Multiplex-genome-editing based rapid directional improvement of complex traits in rice.

Although thousands of genes have been identified or cloned in rice (Oryza sativa) in the last two decades, the majority of them have only been separately characterized in specific varieties or single-gene modified backgrounds, thus limiting their practical application. We developed an optimized multiplex genome editing (MGE) toolbox that can efficiently assemble and stably express up to twelve sgRNA targets in a single plant expression vector. In this study, we established the MGE-based Rapid Directional Improvement (MRDI) strategy for directional improvement of complex agronomic traits in one small-scale rice transformation. This approach provides a rapid and practical procedure, encompassing sgRNA assembly, transgene-free screening and the creation of promising germplasm, by combining the precision of gene editing with phenotype-based field breeding. The MRDI strategy was used to generate the full diversity of twelve main agronomic genes in rice cultivar FXZ for the directional improvement of its growth duration and plant architecture. After applying the MRDI to FXZ, ideal plants with the desired traits of early heading date reduced plant height, and more effective panicles were generated without compromising yield, blast resistance and grain quality. Furthermore, the results of whole-genome sequencing (WGS), including the analysis of structural variations (SVs) and single nucleotide variations (SNVs) in the MGE plants, confirmed the high specificity and low frequency of unwanted mutations associated with this strategy. The MRDI breeding strategy would be a robust approach for exploring and applying crucial agronomic genes, as well as for generating novel elite germplasm in the future.

Prognostic implications of systemic immune-inflammation index in myocardial infarction patients with and without diabetes: insights from the NOAFCAMI-SH registry.

BACKGROUND: It is well-known that systemic inflammation plays a crucial role in the pathogenesis and prognosis of acute myocardial infarction (AMI). The systemic immune-inflammation index (SII, platelet × neutrophil/lymphocyte ratio) is a novel index that is used for the characterization of the severity of systemic inflammation. Recent studies have identified the high SII level as an independent predictor of poor outcomes in patients with AMI. We aimed to investigate the prognostic implications of SII in AMI patients with and without diabetes mellitus (DM). METHODS: We included 2111 patients with AMI from February 2014 to March 2018. Multivariable Cox regression analyses were performed to estimate the hazard ratios (HRs) and 95% confidence intervals (CIs) of all-cause death and cardiovascular (CV) death. Multiple imputation was used for missing covariates. RESULTS: Of 2111 patients (mean age: 65.2 ± 12.2 years, 77.5% were males) analyzed, 789 (37.4%) had DM. Generalized additive model analyses showed that as the SII increased, the C-reactive protein and peak TnT elevated while the LVEF declined, and these associations were similar in patients with and without DM. During a median of 2.5 years of follow-up, 210 all-cause deaths and 154 CV deaths occurred. When treating the SII as a continuous variable, a higher log-transformed SII was significantly associated with increased all-cause mortality (HR: 1.57, 95%CI: 1.02-2.43) and CV mortality (HR: 1.85, 95%CI 1.12-3.05), and such an association was also significant in the diabetics (HRs and 95%CIs for all-cause death and CV death were 2.90 [1.40-6.01] and 3.28 [1.43-7.57], respectively) while not significant in the nondiabetics (Pinteraction for all-cause death and CV death were 0.019 and 0.049, respectively). Additionally, compared to patients with the lowest tertiles of SII, those with the highest tertiles of SII possessed significantly higher all-cause mortality (HR: 1.82, 95%CI 1.19-2.79) and CV mortality (HR: 1.82, 95%CI 1.19-2.79) after multivariable adjustment, and this relationship remained pronounced in the diabetics (HRs and 95%CIs for all-cause death and CV death were 2.00 [1.13-3.55] and 2.09 [1.10-3.98], respectively) but was not observed in the nondiabetics (HRs and 95%CIs for all-cause death and CV death were 1.21 [0.75-1.97] and 1.60 [0.89-2.90], respectively). Our restricted cubic splines analyses indicated a pronounced linear association between SII and mortality only in diabetics. CONCLUSIONS: In AMI patients with DM, high SII is an independent predictor of poor survival and may be helpful for patient's risk stratification.

Association of stress hyperglycemia ratio with in-hospital new-onset atrial fibrillation and long-term outcomes in patients with acute myocardial infarction.

