HLA-corrected tumor mutation burden and homologous recombination deficiency for the prediction of response to PD-(L)1 blockade in advanced non-small-cell lung cancer patients.

BACKGROUND: Immune checkpoint inhibitors (ICIs) have been shown to be beneficial for some patients with advanced non-small-cell lung cancer (NSCLC). However, the underlying mechanisms mediating the limited response to ICIs remain unclear. PATIENTS AND METHODS: We carried out whole-exome sequencing on 198 advanced NSCLC tumors that had been sampled before anti-programmed cell death 1 (anti-PD-1)/programmed death-ligand 1 (PD-L1) therapy. Detailed clinical characteristics were collected on these patients. We designed a new method to estimate human leukocyte antigen (HLA)-corrected tumor mutation burden (TMB), a modification which considers the loss of heterozygosity of HLA from conventional TMB. We carried out external validation of our findings utilizing 89 NSCLC samples and 110 melanoma samples from two independent cohorts of immunotherapy-treated patients. RESULTS: Homology-dependent recombination deficiency was identified in 37 patients (18.7%) and was associated with longer progression-free survival (PFS; P = 0.049). Using the HLA-corrected TMB, non-responders to ICIs were identified, despite having a high TMB (top 25%). Ten patients (21.3% of the high TMB group) were reclassified from the high TMB group into the low TMB group. The objective response rate (ORR), PFS, and overall survival (OS) were all lower in these patients compared with those of the high TMB group (ORR: 20% versus 59%, P = 0.0363; PFS: hazard ratio = 2.91, P = 0.007; OS: hazard ratio = 3.43, P = 0.004). Multivariate analyses showed that high HLA-corrected TMB was associated with a significant survival advantage (hazard ratio = 0.44, P = 0.015), whereas high conventional TMB was not associated with a survival advantage (hazard ratio = 0.63, P = 0.118). Applying this approach to the independent cohorts of 89 NSCLC patients and 110 melanoma patients, TMB-based survival prediction was significantly improved. CONCLUSION: HLA-corrected TMB can reconcile the observed disparity in relationships between TMB and ICI responses, and is of predictive and prognostic value for ICI therapies.

Simultaneous determination of water-soluble whitening ingredients and adenosine in different cosmetic formulations by high-performance liquid chromatography coupled with photodiode array detection.

OBJECTIVE: The Korean Cosmetic Act regulates the use of functional cosmetics) by the law. Four functional cosmetic groups, whitening, anti-wrinkle, UV protection and combination of whitening and anti-wrinkle, were categorized according to the Korean Cosmetic Act and Functional Cosmetics Codex. In this study, high-performance liquid chromatography (HPLC) coupled with photodiode array detection (DAD) was employed for the simultaneous detection of arbutin (and its decomposition product, hydroquinone), niacinamide, ascorbyl glucoside, ethyl ascorbyl ether and adenosine in functional cosmetic products such as creams, emulsions and lotions. METHODS: Separation by HPLC-DAD was conducted using a C18 column with a gradient elution of 5 mm KH2PO4 buffer (containing 0.1% phosphoric acid) and methanol (containing 0.1% phosphoric acid). The wavelengths for the detection of arbutin, hydroquinone, niacinamide, adenosine, ascorbyl glucoside and ethyl ascorbyl ether were 283, 289, 261, 257, 238 and 245 nm, respectively. RESULTS: This method exhibited good linearity (R(2) ≥ 0.999), precision (expressed as relative standard deviation (RSD) < 2%) and mean recoveries (89.42-104.89%). The results obtained by monitoring 100 market samples showed that the detected levels of the tested materials are within the acceptable authorized concentration. CONCLUSION: The method developed herein is simple and can be used for market survey and quality control of functional cosmetics.

Radiofrequency ablation as an alternative to hepatic resection for single small hepatocellular carcinomas.

