Real-Time Observation of Conformational Changes and Translocation of Endogenous Cytochrome c within Intact Mitochondria.

Cytochrome c (cyt c) is a multifunctional protein with varying conformations. However, the conformation of cyt c in its native environment, mitochondria, is still unclear. Here, we applied NMR spectroscopy to investigate the conformation and location of endogenous cyt c within intact mitochondria at natural isotopic abundance, mainly using widespread methyl groups as probes. By monitoring time-dependent chemical shift perturbations, we observed that most cyt c is located in the inner mitochondrial membrane and partially unfolded, which is distinct from its native conformation in solution. When suffering oxidative stress, cyt c underwent oxidative modifications due to increasing reactive oxygen species (ROS), weakening electrostatic interactions with the membrane, and gradually translocating into the inner membrane spaces of mitochondria. Meanwhile, the lethality of oxidatively modified cyt c to cells was reduced compared with normal cyt c. Our findings significantly improve the understanding of the molecular mechanisms underlying the regulation of ROS by cyt c in mitochondria. Moreover, it highlights the potential of NMR to monitor high-concentration molecules at a natural isotopic abundance within intact cells or organelles.

Real-Time NMR-Based Drug Discovery to Identify Inhibitors against Fatty Acid Synthesis in Living Cancer Cells.

Abnormal fatty acid metabolism is recognized as a key driver of tumor development and progression. Although numerous inhibitors have been developed to target this pathway, finding drugs with high specificity that do not disrupt normal cellular metabolism remains a formidable challenge. In this paper, we introduced a novel real-time NMR-based drug screening technique that operates within living cells. This technique provides a direct way to putatively identify molecular targets involved in specific metabolic processes, making it a powerful tool for cell-based drug screening. Using 2-13C acetate as a tracer, combined with 3D cell clusters and a bioreactor system, our approach enables real-time detection of inhibitors that target fatty acid metabolism within living cells. As a result, we successfully demonstrated the initial application of this method in the discovery of traditional Chinese medicines that specifically target fatty acid metabolism. Elucidating the mechanisms behind herbal medicines remains challenging due to the complex nature of their compounds and the presence of multiple targets. Remarkably, our findings demonstrate the significant inhibitory effect of P. cocos on fatty acid synthesis within cells, illustrating the potential of this approach in analyzing fatty acid metabolism events and identifying drug candidates that selectively inhibit fatty acid synthesis at the cellular level. Moreover, this systematic approach represents a valuable strategy for discovering the intricate effects of herbal medicine.

A comprehensive investigation on the chemical changes of traditional Chinese medicine with classic processing technology: Polygonum multiflorum under nine cycles of steaming and sunning as a case study.

The processing of traditional Chinese medicine (TCM) plays an important role in the clinical application, which usually has the function of "increasing efficiency and reducing toxicity". Polygonum multiflorum (PM) has been reported to induce hepatotoxicity, while it is believed that the toxicity is reduced after processing. Studies have shown that the hepatotoxicity of PM is closely related to the changes in chemical components before and after processing. However, there is no comprehensive investigation on the chemical changes of PM during the processing progress. In this research, we established a comprehensive method to profile both small molecule compounds and polysaccharides from raw and different processed PM samples. In detail, an online two-dimensional liquid chromatography coupled with quadrupole-orbitrap mass spectrometry (2D-LC/Q-Orbitrap MS) was utilized to investigate the small molecules, and a total of 150 compounds were characterized successfully. After multivariate statistical analysis, 49 differential compounds between raw and processed products were screened out. Furthermore, an accurate and comprehensive method for quantification of differential compounds in PM samples was established based on ultra-high performance liquid chromatography/Q-Orbitrap-MS (UHPLC/Q-Orbitrap-MS) within 16 min. In addition, the changes of polysaccharides in different PM samples were analyzed, and it was found that the addition of black beans and steaming times would affect the content and composition of polysaccharides in PM significantly. Our work provided a reference basis for revealing the scientific connotation of the processing technology and increasing the quality control and safety of PM.

MAL, a potential immunotherapy target, is associated with poor prognosis in cancer patients.

