Temporal phenotypic variation of spinach root traits and its relation to shoot performance.

The root system is important for the growth and development of spinach. To reveal the temporal variability of the spinach root system, root traits of 40 spinach accessions were measured at three imaging times (20, 30, and 43 days after transplanting) in this study using a non-destructive and non-invasive root analysis system. Results showed that five root traits were reliably measured by this system (RootViz FS), and two of which were highly correlated with manually measured traits. Root traits had higher variations than shoot traits among spinach accessions, and the trait of mean growth rate of total root length had the largest coefficients of variation across the three imaging times. During the early stage, only tap root length was weakly correlated with shoot traits (plant height, leaf width, and object area (equivalent to plant surface area)), whereas in the third imaging, root fresh weight, total root length, and root area were strongly correlated with shoot biomass-related traits. Five root traits (total root length, tap root length, total root area, root tissue density, and maximal root width) showed high variations with coefficients of variation values (CV ≥ 0.3, except maximal root width) and high heritability (H2 > 0.6) among the three stages. The 40 spinach accessions were classified into five subgroups with different growth dynamics of the primary and lateral roots by cluster analysis. Our results demonstrated the potential of in-situ phenotyping to assess dynamic root growth in spinach and provide new perspectives for biomass breeding based on root system ideotypes.

Are vehicle tires major contributors to microplastic emissions into the China seas? A simple model perspective.

Microplastics pose a substantial threat to our environment. Given China's large population and rapid economic growth, it is urgent to estimate the annual emissions of microplastics into its marine environment. The microplastics show a significant variation in their source emissions as well as in their physical and chemical properties, leading to differences in their transport and fate in aquatic environments. To account for these variations, we developed a process-oriented model that considers microplastics from different sources and the inter-provincial variation in their retention rate to assess annual microplastic emissions into the China seas. On a national scale, of the microplastics emitted, 36.05 % are from household laundry activities, 27.26 % are from the wear and tear of vehicle tires, and 24.04 % are from the abrasion of plastic household items. After emission, 60.21 % are removed by wastewater treatment plants. The overall proportion of microplastics that end up in the marine environment highly depends on the specific riverine retention rate of microplastics from vehicle tires. Including the high settling rate of these microplastics, this proportion drops from 9.96 % to 3.29 %, rendering vehicle tires a minor contributor to microplastic emissions into the China seas compared to other sources. Moreover, when using the density-dependent approach and considering the east/west dimension of each province, the microplastic emissions from vehicle tires into the China seas decrease from 71 % to 5.27 %. This underscores the urgent need for global and regional models to account for the detailed riverine transport process of microplastics from vehicle tires in order to enhance the accuracy of their emission estimates into coastal waters.

Genome-driven discovery of new serrawettin W2 analogues from Serratia fonticola DSM 4576.

By expressing a multimodular NRPS gene sefA from Serratia fonticola DSM 4576 in E. coli, four new serrawettin W2 analogues, namely sefopeptides A-D (1-4), were isolated and structurally characterized and their biosynthesis was proposed. A bioactivity assay showed that sefopeptide C (3) exhibits moderate cytotoxic activity against acute promyelocytic leukemia NB4 cells.

Intracranial large artery embolism due to carotid thrombosis caused by a neck massager: A case report.

BACKGROUND: There are few reported cases of intracranial large artery embolism due to carotid thrombosis caused by a neck massager. Herein we report such a case. CASE SUMMARY: A 49-year-old woman presented with left limb weakness and dysarthria after a history of neck massage for 1 mo. Neurological examination showed left central facial paralysis and left hemiparesis with a National Institutes of Health Stroke Scale score of 12. Brain magnetic resonance imaging revealed restricted diffusion on diffusion-weighted imaging in the right parietal and temporal lobes. Computed tomography angiography (CTA) indicated M3 segment embolism of the right middle cerebral artery. Neck CTA revealed thrombosis of the bilateral common carotid arteries. Carotid ultrasound showed thrombosis in the bilateral common carotid arteries (approximately 2 cm below the proximal end of the carotid sinus), and contrast-enhanced ultrasound did not suggest enhancement. No hypertension, diabetes, heart disease, vasculitis, or thrombophilia was found after admission. After 1 wk of treatment with aspirin 200 mg and atorvastatin 40 mg, a carotid ultrasound reexamination showed that the thrombosis had significantly reduced. CONCLUSION: Neck massager may cause carotid artery thrombosis.