BACKGROUND: This study aimed to investigate whether radiofrequency ablation (RFA) is an alternative to surgical resection for hepatocellular carcinoma (HCC) within the context of current guidelines. METHODS: This retrospective study included patients with normal portal pressure and serum bilirubin level who initially underwent liver resection or RFA for a single HCC of maximum size 3 cm. Between-group differences in cumulative rates of survival and recurrence specific for HCC were analysed in the entire cohort and in a propensity score-matched cohort. RESULTS: A total of 604 patients were enrolled, 273 in the liver resection group and 331 in the RFA group. The 5- and 10-year HCC-specific survival rates for the resection and RFA groups were 87·6 versus 82·1 per cent and 59·0 versus 61·2 per cent respectively (P = 0·214), whereas overall 5- and 10-year recurrence-free survival rates for the corresponding groups were 60·6 versus 39·4 per cent and 37·5 versus 25·1 per cent respectively (P < 0·001). In the propensity score-matched cohort (152 pairs), there were no differences in HCC-specific survival (hazard ratio (HR) 1·03 for RFA versus resection; P = 0·899), whereas recurrence-free survival again differed between the treatment groups (HR 1·75; P < 0·001). RFA was independently associated with poorer outcomes in terms of treatment-site recurrence-free survival (adjusted HR 1·66; P = 0·026), but not non-treatment-site recurrence-free survival (adjusted HR 1·15; P = 0·354). CONCLUSION: Although RFA carries a higher risk of treatment-site recurrence than hepatic resection, it provides comparable overall survival in patients with a single small HCC without portal hypertension or a raised bilirubin level.

Polycythemia is associated with bone loss and reduced osteoblast activity in mice.

UNLABELLED: Increased fragility has been described in humans with polycythemia vera (PV). Herein, we describe an osteoporotic phenotype associated with decreased osteoblast activity in a mouse model of PV and another mouse of polycythemia and elevated circulating erythropoietin (EPO). Our results are important for patients with PV or those treated with recombinant EPO (rEPO). INTRODUCTION: PV and other myeloproliferative syndromes have been recently associated with an increased risk for fractures. However, the presence of osteoporosis in these patients has not been well documented. EPO, a hormone primarily known to stimulate erythropoiesis, has been shown recently to regulate bone homeostasis in mice. The aim of this study was to examine the bone phenotype of a mouse model of PV and compare it to that of animals with polycythemia caused by elevated circulating EPO. METHODS: Bone mass and remodeling were evaluated by micro-computed tomography and histomorphometry. The JAK2(V617F) knock-in mouse, a model of human PV, manifests polycythemia and low circulating EPO levels. Results from this mouse were compared to wild type (wt) controls and the tg6 transgenic mouse that shows polycythemia caused by increased constitutive expression of EPO. RESULTS: Compared to wt, both JAK2(V617F) and tg6 mice had a decrease in trabecular bone mass. Tg6 mice showed an additional modest decrease in cortical thickness and cortical bone volume per tissue volume (P < 0.01) suggesting a more severe bone phenotype than JAK2(V617F). Decreased osteoblast numbers and bone formation along with normal osteoclast numbers and activity were found in both mice. CONCLUSIONS: This study indicates that PV is associated with low bone mass and decreased osteoblast activity in mice. Our results support future studies of osteoporosis in affected humans. Polycythemia caused by chronically elevated circulating EPO also results in bone loss, and implications on patients treated with rEPO should be evaluated.

Acute bleeding in the head and neck: angiographic findings and endovascular management.

BACKGROUND AND PURPOSE: Life-threatening bleeding in the head and neck requires urgent management. This study evaluated the angiographic findings related to head and neck bleeding and presents endovascular management techniques. MATERIAL AND METHODS: Sixty-one consecutive patients who presented with acute bleeding in the head and neck areas and subsequently underwent endovascular therapy between January 2002 and October 2012 were included in our study. We evaluated the angiographic findings, techniques, and results of endovascular management. RESULTS: Contrast leakage (n = 10), pseudoaneurysm (n = 20), or both (n = 10) were the most common life-threatening angiographic findings (66%) and were the foci of immediate embolization or endoluminal vessel reconstruction. Seventeen patients (28%) had hypervascular staining of the tumor or mucosa, and 4 patients (6%) did not have any abnormal findings. The acute bleeding was successfully controlled by endovascular management according to the bleeding foci. Carotid arterial lesions, so-called "carotid blowout," required reconstructive or deconstructive therapy. Bleeding of the external carotid artery required specific branch embolization by a combination of various embolic materials. No procedure-related complications occurred except in 1 patient who experienced acute infarction caused by thromboemboli from the covered stent. Seventeen patients (28%) were retreated due to rebleeding after the mean 20-month follow-up. CONCLUSIONS: Contrast leakage or a pseudoaneurysm or both seen on angiography are active bleeding foci and targets for therapy in patients with acute bleeding in the head and neck area. Despite different bleeding-control strategies according to vessel involvement, endovascular treatment is safe and effective for controlling hemorrhage.

