Shikonin reactivates TSGs GADD45B and PPP3CC to block NSCLC cell proliferation and migration through JNK/P38/MAPK signaling pathways.

BACKGROUND: Shikonin, a natural naphthoquinone compound extracted from the Chinese traditional herbal medicine "Lithospermum erythrorhizon", possesses antitumor activity against various cancer types. Tumor-suppressor genes (TSGs) negatively regulate cell growth, proliferation, and differentiation, thereby inhibiting tumor formation. However, the molecular mechanism of action of shikonin on TSGs in non-small-cell lung cancer (NSCLC) remains unclear. METHODS: The inhibitory effect of shikonin on the proliferation and migration abilities of lung cancer cells were measured by Cell Counting Kit 8 (CCK8) and wound healing assays. The alteration of genes by shikonin treatment was detected by mRNA high-throughput sequencing and further confirmed by qPCR and western blotting experiments. The dominant functions of the upregulated genes were analyzed by GO and KEGG profiling. RESULTS: Shikonin inhibited the proliferation and migration of A549 and H1299 NSCLC cells in a dose-dependent manner. mRNA high-throughput sequencing revealed a total of 1794 upregulated genes in shikonin-treated NSCLC cells. Moreover, bioinformatic analysis of GO and KEGG profiling revealed that the up-regulated genes were mostly involved in the JNK/P38/MAPK signaling pathway, among which the expression of GADD45B and PPP3CC was significantly enhanced. Finally, we confirmed that GADD45B and PPP3CC were indeed upregulated in JNK/P38/MAPK pathway. CONCLUSIONS: Taken together, these results suggested that shikonin might affect the expression of GADD45B and PPP3CC through the JNK/P38/MAPK pathway, therefore exerting an inhibitory effect on the proliferation and migration of cancer cells. To our knowledge, this is the first study reporting the role of shikonin in upregulating TSGs to activate the JNK/P38/MAPK signaling pathways in NSCLC.

Influence of Maitake (Grifola frondosa) Particle Sizes on Human Mesenchymal Stem Cells and In Vivo Evaluation of Their Therapeutic Potential.

Maitake (Grifola frondosa) mushroom has received an enormous amount of attention as a dietary supplement due to its high nutritional values. The particle sizes of G. frondosa mushrooms were monitored by a classifying mill. ß-Glucans are the bioactive component of the mushroom, and it was revealed through Fourier transform infrared spectroscopy (FTIR), proton and carbon nuclear magnetic resonance (1H and 13C-NMR), matrix-assisted laser desorption/ionization, and time-of-flight (MALDI-TOF) spectrometry. The biocompatibility of G. frondosa particles, as well as induced osteogenesis of hMSCs, was evaluated through WST-1 assay and alizarin staining (ARS) technique, respectively. Notably, enhanced cell viability was noted in the presence of G. frondosa. Significantly improved calcium deposition has observed from hMSCs with G. frondosa, suggesting to their mineralization potential. The expression of osteogenic related gene markers was examined in the presence of G. frondosa through real-time polymerase chain reaction (qPCR) technique. The upregulation of osteogenic gene markers in the presence of G. frondosa particles was indicating their superior osteogenic potential. Besides, G. frondosa also activated the secretion of various kinds of proteins from the hMSCs indicating their potential for tissue engineering applications. Enhanced secretion of different immunoglobulins was observed in rat serum in the presence of G. frondosa, further demonstrating their therapeutic nature. Therefore, G. frondosa is effective for enhanced osteogenesis and can be utilized as a natural, edible, and osteogenic agent.

Differentiation of neonate mouse spermatogonial stem cells on three-dimensional agar/polyvinyl alcohol nanofiber scaffold.