20-nor-Isopimarane and isopimarane diterpenoids produced by Aspergillus sp. WT03.

Five new uncommon 20-nor-isopimarane diterpenoids, aspewentins N-R (1-5), and three related known congeners (6-8), along with an isopimarane diterpenoid, sphaeropsidin C (9), were isolated from a Coptis chinensis Franch. rhizosphere soil-derived fungal strain, Aspergillus sp. WT03. The structures of compounds 1-9 were characterized based on the comprehensive analysis of the spectroscopic data, and their absolute configurations were elucidated by single crystal X-ray diffraction and time-dependent density functional theory (TDDFT)-ECD calculations. Compounds 1-5 represent rare examples of 20-nor-isopimarane analogues featuring a 1,2,3,4-tetrahydronaphthalene and 3,4-dihydronaphthalen-1(2H)-one moiety. The biosynthetic pathway of these diterpenoids was also proposed. Additionally, compounds 1, 3 and 4 showed moderate cytotoxic activity against MCF-7, A549 and HT-29 cell lines.

Engineering of Specific Single-Module Nonribosomal Peptide Synthetases of the RXP Type for the Production of Defined Peptides.

Rhabdopeptide/xenortide-like peptide (RXP) nonribosomal peptide synthetases (NRPSs) derived from entomophathogenic Xenorhabdus and Photorhabdus bacteria often produce libraries of different peptides varying in amino acid composition, number and degree of methylation, which mainly is a result of promiscuous docking domains (DDs) mediating protein-protein interactions between the different NRPS subunits. In this study, we present two specific RXP-NRPS systems with rather specific DDs that were used as platforms to generate a series of defined RXPs via the exchange of adenylation/methyltransferase (A-MT) domains in the systems followed by heterologous expression in Escherichia coli. Additionally, these results suggest that NRPS subunit interaction is not only exclusively dependent on DDs but at least partially also on A domains.

The Relationship Between Aortic Arch Calcification and Recurrent Stroke in Patients With Embolic Stroke of Undetermined Source-A Case-Control Study.

Background: Aortic arch calcification (AoAC) is associated with plaque development and cardiovascular events. We aimed to estimate the predictive value of AoAC for stroke recurrence in patients with embolic stroke of undetermined source (ESUS). Methods: Consecutive patients with ESUS who were admitted to our center between October 2019 and October 2020 and who had a 1-year follow-up of stroke recurrence were retrospectively reviewed. According to our AoAC grading scale (AGS), AoAC was classified into four grades based on chest computed tomography (CT) findings: no visible calcification (grade 0), spotty calcification (grade 1), lamellar calcification (grade 2), and circular calcification (grade 3). Results: Of the 158 patients with ESUS (age, 62.1 ± 14.5 years; 120 men) enrolled, 24 (15.2%) had recurrent stroke within a 1-year follow-up. The Cox regression analysis showed that stroke history [hazard ratio (HR), 4.625; 95% confidence interval (CI), 1.828-11.700, p = 0.001] and AoAC (HR, 2.672; 95% CI, 1.129-6.319; p = 0.025) predicted recurrent stroke. AGS grade 1 was associated with a significantly higher risk of stroke recurrence than AGS grade 0 (HR, 5.033; 95% CI, 1.858-13.635, p = 0.001) and AGS grade 2 plus 3 (HR, 3.388; 95% CI, 1.124-10.206, p = 0.030). In patients with AoAC, receiver operating characteristic (ROC) analysis showed that AGS had a good value in predicting stroke recurrence in patients with ESUS, with an area under curve (AUC) of 0.735 (95% CI = 0.601-0.869, p = 0.005). Conclusions: Aortic arch calcification, especially spotty calcification, had a good predictive value for stroke recurrence in patients with ESUS.

