Glaucousness is described as the scattering effect of visible light from wax deposited on the cuticle of plant aerial organs. In wheat, two dominant genes lead to non-glaucous phenotypes: Inhibitor of wax 1 (Iw1) and Iw2. The molecular mechanisms and the exact extent (beyond visual assessment) by which these genes affect the composition and quantity of cuticular wax is unclear. To describe the Iw1 locus we used a genetic approach with detailed biochemical characterization of wax compounds. Using synteny and a large number of F2 gametes, Iw1 was fine-mapped to a sub-cM genetic interval on wheat chromosome arm 2BS, which includes a single collinear gene from the corresponding Brachypodium and rice physical maps. The major components of flag leaf and peduncle cuticular waxes included primary alcohols, β-diketones and n-alkanes. Small amounts of C19-C27 alkyl and methylalkylresorcinols that have not previously been described in wheat waxes were identified. Using six pairs of BC2 F3 near-isogenic lines, we show that Iw1 inhibits the formation of β- and hydroxy-β-diketones in the peduncle and flag leaf blade cuticles. This inhibitory effect is independent of genetic background or tissue, and is accompanied by minor but consistent increases in n-alkanes and C24 primary alcohols. No differences were found in cuticle thickness and carbon isotope discrimination in near-isogenic lines differing at Iw1.

The receptor tyrosine kinase Flt3 is an important growth factor receptor in hematopoiesis, and gain-of-function mutations of the receptor contribute to the transformation of acute myeloid leukemia. SOCS6 (suppressor of cytokine signaling 6) is a member of the SOCS family of E3 ubiquitin ligases that can regulate receptor tyrosine kinase signal transduction. In this study, we analyzed the role of SOCS6 in Flt3 signal transduction. The results show that ligand stimulation of Flt3 can induce association of SOCS6 and Flt3 and tyrosine phosphorylation of SOCS6. Phosphopeptide fishing indicated that SOCS6 binds directly to phosphotyrosines 591 and 919 of Flt3. By using stably transfected Ba/F3 cells with Flt3 and/or SOCS6, we show that the presence of SOCS6 can enhance ubiquitination of Flt3, as well as internalization and degradation of the receptor. The presence of SOCS6 also induces weaker activation of Erk1/2, but not Akt, in transfected Ba/F3 and UT-7 cells and in OCI-AML-5 cells. The absence of SOCS6 promotes Ba/F3 and UT-7 cell proliferation induced by oncogenic internal tandem duplications of Flt3. Taken together, these results suggest that SOCS6 negatively regulates Flt3 activation, the downstream Erk signaling pathway, and cell proliferation.

OBJECTIVE

Recent findings in hepatitis C virus (HCV)-monoinfected patients have shown a correlation between low serum levels of 25-OH vitamin D3 [25(OH)D3] and severe liver fibrosis and low sustained virologic response to therapy. Data are lacking in HIV-HCV coinfected patients.

METHODS

One hundred and eighty nine HIV-HCV coinfected patients, who received ≥80% of interferon (IFN) plus ribavirin therapy, were analyzed for baseline serum 25(OH)D3 levels. Correlations between serum 25(OH)D3 levels, chronic hepatitis C features, HCV virologic response to antiviral therapy, and HIV infection characteristics were analyzed.

RESULTS

Mean serum 25(OH)D3 level was 18.5 ± 9.8 ng/ml, including 162 (85%) patients with level ≤30 ng/ml. Serum 25(OH)D3 levels were significantly correlated with the histological Metavir fibrosis score (r = -0.16; p = 0.027). Patients with severe fibrosis (Metavir F3/F4) had lower serum 25(OH)D3 levels compared to F2 and F1 patients (16.2 ± 10.0 vs. 18.9 ± 8.5 and 20.9 ± 11.1 ng/ml, respectively; p = 0.06). In multivariate analysis, low serum 25(OH)D levels were independently associated with severe liver fibrosis (p = 0.04) and cold season (p = 0.0002). Serum levels of 25(OH)D3 were also significantly correlated with liver fibrosis as assessed by FibroTest® (r = -0.22; p = 0.008) and serum α2-macroglobulin levels (r = -0.23; p = 0.006). In contrast, no correlation was found between 25(OH)D3 levels and HCV sustained virologic response to IFN-based therapy [OR 0.98 (0.95-1.01); p = 0.22]. No association was found between 25(OH)D3 levels and markers of HIV-related immunodeficiency.

