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In obesity, elevated insulin causes fatty liver by activating the gene encoding SREBP-1c, a transcription factor that enhances fatty acid synthesis. Two transcription factors, LXRα and C/EBPβ, are necessary but not sufficient for insulin induction of hepatic SREBP-1c mRNA. Here, we show that a third transcription factor, BHLHE40, is required. Immunoprecipitation revealed that BHLHE40 binds to C/EBPβ and LXRα in livers of rats that had fasted and then refed. Hepatic BHLHE40 mRNA rises rapidly when fasted rats are refed and when rat hepatocytes are incubated with insulin. Preventing this rise by gene knockout in mice or siRNAs in hepatocytes reduces the insulin-induced rise in SREBP-1c mRNA. Although BHLHE40 is necessary for insulin induction of SREBP-1c, it is not sufficient as demonstrated by failure of lentiviral BHLHE40 overexpression to increase hepatocyte SREBP-1c mRNA in the absence of insulin. Thus, an additional event is required for insulin to increase SREBP-1c mRNA.

CD11c, also known as integrin alpha X, is the most widely used defining marker for dendritic cells (DCs). CD11c can bind complement iC3b and mediate phagocytosis in vitro, for which it is also referred to as complement receptor 4. However, the functions of this prominent marker protein in DCs, especially in vivo, remain poorly defined. Here, in the process of studying DC activation and immune responses induced by cells lacking self-CD47, we found that DC capture of CD47-deficient cells and DC activation was dependent on the integrin-signaling adaptor Talin1. Specifically, CD11c and its partner Itgb2 were required for DC capture of CD47-deficient cells. CD11b was not necessary for this process but could partially compensate in the absence of CD11c. Mice with DCs lacking Talin1, Itgb2, or CD11c were defective in supporting T-cell proliferation and differentiation induced by CD47-deficient cell associated antigen. These findings establish a critical role for CD11c in DC antigen uptake and activation in vivo. They may also contribute to understanding the functional mechanism of CD47-blockade therapies.

Alzheimer's disease (AD) is the most common type of dementia and is characterized by the accumulation of amyloid (Aβ) plaques and neurofibrillary tangles in the brain. Much attention has been given to develop AD treatments based on the amyloid cascade hypothesis; however, none of these drugs had good efficacy at improving cognitive functions in AD patients suggesting that Aβ might not be the disease origin. Thus, there are urgent needs for the development of new therapies that target on the proximal cause of AD. Cellular calcium (Ca2+) signals regulate important facets of neuronal physiology. An increasing body of evidence suggests that age-related dysregulation of neuronal Ca2+ homeostasis may play a proximal role in the pathogenesis of AD as disrupted Ca2+ could induce synaptic deficits and promote the accumulation of Aβ plaques and neurofibrillary tangles. Given that Ca2+ disruption is ubiquitously involved in all AD pathologies, it is likely that using chemical agents or small molecules specific to Ca2+ channels or handling proteins on the plasma membrane and membranes of intracellular organelles to correct neuronal Ca2+ dysregulation could open up a new approach to AD prevention and treatment. This review summarizes current knowledge on the molecular mechanisms linking Ca2+ dysregulation with AD pathologies and discusses the possibility of correcting neuronal Ca2+ disruption as a therapeutic approach for AD.

Deep learning recently becomes popular because it brings significant improvements on a wide variety of classification and recognition tasks. However, with the population and increasing usage of deep learning based models, not many people take into account the potential security risks which are likely to cause accidents in them. This paper mainly studies on the potential safety hazards in the obstacle recognition and processing system (ORPS) of the self-driving cars, which is constructed by deep learning architecture. We perform an attack that embeds a backdoor in the Mask R-CNN in ORPS by poisoning the dataset. The experiment result shows that it is possible to embed a backdoor in ORPS. We can see that the backdoored network can accurately recognize and trigger the backdoors in the poisoned dataset, which obviously change the size of bounding box and corresponding mask of those poisoned instances. But on the other hand, embedding a backdoor in the deep learning based model will only slightly affect the accuracy of detecting objects without backdoor triggers, which is imperceptible for users. Furthermore, in order to study the working mode of the backdoor and the possibility of detecting the backdoor in the network, we visualize the weights matrices in the backdoored network and try to modify them, but the results show that the existence of the backdoor in network is very cryptic, so it is difficult for users to detect and filter it. Eventually, we hope that our simple work can arouse people’s attention to the self-driving technology and even other deep learning based models.

