A functional study reveals CsNAC086 regulated the biosynthesis of flavonols in Camellia sinensis.

MAIN CONCLUSION: CsNAC086 was found to promote the expression of CsFLS, thus promoting the accumulation of flavonols in Camellia sinensis. Flavonols, the main flavonoids in tea plants, play an important role in the taste and quality of tea. In this study, a NAC TF gene CsNAC086 was isolated from tea plants and confirmed its regulatory role in the expression of flavonol synthase which is a key gene involved in the biosynthesis of flavonols in tea plant. Yeast transcription-activity assays showed that CsNAC086 has self-activation activity. The transcriptional activator domain of CsNAC086 is located in the non-conserved C-terminal region (positions 171-550), while the conserved NAC domain (positions 1-170) does not have self-activation activity. Silencing the CsNAC086 gene using antisense oligonucleotides significantly decreased the expression of CsFLS. As a result, the concentration of flavonols decreased significantly. In overexpressing CsNAC086 tobacco leaves, the expression of NtFLS was significantly increased. Compared with wild-type tobacco, the flavonols concentration increased. Yeast one-hybrid assays showed CsNAC086 did not directly regulate the gene expression of CsFLS. These findings indicate that CsNAC086 plays a role in regulating flavonols biosynthesis in tea plants, which has important implications for selecting and breeding of high-flavonols-concentration containing tea-plant cultivars.

Transcriptome Analysis Reveals Possible Immunomodulatory Activity Mechanism of Chlorella sp. Exopolysaccharides on RAW264.7 Macrophages.

In this study, the exopolysaccharides of Chlorella sp. (CEP) were isolated to obtain the purified fraction CEP4. Characterization results showed that CEP4 was a sulfated heteropolysaccharide. The main monosaccharide components of CEP4 are glucosamine hydrochloride (40.8%) and glucuronic acid (21.0%). The impact of CEP4 on the immune activity of RAW264.7 macrophage cytokines was detected, and the results showed that CEP4 induced the production of nitric oxide (NO), TNF-α, and IL-6 in a dose-dependent pattern within a range of 6 µg/mL. A total of 4824 differentially expressed genes (DEGs) were obtained from the results of RNA-seq. Gene enrichment analysis showed that immune-related genes such as NFKB1, IL-6, and IL-1ß were significantly upregulated, while the genes RIPK1 and TLR4 were significantly downregulated. KEGG pathway enrichment analysis showed that DEGs were significantly enriched in immune-related biological processes, including toll-like receptor (TLR) signaling pathway, cytosolic DNA-sensing pathway, and C-type lectin receptor signaling pathway. Protein-protein interaction (PPI) network analysis showed that HSP90AB1, Rbx1, ISG15, Psmb6, Psmb3, Psmb8, PSMA7, Polr2f, Rpsa, and NEDD8 were the hub genes with an essential role in the immune activity of CEP4. The preliminary results of the present study revealed the potential mechanism of CEP4 in the immune regulation of RAW264.7 macrophages, suggesting that CEP4 is a promising immunoregulatory agent.

A new N-methoxyl-carbonyl benzylisoquinoline from Litsea cubeba.

A new benzylisoquinoline alkaloid (•)-N-methoxycarbonyl-norjuziphine (1) was isolated from Litsea cubeba. Its structure was identified by extensively spectroscopic techniques and confirmed by the single-crystal X-ray diffraction analysis. Compound 1 showed cytotoxicity against HL-60 and MCF-7 cells, with IC50 values of 18.1 and 15.0 µM, respectively, comparable to 3.1 and 17.5 µM of the cisplatin (positive control).

Optimization of monomethoxy polyethyleneglycol-modified oxalate decarboxylase by response surface methodology.

