Integrin-dependent adhesion sites consist of clustered integrins that transmit mechanical forces and provide signaling required for cell survival and morphogenesis. Despite their importance, the regulation of integrin clustering by the cytoplasmic adapter protein talin (Tal) and phosphatidylinositol (PI)-4,5-biphosphate (PI(4,5)P(2)) lipids nor their dynamic coupling to the actin cytoskeleton is fully understood. By using a Tal-dependent integrin clustering assay in intact cells, we identified a PI(4,5)P(2)-binding basic ridge spanning across the F2 and F3 domains of the Tal head that regulates integrin clustering. Clustering requires a new PI(4,5)P(2)-binding site in F2 and is negatively regulated by autoinhibitory interactions between F3 and the Tal rod (Tal-R). The release of the Tal-R exposes a new beta3-integrin-binding site in F3, enabling interaction with a membrane proximal acidic motif, which involves the formation of salt bridges between K(316) and K(324) with E(726) and D(723), respectively. This interaction shields the beta-integrin tail from reassociation with its alpha subunit, thereby maintaining the integrin in a substrate-binding and clustering-competent form.

In Philadelphia chromosome-positive human leukemias, which include chronic myelogenous leukemia and some acute lymphocytic leukemias, the c-abl proto-oncogene on chromosome 9 becomes fused to the bcr gene on chromosome 22, and Bcr-Abl fusion proteins are produced. The Bcr sequences activate the Abl tyrosine kinase which is required for the transforming function of Bcr-Abl. The Bcr sequences also enhance an F-actin-binding activity associated with c-Abl. Here, we show that binding of c-Abl and Bcr-Abl proteins to actin filaments in vivo and in vitro is mediated by an evolutionarily conserved domain at the C-terminal end of c-Abl. The c-Abl F-actin-binding domain contains a consensus motif found in several other actin-crosslinking proteins. Mutations in the consensus motif are shown to abolish binding to F-actin. Bcr-Abl proteins unable to associate with F-actin have a reduced ability to transform Rat-1 fibroblasts and to abrogate the requirement for interleukin-3 in the lymphoblastoid cell line Ba/F3. In transformed cells, Bcr-Abl induces a redistribution of F-actin into punctate, juxtanuclear aggregates. The binding to actin filaments has important implications for the pathogenic and physiological functions of the Bcr-Abl and c-Abl proteins.

Water deficits consistently promote higher concentrations of anthocyanins in red winegrapes and their wines. However, controversy remains as to whether there is any direct effect on berry metabolism other than inhibition of growth. Early (ED) and late (LD) season water deficits, applied before or after the onset of ripening (veraison), were imposed on field grown Vitis vinifera "Cabernet Sauvignon", and the responses of gene expression in the flavonoid pathway and their corresponding metabolites were determined. ED accelerated sugar accumulation and the onset of anthocyanin synthesis. Both ED and LD increased anthocyanin accumulation after veraison. Expression profiling revealed that the increased anthocyanin accumulation resulted from earlier and greater expression of the genes controlling flux through the anthocyanin biosynthetic pathway, including F3H, DFR, UFGT and GST. Increases in total anthocyanins resulted predominantly from an increase of 3'4'5'-hydroxylated forms through the differential regulation of F3'H and F3'5'H. There were limited effects on proanthocyanidin, other flavonols, and on expression of genes committed to their synthesis. These results demonstrate that manipulation of abiotic stress through applied water deficits not only modulates compositional changes during berry ripening, but also alters the timing of particular aspects of the ripening process.

The activation of integrin adhesion receptors from low to high affinity in response to intracellular cues controls cell adhesion and signaling. Binding of the cytoskeletal protein talin to the beta3 integrin cytoplasmic tail is required for beta3 activation, and the integrin-binding PTB-like F3 domain of talin is sufficient to activate beta3 integrins. Here we report that, whereas the conserved talin-integrin interaction is also required for beta1 activation, and talin F3 binds beta1 and beta3 integrins with comparable affinity, expression of the talin F3 domain is not sufficient to activate beta1 integrins. beta1 integrin activation could, however, be detected following expression of larger talin fragments that included the N-terminal and F1 domains, and mutagenesis indicates that these domains cooperate with talin F3 to mediate beta1 activation. This effect is not due to increased affinity for the integrin beta tail and we hypothesize that the N-terminal domains function by targeting or orienting talin in such a way as to optimize the interaction with the integrin tail. Analysis of beta3 integrin activation indicates that inclusion of the N-terminal and F1 domains also enhances F3-mediated beta3 activation. Our results therefore reveal a role for the N-terminal and F1 domains of talin during integrin activation and highlight differences in talin-mediated activation of beta1 and beta3 integrins.

