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To clarify the roles of specific isoforms of PKC in regulating growth and cell cycle progression of the HC11 mammary epithelial cell line, we investigated the effects of activating endogenous PKC isoforms with the phorbol ester tumor promoter TPA, and also the effects of TPA on genetically engineered cells containing increased levels of individual PKC isoforms. We found that TPA treatment of HC11 cells induced a transient cell cycle arrest in G0/G1. Western blot analyses of the TPA treated cells provided evidence that the endogenous PKC alpha present in these cells mediated these effects. Indeed, derivatives of the HC11 cell line that inducibly overexpress an exogenous PKC alpha or ectopic PKC beta 1 exhibited more marked growth inhibition by TPA than control cells. Immunohistochemical staining of cells following treatment with TPA revealed selective translocation of PKC alpha into the nucleus, whereas PKC beta 1 remained in the cytoplasm. The transient arrest of HC11 cells following treatment with TPA was associated with marked induction of both p21cip1 mRNA and protein. This induction was exaggerated in the derivatives that overexpressed either PKC alpha or PKC beta 1. Therefore, in mouse mammary epithelial cells activation of the endogenous PKC alpha can transiently arrest cells in G0/G1 which may be due, at least in part, to induction of the transcription of p21cip1.

We examined the activity of two metabolites of sulindac (a nonsteroidal anti-inflammatory drug), sulindac sulfide and sulindac sulfone (exisulind, Prevatec), and a novel highly potent analog of exisulind (CP248) on a series of human prostate epithelial cell lines. Marked growth inhibition was seen with the BPH-1, LNCaP, and PC3 cell lines with IC50 values of about 66 microM, 137 microM, and 64 nM for sulindac sulfide, exisulind, and CP248, respectively. DNA flow cytometry and 4',6'-diamido-2-phenylindole (DAPI) staining indicated that these three compounds also induced apoptosis in all of these cell lines. Similar growth inhibition also was seen with the PrEC normal human prostate epithelial cell line, but these cells were resistant to induction of apoptosis at concentrations up to 300 microM, 1 mM, and 750 nM of sulindac sulfide, exisulind, and CP248, respectively. Derivatives of LNCaP cells that stably overexpress bcl-2 remained sensitive to growth inhibition and induction of apoptosis by these compounds. In vitro enzyme assays indicated that despite its high potency in inhibiting growth and inducing apoptosis, CP248, like exisulind, lacked cyclooxygenase (COX-1 and COX-2) inhibitory activity even at concentrations up to 10 mM. Moreover, despite variations of COX-1 and COX-2 expression, the three benign and malignant prostate cell lines showed similar sensitivity to growth inhibition and induction of apoptosis by these three compounds. Therefore, sulindac derivatives can cause growth inhibition and induce apoptosis in human prostate cancer cells by a COX-1 and -2 independent mechanism, and this occurs irrespective of androgen sensitivity or increased expression of bcl-2. These compounds may be useful in the prevention and treatment of human prostate cancer.

It is now apparent that apoptosis is closely linked to the control of cell cycle progression. During the G1 to S progression, cyclin D1, p53, and the cyclin dependent kinase inhibitors p21WAF1 and p27kip1 can play roles in induction of apoptosis. During the G2 and M phases, premature activation of Cdk1 can cause cells to enter mitotic catastrophe, which results in apoptosis. In this review we focus on factors acting during G1 and S, particularly cyclin D1, and their effects on cell growth, senescence and apoptosis. We emphasize that cyclin D1 can have diverse effects on cells depending on its level of expression, the specific cell type, the cell context and other factors. Possible mechanisms by which cyclin D1 exerts these diverse effects, via cyclin dependent kinase-dependent and -independent pathways, are discussed.

Magnetic force microscopy (MFM) can be used to image current distributions in current leads of sub-micron dimensions. Here we present a systematic study about the spatial and force resolution of such currents. In the case of force resolution, we studied the least measurable magnetic force of MFM for different sample currents. The analysis of images from parallel Al conducting plates are combined with those from force-distance curves and finite element calculations. Several interacting regimes between the magnetic tip and the currents are found and interpreted. It is shown that model calculations are necessary even for qualitative image interpretation. Then spatial resolution in the range of 100nm can well be obtained and quantitative studies of current distribution on widths of 10nm resolution are possible in special cases. The approach is demonstrated in imaging the current distribution in superconducting Bi2Sr2CaCu2Ox single crystals.

In previous studies, our laboratory demonstrated that Rat 6 (R6) fibroblasts which stably overproduce high levels of PKCepsilon display abnormalities in growth control that are characteristic of malignant transformation (Cacace et al., 1993, Oncogene, 8:2095-2104). The R6-PKCepsilon overproducing cell lines also exhibited a decreased growth factor requirement. The present study demonstrates that conditioned medium (CM) from two individual clones, R6-PKCepsilon 10 and 30, stimulates DNA synthesis in control R6-C1 cells. Maximal DNA synthesis and morphologic transformation was achieved in control cells when they were treated with medium from R6-PKCepsilon cells grown in the presence of TPA (TPA-CM). Size fractionation of the TPA-CM from PKCepsilon 30 cells revealed that this activity is due to a factor(s) that has an apparent molecular weight in the range of 10-30 kD and is heat and acid stable. This factor, like TGFbeta1, stimulated anchorage-independent growth of NRK cells. Western blot analysis (under nonreducing conditions) of the TPA-CM from R6-PKCepsilon 30 and R6-PKCepsilon 10 cells revealed the presence of the 25 kD active forms of TGFbeta2 and 3. These active forms of TGFbeta were not found in the CM of control R6 cells, or R6 cells that overexpress PKCalpha or PKCbeta1. The addition of a pan-specific TGFbeta antibody to NRK cells treated with the 10-30 kD fraction of TPA-CM from PKCepsilon 30 cells blocked the ability of this material to stimulate thymidine incorporation. Taken together, these studies suggest that the oncogenic activity of PKCepsilon in R6 cells is due, at least in part, to its ability to induce production of the active forms of TGFbeta2 and 3.

