In response to cytokine stimuli, Stats are phosphorylated and translocated to the nucleus to activate target genes. Then, most are dephosphorylated and returned to the cytoplasm. Using Ba/F3 cells, we found that the nuclear export of Stat5B by cytokine depletion was inhibited by leptomycin B (LMB), a specific inhibitor of nuclear export receptor chromosome region maintenance 1. Interestingly, LMB treatment in the absence of cytokine led to the accumulation of Stat5B in the nucleus, suggesting that Stat5B shuttles between the nucleus and the cytoplasm as a monomer without cytokine stimulation. This notion is supported by the observation that LMB-induced accumulation of Stat5B in the nucleus was also observed with Stat5B having a mutated tyrosine 699, which is essential for dimer formation. Using a series of mutant Stat5Bs, we identified a part of the coiled coil domain to be a critical region for monomer nuclear import and a more N-terminal region to be critical for the cytokine stimulation dependent import of Stat5B. Taken together, we propose a model in which Stat5B shuttles between the nucleus and cytoplasm by two different mechanisms, one being a factor-independent constitutive shuttling by monomeric form, and the other, a factor stimulation-dependent one regulated by tyrosine phosphorylation and subsequent dimerization.

Males have strikingly increased risk of advanced liver disease. However, the association between testosterone and risk of hepatitis C virus (HCV)-related advanced liver disease is unknown. We performed a cross-sectional study in male veterans with chronic HCV. Blood samples were obtained to measure total serum testosterone and perform the FibroSURE-ActiTest. Other risk-factor data were obtained through systematic questionnaires (e.g., alcohol), physical measurements (e.g., body mass index), and serological tests (e.g., viral load). The association between total testosterone and risk of advanced hepatic fibrosis (F3 and F3/F4) and inflammatory activity (A3 and A2/3) measured by the FibroSURE-ActiTest was evaluated with logistic regression. A total of 308 eligible study participants were prospectively recruited (mean age: 57; 52% African-American). There were 105 cases with advanced fibrosis and 203 mild fibrosis controls as well as 88 cases with advanced inflammatory activity and 220 mild activity controls. Mean total serum testosterone was significantly higher in advanced fibrosis cases as well as advanced inflammatory activity cases, compared to mild disease controls (6.0 versus 5.3 ng/mL and 5.9 versus 5.4 ng/mL, respectively). We observed a significant 25% increase in advanced fibrosis risk and 15% increase in advanced inflammatory activity risk for each 1-ng/mL increase in total serum testosterone. Total testosterone in the upper tertile was associated with an even greater excess risk of advanced fibrosis than advanced inflammatory activity (odds ratio [OR](adjusted advanced fibrosis) = 3.74; 95% CI: 1.86-6.54 versus OR(adjusted advanced inflammatory activity) = 2.23; 95% CI: 1.07-4.93, respectively).

CONCLUSIONS

Total serum testosterone is associated with an increased risk of both advanced hepatic fibrosis and advanced hepatic inflammatory activity in HCV-infected men. Testosterone may be important in the pathogenesis of HCV-related advanced liver disease in males.

OBJECTIVE

To synthesize and complete in vitro characterization of a novel, tumor-targeted nanodevice for visible intraoperative delineation of brain tumors.

METHODS

The ability of dye-loaded polyacrylamide nanoparticles (NP) containing methylene blue, Coomassie blue, or indocyanine green to cause color change in the 9L glioma cell lines was evaluated. Cells were incubated with dye-loaded NPs, photographed, and analyzed colorimetrically. Confocal microscopy was used to determine subcellular localization of NPs in treated cells.

RESULTS

Incubation of glioma cell lines with dye-loaded NPs resulted in clearly visible, quantifiable cell tagging in a dose- and time-dependent manner. Dye-loaded NPs were observed to bind to the surface and become internalized by glioma cells. Coating the NP surface with F3, a peptide that binds to the tumor cell surface receptor nucleolin, significantly increased NP affinity for glioma cells. F3 targeting also significantly increased the rate of cell tagging by dye-loaded NPs. Finally, F3-targeted NPs demonstrated specificity for targeting various cancer cell lines based on their surface expression of cell surface nucleolin.

CONCLUSIONS

F3-targeted dye-loaded NPs efficiently cause definitive color change in glioma cells. This report represents the first use of targeted NPs to cause a visible color change in tumor cell lines. Similar nanodevices may be used in the future to enable visible intraoperative tumor delineation during tumor resection.

BACKGROUND

Primary aldosteronism (PA) is associated with vascular end organ damage.

OBJECTIVE

We evaluated the newly established German Conn's Registry for evidence of renal impairment and compared the data with those from hypertensive subjects of a population-based survey.

METHODS

We conducted a case-control study.