The Nuclear Factor (NF)-kappaB family of transcription factors controls expression of genes which promote cell growth, survival, and neoplastic transformation. Recently we demonstrated aberrant constitutive activation of NF-kappaB in primary human and rat breast cancer specimens and in cell lines. Overexpression of the epidermal growth factor receptor (EGFR) family member Her-2/neu, seen in approximately 30% of breast cancers, is associated with poor prognosis. Previously, Her-2/neu has been shown to signal via a phosphatidylinositol 3 (PI3)-kinase to Akt/protein kinase B (PKB) pathway. Since this signaling pathway was recently shown to activate NF-kappaB, here we have tested the hypothesis that Her-2/neu can activate NF-kappaB in breast cancer. Overexpression of Her-2/neu and EGFR-4 in Ba/F3 cells led to constitutive PI3- and Akt kinase activities, and induction of classical NF-kappaB (p50/p65). Similarly, a tumor cell line and tumors derived from MMTV-Her-2/neu transgenic mice displayed elevated levels of classical NF-kappaB. Engagement of Her-2/neu receptor downregulated the level of NF-kappaB. NF-kappaB binding and activity in the cultured cells was reduced upon inhibition of the PI3- to Akt kinase signaling pathway via ectopic expression of kinase inactive mutants, incubation with wortmannin, or expression of the tumor suppressor phosphatase PTEN. Inhibitors of calpain, but not the proteasome, blocked IkappaB-alpha degradation. Inhibition of Akt did not affect IKK activity. These results indicate that Her-2/neu activates NF-kappaB via a PI3- to Akt kinase signaling pathway that can be inhibited via the tumor suppressor PTEN, and is mediated by calpain rather than the IkappaB kinase complex.

The c-kit receptor tyrosine kinase (KIT) is activated upon ligand binding, thereby leading to a variety of signaling events that play a fundamental role in hematopoiesis. In addition to ligand-dependent activation, we have previously shown that KIT is constitutively activated in a ligand-independent manner by two point mutations, Val-559->>Gly (G559) mutation in the juxtamembrane domain and Asp-814->>Val (V814) mutation in the phosphotransferase domain. To investigate the biochemical consequence and biologic significance of these mutations, retroviral vectors encoding KITG559 or KITV814 were introduced into murine pro-B-type Ba/F3 cells and myeloid FDC-P1 cells, both of which require interleukin-3 (IL-3) for their growth and survival. In the cells, KITG559 or KITV814 were found to be constitutively phophorylated on tyrosine in the absence of stem cell factor (SCF) that is a ligand for KIT. Chemical cross-linking analysis showed that a substantial fraction of the phosphorylated KITG559 underwent dimerization even in the absence of SCF, whereas the phosphorylated KITV814 did not, suggesting the distinct mechanisms underlying constitutive activation of KIT by G559 and V814 mutations. Furthermore, the cells expressing either KITG559 or KITV814 were found to show a factor-independent growth, whereas the cells expressing wild-type KIT (KITWT) proliferated in response to SCF as well as IL-3. Moreover, subcutaneous injection of Ba/F3 cells expressing KITG559 or KITV814 into nude mice resulted in production of large tumors at all sites of the injection within 2 weeks, and all nude mice quickly succumbed to leukemia and died. These results suggest that, although the mechanisms underlying constitutive activation of KITG559 or KITV814 may be different, both of the activating mutations have a function to induce a factor-independent and tumorigenic phenotype. Also, the data of this study raise the possibility that the constitutively activating mutations of c-kit may play a causal role in development of hematologic malignancies.

Two families of small peptides that bind to the human thrombopoietin receptor and compete with the binding of the natural ligand thrombopoietin (TPO) were identified from recombinant peptide libraries. The sequences of these peptides were not found in the primary sequence of TPO. Screening libraries of variants of one of these families under affinity-selective conditions yielded a 14-amino acid peptide (Ile-Glu-Gly-Pro-Thr-Leu-Arg-Gln-Trp-Leu-Ala-Ala-Arg-Ala) with high affinity (dissociation constant approximately 2 nanomolar) that stimulates the proliferation of a TPO-responsive Ba/F3 cell line with a median effective concentration (EC50) of 400 nanomolar. Dimerization of this peptide by a carboxyl-terminal linkage to a lysine branch produced a compound with an EC50 of 100 picomolar, which was equipotent to the 332-amino acid natural cytokine in cell-based assays. The peptide dimer also stimulated the in vitro proliferation and maturation of megakaryocytes from human bone marrow cells and promoted an increase in platelet count when administered to normal mice.

