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Immune checkpoint therapy (ICT) shows encouraging results in a subset of patients with metastatic castration-resistant prostate cancer (mCRPC) but still elicits a sub-optimal response among those with bone metastases. Analysis of patients' bone marrow samples revealed increased Th17 instead of Th1 subsets after ICT. To further evaluate the different tumor microenvironments, we injected mice with prostate tumor cells either subcutaneously or intraosseously. ICT in the subcutaneous CRPC model significantly increases intra-tumoral Th1 subsets and improves survival. However, ICT fails to elicit an anti-tumor response in the bone CRPC model despite an increase in the intra-tumoral CD4 T cells, which are polarized to Th17 rather than Th1 lineage. Mechanistically, tumors in the bone promote osteoclast-mediated bone resorption that releases TGF-ß, which restrains Th1 lineage development. Blocking TGF-ß along with ICT increases Th1 subsets and promotes clonal expansion of CD8 T cells and subsequent regression of bone CRPC and improves survival.
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Linaje de la Célula , Inmunoterapia , Linfocitos T Colaboradores-Inductores/citología , Microambiente Tumoral , Animales , Antígenos/metabolismo , Neoplasias Óseas/secundario , Antígeno CTLA-4/metabolismo , Linaje de la Célula/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Células Clonales , Citocinas/metabolismo , Modelos Animales de Enfermedad , Memoria Inmunológica/efectos de los fármacos , Ipilimumab/farmacología , Masculino , Ratones , Osteoclastos/efectos de los fármacos , Osteoclastos/metabolismo , Receptor de Muerte Celular Programada 1/metabolismo , Neoplasias de la Próstata Resistentes a la Castración/inmunología , Neoplasias de la Próstata Resistentes a la Castración/patología , Análisis de Supervivencia , Linfocitos T Colaboradores-Inductores/efectos de los fármacos , Células TH1/efectos de los fármacos , Factor de Crecimiento Transformador beta/metabolismo , Microambiente Tumoral/efectos de los fármacosRESUMEN
Developing efficient blue emitters with high performance and low cost is crucial for the further development of organic light-emitting diodes (OLEDs). Based on the two experimentally reported green thermally activated delayed fluorescence (TADF) emitters, which are thioxanthone derivatives consisting of carbazole as an electron donor and 9H-thioxanthen-9-one-S,S-dioxide (SOXO) as an electron acceptor with donor-acceptor (D-A) or donor-acceptor-donor (D-A-D) structures, two new blue TADF emitters are designed by simply inserting a phenyl ring between D and A units. The TADF processes of the four thioxanthone derivatives are studied systematically through first-principles calculations. The role of the introduced phenyl ring in the excited state properties of the designed molecules is explored by analyzing the changes in molecular geometries, frontier molecular orbital distributions, the lowest singlet-triplet energy splitting (ΔEST), the spin orbit coupling (SOC) constants, the radiative decay rates (kr) and the nonradiative decay rates (knr), as well as the intersystem crossing rates (kISC) and reverse intersystem crossing rates (kRISC). The results show that when incorporating phenyl units into the D-A and D-A-D structures, both high kr and enhanced kRISC are achieved in Cz-Ph-SOXO and DCz-DPh-SOXO, demonstrating that incorporating the phenyl unit in D-A and D-A-D structures is an efficient way for developing new SOXO-based TADF molecules. It is worth noting that the kRISC values for Cz-Ph-SOXO and DCz-DPh-SOXO are significantly increased with respect to those of the experimental molecules. The present results would provide helpful guidelines for developing new SOXO-based TADF molecules experimentally.
