Akkermansia muciniphila isolated from forest musk deer ameliorates diarrhea in mice via modification of gut microbiota.

BACKGROUND: The forest musk deer, a rare fauna species found in China, is famous for its musk secretion which is used in selected Traditional Chinese medicines. However, over-hunting has led to musk deer becoming an endangered species, and their survival is also greatly challenged by various high incidence and high mortality respiratory and intestinal diseases such as septic pneumonia and enteritis. Accumulating evidence has demonstrated that Akkermannia muciniphila (AKK) is a promising probiotic, and we wondered whether AKK could be used as a food additive in animal breeding programmes to help prevent intestinal diseases. METHODS: We isolated one AKK strain from musk deer feces (AKK-D) using an improved enrichment medium combined with real-time PCR. After confirmation by 16S rRNA gene sequencing, a series of in vitro tests was conducted to evaluate the probiotic effects of AKK-D by assessing its reproductive capability, simulated gastrointestinal fluid tolerance, acid and bile salt resistance, self-aggregation ability, hydrophobicity, antibiotic sensitivity, hemolysis, harmful metabolite production, biofilm formation ability, and bacterial adhesion to gastrointestinal mucosa. RESULTS: The AKK-D strain has a probiotic function similar to that of the standard strain in humans (AKK-H). An in vivo study found that AKK-D significantly ameliorated symptoms in the enterotoxigenic Escherichia coli (ETEC)-induced murine diarrhea model. AKK-D improved organ damage, inhibited inflammatory responses, and improved intestinal barrier permeability. Additionally, AKK-D promoted the reconstitution and maintenance of the homeostasis of gut microflora, as indicated by the fact that AKK-D-treated mice showed a decrease in Bacteroidetes and an increase in the proportion of other beneficial bacteria like Muribaculaceae, Muribaculum, and unclassified f_Lachnospiaceae compared with the diarrhea model mice. CONCLUSION: Taken together, our data show that this novel AKK-D strain might be a potential probiotic for use in musk deer breeding, although further extensive systematic research is still needed.

Blockade of PI3K/AKT signaling pathway by Astragaloside IV attenuates ulcerative colitis via improving the intestinal epithelial barrier.

BACKGROUND: The specific pathogenesis of UC is still unclear, but it has been clear that defects in intestinal barrier function play an important role in it. There is a temporary lack of specific drugs for clinical treatment. Astragaloside IV (AS-IV) is one of the main active ingredients extracted from Astragalus root and is a common Chinese herbal medicine for the treatment of gastrointestinal diseases. This study aimed to determine whether AS-IV has therapeutic value for DSS or LPS-induced intestinal epithelial barrier dysfunction in vivo and in vitro and its potential molecular mechanisms. METHODS: The intestinal tissues from UC patients and colitis mice were collected, intestinal inflammation was observed by colonoscopy, and mucosal barrier function was measured by immunofluorescence staining. PI3K/AKT signaling pathway activator YS-49 and inhibitor LY-29 were administered to colitic mice to uncover the effect of this pathway on gut mucosal barrier modulation. Then, network pharmacology was used to screen Astragaloside IV (AS-IV), a core active component of the traditional Chinese medicine Astragalus membranaceus. The potential of AS-IV for intestinal barrier function repairment and UC treatment through blockade of the PI3K/AKT pathway was further confirmed by histopathological staining, FITC-dextran, transmission electron microscopy, ELISA, immunofluorescence, qRT-PCR, and western blotting. Finally, 16 S rRNA sequencing was performed to uncover whether AS-IV can ameliorate UC by regulating gut microbiota homeostasis. RESULTS: Mucosal barrier function was significantly damaged in UC patients and murine colitis, and the activated PI3K/AKT signaling pathway was extensively involved. Both in vivo and vitro showed that the AS-IV-treated group significantly relieved inflammation and improved intestinal epithelial permeability by inhibiting the activation of the PI3K/AKT signaling pathway. In addition, microbiome data found that gut microbiota participates in AS-IV-mediated intestinal barrier recovery as well. CONCLUSIONS: Our study highlights that AS-IV exerts a protective effect on the integrality of the mucosal barrier in UC based on the PI3K/AKT pathway, and AS-IV may serve as a novel AKT inhibitor to provide a potential therapy for UC.

