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Virology
Haematopoietic stem and progenitor cells from human pluripotent stem cells
May 17, 2017   Nature Add nature.com free-link Cancel
Sugimura R, Jha DK, Han A, Soria-Valles C, da Rocha EL,   . . . . . .   , Keller G, Engelman AN, Snapper SB, Doulatov S, Daley GQ
Haematopoietic stem and progenitor cells from human pluripotent stem cells
May 17, 2017
Nature
A variety of tissue lineages can be differentiated from pluripotent stem cells by mimicking embryonic development through stepwise exposure to morphogens, or by conversion of one differentiated cell type into another by enforced expression of master transcription factors. Here, to yield functional human haematopoietic stem cells, we perform morphogen-directed differentiation of human pluripotent stem cells into haemogenic endothelium followed by screening of 26 candidate haematopoietic stem-cell-specifying transcription factors for their capacity to promote multi-lineage haematopoietic engraftment in mouse hosts. We recover seven transcription factors (ERG, HOXA5, HOXA9, HOXA10, LCOR, RUNX1 and SPI1) that are sufficient to convert haemogenic endothelium into haematopoietic stem and progenitor cells that engraft myeloid, B and T cells in primary and secondary mouse recipients. Our combined approach of morphogen-driven differentiation and transcription-factor-mediated cell fate conversion produces haematopoietic stem and progenitor cells from pluripotent stem cells and holds promise for modelling haematopoietic disease in humanized mice and for therapeutic strategies in genetic blood disorders.
Infectious disease ward admission positively influences P. jiroveci pneumonia (PjP) outcome: A retrospective analysis of 116 HIV-positive and HIV-negative immunocompromised patients
May 15, 2017   PloS One
Ricciardi A, Gentilotti E, Coppola L, Maffongelli G, Cerva C,   . . . . . .   , Toschi N, Di Cave D, Parisi SG, Andreoni M, Sarmati L
Infectious disease ward admission positively influences P. jiroveci pneumonia (PjP) outcome: A retrospective analysis of 116 HIV-positive and HIV-negative immunocompromised patients
May 15, 2017
PloS One
P. jiroveci (Pj) causes a potentially fatal pneumonia in immunocompromised patients and the factors associated with a bad outcome are poorly understood. A retrospective analysis on Pj pneumonia (PjP) cases occurring in Tor Vergata University Hospital, Italy, during the period 2011-2015. The patients' demographic, clinical and radiological characteristics and the Pj genotypes were considered. The study population included 116 patients, 37.9% of whom had haematological malignancy or underwent haematological stem cell transplantation (HSCT), 22.4% had HIV infection, 16.4% had chronic lung diseases (CLD), 7.8% had a solid cancer, and 3.4% underwent a solid organ transplant (SOT). The remaining 12.1% had a miscellaneous other condition. At univariate analysis, being older than 60 years was significantly correlated with a severe PjP (OR [95%CI] 2.52 [0.10-5.76]; p = 0.031) and death (OR [95%CI] 2.44 [1.05-5.70]; p = 0.036), while a previous trimethoprim/sulfamethoxazole (TMP/SMX) prophylaxis were significantly associated with a less severe pneumonia (OR[95%CI] 0.35 [0.15-0.84], p = 0.023); moreover, death due to PjP was significantly more frequent in patients with CLD (OR[95%CI] 3.26 [1.17-9.05]; p = 0.019) while, admission to the Infectious Diseases Unit was significantly associated with fewer deaths (OR[95%CI] 0.10 [0.03-0.36], p = 0.002). At multivariate analysis, a better PjP outcome was observed in patients taking TMP/SMX prophylaxis and that were admitted to the Infectious Diseases Unit (OR[95%CI] 0.27 [0.07-1.03], p = 0.055, OR[95%CI] 0.16 [0.05-0.55]; p = 0.004, respectively). In conclusion, in our study population, TMP/SMX prophylaxis and infectious disease specialist approach were variables correlated with a better PjP outcome.
Frontline Science: Nasal epithelial GM-CSF contributes to TLR5-mediated modulation of airway dendritic cells and subsequent IgA response
May 19, 2017   Journal Of Leukocyte Biology
Cao Y, Zhang E, Yang J, Yang Y, Yu J, Xiao Y, Li W, Zhou D, Li Y, Zhao B, Yan H, Lu M, Zhong M, Yan H
Frontline Science: Nasal epithelial GM-CSF contributes to TLR5-mediated modulation of airway dendritic cells and subsequent IgA response
May 19, 2017
Journal Of Leukocyte Biology
Flagellin, as a TLR5 agonist, is an established mucosal adjuvant for enhancing mucosal IgA responses by i.n. immunization. Nasal epithelial cells (NECs) are the first sentinel cells to be exposed to antigen and adjuvant in i.n. immunization, and it is suggested that they play an important role in the mucosal adjuvant activity of flagellin. However, the molecular mechanism leading to modulation and the response by flagellin-activated NECs remain unknown. We aimed to identify the soluble mediator(s) from flagellin-activated NECs that modulate the functions of airway dendritic cells (DCs) and enhance subsequent IgA response. In vitro studies showed that compared with the TLR4 agonist LPS, flagellin directly triggered slight up-regulation of CD80 on airway DCs but was insufficient to affect CD86 expression and DC-mediated IgA response. With the use of an in vitro system for culturing mouse primary NECs (mNECs), we demonstrated that flagellin-activated mNECs could functionally modulate airway DCs, which manifested as significant up-regulation of CD80/CD86 and enhancement of IgA production. The functional modulation of airway DCs was dependent on TLR5 activation of mNECs rather than direct TLR5 activation of airway DCs. With the use of cytokine array and antibody-blocking assays, we further identified that GM-CSF, a cytokine secreted from TLR5-activated mNECs, contributes to the activation of mNECs to airway DCs and subsequent IgA enhancement. In vivo blocking experiments confirmed that GM-CSF is an important factor in recombinant flagellin derived from Salmonella typhi (FliC)-induced airway DC activation and antigen-specific IgA enhancement. Our data directly demonstrate that nasal epithelial GM-CSF contributes to TLR5-mediated modulation of airway DCs and a subsequent IgA response. © Society for Leukocyte Biology.
