A Natural Allele of a Transcription Factor in Rice Confers Broad-Spectrum Blast Resistance.

Rice feeds half the world's population, and rice blast is often a destructive disease that results in significant crop loss. Non-race-specific resistance has been more effective in controlling crop diseases than race-specific resistance because of its broad spectrum and durability. Through a genome-wide association study, we report the identification of a natural allele of a C2H2-type transcription factor in rice that confers non-race-specific resistance to blast. A survey of 3,000 sequenced rice genomes reveals that this allele exists in 10% of rice, suggesting that this favorable trait has been selected through breeding. This allele causes a single nucleotide change in the promoter of the bsr-d1 gene, which results in reduced expression of the gene through the binding of the repressive MYB transcription factor and, consequently, an inhibition of H2O2 degradation and enhanced disease resistance. Our discovery highlights this novel allele as a strategy for breeding durable resistance in rice.

Continuous Strain Regulation of Palladium-Gold at the Atomic Level.

Revealing the effect of surface structure changes on the electrocatalytic performance is beneficial to the development of highly efficient catalysts. However, precise regulation of the catalyst surface at the atomic level remains challenging. Here, we present a continuous strain regulation of palladium (Pd) on gold (Au) via a mechanically controllable surface strain (MCSS) setup. It is found that the structural changes induced by the strain setup can accelerate electron transfer at the solid-liquid interface, thus achieving a significantly improved performance toward hydrogen evolution reaction (HER). In situ X-ray diffraction (XRD) experiments further confirm that the enhanced activity is attributed to the increased interplanar spacing resulting from the applied strain. Theoretical calculations reveal that the tensile strain modulates the electronic structure of the Pd active sites and facilitates the desorption of the hydrogen intermediates. This work provides an effective approach for revealing the relationships between the electrocatalyst surface structure and catalytic activity.

Engineering and Characterization of Avian Coronavirus Mutants Expressing Fluorescent Reporter Proteins from the Replicase Gene.

Infectious bronchitis virus (IBV) is an avian coronavirus that causes infectious bronchitis, an acute and highly contagious respiratory disease of chickens. IBV evolution under the pressure of comprehensive and widespread vaccination requires surveillance for vaccine resistance, as well as periodic vaccine updates. Reverse genetics systems are very valuable tools in virology, as they facilitate rapid genetic manipulation of viral genomes, thereby advancing basic and applied research. We report here the construction of an infectious clone of IBV strain Beaudette as a bacterial artificial chromosome (BAC). The engineered full-length IBV clone allowed the rescue of an infectious virus that was phenotypically indistinguishable from the parental virus. We used the infectious IBV clone and examined whether an enhanced green fluorescent protein (EGFP) can be produced by the replicase gene ORF1 and autocatalytically released from the replicase polyprotein through cleavage by the main coronavirus protease. We show that IBV tolerates insertion of the EGFP ORF at the 3' end of the replicase gene, between the sequences encoding nsp13 and nsp16 (helicase, RNA exonuclease, RNA endonuclease, and RNA methyltransferase). We further show that EGFP is efficiently cleaved from the replicase polyprotein and can be localized in double-membrane vesicles along with viral RNA polymerase and double-stranded RNA, an intermediate of IBV genome replication. One of the engineered reporter EGFP viruses were genetically stable during passage in cultured cells. We demonstrate that the reporter EGFP viruses can be used to study virus replication in host cells and for antiviral drug discovery and development of diagnostic assays. IMPORTANCE Reverse genetics systems based on bacterial artificial chromosomes (BACs) are the most valuable systems in coronavirus research. Here, we describe the establishment of a reverse genetics system for the avian coronavirus strain Beaudette, the most intensively studied strain. We cloned a copy of the avian coronavirus genome into a BAC vector and recovered infectious virus in permissive cells. We used the new system to construct reporter viruses that produce enhanced green fluorescent protein (EGFP). The EGFP coding sequence was inserted into 11 known cleavage sites of the major coronavirus protease in the replicase gene ORF1. Avian coronavirus tolerated the insertion of the EGFP coding sequence at three sites. The engineered reporter viruses replicated with parental efficiency in cultured cells and were sufficiently genetically stable. The new system facilitates functional genomics of the avian coronavirus genome but can also be used for the development of novel vaccines and anticoronaviral drugs.