AIMS: To investigate the predictive value and prognostic impact of stress hyperglycemia ratio (SHR) for new-onset atrial fibrillation (NOAF) complicating acute myocardial infarction (AMI). MATERIALS AND METHODS: This retrospective study included 2145 AMI patients without AF history between February 2014 and March 2018. SHR was calculated using fasting blood glucose (mmol/L)/[1.59*HbA1c (%)-2.59]. The association between SHR and post-MI NOAF was assessed with multivariable logistic regression analyses. The primary outcome was a composite of cardiac death, heart failure hospitalisation, recurrent MI, and ischaemic stroke (MACE). Cox regression-adjusted hazard ratios with 95% confidence intervals (CI) were estimated for MACE. RESULTS: A total of 245 (11.4%) patients developed NOAF. In the multivariable logistic regression analyses, SHR (each 10% increase) was significantly associated with increased risks of NOAF in the whole population (OR: 1.05, 95% CI: 1.01-1.10), particularly in non-diabetic individuals (OR:1.08, 95% CI: 1.01-1.17). During a median follow-up of 2.7 years, 370 (18.5%) MACEs were recorded. The optimal cut-off value of SHR for MACE prediction was 1.119. Patients with both high SHR (≥1.119) and NOAF possessed the highest risk of MACE compared to those with neither high SHR nor NOAF after multivariable adjustment (HR: 2.18, 95% CI: 1.39-3.42), especially for diabetics (HR: 2.63, 95% CI: 1.41-4.91). Similar findings were observed using competing-risk models. CONCLUSIONS: SHR is an independent predictor of post-MI NOAF in non-diabetic individuals. Diabetic patients with both high SHR and NOAF had the highest risk of MACE, suggesting that therapies targeting SHR may be considered in these patients. TRIAL REGISTRATION: ClinicalTrials.gov, NCT03533543.

Analysis of co-expression and gene regulatory networks associated with sterile lemma development in rice.

BACKGROUND: The sterile lemma is a unique organ of the rice (Oryza sativa L.) spikelet. However, the characteristics and origin of the rice sterile lemma have not been determined unequivocally, so it is important to elucidate the molecular mechanism of the development of the sterile lemma. RESULTS: In the paper, we outline the regulatory mechanism of sterile lemma development by LONG STERILE LEMMA1 (G1), which has been identified as the gene controlling sterile lemma development. Based on the comprehensive analyses of transcriptome dynamics during sterile lemma development with G1 alleles between wild-type (WT) and mutant (MT) in rice, we obtained co-expression data and regulatory networks related to sterile lemma development. Co-transfection assays of rice protoplasts confirmed that G1 affects the expression of various phytohormone-related genes by regulating a number of critical transcription factors, such as OsLBD37 and OSH1. The hormone levels in sterile lemmas from WT and MT of rice supports the hypotheses that lower auxin, lower gibberellin, and higher cytokinin concentrations are required to maintain a normal phenotype of sterile lemmas. CONCLUSION: The regulatory networks have considerable reference value, and some of the regulatory relationships exhibiting strong correlations are worthy of further study. Taken together, these work provided a detailed guide for further studies into the molecular mechanism of sterile lemma development.

IPA1 improves drought tolerance by activating SNAC1 in rice.

Drought is a major abiotic stress to rice (Oryza sativa) during growth. Ideal Plant Architecture (IPA1), the first cloned gene controlling the ideal plant type in rice, has been reported to function in both ideal rice plant architecture and biotic resistance. Here, we report that the IPA1/OsSPL14, encoding a transcriptional factor, positively regulates drought tolerance in rice. The IPA1 is constitutively expressed and regulated by H2O2, abscisic acid, NaCl and polyethylene glycol 6000 treatments in rice. Furthermore, the IPA1-knockout plants showed much greater accumulation of H2O2 as measured by 3,3'-diaminobenzidine staining in leaves compared with WT plants. Yeast one-hybrid, dual-luciferase and electrophoretic mobility shift assays indicated that the IPA1 directly activates the promoter of SNAC1. Expression of SNAC1 is significantly down-regulated in IPA1 knockout plants. Further investigation indicated that the IPA1 plays a positive role in drought-stress tolerance by inducing reactive oxygen species scavenging in rice. Together, these findings indicated that the IPA1 played important roles in drought tolerance by regulating SNAC1, thus activating the antioxidant system in rice.