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BACKGROUND: Exploring a simple and versatile technique for direct immobilization of target enzymes from cell lysate without prior purification is urgently needed. Thus, a novel all-in-one strategy for purification and immobilization of ß-1,3-xylanase was proposed, the target enzymes were covalently immobilized on silica nanoparticles via elastin-like polypeptides (ELPs)-based biomimetic silicification and SpyTag/SpyCatcher spontaneous reaction. Thus, the functional carriers that did not require the time-consuming surface modification step were quickly and efficiently prepared. These carriers could specifically immobilize the SpyTag-fused target enzymes from the cell lysate without pre-purification. RESULTS: The ELPs-SpyCatcher hardly leaked from the carriers (0.5%), and the immobilization yield of enzyme was up to 96%. Immobilized enzyme retained 85.6% of the initial activity and showed 88.6% of the activity recovery. Compared with free ones, the immobilized ß-1,3-xylanase showed improved thermal stability, elevated storage stability and good pH tolerance. It also retained more than 70.6% of initial activity after 12 reaction cycles, demonstrating its excellent reusability. CONCLUSIONS: The results clearly highlighted the effectiveness of the novel enzyme immobilization method proposed here due to the improvement of overall performance of immobilized enzyme in respect to free form for the hydrolysis of macromolecular substrates. Thus, it may have great potential in the conversion of algae biomass as well as other related fields.
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Proteínas Bacterianas/química , Bacteroidetes/enzimología , Biocatálisis , Endo-1,4-beta Xilanasas/química , Enzimas Inmovilizadas/química , Nanoestructuras/química , Mezclas Complejas/químicaRESUMEN
Proteomic profiling on subcellular fractions provides invaluable information regarding both protein abundance and subcellular localization. When integrated with other data sets, it can greatly enhance our ability to predict gene function genome-wide. In this study, we performed a comprehensive proteomic analysis on the light-sensing compartment of photoreceptors called the outer segment (OS). By comparing with the protein profile obtained from the retina tissue depleted of OS, an enrichment score for each protein is calculated to quantify protein subcellular localization, and 84% accuracy is achieved compared with experimental data. By integrating the protein OS enrichment score, the protein abundance, and the retina transcriptome, the probability of a gene playing an essential function in photoreceptor cells is derived with high specificity and sensitivity. As a result, a list of genes that will likely result in human retinal disease when mutated was identified and validated by previous literature and/or animal model studies. Therefore, this new methodology demonstrates the synergy of combining subcellular fractionation proteomics with other omics data sets and is generally applicable to other tissues and diseases.
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Proteínas del Ojo , Perfilación de la Expresión Génica , Proteómica , Enfermedades de la Retina , Transcriptoma , Animales , Proteínas del Ojo/genética , Proteínas del Ojo/metabolismo , Humanos , Ratones , Células Fotorreceptoras , Enfermedades de la Retina/genética , Enfermedades de la Retina/metabolismoRESUMEN
PURPOSE: To characterize the phenotypic variability and report the genetic defects in a cohort of Chinese patients with biallelic variants of the retinol dehydrogenase 12 (RDH12) gene. METHODS: The study included 38 patients from 38 unrelated families with biallelic pathogenic RDH12 variants. Systematic next-generation sequencing data analysis, Sanger sequencing validation, and segregation analysis were used to identify the pathogenic mutations. Detailed ophthalmic examinations, including electroretinogram, fundus photography, fundus autofluorescence and optical coherence tomography, and statistical analysis were performed to evaluate phenotype variability. RESULTS: Twenty-five different mutations of RDH12 were identified in the 38 families. Six of these variants were novel. Val146Asp was observed at the highest frequency (23.7%), and it was followed by Arg62Ter (14.5%) and Thr49Met (9.2%). Twenty-three probands were diagnosed with early-onset severe retinal dystrophy, 6 with Leber congenital amaurosis, 7 with autosomal recessive retinitis pigmentosa, and 2 with cone-rod dystrophy. Self-reported nyctalopia occurred in about a half of patients (55.3%) and was significantly more common among older patients (P < 0.01). Nyctalopia was not significantly associated with best-corrected visual acuity (P = 0.72), but older patients had significantly greater best-corrected visual acuity loss (P < 0.01). Only 15.8% of the patients had nystagmus, which was significantly more likely to occur among 36.8% of the patients with hyperopia >3D (P < 0.01) and/or in cases of reduced best-corrected visual acuity (P = 0.01), but was not associated with age (P = 0.87). CONCLUSION: Several high-frequency RDH12 variants were identified in patients with inherited retinal dystrophies, most of which were missense mutations. Variable but characteristic phenotypes of a progressive nature was observed. Overall, the findings indicated that biallelic RDH12 mutations are a common cause of early-onset retinal dystrophy and a rare cause of cone-rod dystrophy.
