Biochemical characterization of a virus-induced osteosarcoma-like osseous lesionin vitro

Summary Chondroprogenitor cells present in the apical and lateral parts of the mandibular condyle from neonatal mice differentiate towards the osteoblastic lineage and form bone within 7 days in culture. Infection of condylar explants with the FBR osteosarcoma virus (FBR MSV) results in the transformation of cells in the progenitor zone, previously identified as the target for the virus [1], and the formation of a transplantable osteosarcoma-like lesion. Morphiological and biochemical changes in this sytem were investigated in the course of tumor development. Virus infection was followed by a significant increase in cell density and³H-thymidine incorporation within the progenitor zone at the early stage of culture. In later stages, cell density and³H-thymidine incorporation were lower than in control tissue. The³H-thymidine labeling index gave similar results in infected and control tissues until day 7. Then, a significantly higher labeling index was found in the progenitor zone of infected condyles. At this stage, the proliferative effect of the virus even affected the cartilagenous core of the tissue. Quantitative alkaline phosphatase activity increased between day 3 and day 7 and was particularly high in the zone of infected cells. In addition, infected tissues consistently revealed a higher uptake of⁴⁵Ca, and deposition of the radioisotope along irregularly formed bone trabecules in the transformed tissue. The results suggest that there is an enhancement of tissue maturation following infection with the FBR osteosarcoma virus. Although biochemical investigations of whole condyles showed few diffrences in the total values of alkaline phosphatase activity,³H-thymidine incorporation, DNA content, and⁴⁵Ca uptake, the histochemical assays revealed clear differences in the distributional pattern of these parameters within infected and control condyles.     

Source apportionment of methane escaping the subsea permafrost system in the outer Eurasian Arctic Shelf

The East Siberian Arctic Shelf holds large amounts of inundated carbon and methane (CH₄). Holocene warming by overlying seawater, recently fortified by anthropogenic warming, has caused thawing of the underlying subsea permafrost. Despite extensive observations of elevated seawater CH₄ in the past decades, relative contributions from different subsea compartments such as early diagenesis, subsea permafrost, methane hydrates, and underlying thermogenic/ free gas to these methane releases remain elusive. Dissolved methane concentrations observed in the Laptev Sea ranged from 3 to 1,500 nM (median 151 nM; oversaturation by ∼3,800%). Methane stable isotopic composition showed strong vertical and horizontal gradients with source signatures for two seepage areas of δ¹³C-CH₄ = (−42.6 ± 0.5)/(−55.0 ± 0.5) ‰ and δD-CH₄ = (−136.8 ± 8.0)/(−158.1 ± 5.5) ‰, suggesting a thermogenic/natural gas source. Increasingly enriched δ¹³C-CH₄ and δD-CH₄ at distance from the seeps indicated methane oxidation. The Δ¹⁴C-CH₄ signal was strongly depleted (i.e., old) near the seeps (−993 ± 19/−1050 ± 89‰). Hence, all three isotope systems are consistent with methane release from an old, deep, and likely thermogenic pool to the outer Laptev Sea. This knowledge of what subsea sources are contributing to the observed methane release is a prerequisite to predictions on how these emissions will increase over coming decades and centuries.