USP13 negatively regulates antiviral responses by deubiquitinating STING
May 23, 2017   Nature Communications
Sun H, Zhang Q, Jing YY, Zhang M, Wang HY,   . . . . . .   , Zhu QY, Yao J, Shu HB, Lin D, Zhong B
USP13 negatively regulates antiviral responses by deubiquitinating STING
May 23, 2017
Nature Communications
STING (also known as MITA) is critical for host defence against viruses and the activity of STING is regulated by ubiquitination. However, the deubiquitination of STING is not fully understood. Here, we show that ubiquitin-specific protease 13 (USP13) is a STING-interacting protein that catalyses deubiquitination of STING. Knockdown or knockout of USP13 potentiates activation of IRF3 and NF-κB and expression of downstream genes after HSV-1 infection or transfection of DNA ligands. USP13 deficiency results in impaired replication of HSV-1. Consistently, USP13 deficient mice are more resistant than wild-type littermates to lethal HSV-1 infection. Mechanistically, USP13 deconjugates polyubiquitin chains from STING and prevents the recruitment of TBK1 to the signalling complex, thereby negatively regulating cellular antiviral responses. Our study thus uncovers a function of USP13 in innate antiviral immunity and provides insight into the regulation of innate immunity.
A high quantum yield molecule-protein complex fluorophore for near-infrared II imaging
May 19, 2017   Nature Communications
Antaris AL, Chen H, Diao S, Ma Z, Zhang Z,   . . . . . .   , Zhu M, Cheng K, Hong X, Dai H, Cheng Z
A high quantum yield molecule-protein complex fluorophore for near-infrared II imaging
May 19, 2017
Nature Communications
Fluorescence imaging in the second near-infrared window (NIR-II) allows visualization of deep anatomical features with an unprecedented degree of clarity. NIR-II fluorophores draw from a broad spectrum of materials spanning semiconducting nanomaterials to organic molecular dyes, yet unfortunately all water-soluble organic molecules with >1,000 nm emission suffer from low quantum yields that have limited temporal resolution and penetration depth. Here, we report tailoring the supramolecular assemblies of protein complexes with a sulfonated NIR-II organic dye (CH-4T) to produce a brilliant 110-fold increase in fluorescence, resulting in the highest quantum yield molecular fluorophore thus far. The bright molecular complex allowed for the fastest video-rate imaging in the second NIR window with ∼50-fold reduced exposure times at a fast 50 frames-per-second (FPS) capable of resolving mouse cardiac cycles. In addition, we demonstrate that the NIR-II molecular complexes are superior to clinically approved ICG for lymph node imaging deep within the mouse body.
Hit and go CAS9 delivered through a lentiviral based self-limiting circuit
May 22, 2017   Nature Communications
Petris G, Casini A, Montagna C, Lorenzin F, Prandi D, Romanel A, Zasso J, Conti L, Demichelis F, Cereseto A
Hit and go CAS9 delivered through a lentiviral based self-limiting circuit
May 22, 2017
Nature Communications
In vivo application of the CRISPR-Cas9 technology is still limited by unwanted Cas9 genomic cleavages. Long-term expression of Cas9 increases the number of genomic loci non-specifically cleaved by the nuclease. Here we develop a Self-Limiting Cas9 circuit for Enhanced Safety and specificity (SLiCES) which consists of an expression unit for Streptococcus pyogenes Cas9 (SpCas9), a self-targeting sgRNA and a second sgRNA targeting a chosen genomic locus. The self-limiting circuit results in increased genome editing specificity by controlling Cas9 levels. For its in vivo utilization, we next integrate SLiCES into a lentiviral delivery system (lentiSLiCES) via circuit inhibition to achieve viral particle production. Upon delivery into target cells, the lentiSLiCES circuit switches on to edit the intended genomic locus while simultaneously stepping up its own neutralization through SpCas9 inactivation. By preserving target cells from residual nuclease activity, our hit and go system increases safety margins for genome editing.
Ebolavirus's Foibles
May 19, 2017   Cell
Yamayoshi S, Kawaoka Y
Ebolavirus's Foibles
May 19, 2017
Cell
Ebola virus disease poses a global health threat. Here, two studies by Wec et al. and Zhao et al. identified vulnerability in an internal fusion loop of an ebolavirus glycoprotein. Monoclonal antibodies elicited from immunization and isolated from a human survivor that recognized epitopes in this area neutralized all five ebolaviruses, guiding the development of a pan-ebolavirus immunotherapy. Copyright © 2017 Elsevier Inc. All rights reserved.
