Molecular Changes Following Induction of Hepatocellular Carcinoma by Diethylnitrosamine and Thioacetamide, and Subsequent Treatment with Dioscorea membranacea Extract.

Hepatocellular carcinoma (HCC) is a primary liver cancer commonly found in adults. Previously, we showed the anticancer effects of Thai herbal plant extract, Dioscorea membranacea Pierre (DM), in HCC-bearing rats. In the present study, we further examined the proposed mechanism of DM, including apoptosis and antioxidant activity. Moreover, we used RNA sequencing (RNA-seq) to analyze molecular pathways in the rat model in which HCC was induced by diethylnitrosamine (DEN) and thioacetamide (TAA). The HCC-bearing rats were then treated with 40 mg/kg of DM for 8 weeks, after which experimental and control rats were sacrificed and liver tissues were collected. The RNA-seq data of DEN/TAA-treated rats exhibited upregulation of 16 hallmark pathways, including epithelial mesenchymal transition, inflammatory responses, and angiogenesis (p<0.01). DM extract expanded the Bax protein-positive pericentral zone in the tumor areas and decreased hepatic malondialdehyde levels, implying a decrease in lipid peroxidation in liver. However, DM treatment did not ameliorate the molecular pathways induced in DEN/TAA-treated livers. Our findings indicate that DM extract has antioxidant activity and exerts its pro-apoptotic effect on rat HCCs in vivo at the (post-)translational level.

Microcirculatory Function during Endotoxemia-A Functional Citrulline-Arginine-NO Pathway and NOS3 Complex Is Essential to Maintain the Microcirculation.

Competition for the amino acid arginine by endothelial nitric-oxide synthase (NOS3) and (pro-)inflammatory NO-synthase (NOS2) during endotoxemia appears essential in the derangement of the microcirculatory flow. This study investigated the role of NOS2 and NOS3 combined with/without citrulline supplementation on the NO-production and microcirculation during endotoxemia. Wildtype (C57BL6/N background; control; n = 36), Nos2-deficient, (n = 40), Nos3-deficient (n = 39) and Nos2/Nos3-deficient mice (n = 42) received a continuous intravenous LPS infusion alone (200 µg total, 18 h) or combined with L-citrulline (37.5 mg, last 6 h). The intestinal microcirculatory flow was measured by side-stream dark field (SDF)-imaging. The jejunal intracellular NO production was quantified by in vivo NO-spin trapping combined with electron spin-resonance (ESR) spectrometry. Amino-acid concentrations were measured by high-performance liquid chromatography (HPLC). LPS infusion decreased plasma arginine concentration in control and Nos3-/- compared to Nos2-/- mice. Jejunal NO production and the microcirculation were significantly decreased in control and Nos2-/- mice after LPS infusion. No beneficial effects of L-citrulline supplementation on microcirculatory flow were found in Nos3-/- or Nos2-/-/Nos3-/- mice. This study confirms that L-citrulline supplementation enhances de novo arginine synthesis and NO production in mice during endotoxemia with a functional NOS3-enzyme (control and Nos2-/- mice), as this beneficial effect was absent in Nos3-/- or Nos2-/-/Nos3-/- mice.

Pseudolesion in the right parafissural liver parenchyma on CT: The base is found in embryology and collagen content.

BACKGROUND: Computed tomography (CT) images of livers may show a hypo-attenuated structure alongside the falciform ligament, which can be a focal fatty pseudolesion and can mimic a malignancy. The preferred location is on the right parafissural site, ventral in segment IVa/b. The etiology is not clear, nor is it known how the histology of this location develops. These are evaluated in this study. METHODS: 40 adult cadavers with autopsy and / or postmortem CT in a university hospital and a forensic center were included. Liver biopsies were taken at the left side of the falciform ligament as control, and at the right side as the possible precursor of a pseudolesion; these were examined for collagen and fat content. Cadavers with steatotic (>5% fat) or fibrotic (>2% collagen) control samples were excluded. RESULTS: Significantly more collagen was present in the right parafissural liver parenchyma: median 0.68% (IQR: 0.32-1.17%), compared to the left side 0.48% (IQR: 0.21-0.75%) (p 0.008), with equal fat content and CT attenuation values. The etiophysiology goes back to the demise of the umbilical venes in the early embryonic and neonatal period. CONCLUSIONS: The right parafissural area contains more collagen and an equal amount of fat compared to the control left side. This supports the hypothesis of delayed, 'third' inflow: the postnatal change in blood supply from umbilical to portal leaves the downstream parafissural area hypoperfused leading to hypoxia which in turn results in collagen accumulation and the persistence of paraumbilical veins of Sappey.

