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BACKGROUND: Popillia japonica Newman is a scarab beetle native to Japan that is considered a serious pest outside its native range. It can feed on more than 400 host plants and spread about 10 km per year in invaded territories, therefore it is considered the second most important quarantine pest in Europe. Both chemical and biological insecticides have been used for control, with variable results. Despite ongoing efforts, P. japonica remains a threat in invaded countries, and it is necessary to test more effective and sustainable solutions in the context of integrated pest management. Here we present a study on long-lasting insecticide-treated nets (LLINs) assembled in semiochemical-baited attract-and-kill devices (A&Ks) as a means to control adults of P. japonica with low environmental impact. This study complements previous ones in which we first evaluated the effectiveness of the LLINs in the laboratory and then tested both effectiveness and duration in field-exposed A&Ks against P. japonica. In the present work we compared the effectiveness of three different densities of A&Ks per hectare in areas where the population of P. japonica was numerically homogeneous. RESULTS: The different densities of A&K (1, 2, 4 A&Ks per ha) resulted in an overall reduction of the population of P. japonica by about two thirds in comparison to the control area. CONCLUSIONS: This study suggests that the use of one A&K per hectare, requiring minimal management effort, is an effective ratio for reducing local populations of P. japonica. © 2024 The Author(s). Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.
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Antibody-mediated receptor activation is successfully used to develop medical treatments. If the activation induces a pathological response, such antibodies are also excellent tools for defining molecular mechanisms of target receptor malfunction and designing rescue therapies. Prominent examples are naturally occurring autoantibodies inducing the severe blistering disease pemphigus vulgaris (PV). In the great majority of patients, the antibodies bind to the adhesion receptor desmoglein 3 (Dsg3) and interfere with cell signaling to provoke severe blistering in the mucous membranes and/or skin. The identification of a comprehensive causative signaling network downstream of antibody-targeted Dsg3 receptors (e.g., shown by pharmacological activators or inhibitors) is currently being discussed as a basis to develop urgently needed first-line treatments for PV patients. Although polyclonal PV IgG antibodies have been used as proof of principle for pathological signal activation, monospecific anti-Dsg3 antibodies are necessary and have been developed to identify pathological Dsg3 receptor-mediated signal transduction. The experimental monospecific PV antibody AK23, produced from hybridoma cells, was extensively tested in our laboratory in both in vitro and in vivo models for PV and proved to recapitulate the clinicopathological features of PV when generated using the standardized production and purification protocols described herein. © 2024 The Author(s). Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Bovine IgG stripping from FBS and quality control Basic Protocol 2: AK23 hybridoma expansion and IgG production Basic Protocol 3: AK23 IgG purification Basic Protocol 4: AK23 IgG quality control Support Protocol 1: Detection of endotoxin levels Support Protocol 2: Detection and removal of mycoplasma.
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Desmogleína 3 , Pénfigo , Pénfigo/inmunología , Pénfigo/patología , Desmogleína 3/inmunología , Animales , Humanos , Ratones , Autoanticuerpos/inmunología , Investigación Biomédica TraslacionalRESUMEN
Cutaneous squamous cell carcinoma (cSCC) manifests through the complex interactions of UV-induced DNA damage, genetic mutations, and alterations in the tumor microenvironment. A high mutational burden is present in cSCC, as well as both cSCC precursors and normal skin, making driver genes difficult to differentiate. Despite this, several key driver genes have been identified, including TP53, the NOTCH family, CDKN2A, PIK3CA, and EGFR. In addition to mutations, the tumor microenvironment and the manipulation and evasion of the immune system play a critical role in cSCC progression. Novel therapeutic approaches, such as immunotherapy and EGFR inhibitors, have been used to target these dysregulations, and have shown promise in treating advanced cSCC cases, emphasizing the need for targeted interventions considering both genetic and microenvironmental factors for improved patient outcomes.
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Exploration of factors originating from brown adipose tissue that govern the thermogenic adipocyte differentiation is imperative for comprehending the regulatory framework underlying brown fat biogenesis and for devising therapeutic approaches for metabolic disorders associated with obesity. Prior evidence has illuminated the pivotal role of long noncoding RNAs (lncRNAs) in orchestrating thermogenesis within adipose tissue. Here, we aimed to explore and identify the critical lncRNA that could promote thermogenic adipocyte differentiation and to provide a novel strategy to treat obesity-related metabolic diseases in the future. In this study, through amalgamation with our previous lncRNA microarray data from small extracellular vesicles derived from BAT (sEV-BAT), we have identified sEV-BAT-enriched lncRNA AK029592 as a critical constituent of the thermogenic program, which actively fostered beige adipocyte differentiation and enhanced the thermogenic capacities of adipose tissue. Moreover, lncRNA AK029592 could sponge miR-199a-5p in adipocytes to stimulate thermogenic gene expression. Consequently, we concluded lncRNA AK029592 as a crucial lncRNA component of the thermogenic program that regulated beige adipocyte differentiation and white adipose tissue browning, thereby providing a novel therapeutic target and strategy in combating obesity and related metabolic diseases.