Characterization of Multifunctional and Non-stereoselective Oxidoreductase RubE7/IstO, Expanding the Functional Diversity of the Flavoenzyme Superfamily.

Flavin-dependent enzymes enable a broad range of redox transformations and generally act as monofunctional and stereoselective catalysts. Herein, we report the investigation of a multifunctional and non-stereoselective FMN-dependent oxidoreductase RubE7 from the rubrolone biosynthetic pathway. Our study outlines a single RubE7-catalysed sequential reduction of three spatially distinct bonds in a tropolone ring and a reversible double-bond reduction and dehydrogenation. The crystal structure of IstO (a RubE7 homologue) with 2.0 Šresolution reveals the location of the active site at the interface of two monomers, and the size of active site is large enough to permit both flipping and free rotation of the substrate, resulting in multiple nonselective reduction reactions. Molecular docking and site mutation studies demonstrate that His106 is oriented towards the substrate and is important for the reverse dehydrogenation reaction.

Antithrombin protects against Plasmodium falciparum histidine-rich protein II-mediated inflammation and coagulation.

Plasmodium falciparum-derived histidine-rich protein II (HRPII) has been shown to inhibit heparin-dependent anticoagulant activity of antithrombin (AT) and induce inflammation in vitro and in vivo. In a recent study, we showed that HRPII interacts with the AT-binding vascular glycosaminoglycans (GAGs) not only to disrupt the barrier-permeability function of endothelial cells but also to inhibit the antiinflammatory signaling function of AT. Here we investigated the mechanisms of the proinflammatory function of HRPII and the protective activity of AT in cellular and animal models. We found that AT competitively inhibits the GAG-dependent HRPII-mediated activation of NF-κB and expression of intercellular cell adhesion molecule 1 (ICAM1) in endothelial cells. Furthermore, AT inhibits HRPII-mediated histone H3 citrullination and neutrophil extracellular trap (NET) formation in HL60 cells and freshly isolated human neutrophils. In vivo, HRPII induced Mac1 expression on blood neutrophils, MPO release in plasma, neutrophil infiltration, and histone H3 citrullination in the lung tissues. HRPII also induced endothelial cell activation as measured by increased ICAM1 expression and elevated vascular permeability in the lungs. AT effectively inhibited HRPII-mediated neutrophil infiltration, NET formation, and endothelial cell activation in vivo. AT also inhibited HRPII-meditated deposition of platelets and fibrin(ogen) in the lungs and circulating level of von Willebrand factor in the plasma. We conclude that AT exerts protective effects against pathogenic effects of P falciparum-derived HRPII in both cellular and animal models.

Genomic analyses provide insights into spinach domestication and the genetic basis of agronomic traits.

Spinach is a nutritious leafy vegetable belonging to the family Chenopodiaceae. Here we report a high-quality chromosome-scale reference genome assembly of spinach and genome resequencing of 305 cultivated and wild spinach accessions. Reconstruction of ancestral Chenopodiaceae karyotype indicates substantial genome rearrangements in spinach after its divergence from ancestral Chenopodiaceae, coinciding with high repeat content in the spinach genome. Population genomic analyses provide insights into spinach genetic diversity and population differentiation. Genome-wide association studies of 20 agronomical traits identify numerous significantly associated regions and candidate genes for these traits. Domestication sweeps in the spinach genome are identified, some of which are associated with important traits (e.g., leaf phenotype, bolting and flowering), demonstrating the role of artificial selection in shaping spinach phenotypic evolution. This study provides not only insights into the spinach evolution and domestication but also valuable resources for facilitating spinach breeding.

Extracellular Histones Bind Vascular Glycosaminoglycans and Inhibit the Anti-Inflammatory Function of Antithrombin.

