The entry reaction of the plant shikimate pathway is subjected to highly complex metabolite-mediated regulation.

The plant shikimate pathway directs bulk carbon flow toward biosynthesis of aromatic amino acids (AAAs, i.e. tyrosine, phenylalanine, and tryptophan) and numerous aromatic phytochemicals. The microbial shikimate pathway is feedback inhibited by AAAs at the first enzyme, 3-deoxy-d-arabino-heptulosonate 7-phosphate synthase (DHS). However, AAAs generally do not inhibit DHS activities from plant extracts and how plants regulate the shikimate pathway remains elusive. Here, we characterized recombinant Arabidopsis thaliana DHSs (AthDHSs) and found that tyrosine and tryptophan inhibit AthDHS2, but not AthDHS1 or AthDHS3. Mixing AthDHS2 with AthDHS1 or 3 attenuated its inhibition. The AAA and phenylpropanoid pathway intermediates chorismate and caffeate, respectively, strongly inhibited all AthDHSs, while the arogenate intermediate counteracted the AthDHS1 or 3 inhibition by chorismate. AAAs inhibited DHS activity in young seedlings, where AthDHS2 is highly expressed, but not in mature leaves, where AthDHS1 is predominantly expressed. Arabidopsis dhs1 and dhs3 knockout mutants were hypersensitive to tyrosine and tryptophan, respectively, while dhs2 was resistant to tyrosine-mediated growth inhibition. dhs1 and dhs3 also had reduced anthocyanin accumulation under high light stress. These findings reveal the highly complex regulation of the entry reaction of the plant shikimate pathway and lay the foundation for efforts to control the production of AAAs and diverse aromatic natural products in plants.

Derivation of pancreatic acinar cell carcinoma cell line HS-1 as a patient-derived tumor organoid.

Acinar cell carcinoma (ACC) of the pancreas is a malignant tumor of the exocrine cell lineage with a poor prognosis. Due to its rare incidence and technical difficulties, few authentic human cell lines are currently available, hampering detailed investigations of ACC. Therefore, we applied the organoid culture technique to various types of specimens, such as bile, biopsy, and resected tumor, obtained from a single ACC patient. Despite the initial propagation, none of these organoids achieved long-term proliferation or tolerated cryopreservation, confirming the challenging nature of establishing ACC cell lines. Nevertheless, the biopsy-derived early passage organoid developed subcutaneous tumors in immunodeficient mice. The xenograft tumor histologically resembled the original tumor and gave rise to infinitely propagating organoids with solid features and high levels of trypsin secretion. Moreover, the organoid stained positive for carboxylic ester hydrolase, a specific ACC marker, but negative for the duct cell marker CD133 and the endocrine lineage marker synaptophysin. Hence, we concluded the derivation of a novel ACC cell line of the pure exocrine lineage, designated HS-1. Genomic analysis revealed extensive copy number alterations and mutations in EP400 in the original tumor, which were enriched in primary organoids. HS-1 displayed homozygous deletion of CDKN2A, which might underlie xenograft formation from organoids. Although resistant to standard cytotoxic agents, the cell line was highly sensitive to the proteasome inhibitor bortezomib, as revealed by an in vitro drug screen and in vivo validation. In summary, we document a novel ACC cell line, which could be useful for ACC studies in the future.

The utility of the self-controlled study design for pharmacoepidemiological studies without an active comparator medication using a medical information database: An application to assess the risk of varenicline on cardiovascular outcomes.

