The involvement of NLRP3 inflammasome in CUMS-induced AD-like pathological changes and related cognitive decline in mice.

BACKGROUND: Numerous studies have found that inhibiting the expression of NLRP3 inflammasome can significantly improve depressive-like behaviors in mice, but the research on its effect on cognitive decline in depression and its mechanism is still lacking. This study aimed to elucidate the role of NLRP3 inflammasome in cognitive decline in depression and explore the common neuro-immunological mechanisms of depression and Alzheimer's disease (AD). METHODS: Male C57BL/6 mice were subjected to chronic unpredictable mild stress (CUMS) for 5 weeks, treatment group was administered with the NLRP3 inhibitor MCC950 (10 mg/kg, i.p.), fluoxetine served as positive control. Then, the mice were assessed for cognitive behaviors and depression-like behaviors, and changes of microglia and neurons in hippocampus and levels of Aß metabolic pathway and tau protein were measured. To explore the mechanism of NLRP3 activation on neurons, we performed in vitro studies using BV2 microglia and mouse primary neurons. Furthermore, we focused on the role of NLRP3 inflammasome in the function of neurons and the expression of AD pathological indicators. RESULTS: CUMS induced depressive-like behaviors and cognitive decline in mice, which could be reversed by inhibiting NLRP3 inflammasome. MCC950, a specific NLRP3 inhibitor, alleviated CUMS-induced neuron injury and AD-like pathological changes, including the abnormal expression of Aß metabolic pathway and the hyper-phosphorylation of tau protein. LPS (1 µg/mL) + ATP (1 mM) treatment activated the expression of NLRP3 inflammasome and IL-1ß in vitro. In vitro experiment also proved that inhibiting the expression of NLRP3 inflammasome in microglia can restore the Aß metabolic pathway to normal, decrease neuronal tau protein phosphorylation and protect neurons. CONCLUSIONS: Inhibition of NLRP3 inflammasome effectively alleviated CUMS-induced depressive-like behaviors and cognitive decline in mice, and inhibited the activation of AD physiological indicators.

Long-stranded non-coding RNA HCG11 regulates glioma cell proliferation, apoptosis and drug resistance via the sponge MicroRNA-144COX-2 axis.

In the current study, we analyzed long non-coding RNA HCG11 regulates the proliferation, apoptosis and drug resistance of glioma cells by spongy microRNA-144COX-2 axis. For this purpose, glioma cells at the logarithmic growth stage were divided into blank, up-regulated and down-regulated groups. The blank group did not undergo any treatment. Bacteria were inserted into the complete culture medium of the up-regulated group and down-regulated group for co-culture for 24 h. The down-regulated group was transfected with Mir-HCG11 inhibitor. The expressions of Mir-HCG11, Mir-144-3p and COX-2 in each group were observed, and the proliferation and apoptosis of glioma cells were analyzed, and their drug resistance was analyzed. Results showed that compared with the blank group, the expression of Mir-HCG11 and Mir-144 was increased and the expression of COX-2 was decreased in the up-regulated group (P < 0.05). Compared with the up-regulated group, the down-regulated group increased the expression of Mir-HCG11 and Mir-144 and decreased the expression of COX-2 (P < 0.05). Compared with the blank group, the proliferation rate of glioma cells in the up-regulated group (24h, 48h, 72h) was increased (P < 0.05); Compared with the up-regulated group, the proliferation rate of glioma cells in the down-regulated group (24h, 48h, 72h) was decreased (P < 0.05); Compared with the blank group, apoptosis rate of glioma cells in the up-regulated group (24h, 48h, 72h) was decreased (P < 0.05); Compared with the up-regulated group, the apoptosis rate of glioma cells in the down-regulated group (24h, 48h, 72h) was increased (P < 0.05); The IC50 values of Imatinib, VP-16 and TMZ in blank group and up-regulated group was compared (P > 0.05). Compared with the blank group and up-regulated group, the IC50 values of Imatinib, VP-16 and TMZ in the down-regulated group were decreased (P < 0.05). In general, down-regulation of long non-coding RNA-HCG11 can regulate the microRNA-144COX-2 axis in glioma, thus reducing the proliferation rate of glioma cells and improving the apoptosis rate of glioma cells. In addition, down-regulation of long non-coding RNA-HCG11 is also involved in the drug resistance mechanism of Imatinib, VP-16 and TMZ chemotherapy drugs.

