query: Mice are incapable of producing neutralizing antibodies in reponse to the Chikungunya virus (CHIKV) produced in mosquitos.

title: A chikungunya fever vaccine utilizing an insect-specific virus platform

text: Traditionally, vaccine development involves tradeoffs between immunogenicity and safety. Live-attenuated vaccines typically offer rapid and durable immunity but have reduced safety when compared to inactivated vaccines. In contrast, the inability of inactivated vaccines to replicate enhances safety at the expense of immunogenicity, often necessitating multiple doses and boosters. To overcome these tradeoffs, we developed the insect-specific alphavirus, Eilat virus (EILV), as a vaccine platform. To address the chikungunya fever (CHIKF) pandemic, we used an EILV cDNA clone to design a chimeric virus containing the chikungunya virus (CHIKV) structural proteins. The recombinant EILV/CHIKV was structurally identical at 10 Å to wild-type CHIKV, as determined by single-particle cryo-electron microscopy, and it mimicked the early stages of CHIKV replication in vertebrate cells from attachment and entry to viral RNA delivery. Yet the recombinant virus remained completely defective for productive replication, providing a high degree of safety. A single dose of EILV/CHIKV produced in mosquito cells elicited rapid (within 4 d) and long-lasting (>290 d) neutralizing antibodies that provided complete protection in two different mouse models. In nonhuman primates, EILV/CHIKV elicited rapid and robust immunity that protected against viremia and telemetrically monitored fever. Our EILV platform represents the first structurally native application of an insect-specific virus in preclinical vaccine development and highlights the potential application of such viruses in vaccinology.

title: Effects of an opal termination codon preceding the nsP4 gene sequence in the O'Nyong-Nyong virus genome on Anopheles gambiae infectivity.

text: The genomic RNA of an alphavirus encodes four different nonstructural proteins, nsP1, nsP2, nsP3, and nsP4. The polyprotein P123 is produced when translation terminates at an opal termination codon between nsP3 and nsP4. The polyprotein P1234 is produced when translational readthrough occurs or when the opal termination codon has been replaced by a sense codon in the alphavirus genome. Evolutionary pressures appear to have maintained genomic sequences encoding both a stop codon (opal) and an open reading frame (arginine) as a general feature of the O'nyong-nyong virus (ONNV) genome, indicating that both are required at some point. Alternate replication of ONNVs in both vertebrate and invertebrate hosts may determine predominance of a particular codon at this locus in the viral quasispecies. However, no systematic study has previously tested this hypothesis in whole animals. We report here the results of the first study to investigate in a natural mosquito host the functional significance of the opal stop codon in an alphavirus genome. We used a full-length cDNA clone of ONNV to construct a series of mutants in which the arginine between nsP3 and nsP4 was replaced with an opal, ochre, or amber stop codon. The presence of an opal stop codon upstream of nsP4 nearly doubled (75.5%) the infectivity of ONNV over that of virus possessing a codon for the amino acid arginine at the corresponding position (39.8%). Although the frequency with which the opal virus disseminated from the mosquito midgut did not differ significantly from that of the arginine virus on days 8 and 10, dissemination did began earlier in mosquitoes infected with the opal virus. Although a clear fitness advantage is provided to ONNV by the presence of an opal codon between nsP3 and nsP4 in Anopheles gambiae, sequence analysis of ONNV RNA extracted from mosquito bodies and heads indicated codon usage at this position corresponded with that of the virus administered in the blood meal. These results suggest that while selection of ONNV variants is occurring, de novo mutation at the position between nsP3 and nsP4 does not readily occur in the mosquito. Taken together, these results suggest that the primary fitness advantage provided to ONNV by the presence of an opal codon between nsP3 and nsP4 is related to mosquito infectivity.

title: Treatment with cyclooxygenase-2 inhibitors enables repeated administration of vaccinia virus for control of ovarian cancer.

text: Metastatic ovarian cancer is the leading cause of death among women with gynecologic malignancies in the United States. The lack of effective treatment for patients with advanced ovarian cancer warrants development of innovative therapies. Cancer therapy using oncolytic viruses represents a promising new approach for controlling tumors. Vaccinia virus has been shown to preferentially infect tumor cells but not normal tissue. However, oncolytic therapy using recombinant viruses faces the limitation of viral clearance due to generation of neutralizing antibodies. In the current study, we found that cyclooxygenase-2 (Cox-2) inhibitors circumvented this limitation, enabling repeated administration of vaccinia virus without losing infectivity. We quantified the antivaccinia antibody response using enzyme-linked immunosorbent assay (ELISA) and neutralization assays to show that treatment of Cox-2 inhibitors inhibited the generation of neutralizing antibodies. Furthermore, we showed that combination treatment of Cox-2 inhibitors with vaccinia virus was more effective that either treatment alone in treating MOSEC/luc tumor-bearing mice. Thus, the combination of Cox-2 inhibitors and vaccinia virus represents a potential innovative approach to controlling ovarian tumors.

title: Antibody-Based HIV-1 Vaccines: Recent Developments and Future Directions

text: The Global HIV Vaccine Enterprise convened a two-day workshop in May of 2007 to discuss humoral immune responses to HIV and approaches to design vaccines that induce viral neutralizing and other potentially protective antibody responses. The goals of this workshop were to identify key scientific issues, gaps, and opportunities that have emerged since the Enterprise Strategic Plan was first published in 2005 [1], and to make recommendations that Enterprise stakeholders can use to plan new activities.     Most effective viral vaccines work, at least in part, by generating antibodies that inactivate or neutralize the invading virus, and the existing data strongly suggest that an optimally effective HIV-1 vaccine should elicit potent antiviral neutralizing antibodies. However, unlike acute viral pathogens, HIV-1 chronically replicates in the host and evades the antibody response. This immune evasion, along with the large genetic variation among HIV-1 strains worldwide, has posed major obstacles to vaccine development. Current HIV vaccine candidates do not elicit neutralizing antibodies against most circulating virus strains, and thus the induction of a protective antibody response remains a major priority for HIV-1 vaccine development. For an antibody-based HIV-1 vaccine, progress in vaccine design is generally gauged by in vitro assays that measure the ability of vaccine-induced antibodies to neutralize a broad spectrum of viral isolates representing the major genetic subtypes (clades) of HIV-1 [2]. Although it is not known what magnitude and breadth of neutralization will predict protection in vaccine recipients, it is clear that current vaccine immunogens elicit antibodies that neutralize only a minority of circulating isolates. Thus, much progress needs to be made in this area. Also, though virus neutralization is considered a critical benchmark for a vaccine, this may not be the only benchmark for predicting success with antibody-based HIV-1 vaccine immunogens.     The main targets for neutralizing antibodies to HIV-1 are the surface gp120 and trans-membrane gp41 envelope glycoproteins (Env) that mediate receptor and coreceptor binding and the subsequent membrane fusion events that allow the virus to gain entry into cells [3]. Antibodies neutralize the virus by binding these viral spikes and blocking virus entry into susceptible cells, such as CD4+ T cells [4,5]. In order to chronically replicate in the host, the virus exploits several mechanisms to shield itself against antibody recognition, including a dense outer coating of sugar molecules (N-linked glycans) and the strategic positioning of cysteine–cysteine loop structures on the gp120 molecule [6–8]. These shielding mechanisms, although highly effective, have vulnerabilities imposed by fitness constraints. Information on the precise location and molecular structure of these vulnerable regions could be valuable for the rational design of improved vaccine immunogens.     Participants in the workshop identified four areas that, if given proper attention, could provide key information that would bring the field closer to an effective antibody-based HIV-1 vaccine: (1) structure-assisted immunogen design, (2) role of Fc receptors and complement, (3) assay standardization and validation, and (4) immunoregulation of B cell responses.

title: Pancreatic stellate cells: partners in crime with pancreatic cancer cells.

text: Pancreatic stellate cells (PSC) produce the stromal reaction in pancreatic cancer, but their role in cancer progression is not fully elucidated. We examined the influence of PSCs on pancreatic cancer growth using (a) an orthotopic model of pancreatic cancer and (b) cultured human PSCs (hPSC) and human pancreatic cancer cell lines MiaPaCa-2 and Panc-1. Athymic mice received an intrapancreatic injection of saline, hPSCs, MiaPaCa-2 cells, or hPSCs + MiaPaCa-2. After 7 weeks, tumor size, metastases, and tumor histology were assessed. In vitro studies assessed the effect of cancer cell secretions on PSC migration and the effect of hPSC secretions on cancer cell proliferation, apoptosis, and migration. Possible mediators of the effects of hPSC secretions on cancer cell proliferation were examined using neutralizing antibodies. Compared with mice receiving MiaPaCa-2 cells alone, mice injected with hPSCs + MiaPaCa-2 exhibited (a) increased tumor size and regional and distant metastasis, (b) fibrotic bands (desmoplasia) containing activated PSCs within tumors, and (c) increased tumor cell numbers. In vitro studies showed that, in the presence of pancreatic cancer cells, PSC migration was significantly increased. Furthermore, hPSC secretions induced the proliferation and migration, but inhibited the apoptosis, of MiaPaCa-2 and Panc-1 cells. The proliferative effect of hPSC secretions on pancreatic cancer cells was inhibited in the presence of neutralizing antibody to platelet-derived growth factor. Our studies indicate a significant interaction between pancreatic cancer cells and stromal cells (PSCs) and imply that pancreatic cancer cells recruit stromal cells to establish an environment that promotes cancer progression.

