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BackgroundThe major histocompatibility complex (MHC) and the killer cell immunoglobulin-like receptors (KIR) are key regulators of immune responses. The cynomolgus macaque, an Old World monkey species, can be applied as an important preclinical model for studying human diseases, including coronavirus disease 2019 (COVID-19). Several MHC-KIR combinations have been associated with either a poor or good prognosis. Therefore, macaques with a well-characterized immunogenetic profile may improve drug evaluation and speed up vaccine development. At present, a complete overview of the MHC and KIR haplotype organizations in cynomolgus macaques is lacking, and characterization by conventional techniques is hampered by the extensive expansion of the macaque MHC-B region that complicates the discrimination between genes and alleles.MethodsWe assembled complete MHC and KIR genomic regions of cynomolgus macaque using third-generation long-read sequencing approach. We identified functionalMafa-Bloci at the transcriptome level using locus-specific amplification in a cohort of 33 Vietnamese cynomolgus macaques.ResultsThis is the first physical mapping of completeMHCandKIRgene regions in a Vietnamese cynomolgus macaque. Furthermore, we identified four functionalMafa-Bloci (B2,B3,B5, andB6) and showed that alleles of theMafa-I*01,-B*056,-B*034, and-B*001functional lineages, respectively, are highly frequent in the Vietnamese cynomolgus macaque population.ConclusionThe insights into the MHC and KIR haplotype organizations and the level of diversity may refine the selection of animals with specific genetic markers for future medical research.

Background: Synaptic abnormalities in synaptic proteins are the initial hallmarks of Alzheimer’s disease (AD). The higher level of palmitoylation of synaptic proteins was closely associated with amyloid-β (Aβ) in AD. Cattle encephalon glycoside and ignotin (CEGI) have been shown to act as multitarget neurotrophic agents in APPswe/PS1dE9 (APP/PS1) transgenic AD mice. However, it is not clear whether CEGI can influence Aβ deposition or whether it does so by the regulation of protein palmitoylation and expression of synaptic proteins in transgenic AD mice. Objective: In this study, we investigated the roles of CEGI in modulating postsynaptic density protein 95 (PSD-95) palmitoylation, Aβ pathologies, and expression of synaptic-associated proteins in APP/PS1 mice. Methods: Five-month-old APP/PS1 mice were treated intraperitoneally with 6.6 mL/kg of CEGI for 6 weeks. At the end of the treatment period, APP/PS1 mice were subjected to Morris water maze to test their cognitive functions. Acyl-biotinyl exchange (ABE) for PSD-95 palmitoylation, immunofluorescent staining for expression of PSD-95, N-methyl-D-aspartic acid receptor subunit 2B (NR2B), and synaptotagmin 1 (SYT1) were assessed in mouse brain sections. Results: CEGI treatment in APP/PS1 mice significantly reduced Aβ deposition, relieved memory deficits, and decreased PSD-95 palmitoylation while markedly increasing the expression of PSD-95, NR2B, and SYT1 in the frontal cortex. There was a significant correlation between Aβ expression and PSD-95 palmitoylation in APP/PS1 mice. Conclusion: Our findings demonstrate that CEGI improved AD-like neuropathology, possibly by inhibiting PSD-95 palmitoylation, improving learning memory, and enhancing expression of synaptic-associated proteins, representing a potential therapy for AD treatment.

Donor T cells mediate both graft-versus-leukemia (GVL) activity and graft-versus-host disease (GVHD) after allogeneic hematopoietic cell transplantation (allo-HCT). Development of methods that preserve GVL activity while preventing GVHD remains a long-sought goal. Tolerogenic anti–interleukin-2 (IL-2) monoclonal antibody (JES6-1) forms anti–IL-2/IL-2 complexes that block IL-2 binding to IL-2Rβ and IL-2Rγ on conventional T cells that have low expression of IL-2Rα. Here, we show that administration of JES6 early after allo-HCT in mice markedly attenuates acute GVHD while preserving GVL activity that is dramatically stronger than observed with tacrolimus (TAC) treatment. The anti–IL-2 treatment downregulated activation of the IL-2-Stat5 pathway and reduced production of granulocyte-macrophage colony-stimulating factor (GM-CSF). In GVHD target tissues, enhanced T-cell programmed cell death protein 1 (PD-1) interaction with tissue–programmed cell death-ligand 1 (PD-L1) led to reduced activation of protein kinase–mammalian target of rapamycin pathway and increased expression of eomesodermin and B-lymphocyte-induced maturation protein-1, increased T-cell anergy/exhaustion, expansion of Foxp3–IL-10–producing type 1 regulatory (Tr1) cells, and depletion of GM-CSF–producing T helper type 1 (Th1)/cytotoxic T cell type 1 (Tc1) cells. In recipient lymphoid tissues, lack of donor T-cell PD-1 interaction with tissue PD-L1 preserved donor PD-1+TCF-1+Ly108+CD8+ T memory progenitors and functional effectors that have strong GVL activity. Anti–IL-2 and TAC treatments have qualitatively distinct effects on donor T cells in the lymphoid tissues, and CD8+ T memory progenitor cells are enriched with anti–IL-2 treatment compared with TAC treatment. We conclude that administration of tolerogenic anti–IL-2 monoclonal antibody early after allo-HCT represents a novel approach for preventing acute GVHD while preserving GVL activity.

Aims Apelin is the endogenous ligand of G protein-coupled receptor APJ and play an important role in the regulation of cardiovascular homeostasis. We aimed to investigate whether apelin ameliorates vascular calcification (VC) by inhibition of endoplasmic reticulum stress (ERS). Methods and results VC model in rats was induced by nicotine plus vitamin D, while calcification of vascular smooth muscle cell (VSMC) was induced by beta-glycerophosphate. Alizarin Red S staining showed dramatic calcium deposition in the aorta of rats with VC, while calcium contents and ALP activity also increased in calcified aorta. Protein levels of apelin and APJ were decreased in the calcified aorta. In rats with VC, apelin treatment significantly ameliorated aortic calcification, compliance and stimulation of ERS. The ameliorative effect of apelin on VC and ERS was also observed in calcified VSMCs. ERS stimulator (tunicamycin or DTT) blocked the beneficial effect of apelin. Apelin treatment activated the PI3K/Akt signaling, blockage of which by wortmannin or inhibitor IV prevented the ameliorative effect of apelin, while ERS inhibitor 4-PBA rescued the blockade effect of wortmannin. Akt-induced GSK inhibition prevented the phosphorylation of PERK and IRE1, and the activation of these two major ERS branches. F13A blocked the ameliorative effect of apelin on VC and ERS, which was reversed by treatment with 4-PBA or Akt activator SC79 Conclusions Apelin ameliorated VC by binding to APJ and then prevented ERS activation by stimulating Akt signaling. These results might provide new target for therapy and prevention of VC.

This study aimed to investigate whether inhibition of endoplasmic reticulum stress (ERS) mediated the ameliorative effect of apelin on acute heart failure (AHF). Rabbit model of AHF was induced by sodium pentobarbital. Cardiac dysfunction and injury were detected in the rabbit models of AHF, including impaired hemodynamic parameters and increased levels of CK-MB and cTnI. Apelin treatment dramatically improved cardiac impairment caused by AHF. ERS, indexed by increased GRP78, CHOP, and cleaved-caspase12 protein levels, was simultaneously attenuated by apelin. Apelin also could ameliorate increased protein levels of cleaved-caspase3 and Bax, and improved decreased protein levels of Bcl-2. Two common ERS stimulators, tunicamycin (Tm) and dithiothreitol (DTT) blocked the ameliorative effect of apelin on AHF. Phosphorylated Akt levels increased after apelin treatment in the rabbit models of AHF. The Akt signaling inhibitors wortmannin and LY294002 could block the cardioprotective effect of apelin, which could be relieved by ERS inhibitor 4-phenyl butyric acid (4-PBA). The aforementioned beneficial effects of apelin could all be blocked by APJ receptor antagonist F13A. 4-PBA and SC79, an Akt activator, can restore the ameliorative effect of apelin on AHF blocked by F13A. Apelin treatment dramatically ameliorated cardiac impairment caused by AHF, which might be mediated by APJ/Akt/ERS signaling pathway. These results will shed new light on AHF therapy.

