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Supplementary Figures S1-S6, Table S1-S2, and Materials and Methods. Figure S1 shows the distribution of macrophages and ISG15 expression in tissue from patient PDAC tumors, analyzed by IHC, and the expression of the CSC cell surface marker CD133 on PDAC cells cultured as adherent monolayers or as spheres. Figure S2 shows the growth of PDAC cells in vivo alone or with polarized human macrophages. Figure S3-S5 is a comprehensive IHC or IF analysis of ISG15 expression in multiple PDAC, breast and prostate cancer patient tumors. Figure S6 shows the migratory capacity of murine PDAC CSCs primed with control media or CM from M2-polarized ISG15+/+ or ISG15-/- murine macrophages. Table S1 lists the antibodies used in this study. Table S2 lists the sequences of the primers used for RTqPCR analysis.

SummaryThe ubiquitin-like protein, ISG15, can act as a cytokine or can covalently modify host and pathogen-derived proteins. The consequences of ISG15 modification on substrate fate remain unknown. Here we reveal that ISGylation of the Arp2/3 complex slows actin filament formation and stabilizes Arp2/3 dependent structures including cortical actin and lamella. When properly controlled, this serves as an antibacterial and antiviral host defense strategy to directly restrict actin-mediated pathogen spread. However,Listeria monocytogenestakes advantage in models of dysregulated ISGylation, leading to increased mortality due to augmented spread. The underlying molecular mechanism responsible for the ISG15-dependent impact on actin-based motility is due to failed bacterial separation after division. This promotes spread by enabling the formation of multi-headed bacterial “bazookas” with stabilized comet tails that can disseminate deeper into tissues. A bacterial mutant that cannot recruit Arp2/3 or a non-ISGylatable mutant of Arp3 is sufficient to rescue slowed comet tail speed and restrict spread. Importantly, ISG15-deficient neonatal mice have aberrant epidermal epithelia characterized by keratinocytes with diffuse cortical actin, which could underlie observed defects in wound healing in human patients who lack ISG15. Ultimately, our discovery links host innate immune responses to cytoskeletal dynamics with therapeutic implications for viral infection and metastasis.

The role of ISG15 (Interferon-Stimulated Gene 15) is becoming increasingly acknowledged in cancer, pathogen infection and inflammatory immune diseases. ISG15 expression increases in human psoriatic skin, and genetic mutations of ISG15 cause dermatological alterations. Here, we show that ISG15 deficiency leads to reduced inflammation and swelling in a murine model of allergic contact dermatitis (ACD). Bone marrow transplantation and adoptive transfer approaches demonstrate that ISG15 alters dendritic cell (DC) responses in the ACD mouse model. Consistently, Isg15-deficient DCs secrete reduced amounts of pro-inflammatory cytokines, including IL-1β and IL-12. This study opens new avenues to potentiate immunotherapies to treat immune-related dermatological disorders.

Viruses have developed many different strategies to counteract immune responses, andVaccinia virus(VACV) is one of a kind in this aspect. To ensure an efficient infection, VACV undergoes a complex morphogenetic process resulting in the production of two types of infective virions: intracellular mature virus (MV) and extracellular enveloped virus (EV), whose spread depends on different dissemination mechanisms. MVs disseminate after cell lysis, whereas EVs are released or propelled in actin tails from living cells. Here we show that ISG15 participates in the control of VACV dissemination. Infection ofIsg15-/-mouse embryonic fibroblasts with VACV International Health Department-J (IHD-J) strain resulted in decreased EV production, concomitant with reduced induction of actin tails and the abolition of comet-shaped plaque formation, comparing withIsg15+/+cells. Transmission electron microscopy revealed accumulation of intracellular and a decrease in extracellular virus particles in the absence of Interferon Stimulated Gene 15 (ISG15), consistent with altered virus egress. Immunoblot and quantitative proteomic analysis of sucrose gradient-purified virions from both genotypes reported differences in protein levels and composition of viral proteins present on virions, suggesting an ISG15-mediated control of viral proteome. Last, the generation of a recombinant IHD-J expressing V5-tagged ISG15 (IHD-J-ISG15) allowed us to identify several viral proteins as potential ISG15 targets, highlighting the proteins A34 and A36, essential for EV formation. Altogether, our results indicate that ISG15 is an important host factor in the regulation of VACV dissemination.Author SummaryViral infections are a constant battle between the virus and the host. While the host’s only goal is victory, the main purpose of the virus is to spread and conquer new territories at the expense of the host’s resources. Along millions of years of incessant encounters, Poxviruses have developed a unique strategy consisting in the production two specialized “troops”: intracellular mature virions (MVs) and extracellular virions (EVs). MVs mediate transmission between hosts, and EVs ensure advance on the battlefield mediating the long-range dissemination.The mechanism by which the virus ‘decides’ to shed from the primary site of infection and its significant impact in viral transmission is not yet fully established. Here, we demonstrate that this process is finely regulated by ISG15/ISGylation, an interferon-induced ubiquitin-like protein with broad antiviral activity. Studying the mechanism that viruses use during infection could result in new ways of understanding our perpetual war against disease and how we might win the next great battle.