Image Segmentation of Fiducial Marks with Complex Backgrounds Based on the mARU-Net.

Circuits on different layers in a printed circuit board (PCB) must be aligned according to high-precision fiducial mark images during exposure processing. However, processing quality depends on the detection accuracy of fiducial marks. Precise segmentation of fiducial marks from images can significantly improve detection accuracy. Due to the complex background of PCB images, there are significant challenges in the segmentation and detection of fiducial mark images. In this paper, the mARU-Net is proposed for the image segmentation of fiducial marks with complex backgrounds to improve detection accuracy. Compared with some typical segmentation methods in customized datasets of fiducial marks, the mARU-Net demonstrates good segmentation accuracy. Experimental research shows that, compared with the original U-Net, the segmentation accuracy of the mARU-Net is improved by 3.015%, while the number of parameters and training times are not increased significantly. Furthermore, the centroid method is used to detect circles in segmentation results, and the deviation is kept within 30 microns, with higher detection efficiency. The detection accuracy of fiducial mark images meets the accuracy requirements of PCB production.

Construction of drought stress regulation networks in potato based on SMRT and RNA sequencing data.

BACKGROUND: Potato (Solanum tuberosum) is the fourth most important food crop in the world and plays an important role in food security. Drought stress has a significantly negative impact on potato growth and production. There are several publications involved drought stress in potato, this research contributes to enrich the knowledge. RESULTS: In this study, next-generation sequencing (NGS) and single-molecule real-time (SMRT) sequencing technology were used to study the transcription profiles in potato in response to 20%PEG6000 simulates drought stress. The leaves of the variety "Désirée" from in vitro plantlets after drought stress at six time points from 0 to 48 hours were used to perform NGS and SMRT sequencing. According to the sequencing data, a total of 12,798 differentially expressed genes (DEGs) were identified in six time points. The real-time (RT)-PCR results are significantly correlated with the sequencing data, confirming the accuracy of the sequencing data. Gene ontology and KEGG analysis show that these DEGs participate in response to drought stress through galactose metabolism, fatty acid metabolism, plant-pathogen interaction, glutathione metabolism and other pathways. Through the analysis of alternative splicing of 66,888 transcripts, the functional pathways of these transcripts were enriched, and 51,098 transcripts were newly discovered from alternative splicing events and 47,994 transcripts were functionally annotated. Moreover, 3445 lncRNAs were predicted and enrichment analysis of corresponding target genes was also performed. Additionally, Alternative polyadenylation was analyzed by TADIS, and 26,153 poly (A) sites from 13,010 genes were detected in the Iso-Seq data. CONCLUSION: Our research greatly enhanced potato drought-induced gene annotations and provides transcriptome-wide insights into the molecular basis of potato drought resistance.

The transcription factor StTINY3 enhances cold-induced sweetening resistance by coordinating starch resynthesis and sucrose hydrolysis in potato.

The accumulation of reducing sugars in cold-stored tubers, known as cold-induced sweetening (CIS), negatively affects potato processing quality. The starch to sugar interconversion pathways that are altered in cold-stored CIS tubers have been elucidated, but the mechanism that regulates them remains largely unknown. This study identified a CBF/DREB transcription factor (StTINY3) that enhances CIS resistance by both activating starch biosynthesis and repressing the hydrolysis of sucrose to reducing sugars in detached cold-stored tubers. Silencing StTINY3 in a CIS-resistant genotype decreased CIS resistance, while overexpressing StTINY3 in a CIS-sensitive genotype increased CIS resistance, and altering StTINY3 expression was associated with expression changes in starch resynthesis-related genes. We showed first that overexpressing StTINY3 inhibited sucrose hydrolysis by enhancing expression of the invertase inhibitor gene StInvInh2, and second that StTINY3 promoted starch resynthesis by up-regulating a large subunit of the ADP-glucose pyrophosphorylase gene StAGPaseL3, and the glucose-6-phosphate transporter gene StG6PT2. Using electrophoretic mobility shift assays, we revealed that StTINY3 is a nuclear-localized transcriptional activator that directly binds to the dehydration-responsive element/CRT cis-element in the promoters of StInvInh2 and StAGPaseL3. Taken together, these findings established that StTINY3 influences CIS resistance in cold-stored tubers by coordinately modulating the starch to sugar interconversion pathways and is a good target for improving potato processing quality.

Identification of four insertion sites for foreign genes in a pseudorabies virus vector.

