The C4 photosynthesis bifunctional enzymes, PDRPs, of maize are co-opted to cytoplasmic viral replication complexes to promote infection of a prevalent potyvirus sugarcane mosaic virus.

In maize, two pyruvate orthophosphate dikinase (PPDK) regulatory proteins, ZmPDRP1 and ZmPDRP2, are respectively specific to the chloroplast of mesophyll cells (MCs) and bundle sheath cells (BSCs). Functionally, ZmPDRP1/2 catalyse both phosphorylation/inactivation and dephosphorylation/activation of ZmPPDK, which is implicated as a major rate-limiting enzyme in C4 photosynthesis of maize. Our study here showed that maize plants lacking ZmPDRP1 or silencing of ZmPDRP1/2 confer resistance to a prevalent potyvirus sugarcane mosaic virus (SCMV). We verified that the C-terminal domain (CTD) of ZmPDRP1 plays a key role in promoting viral infection while independent of enzyme activity. Intriguingly, ZmPDRP1 and ZmPDRP2 re-localize to cytoplasmic viral replication complexes (VRCs) following SCMV infection. We identified that SCMV-encoded cytoplasmic inclusions protein CI targets directly ZmPDRP1 or ZmPDRP2 or their CTDs, leading to their re-localization to cytoplasmic VRCs. Moreover, we found that CI could be degraded by the 26S proteasome system, while ZmPDRP1 and ZmPDRP2 could up-regulate the accumulation level of CI through their CTDs by a yet unknown mechanism. Most importantly, with genetic, cell biological and biochemical approaches, we provide evidence that BSCs-specific ZmPDRP2 could accumulate in MCs of Zmpdrp1 knockout (KO) lines, revealing a unique regulatory mechanism crossing different cell types to maintain balanced ZmPPDK phosphorylation, thereby to keep maize normal growth. Together, our findings uncover the genetic link of the two cell-specific maize PDRPs, both of which are co-opted to VRCs to promote viral protein accumulation for robust virus infection.

Clinical outcomes of combined catheter ablation and left atrial appendage closure in elderly patients with nonvalvular atrial fibrillation.

BACKGROUND: Catheter ablation (CA) combined with left atrial appendage closure (LAAC) has emerged as a promising method to relieve symptoms while reducing the incidence of stroke in selected high-risk patients with atrial fibrillation (AF). HYPOTHESIS: We aimed to investigate the clinical outcomes of combined CA and LAAC in elderly patients. METHODS: A total of 316 patients with symptomatic drug-refractory AF who underwent combined CA and LAAC between January 2016 and December 2020 were retrospectively included. Baseline characteristics, periprocedural complications, and clinical events during follow-up were recorded and compared between patients aged ≥ 75 years (n = 66) and <75 years (n = 250). RESULTS: Pulmonary vein isolation and satisfactory LAAC were achieved in all patients. No patients experienced death or stroke/transient ischemic stroke periprocedurally. After a median follow-up of 12.2 (6.7-24.4) months and 11.9 (5.5-23.6) months, the rate of sinus rhythm maintenance was comparable between the two groups (≥75 years: 78.8% vs. <75 years: 80.8%; log-rank test, p = 0.674). The median follow-up periods for clinical outcomes were 27.9 (9.3-44.8) months and 25.2 (10.8-45.7) months, respectively. In patients aged ≥ 75 years, one suffered ischemic stroke, and one experienced major bleeding event. In patients aged < 75 years, four had ischemic stroke, and eight had major bleeding events. Two patients aged < 75 years died during follow-up, while none of the patients aged ≥ 75 years died. CONCLUSIONS: Combining CA and LAAC was feasible, safe and effective in elderly patients with AF.

PRMT5 knockdown enhances cell viability and suppresses cell apoptosis, oxidative stress, inflammation and endothelial dysfunction in ox-LDL-induced vascular endothelial cells via interacting with PDCD4.

