Highly pathogenic avian influenza viruses do not inhibit interferon synthesis in infected chickens but can override the interferon-induced antiviral state

From infection studies with cultured chicken cells and experimental mammalian hosts, it is well known that influenza viruses use the nonstructural protein 1 (NS1) to suppress the synthesis of interferon (IFN). However, our current knowledge regarding the in vivo role of virus-encoded NS1 in chickens is much more limited. Here, we report that highly pathogenic avian influenza viruses of subtypes H5N1 and H7N7 lacking fully functional NS1 genes were attenuated in 5-week-old chickens. Surprisingly, in diseased birds infected with NS1 mutants, the IFN levels were not higher than in diseased birds infected with wild-type virus, suggesting that NS1 cannot suppress IFN gene expression in at least one cell population of infected chickens that produces large amounts of the cytokine in vivo. To address the question of why influenza viruses are highly pathogenic in chickens although they strongly activate the innate immune system, we determined whether recombinant chicken alpha interferon (IFN-α) can inhibit the growth of highly pathogenic avian influenza viruses in cultured chicken cells and whether it can ameliorate virus-induced disease in 5-week-old birds. We found that IFN treatment failed to confer substantial protection against challenge with highly pathogenic viruses, although it was effective against viruses with low pathogenic potential. Taken together, our data demonstrate that preventing the synthesis of IFN is not the primary role of the viral NS1 protein during infection of chickens. Our results further suggest that virus-induced IFN does not contribute substantially to resistance of chickens against highly pathogenic influenza viruses.     

Localization of endothelin receptors in bleomycin-induced pulmonary fibrosis in the rat

Pulmonary fibrosis is characterized by excessive extracellular matrix deposition with concomitant loss of gas exchange units, and endothelin-1 (ET-1) has been implicated in its pathogenesis. Increased levels of ET-1 from tissues and bronchoalveolar lavage have been reported in patients with pulmonary fibrosis and in animal models after intratracheal bleomycin. We characterized the cellular distribution of alveolar ET receptors by immunohistochemistry in bleomycin-induced pulmonary fibrosis in the rat and determined the regulation by bleomycin of ET receptor mRNA expression in isolated alveolar macrophages and rat lung fibroblasts. We found significant increases in the numbers of fibroblasts and macrophages at day 7 compared to day 28 and control animals. ET_B receptor immunoreactivity was observed on fibroblasts and invading monocytes. Isolated fibroblasts expressed both ET_A and ET_B receptor mRNA, and ET_A receptor mRNA was upregulated by bleomycin. Isolated resident alveolar macrophages expressed neither ET_A nor ET_B receptor mRNA which were also not induced by bleomycin. We conclude that, while ET_B receptor stimulation of fibroblasts and monocytes recruited during bleomycin-induced lung injury exerts antagonistic effects on fibroblast collagen synthesis, the observed increase in the number of fibroblasts in vivo and upregulation of fibroblast ET_A receptor mRNA by bleomycin in vitro point to a predominance of the profibrotic effects of ET receptor engagement.     

Functional Characterization of the Microbial Community in Geothermally Heated Marine Sediments

The microbial population of geothermally heated sediments in a shallow bay of Vulcano Island (Italy) was characterized with respect to metabolic activities and the putatively catalyzing hyperthermophiles. Site-specific anoxic culturing media, most of which were amended with combinations of electron donors (glucose or carboxylic acids) and acceptors (sulfate), were used for selective enrichment of metabolically defined subpopulations. The mostly archaeal chemoautotrophs produced formate at rates of 3.25 and 0.46 fmol cell⁻¹ day⁻¹ with and without sulfate, respectively. The glucose fermenting heterotrophs produced acetate (18 fmol cell⁻¹ day⁻¹) and lactate (2.6 fmol cell⁻¹ day⁻¹) and were identified as predominantly Thermus sp. and coccoid archaea. These archaeal cells also metabolized lactate (5.6 fmol cell⁻¹ day⁻¹), but neither formate nor acetate. The heterotrophic culture enriched on formate/acetate/propionate/sulfate utilized mainly formate (27 fmol cell⁻¹ day⁻¹) and lactate (89–195 fmol cell⁻¹ day⁻¹), and consumed sulfate (38–68 fmol cell⁻¹ day⁻¹). These formate or lactate consuming sulfate reducers were dominated by Archaeoglobales (7% in situ) and unidentified Archaea. The in situ benthic community comprised 15% Crenarchaeota, a significant group only in the autotrophic cultures, and 3% Thermus sp., the putatively predominant group involved in fermentative metabolism. The role of Thermoccales (4% in situ) remained undisclosed in our experiments. This first comprehensive data set established plausible links between several groups of hyperthermophiles in shallow marine hydrothermal systems, their metabolic function within the benthic microbial community, and biogeochemical turnover rates.     