This paper mainly studies on the potential safety hazards in the obstacle recognition and processing system (ORPS) of the self-driving cars, which is constructed by deep learning architecture. We perform an attack that embeds a backdoor in the Mask R-CNN in ORPS by poisoning the dataset. Under normal circumstances, the backdoored model can accurately identify obstacles (vehicles). However, under certain circumstances, triggering the backdoor in the backdoored model may lead to change the size (bounding box and mask) and confidence of the detected obstacles, which may cause serious accidents. The experiment result shows that it is possible to embed a backdoor in ORPS. We can see that the backdoored network can obviously change the size of bounding box and corresponding mask of those poisoned instances. But on the other hand, embedding a backdoor in the deep learning based model will only slightly affect the accuracy of detecting objects without backdoor triggers, which is imperceptible for users. Eventually, we hope that our simple work can arouse people’s attention to the self-driving technology and even other deep learning based models. It brings motivation about how to judge or detect the existence of the backdoors in these systems.

Significance The presence of cytosolic DNA in mammalian cells signifies microbial invasions and triggers the DNA sensor protein cGAS to produce the second messenger molecule 2′3′-cGAMP, which elicits innate immune responses by binding to and activating the homodimerized adaptor protein STING. Here we show that the high affinity of the asymmetric ligand 2′3′-cGAMP to the symmetric dimer of STING originates from its unique mixed phosphodiester linkages. 2′3′-cGAMP, but not its linkage isomers, adopts an organized free-ligand conformation that resembles the STING-bound conformation and pays low energy costs in changing into the active conformation. Whereas biological structural studies have focused on analyses of protein conformations, our results demonstrate that analyses of free-ligand conformations can be equally important in understanding protein–ligand interactions.

A national research agenda focused on gifted/creative/talented Native American students is needed, as this population remains one of the least researched, most overlooked, and most underserved in the field. Literature-based assumptions surrounding Native American students’ talent development, culture and traditions, cognitive styles and learning preferences, and communication were generated and then reviewed by educators and tribal members for relevance and accurace. This article has three purposes. The first is to analyze the literature-based assumptions concerning gifted education in three Native American communities—Diné, Lakota, and Ojibwe. The second is to call on gifted education researchers to include Native American students in their research. The third is to suggest a research agenda based on data gathered within these communities

In recent years, we have seen significant advancement in technologies to bring about smarter cities worldwide. The interconnectivity of things is the key enabler in these initiatives. An important building block is smart mobility, and it revolves around resolving land transport challenges in cities with dense populations. A transformative direction that global stakeholders are looking into is autonomous vehicles and the transport infrastructure to interconnect them to the traffic management system (that is, vehicle to infrastructure connectivity), as well as to communicate with one another (that is, vehicle to vehicle connectivity) to facilitate better awareness of road conditions. A number of countries had also started to take autonomous vehicles to the roads to conduct trials and are moving towards the plan for larger scale deployment. However, an important consideration in this space is the security of the autonomous vehicles. There has been an increasing interest in the attacks and defences of autonomous vehicles as these vehicles are getting ready to go onto the roads. In this paper, we aim to organize and discuss the various methods of attacking and defending autonomous vehicles, and propose a comprehensive attack and defence taxonomy to better categorize each of them. Through this work, we hope that it provides a better understanding of how targeted defences should be put in place for targeted attacks, and for technologists to be more mindful of the pitfalls when developing architectures, algorithms and protocols, so as to realise a more secure infrastructure composed of dependable autonomous vehicles.

