Down-regulation of MANNANASE7 gene in Brassica napus L. enhances silique dehiscence-resistance.

KEY MESSAGE: MANNANASE7 gene in Brassica napus L. encodes a hemicellulose which located at cell wall or extracellular space and dehiscence-resistance can be manipulated by altering the expression of MANNANASE7. Silique dehiscence is an important physiological process in plant reproductive development, but causes heavy yield loss in crops. The lack of dehiscence-resistant germplasm limits the application of mechanized harvesting and greatly restricts the rapeseed (Brassica napus L.) production. Hemicellulases, together with cellulases and pectinases, play important roles in fruit development and maturation. The hemicellulase gene MANNANASE7 (MAN7) was previously shown to be involved in the development and dehiscence of Arabidopsis (Arabidopsis thaliana) siliques. Here, we cloned BnaA07g12590D (BnMAN7A07), an AtMAN7 homolog from rapeseed, and demonstrate its function in the dehiscence of rapeseed siliques. We found that BnMAN7A07 was expressed in both vegetative and reproductive organs and significantly highly expressed in leaves, flowers and siliques where the abscission or dehiscence process occurs. Subcellular localization experiment showed that BnMAN7A07 was localized in the cell wall. The biological activity of the BnMAN7A07 protein isolated and purified through prokaryotic expression system was verified to catalyse the decomposition of xylan into xylose. Phenotypic studies of RNA interference (RNAi) lines revealed that down-regulation of BnMAN7A07 in rapeseed could significantly enhance silique dehiscence-resistance. In addition, the expression of upstream silique development regulators is altered in BnMAN7A07-RNAi plants, suggesting that a possible feedback regulation mechanism exists in the regulation network of silique dehiscence. Our results demonstrate that dehiscence-resistance can be manipulated by altering the expression of hemicellulase gene BnMAN7A07, which could provide an available genetic resource for breeding practice in rapeseed which is beneficial to mechanized harvest.

Arabidopsis GDSL1 overexpression enhances rapeseed Sclerotinia sclerotiorum resistance and the functional identification of its homolog in Brassica napus.

Sclerotinia stem rot (SSR) caused by Sclerotinia sclerotiorum is a devastating disease of rapeseed (Brassica napus L.). To date, the genetic mechanisms of rapeseed' interactions with S. sclerotiorum are not fully understood, and molecular-based breeding is still the most effective control strategy for this disease. Here, Arabidopsis thaliana GDSL1 was characterized as an extracellular GDSL lipase gene functioning in Sclerotinia resistance. Loss of AtGDSL1 function resulted in enhanced susceptibility to S. sclerotiorum. Conversely, overexpression of AtGDSL1 in B. napus enhanced resistance, which was associated with increased reactive oxygen species (ROS) and salicylic acid (SA) levels, and reduced jasmonic acid levels. In addition, AtGDSL1 can cause an increase in lipid precursor phosphatidic acid levels, which may lead to the activation of downstream ROS/SA defence-related pathways. However, the rapeseed BnGDSL1 with highest sequence similarity to AtGDSL1 had no effect on SSR resistance. A candidate gene association study revealed that only one AtGDSL1 homolog from rapeseed, BnaC07g35650D (BnGLIP1), significantly contributed to resistance traits in a natural B. napus population, and the resistance function was also confirmed by a transient expression assay in tobacco leaves. Moreover, genomic analyses revealed that BnGLIP1 locus was embedded in a selected region associated with SSR resistance during the breeding process, and its elite allele type belonged to a minor allele in the population. Thus, BnGLIP1 is the functional equivalent of AtGDSL1 and has a broad application in rapeseed S. sclerotiorum-resistance breeding.

Genome-wide identification of the NPR1-like gene family in Brassica napus and functional characterization of BnaNPR1 in resistance to Sclerotinia sclerotiorum.

