RESUMEN
OBJECTIVE: To investigate the effects of excessive light exposure during gestation on intrauterine development and early growth of neonates in rats. METHODS: Pregnant rats were randomly allocated to three groups: the constant light exposure group, non-light exposure group and control group. Blood samples were collected from the tail vein to analyze melatonin and cortisol levels. Weight, daily food and water consumption were recorded. Uterine weight, placental weight and placental diameter were measured on gestational day 19. Natural birth and neonate growth were also monitored. The expression of NR1D1(nuclear receptor subfamily 1 group D member 1) in offspring's SCN (suprachiasmatic nuclei), liver and adipose tissue was measured. Expression of NR1D1, MT1(melatonin 1â¯A receptor) and 11ß-HSD2 (placental 11ß-hydroxysteroid dehydrogenase type 2) in placenta was also measured. Finally, the expression of MT1 and 11ß-HSD2 in NR1D1 siRNA transfected JEG-3 cells was evaluated. RESULTS: There were no significant differences in maternal weight gain, pregnancy duration, uterine weight, placental body weight, placental diameter, fetal number among three groups. There were no significant differences in weights or lengths of offspring at birth. Compared to other two groups, constant light exposure group showed significantly more rapid growth of offspring in 21st day post-birth. The expression of NR1D1 in SCN, liver and adipose tissues of offspring was not significantly different among three groups. The maternal serum melatonin and cortisol levels of the constant light exposure group were lower and higher than other two groups, respectively. The expressions of NR1D1, MT1 and 11ß-HSD2 were all decreased in placenta of the constant light exposure group. The expression of MT1 and 11ß-HSD2 in JEG-3 cells were decreased after NR1D1 siRNA transfection. CONCLUSION: Excessive light exposure during pregnancy results in elevated cortisol and reduced melatonin exposure to fetuses in uterus, potentially contributing to an accelerated early growth of offspring in rats.
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Luz , Melatonina , Placenta , Animales , Femenino , Embarazo , Ratas , Placenta/efectos de la radiación , 11-beta-Hidroxiesteroide Deshidrogenasa de Tipo 2 , Desarrollo Fetal/efectos de la radiación , Ratas Sprague-Dawley , Hidrocortisona/sangre , Miembro 1 del Grupo D de la Subfamilia 1 de Receptores Nucleares/metabolismo , Efectos Tardíos de la Exposición Prenatal , Receptor de Melatonina MT1/metabolismo , Animales Recién Nacidos , Exposición Materna , MasculinoRESUMEN
Utilization of lipids as energy substrates after birth causes cardiomyocyte (CM) cell-cycle arrest and loss of regenerative capacity in mammalian hearts. Beyond energy provision, proper management of lipid composition is crucial for cellular and organismal health, but its role in heart regeneration remains unclear. Here, we demonstrate widespread sphingolipid metabolism remodeling in neonatal hearts after injury and find that SphK1 and SphK2, isoenzymes producing the same sphingolipid metabolite sphingosine-1-phosphate (S1P), differently regulate cardiac regeneration. SphK2 is downregulated during heart development and determines CM proliferation via nuclear S1P-dependent modulation of histone acetylation. Reactivation of SphK2 induces adult CM cell-cycle re-entry and cytokinesis, thereby enhancing regeneration. Conversely, SphK1 is upregulated during development and promotes fibrosis through an S1P autocrine mechanism in cardiac fibroblasts. By fine-tuning the activity of each SphK isoform, we develop a therapy that simultaneously promotes myocardial repair and restricts fibrotic scarring to regenerate the infarcted adult hearts.
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Corazón , Lisofosfolípidos , Esfingolípidos , Esfingosina/análogos & derivados , Animales , Esfingolípidos/metabolismo , Isoenzimas , Mamíferos/metabolismo , Fosfotransferasas (Aceptor de Grupo Alcohol)/metabolismoRESUMEN
Cardiac development involves large-scale rearrangements of the proteome. How the developing cardiac cells maintain the integrity of the proteome during the rapid lineage transition remains unclear. Here it is shown that proteotoxic stress visualized by the misfolded and/or aggregated proteins appears during early cardiac differentiation of human pluripotent stem cells and is resolved by activation of the PERK branch of unfolded protein response (UPR). PERK depletion increases misfolded and/or aggregated protein accumulation, leading to pluripotency exit defect and impaired mesendoderm specification of human pluripotent stem cells. Mechanistically, it is found that PERK safeguards mesendoderm specification through its conserved downstream effector ATF4, which subsequently activates a novel transcriptional target WARS1, to cope with the differentiation-induced proteotoxic stress. The results indicate that protein quality control represents a previously unrecognized core component of the cardiogenic regulatory network. Broadly, these findings provide a framework for understanding how UPR is integrated into the developmental program by activating the PERK-ATF4-WARS1 axis.