Antitumor activity of IL-32ß through the activation of lymphocytes, and the inactivation of NF-κB and STAT3 signals.

Cytokine and activation of lymphocytes are critical for tumor growth. We investigated whether interleukin (IL)-32ß overexpression changes other cytokine levels and activates cytotoxic lymphocyte, and thus modify tumor growth. Herein, IL-32ß inhibited B16 melanoma growth in IL-32ß-overexpressing transgenic mice (IL-32ß mice), and downregulated the expressions of anti-apoptotic proteins (bcl-2, IAP, and XIAP) and cell growth regulatory proteins (Ki-67 antigen (Ki-67) and proliferating cell nuclear antigen (PCNA)), but upregulated the expressions of pro-apoptotic proteins (bax, cleaved caspase-3, and cleaved caspase-9). IL-32ß also inhibited colon and prostate tumor growth in athymic nude mice inoculated with IL-32ß-transfected SW620 colon or PC3 prostate cancer cells. The forced expression of IL-32ß also inhibited cell growth in cultured colon and prostate cancer cells, and these inhibitory effects were abolished by IL-32 small interfering RNA (siRNA). IL-10 levels were elevated, but IL-1ß, IL-6, and tumor necrosis factor-alpha (TNF-α) levels were reduced in the tumor tissues and spleens of IL-32ß mice, and athymic nude mice. The number of cytotoxic T (CD8(+)) and natural killer (NK) cells in tumor tissues, spleen, and blood was significantly elevated in IL-32ß mice and athymic nude mice inoculated with IL-32ß-transfected cancer cells. Constituted activated NF-κB and STAT3 levels were reduced in the tumor tissues of IL-32ß mice and athymic nude mice, as well as in IL-32ß-transfected cultured cancer cells. These findings suggest that IL-32ß inhibits tumor growth by increasing cytotoxic lymphocyte numbers, and by inactivating the NF-κB and STAT3 pathways through changing of cytokine levels in tumor tissues.

Stylet- or forceps-guided tube exchanger to facilitate GlideScope intubation in simulated difficult intubations--a randomised controlled trial.

The GlideScope videolaryngoscope is widely used in the management of the difficult airway. However, passing the tracheal tube through the vocal cords can be awkward, and the use of a stylet to guide insertion is recommended. This randomised controlled trial evaluated a forceps-guided tube exchanger as an alternative to the stylet to aid intubation with the GlideScope in patients undergoing anaesthesia, with a simulated difficult airway created by the application of a semi-rigid cervical collar. Data were analysed from 178 patients randomly assigned to undergo intubation using either the stylet (n = 88) or a forceps-guided tube exchanger (n = 90). All intubations were completed successfully, with first attempt rates of 93.2% using the stylet and 94.4% using the exchanger (p = 0.597). The mean (SD) intubation time was 67.8 (28.7) s in the stylet group and 66.1 (15.5) s in the forceps-guided tube exchanger group (p = 0.11). The frequency of sore throat 1 h after extubation was 34.1% in the stylet group and 2.2% in the tube exchanger group (p < 0.001); 24 h after extubation the corresponding figures were 40.0% and 11.1% (p < 0.001). Using a forceps-guided tube exchanger may offer an advantage over a stylet in guiding tracheal intubation when the GlideScope is used.

Long-term results of simple hemiresection arthroplasty in the rheumatoid distal radio-ulnar joint.