Changes of Gastric Juice Microenvironment in Patients with Fundic Gland Polyp and Hyperplastic Polyp.

INTRODUCTION: The aim of this study was to study the relationship between the formation of gastric fundic gland polyp and gastric hyperplastic polyp (HP) and the changes of gastric juice microenvironment. METHODS: The proton-pump inhibitor (PPI) applications to patients were recorded. Gastric juices and biopsy polyps were collected for pathological examination, H. pylori tests, biomarkers, and MUC1, MUC2, MUC5AC expression measurement. RESULTS: Among 34,892 patients, the detection rate of gastric fundic gland polyps was significantly higher than that of gastric HPs (p < 0.01). The incidence rate of gastric fundic gland polyp and gastric HP in PPI users (n = 3,886) was higher than that of non-PPI users (p < 0.01). The occurrence of polyp was positively related to the duration of PPI application and the H. pylori-positive rate. The bile reflux rate between fundic gland polys group (17.61%) and HPs (28.67%) was significantly different (p < 0.01). The levels of gastric juice Gastrin-17, epidermal growth factor (EGF) and MUC2 from patients with gastric fundic gland polyps and gastric HPs were higher than those in the control group (p < 0.01). However, patients with gastric fundic gland polyps and HPs had significantly lower gastric juice PGE2 and MUC5AC (p < 0.01). CONCLUSION: PPI application, H. pylori infection, and bile reflux are the potential risk factors for formation of fundic gland polyps and HPs. The potential mechanism of polyps' formation can be related to the levels of Gastrin-17, EGF, MUC2, PGE2, and MUC5AC in gastric juice.

The short-term safety and effectiveness of a new distal perforating stent graft in Type B aortic dissection: a retrospective study.

BACKGROUND: Spinal artery ischemia (SCI) events can result from over coverage of the descending thoracic aorta with a coated stent during Thoracic Endovascular Aortic Repair (TEVAR). The aim of this study was to determine whether a new distal perforating stent could reduce the incidence of spinal cord ischemia while remodeling the true lumen. METHODS: TBAD patients treated with Talos stent in the vascular surgery Department of Yan 'an Hospital affiliated to Kunming Medical University between December 2017 and October 2019 were retrospectively analyzed to investigate the short-term safety and effectiveness of Talos stent. RESULTS: A total of the 20 patients, including 14 males and 6 females, with an average age of 52.65 ± 8.98 years (range 37-68 years), were included in the analysis. Stent-grafts were successfully implanted in all patients under local anesthesia, with a technical success rate of 100%. The average operation time was 50.75 ± 13.01 min. A total of 2 cases (10%) presented chest pain associated with intercostal artery ischemia that was relieved on the 3rd and 5th postoperative day, respectively. Postoperative mean follow-up was 16.15 ± 3.99 months. No paraplegia or other complications occurred. And stenting did not induce new tears. No migration, deformation, or fracture of the stents occurred. There was a significant difference in the remolding of the true lumen preoperatively and at 12 months postoperatively (P < 0.05). CONCLUSIONS: Talos stent has achieved satisfactory clinical treatment results in short term.

COVID-19 pandemic related long-term chronic stress on the prevalence of depression and anxiety in the general population.