The MAL gene encodes Myelin and Lymphocyte Protein, mainly expressed in T cells with immunomodulatory effects, showing the potential as a target for immunotherapy. However, the mechanism of MAL in the regulation of immune infiltration and its association with the prognosis in pan-cancer patients remain elusive. We used the TCGA, TIMER2.0, GTEx, UCSC, and TISCH databases and the R programming tool to explore the role of MAL in cancers. MAL was differently expressed in the majority of malignancies relative to the matched healthy controls. Patients with low MAL levels had adverse survival outcomes in the BRCA and LUAD cohorts. In all cancer types, MAL showed a significant correlation to specific immune-subpopulation abundance in particular T cells as well as B cells. MAL was also implicated in immunological pathways in BRCA and LUAD, suggesting the important role of MAL in cancer immune regulation. In conclusion, the pan-cancer study indicates that MAL with excellent prognostic value is a potential immunotherapy target in multiple cancers.

Integrated metabolic and transcriptomic profiles reveal the germination-associated dynamic changes for the seeds of Cassia obtusifolia L.

INTRODUCTION: The seeds of Cassia obtusifolia L. (Cassiae [C.] semen) have been widely used as both food and traditional Chinese medicine in China. OBJECTIVES: We aimed to analyze the metabolic mechanisms underlying C. semen germination. MATERIALS AND METHODS: Different samples of C. semen at various germination stages were collected. These samples were subjected to 1 H-NMR and UHPLC/Q-Orbitrap-MS-based untargeted metabolomics analysis together with transcriptomics analysis. RESULTS: A total of 50 differential metabolites (mainly amino acids and sugars) and 20 key genes involved in multiple pathways were identified in two comparisons of different groups (36 h vs 12 h and 84 h vs 36 h). The metabolite-gene network for seed germination was depicted. In the germination of C. semen, fructose and mannose metabolism was activated in the testa rupture period, indicating more energy was needed (36 h). In the embryonic axis elongation period (84 h), the pentose and glucuronate interconversions pathway and the phenylpropanoid biosynthesis pathway were activated, which suggested some nutrient sources (nitrogen and sugar) were in demand. Furthermore, oxygen, energy, and nutrition should be supplied throughout the whole germination process. These global views open up an integrated perspective for understanding the complex biological regulatory mechanisms during the germination process of C. semen.

Metabolomics safety assessments of microcystin exposure via drinking water in rats.

Drinking water exposure to microcystin-leucine-arginine (MC-LR), the most widely occurring cyanotoxins, poses a highly potential risk for human health. However, the health risk of MC-LR exposure at current guideline value in drinking water has not yet entirely evaluated. In the current study, we used 1H NMR-based metabolomics combined with targeted metabolic profiling by GC/LC-MS to explore the toxic effects of MC-LR exposure at environmentally relevant concentrations via drinking water in rats. The results revealed that multiple biological consequences of MC-LR exposure on host metabolism in rats. Both relatively low and high doses of MC-LR used here induced hepatic lipogenesis and inflammation. While only relatively high dose MC-LR (10 µg/L) in drinking water caused more metabolic disorders including inhibition of gluconeogenesis and promotion of ß-oxidation of fatty acid. Although the dose of 1.0 µg/L MC-LR is extremely low for rats, alterations of metabolic profiles were unexpectedly found in rat liver and serum, alarming potential health risk of MC-LR at the WHO guideline level.

The composition differences between small black beans and big black beans from different habitats and its effects on the processing of Polygonum multiflorum.

