Coupling dynamics of respiration, gas exchange, and Pseudomonas fluorescens growth on fresh-cut cucumber (Cucumis sativus L.) in passive modified atmosphere packing.

This study established microbial growth models for fresh-cut cucumber packaged with different O2 transmission rate (OTR) films. Biaxially oriented polyamide/low-density polyethylene (BOPA/LDPE) film (Ⅰ: OTR5, Ⅳ: OTR48) and polyethylene (PE) film (Ⅱ: OTR2058, Ⅲ: OTR3875) were used to construct a passive modified atmosphere packaging (MAP). Mathematic models have been established to account for dynamic variations in the O2/CO2 concentration and their impacts on Pseudomonas fluorescens growth. The coupling models included: 1) respiration models of cucumber and P. fluorescens based on Michaëlis-Menten equation, 2) coupling gas exchange models based on Fick's law that contained models of P. fluorescens growth and respiration, 3) coupling microbial growth models contained respiration and gas exchange models. Coupling model with Baranyi function successfully fitted variations of O2/CO2 concentration and P. fluorescens growth in the two packaging. In addition, quality properties of packed fresh-cut cucumber were determined. The film Ⅳ (OTR48) as a high barrier film showed the highest inhibition of P. fluorescens growth, adequately retained its colour, firmness and total soluble solid (TSS) concentration in contrast to the PE films packaging. The constructed coupling models can be utilized for assessing the shelf life and microbial growth of fresh-cut vegetables with spoilage dominated by pseudomonads.

Effect of Kangfuxiaomi suppository on pelvic inflammatory disease in rats.

Pelvic inflammatory disease (PID) is commonly encountered in gynecological practice. Kangfuxiaomi suppository, made from the compound extract of Periplaneta Americana, is a Traditional Chinese Medicine remedy widely used for the treatment of gynecological disorders. This study aimed to preliminarily explore the therapeutic effect of Kangfuxiaomi suppository in a rat model of PID established by chemical injury and pathogen infection. The key parameters assessed were vulvar inflammation score, vaginal + uterine organ index, and serum levels of interleukin (IL)- 8; tumor necrosis factor (TNF)-α; C-reactive protein (CRP); superoxide dismutase (SOD); and malondialdehyde (MDA). In addition, levels of IL-6, cyclooxygenase (COX)- 2, and IL-2 in cervical tissues as well as that of IL-1ß and prostaglandin E-2 (PGE2) in uterine tissues were measured. The expression levels of nuclear factor-kappa B (NF-κB) p65 and Toll-like receptor 4 (TLR4) in uterine tissues were detected by immunohistochemical method. After Kangfuxiaomi suppository treatment, the vulva inflammation score and histopathological score of PID rats showed a tendency to decrease. Serum IL-8, TNF-α, CRP, and MDA levels were reduced, while SOD levels were significantly increased. Levels of IL-6, IL-2, and COX-2 in cervical tissues were somewhat decreased, and PGE2 and IL-1ß levels in uterine tissue were significantly decreased. Moreover, the levels of NF-κB p65 and TLR4 protein expression were also decreased. These findings demonstrated the therapeutic effect of Kangfuxiaomi suppository in PID rats. The underlying mechanism may involve enhanced antioxidant capacity and decreased secretion of proinflammatory factors via the NF-κB/TLR4 signaling pathway.

Quantitative parameters in novel spectral computed tomography for assessing gastric cancer and cell proliferation.

OBJECTIVES: To investigate the diagnostic ability of novel spectral CT-derived parameters for gastric cancer histological types and Ki-67 expression. METHODS: A total of 72 patients with histologically proven gastric cancer (GC) were retrospectively included in this study. All patients underwent dual-phase enhanced abdominal spectral CT. The arterial (AP) and venous phase (VP) slope of the spectral curve (λHU), iodine concentration (IC), normalized IC (NIC), effective atomic number (Zeff) and iodine-no-water concentration were retrospectively compared between patients with low and high Ki-67 expression levels and with different histological types in GC patients. The ROI was outlined independently by two senior physicians, and the average of three measurements at the largest level was taken. In addition, interobserver reproducibility was assessed by Bland-Altman analysis. Correlations between quantitative parameters and Ki-67 expression levels were assessed by Spearman's correlation coefficients. RESULTS: The values between the mucinous group and nonmucinous carcinoma group were significantly different in both phases. The IC, NIC, and iodine-no-water concentration in the VP were significantly different among the Ki-67_L, Ki-67_M, and Ki-67_H groups. Spearman rank correlation analysis demonstrated a positive correlation between Ki-67 expression levels and IC, NIC, and iodine-no-water concentration in the VP, with correlation coefficients of 0.304, 0.424, and 0.322, respectively. CONCLUSION: Quantitative spectral parameters can discriminate between low and high Ki-67 expression and different histological types in GC. The NIC, IC and iodine-no-water concentration can be useful parameters for evaluating of Ki-67 expression levels.

