An adult mouse liver cDNA library was screened with oligonucleotides corresponding to the conserved WSXWS motif of the haemopoietin receptor family. Using this method, cDNA clones encoding a novel receptor were isolated. The new receptor, named NR1, was most similar in sequence and predicted structure to the alpha-chain of the IL-6 receptor and mRNA was expressed in the 3T3-L1 pre-adipocytic cell line and in a range of primary tissues. Expression of NR1 in the factor-dependent haemopoietic cell line Ba/F3 resulted in the generation of low affinity receptors for IL-11 (Kd approximately 10 nM). The capacity to bind IL-11 with high affinity (Kd = 300-800 pM) appeared to require coexpression of both NR1 and gp130, the common subunit of the IL-6, leukaemia inhibitory factor (LIF), oncostatin M (OSM) and ciliary neurotrophic factor (CNTF) receptors. The expression of both NR1 and gp130 was also necessary for Ba/F3 cells to proliferate and M1 cells to undergo macrophage differentiation in response to IL-11.

Focal adhesion kinase (FAK) is a non-receptor tyrosine kinase that localizes to focal adhesions in adherent cells. Through phosphorylation of proteins assembled at the cytoplasmic tails of integrins, FAK promotes signaling events that modulate cellular growth, survival, and migration. The amino-terminal region of FAK contains a region of sequence homology with band 4.1 and ezrin/radixin/moesin (ERM) proteins termed a FERM domain. FERM domains are found in a variety of signaling and cytoskeletal proteins and are thought to mediate intermolecular interactions with partner proteins and phospholipids at the plasma membrane and intramolecular regulatory interactions. Here we report two crystal structures of an NH2-terminal fragment of avian FAK containing the FERM domain and a portion of the regulatory linker that connects the FERM and kinase domains. The tertiary folds of the three subdomains (F1, F2, and F3) are similar to those of known FERM structures despite low sequence conservation. Differences in the sequence and relative orientation of the F3 subdomain alters the nature of the interdomain interface, and the phosphoinositide binding site found in ERM family FERM domains is not present in FAK. A putative protein interaction site on the F3 lobe is masked by the proximal region of the linker. Additionally, in one structure the adjacent Src SH3 and SH2 binding sites in the linker associate with the surfaces of the F3 and F1 lobes, respectively. These structural features suggest the possibility that protein interactions of the FAK FERM domain can be regulated by binding of Src kinases to the linker segment.

Thrombopoietin (TPO) is a recently cloned cytokine that binds to its receptor, Mpl, and promotes hematopoietic expansion and maturation, primarily of the megakaryocyte lineage. The signaling pathways responsible for these events are thought to involve the Janus family of nonreceptor tyrosine kinases (JAKs) and the signal transducers and activators of transcription (STATs), which are activated by tyrosine phosphorylation. Previous investigators have studied these molecules in engineered and naturally occurring cell lines. To investigate the molecular basis for TPO signal transduction in a more physiologic target, we determined the pattern of JAK and STAT activation in purified, normal urine megakaryocytes. These results are compared with those of established cell lines that only proliferate (Ba/F3- mMPL and DA-1-TPO) or only differentiate (L8057) in response to TPO. From these findings, a model is proposed to explain the physiologic roles of JAK2, TYK2, STAT3, and STAT5 in TPO signaling. Furthermore, previous studies of the physical interaction between Mpl and the JAKs are extended, showing a difference in the association of JAK2 and TYK2 with the TPO receptor. Finally, we show that, in the cell line Ba/F3-mMPL, the closely related proteins STAT5A and STAT5B are both activated by TPO stimulation and are capable of heterodimerization. Together, these results further our understanding of the early stages of megakaryocyte and platelet development.