KEY MESSAGE: The BnaNPR1-like gene family was identified in B. napus, and it was revealed that repression of BnaNPR1 significantly reduces resistance toS. sclerotiorum, intensifies ROS accumulation, and changes the expression of genes associated with SA and JA/ET signaling in response to this pathogen. The NONEXPRESSOR OF PATHOGENESIS-RELATED GENES1 (NPR1) and related NPR1-like genes play an important role in regulating plant defense. Oilseed rape (Brassica napus L.) is an important oilseed crop; however, little is known about the B. napus (Bna) NPR1-like gene family. Here, a total of 19 BnaNPR1-like genes were identified in the B. napus genome, and then named according to their respective best match in Arabidopsis thaliana (At), which led to the determination of B. napus homologs of every AtNPR1-like gene. Analysis of important protein domains and functional motifs indicated the conservation and variation among these homologs. Phylogenetic analysis of these BnaNPR1-like proteins and their Arabidopsis homologs revealed six distinct sub-clades, consequently indicating that their name classification totally conformed to their phylogenetic relationships. Further, B. napus transcriptomic data showed that the expression of three BnaNPR1s was significantly down-regulated in response to infection with Sclerotinia sclerotiorum, the most important pathogen of this crop, whereas BnaNPR2/3/4/5/6s did not show the expression differences in general. Further, we generated B. napus BnaNPR1-RNAi lines to interpret the effect of the down-regulated expression of BnaNPR1s on resistance to S. sclerotiorum. The results showed that BnaNPR1-RNAi significantly decreased this resistance. Further experiments revealed that BnaNPR1-RNAi intensified ROS production and changed defense responses in the interaction of plants with this pathogen. These results indicated that S. sclerotiorum might use BnaNPR1 to regulate specific physiological processes of B. napus, such as ROS production and SA defense response, for the infection.

Improving seed germination and oil contents by regulating the GDSL transcriptional level in Brassica napus.

KEY MESSAGE: Seed germination rate and oil content can be regulated at theGDSL transcriptional level by eitherAtGDSL1 orBnGDSL1 inB. napus. Gly-Asp-Ser-Leu (GDSL)-motif lipases represent an important subfamily of lipolytic enzymes, which play important roles in lipid metabolism, seed development, abiotic stress, and pathogen defense. In the present study, two closely related GDSL-motif lipases, Brassica napus GDSL1 and Arabidopsis thaliana GDSL1, were characterized as functioning in regulating germination rate and seed oil content in B. napus. AtGDSL1 and BnGDSL1 overexpression lines showed an increased seed germination rate and improved seedling establishment compared with wild type. Meanwhile, the constitutive overexpression of AtGDSL1 and BnGDSL1 promoted lipid catabolism and decreased the seed oil content. While RNAi-mediated suppression of BnGDSL1 (Bngdsl1) in B. napus improved the seed oil content and decreased seed germination rate. Moreover, the Bngdsl1 transgenic seeds showed changes in the fatty acid (FA) composition, featuring an increase in C18:1 and a decrease in C18:2 and C18:3. The transcriptional levels of six related core enzymes involved in FA mobilization were all elevated in the AtGDSL1 and BnGDSL1 overexpression lines, but strongly suppressed in the Bngdsl1 transgenic line. These results suggest that improving the seed germination and seed oil content in B. napus could be achieved by regulating the GDSL transcriptional level.

Down-regulation of BnDA1, whose gene locus is associated with the seeds weight, improves the seeds weight and organ size in Brassica napus.

Brassica napus L. is an important oil crop worldwide and is the main raw material for biofuel. Seed weight and seed size are the main contributors to seed yield. DA1 (DA means big in Chinese) is an ubiquitin receptor and negatively regulates seed size. Down-regulation of AtDA1 in Arabidopsis leads to larger seeds and organs by increasing cell proliferation in integuments. In this study, BnDA1 was down-regulated in B. napus by over expressed of AtDA1R358K , which is a functional deficiency of DA1 with an arginine-to-lysine mutation at the 358th amino acid. The results showed that the biomass and size of the seeds, cotyledons, leaves, flowers and siliques of transgenic plants all increased significantly. In particular, the 1000 seed weight increased 21.23% and the seed yield per plant increased 13.22% in field condition. The transgenic plants had no negative traits related to yield. The candidate gene association analysis demonstrated that the BnDA1 locus was contributed to the seeds weight. Therefore, our study showed that regulation of DA1 in B. napus can increase the seed yield and biomass, and DA1 is a promising target for crop improvement.

Up-regulation of programmed cell death 1 ligand 1 on neutrophils may be involved in sepsis-induced immunosuppression: an animal study and a prospective case-control study.