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Células Madre Pluripotentes , eIF-2 Quinasa , Humanos , eIF-2 Quinasa/genética , eIF-2 Quinasa/metabolismo , Células Madre Pluripotentes/metabolismo , Proteoma/metabolismo , Proteostasis , Respuesta de Proteína DesplegadaRESUMEN
A major hurdle in cardiac cell therapy is the lack of a bona fide autologous stem-cell type that can be expanded long-term and has authentic cardiovascular differentiation potential. Here we report that a proliferative cell population with robust cardiovascular differentiation potential can be generated from mouse or human fibroblasts via a combination of six small molecules. These chemically induced cardiovascular progenitor cells (ciCPCs) self-renew long-term in fully chemically defined and xeno-free conditions, with faithful preservation of the CPC phenotype and of cardiovascular differentiation capacity in vitro and in vivo. Transplantation of ciCPCs into infarcted mouse hearts improved animal survival and cardiac function up to 13 weeks post-infarction. Mechanistically, activated fibroblasts revert to a plastic state permissive to cardiogenic signals, enabling their reprogramming into ciCPCs. Expanded autologous cardiovascular cells may find uses in drug discovery, disease modelling and cardiac cell therapy.
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Reprogramación Celular , Fibroblastos , Animales , Diferenciación Celular , Corazón , Ratones , Células MadreRESUMEN
N6-methyladenosine (m6A) plays important role in lineage specifications of embryonic stem cells. However, it is still difficult to systematically dissect the specific m6A sites that are essential for early lineage differentiation. Here, we develop an adenine base editor-based strategy to systematically identify functional m6A sites that control lineage decisions of human embryonic stem cells. We design 7999 sgRNAs targeting 6048 m6A sites to screen for m6A sites that act as either boosters or barriers to definitive endoderm specification of human embryonic stem cells. We identify 78 sgRNAs enriched in the non-definitive endoderm cells and 137 sgRNAs enriched in the definitive endoderm cells. We successfully validate two definitive endoderm promoting m6A sites on SOX2 and SDHAF1 as well as a definitive endoderm inhibiting m6A site on ADM. Our study provides a functional screening of m6A sites and paves the way for functional studies of m6A at individual m6A site level.
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Adenosina/análogos & derivados , Diferenciación Celular , Linaje de la Célula , Endodermo/citología , Regulación del Desarrollo de la Expresión Génica , Células Madre Embrionarias Humanas/metabolismo , Factores de Transcripción SOXB1/metabolismo , Adenosina/genética , Adenosina/metabolismo , Adrenomedulina/genética , Adrenomedulina/metabolismo , Sistemas CRISPR-Cas/genética , Células Cultivadas , Endodermo/metabolismo , Humanos , Proteínas/genética , Proteínas/metabolismo , Factores de Transcripción SOXB1/genéticaRESUMEN
A major clinical challenge for treating patients with pancreatic ductal adenocarcinoma (PDAC) is identifying those that may benefit from adjuvant chemotherapy versus those that will not. Thus, there is a need for a robust and convenient biomarker for predicting chemotherapy response in PDAC patients. In this study, network inference was conducted by integrating the differentially expressed cell cycle signatures and target genes between the basal-like subtype and classical subtype of PDAC. As a result from this statistical analysis, two dominant cell cycle genes, RASAL2 and ASPM, were identified. Based on the expression levels of these two genes, we constructed a "Enhanced Cell Cycle" scoring system (ECC score). Patients were given an ECC score, and respectively divided into ECC-high and ECC-low groups. Survival, pathway enrichment, immune environment characteristics, and chemotherapy response analysis' were performed between the two groups in a total of 891 patients across 5 cohorts. ECC-high patients exhibited shortened recurrence-free survival (RFS) and overall survival (OS) rates. In addition, it was found that adjuvant chemotherapy could significantly improve the outcome of the ECC-high patients while ECC-low patients did not benefit from adjuvant chemotherapy. It was also found that there was less CD8+ T cell, natural killer (NK) cell, M1 macrophage, and plasma cell infiltration in ECC-high patients when compared to ECC-low patients. Also, the expression of CD73, an immune suppressor gene, and it's related hypoxia pathway were elevated in the ECC-high group when compared to the ECC-low group. In conclusion, this study showed that patients characterized as ECC-high not only had reduced RFS and OS rates, but were also more sensitive to adjuvant chemotherapy and could potentially be less sensitive to immune checkpoint inhibitors. Being able to characterize patients by these parameters would allow doctors to make more informed decisions on patient treatment regimens.