ACSL4-dependent ferroptosis does not represent a tumor-suppressive mechanism but ACSL4 rather promotes liver cancer progression

Ferroptosis is a novel type of programmed cell death that differs from apoptosis in that it involves iron-dependent peroxidation of membrane phospholipids. Its role in a variety of human disorders, including cancer has been hypothesized in recent years. While it may function as an endogenous tumor suppressor in a variety of cancers, its role during initiation and progression of liver cancer, particularly hepatocellular carcinoma (HCC), is yet unknown. Because HCC is most commonly found in chronically injured livers, we utilized two well-established mouse models of chronic injury-dependent HCC formation: Treatment with streptozotocin and high-fat diet as metabolic injury model, as well as treatment with diethylnitrosamine and carbon tetrachloride as toxic injury model. We used mice with hepatocyte-specific deletion of Acsl4, a key mediator of ferroptosis, to explore the significance of ferroptotic cell death in hepatocytes, the cell type of origin for HCC. Surprisingly, preventing ferroptotic cell death in hepatocytes by deleting Acsl4 does not increase the formation of HCC. Furthermore, Acsl4-deficient livers display less fibrosis and proliferation, especially in the HCC model of toxic damage. Intriguingly, in this model, the absence of ACSL4-dependent processes such as ferroptosis significantly slow down the growth of HCC. These findings suggest that during HCC formation in a chronically injured liver, ferroptotic cell death is not an endogenous tumor-suppressive mechanism. Instead, we find that ACSL4-dependent processes have an unanticipated cancer-promoting effect during HCC formation, which is most likely due to aggravated liver damage as demonstrated by increased hepatic fibrosis. Previous studies suggested that ferroptosis might have beneficial effects for patients during HCC therapy. As a result, during HCC progression and therapy, ferroptosis may have both cancer-promoting and cancer-inhibitory effects, respectively.Subject terms: Cancer models, Cancer genetics, Cell death     

Adsorption of GST-PI3Kγ at the Air-Buffer Interface and at Substrate and Nonsubstrate Phospholipid Monolayers

The recruitment of phosphoinositide 3-kinase γ (PI3Kγ) to the cell membrane is a crucial requirement for the initiation of inflammation cascades by second-messenger production. In addition to identifying other regulation pathways, it has been found that PI3Kγ is able to bind phospholipids directly. In this study, the adsorption behavior of glutathione S-transferase (GST)-PI3Kγ to nonsubstrate model phospholipids, as well as to commercially available substrate inositol phospholipids (phosphoinositides), was investigated by use of infrared reflection-absorption spectroscopy (IRRAS). The nonsubstrate phospholipid monolayers also yielded important information about structural requirements for protein adsorption. The enzyme did not interact with condensed zwitterionic or anionic monolayers; however, it could penetrate into uncompressed fluid monolayers. Compression to values above its equilibrium pressure led to a squeezing out and desorption of the protein. Protein affinity for the monolayer surface increased considerably when the lipid had an anionic headgroup and contained an arachidonoyl fatty acyl chain in sn-2 position. Similar results on a much higher level were observed with substrate phosphoinositides. No structural response of GST-PI3Kγ to lipid interaction was detected by IRRAS. On the other hand, protein adsorption caused a condensing effect in phosphoinositide monolayers. In addition, the protein reduced the charge density at the interface probably by shifting the pK values of the phosphate groups attached to the inositol headgroups. Because of their strongly polar headgroups, an interaction of the inositides with the water molecules of the subphase can be expected. This interaction is disturbed by protein adsorption, causing the ionization state of the phosphates to change.     