Skeletal muscle contains estrogens and estrogen synthesis-related enzymes. However, it has not been reported whether myoblasts cultured in vitro also express these enzymes. The purpose of the current study was to address these issues and to explore the effects of mechanical stretch on the enzyme system. The in vitro cultured C2C12 mouse myoblasts were divided into the control, stretch, testosterone and stretch plus testosterone groups. Cells in the stretch and stretch plus testosterone groups were mechanically stretched with the Flexercell cell stress loading device at an amplitude of 10% and in a frequency of 0.5 Hz for 8 h. Cells in the testosterone and stretch plus testosterone groups were incubated with 100 nM testosterone for 24 h before distraction. Following the treatments, cell proliferation and estradiol levels, as well as the expressions of 17β-hydroxysteroid (17β-HSD), 3β-hydroxysteroid (3β-HSD) and aromatase were analyzed. Compared to the control, the cell proliferation in all experimental groups increased significantly, the estradiol levels in the mechanically stretched groups were significantly higher, and, moreover, the estradiol levels were positively correlated with the cell proliferation (r = 0.615, p

Summary The presence of microbial or self DNA in the cytoplasm of mammalian cells is a danger signal detected by the DNA sensor cyclic-GMP-AMP (cGAMP) synthase (cGAS), which catalyzes the production of cGAMP that in turn serves as a second messenger to activate innate immune responses. Here we show that endogenous cGAMP in mammalian cells contains two distinct phosphodiester linkages, one between 2′-OH of GMP and 5′-phosphate of AMP, and the other between 3′-OH of AMP and 5′-phosphate of GMP. This molecule, termed 2′3′-cGAMP, is unique in that it binds to the adaptor protein STING with a much greater affinity than cGAMP molecules containing other combinations of phosphodiester linkages. The crystal structure of STING bound to 2′3′-cGAMP revealed the structural basis of this high-affinity binding and a ligand-induced conformational change in STING that may underlie its activation.

Neurons from the brains of Alzheimer’s disease (AD) and related tauopathy patients contain neurofibrillary tangles composed of hyperphosphorylated tau protein. Tau normally stabilizes microtubules (MTs); however, tau hyperphosphorylation leads to loss of this function with consequent MT destabilization and neuronal dysfunction. Accordingly, MT-stabilizing drugs such as paclitaxel and epothilone D have been shown as possible therapies for AD and related tauopathies. However, MT-stabilizing drugs have common side effects such as neuropathy and neutropenia. To find previously undescribed suppressors of tau-induced MT defects, we established a Drosophila model ectopically expressing human tau in muscle cells, which allow for clear visualization of the MT network. Overexpressed tau was hyperphosphorylated and resulted in decreased MT density and greater fragmentation, consistent with previous reports in AD patients and mouse models. From a genetic screen, we found that a histone deacetylase 6 ( HDAC6 ) null mutation rescued tau-induced MT defects in both muscles and neurons. Genetic and pharmacological inhibition of the tubulin-specific deacetylase activity of HDAC6 indicates that the rescue effect may be mediated by increased MT acetylation. These findings reveal HDAC6 as a unique potential drug target for AD and related tauopathies.

The presence of DNA in the cytoplasm of mammalian cells is a danger signal that triggers host immune responses such as the production of type I interferons. Cytosolic DNA induces interferons through the production of cyclic guanosine monophosphate–adenosine monophosphate (cyclic GMP-AMP, or cGAMP), which binds to and activates the adaptor protein STING. Through biochemical fractionation and quantitative mass spectrometry, we identified a cGAMP synthase (cGAS), which belongs to the nucleotidyltransferase family. Overexpression of cGAS activated the transcription factor IRF3 and induced interferon-β in a STING-dependent manner. Knockdown of cGAS inhibited IRF3 activation and interferon-β induction by DNA transfection or DNA virus infection. cGAS bound to DNA in the cytoplasm and catalyzed cGAMP synthesis. These results indicate that cGAS is a cytosolic DNA sensor that induces interferons by producing the second messenger cGAMP.

Cytosolic DNA induces type I interferons and other cytokines that are important for antimicrobial defense but can also result in autoimmunity. This DNA signaling pathway requires the adaptor protein STING and the transcription factor IRF3, but the mechanism of DNA sensing is unclear. We found that mammalian cytosolic extracts synthesized cyclic guanosine monophosphate-adenosine monophosphate (cyclic GMP-AMP, or cGAMP) in vitro from adenosine triphosphate and guanosine triphosphate in the presence of DNA but not RNA. DNA transfection or DNA virus infection of mammalian cells also triggered cGAMP production. cGAMP bound to STING, leading to the activation of IRF3 and induction of interferon-β. Thus, cGAMP functions as an endogenous second messenger in metazoans and triggers interferon production in response to cytosolic DNA.