Monkeypox virus (MPXV) is a zoonotic orthopoxvirus within the Poxviridae family. MPXV is endemic to Central and West Africa. However, the world is currently witnessing an international outbreak with no clear epidemiological links to travel or animal exposure and with ever-increasing numbers of reported cases worldwide. Here, we evaluated and validated a new, sensitive, and specific real-time PCR-assay for MPXV diagnosis in humans and compare the performance of this novel assay against a FoodDrug Administration-cleared pan-Orthopox RT-PCR assay. We determined specificity, sensitivity, and analytic performance of the PKamp™ Monkeypox Virus RT-PCR assay targeting the viral F3L-gene. In addition, we further evaluated MPXV-PCR-positive specimens by viral culture, electron microscopy, and viral inactivation assays. The limit of detection was established at 7.2 genome copies/reaction, and MPXV was successfully identified in 20 clinical specimens with 100% correlation against the reference method with 100% sensitivity and specificity. Our results demonstrated the validity of this rapid, robust, and reliable RT-PCR assay for specific and accurate diagnosis of MPXV infection in human specimens collected both as dry swabs and in viral transport media. This assay has been approved by NYS Department of Health for clinical use.

SUMMARYThe currently expanding monkeypox epidemic is caused by a subclade IIb descendant of a monkeypox virus (MPXV) lineage traced back to Nigeria in 1971. In contrast to monkeypox cases caused by clade I and subclade IIa MPXV, the prognosis of current cases is generally favorable, but person-to-person transmission is much more efficient. MPXV evolution is driven by selective pressure from hosts and loss of virus–host interacting genes. However, there is no satisfactory genetic explanation using single-nucleotide polymorphisms (SNPs) for the observed increased MPXV transmissibility. We hypothesized that key genomic changes may occur in the genome’s low-complexity regions (LCRs), which are highly challenging to sequence and have been dismissed as uninformative. Using a combination of highly sensitive techniques, we determined a first high-quality MPXV genome sequence of a representative of the current epidemic with LCRs resolved at unprecedented accuracy. This effort revealed significant variation in short-tandem repeats within LCRs. We demonstrate that LCR entropy in the MPXV genome is significantly higher than that of SNPs and that LCRs are not randomly distributed.In silicoanalyses indicate that expression, translation, stability, or function of MPXV orthologous poxvirus genes (OPGs) 153, 204, and 208 could be affected in a manner consistent with the established “genomic accordion” evolutionary strategies of orthopoxviruses. Consequently, we posit that genomic studies focusing on phenotypic MPXV clade-/subclade-/lineage-/strain differences should change their focus to the study of LCR variability instead of SNP variability.

PrimPol is the second primase in human cells, the first with the ability to start DNA chains with dNTPs. PrimPol contributes to DNA damage tolerance by restarting DNA synthesis beyond stalling lesions, acting as a TLS primase. Multiple alignment of eukaryotic PrimPols allowed us to identify a highly conserved motif, WxxY near the invariant motif A, which contains two active site metal ligands in all members of the archeo-eukaryotic primase (AEP) superfamily. In vivo and in vitro analysis of single variants of the WFYY motif of human PrimPol demonstrated that the invariant Trp87 and Tyr90 residues are essential for both primase and polymerase activities, mainly due to their crucial role in binding incoming nucleotides. Accordingly, the human variant F88L, altering the WFYY motif, displayed reduced binding of incoming nucleotides, affecting its primase/polymerase activities especially during TLS reactions on UV-damaged DNA. Conversely, the Y89D mutation initially associated with High Myopia did not affect the ability to rescue stalled replication forks in human cells. Collectively, our data suggest that the WFYY motif has a fundamental role in stabilizing the incoming 3′-nucleotide, an essential requisite for both its primase and TLS abilities during replication fork restart., BFU2015-65880-P (MINECO) and PGC2018-093576-B.C21 (MCI/AEI/FEDER,UE) to L.B., BFU2016-80402-R (MINECO/FEDER, UE) and PID2019-106707RB-100 (AEI/10.13039/501100011033) to J.M., Kempe JCK 1831 to G.S, Knut och Alice Wallenbergs Foundation KAW 2019.0307, VR-2018-02781, to S.W., and by institutional grants from Fundación Ramón Areces and Banco de Santander