Engineered Extracellular Vesicle-Delivered CRISPR/Cas9 for Radiotherapy Sensitization of Glioblastoma.

Radiotherapy is a mainstay of glioblastoma (GBM) treatment; however, the development of therapeutic resistance has hampered the efficacy of radiotherapy, suggesting that additional treatment strategies are needed. Here, an in vivo loss-of-function genome-wide CRISPR screen was carried out in orthotopic tumors in mice subjected to radiation treatment to identify synthetic lethal genes associated with radiotherapy. Using functional screening and transcriptome analyses, glutathione synthetase (GSS) was found to be a potential regulator of radioresistance through ferroptosis. High GSS levels were closely related to poor prognosis and relapse in patients with glioma. Mechanistic studies demonstrated that GSS was associated with the suppression of radiotherapy-induced ferroptosis in glioma cells. The depletion of GSS resulted in the disruption of glutathione (GSH) synthesis, thereby causing the inactivation of GPX4 and iron accumulation, thus enhancing the induction of ferroptosis upon radiotherapy treatment. Moreover, to overcome the obstacles to broad therapeutic translation of CRISPR editing, we report a previously unidentified genome editing delivery system, in which Cas9 protein/sgRNA complex was loaded into Angiopep-2 (Ang) and the trans-activator of the transcription (TAT) peptide dual-modified extracellular vesicle (EV), which not only targeted the blood-brain barrier (BBB) and GBM but also permeated the BBB and penetrated the tumor. Our encapsulating EVs showed encouraging signs of GBM tissue targeting, which resulted in high GSS gene editing efficiency in GBM (up to 67.2%) with negligible off-target gene editing. These results demonstrate that a combination of unbiased genetic screens, and CRISPR-Cas9-based gene therapy is feasible for identifying potential synthetic lethal genes and, by extension, therapeutic targets.

Histopathological changes of the dural myeloid cells and lymphatic vessels in a mouse model of sepsis-associated encephalopathy.

As a common diffuse encephalopathy caused by sepsis, sepsis-associated encephalopathy (SAE) is closely associated with increased mortality, severe cognition dysfunction and increased cost of health care in patients of sepsis. Accumulating evidence suggests that the dura mater, the outermost meninges of the central nervous system (CNS), plays an important role in CNS immunity, especially with the discovery of meningeal lymphatic vessels (mLVs), as well as a plentiful array of resident or infiltrating immune cells harbored in the dura. Although these findings have significantly enhanced our understanding of the immune function of dura under both steady-state and pathological condition of CNS, whether and how the immune cells and mLVs within dura response to SAE still remains largely unexplored. Here, we established lipopolysaccharide (LPS) intraperitoneal injection-induced SAE model and examined the dural resident immune cells and mLVs. We analysed the histological change in dura by performing hematoxylin and eosin (H&E) and immunofluorescence staining. Results showed that systemic exposure to LPS induced neutrophils recruitment, exudation and gathering around the dural blood vessels. Moreover, resident macrophage altered its shape as well as location, and downregulated major histocompatibility (MHC) class II expression following LPS injection. We also found that LPS exposure induced dorsal meningeal lymphangiogenesis. Together, these findings collectively demonstrated that LPS-induced SAE can stimulate immune cells and mLVs within dura and provided more information about the immune response of the dura in sepsis.

Deficiency of microRNA-10b promotes DSS-induced inflammatory response via impairing intestinal barrier function.