MicroRNAs (miRNAs) perform critical functions in normal physiology and disease by associating with Argonaute proteins and downregulating partially complementary messenger RNAs (mRNAs). Here we use clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated protein 9 (Cas9) genome-wide loss-of-function screening coupled with a fluorescent reporter of miRNA activity in human cells to identify new regulators of the miRNA pathway. By using iterative rounds of screening, we reveal a novel mechanism whereby target engagement by Argonaute 2 (AGO2) triggers its hierarchical, multi-site phosphorylation by CSNK1A1 on a set of highly conserved residues (S824-S834), followed by rapid dephosphorylation by the ANKRD52-PPP6C phosphatase complex. Although genetic and biochemical studies demonstrate that AGO2 phosphorylation on these residues inhibits target mRNA binding, inactivation of this phosphorylation cycle globally impairs miRNA-mediated silencing. Analysis of the transcriptome-wide binding profile of non-phosphorylatable AGO2 reveals a pronounced expansion of the target repertoire bound at steady-state, effectively reducing the active pool of AGO2 on a per-target basis. These findings support a model in which an AGO2 phosphorylation cycle stimulated by target engagement regulates miRNA:target interactions to maintain the global efficiency of miRNA-mediated silencing.

We have proposed that the low-fidelity compensatory backup alternative DNA repair pathways drive multistep carcinogenesis. Here, we apply it to interpret the clinical features of cancer, such as mutator phenotype, tissue specificity, age specificity, diverse types of cancers originated from the same type of tissue, cancer susceptibility of patients with DNA repair-defective syndromes, development of cancer only for a selected number of individuals among those that share the same genetic defect, invasion and metastasis. Clinically, the theory predicts that to improve the efficacy of molecular targeted or synthetic lethal therapy, it may be crucial to inhibit the low-fidelity compensatory alternative DNA repair either directly or by blocking the signal transducers of the sustained microenvironmental stress.

Innate immune receptor signaling, united Innate immune receptors such as RIG-I, cGAS, and Toll-like receptors bind microbial fragments and alert the immune system to an infection. Each receptor type signals through a different adapter protein. These signals activate the protein kinase TBK1 and the transcription factor IRF3, which tells cells to secrete interferon proteins (IFNs) important for host defense. Liu et al. now report a common signaling mechanism used by all three types of innate immune receptor-adaptor protein pairs to activate IRF3 and generate IFNs. This is important because cells must regulate their IFN production carefully to avoid inflammation and autoimmunity. Science , this issue 10.1126/science.aaa2630

A novel karenitecin, BNP1350, is a topoisomerase I-targeting anticancer agent with significant antitumor activity in vitro and in vivo. A BNP1350-resistant human head and neck carcinoma A253 cell line, denoted A253/BNPR, was developed. The A253/BNPR cell line was approximately 9-fold resistant to BNP1350 and 4-fold cross-resistant to another topoisomerase I inhibitor SN-38, the active metabolite of irinotecan. After drug treatment with equimolar concentrations of BNP1350 (0.7 microM) for 2h, activation of the DNA double-strand break repair protein complexes was similar in the two cell lines, suggesting that DNA dsb repair is not attributable to resistance to BNP1350 in the A253/BNPR cells. Cell cycle analysis indicates that the A253 cell line accumulated primarily in S phase, but G(2) phase accumulation was observed in the A253/BNPR cell line at 48 h after drug removal. Elevated chk1 phosphorylation at Ser(345) following DNA damage induced by BNP1350 was accompanied by G(2) accumulation in the A253/BNPR cell line, while exposure to equimolar concentrations of BNP1350 (0.7 microM) induced S-phase arrest and no increased phosphorylation of chk1 at Ser(345) in the A253 cell line. Under the same conditions, increased chk1 activity was observed in the A253/BNPR cell line, but not in the A253 cell line. Moreover, stimulated binding of 14-3-3 proteins to chk1 was observed in BNP1350-treated A253/BNPR cells. To confirm relationship between chk1 expression/phosphorylation and drug resistance to topo I poisons, we examined the effects of chk1 or chk2 antisense oligonucleotides on the cellular growth inhibition. Chk1 antisense oligonucleotide can sensitize the A253/BNPR cells to killing by topo I inhibitor BNP1350, but no significant sensitization of BNP1350-induced growth inhibition was observed in the drug-sensitive cell line. Chk2 antisense oligonucleotide has only a small sensitization effect on BNP1350-induced growth inhibition in both cell lines. The data indicate that the chk1 signaling pathways that mediate cell cycle checkpoint are associated with cellular resistance to BNP1350 in the A253/BNPR cell line.