Interactive three-dimensional teaching models of the female and male pelvic floor.

Controversies regarding structure and function of the pelvic floor persist because of its poor accessibility and complex anatomical architecture. Most data are based on dissection. This "surgical" approach requires profound prior knowledge, because applying the scalpel precludes a "second look." The "sectional" approach does not entail these limitations, but requires segmentation of structures and three-dimensional reconstruction. This approach has produced several "Visible Human Projects." We dealt with limited spatial resolution and difficult-to-segment structures by proceeding from clear-cut to more fuzzy boundaries and comparing segmentation between investigators. We observed that the bicipital levator ani muscle consisted of pubovisceral and puborectal portions; that the pubovisceral muscle formed, together with rectococcygeal and rectoperineal muscles, a rectal diaphragm; that the external anal sphincter consisted of its subcutaneous portion and the puborectal muscle only; that the striated urethral sphincter had three parts, of which the middle (urethral compressor) was best developed in females and the circular lower ("membranous") best in males; that the rectourethral muscle, an anterior extension of the rectal longitudinal smooth muscle, developed a fibrous node in its center (perineal body); that the perineal body was much better developed in females than males, so that the rectourethral subdivision into posterior rectoperineal and anterior deep perineal muscles was more obvious in females; that the superficial transverse perineal muscle attached to the fibrous septa of the ischioanal fat; and that the uterosacral ligaments and mesorectal fascia colocalized. To facilitate comprehension of the modified topography we provide interactive 3D-PDFs that are freely available for teaching purposes. Clin. Anat. 33:275-285, 2020. © 2019 Wiley Periodicals, Inc.

Glutamine Anabolism Plays a Critical Role in Pancreatic Cancer by Coupling Carbon and Nitrogen Metabolism.

Glutamine is thought to play an important role in cancer cells by being deaminated via glutaminolysis to α-ketoglutarate (aKG) to fuel the tricarboxylic acid (TCA) cycle. Supporting this notion, aKG supplementation can restore growth/survival of glutamine-deprived cells. However, pancreatic cancers are often poorly vascularized and limited in glutamine supply, in alignment with recent concerns on the significance of glutaminolysis in pancreatic cancer. Here, we show that aKG-mediated rescue of glutamine-deprived pancreatic ductal carcinoma (PDAC) cells requires glutamate ammonia ligase (GLUL), the enzyme responsible for de novo glutamine synthesis. GLUL-deficient PDAC cells are capable of the TCA cycle but defective in aKG-coupled glutamine biosynthesis and subsequent nitrogen anabolic processes. Importantly, GLUL expression is elevated in pancreatic cancer patient samples and in mouse PDAC models. GLUL ablation suppresses the development of KrasG12D-driven murine PDAC. Therefore, GLUL-mediated glutamine biosynthesis couples the TCA cycle with nitrogen anabolism and plays a critical role in PDAC.

Arginase 1 deletion in myeloid cells affects the inflammatory response in allergic asthma, but not lung mechanics, in female mice.

BACKGROUND: (Over-)expression of arginase may limit local availability of arginine for nitric oxide synthesis. We investigated the significance of arginase1 (ARG1) for the development of airway hyperresponsiveness (AHR) and lung inflammation in female mice with ovalbumin (OVA)-induced allergic asthma. METHODS: Arg1 was ablated in the lung by crossing Arg1 fl/fl and Tie2Cre tg/- mice. OVA sensitization and challenge were conducted, and AHR to methacholine was determined using the Flexivent system. Changes in gene expression, chemokine and cytokine secretion, plasma IgE, and lung histology were quantified using RT-qPCR, ELISA, and immunohistochemistry, respectively. RESULTS: Arg1 ablation had no influence on the development of OVA-induced AHR, but attenuated OVA-induced increases in expression of Arg2 and Nos2, Slc7a1, Slc7a2, and Slc7a7 (arginine transporters), Il4, Il5 and Il13 (TH2-type cytokines), Ccl2 and Ccl11 (chemokines), Ifng (TH1-type cytokine), Clca3 and Muc5ac (goblet cell markers), and OVA-specific IgE. Pulmonary IL-10 protein content increased, but IL-4, IL-5, IL-13, TNFα and IFNγ content, and lung histopathology, were not affected. Arg1 elimination also decreased number and tightness of correlations between adaptive changes in lung function and inflammatory parameters in OVA/OVA-treated female mice. OVA/OVA-treated female mice mounted a higher OVA-IgE response than males, but the correlation between lung function and inflammation was lower. Arg1-deficient OVA/OVA-treated females differed from males in a more pronounced decline of arginine-metabolizing and -transporting genes, higher plasma arginine levels, a smaller OVA-specific IgE response, and no improvement of peripheral lung function. CONCLUSION: Complete ablation of Arg1 in the lung affects mRNA abundance of arginine-transporting and -metabolizing genes, and pro-inflammatory genes, but not methacholine responsiveness or accumulation of inflammatory cells.