BACKGROUND: Pseudorabies virus (PRV) is a preferred vector for recombinant vaccine construction. Previously, we generated a TK&gE-deleted PRV (PRVΔTK&gE-AH02) based on a virulent PRV AH02LA strain. It was shown to be safe for 1-day-old piglets with maternal PRV antibodies and 4 ~ 5 week-old PRV antibody negative piglets and provide rapid and 100 % protection in weaned pigs against lethal challenge with the PRV variant strain. It suggests that PRVTK&gE-AH02 may be a promising live vaccine vector for construction of recombinant vaccine in pigs. However, insertion site, as a main factor, may affect foreign gene expression. RESULTS: In this study, we constructed four recombinant PRV-S bacterial artificial chromosomes (BACs) carrying the same spike (S) expression cassette of a variant porcine epidemic diarrhea virus strain in different noncoding regions (UL11-10, UL35-36, UL46-27 or US2-1) from AH02LA BAC with TK, gE and gI deletion. The successful expression of S gene (UL11-10, UL35-36 and UL46-27) in recombinant viruses was confirmed by virus rescue, PCR, real-time PCR and indirect immunofluorescence. We observed higher S gene mRNA expression level in swine testicular cells infected with PRV-S(UL11-10)ΔTK/gE and PRV-S(UL35-36)ΔTK/gE compared to that of PRV-S(UL46-27)ΔTK/gE at 6 h post infection (P < 0.05). Moreover, at 12 h post infection, cells infected with PRV-S(UL11-10)ΔTK/gE exhibited higher S gene mRNA expression than those infected with PRV-S(UL35-36)ΔTK/gE (P = 0.097) and PRV-S(UL46-27)ΔTK/gE (P < 0.05). Recovered vectored mutant PRV-S (UL11-10, UL35-36 and UL46-27) exhibited similar growth kinetics to the parental virus (PRVΔTK&gE-AH02). CONCLUSIONS: This study focuses on identification of suitable sites for insertion of foreign genes in PRV genome, which laids a foundation for future development of recombinant PRV vaccines.

Loss of function of a rice TPR-domain RNA-binding protein confers broad-spectrum disease resistance.

Crops carrying broad-spectrum resistance loci provide an effective strategy for controlling infectious disease because these loci typically confer resistance to diverse races of a pathogen or even multiple species of pathogens. Despite their importance, only a few crop broad-spectrum resistance loci have been reported. Here, we report the identification and characterization of the rice bsr-k1 (broad-spectrum resistance Kitaake-1) mutant, which confers broad-spectrum resistance against Magnaporthe oryzae and Xanthomonas oryzae pv oryzae with no major penalty on key agronomic traits. Map-based cloning reveals that Bsr-k1 encodes a tetratricopeptide repeats (TPRs)-containing protein, which binds to mRNAs of multiple OsPAL (OsPAL1-7) genes and promotes their turnover. Loss of function of the Bsr-k1 gene leads to accumulation of OsPAL1-7 mRNAs in the bsr-k1 mutant. Furthermore, overexpression of OsPAL1 in wild-type rice TP309 confers resistance to M. oryzae, supporting the role of OsPAL1 Our discovery of the bsr-k1 allele constitutes a significant conceptual advancement and provides a valuable tool for breeding broad-spectrum resistant rice.

Myocardial infarction induces bone marrow megakaryocyte proliferation, maturation and platelet production.

Platelet, apart from its classic role of homeostasis, serves also as a crucial immune cell component that contributes to the aggravation of atherosclerosis. It has been reported that myocardial infarction (MI) triggers leukocytosis in the bone marrow and spleen, which accelerates post-MI atherosclerosis. However, it remains unclear whether thrombopoiesis is also enhanced after MI. Here, using flow cytometry and bone marrow whole-mount immunofluorescence staining combined with three-dimensional (3D) reconstruction, we for the first time demonstrated an enhanced thrombopoiesis and megakaryopoiesis in a mouse model of coronary artery ligation as a mimic of MI. We showed that MI leads to increasing number of peripheral platelets, as well as elevating number and larger size of bone marrow MKs. We also observed more proplatelets and fragmented MKs, and a closer spatial distribution of MK populations to the bone marrow vascular niche after MI. This study provides direct evidence that MI induces bone marrow megakaryocyte proliferation, maturation and platelet production. It opens a new scope that targeting platelet production might become a novel therapeutic approach for attenuating post-MI atherosclerosis.

