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PLOS_five_shot_dy0 | ***TASK***
the task is to simplify the input abstract of a biomedical literature
***INPUT***
the input is the abstract of a biomedical literature
***OUTPUT***
the output is the simplified abstract for the input abstract of a biomedical literature
***DOCUMENTATION***
***EXAMPLES***
INPUT: CpG Island Methylation in a Mouse Model of Lymphoma Is Driven by the Genetic Configuration of Tumor CellsHypermethylation of CpG islands is a common epigenetic alteration associated with cancer. Global patterns of hypermethylation are tumor-type specific and nonrandom. The biological significance and the underlying mechanisms of tumor-specific aberrant promoter methylation remain unclear, but some evidence suggests that this specificity involves differential sequence susceptibilities, the targeting of DNA methylation activity to specific promoter sequences, or the selection of rare DNA methylation events during disease progression. Using restriction landmark genomic scanning on samples derived from tissue culture and in vivo models of T cell lymphomas, we found that MYC overexpression gave rise to a specific signature of CpG island hypermethylation. This signature reflected gene transcription profiles and was detected only in advanced stages of disease. The further inactivation of the Pten, p53, and E2f2 tumor suppressors in MYC-induced lymphomas resulted in distinct and diagnostic CpG island methylation signatures. Our data suggest that tumor-specific DNA methylation in lymphomas arises as a result of the selection of rare DNA methylation events during the course of tumor development. This selection appears to be driven by the genetic configuration of tumor cells, providing experimental evidence for a causal role of DNA hypermethylation in tumor progression and an explanation for the tremendous epigenetic heterogeneity observed in the evolution of human cancers. The ability to predict genome-wide epigenetic silencing based on relatively few genetic alterations will allow for a more complete classification of tumors and understanding of tumor cell biology.
OUTPUT: Genetic and epigenetic alterations of the genome are common features of cancers. The relationship between these two types of alterations, however, remains unclear. One type of epigenetic modification—DNA methylation in promoter sequences of genes—is of particular interest, since tumor cells have different patterns of promoter methylation than normal cells. Previous studies on human tumor samples have suggested a link between genetic alterations and the induction of aberrant DNA methylation; however, this link has been difficult to rigorously assess because of the incredible genetic heterogeneity found in human cancer. In this study, a mouse model of T cell lymphoma was used to explore the relationship between genetic and epigenetic modifications experienced by tumor cells. By introducing defined genetic changes into preneoplastic T cells of mice, such as the overexpression of the MYC oncogene and the ablation of tumor suppressor genes, we could carefully evaluate how these genetic changes impacted promoter methylation profiles during development of lymphomas in vivo. We found that the introduction of different genetic insults resulted in unique and diagnostic profiles of promoter methylation. Understanding how these methylation signatures contribute to tumor progression could eventually have diagnostic, prognostic, and therapeutic value for human cancers.
INPUT: DNA Methylation Analysis of Chromosome 21 Gene Promoters at Single Base Pair and Single Allele ResolutionDifferential DNA methylation is an essential epigenetic signal for gene regulation, development, and disease processes. We mapped DNA methylation patterns of 190 gene promoter regions on chromosome 21 using bisulfite conversion and subclone sequencing in five human cell types. A total of 28,626 subclones were sequenced at high accuracy using (long-read) Sanger sequencing resulting in the measurement of the DNA methylation state of 580427 CpG sites. Our results show that average DNA methylation levels are distributed bimodally with enrichment of highly methylated and unmethylated sequences, both for amplicons and individual subclones, which represent single alleles from individual cells. Within CpG-rich sequences, DNA methylation was found to be anti-correlated with CpG dinucleotide density and GC content, and methylated CpGs are more likely to be flanked by AT-rich sequences. We observed over-representation of CpG sites in distances of 9, 18, and 27 bps in highly methylated amplicons. However, DNA sequence alone is not sufficient to predict an amplicon's DNA methylation status, since 43% of all amplicons are differentially methylated between the cell types studied here. DNA methylation in promoter regions is strongly correlated with the absence of gene expression and low levels of activating epigenetic marks like H3K4 methylation and H3K9 and K14 acetylation. Utilizing the single base pair and single allele resolution of our data, we found that i) amplicons from different parts of a CpG island frequently differ in their DNA methylation level, ii) methylation levels of individual cells in one tissue are very similar, and iii) methylation patterns follow a relaxed site-specific distribution. Furthermore, iv) we identified three cases of allele-specific DNA methylation on chromosome 21. Our data shed new light on the nature of methylation patterns in human cells, the sequence dependence of DNA methylation, and its function as epigenetic signal in gene regulation. Further, we illustrate genotype–epigenotype interactions by showing novel examples of allele-specific methylation.
OUTPUT: Epigenetics is defined as the inheritance of changes in gene function without changing the DNA sequence. Epigenetic signals comprise methylation of cytosine bases of the DNA and chemical modifications of the histone proteins. DNA methylation plays important roles in development and disease processes. To investigate the biological role of DNA methylation, we analyzed DNA methylation patterns of 190 gene promoter regions on chromosome 21 in five human cell types. Our results show that average DNA methylation levels are distributed bimodally with enrichment of highly methylated and unmethylated sequences, indicating that DNA methylation acts in a switch-like manner. Consistent with the well-established role of DNA methylation in gene silencing, we found DNA methylation in promoter regions strongly correlated with absence of gene expression and low levels of additional activating epigenetic marks. Although methylation levels of individual cells in one tissue are very similar, we observed differences in DNA methylation when comparing different cell types in 43% of all regions analyzed. This finding is in agreement with a role of DNA methylation in cellular development. We identified three cases of genes that are differentially methylated in both alleles that illustrate the tight interplay of genetic and epigenetic processes.
INPUT: Dynamics of DNA methylomes underlie oyster developmentDNA methylation is a critical epigenetic regulator of development in mammals and social insects, but its significance in development outside these groups is not understood. Here we investigated the genome-wide dynamics of DNA methylation in a mollusc model, the oyster Crassostrea gigas, from the egg to the completion of organogenesis. Large-scale methylation maps reveal that the oyster genome displays a succession of methylated and non methylated regions, which persist throughout development. Differentially methylated regions (DMRs) are strongly regulated during cleavage and metamorphosis. The distribution and levels of methylated DNA within genomic features (exons, introns, promoters, repeats and transposons) show different developmental lansdscapes marked by a strong increase in the methylation of exons against introns after metamorphosis. Kinetics of methylation in gene-bodies correlate to their transcription regulation and to distinct functional gene clusters, and DMRs at cleavage and metamorphosis bear the genes functionally related to these steps, respectively. This study shows that DNA methylome dynamics underlie development through transcription regulation in the oyster, a lophotrochozoan species. To our knowledge, this is the first demonstration of such epigenetic regulation outside vertebrates and ecdysozoan models, bringing new insights into the evolution and the epigenetic regulation of developmental processes.
OUTPUT: Elucidating the mechanisms which govern the development of multicellular animals and their evolution is a fundamental task. Epigenetic mechanisms like DNA methylation have recently emerged as critical regulators of mammalian development through the control of genes that determine the identity of cells and the transmission of parental imprints. In invertebrates however, DNA is mostly unmethylated and does not play a role in development except in the peculiar case of social insects. Therefore the significance of DNA methylation in development is thought to be restricted to vertebrates, and thereby considered a recent evolutionary acquisition, and the situation in more distant organisms is unknown. Here we investigated the dynamics of genome-wide DNA methylation patterns in a mollusc, the oyster C. gigas, throughout its development. We found that the dynamics of DNA methylation correspond to the expression dynamics of distinct functional gene clusters that control two critical development steps, cleavage and metamorphosis, and we provide insights into the underlying molecular mechanisms in a non-vertebrate species. These findings challenge the present considerations on the evolution of developmental processes and their epigenetic regulation, and open a new area of research in molecular and developmental biology in invertebrates.
INPUT: Integrative Analysis of DNA Methylation and Gene Expression Data Identifies EPAS1 as a Key Regulator of COPDChronic Obstructive Pulmonary Disease (COPD) is a complex disease. Genetic, epigenetic, and environmental factors are known to contribute to COPD risk and disease progression. Therefore we developed a systematic approach to identify key regulators of COPD that integrates genome-wide DNA methylation, gene expression, and phenotype data in lung tissue from COPD and control samples. Our integrative analysis identified 126 key regulators of COPD. We identified EPAS1 as the only key regulator whose downstream genes significantly overlapped with multiple genes sets associated with COPD disease severity. EPAS1 is distinct in comparison with other key regulators in terms of methylation profile and downstream target genes. Genes predicted to be regulated by EPAS1 were enriched for biological processes including signaling, cell communications, and system development. We confirmed that EPAS1 protein levels are lower in human COPD lung tissue compared to non-disease controls and that Epas1 gene expression is reduced in mice chronically exposed to cigarette smoke. As EPAS1 downstream genes were significantly enriched for hypoxia responsive genes in endothelial cells, we tested EPAS1 function in human endothelial cells. EPAS1 knockdown by siRNA in endothelial cells impacted genes that significantly overlapped with EPAS1 downstream genes in lung tissue including hypoxia responsive genes, and genes associated with emphysema severity. Our first integrative analysis of genome-wide DNA methylation and gene expression profiles illustrates that not only does DNA methylation play a ‘causal’ role in the molecular pathophysiology of COPD, but it can be leveraged to directly identify novel key mediators of this pathophysiology.
OUTPUT: Chronic Obstructive Pulmonary Disease (COPD) is a common lung disease. It is the fourth leading cause of death in the world and is expected to be the third by 2020. COPD is a heterogeneous and complex disease consisting of obstruction in the small airways, emphysema, and chronic bronchitis. COPD is generally caused by exposure to noxious particles or gases, most commonly from cigarette smoking. However, only 20–25% of smokers develop clinically significant airflow obstruction. Smoking is known to cause epigenetic changes in lung tissues. Thus, genetics, epigenetic, and their interaction with environmental factors play an important role in COPD
INPUT: DNA–Methylome Analysis of Mouse Intestinal Adenoma Identifies a Tumour-Specific Signature That Is Partly Conserved in Human Colon CancerAberrant CpG methylation is a universal epigenetic trait of cancer cell genomes. However, human cancer samples or cell lines preclude the investigation of epigenetic changes occurring early during tumour development. Here, we have used MeDIP-seq to analyse the DNA methylome of APCMin adenoma as a model for intestinal cancer initiation, and we present a list of more than 13,000 recurring differentially methylated regions (DMRs) characterizing intestinal adenoma of the mouse. We show that Polycomb Repressive Complex (PRC) targets are strongly enriched among hypermethylated DMRs, and several PRC2 components and DNA methyltransferases were up-regulated in adenoma. We further demonstrate by bisulfite pyrosequencing of purified cell populations that the DMR signature arises de novo in adenoma cells rather than by expansion of a pre-existing pattern in intestinal stem cells or undifferentiated crypt cells. We found that epigenetic silencing of tumour suppressors, which occurs frequently in colon cancer, was rare in adenoma. Quite strikingly, we identified a core set of DMRs, which is conserved between mouse adenoma and human colon cancer, thus possibly revealing a global panel of epigenetically modified genes for intestinal tumours. Our data allow a distinction between early conserved epigenetic alterations occurring in intestinal adenoma and late stochastic events promoting colon cancer progression, and may facilitate the selection of more specific clinical epigenetic biomarkers.
OUTPUT:
| The formation and progression of tumours to metastatic disease is driven by two major mechanisms, i.e. genetic alterations that activate oncogenes or inactivate tumour suppressor genes, and changes in the epigenome that cause variations in the expression of the genetic information. A deeper understanding of the interaction between the genetic and epigenetic mechanisms is critical for the selection of tumour biomarkers and for the future development of therapies. Human tumour specimens and cell lines contain a plethora of genetic and epigenetic changes, which complicate data analysis. In contrast, mouse tumour models such as the APCMin mouse used in this study arise by a single initiating genetic mutation, yet share key traits with human cancer. Here we show that mouse adenomas acquire a multitude of epigenetic alterations, which are recurring in mouse adenoma and in human colon cancer, representing early and advanced tumours, respectively. The use of a mouse model thus allowed us to uncover a sequence of epigenetic changes occurring in tumours, which may facilitate the identification of novel clinical colon cancer biomarkers.
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PLOS_five_shot_dy1 | ***TASK***
the task is to simplify the input abstract of a biomedical literature
***INPUT***
the input is the abstract of a biomedical literature
***OUTPUT***
the output is the simplified abstract for the input abstract of a biomedical literature
***DOCUMENTATION***
***EXAMPLES***
INPUT: Early Myeloid Dendritic Cell Dysregulation is Predictive of Disease Progression in Simian Immunodeficiency Virus InfectionMyeloid dendritic cells (mDC) are lost from blood in individuals with human immunodeficiency virus (HIV) infection but the mechanism for this loss and its relationship to disease progression are not known. We studied the mDC response in blood and lymph nodes of simian immunodeficiency virus (SIV)-infected rhesus macaques with different disease outcomes. Early changes in blood mDC number were inversely correlated with virus load and reflective of eventual disease outcome, as animals with stable infection that remained disease-free for more than one year had average increases in blood mDC of 200% over preinfection levels at virus set-point, whereas animals that progressed rapidly to AIDS had significant loss of mDC at this time. Short term antiretroviral therapy (ART) transiently reversed mDC loss in progressor animals, whereas discontinuation of ART resulted in a 3.5-fold increase in mDC over preinfection levels only in stable animals, approaching 10-fold in some cases. Progressive SIV infection was associated with increased CCR7 expression on blood mDC and an 8-fold increase in expression of CCL19 mRNA in lymph nodes, consistent with increased mDC recruitment. Paradoxically, lymph node mDC did not accumulate in progressive infection but rather died from caspase-8-dependent apoptosis that was reduced by ART, indicating that increased recruitment is offset by increased death. Lymph node mDC from both stable and progressor animals remained responsive to exogenous stimulation with a TLR7/8 agonist. These data suggest that mDC are mobilized in SIV infection but that an increase in the CCR7-CCL19 chemokine axis associated with high virus burden in progressive infection promotes exodus of activated mDC from blood into lymph nodes where they die from apoptosis. We suggest that inflamed lymph nodes serve as a sink for mDC through recruitment, activation and death that contributes to AIDS pathogenesis.
OUTPUT: Myeloid dendritic cells (mDC) are essential innate immune system cells that are lost from blood in human immunodeficiency virus infection through an ill-defined mechanism. We studied the kinetics of the mDC response in blood and lymph nodes of rhesus macaques infected with the closely related simian immunodeficiency virus. We found that differences in the number of blood mDC correlated with eventual disease outcome, as at virus set-point mDC were increased in blood in animals remaining disease free but lost from blood in animals that progressed rapidly to AIDS. mDC loss was linked to an increase in the chemokine axis responsible for mDC recruitment to lymph nodes; however, mDC did not accumulate in tissues but rather died from apoptosis. Lymph node mDC remained responsive to stimulation with a TLR7/8 agonist during infection. Importantly, mDC dysregulation was partially reversed by antiretroviral therapy. These data indicate that chronic mDC recruitment, activation and death within lymph nodes precede development of disease in SIV infected monkeys and may play a role in AIDS pathogenesis.
INPUT: The Human Fungal Pathogen Cryptococcus neoformans Escapes Macrophages by a Phagosome Emptying Mechanism That Is Inhibited by Arp2/3 Complex-Mediated Actin PolymerisationThe lysis of infected cells by disease-causing microorganisms is an efficient but risky strategy for disseminated infection, as it exposes the pathogen to the full repertoire of the host's immune system. Cryptococcus neoformans is a widespread fungal pathogen that causes a fatal meningitis in HIV and other immunocompromised patients. Following intracellular growth, cryptococci are able to escape their host cells by a non-lytic expulsive mechanism that may contribute to the invasion of the central nervous system. Non-lytic escape is also exhibited by some bacterial pathogens and is likely to facilitate long-term avoidance of the host immune system during latency. Here we show that phagosomes containing intracellular cryptococci undergo repeated cycles of actin polymerisation. These actin ‘flashes’ occur in both murine and human macrophages and are dependent on classical WASP-Arp2/3 complex mediated actin filament nucleation. Three dimensional confocal imaging time lapse revealed that such flashes are highly dynamic actin cages that form around the phagosome. Using fluorescent dextran as a phagosome membrane integrity probe, we find that the non-lytic expulsion of Cryptococcus occurs through fusion of the phagosome and plasma membranes and that, prior to expulsion, 95% of phagosomes become permeabilised, an event that is immediately followed by an actin flash. By using pharmacological agents to modulate both actin dynamics and upstream signalling events, we show that flash occurrence is inversely related to cryptococcal expulsion, suggesting that flashes may act to temporarily inhibit expulsion from infected phagocytes. In conclusion, our data reveal the existence of a novel actin-dependent process on phagosomes containing cryptococci that acts as a potential block to expulsion of Cryptococcus and may have significant implications for the dissemination of, and CNS invasion by, this organism.
OUTPUT: Cryptococcus neoformans is fatal fungal pathogen of HIV-positive and other immunocompromised patients that causes an estimated 650 000 deaths per annum. Cryptococcus is able to undermine our immune system by growing within and escaping from immune cells called macrophages. In this study we describe how macrophage cells may be able to prevent this escape by forming a transient ‘cage’ of the protein actin around the intracellular pathogen. Blocking escape from within the macrophage in this way may help prevent the spread of disease around the body, especially into the brain. Thus actin flashes may represent an important host defence against diverse human pathogens.
INPUT: The human cytomegalovirus glycoprotein pUL11 acts via CD45 to induce T cell IL-10 secretionHuman Cytomegalovirus (HCMV) is a widespread pathogen, infection with which can cause severe disease for immunocompromised individuals. The complex changes wrought on the host’s immune system during both productive and latent HCMV infection are well known. Infected cells are masked and manipulated and uninfected immune cells are also affected; peripheral blood mononuclear cell (PBMC) proliferation is reduced and cytokine profiles altered. Levels increase of the anti-inflammatory cytokine IL-10, which may be important for the establishment of HCMV infections and is required for the development of high viral titres by murine cytomegalovirus. The mechanisms by which HCMV affects T cell IL-10 secretion are not understood. We show here that treatment of PBMC with purified pUL11 induces IL-10 producing T cells as a result of pUL11 binding to the CD45 phosphatase on T cells. IL-10 production induced by HCMV infection is also in part mediated by pUL11. Supernatants from pUL11 treated cells have anti-inflammatory effects on untreated PBMC. Considering the mechanism, CD45 can be a positive or negative regulator of TCR signalling, depending on its expression level, and we show that pUL11 also has concentration dependent activating or inhibitory effects on T cell proliferation and on the kinase function of the CD45 substrate Lck. pUL11 is therefore the first example of a viral protein that can target CD45 to induce T cells with anti-inflammatory properties. It is also the first HCMV protein shown to induce T cell IL-10 secretion. Understanding the mechanisms by which pUL11-induced changes in signal strength influence T cell development and function may provide the basis for the development of novel antiviral treatments and therapies against immune pathologies.
OUTPUT: Human cytomegalovirus (HCMV) infects from 45% to 100% of people worldwide, depending on local socio-economic factors. Although usually harmless in healthy individuals, infection with HCMV can cause severe disease in people with weakened or immature immune systems such as transplant recipients and newborns. The establishment and maintenance of life-long infections by HCMV are greatly aided by its ability to modulate the host’s immune system during both active and latent infection; infected cells are masked and both infected and uninfected immune cells have their functions manipulated. One effect of HCMV infection is the induction of the cytokine IL-10, a secreted protein that suppresses many antiviral responses. Here, we identify a viral protein, pUL11, which can induce IL-10 expression by T cells and reduce the production of mediators of inflammation. pUL11 interacts with CD45, an immune system regulator that controls the sensitivity of T cells and has been linked to IL-10 production. We show that pUL11 can likewise affect T cell responses to stimuli, depending on its concentration, and suggest that this underlies its functions. pUL11 is the first viral protein known with this mechanism and further understanding of its effects may lead to the development of novel antiviral therapies and also help in the treatment of immune system disorders.
