RESUMEN
Gain-of-function mutations in the dsDNA sensing adaptor STING lead to a severe autoinflammatory syndrome known as STING-associated vasculopathy with onset in infancy (SAVI). Patients with SAVI develop interstitial lung disease (ILD) and produce autoantibodies that are commonly associated with systemic autoimmune diseases. Mice expressing the most common SAVI mutation, STING V154M (VM), similarly develop ILD but exhibit severe T and B cell lymphopenia and low serum Ig titers, and they lack autoantibodies. Importantly, lethally irradiated VM hosts reconstituted with WT stem cells (WTâVM) still develop ILD. In this study, we find that WTâVM chimeras had restored B cell function, produced autoantibodies, and thereby recapitulated the loss of tolerance seen in patients with SAVI. Lymphocytes derived from both WT and BCR or TCR transgenic (Tg) donors accumulated in the extravascular lung tissue of WT+TgâVM mixed chimeras, but lymphocyte activation and germinal center formation required WT cells with a diverse repertoire. Furthermore, when T cells isolated from the WTâVM chimeras were adoptively transferred to naive Rag1-deficient secondary hosts, they trafficked to the lung and recruited neutrophils. Overall, these findings indicated that VM expression by radioresistant cells promoted the activation of autoreactive B cells and T cells that then differentiated into potentially pathogenic effector subsets.
Asunto(s)
Mutación con Ganancia de Función , Activación de Linfocitos , Proteínas de la Membrana , Animales , Ratones , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Activación de Linfocitos/inmunología , Pulmón/inmunología , Pulmón/patología , Autoanticuerpos/inmunología , Linfocitos B/inmunología , Linfocitos B/metabolismo , Enfermedades Pulmonares Intersticiales/genética , Enfermedades Pulmonares Intersticiales/inmunología , Tolerancia a Radiación/genética , Tolerancia a Radiación/inmunología , Ratones Transgénicos , Linfocitos T/inmunología , Linfocitos T/metabolismo , Femenino , Humanos , Ratones Endogámicos C57BL , Modelos Animales de EnfermedadRESUMEN
Pinibarrenia chlamydospora, sp. nov. isolated from the roots of highbush blueberry in the New Jersey Pine Barrens, is described and illustrated. Based on multigene phylogenetic analysis, as well as morphological and ecological characteristics, Pinibarreniales and Pinibarreniaceae are established to accommodate this novel lineage in Sordariomycetidae, Sordariomycetes. Pinibarreniales, Tracyllalales, and Vermiculariopsiellales are proposed to be included in the subclass Sordariomycetidae. Pinibarreniales likely have a wide distribution and forms association with Ericaceae plants that live in acidic and oligotrophic environments because its DNA barcode matches with environmental sequences from other independent ecological studies. The plant-fungal interaction experiment revealed negative impacts on Arabidopsis, indicating its pathogenicity. This uncovered new fungal lineage will contribute to a better understanding of the diversity and systematics of Sordariomycetes.
RESUMEN
Patients with biallelic hypomorphic mutation in DNASE2 develop systemic autoinflammation and early-onset liver fibrosis. Prior studies showed that Dnase2 -/- Ifnar -/- double knockout (DKO) mice develop Type I IFN-independent liver inflammation, but immune mechanisms were unclear. We now show that DKO mice recapitulate many features of human autoimmune hepatitis (AIH), including periportal and interstitial inflammation and fibrosis and elevated ALT. Infiltrating cells include CD8+ tissue resident memory T cells, type I innate lymphoid cells, and inflammatory monocyte/macrophage cells that replace the Kupffer cell pool. Importantly, TLR9 expression by bone marrow-derived cells is required for the the development of AIH. TLR9 is highly expressed by inflammatory myeloid cells but not long-lived Kupffer cells. Furthermore, the initial recruitment of TLR9 expressing monocytes and subsequent activation of lymphocytes requires IFNγ signaling. These findings highlight a critical role of feed forward loop between TLR9 expressing monocyte-lineage cells and IFNg producing lymphocytes in autoimmune hepatitis.
