RESUMO
Chronic nonbacterial osteomyelitis (CNO), an autoinflammatory bone disease primarily affecting children, can cause pain, hyperostosis and fractures, affecting quality-of-life and psychomotor development. This study investigated CNO-associated variants in P2RX7, encoding for the ATP-dependent trans-membrane K+ channel P2X7, and their effects on NLRP3 inflammasome assembly. Whole exome sequencing in two related transgenerational CNO patients, and target sequencing of P2RX7 in a large CNO cohort (N = 190) were conducted. Results were compared with publicly available datasets and regional controls (N = 1873). Findings were integrated with demographic and clinical data. Patient-derived monocytes and genetically modified THP-1 cells were used to investigate potassium flux, inflammasome assembly, pyroptosis, and cytokine release. Rare presumably damaging P2RX7 variants were identified in two related CNO patients. Targeted P2RX7 sequencing identified 62 CNO patients with rare variants (32.4%), 11 of which (5.8%) carried presumably damaging variants (MAF <1%, SIFT "deleterious", Polyphen "probably damaging", CADD >20). This compared to 83 of 1873 controls (4.4%), 36 with rare and presumably damaging variants (1.9%). Across the CNO cohort, rare variants unique to one (Median: 42 versus 3.7) or more (≤11 patients) participants were over-represented when compared to 190 randomly selected controls. Patients with rare damaging variants more frequently experienced gastrointestinal symptoms and lymphadenopathy while having less spinal, joint and skin involvement (psoriasis). Monocyte-derived macrophages from patients, and genetically modified THP-1-derived macrophages reconstituted with CNO-associated P2RX7 variants exhibited altered potassium flux, inflammasome assembly, IL-1ß and IL-18 release, and pyroptosis. Damaging P2RX7 variants occur in a small subset of CNO patients, and rare P2RX7 variants may represent a CNO risk factor. Observations argue for inflammasome inhibition and/or cytokine blockade and may allow future patient stratification and individualized care.
Assuntos
Inflamassomos , Osteomielite , Humanos , Citocinas , Inflamassomos/genética , Inflamassomos/metabolismo , Osteomielite/genética , Potássio , Piroptose , Receptores Purinérgicos P2X7/genéticaRESUMO
The monkeypox epidemic has attracted global attention to poxviruses. The cytoplasmic replication of poxviruses requires extensive protein synthesis, challenging the capacity of the endoplasmic reticulum (ER). However, the role of the ER in the life cycle of poxviruses is unclear. In this study, we demonstrate that infection with the lumpy skin disease virus (LSDV), a member of the poxvirus family, causes ER stress in vivo and in vitro, further facilitating the activation of the unfolded protein response (UPR). Although UPR activation aids in the restoration of the cellular environment, its significance in the LSDV life cycle remains unclear. Furthermore, the significance of ER imbalance for viral replication is also unknown. We show that LSDV replication is hampered by an unbalanced ER environment. In addition, we verify that the LSDV replication depends on the activation of PERK-eIF2α and IRE1-XBP1 signaling cascades rather than ATF6, implying that global translation and reduced XBP1 cleavage are deleterious to LSDV replication. Taken together, these findings indicate that LSDV is involved in the repression of global translational signaling, ER chaperone transcription, and ATF6 cleavage from the Golgi into the nucleus, thereby maintaining cell homeostasis; moreover, PERK and IRE1 activation contribute to LSDV replication. Our findings suggest that targeting UPR elements may be applied in response to infection from LSDV or even other poxviruses, such as monkeypox.
Assuntos
Vírus da Doença Nodular Cutânea , Mpox , Animais , Bovinos , Humanos , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Vírus da Doença Nodular Cutânea/metabolismo , Mpox/metabolismo , Transdução de Sinais , Resposta a Proteínas não Dobradas , Estresse do Retículo Endoplasmático/fisiologia , Retículo Endoplasmático/metabolismo , Fator 6 Ativador da Transcrição/metabolismoRESUMO
Goatpoxvirus (GTPV), sheeppoxvius (SPPV), and the Lumpy skin disease virus (LSDV) is a Capripoxvirus belonging to the family poxviridae. They can cause significant economic losses in countries where this disease are endemic. However, effective and convenient diagnostic tools against sera antibody are not readily available until now. Toward this goal, a polyclonal antibody competitive enzyme-linked immunosorbent assay (c-ELISA) of detecting serogroup-specific antibody is established based on major LSDV antigen A33. Serum samples (n = 605) were collected to optimize the c-ELISA from different areas. The cut-off value for the c-ELISA was estimate using percent inhibition (PI) values. The diagnostic performance of test including sensitivity (sn) and specificity (sp) were obtained by receiver operator characteristic (ROC) analysis. Among these analysis, > 57.61% PI value was accepted as cut-off of the c-ELISA, the diagnostic sn an diagnostic sp were reached to 96.4% and 98.5%, at > 95% confidence interval. These results show that the developed competitive ELISA is sensitive, specific, and reliable, which make it appropriate for serological investigation.
