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We are captivated by the promise of autonomous systems in our everyday life. However, ensuring that these systems act safely is an immense challenge: introducing complex systems into real-world uncertain environments while guaranteeing safety at all times is impossible in applications like self-driving vehicles, collaborative factory robots, and assistive robots. These systems will inevitably need to make real-time decisions with limited computational resources, and incomplete knowledge of the environment and other agents. This dissertation is an effort towards achieving trustworthy real-world autonomy by enabling autonomous systems to: (1) make theoretical safety guarantees efficiently based on known information, and(2) bridge the safety gap between this theory and the real world by reasoning about uncertainty in the environment and other agents.Towards this goal this dissertation covers various methods for scalable safety and real-time decision-making that draw from control theory, cognitive science, and learning, and are backed by both rigorous theory and physical testing on robotic platforms. We begin with an overview of reachability analysis and its applications for optimal control with safety guarantees. We then tackle the curse of dimensionality associated with reachability computation by decomposing systems or updating previous solutions in a warm-starting fashion. Next we explore planning in a simplified, low-dimensional space online with precomputed safety guarantees and tracking controls offline. This is extended to meta-planning, in which the online algorithm switches between faster/conservative modes and slower/accurate modes. Then we apply all of these tools towards navigating in uncertain environments among hard-to-predict agents such as human pedestrians. We use Bayesian machine learning methods to reason about human intention and navigate in a probabilistically safe manner. Finally, the dissertation ends with information about code bases and reachability tutorial examples.

The response rate to immune checkpoint inhibitor therapy for non-small-cell lung cancer (NSCLC) is just 20%. To improve this figure, several early phase clinical trials combining novel immunotherapeutics with immune checkpoint blockade have been initiated. Unfortunately, these trials have been designed without a strong foundational knowledge of the immune landscape present in NSCLC. Here, we use a flow cytometry panel capable of measuring 51 immune cell populations to comprehensively identify the immune cell composition and function in NSCLC. The results show that the immune cell composition is fundamentally different in lung adenocarcinoma as compared with lung squamous cell carcinoma, and that neutrophils are the most prevalent immune cell type. Using T-cell receptor-β sequencing and tumour reactivity assays, we predict that tumour reactive T cells are frequently present in NSCLC. These results should help to guide the design of clinical trials and the direction of future research in this area.

Chapuis et al. demonstrate that the combination of adoptive cellular therapy with CTLA4 blockade induces long-term remission in a melanoma patient resistant to both modalities administered serially and individually., Adoptive transfer of peripheral blood–derived, melanoma-reactive CD8+ cytotoxic T lymphocytes (CTLs) alone is generally insufficient to eliminate bulky tumors. Similarly, monotherapy with anti-CTLA4 infrequently yields sustained remissions in patients with metastatic melanoma. We postulated that a bolus of enhanced IL-21–primed polyclonal antigen-specific CTL combined with CTLA4 blockade might boost antitumor efficacy. In this first-in-human case study, the combination successfully led to a durable complete remission (CR) in a patient whose disease was refractory to both monoclonal CTL and anti-CTLA4. Long-term persistence and sustained anti-tumor activity of transferred CTL, as well as responses to nontargeted antigens, confirmed mutually beneficial effects of the combined treatment. In this first-in-human study, Chapuis et al. demonstrate that the combination of adoptive cellular therapy with CTLA4 blockade induces long-term remission in a melanoma patient resistant to both modalities administered serially and individually.