Reply to Padmanabhan and Dixit: Hepatitis C virus entry inhibitors for optimally boosting direct-acting antiviral-based treatments
May 17, 2017   Proceedings Of The National Academy Of Sciences Of The United States Of America
Ohashi H, Koizumi Y, Fukano K, Wakita T, Perelson AS, Iwami S, Watashi K
The pentameric complex drives immunologically covert cell-cell transmission of wild-type human cytomegalovirus
May 23, 2017   Proceedings Of The National Academy Of Sciences Of The United States Of America
Murrell I, Bedford C, Ladell K, Miners KL, Price DA, Tomasec P, Wilkinson GWG, Stanton RJ
The pentameric complex drives immunologically covert cell-cell transmission of wild-type human cytomegalovirus
May 23, 2017
Proceedings Of The National Academy Of Sciences Of The United States Of America
Human cytomegalovirus (HCMV) strains that have been passaged in vitro rapidly acquire mutations that impact viral growth. These laboratory-adapted strains of HCMV generally exhibit restricted tropism, produce high levels of cell-free virus, and develop susceptibility to natural killer cells. To permit experimentation with a virus that retained a clinically relevant phenotype, we reconstructed a wild-type (WT) HCMV genome using bacterial artificial chromosome technology. Like clinical virus, this genome proved to be unstable in cell culture; however, propagation of intact virus was achieved by placing the RL13 and UL128 genes under conditional expression. In this study, we show that WT-HCMV produces extremely low titers of cell-free virus but can efficiently infect fibroblasts, epithelial, monocyte-derived dendritic, and Langerhans cells via direct cell-cell transmission. This process of cell-cell transfer required the UL128 locus, but not the RL13 gene, and was significantly less vulnerable to the disruptive effects of IFN, cellular restriction factors, and neutralizing antibodies compared with cell-free entry. Resistance to neutralizing antibodies was dependent on high-level expression of the pentameric gH/gL/gpUL128-131A complex, a feature of WT but not passaged strains of HCMV.
Prognostic and biological significance of the proangiogenic factor EGFL7 in acute myeloid leukemia
May 23, 2017   Proceedings Of The National Academy Of Sciences Of The United States Of America
Papaioannou D, Shen C, Nicolet D, McNeil B, Bill M,   . . . . . .   , Croce CM, Caligiuri MA, Bloomfield CD, Garzon R, Dorrance AM
Prognostic and biological significance of the proangiogenic factor EGFL7 in acute myeloid leukemia
May 23, 2017
Proceedings Of The National Academy Of Sciences Of The United States Of America
Epithelial growth factor-like 7 (EGFL7) is a protein that is secreted by endothelial cells and plays an important role in angiogenesis. Although EGFL7 is aberrantly overexpressed in solid tumors, its role in leukemia has not been evaluated. Here, we report that levels of both EGFL7 mRNA and EGFL7 protein are increased in blasts of patients with acute myeloid leukemia (AML) compared with normal bone marrow cells. High EGFL7 mRNA expression associates with lower complete remission rates, and shorter event-free and overall survival in older (age ≥60 y) and younger (age
Absence of Specific Chlamydia trachomatis Inclusion Membrane Proteins Triggers Premature Inclusion Membrane Lysis and Host Cell Death
May 17, 2017   Cell Reports
Weber MM, Lam JL, Dooley CA, Noriea NF, Hansen BT, Hoyt FH, Carmody AB, Sturdevant GL, Hackstadt T
Absence of Specific Chlamydia trachomatis Inclusion Membrane Proteins Triggers Premature Inclusion Membrane Lysis and Host Cell Death
May 17, 2017
Cell Reports
Chlamydia trachomatis is a human pathogen associated with significant morbidity worldwide. As obligate intracellular parasites, chlamydiae must survive within eukaryotic cells for sufficient time to complete their developmental cycle. To promote host cell survival, chlamydiae express poorly understood anti-apoptotic factors. Using recently developed genetic tools, we show that three inclusion membrane proteins (Incs) out of eleven examined are required for inclusion membrane stability and avoidance of host cell death pathways. In the absence of specific Incs, premature inclusion lysis results in recognition by autophagolysosomes, activation of intrinsic apoptosis, and premature termination of the chlamydial developmental cycle. Inhibition of autophagy or knockdown of STING prevented host cell death and activation of intrinsic apoptosis. Significantly, these findings emphasize the importance of Incs in the establishment of a replicative compartment that sequesters the pathogen from host surveillance systems. Published by Elsevier Inc.
A Temporal Proteomic Map of Epstein-Barr Virus Lytic Replication in B Cells
May 17, 2017   Cell Reports
Ersing I, Nobre L, Wang LW, Soday L, Ma Y, Paulo JA, Narita Y, Ashbaugh CW, Jiang C, Grayson NE, Kieff E, Gygi SP, Weekes MP, Gewurz BE
A Temporal Proteomic Map of Epstein-Barr Virus Lytic Replication in B Cells
May 17, 2017
Cell Reports
Epstein-Barr virus (EBV) replication contributes to multiple human diseases, including infectious mononucleosis, nasopharyngeal carcinoma, B cell lymphomas, and oral hairy leukoplakia. We performed systematic quantitative analyses of temporal changes in host and EBV proteins during lytic replication to gain insights into virus-host interactions, using conditional Burkitt lymphoma models of type I and II EBV infection. We quantified profiles of >8,000 cellular and 69 EBV proteins, including >500 plasma membrane proteins, providing temporal views of the lytic B cell proteome and EBV virome. Our approach revealed EBV-induced remodeling of cell cycle, innate and adaptive immune pathways, including upregulation of the complement cascade and proteasomal degradation of the B cell receptor complex, conserved between EBV types I and II. Cross-comparison with proteomic analyses of human cytomegalovirus infection and of a Kaposi-sarcoma-associated herpesvirus immunoevasin identified host factors targeted by multiple herpesviruses. Our results provide an important resource for studies of EBV replication. Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.