Background The cyclin D1 gene is amplified and/or overexpressed in several types of human cancer, including cancers of the breast, esophagus, head, and neck. However, the role of cyclin D1 in prostate cancer has not been previously studied in detail. Methods Six human prostate cancer cell lines and cultures of normal human prostate cells were examined by Western and Northern blot analyses for levels of expression of the cyclin D1 protein and mRNA, respectively. Southern blot analyses were performed to examine possible amplification of this gene. Immunostaining for cyclin D1 was performed on 50 primary prostate cancer samples. Results Cyclin D1 protein was expressed at relatively high levels in all of the six human prostate cancer cell lines examined, but was not detected in the cultures of normal human prostate cells. The ALVA 41 cell line expressed the highest level of this protein. Relatively high levels of cyclin D1 mRNA were also found in all of the prostate cancer cell lines. Nevertheless, none of these cell lines revealed amplification of the cyclin D1 gene. Twelve of the 50 primary prostate cancer samples (24%) revealed regions of moderate to strongly positive staining for cyclin D1. Conclusions The increased expression of cyclin D1 in several prostate cancer cell lines and in a subset of primary prostate cancer samples suggests that further studies on the expression of this gene and related genes may be of interest in understanding the pathogenesis of prostate cancer.

BACKGROUND & AIMS: Mutations of c-K-ras occur commonly in colonic neoplasms. The aim of this study was to determine how c-K-ras mutations alter the responses to the chemopreventive agent sulindac. METHODS: The parental rat intestinal cell line IEC-18 and c-K-ras-transformed derivatives were treated with sulindac sulfide. Cell cycle distribution was determined by flow-cytometric analysis (fluorescence-activated cell sorter), apoptosis by DNA fragmentation (laddering), flow cytometry, and microscopy, and changes in gene expression by immunoblotting. RESULTS: Sulindac sulfide inhibited cell growth and induced apoptosis in a time- and dose-dependent manner more rapidly in and at lower concentrations in parental cells than ras-transformed cells. Expression of the sulindac sulfide arrested cells in G0/G1, but cells entered apoptosis throughout the cell cycle. Proapoptotic protein Bak was relatively high in untreated parental cells and increased markedly after sulindac sulfide but was low in untreated ras-transformed cells and did not increase after sulindac sulfide. Expression of other Bcl-2 family members was unchanged after sulindac sulfide. However, sulindac sulfide reduced levels of cyclin D1 protein and cyclin E- and cyclin D1- associated kinase activity. CONCLUSIONS: c-K-ras-transformed enterocytes are relatively resistant to sulindac sulfide-induced growth inhibition and apoptosis, which may result from specific reduction of bak expression. (Gastroenterology 1997 Dec;113(6):1892-900)

It is possible to overcome Cu void issues beyond 10nm node device by adapting CVD-Ru liner instead of conventional PVD Ta liner. However, CVD Ru liner integration degrades TDDB performance without optimizing its scheme. In this paper, superior gap-fill performance without TDDB performance degradation will be described in our optimized integration scheme along with a proposal for the mechanism of TDDB degradation in the Ru integration scheme. CVD-Ru liner is the prime candidate for Cu metallization at 10nm node and beyond.

1 Poisoning is a common reason for presentation to hospital and hospital admission but there is no agreed policy for managing these patients. This study exam ined the management of patients presenting with poisoning and the factors affecting the probability of hospital admission and prolonged stay. 2 Data on all cases of poisoning presenting to six Accident and Emergency departments in the North East of England over 12 weeks in 1994 was collected prospectively from A&E notes. Length of stay and outcome were recorded from hospital computer records. 3 Overall, 73% of patients were admitted to a medical ward. Probability of admission was not independently affected by age or gender but was increased in those with intentional poisoning (Odds Ratio (OR) 3.3 [95% CI 1.8, 6.1]), a history of self harm (OR 1.7, [1.0, 2.9]) or potentially hazardous poisoning (OR 3.7 [2.1, 6.6]). There were significant variations between hospitals (50 - 80%) which could not be attributed to case mix. 4 Prolonged stay ( > 2 nights) was more common in patients over 65 years (OR 6.8 [2.9, 16.1]), those with intentional poisoning (OR 2.7 [1.1, 6.6]) and those with potentially hazardous poisoning (OR 2.6 [1.4, 4.9]). Mean hospital stay was 1.5 days and varied signifi cantly between hospitals from 0.8 to 2.1 days and this was independent of case mix. 5 There are appreciable variations in the management of poisoning between hospitals which are not explained by patient characteristics. Savings would occur if rates of admission and duration of stay were reduced by those hospitals where admission is more frequent or hospital stay is longer. However, the impact of this on long term morbidity is unknown.

Epidemiological studies have shown that inhalation of radon is associated with an increased risk for lung cancer among underground miners. The cellular and molecular mechanisms for radon-induced carcinogenesis are not clear. By using a human papillomavirus-immortalized human bronchial epithelial cell line, we show here that a single 30 cGy dose of these high-energy particles induced tumorigenic conversion of the cells 3–4 months postirradiation at frequencies estimated to be ∼4 × 10−7. Transformed cells arise through a series of sequential steps, including altered growth kinetics, loss of response to serum-induced terminal differentiation, anchorage-independent growth, and rumorigenesis, with each preceding stage representing a necessary yet insufficient step toward the later, more malignant phase. Northern and Western blot analyses showed overexpression of the G1 cell-cycle-control kinase, cyclin D1. Analysis of genomic DNA from the tumorigenic cell lines using polymerase chain reaction amplification and restriction enzyme analysis demonstrated no mutation in the K-ras oncogene, which has frequently been found to be mutated in human lung cancers. Western blot analysis showed that both control BEP2D cells and their tumorigenic variants contained phosphorylated Rb proteins. This model provides an opportunity to study the cellular and molecular changes at the various stages in radiation carcinogenesis involving human cells. © 1995 Wiley-Liss, Inc.