We retrospectively studied the radiological and clinical results of a simple hemiresection arthroplasty, a modification of previous oblique distal ulnar resections, in 62 patients with rheumatoid arthritis (68 wrists) over a mean follow-up of 103 (range 60-173) months. Fourteen wrists (20%) became fused at the radiocarpal joint and 28 wrists fused (41%) at the midcarpal joints during follow-up. Severe ulnar translocation of the carpus was observed in two wrists (3%), radio-ulnar convergence in five wrists (7%), as well as a significant decrease of carpal height ratio, without any radiological findings of stylocarpal impingement. Improvements in forearm pronation and supination, and reduction in pain were maintained at the last follow-up. Grip strength was not significantly improved. The Jebsen-Taylor hand function test showed improvements in writing, card turning, and simulated feeding. Fifty-two patients (58 wrists, 85%) were satisfied with the results at the last follow-up. Simple hemiresection arthroplasty showed good long-term results with few complications.

4-O-methylhonokiol, a PPARγ agonist, inhibits prostate tumour growth: p21-mediated suppression of NF-κB activity.

BACKGROUND AND PURPOSE: The effects of 4-O-methylhonokiol (MH), a constituent of Magnolia officinalis, were investigated on human prostate cancer cells and its mechanism of action elucidated. EXPERIMENTAL APPROACH: The anti-cancer effects of MH were examined in prostate cancer and normal cells. The effects were validated in vivo using a mouse xenograft model. KEY RESULTS: MH increased the expression of PPARγ in prostate PC-3 and LNCap cells. The pull-down assay and molecular docking study indicated that MH directly binds to PPARγ. MH also increased transcriptional activity of PPARγ but decreased NF-κB activity. MH inhibited the growth of human prostate cancer cells, an effect attenuated by the PPARγ antagonist GW9662. MH induced apoptotic cell death and this was related to G(0) -G(1) phase cell cycle arrest. MH increased the expression of the cell cycle regulator p21, and apoptotic proteins, whereas it decreased phosphorylation of Rb and anti-apoptotic proteins. Transfection of PC3 cells with p21 siRNA or a p21 mutant plasmid on the cyclin D1/ cycline-dependent kinase 4 binding site abolished the effects of MH on cell growth, cell viability and related protein expression. In the animal studies, MH inhibited tumour growth, NF-κB activity and expression of anti-apoptotic proteins, whereas it increased the transcriptional activity and expression of PPARγ, and the expression of apoptotic proteins and p21 in tumour tissues. CONCLUSIONS AND IMPLICATION: MH inhibits growth of human prostate cancer cells through activation of PPARγ, suppression of NF-κB and arrest of the cell cycle. Thus, MH might be a useful tool for treatment of prostate cancer.

AMP-activated protein kinase-α1 as an activating kinase of TGF-ß-activated kinase 1 has a key role in inflammatory signals.

Although previous studies have proposed plausible mechanisms of the activation of transforming growth factor-ß-activated kinase 1 (TAK1) in inflammatory signals, including Toll-like receptors (TLRs), its activating kinase still remains to be unclear. In the present study, we have provided evidences that AMP-activated protein kinase (AMPK)-α1 has a pivotal role for activating TAK1, and thereby regulate NF-κB-dependent gene expressions in inflammatory signaling mediated by TLR4 and TNF-α stimulation. AMPK-α1 specifically interacts with TAK1 and reciprocally regulates their kinase activities. Upon the stimulation of lipopolysaccharide, AMPK-α1-knockdown (AMPK-α1(KD)) or TAK1-knockdown human monocytic THP-1 cells exhibit a dramatic reduction in the TAK1 or AMPK-α1 kinase activity, respectively, and subsequent suppressions of its downstream signaling cascades, which further leads to inhibitions of NF-κB and thereby productions of proinflammatory cytokines, such as TNF-α, IL-1ß, and IL-6. Importantly, the microarray analysis of AMPK-α1(KD) cells revealed a dramatic reduction in the NF-κB-dependent genes induced by TLR4 and TNF-α stimulation, and the observation was in significant correlation with the results of quantitative real-time PCR. Moreover, AMPK-α1(KD) cells are highly sensitive to the TNF-α-induced apoptosis, which is accompanied with dramatic reductions in the NF-κB-dependent and anti-apoptotic genes. As a result, our data demonstrate that AMPK-α1 as an activating kinase of TAK1 has a key role in mediating inflammatory signals triggered by TLR4 and TNF-α.

