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[ { "docid": "1049501", "text": "Neutrophil extracellular traps (NETs) are implicated in autoimmunity, but how they are generated and their roles in sterile inflammation remain unclear. Ribonucleoprotein immune complexes (RNP ICs), inducers of NETosis, require mitochondrial reactive oxygen species (ROS) for maximal NET stimulation. After RNP IC stimulation of neutrophils, mitochondria become hypopolarized and translocate to the cell surface. Extracellular release of oxidized mitochondrial DNA is proinflammatory in vitro, and when this DNA is injected into mice, it stimulates type I interferon (IFN) signaling through a pathway dependent on the DNA sensor STING. Mitochondrial ROS are also necessary for spontaneous NETosis of low-density granulocytes from individuals with systemic lupus erythematosus. This was also observed in individuals with chronic granulomatous disease, who lack NADPH oxidase activity but still develop autoimmunity and type I IFN signatures. Mitochondrial ROS inhibition in vivo reduces disease severity and type I IFN responses in a mouse model of lupus. Together, these findings highlight a role for mitochondria in the generation not only of NETs but also of pro-inflammatory oxidized mitochondrial DNA in autoimmune diseases.", "title": "Neutrophil extracellular traps enriched in oxidized mitochondrial DNA are interferogenic and contribute to lupus-like disease" } ]