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Carcinoma Ductal Pancreático , Ciclo Celular , Neoplasias Pancreáticas , Carcinoma Ductal Pancreático/tratamiento farmacológico , Carcinoma Ductal Pancreático/genética , Ciclo Celular/efectos de los fármacos , Ciclo Celular/genética , Quimioterapia Adyuvante , Terapia Combinada , Proteínas Activadoras de GTPasa , Humanos , Neoplasias Pancreáticas/tratamiento farmacológico , Neoplasias Pancreáticas/genéticaRESUMEN
Deficiency of the N6 -methyladenosine (m6 A) methyltransferase complex results in global reduction of m6 A abundance and defective cell development in embryonic stem cells (ESCs). However, it's unclear whether regional m6 A methylation affects cell fate decisions due to the inability to modulate individual m6 A modification in ESCs with precise temporal control. Here, a targeted RNA m6 A erasure (TRME) system is developed to achieve site-specific demethylation of RNAs in human ESCs (hESCs). TRME, in which a stably transfected, doxycycline-inducible dCas13a is fused to the catalytic domain of ALKBH5, can precisely and reversibly demethylate the targeted m6 A site of mRNA and increase mRNA stability with limited off-target effects. It is further demonstrated that temporal m6 A erasure on a single site of SOX2 is sufficient to control the differentiation of hESCs. This study provides a versatile toolbox to reveal the function of individual m6 A modification in hESCs, enabling cell fate control studies at the epitranscriptional level.
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Adenosina/análogos & derivados , Desmetilasa de ARN, Homólogo 5 de AlkB/genética , Diferenciación Celular/genética , Factores de Transcripción SOXB1/genética , Adenosina/genética , Caspasas/genética , Dominio Catalítico/genética , Linaje de la Célula/genética , Proliferación Celular/genética , Desmetilación , Células Madre Embrionarias/citología , Células Madre Embrionarias/metabolismo , Humanos , Metilación , Metiltransferasas/genética , Células Madre Pluripotentes/metabolismo , Estabilidad del ARN/genética , ARN Mensajero/genéticaRESUMEN
BACKGROUND: Canonical nonsense-mediated decay (NMD) is an important splicing-dependent process for mRNA surveillance in mammals. However, processed pseudogenes are not able to trigger NMD due to their lack of introns. It is largely unknown whether they have evolved other surveillance mechanisms. RESULTS: Here, we find that the RNAs of pseudogenes, especially processed pseudogenes, have dramatically higher m6A levels than their cognate protein-coding genes, associated with de novo m6A peaks and motifs in human cells. Furthermore, pseudogenes have rapidly accumulated m6A motifs during evolution. The m6A sites of pseudogenes are evolutionarily younger than neutral sites and their m6A levels are increasing, supporting the idea that m6A on the RNAs of pseudogenes is under positive selection. We then find that the m6A RNA modification of processed, rather than unprocessed, pseudogenes promotes cytosolic RNA degradation and attenuates interference with the RNAs of their cognate protein-coding genes. We experimentally validate the m6A RNA modification of two processed pseudogenes, DSTNP2 and NAP1L4P1, which promotes the RNA degradation of both pseudogenes and their cognate protein-coding genes DSTN and NAP1L4. In addition, the m6A of DSTNP2 regulation of DSTN is partially dependent on the miRNA miR-362-5p. CONCLUSIONS: Our discovery reveals a novel evolutionary role of m6A RNA modification in cleaning up the unnecessary processed pseudogene transcripts to attenuate their interference with the regulatory network of protein-coding genes.