INTRODUCTION: The roots of Polygonum multiflorum (PM) serve as a classical traditional Chinese medicine (TCM), which has multiple biological activities. However, many cases of hepatotoxicity in PM have been reported in recent years. Processing PM with black beans decoction is one of the typical processing methods to reduce the hepatotoxicity of PM since ancient times. OBJECTIVES: To find potential effective constituents, as well as the optimal variety and origin of black beans for the processing of PM. METHODS: Based on ultrahigh-performance liquid chromatography Q-Orbitrap mass spectrometry (UHPLC-Q-Orbitrap-MS) analysis, we measured the contents of the two potential toxic compounds (emodin-8-O-glucoside and torachrysone-O-hexose) in raw PM (R-PM), PM processed with big black beans (B-PM) and PM processed with small black beans (S-PM). The flow cytometry method analysed the effects of different processed products of PM on apoptosis of L02 cells in different drug concentration. Proton nuclear magnetic resonance (1 H-NMR) and UHPLC-Q-Orbitrap-MS together with multivariate statistical analysis were used to systematically analyse the different components between small black beans (Small-BB) and big black beans (Big-BB) from 30 different habitats. RESULTS: The toxicity was ranked from small to large: S-PM < B-PM < R-PM. Processing PM with black beans could significantly decrease the apoptosis rate of L02 cells, especially when the drug concentration is 80 µg/mL. Besides, we find five differential compounds (α-arabinose, α-galactose, proline, isomer of daidzein and isomer of genistein) may be potential active ingredients. In terms of the black beans collected from 30 producing areas, we find that Small-BB from Weifang in Shandong province was optimum to processing PM, followed by Shangqiu in Henan province, Jilin and Liaoning province. CONCLUSION: The ingredients that affect the processing of PM may be attributed to α-arabinose, α-galactose, proline, isomer of daidzein and isomer of genistein in black beans. When the drug concentration is higher, the effect of reducing the hepatotoxicity of PM is better. Besides, Small-BB was more effective than Big-BB for reducing the toxicity of PM, especially Small-BB from Weifang in Shandong, Shangqiu in Henan province and northeast China.

Metabolic Changes of Fusarium graminearum Induced by TPS Gene Deletion.

Fusarium head blight (FHB) mainly resulting from Fusarium graminearum (Fg) Schwabe is a notorious wheat disease causing huge losses in wheat production globally. Fg also produces mycotoxins, which are harmful to human and domestic animals. In our previous study, we obtained two Fg mutants, TPS1- and TPS2-, respectively, with a single deletion of trehalose 6-phosphate synthase (TPS1) and trehalose 6-phosphate phosphatase (TPS2) compared with the wild type (WT). Both mutants were unable to synthesize trehalose and produced fewer mycotoxins. To understand the other biochemical changes induced by TPS gene deletion in Fg, we comprehensively analyzed the metabolomic differences between TPS- mutants and the WT using NMR together with gas chromatography-flame ionization detection/mass spectrometry. The expression of some relevant genes was also quantified. The results showed that TPS1- and TPS2- mutants shared some common metabolic feature such as decreased levels for trehalose, Val, Thr, Lys, Asp, His, Trp, malonate, citrate, uridine, guanosine, inosine, AMP, C10:0, and C16:1 compared with the WT. Both mutants also shared some common expressional patterns for most of the relevant genes. This suggests that apart from the reduced trehalose biosynthesis, both TPS1 and TPS2 have roles in inhibiting glycolysis and the tricarboxylic acid cycle but promoting the phosphopentose pathway and nucleotide synthesis; the depletion of either TPS gene reduces the acetyl-CoA-mediated mycotoxin biosynthesis. TPS2- mutants produced more fatty acids than TPS1- mutants, suggesting different roles for TPS1 and TPS2, with TPS2- mutants having impaired trehalose biosynthesis and trehalose 6-phosphate accumulation. This may offer opportunities for developing new fungicides targeting trehalose biosynthesis in Fg for FHB control and mycotoxin reduction in the FHB-affected cereals.

Dynamic metabolic responses of brown planthoppers towards susceptible and resistant rice plants.

Brown planthopper (Nilaparvata lugens Stål, BPH) causes huge economic losses in rice-growing regions, and new strategies for combating BPH are required. To understand how BPHs respond towards BPH-resistant plants, we systematically analysed the metabolic differences between BPHs feeding on the resistant and susceptible plants using NMR and GC-FID/MS. We also measured the expression of some related genes involving glycolysis and biosyntheses of trehalose, amino acids, chitin and fatty acids using real-time PCR. BPH metabonome was dominated by more than 60 metabolites including fatty acids, amino acids, carbohydrates, nucleosides/nucleotides and TCA cycle intermediates. After initial 12 h, BPHs feeding on the resistant plants had lower levels of amino acids, glucose, fatty acids and TCA cycle intermediates than on the susceptible ones. The levels of these metabolites recovered after 24 h feeding. This accompanied with increased level in trehalose, choline metabolites and nucleosides/nucleotides compared with BPH feeding on the susceptible plants. Decreased levels of BPH metabolites at the early feeding probably resulted from less BPH uptakes of sap from resistant plants and recovery of BPH metabolites at the later stage probably resulted from their adaptation to the adverse environment with their increased hopping frequency to ingest more sap together with contributions from yeast-like symbionts in BPHs. Throughout 96 h, BPH feeding on the resistant plants showed significant up-regulation of chitin synthase catalysing biosynthesis of chitin for insect exoskeleton, peritrophic membrane lining gut and tracheae. These findings provided useful metabolic information for understanding the BPH-rice interactions and perhaps for developing new BPH-combating strategies.