[ { "docid": "43054703", "text": "Neutrophil extracellular traps (NETs) are webs of DNA covered with antimicrobial molecules that constitute a newly described killing mechanism in innate immune defense. Previous publications reported that NETs take up to 3-4 h to form via an oxidant-dependent event that requires lytic death of neutrophils. In this study, we describe neutrophils responding uniquely to Staphylococcus aureus via a novel process of NET formation that did not require neutrophil lysis or even breach of the plasma membrane. The multilobular nucleus rapidly became rounded and condensed. During this process, we observed the separation of the inner and outer nuclear membranes and budding of vesicles, and the separated membranes and vesicles were filled with nuclear DNA. The vesicles were extruded intact into the extracellular space where they ruptured, and the chromatin was released. This entire process occurred via a unique, very rapid (5-60 min), oxidant-independent mechanism. Mitochondrial DNA constituted very little if any of these NETs. They did have a limited amount of proteolytic activity and were able to kill S. aureus. With time, the nuclear envelope ruptured, and DNA filled the cytoplasm presumably for later lytic NET production, but this was distinct from the vesicular release mechanism. Panton-Valentine leukocidin, autolysin, and a lipase were identified in supernatants with NET-inducing activity, but Panton-Valentine leukocidin was the dominant NET inducer. We describe a new mechanism of NET release that is very rapid and contributes to trapping and killing of S. aureus.", "title": "A novel mechanism of rapid nuclear neutrophil extracellular trap formation in response to Staphylococcus aureus." }, { "docid": "14853989", "text": "Autoantibodies to DNA and histones (chromatin) are the defining antigen specificity in systemic lupus erythematosus (SLE) and related musculoskeletal disorders but the mechanisms responsible for their induction remain mysterious. That situation rapidly changed once neutrophil extracellular chromatin traps (NETs) were discovered and observed to play a conserved role in innate immune responses to a broad variety of microbial pathogens. At the center of an infectious process, neutrophils exert various antimicrobial defenses, including the release of nuclear chromatin into the extracellular space. The externalized NETs, a complex meshwork of nuclear chromatin and antimicrobial proteins, serve to immobilize and degrade microbial pathogens. Here, we critically evaluate the evidence supporting NETs versus apoptotic bodies as a source for nuclear antigens in autoimmunity. We also discuss the possibility that NET chromatin forms an essential component of immune deposits in the pathogenesis of glomerulonephritis in SLE and other autoimmune immune complex diseases.", "title": "Neutrophil extracellular chromatin traps connect innate immune response to autoimmunity" }, { "docid": "42873134", "text": "Type 1 and type 2 diabetes are characterized by progressive beta-cell failure. Apoptosis is probably the main form of beta-cell death in both forms of the disease. It has been suggested that the mechanisms leading to nutrient- and cytokine-induced beta-cell death in type 2 and type 1 diabetes, respectively, share the activation of a final common pathway involving interleukin (IL)-1beta, nuclear factor (NF)-kappaB, and Fas. We review herein the similarities and differences between the mechanisms of beta-cell death in type 1 and type 2 diabetes. In the insulitis lesion in type 1 diabetes, invading immune cells produce cytokines, such as IL-1beta, tumor necrosis factor (TNF)-alpha, and interferon (IFN)-gamma. IL-1beta and/or TNF-alpha plus IFN-gamma induce beta-cell apoptosis via the activation of beta-cell gene networks under the control of the transcription factors NF-kappaB and STAT-1. NF-kappaB activation leads to production of nitric oxide (NO) and chemokines and depletion of endoplasmic reticulum (ER) calcium. The execution of beta-cell death occurs through activation of mitogen-activated protein kinases, via triggering of ER stress and by the release of mitochondrial death signals. Chronic exposure to elevated levels of glucose and free fatty acids (FFAs) causes beta-cell dysfunction and may induce beta-cell apoptosis in type 2 diabetes. Exposure to high glucose has dual effects, triggering initially \"glucose hypersensitization\" and later apoptosis, via different mechanisms. High glucose, however, does not induce or activate IL-1beta, NF-kappaB, or inducible nitric oxide synthase in rat or human beta-cells in vitro or in vivo in Psammomys obesus. FFAs may cause beta-cell apoptosis via ER stress, which is NF-kappaB and NO independent. Thus, cytokines and nutrients trigger beta-cell death by fundamentally different mechanisms, namely an NF-kappaB-dependent mechanism that culminates in caspase-3 activation for cytokines and an NF-kappaB-independent mechanism for nutrients. This argues against a unifying hypothesis for the mechanisms of beta-cell death in type 1 and type 2 diabetes and suggests that different approaches will be required to prevent beta-cell death in type 1 and type 2 diabetes.", "title": "Mechanisms of pancreatic beta-cell death in type 1 and type 2 diabetes: many differences, few similarities." }, { "docid": "9878167", "text": "Neutrophil extracellular traps (NETs) represent extracellular structures able to bind and kill microorganisms. It is believed that they are generated by neutrophils undergoing cell death, allowing these dying or dead cells to kill microbes. We show that, following priming with granulocyte/macrophage colony-stimulating factor (GM-CSF) and subsequent short-term toll-like receptor 4 (TLR4) or complement factor 5a (C5a) receptor stimulation, viable neutrophils are able to generate NETs. Strikingly, NETs formed by living cells contain mitochondrial, but no nuclear, DNA. Pharmacological or genetic approaches to block reactive oxygen species (ROS) production suggested that NET formation is ROS dependent. Moreover, neutrophil populations stimulated with GM-CSF and C5a showed increased survival compared with resting neutrophils, which did not generate NETs. In conclusion, mitochondrial DNA release by neutrophils and NET formation do not require neutrophil death and do also not limit the lifespan of these cells.", "title": "Viable neutrophils release mitochondrial DNA to form neutrophil extracellular traps" }, { "docid": "28015516", "text": "Systemic lupus erythematosus (SLE) is a systemic autoimmune disease characterized by a breakdown of tolerance to nuclear antigens and the development of immune complexes. Genomic approaches have shown that human SLE leukocytes homogeneously express type I interferon (IFN)-induced and neutrophil-related transcripts. Increased production and/or bioavailability of IFN-α and associated alterations in dendritic cell (DC) homeostasis have been linked to lupus pathogenesis. Although neutrophils have long been shown to be associated with lupus, their potential role in disease pathogenesis remains elusive. Here, we show that mature SLE neutrophils are primed in vivo by type I IFN and die upon exposure to SLE-derived anti-ribonucleoprotein antibodies, releasing neutrophil extracellular traps (NETs). SLE NETs contain DNA as well as large amounts of LL37 and HMGB1, neutrophil proteins that facilitate the uptake and recognition of mammalian DNA by plasmacytoid DCs (pDCs). Indeed, SLE NETs activate pDCs to produce high levels of IFN-α in a DNA- and TLR9 (Toll-like receptor 9)-dependent manner. Our results reveal an unsuspected role for neutrophils in SLE pathogenesis and identify a novel link between nucleic acid-recognizing antibodies and type I IFN production in this disease.", "title": "Netting neutrophils are major inducers of type I IFN production in pediatric systemic lupus erythematosus." }, { "docid": "1605392", "text": "Antigen stimulation of immune cells triggers Ca2+ entry through Ca2+ release-activated Ca2+ (CRAC) channels, promoting the immune response to pathogens by activating the transcription factor NFAT. We have previously shown that cells from patients with one form of hereditary severe combined immune deficiency (SCID) syndrome are defective in store-operated Ca2+ entry and CRAC channel function. Here we identify the genetic defect in these patients, using a combination of two unbiased genome-wide approaches: a modified linkage analysis with single-nucleotide polymorphism arrays, and a Drosophila RNA interference screen designed to identify regulators of store-operated Ca2+ entry and NFAT nuclear import. Both approaches converged on a novel protein that we call Orai1, which contains four putative transmembrane segments. The SCID patients are homozygous for a single missense mutation in ORAI1, and expression of wild-type Orai1 in SCID T cells restores store-operated Ca2+ influx and the CRAC current (ICRAC). We propose that Orai1 is an essential component or regulator of the CRAC channel complex.", "title": "A mutation in Orai1 causes immune deficiency by abrogating CRAC channel function" }, { "docid": "20459964", "text": "Neutrophil is a key cell in pathophysiology of granulomatosis with polyangiitis. Recently, neutrophil extracellular traps were described in this disease. Mitochondrial DNA is also released during traps formation. We measured circulating cell-free mitochondrial and genomic DNA in serum of patients with granulomatosis with polyangiitis. Subjects with the disease (14 active and 11 in remission stage) and 10 healthy controls were enrolled. Quantitative real-time polymerase chain reaction (PCR) was used to measure 79 base pairs (bp) and 230 bp mtDNA fragments. Alu repeats were quantified to evaluate abundance of nuclear DNA in serum at the presence of plasmid control. Both fragments of mtDNA (79 bp and 230 bp) and genomic DNA were elevated significantly in granulomatosis with polyangiitis compared to controls. Only the shorter 79 bp mtDNA correlated with active stage of granulomatosis with polyangiitis and clinical symptoms. A mechanism of extracellular release of mitochondrial DNA accompanies the active stage of the disease. Circulating mtDNA is extremely high in untreated patients. This suggests that biomarker properties of mtDNA are useful for monitoring of treatment.", "title": "Circulating mitochondrial DNA in serum of patients with granulomatosis with polyangiitis." }, { "docid": "29399239", "text": "Neutrophil extracellular traps (NETs) are made of processed chromatin bound to granular and selected cytoplasmic proteins. NETs are released by white blood cells called neutrophils, maybe as a last resort, to control microbial infections. This release of chromatin is the result of a unique form of cell death, dubbed \"NETosis. \" Here we review our understanding of how NETs are made, their function in infections and as danger signals, and their emerging importance in autoimmunity and coagulation.", "title": "Neutrophil extracellular traps: Is immunity the second function of chromatin?" }, { "docid": "17741440", "text": "Small-vessel vasculitis (SVV) is a chronic autoinflammatory condition linked to antineutrophil cytoplasm autoantibodies (ANCAs). Here we show that chromatin fibers, so-called neutrophil extracellular traps (NETs), are released by ANCA-stimulated neutrophils and contain the targeted autoantigens proteinase-3 (PR3) and myeloperoxidase (MPO). Deposition of NETs in inflamed kidneys and circulating MPO-DNA complexes suggest that NET formation triggers vasculitis and promotes the autoimmune response against neutrophil components in individuals with SVV.", "title": "Netting neutrophils in autoimmune small-vessel vasculitis" }, { "docid": "17231273", "text": "Energy deficiency and dysfunction of the Na+, K+-ATPase are common consequences of many pathological insults. The nature and mechanism of cell injury induced by impaired Na+, K+-ATPase, however, are not well defined. We used cultured cortical neurons to examine the hypothesis that blocking the Na+, K+-ATPase induces apoptosis by depleting cellular K+ and, concurrently, induces necrotic injury in the same cells by increasing intracellular Ca2+ and Na+. The Na+, K+-ATPase inhibitor ouabain induced concentration-dependent neuronal death. Ouabain triggered transient neuronal cell swelling followed by cell shrinkage, accompanied by intracellular Ca2+ and Na+ increase, K+ decrease, cytochrome c release, caspase-3 activation, and DNA laddering. Electron microscopy revealed the coexistence of ultrastructural features of both apoptosis and necrosis in individual cells. The caspase inhibitor Z-Val-Ala-Asp(OMe)-fluoromethyl ketone (Z-VAD-FMK) blocked >50% of ouabain-induced neuronal death. Potassium channel blockers or high K+ medium, but not Ca2+ channel blockade, prevented cytochrome c release, caspase activation, and DNA damage. Blocking of K+, Ca2+, or Na+ channels or high K+ medium each attenuated the ouabain-induced cell death; combined inhibition of K+ channels and Ca2+ or Na+ channels resulted in additional protection. Moreover, coapplication of Z-VAD-FMK and nifedipine produced virtually complete neuroprotection. These results suggest that the neuronal death associated with Na+, K+-pump failure consists of concurrent apoptotic and necrotic components, mediated by intracellular depletion of K+ and accumulation of Ca2+ and Na+, respectively. The ouabain-induced hybrid death may represent a distinct form of cell death related to the brain injury of inadequate energy supply and disrupted ion homeostasis.", "title": "Ionic mechanism of ouabain-induced concurrent apoptosis and necrosis in individual cultured cortical neurons" }, { "docid": "4407455", "text": "Inflammatory caspases (caspase-1, -4, -5 and -11) are critical for innate defences. Caspase-1 is activated by ligands of various canonical inflammasomes, and caspase-4, -5 and -11 directly recognize bacterial lipopolysaccharide, both of which trigger pyroptosis. Despite the crucial role in immunity and endotoxic shock, the mechanism for pyroptosis induction by inflammatory caspases is unknown. Here we identify gasdermin D (Gsdmd) by genome-wide clustered regularly interspaced palindromic repeat (CRISPR)-Cas9 nuclease screens of caspase-11- and caspase-1-mediated pyroptosis in mouse bone marrow macrophages. GSDMD-deficient cells resisted the induction of pyroptosis by cytosolic lipopolysaccharide and known canonical inflammasome ligands. Interleukin-1β release was also diminished in Gsdmd−/− cells, despite intact processing by caspase-1. Caspase-1 and caspase-4/5/11 specifically cleaved the linker between the amino-terminal gasdermin-N and carboxy-terminal gasdermin-C domains in GSDMD, which was required and sufficient for pyroptosis. The cleavage released the intramolecular inhibition on the gasdermin-N domain that showed intrinsic pyroptosis-inducing activity. Other gasdermin family members were not cleaved by inflammatory caspases but shared the autoinhibition; gain-of-function mutations in Gsdma3 that cause alopecia and skin defects disrupted the autoinhibition, allowing its gasdermin-N domain to trigger pyroptosis. These findings offer insight into inflammasome-mediated immunity/diseases and also change our understanding of pyroptosis and programmed necrosis.", "title": "Cleavage of GSDMD by inflammatory caspases determines pyroptotic cell death" }, { "docid": "4911006", "text": "Apoptotic cells have long been considered as intrinsically tolerogenic or unable to elicit immune responses specific for dead cell-associated antigens. However, multiple stimuli can trigger a functionally peculiar type of apoptotic demise that does not go unnoticed by the adaptive arm of the immune system, which we named \"immunogenic cell death\" (ICD). ICD is preceded or accompanied by the emission of a series of immunostimulatory damage-associated molecular patterns (DAMPs) in a precise spatiotemporal configuration. Several anticancer agents that have been successfully employed in the clinic for decades, including various chemotherapeutics and radiotherapy, can elicit ICD. Moreover, defects in the components that underlie the capacity of the immune system to perceive cell death as immunogenic negatively influence disease outcome among cancer patients treated with ICD inducers. Thus, ICD has profound clinical and therapeutic implications. Unfortunately, the gold-standard approach to detect ICD relies on vaccination experiments involving immunocompetent murine models and syngeneic cancer cells, an approach that is incompatible with large screening campaigns. Here, we outline strategies conceived to detect surrogate markers of ICD in vitro and to screen large chemical libraries for putative ICD inducers, based on a high-content, high-throughput platform that we recently developed. Such a platform allows for the detection of multiple DAMPs, like cell surface-exposed calreticulin, extracellular ATP and high mobility group box 1 (HMGB1), and/or the processes that underlie their emission, such as endoplasmic reticulum stress, autophagy and necrotic plasma membrane permeabilization. We surmise that this technology will facilitate the development of next-generation anticancer regimens, which kill malignant cells and simultaneously convert them into a cancer-specific therapeutic vaccine.", "title": "Consensus guidelines for the detection of immunogenic cell death." }, { "docid": "32665136", "text": "Traumatic spinal cord injury triggers a complex local inflammatory reaction capable of enhancing repair and exacerbating pathology. The composition and effector potential of the post-injury cellular and molecular immune cascade changes as a function of time and distance from the lesion. Production along this time-space continuum of cytokines, proteases, and growth factors establishes dynamic environments that lead to the death, damage, repair or growth of affected neurons and glia. Microenvironmental cues, therefore, generated by the cells therein, may determine these distinct fates of repair versus pathology. To harness repair, it is necessary to manipulate the assembly and phenotype of cells that comprise the neuroinflammatory response to injury. Here, the potential of the neuroinflammatory response to cause outcomes such as pain, regeneration, and functional recovery is reviewed.", "title": "Neuroinflammation in spinal cord injury: therapeutic targets for neuroprotection and regeneration." }, { "docid": "7681810", "text": "Mitotic spindle assembly is mediated by two processes: a centrosomal and a chromosomal pathway. RanGTP regulates the latter process by releasing microtubule-associated proteins from inhibitory complexes. NuSAP, a microtubule- and DNA-binding protein, is a target of RanGTP and promotes the formation of microtubules near chromosomes. However, the contribution of NuSAP to cell proliferation in vivo is unknown. Here, we demonstrate that the expression of NuSAP highly correlates with cell proliferation during embryogenesis and adult life, making it a reliable marker of proliferating cells. Additionally, we show that NuSAP deficiency in mice leads to early embryonic lethality. Spindle assembly in NuSAP-deficient cells is highly inefficient and chromosomes remain dispersed in the mitotic cytoplasm. As a result of sustained spindle checkpoint activity, the cells are unable to progress through mitosis, eventually leading to caspase activation and apoptotic cell death. Together, our findings demonstrate that NuSAP is essential for proliferation of embryonic cells and, simultaneously, they underscore the importance of chromatin-induced spindle assembly.", "title": "NuSAP is essential for chromatin-induced spindle formation during early embryogenesis." }, { "docid": "6144969", "text": "Virally induced inflammatory responses, beta cell destruction and release of beta cell autoantigens may lead to autoimmune reactions culminating in type 1 diabetes. Therefore, viral capability to induce beta cell death and the nature of virus-induced immune responses are among key determinants of diabetogenic viruses. We hypothesised that enterovirus infection induces a specific gene expression pattern that results in islet destruction and that such a host response pattern is not shared among all enterovirus infections but varies between virus strains. The changes in global gene expression and secreted cytokine profiles induced by lytic or benign enterovirus infections were studied in primary human pancreatic islet using DNA microarrays and viral strains either isolated at the clinical onset of type 1 diabetes or capable of causing a diabetes-like condition in mice. The expression of pro-inflammatory cytokine genes (IL-1-α, IL-1-β and TNF-α) that also mediate cytokine-induced beta cell dysfunction correlated with the lytic potential of a virus. Temporally increasing gene expression levels of double-stranded RNA recognition receptors, antiviral molecules, cytokines and chemokines were detected for all studied virus strains. Lytic coxsackievirus B5 (CBV-5)-DS infection also downregulated genes involved in glycolysis and insulin secretion. The results suggest a distinct, virus-strain-specific, gene expression pattern leading to pancreatic islet destruction and pro-inflammatory effects after enterovirus infection. However, neither viral replication nor cytotoxic cytokine production alone are sufficient to induce necrotic cell death. More likely the combined effect of these and possibly cellular energy depletion lie behind the enterovirus-induced necrosis of islets.", "title": "Enterovirus-induced gene expression profile is critical for human pancreatic islet destruction" }, { "docid": "4319844", "text": "Alternative lengthening of telomeres (ALT) is a telomerase-independent telomere maintenance mechanism that occurs in a subset of cancers. By analyzing telomerase-positive cells and their human TERC knockout-derived ALT human cell lines, we show that ALT cells harbor more fragile telomeres representing telomere replication problems. ALT-associated replication defects trigger mitotic DNA synthesis (MiDAS) at telomeres in a RAD52-dependent, but RAD51-independent, manner. Telomeric MiDAS is a conservative DNA synthesis process, potentially mediated by break-induced replication, similar to type II ALT survivors in Saccharomyces cerevisiae Replication stresses induced by ectopic oncogenic expression of cyclin E, G-quadruplexes, or R-loop formation facilitate the ALT pathway and lead to telomere clustering, a hallmark of ALT cancers. The TIMELESS/TIPIN complex suppresses telomere clustering and telomeric MiDAS, whereas the SMC5/6 complex promotes them. In summary, ALT cells exhibit more telomere replication defects that result in persistent DNA damage responses at telomeres, leading to the engagement of telomeric MiDAS (spontaneous mitotic telomere synthesis) that is triggered by DNA replication stress, a potential driver of genomic duplications in cancer.", "title": "Alternative Lengthening of Telomeres Mediated by Mitotic DNA Synthesis Engages Break-Induced Replication Processes." }, { "docid": "13106686", "text": "Immune sensing of DNA is critical for antiviral immunity but can also trigger autoimmune diseases such as lupus erythematosus (LE). Here we have provided evidence for the involvement of a damage-associated DNA modification in the detection of cytosolic DNA. The oxidized base 8-hydroxyguanosine (8-OHG), a marker of oxidative damage in DNA, potentiated cytosolic immune recognition by decreasing its susceptibility to 3' repair exonuclease 1 (TREX1)-mediated degradation. Oxidizative modifications arose physiologically in pathogen DNA during lysosomal reactive oxygen species (ROS) exposure, as well as in neutrophil extracellular trap (NET) DNA during the oxidative burst. 