title: Neutralizing antibody responses in acute human immunodeficiency virus type 1 subtype C infection.

text: The study of the evolution and specificities of neutralizing antibodies during the course of human immunodeficiency virus type 1 (HIV-1) infection may be important in the discovery of possible targets for vaccine design. In this study, we assessed the autologous and heterologous neutralization responses of 14 HIV-1 subtype C-infected individuals, using envelope clones obtained within the first 2 months postinfection. Our data show that potent but relatively strain-specific neutralizing antibodies develop within 3 to 12 months of HIV-1 infection. The magnitude of this response was associated with shorter V1-to-V5 envelope lengths and fewer glycosylation sites, particularly in the V1-V2 region. Anti-MPER antibodies were detected in 4 of 14 individuals within a year of infection, while antibodies to CD4-induced (CD4i) epitopes developed to high titers in 12 participants, in most cases before the development of autologous neutralizing antibodies. However, neither anti-MPER nor anti-CD4i antibody specificity conferred neutralization breadth. These data provide insights into the kinetics, potency, breadth, and epitope specificity of neutralizing antibody responses in acute HIV-1 subtype C infection.

title: Broad neutralization coverage of HIV by multiple highly potent antibodies

text: Broadly neutralizing antibodies against highly variable viral pathogens are much sought after to treat or protect against global circulating viruses. Here we probed the neutralizing antibody repertoires of four human immunodeficiency virus (HIV)-infected donors with remarkably broad and potent neutralizing responses and rescued 17 new monoclonal antibodies that neutralize broadly across clades. Many of the new monoclonal antibodies are almost tenfold more potent than the recently described PG9, PG16 and VRC01 broadly neutralizing monoclonal antibodies and 100-fold more potent than the original prototype HIV broadly neutralizing monoclonal antibodies. The monoclonal antibodies largely recapitulate the neutralization breadth found in the corresponding donor serum and many recognize novel epitopes on envelope (Env) glycoprotein gp120, illuminating new targets for vaccine design. Analysis of neutralization by the full complement of anti-HIV broadly neutralizing monoclonal antibodies now available reveals that certain combinations of antibodies should offer markedly more favourable coverage of the enormous diversity of global circulating viruses than others and these combinations might be sought in active or passive immunization regimes. Overall, the isolation of multiple HIV broadly neutralizing monoclonal antibodies from several donors that, in aggregate, provide broad coverage at low concentrations is a highly positive indicator for the eventual design of an effective antibody-based HIV vaccine.

title: Phenotypic deficits in the HIV-1 envelope are associated with the maturation of a V2-directed broadly neutralizing antibody lineage

text: Broadly neutralizing antibodies (bnAbs) to HIV-1 can evolve after years of an iterative process of virus escape and antibody adaptation that HIV-1 vaccine design seeks to mimic. To enable this, properties that render HIV-1 envelopes (Env) capable of eliciting bnAb responses need to be defined. Here, we followed the evolution of the V2 apex directed bnAb lineage VRC26 in the HIV-1 subtype C superinfected donor CAP256 to investigate the phenotypic changes of the virus populations circulating before and during the early phases of bnAb induction. Longitudinal viruses that evolved from the VRC26-resistant primary infecting (PI) virus, the VRC26-sensitive superinfecting (SU) virus and ensuing PI-SU recombinants revealed substantial phenotypic changes in Env, with a switch in Env properties coinciding with early resistance to VRC26. Decreased sensitivity of SU-like viruses to VRC26 was linked with reduced infectivity, altered entry kinetics and lower sensitivity to neutralization after CD4 attachment. VRC26 maintained neutralization activity against cell-associated CAP256 virus, indicating that escape through the cell-cell transmission route is not a dominant escape pathway. Reduced fitness of the early escape variants and sustained sensitivity in cell-cell transmission are both features that limit virus replication, thereby impeding rapid escape. This supports a scenario where VRC26 allowed only partial viral escape for a prolonged period, possibly increasing the time window for bnAb maturation. Collectively, our data highlight the phenotypic plasticity of the HIV-1 Env in evading bnAb pressure and the need to consider phenotypic traits when selecting and designing Env immunogens. Combinations of Env variants with differential phenotypic patterns and bnAb sensitivity, as we describe here for CAP256, may maximize the potential for inducing bnAb responses by vaccination.

title: A C-Type Lectin Collaborates with a CD45 Phosphatase Homolog to Facilitate West Nile Virus Infection of Mosquitoes

text: West Nile virus (WNV) is the most common arthropod-borne flavivirus in the United States; however, the vector ligand(s) that participate in infection are not known. We now show that an Aedes aegypti C-type lectin, mosGCTL-1, is induced by WNV, interacts with WNV in a calcium-dependent manner, and facilitates infection in vivo and in vitro. A mosquito homolog of human CD45 in A. aegypti, designated mosPTP-1, recruits mosGCTL-1 to enable viral attachment to cells and to enhance viral entry. In vivo experiments show that mosGCTL-1 and mosPTP-1 function as part of the same pathway and are critical for WNV infection of mosquitoes. A similar phenomenon was also observed in Culex quinquefasciatus, a natural vector of WNV, further demonstrating that these genes participate in WNV infection. During the mosquito blood-feeding process, WNV infection was blocked in vivo with mosGCTL-1 antibodies. A molecular understanding of flaviviral-arthropod interactions may lead to strategies to control viral dissemination in nature.

title: Evolution of an HIV glycan–dependent broadly neutralizing antibody epitope through immune escape

text: Neutralizing antibodies are likely to play a crucial part in a preventative HIV-1 vaccine. Although efforts to elicit broadly cross-neutralizing (BCN) antibodies by vaccination have been unsuccessful, a minority of individuals naturally develop these antibodies after many years of infection. How such antibodies arise, and the role of viral evolution in shaping these responses, is unknown. Here we show, in two HIV-1–infected individuals who developed BCN antibodies targeting the glycan at Asn332 on the gp120 envelope, that this glycan was absent on the initial infecting virus. However, this BCN epitope evolved within 6 months, through immune escape from earlier strain-specific antibodies that resulted in a shift of a glycan to position 332. Both viruses that lacked the glycan at amino acid 332 were resistant to the Asn332-dependent BCN monoclonal antibody PGT128 (ref. 8), whereas escaped variants that acquired this glycan were sensitive. Analysis of large sequence and neutralization data sets showed the 332 glycan to be significantly under-represented in transmitted subtype C viruses compared to chronic viruses, with the absence of this glycan corresponding with resistance to PGT128. These findings highlight the dynamic interplay between early antibodies and viral escape in driving the evolution of conserved BCN antibody epitopes.

title: Persistent LCMV infection is controlled by blockade of type I interferon signaling.

text: During persistent viral infections, chronic immune activation, negative immune regulator expression, an elevated interferon signature, and lymphoid tissue destruction correlate with disease progression. We demonstrated that blockade of type I interferon (IFN-I) signaling using an IFN-I receptor neutralizing antibody reduced immune system activation, decreased expression of negative immune regulatory molecules, and restored lymphoid architecture in mice persistently infected with lymphocytic choriomeningitis virus. IFN-I blockade before and after establishment of persistent virus infection resulted in enhanced virus clearance and was CD4 T cell-dependent. Hence, we demonstrate a direct causal link between IFN-I signaling, immune activation, negative immune regulator expression, lymphoid tissue disorganization, and virus persistence. Our results suggest that therapies targeting IFN-I may help control persistent virus infections.

title: Eosinophil pathogenicity mechanisms and therapeutics in neuromyelitis optica.

text: Eosinophils are abundant in inflammatory demyelinating lesions in neuromyelitis optica (NMO). We used cell culture, ex vivo spinal cord slices, and in vivo mouse models of NMO to investigate the role of eosinophils in NMO pathogenesis and the therapeutic potential of eosinophil inhibitors. Eosinophils cultured from mouse bone marrow produced antibody-dependent cell-mediated cytotoxicity (ADCC) in cell cultures expressing aquaporin-4 in the presence of NMO autoantibody (NMO-IgG). In the presence of complement, eosinophils greatly increased cell killing by a complement-dependent cell-mediated cytotoxicity (CDCC) mechanism. NMO pathology was produced in NMO-IgG-treated spinal cord slice cultures by inclusion of eosinophils or their granule toxins. The second-generation antihistamines cetirizine and ketotifen, which have eosinophil-stabilizing actions, greatly reduced NMO-IgG/eosinophil-dependent cytotoxicity and NMO pathology. In live mice, demyelinating NMO lesions produced by continuous intracerebral injection of NMO-IgG and complement showed marked eosinophil infiltration. Lesion severity was increased in transgenic hypereosinophilic mice. Lesion severity was reduced in mice made hypoeosinophilic by anti-IL-5 antibody or by gene deletion, and in normal mice receiving cetirizine orally. Our results implicate the involvement of eosinophils in NMO pathogenesis by ADCC and CDCC mechanisms and suggest the therapeutic utility of approved eosinophil-stabilizing drugs.