INPUT: Specific Humoral Immunity versus Polyclonal B Cell Activation in Trypanosoma cruzi Infection of Susceptible and Resistant MiceThe etiologic agent of Chagas Disease is Trypanosoma cruzi. Acute infection results in patent parasitemia and polyclonal lymphocyte activation. Polyclonal B cell activation associated with hypergammaglobulinemia and delayed specific humoral immunity has been reported during T. cruzi infection in experimental mouse models. Based on preliminary data from our laboratory we hypothesized that variances in susceptibility to T. cruzi infections in murine strains is related to differences in the ability to mount parasite-specific humoral responses rather than polyclonal B cell activation during acute infection.
Relatively susceptible Balb/c and resistant C57Bl/6 mice were inoculated with doses of parasite that led to similar timing and magnitude of initial parasitemia. Longitudinal analysis of parasite-specific and total circulating antibody levels during acute infection demonstrated that C57Bl/6 mice developed parasite-specific antibody responses by 2 weeks post-infection with little evidence of polyclonal B cell activation. The humoral response in C57Bl/6 mice was associated with differential activation of B cells and expansion of splenic CD21highCD23low Marginal Zone (MZ) like B cells that coincided with parasite-specific antibody secreting cell (ASC) development in the spleen. In contrast, susceptible Balb/c mice demonstrated early activation of B cells and early expansion of MZ B cells that preceded high levels of ASC without apparent parasite-specific ASC formation. Cytokine analysis demonstrated that the specific humoral response in the resistant C57Bl/6 mice was associated with early T-cell helper type 1 (Th1) cytokine response, whereas polyclonal B cell activation in the susceptible Balb/c mice was associated with sustained Th2 responses and delayed
INPUT: Differential Activation of Human Monocytes and Lymphocytes by Distinct Strains of Trypanosoma cruziTrypanosoma cruzi strains are currently classified into six discrete typing units (DTUs) named TcI to VI. It is known that these DTUs have different geographical distribution, as well as biological features. TcI and TcII are major DTUs found in patients from northern and southern Latin America, respectively. Our hypothesis is that upon infection of human peripheral blood cells, Y strain (Tc II) and Col cl1.7 (Tc I), cause distinct immunological changes, which might influence the clinical course of Chagas disease.
We evaluated the infectivity of CFSE-stained trypomastigotes of Col cl1.7 and Y strain in human monocytes for 15 and 72 hours, and determined the immunological profile of lymphocytes and monocytes exposed to the different isolates using multiparameter flow cytometry. Our results showed a similar percentage and intensity of monocyte infection by Y and Col cl1.7. We also observed an increased expression of CD80 and CD86 by monocytes infected with Col cl1.7, but not Y strain. IL-10 was significantly higher in monocytes infected with Col cl1.7, as compared to Y strain. Moreover, infection with Col cl1.7, but not Y strain, led to an increased expression of IL-17 by CD8+ T cells. On the other hand, we observed a positive correlation between the expression of TNF-alpha and granzyme A only after infection with Y strain.
Our study shows that while Col cl1.7 induces higher monocyte activation and, at the same time, production of IL-10, infection with Y strain leads to a lower monocyte activation but higher inflammatory profile. These results show that TcI and TcII have a distinct immunological impact on human cells during early infection, which might influence disease progression.
OUTPUT:
| Chagas disease remains a major public health problem in Latin America with over 13 million people infected. It is believed that the host immune response and genetic diversity of the parasite play an important role in the progression of Chagas disease, which presents a variety of clinical forms ranging from indeterminate to cardiac and digestive forms. Since parasite genetic diversity may influence the development of Chagas disease, our study aims to understand the immune response of human peripheral blood cells upon infection with two T. cruzi strains with different genetic backgrounds (Col cl1.7 – Tc I, and Y strain – TcII). Our study showed differences in the expression of cytokines and activation molecules between cells infected with strains from Tc I (Col cl1.7) and Tc II (Y strain). These data show the importance of parasite strain in the development of the host response early in infection, which may influence the clinical progression of Chagas disease.
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PLOS_five_shot_dy2 | ***TASK***
the task is to simplify the input abstract of a biomedical literature
***INPUT***
the input is the abstract of a biomedical literature
***OUTPUT***
the output is the simplified abstract for the input abstract of a biomedical literature
***DOCUMENTATION***
***EXAMPLES***
INPUT: Drosophila DNA polymerase theta utilizes both helicase-like and polymerase domains during microhomology-mediated end joining and interstrand crosslink repairDouble strand breaks (DSBs) and interstrand crosslinks (ICLs) are toxic DNA lesions that can be repaired through multiple pathways, some of which involve shared proteins. One of these proteins, DNA Polymerase θ (Pol θ), coordinates a mutagenic DSB repair pathway named microhomology-mediated end joining (MMEJ) and is also a critical component for bypass or repair of ICLs in several organisms. Pol θ contains both polymerase and helicase-like domains that are tethered by an unstructured central region. While the role of the polymerase domain in promoting MMEJ has been studied extensively both in vitro and in vivo, a function for the helicase-like domain, which possesses DNA-dependent ATPase activity, remains unclear. Here, we utilize genetic and biochemical analyses to examine the roles of the helicase-like and polymerase domains of Drosophila Pol θ. We demonstrate an absolute requirement for both polymerase and ATPase activities during ICL repair in vivo. However, similar to mammalian systems, polymerase activity, but not ATPase activity, is required for ionizing radiation-induced DSB repair. Using a site-specific break repair assay, we show that overall end-joining efficiency is not affected in ATPase-dead mutants, but there is a significant decrease in templated insertion events. In vitro, Pol θ can efficiently bypass a model unhooked nitrogen mustard crosslink and promote DNA synthesis following microhomology annealing, although ATPase activity is not required for these functions. Together, our data illustrate the functional importance of the helicase-like domain of Pol θ and suggest that its tethering to the polymerase domain is important for its multiple functions in DNA repair and damage tolerance.
OUTPUT: Error-prone DNA Polymerase θ (Pol θ) plays a conserved role in a mutagenic DNA double-strand break repair mechanism called microhomology-mediated end joining (MMEJ). In many organisms, it also participates in a process crucial to the removal/repair of DNA interstrand crosslinks. The exact mechanism by which Pol θ promotes these processes is unclear, but a clue may lie in its dual-domain structure. While the role of its polymerase domain has been well-studied, the function of its helicase-like domain remains an open question. Here we report an absolute requirement for ATPase activity of the helicase-like domain during interstrand crosslink repair in Drosophila melanogaster. We also find that although end joining frequency does not decrease in ATPase-dead mutants, ATPase activity is critical for generating templated insertions. Using purified Pol θ protein, we show that it can bypass synthetic substrates mimicking interstrand crosslink intermediates and can promote MMEJ-like reactions with partial double-stranded and single-stranded DNA. Together, these data demonstrate a novel function for the helicase-like domain of Pol θ in both interstrand crosslink repair and MMEJ and provide insight into why the dual-domain structure has been conserved throughout evolution.
INPUT: The translesion DNA polymerases Pol ζ and Rev1 are activated independently of PCNA ubiquitination upon UV radiation in mutants of DNA polymerase δReplicative DNA polymerases cannot insert efficiently nucleotides at sites of base lesions. This function is taken over by specialized translesion DNA synthesis (TLS) polymerases to allow DNA replication completion in the presence of DNA damage. In eukaryotes, Rad6- and Rad18-mediated PCNA ubiquitination at lysine 164 promotes recruitment of TLS polymerases, allowing cells to efficiently cope with DNA damage. However, several studies showed that TLS polymerases can be recruited also in the absence of PCNA ubiquitination. We hypothesized that the stability of the interactions between DNA polymerase δ (Pol δ) subunits and/or between Pol δ and PCNA at the primer/template junction is a crucial factor to determine the requirement of PCNA ubiquitination. To test this hypothesis, we used a structural mutant of Pol δ in which the interaction between Pol3 and Pol31 is inhibited. We found that in yeast, rad18Δ-associated UV hypersensitivity is suppressed by pol3-ct, a mutant allele of the POL3 gene that encodes the catalytic subunit of replicative Pol δ. pol3-ct suppressor effect was specifically dependent on the Rev1 and Pol ζ TLS polymerases. This result strongly suggests that TLS polymerases could rely much less on PCNA ubiquitination when Pol δ interaction with PCNA is partially compromised by mutations. In agreement with this model, we found that the pol3-FI allele suppressed rad18Δ-associated UV sensitivity as observed for pol3-ct. This POL3 allele carries mutations within a putative PCNA Interacting Peptide (PIP) motif. We then provided molecular and genetic evidence that this motif could contribute to Pol δ-PCNA interaction indirectly, although it is not a bona fide PIP. Overall, our results suggest that the primary role of PCNA ubiquitination is to allow TLS polymerases to outcompete Pol δ for PCNA access upon DNA damage.
OUTPUT: Replicative DNA polymerases have the essential role of replicating genomic DNA during the S phase of each cell cycle. DNA replication occurs smoothly and accurately if the DNA to be replicated is undamaged. Conversely, replicative DNA polymerases stall abruptly when they encounter a damaged base on their template. In this case, alternative specialized DNA polymerases are recruited to insert nucleotides at sites of base lesions. However, these translesion polymerases are not processive and they are poorly accurate. Therefore, they need to be tightly regulated. This is achieved by the covalent binding of the small ubiquitin peptide to the polymerase cofactor PCNA that subsequently triggers the recruitment of translesion polymerases at sites of DNA damage. Yet, recruitment of translesion polymerases independently of PCNA ubiquitination also has been documented, although the underlying mechanism is not known. Moreover, this observation makes more difficult to understand the exact role of PCNA ubiquitination. Here, we present strong genetic evidence in Saccharomyces cerevisiae implying that the replicative DNA polymerase δ (Pol δ) prevents the recruitment of the translesion polymerases Pol ζ and Rev1 following UV irradiation unless PCNA is ubiquitinated. Thus, the primary role of PCNA ubiquitination would be to allow translesion polymerases to outcompete Pol δ upon DNA damage. In addition, our results led us to propose that translesion polymerases could be recruited independently of PCNA ubiquitination when Pol δ association with PCNA is challenged, for instance at difficult-to-replicate loci.
INPUT: Replicative DNA Polymerase δ but Not ε Proofreads Errors in Cis and in TransIt is now well established that in yeast, and likely most eukaryotic organisms, initial DNA replication of the leading strand is by DNA polymerase ε and of the lagging strand by DNA polymerase δ. However, the role of Pol δ in replication of the leading strand is uncertain. In this work, we use a reporter system in Saccharomyces cerevisiae to measure mutation rates at specific base pairs in order to determine the effect of heterozygous or homozygous proofreading-defective mutants of either Pol ε or Pol δ in diploid strains. We find that wild-type Pol ε molecules cannot proofread errors created by proofreading-defective Pol ε molecules, whereas Pol δ can not only proofread errors created by proofreading-defective Pol δ molecules, but can also proofread errors created by Pol ε-defective molecules. These results suggest that any interruption in DNA synthesis on the leading strand is likely to result in completion by Pol δ and also explain the higher mutation rates observed in Pol δ-proofreading mutants compared to Pol ε-proofreading defective mutants. For strains reverting via AT→GC, TA→GC, CG→AT, and GC→AT mutations, we find in addition a strong effect of gene orientation on mutation rate in proofreading-defective strains and demonstrate that much of this orientation dependence is due to differential efficiencies of mispair elongation. We also find that a 3′-terminal 8 oxoG, unlike a 3′-terminal G, is efficiently extended opposite an A and is not subject to proofreading. Proofreading mutations have been shown to result in tumor formation in both mice and humans; the results presented here can help explain the properties exhibited by those proofreading mutants.
OUTPUT: Many DNA polymerases are able to proofread their errors: after incorporation of a wrong base, the resulting mispair invokes an exonuclease activity of the polymerase that removes the mispaired base and allows replication to continue. Elimination of the proofreading activity thus results in much higher mutation rates. We demonstrate that the two major replicative DNA polymerases in yeast, Pol δ and Pol ε, have different proofreading abilities. In diploid cells, Pol ε is not able to proofread errors created by other Pol ε molecules, whereas Pol δ can proofread not only errors created by other Pol δ molecules but also errors created by Pol ε molecules. We also find that mispaired bases not corrected by proofreading have much different likelihoods of being extended, depending on the particular base-base mismatch. In humans, defects in Pol δ or Pol ε proofreading can lead to cancer, and these results help explain the formation of those tumors and the finding that Pol ε mutants seem to be found as frequently, or more so, in human tumors as Pol δ mutants.
INPUT: DNA Polymerase ζ-Dependent Lesion Bypass in Saccharomyces cerevisiae Is Accompanied by Error-Prone Copying of Long Stretches of Adjacent DNATranslesion synthesis (TLS) helps cells to accomplish chromosomal replication in the presence of unrepaired DNA lesions. In eukaryotes, the bypass of most lesions involves a nucleotide insertion opposite the lesion by either a replicative or a specialized DNA polymerase, followed by extension of the resulting distorted primer terminus by DNA polymerase ζ (Polζ). The subsequent events leading to disengagement of the error-prone Polζ from the primer terminus and its replacement with an accurate replicative DNA polymerase remain largely unknown. As a first step toward understanding these events, we aimed to determine the length of DNA stretches synthesized in an error-prone manner during the Polζ-dependent lesion bypass. We developed new in vivo assays to identify the products of mutagenic TLS through a plasmid-borne tetrahydrofuran lesion and a UV-induced chromosomal lesion. We then surveyed the region downstream of the lesion site (in respect to the direction of TLS) for the presence of mutations indicative of an error-prone polymerase activity. The bypass of both lesions was associated with an approximately 300,000-fold increase in the mutation rate in the adjacent DNA segment, in comparison to the mutation rate during normal replication. The hypermutated tract extended 200 bp from the lesion in the plasmid-based assay and as far as 1 kb from the lesion in the chromosome-based assay. The mutation rate in this
INPUT: Analysis of DNA polymerase ν function in meiotic recombination, immunoglobulin class-switching, and DNA damage toleranceDNA polymerase ν (pol ν), encoded by the POLN gene, is an A-family DNA polymerase in vertebrates and some other animal lineages. Here we report an in-depth analysis of pol ν–defective mice and human cells. POLN is very weakly expressed in most tissues, with the highest relative expression in testis. We constructed multiple mouse models for Poln disruption and detected no anatomic abnormalities, alterations in lifespan, or changed causes of mortality. Mice with inactive Poln are fertile and have normal testis morphology. However, pol ν–disrupted mice have a modestly reduced crossover frequency at a meiotic recombination hot spot harboring insertion/deletion polymorphisms. These polymorphisms are suggested to generate a looped-out primer and a hairpin structure during recombination, substrates on which pol ν can operate. Pol ν-defective mice had no alteration in DNA end-joining during immunoglobulin class-switching, in contrast to animals defective in the related DNA polymerase θ (pol θ). We examined the response to DNA crosslinking agents, as purified pol ν has some ability to bypass major groove peptide adducts and residues of DNA crosslink repair. Inactivation of Poln in mouse embryonic fibroblasts did not alter cellular sensitivity to mitomycin C, cisplatin, or aldehydes. Depletion of POLN from human cells with shRNA or siRNA did not change cellular sensitivity to mitomycin C or alter the frequency of mitomycin C-induced radial chromosomes. Our results suggest a function of pol ν in meiotic homologous recombination in processing specific substrates. The restricted and more recent evolutionary appearance of pol ν (in comparison to pol θ) supports such a specialized role.
OUTPUT:
| The work described here fills a current gap in the study of the 16 known DNA polymerases in vertebrate genomes. Until now, experiments with genetically disrupted mice have been reported for all but pol ν, encoded by the POLN gene. To intensively analyze the role of mammalian pol ν we generated multiple Poln-deficient murine models. We discovered that Poln is uniquely upregulated during testicular development and that it is enriched in spermatocytes. This, and phylogenetic analysis indicate a testis-specific function. We observed a modest reduction in meiotic recombination at a recombination hotspot in Poln-deficient mice. Pol ν has been suggested to function in DNA crosslink repair. However, we found no increased DNA crosslink sensitivity in Poln-deficient mice or POLN-depleted human cells. This is a major difference from some previous findings, and we support our conclusion by multiple experimental approaches, and by the very low or absent expression of functional pol ν in mammalian somatic cells. The present work represents the first description and comprehensive analysis of mice deficient in pol ν, and the first thorough phenotypic analysis in human cells.
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PLOS_five_shot_dy3 | ***TASK***
the task is to simplify the input abstract of a biomedical literature
***INPUT***
the input is the abstract of a biomedical literature
***OUTPUT***
the output is the simplified abstract for the input abstract of a biomedical literature
***DOCUMENTATION***
***EXAMPLES***
INPUT: Dynamic remodeling of lipids coincides with dengue virus replication in the midgut of Aedes aegypti mosquitoesWe describe the first comprehensive analysis of the midgut metabolome of Aedes aegypti, the primary mosquito vector for arboviruses such as dengue, Zika, chikungunya and yellow fever viruses. Transmission of these viruses depends on their ability to infect, replicate and disseminate from several tissues in the mosquito vector. The metabolic environments within these tissues play crucial roles in these processes. Since these viruses are enveloped, viral replication, assembly and release occur on cellular membranes primed through the manipulation of host metabolism. Interference with this virus infection-induced metabolic environment is detrimental to viral replication in human and mosquito cell culture models. Here we present the first insight into the metabolic environment induced during arbovirus replication in Aedes aegypti. Using high-resolution mass spectrometry, we have analyzed the temporal metabolic perturbations that occur following dengue virus infection of the midgut tissue. This is the primary site of infection and replication, preceding systemic viral dissemination and transmission. We identified metabolites that exhibited a dynamic-profile across early-, mid- and late-infection time points. We observed a marked increase in the lipid content. An increase in glycerophospholipids, sphingolipids and fatty acyls was coincident with the kinetics of viral replication. Elevation of glycerolipid levels suggested a diversion of resources during infection from energy storage to synthetic pathways. Elevated levels of acyl-carnitines were observed, signaling disruptions in mitochondrial function and possible diversion of energy production. A central hub in the sphingolipid pathway that influenced dihydroceramide to ceramide ratios was identified as critical for the virus life cycle. This study also resulted in the first reconstruction of the sphingolipid pathway in Aedes aegypti. Given conservation in the replication mechanisms of several flaviviruses transmitted by this vector, our results highlight biochemical choke points that could be targeted to disrupt transmission of multiple pathogens by these mosquitoes.
OUTPUT: The Aedes aegypti mosquito transmits arboviruses that cause dengue, Zika, chikungunya and yellow fever. These viruses are endemic in tropical and subtropical regions of the world placing 2.5 billion people at risk of infection. Transmission is critically dependent upon the replication of these viruses in both human and mosquito hosts. Successful viral replication is greatly influenced by the biochemical environment of the host cell or tissue and flaviviruses rearrange this environment to benefit their needs. Host-cell derived metabolites such as lipids, sugars and amino acids are utilized to produce progeny virions, help evade the host immune system and enable successful completion of the life cycle. In this study, we applied high-resolution mass spectrometry to understand the alteration of the biochemical landscape of the mosquito during infection by dengue virus. We focused on the mosquito midgut, as this is the initial site of infection. We identified several metabolites that exhibited dynamic profiles during the course of viral infection and replication. By pinpointing biochemical “choke points” required for viral replication, we can devise strategies that will stall virus replication in the mosquito and prevent its transmission to humans.