Two New Eudesmane-Type Sesquiterpene from Clonostachys sp. Y6-1and Their Cytotoxic Activity.

Two undescribed eudesmane-type sesquiterpenoids together with four known compounds were isolated from Clonostachys sp. Y6-1 associated. Their chemical structures were unambiguously determined by NMR, mass spectrometry, and 13 C-NMR calculation as well as DP4+ probability analyses. The absolute configurations of compounds 1 and 2 were determined by ECD calculation and X-ray single-crystal diffraction methods. Furthermore, all isolates were evaluated for in vitro cytotoxic activities against MCF-7, HCT-116, MDA-MB-231, and SW620 cancer cells. Among them, bioactivity evaluation of compound 5 revealed that weak activity (IC50 =66.55±0.82 µM) against SW620.

Crosstalk between KIF1C and PRKAR1A in left atrial myxoma.

Cardiac myxoma (CM) is the most common benign cardiac tumor, and most CMs are left atrial myxomas (LAMs). Six variations of KIF1C, c.899 A > T, c.772 T > G, c.352 A > T, c.2895 C > T, c.3049 G > A, and c.*442_*443dup in left atrial myxoma tissues are identified by whole-exome sequencing (WES) and Sanger sequencing. RNA-seq and function experiments show the reduction of the expression of KIF1C and PRKAR1A caused by rare variations of KIF1C. KIF1C is observed to be located in the nucleus, bind to the promoter region of PRKAR1A, and regulate its transcription. Reduction of KIF1C decreases PRKAR1A expression and activates the PKA, which causes an increase in ERK1/2 phosphorylation and SRC-mediated STAT3 activation, a reduction of CDH1, TP53, CDKN1A, and BAX, and eventually promotes tumor formation both in vitro and in vivo. The results suggest that inhibition of KIF1C promotes the pathogenesis of LAM through positive feedback formed by the crosstalk between KIF1C and PRKAR1A.

BCKDHA contributes to melanoma progression by promoting the expressions of lipogenic enzymes FASN and ACLY.

The dysregulation of branched-chain amino acid (BCAA) metabolism and related enzymes has been greatly implicated in the progression of multiple types of cancer, whereas remains far from understood in melanoma. Here, we explored the role of the BCAA metabolism enzyme BCKDHA in melanoma pathogenesis and elucidated the underlying mechanisms. In vitro cell biology experiments and in vivo pre-clinical mice model experiments were performed to investigate the role of BCKDHA in melanoma progression. RNA sequencing, immunohistochemical/immunofluorescence staining and bioinformatics analysis were used to examine the underlying mechanism. BCKDHA expression was prominently increased in both melanoma tissues and cell lines. The up-regulation of BCKDHA promoted long-term tumour cell proliferation, invasion and migration in vitro and tumour growth in vivo. Through RNA-sequencing technology, it was found that BCKDHA regulated the expressions of lipogenic fatty acid synthase (FASN) and ATP-citrate lyase (ACLY), which was thereafter proved to mediate the oncogenic role of BCKDHA in melanoma. Our results demonstrate that BCKDHA promotes melanoma progression by regulating FASN and ACLY expressions. Targeting BCKDHA could be exploited as a promising strategy to restrain tumour progression in melanoma.

Discovery of 2H-Indazole-3-carboxamide Derivatives as Novel Potent Prostanoid EP4 Receptor Antagonists for Colorectal Cancer Immunotherapy.