In order to improve the stability of oxalate decarboxylase (Oxdc), response surface methodology (RSM), based on a four-factor three-level Box-Behnken central composite design was used to optimize the reaction conditions of oxalate decarboxylase (Oxdc) modified with monomethoxy polyethyleneglycol (mPEG5000). Four independent variables such as the ratio of mPEG-aldehyde to Oxdc, reaction time, temperature, and reaction pH were investigated in this work. The structure of modified Oxdc was identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Fourier transform infrared (FTIR) spectroscopy, the stability of the modified Oxdc was also investigated. The optimal conditions were as follows: the mole ratio of mPEG-aldehyde to Oxdc of 1:47.6, time of 13.1 h, temperature at 29.9 °C, and the reaction pH of 5.3. Under optimal conditions, experimental modified rate (MR = 73.69%) and recovery rate (RR = 67.58%) were matched well with the predicted value (MR = 75.11%) and (RR = 69.17%). SDS-PAGE and FTIR analysis showed that mPEG was covalently bound to the Oxdc. Compared with native Oxdc, the modified Oxdc (mPEG-Oxdc) showed higher thermal stability and better tolerance to trypsin or different pH treatment. This work will provide a further theoretical reference for enzyme modification and conditional optimization.

[In Vitro Evaluation Method for Drug Release and Particle Release from Drug Coated Balloon].

OBJECTIVES: Development in vitro test methods for drug release and particle release from drug coated balloon to address the disadvantages and inadequacy of existing assessment methods. METHODS: In vitro drug transfer test were carried out using the isolated porcine vessel as target site in the self-designed iliofemoral artery model, drug loss experiment during dilatation and retraction were added in drug release test, the particle test channel was subdivided and the size and number of the large particles were measured by microscopy combined with Image-Pro Plus software. RESULTS: The result shows that the method is feasible and the discrimination is good. The material balance can be achieved. More detailed data can be obtained. CONCLUSIONS: The developed in vitro evaluation method provides a reference for formulation screening, in vitro bench testing and in vivo pre-clinical animal testing.

Role of naturally occurring genome segment reassortment in the pathogenicity of IBDV field isolates in Three-Yellow chickens.

Reassortment among genome segments of infectious bursal disease virus (IBDV) field isolates was reported frequently worldwide, however the pathogenicity of the reassortant field IBDV is poorly understood. In this paper, a pathogenicity study on four representative IBDV field strains isolated from Southern China between 2005 and 2011 was conducted. Twenty-eight-day-old Three-Yellow chickens were divided into four groups and were inoculated intraocularly with one of the four field IBDV strains, namely NN1172, NN1005, GD10111 and JS7, respectively. The mortality and relative weight of bursa and thymus were subsequently determined in the acute phase of infection. In addition, B cells, T cells (CD4(+) and CD8(+)) and virus were quantified in the bursa of Fabricius and thymus, respectively, by flow cytometry and real-time reverse transcription-polymerase chain reaction. The results showed that isolate NN1172, of which parts of segment A and B encoding the hypervariable (v) region of viral protein (VP2) and VP1, respectively, derived from vvIBDV strains, showed the most severe pathogenicity, and caused the most severe bursal B cell depletion as well as CD4(+) and CD8(+) T cell infiltration in the bursa of Fabricius. However, the virus induced the strongest decrease in CD4(+) and CD8(+) T cells in the thymus and exhibited the most efficient viral replication in the target organs. Isolate NN1005, whose vVP2 derived from vvIBDV and VP1 from unidentified origin, exhibited relatively lower pathogenicity compared to NN1172. The other two isolates, JS7 and GD10111, of which the vVP2 derived from vvIBDV and intermediate IBDV, and VP1 from 002-73 and attenuated IBDV, respectively, showed the lowest level of virulence. Our results suggest that various IBDV field isolates with different natural segment reassortments exhibit differential pathogenicity after infection of commercial Three-Yellow chickens.

Polymorphisms of Toll-like receptor 9 are associated with nasopharyngeal carcinoma susceptibility.