INPUT: Protective versus pathologic pre-exposure cytokine profiles in dengue virus infectionHyperendemic circulation of all four types of dengue virus (DENV-1-4) has expanded globally, fueling concern for increased incidence of severe dengue. While the majority of DENV infections are subclinical, epidemiologic studies suggest that type-cross-reactive immunity can influence disease outcome in subsequent infections. The mechanisms controlling these differential clinical outcomes remain poorly defined.
Blood samples were collected from a cohort of school-aged Thai children who subsequently experienced a subclinical DENV infection or developed dengue illness. PBMC collected prior to infection were stimulated in vitro with DENV and the secretion of 30 cytokines was measured using a multiplexed, bead-based array. Significant differences were found in cytokine production based on both the type of DENV used for stimulation and the occurrence of clinical illness. Secretion of IL-15 and MCP-1 was significantly higher by PBMC of subjects who later developed symptomatic DENV infection. In addition, IL-6 was produced by PBMC from all subjects who subsequently developed symptomatic infection, versus 59% of subjects who had subclinical infection. Secretion of IL-12, IL-2R, MIP-1α, RANTES, GM-CSF, and TNFα was significantly lower by PBMC from subjects with symptomatic infection.
These data demonstrate significant differences in pre-existing immune responses to DENV associated with the clinical outcome of subsequent infection. The finding of higher levels of some cytokines in subjects with symptomatic infection and higher levels of other cytokines in subjects with subclinical infection supports the existence of both protective and pathologic immune profiles. Clinical-immunological correlations identified in the context of natural DENV infection may be useful for evaluating immune responses to dengue vaccines.
OUTPUT: Dengue is one of the most prevalent mosquito-borne infectious diseases worldwide. It is caused by one of four viruses, types 1–4, and ranges in severity from subclinical (mild or no symptoms) to dengue fever (febrile illness with headache and bone pain), or less frequently, dengue hemorrhagic fever, where patients experience leaky blood vessels, sometimes significant bleeding, and may be severe enough to cause death. While many risk factors have been associated with development of severe dengue, sequential infection with different virus types is a major factor, implying that the immune response generated after an initial infection is partly responsible for making subsequent exposure clinically worse. This study sought to identify profiles of immune markers that correlate with increased or decreased risk of dengue. Using samples from individuals ~5 months prior to dengue virus infection, who later experienced either subclinical infection or overt disease, we modeled virus exposure in vitro and compared the production of various immune proteins between the two outcome groups. Three of the proteins studied were produced at higher levels by subjects who subsequently had dengue, and six of the proteins were produced at higher levels by subjects who subsequently had subclinical infection. These results help define what constitutes beneficial versus potentially harmful immune responses, aiding in the design of effective dengue vaccines.
INPUT: Dengue Virus Capsid Protein Usurps Lipid Droplets for Viral Particle FormationDengue virus is responsible for the highest rates of disease and mortality among the members of the Flavivirus genus. Dengue epidemics are still occurring around the world, indicating an urgent need of prophylactic vaccines and antivirals. In recent years, a great deal has been learned about the mechanisms of dengue virus genome amplification. However, little is known about the process by which the capsid protein recruits the viral genome during encapsidation. Here, we found that the mature capsid protein in the cytoplasm of dengue virus infected cells accumulates on the surface of ER-derived organelles named lipid droplets. Mutagenesis analysis using infectious dengue virus clones has identified specific hydrophobic amino acids, located in the center of the capsid protein, as key elements for lipid droplet association. Substitutions of amino acid L50 or L54 in the capsid protein disrupted lipid droplet targeting and impaired viral particle formation. We also report that dengue virus infection increases the number of lipid droplets per cell, suggesting a link between lipid droplet metabolism and viral replication. In this regard, we found that pharmacological manipulation of the amount of lipid droplets in the cell can be a means to control dengue virus replication. In addition, we developed a novel genetic system to dissociate cis-acting RNA replication elements from the capsid coding sequence. Using this system, we found that mislocalization of a mutated capsid protein decreased viral RNA amplification. We propose that lipid droplets play multiple roles during the viral life cycle; they could sequester the viral capsid protein early during infection and provide a scaffold for genome encapsidation.
OUTPUT: Dengue virus is the single most significant arthropod-borne virus pathogen in humans. In spite of the urgent medical need to control dengue infections, vaccines are still unavailable, and many aspects of dengue virus biology and pathogenesis remain elusive. We discovered a link between dengue virus replication and ER-derived organelles known as lipid droplets (LDs). Dengue infection increases the amount of LDs per cell and pharmacological inhibition of LD formation greatly reduces dengue virus replication. In addition, we have found that the viral capsid protein in infected cells accumulates on the surface of LDs. Manipulation of infectious clones and generation of new reporter dengue viruses allowed us to define the molecular basis of capsid protein association to LDs. Specific amino acids on the α2 helix, located in the center of the capsid protein, were found to be crucial for both accumulation of capsid protein on LDs and dengue virus infectious particle formation. We propose that LDs facilitate viral replication providing a platform for nucleocapsid formation during encapsidation. Our findings begin to unravel the complex mechanism by which dengue virus usurps cellular organelles to coordinate different steps of the viral life cycle.
INPUT: Potent neutralizing antibodies elicited by dengue vaccine in rhesus macaque target diverse epitopesThere is still no safe and effective vaccine against dengue virus infection. Epidemics of dengue virus infection are increasingly a threat to human health around the world. Antibodies generated in response to dengue infection have been shown to impact disease development and effectiveness of dengue vaccine. In this study, we investigated monoclonal antibody responses to an experimental dengue vaccine in rhesus macaques. Variable regions of both heavy chain (VH) and light chain (VL) were cloned from single antibody-secreting B cells. A total of 780 monoclonal antibodies (mAbs) composed of paired VH and VL were characterized. Results show that the vaccination induces mAbs with diverse germline sequences and a wide range of binding affinities. Six potent neutralizing mAbs were identified among 130 dengue envelope protein binders. Critical amino acids for each neutralizing antibody binding to the dengue envelope protein were identified by alanine scanning of mutant libraries. Diverse epitopes were identified, including epitopes on the lateral ridge of DIII, the I-III hinge, the bc loop adjacent to the fusion loop of DII, and the β-strands and loops of DI. Significantly, one of the neutralizing mAbs has a previously unknown epitope in DII at the interface of the envelope and membrane protein and is capable of neutralizing all four dengue serotypes. Taken together, the results of this study not
INPUT: Lipidated Dengue-2 Envelope Protein Domain III Independently Stimulates Long-Lasting Neutralizing Antibodies and Reduces the Risk of Antibody-Dependent EnhancementDengue virus is a mosquito-transmitted virus that can cause self-limiting dengue fever, severe life-threatening dengue hemorrhagic fever and dengue shock syndrome. The existence of four serotypes of dengue virus has complicated the development of an effective and safe dengue vaccine. Recently, a clinical phase 2b trial of Sanofi Pasteur's CYD tetravalent dengue vaccine revealed that the vaccine did not confer full protection against dengue-2 virus. New approaches to dengue vaccine development are urgently needed. Our approach represents a promising method of dengue vaccine development and may even complement the deficiencies of the CYD tetravalent dengue vaccine.
Two important components of a vaccine, the immunogen and immunopotentiator, were combined into a single construct to generate a new generation of vaccines. We selected dengue-2 envelope protein domain III (D2ED III) as the immunogen and expressed this protein in lipidated form in Escherichia coli, yielding an immunogen with intrinsic immunopotentiation activity. The formulation containing lipidated D2ED III (LD2ED III) in the absence of exogenous adjuvant elicited higher D2ED III-specific antibody responses than those obtained from its nonlipidated counterpart, D2ED III, and dengue-2 virus. In addition, the avidity and neutralizing capacity of the antibodies induced by LD2ED III were higher than those elicited by D2ED III and dengue-2 virus. Importantly, we showed that after lipidation, the subunit candidate LD2ED III exhibited increased immunogenicity while reducing the potential risk of antibody-dependent enhancement of infection in mice.
Our study suggests that the lipidated subunit vaccine approach could be applied to other serotypes of dengue virus and other pathogens.
OUTPUT:
| Vaccines are considered a cost-effective way to control infectious diseases. To rationally design vaccines, antigens and, frequently, adjuvants must be selected to trigger appropriate immune responses against a specific pathogen. We selected dengue-2 envelope protein domain III as a dengue vaccine candidate and expressed this candidate in the lipidated form in an Escherichia coli-based system. Dengue envelope protein domain III mediates binding of the dengue virus to the host cellular receptor. The lipid moiety of the bacterial-derived lipoprotein can activate the innate immune system to elicit an appropriate adaptive immune response. We demonstrated that lipidated dengue-2 envelope protein domain III is more immunogenic than nonlipidated dengue-2 envelope protein domain III. Most importantly, the lipidated dengue-2 envelope protein domain III alone triggered a durable neutralizing antibody response with a low risk of severe side effects. Lipidated subunit vaccines are non-replicating and thus may be less susceptible to replication interference than live attenuated vaccines. Our study suggests that the lipidated subunit vaccine approach could be applied to other serotypes of dengue virus as well as other pathogens.
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PLOS_five_shot_dy4 | ***TASK***
the task is to simplify the input abstract of a biomedical literature
***INPUT***
the input is the abstract of a biomedical literature
***OUTPUT***
the output is the simplified abstract for the input abstract of a biomedical literature
***DOCUMENTATION***
***EXAMPLES***
INPUT: Protein Conservation and Variation Suggest Mechanisms of Cell Type-Specific Modulation of Signaling PathwaysMany proteins and signaling pathways are present in most cell types and tissues and yet perform specialized functions. To elucidate mechanisms by which these ubiquitous pathways are modulated, we overlaid information about cross-cell line protein abundance and variability, and evolutionary conservation onto functional pathway components and topological layers in the pathway hierarchy. We found that the input (receptors) and the output (transcription factors) layers evolve more rapidly than proteins in the intermediary transmission layer. In contrast, protein expression variability decreases from the input to the output layer. We observed that the differences in protein variability between the input and transmission layer can be attributed to both the network position and the tendency of variable proteins to physically interact with constitutively expressed proteins. Differences in protein expression variability and conservation are also accompanied by the tendency of conserved and constitutively expressed proteins to acquire somatic mutations, while germline mutations tend to occur in cell type-specific proteins. Thus, conserved core proteins in the transmission layer could perform a fundamental role in most cell types and are therefore less tolerant to germline mutations. In summary, we propose that the core signal transmission machinery is largely modulated by a variable input layer through physical protein interactions. We hypothesize that the bow-tie organization of cellular signaling on the level of protein abundance variability contributes to the specificity of the signal response in different cell types.
OUTPUT: Cell function is determined by highly organized networks of biological molecules. An important class of protein pathways maintains the transmission of signals from the cell membrane to the nucleus. These signaling pathways are reused for different purposes at an evolutionary scale and in different cell types of the same organism. However, it is largely unknown how this flexibility is achieved and how this flexibility is balanced with the high degree of evolutionary conservation of some signaling proteins and the need for robustness against intra- and extra-cellular perturbations.We show how functional roles of signaling proteins determine patterns of evolutionary conservation, protein abundance (the average over different human cell lines and its variability) and disease mutations. Projecting pathway annotations on protein-protein interaction (PPI) networks, a picture emerges in which PPIs between variable and less conserved receptors and stable and conserved proteins of the core signal transmission machinery largely modulate signaling activity in a tissue-specific manner. This has important implications for the distribution of disease mutations in signaling pathways, which need to be considered for the understanding of their effect.
INPUT: Intercellular Variability in Protein Levels from Stochastic Expression and Noisy Cell Cycle ProcessesInside individual cells, expression of genes is inherently stochastic and manifests as cell-to-cell variability or noise in protein copy numbers. Since proteins half-lives can be comparable to the cell-cycle length, randomness in cell-division times generates additional intercellular variability in protein levels. Moreover, as many mRNA/protein species are expressed at low-copy numbers, errors incurred in partitioning of molecules between two daughter cells are significant. We derive analytical formulas for the total noise in protein levels when the cell-cycle duration follows a general class of probability distributions. Using a novel hybrid approach the total noise is decomposed into components arising from i) stochastic expression; ii) partitioning errors at the time of cell division and iii) random cell-division events. These formulas reveal that random cell-division times not only generate additional extrinsic noise, but also critically affect the mean protein copy numbers and intrinsic noise components. Counter intuitively, in some parameter regimes, noise in protein levels can decrease as cell-division times become more stochastic. Computations are extended to consider genome duplication, where transcription rate is increased at a random point in the cell cycle. We systematically investigate how the timing of genome duplication influences different protein noise components. Intriguingly, results show that noise contribution from stochastic expression is minimized at an optimal genome-duplication time. Our theoretical results motivate new experimental methods for decomposing protein noise levels from synchronized and asynchronized single-cell expression data. Characterizing the contributions of individual noise mechanisms will lead to precise estimates of gene expression parameters and techniques for altering stochasticity to change phenotype of individual cells.
OUTPUT: Inside individual cells, gene products often occur at low molecular counts and are subject to considerable stochastic fluctuations (noise) in copy numbers over time. An important consequence of noisy expression is that the level of a protein can vary considerably even among genetically identical cells exposed to the same environment. Such non-genetic phenotypic heterogeneity is physiologically relevant and critically influences diverse cellular processes. In addition to noise sources inherent in gene product synthesis, recent experimental studies have uncovered additional noise mechanisms that critically effect expression. For example, the time within the cell cycle when a gene duplicates, and the time taken to complete cell cycle are governed by random processes. The key contribution of this work is development of novel mathematical results quantifying how cell cycle-related noise sources combine with stochastic expression to drive intercellular variability in protein molecular counts. Derived formulas lead to many counterintuitive results, such as increasing randomness in the timing of cell division can lower noise in the level of a protein. Finally, these results inform experimental strategies to systematically dissect the contributions of different noise sources in the expression of a gene of interest.
INPUT: A computational model for how cells choose temporal or spatial sensing during chemotaxisCell size is thought to play an important role in choosing between temporal and spatial sensing in chemotaxis. Large cells are thought to use spatial sensing due to large chemical difference at its ends whereas small cells are incapable of spatial sensing due to rapid homogenization of proteins within the cell. However, small cells have been found to polarize and large cells like sperm cells undergo temporal sensing. Thus, it remains an open question what exactly governs spatial versus temporal sensing. Here, we identify the factors that determines sensing choices through mathematical modeling of chemotactic circuits. Comprehensive computational search of three-node signaling circuits has identified the negative integral feedback (NFB) and incoherent feedforward (IFF) circuits as capable of adaptation, an important property for chemotaxis. Cells are modeled as one-dimensional circular system consisting of diffusible activator, inactivator and output proteins, traveling across a chemical gradient. From our simulations, we find that sensing outcomes are similar for NFB or IFF circuits. Rather than cell size, the relevant parameters are the 1) ratio of cell speed to the product of cell diameter and rate of signaling, 2) diffusivity of the output protein and 3) ratio of the diffusivities of the activator to inactivator protein. Spatial sensing is favored when all three parameters are low. This corresponds to a cell moving slower than the time it takes for signaling to propagate across the cell diameter, has an output protein that is polarizable and has a local-excitation global-inhibition system to amplify the chemical gradient. Temporal sensing is favored otherwise. We also find that temporal sensing is more robust to noise. By performing extensive literature search, we find that our prediction agrees with observation in a wide range of species and cell types ranging from E. coli to human Fibroblast cells and propose that our result is universally applicable.
OUTPUT: Unicellular organisms and other single cells often have to migrate towards food sources or away from predators by sensing chemicals present in the environment. There are two ways for a cell to sense these external chemicals: temporal sensing, where the cell senses the external chemical at two different time points after it has moved through a certain distance, or spatial sensing, where the cell senses the external chemical at two different locations on its cellular surface (e.g., the front and rear of the cell) simultaneously. It has been thought that small unicellular organisms employ temporal sensing as their small size prohibits sensing at two different locations on the cellular surface. Using computational modeling, we find that the choice between temporal and spatial sensing is determined by the ratio of cell velocity to the product of cell diameter and rate of signaling, as well as the diffusivities of the signaling proteins. Predictions from our model agree with experimental observations over a wide range of cells, where a fast-moving, small cell performs better comparing the chemoattractant at different times in its trajectory; whereas, a slow-moving, big cell performs better by comparing the chemoattractant concentration at its two ends.
INPUT: Robust Inference of Cell-to-Cell Expression Variations from Single- and K-Cell ProfilingQuantifying heterogeneity in gene expression among single cells can reveal information inaccessible to cell-population averaged measurements. However, the expression level of many genes in single cells fall below the detection limit of even the most sensitive technologies currently available. One proposed approach to overcome this challenge is to measure random pools of k cells (e.g., 10) to increase sensitivity, followed by computational “deconvolution” of cellular heterogeneity parameters (CHPs), such as the biological variance of single-cell expression levels. Existing approaches infer CHPs using either single-cell or k-cell data alone, and typically within a single population of cells. However, integrating both single- and k-cell data may reap additional benefits, and quantifying differences in CHPs across cell populations or conditions could reveal novel biological information. Here we present a Bayesian approach that can utilize single-cell, k-cell, or both simultaneously to infer CHPs within a single condition or their differences across two conditions. Using simulated as well as experimentally generated single- and k-cell data, we found situations where each data type would offer advantages, but using both together can improve precision and better reconcile CHP information contained in single- and k-cell data. We illustrate the utility of our approach by applying it to jointly generated single- and k-cell data to reveal CHP differences in several key inflammatory genes between resting and inflammatory cytokine-activated human macrophages, delineating differences in the distribution of ‘ON’ versus ‘OFF’ cells and in continuous variation of expression level among cells. Our approach thus offers a practical and robust framework to assess and compare cellular heterogeneity within and across biological conditions using modern multiplexed technologies.
OUTPUT: Different cells can make different amounts of biomolecules such as RNA transcripts of genes. New technologies are emerging to measure the transcript level of many genes in single cells. However, accurate quantification of the biological variation from cell to cell can be challenging due to the low transcript level of many genes and the presence of substantial measurement noise. Here we present a flexible, novel computational approach to quantify biological cell-to-cell variation that can use different types of data, namely measurements directly obtained from single cells, and/or those from random pools of k-cells (e.g., k = 10). Assessment of these different inputs using simulated and real data revealed that each data type can offer advantages under different scenarios, but combining both single- and k-cell measurements tend to offer the best of both. Application of our approach to single- and k-cell data obtained from resting and inflammatory macrophages, an important type of immune cells implicated in diverse diseases, revealed interesting changes in cell-to-cell variation in transcript levels upon inflammatory stimulation, thus suggesting that inflammation can shape not only the average expression level of a gene but also the gene’s degree of expression variation among single cells.
INPUT: Exploring the Contextual Sensitivity of Factors that Determine Cell-to-Cell Variability in Receptor-Mediated ApoptosisStochastic fluctuations in gene expression give rise to cell-to-cell variability in protein levels which can potentially cause variability in cellular phenotype. For TRAIL (TNF-related apoptosis-inducing ligand) variability manifests itself as dramatic differences in the time between ligand exposure and the sudden activation of the effector caspases that kill cells. However, the contribution of individual proteins to phenotypic variability has not been explored in detail. In this paper we use feature-based sensitivity analysis as a means to estimate the impact of variation in key apoptosis regulators on variability in the dynamics of cell death. We use Monte Carlo sampling from measured protein concentration distributions in combination with a previously validated ordinary differential equation model of apoptosis to simulate the dynamics of receptor-mediated
INPUT: Determinants of Cell-to-Cell Variability in Protein Kinase SignalingCells reliably sense environmental changes despite internal and external fluctuations, but the mechanisms underlying robustness remain unclear. We analyzed how fluctuations in signaling protein concentrations give rise to cell-to-cell variability in protein kinase signaling using analytical theory and numerical simulations. We characterized the dose-response behavior of signaling cascades by calculating the stimulus level at which a pathway responds (‘pathway sensitivity’) and the maximal activation level upon strong stimulation. Minimal kinase cascades with gradual dose-response behavior show strong variability, because the pathway sensitivity and the maximal activation level cannot be simultaneously invariant. Negative feedback regulation resolves this trade-off and coordinately reduces fluctuations in the pathway sensitivity and maximal activation. Feedbacks acting at different levels in the cascade control different aspects of the dose-response curve, thereby synergistically reducing the variability. We also investigated more complex, ultrasensitive signaling cascades capable of switch-like decision making, and found that these can be inherently robust to protein concentration fluctuations. We describe how the cell-to-cell variability of ultrasensitive signaling systems can be actively regulated, e.g., by altering the expression of phosphatase(s) or by feedback/feedforward loops. Our calculations reveal that slow transcriptional negative feedback loops allow for variability suppression while maintaining switch-like decision making. Taken together, we describe design principles of signaling cascades that promote robustness. Our results may explain why certain signaling cascades like the yeast pheromone pathway show switch-like decision making with little cell-to-cell variability.