8-OHG was also abundant in UV-exposed skin lesions of LE patients and colocalized with type I interferon (IFN). Injection of oxidized DNA in the skin of lupus-prone mice induced lesions that closely matched respective lesions in patients. Thus, oxidized DNA represents a prototypic damage-associated molecular pattern (DAMP) with important implications for infection, sterile inflammation, and autoimmunity.", "title": "Oxidative damage of DNA confers resistance to cytosolic nuclease TREX1 degradation and potentiates STING-dependent immune sensing." }, { "docid": "14692646", "text": "Extracellular vesicles, including exosomes, are small membrane vesicles derived from multivesicular bodies or from the plasma membrane. Most, if not all, cell types release extracellular vesicles, which then enter the bodily fluids. These vesicles contain a subset of proteins, lipids and nucleic acids that are derived from the parent cell. It is thought that extracellular vesicles have important roles in intercellular communication, both locally and systemically, as they transfer their contents, including proteins, lipids and RNAs, between cells. Extracellular vesicles are involved in numerous physiological processes, and vesicles from both non-immune and immune cells have important roles in immune regulation. Moreover, extracellular vesicle-based therapeutics are being developed and clinically tested for the treatment of inflammatory diseases, autoimmune disorders and cancer. Given the tremendous therapeutic potential of extracellular vesicles, this Review focuses on their role in modulating immune responses, as well as their potential therapeutic applications.", "title": "Regulation of immune responses by extracellular vesicles" }, { "docid": "28107602", "text": "The oncogene MDMX is overexpressed in many cancers, leading to suppression of the tumor suppressor p53. Inhibitors of the oncogene product MDMX therefore might help reactivate p53 and enhance the efficacy of DNA-damaging drugs. However, we currently lack a quantitative understanding of how MDMX inhibition affects the p53 signaling pathway and cell sensitivity to DNA damage. Live cell imaging showed that MDMX depletion triggered two distinct phases of p53 accumulation in single cells: an initial postmitotic pulse, followed by low-amplitude oscillations. The response to DNA damage was sharply different in these two phases; in the first phase, MDMX depletion was synergistic with DNA damage in causing cell death, whereas in the second phase, depletion of MDMX inhibited cell death. Thus a quantitative understanding of signal dynamics and cellular states is important for designing an optimal schedule of dual-drug administration.", "title": "Schedule-dependent interaction between anticancer treatments" }, { "docid": "1834762", "text": "Research on the human microbiome has established that commensal and pathogenic bacteria can influence obesity, cancer, and autoimmunity through mechanisms mostly unknown. We found that a component of bacterial biofilms, the amyloid protein curli, irreversibly formed fibers with bacterial DNA during biofilm formation. This interaction accelerated amyloid polymerization and created potent immunogenic complexes that activated immune cells, including dendritic cells, to produce cytokines such as type I interferons, which are pathogenic in systemic lupus erythematosus (SLE). When given systemically, curli-DNA composites triggered immune activation and production of autoantibodies in lupus-prone and wild-type mice. We also found that the infection of lupus-prone mice with curli-producing bacteria triggered higher autoantibody titers compared to curli-deficient bacteria. These data provide a mechanism by which the microbiome and biofilm-producing enteric infections may contribute to the progression of SLE and point to a potential molecular target for treatment of autoimmunity.", "title": "Amyloid-DNA Composites of Bacterial Biofilms Stimulate Autoimmunity." }, { "docid": "10607877", "text": "Cell surface receptors have been extensively studied because they initiate and regulate signal transduction cascades leading to a variety of functional cellular outcomes. An important class of immune receptors (e.g., T-cell antigen receptors) whose ligands are anchored to the surfaces of other cells remain poorly understood. The mechanism by which ligand binding initiates receptor phosphorylation, a process termed \"receptor triggering\", remains controversial. Recently, direct measurements of the (two-dimensional) receptor-ligand complex lifetimes at cell-cell interface were found to be smaller than (three-dimensional) lifetimes in solution but the underlying mechanism is unknown. At the cell-cell interface, the receptor-ligand complex spans a short intermembrane distance (15 nm) compared to long surface molecules (LSMs) whose ectodomains span >40 nm and these LSMs include phosphatases (e.g., CD45) that dephosphorylate the receptor. It has been proposed that size-based segregation of LSMs from a receptor-ligand complex is a mechanism of receptor triggering but it is unclear whether the mechanochemistry supports such small-scale segregation. Here we present a nanometer-scale mathematical model that couples membrane elasticity with the compressional stiffness and lateral mobility of LSMs. We find robust supradiffusive segregation of LSMs from a single receptor-ligand complex. The model predicts that LSM redistribution will result in a time-dependent tension on the complex leading to a decreased two-dimensional lifetime. Interestingly, the model predicts a nonlinear relationship between the three- and two-dimensional lifetimes, which can enhance the ability of receptors to discriminate between similar ligands.", "title": "Mechanical modulation of receptor-ligand interactions at cell-cell interfaces." }, { "docid": "40667066", "text": "Steroid hormones, thyroid hormones, retinoic acids, and vitamin D bind to their receptors, which are now called steroid/nuclear receptors, and liganded receptors translocate either intracellularly or intranuclearly and form large protein complexes with cofactors to induce or repress gene transcription. Therefore, steroid/nuclear receptors are ligand-dependent transcription factors. With the advent of green fluorescent protein (GFP) and its color variants, the subcellular distribution of many steroid/nuclear receptors has been found to be much more dynamic than previously thought, with some of the receptors shuttling between the cytoplasm and nucleus. Steroid/nuclear receptors can be divided into three categories based on their unliganded distribution: those that are primarily in the nucleus, those in the cytoplasm, and those with mixed cytoplasmic and nuclear distributions. However, in all cases, the addition of a ligand leads to almost complete nuclear translocation of the receptors. Hormonal stimulation induces intranuclear receptor distribution from a homogeneous pattern to a heterogeneous dot-like image. Ligand binding to steroid/nuclear receptors leads to the recruitment of many proteins including cofactors to provoke the redistribution of receptor complexes in the nucleus. This focal organization could involve more complex events than simple DNA binding sites for transcription. Protein activities and interactions of steroid/nuclear receptors can be imaged and localized in a single cell.", "title": "Subcellular steroid/nuclear receptor dynamics." }, { "docid": "7764903", "text": "Both eukaryotic and prokaryotic cells release small, phospholipid-enclosed vesicles into their environment. Why do cells release vesicles? Initial studies showed that eukaryotic vesicles are used to remove obsolete cellular molecules. Although this release of vesicles is beneficial to the cell, the vesicles can also be a danger to their environment, for instance in blood, where vesicles can provide a surface supporting coagulation. Evidence is accumulating that vesicles are cargo containers used by eukaryotic cells to exchange biomolecules as transmembrane receptors and genetic information. Because also bacteria communicate to each other via extracellular vesicles, the intercellular communication via extracellular cargo carriers seems to be conserved throughout evolution, and therefore vesicles are likely to be a highly efficient, robust, and economic manner of exchanging information between cells. Furthermore, vesicles protect cells from accumulation of waste or drugs, they contribute to physiology and pathology, and they have a myriad of potential clinical applications, ranging from biomarkers to anticancer therapy. Because vesicles may pass the blood-brain barrier, they can perhaps even be considered naturally occurring liposomes. Unfortunately, pathways of vesicle release and vesicles themselves are also being used by tumors and infectious diseases to facilitate spreading, and to escape from immune surveillance. In this review, the different types, nomenclature, functions, and clinical relevance of vesicles will be discussed.", "title": "Classification, functions, and clinical relevance of extracellular vesicles." }, { "docid": "38131471", "text": "DNA damage is a relatively common event in the life of a cell and may lead to mutation, cancer, and cellular or organismic death. Damage to DNA induces several cellular responses that enable the cell either to eliminate or cope with the damage or to activate a programmed cell death process, presumably to eliminate cells with potentially catastrophic mutations. These DNA damage response reactions include: (a) removal of DNA damage and restoration of the continuity of the DNA duplex; (b) activation of a DNA damage checkpoint, which arrests cell cycle progression so as to allow for repair and prevention of the transmission of damaged or incompletely replicated chromosomes; (c) transcriptional response, which causes changes in the transcription profile that may be beneficial to the cell; and (d) apoptosis, which eliminates heavily damaged or seriously deregulated cells. DNA repair mechanisms include direct repair, base excision repair, nucleotide excision repair, double-strand break repair, and cross-link repair. The DNA damage checkpoints employ damage sensor proteins, such as ATM, ATR, the Rad17-RFC complex, and the 9-1-1 complex, to detect DNA damage and to initiate signal transduction cascades that employ Chk1 and Chk2 Ser/Thr kinases and Cdc25 phosphatases. The signal transducers activate p53 and inactivate cyclin-dependent kinases to inhibit cell cycle progression from G1 to S (the G1/S checkpoint), DNA replication (the intra-S checkpoint), or G2 to mitosis (the G2/M checkpoint). In this review the molecular mechanisms of DNA repair and the DNA damage checkpoints in mammalian cells are analyzed.", "title": "Molecular mechanisms of mammalian DNA repair and the DNA damage checkpoints." }, { "docid": "20610557", "text": "In recent years, the immune-potentiating effects of some widely used chemotherapeutic agents have been increasingly appreciated. This provides a rationale for combining conventional chemotherapy with immunotherapy strategies to achieve durable therapeutic benefits. Previous studies have implicated the immunomodulatory effects of melphalan, an alkylating agent commonly used to treat multiple myeloma, but the underlying mechanisms remain obscure. In the present study, we investigated the impact of melphalan on endogenous immune cells as well as adoptively transferred tumor-specific CD4(+) T cells in tumor-bearing mice. We showed that melphalan treatment resulted in a rapid burst of inflammatory cytokines and chemokines during the cellular recovery phase after melphalan-induced myelodepletion and leukodepletion. After melphalan treatment, tumor cells exhibited characteristics of immunogenic cell death, including membrane translocation of the endoplasmic reticulum-resident calreticulin and extracellular release of high-mobility group box 1. Additionally, there was enhanced tumor Ag uptake by dendritic cells in the tumor-draining lymph node. Consistent with these immunomodulatory effects, melphalan treatment of tumor-bearing mice led to the activation of the endogenous CD8(+) T cells and, more importantly, effectively drove the clonal expansion and effector differentiation of adoptively transferred tumor-specific CD4(+) T cells. Notably, the combination of melphalan and CD4(+) T cell adoptive cell therapy was more efficacious than either treatment alone in prolonging the survival of mice with advanced B cell lymphomas or colorectal tumors. These findings provide mechanistic insights into melphalan's immunostimulatory effects and demonstrate the therapeutic potential of combining melphalan with adoptive cell therapy utilizing antitumor CD4(+) T cells.", "title": "Alkylating agent melphalan augments the efficacy of adoptive immunotherapy using tumor-specific CD4+ T cells." }, { "docid": "12909503", "text": "DNA damage encountered by DNA replication forks poses risks of genome destabilization, a precursor to carcinogenesis. Damage checkpoint systems cause cell cycle arrest, promote repair and induce programed cell death when damage is severe. Checkpoints are critical parts of the DNA damage response network that act to suppress cancer. DNA damage and perturbation of replication machinery causes replication stress, characterized by accumulation of single-stranded DNA bound by replication protein A (RPA), which triggers activation of ataxia telangiectasia and Rad3 related (ATR) and phosphorylation of the RPA32, subunit of RPA, leading to Chk1 activation and arrest. DNA-dependent protein kinase catalytic subunit (DNA-PKcs) [a kinase related to ataxia telangiectasia mutated (ATM) and ATR] has well characterized roles in DNA double-strand break repair, but poorly understood roles in replication stress-induced RPA phosphorylation. We show that DNA-PKcs mutant cells fail to arrest replication following stress, and mutations in RPA32 phosphorylation sites targeted by DNA-PKcs increase the proportion of cells in mitosis, impair ATR signaling to Chk1 and confer a G2/M arrest defect. Inhibition of ATR and DNA-PK (but not ATM), mimic the defects observed in cells expressing mutant RPA32. Cells expressing mutant RPA32 or DNA-PKcs show sustained H2AX phosphorylation in response to replication stress that persists in cells entering mitosis, indicating inappropriate mitotic entry with unrepaired damage.", "title": "Distinct roles for DNA-PK, ATM and ATR in RPA phosphorylation and checkpoint activation in response to replication stress" }, { "docid": "12641252", "text": "Anthracyclin-treated tumor cells are particularly effective in eliciting an anticancer immune response, whereas other DNA-damaging agents such as etoposide and mitomycin C do not induce immunogenic cell death. Here we show that anthracyclins induce the rapid, preapoptotic translocation of calreticulin (CRT) to the cell surface. Blockade or knockdown of CRT suppressed the phagocytosis of anthracyclin-treated tumor cells by dendritic cells and abolished their immunogenicity in mice. The anthracyclin-induced CRT translocation was mimicked by inhibition of the protein phosphatase 1/GADD34 complex. Administration of recombinant CRT or inhibitors of protein phosphatase 1/GADD34 restored the immunogenicity of cell death elicited by etoposide and mitomycin C, and enhanced their antitumor effects in vivo. These data identify CRT as a key feature determining anticancer immune responses and delineate a possible strategy for immunogenic chemotherapy.", "title": "Calreticulin exposure dictates the immunogenicity of cancer cell death" }, { "docid": "6853699", "text": "In atherosclerosis, the accumulation of apolipoprotein B-lipoproteins in the matrix beneath the endothelial cell layer of blood vessels leads to the recruitment of monocytes, the cells of the immune system that give rise to macrophages and dendritic cells. Macrophages derived from these recruited monocytes participate in a maladaptive, nonresolving inflammatory response that expands the subendothelial layer due to the accumulation of cells, lipid, and matrix. Some lesions subsequently form a necrotic core, triggering acute thrombotic vascular disease, including myocardial infarction, stroke, and sudden cardiac death. This Review discusses the central roles of macrophages in each of these stages of disease pathogenesis.", "title": "Macrophages in the Pathogenesis of Atherosclerosis" }, { "docid": "3930020", "text": "Epidermal Langerhans cells (LCs) play a key role in immune defense mechanisms and in numerous immunological disorders. In this report, we show that percutaneous infection of C57BL/6 mice with the helminth parasite Schistosoma mansoni leads to the activation of LCs but, surprisingly, to their retention in the epidermis. Moreover, using an experimental model of LC migration induced by tumor necrosis factor (TNF)-α, we show that parasites transiently impair the departure of LCs from the epidermis and their subsequent accumulation as dendritic cells in the draining lymph nodes. The inhibitory effect is mediated by soluble lipophilic factors released by the parasites and not by host-derived antiinflammatory cytokines, such as interleukin-10. We find that prostaglandin (PG)D2, but not the other major eicosanoids produced by the parasites, specifically impedes the TNF-α–triggered migration of LCs through the adenylate cyclase–coupled PGD2 receptor (DP receptor). Moreover, the potent DP receptor antagonist BW A868C restores LC migration in infected mice. Finally, in a model of contact allergen-induced LC migration, we show that activation of the DP receptor not only inhibits LC emigration but also dramatically reduces the contact hypersensitivity responses after challenge. Taken together, we propose that the inhibition of LC migration could represent an additional stratagem for the schistosomes to escape the host immune system and that PGD2 may play a key role in the control of cutaneous immune responses.", "title": "Role of the Parasite-Derived Prostaglandin D2 in the Inhibition of Epidermal Langerhans Cell Migration during Schistosomiasis Infection" }, { "docid": "6000423", "text": "Despite genetic heterogeneity, myelodysplastic syndromes (MDSs) share features of cytological dysplasia and ineffective hematopoiesis. We report that a hallmark of MDSs is activation of the NLRP3 inflammasome, which drives clonal expansion and pyroptotic cell death. Independent of genotype, MDS hematopoietic stem and progenitor cells (HSPCs) overexpress inflammasome proteins and manifest activated NLRP3 complexes that direct activation of caspase-1, generation of interleukin-1β (IL-1β) and IL-18, and pyroptotic cell death. Mechanistically, pyroptosis is triggered by the alarmin S100A9 that is found in excess in MDS HSPCs and bone marrow plasma. Further, like somatic gene mutations, S100A9-induced signaling activates NADPH oxidase (NOX), increasing levels of reactive oxygen species (ROS) that initiate cation influx, cell swelling, and β-catenin activation. Notably, knockdown of NLRP3 or caspase-1, neutralization of S100A9, and pharmacologic inhibition of NLRP3 or NOX suppress pyroptosis, ROS generation, and nuclear β-catenin in MDSs and are sufficient to restore effective hematopoiesis. Thus, alarmins and founder gene mutations in MDSs license a common redox-sensitive inflammasome circuit, which suggests new avenues for therapeutic intervention.", "title": "The NLRP3 inflammasome functions as a driver of the myelodysplastic syndrome phenotype." } ]

[ { "docid": "16284655", "text": "Human astrocytes are larger and more complex than those of infraprimate mammals, suggesting that their role in neural processing has expanded with evolution. To assess the cell-autonomous and species-selective properties of human glia, we engrafted human glial progenitor cells (GPCs) into neonatal immunodeficient mice. Upon maturation, the recipient brains exhibited large numbers and high proportions of both human glial progenitors and astrocytes. The engrafted human glia were gap-junction-coupled to host astroglia, yet retained the size and pleomorphism of hominid astroglia, and propagated Ca2+ signals 3-fold faster than their hosts. Long-term potentiation (LTP) was sharply enhanced in the human glial chimeric mice, as was their learning, as assessed by Barnes maze navigation, object-location memory, and both contextual and tone fear conditioning. Mice allografted with murine GPCs showed no enhancement of either LTP or learning. These findings indicate that human glia differentially enhance both activity-dependent plasticity and learning in mice.", "title": "Forebrain engraftment by human glial progenitor cells enhances synaptic plasticity and learning in adult mice." } ]