Atherosclerosis (AS) is an important pathological basis of cardiovascular disease (CVD). The development of AS commences with endothelial dysfunction due to vascular endothelial cell injury. It is well documented that protein arginine methyltransferase 5 (PRMT5) is highly related to cardiovascular events. BioGRID database analysis indicates that PRMT5 may interact with programmed cell death 4 (PDCD4), which is reported to be involved in AS progression. This present research was formulated to elucidate the biological roles of PRMT5/PDCD4 in vascular endothelial cell injury during AS. In this current work, HUVECs were stimulated with 100 mg/L ox-LDL for 48 h to construct an in vitro AS model. Expression levels of PRMT5 and PDCD4 were analyzed by performing RT-qPCR and western blot. The viability and apoptosis of HUVECs were determined using CCK-8, flow cytometry and western blot assays. The status of oxidative stress and inflammation was assessed via commercial detection kits and ELISA assay, respectively. Besides, biomarkers of endothelial dysfunction were detected via commercial detection kit and western blot assay. In addition, the interacting relationship between PRMT5 and PDCD4 was verified by Co-IP assay. Highly expressed PRMT5 was observed in ox-LDL-stimulated HUVECs. Knockdown of PRMT5 enhanced the viability and inhibited the apoptosis of ox-LDL-induced HUVECs as well as alleviated ox-LDL-triggered oxidative stress, inflammation and endothelial dysfunction in HUVECs. PRMT5 interacted and bound with PDCD4. Furthermore, the enhancing effect on cell viability as well as the suppressing effects on cell apoptosis, oxidative stress, inflammation and endothelial dysfunction of PRMT5 knockdown in ox-LDL-induced HUVECs were partially abolished upon up-regulation of PDCD4. To conclude, down-regulation of PRMT5 might exert protective effects against vascular endothelial cell injury during AS by suppressing PDCD4 expression.

Effects of food-derived oligopeptide iron chelates on liver injury and gut microbiota homeostasis in iron-deficiency anemia female rats: a pilot study.

Iron deficiency (ID) is the biggest cause of anemia. This pilot study aimed to investigate the effects of food-derived oligopeptide iron chelates on ameliorating liver injury and restoring gut microbiota homeostasis in iron-deficiency anemia (IDA) female rats. Female Sprague-Dawley rats at 21 days old were selected and randomly divided into a control group (N = 4) and an ID model group (N = 16). The ID model group was fed an iron-deficient diet containing 4 mg kg-1 iron for 28 days to generate the IDA rat model and then randomly subdivided into four groups (N = 4 for each group): ID group, ferrous sulfate group, marine fish oligopeptide iron chelate (MCOP-Fe) group, and whey protein oligopeptide iron chelate (WPP-Fe) group. Iron supplements were given to rats in the three intervention groups once per day via intragastric administration for three weeks. After iron supplementation, the hemoglobin levels in the three intervention groups were significantly improved, with the MCOP-Fe and WPP-Fe groups returning to normal. The ALT and AST levels in the ID group increased significantly, while levels in all intervention groups decreased to normal levels. Liver glutathione in the WPP-Fe group was increased, while the activity of superoxide dismutase also tended to be higher. In addition, 16S rRNA gene sequencing showed that IDA resulted in changes to intestinal microbiota. After intervention, the WPP-Fe group showed increased alpha diversity of intestinal microbes. Therefore, MCOP-Fe and WPP-Fe may improve the iron status of IDA female rats as well as ameliorate liver damage, with WPP-Fe showing a greater potential in improving gut microbiota imbalance.

HEAT SHOCK PROTEIN 90.6 interacts with carbon and nitrogen metabolism components during seed development.

Carbon and nitrogen are the two main nutrients in maize (Zea mays L.) kernels, and kernel filling and metabolism determine seed formation and germination. However, the molecular mechanisms underlying the relationship between kernel filling and corresponding carbon and nitrogen metabolism remain largely unknown. Here, we found that HEAT SHOCK PROTEIN 90.6 (HSP90.6) is involved in both seed filling and the metabolism processes of carbon and nitrogen. A single-amino acid mutation within the HATPase_c domain of HSP90.6 led to small kernels. Transcriptome profiling showed that the expression of amino acid biosynthesis- and carbon metabolism-related genes was significantly downregulated in the hsp90.6 mutant. Further molecular evidence showed strong interactions between HSP90.6 and the 26S proteasome subunits REGULATORY PARTICLE NON-ATPASE6 (RPN6) and PROTEASOME BETA SUBUNITD2 (PBD2). The mutation of hsp90.6 significantly reduced the activity of the 26S proteasome, resulting in the accumulation of ubiquitinated proteins and defects in nitrogen recycling. Moreover, we verified that HSP90.6 is involved in carbon metabolism through interacting with the 14-3-3 protein GENERAL REGULATORY FACTOR14-4 (GF14-4). Collectively, our findings revealed that HSP90.6 is involved in seed filling and development by interacting with the components controlling carbon and nitrogen metabolism.