Assuntos
Anticorpos Antivirais , Antígenos Virais , Ensaio de Imunoadsorção Enzimática , Sensibilidade e Especificidade , Ensaio de Imunoadsorção Enzimática/métodos , Anticorpos Antivirais/sangue , Animais , Antígenos Virais/imunologia , Capripoxvirus/imunologia , Curva ROC , Cabras , Infecções por Poxviridae/diagnóstico , Infecções por Poxviridae/veterinária , Infecções por Poxviridae/imunologia , Infecções por Poxviridae/virologiaRESUMO
Paramphistomosis, caused by the rumen fluke, Calicophoron daubneyi, is a parasitic infection of ruminant livestock, which has seen a rapid rise in prevalence throughout Western Europe in recent years. After ingestion of metacercariae (parasite cysts) by the mammalian host, newly excysted juveniles (NEJs) emerge and invade the duodenal submucosa, which causes significant pathology in heavy infections. The immature flukes then migrate upward, along the gastrointestinal tract, and enter the rumen where they mature and begin to produce eggs. Despite their emergence, and sporadic outbreaks of acute disease, we know little about the molecular mechanisms used by C. daubneyi to establish infection, acquire nutrients, and avoid the host immune response. Here, transcriptome analysis of four intramammalian life-cycle stages, integrated with secretome analysis of the NEJ and adult parasites (responsible for acute and chronic diseases, respectively), revealed how the expression and secretion of selected families of virulence factors and immunomodulators are regulated in accordance with fluke development and migration. Our data show that while a family of cathepsins B with varying S2 subsite residues (indicating distinct substrate specificities) is differentially secreted by NEJs and adult flukes, cathepsins L and F are secreted in low abundance by NEJs only. We found that C. daubneyi has an expanded family of aspartic peptidases, which is upregulated in adult worms, although they are under-represented in the secretome. The most abundant proteins in adult fluke secretions were helminth defense molecules that likely establish an immune environment permissive to fluke survival and/or neutralize pathogen-associated molecular patterns such as bacterial lipopolysaccharide in the microbiome-rich rumen. The distinct collection of molecules secreted by C. daubneyi allowed the development of the first coproantigen-based ELISA for paramphistomosis which, importantly, did not recognize antigens from other helminths commonly found as coinfections with rumen fluke.
Assuntos
Proteínas de Helminto/genética , Proteínas de Helminto/metabolismo , Paramphistomatidae/genética , Paramphistomatidae/metabolismo , Animais , Antígenos de Helmintos/genética , Antígenos de Helmintos/imunologia , Antígenos de Helmintos/metabolismo , Bovinos , Cisteína Proteases/genética , Cisteína Proteases/metabolismo , Fezes/parasitologia , Proteínas de Helminto/imunologia , Estágios do Ciclo de Vida , Paramphistomatidae/crescimento & desenvolvimento , Rúmen/parasitologia , Secretoma , Transcriptoma , Infecções por Trematódeos/diagnóstico , Infecções por Trematódeos/imunologia , Infecções por Trematódeos/parasitologiaRESUMO
Intestinal organoids have emerged as powerful model systems for studying the complex structure and function of the intestine. However, there is a lack of widely applicable methods for the collection, labeling, and imaging of intestinal organoids. In this study, we developed a novel method for loading and labeling intestinal organoids, a method that efficiently collects the organoids and facilitates imaging of their three-dimensional (3D) structure. Based on this strainer platform, mouse intestinal organoids were adequately collected and immobilized, facilitating the immunolabeling workflow to target proteins of the organoids. After evaluation, the strainer size of 40 µm was considered to be more conducive to the collection and labeling of mouse intestinal organoids. More extensive research on organoids of multiple types and species origins will contribute to broadening the applicability of the methodology. Overall, our study proposes an innovative workflow for loading and analyzing intestinal organoids. The combination of a strainer-based collection method, fluorescent labeling, and 3D reconstruction provides valuable insights into the organization and complexity of these tissue models, thereby offering new avenues for investigating intestinal development, disease modeling, and drug discovery.
