33,590 research outputs found

    Induction of Metamorphosis in the Marine Gastropod Ilyanassa obsoleta: 5HT, NO and Programmed Cell Death

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    The central nervous system (CNS) of a metamorphically competent larva of the caenogastropod Ilyanassa obsoleta contains a medial, unpaired apical ganglion (AG) of approximately 25 neurons that lies above the commissure connecting the paired cerebral ganglia. The AG, also known as the cephalic or apical sensory organ (ASO), contains numerous sensory neurons and innervates the ciliated velar lobes, the larval swimming and feeding structures. Before metamorphosis, the AG contains 5 serotonergic neurons and exogenous serotonin can induce metamorphosis in competent larvae. The AG appears to be a purely larval structure as it disappears within 3 days of metamorphic induction. In competent larvae, most neurons of the AG display nitric oxide synthase (NOS)-like immunoreactivity and inhibition of NOS activity can induce larval metamorphose. Because nitric oxide (NO) can prevent cells from undergoing apoptosis, a form of programmed cell death (PCD), we hypothesize that inhibition of NOS activity triggers the loss of the AG at the beginning of the metamorphic process. Within 24 hours of metamorphic induction, cellular changes that are typical of the early stages of PCD are visible in histological sections and results of a terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay in metamorphosing larvae show AG nuclei containing fragmented DNA, supporting our hypothesis

    Pigment epithelium-derived factor (PEDF) alone or in combination with an anti-VEGF drug as a possible treatment of choroidal neovascularization and ischemic ocular diseases