Toll-like receptor 9 (TLR9) plays a pivotal role in sensing a wide range of pathogens, including bacteria, fungi, and viruses. A dysregulation of TLR9 signaling may contribute to a higher risk of developing cancers. A hospital-based case-control study, including 356 nasopharyngeal carcinoma (NPC) cases and 356 controls, was conducted to assess the relationship between TLR9 -1237T/C, -1486T/C, and 2848G/A polymorphisms and NPC risk as well as clinical characteristics. The genotypes were determined by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). Protein level of transforming growth factor-ß1 (TGF-ß1), vascular endothelial growth factor (VEGF), and interleukin-6 (IL-6) in NPC biopsies was measured by enzyme-linked immunosorbent assay (ELISA). We found that -1486T/C CC genotype had an increased NPC risk at odds ratio (OR) = 1.808 with 95% confidence interval (CI) 1.169 ∼ 2.798 (P = 0.008). The patients with -1486 CC genotype are inclined to advanced tumor stage and lymph node metastasis. In addition, protein concentration of VEGF in NPC biopsies with -1486 CC genotype was significantly increased compared patients with -1486 TT genotype. For the first time, our data suggested that TLR9 -1486T/C may be a risk biomarker of NPC.

HDAC9-mediated calmodulin deacetylation induces memory impairment in Alzheimer's disease.

AIMS: Alzheimer's disease (AD) is a neurodegenerative disease characterized by progressive cognitive dysfunction and memory impairment. AD pathology involves protein acetylation. Previous studies have mainly focused on histone acetylation in AD, however, the roles of nonhistone acetylation in AD are less explored. METHODS: The protein acetylation and expression levels were detected by western blotting and co-immunoprecipitation. The stoichiometry of acetylation was measured by home-made and site-specific antibodies against acetylated-CaM (Ac-CaM) at K22, K95, and K116. Hippocampus-dependent learning and memory were evaluated by using the Morris water maze, novel object recognition, and contextual fear conditioning tests. RESULTS: We showed that calmodulin (CaM) acetylation is reduced in plasma of AD patients and mice. CaM acetylation and its target Ca2+ /CaM-dependent kinase II α (CaMKIIα) activity were severely impaired in AD mouse brain. The stoichiometry showed that Ac-K22, K95-CaM acetylation were decreased in AD patients and mice. Moreover, we screened and identified that lysine deacetylase 9 (HDAC9) was the main deacetylase for CaM. In addition, HDAC9 inhibition increased CaM acetylation and CaMKIIα activity, and hippocampus-dependent memory in AD mice. CONCLUSIONS: HDAC9-mediated CaM deacetylation induces memory impairment in AD, HDAC9, or CaM acetylation may become potential therapeutic targets for AD.

Biomechanics of Dental Implantation in the Giant Panda (Ailuropoda melanoleuca): A Comparative Study Using Finite Element Analysis.

Giant pandas have a high incidence of tooth wear, loss, and fracture since their diet is specifically bamboo. Dental implantation is a common treatment for tooth loss in humans while rarely reported in wild animals. To explore the applicability of dental implantation in giant pandas, this study measured mandible parameters of the giant panda, from an adult skeletal specimen. The mandible bone block model was developed using computer-aided design 3D mechanical drawing software. Implants of different radius and thread types of the third premolar tooth (PM3) were assembled and imported into an analysis software system for finite element analysis. As a result, the reverse buttress implant with a radius of 7.5 mm and 8.3 mm, and a length of 15 mm was found to be the most suitable implant for use in the giant panda PM3. This study provides a reference for appropriate clinical giant panda dental implantation, although, the feasibility of giant panda dental implantation needs to be studied further.

Effects of Phosphate-solubilizing Bacteria on Soil Phosphorus Fractions and Supply to Maize Seedlings Grown in Lateritic Red Earths and Cinnamon Soils.

Phosphorus (P) is often the limiting factor for plant growth because of its low mobility and availability in soils. Phosphate-solubilizing bacteria (PSB) have been shown to increase the availability of soil P fractions, thereby promoting plant growth. We herein investigated the effects of PSB on P availability in two important Chinese soil types: Lateritic red earths (La) and Cinnamon soils (Ci). We initially isolated 5 PSB strains and assessed their effects on soil P fractions. PSB mainly increased moderately labile P in La and labile P in Ci. We then selected the most promising PSB isolate (99% similarity with Enterobacter chuandaensis) and examined its effects on P accumulation in maize seedlings. The results obtained showed that plant P accumulation increased in response to a PSB inoculation in both soil types and the combination of the PSB inoculation and tricalcium phosphate fertilization in La significantly enhanced P accumulation in plant shoots. The present study demonstrated that the PSB isolates tested differed in their ability to mobilize P from distinct P fertilizers and that PSB isolates have potential as a valuable means of sustainably enhancing seedling growth in Chinese agricultural soils.