Nowadays, small-molecule drugs have become an indispensable part of tumor immunotherapy. Accumulating evidence has indicated that specifically blocking PGE2/EP4 signaling to induce robust antitumor immune response represents an attractive immunotherapy strategy. Herein, a 2H-indazole-3-carboxamide containing compound 1 was identified as a EP4 antagonist hit by screening our in-house small-molecule library. Systematic structure-activity relationship exploration leads to the discovery of compound 14, which displayed single-nanomolar EP4 antagonistic activity in a panel of cell functional assays, high subtype selectivity, and favorable drug-like profiles. Moreover, compound 14 profoundly inhibited the up-regulation of multiple immunosuppression-related genes in macrophages. Oral administration of compound 14, either as monotherapy or in combination with an anti-PD-1 antibody, significantly impaired tumor growth via enhancing cytotoxic CD8+ T cell-mediated antitumor immunity in a syngeneic colon cancer model. Thus, these results demonstrate the potential of compound 14 as a candidate for developing novel EP4 antagonists for tumor immunotherapy.

Bavachin induces apoptosis in colorectal cancer cells through Gadd45a via the MAPK signaling pathway.

Bavachin is a dihydroflavonoid compound isolated from Psoralea corylifolia, and exhibits anti-bacterial, anti-inflammatory, anti-tumor and lipid-lowering activities. Recent attention has gradually drawn on bavachin-induced apoptosis in many human cancer cell lines. However, the anti-cancer effects and related mechanisms in colorectal cancer remain unknown. Here, we investigated the effects of bavachin on colorectal cancer in vivo and in vitro. The results showed that bavachin inhibited the proliferation of human colorectal cancer cells and induce apoptosis. These changes were mediated by activating the MAPK signaling pathway, which significantly up-regulated the expression of Gadd45a. Furthermore, Gadd45a silencing obviously attenuated bavachin-mediated cell apoptosis. Inhibition of the MAPK signaling pathway by JNK/ERK/p38 inhibitors also weakened the up-regulation of Gadd45a by bavachin. The anticancer effect of bavachin was also validated using a mouse xenograft model of human colorectal cancer. In conclusion, these findings suggest that bavachin induces the apoptosis of colorectal cancer cells through activating the MAPK signaling pathway.

Inflammatory Cell-Inspired Cascade Nanozyme Induces Intracellular Radical Storm for Enhanced Anticancer Therapy.

Manipulating intracellular levels of reactive oxygen and nitrogen species (RONS) is of great potential for cancer treatment. Inspired by the natural mechanism of a radical storm in inflammatory cells via activated and regulatable biocatalysis, the authors herein report a self-powered nanozyme that can enable RONS production in tumor cells via cascade reactions. The nanozymes are constructed via glucose oxidase (GOx)-templated inverse microemulsion polymerization from acrylamide, arginine-acrylamide, ferrocene-acrylate, and N,N'-bis(acryloyl)cystamine, followed by surface coating with hyaluronic acid. After targeted delivery into cancer cells, the nanozymes are dissociated by intracellular glutathione to release GOx, which decomposed glucose to generate gluconic acid and H2 O2 . Under such acidified conditions, H2 O2 efficiently oxidized pendant arginine residues to produce nitric oxide , transformed into a highly toxic hydroxyl radical and superoxide anion via ferrocene-mediated Fenton reaction and Haber-Weiss cycle, and simultaneously generated peroxynitrite anion via reaction between NO and ·O2 - , thus provoking the RONS radical storm to effectively eradicate A549 tumor cells both in vitro and in vivo. This nature-inspired enzyme-chemical dynamic therapy may provide a promising modality for anti-cancer treatment.

Adenosine A2A receptor controls the gateway of the choroid plexus.

The choroid plexus (CP) is one of the key gateways regulating the entry of peripheral immune cells into the CNS. However, the neuromodulatory mechanisms of maintaining its gateway activity are not fully understood. Here, we identified adenosine A2A receptor (A2AR) activity as a regulatory signal for the activity of CP gateway under physiological conditions. In association with a tightly closed CP gateway, we found that A2AR was present at low density in the CP. The RNA-seq analysis revealed that the A2AR antagonist KW6002 affected the expression of the cell adhesion molecules' (CAMs) pathway and cell response to IFN-γ in the CP. Furthermore, blocking or activating A2AR signaling in the CP resulted in a decreased and an increased, respectively, expression of lymphocyte trafficking determinants and disruption of the tight junctions (TJs). Furthermore, A2AR signaling regulates the CP permeability. Thus, A2AR activity in the CP may serve as a therapeutic target for remodeling the immune homeostasis in the CNS with implications for the treatment of neuroimmunological disorders.