Resumen Introducción: el objetivo de este estudio fue identificar los factores condicionantes de la desigualdad en el acceso al mercado laboral y remuneración en los profesionales de salud del Perú. Materiales y métodos: se realizó un estudio observacional basado en el análisis secundario de los principales resultados de la Encuesta Nacional a Egresados Universitarios y Universidades, 2014 con una muestra de 1974 profesionales de la salud sometida a expansión para realizar el cálculo de chi cuadrado, odds ratio crudo (ORC) y odds ratio ajustado (oita) mediante regresión logística, con sus intervalos de confianza al 95 %. Resultados: existió mayor riesgo de desocupación en mujeres (ORa: 1.574; IC95 %: 1.456-1.702), en universidades públicas (ORa: 1.137; IC95 %: 1.068-1.210) o ubicadas en provincias diferentes a Lima y Callao (ORa: 1.552; IC95 %: 1.452-1.661). La autoidentificación como mestizos (ORa: 0.704; IC95 %: 0.621-0.798), quechuas (ORa: 0.653; IC95 %: 0.556-0.767), aymaras (ORa: 0.679; IC95 %: 0.511-0.902) y otros (ORa: 0.549; IC95 %: 0.432-0.698) tuvo un efecto protector frente a la desocupación. Las mujeres (ORa: 1.580; IC95 %: 1.4841.682), los quechuas (ORa: 1.259; IC95 %: 1.092-1.451) y los egresados de universidades ubicadas fuera de Lima y Callao (ORa: 2.601; IC95 %: 2.455-2.756) presentaron mayor riesgo de remuneración inferior a los 1000 soles; en cambio, el egreso de una universidad pública (ORa: 0.784; IC95 %: 0.744-0.828) disminuyó la probabilidad de remuneración menor a 1000 soles. Conclusión: el sexo, la etnia, el centralismo y el nivel socioeconómico estarían condicionando la desigualdad en el mercado laboral, a pesar del mayor nivel de instrucción obtenido por los profesionales de salud. Abstract Introduction: This study aimed to identify the conditioning factors of the disparity in the access to the labor market and remuneration for health professionals from Peru. Materials and Methods: An observational study was conducted based on the secondary analysis from the main results of the National Survey of University Graduates and Universities, 2014 involving 1974 health professionals undergoing expansion using chi-square test, crude odds ratio (COR), and adjusted odds ratio (aOR) by logistic regression with 95% confidence intervals (cis). Results: There was greater risk of unemployment in women (aOR: 1.574; 95% ci: 1.456-1.702) and those who graduated from public universities (aOR: 1.137; 95% ci: 1.068-1.210) or from provinces other than Lima and Callao (aOR: 1.552; 95% ci: 1.452-1.661). Self-identification as mestizos (aOR: 0.704; 95% ci: 0.621-0.798), Quechua (aOR: 0.653; 95%ci: 0.556-0.767), Aymara (aOR: 0.679; 95% ci: 0.511-0.902), and others (aOR: 0.549; 95% ci: 0.432-0.698) had a protective effect against unemployment. Women (aOR: 1.580; 95% ci: 1.484-1.682), Quechua (aOR: 1.259; 95% ci: 1.092-1.451), and university graduates who studied outside of Lima and Callao (aOR: 2.601; 95% ci: 2.455-2.756) presented a higher risk of remuneration of less than 1000 soles. Moreover, graduating from a public university (aOR: 0.784; 95% ci: 0.744-0.828) decreased the probability of remuneration of less than 1000 soles. Conclusion: Sex, ethnicity, centralism, and socioeconomic level are conditioning factors of the disparity in the access to the labor market despite the higher level of education obtained by health professionals. Resumo Introdução: O objetivo deste estudo foi identificar os fatores condicionantes da desigualdade no acesso ao mercado laboral e remuneração nos profissionais da saúde do Peru. Materiais e métodos: se realizou um estudo observacional baseado na análise secundária dos principais resultados do Inquérito Nacional de Graduados Universitários e Universidades, 2014 com uma amostra de 1974 profissionais da saúde submetida a expansão para realizar o cálculo de qui-quadrado, odds ratio cru (ORC) e odds ratio ajustado (ORa) mediante regressão logística, com seus intervalos de confiança ao 95%. Resultados: existiu maior risco de desocupação em mulheres (ORa: 1,574; IC95%: 1,456-1,702), de universidades públicas (ORa: 1,137; IC95%: 1,068-1,210) oo localizadas em províncias diferentes a Lima e Callao (ORa: 1,552; IC95%: 1,4521,661). A auto-identificação como mestiços (ORa: 0,704; IC95%: 0,621-0,798), quéchuas (ORa: 0,653; IC95%: 0,556-0,767), aymaras (ORa: 0,679; IC95%: 0,511-0,902) e outros (ORa: 0,549; IC95%: 0,432-0,698) teve um efeito protetor frente ao desemprego. As mulheres (ORa: 1,580; IC95%: 1,484-1,682), os quéchuas (ORa: 1,259; IC95%: 1,092-1,451) e os graduados de universidades localizadas fora de Lima e Callao (ORa: 2,601; IC95%: 2,455-2,756) apresentaram maior risco de remuneração inferior aos 1000 soles; em contraste, a graduação de uma universidade pública (ORa: 0,784; IC95%: 0,744-0,828) diminuiu a probabilidade de remuneração menor a 1000 soles. Conclusões: o sexo, a etnia, o centralismo e o nível socioeconómico estariam condicionando a desigualdade no mercado laboral apesar do maior nível de instrução obtido pelos profissionais de saúde.

