BrdU Uptake Research Articles

Therapeutic Senolysis of Axitinib-Induced Senescent Human Lung Cancer Cells.

Tyrosine kinase inhibitors (TKIs) inhibit receptor-mediated signals in cells. Axitinib is a TKI with high specificity for vascular endothelial growth factor receptors (VEGFRs). We determined whether axitinib could induce senescence in human cancer cells and be lysed by the senolytic drug ABT-263. Human lung and breast adenocarcinoma cell lines were used. These cells were cultured with axitinib or a multi-target TKI lenvatinib. The expression of β-galactosidase, VEGFRs, Ki-67, reactive oxygen species (ROS) of cancer cells, and their BrdU uptake were evaluated by flow cytometry. The mRNA expression of p21 and IL-8 was examined by quantitative PCR. The effects of TKIs on phosphorylation of Akt and Erk1/2, as downstream molecules of VEGFR signaling, were examined by immunoblot. The in vivo anti-cancer effect was examined using a xenograft mice model. Axitinib, but not lenvatinib, induced cellular senescence (increased cell size and enhanced expression of β-galactosidase) in all adenocarcinoma cell lines. Axitinib-induced senescence was unrelated to the expression of VEGFRs on cancer cells. ROS were involved in axitinib-induced senescence. Axitinib-induced senescent lung adenocarcinoma A549 cells were drastically lysed by ABT-263. In A549-xenografted mice, combination therapy with axitinib and ABT-263 significantly suppressed tumor growth with the induction of apoptotic cancer cells.

Majority of human circulating IgG plasmablasts stop blasting in a cell-free pro-survival culture

Following infection or vaccination, early-minted antibody secreting cells (ASC) or plasmablasts appear in circulation transiently, and a small fraction migrates to the spleen or bone marrow (BM) to mature into long-lived plasma cells (LLPC). While LLPC, by definition, are quiescent or non-dividing, the majority of blood ASC are thought to be “blasting” or proliferative. In this study, we find > 95% nascent blood ASC in culture express Ki-67 but only 6–12% incorporate BrdU after 4 h or 24 h labeling. In contrast, < 5% BM LLPC in culture are Ki-67+ with no BrdU uptake. Due to limitations of traditional flow cytometry, we utilized a novel optofluidic technology to evaluate cell division with simultaneous functional IgG secretion. We find 11% early-minted blood ASC undergo division, and none of the terminally differentiated BM LLPC (CD19−CD38hiCD138+) divide during the 7–21 days in culture. While BM LLPC undergo complete cell cycle arrest, the process of differentiation into an ASC or plasmablasts also discourages entry into S phase. Since the majority of Ki-67+ nascent blood ASC have exited cell cycle and are no longer actively “blasting”, the term “plasmablast”, which traditionally refers to an ASC that still has the capacity to divide, may probably be a misnomer.

Hellebrigenin triggers death of promyelocytic leukemia cells by non-genotoxic ways

Bufadienolides are digitalis-like aglycones mainly found in skin secretions of toads. Among their biological properties, the mechanisms of antiproliferative action on tumor cells remain unclear for many compounds, including against leukemia cells. Herein, it was evaluated the mechanisms involved in the antiproliferative and genotoxic actions of hellebrigenin on tumor cell lines and in silico capacity to inhibit the human topoisomerase IIa enzyme. Firstly, its cytotoxic action was investigated by colorimetric assays in human tumor and peripheral blood mononuclear cells (PBMC). Next, biochemical and morphological studies were detailed by light microscopy (trypan blue dye exclusion), immunocytochemistry (BrdU uptake), flow cytometry and DNA/chromosomal damages (Cometa and aberrations). Finally, computational modelling was used to search for topoisomerase inhibition. Hellebrigenin reduced proliferation, BrdU incorporation, viability, and membrane integrity of HL-60 leukemia cells. Additionally, it increased G2/M arrest, internucleosomal DNA fragmentation, mitochondrial depolarization, and phosphatidylserine externalization in a concentration-dependent manner. In contrast to doxorubicin, hellebrigenin did not cause DNA strand breaks in HL-60 cell line and lymphocytes, and it interacts with ATPase domain residues of human topoisomerase IIa, generating a complex of hydrophobic and van der Waals interactions and hydrogen bonds. So, hellebrigenin presented potent anti-leukemic activity at concentrations as low as 0.06 μM, a value comparable to the clinical anticancer agent doxorubicin, and caused biochemical changes suggestive of apoptosis without genotoxic/clastogenic-related action, but it probably triggers catalytic inhibition of topoisomerase II. These findings also emphasize toad steroid toxins as promising lead antineoplasic compounds with relatively low cytotoxic action on human normal cells.