CONCLUSIONS

In HIV-HCV coinfected patients, low serum 25(OH)D3 levels correlate with severe liver fibrosis. In contrast, serum 25(OH)D3 levels are not linked to HCV virologic response to therapy or severity of immunodeficiency.

OBJECTIVE

Although tyrosine kinase inhibitors have improved survival in advanced gastrointestinal stromal tumor (GIST), complete response is rare and most patients eventually fail the first-line treatment with imatinib. Sunitinib malate is the only approved second-line therapy for patients with imatinib-resistant or imatinib-intolerant GIST. The clinical benefit of sunitinib is genotype-dependent in regards to both primary and secondary mutations, with GIST patients harboring the KIT(AY502-3ins) exon 9 mutation being the most sensitive.

METHODS

As sunitinib resistance is now emerging, our goal was to investigate mechanisms of progression and to test the efficacy of novel tyrosine kinase inhibitor on these resistant mutants in vitro. N-ethyl-N-nitrosourea mutagenesis of Ba/F3 cells expressing the KIT(AY502-3ins) mutant was used to investigate novel patterns of resistant mutations evolving in the presence of sunitinib.

RESULTS

Tumors from patients who developed sunitinib resistance after at least 1 year of radiographic response were analyzed, showing similar findings of a primary KIT(AY502-3ins) mutation and a secondary mutation in the KIT activation loop. Ba/F3 cells expressing these sunitinib-resistant double mutants showed sensitivity to both dasatinib and nilotinib.

CONCLUSIONS

Sunitinib resistance in GIST shares similar pathogenetic mechanisms identified in imatinib failure, with acquisition of secondary mutations in the activation domain after an extended initial response to the drug. Moreover, in vitro mutagenesis with or without N-ethyl-N-nitrosourea of Ba/F3 cells expressing KIT(AY502-3ins) showed acquisition of secondary mutations restricted to the second kinase domain of KIT. In contrast, in vitro resistance to imatinib produces a broader spectrum of secondary mutations including mutations in both KIT kinase domains.

Dorsal root ganglion neurons project axons to specific target layers in the gray matter of the spinal cord, according to their sensory modality. Using an in vivo approach, we demonstrate an involvement of the two immunoglobulin superfamily cell adhesion molecules axonin-1/TAG-1 and F11/F3/contactin in subpopulation-specific sensory axon guidance. Proprioceptive neurons, which establish connections with motoneurons in the ventral horn, depend on F11 interactions. Nociceptive fibers, which target to layers in the dorsal horn, require axonin-1 for pathfinding. In vitro NgCAM and NrCAM were shown to bind to both axonin-1 and F11. However, despite this fact and despite their ubiquitous expression in the spinal cord, NgCAM and NrCAM are selective binding partners for axonin-1 and F11 in sensory axon guidance. Whereas nociceptive pathfinding depends on NgCAM and axonin-1, proprioceptive fibers require NrCAM and F11.

In search of an adequate model for the human metabolic syndrome, the metabolic characteristics of Wistar rats were analysed after being submitted to different protocols of high fructose ingestion. First, two adult rat groups (aged 90 d) were studied: a control group (C1; n 6) received regular rodent chow (Labina, Purina) and a fructose group (F1; n 6) was fed on regular rodent chow. Fructose was administered as a 10 % solution in drinking water. Second, two adult rat groups (aged 90 d) were evaluated: a control group (C2; n 6) was fed on a balanced diet (AIN-93G) and a fructose group (F2; n 6) was fed on a purified 60 % fructose diet. Finally, two young rat groups (aged 28 d) were analysed: a control group (C3; n 6) was fed on the AIN-93G diet and a fructose group (F3; n 6) was fed on a 60 % fructose diet. After 4-8 weeks, the animals were evaluated. Glucose tolerance, peripheral insulin sensitivity, blood lipid profile and body fat were analysed. In the fructose groups F2 and F3 glucose tolerance and insulin sensitivity were lower, while triacylglycerolaemia was higher than the respective controls C2 and C3 (P < 0.05). Blood total cholesterol, HDL and LDL as well as body fat showed change only in the second protocol. In conclusion, high fructose intake is more effective at producing the signs of the metabolic syndrome in adult than in young Wistar rats. Additionally, diet seems to be a more effective way of fructose administration than drinking water.