Introdução: O objetivo deste estudo foi identificar os fatores condicionantes da desigualdade no acesso ao mercado laboral e remuneração nos profissionais da saúde do Peru. Materiais e métodos: se realizou um estudo observacional baseado na análise secundária dos principais resultados do Inquérito Nacional de Graduados Universitários e Universidades, 2014 com uma amostra de 1974 profissionais da saúde submetida a expansão para realizar o cálculo de qui-quadrado, odds ratio cru (ORC) e odds ratio ajustado (ORA) mediante regressão logística, com seus intervalos de confiança ao 95%. Resultados: existiu maior risco de desocupação em mulheres (ORA: 1,574; IC95%: 1,456-1,702), de universidades públicas (ORA: 1,137; ic95%: 1,068-1,210) oo localizadas em províncias diferentes a Lima e Callao (ORA: 1,552; IC95%: 1,452-1,661). A auto-identificação como mestiços (ORA: 0,704; IC95%: 0,621-0,798), quéchuas (ORA: 0,653; IC95%: 0,556-0,767), aymaras (ORA: 0,679; IC95%: 0,511-0,902) e outros (ORA: 0,549; ic95%: 0,432-0,698) teve um efeito protetor frente ao desemprego. As mulheres (ORA: 1,580; IC95%: 1,484-1,682), os quéchuas (ORA: 1,259; IC95%: 1,092-1,451) e os graduados de universidades localizadas fora de Lima e Callao (ORA: 2,601; IC95%: 2,455-2,756) apresentaram maior risco de remuneração inferior aos 1000 soles; em contraste, a graduação de uma universidade pública (ORA: 0,784; IC95%: 0,744-0,828) diminuiu a probabilidade de remuneração menor a 1000 soles. Conclusões: o sexo, a etnia, o centralismo e o nível socioeconômico estariam condicionando a desigualdade no mercado laboral apesar do maior nível de instrução obtido pelos profissionais de saúde.

Induction of the endogenous innate immune system by interferon (IFN) triggers the expression of many proteins that serve like alarm bells in the body, activating an immune response. After a viral infection, one of the genes activated by IFN induction is the IFN-stimulated gene 15 (ISG15), which encodes a ubiquitin-like protein that undergoes a reversible posttranslational modification (ISGylation). ISG15 protein can also act unconjugated, intracellularly and secreted, acting as a cytokine. Although ISG15 has an essential role in host defense responses to microbial infection, its role as an immunomodulator in the vaccine field remains to be defined. In this investigation, we showed that ISG15 exerts an immunomodulatory role in human immunodeficiency virus (HIV) vaccines. In mice, after priming with a DNA-ISG15 vector mixed with a DNA expressing HIV-1 gp120 (DNA-gp120), followed by a booster with a modified vaccinia virus Ankara (MVA) vector expressing HIV-1 antigens, both wild-type ISG15-conjugated (ISG15-wt) and mutant unconjugated (ISG15-mut) proteins act as immune adjuvants by increasing the magnitude and quality of HIV-1-specific CD8 T cells, with ISG15-wt providing better immunostimulatory activity than ISG15-mut. The HIV-1 Env-specific CD8 T cell responses showed a predominant T effector memory (TEM) phenotype in all groups. Moreover, the amount of DNA-gp120 used to immunize mice could be reduced 5-fold after mixing with DNA-ISG15 without affecting the potency and the quality of the HIV-1 Env-specific immune responses. Our study clearly highlights the potential use of the IFN-induced ISG15 protein as immune adjuvant to enhance immune responses to HIV antigens, suggesting that this molecule might be exploitable for prophylactic and therapeutic vaccine approaches against pathogens. IMPORTANCE Our study described the potential role of ISG15 as an immunomodulatory molecule in the optimization of HIV/AIDS vaccine candidates. Using a DNA prime–MVA boost immunization protocol, our results indicated an increase in the potency and the quality of the HIV-1 Env-specific CD8 T cell response. These results highlight the adjuvant potency of ISG15 to elicit improved viral antigen presentation to the immune system, resulting in an enhanced HIV-1 vaccine immune response. The DNA-ISG15 vector could find applicability in the vaccine field in combination with other nucleic acid-based vector vaccines.