Suppression of tumour growth from transplanted astrocytoma cells transfected with luciferase in mice by bioluminescence mediated, systemic, photodynamic therapy

BackgroundGrade 4 astrocytomas are usually incurable due to their diffusely infiltrative nature. Photodynamic therapy (PDT) is a promising therapeutic option, but external light delivery is impractical when cancer cells infiltrate unknown areas of normal brain. Hence the search for endogenous sources to generate light at cancer cells. In vitro, astrocytoma cells, transfected with firefly luciferase, can be killed by bioluminescence-mediated PDT (bPDT). This study asks if bPDT can suppress tumour growth In vivo, when all components of treatment are administered systemically. MethodsTransfected astrocytoma cells were injected subcutaneously or intra-cranially in athymic CD1 nu/nu mice. bPDT required ip bolus of mTHPC (photosensitiser) and delivery of the d-luciferin substrate over 7 days via an implanted osmotic pump. Control animals had no treatment, photosensitiser only or d-luciferin only. For subcutaneous tumours, size and BLI (light emitted after d-luciferin bolus) were measured before and every 2 days after PDT. For intracranial tumours, monitoring was weekly BLI. ResultsFor subcutaneous tumours, there was significant suppression of the tumour growth rate (P<0.05), and absolute tumour size (P<0.01) after bPDT. Proliferation of subcutaneous and intracranial tumours (monitored by BrdU uptake) was significantly reduced in treated mice. (P<0.001) ConclusionsThis study reports bPDT suppression of tumour growth from luciferase transfected astrocytoma cells with all components of treatment given systemically, as required for effective management of recurrent astrocytomas in unknown sites. However, research on systemic bPDT is needed to establish whether effects on non-transfected tumours can be achieved without any unacceptable effects on normal tissues.

Perfluorooctanoic acid (PFOA) affects steroidogenesis and antioxidant defence in granulosa cells from swine ovary

PFOA is mainly employed in products with water and oil repellent properties. Due to its persistence, bioaccumulation and critical effects on health, its use has been restricted in several countries. This research was intended to explore PFOA action on the main functions of swine ovarian granulosa cells, a valuable model for translational medicine. Moreover, since we previously demonstrated a disruptive effect on free radical generation we sought to explore PFOA effects on the main antioxidant enzymes. PFOA inhibited cell proliferation (p < 0.001), assessed by BrdU uptake. Steroidogenesis was disrupted: PFOA also stimulated 17β-estradiol production (p < 0.05), increased progesterone production (p < 0.05) at the lowest dose while it displayed an inhibitory effect at higher concentrations (p < 0.05). SOD (p < 0.001), catalase (p < 0.05) and peroxidase (p < 0.01) activities were stimulated. Therefore, our study supports a disruptive effect of PFOA in cultured swine granulosa cells.

SPOCK1 Overexpression Suggests Poor Prognosis of Ovarian Cancer

Sparc/osteonectin, cwcv, and kazal-like domains proteoglycan 1 (SPOCK1) has been found in a variety of malignant tumors and is associated with a poor prognosis. We aimed to explore the role of SPOCK1 in ovarian cancer. Ovarian cancer cell lines SKOV3 and SW626 were transfected with SPOCK1 overexpressing or empty vector using electroporation. Cells were studied by immunostaining and an automated Western blotting system. BrdU uptake and wound healing assays assessed cell proliferation and migration. SPOCK1 expression in human ovarian cancer tissues and in blood samples were studied by immunostaining and ELISA. Survival of patients with tumors exhibiting low and high SPOCK1 expression was analyzed using online tools. Both transfected cell lines synthesized different SPOCK1 variants; SKOV3 cells also secreted the proteoglycan. SPOCK1 overexpression stimulated DNA synthesis and cell migration involving p21CIP1. Ovarian cancer patients had increased SPOCK1 serum levels compared to healthy controls. Tumor cells of tissues also displayed abundant SPOCK1. Moreover, SPOCK1 levels were higher in untreated ovarian cancer serum and tissue samples and lower in recipients of chemotherapy. According to in silico analyses, high SPOCK1 expression was correlated with shorter survival. Our findings suggest SPOCK1 may be a viable anti-tumor therapeutic target and could be used for monitoring ovarian cancer.

Effects of the Myokine Irisin on Stromal Cells from Swine Adipose Tissue.

Irisin is a hormone able to reproduce some of the positive effects of physical activity and diet. Recently, we demonstrated the presence of Irisin at the ovarian level as a potential physiological regulator of follicular function. Adipose tissue is crucial for reproductive function through its metabolic activity and the production of adipokines. At present, the exact nature of adipocyte precursors is still under debate, but an important role has been assigned to the population of adipose tissue mesenchymal stromal cells (ASCs) of perivascular origin. It should be noted that, when appropriately stimulated, ASCs can differentiate into preadipocytes and, subsequently, adipocytes. Therefore, this present study was undertaken to explore the potential effect of Irisin on ASCs, known for their high differentiative potential. Since Irisin expression in ASCs was confirmed by PCR, we tested its potential effects on the main functional activities of these cells, including proliferation (BrdU uptake); metabolic activity (ATP production); redox status, evaluated as the generation of free molecules such as superoxide anion and nitric oxide; and scavenger activities, assessed as both enzymatic (superoxide dismutase) and non-enzymatic antioxidant power. Moreover, we tested the effect of Irisin on ASCs adipogenic differentiation. BrdU uptake was significantly (p &lt; 0.001) inhibited by Irisin, while ATP production was significantly (p &lt; 0.05) increased. Both superoxide anion and nitric oxide generation were significantly increased (p &lt; 0.001) by Irisin, while scavenger activity was significantly reduced (p &lt; 0.05). Irisin was found to significantly (p &lt; 0.05) inhibit ASCs adipogenic differentiation. Taken together, the present results suggest a potential local role of Irisin in the regulation of adipose tissue function.