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    Ocular neovascular diseases such as choroidal neovascularization (CNV) and age-related macular degeneration (AMD) are today the most common causes of visual loss and blindness. Although intravitreal anti-vascular endothelial growth factor (VEGF) therapy has achieved major breakthroughs in the treatment of CNV and wet AMD, it does not always successfully suppress CNV, and no efficacious treatment is yet available to prevent photoreceptor degeneration. The formation of CNV is a compensatory response to ocular ischemia. If CNV can be stabilized, it may be helpful for photoreceptor survival and maintaining the normal physiological function of the retina. With a wide use of optical coherence tomography angiography (OCTA) in the clinic, quiescent CNVs without exudation are much more frequently found than previously assumed. In this study, I examined the efficacy of a pigment epithelium derived factor (PEDF) protein alone or combined with a VEGF antagonist (Avastin) in the treatment of rat quiescent CNV in vivo. The rat quiescent CNV was established by subretinal injection of an adeno-associated viral (AAV)-VEGF-A165 vector, which expresses VEGF and leads to vascular growth from the choroid into the subretinal space. The fluorescein angiography (FA), indocyanine green angiography (ICG) and optical coherence tomography (OCT) were performed at the third week, fourth week, sixth week and seventh week after subretinal injection of the AAV-VEGF-A165 vector. The maximal thickness of CNV was measured in the OCT images. Picrosirius red stain was used to quantify mature and immature collagen content in the formalin-fixed, paraffin-embedded quiescent CNV samples. The collagen Ⅳ, VEGF and PEDF were examined in the quiescent CNV samples by immunohistochemistry (IHC), and the percentage of positive staining area to the total CNV area was calculated by ImageJ software. To analyze the effect of PEDF on retinal photoreceptor cells, the outer nuclear layer (ONL) area as a percentage of the corresponding CNV area was quantified. The apoptosis of ONL cells above the CNV area was detected by TUNEL staining. PEDF combined with anti-VEGF therapy significantly inhibits VEGF expression in quiescent CNV. The ONL area as a percentage of the corresponding CNV area is increased in the PEDF treatment group (92.29, 120.27) and decreased in the Avastin treatment group (55.11, 75.59) (P ﹤0.001, Wilcoxon test with Kruskal-Wallis test). The apoptosis of ONL cells above the CNV area is reduced in the PEDF treatment group (0.95 ± 0.60, n/1000μm) compared to the vehicle treatment group (2.26 ± 0.95, n/1000μm) (P ﹤0.001, one-way ANOVA test). Thus, PEDF shows a neuroprotective effect for the retinal photoreceptor cells. In this in vivo experiment, a VEGF antagonist (Avastin) can significantly inhibit the formation of CNV in this rat quiescent CNV model and reduce its thickness. However, quiescent CNV can supply oxygen to the photoreceptor cells and protect photoreceptor cells from degeneration, thus maintaining vision. Although anti-VEGF (Avastin) treatment alone inhibits CNV, it also accelerates photoreceptor cell degeneration in this in vivo quiescent CNV model. In the clinic, development of macular geographic atrophy and fibrosis are found in patients with wet AMD under long term anti-VEGF treatment. PEDF has no effect on mature and immature collagen in this quiescent CNV model. The information on the therapeutic effect is shown in Table 1. PEDF is a potential vascular stabilizing and neuroprotective factor for stabilizing CNV and maintaining vision. Ischemic/hypoxic retinopathy is a common condition that can cause visual impairment and blindness. However, the changes of the choroidal blood vessels under ischemic/hypoxic conditions are not clearly known. The apoptosis of retinal ganglion cells (RGCs) and the retinal degeneration under ischemic/hypoxic conditions cannot be treated. In this study, I established an ischemic/hypoxic ex vivo eye model by incubating the freshly enucleated rat eyes in Dulbecco's modified eagle medium (DMEM) at 4 °C for 14 hours. After 14 hours, eyes (including control eyes, eyes intravitreally injected with vehicle or PEDF protein) were fixed for electron microscopy (EM) and immunohistochemistry (IHC) respectively. The area of lumen (μm2/μm of Bruch’s membrane) and the area of the whole vessel (μm2/μm of Bruch’s membrane) were analyzed and immunohistochemical staining for VEGF and PEDF in the retina and choroidal vessels was performed. The effects of PEDF on the choriocapillaris and retinal neural cells under ischemia/hypoxia were investigated. In addition, the oxygen concentration within the vitreous was measured by an oxygen-sensitive microsensor in both the ischemic/hypoxic ex vivo eye model and the living rats under anesthesia. TUNEL staining was performed and the apoptosis of retinal neural cells was analyzed. In the living rats, the concentration of oxygen within the vitreous was on average 16.4 % of the oxygen concentration in the air. In the ischemic/hypoxic ex vivo eyes, the oxygen concentration within the vitreous was gradually decreased and the concentration was about 50% of the value in the eyes immediately after enucleation after about 400 minutes of incubation, indicating mild hypoxia during this process. After 14 hours of ischemia/hypoxia, the endothelial cells of the choroidal vessel were ultra-structurally similar with respect to cell organelles compared to the immediately fixed control eyes, but the morphology of the choroidal vessels changed dramatically. In the ischemic/hypoxic eyes, filopodia-like projections filled out the choroidal vessel lumen and appeared identical to the labyrinth-capillaries found in surgically extracted choroidal neovascular membranes from patients with wet AMD. The structural changes within the choriocapillaris in this ischemic/hypoxic eye model can mimic early changes in the process of pathological angiogenesis as observed in patients with CNV or wet AMD. This ex vivo eye model can be used to investigate short term drug effects on the choriocapillaris after ischemia/hypoxia. PEDF inhibited the filopodia-like projection formation and kept the choroidal lumen open as in vivo. The area of lumen is significantly reduced in the ischemic/hypoxic group (0.308±0.087μm2/μm) compared to the PEDF-treated group (1.034±0.077μm2/μm) (P<0.001, t test). The area of vessel is significantly reduced in the ischemic/hypoxic group (0.675±0.048 μm2/μm) compared to the PEDF-treated group (1.583±0.094μm2/μm) (P<0.001, t test). The intravitreally injected PEDF protein located in the whole retina and the choroidal vessels, indicating that PEDF protein can penetrate the retina and is transported into the choroid. The apoptosis of RGCs (2.36, 2.89; percentage of positive cells per 100μm) and inner nuclear cells (15.81, 22.89; number of positive cells per 1000μm) was significantly reduced in the PEDF-treated eyes compared to the ischemic/hypoxic eyes [ RGCs (3.14, 3.75; percentage of positive cells per 100μm) (P = 0.015), inner nuclear cells (33.69, 45.22; number of positive cells per 1000μm) (P = 0.001) Wilcoxon test]. The detailed information is shown in Table 2. Thus, PEDF is a promising candidate for treating ischemic/hypoxic retinopathy or human CNV alone or combined with other drugs. In short, PEDF protected retinal neural cells in both the in vivo quiescent CNV model and the ex vivo ischemic/hypoxic eye model. PEDF inhibits the formation of filopodia-like projections and keeps the choroidal vessel lumen open in this ex vivo ischemic eye model.Dissertation ist gesperrt bis 17.02.202

