola. Similarly, crosses with other endomembrane marker fusions identified mislocalization to aggregate structures, indicating a general defect in protein trafficking. Map-based cloning showed that the mvp1-1 mutation altered a gene encoding a putative myrosinase-associated protein, and glutathione S-transferase pull-down assays demonstrated that MVP1 interacted specifically with the Arabidopsis myrosinase protein, THIOGLUCOSIDE GLUCOHYDROLASE2 (TGG2), but not TGG1. Moreover, the mvp1-1 mutant showed increased nitrile production during glucosinolate hydrolysis, suggesting that MVP1 may play a role in modulation of myrosinase activity. We propose that MVP1 is a myrosinase-associated protein that functions, in part, to correctly localize the myrosinase TGG2 and prevent inappropriate glucosinolate hydrolysis that could generate cytotoxic molecules.The plant endomembrane system is a complex network of subcellular compartments that includes the endoplasmic reticulum (ER), Golgi apparatus, vacuole, plasma membrane, secretory vesicles, and numerous intermediary compartments. Protein trafficking through the endomembrane system requires specific cargo recognition and delivery mechanisms that are mediated by a series of highly specific targeting signals (Surpin and Raikhel, 2004), whose proper recognition is critical for the function of numerous downstream processes, such as floral development (Sohn et al., 2007), gravitropism (Kato et al., 2002; Surpin et al., 2003; Yano et al., 2003), abiotic stress tolerance (Zhu et al., 2002), autophagy (Surpin et al., 2003; Bassham., 2007), pathogen defense (Robatzek, 2007), and turgor pressure and growth (De, 2000).The importance of protein trafficking for plant survival was demonstrated by the identification of the essential Arabidopsis (Arabidopsis thaliana) gene VACUOLELESS1 (VCL1; Rojo et al., 2001). VCL1 was identified as a homolog of Saccharomyces cerevisiae VPS16, which is critical for yeast vacuole biogenesis. Knockouts of yeast VPS16 lack discernible vacuoles but survive despite their severe phenotype. The absence of vacuoles in Arabidopsis vcl1-1 mutants results in embryo lethality (Rojo et al., 2001). The essential nature of trafficking in plants was also demonstrated by insertional mutagenesis of syntaxin genes, where lethality was observed after disruption of single genes in families with highly homologous members (Lukowitz et al., 1996; Sanderfoot et al., 2001). Thus, despite large families of endomembrane components with many homologous genes, many are not redundant in Arabidopsis.Although embryo-lethal mutations provide critical data, it is difficult to obtain additional information. Less severe mutations have proven successful for functional genetics studies of endomembrane trafficking proteins. For example, point mutations in the KATAMARI1/MURUS3 (KAM1/MUR3; Tamura et al., 2005) and KATAMARI2/GRAVITROPISM DEFECTIVE2 (KAM2/GRV2; Tamura et al., 2007; Silady et al., 2008) genes lead to disruption of endomembranes, resulting in the formation of perinuclear aggregates containing organelles. Nonlethal trafficking disruptions have also been generated using chemical genomics, where small molecules were used to perturb trafficking of a soluble cargo protein (Zouhar et al., 2004) and localization of endomembrane markers (Surpin et al., 2005; Robert et al., 2008). Such studies have provided valuable clues about these essential cellular processes.In order to obtain less severe, viable mutants with defects in endomembrane protein trafficking, we previously identified point mutants with defects in localization of a tonoplast reporter protein, GFP:δ-TIP (Avila et al., 2003). Two hundred one putative mutants were grouped into four categories based on the nature of their defects. One unique mutant, cell shape phenotype1, was recently characterized as a trehalose-6-phosphate synthase with roles in regulation of plant architecture, epidermal pavement cell shape, and trichome branching (Chary et al., 2008).Here, we describe an endomembrane trafficking mutant categorized by perinuclear aggregates of GFP:δ-TIP fluorescence (Avila et al., 2003). We refer to this mutant as modified vacuole phenotype1-1 (mvp1-1). At least five endomembrane fusion proteins are partially relocalized to these structures. Positional cloning identified MVP1 as a myrosinase-associated protein (MyAP) localized previously to the tonoplast by proteomics (Carter et al., 2004). mvp1-1 mutants showed reduced endomembrane system functionality, as demonstrated by defects in growth, utilization of stored carbon, gravitropic responsiveness, salt sensitivity, and increased susceptibility to a fungal necrotroph. MVP1 interacted specifically with THIOGLUCOSIDE GLUCOHYDROLASE2 (TGG2), a known myrosinase protein in Arabidopsis, and the mvp1-1 mutation had a significant effect on nitrile production during glucosinolate hydrolysis, suggesting a role in myrosinase function. Furthermore, MVP1 may function in quality control of glucosinolate hydrolysis by contributing to the proper tonoplast localization of TGG2.     

