Metabolomics and transcriptomics analyses for characterizing the alkaloid metabolism of Chinese jujube and sour jujube fruits.

Introduction: Jujube is an important economic forest tree whose fruit is rich in alkaloids. Chinese jujube (Ziziphus jujuba Mill.) and sour jujube (Ziziphus spinosa Hu.) are the two most important species of the jujube genus. However, the mechanisms underlying the synthesis and metabolism of alkaloids in jujube fruits remain poorly understood. Methods: In this study, the fruits of Ziziphus jujuba 'Hupingzao' and Ziziphus spinosa 'Taigusuanzao' in different harvest stages were used as test materials, we first integrated widely targeted metabolomics and transcriptomics analyses to elucidate the metabolism of alkaloids of jujube fruits. Results: In the metabolomics analysis, 44 alkaloid metabolites were identified in 4 samples, 3 of which were unique to sour jujube fruit. The differential alkaloid metabolites (DAMs) were more accumulated in sour jujube than in Chinese jujube; further, they were more accumulated in the white ripening stage than in the red stage. DAMs were annotated to 12 metabolic pathways. Additionally, transcriptomics data revealed 259 differentially expressed genes (DEGs) involved in alkaloid synthesis and metabolism. By mapping the regulatory networks of DAMs and DEGs, we screened out important metabolites and 11 candidate genes. Discussion: This study preliminarily elucidated the molecular mechanism of jujube alkaloid synthesis. The candidate genes regulated the synthesis of key alkaloid metabolites, but the specific regulation mechanism is unclear. Taken together, our results provide insights into the metabolic networks of alkaloid synthesis in Chinese jujube and sour jujube fruits at different harvest stages, thereby providing a theoretical reference for further research on the regulatory mechanism of jujube alkaloids and their development and utilization.

Sericin Ser3 Ectopic Expressed in Posterior Silk Gland Affects Hemolymph Immune Melanization Response via Reducing Melanin Synthesis in Silkworm.

The transgenesis of silkworms is an important way to innovate genetic resources and silk function. However, the silk-gland (SG) of transgenic silkworms, which is the most concerned target tissue of sericulture, often suffers from low vitality, stunting and other problems, and the reasons are still unknown. This study trans engineered recombinant Ser3, a middle silk gland (MSG) specific expression gene, in the posterior silk gland (PSG) of the silkworm, and studied hemolymph immune melanization response changes in mutant pure line SER (Ser3+/+). The results showed that although the mutant had normal vitality, the melanin content and phenoloxidase (PO) activity in hemolymph related to humoral immunity were significantly reduced, and caused significantly slower blood melanization and weaker sterilization ability. The mechanism investigation showed that the mRNA levels and enzymatic activities of phenylalanine hydroxylase (PAH), tyrosine hydroxylase (TH) and dopamine decarboxylase (DDC) in the melanin synthesis pathway in mutant hemolymph, as well as the transcription levels of the PPAE, SP21 and serpins genes in the serine protease cascade were significantly affected. Moreover, the total antioxidant capacity, superoxide anion inhibition capacity and catalase (CAT) level related to the redox metabolic capacity of hemolymph were significantly increased, while the activities of superoxide dismutase (SOD) and glutathione reductase (GR), as well as the levels of hydrogen peroxide (H2O2) and glutathione (GSH), were significantly decreased. In conclusion, the anabolism of melanin in the hemolymph of PSG transgenic silkworm SER was inhibited, while the basic response level of oxidative stress was increased, and the hemolymph immune melanization response was decreased. The results will significantly improve the safe assessment and development of genetically modified organisms.

Genomic C-Value Variation Analysis in Jujube (Ziziphus jujuba Mill.) in the Middle Yellow River Basin.

Chinese jujube (Ziziphus jujuba Mill.) originated in the Yellow River basin (YRB) of the Shanxi-Shaanxi region. The genomic C-value is a crucial indicator for plant breeding and germplasm evaluation. In this study, we used flow cytometry to determine the genomic C-values of jujube germplasms in the YRB of the Shanxi-Shaanxi region and evaluated their differences in different sub-regions. Of the 29 sub-regions, the highest and lowest variations were in Linxian and Xiaxian, respectively. The difference between jujube germplasms was highly significant (F = 14.89, p < 0.0001) in Linxian. Cluster analysis showed that both cluster 2 and 4 belonged to Linxian, which were clearly separated from other taxa but were cross-distributed in them. Linxian County is an important gene exchange center in the YRB of the Shanxi-Shaanxi region. Principal component analysis showed that cluster 1 had low genomic C-values and single-fruit weights and cluster 2 had high genomic C-values and vitamin C contents. The genomic C-value was correlated with single-fruit weight and vitamin C content. In addition, the genomic C-value was used to predict fruit agronomic traits, providing a reference for shortening the breeding cycle and genetic diversity-related studies of jujube germplasm.

