Concurrent induction of apoptosis and necroptosis in apigenin­treated malignant mesothelioma cells: Reversal of Warburg effect through Akt inhibition and p53 upregulation.

A high dependence on aerobic glycolysis, known as the Warburg effect, is one of the metabolic features exhibited by tumor cells. Therefore, targeting glycolysis is becoming a very promising strategy for the development of anticancer drugs. In the present study, it was investigated whether pre­adaptation of malignant mesothelioma (MM) cells to an acidic environment was associated with a metabolic shift to the Warburg phenotype in energy production, and whether apigenin targets acidosis­driven metabolic reprogramming. Cell viability, glycolytic activity, Annexin V­PE binding activity, reactive oxygen species (ROS) levels, mitochondrial membrane potential, ATP content, western blot analysis and spheroid viability were assessed in the present study. MM cells pre­adapted to lactic acid were resistant to the anticancer drug gemcitabine, increased Akt activation, downregulated p53 expression, and upregulated rate­limiting enzymes in glucose metabolism compared with their parental cells. Apigenin treatment increased cytotoxicity, Akt inactivation and p53 upregulation. Apigenin also reduced glucose uptake along with downregulation of key regulatory enzymes in glycolysis, increased ROS levels with loss of mitochondrial membrane potential, and downregulated the levels of complexes I, III and IV in the mitochondrial electron transport chain with intracellular ATP depletion, resulting in upregulation of molecules mediating apoptosis and necroptosis. Apigenin­induced alterations of cellular responses were similar to those of Akt inactivation by Ly294002. Overall, the present results provide mechanistic evidence supporting the anti­glycolytic and cytotoxic role of apigenin via inhibition of the PI3K/Akt signaling pathway and p53 upregulation.

Anion-Dependent Polarization and Piezoelectric Power Generation in Hybrid Halide MAPbX3 (X = I, Br, and Cl) Thin Films with Out-of-Plane Structural Adjustments.

Anion-dependent differences in the electromechanical energy harvesting capability of perovskite halides have not been experimentally demonstrated thus far. Herein, anion-dependent piezoelectricity and bending-driven power generation in high-quality methylammonium lead halide MAPbX3 (X = I, Br, and Cl) thin films are explored; additionally, anisotropic in situ strain is imposed to improve energy harvesting under tensile bending. After applying the maximum in situ strain of -0.73% for all the halide thin films, the MAPbI3 thin-film harvester exhibited a peak voltage/current of ≈23.1 V/≈1703 nA as the best values, whereas MAPbBr3 and MAPbCl3 demonstrated ≈5.6 V/≈176 nA and ≈3.3 V/≈141 nA, respectively, under identical bending conditions. Apart from apparent ferroelectricity of tetragonal MAPbI3 , origin of the piezoelectricity in both cubic MAPbBr3 and MAPbCl3 is explored as being related to organic-inorganic hydrogen bonding, lattice distortion, and ionic migration, with experimental supports of effective piezoelectric coefficient and grain boundary potential. Conclusively, piezoelectricity of the cubic halides is assumed to be due to their soft polarity modes and relatively low elastic modulus with vacancies contributing to space-charge polarization. In the case of ferroelectric MAPbI3 , the distortion of PbI6 octahedra and atomic displacement within each octahedron are quantitatively estimated.

Origin of the anisotropic-strain-driven photoresponse enhancement in inorganic halide-based self-powered flexible photodetectors.

Strain engineering has been recognized as a critical strategy in modulating the optoelectronic properties of perovskite halide materials. Here, we demonstrate a self-powered, flexible photodetector based on CsPbBr3 thin films with controllable compressive or tensile strain of up to ±0.81%, which was produced in situ via a sequential two-step deposition on bent polymer substrates. The best photoresponsivity of ∼121.5 mA W-1 with a photocurrent of 5.15 µA was achieved at zero bias under a power intensity of 0.47 mW cm-2 for the maximum tensile strain of +0.81%, which corresponds to a ∼100.2% increase relative to that of the unstrained case. The in situ tensile strain adjusted the band alignments, making them favorable for enhanced charge transport and thus a higher photoresponse. The structural origin of this superlative balanced photodetection performance was systematically revealed to be associated with the distortion of coupled PbBr6 octahedra and the atomic displacement within the octahedron.

PKC-ß modulates Ca2+ mobilization through Stim1 phosphorylation.