Ubiquitylomes and proteomes analyses provide a new interpretation of the molecular mechanisms of rice leaf senescence.

MAIN CONCLUSION: We identified a typical rice premature senescence leaf mutant 86 (psl86) and exhibited the first global ubiquitination data during rice leaf senescence. Premature leaf senescence affects the yield and quality of rice, causing irreparable agricultural economic losses. In this study, we reported a rice premature senescence leaf mutant 86 (psl86) in the population lines of rice (Oryza sativa) japonica cultivar 'Yunyin' (YY) mutagenized using ethyl methane sulfonate (EMS) treatment. Immunoblotting analysis revealed that a higher ubiquitination level in the psl86 mutant compared with YY. Thus, we performed the proteome and ubiquitylome analyses to identify the differential abundance proteins and ubiquitinated proteins (sites) related to leaf senescence. Among 885 quantified lysine ubiquitination (Kub) sites in 492 proteins, 116 sites in 94 proteins were classified as up-regulated targets and seven sites in six proteins were classified as down-regulated targets at a threshold of 1.5. Proteins with up-regulated Kub sites were mainly enriched in the carbon fixation in photosynthetic organisms, glycolysis/gluconeogenesis and the pentose phosphate pathway. Notably, 14 up-regulated Kub sites in 11 proteins were enriched in the carbon fixation in photosynthetic organism pathway, and seven proteins (rbcL, PGK, GAPA, FBA5, ALDP, CFBP1 and GGAT) were down-regulated, indicating this pathway is tightly regulated by ubiquitination during leaf senescence. To our knowledge, we present the first global data on ubiquitination during rice leaf senescence.

RNA-seq profiling of primary calli induced by different media and photoperiods for japonica rice 'Yunyin'.

The induction of embryogenic calli plays a vital role in the genetic transformation and regeneration of rice (Oryza sativa L.). Despite progress in rice tissue culture, the molecular mechanisms of embryogenic callus induction remain unknown. In this study, gene expression profiles associated with calli were comprehensively analyzed during callus induction of japonica rice 'Yunyin'. We first confirmed that NMB medium with 24 h of light and 0 h of dark (NMB-L) was the optimal condition for 'Yunyin' callus induction, while J3 medium with 0 h of light and 24 h of dark (J3-D) was the worst condition. After transcriptome analysis, 33,597 unigenes were assembled, among which we identified 6,063 DEGs (Differentially Expressed Genes) related to media and seven DEGs related to photoperiod. Phenylpropanoid biosynthesis, plant hormone signal, and starch and sucrose metabolism were the top three pathways affected by media, while the circadian rhythm-plant pathway was associated with photoperiod. Furthermore, we identified two candidate genes, Os01g0965900 and Os12g0555200, affected by both medium and photoperiod. Statistical analysis of RNA-seq libraries showed that the expression levels of these two genes in J3-D calli were over 2.5 times higher than those in NMB-L calli, which was further proved by RT-qPCR analysis. Based on FPKM (Fragments Per Kilobase of transcript Per Million mapped reads), unigenes belonging to the NMB-L group were mainly assigned to ribosome, carbon metabolism, biosynthesis of amino acids, protein processing in endoplasmic reticulum, and plant hormone signal transduction pathways. We transformed Os12g0555200Nip and Os12g05552009311 into 'Nipponbare' calli and observed their effects on the growth and development process of rice calli using TEM (Transmission Electron Microscopy) and SEM (Scanning Electron Microscopy). Observations showed that Os12g05552009311 was more disadvantageous to rice callus growth than Os12g0555200Nip. Our results reveal that the Os12g0555200, identified from transcriptomic profiles, has a negative influence during 'Yunyin' callus induction. Supplementary information: The online version contains supplementary material available at 10.1007/s11032-022-01283-y.

Prognostic impact of stress hyperglycemia ratio in acute myocardial infarction patients with and without diabetes mellitus.