BACKGROUND/AIMS: Binding of histones to molecular pattern recognition receptors on endothelial cells and leukocytes provokes proinflammatory responses and promotes activation of coagulation. Histones also bind therapeutic heparins, thereby neutralizing their anticoagulant functions. The aim of this study was to test the hypothesis that histones can interact with the antithrombin (AT)-binding vascular glycosaminoglycans (GAGs) to induce inflammation and inhibit the anti-inflammatory function of AT. METHODS: We evaluated the heparin-binding function of histones by an AT-dependent protease-inhibition assay. Furthermore, we treated endothelial cells with histones in the absence and presence of AT and monitored cellular phenotypes employing established signaling assays. RESULTS: Histones neutralized AT-dependent anticoagulant function of heparin in both purified protease-inhibition and plasma-based assays. Histones also disrupted endothelial cell barrier-permeability function by a GAG-dependent mechanism as evidenced by the GAG-antagonist, surfen, abrogating their disruptive effects. Further studies revealed histones and AT compete for overlapping binding-sites on GAGs, thus increasing concentrations of one protein abrogated effects of the other. Histones elicited proapoptotic effects by inducing nuclear localization of PKC-δ in endothelial cells and barrier-disruptive effects by destabilizing VE-cadherin, which were inhibited by AT, but not by a D-helix mutant of AT incapable of interacting with GAGs. Finally, histones induced release of Weibel-Palade body contents, VWF and angiopoietin-2, and promoted expression of cell adhesion molecules on endothelial cells, which were all downregulated by AT but not by D-helix mutant of AT. CONCLUSION: We conclude that histones and AT compete for overlapping binding sites on vascular GAGs to modulate coagulation and inflammation.

Epsins Negatively Regulate Aortic Endothelial Cell Function by Augmenting Inflammatory Signaling.

Background: The endothelial epsin 1 and 2 endocytic adaptor proteins play an important role in atherosclerosis by regulating the degradation of the calcium release channel inositol 1,4,5-trisphosphate receptor type 1 (IP3R1). In this study, we sought to identify additional targets responsible for epsin-mediated atherosclerotic endothelial cell activation and inflammation in vitro and in vivo. Methods: Atherosclerotic ApoE-/- mice and ApoE-/- mice with an endothelial cell-specific deletion of epsin 1 on a global epsin 2 knock-out background (EC-iDKO/ApoE-/-), and aortic endothelial cells isolated from these mice, were used to examine inflammatory signaling in the endothelium. Results: Inflammatory signaling was significantly abrogated by both acute (tumor necrosis factor-α (TNFα) or lipopolysaccharide (LPS)) and chronic (oxidized low-density lipoprotein (oxLDL)) stimuli in EC-iDKO/ApoE-/- mice and murine aortic endothelial cells (MAECs) isolated from epsin-deficient animals when compared to ApoE-/- controls. Mechanistically, the epsin ubiquitin interacting motif (UIM) bound to Toll-like receptors (TLR) 2 and 4 to potentiate inflammatory signaling and deletion of the epsin UIM mitigated this interaction. Conclusions: The epsin endocytic adaptor proteins potentiate endothelial cell activation in acute and chronic models of atherogenesis. These studies further implicate epsins as therapeutic targets for the treatment of inflammation of the endothelium associated with atherosclerosis.

Ultranarrow and Tunable Fano Resonance in Ag Nanoshells and a Simple Ag Nanomatryushka.