BACKGROUND: Calcium ions play a pivotal role in cell proliferation, differentiation, and migration. Under basal conditions, the calcium level is tightly regulated; however, cellular activation by growth factors increase the ion level through calcium pumps in the plasma membrane and endoplasmic reticulum for calcium signaling. Orai1 is a major calcium channel in the cell membrane of non-excitable cells, and its activity depends on the stromal interaction molecule 1 (Stim1). Several groups reported that the store-operated calcium entry (SOCE) can be modulated through phosphorylation of Stim1 by protein kinases such as extracellular signal-regulated kinase (ERK), protein kinase A (PKA), and p21-activated kinase (PAK). PKC is a protein kinase that is activated by calcium and diacylglycerol (DAG), but it remains unclear what role activated PKC plays in controlling the intracellular calcium pool. OBJECTIVES: Here, we investigated whether PKC-ß controls intracellular calcium dynamics through Stim1. METHODS: Several biochemical methods such as immune-precipitation, site directed mutagenesis, in vitro kinase assay were employed to investigate PKC interaction with and phosphorylation of Stim1. Intracellular calcium mobilization, via Stim1 mediated SOCE channel, were studied using in the presence of PKC activator or inhibitor under a confocal microscope. RESULTS: Our data demonstrate that PKC interacts with and phosphorylates Stim1 in vitro. phosphorylation of Stim1 at its C-terminal end appears to be important in the regulation of SOCE activity in HEK293 and HeLa cells. Additionally, transient intracellular calcium mobilization assays demonstrate that the SOCE activity was inhibited by PKC activators or activated by PKC inhibitors. CONCLUSION: In sum, our data suggest a repressive role of PKC in regulating calcium entry through SOCE.

Curcumin Targets Both Apoptosis and Necroptosis in Acidity-Tolerant Prostate Carcinoma Cells.

OBJECTIVE: Curcumin, a major bioactive curcuminoid derived from the rhizome of Curcuma longa, is known to have anticancer potential and is still under investigation. In this study, we investigated the cytotoxic mechanism(s) of curcumin against acidity-tolerant prostate cancer PC-3AcT cells in lactic acid-containing medium. METHODS: Using 2D-monolyer and 3D spheroid culture models, MTT assay, annexin V-PE binding assay, flow cytometric analysis, measurement of ATP content, and Western blot analysis were used for this study. RESULTS: At nontoxic concentrations in normal prostate epithelial RWPE-1 and HPrEC cells, curcumin led to strong cytotoxicity in PC-3AcT cells, including increases in sub-G0/G1 peak, annexin V-PE-positive cells, and ROS levels; loss of mitochondrial membrane potential; reduction of cellular ATP content; DNA damage; and concurrent induction of apoptosis and necroptosis. A series of changes induced by curcumin were effectively reversed by reducing ROS levels or replenishing ATP. Pretreatment with apoptosis inhibitor Q-VD-Oph-1 or necroptosis inhibitor necrostatin-1 restored cell viability inhibited by curcumin. Treatment of 3D spheroids with curcumin decreased cell viability, accompanied by an increase in mediators of apoptosis and necroptosis, including cleaved caspase-3 and cleaved PARP, phospho (p)-RIP3, and p-MLKL proteins. CONCLUSION: This study shows that curcumin simultaneously induces apoptosis and necroptosis by oxidative mitochondrial dysfunction and subsequent ATP depletion, providing a mechanistic basis for understanding the novel role of curcumin for prostate carcinoma cells.

Apigenin causes necroptosis by inducing ROS accumulation, mitochondrial dysfunction, and ATP depletion in malignant mesothelioma cells.

Apigenin, a naturally occurring flavonoid, is known to exhibit significant anticancer activity. This study was designed to determine the effects of apigenin on two malignant mesothelioma cell lines, MSTO-211H and H2452, and to explore the underlying mechanism(s). Apigenin significantly inhibited cell viability with a concomitant increase in intracellular reactive oxygen species (ROS) and caused the loss of mitochondrial membrane potential (Δ𝚿m), and ATP depletion, resulting in apoptosis and necroptosis in monolayer cell culture. Apigenin upregulated DNA damage response proteins, including the DNA double strand break marker phospho (p)- histone H2A.X. and caused a transition delay at the G2/M phase of cell cycle. Western blot analysis showed that apigenin treatment upregulated protein levels of cleaved caspase-3, cleaved PARP, p-MLKL, and p-RIP3 along with an increased Bax/Bcl-2 ratio. ATP supplementation restored cell viability and levels of DNA damage-, apoptosisand necroptosis-related proteins that apigenin caused. In addition, N-acetylcysteine reduced ROS production and improved Δ𝚿m loss and cell death that were caused by apigenin. In a 3D spheroid culture model, ROS-dependent necroptosis was found to be a mechanism involved in the anti-cancer activity of apigenin against malignant mesothelioma cells. Taken together, our findings suggest that apigenin can induce ROS-dependent necroptotic cell death due to ATP depletion through mitochondrial dysfunction. This study provides us a possible mechanism underlying why apigenin could be used as a therapeutic candidate for treating malignant mesothelioma.