OBJECTIVE

Transient elastography (TE) is a validated non-invasive tool to evaluate hepatic fibrosis in patients with hepatitis C virus (HCV) infection. Whether TE may sense changes of liver fibrosis following therapeutic HCV eradication has never been evaluated.

METHODS

37 HCV cirrhotics with paired pre- and post-sustained virological response (SVR) liver biopsies (LB) underwent TE at the time of post-SVR LB. Liver fibrosis was staged with the METAVIR scoring system and the area of fibrosis (%) was assessed morphometrically.

RESULTS

Thirty-three patients had valid TE measurements after 61 (48-104) months from an SVR, and 20 (61%) of them had cirrhosis regression. On post-SVR LB, the median area of fibrosis was 2.3%, being significantly reduced from baseline (p<0.0001). Median TE value was 9.8 kPa being lower in regressed vs. not regressed patients (9.1 kPa vs. 12.9 kPa, p=0.01). TE was <12 kPa in 5 (38%) F4 patients and in 19 (95%) ≤F3 patients (p=0.0007). The diagnostic accuracy of TE for diagnosing F4 after treatment was 61% sensitivity, 95% specificity, 12.3 LR+, 0.4 LR-, and AUROC 0.77. A significant correlation was found between TE and both fibrosis stage (r=0.56; p=0.001) and morphometry (r=0.56, p=0.001) as well as between fibrosis stage and area of fibrosis (r=0.72, p=0001).

CONCLUSIONS

Following therapeutic eradication of HCV, the predictive power of the viremic cut-off of 12 kPa was low as a consequence of liver remodelling and fibrosis reabsorption. LB still remains the only reliable approach to stage liver fibrosis following an SVR.

The leptin receptor (LR), a member of the class I cytokine receptor family, is composed of a single subunit. Its extracellular domain consists of two so-called cytokine receptor homology domains, separated by an Ig-like domain, and two additional fibronectin type III modules. Requirements for LR activation were examined using a complementation strategy. Two LR mutants, LR-FFY-Deltabox 1 and LR-F3, deficient in Janus kinase or signal transducer and activator of transcription (STAT) activation, respectively, were only able to generate a STAT3-dependent signal when coexpressed. Based on the requirements for Janus kinase/STAT signaling, and on the lack of complementation with similar receptor constructs, but containing the extracellular domain of the homodimeric erythropoietin receptor, this observation can be explained only by higher order LR clustering. Using a panel of deletion mutants we were able to define a role for the cytokine receptor homology 1 and Ig-like domains in leptin signaling. Moreover, we demonstrate a nonredundant function for the individual receptor chains within the homomeric LR complex. Based on these data, we propose a possible model for LR clustering.

The leptin receptor gene (LEPR) is a candidate for traits related to growth and body composition, and is located on SSC6 in a region where fatness and meat composition quantitative trait loci (QTL) have previously been detected in several F2 experimental designs. The aims of this work were: (i) to fine map these QTL on a larger sample of animals and generations (F3 and backcross) of an Iberian x Landrace intercross and (ii) to examine the effects of LEPR alleles on body composition traits. Eleven single nucleotide polymorphisms (SNPs) were detected by sequencing LEPR coding regions in Iberian and Landrace pig samples. Three missense polymorphisms were genotyped by pyrosequencing in 33 F0, 70 F1, 418 F2, 86 F3 and 128 individuals coming from the backcross of four F2 males with 24 Landrace females. Thirteen microsatellites and one SNP were also genotyped. Traits analysed were: backfat thickness at different locations (BF(T)), intramuscular fat percentage (IMF(P)), eye muscle area (EM(A)), loin depth (LO(D)), weight of shoulder (SH(W)), weight of ribs (RIB(W)) and weight of belly bacon (BB(W)). Different statistical models were applied in order to evaluate the number and effects of QTL on chromosome 6 and the possible causality of the LEPR gene variants with respect to the QTL. The results support the presence of two QTL on SSC6. One, at position 60-100 cM, affects BF(T) and RIB(W). The other and more significant maps in a narrow region (130-132 cM) and affects BF(T), IMF(P), EM(A), LO(D), SH(W), RIB(W) and BB(W). Results also support the association between LEPR alleles and BF(T) traits. The possible functional implications of the analysed polymorphisms are considered.