RESUMEN
Patients afflicted with Stimulator of interferon gene (STING) gain-of-function mutations frequently present with debilitating interstitial lung disease (ILD) that is recapitulated in mice expressing the STINGV154M mutation (VM). Prior radiation chimera studies revealed an unexpected and critical role for non-hematopoietic cells in initiating ILD. To identify STING-expressing non-hematopoietic cell types required for the development of ILD, we use a conditional knockin (CKI) model and direct expression of the VM allele to hematopoietic cells, fibroblasts, epithelial cells, or endothelial cells. Only endothelial cell-targeted VM expression results in enhanced recruitment of immune cells to the lung associated with elevated chemokine expression and the formation of bronchus-associated lymphoid tissue, as seen in the parental VM strain. These findings reveal the importance of endothelial cells as instigators of STING-driven lung disease and suggest that therapeutic targeting of STING inhibitors to endothelial cells could potentially mitigate inflammation in the lungs of STING-associated vasculopathy with onset in infancy (SAVI) patients or patients afflicted with other ILD-related disorders.
Asunto(s)
Células Endoteliales , Mutación con Ganancia de Función , Pulmón , Proteínas de la Membrana , Animales , Proteínas de la Membrana/metabolismo , Proteínas de la Membrana/genética , Células Endoteliales/metabolismo , Células Endoteliales/patología , Ratones , Pulmón/patología , Pulmón/metabolismo , Linfocitos/metabolismo , Enfermedades Pulmonares Intersticiales/patología , Enfermedades Pulmonares Intersticiales/genética , Enfermedades Pulmonares Intersticiales/metabolismo , Ratones Endogámicos C57BL , HumanosRESUMEN
Gain-of-function mutations in the dsDNA sensing adaptor STING lead to a severe autoinflammatory syndrome known as STING-associated vasculopathy with onset in Infancy (SAVI). SAVI patients develop interstitial lung disease (ILD) and commonly produce anti-nuclear antibodies (ANAs), indicative of concomitant autoimmunity. Mice heterozygous for the most common SAVI mutation, V154M (VM), also develop ILD, triggered by nonhematopoietic VM cells, but exhibit severe peripheral lymphopenia, low serum Ig titers and fail to produce autoantibodies. In contrast, we now show that lethally irradiated VM mice reconstituted with WT stem cells (WTâVM chimeras) develop ANAs and lung-reactive autoantibodies associated with accumulation of activated lymphocytes and formation of germinal centers in lung tissues. Moreover, when splenocytes from WTâVM chimeras were adoptively transferred into unmanipulated Rag1 -/- mice, donor T cells accumulated in the lung. Overall, these findings demonstrate that expression of the VM mutation in non-hematopoietic cells can promote the activation of immunocompetent autoreactive lymphocytes. Summary: Chimeric mice expressing STING only in non-hematopoietic cells develop systemic and lung directed autoimmunity which recapitulates what is seen in pediatric patients with SAVI disease.
RESUMEN
Patients afflicted with STING gain-of-function mutations frequently present with debilitating interstitial lung disease ( ILD ) that is recapitulated in mice expressing the STING V154M mutation ( VM ). Prior radiation chimera studies revealed an unexpected and critical role for non-hematopoietic cells in the initiation of ILD. To identify STING-expressing non-hematopoietic cell types relevant to ILD, we generated a conditional knock-in ( CKI ) model in which expression of the VM allele was directed to hematopoietic cells, fibroblasts, epithelial cells, or endothelial cells. Only endothelial cell-targeted expression of the mutant allele resulted in the recruitment of immune cells to the lung and the formation of bronchus-associated lymphoid tissue, as seen in the parental VM strain. These findings reveal the importance of endothelial cells as instigators of STING-driven lung disease and suggest that therapeutic targeting of STING inhibitors to endothelial cells could potentially mitigate inflammation in the lungs of SAVI patients or patients afflicted with other ILD-related disorders. Summary: Patients with STING gain-of-function (GOF) mutations develop life-threatening lung autoinflammation. In this study, Gao et al. utilize a mouse model of conditional STING GOF to demonstrate a role for endothelial STING GOF in initiating immune cell recruitment into lung tissues of SAVI mice.
