Genome-wide identification and genetic characterization of the CaMYB family and its response to five types of heavy metal stress in hot pepper (Capsicum annuum cv. CM334).

MYB proteins play a crucial role in plant growth and development and stress responses. In this study, 160 members of the MYB gene family from the pepper genome database were used to analyze gene structures, chromosome localization, collinearity, genetic affinity and expression in response to heavy metals. The results identified R2R3-MYB members and further phylogenetically classified them into 35 subgroups based on highly conserved gene structures and motifs. Collinearity analysis showed that segmental duplication events played a crucial role in the functional expansion of the CaMYB gene family by intraspecific collinearity, and at least 12 pairs of CaMYB genes existed between species prior to the differentiation between monocots and dicots. Moreover, the upstream CaMYB genes were mainly localized to the phytohormone elements ABRE and transcription factor elements MYB and MYC. Further analysis revealed that MYB transcription factors were closely associated with a variety of abiotic stress-related proteins (e.g., MAC-complex and SKIP). Under the stress of five metal ions, Cd2+, Cu2+, Pb2+, Zn2+, and Fe3+, the expression levels of some CaMYB family genes were upregulated. Of these genes, pairing homologous 1 (PH-1), PH-13, and PH-15 in the roots of Capsicum annuum were upregulated to the greatest extent, indicating that these three MYB family members are particularly sensitive to these five metals. This study provides a theoretical reference for the analysis of the molecular regulatory mechanism of MYB family genes in mediating the response to heavy metals in plants. This study reveals the mode of interaction between MYB and a variety of abiotic stress proteins and clarifies the biological functions of CaMYB family members in the regulation of heavy metal stress.

miR-15a regulates oxygen glucose deprivation/reperfusion (OGD/R)-induced neuronal injury by targeting BDNF.

Multiple microRNAs (miRs) have also been implicated in ischemic brain injury. This research intended to probe the regulatory function and the mechanism of miR-15a on the ischemic brain injury induced by oxygen-glucose deprivation/reoxygenation (OGD/R) in neurons of rats. The OGD/R model was established with the cortical neurons separated from rats. After transfection with miR-15a mimic negative control (NC), miR-15a mimic, miR-15a inhibitor NC and miR-15a inhibitor, the OGD/R-induced apoptosis were detected. Using bioinformatic softwares including TargetScan, miRanda, and miRWalk to predict the underlying targets of miR-15a, and the binding of miR-15a with brain-derived neurotrophic factor (BDNF) were validated with double-fluorescein reporter assay system. The expression levels of BDNF mRNA and protein were detected with qRT-PCR and western blot. The effect of miR-15a on PI3K/AKT pathway in neurons submitted to OGD/R was also investigated. The findings showed that miR-15a may mediate the apoptosis of neurons submitted to OGD/R, and lower expression of Bcl-2 and higher expression of Bax and cleaved caspase-3 were observed. BDNF was screened as the candidate target, and the direct binding of miR-15a with 3'-UTR of BDNF were verified. Further research showed that miR-15a downregulated the expression of BDNF mRNA and protein, thus exerted negative regulatory effect on the OGD/R injury. PI3K/AKT pathway may be related to the regulatory effect of miR-15a. Our findings contribute to uncovering novel pathogenesis for ischemic brain injury.

Atrial septal defect in a patient with congenital disorder of glycosylation type 1a: a case report.

BACKGROUND: Atrial septal defect often become more severe when encountered in genetic syndromes. Congenital disorder of glycosylation type 1a is an inherited metabolic disorder associated with mutations in PMM2 gene and can affect almost all organs. Cardiac abnormalities vary greatly in congenital disorder of glycosylation type 1a and congenital heart defects have already been reported, but there is little knowledge about the effect of this inherited disorder on an existing congenital heart defect. Herein we report for the first time on a baby with congenital disorder of glycosylation type 1a with atrial septal defect and make a comparison of changes in atrial septal defect by follow-ups to the age of 3. CASE PRESENTATION: Our patient was an 8-month-old Han Chinese boy. At the initial visit, he presented with recurrent lower respiratory infection, heart murmur, psychomotor retardation, inverted nipples, and cerebellar atrophy. Echocardiography revealed a 8 mm secundum atrial septal defect with left-to-right shunt (Qp/Qs ratio 1.6). Enzyme testing of phosphomannomutase 2 demonstrated decreased levels of phosphomannomutase 2 activities in fibroblasts. Whole exon sequencing showed he was heterozygous for a frameshift mutation (p.I153X) and a missense mutation (p.I132T) in PMM2 gene. The diagnosis of congenital disorder of glycosylation type 1a with atrial septal defect was issued. Now, he is 3-years old at the time of this writing, with the development of congenital disorder of glycosylation type 1a (cerebellar atrophy become more severe and the symptom of nystagmus emerged), the size of atrial septal defect increased to 10 mm and the Qp/Qs ratio increased to 1.9, which suggested exacerbation of the atrial septal defect. Congenital heart defect-associated gene sequencing is then performed and shows there are no pathogenic mutations, which suggested intrinsic cardiac factors are not the cause of exacerbation of the atrial septal defect in our patient and it is reasonable to assume congenital disorder of glycosylation type 1a can worsen the situation of the existing atrial septal defect. CONCLUSIONS: This report highlights the view that congenital disorders of glycosylation type 1a should be excluded when faced with congenital heart defect with cerebellar atrophy or neurodevelopmental delay, especially when the situation of congenital heart defect becomes more and more severe.