Dysregulation of cyclin expression has been reported for several human malignancies, including breast cancer. To further investigate the role of cyclin genes in mammary tumorigenesis we analyzed the expression of cyclins D1, E and A and other cell cycle-related proteins in a series of nine N-methyl-N-nitrosourea-induced primary rat mammary tumors. Western blot analysis revealed a 10- to 15-fold increase in the level of cyclin D1 protein in most (7/9) of the tumors, when compared with normal rat mammary gland. The two tumors that did not show this increase also displayed negligible levels of the retinoblastoma protein. A moderate increase, 1.5- to 2-fold, in the level of cyclin E was observed in four tumors and three tumors displayed abnormal low molecular weight cyclin E-related proteins. None of the tumors showed amplification of the cyclin D1 or E genes when studied by Southern blot analysis. All nine tumors showed a 2- to 6-fold increase in the level of cyclin A protein. Most of the tumors also displayed a marked increase in levels of the CDK2 and CDK4 proteins. These changes did not appear to be simply a consequence of increased cell proliferation, as assessed by proliferating cell nuclear antigen analysis. Thus, aberrant expression of cyclins and other cyclin-related genes occurs frequently in mammary tumorigenesis in both rodents and humans.

A multistep pulsed-laser deposition (PLD) process is presented for epitaxial, nominally undoped ZnO thin films of total thickness of 1 to 2 μm on c-plane sapphire substrates. We obtain reproducibly high electron mobilities from 115 up to 155 cm2/V s at 300 K in a narrow carrier concentration range from 2 to 5×1016 cm−3. The key issue of the multistep PLD process is the insertion of 30-nm-thin ZnO relaxation layers deposited at reduced substrate temperature. The high-mobility samples show atomically flat surface structure with grain size of about 0.5–1 μm, whereas the surfaces of low-mobility films consist of clearly resolved hexagonally faceted columnar grains of only 200-nm size, as shown by atomic force microscopy. Structurally optimized PLD ZnO thin films show narrow high-resolution x-ray diffraction peak widths of the ZnO(0002) ω- and 2Θ-scans as low as 151 and 43 arcsec, respectively, and narrow photoluminescence linewidths of donor-bound excitons of 1.7 meV at 2 K.

To assess the association between high body mass index (BMI) and treatment response in recent-onset rheumatoid arthritis.In the Behandelstrategieën voor Reumatoide Artritis (Treatment Strategies for Rheumatoid Arthritis) study, 508 patients were randomized to initial monotherapy or combination therapy with prednisone or infliximab (IFX). The response to Disease Activity Score (DAS) ≤2.4-steered treatment (first dose and after 1 year) was compared between patients with a BMI25 kg/m(2) and ≥25 kg/m(2) , using relative risk (RR) regression analyses. DAS, components of DAS, and functional ability during the first year were compared using linear mixed models.High BMI was independently associated with failure to achieve a DAS ≤2.4 on initial therapy (RR 1.20 [95% confidence interval (95% CI) 1.05, 1.37]). The effect for combination therapy with prednisone was RR 1.55 (95% CI 1.06, 2.28) and for combination therapy with IFX 1.42 (95% CI 0.98, 2.06). The RRs for failure after 1 year were 1.46 (95% CI 0.75, 2.83) and 2.20 (95% CI 0.99, 4.92), respectively. High BMI was also associated with failure on delayed combination therapy with IFX, after adjustment for selection bias related to previous failure on disease-modifying antirheumatic drugs. No significant association was observed in the initial monotherapy groups. In the first year, patients with a high BMI had higher DAS and worse functional ability, with more tender joints and a higher visual analog scale global health, but not more swollen joints and similar systemic inflammation.High BMI was independently associated with failure to achieve low DAS on initial combination therapy with prednisone and on initial and delayed treatment with IFX. Patients with a high BMI experienced more pain, but not more swelling or systemic inflammation.

The endocannabinoid system is important in the pathogenesis of obesity-related metabolic disorders. However, the effect of inhibiting the endocannabinoid system in type 2 diabetic nephropathy is unclear. Therefore, we examined the effect of the cannabinoid (CB)1 receptor antagonist, SR141716, on insulin resistance and diabetic nephropathy in db/db mice. Six-week-old db/db mice were treated with the CB1-specific antagonist SR141716 (10 mg/kg · d) for 3 months. Treatment with SR141716 significantly improved insulin resistance and lipid abnormalities. Concomitantly, CB1 antagonism improved cardiac functional and morphological abnormality, hepatic steatosis, and phenotypic changes of adipocytes into small differentiated forms, associated with increased adiponectin expression and decreased lipid hydroperoxide levels. CB1 receptor was overexpressed in diabetic kidneys, especially in podocytes. Treatment with the SR141716 markedly decreased urinary albumin excretion and mesangial expansion and suppressed profibrotic and proinflammatory cytokine synthesis. Furthermore, SR141716 improved renal lipid metabolism and decreased urinary 8-isoprostane levels, renal lipid hydroperoxide content, and renal lipid content. In cultured podocytes, high-glucose stimulation increased CB1 receptor expression, and SR141716 treatment abolished high-glucose-induced up-regulation of collagen and plasminogen activator inhibitor-1 synthesis. Additionally, knockdown of CB1 receptor expression by stealth small interfering RNA abolished high-glucose-induced sterol-regulatory element-binding protein-1 expression in podocytes. These findings suggest that CB1 blockade improves insulin resistance and protect against renal injury through both metabolic and antifibrotic effects in type 2 diabetic nephropathy. Targeting CB1 blockade could therefore provide a new therapeutic target to prevent type 2 diabetic nephropathy.

