miR-93/106b and their host gene minichromosome maintenance complex component 7 (MCM7) reside at chr7q22, a region frequently rearranged in leiomyomas. We explored the expression of miR-93/106b in leiomyoma and paired myometrium (n = 63) from untreated and patients exposed to hormonal therapies (GnRH agonist, Depo-Provera, and oral contraceptives) from African-Americans and Caucasians and their regulatory functions in isolated paired (n = 15) leiomyoma and myometrial smooth muscle cells and the leiomyosarcoma cell line. At tissue level leiomyomas expressed significantly lower levels of miR-93 and elevated MCM7 as compared with myometrium with limited racial influence or hormonal exposure on their expression. Assessing the regulatory function of miR-93/106b through doxycycline-inducible lentiviral transduction in a microarray analysis, tissue factor (F3) and IL8 were identified as their possible targets. At the tissue level, leiomyomas expressed a significantly lower level of F3 and an elevated IL-8 level, which exhibited an inverse relationship with miR-93 but with limited racial or hormonal influences. The gain of function of miR-93/106b in leiomyoma smooth muscle cells, myometrial smooth muscle cells, and the leiomyosarcoma cell line dose dependently repressed F3 and IL8 through direct interactions with their respective 3'-untranslated region and indirectly through F3 repression inhibited IL8, CTGF, and PAI-1 expression, confirmed by using small interfering RNA silencing or factor Vlla (FVIIa) activation of F3, as well as reducing the rate of proliferation, while increasing caspase-3/7 activity. We concluded that differential expression of miR-93/106b and their direct and/or indirect regulatory functions on F3, IL8, CTGF, and PAI-1 expression, with key roles in inflammation and tissue turnover may be of significance in the outcome of leiomyoma growth and associated symptoms.

Tomato (Lycopersicon esculentum) is susceptible to the powdery mildew Oidium lycopersici, but several wild relatives such as Lycopersicon parviflorum G1.1601 are completely resistant. An F2 population from a cross of Lycopersicon esculentum cv. Moneymaker x Lycopersicon parviflorum G1.1601 was used to map the O. lycopersici resistance by using amplified fragment length polymorphism markers. The resistance was controlled by three quantitative trait loci (QTLs). Ol-qtl1 is on chromosome 6 in the same region as the Ol-1 locus, which is involved in a hypersensitive resistance response to O. lycopersici. Ol-qtl2 and Ol-qtl3 are located on chromosome 12, separated by 25 cM, in the vicinity of the Lv locus conferring resistance to another powdery mildew species, Leveillula taurica. The three QTLs, jointly explaining 68% of the phenotypic variation, were confirmed by testing F3 progenies. A set of polymerase chain reaction-based cleaved amplified polymorphic sequence and sequence characterized amplified region markers was generated for efficient monitoring of the target QTL genomic regions in marker assisted selection. The possible relationship between genes underlying major and partial resistance for tomato powdery mildew is discussed.

BACKGROUND

Most people suffering from severe mental illness (SMI) lack paid employment. This study investigates the relationship between work status and objective as well as subjective quality of life (QoL) in people with SMI.

METHODS

The sample consists of 261 subjects (102 women, 159 men) aged 35 (men) and 38 (women) years on average, of whom 158 suffer from a schizophrenic disorder (ICD-10: F2) and 103 were diagnosed as having an affective disorder (ICD-10: F3). Subjective QoL was assessed with the WHOQOL-BREF scale.

RESULTS

Subjects with an occupation in general have a larger social network at their disposal and receive more social support. With regard to income, few (12%) of the subjects with a job on the open labour market live below the poverty level, but many (28-38%) of those engaged in sheltered or other work-like activities do. Occupation ameliorates satisfaction with life domains referring to social integration (social relationships, environment), whereas the individual's well-being (psychological, physical) is hardly affected. Social support is an important mediator of the relationship between occupation and subjective QoL. Income is weakly and negatively related to subjective QoL.

CONCLUSIONS

Supportive relationships to colleagues at the workplace mainly explain the better subjective QoL of SMI people with an occupation. When designing specific employment possibilities for people with SMI, we should take notice of the social support dimension at the workplace. Mentally ill people have a substantial poverty risk, even when they are working. In particular, payment for sheltered work should be ameliorated.

BACKGROUND

Pandemic and seasonal respiratory viruses are a major global health concern. Given the genetic diversity of respiratory viruses and the emergence of drug resistant strains, the targeted disruption of human host-virus interactions is a potential therapeutic strategy for treating multi-viral infections. The availability of large-scale genomic datasets focused on host-pathogen interactions can be used to discover novel drug targets as well as potential opportunities for drug repositioning.