Cathepsin F is a potential marker for senescent human skin fibroblasts and keratinocytes associated with skin aging

Cellular senescence is characterized by cell cycle arrest and the senescence-associated secretory phenotype (SASP) and can be triggered by a variety of stimuli, including deoxyribonucleic acid (DNA) damage, oxidative stress, and telomere exhaustion. Cellular senescence is associated with skin aging, and identification of specific markers of senescent cells is essential for development of targeted therapies. Cathepsin F (CTSF) has been implicated in dermatitis and various cancers and participates in cell immortalization through its association with Bcl family proteins. It is a candidate therapeutic target to specifically label and eliminate human skin fibroblasts and keratinocytes immortalized by aging and achieve skin rejuvenation. In this study, we investigated whether CTSF is associated with senescence in human fibroblasts and keratinocytes. In senescence models, created using replicative aging, ionizing radiation exposure, and the anticancer drug doxorubicin, various senescence markers were observed, such as senescence-associated β-galactosidase (SA-β-gal) activity, increased SASP gene expression, and decreased uptake of the proliferation marker BrdU. Furthermore, CTSF expression was elevated at the gene and protein levels. In addition, CTSF-positive cells were abundant in aged human epidermis and in some parts of the dermis. In the population of senescent cells with arrested division, the number of CTSF-positive cells was significantly higher than that in the proliferating cell population. These results suggest that CTSF is a candidate for therapeutic modalities targeting aging fibroblasts and keratinocytes.

Improving the metabolic efficiency of human T cells through AMPK agonist treatment

Abstract BACKGROUND Adoptive immunotherapies are limited by the increased differentiation and metabolic exhaustion of the T cell product. The cellular energy sensor AMP-activated protein kinase (AMPK) controls many metabolic pathways. We hypothesized that increasing AMPK signaling during T cell expansion would create more metabolically efficient T cells. RESULTS Human T cells were stimulated in vitro, expanded with or without AMPK agonist A769662, and restimulated under varying glucose concentrations. Agonist treatment increased T cell expansion by manual cell counts (1.5× increase in cell number, p&amp;lt;0.01), and produced a higher percentage of CD62L+CD27+ cells by flow cytometry (10% increase, p&amp;lt;0.01). Twenty-four hours after re-stimulation, agonist-treated cells exhibited decreased basal oxygen consumption and extracellular acidification rates (45% and 20% decrease, p&amp;lt;0.001) but increased spare respiratory capacity (SRC) (97% increase, p&amp;lt;0.001). At 72 hours, control cells decreased proliferation in a glucose dose-dependent manner, as measured by BrDU uptake, whereas treated cells maintained a higher level of proliferation despite limited glucose availability. DISCUSSION Increasing AMPK signaling during T cell expansion improves doubling time while maintaining higher CD27 positivity, suggesting a greater yield of less differentiated cells. Upon re-stimulation, treated cells initiate less metabolic activity but maintain greater proliferation and SRC under glucose-limiting conditions. These results suggest that greater metabolic efficiency in agonist-treated cells allows for continued function despite nutrient restriction, a trait which could confer significant advantages to adoptive immunotherapies. Supported by grants from NIH (5K12 HD052892) and the St. Baldrick's Foundation (St. Baldrick's Fellowship Grant)

Effect of two homeopathic remedies at different degrees of dilutions on the wound closure of 3T3 fibroblasts in in vitro scratch assay

Background: Since ancient times, preparations from traditional medicinal plants e.g. Arnica montana, Calendula officinalis or Hypericum perforatum have been used for different wound healing purposes. The aim of this study was to investigate the efficacy of the commercial low dilution homeopathic remedy Similasan® Arnica plus Spray, a preparation of Arnica montana 4x, Calendula officinalis 4x, Hypericum perforatum 4x and Symphytum officinale 6x (0712-2) and medium diluted SIM WuS (Petroleum 15x, Arnica montana 15x, Calcium fluoratum 12x, Calendula officinalis 12x, Hepar sulfuris 12x and Mercurius solubilis 15x; 1101-4), on the wound healing in cultured NIH 3T3 fibroblasts. Both remedies were from Similasan AG (Jonen, Switzerland) and prepared according the German Homoeopathic Pharmacopoeia (GHP) following descriptions 4a for arnica, 3a for marigold and St. John’s wort, 2a for comfrey, 5a for petroleum, and 6 for calcium fluoride, hepar sulfuris and mercurius solubilis.&#x0D; Materials and Methods: Cell proliferation, migration and wound closure promoting effect of the preparations (0712-2, 1101- 4) and their succussed solvents (0712-1, 1101-3) were investigated on mouse NIH 3T3 fibroblasts. Cell viability was determined by WST-1 assay, cell growth using BrdU uptake, cell migration by chemotaxis assay and wound closure by CytoSelect ™Wound Healing Assay Kit which generated a defined wound area. All assays were performed in three independent controlled experiments. In some experiments diluted unsuccussed alcohol (0712-3) was also investigated.&#x0D; Results: Preparations (0712-1), (0712-2), (0712-3), (1101-3) and (1101-4) were investigated at decimal dilution steps from 1x to 4x. Cell viabilty was not affected by any of the substances and (0712-1) and (0712-2) showed no stimulating effect on cell proliferation. Preparation (0712-2) exerted a stimulating effect on fibroblast migration (31.7%) vs 15% with succussed solvent (0712-1) at 1:100 dilutions (p0.05). Positive control 2 ng/ml EGF increased migratory activity of cells by 49.8%. Preparation (0712-2) at a dilution of 1:100 promoted in vitro wound closure by 59.5% and differed significantly (p