METHODS

A total of 408 patients with PA from the Conn's registry treated in five German centers were matched for age, sex, and body mass index in a 1:1 ratio with 408 hypertensive control subjects from the population-based F3 survey of the Kooperative Gesundheitsforschung in the region of Augsburg (KORA).

METHODS

We measured serum creatinine and calculated glomerular filtration rate (GFR).

RESULTS

The percentage of patients with a serum creatinine concentration above the normal range of 1.25 mg/dl was higher in patients with PA than in hypertensive controls (29 vs. 10%; P < 0.001). Regression analysis showed that age, male sex, low potassium, and high aldosterone concentrations were independent predictors of a lower GFR. Adrenalectomy reduced systolic blood pressure from a mean of 160 to 144 mm Hg. In parallel, we observed an increase in serum creatinine and a decrease of GFR from 71 to 64 ml/min (P < 0.001). A similar trend was seen after spironolactone treatment.

CONCLUSIONS

In a large cohort of patients with PA, markers of disease activity such as plasma aldosterone and serum potassium are independent predictors of a lower GFR. Specific interventions, such as adrenalectomy or spironolactone treatment, are associated with a further decline in GFR.

To investigate the brain topography of the human sleep EEG along the antero-posterior axis, spectra (0.25-25 Hz; 1 Hz bins) were computed from all-night EEG recordings (n = 20 subjects) obtained from an anterior F3-C3) and a posterior (P3-O1) derivation. State-dependent and frequency-dependent topographic differences were observed. In non-rapid eye movement (REM) sleep, power in the anterior derivation was higher than in the posterior derivation in the 2 Hz bin, and lower in the 4-10 Hz bins. In REM sleep, a posterior dominance was present in most bins below 18 Hz. The 2-6 Hz bins exhibited an antero-posterior shift of power over consecutive non-REM sleep episodes. Consistent shifts of power were also present within non-REM sleep episodes. The results suggest that anterior and posterior cortical regions may be differently involved in the sleep process.

FLT3 is a receptor tyrosine kinase that may play a role in a significant proportion of leukemias. In addition to being aberrantly expressed in acute leukemias, activating mutations of the FLT3 gene have been found in patients with AML, myelodysplastic syndrome (MDS) and more rarely, ALL. Internal tandem duplications (ITDs) of the FLT3 gene have been detected in 17-34% of patients with AML and portend a poor prognosis for these patients. FLT3 receptors containing ITD mutations (FLT3/ITDs) are constitutively activated in the absence of FLT3 ligand (FL) stimulation leading to the activation of downstream signaling proteins, including ERK and STAT 5. FLT3 activity, therefore, is a logical target for therapeutic intervention. AG1296 is a tyrosine kinase inhibitor of the tyrphostin class that shows inhibitory activity for wild-type FLT3, in addition to the PDGF and c-KIT receptors. We examined the inhibitory effects of AG1296 on FLT3/ITDs isolated from AML patients in the IL-3-dependent cell line, Ba/F3, as well as in primary leukemia samples from AML patients. Immunoprecipitation and immunoblotting analyses demonstrated that FLT3/ITDs were constitutively phosphorylated in the absence of FL. The auto-phosphorylation of FLT3/ITDs was inhibited by AG1296 with an IC(50) of approximately 1 microM. FLT3/ITDs were associated with constitutive phosphorylation of ERK, STAT 5A, STAT 5B, CBL, VAV and SHP2 in Ba/F3 cells. The phosphorylation of these downstream signaling molecules was suppressed in a dose-responsive fashion by AG1296. AG1296 inhibited IL-3 independent growth and induced apoptosis in Ba/F3 cells transformed by FLT3/ITDs. AG1296 also inhibited FLT3 auto-phosphorylation, and induced a cytotoxic effect, in primary AML cells. These findings suggest that inhibiting the activity of FLT3 may have a therapeutic value in some leukemias expressing FLT3/ITDs.

The mode of activation of glycoprotein 130 kDa (gp130) and the transmission of the activation status through the plasma membrane are incompletely understood. In particular, the molecular function of the three juxtamembrane fibronectin III-like domains of gp130 in signal transmission remains unclear. To ask whether forced dimerization of gp130 is sufficient for receptor activation, we replaced the entire extracellular portion of gp130 with the c-jun leucine zipper region in the chimeric receptor protein L-gp130. On expression in cells, L-gp130 stimulates ligand-independent signal transducer and activator of transcription (STAT) 3 and extracellular signal-regulated kinase 1/2 phosphorylation. gp130 activation could be abrogated by the addition of a competing peptide comprising the leucine zipper region of c-fos. When stably expressed in the interleukin-3-dependent Ba/F3 murine pre-B-cells, these cells showed constitutive STAT3 activation and cytokine-independent growth over several months. Because gp130 stimulation completely suppressed differentiation of murine embryonic stem cells in vitro, we also stably expressed L-gp130 in these cells, which completely blocked their differentiation in the absence of cytokine stimulation and was consistent with high constitutive expression levels of the stem cell factor OCT-4. Thus, L-gp130 can be used in vitro and in vivo to mimic constitutive and ligand-independent activation of gp130 and STAT3, the latter of which is frequently observed in neoplastic diseases.