RESULTS

In this study, we performed a large-scale analysis of microarray datasets involving host response to infections by influenza A virus, respiratory syncytial virus, rhinovirus, SARS-coronavirus, metapneumonia virus, coxsackievirus and cytomegalovirus. Common genes and pathways were found through a rigorous, iterative analysis pipeline where relevant host mRNA expression datasets were identified, analyzed for quality and gene differential expression, then mapped to pathways for enrichment analysis. Possible repurposed drugs targets were found through database and literature searches. A total of 67 common biological pathways were identified among the seven different respiratory viruses analyzed, representing fifteen laboratories, nine different cell types, and seven different array platforms. A large overlap in the general immune response was observed among the top twenty of these 67 pathways, adding validation to our analysis strategy. Of the top five pathways, we found 53 differentially expressed genes affected by at least five of the seven viruses. We suggest five new therapeutic indications for existing small molecules or biological agents targeting proteins encoded by the genes F3, IL1B, TNF, CASP1 and MMP9. Pathway enrichment analysis also identified a potential novel host response, the Parkin-Ubiquitin Proteasomal System (Parkin-UPS) pathway, which is known to be involved in the progression of neurodegenerative Parkinson's disease.

CONCLUSIONS

Our study suggests that multiple and diverse respiratory viruses invoke several common host response pathways. Further analysis of these pathways suggests potential opportunities for therapeutic intervention.

OBJECTIVE

Lung cancers harboring common EGFR mutations respond to EGFR tyrosine kinase inhibitors (TKI), whereas exon 20 insertions (Ins20) are resistant to them. However, little is known about mutations in exon 18.

METHODS

Mutational status of lung cancers between 2001 and 2015 was reviewed. Three representative mutations in exon 18, G719A, E709K, and exon 18 deletion (Del18: delE709_T710insD) were retrovirally introduced into Ba/F3 and NIH/3T3 cells. The 90% inhibitory concentrations (IC90s) of first-generation (1G; gefitinib and erlotinib), second-generation (2G; afatinib, dacomitinib, and neratinib), and third-generation TKIs (3G; AZD9291 and CO1686) were determined.

RESULTS

Among 1,402 EGFR mutations, Del19, L858R, and Ins20 were detected in 40%, 47%, and 4%, respectively. Exon 18 mutations, including G719X, E709X, and Del18, were present in 3.2%. Transfected Ba/F3 cells grew in the absence of IL3, and NIH/3T3 cells formed foci with marked pile-up, indicating their oncogenic abilities. IC90s of 1G and 3G TKIs in G719A, E709K, and Del18 were much higher than those in Del19 (by >11-50-fold), whereas IC90s of afatinib were only 3- to 7-fold greater than those for Del19. Notably, cells transfected with G719A and E709K exhibited higher sensitivity to neratinib (by 5-25-fold) than those expressing Del19. Patients with lung cancers harboring G719X exhibited higher response rate to afatinib or neratinib (∼ 80%) than to 1G TKIs (35%-56%) by compilation of data in the literature.

CONCLUSIONS

Lung cancers harboring exon 18 mutations should not be overlooked in clinical practice. These cases can be best treated with afatinib or neratinib, although the currently available in vitro diagnostic kits cannot detect all exon 18 mutations.

The HIV-1 Vif protein inactivates the cellular antiviral cytidine deaminase APOBEC3F (A3F) in virus-infected cells by specifically targeting it for proteasomal degradation. Several studies identified Vif sequence motifs involved in A3F interaction, whereas a Vif-binding A3F interface was proposed based on our analysis of highly similar APOBEC3C (A3C). However, the structural mechanism of specific Vif-A3F recognition is still poorly understood. Here we report structural features of interaction interfaces for both HIV-1 Vif and A3F molecules. Alanine-scanning analysis of Vif revealed that six residues located within the conserved Vif F1-, F2-, and F3-box motifs are essential for both A3C and A3F degradation, and an additional four residues are uniquely required for A3F degradation. Modeling of the Vif structure on an HIV-1 Vif crystal structure revealed that three discontinuous flexible loops of Vif F1-, F2-, and F3-box motifs sterically cluster to form a flexible A3F interaction interface, which represents hydrophobic and positively charged surfaces. We found that the basic Vif interface patch (R17, E171, and R173) involved in the interactions with A3C and A3F differs. Furthermore, our crystal structure determination and extensive mutational analysis of the A3F C-terminal domain demonstrated that the A3F interface includes a unique acidic stretch (L291, A292, R293, and E324) crucial for Vif interaction, suggesting additional electrostatic complementarity to the Vif interface compared with the A3C interface. Taken together, these findings provide structural insights into the A3F-Vif interaction mechanism, which will provide an important basis for development of novel anti-HIV-1 drugs using cellular cytidine deaminases.