Overexpression of the OsIMP Gene Increases the Accumulation of Inositol and Confers Enhanced Cold Tolerance in Tobacco through Modulation of the Antioxidant Enzymes' Activities.

Inositol is a cyclic polyol that is involved in various physiological processes, including signal transduction and stress adaptation in plants. l-myo-inositol monophosphatase (IMPase) is one of the metal-dependent phosphatase family members and catalyzes the last reaction step of biosynthesis of inositol. Although increased IMPase activity induced by abiotic stress has been reported in chickpea plants, the role and regulation of the IMP gene in rice (Oryza sativa L.) remains poorly understood. In the present work, we obtained a full-length cDNA sequence coding IMPase in the cold tolerant rice landraces in Gaogonggui, which is named as OsIMP. Multiple alignment results have displayed that this sequence has characteristic signature motifs and conserved enzyme active sites of the phosphatase super family. Phylogenetic analysis showed that IMPase is most closely related to that of the wild rice Oryza brachyantha, while transcript analysis revealed that the expression of the OsIMP is significantly induced by cold stress and exogenous abscisic acid (ABA) treatment. Meanwhile, we cloned the 5' flanking promoter sequence of the OsIMP gene and identified several important cis-acting elements, such as LTR (low-temperature responsiveness), TCA-element (salicylic acid responsiveness), ABRE-element (abscisic acid responsiveness), GARE-motif (gibberellin responsive), MBS (MYB Binding Site) and other cis-acting elements related to defense and stress responsiveness. To further investigate the potential function of the OsIMP gene, we generated transgenic tobacco plants overexpressing the OsIMP gene and the cold tolerance test indicated that these transgenic tobacco plants exhibit improved cold tolerance. Furthermore, transgenic tobacco plants have a lower level of hydrogen peroxide (H2O2) and malondialdehyde (MDA), and a higher content of total chlorophyll as well as increased antioxidant enzyme activities of superoxide dismutase (SOD), catalase (CAT) and peroxidase (POD), when compared to wild type (WT) tobacco plants under normal and cold stress conditions.

RNA interference of the nicotine demethylase gene CYP82E4v1 reduces nornicotine content and enhances Myzus persicae resistance in Nicotiana tabacum L.

The CYP82E4v1 gene was identified to encode nicotine demethylase, which catalyzed the conversion of nicotine to nornicotine. In this study, we constructed CYP82E4v1-RNAi vector and genetically transformed tobacco variety K326. The determination results of nicotine and nornicotine content via HPLC demonstrated that there was significant increase of nicotine content and reduction of nornicotine content in transgenic plants compared with those in wild-type plants. Exogenous application of IAA or GA3 could reduce the nicotine content in tobaccos, while ABA or 6-BA could increase the content of nicotine. And the more significant difference of nicotine content change in transgenic plants. Aphid-inoculation experiment demonstrated the number of aphid population in transgenic plants was significantly lower than wild-type plants at 12 d after aphid-inoculation. Meanwhile, the activity of AOEs and PAL in transgenic and wild-type tobacco plants after aphid-inoculation was measured. At 3 d after aphid-inoculation, both AOEs and PAL activity were significantly higher than controls, including wild-type plants with aphid-inoculation and transgenic plants with mock-inoculation. Also, the relative expression of these genes involved in salicylic acid/jasmonic acid (SA/JA) signaling pathways was analyzed at different stages after aphid-inoculation and the results demonstrated that there was significantly higher expression of JA-induced LOX gene in both transgenic and wild-type plants inoculated by aphid than the non-inoculated ones while no significant difference in the expression of SA-induced PR-1a gene among them was found, which indicated the JA-mediated resistance response was activated during aphid infestation. Moreover, although the expression level of BGL (another JA-induced gene) was less significant between the two inoculated tobaccos, it was significantly higher than the plant without inoculation, which was 1.4 and 2.2 folds higher than the non-inoculated controls respectively. To sum up, the improvement of aphid-resistance in transgenic tobaccos was based on nicotine accumulation which might cause nerve and antifeed toxicity and JA-mediated resistance response by enhancing the activities of AOEs and PAL.

Selectable marker-free co-expression of Nicotiana rustica CN and Nicotiana tabacum HAK1 genes improves resistance to tobacco mosaic virus in tobacco.