BACKGROUND: Recent studies have shown that neutrophils may display an antigen-presenting function and inhibit lymphocyte proliferation by expressing programmed cell death 1 ligand 1 (PD-L1). The current study was performed to investigate the effect of neutrophils and their pathophysiological significance during sepsis. METHODS: Neutrophil PD-L1 expression was determined in both septic mice (n = 6) and patients (n = 41). Neutrophils from septic mice were subtyped into PD-L1 and PD-L1 populations to determine their phenotypes and functions. Septic neutrophils were cocultured with lymphocytes to observe the effect of septic neutrophils on lymphocyte apoptosis. RESULTS: The PD-L1 level on neutrophils from septic mice was significantly up-regulated (21.41 ± 4.76%). This level increased with the progression of sepsis and the migration of neutrophils from the bone marrow to the blood and peritoneal cavity. The percentages of CD11a, CD62L, and C-C chemokine receptor type 2 were lower, whereas the percentages of CD16 and CD64 were higher on PD-L1 neutrophils than on PD-L1 neutrophils. The migratory capacity of PD-L1 neutrophils was compromised. Septic neutrophils induced lymphocyte apoptosis via a contact mechanism, and this process could be reversed by anti-PD-L1 antibody. PD-L1 was also up-regulated on neutrophils from patients with severe sepsis (14.6% [3.75%, 42.1%]). The levels were negatively correlated with the monocyte human leukocyte antigen-DR level and positively correlated with the severity of septic patients. Neutrophil PD-L1 was a predictor for the prognosis of severe sepsis, with an area of 0.74 under the receiver operating curve. CONCLUSIONS: PD-L1 is up-regulated on neutrophils during sepsis, which may be related to sepsis-induced immunosuppression.

Research progress on the physiological response and molecular mechanism of cold response in plants.

Low temperature is a critical environmental stress factor that restricts crop growth and geographical distribution, significantly impacting crop quality and yield. When plants are exposed to low temperatures, a series of changes occur in their external morphology and internal physiological and biochemical metabolism. This article comprehensively reviews the alterations and regulatory mechanisms of physiological and biochemical indices, such as membrane system stability, redox system, fatty acid content, photosynthesis, and osmoregulatory substances, in response to low-temperature stress in plants. Furthermore, we summarize recent research on signal transduction and regulatory pathways, phytohormones, epigenetic modifications, and other molecular mechanisms mediating the response to low temperatures in higher plants. In addition, we outline cultivation practices to improve plant cold resistance and highlight the cold-related genes used in molecular breeding. Last, we discuss future research directions, potential application prospects of plant cold resistance breeding, and recent significant breakthroughs in the research and application of cold resistance mechanisms.

Nitric oxide and ATP-sensitive potassium channels mediate lipopolysaccharide-induced depression of central respiratory-like activity in brain slices.

Infection may result in early abnormalities in respiratory movement, and the mechanism may involve central and peripheral factors. Peripheral mechanisms include lung injury and alterations in electrolytes and body temperature, but the central mechanisms remain unclear. In the present study, brainstem slices harvested from rats were stimulated with lipopolysaccharide at different doses. Central respiratory activities as demonstrated by electrophysiological activity of the hypoglossal rootlets were examined and the mechanisms were investigated by inhibiting nitric oxide synthase and ATP-sensitive potassium channels. As a result, 0.5 µg/ml lipopolysaccharide mainly caused inhibitory responses in both the frequency and the output intensity, while 5 µg/ml lipopolysaccharide caused an early frequency increase followed by delayed decreases in both the frequency and the output intensity. At both concentrations the inhibitory responses were fully reversed by inhibition of nitric oxide synthase with Nω-nitro-L-arginine methyl ester hydrochloride (20 µM), and by inhibition of ATP- sensitive potassium channels with glybenclamide (100 µM). These results show that direct lipopolysaccharide challenge altered central respiratory activity in dose- and time- related manners. Nitric oxide synthase and ATP-sensitive potassium channels may be involved in the respiratory changes.

Genome-Wide Prediction, Functional Divergence, and Characterization of Stress-Responsive BZR Transcription Factors in B. napus.

