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1.
J Transl Med ; 21(1): 829, 2023 11 17.
Artículo en Inglés | MEDLINE | ID: mdl-37978386

RESUMEN

Coronavirus disease 2019 (COVID-19) is an infectious disease caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Since the start of the pandemic, olfactory dysfunction (OD) has been reported as a common symptom of COVID-19. In some asymptomatic carriers, OD is often the first and even the only symptom. At the same time, persistent OD is also a long-term sequela seen after COVID-19 that can have a serious impact on the quality of life of patients. However, the pathogenesis of post-COVID-19 OD is still unclear, and there is no specific treatment for its patients. The aim of this paper was to review the research on OD caused by SARS-CoV-2 infection and to summarize the mechanism of action, the pathogenesis, and current treatments.


Asunto(s)
COVID-19 , Trastornos del Olfato , Humanos , COVID-19/complicaciones , SARS-CoV-2 , Calidad de Vida , Trastornos del Olfato/complicaciones , Trastornos del Olfato/terapia , Olfato
2.
Front Immunol ; 13: 996721, 2022.
Artículo en Inglés | MEDLINE | ID: mdl-36389765

RESUMEN

Interpreting the mechanisms and principles that govern gene activity and how these genes work according to -their cellular distribution in organisms has profound implications for cancer research. The latest technological advancements, such as imaging-based approaches and next-generation single-cell sequencing technologies, have established a platform for spatial transcriptomics to systematically quantify the expression of all or most genes in the entire tumor microenvironment and explore an array of disease milieus, particularly in tumors. Spatial profiling technologies permit the study of transcriptional activity at the spatial or single-cell level. This multidimensional classification of the transcriptomic and proteomic signatures of tumors, especially the associated immune and stromal cells, facilitates evaluation of tumor heterogeneity, details of the evolutionary trajectory of each tumor, and multifaceted interactions between each tumor cell and its microenvironment. Therefore, spatial profiling technologies may provide abundant and high-resolution information required for the description of clinical-related features in immuno-oncology. From this perspective, the present review will highlight the importance of spatial transcriptomic and spatial proteomics analysis along with the joint use of other sequencing technologies and their implications in cancers and immune-oncology. In the near future, advances in spatial profiling technologies will undoubtedly expand our understanding of tumor biology and highlight possible precision therapeutic targets for cancer patients.


Asunto(s)
Neoplasias , Microambiente Tumoral , Humanos , Microambiente Tumoral/genética , Proteómica , Oncología Médica , Secuenciación de Nucleótidos de Alto Rendimiento
3.
Ther Clin Risk Manag ; 18: 185-195, 2022.
Artículo en Inglés | MEDLINE | ID: mdl-35281777

RESUMEN

The olfactory nerve (ON) is the only cranial nerve exposed to the external environment. Hence, it is susceptible to damage from head trauma, viral infection, inflammatory stimulation, and chemical toxins, which can lead to olfactory dysfunction. However, compared with all other cranial nerves, the ON is unique due to its inherent ability to regenerate. This characteristic provides a theoretical basis for treatment of olfactory dysfunction. Olfactory training (OT) is one of the main treatments for olfactory dysfunction. It is easy to apply and has few side-effects, and has been shown to be efficacious for patients with olfactory dysfunction of various causes. To further understand the application value of ON regeneration and OT on olfactory dysfunction, we review the research progress on the mechanism of ON regeneration and OT.

4.
Mol Cancer ; 19(1): 137, 2020 09 04.
Artículo en Inglés | MEDLINE | ID: mdl-32887624

RESUMEN

An amendment to this paper has been published and can be accessed via the original article.