A gD&gC-substituted pseudorabies virus vaccine strain provides complete clinical protection and is helpful to prevent virus shedding against challenge by a Chinese pseudorabies variant.

BACKGROUND: Since 2011, pseudorabies caused by a variant PRV has re-emerged in many Chinese Bartha-K61-vaccinated pig farms. An efficacious vaccine is necessary to control this disease. We described the construction of a gD&gC-substituted pseudorabies virus (PRV B-gD&gCS) from the Bartha-K61 (as backbone) and AH02LA strain (as template for gD and gC genes) through bacterial artificial chromosome (BAC) technology using homologous recombination. The growth kinetics of PRV B-gD&gCS was compared with Bartha-K61. Its safety was evaluated in 28-day-old piglets. Protection efficacy was tested in piglets by lethal challenge with AH02LA at 7 days post vaccination, including body temperature, clinical symptoms, virus shedding, mortality rate, and lung lesions. RESULTS: The results showed that a BAC clone of Bartha-K61 and a B-gD&gCS clone were successfully generated. The growth kinetics of PRV B-gD&gCS strain on ST (Swine testicular) cells was similar to that of the Bartha-K61 strain. No piglets inoculated intramuscularly with PRV B-gD&gCS strain exhibited any clinical symptoms or virus shedding. After AH02LA challenge, all piglets in PRV B-gD&gCS and Bartha-K61 groups (n = 5 each) survived without exhibiting any clinical symptoms and high body temperature. More importantly, PRV B-gD&gCS strain completely prevented virus shedding in 2 piglets and reduced virus shedding post challenge in the other 3 piglets as compared with Bartha-K61 group. CONCLUSIONS: Our results suggest that PRV B-gD&gCS strain is a promising vaccine candidate for the effective control of current severe epidemic pseudorabies in China.

The Multivesicular Bodies (MVBs)-Localized AAA ATPase LRD6-6 Inhibits Immunity and Cell Death Likely through Regulating MVBs-Mediated Vesicular Trafficking in Rice.

Previous studies have shown that multivesicular bodies (MVBs)/endosomes-mediated vesicular trafficking may play key roles in plant immunity and cell death. However, the molecular regulation is poorly understood in rice. Here we report the identification and characterization of a MVBs-localized AAA ATPase LRD6-6 in rice. Disruption of LRD6-6 leads to enhanced immunity and cell death in rice. The ATPase activity and homo-dimerization of LRD6-6 is essential for its regulation on plant immunity and cell death. An ATPase inactive mutation (LRD6-6E315Q) leads to dominant-negative inhibition in plants. The LRD6-6 protein co-localizes with the MVBs marker protein RabF1/ARA6 and interacts with ESCRT-III components OsSNF7 and OsVPS2. Further analysis reveals that LRD6-6 is required for MVBs-mediated vesicular trafficking and inhibits the biosynthesis of antimicrobial compounds. Collectively, our study shows that the AAA ATPase LRD6-6 inhibits plant immunity and cell death most likely through modulating MVBs-mediated vesicular trafficking in rice.

Overexpression of miR169o, an Overlapping MicroRNA in Response to Both Nitrogen Limitation and Bacterial Infection, Promotes Nitrogen Use Efficiency and Susceptibility to Bacterial Blight in Rice.

Limiting nitrogen (N) supply contributes to improved resistance to bacterial blight (BB) caused by Xanthomonas oryzae pv. oryzae (Xoo) in susceptible rice (Oryza sativa). To understand the regulatory roles of microRNAs (miRNAs) in this phenomenon, 63 differentially expressed overlapping miRNAs in response to Xoo infection and N limitation stress in rice were identified through deep RNA sequencing and stem-loop quantitative real-time PCR. Among these, miR169o was further assessed as a typical overlapping miRNA through the overexpression of the miR169o primary gene. Osa-miR169o-OX plants were taller, and had more biomass accumulation with significantly increased nitrate and total amino acid contents in roots than the wild type (WT). Transcript level assays showed that under different N supply conditions, miR169o oppositely regulated NRT2, and this is reduced under normal N supply conditions but remarkably induced under N-limiting stress. On the other hand, osa-miR169o-OX plants also displayed increased disease lesion lengths and reduced transcriptional levels of defense gene (PR1b, PR10a, PR10b and PAL) compared with the WT after inoculation with Xoo. In addition, miR169o impeded Xoo-mediated NRT transcription. Therefore, the overlapping miR169o contributes to increase N use efficiency and negatively regulates the resistance to BB in rice. Consistently, transient expression of NF-YA genes in rice protoplasts promoted the transcripts of PR genes and NRT2 genes, while it reduced the transcripts of NRT1 genes. Our results provide novel and additional insights into the co ordinated regulatory mechanisms of cross-talk between Xoo infection and N deficiency responses in rice.