title: AgDscam, a Hypervariable Immunoglobulin Domain-Containing Receptor of the       Anopheles gambiae Innate Immune System

text: Activation of the insect innate immune system is dependent on a limited number of pattern recognition receptors (PRRs) capable of interacting with pathogen-associated molecular pattern. Here we report a novel role of an alternatively spliced hypervariable immunoglobulin domain-encoding gene, Dscam, in generating a broad range of PRRs implicated in immune defense in the malaria vector Anopheles gambiae. The mosquito Down syndrome cell adhesion molecule gene, AgDscam, has a complex genome organization with 101 exons that can produce over 31,000 potential alternative splice forms with different combinations of adhesive domains and interaction specificities. AgDscam responds to infection by producing pathogen challenge-specific splice form repertoires. Transient silencing of AgDscam compromises the mosquito's resistance to infections with bacteria and the malaria parasite Plasmodium. AgDscam is mediating phagocytosis of bacteria with which it can associate and defend against in a splice form–specific manner. AgDscam is a hypervariable PRR of the A. gambiae innate immune system.

title: Alphavirus-derived small RNAs modulate pathogenesis in disease vector mosquitoes.

text: Mosquito-borne viruses cause significant levels of morbidity and mortality in humans and domesticated animals. Maintenance of mosquito-borne viruses in nature requires a biological transmission cycle that involves alternating virus replication in a susceptible vertebrate and mosquito host. Although the vertebrate infection is acute and often associated with disease, continual transmission of these viruses in nature depends on the establishment of a persistent, nonpathogenic infection in the mosquito vector. An antiviral RNAi response has been shown to limit the replication of RNA viruses in flies. However, the importance of the RNAi pathway as an antiviral defense in mammals is unclear. Differences in the immune responses of mammals and mosquitoes may explain why these viruses are not generally associated with pathology in the invertebrate host. We identified virus-derived small interfering RNAs (viRNAs), 21 nt in length, in Aedes aegypti infected with the mosquito-borne virus, Sindbis (SINV). viRNAs had an asymmetric distribution that spanned the length of the SINV genome. To determine the role of viRNAs in controlling pathogenic potential, mosquitoes were infected with recombinant alphaviruses expressing suppressors of RNA silencing. Decreased survival was observed in mosquitoes in which the accumulation of viRNAs was suppressed. These results suggest that an exogenous siRNA pathway is essential to the survival of mosquitoes infected with alphaviruses and, thus, the maintenance of these viruses in nature.

title: Thrombopoietin/MPL signaling regulates hematopoietic stem cell quiescence and interaction with the osteoblastic niche.

text: Maintenance of hematopoietic stem cells (HSCs) depends on interaction with their niche. Here we show that the long-term (LT)-HSCs expressing the thrombopoietin (THPO) receptor, MPL, are a quiescent population in adult bone marrow (BM) and are closely associated with THPO-producing osteoblastic cells. THPO/MPL signaling upregulated beta1-integrin and cyclin-dependent kinase inhibitors in HSCs. Furthermore, inhibition and stimulation of THPO/MPL pathway by treatments with anti-MPL neutralizing antibody, AMM2, and with THPO showed reciprocal regulation of quiescence of LT-HSC. AMM2 treatment reduced the number of quiescent LT-HSCs and allowed exogenous HSC engraftment without irradiation. By contrast, exogenous THPO transiently increased quiescent HSC population and subsequently induced HSC proliferation in vivo. Altogether, these observations suggest that THPO/MPL signaling plays a critical role of LT-HSC regulation in the osteoblastic niche.

title: the Development of Autoimmune Myocarditis in Mice by an

text:
BACKGROUND Interleukin (IL)-12 exerts a potent proinflammatory effect by stimulating T-helper (Th) 1 responses. This effect is believed to be mediated primarily through the activation of STAT4 and subsequent production of interferon (IFN)-gamma. Methods and Results- We examined the role of IL-12 receptor (IL-12R) signaling in the development of murine experimental autoimmune myocarditis (EAM) induced by cardiac myosin immunization. Both IL-12Rbeta1-deficient mice and STAT4-deficient mice were resistant to the induction of myocarditis. Treatment with exogenous IL-12 exacerbated disease. We questioned whether IFN-gamma is required for the disease-promoting activity of IL-12. On the contrary, we found that IFN-gamma suppresses EAM. Lack of IFN-gamma due to either depletion with an antibody or a genetic deficiency exacerbated myocarditis. Spleens from IFN-gamma-deficient mice immunized with cardiac myosin showed increased cellularity; greater numbers of CD3+, CD4+, CD8+, and IL-2-producing cells; and heightened ability to produce cytokines on stimulation in vitro. Treatment of mice with recombinant IFN-gamma suppressed the development of myocarditis.
 CONCLUSIONS IL-12/IL-12R/STAT4 signaling promotes the development of EAM. In contrast, IFN-gamma plays a protective role. The disease-limiting effects of IFN-gamma might be explained by its ability to control the expansion of activated T lymphocytes.

title: Characteristics of the Earliest Cross-Neutralizing Antibody Response to HIV-1

text: Recent cross-sectional analyses of HIV-1+ plasmas have indicated that broadly cross-reactive neutralizing antibody responses are developed by 10%-30% of HIV-1+ subjects. The timing of the initial development of such anti-viral responses is unknown. It is also unknown whether the emergence of these responses coincides with the appearance of antibody specificities to a single or multiple regions of the viral envelope glycoprotein (Env). Here we analyzed the cross-neutralizing antibody responses in longitudinal plasmas collected soon after and up to seven years after HIV-1 infection. We find that anti-HIV-1 cross-neutralizing antibody responses first become evident on average at 2.5 years and, in rare cases, as early as 1 year following infection. If cross-neutralizing antibody responses do not develop during the first 2-3 years of infection, they most likely will not do so subsequently. Our results indicate a potential link between the development of cross-neutralizing antibody responses and specific activation markers on T cells, and with plasma viremia levels. The earliest cross-neutralizing antibody response targets a limited number of Env regions, primarily the CD4-binding site and epitopes that are not present on monomeric Env, but on the virion-associated trimeric Env form. In contrast, the neutralizing activities of plasmas from subjects that did not develop cross-neutralizing antibody responses target epitopes on monomeric gp120 other than the CD4-BS. Our study provides information that is not only relevant to better understanding the interaction of the human immune system with HIV but may guide the development of effective immunization protocols. Since antibodies to complex epitopes that are present on the virion-associated envelope spike appear to be key components of earliest cross-neutralizing activities of HIV-1+ plasmas, then emphasis should be made to elicit similar antibodies by vaccination.

title: Loss of DExD/H box RNA helicase LGP2 manifests disparate antiviral responses.

text: The DExD/H box RNA helicase retinoic acid-inducible gene I (RIG-I) and the melanoma differentiation-associated gene 5 (MDA5) are key intracellular receptors that recognize virus infection to produce type I IFN. A third helicase gene, Lgp2, is homologous to Rig-I and Mda5 but lacks a caspase activation and recruitment domain. We generated Lgp2-deficient mice and report that the loss of this gene greatly sensitizes cells to cytosolic polyinosinic/polycytidylic acid-mediated induction of type I IFN. However, negative feedback inhibition of IFN-beta transcription was found to be normal in the absence of LGP2, indicating that LGP2 is not the primary negative regulator of type I IFN production. Our data further indicate that Lgp2-/- mice exhibited resistance to lethal vesicular stomatitis virus infection, a virus whose replicative RNA intermediates are recognized specifically by RIG-I rather than by MDA5 to trigger the production of type I IFN. However, mice lacking LGP2 were observed to exhibit a defect in type I IFN production in response to infection by the encephalomyocarditis virus, the replication of which activates MDA5-dependent innate immune responses. Collectively, our data indicate a disparate regulatory role for LGP2 in the triggering of innate immune signaling pathways following RNA virus infection.

title: Rational design of a flavivirus vaccine by abolishing viral RNA 2'-O methylation.

text: Viruses that replicate in the cytoplasm cannot access the host nuclear capping machinery. These viruses have evolved viral methyltransferase(s) to methylate N-7 and 2'-O cap of their RNA; alternatively, they "snatch" host mRNA cap to form the 5' end of viral RNA. The function of 2'-O methylation of viral RNA cap is to mimic cellular mRNA and to evade host innate immune restriction. A cytoplasmic virus defective in 2'-O methylation is replicative, but its viral RNA lacks 2'-O methylation and is recognized and eliminated by the host immune response. Such a mutant virus could be rationally designed as a live attenuated vaccine. Here, we use Japanese encephalitis virus (JEV), an important mosquito-borne flavivirus, to prove this novel vaccine concept. We show that JEV methyltransferase is responsible for both N-7 and 2'-O cap methylations as well as evasion of host innate immune response. Recombinant virus completely defective in 2'-O methylation was stable in cell culture after being passaged for >30 days. The mutant virus was attenuated in mice, elicited robust humoral and cellular immune responses, and retained the engineered mutation in vivo. A single dose of immunization induced full protection against lethal challenge with JEV strains in mice. Mechanistically, the attenuation phenotype was attributed to the enhanced sensitivity of the mutant virus to the antiviral effects of interferon and IFIT proteins. Collectively, the results demonstrate the feasibility of using 2'-O methylation-defective virus as a vaccine approach; this vaccine approach should be applicable to other flaviviruses and nonflaviviruses that encode their own viral 2'-O methyltransferases.