RESUMEN
The cyclic cyclic gaunosine monophosphate adenosine monophosphate (GMP-AMP) synthase-stimulator of interferon genes (cGAS-STING) pathway senses cytosolic dsDNA and initiates immune responses against pathogens. It is also implicated in several auto-inflammatory diseases known as monogenic interferonopathies, specifically Three prime repair exonuclease 1 (Trex1) loss-of-function (LOF), Dnase2 LOF, and stimulator of interferon genes-associated-vasculopathy-with-onset-in-infancy (SAVI). Although monogenic interferonopathies have diverse clinical presentations, they are distinguished by the elevation of type-1 interferons (T1IFNs). However, animal models have demonstrated that T1IFNs contribute to only some disease outcomes and that cGAS-STING activation also promotes T1IFN-independent pathology. For example, while T1IFNs drive the immunopathology associated with Trex1 LOF, disease in Dnase2 LOF is partially independent of T1IFNs, while disease in SAVI appears to occur entirely independent of T1IFNs. Additionally, while the cGAS-STING pathway is well characterized in hematopoietic cells, these animal models point to important roles for STING activity in nonhematopoietic cells in disease. Together, these models illustrate the complex role that cGAS-STING-driven responses play in the pathogenesis of inflammatory diseases across tissues.
Asunto(s)
Interferón Tipo I , Animales , Interferón Tipo I/genética , Nucleótidos Cíclicos , Nucleotidiltransferasas/metabolismo , Inflamación/genéticaRESUMEN
Pediatric patients with constitutively active mutations in the cytosolic double-stranded-DNA-sensing adaptor STING develop an autoinflammatory syndrome known as STING-associated vasculopathy with onset in infancy (SAVI). SAVI patients have elevated interferon-stimulated gene expression and suffer from interstitial lung disease (ILD) with lymphocyte predominate bronchus-associated lymphoid tissue (BALT). Mice harboring SAVI mutations (STING V154M [VM]) that recapitulate human disease also develop lymphocyte-rich BALT. Ablation of either T or B lymphocytes prolongs the survival of SAVI mice, but lung immune aggregates persist, indicating that T cells and B cells can independently be recruited as BALT. VM T cells produced IFNγ, and IFNγR deficiency prolonged the survival of SAVI mice; however, T-cell-dependent recruitment of infiltrating myeloid cells to the lung was IFNγ independent. Lethally irradiated VM recipients fully reconstituted with wild type bone-marrow-derived cells still developed ILD, pointing to a critical role for VM-expressing radioresistant parenchymal and/or stromal cells in the recruitment and activation of pathogenic lymphocytes. We identified lung endothelial cells as radioresistant cells that express STING. Transcriptional analysis of VM endothelial cells revealed up-regulation of chemokines, proinflammatory cytokines, and genes associated with antigen presentation. Together, our data show that VM-expressing radioresistant cells play a key role in the initiation of lung disease in VM mice and provide insights for the treatment of SAVI patients, with implications for ILD associated with other connective tissue disorders.