Assuntos
Corantes , Descoberta de Drogas , Animais , Camundongos , Modelos Biológicos , Organoides , Fluxo de TrabalhoRESUMO
The recent spread of the monkeypox virus among humans has heightened concerns regarding orthopoxvirus infections. Consequently, conducting a comprehensive study on the immunobiology of the monkeypox virus is imperative for the development of effective therapeutics. Ectromelia virus (ECTV) closely resembles the genetic and disease characteristics of monkeypox virus, making it a valuable research tool for studying orthopoxvirus-host interactions. Guanylate-binding proteins (GBPs), highly expressed interferon-stimulated genes (ISGs), have antagonistic effects against various intracellular pathogenic microorganisms. Our previous research has shown that GBP2 has a mild but statistically significant inhibitory effect on ECTV infection. The presence of a significant number of molecules in the poxvirus genome that encode the host immune response raises questions about whether it also includes proteins that counteract the antiviral activity of GBP2. Using IP/MS and co-IP technology, we discovered that the poly(A) polymerase catalytic subunit (PAPL) protein of ECTV is a viral regulatory molecule that interacts with GBP2. Further studies have shown that PAPL antagonizes the antiviral activity of GBP2 by reducing its protein levels. Knocking out the PAPL gene of ECTV with the CRISPR/Cas9 system significantly diminishes the replication ability of the virus, indicating the indispensable role of PAPL in the replication process of ECTV. In conclusion, our study presents preliminary evidence supporting the significance of PAPL as a virulence factor that can interact with GBP2.
Assuntos
Vírus da Ectromelia , Ectromelia Infecciosa , Animais , Camundongos , Humanos , Vírus da Ectromelia/genética , Proteínas Virais/genética , Proteínas Virais/metabolismo , Polinucleotídeo Adenililtransferase/metabolismo , Domínio Catalítico , Antivirais/farmacologiaRESUMO
BACKGROUND AND AIMS: The transcription factor nuclear factor erythroid 2-related factor 2 (Nrf2) regulates an array of cytoprotective genes, yet studies in transgenic mice have led to conflicting reports on its role in liver regeneration. We aimed to test the hypothesis that pharmacological activation of Nrf2 would enhance liver regeneration. APPROACH AND RESULTS: Wild-type and Nrf2 null mice were administered bardoxolone methyl (CDDO-Me), a potent activator of Nrf2 that has entered clinical development, and then subjected to two-thirds partial hepatectomy. Using translational noninvasive imaging techniques, CDDO-Me was shown to enhance the rate of restoration of liver volume (MRI) and improve liver function (multispectral optoacoustic imaging of indocyanine green clearance) in wild-type, but not Nrf2 null, mice following partial hepatectomy. Using immunofluorescence imaging and whole transcriptome analysis, these effects were found to be associated with an increase in hepatocyte hypertrophy and proliferation, the suppression of immune and inflammatory signals, and metabolic adaptation in the remnant liver tissue. Similar processes were modulated following exposure of primary human hepatocytes to CDDO-Me, highlighting the potential relevance of our findings to patients. CONCLUSIONS: Our results indicate that pharmacological activation of Nrf2 is a promising strategy for enhancing functional liver regeneration. Such an approach could therefore aid the recovery of patients undergoing liver surgery and support the treatment of acute and chronic liver disease.