Postmenopausal osteoporosis (PMOP) is an estrogen deficiency-induced bone loss, which has been shown an association with an altered gut microbiota (GM). Gut microbiota-bone axis has been recognized as a crucial mediator for bone homeostasis. Icariin (ICA) is an effective agent to delay bone loss by regulating the bone homeostasis. Thus, we hypothesize that ICA can prevent bone loss by modulating GM and regulating metabolite alterations. The effects of ICA on bone metabolism improvement in ovariectomized (OVX) rats and their relationships with the GM and fecal metabolites were investigated. Micro-computed tomography (micro-CT) and hematoxylin-eosin (HE) staining showed a typical bone boss in OVX group, while ICA or estradiol (E2) administration exhibited positive effects on bone micro-architecture improvement. The GM such as Actinobacteria, Gammaproteobacteria, Erysipelotrichi, Erysipelotrichales, Enterobacteriales, Actinomycetales, Ruminococcus and Oscillospira significantly correlated to serum bone Gla-protein (BGP), receptor activator of nuclear factor-κB (RANK), receptor activator of nuclear factor-κB ligand (RANKL), osteoprotegerin (OPG) and tartrate resistant acid phosphatase (TRACP). Further t-test revealed a substantial variation of the GM and fecal metabolites in different treatments. Among them, Lachnoclostridium, Butyricimonas, Rikenella, Paraprevolla, Adlercreutzia, Enterorhabdus, Anaerovorax, Allobaculum, Elusimicrobium, Lactococcus, Globicatella and Lactobacillus were probably the key microbial communities driving the change of bile acid, amino acid and fatty acid, thereby leading to an improvement of PMOP. The significant up-regulation of L-Saccharopine, 1-Aminocyclohexadieneacid and linoleic acid after ICA administration suggested important contributions of amino acid and fatty acid metabolisms in the prevention and treatment of PMOP. Taken together, our study has provided new perspectives to better understand the effects of ICA on PMOP improvement by regulating GM and the associated fecal metabolites. Our findings contribute to develop ICA as a potential therapy for PMOP.

Hair cells play key roles in hearing and balance, and hair cell loss would result in hearing loss or vestibular dysfunction. Cellular and molecular research in hair cell biology provides us a better understanding of hearing and deafness. Zebrafish, owing to their hair cell-enriched organs, have been widely applied in hair cell-related research worldwide. Similar to mammals, zebrafish have inner ear hair cells. In addition, they also have lateral line neuromast hair cells. These different types of hair cells vary in morphology and function. However, systematic analysis of their molecular characteristics remains lacking. In this study, we analyzed the GFP+ cells isolated from Tg(Brn3c:mGFP) larvae with GFP expression in all hair cells using single-cell RNA-sequencing (scRNA-seq). Three subtypes of hair cells, namely macula hair cell (MHC), crista hair cell (CHC), and neuromast hair cell (NHC), were characterized and validated by whole-mount in situ hybridization analysis of marker genes. The hair cell scRNA-seq data revealed hair cell-specific genes, including hearing loss genes that have been identified in humans and novel genes potentially involved in hair cell formation and function. Two novel genes were discovered to specifically function in NHCs and MHCs, corresponding to their specific expression in NHCs and MHCs. This study allows us to understand the specific genes in hair cell subpopulations of zebrafish, which will shed light on the genetics of both human vestibular and cochlear hair cell function.

Euthyroidism is of profound importance for lifetime health. However, the early diagnosis or therapeutics of thyroid developmental defects has not been established, mainly due to limited understanding of human thyroid development and a lack of recapitulating research model. Herein, the authors elaborate the cell atlas and potential regulatory signaling of the evolution of heterogeneous thyrocyte population from 12 to 16 gestational weeks. Moreover, they establish a long-term culture of human fetal thyroid organoids (hFTOs) system, which retains the fetal thyroid lineages and molecular signatures, as well as the ability to generate functional human thyroid follicles post mice renal transplantation. Notably, cAMP signaling activation in hFTOs by forskolin boosts the maturation of follicle and thus thyroid hormone T4 secretion, which recapitulates the key developmental events of fetal thyroid. Employing this ex vivo system, it is found that enhanced chromatin accessibility at thyroid maturation genes (such as TPO and TG) loci permits the transcription for hormone production. This study provides the cell atlas of and an organoid model for human thyroid development, which will facilitate thyroid research and prospective medicine.

Knee osteoarthritis (KOA) is a common disease with no specific treatment. Icariin (ICA) is considered an agent for KOA. This study aimed to confirm the pain-related neuromodulation mechanisms of ICA on KOA. Three experiments were designed: (1) verifying the therapeutic effects of ICA in vivo and in vitro, (2) exploring the potential pain-related neuromodulation pathways involved in ICA treatment by functional magnetic resonance imaging (fMRI) and virus retrograde tracing (VRT) and (3) confirming the pain-related targets by tandem mass tag (TMT)-based quantitative proteomics and bioinformatic analyses. Experiment 1 verified the efficacy of ICA in OA animal and cell models. Experiment 2 found a series of brain regions associated with KOA reversed by ICA treatment, indicating that a pain-related hypothalamic-mediated neuromodulation pathway and an endocannabinoid (EC)-related pathway contribute to ICA mechanisms. Experiment 3 explored and confirmed four pain-related genes involved in KOA and ICA treatment. We confirmed the key role of pain-related neuromodulation mechanisms in ICA treatment associated with its analgesic effect. Our findings contribute to considering ICA as a novel therapy for KOA.

Epimedokoreanin B (EKB) is a prenylated flavonoid isolated from Epimedium koreanum. In this article, we described the anti-cancerous effects of EKB and its underlying mechanism in human non-small cell lung cancer (NSCLC) A549 and NCI-H292 cells. EKB treatment inhibited cell proliferation and migration accompanied by cytoplasmic vacuolation in both cell lines. The cell death induced by EKB lacked the features of apoptosis like chromatin condensation, phosphatidyl serine exposure and caspase cleavage. The vacuoles stimulated by EKB predominantly derived from endoplasmic reticulum (ER) and mitochondria dilation, which are the characteristics of paraptosis. Down-regulation of Alix and up-regulation of ER stress-related proteins after EKB treatment further supported the occurrence of paraptosis. ER stress inhibitor 4-phenylbutyric acid (4-PBA) and protein synthesis inhibitor cycloheximide (CHX) treatment antagonized the vacuoles formation as well as cell death induced by EKB, indicating that ER stress was involved in EKB induced paraptosis. In addition, autophagosome accumulation accompanied with autophagy flux blocking was observed in EKB treated cells, this was consistent with the occurrence of ER stress. Collectively, EKB was demonstrated as a paraptosis-like cell death inducer in A549 and NCI-H292 cells. The inhibitory effect of EKB on lung cancer cell proliferation was further demonstrated in a zebrafish xenograft model. These findings raise the possibility that paraptosis inducers may be considered as alternative choices for lung cancer therapy.

Senescent astrocytes accumulate with aging and contribute to brain dysfunction and diseases such as Alzheimer's disease (AD), however, the mechanisms underlying the senescence of astrocytes during aging remain unclear. In the present study, we found that Yes‐associated Protein (YAP) was downregulated and inactivated in hippocampal astrocytes of aging mice and AD model mice, as well as in D‐galactose and paraquat‐induced senescent astrocytes, in a Hippo pathway‐dependent manner. Conditional knockout of YAP in astrocytes significantly promoted premature senescence of astrocytes, including reduction of cell proliferation, hypertrophic morphology, increase in senescence‐associated β‐galactosidase activity, and upregulation of several senescence‐associated genes such as p16, p53 and NF‐κB, and downregulation of Lamin B1. Further exploration of the underlying mechanism revealed that the expression of cyclin‐dependent kinase 6 (CDK6) was decreased in YAP knockout astrocytes in vivo and in vitro, and ectopic overexpression of CDK6 partially rescued YAP knockout‐induced senescence of astrocytes. Finally, activation of YAP signaling by XMU‐MP‐1 (an inhibitor of Hippo kinase MST1/2) partially rescued the senescence of astrocytes and improved the cognitive function of AD model mice and aging mice. Taken together, our studies identified unrecognized functions of YAP‐CDK6 pathway in preventing astrocytic senescence in vitro and in vivo, which may provide further insights and new targets for delaying brain aging and aging‐related neurodegenerative diseases such as AD., YAP signaling is downregulated and inactivated in hippocampal astrocytes of aging mice and AD model mice, as well as in D‐gal‐ and PQ‐induced senescent astrocytes, in a Hippo pathway‐dependent manner. Mechanistically, YAP prevents the premature senescence of astrocytes through CDK6 signaling. Activation of YAP by XMU‐MP‐1 delays the senescence of astrocytes, and thereby improving the cognitive function of aged mice and AD model mice.