Background Various cytokines and inflammatory mediators are known to be involved in the pathogenesis of rheumatoid arthritis (RA). We hypothesized that polymorphisms in selected inflammatory response and tissue repair genes contribute to the susceptibility to and severity of RA. Methods Polymorphisms in TNFA, IL1B, IL4, IL6, IL8, IL10, PAI1, NOS2a, C1INH, PARP, TLR2 and TLR4 were genotyped in 376 Caucasian RA patients and 463 healthy Caucasian controls using single base extension. Genotype distributions in patients were compared with those in controls. In addition, the association of polymorphisms with the need for anti-TNF-α treatment as a marker of RA severity was assessed. Results The IL8 781 CC genotype was associated with early onset of disease. The TNFA -238 G/A polymorphism was differentially distributed between RA patients and controls, but only when not corrected for age and gender. None of the polymorphisms was associated with disease severity. Conclusions We here report an association between IL8 781 C/T polymorphism and age of onset of RA. Our findings indicate that there might be a role for variations in genes involved in the immune response and in tissue repair in RA pathogenesis. Nevertheless, additional larger genomic and functional studies are required to further define their role in RA.

P-glycoprotein is phosphorylated in cells, and it has been suggested that phosphorylation may regulate the drug transport activity of P-glycoprotein. Domain mapping, utilizing a combination of cyanogen bromide digestion and immunoblot analysis, was used to reveal the major phosphorylation sites in murine mdr1b P-glycoprotein. After labeling of J7.V1-1 cells with [32P]Pi, or labeling membranes with [gamma-32P]ATP and either protein kinase A or protein kinase C, it was found that the majority of the label was contained within a single cyanogen bromide fragment (amino acid 627-682) that encompassed the majority of the linker region. The in vitro protein kinase C phosphorylation sites within this fragment were analyzed by a combination of fast atom bombardment mass spectrometry (FABMS) and two-dimensional phosphopeptide mapping. FABMS analysis of a protein kinase C-phosphorylated synthetic peptide, corresponding to a segment of the linker region of P-glycoprotein, identified serine 669 as the single site of phosphorylation. Comparison of two-dimensional tryptic phosphopeptide maps prepared from synthetic peptide and P-glycoprotein, both of which were phosphorylated in vitro with protein kinase C, revealed that serine 669 was also the major phosphorylation site in the intact glycoprotein. The in vitro protein kinase A phosphorylation site was identified as serine 681 by site-directed mutagenesis. Inspection of the gene organization and the deduced amino acid sequence of mdr1b P-glycoprotein revealed that the linker region, although shorter than the R domain (55 versus 241 amino acids), fits the operational definition of the R domain of cystic fibrosis conductance regulator. Like the R domain, the linker region is encoded by a single exon, is highly charged with alternating acidic and basic side chains, and contains several protein kinase A/protein kinase C consensus phosphorylation sites. Since the R domain is believed to be involved in the regulation of cystic fibrosis conductance regulator function by phosphorylation, it is possible that the linker region plays a similar regulatory role in P-glycoprotein function.

STUDY OBJECTIVE--To investigate the numbers and characteristics of patients with ophthalmological accidents and emergencies presenting to hospitals. DESIGN--Prospective survey over eight weeks. SETTING--Two general and one ophthalmic accident and emergency departments, two general outpatient departments, and an eye hospital ward consulting room (all in two teaching hospitals) in Newcastle upon Tyne. MEASUREMENTS AND MAIN RESULTS--Consultation numbers by age, sex, health district of residence, source of referral, diagnosis, and disposal were determined. An average of 37 ophthalmological emergency patients were seen daily. The all cause consultation rate per 1000 population for Newcastle residents was 2.64 (17.2 per year); for injuries it was 1.10 (7.2 per year) and for inflammations the rate was 0.91 (5.9 per year). Consultation rates per 1000 were 3.5 for males and 1.8 for females, the excess being explained by the higher risk of injury to men. Most patients were self-referred (58%), consulted during office hours (79.6%), were attended by senior house officers working alone (83.9%), and were asked to return for follow up (66.1%). Patients in an accident and emergency department seldom saw a consultant in their initial management. The diagnoses of patients from outside Newcastle were little different from those who lived within the city. The 10 commonest problems accounted for 68% of all cases. Injuries were the commonest problem (40.9% of all diagnoses). CONCLUSION--Ophthalmological accident and emergencies are an important component of an accident and emergency department workload. These patients are usually seen by junior doctors, some untrained in ophthalmology. The wide range of presenting problems poses a challenge for training and the organisation of effective referral chains, while the gender difference in injury rates points to the potential for prevention.

The objectives of this study were to determine volatile organic compound (VOC) emission signatures of nine typical building materials by using proton transfer reaction-mass spectrometry (PTR-MS) and to explore the correlation between the PTR-MS measurements and the measurements of acceptability by human subjects. VOC emissions from each material were measured in a 50-l small-scale chamber. Chamber air was sampled by PTR-MS to determine emission signatures. Sorbent tube sampling and TD-GC/MS analysis were also performed to identify the major VOCs emitted and to compare the resulting data with the PTR-MS emission signatures. The data on the acceptability of air quality assessed by human subjects were obtained from a previous experimental study in which the emissions from the same batch of materials were determined under the same area-specific ventilation rates as in the case of the measurements with PTR-MS. Results show that PTR-MS can be an effective tool for establishing VOC emission signatures of material types and that there were reasonable correlations between the PTR-MS measurements and the acceptability of air quality for the nine materials tested when the sum of selected major individual VOC odor indices was used to represent the emission level measured by PTR-MS.The study shows that unique emission patterns may exist for different types of building materials. These patterns, or signatures, can be established by using PTR-MS, an online monitoring device. The sum of selected major individual VOC odor indices determined by PTR-MS correlates well with the acceptability of air quality assessed by human subjects, and hence provides a feasible approach to assessing perceived indoor air quality. This online assessment will open a new gate in understanding the role of VOC emissions from building materials on perceived air quality, forming a good foundation to develop real-time or near real-time methods for standard material emission testing and labeling, quality control of emissions from materials, and assessing the acceptability of air quality in buildings.