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Adenosina/análogos & derivados , Genoma Humano , Seudogenes , Empalme del ARN , ARN Mensajero/genética , Selección Genética , Adenosina/genética , Adenosina/metabolismo , Línea Celular , Línea Celular Transformada , Destrina/genética , Destrina/metabolismo , Células HEK293 , Proyecto Mapa de Haplotipos , Células Madre Embrionarias Humanas/citología , Células Madre Embrionarias Humanas/metabolismo , Humanos , Linfocitos/citología , Linfocitos/metabolismo , MicroARNs/genética , MicroARNs/metabolismo , Degradación de ARNm Mediada por Codón sin Sentido , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , ARN Mensajero/metabolismoRESUMEN
N6 -methyladenosine (m6 A) modification of mRNA mediates diverse cellular and viral functions. Infection with Epstein-Barr virus (EBV) is causally associated with nasopharyngeal carcinoma (NPC), 10% of gastric carcinoma, and various B-cell lymphomas, in which the viral latent and lytic phases both play vital roles. Here, we show that EBV transcripts exhibit differential m6 A modification in human NPC biopsies, patient-derived xenograft tissues, and cells at different EBV infection stages. m6 A-modified EBV transcripts are recognized and destabilized by the YTHDF1 protein, which leads to the m6 A-dependent suppression of EBV infection and replication. Mechanistically, YTHDF1 hastens viral RNA decapping and mediates RNA decay by recruiting RNA degradation complexes, including ZAP, DDX17, and DCP2, thereby post-transcriptionally downregulating the expression of EBV genes. Taken together, our results reveal the critical roles of m6 A modifications and their reader YTHDF1 in EBV replication. These findings contribute novel targets for the treatment of EBV-associated cancers.
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Infecciones por Virus de Epstein-Barr , Neoplasias Nasofaríngeas , Adenosina/análogos & derivados , Proteínas Portadoras , Herpesvirus Humano 4/genética , Humanos , Estabilidad del ARN , Proteínas de Unión al ARN/genética , Replicación ViralRESUMEN
N6-methyladenosine (m6A) is a reversible and dynamic RNA modification in eukaryotes. However, how cells establish cell-specific m6A methylomes is still poorly understood. Here, we developed a computational framework to systematically identify cell-specific trans regulators of m6A through integrating gene expressions, binding targets and binding motifs of large number of RNA binding proteins (RBPs) with a co-methylation network constructed using large-scale m6A methylomes across diverse cell states. We applied the framework and successfully identified 32 high-confidence m6A regulators that modulated the variable m6A sites away from stop codons in a cell-specific manner. To validate them, we knocked down three regulators respectively and found two of them (TRA2A and CAPRIN1) selectively promoted the methylations of the m6A sites co-localized with their binding targets on RNAs through physical interactions with the m6A writers. Knockdown of TRA2A increased the stabilities of the RNAs with TRA2A bound near the m6A sites and decreased the viability of cells. The successful identification of m6A regulators demonstrates a powerful and widely applicable strategy to elucidate the cell-specific m6A regulators. Additionally, our discovery of pervasive trans-acting regulating of m6A provides novel insights into the mechanisms by which spatial and temporal dynamics of m6A methylomes are established.
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Adenosina/análogos & derivados , Proteínas de Ciclo Celular/genética , Proteínas de Unión al ARN/genética , ARN/genética , Adenosina/química , Adenosina/genética , Linaje de la Célula/genética , Supervivencia Celular/genética , Regulación de la Expresión Génica/genética , Técnicas de Silenciamiento del Gen/métodos , Células Hep G2 , Humanos , Metilación , Procesamiento Postranscripcional del ARN/genéticaRESUMEN
The N6-methyladenosine (m6A) modification influences various mRNA metabolic events and tumorigenesis, however, its functions in nonsense-mediated mRNA decay (NMD) and whether NMD detects induced carcinogenesis pathways remain undefined. Here, we showed that the m6A methyltransferase METTL3 sustained its oncogenic role by modulating NMD of splicing factors and alternative splicing isoform switches in glioblastoma (GBM). Methylated RNA immunoprecipitation-seq (MeRIP-seq) analyses showed that m6A modification peaks were enriched at metabolic pathway-related transcripts in glioma stem cells (GSC) compared with neural progenitor cells. In addition, the clinical aggressiveness of malignant gliomas was associated with elevated expression of METTL3. Furthermore, silencing METTL3 or overexpressing dominant-negative mutant METTL3 suppressed the growth and self-renewal of GSCs. Integrated transcriptome and MeRIP-seq analyses revealed that downregulating the expression of METTL3 decreased m6A modification levels of serine- and arginine-rich splicing factors (SRSF), which led to YTHDC1-dependent NMD of SRSF transcripts and decreased SRSF protein expression. Reduced expression of SRSFs led to larger changes in alternative splicing isoform switches. Importantly, the phenotypes mediated by METTL3 deficiency could be rescued by downregulating BCL-X or NCOR2 isoforms. Overall, these results establish a novel function of m6A in modulating NMD and uncover the mechanism by which METTL3 promotes GBM tumor growth and progression. SIGNIFICANCE: These findings establish the oncogenic role of m6A writer METTL3 in glioblastoma stem cells.