The innate immune system utilizes pattern-recognition receptors (PRRs) to detect the invasion of pathogens and initiate host antimicrobial responses such as the production of type I interferons and proinflammatory cytokines. Nucleic acids, which are essential genetic information carriers for all living organisms including viral, bacterial, and eukaryotic pathogens, are major structures detected by the innate immune system. However, inappropriate detection of self nucleic acids can result in autoimmune diseases. PRRs that recognize nucleic acids in cells include several endosomal members of the Toll-like receptor family and several cytosolic sensors for DNA and RNA. Here, we review the recent advances in understanding the mechanism of nucleic acid sensing and signaling in the cytosol of mammalian cells as well as the emerging role of cytosolic nucleic acids in autoimmunity.

Topoisomerase I (TOP1)-mediated DNA damage induced by camptothecin (CPT) in the presence of active transcription has been studied using purified calf thymus TOP1 and T7 RNA polymerase. CPT-stabilized TOP1 cleavable complexes located on the template strand within the transcribed region were found to be converted into irreversible strand breaks by the elongating RNA polymerase. By contrast, CPT-stabilized TOP1 cleavable complexes located on the non-template strand within the transcribed region was unaffected by the elongating RNA polymerase. Previous studies have demonstrated that the elongating T7 RNA polymerase is arrested by TOP1 cleavable complexes located on the template but not the non-template strand [Bendixen et al ., (1990) Biochemistry , 29, 5613-5619]. Together, these results suggest a model in which collision between the TOP1-cleavable complexes located on the template strand and the elongating RNA polymerase results in transcription arrest and conversion of TOP1 cleavable complexes into 'irreversible' strand breaks. The implication of the transcription collision model in DNA damage and repair, as well as cell killing, is discussed.

Alarm Bells The presence of DNA in the cytosol of mammalian cells is a danger signal, indicating, for example, that a DNA-containing virus has infected the cell. This signal triggers an innate immune response, which involves the expression of type I interferons, and is critical for antiviral immunity and responses to DNA vaccines. Cyclic GMP-AMP synthase (cGAS) was recently identified as a sensor of cytosolic DNA. Li et al. (p. 1390 , published online 29 August) now use knockout mice to provide genetic evidence that, in multiple cell types, cGAS is the primary DNA sensor required for the type I interferon response in vivo.

HIV Detection Is a (c)GAS Despite it being one of the most highly studied viruses, there are still many unknowns when it comes to HIV—including how it triggers the innate immune response. Gao et al. (p. 903 , published 8 August) now demonstrate that the DNA sensor cyclic GMP-AMP synthase (cGAS) detects HIV infection. Reverse-transcribed HIV DNA triggers cGAS and downstream activation of antiviral immunity. Detection of HIV, as well as the retroviruses simian immunodeficiency virus and murine leukemia virus, was abrogated in mouse and human cells deficient in cGAS—suggesting that cGAS may be a critical activator of innate immunity in response to retroviral infection.

