PKM2 Is a Potential Diagnostic and Therapeutic Target for Retinitis Pigmentosa

Retinitis pigmentosa (RP) is a major cause of blindness that is difficult to diagnose and treat. PKM2, a subtype of pyruvate kinase, is strongly associated with oxidative stress and is expressed in photoreceptors. We investigated whether PKM2 reduces photoreceptor cell apoptosis and evaluated possible antiapoptotic mechanisms in RP. We established RP models by exposing 661W cells to blue light and modulated PKM2 activity using a PKM2 inhibitor. We measured the apoptosis rates using calcein-acetoxymethyl ester/propidium iodide double staining and Cell Counting Kit-8, the oxidative stress levels using a reactive oxygen species assay, and the changes in protein expression by western blotting. Photodamage increased PKM2 expression, cellular oxidative stress, and apoptosis of 661W cells. PKM2 inhibition significantly reduced the levels of apoptosis and oxidative stress induced by photodamage. Our data suggest that PKM2 is a potential disease marker and therapeutic target for RP.

Molecular and Cellular Reports A Pharmacological Study of the Action of Esculetin on Human Prostate Cancer Cells

Esculetin is a derivative of coumarin, and is the dominant, vigorous component of the conventional Chinese medicine Cortex Fraxini. Recently, the molecular pathway study and clinical use of Cortex Fraxini and esculetin are becoming intensive. In vitro, esculetin has been shown to provoke apoptotic responses via mitochondrial routes and other cellular responses in diverse cell types. The action of esculetin on cytosolic Ca2+ concentration ([Ca2+]i) in prostate cells is unknown. [Ca2+]i were assayed by applying fura-2, a fluorescent Ca2+-sensitive probe. WST-1 was used to measure cell death. Esculetin at doses of 25–100 µM provoked [Ca2+]i raises. Removing external Ca2+ decreased the response by 15%. Esculetin (100 µM) provoked Mn2+ entry implying Ca2+ influx. Esculetin-provoked Ca2+ influx was suppressed by half by protein kinase C (PKC) activator (phorbol 12-myristate 13 acetate, PMA) and inhibitor (GF109203X); and by three inhibitors of store-operated Ca2+ channels: nifedipine, econazole and SKF96365. In the absence of Ca2+, pretreatment with the endoplasmic reticulum (ER) Ca2+ pump suppressor thapsigargin completely suppressed esculetin-provoked [Ca2+]i raises. Suppression of phospholipase C (PLC) with U73122 eliminated esculetin-provoked [Ca2+]i raises. Esculetin at 20–70 µM caused death of cells, which was not prevented by incubation with the Ca2+ binder 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid-acetoxymethyl ester (BAPTA/AM). In sum, in PC3 prostate cells, esculetin provoked [Ca2+]i raises by provoking PLC-associated Ca2+ discharge from ER and Ca2+ influx via PKC-sensitive store-operated Ca2+ influx. Additionally, esculetin provoked Ca2+-dissociated cell death.

17β-Estradiol Increases APE1/Ref-1 Secretion in Vascular Endothelial Cells and Ovariectomized Mice: Involvement of Calcium-Dependent Exosome Pathway

Apurinic/apyrimidinic endonuclease-1/redox factor-1 (APE1/Ref-1) is a multifunctional protein that can be secreted, and recently suggested as new biomarker for vascular inflammation. However, the endogenous hormones for APE1/Ref-1 secretion and its underlying mechanisms are not defined. Here, the effect of twelve endogenous hormones on APE1/Ref-1 secretion was screened in cultured vascular endothelial cells. The endogenous hormones that significantly increased APE1/Ref-1 secretion was 17β-estradiol (E2), 5?-dihydrotestosterone, progesterone, insulin, and insulin-like growth factor. The most potent hormone inducing APE1/Ref-1 secretion was E2, which in cultured endothelial cells, E2 for 24 h increased APE1/Ref-1 secretion level of 4.56 ± 1.16 ng/mL, compared to a basal secretion level of 0.09 ± 0.02 ng/mL. Among the estrogens, only E2 increased APE1/Ref-1 secretion, not estrone and estriol. Blood APE1/Ref-1 concentrations decreased in ovariectomized (OVX) mice but were significantly increased by the replacement of E2 (0.39 ± 0.09 ng/mL for OVX vs. 4.67 ± 0.53 ng/mL for OVX + E2). E2-induced APE1/Ref-1secretion was remarkably suppressed by the estrogen receptor (ER) blocker fulvestrant and intracellular Ca2+ chelator 1,2-Bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid tetrakis (acetoxymethyl ester) (BAPTA-AM), suggesting E2-induced APE1/Ref-1 secretion was dependent on ER and intracellular calcium. E2-induced APE1/Ref-1 secretion was significantly inhibited by exosome inhibitor GW4869. Furthermore, APE1/Ref-1 level in CD63-positive exosome were increased by E2. Finally, fluorescence imaging data showed that APE1/Ref-1 co-localized with CD63-labled exosome in the cytoplasm of cells upon E2 treatment. Taken together, E2 was the most potent hormone for APE1/Ref-1 secretion, which appeared to occur through exosomes that were dependent on ER and intracellular Ca2+. Furthermore, hormonal effects should be considered when analyzing biomarkers for vascular inflammation.