Role of RS-1 derivatives in homology-directed repair at the human genome ATG5 locus.

Genome editing is a useful tool in basic and clinical research. Among the several approaches used in genome editing, the CRISPR-Cas9 system using clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated protein 9 (Cas9) along with a guide RNA has been developed recently. The CRISPR/Cas9 system induces site-specific double-stranded DNA breaks, which result in DNA repair via non-homologous end joining (NHEJ) or homology-directed repair (HDR). However, HDR efficiency is lower than that of NHEJ and accordingly poses a challenge in genome modification studies. Several chemical compounds including RS-1 have been shown to enhance the HDR knock-in process by two- to six-fold in HEK 293 cells and rabbit embryos. Based on this finding, we developed an antibiotic resistance system to screen RS-1 chemical derivatives, which may promote efficient HDR. In this study, we report several chemical compounds with high knock-in efficiency at the ATG5 gene locus, using HeLa cell-based assays.

Pifithrin-µ induces necroptosis through oxidative mitochondrial damage but accompanies epithelial-mesenchymal transition-like phenomenon in malignant mesothelioma cells under lactic acidosis.

Heat shock protein 70 (HSP70), a chaperone protein associated with tumorigenesis and chemoresistance, has attracted significant attention as a potential therapeutic target for the development of anticancer drugs. Here, the effects of pifithrin-µ, an effective dual inhibitor of HSP70 and p53, on anticancer activities and epithelial-mesenchymal transition (EMT) were investigated in malignant mesothelioma (MM) cells. MSTO-211HAcT cells, pre-incubated in a medium containing lactic acid, showed more potent resistance to cisplatin and gemcitabine, compared with their acid-sensitive parental MSTO-211H cells. Pifithrin-µ treatment induced both apoptosis and necroptosis, which were accompanied by an EMT-like phenomenon, as evidenced by an elongated cell morphology, decreased levels of epithelial cell markers including E-cadherin, claudin-1, and ß-catenin, increased levels of mesenchymal markers including Snail, Slug, and vimentin, and increased cell migratory property. Moreover, pifithrin-µ increased intracellular ROS levels, which is associated with mitochondrial dysfunction and decreased cellular ATP content. A series of changes caused by pifithrin-µ treatment were effectively restored by lowering the ROS level through pretreatment with N-acetylcysteine. Collectively, our results suggest that pifithrin-µ may promote the metastatic behavior of surviving cells by triggering the EMT, despite its effective cell-killing action against MM cells, possibly linked to oxidative mitochondrial dysfunction and ATP depletion.

LAMP-3 (Lysosome-Associated Membrane Protein 3) Promotes the Intracellular Proliferation of Salmonella typhimurium.

Lysosomes are cellular organelles containing diverse classes of catabolic enzymes that are implicated in diverse cellular processes including phagocytosis, autophagy, lipid transport, and aging. Lysosome-associated membrane proteins (LAMP-1 and LAMP-2) are major glycoproteins important for maintaining lysosomal integrity, pH, and catabolism. LAMP-1 and LAMP-2 are constitutively expressed in Salmonella-infected cells and are recruited to Salmonella-containing vacuoles (SCVs) as well as Salmonella-induced filaments (Sifs) that promote the survival and proliferation of the Salmonella. LAMP-3, also known as DC-LAMP/CD208, is a member of the LAMP family of proteins, but its role during Salmonella infection remains unclear. DNA microarray analysis identified LAMP-3 as one of the genes responding to LPS stimulation in THP-1 macrophage cells. Subsequent analyses reveal that LPS and Salmonella induced the expression of LAMP-3 at both the transcriptional and translational levels. Confocal Super resolution N-SIM imaging revealed that LAMP-3, like LAMP-2, shifts its localization from the cell surface to alongside Salmonella. Knockdown of LAMP-3 by specific siRNAs decreased the number of Salmonella recovered from the infected cells. Therefore, we conclude that LAMP-3 is induced by Salmonella infection and recruited to the Salmonella pathogen for intracellular proliferation.

The effect of cuff pressure on postoperative sore throat after Cobra perilaryngeal airway.