Identification of two new QTLs of maize (Zea mays L.) underlying kernel row number using the HNAU-NAM1 population.

BACKGROUND: Maize kernel row number (KRN) is one of the most important yield traits and has changed greatly during maize domestication and selection. Elucidating the genetic basis of KRN will be helpful to improve grain yield in maize. RESULTS: Here, we measured KRN in four environments using a nested association mapping (NAM) population named HNAU-NAM1 with 1,617 recombinant inbred lines (RILs) that were derived from 12 maize inbred lines with a common parent, GEMS41. Then, five consensus quantitative trait loci (QTLs) distributing on four chromosomes were identified in at least three environments along with the best linear unbiased prediction (BLUP) values by the joint linkage mapping (JLM) method. These QTLs were further validated by the separate linkage mapping (SLM) and genome-wide association study (GWAS) methods. Three KRN genes cloned through the QTL assay were found in three of the five consensus QTLs, including qKRN1.1, qKRN2.1 and qKRN4.1. Two new QTLs of KRN, qKRN4.2 and qKRN9.1, were also identified. On the basis of public RNA-seq and genome annotation data, five genes highly expressed in ear tissue were considered candidate genes contributing to KRN. CONCLUSIONS: This study carried out a comprehensive analysis of the genetic architecture of KRN by using a new NAM population under multiple environments. The present results provide solid information for understanding the genetic components underlying KRN and candidate genes in qKRN4.2 and qKRN9.1. Single-nucleotide polymorphisms (SNPs) closely linked to qKRN4.2 and qKRN9.1 could be used to improve inbred yield during molecular breeding in maize.

The RppC-AvrRppC NLR-effector interaction mediates the resistance to southern corn rust in maize.

Southern corn rust (SCR), caused by the fungal pathogen Puccinia polysora, is a major threat to maize production worldwide. Efficient breeding and deployment of resistant hybrids are key to achieving durable control of SCR. Here, we report the molecular cloning and characterization of RppC, which encodes an NLR-type immune receptor and is responsible for a major SCR resistance quantitative trait locus. Furthermore, we identified the corresponding avirulence effector, AvrRppC, which is secreted by P. polysora and triggers RppC-mediated resistance. Allelic variation of AvrRppC directly determines the effectiveness of RppC-mediated resistance, indicating that monitoring of AvrRppC variants in the field can guide the rational deployment of RppC-containing hybrids in maize production. Currently, RppC is the most frequently deployed SCR resistance gene in China, and a better understanding of its mode of action is critical for extending its durability.

Loss-of-function alleles of ZmPLD3 cause haploid induction in maize.

Doubled haploid technology has been widely applied to multiple plant species and is recognized as one of the most important technologies for improving crop breeding efficiency. Although mutations in MATRILINEAL/Zea mays PHOSPHOLIPASE A1/NOT LIKE DAD (MTL/ZmPLA1/NLD) and Zea mays DOMAIN OF UNKNOWN FUNCTION 679 MEMBRANE PROTEIN (ZmDMP) have been shown to generate haploids in maize, knowledge of the genetic basis of haploid induction (HI) remains incomplete. Therefore, cloning of new genes underlying HI is important for further elucidating its genetic architecture. Here, we found that loss-of-function mutations of Zea mays PHOSPHOLIPASE D3 (ZmPLD3), one of the members from the phospholipase D subfamily, could trigger maternal HI in maize. ZmPLD3 was identified through a reverse genetic strategy based on analysis of pollen-specifically expressed phospholipases, followed by validation through the clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR-Cas9) system. Mutations of ZmPLD3 resulted in a haploid induction rate (HIR) similar to that of mtl/zmpla1/nld and showed synergistic effects rather than functional redundancy on tripling the HIR (from 1.19% to 4.13%) in the presence of mtl/zmpla1/nld. RNA-seq profiling of mature pollen indicated that a large number of pollen-specific differentially expressed genes were enriched in processes related to gametogenesis development, such as pollen tube development and cell communication, during the double-fertilization process. In addition, ZmPLD3 is highly conserved among cereals, highlighting the potential application of these in vivo haploid-inducer lines for other important crop plant species. Collectively, our discovery identifies a novel gene underlying in vivo maternal HI and provides possibility of breeding haploid inducers with further improved HIR.