BRASSINAZOLE RESISTANT (BZR) are transcriptional factors that bind to the DNA of targeted genes to regulate several plant growth and physiological processes in response to abiotic and biotic stresses. However, information on such genes in Brassica napus is minimal. Furthermore, the new reference Brassica napus genome offers an excellent opportunity to systematically characterize this gene family in B. napus. In our study, 21 BnaBZR genes were distributed across 19 chromosomes of B. napus and clustered into four subgroups based on Arabidopsis thaliana orthologs. Functional divergence analysis among these groups evident the shifting of evolutionary rate after the duplication events. In terms of structural analysis, the BnaBZR genes within each subgroup are highly conserved but are distinctive within groups. Organ-specific expression analyses of BnaBZR genes using RNA-seq data and quantitative real-time polymerase chain reaction (qRT-PCR) revealed complex expression patterns in plant tissues during stress conditions. In which genes belonging to subgroups III and IV were identified to play central roles in plant tolerance to salt, drought, and Sclerotinia sclerotiorum stress. The insights from this study enrich our understanding of the B. napus BZR gene family and lay a foundation for future research in improving rape seed environmental adaptability.

Serum miR-146a and miR-223 as potential new biomarkers for sepsis.

OBJECTIVE: Current biomarkers cannot completely distinguish sepsis from systemic inflammatory response syndrome (SIRS) caused by other non-infectious diseases. Circulating microRNAs (miRNAs) are promising biomarkers for several diseases, but their correlation with sepsis is not totally clarified. METHODS: Seven miRNAs related to inflammation or infection were included in the present study. Serum miRNA expression was investigated in 50 patients diagnosed with sepsis, 30 patients with SIRS and 20 healthy controls to evaluate the diagnostic and prognostic value. Expression levels of serum miRNAs were determined by quantitative PCR using the Qiagen miScript system. Serum CRP and IL-6 levels were determined by enzyme linked immunosorbent assay. RESULTS: Serum miR-146a and miR-223 were significantly reduced in septic patients compared with SIRS patients and healthy controls. The areas under the receiver operating characteristic curve of miR-146a, miR-223 and IL-6 were 0.858, 0.804 and 0.785, respectively. CONCLUSION: Serum miR-146a and miR-223 might serve as new biomarkers for sepsis with high specificity and sensitivity. (ClinicalTrials.gov number, NCT00862290.).

Association between inflammatory genetic polymorphism and acute lung injury after cardiac surgery with cardiopulmonary bypass.

BACKGROUND: Recently, single nucleotide polymorphisms were proposed as potentially new predictors for perioperative risks, such as myocardial infarction and organ dysfunction. The objectives of this study were to investigate whether IL-6 -572C/G, IL-10 -1082A/G, and TNF-alpha -308G/A were associated with acute lung injury after cardiac surgery with cardiopulmonary bypass. MATERIAL/METHODS: One hundred patients with acute lung injury at 24 hours after cardiac surgery with cardiopulmonary bypass and 112 patients without acute lung injury as controls were included. Genotyping assay was performed with real-time fluorescence-based allele-specific PCR. Serum levels of IL-6, IL-10, and TNF-alpha were also determined by ELISA. Associations between these polymorphisms and acute lung injury, as well as serum cytokine levels, were analyzed. All patients were genotyped for IL-6 -572C/G, IL-10 -1082A/G, and TNF-alpha -308G/A. Circulating level of these cytokines were also determined. RESULTS: Acute lung injury after cardiac surgery with cardiopulmonary bypass was associated with IL-6 -572C/G polymorphism, but not IL-10 -1082A/G or TNF-alpha -308G/A. This functional polymorphism was further confirmed by multivariate analyses. The ratio of circulating concentrations of IL-10/IL-6 was associated with IL-6 genotypes and incidence of acute lung injury as well. CONCLUSIONS: The IL-6 -572 polymorphism was associated with acute lung injury after cardiac surgery with cardiopulmonary bypass. Proinflammatory and anti-inflammatory imbalance might be the clinical significance of IL-6 polymorphism (ClinicalTrials.gov number, NCT00826072).

BnaMPK6 is a determinant of quantitative disease resistance against Sclerotinia sclerotiorum in oilseed rape.