PrimPol is the second primase discovered in eukaryotic cells, whose function is to restart the stalled replication forks during both mitochondrial and nuclear DNA replication. This chapter revises our current knowledge about the mechanism of synthesis of DNA primers by human PrimPol, and the importance of its distinctive Zn-finger domain (ZnFD). After PrimPol forms a binary complex with ssDNA, the formation of the pre-ternary complex strictly requires the presence of Mn

O preconceito em relacao a pobreza, e especialmente em relacao a pobreza infantil, construido com base em pesados estereotipos de classe, carrega uma serie de imagens e representacoes negativas associadas. Demasiadas vezes associada a errância e a delinquencia, a infância pobre e vista como uma ameaca a sociedade, e configura um objeto incomodo sobre o qual se tem dificuldade em olhar, porque da visibilidade a um lado perturbador da nossa realidade social. O presente trabalho propoe que nos detenhamos sobre o modo como a infância em estado de excecao foi representada pelo cinema. A partir de Os Esquecidos, de Luis Bunuel e Os Incompreendidos, de Francois Truffaut, tentaremos entender como a pobreza infantil nos e dada a ver, ao mesmo tempo que questionamos o papel do cinema no exercicio da resistencia contra o fenomeno da invisibilidade da pobreza, bem como o alcance e os limites da nossa reacao as imagens que projeta. Palavras-chave : Estado de excecao - Infância - Cinema - Pobreza CHILDHOOD AND POVERTY: CHILDREN CARING FOR THE CINEMA Prejudice against poverty, and especially child poverty, built on heavy class stereotypes, carries a series of associated negative images and representations. Too often associated with wandering and delinquency, poor childhood is seen as a threat to society, and it is an uncomfortable object to look at because it gives visibility to a disturbing side of our social reality. This paper proposes that we focus on the way in which childhood in an exception state was represented by cinema. From Luis Bunuel's “Os Esquecidos” and Francois Truffaut's “Os Incompreendidos”, we will try to understand how child poverty is given to us, while questioning the role of cinema in the exercise of resistance against the phenomenon of invisibility of poverty, as well as the range and limits of our reaction to the images we project. Keywords : State of exception - Childhood - Cinema - Poverty.

PrimPol is a human primase/polymerase specialized in downstream repriming of stalled forks during both nuclear and mitochondrial DNA replication. Like most primases and polymerases, PrimPol requires divalent metal cations, as Mg2+ or Mn2+, used as cofactors for catalysis. However, little is known about the consequences of using these two metal cofactors in combination, which would be the most physiological scenario during PrimPolmediated reactions, and the individual contribution of the putative carboxylate residues (Asp114, Glu116 and Asp280) acting as metal ligands. By site-directed mutagenesis in human PrimPol, we confirmed the catalytic relevance of these three carboxylates, and identified Glu116 as a relevant enhancer of distinctive PrimPol reactions, which are highly dependent on Mn2+. Herein, we evidenced that PrimPol Glu116 contributes to errorprone tolerance of 8oxodG more markedly when both Mg2+ and Mn2+ ions are present. Moreover, Glu116 was important for TLS events mediated by primer/template realignments, and crucial to achieving an optimal primase activity, processes in which Mn2+ is largely preferred. EMSA analysis of PrimPol:ssDNA:dNTP pre-ternary complex indicated a critical role of each metal ligand, and a significant impairment when Glu116 was changed to a more conventional aspartate. These data suggest that PrimPol active site requires a specific motif A (DxE) to favor the use of Mn2+ ions in order to achieve optimal incoming nucleotide stabilization, especially required during primer synthesis., Spanish Ministry of Economy and Competitiveness (BFU2012−3769 and BFU2015−65880-P) to L.B. P.A.C. was the recipient of a FPI-predoctoral fellowship from Spanish Ministry of Economy and Competitiveness. G.S.M. received a JAE-predoctoral fellowship from the Spanish Research Council (CSIC).