A novel animal model for vulnerable atherosclerotic plaque: dehydrated ethanol lavage in the carotid artery of rabbits fed a Western diet.

BACKGROUND: The study of unstable atherosclerotic plaques is limited by the absence of ideal animal models to reproduce the plaque instability observed in humans. In this study, we attempted to develop a novel animal model for vulnerable atherosclerotic plaques using dehydrated ethanol lavage in rabbits fed a Western diet (WD). METHODS: A total of 30 New Zealand White (NZW) rabbits were randomized to 5 groups, including a control group with or without WD, a balloon injury with WD group, and an ethanol injury with or without WD group. Operations were conducted using the right common carotid artery as the target vessel. All animals were followed up for 3 months unless a vascular event occurred. Blood samples and carotid artery specimens were ultimately collected for analysis of atherogenesis. RESULTS: Compared to rabbits in which lesions were induced by balloon injury, those subjected to an ethanol lavage with high cholesterol diet showed progressive atherosclerotic lesions in all carotid artery segments, which were characterized by greater plaque burden, smaller minimum lumen area (MLA), and increased vulnerability as indicated by abundant macrophages, scattered smooth muscle cell (SMC) composition, higher matrix metalloproteinase-9 (MMP-9) expression in plaques, thinner fibrous cap thickness, and higher possibility of stroke event (50% vs. 0%). Meanwhile, the serum interleukin-1ß (IL-1ß) and monocyte chemoattractant protein-1 (MCP-1) levels in the ethanol injury group with a high-cholesterol diet were significantly higher than those in the balloon injury group after 3 months (all P<0.01). CONCLUSIONS: We successfully established a novel animal model for vulnerable atherosclerosis by ethanol exposure of the carotid segment that has a higher predictive value for the probability of ischemic events than the balloon injury model. Therefore, it may represent a promising animal model for investigating new therapeutic approaches, novel imaging modalities, and underlying mechanisms for vulnerable atherosclerotic plaque.

RNA-guided Cas9 as an in vivo desired-target mutator in maize.

The RNA-guided Cas9 system is a versatile tool for genome editing. Here, we established a RNA-guided endonuclease (RGEN) system as an in vivo desired-target mutator (DTM) in maize to reduce the linkage drag during breeding procedure, using the LIGULELESS1 (LG1) locus as a proof-of-concept. Our system showed 51.5%-91.2% mutation frequency in T0 transgenic plants. We then crossed the T1 plants stably expressing DTM with six diverse recipient maize lines and found that 11.79%-28.71% of the plants tested were mutants induced by the DTM effect. Analysis of successive F2 plants indicated that the mutations induced by the DTM effect were largely heritable. Moreover, DTM-generated hybrids had significantly smaller leaf angles that were reduced more than 50% when compared with that of the wild type. Planting experiments showed that DTM-generated maize plants can be grown with significantly higher density and hence greater yield potential. Our work demonstrate that stably expressed RGEN could be implemented as an in vivoDTM to rapidly generate and spread desired mutations in maize through hybridization and subsequent backcrossing, and hence bypassing the linkage drag effect in convention introgression methodology. This proof-of-concept experiment can be a potentially much more efficient breeding strategy in crops employing the RNA-guided Cas9 genome editing.

Association of P2Y12 gene promoter DNA methylation with the risk of clopidogrel resistance in coronary artery disease patients.