Pharmacologic or Genetic Targeting of Glutamine Synthetase Skews Macrophages toward an M1-like Phenotype and Inhibits Tumor Metastasis.

Glutamine-synthetase (GS), the glutamine-synthesizing enzyme from glutamate, controls important events, including the release of inflammatory mediators, mammalian target of rapamycin (mTOR) activation, and autophagy. However, its role in macrophages remains elusive. We report that pharmacologic inhibition of GS skews M2-polarized macrophages toward the M1-like phenotype, characterized by reduced intracellular glutamine and increased succinate with enhanced glucose flux through glycolysis, which could be partly related to HIF1α activation. As a result of these metabolic changes and HIF1α accumulation, GS-inhibited macrophages display an increased capacity to induce T cell recruitment, reduced T cell suppressive potential, and an impaired ability to foster endothelial cell branching or cancer cell motility. Genetic deletion of macrophagic GS in tumor-bearing mice promotes tumor vessel pruning, vascular normalization, accumulation of cytotoxic T cells, and metastasis inhibition. These data identify GS activity as mediator of the proangiogenic, immunosuppressive, and pro-metastatic function of M2-like macrophages and highlight the possibility of targeting this enzyme in the treatment of cancer metastasis.

Effect of Hyperglycemia on Gene Expression during Early Organogenesis in Mice.

BACKGROUND: Cardiovascular and neural malformations are common sequels of diabetic pregnancies, but the underlying molecular mechanisms remain unknown. We hypothesized that maternal hyperglycemia would affect the embryos most shortly after the glucose-sensitive time window at embryonic day (ED) 7.5 in mice. METHODS: Mice were made diabetic with streptozotocin, treated with slow-release insulin implants and mated. Pregnancy aggravated hyperglycemia. Gene expression profiles were determined in ED8.5 and ED9.5 embryos from diabetic and control mice using Serial Analysis of Gene Expression and deep sequencing. RESULTS: Maternal hyperglycemia induced differential regulation of 1,024 and 2,148 unique functional genes on ED8.5 and ED9.5, respectively, mostly in downward direction. Pathway analysis showed that ED8.5 embryos suffered mainly from impaired cell proliferation, and ED9.5 embryos from impaired cytoskeletal remodeling and oxidative phosphorylation (all P ≤ E-5). A query of the Mouse Genome Database showed that 20-25% of the differentially expressed genes were caused by cardiovascular and/or neural malformations, if deficient. Despite high glucose levels in embryos with maternal hyperglycemia and a ~150-fold higher rate of ATP production from glycolysis than from oxidative phosphorylation on ED9.5, ATP production from both glycolysis and oxidative phosphorylation was reduced to ~70% of controls, implying a shortage of energy production in hyperglycemic embryos. CONCLUSION: Maternal hyperglycemia suppressed cell proliferation during gastrulation and cytoskeletal remodeling during early organogenesis. 20-25% of the genes that were differentially regulated by hyperglycemia were associated with relevant congenital malformations. Unexpectedly, maternal hyperglycemia also endangered the energy supply of the embryo by suppressing its glycolytic capacity.

3D Topography of the Young Adult Anal Sphincter Complex Reconstructed from Undeformed Serial Anatomical Sections.