The main utility of QSAR models is their ability to predict activities/properties for new chemicals, and this external prediction ability is evaluated by means of various validation criteria. As a measure for such evaluation the OECD guidelines have proposed the predictive squared correlation coefficient Q(2)(F1) (Shi et al.). However, other validation criteria have been proposed by other authors: the Golbraikh-Tropsha method, r(2)(m) (Roy), Q(2)(F2) (Schüürmann et al.), Q(2)(F3) (Consonni et al.). In QSAR studies these measures are usually in accordance, though this is not always the case, thus doubts can arise when contradictory results are obtained. It is likely that none of the aforementioned criteria is the best in every situation, so a comparative study using simulated data sets is proposed here, using threshold values suggested by the proponents or those widely used in QSAR modeling. In addition, a different and simple external validation measure, the concordance correlation coefficient (CCC), is proposed and compared with other criteria. Huge data sets were used to study the general behavior of validation measures, and the concordance correlation coefficient was shown to be the most restrictive. On using simulated data sets of a more realistic size, it was found that CCC was broadly in agreement, about 96% of the time, with other validation measures in accepting models as predictive, and in almost all the examples it was the most precautionary. The proposed concordance correlation coefficient also works well on real data sets, where it seems to be more stable, and helps in making decisions when the validation measures are in conflict. Since it is conceptually simple, and given its stability and restrictiveness, we propose the concordance correlation coefficient as a complementary, or alternative, more prudent measure of a QSAR model to be externally predictive.

A human neural stem cell (NSC) line over-expressing human choline acetyltransferase (ChAT) gene was generated and these F3.ChAT NSCs were transplanted into the brain of rat Alzheimer disease (AD) model which was induced by application of ethylcholine mustard aziridinium ion (AF64A) that specifically denatures cholinergic nerves and thereby leads to memory deficit as a salient feature of AD. Transplantation of F3.ChAT human NSCs fully recovered the learning and memory function of AF64A animals, and induced elevated levels of acetylcholine (ACh) in cerebrospinal fluid (CSF). Transplanted F3.ChAT human NSCs were found to migrate to various brain regions including cerebral cortex, hippocampus, striatum and septum, and differentiated into neurons and astrocytes. The present study demonstrates that brain transplantation of human NSCs over-expressing ChAT ameliorates complex learning and memory deficits in AF64A-cholinotoxin-induced AD rat model.

Currently all approved anti-cancer therapeutic monoclonal antibodies (mAbs) are of the IgG isotype, which rely on Fcgamma receptors (FcγRs) to recruit cellular effector functions. In vitro studies showed that targeting of FcαRI (CD89) by bispecific antibodies (bsAbs) or recombinant IgA resulted in more effective elimination of tumour cells by myeloid effector cells than targeting of FcγR. Here we studied the in vivo anti-tumour activity of IgA EGFR antibodies generated using the variable sequences of the chimeric EGFR antibody cetuximab. Using FcαRI transgenic mice, we demonstrated significant in vivo anti-tumour activity of IgA2 EGFR against A431 cells in peritoneal and lung xenograft models, as well as against B16F10-EGFR cells in a lung metastasis model in immunocompetent mice. IgA2 EGFR was more effective than cetuximab in a short-term syngeneic peritoneal model using EGFR-transfected Ba/F3 target cells. The in vivo cytotoxic activity of IgA2 EGFR was mediated by macrophages and was significantly decreased in the absence of FcαRI. These results support the potential of targeting FcαRI for effective antibody therapy of cancer.