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Adenosina/análogos & derivados , Glioblastoma/metabolismo , Degradación de ARNm Mediada por Codón sin Sentido/fisiología , ARN Mensajero/metabolismo , Adenosina/metabolismo , Empalme Alternativo/fisiología , Animales , Carcinogénesis/metabolismo , Línea Celular Tumoral , Proliferación Celular/fisiología , Femenino , Regulación Neoplásica de la Expresión Génica/fisiología , Glioma/metabolismo , Humanos , Metiltransferasas/metabolismo , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , Células-Madre Neurales/metabolismo , Transcriptoma/fisiologíaRESUMEN
Toxoplasma gondii infection evokes a strong Th1-type response with interleukin (IL)-12 and interferon (IFN)-γ secretion. Recent studies suggest that the infection of pregnant mice with T. gondii may lead to adverse pregnancy results caused by subversion of physiological immune tolerance at maternofetal interface rather than direct invasion of the parasite. Genotype-associated dense granule protein GRA15II tends to induce classically activated macrophage (M1) differentiation and subsequently activating NK, Th1, and Th17 cells whereas rhoptry protein ROP16I/III drives macrophages to alternatively activated macrophage (M2) polarization and elicits Th2 immune response. Unlike the archetypal strains of types I, II, and III, type Chinese 1 strains possess both GRA15II and ROP16I/III, suggesting a distinct pathogenesis of Toxoplasma-involved adverse pregnancies. We constructed T. gondii type Chinese 1 strain of WH3Δrop16 based on CRISPR/Cas9 technology to explore the ROP16I/III-deficient/GRA15II-dominant parasites in induction of trophoblast apoptosis in vitro and abnormal pregnant outcomes of mice in vivo. Our study showed that Toxoplasma WH3Δrop16 remarkably induced apoptosis of trophoblasts. C57BL/6 pregnant mice injected with the tachyzoites of WH3Δrop16 presented increased absorptivity of fetuses in comparison with the mice infected with WH3 wild type (WH3 WT) parasites although no remarkable difference of virulence to mice was seen between the two strains. Additionally, the mice inoculated with WH3Δrop16 tachyzoites exhibited a notable expression of both IL-17A and IFN-γ, while the percentage of CD4+CD25+FoxP3 [T regulatory cells (Tregs)] were diminished in splenocytes and placenta tissues compared to those infected with WH3 WT parasites. Accordingly, expressions of IL-4, IL-10, and transforming growth factor beta 1, the pivotal cytokines of Th2 and Tregs response, were significantly dampened whereas IFN-γ and IL-12 expressions were upregulated in WH3Δrop16-infected mice, which gave rise to more prominent outcomes of abnormal pregnancies. Our results indicated that the WH3Δrop16 parasites with gra15II background of T. gondii type Chinese 1 strains may cause miscarriage and stillbirth due to subversion of the maternal immune tolerance and system immunity of the animals and the GRA15II effector contributes to the process of adverse pregnant consequences.