Sclerotinia sclerotiorum causes a devastating disease in oilseed rape (Brassica napus), resulting in major economic losses. Resistance response of B. napus against S. sclerotiorum exhibits a typical quantitative disease resistance (QDR) characteristic, but the molecular determinants of this QDR are largely unknown. In this study, we isolated a B. napus mitogen-activated protein kinase gene, BnaMPK6, and found that BnaMPK6 expression is highly responsive to infection by S. sclerotiorum and treatment with salicylic acid (SA) or jasmonic acid (JA). Moreover, overexpression (OE) of BnaMPK6 significantly enhances resistance to S. sclerotiorum, whereas RNAi in BnaMPK6 significantly reduces this resistance. These results showed that BnaMPK6 plays an important role in defense to S. sclerotiorum. Furthermore, expression of defense genes associated with SA-, JA- and ethylene (ET)-mediated signaling was investigated in BnaMPK6-RNAi, WT and BnaMPK6-OE plants after S. sclerotiorum infection, and consequently, it was indicated that the activation of ET signaling by BnaMPK6 may play a role in the defense. Further, four BnaMPK6-encoding homologous loci were mapped in the B. napus genome. Using the allele analysis and expression analysis on the four loci, we demonstrated that the locus BnaA03.MPK6 makes an important contribution to QDR against S. sclerotiorum. Our data indicated that BnaMPK6 is a previously unknown determinant of QDR against S. sclerotiorum in B. napus.

A systematic review of angiotensin receptor blockers in preventing stroke.

BACKGROUND AND PURPOSE: Angiotensin receptor blockers are widely used in patients at high risk of cardiocerebrovascular events. The aim of this meta-analysis was to investigate the effects of angiotensin receptor blockers on the risk of stroke. METHODS: Electronic searches of MEDLINE, EMBASE, and the Cochrane central register of controlled trials were performed. A total of 20 randomized clinical trials with 108 286 patients reporting stroke were available for this clinical outcome analysis. RESULTS: Angiotensin receptor blockers were associated with a significant reduction in the risk of stroke than placebo with an OR of 0.91 (0.84 to 0.98). Angiotensin receptor blockers were associated with no significant reduction in the risk of stroke compared with angiotensin-converting enzyme inhibitors (OR, 0.93; 0.84 to 1.03) and calcium antagonists (OR, 1.16; 0.91 to 1.48). CONCLUSIONS: Evidence of the benefit of angiotensin receptor blockers on the risk of stroke is provided when compared with placebo. There was no evidence of the benefit when comparing angiotensin receptor blockers with angiotensin-converting enzyme inhibitors and with calcium antagonists.

Recent advances in enhancement of oil content in oilseed crops.

Plant oils are very valuable agricultural commodity. The manipulation of seed oil composition to deliver enhanced fatty acid compositions, which are appropriate for feed or fuel, has always been a main objective of metabolic engineers. The last two decennary have been noticeable by numerous significant events in genetic engineering for identification of different gene targets to improve oil yield in oilseed crops. Particularly, genetic engineering approaches have presented major breakthrough in elevating oil content in oilseed crops such as Brassica napus and soybean. Additionally, current research efforts to explore the possibilities to modify the genetic expression of key regulators of oil accumulation along with biochemical studies to elucidate lipid biosynthesis will establish protocols to develop transgenic oilseed crops along much improved oil content. In this review, we describe current distinct genetic engineering approaches investigated by researchers for ameliorating oil content and its nutritional quality. Moreover, we will also discuss some auspicious and innovative approaches and challenges for engineering oil content to yield oil at much higher rate in oilseed crops.

Recent Advances in Mechanisms of Plant Defense to Sclerotinia sclerotiorum.

Sclerotinia sclerotiorum (Lib.) de Bary is an unusual pathogen which has the broad host range, diverse infection modes, and potential double feeding lifestyles of both biotroph and necrotroph. It is capable of infecting over 400 plant species found worldwide and more than 60 names have agriculturally been used to refer to diseases caused by this pathogen. Plant defense to S. sclerotiorum is a complex biological process and exhibits a typical quantitative disease resistance (QDR) response. Recent studies using Arabidopsis thaliana and crop plants have obtained new advances in mechanisms used by plants to cope with S. sclerotiorum infection. In this review, we focused on our current understanding on plant defense mechanisms against this pathogen, and set up a model for the defense process including three stages: recognition of this pathogen, signal transduction and defense response. We also have a particular interest in defense signaling mediated by diverse signaling molecules. We highlight the current challenges and unanswered questions in both the defense process and defense signaling. Essentially, we discussed candidate resistance genes newly mapped by using high-throughput experiments in important crops, and classified these potential gene targets into different stages of the defense process, which will broaden our understanding of the genetic architecture underlying quantitative resistance to S. sclerotiorum. We proposed that more powerful mapping population(s) will be required for accurate and reliable QDR gene identification.

