Tacaribe virus but not junin virus infection induces cytokine release from primary human monocytes and macrophages

The mechanisms underlying the development of disease during arenavirus infection are poorly understood. However, common to all hemorrhagic fever diseases is the involvement of macrophages as primary target cells, suggesting that the immune response in these cells may be of paramount importance during infection. Thus, in order to identify features of the immune response that contribute to arenavirus pathogenesis, we have examined the growth kinetics and cytokine profiles of two closely related New World arenaviruses, the apathogenic Tacaribe virus (TCRV) and the hemorrhagic fever-causing Junin virus (JUNV), in primary human monocytes and macrophages. Both viruses grew robustly in VeroE6 cells; however, TCRV titres were decreased by approximately 10 fold compared to JUNV in both monocytes and macrophages. Infection of both monocytes and macrophages with TCRV also resulted in the release of high levels of IL-6, IL-10 and TNF-α, while levels of IFN-α, IFN-β and IL-12 were not affected. However, we could show that the presence of these cytokines had no direct effect on growth of either TCRV of JUNV in macrophages. Further analysis also showed that while the production of IL-6 and IL-10 are dependent on viral replication, production of TNF-α also occurs after exposure to UV-inactivated TCRV particles and is thus independent of productive virus infection. Surprisingly, JUNV infection did not have an effect on any of the cytokines examined indicating that, in contrast to other viral hemorrhagic fever viruses, macrophage-derived cytokine production is unlikely to play an active role in contributing to the cytokine dysregulation observed in JUNV infected patients. Rather, these results suggest that an early, controlled immune response by infected macrophages may be critical for the successful control of infection of apathogenic viruses and prevention of subsequent disease, including systemic cytokine dysregulation.     

Disrupted‐in‐schizophrenia 1 functional polymorphisms and D2/D3 receptor availability: A [11C]‐(+)‐PHNO imaging study

The disrupted‐in‐schizophrenia 1 (DISC1) protein has been implicated in a range of biological mechanisms underlying chronic mental disorders such as schizophrenia. Schizophrenia is associated with abnormal striatal dopamine signalling, and all antipsychotic drugs block striatal dopamine 2/3 receptors (D_2/3Rs). Importantly, the DISC1 protein directly interacts and forms a protein complex with the dopamine D₂ receptor (D₂R) that inhibits agonist‐induced D₂R internalisation. Moreover, animal studies have found large striatal increases in the proportion of D₂R receptors in a high affinity state (D₂^highR) in DISC1 rodent models. Here, we investigated the relationship between the three most common polymorphisms altering the amino‐acid sequence of the DISC1 protein (Ser704Cys (rs821616), Leu607Phe (rs6675281) and Arg264Gln (rs3738401)) and striatal D_2/3R availability in 41 healthy human volunteers, using [¹¹C]‐(+)‐PHNO positron emission tomography. We found no association between DISC1 polymorphisms and D_2/3R availability in the striatum and D₂R availability in the caudate and putamen. Therefore, despite a direct interaction between DISC1 and the D₂R, none of its main functional polymorphisms impact striatal D_2/3R binding potential, suggesting DISC1 variants act through other mechanisms.     

Dissection of the NKG2C NK cell response against Puumala Orthohantavirus

BackgroundInfections with the Puumala orthohantavirus (PUUV) in humans may cause hemorrhagic fever with renal syndrome (HFRS), known as nephropathia epidemica (NE), which is associated with acute renal failure in severe cases. In response to PUUV-infections, a subset of potent antiviral NKG2C⁺ NK cells expand, whose role in virus defence and pathogenesis of NE is unclear. NKG2C⁺ NK cell proliferation is mediated by binding of NKG2C/CD94 to HLA-E on infected cells. The proliferation and activation of NKG2C⁺ NK cells via the NKG2C/HLA-E axis is affected by different NKG2C (NKG2C^wt/del) and HLA-E (HLA-E*0101/0103) alleles, which naturally occur in the human host. Homozygous (NKG2C^del/del) and heterozygous (NKG2C^wt/del) deletions of the NKG2C receptor results in an impaired NKG2C/CD94 mediated proliferation and activation of NKG2C⁺ cells. We therefore analyzed the PUUV-mediated NKG2C⁺ NK cell responses and the impact of different NKG2C and HLA-E alleles in NE patients.Methodology/Principal findingsNKG2C⁺ NK cell expansion and effector functions in PUUV-infected cells were investigated using flow cytometry and it was shown that PUUV-infected endothelial cells led to a NKG2C/CD94 mediated NKG2C⁺ NK cell activation and expansion, dependent on the HLA-G-mediated upregulation of HLA-E. Furthermore, the NKG2C^del and HLA-E*0101/0103 alleles were determined in 130 NE patients and 130 matched controls, and it was shown that in NE patients the NKG2C^wt/del allele was significantly overrepresented, compared to the NKG2C^wt/wt variant (p = 0.01). In addition, in vitro analysis revealed that NKG2C^wt/del NK cells exhibited on overall a lower proliferation (p = 0.002) and lower IFNγ expression (p = 0.004) than NKG2C^wt/wt NK cells.Conclusions/SignificanceOur results corroborate the substantial impact of the NKG2C/HLA-E axis on PUUV-specific NK cell responses. A weak NKG2C⁺ NK cell response, as reflected by NKG2C^wt/del variant, may be associated with a higher risk for a severe hantavirus infections.     