Lipid metabolism that correlates tightly to the glucose metabolic regulation in malignant cells includes hepatocellular carcinoma (HCC) cells. The transcription factor Sterol Regulatory Element Binding Protein 1 (SREBP-1), a regulator of fatty acid synthesis, has been shown to pivotally regulate the proliferation and metastasis of HCC cells. However, the intrinsic mechanism by which SREBP-1 regulates the survival of HCC cells remains unclear. In this study, among HCC patients who had dismal responses to Sorafenib, a high SREBP-1 level was found in the tumors and correlated to poor survival. This observation suggested the negative role of SREBP-1 in clinical HCC prognosis. Our mechanistical studies reveal that the inhibition of SREBP-1 via its inhibitor Betulin suppresses cellular glucose metabolism. In addition to the reduced glycolytic activity, a thwarted metastatic potential was observed in HCC cells upon Betulin administration. Moreover, our data show that SREBP-1 inhibition facilitated the antitumor effects of Sorafenib on HCC cells and xenograft tumors.

Background Cisplatin resistance is a major challenge for advanced head and neck cancer (HNC). Understanding the underlying mechanisms and developing effective strategies against cisplatin resistance are highly desired in the clinic. However, how tumor stroma modulates HNC growth and chemoresistance is unclear. Results We show that cancer-associated fibroblasts (CAFs) are intrinsically resistant to cisplatin and have an active role in regulating HNC cell survival and proliferation by delivering functional miR-196a from CAFs to tumor cells via exosomes. Exosomal miR-196a then binds novel targets, CDKN1B and ING5, to endow HNC cells with cisplatin resistance. Exosome or exosomal miR-196a depletion from CAFs functionally restored HNC cisplatin sensitivity. Importantly, we found that miR-196a packaging into CAF-derived exosomes might be mediated by heterogeneous nuclear ribonucleoprotein A1 (hnRNPA1). Moreover, we also found that high levels of plasma exosomal miR-196a are clinically correlated with poor overall survival and chemoresistance. Conclusions The present study finds that CAF-derived exosomal miR-196a confers cisplatin resistance in HNC by targeting CDKN1B and ING5, indicating miR-196a may serve as a promising predictor of and potential therapeutic target for cisplatin resistance in HNC. Electronic supplementary material The online version of this article (10.1186/s13059-018-1604-0) contains supplementary material, which is available to authorized users.

Dendrobium mixture (DM) is a patent Chinese herbal formulation consisting of Dendrobii Caulis, Astragali Radix, Rehmanniae Radix as the main ingredients. DM has been shown to alleviate diabetic related symptoms attributed to its anti-hyperglycaemic and anti-inflammatory activities. However, the effect on diabetic induced cognitive dysfunction has not been investigated. This study aims to investigate the effect of DM in improving diabetic cognitive impairment and associated mechanisms. Our study confirmed the anti-hyperglycaemic effect of DM and showed its capacity to restore the cognitive and memory function in high fat/high glucose and streptozotocin-induced diabetic rats. The neuroprotective effect was manifested as improved learning and memory behaviours, restored blood-brain barrier tight junction, and enhanced expressions of neuronal survival related biomarkers. DM protected the colon tight junction, and effectively lowered the circulated proinflammatory mediators including tumour necrosis factor-α, interleukin-6 and lipopolysaccharides. In the gut microbiota, DM corrected the increase in the abundance of Firmicutes, the increase in the ratio of Firmicutes/Bacteroidetes, and the decrease in the abundance of Bacteroidetes in diabetic rats. It also reversed the abundance of Lactobacillus, Ruminococcus and Allobaculum genera. Short chain fatty acids, isobutyric acid and ethylmethylacetic acid, were negatively and significantly correlated to Ruminococcus and Allobaculum. Isovaleric acid was positively and significantly correlated with Lactobacillus, which all contributing to the improvement in glucose level, systemic inflammation and cognitive function in diabetic rats. Our results demonstrated the potential of DM as a promising therapeutic agent in treating diabetic cognitive impairment and the underlying mechanism may be associated with regulating gut microbiota.

BACKGROUND Acute myocardial infarction is the leading cause of mortality among adults worldwide. The present study aimed to investigate the role and mechanism of thrombin and SIRT1 in hypoxia/reoxygenation (H/R) injury. MATERIAL AND METHODS H9c2 cardiomyocytes were used to create an H/R model to simulate in vivo ischemia/reperfusion injury. The MTT assay was used to measure cell viability, qRT-PCR was used to detect the level of SIRT1, thrombin, and PAR-1, and western blot analysis was conducted for evaluation of thrombin, PAR-1, SIRT1, LC3I, LC3II, and Beclin1. ELISA was applied for determination of IL-1ß, IL-6, TNF-alpha, MMP-9, and ICAM-1. After the establishment of the H/R model, superoxide dismutase (SOD) activity was evaluated by the xanthine oxidase method, malondialdehyde content was detected by thiobarbituric acid assay, and reactive oxygen species generation was measured by CM-H2DCFDA. RESULTS The findings showed that thrombin enhanced inflammatory factor secretion and oxidative stress but inhibited cell viability in H/R-injured cardiomyocytes. We also observed that thrombin promoted autophagy in H/R-injured cardiomyocytes. In addition, thrombin enhanced the upregulation of SIRT1 expression by H/R. However, it was found that inhibition of SIRT1 could suppress the effect of thrombin on inflammatory factor secretion, oxidative stress, and cell viability. Moreover, downregulation of SIRT1 suppressed the inhibitory effect of thrombin on autophagy in H/R injury. CONCLUSIONS Thrombin aggravates H/R injury of cardiomyocytes by activating an autophagy pathway mediated by SIRT1. These findings might provide a potential target therapy for the treatment of ischemia/reperfusion injury in future clinical work.

Enveloped viruses hijack not only the host translation processes, but also its glycosylation machinery, and to a variable extent cover viral surface proteins with tolerogenic host-like structures. SARS-CoV-2 surface protein S presents as a trimer on the viral surface and is covered by a dense shield of N-linked glycans, and a few O-glycosites have been reported. The location of O-glycans is controlled by a large family of initiating enzymes with variable expression in cells and tissues and hence difficult to predict. Here, we used our well-established O-glycoproteomic workflows to map the precise positions of O-linked glycosylation sites on three different entities of protein S – insect cell or human cell-produced ectodomains, or insect cell derived receptor binding domain (RBD). In total 25 O-glycosites were identified, with similar patterns in the two ectodomains of different cell origin, and a distinct pattern of the monomeric RBD. Strikingly, 16 out of 25 O-glycosites were located within three amino acids from known N-glycosites. However, O-glycosylation was primarily found on peptides that were unoccupied by N-glycans, and otherwise had low overall occupancy. This suggests possible complementary functions of O-glycans in immune shielding and negligible effects of O-glycosylation on subunit vaccine design for SARS-CoV-2.

Efforts to improve the prognosis of steroid-resistant gut acute graft-versus-host-disease (SR-Gut-aGVHD) have suffered from poor understanding of its pathogenesis. Here we show that the pathogenesis of SR-Gut-aGVHD is associated with reduction of IFN-γ+ Th/Tc1 cells and preferential expansion of IL-17−IL-22+ Th/Tc22 cells. The IL-22 from Th/Tc22 cells causes dysbiosis in a Reg3γ-dependent manner. Transplantation of IFN-γ-deficient donor CD8+ T cells in the absence of CD4+ T cells produces a phenocopy of SR-Gut-aGVHD. IFN-γ deficiency in donor CD8+ T cells also leads to a PD-1-dependent depletion of intestinal protective CX3CR1hi mononuclear phagocytes (MNP), which also augments expansion of Tc22 cells. Supporting the dual regulation, simultaneous dysbiosis induction and depletion of CX3CR1hi MNP results in full-blown Gut-aGVHD. Our results thus provide insights into SR-Gut-aGVHD pathogenesis and suggest the potential efficacy of IL-22 antagonists and IFN-γ agonists in SR-Gut-aGVHD therapy., Pathogenesis of steroid-resistant gut acute graft-versus-host-disease (SR-Gut-aGVHD) remains unclear., Here the authors show in mouse models that dysbiosis caused by the expansion of Th/Tc22, as well as depletion of CX3CR1hi mononuclear phagocytes resulted from the reduction of Th/Tc1, contributes to SR-Gut-aGVHD onset.