RNA virus infections are detected by the RIG-I family of receptors, which induce type-I interferons through the mitochondrial protein MAVS. MAVS forms large prion-like polymers that activate the cytosolic kinases IKK and TBK1, which in turn activate NF-κB and IRF3, respectively, to induce interferons. Here we show that MAVS polymers recruit several TRAF proteins, including TRAF2, TRAF5, and TRAF6, through distinct TRAF-binding motifs. Mutations of these motifs that disrupted MAVS binding to TRAFs abrogated its ability to activate IRF3. IRF3 activation was also abolished in cells lacking TRAF2, 5, and 6. These TRAF proteins promoted ubiquitination reactions that recruited NEMO to the MAVS signaling complex, leading to the activation of IKK and TBK1. These results delineate the mechanism of MAVS signaling and reveal that TRAF2, 5, and 6, which are normally associated with NF-κB activation, also play a crucial role in IRF3 activation in antiviral immune responses. DOI: http://dx.doi.org/10.7554/eLife.00785.001, eLife digest The innate immune system can detect and destroy viruses, bacteria and other pathogens that enter the human body. In particular, inside cells, viral RNA can bind to and activate a protein called RIG-I. This protein switches on another protein, called MAVS, which can activate other copies of itself. These MAVS molecules then aggregate together on the membrane of mitochondria and send a signal that leads to the production of small proteins, called cytokines, which stimulate an inflammatory response and ultimately neutralize the virus. Although many of the proteins that are activated by MAVS in the innate immunity signaling pathway have been identified, precisely how MAVS transmits this signal is unknown. Now, Liu et al. explore how this protein can propagate signals in the innate immune response by monitoring activation of the transcription factors IRF3 and NF-κB, which transcribe cytokine genes. Previous studies have suggested that a protein known as ubiquitin is needed to activate RIG-I, and that this protein collaborates with MAVS to signal through the innate immunity pathway. Liu et al. found that a group of proteins including TRAF2, TRAF5, TRAF6 and LUBAC relay the antiviral signal by binding to MAVS. These so-called ‘E3 ligases’ string ubiquitin together in chains called polyubiquitin, which is essential for activating signaling after, or downstream of, MAVS; however, the association of these E3 ligases with MAVS also requires that multiple copies of MAVS cluster together. MAVS, the TRAF proteins and LUBAC collectively recruit other innate immunity pathway proteins to activate IRF3 and NF-κB, and thus transcription of the genes that control the innate immunity response. Together, these results show the intricate interplay of proteins needed to eliminate viruses from the body. DOI: http://dx.doi.org/10.7554/eLife.00785.002

This paper addresses the consensus of second-order multiagent systems with general topology and time delay based on the nearest neighbor rule. By using the Laplace transform technique, it is proved that the second-order multi-agent system in the presence of time-delay can reach consensus if the network topology contains a globally reachable node and time delay is bounded. The bound of time-delay only depends on eigenvalues of the Laplacian matrix of the system. The main contribution of this paper is that the accurate state of the consensus center and the upper bound of the communication delay to make the agents reach consensus are given. Some numerical simulations are given to illustrate the theoretical results.

In this paper, we address the consensus of second-order multi-agent dynamical system. We also build a second-order multi-agent model with a general topology. We can obtain a special condition to ensure the consensus of the second-order system. Introducing the Laplace transform, we can have the consensus of the system. Moreover, we obtain the accurate solution of the system. Finally, numerical simulations are presented to verify the theoretical results.

Pentraxin 3 (PTX3) was identified as a marker of the inflammatory response and overexpressed in various tissues and cells related to cardiovascular disease. Honokiol, an active component isolated from the Chinese medicinal herb Magnolia officinalis, was shown to have a variety of pharmacological activities. In the present study, we aimed to investigate the effects of honokiol on palmitic acid (PA)-induced dysfunction of human umbilical vein endothelial cells (HUVECs) and to elucidate potential regulatory mechanisms in this atherosclerotic cell model. Our results showed that PA significantly accelerated the expression of PTX3 in HUVECs through the IκB kinase (IKK)/IκB/nuclear factor-κB (NF-κB) pathway, reduced cell viability, induced cell apoptosis and triggered the inflammatory response. Knockdown of PTX3 supported cell growth and prevented apoptosis by blocking PA-inducted nitric oxide (NO) overproduction. Honokiol significantly suppressed the overexpression of PTX3 in PA-inducted HUVECs by inhibiting IκB phosphorylation and the expression of two NF-κB subunits (p50 and p65) in the IKK/IκB/NF-κB signaling pathway. Furthermore, honokiol reduced endothelial cell injury and apoptosis by regulating the expression of inducible NO synthase and endothelial NO synthase, as well as the generation of NO. Honokiol showed an anti-inflammatory effect in PA-inducted HUVECs by significantly inhibiting the generation of interleukin-6 (IL-6), IL-8 and monocyte chemoattractant protein-1. In summary, honokiol repaired endothelial dysfunction by suppressing PTX3 overexpression in an atherosclerotic cell model. PTX3 may be a potential therapeutic target for atherosclerosis.