PURPOSE: To illustrate the utility of the self-controlled study design for studies without an active comparator, we compared the results of a cohort design study with a non-user comparator with those of a self-controlled design study in evaluating the risk of varenicline on cardiovascular outcomes, using a Japanese medical claims database. METHODS: The participating smokers were identified from health-screening results collected between May 2008 and April 2017. Using a non-user-comparator cohort study design, we estimated the hazard ratios (HRs) and 95% confidence intervals (CIs) of varenicline on initial hospitalization with cardiovascular outcomes using Cox's model adjusted for patients' sex, age, medical history, medication history, and health-screening results. Using a self-controlled study design, the within-subject HR was estimated using a stratified Cox's model adjusted for medical history, medication history, and health-screening results. The estimate from a recent meta-analysis was considered the gold standard (risk ratio: 1.03). RESULTS: We identified 460 464 smokers (398 694 males [86.6%]; mean (standard deviation) age: 42.9 [10.8] years) in the database. Of these, 11 561 had been dispensed varenicline at least once, and 4511 had experienced cardiovascular outcomes. The estimate of the non-user-comparator cohort study design exceeded the gold standard (HR [95% CI]: 2.04 [1.22-3.42]), whereas that of the self-controlled study design was close to the gold standard (within-subject HR [95% CI]: 1.12 [0.27-4.70]). CONCLUSIONS: The self-controlled study design is useful alternative to a non-user-comparator cohort design when evaluating the risk of medications relative to their non-use, based on a medical information database.

Prevalence and Risk Factors of Silent Cerebral Microbleeds in Patients with Coronary Artery Disease.

OBJECTIVES: Cerebral microbleeds (CMBs), which can be detected by gradient-echo T2*-weighted magnetic resonance imaging (MRI), represent small chronic brain hemorrhages caused by structural abnormalities in cerebral small vessels. CMBs are known to be a potential predictor of future stroke, and are associated with age, various cardiovascular risk factors, cognitive impairment, and the use of antithrombotic drugs. Patients with coronary artery disease (CAD) are at potentially high risk of CMBs due to the presence of coexistent conditions. However, little is known about CMBs in patients with CAD. We aimed to identify the factors associated with the presence of CMBs among patients with CAD. METHODS: We evaluated 356 consecutive patients [mean age, 72 ± 10 years; men = 276 (78%)] with angiographically proven CAD who underwent T2*-weighted brain MRI. The brain MRI was assessed by researchers blinded to the patients' clinical details. RESULTS: CMBs were found in 128 (36%) patients. Among 356 patients, 119 (33%) had previously undergone percutaneous coronary intervention (PCI), and 26 (7%) coronary artery bypass grafting (CABG). There was no significant relationship between CMBs and sex, hypertension, dyslipidemia, diabetes mellitus, anticoagulation therapy, antiplatelet therapy, or prior PCI. CMBs were significantly associated with advanced age, previous CABG, eGFR, non-HDL cholesterol, carotid artery disease, long-term antiplatelet therapy, and long-term dual antiplatelet therapy (DAPT) using univariate logistic regression analysis. The multivariate logistic regression analysis showed that long-term antiplatelet therapy (odds ratio, 1.73; 95% CI, 1.06 - 2.84; P = 0.03) or long-term DAPT (odds ratio, 2.92; 95% CI, 1.39 - 6.17; P = 0.004) was significantly associated with CMBs after adjustment for confounding variables. CONCLUSIONS: CMBs were frequently observed in patients with CAD and were significantly associated with long-term antiplatelet therapy, especially long-term DAPT.

Grana-Localized Proteins, RIQ1 and RIQ2, Affect the Organization of Light-Harvesting Complex II and Grana Stacking in Arabidopsis.

Grana are stacked thylakoid membrane structures in land plants that contain PSII and light-harvesting complex II proteins (LHCIIs). We isolated two Arabidopsis thaliana mutants, reduced induction of non-photochemical quenching1 (riq1) and riq2, in which stacking of grana was enhanced. The curvature thylakoid 1a (curt1a) mutant was previously shown to lack grana structure. In riq1 curt1a, the grana were enlarged with more stacking, and in riq2 curt1a, the thylakoids were abnormally stacked and aggregated. Despite having different phenotypes in thylakoid structure, riq1, riq2, and curt1a showed a similar defect in the level of nonphotochemical quenching of chlorophyll fluorescence (NPQ). In riq curt1a double mutants, NPQ induction was more severely affected than in either single mutant. In riq mutants, state transitions were inhibited and the PSII antennae were smaller than in wild-type plants. The riq defects did not affect NPQ induction in the chlorophyll b-less mutant. RIQ1 and RIQ2 are paralogous and encode uncharacterized grana thylakoid proteins, but despite the high level of identity of the sequence, the functions of RIQ1 and RIQ2 were not redundant. RIQ1 is required for RIQ2 accumulation, and the wild-type level of RIQ2 did not complement the NPQ and thylakoid phenotypes in riq1 We propose that RIQ proteins link the grana structure and organization of LHCIIs.