PrimPol is a human primase/polymerase specialized in re-starting stalled forks by repriming beyond lesions such as pyrimidine dimers, and replication-perturbing structures including G-quadruplexes and R-loops. Unlike most conventional primases, PrimPol proficiently discriminates against ribonucleotides (NTPs), being able to start synthesis using deoxynucleotides (dNTPs), yet the structural basis and physiological implications for this discrimination are not understood. In silico analyses based on the three-dimensional structure of human PrimPol and related enzymes enabled us to predict a single residue, Tyr, as the main effector of sugar discrimination in human PrimPol and a change of Tyr to histidine to boost the efficiency of NTP incorporation. We show here that the Y100H mutation profoundly stimulates NTP incorporation by human PrimPol, with an efficiency similar to that for dNTP incorporation during both primase and polymerase reactions in vitro. As expected from the higher cellular concentration of NTPs relative to dNTPs, Y100H expression in mouse embryonic fibroblasts and U2OS osteosarcoma cells caused enhanced resistance to hydroxyurea, which decreases the dNTP pool levels in S-phase. Remarkably, the Y100H PrimPol mutation has been identified in cancer, suggesting that this mutation could be selected to promote survival at early stages of tumorigenesis, which is characterized by depleted dNTP pools., Spanish Ministry of Economy and Competitiveness (MINECO; BFU2012–3769, BFU2014–51672-REDC and BFU2015–65880-P (co-funded with European Union FEDER funds) to L.B.; BFU2013–49153-P and BFU2016–80402-R (co-funded with European Union FEDER funds) to J.M.). A.D.T., P.A.C. and D.G.A. are recipients of MINECO FPI-predoctoral fellowships. M.D. was the recipient of a FPI-predoctoral fellowship from Programa de Excelencia “Severo Ochoa” (CNIO-MINECO)

High-throughput genomics technologies are currently being used to study a wide variety of viral infections, providing insight into which cellular genes and pathways are regulated after infection, and how these changes are related, or not, to efficient elimination of the pathogen. This article will focus on how gene expression studies of infections with non-replicative poxviruses currently used as vaccine vectors provide a global perspective of the molecular events associated with the viral infection in human cells. These high-throughput genomics approaches have the potential to lead to the identification of specific new properties of the viral vector or novel cellular targets that may aid in the development of more effective pox-derived vaccines and antivirals.

PrimPol is the second primase discovered in eukaryotic cells, whose function is to restart the stalled replication forks during both mitochondrial and nuclear DNA replication. This chapter revises our current knowledge about the mechanism of synthesis of DNA primers by human PrimPol, and the importance of its distinctive Zn-finger domain (ZnFD). After PrimPol forms a binary complex with ssDNA, the formation of the pre-ternary complex strictly requires the presence of Mn2+ ions to stabilize the interaction of the incoming deoxynucleotide at the 3'-site. The capacity to bind both ssDNA template and 3'-deoxynucleotide was shown to reside in the AEP core of PrimPol, with ZnFD being dispensable at these two early steps of the primase reaction. Sugar selection favoring dNTPs versus NTPs at the 3' site is mediated by a specific tyrosine (Tyr100) acting as a steric gate. Besides, a specific glutamate residue (Glu116) conforming a singular A motif (DxE) promotes the use of Mn2+ to stabilize the pre-ternary complex. Mirroring the function of the PriL subunit of dimeric AEP primases, the ZnFD of PrimPol is crucial to stabilize the initiating 5'-nucleotide, specifically interacting with the gamma-phosphate. Such an interaction is crucial to optimize dimer formation and the subsequent translocation events leading to the processive synthesis of a mature DNA primer. Finally, the capacity of PrimPol to tolerate lesions is discussed in the context of its DNA primase function, and its potential as a TLS primase.

Human PrimPol is a monomeric enzyme whose DNA primase activity is required to rescue stalled replication forks during nuclear and mitochondrial DNA replication. PrimPol contains an Archeal-Eukaryotic Primases (AEP) core followed by a C-terminal Zn finger-containing domain (ZnFD), that is exclusively required for primer formation and for PrimPol function in vivo. The present study describes the sequential substrate interactions of human PrimPol during primer synthesis, and the relevance of the ZnFD at each individual step. Both the formation of a PrimPol:ssDNA binary complex and the upcoming interaction with the 3-nucleotide (pre-ternary complex) remained intact when lacking the ZnFD. Conversely, the ZnFD was required for the subsequent binding and selection of the 5-nucleotide that will become the first nucleotide of the new primer strand. Providing different 5-site nucleotides, we can conclude that the ZnFD of PrimPol most likely interacts with the - phosphate moiety of the 5-site nucleotide, optimizing formation of the initial dimer. Moreover, the ZnFD also contributes to recognize the cryptic G at the preferred priming sequence 3GTC5. Dimer elongation to obtain long DNA primers occurs processively and is facilitated by the 5-terminal triphosphate, indicating that the ZnFD is also essential in the subsequent translocation/elongation events during DNA primer synthesis., Spanish Ministry of Economy and Competitiveness [BFU2012-3769, BFU2014-51672-REDC, BFU2015-65880-P] and Comunidad de Madrid [S2010/BMD-2361]