Multiple asymptotical ω-periodicity of fractional-order delayed neural networks under state-dependent switching.

This paper presents theoretical results on multiple asymptotical ω-periodicity of a state-dependent switching fractional-order neural network with time delays and sigmoidal activation functions. Firstly, by combining the geometrical properties of activation functions with the range of switching threshold, a partition of state space is given. Then, the conditions guaranteeing that the solutions can approach each other infinitely in each positive invariant set are derived. Furthermore, the S-asymptotical ω-periodicity and the convergence of solutions in positive invariant sets are discussed. It is worth noting that the number of attractors increases to 3n from 2n in a neural network without switching. Finally, three numerical examples are given to substantiate the theoretical results.

Synchronization of coupled switched neural networks subject to hybrid stochastic disturbances.

In this paper, the theoretical analysis on exponential synchronization of a class of coupled switched neural networks suffering from stochastic disturbances and impulses is presented. A control law is developed and two sets of sufficient conditions are derived for the synchronization of coupled switched neural networks. First, for desynchronizing stochastic impulses, the synchronization of coupled switched neural networks is analyzed by Lyapunov function method, the comparison principle and a impulsive delay differential inequality. Then, for general stochastic impulses, by partitioning impulse interval and using the convex combination technique, a set of sufficient condition on the basis of linear matrix inequalities (LMIs) is derived for the synchronization of coupled switched neural networks. Eventually, two numerical examples and a practical application are elaborated to illustrate the effectiveness of the theoretical results.

Autism-Risk Gene necab2 Regulates Psychomotor and Social Behavior as a Neuronal Modulator of mGluR1 Signaling.

Background: De novo deletion of the neuronal calcium-binding protein 2 (NECAB2) locus is associated with idiopathic autism spectrum disorders (ASDs). The in vivo function of NECAB2 in the brain remains largely elusive. Methods: We investigated the morphological and behavioral profiles of both necab2 knock-out and overexpression zebrafish models. The expression pattern and molecular role of necab2 were probed through a combination of in vitro and in vivo assays. Results: We show that Necab2 is a neuronal specific, cytoplasmic, and membrane-associated protein, abundantly expressed in the telencephalon, habenula, and cerebellum. Necab2 is distributed peri-synaptically in subsets of glutamatergic and GABAergic neurons. CRISPR/Cas9-generated necab2 knock-out zebrafish display normal morphology but exhibit a decrease in locomotor activity and thigmotaxis with impaired social interaction only in males. Conversely, necab2 overexpression yields behavioral phenotypes opposite to the loss-of-function. Proteomic profiling uncovers a role of Necab2 in modulating signal transduction of G-protein coupled receptors. Specifically, co-immunoprecipitation, immunofluorescence, and confocal live-cell imaging suggest a complex containing NECAB2 and the metabotropic glutamate receptor 1 (mGluR1). In vivo measurement of phosphatidylinositol 4,5-bisphosphate further substantiates that Necab2 promotes mGluR1 signaling. Conclusions: Necab2 regulates psychomotor and social behavior via modulating a signaling cascade downstream of mGluR1.

Transcriptome analysis reveals possible antitumor mechanism of Chlorella exopolysaccharide.