BACKGROUND: The COVID-19 pandemic has lasted for more than 1 year, causing far-reaching and unprecedented changes in almost all aspects of society. This study aimed to evaluate the long-term consequences of the COVID-19 pandemic on depression and anxiety, and explore the factors associated with it. METHODS: A cross-sectional study using an online survey was conducted to assess mental health problems from February 2 to February 9, 2021 by using patient health questionnaire-9 (PHQ-9) and generalized anxiety disorder-7 (GAD-7). The insomnia severity index (ISI), demographic data and COVID-19 related variables were measured by a self-designed questionnaire. The factors associated with depressive and anxiety symptoms were identified by Pearson chi-square test and binary logistic regression analysis. RESULTS: In the study that 1171 participants enrolled, the overall prevalence of depressive and anxiety symptoms among general people was 22.6 and 21.4% respectively in the present study. Living alone was a potential risk factor for depressive symptoms, while regular exercises was a potential protective factor. The prevalence of depressive and anxiety symptoms was significantly associated with the severity of insomnia symptoms and the negative feelings about pandemic. CONCLUSION: COVID-19 pandemic- related chronic stress has brought about profound impacts on long-term mental health in the general population. The level of insomnia and a negative attitude towards the pandemic are significantly correlated with unfavorable mental health. However, we failed to found a significant association of age and gender with the mental health symptoms, although they were recognized as well-established risk factors during the outbreak by some other studies. This discrepancy may be because the acute and chronic effects of the pandemic are influenced by different factors, which reminds that more attention should be paid to the intrinsic psychological factors and physical reactions towards COVID-19.

Clinical evaluation of modified invaginated pancreaticojejunostomy for pancreaticoduodenectomy.

BACKGROUND: Pancreaticoduodenectomy (PD) remains the major curative operation for malignant neoplasm of pancreas or cancerous tumors near the pancreas. Despite advancements in recent years, the postoperative recurrence rate of these neoplasms and tumors remains high. Moreover, overall morbidity remains high due to clinically relevant postoperative pancreatic fistula (POPF). METHODS: To compare the clinical outcomes of modified invaginated anastomosis and mucosa-to-mucosa anastomosis, this retrospective study included 343 patients who underwent PD from January 2008 to January 2019 at Beijing Friendship Hospital, Capital Medical University. The patients' general conditions and disease status were preoperatively evaluated. The surgical procedure was recorded, and operative management was appropriately performed. RESULTS: Compared with mucosa-to-mucosa anastomosis, modified invaginated anastomosis resulted in a higher intraoperative blood transfusion rate (P < 0.001) and lower hospitalization expenses (P = 0.049). However, no significant differences were found in operation time (P = 0.790), intraoperative bleeding (P = 0.428), postoperative recovery exhaust time (P = 0.442), time to normal flow of food (P = 0.163), and hospitalization time (P = 0.567). Operation time was a risk factor for POPF (odds ratio 1.010; 95% confidence interval 1.003-1.016; P = 0.003). The incidence of pancreatic fistula (grades B and C) was lower in the patients who underwent modified invaginated anastomosis (14.1%) than in those who underwent mucosa-to-mucosa anastomosis (15.3%). The operation time was greater in the POPF group than in the non POPF group among the patients who received modified invaginated anastomosis (P = 0.003) and mucosa-to-mucosa anastomosis (P = 0.002). CONCLUSION: Modified invaginated pancreaticojejunostomy for PD resulted in a decreased incidence of POPF; it may serve as a new approach for PD while managing patients who have undergone PD.

Long non-coding RNA HCG11 suppresses the growth of glioma by cooperating with the miR-4425/MTA3 axis.