Asunto(s)
Células Endoteliales , Enfermedades Pulmonares Intersticiales , Proteínas de la Membrana , Linfocitos T , Enfermedades Vasculares , Animales , Niño , Células Endoteliales/inmunología , Células Endoteliales/efectos de la radiación , Mutación con Ganancia de Función , Humanos , Interferón gamma/genética , Interferón gamma/metabolismo , Enfermedades Pulmonares Intersticiales/genética , Enfermedades Pulmonares Intersticiales/inmunología , Depleción Linfocítica , Tejido Linfoide/inmunología , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Ratones , Tolerancia a Radiación , Linfocitos T/inmunología , Enfermedades Vasculares/genética , Enfermedades Vasculares/inmunologíaRESUMEN
The swelling of univalent and multivalent charged polymeric networks in electrolytic solutions is studied using a classical thermodynamic model. Such systems were first modeled by Donnan, who derived an expression for the chemical potential of the ions by introducing an electric potential that is commonly referred to as the Donnan potential. This well-established theory leads to a simple quadratic relationship for the partitioning of ions between the network and the external solution. When the concentration of fixed charges in the swollen gel is large enough, the electrolyte in the external solution is "excluded" from the gel (commonly referred to as Donnan exclusion). In the standard Donnan theory, and in virtually all subsequent theories, the magnitude of Donnan exclusion decreases with increasing electrolyte concentration in the external solution. Our model predicts this is not necessarily true; we show that the magnitude of Donnan exclusion increases with increasing electrolyte concentration over a broad range of parameter space (average chain length between crosslinks, fraction of charged monomers in the network, the nature of the interactions between the ions, solvent molecules and polymer chains, and ion concentration in the external solution). We also present explicit bounds for the validity of Donnan's original theory. Model predictions are compared to simulations and experimental data obtained for a cationic gel immersed in electrolytic solutions of salts containing univalent and bivalent cations.
RESUMEN
BACKGROUNDInfluenza A virus (IAV) and SARS-CoV-2 are pandemic viruses causing millions of deaths, yet their clinical manifestations are distinctly different.METHODSWith the hypothesis that upper airway immune and epithelial cell responses are also distinct, we performed single-cell RNA sequencing (scRNA-Seq) on nasal wash cells freshly collected from adults with either acute COVID-19 or influenza or from healthy controls. We focused on major cell types and subtypes in a subset of donor samples.ResultsNasal wash cells were enriched for macrophages and neutrophils for both individuals with influenza and those with COVID-19 compared with healthy controls. Hillock-like epithelial cells, M2-like macrophages, and age-dependent B cells were enriched in COVID-19 samples. A global decrease in IFN-associated transcripts in neutrophils, macrophages, and epithelial cells was apparent in COVID-19 samples compared with influenza samples. The innate immune response to SARS-CoV-2 appears to be maintained in macrophages, despite evidence for limited epithelial cell immune sensing. Cell-to-cell interaction analyses revealed a decrease in epithelial cell interactions in COVID-19 and highlighted differences in macrophage-macrophage interactions for COVID-19 and influenza.ConclusionsOur study demonstrates that scRNA-Seq can define host and viral transcriptional activity at the site of infection and reveal distinct local epithelial and immune cell responses for COVID-19 and influenza that may contribute to their divergent disease courses.FundingMassachusetts Consortium on Pathogen Readiness, the Mathers Foundation, and the Department of Defense (W81XWH2110029) "COVID-19 Expansion for AIRe Program."