Polycystic ovary syndrome (PCOS) is the most common female endocrine disorder. Ovarian changes in PCOS women are well characterized by ultrasound. However, the ovarian pathophysiology is not fully understood. The aim of this study was to characterize the expression, in both the central ovarian stroma and in granulosa cells (GCs), of a number of genes, including several inflammation-related genes, which have been hypothesized to be involved in the pathophysiology of PCOS. Biopsies of the central ovarian stroma were obtained from PCOS women (Rotterdam criteria) and from normally ovulating women in follicular phase. GCs were retrieved from PCOS-women and non-PCOS women, undergoing in vitro maturation. The expressions of 57 genes were analyzed by quantitative-PCR using a low-density-gene array. The main outcome measures were over-expression or under-expression of the specific genes. The results showed that in the central stroma of PCOS ovaries, five inflammation-related genes (CCL2, IL1R1, IL8, NOS2, TIMP1), the leukocyte marker CD45, the inflammation-related transcription factor RUNX2 and the growth factor AREG were under-expressed. The growth factor DUSP12 and the coagulation factor TFPI2 were over-expressed. In the GC of PCOS, all of the differentially expressed genes were over-expressed; the inflammation-related IL1B, IL8, LIF, NOS2 and PTGS2, the coagulation-related F3 and THBS1, the growth factors BMP6 and DUSP12, the permeability-related AQ3 and the growth-arrest-related GADD45A. In conclusion, the results indicate major alterations in the local ovarian immune system of PCOS ovaries. This may have implications for the PCOS-related defects in the inflammation-like ovulatory process and for the susceptibility to acquire the inflammatory state of ovarian hyperstimulation syndrome.

In maize, mutations in the pr1 locus lead to the accumulation of pelargonidin (red) rather than cyanidin (purple) pigments in aleurone cells where the anthocyanin biosynthetic pathway is active. We characterized pr1 mutation and isolated a putative F3'H encoding gene (Zmf3'h1) and showed by segregation analysis that the red kernel phenotype is linked to this gene. Genetic mapping using SNP markers confirms its position on chromosome 5L. Furthermore, genetic complementation experiments using a CaMV 35S::ZmF3'H1 promoter-gene construct established that the encoded protein product was sufficient to perform a 3'-hydroxylation reaction. The Zmf3'h1-specific transcripts were detected in floral and vegetative tissues of Pr1 plants and were absent in pr1. Four pr1 alleles were characterized: two carry a 24 TA dinucleotide repeat insertion in the 5'-upstream promoter region, a third has a 17-bp deletion near the TATA box, and a fourth contains a Ds insertion in exon1. Genetic and transcription assays demonstrated that the pr1 gene is under the regulatory control of anthocyanin transcription factors red1 and colorless1. The cloning and characterization of pr1 completes the molecular identification of all genes encoding structural enzymes of the anthocyanin pathway of maize.

Adiponectin and its receptors (AdipoR1 and AdipoR2) mRNAs are expressed in various chicken tissues including ovary. However, the cellular expression and the role of adiponectin system have never been investigated in chicken ovary. Here, we have shown that the level of adiponectin mRNA is about 10- to 30-fold higher (p<0.001) in theca cells than in granulosa cells from each hierarchical yellow follicle studied (F4-F1). In contrast, the level of AdipoR1 mRNA expression was about two-fold lower in theca cells than in granulosa cells (p<0.05) whereas those of AdipoR2 was similar in both ovarian cells. Whereas expression of adiponectin mRNA increased with follicular differentiation in theca cells, it decreased in granulosa cells. In contrast, mRNA expression of AdipoR1 and AdipoR2 in both theca and granulosa cells remained stable during yellow follicle development. To determine whether adiponectin is involved in the ovarian steroidogenesis, LH (100 ng/ml)-, FSH (100 ng/ml)- and IGF-1 (100 ng/ml)-induced progesterone production was measured in absence or presence of human recombinant adiponectin (10 microg/ml) for 36 h in cultured granulosa cells from F1, F2 and mixed F3 and F4 follicles. In absence of LH, FSH and IGF-1, adiponectin treatment had no effects on progesterone production whatever vitollegenic follicle studied. However, it increased by about two-fold IGF-1-induced progesterone secretion in F2 and F3/4 follicles whereas it halved progesterone production in response to gonadotropins (LH and FSH) in F3/4 follicles. Thus, in chicken, adiponectin, mainly expressed in theca cells, could exert paracrine or autocrine effect on the ovarian steroidogenesis.