RESUMEN Fundamento: Las anomalías cromosómicas pueden ser numéricas o estructurales, esta última puede producirse por duplicación parcial o total de un cromosoma, como se describe en la trisomía parcial 9p. Los afectados por esta cromosopatía, se caracterizan por hipotonía, discapacidad intelectual, retraso sicomotor, malformaciones craneofaciales distintivas, anomalías de manos y pies. Objetivo: Ilustrar debido a su rareza un caso de cromosopatía. Presentación de caso: Se describen las manifestaciones fenotípicas de un niño de dos años, con diagnóstico clínico, de una trisomía parcial 9p, con un cariotipo no balanceado definido por la siguiente fórmula: 47,XY+(mar). Conclusiones: Se concluyen con los estudios realizados que este paciente presenta una trisomía de novo en línea pura; aún sin diagnóstico confirmado por estudio molecular por hibridación in situ fluorescente (FISH), fue necesario el diagnóstico clínico precoz para intervención temprana y brindar asesoramiento genético a la familia. Palabras clave: Trisomía, trisomía cromosoma 9p, aberraciones cromosómicas, síndrome de Rethore, trastornos de los cromosomas; síndrome de Down. DeSC: TRISOMÍA; CROMOSOMAS HUMANOS PAR 9; ABERRACIONES CROMOSÓMICAS; TRASTORNOS DE LOS CROMOSOMAS; SÍNDROME DE DOWN.

Self/non-self-discrimination by the innate immune system relies on germline-encoded, non-rearranging receptors expressed by innate immune cells recognizing conserved pathogen-associated molecular patterns. The natural killer group 2D (NKG2D) receptor is a potent immune-activating receptor that binds human genome-encoded ligands, whose expression is negligible in normal tissues, but increased in stress and disease conditions for reasons that are incompletely understood. Here it is not clear how the immune system reconciles receptor binding of self-proteins with self/non-self-discrimination to avoid autoreactivity. We now report that increased expression of NKG2D ligands after virus infection depends on interferon response factors activated by the detection of viral double-stranded RNA by pattern-recognition receptors (RIG-I/MDA-5) and that NKG2D ligand up-regulation can be blocked by the expression of viral dsRNA-binding proteins. Thus, innate immunity-mediated recognition of viral nucleic acids triggers the infected cell to release interferon for NK cell recruitment and to express NKG2D ligands to become more visible to the immune system. Finally, the observation that NKG2D-ligand induction is a consequence of signaling by pattern-recognition receptors that have been selected over evolutionary time to be highly pathogen-specific explains how the risks of autoreactivity in this system are minimized.

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 infection, This 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.-S

Interferon stimulated gene 15 (ISG15) is an ubiquitin-like protein whose expression and conjugation to targets (ISGylation) is induced by infection, interferon (IFN)-α and -β, ischemia, DNA damage and aging. Attention has historically focused on the antiviral effects of ISGylation, which blocks the entry, replication or release of different intracellular pathogens. However, recently, new functions of ISGylation have emerged that implicate it in multiple cellular processes, such as DNA repair, autophagy, protein translation and exosome secretion. In this Review, we discuss the induction and conjugation of ISG15, as well as the functions of ISGylation in the prevention of infections and in cancer progression. We also offer a novel perspective with regard to the latest findings on this pathway, with special attention to the role of ISGylation in the inhibition of exosome secretion, which is mediated by fusion of multivesicular bodies with lysosomes. Finally, we propose that under conditions of stress or infection, ISGylation acts as a defense mechanism to inhibit normal protein translation by modifying protein kinase R (PKR, also known as EIF2AK2), while any newly synthesized proteins are being tagged and thus marked as potentially dangerous. Then, the endosomal system is re-directed towards protein degradation at the lysosome, to effectively ‘lock’ the cell gates and thus prevent the spread of pathogens, prions and deleterious aggregates through exosomes.

Vaccinia virus (VACV) is one of the most extensively-studied viruses of the Poxviridae family. It is easy to genetically modify, so it has become a key tool for many applications. In this context, reporter genes facilitate the study of the role of foreign genes introduced into the genome of VACV. In this review, we describe the type of reporter genes that have been used to generate reporter-expressing VACV and the applications of the recombinant viruses obtained. Reporter-expressing VACV are currently employed in basic and immunology research, in the development of vaccines and cancer treatment., This work is supported by Grant FIS2011-00127 and Reference SAF2014-54623-R to SG