Electrospun nanofiber matrices sufficiently mimic the structural morphology of natural extracellular matrix. In this study, we aimed to examine the effects of agar/polyvinyl alcohol nanofiber (PVA) scaffold on the proliferation efficiency and differentiation potential of neonate mouse spermatogonial stem cells (SCCs). Testicular cells were isolated from testes of 40 mouse pups and were seeded in: 1) 2D cell culture plates in the absence (2D/-GF) or presence (2D/+GF) of growth factors and 2) onto agar/PVA scaffold in the absence (3D/-GF) or presence (3D/+GF) of growth factors. The cells were subsequently cultured for 4 weeks. First 2 weeks were dedicated to proliferative phase, whereas the next 2 weeks emphasized the differentiation phase. The identity of the SCCs was investigated at different time-points by flow cytometry and quantitative reverse transcription PCR (qRT-PCR) analyses against the germ cell markers, including PLZF, Id-4, Gfrα-1, Tekt-1, and Sycp-3. After 2 weeks of culture, the 3D/+GF group showed the highest percentage of PLZF-positive cells among culture systems (P < 0.05). The expression levels of pre-meiotic markers (Id-4 and Gfrα-1) decreased significantly in all groups, particularly in 3D/+GF group after 28 days of culture. Additionally, the cells in the 3D/+GF group displayed the highest expression of meiotic (Sycp-3) and post-meiotic markers (Tekt-1) 14 days after differentiation induction. Seemingly, the combination of the agar/PVA scaffold and growth factor-supplemented medium synergistically increased the differentiation rate of mouse SSCs into meiotic and post-meiotic cells. Thus, agar/PVA nanofiber scaffolds may have the potential for applications in the restoration of infertility, especially in azoospermic males. ABBREVIATIONS: 2D: two dimentional; 3D: three dimentional; bFGF: basic fibroblast growth factor; BMP-4: bone morphogenetic protein 4; DMEM: Dulbecco's modified Eagle's medium; ECM: extracellular matrix; FCS: fetal calf serum; FTIR: Fourier-transform infrared spectroscopy; GDNF: glial cell line-derived neurotrophic factor; GF: growth factors; Gfrα-1, GDNF family co-receptor α1; Id-4, Inhibitor of DNA Binding 4; MTT: methylthiazoltetrazolium; PLZF: promyelocytic leukemia zinc finger; PVA: polyvinyl alcohol; qRT-PCR: quantitative reverse transcription PCR; RA: retinoic acid; SACS: soft agar culture system; SD: standard deviation; SEM: scanning electron microscope; SSCs: spermatogonial stem cells; Sycp-3, Synaptonemal complex protein 3; Tekt-1, Tektin 1.

Centella asiatica L. Phytosome Improves Cognitive Performance by Promoting Bdnf Expression in Rat Prefrontal Cortex.

A wide range of people in the world use natural remedies as primary approaches against illnesses. Accordingly, understanding the mechanisms of action of phytochemicals has become of great interest. In this context, Centella asiatica L. is extensively used, not only as anti-inflammatory or antioxidant agent but also as brain tonic. On this basis, the purpose of this study was to evaluate whether the chronic administration of C. asiatica L. to adult male rats was able to improve the expression of Bdnf, one of the main mediators of brain plasticity. Moreover, we assessed whether the treatment could affect the cognitive performance in the novel object recognition (NOR) test. We confirmed the presence of the main compounds in the plasma. Furthermore, C. asiatica L. administration induced an increase of Bdnf in the prefrontal cortex, and the administration of the higher dose of the extract was able to improve cognitive performance. Finally, the increase in the preference index in the NOR test was paralleled by a further increase in Bdnf expression. Overall, we highlight the ability of C. asiatica L. to affect brain functions by increasing Bdnf expression and by enhancing the cognitive performance.

Yinchenhao decoction attenuates obstructive jaundice-induced liver injury and hepatocyte apoptosis by suppressing protein kinase RNA-like endoplasmic reticulum kinase-induced pathway.