Persimmon fruits accumulate a large amount of proanthocyanidin (PA) during development. Fruits of pollination-constant and non-astringent (PCNA) type mutants lose their ability to produce PA at an early stage of fruit development, while fruits of the normal (non-PCNA) type remain rich in PA until fully ripened. To understand the molecular mechanism for this difference, we isolated the genes involved in PA accumulation that are differentially expressed between PCNA and non-PCNA, and confirmed their correlation with PA content and composition. The expression of structural genes of the shikimate and flavonoid biosynthetic pathways and genes encoding transferases homologous to those involved in the accumulation of phenolic compounds were downregulated coincidentally only in the PCNA type. Analysis of PA composition using the phloroglucinol method suggested that the amounts of epigallocatechin and its 3-O-gallate form were remarkably low in the PCNA type. In the PCNA type, the genes encoding flavonoid 3'5' hydroxylase (F3'5'H) and anthocyanidin reductase (ANR) for epigallocatechin biosynthesis showed remarkable downregulation, despite the continuous expression level of their competitive genes, flavonoid 3' hydroxylation (F3'H) and leucoanthocyanidin reductase (LAR). We also confirmed that the relative expression levels of F3'5'H to F3'H, and ANR to LAR, were considerably higher, and the PA composition corresponded to the seasonal expression balances in both types. These results suggest that expressions of F3'5'H and ANR are important for PA accumulation in persimmon fruit. Lastly, we tested enzymatic activity of recombinant DkANR in vitro, which is thought to be an important enzyme for PA accumulation in persimmon fruits.

BACKGROUND

Noninvasive risk factors are required for predicting the development of hepatocellular carcinoma (HCC) not only in patients with cirrhosis but also in those with chronic hepatitis who are infected with hepatitis C virus (HCV).

METHODS

A total of 707 patients with chronic HCV infection without other risks were evaluated for the predictive value of noninvasive risk factors for HCC, including age, sex, viral load, genotype, fibrosis stage, aspartate and alanine aminotransferase levels, bilirubin, albumin, platelet count, and alpha-fetoprotein (AFP) at entry to the study, as well as interferon (IFN) therapy they received.

RESULTS

The ten-year cumulative incidence rates of HCC for patients with fibrosis stages F0/F1, F2, F3, and F4 were 2.5, 12.8, 19.3, and 55.9%, respectively. Multivariate analysis identified age ≥57 years [hazard ratio (HR) 2.026, P = 0.004], fibrosis stage F4 (HR 3.957, P < 0.001), and AFP 6-20 ng/mL (HR 1.942, P = 0.030) and ≥20 ng/mL (HR 3.884, P < 0.001), as well as the response to IFN [relative risk (RR) 0.099, P < 0.001], as independent risk factors for the development of HCC. The ten-year cumulative incidence rates of HCC in the patients with AFP levels of <6, 6-20, and ≥20 ng/mL at entry were 6.0, 24.6, and 47.3%, respectively.

CONCLUSIONS

Not only high (>20 ng/mL), but also even slightly elevated (6-20 ng/mL) AFP levels, could serve as a risk factor for HCC to complement the fibrosis stage. In contrast, AFP levels <6 ng/mL indicate a low risk of HCC development in patients infected with HCV, irrespective of the fibrosis stage.

Rhabdomyosarcoma (RMS) is the most common childhood soft tissue sarcoma. Despite advances in modern therapy, patients with relapsed or metastatic disease have a very poor clinical prognosis. Fibroblast Growth Factor Receptor 4 (FGFR4) is a cell surface tyrosine kinase receptor that is involved in normal myogenesis and muscle regeneration, but not commonly expressed in differentiated muscle tissues. Amplification and mutational activation of FGFR4 has been reported in RMS and promotes tumor progression. Therefore, FGFR4 is a tractable therapeutic target for patients with RMS. In this study, we used a chimeric Ba/F3 TEL-FGFR4 construct to test five tyrosine kinase inhibitors reported to specifically inhibit FGFRs in the nanomolar range. We found ponatinib (AP24534) to be the most potent FGFR4 inhibitor with an IC50 in the nanomolar range. Ponatinib inhibited the growth of RMS cells expressing wild-type or mutated FGFR4 through increased apoptosis. Phosphorylation of wild-type and mutated FGFR4 as well as its downstream target STAT3 was also suppressed by ponatinib. Finally, ponatinib treatment inhibited tumor growth in a RMS mouse model expressing mutated FGFR4. Therefore, our data suggests that ponatinib is a potentially effective therapeutic agent for RMS tumors that are driven by a dysregulated FGFR4 signaling pathway.