Shotgun glycomics: a microarray strategy for functional glycomics

Major challenges of glycomics are to characterize a glycome and identify functional glycans as ligands for glycan-binding proteins (GBPs). To address these issues we developed a general strategy termed shotgun glycomics. We focus on glycosphingolipids (GSLs), a class of glycoconjugates that is challenging to study, recognized by toxins, antibodies and GBPs. We derivatized GSLs extracted from cells with a heterobifunctional fluorescent tag suitable for covalent immobilization. We separated fluorescent GSLs by multidimensional chromatography, quantified them and coupled them to glass slides to create GSL shotgun microarrays. Then we interrogated the microarrays with cholera toxin, antibodies and sera from individuals with Lyme disease to identify biologically relevant GSLs that we subsequently characterized by mass spectrometry. Shotgun glycomics incorporating GSLs and potentially glycoprotein-derived glycans is an approach for accessing the complex glycomes of animal cells and is a strategy for focusing structural analyses on functionally important glycans.     

Risk of hospitalization for upper gastrointestinal bleeding in Helicobacter pylori eradicated patients newly started on warfarin or direct oral anticoagulants: A population-based cohort study

Background

To investigate risks of hospitalization for upper gastrointestinal bleeding (UGIB) in H. pylori-eradicated patients newly started on warfarin or direct oral anti-coagulants (DOACs).

Methods

We identified all patients who had previously received H. pylori eradication therapy or were found to have no H. pylori on endoscopy and were then newly started on warfarin or DOACs from a population-based electronic healthcare database. Primary analysis was the risk of UGIB between warfarin and DOACs users in H. pylori-eradicated patients. Secondary analysis included the UGIB risk between H. pylori-eradicated and H. pylori-negative patients who were newly started on warfarin or DOACs. The hazard ratio (HR) of UGIB was approximated by pooled logistic regression model incorporating the inverse propensity of treatment weightings with time-varying covariables.

Results

Among H. pylori-eradicated patients, DOACs had a significantly lower risk of UGIB (HR: 0.26, 95% CI 0.09–0.71) compared with warfarin. In particular, lower UGIB risks with DOACs were observed among older ( ≥ 65 years) patients, female, those without a history of UGIB or peptic ulcer, or ischemic heart disease, and non-users of acid-suppressive agents or aspirin. Secondary analysis showed no significant difference in UGIB risk between H. pylori-eradicated and H. pylori-negative patients newly started on warfarin (HR: 0.63,95% CI 0.33–1.19) or DOACs (HR: 1.37, 95% CI 0.45–4.22).

Conclusions

In H. pylori-eradicated patients, new users of DOACs had a significantly lower risk of UGIB than new warfarin users. Furthermore, the risk of UGIB in new warfarin or DOACs users was comparable between H. pylori-eradicated and H. pylori-negative patients.     