BACKGROUND: Clopidogrel inhibits the ADP receptor P2Y12 to keep down the platelet aggregation. The goal of our study is to investigate the contribution of P2Y12 promoter DNA methylation to the risk of clopidogrel resistance (CR). METHODS: The platelet functions were measured by the VerifyNow P2Y12 assay. Applying the bisulfite pyrosequencing technology, DNA methylation levels of two CpG dinucleotides on P2Y12 promoter were tested among 49 CR cases and 57 non-CR controls. We also investigated the association among P2Y12 DNA methylation, various biochemical characteristics, and CR. RESULT: Lower methylation of two CpGs indicated the poorer clopidogrel response (CpG1, P=0.009; CpG2, P=0.022) in alcohol abusing status. Meanwhile CpG1 methylation was inversely correlated with CR in smoking patients (P=0.026) and in subgroup of Albumin<35 (P=0.002). We observed that the level of DNA methylation might be affected by some clinical markers, such as TBIL, LEVF, Albumin, AST. The results also showed that the quantity of stent, fasting blood-glucose, and lower HbAC1 were the predictors of CR. CONCLUSIONS: The evidence from our study indicates that P2Y12 methylation may bring new hints to elaborate the pathogenesis of CR.

High-dose atorvastatin reloading before percutaneous coronary intervention increased circulating endothelial progenitor cells and reduced inflammatory cytokine expression during the perioperative period.

OBJECTIVE: We investigated atorvastatin reloading effects on endothelial progenitor cell (EPC) count and inflammatory cytokine expression after percutaneous coronary intervention (PCI) in patients with stable angina pectoris who had previously received long-term statin treatments. METHODS: Patients with stable angina pectoris were treated with 80 mg atorvastatin 12 hours and 40 mg atorvastatin 2 hours before coronary angioplasty (n = 15) or preoperatively with 40 mg/d atorvastatin for 7 days (n = 15) or did not receive atorvastatin (n = 15). CD45-/133+/34+, CD45-/CD34+/kinase insert domain receptor (KDR)+, and CD45-/CD144+/KDR+ EPCs in the peripheral blood were determined by flow cytometry 1 hour before as well as 1 hour, 6 hours, and 24 hours after PCI. Soluble intercellular adhesion molecule 1 (sICAM-1), hypersensitive C-reactive protein (hCRP), and troponin-I (TnI) serum concentrations were analyzed immediately prior to and 24 hours after PCI. RESULTS: In the 40mg Atorvastatin and control groups, none of the analyzed EPC blood concentrations changed significantly from 1h before operation to 1h and 6 h postoperative values. In contrast, the number of circulating early differentiation stage EPCs CD45-/133+/34+ and CD45-/CD34+/ KDR+ raised significantly from 1 h preoperative values (57.3±9.3; 57.3 ± 10.7) to 1 h postoperative ((74.4 ± 11.4; 78.8 ± 16.2), (p < 0.05)) and 6 h postoperative ((93 ± 16.9; 99.7 ± 11.9), (p < 0.05)) concentrations after coronary angioplasty in the 80mg Atorvastatin medication patients. In the control group, the sICAM-1 (174.55 ± 38.91 vs 204.11 ± 58.24) and hCRP (1.89 ± 1.93 vs 9.0 ± 11.1) serum concentrations at 24 hours after PCI were significantly elevated (P < .05) compared to preoperative values, whereas the increases in the 2 groups treated with atorvastatin were not significant. In addition, the rise in serum TnI concentration level from pre- to postoperative in the 80-mg (0.02 ± 0.02 vs 0.09 ± 0.08) and the 40-mg (0.01 ± 0.03 vs 1.2 ± 2.59) reloading groups was less than that of the controls (0.01 ± 0.02 vs 1.75 ± 3.09) (p < 0.05). CONCLUSION: Our results suggested that high-dose atorvastatin application before PCI triggered early EPC circulation. Furthermore, postoperative inflammatory cytokine sICAM-1 as well as hCRP serum levels were reduced, while postinterventional myocardial injury marker TnI elevations were inversely correlated with statin reloadings.