BnaMPK3 Is a Key Regulator of Defense Responses to the Devastating Plant Pathogen Sclerotinia sclerotiorum in Oilseed Rape.

The disease caused by Sclerotinia sclerotiorum has traditionally been difficult to control, resulting in tremendous economic losses in oilseed rape (Brassica napus). Identification of important genes in the defense responses is critical for molecular breeding, an important strategy for controlling the disease. Here, we report that a B. napus mitogen-activated protein kinase gene, BnaMPK3, plays an important role in the defense against S. sclerotiorum in oilseed rape. BnaMPK3 is highly expressed in the stems, flowers and leaves, and its product is localized in the nucleus. Furthermore, BnaMPK3 is highly responsive to infection by S. sclerotiorum and treatment with jasmonic acid (JA) or the biosynthesis precursor of ethylene (ET), but not to treatment with salicylic acid (SA) or abscisic acid. Moreover, overexpression (OE) of BnaMPK3 in B. napus and Nicotiana benthamiana results in significantly enhanced resistance to S. sclerotiorum, whereas resistance is diminished in RNAi transgenic plants. After S. sclerotiorum infection, defense responses associated with ET, JA, and SA signaling are intensified in the BnaMPK3-OE plants but weakened in the BnaMPK3-RNAi plants when compared to those in the wild type plants; by contrast the level of both H2O2 accumulation and cell death exhibits a reverse pattern. The candidate gene association analyses show that the BnaMPK3-encoding BnaA06g18440D locus is a cause of variation in the resistance to S. sclerotiorum in natural B. napus population. These results suggest that BnaMPK3 is a key regulator of multiple defense responses to S. sclerotiorum, which may guide the resistance improvement of oilseed rape and related economic crops.

Retrospective analysis on acute respiratory distress syndrome in ICU.

OBJECTIVE: To assess the incidence, etiology, physiological and clinical features, mortality, and predictors of acute respiratory distress syndrome (ARDS) in intensive care unit (ICU). METHODS: A retrospective analysis of 5 314 patients admitted to the ICU of our hospital from April 1994 to December 2003 was performed in this study. The ARDS patients were identified with the criteria of the American-European Consensus Conference (AECC). Acute physiology and chronic health evaluation III (APACHE III), multiple organ dysfunction syndrome score (MODS score), and lung injury score (LIS) were determined on the onset day of ARDS for all the patients. Other recorded variables included age, sex, biochemical indicators, blood gas analysis, length of stay in ICU, length of ventilation, presence or absence of tracheostomy, ventilation variables, elective operation or emergency operation. RESULTS: Totally, 131 patients (2.5%) developed ARDS, among whom, 12 patients were excluded from this study because they died within 24 hours and other 4 patients were also excluded for their incomplete information. Therefore, there were only 115 cases (62 males and 53 females, aged 22-75 years, 58 years on average) left, accounting for 2.2% of the total admitted patients. Their average ICU stay was (11.27+/-7.24) days and APACHE III score was 17.23+/-7.21. Pneumonia and sepsis were the main cause of ARDS. The non-survivors were obviously older and showed significant difference in the ICU length of stay and length of ventilation as compared with the survivors. On admission, the non-survivors had significantly higher MODS and lower BE (base excess). The hospital mortality was 55.7%. The main cause of death was multiple organ failure. Predictors of death at the onset of ARDS were advanced age, MODS > or = to 8, and LIS > or = 2.76. CONCLUSIONS: ARDS is a frequent syndrome in this cohort. Sepsis and pneumonia are the most common risk factors. The main cause of death is multiple organ failure. The mortality is high but similar to most recent series including severe comorbidities. Based on this patient population, advanced age, MODS score, and LIS may be the important prognostic indicators for ARDS.