Fourteen undescribed compounds, including five 2,5-diarylcyclopentenones xylariaones A1-B2, seven α-pyrone derivatives xylaripyones A-G, one γ-pyrone derivative xylaripyone H, one diketopiperazine cyclo-(L-Leu-N-ethyl-L-Glu), and two known diketopiperazines, were isolated from cultures of the endophytic fungus Xylaria sp., which was separated from Cudrania tricuspidata Bureau ex Lavallée. Their structures were determined by analysing extensive spectroscopic data (HRESIMS and NMR) and electronic circular dichroism (ECD) calculations. Furthermore, these compounds were evaluated for potential antiproliferative activity against the human tumour cell lines PC3 and A549, and the results showed that xylaripyone D exhibited moderate inhibitory activity against the proliferation of PC3 cell lines with an IC

Gei Herba, Chinese named Lanbuzheng (LBZ), is a traditional Chinese medicine promotes hematopoiesis, yet the underlying mechanism for this effect remains largely unknown. In the present study, a novel approach combining LC-MS metabolomics and molecular pharmacology was developed to investigate the hematopoietic effect and mechanism of LBZ on hematopoietic dysfunction (HD) caused by cyclophosphamide (CTX) in treated mice. The results show that LBZ can reduce damage in the spleen, a result consistent with the peripheral hemogram. Fourteen potential biomarkers were identified in the spleen by metabolic profiles analysis, including 5-hydroxymethyluracil, ascorbalamic acid, adenosine 5'-monophosphate, menadiol disulfate, l-homocysteine sulfonic acid and l-carnitine. Change in biomarker levels suggest that LBZ mainly affects β-oxidation of very-long-chain fatty acids, oxidation of branched chain fatty acids and carnitine synthesis, and those metabolites produced along with related metabolic pathways are closely associated with anti-apoptosis. A molecular pharmacology approach was simultaneously developed to examine accompanying cellular signaling mechanisms. LBZ activates PI3K/Akt signaling pathways and granulocyte-colony-stimulating-factor (G-CSF)-mediated Janus kinase 2 (JAK2)/transcription 3 (STAT3), resulting in inhibiting the release of cytochrome c. Further, LBZ inhibits caspase-mediated mitochondrial-dependent apoptosis mediated by caspase-9 and caspase-3. LBZ can thus reduce CTX-induced HD via G-CSF-mediated JAK2/STAT3 signaling and PI3K/Akt mitochondrial-dependent apoptotic pathways. The present study combines metabolomic and molecular pharmacological methods to elucidate mechanisms for the protective effect of LBZ on mouse HD following CTX-induced damage. This approach may be useful for exploring mechanisms of action of other drugs.

Background Hepatic encephalopathy (HE), a severe neuropsychiatric complication, is associated with increased blood levels of ammonia and bile acids (BAs). We sought to determine (1) whether abnormally increased blood BAs in liver cirrhotic patients with HE is caused by elevation of apical sodium-dependent BA transporter (ASBT)-mediated BA reabsorption; and (2) whether increased BA reabsorption would exacerbate ammonia-induced brain injuries. Methods We quantitatively measured blood BA and ammonia levels in liver cirrhosis patients with or without HE and healthy controls. We characterized ASBT expression, BA profiles, and ammonia concentrations in a chronic liver disease (CLD) mouse model induced by streptozotocin-high fat diet (STZ-HFD) and an azoxymethane (AOM) - induced acute liver failure (ALF) mouse model. These two mouse models were treated with SC-435 (ASBT inhibitor) and budesonide (ASBT activator), respectively. Findings Blood concentrations of ammonia and conjugated BAs were substantially increased in cirrhotic patients with HE (n = 75) compared to cirrhotic patients without HE (n = 126). Pharmacological inhibition of the enterohepatic BA circulation using a luminal- restricted ASBT inhibitor, SC-435, in mice with AOM-induced ALF and STZ-HFD -induced CLD effectively reduced BA and ammonia concentrations in the blood and brain, and alleviated liver and brain damages. Budesonide treatment induced liver and brain damages in normal mice, and exacerbated these damages in AOM-treated mice. Interpretation ASBT mediated BA reabsorption increases intestinal luminal pH and facilitates conversion of intestinal ammonium to ammonia, leading to abnormally high levels of neurotoxic ammonia and cytotoxic BAs in the blood and brain. Inhibition of intestinal ASBT with SC-435 can effectively remove neurotoxic BAs and ammonia from the bloodstream and thus, mitigate liver and brain injuries resulting from liver failure.

Uncontrolled cardiac fibrosis following myocardial infarction (MI) is a critical pathological change leading to heart failure. Current pharmacotherapies are limited by unsatisfactory efficacy and undesired systemic side effects. Astaxanthin (ASX) is a natural carotenoid with strong antioxidant and anti-inflammatory activities. The effects of ASX on MI-induced cardiac fibrosis and the underlying mechanisms remain largely unknown. In this study, after the establishment of MI model, mice were administrated with ASX (200 mg/kg⋅d) for 4 weeks. We found that ASX treatment attenuated cardiac fibrosis and improved heart function following MI, as evidenced by reduced collagen I/III ratio, hydroxyproline content and left ventricular end diastolic pressure (LVEDP). Lipidomic analysis revealed the overaccumulation of myocardial ceramides in mice with cardiac fibrosis, which was normalized by ASX treatment. Molecular docking analysis showed that ASX produced a tight fit in the pocket of sphingomyelin phosphodiesterase 1 (SMPD1), a key enzyme in the production of ceramides. Western blot analysis confirmed the significant inhibition of SMPD1 expression by ASX. Furthermore, MI-induced overexpression of transforming growth factor β1 (TGF-β1) and phosphorylated SMAD2/3 were attenuated by ASX administration. SMPD1 knockout (KO) abrogated the beneficial effect of ASX. Taken together, our results suggest that the cardioprotective effects of ASX are mediated by SMPD1 through the indirection inhibition of TGF- β1/SMAD signaling cascade.

Osteopontin (OPN), a chemokine-like protein, plays a crucial role in the proliferation and metastasis of various cancers. However, how tumor stroma modulates the expression of neoplastic OPN and the multifaceted roles of OPN in head and neck cancer (HNC) are unclear. In this study, we tried to investigate the bridging role of OPN between tumor stroma and cancer cells. Methods: Immunohistochemical staining and quantitative real-time PCR were used to detect OPN expression in HNC tissues, and the correlations between OPN expression and clinicopathologic features were then analyzed. We used a co-culture assay to study the modulatory role of IL-6 on OPN expression and immunoprecipitation analysis was used to determine the endogenous interaction between OPN and integrin αvβ3. Furthermore, a xenograft assay was carried out to confirm the tumor-promoting role and the potential therapeutic value of OPN in HNC. Results: We found that OPN was significantly up-regulated in HNCs, and the elevated OPN was correlated with poor prognosis. Moreover, we identified IL-6 secreted by cancer-associated fibroblasts (CAFs) as the major upstream molecule that triggers the induction of neoplastic OPN. As such, during the interaction of fibroblasts and cancer cells, the increased neoplastic OPN induced by stromal IL-6 accelerated the growth, migration and invasion of cancer cells. More importantly, we also showed that soluble OPN could promote HNC progression via the integrin αvβ3-NF-kappa B pathway, and the combination of OPN and IL-6 had a better prognostic and diagnostic performance in HNC than either molecule alone. Conclusion: Our study identified a novel modulatory role for OPN in HNC progression and further demonstrated that the combination of OPN and IL-6 might be a promising prognostic and diagnostic indicator as well as a potential cancer therapeutic target.

Background Preeclampsia (PE) is a common pregnancy‐related syndrome characterized by hypertension and proteinuria, and a major cause of maternal mortality. Therefore, there is an urgent need to identify early biomarkers of PE. The aim of the present study was to identify the functions of miR‐576‐5p in PE. Methods Effects of miR‐576‐5p and transcription factor AP‐2α (TFAP2A) on invasion of human trophoblast HTR8/SVneo cells were investigated. Real‐time quantitative polymerase chain reaction (RT‐qPCR) and western blotting were used to assess the expression of miR‐576‐5p, TFAP2A, E‐cad, and Vimentin in PE tissues and cells. Additionally, immunofluorescence was used to detect the expression of TFAP2A in PE trophoblastic tissue. Subsequently, constructed miR‐576‐5p mimics, miR‐576‐5p inhibitor, and siRNA‐TFAP2A plasmids were transfected into HTR8/SVneo cells for further experiments, including a CCK‐8 assay for cell proliferation, Transwell assay for cell invasion and the luciferase reporter gene system was employed for target verification. Results A lower expression of miR‐576‐5p and a higher expression of TFAP2A were identified in PE rats. E‐cadherin was highly expressed while Vimentin was downregulated. Further statistical analysis indicated that cell proliferation of HTR8/SVneo cells decreased in the miR‐576‐5p inhibitor group and increased in the miR‐576‐5p mimics and siRNA‐TFAP2A groups. miR‐576‐5p inhibitor suppressed cell invasion, and miR‐576‐5p mimics and siRNA‐TFAP2A improved cell invasion. The analysis of luciferase reporter demonstrated a decreased luciferase activity in miR‐576‐5p mimics group compared with control group, which indicates that TFAP2A may be a target of miR‐576‐5p. Interference of TFAP2A could downregulate E‐cadherin and upregulate Vimentin expression. Conclusion Overexpression of miR‐576‐5p and knockdown of TFAP2A may elevate cell proliferation and invasion of human trophoblast cells in vitro. Therefore, miR‐576‐5p may be used as a notable biomarker for the diagnosis, prevention, and treatment of PE. miR‐576‐5p targeting TFAP2A deserve further investigation in order to explore their potential role in PE., Preeclampsia (PE) is a common pregnancy‐related disease characterized by hypertension and proteinuria, and a major cause of maternal mortality. Therefore, there is an urgent need to identify early biomarkers of PE. The aim of the present study was to identify the functions of miR‐576‐5p in PE.