PURPOSE: The cuff volume of the Cobra perilaryngeal airway (CobraPLA) is larger than that of other alternative airway devices and makes it difficult to predict the effect of cuff pressure on the perilaryngeal mucosa. We tested the hypothesis that adjustment of the cuff pressure of the CobraPLA could reduce the incidence of postoperative sore throat (POST). METHODS: After induction of general anesthesia and insertion of the CobraPLA by standardized method, the cuff pressure was set to 60 cmH(2)O (group C, n = 87) or adjusted to minimal seal-up pressure +5 cmH(2)O (group A, n = 87). The frequency and severity (0, none; 1, mild; 2, moderate; 3, severe) of throat soreness, pain, discomfort, and adverse effects were evaluated 1 and 24 h after removal of the CobraPLA. RESULTS: Incidence of moderate POST in group C was higher than that in group A (11% vs. 2%, P = 0.021) whereas the overall POST incidence was not different between the two groups (31% vs. 20%, P = 0.092). The inflated air volume of group A was different from that of group C (41 vs. 50 ml, P = 0.009). CONCLUSIONS: Adjustment of cuff pressure reduces the incidence of moderate POST after use of the CobraPLA.

[Involvement of splenic hemangioma and rectal varices in a patient with klippel: trenaunay syndrome].

Klippel - Trenaunay syndrome (KTS) is characterized by a cutaneous vascular nevus of the involved extremity, bone and soft tissue hypertrophy of the extremity and venous malformations. We present a case of KTS with splenic hemangiomas and rectal varices. A 29-year-old woman was referred for intermittent hematochezia for several years. She had history with a number of operations for cutaneous and soft tissue hamangiomas since the age of one year old and for increased circumference of her left thigh during the last few months. Abdominal CT revealed multiple hemangiomas in the spleen, fusiform aneurysmal dilatation of the deep veins and soft tissue hemangiomas. There was no evidence of hepatosplenomegaly or liver cirrhosis. Colonoscopy revealed hemangiomatous involvement in the rectum. There were rectal varices without evidence of active bleeding. Upon venography of the left leg, we also found infiltrative dilated superficial veins in the subcutaneous tissue and aneurysmal dilatation of the deep veins. The patient was finally diagnosed with KTS, and treated with oral iron supplementation only, which has been tolerable to date. Intervention or surgery is not required. When gastrointestinal varices or hemangiomatous mucosal changes are detected in a young patient without definite underlying cause, KTS should be considered.

Tongue bite in a patient with tracheostomy after prone position -A case report-.

A 22-year-old man underwent an operation for posterolateral fusion of the lumbar spine at L3-5. He was ventilated via a tracheostomy site in a prone position for 210 minutes. Ventilator function and eyeballs were checked periodically. After changing his position to supine for the wake-up test, it was noticed that his tongue was self-inflicted and looked to be cut unless immediate decompression was applied. After several manual attempts to open the mouth failed, anesthesia depth was deepened with thiopental sodium and neuromuscular blocker to decompress and reposition the tongue into the intraoral cavity. Minimal teeth marks and scarring remained after seven months without any complications.

The effect of airway obstruction on systolic arterial and central venous pressure during sedation in patients undergoing total knee replacement.

BACKGROUND: Severe respiratory variations of systolic arterial and central venous pressure (CVP) may increase the risk of embolic event in orthopedic patient. As airway obstruction during sedation can cause this respiratory variation, we evaluated the degree of variations of systolic blood (SBP) and CVP during airway obstruction period. METHODS: Fifteen females who had obstructed airway during total knee replacement (TKR) were included for the study. After regional anesthesia were established, SBP and CVP variations were analyzed according to the three periods; baseline, obstruction, and airway, respectively. Calculated CVP variables were similar to SBP variables as below: DeltaSBP = Expmax (maximal value at expiration) - Inspnadir (minimal value at inspiration), %DeltaSBP = (DeltaSBP/ Exp(max)) x 100. The frequencies of pulsus paradoxus (PP) and negative inspiratory CVP (NIC) were also measured. RESULTS: At obstruction period, DeltaSBP was 21.7 mmHg and 93.3% of patient had PP. Also, DeltaCVP was 19.3 mmHg and 100% of patient showed NIC. %DeltaCVP (140%) was larger than %DeltaSBP (16%). And DeltaCVP was inversely correlated with baseline and obstruction SBP and %DeltaCVP was also inversely correlated with baseline CVP at obstruction period. CONCLUSIONS: During airway obstruction in sedated TKR patients, variations of CVP are larger than those of SBP. So we have to monitor CVP continuously as well as SBP so as not to increase the possible risk of respiratory of variation.

[Comparison of transient elastography and hepatic fibrosis assessed by histology in chronic liver disease].