Creative Commons Attribution Non-Commerical Share Alike 3.0 License.-- et al., Cot/tpl2 is the only MAP3K that activates MKK1/2-Erk1/2 in Toll-like receptor-activated macrophages. Here we show that Cot/tpl2 regulates RSK, S6 ribosomal protein, and 4E-BP phosphorylation after stimulation of bone marrow-derived macrophages with lipopolysaccharide (LPS), poly I:C, or zymosan. The dissociation of the 4E-BP-eIF4E complex, a key event in the cap-dependent mRNA translation initiation, is dramatically reduced in LPS-stimulated Cot/tpl2-knockout (KO) macrophages versus LPS-stimulated wild-type (Wt) macrophages. Accordingly, after LPS activation, increased cap-dependent translation is observed in Wt macrophages but not in Cot/tpl2 KO macrophages. In agreement with these data, Cot/tpl2 increases the polysomal recruitment of the 5´ TOP eEF1α and eEF2 mRNAs, as well as of inflammatory mediator gene-encoding mRNAs, such as tumor necrosis factor α (TNFα), interleukin-6 (IL-6), and KC in LPS-stimulated macrophages. In addition, Cot/tpl2 deficiency also reduces total TNFα, IL-6, and KC mRNA expression in LPS-stimulated macrophages, which is concomitant with a decrease in their mRNA half-lives. Macrophages require rapid fine control of translation to provide an accurate and not self-damaging response to host infection, and our data show that Cot/tpl2 controls inflammatory mediator gene-encoding mRNA translation in Toll-like receptor-activated macrophages., This work was supported by SAF 2011-24481 and Mutua Madrileña grants to S.A. and by FIS 2009-80145 and Universidad Autónoma de Madrid CM-CCG10-4911 grants to G.S. PD 0325901 was a gift from Philip Cohen, rapamycin was from Victor Calvo, and the biscitronic pcDNA3rLuc-polIRESfLuc plasmid was generously provided by Alexey Benyumov. M.L.P. is a recipient of an FPU Fellowship from the Universidad Autónoma de Madrid. S.G. holds a research contract from the Ramón y Cajal Program of Spain.

A question that raises the study of Asia history is that it is built, in a way, from the memory that West has from it, a partial memory that implies a strongly marked orientalist perspective. Taking the analysis of two Hollywood productions (“Anna and the King of Siam” and “The King and I”), we will see in what measure cinema can operate historically on the level of the ideological justification of colonialism, placing on stage an individual history (the formative, romantic relationship between Anna, English governess at the Siamese court of the 19th century, and the king of Siam) as the happy draw of a political conflict which, in its secret violences, it comes to cover up, making natural such a unequal balance of power – Siam´s forced westernization in a moment that british imperialism was to be feel in full in Southeast Asia at its highest.

Viruses are responsible for the majority of infectious diseases, from the common cold to HIV/AIDS or hemorrhagic fevers, the latter with devastating effects on the human population. Accordingly, the development of efficient antiviral therapies is a major goal and a challenge for the scientific community, as we are still far from understanding the molecular mechanisms that operate after virus infection. Interferon-stimulated gene 15 (ISG15) plays an important antiviral role during viral infection. ISG15 catalyzes a ubiquitin-like post-translational modification termed ISGylation, involving the conjugation of ISG15 molecules to de novo synthesized viral or cellular proteins, which regulates their stability and function. Numerous biomedically relevant viruses are targets of ISG15, as well as proteins involved in antiviral immunity. Beyond their role as cellular powerhouses, mitochondria are multifunctional organelles that act as signaling hubs in antiviral responses. In this review, we give an overview of the biological consequences of ISGylation for virus infection and host defense. We also compare several published proteomic studies to identify and classify potential mitochondrial ISGylation targets. Finally, based on our recent observations, we discuss the essential functions of mitochondria in the antiviral response and examine the role of ISG15 in the regulation of mitochondrial processes, specifically OXPHOS and mitophagy.

The aim of this study was to define the effects on antigen-presenting cells of the expression of HIV antigens from an attenuated poxvirus vector. We have analyzed the transcriptional changes in gene expression following infection of human immature monocyte-derived dendritic cells (DC) with recombinant modified vaccinia virus Ankara (MVA) expressing the genes encoding the gp120 and Gag-Pol-Nef antigens of HIV type 1 clade B (referred to as MVA-B) versus parental MVA infection. Using microarray technology and real-time reverse transcription-PCR, we demonstrated that the HIV proteins induced the expression of cytokines, cytokine receptors, chemokines, chemokine receptors, and molecules involved in antigen uptake and processing, including major histocompatibility complex (MHC) genes. Levels of mRNAs for interleukin-1, beta interferon, CCR8, and SCYA20 were higher after HIV antigen production. MVA-B infection also modulated the expression of antigen processing and presentation genes: the gene for MICA was upregulated, whereas those for HLA-DRA and HSPA5 were downregulated. Indeed, the increased expression of the gene for MICA, a glycoprotein related to major histocompatibility complex class I molecules, was shown to enhance the interaction between MVA-B-infected target cells and cytotoxic lymphocytes. The expression profiles of the genes for protein kinases such as JAK1 and IRAK2 were activated after HIV antigen expression. Several genes included in the JAK-STAT and mitogen-activated protein kinase signaling pathways were regulated after HIV antigen expression. Our findings provide the first gene signatures in DC of a candidate MVA-B vaccine expressing four HIV antigens and identified the biological roles of some of the regulatory genes, like that for MICA, which will help in the design of more effective MVA-derived vaccines.