BACKGROUND: Chronic biliary obstruction results in ischemia and hypoxia of hepatocytes, and leads to apoptosis. Apoptosis is very important in regulating the homeostasis of the hepatobiliary system. Endoplasmic reticulum (ER) stress is one of the signaling pathways that induce apoptosis. Moreover, the protein kinase RNA-like endoplasmic reticulum kinase (PERK)-induced apoptotic pathway is the main way; but its role in liver injury remains unclear. Yinchenhao decoction (YCHD) is a traditional Chinese medicine formula that alleviates liver injury and apoptosis, yet its mechanism is unknown. We undertook this study to investigate the effects of YCHD on the expression of ER stress proteins and hepatocyte apoptosis in rats with obstructive jaundice (OJ). AIM: To investigate whether YCHD can attenuate OJ-induced liver injury and hepatocyte apoptosis by inhibiting the PERK-CCAAT/enhancer-binding protein homologous protein (CHOP)-growth arrest and DNA damage-inducible protein 34 (GADD34) pathway and B cell lymphoma/leukemia-2 related X protein (Bax)/B cell lymphoma/leukemia-2 (Bcl-2) ratio. METHODS: For in vivo experiments, 30 rats were divided into three groups: control group, OJ model group, and YCHD-treated group. Blood was collected to detect the indicators of liver function, and liver tissues were used for histological analysis. For in vitro experiments, 30 rats were divided into three groups: G1, G2, and G3. The rats in group G1 had their bile duct exposed without ligation, the rats in group G2 underwent total bile duct ligation, and the rats in group G3 were given a gavage of YCHD. According to the serum pharmacology, serum was extracted and centrifuged from the rat blood to cultivate the BRL-3A cells. Terminal deoxynucleotidyl transferase mediated dUTP nick end-labelling (TUNEL) assay was used to detect BRL-3A hepatocyte apoptosis. Alanine aminotransferase (ALT) and aspartate transaminase (AST) levels in the medium were detected. Western blot and quantitative real-time polymerase chain reaction (qRT-PCR) analyses were used to detect protein and gene expression levels of PERK, CHOP, GADD34, Bax, and Bcl-2 in the liver tissues and BRL-3A cells. RESULTS: Biochemical assays and haematoxylin and eosin staining suggested severe liver function injury and liver tissue structure damage in the OJ model group. The TUNEL assay showed that massive BRL-3A rat hepatocyte apoptosis was induced by OJ. Elevated ALT and AST levels in the medium also demonstrated that hepatocytes could be destroyed by OJ. Western blot or qRT-PCR analyses showed that the protein and mRNA expression levels of PERK, CHOP, and GADD34 were significantly increased both in the rat liver tissue and BRL-3A rat hepatocytes by OJ. The Bax and Bcl-2 levels were increased, and the Bax/Bcl-2 ratio was also increased. When YCHD was used, the PERK, CHOP, GADD34, and Bax levels quickly decreased, while the Bcl-2 levels increased, and the Bax/Bcl-2 ratio decreased. CONCLUSION: OJ-induced liver injury and hepatocyte apoptosis are associated with the activation of the PERK-CHOP-GADD34 pathway and increased Bax/Bcl-2 ratio. YCHD can attenuate these changes.

Embryonic Chimeras with Human Pluripotent Stem Cells.

Human pluripotent stem (PS) cells can be isolated from preimplantation embryos or by reprogramming of somatic cells or germline progenitors. Human PS cells are considered the "holy grail" of regenerative medicine because they have the potential to form all cell types of the adult body. Because of their similarity to humans, nonhuman primate (NHP) PS cells are also important models for studying human biology and disease, as well as for developing therapeutic strategies and test bed for cell replacement therapy. This chapter describes adjusted methods for cultivation of PS cells from different primate species, including African green monkey, rhesus monkey, chimpanzee, and human. Supplementation of E8 medium and inhibitors of the Tankyrase and GSK3 kinases to various primate PS cell media reduce line-dependent predisposition for spontaneous differentiation in conventional PS cell cultures. We provide methods for basic characterization of primate PS cell lines, which include immunostaining for pluripotency markers such as OCT4 and TRA-1-60, as well as in vivo teratoma formation assay. We provide methods for generating alternative PS cells including region-selective primed PS cells, two different versions of naïve-like cells, and recently reported extended pluripotent stem (EPS) cells. These derivations are achieved by acclimation of conventional PS cells to target media, episomal reprogramming of somatic cells, or resetting conventional PS cells to a naïve-like state by overexpression of KLF2 and NANOG. We also provide methods for isolation of PS cells from human blastocysts. We describe how to generate interspecies primate-mouse chimeras at the blastocyst and postimplantation embryo stages. Systematic evaluation of the chimeric competency of human and primate PS cells will aid in efforts to overcome species barriers and achieve higher grade chimerism in postimplantation conceptuses that could enable organ-specific enrichment of human xenogeneic PS cell derivatives in large animals such as pigs and sheep.

Olive leaf extract counteracts epithelial to mesenchymal transition process induced by peritoneal dialysis, through the inhibition of TGFß1 signaling.

The mesothelial cells (MCs) play an important role in the morpho-functional alterations of the peritoneal membrane (PM) undergoing peritoneal dialysis (PD). MCs, through the epithelial-mesenchymal transition process (EMT), progressively acquire a myofibroblast-like phenotype, promoting peritoneal fibrosis (PF) and failure of peritoneal membrane function. Transforming growth factor ß1 (TGFß1), through canonical and non-canonical pathways, promotes the epithelial-mesenchymal transition (EMT) process leading to PF. To investigate the therapeutic potential of an olive leaf extract (OLE) on preserving peritoneal membrane function, we evaluated the effect of OLE on the TGFß1-induced EMT in mesothelial cells, Met5A, and elucidated the underlying molecular mechanisms. As assessed by changes in the expression of epithelial, mesenchymal, and fibrotic cell markers (such as E-cadherin, N-cadherin, α-SMA, fibronectin, vimentin), levels of matrix metalloproteinases (MMP2 and MMP9), and cell migration, OLE inhibited the TGFß1-induced EMT. Importantly, the beneficial effect of OLE was mediated by reduction of the TGFß1-induced activation of Smad2/3 signaling and the mitigation of extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK), and p38 mitogen-activated protein kinase (MAPK) phosphorylation. Smad/non-Smad signaling pathways, activated by TGFß1, both reduce expression of epithelial marker E-cadherin which has a crucial role in EMT initiation. Interestingly, we observed that in presence of OLE activity of the E-cadherin, promoter was increased and concomitantly OLE reduced the nuclear content of its co-repressor SNAIL. Our results suggest the potential therapeutic of OLE to counteract fibrotic process in peritoneal dialysis patients.