We assessed the cost-effectiveness of sofosbuvir (SOF)-based triple therapy (TT) compared with boceprevir (BOC)- and telaprevir (TVR)-based TT in untreated genotype 1 (G1) chronic hepatitis C (CHC) patients discriminated according to IL28B genotype, severity of liver fibrosis, and G1 subtype. The available published literature provided the data source. The target population was made up of untreated Caucasian patients, aged 50 years, with G1CHC and these were evaluated over a lifetime horizon by Markov model. The study was carried out from the perspective of the Italian National Health Service. Outcomes included discounted costs (in euros at 2013 value), life-years gained (LYG), quality-adjusted life year (QALY), and incremental cost-effectiveness ratio (ICER). Cost of SOF was assumed to be € 3,500 per week, i.e., the price generating a willingness-to-pay threshold of € 25,000 per LYG compared with TVR in the entire population of untreated G1 patients. The robustness of the results was evaluated by one-way deterministic and multivariate probabilistic sensitivity analyses. SOF was cost-effective compared with BOC in all strategies with the exception of cirrhosis and IL28B CC patients. In comparison with TVR-based strategies, SOF was cost-effective in IL28B CT/TT (ICER per LYG € 22,229) and G1a (€ 19,359) patients, not cost-effective in IL28B CC (€45,330), fibrosis F0-F3 (€ 26,444), and in cirrhosis (€ 34,906) patients, and dominated in G1b patients. The models were sensitive to SOF prices and to likelihood of sustained virological response.

CONCLUSIONS

In untreated G1 CHC patients, SOF-based TT may be a cost-effective alternative to first-generation protease inhibitors depending on pricing. The cost-effectiveness of SOF improved in IL28B CT/TT and G1a patients. SOF was dominated by TVR in G1b patients even if, in clinical practice, this issue could be counterbalanced by the good tolerability profile of SOF and by the shorter treatment duration.

The study of QTL x environment interaction (QEI) is important for understanding genotype x environment interaction (GEI) in many quantitative traits. For modeling GEI and QEI, factorial regression (FR) models form a powerful class of models. In FR models, covariables (contrasts) defined on the levels of the genotypic and/or environmental factor(s) are used to describe main effects and interactions. In FR models for QTL expression, considerable numbers of genotypic covariables can occur as for each putative QTL an additional covariable needs to be introduced. For large numbers of genotypic and/or environmental covariables, least square estimation breaks down and partial least squares (PLS) estimation procedures become an attractive alternative. In this paper we develop methodology for analyzing QEI by FR for estimating effects and locations of QTLs and QEI and interpreting QEI in terms of environmental variables. A randomization test for the main effects of QTLs and QEI is presented. A population of F2 derived F3 families was evaluated in eight environments differing in drought stress and soil nitrogen content and the traits yield and anthesis silking interval (ASI) were measured. For grain yield, chromosomes 1 and 10 showed significant QEI, whereas in chromosomes 3 and 8 only main effect QTLs were observed. For ASI, QTL main effects were observed on chromosomes 1, 2, 6, 8, and 10, whereas QEI was observed only on chromosome 8. The assessment of the QEI at chromosome 1 for grain yield showed that the QTL main effect explained 35.8% of the QTL + QEI variability, while QEI explained 64.2%. Minimum temperature during flowering time explained 77.6% of the QEI. The QEI analysis at chromosome 10 showed that the QTL main effect explained 59.8% of the QTL + QEI variability, while QEI explained 40.2%. Maximum temperature during flowering time explained 23.8% of the QEI. Results of this study show the possibilities of using FR for mapping QTL and for dissecting QEI in terms of environmental variables. PLS regression is efficient in accounting for background noise produced by other QTLs.

JAK2(V617F) is a gain of function mutation that promotes cytokine-independent growth of myeloid cells and accounts for a majority of myeloproliferative neoplasms (MPN). Mutations in p53 are rarely found in these diseases before acute leukemia transformation, but this does not rule out a role for p53 deregulation in disease progression. Using Ba/F3-EPOR cells and ex vivo cultured CD34(+) cells from MPN patients, we demonstrate that expression of JAK2(V617F) affected the p53 response to DNA damage. We show that E3 ubiquitin ligase MDM2 accumulated in these cells, due to an increased translation of MDM2 mRNA. Accumulation of the La autoantigen, which interacts with MDM2 mRNA and promotes its translation, was responsible for the increase in MDM2 protein level and the subsequent degradation of p53 after DNA damage. Downregulation of La protein or cell treatment with nutlin-3, a MDM2 antagonist, restored the p53 response to DNA damage and the cytokine-dependence of Ba/F3-EPOR-JAK2(V617F) cells. Altogether, these data indicate that the JAK2(V617F) mutation affects p53 response to DNA damage through the upregulation of La antigen and accumulation of MDM2. They also suggest that p53 functional inactivation accounts for the cytokine hypersensitivity of JAK2(V617F) MPN and might have a role in disease progression.