Understanding source-sink interactions: Progress in model plants and translational research to crops.

Agriculture is facing a massive increase in demand per hectare as a result of an ever-expanding population and environmental deterioration. While we have learned much about how environmental conditions and diseases impact crop yield, until recently considerably less was known concerning endogenous factors, including within-plant nutrient allocation. In this review, we discuss studies of source-sink interactions covering both fundamental research in model systems under controlled growth conditions and how the findings are being translated to crop plants in the field. In this respect we detail efforts aimed at improving and/or combining C3, C4, and CAM modes of photosynthesis, altering the chloroplastic electron transport chain, modulating photorespiration, adopting bacterial/algal carbon-concentrating mechanisms, and enhancing nitrogen- and water-use efficiencies. Moreover, we discuss how modulating TCA cycle activities and primary metabolism can result in increased rates of photosynthesis and outline the opportunities that evaluating natural variation in photosynthesis may afford. Although source, transport, and sink functions are all covered in this review, we focus on discussing source functions because the majority of research has been conducted in this field. Nevertheless, considerable recent evidence, alongside the evidence from classical studies, demonstrates that both transport and sink functions are also incredibly important determinants of yield. We thus describe recent evidence supporting this notion and suggest that future strategies for yield improvement should focus on combining improvements in each of these steps to approach yield optimization.

Coordinated regulation of the entry and exit steps of aromatic amino acid biosynthesis supports the dual lignin pathway in grasses.

Vascular plants direct large amounts of carbon to produce the aromatic amino acid phenylalanine to support the production of lignin and other phenylpropanoids. Uniquely, grasses, which include many major crops, can synthesize lignin and phenylpropanoids from both phenylalanine and tyrosine. However, how grasses regulate aromatic amino acid biosynthesis to feed this dual lignin pathway is unknown. Here we show, by stable-isotope labeling, that grasses produce tyrosine >10-times faster than Arabidopsis without compromising phenylalanine biosynthesis. Detailed in vitro enzyme characterization and combinatorial in planta expression uncovered that coordinated expression of specific enzyme isoforms at the entry and exit steps of the aromatic amino acid pathway enables grasses to maintain high production of both tyrosine and phenylalanine, the precursors of the dual lignin pathway. These findings highlight the complex regulation of plant aromatic amino acid biosynthesis and provide novel genetic tools to engineer the interface of primary and specialized metabolism in plants.

Construction of a URA3 deletion strain from the allotetraploid bottom-fermenting yeast Saccharomyces pastorianus.

The bottom-fermenting lager yeast Saccharomyces pastorianus has been proposed to be allotetraploid, containing two S. cerevisiae (Sc)-type and two S. bayanus (Sb)-type chromosomes. This chromosomal constitution likely explains why recessive mutants of S. pastorianus have not previously been reported. Here we describe the construction of a ura3 deletion strain derived from the lager strain Weihenstephan34/70 by targeted transformation and subsequent loss of heterozygosity (LOH). Initially, deletion constructs of the Sc and Sb types of URA3 were constructed in laboratory yeast strains in which a TDH3p-hygro allele conferring hygromycin B resistance replaced ScURA3 and a KanMX cassette conferring G-418 resistance replaced SbURA3. The lager strain was then transformed with these constructs to yield a heterozygous URA3 disruptant (ScURA3⁺/Scura3Δ::TDH3p-hygro, SbURA3⁺/Sbura3Δ::KanMX), which was plated on 5-fluoroorotic acid (5-FOA) plates to generate the desired Ura⁻ homozygous disruptant (Scura3Δ::TDH3p-hygro/Scura3Δ::TDH3p-hygro Sbura3Δ::KanMX/Sbura3Δ::KanMX) through LOH. This ura3 deletion strain was then used to construct a bottom-fermenting yeast transformant overexpressing ATF1 that encodes an enzyme that produces acetate esters. The ATF1-overexpressing transformant produced significantly more acetate esters than the parent strain. The constructed ura3∆ lager strain will be a useful host for constructing strains of relevance to brewing.

