OsWRKY62 and OsWRKY76 Interact with Importin α1s for Negative Regulation of Defensive Responses in Rice Nucleus.

Background OsWRKY62 and OsWRKY76, two close members of WRKY transcription factors, function together as transcriptional repressors. OsWRKY62 is predominantly localized in the cytosol. What are the regulatory factors for OsWRKY62 nuclear translocation? Results In this study, we characterized the interaction of OsWRKY62 and OsWRKY76 with rice importin, OsIMα1a and OsIMα1b, for nuclear translocation. Chimeric OsWRKY62.1-GFP, which is predominantly localized in the cytoplasm, was translocated to the nucleus of Nicotiana benthamiana leaf cells in the presence of OsIMα1a or OsIMαΔIBB1a lacking the auto-inhibitory importin ß-binding domain. OsIMαΔIBB1a interacted with the WRKY domain of OsWRKY62.1, which has specific bipartite positively charged concatenated amino acids functioning as a nuclear localization signal (NLS). Similarly, we found that OsIMαΔIBB1a interacted with the AvrPib effector of rice blast fungus Magnaporthe oryzae, which contains a scattered distribution of positively charged amino acids. Furthermore, we identified a nuclear export signal (NES) in OsWRKY62.1 that inhibited nuclear transportation. Overexpression of OsIMα1a or OsIMα1b enhanced resistance to M. oryzae, whereas knockout mutants decreased resistance to the pathogen. However, overexpressing both OsIMα1a and OsWRKY62.1 were slightly more susceptible to M. oryzae than OsWRKY62.1 alone. Ectopic overexpression of OsWRKY62.1-NES fused gene compromised the enhanced susceptibility of OsWRKY62.1 to M. oryzae. Conclusion These results revealed the existence of NLS and NES in OsWRKY62. OsWRKY62, OsWRKY76, and AvrPib effector translocate to nucleus in association with importin α1s through new types of nuclear localization signals for negatively regulating defense responses.

Research progress on febrile non-hemolytic transfusion reaction: a narrative review.

Background and Objective: About 1% of patients who receive blood transfusions will develop transfusion reactions. Febrile non-hemolytic transfusion reaction (FNHTR) is the most common type of transfusion reaction. It not only leads to misdiagnosis and delayed treatment, but also incurs a huge economic burden. This article reviews FNHTR systematically, aiming to make clinicians have a more comprehensive understanding of FNHTR and reduce the occurrence of this side effect. Methods: A comprehensive search of the PubMed, Embase, and Cochrane Library databases was performed. Medical Subject Headings (MeSH) included Blood Transfusion, Transfusion Reaction, and Febrile Non-Hemolytic Transfusion Reaction. The searches and literature screening were performed by 2 researchers; any differences of opinion or results were resolved through negotiation. Key Content and Findings: The pathophysiological mechanisms of FNHTR mainly included immune and non-immune pathways. The former was associated with antibodies against human leukocyte antigen (HLA) produced in transfused patients, while the latter was associated with cytokines released from blood products during storage. Women with a reproductive history and those patients with multiple blood transfusions were more likely to experience FNHTR. Primary hematologic disease, malignant disease, and transfusion with over 6 units of leukocyte-depleted packed red blood cells were independent risk factors for the development of FNHTR. FNHTR could be diagnosed by accompaniment of the fever symptom (body temperature ≥38 ℃, maybe an increase of body temperature of more than 1 ℃ compared with that before blood transfusion) during or within 4 hours after transfusion, or the presence of chills, shakes, headache, and nausea, among other symptoms. FNHTR should be mainly differentiated from other types of transfusion reactions with similar symptoms. Prophylactic strategies for the routine use of antipyretic drugs before transfusion remain controversial. Removal of leukocyte components from blood could reduce the incidence of FNHTR significantly. Conclusions: The pathogenesis of FNHTR is mainly associated with anti-HLA antibodies and cytokines released from blood products during storage. Specific markers and effective detection methods for FNHTR are still lacking. Treatment for FNHTR is currently limited to antipyretic drugs, sedation, and other symptomatic treatment measures. More studies are warranted to focus on the pathological mechanism of FNHTR.