Phospholipase Cgamma Pathway-Dependent mTOR Signaling Controls IL-7-Mediated Early B Cell Development

Early B cell lymphopoiesis is intricately regulated by signals from various receptors including the pre-B cell receptor (pre-BCR) and the interleukin-7 receptor (IL-7R), but the molecular mechanism underlying the regulation is not fully understood. The Phospholipase Cγ (PLCγ) signaling pathway plays a critical role in antigen receptor-mediated activation of mature B cells, but the role of this pathway in cytokine function and early B cell development is completely unknown. Here we generated inducible PLCγ1/PLCγ2 double-deficient mice and found that the mutant mice exhibited a complete blockage of early B cell development at the pre-pro-B cell stage, similar to IL-7R-deficient but not pre-BCR-deficient mice. Of note, PLCγ1/PLCγ2 double-deficient mice had normal numbers of the HSC-containing Lin-c-Kit+Sca1+ (LSK) cells, myeloid progenitor-containing Lin-c-Kit+Sca1- (LK) cells and myeloid lineage cells compared to wild-type mice. PLCγ1/PLCγ2 double-deficient mice displayed a marked reduction in IL-7-induced rearrangement of the distal VHJ558 family gene segments. In addition, PLCγ1/PLCγ2 double deficiency altered the expression of lymphoid-lineage-affiliated genes in common lymphoid progenitors (CLPs) and the expression of B cell lineage-affiliated genes, IL-7 signaling pathway-regulated genes and cell cycle pathway-related genes in B cell progenitors. Further, PLCγ1/PLCγ2 double-deficient Lin- BM progenitors co-cultured with OP-9 stromal cells plus IL-7 failed to give rise to B cells whereas wild-type control BM progenitors generated B cells. PLCγ1/PLCγ2 double-deficient B progenitors exhibited a decrease in the level of IL-7-induced BrdU uptake compared to the corresponding wild-type control cells. Biochemically, the PLCγ pathway was activated upon IL-7 engagement in Rag1-deficient pro-B cells that lack the pre-BCR. PLCγ1/PLCγ2-double deficiency or inhibition of PLCγ pathway by PLC inhibitors impaired IL-7-induced activation of mTORC1 but not Stat5. Consistently, inhibition of the PLCγ pathway impaired IL-7-mediated metabolism of B cell progenitors. It's known that PLCγ activates the DAG/PKC-dependent and IP3/Ca2+-dependent signaling pathways, the two major downstream signaling cascades. We found that inhibition of the PKC-dependent, but not the Ca2+-dependent, pathway impaired IL-7-mediated B cell production and mTORC1 but not Stat5 activation. In addition, the PKC agonist PDBu induced mTORC1 activation in wild-type B cell progenitors, and stimulated 3H-thymidine incorporation in PLCγ1/PLCγ2 double-deficient B cell progenitors to the same extent as it did in wild-type control cells. Moreover, disruption of conventional Akt/TSC/Rheb signaling axis did not affect IL-7-controlled mTORC1 activation or early B cell development. Taken together, our current study for the first time demonstrates that the PLCγ pathway is critical for cytokine-mediated biological function, such as IL-7R-regulated early B lymphopoiesis, and this pathway directs IL-7R-mediated early B cell development through a novel and thitherto-unknown PLCγ/DAG/PKC-dependent but not conventional Akt/Rheb-dependent activation of mTORC1. DisclosuresNo relevant conflicts of interest to declare.