The NEDD8-activating enzyme inhibitor MLN4924 reduces ischemic brain injury in mice.

Blood-brain barrier (BBB) breakdown and inflammation occurring at the BBB have a key, mainly a deleterious role in the pathophysiology of ischemic stroke. Neddylation is a ubiquitylation-like pathway that is critical in various cellular functions by conjugating neuronal precursor cell-expressed developmentally down-regulated protein 8 (NEDD8) to target proteins. However, the roles of neddylation pathway in ischemic stroke remain elusive. Here, we report that NEDD8 conjugation increased during acute phase after ischemic stroke and was present in intravascular and intraparenchymal neutrophils. Inhibition of neddylation by MLN4924, also known as pevonedistat, inactivated cullin-RING E3 ligase (CRL), and reduced brain infarction and improved functional outcomes. MLN4924 treatment induced the accumulation of the CRL substrate neurofibromatosis 1 (NF1). By using virus-mediated NF1 silencing, we show that NF1 knockdown abolished MLN4924-dependent inhibition of neutrophil trafficking. These effects were mediated through activation of endothelial P-selectin and intercellular adhesion molecule-1 (ICAM-1), and blocking antibodies against P-selectin or anti-ICAM-1 antibodies reversed NF1 silencing-induced increase in neutrophil infiltration in MLN4924-treated mice. Furthermore, we found that NF1 silencing blocked MLN4924-afforded BBB protection and neuroprotection through activation of protein kinase C δ (PKCδ), myristoylated alanine-rich C-kinase substrate (MARCKS), and myosin light chain (MLC) in cerebral microvessels after ischemic stroke, and treatment of mice with the PKCδ inhibitor rottlerin reduced this increased BBB permeability. Our study demonstrated that increased neddylation promoted neutrophil trafficking and thus exacerbated injury of the BBB and stroke outcomes. We suggest that the neddylation inhibition may be beneficial in ischemic stroke.

Potential Value of Expiratory CT in Quantitative Assessment of Pulmonary Vessels in COPD.

Objective: To investigate the associations between intrapulmonary vascular volume (IPVV) depicted on inspiratory and expiratory CT scans and disease severity in COPD patients, and to determine which CT parameters can be used to predict IPVV. Methods: We retrospectively collected 89 CT examinations acquired on COPD patients from an available database. All subjects underwent both inspiratory and expiratory CT scans. We quantified the IPVV, airway wall thickness (WT), the percentage of the airway wall area (WA%), and the extent of emphysema (LAA%-950) using an available pulmonary image analysis tool. The underlying relationship between IPVV and COPD severity, which was defined as mild COPD (GOLD stage I and II) and severe COPD (GOLD stage III and IV), was analyzed using the Student's t-test (or Mann-Whitney U-test). The correlations of IPVV with pulmonary function tests (PFTs), LAA%-950, and airway parameters for the third to sixth generation bronchus were analyzed using the Pearson or Spearman's rank correlation coefficients and multiple stepwise regression. Results: In the subgroup with only inspiratory examinations, the correlation coefficients between IPVV and PFT measures were -0.215 ~ -0.292 (p < 0.05), the correlation coefficients between IPVV and WT3-6 were 0.233 ~ 0.557 (p < 0.05), and the correlation coefficient between IPVV and LAA%-950 were 0.238 ~ 0.409 (p < 0.05). In the subgroup with only expiratory scan, the correlation coefficients between IPVV and PFT measures were -0.238 ~ -0.360 (p < 0.05), the correlation coefficients between IPVV and WT3-6 were 0.260 ~ 0.566 (p < 0.05), and the correlation coefficient between IPVV and LAA%-950 were 0.241 ~ 0.362 (p < 0.05). The multiple stepwise regression analyses demonstrated that WT were independently associated with IPVV (P < 0.05). Conclusion: The expiratory CT scans can provide a more accurate assessment of COPD than the inspiratory CT scans, and the airway wall thickness maybe an independent predictor of pulmonary vascular alteration in patients with COPD.

Neutrophil extracellular traps promote tPA-induced brain hemorrhage via cGAS in mice with stroke.