title: Broad and potent neutralization of HIV-1 by a gp41-specific human antibody

text: Characterization of human monoclonal antibodies is providing considerable insight into mechanisms of broad HIV-1 neutralization. Here we report an HIV-1 gp41 membrane-proximal external region (MPER)-specific antibody, named 10E8, which neutralizes ∼98% of tested viruses. An analysis of sera from 78 healthy HIV-1-infected donors demonstrated that 27% contained MPER-specific antibodies and 8% contained 10E8-like specificities. In contrast to other neutralizing MPER antibodies, 10E8 did not bind phospholipids, was not autoreactive, and bound cell-surface envelope. The structure of 10E8 in complex with the complete MPER revealed a site of vulnerability comprising a narrow stretch of highly conserved gp41-hydrophobic residues and a critical arginine or lysine just before the transmembrane region. Analysis of resistant HIV-1 variants confirmed the importance of these residues for neutralization. The highly conserved MPER is a target of potent, non-self-reactive neutralizing antibodies, suggesting that HIV-1 vaccines should aim to induce antibodies to this region of HIV-1 envelope glycoprotein.


query: Mice defective for deoxyribonucleic acid (DNA) polymerase I (polI) fail to reveal increased sensitivity to ionizing radiation (IR).

title: Nonoverlapping functions of DNA polymerases mu, lambda, and terminal deoxynucleotidyltransferase during immunoglobulin V(D)J recombination in vivo.

text: DNA polymerases mu (pol mu), lambda (pol lambda), and terminal deoxynucleotidyltransferase (TdT) are enzymes of the pol X family that share homology in sequence and functional domain organization. We showed previously that pol mu participates in light chain but surprisingly not heavy chain gene rearrangement. We show here that immunoglobulin heavy chain junctions from pol lambda-deficient animals have shorter length with normal N-additions, thus indicating that pol lambda is recruited during heavy chain rearrangement at a step that precedes the action of TdT. In contrast to previous in vitro studies, analysis of animals with combined inactivation of these enzymes revealed no overlapping or compensatory activities for V(D)J recombination between pol mu, pol lambda, and TdT. This complex usage of polymerases with distinct catalytic specificities may correspond to the specific function that the third hypervariable region assumes for each immunoglobulin chain, with pol lambda maintaining a large heavy chain junctional heterogeneity and pol mu ensuring a restricted light chain junctional variability.

title: Deregulated DNA polymerase β strengthens ionizing radiation-induced nucleotidic and chromosomal instabilities

text: DNA polymerase β (Pol β) is an error-prone enzyme which has been found to be overexpressed in several human tumors. By using a couple of recombinant CHO cells differing only from the exogenous expression of Pol β, we showed here that cells overexpressing Pol β are much more sensitive to IR treatments by increasing apoptosis. We also found that the surviving cells displayed an hypermutator phenotype which could be explained by different pathways involving Pol β, such as (i) an increased capacity to incorporate into DNA the mutagenic dGTP analog, 8-oxo-dGTP, one of the most abundant purine-derived nucleotides exposed to γ-irradiation, (ii) the induction of IR-induced DNA breaks and (iii) accumulation of chromosome aberrations induced by radiation. Alteration of Pol β expression in irradiated cells thus appears to strengthen both cell death and genetic changes associated with a malignant phenotype. These data provide new insights into the cellular response to radiations and the associated carcinogenic consequences.

title: The DNA polymerase λ is required for the repair of non-compatible DNA double strand breaks by NHEJ in mammalian cells

text: DNA polymerase lambda (polλ) is a recently identified DNA polymerase whose cellular function remains elusive. Here we show, that polλ participates at the molecular level in a chromosomal context, in the repair of DNA double strand breaks (DSB) via non-homologous end joining (NHEJ) in mammalian cells. The expression of a catalytically inactive form of polλ (polλDN) decreases the frequency of NHEJ events in response to I-Sce-I-induced DSB whereas inactivated forms of its homologues polβ and polμ do not. Only events requiring DNA end processing before ligation are affected; this defect is associated with large deletions arising in the vicinity of the induced DSB. Furthermore, polλDN-expressing cells exhibit increased sensitization and genomic instability in response to ionizing radiation similar to that of NHEJ-defective cells. Our data support a requirement for polλ in repairing a subset of DSB in genomic DNA, thereby contributing to the maintenance of genetic stability mediated by the NHEJ pathway.

title: Association of DNA polymerase mu (pol mu) with Ku and ligase IV: role for pol mu in end-joining double-strand break repair.

text: Mammalian DNA polymerase mu (pol mu) is related to terminal deoxynucleotidyl transferase, but its biological role is not yet clear. We show here that after exposure of cells to ionizing radiation (IR), levels of pol mu protein increase. pol mu also forms discrete nuclear foci after IR, and these foci are largely coincident with IR-induced foci of gammaH2AX, a previously characterized marker of sites of DNA double-strand breaks. pol mu is thus part of the cellular response to DNA double-strand breaks. pol mu also associates in cell extracts with the nonhomologous end-joining repair factor Ku and requires both Ku and another end-joining factor, XRCC4-ligase IV, to form a stable complex on DNA in vitro. pol mu in turn facilitates both stable recruitment of XRCC4-ligase IV to Ku-bound DNA and ligase IV-dependent end joining. In contrast, the related mammalian DNA polymerase beta does not form a complex with Ku and XRCC4-ligase IV and is less effective than pol mu in facilitating joining mediated by these factors. Our data thus support an important role for pol mu in the end-joining pathway for repair of double-strand breaks.

title: Ionizing radiation-induced long-term expression of senescence markers in mice is independent of p53 and immune status.

text: Exposure to IR has been shown to induce the formation of senescence markers, a phenotype that coincides with lifelong delayed repair and regeneration of irradiated tissues. We hypothesized that IR-induced senescence markers could persist long-term in vivo, possibly contributing to the permanent reduction in tissue functionality. Here, we show that mouse tissues exposed to a sublethal dose of IR display persistent (up to 45 weeks, the maximum time analyzed) DNA damage foci and increased p16(INK4a) expression, two hallmarks of cellular senescence and aging. BrdU-labeling experiments revealed that IR-induced damaged cells are preferentially eliminated, at least partially, in a tissue-dependent manner. Unexpectedly, the accumulation of damaged cells was found to occur independent from the DNA damage response modulator p53, and from an intact immune system, as their levels were similar in wild-type and Rag2(-/-) gammaC(-/-) mice, the latter being deficient in T, B, and NK cells. Together, our results provide compelling evidence that exposure to IR induces long-term expression of senescence markers in vivo, an effect that may contribute to the reduced tissue functionality observed in cancer survivors.

title: Defective replication stress response inhibits lymphomagenesis and impairs lymphocyte reconstitution

text: DNA replication stress promotes genome instability in cancer. However, the contribution of the replication stress response to the development of malignancies remains unresolved. The DNA replication stress response protein SMARCAL1 stabilizes DNA replication forks and prevents replication fork collapse, a cause of DNA breaks and apoptosis. While the fork regression/remodeling functions of SMARCAL1 have been investigated, its in vivo functions in replication stress and cancer are unclear. Using a gamma radiation (IR)-induced replication stress T-cell lymphoma mouse model, we observed a significant inhibition of lymphomagenesis in mice lacking one or both alleles of Smarcal1. Notably, a quarter of the Smarcal1-deficient mice did not develop tumors. Moreover, hematopoietic stem/progenitor cells (HSPCs) and developing thymocytes in Smarcal1-deficient mice showed increased DNA damage and apoptosis during the proliferation burst following IR and an impaired ability to repopulate the thymus after IR. Additionally, mice lacking Smarcal1 showed significant HSPC defects when challenged to respond to other replication stress stimuli. Thus, our data reveal the critical function of the DNA replication stress response and, specifically, Smarcal1 in hematopoietic cell survival and tumor development. Our results also provide important insight into the immunodeficiency observed in individuals with mutations in SMARCAL1 by suggesting that it is an HSPC defect.

title: Surfactant Protein B Suppresses Lung Cancer Progression by Inhibiting Secretory Phospholipase A2 Activity and Arachidonic Acid Production

text: Background/Aims: Radiotherapy is applied to patients with inoperable cancer types including advanced stage non-small cell lung cancer (NSCLC) and radioresistance functions as a critical obstacle in radiotherapy. This study was aimed to investigate the mechanism of radioresistance regulated by surfactant protein B (SP-B). Methods: To investigate the role of SP-B in radioresistance, ΔSFTPB A549 cell line was established and SP-B expression was analyzed. In response to ionizing radiation (IR), the change of SP-B expression was analyzed in A549 and NCI-H441 cell lines. Conditioned media (CM) from NSCLC cells were utilized to evaluate the downstream signaling pathway. The in vivo effects of SP-B were assessed through mouse xenograft model with intratumoral injection of CM. Results: In response to IR, NSCLC cell lines showed decreased SP-B regulated by the TGF-β signaling and decreased SP-B stimulated cell survival and epithelial-mesenchymal transition. Treatment with CM from irradiated cells activated sPLA2, enhanced protein kinase Cδ-MAPKs signaling pathway, and increased arachidonic acid production. We confirmed the in vivo roles of SP-B through mouse xenograft model. Conclusion: Our results revealed that down-regulation of SP-B was involved in the radiation-induced metastatic conversion of NSCLC and provided evidence that SP-B acted as a suppressor of NSCLC progression.

