ISG15 is counteracted by vaccinia virus E3 protein and controls the proinflammatory response against viral infection

Conjugation of ISG15 inhibits replication of several viruses. Here, using an expression system for assaying human and mouse ISG15 conjugations (ISGylations), we have demonstrated that vaccinia virus E3 protein binds and antagonizes human and mouse ISG15 modification. To study ISGylation importance in poxvirus infection, we used a mouse model that expresses deconjugating proteases. Our results indicate that ISGylation restricts in vitro replication of the vaccinia virus VV E3L mutant but unconjugated ISG15 is crucial to counteract the inflammatory response produced after VV E3L infectionThis work was supported by grants from the Spanish Ministry of Health, FIS2011-00127, and UAM-Banco de Santander to S.G. and was also partly supported by NIAID grant U19AI083025 to A.G.-

Differential induction of apoptosis, interferon signaling, and phagocytosis in macrophages infected with a panel of attenuated and nonattenuated poxviruses

Due to the essential role macrophages play in antiviral immunity, it is important to understand the intracellular and molecular processes that occur in macrophages following infection with various strains of vaccinia virus, particularly those used as vaccine vectors. Similarities as well as differences were found in macrophages infected with different poxvirus strains, particularly at the level of virus-induced apoptosis and the expression of immunomodulatory genes, as determined by microarray analyses. Interestingly, the attenuated modified vaccinia Ankara virus (MVA) was particularly efficient in triggering apoptosis and beta interferon (IFN- ) secretion and in inducing changes in the expression of genes associated with increased activation of innate immunity, setting it apart from the other five vaccinia virus strains tested. Taken together, these results increase our understanding of how these viruses interact with human macrophages, at the cellular and molecular levels, and suggest mechanisms that may underlie their utility as recombinant vaccine vectorsThis work was supported by grants from the Spanish Ministry of Health, FIS2011-00127 (S.G.) and FISPI11/00350 (E.L.-C), and by the Universidad Autónoma de Madrid (UAM)-Banco de Santander (S.G.

PimT, an amino acid exporter controls polyene production via secretion of the quorum sensing pimaricin-inducer PI-factor in Streptomyces natalensis

<p>Abstract</p> <p>Background</p> <p>Polyenes represent a major class of antifungal agents characterised by the presence of a series of conjugated double bonds in their planar hydroxylated macrolide ring structure. Despite their general interest, very little is known about the factors that modulate their biosynthesis. Among these factors, we have recently discovered a new inducing compound (PI-factor) in the pimaricin producer <it>Streptomyces natalensis</it>, which elicits polyene production in a manner characteristic of quorum sensing. Here, we describe the involvement of an amino-acid exporter from <it>S. natalensis </it>in modulating the expression of pimaricin biosynthetic genes via secretion of the quorum-sensing pimaricin-inducer PI-factor.</p> <p>Results</p> <p>Adjacent to the pimaricin gene cluster lies a member of the RhtB family of amino-acid exporters. Gene deletion and complementation experiments provided evidence for a role for PimT in the export of L-homoserine, L-serine, and L-homoserine lactone. Expression of the gene was shown to be induced by homoserine and by the quorum-sensing pimaricin-inducer PI-factor. Interestingly, the mutant displayed 65% loss of pimaricin production, and also 50% decrease in the production of PI, indicating that PimT is used as PI-factor exporter, and suggesting that the effect in antifungal production might be due to limited secretion of the inducer.</p> <p>Conclusion</p> <p>This report describes the involvement of an amino acid exporter (encoded by <it>pimT </it>in the vicinity of the pimaricin cluster) in modulating the expression of antibiotic biosynthetic genes via secretion of the quorum-sensing pimaricin-inducer PI-factor. The discovery of the participation of amino acid exporters in a signal transduction cascade for the production of polyene macrolides is unexpected, and represents an important step forward towards understanding the regulatory network for polyene regulation. Additionally, this finding constitutes the first detailed characterization of an amino-acid exporter in an Actinomycete, and to our knowledge, the first evidence for the implication of this type of exporters in quorum sensing.</p

ISG15 Is a Novel Regulator of Lipid Metabolism during Vaccinia Virus Infection.