Structural insights into reptarenavirus cap-snatching machinery
May 15, 2017   PLoS Pathogens
Rosenthal M, Gogrefe N, Vogel D, Reguera J, Rauschenberger B, Cusack S, Günther S, Reindl S
Structural insights into reptarenavirus cap-snatching machinery
May 15, 2017
PLoS Pathogens
Cap-snatching was first discovered in influenza virus. Structures of the involved domains of the influenza virus polymerase, namely the endonuclease in the PA subunit and the cap-binding domain in the PB2 subunit, have been solved. Cap-snatching endonucleases have also been demonstrated at the very N-terminus of the L proteins of mammarena-, orthobunya-, and hantaviruses. However, a cap-binding domain has not been identified in an arena- or bunyavirus L protein so far. We solved the structure of the 326 C-terminal residues of the L protein of California Academy of Sciences virus (CASV), a reptarenavirus, by X-ray crystallography. The individual domains of this 37-kDa fragment (L-Cterm) as well as the domain arrangement are structurally similar to the cap-binding and adjacent domains of influenza virus polymerase PB2 subunit, despite the absence of sequence homology, suggesting a common evolutionary origin. This enabled identification of a region in CASV L-Cterm with similarity to a cap-binding site; however, the typical sandwich of two aromatic residues was missing. Consistent with this, cap-binding to CASV L-Cterm could not be detected biochemically. In addition, we solved the crystal structure of the corresponding endonuclease in the N-terminus of CASV L protein. It shows a typical endonuclease fold with an active site configuration that is essentially identical to that of known mammarenavirus endonuclease structures. In conclusion, we provide evidence for a presumably functional cap-snatching endonuclease in the N-terminus and a degenerate cap-binding domain in the C-terminus of a reptarenavirus L protein. Implications of these findings for the cap-snatching mechanism in arenaviruses are discussed.
A Role for the Host DNA Damage Response in Hepatitis B Virus cccDNA Formation-and Beyond?
May 22, 2017   Viruses
Schreiner S, Nassal M
A Role for the Host DNA Damage Response in Hepatitis B Virus cccDNA Formation-and Beyond?
May 22, 2017
Viruses
Chronic hepatitis B virus (HBV) infection puts more than 250 million people at a greatly increased risk to develop end-stage liver disease. Like all hepadnaviruses, HBV replicates via protein-primed reverse transcription of a pregenomic (pg) RNA, yielding an unusually structured, viral polymerase-linked relaxed-circular (RC) DNA as genome in infectious particles. Upon infection, RC-DNA is converted into nuclear covalently closed circular (ccc) DNA. Associating with cellular proteins into an episomal minichromosome, cccDNA acts as template for new viral RNAs, ensuring formation of progeny virions. Hence, cccDNA represents the viral persistence reservoir that is not directly targeted by current anti-HBV therapeutics. Eliminating cccDNA will thus be at the heart of a cure for chronic hepatitis B. The low production of HBV cccDNA in most experimental models and the associated problems in reliable cccDNA quantitation have long hampered a deeper understanding of cccDNA molecular biology. Recent advancements including cccDNA-dependent cell culture systems have begun to identify select host DNA repair enzymes that HBV usurps for RC-DNA to cccDNA conversion. While this list is bound to grow, it may represent just one facet of a broader interaction with the cellular DNA damage response (DDR), a network of pathways that sense and repair aberrant DNA structures and in the process profoundly affect the cell cycle, up to inducing cell death if repair fails. Given the divergent interactions between other viruses and the DDR it will be intriguing to see how HBV copes with this multipronged host system.
A Japanese Encephalitis Virus Vaccine Inducing Antibodies Strongly Enhancing In Vitro Infection Is Protective in Pigs
May 22, 2017   Viruses
García-Nicolás O, Ricklin ME, Liniger M, Vielle NJ, Python S, Souque P, Charneau P, Summerfield A
A Japanese Encephalitis Virus Vaccine Inducing Antibodies Strongly Enhancing In Vitro Infection Is Protective in Pigs
May 22, 2017
Viruses
The Japanese encephalitis virus (JEV) is responsible for zoonotic severe viral encephalitis transmitted by Culex mosquitoes. Although birds are reservoirs, pigs play a role as amplifying hosts, and are affected in particular through reproductive failure. Here, we show that a lentiviral JEV vector, expressing JEV prM and E proteins (TRIP/JEV.prME), but not JEV infection induces strong antibody-dependent enhancement (ADE) activities for infection of macrophages. Such antibodies strongly promoted infection via Fc receptors. ADE was found at both neutralizing and non-neutralizing serum dilutions. Nevertheless, in vivo JEV challenge of pigs demonstrated comparable protection induced by the TRIP/JEV.prME vaccine or heterologous JEV infection. Thus, either ADE antibodies cause no harm in the presence of neutralizing antibodies or may even have protective effects in vivo in pigs. Additionally, we found that both pre-infected and vaccinated pigs were not fully protected as low levels of viral RNA were found in lymphoid and nervous system tissue in some animals. Strikingly, the virus from the pre-infection persisted in the tonsils throughout the experiment. Finally, despite the vaccination challenge, viral RNA was detected in the oronasal swabs in all vaccinated pigs. These latter data are relevant when JEV vaccination is employed in pigs.