Safety and immunogenicity of an attenuated Chinese pseudorabies variant by dual deletion of TK&gE genes.

BACKGROUND: Since the outbreak of a new emerging virulent pseudorabies virus mutant in Chinese pig herds, intensive research has been focused on the construction of novel gene deletion vaccine based on the variant virulent viruses. An ideal vaccine candidate is expected to have a balanced safety and immunogenicity. RESULTS: From the infectious clone of PRV AH02LA strain, a TK deletion mutant was generated through two-step Red mutagenesis. After homologous recombination with a transfer vector, a TK&gE dual deficient mutant PRV (PRVΔTK&gE-AH02) was generated, and its structure verified by PCR, RFLP and sequencing. Growth kinetics test showed that PRVΔTK&gE-AH02 reached a titer of 107.5 TCID50 /mL on ST cells. The PRVΔTK&gE-AH02 at a dose of 106.0 TCID50 /animal was not virulent in mice or 1-day-old piglets with maternal PRV antibodies. No clinical signs or virus shedding were detected in 28~ 35-day-old piglets without maternal PRV antibodies after nasal or intramuscular administration with a dose of 106.0 TCID50, although it caused one death of four 1-day-old piglets without maternal PRV antibodies. In the efficiency test of PRVΔTK&gE-AH02, all four 28~ 35-day-old piglets without PRV antibody in the challenge control showed typical clinical symptoms and virus shedding, and two died at 4~ 5 days post challenge. All piglets in 105.0, 104.0 and 103.0 TCID50/dose PRVΔTK&gE-AH02 groups provided complete protection against challenge at only 7 days post intramuscular vaccination. More importantly, PRVΔTK&gE-AH02 stopped virus shedding in these piglets. In contrast, all four piglets in PRV Bartha K61 vaccine group developed high body temperature (≥40.5 °C) and viral shedding, despite they had mild or even no clinical symptoms. CONCLUSIONS: The constructed TK&gE dual deletion mutant PRVΔTK&gE-AH02 can reach high titers on ST cells. The live vaccine of PRVΔTK&gE-AH02 is highly safe, and can not only provide clinical protection but also stops virus shedding. This study suggests that PRVΔTK&gE-AH02 might work as a promising vaccine candidate to combat the PRV variant emerging in Chinese herds since 2011.

Short-Term Hesperidin Pretreatment Attenuates Rat Myocardial Ischemia/Reperfusion Injury by Inhibiting High Mobility Group Box 1 Protein Expression via the PI3K/Akt Pathway.

BACKGROUND/AIMS: Hesperidin pretreatment has been shown to protect against myocardial ischemia/reperfusion (I/R) injury, but the underlying mechanism is poorly understood. This study aimed to investigate the cardioprotective effects of a 3-day hesperidin pretreatment on I/R injury and to further explore whether its mechanism of action was associated with the inhibition of high mobility group box 1 protein (HMGB1) expression via the PI3K/Akt pathway. METHODS: In a fixed-dose study, hematoxylin and eosin staining and myocardial enzyme measurements were used to determine the optimal dose of hesperidin that elicited the best cardioprotective effects against I/R injury. Furthermore, rats were pretreated with 200 mg/kg hesperidin, and infarct size and the levels of myocardial enzymes, apoptosis, inflammatory and oxidative indices, and HMGB1 and p-Akt expression were measured. RESULTS: Our results indicated that while different 3-day hesperidin pretreatment doses promoted histopathological changes and reduced myocardial enzymes induced by I/R the optimal dose was 200 mg/kg. Moreover, the 200 mg/kg hesperidin pretreatment not only significantly decreased the infarct size as well as myocardial enzyme levels but also inhibited myocardial apoptosis, the inflammatory response and oxidative stress. Additionally, hesperidin downregulated HMGB1 expression and upregulated p-Akt expression in the myocardium. LY294002, a specific PI3K inhibitor, partially reversed the decreased HMGB1 expression, increased p-Akt expression induced by hesperidin and abolished the anti-apoptotic, anti-inflammatory and anti-oxidative effects of hesperidin. CONCLUSION: These findings suggest that short-term pretreatment with hesperidin protects against myocardial I/R injury by suppressing myocardial apoptosis, the inflammatory response and oxidative stress via PI3K/Akt pathway activation and HMGB1 inhibition.