5.
Cell Death Dis ; 11(1): 17, 2020 01 06.
Artículo en Inglés | MEDLINE | ID: mdl-31907348

RESUMEN

Endoplasmic reticulum (ER) stress-associated cell death is prevalent in various liver diseases. However, the determinant mechanism how hepatocytes survive unresolved stress was still unclear. Interleukin-24 (IL-24) was previously found to promote ER stress-mediated cell death, and yet its expression and function in the liver remained elusive. Here we identified an antiapoptotic role of IL-24, which transiently accumulated within ER-stressed hepatocytes in a X-box binding protein 1 (XBP1)-dependent manner. Disruption of IL-24 increased cell death in the CCL4- or APAP-challenged mouse liver or Tm-treated hepatocytes. In contrast, pharmaceutical blockade of eukaryotic initiation factor 2α (eIF2α) or genetical ablation of C/EBP homologous protein (CHOP) restored hepatocyte function in the absence of IL-24. In a clinical setting, patients with acute liver failure manifested a profound decrease of hepatic IL-24 expression, which was associated with disease progression. In conclusion, intrinsic hepatocyte IL-24 maintains ER homeostasis by restricting the eIF2α-CHOP pathway-mediated stress signal, which might be exploited as a bio-index for prognosis or therapeutic intervention in patients with liver injury.


Asunto(s)
Apoptosis , Interleucinas/metabolismo , Espacio Intracelular/metabolismo , Hígado/metabolismo , Hígado/patología , Transducción de Señal , Respuesta de Proteína Desplegada , Proteína 1 de Unión a la X-Box/metabolismo , Animales , Estrés del Retículo Endoplásmico , Factor 2 Eucariótico de Iniciación , Hepatocitos/metabolismo , Hepatocitos/patología , Homeostasis , Interleucinas/deficiencia , Hígado/lesiones , Ratones Endogámicos C57BL , Modelos Biológicos , Factor de Transcripción CHOP/metabolismo , eIF-2 Quinasa/metabolismo
6.
Mol Cancer ; 18(1): 163, 2019 11 18.
Artículo en Inglés | MEDLINE | ID: mdl-31735169

RESUMEN

BACKGROUND: Dynamic N6-methyladenosine (m6A) modification was previously identified as a ubiquitous post-transcriptional regulation that affected mRNA homeostasis. However, the m6A-related epitranscriptomic alterations and functions remain elusive in human cancer. Here we aim to identify the profile and outcome of m6A-methylation in hepatocellular carcinoma (HCC). RESULTS: Using liquid chromatography-tandem mass spectrometry and m6A-immunoprecipitation in combination with high-throughput sequencing, we determined the m6A-mRNA levels in human HCC. Human HCC exhibited a characteristic gain of m6A modification in tandem with an increase of mRNA expression, owing to YTH domain family 2 (YTHDF2) reduction. The latter predicted poor classification and prognosis of HCC patients, and highly correlated with HCC m6A landscape. YTHDF2 silenced in human HCC cells or ablated in mouse hepatocytes provoked inflammation, vascular reconstruction and metastatic progression. Mechanistically, YTHDF2 processed the decay of m6A-containing interleukin 11 (IL11) and serpin family E member 2 (SERPINE2) mRNAs, which were responsible for the inflammation-mediated malignancy and disruption of vascular normalization. Reciprocally, YTHDF2 transcription succumbed to hypoxia-inducible factor-2α (HIF-2α). Administration of a HIF-2α antagonist (PT2385) restored YTHDF2-programed epigenetic machinery and repressed liver cancer. CONCLUSION: Our results have characterized the m6A-mRNA landscape in human HCC and revealed YTHDF2 as a molecular 'rheostat' in epitranscriptome and cancer progression.


Asunto(s)
Carcinoma Hepatocelular/etiología , Carcinoma Hepatocelular/patología , Inflamación/complicaciones , Inflamación/genética , Neoplasias Hepáticas/etiología , Neoplasias Hepáticas/patología , Neovascularización Patológica/genética , Proteínas de Unión al ARN/genética , Adenosina/análogos & derivados , Animales , Epigénesis Genética , Humanos , Metilación , Ratones , Modelos Biológicos , ARN Mensajero/genética , ARN Mensajero/metabolismo , Proteínas de Unión al ARN/metabolismo , Transcriptoma
7.
Mol Ther Nucleic Acids ; 16: 494-504, 2019 Jun 07.
Artículo en Inglés | MEDLINE | ID: mdl-31051334