Functional inactivation of UDP-N-acetylglucosamine pyrophosphorylase 1 (UAP1) induces early leaf senescence and defence responses in rice.

Plant leaf senescence and defence responses are important biological processes, but the molecular mechanisms involved are not well understood. This study identified a new rice mutant, spotted leaf 29 (spl29). The SPL29 gene was identified by map-based cloning, and SPL29 was confirmed as UDP-N-acetylglucosamine pyrophosphorylase 1 (UAP1) by enzymatic analysis. The mutant spl29 lacks UAP activity. The biological phenotypes for which UAP is responsible have not previously been reported in plants. The spl29 mutant displayed early leaf senescence, confirmed by chlorophyll loss and photosystem II decline as physiological indicators, chloroplast degradation as a cellular characteristic, and both upregulation of senescence transcription factors and senescence-associated genes, and downregulation of photosynthesis-related genes, as molecular evidence. Defence responses were induced in the spl29 mutant, shown by enhanced resistance to bacterial blight inoculation and upregulation of defence response genes. Reactive oxygen species, including O2 (-) and H2O2, accumulated in spl29 plants; there was also increased malondialdehyde content. Enhanced superoxide dismutase activity combined with normal catalase activity in spl29 could be responsible for H2O2 accumulation. The plant hormones jasmonic acid and abscisic acid also accumulated in spl29 plants. ROS and plant hormones probably play important roles in early leaf senescence and defence responses in the spl29 mutant. Based on these findings, it is suggested that UAP1 is involved in regulating leaf senescence and defence responses in rice.

Proteomics Analysis Reveals the Underlying Factors of Mucilage Disappearance in Brasenia schreberi and Its Influence on Nutrient Accumulation.

Brasenia schreberi J.F. Gmel (BS) is rich in mucilage, which has diverse biological activities, and is utilized in the food and pharmaceutical industries due to its nutritional value. Proteomics analysis was employed to investigate the cause of mucilage disappearance in BS and its effect on nutrient accumulation. Among the 2892 proteins identified, 840 differentially expressed proteins (DEPs) were found to be involved in mucilage development. By comparing the expression patterns and functions and pathway enrichment, the DEPs mainly contributed to carbon and energy metabolism, polysaccharide metabolism, and photosynthesis. Our study also revealed positive correlations between mucilage accumulation and tryptophan metabolism, with high levels of indole-3-acetic (IAA) contributing to mucilage accumulation. Furthermore, environmental changes and particularly excessive nutrients were found to be detrimental to mucilage synthesis. Overall, in the absence of various stimuli in the growing environment, BS accumulates more nutrients within the plant itself instead of producing mucilage.

Combined NMR and MS-based metabonomics and real-time PCR analyses reveal dynamic metabolic changes of Ganoderma lucidum during fruiting body growing.

Ganoderma lucidum (G. lucidum) is a rare medicinal fungus with various beneficial properties. One of its main components, ganoderic acids (GAs), are important triterpenoids known for their sedative and analgesic, hepatoprotective, and anti-tumor activities. Understanding the growth and development of the G. lucidum fruiting body is crucial for determining the optimal time to harvest them. In this study, we used nuclear magnetic resonance (NMR) spectroscopy to systematically characterize the metabolites of G. lucidum at seven distinct developmental stages. We also measured the contents of seven kinds of GAs using LC-MS/MS. A total of 49 metabolites were detected in G. lucidum, including amino acids, sugars, organic acids and GAs. During the transition from the bud development period (I) to the budding period (II), we observed a rapid accumulation of glucose, tyrosine, nicotinamide ribotide, inosine and GAs. After the budding period, the contents of most metabolites decreased until the mature period (VII). In addition, the contents of GAs showed an initial raising, followed by a decline during the elongation period, except for GAF, which exhibited a rapid raise during the mature stage. We also detected the expression of several genes involved in GA synthesis, finding that most genes including 16 cytochrome P450 monooxygenase were all down-regulated during periods IV and VII compared to period I. These findings provide valuable insights into the dynamic metabolic profiles of G. lucidum throughout its growth stage, and it is recommended to harvest G. lucidum at period IV.