Intracerebral hemorrhage associated with thrombolytic therapy with tissue plasminogen activator (tPA) in acute ischemic stroke continues to present a major clinical problem. Here, we report that infusion of tPA resulted in a significant increase in markers of neutrophil extracellular traps (NETs) in the ischemic cortex and plasma of mice subjected to photothrombotic middle cerebral artery occlusion. Peptidylarginine deiminase 4 (PAD4), a critical enzyme for NET formation, is also significantly upregulated in the ischemic brains of tPA-treated mice. Blood-brain barrier (BBB) disruption after ischemic challenge in an in vitro model of BBB was exacerbated after exposure to NETs. Importantly, disruption of NETs by DNase I or inhibition of NET production by PAD4 deficiency restored tPA-induced loss of BBB integrity and consequently decreased tPA-associated brain hemorrhage after ischemic stroke. Furthermore, either DNase I or PAD4 deficiency reversed tPA-mediated upregulation of the DNA sensor cyclic GMP-AMP (cGAMP) synthase (cGAS). Administration of cGAMP after stroke abolished DNase I-mediated downregulation of the STING pathway and type 1 interferon production and blocked the antihemorrhagic effect of DNase I in tPA-treated mice. We also show that tPA-associated brain hemorrhage after ischemic stroke was significantly reduced in cGas-/- mice. Collectively, these findings demonstrate that NETs significantly contribute to tPA-induced BBB breakdown in the ischemic brain and suggest that targeting NETs or cGAS may ameliorate thrombolytic therapy for ischemic stroke by reducing tPA-associated hemorrhage.

Neutrophil extracellular traps released by neutrophils impair revascularization and vascular remodeling after stroke.

Neovascularization and vascular remodeling are functionally important for brain repair after stroke. We show that neutrophils accumulate in the peri-infarct cortex during all stages of ischemic stroke. Neutrophils producing intravascular and intraparenchymal neutrophil extracellular traps (NETs) peak at 3-5 days. Neutrophil depletion reduces blood-brain barrier (BBB) breakdown and enhances neovascularization at 14 days. Peptidylarginine deiminase 4 (PAD4), an enzyme essential for NET formation, is upregulated in peri-ischemic brains. Overexpression of PAD4 induces an increase in NET formation that is accompanied by reduced neovascularization and increased BBB damage. Disruption of NETs by DNase 1 and inhibition of NET formation by genetic ablation or pharmacologic inhibition of PAD increases neovascularization and vascular repair and improves functional recovery. Furthermore, PAD inhibition reduces stroke-induced STING-mediated production of IFN-ß, and STING knockdown and IFN receptor-neutralizing antibody treatment reduces BBB breakdown and increases vascular plasticity. Collectively, our results indicate that NET release impairs vascular remodeling during stroke recovery.

Impact of adding glucose-coated water-soluble silver nanoparticles to the silkworm larval diet on silk protein synthesis and related properties.

Biological modifications of the silk fibroin (silk) material have broad applications in textiles, biomedical materials and other industrial materials. It is economical to incorporate nanoparticles to the biosynthesis of silk fibroin by adding them to silkworm larval diets. This strategy may result in the rapid stable production of modified silk. Glucose-coated silver nanoparticles (AgNPs) were used to improve the AgNPs' biocompatibility, and the AgNPs were efficiently incorporated into silk by feeding. Larvae fed with AgNPs produced silk with significantly improved antibacterial properties and altered silk secondary structures. Both positive and negative effects on the growth and synthesis of silk proteins were observed after different AgNPs doses. Larvae feeding with low concentration of 0.02% and medium 0.20% AgNPs have greater transfer efficiencies of AgNPs to silk compared with feeding high concentration of 2.00% AgNPs. In addition, the elongation and tensile strength of the produced silk fibers were also significantly increased, with greater mammalian cell compatibility. The appropriate AgNPs concentration in the diet of silkworms can promote the synthesis of silk proteins, enhance their mechanical properties, improve their antibacterial property and inhibit the presence of Gram-negative bacteria.

ADAMTS13 maintains cerebrovascular integrity to ameliorate Alzheimer-like pathology.