title: Suppression of Myc oncogenic activity by ribosomal protein haploinsufficiency

text: The Myc oncogene regulates the expression of several components of the protein synthetic machinery, including ribosomal proteins, initiation factors of translation, RNA polymerase III and ribosomal DNA. Whether and how increasing the cellular protein synthesis capacity affects the multistep process leading to cancer remains to be addressed. Here we use ribosomal protein heterozygote mice as a genetic tool to restore increased protein synthesis in Emu-Myc/+ transgenic mice to normal levels, and show that the oncogenic potential of Myc in this context is suppressed. Our findings demonstrate that the ability of Myc to increase protein synthesis directly augments cell size and is sufficient to accelerate cell cycle progression independently of known cell cycle targets transcriptionally regulated by Myc. In addition, when protein synthesis is restored to normal levels, Myc-overexpressing precancerous cells are more efficiently eliminated by programmed cell death. Our findings reveal a new mechanism that links increases in general protein synthesis rates downstream of an oncogenic signal to a specific molecular impairment in the modality of translation initiation used to regulate the expression of selective messenger RNAs. We show that an aberrant increase in cap-dependent translation downstream of Myc hyperactivation specifically impairs the translational switch to internal ribosomal entry site (IRES)-dependent translation that is required for accurate mitotic progression. Failure of this translational switch results in reduced mitotic-specific expression of the endogenous IRES-dependent form of Cdk11 (also known as Cdc2l and PITSLRE), which leads to cytokinesis defects and is associated with increased centrosome numbers and genome instability in Emu-Myc/+ mice. When accurate translational control is re-established in Emu-Myc/+ mice, genome instability is suppressed. Our findings demonstrate how perturbations in translational control provide a highly specific outcome for gene expression, genome stability and cancer initiation that have important implications for understanding the molecular mechanism of cancer formation at the post-genomic level.

title: Genotoxic Stress Abrogates Renewal of Melanocyte Stem Cells by Triggering Their Differentiation

text: Somatic stem cell depletion due to the accumulation of DNA damage has been implicated in the appearance of aging-related phenotypes. Hair graying, a typical sign of aging in mammals, is caused by the incomplete maintenance of melanocyte stem cells (MSCs) with age. Here, we report that irreparable DNA damage, as caused by ionizing radiation, abrogates renewal of MSCs in mice. Surprisingly, the DNA-damage response triggers MSC differentiation into mature melanocytes in the niche, rather than inducing their apoptosis or senescence. The resulting MSC depletion leads to irreversible hair graying. Furthermore, deficiency of Ataxia-telangiectasia mutated (ATM), a central transducer kinase of the DNA-damage response, sensitizes MSCs to ectopic differentiation, demonstrating that the kinase protects MSCs from their premature differentiation by functioning as a "stemness checkpoint" to maintain the stem cell quality and quantity.

title: The Ino80 chromatin-remodeling complex restores chromatin structure during UV DNA damage repair

text: Chromatin structure is modulated during deoxyribonucleic acid excision repair, but how this is achieved is unclear. Loss of the yeast Ino80 chromatin-remodeling complex (Ino80-C) moderately sensitizes cells to ultraviolet (UV) light. In this paper, we show that INO80 acts in the same genetic pathway as nucleotide excision repair (NER) and that the Ino80-C contributes to efficient UV photoproduct removal in a region of high nucleosome occupancy. Moreover, Ino80 interacts with the early NER damage recognition complex Rad4-Rad23 and is recruited to chromatin by Rad4 in a UV damage-dependent manner. Using a modified chromatin immunoprecipitation assay, we find that chromatin disruption during UV lesion repair is normal, whereas the restoration of nucleosome structure is defective in ino80 mutant cells. Collectively, our work suggests that Ino80 is recruited to sites of UV lesion repair through interactions with the NER apparatus and is required for the restoration of chromatin structure after repair.

title: Increased susceptibility to UV-induced skin carcinogenesis in polymerase eta-deficient mice.

text: Xeroderma pigmentosum variant (XPV) patients with mutations in the DNA polymerase eta (pol eta) gene are hypersensitive to sunlight and have greatly increased susceptibility to sunlight-induced skin cancer. Consistent with the ability of Pol eta to efficiently bypass UV light-induced cyclobutane pyrimidine dimers, XPV cells lacking Pol eta have diminished capacity to replicate UV-damaged DNA and are sensitive to UV light-induced killing and mutagenesis. To better understand these and other Pol eta functions, we generated Pol eta-deficient mice. Mice homozygous for a null mutation in pol eta are viable, fertile, and do not show any obvious spontaneous defects during the first year of life. However, fibroblasts derived from these mutant mice are sensitive to killing by exposure to UV light, and all Pol eta-deficient mice develop skin tumors after UV irradiation, in contrast to the wild-type littermate controls that did not develop such tumors. These results and biochemical studies of translesion synthesis by mouse Pol eta indicate that Pol eta-dependent bypass of cyclobutane pyrimidine dimers suppresses UV light-induced skin cancer in mice. Moreover, 37.5% of pol eta heterozygous mice also developed skin cancer during 5 months after a 5-month exposure to UV light, suggesting that humans who are heterozygous for mutations in pol eta may also have an increased risk of skin cancer.

title: The BLM helicase contributes to telomere maintenance through processing of late-replicating intermediate structures

text: Werner's syndrome (WS) and Bloom's syndrome (BS) are cancer predisposition disorders caused by loss of function of the RecQ helicases WRN or BLM, respectively. BS and WS are characterized by replication defects, hyperrecombination events and chromosomal aberrations, which are hallmarks of cancer. Inefficient replication of the G-rich telomeric strand contributes to chromosome aberrations in WS cells, demonstrating a link between WRN, telomeres and genomic stability. Herein, we provide evidence that BLM also contributes to chromosome-end maintenance. Telomere defects (TDs) are observed in BLM-deficient cells at an elevated frequency, which is similar to cells lacking a functional WRN helicase. Loss of both helicases exacerbates TDs and chromosome aberrations, indicating that BLM and WRN function independently in telomere maintenance. BLM localization, particularly its recruitment to telomeres, changes in response to replication dysfunction, such as in WRN-deficient cells or after aphidicolin treatment. Exposure to replication challenge causes an increase in decatenated deoxyribonucleic acid (DNA) structures and late-replicating intermediates (LRIs), which are visible as BLM-covered ultra-fine bridges (UFBs) in anaphase. A subset of UFBs originates from telomeric DNA and their frequency correlates with telomere replication defects. We propose that the BLM complex contributes to telomere maintenance through its activity in resolving LRIs.

title: PHD3-dependent hydroxylation of HCLK2 promotes the DNA damage response.

text: The DNA damage response (DDR) is a complex regulatory network that is critical for maintaining genome integrity. Posttranslational modifications are widely used to ensure strict spatiotemporal control of signal flow, but how the DDR responds to environmental cues, such as changes in ambient oxygen tension, remains poorly understood. We found that an essential component of the ATR/CHK1 signaling pathway, the human homolog of the Caenorhabditis elegans biological clock protein CLK-2 (HCLK2), associated with and was hydroxylated by prolyl hydroxylase domain protein 3 (PHD3). HCLK2 hydroxylation was necessary for its interaction with ATR and the subsequent activation of ATR/CHK1/p53. Inhibiting PHD3, either with the pan-hydroxylase inhibitor dimethyloxaloylglycine (DMOG) or through hypoxia, prevented activation of the ATR/CHK1/p53 pathway and decreased apoptosis induced by DNA damage. Consistent with these observations, we found that mice lacking PHD3 were resistant to the effects of ionizing radiation and had decreased thymic apoptosis, a biomarker of genomic integrity. Our identification of HCLK2 as a substrate of PHD3 reveals the mechanism through which hypoxia inhibits the DDR, suggesting hydroxylation of HCLK2 is a potential therapeutic target for regulating the ATR/CHK1/p53 pathway.

title: Acetylation of apurinic/apyrimidinic endonuclease-1 regulates Helicobacter pylori-mediated gastric epithelial cell apoptosis.

text:
BACKGROUND & AIMS Helicobacter pylori-induced gastric epithelial cell (GEC) apoptosis is a complex process that includes activation of the tumor suppressor p53. p53-mediated apoptosis involves p53 activation, bax transcription, and cytochrome c release from mitochondria. Apurinic/apyrimidinic endonuclease-1 (APE-1) regulates transcriptional activity of p53, and H pylori induce APE-1 expression in human GECs. H pylori infection increases intracellular calcium ion concentration [Ca2+]i of GECs, which induces APE-1 acetylation. We investigated the effects of H pylori infection and APE-1 acetylation on GEC apoptosis.
 METHODS AGS cells (wild-type or with suppressed APE-1), KATO III cells, and cells isolated from gastric biopsy specimens were infected with H pylori. Effects were examined by immunoblotting, real-time reverse-transcription polymerase chain reaction, immunoprecipitation, immunofluorescence microscopy, chromatin immunoprecipitation, mobility shift, DNA binding, and luciferase assays.
 RESULTS H pylori infection increased [Ca2+]i and acetylation of APE-1 in GECs, but the acetylation status of APE-1 did not affect the transcriptional activity of p53. In GECs, expression of a form of APE-1 that could not be acetylated increased total and mitochondrial levels of Bax and induced release of cytochrome c and fragmentation of DNA; expression of wild-type APE-1 reduced these apoptotic events. We identified a negative calcium response element in the human bax promoter and found that poly (adenosine diphosphate-ribose) polymerase 1 recruited the acetylated APE-1/histone deacetylase-1 repressor complex to bax nCaRE.
 CONCLUSIONS H pylori-mediated acetylation of APE-1 suppresses Bax expression; this prevents p53-mediated apoptosis when H pylori infect GECs.