BACKGROUND: Glioma is a type of malignant tumor that occurs in the central nervous system of adults. Long non-coding RNAs (lncRNAs) that potentially participate in the initiation and progression of glioma have been widely reported. As a now-found lncRNA, HLA complex group 11 (HCG11) has not yet been studied in glioma. The present study aimed to determine the role of HCG11 in the tumorigenesis of glioma. METHODS: A quantitative real-time polymerase chain reaction assay was performed to examine the expression pattern of HCG11 in 84 glioma tissues and cell lines. The overall survival rate of glioma patients with a high or low level of HCG11 or metastasis-associated 1 family member 3 (MTA3) was analyzed by Kaplan-Meier analysis. The effect of HCG11 on glioma cell growth was determined by in vitro and in vivo experiments. MicroRNAs (miRNAs) that potentially interact with HCG11 were searched and determined by bioinformatics analysis and a luciferase reporter assay. Similarly, the target of miRNA-4425 was identified. Finally, rescue assays were conducted to determine the bio-function of the competing endogenous RNA pathway. RESULTS: HCG11 was downregulated in 84 pairs of glioma tissues and cell lines. Moreover, a low level of HCG11 indicted the lower overall survival rate of glioma patients. Regarding the mechanism, HCG11 was abundant in the cytoplasm of glioma cells and interacted with miR-4425 to release the expression of MTA3. miR-4425 and MTA3 participated in HCG11-mediated glioma growth. CONCLUSIONS: LncRNA HCG11 suppresses the growth of glioma by cooperating with the miR-4425/MTA3 axis.

Molecular identification of BrHAB2a, one of the two AtHAB2-like proteins in Brassica rapa, is an important component of ABA signaling.

Abscisic acid (ABA) signaling is a vital physiological step that is used by many land plants to fight against environmental stress. As components of the linear ABA signaling pathway, clade A protein phosphatases type 2C (PP2C-As) are mainly inhibited by PYRABACTIN RESISTANCE1/PYR1-LIKE/REGULATORY COMPONENTS OF ABA RECEPTORS (PYLs)-type receptors upon their binding to ABA. Here, we show that the genome of Brassica rapa encodes 14 putative clade A PP2C-like proteins (BrPP2C-As). Two of these BrPP2C-As, Bra025964 and Bra016595, show high similarity to the HAB2 (Homology to ABI2) protein in Arabidopsis. RNAseq data reveal that nearly all BrPP2C-As, like BrHAB2a (Bra025964) and BrHAB2b (Bra016595), were highly expressed in at least one tissue. Overexpression of BrHAB2a conferred ABA insensitivity to Arabidopsis thaliana seedlings. Furthermore, the phosphatase activity of BrHAB2a could be inhibited by AtPYL1 or BrPYL1 in the presence of ABA. Overall, these results suggest that BrHAB2a is a functional PP2C-A like protein phosphotase and a key component of ABA signaling in Brassica rapa.

Molecular characterization of an AtPYL1-like protein, BrPYL1, as a putative ABA receptor in Brassica rapa.

Abscisic acid (ABA)-induced physiological changes are conserved in many land plants and underlie their responses to environmental stress and pathogens. The PYRABACTIN RESISTANCE1/PYR1-LIKE/REGULATORY COMPONENTS OF ABA RECEPTORS (PYLs)-type receptors perceive the ABA signal and initiate signal transduction. Here, we show that the genome of Brassica rapa encodes 24 putative AtPYL-like proteins. The AtPYL-like proteins in Brassica rapa (BrPYLs) can also be classified into 3 subclasses. We found that nearly all BrPYLs displayed high expression in at least one tissue. Overexpression of BrPYL1 conferred ABA hypersensitivity to Arabidopsis. Further, ABA activated the expression of an ABA-responsive reporter in Arabidopsis protoplasts expressing BrPYL1. Overall, these results suggest that BrPYL1 is a putative functional ABA receptor in Brassica rapa.

SOS2-LIKE PROTEIN KINASE5, an SNF1-RELATED PROTEIN KINASE3-Type Protein Kinase, Is Important for Abscisic Acid Responses in Arabidopsis through Phosphorylation of ABSCISIC ACID-INSENSITIVE5.