Tooth Removal in the Leopard Gecko and the de Novo Formation of Replacement Teeth

Introduction and Objectives: The ability to replace teeth throughout life is a trait found in most vertebrates and was present in ancient mammals. Modern mammals including humans can only replace their teeth once and much effort is currently being devoted to uncovering stem cells to be used in tooth regeneration. Work from our lab and others has identified some of the differences in the reptilian dentition that could underlie the basis of polyphyodonty. In lizards, the dental lamina, a source of putative dental epithelial progenitor cells, persists until adulthood. In contrast, in humans there are very few remnants of the dental lamina remaining once the permanent dentition is complete. In this project we use the leopard gecko (Eublepharis macularius) and surgical treatments to trace the contribution of label-retaining cells to the next generation of teeth. We hypothesized that premature removal of unerupted second generation teeth will stimulate populations of quiescent label-retaining cells to proliferate. Then by following the labeled cells for several months we will identify progeny of the epithelial progenitor cells in newly formed teeth. Materials and Methods: Second generation, unerupted teeth were removed from geckos under sedation. A pulse of BrdU was administered over 3 sequential days. Animals were euthanized at 1 week post surgery (immediately after the BrdU pulse or time 0) and 1 or 2 months post surgery (N = 3 animals per timepoint). Animals were processed for immunofluorescence staining with BrdU antibodies. We counted the proportion of nuclei in the dental lamina that stained positive for BrdU. Results: One week after the surgery, we identified statistically significant higher uptake of BrdU in the dental epithelium of treated as compared to the control, non-manipulated regions (P < 0.01). One month after the surgery, there was a significant decrease in BrdU label in the dental lamina (P< 0.001), decreasing to the same level as controls at time zero The newly formed tooth buds regenerating in the sites of tooth removal contained BrdU labeled cells in all parts of the enamel organ. The original label-retaining cells divided several times since the original pulse, based on the fragmented appearance of the BrdU in the nuclei. Conclusion: In our previous work, it was difficult to label the quiescent cells of the dental lamina. Here the removal of the successional teeth stimulates proliferation of the dental lamina. With a larger population of label-retaining cells that could be tracked we were able to find transit amplifying cells that divide and contribute to the enamel organ. Significance: We have developed an injury model that will allow us to test the ability of different signals to regulate the activity of stem cells. Moreover label-retaining cells are multipotent and can self-renew, thereby possessing some of the properties of progenitor cells.

Prevention of postoperative intrapericardial adhesion by dextrin hydrogel

ObjectivePostoperative intrapericardial adhesion increases the risk of complications in patients undergoing reoperation. We investigated the effect of a bioabsorbable dextrin hydrogel (DHG) on the formation of intrapericardial adhesions.MethodsIntrapericardial adhesion was surgically induced in Japanese white rabbits with DHG treatment (Adh + DHG) or without DHG treatment (Adh). The sham group was not treated with DHG and intrapericardial adhesion was not induced. The extent of intrapericardial adhesion was assessed by adhesion scoring and crystal violet staining of the pericardial cavity. Bromodeoxyuridine (BrdU) uptake assay was performed to assess the proliferative response to the injury in the tissue beneath the intrapericardial adhesion.ResultsThe Adh + DHG group showed looser intrapericardial adhesions compared to the Adh group. The adhesion area of the Adh + DHG group was 4.6 ± 2.2%, whereas that of the Adh group was 32.6 ± 6.4% at the end of the 28-day observation period (p < 0.01). The induction of intrapericardial adhesion resulted in a proliferative response mainly in the cardiac tissue just beneath the adhesion. There were 48.6 ± 10.7 cells/0.1 mm2 BrdU-positive cells in the Adh + DHG group and 135.7 ± 23.8 cells/0.1 mm2 BrdU-positive cells in the Adh group on day 28 (p < 0.05).ConclusionThese findings indicate that DHG effectively prevented intrapericardial adhesion in this model.

Glucose Stress Induces Early Onset of Metastasis in Hormone-Sensitive Breast Cancer Cells

Cell adhesion is the backbone of many events in the cancer cell life cycle, including proliferation, metastasis, migration, invasion and even cell survival. In a tumor, usually the cells in the core have high migratory potential though they constantly suffer from glucose starvation. Our study was aimed at understanding events such as attachment to the surfaces at one site and then mobility to the secondary sites during progression of cancer in the hormone sensitive breast cancer cells MCF7, following their exposure to different concentrations of glucose in the environment. We have shown that low glucose availability is detected within 3 h of shortage which is then translated into variable expression of genes for cell-to-cell adhesion such as cadherins and Ig-like cell adhesion molecules, and matrix-associated genes such as integrins and metalloproteases. We also found that low glucose concentrations induced cell adhesion, whereas higher concentrations stimulated cell migration. In addition, several regulatory molecules involved in mitochondrial metabolism, proliferation, and glucose uptake as demonstrated respectively by MTT assay, BrdU uptake, glucose uptake and pyruvate kinase activity showed varied expression during epithelial to mesenchymal transition. Cytoskeleton staining demonstrated development of lamellipodia in glucose starved medium indicating cascade of physiological and molecular events in the cells to find a more nutrient-rich environment for the development of secondary tumor. Further studies on protein markers with a 3D spheroid culturing approach are likely to expand our understanding of onset of metastasis in tumor tissues.