title: Hyperglycemia is associated with increased bone mineral density and decreased trabecular bone score in elderly Japanese men: The Fujiwara-kyo osteoporosis risk in men (FORMEN) study.

text:
PURPOSE Patients with type 2 diabetes mellitus (T2DM) have an increased fracture risk despite having higher areal bone mineral density (aBMD). This study aimed to clarify the association between glycemic and insulin resistance status and bone microarchitecture, and whether pentosidine and bone turnover markers play any roles in the association.
 METHODS A total of 2012 community-dwelling men aged ≥65years completed baseline measurements of spine aBMD, fasting plasma glucose (FPG) and serum insulin, hemoglobin A1c (HbA1c), osteocalcin, type I procollagen N-terminal propeptide, type I collagen C-terminal crosslinking telopeptide, tartrate-resistant acid phosphatase isoenzyme 5b, pentosidine, height and weight and an interview regarding past disease history. Homeostasis model assessment-insulin resistance (HOMA-IR) was also calculated. T2DM was defined as physician-diagnosed middle age or elderly-onset diabetes mellitus, or according to biochemical test results. To evaluate bone microarchitecture, trabecular bone score (TBS) was calculated at the same vertebrae as those used for aBMD measurement.
 RESULTS After excluding participants who had a disease history and/or were taking medications affecting bone metabolism, 1683 men (age, 72.9±5.2years) were analyzed. Men with T2DM had significantly higher aBMD compared to those without T2DM. There was no significant difference in TBS. However, FPG, HbA1c and HOMA-IR levels were significantly inversely correlated with TBS after adjusting for age, BMI and aBMD. Multivariate linear regression analyses revealed that glycemic indices (FPG and HbA1c) were significantly associated with increased aBMD and decreased TBS, and that HOMA-IR was associated only with TBS. These associations did not change after further adjusting for bone turnover makers and pentosidine levels.
 CONCLUSIONS Hyperglycemia and elevated insulin-resistance were associated with low TBS independently of bone turnover and pentosidine levels.

title: Ribosomal DNA copy loss and repeat instability in ATRX-mutated cancers

text: ATRX (alpha thalassemia/mental retardation X-linked) complexes with DAXX to deposit histone variant H3.3 into repetitive heterochromatin. Recent genome sequencing studies in cancers have revealed mutations in ATRX and their association with ALT (alternative lengthening of telomeres) activation. Here we report depletion of ATRX in mouse ES cells leads to selective loss in ribosomal RNA gene (rDNA) copy number. Supporting this, ATRX-mutated human ALT-positive tumors also show a substantially lower rDNA copy than ALT-negative tumors. Further investigation shows that the rDNA copy loss and repeat instability are caused by a disruption in H3.3 deposition and thus a failure in heterochromatin formation at rDNA repeats in the absence of ATRX. We also find that ATRX-depleted cells are reduced in ribosomal RNA transcription output and show increased sensitivity to RNA polymerase I (Pol I) transcription inhibitor CX5461. In addition, human ALT-positive cancer cell lines are also more sensitive to CX5461 treatment. Our study provides insights into the contribution of ATRX loss of function to tumorigenesis through the loss of rDNA stability and suggests the therapeutic potential of targeting Pol I transcription in ALT cancers.

title: Visualization of recombination intermediates produced by RAD52-mediated single-strand annealing.

text: Double-strand breaks (DSBs) occur frequently during DNA replication. They are also caused by ionizing radiation, chemical damage or as part of the series of programmed events that occur during meiosis. In yeast, DSB repair requires RAD52, a protein that plays a critical role in homologous recombination. Here we describe the actions of human RAD52 protein in a model system for single-strand annealing (SSA) using tailed (i.e. exonuclease resected) duplex DNA molecules. Purified human RAD52 protein binds resected DSBs and promotes associations between complementary DNA termini. Heteroduplex intermediates of these recombination reactions have been visualized by electron microscopy, revealing the specific binding of multiple rings of RAD52 to the resected termini and the formation of large protein complexes at heteroduplex joints formed by RAD52-mediated annealing.

title: Cancer risks attributable to low doses of ionizing radiation: assessing what we really know.

text: High doses of ionizing radiation clearly produce deleterious consequences in humans, including, but not exclusively, cancer induction. At very low radiation doses the situation is much less clear, but the risks of low-dose radiation are of societal importance in relation to issues as varied as screening tests for cancer, the future of nuclear power, occupational radiation exposure, frequent-flyer risks, manned space exploration, and radiological terrorism. We review the difficulties involved in quantifying the risks of low-dose radiation and address two specific questions. First, what is the lowest dose of x- or gamma-radiation for which good evidence exists of increased cancer risks in humans? The epidemiological data suggest that it is approximately 10-50 mSv for an acute exposure and approximately 50-100 mSv for a protracted exposure. Second, what is the most appropriate way to extrapolate such cancer risk estimates to still lower doses? Given that it is supported by experimentally grounded, quantifiable, biophysical arguments, a linear extrapolation of cancer risks from intermediate to very low doses currently appears to be the most appropriate methodology. This linearity assumption is not necessarily the most conservative approach, and it is likely that it will result in an underestimate of some radiation-induced cancer risks and an overestimate of others.

title: Kinetics and Fidelity of the Repair of Cas9-Induced Double-Strand DNA Breaks

text: The RNA-guided DNA endonuclease Cas9 is a powerful tool for genome editing. Little is known about the kinetics and fidelity of the double-strand break (DSB) repair process that follows a Cas9 cutting event in living cells. Here, we developed a strategy to measure the kinetics of DSB repair for single loci in human cells. Quantitative modeling of repaired DNA in time series after Cas9 activation reveals variable and often slow repair rates, with half-life times up to ∼10 hr. Furthermore, repair of the DSBs tends to be error prone. Both classical and microhomology-mediated end joining pathways contribute to the erroneous repair. Estimation of their individual rate constants indicates that the balance between these two pathways changes over time and can be altered by additional ionizing radiation. Our approach provides quantitative insights into DSB repair kinetics and fidelity in single loci and indicates that Cas9-induced DSBs are repaired in an unusual manner.

title: The structure of DdrB from Deinococcus: a new fold for single-stranded DNA binding proteins

text: Deinococcus spp. are renowned for their amazing ability to recover rapidly from severe genomic fragmentation as a result of exposure to extreme levels of ionizing radiation or desiccation. Despite having been originally characterized over 50 years ago, the mechanism underlying this remarkable repair process is still poorly understood. Here, we report the 2.8 A structure of DdrB, a single-stranded DNA (ssDNA) binding protein unique to Deinococcus spp. that is crucial for recovery following DNA damage. DdrB forms a pentameric ring capable of binding single-stranded but not double-stranded DNA. Unexpectedly, the crystal structure reveals that DdrB comprises a novel fold that is structurally and topologically distinct from all other single-stranded binding (SSB) proteins characterized to date. The need for a unique ssDNA binding function in response to severe damage, suggests a distinct role for DdrB which may encompass not only standard SSB protein function in protection of ssDNA, but also more specialized roles in protein recruitment or DNA architecture maintenance. Possible mechanisms of DdrB action in damage recovery are discussed.


query: Mice defective for deoxyribonucleic acid (DNA) polymerase m (polm) fail to reveal increased sensitivity to ionizing radiation (IR).

title: Nonoverlapping functions of DNA polymerases mu, lambda, and terminal deoxynucleotidyltransferase during immunoglobulin V(D)J recombination in vivo.

text: DNA polymerases mu (pol mu), lambda (pol lambda), and terminal deoxynucleotidyltransferase (TdT) are enzymes of the pol X family that share homology in sequence and functional domain organization. We showed previously that pol mu participates in light chain but surprisingly not heavy chain gene rearrangement. We show here that immunoglobulin heavy chain junctions from pol lambda-deficient animals have shorter length with normal N-additions, thus indicating that pol lambda is recruited during heavy chain rearrangement at a step that precedes the action of TdT. In contrast to previous in vitro studies, analysis of animals with combined inactivation of these enzymes revealed no overlapping or compensatory activities for V(D)J recombination between pol mu, pol lambda, and TdT. This complex usage of polymerases with distinct catalytic specificities may correspond to the specific function that the third hypervariable region assumes for each immunoglobulin chain, with pol lambda maintaining a large heavy chain junctional heterogeneity and pol mu ensuring a restricted light chain junctional variability.

title: Deregulated DNA polymerase β strengthens ionizing radiation-induced nucleotidic and chromosomal instabilities

text: DNA polymerase β (Pol β) is an error-prone enzyme which has been found to be overexpressed in several human tumors. By using a couple of recombinant CHO cells differing only from the exogenous expression of Pol β, we showed here that cells overexpressing Pol β are much more sensitive to IR treatments by increasing apoptosis. We also found that the surviving cells displayed an hypermutator phenotype which could be explained by different pathways involving Pol β, such as (i) an increased capacity to incorporate into DNA the mutagenic dGTP analog, 8-oxo-dGTP, one of the most abundant purine-derived nucleotides exposed to γ-irradiation, (ii) the induction of IR-induced DNA breaks and (iii) accumulation of chromosome aberrations induced by radiation. Alteration of Pol β expression in irradiated cells thus appears to strengthen both cell death and genetic changes associated with a malignant phenotype. These data provide new insights into the cellular response to radiations and the associated carcinogenic consequences.

title: Association of DNA polymerase mu (pol mu) with Ku and ligase IV: role for pol mu in end-joining double-strand break repair.