OBJECTIVE

HIV-1 Vif targets cellular antiviral APOBEC3F (A3F) enzyme for degradation. However, the details on the structural mechanism for specific A3F recognition remain unclear. This study reports structural features of interaction interfaces for both HIV-1 Vif and A3F molecules. Three discontinuous sequence motifs of Vif, F1, F2, and F3 boxes, assemble to form an A3F interaction interface. In addition, we determined a crystal structure of the wild-type A3F C-terminal domain responsible for the Vif interaction. These results demonstrated that both electrostatic and hydrophobic interactions are the key force driving Vif-A3F binding and that the Vif-A3F interfaces are larger than the Vif-A3C interfaces. These findings will allow us to determine the configurations of the Vif-A3F complex and to construct a structural model of the complex, which will provide an important basis for inhibitor development.

We identified mutations in the IL7Ra gene or in genes encoding the downstream signaling molecules JAK1, JAK3, STAT5B, N-RAS, K-RAS, NF1, AKT and PTEN in 49% of patients with pediatric T-cell acute lymphoblastic leukemia (T-ALL). Strikingly, these mutations (except RAS/NF1) were mutually exclusive, suggesting that they each cause the aberrant activation of a common downstream target. Expressing these mutant signaling molecules-but not their wild-type counterparts-rendered Ba/F3 cells independent of IL3 by activating the RAS-MEK-ERK and PI3K-AKT pathways. Interestingly, cells expressing either IL7Ra or JAK mutants are sensitive to JAK inhibitors, but respond less robustly to inhibitors of the downstream RAS-MEK-ERK and PI3K-AKT-mTOR pathways, indicating that inhibiting only one downstream pathway is not sufficient. Here, we show that inhibiting both the MEK and PI3K-AKT pathways synergistically prevents the proliferation of BaF3 cells expressing mutant IL7Ra, JAK and RAS. Furthermore, combined inhibition of MEK and PI3K/AKT was cytotoxic to samples obtained from 6 out of 11 primary T-ALL patients, including 1 patient who had no mutations in the IL7R signaling pathway. Taken together, these results suggest that the potent cytotoxic effects of inhibiting both MEK and PI3K/AKT should be investigated further as a therapeutic option using leukemia xenograft models.

A number of leishmaniasis vaccine candidates are at various stages of pre-clinical and clinical development. Leishmaniasis is a vector-borne neglected tropical disease (NTD) caused by a protozoan parasite of the genus Leishmania and transmitted to humans by the bite of a sand fly. Visceral leishmaniasis (VL, kala-azar) is a high mortality NTD found mostly in South Asia and East Africa, while cutaneous leishmaniasis (CL) is a disfiguring NTD highly endemic in the Middle East, Central Asia, North Africa, and the Americas. Estimates attribute 50,000 annual deaths and 3.3 million disability-adjusted life years to leishmaniasis. There are only a few approved drug treatments, no prophylactic drug and no vaccine. Ideally, an effective vaccine against leishmaniasis will elicit long-lasting immunity and protect broadly against VL and CL. Vaccines such as Leish-F1, F2 and F3, developed at IDRI and designed based on selected Leishmania antigen epitopes, have been in clinical trials. Other groups, including the Sabin Vaccine Institute in collaboration with the National Institutes of Health are investigating recombinant Leishmania antigens in combination with selected sand fly salivary gland antigens in order to augment host immunity. To date, both VL and CL vaccines have been shown to be cost-effective in economic modeling studies.