Psoriatic skin inflammation induces a pre-diabetic phenotype via the endocrine actions of skin secretome

ObjectivePsoriasis is a chronic inflammatory skin disease that is thought to affect ∼2% of the global population. Psoriasis has been associated with ∼30% increased risk of developing type 2 diabetes (T2D), with numerous studies reporting that psoriasis is an independent risk-factor for T2D, separate from underlying obesity. Separately, studies of skin-specific transgenic mice have reported altered whole-body glucose homeostasis in these models. These studies imply a direct role for skin inflammation and dysfunction in mediating the onset of T2D in psoriasis patients, potentially via the endocrine effects of the skin secretome on key metabolic tissues. We used a combination of in vivo and ex vivo mouse models and ex vivo human imiquimod (IMQ) models to investigate the effects of psoriasis-mediated changes in the skin secretome on whole-body metabolic function. MethodsTo induce psoriatic skin inflammation, mice were topically administered 75 mg of 5% IMQ cream (or Vaseline control) to a shaved dorsal region for 4 consecutive days. On day 5, mice were fasted for glucose and insulin tolerance testing, or sacrificed in the fed state with blood and tissues collected for analysis. To determine effects of the skin secretome, mouse skin was collected at day 5 from IMQ mice and cultured for 24 h. Conditioned media (CM) was collected and used 1:1 with fresh media to treat mouse explant subcutaneous adipose tissue (sAT) and isolated pancreatic islets. For human CM experiments, human skin was exposed to 5% IMQ cream for 20 min, ex vivo, to induce a psoriatic phenotype, then cultured for 24 h. CM was collected, combined 1:1 with fresh media and used to treat human sAT ex vivo. Markers of tissue inflammation and metabolic function were determined by qPCR. Beta cell function in isolated islets was measured by dynamic insulin secretion. Beta-cell proliferation was determined by measurement of Ki67 immunofluorescence histochemistry and BrDU uptake, whilst islet apoptosis was assessed by caspase 3/7 activity. All data is expressed as mean ± SEM. ResultsTopical treatment with IMQ induced a psoriatic-like phenotype in mouse skin, evidenced by thickening, erythema and inflammation of the skin. Topical IMQ treatment induced inflammation and signs of metabolic dysfunction in sub-cutaneous and epidydimal adipose tissue, liver, skeletal muscle and gut tissue. However, consistent with islet compensation and a pre-diabetic phenotype, IMQ mice displayed improved glucose tolerance, increased insulin and c-peptide response to glucose, and increased beta cell proliferation. Treatment of sAT with psoriatic mouse or human skin-CM replicated the in vivo phenotype, leading to increased inflammation and metabolic dysfunction in mouse and human sAT. Treatment of pancreatic islets with psoriatic mouse skin-CM induced increases in beta-proliferation and apoptosis, thus partially replicating the in vivo phenotype. ConclusionsPsoriasis-like skin inflammation induces a pre-diabetic phenotype, characterised by tissue inflammation and markers of metabolic dysfunction, together with islet compensation in mice. The in vivo phenotype is partially replicated by exposure of sAT and pancreatic islets to psoriatic-skin conditioned media. These results support the hypothesis that psoriatic skin inflammation, potentially via the endocrine actions of the skin secretome, may constitute a novel pathophysiological pathway mediating the development of T2D.

Antidepressant effect of BE360, a new selective estrogen receptor modulator, activated via CREB/BDNF, Bcl-2 signaling pathways in ovariectomized mice

We have previously reported that the carborane compound BE360, a novel selective estrogen receptor modulator, has a therapeutic potential against dementia. This study aimed to explore the effects and underlying mechanisms of BE360 on depression-like behaviors in ovariectomized (OVX) mice subjected to subchronic stress, which are postmenopausal depression models. BE360 was subcutaneously administrated using a mini-osmotic pump, for 2 weeks. Depression-like behaviors were evaluated using the forced swimming test. Neurogenesis in the hippocampal dentate gyrus (DG) was measured by analyzing cells expressing doublecortin (DCX) following 5-bromo-2’-deoxyuridine (BrdU) uptake. The levels of phosphorylated cyclic-AMP response element-binding protein (p-CREB), brain-derived neurotrophic factor (BDNF), and Bcl-2 were measured using immunohistochemistry or immunoblotting. Depression-like behaviors in OVX + Stress-exposed mice improved after chronic treatment with BE360. BE360 treatment in OVX + Stress-exposed mice increased p-CREB, BDNF, and Bcl-2 expressions in the hippocampus. Immunohistochemistry showed that the number of BrdU/DCX double-positive cells in the DG of the hippocampus, which decreased significantly in OVX + Stress-exposed mice, increased after subchronic treatment with BE360. The present study demonstrates that BE360 exerts antidepressant effects via hippocampal neurogenesis, potentially activated through CREB/BDNF, Bcl-2 signaling pathways. These results indicate that BE360 may have therapeutic potential against postmenopausal depression.