Callicarpanes A-L, Twelve New Clerodane Diterpenoids with NLRP3 Inflammasome Inhibitory Activity from Callicarpa integerrima.

Twelve new clerodane diterpenoids named callicarpanes A-L (1-12), together with eight known compounds (13-20), were isolated from Callicarpa integerrima. Their structures were determined by comprehensive spectroscopic data. The calculated chemical shifts were used to identify relative configurations using DP4+ analysis. The absolute configurations (AC) were assigned based on quantum chemical calculations and X-ray single-crystal diffraction methods. Compounds 1, 3, 5, 9, 10, 12, 15, 16, and 19 showed significant inhibitory activity for NLRP3 inflammasome activation, with the IC50 against lactate dehydrogenase (LDH) release ranging from 0.08 to 4.78 µM. Further study revealed that compound 10 repressed IL-1ß secretion and caspase-1 maturation in J774A.1 cell as well as blocked macrophage pyroptosis.

Discerning the Contributions of Gold Species in Butadiene Hydrogenation: From Single Atoms to Nanoparticles.

Identification of the roles of different active sites is vital for the rational design of catalysts. We present a cutting-edge strategy to discern the contributions of different single-atom gold species and nanoparticles in 1,3-butadiene hydrogenation, through coupling of advanced spectroscopic techniques, electron microscopy-based automated image analyses, and steady-state and kinetic studies. While all the carbon-hosted single gold atoms display negligible initial activity, the in situ-evolved gold nanoparticles are highly active. Full metal-species quantification is realized by combining electron-microscopy-based atom recognition statistics and deep-learning-driven nanoparticle segmentation algorithm, allowing the structure-activity correlations for the hybrid catalysts containing different Au architectures to be established. Surface exposure density of Au nanoparticles, as revealed by electron-microscopy-based statistics, is revealed as a new and reliable activity descriptor.

Reactive oxygen species-responsive branched poly (ß-amino ester) with robust efficiency for cytosolic protein delivery.

Protein therapy targeting the intracellular machinery holds great potentials for disease treatment, and therefore, effective cytosolic protein delivery technologies are highly demanded. Herein, we developed reactive oxygen species (ROS)-degradable, branched poly(ß-amino ester) (PBAE) with built-in phenylboronic acid (PBA) in the backbone and terminal-pendent arginine for the efficient cytosolic protein delivery. The PBAE could form stable and cell-ingestible nanocomplexes (NCs) with proteins via electrostatic interaction, nitrogen-boronate (N-B) coordination, and hydrogen bonding, while it can be degraded into small segments by the over-produced H2O2 in tumor cells to enable cytoplasmic protein release. As thus, PBAE exhibited high efficiency in delivering varieties of proteins with distinct molecular weights (12.4-430 kDa) and isoelectric points (4.7-10.5) into tumor cells, including enzymes, toxins, and antibodies. Moreover, PBAE mediated efficient delivery of saporin into tumor cells in vivo, provoking pronounced anti-tumor outcomes. This study provides a robust and versatile platform for cytosolic protein delivery, and the elaborately tailored PBAE may find promising applications for protein-based biological research and disease management. STATEMENT OF SIGNIFICANCE: Cytosolic delivery of native proteins holds great therapeutic potentials, which however, is limited by the lack of robust delivery carriers that can simultaneously feature strong protein encapsulation yet effective intracellular protein release. Herein, ROS-degradable, branched poly(ß-amino ester) (PBAE) with backbone-embedded phenylboronic acid (PBA) and terminal-pendent arginine was developed to synchronize these two processes. PBA and arginine moieties allowed PBAE to encapsulate proteins via N-B coordination, electrostatic interaction, hydrogen bonding, and salt bridging, while PBA could be oxidized by over-produced H2O2 inside cancer cells to trigger PBAE degradation and intracellular protein release. As thus, the top-performing PBAE mediated efficient cytosolic delivery of various proteins including enzymes, toxins, and antibodies. This study provides a powerful platform for cytosolic protein delivery, and may find promising utilities toward intracellular protein therapy against cancer and other diseases such as inflammation.

Ten new prenylated flavonoids from Macaranga denticulata and their antitumor activities.