BACKGROUND AND AIMS: Stress hyperglycemia ratio (SHR) is associated with increased in-hospital morbidity and mortality in patients with acute myocardial infarction (AMI). We aimed to investigate the impact of stress "hyperglycemia" on long-term mortality after AMI in patients with and without diabetes mellitus (DM). METHODS AND RESULTS: We included 2089 patients with AMI between February 2014 and March 2018. SHR was measured with the fasting glucose divided by the estimated average glucose derived from glycosylated hemoglobin (HbA1c). The primary endpoint was all-cause death. Of 2 089 patients (mean age: 65.7 ± 12.4, 76.7% were men) analyzed, 796 (38.1%) had DM. Over a median follow-up of 2.7 years, 141 (6.7%) and 150 (7.2%) all-cause deaths occurred in the diabetic and nondiabetic cohorts, respectively. Compared with participants with low SHR (<1.24 in DM; <1.14 in non-DM), the hazard ratios and 95% confidence intervals for those with high SHR (≥1.24 in DM; ≥1.14 in non-DM) for all-cause mortality were 2.23 (1.54-3.23) and 1.79 (1.15-2.78); for cardiovascular mortality were 2.42 (1.63-3.59) and 2.10 (1.32-3.35) in DM and non-DM subjects, respectively. The mortality prediction was improved in the diabetic individuals with the incorporation of SHR into the Global Registry of Acute Coronary Events (GRACE) score, showing an increase in a continuous net reclassification index of 0.184 (95%CI: 0.003-0.365) and an absolute integrated discrimination improvement of 0.014 (95%CI: 0.002-0.025). CONCLUSION: The improvement in the prediction of long-term mortality beyond the GRACE score indicates the potential of SHR as a biomarker for post-MI risk stratification among patients with DM. REGISTRATION NUMBER FOR CLINICAL TRIALS: NCT03533543.

ZPI prevents ox-LDL-mediated endothelial injury leading to inhibition of EndMT, inflammation, apoptosis, and oxidative stress through activating Pi3k/Akt signal pathway.

Oxidized low-density lipoprotein (ox-LDL)-mediated endothelial dysfunction exerts an essential role in the development of atherosclerosis. Protein Z-dependent protease inhibitor (ZPI), a member of the serine protease inhibitor superfamily, could inhibit the function of activated coagulation factor X (FXa) via interaction with protein Z (PZ). Studies have pointed out that ZPI was statistically related to atherosclerotic diseases, which may have a robust cardiovascular protective effect. However, the underlying mechanism of ZPI on ox-LDL-mediated endothelial injury requires further elucidation. Human umbilical vein endothelial cells (HUVECs) were treated with ox-LDL (100 µg/ml) and ZPI (10 µg/ml). Cell viability was measured by the Cell Counting Kit-8 (CCK-8) assay. Cell apoptosis, oxidative stress, and endothelial-to-mesenchymal transition (EndMT) were analyzed by immunofluorescence (IF). Cell migration was measured using a wound-healing assay. Quantitative real-time polymerase chain reaction and western blot analysis were performed to determine messenger RNA and protein expression. Ox-LDL (100 µg/ml, 48 h) significantly reduced cell viability and migration, increased EndMT, inflammation, apoptosis, and oxidative stress. The related protein expression of phosphatidylinositol 3 kinase/protein kinase B (Pi3k/Akt) signal pathway in HUVECs was also simultaneously decreased. We also discovered that ZPI treatment could prevent ox-LDL-mediated endothelial injury through the improvement of cell viability and alleviation of apoptosis, oxidative stress, EndMT, and inflammation. Thus, the protective effect of ZPI on HUVECs may be mediated by activation of the Pi3k/Akt signal pathway. ZPI may exert an important protective role in HUVECs dysfunction triggered by ox-LDL via activation of the Pi3k/Akt signal pathway. Therefore, ZPI may possess potential therapeutic effects on atherosclerotic endothelial injury-related diseases.

GC-MS-based metabolite profiling of key differential metabolites between superior and inferior spikelets of rice during the grain filling stage.