Spinal matrix metalloproteinase 3 mediates inflammatory hyperalgesia via a tumor necrosis factor-dependent mechanism.

Matrix metalloproteinases (MMPs) have been implicated in the modulation of synaptic plasticity, glial activation, and long-term potentiation in the CNS. Here we demonstrate for the first time a mechanism for the regulation of nociceptive processing by spinal MMP-3 during peripheral inflammation. We first determined by western blotting that the catalytic (active) form of MMP-3 (cMMP-3) is increased in lumbar spinal cord following peripheral inflammation in rats. The peripheral inflammation-induced thermal hyperalgesia and tactile hypersensitivity was transiently (2-3 h) attenuated by intrathecal (IT) pretreatment with either an MMP-3 inhibitor (NNGH), or a broad spectrum MMP inhibitor (GM6001). In addition, IT delivery of cMMP-3 evoked hypersensitivity, whereas the pro (enzymatically inactive) form of MMP-3 did not. This suggests a pro-algesic effect of spinal MMP-3 mediated by an enzymatic mechanism. This cMMP-3-induced hypersensitivity is concurrent with increased tumor necrosis factor (TNF) in the spinal cord. The hypersensitivity behavior is prevented by intrathecal etanercept (TNF blockade). Treatment with cMMP-3 resulted in an increase in TNF release from spinal primary microglial, but not astrocyte cultures. These findings thus present direct evidence implicating MMP-3 in the coordination of spinal nociceptive processing via a spinal TNF-dependent mechanism.

Inhibition of myeloid cell leukemia-1 by tolfenamic acid induces apoptosis in mucoepidermoid carcinoma.

OBJECTIVES: The aim of this study was to evaluate the role of tolfenamic acid (Tol) and ampiroxicam (Amp) in the apoptotic regulation of YD-15 salivary mucoepidermoid carcinoma (MEC). MATERIALS AND METHODS: The effect of Tol on apoptosis and its mechanism were examined using a 3-(4,5-dimethylthiazol-2-yl)-5-(2,4-disulfophenyl)-2-(4-sulfophenyl)-2H-tetrazolium assay, Sub-G(1) population, Western blot analysis, 4'-6-Diamidino-2-phenylindole staining, reverse transcriptase polymerase chain reaction, immunostaining and small interfering RNA transfection. RESULTS: Tol inhibited cell growth of YD-15 cells but Amp did not. Tol induces apoptosis in YD-15 cells as evidenced by nuclear fragmentation, accumulation of the sub-G1 phase and the activation of caspase 3. Tol inhibited myeloid cell leukemia-1 (MCL-1) at the protein and mRNA levels. The treatment of MCL-1 siRNA to YD-15 cells resulted in the activation of caspase 3 and the inhibition of cell growth. Moreover, MCL-1 was regulated by specificity protein 1, but not by mitogen-activated protein kinases. CONCLUSION: These results suggest that Tol could be a potent anti-cancer drug for YD-15 MEC cells that acts by regulating the MCL-1 protein.

IL-32γ inhibits cancer cell growth through inactivation of NF-κB and STAT3 signals.