Near infrared light fluorescence imaging-guided biomimetic nanoparticles of extracellular vesicles deliver indocyanine green and paclitaxel for hyperthermia combined with chemotherapy against glioma

Background We investigated the therapeutic effect of targeting extracellular vesicles (EVs) loaded with indocyanine green (ICG) and paclitaxel (PTX) on glioma. Methods Raw264.7 cells were harvested to extract EVs for the preparation of ICG/PTX@RGE-EV by electroporation and click chemistry. We evaluated the success of modifying Neuropilin-1 targeting peptide (RGE) on the EV membrane of ICG/PTX@RGE-EV using super-resolution fluorescence microscopy and flow cytometry. Spectrophotometry and high performance liquid chromatography (HPLC) were implemented for qualitative and quantitative analysis of the ICG and PTX loaded in EVs. Photothermal properties of the vesicles were evaluated by exposing to 808-nm laser light. Western blot analysis, cell counting kit 8 (CCK-8), Calcein Acetoxymethyl Ester/propidium iodide (Calcein-AM/PI) staining, and flow cytometry were utilized for assessing effects of vesicle treatment on cellular behaviors. A nude mouse model bearing glioma was established to test the targeting ability and anti-tumor action of ICG/PTX@RGE-EV in vivo. Results Under exposure to 808-nm laser light, ICG/PTX@RGE-EV showed good photothermal properties and promotion of PTX release from EVs. ICG/PTX@RGE-EV effectively targeted U251 cells, with activation of the Caspase-3 pathway and elevated apoptosis in U251 cells through chemotherapy combined with hyperthermia. The anti-tumor function of ICG/PTX@RGE-EV was confirmed in the glioma mice via increased accumulation of PTX in the ICG/PTX@RGE-EV group and an increased median survival of 48 days in the ICG/PTX@RGE-EV group as compared to 25 days in the PBS group. Conclusion ICG/PTX@RGE-EV might actively target glioma to repress tumor growth by accelerating glioma cell apoptosis through combined chemotherapy-hyperthermia. Graphic Abstract

Permanent ectoplasmic structures in deep-sea <i>Cibicides</i> and <i>Cibicidoides</i> taxa – long-term observations at in situ pressure

Deep-sea Cibicidoides pachyderma (forma mundulus) and related Cibicidoides spp. were cultured at in situ pressure for 1–2 d, or 6 weeks to 3 months. During that period, fluorescence analyses following BCECF-AM (2′,7′-bis(2-carboxyethyl)-5-(and-6)-carboxyfluorescein acetoxymethyl ester) or calcein (bis[N,N-bis(carboxymethyl)aminomethyl]-fluorescein) labelling revealed a persisting cytoplasmic sheet or envelope surrounding the Cibicidoides tests. Thus, the Cibicidoides shell can be considered as an internal rather than an external cell structure. A couple of days to a week after being transferred into high-pressure aquaria and adjusted to a pressure of 115 bar, the foraminifera changed from a mobile to a more or less sessile living mode. During this quasi-sessile way of life, a series of comparably thick static ectoplasmic structures developed that were not resorbed or remodelled but, except for occasional further growth, remained unchanged throughout the experiments. Three different types of these permanent structures were observed. (a) Ectoplasmic “roots” were common in adult C. pachyderma, C. lobatulus, and C. wuellerstorfi specimens. In our experiments single ectoplasmic roots grew to a maximum of 700 times the individuals' shell diameter and were presumably used to anchor the specimen in an environment with strong currents. (b) Ectoplasmic “trees” describe rigid ectoplasmic structures directed into the aquarium's water body and were used by the foraminifera to climb up and down these ectoplasmic structures. Ectoplasmic trees have so far only been observed in C. pachyderma and enabled the tree-forming foraminifera to elevate itself above ground. (c) Ectoplasmic “twigs” were used to guide and hold the more delicate pseudopodial network when distributed into prevailing currents and were, in our experiments, also only developed in C. pachyderma specimens. Relocation of a specimen usually required it to tear apart and leave behind the rigid ectoplasmic structures and eventually also the envelope surrounding the test. Apparently, these rigid structures could not be resorbed or reused.