Recent reports have demonstrated fusion of the TEL gene on 12p13 to the JAK2 gene on 9p24 in human leukemias. Three variants have been identified that fuse the TEL pointed (PNT) domain to (i) the JAK2 JH1-kinase domain, (ii) part of and (iii) all of the JH2 pseudokinase domain. We report that all of the human TEL/JAK2 variants, and a human/mouse chimeric hTEL/mJAK2(JH1) fusion gene, transform the interleukin-3 (IL-3)-dependent murine hematopoietic cell line Ba/F3 to IL-3-independent growth. Transformation requires both the TEL PNT domain and JAK2 kinase activity. Furthermore, all TEL/JAK2 variants strongly activated STAT 5 by phosphotyrosine Western blots and by electrophoretic mobility shift assays (EMSA). Mice (n = 40) transplanted with bone marrow infected with the MSCV retrovirus containing either the hTEL/mJAK2(JH1) fusion or its human counterpart developed a fatal mixed myeloproliferative and T-cell lymphoproliferative disorder with a latency of 2-10 weeks. In contrast, mice transplanted with a TEL/JAK2 mutant lacking the TEL PNT domain (n = 10) or a kinase-inactive TEL/JAK2(JH1) mutant (n = 10) did not develop the disease. We conclude that all human TEL/JAK2 fusion variants are oncoproteins in vitro that strongly activate STAT 5, and cause lethal myelo- and lymphoproliferative syndromes in murine bone marrow transplant models of leukemia.

Activating mutations in either BRAF or NRAS are seen in a significant number of malignant melanomas, but their incidence appears to be dependent to ultraviolet light exposure. Thus, BRAF mutations have the highest incidence in non-chronic sun damaged (CSD), and are uncommon in acral, mucosal and CSD melanomas. More recently, activating KIT mutations have been described in rare cases of metastatic melanoma, without further reference to their clinical phenotypes. This finding is intriguing since KIT expression is downregulated in most melanomas progressing to more aggressive lesions. In this study, we investigated a group of anal melanomas for the presence of BRAF, NRAS, KIT and PDGFRA mutations. A heterozygous KIT exon 11 L576P substitution was identified in 3 of 20 cases tested. The 3 KIT mutation-carrying tumors were strongly immunopositive for KIT protein. No KIT mutations were identified in tumors with less than 4+ KIT immunostaining. NRAS mutation was identified in one tumor. No BRAF or PDGFRA mutations were identified in either KIT positive or negative anal melanomas. In vitro drug testing of stable transformant Ba/F3 KIT(L576P) mutant cells showed sensitivity for dasatinib (previously known as BMS-354825), a dual SRC/ABL kinase inhibitor, and imatinib. However, compared to an imatinib-sensitive KIT mutant, dasatinib was potent at lower doses than imatinib in the KIT(L576P) mutant. These results suggest that a subset of anal melanomas show activating KIT mutations, which are susceptible for therapy with specific kinase inhibitors.

Progress toward understanding the biological roles of carbohydrates has been remarkably slow, and efforts to exploit this class of biopolymers as diagnostic and therapeutic targets have proven extremely challenging. Both basic and clinical research rely heavily on identifying and monitoring expression levels of carbohydrates. Over the last 30 years, the majority of expression information has been derived from antibody- and lectin-binding studies. Using a carbohydrate microarray containing 80 different glycans and glycoproteins, the specificities of 27 antiglycan antibodies were evaluated, including antibodies to histo-blood group A, B, and H antigens (81FR2.2, CLCP-19B, B389, 92FR-A2, B480, B460, B376, and B393), Lewis antigens (7LE, 15C02, 28, ZC-18C, 121SLE, CA199.02, PR.5C5, 2-25LE, BR55, T174, T218, F3, A70-C/C8, FR4A5, and K21), and other tumor-associated antigens (B389, 1A4, B1.1, and 5B5). In total, evaluation of over 2000 individual carbohydrate-protein interactions was carried out. More than half of the antibodies considered to be specific for their designated antigen were found to cross-react with other glycans. The cross-reactive glycans could be mistaken for the designated antigen in biopsy samples or other biological samples, leading to inaccurate conclusions.

BACKGROUND

Many studies have reported beneficial effects from the application of near-infrared (NIR) light photobiomodulation (PBM) to the body, and one group has reported beneficial effects applying it to the brain in stroke patients. We have reported that the measurement of a patient's left and right hemispheric emotional valence (HEV) may clarify data and guide lateralized treatments. We sought to test whether a NIR treatment could 1. improve the psychological status of patients, 2. show a relationship between immediate psychological improvements when HEV was taken into account, and 3. show an increase in frontal pole regional cerebral blood flow (rCBF), and 4. be applied without side effects.