We study theoretically the Fano resonances (FRs) produced by the near-field coupling between the lowest-order (dipolar) sphere plasmon resonance and the dipolar cavity plasmon mode supported by an Ag nanoshell or the hybrid mode in a simple three-layered Ag nanomatryushka constructed by incorporating a solid Ag nanosphere into the center of Ag nanoshell. We find that the linewidth of dipolar cavity plasmon resonance or hybrid mode induced FR is as narrow as 6.8 nm (corresponding to a high Q-factor of ~160 and a long dephasing time of ~200 fs) due to the highly localized feature of the electric-fields. In addition, we attribute the formation mechanisms of typical asymmetrical Fano line profiles in the extinction spectra to the constructive (Fano peak) and the destructive interferences (Fano dip) arising from the symmetric and asymmetric charge distributions between the dipolar sphere and cavity plasmon or hybrid modes. Interestingly, by simply adjusting the structural parameters, the dielectric refractive index required for the strongest FR in the Ag nanomatryushka can be reduced to be as small as 1.4, which largely reduces the restriction on materials, and the positions of FR can also be easily tuned across a broad spectral range. The ultranarrow linewidth, highly tunability together with the huge enhancement of electric fields at the FR may find important applications in sensing, slow light, and plasmon rulers.

A single-domain small protein Med-ORF10 regulates the production of antitumour agent medermycin in Streptomyces.

Med-ORF10, a single-domain protein with unknown function encoded by a gene located in a gene cluster responsible for the biosynthesis of a novel antitumour antibiotic medermycin, shares high homology to a group of small proteins widely distributed in many aromatic polyketide antibiotic pathways. This group of proteins contain a nuclear transport factor-2 (NTF-2) domain and appear to undergo an evolutionary divergence in their functions. Gene knockout and interspecies complementation suggested that Med-ORF10 plays a regulatory role in medermycin biosynthetic pathway. Overexpression of med-ORF10 in its wild-type strain led to significant increase of medermycin production. It was also shown by qRT-PCR and Western blot that Med-ORF10 controls the expression of genes encoding tailoring enzymes involved in medermycin biosynthesis. Transcriptome analysis and qRT-PCR revealed that Med-ORF10 has pleiotropic effects on more targets. However, there is no similar conserved domain available in Med-ORF10 compared to those of mechanistically known regulatory proteins; meanwhile, no direct interaction between Med-ORF10 and its target promoter DNA was detected via gel shift assay. All these studies suggest that Med-ORF10 regulates medermycin biosynthesis probably via an indirect mode.

Identification of a C-Glycosyltransferase Involved in Medermycin Biosynthesis.

C-Glycosylation in the biosynthesis of bioactive natural products is quite unique, which has not been studied well. Medermycin, as an antitumor agent in the family of pyranonaphthoquinone antibiotics, is featured with unique C-glycosylation. Here, a new C-glycosyltransferase (C-GT) Med-8 was identified to be essential for the biosynthesis of medermycin, as the first example of C-GT to recognize a rare deoxyaminosugar (angolosamine). med-8 and six genes (med-14, -15, -16, -17, -18, and -20 located in the medermycin biosynthetic gene cluster) predicted for the biosynthesis of angolosamine were proved to be functional and sufficient for C-glycosylation. A C-glycosylation cassette composed of these seven genes could convert a proposed substrate into a C-glycosylated product. In conclusion, these genes involved in the C-glycosylation of medermycin were functionally identified and biosynthetically engineered, and they provided the possibility of producing new C-glycosylated compounds.

Thrombomodulin is essential for maintaining quiescence in vascular endothelial cells.

Thrombomodulin (TM) is a thrombin receptor on endothelial cells that is involved in promoting activation of the anticoagulant protein C pathway during blood coagulation. TM also exerts protective anti-inflammatory properties through a poorly understood mechanism. In this study, we investigated the importance of TM signaling to cellular functions by deleting it from endothelial cells by CRISPR-Cas9 technology and analyzed the resultant phenotype of TM-deficient (TM-/- ) cells. Deficiency of TM in endothelial cells resulted in increased basal permeability and hyperpermeability when stimulated by thrombin and TNF-α. The loss of the basal barrier permeability function was accompanied by increased tyrosine phosphorylation of VE-cadherin and reduced polymerization of F-actin filaments at cellular junctions. A significant increase in basal NF-κB signaling and expression of inflammatory cell adhesion molecules was observed in TM-/- cells that resulted in enhanced adhesion of leukocytes to TM-/- cells in flow chamber experiments. There was also a marked increase in expression, storage, and release of the von Willebrand factor (VWF) and decreased storage and release of angiopoietin-2 in TM-/- cells. In a flow chamber assay, isolated platelets adhered to TM-/- cells, forming characteristic VWF-platelet strings. Increased VWF levels and inflammatory foci were also observed in the lungs of tamoxifen-treated ERcre-TMf/f mice. Reexpression of the TM construct in TM-/- cells, but not treatment with soluble TM, normalized the cellular phenotype. Based on these results, we postulate cell-bound TM endows a quiescent cellular phenotype by tightly regulating expression of procoagulant, proinflammatory, and angiogenic molecules in vascular endothelial cells.