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Oxidorreductasas de Alcohol/genética , Enfermedades Hereditarias del Ojo/genética , Mutación , Distrofias Retinianas/genética , Agudeza Visual , Adolescente , Adulto , Oxidorreductasas de Alcohol/metabolismo , Variación Biológica Poblacional , Niño , Preescolar , Análisis Mutacional de ADN , Electrorretinografía , Enfermedades Hereditarias del Ojo/diagnóstico , Enfermedades Hereditarias del Ojo/metabolismo , Femenino , Genotipo , Humanos , Masculino , Persona de Mediana Edad , Linaje , Fenotipo , Distrofias Retinianas/diagnóstico , Distrofias Retinianas/metabolismo , Adulto JovenRESUMEN
Elastin-Like polypeptides (ELPs), as well-known temperature-controlled bio-macromolecules, are widely used. However, little is known about the interactions between ELPs and macromolecules, which is an important yet neglected problem. Here, the phase transition characteristics of an ELPs-SpyCatcher fusion protein (E-C) in the presence of polyethylene glycol (PEG) in single salts (Na2CO3, Na2SO4, NaCl) solutions were investigated using a UV spectrophotometer, DLC, and fluorescence spectroscopy, and we got some interesting results. The phases transition of E-C occurred at a concentration lower than 0.5 mol/L Na2CO3/PEG2000, while in single Na2CO3 (<0.5 mol/L), the phase transition of E-C did not occur. In the Na2CO3/PEG solution, we observed a unique two-step phase transition of E-C when the Na2CO3 concentration was 0.5 mol/L and PEG2000 concentration was less than 0.15 g/mL, respectively. In the Na2CO3/PEG2000 solution, the phase-transition temperature of E-C decreased with the increase of PEG concentration, but increased in the Na2SO4/PEG2000 solution, while it remained unchanged in the NaCl/PEG2000 solution. However, the phase-transition temperature of the linear ELPs40 decreased under the same salts/PEG2000 solutions. We also addressed the possible molecular mechanism of the interesting results. In contrast to the current well-understood salts-ELPs interactions, this work provides some new insights into the interaction between the PEG-salts-ELPs in solution.
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Elastina/química , Péptidos/química , Polietilenglicoles/química , Transición de Fase , Espectrometría de Fluorescencia , Espectrofotometría Ultravioleta , Temperatura de TransiciónRESUMEN
Drosophila eye development is a complex process that involves many transcription factors (TFs) and interactions with their cofactors and targets. The TF Sine oculis (So) and its cofactor Eyes absent (Eya) are highly conserved and are both necessary and sufficient for eye development. Despite their many important roles during development, the direct targets of So are still largely unknown. Therefore the So-dependent regulatory network governing eye determination and differentiation is poorly understood. In this study, we intersected gene expression profiles of so or eya mutant eye tissue prepared from three different developmental stages and identified 1731 differentially expressed genes across the Drosophila genome. A combination of co-expression analyses and motif discovery identified a set of twelve putative direct So targets, including three known and nine novel targets. We also used our previous So ChIP-seq data to assess motif predictions for So and identified a canonical So binding motif. Finally, we performed in vivo enhancer reporter assays to test predicted enhancers from six candidate target genes and find that at least one enhancer from each gene is expressed in the developing eye disc and that their expression patterns overlap with that of So. We furthermore confirmed that the expression level of predicted direct So targets, for which antibodies are available, are reduced in so or eya post-mitotic knockout eye discs. In summary, we expand the set of putative So targets and show for the first time that the combined use of expression profiling of so with its cofactor eya is an effective method to identify novel So targets. Moreover, since So is highly conserved throughout the metazoa, our results provide the basis for future functional studies in a wide variety of organisms.