METHODS

We gave 10 patients, (5 M/5 F) with major depression, including 9 with anxiety, 7 with a past history of substance abuse (6 with an opiate abuse and 1 with an alcohol abuse history), and 3 with post traumatic stress disorder, a baseline standard diagnostic interview, a Hamilton Depression Rating Scale (HAM-D), a Hamilton Anxiety Rating Scale (HAM-A), and a Positive and Negative Affect Scale (PANAS). We then gave four 4-minute treatments in a random order: NIR to left forehead at F3, to right forehead at F4, and placebo treatments (light off) at the same sites. Immediately following each treatment we repeated the PANAS, and at 2-weeks and at 4-weeks post treatment we repeated all 3 rating scales. During all treatments we recorded total hemoglobin (cHb), as a measure of rCBF with a commercial NIR spectroscopy device over the left and the right frontal poles of the brain.

RESULTS

At 2-weeks post treatment 6 of 10 patients had a remission (a score </= 10) on the HAM-D and 7 of 10 achieved this on the HAM-A. Patients experienced highly significant reductions in both HAM-D and HAM-A scores following treatment, with the greatest reductions occurring at 2 weeks. Mean rCBF across hemispheres increased from 0.011 units in the off condition to 0.043 units in the on condition, for a difference of 0.032 (95% CI: -0.016, 0.080) units, though this result did not reach statistical significance. Immediately after treatment the PANAS improved to a significantly greater extent with NIR "on" relative to NIR "off" when a hemisphere with more positive HEV was treated than when one with more negative HEV was treated. We observed no side effects.

CONCLUSIONS

This small feasibility study suggests that NIR-PBM may have utility for the treatment of depression and other psychiatric disorders and that double blind randomized placebo-controlled trials are indicated.

BACKGROUND

ClinicalTrials.gov Identifier: NCT00961454.

OBJECTIVE

ALK rearrangement-positive lung cancers can be effectively treated with ALK inhibitors. However, the magnitude and duration of response is heterogeneous. In addition, acquired resistance limits the efficacy of ALK inhibitors, with most upfront resistance mechanisms being unknown.

METHODS

By making use of the Ba/F3 cell line model, we analyzed the cytotoxic efficacy of ALK kinase inhibitors as a function of different EML4-ALK fusion variants v1, v2, v3a, and v3b as well as of three artificially designed EML4-ALK deletion constructs and the ALK fusion genes KIF5b-ALK and NPM1-ALK. In addition, the intracellular localization, the sensitivity to HSP90 inhibition and the protein stability of ALK fusion proteins were studied.

RESULTS

Different ALK fusion genes and EML4-ALK variants exhibited differential sensitivity to the structurally diverse ALK kinase inhibitors crizotinib and TAE684. In addition, differential sensitivity correlated with differences in protein stability in EML4-ALK-expressing cells. Furthermore, the sensitivity to HSP90 inhibition also varied depending on the ALK fusion partner but differed from ALK inhibitor sensitivity patterns. Finally, combining inhibitors of ALK and HSP90 resulted in synergistic cytotoxicity.

CONCLUSIONS

Our results might explain some of the heterogeneous responses of ALK-positive tumors to ALK kinase inhibition observed in the clinic. Thus, targeted therapy of ALK-positive lung cancer should take into account the precise ALK genotype. Furthermore, combining ALK and HSP90 inhibitors might enhance tumor shrinkage in EML4-ALK-driven tumors.

Studies assessing the role of Stat5 in the IL-2 proliferative signal have produced contradictory, and thus inconclusive, results. One factor confounding many of these studies is the ability of IL-2R to deliver redundant mitogenic signals from different cytoplasmic tyrosines on the IL-2R beta-chain (IL-2Rbeta). Therefore, to assess the role of Stat5 in mitogenic signaling independent of any redundant signals, all cytoplasmic tyrosines were deleted from IL-2Rbeta except for Tyr510, the most potent Stat5-activating site. This deletion mutant retained the ability to induce Stat5 activation and proliferation in the T cell line CTLL-2 and the pro-B cell line BA/F3. A set of point mutations at or near Tyr510 that variably compromised Stat5 activation also compromised the proliferative signal and revealed a quantitative correlation between the magnitude of Stat5 activation and proliferation. Proliferative signaling by a receptor mutant with a weak Stat5 activating site could be rescued by overexpression of wt Stat5a or b. Additionally, the ability of this receptor mutant to induce c-myc, bcl-x, and bcl-2 was enhanced by overexpression of wt Stat5. By contrast, overexpression of a version of Stat5a lacking the C-terminal trans-activation domain inhibited the induction of these genes and cell proliferation. Thus, Stat5 is a critical component of the proliferative signal from Tyr510 of the IL-2R and regulates expression of both mitogenic and survival genes through its trans-activation domain.