    Inhibition of Stimulator of Interferon Genes Protects Against Myocardial Ischemia-Reperfusion Injury in Diabetic Mice

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    Background: Although the past decade has witnessed substantial scientific progress with the advent of cardioprotective pharmacological agents, most have failed to protect against myocardial ischemia/reperfusion (I/R) injury in diabetic hearts. This study was aimed at investigating the role of stimulator of interferon genes (STING) in I/R injury in diabetic mice and further exploring the underlying mechanisms. Methods: Type 2 diabetic mice were subjected to I/R or sham operation to investigate the role of STING. STING knockout mice were subjected to 30 minutes of ischemia followed by reperfusion for 24 hours. Finally, myocardial injury, cardiac function, and inflammation levels were assessed. Results: STING pathway activation was observed in diabetic I/R hearts, as evidenced by increased p-TBK and p-IRF3 expression. STING knockout significantly decreased the ischemic area and improved cardiac function after I/R in diabetic mice. STING knockout also elicited cardio-protective effects by decreasing serum cardiac troponin T and lactate dehydrogenase levels, thus diminishing the inflammatory response in the heart after I/R in diabetic mice. In vitro , STING inhibition decreased the expression of hypoxia-re-oxygenation-induced inflammatory cytokines. Conclusions: Targeting STING inhibits inflammation and prevents I/R injury in diabetic mice. Thus, STING may be a potential novel therapeutic target against myocardial I/R injury in diabetes

    Bilobetin induces apoptosis in human hepatocellular carcinoma cells via ROS level elevation and inhibition of CYP2J2

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    Bilobetin is a biflavonoid isolated from the leaves of Ginkgo biloba. Bilobetin displays several biological effects, however, the activities of bilobetin against cancer have been solely demonstrated. Thus, the aim of this study is investigating the apoptotic effects of and anticancer mechanism of bilobetin in Huh7 and HepG2 cells, both of human hepatocellular carcinoma (HCC) cell lines. MTT, cell counting, and colony formation assay showed that anti-proliferation effect of bilobetin. Cell cycle analysis revealed that bilobetin induces increase of population of the sub G1 phase. Annexin V/PI staining and TUNEL assay showed that bilobetin treatment promotes apoptotic cell death and DNA fragmentation. Bilobetin also induces ROS elevation and DNA damage in HCC cells. Western blot analysis elucidated that bilobetin displays apoptotic signaling pathway in HCC cells via upregulating cleaved PARP, cleaved caspase3 and Bax and downregulating CYP2J2. Bilobetin inhibited CYP2J2-catalyzed terfenadine and ebastine hydroxylase activities with IC50 values of 0.81 and 2.21 μM, respectively. Transfection of CYP2J2 siRNA increased the anticancer effect of bilobetin in HCC cells through the inhibition of CYP2J2 expression. Taken together, this study suggests that bilobetin induces apoptosis against HCC cells via ROS level elevation and inhibition of CYP2J2

    Studying the interplay between ageing and Parkinson's disease using the zebrafish model