Neural stem cells and progenitor cells migrate selectively to tumor loci in vivo. We exploited the tumor-tropic properties of HB1.F3.C1 cells, an immortalized cell line derived from human fetal telencephalon, to deliver the cDNA encoding a secreted form of rabbit carboxylesterase (rCE) to disseminated neuroblastoma tumors in mice. This enzyme activates the prodrug CPT-11 more efficiently than do human enzymes. Mice bearing multiple tumors were treated with rCE-expressing HB1.F3.C1 cells and schedules of administration of CPT-11 that produced levels of active drug (SN-38) tolerated by patients. Both HB1.F3.C1 cells and CPT-11 were given i.v. None of the untreated mice and 30% of mice that received only CPT-11 survived long term. In contrast, 90% of mice treated with rCE-expressing HB1.F3.C1 cells and 15 mg/kg CPT-11 survived for 1 year without detectable tumors. Plasma carboxylesterase activity and SN-38 levels in mice receiving both rCE-expressing HB1.F3.C1 cells (HB1.F3.C1/AdCMVrCE) and CPT-11 were comparable with those in mice receiving CPT-11 only. These data support the hypothesis that the antitumor effect of the described neural stem/progenitor cell-directed enzyme prodrug therapy (NDEPT) is mediated by production of high concentrations of active drug selectively at tumor sites, thereby maximizing the antitumor effect of CPT-11. NDEPT approaches merit further investigation as effective, targeted therapy for metastatic tumors. We propose that the described approach may have greatest use for eradicating minimum residual disease.

mcl-1, a bcl-2 family member, was originally identified as an early gene induced during differentiation of ML-1 myeloid leukemia cells. In the present study, we demonstrate that Mcl-1 is tightly regulated by the granulocyte-macrophage colony-stimulating factor (GM-CSF) signaling pathway. Upon deprivation of survival factor from TF-1 myeloid progenitor cells, Mcl-1 levels quickly dropped prior to visible detection of apoptosis of these cells. Upon restimulation of these deprived cells with GM-CSF, the mcl-1 mRNA was immediately induced and its protein product was accordingly resynthesized. Analysis with Ba/F3 cells expressing various truncation mutants of the GM-CSF receptor revealed that the membrane distal region between amino acids 573 and 755 of the receptor beta chain was required for mcl-1 induction. Transient-transfection assays with luciferase reporter genes driven by various regions of the mcl-1 promoter demonstrated that the upstream sequence between -197 and -69 is responsible for cytokine activation of the mcl-1 gene. Overexpression of mcl-1 delayed but did not completely prevent apoptosis of cells triggered by cytokine withdrawal. Its down regulation by antisense constructs overcame, at least partially, the survival activity of GM-CSF and induced the apoptosis of TF-1 cells. Taken together, these results suggest that mcl-1 is an immediate-early gene activated by the cytokine receptor signaling pathway and is one component of the GM-CSF viability response.

Environmental compounds including fungicides, plastics, pesticides, dioxin and hydrocarbons can promote the epigenetic transgenerational inheritance of adult-onset disease in future generation progeny following ancestral exposure during the critical period of fetal gonadal sex determination. This study examined the actions of the pesticide methoxychlor to promote the epigenetic transgenerational inheritance of adult-onset disease and associated differential DNA methylation regions (i.e. epimutations) in sperm. Gestating F0 generation female rats were transiently exposed to methoxychlor during fetal gonadal development (gestation days 8 to 14) and then adult-onset disease was evaluated in adult F1 and F3 (great-grand offspring) generation progeny for control (vehicle exposed) and methoxychlor lineage offspring. There were increases in the incidence of kidney disease, ovary disease, and obesity in the methoxychlor lineage animals. In females and males the incidence of disease increased in both the F1 and the F3 generations and the incidence of multiple disease increased in the F3 generation. There was increased disease incidence in F4 generation reverse outcross (female) offspring indicating disease transmission was primarily transmitted through the female germline. Analysis of the F3 generation sperm epigenome of the methoxychlor lineage males identified differentially DNA methylated regions (DMR) termed epimutations in a genome-wide gene promoters analysis. These epimutations were found to be methoxychlor exposure specific in comparison with other exposure specific sperm epimutation signatures. Observations indicate that the pesticide methoxychlor has the potential to promote the epigenetic transgenerational inheritance of disease and the sperm epimutations appear to provide exposure specific epigenetic biomarkers for transgenerational disease and ancestral environmental exposures.