Interquartile range/median value (IQR/M) of liver stiffness measurement (LSM) is a factor in chronic hepatitis C (CHC) leading to over estimation of fibrosis by Fibroscan.To investigate factors that affect the accuracy of LSM in chronic hepatitis B (CHB).One hundred and ninety-nine patients were enrolled. Only procedures yieldingor =10 valid measurements were considered reliable. Liver fibrosis was evaluated using the Batts and Ludwig system. Liver biopsy (LB) specimens15 mm were considered ineligible.The mean age (142 men and 57 women) was 40.1 years. A significant discordance (discordance of at least two stages between LB and LSM) was identified in 38 (19.1%) and 47 (23.6%) patients respectively, according to Marcellin et al. and Chan et al.'s cutoff values. In multivariate analyses, BMI and fibrosis stage (F0-2 vs. F3-4) were identified as independent predictors for significant discordance (P = 0.040; hazard ratio [HR], 1.126; 95% confidence interval [CI], 1.005-1.261 and P = 0.036; HR, 0.450; 95% CI, 0.213-0.949 respectively) with Marcellin et al.'s cutoffs, whereas fibrosis stage was the only independent predictor (P = 0.004; HR, 0.300; 95% CI, 0.131-0.685) with Chan's cutoffs.Success rate and IQR/M were not predictive factors of the accuracy for diagnosing liver fibrosis by Fibroscan in CHB. Fibrosis stage (F0-2) was the only factor to predict significant discordance between LB and LSM.

This paper is focused on the design, fabrication and characterization of a continuous flow magnetophoretic microseparator for the separation of selectively tagged cells using para-magnetophoresis. The microseparator was characterized experimentally using head and neck cells lines 212LN and 686LN-M4E as a case study. The microfluidic separation system can separate the selectively tagged cancer cells depending on their membrane protein expression level. Experimental results show that at flow rates of 100 µL/hr and 200 µL/hr, 86.3% and 79.0% of tagged cancer cells were attracted toward center channel respectively, while 95.1% and 87.2% of non-tagged cells remained in the side channels. Additionally, the experimental results of separating tagged cancerous cells from human blood were presented.

The focal adhesion kinase (FAK) is a non-receptor tyrosine kinase that localizes to the points of cell contact with the extracellular matrix, called focal adhesions. FAK is involved in several cellular processes including invasion, motility, proliferation and apoptosis. In in vivo animal studies, FAK has been shown to contribute to tumor development and malignancy. Furthermore, FAK expression was shown to be elevated in a number of human cancers. Increased FAK expression and activity are correlated with malignant phenotype and poor prognosis in patients. Taken together, these studies suggest that FAK is a potentially good target for drug discovery. In this review, FAK and its relationship to cancer, as well as approaches to therapeutic intervention of FAK will be discussed.

Rapamycin, a natural product inhibitor of the Raptor-mammalian target of rapamycin complex (mTORC1), is known to induce Protein kinase B (Akt/PKB) Ser-473 phosphorylation in a subset of human cancer cell lines through inactivation of S6K1, stabilization of insulin receptor substrate (IRS)-1, and increased signaling through the insulin/insulin-like growth factor-I/phosphatidylinositol 3-kinase (PI3K) axis. We report that A-443654, a potent small-molecule inhibitor of Akt serine/threonine kinases, induces Akt Ser-473 phosphorylation in all human cancer cell lines tested, including PTEN- and TSC2-deficient lines. This phenomenon is dose-dependent, manifests coincident with Akt inhibition and likely represents an alternative, rapid-feedback pathway that can be functionally dissociated from mTORC1 inhibition. Experiments performed in TSC2-/- cells indicate that TSC2 and IRS-1 cooperate with, but are dispensable for, A-443654-mediated Akt phosphorylation. This feedback event does require PI3K activity, however, as it can be inhibited by LY294002 or wortmannin. Small interfering RNA-mediated knockdown of mTOR or Rictor, components of the rapamycin-insensitive mTORC2 complex, but not the mTORC1 component Raptor, also inhibited Akt Ser-473 phosphorylation induced by A-443654. Our data thus indicate that Akt phosphorylation and activity are coupled in a manner not previously appreciated and provide a novel mode of Akt regulation that is distinct from the previously described rapamycin-induced IRS-1 stabilization mechanism.