3-Deoxy-D-arabino-heptulosonate 7-phosphate synthase as the gatekeeper of plant aromatic natural product biosynthesis.

The shikimate pathway connects the central carbon metabolism with the biosynthesis of aromatic amino acids-l-tyrosine, l-phenylalanine, and l-tryptophan-which play indispensable roles as precursors of numerous aromatic phytochemicals. Despite the importance of the shikimate pathway-derived products for both plant physiology and human society, the regulatory mechanism of the shikimate pathway remains elusive. This review summarizes the recent progress and current understanding on the plant 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase (DAHP synthase or DHS) enzymes that catalyze the committed reaction of the shikimate pathway. We particularly focus on how the DHS activity is regulated in plants in comparison to those of microbes and discuss potential roles of DHS as the critical gatekeeper for the production of plant aromatic compounds.

Point mutations that boost aromatic amino acid production and CO2 assimilation in plants.

Aromatic compounds having unusual stability provide high-value chemicals and considerable promise for carbon storage. Terrestrial plants can convert atmospheric CO2 into diverse and abundant aromatic compounds. However, it is unclear how plants control the shikimate pathway that connects the photosynthetic carbon fixation with the biosynthesis of aromatic amino acids, the major precursors of plant aromatic natural products. This study identified suppressor of tyra2 (sota) mutations that deregulate the first step in the plant shikimate pathway by alleviating multiple effector-mediated feedback regulation in Arabidopsis thaliana. The sota mutant plants showed hyperaccumulation of aromatic amino acids accompanied by up to a 30% increase in net CO2 assimilation. The identified mutations can be used to enhance plant-based, sustainable conversion of atmospheric CO2 to high-energy and high-value aromatic compounds.

A new mechanism for reduced cell adhesion: Adsorption dynamics of collagen on a nanoporous gold surface.

Nanostructured materials such as nanoparticles and nanoporous materials strongly affect cell behaviors such as cell viability. Because cellular uptake of nanoporous materials does not occur, mechanisms for the effects of nanoporous materials on cells are different from those of nanoparticles. The effects of nanoporous materials on cells are thought to result from large conformational changes in the extracellular matrix (ECM) induced by the nanoporous materials, although the mechanotransduction and the critical focal adhesion cluster size also have an effect on the cell response. However, we show that the adhesion of mesenchymal stem cells to a gold surface is reduced for nanoporous gold (NPG), despite the conformational changes in collagen induced by NPG being below the detection limits of the experimental analyses. The adsorption dynamics of collagen on NPG are investigated by molecular dynamics simulations to determine the origin of the reduced cell adhesion to NPG. The adsorption energy of collagen on NPG is lower than that on flat gold (FG) despite there being little difference between the global conformation of collagen segments adsorbed on NPG compared with FG. This finding is related to the surface strain of NPG and the limited movement of collagen amino acids owing to interchain hydrogen bonds. The results obtained in this study provide new insight into the interactions between nanostructured materials and the ECM.

Characterization of multiple alternative forms of heterogeneous nuclear ribonucleoprotein K by phosphate-affinity electrophoresis.

The phosphorylation of heterogeneous nuclear ribonucleoprotein K (hnRNP K) is thought to play an important role in cell regulation and signal transduction. However, the relationship between hnRNP K phosphorylation and cellular events has only been indirectly examined, and the phosphorylated forms of endogenous hnRNP K have not been biochemically characterized in detail. In this study, we extensively examined the phosphorylated forms of endogenous hnRNP K by direct protein-chemical characterization using phosphate-affinity electrophoresis followed by immunoblotting and MS. Phosphate-affinity electrophoresis enabled us to sensitively detect and separate the phosphorylated forms of hnRNP K. When we used 2-DE with phosphate-affinity SDS-PAGE in the second dimension, the nuclear fraction contained more than 20 spots of endogenous hnRNP K on the 2-D map. We determined that the multiple forms of hnRNP K were produced mainly by alternative splicing of the single hnRNP K gene and phosphorylation of Ser116 and/or Ser284. Furthermore, the subcellular localization of these proteins revealed by the 2-D gel correlated with their phosphorylation states and alternative splicing patterns. The results also indicated that the multiple forms of hnRNP K were differentially modulated in response to external stimulation with bacterial lipopolysaccharide or serum.