The inheritance and genetic linkage analysis for seed dormancy and preharvest sprouting (PHS) resistance were carried out in an F8 recombinant inbred lines (RILs) derived from the cross between "CN19055" (white-grained, PHS-resistant) with locally adapted Australian cultivar "Annuello" (white-grained, PHS-susceptible). Seed dormancy was assessed as germination index (GI7) while assessment for preharvest sprouting resistance was based on whole head assay (sprouting index, SI) and visibly sprouted seeds (VI). Segregation analysis of the F2, F3 data from the glasshouse and the RIL population in 2004 and 2005 field data sets indicated that seed dormancy and PHS resistance in CN19055 is controlled by at least two genes. Heritabilities for GI7 and VI were high and moderate for SI. The most accurate method for assessing PHS resistance was achieved using VI and GI7 while SI exhibited large genotype by environment interaction. Two quantitative trait loci (QTLs) QPhs.dpivic.4A.1 and QPhs.dpivic.4A.2 were identified. On pooled data across four environments, the major QTL, QPhs.dpivic.4A.2, explained 45% of phenotypic variation for GI7, 43% for VI and 20% for SI, respectively. On the other hand, QPhs.dpivic.4A.1 which accounted for 31% of the phenotypic variation in GI7 in 2004 Horsham field trial, was not stable across environments. Physical mapping of two SSR markers, Xgwm937 and Xgwm894 linked to the major QTL for PHS resistance, using Chinese Spring deletions lines for chromosome 4AS and 4AL revealed that the markers were located in the deletion bins 4AL-12 and 4AL-13. The newly identified SSR markers (Xgwm937/Xgwm894) showed strong association with seed dormancy and PHS resistance in a range of wheat lines reputed to possess PHS resistance. The results suggest that Xgwm937/Xgwm894 could be used in marker-assisted selection (MAS) for incorporating preharvest sprouting resistance into elite wheat cultivars susceptible to PHS.

We identified amplification of RICTOR, a key component of the mTOR complex 2 (mTORC2), as the sole actionable genomic alteration in an 18-year-old never-smoker with lung adenocarcinoma. Amplification of RICTOR occurs in 13% of lung cancers (1,016 cases) in The Cancer Genome Atlas and at a similar frequency in an independent cohort of 1,070 patients identified by genomic profiling. In the latter series, 11% of cases harbored RICTOR amplification as the only relevant genomic alteration. Its oncogenic roles were suggested by decreased lung cancer cell growth both in vitro and in vivo with RICTOR ablation, and the transforming capacity of RICTOR in a Ba/F3-cell system. The mTORC1/2 inhibitors were significantly more active against RICTOR-amplified lung cancer cells as compared with other agents targeting the PI3K-AKT-mTOR pathway. Moreover, an association between RICTOR amplification and sensitivities to mTORC1/2 inhibitors was observed. The index patient has been treated with mTORC1/2 inhibitors that led to tumor stabilization for more than 18 months.

CONCLUSIONS

RICTOR amplification may define a novel and unique molecular subset of patients with lung cancer who may benefit from treatment with mTORC1/2 inhibitors.

Mandibular lateral incisor-canine (Mn.I2.C) transposition is a rare developmental disturbance of tooth order characterized by positional interchange of the two teeth. In children with Mn.I2.C anomaly, the mandibular lateral incisor shows distal ectopic eruption and the adjacent canine subsequently erupts mesial to it. A sample of 60 orthodontic patients with Mn.I2.C transposition was studied using roentgenograms taken at the time of diagnosis. Two age-related phenotypes of the anomaly were identified: early-stage (median age, 9 years) and mature-stage (median age, 12 years). Mn.I2.C transposition occurred bilaterally in 10 subjects (17%) and favored female expression (sex ratio, M1:F3) and right-side occurrence (68% of unilateral cases). Statistically significant associations were found between Mn.I2.C transposition and increased frequency of tooth agenesis (M3, p < 0.01; MnP2, p < 0.01) and peg-shaped maxillary lateral incisors (p < 0.0001). The results from this study and the analysis of 50 previously published cases provide evidence that Mn.I2.C transposition is a disturbance of tooth order and eruptive position probably caused by genetic influences. The Mn.I2.C anomaly likely results from genetic mechanisms similar to those responsible for occurrences of its associated dental anomalies, such as tooth agenesis and peg-shaped maxillary lateral incisors. In an appendix, clinical orthodontic management of Mn.I2.C transposition is discussed, based on treatment data derived from the study sample.

The availability of information on the genetic diversity and population structure in wheat (Triticum aestivum L.) breeding lines will help wheat breeders to better use their genetic resources and manage genetic variation in their breeding program. The recent advances in sequencing technology provide the opportunity to identify tens or hundreds of thousands of single nucleotide polymorphism (SNPs) in large genome species (e.g., wheat). These SNPs can be utilized for understanding genetic diversity and performing genome wide association studies (GWAS) for complex traits. In this study, the genetic diversity and population structure were investigated in a set of 230 genotypes (F3:6) derived from various crosses as a prerequisite for GWAS and genomic selection. Genotyping-by-sequencing provided 25,566 high-quality SNPs. The polymorphism information content (PIC) across chromosomes ranged from 0.09 to 0.37 with an average of 0.23. The distribution of SNPs markers on the 21 chromosomes ranged from 319 on chromosome 3D to 2,370 on chromosome 3B. The analysis of population structure revealed three subpopulations (G1, G2, and G3). Analysis of molecular variance identified 8% variance among and 92% within subpopulations. Of the three subpopulations, G2 had the highest level of genetic diversity based on three genetic diversity indices: Shannon's information index (I) = 0.494, diversity index (h) = 0.328 and unbiased diversity index (uh) = 0.331, while G3 had lowest level of genetic diversity (I = 0.348, h = 0.226 and uh = 0.236). This high genetic diversity identified among the subpopulations can be used to develop new wheat cultivars.