text: Mammalian DNA polymerase mu (pol mu) is related to terminal deoxynucleotidyl transferase, but its biological role is not yet clear. We show here that after exposure of cells to ionizing radiation (IR), levels of pol mu protein increase. pol mu also forms discrete nuclear foci after IR, and these foci are largely coincident with IR-induced foci of gammaH2AX, a previously characterized marker of sites of DNA double-strand breaks. pol mu is thus part of the cellular response to DNA double-strand breaks. pol mu also associates in cell extracts with the nonhomologous end-joining repair factor Ku and requires both Ku and another end-joining factor, XRCC4-ligase IV, to form a stable complex on DNA in vitro. pol mu in turn facilitates both stable recruitment of XRCC4-ligase IV to Ku-bound DNA and ligase IV-dependent end joining. In contrast, the related mammalian DNA polymerase beta does not form a complex with Ku and XRCC4-ligase IV and is less effective than pol mu in facilitating joining mediated by these factors. Our data thus support an important role for pol mu in the end-joining pathway for repair of double-strand breaks.

title: The DNA polymerase λ is required for the repair of non-compatible DNA double strand breaks by NHEJ in mammalian cells

text: DNA polymerase lambda (polλ) is a recently identified DNA polymerase whose cellular function remains elusive. Here we show, that polλ participates at the molecular level in a chromosomal context, in the repair of DNA double strand breaks (DSB) via non-homologous end joining (NHEJ) in mammalian cells. The expression of a catalytically inactive form of polλ (polλDN) decreases the frequency of NHEJ events in response to I-Sce-I-induced DSB whereas inactivated forms of its homologues polβ and polμ do not. Only events requiring DNA end processing before ligation are affected; this defect is associated with large deletions arising in the vicinity of the induced DSB. Furthermore, polλDN-expressing cells exhibit increased sensitization and genomic instability in response to ionizing radiation similar to that of NHEJ-defective cells. Our data support a requirement for polλ in repairing a subset of DSB in genomic DNA, thereby contributing to the maintenance of genetic stability mediated by the NHEJ pathway.

title: DNA replication is required To elicit cellular responses to psoralen-induced DNA interstrand cross-links.

text: Following introduction of DNA interstrand cross-links (ICLs), mammalian cells display chromosome breakage or cell cycle delay with a 4N DNA content. To further understand the nature of the delay, previously described as a G(2)/M arrest, we developed a protocol to generate ICLs during specific intervals of the cell cycle. Synchronous populations of G(1), S, and G(2) cells were treated with photoactivated 4'-hydroxymethyl-4,5',8-trimethylpsoralen (HMT) and scored for normal passage into mitosis. In contrast to what was found for ionizing radiation, ICLs introduced during G(2) did not result in a G(2)/M arrest, mitotic arrest, or chromosome breakage. Rather, subsequent passage through S phase was required to trigger both chromosome breakage and arrest in the next cell cycle. Similarly, ICLs introduced during G(1) did not cause a G(1)/S arrest. We conclude that DNA replication is required to elicit the cellular responses of cell cycle arrest and genomic instability after psoralen-induced ICLs. In primary human fibroblasts, the 4N DNA content cell cycle arrest triggered by ICLs was long lasting but reversible. Kinetic analysis suggested that these cells could remove up to approximately 2,500 ICLs/genome at an average rate of 11 ICLs/genome/h.

title: A vertebrate gene, ticrr, is an essential checkpoint and replication regulator.

text: Eukaryotes have numerous checkpoint pathways to protect genome fidelity during normal cell division and in response to DNA damage. Through a screen for G2/M checkpoint regulators in zebrafish, we identified ticrr (for TopBP1-interacting, checkpoint, and replication regulator), a previously uncharacterized gene that is required to prevent mitotic entry after treatment with ionizing radiation. Ticrr deficiency is embryonic-lethal in the absence of exogenous DNA damage because it is essential for normal cell cycle progression. Specifically, the loss of ticrr impairs DNA replication and disrupts the S/M checkpoint, leading to premature mitotic entry and mitotic catastrophe. We show that the human TICRR ortholog associates with TopBP1, a known checkpoint protein and a core component of the DNA replication preinitiation complex (pre-IC), and that the TICRR-TopBP1 interaction is stable without chromatin and requires BRCT motifs essential for TopBP1's replication and checkpoint functions. Most importantly, we find that ticrr deficiency disrupts chromatin binding of pre-IC, but not prereplication complex, components. Taken together, our data show that TICRR acts in association with TopBP1 and plays an essential role in pre-IC formation. It remains to be determined whether Ticrr represents the vertebrate ortholog of the yeast pre-IC component Sld3, or a hitherto unknown metazoan replication and checkpoint regulator.

title: Distinct roles for DNA-PK, ATM and ATR in RPA phosphorylation and checkpoint activation in response to replication stress

text: DNA damage encountered by DNA replication forks poses risks of genome destabilization, a precursor to carcinogenesis. Damage checkpoint systems cause cell cycle arrest, promote repair and induce programed cell death when damage is severe. Checkpoints are critical parts of the DNA damage response network that act to suppress cancer. DNA damage and perturbation of replication machinery causes replication stress, characterized by accumulation of single-stranded DNA bound by replication protein A (RPA), which triggers activation of ataxia telangiectasia and Rad3 related (ATR) and phosphorylation of the RPA32, subunit of RPA, leading to Chk1 activation and arrest. DNA-dependent protein kinase catalytic subunit (DNA-PKcs) [a kinase related to ataxia telangiectasia mutated (ATM) and ATR] has well characterized roles in DNA double-strand break repair, but poorly understood roles in replication stress-induced RPA phosphorylation. We show that DNA-PKcs mutant cells fail to arrest replication following stress, and mutations in RPA32 phosphorylation sites targeted by DNA-PKcs increase the proportion of cells in mitosis, impair ATR signaling to Chk1 and confer a G2/M arrest defect. Inhibition of ATR and DNA-PK (but not ATM), mimic the defects observed in cells expressing mutant RPA32. Cells expressing mutant RPA32 or DNA-PKcs show sustained H2AX phosphorylation in response to replication stress that persists in cells entering mitosis, indicating inappropriate mitotic entry with unrepaired damage.

title: in

text: Transglutaminase 2 (TGase 2) is a Ca+2-dependent enzyme that catalyzes both intracellular and extracellular cross-linking reactions by transamidation of specific glutamine residues. TGase 2 is known to be involved in the membrane-mediated events required for glucose-stimulated insulin release from the pancreatic beta cells. Here we show that targeted disruption of TGase 2 impairs glucose-stimulated insulin secretion. TGase 2-/- mice show glucose intolerance after intraperitoneal glucose loading. TGase 2-/- mice manifest a tendency to develop hypoglycemia after administration of exogenous insulin as a consequence of enhanced insulin receptor substrate 2 (IRS-2) phosphorylation. We suggest that the increased peripheral sensitivity to insulin partially compensates for the defective secretion in this animal model. TGase 2-/- mouse phenotype resembles that of the maturity-onset diabetes of young (MODY) patients. In the course of screening for human TGase 2 gene in Italian subjects with the clinical features of MODY, we detected a missense mutation (N333S) in the active site of the enzyme. Collectively, these results identify TGase 2 as a potential candidate gene in type 2 diabetes.


query: Mice defective for deoxyribonucleic acid (DNA) polymerase m (polm) reveal increased sensitivity to ionizing radiation (IR).

title: Ionizing radiation-induced long-term expression of senescence markers in mice is independent of p53 and immune status.

text: Exposure to IR has been shown to induce the formation of senescence markers, a phenotype that coincides with lifelong delayed repair and regeneration of irradiated tissues. We hypothesized that IR-induced senescence markers could persist long-term in vivo, possibly contributing to the permanent reduction in tissue functionality. Here, we show that mouse tissues exposed to a sublethal dose of IR display persistent (up to 45 weeks, the maximum time analyzed) DNA damage foci and increased p16(INK4a) expression, two hallmarks of cellular senescence and aging. BrdU-labeling experiments revealed that IR-induced damaged cells are preferentially eliminated, at least partially, in a tissue-dependent manner. Unexpectedly, the accumulation of damaged cells was found to occur independent from the DNA damage response modulator p53, and from an intact immune system, as their levels were similar in wild-type and Rag2(-/-) gammaC(-/-) mice, the latter being deficient in T, B, and NK cells. Together, our results provide compelling evidence that exposure to IR induces long-term expression of senescence markers in vivo, an effect that may contribute to the reduced tissue functionality observed in cancer survivors.

title: Defective replication stress response inhibits lymphomagenesis and impairs lymphocyte reconstitution

text: DNA replication stress promotes genome instability in cancer. However, the contribution of the replication stress response to the development of malignancies remains unresolved. The DNA replication stress response protein SMARCAL1 stabilizes DNA replication forks and prevents replication fork collapse, a cause of DNA breaks and apoptosis. While the fork regression/remodeling functions of SMARCAL1 have been investigated, its in vivo functions in replication stress and cancer are unclear. Using a gamma radiation (IR)-induced replication stress T-cell lymphoma mouse model, we observed a significant inhibition of lymphomagenesis in mice lacking one or both alleles of Smarcal1. Notably, a quarter of the Smarcal1-deficient mice did not develop tumors. Moreover, hematopoietic stem/progenitor cells (HSPCs) and developing thymocytes in Smarcal1-deficient mice showed increased DNA damage and apoptosis during the proliferation burst following IR and an impaired ability to repopulate the thymus after IR. Additionally, mice lacking Smarcal1 showed significant HSPC defects when challenged to respond to other replication stress stimuli. Thus, our data reveal the critical function of the DNA replication stress response and, specifically, Smarcal1 in hematopoietic cell survival and tumor development. Our results also provide important insight into the immunodeficiency observed in individuals with mutations in SMARCAL1 by suggesting that it is an HSPC defect.