BACKGROUND: The asynchronous filling between superior spikelets (SS) and inferior spikelets (IS) in rice has become a research hotspot. The stagnant development and poor grain filling of IS limit yields and the formation of good quality rice. A large number of studies on this phenomenon have been carried out from the genome, transcriptome and proteome level, indicating that asynchronous filling of SS and IS filling is a complex, but orderly physiological and biochemical process involving changes of a large number of genes, protein expression and modification. However, the analysis of metabolomics differences between SS and IS is rarely reported currently. RESULTS: This study utilized untargeted metabolomics and identified 162 metabolites in rice spikelets. Among them, 17 differential metabolites associated with unsynchronized grain filling between SS and IS, 27 metabolites were related to the stagnant development of IS and 35 metabolites related to the lower maximum grain-filling rate of IS compared with the SS. We found that soluble sugars were an important metabolite during grain filling for SS and IS. Absolute quantification was used to further analyze the dynamic changes of 4 types of soluble sugars (sucrose, fructose, glucose, and trehalose) between SS and IS. The results showed that sucrose and trehalose were closely associated with the dynamic characteristics of grain filling between SS and IS. The application of exogenous sugar showed that trehalose functioned as a key sugar signal during grain filling of IS. Trehalose regulated the expression of genes related to sucrose conversion and starch synthesis, thereby promoting the conversion of sucrose to starch. The difference in the spatiotemporal expression of TPS-2 and TPP-1 between SS and IS was an important reason that led to the asynchronous change in the trehalose content between SS and IS. CONCLUSIONS: The results from this study are helpful for understanding the difference in grain filling between SS and IS at the metabolite level. In addition, the present results can also provide a theoretical basis for the next step of using metabolites to regulate the filling of IS.

PEPC of sugarcane regulated glutathione S-transferase and altered carbon-nitrogen metabolism under different N source concentrations in Oryza sativa.

BACKGROUND: Phosphoenolpyruvate carboxylase (PEPC) plays an important role in the primary metabolism of higher plants. Several studies have revealed the critical importance of PEPC in the interaction of carbon and nitrogen metabolism. However, the function mechanism of PEPC in nitrogen metabolism is unclear and needs further investigation. RESULTS: This study indicates that transgenic rice expressing the sugarcane C4-PEPC gene displayed shorter primary roots and fewer crown roots at the seedling stage. However, total nitrogen content was significantly higher in transgenic rice than in wild type (WT) plants. Proteomic analysis revealed that there were more differentially expressed proteins (DEPs) responding to nitrogen changes in transgenic rice. In particular, the most enriched pathway "glutathione (GSH) metabolism", which mainly contains GSH S-transferase (GST), was identified in transgenic rice. The expression of endogenous PEPC, GST and several genes involved in the TCA cycle, glycolysis and nitrogen assimilation changed in transgenic rice. Correspondingly, the activity of enzymes including GST, citrate synthase, 6-phosphofructokinase, pyruvate kinase and ferredoxin-dependent glutamate synthase significantly changed. In addition, the levels of organic acids in the TCA cycle and carbohydrates including sucrose, starch and soluble sugar altered in transgenic rice under different nitrogen source concentrations. GSH that the substrate of GST and its components including glutamic acid, cysteine and glycine accumulated in transgenic rice. Moreover, the levels of phytohormones including indoleacetic acid (IAA), zeatin (ZT) and isopentenyladenosine (2ip) were lower in the roots of transgenic rice under total nutrients. Taken together, the phenotype, physiological and biochemical characteristics of transgenic rice expressing C4-PEPC were different from WT under different nitrogen levels. CONCLUSIONS: Our results revealed the possibility that PEPC affects nitrogen metabolism through regulating GST, which provide a new direction and concepts for the further study of the PEPC functional mechanism in nitrogen metabolism.

Long-term impact of the burden of new-onset atrial fibrillation in patients with acute myocardial infarction: results from the NOAFCAMI-SH registry.

AIMS: We aimed to investigate the prognostic impact of the burden of new-onset atrial fibrillation (NOAF) on long-term cardiovascular outcomes in patients with acute myocardial infarction (AMI). METHODS AND RESULTS: This retrospective analysis consecutively included patients without a documented atrial fibrillation (AF) history who admitted for AMI at Shanghai Tenth People's Hospital between February 2014 and March 2018. Atrial fibrillation burden was measured as the percentage of time spent in AF, and its optimal cut-off value of 10.87% was identified by X-tile software. Of 2399 patients (mean age: 65.8 years, 76.6% of men), 278 (11.6%) developed NOAF during hospitalization. During a median follow-up of 2.7 years, the incidence of all-cause death was 3.19, 9.00, and 17.41 per 100 person-years in the sinus rhythm (SR), low-burden (AF burden ≤ 10.87%), and high-burden (AF burden > 10.87%) groups, respectively. After adjustment for confounders, it was the high-burden NOAF [hazard ratio (HR): 1.94, 95% confidence interval (CI): 1.28-2.95] rather than the low-burden one (HR: 1.47, 95% CI: 0.97-2.21) that was significantly associated with increased mortality compared with SR. Concordant results were obtained in our propensity score-matched analyses [2.55 (1.57-4.16) and 1.32 (0.85-2.05) for high- and low-burden NOAF, respectively). In addition, post-myocardial infarction NOAF was associated with an increased risk of heart failure irrespective of its burden. Only those high-burden individuals were at heightened risk of ischaemic stroke. The restricted cubic spline curves illustrated a dose-response relationship of NOAF burden with outcomes. CONCLUSION: In patients with NOAF complicating AMI, high AF burden was strongly associated with long-term outcomes.