OUTPUT:
| Cells sense their surroundings and respond to soluble factors in the extracellular space. Extracellular factors frequently induce heterogeneous responses, thereby restricting the biological outcome to a fraction of the cell population. However, the question arises how such cell-to-cell variability can be controlled, because some cellular systems show a very homogenous response at a defined level of an extracellular stimulus. We derived an analytical framework to systematically characterize the cell-to-cell variability of intracellular signaling pathways which transduce external signals. We analyzed how heterogeneity arises from fluctuations in the total concentrations of signaling proteins because this is the main source of variability in eukaryotic systems. We find that signaling pathways can be highly variable or inherently invariant, depending on the kinetic parameters and the structural features of the cascade. Our results indicate that the cell-to-cell variability can be reduced by negative feedback in the cascade or by signaling crosstalk between parallel pathways. We precisely define the role of negative feedback loops in variability suppression, and show that different aspects of the dose-response curve can be controlled, depending on the feedback kinetics and site of action in the cascade. This work constitutes a first step towards a systematic understanding of cell-to-cell variability in signal transduction.
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PLOS_five_shot_dy5 | ***TASK***
the task is to simplify the input abstract of a biomedical literature
***INPUT***
the input is the abstract of a biomedical literature
***OUTPUT***
the output is the simplified abstract for the input abstract of a biomedical literature
***DOCUMENTATION***
***EXAMPLES***
INPUT: Combining Regulatory T Cell Depletion and Inhibitory Receptor Blockade Improves Reactivation of Exhausted Virus-Specific CD8+ T Cells and Efficiently Reduces Chronic Retroviral LoadsChronic infections with human viruses, such as HIV and HCV, or mouse viruses, such as LCMV or Friend Virus (FV), result in functional exhaustion of CD8+ T cells. Two main mechanisms have been described that mediate this exhaustion: expression of inhibitory receptors on CD8+ T cells and expansion of regulatory T cells (Tregs) that suppress CD8+ T cell activity. Several studies show that blockage of one of these pathways results in reactivation of CD8+ T cells and partial reduction in chronic viral loads. Using blocking antibodies against PD-1 ligand and Tim-3 and transgenic mice in which Tregs can be selectively ablated, we compared these two treatment strategies and combined them for the first time in a model of chronic retrovirus infection. Blocking inhibitory receptors was more efficient than transient depletion of Tregs in reactivating exhausted CD8+ T cells and reducing viral set points. However, a combination therapy was superior to any single treatment and further augmented CD8+ T cell responses and resulted in a sustained reduction in chronic viral loads. These results demonstrate that Tregs and inhibitory receptors are non-overlapping factors in the maintenance of chronic viral infections and that immunotherapies targeting both pathways may be a promising strategy to treat chronic infectious diseases.
OUTPUT: A loss of function, the so-called ‘exhaustion’ of CD8+ T cells, is a hallmark of many chronic infections. The T cell exhaustion is mediated by two main mechanisms, the expression of inhibitory receptors on CD8+ T cells and virus-induced expansion of regulatory T cells (Tregs), which suppress CD8+ T cell activity. Several mouse studies revealed a reactivation of CD8+ T cells and reduction in chronic viral loads after blockage of one of these pathways. These results initiated a number of clinical studies mainly with cancer patients, in which blocking antibodies were used to interfere with inhibitory receptor signaling or drugs that deplete Tregs. For the first time we combined the two therapeutic approaches by using transgenic mice in which Tregs can be selectively ablated and injection of blocking antibodies in a chronic retroviral infection. The results indicate that the combination therapy was superior to any single treatment in further augmenting CD8+ T cell responses and reducing chronic viral loads. Our findings demonstrate that Tregs and inhibitory receptors are non-overlapping factors in the maintenance of chronic viral infections and that immunotherapies targeting both pathways may be a promising new strategy to treat chronic infectious diseases.
INPUT: Varicella zoster virus productively infects human peripheral blood mononuclear cells to modulate expression of immunoinhibitory proteins and blocking PD-L1 enhances virus-specific CD8+ T cell effector functionVaricella zoster virus (VZV) is a lymphotropic alpha-herpesvirinae subfamily member that produces varicella on primary infection and causes zoster, vascular disease and vision loss upon reactivation from latency. VZV-infected peripheral blood mononuclear cells (PBMCs) disseminate virus to distal organs to produce clinical disease. To assess immune evasion strategies elicited by VZV that may contribute to dissemination of infection, human PBMCs and VZV-specific CD8+ T cells (V-CD8+) were mock- or VZV-infected and analyzed for immunoinhibitory protein PD-1, PD-L1, PD-L2, CTLA-4, LAG-3 and TIM-3 expression using flow cytometry. All VZV-infected PBMCs (monocytes, NK, NKT, B cells, CD4+ and CD8+ T cells) and V-CD8+ showed significant elevations in PD-L1 expression compared to uninfected cells. VZV induced PD-L2 expression in B cells and V-CD8+. Only VZV-infected CD8+ T cells, NKT cells and V-CD8+ upregulated PD-1 expression, the immunoinhibitory receptor for PD-L1/PD-L2. VZV induced CTLA-4 expression only in V-CD8+ and no significant changes in LAG-3 or TIM-3 expression were observed in V-CD8+ or PBMC T cells. To test whether PD-L1, PD-L2 or CTLA-4 regulates V-CD8+ effector function, autologous PBMCs were VZV-infected and co-cultured with V-CD8+ cells in the presence of blocking antibodies against PD-L1, PD-L2 or CTLA-4; ELISAs revealed significant elevations in IFNγ only upon blocking of PD-L1. Together, these results identified additional immune cells that are permissive to VZV infection (monocytes, B cells and NKT cells); along with a novel mechanism for inhibiting CD8+ T cell effector function through induction of PD-L1 expression.
OUTPUT: The burden of disease produced by VZV is significant, since 90% of the world population harbors latent virus. At least 50% of infected individuals will reactivate by 85 years of age to develop zoster, which is an established risk factor for stroke and myocardial infarction, as well as multisystem diseases with or without rash. VZV-infected PBMCs disseminate virus to distal organs to produce clinical disease. PD-L1 is an immunoinhibitory protein that interacts with PD-1, its receptor, expressed mainly on T cells to prevent their activation and subsequent clearance of virus- or malignantly transformed cells. We show here that all peripheral blood mononuclear cells have a dramatic induction of PD-L1 expression upon infection with VZV, which is combined with the induction of PD-1 expression on CD8+ T cells, NKT cells and VZV-specific CD8+ T cells. Blocking PD-L1 during co-culturing of VZV-specific CD8+ T cells with autologous VZV-infected PBMCs enhanced IFNγ levels almost 2-fold compared to isotype controls. These results indicate that blocking PD-L1 expression during varicella or zoster may restore CD8+ T cell effector function, enabling effective clearance of virus-infected cells to reduce viral spread and multisystem disease.
INPUT: CD8+ T Cells from a Novel T Cell Receptor Transgenic Mouse Induce Liver-Stage Immunity That Can Be Boosted by Blood-Stage Infection in Rodent MalariaTo follow the fate of CD8+ T cells responsive to Plasmodium berghei ANKA (PbA) infection, we generated an MHC I-restricted TCR transgenic mouse line against this pathogen. T cells from this line, termed PbT-I T cells, were able to respond to blood-stage infection by PbA and two other rodent malaria species, P. yoelii XNL and P. chabaudi AS. These PbT-I T cells were also able to respond to sporozoites and to protect mice from liver-stage infection. Examination of the requirements for priming after intravenous administration of irradiated sporozoites, an effective vaccination approach, showed that the spleen rather than the liver was the main site of priming and that responses depended on CD8α+ dendritic cells. Importantly, sequential exposure to irradiated sporozoites followed two days later by blood-stage infection led to augmented PbT-I T cell expansion. These findings indicate that PbT-I T cells are a highly versatile tool for studying multiple stages and species of rodent malaria and suggest that cross-stage reactive CD8+ T cells may be utilized in liver-stage vaccine design to enable boosting by blood-stage infections.
OUTPUT: Malaria is a disease caused by Plasmodium species, which have a highly complex life cycle involving both liver and blood stages of mammalian infection. To prevent disease, one strategy has been to induce CD8+ T cells against liver-stage parasites, usually by immunization with stage-specific antigens. Here we describe a T cell receptor specificity that recognizes an antigen expressed in both the liver and blood stages of several rodent Plasmodium species. We generated a T cell receptor transgenic mouse with this specificity and showed that T cells from this line could protect against liver-stage infection. We used this novel tool to identify the site and cell-type involved in priming to a recently developed intravenous attenuated sporozoite vaccine shown to have efficacy in humans. We showed that CD8+ T cells with this specificity could protect against liver-stage infection while causing pathology to the blood stage. Finally, we provided evidence that T cells with cross-stage specificity can be primed and boosted on alternative stages, raising the possibility that antigens expressed in multiple stages might be ideal vaccine candidates for generating strong immunity to liver-stage parasites.
INPUT: Synergistic Reversal of Intrahepatic HCV-Specific CD8 T Cell Exhaustion by Combined PD-1/CTLA-4 BlockadeViral persistence is associated with hierarchical antiviral CD8 T cell exhaustion with increased programmed death-1 (PD-1) expression. In HCV persistence, HCV-specific CD8 T cells from the liver (the site of viral replication) display increased PD-1 expression and a profound functional impairment that is not reversed by PD-1 blockade alone. Here, we report that the inhibitory receptor cytotoxic T lymphocyte-associated antigen-4 (CTLA-4) is preferentially upregulated in PD-1+ T cells from the liver but not blood of chronically HCV-infected patients. PD-1/CTLA-4 co-expression in intrahepatic T cells was associated with a profound HCV-specific effector dysfunction that was synergistically reversed by combined PD-1/CTLA-4 blockade in vitro, but not by blocking PD-1 or CTLA-4 alone. A similar effect was observed in circulating HCV-specific CD8 T cells with increased PD-1/CTLA-4 co-expression during acute hepatitis C. The functional response to combined blockade was directly associated with CTLA-4 expression, lost with CD28-depletion and CD4-independent (including CD4+FoxP3+ Tregs). We conclude that PD-1 and CTLA-4 pathways both contribute to virus-specific T cell exhaustion at the site of viral replication by a redundant mechanism that requires combined PD-1/CTLA-4 blockade to reverse. These findings provide new insights into the mechanisms of virus-specific T cell dysfunction, and suggest that the synergistic effect by combined inhibitory receptor blockade might have a therapeutic application against chronic viral infection in vivo, provided that it does not induce autoimmunity.
OUTPUT: Hepatitis C virus (HCV) is an important human pathogen with a high rate of persistence associated with chronic liver disease that can progress to cirrhosis and hepatocellular carcinoma. Chronic HCV infection occurs in the setting of impaired antiviral T cells that over-express an inhibitory receptor PD-1 (programmed death-1 receptor). Recent studies showed that in vitro inhibition of the PD-1 pathway via an inhibitory antibody can reverse the functional impairment in HCV-specific CD8 T cells from blood but not the liver (the site of viral infection and disease progression). In this study, we show that a second co-inhibitory receptor, CTLA-4, is upregulated in HCV-specific CD8 T cells from the
INPUT: B7-H1 Blockade Increases Survival of Dysfunctional CD8+ T Cells and Confers Protection against Leishmania donovani InfectionsExperimental visceral leishmaniasis (VL) represents an exquisite model to study CD8+ T cell responses in a context of chronic inflammation and antigen persistence, since it is characterized by chronic infection in the spleen and CD8+ T cells are required for the development of protective immunity. However, antigen-specific CD8+ T cell responses in VL have so far not been studied, due to the absence of any defined Leishmania-specific CD8+ T cell epitopes. In this study, transgenic Leishmania donovani parasites expressing ovalbumin were used to characterize the development, function, and fate of Leishmania-specific CD8+ T cell responses. Here we show that L. donovani parasites evade CD8+ T cell responses by limiting their expansion and inducing functional exhaustion and cell death. Dysfunctional CD8+ T cells could be partially rescued by in vivo B7-H1 blockade, which increased CD8+ T cell survival but failed to restore cytokine production. Nevertheless, B7-H1 blockade significantly reduced the splenic parasite burden. These findings could be exploited for the design of new strategies for immunotherapeutic interventions against VL.
OUTPUT:
| The protozoan parasite Leishmania donovani is the cause of visceral leishmaniasis, a chronic disease that currently affects 12 million people worldwide. We are interested in understanding the immune mechanisms that can control infection. Preliminary studies suggested that CD8+ T cells can kill parasites and limit disease; however, studying these important killer cells has been hindered, because we do not know what parasite molecules they recognize. To overcome this, we engineered parasites to express ovalbumin. Since many tools exist to track and measure immune cells targeted at ovalbumin, we can now track the specific CD8+ T cell responses that develop upon infection with Leishmania. We found that Leishmania initially induced CD8+ T cells to divide and produce molecules such as IFN-gamma that may help them to kill parasites. However, the CD8+ T cells rapidly lost their effector function and died off as infection progressed. More encouragingly, though, we were able to recover some CD8+ T cell function by blocking immune inhibitory molecules that are induced by parasite infection. The recovered T cells killed parasites and controlled infection. These results are important as they could be exploited for the design of new therapeutic vaccine strategies aimed at inducing protective CD8+ T cells.
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PLOS_five_shot_dy6 | ***TASK***
the task is to simplify the input abstract of a biomedical literature
***INPUT***
the input is the abstract of a biomedical literature
***OUTPUT***
the output is the simplified abstract for the input abstract of a biomedical literature
***DOCUMENTATION***
***EXAMPLES***
INPUT: Prion Protein Amino Acid Determinants of Differential Susceptibility and Molecular Feature of Prion Strains in Mice and VolesThe bank vole is a rodent susceptible to different prion strains from humans and various animal species. We analyzed the transmission features of different prions in a panel of seven rodent species which showed various degrees of phylogenetic affinity and specific prion protein (PrP) sequence divergences in order to investigate the basis of vole susceptibility in comparison to other rodent models. At first, we found a differential susceptibility of bank and field voles compared to C57Bl/6 and wood mice. Voles showed high susceptibility to sheep scrapie but were resistant to bovine spongiform encephalopathy, whereas C57Bl/6 and wood mice displayed opposite features. Infection with mouse-adapted scrapie 139A was faster in voles than in C57Bl/6 and wood mice. Moreover, a glycoprofile change was observed in voles, which was reverted upon back passage to mice. All strains replicated much faster in voles than in mice after adapting to the new species. PrP sequence comparison indicated a correlation between the transmission patterns and amino acids at positions 154 and 169 (Y and S in mice, N and N in voles). This correlation was confirmed when inoculating three additional rodent species: gerbils, spiny mice and oldfield mice with sheep scrapie and 139A. These rodents were chosen because oldfield mice do have the 154N and 169N substitutions, whereas gerbil and spiny mice do not have them. Our results suggest that PrP residues 154 and 169 drive the susceptibility, molecular phenotype and replication rate of prion strains in rodents. This might have implications for the assessment of host range and molecular traceability of prion strains, as well as for the development of improved animal models for prion diseases.
OUTPUT: Prions are unconventional infectious agents that cause fatal neurodegenerative diseases in animals and humans. A pathological form of the cellular prion protein (PrPC), named PrPSc, appears to be the major or the sole component of prions. These agents are transmitted by inducing the conversion of host PrPC into PrPSc that accumulates in the brain of affected individuals. Different factors are believed to modulate such events, which explains the variable transmission efficiency observed under inter-species experimental inoculation. These factors are still fairly unknown, although evidence exists that some kind of structural compatibility between PrPSc of the infectious inoculum and PrPC of the host has a role in making transmission more or less efficient. We investigated the transmission of prions to different rodents and showed that specific amino acid substitutions (Y154N and S169N) in the prion protein are major determinants of susceptibility to prions. In particular, we showed that these specific variations i) direct the transmission rate of prions between different species in a way that is dependent on the prion strain, ii) affect the molecular characteristics of prions, and iii) influence their replication efficiency.
INPUT: PPARα siRNA–Treated Expression Profiles Uncover the Causal Sufficiency Network for Compound-Induced Liver HypertrophyUncovering pathways underlying drug-induced toxicity is a fundamental objective in the field of toxicogenomics. Developing mechanism-based toxicity biomarkers requires the identification of such novel pathways and the order of their sufficiency in causing a phenotypic response. Genome-wide RNA interference (RNAi) phenotypic screening has emerged as an effective tool in unveiling the genes essential for specific cellular functions and biological activities. However, eliciting the relative contribution of and sufficiency relationships among the genes identified remains challenging. In the rodent, the most widely used animal model in preclinical studies, it is unrealistic to exhaustively examine all potential interactions by RNAi screening. Application of existing computational approaches to infer regulatory networks with biological outcomes in the rodent is limited by the requirements for a large number of targeted permutations. Therefore, we developed a two-step relay method that requires only one targeted perturbation for genome-wide de novo pathway discovery. Using expression profiles in response to small interfering RNAs (siRNAs) against the gene for peroxisome proliferator-activated receptor α (Ppara), our method unveiled the potential causal sufficiency order network for liver hypertrophy in the rodent. The validity of the inferred 16 causal transcripts or 15 known genes for PPARα-induced liver hypertrophy is supported by their ability to predict non-PPARα–induced liver hypertrophy with 84% sensitivity and 76% specificity. Simulation shows that the probability of achieving such predictive accuracy without the inferred causal relationship is exceedingly small (p < 0.005). Five of the most sufficient causal genes have been previously disrupted in mouse models; the resulting phenotypic changes in the liver support the inferred causal roles in liver hypertrophy. Our results demonstrate the feasibility of defining pathways mediating drug-induced toxicity from siRNA-treated expression profiles. When combined with phenotypic evaluation, our approach should help to unleash the full potential of siRNAs in systematically unveiling the molecular mechanism of biological events.
OUTPUT: Approaches for discovering mechanisms of action and for identifying molecular biomarkers in biomedical research are evolving today, as the growing symbiosis with computational sciences becomes more widely appreciated. In fact, the combination of various new technologies has been pushing forward both frontiers. Here, we present an example of the combined use of in vivo siRNA knock-down technology, genome-wide gene expression profiling, and computational reasoning to unveil regulatory causal relationships and the sufficiency network of identified genes for compound-induced toxicity. Unlike previously reported approaches, our method requires only one targeted perturbation for genome-wide de novo pathway discovery. Hence, our method can be directly applied to animal models in which it is still technically challenging to perform genome-wide genetic perturbation or RNAi screening. The independent application of our derived model to compounds with unrelated mechanisms of action suggests the existence of a universal molecular module that mediates liver hypertrophy. The resulting sufficiency network for induction of liver hypertrophy will have an immediate impact on the progress of toxicogenomics. When combined with phenotypic evaluation, our approach should help to unleash the full potential of siRNAs in systematically unveiling the molecular mechanisms of biological events.