[ { "docid": "14192687", "text": "The long-term goal of nuclear transfer or alternative reprogramming approaches is to create patient-specific donor cells for transplantation therapy, avoiding immunorejection, a major complication in current transplantation medicine. It was recently shown that the four transcription factors Oct4, Sox2, Klf4, and c-Myc induce pluripotency in mouse fibroblasts. However, the therapeutic potential of induced pluripotent stem (iPS) cells for neural cell replacement strategies remained unexplored. Here, we show that iPS cells can be efficiently differentiated into neural precursor cells, giving rise to neuronal and glial cell types in culture. Upon transplantation into the fetal mouse brain, the cells migrate into various brain regions and differentiate into glia and neurons, including glutamatergic, GABAergic, and catecholaminergic subtypes. Electrophysiological recordings and morphological analysis demonstrated that the grafted neurons had mature neuronal activity and were functionally integrated in the host brain. Furthermore, iPS cells were induced to differentiate into dopamine neurons of midbrain character and were able to improve behavior in a rat model of Parkinson's disease upon transplantation into the adult brain. We minimized the risk of tumor formation from the grafted cells by separating contaminating pluripotent cells and committed neural cells using fluorescence-activated cell sorting. Our results demonstrate the therapeutic potential of directly reprogrammed fibroblasts for neuronal cell replacement in the animal model.", "title": "Neurons derived from reprogrammed fibroblasts functionally integrate into the fetal brain and improve symptoms of rats with Parkinson's disease." }, { "docid": "803312", "text": "The complexity of the human brain has made it difficult to study many brain disorders in model organisms, highlighting the need for an in vitro model of human brain development. Here we have developed a human pluripotent stem cell-derived three-dimensional organoid culture system, termed cerebral organoids, that develop various discrete, although interdependent, brain regions. These include a cerebral cortex containing progenitor populations that organize and produce mature cortical neuron subtypes. Furthermore, cerebral organoids are shown to recapitulate features of human cortical development, namely characteristic progenitor zone organization with abundant outer radial glial stem cells. Finally, we use RNA interference and patient-specific induced pluripotent stem cells to model microcephaly, a disorder that has been difficult to recapitulate in mice. We demonstrate premature neuronal differentiation in patient organoids, a defect that could help to explain the disease phenotype. Together, these data show that three-dimensional organoids can recapitulate development and disease even in this most complex human tissue.", "title": "Cerebral organoids model human brain development and microcephaly" }, { "docid": "27555165", "text": "Human cytomegalovirus (HCMV) is a ubiquitous herpesvirus that causes life-threatening disease in patients who are immunosuppressed for bone marrow or tissue transplantation or who have AIDS (ref. 1). HCMV establishes lifelong latent infections and, after periodic reactivation from latency, uses a panel of immune evasion proteins to survive and replicate in the face of robust, fully primed host immunity. Monocyte/macrophages are important host cells for HCMV, serving as a latent reservoir and as a means of dissemination throughout the body. Macrophages and other HCMV-permissive cells, such as endothelial and glial cells, can express MHC class II proteins and present antigens to CD4+ T lymphocytes. Here, we show that the HCMV protein US2 causes degradation of two essential proteins in the MHC class II antigen presentation pathway: HLA-DR-α and DM-α. This was unexpected, as US2 has been shown to cause degradation of MHC class I (refs. 5,6), which has only limited homology with class II proteins. Expression of US2 in cells reduced or abolished their ability to present antigen to CD4+ T lymphocytes. Thus, US2 may allow HCMV-infected macrophages to remain relatively 'invisible' to CD4+ T cells, a property that would be important after virus reactivation.", "title": "Cytomegalovirus US2 destroys two components of the MHC class II pathway, preventing recognition by CD4+ T cells" }, { "docid": "16572581", "text": "Type 1 diabetes (T1D) is characterized by the destruction of the insulin-producing β-cells of pancreatic islets. Genetic and environmental factors both contribute to T1D development. Viral infection with enteroviruses is a suspected trigger for T1D, but a causal role remains unproven and controversial. Studies in animals are problematic because of species-specific differences in host cell susceptibility and immune responses to candidate viral pathogens such as coxsackievirus B (CVB). In order to resolve the controversial role of viruses in human T1D, we developed a viral infection model in immunodeficient mice bearing human islet grafts. Hyperglycemia was induced in mice by specific ablation of native β-cells. Human islets, which are naturally susceptible to CVB infection, were transplanted to restore normoglycemia. Transplanted mice were infected with CVB4 and monitored for hyperglycemia. Forty-seven percent of CVB4-infected mice developed hyperglycemia. Human islet grafts from infected mice contained viral RNA, expressed viral protein, and had reduced insulin levels compared with grafts from uninfected mice. Human-specific gene expression profiles in grafts from infected mice revealed the induction of multiple interferon-stimulated genes. Thus, human islets can become severely dysfunctional with diminished insulin production after CVB infection of β-cells, resulting in diabetes.", "title": "Viral Infection of Engrafted Human Islets Leads to Diabetes" }, { "docid": "3952288", "text": "Aire-expressing medullary thymic epithelial cells (mTECs) play a key role in preventing autoimmunity by expressing tissue-restricted antigens to help purge the emerging T cell receptor repertoire of self-reactive specificities. Here we demonstrate a novel role for a CD4+3− inducer cell population, previously linked to development of organized secondary lymphoid structures and maintenance of T cell memory in the functional regulation of Aire-mediated promiscuous gene expression in the thymus. CD4+3− cells are closely associated with mTECs in adult thymus, and in fetal thymus their appearance is temporally linked with the appearance of Aire+ mTECs. We show that RANKL signals from this cell promote the maturation of RANK-expressing CD80−Aire− mTEC progenitors into CD80+Aire+ mTECs, and that transplantation of RANK-deficient thymic stroma into immunodeficient hosts induces autoimmunity. Collectively, our data reveal cellular and molecular mechanisms leading to the generation of Aire+ mTECs and highlight a previously unrecognized role for CD4+3−RANKL+ inducer cells in intrathymic self-tolerance.", "title": "RANK signals from CD4+3− inducer cells regulate development of Aire-expressing epithelial cells in the thymic medulla" }, { "docid": "36637129", "text": "Reprogramming of somatic cells into pluripotency stem cell state has opened new opportunities in cell replacement therapy and disease modeling in a number of neurological disorders. It still remains unknown, however, to what degree the grafted human-induced pluripotent stem cells (hiPSCs) differentiate into a functional neuronal phenotype and if they integrate into the host circuitry. Here, we present a detailed characterization of the functional properties and synaptic integration of hiPSC-derived neurons grafted in an in vitro model of hyperexcitable epileptic tissue, namely organotypic hippocampal slice cultures (OHSCs), and in adult rats in vivo. The hiPSCs were first differentiated into long-term self-renewing neuroepithelial stem (lt-NES) cells, which are known to form primarily GABAergic neurons. When differentiated in OHSCs for 6 weeks, lt-NES cell-derived neurons displayed neuronal properties such as tetrodotoxin-sensitive sodium currents and action potentials (APs), as well as both spontaneous and evoked postsynaptic currents, indicating functional afferent synaptic inputs. The grafted cells had a distinct electrophysiological profile compared to host cells in the OHSCs with higher input resistance, lower resting membrane potential, and APs with lower amplitude and longer duration. To investigate the origin of synaptic afferents to the grafted lt-NES cell-derived neurons, the host neurons were transduced with Channelrhodopsin-2 (ChR2) and optogenetically activated by blue light. Simultaneous recordings of synaptic currents in grafted lt-NES cell-derived neurons using whole-cell patch-clamp technique at 6 weeks after grafting revealed limited synaptic connections from host neurons. Longer differentiation times, up to 24 weeks after grafting in vivo, revealed more mature intrinsic properties and extensive synaptic afferents from host neurons to the lt-NES cell-derived neurons, suggesting that these cells require extended time for differentiation/maturation and synaptogenesis. However, even at this later time point, the grafted cells maintained a higher input resistance. These data indicate that grafted lt-NES cell-derived neurons receive ample afferent input from the host brain. Since the lt-NES cells used in this study show a strong propensity for GABAergic differentiation, the host-to-graft synaptic afferents may facilitate inhibitory neurotransmitter release, and normalize hyperexcitable neuronal networks in brain diseases, for example, such as epilepsy.", "title": "Optogenetics reveal delayed afferent synaptogenesis on grafted human-induced pluripotent stem cell-derived neural progenitors." }, { "docid": "3360421", "text": "We describe the derivation of pluripotent embryonic stem (ES) cells from human blastocysts. Two diploid ES cell lines have been cultivated in vitro for extended periods while maintaining expression of markers characteristic of pluripotent primate cells. Human ES cells express the transcription factor Oct-4, essential for development of pluripotential cells in the mouse. When grafted into SCID mice, both lines give rise to teratomas containing derivatives of all three embryonic germ layers. Both cell lines differentiate in vitro into extraembryonic and somatic cell lineages. Neural progenitor cells may be isolated from differentiating ES cell cultures and induced to form mature neurons. Embryonic stem cells provide a model to study early human embryology, an investigational tool for discovery of novel growth factors and medicines, and a potential source of cells for use in transplantation therapy.", "title": "Embryonic stem cell lines from human blastocysts: somatic differentiation in vitro" }, { "docid": "33535222", "text": "CD4+CD25+ regulatory T cells (Treg's) play a pivotal role in preventing organ-specific autoimmune diseases and in inducing tolerance to allogeneic organ transplants. We and others recently demonstrated that high numbers of Treg's can also modulate graft-versus-host disease (GVHD) if administered in conjunction with allogeneic hematopoietic stem cell transplantation in mice. In a clinical setting, it would be impossible to obtain enough freshly purified Treg's from a single donor to have a therapeutic effect. Thus, we performed regulatory T cell expansion ex vivo by stimulation with allogeneic APCs, which has the additional effect of producing alloantigen-specific regulatory T cells. Here we show that regulatory T cells specific for recipient-type alloantigens control GVHD while favoring immune reconstitution. Irrelevant regulatory T cells only mediate a partial protection from GVHD. Preferential survival of specific regulatory T cells, but not of irrelevant regulatory T cells, was observed in grafted animals. Additionally, the use of specific regulatory T cells was compatible with some form of graft-versus-tumor activity. These data suggest that recipient-type specific Treg's could be preferentially used in the control of GVHD in future clinical trials.", "title": "Recipient-type specific CD4+CD25+ regulatory T cells favor immune reconstitution and control graft-versus-host disease while maintaining graft-versus-leukemia." }, { "docid": "37699461", "text": "Beta-cell replacement is considered to be the most promising approach for treatment of type 1 diabetes. Its application on a large scale is hindered by a shortage of cells for transplantation. Activation of insulin expression, storage, and regulated secretion in stem/progenitor cells offers novel ways to overcome this shortage. We explored whether fetal human progenitor liver cells (FH) could be induced to differentiate into insulin-producing cells after expression of the pancreatic duodenal homeobox 1 (Pdx1) gene, which is a key regulator of pancreatic development and insulin expression in beta cells. FH cells possess a considerable replication capacity, and this was further extended by introduction of the gene for the catalytic subunit of human telomerase. Immortalized FH cells expressing Pdx1 activated multiple beta-cell genes, produced and stored considerable amounts of insulin, and released insulin in a regulated manner in response to glucose. When transplanted into hyperglycemic immunodeficient mice, the cells restored and maintained euglycemia for prolonged periods. Quantitation of human C-peptide in the mouse serum confirmed that the glycemia was normalized by the transplanted human cells. This approach offers the potential of a novel source of cells for transplantation into patients with type 1 diabetes.", "title": "Reversal of hyperglycemia in mice by using human expandable insulin-producing cells differentiated from fetal liver progenitor cells." }, { "docid": "21232018", "text": "We investigated the capacity of young ovaries, transplanted into old ovariectomized CBA mice, to improve remaining life expectancy of the hosts. Donor females were sexually mature 2-month-olds; recipients were prepubertally ovariectomized at 3 weeks and received transplants at 5, 8 or 11 months of age. Relative to ovariectomized control females, life expectancy at 11 months was increased by 60% in 11-month recipient females and by 40% relative to intact control females. Only 20% of the 11-month transplant females died in the 300-day period following ovarian transplantation, whereas nearly 65% of the ovariectomized control females died during this same period. The 11-month-old recipient females resumed oestrus and continued to cycle up to several months beyond the age of control female reproductive senescence. Across the three recipient age groups, transplantation of young ovaries increased life expectancy in proportion to the relative youth of the ovary. Our results relate to recent findings on the gonadal input upon aging in Caenorhabditis elegans and may suggest how the mammalian gonad, including that of humans, could regulate aging and determine longevity.", "title": "Age of ovary determines remaining life expectancy in old ovariectomized mice." }, { "docid": "9412420", "text": "Mesenchymal stem cells (MSCs) commonly defined by in vitro functions have entered clinical application despite little definition of their function in residence. Here, we report genetic pulse-chase experiments that define osteoblastic cells as short-lived and nonreplicative, requiring replenishment from bone-marrow-derived, Mx1(+) stromal cells with \"MSC\" features. These cells respond to tissue stress and migrate to sites of injury, supplying new osteoblasts during fracture healing. Single cell transplantation yielded progeny that both preserve progenitor function and differentiate into osteoblasts, producing new bone. They are capable of local and systemic translocation and serial transplantation. While these cells meet current definitions of MSCs in vitro, they are osteolineage restricted in vivo in growing and adult animals. Therefore, bone-marrow-derived MSCs may be a heterogeneous population with the Mx1(+) population, representing a highly dynamic and stress responsive stem/progenitor cell population of fate-restricted potential that feeds the high cell replacement demands of the adult skeleton.", "title": "Endogenous bone marrow MSCs are dynamic, fate-restricted participants in bone maintenance and regeneration." }, { "docid": "188911", "text": "Antigen-presenting, major histocompatibility complex (MHC) class II-rich dendritic cells are known to arise from bone marrow. However, marrow lacks mature dendritic cells, and substantial numbers of proliferating less-mature cells have yet to be identified. The methodology for inducing dendritic cell growth that was recently described for mouse blood now has been modified to MHC class II-negative precursors in marrow. A key step is to remove the majority of nonadherent, newly formed granulocytes by gentle washes during the first 2-4 d of culture. This leaves behind proliferating clusters that are loosely attached to a more firmly adherent \"stroma. \" At days 4-6 the clusters can be dislodged, isolated by 1-g sedimentation, and upon reculture, large numbers of dendritic cells are released. The latter are readily identified on the basis of their distinct cell shape, ultrastructure, and repertoire of antigens, as detected with a panel of monoclonal antibodies. The dendritic cells express high levels of MHC class II products and act as powerful accessory cells for initiating the mixed leukocyte reaction. Neither the clusters nor mature dendritic cells are generated if macrophage colony-stimulating factor rather than granulocyte/macrophage colony-stimulating factor (GM-CSF) is applied. Therefore, GM-CSF generates all three lineages of myeloid cells (granulocytes, macrophages, and dendritic cells). Since > 5 x 10(6) dendritic cells develop in 1 wk from precursors within the large hind limb bones of a single animal, marrow progenitors can act as a major source of dendritic cells. This feature should prove useful for future molecular and clinical studies of this otherwise trace cell type.", "title": "Generation of large numbers of dendritic cells from mouse bone marrow cultures supplemented with granulocyte/macrophage colony-stimulating factor" }, { "docid": "16375102", "text": "The simple yet powerful technique of induced pluripotency may eventually supply a wide range of differentiated cells for cell therapy and drug development. However, making the appropriate cells via induced pluripotent stem cells (iPSCs) requires reprogramming of somatic cells and subsequent redifferentiation. Given how arduous and lengthy this process can be, we sought to determine whether it might be possible to convert somatic cells into lineage-specific stem/progenitor cells of another germ layer in one step, bypassing the intermediate pluripotent stage. Here we show that transient induction of the four reprogramming factors (Oct4, Sox2, Klf4, and c-Myc) can efficiently transdifferentiate fibroblasts into functional neural stem/progenitor cells (NPCs) with appropriate signaling inputs. Compared with induced neurons (or iN cells, which are directly converted from fibroblasts), transdifferentiated NPCs have the distinct advantage of being expandable in vitro and retaining the ability to give rise to multiple neuronal subtypes and glial cells. Our results provide a unique paradigm for iPSC-factor-based reprogramming by demonstrating that it can be readily modified to serve as a general platform for transdifferentiation.", "title": "Direct reprogramming of mouse fibroblasts to neural progenitors." }, { "docid": "13757347", "text": "GABAergic interneurons of the parvalbumin-positive fast-spiking basket cells subtype (PV INs) are important regulators of cortical network excitability and of gamma oscillations, involved in signal processing and cognition. Impaired development or function of PV INs has been associated with epilepsy in various animal models of epilepsy, as well as in some genetic forms of epilepsy in humans. In this review, we provide an overview of some of the experimental data linking PV INs dysfunction with epilepsy, focusing on disorders of the specification, migration, maturation, synaptic function, or connectivity of PV INs. Furthermore, we reflect on the potential therapeutic use of cell-type specific stimulation of PV INs within active networks and on the transplantation of PV INs precursors in the treatment of epilepsy and its comorbidities.", "title": "Involvement of cortical fast-spiking parvalbumin-positive basket cells in epilepsy." }, { "docid": "21932050", "text": "Human cytomegalovirus (HCMV) is one of the largest known DNA viruses. It is ubiquitous, and following resolution of primary productive infection, it persists in the human host by establishing a lifelong latent infection in myeloid lineage cells such as monocytes and their progenitors. Most adults with HCMV infection are healthy but it can cause neurologic deficits in infants, and remains an important cause of morbidity and mortality in the immunosuppressed patient. Microarray-based studies of HCMV have provided useful information about genes that are transcriptionally active during both productive and latent phases of infection. This chapter describes how to study genes in HCMV using microarrays and two cell types (productively infected human foreskin fibroblasts, and latently infected primary human myeloid progenitor cells).", "title": "Microarrays for the study of viral gene expression during human cytomegalovirus latent infection." }, { "docid": "4388470", "text": "In the mammalian model of sex determination, embryos are considered to be sexually indifferent until the transient action of a sex-determining gene initiates gonadal differentiation. Although this model is thought to apply to all vertebrates, this has yet to be established. Here we have examined three lateral gynandromorph chickens (a rare, naturally occurring phenomenon in which one side of the animal appears male and the other female) to investigate the sex-determining mechanism in birds. These studies demonstrated that gynandromorph birds are genuine male:female chimaeras, and indicated that male and female avian somatic cells may have an inherent sex identity. To test this hypothesis, we transplanted presumptive mesoderm between embryos of reciprocal sexes to generate embryos containing male:female chimaeric gonads. In contrast to the outcome for mammalian mixed-sex chimaeras, in chicken mixed-sex chimaeras the donor cells were excluded from the functional structures of the host gonad. In an example where female tissue was transplanted into a male host, donor cells contributing to the developing testis retained a female identity and expressed a marker of female function. Our study demonstrates that avian somatic cells possess an inherent sex identity and that, in birds, sexual differentiation is substantively cell autonomous.", "title": "Somatic sex identity is cell-autonomous in the chicken" }, { "docid": "8790729", "text": "BACKGROUND There is a widespread interest in developing renewable sources of islet-replacement tissue for type I diabetes mellitus. Human mesenchymal cells isolated from the Wharton's jelly of the umbilical cord (HUMSCs), which can be easily obtained and processed compared with embryonic and bone marrow stem cells, possess stem cell properties. HUMSCs may be a valuable source for the generation of islets. METHODOLOGY AND PRINCIPAL FINDINGS HUMSCs were induced to transform into islet-like cell clusters in vitro through stepwise culturing in neuron-conditioned medium. To assess the functional stability of the islet-like cell clusters in vivo, these cell clusters were transplanted into the liver of streptozotocin-induced diabetic rats via laparotomy. Glucose tolerance was measured on week 12 after transplantation accompanied with immunohistochemistry and electron microscopy analysis. These islet-like cell clusters were shown to contain human C-peptide and release human insulin in response to physiological glucose levels. Real-time RT-PCR detected the expressions of insulin and other pancreatic beta-cell-related genes (Pdx1, Hlxb9, Nkx2.2, Nkx6.1, and Glut-2) in these islet-like cell clusters. The hyperglycemia and glucose intolerance in streptozotocin-induced diabetic rats was significantly alleviated after xenotransplantation of islet-like cell clusters, without the use of immunosuppressants. In addition to the existence of islet-like cell clusters in the liver, some special fused liver cells were also found, which characterized by human insulin and nuclei-positive staining and possessing secretory granules. CONCLUSIONS AND SIGNIFICANCE In this study, we successfully differentiate HUMSCs into mature islet-like cell clusters, and these islet-like cell clusters possess insulin-producing ability in vitro and in vivo. HUMSCs in Wharton's Jelly of the umbilical cord seem to be the preferential source of stem cells to convert into insulin-producing cells, because of the large potential donor pool, its rapid availability, no risk of discomfort for the donor, and low risk of rejection.", "title": "Islet-Like Clusters Derived from Mesenchymal Stem Cells in Wharton's Jelly of the Human Umbilical Cord for Transplantation to Control Type 1 Diabetes" }, { "docid": "33986200", "text": "Probing a wide range of cellular phenotypes in neurodevelopmental disorders using patient-derived neural progenitor cells (NPCs) can be facilitated by 3D assays, as 2D systems cannot entirely recapitulate the arrangement of cells in the brain. Here, we developed a previously unidentified 3D migration and differentiation assay in layered hydrogels to examine how these processes are affected in neurodevelopmental disorders, such as Rett syndrome. Our soft 3D system mimics the brain environment and accelerates maturation of neurons from human induced pluripotent stem cell (iPSC)-derived NPCs, yielding electrophysiologically active neurons within just 3 wk. Using this platform, we revealed a genotype-specific effect of methyl-CpG-binding protein-2 (MeCP2) dysfunction on iPSC-derived neuronal migration and maturation (reduced neurite outgrowth and fewer synapses) in 3D layered hydrogels. Thus, this 3D system expands the range of neural phenotypes that can be studied in vitro to include those influenced by physical and mechanical stimuli or requiring specific arrangements of multiple cell types.", "title": "Layered hydrogels accelerate iPSC-derived neuronal maturation and reveal migration defects caused by MeCP2 dysfunction." }, { "docid": "9122283", "text": "RATIONALE Multiple biological mechanisms contribute to the efficacy of cardiac cell therapy. Most prominent among these are direct heart muscle and blood vessel regeneration from transplanted cells, as opposed to paracrine enhancement of tissue preservation and/or recruitment of endogenous repair. OBJECTIVE Human cardiac progenitor cells, cultured as cardiospheres (CSps) or as CSp-derived cells (CDCs), have been shown to be capable of direct cardiac regeneration in vivo. Here we characterized paracrine effects in CDC transplantation and investigated their relative importance versus direct differentiation of surviving transplanted cells. METHODS AND RESULTS In vitro, many growth factors were found in media conditioned by human adult CSps and CDCs; CDC-conditioned media exerted antiapoptotic effects on neonatal rat ventricular myocytes, and proangiogenic effects on human umbilical vein endothelial cells. In vivo, human CDCs secreted vascular endothelial growth factor, hepatocyte growth factor, and insulin-like growth factor 1 when transplanted into the same SCID mouse model of acute myocardial infarction where they were previously shown to improve function and to produce tissue regeneration. Injection of CDCs in the peri-infarct zone increased the expression of Akt, decreased apoptotic rate and caspase 3 level, and increased capillary density, indicating overall higher tissue resilience. Based on the number of human-specific cells relative to overall increases in capillary density and myocardial viability, direct differentiation quantitatively accounted for 20% to 50% of the observed effects. CONCLUSIONS Together with their spontaneous commitment to cardiac and angiogenic differentiation, transplanted CDCs serve as \"role models,\" recruiting endogenous regeneration and improving tissue resistance to ischemic stress. The contribution of the role model effect rivals or exceeds that of direct regeneration.", "title": "Relative roles of direct regeneration versus paracrine effects of human cardiosphere-derived cells transplanted into infarcted mice." }, { "docid": "12827098", "text": "Despite accumulating evidence suggesting local self-maintenance of tissue macrophages in the steady state, the dogma remains that tissue macrophages derive from monocytes. Using parabiosis and fate-mapping approaches, we confirmed that monocytes do not show significant contribution to tissue macrophages in the steady state. Similarly, we found that after depletion of lung macrophages, the majority of repopulation occurred by stochastic cellular proliferation in situ in a macrophage colony-stimulating factor (M-Csf)- and granulocyte macrophage (GM)-CSF-dependent manner but independently of interleukin-4. We also found that after bone marrow transplantation, host macrophages retained the capacity to expand when the development of donor macrophages was compromised. Expansion of host macrophages was functional and prevented the development of alveolar proteinosis in mice transplanted with GM-Csf-receptor-deficient progenitors. Collectively, these results indicate that tissue-resident macrophages and circulating monocytes should be classified as mononuclear phagocyte lineages that are independently maintained in the steady state.", "title": "Tissue-resident macrophages self-maintain locally throughout adult life with minimal contribution from circulating monocytes." }, { "docid": "29231620", "text": "Cortical malformations are commonly associated with intractable epilepsy and other developmental disorders. Our studies utilize the tish rat, a spontaneously occurring genetic model of subcortical band heterotopia (SBH) associated with epilepsy, to evaluate the developmental events underlying SBH formation in the neocortex. Our results demonstrate that Pax6(+) and Tbr2(+) progenitors are mislocalized in tish(+/-) and tish(-/-)- neocortex throughout neurogenesis. In addition, mislocalized tish(-/-) progenitors possess a longer cell cycle than wild type or normally-positioned tish(-/-) progenitors, owing to a lengthened G(2)+M+G(1) time. This mislocalization is not associated with adherens junction breakdown or loss of radial glial polarity in the ventricular zone (VZ), as assessed by immunohistochemistry against phalloidin (to identify F-actin), aPKC-λ and Par3. However, vimentin immunohistochemistry indicates that the radial glial scaffold is disrupted in the region of the tish(-/-) heterotopia. Moreover, lineage tracing experiments using in utero electroporation in tish(-/-) neocortex demonstrate that mislocalized progenitors do not retain contact with the ventricular surface and that ventricular/subventricular zone (SVZ) progenitors produce neurons that migrate into both the heterotopia and cortical plate (CP). Taken together, these findings define a series of developmental errors contributing to SBH formation that differs fundamentally from a primary error in neuronal migration.", "title": "Disturbances in the positioning, proliferation and apoptosis of neural progenitors contribute to subcortical band heterotopia formation." }, { "docid": "52805891", "text": "Environmental factors and host genetics interact to control the gut microbiota, which may have a role in the development of obesity and insulin resistance. TLR2-deficient mice, under germ-free conditions, are protected from diet-induced insulin resistance. It is possible that the presence of gut microbiota could reverse the phenotype of an animal, inducing insulin resistance in an animal genetically determined to have increased insulin sensitivity, such as the TLR2 KO mice. In the present study, we investigated the influence of gut microbiota on metabolic parameters, glucose tolerance, insulin sensitivity, and signaling of TLR2-deficient mice. We investigated the gut microbiota (by metagenomics), the metabolic characteristics, and insulin signaling in TLR2 knockout (KO) mice in a non-germ free facility. Results showed that the loss of TLR2 in conventionalized mice results in a phenotype reminiscent of metabolic syndrome, characterized by differences in the gut microbiota, with a 3-fold increase in Firmicutes and a slight increase in Bacteroidetes compared with controls. These changes in gut microbiota were accompanied by an increase in LPS absorption, subclinical inflammation, insulin resistance, glucose intolerance, and later, obesity. In addition, this sequence of events was reproduced in WT mice by microbiota transplantation and was also reversed by antibiotics. At the molecular level the mechanism was unique, with activation of TLR4 associated with ER stress and JNK activation, but no activation of the IKKβ-IκB-NFκB pathway. Our data also showed that in TLR2 KO mice there was a reduction in regulatory T cell in visceral fat, suggesting that this modulation may also contribute to the insulin resistance of these animals. Our results emphasize the role of microbiota in the complex network of molecular and cellular interactions that link genotype to phenotype and have potential implications for common human disorders involving obesity, diabetes, and even other immunological disorders.", "title": "Gut Microbiota Is a Key Modulator of Insulin Resistance in TLR 2 Knockout Mice" }, { "docid": "19204979", "text": "Cells derived from blood vessels of human skeletal muscle can regenerate skeletal muscle, similarly to embryonic mesoangioblasts. However, adult cells do not express endothelial markers, but instead express markers of pericytes, such as NG2 proteoglycan and alkaline phosphatase (ALP), and can be prospectively isolated from freshly dissociated ALP+ cells. Unlike canonical myogenic precursors (satellite cells), pericyte-derived cells express myogenic markers only in differentiated myotubes, which they form spontaneously with high efficiency. When transplanted into severe combined immune deficient–X-linked, mouse muscular dystrophy (scid–mdx) mice, pericyte-derived cells colonize host muscle and generate numerous fibres expressing human dystrophin. Similar cells isolated from Duchenne patients, and engineered to express human mini-dystrophin, also give rise to many dystrophin-positive fibres in vivo. These data show that myogenic precursors, distinct from satellite cells, are associated with microvascular walls in the human skeletal muscle, may represent a correlate of embryonic 'mesoangioblasts' present after birth and may be a promising candidate for future cell-therapy protocols in patients.", "title": "Pericytes of human skeletal muscle are myogenic precursors distinct from satellite cells" }, { "docid": "18399038", "text": "Glioma tumour-initiating cells (GTICs) can originate upon the transformation of neural progenitor cells (NPCs). Studies on GTICs have focused on primary tumours from which GTICs could be isolated and the use of human embryonic material. Recently, the somatic genomic landscape of human gliomas has been reported. RTK (receptor tyrosine kinase) and p53 signalling were found dysregulated in ∼90% and 86% of all primary tumours analysed, respectively. Here we report on the use of human-induced pluripotent stem cells (hiPSCs) for modelling gliomagenesis. Dysregulation of RTK and p53 signalling in hiPSC-derived NPCs (iNPCs) recapitulates GTIC properties in vitro. In vivo transplantation of transformed iNPCs leads to highly aggressive tumours containing undifferentiated stem cells and their differentiated derivatives. Metabolic modulation compromises GTIC viability. Last, screening of 101 anti-cancer compounds identifies three molecules specifically targeting transformed iNPCs and primary GTICs. Together, our results highlight the potential of hiPSCs for studying human tumourigenesis.", "title": "Establishment of human iPSC-based models for the study and targeting of glioma initiating cells" }, { "docid": "24612804", "text": "IL-17 is a novel, CD4+ T cell-restricted cytokine. In vivo, it stimulates hematopoiesis and causes neutrophilia consisting of mature granulocytes. In this study, we show that IL-17-mediated granulopoiesis requires G-CSF release and the presence or induction of the transmembrane form of stem cell factor (SCF) for optimal granulopoiesis. However, IL-17 also protects mice from G-CSF neutralization-induced neutropenia. G-CSF neutralization completely reversed IL-17-induced BM progenitor expansion, whereas splenic CFU-GM/CFU-granulocyte-erythrocyte-megakaryocyte-monocyte was only reduced by 50% in both Sl/Sld and littermate control mice. Thus, there remained a significant SCF/G-CSF-independent effect of IL-17 on splenic granulopoiesis, resulting in a preservation of mature circulating granulocytes. IL-17 is a cytokine that potentially interconnects lymphocytic and myeloid host defense and may have potential for therapeutic development.", "title": "Requirement of endogenous stem cell factor and granulocyte-colony-stimulating factor for IL-17-mediated granulopoiesis." }, { "docid": "12742164", "text": "Stem cells, which are clonogenic cells with self-renewal and multilineage differentiation properties, have the potential to replace or repair damaged tissue. We have directly isolated clonogenic human central nervous system stem cells (hCNS-SC) from fresh human fetal brain tissue, using antibodies to cell surface markers and fluorescence-activated cell sorting. These hCNS-SC are phenotypically 5F3 (CD133)(+), 5E12(+), CD34(-), CD45(-), and CD24(-/lo). Single CD133(+) CD34(-) CD45(-) sorted cells initiated neurosphere cultures, and the progeny of clonogenic cells could differentiate into both neurons and glial cells. Single cells from neurosphere cultures initiated from CD133(+) CD34(-) CD45(-) cells were again replated as single cells and were able to reestablish neurosphere cultures, demonstrating the self-renewal potential of this highly enriched population. Upon transplantation into brains of immunodeficient neonatal mice, the sorted/expanded hCNS-SC showed potent engraftment, proliferation, migration, and neural differentiation.", "title": "Direct isolation of human central nervous system stem cells." }, { "docid": "4427392", "text": "The functional heart is comprised of distinct mesoderm-derived lineages including cardiomyocytes, endothelial cells and vascular smooth muscle cells. Studies in the mouse embryo and the mouse embryonic stem cell differentiation model have provided evidence indicating that these three lineages develop from a common Flk-1+ (kinase insert domain protein receptor, also known as Kdr) cardiovascular progenitor that represents one of the earliest stages in mesoderm specification to the cardiovascular lineages. To determine whether a comparable progenitor is present during human cardiogenesis, we analysed the development of the cardiovascular lineages in human embryonic stem cell differentiation cultures. Here we show that after induction with combinations of activin A, bone morphogenetic protein 4 (BMP4), basic fibroblast growth factor (bFGF, also known as FGF2), vascular endothelial growth factor (VEGF, also known as VEGFA) and dickkopf homolog 1 (DKK1) in serum-free media, human embryonic-stem-cell-derived embryoid bodies generate a KDRlow/C-KIT(CD117)neg population that displays cardiac, endothelial and vascular smooth muscle potential in vitro and, after transplantation, in vivo. When plated in monolayer cultures, these KDRlow/C-KITneg cells differentiate to generate populations consisting of greater than 50% contracting cardiomyocytes. Populations derived from the KDRlow/C-KITneg fraction give rise to colonies that contain all three lineages when plated in methylcellulose cultures. Results from limiting dilution studies and cell-mixing experiments support the interpretation that these colonies are clones, indicating that they develop from a cardiovascular colony-forming cell. Together, these findings identify a human cardiovascular progenitor that defines one of the earliest stages of human cardiac development.", "title": "Human cardiovascular progenitor cells develop from a KDR+ embryonic-stem-cell-derived population" }, { "docid": "42787108", "text": "Lineage-specific differentiation potential varies among different human pluripotent stem cell (hPSC) lines, becoming therefore highly desirable to prospectively know which hPSC lines exhibit the highest differentiation potential for a certain lineage. We have compared the hematopoietic potential of 14 human embryonic stem cell (hESC)/induced pluripotent stem cell (iPSC) lines. The emergence of hemogenic progenitors, primitive and mature blood cells, and colony-forming unit (CFU) potential was analyzed at different time points. Significant differences in the propensity to differentiate toward blood were observed among hPSCs: some hPSCs exhibited good blood differentiation potential, whereas others barely displayed blood-differentiation capacity. Correlation studies revealed that the CFU potential robustly correlates with hemogenic progenitors and primitive but not mature blood cells. Developmental progression of mesoendodermal and hematopoietic transcription factors expression revealed no correlation with either hematopoietic initiation or maturation efficiency. Microarray studies showed distinct gene expression profile between hPSCs with good versus poor hematopoietic potential. Although neuroectoderm-associated genes were downregulated in hPSCs prone to hematopoietic differentiation many members of the Nodal/Activin signaling were upregulated, suggesting that this signaling predicts those hPSC lines with good blood-differentiation potential. The association between Nodal/Activin signaling and the hematopoietic differentiation potential was confirmed using loss- and gain-of-function functional assays. Our data reinforce the value of prospective comparative studies aimed at determining the lineage-specific differentiation potential among different hPSCs and indicate that Nodal/Activin signaling seems to predict those hPSC lines prone to hematopoietic specification.", "title": "Nodal/Activin signaling predicts human pluripotent stem cell lines prone to differentiate toward the hematopoietic lineage." }, { "docid": "11527199", "text": "Background Current criteria for the selection of unrelated donors for hematopoietic cell transplantation (HCT) include matching for the alleles of each human leukocyte antigen (HLA) locus within the major histocompatibility complex (MHC). Graft-versus-host disease (GVHD), however, remains a significant and potentially life-threatening complication even after HLA-identical unrelated HCT. The MHC harbors more than 400 genes, but the total number of transplantation antigens is unknown. Genes that influence transplantation outcome could be identified by using linkage disequilibrium (LD)-mapping approaches, if the extended MHC haplotypes of the unrelated donor and recipient could be defined.", "title": "MHC Haplotype Matching for Unrelated Hematopoietic Cell Transplantation" }, { "docid": "36398420", "text": "The purpose of this study was to determine the lineage progression of human and murine very small embryonic-like (HuVSEL or MuVSEL) cells in vitro and in vivo. In vitro, HuVSEL and MuVSEL cells differentiated into cells of all three embryonic germ layers. HuVSEL cells produced robust mineralized tissue of human origin compared with controls in calvarial defects. Immunohistochemistry demonstrated that the HuVSEL cells gave rise to neurons, adipocytes, chondrocytes, and osteoblasts within the calvarial defects. MuVSEL cells were also able to differentiate into similar lineages. First round serial transplants of MuVSEL cells into irradiated osseous sites demonstrated that ∼60% of the cells maintained their VSEL cell phenotype while other cells differentiated into multiple tissues at 3 months. Secondary transplants did not identify donor VSEL cells, suggesting limited self renewal but did demonstrate VSEL cell derivatives in situ for up to 1 year. At no point were teratomas identified. These studies show that VSEL cells produce multiple cellular structures in vivo and in vitro and lay the foundation for future cell-based regenerative therapies for osseous, neural, and connective tissue disorders.", "title": "Human and murine very small embryonic-like cells represent multipotent tissue progenitors, in vitro and in vivo." } ]