Human Beta Defensin 2 Selectively Inhibits HIV-1 in Highly Permissive CCR6⁺CD4⁺ T Cells
May 16, 2017   Viruses
Lafferty MK, Sun L, Christensen-Quick A, Lu W, Garzino-Demo A
Human Beta Defensin 2 Selectively Inhibits HIV-1 in Highly Permissive CCR6⁺CD4⁺ T Cells
May 16, 2017
Viruses
Chemokine receptor type 6 (CCR6)⁺CD4⁺ T cells are preferentially infected and depleted during HIV disease progression, but are preserved in non-progressors. CCR6 is expressed on a heterogeneous population of memory CD4⁺ T cells that are critical to mucosal immunity. Preferential infection of these cells is associated, in part, with high surface expression of CCR5, CXCR4, and α4β7. In addition, CCR6⁺CD4⁺ T cells harbor elevated levels of integrated viral DNA and high levels of proliferation markers. We have previously shown that the CCR6 ligands MIP-3α and human beta defensins inhibit HIV replication. The inhibition required CCR6 and the induction of APOBEC3G. Here, we further characterize the induction of apolipoprotein B mRNA editing enzyme (APOBEC3G) by human beta defensin 2. Human beta defensin 2 rapidly induces transcriptional induction of APOBEC3G that involves extracellular signal-regulated kinases 1/2 (ERK1/2) activation and the transcription factors NFATc2, NFATc1, and IRF4. We demonstrate that human beta defensin 2 selectively protects primary CCR6⁺CD4⁺ T cells infected with HIV-1. The selective protection of CCR6⁺CD4⁺ T cell subsets may be critical in maintaining mucosal immune function and preventing disease progression.
Sex Bias in Pathogenesis of Autoimmune Neuroinflammation: Relevance for Dimethyl Fumarate Immunomodulatory/Anti-oxidant Action
May 23, 2017   Molecular Neurobiology
Stojić-Vukanić Z, Kotur-Stevuljević J, Nacka-Aleksić M, Kosec D, Vujnović I, Pilipović I, Dimitrijević M, Leposavić G
Sex Bias in Pathogenesis of Autoimmune Neuroinflammation: Relevance for Dimethyl Fumarate Immunomodulatory/Anti-oxidant Action
May 23, 2017
Molecular Neurobiology
In the present study, upon showing sexual dimorphism in dimethyl fumarate (DMF) efficacy to moderate the clinical severity of experimental autoimmune encephalomyelitis (EAE) in Dark Agouti rats, cellular and molecular substrate of this dimorphism was explored. In rats of both sexes, DMF administration from the day of immunization attenuated EAE severity, but this effect was more prominent in males leading to loss of the sexual dimorphism observed in vehicle-administered controls. Consistently, in male rats, DMF was more efficient in diminishing the number of CD4+ T lymphocytes infiltrating spinal cord (SC) and their reactivation, the number of IL-17+ T lymphocytes and particularly cellularity of their highly pathogenic IFN-γ+GM-CSF+IL-17+ subset. This was linked with changes in SC CD11b+CD45+TCRαβ- microglia/proinflammatory monocyte progeny, substantiated in a more prominent increase in the frequency of anti-inflammatory phygocyting CD163+ cells and the cells expressing high surface levels of immunoregulatory CD83 molecule (associated with apoptotic cells phagocytosis and implicated in downregulation of CD4+ T lymphocyte reactivation) among CD11b+CD45+TCRαβ- cells in male rat SC. These changes were associated with greater increase in the nuclear factor (erythroid-derived 2)-like 2 expression in male rats administered with DMF. In accordance with the previous findings, DMF diminished reactive nitrogen and oxygen species generation and consistently, SC level of advanced oxidation protein products, to the greater extent in male rats. Overall, our study indicates sex-specificity in the sensitivity of DMF cellular and molecular targets and encourages sex-based clinical research to define significance of sex for action of therapeutic agents moderating autoimmune neuroinflammation-/oxidative stress-related nervous tissue damage.
Effect of Lipoic Acid on the Biochemical Mechanisms of Resistance to Bortezomib in SH-SY5Y Neuroblastoma Cells
May 12, 2017   Molecular Neurobiology
Tibullo D, Giallongo C, Puglisi F, Tomassoni D, Camiolo G, Cristaldi M, Brundo MV, Anfuso CD, Lupo G, Stampone T, Li Volti G, Amenta F, Avola R, Bramanti V
Effect of Lipoic Acid on the Biochemical Mechanisms of Resistance to Bortezomib in SH-SY5Y Neuroblastoma Cells
May 12, 2017
Molecular Neurobiology
Neuroblastoma (NB) is an extracranial solid cancer and the most common cancer in infancy. Despite the standard treatment for NB is based on the combination of chemotherapeutic drugs such as doxorubicin, vincristine, cyclophosphamide, and cisplatin, chemoresistance occurs over the time. The aim of the present research was to evaluate the effect of bortezomib (BTZ) (50 nM) on NB cell viability and how lipoic acid (ALA) (100 μM) modifies pharmacological response to this chemotherapeutic agent. Cell viability was assessed by ATP luminescence assay whereas expression of oxidative stress marker (i.e., heme oxygenase-1) and endoplasmic reticulum stress proteins was performed by real-time PCR, western blot, and immunofluorescence. Our data showed that BTZ treatment significantly reduced cell viability when compared to untreated cultures (about 40%). Interestingly, ALA significantly reduced the efficacy of BTZ (about 30%). Furthermore, BTZ significantly induced heme oxygenase-1 as a result of increased oxidative stress and such overexpression was prevented by concomitant treatment with ALA. Similarly, ALA significantly reduced BTZ-mediated endoplasmic reticulum stress as measured by reduction in BiP1 and IRE1α, ERO1α, and PDI expression. In conclusion, our data suggest that BTZ efficacy is dependent on cellular redox status and such mechanisms may be responsible of chemoresistance to this chemotherapeutic agent.