INPUT: Trans-Dominant Inhibition of Prion Propagation In Vitro Is Not Mediated by an Accessory CofactorPrevious studies identified prion protein (PrP) mutants which act as dominant negative inhibitors of prion formation through a mechanism hypothesized to require an unidentified species-specific cofactor termed protein X. To study the mechanism of dominant negative inhibition in vitro, we used recombinant PrPC molecules expressed in Chinese hamster ovary cells as substrates in serial protein misfolding cyclic amplification (sPMCA) reactions. Bioassays confirmed that the products of these reactions are infectious. Using this system, we find that: (1) trans-dominant inhibition can be dissociated from conversion activity, (2) dominant-negative inhibition of prion formation can be reconstituted in vitro using only purified substrates, even when wild type (WT) PrPC is pre-incubated with poly(A) RNA and PrPSc template, and (3) Q172R is the only hamster PrP mutant tested that fails to convert into PrPSc and that can dominantly inhibit conversion of WT PrP at sub-stoichiometric levels. These results refute the hypothesis that protein X is required to mediate dominant inhibition of prion propagation, and suggest that PrP molecules compete for binding to a nascent seeding site on newly formed PrPSc molecules, most likely through an epitope containing residue 172.
OUTPUT: Over the past two decades, various investigators have observed that heterozygous animals possessing two different forms of the gene encoding the prion protein (PrP) are more difficult to infect with some strains of infectious prions than homozygous animals possessing only the most commonly occurring form of the gene encoding PrP for that species. In 1995, it was hypothesized that the inhibition of prion infection in heterozygous animals might be caused by competition between the two different types of PrP molecules for binding to a common cofactor required for prion propagation, provisionally named “protein X,” through a specific portion of the PrP molecule. Here, we report that mixing different purified PrP molecules together in test tube reactions lacking accessory proteins can also interfere with prion propagation. We also found that some mutations of the putative protein X binding site do not inhibit the formation of hamster prions in chemical reactions. Our work suggests that different PrP molecules most likely compete for binding to newly formed prions rather than an accessory protein cofactor, and argues against the existence of protein X.
INPUT: Characterizing the Syphilis-Causing Treponema pallidum ssp. pallidum Proteome Using Complementary Mass SpectrometryThe spirochete bacterium Treponema pallidum ssp. pallidum is the etiological agent of syphilis, a chronic multistage disease. Little is known about the global T. pallidum proteome, therefore mass spectrometry studies are needed to bring insights into pathogenicity and protein expression profiles during infection.
To better understand the T. pallidum proteome profile during infection, we studied T. pallidum ssp. pallidum DAL-1 strain bacteria isolated from rabbits using complementary mass spectrometry techniques, including multidimensional peptide separation and protein identification via matrix-assisted laser desorption ionization-time of flight (MALDI-TOF/TOF) and electrospray ionization (ESI-LTQ-Orbitrap) tandem mass spectrometry. A total of 6033 peptides were detected, corresponding to 557 unique T. pallidum proteins at a high level of confidence, representing 54% of the predicted proteome. A previous gel-based T. pallidum MS proteome study detected 58 of these proteins. One hundred fourteen of the detected proteins were previously annotated as hypothetical or uncharacterized proteins; this is the first account of 106 of these proteins at the protein level. Detected proteins were characterized according to their predicted biological function and localization; half were allocated into a wide range of functional categories. Proteins annotated as potential membrane proteins and proteins with unclear functional annotations were subjected to an additional bioinformatics pipeline analysis to facilitate further characterization. A total of 116 potential membrane proteins were identified, of which 16 have evidence supporting outer membrane localization. We found 8/12 proteins related to the paralogous tpr gene family: TprB, TprC/D, TprE, TprG, TprH, TprI and TprJ. Protein abundance was semi-quantified using label-free spectral counting methods. A low correlation (r = 0.26) was found between previous microarray signal data and protein abundance.
This is the most comprehensive description of the global T. pallidum proteome to date. These data provide valuable insights into in vivo T. pallidum protein expression, paving the way for improved understanding of the pathogenicity of this enigmatic organism.
OUTPUT: Syphilis remains a major cause of morbidity and mortality worldwide. The bacterium causing syphilis, Treponema pallidum ssp. pallidum, has evolved into a highly distinctive organism that is only able survive (and be propagated) in mammals. In humans it can evade the immune system for decades with devastating consequences. Much remains to be learned about how it accomplishes this. Only a minority of its predicted proteins have been detected experimentally thus far. We aimed to more comprehensively characterize the
INPUT: Two Misfolding Routes for the Prion Protein around pH 4.5Using molecular dynamics simulations, we show that the prion protein (PrP) exhibits a dual behavior, with two possible transition routes, upon protonation of H187 around pH 4.5, which mimics specific conditions encountered in endosomes. Our results suggest a picture in which the protonated imidazole ring of H187 experiences an electrostatic repulsion with the nearby guanidinium group of R136, to which the system responds by pushing either H187 or R136 sidechains away from their native cavities. The regions to which H187 and R136 are linked, namely the C-terminal part of H2 and the loop connecting S1 to H1, respectively, are affected in a different manner depending on which pathway is taken. Specific in vivo or in vitro conditions, such as the presence of molecular chaperones or a particular experimental setup, may favor one transition pathway over the other, which can result in very different monomers. This has some possible connections with the observation of various fibril morphologies and the outcome of prion strains. In addition, the finding that the interaction of H187 with R136 is a weak point in mammalian PrP is supported by the absence of the residue pair in non-mammalian species that are known to be resistant to prion diseases.
OUTPUT:
| Transmissible spongiform encephalopathies, which include the “mad cow” disease and the Creutzfeldt-Jakob disease, are related to the abnormal folding of a host protein termed the prion protein (PrP). Many aspects of the underlying molecular mechanism still remain elusive. Among the hypotheses that have been put forward in the past few years, it has been suggested that PrP could be destabilized by the protonation of a specific residue, H187, when the protein passes through acidic cell organelles. We have modeled PrP at the atomistic level, with the neutral and protonated forms of H187. Our simulations show that the destabilization process can follow two alternative pathways that could lead to different final structures. This discovery may shed some light on one of the most puzzling aspect of prion diseases, the fact that they exhibit various strains encoded in the structure of misfolded PrP. In addition, the atomistic details provided by our model highlight a key interactions partner in the destabilization process, R136. The residue pair is not present in non-mammalian species that do not develop prion diseases.
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PLOS_five_shot_dy7 | ***TASK***
the task is to simplify the input abstract of a biomedical literature
***INPUT***
the input is the abstract of a biomedical literature
***OUTPUT***
the output is the simplified abstract for the input abstract of a biomedical literature
***DOCUMENTATION***
***EXAMPLES***
INPUT: The Outcome of Trachomatous Trichiasis Surgery in Ethiopia: Risk Factors for RecurrenceOver 1.2 million people are blind from trachomatous trichiasis (TT). Lid rotation surgery is the mainstay of treatment, but recurrence rates can be high. We investigated the outcomes (recurrence rates and other complications) of posterior lamellar tarsal rotation (PLTR) surgery, one of the two most widely practised TT procedures in endemic settings.
We conducted a two-year follow-up study of 1300 participants who had PLTR surgery, conducted by one of five TT nurse surgeons. None had previously undergone TT surgery. All participants received a detailed trachoma eye examination at baseline and 6, 12, 18 and 24 months post-operatively. The study investigated the recurrence rates, other complications and factors associated with recurrence. Recurrence occurred in 207/635 (32.6%) and 108/641 (16.9%) of participants with pre-operative major (>5 trichiatic lashes) and minor (<5 lashes) TT respectively. Of the 315 recurrences, 42/315 (3.3% overall) had >5 lashes (major recurrence). Recurrence was greatest in the first six months after surgery: 172 cases (55%) occurring in this period. Recurrence was associated with major TT pre-operatively (OR 2.39, 95% CI 1.83–3.11), pre-operative entropic lashes compared to misdirected/metaplastic lashes (OR 1.99, 95% CI 1.23–3.20), age over 40 years (OR 1.59, 95% CI 1.14–2.20) and specific surgeons (surgeon recurrence risk range: 18%–53%). Granuloma occurred in 69 (5.7%) and notching in 156 (13.0%).
Risk of recurrence is high despite high volume, highly trained surgeons. However, the vast majority are minor recurrences, which may not have significant corneal or visual consequences. Inter-surgeon variation in recurrence is concerning; surgical technique, training and immediate post-operative lid position require further investigation.
OUTPUT: Trachoma is the most common infectious cause of blindness worldwide. It causes trichiasis (inturning of the eyelashes to touch the eye), which can cause visual loss. Trachomatous trichiasis (TT) affects over eight million people, 1.2 of whom live in Ethiopia – the most affected country worldwide. Surgery is the mainstay of treatment for TT. However, results of surgery in the field are often very mixed. We investigated the surgical outcomes of one of the two most widely used surgical techniques (posterior lamellar rotation), in 1300 individuals in the Amhara Region of Ethiopia. We found that recurrence occurred frequently: 315/1276 (24.7%) participants. However, recurrence was rarely severe (greater than 5 lashes): 42 participants (3.3%). Recurrence occurred much more frequently in participants who had severe pre-operative disease and with specific surgeons. The high recurrence rates and inter-surgeon variation is concerning. Further research will be required to investigate factors such as surgical technique, surgeon training and immediate post-operative lid position, in order to improve surgical outcomes.
INPUT: Predicting Surgery Targets in Temporal Lobe Epilepsy through Structural Connectome Based SimulationsTemporal lobe epilepsy (TLE) is a prevalent neurological disorder resulting in disruptive seizures. In the case of drug resistant epilepsy resective surgery is often considered. This is a procedure hampered by unpredictable success rates, with many patients continuing to have seizures even after surgery. In this study we apply a computational model of epilepsy to patient specific structural connectivity derived from diffusion tensor imaging (DTI) of 22 individuals with left TLE and 39 healthy controls. We validate the model by examining patient-control differences in simulated seizure onset time and network location. We then investigate the potential of the model for surgery prediction by performing in silico surgical resections, removing nodes from patient networks and comparing seizure likelihood post-surgery to pre-surgery simulations. We find that, first, patients tend to transit from non-epileptic to epileptic states more often than controls in the model. Second, regions in the left hemisphere (particularly within temporal and subcortical regions) that are known to be involved in TLE are the most frequent starting points for seizures in patients in the model. In addition, our analysis also implicates regions in the contralateral and frontal locations which may play a role in seizure spreading or surgery resistance. Finally, the model predicts that patient-specific surgery (resection areas chosen on an individual, model-prompted, basis and not following a predefined procedure) may lead to better outcomes than the currently used routine clinical procedure. Taken together this work provides a first step towards patient specific computational modelling of epilepsy surgery in order to inform treatment strategies in individuals.
OUTPUT: Temporal lobe epilepsy (TLE) is a disorder characterised by unpredictable seizures, where surgical removal of brain tissue is often the final treatment option. In roughly 30% of cases surgery procedures are unsuccessful at preventing future seizures. This paper shows the application of a computational model which uses patient derived brain connectivity to predict the success rates of surgery in people with TLE. We consider the brains of 22 patients as networks, with brain regions as nodes and the white matter connections between them as edges. The brain network is unique to each subject and produced from brain imaging scans of 22 patients and 39 controls. Seizures are simulated before and after surgery, where surgery in the model is the removal of nodes from the network. The model successfully identifies regions known to be involved in TLE, and its predicted success rates for surgery are close to the results found in reality. The model additionally provides patient specific recommendations for surgical procedures, which in simulations show improved results compared to standard surgery in every case. This is a first step towards designing personalised surgery procedures in order to improve surgery success rates.
INPUT: Determinants for not utilizing trachomatous trichiasis surgery among trachomatous trichiasis patients in Mehalsayint District, North-East EthiopiaGlobally, trachoma is the leading cause of infectious blindness. In Ethiopia, the overall Trachomatous Trichiasis (TT) surgical coverage is 41%. Identifying determinants for not utilizing TT surgery among TT patients is important to design and monitor effective intervention programs. Therefore, this study aimed to identify determinants for not utilizing TT surgery among TT patients in Mehalsayint District, North East Ethiopia.
A community based unmatched case control study was employed from March 30, 2017 to April 13, 2017. A total of 482 study participants (241 cases and 241 controls) with age of ≥15 years were included in the study. The data were entered with Epi info version 7.2 software and exported to SPSS version 20 for analysis. Bivariate analysis was fitted to screen candidate variables with p<0.2 for the final model. Finally, multivariable logistic regression analysis was employed to identify significant factors (p<0.05) for not utilizing TT surgery. Respondents’ age of 16–30 years (AOR: 10.11; 95% CI: 2.72, 37.59) and widowed respondents (AOR: 0.40; 95% CI: 0.21, 0.77), time to reach the service (AOR: 0.46; 95% CI: 0.24, 0.87), unavailability of TT surgeon (AOR: 5.00; 95% CI: 1.16, 21.38), symptoms of trichiasis (AOR: 7.49; 95% CI: 2.41, 23.26), duration of the problem (AOR: 2.56; 95% CI: 1.44, 4.54), the affected eye (AOR: 2.16; 95% CI: 1.23, 3.80), epilation practice (AOR: 3.22; 95% CI: 1.84, 5.64), and place of TT surgery given (AOR: 4.21; 95% CI: 2.48, 7.14) were significant determinants for not utilizing TT surgical services.
In this study, TT surgery against trachoma is very low and TT remains public health problem in the district. Being younger age and widowed, time taken to reach the service, absence of TT surgeon, symptoms of trichiasis, duration of problem, the affected eye, epilation practice, and service place were determinants for the inability of TT surgical services. The findings of this study would help in designing effective interventions to reduce trachoma in that district.
OUTPUT: Trachoma is the common ophthalmic infection and cause of blindness worldwide. It is caused by ocular infections with causative agent of Chlamydia trachomatis that might effect in chronic inflammation of the eyelids, which produces scarring of the conjunctiva that can consequently cause entropion trichiasis, resulting in interned eyelashes. The interned eyelashes as well as other changes of the eye, harm the cornea causing severe pain, corneal opacity and resulting vision loss. Over a million people in Ethiopia are estimated to have Trachomatous trichiasis (TT). Trachomatous trichiasis surgery is the backbone treatment option. Though the provision of free surgical services in the country exists, utilization rates are very low. Identifying the determinants for not utilizing the service is mandatory to take measures towards surgical uptake. A total of 482 study participants (241 cases and 241 controls) with age of ≥15 years were included in the study. The determinants for not use of surgical services were respondents in the younger age group (16–30 years) and widowed participants, lengthy distance from the service, unavailability of TT surgeon, no trichiasis symptoms, long time knowing the problem, right/left eye affected, no experience of epilation practice, and participants who knew place of service was given at health center.
INPUT: Eyelash Epilation in the Absence of Trichiasis: Results of a Population-Based Prevalence Survey in the Western Division of FijiThe WHO definition of trachomatous trichiasis (TT) is “at least one eyelash touching the globe, or evidence of recent epilation of in-turned eyelashes”, reflecting the fact that epilation is
INPUT: Epilation for Minor Trachomatous Trichiasis: Four-Year Results of a Randomised Controlled TrialTrachomatous trichiasis (TT) needs to be managed to reduce the risk of vision loss. The long-term impact of epilation (a common traditional practice of repeated plucking of lashes touching the eye) in preventing visual impairment and corneal opacity from TT is unknown. We conducted a randomized controlled trial of epilation versus surgery for the management of minor TT (fewer than six lashes touching the eye) in Ethiopia. Here we report the four-year outcome and the effect on vision and corneal opacity.
1300 individuals with minor TT were recruited and randomly assigned to quality trichiasis surgery or repeated epilation using high quality epilation forceps by a trained person with good near vision. Participants were examined six-monthly for two-years, and then at four-years after randomisation. At two-years all epilation arm participants were offered free surgery. At four-years 1151 (88.5%) were re-examined: 572 (88%) and 579 (89%) from epilation and surgery arms, respectively. At that time, 21.1% of the surgery arm participants had recurrent TT; 189/572 (33%) of the epilation arm had received surgery, while 383 (67%) declined surgery and had continued epilating (“epilation-only”). Among the epilation-only group, 207 (54.1%) fully controlled their TT, 166 (43.3%) had minor TT and 10 (2.6%) had major TT (>5 lashes). There were no differences between participants in the epilation-only, epilation-to-surgery and surgery arm participants in changes in visual acuity and corneal opacity between baseline and four-years.
Most minor TT participants randomised to the epilation arm continued epilating and controlled their TT. Change in vision and corneal opacity was comparable between surgery and epilation-only participants. This suggests that good quality epilation with regular follow-up is a reasonable second-line alternative to surgery for minor TT for individuals who either decline surgery or do not have immediate access to surgical treatment.
OUTPUT:
| Trachoma causes visual impairment through the effect of in-turned eyelashes (trichiasis) on the surface of the eye. Epilation is a common traditional practice of intermittent plucking of lashes touching the eye, however, its long-term effectiveness in preventing visual impairment is unknown. We conducted a randomized controlled trial of epilation versus eyelid surgery (the main treatment option) in 1300 people with mild trichiasis in Ethiopia. We defined mild trichiasis as fewer than six lashes touching the eye. We have previously reported results to two years and have now re-assessed these individuals at four years. Overall, we found no difference between the epilation and surgery groups in terms of change in vision and corneal opacity between baseline and four years. Most mild trichiasis participants randomised to the epilation arm continued epilating and controlled their trichiasis. This suggests that good quality epilation is a reasonable second-line alternative to surgery for mild trichiasis for individuals who either decline surgery or do not have immediate access to surgical treatment.
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PLOS_five_shot_dy8 | ***TASK***
the task is to simplify the input abstract of a biomedical literature
***INPUT***
the input is the abstract of a biomedical literature
***OUTPUT***
the output is the simplified abstract for the input abstract of a biomedical literature
***DOCUMENTATION***
***EXAMPLES***
INPUT: Human macrophages differentiated in the presence of vitamin D3 restrict dengue virus infection and innate responses by downregulating mannose receptor expressionSevere dengue disease is associated with high viral loads and overproduction of pro-inflammatory cytokines, suggesting impairment in the control of dengue virus (DENV) and the mechanisms that regulate cytokine production. Vitamin D3 has been described as an important modulator of immune responses to several pathogens. Interestingly, increasing evidence has associated vitamin D with decreased DENV infection and early disease recovery, yet the molecular mechanisms whereby vitamin D reduces DENV infection are not well understood.
Macrophages represent important cell targets for DENV replication and consequently, they are key drivers of dengue disease. In this study we evaluated the effect of vitamin D3 on the differentiation of monocyte-derived macrophages (MDM) and their susceptibility and cytokine response to DENV. Our data demonstrate that MDM differentiated in the presence of vitamin D3 (D3-MDM) restrict DENV infection and moderate the classical inflammatory cytokine response. Mechanistically, vitamin D3-driven differentiation led to reduced surface expression of C-type lectins including the mannose receptor (MR, CD206) that is known to act as primary receptor for DENV attachment on macrophages and to trigger of immune signaling. Consequently, DENV bound less efficiently to vitamin D3-differentiated macrophages, leading to lower infection. Interestingly, IL-4 enhanced infection was reduced in D3-MDM by restriction of MR expression. Moreover, we detected moderate secretion of TNF-α, IL-1β, and IL-10 in D3-MDM, likely due to less MR engagement during DENV infection.
Our findings reveal a molecular mechanism by which vitamin D counteracts DENV infection and progression of severe disease, and indicates its potential relevance as a preventive or therapeutic candidate.