Tri-methylation on histone H3 lysine 4 (H3K4me3) is associated with active gene expression but its regulatory role in transcriptional activation is unclear. Here we used Caenorhabditis elegans to investigate the connection between H3K4me3 and gene expression regulation during aging. We uncovered around 30% of H3K4me3 enriched regions to show significant and reproducible changes with age. We further showed that these age-dynamic H3K4me3 regions largely mark gene-bodies and are acquired during adult stages. We found that these adult-specific age-dynamic H3K4me3 regions are correlated with gene expression changes with age. In contrast, H3K4me3 marking established during developmental stages remained largely stable with age, even when the H3K4me3 associated genes exhibited RNA expression changes during aging. Importantly, the genes associated with changes in H3K4me3 and RNA levels with age are enriched for functional groups commonly implicated in aging biology. Therefore, our findings suggested divergent roles of H3K4me3 in gene expression regulation during aging, with important implications on aging-dependent pathophysiologies., Author summary Histone modifications, the specific chemical modifications on histone proteins, are key for regulating the packing of DNA, and thus have important influence on diverse biological processes. An intensely studied function of histone modifications is their contribution to regulating gene expression. Recent studies in diverse model organisms demonstrated that the global alterations of particular histone modifications, for instance H3K4me3, extend the lifespan of the organism. However, the underlying molecular mechanisms remain largely unclear. In this study, we monitored whether and how the genome-wide pattern of the histone modification H3K4me3 changes during aging in the somatic cells of the model organism C. elegans. We identified interesting and non-conventional patterns of H3K4me3, which span gene-bodies and are acquired during adulthood, that are particularly prone to changes with aging. This is contrasted to the well-studied H3K4me3 patterns that span transcriptional start sites and 5’ promoter regions and are established early during development, which remain stable with age. Consistent with the close association between H3K4me3 marking and active transcription, we observed that the age-dynamic H3K4me3 markings are highly correlated with corresponding RNA expression changes. Importantly, the genes that are associated with both H3K4me3 and RNA expression changes with age are over-represented for functional groups commonly implicated in aging biology. In summary, our findings revealed a lesser known pattern of H3K4me3 modification that can have important biological roles in aging.

Notes that research has not isolated the effects of sexist reading material on the reader. (MKM)

Regular consumption of fruits and vegetables is associated with reduced risk of age-related functional decline and chronic diseases such as cancer and cardiovascular disease. These effects are primarily attributed to phytochemicals, plant compounds with a wide range of biological activities and health benefits. Apples, the top contributor of fruit phenolics in American diets, have high antioxidant, antiproliferative and chemopreventive activity in vitro and in vivo. However, little is known about their effects on aging. The objectives of this study were to determine the effects of whole apple phytochemical extracts on lifespan, healthspan and resistance to various stresses in vivo using Caenorhabditis elegans as a model. The mean and maximum lifespan of animals treated with 2.5, 5 and 10 mg/ml whole apple extracts increased significantly in a dose-dependent manner by up to 39% and 25%, respectively. Healthspan also significantly improved as indicated by improved motility and reduced lipofuscin accumulation. Animals pre-treated with whole apple extracts were more resistant to stresses such as heat, UV radiation, paraquat-induced oxidative stress, and pathogenic infection, suggesting that cellular defense and immune system functions also improved. Our findings indicate that, in C. elegans, whole apple extracts slow aging, extend lifespan, improve healthspan, and enhance resistance to stress.

The 29 th annual meeting of the Society for Immunotherapy of Cancer (SITC) was held November 7-9, 2014 in National Harbor, MD and was organized by Dr. Arthur A. Hurwitz (National Cancer Institute), Dr. Kim A. Margolin (Stanford University), Dr. Daniel E. Speiser (Ludwig Center for Cancer Research, University of Lausanne) and Dr. Walter J. Urba (Earle A. Chiles Research Institute, Providence Cancer Center). This meeting included over 1,600 registered participants from 28 separate countries, making it the largest SITC meeting held to date. It highlighted significant worldwide progress in the development and application of cancer immunology to the practice of clinical oncology, including advances in diagnosis, prognosis and therapy, utilizing several immunological pathways and mechanisms for a variety of oncologic conditions. Presentations and posters demonstrated that many concepts that had been pursued preclinically in the past are now being translated into clinical practice, with clear benefits for patients.