[Treatment of chronic primary glomerulopathy patients of Shen deficiency and dampness heat syndrome by yishen qingli granule combined low-dose Tripterygium wilfordii multiglycoside tablet: a clinical efficacy observation].

OBJECTIVE: To evaluate the clinical efficacy and safety of treatment of chronic primary glomerulopathy (CPG) patients of Shen deficiency and dampness heat syndrome (SDDHS) by Yishen Qingli Granule (YQG) combined with low-dose Tripterygium Wilfordii multiglycoside Tablet (TWT). METHODS: Totally 231 CPG patients of SDDHS were enrolled in this study (including 60 patients from First Affiliated Hospital of Nanjing University of Chinese Medicine, 58 from First Affiliated Hospital of Nanjing Medical University, 46 from Xinqiao Hospital of Third Military Medical University, 35 from First Affiliated Hospital of Guangzhou University of Chinese Medicine, 14 from First Affiliated Hospital of Soochow University, and 18 from Wuxi Affiliated Hospital of Nanjing University of Chinese Medicine). They were randomly assigned to the control group (116 cases) and the trial group (115 cases) according to block group method. There were 217 cases in the safety analysis set (109 cases in the trial group vs 108 cases in the control group), and 203 cases in the full analysis set (99 cases in the trial group vs 104 cases in the control group). All patients received basic treatment such as ACEI/ARB. Furthermore, YQG (consisting of raw astragalus 10 g, prepared Polygonum Multiflorum 10 g, Pyrrosia 10 g, 1.5 g each package, containing 10 g of crude drugs) was additionally given to patients in the trial group, each package, twice daily. The TWT (10 mg) was given, twice a day. The TWT dose was adjusted according to 24 h urinary total protein (UTP). The placebos of YQG and TWT were administered to those in the control group. The treatment course consisted of 24 weeks and the follow-up visit lasted for 24 weeks. The biochemical indices were observed before and after treatment including 24 h UTP, urine red cell count (U(RBC)), renal functions (BUN, SCr), blood routine test (WBC), and liver functions (SGPT, SGOT). Reverse reactions such as gastrointestinal discomfort, skin rash, and irregular menstruation were also observed. RESULTS: Compared with the control group, the total effective rate was better in the trial group (82.83% vs 61.54%, P < 0.01). Results of stratified comparison of UTP showed better efficacy in the trial group (0.8-3.0 g/24 h, P < 0.01). The UTP decline occurred in the trial group after 8 weeks of treatment, with stable action, showing statistical difference when compared with the control group (P < 0.01). In the trial group, U(RBC) level decreased after treatment but changed more significantly. But there was no statistical difference in the changes when compared with the control group (P > 0.05). After treatment, there were no statistical difference in safety indicators such as WBC, SGPT, and SGOT between the two groups after treatment (P > 0.05). CONCLUSION: On the basis of basic treatment such as ACEI/ARB, application of YQG combined with low-dose TWT had better effect in controlling proteinuria of CPG patients, and could help stabilizing their conditions with less adverse reactions.

Combination of peak-picking and binning for NMR-based untargeted metabonomics study.