BACKGROUND: Pelvic-floor anatomy is usually studied by artifact-prone dissection or imaging, which requires prior anatomical knowledge. We used the serial-section approach to settle contentious issues and an interactive 3D-pdf to make the results widely accessible. METHOD: 3D reconstructions of undeformed thin serial anatomical sections of 4 females and 2 males (21-35y) of the Chinese Visible Human database. FINDINGS: Based on tendinous septa and muscle-fiber orientation as segmentation guides, the anal-sphincter complex (ASC) comprised the subcutaneous external anal sphincter (EAS) and the U-shaped puborectal muscle, a part of the levator ani muscle (LAM). The anococcygeal ligament fixed the EAS to the coccygeal bone. The puborectal-muscle loops, which define the levator hiatus, passed around the anorectal junction and inserted anteriorly on the perineal body and pubic bone. The LAM had a common anterior attachment to the pubic bone, but separated posteriorly into puborectal and "pubovisceral" muscles. This pubovisceral muscle was bilayered: its internal layer attached to the conjoint longitudinal muscle of the rectum and the rectococcygeal fascia, while its outer, patchy layer reinforced the inner layer. ASC contraction makes the ano-rectal bend more acute and lifts the pelvic floor. Extensions of the rectal longitudinal smooth muscle to the coccygeal bone (rectococcygeal muscle), perineal body (rectoperineal muscle), and endopelvic fascia (conjoint longitudinal and pubovisceral muscles) formed a "diaphragm" at the inferior boundary of the mesorectum that suspended the anorectal junction. Its contraction should straighten the anorectal bend. CONCLUSION: The serial-section approach settled contentious topographic issues of the pelvic floor. We propose that the ASC is involved in continence and the rectal diaphragm in defecation.

FOXO1 activates glutamine synthetase gene in mouse skeletal muscles through a region downstream of 3'-UTR: possible contribution to ammonia detoxification.

Skeletal muscle is a reservoir of energy in the form of protein, which is degraded under catabolic conditions, resulting in the formation of amino acids and ammonia as a byproduct. The expression of FOXO1, a forkhead-type transcription factor, increases during starvation and exercise. In agreement, transgenic FOXO1-Tg mice that overexpress FOXO1 in skeletal muscle exhibit muscle atrophy. The aim of this study was to examine the role of FOXO1 in amino acid metabolism. The mRNA and protein expressions of glutamine synthetase (GS) were increased in skeletal muscle of FOXO1-Tg mice. Fasting induced FOXO1 and GS expression in wild-type mice but hardly increased GS expression in muscle-specific FOXO1 knockout (FOXO1-KO) mice. Activation of FOXO1 also increased GS mRNA and protein expression in C2C12 myoblasts. Using a transient transfection reporter assay, we observed that FOXO1 activated the GS reporter construct. Mutation of a putative FOXO1-binding consensus sequence in the downstream genomic region of GS decreased basal and FOXO1-dependent reporter activity significantly. A chromatin immunoprecipitation assay showed that FOXO1 was recruited to the 3' region of GS in C2C12 myoblasts. These results suggest that FOXO1 directly upregulates GS expression. GS is considered to mediate ammonia clearance in skeletal muscle. In agreement, an intravenous ammonia challenge increased blood ammonia concentrations to a twofold higher level in FOXO1-KO than in wild-type mice, demonstrating that the capacity for ammonia disposal correlated inversely with the expression of GS in muscle. These data indicate that FOXO1 plays a role in amino acid metabolism during protein degradation in skeletal muscle.

Arginase-1 deficiency regulates arginine concentrations and NOS2-mediated NO production during endotoxemia.

RATIONALE AND OBJECTIVE: Arginase-1 is an important component of the intricate mechanism regulating arginine availability during immune responses and nitric oxide synthase (NOS) activity. In this study Arg1(fl/fl)/Tie2-Cre(tg/-) mice were developed to investigate the effect of arginase-1 related arginine depletion on NOS2- and NOS3-dependent NO production and jejunal microcirculation under resting and endotoxemic conditions, in mice lacking arginase-1 in endothelial and hematopoietic cells. METHODS AND RESULTS: Arginase-1-deficient mice as compared with control mice exhibited higher plasma arginine concentration concomitant with enhanced NO production in endothelial cells and jejunal tissue during endotoxemia. In parallel, impaired jejunal microcirculation was observed in endotoxemic conditions. Cultured bone-marrow-derived macrophages of arginase-1 deficient animals also presented a higher inflammatory response to endotoxin than control littermates. Since NOS2 competes with arginase for their common substrate arginine during endotoxemia, Nos2 deficient mice were also studied under endotoxemic conditions. As Nos2(-/-) macrophages showed an impaired inflammatory response to endotoxin compared to wild-type macrophages, NOS2 is potentially involved. A strongly reduced NO production in Arg1(fl/fl)/Tie2-Cre(tg/-) mice following infusion of the NOS2 inhibitor 1400W further implicated NOS2 in the enhanced capacity to produce NO production Arg1(fl/fl)/Tie2-Cre(tg/-) mice. CONCLUSIONS: Reduced arginase-1 activity in Arg1(fl/fl)/Tie2-Cre(tg/-) mice resulted in increased inflammatory response and NO production by NOS2, accompanied by a depressed microcirculatory flow during endotoxemia. Thus, arginase-1 deficiency facilitates a NOS2-mediated pro-inflammatory activity at the expense of NOS3-mediated endothelial relaxation.