Flavonoids from Scutellaria baicalensis inhibit senescence-associated secretory phenotype production by interrupting IκBζ/C/EBPβ pathway: Inhibition of age-related inflammation

BackgroundProlonged exposure to the senescence-associated secretory phenotype (SASP) with age leads to chronic low-grade inflammation in neighboring cells and tissues, causing many chronic degenerative diseases. PurposeThe effects on SASP production of the ethanol extract from Scutellaria radix and 17 isolated flavonoid constituents were examined in vitro and in vivo. MethodsCellular senescence was induced by bleomycin. Expression of the SASP and cell signaling molecules was detected using ELISA, RT-qPCR, Western blotting, and immunofluorescence staining. To investigate the in vivo effects, 21-month-old aged rats were used. ResultsThe ethanol extract and 5 compounds including 1 (Oroxylin A; 5,7-dihydroxy-6-methoxyflavone), 5 (2′,6′,5,7-tetrahydroxy-8-methoxyflavone), 8 (2′,5,7-trihydroxyflavone), 10 (2′,5,7-trihydroxy-8-methoxyflavone) and 11 (2′,5,7-trihydroxy-6-methoxyflavone) potently reduced IL-6 and IL-8 production and gene expression of the SASP, including IL-1α, IL-1β, IL-6, IL-8, GM-CSF, CXCL1, MCP-2, and MMP-3. This finding indicates the important role of the B-ring 2′‑hydroxyl group in flavonoid molecules. Furthermore, compounds 8 and 11, the strongest SASP inhibitors, decreased the expression of IκBζ and C/EBPβ protein without affecting either BrdU uptake or the expression of senescence markers, such as pRb and p21. Finally, the oral administration of compound 8 to aged rats at 2 and 4 mg/kg/day for 10 days significantly inhibited the gene expression of SASP and IκBζ in kidneys. This is the first report of the strong SASP inhibitory action of flavonoids from Scutellaria radix on in vitro and in vivo senescence models. The inhibitory action was shown to be mediated mainly by interfering with the IκBζ/C/EBPβ signaling pathway. ConclusionTargeting production of the SASP using flavonoids from Scutellaria radix or its extract might help reduce low-grade sterile inflammation and control age-related diseases.

The human amniotic fluid stem cell secretome exerts cardio-active paracrine potential for myocardial repair and regeneration

Background & Aim Functional replacement of cardiomyocytes following injury is the sine qua non condition for heart regeneration. We previously reported that human amniotic fluid-derived stem cells (hAFS) are cardioprotective on rodent ischemic myocardium and cardiac cells exposed to cardiotoxicity. Here we investigated whether hAFS secretome (all paracrine factors released in the cell-conditioned medium, hAFS-CM, including extracellular vesicles, hAFS-EV) can trigger cardiomyocyte cell cycle re-entry. We also evaluated gestational age influence on the secretome of II trimester hAFS from leftover samples of prenatal amniocentesis (fetal hAFS) over III trimester cells from clinical waste of scheduled C-section delivery (perinatal hAFS). Methods, Results & Conclusion c-KIT+ hAFS were isolated as previously described and preconditioned by 1% O2 for 24h. A preclinical mouse model of myocardial infarction (MI) was treated with single intra-myocardial injection of fetal hAFS-CM over hAFS-EV to assess cardiac repair and cardiomyocyte cell cycle progression by bromodeoxyuridine (BrdU) uptake 4 weeks post-MI. Neonatal mouse ventricular cardiomyocytes (mNVCM) were isolated from R26pFUCCI2 transgenic pups with a nuclear probe system to fluorescently tag cell cycle stages (G1 by mCherry; S-G2-M by mVenus/GFP) and stimulated by fetal versus perinatal hAFS-CM to investigate cardiomyocyte renewal. Fetal hAFS-CM injection soon after MI counteracted scarring, supported local angiogenesis and reactivation of epicardial progenitors. Fetal hAFS-EV recapitulated all the beneficial effects of whole hAFS-CM, but for vessel formation while significantly counteracted cardiac function worsening. BrdU-positive resident cardiomyocytes were similarly increased by 2.5-fold when primed with either fetal hAFS-CM or -EV over vehicle-treated mice (p Our preliminary findings suggest that hAFS secretome can enhance cardiac repair and sustain endogenous regenerative mechanisms. Further analyses are ongoing to evaluate the effect of perinatal versus fetal hAFS-EV on myocardial renewal, so to define the most cardio-active secretome formulation.

Loss of METTL3 Mediated m6A RNA Modification Results in Double-Stranded RNA Induced Innate Immune Response and Hematopoietic Failure