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    Parkinson’s disease (PD) is a neurodegenerative disorder characterised by the loss of dopaminergic neurons in the substantia nigra. Ageing is the major risk factor for developing PD but the interplay between ageing and PD remains elusive. To investigate the effect of ageing on PD-relevant pathological mechanisms, zebrafish mutant lines harbouring mutations in ageing-associated genes (klotho-/-, sirt1-/-, satb1a-/-, satb1b-/- and satb1a-/-;satb1b-/-) were generated, using CRISPR/Cas9 gene editing. Likewise, a chemical model for SIRT1 deficiency was utilised. klotho-/- zebrafish displayed an accelerated ageing phenotype at 3mpf and reduced survival to 6mpf. Dopaminergic neuron number, MPP+ susceptibility and microglial number were unaffected in klotho-/- larvae. NAD+ levels were decreased in 6mpf klotho-/- brains. However, ATP levels and DNA damage were unaffected. sirt1-/- zebrafish did not display a phenotype through adulthood. il-1β and il-6 were not upregulated in sirt1-/- larvae, and chemical inhibition of sirt1 did not increase microglial number. cdkn1a, il-1β and il-6 were not upregulated in satb1a-/- and satb1b-/- larvae. Dopaminergic neuron number and MPP+ susceptibility were unaffected in satb1a-/- larvae. However, satb1b-/- larvae demonstrated a moderate decrease in dopaminergic neuron number but equal susceptibility to MPP+ as satb1b+/+ larvae. Adult satb1a-/- but not adult satb1b-/- zebrafish were emaciated. satb1a-/-;satb1b-/- zebrafish did not display a phenotype through adulthood. Transgenic zebrafish expressing human wildtype α-Synuclein (Tg(eno2:hsa.SNCA-ires-EGFP)) were crossed with klotho-/- and sirt1-/- zebrafish, and treated with a sirt1-specific inhibitor. Neither genetic cross affected survival. The klotho mutation did not increase microglial number in Tg(eno2:hsa.SNCA-ires-EGFP) larvae. Likewise, sirt1 inhibition did not induce motor impairment or cell death in Tg(eno2:hsa.SNCA-ires-EGFP) larvae. In conclusion, the suitability of zebrafish for studying ageing remains elusive, as only 1 ageing-associated mutant line displayed accelerated ageing. However, zebrafish remain an effective model for studying PD-relevant pathological mechanisms due to the availability of CRISPR/Cas9 gene editing, neuropathological and neurobehavioral tools

    Ferroptosis: new insight into the mechanisms of diabetic nephropathy and retinopathy

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    Diabetic nephropathy (DN) and diabetic retinopathy (DR) are the most serious and common diabetes-associated complications. DN and DR are all highly prevalent and dangerous global diseases, but the underlying mechanism remains to be elucidated. Ferroptosis, a relatively recently described type of cell death, has been confirmed to be involved in the occurrence and development of various diabetic complications. The disturbance of cellular iron metabolism directly triggers ferroptosis, and abnormal iron metabolism is closely related to diabetes. However, the molecular mechanism underlying the role of ferroptosis in DN and DR is still unclear, and needs further study. In this review article, we summarize and evaluate the mechanism of ferroptosis and its role and progress in DN and DR, it provides new ideas for the diagnosis and treatment of DN and DR

    Evaluation of the Anticancer Activity of Calcium Ions Introduced into Human Breast Adenocarcinoma Cells MCF-7/WT and MCF-7/DOX by Electroporation

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    Breast cancer ranks among the top three most common malignant neoplasms in Poland. The use of calcium ion-assisted electroporation is an alternative approach to the classic treatment of this disease. The studies conducted in recent years confirm the effectiveness of electroporation with calcium ions. Electroporation is a method that uses short electrical pulses to create transitional pores in the cell membrane to allow the penetration of certain drugs. The aim of this study was to investigate the antitumor effects of electroporation alone and calcium ion-assisted electroporation on human mammary adenocarcinoma cells that are sensitive (MCF-7/WT) and resistant to doxorubicin (MCF-7/DOX). The cell viability was assessed using independent tests: MTT and SRB. The type of cell death after the applied therapy was determined by TUNEL and flow cytometry (FACS) methods. The expression of Cav3.1 and Cav3.2 proteins of T-type voltage-gated calcium channels was assessed by immunocytochemistry, and changes in the morphology of CaEP-treated cells were visualized using a holotomographic microscope. The obtained results confirmed the effectiveness of the investigated therapeutic method. The results of the work constitute a good basis for planning research at the in vivo level and in the future to develop a more effective and safer method of breast cancer treatment for patients

    Efferocytes release extracellular vesicles to resolve inflammation and tissue injury via prosaposin-GPR37 signaling.