Inflammatory bowel disease (IBD) is a non-specific inflammatory disease of the intestine with the pathogenesis to be largely unknown. We found that microRNA (miR)-10b knock-out mice displayed mild IBD symptoms, suggesting that miR-10b may be involved in the onset and development of IBD. This study focuses on elucidating the role of miR-10b in IBD. The colitis model was induced by feeding the mice with 2.5% dextran sodium sulfate (DSS), and the expression levels of miR-10b in colon tissue and blood samples were examined. The severity of colitis was assessed by disease activity index, colon length, histopathological damage, intestinal permeability and ELISA. Then, after transfection of Caco-2 cells with miR-10b mimic and inhibitor, qRT-PCR was used to detect the expression levels of intestinal barrier related genes in colon tissues and cells. miR-10b levels were significantly reduced in mice with DSS-induced acute colitis. Compared with wild-type (WT) mice, miR-10b knockout mice were more sensitive to DSS-induced colitis characterized by increased inflammatory cell infiltration and more severe disruption of colonic barrier function. In addition, by inhibiting miR-10b and thus increasing intestinal barrier gene expression in Caco-2 cells, we found that miR-10b suppressed inflammatory responses and enhanced intestinal barrier function both in vivo and in vitro. miR-10b inhibits the inflammatory response in DSS-induced acute colitis mice in vivo and enhances intestinal barrier function in vitro, suggesting that miR-10b plays a key role in the developmental process of IBD. Thus, miR-10b may be expected to be a new target for the treatment of IBD.

Proteomic and Metabolomic Characterization of SARS-CoV-2-Infected Cynomolgus Macaque at Early Stage.

Although tremendous effort has been exerted to elucidate the pathogenesis of severe COVID-19 cases, the detailed mechanism of moderate cases, which accounts for 90% of all patients, remains unclear yet, partly limited by lacking the biopsy tissues. Here, we established the COVID-19 infection model in cynomolgus macaques (CMs), monitored the clinical and pathological features, and analyzed underlying pathogenic mechanisms at early infection stage by performing proteomic and metabolomic profiling of lung tissues and sera samples from COVID-19 CMs models. Our data demonstrated that innate immune response, neutrophile and platelet activation were mainly dysregulated in COVID-19 CMs. The symptom of neutrophilia, lymphopenia and massive "cytokines storm", main features of severe COVID-19 patients, were greatly weakened in most of the challenged CMs, which are more semblable as moderate patients. Thus, COVID-19 model in CMs is rational to understand the pathogenesis of moderate COVID-19 and may be a candidate model to assess the safety and efficacy of therapeutics and vaccines against SARS-CoV-2 infection.

HPLC-DVD combined with chemometrics to analyze the correlation between the Q-marker content and color of Corni Fructus.

Although Corni Fructus (CF) is a fruit with great economic value and development potential in medicine and food, too much reliance on personal experience for quality evaluation seriously limits the trading and circulation of CF. In the present study, through the research on the correlation between the chemical composition and the appearance color, a standard colorimetric card related to CF quality was established, which simplified the quality evaluation process and improved the accuracy of the visual evaluation of CF. Firstly, a total of 29 batches of CF from different places were collected. Then, "imread" in the MATLAB software was used to convert the color of all samples into RGB values, and HPLC-DVD was used to measure the content of the main chemical components in CF. Thereafter, the correlation between the content and color was studied by using MLR, BP-ANNs and SVM chemometric tools to screen the Q-marker of CF, which was further confirmed by in vivo and in vitro experiments. Finally, the Q-marker standard colorimetric card with the best fitting degree is established according to the prediction model. Thus, this study provides an auxiliary reference for the color evaluation of CF and a reference for the standardization and quantification of the macro characteristics of traditional Chinese medicine and food.

Genome-wide CRISPR screen identified Rad18 as a determinant of doxorubicin sensitivity in osteosarcoma.

BACKGROUND: Osteosarcoma (OS) is a malignant bone tumor mostly occurring in children and adolescents, while chemotherapy resistance often develops and the mechanisms involved remain challenging to be fully investigated. METHODS: Genome-wide CRISPR screening combined with transcriptomic sequencing were used to identify the critical genes of doxorubicin resistance. Analysis of clinical samples and datasets, and in vitro and in vivo experiments (including CCK-8, apoptosis, western blot, qRT-PCR and mouse models) were applied to confirm the function of these genes. The bioinformatics and IP-MS assays were utilized to further verify the downstream pathway. RGD peptide-directed and exosome-delivered siRNA were developed for the novel therapy strategy. RESULTS: We identified that E3 ubiquitin-protein ligase Rad18 (Rad18) contributed to doxorubicin-resistance in OS. Further exploration revealed that Rad18 interact with meiotic recombination 11 (MRE11) to promote the formation of the MRE11-RAD50-NBS1 (MRN) complex, facilitating the activation of the homologous recombination (HR) pathway, which ultimately mediated DNA damage tolerance and leaded to a poor prognosis and chemotherapy response in patients with OS. Rad18-knockout effectively restored the chemotherapy response in vitro and in vivo. Also, RGD-exosome loading chemically modified siRad18 combined with doxorubicin, where exosome and chemical modification guaranteed the stability of siRad18 and the RGD peptide provided prominent targetability, had significantly improved antitumor activity of doxorubicin. CONCLUSIONS: Collectively, our study identifies Rad18 as a driver of OS doxorubicin resistance that promotes the HR pathway and indicates that targeting Rad18 is an effective approach to overcome chemotherapy resistance in OS.