Inhibition of miR-25 attenuates doxorubicin-induced apoptosis, reactive oxygen species production and DNA damage by targeting PTEN.

Background: Doxorubicin (DOX) is one of the widely used anti-cancer drugs, whereas it can induce irreversible cardiac injury in a dose-dependent manner which limits its utility in clinic. Our study aimed to investigate the relationship between miR-25 and DOX-induced cardiac injury and its underlying mechanism. Methods: Mice and H9c2 cells were exposed to DOX. The overexpressed or knockdown of miR-25 in H9c2 cells was achieved by miR-25 mimic or inhibitor and the efficiency of transfection was identified by qRT-PCR or Western blotting. Cell viability, apoptotic cell rate, and levels of apoptosis-related proteins were determined by CCK-8, flow cytometry, and Western blotting, respectively. Furthermore, Western blotting and immunofluorescence staining (IF) were performed to assess the expression levels of reactive oxygen species and degree of DNA damage. Results: As a result, DOX significantly upregulated miR-25 expression in mice and H9c2 cells and reduced cell viability and increased cell apoptosis in vitro and in vivo. miR-25 overexpression expedited cell injury induced by DOX in H9c2 cells demonstrated by the increased cell apoptosis and reactive oxygen species (ROS) production, whereas miR-25 inhibition attenuated the cell injury. Furthermore, miR-25 negatively controlled the expression of phosphatase and tensin homolog deleted on chromosome 10 (PTEN). Intervention the expression of PTEN using si-PTEN reversed the beneficial effects of miR-25 inhibition on DOX-injured H9c2 cells. Conclusion: In conclusion, this study demonstrated that miR-25 is involved in DOX-induced cell damage through the regulation of PTEN expression.

MicroRNA-320 targeting neuropilin 1 inhibits proliferation and migration of vascular smooth muscle cells and neointimal formation.

This study shows that microRNA-320 (miR-320) is associated with many important cell functions, including cell differentiation, proliferation, migration, and apoptosis. However, the role of miR-320 in vascular smooth muscle cells (VSMCs) and proliferative vascular diseases is still completely unclear. In our study, we found that the expression of miR-320 in human VSMCs after PDGF stimulation was significantly down-regulated in time- and dose-dependent manner. Function analyses identified that miR-320 could inhibit the proliferation and migration of VSMCs in both basal and PDGF-stimulated conditions. Furthermore, Neuropilin 1 (NRP1) was demonstrated as a direct target of miR-320 in Luciferase reporter assays and miR-320 overexpression inhibited the expression of NRP1 with or without PDGF treatment. Finally, miR-320 was markedly decreased in mice carotid arteries after ligated injury, while the restoration of miR-320 via Ad-miR-320 attenuated neointimal hyperplasia by declining the NRP1 expression. The results confirmed that miR-320 regulated proliferation and migration of VSMCs and neointimal formation by targeting NRP1. These novel findings implied that the regulation of NRP1 expression by miR-320 has important significance in the early diagnosis and treatment of proliferation vascular diseases.

Lipid oxidation inactivates the anticoagulant function of protein Z-dependent protease inhibitor (ZPI).