We used high-frequency repetitive transcranial magnetic stimulation (rTMS) to study the role of the mesial frontocentral cortex (including the supplementary motor area) in the organization of sequential finger movements of different complexity in humans. In 15 subjects, rTMS was randomly applied to the scalp overlying the region of the supplementary motor area and over other positions, including the contralateral primary motor cortex (hand area) during the performance of three overlearned finger sequences on an electronic piano. In all trials, rTMS (frequency 15-20 Hz) started 2 s after the first key press and lasted for approximately 2 s. All sequences were metronome-paced at 2 Hz and retrieved from memory. The 'simple' sequence consisted of 16 repeated index finger key presses, the 'scale' sequence of four times four sequential key presses of the little, ring, middle and index fingers, and the 'complex' sequence of a much less systematic and, therefore, more difficult series of 16 key presses. To measure the effects of rTMS interference with regional cortical function, we analysed rTMS-induced accuracy errors in the movement sequences. Stimulation over the supplementary motor area induced accuracy errors only in the complex sequence, while stimulation over the primary motor cortex induced errors in both the complex and scale sequences, and stimulation over other positions (e.g. F3, F4, FCz, P3, P4) did not interfere with sequence performance at all. Stimulation over the supplementary motor area interfered with the organization of subsequent elements in the complex sequence of movements, with error induction occurring approximately 1 s later than with stimulation over the primary motor cortex. Our findings are in keeping with recent results in non-human primates (Tanji J, Shima K. Nature, 1994; 371: 413-6) indicating a critical role of the supplementary motor area in the organization of forthcoming movements in complex motor sequences that are rehearsed from memory and fit into a precise timing plan.

OBJECTIVE

We measured the accuracy of magnetic resonance elastography (MRE) for the detection and staging of liver fibrosis in chronic hepatitis B (CHB) and compared it with serum fibrosis markers.

METHODS

Prospective comparison of MRE and routine serum fibrosis markers, namely serum alanine aminotransferase (ALT), serum aspartate aminotransferase (AST), ALT/AST ratio (AAR), AST to platelet ratio index (APRI) and prothrombin index (PI), was performed in 63 consecutive CHB patients who underwent MRE and histological confirmation of liver fibrosis within a 6-month interval. Diagnostic performance of MRE and serum markers for staging fibrosis (≥F1), significant fibrosis (≥F2), advanced fibrosis (≥F3) and cirrhosis (F4) was compared.

RESULTS

The study group comprised 63 patients (19 female; mean age ± SD, 50 ± 11.9 years). MRE (ρ = 0.94, P < 0.0001), APRI (ρ = 0.42, P = 0.0006), PI (ρ = 0.42, P = 0.0006) and AST (ρ = 0.28, P = 0.028) results correlated significantly with fibrosis stage. MRE was significantly more accurate than serum fibrosis markers for the detection of significant fibrosis (0.99 vs. 0.55-0.73) and cirrhosis (0.98 vs. 0.53-0.77). Sensitivity, specificity, positive predictive and negative predictive values for MRE for significant fibrosis and cirrhosis were 97.4 %, 100 %, 100 % and 96 %, and 100 %, 95.2 %, 91.3 % and 100 %, respectively.

CONCLUSIONS

MRE is an accurate non-invasive technique for the detection and staging of liver fibrosis in CHB.

CONCLUSIONS

• Magnetic resonance elastography is accurate for liver fibrosis detection and staging. • MR elastography is more accurate than serum tests for staging liver fibrosis. • MR elastography can potentially replace liver biopsy in chronic hepatitis B.

We isolated cDNAs encoding a mouse interleukin 10 receptor (mIL-10R) from mouse mast cell and macrophage cell lines. The two cDNAs are substantially identical and express an approximately 110-kDa polypeptide in COS7 cells, which binds mIL-10 specifically. A mouse pro-B-cell line (Ba/F3) expressing transfected recombinant mIL-10R binds IL-10 with high affinity (approximately 70 pM) and proliferates in response to mIL-10. mIL-10R is structurally related to interferon receptors (IFNRs). Since IL-10 inhibits macrophage activation by IFN-gamma, a possible implication of this relationship interaction of IL-10R and IFN-gamma R or their signaling pathways.