OBJECTIVE

Liver stiffness measurement (LSM) by transient elastography is a noninvasive test of liver fibrosis, but cannot be performed in a significant proportion of obese patients. The aim of this study was to evaluate the performance of the new XL probe in patients with nonalcoholic fatty liver disease (NAFLD).

METHODS

Liver biopsy and paired LSM by both the original M probe and XL probe were performed on 193 consecutive NAFLD patients in France and Hong Kong.

RESULTS

Compared with M probe, XL probe was more likely to achieve 10 valid measurements (95% vs. 81%; P<0.001) and a success rate of over 60% (90% vs. 74%; P<0.001). The areas under receiver operating characteristics curves of XL probe for F2, F3, and F4 disease were 0.80, 0.85, and 0.91, respectively. XL probe tended to generate lower LSM than M probe in the same patient. At a cutoff of 7.2 kPa, the sensitivity, specificity, positive, and negative predictive values for F3 or greater disease were 78%, 78%, 60%, and 89%, respectively. Discordance of at least two stages between XL probe and histology was observed in 16 (9%) patients. Body mass index (BMI) over 35 kg/m(2) was independently associated with discordance (adjusted odds ratio 9.09; 95% confidence interval 1.10-75.43). Reliable measurements by XL probe were obtained in 75% of the overall population and 65% of patients with BMI over 30 kg/m(2).

CONCLUSIONS

LSM by XL probe can be performed successfully in most NAFLD patients, but obesity is associated with less accurate and reliable measurements.

Type 2 diabetes is an important risk factor for non-alcoholic fatty liver disease (NAFLD), but current guidelines provide conflicting recommendations on whether diabetic patients should be screened for NAFLD. We therefore studied the strategy of screening diabetic patients by FibroScan.

Liver fat and fibrosis were assessed by controlled attenuation parameter (CAP) and liver stiffness measurements (LSM) by FibroScan at a diabetic centre for patients from primary care and hospital clinics. Probe-specific LSM cut-offs were used to detect advanced fibrosis.

Of 1918 patients examined, 1799 (93.8%) had valid CAP and 1884 (98.2%) had reliable LSM (1770 with the M probe and 114 with the XL probe). The proportion of patients with increased CAP and LSM was 72.8% (95% CI 70.7% to 74.8%) and 17.7% (95% CI 16.0% to 19.5%), respectively. By multivariable analysis, female gender, higher body mass index, triglycerides, fasting plasma glucose and alanine aminotransferase (ALT) and non-insulin use were associated with increased CAP. Longer duration of diabetes, higher body mass index, increased ALT and spot urine albumin:creatinine ratio and lower high-density lipoprotein-cholesterol were associated with increased LSM. Ninety-four patients (80% had increased LSM) underwent liver biopsy: 56% had steatohepatitis and 50% had F3-4 disease.

Diabetic patients have a high prevalence of NAFLD and advanced fibrosis. Those with obesity and dyslipidaemia are at particularly high risk and may be the target for liver assessment. Our data support screening for NAFLD and/or advanced fibrosis in patients with type 2 diabetes.

One Japanese pedigree of familial essential thrombocythemia (FET) inherited in an autosomal-dominant manner is presented. A unique point mutation, serine 505 to asparagine 505 (Ser505Asn), was identified in the transmembrane domain of the c-MPL gene in all of the 8 members with thrombocythemia, but in none of the other 8 unaffected members in this FET family. The Ba/F3 cells expressing the mutant Asn505 acquired interleukin 3 (IL-3)-independent survival capacity, whereas those expressing wild-type Ser505 did not. The autonomous phosphorylation of Mek1/2 and Stat5b was observed in the mutant Ba/F3 cells in the absence of IL-3. The former was also found in platelets derived from the affected individual in the absence of thrombopoietin. These results show that the Asn505 is an activating mutation with respect to the intracellular signaling and survival of the cells. This is the first report of FET deriving from a dominant-positive activating mutation of the c-MPL gene.