Four receptor-like cytoplasmic kinases regulate development and immunity in rice.

Receptor-like cytoplasmic kinases (RLCKs) represent a large family of proteins in plants. However, few RLCKs have been well characterized. Here, we report the functional characterization of four rice RLCKs - OsRLCK57, OsRLCK107, OsRLCK118 and OsRLCK176 from subfamily VII. These OsRLCKs interact with the rice brassinosteroid receptor, OsBRI1 in yeast cell, but not the XA21 immune receptor. Transgenic lines silenced for each of these genes have enlarged leaf angles and are hypersensitive to brassinolide treatment compared to wild type rice. Transgenic plants silenced for OsRLCK57 had significantly fewer tillers and reduced panicle secondary branching, and lines silenced for OsRLCK107 and OsRLCK118 produce fewer seeds. Silencing of these genes decreased Xa21 gene expression and compromised XA21-mediated immunity to Xanthomonas oryzae pv. oryzae. Our study demonstrates that these OsRLCKs negatively regulate BR signalling, while positively regulating immune responses by contributing to the expression of the immune receptor XA21.

An inactivated gE-deleted pseudorabies vaccine provides complete clinical protection and reduces virus shedding against challenge by a Chinese pseudorabies variant.

BACKGROUND: Since the end of 2011 an outbreak of pseudorabies affected Chinese pig herds that had been vaccinated with the commercial vaccine made of Bartha K61 strain. It is now clear that the outbreak was caused by an emergent PRV variant. Even though vaccines made of PRV Bartha K61 strain can confer certain cross protection against PRV variants based on experimental data, less than optimal clinical protection and virus shedding reduction were observed, making the control or eradication of this disease difficult. RESULTS: An infectious clone of PRV AH02LA strain was constructed to generate a gE deletion mutant PRV(LA-AB) strain. PRV(LA-AB) strain can reach a titer of 108.43 TCID50 /mL (50% tissue culture infectious dose) on BHK-21 cells. To evaluate the efficiency of the inactivated vaccine made of PRV(LA-AB) strain, thirty 3-week-old PRV-negative piglets were divided randomly into six groups for vaccination and challenge test. All five piglets in the challenge control showed typical clinical symptoms of pseudorabies post challenge. Sneezing and nasal discharge were observed in four and three piglets in groups C(vaccinated with inactivated PRV Bartha K61 strain vaccine) and D(vaccinated with live PRV Bartha K61 strain vaccine) respectively. In contrast, piglets in both groups A(vaccinated with inactivated PRV LA-AB strain vaccine) and B(vaccinated with inactivated PRV LA-AB strain vaccine with adjuvant) presented mild or no clinical symptoms. Moreover, viral titers detected via nasal swabs were approximately 100 times lower in group B than in the challenge control, and the duration of virus shedding (3-4 days) was shorter than in either the challenge control (5-10 days) or groups C and D (5-6 days). CONCLUSIONS: The infectious clone constructed in this study harbors the whole genome of the PRV variant AH02LA strain. The gE deletion mutant PRV(LA-AB)strain generated from PRV AH02LA strain can reach a high titer on BHK-21 cells. An inactivated vaccine of PRV LA-AB provides clinical protection and significantly reduces virus shedding post challenge, especially if accompanied by the adjuvant CVC1302. While Inactivated or live vaccines made of PRV Barth K61 strain can provide only partial protection in this test.

Bacillus Calmette-Guérin-inoculation at different time points influences the outcome of C57BL/6 mice infected with Plasmodium chabaudi chabaudi AS.