T-cell prolymphocytic leukemia (T-PLL) is a rare form of mature T-cell leukemia associated with chromosomal rearrangements implicating MTCP1 or TCL1 genes. These genes encode two homologous proteins, p13(MTCP1) and p14(TCL1), which share no similarity with other known protein. To determine the oncogenic role of MTCP1, mice transgenic for MTCP1 under the control of CD2 regulatory regions (CD2-p13 mice) were generated. No abnormality was detected during the first year after birth. A late effect of the transgene was searched for in a cohort of 48 CD2-p13 mice aged 15 to 20 months, issued from 3 independent founders. Lymphoid hemopathies, occurring in the three transgenic lines, were characterized by lymphoid cells with an irregular nucleus, a unique and prominent nucleolus, condensed chromatin, a basophilic cytoplasm devoid of granules, and an immunophenotype of mature T cells. The molecular characterization of Tcrb rearrangements demonstrated the monoclonal origin of these populations. Histopathological analysis of the cohort demonstrated early splenic and hepatic infiltrations, whereas lymphocytosis and medullar infiltrations were found infrequently. The engraftment of these proliferations in H2-matched animals demonstrated their malignant nature. Cumulative incidence of the disease at 20 months was 100%, 50%, and 21% in F3, F4, and F7 lines, respectively, and null in the control group. The level of expression of the transgene, as estimated by Western blotting in the transgenic lines correlated with the tumoral incidence, with the highest expression of p13(MTCP1) being found in F3 mice. CD2-p13 transgenic mice developed an hemopathy similar to human T-PLL. These data demonstrate that p13(MTCP1) is an oncoprotein and that CD2-p13 transgenic mice represent the first animal model for mature T-PLL.

Cripto-1 (CR-1), a recently discovered protein of the epidermal growth factor (EGF) family, was found to interact with a high affinity, saturable binding site(s) on HC-11 mouse mammary epithelial cells and on several different human breast cancer cell lines. This receptor exhibits specificity for CR-1, since other EGF-related peptides including EGF, transforming growth factor alpha, heparin-binding EGF-like growth factor, amphiregulin, epiregulin, betacellulin, or heregulin beta1 that bind to either the EGF receptor or to other type 1 receptor tyrosine kinases such as erb B-3 or erb B-4 fail to compete for binding. Conversely, CR-1 was found not to directly bind to or to activate the tyrosine kinases associated with the EGFR, erb B-2, erb B-3, or erb B-4 either alone or in various pairwise combinations which have been ectopically expressed in Ba/F3 mouse pro-B lymphocyte cells. However, exogenous CR-1 could induce an increase in the tyrosine phosphorylation of 185- and 120-kDa proteins and a rapid (within 3-5 min) increase in the tyrosine phosphorylation of the SH2-containing adaptor proteins p66, p52, and p46 Shc in mouse mammary HC-11 epithelial cells and in human MDA-MB-453 and SKBr-3 breast cancer cells. CR-1 was also found to promote an increase in the association of the adaptor Grb2-guanine nucleotide exchange factor-mouse son of sevenless (mSOS) signaling complex with tyrosine-phosphorylated Shc in HC-11 cells. Finally, CR-1 was able to increase p42(erk-2) mitogen-activated protein kinase (MAPK) activity in HC-11 cells within 5-10 min of treatment. These data demonstrate that CR-1 can function through a receptor which activates intracellular components in the ras/raf/MEK/MAPK pathway.

BACKGROUND

The serogroup A conjugate meningococcal vaccine, MenAfriVac, was introduced in mass vaccination campaigns in December 2010 in Burkina Faso, Mali and Niger. In the coming years, vaccination will be extended to other African countries at risk of epidemics. To document the molecular characteristics of disease-causing meningococcal strains circulating in the meningitis belt of Africa before vaccine introduction, the World Health Organization Collaborating Centers on Meningococci in Europe and United States established a common strain collection of 773 isolates from cases of invasive meningococcal disease collected between 2004 and 2010 from 13 sub-Saharan countries.

METHODS

All isolates were characterized by multilocus sequence typing, and 487 (62%) were also analyzed for genetic variation in the surface antigens PorA and FetA. Antibiotic susceptibility was tested for part of the collection.

RESULTS

Only 19 sequence types (STs) belonging to 6 clonal complexes were revealed. ST-5 clonal complex dominated with 578 (74.8%) isolates. All ST-5 complex isolates were remarkably homogeneous in their PorA (P1.20,9) and FetA (F3-1) and characterized the serogroup A strains which have been responsible for most epidemics during this time period. Sixty-eight (8.8%) of the 773 isolates belonged to the ST-11 clonal complex which was mainly represented by serogroup W135, while an additional 38 (4.9%) W135 isolates belonged to the ST-175 complex. Forty-eight (6.2%) serogroup X isolates from West Africa belonged to the ST-181 complex, while serogroup X cases in Kenya and Uganda were caused by an unrelated clone, ST-5403. Serogroup X, ST-181, emerged in Burkina Faso before vaccine introduction.