Isoelectric focusing of high-molecular-weight protein complex under native conditions using agarose gel.

The isolation and characterization of protein complexes are essential steps toward understanding cellular functions. A method for separating and characterizing high-molecular-weight protein complexes using two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) with native agarose gel isoelectric focusing (IEF) is described. Using this method, fractions containing high-molecular-weight protein complexes were analyzed. The advantages of using native agarose gel IEF include the ability to concentrate the protein complexes and the ease of handling when performing 2D separations. Although limited with respect to the size of molecules and particles that may be separated, this method is useful for the isolation and characterization of high-molecular-weight protein complexes.

Cardiac Regeneration by Statin-Polymer Nanoparticle-Loaded Adipose-Derived Stem Cell Therapy in Myocardial Infarction.

Clinical trials with autologous adipose-derived stem cell (AdSC) therapy for ischemic heart diseases (IHDs) are ongoing. However, little is known about combinational therapeutic effect of AdSCs and statin poly(lactic-co-glycolic) acid (PLGA) nanoparticles on the ischemic myocardium. We investigated the hypothesis that statins, which have pleiotropic effects, augment the therapeutic potential of AdSCs and that AdSCs also act as drug delivery tools. Simvastatin-conjugated nanoparticles (SimNPs) significantly promoted migration activity without changing proliferation activity and upregulated growth factor gene expression in vitro. A small number of intravenously administered SimNP-loaded AdSCs (10,000 cells per mouse) improved cardiac function following myocardial infarction, inducing endogenous cardiac regeneration in the infarcted myocardium. The de novo regenerated myocardium was thought to be derived from epicardial cells, which were positive for Wilms' tumor protein 1 expression. These findings were attributed to the sustained, local simvastatin release from the recruited SimNP-loaded AdSCs in the infarcted myocardium rather than to the direct contribution of recruited AdSCs to tissue regeneration. SimNP-loaded AdSCs may lead to a novel somatic stem cell therapy for IHDs. Stem Cells Translational Medicine 2019;8:1055-1067.

Genome-wide identification and characterization of transcripts translationally regulated by bacterial lipopolysaccharide in macrophage-like J774.1 cells.

Although Escherichia coli LPS is known to elicit various proinflammatory responses in macrophages, its effect on the translational states of transcripts has not yet been explored on a genome-wide scale. To address this, we investigated the mRNA profiles in polysomal and free messenger ribonucleoprotein particle (mRNP) fractions of mouse macrophage-like J774.1 cells, using Affymetrix Mouse Genome 430 2.0 GeneChips. Comparison of the mRNA profiles in total cellular, polysomal, and free mRNP fractions enabled us to identify transcripts that were modulated at the translational level by LPS: among 19,791 transcripts, 115 and 418 were up- and downregulated at 1, 2, or 4 h after LPS stimulation (100 ng/ml) in a translation-dependent manner. Interestingly, gene ontology-based analysis suggested that translation-dependent downregulated genes frequently include those encoding proteins in the mitochondrial respiratory chain. In fact, the mRNA levels of some transcripts for complexes I, IV, and V in the mitochondrial respiratory chain were translationally downregulated, eventually contributing to the decline of their protein levels. Moreover, the amount of metabolically labeled cytochrome oxidase subunit Va in complex IV was decreased without any change of its mRNA level in total cellular fraction after LPS stimulation. Consistently, the total amounts and activities of complexes I and IV were attenuated by LPS stimulation, and the attenuation was independent of nitric oxide. These results demonstrated that translational suppression may play a critical role in the LPS-mediated attenuation of mitochondrial oxidative phosphorylation in a nitric oxide-independent manner in J774.1 cells.