The East Siberian Arctic Shelf (ESAS) is the world’s largest and shallowest shelf sea system, formed through inundation of northeast Siberia during sea level transgression in the early Holocene. The ESAS holds substantial but poorly constrained amounts of organic carbon and methane (CH₄). These carbon/methane stores are contained in unknown partitions as gas hydrates, unfrozen sediment, subsea permafrost, gas pockets within and below the subsea permafrost, and as underlying thermogenic gas (1–3). Methane release to the atmosphere from these compartments could potentially have significant effects on the global climate (4, 5), yet there are large uncertainties regarding the size and the vulnerability toward remobilization of these inaccessible and elusive subsea carbon/methane pools. Conceptual development and modeling have predicted that warming of the ESAS system by a combination of geothermal heat and climate-driven Holocene heat flux from overlying seawater, recently further enhanced by Anthropocene warming, may lead to thawing of subsea permafrost (6, 7). Subsea permafrost drilling in the Laptev Sea, in part at the same sites as 30 y ago, has recently confirmed that the subsea permafrost has indeed come near the point of thawing (8). In addition to mobilization of the carbon/methane stored within the subsea permafrost, its degradation can also lead to the formation of pathways for gaseous methane from underlying reservoirs, allowing further methane release to the overlying water column (3, 9).Near-annual ship-based expeditions to the ESAS over the past two decades have documented widespread seep locations with extensive methane releases to the water column (3, 10). Methane levels are often found to be 10 to 100 times higher than the atmospheric equilibrium and are particularly elevated in areas of strong ebullition from subsea gas seeps (“methane hotspots”). Similarly, elevated dissolved methane concentrations in bottom waters appear to be spatially related to the thermal state of subsea permafrost as deduced from modeling results and/or geophysical surveys (7, 9). Currently, we lack critical knowledge on the quantitative or even relative contributions of the different subsea pools to the observed methane release, a prerequisite for robust predictions on how these releases will develop. An important distinction needs to be made between pools that release methane gradually, such as methane produced microbially in shallow sediments during early diagenesis or in thawing subsea permafrost, versus pools with preformed methane that may release more abruptly once pathways are available, such as from disintegrating methane hydrates and pools of thermogenic (natural) gas below the subsea permafrost. Multidimensional isotope analysis offers a useful means to disentangle the relative importance of these different subsea sources of methane to the ESAS: Stable isotope data (δ¹³C-CH₄ and δD-CH₄) provide useful information on methane formation and removal pathways, and the radiocarbon content of methane (Δ¹⁴C-CH₄) helps to determine the age and methane source reservoir (see SI Appendix, text S1 for details on these isotope systematics and typical isotopic signatures for the ESAS subsea system).Here, we present triple-isotope–based source apportionment of methane conducted as part of the Swedish–Russian–US investigation of carbon–climate–cryosphere interactions in the East Siberian Arctic Ocean (SWERUS-C3) program. To this end, the distribution of dissolved methane, its stable carbon and hydrogen isotope composition, as well as natural radiocarbon abundance signature, were investigated with a focus on the isotopic fingerprint of methane escaping the seabed to pinpoint the subsea sources of elevated methane in the outer Laptev Sea.     

Prophylaxis of ventricular fibrillation after acute experimental coronary occlusion by chronic beta-adrenoceptor blockade with atenolol

Acute occlusions of the left circumflex coronary artery were performed in open-chest dogs. A control group (n = 19) was compared with three groups (total n = 17) pretreated once daily with different doses of the cardioselective beta-blocking drug atenolol (ICI 66 082) given by mouth for 5 days. Only animals without coronary collateral vessels were examined, having a mortality rate of 100% in the control group. Arrhythmias and ventricular fibrillation during the first 30 min after coronary occlusion showed a biphasic distribution in time (phase 1a and 1b). A lower degree of beta-adrenoceptor blockade reduced the incidence of arrhythmias and ventricular fibrillation in phase 1a, but fibrillation occurred in all animals during phase 1b. A higher dose of the beta-blocking drug protected the animals from ventricular fibrillation, and arrhythmias in phase 1a were greatly reduced. At all times the ventricular fibrillation threshold in the group pretreated with atenolol was significantly higher than in the control group. In both groups a significant decrease in ventricular fibrillation threshold was found only during phase 1a. The greater sensitivity of phase 1a arrhythmias to beta-blockade and the lack of a decrease in ventricular fibrillation threshold during phase 1b might indicate differences in the genesis of arrhythmias and fibrillation in phases 1a and 1b.     

Lifespan extension of Drosophila melanogaster through hormesis by repeated mild heat stress

We assessed the impact of repeated episodes of a mild heat stress on lifespan, fecundity, heat stress resistance and Hsp70 expression in Drosophila melanogaster. There was a significant increase in lifespan of females repeatedly exposed to a mild heat stress when measured in both a pair and a group situation. There was no effect on fecundity when the flies were first exposed to the mild heat stress at an age later than 3 days old, but when it did occur on day 3, there was a significant effect on cumulative fecundity levels over 18 days. The negative fitness effect appears to be the result of a direct cessation or reduction of oviposition during the first bout of stress exposure, and is influenced by the age at which this first exposure occurs. The mild heat stress had no impacton egg viability. The mild heat stress exposures increased resistance to potentially lethal heat stress and levels of Hsp70 expression in heat-exposed flies were higher than those in controls. This revised version was published online in July 2006 with corrections to the Cover Date.     