OBJECTIVE

The purpose of this study was to evaluate the amount of femoral tunnel widening that occurred after anterior cruciate ligament reconstruction using quadrupled hamstring autografts and to determine the clinical significance of any such tunnel enlargement.

METHODS

Retrospective clinical analysis.

METHODS

Twenty-nine patients who had undergone reconstruction of a torn anterior cruciate ligament with quadrupled hamstring autograft and cross pin femoral fixation were evaluated to determine the incidence and significance of postoperative femoral tunnel widening. A single surgeon performed all procedures, and average follow-up was 18.4 months (range, 12 to 31.5 months). All patients underwent flexion posteroanterior and lateral radiographs, an examination for determination of an International Knee Documentation Committee (IKDC) rating, had KT-1000 data collected, and completed Lysholm and Knee Outcome Survey functional questionnaires. Femoral tunnels were clearly seen in 27 patients. The tunnel diameters were measured at the opening of the tunnel, at the widest part of the tunnel, and just proximal to the cross pin. The amount of tunnel widening for each patient was then compared with the individual's KT-1000 data, IKDC rating, and Lysholm and knee outcome survey scores to assess correlation.

RESULTS

Four different tunnel morphologies were noted, with the linear type being the most common. The widening at the greatest tunnel diameter was 65.5% on average. Side-to-side KT-1000 differences averaged 1.04 mm at 30 lb, and 1.10 mm at manual maximum. Eleven patients had IKDC overall ratings of normal, 13 had ratings that were nearly normal, and 2 had abnormal. Average Lysholm and knee outcome survey scores were 92.6 and 93.9, respectively. A significant correlation was found only between F2 and F3 widening with Lysholm scores. However, the significance was eliminated with removal of 2 outliers.

CONCLUSIONS

The exact etiology of postoperative anterior cruciate ligament tunnel widening remains unknown. The present study reveals that significant tunnel widening occurs with quadrupled hamstring autografts and femoral cross pin fixation. However, the widening does not appear to have a significant effect on postoperative ligament laxity or functional knee scores, at least in the short term.

The aim of the present study was to define the origin of the thalamocortical projections to each of the mesial and superior area 6 areas. To this purpose, restricted injections of neuronal tracers were made into areas F3, F6, F2, and F7 after physiological identification of the injection sites. The results showed that each of these areas receives afferents from a set of thalamic nuclei and that this set differs, qualitatively and quantitatively, according to the injected area. The main inputs to F3 [supplementary motor area properly defined (SMA-proper)] originate in the nuclei ventral lateral, pars oralis (VLo), ventral posterior lateral, pars oralis (VPLo), and ventral lateral, pars caudalis (VLc) as well as in caudal parts of the VPLo and VLc (VPLo/VLc complex). F6 (pre-SMA) is mainly the target of nucleus ventral anterior, pars parvocellularis (VApc), and area X of Olszewski. The input to F2 originates mainly in the VPLo/VLc complex, in VLc, and in VLo. The dorsal part of F7 (supplementary eye field) mainly receives from area X, VApc, and nucleus ventral anterior, pars magnocellularis (VAmc), whereas the ventral F7 is connected with VApc, area X, VLc, and the VPLo/VLc complex. All of the injected areas receive a strong projection from the medial dorsal nucleus (MD). It is concluded that each cortical area is a target of both cerebellar and basal ganglia circuits. F3 and F2 are targets of the so-called "motor" basal ganglia circuit and a cerebellar circuit originating in dorsorostral sectors of dentate and interpositus nuclei. In contrast, F6 and ventral F7 receive a basal ganglia input mainly from the so-called "complex" circuit and a cerebellar input originating in the ventrocaudal sectors of dentate and interpositus nuclei. Finally, with respect to the rest of F7, dorsal F7 also receives a basal ganglia input from the "oculomotor circuit."