Intracellular Staphylococcus aureus eludes selective autophagy by activating a host cell kinase

Autophagy, a catabolic pathway of lysosomal degradation, acts not only as an efficient recycle and survival mechanism during cellular stress, but also as an anti-infective machinery. The human pathogen Staphylococcus aureus (S. aureus) was originally considered solely as an extracellular bacterium, but is now recognized additionally to invade host cells, which might be crucial for persistence. However, the intracellular fate of S. aureus is incompletely understood. Here, we show for the first time induction of selective autophagy by S. aureus infection, its escape from autophagosomes and proliferation in the cytoplasm using live cell imaging. After invasion, S. aureus becomes ubiquitinated and recognized by receptor proteins such as SQSTM1/p62 leading to phagophore recruitment. Yet, S. aureus evades phagophores and prevents further degradation by a MAPK14/p38α MAP kinase-mediated blockade of autophagy. Our study demonstrates a novel bacterial strategy to block autophagy and secure survival inside the host cell.     

Proteomic response of the marine ammonia-oxidising archaeon Nitrosopumilus maritimus to iron limitation reveals strategies to compensate for nutrient scarcity

Dissolved iron (Fe) is vanishingly low in the oceans, with ecological success conferred to microorganisms that can restructure their biochemistry to maintain high growth rates during Fe scarcity. Chemolithoautotrophic ammonia-oxidising archaea (AOA) are highly abundant in the oceans, constituting ~30% of cells below the photic zone. Here we examine the proteomic response of the AOA isolate Nitrosopumilus maritimus to growth-limiting Fe concentrations. Under Fe limitation, we observed a significant reduction in the intensity of Fe-dense ferredoxins associated with respiratory complex I whilst complex III and IV proteins with more central roles in the electron transport chain remain unchanged. We concomitantly observed an increase in the intensity of Fe-free functional alternatives such as flavodoxin and plastocyanin, thioredoxin and alkyl hydroperoxide which are known to mediate electron transport and reactive oxygen species detoxification, respectively. Under Fe limitation, we found a marked increase in the intensity of the ABC phosphonate transport system (Phn), highlighting an intriguing link between Fe and P cycling in N. maritimus. We hypothesise that an elevated uptake of exogenous phosphonates under Fe limitation may either supplement N. maritimus' endogenous methylphosphonate biosynthesis pathway - which requires Fe - or enhance the production of phosphonate-containing exopolysaccharides known to efficiently bind environmental Fe.     

From Mendelian to molecular genetics: the Xiphophorus melanoma model

In human tumor biopsies it is almost impossible to pinpoint the particular molecular abnormalities that determine neoplasia. In animal models where tumorigenesis is initiated by clearly defined genetic events, it is possible to study the genes and their functions that make a normal cell become a fully malignant cancer cell. In the fish Xiphophorus, melanoma can be initiated by simple crossings, and the signaling pathways that govern tumor growth and progression can be delineated. This model offers the prospect of obtaining a complete picture of the molecular changes and regulatory networks underlying tumor formation, which should contribute to a better understanding of some general principles of cancer biology, and identify new targets for melanoma research in particular.     

Adipose stem cell-derived extracellular matrix represents a promising biomaterial by inducing spontaneous formation of prevascular-like structures by mvECs

Tissue constructs of physiologically relevant scale require a vascular system to maintain cell viability. However, in vitro vascularization of engineered tissues is still a major challenge. Successful approaches are based on a feeder layer (FL) to support vascularization. Here, we investigated whether the supporting effect on the self-assembled formation of prevascular-like structures by microvascular endothelial cells (mvECs) originates from the FL itself or from its extracellular matrix (ECM). Therefore, we compared the influence of ECM, either derived from adipose-derived stem cells (ASCs) or adipogenically differentiated ASCs, with the classical cell-based FL. All cell-derived ECM (cdECM) substrates enabled mvEC growth with high viability. Prevascular-like structures were visualized by immunofluorescence staining of endothelial surface protein CD31 and could be observed on all cdECM and FL substrates but not on control substrate collagen I. On adipogenically differentiated ECM, longer and higher branched structures could be found compared with stem cell cdECM. An increased concentration of proangiogenic factors was found in cdECM substrates and FL approaches compared with controls. Finally, the expression of proteins associated with tube formation (E-selectin and thrombomodulin) was confirmed. These results highlight cdECM as promising biomaterial for adipose tissue engineering by inducing the spontaneous formation of prevascular-like structures by mvECs.     