The Snail family of transcription factors are core inducers of epithelial-to-mesenchymal transition (EMT). Here we show that the F-box protein FBXO11 recognizes and promotes ubiquitin-mediated degradation of multiple Snail family members including Scratch. The association between FBXO11 and Snai1 in vitro is independent of Snai1 phosphorylation. Overexpression of FBXO11 in mesenchymal cells reduces Snail protein abundance and cellular invasiveness. Conversely, depletion of endogenous FBXO11 in epithelial cancer cells causes Snail protein accumulation, EMT, and tumor invasion, as well as loss of estrogen receptor expression in breast cancer cells. Expression of FBXO11 is downregulated by EMT-inducing signals TGFβ and nickel. In human cancer, high FBXO11 levels correlate with expression of epithelial markers and favorable prognosis. The results suggest that FBXO11 sustains the epithelial state and inhibits cancer progression. Inactivation of FBXO11 in mice leads to neonatal lethality, epidermal thickening, and increased Snail protein levels in epidermis, validating that FBXO11 is a physiological ubiquitin ligase of Snail. Moreover, in C. elegans, the FBXO11 mutant phenotype is attributed to the Snail factors as it is suppressed by inactivation/depletion of Snail homologs. Collectively, these findings suggest that the FBXO11-Snail regulatory axis is evolutionarily conserved and critically governs carcinoma progression and mammalian epidermal development.

The transcriptional co-regulator host cell factor-1 (HCF-1) plays critical roles in promoting cell cycle progression in diverse cell types, and in maintaining self-renewal of embryonic stem cells, but its role in pancreatic β-cell function has not been investigated. Immunhistochemistry of mouse pancreas revealed nuclear expression of HCF-1 in pancreatic islets. Reducing HCF-1 expression in the INS-1 pancreatic β-cell line resulted in reduced cell proliferation, reduced glucose-stimulated insulin secretion, and reduced expression of the critical β-cell transcription factor Pdx1. HCF-1 is a known co-activator of the E2F1 transcription factor, and loss of E2F1 results in pancreatic β-cell dysfunction and reduced expression of Pdx1. Therefore we wondered whether HCF-1 might be required for E2F1 regulation of Pdx1. Chromatin immunoprecipitation experiments revealed that HCF-1 and E2F1 co-localize to the Pdx1 promoter. These results indicate that HCF-1 represents a novel transcriptional regulator required for maintaining pancreatic β-cell function.

Larvae of the nematode Caenorhabditis elegans must choose between reproductive development and dauer diapause. This decision is based on sensing of environmental inputs and dauer pheromone, a small molecule signal that serves to monitor population density. These signals are integrated via conserved neuroendocrine pathways that converge on steroidal ligands of the nuclear receptor DAF-12, a homolog of the mammalian vitamin D receptor and liver X receptor. DAF-12 acts as the main switch between gene expression programs that drive either reproductive development or dauer entry. Extensive studies in the past two decades demonstrated that biosynthesis of two bile acid-like DAF-12 ligands, named dafachronic acids (DA), controls developmental fate. In this issue of PLoS Biology, Wollam et al. showed that a conserved steroid-modifying enzyme, DHS-16, introduces a key feature in the structures of the DAF-12 ligands, closing a major gap in the DA biosynthesis pathway. The emerging picture of DA biosynthesis in C. elegans enables us to address a key question in the field: how are complex environmental signals integrated to enforce binary, organism-wide decisions on developmental fate? Schaedel et al. demonstrated that pheromone and DA serve as competing signals, and that a positive feedback loop based on regulation of DA biosynthesis ensures organism-wide commitment to reproductive development. Considering that many components of DA signaling are highly conserved, ongoing studies in C. elegans may reveal new aspects of bile acid function and lifespan regulation in mammals.