OUTPUT: Dengue represents a major worldwide concern for public health. Clinical complications rely on vascular leak of fluids and molecules from the bloodstream that leads to a potentially fatal hemodynamic compromise. Disease progression has been related to poor control of dengue virus (DENV) dissemination and excessive production of pro-inflammatory mediators that affect the endothelial function. Vitamin D has been shown to modulate immune responses and to alleviate dengue disease. Here, we studied how addition of vitamin D during macrophage differentiation modulates the functional features of these cells in the context of DENV infection. We observed that vitamin D reduced susceptibility of these cells to DENV infection and down-regulated the virus-induced cytokine response. This phenotype was attributed to downregulation of MR, a molecule hijacked by the virus to gain entry into the cells and a key receptor of the MR/CLEC5A complex that links binding and immune activation during DENV infection. Our study sheds light on the mechanism by which vitamin D can restrict DENV dissemination and the cytokine response in macrophages, indicating the potential relevance of this hormone as a preventive and therapeutic candidate.
INPUT: Human Macrophage Response to L. (Viannia) panamensis: Microarray Evidence for an Early Inflammatory ResponsePrevious findings indicate that susceptibility to Leishmania (Viannia) panamensis infection of monocyte-derived macrophages from patients and asymptomatically infected individuals were associated with the adaptive immune response and clinical outcome.
To understand the basis for this difference we examined differential gene expression of human monocyte-derived macrophages following exposure to L. (V.) panamensis. Gene activation profiles were determined using macrophages from healthy volunteers cultured with or without stationary phase promastigotes of L. (V.) panamensis. Significant changes in expression (>1.5-fold change; p<0.05; up- or down-regulated) were identified at 0.5, 4 and 24 hours. mRNA abundance profiles varied over time, with the highest level of activation occurring at earlier time points (0.5 and 4 hrs). In contrast to observations for other Leishmania species, most significantly changed mRNAs were up- rather than down-regulated, especially at early time points. Up-regulated transcripts over the first 24 hours belonged to pathways involving eicosanoid metabolism, oxidative stress, activation of PKC through G protein coupled receptors, or mechanism of gene regulation by peroxisome proliferators via PPARα. Additionally, a marked activation of Toll-receptor mediated pathways was observed. Comparison with published microarray data from macrophages infected with L. (Leishmania) chagasi indicate differences in the regulation of genes involved in signaling, motility and the immune response.
Results show that the early (0.5 to 24 hours) human monocyte-derived macrophage response to L. (Viannia) panamensis is not quiescent, in contrast to published reports examining later response times (48–96 hours). Early macrophage responses are important for the developing cellular response at the site of infection. The kinetics and the mRNA abundance profiles induced by L. (Viannia) panamensis illustrate the dynamics of these interactions and the distinct biologic responses to different Leishmania species from the outset of infection within their primary host cell.
OUTPUT: Leishmania parasites cause a spectrum of diseases (cutaneous, visceral and the deforming forms—chronic cutaneous and mucocutaneous) known as leishmaniasis. The macrophage, a key cell in the immune system, is the cellular target of Leishmania parasites in the mammalian host. Previous studies showed the responses of monocytederived macrophages from naturally infected humans to infection with Leishmania (Viannia) panamensis were key to adaptive immune responses and clinical outcome. Consequently, an mRNA microarray approach was employed to assess the changes in macrophage gene expression over time (0.5 to 24 hours) induced by L. panamensis. The highest level of gene expression induction occurred early (0.5–4 hours); the early pathways (groups of genes) activated included those involved in the innate immune response (signaling, phagocytosis, TLR activation, and inflammatory). Early gene activation is presumed to be important for the developing cellular milieu at the site of infection. By 24 hours post-infection the dominant pathways involved metabolic functions. However, a comparison of the macrophage response to L. (V.) panamensis to that of L. (L.) chagasi (causative agent of visceral leishmaniasis) at 24 hours revealed a differential up-regulation of genes (cell adhesion, signaling, and inflammation) in response to these species. These observations underscore the distinct biology of different Leishmania species from the outset of infection.
INPUT: Dengue Virus Nonstructural Protein 1 Induces Vascular Leakage through Macrophage Migration Inhibitory Factor and AutophagyDengue virus (DENV) is the most common mosquito-borne flavivirus; it can either cause mild dengue fever or the more severe dengue hemorrhagic fever (DHF) and dengue shock syndrome (DSS). One of the characteristic features of DHF/DSS is vascular leakage; although DENV nonstructural protein 1 (NS1) has been proved to induce vascular leakage after binding to Toll-like receptor 4, the down-stream mechanism has not yet been fully understood. In the sera of DENV-infected patients, the concentrations of DENV NS1 and inflammatory cytokine macrophage migration inhibitory factor (MIF) are positively correlated with disease severity, but whether DENV NS1 induces vascular leakage through MIF secretion remains unknown. We demonstrated that recombinant NS1 induced vascular leakage and MIF secretion both in human endothelial cell line HMEC-1 and in mice. Furthermore, these phenomena were inhibited in the presence of anti-NS1 antibodies both in vitro and in vivo. DENV NS1 also induced LC3-I to LC3-II conversion and p62 degradation in endothelial cell line, which indicated the formation of autophagy. To clarify whether MIF or autophagy mediated DENV NS1-induced vascular leakage, various inhibitors were applied. The results showed that DENV NS1-induced vascular leakage and VE-cadherin disarray were blocked in the presence of MIF inhibitors, anti-MIF-antibodies or autophagy inhibitors. An Atg5 knockdown clone further confirmed that autophagy formation of endothelial cells was required in NS1-induced vascular leakage. Furthermore, DENV NS1-induced LC3 puncta were also decreased in the presence of MIF inhibitors, indicating that MIF mediated DENV NS1-induced autophagy. Taken together, the results suggest a potential mechanism of DENV-induced vascular leakage and provide possible therapeutic targets against DHF/DSS.
OUTPUT: Dengue is a viral disease transmitted by mosquitoes. The symptoms of dengue are often mild; however, severe dengue is one of the leading causes of hospitalization and death among children in Asian and Latin American countries. A symptom of severe dengue is vascular leakage, which can result in fluid accumulation, hypotension, circulatory collapse, and even death. For dengue and severe dengue, there is no specific treatment, and the only supportive treatment is to maintain a patient’s body fluids at normal levels. As a result, investigating the mechanism of how dengue virus (DENV) causes vascular leakage is an important and urgent issue. In this study, we demonstrated that DENV nonstructural protein 1 (NS1) induced vascular leakage through the secretion of macrophage migration inhibitory factor (MIF) and the formation of autophagy. Inhibition of MIF or autophagy formation effectively reversed NS1-induced vascular leakage both in vitro and in mice. These results provide possible therapeutic targets for treating vascular leakage in severe dengue.
INPUT: Brugia malayi Microfilariae Induce a Regulatory Monocyte/Macrophage Phenotype That Suppresses Innate and Adaptive Immune ResponsesMonocytes and macrophages contribute to the dysfunction of immune responses in human filariasis. During patent infection monocytes encounter microfilariae in the blood, an event that occurs in asymptomatically infected filariasis patients that are immunologically hyporeactive.
To determine whether blood microfilariae directly act on blood monocytes and in vitro generated macrophages to induce a regulatory phenotype that interferes with innate and adaptive responses.
Monocytes and in vitro generated macrophages from filaria non-endemic normal donors were stimulated in vitro with Brugia malayi microfilarial (Mf) lysate. We could show that monocytes stimulated with Mf lysate develop a defined regulatory phenotype, characterised by expression of the immunoregulatory markers IL-10 and PD-L1. Significantly, this regulatory phenotype was recapitulated in monocytes from Wuchereria bancrofti asymptomatically infected patients but not patients with pathology or endemic normals. Monocytes from non-endemic donors stimulated with Mf lysate directly inhibited CD4+ T cell proliferation and cytokine production (IFN-γ, IL-13 and IL-10). IFN-γ responses were restored by neutralising IL-10 or PD-1. Furthermore, macrophages stimulated with Mf lysate expressed high levels of IL-10 and had suppressed phagocytic abilities. Finally Mf lysate applied during the differentiation of macrophages in vitro interfered with macrophage abilities to respond to subsequent LPS stimulation in a selective manner.
Conclusively, our study demonstrates that Mf lysate stimulation of monocytes from healthy donors in vitro induces a regulatory phenotype, characterized by expression
INPUT: The Mannose Receptor Mediates Dengue Virus Infection of MacrophagesMacrophages (MØ) and mononuclear phagocytes are major targets of infection by dengue virus (DV), a mosquito-borne flavivirus that can cause haemorrhagic fever in humans. To our knowledge, we show for the first time that the MØ mannose receptor (MR) binds to all four serotypes of DV and specifically to the envelope glycoprotein. Glycan analysis, ELISA, and blot overlay assays demonstrate that MR binds via its carbohydrate recognition domains to mosquito and human cell–produced DV antigen. This binding is abrogated by deglycosylation of the DV envelope glycoprotein. Surface expression of recombinant MR on NIH3T3 cells confers DV binding. Furthermore, DV infection of primary human MØ can be blocked by anti-MR antibodies. MR is a prototypic marker of alternatively activated MØ, and pre-treatment of human monocytes or MØ with type 2 cytokines (IL-4 or IL-13) enhances their susceptibility to productive DV infection. Our findings indicate a new functional role for the MR in DV infection.
OUTPUT:
| Dengue disease and its severe manifestations are a growing public health concern, with a third to half the world's population living in dengue-endemic areas. In recent years there have been significant advances in understanding dengue virus (DV) interactions with target cells such as macrophages, dendritic cells, hepatocytes, and endothelial cells. Interaction with and infection of these cells leads to the production of new virions as well as immune mediators, which can shape the course of the subsequent immune response. The vascular leakage associated with dengue haemorrhagic fever is believed to be immune mediated. Our work on the interaction of DV with human macrophages has led to two major findings; first, we have identified that the macrophage mannose receptor is important for mediating the infection of human macrophages by DV, and second, that the type 2 cytokines IL-4 and IL-13 enhance macrophage susceptibility to DV infection. DV–receptor interactions are of critical importance for understanding not only the mechanisms of entry, but also the biology of infection and the pathogenesis. Understanding the immunopathogenesis of dengue disease is crucial to the development of both a safe dengue vaccine and therapeutic inhibitors of early DV replication.
|
PLOS_five_shot_dy9 | ***TASK***
the task is to simplify the input abstract of a biomedical literature
***INPUT***
the input is the abstract of a biomedical literature
***OUTPUT***
the output is the simplified abstract for the input abstract of a biomedical literature
***DOCUMENTATION***
***EXAMPLES***
INPUT: Where’s the Noise? Key Features of Spontaneous Activity and Neural Variability Arise through Learning in a Deterministic NetworkEven in the absence of sensory stimulation the brain is spontaneously active. This background “noise” seems to be the dominant cause of the notoriously high trial-to-trial variability of neural recordings. Recent experimental observations have extended our knowledge of trial-to-trial variability and spontaneous activity in several directions: 1. Trial-to-trial variability systematically decreases following the onset of a sensory stimulus or the start of a motor act. 2. Spontaneous activity states in sensory cortex outline the region of evoked sensory responses. 3. Across development, spontaneous activity aligns itself with typical evoked activity patterns. 4. The spontaneous brain activity prior to the presentation of an ambiguous stimulus predicts how the stimulus will be interpreted. At present it is unclear how these observations relate to each other and how they arise in cortical circuits. Here we demonstrate that all of these phenomena can be accounted for by a deterministic self-organizing recurrent neural network model (SORN), which learns a predictive model of its sensory environment. The SORN comprises recurrently coupled populations of excitatory and inhibitory threshold units and learns via a combination of spike-timing dependent plasticity (STDP) and homeostatic plasticity mechanisms. Similar to balanced network architectures, units in the network show irregular activity and variable responses to inputs. Additionally, however, the SORN exhibits sequence learning abilities matching recent findings from visual cortex and the network’s spontaneous activity reproduces the experimental findings mentioned above. Intriguingly, the network’s behaviour is reminiscent of sampling-based probabilistic inference, suggesting that correlates of sampling-based inference can develop from the interaction of STDP and homeostasis in deterministic networks. We conclude that key observations on spontaneous brain activity and the variability of neural responses can be accounted for by a simple deterministic recurrent neural network which learns a predictive model of its sensory environment via a combination of generic neural plasticity mechanisms.
OUTPUT: Neural recordings seem very noisy. If the exact same stimulus is shown to an animal multiple times, the neural response will vary substantially. In fact, the activity of a single neuron shows many features of a random process. Furthermore, the spontaneous activity occurring in the absence of any sensory stimulus, which is usually considered a kind of background noise, often has a magnitude comparable to the activity evoked by stimulus presentation and interacts with sensory inputs in interesting ways. Here we show that the key features of neural variability and spontaneous activity can all be accounted for by a simple and completely deterministic neural network learning a predictive model of its sensory inputs. The network’s deterministic dynamics give rise to structured but variable responses matching key experimental findings obtained in different mammalian species with different recording techniques. Our results suggest that the notorious variability of neural recordings and the complex features of spontaneous brain activity could reflect the dynamics of a largely deterministic but highly adaptive network learning a predictive model of its sensory environment.
INPUT: Homeostatic Scaling of Excitability in Recurrent Neural NetworksNeurons adjust their intrinsic excitability when experiencing a persistent change in synaptic drive. This process can prevent neural activity from moving into either a quiescent state or a saturated state in the face of ongoing plasticity, and is thought to promote stability of the network in which neurons reside. However, most neurons are embedded in recurrent networks, which require a delicate balance between excitation and inhibition to maintain network stability. This balance could be disrupted when neurons independently adjust their intrinsic excitability. Here, we study the functioning of activity-dependent homeostatic scaling of intrinsic excitability (HSE) in a recurrent neural network. Using both simulations of a recurrent network consisting of excitatory and inhibitory neurons that implement HSE, and a mean-field description of adapting excitatory and inhibitory populations, we show that the stability of such adapting networks critically depends on the relationship between the adaptation time scales of both neuron populations. In a stable adapting network, HSE can keep all neurons functioning within their dynamic range, while the network is undergoing several (patho)physiologically relevant types of plasticity, such as persistent changes in external drive, changes in connection strengths, or the loss of inhibitory cells from the network. However, HSE cannot prevent the unstable network dynamics that result when, due to such plasticity, recurrent excitation in the network becomes too strong compared to feedback inhibition. This suggests that keeping a neural network in a stable and functional state requires the coordination of distinct homeostatic mechanisms that operate not only by adjusting neural excitability, but also by controlling network connectivity.
OUTPUT: The central nervous system is continuously adapting to a wide variety of input signals. Single neurons receive from one to thousands of input signals and need mechanisms to prevent their output activity from locking up in quiescence or saturation. One experimentally observed mechanism is homeostatic scaling of neuronal excitability (HSE), which adapts neuronal responsiveness at the time scale of minutes. Most neurons function in networks of excitatory and inhibitory cells. Maintaining stability of activity in such networks is highly relevant, because deviations can result in pathologies like epilepsy. Can HSE control output activity of single neurons without interfering with network stability? To address this question we implement HSE in a neuronal network model. We show that stable functioning of HSE requires that the adaptation rate of the inhibitory cells is slower than that of the excitatory cells. We subsequently investigate various changes in network organization that demand adaptation by HSE, showing that HSE can successfully control activity levels as long as feedback excitation is not stronger than feedback inhibition. This suggests that maintaining stable, functional networks requires the coordination of distinct homeostatic mechanisms, acting not only through adjustments of single cell responsiveness, but also by controlling network connectivity.
INPUT: Task-Dependent Changes in Cross-Level Coupling between Single Neurons and Oscillatory Activity in Multiscale NetworksUnderstanding the principles governing the dynamic coordination of functional brain networks remains an important unmet goal within neuroscience. How do distributed ensembles of neurons transiently coordinate their activity across a variety of spatial and temporal scales? While a complete mechanistic account of this process remains elusive, evidence suggests that neuronal oscillations may play a key role in this process, with different rhythms influencing both local computation and long-range communication. To investigate this question, we recorded multiple single unit and local field potential (LFP) activity from microelectrode arrays implanted bilaterally in macaque motor areas. Monkeys performed a delayed center-out reach task either manually using their natural arm (Manual Control, MC) or under direct neural control through a brain-machine interface (Brain Control, BC). In accord with prior work, we found that the spiking activity of individual neurons is coupled to multiple aspects of the ongoing motor beta rhythm (10–45 Hz) during both MC and BC, with neurons exhibiting a diversity of coupling preferences. However, here we show that for identified single neurons, this beta-to-rate mapping can change in a reversible and task-dependent way. For example, as beta power increases, a given neuron may increase spiking during MC but decrease spiking during BC, or exhibit a reversible shift in the preferred phase of firing. The within-task stability of coupling, combined with the reversible cross-task changes in coupling, suggest that task-dependent changes in the beta-to-rate mapping play a role in the transient functional reorganization of neural ensembles. We characterize the range of task-dependent changes in the mapping from beta amplitude, phase, and inter-hemispheric phase differences to the spike rates of an ensemble of simultaneously-recorded neurons, and discuss the potential implications that dynamic remapping from oscillatory activity to spike rate and timing may hold for models of computation and communication in distributed functional brain networks.
OUTPUT: How is the functional role of a particular neuron established within an ensemble? The concept of a neural tuning curve – the mapping from input variables such as movement direction to output firing rate – has proven useful in investigating neural function. However, prior work shows that tuning curves are not fixed but may be remapped as a function of task demands – presumably via high-level mechanisms of cognitive control. How is this accomplished? Brain rhythms may play a causal role in this process, but the coupling of single cells to network activity remains poorly understood. We investigated the coupling between rhythmic beta activity and spiking as macaques performed two different tasks. This coupling can be described in terms of a function that maps oscillatory amplitude and phase to instantaneous spike rate. Similarly to direction tuning, this “internal” tuning curve also exhibits task-dependent changes. We characterize these changes across a large ensemble of simultaneously-recorded cells, and consider some of the neuro-computational implications presented by cross-level coupling between single cells and large-scale networks. In particular, relative to the slow time-scale of behavior, the observed beta-to-rate mappings may prove useful for modulating winner-take-all dynamics on intermediate time-scales and relative spike timing on fast time-scales.
INPUT: Neuronal Firing Sensitivity to Morphologic and Active Membrane ParametersBoth the excitability of a neuron's membrane, driven by active ion channels, and dendritic morphology contribute to neuronal firing dynamics, but the relative importance and interactions between these features remain poorly understood. Recent modeling studies have shown that different combinations of active conductances can evoke similar firing patterns, but have neglected how morphology might contribute to homeostasis. Parameterizing the morphology of a cylindrical dendrite, we introduce a novel application of mathematical sensitivity analysis that quantifies how dendritic length, diameter, and surface area influence neuronal firing, and compares these effects directly against those of active parameters. The method was applied to a model of neurons from goldfish Area II. These neurons exhibit, and likely contribute to, persistent activity in eye velocity storage, a simple model of working memory. We introduce sensitivity landscapes, defined by local sensitivity analyses of firing rate and gain to each parameter, performed globally across the parameter space. Principal directions over which sensitivity to all parameters varied most revealed intrinsic currents that most controlled model output. We found domains where different groups of parameters had the highest sensitivities, suggesting that interactions within each group shaped firing behaviors within each specific domain. Application of our method, and its characterization of which models were sensitive to general morphologic features, will lead to advances in understanding how realistic morphology participates in functional homeostasis. Significantly, we can predict which active conductances, and how many of them, will compensate for a given age- or development-related structural change, or will offset a morphologic perturbation resulting from trauma or neurodegenerative disorder, to restore normal function. Our method can be adapted to analyze any computational model. Thus, sensitivity landscapes, and the quantitative predictions they provide, can give new insight into mechanisms of homeostasis in any biological system.