Asunto(s)
COVID-19 , Inmunidad Innata , Virus de la Influenza A , Gripe Humana , Macrófagos , RNA-Seq , SARS-CoV-2 , Adulto , COVID-19/genética , COVID-19/inmunología , Femenino , Humanos , Virus de la Influenza A/genética , Virus de la Influenza A/inmunología , Gripe Humana/genética , Gripe Humana/inmunología , Macrófagos/inmunología , Macrófagos/virología , Masculino , Lavado Nasal (Proceso) , SARS-CoV-2/genética , SARS-CoV-2/inmunologíaRESUMEN
Detection of DNA is an important determinant of host-defense but also a driver of autoinflammatory and autoimmune diseases. Failure to degrade self-DNA in DNAseII or III(TREX1)-deficient mice results in activation of the cGAS-STING pathway. Deficiency of cGAS or STING in these models ameliorates disease manifestations. However, the contribution of the cGAS-STING pathway, relative to endosomal TLRs, in systemic lupus erythematosus (SLE) is controversial. In fact, STING deficiency failed to rescue, and actually exacerbated, disease manifestations in Fas-deficient SLE-prone mice. We have now extended these observations to a chronic model of SLE induced by the i.p. injection of TMPD (pristane). We found that both cGAS- and STING-deficiency not only failed to rescue mice from TMPD-induced SLE, but resulted in increased autoantibody production and higher proteinuria levels compared to cGAS STING sufficient mice. Further, we generated cGASKOFaslpr mice on a pure MRL/Faslpr background using Crispr/Cas9 and found slightly exacerbated, and not attenuated, disease. We hypothesized that the cGAS-STING pathway constrains TLR activation, and thereby limits autoimmune manifestations in these two models. Consistent with this premise, mice lacking cGAS and Unc93B1 or STING and Unc93B1 developed minimal systemic autoimmunity as compared to cGAS or STING single knock out animals. Nevertheless, TMPD-driven lupus in B6 mice was abrogated upon AAV-delivery of DNAse I, implicating a DNA trigger. Overall, this study demonstrated that the cGAS-STING pathway does not promote systemic autoimmunity in murine models of SLE. These data have important implications for cGAS-STING-directed therapies being developed for the treatment of systemic autoimmunity.
Asunto(s)
Autoinmunidad , Susceptibilidad a Enfermedades , Lupus Eritematoso Sistémico/etiología , Proteínas de la Membrana/metabolismo , Nucleotidiltransferasas/metabolismo , Transducción de Señal , Animales , Autoanticuerpos/inmunología , Autoinmunidad/genética , Biomarcadores , Modelos Animales de Enfermedad , Expresión Génica , Inmunofenotipificación , Lupus Eritematoso Sistémico/metabolismo , Lupus Eritematoso Sistémico/patología , Macrófagos/inmunología , Macrófagos/metabolismo , Proteínas de la Membrana/genética , Ratones , Ratones Noqueados , Nucleotidiltransferasas/genéticaRESUMEN
This study shows that it is possible to obtain homogeneous mixtures of two chemically distinct polymers with a lithium salt for electrolytic applications. This approach is motivated by the success of using mixtures of organic solvents in modern lithium-ion batteries. The properties of mixtures of a polyether, poly(ethylene oxide) (PEO), a poly(ether-acetal), poly(1,3,6-trioxocane) (P(2EO-MO)), and lithium bis(trifluoromethanesulfonyl)imide (LiTFSI) salt were studied by small-angle neutron scattering (SANS) and electrochemical characterization in symmetric cells. The SANS data are used to determine the miscibility window and quantify the effect of added salt on the thermodynamic interactions between the polymers. In the absence of salt, PEO/P(2EO-MO) blends are homogeneous and characterized by attractive interactions, i.e., a negative Flory-Huggins interaction parameter, χ. The addition of small amounts of salt results in a positive effective Flory-Huggins interaction parameter, χ eff, and macrophase separation. Surprisingly, miscible blends and negative χ eff parameters are obtained when the salt concentration is increased beyond a critical value. The electrochemical properties of PEO/P(2EO-MO)/LiTFSI blends at a given salt concentration were close to those obtained in PEO/LiTFSI electrolytes at the same salt concentration. This suggests that in the presence of PEO the electrochemical properties exhibited by P(2EO-MO) chains are similar to those of PEO chains. This work opens the door to a new direction for creating new and improved polymer electrolytes either by combining existing polymers and salt or by synthesizing new polymers with the specific aim of including them in miscible polymer blend electrolytes.