Ten new prenylated flavonoids, named denticulains A-J (1-10), together with seven known prenylated flavonoids (11-17) were isolated from Macaranga denticulata. Their structures were elucidated on the basis of detailed spectroscopic analysis and by comparison with literature data. In addition, compounds 1 and 14 inhibited the proliferation of SW620 and HCT-116 cell lines with an IC50 value of 46.08 µM and 56.83 µM, respectively.

Host-induced alteration of the neighbors of single platinum atoms enables selective and stable hydrogenation of butadiene.

Tuning the coordination neighbors of the metal center is emerging as an elegant approach to manipulating the performance of supported single-atom catalysts in heterogeneous catalysis. Herein, atomically dispersed Pt species with different coordination neighbors hosted on nitrogen-doped carbon (NC) and graphitic carbon nitride (C3N4) are constructed through an impregnation-activation approach. Advanced characterization techniques including X-ray electron microscopy, X-ray absorption spectroscopy, and high angle annular dark-field scanning transmission electron microscopy reveal the different nature of active sites induced by the hosts: i.e., the Pt-Nx configuration in NC but both Pt-N and Pt-O coordinations in C3N4. H2-D2 exchange experiments and electron microscopy further evidence that Pt/NC exhibits a high propensity for H2 splitting and high thermal stability of the Pt species against agglomeration, whereas Pt/C3N4 cannot dissociate H2 and the Pt atoms easily aggregate in the reductive stream. Consequently, when applied in the selective hydrogenation of 1,3-butadiene, Pt/NC exhibits higher selectivity to butenes and excellent stability, but Pt/C3N4 behaves as a nanoparticle analogue favoring deep hydrogenation. The superior selectivity patterns of the single Pt atoms over Pt nanoparticles are rationalized by the inversed adsorption strength between the H2 and 1,3-butadiene molecules at different metal sites, which is substantiated by the kinetic studies.

Stem-end Rot Caused by Lasiodiplodia brasiliense on Tobacco in China.

Tobacco (Nicotiana tabacum L.), is a major cash crop grown worldwide for its leaves, which are dried and fermented before being put in tobacco products. Tobacco production is seriously threatened by numerous diseases (Qiu et al. 2021). In August 2021, plants with stem-end rot were observed in a tobacco field in Zunyi City, Guizhou Province, China. Surveys indicated a 22 to 35% disease incidence in five counties. Symptoms of black necrosis appeared at the base of stems, and leaves turned yellow. To isolate the pathogen, diseased stems were cut into small segments and placed on potato dextrose agar (PDA) at 25°C in darkness for 3 to 5 days. To obtain pure cultures, hyphal tips from colonies were transferred to fresh PDA plates. A representative strain, GZAX 110, was used for further identification. The fungal colonies were initially gray, later deepening to smoke-gray. Conidiogenous cells were fully divided, discrete, transparent, thin-walled, smooth and cylindrical. Conidia matured slowly, were ellipsoid to ovoid, containing granular content, with a rounded apex. The base was largely truncated, and conidia became dark brown with one central septum, 21.0-30.0 × 12.0-18.0 µm. On water-agar medium, teleomorph structures were not observed. These characteristics suggested the fungus was Lasiodiplodia sp. (Netto et al. 2014). For further confirmation, genomic DNA was extracted using the CTAB method (Watanabe et al. 2010); and the ITS (internal transcribed spacer region of rDNA) and EF-1α (translation-elongation factor) regions were amplified with primer pairs ITS1/ITS4 and EF1-688F/EF1-1251R (Cruywagen et al. 2017), respectively. The ITS and EF-1α sequences (OM534558 and OM632673) were analyzed by BLASTN searches. The ITS sequence showed 100% identity (490/490 bp) to L. brasiliense strain AGQMy0011 (MW274145) and the EF-1ɑ sequence showed 100% identity (551/551 bp) to L. brasiliense strain EX1 (MF580811). A multilocus phylogenetic tree was constructed via the Maximum-likelihood (RAxML v.7.2.8) and Bayesian Inference (MrBayes v.3.2.1) analyses (Elsie et al. 2017) using combined ITS and EF1-α reference sequences of Lasiodiplodia species. Phylogenetic analysis showed that GZAX 110 clustered monophyletically with strains of L. brasiliense. Thus, the isolate GZAX 110 was confirmed as L. brasiliense. Pathogenicity of GZAX 110 was tested on tobacco plants at the eight leaf stage by attaching mycelial plugs (5 mm in diameter) to stems and leaves according to Cruywagen et al. (2017). Inoculated plants were kept in a greenhouse (16 h light/8 h darkness, 22℃, relative humidity >85%). Control plants were inoculated with PDA plugs. The experiment was repeated three times with five plants. Seven days after inoculation, dark brown necrosis was observed at inoculation sites on stems and leaves, while the control plants remained healthy. The pathogen was re-isolated from the inoculated sites and further validated as the same fungus through morphological and phylogenetic analyses. Previously, this fungus has been reported on Mangifera indica (mango) in China (Zhang et al. 2018), and apple (Martins et al. 2018) and papaya (Netto et al. 2014) in Brazil. However, to our knowledge, this is the first worldwide report of L. brasiliense causing stem-end rot on tobacco. This report provides information for future diagnosis and management of the disease.