Polymeric ionically conductive composite matrices and electrical stimulation strategies for nerve regeneration: In vitro characterization.

Stem cell strategies and the use of electrical stimulation (ES) represent promising new frontiers for peripheral nerve regeneration. Composite matrices were fabricated by coating electrospun polycaprolactone/cellulose acetate micro-nanofibers with chitosan and ionically conductive (IC) polymers including, sulfonated polyaniline, and lignin sulfonate. These composite matrices were characterized for surface morphology, coating uniformity, ionic conductivity, and mechanical strength to explore as scaffold materials for nerve regeneration in conjunction with ES. Composite matrices measured conductivity in the range of 0.0049-0.0068 mS/m due to the uniform coating of sulfonated polymers on the micro-nanofibers. Thin films (2D) and composite fiber matrices (3D) of IC polymers seeded with human mesenchymal stem cells (hMSCs) were electrically stimulated at 0.5 V, 20 Hz for 1 h daily for 14 days to study the changes in cell viability, morphology, and expression of the neuronal-like phenotype. In vitro ES lead to changes in hMSCs' fibroblast morphology into elongated neurite-like structures with cell bodies for ES-treated and positive control growth factor-treated groups. Immunofluorescent staining revealed the presence of neuronal markers including ß3-tubulin, microtubule-associated protein 2, and nestin in response to ES. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater 107B: 1792-1805, 2019.

Development of Bispecific Antibody Derivatives for Cancer Immunotherapy.

Development of antibody-based immunotherapeutics has progressed from direct tumor-targeting, with antibodies such as rituximab, to blocking of immune checkpoints to reactivate antitumor immunity. In addition, bispecific antibodies/antibody fragments are also of great interest in cancer therapy, as these constructs have the ability to redirect immune effector cells to cancer targets and, thereby, enhance therapeutic efficacy. A number of bispecific antibody formats have been reported, with the first FDA-approved bispecific antibody being blinatumomab, a so-called bispecific T cell engager (BiTE), which redirects and potently activates T cell immune responses. Recently, we described an additional novel bispecific antibody derivative, termed RTX-CD47, which was designed to inhibit the innate immune checkpoint CD47-SIRPα only on -positive cancer cells. RTX-CD47 contains two antibody fragments in tandem and has monovalent binding specificity for CD47 and . Only upon dual binding to and CD47 RTX-CD47 blocks CD47 "Don't eat me" signaling. Here, we provide a detailed protocol for the construction and functional evaluation of such a bispecific antibody derivative.

Suppression of miR-21 and miR-155 of macrophage by cinnamaldehyde ameliorates ulcerative colitis.

Ulcerative colitis (UC) is a major form of inflammatory bowel disease which involved mucosal immune dysfunction. Cinnamaldehyde (CA) is major active compound from cinnamon, a useful traditional medicine in Asia which shows superior antibacterial and anti-inflammatory activity. In this study, we investigated the effects of CA on UC both in vivo and in vitro. We showed that CA attenuated the symptoms of DSS-induced colitis, including loss of body weights, disease activity index (DAI), shortening of the colon lengths and infiltration of inflammatory cells. Moreover, CA decreased the pro-inflammatory cytokines and NLRP3 inflammasome, miR-21 and miR-155 in colon tissues, in addition, the percentage of macrophages was reduced based on the surface marker F4/80 and IL-10 secretion in CA-treated group, suggesting that the CA ameliorate the UC via activation of macrophage. Herein, the effects of CA on macrophage cells were examined in vitro. We found that CA reduced the level of proinflammatory cytokines, such as TNF-α, IL-1ß, IL-6, in the activation of RAW264.7, human macrophage-like cells U937, and primary peritoneal macrophages. Furthermore, the suppression of NLRP3 inflammasome, miR-21 and miR-155 was also found in CA-treated LPS-stimulated RAW264.7 cells. CA also reduced the production of reactive oxygen species, the phosphorylation of AKT, mTOR and COX2 protein level in the RAW264.7. Meanwhile, data revealed that transferred miR-21 or miR-155 inhibitor suppressed levels of IL-1ß and IL-6, whereas miR-21 or miR-155 mimics increased expressions of these, and CA suppressed these expressions. Our results indicate that CA could ameliorate DSS-induced colitis through inhibition of NLRP3 inflammasome activation and miR-21 and miR-155 levels in colons and macrophage, suggesting that CA might be a potentially effective drug for UC.