Bacillus Calmette-Guérin (BCG) is an attenuated Mycobacterium tuberculosis vaccine. We performed a series of co-infection experiments with BCG-Plasmodium chabaudi chabaudi Landau, 1965 AS using C57BL/6 mice to analyse whether BCG can affect the development of protective immunity to infection with Plasmodium spp. and the mechanism of this protection. We divided mice into four groups: BCG-inoculation 4 weeks prior to P. c. chabaudi AS infection (B-4w-Pc); simultaneous BCG-inoculation and P. c. chabaudi AS infection (Pc+B); BCG-inoculation 3 days post P. c. chabaudi AS (Pc-3-B) infection; and mono-P. c. chabaudi AS infection as control (Pc). The parasitemia level in the B-4w-Pc group was noticeably higher than control group at 6-19 days post infection (dpi). Compared with the control group, the proportion of CD4(+)CD69(+) T cells was significantly reduced 5, 8 and 12 dpi, but the proportion of CD4(+)CD25(+)Foxp3(+) Tregs was significantly increased in the B-4w-Pc group on 5 and 8 dpi. The B-4w-Pc group also demonstrated reduced levels of IFN-γ and TNF-α on 5 and 8 dpi and significantly elevated level of IL-10 on 12 dpi. There were significantly fewer mDCs (CD11c(+)CD11b(+)) and pDCs (CD11c(+)B220(+)) in the B-4w-Pc group than the control group at all the time points post infection and the expression of MHC II was noticeably reduced on day 8 pi. Our findings confirmed that BCG inoculation prior to Plasmodium infection resulted in excessive activation and proliferation of Tregs and upregulation of anti-inflammatory mediators, which inhibited establishment of a Th1-dominant immune response during the early stages of Plasmodium infection by inhibiting dendritive cells response. BCG inoculation prior to P. c. chabaudi AS infection may contribute to overgrowth of parasites as well as mortality in mice.

Beta-1-adrenergic receptors mediate Nrf2-HO-1-HMGB1 axis regulation to attenuate hypoxia/reoxygenation-induced cardiomyocytes injury in vitro.

UNLABELLED: BACKGROUD/AIMS: The aim of the study was to evaluate the effects of beta1-adrenergic receptors (ß1-ARs) -mediated nuclear factor erythroid 2-related factor 2 (Nrf2)-heme oxygenase-1 (HO-1)-high mobility group box 1 protein (HMGB1) axis regulation in hypoxia/reoxygenation (H/R)-induced neonatal rat cardiomyocytes. METHODS: The neonatal cultured cardiomyocytes were concentration-dependently pretreated by dobutamine (DOB), a selective ß1-adrenergic receptor agonist, in the absence and/or presence of LY294002 (a phosphatidylinositol 3-kinase (PI3K) inhibitor), SB203580 (a p38mitogen-activated-protein kinase (p38MAPK) inhibitor), Nrf2siRNA and HO-1siRNA, respectively, and then treated by H/R. The effects and mechanisms by which H/R-induced cardiomyocytes injury were evaluated. RESULTS: Significant increase of HO-1 was found in neonatal cultured cardiomyocytes treated with DOB, when compared to the control group. Significant change for Nrf2 translocation was also revealed in neonatal cultured cardiomyocytes treated with DOB. Insignificant decreases of NF-kappaB p65 activation and HMGB1 release were observed in H/R-induced neonatal cultured cardiomyocytes treated with DOB, when compared to the control group. Importantly, DOB treatment significantly increased the cell viability and decreased the levels of LDH and MDA in H/R-induced cardiomyocytes injury. However, DOB failed to increase HO-1, inhibit NF-kappaB p65 activation, prevent HMGB1 release and attenuate H/R-induced cardiomyocytes injury when the cultured cardiomyocytes were pretreated by Nrf2siRNA, HO-1siRNA, PI3K inhibitor (LY294002) and p38MAPK inhibitor (SB203580), respectively. CONCLUSIONS: ß1-ARs-mediated Nrf2-HO-1-HMGB1 axis regulation plays a critical protective role in H/R-induced neonatal rat cardiomyocytes injury in vitro via PI3K/p38MAPK signaling pathway.

Construction of a recombinant duck enteritis virus (DEV) expressing hemagglutinin of H5N1 avian influenza virus based on an infectious clone of DEV vaccine strain and evaluation of its efficacy in ducks and chickens.