RESUMEN
OBJECTIVE: Operators are mindful of the balloon-to-aortic annulus ratio when performing balloon aortic valvuloplasty. The method of measurement of the aortic valve annulus has not been standardised. METHODS AND RESULTS: Patients who underwent aortic valvuloplasty at two paediatric centres between 2007 and 2014 were included. The valve annulus measured by echocardiography and angiography was used to calculate the balloon-to-aortic annulus ratio and measurements were compared. The primary endpoint was an increase in aortic insufficiency by ≥2 degrees. Ninety-eight patients with a median age at valvuloplasty of 2.1 months (Interquartile range (IQR): 0.2-105.5) were included. The angiographic-based annulus was 8.2 mm (IQR: 6.8-16.0), which was greater than echocardiogram-based annulus of 7.5 mm (IQR: 6.1-14.8) (p < 0.001). This corresponded to a significantly lower angiographic balloon-to-aortic annulus ratio of 0.9 (IQR: 0.9-1.0), compared to an echocardiographic ratio of 1.1 (IQR: 1.0-1.1) (p < 0.001). The degree of discrepancy in measured diameter increased with smaller valve diameters (p = 0.041) and in neonates (p = 0.044). There was significant disagreement between angiographic and echocardiographic balloon-to-aortic annulus ratio measures regarding "High" ratio of >1.2, with angiographic ratio flagging only 2/12 (16.7%) of patients flagged by echocardiographic ratio as "High" (p = 0.012). Patients who had an increase in the degree of aortic insufficiency post valvuloplasty, only 3 (5.5%) had angiographic ratio > 1.1, while 21 (38%) had echocardiographic ratio >1.1 (p < 0.001). Patients with resultant ≥ moderate insufficiency more often had an echocardiographic ratio of >1.1 than angiographic ratio of >1.1 There was no association between increase in balloon-to-aortic annulus ratio and gradient reduction. CONCLUSIONS: Angiographic measurement is associated with a greater measured aortic valve annulus and the development of aortic insufficiency. Operators should use caution when relying solely on angiographic measurement when performing balloon aortic valvuloplasty.
Asunto(s)
Estenosis de la Válvula Aórtica , Valvuloplastia con Balón , Angiografía , Válvula Aórtica/diagnóstico por imagen , Válvula Aórtica/cirugía , Estenosis de la Válvula Aórtica/diagnóstico , Estenosis de la Válvula Aórtica/cirugía , Niño , Ecocardiografía , Humanos , Lactante , Recién Nacido , Estudios Retrospectivos , Resultado del TratamientoRESUMEN
Genome-wide association studies have identified common genetic variants impacting human diseases; however, there are indications that the functional consequences of genetic polymorphisms can be distinct depending on cell type-specific contexts, which produce divergent phenotypic outcomes. Thus, the functional impact of genetic variation and the underlying mechanisms of disease risk are modified by cell type-specific effects of genotype on pathological phenotypes. In this study, we extend these concepts to interrogate the interdependence of cell type- and stimulation-specific programs influenced by the core autophagy gene Atg16L1 and its T300A coding polymorphism identified by genome-wide association studies as linked with increased risk of Crohn's disease. We applied a stimulation-based perturbational profiling approach to define Atg16L1 T300A phenotypes in dendritic cells and T lymphocytes. Accordingly, we identified stimulus-specific transcriptional signatures revealing T300A-dependent functional phenotypes that mechanistically link inflammatory cytokines, IFN response genes, steroid biosynthesis, and lipid metabolism in dendritic cells and iron homeostasis and lysosomal biogenesis in T lymphocytes. Collectively, these studies highlight the combined effects of Atg16L1 genetic variation and stimulatory context on immune function.