Multiple EGF-like domains 10 (Megf10) is a class F scavenger receptor (SR-F3) expressed on astrocytes and myosatellite cells, and recessive mutations in humans result in early-onset myopathy, areflexia, respiratory distress, and dysphagia (EMARDD). Here we report that Megf10-deficient mice have increased apoptotic cells in the developing cerebellum and have impaired phagocytosis of apoptotic cells by astrocytes ex vivo We also report that cells transfected with Megf10 gain the ability to phagocytose apoptotic neurons and that Megf10 binds with high affinity to C1q, an eat-me signal for apoptotic cells. In contrast, cells expressing Megf10 with EMARDD mutations have impaired apoptotic cell clearance and impaired binding to C1q. Our studies reveal that Megf10 is a receptor for C1q and identify a novel role for Megf10 in clearance of apoptotic cells in the mammalian developing brain with potential relevance to EMARDD patients and other CNS disorders.

Apoptosis is a universal homeostatic process and occurs in many disease conditions. Multiple EGF-like domains 10 (Megf10) is emerging as an essential receptor for synaptic pruning, clearance of neuronal debris, and for muscle differentiation. Here we define a novel Megf10-dependent pathway for apoptotic cell clearance and show that Megf10 is a receptor for C1q, an eat-me signal, that binds phosphatidylserine expressed on the surface of apoptotic cells. Understanding the pathways by which apoptotic cells are cleared in the CNS is relevant to many physiological and pathological conditions of the CNS.

Low temperature is a major limiting factor in rice growth and development. Mapping of quantitative trait loci (QTLs) controlling cold tolerance is important for rice breeding. Recent studies have suggested that bulked segregant analysis (BSA) combined with next-generation sequencing (NGS) can be an efficient and cost-effective way for QTL mapping. In this study, we employed NGS-assisted BSA to map QTLs conferring cold tolerance at the seedling stage in rice. By deep sequencing of a pair of large DNA pools acquired from a very large F3 population (10,800 individuals), we obtained ∼450,000 single nucleotide polymorphisms (SNPs) after strict screening. We employed two statistical methods for QTL analysis based on these SNPs, which yielded consistent results. Six QTLs were mapped on chromosomes 1, 2, 5, 8 and 10. The three most significant QTLs on chromosomes 1, 2 and 8 were validated by comparison with previous studies. Two QTLs on chromosomes 2 and 5 were also identified previously, but at the booting stage rather than the seedling stage, suggesting that some QTLs may function at different developmental stages, which would be useful for cold tolerance breeding in rice. Compared with previously reported QTL mapping studies for cold tolerance in rice based on the traditional approaches, the results of this study demonstrated the advantages of NGS-assisted BSA in both efficiency and statistical power.

Between 30% and 50% of patients with advanced-stage anaplastic large-cell lymphoma (ALCL) harbor the balanced chromosomal rearrangement t(2;5)(p23;q35), which results in the generation of the fusion protein nucleophosmin-anaplastic lymphoma kinase (NPM-ALK). To further study survival signaling by NPMALK, we generated Ba/F3 cell lines with either inducible or constitutive expression of NPM-ALK and examined the regulation of the AKT target FOXO3a. We hypothesized that NPM-ALK signaling through phosphoinositol 3-kinase (PI 3-kinase) and AKT would regulate FOXO3a, a member of the forkhead family of transcription factors, thereby stimulating proliferation and blocking programmed cell death in NPM-ALK-transformed cells. In Ba/F3 cells with induced or constitutive expression of NPM-ALK, concomitant AKT activation and phosphorylation of its substrate, FOXO3a, was observed. In addition, transient expression of NPM-ALK in U-20S cells inhibited FOXO3a-mediated transactivation of reporter gene expression. Furthermore, NPM-ALK-induced FOXO3a phosphorylation in Ba/F3 cells resulted in nuclear exclusion of this transcriptional regulator, up-regulation of cyclin D2 expression, and down-regulation of p27(kip1) and Bim-1 expression. NPMALK reversal of proliferation arrest and of p27(kip1) induction was dependent on the phosphorylation of FOXO3a. Thus, FOXO3a is a barrier to hematopoietic transformation that is overcome by phosphorylation and cytoplasmic relocalization induced by the expression of NPM-ALK.