RESUMEN

MicroRNAs (miRNAs) act as regulators of aging at the tissue or organism level or as regulators of cellular senescence. Targeted deletion of miR-126 in mice causes partial embryonic lethality, but its biological function in the liver is still largely unknown. Here, we deleted miR-126a, using the CRISPR/Cas9 system in vitro and in vivo. miR-126a was reduced in the aging livers, and disruption of miR-126a in bone mesenchymal stem cells (BMSCs) induced age-associated telomere shortening, DNA damage responses, and proinflammatory cytokines. Moreover, disruption of miR-126a in mice caused hepatocyte senescence, inflammation, and metabolism deficiency. In addition, disruption of miR-126a via BMSC transplantation aggravated the severity of liver defects induced by cholestasis compared with that in the functional miR-126a BMSC group. Mechanistically, we identified versican (VCAN) as a novel direct miR-126a-5p target that induces telomere shortening, BMSC senescence, and nuclear factor κB (NF-κB) pathway activation. This study identified aging-related reduced expression of miR-126a and promotion of its target VCAN as a key mechanism in the regulation of hepatic metabolic function during aging and hepatic damage by inducing NF-κB pathway activation, DNA repair function disorder, and telomere attrition. The findings indicate that miR-126a may be a drug target for the treatment of hepatic failure.

8.
Hum Gene Ther ; 30(4): 446-458, 2019 04.
Artículo en Inglés | MEDLINE | ID: mdl-29706119

RESUMEN

CRISPR/Cas9-mediated programmed cell death protein 1 (PD-1) disruption in chimeric antigen receptor (CAR) T cells could be an appealing choice to improve the therapeutic efficacy of CAR T cells in an immunosuppressive tumor microenvironment. In most of the reported cases, Cas9 was delivered into T cells by way of electroporation with RNA or protein. However, transient expression of Cas9 by transfection with a plasmid encoding its gene is apparently simpler, as it avoids the steps of in vitro transcription of DNA or protein production. This study tried nucleofection into human primary T cells of plasmids encoding both CRISPR/Cas9 for disrupting the PD-1 gene and the piggyBac transposon system for expressing CD133-specific CAR in one reaction. Based on drug selection, CD133-specific CAR T cells were obtained in which, on average, 91.5% of the PD-1 gene sites were disrupted, but almost no Cas9 gene expression was found in the final engineered CAR T cells. The PD-1-deficient CD133-specific CAR T cells showed similar levels of cytokine secretion and improved proliferation and cytotoxicity in vitro, and enhanced inhibition of tumor growth in an orthotopic mouse model of glioma, compared to conventional CD133-CAR T cells. The described method could be useful for the production of PD-1-deficient CAR T cells for cancer immunotherapy.


Asunto(s)
Antígeno AC133/inmunología , Sistemas CRISPR-Cas , Silenciador del Gen , Plásmidos/genética , Receptor de Muerte Celular Programada 1/genética , Receptores Quiméricos de Antígenos/metabolismo , Linfocitos T/inmunología , Linfocitos T/metabolismo , Antígeno AC133/antagonistas & inhibidores , Animales , Biomarcadores , Línea Celular Tumoral , Citotoxicidad Inmunológica , Modelos Animales de Enfermedad , Edición Génica , Técnicas de Silenciamiento del Gen , Ingeniería Genética , Humanos , Inmunofenotipificación , Inmunoterapia Adoptiva , Activación de Linfocitos , Ratones , Receptor de Muerte Celular Programada 1/deficiencia , Receptor de Muerte Celular Programada 1/metabolismo , Receptores Quiméricos de Antígenos/genética , Especificidad del Receptor de Antígeno de Linfocitos T , Transfección/métodos , Ensayos Antitumor por Modelo de Xenoinjerto
9.
J Cell Biochem ; 120(2): 1122-1132, 2019 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-30256444