The essential differences in microbial and chemical components of musk of different qualities secreted by captive male forest musk deer (Moschus berezovskii).

Musk is a precious raw material and ingredient in Chinese traditional medicine. The production of unqualified musk has become a puzzling problem in forest musk deer (FMD) breeding. However, what the essential differences between so-called unqualified musk and mature qualified musk have not yet been elucidated. In this study, 12 musk samples were collected and separated into two groups according to their external properties. One group is white or black cream-like secretion with sour or unpleasant odour (MM); the other group is brown or blackish brown solid secretion with pleasant fragrance (DM). Next-generation sequencing and gas chromatography-mass spectrometry were used to explore the essential differences between the DM and MM groups in microbial and chemical compositions. The results indicate that the DM group has more heterogenous microbial structure but simpler relationships among microbial communities. LEfSe analysis showed that 14 taxa at the genus level could be used to distinguish the DM and MM groups and Bacillus, Paracoccus, tenoteophomonas, Mycobacterium and Leuconostoc were more abundant in the DM group (P < 0.05). In addition, six compounds were identified to specifically distinguish the DM and MM groups under the OPLS-DA model. PICRUSt analysis revealed that metabolic pathways such as carbohydrate metabolism, nucleotide metabolism, energy metabolism, transport and catabolism were enriched in the DM group. All these findings of differences in microbiota and chemical compositions would provide potential clues for MM quality improvement and new evidence for the scientific establishment of a quality evaluation standard for musk.

Targeting radiation-tolerant persister cells as a strategy for inhibiting radioresistance and recurrence in glioblastoma.

BACKGROUND: Compelling evidence suggests that glioblastoma (GBM) recurrence results from the expansion of a subset of tumor cells with robust intrinsic or therapy-induced radioresistance. However, the mechanisms underlying GBM radioresistance and recurrence remain elusive. To overcome obstacles in radioresistance research, we present a novel preclinical model ideally suited for radiobiological studies. METHODS: With this model, we performed a screen and identified a radiation-tolerant persister (RTP) subpopulation. RNA sequencing was performed on RTP and parental cells to obtain mRNA and miRNA expression profiles. The regulatory mechanisms among NF-κB, YY1, miR-103a, XRCC3, and FGF2 were investigated by transcription factor activation profiling array analysis, chromatin immunoprecipitation, western blot analysis, luciferase reporter assays, and the MirTrap system. Transferrin-functionalized nanoparticles (Tf-NPs) were employed to improve blood-brain barrier permeability and RTP targeting. RESULTS: RTP cells drive radioresistance by preferentially activating DNA damage repair and promoting stemness. Mechanistic investigations showed that continual radiation activates the NF-κB signaling cascade and promotes nuclear translocation of p65, leading to enhanced expression of YY1, the transcription factor that directly suppresses miR-103a transcription. Restoring miR-103a expression under these conditions suppressed the FGF2-XRCC3 axis and decreased the radioresistance capability. Moreover, Tf-NPs improved radiosensitivity and provided a significant survival benefit. CONCLUSIONS: We suggest that the NF-κB-YY1-miR-103a regulatory axis is indispensable for the function of RTP cells in driving radioresistance and recurrence. Thus, our results identified a novel strategy for improving survival in patients with recurrent/refractory GBM.