The cAMP-responsive element binding protein (CREB) is a 43-kDa nuclear transcription factor that regulates cell growth, memory, and glucose homeostasis. We showed previously that CREB is amplified in myeloid leukemia blasts and expressed at higher levels in leukemia stem cells from patients with myeloid leukemia. CREB transgenic mice develop myeloproliferative disease after 1 year, but not leukemia, suggesting that CREB contributes to but is not sufficient for leukemogenesis. Here, we show that CREB is most highly expressed in lineage negative hematopoietic stem cells (HSCs). To understand the role of CREB in hematopoietic progenitors and leukemia cells, we examined the effects of RNA interference (RNAi) to knock down CREB expression in vitro and in vivo. Transduction of primary HSCs or myeloid leukemia cells with lentiviral CREB shRNAs resulted in decreased proliferation of stem cells, cell- cycle abnormalities, and inhibition of CREB transcription. Mice that received transplants of bone marrow transduced with CREB shRNA had decreased committed progenitors compared with control mice. Mice injected with Ba/F3 cells expressing either Bcr-Abl wild-type or T315I mutation with CREB shRNA had delayed leukemic infiltration by bioluminescence imaging and prolonged median survival. Our results suggest that CREB is critical for normal myelopoiesis and leukemia cell proliferation.

The thrombopoietin receptor (TpoR) regulates hematopoietic stem cell renewal, megakaryocyte differentiation, and platelet formation. TpoR signals by activating Janus kinases JAK2 and Tyk2. Here we show that, in addition to signaling downstream from the activated TpoR, JAK2 and Tyk2 strongly promote cell surface localization and enhance total protein levels of the TpoR. This effect is caused by stabilization of the mature endoglycosidase H-resistant form of the receptor. Confocal microscopy indicates that TpoR colocalizes partially with recycling transferrin in Ba/F3 cells. The interaction with JAK2 or Tyk2 appears to protect the receptor from proteasome degradation. Sequences encompassing Box1 and Box2 regions of the receptor cytosolic domain and an intact JAK2 or Tyk2 FERM domain are required for these effects. We discuss the relevance of our results to the reported defects of TpoR processing in myeloproliferative diseases and to the mechanisms of Tpo signaling and clearance via the TpoR.

Aberrant expression of FLT3 has been found in most cases of B-lineage ALL and AML, and subsets of T cell ALL, CML in blast crisis and CLL. In 20% of patients with AML the receptor has small internal tandem duplications of the juxtamembrane region which appear to contitutively activate the receptor. To investigate whether FLT3 activation could play a role in leukemia, we generated a constitutively activated FLT3 by fusing its cytoplasmic domain to the helix-loop-helix domain of TEL in analogy to the fusion that occurs with TEL-PDGFR in CMML. In vitro translation assays demonstrated oligomerization and intrinsic tyrosine kinase activity of the TEL-FLT3 chimeric receptor. Constitutively activated TEL-FLT3 conferred IL-3 independence and long-term proliferation to transfected Ba/F3 cells. Immunoblot analyses showed that JAK 2, STAT 3, STAT 5a, STAT 5b and CBL were tyrosine-phosphorylated in TEL-FLT3 expressing Ba/F3 cells in the absence of IL-3. These data suggest a possible role for the JAK/STAT pathway in FLT3 signaling. Transplantation of TEL-FLT3 expressing Ba/F3 cells into syngeneic mice caused mortality in all mice by 3 weeks after injection. Histopathologic analysis demonstrated a massive infiltration of mononuclear cells in the liver, spleen and bone marrow. The mimicking of naturally occurring TEL fusions provides an approach to assess aspects of the biology of activated FLT3, or other receptor-type tyrosine kinases (RTKs) in leukemic transformation.