Assuntos
Regeneração Hepática/efeitos dos fármacos , Fígado/efeitos dos fármacos , Fator 2 Relacionado a NF-E2/agonistas , Ácido Oleanólico/análogos & derivados , Adulto , Idoso de 80 Anos ou mais , Animais , Células Cultivadas , Feminino , Regulação da Expressão Gênica/efeitos dos fármacos , Hepatectomia , Hepatócitos , Humanos , Fígado/fisiologia , Fígado/cirurgia , Regeneração Hepática/genética , Masculino , Camundongos , Camundongos Knockout , Pessoa de Meia-Idade , Fator 2 Relacionado a NF-E2/genética , Fator 2 Relacionado a NF-E2/metabolismo , Ácido Oleanólico/administração & dosagem , Cultura Primária de CélulasRESUMO
Post copulatory interactions between the sexes in internally fertilizing species elicits both sexual conflict and sexual selection. Macroevolutionary and comparative studies have linked these processes to rapid transcriptomic evolution in sex-specific tissues and substantial transcriptomic post mating responses in females, patterns of which are altered when mating between reproductively isolated species. Here, we tested multiple predictions arising from sexual selection and conflict theory about the evolution of sex-specific and tissue-specific gene expression and the post mating response at the microevolutionary level. Following over 150 generations of experimental evolution under either reduced (enforced monogamy) or elevated (polyandry) sexual selection in Drosophila pseudoobscura, we found a substantial effect of sexual selection treatment on transcriptomic divergence in virgin male and female reproductive tissues (testes, male accessory glands, the female reproductive tract and ovaries). Sexual selection treatment also had a dominant effect on the post mating response, particularly in the female reproductive tract - the main arena for sexual conflict - compared to ovaries. This effect was asymmetric with monandry females typically showing more post mating responses than polyandry females, with enriched gene functions varying across treatments. The evolutionary history of the male partner had a larger effect on the post mating response of monandry females, but females from both sexual selection treatments showed unique patterns of gene expression and gene function when mating with males from the alternate treatment. Our microevolutionary results mostly confirm comparative macroevolutionary predictions on the role of sexual selection on transcriptomic divergence and altered gene regulation arising from divergent coevolutionary trajectories between sexual selection treatments.
Assuntos
Comportamento Sexual Animal , Seleção Sexual , Animais , Evolução Biológica , Drosophila/genética , Feminino , Masculino , Reprodução/genética , Comportamento Sexual Animal/fisiologia , Transcriptoma/genéticaRESUMO
Nerve regeneration is blocked after spinal cord injury (SCI) by a complex myelin-associated inhibitory (MAI) microenvironment in the lesion site; however, the underlying mechanisms are not fully understood. During the process of neural stem cell (NSC) differentiation, pathway inhibitors were added to quantitatively assess the effects on neuronal differentiation. Immunoprecipitation and lentivirus-induced overexpression were used to examine effects in vitro. In vivo, animal experiments and lineage tracing methods were used to identify nascent neurogenesis after SCI. In vitro results indicated that myelin inhibited neuronal differentiation by activating the epidermal growth factor receptor (EGFR)-extracellular-regulated kinase (ERK) signaling cascade. Subsequently, we found that tripartite motif (TRIM) 32, a neuronal fate-determining factor, was inhibited. Moreover, inhibition of EGFR-ERK promoted TRIM32 expression and enhanced neuronal differentiation in the presence of myelin. We further demonstrated that ERK interacts with TRIM32 to regulate neuronal differentiation. In vivo results indicated that EGFR-ERK blockade increased TRIM32 expression and promoted neurogenesis in the injured area, thus enhancing functional recovery after SCI. Our results showed that EGFR-ERK blockade antagonized MAI of neuronal differentiation of NSCs through regulation of TRIM32 by ERK. Collectively, these findings may provide potential new targets for SCI repair.
Assuntos
Receptores ErbB/antagonistas & inibidores , MAP Quinases Reguladas por Sinal Extracelular/antagonistas & inibidores , Proteínas de Ligação ao GTP/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Traumatismos da Medula Espinal/metabolismo , Animais , Células Cultivadas , Cetuximab/farmacologia , Receptores ErbB/metabolismo , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Feminino , Flavonoides/farmacologia , Gefitinibe/farmacologia , Camundongos , Camundongos Endogâmicos C57BL , Neurogênese/efeitos dos fármacos , Neurônios/citologia , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Ratos , Ratos Sprague-Dawley , Transdução de Sinais , Regulação para CimaRESUMO
BACKGROUND: Orf virus (ORFV) is a member of the genus Parapoxvirus and family Poxviridae. The virus has a worldwide distribution and infects sheep, goats, humans, and wild animals. However, due to the complex structure of the poxvirus, the underlying mechanism of the entry and infection by ORFV remains largely unknown. ORFV ORF047 encodes a protein named L1R. Poxviral L1R serves as the receptor-binding protein and blocks virus binding and entry independently of glycosaminoglycans (GAGs). The study aimed to identify the host interaction partners of ORFV ORF047. METHODS: Yeast two-hybrid cDNA library of sheep testicular cells was applied to screen the host targets with ORF047 as the bait. ORF047 was cloned into a pBT3-N vector and expressed in the NMY51 yeast strain. Then, the expression of bait proteins was validated by Western blot analysis. RESULTS: Sheep SERP1and PABPC4 were identified as host target proteins of ORFV ORF047, and a Co-IP assay further verified their interaction. CONCLUSIONS: New host cell proteins SERP1and PABPC4 were found to interact with ORFV ORF047 and might involve viral mRNA translation and replication.