BACKGROUND: Highly pathogenic avian influenza virus (AIV) subtype H5N1 remains a threat to poultry. Duck enteritis virus (DEV)-vectored vaccines expressing AIV H5N1 hemagglutinin (HA) may be viable AIV and DEV vaccine candidates. METHODS: To facilitate the generation and further improvement of DEV-vectored HA(H5) vaccines, we first constructed an infectious clone of DEV Chinese vaccine strain C-KCE (DEV(C-KCE)). Then, we generated a DEV-vectored HA(H5) vaccine (DEV-H5(UL55)) based on the bacterial artificial chromosome (BAC) by inserting a synthesized HA(H5) expression cassette with a pMCMV IE promoter and a consensus HA sequence into the noncoding area between UL55 and LORF11. The immunogenicity and protective efficacy of the resulting recombinant vaccine against DEV and AIV H5N1 were evaluated in both ducks and chickens. RESULTS: The successful construction of DEV BAC and DEV-H5(UL55) was verified by restriction fragment length polymorphism analysis. Recovered virus from the BAC or mutants showed similar growth kinetics to their parental viruses. The robust expression of HA in chicken embryo fibroblasts infected with the DEV-vectored vaccine was confirmed by indirect immunofluorescence and western blotting analyses. A single dose of 10(6) TCID50 DEV-vectored vaccine provided 100 % protection against duck viral enteritis in ducks, and the hemagglutination inhibition (HI) antibody titer of AIV H5N1 with a peak of 8.2 log2 was detected in 3-week-old layer chickens. In contrast, only very weak HI titers were observed in ducks immunized with 10(7) TCID50 DEV-vectored vaccine. A mortality rate of 60 % (6/10) was observed in 1-week-old specific pathogen free chickens inoculated with 10(6) TCID50 DEV-vectored vaccine. CONCLUSIONS: We demonstrate the following in this study. (i) The constructed BAC is a whole genome clone of DEV(C-KCE). (ii) The insertion of an HA expression cassette sequence into the noncoding area between UL55 and LORF11 of DEV(C-KCE) affects neither the growth kinetics of the virus nor its protection against DEV. (iii) DEV-H5(UL55) can generate a strong humoral immune response in 3-week-old chickens, despite the virulence of this virus observed in 1-week-old chickens. (iv) DEV-H5(UL55) induces a weak HI titer in ducks. An increase in the HI titers induced by DEV-vectored HA(H5) will be required prior to its wide application.

[Selectively stimulating ß1-adrenergic receptor attenuates rat myocardial ischemia/reperfusion injury in vivo by inhibiting high mobility group box 1 protein release].

OBJECTIVE: To investigate whether selectively stimulating ß1-adrenergic receptor could inhibit high mobility group box 1 protein and attenuate myocardial ischemia/reperfusion(I/R) injury in rats. METHODS: Eighty healthy male Sprague-Dawley (SD) rats were randomly divided into seven groups: (1) Sham operated group (SO); (2) Ischemia/reperfusion (I/R) group; (3) Dobutamine1 (5 µg×kg⁻¹ · min⁻¹) + I/R group; (4) Dobutamine2 (10 µg·kg⁻¹ × min⁻¹) + I/R group; (5) LY294002 (0.3 mg/kg) + Dobutamine2 + I/R group; (6) SB203580 (1 mg/kg) + Dobutamine2 + I/R group; (7) ZnPPIX (10 mg/kg) + Dobutamine2+I/R group. Rats were pretreated by saline, dobutamine, LY294002, SB203580 and ZnPPIX, respectively, then underwent myocardial I/R. Myocardial I/R injury and oxidative stress were assessed, and myocardial HO-1, NF-κB and HMGB1 expressions were measured by Western blot analysis. RESULTS: Dobutamine significantly reduced the myocardial infarct size (P < 0.05), myocardial enzymes (LDH and CK) (P < 0.05) and proinfiammation cytokines (TNF-α and IL-6), reduced oxidative stress (MDA and SOD) in a dose-dependent manner (all P < 0.05). Meanwhile, dobutamine significantly and dose-dependently mediated the induction of HO-1 (P < 0.05), the expression of NF-κB (P < 0.05) and HMGB1 (P < 0.05). However, all the effects could be significantly reversed by co-treatment with LY294002, SB203580 and ZnPPIX (all P < 0.05). CONCLUSIONS: Current study demonstrates that selectively stimulating ß1-adrenergic receptor by dobutasmine could reduce rat myocardial I/R injury in vivo through promoting the induction of HO-1 and inhibiting HMGB1 release.