Novel Loss-of-Function Mutations in NPR2 Cause Acromesomelic Dysplasia, Maroteaux Type.

Acromesomelic dysplasia, Maroteaux type (AMDM) is a rare skeletal dysplasia characterized by severe disproportionate short stature, short hands and feet, normal intelligence, and facial dysmorphism. Homozygous or compound heterozygous mutations in the natriuretic peptide receptor 2 (NPR2) gene produce growth-restricted phenotypes. The current study was designed to identify and characterize NPR2 loss-of-function mutations in patients with AMDM and to explore therapeutic responses to recombinant growth hormone (rhGH). NPR2 was sequenced in two Chinese patients with AMDM via next generation sequencing, and in silico structural analysis or transcript analysis of two novel variants was performed to examine putative protein changes. rhGH treatment was started for patient 1. Three NPR2 mutations were identified in two unrelated cases: two compound heterozygous mutations c.1112G>A p.(Arg371Gln) and c.2887+2T>C in patient 1 and a homozygous mutation c.329G>A p.(Arg110His) in patient 2, yielding distinct phenotypes. RNA extracted from peripheral blood cells of patient 1 showed alternatively spliced transcripts not present in control cells. Homology modeling analyses suggested that the c.1112G>A p.(Arg371Gln) mutation disrupted the binding of NPR-B homodimer to its ligand (C-type natriuretic peptide) in the extracellular domain as a result of global allosteric effects on homodimer formation. Thus, c.2887+2T>C and c.1112G>A p.(Arg371Gln) in NPR2 were loss-of-function mutations. Furthermore, rhGH therapy in patient 1 increased the patient's height by 0.6SDS over 15 months without adversely affecting the trunk-leg proportion. The short-term growth-promoting effect was equivalent to that reported for idiopathic short stature. Overall, our findings broadened the genotypic spectrum of NPR2 mutations in individuals with AMDM and provided insights into the efficacy of rhGH in these patients.

Choroid plexus-selective inactivation of adenosine A2A receptors protects against T cell infiltration and experimental autoimmune encephalomyelitis.

BACKGROUND: Multiple sclerosis (MS) is one of the most common autoimmune disorders characterized by the infiltration of immune cells into the brain and demyelination. The unwanted immunosuppressive side effect of therapeutically successful natalizumab led us to focus on the choroid plexus (CP), a key site for the first wave of immune cell infiltration in experimental autoimmune encephalomyelitis (EAE), for the control of immune cells trafficking. Adenosine A2A receptor (A2AR) is emerging as a potential pharmacological target to control EAE pathogenesis. However, the cellular basis for the A2AR-mediated protection remains undetermined. METHODS: In the EAE model, we assessed A2AR expression and leukocyte trafficking determinants in the CP by immunohistochemistry and qPCR analyses. We determined the effect of the A2AR antagonist KW6002 treatment at days 8-12 or 8-14 post-immunization on T cell infiltration across the CP and EAE pathology. We determined the critical role of the CP-A2AR on T cell infiltration and EAE pathology by focal knock-down of CP-A2AR via intracerebroventricular injection of CRE-TAT recombinase into the A2ARflox/flox mice. In the cultured CP epithelium, we also evaluated the effect of overexpression of A2ARs or the A2AR agonist CGS21680 treatment on the CP permeability and lymphocytes migration. RESULTS: We found the specific upregulation of A2AR in the CP associated with enhanced CP gateway activity peaked at day 12 post-immunization in EAE mice. Furthermore, the KW6002 treatment at days 8-12 or 8-14 post-immunization reduced T cell trafficking across the CP and attenuated EAE pathology. Importantly, focal CP-A2AR knock-down attenuated the pathogenic infiltration of Th17+ cells across the CP via inhibiting the CCR6-CCL20 axis through NFκB/STAT3 pathway and protected against EAE pathology. Lastly, activation of A2AR in the cultured epithelium by A2AR overexpression or CGS21680 treatment increased the permeability of the CP epithelium and facilitated lymphocytes migration. CONCLUSION: These findings define the CP niche as one of the primary sites of A2AR action, whereby A2AR antagonists confer protection against EAE pathology. Thus, pharmacological targeting of the CP-A2AR represents a novel therapeutic strategy for MS by controlling immune cell trafficking across CP.