Background The lncRNA LINC00460 plays crucial roles in several epithelial cancers, although its mechanisms of action differ greatly in different cellular contexts. In this study, we aimed to determine the potential clinical applications of LINC00460 and elucidate the mechanisms by which LINC00460 affects the development and progression of head and neck squamous cell carcinoma (HNSCC). Methods The biological functions of LINC00460 were assessed in several epithelial cancer cell lines. The subcellular localization of LINC00460 was evaluated by cell nuclear/cytoplasmic fractionation and fluorescence in situ hybridization. RNA pull-down assays, LS-MS/MS analysis, and RNA and chromatin immunoprecipitation assays were performed to identify the molecular mechanism by which LINC00460 promotes HNSCC progression. The clinical pathological features of LINC00460 and PRDX1 were evaluated in HNSCC tissues and paired adjacent normal tissues. Results LINC00460 enhanced HNSCC cell proliferation and metastasis in vitro and in vivo and induced epithelial–mesenchymal transition (EMT). LINC00460 primarily localized within the cytoplasm of HNSCC cells, physically interacted with PRDX1 and facilitated PRDX1 entry into the nucleus. PRDX1 promoted the transcription of LINC00460, forming a positive feedback loop. In addition, PRDX1 also promoted the transcription of EMT-related genes (such as ZEB1, ZEB2 and VIM) through enrichment on gene promoters in the nucleus. LINC00460 effectively induced HNSCC cell EMT in a PRDX1-dependent manner, and PRDX1 mainly mediated the EMT-promoting effect of LINC00460. High levels of LINC00460 and PRDX1 expression were positively associated with lymph metastasis, pathological differentiation and tumor size in HNSCC patients. Conclusions LINC00460 promoted EMT in HNSCC cells by facilitating PRDX1 entry into the nucleus. LINC00460 and PRDX1 are promising candidate prognostic predictors and potential targets for cancer therapy for HNSCC. Electronic supplementary material The online version of this article (10.1186/s13046-019-1364-z) contains supplementary material, which is available to authorized users.

Background Significantly elevated serum and hepatic bile acid (BA) concentrations have been known to occur in patients with liver fibrosis. However, the roles of different BA species in liver fibrogenesis are not fully understood. Methods We quantitatively measured blood BA concentrations in nonalcoholic steatohepatitis (NASH) patients with liver fibrosis and healthy controls. We characterized BA composition in three mouse models induced by carbon tetrachloride (CCl4), streptozotocin-high fat diet (STZ-HFD), and long term HFD, respectively. The molecular mechanisms underlying the fibrosis-promoting effects of BAs were investigated in cell line models, a 3D co-culture system, and a Tgr5 (HSC-specific) KO mouse model. Findings We found that a group of conjugated 12α-hydroxylated (12α-OH) BAs, such as taurodeoxycholate (TDCA) and glycodeoxycholate (GDCA), significantly increased in NASH patients and liver fibrosis mouse models. 12α-OH BAs significantly increased HSC proliferation and protein expression of fibrosis-related markers. Administration of TDCA and GDCA directly activated HSCs and promoted liver fibrogenesis in mouse models. Blockade of BA binding to TGR5 or inhibition of ERK1/2 and p38 MAPK signaling both significantly attenuated the BA-induced fibrogenesis. Liver fibrosis was attenuated in mice with Tgr5 depletion. Interpretation Increased hepatic concentrations of conjugated 12α-OH BAs significantly contributed to liver fibrosis via TGR5 mediated p38MAPK and ERK1/2 signaling. Strategies to antagonize TGR5 or inhibit ERK1/2 and p38 MAPK signaling may effectively prevent or reverse liver fibrosis. Fundings This study was supported by the National Institutes of Health/National Cancer Institute Grant 1U01CA188387-01A1, the National Key Research and Development Program of China (2017YFC0906800); the State Key Program of National Natural Science Foundation (81430062); the National Natural Science Foundation of China (81974073, 81774196), China Postdoctoral Science Foundation funded project, China (2016T90381), and E-institutes of Shanghai Municipal Education Commission, China (E03008).

Ethnopharmacological relevance Cholestasis caused by bile secretion and excretion disorders is a serious manifestation of liver disease. With limited treatment methods, it affects millions of people worldwide. Huangqi decoction (HQD), an effective traditional Chinese medicine, is used to treat chronic cholestatic liver diseases. However, the action mechanisms of it were not fully elucidated. Aim of the study We aim to investigate the therapeutic effect of HQD, and its active component, astragalosides, against α-naphthylisothiocyanate (ANIT)-induced cholestasis in rats based on targeted metabolomics analysis and revel the potential mechanism. Materials and methods The therapeutic effect of HQD and astragalosides on ANIT-induced cholestasis model rats were evaluated by serum biochemical analysis. Liver damage was identified by histopathology. The levels of bile acids (BAs) and free fatty acids (FFAs) in serum and liver tissues were measured by ultra-high performance liquid chromatography-triple quadrupole mass spectrometry (UPLC-TQMS). qRT-PCR and Western blot analysis were used to measure the expression of nuclear hormone receptor, membrane receptor and BA transporter protein in cholestatic rats before and after HQD and astragalosides treatment. Results The obtained data showed that the administration of ANIT caused obvious cholestasis with significantly increased intrahepatic retention of hydrophobic BAs and altered FFAs, which were consistent with the liver histopathological and serum biochemical findings. HQD and astragalosides treatment were able to attenuate ANIT-induced BAs and FFAs perturbation, ameliorate the impaired liver function, histopathological ductular reaction, and lipid peroxidation damage by ANIT. Elevated mRNA and protein expression of transporters related to BA metabolism and genes related to lipogenesis and lipid oxidation metabolism in cholestasis were attenuated or normalized by HQD and astragalosides treatment. Conclusions Intervention by ANIT can significantly change the homeostasis of BAs and FFAs. HQD and astragalosides exerted a hepatoprotective effect against cholestatic liver injury by restoring the altered BA and FFA metabolism through the improvement of BA transporter, nucleus hormone receptor, and membrane receptor.

We evaluated the effects of administration of 1,25-dihydroxyvitamin D (1,25(OH)2D) during pregnancy on relieving adverse outcomes of preeclampsia and the pathologic and biochemical changes in reduction in uteroplacental perfusion (RUPP) model of rats. On day 1, 7, and 14 of pregnancy, rats in pregnant RUPP plus 1,25(OH)2D (RUPP+VD) group (n = 15) received 120 ng/100 g body weight/week of 1,25(OH)2D by subcutaneous injection, while rats in normal pregnant (n = 12) and the RUPP group (n = 14) received 1,25(OH)2D vehicle (saline solution). On day 19 of pregnancy, after measure of blood pressure and cardiac function and urine collection, rats were euthanized, and fetal and maternal serum, placenta, and heart and kidney were collected. Fetal mortality, urinary protein, glucose, and parameters for kidney function in serum were measured. We evaluated vitamin D receptor expression and pathological and ultrastructural changes in rat heart, kidney, and placenta. Levels of oxidative stress, endoplasmic reticulum (ER) stress, apoptosis, and autophagy were measured in placenta. Compared to RUPP rats, 1,25(OH)2D decreased fetal mortality, mean blood pressure, 24-h urinary protein, urine microalbumin, and hyperglycemia in RUPP+VD rats. These were consistent with the improvements of structure impairment in heart, kidney, and placenta of RUPP rat by 1,25(OH)2D. In placenta of RUPP rat, the decrease in oxidative stress and ER stress by 1,25(OH)2D treatment was accompanied by autophagy activation and apoptosis attenuation. 1,25(OH)2D plays a beneficial effect on preeclampsia at the early gestation and might be used as a potential protective agent for preeclampsia. However, the RUPP model only recapitulated the hypoxic origin of preeclampsia; further randomized controlled trial is expected to be performed for validation and evaluation.