Striated muscle alpha-tropomyosin expressed in Escherichia coli is unacetylated, polymerizes poorly, and binds weakly to F-actin (Hitchcock-DeGregori, S. E., and Heald, R. W. (1987) J. Biol. Chem. 262, 9730-9735). To define the structural requirements of NH2-terminal modification for striated tropomyosin function, an acetylated recombinant tropomyosin and an unacetylated short fusion recombinant tropomyosin were compared. An acetylated recombinant chicken striated muscle alpha-tropomyosin was expressed in insect Sf9 cells using the baculovirus expression vector system. The purified tropomyosin (approximately 15 mg/liter of insect cell suspension) polymerized, comigrated with chicken striated alpha-tropomyosin purified from muscle on two-dimensional polyacrylamide gels, was blocked at the NH2 terminus, and had the same actin affinity as muscle tropomyosin. These results conclusively show the importance of NH2-terminal acetylation for striated tropomyosin function. To learn if a short fusion peptide would substitute for amino-terminal acetylation, tropomyosin with AlaSer-Arg on the NH2 terminus was constructed and expressed in E. coli as an unacetylated protein. This f3-tropomyosin bound to actin with a 10-fold higher affinity than striated muscle alpha-TM and, unlike muscle tropomyosin, exhibited a shear-dependent viscosity. The altered function of f3-tropomyosin shows that the naturally occurring acetylated NH2 terminus is required for full, normal function. It is proposed that a major requirement for cooperative binding of striated muscle tropomyosin to actin is modification of the alpha-amino group of methionine to be an amide, as when acetylated or in a peptide bond in a fusion protein, to make the extreme NH2 terminus more hydrophobic. The results are discussed in terms of known coiled coil structure.

Toll-like receptors (TLR) recognize a variety of microbial products and activate defense responses. Pathogen sensing by TLR2/4 requires accessory molecules, whereas little is known about a molecule required for DNA recognition by TLR9. After endocytosis of microbes, microbial DNA is exposed and recognized by TLR9 in lysosomes. We here show that cathepsins, lysosomal cysteine proteases, are required for TLR9 responses. A cell line Ba/F3 was found to be defective in TLR9 responses despite enforced TLR9 expression. Functional cloning with Ba/F3 identified cathepsin B/L as a molecule required for TLR9 responses. The protease activity was essential for the complementing effect. TLR9 responses were also conferred by cathepsin S or F, but not by cathepsin H. TLR9-dependent B cell proliferation and CD86 upregulation were apparently downregulated by cathepsin B/L inhibitors. Cathepsin B inhibitor downregulated interaction of CpG-B with TLR9 in 293T cells. These results suggest roles for cathepsins in DNA recognition by TLR9.

BACKGROUND

Epigenetic mechanisms might be involved in the regulation of interindividual lipid level variability and thus may contribute to the cardiovascular risk profile. The aim of this study was to investigate the association between genome-wide DNA methylation and blood lipid levels high-density lipoprotein cholesterol, low-density lipoprotein cholesterol, triglycerides, and total cholesterol. Observed DNA methylation changes were also further analyzed to examine their relationship with previous hospitalized myocardial infarction.

RESULTS

Genome-wide DNA methylation patterns were determined in whole blood samples of 1776 subjects of the Cooperative Health Research in the Region of Augsburg F4 cohort using the Infinium HumanMethylation450 BeadChip (Illumina). Ten novel lipid-related CpG sites annotated to various genes including ABCG1, MIR33B/SREBF1, and TNIP1 were identified. CpG cg06500161, located in ABCG1, was associated in opposite directions with both high-density lipoprotein cholesterol (β coefficient=-0.049; P=8.26E-17) and triglyceride levels (β=0.070; P=1.21E-27). Eight associations were confirmed by replication in the Cooperative Health Research in the Region of Augsburg F3 study (n=499) and in the Invecchiare in Chianti, Aging in the Chianti Area study (n=472). Associations between triglyceride levels and SREBF1 and ABCG1 were also found in adipose tissue of the Multiple Tissue Human Expression Resource cohort (n=634). Expression analysis revealed an association between ABCG1 methylation and lipid levels that might be partly mediated by ABCG1 expression. DNA methylation of ABCG1 might also play a role in previous hospitalized myocardial infarction (odds ratio, 1.15; 95% confidence interval=1.06-1.25).

CONCLUSIONS

Epigenetic modifications of the newly identified loci might regulate disturbed blood lipid levels and thus contribute to the development of complex lipid-related diseases.