Assuntos
Interações entre Hospedeiro e Microrganismos , Vírus do Orf/metabolismo , Proteínas do Envelope Viral/metabolismo , Animais , Células Cultivadas , Masculino , Proteínas de Membrana/metabolismo , Vírus do Orf/química , Vírus do Orf/genética , Ligação Proteica , Ovinos/virologia , Testículo/citologia , Proteínas do Envelope Viral/análise , Proteínas do Envelope Viral/genéticaRESUMO
BACKGROUND: Osteoarthritis remains one of the greatest causes of morbidity and mortality in the equine population. The inability to detect pre-clinical changes in osteoarthritis has been a significant impediment to the development of effective therapies against this disease. Synovial fluid represents a potential source of disease-specific small non-coding RNAs (sncRNAs) that could aid in the understanding of the pathogenesis of osteoarthritis. We hypothesised that early stages of osteoarthritis would alter the expression of sncRNAs, facilitating the understanding of the underlying pathogenesis and potentially provide early biomarkers. METHODS: Small RNA sequencing was performed using synovial fluid from the metacarpophalangeal joints of both control and early osteoarthritic horses. A group of differentially expressed sncRNAs was selected for further validation through qRT-PCR using an independent cohort of synovial fluid samples from control and early osteoarthritic horses. Bioinformatic analysis was performed in order to identify putative targets of the differentially expressed microRNAs and to explore potential associations with specific biological processes. RESULTS: Results revealed 22 differentially expressed sncRNAs including 13 microRNAs; miR-10a, miR-223, let7a, miR-99a, miR-23b, miR-378, miR-143 (and six novel microRNAs), four small nuclear RNAs; U2, U5, U11, U12, three small nucleolar RNAs; U13, snoR38, snord96, and one small cajal body-specific RNA; scarna3. Five sncRNAs were validated; miR-223 was significantly reduced in early osteoarthritis and miR-23b, let-7a-2, snord96A and snord13 were significantly upregulated. Significant cellular actions deduced by the differentially expressed microRNAs included apoptosis (P < 0.0003), necrosis (P < 0.0009), autophagy (P < 0.0007) and inflammation (P < 0.00001). A conservatively filtered list of 57 messenger RNA targets was obtained; the top biological processes associated were regulation of cell population proliferation (P < 0.000001), cellular response to chemical stimulus (P < 0.000001) and cell surface receptor signalling pathway (P < 0.000001). CONCLUSIONS: Synovial fluid sncRNAs may be used as molecular biomarkers for early disease in equine osteoarthritic joints. The biological processes they regulate may play an important role in understanding early osteoarthritis pathogenesis. Characterising these dynamic molecular changes could provide novel insights on the process and mechanism of early osteoarthritis development and is critical for the development of new therapeutic approaches.
Assuntos
Doenças dos Cavalos/diagnóstico , Osteoartrite/veterinária , Pequeno RNA não Traduzido/metabolismo , Líquido Sinovial , Animais , Biomarcadores , Cavalos , Osteoartrite/diagnóstico , Osteoartrite/metabolismoRESUMO
The interfascicular matrix (IFM) binds tendon fascicles and contains a population of morphologically distinct cells. However, the role of IFM-localised cell populations in tendon repair remains to be determined. The basement membrane protein laminin-α4 also localises to the IFM. Laminin-α4 is a ligand for several cell surface receptors, including CD146, a marker of pericyte and progenitor cells. We used a needle injury model in the rat Achilles tendon to test the hypothesis that the IFM is a niche for CD146+ cells that are mobilised in response to tendon damage. We also aimed to establish how expression patterns of circulating non-coding RNAs alter with tendon injury and identify potential RNA-based markers of tendon disease. The results demonstrate the formation of a focal lesion at the injury site, which increased in size and cellularity for up to 21 days post injury. In healthy tendon, CD146+ cells localised to the IFM, compared with injury, where CD146+ cells migrated towards the lesion at days 4 and 7, and populated the lesion 21 days post injury. This was accompanied by increased laminin-α4, suggesting that laminin-α4 facilitates CD146+ cell recruitment at injury sites. We also identified a panel of circulating microRNAs that are dysregulated with tendon injury. We propose that the IFM cell niche mediates the intrinsic response to injury, whereby an injury stimulus induces CD146+ cell migration. Further work is required to fully characterise CD146+ subpopulations within the IFM and establish their precise roles during tendon healing.