RESUMEN

The adipokine Chemerin has been reported to regulate differentiation and metabolism of adipocytes, but the mechanism underlying lipolysis is still largely unknown. The purpose of this study was to explore whether ERK1/2 pathway is involved in regulating Chemerin during bovine intramuscular mature adipocyte lipolysis. Intramuscular mature adipocytes of dairy bull calves were cultured in vitro and were treated with Chemerin or U0126, which is an inhibitor of ERK1/2 pathway. The results showed that TG content in cells was significantly decreased, glycerol and free fatty acid were significantly increased in cell culture media, and the expression of phosphorylated ERK1/2 in cells was increased in Chemerin-treated group, suggested that ERK1/2 pathway was involved in regulation of lipolysis by Chemerin. In addition, the expression of lipolytic-related critical factors ATGL, HSL, LPL, PPARα, UCP3, and CPT1 were upregulated, but the expression of adipogenic key factors, including PPARγ and C/EBPα were downregulated by Chemerin. Interestingly, all the effects of Chemerin on genes expression in intramuscular mature adipocytes or fat tissue were inhibited by U0126, showed that the function of Chemerin to promote adipose decomposition will be significantly weakened if the ERK1/2 pathway is suppressed, and confirmed that ERK1/2 pathway is involved in mediate Chemerin-enhanced lipolysis. In conclusion, the study demonstrated that Chemerin induce intramuscular mature adipocytes lipolysis through activation of the ERK1/2 pathway. Our research at least provide partial mechanisms of Chemerin on lipolysis and deposition of intramuscular fat tissue of dairy bull calves.

10.
DNA Res ; 26(2): 105-117, 2019 Apr 01.
Artículo en Inglés | MEDLINE | ID: mdl-30590473

RESUMEN

High-throughput stage-specific transcriptomics provides an unbiased approach for understanding the process of cell development. Here, we report transcriptome analysis of primordial germ cell, female germline stem cell (FGSC), germinal vesicle and mature oocyte by performing RNA sequencing of freshly isolated cells in mice. As expected, these stages and gene-expression profiles are consistent with developmental timing. Analysis of genome-wide DNA methylation during female germline development was used for confirmation. By pathway analysis and blocking experiments, we demonstrate PI3K-AKT pathway is critical for FGSC maintenance. We also identify functional modules with hub genes and lncRNAs, which represent candidates for regulating FGSC self-renewal and differentiation. Remarkably, we note alternative splicing patterns change dramatically during female germline development, with the highest occurring in FGSCs. These findings are invaluable resource for dissecting the molecular pathways and processes into oogenesis and will be wider applications for other types of stem cell research.


Asunto(s)
Metilación de ADN , Células Germinativas/metabolismo , Transducción de Señal , Animales , Diferenciación Celular , Proliferación Celular , Epigenómica , Epistasis Genética , Femenino , Ratones , Oocitos/metabolismo , Óvulo/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Análisis de Secuencia de ARN , Transcriptoma
11.
Front Immunol ; 9: 2101, 2018.
Artículo en Inglés | MEDLINE | ID: mdl-30258450

RESUMEN

Innate lymphoid cells (ILCs) are the most recently identified family of the innate immune system and are hypothesized to modulate immune functions prior to the generation of adaptive immune responses. Subsets of ILCs reside in the mucosa and regulate immune responses to external pathogens; however, their role and the mechanism by which they protect against intracellular bacterial infection is not completely understood. In this report, using S. typhimurium and L. monocytogenes, we found that the levels of group 1 ILCs and NCR+ ILC3s were increased upon infection and that these increases were associated with Runt-related transcription factor 3 (Runx3) expression. Runx3 fl/fl PLZF-cre mice were much more sensitive to infection with the intracellular bacterial pathogens S. typhimurium and L. monocytogenes partially due to abnormal Group 1 ILC and NCR+ILC3 function. We also found that Runx3 directly binds to the Il12Rß2 promoter and intron 8 to accelerate the expression of Il12Rß2 and modulates IFNγ secretion triggered by the IL12/ STAT4 axis. Therefore, we demonstrate that Runx3 influences group 1 ILC- and NCR+ILC3-mediated immune protection against intracellular bacterial infections of both the gut and liver.


Asunto(s)
Subunidad alfa 3 del Factor de Unión al Sitio Principal/inmunología , Inmunidad Innata , Interleucina-12/inmunología , Listeria monocytogenes/inmunología , Listeriosis/inmunología , Linfocitos/inmunología , Infecciones por Salmonella/inmunología , Salmonella typhimurium/inmunología , Transducción de Señal/inmunología , Animales , Subunidad alfa 3 del Factor de Unión al Sitio Principal/genética , Interleucina-12/genética , Listeriosis/genética , Listeriosis/patología , Linfocitos/patología , Ratones , Ratones Transgénicos , Infecciones por Salmonella/genética , Infecciones por Salmonella/patología , Transducción de Señal/genética
12.
J Cell Biol ; 217(11): 3901-3911, 2018 11 05.
Artículo en Inglés | MEDLINE | ID: mdl-30097513