Genetic diversity and biogeography of native and introduced populations of Ulva pertusa (Ulvales,Chlorophyta)

Genetic diversity of native and introduced populations of Ulva pertusa (Ulvales, Chlorophyta) was examined using genetic markers of chloroplast, mitochondria and nuclear non‐coding region sequences. In the preliminary investigations to genetically identify the species for further analyses, U. pertusa was found only from temperate coasts of the more extensive collection sites including tropical coasts suggesting that it is a temperate species and basically not distributed in tropical regions. For chloroplast and mitochondrial sequences, repeating patterns of short tandem repeat sequences and nucleotide substitutions were used to recognize the haplotypes (genetic types). A total of 48 haplotypes based on combinations of chloroplast and mitochondrial haplotypes were recognized in the 244 specimens collected in the presumptive native range (Northeast Asia) and introduced populations (North America, Australia, New Zealand, Chile and Europe). Among them, 46 haplotypes (H1–H8 and H11–H48) were recognized in Northeast Asia, whereas only 1–5 haplotypes were recognized in the other areas. Nuclear microsatellite sequences were also analyzed. The lengths of the PCR products including the nuclear microsatellite region of 234 specimens were determined, and a total of 17 genotypes were recognized. Among them, 14 genotypes were found in Northeast Asia, whereas 1–7 genotypes were recognized in the other areas. Based on the results, the hypothesis that the native range of the species is in Northeast Asia was supported, and the populations outside this range were concluded to be non‐indigenous populations.     

AT 101 induces early mitochondrial dysfunction and HMOX1 (heme oxygenase 1) to trigger mitophagic cell death in glioma cells

In most cases, macroautophagy/autophagy serves to alleviate cellular stress and acts in a pro-survival manner. However, the effects of autophagy are highly contextual, and autophagic cell death (ACD) is emerging as an alternative paradigm of (stress- and drug-induced) cell demise. AT 101 ([-]-gossypol), a natural compound from cotton seeds, induces ACD in glioma cells as confirmed here by CRISPR/Cas9 knockout of ATG5 that partially, but significantly rescued cell survival following AT 101 treatment. Global proteomic analysis of AT 101-treated U87MG and U343 glioma cells revealed a robust decrease in mitochondrial protein clusters, whereas HMOX1 (heme oxygenase 1) was strongly upregulated. AT 101 rapidly triggered mitochondrial membrane depolarization, engulfment of mitochondria within autophagosomes and a significant reduction of mitochondrial mass and proteins that did not depend on the presence of BAX and BAK1. Conversely, AT 101-induced reduction of mitochondrial mass could be reversed by inhibiting autophagy with wortmannin, bafilomycin A₁ and chloroquine. Silencing of HMOX1 and the mitophagy receptors BNIP3 (BCL2 interacting protein 3) and BNIP3L (BCL2 interacting protein 3 like) significantly attenuated AT 101-dependent mitophagy and cell death. Collectively, these data suggest that early mitochondrial dysfunction and HMOX1 overactivation synergize to trigger lethal mitophagy, which contributes to the cell killing effects of AT 101 in glioma cells.

Abbreviations: ACD, autophagic cell death; ACN, acetonitrile; AT 101, (-)-gossypol; BAF, bafilomycin A₁; BAK1, BCL2-antagonist/killer 1; BAX, BCL2-associated X protein; BH3, BCL2 homology region 3; BNIP3, BCL2 interacting protein 3; BNIP3L, BCL2 interacting protein 3 like; BP, Biological Process; CCCP, carbonyl cyanide m-chlorophenyl hydrazone; CC, Cellular Component; Con, control; CQ, chloroquine; CRISPR, clustered regularly interspaced short palindromic repeats; DMEM, Dulbecco’s Modified Eagle Medium; DTT, 1,4-dithiothreitol; EM, electron microscopy; ER, endoplasmatic reticulum; FACS, fluorescence-activated cell sorting; FBS, fetal bovine serum; FCCP, carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone; GO, Gene Ontology; HAcO, acetic acid; HMOX1, heme oxygenase 1; DKO, double knockout; LC-MS/MS, liquid chromatography coupled to tandem mass spectrometry; LPL, lipoprotein lipase, MEFs, mouse embryonic fibroblasts; mPTP, mitochondrial permeability transition pore; MTG, MitoTracker Green FM; mt-mKeima, mito-mKeima; MT-ND1, mitochondrially encoded NADH:ubiquinone oxidoreductase core subunit 1; PBS, phosphate-buffered saline; PE, phosphatidylethanolamine; PI, propidium iodide; PRKN, parkin RBR E3 ubiquitin protein ligase; SDS, sodium dodecyl sulfate; SQSTM1/p62, sequestome 1; STS, staurosporine; sgRNA, single guide RNA; SILAC, stable isotope labeling with amino acids in cell culture; TFA, trifluoroacetic acid, TMRM, tetramethylrhodamine methyl ester perchlorate; WM, wortmannin; WT, wild-type