Abscisic acid (ABA) plays an essential role in seed germination. In this study, we demonstrate that one SNF1-related protein kinase3-type protein kinase, SOS2-like protein kinase5 (PKS5), is involved in ABA signal transduction via the phosphorylation of an interacting protein, abscisic acid-insensitive5 (ABI5). We found that pks5-3 and pks5-4, two previously identified PKS5 superactive kinase mutants with point mutations in the PKS5 FISL/NAF (a conserved peptide that is necessary for interaction with SOS3 or SOS3-like calcium binding proteins) motif and the kinase domain, respectively, are hypersensitive to ABA during seed germination. PKS5 was found to interact with ABI5 in yeast (Saccharomyces cerevisiae), and this interaction was further confirmed in planta using bimolecular fluorescence complementation. Genetic studies revealed that ABI5 is epistatic to PKS5. PKS5 phosphorylates a serine (Ser) residue at position 42 in ABI5 and regulates ABA-responsive gene expression. This phosphorylation was induced by ABA in vivo and transactivated ABI5. Expression of ABI5, in which Ser-42 was mutated to alanine, could not fully rescue the ABA-insensitive phenotypes of the abi5-8 and pks5-4abi5-8 mutants. In contrast, mutating Ser-42 to aspartate rescued the ABA insensitivity of these mutants. These data demonstrate that PKS5-mediated phosphorylation of ABI5 at Ser-42 is critical for the ABA regulation of seed germination and gene expression in Arabidopsis (Arabidopsis thaliana).

Hydrotherapy vs. conventional land-based exercise for improving walking and balance after stroke: a randomized controlled trial.

OBJECTIVE: To investigate the effects of hydrotherapy on walking ability and balance in patients with chronic stroke. DESIGN: Single-blind, randomized controlled pilot trial. SETTING: Outpatient rehabilitation clinic at a tertiary neurological hospital in China. SUBJECTS: A total of 28 participants with impairments in walking and controlling balance more than six months post-stroke. INTERVENTION: After baseline evaluations, participants were randomly assigned to a land-based therapy (control group, n = 14) or hydrotherapy (study group, n = 14). Participants underwent individual sessions for four weeks, five days a week, for 45 minutes per session. MAIN MEASURES: After four weeks of rehabilitation, all participants were evaluated by a blinded assessor. Functional assessments included the Functional Reach Test, Berg Balance Scale, 2-minute walk test, and Timed Up and Go Test. RESULTS: After four weeks of treatment, the Berg Balance Scale, functional reach test, 2-minute walk test, and the Timed Up and Go Test scores had improved significantly in each group (P < 0.05). The mean improvement of the functional reach test and 2-minute walk test were significantly higher in the aquatic group than in the control group (P < 0.01). The differences in the mean values of the improvements in the Berg Balance Scale and the Timed Up and Go Test were not statistically significant. CONCLUSION: The results of this study suggest that a relatively short programme (four weeks) of hydrotherapy exercise resulted in a large improvement in a small group (n = 14) of individuals with relatively high balance and walking function following a stroke.

A qualitative comparative analysis of the evolution path of postgraduate enrollment expansion policies and social development in China.

Postgraduate enrollment expansion policies in China have formed a development system of postgraduate education with an expansion of scale and increase of categories. Taking the institutional equilibrium theory as the prerequisite of assumption, this study evaluates 30 postgraduate enrollment expansion policies using the fuzzy set qualitative comparative analysis method (fsQCA method) and explores the matching degree between policy changing path and social development. It is found that path dependence is the main cause and trend of progressive policy change, the construction of the national strategy determines the direction and content of policy change, and the environment induces the abrupt logic and gives early warning to policy change. Therefore, the postgraduate enrollment expansion policy system respects the law of education itself, has contingency and flexibility, adjusts the training structure, and improves the competence of highly specialized talents to serve society. However, it is still necessary to closely follow the social demand to clarify the dialectical relationship between the scale and structure of postgraduate enrollment and highlight the strategic advantages of the government's decision.