Ablation of Arg1 in hematopoietic cells improves respiratory function of lung parenchyma, but not that of larger airways or inflammation in asthmatic mice.

Asthma is a chronic inflammatory disease of the small airways, with airway hyperresponsiveness (AHR) and inflammation as hallmarks. Recent studies suggest a role for arginase in asthma pathogenesis, possibly because arginine is the substrate for both arginase and NO synthase and because NO modulates bronchial tone and inflammation. Our objective was to investigate the importance of increased pulmonary arginase 1 expression on methacholine-induced AHR and lung inflammation in a mouse model of allergic asthma. Arginase 1 expression in the lung was ablated by crossing Arg1(fl/fl) with Tie2Cre(tg/-) mice. Mice were sensitized and then challenged with ovalbumin. Lung function was measured with the Flexivent. Adaptive changes in gene expression, chemokine and cytokine secretion, and lung histology were quantified with quantitative PCR, ELISA, and immunohistochemistry. Arg1 deficiency did not affect the allergic response in lungs and large-airway resistance, but it improved peripheral lung function (tissue elastance and resistance) and attenuated adaptive increases in mRNA expression of arginine-catabolizing enzymes Arg2 and Nos2, arginine transporters Slc7a1 and Slc7a7, chemokines Ccl2 and Ccl11, cytokines Tnfa and Ifng, mucus-associated epithelial markers Clca3 and Muc5ac, and lung content of IL-13 and CCL11. However, expression of Il4, Il5, Il10, and Il13 mRNA; lung content of IL-4, IL-5, IL-10, TNF-α, and IFN-γ protein; and lung pathology were not affected. Correlation analysis showed that Arg1 ablation disturbed the coordinated pulmonary response to ovalbumin challenges, suggesting arginine (metabolite) dependence of this response. Arg1 ablation in the lung improved peripheral lung function and affected arginine metabolism but had little effect on airway inflammation.

Fibroblast growth factor 21 is induced by endoplasmic reticulum stress.

Increased hepatic expression is held responsible for elevated serum levels of fibroblast growth factor 21 (FGF21) in non-alcoholic fatty liver disease (NAFLD) but the underlying molecular mechanism is unclear. In the present study we tested the postulate that the metabolic hormone FGF21 is regulated by endoplasmic reticulum (ER) stress, a condition that is observed in a number of diseases including NAFLD and results in activation of an adaptive response known as the unfolded protein response (UPR). ER stress stimuli were found to induce expression of Fgf21 mRNA in H4IIE hepatoma cells and in isolated rat hepatocytes. Moreover, intraperitoneal injection of the ER stressor tunicamycin induced hepatic Fgf21 expression in mice and resulted in marked elevation of serum FGF21 levels. The effect of ER stress on FGF21 expression could be mimicked by overexpression of ATF4, a transcriptional effector of the PERK-branch of the UPR. In silico analysis revealed the presence of two binding sites for ATF4 in the FGF21 promoter region. Combined disruption of these elements, abrogated FGF21 promoter activity induced by ER stress or ATF4 overexpression. These findings implicate the PERK/eIF2alpha/ATF4 cascade in ER stress regulation of FGF21. A consequence of this notion is that other intracellular stress signaling pathways that converge at eIF2alpha, can regulate FGF21 expression. Indeed, both nutrient (amino acid deprivation) and oxidative stress (arsenite) were found to induce Fgf21 expression in hepatoma cells and isolated rat hepatocytes. In conclusion, FGF21 expression is regulated by ER stress and additional intracellular stress signaling pathways. Our findings suggest that increased cellular stress in fatty livers may underlie the elevated FGF21 levels observed in patients with NAFLD.