Because of the importance of oscillations as a general phenomenon of neuronal activity the use of EEG spectral analysis is among the most important approaches for studying human information processing. Usually, oscillations at different frequencies occur simultaneously during information processing. Thus, the question for synchronisation of different frequencies by phase coupling and its possible functional significance is of primary importance. An answer may be given by bispectral analysis. Estimation of the (cross-) bispectrum allows to identify synchronised frequencies and possibly, the existence of non-linear phase coupling of different oscillators. Previous studies have demonstrated the simultaneous occurrence of slow (4-7 Hz) and fast (20-30 Hz) oscillations at frontal and prefrontal electrode positions during memory processing. However, interrelations between these rhythms have not been investigated up to now. In order to test short-term memory, the Sternberg task with random figures and number words was carried out on 10 female subjects. During the task EEG was recorded. Power and bispectral analyses from frontal, prefrontal and frontopolar regions were performed off-line. Increased power was found in both the theta and the gamma bands. Strong phase-coupling between theta at Fz and gamma at F3 and at Fp1, respectively, was shown for memorising number words by means of cross-bicoherence. A possible reason for this is an amplitude modulation of gamma frequencies by slow oscillations. The correspondent coherence analysis between the envelope of gamma frequencies at Fp1 and the raw EEG at Fz supports this presumption. This finding is interpreted as an EEG aspect of the functional linking between the prefrontal areas and the G.cinguli (as part of the limbic system), which are both extremely important for memory functions.

Flavonoids are important secondary metabolites in many fruits, and their hydroxylation pattern determines their color, stability, and antioxidant capacity. Hydroxylation of the B-ring of flavonoids is catalyzed by flavonoid 3'-hydroxylase (F3'H) and flavonoid 3',5'-hydroxylase (F3'5'H), and may also require cytochrome b5. We report the identification of genes encoding F3'H, F3'5'H, and a putative cytochrome b5 from grapevine (Vitis vinifera L. cv Shiraz) and their transcriptional regulation in fruit. Functionality of the genes VvF3'H and VvF3'5'H1 was demonstrated by ectopic expression in petunia (Petunia hybrida), which altered flower color and flavonoid composition as expected. VvF3'H was expressed in grapes before flowering, when 3'-hydroxylated flavonols are made, and all three genes were expressed after flowering, when proanthocyanidins (PAs) are synthesized. In berry skin, expression of all three genes was low at the onset of ripening (véraison) but increased after véraison concomitant with the accumulation of 3'- and 3',5'-hydroxylated anthocyanins. VvF3'H and VvCytoB5 were expressed in seeds but not VvF3'5'H1, consistent with the accumulation of 3'-hydroxylated PAs in this tissue. VvCytoB5 expression was correlated with expression of both VvF3'H and VvF3'5'H1 in the different grape tissues. In contrast to red grapes, where VvF3'H, VvF3'5'H1, and VvCytoB5 were highly expressed during ripening, the expression of VvF3'5'H1 and VvCytoB5 in white grapes during ripening was extremely low, suggesting a difference in transcriptional regulation. Our results show that temporal and tissue-specific expression of VvF3'H, VvF3'5'H1, and VvCytoB5 in grapes is coordinated with the accumulation of the respective hydroxylated flavonols and PAs, as well as anthocyanins. Understanding the regulation of flavonoid hydroxylases could be used to modify flavonoid composition of fruits.

Cannabinoids present in Cannabis sativa (marijuana) exert biological effects via cannabinoid receptors CB1 and CB2. We recently demonstrated that CB1 and CB2 receptors regulate progression of experimental liver fibrosis. We therefore investigated the impact of cannabis smoking on fibrosis progression rate in patients with chronic hepatitis C (CHC). Two hundred seventy consecutive untreated patients with CHC of known duration undergoing liver biopsy were studied. Demographic, epidemiological, metabolic, and virological data were recorded, and detailed histories of cannabis, alcohol, and tobacco use over the span of hepatitis C virus infection were obtained. Fibrosis stage, steatosis, and activity grades were scored according to Metavir system. Patients were categorized as noncannabis users (52.2%), occasional users (14.8%), or daily users (33.0%), and the relationship between cannabis use and fibrosis progression rate (FPR) or fibrosis stage was assessed. On multivariate analysis, six factors were independently related to a FPR greater than 0.074 (median value of the cohort): daily cannabis use (OR = 3.4 [1.5-7.4]), Metavir activity grade A2 or higher (OR = 5.4 [2.9-10.3]), age at contamination of more than 40 years (OR = 10.5 [3.0-37.1]), genotype 3 (OR = 3.4 [1.5-7.7]), excessive alcohol intake (OR = 2.2 [1.1-4.5]), and steatosis (OR = 2.0 [1.0-4.1]). Daily cannabis use was also an independent predictor of a rapid FPR (>0.15) (OR = 3.6 [1.5-7.5]). Finally, severe fibrosis >> or =F3) was also predicted by daily cannabis use (OR = 2.5 [1.1-5.6]; P = .034), independently of Metavir activity grade, excessive alcohol intake, age at liver biopsy, steatosis, and tobacco smoking. In conclusion, daily cannabis smoking is significantly associated with fibrosis progression during CHC. Patients with ongoing CHC should be advised to refrain from regular cannabis use.