To gain insights into the mechanisms of endothelial nitric oxide synthase (eNOS) gene expression, we have cloned the eNOS promoter and fused it to a luciferase reporter gene to map regions of the promoter important for basal transcription in bovine aortic endothelial cells (BAEC). Transfection of BAEC with F1 luciferase (LUC) (-1600 to +22 nucleotides) yielded a 35-fold increase in promoter. Progressive deletion from -1600 to -1033 (F2 and F3 LUC) did not significantly influence eNOS promoter activity. Further deletion from -1033 to -779 (F4 LUC) resulted in an approximate 40% reduction in basal promoter activity, and still further deletion from -779 to -494 (F5 LUC) did not markedly influence activity. Deletion from -494 to -166 (F6 LUC) reduced eNOS promoter activity by 40-50%. Specific mutation of the consensus GATA site (-230) in the F3 LUC construct reduced luciferase activity (by 25-30%). Gel shift analysis and antibody depletion using BAEC nuclear extracts demonstrated in vitro binding of GATA-2 to the oligonucleotide sequence containing the -230 GATA site. Next, we mutated the Sp1 site (-103) in the F3 and F6 LUC constructs and in the F3 GATA mutant construct. Expression of these Sp1 mutants in BAEC resulted in a 85-90% reduction in normalized luciferase activity. Gel shift and antibody supershift analysis using a BAEC nuclear extracts demonstrated four specific, DNA-protein complexes binding to the eNOS Sp-1 site, with the slowest migrating form composed of Sp1 and another nuclear protein. These data demonstrate that the Sp1 site is an important cis-element in the core eNOS promoter.

Fucus vesiculosus was sequentially extracted with water at 22 degrees C (fraction 1 (F1)) and 60 degrees C (F2), and with 0.1 M HCl (F3) and 2 M KOH (F4) at 37 degrees C. Soluble fractions (42.3% yield) were composed of neutral sugars (18.9-48 g/100 g), uronic acids (8.8-52.8 g/100 g), sulfate (2.4-11.5 g/100 g), small amounts of protein (< 1-6.1 g/100 g), and nondialyzable polyphenols (0.1-2.7 g/100 g). The main neutral sugars were fucose, glucose, galactose, and xylose. Infrared (IR) spectra of the fractions showed absorption bands at 820-850 and 1225-1250 cm(-1) for sulfate. F1, F2, and F4 also exhibited an absorption band at 1425 cm(-1), due to uronic acids, and their IR spectra resembled that of alginate. F3 had an IR spectrum similar to that of fucoidan with an average molecular weight of 1.6 x 10(6) Da, calculated by molecular exclusion high-performance liquid chromatography. The presence of fucose in this polysaccharide was confirmed by (1)H NMR spectroscopy. This fraction showed the highest potential to be antioxidant by the ferric reducing antioxidant power (FRAP) assay, followed by the alkali- and water-soluble fractions. Sulfated polysaccharides from edible seaweeds potentially could be used as natural antioxidants by the food industry.

Although known for almost 80 years, the physiological role of plasmalogens (PLs), the major mammalian ether lipids (ELs), is still enigmatic. Humans that lack ELs suffer from rhizomelic chondrodysplasia punctata (RCDP), a peroxisomal disorder usually resulting in death in early childhood. In order to learn more about the functions of ELs, we generated a mouse model for RCDP by a targeted disruption of the dihydroxyacetonephosphate acyltransferase gene. The mutant mice revealed multiple abnormalities, such as male infertility, defects in eye development, cataract and optic nerve hypoplasia, some of which were also observed in RCDP. Mass spectroscopic analysis demonstrated the presence of highly unsaturated fatty acids including docosahexaenoic acid (DHA) in brain PLs and the occurrence of PLs in lipid raft microdomains (LRMs) isolated from brain myelin. In mutants, PLs were completely absent and the concentration of brain DHA was reduced. The marker proteins flotillin-1 and F3/contactin were found in brain LRMs in reduced concentrations. In addition, the gap junctional protein connexin 43, known to be recruited to LRMs and essential for lens development and spermatogenesis, was down-regulated in embryonic fibroblasts of the EL-deficient mice. Free cholesterol, an important constituent of LRMs, was found in these fibroblasts to be accumulated in a perinuclear compartment. These data suggest that the EL-deficient mice allow the identification of new phenotypes not related so far to EL-deficiency (male sterility, defects in myelination and optic nerve hypoplasia) and indicate that PLs are required for the correct assembly and function of LRMs.