RESUMEN

Mammalian oocytes are arrested at the prophase of the first meiotic division for months and even years, depending on species. Meiotic resumption of fully grown oocytes requires activation of M-phase-promoting factor (MPF), which is composed of Cyclin B1 and cyclin-dependent kinase 1 (CDK1). It has long been believed that Cyclin B1 synthesis/accumulation and its interaction with CDK1 is a prerequisite for MPF activation in oocytes. In this study, we revealed that oocyte meiotic resumption occurred in the absence of Cyclin B1. Ccnb1-null oocytes resumed meiosis and extruded the first polar body. Without Cyclin B1, CDK1 could be activated by up-regulated Cyclin B2. Ccnb1 and Ccnb2 double knockout permanently arrested the oocytes at the prophase of the first meiotic division. Oocyte-specific Ccnb1-null female mice were infertile due to failed MPF activity elevation and thus premature interphase-like stage entry in the second meiotic division. These results have revealed a hidden compensatory mechanism between Cyclin B1 and Cyclin B2 in regulating MPF and oocyte meiotic resumption.


Asunto(s)
Ciclina B1/metabolismo , Ciclina B2/metabolismo , Factor Promotor de Maduración/metabolismo , Meiosis , Oocitos/metabolismo , Animales , Línea Celular , Ciclina B1/genética , Ciclina B2/genética , Femenino , Factor Promotor de Maduración/genética , Mesotelina , Ratones , Ratones Noqueados , Células Madre Embrionarias de Ratones , Oocitos/citología
13.
Nat Struct Mol Biol ; 25(1): 45-52, 2018 01.
Artículo en Inglés | MEDLINE | ID: mdl-29323274

RESUMEN

The APOBEC-AID family of cytidine deaminase prefers single-stranded nucleic acids for cytidine-to-uridine deamination. Single-stranded nucleic acids are commonly involved in the DNA repair system for breaks generated by CRISPR-Cas9. Here, we show in human cells that APOBEC3 can trigger cytidine deamination of single-stranded oligodeoxynucleotides, which ultimately results in base substitution mutations in genomic DNA through homology-directed repair (HDR) of Cas9-generated double-strand breaks. In addition, the APOBEC3-catalyzed deamination in genomic single-stranded DNA formed during the repair of Cas9 nickase-generated single-strand breaks in human cells can be further processed to yield mutations mainly involving insertions or deletions (indels). Both APOBEC3-mediated deamination and DNA-repair proteins play important roles in the generation of these indels. Therefore, optimizing conditions for the repair of CRISPR-Cas9-generated DNA breaks, such as using double-stranded donors in HDR or temporarily suppressing endogenous APOBEC3s, can repress these unwanted mutations in genomic DNA.


Asunto(s)
Sistemas CRISPR-Cas , Citosina Desaminasa/química , Roturas del ADN de Doble Cadena , Reparación del ADN , Mutación , Desaminasas APOBEC , Citidina/química , Citidina Desaminasa/genética , ADN de Cadena Simple , Células HEK293 , Células HeLa , Humanos , Mutación INDEL , Oligonucleótidos/genética , ARN Interferente Pequeño/metabolismo , Reparación del ADN por Recombinación , Análisis de Secuencia de ADN
14.
Oncotarget ; 8(58): 98823-98836, 2017 Nov 17.
Artículo en Inglés | MEDLINE | ID: mdl-29228730