Summary Hemagglutinins (HAs) from human influenza viruses adapt to bind α2-6-linked sialosides, overcoming a receptor-defined species barrier distinct from the α2-3 specificity of avian virus progenitors. Additionally, human-adapted HAs gain glycosylation sites over time, although their biological function is poorly defined. Using quantitative glycomic analysis, we show that HAs from human pandemic viruses exhibit significant proportions of high-mannose type N-linked glycans throughout the head domain. By contrast, poorly adapted avian-origin HAs contain predominately complex-type glycans, which have greater structural diversity. Although oligomannose levels vary, they are present in all tested recombinant HAs and whole viruses and can be specifically targeted for universal detection. The positions of high-mannose glycosites on the HA of human H1N1 and H3N2 strains are conserved. Additionally, high-mannose-binding lectins possess a broad capacity to neutralize and prevent infection with contemporary H3N2 strains. These findings reveal the biological significance of HA glycosylation and therapeutic potential of targeting these structures.

Poly(N-acryloyl-glucosamine-co-methylenebisacrylamide) (poly(AGA-co-MBA))-modified Fe3O4 nanoparticles were prepared via simple aqueous surface polymerization. The resultant Fe3O4@poly(AGA-co-MBA) nanoparticles were used as hydrophilic magnetic solid phase extraction sorbents. Ten aminoglycosides were selected as model analytes to evaluate the extraction performance and mechanism of the sorbents. The prepared magnetic nanoparticles were characterized by Fourier transformed infrared spectroscopy, elemental analysis, a vibrating sample magnetometer, and so on. The sorbents exhibited efficient extraction towards model analytes in hydrophilic interaction chromatography. Only 10.0 mg of sorbent was needed to adsorb analytes from 10.0 mL of a sample solution within a short time (5.0 min). Under optimized conditions, Fe3O4@poly(AGA-co-MBA) nanoparticles were applied in the sample preparation for the analysis of 10 aminoglycosides in meat samples by high-performance liquid chromatography-tandem mass spectrometry. The limits of detection (LODs) for the 10 aminoglycosides were in the range of 0.6–23.6 µg kg−1. Satisfactory recoveries of spiked meat samples from 84.6–98.3% with acceptable relative standard deviations from 3.1% to 7.8% were achieved. Compared to other reported methods for the analysis of aminoglycosides, the present work had comparable or higher sensitivities with a simple extraction procedure. The proposed hydrophilic interaction extraction sorbents are promising as powerful alternatives for extracting and enriching aminoglycosides in animal-derived food samples.

MicroRNAs (miRNAs) are single-stranded small non-coding RNAs, generally 18-25 nucleotides in length, that act as repressors of gene expression. miRNAs are encoded by independent genes or processed from a variety of different RNA species. So far, there is no evidence showing that the ribosomal DNA-hosted microRNA is implicated in vertebrate development. Currently, we found a highly expressed small RNA hosted in ribosomal DNA was predicted as a novel miRNA, named miR-ntu1, in zebrafish endothelial cells by deep sequencing analysis. The miRNA was validated by custom-designed Taqman PCR, Northern Blot, and in silico analysis. Furthermore, we demonstrated that miR-ntu1 played a crucial role in zebrafish angiogenesis via modulation of Notch signaling. Our findings provide a notable case that a miRNA hosted in ribosomal DNA is involved in vertebrate development.

Oxidative stress has been suggested to play an important role in neuronal injury. Ethyl gallate (EG) is the ethyl ester of gallic acid which has been acknowledged as an antioxidant. We previously demonstrated that EG effectively inhibited H

Protein delta homolog 1 (DLK1) regulates the odontoblastic differentiation of human dental pulp stem cells. It was hypothesized that DLK1 may exert regulatory effects on epithelial‑mesenchymal interactions in tooth development. The present study investigated the expression of DLK1 during the development of mouse enamel and its role in the proliferation and differentiation of ameloblast‑lineage cells (ALCs). DLK1 expression was upregulated in ameloblasts in the first mandibular molar during the entire process of enamel development. The mRNA and protein levels of DLK1 were significantly upregulated following ameloblastic induction in ALCs. In addition, overexpression of DLK1 promoted the proliferation of ALCs, inhibited ameloblastic differentiation, upregulated the expression of amelogenin and enamelin, and downregulated the expression of odontogenic ameloblast‑associated protein and kallikrein 4. The results of the present study suggested that DLK1 may be a potent regulator of ameloblast proliferation and differentiation, and may regulate enamel formation during tooth development.

A functional electrospun nanofibers-based electrochemiluminescence (ECL) immunosensor for detection of the tumor suppressor protein p53 (TSP

While targeted agents are an important part of the treatment arsenal for colorectal cancer, there is still a lack of efficient small-molecule targeted agents based on the understanding of pathogenic molecular mechanisms. In this study, curcumin analog RL71 displayed potent cytotoxicity towards human colon cancer cells with an IC50 of 0.8 µM in SW480 cells and inhibited xenotransplanted tumor growth in a dose-dependent manner. Using affinity chromatography, we identified sarco/endoplasmic reticulum calcium-ATPase (SERCA) 2 as the binding target of RL71. Most notably, RL71 demonstrated special binding to SERCA2 at a novel site with the lowest estimated free energy -8.89 kcal mol(-1), and the SERCA2 residues critical for RL71 binding were identified. RL71 suppressed the Ca(2+)-ATPase activity of SERCA2 both in vitro and in vivo, accompanied by the induction of endoplasmic reticulum stress leading to apoptosis and G2/M cycle arrest in SW480 cells. In addition, RL71 showed synergistic cytotoxicity with the pan-SERCA inhibitor thapsigargin. These results suggest that RL71 could be a selective small-molecule inhibitor of SERCA2, and that it may serve as a lead compound for the study of targeted colorectal cancer therapy.

The purpose of this study was to investigate the effect of combretastatin A4 phosphate (CA4P) on vasculogenic mimicry (VM) channel formation in vitro and in vivo after a single-dose treatment and the underlying mechanism involved in supporting VM. In vitro model of three-dimensional cultures was used to test the effect of CA4P on the tube formation of Walker 256 cells. Western blot analysis was conducted to assess the expression of hypoxia-inducible factor (HIF)-1α and VM-associated markers. W256 tumor-bearing rat model was established to demonstrate the effect of CA4P on VM formation and tumor hypoxia by double staining and a hypoxic marker pimonidazole. Anti-tumor efficacy of CA4P treatment was evaluated by tumor growth curve. Under hypoxic conditions for 48 h in vitro, W256 cells formed VM network associated with increased expression of VM markers. Pretreatment with CA4P did not influence the amount of VM in 3-D culture as well as the expression of these key molecules. In vivo, W256 tumors showed marked intratumoral hypoxia after CA4P treatment, accompanied by increased VM formation. CA4P exhibited only a delay in tumor growth within 2 days but rapid tumor regrowth afterward. VM density was positively related to tumor volume and tumor weight at day 8. CA4P causes hypoxia which induces VM formation in W256 tumors through HIF-1α/EphA2/PI3K/matrix metalloproteinase (MMP) signaling pathway, resulting in the consequent regrowth of the damaged tumor.

Based on the breast cancer cells and the vascular endothelial cells are both estrogen-sensitive, we proposed a close reciprocity existed between them in the tumor microenvironment, via shared molecular mechanism affected by environmental endocrine disruptors (EDCs). In this study, bisphenol A (BPA), via triggering G-protein estrogen receptor (GPER), stimulated cell proliferation and migration of bovine vascular endothelial cells (BVECs) and breast cancer cells (SkBr-3 and MDA-MB-231) and enhanced tumor growth in vivo. Moreover, the expression of both hypoxia inducible factor-1 alpha (HIF-1α) and vascular endothelial growth factor (VEGF) were up-regulated in a GPER-dependent manner by BPA treatment under hypoxic condition, and the activated GPER induced the HIF-1α expression by competitively binding to caveolin-1 (Cav-1) and facilitating the release of heat shock protein 90 (HSP90). These findings show that in a hypoxic microenvironment, BPA promotes HIF-1α and VEGF expressions through a shared GPER/Cav-1/HSP90 signaling cascade. Our observations provide a probable hypothesis that the effects of BPA on tumor development are copromoting relevant biological responses in both vascular endothelial and breast cancer cells.