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Ojo Compuesto de los Artrópodos/crecimiento & desarrollo , Proteínas de Drosophila/fisiología , Drosophila melanogaster/genética , Proteínas del Ojo/fisiología , Regulación del Desarrollo de la Expresión Génica , Estudio de Asociación del Genoma Completo , Proteínas de Homeodominio/fisiología , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Animales , Inmunoprecipitación de Cromatina , Ojo Compuesto de los Artrópodos/ultraestructura , Secuencia de Consenso , Drosophila melanogaster/crecimiento & desarrollo , Ontología de Genes , Estudios de Asociación Genética , Discos Imaginales/metabolismo , Larva , Pupa , ARN Mensajero/genética , Transcripción Genética , TranscriptomaRESUMEN
Histone acetyltransferase 1 is an evolutionarily conserved type B histone acetyltransferase that is thought to be responsible for the diacetylation of newly synthesized histone H4 on lysines 5 and 12 during chromatin assembly. To understand the function of this enzyme in a complex organism, we have constructed a conditional mouse knockout model of Hat1. Murine Hat1 is essential for viability, as homozygous deletion of Hat1 results in neonatal lethality. The lungs of embryos and pups genetically deficient in Hat1 were much less mature upon histological evaluation. The neonatal lethality is due to severe defects in lung development that result in less aeration and respiratory distress. Many of the Hat1(-/-) neonates also display significant craniofacial defects with abnormalities in the bones of the skull and jaw. Hat1(-/-) mouse embryonic fibroblasts (MEFs) are defective in cell proliferation and are sensitive to DNA damaging agents. In addition, the Hat1(-/-) MEFs display a marked increase in genome instability. Analysis of histone dynamics at sites of replication-coupled chromatin assembly demonstrates that Hat1 is not only responsible for the acetylation of newly synthesized histone H4 but is also required to maintain the acetylation of histone H3 on lysines 9, 18, and 27 during replication-coupled chromatin assembly.
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Desarrollo Embrionario/genética , Inestabilidad Genómica , Histona Acetiltransferasas/genética , Histonas/genética , Acetilación , Animales , Proliferación Celular , Supervivencia Celular/genética , Ensamble y Desensamble de Cromatina/genética , Replicación del ADN/genética , Fibroblastos/citología , Fibroblastos/metabolismo , Humanos , Histona Demetilasas con Dominio de Jumonji/genética , Ratones , Ratones NoqueadosRESUMEN
Leber congenital amaurosis (LCA) and retinitis pigmentosa (RP) are two genetically heterogeneous retinal degenerative disorders. Despite the identification of a number of genes involved in LCA and RP, the genetic etiology remains unknown in many patients. In this study, we aimed to identify novel disease-causing genes of LCA and RP. Retinal capture sequencing was initially performed to screen mutations in known disease-causing genes in different cohorts of LCA and RP patients. For patients with negative results, we performed whole exome sequencing and applied a series of variant filtering strategies. Sanger sequencing was done to validate candidate causative IFT140 variants. Exome sequencing data analysis led to the identification of IFT140 variants in multiple unrelated non-syndromic LCA and RP cases. All the variants are extremely rare and predicted to be damaging. All the variants passed Sanger validation and segregation tests provided that the family members' DNA was available. The results expand the phenotype spectrum of IFT140 mutations to non-syndromic retinal degeneration, thus extending our understanding of intraflagellar transport and primary cilia biology in the retina. This work also improves the molecular diagnosis of retinal degenerative disease.