Using phylogenetic analysis and pairwise comparison of 670 complete hepatitis B virus (HBV) genomes, we demonstrated that nucleotide divergence greater than 7.5% can be used to separate strains into genotypes A-H. Strains can be separated into subgenotypes when two criteria are met: nucleotide divergence of about 4% but less than 7.5% and good bootstrap support. There is a highly statistically significant association between serological subtypes and genotypes (chi2-test for association, P < 0.0001): adw is associated with genotypes A, B, F, G, and H, adr with C and ayw with D and E. The logistic regression method showed that 1802-1803CG are characteristic of genotypes A, D, and E whereas 1802-1803TT are characteristic of genotypes B, C, and F. 1858C is positively associated with genotypes A, F, and H and 1858T with genotypes B, D, and E. Subgenotypes C2, F1/F4 can be differentiated from subgenotypes C1, F2/F3, respectively, because the latter have 1858C as opposed to 1858T in the former. 1888A was positively associated with subgenotype A1 and TAA at 1817 with genotype G. The Haploplot method revealed high linkage between loci 1858 and 1896 but strong evidence of recombination between loci 1862 and 1896. Loci 1809-1812, 1862, and 1888 may have co-evolved. Using a computer program, we showed that serological subtype deduced from the S region (position 155-835) and mutations/variations within the basic core promoter/precore region (1653-1900), allowed genotyping of HBV with 97% sensitivity and 99% specificity. Certain subgenotypes or subgenotype groups could also be differentiated.

Environmental compounds are known to promote epigenetic transgenerational inheritance of disease. The current study was designed to determine if a "pesticide mixture" (pesticide permethrin and insect repellent N,N-diethyl-meta-toluamide, DEET) promotes epigenetic transgenerational inheritance of disease and associated DNA methylation epimutations in sperm. Gestating F0 generation female rats were exposed during fetal gonadal sex determination and the incidence of disease evaluated in F1 and F3 generations. There were significant increases in the incidence of total diseases in animals from pesticide lineage F1 and F3 generation animals. Pubertal abnormalities, testis disease, and ovarian disease (primordial follicle loss and polycystic ovarian disease) were increased in F3 generation animals. Analysis of the pesticide lineage F3 generation sperm epigenome identified 363 differential DNA methylation regions (DMR) termed epimutations. Observations demonstrate that a pesticide mixture (permethrin and DEET) can promote epigenetic transgenerational inheritance of adult onset disease and potential sperm epigenetic biomarkers for ancestral environmental exposures.

OBJECTIVE

To document utility of shear-wave (SW) elastography for assessing liver fibrosis in chronic hepatitis B and to compare its performance with that of transient elastography.

METHODS

Ethics committee approved the study, and informed consent was obtained. Patients with liver biopsy correlation (n = 226) and healthy patients (n = 171) were analyzed. Results of SW elastography of liver, SW elastography of spleen, and transient elastography of liver were compared and correlated according to METAVIR scores. Areas under the receiver operating characteristic curve (AUCs), binary logistic regression, and Delong test were used.

RESULTS

AUC for SW elastography of liver, transient elastography of liver, and SW elastography of spleen was, respectively, 0.86, 0.80, and 0.81 for fibrosis (≥ F1 stage); 0.88, 0.78, and 0.82 for moderate fibrosis (≥ F2 stage); 0.93, 0.83, and 0.83 for severe fibrosis (≥ F3 stage); and 0.98, 0.92, and 0.84 for cirrhosis (F4 stage). SW elastography of liver showed significantly higher accuracy than transient elastography of liver and SW elastography of spleen in all fibrosis stages (P = .01-.04). SW elastography of spleen showed similar accuracy with transient elastography of liver (P = .21-.99). Combination SW elastography of liver and SW elastography of spleen to predict fibrosis staging showed diagnostic accuracy not further improved compared with SW elastography of liver alone (similar AUC; ≥ F1, P = .87; ≥ F2, P = .81; ≥ F3, P = .84; ≥ F4, P = .88). SW elastography of liver had higher successful rate than transient elastography of liver (98.9% vs 89.6%). Prevalence of discordance in at least two stages with liver histologic staging was 10.2% (23 of 226) for SW elastography of liver and 28.2% (58 of 206) for SW elastography of spleen.