OBJECTIVE

Amantadine may augment virological response rates to interferon-based therapy in chronic hepatitis C patients. Using a novel design, amantadine was studied in naïve genotype 1 patients treated in combination with peginterferon alfa-2a (40KD)/ribavirin.

METHODS

Patients enrolled in this randomized, placebo-controlled multicenter trial were stratified by single-dose interferon sensitivity (stratum I, 24-h HCV-RNA decline >1.4-log10; II, 0.8-1.39-log10; III, <0.8-log10; a reliable means of identifying nonresponders to interferon/ribavirin) and fibrosis grade (F0/1/2 vs. F3/4) at baseline. All patients received peginterferon alfa-2a (40KD) 180 microg/week plus ribavirin 1000-1200 mg/day and were randomized to receive amantadine 100 mg twice daily (N = 114) or placebo (N = 95) for 48 weeks.

RESULTS

Week-24 virological response rates in strata II and III, the primary outcome, were similar in patients treated with amantadine (63.7%) or placebo (65.7%), as were sustained virological response rates at week 72 (46.5 and 51.6%, respectively). Adverse event profiles were similar and amantadine did not improve health-related quality of life compared with placebo. Interferon sensitivity was the only significant predictor of treatment outcome.

CONCLUSIONS

Adding amantadine to peginterferon alfa-2a (40KD)/ribavirin combination therapy does not augment virological response rates in genotype 1 patients. Virological response was almost exclusively determined by interferon sensitivity at baseline.

To assess nonlinear EEG activity in patients with Alzheimer's disease (AD) and vascular dementia (VaD), the authors estimated the correlation dimension (D2) and the first positive Lyapunov exponent (L1) of the EEGs in both patients and age-matched healthy control subjects. EEGs were recorded in 15 electrodes from 12 AD patients, 12 VaD patients, and 14 healthy subjects. The AD patients had significantly lower D2 values than the normal control subjects, (P < H>> 0.05), except at the F7 and the O1 electrodes, and the VaD patients, except at the C3 and the C4 electrodes. The VaD patients had relatively increased values of D2 and L1 compared with the AD patients, and rather higher values of D2 than the normal control subjects at the F7, F4, F8, Fp2, O1, and O2 electrodes. The L1 values of the EEGs were also lower for the AD patients than for the normal control subjects, except in the O1 and the O2 channels, and for the VaD patients at all electrodes. The L1 values were higher for the VaD patients than for the normal control subjects (F3, F4, F8, O1, and O2). In addition, the authors detected that the VaD patients had an uneven distribution of D2 values over the regions than the AD patients and the normal control subjects, although the statistics do not confirm this. By contrast, AD patients had uniformly lower D2 values in most regions, indicating that AD patients have less complex temporal characteristics of the EEG in entire regions. These nonlinear analyses of the EEG may be helpful in understanding the nonlinear EEG activity in AD and VaD.

BACKGROUND

Transient elastography (FibroScan) is a simple and noninvasive method to assess liver fibrosis by measuring liver stiffness and therefore can be a promising tool to evaluate liver fibrosis and avoid liver biopsy. We prospectively assessed the performance of transient elastography in patients with recurrent hepatitis C virus after living donor liver transplantation, in comparison with the surrogate serum markers.

METHODS

Fifty-six patients with recurrent hepatitis C virus after living donor liver transplantation, who underwent both liver biopsy and transient elastography were included in this study. The grade of liver fibrosis (the Scheuer classification) obtained by biopsy was compared to liver stiffness measured by the transient elastography.

RESULTS

The fibrosis grades were as follows: F0, n=22; F1, n=13; F2, n=9; F3, n=7; and F4, n=5. Liver stiffness values ranged from 2.9 to 72.0 kPa. The optimal cutoff values were 8.8 kPa for F>or=1, 9.9 kPa for F>or=2, 15.4 kPa for F>or=3, and 26.5 kPa for F>or=4. The area under the receiver operator characteristic curve for the diagnosis of fibrosis (F>or=2) by transient elastography was 0.92, while that by hyaluronic acid, type 4 collagen, alanine aminotransferase, and the aspartate transaminase to platelets ratio index were 0.52, 0.62, 0.64, and 0.70, respectively.

CONCLUSIONS

These data suggest that transient elastography is a simple, noninvasive and reliable tool to assess liver fibrosis in patients with recurrent hepatitis C virus after living donor liver transplantation.