Assuntos
Antígeno CD146/metabolismo , Matriz Extracelular/metabolismo , Laminina/metabolismo , Traumatismos dos Tendões/metabolismo , Tendões/metabolismo , Tendão do Calcâneo/metabolismo , Tendão do Calcâneo/patologia , Animais , Antígeno CD146/genética , Modelos Animais de Doenças , Suscetibilidade a Doenças , Feminino , Imunofluorescência , Expressão Gênica , Ligantes , Ligação Proteica , Ratos , Traumatismos dos Tendões/etiologia , Traumatismos dos Tendões/patologia , Tendões/patologiaRESUMO
Ageing is a leading risk factor predisposing cartilage to osteoarthritis. However, little research has been conducted on the effect of ageing on the expression of small non-coding RNAs (sncRNAs). RNA from young and old chondrocytes from macroscopically normal equine metacarpophalangeal joints was extracted and subjected to small RNA sequencing (RNA-seq). Differential expression analysis was performed in R using package DESeq2. For transfer RNA (tRNA) fragment analysis, tRNA reads were aligned to horse tRNA sequences using Bowtie2 version 2.2.5. Selected microRNA (miRNAs or miRs) and small nucleolar RNA (snoRNA) findings were validated using real-time quantitative Polymerase Chain Reaction (qRT-PCR) in an extended cohort of equine chondrocytes. tRNA fragments were further investigated in low- and high-grade OA human cartilage tissue. In total, 83 sncRNAs were differentially expressed between young and old equine chondrocytes, including miRNAs, snoRNAs, small nuclear RNAs (snRNAs), and tRNAs. qRT-PCR analysis confirmed findings. tRNA fragment analysis revealed that tRNA halves (tiRNAs), tiRNA-5035-GluCTC and tiRNA-5031-GluCTC-1 were reduced in both high grade OA human cartilage and old equine chondrocytes. For the first time, we have measured the effect of ageing on the expression of sncRNAs in equine chondrocytes. Changes were detected in a number of different sncRNA species. This study supports a role for sncRNAs in ageing cartilage and their potential involvement in age-related cartilage diseases.
Assuntos
Senescência Celular/genética , Condrócitos/metabolismo , Pequeno RNA não Traduzido/metabolismo , Envelhecimento/genética , Animais , Cartilagem Articular/patologia , Condrócitos/patologia , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Cavalos/genética , Humanos , MicroRNAs/genética , MicroRNAs/metabolismo , Osteoartrite/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA de Transferência/genética , RNA de Transferência/metabolismo , Reação em Cadeia da Polimerase em Tempo Real , Reprodutibilidade dos Testes , Análise de Sequência de RNARESUMO
The transcription factor NRF2, governed by its repressor KEAP1, protects cells against oxidative stress. There is interest in modelling the NRF2 response to improve the prediction of clinical toxicities such as drug-induced liver injury (DILI). However, very little is known about the makeup of the NRF2 transcriptional network and its response to chemical perturbation in primary human hepatocytes (PHH), which are often used as a translational model for investigating DILI. Here, microarray analysis identified 108 transcripts (including several putative novel NRF2-regulated genes) that were both downregulated by siRNA targeting NRF2 and upregulated by siRNA targeting KEAP1 in PHH. Applying weighted gene co-expression network analysis (WGCNA) to transcriptomic data from the Open TG-GATES toxicogenomics repository (representing PHH exposed to 158 compounds) revealed four co-expressed gene sets or 'modules' enriched for these and other NRF2-associated genes. By classifying the 158 TG-GATES compounds based on published evidence, and employing the four modules as network perturbation metrics, we found that the activation of NRF2 is a very good indicator of the intrinsic biochemical reactivity of a compound (i.e. its propensity to cause direct chemical stress), with relatively high sensitivity, specificity, accuracy and positive/negative predictive values. We also found that NRF2 activation has lower sensitivity for the prediction of clinical DILI risk, although relatively high specificity and positive predictive values indicate that false positive detection rates are likely to be low in this setting. Underpinned by our comprehensive analysis, activation of the NRF2 network is one of several mechanism-based components that can be incorporated into holistic systems toxicology models to improve mechanistic understanding and preclinical prediction of DILI in man.