Effects of brief mindfulness meditation training on attention and dispositional mindfulness in young adult males.

This study examined the impact of brief mindfulness meditation (BMM) training on attention function and dispositional mindfulness in young males. 126 male participants aged 18-26 from the security industry were recruited, with 66 participants (M = 22.84, SD = 2.41) undergoing 4-week mindfulness meditation training and 60 participants (M = 23.07, SD = 2.29) in the active control group. The intervention was integrated into the participants' schedules. Measures included Five Facets Mindfulness Questionnaires (FFMQ), concentration and assignment attention tasks, Attention Network Test (ANT), and saliva cortisol concentration. Findings indicate that brief mindfulness meditation training led to significant improvements in participants' FFMQ scores), with marginally significant enhancements in the executive control network. However, it had no discernible effect on alertness and orientation networks. Additionally, brief mindfulness meditation training enhanced attention allocation to light stimulation and prolonged individual attention. Surprisingly, there was no observed decrease in saliva cortisol concentration among meditation training participants. However, this study did not find a decrease in saliva cortisol concentration in the brief mindfulness meditation group. In conclusion, this study highlights the potential of a 4-week brief mindfulness meditation training program to enhance dispositional mindfulness and specific aspects of attention function in young men, offering practical insights into the benefits of mindfulness meditation practices for this demographic.

Increased water content in multifidus muscles of young adults with chronic nonspecific low back pain detected by dual-energy CT and MRI.

OBJECTIVES: To demonstrate the feasibility of better diagnosing young adults with chronic nonspecific low back pain (CNLBP) by measuring water content in paraspinal muscles using water-muscle decomposition technique in dual-energy CT (DECT) and T2-mapping in MRI. METHODS: This prospective cross-sectional study included 110 young individuals (56 with CNLBP at age of 25.7 ± 2.0 years and 54 of asymptomatic at age of 25.1 ± 1.9 years) who underwent both MRI and DECT on the spine. T2 values on T2 mapping in MRI and water density (WD) value on water(muscle) images in DECT were generated at the L1-L4 levels for erector spinae muscle and L2-L5 for multifidus muscle. Pain duration time, Oswestry Disability Index (ODI), Visual Analogue Scale (VAS) were recorded for CNLBP patients. Difference of T2 value and WD between the two patient groups, and correlations between T2 value and WD, and T2 value and WD with clinical indicators were analyzed. RESULTS: Compared with asymptomatic participants, the mean WD of multifidus muscle at L4-L5 and mean T2 values of multifidus muscle at L5 were significantly higher in CNLBP patients (all P < 0.05). T2 values had moderate to strong positive correlations (r = 0.34-0.60, all P < 0.05) with DECT WD in CNLBP patients and healthy volunteers. There was a weak correlation between VAS and WD in L5-level multifidus muscle (r = 0.29, P < 0.05). CONCLUSIONS: The T2 values in MRI and WD in DECT are higher in multifidus muscles of lower vertebra levels for young CNLBP patients, and there exists positive correlation between WD and T2 values, providing useful information for diagnosing CNLBP.

Molecular characterization of an ABA insensitive 5 orthologue in Brassica oleracea.

ABI5 (ABA insensitive 5), a bZIP (Basic leucine zipper) transcription factor, has been shown to be a major mediator of plant ABA responses during seed germination. Although the molecular basis of ABI5-modulated processes has been well demonstrated in Arabidopsis thaliana, its identity and function in cabbage (Brassica oleracea var. capitata L.) remain elusive. Here, we describe our identification of BolABI5 (an ABI5 orthologue in B.oleracea) as a functional bZIP transcription factor in the modulation of plant ABA responses. Expression of BolABI5 was dramatically induced by drought stress and exogenous ABA. Heterogeneous expression of BolABI5 rescued the insensitive phenotype of Arabidopsis abi5-1 to ABA during seed germination. Subcellular localization and trans-activation assays revealed that BolABI5 was localized in the nucleus and possessed DNA binding and trans-activation activities. Deletion of the bZIP domain generated BolABI5ΔbZIP, which no longer localized exclusively in the nucleus and had almost no detectable DNA-binding or trans-activation activities. Overall, these results suggest that BolABI5 may function as ABI5 in the positive regulation of plant ABA responses.