Interferon-stimulated gene 15 (ISG15) is a 15-kDa ubiquitin-like modifier that binds to target proteins in a process termed ISGylation. ISG15, first described as an antiviral molecule against many viruses, participates in numerous cellular processes, from immune modulation to the regulation of genome stability. Interestingly, the role of ISG15 as a regulator of cell metabolism has recently gained strength. We previously described ISG15 as a regulator of mitochondrial functions in bone marrow-derived macrophages (BMDMs) in the context of Vaccinia virus (VACV) infection. Here, we demonstrate that ISG15 regulates lipid metabolism in BMDMs and that ISG15 is necessary to modulate the impact of VACV infection on lipid metabolism. We show that Isg15-/- BMDMs demonstrate alterations in the levels of several key proteins of lipid metabolism that result in differences in the lipid profile compared with Isg15+/+ (wild-type [WT]) BMDMs. Specifically, Isg15-/- BMDMs present reduced levels of neutral lipids, reflected by decreased lipid droplet number. These alterations are linked to increased levels of lipases and are independent of enhanced fatty acid oxidation (FAO). Moreover, we demonstrate that VACV causes a dysregulation in the proteomes of BMDMs and alterations in the lipid content of these cells, which appear exacerbated in Isg15-/- BMDMs. Such metabolic changes are likely caused by increased expression of the metabolic regulators peroxisome proliferator-activated receptor-γ (PPARγ) and PPARγ coactivator-1α (PGC-1α). In summary, our results highlight that ISG15 controls BMDM lipid metabolism during viral infections, suggesting that ISG15 is an important host factor to restrain VACV impact on cell metabolism. IMPORTANCE The functions of ISG15 are continuously expanding, and growing evidence supports its role as a relevant modulator of cell metabolism. In this work, we highlight how the absence of ISG15 impacts macrophage lipid metabolism in the context of viral infections and how poxviruses modulate metabolism to ensure successful replication. Our results open the door to new advances in the comprehension of macrophage immunometabolism and the interaction between VACV and the host.We thank the expert technical assistance of Sara Sandoval. We are grateful to Miguel Sánchez-Álvarez who has kindly provided several commercial reagents. We would like to thank the Spanish National Plan for Scientific and Technical Research and Innovation (Plan Estatal de Investigación Científica y Técnica y de Innovación), (Ministry of Health of Spain, State Secretary of R1D and FEDER/FSE).S

Subcellular forms and biochemical events triggered in human cells by HCV polyprotein expression from a viral vector

To identify the subcellular forms and biochemical events induced in human cells after HCV polyprotein expression, we have used a robust cell culture system based on vaccinia virus (VACV) that efficiently expresses in infected cells the structural and nonstructural proteins of HCV from genotype 1b (VT7-HCV7.9). As determined by confocal microscopy, HCV proteins expressed from VT7-HCV7.9 localize largely in a globular-like distribution pattern in the cytoplasm, with some proteins co-localizing with the endoplasmic reticulum (ER) and mitochondria. As examined by electron microscopy, HCV proteins induced formation of large electron-dense cytoplasmic structures derived from the ER and containing HCV proteins. In the course of HCV protein production, there is disruption of the Golgi apparatus, loss of spatial organization of the ER, appearance of some "virus-like" structures and swelling of mitochondria. Biochemical analysis demonstrate that HCV proteins bring about the activation of initiator and effector caspases followed by severe apoptosis and mitochondria dysfunction, hallmarks of HCV cell injury. Microarray analysis revealed that HCV polyprotein expression modulated transcription of genes associated with lipid metabolism, oxidative stress, apoptosis, and cellular proliferation. Our findings demonstrate the uniqueness of the VT7-HCV7.9 system to characterize morphological and biochemical events related to HCV pathogenesis

Em memória dos que partiram: análise retrospetiva dos últimos 30 dias de vida, na ULDM da SCM de Sernancelhe

info:eu-repo/semantics/publishedVersio

Response to a Salmonella Typhimurium challenge in piglets supplemented with protected sodium butyrate or Bacillus licheniformis : effects on performance, intestinal health and behavior

Salmonella spp. is one of the worldwide leading causes of food-borne illnesses for which the inclusion of probiotics or organic acids in animal feeds can be useful control methods. Experimental models are utilized to test the efficacy of strategies against pathogens, but they exhibit limitations which may preclude finding sensible evaluation parameters. The objective of this work is to evaluate the efficacy of 2 different feed additives; a Bacillus licheniformis based probiotic and a protected sodium butyrate (SB) salt, using an experimental model of salmonellosis and, second, to explore if behavior analysis can be used as a sensible evaluation tool for additives evaluation. A total of 78 piglets weaned at 24 d, 8.3 kg BW, were used. Seventy-two were placed in 3 rooms of 8 pens (3 animals/pen) with evenly distributed treatments (n = 8): CON, control group with plain diet; PRO, plain diet with 1 kg/t of Proporc (10 9 cfu of B. licheniformis /kg of feed), and BUT, plain diet with 3 kg/t of Gustor BP70 (2.1 g of partially protected SB salt/kg of feed). Remaining piglets (n = 6) were separated and used as a challenge negative control. The experiment lasted 16 d. After 1 wk of adaptation, animals were challenged with 5 × 10 8 cfu of Salmonella Typhimurium. One pig per pen was euthanized and sampled at d 4 and 8 post-inoculation (PI). There were no significant differences among treatments for ADFI, ADG, G:F, rectal temperature, fecal consistency, pH, ammonia, short-chain fatty acids and lactic acid concentrations, cytokine TNF-α, Pig-MAP acute-phase proteins and histological parameters. However, both products were equally able to reduce colonization and shedding of Salmonella (P = 0.016 for PRO and BUT vs. CON). In addition, PRO treatment had a positive effect on behavioral displays, particularly exploring (P < 0.05 vs. CON), feeding (P < 0.05 vs. CON and BUT) and other active behaviors (P < 0.05 vs. CON and BUT) in the morning period (0830 to 1030 h). In the afternoon (1400 to 1600 h), the challenge effect was most significant. Pigs were less active after the challenge (P < 0.001), with a decrease in positive contacts (P = 0.004), exploration (P < 0.001) and feeding behaviors (P < 0.001) on d 3 PI, in comparison with before the challenge. Accordingly, many lying conducts increased at d 3 PI (P < 0.05). In conclusion, both treatments had positive effects against Salmonella, and behavior analysis appears to be a sensible tool to be considered