Frondanol, a Nutraceutical Extract from Cucumaria frondosa, Attenuates Colonic Inflammation in a DSS-Induced Colitis Model in Mice.

Frondanol is a nutraceutical lipid extract of the intestine of the edible Atlantic sea cucumber, Cucumaria frondosa, with potent anti-inflammatory effects. In the current study, we investigated Frondanol as a putative anti-inflammatory compound in an experimental model of colonic inflammation. C57BL/6J male black mice (C57BL/6J) were given 3% dextran sodium sulfate (DSS) in drinking water for 7 days to induce colitis. The colitis group received oral Frondanol (100 mg/kg body weight/per day by gavage) and were compared with a control group and the DSS group. Disease activity index (DAI) and colon histology were scored for macroscopic and microscopic changes. Colonic tissue length, myeloperoxidase (MPO) concentration, neutrophil and macrophage marker mRNA, pro-inflammatory cytokine proteins, and their respective mRNAs were measured using ELISA and real-time RT-PCR. The tissue content of leukotriene B4 (LTB4) was also measured using ELISA. Frondanol significantly decreased the DAI and reduced the inflammation-associated changes in colon length as well as macroscopic and microscopic architecture of the colon. Changes in tissue MPO concentrations, neutrophil and macrophage mRNA expression (F4/80 and MIP-2), and pro-inflammatory cytokine content (IL-1β, IL-6 and TNF-α) both at the protein and mRNA level were significantly reduced by Frondanol. The increase in content of the pro-inflammatory mediator leukotriene B4 (LTB4) induced by DSS was also significantly inhibited by Frondanol. It was thus found that Frondanol supplementation attenuates colon inflammation through its potent anti-inflammatory activity.

A membrane-type surfactant protein D (SP-D) suppresses macrophage-mediated cytotoxicity in swine endothelial cells.

OBJECTIVE: Surfactant protein D (SP-D), which is secreted mainly in the lung, is an oligometric C type lectin that promotes phagocytosis by binding to carbohydrates on microbial surfaces. SP-D can also bind SIRPα, leading to a decrease in cytokine production by monocytes/macrophages. In the present study, we examined the possibility that SP-D suppresses macrophage-mediated xenogeneic cytotoxicity, by creating a membrane-type SP-D. METHODS: The cDNA for the carbohydrate recognition domain (CRD) of human SP-D was switched to that of a membrane-type protein, collectin placenta 1 (CL-P1), with a Flag-tag. The cDNA of CD47 was prepared as a control. The suppressive function of the membrane-type protein of the hybrid molecule, CL-SP-D, to monocytes/macrophages was then studied and the results compared with that for CD47. RESULTS: The expression of Flag-tagged CL-SP-D on the transfected SECs and the SIRPα on monocyte-like cells, THP-1 cells, was confirmed by FACS using anti-Flag Ab and anti-CD172a, respectively. The molecular size of the hybrid protein was next assessed by western blot. While significant cytotoxicity against SEC was induced in differentiated THP-1 cells, CL-SP-D significantly reduced THP-1-mediated cytotoxicity. In addition, phosphorylated SHP-1 was clearly detected in SEC/CL-SP-D in western blots. Moreover, IL-10 production was upregulated and IL-1ß production was suppressed in the case of THP-1 and SEC/CL-SP-D, compared with naïve SEC. Next, the cytotoxicity caused by the in vitro generated macrophage was assessed under the same conditions as were used for THP-1. CL-SP-D also showed the significant down-regulation on the macrophage. In addition, changes in IL-10 production by the macrophage confirmed the results. CONCLUSIONS: These findings indicate that the membrane-type SP-D serve as an effective therapeutic strategy for inhibiting macrophage-mediated xenograft rejection in xenotransplantation.

Elevation of tumour markers TGF-ß, M2-PK, OV-6 and AFP in hepatocellular carcinoma (HCC)-induced rats and their suppression by microalgae Chlorella vulgaris.