BACKGROUND/AIMS: Transient elastography (Fibroscan, Echosens, France) is a new, simple, and non-invasive method to assess the degree of hepatic fibrosis by measuring liver stiffness. Recent reports have shown that liver stiffness measurement using Fibroscan allowed accurate prediction of hepatic fibrosis in patient with chronic hepatitis C. The aim of this study was to evaluate accuracy of Fibroscan for the detection of hepatic fibrosis in Korea with various etiologies of chronic liver disease by comparison with fibrosis assessed by histologic examination. METHODS: Fifty-four patients with chronic liver diseases, which were histologically confirmed within recent 6 months were enrolled. Etiologies were HBV, HCV infection, autoimmune hepatitis, and non alcoholic steatohepatitis. Hepatic fibrosis was graded on the basis of standard guideline proposed by the Korean Study Group for the Pathology of Digestive Diseases. RESULTS: Fibroscan values were significantly higher in F3 (16.96 kPa) and F4 (19.86 kPa) than others (p=0.003). Liver stiffness measurement was significantly correlated to the fibrosis stage (r=0.614, p<0.0001). CONCLUSIONS: Liver stiffness measurement by Fibroscan is a promising method for the assessment of hepatic fibrosis in chronic liver disease because it accompanies no complication.

Prevention of rocuronium-induced withdrawal movement in children: a comparison of remifentanil with alfentanil.

BACKGROUND: This study was designed to compare the efficacy of remifentanil and alfentanil without the venous occlusion technique in preventing the withdrawal response associated with rocuronium injection in children. METHODS: One hundred and twenty children aged between 3 and 10 years were randomly allocated into one of four groups to receive either i.v. remifentanil 0.5 microg.kg(-1) (remi 0.5 group), remifentanil 1 microg.kg(-1) (remi 1.0 group), alfentanil 15 microg.kg(-1) (alfentanil group) or saline 5 ml (saline group). Anesthesia was induced with 2.5% thiopental sodium 5 mg.kg(-1) and the test drug was injected over 30 s. One minute later, 1% rocuronium 0.6 mg.kg(-1) was injected over 5 s and the response was recorded. Mean arterial pressure (MAP) and heart rate (HR) were recorded on arrival in the operating room, before and 1 min after tracheal intubation. RESULTS: The incidence of withdrawal movement in the saline group (93%) was significantly higher than that in the remi 0.5, remi 1.0, and alfentanil groups (53%, 17%, and 20%, respectively) (P < 0.05). The incidence in the remi 1.0 and alfentanil groups was significantly less than that in the remi 0.5 group (P < 0.05). After intubation, MAP and HR were significantly higher in the saline group than that in remi 1.0 and alfentanil groups. CONCLUSIONS: Both remifentanil 1 microg.kg(-1) and alfentanil 15 microg.kg(-1) can be used to prevent rocuronium-associated withdrawal movement in children because they are equally effective and attenuate the increase in MAP and HR after intubation.

GABA and glycine in synaptic microcircuits associated with physiologically characterized primary afferents of cat trigeminal principal nucleus.

Previous studies suggest that sensory information conveyed through trigeminal afferents is more strongly controlled at the level of the first synapse by GABA-mediated presynaptic mechanisms in the trigeminal principal sensory nucleus (Vp) than other sensory nuclei. However, it is unknown if such a mechanism is common to functionally different classes of primary afferent in the same nucleus or across the nuclei. To address these issues, the present study focused on synaptic microcircuits associated with slowly adapting (SA) mechanosensory afferents innervating the periodontal ligaments in the cat Vp and attempted to examine GABA, glycine, and glutamate immunoreactivity in axon terminals involved in the circuits. Afferents were physiologically characterized before injection of horseradish peroxidase (HRP) and preparation for electron microscopy. HRP-labeled afferent boutons were serially sectioned and immunostained with antibodies against GABA, glycine, and glutamate using a postembedding immunogold method. All the afferent boutons examined contacted non-primary dendrites and they were frequently postsynaptic to unlabeled axons (p-endings). Axodendritic and axoaxonic contacts per afferent bouton were 1.3 (46/35) and 2.0 (70/35), respectively. Most p-endings were immunoreactive for GABA (63/70) and also glycine was co-stained in the majority of the p-endings (49/63). Thirty percent of p-endings with the colocalization of GABA and glycine participated in synaptic triads where a p-ending formed a synapse with the same dendrite as the afferent bouton. None of the p-endings was immunoreactive for glutamate. Most afferent boutons were enriched with glutamate but were immunonegative for GABA and glycine. This study provides evidence suggesting that transmission from SA afferents is strongly controlled presynaptically by GABAergic interneurons with colocalized glycine, and that a proportion of these interneurons, involved in synaptic triads, may also have postsynaptic inhibitory actions on target neurons of the SA afferents.