Molecular profiling and feasibility using a comprehensive hybrid capture panel on a consecutive series of non-small-cell lung cancer patients from a single centre

Background: Targeted next-generation sequencing (NGS) is recommended to screen actionable genomic alterations (GAs) in patients with non-small-cell lung cancer (NSCLC). We determined the feasibility to detect actionable GAs using TruSight™ Oncology 500 (TSO500) in 200 consecutive patients with NSCLC. Materials and methods: DNA and RNA were sequenced on an Illumina® NextSeq 550 instrument and processed using the TSO500 Docker pipeline. Clinical actionability was defined within the molecular tumour board following European Society for Medical Oncology (ESMO) guidelines for oncogene-addicted NSCLC. Overall survival (OS) was estimated as per the presence of druggable GAs and treatment with targeted therapy. Results: Most patients were males (69.5%) and former or current smokers (86.5%). Median age was 64 years. The most common histological type and tumour stage were lung adenocarcinoma (81%) and stage IV (64%), respectively. Sequencing was feasible in most patients (93.5%) and actionable GAs were found in 26.5% of patients. A high concordance was observed between single-gene testing and TSO500 NGS panel. Patients harbouring druggable GAs and receiving targeted therapy achieved longer OS compared to patients without druggable GAs. Conversely, patients with druggable GAs not receiving targeted therapy had a trend toward shorter OS compared with driver-negative patients. Conclusions: Hybrid capture sequencing using TSO500 panel is feasible to analyse clinical samples from patients with NSCLC and is an efficient tool for screening actionable GAs

Interferon-stimulated gene 15 pathway is a novel mediator of endothelial dysfunction and aneurysms development in angiotensin II infused mice through increased oxidative stress

AIMS: Interferon-stimulated gene 15 (ISG15) encodes a ubiquitin-like protein that induces a reversible post-translational modification (ISGylation) and can also be secreted as a free form. ISG15 plays an essential role as host-defence response to microbial infection; however, its contribution to vascular damage associated with hypertension is unknown. METHODS AND RESULTS: Bioinformatics identified ISG15 as a mediator of hypertension-associated vascular damage. ISG15 expression positively correlated with systolic and diastolic blood pressure and carotid intima-media thickness in human peripheral blood mononuclear cells. Consistently, Isg15 expression was enhanced in aorta from hypertension models and in angiotensin II (AngII)-treated vascular cells and macrophages. Proteomics revealed differential expression of proteins implicated in cardiovascular function, extracellular matrix and remodelling, and vascular redox state in aorta from AngII-infused ISG15-/- mice. Moreover, ISG15-/- mice were protected against AngII-induced hypertension, vascular stiffness, elastin remodelling, endothelial dysfunction, and expression of inflammatory and oxidative stress markers. Conversely, mice with excessive ISGylation (USP18C61A) show enhanced AngII-induced hypertension, vascular fibrosis, inflammation and reactive oxygen species (ROS) generation along with elastin breaks, aortic dilation, and rupture. Accordingly, human and murine abdominal aortic aneurysms showed augmented ISG15 expression. Mechanistically, ISG15 induces vascular ROS production, while antioxidant treatment prevented ISG15-induced endothelial dysfunction and vascular remodelling. CONCLUSION: ISG15 is a novel mediator of vascular damage in hypertension through oxidative stress and inflammation.This work was supported by the Ministerio de Ciencia e Innovación and Fondo Europeo de Desarrollo Regional (FEDER)/FSE (SAF2016-80305P; SAF2017-88089-R; SAF2016-79151-R; RTI2018-099246-B-I00), Ministerio de Innovación, Cultura y Deportes (PGC2018-097019-B-I00), Instituto de Salud Carlos III (ISCIII; FIS PI18/0919); Comunidad de Madrid (CM) (AORTASANA B2017/BMD-3676) FEDER-a way to build Europe, Bayer AG (2019-09-2433), CM-Universidad Autónoma de Madrid (SI1-PJI-2019-00321), and British Heart Foundation (CH/12/4/29762; RE//18/6/34217). M.G.-A. was supported by an FPI-UAM fellowship, R.R.-D. by a Juan de la Cierva contract (IJCI-2017-31399), and A.C.M. by a Walton Fellowship, University of Glasgow. The CNIC is supported by ISCIII, the Ministerio de Ciencia e Innovación, and the Pro CNIC Foundation, and is a Severo Ochoa Center of Excellence (SEV-2015-0505)