Cholestasis is a clinical disorder defined as an impairment of bile flow, and that leads to toxic bile acid (BA) accumulation in hepatocytes. Here, we investigated the hepatoprotective effect of Yinchenhaotang (YCHT), a well-known formulae for the treatment of jaundice and liver disorders, against the cholestasis using the α-naphthylisothiocyanate (ANIT)-induced cholestasis in male Wistar rats. ANIT feeding induced significant cholestasis with substantially increased intrahepatic retention of hydrophobic BAs. The dynamic changes of serum and liver BAs indicated that YCHT was able to attenuate ANIT-induced BA perturbation, which is consistent with the histopathological findings that YCHT significantly decreased the liver damage. YCHT treatment substantially reduced serum alanine aminotransferase (ALT), alkaline phosphatase (AST), total bilirubin (TBIL) and direct bilirubin (DBIL) with minimal bile duct damage in the ANIT treated rats. Elevated mRNA expression of liver IL-6, IL-17A, IL-17F, TGF-β1, α-SMA, TGR5, NTCP, OATP1a1, and ileum ASBT and decreased liver IL-10, FXR, CAR, VDR, BSEP, MRP2, MRP3, MRP4 was also observed in ANIT-induced cholestasis but were attenuated or normalized by YCHT. Our results demonstrated that the BA profiles were significantly altered with ANIT intervention and YCHT possesses the hepatoprotective potential against cholestatic liver injury induced by hepatotoxin such as ANIT.

Emerging evidence points to a strong association between sex and gut microbiota, bile acids (BAs), and gastrointestinal cancers. Here, we investigated the mechanistic link between microbiota and hepatocellular carcinogenesis using a streptozotocin-high fat diet (STZ-HFD) induced nonalcoholic steatohepatitis-hepatocellular carcinoma (NASH-HCC) murine model and compared results for both sexes. STZ-HFD feeding induced a much higher incidence of HCC in male mice with substantially increased intrahepatic retention of hydrophobic BAs and decreased hepatic expression of tumor-suppressive microRNAs. Metagenomic analysis showed differences in gut microbiota involved in BA metabolism between normal male and female mice, and such differences were amplified when mice of both sexes were exposed to STZ-HFD. Treating STZ-HFD male mice with 2% cholestyramine led to significant improvement of hepatic BA retention, tumor-suppressive microRNA expressions, microbial gut communities, and prevention of HCC. Additionally the sex-dependent differences in BA profiles in the murine model can be correlated to the differential BA profiles between men and women during the development of HCC. These results uncover distinct male and female profiles for gut microbiota, BAs, and microRNAs that may contribute to sex-based disparity in liver carcinogenesis, and suggest new possibilities for preventing and controlling human obesity-related gastrointestinal cancers that often exhibit sex differences.

Hypericin (Hyp) is newly recognized as a necrosis avid agent, but its poor solubility imposes a great hindrance in clinical application. The aim of this paper was to explore sodium cholate (NaCh) as a potential solvent for Hyp and assess the targetability of (131)I-Hyp in rat necrosis models. Hyp solubility in NaCh solutions was evaluated by equilibrium solubility measurement. Biodistribution of (131)I-Hyp in NaCh solutions and mixed organic solvents was investigated in rat models of liver and muscle necrosis examined with MRI and SPECT/CT in vivo. In addition, pharmacokinetics of (131)I-Hyp in NaCh solutions was studied in healthy rats. Results showed NaCh could improve Hyp solubility and (131)I-Hyp incubated in NaCh solutions/rat plasma was stable up to 120 h. On SPECT/CT images at 24 h post injection, liver infarction location appeared as hot spots. Liver necrosis-to-liver ratios were 12.2, 10.0, 9.6 and 8.2 in 60, 15, 2 mmol/L of NaCh solutions and organic solvents, and muscle necrosis-to-liver ratios were 11.1, 10.1, 7.7 and 7.4, respectively. Pharmacokinetics study revealed t(1/2)z (11.93, 8.96 h, p > 0.05) and AUC (0-∞) (421.21, 553.34 MBq/L h, p

High-frequency alleles and/or co-occurring human leukocyte antigen alleles across loci appear to be more important than individual alleles as markers of disease risk and have clinical value as biomarkers for targeted screening or the development of new disease therapies. To better elucidate the major histocompatibility complex (MHC) background and to facilitate the experimental use of cynomolgus macaques, Mafa-DPA1, Mafa-DQA1, Mafa-DRA, and Mafa-DOA alleles were characterized, and their combinations were investigated in 30 Vietnamese macaques by gene cloning and sequencing. A total of 26 Mafa-DPA1, 18 Mafa-DQA1, 9 Mafa-DRA, and 15 Mafa-DOA alleles, including 7 high-frequency alleles, were identified in this study, respectively. In addition, 15 Mafa-DQA1, 17 Mafa-DPA1, 15 Mafa-DOA, and 2 Mafa-DRA alleles represented novel sequences that had not been documented in earlier studies. Our results also showed that the Vietnamese macaques might be valuable because no less than 30 % of the test animals possessed Mafa-DRA*01:02:01 (90 %), -DQA1*26:01:03 (37 %), -DOA*01:02:07 (34 %), and -DQA1*01:03:03 (30 %). We previously reported that the combinations of MHC class II alleles, including the combination of DOA*01:02:07-DPA1*02:09 and DOA*01:02:07-DQA1*01:03:03, were detected in 17 and 14 % of the animals, respectively. Interestingly, more than two Mafa-DQA1 and Mafa-DPA1 alleles were detected in one animal in this study, which suggested that they might be caused by a chromosomal duplication. If our findings can be validated by other studies, it will further enrich the number of known Mafa-DPA1 and Mafa-DQA1 polymorphisms. Our results identified the co-occurring MHC alleles across loci in a cohort of Vietnamese cynomolgus macaques, which emphasized the value of this species as a model for biomedical research.

Cancers are often with spontaneous or therapeutic necrosis that could be utilized as a generic target for developing new treatments. The purpose of this study was to investigate the biodistribution and pharmacokinetics of radioiodinated hypericin (Hyp), a naturally occurring compound, after intravenous (i.v.) injection in a rat model of liver and muscle necrosis (n = 42), and evaluate its necrosis affinity. Hyp was labeled with (131)I with labeling efficiency99%. After incubating in solution/rat plasma for 8 days, radiochemical purity of (131)I-Hyp remained 98.1 and 97.1%, respectively, indicating good in vitro stability. SPECT-CT images at 24 h after i.v. injection of (131)I-Hyp in rats with induced liver and muscle necrosis showed obvious tracer absorption in necrotic tissues. Biodistribution studies revealed that the percentage of the injected dose per gram of tissue (%ID/g) evolved from 1.9 %ID/g at 6 h, through a maximum 3.0 %ID/g at 12 h, to 1.0 %ID/g at 192 h in necrotic liver. Pharmacokinetics studies revealed that the terminal elimination half-life, total body clearance and area under the curve of (131)I-Hyp were 32.7 h, 9.2 L/h/kg and 1.6 MBq/L*h, respectively. These results demonstrated that (131)I-Hyp features a long blood circulation in animals and persistent retention in necrotic tissues. Therefore, (131)I-labeled Hyp could be a broad-spectrum anti-tumor agent with a cost much cheaper relative to the biological agents such as monoclonal antibodies.

A high-throughput cell-based method was developed for screening traditional Chinese herbal medicines (TCHMs) for potential stem cell growth promoters. Mouse embryonic stem (mES) cells expressing enhanced green fluorescent protein (EGFP) were cultured in growth media supplemented with various TCHM extracts. The dosage-dependent effects of TCHM extracts on cell growth, including proliferation and cytotoxicity, were assessed via EGFP fluorescence measurement. Seven TCHMs were investigated, and among them Panax notoginseng (PN), Rhizoma Atractylodis macrocephalae, Rhizoma chuanxiong, and Ganoderma lucidum spores (GLS) showed potential to improve mES cell proliferation. Eleven mixtures of these four TCHMs were then studied, and the results showed that the mixture of PN and GLS had the strongest growth promoting effect, increasing the specific growth rate of mES cells by 29.5% at a low dosage of 0.01% (wt/vol) PN/GLS (P

Spinal cord injury (SCI) is one of the most severe traumatic injuries that results in dysfunction of limbs and trunk below the damaged section. Recent studies have shown that gastrodin (GAS) could improve the recovery of SCI. In the current study, we aimed to examine the possible mechanism underlying the effect of GAS on recovery of SCI in rats. In rats with SCI, GAS improved locomotor functions and decreased permeability of blood-spinal cord barrier, as illustrated by increase of Basso-Beattie-Bresnahan scores and decrease of Evans blue leakage. In addition, GAS inhibited inflammation, as evidenced by decrease of proinflammatory cytokines, including tumor necrosis factor α (TNFα) and interleukin-1β (IL-1β) in rats following SCI. Moreover, increase of TBARS content and decrease of glutathione (GSH) content and superoxide dismutase (SOD) activities in SCI rats were inhibited by GAS. Furthermore, GAS enhanced mRNA expression of nuclear factor (erythroid-derived 2)-like 2 (Nrf2), catalytic subunit of γ-glutamylcysteine ligase (GCLc) and modified subunit of γ-glutamylcysteine ligase (GCLm). The data suggested that GAS may promote the recovery of SCI through the enhancement of Nrf2-GCLc/GCLm signaling pathway, and subsequent improvement of oxidative stress and inflammation, resulting in decrease of permeability of BSCB and improved recovery of locomotor function in rats with SCI. The results have provided novel insights into GAS-related therapy of SCI and associated neurodegenerative diseases.