Citrus sinensis MYB Transcription Factor CsMYB85 Induce Fruit Juice Sac Lignification Through Interaction With Other CsMYB Transcription Factors.

Varieties of Citrus are commercially important fruits that are cultivated worldwide and are valued for being highly nutritious and having an appealing flavor. Lignification of citrus fruit juice sacs is a serious physiological disorder that occurs during postharvest storage, for which the underlying transcriptional regulatory mechanisms remain unclear. In this study, we identified and isolated a candidate MYB transcription factor, CsMYB85, that is involved in the regulation of lignin biosynthesis in Citrus sinensis, which has homologs in Arabidopsis and other plants. We found that during juice sac lignification, CsMYB85 expression levels increase significantly, and therefore, suspected that this gene may control lignin biosynthesis during the lignification process. Our results indicated that CsMYB85 binds the CsMYB330 promoter, regulates its expression, and interacts with CsMYB308 in transgenic yeast and tobacco. A transient expression assay indicated that Cs4CL1 expression levels and lignin content significantly increased in fruit juice sacs overexpressing CsMYB85. At4CL1 expression levels and lignin content were also significantly increased in Arabidopsis overexpressing CsMYB85. We accordingly present convincing evidence for the participation of the CsMYB85 transcription factor in fruit juice sac lignification, and thereby provide new insights into the transcriptional regulation of this process in citrus fruits.

Determination of hemolysis index thresholds for biochemical tests on Siemens Advia 2400 chemistry analyzer.

BACKGROUND: In vitro hemolysis is still the most common source of pre-analytical nonconformities. This study aimed to investigate the hemolytic effects on commonly used biochemical tests as well as to determine the hemolysis index (HI) thresholds on Siemens Advia 2400 chemistry analyzer. METHODS: Peripheral blood samples were collected from forty healthy volunteers. Hemolysis was achieved using syringes. Five hemolysis levels were produced including the no hemolysis group, slight hemolysis group, mild hemolysis group, moderate hemolysis group, and heavy hemolysis group. We then used the bias from baseline (no hemolysis) and HI to construct regression functions. The HI corresponding to the bias limits was considered as HI thresholds. We chose the total allowable error (TAE) as the bias limit. RESULTS: Of the twenty-eight analytes, ten analytes had clinical significance. Creatine kinase-MB, creatine kinase, potassium, aspartate aminotransferase, and hydroxybutyrate dehydrogenase were all positively affected; the corresponding HI threshold was 45.2, 99.96, 4.07, 10.16, and 7.94, respectively. Lactate dehydrogenase was also positively interfered, but we failed to calculate the HI threshold. Total bile acid, uric acid, and sodium were all negatively affected, and the HI threshold was 42.23, 500 and 501.8, respectively. Glucose was also negatively interfered, but it failed to achieve the HI threshold. CONCLUSIONS: When the HI value was higher than its threshold, the corresponding analyte was considered inappropriate for reporting. The implementation of the assay-specific HI thresholds could provide an accurate method to identify analytes interfered by hemolysis, which would improve clinical interpretations and further boost laboratory quality by reducing errors associated with hemolysis.

Citrus sinensis MYB transcription factors CsMYB330 and CsMYB308 regulate fruit juice sac lignification through fine-tuning expression of the Cs4CL1 gene.

Lignin is one of the most important components of the plant cell wall, and the expression and transcriptional regulation of lignin biosynthesis-related genes have been studied widely in Arabidopsis and other plants. Citrus fruit juice sacs often undergo lignification, particularly during fruit ripening and storage periods; however, the underlying genetic mechanisms have been little investigated. In this study, we isolated and identified CsMYB330 and CsMYB308 transcription factors, and found that their expression levels are significantly altered during the lignification of citrus fruit juice sacs. We found that CsMYB330 and CsMYB308 can recognize and bind AC elements in the Cs4CL1 promoter and finely regulate expression of the Cs4CL1 gene. In this regulatory process, CsMYB330 was identified as a transcriptional activator, whereas CsMYB308 appears to be a transcriptional repressor. In addition, using a transient assay, we demonstrated that expression of the Cs4CL1 gene is significantly altered in fruit juice sacs overexpressing these two transcription factors. These results indicate that the transcription factors CsMYB330 and CsMYB308 play important roles in the lignification of citrus fruit juice sacs and provide novel insights into the transcriptional regulation associated with fruit juice sac lignification.