CONCLUSIONS

In the seven years preceding introduction of a new serogroup A conjugate vaccine, serogroup A of the ST-5 clonal complex was identified as the predominant disease-causing strain.

The cell membrane is an important interface for communication with extracellular events, and incorporation of bioactive substances, such as antibodies and receptors, into the cell membrane may enhance the potential abilities of cells. Gene manipulation, chemical modification of membrane proteins, and cell surface painting using a GPI anchor have been performed to introduce substances into cell membranes. Furthermore, many lipid anchors have also been used to modify lipid membranes such as liposomes. In this study, we have focused on developing an easy and rapid method for anchoring of substances including macromolecular proteins into the membranes of living mammalian cells. We employed a single oleyl chain derivative coupled with hydrophilic poly(ethylene glycol) (PEG90, the ethyleneoxide (EO) unit is 90) to facilitate solubilization in water. This water-soluble derivative was designated Biocompatible Anchor for Membrane (BAM). Some proteins (streptavidin, EGFP and an antibody) were coupled with BAM90 at the distal terminal of PEG and rapidly (within a few minutes) anchored into the membranes of various cells (NIH3T3, 32D, Ba/F3, hybridoma 9E10). However, the anchored BAM90 disappeared from the cell membranes within 4-5 h in serum-free culture media, and moreover, the retention time of anchoring was shortened (1-2 h) in culture medium containing 10% FBS. We further prepared a dioleylphosphatidylethanolamine (DOPE)-PEG derivative (DOPE-BAM80, the EO unit is 80) as a double oleyl chain derivative for comparison with the single oleyl chain derivative, BAM90. The retention time of anchored DOPE-BAM80 was longer than that of BAM90 and more than 8 h in culture media with and without 10% serum. Furthermore, the treatment time of DOPE-BAM80 for anchoring was nearly as short (within a few minutes) as that of BAM90. In addition, both types of BAMs, BAM90 and DOPE-BAM80, showed no cytotoxicity. Therefore, DOPE-BAM80 is useful for protein anchoring to cells. Although the utilization of BAM90 is considered to be limited, it is expected to useful in restricted environments, for example, in tissues such as the cornea, peritoneum, bladder, and various mucosae, which are less exposed to serum. Thus, we suggest the possibility that both types of BAM can be applied to cell surface engineering.

Transcranial direct current stimulation (tDCS) is a neuromodulatory technique which involves passing a mild electric current to the brain through electrodes placed on the scalp. Several clinical studies suggest that tDCS may have clinically meaningful efficacy in the treatment of depression. The objective of this study was to simulate and compare the effects of several tDCS montages either used in clinical trials or proposed, for the treatment of depression, in different high-resolution anatomically-accurate head models. Detailed segmented finite element head models of two subjects were presented, and a total of eleven tDCS electrode montages were simulated. Sensitivity analysis on the effects of changing the size of the anode, rotating both electrodes and displacing the anode was also conducted on selected montages. The F3-F8 and F3-F4 montages have been used in clinical trials reporting significant antidepressant effects and both result in relatively high electric fields in dorsolateral prefrontal cortices. Other montages using a fronto-extracephalic or fronto-occipital approach result in greater stimulation of central structures (e.g. anterior cingulate cortex) which may be advantageous in treating depression, but their efficacy has yet to be tested in randomised controlled trials. Results from sensitivity analysis suggest that electrode position and size may be adjusted slightly to accommodate other priorities, such as skin discomfort and damage.

Internal tandem duplication mutations in the Flt3 tyrosine kinase gene (ITD-Flt3) and overexpression of Survivin are frequently found in patients with acute myeloid leukemia (AML). We investigated whether Survivin mediates the enhanced survival of primary hematopoietic progenitor cells (HPCs) resulting from ITD-Flt3 signaling. Ectopic ITD-Flt3 mutants increased Survivin expression in Ba/F3 cells downstream of PI3-kinase/Akt. Treatment of ITD-Flt3(+) human MV4-11 leukemia cells with the ITD-Flt3 inhibitor SU5416 reduced Survivin expression and inhibited cell proliferation. ITD-Flt3 dramatically increased the number of primary mouse marrow c-kit(+), Sca-1(+), Lin(Neg) cells and colony-forming unit granulocyte-macrophages (CFU-GMs) able to proliferate in the absence of growth factors, whereas Survivin deletion significantly reduced growth factor-independent proliferation and increased apoptosis, which was further accentuated by SU5416. Ectopic ITD-Flt3 reduced differentiation of Lin(Neg) marrow cells cultured with granulocyte-macrophage colony-stimulating factor (GM-CSF) plus stem cell factor, which was partially blocked by Survivin deletion. In addition, Survivin deletion decreased secondary colony formation induced by ITD-Flt3. Dominant-negative (dn)-Survivin delayed development of acute leukemia in mice that received a transplant of Ba/F3 cells expressing ITD-Flt3. These results suggest that Survivin regulates expansion of ITD-Flt3-transformed HPCs with self-renewal capability and development of ITD-Flt3(+) acute leukemia and that antagonizing Survivin may provide therapeutic benefit for patients with acute leukemia expressing ITD-Flt3.