Flagellar Hook Protein FlgE Induces Microvascular Hyperpermeability via Ectopic ATP Synthase ß on Endothelial Surface.

We previously demonstrated the immunostimulatory efficacy of Pseudomonas aeruginosa flagellar hook protein FlgE on epithelial cells, presumably via ectopic ATP synthases or subunits ATP5B on cell membranes. Here, by using recombinant wild-type FlgE, mutant FlgE (FlgEM; bearing mutations on two postulated critical epitopes B and F), and a FlgE analog in pull-down assay, Western blotting, flow cytometry, and ELISA, actual bindings of FlgE proteins or epitope B/F peptides with ATP5B were all confirmed. Upon treatment with FlgE proteins, human umbilical vein endothelial cells (HUVECs) and SV40-immortalized murine vascular endothelial cells manifested decreased proliferation, migration, tube formation, and surface ATP production and increased apoptosis. FlgE proteins increased the permeability of HUVEC monolayers to soluble large molecules like dextran as well as to neutrophils. Immunofluorescence showed that FlgE induced clustering and conjugation of F-actin in HUVECs. In Balb/c-nude mice bearing transplanted solid tumors, FlgE proteins induced a microvascular hyperpermeability in pinna, lungs, tumor mass, and abdominal cavity. All effects observed in FlgE proteins were partially or completely impaired in FlgEM proteins or blocked by pretreatment with anti-ATP5B antibodies. Upon coculture of bacteria with HUVECs, FlgE was detectable in the membrane and cytosol of HUVECs. It was concluded that FlgE posed a pathogenic ligand of ectopic ATP5B that, upon FlgE-ATP5B coupling on endothelial cells, modulated properties and increased permeability of endothelial layers both in vitro and in vivo. The FlgE-ectopic ATP5B duo might contribute to the pathogenesis of disorders associated with bacterial infection or ectopic ATP5B-positive cells.

Matrine treatment induced an A2 astrocyte phenotype and protected the blood-brain barrier in CNS autoimmunity.

Type 1 astrocytes (A1), which are highly proinflammatory and neurotoxic, are prevalent in multiple sclerosis (MS). In addition, in MS and its animal model, experimental autoimmune encephalomyelitis (EAE), immune cells must cross the blood-brain barrier (BBB) and infiltrate into the parenchyma of the central nervous system (CNS) in order to induce neurological deficits. We have previously reported that treatment of EAE with matrine (MAT), a quinazine alkaloid derived from Sophorae Flavescens, effectively inhibited CNS inflammation and promoted neuroregeneration. However, the impact of MAT treatment on astrocyte phenotype is not known. In the present study, we showed that MAT treatment inhibited the generation of neurotoxic A1 astrocytes and promoted neuroprotective A2 astrocytes in the CNS of EAE, most likely by inhibiting production of the A1-inducing cytokine cocktail. MAT also downregulated the expression of vascular endothelial growth factor-A (VEGF-A) and upregulated tight junction proteins Claudin 5 and Occludin, thus protecting the BBB from CNS inflammation-induced damage. Moreover, MAT treatment promotes the formation of astrocyte tight junctions at glia limitans, thereby limiting parenchymal invasion of the CNS by immune cells. Taken together, the inhibition of A1 astrogliogenesis, and the dual effects on the BBB and astrocytic glia limitans, may be the mechanisms whereby MAT significantly improves EAE clinical scores and neuroprotection.