m6A demethylase FTO facilitates tumor progression in lung squamous cell carcinoma by regulating MZF1 expression.

N6-Methyladenosine (m6A) represents the most prevalent internal modification in mammalian mRNAs. Emerging evidences suggest that m6A modification is profoundly implicated in many biological processes, including cancer development. However, limited knowledge is available about the functional importance of m6A in lung cancer. In this study, by data mining The Cancer Genome Atlas (TCGA) database, we first identified fat mass- and obesity-associated protein (FTO) as a prognostic factor for lung squamous cell carcinoma (LUSC). Then we showed that FTO, but not other m6A modification genes including METTL3, METTL14 and ALKBH5, was the major dysregulated factor responsible for aberrant m6A modification in LUSC. Loss-of-function studies suggested that FTO knockdown effectively inhibited cell proliferation and invasion, while promoted cell apoptosis of L78 and NCI-H520 cells. Furthermore, overexpression of FTO, but not its mutant form, facilitated the malignant phenotypes of CHLH-1 cells. Mechanistically, FTO enhanced MZF1 expression by reducing m6A levels and mRNA stability in MZF1 mRNA transcript, leading to oncogenic functions. Taken together, our study demonstrates the functional importance of FTO in the tumor progression of LUSC and provides a potential therapeutic target for LUSC treatment.

Alternative Splicing of Rice WRKY62 and WRKY76 Transcription Factor Genes in Pathogen Defense.

The WRKY family of transcription factors (TFs) functions as transcriptional activators or repressors in various signaling pathways. In this study, we discovered that OsWRKY62 and OsWRKY76, two genes of the WRKY IIa subfamily, undergo constitutive and inducible alternative splicing. The full-length OsWRKY62.1 and OsWRKY76.1 proteins formed homocomplexes and heterocomplexes, and the heterocomplex dominates in the nuclei when analyzed in Nicotiana benthamiana leaves. Transgenic overexpression of OsWRKY62.1 and OsWRKY76.1 in rice (Oryza sativa) enhanced plant susceptibility to the blast fungus Magnaporthe oryzae and the leaf blight bacterium Xanthomonas oryzae pv oryzae, whereas RNA interference and loss-of-function knockout plants exhibited elevated resistance. The dsOW62/76 and knockout lines of OsWRKY62 and OsWRKY76 also showed greatly increased expression of defense-related genes and the accumulation of phytoalexins. The ratio of full-length versus truncated transcripts changed in dsOW62/76 plants as well as in response to pathogen infection. The short alternative OsWRKY62.2 and OsWRKY76.2 isoforms could interact with each other and with full-length proteins. OsWRKY62.2 showed a reduced repressor activity in planta, and two sequence determinants required for the repressor activity were identified in the amino terminus of OsWRKY62.1. The amino termini of OsWRKY62 and OsWRKY76 splice variants also showed reduced binding to the canonical W box motif. These results not only enhance our understanding of the DNA-binding property, the repressor sequence motifs, and the negative feedback regulation of the IIa subfamily of WRKYs but also provide evidence for alternative splicing of WRKY TFs during the plant defense response.

Identification and expression analysis of NADH-cytochrome b5 reductase gene in the cotton bollworm, Helicoverpa armigera.