In NMR-based untargeted metabolomic studies, 1H NMR spectra are usually divided into equal bins/buckets to diminish the effects of peak shift caused by sample status or instrument instability, and to reduce the number of variables used as input for the multivariate statistical analysis. It was noticed that the peaks near bin boundaries may cause significant changes in integral values of adjacent bins, and the weaker peak may be obscured if it is allocated in the same bin with intense peaks. Several efforts have been taken to improve the performance of binning. Here we propose an alternative method, named P-Bin, based on the combination of the classic peak-picking and binning procedures. The location of each peak defined by peak-picking is used as the center of the individual bin. P-Bin is expected to keep all spectral information associated with the peaks and significantly reduce the data size as the spectral regions without peaks are not considered. In addition, both peak-picking and binning are routine procedures, making P-Bin easy to be implemented. To verify the performance, two sets of experimental data from human plasma and Ganoderma lucidum (G. lucidum) extracts were processed using the conventional binning method and the proposed method, before the principal component analysis (PCA) and the orthogonal projection to latent structures discriminant analysis (OPLS-DA). The results indicate that the proposed method has improved both the clustering performance of PCA score plots and the interpretability of OPLS-DA loading plots, and P-Bin could be an improved version of data preparation for metabonomic study.

Identification of ALG3 as a potential prognostic biomarker in lung adenocarcinoma.

Background: The abnormal expression of Alpha-1,3-mannosyltransferase (ALG3) has been implicated in tumor promotion. However, the clinical significance of ALG3 in Lung Adenocarcinoma (LUAD) remains poorly understood. Therefore, we aimed to assess the prognostic value of ALG3 and its association with immune infiltrates in LUAD. Methods: The transcriptional expression profiles of ALG3 were obtained from the Cancer Genome Atlas (TCGA), comparing lung adenocarcinoma tissue with normal tissues. To determine the prognostic significance of AGL3, Kaplan-Meier plotter, and Cox regression analysis were employed. Logistic regression was utilized to analyze the association between ALG3 expression and clinical characteristics. Additionally, a receiver operating characteristic (ROC) curve and a nomogram were constructed. To explore the underlying mechanisms, the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis and gene set enrichment analysis (GSEA) was conducted. The relationship between AGL3A mRNA expression and immune infiltrates was investigated using the tumor immune estimation resource (TIMER) and tumor-immune system interaction database (TISIDB). Furthermore, an in vitro experiment was performed to assess the impact of ALG3 mRNA on lung cancer stemness abilities and examine key signaling pathway proteins. Results: Our results revealed the ALG3 mRNA and protein expression in patients with LUAD was much higher than that in adjacent normal tissues. High expression of ALG3 was significantly associated with N stage (N0, HR = 1.98, P = 0.002), pathological stage (stage I, HR = 2.09, P = 0.003), and the number of pack years (<40, HR = 2.58, P = 0.001). Kaplan-Meier survival analysis showed that high expression of ALG3 was associated with poor overall survival (P < 0.001), disease-free survival (P < 0.001), and progression-free interval (P = 0.007). Through multivariate analysis, it was determined that elevated ALG3 expression independently impacted overall survival (HR = 1.325, P = 0.04). The Tumor Immune Estimation Resource discovered a link between ALG3 expression and tumor-infiltrating immune cells in LUAD. Additionally, ROC analysis proved that ALG3 is a reliable diagnostic marker for LUAD (AUC:0.923). Functional pathways analysis identified that ALG3 is negatively correlated with FAT4. We performed qRT-PCR to assess that knockdown ALG3 expression significantly upregulated FAT4 expression. Spheroid assay and flow cytometry analysis results showed that downregulated of ALG3 inhibited H1975 cell line stemness. Western blot analysis revealed that decreased ALG3 inhibited the YAP/TAZ signal pathway. Conclusion: High expression of ALG3 is strongly associated with poor prognosis and immune infiltrates in LUAD.

The Bphi008a gene interacts with the ethylene pathway and transcriptionally regulates MAPK genes in the response of rice to brown planthopper feeding.