Classic Spotlight, 1988 and 1989: Articles of Significant Interest Selected from the Journal of Virology Archives by the Editors
May 13, 2017   Journal Of Virology
The influenza A virus NS1 protein promotes efficient nuclear export of unspliced viral M1 mRNA
May 18, 2017   Journal Of Virology
Pereira CF, Read EKC, Wise HM, Amorim MJ, Digard P
The influenza A virus NS1 protein promotes efficient nuclear export of unspliced viral M1 mRNA
May 18, 2017
Journal Of Virology
Influenza A virus mRNAs are transcribed by the viral RNA-dependent RNA polymerase in the cell nucleus before being exported to the cytoplasm for translation. Segment 7 produces two major transcripts: an unspliced mRNA that encodes the M1 matrix protein and a spliced transcript that encodes the M2 ion channel. Export of both mRNAs is dependent on the cellular NXF1/TAP pathway but it is unclear how they are recruited to the export machinery or how the intron-containing but unspliced M1 mRNA bypasses the normal quality control checkpoints. Using fluorescent in situ hybridization to monitor segment 7 mRNA localisation, we found that cytoplasmic accumulation of unspliced M1 mRNA was inefficient in the absence of NS1, both in the context of segment 7 RNPs reconstituted by plasmid transfection and in mutant virus-infected cells. This effect was independent of any major effect on steady-state levels of segment 7 mRNA or splicing, but corresponded to a ∼ 5-fold reduction in the accumulation of M1. A similar defect in intronless HA mRNA nuclear export was seen with an NS1 mutant virus. Efficient export of M1 mRNA required both an intact NS1 RNA-binding domain and effector domain. Furthermore, while wildtype NS1 interacted with cellular NXF1 and also increased the interaction of segment 7 mRNA with NXF1, mutant NS1 polypeptides unable to promote mRNA export did neither. Thus we propose that NS1 facilitates late viral gene expression by acting as an adaptor between viral mRNAs and the cellular nuclear export machinery to promote their nuclear export.IMPORTANCE Influenza A virus is a major pathogen of a wide variety of mammalian and avian species that threatens public health and food security. A fuller understanding of the virus life cycle is important to aid control strategies. The virus has a small genome that encodes for relatively few proteins that are often multifunctional. Here, we characterise a new function for the NS1 protein, showing that as well as previously identified roles in antagonising the innate immune defenses of the cell and directly upregulating translation of viral mRNAs, it also promotes the nuclear export of the viral late gene mRNAs by acting as an adaptor between the viral mRNAs and the cellular mRNA nuclear export machinery. Copyright © 2017 Pereira et al.
Natural variation of Epstein-Barr virus genes, proteins and pri-miRNA (revised)
May 18, 2017   Journal Of Virology
Correia S, Palser A, Elgueta Karstegl C, Middeldorp JM, Ramayanti O, Cohen JI, Hildesheim A, Fellner MD, Wiels J, White RE, Kellam P, Farrell PJ
Natural variation of Epstein-Barr virus genes, proteins and pri-miRNA (revised)
May 18, 2017
Journal Of Virology
Viral gene sequences from an enlarged set of about 200 Epstein-Barr virus (EBV) strains including many primary isolates have been used to investigate variation in key viral genetic regions, particularly LMP1, Zp, gp350, EBNA1 and the BART miRNA cluster 2. Determination of type 1 and type 2 EBV in saliva samples from people from a wide range of geographic and ethnic backgrounds demonstrates a small percentage of healthy white Caucasian British people carrying predominantly type 2 EBV. Linkage of Zp and gp350 variants to type 2 EBV is likely to be due to their genes being adjacent to the EBNA3 locus, which is one of the major determinants of the type 1/type 2 distinction. A novel classification of EBNA1 DNA binding domains named QCIGP results from phylogeny analysis of their protein sequences but is not linked to the type 1/type 2 classification. The BART cluster 2 miRNA region is classified into three major variants through SNPs in the pri-miRNA outside of the mature miRNA sequences. These SNPs can result in altered levels of expression of some miRNAs from the BART variant frequently present in Chinese and Indonesian nasopharyngeal carcinoma (NPC) samples. The EBV genetic variants identified here provide a basis for future more directed analysis of association of specific EBV variation with EBV biology and EBV associated diseases.IMPORTANCE Incidence of diseases associated with EBV varies greatly in different parts of the world. Relationships between EBV genome sequence variation and health, disease, geography and ethnicity of the host may thus be important for understanding the role of EBV in diseases and for development of an effective EBV vaccine. This paper provides the most comprehensive analysis so far of variation in specific EBV genes relevant to these diseases and proposed EBV vaccines. By focussing on variation in LMP1, Zp, gp350, EBNA1 and the BART miRNA cluster 2, new relationships to the known type 1/type 2 strains are demonstrated and novel classification of EBNA1 and the BART miRNAs is proposed. Copyright © 2017 Correia et al.