[ { "docid": "14637235", "text": "Histone levels are tightly regulated to prevent harmful effects such as genomic instability and hypersensitivity to DNA-damaging agents due to the accumulation of these highly basic proteins when DNA replication slows down or stops. Although chromosomal histones are stable, excess (non-chromatin bound) histones are rapidly degraded in a Rad53 (radiation sensitive 53) kinase-dependent manner in Saccharomyces cerevisiae. Here we demonstrate that excess histones associate with Rad53 in vivo and seem to undergo modifications such as tyrosine phosphorylation and polyubiquitylation, before their proteolysis by the proteasome. We have identified the Tyr 99 residue of histone H3 as being critical for the efficient ubiquitylation and degradation of this histone. We have also identified the ubiquitin conjugating enzymes (E2) Ubc4 and Ubc5, as well as the ubiquitin ligase (E3) Tom1 (temperature dependent organization in mitotic nucleus 1), as enzymes involved in the ubiquitylation of excess histones. Regulated histone proteolysis has major implications for the maintenance of epigenetic marks on chromatin, genomic stability and the packaging of sperm DNA.", "title": "Histone levels are regulated by phosphorylation and ubiquitylation dependent proteolysis" } ]

[ { "docid": "7137057", "text": "BACKGROUND & AIMS HBV covalently closed circular DNA (cccDNA), the replicative intermediate responsible for persistent HBV infection of hepatocytes, is the template for transcription of all viral mRNAs. Nuclear cccDNA accumulates as a stable episome organized into minichromosomes by histone and nonhistone proteins. In this study we investigated, by a newly developed sensitive and specific assay, the relationship between viral replication and HBV chromatin assembly, transcription, and interaction with viral and cellular regulatory proteins. METHODS To achieve this aim we coupled a quantitative chromatin immunoprecipitation (ChIP) technique to an established method that allows the amplification of virion-encapsidated HBV genomes after transfection of linear HBV DNA into human hepatoma HuH7 cells. The cccDNA-ChIP technique was also applied to study HBV minichromosome transcriptional regulation in liver tissue from HBV-infected patients. RESULTS The use of anti-acetyl-H4/-H3 specific antibodies to immunoprecipitate transcriptionally active chromatin revealed that HBV replication is regulated by the acetylation status of the cccDNA-bound H3/H4 histones. Class I histone deacetylases inhibitors induced an evident increase of both cccDNA-bound acetylated H4 and HBV replication. Finally, histones hypoacetylation and histone deacetylase 1 recruitment onto the cccDNA in liver tissue correlated with low HBV viremia in hepatitis B patients. CONCLUSIONS We developed a ChIP-based assay to analyze, in vitro and ex vivo, the transcriptional regulation of HBV cccDNA minichromosome. Our results provide new insights on the regulation of HBV replication and identify the enzymatic activities that modulate the acetylation of cccDNA-bound histones as new therapeutic targets for anti-HBV drugs.", "title": "Hepatitis B virus replication is regulated by the acetylation status of hepatitis B virus cccDNA-bound H3 and H4 histones." }, { "docid": "12588500", "text": "Chromatin assembly factor 1 (CAF-1) and Rtt106 participate in the deposition of newly synthesized histones onto replicating DNA to form nucleosomes. This process is critical for the maintenance of genome stability and inheritance of functionally specialized chromatin structures in proliferating cells. However, the molecular functions of the acetylation of newly synthesized histones in this DNA replication-coupled nucleosome assembly pathway remain enigmatic. Here we show that histone H3 acetylated at lysine 56 (H3K56Ac) is incorporated onto replicating DNA and, by increasing the binding affinity of CAF-1 and Rtt106 for histone H3, H3K56Ac enhances the ability of these histone chaperones to assemble DNA into nucleosomes. Genetic analysis indicates that H3K56Ac acts in a nonredundant manner with the acetylation of the N-terminal residues of H3 and H4 in nucleosome assembly. These results reveal a mechanism by which H3K56Ac regulates replication-coupled nucleosome assembly mediated by CAF-1 and Rtt106.", "title": "Acetylation of Histone H3 Lysine 56 Regulates Replication-Coupled Nucleosome Assembly" }, { "docid": "14155726", "text": "Nuclear actin-related proteins (Arps) are subunits of several chromatin remodelers, but their molecular functions within these complexes are unclear. We report the crystal structure of the INO80 complex subunit Arp8 in its ATP-bound form. Human Arp8 has several insertions in the conserved actin fold that explain its inability to polymerize. Most remarkably, one insertion wraps over the active site cleft and appears to rigidify the domain architecture, while active site features shared with actin suggest an allosterically controlled ATPase activity. Quantitative binding studies with nucleosomes and histone complexes reveal that Arp8 and the Arp8-Arp4-actin-HSA sub-complex of INO80 strongly prefer nucleosomes and H3-H4 tetramers over H2A-H2B dimers, suggesting that Arp8 functions as a nucleosome recognition module. In contrast, Arp4 prefers free (H3-H4)(2) over nucleosomes and may serve remodelers through binding to (dis)assembly intermediates in the remodeling reaction.", "title": "Structure of Actin-related protein 8 and its contribution to nucleosome binding" }, { "docid": "19485243", "text": "The transcription factors HNF3 (FoxA) and GATA-4 are the earliest known to bind the albumin gene enhancer in liver precursor cells in embryos. To understand how they access sites in silent chromatin, we assembled nucleosome arrays containing albumin enhancer sequences and compacted them with linker histone. HNF3 and GATA-4, but not NF-1, C/EBP, and GAL4-AH, bound their sites in compacted chromatin and opened the local nucleosomal domain in the absence of ATP-dependent enzymes. The ability of HNF3 to open chromatin is mediated by a high affinity DNA binding site and by the C-terminal domain of the protein, which binds histones H3 and H4. Thus, factors that potentiate transcription in development are inherently capable of initiating chromatin opening events.", "title": "Opening of compacted chromatin by early developmental transcription factors HNF3 (FoxA) and GATA-4." }, { "docid": "502591", "text": "E2F proteins can either activate or repress transcription. Following mitogenic stimulation, repressive E2F4-p130-histone deacetylase complexes dissociate from, while activating species (E2F1, -2, and -3) associate with, target promoters. Histones H3 and H4 simultaneously become hyperacetylated, but it remains unclear whether this is a prerequisite or a consequence of E2F binding. Here, we show that activating E2F species are required for hyperacetylation of target chromatin in human cells. Overexpression of a dominant-negative (DN) E2F1 mutant in serum-stimulated T98G cells blocked all E2F binding, H4 acetylation, and, albeit partially, H3 acetylation. Target gene activation and S-phase entry were also blocked by DN E2F1. Conversely, ectopic activation of E2F1 rapidly induced H3 and H4 acetylation, demonstrating a direct role for E2F in these events. E2F1 was previously shown to bind the histone acetyltransferases (HATs) p300/CBP and PCAF/GCN5. In our hands, ectopically expressed E2F1 also bound the unrelated HAT Tip60 and induced recruitment of five subunits of the Tip60 complex (Tip60, TRRAP, p400, Tip48, and Tip49) to target promoters in vivo. Moreover, E2F-dependent recruitment of Tip60 to chromatin occurred in late G(1) following serum stimulation. We speculate that the activities of multiple HAT complexes account for E2F-dependent acetylation, transcription, and S-phase entry.", "title": "E2F-dependent histone acetylation and recruitment of the Tip60 acetyltransferase complex to chromatin in late G1." }, { "docid": "13907427", "text": "Poly(ADP-ribosyl)ation plays a major role in DNA repair, where it regulates chromatin relaxation as one of the critical events in the repair process. However, the molecular mechanism by which poly(ADP-ribose) modulates chromatin remains poorly understood. Here we identify the poly(ADP-ribose)-regulated protein APLF as a DNA-damage-specific histone chaperone. APLF preferentially binds to the histone H3/H4 tetramer via its C-terminal acidic motif, which is homologous to the motif conserved in the histone chaperones of the NAP1L family (NAP1L motif). We further demonstrate that APLF exhibits histone chaperone activities in a manner that is dependent on its acidic domain and that the NAP1L motif is critical for the repair capacity of APLF in vivo. Finally, we identify structural analogs of APLF in lower eukaryotes with the ability to bind histones and localize to the sites of DNA-damage-induced poly(ADP-ribosyl)ation. Collectively, these findings define the involvement of histone chaperones in poly(ADP-ribose)-regulated DNA repair reactions.", "title": "DNA repair factor APLF is a histone chaperone." }, { "docid": "14338915", "text": "The mechanisms ensuring specific incorporation of CENP-A at centromeres are poorly understood. Mis16 and Mis18 are required for CENP-A localization at centromeres and form a complex that is conserved from fission yeast to human. Fission yeast sim1 mutants that alleviate kinetochore domain silencing are defective in Scm3(Sp), the ortholog of budding yeast Scm3(Sc). Scm3(Sp) depends on Mis16/18 for its centromere localization and like them is recruited to centromeres in late anaphase. Importantly, Scm3(Sp) coaffinity purifies with CENP-A(Cnp1) and associates with CENP-A(Cnp1) in vitro, yet localizes independently of intact CENP-A(Cnp1) chromatin and is differentially released from chromatin. While Scm3(Sc) has been proposed to form a unique hexameric nucleosome with CENP-A(Cse4) and histone H4 at budding yeast point centromeres, we favor a model in which Scm3(Sp) acts as a CENP-A(Cnp1) receptor/assembly factor, cooperating with Mis16 and Mis18 to receive CENP-A(Cnp1) from the Sim3 escort and mediate assembly of CENP-A(Cnp1) into subkinetochore chromatin.", "title": "Fission Yeast Scm3: A CENP-A Receptor Required for Integrity of Subkinetochore Chromatin" }, { "docid": "20781656", "text": "Some three decades have passed since the discovery of nucleosomes in 1974 and the first isolation of a histone chaperone in 1978. While various types of histone chaperones have been isolated and functionally analyzed, the elementary processes of nucleosome assembly and disassembly have been less well characterized. Recently, the tertiary structure of a hetero-trimeric complex composed of the histone chaperone CIA/ASF1 and the histone H3-H4 dimer was determined, and this complex was proposed to be an intermediate in nucleosome assembly and disassembly reactions. In addition, CIA alone was biochemically shown to dissociate the histone (H3-H4)2 tetramer into two histone H3-H4 dimers. This activity suggested that CIA regulates the semi-conservative replication of nucleosomes. Here, we provide an overview of prominent histone chaperones with the goal of elucidating the mechanisms that preserve and modify epigenetic information. We also discuss the reactions involved in nucleosome assembly and disassembly.", "title": "Histone chaperones: 30 years from isolation to elucidation of the mechanisms of nucleosome assembly and disassembly" }, { "docid": "29877890", "text": "Recent structures of the nucleosome core particle reveal details of histone-histone and histone-DNA interactions. These structures have now set the stage for understanding chromatin assembly and dynamics during replication and transcription. Histone chaperones and chromatin remodeling complexes are important in both of these processes. The nucleosome and its protein core, the histone octamer, have twofold symmetry, which histone chaperones may use to bind core histones. Recent studies suggest that the nucleoplasmin pentamer may mediate histone storage, sperm chromatin decondensation and nucleosome assembly, by dimerizing to form a decamer. In this model, histone binding on the lateral surface of the chaperone involves stereospecific interactions and a shared twofold axis.", "title": "Histone chaperones and nucleosome assembly." }, { "docid": "175735", "text": "MOTIVATION The nucleosome is the basic repeating unit of chromatin. It contains two copies each of the four core histones H2A, H2B, H3 and H4 and about 147 bp of DNA. The residues of the histone proteins are subject to numerous post-translational modifications, such as methylation or acetylation. Chromatin immunoprecipitiation followed by sequencing (ChIP-seq) is a technique that provides genome-wide occupancy data of these modified histone proteins, and it requires appropriate computational methods. RESULTS We present NucHunter, an algorithm that uses the data from ChIP-seq experiments directed against many histone modifications to infer positioned nucleosomes. NucHunter annotates each of these nucleosomes with the intensities of the histone modifications. We demonstrate that these annotations can be used to infer nucleosomal states with distinct correlations to underlying genomic features and chromatin-related processes, such as transcriptional start sites, enhancers, elongation by RNA polymerase II and chromatin-mediated repression. Thus, NucHunter is a versatile tool that can be used to predict positioned nucleosomes from a panel of histone modification ChIP-seq experiments and infer distinct histone modification patterns associated to different chromatin states. AVAILABILITY The software is available at http://epigen.molgen.mpg.de/nuchunter/.", "title": "Inferring nucleosome positions with their histone mark annotation from ChIP data" }, { "docid": "1259280", "text": "The chromatin architecture of eukaryotic gene promoters is generally characterized by a nucleosome-free region (NFR) flanked by at least one H2A.Z variant nucleosome. Computational predictions of nucleosome positions based on thermodynamic properties of DNA-histone interactions have met with limited success. Here we show that the action of the essential RSC remodeling complex in S. cerevisiae helps explain the discrepancy between theory and experiment. In RSC-depleted cells, NFRs shrink such that the average positions of flanking nucleosomes move toward predicted sites. Nucleosome positioning at distinct subsets of promoters additionally requires the essential Myb family proteins Abf1 and Reb1, whose binding sites are enriched in NFRs. In contrast, H2A.Z deposition is dispensable for nucleosome positioning. By regulating H2A.Z deposition using a steroid-inducible protein splicing strategy, we show that NFR establishment is necessary for H2A.Z deposition. These studies suggest an ordered pathway for the assembly of promoter chromatin architecture.", "title": "Mechanisms that Specify Promoter Nucleosome Location and Identity" }, { "docid": "30041340", "text": "BACKGROUND Histone deimination regulates gene function and contributes to antimicrobial response, allowing the formation of neutrophil extracellular traps (NETs). Deiminated proteins are target of anti-citrullinated peptides antibodies (ACPA) in rheumatoid arthritis (RA). OBJECTIVE The objective of this paper is to test the hypothesis that RA sera react with deiminated histones contained in NETs. METHODS Neutrophils from peripheral blood were stimulated with A23187 and acid treated; NETosis was induced by phorbol myristate acetate, and NET proteins were isolated. Sera were tested by immunoblot on acid extracted proteins from neutrophils and from NETs, and by ELISA on deiminated histone H4 or H4-derived peptides. Bands reactive with RA sera were excised from gels, digested with trypsin and subjected to matrix-assisted laser desorption/ionisation time of flight (MALDI-TOF) analysis, before and after derivatisation to detect citrullinated peptides. RESULTS RA sera reacted with a deiminated antigen of 11 KDa from activated neutrophils, recognised also by anti-H4 and antideiminated H4 antibodies. A similar reactivity was observed with NET proteins. The antigen from neutrophils or NETs was identified as citrullinated H4 by MALDI-TOF analysis. By ELISA, RA sera bound in vitro citrullinated H4. Citrullinated H4 14-34 and 31-50 peptides detected antibodies in 67% and 63% of RA sera and in less than 5% of controls; antibody titre was correlated with anti-CCP2. CONCLUSIONS Citrullinated H4 from activated neutrophils and NETs is a target of antibodies in RA, and synthetic citrullinated H4-derived peptides are a new substrate for ACPA detection. As NETosis can generate antigens for ACPA, these data suggest a novel connection between innate and adaptive immunity in RA.", "title": "Antibodies from patients with rheumatoid arthritis target citrullinated histone 4 contained in neutrophils extracellular traps." }, { "docid": "5966635", "text": "Activation of transcription within chromatin has been correlated with the incorporation of the essential histone variant H2A.Z into nucleosomes. H2A.Z and other histone variants may establish structurally distinct chromosomal domains; however, the molecular mechanism by which they function is largely unknown. Here we report the 2.6 Å crystal structure of a nucleosome core particle containing the histone variant H2A.Z. The overall structure is similar to that of the previously reported 2.8 Å nucleosome structure containing major histone proteins. However, distinct localized changes result in the subtle destabilization of the interaction between the (H2A.Z–H2B) dimer and the (H3–H4)2 tetramer. Moreover, H2A.Z nucleosomes have an altered surface that includes a metal ion. This altered surface may lead to changes in higher order structure, and/or could result in the association of specific nuclear proteins with H2A.Z. Finally, incorporation of H2A.Z and H2A within the same nucleosome is unlikely, due to significant changes in the interface between the two H2A.Z–H2B dimers.", "title": "Crystal structure of a nucleosome core particle containing the variant histone H2A.Z" }, { "docid": "36831892", "text": "Considerable energetic investment is devoted to altering large stretches of chromatin adjacent to DNA double strand breaks (DSBs). Immediately ensuing DSB formation, a myriad of histone modifications are elicited to create a platform for inducible and modular assembly of DNA repair protein complexes in the vicinity of the DNA lesion. This complex signaling network is critical to repair DNA damage and communicate with cellular processes that occur in cis and in trans to the genomic lesion. Failure to properly execute DNA damage inducible chromatin changes is associated with developmental abnormalities, immunodeficiency, and malignancy in humans and in genetically engineered mouse models. This review will discuss current knowledge of DNA damage responsive histone changes that occur in mammalian cells, highlighting their involvement in the maintenance of genome integrity.", "title": "Histone tails: Directing the chromatin response to DNA damage." }, { "docid": "9732010", "text": "Histone acetyltransferases (HATs) and ATP-dependent chromatin remodeling factors (ADCRs) are involved in selective gene regulation via modulation of local chromatin configuration. Activation of the recombination hotspot ade6-M26 of Schizosaccharomyces pombe is mediated by a cAMP responsive element (CRE)-like sequence, M26, and a heterodimeric ATF/CREB transcription factor, Atf1.Pcr1. Chromatin remodeling occurs meiotically around M26. We examined the roles of HATs and ADCRs in chromatin remodeling around M26. Histones H3 and H4 around M26 were hyperacetylated in an M26- and Atf1-dependent manner early in meiosis. SpGcn5, the S. pombe homolog of Gcn5p, was required for the majority of histone H3 acetylation around M26 in vivo. Deletion of gcn5+ caused a significant delay in chromatin remodeling but only partial reduction of M26 meiotic recombination frequency. The snf22+ (a Swi2/Snf2-ADCR homologue) deletion and snf22+ gcn5+ double deletion abolished chromatin remodeling and significant reduction of meiotic recombination around M26. These results suggest that HATs and ADCRs cooperatively alter local chromatin structure, as in selective transcription activation, to activate meiotic recombination at M26 in a site-specific manner.", "title": "Roles of histone acetylation and chromatin remodeling factor in a meiotic recombination hotspot." }, { "docid": "25606339", "text": "TLR3 has been implicated in the pathogenesis of several viral infections, including SIV- and HIV-1-induced inflammation and AIDS. However the molecular mechanisms of these TLR3-mediated effects are not known, and it is not known whether HIV interacts with cellular TLR3 to affect disease process. Here we investigate the effects of TLR3 ligands on HIV-1 transactivation using both primary human macrophages and cells containing integrated copies of the HIV-1 promoter. We demonstrate that TLR3 activation induced upregulation of transcription factors such as c-Jun, CCAAT/enhancer-binding protein alpha (CEBPA), signal transducer and activator of transcription (STAT)-1, STAT-2, RELB, and nuclear factor kappa-B1 (NFκB1), most of which are known to regulate the HIV promoter activity. We also demonstrate that TLR3 activation increased HIV-1 transactivation via the c-Jun N-terminal kinase (JNK) and NFκB pathways. This was associated with epigenetic modifications, including decreased histone deacetylase activity, increased histone acetyl transferase (HAT) activity, and increased acetylation of histones H3 and H4 at lysine residues in the nucleosome-0 and nucleosome-1 of the HIV-1 promoter. However, prolonged TLR3 activation decreased HIV-1 transactivation, decreased HAT activity and Tat transcription, and suppressed viral replication. Overall, data suggests that TLR3 can act as viral sensor to mediate viral transactivation, cellular signaling, innate immune response, and inflammation in HIV-infected humans. Our study provides novel insights into the molecular basis for these TLR3-mediated effects.", "title": "Toll-like receptor-3 mediates HIV-1 transactivation via NFκB and JNK pathways and histone acetylation, but prolonged activation suppresses Tat and HIV-1 replication." }, { "docid": "2817000", "text": "In S. cerevisiae, histone variant H2A.Z is deposited in euchromatin at the flanks of silent heterochromatin to prevent its ectopic spread. We show that H2A.Z nucleosomes are found at promoter regions of nearly all genes in euchromatin. They generally occur as two positioned nucleosomes that flank a nucleosome-free region (NFR) that contains the transcription start site. Astonishingly, enrichment at 5' ends is observed not only at actively transcribed genes but also at inactive loci. Mutagenesis of a typical promoter revealed a 22 bp segment of DNA sufficient to program formation of a NFR flanked by two H2A.Z nucleosomes. This segment contains a binding site of the Myb-related protein Reb1 and an adjacent dT:dA tract. Efficient deposition of H2A.Z is further promoted by a specific pattern of histone H3 and H4 tail acetylation and the bromodomain protein Bdf1, a component of the Swr1 remodeling complex that deposits H2A.Z.", "title": "Histone Variant H2A.Z Marks the 5′ Ends of Both Active and Inactive Genes in Euchromatin" }, { "docid": "22522432", "text": "The stable contact of ISW2 with nucleosomal DNA approximately 20 bp from the dyad was shown by DNA footprinting and photoaffinity labeling using recombinant histone octamers to require the histone H4 N-terminal tail. Efficient ISW2 remodeling also required the H4 N-terminal tail, although the lack of the H4 tail can be mostly compensated for by increasing the incubation time or concentration of ISW2. Similarly, the length of extranucleosomal DNA affected the stable contact of ISW2 with this same internal nucleosomal site, with the optimal length being 70 to 85 bp. These data indicate the histone H4 tail, in concert with a favorable length of extranucleosomal DNA, recruits and properly orients ISW2 onto the nucleosome for efficient nucleosome remodeling. One consequence of this property of ISW2 is likely its previously observed nucleosome spacing activity.", "title": "Regulation of ISW2 by concerted action of histone H4 tail and extranucleosomal DNA." }, { "docid": "3669694", "text": "Generation of induced pluripotent stem cells (iPSCs) by somatic cell reprogramming involves global epigenetic remodelling. Whereas several proteins are known to regulate chromatin marks associated with the distinct epigenetic states of cells before and after reprogramming, the role of specific chromatin-modifying enzymes in reprogramming remains to be determined. To address how chromatin-modifying proteins influence reprogramming, we used short hairpin RNAs (shRNAs) to target genes in DNA and histone methylation pathways, and identified positive and negative modulators of iPSC generation. Whereas inhibition of the core components of the polycomb repressive complex 1 and 2, including the histone 3 lysine 27 methyltransferase EZH2, reduced reprogramming efficiency, suppression of SUV39H1, YY1 and DOT1L enhanced reprogramming. Specifically, inhibition of the H3K79 histone methyltransferase DOT1L by shRNA or a small molecule accelerated reprogramming, significantly increased the yield of iPSC colonies, and substituted for KLF4 and c-Myc (also known as MYC). Inhibition of DOT1L early in the reprogramming process is associated with a marked increase in two alternative factors, NANOG and LIN28, which play essential functional roles in the enhancement of reprogramming. Genome-wide analysis of H3K79me2 distribution revealed that fibroblast-specific genes associated with the epithelial to mesenchymal transition lose H3K79me2 in the initial phases of reprogramming. DOT1L inhibition facilitates the loss of this mark from genes that are fated to be repressed in the pluripotent state. These findings implicate specific chromatin-modifying enzymes as barriers to or facilitators of reprogramming, and demonstrate how modulation of chromatin-modifying enzymes can be exploited to more efficiently generate iPSCs with fewer exogenous transcription factors.", "title": "Chromatin modifying enzymes as modulators of reprogramming" }, { "docid": "8331432", "text": "The transcription factor HNF3 and linker histones H1 and H5 possess winged-helix DNA-binding domains, yet HNF3 and other fork head-related proteins activate genes during development whereas linker histones compact DNA in chromatin and repress gene expression. We compared how the two classes of factors interact with chromatin templates and found that HNF3 binds DNA at the side of nucleosome cores, similarly to what has been reported for linker histone. A nucleosome structural binding site for HNF3 is occupied at the albumin transcriptional enhancer in active and potentially active chromatin, but not in inactive chromatin in vivo. While wild-type HNF3 protein does not compact DNA extending from the nucleosome, as does linker histone, site-directed mutants of HNF3 can compact nucleosomal DNA if they contain basic amino acids at positions previously shown to be essential for nucleosomal DNA compaction by linker histones. The results illustrate how transcription factors can possess special nucleosome-binding activities that are not predicted from studies of factor interactions with free DNA.", "title": "Binding of the winged-helix transcription factor HNF3 to a linker histone site on the nucleosome." }, { "docid": "15960670", "text": "The centromere is a chromatin region that serves as the spindle attachment point and directs accurate inheritance of eukaryotic chromosomes during cell divisions. However, the mechanism by which the centromere assembles and stabilizes at a specific genomic region is not clear. The de novo formation of a human/mammalian artificial chromosome (HAC/MAC) with a functional centromere assembly requires the presence of alpha-satellite DNA containing binding motifs for the centromeric CENP-B protein. We demonstrate here that de novo centromere assembly on HAC/MAC is dependent on CENP-B. In contrast, centromere formation is suppressed in cells expressing CENP-B when alpha-satellite DNA was integrated into a chromosomal site. Remarkably, on those integration sites CENP-B enhances histone H3-K9 trimethylation and DNA methylation, thereby stimulating heterochromatin formation. Thus, we propose that CENP-B plays a dual role in centromere formation, ensuring de novo formation on DNA lacking a functional centromere but preventing the formation of excess centromeres on chromosomes.", "title": "CENP-B Controls Centromere Formation Depending on the Chromatin Context" }, { "docid": "24311787", "text": "Variant histone H2AZ-containing nucleosomes are involved in the regulation of gene expression. In Saccharomyces cerevisiae, chromatin deposition of histone H2AZ is mediated by the fourteen-subunit SWR1 complex, which catalyzes ATP-dependent exchange of nucleosomal histone H2A for H2AZ. Previous work defined the role of seven SWR1 subunits (Swr1 ATPase, Swc2, Swc3, Arp6, Swc5, Yaf9, and Swc6) in maintaining complex integrity and H2AZ histone replacement activity. Here we examined the function of three additional SWR1 subunits, bromodomain containing Bdf1, actin-related protein Arp4 and Swc7, by analyzing affinity-purified mutant SWR1 complexes. We observed that depletion of Arp4 (arp4-td) substantially impaired the association of Bdf1, Yaf9, and Swc4. In contrast, loss of either Bdf1 or Swc7 had minimal effects on overall complex integrity. Furthermore, the basic H2AZ histone replacement activity of SWR1 in vitro required Arp4, but not Bdf1 or Swc7. Thus, three out of fourteen SWR1 subunits, Bdf1, Swc7, and previously noted Swc3, appear to have roles auxiliary to the basic histone replacement activity. The N-terminal region of the Swr1 ATPase subunit is necessary and sufficient to direct association of Bdf1 and Swc7, as well as Arp4, Act1, Yaf9 and Swc4. This same region contains an additional H2AZ-H2B specific binding site, distinct from the previously identified Swc2 subunit. These findings suggest that one SWR1 enzyme might be capable of binding two H2AZ-H2B dimers, and provide further insight on the hierarchy and interdependency of molecular interactions within the SWR1 complex.", "title": "N terminus of Swr1 binds to histone H2AZ and provides a platform for subunit assembly in the chromatin remodeling complex." }, { "docid": "29788648", "text": "NuA4, the major H4 lysine acetyltransferase (KAT) complex in Saccharomyces cerevisiae, is recruited to promoters and stimulates transcription initiation. NuA4 subunits contain domains that bind methylated histones, suggesting that histone methylation should target NuA4 to coding sequences during transcription elongation. We show that NuA4 is cotranscriptionally recruited, dependent on its physical association with elongating polymerase II (Pol II) phosphorylated on the C-terminal domain by cyclin-dependent kinase 7/Kin28, but independently of subunits (Eaf1 and Tra1) required for NuA4 recruitment to promoters. Whereas histone methylation by Set1 and Set2 is dispensable for NuA4's interaction with Pol II and targeting to some coding regions, it stimulates NuA4-histone interaction and H4 acetylation in vivo. The NuA4 KAT, Esa1, mediates increased H4 acetylation and enhanced RSC occupancy and histone eviction in coding sequences and stimulates the rate of transcription elongation. Esa1 cooperates with the H3 KAT in SAGA, Gcn5, to enhance these functions. Our findings delineate a pathway for acetylation-mediated nucleosome remodeling and eviction in coding sequences that stimulates transcription elongation by Pol II in vivo.", "title": "NuA4 lysine acetyltransferase Esa1 is targeted to coding regions and stimulates transcription elongation with Gcn5." }, { "docid": "20630805", "text": "Histone posttranslational modifications are key components of diverse processes that modulate chromatin structure. These marks function as signals during various chromatin-based events, and act as platforms for recruitment, assembly or retention of chromatin-associated factors. The best-known function of histone phosphorylation takes place during cellular response to DNA damage, when phosphorylated histone H2A(X) demarcates large chromatin domains around the site of DNA breakage. However, multiple studies have also shown that histone phosphorylation plays crucial roles in chromatin remodeling linked to other nuclear processes. In this review, we summarize the current knowledge of histone phosphorylation and describe the many kinases and phosphatases that regulate it. We discuss the key roles played by this histone mark in DNA repair, transcription and chromatin compaction during cell division and apoptosis. Additionally, we describe the intricate crosstalk that occurs between phosphorylation and other histone modifications and allows for sophisticated control over the chromatin remodeling processes.", "title": "Histone phosphorylation: a chromatin modification involved in diverse nuclear events." }, { "docid": "20368353", "text": "The basic determinant of chromosome inheritance, the centromere, is specified in many eukaryotes by an epigenetic mark. Using gene targeting in human cells and fission yeast, chromatin containing the centromere-specific histone H3 variant CENP-A is demonstrated to be the epigenetic mark that acts through a two-step mechanism to identify, maintain and propagate centromere function indefinitely. Initially, centromere position is replicated and maintained by chromatin assembled with the centromere-targeting domain (CATD) of CENP-A substituted into H3. Subsequently, nucleation of kinetochore assembly onto CATD-containing chromatin is shown to require either the amino- or carboxy-terminal tail of CENP-A for recruitment of inner kinetochore proteins, including stabilizing CENP-B binding to human centromeres or direct recruitment of CENP-C, respectively.", "title": "A two-step mechanism for epigenetic specification of centromere identity and function" }, { "docid": "46248894", "text": "Long intergenic noncoding RNAs (lincRNAs) regulate chromatin states and epigenetic inheritance. Here, we show that the lincRNA HOTAIR serves as a scaffold for at least two distinct histone modification complexes. A 5' domain of HOTAIR binds polycomb repressive complex 2 (PRC2), whereas a 3' domain of HOTAIR binds the LSD1/CoREST/REST complex. The ability to tether two distinct complexes enables RNA-mediated assembly of PRC2 and LSD1 and coordinates targeting of PRC2 and LSD1 to chromatin for coupled histone H3 lysine 27 methylation and lysine 4 demethylation. Our results suggest that lincRNAs may serve as scaffolds by providing binding surfaces to assemble select histone modification enzymes, thereby specifying the pattern of histone modifications on target genes.", "title": "Long noncoding RNA as modular scaffold of histone modification complexes" }, { "docid": "36547290", "text": "IL-6 is an immunoregulatory cytokine with multiple functions in hemopoiesis, proliferation, and tumorigenesis. IL-6 triggers phosphorylation, dimerization, and nuclear translocation of STAT3, which binds to target promoters and activates transcription. Brahma-related gene 1 (BRG1), the enzymatic engine of the yeast-mating type-switching and sucrose-nonfermenting chromatin-remodeling complex, is essential for recruitment of STAT1 or STAT1/STAT2-containing complexes to IFN targets. We hypothesized that BRG1 might also be required for STAT3 recruitment. In this study, we show that induction of a subset of human IL-6-responsive genes is BRG1 dependent. BRG1 is constitutively present at these targets and is required for STAT3 recruitment, downstream histone modifications, and IL-6-induced chromatin remodeling. IL-6-induced recruitment of STAT3 to the IFN regulatory factor 1 promoter and subsequent mRNA synthesis is BRG1 dependent, even though IFN-gamma-mediated STAT1 recruitment to this locus is BRG1 independent. BRG1 also increased basal expression of IFN-induced transmembrane protein 3 and IFN-gamma-induced protein 16, and the basal chromatin accessibility at the promoter of IFN regulatory factor 1. The effect on basal expression was STAT3 independent, as revealed by small interfering RNA knockdown. Together with prior observations, these data reveal that BRG1 has a broad role in mediating STAT accessibility at multiple cytokine-responsive promoters and exposes promoter specific differences in both the effect of BRG1 on basal chromatin accessibility and on access of different STAT proteins to the same target.", "title": "Brahma-related gene 1-dependent STAT3 recruitment at IL-6-inducible genes." }, { "docid": "25041967", "text": "Human artificial chromosomes have been used to model requirements for human chromosome segregation and to explore the nature of sequences competent for centromere function. Normal human centromeres require specialized chromatin that consists of alpha satellite DNA complexed with epigenetically modified histones and centromere-specific proteins. While several types of alpha satellite DNA have been used to assemble de novo centromeres in artificial chromosome assays, the extent to which they fully recapitulate normal centromere function has not been explored. Here, we have used two kinds of alpha satellite DNA, DXZ1 (from the X chromosome) and D17Z1 (from chromosome 17), to generate human artificial chromosomes. Although artificial chromosomes are mitotically stable over many months in culture, when we examined their segregation in individual cell divisions using an anaphase assay, artificial chromosomes exhibited more segregation errors than natural human chromosomes (P < 0.001). Naturally occurring, but abnormal small ring chromosomes derived from chromosome 17 and the X chromosome also missegregate more than normal chromosomes, implicating overall chromosome size and/or structure in the fidelity of chromosome segregation. As different artificial chromosomes missegregate over a fivefold range, the data suggest that variable centromeric DNA content and/or epigenetic assembly can influence the mitotic behavior of artificial chromosomes.", "title": "Human artificial chromosomes with alpha satellite-based de novo centromeres show increased frequency of nondisjunction and anaphase lag." }, { "docid": "1684489", "text": "BACKGROUND Production of the GTP-bound form of the Ran GTPase (RanGTP) around chromosomes induces spindle assembly by activating nuclear localization signal (NLS)-containing proteins. Several NLS proteins have been identified as spindle assembly factors, but the complexity of the process led us to search for additional proteins with distinct roles in spindle assembly. RESULTS We identify a chromatin-remodeling ATPase, CHD4, as a RanGTP-dependent microtubule (MT)-associated protein (MAP). MT binding occurs via the region containing an NLS and chromatin-binding domains. In Xenopus egg extracts and cultured cells, CHD4 largely dissociates from mitotic chromosomes and partially localizes to the spindle. Immunodepletion of CHD4 from egg extracts significantly reduces the quantity of MTs produced around chromatin and prevents spindle assembly. CHD4 RNAi in both HeLa and Drosophila S2 cells induces defects in spindle assembly and chromosome alignment in early mitosis, leading to chromosome missegregation. Further analysis in egg extracts and in HeLa cells reveals that CHD4 is a RanGTP-dependent MT stabilizer. Moreover, the CHD4-containing NuRD complex promotes organization of MTs into bipolar spindles in egg extracts. Importantly, this function of CHD4 is independent of chromatin remodeling. CONCLUSIONS Our results uncover a new role for CHD4 as a MAP required for MT stabilization and involved in generating spindle bipolarity.", "title": "CHD4 Is a RanGTP-Dependent MAP that Stabilizes Microtubules and Regulates Bipolar Spindle Formation" }, { "docid": "18038955", "text": "INO80 is an evolutionarily conserved, ATP-dependent chromatin-remodeling enzyme that plays roles in transcription, DNA repair, and replication. Here, we show that yeast INO80 facilitates these diverse processes at least in part by controlling genome-wide distribution of the histone variant H2A.Z. In the absence of INO80, H2A.Z nucleosomes are mislocalized, and H2A.Z levels at promoters show reduced responsiveness to transcriptional changes, suggesting that INO80 controls H2A.Z dynamics. Additionally, we demonstrate that INO80 has a histone-exchange activity in which the enzyme can replace nucleosomal H2A.Z/H2B with free H2A/H2B dimers. Genetic interactions between ino80 and htz1 support a model in which INO80 catalyzes the removal of unacetylated H2A.Z from chromatin as a mechanism to promote genome stability.", "title": "Global Regulation of H2A.Z Localization by the INO80 Chromatin-Remodeling Enzyme Is Essential for Genome Integrity" } ]