NADH-cytochrome b(5) reductase (CBR) is one of the most important components of cytochrome P450s, which play an essential role in the detoxification of xenobiotics as well as insecticide resistance in insect pest. In the present study, two novel full-length cDNAs of CBR of the cotton bollworm, Helicoverpa armigera (Hübner) were amplified by means of reverse transcription-polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends (RACE) techniques. The sequencing results showed that the transcripts were 1809bp and 1518bp for HaCBR1 and HaCBR2, respectively, including 969bp and 939bp of complete open reading frame (ORF), which encoded 322 and 312 amino acids respectively. The putative structure and function of HaCBR1 and HaCBR2 were preliminarily analyzed by SMART program. HaCBR1 and HaCBR2 (GenBank accession numbers: HQ638220 and HQ190046HQ638220HQ190046) showed high identities with CBRs of other species. The expression of HaCBR1 and HaCBR2 mRNA was detected by real-time quantitative polymerase chain reaction (RT-qPCR) in most developmental stages of H. armigera with the exception of eggs, as well as in tissues such as cuticle, fatbody and midgut. The expression level of the two genes was significantly induced by phenobarbital (PB). These results would contribute to the understanding of CBR function in H. armigera and provide information for further study on the interactions of different components of cytochrome P450 enzyme systems.

The rice ERF transcription factor OsERF922 negatively regulates resistance to Magnaporthe oryzae and salt tolerance.

Rice OsERF922, encoding an APETELA2/ethylene response factor (AP2/ERF) type transcription factor, is rapidly and strongly induced by abscisic acid (ABA) and salt treatments, as well as by both virulent and avirulent pathovars of Magnaporthe oryzae, the causal agent of rice blast disease. OsERF922 is localized to the nucleus, binds specifically to the GCC box sequence, and acts as a transcriptional activator in plant cells. Knockdown of OsERF922 by means of RNAi enhanced resistance against M. oryzae. The elevated disease resistance of the RNAi plants was associated with increased expression of PR, PAL, and the other genes encoding phytoalexin biosynthetic enzymes and without M. oryzae infection. In contrast, OsERF922-overexpressing plants showed reduced expression of these defence-related genes and enhanced susceptibility to M. oryzae. In addition, the OsERF922-overexpressing lines exhibited decreased tolerance to salt stress with an increased Na(+)/K(+) ratio in the shoots. The ABA levels were found increased in the overexpressing lines and decreased in the RNAi plants. Expression of the ABA biosynthesis-related genes, 9-cis-epoxycarotenoid dioxygenase (NCED) 3 and 4, was upregulated in the OsERF922-overexpressing plants, and NCED4 was downregulated in the RNAi lines. These results suggest that OsERF922 is integrated into the cross-talk between biotic and abiotic stress-signalling networks perhaps through modulation of the ABA levels.

Thymocytes are activated by toluene inhalation through the transcription factors NF-κB, STAT5 and NF-AT.

Toluene has been extensively examined for effects on the central nervous system. To investigate the influence of low-level inhalation of toluene on the naive immune cells, male C3H/HeN mice were exposed to filtered air (control) and 50 ppm of toluene for 3 weeks. Low-level exposure resulted in (1) increased proliferation of thymocytes, (2) IL-2 production induced in thymocytes and (3) activation of the transcription factors NF-κB, STAT5 and NF-AT in thymocytes. These results suggest that thymocytes are sensitive cells and T cell activators are candidates for biomarkers for low-level exposure to toluene on naive immune cells.

[Relationship between red cell volume distribution width and cardiac function in acute coronary syndrome patients].

OBJECTIVE: To evaluate the relationship between red cell volume distribution width (RDW) and cardiac function in acute coronary syndrome (ACS) patients. METHODS: We analyzed the baseline clinical data of 228 ACS patients consecutively admitted into our hospital according to the classification of RDW and NYHA function class. Correlation analysis was made respectively between RDW and plasma creatinine and left ventricular ejection fraction (LVEF). RESULTS: There were significant differences of age [Group Q(1) to Group Q(4): (60 ± 12), (65 ± 11), (64 ± 11), (71 ± 12) years old], plasma creatinine [Group Q(1) to Group Q(4): (70 ± 19), (76 ± 24), (74 ± 20), (85 ± 26) µmol/L] and LVEF [Group Q(1) to Group Q(4): (57 ± 9)%, (55 ± 10)%, (53 ± 11)%, (49 ± 11)%] among different RDW groups (P = 0.0000, 0.0054, 0.0002, respectively). Comparing to NYHA function I, patients of NYHA function IV had a higher RDW (13.9% ± 1.2% vs 13.1% ± 5.7%, P < 0.05). There were significant negative correlations between RDW and LVEF (r = -0.30, P < 0.01). CONCLUSION: RDW may be significantly related to cardiac function in Chinese ACS patients.