Ultrasound-assisted enzymatic extraction of Corni Fructus alpha-glucosidase inhibitors improves insulin resistance in HepG2 cells.

Corni Fructus (CF) is a traditional medicine and beneficial food with multifaceted protective effects against diabetes and its complications. Since alpha-glucosidase inhibitors (GIs) are promising first-choice oral antihyperglycemic drugs for diabetes, we examined whether GIs from CF (GICF) are useful for diabetes treatment. Therefore, GICF was extracted by ultrasound-assisted enzymatic extraction (UAEE) that is optimized by a three-level, four-factor Box-Behnken design and determined by ultra-performance liquid chromatography. Compared to 36.31 mg g-1 without enzyme treatment, the GICF yield increased to 70.44 mg g-1via UAEE under optimum conditions (0.5% compound enzyme extracted in 23 min at 46 °C and pH 4.8). The activity (91.99%) of GICF was as predicted (93.28%). When GICF was used in an insulin-resistant HepG2 cell model, it significantly ameliorated the glucose metabolism in a dose-dependent manner. Our findings indicate that UAEE may be an innovative method for functional food extraction and a potential strategy for high-quality food ingredient (such as GI) production with high efficiency and productivity.

Total Saponins Isolated from Corni Fructus via Ultrasonic Microwave-Assisted Extraction Attenuate Diabetes in Mice.

Saponins have been extensively used in the food and pharmaceutical industries because of their potent bioactive and pharmacological functions including hypolipidemic, anti-inflammatory, expectorant, antiulcer and androgenic properties. A lot of saponins-containing foods are recommended as nutritional supplements for diabetic patients. As a medicine and food homologous material, Corni Fructus (CF) contains various active ingredients and has the effect of treating diabetes. However, whether and how CF saponins attenuate diabetes is still largely unknown. Here, we isolated total saponins from CF (TSCF) using ultrasonic microwave-assisted extraction combined with response surface methodology. The extract was further purified by a nonpolar copolymer styrene type macroporous resin (HPD-300), with the yield of TSCF elevated to 13.96 mg/g compared to 10.87 mg/g obtained via unassisted extraction. When used to treat high-fat diet and streptozotocin-induced diabetic mice, TSCF significantly improved the glucose and lipid metabolisms of T2DM mice. Additionally, TSCF clearly ameliorated inflammation and oxidative stress as well as pancreas and liver damages in the diabetic mice. Mechanistically, TSCF potently regulated insulin receptor (INSR)-, glucose transporter 4 (GLUT4)-, phosphatidylinositol 3-kinase (PI3K)-, and protein kinase B (PKB/AKT)-associated signaling pathways. Thus, our data collectively demonstrated that TSCF could be a promising functional food ingredient for diabetes improvement.

Protective Effects of Iridoid Glycoside from Corni Fructus on Type 2 Diabetes with Nonalcoholic Fatty Liver in Mice.

Type 2 diabetes mellitus (T2DM) is a common chronic metabolic disease. Accumulating evidence has demonstrated that nonalcoholic fatty liver disease (NAFLD) shares common typical features with T2DM, and they affect each other extensively. Thus, NAFLD has emerged as a novel target for T2DM prevention and care. Although Corni Fructus (CF) and its extracts have a therapeutic effect on T2DM, its effects and mechanisms on T2DM with NAFLD are far from elucidated. In this study, a mouse model of T2DM with NAFLD complication was established in ICR mice by feeding a high-fat, high-sugar (HFHS) diet and intraperitoneally injecting with a low dose of streptozotocin (STZ). Then, the effects of iridoid glycosides (IG) extracted from CF on this mouse model were investigated. We found that 4-week IG administration remarkably alleviated hyperglycemia and insulin resistance and significantly reduced inflammation, oxidative stress, and fat accumulation in the liver of T2DM with NAFLD mice. Further studies showed that IG inhibited the NF-κB but enhanced the PI3K-AKT signaling pathway. In summary, these results indicated that the IG from CF has potential therapeutic effects on T2DM with NAFLD.

Ultrasonic-microwave assisted extraction of total triterpenoid acids from Corni Fructus and hypoglycemic and hypolipidemic activities of the extract in mice.