RESUMEN

Liver regeneration/repair is a compensatory regrowth following acute liver failure, and bone marrow-derived mesenchyme stem cell (BMSC) transplantation is an effective therapy that promotes liver regeneration/repair. Wnt1 inducible signaling pathway protein 2 (Wisp2) is highly expressed in BMSCs, however, its function remains unclear. In this work, we used clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein -9 nuclease (CRISPR/Cas9) genome editing technology to knockdown Wisp2 in BMSCs, and these modified cells were then transplanted into rats which were induced by the 2-AAF/PH. By linking the expression of Cas9 to green fluorescent protein (GFP), we tracked BMSCs in the rats. Disruption of Wisp2 inhibited the homing of BMSCs to injured liver and aggravated liver damage as indicated by remarkably high levels of ALT and AST. Moreover, the key factor in BMSC transplantation, C-X-C chemokine receptor type 4 (Cxcr4), was down-regulated in the Wisp2 depleted BMSCs and had a lower expression in the livers of the corresponding rats. By tracing the GFP marker, more BMSCs were observed to differentiate into CD31 positive endothelial cells in the functional Wisp2 cells but less in the Wisp2 gene disrupted cells. In summary, Wisp2 promotes the homing of BMSCs through Cxcr4 related signaling during liver repair in rats.

15.
Cell Death Dis ; 8(10): e3142, 2017 10 26.
Artículo en Inglés | MEDLINE | ID: mdl-29072697

RESUMEN

Spermatogenesis, which involves mitosis and meiosis of male germ cells, is a highly complicated and coordinately ordered process. Cyclin B1 (CCNB1), an important regulator in cell cycle machinery, is proved essential for mouse embryonic development. However, the role of CCNB1 in mammalian spermatogenesis remains unclear. Here we tested the requirement for CCNB1 using conditional knockout mice lacking CCNB1 in male germ cells. We found that ablation of CCNB1 in gonocytes and spermatogonia led to mouse sterile caused by the male germ cells' depletion. Gonocyte and spermatogonia without CCNB1 is unable to proliferate normally and apoptosis increased. Moreover, CCNB1 ablation in spermatogonia may promote their differentiation by downregulating Lin28a and upregulating let-7 miRNA. However, ablation of CCNB1 in premeiotic male germ cells did not have an effect on meiosis of spermatocytes and male fertility, suggesting that CCNB1 may be dispensable for meiosis of spermatocytes. Collectively, these results indicate that CCNB1 is critically required for the proliferation of gonocytes and spermatogonia but may be redundant in meiosis of spermatocytes in mouse spermatogenesis.


Asunto(s)
Ciclina B1/fisiología , Espermatogénesis/fisiología , Animales , Diferenciación Celular , Ciclina B1/deficiencia , Ciclina B1/genética , Ciclina B1/metabolismo , Masculino , Ratones , Ratones Noqueados
16.
Oncoimmunology ; 6(1): e1249558, 2017.
Artículo en Inglés | MEDLINE | ID: mdl-28197365

RESUMEN

The successful use of immune cell checkpoint inhibitors PD-1 and PD-L1, over the past 5 y has raised the concern of using immunotherapy to treat various cancers. Epstein-Barr virus-associated gastric cancer (EBVaGC) exhibits high infiltration of lymphocytes and high amplification of immune-related genes including PD-L1 as distinguished from Epstein-Barr virus-non-associated gastric cancer (EBVnGC). Here, we presume that this PD-1/PD-L1 pathway may hinder the efficacy of adoptive T cell therapy toward EBVaGC. These studies reveal possibility of generating PD-1-disrupted CTL by CRISPR-Cas9 system and demonstrate enhanced immune response of these PD-1-disrupted CTLs to the EBV-LMP2A antigen and superior cytotoxicity to the EBV-positive gastric cancer cell. In addition, when combined with low-dose radiotherapy, these PD-1-disrupted CTLs mediated an impressive antitumor effect in a xenograft mouse model of EBVaGC. Taken together, these studies illustrate PD-1/PD-L1-mediated immune tolerance of EBVaGC and provide a new strategy for targeting immune checkpoints to break the tolerance for the T cell-based adoptive therapy.