CONCLUSIONS

SW elastography provides more accurate correlation of liver elasticity with liver fibrosis stage compared with transient elastography, especially in identification of stage F2 or greater.

The purpose of this study was to explore human development of EEG coherence and phase differences over the period from infancy to 16 years of age. The electroencephalogram (EEG) was recorded from 19 scalp locations from 458 subjects ranging in age from 2 months to 16.67 years. EEG coherence and EEG phase differences were computed for the left and right hemispheres in the posterior-to-anterior direction (O1/2-P3/4, O1/2-C3/4, O1/2-F3/4, and O1/2-Fp1/2) and the anterior-to- posterior direction (Fp1/2-F3/4, Fp1/2-C3/4, Fp1/2-P3/4, and Fp1/2-O1/2) in the beta frequency band (13-25 Hz). Sliding averages of EEG coherence and phase were computed using 1 year averages and 9 month overlapping that produced 64 means from 0.44 years of age to 16.22 years of age. Rhythmic oscillations in coherence and phase were noted in all electrode combinations. Different developmental trajectories were present for coherence and phase differences and for anterior-to-posterior and posterior-to-anterior directions and inter-electrode distance. Large changes in EEG coherence and phase were present from approximately 6 months to 4 years of age followed by a significant linear trend to higher coherence in short distance inter-electrode distances and longer phase delays in long inter-electrode distances. The results are consistent with a genetic model of rhythmic long term connection formation that occurs in cycles along a curvilinear trajectory toward adulthood. Competition for dendritic space, development of complexity, and nonlinear dynamic oscillations are discussed.

Embryonic exposure to the endocrine disruptor vinclozolin during gonadal sex determination appears to promote an epigenetic reprogramming of the male germ line that is associated with transgenerational adult-onset disease states. Transgenerational effects on the embryonic day 16 (E16) testis demonstrated reproducible changes in the testis transcriptome for multiple generations (F1-F3). The expression of 196 genes was found to be influenced, with the majority of gene expression being decreased or silenced. Dramatic changes in the gene expression of methyltransferases during gonadal sex determination were observed in the F1 and F2 vinclozolin generation (E16) embryonic testis, but the majority returned to control-generation levels by the F3 generation. The most dramatic effects were on the germ-line-associated Dnmt3A and Dnmt3L isoforms. Observations demonstrate that an embryonic exposure to vinclozolin appears to promote an epigenetic reprogramming of the male germ line that correlates with transgenerational alterations in the testis transcriptome in subsequent generations.

BACKGROUND

Patients diagnosed with metastatic cancer have almost uniformly poor prognoses. The treatments available for patients with disseminated disease are usually not curative and have side effects that limit the therapy that can be given. A treatment that is selectively toxic to tumors would maximize the beneficial effects of therapy and minimize side effects, potentially enabling effective treatment to be administered.

RESULTS

We postulated that the tumor-tropic property of stem cells or progenitor cells could be exploited to selectively deliver a therapeutic gene to metastatic solid tumors, and that expression of an appropriate transgene at tumor loci might mediate cures of metastatic disease. To test this hypothesis, we injected HB1.F3.C1 cells transduced to express an enzyme that efficiently activates the anti-cancer prodrug CPT-11 intravenously into mice bearing disseminated neuroblastoma tumors. The HB1.F3.C1 cells migrated selectively to tumor sites regardless of the size or anatomical location of the tumors. Mice were then treated systemically with CPT-11, and the efficacy of treatment was monitored. Mice treated with the combination of HB1.F3.C1 cells expressing the CPT-11-activating enzyme and this prodrug produced tumor-free survival of 100% of the mice for >6 months (P<0.001 compared to control groups).

CONCLUSIONS

The novel and significant finding of this study is that it may be possible to exploit the tumor-tropic property of stem or progenitor cells to mediate effective, tumor-selective therapy for metastatic tumors, for which no tolerated curative treatments are currently available.