BACKGROUND: Chlorella vulgaris (ChV), a unicellular green algae has been reported to have anticancer and antioxidant effects. The aim of this study was to determine the chemopreventive effect of ChV on liver cancer induced rats by determining the level and expression of several liver tumour markers. METHODS: Male Wistar rats (200-250 g) were divided into 4 groups according to the diet given: control group (normal diet), ChV group with three different doses (50, 150 and 300 mg/kg body weight), liver cancer- induced group (choline deficient diet + 0.1% ethionine in drinking water or CDE group), and the treatment group (CDE group treated with three different doses of ChV). Rats were killed at 0, 4, 8 and 12 weeks of experiment and blood and tissue samples were taken from all groups for the determination of tumour markers expression alpha-fetoprotein (AFP), transforming growth factor-ß (TGF-ß), M2-pyruvate kinase (M2-PK) and specific antigen for oval cells (OV-6). RESULTS: Serum level of TGF-ß increased significantly (p < 0.05) in CDE rats. However, ChV at all doses managed to decrease (p < 0.05) its levels to control values. Expressions of liver tumour markers AFP, TGF-ß, M2-PK and OV-6 were significantly higher (p < 0.05) in tissues of CDE rats when compared to control showing an increased number of cancer cells during hepatocarcinogenesis. ChV at all doses reduced their expressions significantly (p < 0.05). CONCLUSIONS: Chlorella vulgaris has chemopreventive effect by downregulating the expression of tumour markers M2-PK, OV-6, AFP and TGF-ß, in HCC-induced rats.

Specific suppression of microgliosis cannot circumvent the severe neuropathology in peroxisomal ß-oxidation-deficient mice.

An important hallmark of various neurodegenerative disorders is the proliferation and activation of microglial cells, the resident immune cells of the central nervous system (CNS). Mice that lack multifunctional protein-2 (MFP2), the key enzyme in peroxisomal ß-oxidation, develop excessive microgliosis that positively correlates with behavioral deficits whereas no neuronal loss occurs. However, the precise contribution of neuroinflammation to the fatal neuropathology of MFP2 deficiency remains largely unknown. Here, we first attempted to suppress the inflammatory response by administering various anti-inflammatory drugs but they failed to reduce microgliosis. Subsequently, Mfp2-/- mice were treated with the selective colony-stimulating factor 1 receptor (CSF1R) inhibitor PLX5622 as microglial proliferation and survival is dependent on CSF1R signaling. This resulted in the elimination of >95% of microglia from control mice but only 70% of the expanded microglial population from Mfp2-/- mice. Despite microglial diminution in Mfp2-/- brain, inflammatory markers remained unaltered and residual microglia persisted in a reactive state. CSF1R inhibition did not prevent neuronal dysfunction, cognitive decline and clinical deterioration of Mfp2-/- mice. Collectively, the unaltered inflammatory profile despite suppressed microgliosis concurrent with persevering clinical decline strengthens our hypothesis that neuroinflammation importantly contributes to the Mfp2-/- phenotype.

Frying oils with high natural or added antioxidants content, which protect against postprandial oxidative stress, also protect against DNA oxidation damage.

PURPOSE: Using sunflower oil as frying oil increases postprandial oxidative stress, which is considered the main endogenous source of DNA oxidative damage. We aimed to test whether the protective effect of virgin olive oil and oil models with added antioxidants against postprandial oxidative stress may also protect against DNA oxidative damage. METHODS: Twenty obese people received four breakfasts following a randomized crossover design consisting of different oils [virgin olive oil (VOO), sunflower oil (SFO), and a mixed seed oil (SFO/canola oil) with added dimethylpolysiloxane (SOX) or natural antioxidants from olives (SOP)], which were subjected to 20 heating cycles. RESULTS: We observed the postprandial increase in the mRNA levels of p53, OGG1, POLB, and GADD45b after the intake of the breakfast prepared with SFO and SOX, and an increase in the expression of MDM2, APEX1, and XPC after the intake of the breakfast prepared with SFO, whereas no significant changes at the postprandial state were observed after the intake of the other breakfasts (all p values <0.05). We observed lower 8-OHdG postprandial levels after the intake of the breakfast prepared with VOO and SOP than after the intake of the breakfast prepared with SFO and SOX (all p values <0.05). CONCLUSIONS: Our results support the beneficial effect on DNA oxidation damage of virgin olive oil and the oil models with added antioxidants, as compared to the detrimental use of sunflower oil, which induces p53-dependent DNA repair pathway activation.

Lifelong quercetin enrichment and cardioprotection in Mdx/Utrn+/- mice.