Several studies have shown physiological functions of interleukin (IL)-32, a novel cytokine. However, the role of IL-32 in cancer development has not been reported. In this study, we showed that IL-32γ inhibited tumor growth in IL-32γ-overexpressing transgenic mice inoculated with melanoma as well as colon tumor growth in xenograft nude mice inoculated with IL-32γ-transfected colon cancer cells (SW620). The inhibitory effect of IL-32γ on tumor growth was associated with the inhibition of constitutive activated nuclear transcription factor-κB (NF-κB) and of signal transducer and activator of transcription 3 (STAT3). The expression of antiapoptotic, cell proliferation and tumor-promoting genes (bcl-2, X-chromosome inhibitor of apoptosis protein (IAP), cellular IAP and cellular FADD-like IL-1ß-converting enzyme-inhibitory protein, cyclin D), cyclin-dependent kinase 4, cycolooxygenase-2 and inducible nitric oxide synthase was decreased, whereas the expression of apoptotic target genes (caspase-3 and -9, bax) increased. In tumor, spleen and blood, the number of cytotoxic CD8(+) T cells and CD57(+) natural killer cells and the levels of IL-10 increased, but that of tumor necrosis factor-α (TNF-α), IL-1ß and IL-6 decreased. We also found that forced overexpression of IL-32γ inhibited colon cancer cell (SW620 and HCT116) growth accompanied with the inhibition of activated NF-κB and STAT3 in vitro. In addition, when IL-32γ was knocked down by small interfering RNA (siRNA) or neutralized with an anti-IL-32γ antibody, IL-32γ-induced colon cancer cell growth inhibition, the IL-32γ-induced decrease of TNF-α, IL-1 and IL-6 production, and the increase of IL-10 production were abolished. However, siRNA of NF-κB and STAT3 augmented IL-32γ-induced colon cancer cell growth inhibition. These findings indicate significant pathophysiological roles of IL-32γ in cancer development.

Apoptotic effect of Polygonum Cuspidatum in oral cancer cells through the regulation of specificity protein 1.

OBJECTIVES: The aim of this study was to evaluate the growth inhibitory and apoptosis-inducing effects and mechanisms of Polygonum cuspidatum root in oral cancer cells. MATERIALS AND METHODS: The testing materials were separated by normal-phase silica gel liquid chromatography. The effect of P. cuspidatum root on apoptotsis and its mechanism were performed using 3-(4,5-dimethylthiazol-20yl)-(3-carboxymethoxyphenyl)-2-(4-sulphophenyl)-2H-tetrazolium) (MTS) assay, western blot analysis, RT-PCR, promoter assay, and (4'-6-Diamidino-2-phenylindole) (DAPI) staining. RESULTS: The methanol extract of P. cuspidatum (MEPC) inhibited the proliferation of oral cancer cells by inducing caspase-dependent apoptosis. Protein and mRNA expression levels and the transactivation of Specificity protein 1 (Sp1) were markedly decreased in KB cells treated with MEPC. Ethyl acetate fraction (EA) from MEPC was more potent than aqueous fraction (AQ) from MEPC to induce apoptosis. F2, F3, and F4 from EA differentially inhibited the growth of KB cells, and it depends on the amount of Emodin in F2, F3, and F4. Moreover, Emodin inhibited oral cancer cell growth and induced caspase-dependent apoptosis by decreasing Sp1. MEPC also decreased an apoptosis-related downstream target of Sp1 protein, survivin. CONCLUSION: The results from this study strongly suggest that MEPC, its fraction, and Emodin may be potential bioactive materials to cause apoptosis mechanism via the down-regulation of Sp1 in oral cancer cells.

Physicochemical properties, and antioxidant and antimicrobial effects of garlic and onion powder in fresh pork belly and loin during refrigerated storage.

Pork loin and belly cuts were pumped to 110% of their original weight with solutions containing 5% of various ingredients (sodium ascorbate, garlic, and onion powder), and evaluated the physicochemical properties, and antioxidant and antimicrobial activities during refrigerated storage at 8 degrees C. The addition of garlic and onion powder tended to increase redness (a) and yellowness (b) in both the belly lean and loin with the exception of a few cases. Free fatty acid values in both pork belly and loin cuts were reduced with the addition of these ingredients, as compared to the control. Significant differences in peroxide values were observed in sodium ascorbate and garlic-injected belly, and in sodium ascorbate and onion-injected loin, as compared to the control. Thiobarbituric acid reactive substance values in the pork belly with garlic or onion powder were significantly lower than in the belly without these ingredients or with sodium ascorbate (P < 0.05). Total plate counts were lower in both the belly and loin containing garlic and onion powder, as compared to the control. In both the belly and loin cuts, the content of oxidative products (volatile compounds) was reduced with the addition of garlic and onion powder, particularly the aldehydes (hexanal). Overall, garlic and onion in enhanced meats showed an antioxidant activity as effective as that of sodium ascorbate and also an antimicrobial effect to inhibit the growth of total bacteria and Enterobacteriaceae.