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Polycomb proteins are a fundamental repressive system that plays crucial developmental roles by orchestrating cell-type-specific transcription programs that govern cell identity. Direct alterations of Polycomb activity are indeed implicated in human pathologies, including developmental disorders and cancer. General Polycomb repression is coordinated by three distinct activities that regulate the deposition of two histone post-translational modifications: tri-methylation of histone H3 lysine 27 (H3K27me3) and histone H2A at lysine 119 (H2AK119ub1). These activities exist in large and heterogeneous multiprotein ensembles consisting of common enzymatic cores regulated by heterogeneous non-catalytic modules composed of a large number of accessory proteins with diverse biochemical properties. Here, we have analyzed the current molecular knowledge, focusing on the functional interaction between the core enzymatic activities and their regulation mediated by distinct accessory modules. This provides a comprehensive analysis of the molecular details that control the establishment and maintenance of Polycomb repression, examining their underlying coordination and highlighting missing information and emerging new features of Polycomb-mediated transcriptional control.
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Maternal inflammation can lead to premature birth and fetal brain damage. CircRNA_19038 and lncRNA-AK016022 have been shown to be significantly reduced in brain tissues of preterm mice, while whether they are involved in the regulation of preterm white matter injury remains to be explored. Pregnant mice were intraperitoneally injected with lipopolysaccharide (LPS) to establish a preterm brain injury model. Healthy mice born at term served as controls. Lentivirus-mediated circ_19038 overexpression vector (LV-circ_19038), LV-lnc-AK016022, LV-Sirt1 and LV-sh-Sirt1 were administered to preterm mice through the ventricles. The expression levels of circ_19038, lnc-AK016022 and Sirt1 in the brain tissues of preterm mice were significantly lower than those of full-term healthy mice, and circ_19038 and lnc-AK016022 were co-localized in the brain tissues. Upregulation of circ_19038 or/and lnc-AK016022 promoted remyelination and alleviated white matter structural damage, neuroinflammation, and long-term cognitive and motor deficits in preterm mice, and the combined effect of circ_19038 and lnc-AK016022 showed better results. Primary mouse neuronal cells were isolated to investigate the regulatory effects of circ_19038 and lnc-AK016022 on Sirt1. Circ_19038 and lnc-AK016022 jointly promoted the expression of Sirt1 by adsorbing miR-1b and miR-328, respectively. Moreover, silencing Sirt1 antagonized the beneficial effects of circ_19038 or/and lnc-AK016022 on brain white matter injury in preterm mice. In conclusion, circ_19038 and lnc-AK016022 synergistically regulated Sirt1 expression to promote remyelination and alleviate white matter injury in preterm mice.
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ARN Circular , ARN Largo no Codificante , Sirtuina 1 , Sustancia Blanca , Animales , Sirtuina 1/metabolismo , Sirtuina 1/genética , ARN Largo no Codificante/genética , ARN Largo no Codificante/metabolismo , Ratones , Sustancia Blanca/metabolismo , Sustancia Blanca/patología , Sustancia Blanca/lesiones , Femenino , Embarazo , ARN Circular/genética , ARN Circular/metabolismo , Remielinización , Ratones Endogámicos C57BL , Lipopolisacáridos/toxicidad , Nacimiento Prematuro , Encéfalo/metabolismo , Encéfalo/patologíaRESUMEN
The pathogenesis of non-alcoholic fatty liver disease (NAFLD) is influenced by a number of variables, including endoplasmic reticulum stress (ER). Thioredoxin domain-containing 5 (TXNDC5) is a member of the protein disulfide isomerase family and acts as an endoplasmic reticulum (ER) chaperone. Nevertheless, the function of TXNDC5 in hepatocytes under ER stress remains largely uncharacterized. In order to identify the role of TXNDC5 in hepatic wild-type (WT) and TXNDC5-deficient (KO) AML12 cell lines, tunicamycin, palmitic acid, and thapsigargin were employed as stressors. Cell viability, mRNA, protein levels, and mRNA splicing were then assayed. The protein expression results of prominent ER stress markers indicated that the ERN1 and EIF2AK3 proteins were downregulated, while the HSPA5 protein was upregulated. Furthermore, the ATF6 protein demonstrated no significant alterations in the absence of TXNDC5 at the protein level. The knockout of TXNDC5 has been demonstrated to increase cellular ROS production and its activity is required to maintain normal mitochondrial function during tunicamycin-induced ER stress. Tunicamycin has been observed to disrupt the protein levels of HSPA5, ERN1, and EIF2AK3 in TXNDC5-deficient cells. However, palmitic acid has been observed to disrupt the protein levels of ATF6, HSPA5, and EIF2AK3. In conclusion, TXNDC5 can selectively activate distinct ER stress pathways via HSPA5, contingent on the origin of ER stress. Conversely, the absence of TXNDC5 can disrupt the EIF2AK3 cascade.