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Proteínas Portadoras/genética , Amaurosis Congénita de Leber/genética , Retinitis Pigmentosa/genética , Adulto , Secuencia de Aminoácidos , Niño , Femenino , Estudios de Asociación Genética , Humanos , Masculino , Persona de Mediana Edad , Mutación Missense , LinajeRESUMEN
Somatic nuclear autoantigenic sperm protein (sNASP) is a human homolog of the N1/N2 family of histone chaperones. sNASP contains the domain structure characteristic of this family, which includes a large acidic patch flanked by several tetratricopeptide repeat (TPR) motifs. sNASP possesses a unique binding specificity in that it forms specific complexes with both histone H1 and histones H3/H4. Based on the binding affinities of sNASP variants to histones H1, H3.3, H4 and H3.3/H4 complexes, sNASP uses distinct structural domains to interact with linker and core histones. For example, one of the acidic patches of sNASP was essential for linker histone binding but not for core histone interactions. The fourth TPR of sNASP played a critical role in interactions with histone H3/H4 complexes, but did not influence histone H1 binding. Finally, analysis of cellular proteins demonstrated that sNASP existed in distinct complexes that contained either linker or core histones.
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Autoantígenos/metabolismo , Histonas/metabolismo , Proteínas Nucleares/metabolismo , Autoantígenos/química , Línea Celular , Humanos , Modelos Moleculares , Proteínas Nucleares/química , Dominios y Motivos de Interacción de Proteínas , Isoformas de Proteínas/química , Isoformas de Proteínas/metabolismoRESUMEN
We have previously demonstrated synergy between ICOS costimulation (IVAX; ICOSL-transduced B16-F10 cellular vaccine) and CTLA-4 blockade in antitumor therapy. In this study, we employed CyTOF and single-cell RNA sequencing and observed significant remodeling of the lymphoid and myeloid compartments in combination therapy. Compared with anti-CTLA-4 monotherapy, the combination therapy enriched Th1 CD4 T cells, effector CD8 T cells, and M1-like antitumor proinflammatory macrophages. These macrophages were critical to the therapeutic efficacy of anti-CTLA-4 combined with IVAX or anti-PD-1. Macrophage depletion with clodronate reduced the tumor-infiltrating effector CD4 and CD8 T cells, impairing their antitumor functions. Furthermore, the recruitment and polarization of M1-like macrophages required IFN-γ. Therefore, in this study, we show that there is a positive feedback loop between intratumoral effector T cells and tumor-associated macrophages (TAMs), in which the IFN-γ produced by the T cells polarizes the TAMs into M1-like phenotype, and the TAMs, in turn, reshape the tumor microenvironment to facilitate T cell infiltration, immune function, and tumor rejection.
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Neoplasias , Macrófagos Asociados a Tumores , Humanos , Antígeno CTLA-4 , Neoplasias/terapia , Linfocitos T CD8-positivos , Fenotipo , Microambiente Tumoral , Proteína Coestimuladora de Linfocitos T InduciblesRESUMEN
Chromatin is disassembled and reassembled during DNA repair. To assay chromatin reassembly accompanying DNA double strand break repair, ChIP analysis can be used to monitor the presence of histone H3 near the lesion. The chromatin assembly factor Asf1p, as well as the acetylation of histone H3 lysine 56, have been shown to promote chromatin reassembly when DNA double strand break repair is complete. Using Gal-HO-mediated double strand break repair, we have tested each of the components of the nuclear Hat1p-containing type B histone acetyltransferase complex (NuB4) and have found that they can affect repair-linked chromatin reassembly but that their contributions are not equivalent. In particular, deletion of the catalytic subunit, Hat1p, caused a significant defect in chromatin reassembly. In addition, loss of the histone chaperone Hif1p, when combined with an allele of H3 that mutates lysines 14 and 23 to arginine, has a pronounced effect on chromatin reassembly that is similar to that observed in an asf1Δ. The role of Hat1p and Hif1p is at least partially redundant with the role of Asf1p. Consistent with a more prominent role for Hif1p in chromatin reassembly than either Hat1p or Hat2p, Hif1p exists in complex(es) independent of Hat1p and Hat2p and influences the activity of an H3-specific histone acetyltransferase activity. Our data directly demonstrate the role of the nuclear HAT1 complex (NuB4) components in DNA repair-linked chromatin reassembly.