Hematopoietic stem cell (HSC) self-renewal and lineage output are orchestrated by multiple regulatory layers, including RNA modifications. N6-methyladenosine (m6A) is an abundant modification found in RNAs which affects the translation and stability of modified transcripts. The effects of m6A are determined by m6A writers (install m6A), erasers (remove m6A) and readers (recognize m6A). In embryonic stem cells, deletion of the m6A writer METTL3 enforces a naïve pluripotent state. This raises the question of whether m6A RNA methylation analogously regulates stem cell self-renewal and differentiation in somatic stem cells such as HSCs. Using a Vav-Cre/Mettl3 (VCM3) hematopoietic-specific knockout mouse model, we show that loss of the RNA m6A writer METTL3 in fetal HSCs results in hematopoietic failure and perinatal lethality. At E14.5 (FL) hematopoiesis loss of Mettl3/m6A results in hematopoietic failure with expansion of Lin-Sca-1+c-Kit+ (LSK) hematopoietic stem and progenitor cells (HSPCs) that are defective in the production of progenitors and mature blood cells, as evidenced by failure to rescue lethally irradiated congenic recipient mice in transplant experiments. The relative defect I hematopoiesis was further demonstrated by competitive transplant experiments, in which transplanted KO FL were consistently out-competed by WT FL. Interestingly, BrdU/7AAD labeling reveals a significant proliferative defect with reduced BrdU uptake in VCM3 KO FL cells, and specifically in Lin-c-Kit+Sca-1- (LK) progenitor cells. RNA-seq analysis of FL LSK cells reveals that loss of m6A results in upregulation of multiple 2'-5'-oligoadenylate synthetase (OAS) family genes. Interestingly, the majority of OAS family genes are not m6A modified in several m6A sequencing data sets. We therefore hypothesized that the OAS genes might be regulated at the transcriptional level. We performed cleavage under targets and release using nuclease (CUT&amp;RUN) analysis and found that OAS family genes are transcriptionally activated, as evidenced by significant increase in H3K4 trimethylation (H3K4me3) at their respective promoter regions. The OAS family genes are activated by the presence of double stranded RNA (dsRNA), which can arise either endogenously or as a pathogen-associated trigger of the innate immune system in the context of viral infection. The dsRNA response includes three major response mechanisms. First, OAS genes facilitate RNase L dimerization, which mediates cleavage of cellular tRNA and rRNA, resulting in translation and proliferation arrest. Second, activation of the PKR/eIF2a pathway also results in translation arrest. Lastly, activation of the MDA-5/RIG-I/MAVS response induces interferon signaling, which further limits cellular proliferation. Staining of VCM3 WT &amp; KO FL cells with the dsRNA-specific J2 antibody directly demonstrated significant accumulation of dsRNA in KO FL cells. We further demonstrated activation of the OAS/RNaseL axis in KO cells by quantification of tRNA-His-36 cleavage, which acts as a sensitive marker of RNase L activity. Phosphorylation of both PKR and eIF2a in KO cells demonstrated enhanced activity of this pathway. Lastly, we found a significant upregulation of interferon pathway genes in KO cells, and demonstrated that CRISPR mediated knockout of Mavs in VCM3 KO FL cells diminishes the activity of interferon response genes and rescues the Mettl3 KO phenotype in colony forming unit (CFU) assays. These results suggest that in the absence of m6A, endogenous dsRNA formation is enhanced, resulting in an associated inflammatory response which partially accounts for the hematopoietic defect observed in Mettl3 KO mice. In conclusion, our study suggests a novel, protective role for the m6A RNA modification in preventing endogenous dsRNA formation and aberrant activation of a detrimental innate immune response during mammalian hematopoietic development. Disclosures Fan: IsoPlexis: Other: served on the scientific discovery board, Patents &amp; Royalties; Singleron Biotechnologies: Other: served on the Scientific Advisory Board; BioTechne: Other: served on the Scientific Advisory Board. Flavell:Rheos Biomedicines: Equity Ownership; GSK: Consultancy; Artizan Biosciences: Equity Ownership; Troy: Equity Ownership; SMOC: Equity Ownership; Zai labs: Consultancy.

GENE-21. ROLE OF POT1 MUTATION IN GLIOMA PROLIFERATION AND SEXUAL DIVERGENCE OF SURVIVAL

Abstract Germline POT1 mutations are associated with risk of several cancers including glioma. In The Cancer Genome Atlas, we found that the level of POT1 gene expression in tumors is associated with overall survival in IDH wild-type glioma independent of age and tumor grade (P=0.036). To assess if POT1 expression in a native mouse model of IDH wild-type glioma (referred to as C3 tumors) affects survival, we targeted both mouse POT1 orthologs (Pot1a/Pot1b) in C3 tumors using CRISPR/Cas9, generating C5 tumors. Interestingly, we noted a sexual divergence of survival, with female C5 mice dying faster than males (P=0.0034), whereas C3 mice show no sexual divergence in survival. To assess the genes and pathways underlying this divergence, we performed RNAseq profiling on C3 and C5 tumors and normalized the sex differences in the C5 profile for baseline sex differences in the C3 profile. Compared to males, female C5 tumors had lower enrichment of numerous gene sets related to immune surveillance. To specifically assess the effects of glioma-associated human POT1 variants, we expressed these variants in a mouse tumor sphere culture. We noted that expression of POT1-G95C variant increased the rate of sphere formation in vitro. In vivo expression of this variant in embryonic mouse brain during the gliogenic period increased the proliferative rate as assessed by BrdU uptake (P=0.008). These findings suggest a potential role for human POT1 variants in growth and proliferation of glial progenitor cells as well as glioma tumor cells. Additionally, the sexual divergence of survival in C5 tumors points to a differential interaction between POT1 loss and sex in regulation of immune response to these tumors.