18.
Biol Open ; 5(7): 979-86, 2016 Jul 15.
Artículo en Inglés | MEDLINE | ID: mdl-27378433

RESUMEN

The low-density lipoprotein receptor (LDLR) plays a critical role in the liver for the clearance of plasma low-density lipoprotein (LDL). Its deficiency causes hypercholesterolemia in many models. To facilitate the usage of rats as animal models for the discovery of cholesterol-lowering drugs, we took a genetic approach to delete the LDLR in rats aiming to increase plasma LDL cholesterol (LDL-C). An LDLR knockout rat was generated via zinc-finger nuclease technology, which harbors a 19-basepair deletion in the seventh exon of the ldlr gene. As expected, deletion of the LDLR elevated total cholesterol and total triglyceride in the plasma, and caused a tenfold increase of plasma LDL-C and a fourfold increase of plasma very low-density lipoprotein (VLDL-C). A lipidomics analysis revealed that deletion of the LDLR affected hepatic lipid metabolism, particularly lysophosphatidylcholines, free fatty acids and sphingolipids in the liver. Cholesterol ester (CE) 20:4 also displayed a significant increase in the LDLR knockout rats. Taken together, the LDLR knockout rat offers a new model of hypercholesterolemia, and the lipidomics analysis reveals hepatic lipid signatures associating with deficiency of the LDL receptor.

19.
PLoS One ; 11(4): e0154364, 2016.
Artículo en Inglés | MEDLINE | ID: mdl-27119535

RESUMEN

The CRISPR/Cas9 system has been developed as an easy-handle and multiplexable approach for engineering eukaryotic genomes by zygote microinjection of Cas9 and sgRNA, while preparing Cas9 for microinjection is laborious and introducing inconsistency into the experiment. Here, we describe a modified strategy for gene targeting through using oocyte-specific Cas9 transgenic mouse. With this mouse line, we successfully achieve precise gene targeting by injection of sgRNAs only into one-cell-stage embryos. Through comprehensive analysis, we also show allele complexity and off-target mutagenesis induced by this strategy is obviously lower than Cas9 mRNA/sgRNA injection. Thus, injection of sgRNAs into oocyte-specific Cas9 transgenic mouse embryo provides a convenient, efficient and reliable approach for mouse genome editing.


Asunto(s)
Sistemas CRISPR-Cas , Marcación de Gen , Ratones Transgénicos , Oocitos/citología , Alelos , Animales , Cartilla de ADN/genética , Femenino , Edición Génica , Genoma , Masculino , Ratones , Ratones Endogámicos C57BL , Mutagénesis , Proteína con Dominio Pirina 3 de la Familia NLR/genética , Reacción en Cadena de la Polimerasa , ARN Guía de Kinetoplastida/genética , Receptores Androgénicos/genética , Análisis de Secuencia de ADN , Glicoproteínas de la Zona Pelúcida/genética
20.
Sci Rep ; 6: 20070, 2016 Jan 28.
Artículo en Inglés | MEDLINE | ID: mdl-26818188

RESUMEN

Strategies that enhance the function of T cells are critical for immunotherapy. One negative regulator of T-cell activity is ligand PD-L1, which is expressed on dentritic cells (DCs) or some tumor cells, and functions through binding of programmed death-1 (PD-1) receptor on activated T cells. Here we described for the first time a non-viral mediated approach to reprogram primary human T cells by disruption of PD-1. We showed that the gene knockout of PD-1 by electroporation of plasmids encoding sgRNA and Cas9 was technically feasible. The disruption of inhibitory checkpoint gene PD-1 resulted in significant reduction of PD-1 expression but didn't affect the viability of primary human T cells during the prolonged in vitro culture. Cellular immune response of the gene modified T cells was characterized by up-regulated IFN-γ production and enhanced cytotoxicity. These results suggest that we have demonstrated an approach for efficient checkpoint inhibitor disruption in T cells, providing a new strategy for targeting checkpoint inhibitors, which could potentialy be useful to improve the efficacy of T-cell based adoptive therapies.


Asunto(s)
Sistemas CRISPR-Cas , Neoplasias/genética , Receptor de Muerte Celular Programada 1/genética , Subgrupos de Linfocitos T/metabolismo , Secuencia de Bases , Células Cultivadas , Citocinas/biosíntesis , Citotoxicidad Inmunológica , Edición Génica , Técnicas de Inactivación de Genes , Orden Génico , Marcación de Gen , Técnicas de Transferencia de Gen , Sitios Genéticos , Vectores Genéticos/genética , Humanos , Activación de Linfocitos , Neoplasias/inmunología , Neoplasias/metabolismo , Neoplasias/patología , Fenotipo , Receptor de Muerte Celular Programada 1/deficiencia , ARN Guía de Kinetoplastida/genética , Subgrupos de Linfocitos T/inmunología , Transfección
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