Spatial heterogeneity of myocardial perfusion predicts local potassium channel expression and action potential duration

AIMS: In the heart, there is not only a transmural gradient of left ventricular perfusion and action potential duration (APD), but also spatial heterogeneity within each myocardial layer, where local blood flow and energy turnover vary more than three-fold between individual regions. We analysed at high spatial resolution whether a corresponding heterogeneity also extends to ion channel gene expression and APD. METHODS AND RESULTS: In the open-chest beagle dog, left ventricular 300 microL samples of very low or high flow were identified by radioactive microspheres and expression levels determined by quantitative PCR. The distribution of epicardial APD was assessed by mapping local activation repolarization intervals (ARIs) and QT interval (QT). ERG, the potassium channel mediating IKr, and KChIP2, the interacting protein modulating Ito, were increased in Low flow (3.3- and 2.5-fold, P < 0.001 and <0.05, respectively; n = 6 hearts, 30-31 samples each) as compared with High flow areas. This suggested enhanced repolarizing currents in Low flow areas, and in consequence, mathematical model analysis predicted a shorter local APD upon enhanced ERG and IKr. Epicardial mapping revealed a patchy, temporally stable APD pattern (n = 11), a small apico-basal gradient and an APD prolongation induced by the ERG blocker dofetilide predominantly in areas of short basal ARI or QT, respectively (n = 9). In addition, in Short QT areas, ERG expression was three-fold increased (P < 0.05, n = 4). CONCLUSION: The spatial pattern of perfusion is matched by the novel patterns of K+ channel expression and APD. Whenever this newly recognized intramural dispersion of APD increases, it may contribute to arrhythmogenesis.     

Fatty acid composition of liver triglycerides in various stages of fat deposition in the parenchyma

Summary In 30 diabetic inpatients the fatty acid pattern of triglycerides in parenchymal liver cells was studied by gas-liquid chromatography. With increasing size of fat droplets, a significant increase in the proportion of palmitic and oleic acid was observed as well as a significant fall of arachidonic (C 20: 4) and eicosapentaenoic acid (C 20: 5).     

Quantitative proteomics and phosphoproteomics on serial tumor biopsies from a sorafenib-treated HCC patient