OBJECTIVE

Activating mutations in FLT3 are known to be a frequent transforming event in acute myeloid leukemia. Small molecule-inhibitor therapy targeting the FLT3 kinase is, therefore, an attractive strategy. FLT3 kinase inhibitors, such as PKC412, have already entered clinical trials. Even though results are encouraging, emergence of primary and secondary resistance does occur in the majority of patients. Thus, it will be crucial to carefully characterize the activity of every single compound against different activating and resistance FLT3-internal tandem duplication (ITD) mutations. Here we tested the efficacy of sunitinib and sorafenib to inhibit primary FLT3 activating mutations (ITD and D835Y) and of secondary resistance mutations.

METHODS

Ba/F3 cell lines stably expressing oncogenic FLT3 mutations were used to calculate cellular IC(50) values for sunitinib and sorafenib using cell proliferation assays. Differential IC(50) values for sorafenib toward FLT3-ITD and FLT3-D835Y were confirmed by Western blotting. Cell death was measured by propidium-iodide staining and flow cytometry.

RESULTS

Sorafenib inhibits FLT3-ITD more potent than FLT3-D835Y, while sunitinib is equally effective against both mutant forms of FLT3. Importantly, sensitivity toward sorafenib and sunitinib varies between the different secondary FLT3-ITD resistance mutations.

CONCLUSIONS

These results establish sensitivity profiles for the FLT3 inhibitors sunitinib and sorafenib. This may help to develop rational treatment strategies for acute myeloid leukemia with these compounds.

There is considerable evidence for non-genomic transmission between generations of phenotypes induced by environmental exposures during development, although the mechanism is poorly understood. We investigated whether alterations in expression of the liver transcriptome induced in F1 offspring by feeding F0 dams a protein-restricted (PR) diet during pregnancy were passed with or without further change to two subsequent generations. The number of genes that differed between adult female offspring of F0 protein-restricted (PR) and protein-sufficient (PS) dams was F1 1,684 genes, F2 1,680 and F3 2,062. 63/113 genes that were altered in all three generations showed directionally opposite differences between generations. There was a trend toward increased proportions of up-regulated genes in F3 compared to F1. KEGG analysis showed that only the Adherens Junctions pathway was altered in all three generations. PR offspring showed altered fasting glucose homeostasis and changes in phosphoenolpyruvate carboxykinase promoter methylation and expression in all three generations. These findings show that dietary challenge during F0 pregnancy induced altered gene expression in all three generations, but relatively few genes showed transmission of altered expression between generations. For the majority of altered genes, these changes were not found in all generations, including some genes that were changed in F3 but not F1, or the direction and magnitude of difference between PR and PS differed between generations. Such variation may reflect differences between generations in the signals received by the fetus from the mother as a consequence of changes in the interaction between her phenotype and the environment.

Immunization with recombinant heat shock protein 60 (rHsp60) from Histoplasma capsulatum or a region of the protein designated fragment 3 (F3) confers protection from a subsequent challenge in mice. To determine the T cell repertoire involved in the response to Hsp60, T cell clones from C57BL/6 mice immunized with rHsp60 were generated and examined for Vbeta usage by flow cytometry and RT-PCR. Vbeta8.1/8.2(+) T cells were preferentially expanded; other clones bore Vbeta4, -6, or -11. When Vbeta8.1/8.2(+) cells were depleted in mice, Vbeta4(+) T cell clones were almost exclusively isolated. Measurement of cytokine production demonstrated that nine of 16 Vbeta8.1/8.2(+) clones were Th1, while only three of 13 non-Vbeta8.1/8.2(+) clones were Th1. In mice immunized with rHsp60, depletion of Vbeta8.1/8.2(+), but not Vbeta6(+) plus Vbeta7(+), T cells completely abolished the protective efficacy of Hsp60 to lethal and sublethal challenges. Examination of the TCR revealed that a subset of Vbeta8.1/2(+) clones that produced IFN-gamma and were reactive to F3 shared a common CDR3 sequence, DGGQG. Transfer of these T cell clones into TCR alpha/beta(-/-) or IFN-gamma(-/-) mice significantly improved survival, while transfer of other Vbeta8.1/8.2(+) clones that were F3 reactive but were Th2 or clones that were not reactive to F3 but were Th1 did not confer protection. These data indicate that a distinct subset of Vbeta8.1/8.2(+) T cells is crucial for the generation of a protective response to rHsp60.