Caspase-3 activation as a key factor for HBx-transformed cell death.

OBJECTIVES: Nuclear factor-kappa B (NF-kappaB) activation has been associated with the tumorigenic growth of hepatitis B virus X protein (HBx)-transformed cells. This study was aimed to find a key target for treatment of HBx-mediated cancers. MATERIALS AND METHODS: NF-kappaB activation, endoplasmic reticulum-stress (ER-stress), caspase-3 activation, and cell proliferation were evaluated after Chang/HBx cells permanently expressing HBx viral protein were treated with inhibitors of NF-kappaB, proteasome and DNA topoisomerase. RESULTS: Inhibition of NF-kappaB transcriptional activity by transient transfection with mutant plasmids encoding Akt1 and glycogen synthase kinase-3beta (GSK-3beta), or by treatment with chemical inhibitors, wortmannin and LY294002, showed little effect on the survival of Chang/HBx cells. Furthermore, IkappaBalpha (S32/36A) mutant plasmid or other NF-kappaB inhibitors, 1-pyrrolidinecarbonidithioic acid and sulphasalazine, were also shown to have little effect on the cell proliferation. By contrast, proteasome inhibitor-1 (Pro1) and MG132 enhanced the HBx-induced ER-stress response and the subsequent activation of caspase-12, -9 and -3 and reduced cell proliferation. Camptothecin (CPT), however, triggered activation of caspase-3 without induction of caspase-12, and reduced cell proliferation. In addition, CPT-induced cell death was reversed by pre-treatment with z-DEVD, a caspase-3-specific inhibitor. CONCLUSIONS: Detailed exploitation of the regulators of caspase-3 activation could open the gate for finding an efficient target for development of anticancer therapeutics against HBx-transformed hepatocellular carcinoma.

Protective effect of oxidative stress in HaCaT keratinocytes expressing E7 oncogene.

In a previous study, we established a stable cell line which constitutively expresses E7 in HaCaT human keratinocyte cell line and identified various relevant factors including oxygen modulators affected by the E7 oncogene. E7-expressing HaCaT cells (HaCaT/E7) appeared to be more resistant to H2O2-induced cell death. Here, we demonstrate how E7 oncogene would modulate oxidative stress-induced cell death. In addition, we verified the increased expression of catalase in the HaCaT/E7 by Western blot analysis. The results suggest that the E7 oncogene would induce higher resistance to ROS-induced cell injury in the E7-infected cells via the upregulation of catalase. To investigate these paradoxical effects of high concentrations of H2O2 (500 microM-1 mM), we examined their effects on receptor mediated apoptosis, cell death via the mitochondrial pathway and modulation of apoptosis related factors. Our results revealed that HaCaT keratinocytes infected with HPV 16 E7 oncogene modulated expressions of catalase, Bcl-xL, IL-18, Fas, Bad, and cytochrome c as well as NF-kappaB, resulting in the resistance to oxidative stress-induced cell death.

Directed differentiation of human embryonic stem cells towards a pancreatic cell fate.

AIMS/HYPOTHESIS: The relative lack of successful pancreatic differentiation of human embryonic stem cells (hESCs) may suggest that directed differentiation of hESCs into definitive endoderm and subsequent commitment towards a pancreatic fate are not readily achieved. The aim of this study was to investigate whether sequential exposure of hESCs to epigenetic signals that mimic in vivo pancreatic development can efficiently generate pancreatic endodermal cells, and whether these cells can be further matured and reverse hyperglycaemia upon transplantation. MATERIALS AND METHODS: The hESCs were sequentially treated with serum, activin and retinoic acid (RA) during embryoid body formation. The patterns of gene expression and protein production associated with embryonic germ layers and pancreatic endoderm were analysed by RT-PCR and immunostaining. The developmental competence and function of hESC-derived PDX1-positive cells were evaluated after in vivo transplantation. RESULTS: Sequential treatment with serum, activin and RA highly upregulated the expression of the genes encoding forkhead box protein A2 (FOXA2), SRY-box containing gene 17 (SOX17), pancreatic and duodenal homeobox 1 (PDX1) and homeobox HB9 (HLXB9). The population of pancreatic endodermal cells that produced PDX1 was significantly increased at the expense of ectodermal differentiation, and a subset of the PDX1-positive cells also produced FOXA2, caudal-type homeobox transcription factor 2 (CDX2), and nestin (NES). After transplantation, the PDX1-positive cells further differentiated into mature cell types producing insulin and glucagon, resulting in amelioration of hyperglycaemia and weight loss in streptozotocin-treated diabetic mice. CONCLUSIONS/INTERPRETATION: Our strategy allows the progressive differentiation of hESCs into pancreatic endoderm capable of generating mature pancreatic cell types that function in vivo. These findings may establish the basis of further investigations for the purification of transplantable islet progenitors derived from hESCs.