The viral disease caused by tobacco mosaic virus (TMV) is the most prevalent viral disease in many tobacco production areas. A breeding strategy based on resistance genes is an effective method for improving TMV resistance in tobacco. Also, the physiological status of plants is also critical to disease resistance improvement. Potassium ion is one of the most abundant inorganic nutrients in plant cells, and mediates plant responses to abiotic and biotic stresses. Improving K+ content in soil by fertilising can enhance diseases resistance of crops. However, the K+ absorption in plants depends mostly on K+ transporters located in cytoplasmic membrane. Therefore, the encoding genes for K+ transporters are putative candidates to target for improving tobacco mosaic virus resistance. In this work, the synergistic effect of a N-like resistance gene CN and a tobacco putative potassium transporter gene HAK1 was studied. The results showed that TMV-resistance in CN-HAK1-containing tobaccos was significantly enhanced though a of strengthening leaf thickness and reduction in the size of necrotic spots compared with only CN-containing plants, indicating the improvement of potassium nutrition in plant cells could increase the tobacco resistance to TMV by reducing the spread of the virus. Quantitative real-time polymerase chain reaction (qRT-PCR) analysis for TMV-CP expression in the inoculated leaf of the transgenic and wild-type plants also supported the conclusion. Further, the results of defence-related determination including antioxidative enzymes (AOEs) activity, salicylic acid (SA) content and the expression of resistance-related genes demonstrated CN with HAK1 synergistically enhanced TMV-resistance in transgenic tobaccos. Additionally, the HAK1- overexpression significantly improved the photosynthesis and K+-enriching ability in trans-CN-HAK1 tobaccos, compared with other counterparts. Finally, this work provides a method for screening new varieties of marker-free and safe transgenic antiviral tobacco.

[Flame atomic absorption spectrometric determination of copper and lead in beer after preconcentration using a rapid coprecipitation technique with 8-oxyquinoline].

A method was proposed for the determination of trace copper and lead in beer with flame atomic absorption spectrometry after preconcentration of copper and lead by rapid coprecipitation technique with 8-oxyquinoline-Mg(II) using manganese as an internal standard at pH 9. The standard addition recovery of lead is between 97.6%-103.0%. The detection limit is 6.28 x 10(-3) microg x mL(-1) for copper and 2.26 x 10(-2) microg x mL(-1) for lead when the sample volume is 100 mL. The effect of matrix can be overcome by the method and the results are satisfying. The method proposed here is rapid and has good reproducibility.

[Therapeutic effectiveness of intravenous immunoglobulin at 1 g/kg and 2 g/kg on Kawasaki disease: a comparative and follow-up study].

OBJECTIVE: To evaluate the effectiveness of intravenous immunoglobulin IVIG, 1 g/kg single intravenous injection in treating and preventing cardiac consequences of Kawasaki disease (KD) in children. METHODS: A total of 242 children with KD disease were enrolled in the study. In the randomized controlled trial, they were randomly divided into two groups: IVIG 1 g/kg group and IVIG 2 g/kg group, with aspirin administered within the first 7 to 10 days of illness. The occurrence and restoration of coronary artery lesion (CAL) in these two groups as well as the clinical and laboratory indexes including total fever duration, restoration of cervical lymphadenopathy, white blood cells count, platelet count, serum immunoglobulin, C reactive protein, erythrocyte sedimentation rate and EKG were observed. The clinical effectiveness of the groups before and after the treatment was analyzed. RESULTS: The age of the 242 children with KD disease ranged from 3 months to 14 years (mean 4.0 +/- 2.8 years old). Male to female ratio was 1.66:1, 83.1% of KD patients were blow 5 years old, 93.4% patients were followed up with echocardiography at the end of the first year and the follow-up period was (38 +/- 18) months, ranging from 4 months to 5.4 years; 86.9% of the cases in 1 g/kg group and 91.7% of the cases in 2 g/kg group had their fever controlled within 48 hours. The difference was not significant (P > 0.05). Serum immunoglobulin level was markedly enhanced after IVIG. Serum immunoglobulin levels in the patients of 2 g/kg group and 1 g/kg group were (26.9 +/- 7.4) g/L and (18.3 +/- 6.9) g/L, respectively (P < 0.01). The average duration of fever in IVIG 1 g/kg group was 10.6 days. After the treatment with 1 g/kg of IVIG, the abnormal white blood cells count, platelet count, C reactive protein, erythrocyte sedimentation rate and abnormal EKG findings were greatly improved (P < 0.001). However, there was no significant difference in the above-mentioned improvement between IVIG 1 g/kg group and IVIG 2 g/kg group (P > 0.05). In IVIG 1 g/kg group the occurrence of CAL was 29.5%. After the one-year follow-up, 87.5% CAL restored, but 12.5% did not, among which 9.4% were those of IVIG non-responders. In IVIG 2 g/kg group the incidence of CAL was 24.2%. After the one-year follow-up, 89.3% CAL restored, but 10.7% did not, all of which were those of IVIG non-responders. There was no significant difference in the incidence of CAL between the two groups (P > 0.05). CONCLUSION: Single intravenous injection of IVIG at 1 g/kg could effectively alleviate the clinical symptoms, decrease the incidence of CAL and reduce the complication of cardiovascular system. In the treatment of KD, the therapeutic effectiveness of IVIG at 1 g/kg was not significantly different from that of single intravenous injection of IVIG at 2 g/kg.