The aim of this paper was to present our experience with seven monoclonal antibodies, six of them were applied in immunohistochemistry and immunoblotting of MyHC isoforms in rats and humans, one of them, 6H1 (Lucas et al., 2000), was tested in human muscle sections only. The four antibodies specific to rat MyHC isoforms, BA-D5,SC-71, BF-35,BF-F3 (Schiaffino et al.,1989) reacted as declared both on muscle sections and immunoblots of rat except SC-71 antibody, which stained MyHC-2a and -2x bands in blots. One of the two commercially available antibodies, A4.74 antibody,reliably marked type 2a fibres of rat,but in blots it weakly stained MyHC-2a and -2x isoforms when used undiluted. The other one, F113.15F4, stained type 2a and 2x fibres and corresponding MyHC bands in blots. Therefore, using this antibody rat MyHC-2x can be additionally confirmed, which can be otherwise demonstrated only on the principle of exclusion with BF-35.Using the same set of antibodies human fast MyHC isoforms can be revealed less clearly. Namely, SC-71 and A4.74 antibodies intensively stained histochemical type 2a,predominantly expressing 2a MyHC transcripts and moderately type 2x fibres, expressing mostly 2x MyHC transcripts, in blots the antibodies recognized both fast isoforms. The 6H1antibody was the only one that selectively labelled type 2x fibres, whereas BF-35 left unstained only a variable proportion of histochemical type 2x fibres and MyHC-2x in blots. F113.15F4 did not distinguish between human fast fibre types and corresponding MyHC isoforms in blots. The negativeresults obtained with BF-F3 in muscle sections and in blots are in agreement with the absence of MyHC-2b in human skeletal muscles. Our results imply that the reactivity of antibodies specific to distinct MyHC isoforms should be carefully evaluated not only among various species but with the two different techniques used as well.

OBJECTIVE

There is controversy about the performance of noninvasive tests such as FibroTest in diagnosing intermediate stages of fibrosis. We investigated whether this controversy results from limitations of biopsy analysis for intermediate-stage fibrosis and inappropriate determination of the standard area under the receiver-operator characteristic curve (AUROC).

METHODS

To determine whether biopsy has a lower diagnostic performance for fibrosis stage F2 (few septa) vs F1 (fibrosis without septa), compared with its performance for F1 vs F0 or F4 vs F3, we determined the fibrotic areas of large surgical samples collected from 20 consecutive patients with chronic liver disease or normal liver tissue that surrounded tumors. We analyzed digitized images of 27,869 virtual biopsies of increasing length and also analyzed data from 6500 patients with interpretable FibroTest results who also underwent biopsy analysis.

RESULTS

The overall performance of biopsy analysis (by Obuchowski measure) increased with biopsy length from 0.885 for 5-mm to 0.912 for 30-mm samples (P < .0001). The performance of biopsy was lower for the diagnosis of F2 vs F1 samples (weighted AUROC [wAUROC] = 0.505) than for F1 vs F0 (wAUROC = 0.773; 53% difference; P < .0001) or F4 vs F3 (wAUROC = 0.700; 39% difference; P < .0001), even when 30-mm biopsy samples were used. The performance of FibroTest was also lower for the diagnosis of F2 vs F1 samples (wAUROC = 0.512) than for F1 vs F0 samples (wAUROC = 0.626; 22% difference; P < .0001) or F4 vs F3 (wAUROC = 0.628; 23% difference; P < .0001). However, the FibroTest had smaller percentage differences among wAUROC values than biopsy.

CONCLUSIONS

Biopsy has a low level of diagnostic performance for fibrosis stages F2 and F1. The recommendation for biopsy analysis, instead of a validated biomarker panel such as FibroTest, for the diagnosis of intermediate stages of fibrosis is therefore misleading.