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Núcleo Celular/metabolismo , Cromatina/metabolismo , Reparación del ADN , Histona Acetiltransferasas/metabolismo , Saccharomyces cerevisiae/metabolismo , Acetilación , Alelos , Catálisis , Inmunoprecipitación de Cromatina , Daño del ADN , Endonucleasas/metabolismo , Histona Acetiltransferasas/química , Chaperonas de Histonas , Histonas/química , Proteínas Nucleares/metabolismo , Unión Proteica , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMEN
Enzyme separation, purification, immobilization, and catalytic performance improvement have been the research hotspots and frontiers as well as the challenges in the field of biocatalysis. Thus, the development of novel methods for enzyme purification, immobilization, and improvement of their catalytic performance and storage are of great significance. Herein, ferritin was fused with the lichenase gene to achieve the purpose. The results showed that the fused gene was highly expressed in the cells of host strains, and that the resulted fusion proteins could self-aggregate into carrier-free active immobilized enzymes in vivo. Through low-speed centrifugation, the purity of the enzymes was up to > 90%, and the activity recovery was 61.1%. The activity of the enzymes after storage for 608 h was higher than the initial activity. After being used for 10 cycles, it still maintained 50.0% of the original activity. The insoluble active lichenase aggregates could spontaneously dissolve back into the buffer and formed the soluble polymeric lichenases with the diameter of about 12 nm. The specific activity of them was 12.09 times that of the free lichenase, while the catalytic efficiency was 7.11 times and the half-life at 50 â was improved 11.09 folds. The results prove that the ferritin can be a versatile tag to trigger target enzyme self-aggregation and oligomerization in vivo, which can simplify the preparation of the target enzymes, improve their catalysis performance, and facilitate their storage.
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Ferritinas , Glicósido Hidrolasas , Biocatálisis , Enzimas Inmovilizadas/metabolismo , Ferritinas/genética , Ferritinas/metabolismo , Glicósido Hidrolasas/genética , Glicósido Hidrolasas/metabolismoRESUMEN
Germline mutations in BRCA1 or BRCA2 exist in ~2-7% of breast cancer patients, which has led to the approval of PARP inhibitors in the advanced setting. We have previously reported a phase II neoadjuvant trial of single agent talazoparib for patients with germline BRCA pathogenic variants with a pathologic complete response (pCR) rate of 53%. As nearly half of the patients treated did not have pCR, better strategies are needed to overcome treatment resistance. To this end, we conducted multi-omic analysis of 13 treatment naïve breast cancer tumors from patients that went on to receive single-agent neoadjuvant talazoparib. We looked for biomarkers that were predictive of response (assessed by residual cancer burden) after 6 months of therapy. We found that all resistant tumors exhibited either the loss of SHLD2, expression of a hypoxia signature, or expression of a stem cell signature. These results indicate that the deep analysis of pre-treatment tumors can identify biomarkers that are predictive of response to talazoparib and potentially other PARP inhibitors, and provides a framework that will allow for better selection of patients for treatment, as well as a roadmap for the development of novel combination therapies to prevent emergence of resistance.
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Pob3p is an essential component of the S. cerevisiae FACT complex (yFACT). Several lines of evidence indicate that the yFACT complex plays an important role in chromatin assembly including the observation that the pob3 Q308K allele is synthetically lethal with an allele of histone H4 that prevents the diacetylation of newly synthesized molecules. We have analyzed the genetic interactions between the Q308K allele of POB3 and mutations in all of the sites of acetylation that have been identified on newly synthesized histones. Genetic interactions were observed between POB3 and sites of acetylation on the NH(2)-terminal tails of H3 and H4. For histone H3, lysine residues 14 and 23 were particularly important when POB3 activity is compromised. Surprisingly, synthetic defects observed when the pob3 Q308K allele was combined with mutations of H4 lysines 5 and 12, were not phenocopied by deletion of HAT1, which encodes the enzyme that is thought to generate this pattern of acetylation on H4. Genetic interactions were also observed between POB3 and sites of acetylation found in the core domain of newly synthesized histones H3 and H4. These include synthetic lethality with an allele of H4 lysine 91 that mimics constitutive acetylation. While the mutations that alter H4 lysines 5, 12 and 91 do not affect binding to Pob3p, mutation of histone H3 lysine 56 decreases the association of histones with Pob3p. These results support the model that the yFACT complex plays a central role in chromatin assembly pathways regulated by acetylation of newly synthesized histones.