Microalgae, one of the most important classes of biomass producers, can produce exopolysaccharides similar to bacteria. The exopolysaccharide from Chlorella (CEPS) displays remarkable anticancer activity the mechanism of which remains to be elucidated. In this study, we analyzed the inhibitory effect of CEPS on the growth of HeLa cells. The results showed that CEPS inhibited the proliferation, decreased the viability, and changed the morphology of HeLa cells. Transcriptome analysis showed that 1894 genes were differentially expressed in the CEPS-treated group compared with the control group, including 1076 genes that were upregulated and 818 genes that were downregulated. The results of gene function enrichment analysis showed that the differentially expressed genes (DEGs) were significantly enriched in apoptosis and tumor-related biological processes and participated in several cancer and apoptosisrelated signaling pathways, including the MAPK signaling pathway, TNF signaling pathway, and the PI3K-Akt signaling pathway. The protein-protein interaction network identified 13 DEGs including PTPN11, RSAD2, ISG15, IFIT1, MX2, IFIT2, OASL, OAS1, JUN, OAS2, XAF1, ISG20, and IRF9 as hub genes. Our results suggest that CEPS is a promising therapeutic drug for the follow-up interventional therapy of cancer.

Inhibition of TGF-ß/Smad3 Signaling Disrupts Cardiomyocyte Cell Cycle Progression and Epithelial-Mesenchymal Transition-Like Response During Ventricle Regeneration.

Unlike mammals, zebrafish can regenerate injured hearts even in the adult stage. Cardiac regeneration requires the coordination of cardiomyocyte (CM) proliferation and migration. The TGF-ß/Smad3 signaling pathway has been implicated in cardiac regeneration, but the molecular mechanisms by which this pathway regulates CM proliferation and migration have not been fully illustrated. Here, we investigated the function of TGF-ß/Smad3 signaling in a zebrafish model of ventricular ablation. Multiple components of this pathway were upregulated/activated after injury. Utilizing a specific inhibitor of Smad3, we detected an increased ratio of unrecovered hearts. Transcriptomic analysis suggested that the TGF-ß/Smad3 signaling pathway could affect CM proliferation and migration. Further analysis demonstrated that the CM cell cycle was disrupted and the epithelial-mesenchymal transition (EMT)-like response was impaired, which limited cardiac regeneration. Altogether, our study reveals an important function of TGF-ß/Smad3 signaling in CM cell cycle progression and EMT process during zebrafish ventricle regeneration.

Corrigendum: Inhibition of TGF-ß/Smad3 Signaling Disrupts Cardiomyocyte Cell Cycle Progression and Epithelial-Mesenchymal Transition-Like Response During Ventricle Regeneration.

[This corrects the article DOI: 10.3389/fcell.2021.632372.].

Breast mucoepidermoid carcinoma: a case report and review of literature.

Breast mucoepidermoid carcinoma (MEC) is clinically rare, with an estimated incidence of 0.2-0.3% of all primary breast tumors. To date, only 41 cases have been reported in the literature. Herein, we present a case of breast MEC diagnosed at our hospital. The clinicopathologic features were preliminarily discussed by reviewing the literature. A 42-year-old Chinese woman presented with a lump in her right breast that was detected approximately three months prior. A microscopic examination showed that the breast MEC was composed of different proportions of mucinous cells, intermediate cells, and epidermoid cells. Most mucinous cells were positive for cytokeratin 7, while the epidermoid and intermediate cells were positive for p63 and cytokeratin 5/6. All tumor cells were negative for other myoepithelial markers, such as calponin. Tumor cells did not express estrogen, progesterone, or the HER-2/neu protein. After the patient underwent mastectomy, she was diagnosed with a low-grade mucoepidermoid carcinoma based on the clinical, histologic, and immuno-phenotypic characteristics. Our findings provide further insight into the pathologic mechanism of MEC, as correct diagnosis is essential for patient management.

MiR-125a-5p Regulates Vitamin D Receptor Expression in a Mouse Model of Experimental Autoimmune Encephalomyelitis.