Lipid oxidation due to oxidative stress plays an important role in the pathogenesis of inflammatory and thrombotic cardiovascular diseases. Several findings suggest that lipid peroxidation can alter the function of coagulation proteins and contribute to a hypercoagulable state, but the molecular mechanisms are unclear. Here, we report that oxidized phospholipids suppress the anticoagulant function of the serpin, protein Z-dependent protease inhibitor (ZPI), a specific inhibitor of membrane-associated factor Xa (FXa) that requires protein Z (PZ), phospholipid, and calcium as cofactors. We found that this suppression arises from a diminished ability of the oxidized membrane to function as a cofactor to promote ZPI inhibition of membrane-bound FXa, due fully or in part to the susceptibility of the bound ZPI-PZ complex to oxidative inactivation. Surprisingly, free ZPI was also susceptible to inactivation by oxidized membrane vesicles in the absence of calcium. Oxidized vesicles containing both phosphatidylserine and polyunsaturated fatty acids were required to promote inactivation of the ZPI-PZ complex or free ZPI, indicating that binding of the PZ-complexed or free ZPI to peroxide-modified phospholipid vesicles mediates the inactivation. Heparin protected the ZPI-PZ complex and free ZPI from inactivation, suggesting that blocking the heparin-binding site on ZPI interferes with ZPI binding to lipid or to PZ. This was confirmed by direct lipid-binding experiments. Native PAGE indicated that oxidization induced dissociation of the ZPI-PZ complex and increased the negative charge of ZPI. We conclude that compromised ZPI anticoagulant function could contribute to thrombus initiation and growth in oxidative stress-induced cardiovascular diseases.

OsPRX2 contributes to stomatal closure and improves potassium deficiency tolerance in rice.

Peroxiredoxins (Prxs) which are thiol-based peroxidases have been implicated in the toxic reduction and intracellular concentration regulation of hydrogen peroxide. In Arabidopsis thaliana At2-CysPrxB (At5g06290) has been demonstrated to be essential in maintaining the water-water cycle for proper H2O2 scavenging. Although the mechanisms of 2-Cys Prxs have been extensively studied in Arabidopsis thaliana, the function of 2-Cys Prxs in rice is unclear. In this study, a rice homologue gene of At2-CysPrxB, OsPRX2 was investigated aiming to characterize the effect of 2-Cys Prxs on the K+-deficiency tolerance in rice. We found that OsPRX2 was localized in the chloroplast. Overexpressed OsPRX2 causes the stomatal closing and K+-deficiency tolerance increasing, while knockout of OsPRX2 lead to serious defects in leaves phenotype and the stomatal opening under the K+-deficiency tolerance. Detection of K+ accumulation, antioxidant activity of transgenic plants under the starvation of potassium, further confirmed that OsPRX2 is a potential target for engineering plants with improved potassium deficiency tolerance.

Rac1 promotes the survival of H9c2 cells during serum deficiency targeting JNK/c-JUN/Cyclin-D1 and AKT2/MCL1 pathways.

Rac1, known as a "molecular switch", plays a crucial role in plenty of cellular processes. Rac1 aggravates the damage of myocardial cells in the process of myocardial ischemia-reperfusion during myocardial infarction through activating the NADPH oxidase and bringing about the reactive oxygen species(ROS) generation. Myocardial ischemia and hypoxia are the basic pathogenesis of myocardial infarction and the underlying mechanisms are intricate and varied. Moreover, the regulatory effect of Rac1 on myocardial cells in the condition of serum starvation and the potential mechanisms are still incompletely undefined. Therefore, heart-derived H9c2 cells cultured in 0% serum were used to mimic ischemic myocardial cells and to clarify the role of Rac1 in H9c2 cells and the underlying mechanisms during serum deficiency. After Rac1 was knocked down using specific siRNA, cell apoptosis was assessed by flow cytometry assay and cell proliferation was detected by CCK-8 assay and EdU assay. In addition, the expression and activation of protein in related signaling pathway were detected by Western blot and siRNAs was used to testify the signaling pathways. Our results indicated that Rac1 inhibited apoptosis, promoted proliferation and cell cycle progression of H9c2 cells during serum deficiency. We concluded that Rac1 inhibited apoptosis in an AKT2/MCL1 dependent way and promoted cell proliferation through JNK/c-JUN/Cyclin-D1.

The influence of individual socioeconomic status on the clinical outcomes in ischemic stroke patients with different neighborhood status in Shanghai, China.