Background Several referral strategies for axial spondyloarthritis (axSpA) have been proposed. The goal of these strategies is to overcome the delay between the first symptoms and the final diagnosis of axSpA by supporting primary care physicians in recognizing potential axSpA patients. All referral strategies are tested in their original study population, however no direct comparison of the performance of different strategies in one study population is performed. Objectives To evaluated six different referral strategies for axSpA in unselected young primary care patients with chronic low back pain (CLBP) and secondly to find the optimal referral strategy for daily practice. Methods The referral strategies were evaluated in a large Dutch primary care population of unselected CLBP patients (18-45 years, CLBP ≥3 months, back pain onset 1 , MASTER 2 , RADAR 3 , the 2-step strategy 4 , the CaFaSpA strategy 5 , and the new ASAS recommendations 6 . For a description of the different strategies see Figure 1. The performance of the different referral models was assessed by sensitivity, specificity, area under the curve (AUC) and positive predictive value (PPV). Results In total 941 primary care CLBP patients participated (58% female, mean age 36.0 years), of those were 181 (19%) identified as axSpA, 54 of the 181 (30%) were newly diagnosed with ankylosing spondylitis. Almost all referral strategies had a good discriminative performance (AUC >0.7). (Table 1) The MASTER referral strategy had the most balanced sensitivity and specificity. The new ASAS proposal has the lowest AUC, lowest specificity and the lowest PPV. Conclusions Almost all referral strategies had a good performance in this primary care cohort of CLBP patients. Strategies including imaging, HLA-B27 and inflammatory back pain (IBP) had the highest AUC and PPV. However imaging and blood tests are not always accessible in primary care, mainly due to high costs. The optimal strategy for primary care should include non-invasive referral parameters without additional costs such as IBP, good response to NSAIDs and family history. References Brandt et al, 2007. Poddubnyy et al, 2011. Sieper et al, 2013. Braun et al, 2013. van Hoeven et al, 2014. Poddubnyy et al, 2014 Acknowledgements An unrestricted research grant was provided by AbbVie. Disclosure of Interest None declared

Background There is a delay between the onset of the first back pain symptoms and the final diagnosis of axial spondyloarthritis (axSpA). This delay can be explained by difficulties for primary care physicians to recognize and subsequently refer potential axSpA patients in the huge number of chronic low back pain (CLBP) patients seen in primary care. At this moment there is no widely accepted referral strategy. Recently the ASAS workgroup proposed recommendations for early referral although these were not yet validated in a primary care setting. Objectives To test the ASAS proposed recommendations for early referral in a primary care setting. Methods Our study population included primary care patients (18-45 years) with CLBP (≥3 months, age at back pain onset Results In total 941 primary care CLBP patients participated (58% female, mean age 36.0 years), of those were 181 (19%) identified as axSpA, 54 of the 181 (30%) were newly diagnosed with ankylosing spondylitis. 773 (82%) patients had at least one parameter present and thus according to the recommendations should be referred to the rheumatologists. The sensitivity of the ASAS recommendation is 100% (181/181), the specificity 22% (168/760) and the positive predictive value 23% (181/773) (Table 1). Conclusions The ASAS recommendation for early referral has a perfect sensitivity in primary care CLBP population. However this comes at the cost of a low specificity, meaning that almost 80% of the referred patients will undergo unnecessary diagnostic work up. A more specific referral strategy will be needed in daily primary care. Acknowledgements An unrestricted research grant was provided by AbbVie. Disclosure of Interest None declared

The Ottawa ankle rules are a clinical instrument calibrated towards a high sensitivity in order not to miss any fractures of the ankle or mid-foot. This is at the cost of a mean specificity of around only 32%. The aim of this study was to determine the suitability of tuning fork testing in combination with existing Ottawa guidance for increasing the specificity in detecting fractures of the lateral malleolus. A prospective pilot study was carried out, in which a single trained investigator examined all patients with already "Ottawa positive" findings for possible lateral malleolus injury by applying a tuning fork (C(o) 128 Hz). The tuning fork test findings were compared with formal reports of plain ankle radiographs using simple cross-table analysis. The observed prevalence of ankle fractures was 5 of 49 (10%). Sensitivity and specificity were calculated as 100% and 61%, respectively, for tuning fork testing on the tip of the lateral malleolus (TLM), and as 100% and 95%, respectively, for testing on the distal fibula shaft (DFS). The associated positive and negative likelihood ratios were 2.59 and 0 (TLM), and 22 and 0 (DFS), respectively. The data were significant, with p = 0.014 (TLM) and p

A series of heteroaryl-pyridine containing inhibitors of Akt are reported. The synthesis and structure–activity relationships are discussed, leading to the discovery of a indazole-pyridine analogue (Ki = 0.16 nM). These compounds bind in the ATP binding site, are potent, ATP competitive, and reversible inhibitors of Akt activity. No selectivity amongst the Akt isoforms is observed for this analogue, but there is good selectivity against an panel of other kinases. It is least selective for other members of the AGC family of kinases but is nonetheless 40-fold selective for Akt over PKA. The compound shows cellular activity and significantly slows tumor growth in vivo.

This chapter studies induced magnetic order by ion irradiation of carbon-based structures. Proton irradiation can trigger magnetic ordering in graphite. The results after proton irradiation show that magnetic ordering exists in a carbon structure without the influence of magnetic ions. There is a negligible influence of the topography onto the magnetic signal, and the magnetic signal depends on the implanted charge, proton current, and probably also on the carbon structure. Hydrogenated nanographite can have a spontaneous magnetization due to different numbers of mono- and dihydrogenated carbon atoms. Theoretical simulations indicate that a magnetic ordered state should be easier to achieve with a mixture of carbon–hydrogen bonds than fluorinated nanographite. Muon spin rotation/relaxation experiments indicate that a positive muon in graphite triggers a local magnetic moment around it. Hydrogen may also play an important role in the magnetic ordering found in fullerenes.