Asunto(s)
Proteínas Relacionadas con la Autofagia/metabolismo , Enfermedad de Crohn/metabolismo , Células Dendríticas/fisiología , Genotipo , Linfocitos T/fisiología , Animales , Proteínas Relacionadas con la Autofagia/genética , Células Cultivadas , Enfermedad de Crohn/genética , Predisposición Genética a la Enfermedad , Humanos , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Especificidad de Órganos , Fenotipo , Polimorfismo Genético , Riesgo , Activación TranscripcionalRESUMEN
OBJECTIVE: Patients with hypomorphic mutations in DNase II develop a severe and debilitating autoinflammatory disease. This study was undertaken to compare the disease parameters in these patients to those in a murine model of DNase II deficiency, and to evaluate the role of specific nucleic acid sensors and identify the cell types responsible for driving the autoinflammatory response. METHODS: To avoid embryonic death, Dnase2-/- mice were intercrossed with mice that lacked the type I interferon (IFN) receptor (Ifnar-/- ). The hematologic changes and immune status of these mice were evaluated using complete blood cell counts, flow cytometry, serum cytokine enzyme-linked immunosorbent assays, and liver histology. Effector cell activity was determined by transferring T cells from Dnase2-/- × Ifnar-/- double-knockout (DKO) mice into Rag1-/- mice, and 4 weeks after cell transfer, induced changes were assessed in the recipient mice. RESULTS: In Dnase2-/- × Ifnar-/- DKO mice, many of the disease features found in DNase II-deficient patients were recapitulated, including cytopenia, extramedullary hematopoiesis, and liver fibrosis. Dnase2+/+ × Rag1-/- mice (n > 22) developed a hematologic disorder that was attributed to the transfer of an unusual IFNγ-producing T cell subset from the spleens of donor Dnase2-/- × Ifnar-/- DKO mice. Autoinflammation in this murine model did not depend on the stimulator of IFN genes (STING) pathway but was highly dependent on the chaperone protein Unc93B1. CONCLUSION: Dnase2-/- × Ifnar-/- DKO mice may be a valid model for exploring the innate and adaptive immune mechanisms responsible for the autoinflammation similar to that seen in DNASE2-hypomorphic patients. In this murine model, IFNγ is required for T cell activation and the development of clinical manifestations. The role of IFNγ in DNASE2-deficient patient populations remains to be determined, but the ability of Dnase2-/- mouse T cells to transfer disease to Rag1-/- mice suggests that T cells may be a relevant therapeutic target in patients with IFN-related systemic autoinflammatory diseases.
Asunto(s)
Enfermedades Autoinmunes/etiología , Endodesoxirribonucleasas/deficiencia , Inflamación/inmunología , Interferón gamma/biosíntesis , Células TH1/metabolismo , Animales , Modelos Animales de Enfermedad , Interferón Tipo I , Ratones , Ratones Endogámicos C57BLRESUMEN
Polymer chain dynamics of a nanostructured block copolymer electrolyte, polystyrene-block-poly(ethylene oxide) (SEO) mixed with lithium bis(trifluoromethanesulfonyl)imide (LiTFSI) salt, are investigated by neutron spin echo (NSE) spectroscopy on the 0.1-100 ns time scale and analyzed using the Rouse model at short times (t ≤ 10 ns) and the reptation tube model at long times (t ≥ 50 ns). In the Rouse regime, the monomeric friction coefficient increases with increasing salt concentration, as seen previously in homopolymer electrolytes. In the reptation regime, the tube diameters, which represent entanglement constraints, decrease with increasing salt concentration. The normalized longest molecular relaxation time, calculated from the NSE results, increases with increasing salt concentration. We argue that quantifying chain motion in the presence of ions is essential for predicting the behavior of polymer-electrolyte-based batteries operating at large currents.