Duchenne Muscular Dystrophy (DMD) is associated with progressive cardiac pathology; however, the SIRT1/PGC1-α activator quercetin may cardioprotect dystrophic hearts. We tested the extent to which long-term 0.2% dietary quercetin enrichment attenuates dystrophic cardiopathology in Mdx/Utrn+/- mice. At 2 mo, Mdx/Utrn+/- mice were fed quercetin-enriched (Mdx/Utrn+/--Q) or control diet (Mdx/Utrn+/-) for 8 mo. Control C57BL/10 (C57) animals were fed a control diet for 10 mo. Cardiac function was quantified by MRI at 2 and 10 mo. Spontaneous physical activity was quantified during the last week of treatment. At 10 mo hearts were excised for histological and biochemical analysis. Quercetin feeding improved various physiological indexes of cardiac function in diseased animals. Mdx/Utrn+/--Q also engaged in more high-intensity physical activity than controls. Histological analyses of heart tissues revealed higher expression and colocalization of utrophin and α-sarcoglycan. Lower abundance of fibronectin, cardiac damage (Hematoxylin Eosin-Y), and MMP9 were observed in quercetin-fed vs. control Mdx/Utrn+/- mice. Quercetin evoked higher protein abundance of PGC-1α, cytochrome c, ETC complexes I-V, citrate synthase, SOD2, and GPX compared with control-fed Mdx/Utrn+/- Quercetin decreased abundance of inflammatory markers including NFκB, TGF-ß1, and F4/80 compared with Mdx/Utrn+/-; however, P-NFκB, P-IKBα, IKBα, CD64, and COX2 were similar between groups. Dietary quercetin enrichment improves cardiac function in aged Mdx/Utrn+/- mice and increases mitochondrial protein content and dystrophin glycoprotein complex formation. Histological analyses indicate a marked attenuation in pathological cardiac remodeling and indicate that long-term quercetin consumption benefits the dystrophic heart. NEW & NOTEWORTHY: The current investigation provides first-time evidence that quercetin provides physiological cardioprotection against dystrophic pathology and is associated with improved spontaneous physical activity. Secondary findings suggest that quercetin-dependent outcomes are in part due to PGC-1α pathway activation.

Nogo receptor complex expression dynamics in the inflammatory foci of central nervous system experimental autoimmune demyelination.

BACKGROUND: Nogo-A and its putative receptor NgR are considered to be among the inhibitors of axonal regeneration in the CNS. However, few studies so far have addressed the issue of local NgR complex multilateral localization within inflammation in an MS mouse model of autoimmune demyelination. METHODS: Chronic experimental autoimmune encephalomyelitis (EAE) was induced in C57BL/6 mice. Analyses were performed on acute (days 18-22) and chronic (day 50) time points and compared to controls. The temporal and spatial expression of the Nogo receptor complex (NgR and coreceptors) was studied at the spinal cord using epifluorescent and confocal microscopy or real-time PCR. Data are expressed as cells/mm2, as mean % ± SEM, or as arbitrary units of integrated density. RESULTS: Animals developed a moderate to severe EAE without mortality, followed by a progressive, chronic clinical course. NgR complex spatial expression varied during the main time points of EAE. NgR with coreceptors LINGO-1 and TROY was increased in the spinal cord in the acute phase whereas LINGO-1 and p75 signal seemed to be dominant in the chronic phase, respectively. NgR was detected on gray matter NeuN+ neurons of the spinal cord, within the white matter inflammatory foci (14.2 ± 4.3 % NgR+ inflammatory cells), and found to be colocalized with GAP-43+ axonal growth cones while no ß-TubIII+, SMI-32+, or APP+ axons were found as NgR+. Among the NgR+ inflammatory cells, 75.6 ± 9.0 % were microglial/macrophages (lectin+), 49.6 ± 14.2 % expressed CD68 (phagocytic ED1+ cells), and no cells were Mac-3+. Of these macrophages/monocytes, only Arginase-1+/NgR+ but not iNOS+/NgR+ were present in lesions both in acute and chronic phases. CONCLUSIONS: Our data describe in detail the expression of the Nogo receptor complex within the autoimmune inflammatory foci and suggest a possible immune action for NgR apart from the established inhibitory one on axonal growth. Its expression by inflammatory macrophages/monocytes could signify a possible role of these cells on axonal guidance and clearance of the lesioned area during inflammatory demyelination.

Epigenetics of Epileptogenesis-Evoked Upregulation of Matrix Metalloproteinase-9 in Hippocampus.