Two sets of samples were synthesized at 3.5 GPa near the point of C60 cage collapse at different annealing times. A clear structural transformation from mixture of C60 polymeric phases to graphite-like hard carbon phase was confirmed by X-ray diffraction and Raman spectroscopy. Magnetic force microscopy and superconducting quantum interference device were used to characterize the magnetic properties of the synthesized samples. We found that the sample preparation conditions used in this study are not suitable to produce bulk magnetic carbon., Comment: 26 pages, 7 figures

AIMS: The expression of TGF beta-inducible gene h3(beta ig-h3) has been used to assess the biological activity of TGF beta in the kidney. In this study, we investigated whether the urinary concentration of beta ig-h3 is associated with diabetic nephropathy in patients with Type 2 diabetes mellitus. We also evaluated the relationship between the urinary concentration of beta ig-3 and proteinuria and microalbuminuria (AER) in a normal healthy population and in Type 2 diabetes patients. METHODS: Four hundred and seventy-nine Type 2 diabetic patients without non-diabetic kidney diseases and 528 healthy control subjects were enrolled. The study subjects were divided into five groups: a non-diabetic healthy control group with normal ACR (n = 443), a non-diabetic healthy control group with microalbuminuria (n = 85), a normoalbuminuric diabetic group (n = 198), a microalbuminuric diabetic group (n = 155) and an overt proteinuria group (n = 126). Urinary levels of beta ig-h3 were measured by enzyme-linked immunosorbent assay. RESULTS: (i) Urinary excretion of beta ig-h3 was significantly higher in the diabetic groups than in the controls, even in the normoalbuminuric stage (25.02 +/- 8.84 vs. 18.67 +/- 6.56, P = 0.03). In diabetic patients, urinary beta ig-h3 levels increased significantly as diabetic nephropathy advanced (25.02 +/- 8.84 vs. 34.06 +/- 24.55 vs. 169.63 +/- 57.33, P < 0.001). (ii) Proteinuria was found to be significantly correlated with urinary beta ig-h3 (healthy control; r = 0.137, P = 0.019, diabetic patients; r = 0.604, P < 0.001). ACR was also found to be significantly related with urinary beta ig-h3 in diabetic patients (r = 0.383, P = 0.006). (iii) In diabetic patients, urinary beta ig-h3 was significantly related with systolic and diastolic blood pressure (systolic blood pressure: r = 0.436, P = 0.024; diastolic blood pressure, r = 0.365, P = 0.042), total cholesterol and HbA(1c) (cholesterol: r = 0.169, P = 0.03, HbA(1c); r = 0.387, P = 0.044). Logistic regression analyses showed that urinary beta ig-h3 was associated with a significant increase in the risk of microalbuminuria and proteinuria in diabetic patients. CONCLUSIONS: Longitudinal monitoring of urinary beta ig-h3 may improve the likelihood of detecting diabetic nephropathy at an earlier stage and beta ig-h3 could be a sensitive marker of diabetic kidney disease progression.

Aims Vascular endothelial growth factor (VEGF) has been implicated in the pathogenesis of diabetes mellitus. We determined whether alterations of plasma and urinary VEGF levels are related to diabetic nephropathy in Type 2 diabetic patients. Methods One hundred and seven patients and 47 healthy controls were studied. Study subjects were divided into four groups using urinary albumin-to-creatinine ratio (ACR): a non-diabetic healthy control group (n = 47), a normoalbuminuric diabetic group (n = 37), a microalbuminuric diabetic group (n = 37) and an overt proteinuric diabetic group (n = 33). VEGF levels were measured by enzyme-linked immunosorbent assay. Results (i) Urinary VEGF concentrations were significantly higher in the diabetic groups, even at the normoalbuminuric stage (log VEGF/Cr, normoalbuminuria; 4.33 ± 1.06 vs. control; 3.53 ± 0.79, P = 0.009). Urinary VEGF excretions increased as diabetic nephropathy advanced. (ii) Plasma and urinary VEGF levels were higher in hypertensive diabetic patients than in the normotensive individuals with diabetes. (iii) In those with diabetes, plasma VEGF levels were found to be positively correlated with plasma urea (r = 0.398, P = 0.039) and urinary ACR (r = 0.251, P = 0.044), and urinary VEGF to be positively correlated with urinary ACR (r = 0.645, P

The microstructural, magnetic, and magnetotransport properties of ${\mathrm{La}}_{0.7}{\mathrm{Ca}}_{0.3}{\mathrm{MnO}}_{3}$ films epitaxially grown on ${\mathrm{SrTiO}}_{3}$ substrates were studied as a function of film thickness. Films with a thickness above 70 nm show bulklike properties consistent with the relaxed film growth. Very thin films are fully strained and are insulating and ferromagnetic in zero field. Under the application of a large magnetic field a strongly hysteretic metal-insulator transition is seen accompanied by a transition to a ferromagnetic state with larger saturation magnetization. The field-induced metallic state is shown to be anisotropic with metallic domains extending along the field direction. An analysis of the microstructure shows that the strain variation across growth islands is far too small to induce strain-related phase separation on length scales of the order of 100 nm. It is concluded that the insulating state is a property of homogeneously strained manganite films and arises from a weakening of the double-exchange interaction due to the ordering of atomic orbitals.

Summary form only given. Granular ferromagnets show a magnetoresistance due to spin-polarized tunnelling between individual grains. Within a simple model Inoue et al. (1996) calculated the magnetoresistance ratio and obtained [R/sub 0/-R(H)]/R(H) = P/sup 2/. Here R(H) denotes the resistance in an applied field H and R/sub 0/ = R(H = 0); since the spin-polarization P is limited, P /spl les/ 1, the magnetoresistance cannot exceed unity. La/sub 0.7/Ca/sub 0.3/MnO/sub 3/ films (LCMO) films with various thicknesses were prepared by pulsed laser deposition onto LaAlO/sub 3/ substrates. Films with a nominal thickness larger than 4 nm were continuous and showed a metal-insulator transition above 200 K. Films with a smaller nominal thickness were discontinuous as evidenced by atomic force microscopy. These films are superparamagnetic and show hopping conduction. The resistance follows a exp[(To/T)/sup 3/5/] law for temperatures T < 200 K; this might be related to a thermally assisted tunnelling process in a two dimensional granular structure formed by surface diffusion. The magnetoresistance [Ro-R(H)]/R(H) of the 2.2 nm film exceeds the theoretical limit. The data can be convincingly fitted by an exponential dependence, exp[J/sub m/H/sup 2//kBT]-1, where J/sub m/H/sup 2/ is an activation energy. This indicates that the intergrain coupling energy plays a major role in the tunnelling process. We discuss this data and present a more detailed model for tunnelling in granular materials.