[ { "docid": "16120395", "text": "Tight regulation of the expression of mRNAs encoding iron uptake proteins is essential to control iron homeostasis and avoid intracellular iron toxicity. We show that many mRNAs encoding iron uptake or iron mobilization proteins are expressed in iron-replete conditions in the absence of the S. cerevisiae RNase III ortholog Rnt1p or of the nuclear exosome component Rrp6p. Extended forms of these mRNAs accumulate in the absence of Rnt1p or of the 5'-->3' exonucleases Xrn1p and Rat1p, showing that multiple degradative pathways contribute to the surveillance of aberrant forms of these transcripts. RNase III-deficient cells are hypersensitive to high iron concentrations, suggesting that Rnt1p-mediated RNA surveillance is required to prevent iron toxicity. These results show that RNA surveillance through multiple ribonucleolytic pathways plays a role in iron homeostasis in yeast to avoid the potentially toxic effects of the expression of the iron starvation response in iron-replete conditions.", "title": "Multiple RNA surveillance pathways limit aberrant expression of iron uptake mRNAs and prevent iron toxicity in S. cerevisiae." } ]

[ { "docid": "8247597", "text": "Mutations and deletions in the mitochondrial genome (mtDNA), as well as instability of the nuclear genome, are involved in multiple human diseases. Here, we report that in Saccharomyces cerevisiae, loss of mtDNA leads to nuclear genome instability, through a process of cell-cycle arrest and selection we define as a cellular crisis. This crisis is not mediated by the absence of respiration, but instead correlates with a reduction in the mitochondrial membrane potential. Analysis of cells undergoing this crisis identified a defect in iron-sulfur cluster (ISC) biogenesis, which requires normal mitochondrial function. We found that downregulation of nonmitochondrial ISC protein biogenesis was sufficient to cause increased genomic instability in cells with intact mitochondrial function. These results suggest mitochondrial dysfunction stimulates nuclear genome instability by inhibiting the production of ISC-containing protein(s), which are required for maintenance of nuclear genome integrity. For a video summary of this article, see the PaperFlick file available with the online Supplemental Data.", "title": "Mitochondrial Dysfunction Leads to Nuclear Genome Instability via an Iron-Sulfur Cluster Defect" }, { "docid": "23664875", "text": "Termination of replication forks at the natural termini of the rDNA of Saccharomyces cerevisiae is controlled in a sequence-specific and polar mode by the interaction of the Fob1p replication terminator protein with the tandem Ter sites located in the nontranscribed spacers. Here we show, by both 2D gel analyses and chromatin immunoprecipitations (ChIP), that there exists a second level of global control mediated by the intra-S-phase checkpoint protein complex of Tof1p and Csm3p that protect stalled forks at Ter sites against the activity of the Rrm3p helicase (\"sweepase\"). The sweepase tends to release arrested forks presumably by the transient displacement of the Ter-bound Fob1p. Consistent with this mechanism, very few replication forks were arrested at the natural replication termini in the absence of the two checkpoint proteins. In the absence of the Rrm3p helicase, there was a slight enhancement of fork arrest at the Ter sites. Simultaneous deletions of the TOF1 (or CSM3), and the RRM3 genes restored fork arrest by removing both the fork-releasing and fork-protection activities. Other genes such as MRC1, WSS1, and PSY2 that are also involved in the MRC1 checkpoint pathway were not involved in this global control. This observation suggests that Tof1p-Csm3p function differently from MRC1 and the other above-mentioned genes. This mechanism is not restricted to the natural Ter sites but was also observed at fork arrest caused by the meeting of a replication fork with transcription approaching from the opposite direction.", "title": "The Tof1p-Csm3p protein complex counteracts the Rrm3p helicase to control replication termination of Saccharomyces cerevisiae." }, { "docid": "46346525", "text": "Mu transposons carrying the chloramphenicol resistance marker have been inserted into the cloned Escherichia coli genes sodA and sodB coding for manganese superoxide dismutase (MnSOD) and iron superoxide dismutase (FeSOD) respectively, creating mutations and gene fusions. The mutated sodA or sodB genes were introduced into the bacterial chromosome by allelic exchange. The resulting mutants were shown to lack the corresponding SOD by activity measurements and immunoblot analysis. Aerobically, in rich medium, the absence of FeSOD or MnSOD had no major effect on growth or sensitivity to the superoxide generator, paraquat. In minimal medium aerobic growth was not affected, but the sensitivity to paraquat was increased, especially in the sodA mutant. A sodA sodB double mutant completely devoid of SOD was also obtained. It was able to grow aerobically in rich medium, its catalase level was unaffected and it was highly sensitive to paraquat and hydrogen peroxide; the double mutant was unable to grow aerobically on minimal glucose medium. Growth could be restored by removing oxygen, by providing an SOD-overproducing plasmid or by supplementing the medium with the 20 amino acids. It is concluded that the total absence of SOD in E. coli creates a conditional sensitivity to oxygen.", "title": "Isolation of superoxide dismutase mutants in Escherichia coli: is superoxide dismutase necessary for aerobic life?" }, { "docid": "26596106", "text": "In the yeast S. cerevisiae, ribosome assembly is linked to environmental conditions by the coordinate transcriptional regulation of genes required for ribosome biogenesis. In this study we show that two nonessential stress-responsive genes, YAR1 and LTV1, function in 40S subunit production. We provide genetic and biochemical evidence that Yar1, a small ankyrin-repeat protein, physically interacts with RpS3, a component of the 40S subunit, and with Ltv1, a protein recently identified as a substoichiometric component of a 43S preribosomal particle. We demonstrate that cells lacking YAR1 or LTV1 are hypersensitive to particular protein synthesis inhibitors and exhibit aberrant polysome profiles, with a reduced absolute number of 40S subunits and an excess of free 60S subunits. Surprisingly, both mutants are also hypersensitive to a variety of environmental stress conditions. Overexpression of RPS3 suppresses both the stress sensitivity and the ribosome biogenesis defect of Deltayar1 mutants, but does not suppress either defect in Deltaltv1 mutants. We propose that YAR1 and LTV1 play distinct, nonessential roles in 40S subunit production. The stress-sensitive phenotypes of strains lacking these genes reveal a hitherto unknown link between ribosome biogenesis factors and environmental stress sensitivity.", "title": "Genetic and biochemical interactions among Yar1, Ltv1 and Rps3 define novel links between environmental stress and ribosome biogenesis in Saccharomyces cerevisiae." }, { "docid": "15551129", "text": "Many species of mycobacteria form structured biofilm communities at liquid–air interfaces and on solid surfaces. Full development of Mycobacterium smegmatis biofilms requires addition of supplemental iron above 1 μM ferrous sulphate, although addition of iron is not needed for planktonic growth. Microarray analysis of the M. smegmatis transcriptome shows that iron-responsive genes – especially those involved in siderophore synthesis and iron uptake – are strongly induced during biofilm formation reflecting a response to iron deprivation, even when 2 μM iron is present. The acquisition of iron under these conditions is specifically dependent on the exochelin synthesis and uptake pathways, and the strong defect of an iron–exochelin uptake mutant suggests a regulatory role of iron in the transition to biofilm growth. In contrast, although the expression of mycobactin and iron ABC transport operons is highly upregulated during biofilm formation, mutants in these systems form normal biofilms in low-iron (2 μM) conditions. A close correlation between iron availability and matrix-associated fatty acids implies a possible metabolic role in the late stages of biofilm maturation, in addition to the early regulatory role. M. smegmatis surface motility is similarly dependent on iron availability, requiring both supplemental iron and the exochelin pathway to acquire it.", "title": "The role of iron in Mycobacterium smegmatis biofilm formation: the exochelin siderophore is essential in limiting iron conditions for biofilm formation but not for planktonic growth" }, { "docid": "12922760", "text": "BACKGROUND G-quadruplexes (G4s) are stable non-canonical DNA secondary structures consisting of stacked arrays of four guanines, each held together by Hoogsteen hydrogen bonds. Sequences with the ability to form these structures in vitro, G4 motifs, are found throughout bacterial and eukaryotic genomes. The budding yeast Pif1 DNA helicase, as well as several bacterial Pif1 family helicases, unwind G4 structures robustly in vitro and suppress G4-induced DNA damage in S. cerevisiae in vivo. RESULTS We determined the genomic distribution and evolutionary conservation of G4 motifs in four fission yeast species and investigated the relationship between G4 motifs and Pfh1, the sole S. pombe Pif1 family helicase. Using chromatin immunoprecipitation combined with deep sequencing, we found that many G4 motifs in the S. pombe genome were associated with Pfh1. Cells depleted of Pfh1 had increased fork pausing and DNA damage near G4 motifs, as indicated by high DNA polymerase occupancy and phosphorylated histone H2A, respectively. In general, G4 motifs were underrepresented in genes. However, Pfh1-associated G4 motifs were located on the transcribed strand of highly transcribed genes significantly more often than expected, suggesting that Pfh1 has a function in replication or transcription at these sites. CONCLUSIONS In the absence of functional Pfh1, unresolved G4 structures cause fork pausing and DNA damage of the sort associated with human tumors.", "title": "The essential Schizosaccharomyces pombe Pfh1 DNA helicase promotes fork movement past G-quadruplex motifs to prevent DNA damage" }, { "docid": "30844602", "text": "PROBLEM/CONDITION Since 1973, CDC has maintained a collaborative surveillance program for collection and periodic reporting of data on the occurrence and causes of foodborne-disease outbreaks (FBDOs) in the United States. REPORTING PERIOD COVERED This summary reviews data from January 1993 through December 1997. DESCRIPTION OF SYSTEM The Foodborne-Disease Outbreak Surveillance System reviews data concerning FBDOs, defined as the occurrence of two or more cases of a similar illness resulting from the ingestion of a common food. State and local public health departments have primary responsibility for identifying and investigating FBDOs. State, local, and territorial health departments use a standard form to report these outbreaks to CDC. RESULTS During 1993-1997, a total of 2,751 outbreaks of foodborne disease were reported (489 in 1993, 653 in 1994, 628 in 1995, 477 in 1996, and 504 in 1997). These outbreaks caused a reported 86,058 persons to become ill. Among outbreaks for which the etiology was determined, bacterial pathogens caused the largest percentage of outbreaks (75%) and the largest percentage of cases (86%). Salmonella serotype Enteritidis accounted for the largest number of outbreaks, cases, and deaths; most of these outbreaks were attributed to eating eggs. Chemical agents caused 17% of outbreaks and 1% of cases; viruses, 6% of outbreaks and 8% of cases; and parasites, 2% of outbreaks and 5% of cases. INTERPRETATION The annual number of FBDOs reported to CDC did not change substantially during this period or from previous years. During this reporting period, S. Enteritidis continued to be a major cause of illness and death. In addition, multistate outbreaks caused by contaminated produce and outbreaks caused by Escherichia coli O157:H7 remained prominent. ACTIONS TAKEN Current methods to detect FBDOs are improving, and several changes to improve the ease and timeliness of reporting FBDO data are occurring (e.g., a revised form to simplify FBDO reporting by state health departments and electronic reporting methods). State and local health departments continue to investigate and report FBDOs as part of efforts to better understand and define the epidemiology of foodborne disease in the United States. At the regional and national levels, surveillance data provide an indication of the etiologic agents, vehicles of transmission, and contributing factors associated with FBDOs and help direct public health actions to reduce illness and death caused by FBDOs.", "title": "Surveillance for foodborne-disease outbreaks--United States, 1993-1997." }, { "docid": "4421742", "text": "Emerging evidence suggests that pulmonary iron accumulation is implicated in a spectrum of chronic lung diseases. However, the mechanism(s) involved in pulmonary iron deposition and its role in the in vivo pathogenesis of lung diseases remains unknown. Here we show that a point mutation in the murine ferroportin gene, which causes hereditary hemochromatosis type 4 (Slc40a1C326S), increases iron levels in alveolar macrophages, epithelial cells lining the conducting airways and lung parenchyma, and in vascular smooth muscle cells. Pulmonary iron overload is associated with oxidative stress, restrictive lung disease with decreased total lung capacity and reduced blood oxygen saturation in homozygous Slc40a1C326S/C326S mice compared to wild-type controls. These findings implicate iron in lung pathology, which is so far not considered a classical iron-related disorder.", "title": "Disruption of the Hepcidin/Ferroportin Regulatory System Causes Pulmonary Iron Overload and Restrictive Lung Disease" }, { "docid": "4300851", "text": "A major goal of biology is to provide a quantitative description of cellular behaviour. This task, however, has been hampered by the difficulty in measuring protein abundances and their variation. Here we present a strategy that pairs high-throughput flow cytometry and a library of GFP-tagged yeast strains to monitor rapidly and precisely protein levels at single-cell resolution. Bulk protein abundance measurements of >2,500 proteins in rich and minimal media provide a detailed view of the cellular response to these conditions, and capture many changes not observed by DNA microarray analyses. Our single-cell data argue that noise in protein expression is dominated by the stochastic production/destruction of messenger RNAs. Beyond this global trend, there are dramatic protein-specific differences in noise that are strongly correlated with a protein's mode of transcription and its function. For example, proteins that respond to environmental changes are noisy whereas those involved in protein synthesis are quiet. Thus, these studies reveal a remarkable structure to biological noise and suggest that protein noise levels have been selected to reflect the costs and potential benefits of this variation.", "title": "Single-cell proteomic analysis of S. cerevisiae reveals the architecture of biological noise" }, { "docid": "516867", "text": "The unicellular eukaryotic organisms represent the popular model systems to understand aging in eukaryotes. Candida albicans, a polymorphic fungus, appears to be another distinctive unicellular aging model in addition to the budding yeast Saccharomyces cerevisiae and fission yeast Schizosaccharomyces pombe. The two types of Candida cells, yeast (blastospore) form and hyphal (filamentous) form, have similar replicative lifespan. Taking the advantage of morphologic changes, we are able to obtain cells of different ages. Old Candida cells tend to accumulate glycogen and oxidatively damaged proteins. Deletion of the SIR2 gene causes a decrease of lifespan, while insertion of an extra copy of SIR2 extends lifespan, indicating that like in S. cerevisiae, Sir2 regulates cellular aging in C. albicans. Interestingly, Sir2 deletion does not result in the accumulation of extra-chromosomal rDNA molecules, but influences the retention of oxidized proteins in mother cells, suggesting that the extra-chromosomal rDNA molecules may not be associated with cellular aging in C. albicans. This novel aging model, which allows efficient large-scale isolation of old cells, may facilitate biochemical characterizations and genomics/proteomics studies of cellular aging, and help to verify the aging pathways observed in other organisms including S. cerevisiae.", "title": "Candida albicans, a distinctive fungal model for cellular aging study" }, { "docid": "23604601", "text": "The IME1 gene of Saccharomyces cerevisiae is required for initiation of meiosis. Transcription of IME1 is detected under conditions which are known to induce initiation of meiosis, namely starvation for nitrogen and glucose, and the presence of MATa1 and MAT alpha 2 gene products. In this paper we show that IME1 is also subject to translational regulation. Translation of IME1 mRNA is achieved either upon nitrogen starvation, or upon G1 arrest. In the presence of nutrients, constitutively elevated transcription of IME1 is also sufficient for the translation of IME1 RNA. Four different conditions were found to cause expression of Ime1 protein in vegetative cultures: elevated transcription levels due to the presence of IME1 on a multicopy plasmid; elevated transcription provided by a Gal-IME1 construct; G1 arrest due to alpha-factor treatment; G1 arrest following mild heat-shock treatment of cdc28 diploids. Using these conditions, we obtained evidence that starvation is required not only for transcription and efficient translation of IME1, but also for either the activation of Ime1 protein or for the induction/activation of another factor that, either alone or in combination with Ime1, induces meiosis.", "title": "Post-transcriptional regulation of IME1 determines initiation of meiosis in Saccharomyces cerevisiae." }, { "docid": "42267740", "text": "Various proteins have been found to play roles in both the repair of UV damaged DNA and heterochromatin-mediated silencing in the yeast Saccharomyces cerevisiae. In particular, factors that are involved in the methylation of lysine-79 of histone H3 by Dot1p have been implicated in both processes, suggesting a bipartite function for this modification. We find that a dot1 null mutation and a histone H3 point mutation at lysine-79 cause increased sensitivity to UV radiation, suggesting that lysine-79 methylation is important for efficient repair of UV damage. Epistasis analysis between dot1 and various UV repair genes indicates that lysine-79 methylation plays overlapping roles within the nucleotide excision, post-replication and recombination repair pathways, as well as RAD9-mediated checkpoint function. In contrast, epistasis analysis with the H3 lysine-79 point mutation indicates that the lysine-to-glutamic acid substitution exerts specific effects within the nucleotide excision repair and post-replication repair pathways, suggesting that this allele only disrupts a subset of the functions of lysine-79 methylation. The overall results indicate the existence of distinct and separable roles of histone H3 lysine-79 methylation in the response to UV damage, potentially serving to coordinate the various repair processes.", "title": "Methylation of histone H3 lysine-79 by Dot1p plays multiple roles in the response to UV damage in Saccharomyces cerevisiae." }, { "docid": "28704738", "text": "The miR-294 and miR-302 microRNAs promote the abbreviated G1 phase of the embryonic stem cell (ESC) cell cycle and suppress differentiation induced by let-7. Here, we evaluated the role of the retinoblastoma (Rb) family proteins in these settings. Under normal growth conditions, miR-294 promoted the rapid G1-S transition independent of the Rb family. In contrast, miR-294 suppressed the further accumulation of cells in G1 in response to nutrient deprivation and cell-cell contact in an Rb-dependent fashion. We uncovered five additional miRNAs (miR-26a, miR-99b, miR-193, miR-199a-5p, and miR-218) that silenced ESC self-renewal in the absence of other miRNAs, all of which were antagonized by miR-294 and miR-302. Four of the six differentiation-inducing miRNAs induced an Rb-dependent G1 accumulation. However, all six still silenced self-renewal in the absence of the Rb proteins. These results show that the miR-294/miR-302 family acts through Rb-dependent and -independent pathways to regulate the G1 restriction point and the silencing of self-renewal, respectively.", "title": "miR-294/miR-302 promotes proliferation, suppresses G1-S restriction point, and inhibits ESC differentiation through separable mechanisms." }, { "docid": "33669399", "text": "Gametophytic self-incompatibility (SI) in plants is a widespread mechanism preventing self-fertilization and the ensuing inbreeding depression, but it often evolves to self-compatibility. We analyze genetic mechanisms for the breakdown of gametophytic SI, incorporating a dynamic model for the evolution of inbreeding depression allowing for partial purging of nearly recessive lethal mutations by selfing, and accounting for pollen limitation and sheltered load linked to the S-locus. We consider two mechanisms for the breakdown of gametophytic SI: a nonfunctional S-allele and an unlinked modifier locus that inactivates the S-locus. We show that, under a wide range of conditions, self-compatible alleles can invade a self-incompatible population. Conditions for invasion are always less stringent for a nonfunctional S-allele than for a modifier locus. The spread of self-compatible genotypes is favored by extremely high or low selfing rates, a small number of S-alleles, and pollen limitation. Observed parameter values suggest that the maintenance of gametophytic SI is caused by a combination of high inbreeding depression in self-incompatible populations coupled with intermediate selfing rates of the self-compatible genotypes and sheltered load linked to the S-locus.", "title": "Loss of gametophytic self-incompatibility with evolution of inbreeding depression." }, { "docid": "2721426", "text": "RNA molecules contain a variety of chemically diverse, posttranscriptionally modified bases. The most abundant modified base found in cellular RNAs, pseudouridine (Ψ), has recently been mapped to hundreds of sites in mRNAs, many of which are dynamically regulated. Though the pseudouridine landscape has been determined in only a few cell types and growth conditions, the enzymes responsible for mRNA pseudouridylation are universally conserved, suggesting many novel pseudouridylated sites remain to be discovered. Here, we present Pseudo-seq, a technique that allows the identification of sites of pseudouridylation genome-wide with single-nucleotide resolution. In this chapter, we provide a detailed description of Pseudo-seq. We include protocols for RNA isolation from Saccharomyces cerevisiae, Pseudo-seq library preparation, and data analysis, including descriptions of processing and mapping of sequencing reads, computational identification of sites of pseudouridylation, and assignment of sites to specific pseudouridine synthases. The approach presented here is readily adaptable to any cell or tissue type from which high-quality mRNA can be isolated. Identification of novel pseudouridylation sites is an important first step in elucidating the regulation and functions of these modifications.", "title": "Pseudo-Seq: Genome-Wide Detection of Pseudouridine Modifications in RNA." }, { "docid": "4993011", "text": "ATRX (alpha thalassemia/mental retardation X-linked) complexes with DAXX to deposit histone variant H3.3 into repetitive heterochromatin. Recent genome sequencing studies in cancers have revealed mutations in ATRX and their association with ALT (alternative lengthening of telomeres) activation. Here we report depletion of ATRX in mouse ES cells leads to selective loss in ribosomal RNA gene (rDNA) copy number. Supporting this, ATRX-mutated human ALT-positive tumors also show a substantially lower rDNA copy than ALT-negative tumors. Further investigation shows that the rDNA copy loss and repeat instability are caused by a disruption in H3.3 deposition and thus a failure in heterochromatin formation at rDNA repeats in the absence of ATRX. We also find that ATRX-depleted cells are reduced in ribosomal RNA transcription output and show increased sensitivity to RNA polymerase I (Pol I) transcription inhibitor CX5461. In addition, human ALT-positive cancer cell lines are also more sensitive to CX5461 treatment. Our study provides insights into the contribution of ATRX loss of function to tumorigenesis through the loss of rDNA stability and suggests the therapeutic potential of targeting Pol I transcription in ALT