Fluctuating valence in a correlated solid and the anomalous properties of delta-plutonium.

Although the nuclear properties of the late actinides (plutonium, americium and curium) are fully understood and widely applied to energy generation, their solid-state properties do not fit within standard models and are the subject of active research. Plutonium displays phases with enormous volume differences, and both its Pauli-like magnetic susceptibility and resistivity are an order of magnitude larger than those of simple metals. Curium is also highly resistive, but its susceptibility is Curie-like at high temperatures and orders antiferromagnetically at low temperatures. The anomalous properties of the late actinides stem from the competition between itinerancy and localization of their f-shell electrons, which makes these elements strongly correlated materials. A central problem in this field is to understand the mechanism by which these conflicting tendencies are resolved in such materials. Here we identify the electronic mechanisms responsible for the anomalous behaviour of late actinides, revisiting the concept of valence using a theoretical approach that treats magnetism, Kondo screening, atomic multiplet effects and crystal field splitting on the same footing. We find that the ground state in plutonium is a quantum superposition of two distinct atomic valences, whereas curium settles into a magnetically ordered single valence state at low temperatures. The f(7) configuration of curium is contrasted with the multiple valences of the plutonium ground state, which we characterize by a valence histogram. The balance between the Kondo screening and magnetism is controlled by the competition between spin-orbit coupling, the strength of atomic multiplets and the degree of itinerancy. Our approach highlights the electronic origin of the bonding anomalies in plutonium, and can be applied to predict generalized valences and the presence or absence of magnetism in other compounds starting from first principles.

Determination of volatile flavor components in danggui cultivars by solvent free injection and hydrodistillation followed by gas chromatographic-mass spectrometric analysis.

The rhizome of Angelica gigas Nakai, Angelica sinensis Diels and Angelica acutiloba Kitagawa (Umbelliferae) were chopped and extracted by hydrodistillation (HD) and solvent free solid injector (SFSI) methods to obtain the volatile compounds that were then characterized and identified by gas chromatography-mass spectrometry (GC/MS). SFSI extraction yielded a generally higher amount of volatile compounds than that produced by HD. A total of 48 components [including terpenes (8), aldehydes (4), alcohol (2), coumarins (9), Phthalide (3), acids (2) and sterols (2)] were identified by SFSI and gas chromatography-mass spectrometry from the three species of danggui cultivars, whereas, 24-essential oil was obtained by HD for Korean danggui only. According to these analyses, several coumarin derivatives such as decursinol angelate (16.83%) and decursin (29.34%) were found to be the dominant ones, followed by lomatin (10.25%) and marmesin (9.33%) in Korean danggui. Furfural and butylidene phthalide were the main components in Japanese in addition to butylidene dihydro-phthalide in Chinese danggui. Some parameters affecting the extraction efficiency such as injector temperature, injection time and pre-heating time were optimized. The results showed that the SFSI-method gave a higher yield of components with higher molecular mass than hydrodistillation. SFSI required little time to prepare the sample, little sample mass and a small quantity of organic solvent was needed. It can be concluded that analysis of volatile flavor compounds by SFSI in combination with gas chromatography/mass spectrometry is a suitable monitoring technique to differentiate danggui cultivars.