Deletion of the Vaccinia B1 Kinase Reveals Essential Functions of this Enzyme Complemented Partly by the Homologous Cellular Kinase VRK2
May 18, 2017   Journal Of Virology
Olson AT, Rico AB, Wang Z, Delhon G, Wiebe MS
Deletion of the Vaccinia B1 Kinase Reveals Essential Functions of this Enzyme Complemented Partly by the Homologous Cellular Kinase VRK2
May 18, 2017
Journal Of Virology
The vaccinia B1 kinase is highly conserved among poxviruses and is essential for the viral lifecycle. B1 exhibits a remarkable degree of similarity to VRKs, a family of cellular kinases, suggesting that the viral enzyme has evolved to mimic VRK activity. Indeed, B1 and VRKs have been demonstrated to target a shared substrate, the DNA binding protein BAF, elucidating a signaling pathway important for both mitosis and the antiviral response. In this study, we further characterize the role of B1 during vaccinia infection to gain novel insights into its regulation and integration with cellular signaling pathways. We begin by describing the construction and characterization of the first B1 deletion virus (vvΔB1) produced using a complementing cell line expressing the viral kinase. Examination of vvΔB1 revealed that B1 is critical for production of infectious virions in various cell types and is sufficient for BAF phosphorylation. Interestingly, the severity of the defect in DNA replication following loss of B1 varied between cell types, leading us to posit that cellular VRKs partly complement for B1 in some cell lines. Using cell lines devoid of either VRK1 or VRK2 we tested this hypothesis and discovered that VRK2 expression facilitates DNA replication and allows later stages of the viral lifecycle to proceed in the absence of B1. Finally, we present evidence that the impact of VRK2 on vaccinia is largely independent of BAF phosphorylation. These data support a model in which B1 and VRK2 share additional substrates important for replication of cytoplasmic poxviruses.IMPORTANCE Viral mimicry of cellular signaling modulators provides clear evidence that the pathogen is targeting an important host pathway during infection. Poxviruses employ numerous viral homologs of cellular proteins, the study of which have yielded insights into signaling pathways used by both virus and cells alike. The vaccinia B1 protein is a homolog of cellular VRKs (vaccinia-related kinases) and is needed for viral DNA replication and likely other stages of the viral lifecycle. However, much remains to be learned about how B1 and VRKs overlap functionally. This study utilizes new tools including a B1 deletion virus and VRK knockout cells to further characterize the functional links between the viral and cellular enzymes. As a result, we have discovered that B1 and VRK2 target a common set of substrates vital to productive infection of this large cytoplasmic DNA virus. Copyright © 2017 American Society for Microbiology.
Cell cycle-dependent expression of AAV2 Rep in HSV-1 co-infections gives rise to a mosaic of cells replicating either AAV2 or HSV-1
May 18, 2017   Journal Of Virology
Franzoso FD, Seyffert M, Vogel R, Yakimovich A, de Andrade Pereira B, Meier AF, Sutter SO, Tobler K, Vogt B, Greber UF, Büning H, Ackermann M, Fraefel C
Cell cycle-dependent expression of AAV2 Rep in HSV-1 co-infections gives rise to a mosaic of cells replicating either AAV2 or HSV-1
May 18, 2017
Journal Of Virology
Adeno-associated virus 2 (AAV2) depends for productive replication on the simultaneous presence of a helper virus such as herpes simplex virus type 1 (HSV-1). At the same time, AAV2 efficiently blocks the replication of HSV-1, which would eventually limit its own replication by diminishing the helper virus reservoir. This discrepancy begs the question how AAV2 and HSV-1 can co-exist in a cell population. Here we show that in co-infected cultures, AAV2 DNA replication takes place almost exclusively in S/G2 cells, while HSV-1 DNA replication is restricted to G1. Live microscopy revealed that not only wtAAV2 replication but also reporter gene expression from both single-stranded and double-stranded (self-complementary) recombinant AAV2 vectors preferentially occurs in S/G2 cells, suggesting that the S/G2 preference is independent of the nature of the viral genome. Interestingly, however, a substantial proportion of the S/G2 cells transduced by the double-stranded but not the single-stranded recombinant AAV2 vectors progressed through mitosis in absence of the helper virus. We conclude that cell cycle-dependent AAV2 rep expression facilitates cell cycle-dependent AAV2 DNA replication, and inhibits HSV-1 DNA replication. This may limit competition for cellular and viral helper factors, and hence, creates a biological niche for either virus to replicate.IMPORTANCE Adeno-associated virus 2 (AAV2) differs from most other viruses, as it requires not only a host cell for replication but also a helper virus such as an adenovirus or a herpes virus. This situation inevitably leads to competition for cellular resources. AAV2 has been shown to efficiently inhibit the replication of helper viruses. Here, we present a new facet of the interaction between AAV2 and one of its helper viruses, herpes simplex virus type 1 (HSV-1). We observed that AAV2 rep gene expression is cell cycle dependent, and gives rise to distinct time controlled windows for HSV-1 replication. High Rep protein levels in S/G2 phases support AAV2 replication and inhibit HSV-1 replication. Conversely, low Rep protein levels in G1 permit HSV-1 replication, but are insufficient for AAV2 replication. This allows both viruses to productively replicate in distinct sets of dividing cells. Copyright © 2017 American Society for Microbiology.