BACKGROUND

Infections during fetal life or neonatal period, including infections with Toxoplasma gondii, may be associated with a risk for schizophrenia and other mental disorders. The objectives of this study were to study the association between serological markers for maternal and neonatal infection and the risk for schizophrenia, related psychoses, and affective disorders in a national cohort of newborns.

METHODS

This study was a cohort-based, case-control study combining data from national population registers and patient registers and a national neonatal screening biobank in Denmark. Patients included persons born in Denmark in 1981 or later followed up through 1999 with respect to inpatient or outpatient treatment for schizophrenia or related disorders (ICD-10 F2) or affective disorders (ICD-10 F3).

RESULTS

Toxoplasma gondii immunoglobulin G (IgG) levels corresponding to the upper quartile among control subjects were significantly associated with schizophrenia risk (odds ratio [OR] = 1.79, p = .045) after adjustment for urbanicity of place of birth, year of birth, gender, and psychiatric diagnoses among first-degree relatives. There was no significant association between any marker of infection and other schizophrenia-like disorders or affective disorders.

CONCLUSIONS

Our study supports an association between Toxoplasma gondii and early-onset schizophrenia. Further studies are needed to establish if the association is causal and if it generalizes to cases with onset after age 18.

BACKGROUND

FibroTest (FT) is a biomarker of liver fibrosis initially validated in patients with chronic hepatitis C (CHC). The aim was to test two hypotheses, one, that the FT diagnostic value was similar in the three other frequent fibrotic diseases: chronic hepatitis B (CHB), alcoholic liver disease (ALD) and non-alcoholic fatty liver disease (NAFLD); and the other, that the FT diagnostic value was similar for intermediate and extreme fibrosis stages.

METHODS

The main end points were the FT area under the ROC curves (AUROCs) for the diagnosis of bridging fibrosis (F2F3F4 vs. F0F1), standardized for the spectrum of fibrosis stages, and the comparison of FT AUROCs between adjacent stages. Two meta-analyses were performed: one combining all the published studies (random model), and one of an integrated data base combining individual data. Sensitivity analysis integrated the independency of authors, lenght of biopsy, prospective design, respect of procedures, comorbidities, and duration between biopsy and serum sampling.

RESULTS

A total of 30 studies were included which pooled 6,378 subjects with both FT and biopsy (3,501 HCV, 1,457 HBV, 267 NAFLD, 429 ALD, and 724 mixed). Individual data were analyzed in 3,282 patients. The mean standardized AUROC was 0.84 (95% CI, 0.83-0.86), without differences between causes of liver disease: HCV 0.85 (0.82-0.87), HBV 0.80 (0.77-0.84), NAFLD 0.84 (0.76-0.92), ALD 0.86 (0.80-0.92), mixed 0.85 (0.80-0.93). The AUROC for the diagnosis of the intermediate adjacent stages F2 vs. F1 (0.66; 0.63-0.68, n = 2,055) did not differ from that of the extreme stages F3 vs. F4 (0.69; 0.65-0.72, n = 817) or F1 vs. F0 (0.62; 0.59-0.65, n = 1788).

CONCLUSIONS

FibroTest is an effective alternative to biopsy in patients with chronic hepatitis C and B, ALD and NAFLD. The FT diagnostic value is similar for the diagnosis of intermediate and extreme fibrosis stages.