Blood-brain barrier (BBB) defects and cerebrovascular dysfunction contribute to amyloid-ß (Aß) brain accumulation and drive Alzheimer disease (AD) pathology. By regulating vascular functions and inflammation in the microvasculature, a disintegrin and metalloprotease with thrombospondin type I motif, member 13 (ADAMTS13) plays a significant protective effect in atherosclerosis and stroke. However, whether ADAMTS13 influences AD pathogenesis remains unclear. Using in vivo multiphoton microscopy, histological, behavioral, and biological methods, we determined BBB integrity, cerebrovascular dysfunction, amyloid accumulation, and cognitive impairment in APPPS1 mice lacking ADAMTS13. We also tested the impact of viral-mediated expression of ADAMTS13 on cerebrovascular function and AD-like pathology in APPPS1 mice. We show that ADAMTS13 deficiency led to an early and progressive BBB breakdown as well as reductions in vessel density, capillary perfusion, and cerebral blood flow in APPPS1 mice. We found that deficiency of ADAMTS13 increased brain plaque load and Aß levels and accelerated cerebral amyloid angiopathy (CAA) by impeding BBB-mediated clearance of brain Aß, resulting in worse cognitive decline in APPPS1 mice. Virus-mediated expression of ADAMTS13 attenuated BBB disruption and increased microvessels, capillary perfusion, and cerebral blood flow in APPPS1 mice already showing BBB damage and plaque deposition. These beneficial vascular effects were reflected by increase in clearance of cerebral Aß, reductions in Aß brain accumulation, and improvements in cognitive performance. Our results show that ADAMTS13 deficiency contributes to AD cerebrovascular dysfunction and the resulting pathogenesis and cognitive deficits and suggest that ADAMTS13 may offer novel therapeutic opportunities for AD.

Growth Differentiation Factor 11 Promotes Neurovascular Recovery After Stroke in Mice.

Background: Growth differentiation factor 11 (GDF11), a member of transforming growth factor-ß (TGF-ß) superfamily, was shown to rejuvenate cardiac and skeletal muscle function and to improve cerebral vasculature and neurogenesis in old mice. However, recent experimental data reported that raising GDF11 levels inhibited skeletal muscle regeneration and had no effect on cardiac hypertrophy. Our aim was to investigate the effects of GDF11 on brain repair during the recovery phase after stroke. Methods: Mice were subjected to distal middle cerebral artery occlusion, and recombinant GDF11 (rGDF11) was injected intraperitoneally once a day during days 7-13 after stroke. Neuronal precursor cells (NPCs) proliferation and angiogenesis were assayed at 14 days. Neuronal regeneration was assayed at 42 days. The beam-walking test and CatWalk were used to evaluate behavioral functions. Downstream pathways of GDF11 were also investigated. Results: GDF11 was upregulated in the ipsilateral peri-infarct cortex and subventricular zone (SVZ) at 14 days after stroke. Treatment with rGDF11 enhanced the number of newborn NPCs and endothelial cells, microvascular length and area, and brain capillary perfusion. Western blots showed that rGDF11 upregulated brain-derived neurotrophic factor (BDNF) and increased the levels of proangiogenic factor angiopoietin-2 (Ang-2) and phosphorylation of vascular endothelial growth factor receptor-2 (VEGFR-2). We also found that rGDF11 upregulated the transcription factors Smad2 and Smad3 phosphorylation, but these activations were blocked by a TGF-ß receptor inhibitor SB431542. Moreover, rGDF11-induced angiogenic remodeling and NPCs proliferation were reversed by injection of SB431542, suggesting that GDF11 may exert its effect via the TGF-ß/Smad2/3 signaling pathway. Finally, treating mice with rGDF11 resulted in a significant increase in neuronal regeneration and functional recovery. Conclusion: GDF11 promoted neurogenesis and angiogenesis and contributed to functional recovery after stroke in mice.

Genetic association between SHANK2 polymorphisms and susceptibility to autism spectrum disorder.

Autism spectrum disorder (ASD), as one of early-onset neurodevelopmental disorders, is characterized by the following symptoms, including repetitive and stereotyped behaviors, impairments in social interaction, and dysfunctions in communication. ASD afflicts ∼1.5% of children aged 8 years in America and ∼4.5‰ of children aged 0-6 years in China. Existing studies suggest that SH3 and multiple ankyrin repeat domains protein 2 (SHANK2) is implicated in ASD. However, associations between SNPs in SHANK2 introns and ASD risk have been less investigated. In this study, on the basis of case-control study (226 cases and 239 controls), we selected nine SNPs (rs76717360, rs11236697, rs74336682, rs77950809, rs17428526, rs35459123, rs75357229, rs61887413, and rs77716438) in SHANK2 introns to investigate genetic associations between SHANK2 polymorphisms and susceptibility to ASD using improved multiple ligase detection reaction (iMLDR). We identified that the polymorphism of rs76717360 was associated with risk of ASD in Chinese population; the haplotype of rs11236697 C (T) or rs74336682 G (A) increased ASD risk; and haplotypes with ≥ five SNPs containing rs11236697 and rs74336682 were associated with risk of ASD. Our results indicate SHANK2 is a susceptibility gene for ASD in Chinese children. © 2018 IUBMB Life, 70(8):763-776, 2018.