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Histonas/genética , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , Factores de Transcripción/genética , Acetilación , Histona Acetiltransferasas/genética , Histona Acetiltransferasas/metabolismo , Histonas/metabolismo , Mutación , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Factores de Transcripción/metabolismoRESUMEN
Immune receptors expressed on TAMs are intriguing targets for tumor immunotherapy. In this study, we found inhibitory receptor LILRB4 on a variety of intratumoral immune cell types in murine tumor models and human cancers, most prominently on TAMs. LILRB4, known as gp49B in mice, is a LILRB family receptor. Human and murine LILRB4 have two extracellular domains but differ in the number of intracellular ITIMs (three versus two). We observed a high correlation in LILRB4 expression with other immune inhibitory receptors. After tumor challenge, LILRB4-/- mice and mice treated with anti-LILRB4 antibody showed reduced tumor burden and increased survival. LILRB4-/- genotype or LILRB4 blockade increased tumor immune infiltrates and the effector (Teff) to regulatory (Treg) T cell ratio and modulated phenotypes of TAMs toward less suppressive, CD4+ T cells to Th1 effector, and CD8+ T cells to less exhausted. These findings reveal that LILRB4 strongly suppresses tumor immunity in TME and that alleviating that suppression provides antitumor efficacy.
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Glicoproteínas de Membrana/inmunología , Neoplasias/inmunología , Neoplasias/terapia , Receptores Inmunológicos/inmunología , Animales , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD8-positivos/inmunología , Línea Celular Tumoral , Movimiento Celular/inmunología , Humanos , Inmunoterapia/métodos , Ratones , Ratones Endogámicos C57BL , Linfocitos T Reguladores/inmunologíaRESUMEN
The relationship between ATR/Chk1 activity and replication stress, coupled with the development of potent and tolerable inhibitors of this pathway, has led to the clinical exploration of ATR and Chk1 inhibitors (ATRi/Chk1i) as anticancer therapies for single-agent or combinatorial application. The clinical efficacy of these therapies relies on the ability to ascertain which patient populations are most likely to benefit, so there is intense interest in identifying predictive biomarkers of response. To comprehensively evaluate the components that modulate cancer cell sensitivity to replication stress induced by Chk1i, we performed a synthetic-lethal drop-out screen in a cell line derived from a patient with triple-negative breast cancer (TNBC), using a pooled barcoded shRNA library targeting ~350 genes involved in DNA replication, DNA damage repair, and cycle progression. In addition, we sought to compare the relative requirement of these genes when DNA fidelity is challenged by clinically relevant anticancer breast cancer drugs, including cisplatin and PARP1/2 inhibitors, that have different mechanisms of action. This global comparison is critical for understanding not only which agents should be used together for combinatorial therapies in breast cancer patients, but also the genetic context in which these therapies will be most effective, and when a single-agent therapy will be sufficient to provide maximum therapeutic benefit to the patient. We identified unique potentiators of response to ATRi/Chk1i and describe a new role for components of the cytosolic iron-sulfur assembly (CIA) pathway, MMS19 and CIA2B-FAM96B, in replication stress tolerance of TNBC.