cancers.", "title": "Ribosomal DNA copy loss and repeat instability in ATRX-mutated cancers" }, { "docid": "18190552", "text": "BACKGROUND Avian influenza (AI) caused by H7 AI viruses (AIVs) of both low pathogenicity (LP) and high pathogenicity (HP) are notifiable poultry diseases. OBJECTIVES Design and validate two RealTime reverse transcriptase polymerase chain reactions (RRT PCRs) for Eurasian H7 AIV detection and pathotyping. METHODS The H7 RRT PCRs amplified within the (i) HA2 and (ii) cleavage site CS regions of the haemagglutinin gene. Both were validated against 65 H7 AIVs, 57 non-H7 AIVs and 259 poultry swabs in comparison to M gene (AI generic) RRT PCR and virus isolation (VI). An additional 38 swabs and 20 tissue specimens extended validation against M gene RRT PCR. RESULTS Both H7 RRT PCRs amplified all 61 Eurasian lineage H7 AIVs and none of 57 non-H7 AIVs. A total of 297 poultry swabs were used to determine diagnostic sensitivity and specificity relative to M gene RRT PCR, sensitivity was 95.4% and 64.6% for the HA2 and CS RRT PCRs respectively, and specificity 97.9% and 99.6% respectively. The H7 HA2 RRT PCR was more sensitive than VI. This was emphasized by analysis of 37 swabs from turkeys infected experimentally with HPAI H7N1 virus sampled at 24 hours post-inoculation and LPAI H7N1 chicken infections sampled at 40-64 hours. Although less sensitive, usefulness of the H7 CS RRT PCR was confirmed by the correct molecular pathotyping for all 61 Eurasian lineage H7 AIVs tested. CONCLUSIONS The high sensitivity of H7 HA2 RRT PCR confirms its suitability for use in poultry surveillance and disease diagnosis. H7 CS RRT PCR provides an opportunity for rapid pathotyping of H7 AIVs.", "title": "Validated RealTime reverse transcriptase PCR methods for the diagnosis and pathotyping of Eurasian H7 avian influenza viruses" }, { "docid": "3553087", "text": "Chronic obstructive pulmonary disease (COPD) is linked to both cigarette smoking and genetic determinants. We have previously identified iron-responsive element-binding protein 2 (IRP2) as an important COPD susceptibility gene and have shown that IRP2 protein is increased in the lungs of individuals with COPD. Here we demonstrate that mice deficient in Irp2 were protected from cigarette smoke (CS)-induced experimental COPD. By integrating RNA immunoprecipitation followed by sequencing (RIP-seq), RNA sequencing (RNA-seq), and gene expression and functional enrichment clustering analysis, we identified Irp2 as a regulator of mitochondrial function in the lungs of mice. Irp2 increased mitochondrial iron loading and levels of cytochrome c oxidase (COX), which led to mitochondrial dysfunction and subsequent experimental COPD. Frataxin-deficient mice, which had higher mitochondrial iron loading, showed impaired airway mucociliary clearance (MCC) and higher pulmonary inflammation at baseline, whereas mice deficient in the synthesis of cytochrome c oxidase, which have reduced COX, were protected from CS-induced pulmonary inflammation and impairment of MCC. Mice treated with a mitochondrial iron chelator or mice fed a low-iron diet were protected from CS-induced COPD. Mitochondrial iron chelation also alleviated CS-induced impairment of MCC, CS-induced pulmonary inflammation and CS-associated lung injury in mice with established COPD, suggesting a critical functional role and potential therapeutic intervention for the mitochondrial-iron axis in COPD.", "title": "Mitochondrial iron chelation ameliorates cigarette-smoke induced bronchitis and emphysema in mice" }, { "docid": "21425864", "text": "Glycosyl phosphatidylinositols (GPIs) anchor many proteins to the surface of eukaryotic cells and may also serve as sorting signals on proteins and participate in signal transduction. We have isolated a Saccharomyces cerevisiae GPI anchoring mutant, gpi1, using a colony screen for cells blocked in [3H]inositol incorporation into protein. The gpi1 mutant is defective in vitro in the synthesis of N-acetylglucosaminyl phosphatidylinositol, the first intermediate in GPI synthesis, and is also temperature-sensitive for growth. Completion of the first step in GPI assembly is therefore required for growth of the unicellular eukaryote S. cerevisiae. GPI synthesis could therefore be exploited as a target for antifungal or antiparasitic agents.", "title": "A conditionally lethal yeast mutant blocked at the first step in glycosyl phosphatidylinositol anchor synthesis." }, { "docid": "27889071", "text": "The high prevalence of microcytosis (defined here as mean cell haemoglobin<27 pg) with no other abnormality is a principal cause of confusion in screening for haemoglobin disorders. Here we report the results of a small pilot study aiming to resolve this confusion by routinely proceeding to plasma ferritin and HPLC assay, using the original sequestrene blood sample, when microcytosis is detected. Participants comprised a random sample of 1,302 people referred for a full blood count by their General Practitioner (GP) to the laboratory of a North London district general hospital serving a multi-ethnic inner-city population. Ethnicity was established by questionnaire. In North Europeans, microcytosis was present in 3% of males (half were iron-deficient) and 11% of females (most were iron-deficient). Among ethnic minorities, microcytosis was present in 35% of males (one tenth were iron-deficient), and 45% of females (less than half were iron-deficient): an exclusion diagnosis of \"probable alpha thalassaemia\" could be made in the remainder. We conclude that when microcytosis is present, routine further analysis of the original sequestrene sample by plasma ferritin assay and haemoglobinopathy screening could lead to a more efficient and cost-effective laboratory service for primary care and maternity services.", "title": "Microcytosis, iron deficiency and thalassaemia in a multi-ethnic community: a pilot study." }, { "docid": "23918031", "text": "The platelet precursor, the megakaryocyte, matures to a polyploid cell as a result of DNA replication in the absence of mitosis (endomitosis). The factors controlling endomitosis are accessible to analysis in our megakaryocytic cell line, MegT, generated by targeted expression of temperature-sensitive simian virus 40 large T antigen to megakaryocytes of transgenic mice. We aimed to define whether endomitosis consists of a continuous phase of DNA synthesis (S) or of S phases interrupted by gaps. Analysis of the cell cycle in MegT cells revealed that, upon inactivation of large T antigen, the cells shifted from a mitotic cell cycle to an endomitotic cell cycle consisting of S/Gap phases. The level of the G1/S cyclin, cyclin A, as well as of the G1 phase cyclin, cyclin D3, were elevated at the onset of DNA synthesis, either in MegT cells undergoing a mitotic cell cycle or during endomitosis. In contrast, the level of the mitotic cyclin, cyclin B1, cycled in cells displaying a mitotic cell cycle while not detectable during endomitosis. Comparable levels of the mitotic kinase protein, Cdc2, were detected during the mitotic cell cycle or during endomitosis; however, cyclin B1-dependent Cdc2 kinase activity was largely abolished in the polyploid cells. Fibroblasts immortalized with the same heat-labile oncogene do not display reduced levels of cyclin B1 upon shifting to high temperature nor do they become polyploid, indicating that reduced levels of cyclin B1 is a property of megakaryocytes and not of the T-antigen mutant. We conclude that cellular programming during endoreduplication in megakaryocytes is associated with reduced levels of cyclin B1.", "title": "The cell cycle in polyploid megakaryocytes is associated with reduced activity of cyclin B1-dependent cdc2 kinase." }, { "docid": "10627801", "text": "The DExD/H box RNA helicase retinoic acid-inducible gene I (RIG-I) and the melanoma differentiation-associated gene 5 (MDA5) are key intracellular receptors that recognize virus infection to produce type I IFN. A third helicase gene, Lgp2, is homologous to Rig-I and Mda5 but lacks a caspase activation and recruitment domain. We generated Lgp2-deficient mice and report that the loss of this gene greatly sensitizes cells to cytosolic polyinosinic/polycytidylic acid-mediated induction of type I IFN. However, negative feedback inhibition of IFN-beta transcription was found to be normal in the absence of LGP2, indicating that LGP2 is not the primary negative regulator of type I IFN production. Our data further indicate that Lgp2-/- mice exhibited resistance to lethal vesicular stomatitis virus infection, a virus whose replicative RNA intermediates are recognized specifically by RIG-I rather than by MDA5 to trigger the production of type I IFN. However, mice lacking LGP2 were observed to exhibit a defect in type I IFN production in response to infection by the encephalomyocarditis virus, the replication of which activates MDA5-dependent innate immune responses. Collectively, our data indicate a disparate regulatory role for LGP2 in the triggering of innate immune signaling pathways following RNA virus infection.", "title": "Loss of DExD/H box RNA helicase LGP2 manifests disparate antiviral responses." }, { "docid": "49208216", "text": "Staphylococcus aureus is a human commensal that can also cause systemic infections. This transition requires evasion of the immune response and the ability to exploit different niches within the host. However, the disease mechanisms and the dominant immune mediators against infection are poorly understood. Previously it has been shown that the infecting S. aureus population goes through a population bottleneck, from which very few bacteria escape to establish the abscesses that are characteristic of many infections. Here we examine the host factors underlying the population bottleneck and subsequent clonal expansion in S. aureus infection models, to identify underpinning principles of infection. The bottleneck is a common feature between models and is independent of S. aureus strain. Interestingly, the high doses of S. aureus required for the widely used \"survival\" model results in a reduced population bottleneck, suggesting that host defences have been simply overloaded. This brings into question the applicability of the survival model. Depletion of immune mediators revealed key breakpoints and the dynamics of systemic infection. Loss of macrophages, including the liver Kupffer cells, led to increased sensitivity to infection as expected but also loss of the population bottleneck and the spread to other organs still occurred. Conversely, neutrophil depletion led to greater susceptibility to disease but with a concomitant maintenance of the bottleneck and lack of systemic spread. We also used a novel microscopy approach to examine abscess architecture and distribution within organs. From these observations we developed a conceptual model for S. aureus disease from initial infection to mature abscess. This work highlights the need to understand the complexities of the infectious process to be able to assign functions for host and bacterial components, and why S. aureus disease requires a seemingly high infectious dose and how interventions such as a vaccine may be more rationally developed.", "title": "Staphylococcus aureus infection dynamics" }, { "docid": "28617573", "text": "More than ever, clinicians need regularly updated reviews given the continuously increasing amount of new information regarding innovative cervical cancer prevention methods. A summary is given from recent meta-analyses and systematic reviews on 3 possible clinical applications of human papillomavirus (HPV) testing: triage of women with equivocal or low-grade cytologic abnormalities; prediction of the therapeutic outcome after treatment of cervical intraepithelial neoplasia (CIN) lesions, and last not but not least, primary screening for cervical cancer and pre-cancer. Consistent evidence is available indicating that HPV-triage with the Hybrid Capture(®) 2 assay (Qiagen Gaithersburg, Inc., MD, USA [previously Digene Corp.] (HC2) is more accurate (higher sensitivity, similar specificity) than repeat cytology to triage women with equivocal Pap smear results. Several other tests show at least similar accuracy but mRNA testing with the APTIMA(®) (Gen-Probe Inc., San Diego, CA, USA) test is similarly sensitive but more specific compared to HC2. In triage of low-grade squamous intraepithelial lesions (LSIL), HC2 is more sensitive but its specificity is substantially lower compared to repeat cytology. The APTIMA(®) test is more specific than HC2 without showing a loss in sensitivity. Identification of DNA of HPV types 16 and/or 18, or RNA from the five most carcinogenic HPV types allow selecting women at highest risk for CIN3+ but the sensitivity and negative predictive value of these markers are lower than full-range high-risk HPV (hrHPV) testing. After conservative treatment of cervical pre-cancer, HPV testing picks up more quickly, with higher sensitivity and not lower specificity, residual or recurrent high-grade CIN than follow-up cytology. Primary screening for hrHPV generally detects more CIN2, CIN3 or cancer compared to cytology at cut-off atypical squamous cells of undetermined significance (ASC-US) or LSIL, but is less specific. Combined HPV and cytology screening provides a further small gain in sensitivity at the expense of a considerable loss in specificity if positive by either test is referred to colposcopy, in comparison with HPV testing only. Randomised trials and follow-up of cohort studies consistently demonstrate a significantly lower cumulative incidence of CIN3+ and even of cancer, in women aged 30 years or older, who were at enrollment hrHPV DNA negative compared to those who were cytologically negative. The difference in cumulative risk of CIN3+ or cancer for double negative (cytology & HPV) versus only HPV-negative women is small. HC2, GP5+/6+ PCR (polymerase chain reaction), cobas(®) 4800 PCR (Roche Molecular Systems Inc., Alameda, CA, USA) and Real Time PCR (Abbott Molecular, Des Plaines, IL, USA) can be considered as clinically validated for use in primary screening. The loss in specificity associated with primary HPV-based screening can be compensated by appropriate algorithms involving reflex cytology and/or HPV genotyping for HPV16 or 18. There exists a substantial evidence base to support that HPV testing is advantageous both in triage of women with equivocal abnormal cytology, in surveillance after treatment of CIN lesions and in primary screening of women aged 30 years or older. However, the possible advantages offered by HPV-based screening require a well organised program with good compliance with screening and triage policies. This article forms part of a special supplement entitled \"Comprehensive Control of HPV Infections and Related Diseases\" Vaccine Volume 30, Supplement 5, 2012.", "title": "Evidence regarding human papillomavirus testing in secondary prevention of cervical cancer." }, { "docid": "44387884", "text": "The abnormal metabolic state that accompanies diabetes renders arteries susceptible to atherosclerosis, being capable of altering the functional properties of multiple cell types, including endothelium and platelets. In particular, an altered platelet metabolism and changes in intraplatelet signaling pathways may contribute to the pathogenesis of atherothrombotic complications of diabetes. A variety of mechanisms may be responsible for enhanced platelet aggregation. Among them, hyperglycemia may represent a causal factor for in vivo platelet activation, and may be responsible for nonenzymatic glycation of platelet glycoproteins, causing changes in their structure and conformation, as well as alterations of membrane lipid dynamics. Furthermore, hyperglycemia-induced oxidative stress is responsible for enhanced peroxidation of arachidonic acid to form biologically active isoprostanes, which represents an important biochemical link between impaired glycemic control and persistent platelet activation. Finally, increased oxidative stress is responsible for activation of transcription factors and expression of redox-sensitive genes leading to a phenotypic switch of endothelium toward an adhesive, pro-thrombotic condition, initial platelet activation, adhesion and subsequent platelet aggregate formation. All this evidence is strengthened by the results of clinical trials documenting the beneficial effects of metabolic control on platelet function, and by the finding that aspirin treatment may even be more beneficial in diabetic than in high-risk non-diabetic patients. Attention to appropriate medical management of diabetic patients will have great impact on long-term outcome in this high-risk population.", "title": "Platelet activation in type 2 diabetes mellitus." }, { "docid": "7225911", "text": "It is well known that upon stress, the level of the tumor suppressor p53 is remarkably elevated. However, despite extensive studies, the underlying mechanism involving important inter-players for stress-induced p53 regulation is still not fully understood. We present evidence that the human lincRNA-RoR (RoR) is a strong negative regulator of p53. Unlike MDM2 that causes p53 degradation through the ubiquitin-proteasome pathway, RoR suppresses p53 translation through direct interaction with the heterogeneous nuclear ribonucleoprotein I (hnRNP I). Importantly, a 28-base RoR sequence carrying hnRNP I binding motifs is essential and sufficient for p53 repression. We further show that RoR inhibits p53-mediated cell cycle arrest and apoptosis. Finally, we demonstrate a RoR-p53 autoregulatory feedback loop where p53 transcriptionally induces RoR expression. Together, these results suggest that the RoR-hnRNP I-p53 axis may constitute an additional surveillance network for the cell to better respond to various stresses.", "title": "The human long non-coding RNA-RoR is a p53 repressor in response to DNA damage" }, { "docid": "12383365", "text": "ABSTRACT Lyme disease (LD) is emerging in Canada because of the northward expansion of the geographic range of the tick vector Ixodes scapularis (Say). Early detection of emerging areas of LD risk is critical to public health responses, but the methods to do so on a local scale are lacking. Passive tick surveillance has operated in Canada since 1990 but this method lacks specificity for identifying areas where tick populations are established because of dispersion of ticks from established LD risk areas by migratory birds. Using data from 70 field sites in Quebec visited previously, we developed a logistic regression model for estimating the risk of I. scapularis population establishment based on the number of ticks submitted in passive surveillance and a model-derived environmental suitability index. Sensitivity-specificity plots were used to select an optimal threshold value of the linear predictor from the model as the signal for tick population establishment. This value was used to produce an “Alert Map” identifying areas where the passive surveillance data suggested ticks were establishing in Quebec. Alert Map predictions were validated by field surveillance at 76 sites: the prevalence of established I. scapularis populations was significantly greater in areas predicted as high-risk by the Alert map (29 out of 48) than in areas predicted as moderate-risk (4 out of 30) (P < 0.001). This study suggests that Alert Maps created using this approach can provide a usefully rapid and accurate tool for early identification of emerging areas of LD risk at a geographic scale appropriate for local disease control and prevention activities.", "title": "Passive Surveillance for I. scapularis Ticks: Enhanced Analysis for Early Detection of Emerging Lyme Disease Risk" }, { "docid": "2601324", "text": "Oligodendrocytes, the myelin-forming glial cells of the central nervous system, maintain long-term axonal integrity. However, the underlying support mechanisms are not understood. Here we identify a metabolic component of axon–glia interactions by generating conditional Cox10 (protoheme IX farnesyltransferase) mutant mice, in which oligodendrocytes and Schwann cells fail to assemble stable mitochondrial cytochrome c oxidase (COX, also known as mitochondrial complex IV). In the peripheral nervous system, Cox10 conditional mutants exhibit severe neuropathy with dysmyelination, abnormal Remak bundles, muscle atrophy and paralysis. Notably, perturbing mitochondrial respiration did not cause glial cell death. In the adult central nervous system, we found no signs of demyelination, axonal degeneration or secondary inflammation. Unlike cultured oligodendrocytes, which are sensitive to COX inhibitors, post-myelination oligodendrocytes survive well in the absence of COX activity. More importantly, by in vivo magnetic resonance spectroscopy, brain lactate concentrations in mutants were increased compared with controls, but were detectable only in mice exposed to volatile anaesthetics. This indicates that aerobic glycolysis products derived from oligodendrocytes are rapidly metabolized within white matter tracts. Because myelinated axons can use lactate when energy-deprived, our findings suggest a model in which axon–glia metabolic coupling serves a physiological function.", "title": "Glycolytic oligodendrocytes maintain myelin and long-term axonal integrity" }, { "docid": "40913091", "text": "Objective: To evaluate the frequency of α -gene, s-gene, and hemoglobin variant numbers in subjects with Microcytic hypochromic anemia. Methodology: In total out of 850, 340 subjects with microcytic hypochromic anemia [MCV<80fl; MCH<27pg] from Southwest part of Iran, were studied in Research Center of Thalassemia and Hemoglobinopathies (RCTH) which is the only center working on hematology and oncology in Southwest (Khuzestan) region of Iran. These include 325 individuals: 171 with Beta-thalassemia trait, 88 with Alpha-thalassemia trait, 13 with thalassemia major, 11 with hemoglobin variants (HbS, HbC, and HbD Punjab ) and 42 with iron-deficiency anemia. The rest 15 patients diagnosed with no definite etiology. Results: Genotyping for -α 3.7 , -α 4.2 , – α PA , - α 5NT and - - MED was done with gap-PCR. The overall frequency of - α 3.7 deletion in 325 individuals is 20%. Genotyping for 23 most known s-gene mutations was done with direct mutation analysis by Amplification Refractory Mutation System (ARMS). The most frequent mutations were CD 36/37, IVS II-I, and IVS I-110 with 9.7%, 11.7%, and 3.5% respected frequencies in 340 patients. There was statistically significant difference between Beta-thalassemia trait and Beta-thalassemia Major in case of MCV (p- value = 0.25) and MCH (P–value =0.23) indices, and also MCH index between Beta-thalassemia trait and Hb Variants (P-value = 0.04). Conclusion: The α -gene and s-gene mutation is quite common in the Southwest part of Iran. Molecular genotyping of α -thalassemia and s-thalassemia help to diagnose unexplained microcytosis, and thus prevent unnecessary iron supplementation.", "title": "GENOTYPING OF THALASSEMIA IN MICROCYTIC HYPOCHROMIC ANEMIA PATIENTS FROM SOUTHWEST REGION OF IRAN" }, { "docid": "44562221", "text": "Endogenous glucocorticoids (GC) play an important role in the termination of the inflammatory response following infection and tissue injury. However, recent findings indicate that stress can impair the anti-inflammatory capacities of these hormones. Lipopolysaccharide (LPS)-stimulated splenocytes of mice that were repeatedly subjected to social disruption (SDR) stress were less sensitive to the immunosuppressive effects of corticosterone (CORT) as demonstrated by an increased production of pro-inflammatory cytokines and enhanced cell survival. Myeloid cells expressing the marker CD11b were shown to play a key role in this process. Here we investigated the role of the bone marrow as a potential source of the GC-insensitive cells. The study revealed that LPS-stimulated bone marrow cells, in the absence of experimental stress, were virtually GC-resistant and retained high levels of cell viability after treatment with CORT. Recurrent exposure to the acute stressor over a period of 2, 4 or 6 days led to an increase in the GC sensitivity of the bone marrow cells. This increase in GC sensitivity was associated with enhanced mRNA expression of granulocyte-macrophage colony-stimulating factor (GM-CSF), an increase in the number of myeloid progenitors, and a decrease in the proportion of mature CD11b+ cells. The changes in the cellular composition of the bone marrow were accompanied by an increase in splenic CD11b+ cell numbers. Simultaneous assessment of the GC sensitivity in bone marrow and spleen revealed a significant negative correlation between both tissues suggesting that social stress causes the redistribution of GC-insensitive myeloid cells from the bone marrow to the spleen.", "title": "Tissue-specific alterations in the glucocorticoid sensitivity of immune cells following repeated social defeat in mice" } ]