Multiple sclerosis (MS) is a chronic and incurable autoimmune neurodegenerative disease of the central nervous system. Although the symptoms of MS can be managed by vitamin D3 treatment alone, this condition cannot be completely eradicated. Thus, there might be unknown factors capable of regulating the vitamin D receptor (VDR). Genome-wide analysis showed that miRNAs were associated with VDRs. We sought to determine the role and mechanism of action of miRNA-125a-5p and VDRs in a model of MS, mice with experimental autoimmune encephalomyelitis (EAE), which was induced by myelin oligodendrocyte glycoprotein 35-55 peptides. EAE mice showed decreased mean body weight but increased mean clinical scores compared with vehicle or control mice. And inflammatory infiltration was found in the lumbosacral spinal cord of EAE mice. In addition, VDR expression was significantly lower while the expression of miR-125a-5p was markedly higher in the spinal ventral horn of EAE mice than in vehicle or control mice. Importantly, activation of VDRs by paricalcitol or inhibition of miR-125a-5p by its antagomir markedly decreased the mean clinical scores in EAE mice. Interestingly, VDR and miR-125a-5p were co-localized in the same neurons of the ventral horn. More importantly, inhibition of miR-125a-5p remarkably blocked the decrease of VDRs in EAE mice. These results support a critical role for miR-125a-5p in modulating VDR activity in EAE and suggest potential novel therapeutic interventions.

Polyphyllin VI Induces Caspase-1-Mediated Pyroptosis via the Induction of ROS/NF-κB/NLRP3/GSDMD Signal Axis in Non-Small Cell Lung Cancer.

Trillium tschonoskii Maxim (TTM), a traditional Chinese medicine, has been demonstrated to have a potent anti-tumor effect. Recently, polyphyllin VI (PPVI), a main saponin isolated from TTM, was reported by us to significantly suppress the proliferation of non-small cell lung cancer (NSCLC) via the induction of apoptosis and autophagy in vitro and in vivo. In this study, we further found that the NLRP3 inflammasome was activated in PPVI administrated A549-bearing athymic nude mice. As is known to us, pyroptosis is an inflammatory form of caspase-1-dependent programmed cell death that plays an important role in cancer. By using A549 and H1299 cells, the in vitro effect and action mechanism by which PPVI induces activation of the NLRP3 inflammasome in NSCLC were investigated. The anti-proliferative effect of PPVI in A549 and H1299 cells was firstly measured and validated by MTT assay. The activation of the NLRP3 inflammasome was detected by using Hoechst33324/PI staining, flow cytometry analysis and real-time live cell imaging methods. We found that PPVI significantly increased the percentage of cells with PI signal in A549 and H1299, and the dynamic change in cell morphology and the process of cell death of A549 cells indicated that PPVI induced an apoptosis-to-pyroptosis switch, and, ultimately, lytic cell death. In addition, belnacasan (VX-765), an inhibitor of caspase-1, could remarkably decrease the pyroptotic cell death of PPVI-treated A549 and H1299 cells. Moreover, by detecting the expression of NLRP3, ASC, caspase-1, IL-1ß, IL-18 and GSDMD in A549 and h1299 cells using Western blotting, immunofluorescence imaging and flow cytometric analysis, measuring the caspase-1 activity using colorimetric assay, and quantifying the cytokines level of IL-1ß and IL-18 using ELISA, the NLRP3 inflammasome was found to be activated in a dose manner, while VX-765 and necrosulfonamide (NSA), an inhibitor of GSDMD, could inhibit PPVI-induced activation of the NLRP3 inflammasome. Furthermore, the mechanism study found that PPVI could activate the NF-κB signaling pathway via increasing reactive oxygen species (ROS) levels in A549 and H1299 cells, and N-acetyl-L-cysteine (NAC), a scavenger of ROS, remarkably inhibited the cell death, and the activation of NF-κB and the NLRP3 inflammasome in PPVI-treated A549 and H1299 cells. Taken together, these data suggested that PPVI-induced, caspase-1-mediated pyroptosis via the induction of the ROS/NF-κB/NLRP3/GSDMD signal axis in NSCLC, which further clarified the mechanism of PPVI in the inhibition of NSCLC, and thereby provided a possibility for PPVI to serve as a novel therapeutic agent for NSCLC in the future.

Nickel-Catalyzed Intramolecular Direct Arylation of Imines toward Diverse Indoles.

An efficient nickel-catalyzed intramolecular direct arylation of imines with challenging aryl chlorides has been developed. The versatile nickel catalysis made use of easily accessible imines and delivered diversely decorated 2-arylindoles of considerable importance to biological and medicinal chemistry.