Triterpene acids, the main component of Corni Fructus, could improve diabetes mellitus, for which the underlying hypoglycemic mechanism is still unclear, in patients. In this study, total triterpenoid acids were extracted by ultrasonic-microwave assisted extraction optimized by the response surface methodology. The extract was then purified with an X-5 macroporous resin, and the yield of total triterpenoid acids increased to 281.24 mg g-1 as compared with the 35.71 mg g-1 obtained by unassisted extraction. The contents of five components were determined by ultrafast performance liquid chromatography. In addition, the hypoglycemic and hypolipidemic activities of total triterpenoid acids in diabetic mice induced by streptozotocin and a high fat diet were studied. The results indicated that all parameters (oral glucose tolerance, insulin resistance and liver damage) related to diabetes were significantly improved by total triterpenoid acids. Furthermore, total triterpenoid acids significantly recovered the expression level of AMP-activated protein kinase and its downstream proteins, including acetyl-CoA carboxylase, carnitine palmityltransferase-1, peroxisome proliferator-activated receptor alpha, sterol regulatory element-binding protein 1c and fatty acid synthase. Altogether, total triterpenoid acids could ameliorate hyperlipidemia and hyperglycemia in diabetic mice, probably by activating the AMP-activated protein kinase-peroxisome proliferator-activated receptor signaling pathway and inhibiting the sterol regulatory element-binding protein 1c and fatty acid synthase signaling pathways. Therefore, total triterpene acids, isolated from Corni Fructus which is a prevailing health food, could be a functional food ingredient with therapeutic and commercial values.

Blockade of Discoidin Domain Receptor 2 as a Strategy for Reducing Inflammation and Joint Destruction in Rheumatoid Arthritis Via Altered Interleukin-15 and Dkk-1 Signaling in Fibroblast-Like Synoviocytes.

OBJECTIVE: This study was undertaken to uncover the pathophysiologic role of discoidin domain receptor 2 (DDR-2), a putative fibrillar collagen receptor, in inflammation promotion and joint destruction in rheumatoid arthritis (RA). METHODS: In synovial tissue from patients with RA and from mice with collagen antibody-induced arthritis (CAIA) (using Ddr2-/- and DBA/1 mice), gene and protein expression levels of DDR-2, interleukin-15 (IL-15), and Dkk-1 were measured by quantitative reverse transcription-polymerase chain reaction, Western blotting, and immunohistochemistry. Gene knockdown of DDR2 in human RA fibroblast-like synoviocytes (FLS) was conducted via small interfering RNA. Interaction between the long noncoding RNA H19 and microRNA 103a (miR-103a) was assessed in RA FLS using RNA pulldown assays. Cellular localization of H19 was examined using fluorescence in situ hybridization assays. Chromatin immunoprecipitation and dual luciferase reporter assays were applied to verify H19 transcriptional and posttranscriptional regulation by miR-103a. RESULTS: DDR2 messenger RNA (mRNA) expression was significantly associated with the levels of IL-15 and Dkk-1 mRNA in the synovial tissue of RA patients (r2 = 0.2022-0.3293, all P < 0.05; n = 33) and with the serum levels of IL-15 and Dkk-1 in mice with CAIA (P < 0.05). In human RA FLS, activated DDR-2 induced the expression of H19 through c-Myc. Moreover, H19 directly interacted with and promoted the degradation of miR-103a. CONCLUSION: These results indicate a novel role for activated DDR-2 in RA FLS, showing that DDR-2 is responsible for regulating the expression of IL-15 and Dkk-1 in RA FLS and is involved in the promotion of inflammation and joint destruction during pathophysiologic development of RA. Moreover, DDR-2 inhibition, acting through the H19-miR-103a axis, leads to reductions in the inflammatory reaction and severity of joint destruction in mice with CAIA, suggesting that inhibition of DDR-2 may be a potential therapeutic strategy for RA.

ΗΙF1α, EGR1 and SP1 co-regulate the erythropoietin receptor expression under hypoxia: an essential role in the growth of non-small cell lung cancer cells.