HIV infection abrogates adaptive immunity by the depletion of CD4 T cells. However, innate immune defense mechanisms against HIV is largely unknown. Here we show that pseudotyped HIV can infect human and mouse cell lines, leading to the production of interferons and other antiviral cytokines. Activation of innate immunity by HIV requires viral cDNA synthesis but not cDNA integration. We show that retrotranscribed HIV cDNA is sensed by the cytosolic DNA sensor cGAS, which then produces the second messenger 2'3'cGAMP to activate the adaptor STING. Importantly, wild type HIV also triggers cGAMP production in human primary macrophages, underscoring the key role of cGAS in HIV sensing. Furthermore, cytosolic sensing of other retroviruses such as murine leukemia virus and simian immunodeficiency virus also depends on cGAS. These results further our understanding of immune detection of HIV and can be potentially applied in HIV vaccine and adjuvant designs.

Background: It is important that serological assays detect antibodies to human immunodeficiency virus (HIV) in all infected individuals, including those infected with less prevalent, more diverse subtypes. Methods: Performance of the ADVIA Centaur® HIV 1/O/2 Enhanced (EHIV) Assay was tested on 1344 samples from HIV-positive subjects, 6061 samples from groups at low-risk for HIV infection, and 1042 samples from groups at high-risk for HIV-1 and HIV-2 infection. Results were compared with those of an FDA-approved predicate assay. Results: The ADVIA Centaur EHIV Assay showed good precision with a diagnostic specificity of 99.9% and diagnostic sensitivity of 100%. HIV seroconversion was detected earlier in 6 panels, at the same time in 13 panels and later in only 1 of the panels when compared to the predicate assay, thereby narrowing the window period between infection and antibody detection. Of clinical significance, a blood donor sample that was indeterminate by HIV-1 Western blot and non-reactive by the predicate assay was repeatedly reactive in the ADVIA Centaur Assay and confirmed as positive by HIV-2 immunoblot. Conclusions: The ADVIA Centaur EHIV Assay is useful as an aid in the diagnosis of individuals infected with HIV-1 and/or HIV-2.

Human head and neck squamous carcinoma cell lines, A253 and FaDu, were utilized to identify mediators associated with response to topoisomerase I poison, SN-38, a metabolite of irinotecan. The drug sensitivity of FaDu cells to SN-38 was significantly higher than that of the A253 cells. In A253 cells, G2/M arrest following drug treatment (0.35 microM SN-38, 2-h exposure) was accompanied by DNA fragmentation in the 50-300 kb range, but FaDu cells accumulated in S-phase concurrently with induction of smaller DNA fragmentation in the 4-80 kb range. Because the critical regulatory step in activating cdc2 during progression into mitosis appears to be dephosphorylation of Tyrosine 15 (Tyr15), we examined the Tyr15 phosphorylation status of cdc2 in both cell lines. Slightly increased levels of cdc2 phosphorylation was observed in the A253 cells, while reduced levels of cdc2 phosphorylation was noted in the FaDu cells, corresponding to the abrogation of the G2-phase arrest. Increased chk1 phosphorylation at Ser345 induced by SN-38 was accompanied by the observed G2 phase arrest in the A253 cell line, while significant downregulation of chk1 and cdc25C phosphorylation, which resulted in the abrogation of G2/M checkpoint arrest, was noted in FaDu cells at this timepoint. These results suggest that alterations of chk1 signaling are associated with the response to topoisomerase I poison SN-38. Furthermore, A253 cells possess higher levels of endogenous hMLH1, compared to FaDu cells. A deficiency in G2 arrest was observed in FaDu cells, suggesting endogenous hMLH1 protein expression is associated with the abrogation of G2/M arrest, subsequently with the response to topoisomerase I poison SN-38.