We examined ways in which the Brown planthopper induced008a (Bphi008a; AY256682) gene of rice (Oryza sativa) enhances the plant's resistance to a specialist herbivore, the brown planthopper (BPH; Nilaparvata lugens). Measurement of the expression levels of ethylene synthases and of ethylene emissions showed that BPH feeding rapidly initiated the ethylene signaling pathway and up-regulated Bphi008a transcript levels after 6 to 96 h of feeding. In contrast, blocking ethylene transduction (using 1-methylcyclopropene) reduced Bphi008a transcript levels in wild-type plants fed upon by BPH. In vitro kinase assays showed that Bphi008a can be phosphorylated by rice Mitogen-activated Protein Kinase5 (OsMPK5), and yeast two-hybrid assays demonstrated that the carboxyl-terminal proline-rich region of Bphi008a interacts directly with this kinase. Furthermore, bimolecular fluorescence complementation assays showed that this interaction occurs in the nucleus. Subsequently, we found that Bphi008a up-regulation and down-regulation were accompanied by different changes in transcription levels of OsMPK5, OsMPK12, OsMPK13, and OsMPK17 in transgenic plants. Immunoblot analysis also showed that the OsMPK5 protein level increased in overexpressing plants and decreased in RNA interference plants after BPH feeding. In transgenic lines, changes in the expression levels of several enzymes that are important components of the defenses against the BPH were also observed. Finally, yeast two-hybrid screening results showed that Bphi008a is able to interact with a b-ZIP transcription factor (OsbZIP60) and a RNA polymerase polypeptide (SDRP).

The Different Metabolic Responses of Resistant and Susceptible Wheats to Fusarium graminearum Inoculation.

Fusarium head blight (FHB) is a serious wheat disease caused by Fusarium graminearum (Fg) Schwabe. FHB can cause huge loss in wheat yield. In addition, trichothecene mycotoxins produced by Fg are harmful to the environment and humans. In our previous study, we obtained two mutants TPS1- and TPS2-. Neither of these mutants could synthesize trehalose, and they produced fewer mycotoxins. To understand the complex interaction between Fg and wheat, we systematically analyzed the metabolic responses of FHB-susceptible and -resistant wheat to ddH2O, the TPS- mutants and wild type (WT) using NMR combined with multivariate analysis. More than 40 metabolites were identified in wheat extracts including sugars, amino acids, organic acids, choline metabolites and other metabolites. When infected by Fg, FHB-resistant and -susceptible wheat plants showed different metabolic responses. For FHB-resistant wheat, there were clear metabolic differences between inoculation with mutants (TPS1-/TPS2-) and with ddH2O/WT. For the susceptible wheat, there were obvious metabolic differences between inoculation with mutant (TPS1-/TPS2-) and inoculation with ddH2O; however, there were no significant metabolic differences between inoculation with TPS- mutants and with WT. Specifically, compared with ddH2O, resistant wheat increased the levels of Phe, p-hydroxy cinnamic acid (p-HCA), and chlorogenic acid in response to TPS- mutants; however, susceptible wheat did not. Shikimate-mediated secondary metabolism was activated in the FHB-resistant wheat to inhibit the growth of Fg and reduce the production of mycotoxins. These results can be helpful for the development of FHB-resistant wheat varieties, although the molecular relationship between the trehalose biosynthetic pathway in Fg and shikimate-mediated secondary metabolism in wheat remains to be further studied.

A rice ß-1,3-glucanase gene Osg1 is required for callose degradation in pollen development.

Plant ß-1,3-glucanases are involved in plant defense and development. In rice (Oryza sativa), 14 genes encoding putative ß-1,3-glucanases have been isolated and sequenced. However, only limited information is available on the function of these ß-1,3-glucanase genes. In this study, we report a detailed functional characterization of one of these genes, Osg1. Osg1 encodes a glucanase carrying no C-terminal extension. Osg1 was found to be expressed throughout the plant and highly expressed in florets, leaf sheaths, and leaf blades. Investigations using real-time PCR, immunocytochemical analysis, and a GUS-reporter gene driven by the Osg1 promoter indicated that Osg1 was mainly expressed at the late meiosis, early microspore, and middle microspore stages in the florets. To elucidate the role of Osg1, we suppressed expression of the Osg1 gene by RNA interference in transgenic rice. The silencing of Osg1 resulted in male sterility. The pollen mother cells appeared to be normal in Osg1-RI plants, but callose degradation was disrupted around the microspores in the anther locules of the Osg1-RI plants at the early microspore stage. Consequently, the release of the young microspores into the anther locules was delayed, and the microspores began to degenerate later. These results provide evidence that Osg1 is essential for timely callose degradation in the process of tetrad dissolution.