Assuntos
Doença Hepática Induzida por Substâncias e Drogas/genética , Redes Reguladoras de Genes/efeitos dos fármacos , Hepatócitos/efeitos dos fármacos , Fator 2 Relacionado a NF-E2/genética , Células Cultivadas , Doença Hepática Induzida por Substâncias e Drogas/patologia , Regulação da Expressão Gênica/efeitos dos fármacos , Hepatócitos/patologia , Humanos , Isotiocianatos/efeitos adversos , Proteína 1 Associada a ECH Semelhante a Kelch/genética , Análise de Sequência com Séries de Oligonucleotídeos , Estresse Oxidativo/efeitos dos fármacos , Estresse Oxidativo/genética , RNA Interferente Pequeno , SulfóxidosRESUMO
Recent theory predicts that increased phenotypic plasticity can facilitate adaptation as traits respond to selection. When genetic adaptation alters the social environment, socially mediated plasticity could cause co-evolutionary feedback dynamics that increase adaptive potential. We tested this by asking whether neural gene expression in a recently arisen, adaptive morph of the field cricket Teleogryllus oceanicus is more responsive to the social environment than the ancestral morph. Silent males (flatwings) rapidly spread in a Hawaiian population subject to acoustically orienting parasitoids, changing the population's acoustic environment. Experimental altering crickets' acoustic environments during rearing revealed broad, plastic changes in gene expression. However, flatwing genotypes showed increased socially mediated plasticity, whereas normal-wing genotypes exhibited negligible expression plasticity. Increased plasticity in flatwing crickets suggests a coevolutionary process coupling socially flexible gene expression with the abrupt spread of flatwing. Our results support predictions that phenotypic plasticity should rapidly evolve to be more pronounced during early phases of adaptation.
Assuntos
Evolução Biológica , Expressão Gênica , Gryllidae , Animais , Genótipo , Gryllidae/genética , Havaí , Masculino , FenótipoRESUMO
The transcription factor Nrf2 exerts protective effects in numerous experimental models of acute kidney injury, and is a promising therapeutic target in chronic kidney disease. To provide a detailed insight into the regulatory roles of Nrf2 in the kidney, we performed integrated transcriptomic and proteomic analyses of kidney tissue from wild-type and Nrf2 knockout mice treated with the Nrf2 inducer methyl-2-cyano-3,12-dioxooleano-1,9-dien-28-oate (CDDO-Me, also known as bardoxolone methyl). After 24 h, analyses identified 2561 transcripts and 240 proteins that were differentially expressed in the kidneys of Nrf2 knockout mice, compared with those of wild-type counterparts, and 3122 transcripts and 68 proteins that were differentially expressed in wild-type mice treated with CDDO-Me, compared with those of vehicle control. In the light of their sensitivity to genetic and pharmacological modulation of renal Nrf2 activity, genes/proteins that regulate xenobiotic disposition, redox balance, the intra/extracellular transport of small molecules, and the supply of NADPH and other cellular fuels were found to be positively regulated by Nrf2 in the kidney. This was verified by qPCR, immunoblotting, pathway analysis, and immunohistochemistry. In addition, the levels of NADPH and glutathione were found to be significantly decreased in the kidneys of Nrf2 knockout mice. Thus, Nrf2 regulates genes that coordinate homeostatic processes in the kidney, highlighting its potential as a novel therapeutic target.
RESUMO
The reconstruction of dynamic magnetic resonance images from incomplete k-space data has sparked significant research interest due to its potential to reduce scan time. However, traditional iterative optimization algorithms fail to faithfully reconstruct images at higher acceleration factors and incur long reconstruction time. Furthermore, end-to-end deep learning-based reconstruction algorithms suffer from large model parameters and lack robustness in the reconstruction results. Recently, unrolled deep learning models, have shown immense potential in algorithm stability and applicability flexibility. In this paper, we propose an unrolled deep learning network based on a second-order Half-Quadratic Splitting(HQS) algorithm, where the forward propagation process of this framework strictly follows the computational flow of the HQS algorithm. In particular, we propose a degradation-sense module by associating random sampling patterns with intermediate variables to guide the iterative process. We introduce the Information Fusion Transformer(IFT) to extract both local and non-local prior information from image sequences, thereby removing aliasing artifacts resulting from random undersampling. Finally, we impose low-rank constraints within the HQS algorithm to further enhance the reconstruction results. The experiments demonstrate that each component module of our proposed model contributes to the improvement of the reconstruction task. Our proposed method achieves comparably satisfying performance to the state-of-the-art methods and it exhibits excellent generalization capabilities across different sampling masks. At the low acceleration factor, there is a 0.7% enhancement in the PSNR. Furthermore, when the acceleration factor reached 8 and 12, the PSNR achieves an improvement of 3.4% and 5.8% respectively.