E2F8 is essential for polyploidization in mammalian cells.

Polyploidization is observed in all mammalian species and is a characteristic feature of hepatocytes, but its molecular mechanism and biological significance are unknown. Hepatocyte polyploidization in rodents occurs through incomplete cytokinesis, starts after weaning and increases with age. Here, we show in mice that atypical E2F8 is induced after weaning and required for hepatocyte binucleation and polyploidization. A deficiency in E2f8 led to an increase in the expression level of E2F target genes promoting cytokinesis and thereby preventing polyploidization. In contrast, loss of E2f1 enhanced polyploidization and suppressed the polyploidization defect of hepatocytes deficient for atypical E2Fs. In addition, E2F8 and E2F1 were found on the same subset of target promoters. Contrary to the long-standing hypothesis that polyploidization indicates terminal differentiation and senescence, we show that prevention of polyploidization through inactivation of atypical E2Fs has, surprisingly, no impact on liver differentiation, zonation, metabolism and regeneration. Together, these results identify E2F8 as a repressor and E2F1 as an activator of a transcriptional network controlling polyploidization in mammalian cells.

Pivotal Advance: Arginase-1-independent polyamine production stimulates the expression of IL-4-induced alternatively activated macrophage markers while inhibiting LPS-induced expression of inflammatory genes.

In macrophages, basal polyamine (putrescine, spermidine, and spermine) levels are relatively low but are increased upon IL-4 stimulation. This Th2 cytokine induces Arg1 activity, which converts arginine into ornithine, and ornithine can be decarboxylated by ODC to produce putrescine, which is further converted into spermidine and spermine. Recently, we proposed polyamines as novel agents in IL-4-dependent E-cadherin regulation in AAMs. Here, we demonstrate for the first time that several, but not all, AAM markers depend on polyamines for their IL-4-induced gene and protein expression and that polyamine dependency of genes relies on the macrophage type. Remarkably, Arg1-deficient macrophages display rather enhanced IL-4-induced polyamine production, suggesting that an Arg1-independent polyamine synthesis pathway may operate in macrophages. On the other side of the macrophage activation spectrum, LPS-induced expression of several proinflammatory genes was increased significantly in polyamine-depleted CAMs. Overall, we propose Arg1 independently produced polyamines as novel regulators of the inflammatory status of the macrophage. Indeed, whereas polyamines are needed for IL-4-induced expression of several AAM mediators, they inhibit the LPS-mediated expression of proinflammatory genes in CAMs.

Possible roles of DLK1 in the Notch pathway during development and disease.

The Delta-Notch pathway is an evolutionarily conserved signaling pathway which controls a broad range of developmental processes including cell fate determination, terminal differentiation and proliferation. In mammals, four Notch receptors (NOTCH1-4) and five activating canonical ligands (JAGGED1, JAGGED2, DLL1, DLL3 and DLL4) have been described. The precise function of noncanonical Notch ligands remains unclear. Delta-like 1 homolog (DLK1), the best studied noncanonical Notch ligand, has been shown to act as an inhibitor of Notch signaling in vitro, but its function in vivo is poorly understood. In this review we summarize Notch signaling during development and highlight recent studies in DLK1expression that reveal new insights into its function.

Comparative study of thin sectional anatomical images from Chinese visible human data set and computed tomography images of superior mediastinum.

The structures of superior mediastinum and their spatial relationships are complex and difficult to master. This study aimed to compare visualization of the superior mediastinum based on computed tomography (CT) images and on the thin sections of the Chinese visible human (CVH) data set to provide a sectional anatomical basis for diagnostic imaging of superior mediastinal pathology. CVH sections of the mediastinum of a 35-year old male were compared with plain and enhanced CT images of a 45-year old male without apparent abnormalities in the upper chest. In addition, a three-dimensional model based on the CVH sections was compared with a model based on CT images. Although CT imaging is noninvasive and can be carried out in many individuals, its weakness is clearly the visualization of small soft tissue structures. In this respect, the sectional anatomical approach of the CVH images is complementary, as it visualizes these small soft tissue structures due to the higher resolution in the plain of sectioning and the color of the different structures in the section. Three-dimensional surface and volume rendering of reconstructions of the CVH data set can help medical students and less experienced thoracic surgeons to familiarize themselves with the topographic anatomy of the superior mediastinal structures and their spatial relationships, and thus with interpreting CT images of patients.