BACKGROUND: Overexpression of erythropoietin (EPO) and EPO receptor (EPO-R) is associated with poor prognosis in non-small-cell lung carcinoma (NSCLC). Hypoxia, a potent EPO inducer, is a major stimulating factor in the growth of solid tumors. However, how EPO-R expression is regulated under hypoxia is largely unknown. METHODS: The role of EPO-R in NSCLC cell proliferation was assessed by RNA interference in vitro. Luciferase reporter assays were performed to map the promoter elements involved in the EPO-R mRNA transcription. Nuclear co-immunoprecipitation and chromatin immunoprecipitation were performed to assess the interaction among transcription factors HIF1α, SP1, and EGR1 in the regulation of EPO-R under hypoxia. The expression of key EPO-R transcription factors in clinical specimens were determined by immunohistochemistry. RESULTS: Hypoxia induced a dosage and time dependent EPO-R mRNA expression in NSCLC cells. Knockdown of EPO-R reduced NSCLC cell growth under hypoxia (P < 0.05). Mechanistically, a SP1-EGR1 overlapped DNA binding sequence was essential to the hypoxia induced EPO-R transcription. In the early phase of hypoxia, HIF1α interacted with EGR1 that negatively regulated EPO-R. With the exit of EGR1 in late phase, HIF1α positively regulated EPO-R expression through additive interaction with SP1. In clinical NSCLC specimen, SP1 was positively while EGR1 was negatively associated with active EPO-R expression (P < 0.05). CONCLUSIONS: HIF1α, SP1 and EGR1 mediated EPO-R expression played an essential role in hypoxia-induced NSCLC cell proliferation. Our study presents a novel mechanism of EPO-R regulation in the tumor cells, which may provide information support for NSCLC diagnosis and treatment.

The protective and anti-inflammatory effects of a modified glucagon-like peptide-2 dimer in inflammatory bowel disease.

Inflammatory bowel disease (IBD) is a chronic, recurrent, and remitting inflammatory disease resulting from immune dysregulation in the gut. As a clinically frequent disease, it can affect individuals throughout their lives, with multiple complications. Glucagon-like peptide 2 (GLP-2) is a potent epithelium-specific intestinal growth factor. However, native GLP-2 has a relatively short half-life in human circulation because of extensive renal clearance and rapid degradation by the proteolytic enzyme dipeptidyl peptidase-IV (DPP-IV). Previously, We prepared a recombinant GLP-2 variant (GLP-2②), which has increased half-life and activity as compared to the [Gly2]GLP-2 monomer. The aim of the present study was to investigate the protective potential of GLP-2② in IBD models. LPS-induced in vitro model and dextran sulfate sodium (DSS)-induced in vivo model were used to study the anti-inflammatory and therapeutic effect of GLP-2②. We found that treated with GLP-2② showed a significantly reduction in the secretion of inflammatory cytokines. Furthermore, GLP-2② alleviated symptoms of DSS-induced colitis. GLP-2② treated mice displayed an increase in body weight, lower colitis scores, and fewer mucosal damage compared with GLP-2 treated mice. MPO activities, protein expression of NLRP3 and COX2 in the colon tissues were significantly reduced in GLP-2② groups. Importantly, the ameliorative effect of GLP-2② was related to anti-apoptosis effect in colon tissues. These findings demonstrated that GLP-2② may offer a superior therapeutic benefit over [Gly2]GLP-2 monomer for treatment of IBD.

A Single Codon Optimization Enhances Recombinant Human TNF-α Vaccine Expression in Escherichia coli.

As a proinflammatory cytokine, tumor necrosis factor-alpha (TNF-α) plays a pivotal role in various autoimmune diseases such as rheumatoid arthritis (RA). Thus, TNF-α has been defined as a therapeutic target for RA. Although some TNF-α antagonists including neutralizing monoclonal antibodies and soluble receptors have been approved to be successful in attenuating symptoms in patients suffering from RA, the long-term use of these passive immunization reagents could cause some problems like a variable degree of immunogenicity. In the present study, in order to wake up active immune responses of RA patients, we developed a recombinant TNF-α therapeutic vaccine (named mrTNF-PADRE) by coupling a 12-amino acid universal Pan HLA-DR Epitope (PADRE) to the protein. Codon optimization was performed to improve the secondary structure of mrTNF-PADRE mRNA to ensure its heterologous expression. As a result, a single codon synonymous mutation greatly elevated recombinant protein expression (about 30% of the total bacteria proteins) in E. coli as compared with the undetectable expression of the unoptimized gene. Although expressed as insoluble inclusion bodies (IBs), the vaccine can be effectively prepared with a purity of over 95% by IBs washing and one-step gel-infiltration chromatography. By this strategy, a stable yield of 5.2 mg purified mrTNF-PADRE per gram of cell paste could be obtained.