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Antecedentes Genéticos , Predisposición Genética a la Enfermedad , Complicaciones Infecciosas del Embarazo/parasitología , Resultado del Embarazo , Toxoplasma/clasificación , Toxoplasma/genética , Toxoplasmosis Animal/parasitología , Animales , Apoptosis , Sistemas CRISPR-Cas , Modelos Animales de Enfermedad , Femenino , Marcación de Gen , Macrófagos Peritoneales/inmunología , Macrófagos Peritoneales/metabolismo , Macrófagos Peritoneales/parasitología , Masculino , Ratones , Placenta , Embarazo , Complicaciones Infecciosas del Embarazo/inmunología , Bazo/inmunología , Bazo/metabolismo , Células TH1/inmunología , Células TH1/metabolismo , Células Th17/inmunología , Células Th17/metabolismo , Toxoplasmosis Animal/inmunología , Trofoblastos/metabolismoRESUMEN
BACKGROUND: Toxoplasma gondii is an opportunistic protozoan apicomplexan and obligate intracellular parasite that infects a wide range of animals and humans. Rhoptry proteins 5 (ROP5), ROP16, ROP18 and dense granules 15 (GRA15) are the important effectors secreted by T. gondii which link to the strain virulence for mice and modulate the host's response to the parasite. Little has been known about these molecules as well as GRA3 in type Chinese 1 strains that show polymorphism among strains of archetypical genotypes. This study examined the genetic diversity of these effectors and its correlated virulence in mice among T. gondii isolates from China. RESULTS: Twenty-one isolates from stray cats were detected, of which 15 belong to Chinese 1, and 6 to ToxoDB #205. Wh6 isolate, a Chinese 1 strain, has an avirulent phenotype. PCR-RFLP results of ROP5 and ROP18 presented few variations among the strains. Genotyping of GRA15 and ROP16 revealed that all the strains belong to type II allele except Xz7 which carries type I allele. ROP16 amino acid alignment at 503 locus demonstrated that 17 isolates are featured as type I or type III (ROP16I/III), and the other 4 as type II (ROP16II). The strains investigated may be divided into four groups based on GRA3 amino acid alignment, and all isolates of type Chinese 1 belong to the µ-1 allele except Wh6 which is identical to type II strain. CONCLUSIONS: PCR-RFLP and sequence alignment analyses of ROP5, ROP16, ROP18, GRA3, and GRA15 in T. gondii revealed that strains with the same genotype may have variations in some of their key genes. GRA3 variation exhibited by Wh6 strain may be associated with the difference in phenotype and pathogenesis.
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Variación Genética , Toxoplasma/genética , Toxoplasma/aislamiento & purificación , Alelos , Animales , Gatos , China , Biología Computacional , Genotipo , Técnicas de Genotipaje , Humanos , Filogenia , Reacción en Cadena de la Polimerasa , Polimorfismo Genético , Polimorfismo de Longitud del Fragmento de Restricción , Proteínas Serina-Treonina Quinasas/genética , Proteínas Tirosina Quinasas/genética , Proteínas Protozoarias/genética , Toxoplasma/clasificación , Toxoplasma/patogenicidad , Toxoplasmosis Animal/parasitología , Factores de Virulencia/genéticaRESUMEN
Toxoplasma gondii is of public health and veterinary importance causing severe diseases in immunocompromised individuals including HIV/AIDS patients and in congenital cases and animals. There is limited information on the epidemiology of T. gondii infection in humans, particularly HIV patients and food animals and the parasite genotypes in Ghana. A total of 394 HIV-infected patients from three hospitals were screened for T. gondii anti-IgG and IgM using ELISA. DNAs from blood samples of seropositve participants and 95 brain tissues of food animals were PCR assayed to detect Toxoplasma gra6. DNA positive samples were genotyped using multilocus nested polymerase chain reaction restriction fragment length polymorphism at 10 loci: sag1, alt.sag2, sag3, btub, gra6, l358, c22-8, c29-2, pk1, and apico. The overall seroprevalence was 74.37% (293/394). Toxoplasma DNAs were detected in 3.07% of the seropositive participants and 9.47% of the animals. Six of the human DNA positive samples were partly typed at sag3: 33.33, 50, and 16.67% isolates had type I, II, and III alleles, respectively. All nine isolates from food animals typed at nine loci except apico were atypical: six isolates were identical to ToxoDB #41 and #145, and one was identical to TgCkBrRj2 all identified in Brazil. The genotype of two isolates has not been reported previously and was named as TgCtGh1. T. gondii seroprevalence is high among the HIV-infected individuals with T. gondii circulating in Ghana being genetically diverse.