Protection of Iron-Induced Oxidative Damage in Neuroblastoma (SH-SY5Y) Cells by Combination of 1-(N-Acetyl-6-aminohexyl)-3-hydroxy-2-methylpyridin-4-one and Green Tea Extract

Iron is a crucial trace element and essential for many cellular processes; however, excessive iron accumulation can induce oxidative stress and cell damage. Neurodegenerative disorders, such as Alzheimer’s disease and Parkinson’s disease, have been associated with altered iron homoeostasis causing altered iron distribution and accumulation in brain tissue. This study aims to investigate the protective effect of 1-(N-acetyl-6-aminohexyl)-3-hydroxy-2-methylpyridin-4-one (CM1) in combination with green tea extract (GTE) on iron-induced oxidative stress in neuroblastoma (SH-SY5Y) cells. Cells were cultured in medium with or without ferric chloride loading. Their viability and mitochondrial activity were assessed using MTT and JC-1 staining methods. Levels of the cellular labile iron pool (LIP), reactive oxygen species (ROS), and lipid-peroxidation products were determined using calcein acetoxymethyl ester, 2′,7′-dichlorohydrofluorescein diacetate, and TBARS-based assays, respectively. The viability of iron-loaded cells was found to be significantly increased after treatment with CM1 (10 µM) for 24 h. CM1 co-treatment with GTE resulted in a greater protective effect than their monotherapy. Combination of CM1 and GTE also reduced mitochondrial disruption and LIP content and ROS and TBARS production. In conclusion, the combination of CM1 and GTE exhibits protection against iron-induced oxidative stress in neuroblastoma cells.

Permanent ectoplasmic structures in deep-sea Cibicides/oides taxa – long-term observations at in situ pressure

Deep-sea Cibicidoides pachyderma (forma mundulus) and related Cibicidoides spp. were cultured at in situ pressure for 1-2 days, or 6 weeks to 3 months. During that period, fluorescence analyses following BCECF-AM (2’,7’-bis(2-carboxyethyl)-5-(and-6)-carboxyfluorescein acetoxymethyl ester) or Calcein AM (4,5-Bis((N,N-bis(carboxymethy)amino)methyl)fluorescein acetoxymethylester) labelling, revealed a persisting cytoplasmic sheet or envelope surrounding the Cibicidoides tests. Thus, the Cibicidoides shell can be considered rather as an internal than an external cell structure. A couple of days to a week after being transferred into high-pressure aquaria and adjusted to a pressure of 115 bar, the foraminifera changed from a mobile to a more or less sessile living mode. During this quasi sessile way of life, a series of comparably thick static ectoplasmic structures developed that were not resorbed or remodelled but, except for occasional further growth, remained unchanged throughout the experiments. Three different types of these ‘permanent structures’ were observed: A) Ectoplasmic ‘roots’ were common in adult C. pachyderma, C. lobatulus and C. wuellerstorfi specimens. In our experiments single ectoplasmic ‘roots’ grew to maximum 700 times the individuals shell diameter and were presumably used to anchor the specimen in an environment with strong currents. B) Ectoplasmic ‘trees’ describe rigid ectoplasmic structures directed into the aquarium’s water body and were used by the foraminifera to climb up and down these ectoplasmic structures. Ectoplasmic ‘trees’ were so far only observed in C. pachyderma and enabled the ‘tree’-forming foraminifera to elevate itself above ground. C) Ectoplasmic ‘twigs’ were used to guide and hold the more delicate pseudopodial network when distributed into prevailing currents, and were, in our experiments, also only developed in C. pachyderma specimens. Relocation of a specimen usually required to tear apart and leave behind the rigid ectoplasmic structures, eventually also the envelope surrounding the test. Apparently, these rigid structures could not be resorbed or reused.