Effects of 1-bromopropane, a substitute for chlorofluorocarbons, on BDNF expression.

1-Bromopropane (1-BP) has been widely used as an alternative to ozone-depleting chlorofluorocarbons in various industries. Although the neurotoxicity of 1-BP has been recently reported, there is little information about the effect of 1-BP on the cells in brain by experimental approach. Here we studied the effect of 1-BP on brain-derived neurotrophic factor (BDNF) expression in astrocytes in vitro. The BDNF mRNA level was remarkably decreased by 1-BP in a human astrocytoma cell line, U251, and in mouse primary astrocytes. The DNA-binding and specific reporter activity of cAMP response element-binding transcription factor (CREB), which is one of the key molecules regulating BDNF expression, were reduced by 1-BP in U251 and/or mouse primary astrocytes. Additionally, protein kinase A (PKA) activity was suppressed by 1-BP in U251. These results suggest that BDNF expression was affected by 1-BP through at least PKA.

The indoor air pollutant 2-ethyl-hexanol activates CD4 cells.

It has been reported that the numbers of people suffering from occupational asthma and skin rashes triggered by various chemicals in indoor air have increased markedly. Two-ethyl-hexanol (2-EH) is known to be an indoor air pollutant and its influence on health is of great concern. However, there are only a few reports regarding its effect on immune cells. Thus, we investigated the effects of 2-EH on immune responses in vitro with respect to effects on regulation of transcription factors as well as on 2-EH induced proliferation of spleen cells in vitro. The production of interleukin (IL)-6 and immunoglobulin were not induced by 2-EH. To characterize the effector cells of 2-EH, we prepared CD4-positive, CD8-positive, and peritoneal exudate cells (PEC). IL-2 was induced by 2-EH in CD4 cells, but not in CD8 cells. CD3-induced IL-2 expression was enhanced by 2-EH in CD4 cells, but not in CD8 cells. Moreover, IL-6 production was not induced by 2-EH in PEC. Nuclear factor-kappa B, nuclear factor of activated T, and signal transducer and activator of transcription-5 were activated by 2-EH in CD4 cells. Taken together, 2-EH activated CD4 cells, where this was accompanied by the activation of transcription factors. This suggested that the indoor pollutant 2-EH could function as a modulator of immune response.

DNA-binding activity of NF-kappaB and phosphorylation of p65 are induced by N-acetylcysteine through phosphatidylinositol (PI) 3-kinase.

N-Acetylcysteine (NAC) has been widely used as an antioxidant in research, however, it has also been found to reduce the binding of TNF to its receptor independent of its antioxidative role. In this study, we investigated the effect of NAC on NF-kappaB activation. In HeLa cells, Hep3B cells, and A549 cells, DNA-binding activity of NF-kappaB was induced by NAC without any other stimulation but not by tetramethylthiourea (TMTU) or vitamin C, suggesting that ROS is not involved in the effect of NAC. The degradation of IkappaBalpha and nuclear translocation of NF-kappaB were not induced by NAC. The phosphorylation of p65 at serine 536 was induced by NAC, which is known to contribute to the enhancement of DNA-binding activity of NF-kappaB, however, NAC did not directly phosphorylate p65. The NAC-induced DNA-binding activity of NF-kappaB and phosphorylation of p65 were sensitive to a phosphatidylinositol (PI) 3-kinase inhibitor, partially sensitive to an IkappaB kinase (IKK) inhibitor, but not sensitive to a Bruton's tyrosine kinase (Btk) inhibitor. Moreover, both the DNA-binding activity and phosphorylation induced by NAC were reduced by the overexpression of a dominant negative Akt in HeLa cells. These results suggest that NAC activates mainly PI3K to phosphorylate p65 and subsequently induces DNA-binding activity of NF-kappaB, independent of its antioxidative function.