Camptothecins demonstrate a broad spectrum of antitumor activity. Although they are known to trap DNA topoisomerase I on DNA, form cleavable complexes, and generate DNA breaks upon collision with DNA or RNA polymerases, the precise mechanisms predictive for antitumor activity remain to be identified. Recent studies using panels of colorectal and breast cancer cell lines indicate that events downstream of cleavable complexes are more relevant. In this study, we chose SN-38, an active metabolite of irinotecan, to characterize DNA double strand breaks and repair mechanisms induced by this type of drugs using a human head and neck squamous cell carcinoma cell line A253. The results showed that 2-h exposure of cells to an IC(50) concentration of SN-38 induces biphasic DNA double-strand break (DSBs): an immediate phase, which was greatly reduced within 8 h, and a lagging phase, culminating 24 h after drug removal. Three DNA double-strand break repair protein complexes were activated: DNA-dependent protein kinase (DNA-PK), NBS1-MRE11-RAD50, and BRCA1. Aphidicolin, a DNA polymerase inhibitor, abolished both phase I DSBs and the activation of repair protein complexes, suggesting that they resulted from the collision between the cleavable complex and DNA polymerase of S-phase cells. This is in contrast to ionizing radiation-induced activation of DNA-PK and NBS1-MRE11-RAD50 complexes that occur predominantly among non-S-phase cells. The trigger for phase II DSBs cannot be abolished by aphidicolin. The data also indicate that DNA fragments in the size of 50 to 200 kilobases were detected in the lagging phase. This suggests that the late DNA DSBs were associated with apoptotic cell death.

The presence of DNA in the cytoplasm of mammalian cells is a danger signal that alerts the host to microbial infection. We have recently identified cyclic GMP-AMP synthase (cGAS) as a cytosolic DNA sensor that triggers innate immune responses. Here, we further investigate the mechanism of cGAS activation by DNA and the function of cGAS in vivo through a combination of structural, biochemical and genetic approaches. Surprisingly, in contrast to several recent structural studies, which described the formation of a 1:1 complex between cGAS and DNA through a single interaction site, our results reveal that cGAS interacts with DNA through two distinct binding sites, forming a complex composed of dimeric cGAS bound to two molecules of DNA. Functional analyses of cGAS mutants demonstrate that both the dimeric interface and the two DNA binding surfaces are critical for cGAS activation. We also show that multiple cell types from cGAS-deficient mice failed to produce type I interferons and other cytokines in response to DNA virus infection. cGas-/- mice were more susceptible to lethal infection with herpes simplex virus 1 (HSV1) than wild-type mice. In addition, cGAS product cGAMP is an adjuvant that boosts antigen-specific T cell activation and antibody production in mice. These results not only provide new insights into the mechanism of DNA sensing by cGAS, but also demonstrate that cGAS is a nonredundant and general cytosolic DNA sensor that activates type I interferon pathway.

International audience; Topoisomerase I (top1) inhibitors (camptothecins and other structurally diverse compounds) are effective and promising anticancer agents. Determinants of selectivity toward cancer cells and resistance are multifactorial. These factors can be separated in three groups. The first is related to alterations in drug distribution and metabolism. The second group includes both quantitative and qualitative (mutations) differences in top I. The third group includes resistance and sensitivity factors downstream from the cleavage complexes. They include DNA repair, cell cycle checkpoints and apoptosis, and are probably key to the relative selectivity of camptothecins toward cancer cells and to clinical resistance. Copyright 1999 Harcourt Publishers Ltd.

The presence of DNA in the cytoplasm of mammalian cells is a danger signal that triggers the host immune responses such as the production of type-I interferons (IFN). This DNA signaling pathway requires the adaptor protein STING and the transcription factor IRF3, but the mechanism of DNA sensing is unclear. Here we showed that mammalian cytosolic extracts synthesized cyclic-GMP-AMP (cGAMP) in vitro from ATP and GTP in the presence of DNA but not RNA. DNA transfection or DNA virus infection of mammalian cells also triggered cGAMP production in vivo. cGAMP bound to STING, leading to the activation of IRF3 and induction of interferon-β. Thus, cGAMP represents the first cyclic di-nucleotide in metazoa and it functions as an endogenous second messenger that triggers interferon production in response to cytosolic DNA. Through biochemical fractionation and quantitative mass spectrometry, we identified a cGAMP synthase (cGAS), which belongs to the nucleotidyltransferase family. Overexpression of cGAS activated the transcription factor IRF3 and induced IFNβ in a STING-dependent manner. Knockdown of cGAS inhibited IRF3 activation and IFNβ induction by DNA transfection or DNA virus infection. cGAS bound to DNA in the cytoplasm and catalyzed cGAMP synthesis. These results indicate that cGAS is a cytosolic DNA sensor that induces interferons by producing the second messenger cGAMP.