Dysregulated bile acids (BAs) are closely associated with liver diseases and attributed to altered gut microbiota. Here, we show that the intrahepatic retention of hydrophobic BAs including deoxycholate (DCA), taurocholate (TCA), taurochenodeoxycholate (TCDCA), and taurolithocholate (TLCA) were substantially increased in a streptozotocin and high fat diet (HFD) induced nonalcoholic steatohepatitis-hepatocellular carcinoma (NASH-HCC) mouse model. Additionally chronic HFD-fed mice spontaneously developed liver tumors with significantly increased hepatic BA levels. Enhancing intestinal excretion of hydrophobic BAs in the NASH-HCC model mice by a 2% cholestyramine feeding significantly prevented HCC development. The gut microbiota alterations were closely correlated with altered BA levels in liver and feces. HFD-induced inflammation inhibited key BA transporters, resulting in sustained increases in intrahepatic BA concentrations. Our study also showed a significantly increased cell proliferation in BA treated normal human hepatic cell lines and a down-regulated expression of tumor suppressor gene CEBPα in TCDCA treated HepG2 cell line, suggesting that several hydrophobic BAs may collaboratively promote liver carcinogenesis.

Necrosis avid agents (NAAs) can be used for diagnose of necrosis-related diseases, evaluation of therapeutic responses and targeted therapeutics of tumor. In order to probe into the effects of molecular skeleton structure on necrosis targeting and clearance properties of radioiodinated dianthrones, four dianthrone compounds with the same substituents but different skeletal structures, namely Hypericin (Hyp), protohypericin (ProHyp), emodin dianthrone mesomer (ED-1) and emodin dianthrone raceme (ED-2) were synthesized and radioiodinated. Then radioiodinated dianthrones were evaluated in vitro for their necrosis avidity in A549 lung cancer cells untreated and treated with H2O2. Their biodistribution and pharmacokinetic properties were determined in rat models of induced necrosis. In vitro cell assay revealed that destruction of rigid skeleton structure dramatically reduced their necrosis targeting ability. Animal studies demonstrated that destruction of rigid skeleton structure dramatically reduced the necrotic tissue uptake and speed up the clearance from the most normal tissues for the studied compounds. Among these 131I-dianthrones, 131I-Hyp exhibited the highest uptake and persistent retention in necrotic tissues. Hepatic infarction could be clearly visualized by SPECT/CT using 131I-Hyp as an imaging probe. The results suggest that the skeleton structure of Hyp is the lead structure for further structure optimization of this class of NAAs.

Human peroxiredoxin 2 (Prx2), which is abundant in erythrocytes, has been shown to play a key role in protecting erythrocytes against oxidative stress by scavenging reactive oxygen species as well as participating in cell signal transduction. Here, human Prx2 gene was successfully cloned into Escherichia coli BL21 (DE3) for Prx2 expression. Sodium dodecyl sulfate polyacrylamide gel electrophoresis analysis suggested that the recombinant protein was expressed mainly in a soluble form. The recombinant protein was purified by one-step Ni-nitrilotriacetic acid chelating affinity chromatography to a purity of up to 91.5%. The peroxidase activity of Prx2 to scavenge H(2)O(2) was determined by a ferrithiocyanate assay. The ability of Prx2 to protect plasmid DNA was tested by using a mixed-function oxidation system, and results showed that Prx2 could prevent DNA from undergoing oxidative stress. Ultraviolet (UV)-induced cell apoptosis assay demonstrated that Prx2 is also able to protect NIH/3T3 cells from UV-induced damage, suggesting its possible applications in cosmetics and other areas.

Luteolin has been shown to have a strong anticancer effect on various cancer models via programmed cell death (apoptosis). However, the fundamental mechanisms of these effects are still unclear. In the present study, we examined the question of whether or not luteolin can inhibit proliferation of pancreatic carcinoma cells, via apoptosis. We used three human pancreatic carcinoma cell lines, PANC-1, CoLo‑357 and BxPC-3 in our study. In luteolin-treated pancreatic carcinoma cells, typical features of apoptosis were observed. Luteolin increased the expression of the pro-apoptotic protein Bax and decreased the expression of the anti-apoptotic protein Bcl-2, with a concomitant increase in the levels of caspase-3 and cleaved PARP after treatment for 24 h. Luteolin inhibited HUVEC proliferation and vessel growth in CAM in vivo. In addition, the concentration of VEGF in the conditioned medium from human pancreatic carcinoma cells was downregulated by luteolin. Pancreatic carcinoma cells, pretreated with luteolin, could decrease the capillary-like structure formation by HUVEC, which was analyzed by a co-culture system. The abatement of VEGF secretion was related to the inhibition of VEGF mRNA expression, which may be regulated by inhibiting the transcription activity of nuclear transcription factor NF-κB.

Clostridium difficile toxin A (TcdA) is one of the main pathogenic factors released by C. difficile. Due to its potent cytotoxic and proinflammatory activities, we investigated the anti-tumor activity of TcdA. CT26 colorectal cancer cells were challenged with recombinant TcdA, and it was found that TcdA could induce apoptosis of CT26 cells. Calreticulin (CRT) exposure to the cell surface during TcdA-induced apoptosis suggested that this apoptosis may correlate with immunogenicity. Moreover, TcdA-treated apoptotic CT26 cells were highly immunogenic since they could stimulate DC activation, T-cell activation, and anti-tumor activity. Furthermore, the anti-tumor immune response generated was specific and long-term. In summary, these studies demonstrate that C. difficile toxin A can induce apoptotic death of CT26 colorectal cancer cells and stimulate potent anti-tumor immunity.

High-frequency alleles and/or co-occurring human leukocyte antigen (HLA) alleles across loci appear to be more important than individual alleles, because they might be markers of disease risk that have clinical value as biomarkers for targeted screening or the development of new therapies. To better elucidate the major histocompatibility complex background and to facilitate the experimental use of cynomolgus macaques, Mafa-B, Mafa-DQB1, and Mafa-DRB alleles were characterized and their combinations were investigated from 30 macaques of Vietnamese origin by cloning and sequencing. A total of 48 Mafa-B, 22 Mafa-DQB1, and 42 Mafa-DRB alleles, were detected in this study, respectively. In addition, two Mafa-DQB1 and eight Mafa-DRB alleles represented novel sequences that had not been documented in earlier studies. Our results also showed that the macaque from Vietnam might be valuable because >30% of the test animals possessed Mafa-DRB*w304 (30%) and -DQB1*0616 (30%). We report that the combination of major histocompatibility complex (MHC) class I and II alleles, including the combination of DRB3*0403-DRB*w304, DRB1*1013-DRB*w304, and Mafa-B*007:01:01-DRB*w304, which was in 17%, 13%, and 13% of the animals, respectively. Interesting, more than two Mafa-DQB1 alleles detected in one animal in this study suggest that Mafa-DQB1, like Mafa-DRB, might be a duplication in the chromosome, which have ever been documented in cynomolgus monkeys but has not yet been observed in rhesus macaques or other primates. Our results for the high frequency of commonly co-occurring MHC alleles across loci in a cohort of the Vietnamese cynomolgus macaque emphasized the value of this species as a model for biomedical research.

The aim of the present study was to develop an oil-free o/w microemulsion, composed of pluronic F68, propylene glycol and saline, which solubilized poorly soluble anesthetic drug propofol for intravenous administration. The ternary diagram was constructed to identify the regions of microemulsions, and the optimal composition of microemulsion was determined by in vitro evaluation such as globule size upon dilution and rheology. The droplet size of the diluent emulsion corresponding to oil-in-water type ranged from 200 to 300nm in diameter. Stability analysis of the microemulsions indicated that they were stable upon storage for at least 6 months. Hemolysis percent of propofol microemulsions was lower than that of commercial lipid emulsion (CLE) at 4h. Acute toxicity test showed that median lethal dose of propofol microemulsion was the same as that of CLE. No significant difference in time for unconsciousness and recovery of righting reflex was observed between the prepared microemulsions and CLE. In conclusion, microemulsion would be a promising intravenous delivery system for propofol.