Piezoelectric (Pb/sub 0.5/Bi/sub 0.5/)(Ti/sub 0.5/(Fe/sub 1-x/Mn/sub x/)/sub 0.5/)O/sub 3/ (x=0.00-0.02) ceramic powder was prepared by alcohol dehydration of an aqueous multicomponent ammonium citrate solution. The tetragonal phase with the desired stoichiometry formed after heat treatment at as low as 800 degrees C. A high tetragonality (c/a ratio of 1.140+or-0.001) was achieved after heat treatment at 1000 degrees C for 1 h. Flexible 0.3 composites prepared from the 1000 degrees C powder (x=0.01) and Eccogel polymer exhibited piezoelectric charge coefficients (d/sub 33/) of 60 pC/N and a voltage coefficients (g/sub 33/) of 205*10/sup -3/ Vm/N. >

A 2.5 V, 72 Mbit packet protocol based SDRAM (PSDRAM) achieving a peak bandwidth of 2.0 GByte/s has been developed with a 0.23 /spl mu/m twin-well, 4-poly, 2-metal CMOS process. An internal Vcc of 2.0 V and V/sub term/ of 1.8 V with 0.8 V signal swing are used in the array to reduce the sensing power and I/O switching power, respectively. The total maximum chip power consumption of 1.80 W, including the average I/O switching power of 0.25 W, has been achieved when internal 16 banks are interleavingly operated with 20 ns interval commands at 2.0 GByte/s, Vcc=2.7 V, and T=25/spl deg/C.

AIM: There are still insufficient clinical reports concerning quality-switched Nd:YAG laser (QSNYL) in the treatment of superficial pigmented lesions in Asians. The purpose of this study was to analyse the efficacy and side effect profiles of QSNYL-assisted pigment removal in brown skin. METHODS: A total of 71 patients, presenting a wide gamut of superficial epidermal lesions, were treated with QSNYL. Clinical responses were assessed by comKeywords: paring photographs that were taken serially in every treatment. RESULTS: Treatment using the QSNYL is reported individually for the various superficial pigmented lesions in the skin of Korean patients. CONCLUSION: QSNYL may be a beneficial alternative tool for the treatment of a number of benign pigmented lesions including freckles, lentigines and unilateral lentiginosis.

A study was carried out to investigate the water balance and losses of nutrients at a paddy plot located at the southern Korea. The observed amount of precipitation, irrigation, runoff, evapotranspiration and infiltration water for the experimental paddy field during cropping period were 1,030, 566, 701, 551, and 310 mm for 1999 and 1,214, 413, 710, 682, and 234 mm for 2000 year, respectively. The measured input quantities of N and P into the paddy field during cropping period of each year were 122 to 140 kg N ha-1 and 12.64 to 13.08 kg P kg ha-1 by chemical fertilizer, 20 to 28 kg N ha-1 and 0.35 to 0.36 kg P ha-1 by precipitation, and 26 to 35 kg N ha-1 and 0.57 to 0.72 kg P ha-1 by irrigation water, respectively. The output amounts of N and P from paddy field were measured as follows. They were 48 to 52 kg N ha-1 and 1.1 to 1.6 kg P ha-1 by runoff water, and 9 to 12 kg N ha-1 and 0.04 to 0.05 kg ha-1 by infiltration water.

We cloned and sequenced a gene, kpcA (Kex2p-like proprotein convertase A), from a genomic library of Aspergillus nidulans. The kpcA gene encodes an 820-residue protein, named KpcA, which contains a putative subtilisin-like catalytic domain (residues 136-466) homologous to that of the subtilisin serine protease family. KpcA shows 56, 73, and 47% amino acid identities with Saccharomyces cerevisiae Kex2p, Aspergillus niger KexB, and mouse furin within the subtilisin-like catalytic domain, respectively. The sequences around the proposed active site Asp, His, and Ser residues of KpcA are similar to those of other Kex2p family members. The KpcA mRNA transcript with an expected size of approximately 2.8 kb was detected in A. nidulans. The substrate specificity of KpcA, expressed in CHO cells, is similar to that of A. niger KexB and yeast Kex2p. We conclude that KpcA is a resident Kex2p-like proprotein that processes endoprotease in A. nidulans.

The ability to reproduce both sexually and asexually is one of the characteristics of the homothalic ascomycete Aspergillus nidulans. Unlike the other Aspergillus species, A. nidulans undergoes sexual development that seems to be regulated by internal and external stimuli. To begin to understand the sexual reproduction of A. nidulans we previously isolated and characterized several NSD (never in sexual development) mutants that failed to produce any sexual reproductive organs, and identified four complementation groups, nsdA, nsdB, nsdC, and nsdD. The nsdD gene has been isolated, and it is predicted to encode a GATA-type transcription factor with the type IVb zinc finger DNA-binding domain. The mRNA of the nsdD gene started to accumulate in the early phase of vegetative growth, and the level increased as sexual development proceeded. However, it decreased during asexual sporulation and no nsdD mRNA was detected in conidia. Deletion of nsdD resulted in no cleistothecia (fruiting bodies) formation, even under the conditions that preferentially promoted sexual development, indicating that nsdD is necessary for sexual development. In contrast, when the nsdD gene was over-expressed, sexual-specific organ (Hülle cell) was formed even in submerged culture, which normally completely blocked sexual development, and the number of cleistothecia was also dramatically increased on solid medium. These results lead us to propose that the nsdD gene functions in activating sexual development of A. nidulans. Multiple copies of the nsdD gene could suppress nsdB5 and veA1, indicating that either nsdD acts downstream of these genes or possibly functions in overlapping pathway(s).