BolOST1, an ortholog of Open Stomata 1 with alternative splicing products in Brassica oleracea, positively modulates drought responses in plants.

Open Stomata 1 (OST1), an ABA-activated sucrose non-fermenting 1 (SNF1)-related protein kinase, is critical for plant drought responses. We investigated the functions of two splicing isoforms of the OST1 ortholog in Brassica oleracea (BolOST1). BolOST1 expression was found to be dramatically induced by drought and high-salt stress, and the ectopic expression of BolOST1 restored the drought-sensitive phenotype of ost1. Subcellular localization revealed that BolOST1 is localized in both the nucleus and cytoplasm. BolOST1 was also demonstrated to phosphorylate the N-terminal fragment of ABI5 (ABA Insensitive 5, ABI5-N). A firefly luciferase complementation assay revealed that BolOST1 interacts with both BolABI5 and an ABI1 ortholog in B. oleracea (BolABI1). Overall, these results suggest that BolOST1 is a functional SnRK2-type protein kinase and that the early ABA signaling network may be conserved between Arabidopsis and cabbage.

Negative regulation of abscisic acid signaling by the Brassica oleracea ABI1 ortholog.

ABI1 (ABA Insensitive 1) is an important component of the core regulatory network in early ABA (Abscisic acid) signaling. Here, we investigated the functions of an ABI1 ortholog in Brassica oleracea (BolABI1). The expression of BolABI1 was dramatically induced by drought, and constitutive expression of BolABI1 confers ABA insensitivity upon the wild-type. Subcellular localization and phosphatase assays reveal that BolABI1 is predominantly localized in the nucleus and harbors phosphatase activity. Furthermore, BolABI1 interacts with a homolog of OST1 (OPEN STOMATA 1) in B. oleracea (BolOST1) and can dephosphorylate ABI5 (ABA Insensitive 5) in vitro. Overall, these results suggest that BolABI1 is a functional PP2C-type protein phosphatase that is involved in the negative modulation of the ABA signaling pathway.

Genome-wide exploration of the GDSL-type esterase/lipase gene family in rapeseed reveals several BnGELP proteins active during early seedling development.

The Gly-Asp-Ser-Leu (GDSL)-type esterase/lipase proteins (GELP) are one of the most important families of lipolytic enzymes and play prominent roles in seed germination and early seedling establishment through mobilizing the lipids stored in seeds. However, there are no comprehensive studies systematically investigating the GELP gene family in Brassica napus (BnGELP), and their biological significance to these physiological processes are far from understood. In the present study, a total of 240 BnGELP genes were identified in B. napus cultivar "Zhongshuang 11" (ZS11), which is nearly 2.3-fold more GELP genes than in Arabidopsis thaliana. The BnGELP genes clustered into 5 clades based on phylogenetic analysis. Ten BnGELPs were identified through zymogram analysis of esterase activity followed by mass spectrometry, among which five clustered into the clade 5. Gene and protein architecture, gene expression, and cis-element analyses of BnGELP genes in clade 5 suggested that they may play different roles in different tissues and in response to different abiotic stresses. BnGELP99 and BnGELP159 were slightly induced by cold, which may be attributed to two low-temperature responsive cis-acting regulatory elements present in their promoters. An increased activity of esterase isozymes by cold was also observed, which may reflect other cold inducible esterases/lipases in addition to the ten identified BnGELPs. This study provides a systemic view of the BnGELP gene family and offers a strategy for researchers to identify candidate esterase/lipase genes responsible for lipid mobilization during seed germination and early seedling establishment.