The LspC3-41I restriction-modification system is the major determinant for genetic manipulations of Lysinibacillus sphaericus C3-41
May 20, 2017   BMC Microbiology
Fu P, Ge Y, Wu Y, Zhao N, Yuan Z, Hu X
The LspC3-41I restriction-modification system is the major determinant for genetic manipulations of Lysinibacillus sphaericus C3-41
May 20, 2017
BMC Microbiology
Lysinibacillus sphaericus has been widely used in integrated mosquito control program and it is one of the minority bacterial species unable to metabolize carbohydrates. In consideration of the high genetic conservation at genomic level and difficulty of genetic horizontal transfer, it is hypothesized that effective restriction-modification (R-M) systems existed in mosquitocidal L. sphaericus. In this study, six type II R-M systems including LspC3-41I were predicted in L. sphaericus C3-41 genome. It was found that the cell free extracts (CFE) from this strain shown similar restriction and methylation activity on exogenous Bacillus/Escherichia coli shuttle vector pBU4 as the HaeIII, which is an isoschizomer of BspRI. The Bsph_0498 (encoding the predicted LspC3-41IR) knockout mutant Δ0498 and the complement strain RC0498 were constructed. It was found that the unmethylated pBU4 can be digested by the CFE of C3-41 and RC0498, but not by that of Δ0498. Furthermore, the exogenous plasmid pBU4 can be transformed at very high efficacy into Δ0498, low efficacy into RC0498, but no transformation into C3-41, indicating that LspC3-41I might be a major determinant for the genetic restriction barrier of strain C3-41. Besides, lspC3-41IR and lspC3-41IM genes are detected in other two strains besides C3-41 of the tested 16 L. sphaericus strains, which all belonging to serotype H5 and MLST sequence type (ST) 1. Furthermore, the three strains are not horizontal transferred, and this restriction could be overcome by in vitro methylation either by the host CFE or by commercial methytransferase M. HaeIII. The results provide an insight to further study the genetic restriction, modification and evolution of mosquitocidal L. sphaericus, also a theoretical basis and a method for the genetic manipulations of L. sphaericus. LspC3-41I is identified as the major determinant for the restriction barrier of L. sphaericus C3-41. Only three strains of the tested 16 L. sphaericus strains, which all belonging to serotype H5 and ST1 by MLST scheme, contain LspC3-41I system. Two different methods can be used to overcome the restriction barrier of the three isolates to get transformants efficiently: 1) to methylate plasmid DNA prior to the electroporation; and 2) to delete the major restriction endonuclease encoding gene lspC3-41IR.
Nasal decontamination for the prevention of surgical site infection in Staphylococcus aureus carriers
May 18, 2017   The Cochrane Database Of Systematic Reviews
Liu Z, Norman G, Iheozor-Ejiofor Z, Wong JK, Crosbie EJ, Wilson P
Nasal decontamination for the prevention of surgical site infection in Staphylococcus aureus carriers
May 18, 2017
The Cochrane Database Of Systematic Reviews
Surgical site infection rates in the month following surgery vary from 1% to 5%. Due to the large number of surgical procedures conducted annually, the costs of these surgical site infections (SSIs) can be considerable in financial and social terms. Nasal decontamination using antibiotics or antiseptics is performed to reduce the risk of SSIs by preventing organisms from the nasal cavity being transferred to the skin where a surgical incision will be made. Staphylococcus aureus (S aureus) colonises the nasal cavity and skin of carriers and can cause infection in open or unhealed surgical wounds. S aureus is the leading nosocomial (hospital-acquired) pathogen in hospitals worldwide. The potential effectiveness of nasal decontamination of S aureus is thought to be dependent on both the antibiotic/antiseptic used and the dose of application; however, it is unclear whether nasal decontamination actually reduces postoperative wound infection in S aureus carriers. To assess the effects of nasal decontamination on preventing surgical site infections (SSIs) in people who are S aureus carriers undergoing surgery. In September 2016 we searched the Cochrane Wounds Specialised Register, the Cochrane Central Register of Controlled Trials (CENTRAL) (the Cochrane Library), Ovid MEDLINE, Ovid MEDLINE (In-Process & Other Non-Indexed Citations), Ovid Embase, and EBSCO CINAHL Plus. We also searched three clinical trial registries and the references of included studies and relevant systematic reviews. There were no restrictions based on language, date of publication or study setting. Randomised controlled trials (RCTs) which enrolled S aureus carriers with any type of surgery and assessed the use of nasal decontamination with antiseptic/antibiotic properties were included in the review. Two review authors independently performed study selection, data extraction, risk of bias assessment and GRADE assessment. We located two studies (291 participants) for inclusion in this review. The trials were clinically heterogeneous with differences in duration of follow-up, and nasal decontamination regimens. One study compared mupirocin (2% contained in a base of polyethylene glycol 400 and polyethylene glycol 3350) with a placebo in elective cardiac surgery patients; and one study compared Anerdian (iodine 0.45% to 0.57% (W/V), chlorhexidine acetate 0.09% to 0.11% (W/V)) with no treatment also in cardiac surgery patients. The trials reported limited outcome data on SSI, adverse events and secondary outcomes (e.g. S aureus SSI, mortality). Mupirocin compared with placeboThis study found no clear difference in SSI risk following use of mupirocin compared with placebo (1 trial, 257 participants); risk ratio (RR) 1.60, 95% confidence interval (CI) 0.79 to 3.25 based on 18/130 events in the mupirocin group and 11/127 in the control group; low-certainty evidence (downgraded twice due to imprecision). Anerdian compared with no treatmentIt is uncertain whether there is a difference in SSI risk following treatment with Anerdian compared with no treatment (1 trial, 34 participants); RR 0.89, 95% CI 0.06 to 13.08 based on 1/18 events in the Anerdian group and 1/16 in the control group; very low certainty evidence (downgraded twice due to imprecision and once due to risk of bias). There is currently limited rigorous RCT evidence available regarding the clinical effectiveness of nasal decontamination in the prevention of SSI. This limitation is specific to the focused question our review addresses, looking at nasal decontamination as a single intervention in participants undergoing surgery who are known S aureus carriers. We were only able to identify two studies that met the inclusion criteria for this review and one of these was very small and poorly reported. The potential benefits and harms of using decontamination for the prevention of SSI in this group of people remain uncertain.

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