Phosphonofluoresceins: Synthesis, Spectroscopy, and Applications

<p>Xanthene fluorophores, like fluorescein, have been versatile molecules across diverse fields of chemistry and life sciences. Despite the ubiquity of 3-carboxy and 3-sulfuonofluorescein for the last 150 years, to date, no reports of 3-phosphonofluorescein exist. Here, we report the synthesis, spectroscopic characterization, and applications of 3-phosphonofluoresceins. The absorption and emission of 3-phosphonofluoresceins remain relatively unaltered from the parent 3-carboxyfluorescein. 3-phosphonofluoresceins show enhanced water solubility compared to 3-carboxyfluorescein and persist in an open, visible light-absorbing state even at low pH and in low dielectric media while 3-carboxyfluoresceins tend to lactonize. In contrast, the spirocyclization tendency of 3-phosphonofluoresceins can be modulated by esterification of the phosphonic acid. The bis-acetoxymethyl ester of 3-phosphonofluorescein readily enters living cells, showing excellent accumulation (>6x) and retention (>11x), resulting in a nearly 70-fold improvement in cellular brightness compared to 3-carboxyfluorescein. In a complementary fashion, the free acid form of 3-phosphonofluorescein does not cross cellular membranes, making it ideally suited for incorporation into a voltage-sensing scaffold. We develop a new synthetic route to functionalized 3-phosphonofluoresceins to enable the synthesis of phosphono-voltage sensitive fluorophores, or phosVF2.1.Cl. Phosphono-VF2.1.Cl shows excellent membrane localization, cellular brightness, and voltage sensitivity (26% ΔF/F per 100 mV), rivalling that of sulfono-based VF dyes. In sum, we develop the first synthesis of 3-phosphonofluoresceins, characterize the spectroscopic properties of this new class of xanthene dyes, and utilize these insights to show the utility of 3-phosphonofluoresceins in intracellular imaging and membrane potential sensing. </p> <p> </p>

Phosphonofluoresceins: Synthesis, Spectroscopy, and Applications

<p>Xanthene fluorophores, like fluorescein, have been versatile molecules across diverse fields of chemistry and life sciences. Despite the ubiquity of 3-carboxy and 3-sulfuonofluorescein for the last 150 years, to date, no reports of 3-phosphonofluorescein exist. Here, we report the synthesis, spectroscopic characterization, and applications of 3-phosphonofluoresceins. The absorption and emission of 3-phosphonofluoresceins remain relatively unaltered from the parent 3-carboxyfluorescein. 3-phosphonofluoresceins show enhanced water solubility compared to 3-carboxyfluorescein and persist in an open, visible light-absorbing state even at low pH and in low dielectric media while 3-carboxyfluoresceins tend to lactonize. In contrast, the spirocyclization tendency of 3-phosphonofluoresceins can be modulated by esterification of the phosphonic acid. The bis-acetoxymethyl ester of 3-phosphonofluorescein readily enters living cells, showing excellent accumulation (>6x) and retention (>11x), resulting in a nearly 70-fold improvement in cellular brightness compared to 3-carboxyfluorescein. In a complementary fashion, the free acid form of 3-phosphonofluorescein does not cross cellular membranes, making it ideally suited for incorporation into a voltage-sensing scaffold. We develop a new synthetic route to functionalized 3-phosphonofluoresceins to enable the synthesis of phosphono-voltage sensitive fluorophores, or phosVF2.1.Cl. Phosphono-VF2.1.Cl shows excellent membrane localization, cellular brightness, and voltage sensitivity (26% ΔF/F per 100 mV), rivalling that of sulfono-based VF dyes. In sum, we develop the first synthesis of 3-phosphonofluoresceins, characterize the spectroscopic properties of this new class of xanthene dyes, and utilize these insights to show the utility of 3-phosphonofluoresceins in intracellular imaging and membrane potential sensing. </p> <p> </p>

Digoxin Combined with Aerobic Interval Training Improved Cardiomyocyte Contractility

Digoxin is a cardiotonic that increases the cardiac output without causing deleterious effects on heart, as well as improves the left ventricular performance during physical exercise. We tested whether the association between chronic digoxin administration and aerobic interval training (AIT) promotes beneficial cardiovascular adaptations by improving the myocardial contractility and calcium (Ca2+) handling. Male Wistar rats were randomly assigned to sedentary control (C), interval training (T), sedentary digoxin (DIGO) and T associated to digoxin (TDIGO). AIT was performed on a treadmill (1h/day, 5 days/week) for 60 days, consisting of successive 8-min periods at 80% and 20% of VO2máx for 2 min. Digoxin was administered by orogastric gavage for 60 days. Left ventricle samples were collected to analysis of Ca2+ handling proteins; contractility and Ca2+ handling were performed on isolated cardiomyocytes. TDIGO group had a greater elevation in fractional shortening (44%) than DIGO, suggesting a cardiomyocyte contractile improvement. In addition, T or TDIGO groups showed no change in cardiomyocytes properties after Fura2-acetoxymethyl ester, as well as in sarcoplasmic reticulum Ca2+-ATPase (SERCA2a), phospholamban and calcineurin expressions. The main findings indicate that association of digoxin and aerobic interval training improved the cardiomyocyte contractile function, but these effects seem to be unrelated to Ca2+ handling.