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ß-hydroxybutyrate (ß-OHB) is an essential metabolic energy source during fasting and functions as a chromatin regulator by lysine ß-hydroxybutyrylation (Kbhb) modification of the core histones H3 and H4. We report that Kbhb on histone H3 (H3K9bhb) is enriched at proximal promoters of critical gene subsets associated with lipolytic and ketogenic metabolic pathways in small intestine (SI) crypts during fasting. Similar Kbhb enrichment is observed in Lgr5+ stem cell-enriched epithelial spheroids treated with ß-OHB in vitro. Combinatorial chromatin state analysis reveals that H3K9bhb is associated with active chromatin states and that fasting enriches for an H3K9bhb-H3K27ac signature at active metabolic gene promoters and distal enhancer elements. Intestinal knockout of Hmgcs2 results in marked loss of H3K9bhb-associated loci, suggesting that local production of ß-OHB is responsible for chromatin reprogramming within the SI crypt. We conclude that modulation of H3K9bhb in SI crypts is a key gene regulatory event in response to fasting.
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Ácido 3-Hidroxibutírico/metabolismo , Ayuno/metabolismo , Histonas/metabolismo , Acetilación , Animales , Cromatina/metabolismo , Ayuno/fisiología , Femenino , Expresión Génica/genética , Regulación de la Expresión Génica/genética , Intestino Delgado/metabolismo , Cuerpos Cetónicos/metabolismo , Lisina/análogos & derivados , Lisina/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Regiones Promotoras Genéticas/genética , Secuencias Reguladoras de Ácidos Nucleicos/genéticaRESUMEN
To elucidate mechanisms by which T cells eliminate leukemia, we study donor lymphocyte infusion (DLI), an established immunotherapy for relapsed leukemia. We model T cell dynamics by integrating longitudinal, multimodal data from 94,517 bone marrow-derived single T cell transcriptomes in addition to chromatin accessibility and single T cell receptor sequencing from patients undergoing DLI. We find that responsive tumors are defined by enrichment of late-differentiated T cells before DLI and rapid, durable expansion of early differentiated T cells after treatment, highly similar to "terminal" and "precursor" exhausted subsets, respectively. Resistance, in contrast, is defined by heterogeneous T cell dysfunction. Surprisingly, early differentiated T cells in responders mainly originate from pre-existing and novel clonotypes recruited to the leukemic microenvironment, rather than the infusion. Our work provides a paradigm for analyzing longitudinal single-cell profiling of scenarios beyond adoptive cell therapy and introduces Symphony, a Bayesian approach to infer regulatory circuitry underlying T cell subsets, with broad relevance to exhaustion antagonists across cancers.
Asunto(s)
Inmunoterapia Adoptiva/métodos , Leucemia/inmunología , Activación de Linfocitos/inmunología , Transfusión de Linfocitos/métodos , Recurrencia Local de Neoplasia/inmunología , Trasplante de Células Madre/métodos , Linfocitos T/inmunología , Evolución Clonal , Humanos , Leucemia/patología , Leucemia/terapia , Estudios Longitudinales , Recurrencia Local de Neoplasia/patología , Recurrencia Local de Neoplasia/terapia , Donantes de Tejidos , Trasplante HomólogoRESUMEN
Human chondrocytes are expanded and used in autologous chondrocyte implantation techniques and are known to rapidly de-differentiate in culture. These chondrocytes, when cultured on tissue culture plastic (TCP), undergo both phenotypical and morphological changes and quickly lose the ability to re-differentiate to produce hyaline-like matrix. Growth on synoviocyte-derived extracellular matrix (SDECM) reduces this de-differentiation, allowing for more than twice the number of population doublings (PD) whilst retaining chondrogenic capacity. The goal of this study was to apply RNA sequencing (RNA-Seq) analysis to examine the differences between TCP-expanded and SDECM-expanded human chondrocytes. Human chondrocytes from three donors were thawed from primary stocks and cultured on TCP flasks or on SDECM-coated flasks at physiological oxygen tension (5%) for 4 passages. During log expansion, RNA was extracted from the cell layer (70â»90% confluence) at passages 1 and 4. Total RNA was column-purified and DNAse-treated before quality control analysis and next-generation RNA sequencing. Significant effects on gene expression were observed due to both culture surface and passage number. These results offer insight into the mechanism of how SDECM provides a more chondrogenesis-preserving environment for cell expansion, the transcriptome-wide changes that occur with culture, and potential mechanisms for further enhancement of chondrogenesis-preserving growth.