Compensatory signaling pathways in tumors confer resistance to targeted therapy, but the pathways and their mechanisms of activation remain largely unknown. We describe a procedure for quantitative proteomics and phosphoproteomics on snap-frozen biopsies of hepatocellular carcinoma (HCC) and matched nontumor liver tissue. We applied this procedure to monitor signaling pathways in serial biopsies taken from an HCC patient before and during treatment with the multikinase inhibitor sorafenib. At diagnosis, the patient had an advanced HCC. At the time of the second biopsy, abdominal imaging revealed progressive disease despite sorafenib treatment. Sorafenib was confirmed to inhibit MAPK signaling in the tumor, as measured by reduced ribosomal protein S6 kinase phosphorylation. Hierarchical clustering and enrichment analysis revealed pathways broadly implicated in tumor progression and resistance, such as epithelial-to-mesenchymal transition and cell adhesion pathways. Thus, we describe a protocol for quantitative analysis of oncogenic pathways in HCC biopsies and obtained first insights into the effect of sorafenib in vivo. This protocol will allow elucidation of mechanisms of resistance and enable precision medicine.Hepatocellular carcinoma (HCC) is a global health concern with an estimated 750,000 new cases per year (1). In more than 80% of cases, HCC arises in a setting of liver cirrhosis mainly of alcoholic or viral origin (2). The prognosis for HCC patients is poor, with less than 30% qualifying for curative treatments such as tumor resection or liver transplantation (2). Median survival time of patients that cannot be treated surgically is less than 1 y. Sorafenib is the only approved targeted therapy for HCC, prolonging median patient survival by ∼3 mo (3). Sorafenib is a multikinase inhibitor of Raf (B and C), vascular endothelial growth factor receptor (VEGFR), and platelet-derived growth factor receptor (PDGFR) (4), which presumably inhibits not only tumor cells but also endothelial cells responsible for tumor vascularization.Resistance to a targeted cancer drug can be intrinsic or adaptive (5). Sorafenib is largely cytostatic (6), suggesting that intrinsic resistance is more common in tumors, although some reports describe tumor shrinkage upon sorafenib treatment (7). Studies involving HCC cell lines or immunohistochemical staining of tumor sections revealed that sorafenib resistance correlates with the up-regulation of several signaling pathways, including the mammalian target of rapamycin (mTOR) pathway as assayed by S6 S235/236 (8) and Akt S473 phosphorylation (9). Other potential resistance mechanisms involve epithelial-to-mesenchymal transition (EMT) and autophagy (10, 11). However, the molecular mechanisms of sorafenib resistance in patients are largely unknown. Understanding the pathways that confer intrinsic or adaptive resistance would allow precision medicine and increase treatment efficacy.Proteomic analysis allows the identification of drug targets for cancer treatment and biomarkers for cancer classification or recurrence. In particular, MS is a powerful tool for resolving the complexity of cancer signaling pathways. With regard to HCC, qualitative proteomics has been performed on resected tumor material (12), laser-capture microdissected material from tissue sections (13, 14), and primary hepatocytes or serum derived from patients (15, 16). These studies (17, 18) identified HCC biomarkers such as glutamine synthetase and heat shock protein 70 (Hsp70) that are currently in use for diagnosis (19, 20). Quantitative proteomics has been performed on HCC resected tissue and serum (21, 22). Recently, proteomics has been performed on tumor biopsies of renal cell carcinoma patients (23). Several studies also have described phosphoproteomic analyses of resected HCC or other cancer material (24–26), in some cases quantifying up to 8,000 phosphorylated sites (hereafter referred to as “phosphosites”) starting with 2 mg of protein (18, 27–30). However, to our knowledge, quantitative proteomics and phosphoproteomics, hereafter collectively referred to as “(phospho)proteomics,” have yet to be performed on tumor biopsies, possibly because biopsy material is nonrenewable and typically provides only a very small amount of protein. Importantly, quantitative (phospho)proteomics on serial biopsies taken before and during treatment has not been described. We note that although a biopsy procedure generates less material than a resection, it has the important advantage of capturing normally dynamic properties of a tumor, such as the phosphorylation status of signaling pathways. Biopsies are immediately snap-frozen upon removal from the patient and, unlike resected tissue, are obtained without causing ischemia or hypoglycemia in the collected tissue. Needle biopsies are taken routinely to diagnose and stage the disease. Another important consideration is a method to perform quantitative (phospho)proteomics, such as super-SILAC (“SILAC” is an acronym for “stable isotope labeling of amino acids in cell culture”), that allows direct comparison of biopsies obtained at different times or from different patients (31).We describe quantitative (phospho)proteomic analyses of needle biopsies of HCC and matched nontumor tissue from a human patient. These analyses provide a global snapshot of signaling pathways in the biopsy material. Analyzing serial biopsies taken from a patient before and during therapy, we measured differences in signaling pathways between tumor and matched nontumor control tissue and the changes in these signaling pathways upon sorafenib treatment. Our findings provide insight into mechanisms of tumor progression and resistance to cancer therapy.     

Anal Ultra Slow Waves (A Smooth Muscle Phenomenon Associated with Dyschezia)

Anal ultra slow waves (USWs) have been describedin a variety of anorectal disorders, all of which may beassociated with constipation. We investigated whetherthey represent a marker for dyschezia and whether their occurrence can be modified.Manometric and endosonographic studies were performed in25 patients with dyschezia, in 25 age- and sex-matchedcontrols, and in an equal number of patients with hemorrhoids. Patients exhibiting ultra slowwaves were repeatedly studied with and without localadministration of isosorbide dinitrate. In addition, wedetermined whether stimulatory maneuvers modify the occurrence of USWs. Anal USWs were persistentlyfound in 56% of patients with dyschezia, in 8% ofpatients with hemorrhoids, and in none of the healthycontrols. They were stimulated by anal squeeze and completely abolished by local administration ofisosorbide dinitrate. Ultra slow waves always occurredin conjunction with an increase in anal resting pressureand were tightly associated with a fluctuation in slow wave amplitude. Anal sphinctermorphology was similar in patients with dyschezia and incontrols. We conclude that anal USWs occur mostfrequently in patients with dyschezia and indicatesmooth muscle dysfunction. Treatment directed atabolishing this motor phenomenon may represent a novelapproach to the management of patients withdyschezia.