During myelination, membrane-specialized domains are generated by complex interactions between axon and glial cells. The cell adhesion molecules caspr/paranodin and F3/contactin play a crucial role in the generation of functional septate-like junctions at paranodes. We have previously demonstrated that association with the glycosylphosphatidylinositol-linked F3/contactin is required for the recruitment of caspr/paranodin into the lipid rafts and its targeting to the cell surface. When transfected alone in neuroblastoma N2a cells, caspr/paranodin is retained in the endoplasmic reticulum (ER). Using chimerical constructs, we show that the cytoplasmic region does not contain any ER retention signal, whereas the ectodomain plays a crucial role in caspr/paranodin trafficking. A series of truncations encompassing the extracellular region of caspr/paranodin was unable to abolish ER retention. We show that N-glycosylation and quality control by the lectin-chaperone calnexin are required for the cell surface delivery of caspr/paranodin. Cell surface transport of F3/contactin and caspr/paranodin is insensitive to brefeldin A and the two glycoproteins are endoglycosidase H-sensitive when associated in complex, recruited into the lipid rafts, and expressed on the cell surface. Our results indicate a Golgi-independent pathway for the paranodal cell adhesion complex that may be implicated in the segregation of axonal subdomains.

The up-regulation of "tissue" transglutaminase (TG2) gene has been shown to occur in various pathologies and can lead to severe liver injury; however, its role in the onset of liver damage has not yet been clarified. To address this issue, we have used two experimental settings: carbon tetrachloride (CCl(4))-induced liver injury in wild-type and TG2 knockout mice; and liver biopsies obtained from a large cohort of hepatitis C virus (HCV)-infected patients. Mice lacking TG2 failed to clear the hepatic necrotic tissue formed in response to prolonged CCl(4) exposure (5 weeks) and 60% of them died before the end of the treatment. By contrast, wild-type mice were able to recover after the toxic insult. CCl(4)-treated TG2 null mice showed a derangement of the hepatic lobular architecture and a progressive accumulation of extracellular matrix (ECM) components and inflammatory cells which were not observed in the liver of control animals. Consistent with this protective role, we observed that TG2 levels were much higher (up to 15-fold) during the initial stages of liver fibrosis in HCV-infected individuals (METAVIR = F2) compared with uninfected controls, in which the enzyme protein localized in the hepatocytes facing the periportal infiltrate. By contrast, the enzyme levels decreased in the advanced stages (METAVIR = F3 and F4) and their localization was limited to the ECM. Our data demonstrate that TG2 plays a protective role in the liver injury by favoring tissue stability and repair.

Activation (affinity regulation) of integrin adhesion receptors controls cell migration and extracellular matrix assembly. Talin connects integrins with actin filaments and influences integrin affinity by binding to the integrins' short cytoplasmic beta-tail. The principal beta-tail binding site in talin is a FERM domain, comprised of three subdomains (F1, F2, and F3). Previous studies of integrin alphaIIbbeta3 have shown that both F2 and F3 bind the beta3 tail, but only F3, or the F2-F3 domain pair, induces activation. Here, talin-induced perturbations of beta3 NMR resonances were examined to explore integrin activation mechanisms. F3 and F2-F3, but not F2, distinctly perturbed the membrane-proximal region of the beta3 tail. All domains also perturbed more distal regions of the beta3 tail that appear to form the major interaction surface, since the beta3(Y747A) mutation suppressed those effects. These results suggest that perturbation of the beta3 tail membrane-proximal region is associated with talin-mediated integrin activation.

The effect of superoxide radical on the azide-insensitive ATP-dependent Ca2+-transport by a plasma membrane (PM)-enriched fraction (F2) and an endoplasmic reticulum (ER)-enriched fraction (F3) isolated from pig coronary artery was examined using xanthine oxidase plus xanthine to generate superoxide ions. A preincubation with xanthine oxidase plus xanthine at 37 degrees C preferentially inactivated the oxalate-stimulated Ca2+ uptake by the F3 fraction rather than the phosphate-stimulated uptake by the F2 fraction, indicating that the Ca2+ pump in the ER was more susceptible to this free radical. The inactivation of the Ca2+ uptake depended on the concentrations of xanthine oxidase and xanthine in the preincubation mixture as well as on the preincubation time. Furthermore, the inclusion of superoxide dismutase in the preincubation mixture prevented the inactivation. Thus the inactivation was caused by superoxide radical. Preincubation with xanthine oxidase plus xanthine, however, altered the half-life of efflux of Ca2+ from these vesicles only marginally. On sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the F3 fraction showed formation of a Ca2+-dependent acid stable phosphoenzyme at 0 degree C predominantly at a protein band corresponding to 100 kDa. The level of the 100-kDa acylphosphate intermediate was inhibited in parallel with the inhibition of the Ca2+ uptake by preincubation with xanthine oxidase plus xanthine. We conclude that superoxide radical inactivates the ER Ca2+ transport by lowering the level of the phosphoenzyme.