Genome-Wide Identification of the Cytochrome P450 Superfamily Genes and Targeted Editing of BnCYP704B1 Confers Male Sterility in Rapeseed.

The cytochrome P450 (CYP450) monooxygenase superfamily, which is involved in the biosynthesis pathways of many primary and secondary metabolites, plays prominent roles in plant growth and development. However, systemic information about CYP450s in Brassica napus (BnCYP450) was previously undiscovered and their biological significance are far from understood. Members of clan 86 CYP450s, such as CYP704Bs, are essential for the formation of pollen exine in plant male reproduction, and the targeted mutagenesis of CYP704B genes has been used to create new male sterile lines in many crops. In the present study, a total of 687 BnCYP450 genes were identified in Brassica napus cultivar "Zhongshuang 11" (ZS11), which has nearly 2.8-fold as many CYP450 members as in Arabidopsis thaliana. It is rationally estimated since Brassica napus is a tetraploid oil plant with a larger genome compared with Arabidopsis thaliana. The BnCYP450 genes were divided into 47 subfamilies and clustered into nine clans. Phylogenetic relationship analysis reveals that CYP86 clan consists of four subfamilies and 109 BnCYP450s. Members of CYP86 clan genes display specific expression profiles in different tissues and in response to ABA and abiotic stresses. Two BnCYP450s within the CYP704 subfamily from CYP86 clan, BnCYP704B1a and BnCYP704B1b, display high similarity to MS26 (Male Sterility 26, also known as CYP704B1). These two BnCYP704B1 genes were specifically expressed in young buds. We then simultaneously knocked-out these two BnCYP704B1 genes through a clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9) genome engineering system. The edited plants displayed a pollenless, sterile phenotype in mature anthers, suggesting that we successfully reproduced genic male sterility (GMS, also known as nuclear male sterility) lines in Brassica napus. This study provides a systemic view of BnCYP450s and offers a strategy to facilitate the commercial utility of the CRISPR/Cas9 system for the rapid generation of GMS in rapeseed via knocking-out GMS controlling genes.

Genome-Wide Analysis of MYB Transcription Factor Gene Superfamily Reveals BjPHL2a Involved in Modulating the Expression of BjCHI1 in Brassica juncea.

Brassica juncea is an economically important vegetable and oilseed crop. The MYB transcription factor superfamily is one of the largest transcription factor families in plants, and plays crucial roles in regulating the expression of key genes involved in a variety of physiological processes. However, a systematic analysis of the MYB transcription factor genes in Brassica juncea (BjMYB) has not been performed. In this study, a total of 502 BjMYB superfamily transcription factor genes were identified, including 23 1R-MYBs, 388 R2R3-MYBs, 16 3R-MYBs, 4 4R-MYBs, 7 atypical MYBs, and 64 MYB-CCs, which is approximately 2.4-fold larger than that of AtMYBs. Phylogenetic relationship analysis revealed that the MYB-CC subfamily consists of 64 BjMYB-CC genes. The expression pattern of members of PHL2 subclade homologous genes in Brassica juncea (BjPHL2) after Botrytis cinerea infection were determined, and BjPHL2a was isolated from a yeast one-hybrid screen with the promoter of BjCHI1 as bait. BjPHL2a was found to localize mainly in the nucleus of plant cells. An EMSA assay confirmed that BjPHL2a binds to the Wbl-4 element of BjCHI1. Transiently expressed BjPHL2a activates expression of the GUS reporter system driven by a BjCHI1 mini-promoter in tobacco (Nicotiana benthamiana) leaves. Taken together, our data provide a comprehensive evaluation of BjMYBs and show that BjPHL2a, one of the members of BjMYB-CCs, functions as a transcription activator by interacting with the Wbl-4 element in the promoter of BjCHI1 for targeted gene-inducible expression.