Undefined xylose media extracted from biorefinery waste for enhanced and eco-friendly production of cephalosporin C by Acremonium chrysogenum M35

Xylose-rich undefined broth, extracted from the dilute acid pretreatment wastes of barley straw, serves as resourceful media for Acremonium chrysogenum M35 culture and production of cephalosporin C (CPC). Concentrating the extract with proper reprocessing enables to prepare various concentrations of xylose broth (2%–8%). The undefined xylose media were prepared for CPC production from A. chrysogenum M35 by the addition of other nutrients. Cell growth and CPC production were the most effective at 6% xylose and additional 2% glycerol, with maximum CPC production of 9.07 g/L after 6 days, which is higher production than that in defined media prepared with laboratory-level nutrients and reagents. Investigation of autotrophic and reverse trans-sulfuration pathways for cysteine synthesis, a limited element of three precursors for CPC synthesis, supports the enhanced CPC production in undefined media. Abundance of xylose ensures a maintained NADPH concentration required for sulfate reduction and synthesis of amino sulfide such as cysteine. Cystathionine-γ-lyase activity profiling indicated more efficient biosynthesis in undefined media than in other cultures use glycerol and glucose, and the biosynthesis pathway of CPC production by the cephalosporin gene cluster (i.e. pcbC and cefG genes) was investigated. The process using undefined xylose media was designed, and process simulation program confirmed our results.     

Human footprints in the Global South accelerate biomass carbon loss in ecologically sensitive regions

Human activities have placed significant pressure on the terrestrial biosphere, leading to ecosystem degradation and carbon losses. However, the full impact of these activities on terrestrial biomass carbon remains unexplored. In this study, we examined changes in global human footprint (HFP) and human-induced aboveground biomass carbon (AGBC) losses from 2000 to 2018. Our findings show an increasing trend in HFP globally, resulting in the conversion of wilderness areas to highly modified regions. These changes have altered global biomes' habitats, particularly in tropical and subtropical regions. We also found accelerated AGBC loss driven by HFP expansion, with a total loss of 19.99 ± 0.196 PgC from 2000 to 2018, especially in tropical regions. Additionally, AGBC is more vulnerable in the Global South than in the Global North. Human activities threaten natural habitats, resulting in increasing AGBC loss even in strictly protected areas. Therefore, scientifically guided planning of future human activities is crucial to protect half of Earth through mitigation and adaptation under future risks of climate change and global urbanization.     

Association between weekend catch-up sleep and the risk of depression among Korean middle-aged adults

Sleep and Biological Rhythms - Determining whether weekend catch-up sleep (CUS) is related to high risk of depression in the general middle-aged population in Korea. This study is a cross-sectional...     

Improvement in the thermostability of D-psicose 3-epimerase from Agrobacterium tumefaciens by random and site-directed mutagenesis

The S213C, I33L, and I33L S213C variants of D-psicose 3-epimerase from Agrobacterium tumefaciens, which were obtained by random and site-directed mutagenesis, displayed increases of 2.5, 5, and 7.5°C in the temperature for maximal enzyme activity, increases of 3.3-, 7.2-, and 29.9-fold in the half-life at 50°C, and increases of 3.1, 4.3, and 7.6°C in apparent melting temperature, respectively, compared with the wild-type enzyme. Molecular modeling suggests that the improvement in thermostability in these variants may have resulted from increased putative hydrogen bonds and formation of new aromatic stacking interactions. The immobilized wild-type enzyme with and without borate maintained activity for 8 days at a conversion yield of 70% (350 g/liter psicose) and for 16 days at a conversion yield of 30% (150 g/liter psicose), respectively. After 8 or 16 days, the enzyme activity gradually decreased, and the conversion yields with and without borate were reduced to 22 and 9.6%, respectively, at 30 days. In contrast, the activities of the immobilized I33L S213C variant with and without borate did not decrease during the operation time of 30 days. These results suggest that the I33L S213C variant may be useful as an industrial producer of D-psicose.