Acetylation and Amination Protect Angiotensin 1-7 from Physiological Hydrolyzation and Therefore Increases Its Antitumor Effects on Lung Cancer.

The recently reported inhibitory effects of angiotensin 1-7 (Ang-(1-7)) on various cancers indicate its potential use as a therapeutic agent for primary and metastatic cancers. However, its extremely short half-life in the circulation greatly compromises its potential applications. Here, we reported an Ang-(1-7) analogue peptide with the amino and carboxy termini protected by acetylation and amination. The in vitro and in vivo degradation of the resulting analogue, Ang-AA, were determined using high-performance liquid chromatography (HPLC). At the same time, small RNA interference and competition studies were performed to evaluate the specific capacity of Ang-AA to bind to the cell surface Mas receptor. Cell Counting Kit-8 (CCK8), wound-healing, and Boyden chamber assays were performed to investigate the inhibitory effects of Ang-AA on A549 cells. Finally, the synergistic inhibitory effects of Ang-AA and paclitaxel (PTX) on A549 xenografts in mice were observed using animal imaging systems and survival observations. The toxicity of Ang-AA in mice was evaluated. Our results showed that acetylation and amination significantly inhibited the hydrolyzation of Ang-(1-7) in vitro and in vivo. The half-life of Ang-(1-7) in rats was prolonged from 2.4 ± 0.6 min to 238.7 ± 61.3 min ( p < 0.001). The specific binding of Ang-AA to the Mas receptor was well preserved, and Ang-AA exerted significantly greater inhibitory effects on the proliferation, migration, and invasion of A549 cells than Ang-(1-7). The combination of Ang-AA and PTX exhibited a significantly greater synergistic inhibitory effect on A549 xenografts than the combination of Ang-(1-7) and PTX. Ang-AA did not display obvious toxicity in mice. Our findings indicate acetylation and amination is a simple and effective method for producing Ang-(1-7) as a bioactive peptide.

PRL-3 is a potential glioblastoma prognostic marker and promotes glioblastoma progression by enhancing MMP7 through the ERK and JNK pathways.

Purpose: Glioblastoma is the most common and aggressive type of primary brain malignancy and is associated with a poor prognosis. Previously, we found that phosphatase of regenerating liver-3 (PRL-3) was significantly up-regulated in glioblastoma as determined by a microarray analysis. However, the function of PRL-3 in glioblastoma remains unknown. We aimed to investigate the clinical relationship between PRL-3 and glioblastoma, and uncover the mechanisms of PRL-3 in the process of glioblastoma. Methods: PRL-3 expression was evaluated in 61 glioblastoma samples and 4 cell lines by RT-qPCR and immunohistochemistry. Kaplan-Meier analysis was performed to evaluate the prognostic value of PRL-3 for overall survival (OS) and progression-free survival (PFS) for glioblastoma patients. Proliferation was evaluated by Cell Counting Kit-8 (CCK-8) assay and EdU proliferation assay, migration and invasion by wound-closure/Transwell assays, and qRT-PCR/immunoblotting/IHC were used for both in vivo and in vitro investigations. Result: A high PRL-3 expression level was closely correlated with unfavorable OS and PFS for glioblastoma patients, and was also significantly correlated with Ki-67 expression. Down-regulation of PRL-3 inhibited glioma cell proliferation, invasion and migration through ERK/JNK/matrix metalloproteinase 7 (MMP7) in vitro and in vivo. Conclusions: PRL-3 expression enhances the invasion and proliferation of glioma cells, highlighting this phosphatase as a novel prognostic candidate and an attractive target for future therapy in glioblastoma.