Endovascular treatment of hemodialysis-induced lower limb artery ischemia: retrospective analysis from a single center.

BACKGROUND: Critical limb artery ischemia is one of common complications after hemodialysis, with endovascular therapy (EVT) having become its first-line treatment. There is no relevant study investigating the relationship between EVT and the prognosis of hemodialysis patients with critical lower limb ischemia, the most common site of vascular dysfunction. METHODS: This was a retrospective, nonrandomized, single-center study. Hemodialysis patients with critical lower limb ischemia between May 2015 and October 2018 were included in this study. Their demographic and clinical data and the results of laboratory test were collected. The outcomes included all-cause mortality, amputation, and revascularization. Kaplan-Meier analysis and log-rank test were used to assess overall survival and amputation-free survival. Univariable and multivariable hazard Cox regression analyses were performed to determine risk factors of amputation and mortality. RESULTS: In all, 67 hemodialysis patients were finally included in this study. The median age of included patients was 69.8±8.7 years, and the median duration of hemodialysis was 44.1±9.2 months. There was no significant difference between patients receiving and not receiving EVT in collected demographic and clinical data except for the duration of hemodialysis (46.1±9.0 vs. 41.7±9.0 months; P=0.048). The level of high-density lipoprotein cholesterol (HDL-C) in patients receiving EVT was 1.4±0.6 mmol/L, which was significantly lower than the 1.9±0.6 mmol/L in patients not receiving EVT (P<0.001). The results from the Kaplan-Meier curves indicated that the incidences of all-cause mortality and amputation were much lower in patients receiving EVT than in those not receiving EVT (P=0.038 and P=0.020). Hazard Cox regression analysis also indicated that EVT played protective role in all-cause mortality and amputation in hemodialysis patients with lower limb ischemia. Age, nutritional risk, stroke, and C-reactive protein (CRP) were also determined as independent risk factors of all-cause mortality according to multivariable analysis. Additionally, duration of hemodialysis and smoking history were identified as independent risk factors of amputation. CONCLUSIONS: EVT could be an efficient treatment for critical lower limb ischemia in hemodialysis patients to reduce all-cause mortality and the incidence of amputation. Moreover, some risk factors, such as malnutritional and stroke, should be avoided to improve the prognosis of hemodialysis patients.

Global Analysis of UDP Glucose Pyrophosphorylase (UDPGP) Gene Family in Plants: Conserved Evolution Involved in Cell Death.

UDP glucose pyrophosphorylase (UDPGP) family genes have been reported to play essential roles in cell death or individual survival. However, a systematic analysis on UDPGP gene family has not been performed yet. In this study, a total of 454 UDPGP proteins from 76 different species were analyzed. The analyses of the phylogenetic tree and orthogroups divided UDPGPs into three clades, including UDP-N-acetylglucosamine pyrophosphorylase (UAP), UDP-glucose pyrophosphorylase (UGP, containing UGP-A and UGP-B), and UDP-sugar pyrophosphorylase (USP). The evolutionary history of the UDPGPs indicated that the members of UAP, USP, and UGP-B were relatively conserved while varied in UGP-A. Homologous sequences of UGP-B and USP were found only in plants. The expression profile of UDPGP genes in Oryza sativa was mainly motivated under jasmonic acid (JA), abscisic acid (ABA), cadmium, and cold treatments, indicating that UDPGPs may play an important role in plant development and environment endurance. The key amino acids regulating the activity of UDPGPs were analyzed, and almost all of them were located in the NB-loop, SB-loop, or conserved motifs. Analysis of the natural variants of UDPGPs in rice revealed that only a few missense mutants existed in coding sequences (CDSs), and most of the resulting variations were located in the non-motif sites, indicating the conserved structure and function of UDPGPs in the evolution. Furthermore, alternative splicing may play a key role in regulating the activity of UDPGPs. The spatial structure prediction, enzymatic analysis, and transgenic verification of UAP isoforms illustrated that the loss of N- and C-terminal sequences did not affect the overall 3D structures, but the N- and C-terminal sequences are important for UAP genes to maintain their enzymatic activity. These results revealed a conserved UDPGP gene family and provided valuable information for further deep functional investigation of the UDPGP gene family in plants.