The products of genes that cause cerebral cavernous malformations (CCM1/KRIT1, CCM2, and CCM3) physically interact. CCM1/KRIT1 links this complex to endothelial cell (EC) junctions and maintains junctional integrity in part by inhibiting RhoA. Heart of glass (HEG1), a transmembrane protein, associates with KRIT1. In this paper, we show that the KRIT1 band 4.1, ezrin, radixin, and moesin (FERM) domain bound the HEG1 C terminus (K(d) = 1.2 µM) and solved the structure of this assembly. The KRIT1 F1 and F3 subdomain interface formed a hydrophobic groove that binds HEG1(Tyr(1,380)-Phe(1,381)), thus defining a new mode of FERM domain-membrane protein interaction. This structure enabled design of KRIT1(L717,721A), which exhibited a >100-fold reduction in HEG1 affinity. Although well folded and expressed, KRIT1(L717,721A) failed to target to EC junctions or complement the effects of KRIT1 depletion on zebrafish cardiovascular development or Rho kinase activation in EC. These data establish that this novel FERM-membrane protein interaction anchors CCM1/KRIT1 at EC junctions to support cardiovascular development.

Adults of Rivulus marmoratus (Cyprinodontidae) contained eggs when dissected or laid eggs when kept alive. They invariably oviposited solo, emitting eggs in various developmental stages. Both juvenescent and senescent fish have functional ovotestes. Progeny (F3) were obtained via a selfed great grandparent, grandparent, and parent, the last two in isolation ab ovo.

The tricorn interacting factor F3 is an 89 kDa zinc aminopeptidase from the archaeon Thermoplasma acidophilum. Together with the tricorn interacting factors F1 and F2, F3 degrades the tricorn protease products and thus completes the proteasomal degradation pathway by generating free amino acids. Here, we present the crystal structures of F3 in three different conformations at 2.3 A resolution. The zinc aminopeptidase is composed of four domains: an N-terminal saddle-like beta-structure domain; a thermolysin-like catalytic domain; a small barrel-like beta-structure domain; and an alpha-helical C-terminal domain, the latter forming a deep cavity at the active site. Three crystal forms provide snapshots of the molecular dynamics of F3 where the C-terminal domain can adapt to form an open, an intermediate and a nearly closed cavity, respectively. With the conserved Zn(2+)-binding motifs HEXXH and NEXFA as well as the N-terminal substrate-anchoring glutamate residues, F3 together with the leukotriene A4 hydrolase, represents a novel gluzincin subfamily of aminoproteases. We discuss the functional implications of these structures with respect to the underlying catalytic mechanism, substrate recognition and processing, and possible component interactions.

BACKGROUND

The hepatotoxicity of highly active antiretroviral therapy (HAART) could enhance liver fibrosis in HIV/Hepatitis C virus (HCV)-coinfected patients. Moreover, HAART-related immune restoration could lessen HCV-associated liver damage. The data on the effect of protease inhibitors (PI) on liver fibrosis are scant and contradictory. No information is available on the relationship between non-nucleoside analogue therapy and liver fibrosis in co-infected patients.

OBJECTIVE

To investigate the associations between the use of different antiretroviral drugs and the liver fibrosis in patients with HIV and HCV infections.

METHODS

Cross-sectional study.

METHODS

All HIV/HCV co-infected patients with an available liver biopsy and known or estimated duration of HCV infection seen at a Infectious Diseases Unit were included in the study. The fibrosis stage and the fibrosis progression rate were evaluated.