OUTPUT: Homeostasis is a process that allows a system to maintain a certain level of output over a long time, even though the inputs controlling the output are changing. Recently, studies of neurons and neuronal networks have shown that the “active” parameters that describe the movement of ions across the cell membrane contribute to homeostasis, since these parameters can be combined in different ways to maintain a specific output. There is also evidence that the physical shape (“morphology”) of the neuron may play a role in homeostasis, but this possibility has not been explored in computational models. We have developed a method that uses sensitivity analysis to evaluate how different kinds of parameters, like active and morphologic ones, affect model output. Across a multi-dimensional parameter space, we identified both local and global trends in parameter sensitivities that indicate regions where different parameters, even morphologic ones, contribute strongly to homeostasis. Significantly, the authors used sensitivities to predict which parameters should change, and by how much, to compensate for changes in another parameter to restore normal function. These predictions may prove important to neuronal aging, disease, and trauma research, but the method can be used to analyze any computational model.
INPUT: Training Excitatory-Inhibitory Recurrent Neural Networks for Cognitive Tasks: A Simple and Flexible FrameworkThe ability to simultaneously record from large numbers of neurons in behaving animals has ushered in a new era for the study of the neural circuit mechanisms underlying cognitive functions. One promising approach to uncovering the dynamical and computational principles governing population responses is to analyze model recurrent neural networks (RNNs) that have been optimized to perform the same tasks as behaving animals. Because the optimization of network parameters specifies the desired output but not the manner in which to achieve this output, “trained” networks serve as a source of mechanistic hypotheses and a testing ground for data analyses that link neural computation to behavior. Complete access to the activity and connectivity of the circuit, and the ability to manipulate them arbitrarily, make trained networks a convenient proxy for biological circuits and a valuable platform for theoretical investigation. However
INPUT: From homeostasis to behavior: Balanced activity in an exploration of embodied dynamic environmental-neural interactionIn recent years, there have been many computational simulations of spontaneous neural dynamics. Here, we describe a simple model of spontaneous neural dynamics that controls an agent moving in a simple virtual environment. These dynamics generate interesting brain-environment feedback interactions that rapidly destabilize neural and behavioral dynamics demonstrating the need for homeostatic mechanisms. We investigate roles for homeostatic plasticity both locally (local inhibition adjusting to balance excitatory input) as well as more globally (regional “task negative” activity that compensates for “task positive”, sensory input in another region) balancing neural activity and leading to more stable behavior (trajectories through the environment). Our results suggest complementary functional roles for both local and macroscale mechanisms in maintaining neural and behavioral dynamics and a novel functional role for macroscopic “task-negative” patterns of activity (e.g., the default mode network).
OUTPUT:
| In recent years, there has been growing interest in using computational models based on the human structural connectome to better understand the brain. These simulations typically investigate spontaneous neural dynamics, in the absence of tasks, sensory input or motor output. Here, we take a different approach, embodying a computational model of spontaneous neural dynamics to control a simulated agent, with sensory input from and motor output to a simulated environment. Embodying the model radically changes how the model operates and changes how we understand the computational mechanisms. We observe interesting brain-environment feedback interactions and observe how different homeostatic systems are needed to compensate for this feedback. We observe this both in the simulated neural dynamics and the behavior of the embodied agent. These findings suggest novel functional roles for homeostatic systems in maintaining neural dynamics and behavior and for the poorly understood default mode network pattern of activity reported in functional neuroimaging in humans and animals.
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PLOS_five_shot_dy10 | ***TASK***
the task is to simplify the input abstract of a biomedical literature
***INPUT***
the input is the abstract of a biomedical literature
***OUTPUT***
the output is the simplified abstract for the input abstract of a biomedical literature
***DOCUMENTATION***
***EXAMPLES***
INPUT: Satellite DNA Modulates Gene Expression in the Beetle Tribolium castaneum after Heat StressNon-coding repetitive DNAs have been proposed to perform a gene regulatory role, however for tandemly repeated satellite DNA no such role was defined until now. Here we provide the first evidence for a role of satellite DNA in the modulation of gene expression under specific environmental conditions. The major satellite DNA TCAST1 in the beetle Tribolium castaneum is preferentially located within pericentromeric heterochromatin but is also dispersed as single repeats or short arrays in the vicinity of protein-coding genes within euchromatin. Our results show enhanced suppression of activity of TCAST1-associated genes and slower recovery of their activity after long-term heat stress relative to the same genes without associated TCAST1 satellite DNA elements. The level of gene suppression is not influenced by the distance of TCAST1 elements from the associated genes up to 40 kb from the genes’ transcription start sites, but it does depend on the copy number of TCAST1 repeats within an element, being stronger for the higher number of copies. The enhanced gene suppression correlates with the enrichment of the repressive histone marks H3K9me2/3 at dispersed TCAST1 elements and their flanking regions as well as with increased expression of TCAST1 satellite DNA. The results reveal transient, RNAi based heterochromatin formation at dispersed TCAST1 repeats and their proximal regions as a mechanism responsible for enhanced silencing of TCAST1-associated genes. Differences in the pattern of distribution of TCAST1 elements contribute to gene expression diversity among T. castaneum strains after long-term heat stress and might have an impact on adaptation to different environmental conditions.
OUTPUT: Non-coding repetitive DNAs constitute a considerable portion of most eukaryotic genomes and their function is intensively investigated. Here we analyse a gene-regulatory role for tandemly repeated satellite DNA which is the major building element of pericentromeric and centromeric heterochromatin in many eukaryotes. We use as a model system the beetle Tribolium castaneum which has a major satellite DNA preferentially located in pericentromeric heterochromatin but satellite repeats are also dispersed in the vicinity of protein-coding genes within euchromatin. Our results demonstrate for the first time the role of satellite DNA in the modulation of protein-gene expression and reveal the molecular mechanism of their gene-regulatory activity. The influence of satellite DNA on neighbouring genes is epigenetic in nature and is induced by specific changes in the environment such as long-term heat stress. Based on this, the impact of satellite DNAs on adaptation to different environmental conditions as well as their role in the evolution of gene regulatory networks is proposed.
INPUT: Probing instructions for expression regulation in gene nucleotide compositionsGene expression is orchestrated by distinct regulatory regions to ensure a wide variety of cell types and functions. A challenge is to identify which regulatory regions are active, what are their associated features and how they work together in each cell type. Several approaches have tackled this problem by modeling gene expression based on epigenetic marks, with the ultimate goal of identifying driving regions and associated genomic variations that are clinically relevant in particular in precision medicine. However, these models rely on experimental data, which are limited to specific samples (even often to cell lines) and cannot be generated for all regulators and all patients. In addition, we show here that, although these approaches are accurate in predicting gene expression, inference of TF combinations from this type of models is not straightforward. Furthermore these methods are not designed to capture regulation instructions present at the sequence level, before the binding of regulators or the opening of the chromatin. Here, we probe sequence-level instructions for gene expression and develop a method to explain mRNA levels based solely on nucleotide features. Our method positions nucleotide composition as a critical component of gene expression. Moreover, our approach, able to rank regulatory regions according to their contribution, unveils a strong influence of the gene body sequence, in particular introns. We further provide evidence that the contribution of nucleotide content can be linked to co-regulations associated with genome 3D architecture and to associations of genes within topologically associated domains.
OUTPUT: Identifying a maximum of DNA determinants implicated in gene regulation will accelerate genetic analyses and precision medicine approaches by identifying key gene features. In that context decoding the sequence-level instructions for gene regulation is of prime importance. Among global efforts to achieve this objective, we propose a novel approach able to explain gene expression in each patient sample using only DNA features. Our approach, which is as accurate as methods based on epigenetics data, reveals a strong influence of the nucleotide content of gene body sequences, in particular introns. In contrast to canonical regulations mediated by specific DNA motifs, our model unveils a contribution of global nucleotide content notably in co-regulations associated with genome 3D architecture and to associations of genes within topologically associated domains. Overall our study confirms and takes advantage of the existence of sequence-level instructions for gene expression, which lie in genomic regions largely underestimated in regulatory genomics but which appear to be linked to chromatin architecture.
INPUT: Two Distinct Repressive Mechanisms for Histone 3 Lysine 4 Methylation through Promoting 3′-End Antisense TranscriptionHistone H3 di- and trimethylation on lysine 4 are major chromatin marks that correlate with active transcription. The influence of these modifications on transcription itself is, however, poorly understood. We have investigated the roles of H3K4 methylation in Saccharomyces cerevisiae by determining genome-wide expression-profiles of mutants in the Set1 complex, COMPASS, that lays down these marks. Loss of H3K4 trimethylation has virtually no effect on steady-state or dynamically-changing mRNA levels. Combined loss of H3K4 tri- and dimethylation results in steady-state mRNA upregulation and delays in the repression kinetics of specific groups of genes. COMPASS-repressed genes have distinct H3K4 methylation patterns, with enrichment of H3K4me3 at the 3′-end, indicating that repression is coupled to 3′-end antisense transcription. Further analyses reveal that repression is mediated by H3K4me3-dependent 3′-end antisense transcription in two ways. For a small group of genes including PHO84, repression is mediated by a previously reported trans-effect that requires the antisense transcript itself. For the majority of COMPASS-repressed genes, however, it is the process of 3′-end antisense transcription itself that is the important factor for repression. Strand-specific qPCR analyses of various mutants indicate that this more prevalent mechanism of COMPASS-mediated repression requires H3K4me3-dependent 3′-end antisense transcription to lay down H3K4me2, which seems to serve as the actual repressive mark. Removal of the 3′-end antisense promoter also results in derepression of sense transcription and renders sense transcription insensitive to the additional loss of SET1. The derepression observed in COMPASS mutants is mimicked by reduction of global histone H3 and H4 levels, suggesting that the H3K4me2 repressive effect is linked to establishment of a repressive chromatin structure. These results indicate that in S. cerevisiae, the non-redundant role of H3K4 methylation by Set1 is repression, achieved through promotion of 3′-end antisense transcription to achieve specific rather than global effects through two distinct mechanisms.
OUTPUT: In eukaryotes, DNA is packaged together with histones into nucleosomes. This packaging has a repressive role on gene expression. The N-termini of histones are subject to multiple modifications that affect DNA–dependent processes. The histone modification that has been predominantly linked with active transcription in all eukaryotes is histone H3 lysine 4 (H3K4) methylation. Here we investigate the functional effects of each H3K4 methylation state on transcription. Removal of the mark that is most characteristic for transcription, H3K4 trimethylation, has no effect on coding gene expression, in steady-state or dynamically changing conditions. Combined loss of H3K4 tri- and di-methylation does have an effect and leads to loss of repression of specific genes, the opposite of what is expected for global marks of active genes. The affected genes have antisense transcription. We show that there are two separate mechanisms through which H3K4 methylation represses transcription of protein-coding genes, one through antisense transcripts and one through the process of antisense transcription. In summary, we show how a general mark of active transcription can have specific repressive effects that are themselves also linked to repression through nucleosomes.
INPUT: Genome Wide Analysis Reveals Zic3 Interaction with Distal Regulatory Elements of Stage Specific Developmental Genes in ZebrafishZic3 regulates early embryonic patterning in vertebrates. Loss of Zic3 function is known to disrupt gastrulation, left-right patterning, and neurogenesis. However, molecular events downstream of this transcription factor are poorly characterized. Here we use the zebrafish as a model to study the developmental role of Zic3 in vivo, by applying a combination of two powerful genomics approaches – ChIP-seq and microarray. Besides confirming direct regulation of previously implicated Zic3 targets of the Nodal and canonical Wnt pathways, analysis of gastrula stage embryos uncovered a number of novel candidate target genes, among which were members of the non-canonical Wnt pathway and the neural pre-pattern genes. A similar analysis in zic3-expressing cells obtained by FACS at segmentation stage revealed a dramatic shift in Zic3 binding site locations and identified an entirely distinct set of target genes associated with later developmental functions such as neural development. We demonstrate cis-regulation of several of these target genes by Zic3 using in vivo enhancer assay. Analysis of Zic3 binding sites revealed a distribution biased towards distal intergenic regions, indicative of a long distance regulatory mechanism; some of these binding sites are highly conserved during evolution and act as functional enhancers. This demonstrated that Zic3 regulation of developmental genes is achieved predominantly through long distance regulatory mechanism and revealed that developmental transitions could be accompanied by dramatic changes in regulatory landscape.
OUTPUT: The Zic3 transcription factor regulates early embryonic patterning, and the loss of its function leads to defects in left-right body asymmetry. Previous studies have only identified a small number of Zic3 targets, which renders the molecular mechanism underlying its activity insufficiently understood. Utilizing two genomics technologies, next generation sequencing and microarray, we profile the genome-wide binding sites of Zic3 and identified its target genes in the developing zebrafish embryo. Our results show that Zic3 regulates its target genes predominantly through regulatory elements located far from promoters. Among the targets of Zic3 are the Nodal and Wnt pathways known to regulate gastrulation and left-right body asymmetry, as well as neural pre-pattern genes regulating proliferation of neural progenitors. Using enhancer activity assay, we further show that genomic regions bound by Zic3 function as enhancers. Our study provides a genome-wide view of the regulatory landscape of Zic3 and its changes during vertebrate development.
INPUT: Function and Evolution of DNA Methylation in Nasonia vitripennisThe parasitoid wasp Nasonia vitripennis is an emerging genetic model for functional analysis of DNA methylation. Here, we characterize genome-wide methylation at a base-pair resolution, and compare these results to gene expression across five developmental stages and to methylation patterns reported in other insects. An accurate assessment of DNA methylation across the genome is accomplished using bisulfite sequencing of adult females from a highly inbred line. One-third of genes show extensive methylation over the gene body, yet methylated DNA is
INPUT: Three distinct mechanisms of long-distance modulation of gene expression in yeastRecent Hi-C measurements have revealed numerous intra- and inter-chromosomal interactions in various eukaryotic cells. To what extent these interactions regulate gene expression is not clear. This question is particularly intriguing in budding yeast because it has extensive long-distance chromosomal interactions but few cases of gene regulation over-a-distance. Here, we developed a medium-throughput assay to screen for functional long-distance interactions that affect the average expression level of a reporter gene as well as its cell-to-cell variability (noise). We ectopically inserted an insulated MET3 promoter (MET3pr) flanked by ~1kb invariable sequences into thousands of genomic loci, allowing it to make contacts with different parts of the genome, and assayed the MET3pr activity in single cells. Changes of MET3pr activity in this case necessarily involve mechanisms that function over a distance. MET3pr has similar activities at most locations. However, at some locations, they deviate from the norm and exhibit three distinct patterns including low expression / high noise, low expression / low noise, and high expression / low noise. We provided evidence that these three patterns of MET3pr expression are caused by Sir2-mediated silencing, transcriptional interference, and 3D clustering. The clustering also occurs in the native genome and enhances the transcription of endogenous Met4-targeted genes. Overall, our results demonstrate that a small fraction of long-distance chromosomal interactions can affect gene expression in yeast.
OUTPUT:
| Eukaryotic transcription occurs within the nucleus where DNA is packaged into high order chromosome structures. Some long-distance chromosomal interactions play an important role in gene regulation in higher eukaryotic species, such as mouse and human. In budding yeast, gene expression is traditionally thought to be regulated over short distances because the upstream regulatory sequences (URSs) are usually located close to the core promoters. However, recent chromosome conformation capture experiments have detected numerous long-distance chromosomal interactions in the yeast genome. The function of these interactions in gene regulation remains unclear. Here, we developed a new assay to screen for long-distance interactions that affect the activity of a reporter gene. We found three regulatory mechanisms that act from a distance: silencing, transcriptional interference, and 3D clustering, which alter expression level of the reporter gene as well as its cell-to-cell variability. Our results demonstrate that transcription in budding yeast, similar to transcription in higher eukaryotes, can be regulated over long distances. We anticipate our assay can be used as a general platform to screen for functional long-distance chromosomal interactions that affect gene expression.
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PLOS_five_shot_dy11 | ***TASK***
the task is to simplify the input abstract of a biomedical literature
***INPUT***
the input is the abstract of a biomedical literature
***OUTPUT***
the output is the simplified abstract for the input abstract of a biomedical literature
***DOCUMENTATION***
***EXAMPLES***
INPUT: Chromosome Axis Defects Induce a Checkpoint-Mediated Delay and Interchromosomal Effect on Crossing Over during Drosophila MeiosisCrossovers mediate the accurate segregation of homologous chromosomes during meiosis. The widely conserved pch2 gene of Drosophila melanogaster is required for a pachytene checkpoint that delays prophase progression when genes necessary for DSB repair and crossover formation are defective. However, the underlying process that the pachytene checkpoint is monitoring remains unclear. Here we have investigated the relationship between chromosome structure and the pachytene checkpoint and show that disruptions in chromosome axis formation, caused by mutations in axis components or chromosome rearrangements, trigger a pch2-dependent delay. Accordingly, the global increase in crossovers caused by chromosome rearrangements, known as the “interchromosomal effect of crossing over,” is also dependent on pch2. Checkpoint-mediated effects require the histone deacetylase Sir2, revealing a conserved functional connection between PCH2 and Sir2 in monitoring meiotic events from Saccharomyces cerevisiae to a metazoan. These findings suggest a model in which the pachytene checkpoint monitors the structure of chromosome axes and may function to promote an optimal number of crossovers.
OUTPUT: Meiosis is a specialized cell division in which diploid organisms form haploid gametes for sexual reproduction. This is accomplished by a single round of replication followed by two consecutive divisions. At the first meiotic division, the segregation of homologous chromosomes in most organisms is dependent upon genetic recombination, or crossing over. Crossing over must therefore be regulated to ensure that every pair of homologous chromosomes receives at least one reciprocal exchange. Homologous chromosomes that do not receive a crossover frequently undergo missegregation, yielding gametes that do not contain the normal chromosome number, conditions frequently associated in humans with infertility and birth defects. The pch2 gene is widely conserved and in Drosophila melanogaster is required for a meiosis-specific checkpoint that delays progression when crossover formation is defective. However, the underlying process that the checkpoint is monitoring remains unclear. Here we show that defects in axis components and homolog alignment are sufficient to induce checkpoint activity and increase crossing over across the genome. Based on these observations, we hypothesize that the checkpoint may monitor the integrity of chromosome axes and function to promote an optimal number of crossovers during meiosis.
INPUT: Arabidopsis PCH2 Mediates Meiotic Chromosome Remodeling and Maturation of CrossoversMeiotic chromosomes are organized into linear looped chromatin arrays by a protein axis localized along the loop-bases. Programmed remodelling of the axis occurs during prophase I of meiosis. Structured illumination microscopy (SIM) has revealed dynamic changes in the chromosome axis in Arabidopsis thaliana and Brassica oleracea. We show that the axis associated protein ASY1 is depleted during zygotene concomitant with synaptonemal complex (SC) formation. Study of an Atpch2 mutant demonstrates this requires the conserved AAA+ ATPase, PCH2, which localizes to the sites of axis remodelling. Loss of PCH2 leads to a failure to deplete ASY1 from the axes and compromizes SC polymerisation. Immunolocalization of recombination proteins in Atpch2 indicates that recombination initiation and CO designation during early prophase I occur normally. Evidence suggests that CO interference is initially functional in the mutant but there is a defect in CO maturation following designation. This leads to a reduction in COs and a failure to form COs between some homologous chromosome pairs leading to univalent chromosomes at metaphase I. Genetic analysis reveals that CO distribution is also affected in some chromosome regions. Together these data indicate that the axis remodelling defect in Atpch2 disrupts normal patterned formation of COs.