Suppressive effect of reactive oxygen species on CD40-induced B cell activation.

Reactive oxygen species (ROS) produced by the innate immune system work as effectors to destroy pathogens and to control cellular responses. However, their role in the adaptive immune response remains unclear. Here we studied the effect of exogenous ROS on CD40-induced B cell activation. H2O2 treatment inhibited CD40-induced immunoglobulin production of B cells, DNA binding of NF-kappaB, IkappaBalpha degradation and IKK phosphorylation. On the other hand, H2O2 treatment did not induce obvious B cell death after 30 min of stimulation. Although the ligation of anti-CD40 antibody was not disturbed by H2O2, TRAF2 recruitment to CD40 was inhibited. These results suggest that exogenous ROS play a negative role in CD40 signaling during B cell activation.

Changes in the function of the inhibitory neurotransmitter system in the rat brain following subchronic inhalation exposure to 1-bromopropane.

1-Bromopropane (1-BP) has been widely used as a cleaning agent and a solvent in industries, but the central neurotoxicity of 1-BP remains to be clarified. In the present study, we investigated the effects of subchronic inhalation exposure to 1-BP vapor on the function of the inhibitory neurotransmitter system mediated by gamma-aminobutyric acid (GABA) in the rat brain. Male Wistar rats were exposed to 1-BP vapor for 12 weeks (6h/day, 5 days/week) at a concentration of 400 ppm, and, in order to investigate the expression and function of brain GABA type A (GABAA) receptors, total/messenger RNA was prepared from the neocortex, hippocampus, and cerebellum of the control and 1-BP-exposed rats. Moreover, hippocampal slices were prepared, and the population spike (PS) amplitude and the slope of the field excitatory postsynaptic potential (fEPSP) were investigated in the paired-pulse configuration of the extracellular recording technique. Using the Xenopus oocyte expression system, we compared GABA concentration-response curves obtained from oocytes injected with brain subregional mRNAs of control and 1-BP exposed rats, and observed no significant differences in apparent GABA affinity. On the other hand, paired-pulse inhibition of PS amplitude was significantly decreased in the hippocampal dentate gyrus (DG) by exposure to 1-BP, without any effect on the paired-pulse ratio of the fEPSP slopes, suggesting neuronal disinhibition in the DG. Moreover, RT-PCR analysis indicated decreased levels of GABAA receptor beta3 and delta subunit mRNAs in the hippocampus of 1-BP-exposed rats. These results demonstrate that subchronic inhalation exposure to 1-BP vapor reduces the function of the hippocampal GABAergic system, which could be due to changes in the expression and function of GABAA receptors, especially the delta subunit-containing GABAA receptors.

[Study on mechanism of the anti-tumor activity of Acanthopanax gracilistylus].

OBJECTIVE: To study the mechanism of anti-tumor activity of Acanthopanax gracilistylus extract (Age). METHODS: The tumor cells proliferation was detected by using (3H)-TdR incorporation method, and the effects of Age on cell cycle of tumor cells, retinoblastoma (Rb) protein and cyclin-dependent kinases (Cdk) were analyzed by flow cytometry and Western blotting assay, respectively. RESULTS: It was indicated by cytoactivity test in vitro that Age only had effect in inhibiting the proliferation of tumor cells, it couldn't lead to death of cells. Under action of Age, the proliferation of tumor cells was halted at G0/G1 stage of cell cycle, and showed no direct cytotoxic effect by Age. Age could induce lowering of the expression of Rb, Cdk2 and Cdk4, cause halt of tumor cell proliferation. CONCLUSION: The tumor inhibitory effect of Age is realized by way of regulating the activity of cell cycle controlling enzymes to suspend the proliferation of tumor cells.