Recent findings indicate that perturbations of the mitochondrial electron transport chain (METC) can cause extended longevity in evolutionarily diverse organisms. To uncover the molecular basis of how altered METC increases lifespan in C. elegans, we performed an RNAi screen and revealed that three predicted transcription factors are specifically required for the extended longevity of mitochondrial mutants. In particular, we demonstrated that the nuclear homeobox protein CEH-23 uniquely mediates the longevity but not the slow development, reduced brood size, or resistance to oxidative stress associated with mitochondrial mutations. Furthermore, we showed that ceh-23 expression levels are responsive to altered METC, and enforced overexpression of ceh-23 is sufficient to extend lifespan in wild-type background. Our data point to mitochondria-to-nucleus communications to be key for longevity determination and highlight CEH-23 as a novel longevity factor capable of responding to mitochondrial perturbations. These findings provide a new paradigm for how mitochondria impact aging and age-dependent diseases., Author Summary Mitochondria have long been associated with aging and age-related diseases. Recent research has shown that a slight dampening of mitochondrial function can dramatically increase the lifespan of a wide range of organisms, suggesting that a similar mechanism likely operates in humans. The molecular basis of this observation is largely unknown, however. Uncovering the genes that allow altered mitochondrial function to impact longevity will give us important new insights into how mitochondria affect the aging process and will pave the way for future therapeutic developments aiming to improve healthy aging and to treat age-related diseases. Here, we used an RNAi screen in the genetic model organism C. elegans, a nematode worm, to uncover how altered mitochondrial function can modulate longevity. We found that in order for mitochondria to affect lifespan, they must communicate with several unique transcription factors in the nucleus. Notably, we discovered that the putative homeobox transcription factor CEH-23, which has not previously been implicated in longevity determination, is able to respond to changes in mitochondrial function and in turn causes an extension in lifespan.

Our understanding of the genetic mechanisms of organismal aging has advanced dramatically during the past two decades. With the development of large-scale RNAi screens, the last few years saw the remarkable identifications of hundreds of new longevity genes in the roundworm Caenorhabditis elegans. The various RNAi screens revealed many biological pathways previously unknown to be related to aging. In this review, we focus on findings from the recent large-scale RNAi longevity screens, and discuss insights they have provided into the complex biological process of aging and considerations of the RNAi technology will continue to have on the future development of the aging field.

The PMK-1 p38 mitogen-activated protein kinase pathway and the DAF-2–DAF-16 insulin signaling pathway control Caenorhabditis elegans intestinal innate immunity. pmk-1 loss-of-function mutants have enhanced sensitivity to pathogens, while daf-2 loss-of-function mutants have enhanced resistance to pathogens that requires upregulation of the DAF-16 transcription factor. We used genetic analysis to show that the pathogen resistance of daf-2 mutants also requires PMK-1. However, genome-wide microarray analysis indicated that there was essentially no overlap between genes positively regulated by PMK-1 and DAF-16, suggesting that they form parallel pathways to promote immunity. We found that PMK-1 controls expression of candidate secreted antimicrobials, including C-type lectins, ShK toxins, and CUB-like genes. Microarray analysis demonstrated that 25% of PMK-1 positively regulated genes are induced by Pseudomonas aeruginosa infection. Using quantitative PCR, we showed that PMK-1 regulates both basal and infection-induced expression of pathogen response genes, while DAF-16 does not. Finally, we used genetic analysis to show that PMK-1 contributes to the enhanced longevity of daf-2 mutants. We propose that the PMK-1 pathway is a specific, indispensable immunity pathway that mediates expression of secreted immune response genes, while the DAF-2–DAF-16 pathway appears to regulate immunity as part of a more general stress response. The contribution of the PMK-1 pathway to the enhanced lifespan of daf-2 mutants suggests that innate immunity is an important determinant of longevity., Synopsis The innate immune system provides the first line of defense against pathogen infection and relies upon pathways conserved across mammals, insects, and nematodes. Here, the authors have analyzed the transcriptional response of the nematode Caenorhabditis elegans to infection by the human pathogen Pseudomonas aeruginosa. They investigated this transcriptional response in the context of two conserved pathways involved in pathogen defense: the PMK-1 p38 mitogen-activated protein kinase (p38 MAPK) pathway and the DAF-2–DAF-16 insulin-signaling pathway. Specifically, the authors found that the p38 MAPK pathway plays a critical role in the infection-induced expression of secreted immune response genes. These genes include C-type lectins, lysozymes, and antimicrobial peptides that fight off infection in many species. In contrast, they found that the DAF-16 pathway is not required for immune response gene expression and may regulate immunity as part of a general stress response that functions in parallel to p38 MAPK. In addition, the authors observed that p38 MAPK contributes to the enhanced longevity of daf-2 mutants, implicating p38 MAPK signaling in the regulation of longevity, possibly through its role in immunity.