Protective Role of Activated Protein C against Viral Mimetic Poly(I:C)-Induced Inflammation.

Activated protein C (APC) is an anticoagulant plasma serine protease which exhibits potent cytoprotective and anti-inflammatory activities. Here, we studied protective effects of APC on the proinflammatory function of polyinosinic:polycytidylic acid [poly(I:C)], a synthetic analog of viral double-stranded RNA, in cellular and animal models. Poly(I:C) induced histone H3 extranuclear translocation via interaction with toll-like receptor 3 in two established endothelial cell lines. Furthermore, poly(I:C) induced histone H3 extranuclear translocation in J774A.1 macrophages and human neutrophils and formation of macrophage and neutrophil extracellular traps (ETs). Mechanistically, poly(I:C) was found to upregulate expression of peptidylarginine deiminase 4 and enhance its interaction with histone H3, thereby leading to increased histone citrullination and neutrophil ET formation. Poly(I:C) elicited proinflammatory signaling responses by inducing nuclear factor kappa B activation and disrupting endothelial cell permeability. In vivo, poly(I:C) enhanced cell surface expression of Mac-1 on neutrophils in mice and facilitated their infiltration to lung tissues. Poly(I:C) also downregulated thrombomodulin expression in mouse tissues and reduced its circulating soluble level in plasma. We demonstrate in this study that APC and a signaling-selective mutant of APC effectively inhibit proinflammatory signaling effects of poly(I:C) in both cellular and animal models. We further demonstrate that unlike the requirement for endothelial protein C receptor on endothelial cells, the integrin Mac-1 is involved in the protease-activated receptor 1-dependent APC inhibition of macrophage ET formation in J774A.1 cells. Taken together, these results support a key role for APC signaling in inhibiting the viral mimetic-induced proinflammatory signaling responses and histone translocation-associated formation of ETs by innate immune cells.

Identification and characterization of the NPF, NRT2 and NRT3 in spinach.

Nitrate transporters (NRTs) participate in nitrate uptake, transport and allocation within the plant. However, this gene family has not been studied thoroughly in spinach. This study provided the general information about spinach SoNRTs and their transcriptional and functional responses to different levels of nitrate supplies. Resultes showed that fifty-seven NPF (also known as NRT1), nine NRT2 and one NRT3 were identified in spinach homologous to characterized Arabidopsis NRT genes. Phylogenetic analysis organized the SoNRT family into three clades: NPF with three subclades, NRT2, and NRT3. The tested SoNRT genes showed the various expression profiles in relation to tissue specificity and nitrate supply, indicating their functional diversity in response to external nitrate supply. Among them, transgenic Arabidopsis plants overexpressing SoNPF30 showed improved biomass, decreased shoot nitrate contents but no significant difference of 15NO3- uptake rates when compared with those of the wild type in response to high N treatment. Under low N treatment, overexpressing of SoNRT3 significantly increased whole plant biomass, root nitrate contents and 15NO3- uptake rates. These demonstrated that SoNPF30 and SoNRT3 confer greater capacity for nitrate translocation or nitrate uptake, and could help to improve the ability of plant nitrogen utilization under those conditions. Our findings provide a valuable basis for future research on this family of genes in spinach.