Age is probably the single most crucial factor determining how humans sleep. Age and level of vigilance significantly influence the electroencephalogram (EEG) and the polysomnogram (PSG). The Pediatric Task Force provide an evidence-based review of the age-related development of the polysomnographic features of sleep in neonates, infants, and children, assessing the reliability and validity of these features, and assessing alternative methods of measurement. We used this annotated supporting text to develop rules for scoring sleep and arousals in infants and children. A pediatric EEG or PSG can only be determined to be normal by assessing whether the EEG patterns are appropriate for maturational age. Sleep in infants at term can be scored as NREM and REM sleep because all the polysomnographic and EEG features of REM sleep are present and quiet sleep, if not NREM sleep, is at least "not REM sleep." The dominant posterior rhythm (DPR) of relaxed wakefulness increases in frequency with age: (1) 3.5-4.5 Hz in 75% of normal infants by 3-4 months post-term; (2) 5-6 Hz in most infants 5-6 months post-term; 3) 6 Hz in 70% of normal children by 2 months of age; and 3) 8 Hz (range 7.5-9.5 Hz) in 82% of normal children age 3 years, 9 Hz in 65% of 9-year-olds, and 10 Hz in 65% of 15-year-old controls. Sleep spindles in children occur independently at two different frequencies and two different scalp locations: 11.0-12.75 Hz over the frontal and 13.0-14.75 Hz over the centroparietal electrodes; these findings are most prominent in children younger than 13 years. Centroparietal spikes are often maximal over the vertex (Cz), less often maximal over the left central (C3) or right central (C4) EEG derivation. About 50% of sleep spindles within a particular infant's PSG are asynchronous before 6 months of age, 30% at 1 year. Based on this, we recommend that: (1) sleep spindles be scored as a polysomnographic signature of NREM stage 2 sleep (N2) at whatever age they are first seen in a PSG, typically present by 2 to 3 months post-term; (2) identify and score sleep spindles from the frontal and centroparietal EEG derivations, especially in infants and children younger than 13 years. NREM sleep in an infant or child can be scored if the dominant posterior rhythm occupies <50% of a 30-second epoch, and one or more of the following EEG patterns appear: (1) a diffuse lower voltage mixed frequency activity; (2) hypnagogic hypersynchrony; (3) rhythmic anterior theta of drowsiness; (4) diffuse high voltage occipital delta slowing; (5) runs or bursts of diffuse, frontal, frontocentral, or occipital maximal rhythmic 3-5 Hz slowing; (6) vertex sharp waves; and/or (7) post-arousal hypersynchrony. K complexes first appear 5 months post-term and are usually present by 6 months post-term, whereas clearly recognizable vertex sharp waves are most often seen 16 months post-term. Vertex sharp waves are best seen over the central (Cz, C3, C4) and K complexes over the frontal (Fz, F3, F4) electrodes. Slow wave activity (SWA) of slow wave sleep (SWS) is first seen as early as 2 to 3 months post-term and is usually present 4 to 4.5 months post-term. SWA of SWS in an infant or child often has a peak-to-peak amplitude of 100 to 400 microV. Based on consensus voting we recommended scoring N1, N2, and N3 corresponding to NREM 1, 2, and SWS whenever it was recognizable in an infant's PSG, usually by 4 to 4.5 months post-term (as early as 2-3 months post-term). Epochs of NREM sleep which contain no sleep spindles, K complexes, or SWA would be scored as N1; those which contain either K complexes or sleep spindles and <20% SWS as N2, and those in which >20% of the 30-second epoch contain 0.5 to 2 Hz >75 microV (usually 100-400 microV) activity as N3. The DPR should be scored in the EEG channel that is best observed, (typically occipital), but DPR reactive to eye opening can be seen in central electrodes. Because sleep spindles occur independently over the frontal and central regions in children, they should be scored whether they occur in the frontal or central regions. Because sleep spindles are asynchronous before age 2 years, simultaneous recording of left and right frontal and central activity may be warranted in children 1-2 years of age. Simultaneous recording of left, right, and midline central electrodes may be appropriate because of the asynchronous nature of sleep spindles before age 2 years, but reliability testing is needed. Evidence has shown that the PSG cannot reliably be used to identify neurological deficits or to predict behavior or outcome in infants because of significant diversity of results, even in normal infants. Normal sleep EEG patterns and architecture are present in the first year of life, even in infants with severe neurological compromise. Increasing evidence suggests that sleep and its disorders play critical roles in the development of healthy children and healthy adults thereafter. Reliability studies comparing head-to-head different scoring criteria, recording techniques, and derivations are needed so that future scoring recommendations can be based on evidence rather than consensus opinion. We need research comparing clinical outcomes with PSG measures to better inform clinicians and families exactly what meaning a PSG has in evaluating a child's suspected sleep disorder.

Expression cloning of cDNAs was first described a decade ago and was based on transient expression of cDNA libraries in COS cells. In contrast to transient transfection of plasmids, retroviral gene transfer delivers genes stably into a wide range of target cells. We utilize a simple packaging system for production of high-titer retrovirus stock from cDNA libraries to establish a cDNA expression cloning system. In two model experiments, murine interleukin (IL)-3-dependent Ba/F3 cells were infected with libraries of retrovirally expressed cDNA derived from human T-cell mRNA or human IL-3-dependent TF-1 cell line mRNA. These infected Ba/F3 cells were selected for the expression of CD2 by flow cytometry or for the alpha subunit of the human IL-3 receptor (hIL-3R alpha) by factor-dependent growth. CD2 (frequency, 1 in 10(4)) and hIL-3R alpha (frequency, 1 in 1.5 x 10(5)) cDNAs were readily detected in small-scale experiments, indicating this retroviral expression cloning system is efficient enough to clone low-abundance cDNAs by their expression or function.