RESUMEN
The CRISPR-Cas9 system provides unprecedented genome editing capabilities. However, off-target effects lead to sub-optimal usage and additionally are a bottleneck in the development of therapeutic uses. Herein, we introduce the first machine learning-based approach to off-target prediction, yielding a state-of-the-art model for CRISPR-Cas9 that outperforms all other guide design services. Our approach, Elevation, consists of two interdependent machine learning models-one for scoring individual guide-target pairs, and another which aggregates these guide-target scores into a single, overall summary guide score. Through systematic investigation, we demonstrate that Elevation performs substantially better than competing approaches on both tasks. Additionally, we are the first to systematically evaluate approaches on the guide summary score problem; we show that the most widely-used method performs no better than random at times, whereas Elevation consistently outperformed it, sometimes by an order of magnitude. We also introduce an evaluation method that balances errors between active and inactive guides, thereby encapsulating a range of practical use cases; Elevation is consistently superior to other methods across the entire range. Finally, because of the large scale and computational demands of off-target prediction, we have developed a cloud-based service for quick retrieval. This service provides end-to-end guide design by also incorporating our previously reported on-target model, Azimuth. (https://crispr.ml:please treat this web site as confidential until publication).
RESUMEN
The Cys-Cys chemokine receptor 6 (CCR6) is a well-established modulator of inflammation. Although several genetic associations have been identified between CCR6 polymorphisms and immune system disorders (e.g., rheumatoid arthritis and Crohn's disease), the pharmacological effects of naturally occurring missense mutations in this receptor have yet to be characterized. In this study, we initially assessed G protein-mediated signaling and observed that wild-type (WT) CCR6 exhibited ligand-independent activity. In addition, we found that the five most frequent CCR6 missense variants (A89T, A150V, R155W, G345S, and A369V) exhibited decreased basal and/or ligand induced Gαi protein signaling. To complement the study of these loss-of-function variants, we engineered a set of constitutively active CCR6 receptors. Selected mutations enhanced basal G protein-mediated signaling up to 3-fold relative to the WT value. Using a bioluminescence resonance energy transfer assay we investigated the ability of each naturally occurring and engineered CCR6 receptor mutant to recruit ß-arrestin. In contrast to G protein-mediated signaling, ß-arrestin mobilization was largely unperturbed by the naturally occurring loss-of-function CCR6 variants. Elevated recruitment of ß-arrestin was observed in one of the engineered constitutively active mutants (T98P). Our results demonstrate that point mutations in CCR6 can result in either a gain or loss of receptor function. These observations underscore the need to explore how CCR6 natural variants may influence immune cell physiology and human disease.
Asunto(s)
Mutación Puntual , Receptores CCR6/genética , Receptores CCR6/metabolismo , Bases de Datos Genéticas , Humanos , Transporte de Proteínas/genética , beta-Arrestinas/metabolismoRESUMEN
OBJECTIVES: We sought to determine the relationship between aortic valve morphology and left ventricular (LV) systolic function in children with aortic stenosis (AS) prior to balloon aortic valvuloplasty (BAV). BACKGROUND: Both aortic valve morphology and LV systolic function have been linked with outcomes in children with congenital AS undergoing BAV. The relationship between aortic valve morphology and LV function is poorly defined despite their importance in regard to outcomes. METHODS: We performed a retrospective multicenter cohort study of 89 AS patients who underwent BAV between 2007-2013. Pre-BAV echocardiograms were analyzed for: aortic valve opening (AVO); aortic valve type (true bicuspid, functionally bicuspid, or unicuspid); maximal raphe length; aortic valve leaflet symmetry; and valve angle of excursion. The primary endpoint was low function, defined as LV shortening fraction (LVSF) <28%. RESULTS: Median patient age was 0.17 years (interquartile range [IQR], 0.10-10.74 years) and the median aortic valve mean gradient was 47.00 mm Hg (IQR, 36.75-56.00 mm Hg). Multivariate analysis demonstrated that low AVO (P=.03) was associated with reduced LV function, independent of age or aortic valve gradient (R² = .652). Bicuspid aortic valve (P=.07) was associated with improved LV function compared with functionally unicuspid aortic valve. Low AVO <0.10 was associated with higher adverse outcome. CONCLUSION: LV systolic function is most significantly influenced by degree of valve stenosis. Qualitative aspects such as valve type may also affect LV systolic function. Further study may elucidate whether aortic valve morphology or LV function is the principal predictor of response to BAV and of late outcomes after BAV.