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    Macrophages release soluble mediators following efferocytic clearance of apoptotic cells to facilitate intercellular communication and promote the resolution of inflammation. However, whether inflammation resolution is modulated by extracellular vesicles (EVs) and vesicular mediators released by efferocytes is not known. We report that efferocyte-derived EVs express prosaposin, which binds to macrophage GPR37 to increase expression of the efferocytosis receptor Tim4 via an ERK-AP1-dependent signaling axis, leading to increased macrophage efferocytosis efficiency and accelerated resolution of inflammation. Neutralization and knockdown of prosaposin or blocking GRP37 abrogates the pro-resolution effects of efferocyte-derived EVs in vivo. Administration of efferocyte-derived EVs in a murine model of atherosclerosis is associated with an increase in lesional macrophage efferocytosis efficiency and a decrease in plaque necrosis and lesional inflammation. Thus, we establish a critical role for efferocyte-derived vesicular mediators in increasing macrophage efferocytosis efficiency and accelerating the resolution of inflammation and tissue injury

    MIEAP、ATG5はBRAFV600E陽性甲状腺癌マウスモデルにおいて腫瘍抑制因子である

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    Mitochondria-eating protein (MIEAP) is a molecule important for non-canonical mitophagy and thought to be a tumor suppressor. Our previous study found that MIEAP expression is defective in thyroid oncocytomas, irrespective of being benign or malignant, and also in non-oncocytic thyroid cancers. Thyroid oncocytomas are composed of large polygonal cells with eosinophilic cytoplasm that is rich in abnormal mitochondria. Thus, our data indicate that, together with increased mitochondrial biogenesis that compensates for the dysfunction of the mitochondria, MIEAP plays a critical role in the accumulation of mitochondria in thyroid oncocytic tumors, whereas a defective MIEAP expression alone is not sufficient for mitochondrial accumulation in non-oncocytic cancers with normal mitochondria. To clarify whether MIEAP is a tumor suppressor in the thyroids and whether MIEAP knockout (KO) alone is sufficient for the oncocytic phenotype and also to extend our effort toward canonical mitophagy (a selective autophagy), we here conducted mouse studies using genetically engineered mice. BrafCA/wt mice developed thyroid cancers 1 year after intrathyroidal injection of adenovirus expressing Cre, while cancer development was observed at 6 months in adenovirus-Cre-injected BrafCA/wt;MieapKO/KO and BrafCA/wt;Atg5flox/flox mice [where autophagy-related 5 (ATG5) is a component of autophagic machinery], although KO of either molecule alone was not sufficient for cancer development. These data demonstrate that MIEAP or ATG5 KO accelerated thyroid cancer development. However, cancers in adenovirus-Cre-injected BrafCA/wt;MieapKO/KO and BrafCA/wt;Atg5flox/flox mice were not oncocytic. In conclusion, we here show that MIEAP and ATG5 are both tumor suppressors in thyroid carcinogenesis, but as we have anticipated from our previous data, KO of either molecule does not confer the oncocytic phenotype to BRAFV600E-positive thyroid cancers. The combination of disruptive mitochondrial function and impaired mitochondrial quality control may be necessary to establish a mouse model of thyroid oncocytoma.長崎大学学位論文 学位記番号:共博(医歯薬)甲第28号 学位授与年月日:令和5年3月20日Author: Koichiro Hamada, Tomomi Kurashige, Mika Shimamura, Hirofumi Arakawa, Yasuyuki Nakamura and Yuji NagayamaCitation: Frontiers in Endocrinology, 13, art. no. 932754; 2022Nagasaki University (長崎大学)課程博
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