Comparison of fecal preservation and extraction methods for steroid hormone metabolite analysis in wild crested macaques

Since the non-invasive field endocrinology techniques were developed, several fecal preservation and extraction methods have been established for a variety of species. However, direct adaptation of methods from previous studies for use in crested macaques should be taken with caution. We conducted an experiment to assess the accuracy and stability of fecal estrogen metabolite (E1C) and glucocorticoid metabolite (GCM) concentrations in response to several preservation parameters: (1) time lag between sample collection and fecal preservation; (2) long-term storage of fecal samples in 80% methanol (MeOH) at ambient temperature; (3) different degrees of feces drying temperature using a conventional oven; and (4) different fecal preservation techniques (i.e., freeze-drying, oven-drying, and field-friendly extraction method) and extraction solvents (methanol, ethanol, and commercial alcohol). The study used fecal samples collected from crested macaques (Macaca nigra) living in the Tangkoko Reserve, North Sulawesi, Indonesia. Samples were assayed using validated E1C and GCM enzyme immunoassays. Concentrations of E1C and GCM in unprocessed feces stored at ambient temperature remained stable for up to 8 h of storage after which concentrations of both E1C and GCM changed significantly compared to controls extracted at time 0. Long-term storage in 80% MeOH at ambient temperature affected hormone concentrations significantly with concentrations of both E1C and GCM increasing after 6 and 4 months of storage, respectively. Drying fecal samples using a conventional oven at 50, 70, and 90 °C did not affect the E1C concentrations, but led to a significant decline for GCM concentrations in samples dried at 90 °C. Different fecal preservation techniques and extraction solvents provided similar results for both E1C and GCM concentrations. Our results confirm previous studies that prior to application of fecal hormone analysis in a new species, several preservation parameters should be evaluated for their effects on hormone metabolite stability. The results also provide several options for fecal preservation, extraction, and storage methods that can be selected depending on the condition of the field site and laboratory.     

Environmental Effects over the First 2½ Rotation Periods of a Fertilised Poplar Short Rotation Coppice

A short rotation coppice (SRC) with poplar was established in a randomised fertilisation experiment on sandy loam soil in Potsdam (Northeast Germany). The main objective of this study was to assess if negative environmental effects as nitrogen leaching and greenhouse gas emissions are enhanced by mineral nitrogen (N) fertiliser applied to poplar at rates of 0, 50 and 75 kg N ha^?1 year^?1 and how these effects are influenced by tree age with increasing number of rotation periods and cycles of organic matter decomposition and tree growth after each harvesting event. Between 2008 and 2012, the leaching of nitrate (NO₃ ^?) was monitored with self-integrating accumulators over 6-month periods and the emissions of the greenhouse gases (GHG) nitrous oxide (N₂O) and carbon dioxide (CO₂) were determined in closed gas chambers. During the first 4 years of the poplar SRC, most nitrogen was lost through NO₃ ^? leaching from the main root zone; however, there was no significant relationship to the rate of N fertilisation. On average, 5.8 kg N ha^?1 year^?1 (13.0 kg CO_2equ) was leached from the root zone. Nitrogen leaching rates decreased in the course of the 4-year study parallel to an increase of the fine root biomass and the degree of mycorrhization. In contrast to N leaching, the loss of nitrogen by N₂O emissions from the soil was very low with an average of 0.61 kg N ha^?1 year^?1 (182 kg CO_2equ) and were also not affected by N fertilisation over the whole study period. Real CO₂ emissions from the poplar soil were two orders of magnitude higher ranging between 15,122 and 19,091 kg CO₂ ha^?1 year^?1 and followed the rotation period with enhanced emission rates in the years of harvest. As key-factors for NO₃ ^? leaching and N₂O emissions, the time after planting and after harvest and the rotation period have been identified by a mixed effects model.     

Matrix protein synthesis by glomerular mesangial cells in culture: Effects of transforming growth factor β (TGFβ) and platelet-derived growth factor (PDGF) on fibronectin and collagen type IV mRNA

The pathogenesis of glomerular scarring is multifactional; recent evidence suggests that transforming growth factor β (TGFβ), a pleiotropic cicatricial mediator, may promote mesangial sclerosis by enhancing the production of extracellular matrix proteins. We studied the effect of TGFβ1 and TFGβ2 on collagen type IV and fibronectin (FN) synthesis in human glomerular mesangial cells in culture (GMC). Two hours after addition of TGFβ, an up to twofold increase in abundance of collagen type IV mRNA was found, which further increased up to fivefold within 24 h. Addition of cycloheximide did not inhibit the TGFβ effect, but caused by itself an up to twofold increase in the abundance of collagen type IV mRNA after 2 h. Together with collagen mRNA, the mRNA for FN and for platelet-derived growth factor (PDGF) was also enhanced. PDGF was found to enhance abundance of the collagen type IV and fibronectin mRNA in GMC. A neutralizing antibody to PDGF or a PDGF-antisense oligonucleotide partly inhibited the TGFβ-induced increase of collagen type IV mRNA, suggesting that TGFβ can affect the collagen type IV synthesis not only directly but also indirectly via the synthesis of PDGF. © 1995 Wiley-Liss, Inc.