OBJECTIVE

Chronic myeloid leukemia (CML) is effectively treated with imatinib. However, reactivation of Bcr-Abl via kinase domain mutations that reduce sensitivity to imatinib can cause relapse. As combination therapy is frequently used to prevent emergence of resistance, the combination of imatinib with an inhibitor of imatinib-resistant Bcr-Abl mutants (e.g., Src/Abl inhibitors AP23848 and BMS-354825) was investigated.

METHODS

To test this approach, cellular proliferation and Bcr-Abl tyrosine phosphorylation assays were done on Ba/F3 cells expressing wild-type (WT) Bcr-Abl and four common imatinib-resistant mutants (Y253F, E255K, T315I, and M351T). Colony-forming assays with primary CML cells were also done.

RESULTS

Both Src/Abl inhibitors retained full inhibitory capacity when coadministered with imatinib at concentrations above typical clinical levels. For cells expressing WT Bcr-Abl or the marginally imatinib-resistant mutant M351T, inclusion of imatinib at therapeutic levels enhanced the effects of the Src/Abl inhibitors. By comparison, for the highly imatinib-resistant mutants Y253F and E255K, inclusion of imatinib at clinical levels resulted in only a slight enhancement beyond the effects of the Src/Abl inhibitors. None of the inhibitors affected Bcr-Abl T315I cells. Colony-forming assays with primary CML cells yielded analogous results.

CONCLUSIONS

Our results indicate that Src/Abl inhibitors are compatible with imatinib and suggest that combined Abl inhibitor therapy is a feasible treatment strategy for patients with CML.

Purpose To develop and validate a deep learning system (DLS) for staging liver fibrosis by using CT images in the liver. Materials and Methods DLS for CT-based staging of liver fibrosis was created by using a development data set that included portal venous phase CT images in 7461 patients with pathologically confirmed liver fibrosis. The diagnostic performance of the DLS was evaluated in separate test data sets for 891 patients. The influence of patient characteristics and CT techniques on the staging accuracy of the DLS was evaluated by logistic regression analysis. In a subset of 421 patients, the diagnostic performance of the DLS was compared with that of the radiologist's assessment, aminotransferase-to-platelet ratio index (APRI), and fibrosis-4 index by using the area under the receiver operating characteristic curve (AUROC) and Obuchowski index. Results In the test data sets, the DLS had a staging accuracy of 79.4% (707 of 891) and an AUROC of 0.96, 0.97, and 0.95 for diagnosing significant fibrosis (F2-4), advanced fibrosis (F3-4), and cirrhosis (F4), respectively. At multivariable analysis, only pathologic fibrosis stage significantly affected the staging accuracy of the DLS (P = .016 and .013 for F1 and F2, respectively, compared with F4), whereas etiology of liver disease and CT technique did not. The DLS (Obuchowski index, 0.94) outperformed the radiologist's interpretation, APRI, and fibrosis-4 index (Obuchowski index range, 0.71-0.81; P ˂ .001) for staging liver fibrosis. Conclusion The deep learning system allows for accurate staging of liver fibrosis by using CT images. © RSNA, 2018 Online supplemental material is available for this article.

OBJECTIVE

To assess the values of liver stiffness (LS) in patients with hepatitis B virus (HBV) chronic hepatitis and to compare them with those in patients with hepatitis C virus (HCV) chronic hepatitis.

METHODS

The study included 140 patients with HBV chronic hepatitis, and 317 patients with HCV chronic hepatitis, in which LS was measured (FibroScan®-Echosens®) and liver biopsy was performed in the same session (assessed according to the Metavir score).

RESULTS

According to the Metavir score of the 140 HBV patients: one had F0, 32 had F1, 67 had F2, 33 had F3 and 7 had F4. Of the 317 HCV patients: 5 had F0, 34 had F1, 146 had F2, 93 had F3 and 39 had F4. For the same severity of fibrosis, the mean values of LS in HBV patients were similar to those in HCV patients: F1, 6.5 ± 1.9 kPa vs 5.8 ± 2.1 kPa (P = 0.0889); F2, 7.1 ± 2 kPa vs 6.9 ± 2.5 kPa (P = 0.3369); F3, 9.1 ± 3.6 kPa vs 9.9 ± 5 kPa (P = 0.7038); F4, 19.8 ± 8.6 kPa vs 17.3 ± 6.1 kPa (P = 0.6574). A significant direct correlation between LS measurements and fibrosis was found in HCV patients (Spearman's r = 0.578, P < 0.0001), as well as in HBV patients (r = 0.408, P < 0.0001). The correlation was more significant in HCV than in HBV patients (Fisher's Z-test, Z = 2.210, P = 0.0271).

CONCLUSIONS

In our group, the mean values of LS in patients with chronic B hepatitis were similar to those in patients with chronic HCV hepatitis, for the same stage of fibrosis. Also, LS was correlated with the severity of fibrosis both in HBV and HCV chronic hepatitis patients.