[{"docid":"15928989","text":"Successful pregnancy requires coordination of an array of signals and f(...TRUNCATED)

[{"docid":"1780819","text":"BACKGROUND Endometrial cancer incidence is continuing to rise in the wak(...TRUNCATED)

[{"docid":"12009265","text":"CONTEXT Many individuals take vitamins in the hopes of preventing chron(...TRUNCATED)

[{"docid":"25513319","text":"Metabolic pathway reprogramming is a hallmark of cancer cell growth and(...TRUNCATED)

[{"docid":"18872233","text":"IMPORTANCE Bariatric surgery is associated with sustained weight loss a(...TRUNCATED)

[{"docid":"43220289","text":"Extreme obesity is associated with severe psychiatric and somatic comor(...TRUNCATED)

[{"docid":"13083189","text":"OBJECTIVES Despite recognition of the important influence of environmen(...TRUNCATED)

[{"docid":"18537148","text":"The purpose of this investigation was to determine whether maximal oxyg(...TRUNCATED)

[{"docid":"19912367","text":"Age-related changes in the niche have long been postulated to impair th(...TRUNCATED)

[{"docid":"20418809","text":"A key determinant of geriatric frailty is sarcopenia, the age-associate(...TRUNCATED)

[{"docid":"8290953","text":"BACKGROUND We have developed techniques to implant angiogenic patches on(...TRUNCATED)

[{"docid":"1122279","text":"BACKGROUND Endothelium-dependent modulation of coronary tone is impaired(...TRUNCATED)