OUTPUT: In the reproductive cells of many eukaryotes, a process called meiosis generates haploid gametes. During meiosis, homologous parental chromosomes (homologs) recombine forming crossovers (CO) that provide genetic variation. CO formation generates physical links called chiasmata, which are essential for accurate homolog segregation. CO control designates a sub-set of recombination precursors that will mature to form at least one chiasma between each homolog pair. Recombination is accompanied by extensive chromosome reorganization. Formation of a proteinaceous axis organizes the pairs of sister chromatids of each homolog into conjoined linear looped chromatin arrays. Pairs of homologs then align and synapse becoming closely associated along their length by a protein structure, the synaptonemal complex (SC). The SC is disassembled at the end of prophase I and recombination is completed. We have investigated the link between recombination and chromosome remodelling by analysing the role of a protein, PCH2, which we show is required for remodelling of the chromosome axis during SC formation. In wild type, immunolocalization reveals depletion of the axis-associated signal of the axis component, ASY1, along synapsed regions of the chromosomes. In the absence of PCH2, the ASY1 signal is not depleted from the chromosome axis and the SC does not form normally. Although this defect in chromosome remodelling has no obvious effect on CO designation, CO maturation is perturbed such that the formation of at least one CO per homolog pair no longer occurs.
INPUT: Multiple Opposing Constraints Govern Chromosome Interactions during MeiosisHomolog pairing and crossing over during meiosis I prophase is required for accurate chromosome segregation to form euploid gametes. The repair of Spo11-induced double-strand breaks (DSB) using a homologous chromosome template is a major driver of pairing in many species, including fungi, plants, and mammals. Inappropriate pairing and crossing over at ectopic loci can lead to chromosome rearrangements and aneuploidy. How (or if) inappropriate ectopic interactions are disrupted in favor of allelic interactions is not clear. Here we used an in vivo “collision” assay in budding yeast to test the contributions of cohesion and the organization and motion of chromosomes in the nucleus on promoting or antagonizing interactions between allelic and ectopic loci at interstitial chromosome sites. We found that deletion of the cohesin subunit Rec8, but not other chromosome axis proteins (e.g. Red1, Hop1, or Mek1), caused an increase in homolog-nonspecific chromosome interaction, even in the absence of Spo11. This effect was partially suppressed by expression of the mitotic cohesin paralog Scc1/Mdc1, implicating Rec8's role in cohesion rather than axis integrity in preventing nonspecific chromosome interactions. Disruption of telomere-led motion by treating cells with the actin polymerization inhibitor Latrunculin B (Lat B) elevated nonspecific collisions in rec8Δ spo11Δ. Next, using a visual homolog-pairing assay, we found that the delay in homolog pairing in mutants defective for telomere-led chromosome motion (ndj1Δ or csm4Δ) is enhanced in Lat B–treated cells, implicating actin in more than one process promoting homolog juxtaposition. We suggest that multiple, independent contributions of actin, cohesin, and telomere function are integrated to promote stable homolog-specific interactions and to destabilize weak nonspecific interactions by modulating the elastic spring-like properties of chromosomes.
OUTPUT: Meiosis is the key stage of gametogenesis, when the diploid genome complement is reduced by one half to form haploid gametes for sexual reproduction. Accurate chromosome segregation requires that homologous chromosomes pair, recombine by crossing over, and segregate from one another during the first meiotic division. Missegregation of homologs leads to the formation of aneuploid gametes, while erroneous crossing over between ectopic chromosomal loci can lead to chromosomal rearrangements such as translocations and deletions. We found that nonspecific interactions between interstitial chromosomal sites can be enabled or prevented through multiple, independent mechanisms during meiosis in budding yeast. These include organization of chromosomes in the nucleus, integrity of the chromosome axis structure, and actin-led chromosome movement. Acting together, these processes can reinforce strong chromosome interactions that promote pairing, while acting in opposition they can eliminate weak nonspecific interactions. These data provide an integrated view of how homologous chromosome pairing is achieved.
INPUT: The pch2Δ Mutation in Baker's Yeast Alters Meiotic Crossover Levels and Confers a Defect in Crossover InterferencePch2 is a widely conserved protein that is required in baker's yeast for the organization of meiotic chromosome axes into specific domains. We provide four lines of evidence suggesting that it regulates the formation and distribution of crossover events required to promote chromosome segregation at Meiosis I. First, pch2Δ mutants display wild-type crossover levels on a small (III) chromosome, but increased levels on larger (VII, VIII, XV) chromosomes. Second, pch2Δ mutants show defects in crossover interference. Third, crossovers observed in pch2Δ require both Msh4-Msh5 and Mms4-Mus81 functions. Lastly, the pch2Δ mutation decreases spore viability and disrupts crossover interference in spo11 hypomorph strains that have reduced levels of meiosis-induced double-strand breaks. Based on these and previous observations, we propose a model in which Pch2 functions at an early step in crossover control to ensure that every homolog pair receives an obligate crossover.
OUTPUT: During meiosis, cells that ultimately become gametes (such as eggs or sperm) undergo a single round of DNA replication followed by two consecutive divisions. In most organisms, the segregation of chromosomes at the first meiotic division is dependent upon genetic exchange, or crossing over, at homologous sites along chromosomes. Crossing over must therefore be regulated to ensure that every pair of matched chromosomes receives at least one crossover. Matched chromosomes that do not receive a crossover frequently undergo missegregation at the first meiotic division, yielding gametes that do not contain the normal chromosome number. Such missegregation events have been linked to human infertility syndromes. We used a genetic approach to study meiotic crossover control in baker's yeast. Our work suggests that Pch2 is required in crossover control during meiosis; mutants lacking Pch2 display altered crossover levels and distribution. Furthermore, pch2 mutations cause enhanced gamete inviability in strains that are mildly defective in initiating recombination. Based on these observations, we hypothesize that Pch2 acts early in crossover control, in steps that occur prior to those proposed for previously characterized crossover-promoting factors.
INPUT: Interplay between Synaptonemal Complex, Homologous Recombination, and Centromeres during Mammalian MeiosisThe intimate synapsis of homologous chromosome pairs (homologs) by synaptonemal complexes (SCs) is an essential feature of meios
INPUT: Pch2 Links Chromosome Axis Remodeling at Future Crossover Sites and Crossover Distribution during Yeast MeiosisSegregation of homologous chromosomes during meiosis I depends on appropriately positioned crossovers/chiasmata. Crossover assurance ensures at least one crossover per homolog pair, while interference reduces double crossovers. Here, we have investigated the interplay between chromosome axis morphogenesis and non-random crossover placement. We demonstrate that chromosome axes are structurally modified at future crossover sites as indicated by correspondence between crossover designation marker Zip3 and domains enriched for axis ensemble Hop1/Red1. This association is first detected at the zygotene stage, persists until double Holliday junction resolution, and is controlled by the conserved AAA+ ATPase Pch2. Pch2 further mediates crossover interference, although it is dispensable for crossover formation at normal levels. Thus, interference appears to be superimposed on underlying mechanisms of crossover formation. When recombination-initiating DSBs are reduced, Pch2 is also required for viable spore formation, consistent with further functions in chiasma formation. pch2Δ mutant defects in crossover interference and spore viability at reduced DSB levels are oppositely modulated by temperature, suggesting contributions of two separable pathways to crossover control. Roles of Pch2 in controlling both chromosome axis morphogenesis and crossover placement suggest linkage between these processes. Pch2 is proposed to reorganize chromosome axes into a tiling array of long-range crossover control modules, resulting in chiasma formation at minimum levels and with maximum spacing.
OUTPUT:
| In the germ line of sexually reproducing organisms, haploid gametes are generated from diploid precursor cells by a specialized cell division called meiosis. Reduction by half of chromosome numbers during the first meiotic division depends on genetic exchange, resulting in the formation of crossovers. Without crossovers, pairs of homologous chromosomes frequently fail to separate, resulting in unbalanced gametes with a surplus or deficit of individual chromosomes. Along a given chromosome, crossovers form in different locations in different cells, but distribution of crossovers within each cell is controlled in two ways: first, at least one crossover is formed along each homolog pair, irrespective of size; second, a crossover in a given interval reduces the frequency of crossovers in adjacent chromosome regions. Here, we identify functions of the evolutionarily conserved protein Pch2 in suppressing additional crossovers in adjacent regions and ensuring homolog segregation under certain conditions. Pch2 further controls the assembly of chromosome axis protein Hop1 at future crossover sites. Our findings reveal that chromosome axes undergo structural changes at the same positions where crossovers occur. Thus, axis remodeling and crossover placement are linked via Pch2.
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PLOS_five_shot_dy12 | ***TASK***
the task is to simplify the input abstract of a biomedical literature
***INPUT***
the input is the abstract of a biomedical literature
***OUTPUT***
the output is the simplified abstract for the input abstract of a biomedical literature
***DOCUMENTATION***
***EXAMPLES***
INPUT: Type III Effector Activation via Nucleotide Binding, Phosphorylation, and Host Target InteractionThe Pseudomonas syringae type III effector protein avirulence protein B (AvrB) is delivered into plant cells, where it targets the Arabidopsis RIN4 protein (resistance to Pseudomonas maculicula protein 1 [RPM1]–interacting protein). RIN4 is a regulator of basal host defense responses. Targeting of RIN4 by AvrB is recognized by the host RPM1 nucleotide-binding leucine-rich repeat disease resistance protein, leading to accelerated defense responses, cessation of pathogen growth, and hypersensitive host cell death at the infection site. We determined the structure of AvrB complexed with an AvrB-binding fragment of RIN4 at 2.3 Å resolution. We also determined the structure of AvrB in complex with adenosine diphosphate bound in a binding pocket adjacent to the RIN4 binding domain. AvrB residues important for RIN4 interaction are required for full RPM1 activation. AvrB residues that contact adenosine diphosphate are also required for initiation of RPM1 function. Nucleotide-binding residues of AvrB are also required for its phosphorylation by an unknown Arabidopsis protein(s). We conclude that AvrB is activated inside the host cell by nucleotide binding and subsequent phosphorylation and, independently, interacts with RIN4. Our data suggest that activated AvrB, bound to RIN4, is indirectly recognized by RPM1 to initiate plant immune system function.
OUTPUT: Many bacterial pathogens use a specialized protein “injection needle” called a type III secretion system to help colonize cells of higher organisms. The type III secretion needle attaches to a host cell and is the delivery conduit for a variety of bacterial proteins that act inside of the host cell. These proteins are called type III effectors. They manipulate host cell biology in order to help the bacterial pathogen colonize the host. We studied one type III effector from plant pathogenic bacteria called Pseudomonas syringae. This effector, termed avirulence protein B (AvrB), is targeted to the inner face of the plant cell plasma membrane, where it interacts with a membrane-bound host protein called RIN4 (resistance to Pseudomonas maculicula protein–interacting protein). RIN4 is phosphorylated in the presence of AvrB and an as-yet-unknown additional host factor. We provide a structural basis for the binding of AvrB to RIN4 and a possible mechanism of action for AvrB inside the host. AvrB activation and its ability to bind RIN4 have evolved to help the pathogen, yet in Arabidopsis, the AvrB-dependent phosphorylation of RIN4 is sensed by the plant immune system, leading to a rapid halt in pathogen growth.
INPUT: The Secreted Antifungal Protein Thionin 2.4 in Arabidopsis thaliana Suppresses the Toxicity of a Fungal Fruit Body Lectin from Fusarium graminearumPlants possess active defense systems and can protect themselves from pathogenic invasion by secretion of a variety of small antimicrobial or antifungal proteins such as thionins. The antibacterial and antifungal properties of thionins are derived from their ability to induce open pore formation on cell membranes of phytopathogens, resulting in release of potassium and calcium ions from the cell. Wheat thionin also accumulates in the cell walls of Fusarium-inoculated plants, suggesting that it may have a role in blocking pathogen infection at the plant cell walls. Here we developed an anti-thionin 2.4 (Thi2.4) antibody and used it to show that Thi2.4 is localized in the cell walls of Arabidopsis and cell membranes of F. graminearum, when flowers are inoculated with F. graminearum. The Thi2.4 protein had an antifungal effect on F. graminearum. Next, we purified the Thi2.4 protein, conjugated it with glutathione-S-transferase (GST) and coupled the proteins to an NHS-activated column. Total protein from F. graminearum was applied to GST-Thi2.4 or Thi2.4-binding columns, and the fungal fruit body lectin (FFBL) of F. graminearum was identified as a Thi2.4-interacting protein. This interaction was confirmed by a yeast two-hybrid analysis. To investigate the biological function of FFBL, we infiltrated the lectin into Arabidopsis leaves and observed that it induced cell death in the leaves. Application of FFBL at the same time as inoculation with F. graminearum significantly enhanced the virulence of the pathogen. By contrast, FFBL-induced host cell death was effectively suppressed in transgenic plants that overexpressed Thi2.4. We found that a 15 kD Thi2.4 protein was specifically expressed in flowers and flower buds and suggest that it acts not only as an antifungal peptide, but also as a suppressor of the FFBL toxicity. Secreted thionin proteins are involved in this dual defense mechanism against pathogen invasion at the plant-pathogen interface.
OUTPUT: Host-pathogen interactions involve a multiplicity of mechanisms that coevolved for successful host resistance to pathogenic invasion or for overcoming host defenses by the pathogen. In our study, we focused on antifungal peptides called thionins that plants use for defense against a broad range of phytopathogens. Recently, a wheat thionin was shown to preferentially accumulate in plant cell walls, suggesting that it might have a novel function there during plant-pathogen interactions. We investigated this possible interaction in the model plant species Arabidopsis thaliana and found that the plant thionin 2.4 (Thi2.4) protein interacted with a secreted protein from the fungal species Fusarium graminearum named the fungal fruiting body lectin (FFBL). FFBL causes cell death in Arabidopsis leaves; however, its effect is largely prevented in Arabidopsis plants overexpressing the Thi2.4 protein, i.e., Thi2.4 can act as an effective trap against FFBL. We also found that inoculating flower buds with F. graminearum and FFBL reduces accumulation of Thi2.4 and that disease symptoms develop in the flower buds 2 days after inoculation. Thus, molecular competition between the two secretory proteins, host Thi2.4 and pathogen FFBL, in extracellular spaces is likely to determine whether or not host plants can prevent invasion by F. graminearum.
INPUT: Norovirus Regulation of the Innate Immune Response and Apoptosis Occurs via the Product of the Alternative Open Reading Frame 4Small RNA viruses have evolved many mechanisms to increase the capacity of their short genomes. Here we describe the identification and characterization of a novel open reading frame (ORF4) encoded by the murine norovirus (MNV) subgenomic RNA, in an alternative reading frame overlapping the VP1 coding region. ORF4 is translated during virus infection and the resultant protein localizes predominantly to the mitochondria. Using reverse genetics we demonstrated that expression of ORF4 is not required for virus replication in tissue culture but its loss results in a fitness cost since viruses lacking the ability to express ORF4 restore expression upon repeated passage in tissue culture. Functional analysis indicated that the protein produced from ORF4 antagonizes the innate immune response to infection by delaying the upregulation of a number of cellular genes activated by the innate pathway, including IFN-Beta. Apoptosis in the RAW264.7 macrophage cell line was also increased during virus infection in the absence of ORF4 expression. In vivo analysis of the WT and mutant virus lacking the ability to express ORF4 demonstrated an important role for ORF4 expression in infection and virulence. STAT1-/- mice infected with a virus lacking the ability to express ORF4 showed a delay in the onset of clinical signs when compared to mice infected with WT virus. Quantitative PCR and histopathological analysis of samples from these infected mice demonstrated that infection with a virus not expressing ORF4 results in a delayed infection in this system. In light of these findings we propose the name virulence factor 1, VF1 for this protein. The identification of VF1 represents the first characterization of an alternative open reading frame protein for the calicivirus family. The immune regulatory function of the MNV VF1 protein provide important perspectives for future research into norovirus biology and pathogenesis.
OUTPUT: This report describes the identification and characterization of a novel protein of unknown function encoded by a mouse virus genetically similar to human noroviruses. This gene is unique to the mouse virus and occupies the same part of the genome that codes for the major capsid protein. The protein that we have described as virulence factor 1 (VF1) is found in all murine norovirus isolates, absent in all human strains but is indeed expressed during infection. Its expression enables MNV-1 to establish efficient infection of its natural host through interference with interferon-mediated response pathways and apoptosis. Our data would indicate that the VF1 protein is multi-functional with an ability to modulate the host's response to infection. Murine noroviruses are frequently used firstly as a model to study human norovirus replication and pathogenesis, studies hampered by their inability to replicate in cell culture. Secondly, persistent infection of laboratory animals with murine norovirus may affect other models of disease using experimental mice. The role of VF1 in infection and pathology in the differential outcome of infection is the source of continued research in our laboratory.
INPUT: The Machinery at Endoplasmic Reticulum-Plasma Membrane Contact Sites Contributes to Spatial Regulation of Multiple Legionella Effector ProteinsThe Dot/Icm system of the intracellular pathogen Legionella pneumophila has the capacity to deliver over 270 effector proteins into host cells during infection. Important questions remain as to spatial and temporal mechanisms used to regulate such a large array of virulence determinants after they have been delivered into host cells. Here we investigated several L. pneumophila effector proteins that contain a conserved phosphatidylinositol-4-phosphate (PI4P)-binding domain first described in the effector DrrA (SidM). This PI4P binding domain was essential for the localization of effectors to the early L. pneumophila-containing vacuole (LCV), and DrrA-mediated recruitment of Rab1 to the LCV required PI4P-binding activity. It was found that the host cell machinery that regulates sites of contact between the plasma membrane (PM) and the endoplasmic reticulum (ER) modulates PI4P dynamics on the LCV to control localization of these effectors. Specifically, phosphatidylinositol-4-kinase IIIα (PI4KIIIα) was important for generating a PI4P signature that enabled L. pneumophila effectors to localize to the PM-derived vacuole, and the ER-associated phosphatase Sac1 was involved in metabolizing the PI4P on the vacuole to promote the dissociation of effectors. A defect in L. pneumophila replication in macrophages deficient in PI4KIIIα was observed, highlighting that a PM-derived PI4P signature is critical for biogenesis of a vacuole that supports intracellular multiplication of L. pneumophila. These data indicate that PI4P metabolism by enzymes controlling PM-ER contact sites regulate the association of L. pneumophila effectors to coordinate early stages of vacuole biogenesis.
OUTPUT: The intracellular pathogen Legionella pneumophila encodes at least 270 effectors that modulate trafficking of the pathogen-occupied vacuole
INPUT: RIN4 Functions with Plasma Membrane H+-ATPases to Regulate Stomatal Apertures during Pathogen AttackPathogen perception by the plant innate immune system is of central importance to plant survival and productivity. The Arabidopsis protein RIN4 is a negative regulator of plant immunity. In order to identify additional proteins involved in RIN4-mediated immune signal transduction, we purified components of the RIN4 protein complex. We identified six novel proteins that had not previously been implicated in RIN4 signaling, including the plasma membrane (PM) H+-ATPases AHA1 and/or AHA2. RIN4 interacts with AHA1 and AHA2 both in vitro and in vivo. RIN4 overexpression and knockout lines exhibit differential PM H+-ATPase activity. PM H+-ATPase activation induces stomatal opening, enabling bacteria to gain entry into the plant leaf; inactivation induces stomatal closure thus restricting bacterial invasion. The rin4 knockout line exhibited reduced PM H+-ATPase activity and, importantly, its stomata could not be re-opened by virulent Pseudomonas syringae. We also demonstrate that RIN4 is expressed in guard cells, highlighting the importance of this cell type in innate immunity. These results indicate that the Arabidopsis protein RIN4 functions with the PM H+-ATPase to regulate stomatal apertures, inhibiting the entry of bacterial pathogens into the plant leaf during infection.
OUTPUT:
| Plants are continuously exposed to microorganisms. In order to resist infection, plants rely on their innate immune system to inhibit both pathogen entry and multiplication. We investigated the function of the Arabidopsis protein RIN4, which acts as a negative regulator of plant innate immunity. We biochemically identified six novel RIN4-associated proteins and characterized the association between RIN4 and the plasma membrane H+-ATPase pump. Our results indicate that RIN4 functions in concert with this pump to regulate leaf stomata during the innate immune response, when stomata close to block the entry of bacterial pathogens into the leaf interior.
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