RESULTS

The inclusion criteria were fulfilled by 152 patients. Age at HCV infection < 20 years [adjusted odds ratio (AOR), 0.39; 95% confidence interval (CI), 0.19-0.82], PI-based HAART (AOR, 0.39; 95% CI, 0.19-0.78) and nevirapine-based HAART (AOR, 2.56; 95% CI, 1.02-6.58) were associated with fibrosis stage>>or= F3. The variables associated with fibrosis progression rate>> 0.2 units/year were age at HCV infection < 20 years (AOR, 0.23; 95% CI, 0.1-0.52), CD4 cell counts < or = 250 x 10/l at liver biopsy (AOR, 2.8; 95% CI, 1.1-7.1), PI-based HAART (AOR, 0.39; 95% CI, 0.2-0.8) and nevirapine-based HAART (AOR, 3.82; 95% CI, 1.9-7.6).

CONCLUSIONS

HAART regimens including nevirapine are associated with faster liver fibrosis progression in HIV-infected patients with chronic hepatitis C. In contrast, patients on PI as the backbone of potent antiretroviral therapy are more likely to show less liver fibrosis.

Recent evidences suggest that the extracts of plant products are able to modulate innate immune responses. A saponin GL and a chalcone ILG are representative components of Glycyrrhiza uralensis, which attenuate inflammatory responses mediated by TLRs. Here, we show that GL and ILG suppress different steps of the LPS sensor TLR4/MD-2 complex signaling at the receptor level. Extract of G. uralensis suppressed IL-6 and TNF-α production induced by lipid A moiety of LPS in RAW264.7 cells. Among various G. uralensis-related components of saponins and flavanones/chalcones, GL and ILG could suppress IL-6 production induced by lipid A in dose-dependent manners in RAW264.7 cells. Furthermore, elevation of plasma TNF-α in LPS-injected mice was attenuated by passive administration of GL or ILG. GL and ILG inhibited lipid A-induced NF-κB activation in Ba/F3 cells expressing TLR4/MD-2 and CD14 and BMMs. These components also inhibited activation of MAPKs, including JNK, p38, and ERK in BMMs. In addition, GL and ILG inhibited NF-κB activation and IL-6 production induced by paclitaxel, a nonbacterial TLR4 ligand. Interestingly, GL attenuated the formation of the LPS-TLR4/MD-2 complexes, resulting in inhibition of homodimerization of TLR4. Although ILG did not affect LPS binding to TLR4/MD-2, it could inhibit LPS-induced TLR4 homodimerization. These results imply that GL and ILG modulate the TLR4/MD-2 complex at the receptor level, leading to suppress LPS-induced activation of signaling cascades and cytokine production, but their effects are exerted at different steps of TLR4/MD-2 signaling.

The effects of the multikinase inhibitor sorafenib (BAY 43-9006), an agent shown previously to induce apoptosis in human leukemia cells through inhibition of myeloid cell leukemia-1 (Mcl-1) translation, have been examined in Bcr/Abl(+) leukemia cells resistant to imatinib mesylate (IM). When administered at pharmacologically relevant concentrations (10-15 microM), sorafenib potently induced apoptosis in imatinib mesylate-resistant cells expressing high levels of Bcr/Abl, cells exhibiting a Bcr/Abl-independent, Lyn-dependent form of resistance, and CD34(+) cells obtained from imatinib-resistant patients. In addition, Ba/F3 cells expressing mutations rendering them resistant to IM (e.g., E255K, M351T) or to IM, dasatinib, and nilotinib (T315I) remained fully sensitive to sorafenib. Induction of apoptosis by sorafenib was associated with rapid and pronounced down-regulation of Mcl-1 and diminished signal transducer and activator of transcription (STAT) 5 phosphorylation and reporter activity but only very modest and delayed inactivation of the Bcr/Abl downstream target Crkl. Moreover, transfection with a constitutively active STAT5 construct partially but significantly protected cells from sorafenib lethality. Ba/F3 cells expressing Bcr/Abl mutations were as sensitive to sorafenib-induced Mcl-1 down-regulation and dephosphorylation of STAT5 and eukaryotic initiation factor 4E as wild-type cells. Finally, stable knockdown of Bcl-2-interacting mediator of cell death (Bim) with short hairpin RNA in K562 cells significantly diminished sorafenib lethality, arguing strongly for a functional role of this proapoptotic Bcl-2 family member in the lethality of this agent. Together, these findings suggest that sorafenib effectively induces apoptosis in highly imatinib-resistant chronic myelogenous leukemia cells, most likely by inhibiting or down-regulating targets (i.e., STAT5 and Mcl-1) downstream or independent of Bcr/Abl.