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Genotipo , Toxoplasmosis/epidemiología , Alelos , Animales , Anticuerpos Antiprotozoarios , Femenino , Variación Genética , Ghana/epidemiología , Infecciones por VIH/complicaciones , Humanos , Inmunoglobulina G , Reacción en Cadena de la Polimerasa/veterinaria , Polimorfismo de Longitud del Fragmento de Restricción , Prevalencia , Estudios Seroepidemiológicos , Toxoplasmosis/parasitologíaRESUMEN
Recombinant SjCystatin (rSjCystatin), a recombinant protein of Schistosoma japonicum cystatin, has been reported to have an effect on immunoregulation mediated by IL-10 induction. Rheumatoid arthritis (RA) is a common autoimmune inflammatory arthropathy, and recombinant immune-modulating drugs for RA treatment are under development. We aimed to study the putative immune regulation of rSjCystatin and its prophylactic/therapeutic effects on murine collagen-induced arthritis (CIA). CIA was induced in DBA/1 mice by inoculation with bovine collagen II (CII). rSjCystatin was administered prior or post development of CIA. The severity of CIA was assessed using established clinical and histopathological scoring systems. The incidence was also determined. The CII-specific antibodies in sera and cytokines in splenocyte culture supernatants were measured by ELISA. Th1/Th2/Th17 cells and Tregs development in splenocytes were monitored by flow cytometry. The inflammatory mediators in the diseased joint were semiquantitated by qPCR. Prophylactic injection of rSjCystatin attenuated paw clinical scores, incidence, and histopathology scores of joints in CIA mice. The arthritis-alleviative effects were closely associated with the augmentation of IL-4, IL-10, and collagen-specific IgG1, and with the distinct reduction of IFN-γ, collagen-specific IgG2a, and the marked decrease of proinflammatory cytokines IL-6, IL-17, and TNF-α and RANKL. The data indicate that rSjCystatin may prevent cartilage destruction and inflammation of joints in CIA mice. The effects are related to the inhibitory modulation of Th1 and Th17 and upregulation of Tregs and Th2 via a shift of cytokines profiling from Th1 to Th2 response.
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Artritis Reumatoide/tratamiento farmacológico , Artritis Reumatoide/inmunología , Colágeno Tipo II/efectos adversos , Cistatinas/administración & dosificación , Proteínas del Helminto/administración & dosificación , Schistosoma japonicum/inmunología , Animales , Artritis Reumatoide/genética , Bovinos , Colágeno Tipo II/inmunología , Cistatinas/inmunología , Proteínas del Helminto/inmunología , Humanos , Interleucina-10/genética , Interleucina-10/inmunología , Interleucina-17/genética , Interleucina-17/inmunología , Interleucina-4/genética , Interleucina-4/inmunología , Interleucina-6/inmunología , Masculino , Ratones , Ratones Endogámicos DBA , Schistosoma japonicum/química , Células Th17/inmunología , Factor de Necrosis Tumoral alfa/genética , Factor de Necrosis Tumoral alfa/inmunologíaRESUMEN
BACKGROUND: Quantitative polymerase chain reaction (qPCR) analysis is a precise and effective method for the study of mRNA expression throughout the field of peripheral blood mononuclear cell (PBMC) research. However, the use of suitable reference genes for data normalization is critical to obtain meaningful and reproducible results. The present study aimed to identify the greatest reference genes for further research in PBMC of Chronic Hepatitis B (CHB) patients. METHODS: We assessed the expression stability of four commonly used reference genes (beta actin, beta-tubulin, 18S rRNA, GAPDH) in PBMC of CHB patients. Then we employed geNorm, BestKeeper, and Normfinder to evaluate the expression stability of these reference genes. RESULTS: All four genes displayed no significant differences between patient and control groups except beta actin and thus beta actin should not be used as a normalizing gene in a discussed experimental setup. GAPDH and beta-tubulin composed the best pair of reference genes for normalization purposes in future studies of gene expression in PBMC of CHB patients according to three algorithms. CONCLUSIONS: GAPDH and beta-tubulin were the best combination of two reference genes in this study for RT-qPCR analysis.