Objective: Socioeconomic status (SES) is being recognized as an important factor in both social and medical problems. The aim of present study is to examine the relationship between SES and ischemic stroke and investigate whether SES is a predictor of clinical outcomes among patients with different neighborhood status from Shanghai, China. Methods: A total of 471 first-ever ischemic stroke patients aged 18-80 years were enrolled in this retrospective study. The personal SES of each patient was evaluated using a summed score derived from his or her educational level, household income, occupation, and medical reimbursement rate. Clinical adverse events and all-cause mortality were analyzed to determine whether SES was a prognostic factor, its prognostic impact was then assessed based on different neighborhood status using multivariable Cox proportional hazard models after adjusting for other covariates. Results: The individual SES showed a significant positive correlation with neighborhood status (r = 0.370; P < 0.001). The incidence of clinical adverse events and mortality were significantly higher in low SES patients compared with middle and high SES patients (P = 0.001 and P = 0.037, respectively). After adjusting other risk factors and neighborhood status, Kaplan-Meier analysis showed clinical adverse events and deaths were still higher in the low SES patients (all P < 0.05). Multivariate Cox regression analysis demonstrated that both personal SES and neighborhood status are independent prognostic factors for ischemic stroke (all P < 0.05). Besides, among patients with low and middle neighborhood status, lower individual SES was significantly associated with clinical adverse events and mortality (all P < 0.05). Conclusion: Both individual SES and neighborhood status are significantly associated with the prognosis after ischemic stroke. A lower personal SES as well as poorer neighborhood status may significantly increase risk for adverse clinical outcomes among ischemic stroke patients.

Protein repair L-isoaspartyl methyltransferase 1 (PIMT1) in rice improves seed longevity by preserving embryo vigor and viability.

Damaged proteins containing abnormal isoaspartyl (isoAsp) accumulate as seeds age and the abnormality is thought to undermine seed vigor. Protein-L-isoaspartyl methyltransferase (PIMT) is involved in isoAsp-containing protein repair. Two PIMT genes from rice (Oryza sativa L.), designated as OsPIMT1 and OsPIMT2, were isolated and investigated for their roles. The results indicated that OsPIMT2 was mainly present in green tissues, but OsPIMT1 largely accumulated in embryos. Confocal visualization of the transient expression of OsPIMTs showed that OsPIMT2 was localized in the chloroplast and nucleus, whereas OsPIMT1 was predominately found in the cytosol. Artificial aging results highlighted the sensitivity of the seeds of OsPIMT1 mutant line when subjected to accelerated aging. Overexpression of OsPIMT1 in transgenic seeds reduced the accumulation of isoAsp-containing protein in embryos, and increased embryo viability. The germination percentage of transgenic seeds overexpressing OsPIMT1 increased 9-15% compared to the WT seeds after 21-day of artificial aging, whereas seeds from the OsPIMT1 RNAi lines overaccumulated isoAsp in embryos and experienced rapid loss of seed germinability. Taken together, these data strongly indicated that OsPIMT1-related seed longevity improvement is probably due to the repair of detrimental isoAsp-containing proteins that over accumulate in embryos when subjected to accelerated aging.

Antisense suppression of LOX3 gene expression in rice endosperm enhances seed longevity.

Lipid peroxidation plays a major role in seed longevity and viability. In rice grains, lipid peroxidation is catalyzed by the enzyme lipoxygenase 3 (LOX3). Previous reports showed that grain from the rice variety DawDam in which the LOX3 gene was deleted had less stale flavour after grain storage than normal rice. The molecular mechanism by which LOX3 expression is regulated during endosperm development remains unclear. In this study, we expressed a LOX3 antisense construct in transgenic rice (Oryza sativa L.) plants to down-regulate LOX3 expression in rice endosperm. The transgenic plants exhibited a marked decrease in LOX mRNA levels, normal phenotypes and a normal life cycle. We showed that LOX3 activity and its ability to produce 9-hydroperoxyoctadecadienoic acid (9-HPOD) from linoleic acid were significantly lower in transgenic seeds than in wild-type seeds by measuring the ultraviolet absorption of 9-HPOD at 234 nm and by high-performance liquid chromatography. The suppression of LOX3 expression in rice endosperm increased grain storability. The germination rate of TS-91 (antisense LOX3 transgenic line) was much higher than the WT (29% higher after artificial ageing for 21 days, and 40% higher after natural ageing for 12 months). To our knowledge, this is the first report to demonstrate that decreased LOX3 expression can preserve rice grain quality during storage with no impact on grain yield, suggesting potential applications in agricultural production.