Enhanced levels of Matrix Metalloproteinase-9 (MMP-9) have been implicated in the pathogenesis of epilepsy in humans and rodents. Lack of Mmp-9 impoverishes, whereas excess of Mmp-9 facilitates epileptogenesis. Epigenetic mechanisms driving the epileptogenesis-related upregulation of MMP-9 expression are virtually unknown. The aim of this study was to reveal these mechanisms. We analyzed hippocampi extracted from adult and pediatric patients with temporal lobe epilepsy as well as from partially and fully pentylenetetrazole kindled rats. We used a unique approach to the analysis of the kindling model results (inclusion in the analysis of rats being during kindling, and not only a group of fully kindled animals), which allowed us to separate the molecular effects exerted by the epileptogenesis from those related to epilepsy and epileptic activity. Consequently, it allowed for a disclosure of molecular mechanisms underlying causes, and not consequences, of epilepsy. Our data show that the epileptogenesis-evoked upregulation of Mmp-9 expression is regulated by removal from Mmp-9 gene proximal promoter of the two, interweaved potent silencing mechanisms-DNA methylation and Polycomb Repressive Complex 2 (PRC2)-related repression. Demethylation depends on a gradual dissociation of the DNA methyltransferases, Dnmt3a and Dnmt3b, and on progressive association of the DNA demethylation promoting protein Gadd45ß to Mmp-9 proximal gene promoter in vivo. The PRC2-related mechanism relies on dissociation of the repressive transcription factor YY1 and the dissipation of the PRC2-evoked trimethylation on Lys27 of the histone H3 from the proximal Mmp-9 promoter chromatin in vivo. Moreover, we show that the DNA hydroxymethylation, a new epigenetic DNA modification, which is localized predominantly in the gene promoters and is particularly abundant in the brain, is not involved in a regulation of MMP-9 expression during the epileptogenesis in the rat hippocampus as well as in the hippocampi of pediatric and adult epileptic patients. Additionally, we have also found that despite of its transient nature, the histone modification H3S10ph is strongly and gradually accumulated during epileptogenesis in the cell nuclei and in the proximal Mmp-9 gene promoter in the hippocampus, which suggests that H3S10ph can be involved in DNA demethylation in mammals, and not only in Neurospora. The study identifies MMP-9 as the first protein coding gene which expression is regulated by DNA methylation in human epilepsy. We present a detailed epigenetic model of the epileptogenesis-evoked upregulation of MMP-9 expression in the hippocampus. To our knowledge, it is the most complex and most detailed mechanism of epigenetic regulation of gene expression ever revealed for a particular gene in epileptogenesis. Our results also suggest for the first time that dysregulation of DNA methylation found in epilepsy is a cause rather than a consequence of this condition.

Sex differences in Gadd45b expression and methylation in the developing rodent amygdala.

Precise spatiotemporal epigenetic regulation of the genome facilitates species-typical development; sexual differentiation of the brain by gonadal hormones and sex chromosomes causes extensive epigenetic reprogramming of many cells in the body, including the brain, and may indirectly predispose males and females to different psychiatric conditions. We and others have demonstrated sex differences in DNA methylation, as well as in the enzymes that form, or 'write', this epigenetic modification. However, while a growing body of evidence suggests that DNA methylation undergoes rapid turnover and is dynamically regulated in vivo, to our knowledge no studies have been done investigating whether sex differences exist in the epigenetic 'erasers' during postnatal development. Here we report sex differences in the expression of growth arrest and DNA damage inducible factor ß (Gadd45b), but not family members α (a) or γ (g), in the neonatal and juvenile rodent amygdala.

A superfusion apparatus for ex vivo human eye irritation investigations.

A superfusion apparatus (SA) was developed to maintain isolated human corneas ex vivo under conditions which mimic the natural eye environment in vivo, including controlled temperature, tear flow and intraocular pressure. The SA was designed, developed and tested for use in ophthalmic pre-clinical research and to test new pharmaceutical formulations. Corneas undergo an equilibration process in the new physiological environment for one day. The test was then initiated by the application of the test substance, incubation, and temporal assessment of corneal damage using various parameters. The effects of mild and severe irritant concentrations of NaOH (2% and 8%, respectively) on corneal opacity, swelling and epithelial integrity were studied, and the inflammatory status assessed using F4/80 and MPO as macrophages and neutrophils markers, respectively. The SA was then used to test new artificial tear formulations supplemented with silver ions as an active constituent, showing different degrees of inflammatory responses as indicated by the migration of MPO and F4/80 positive cells towards the epithelium. The human cornea superfusion apparatus was proposed as a model for acute eye irritation research.