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Perfilación de la Expresión Génica/normas , Gliceraldehído-3-Fosfato Deshidrogenasas/genética , Hepatitis B Crónica/genética , Leucocitos Mononucleares/química , ARN Mensajero/genética , Reacción en Cadena en Tiempo Real de la Polimerasa/normas , Tubulina (Proteína)/genética , Actinas/genética , Algoritmos , Calibración , Estudios de Casos y Controles , Perfilación de la Expresión Génica/métodos , Marcadores Genéticos , Hepatitis B Crónica/sangre , Hepatitis B Crónica/diagnóstico , Humanos , Leucocitos Mononucleares/virología , Valor Predictivo de las Pruebas , ARN Mensajero/sangre , ARN Ribosómico 16S/genética , Estándares de ReferenciaRESUMEN
BACKGROUND: The primary aim of this study is to measure the JAK-STAT signaling in HBV infected peripheral blood mononuclear cells (PBMCs) stimulated by IFN-α and 3-TC and explore the influence of HBV to the JAKSTAT signaling pathways. METHODS: PBMCs were separated from healthy volunteers and patients who had not received any treatment with chronic hepatitis B. PBMCs were divided into the control group, IFN-α stimulation group, Lamivudine stimulation group, and combined treatment group. The expression of molecules of JAK-STAT signal transduction pathway (STAT1, STAT2, IRF9) and the antiviral protein (MxA) were detected by RT-qPCR and Western blot method. RESULTS: The majority of IFN-α inducible genes were expressed. The molecules of JAK-STAT signal transduction pathway (STAT1, STAT2, IRF9) and the antiviral protein (MxA) were highly expressed in IFN-α stimulation group and the combined treatment group. Compared to healthy controls, the expression levels of molecules (STAT1, IRF9) and the antiviral protein (MxA) are significantly lower in the control group, IFN-α stimulation group, and the combined treatment group of the CHB patients. CONCLUSIONS: IFN-α could activate JAK-STAT signaling transduction pathway in PBMCs of HBV-infected patients and HBV might process the activity to antagonize the antiviral activity in HBV infected PBMCs.
Asunto(s)
Antivirales/farmacología , Hepatitis B Crónica/tratamiento farmacológico , Interferón-alfa/farmacología , Lamivudine/farmacología , Leucocitos Mononucleares/efectos de los fármacos , Estudios de Casos y Controles , Células Cultivadas , Quimioterapia Combinada , Hepatitis B Crónica/genética , Hepatitis B Crónica/inmunología , Hepatitis B Crónica/metabolismo , Humanos , Subunidad gamma del Factor 3 de Genes Estimulados por el Interferón/genética , Subunidad gamma del Factor 3 de Genes Estimulados por el Interferón/metabolismo , Quinasas Janus/metabolismo , Leucocitos Mononucleares/inmunología , Leucocitos Mononucleares/metabolismo , Proteínas de Resistencia a Mixovirus/genética , Proteínas de Resistencia a Mixovirus/metabolismo , Factor de Transcripción STAT1/genética , Factor de Transcripción STAT1/metabolismo , Factor de Transcripción STAT2/genética , Factor de Transcripción STAT2/metabolismo , Transducción de Señal/efectos de los fármacos , Regulación hacia ArribaRESUMEN
BACKGROUND: Toxoplasma gondii is an intracellular protozoan that affects most species of endothermic animals including humans with a great infection rate. The vertical transmission of T. gondii causes abortion, constituting a serious threat to humans and leading to great losses in livestock production. Distinct from population structure of T. gondii in North America and Europe, Chinese 1 (ToxoDB #9) is a dominant genotype prevalent in China. Among the isolates of Chinese 1, the Wh3 and Wh6 have different virulence and pathogenicity in mice. However, little has been known about their difference at the genomic level. Thus the next-generation sequencing (NGS) approach was used to discover the association of the phenotypical variations with the genome sequencing data and the expression and polymorphisms of the key effectors. RESULTS: We successfully sequenced the genome of Chinese 1 strains of Wh3 and Wh6. The average sequencing depths were 63.91 and 63.61 for Wh3 and Wh6, respectively. The variations of both isolates were identified in comparison with reference genome of type I GT1 strain. There were 505,645 and 505,856 SNPs, 30,658 and 30,004 indels, 4661 and 2320 SVs, and 1942 and 3080 CNVs for Wh3 and Wh6, respectively. In target search variations of particular factors of T. gondii, the dense granule protein 3 (GRA3) and rhoptry neck protein 3 (RON3) were found to have 35 SNPs, 2 indels and 89 SNPs, 6 indels, respectively. GRA3 and RON3 were both found to have higher expression levels in less virulent Wh6 than in virulent Wh3. Both strains of type Chinese 1 share polymorphic GRA15II and ROPI/III with type I, II, and III strains. CONCLUSIONS: Sequencing of the two strains revealed that genome structure of Chinese 1 and type I strains has considerable genomic variations. Sequencing and qRT-PCR analyses of 26 effectors displayed a remarkable variation that may be associated with phenotype and pathogenic differences.