Association between SHANK3 polymorphisms and susceptibility to autism spectrum disorder.

Autism spectrum disorder (ASD), as one of neurodevelopmental disorders, affects about 1/160 of people worldwide. The etiology and pathogenesis of ASD remain elusive. Synapses are essential components of neurons and basic information transmission unit in the nervous system, adjusting behavior to environmental stimuli and controlling body functions, memories, and emotions. SHANK3 is one of the synapse genes which play important roles in maintaining synaptic structure and function. SHANK3 has been researched as a probably susceptibility gene for ASD. We investigated the association between polymorphisms in SHANK3 and ASD in the Northeast Han Chinese population. A total of 470 subjects (229 cases and 241 controls) were enrolled in our case-control study. Five single nucleotide polymorphisms (SNPs) (rs756638, rs4824116, rs76268556, rs9616915, and rs75767639) in SHANK3 were selected and genotyped. Our study did not identify a significant association of SHANK3 SNPs with ASD in the Northeast Han Chinese population. Future studies need to test more SHANK3 SNPs in large sample to demonstrate the association between SNPs in SHANK3 and ASD.

Risk factors predict frequent hospitalization in patients with acute exacerbation of COPD.

Purpose: COPD is a heterogeneous disease, and the available prognostic indexes are therefore limited. This study aimed to identify the factors associated with acute exacerbation leading to hospitalization. Patients and methods: This was a retrospective study of consecutive patients with COPD (meeting the Global Initiative for Chronic Obstructive Lung Disease [GOLD] diagnostic criteria) hospitalized at the Ninth Hospital of Xi'an Affiliated Hospital of Xi'an Jiaotong University between October 2014 and September 2016. During follow-up after first hospitalization, the patients who had been rehospitalized within 1 year for acute exacerbation were grouped into the frequent exacerbation (FE) group, while the others were grouped into the infrequent exacerbation (IE) group. The baseline demographic, clinical, laboratory, pulmonary function, and imaging data were compared between the two groups. Results: Compared with the IE group, the FE group had lower forced expiratory volume in 1 second (FEV1)/forced vital capacity (FVC) (P=0.005), FEV1%pred (P=0.002), maximal mid-expiratory flow (MMEF25-75%pred) (P=0.003), and ratio of carbon monoxide diffusion capacity to alveolar ventilation (DLCO/VA) (P=0.03) and higher resonant frequency (Fres; P=0.04). According to generations of bronchi, the percentage of the wall area (%WA) of lobes was found to be higher in the FE group. Emphysema index (EI), mean emphysema density (MED)whole and MEDleft lung in the FE group were significantly worse than in the IE group (P<0.05). Using logistic regression, exacerbation hospitalizations in the past year (odds ratio [OR] 14.4, 95% CI 6.1-34.0, P<0.001) and EI >10% (OR 2.9, 95% CI 1.2-7.1, P=0.02) were independently associated with frequent acute exacerbation of COPD (AECOPD) hospitalization. Conclusion: Exacerbation hospitalizations in the past year and imaging features of emphysema (EI) were independently associated with FE hospitalization.

Detection of Ag(+) using graphite carbon nitride nanosheets based on fluorescence quenching.

The graphite carbon nitride (g-C3N4) nanosheets were synthesized and applied for the detection of Ag(+) ion in aqueous solutions. Transmission electron microscopy, Fourier infrared spectroscopy, x-ray diffraction, ultraviolet/visible and photoluminescence spectroscopy were used for characterization of g-C3N4 nanosheets. The fluorescence intensity of g-C3N4 nanosheets decreases with the increase in the concentration of Ag(+). The fluorescence probe can be applied for detection of Ag(+). The results show that it has high selectivity to Ag(+) and exhibits a good linearity over the concentration range 0.020-2.0µM with a detection limit of 27nM. Most cations do not have any interference on the detection of Ag(+). The quenching process is assessed and discussed. Finally, the g-C3N4 nanosheets have been successfully used for the detection of Ag(+) in real water samples. The recoveries of spiked water samples are >97%.