title: Surfactant Protein B Suppresses Lung Cancer Progression by Inhibiting Secretory Phospholipase A2 Activity and Arachidonic Acid Production

text: Background/Aims: Radiotherapy is applied to patients with inoperable cancer types including advanced stage non-small cell lung cancer (NSCLC) and radioresistance functions as a critical obstacle in radiotherapy. This study was aimed to investigate the mechanism of radioresistance regulated by surfactant protein B (SP-B). Methods: To investigate the role of SP-B in radioresistance, ΔSFTPB A549 cell line was established and SP-B expression was analyzed. In response to ionizing radiation (IR), the change of SP-B expression was analyzed in A549 and NCI-H441 cell lines. Conditioned media (CM) from NSCLC cells were utilized to evaluate the downstream signaling pathway. The in vivo effects of SP-B were assessed through mouse xenograft model with intratumoral injection of CM. Results: In response to IR, NSCLC cell lines showed decreased SP-B regulated by the TGF-β signaling and decreased SP-B stimulated cell survival and epithelial-mesenchymal transition. Treatment with CM from irradiated cells activated sPLA2, enhanced protein kinase Cδ-MAPKs signaling pathway, and increased arachidonic acid production. We confirmed the in vivo roles of SP-B through mouse xenograft model. Conclusion: Our results revealed that down-regulation of SP-B was involved in the radiation-induced metastatic conversion of NSCLC and provided evidence that SP-B acted as a suppressor of NSCLC progression.

title: Suppression of Myc oncogenic activity by ribosomal protein haploinsufficiency

text: The Myc oncogene regulates the expression of several components of the protein synthetic machinery, including ribosomal proteins, initiation factors of translation, RNA polymerase III and ribosomal DNA. Whether and how increasing the cellular protein synthesis capacity affects the multistep process leading to cancer remains to be addressed. Here we use ribosomal protein heterozygote mice as a genetic tool to restore increased protein synthesis in Emu-Myc/+ transgenic mice to normal levels, and show that the oncogenic potential of Myc in this context is suppressed. Our findings demonstrate that the ability of Myc to increase protein synthesis directly augments cell size and is sufficient to accelerate cell cycle progression independently of known cell cycle targets transcriptionally regulated by Myc. In addition, when protein synthesis is restored to normal levels, Myc-overexpressing precancerous cells are more efficiently eliminated by programmed cell death. Our findings reveal a new mechanism that links increases in general protein synthesis rates downstream of an oncogenic signal to a specific molecular impairment in the modality of translation initiation used to regulate the expression of selective messenger RNAs. We show that an aberrant increase in cap-dependent translation downstream of Myc hyperactivation specifically impairs the translational switch to internal ribosomal entry site (IRES)-dependent translation that is required for accurate mitotic progression. Failure of this translational switch results in reduced mitotic-specific expression of the endogenous IRES-dependent form of Cdk11 (also known as Cdc2l and PITSLRE), which leads to cytokinesis defects and is associated with increased centrosome numbers and genome instability in Emu-Myc/+ mice. When accurate translational control is re-established in Emu-Myc/+ mice, genome instability is suppressed. Our findings demonstrate how perturbations in translational control provide a highly specific outcome for gene expression, genome stability and cancer initiation that have important implications for understanding the molecular mechanism of cancer formation at the post-genomic level.

title: Genotoxic Stress Abrogates Renewal of Melanocyte Stem Cells by Triggering Their Differentiation

text: Somatic stem cell depletion due to the accumulation of DNA damage has been implicated in the appearance of aging-related phenotypes. Hair graying, a typical sign of aging in mammals, is caused by the incomplete maintenance of melanocyte stem cells (MSCs) with age. Here, we report that irreparable DNA damage, as caused by ionizing radiation, abrogates renewal of MSCs in mice. Surprisingly, the DNA-damage response triggers MSC differentiation into mature melanocytes in the niche, rather than inducing their apoptosis or senescence. The resulting MSC depletion leads to irreversible hair graying. Furthermore, deficiency of Ataxia-telangiectasia mutated (ATM), a central transducer kinase of the DNA-damage response, sensitizes MSCs to ectopic differentiation, demonstrating that the kinase protects MSCs from their premature differentiation by functioning as a "stemness checkpoint" to maintain the stem cell quality and quantity.

title: The Ino80 chromatin-remodeling complex restores chromatin structure during UV DNA damage repair

text: Chromatin structure is modulated during deoxyribonucleic acid excision repair, but how this is achieved is unclear. Loss of the yeast Ino80 chromatin-remodeling complex (Ino80-C) moderately sensitizes cells to ultraviolet (UV) light. In this paper, we show that INO80 acts in the same genetic pathway as nucleotide excision repair (NER) and that the Ino80-C contributes to efficient UV photoproduct removal in a region of high nucleosome occupancy. Moreover, Ino80 interacts with the early NER damage recognition complex Rad4-Rad23 and is recruited to chromatin by Rad4 in a UV damage-dependent manner. Using a modified chromatin immunoprecipitation assay, we find that chromatin disruption during UV lesion repair is normal, whereas the restoration of nucleosome structure is defective in ino80 mutant cells. Collectively, our work suggests that Ino80 is recruited to sites of UV lesion repair through interactions with the NER apparatus and is required for the restoration of chromatin structure after repair.

title: Increased susceptibility to UV-induced skin carcinogenesis in polymerase eta-deficient mice.

text: Xeroderma pigmentosum variant (XPV) patients with mutations in the DNA polymerase eta (pol eta) gene are hypersensitive to sunlight and have greatly increased susceptibility to sunlight-induced skin cancer. Consistent with the ability of Pol eta to efficiently bypass UV light-induced cyclobutane pyrimidine dimers, XPV cells lacking Pol eta have diminished capacity to replicate UV-damaged DNA and are sensitive to UV light-induced killing and mutagenesis. To better understand these and other Pol eta functions, we generated Pol eta-deficient mice. Mice homozygous for a null mutation in pol eta are viable, fertile, and do not show any obvious spontaneous defects during the first year of life. However, fibroblasts derived from these mutant mice are sensitive to killing by exposure to UV light, and all Pol eta-deficient mice develop skin tumors after UV irradiation, in contrast to the wild-type littermate controls that did not develop such tumors. These results and biochemical studies of translesion synthesis by mouse Pol eta indicate that Pol eta-dependent bypass of cyclobutane pyrimidine dimers suppresses UV light-induced skin cancer in mice. Moreover, 37.5% of pol eta heterozygous mice also developed skin cancer during 5 months after a 5-month exposure to UV light, suggesting that humans who are heterozygous for mutations in pol eta may also have an increased risk of skin cancer.

text: Werner's syndrome (WS) and Bloom's syndrome (BS) are cancer predisposition disorders caused by loss of function of the RecQ helicases WRN or BLM, respectively. BS and WS are characterized by replication defects, hyperrecombination events and chromosomal aberrations, which are hallmarks of cancer. Inefficient replication of the G-rich telomeric strand contributes to chromosome aberrations in WS cells, demonstrating a link between WRN, telomeres and genomic stability. Herein, we provide evidence that BLM also contributes to chromosome-end maintenance. Telomere defects (TDs) are observed in BLM-deficient cells at an elevated frequency, which is similar to cells lacking a functional WRN helicase. Loss of both helicases exacerbates TDs and chromosome aberrations, indicating that BLM and WRN function independently in telomere maintenance. BLM localization, particularly its recruitment to telomeres, changes in response to replication dysfunction, such as in WRN-deficient cells or after aphidicolin treatment. Exposure to replication challenge causes an increase in decatenated deoxyribonucleic acid (DNA) structures and late-replicating intermediates (LRIs), which are visible as BLM-covered ultra-fine bridges (UFBs) in anaphase. A subset of UFBs originates from telomeric DNA and their frequency correlates with telomere replication defects. We propose that the BLM complex contributes to telomere maintenance through its activity in resolving LRIs.

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title: Regulation of p53 in response to DNA damage

text: Activation of p53 can occur in response to a number of cellular stresses, including DNA damage, hypoxia and nucleotide deprivation. Several forms of DNA damage have been shown to activate p53, including those generated by ionising radiation (IR), radio-mimetic drugs, ultraviolet light (UV) and chemicals such as methyl methane sulfonate (MMS). Under normal conditions, p53 levels are maintained at a low state by virtue of the extremely short-half life of the polypeptide. In addition to this, p53 normally exists in an largely inactive state that is relatively inefficient at binding to DNA and activating transcription. Activation of p53 in response to DNA damage is associated with a rapid increase in its levels and with an increased ability of p53 to bind DNA and mediate transcriptional activation. This then leads to the activation of a number of genes whose products trigger cell-cycle arrest, apoptosis, or DNA repair. Recent work has suggested that this regulation is brought about largely through DNA damage triggering a series of phosphorylation, de-phosphorylation and acetylation events on the p53 polypeptide. Here, we discuss the nature of these modifications, the enzymes that bring them about, and how changes in p53 modification lead to p53 activation.