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Anthropogenic endocrine disruptors now contaminate all environments globally, with concomitant deleterious effects across diverse taxa. While most studies on endocrine disruption (ED) have focused on vertebrates, the superimposition of male sexual characteristics in the female dogwhelk, Nucella lapillus (imposex), caused by organotins, provides one of the most clearcut ecological examples of anthropogenically induced ED in aquatic ecosystems. To identify the underpinning mechanisms of imposex for this 'nonmodel' species, we combined Roche 454 pyrosequencing with custom oligoarray fabrication inexpensively to both generate gene models and identify those responding to chronic tributyltin (TBT) treatment. The results supported the involvement of steroid, neuroendocrine peptide hormone dysfunction and retinoid mechanisms, but suggested additionally the involvement of putative peroxisome proliferator-activated receptor (PPAR) pathways. Application of rosiglitazone, a well-known vertebrate PPARγ ligand, to dogwhelks induced imposex in the absence of TBT. Thus, while TBT-induced imposex is linked to the induction of many genes and has a complex phenotype, it is likely also to be driven by PPAR-responsive pathways, hitherto not described in invertebrates. Our findings provide further evidence for a common signalling pathway between invertebrate and vertebrate species that has previously been overlooked in the study of endocrine disruption.
Assuntos
Transtornos do Desenvolvimento Sexual/induzido quimicamente , Disruptores Endócrinos/toxicidade , Monitoramento Ambiental/métodos , Gastrópodes/efeitos dos fármacos , Transcriptoma , Compostos de Trialquitina/toxicidade , Animais , Feminino , Gastrópodes/genética , Gastrópodes/crescimento & desenvolvimento , Biblioteca Gênica , Masculino , Análise de Sequência com Séries de Oligonucleotídeos , Receptores Ativados por Proliferador de Peroxissomo/metabolismo , Rosiglitazona , Análise de Sequência de DNA , Tiazolidinedionas/toxicidade , Poluentes Químicos da Água/toxicidadeRESUMO
The tailing genome walking strategies are simple and efficient. However, they sometimes can be restricted due to the low stringency of homo-oligomeric primers. Here we modified their conventional tailing step by adding polythymidine and polyguanine to the target single-stranded DNA (ssDNA). The tailed ssDNA was then amplified exponentially with a specific primer in the known region and a primer comprising 5' polycytosine and 3' polyadenosine. The successful application of this novel method for identifying integration sites mediated by φC31 integrase in goat genome indicates that the method is more suitable for genomes with high complexity and local GC content.
Assuntos
DNA de Cadeia Simples/química , DNA de Cadeia Simples/genética , Genoma , Animais , Composição de Bases , Sequência de Bases , Primers do DNA/química , Primers do DNA/genética , DNA de Cadeia Simples/metabolismo , Cabras , Guanina/química , Integrases/metabolismo , Técnicas de Amplificação de Ácido Nucleico , Timidina/químicaRESUMO
The enzyme φC31 integrase from Streptomyces phage has been documented as functional in mammalian cells and, therefore, has the potential to be a powerful gene manipulation tool. However, the activity of this enzyme is cell-type dependent. The more active mutant forms of φC31 integrase are required. Therefore, a rapid and effective method should be developed to detect the intracellular activity of φC31 integrase. We devised in this study an integrase-inversion cassette that contains the enhanced green fluorescent protein (EGFP) gene and the reverse complementary DsRed gene, which are flanked by attB and reverse complementary attP. This cassette can be inverted by φC31 integrase, thereby altering the fluorescent protein expression. Thus, φC31 integrase activity can be qualitatively or quantitatively evaluated based on the detected fluorescence. Furthermore, this cassette-based method was applied to several cell types, demonstrating that it is an efficient and reliable tool for measuring φC31 integrase activity in mammalian cells.