DLK1, a serum marker for hepatoblastoma in young infants.

Hepatoblastoma is a malignant pediatric liver tumor. The currently used diagnostic serum marker for hepatoblastoma, α-fetoprotein (AFP), is not always reliable in infants with hepatoblastoma, due to the physiologically elevated levels of AFP in this age group. In this report, we show that Delta-like 1 homolog (DLK1), a protein highly expressed during fetal development, but almost completely absent after birth, and an established liver-stem cell marker, is a new candidate serum marker of hepatoblastoma, especially in young infants.

The HepaRG cell line is suitable for bioartificial liver application.

For bioartificial liver application, cells should meet the following minimal requirements: ammonia elimination, drug metabolism and blood protein synthesis. Here we explore the suitability of HepaRG cells, a human cell line reported to differentiate into hepatocyte clusters and surrounding biliary epithelial-like cells at high density and after exposure to dimethyl sulfoxide (DMSO). The effect of carbamoyl-glutamate (CG), an activator of urea cycle enzyme carbamoylphosphate synthetase (CPS) was studied additionally. The effects of DMSO and/or CG were assessed in presence of (15)NH(4)Cl on HepaRG cells in monolayer. We tested hepatocyte-specific functions at transcript and biochemical level, cell damage parameters and performed immunostainings. Ureagenesis, ammonia/galactose elimination and albumin, glutamine synthetase and CPS transcript levels were higher in -DMSO than +DMSO cultures, probably due to a higher cell content and/or cluster-neighbouring regions contributing to their functionality. DMSO treatment increased cytochrome P450 (CYP) transcript levels and CYP3A4 activity, but also cell damage and repressed hepatic functionality in cluster-neighbouring regions. The levels of ammonia elimination, apolipoprotein A-1 production, and transcription of CYP3A4, CYP2B6 and albumin reached those of primary hepatocytes in either the + or -DMSO cultures. Preconditioning with CG increased conversion of (15)NH(4)Cl into (15)N-urea 4-fold only in -DMSO cultures. Hence, HepaRG cells show high metabolic and synthetic functionality in the absence of DMSO, however, their drug metabolism is only high in the presence of DMSO. An unparalleled broad hepatic functionality, suitable for bioartificial liver application, can be accomplished by combining CG treated -DMSO cultures with +DMSO cultures.

Hypoxia induces dilated cardiomyopathy in the chick embryo: mechanism, intervention, and long-term consequences.

BACKGROUND: Intrauterine growth restriction is associated with an increased future risk for developing cardiovascular diseases. Hypoxia in utero is a common clinical cause of fetal growth restriction. We have previously shown that chronic hypoxia alters cardiovascular development in chick embryos. The aim of this study was to further characterize cardiac disease in hypoxic chick embryos. METHODS: Chick embryos were exposed to hypoxia and cardiac structure was examined by histological methods one day prior to hatching (E20) and at adulthood. Cardiac function was assessed in vivo by echocardiography and ex vivo by contractility measurements in isolated heart muscle bundles and isolated cardiomyocytes. Chick embryos were exposed to vascular endothelial growth factor (VEGF) and its scavenger soluble VEGF receptor-1 (sFlt-1) to investigate the potential role of this hypoxia-regulated cytokine. PRINCIPAL FINDINGS: Growth restricted hypoxic chick embryos showed cardiomyopathy as evidenced by left ventricular (LV) dilatation, reduced ventricular wall mass and increased apoptosis. Hypoxic hearts displayed pump dysfunction with decreased LV ejection fractions, accompanied by signs of diastolic dysfunction. Cardiomyopathy caused by hypoxia persisted into adulthood. Hypoxic embryonic hearts showed increases in VEGF expression. Systemic administration of rhVEGF(165) to normoxic chick embryos resulted in LV dilatation and a dose-dependent loss of LV wall mass. Lowering VEGF levels in hypoxic embryonic chick hearts by systemic administration of sFlt-1 yielded an almost complete normalization of the phenotype. CONCLUSIONS/SIGNIFICANCE: Our data show that hypoxia causes a decreased cardiac performance and cardiomyopathy in chick embryos, involving a significant VEGF-mediated component. This cardiomyopathy persists into adulthood.