A general theme that has emerged from studies of DNA tumor viruses is that otherwise unrelated oncoproteins encoded by these viruses often target the same important cellular factors. Major oncogenic determinants for human adenovirus type 9 (Ad9) and high-risk human papillomaviruses (HPV) are the E4-ORF1 and E6 oncoproteins, respectively, and although otherwise unrelated, both of these viral proteins possess a functional PDZ domain-binding motif that is essential for their transforming activity and for binding to the PDZ domain-containing and putative tumor suppressor protein DLG. We report here that the PDZ domain-binding motifs of Ad9 E4-ORF1 and high-risk HPV-18 E6 also mediate binding to the widely expressed cellular factor MUPP1, a large multi-PDZ domain protein predicted to function as an adapter in signal transduction. With regard to the consequences of these interactions in cells, we showed that Ad9 E4-ORF1 aberrantly sequesters MUPP1 within the cytoplasm of cells whereas HPV-18 E6 targets this cellular protein for degradation. These effects were specific because mutant viral proteins unable to bind MUPP1 lack these activities. From these results, we propose that the multi-PDZ domain protein MUPP1 is involved in negatively regulating cellular proliferation and that the transforming activities of two different viral oncoproteins depend, in part, on their ability to inactivate this cellular factor.

Human chorionic gonadotropin (hCG) preparations contain activity against HIV type 1 (HIV-1). However, there has been controversy about whether some biological activities of hCG beta-subunit (hCGbeta) preparations are caused by the beta-subunit itself or other proteins present in the preparations. We report here the purification, characterization, and identification of three enzymes with anti-HIV activity present in the beta-core fraction of hCGbeta prepared from the urine of pregnant women. The N-terminal amino acid sequence of one protein is identical to human urinary lysozyme C, and those of the other two are identical to human RNase A and urinary RNase U. We thus refer to these proteins as AVL (antiviral lysozyme) and AVR (antiviral RNases). In addition to HIV-1 inhibition, AVL is capable of lysing Micrococcus lysodeikticus. AVR digests a variety of RNA substrates, including RNA from HIV-1-infected cells. We also find that lysozyme from chicken egg white, human milk, and human neutrophils and RNase A from bovine pancreas possess activity against HIV-1. These findings may offer additional strategies for the treatment of HIV-1 infection., published_or_final_version

Background Adoptive T cell therapy represents an attractive modality for the treatment of patients with cancer. Peripheral blood mononuclear cells have been used as a source of antigen specific T cells but the very low frequency of T cells recognizing commonly expressed antigens such as NY-ESO-1 limit the applicability of this approach to other solid tumors. To overcome this, we tested a strategy combining IL-21 modulation during in vitro stimulation with first-in-class use of tetramer-guided cell sorting to generate NY-ESO-1 specific cytotoxic T lymphocytes (CTL). Methods CTL generation was evaluated in 6 patients with NY-ESO-1 positive sarcomas, under clinical manufacturing conditions and characterized for phenotypic and functional properties. Results Following in vitro stimulation, T cells stained with NY-ESO-1 tetramer were enriched from frequencies as low as 0.4% to >90% after single pass through a clinical grade sorter. NY-ESO-1 specific T cells were generated from all 6 patients. The final products expanded on average 1200-fold to a total of 36 billion cells, were oligoclonal and contained 67-97% CD8+, tetramer+ T cells with a memory phenotype that recognized endogenous NY-ESO-1. Conclusion This study represents the first series using tetramer-guided cell sorting to generate T cells for adoptive therapy. This approach, when used to target more broadly expressed tumor antigens such as WT-1 and additional Cancer-Testis antigens will enhance the scope and feasibility of adoptive T cell therapy. Electronic supplementary material The online version of this article (doi:10.1186/s40425-014-0036-y) contains supplementary material, which is available to authorized users.