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Chaperón BiP del Retículo Endoplásmico , Estrés del Retículo Endoplásmico , Retículo Endoplásmico , Hepatocitos , Proteína Disulfuro Isomerasas , Transducción de Señal , Tunicamicina , Chaperón BiP del Retículo Endoplásmico/metabolismo , Proteína Disulfuro Isomerasas/metabolismo , Proteína Disulfuro Isomerasas/genética , Hepatocitos/metabolismo , Animales , Tunicamicina/farmacología , Retículo Endoplásmico/metabolismo , Ratones , Especies Reactivas de Oxígeno/metabolismo , Factor de Transcripción Activador 6/metabolismo , Factor de Transcripción Activador 6/genética , Línea Celular , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Serina-Treonina Quinasas/genética , Proteínas de Choque Térmico/metabolismo , Proteínas de Choque Térmico/genética , Endorribonucleasas/metabolismo , Endorribonucleasas/genética , Ácido Palmítico/farmacología , Ácido Palmítico/metabolismo , Tapsigargina/farmacología , Humanos , Enfermedad del Hígado Graso no Alcohólico/metabolismo , Enfermedad del Hígado Graso no Alcohólico/genética , Enfermedad del Hígado Graso no Alcohólico/patología , Tiorredoxinas/metabolismo , Tiorredoxinas/genética , Supervivencia Celular/efectos de los fármacosRESUMEN
From the fruits of Cordia dichotoma, 11 new phenolic compounds, dichotomins A-K, were isolated, together with 19 known compounds. Through the analysis of detailed NMR data and HRESIMS data, the planar structures of all compounds were confirmed. Using NMR calculations, the absolute configuration of dichotomins A-K was elucidated by comparing their observed and computed electronic circular dichroism (ECD) spectra. Dichotomin H (8) and dichotomin I (9) were determined as two pairs of enantiomers. The enantiomers of compounds 8 and 9 were separated using chiral-phase high-performance liquid chromatography (HPLC), and the stereostructure of each enantiomer was determined by similarly calculating the ECD. Compounds 3, 5, 7, 17, 18, 23-25, and 27-30 increased glucose uptake by 1.04- to 2.85-folds at concentrations of 30 µg/mL. Further studies revealed that compounds 3 and 5 had a moderate effect on glucose transporter 4 (GLUT4) translocation activity in L6 cells. At 30 µg/mL, compound 3 significantly enhanced AMPK phosphorylation and GLUT4 expression. As a whole, compound 3 has the potential to be a drug candidate for the treatment of type 2 diabetes mellitus (T2DM).
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Frutas , Transportador de Glucosa de Tipo 4 , Glucosa , Fenoles , Extractos Vegetales , Transportador de Glucosa de Tipo 4/metabolismo , Transportador de Glucosa de Tipo 4/genética , Frutas/química , Glucosa/metabolismo , Fenoles/química , Fenoles/farmacología , Fenoles/metabolismo , Animales , Ratas , Extractos Vegetales/química , Extractos Vegetales/farmacología , Extractos Vegetales/metabolismo , Transporte Biológico/efectos de los fármacos , Estructura Molecular , Línea Celular , Transporte de Proteínas , Humanos , Proteínas Quinasas Activadas por AMP/metabolismo , Proteínas Quinasas Activadas por AMP/químicaRESUMEN
BACKGROUND: Numerous meta-analyses and clinical studies have shown that subtypes of immune cells are associated with the development of skin cancer, but it is not clear whether this association is causal or biased. Mendelian randomization (MR) analysis reduces the effect of confounding factors and improves the accuracy of the results when compared to traditional studies. Thus, in order to examine the causal relationship between various immune cell and skin cancer, this study employs two-sample MR. METHODS: This study assesses the causal association between 731 immune cell characteristics and skin cancer using a two-sample Mendel randomization (MR) methodology. Multiple MR methods were used to bias and to derive reliable estimates of causality between instrumental variables and outcomes. Comprehensive sensitivity analyses were used to validate the stability, heterogeneity and horizontal multiplicity of the results. RESULTS: We discovered that potential causal relationships between different types of immune cells and skin cancer disease. Specifically, one type of immune cell as potentially causal to malignant melanoma of skin (MM), eight different types of immune cells as potentially causal to basal cell carcinoma (BCC), four different types of immune cells as potentially causal to actinic keratosis (AK), and no different types of immune cells were found to have a potential causal association with squamous cell carcinoma(SCC), with stability in all of the results. CONCLUSION: This study demonstrates the close connection between immune cells and skin cancer disease by genetic means, which enriches the current knowledge about the role of immune cells in skin cancer and also contributes to the design of therapeutic strategies from an immunological perspective.
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Melanoma , Análisis de la Aleatorización Mendeliana , Neoplasias Cutáneas , Humanos , Neoplasias Cutáneas/genética , Neoplasias Cutáneas/inmunología , Melanoma/genética , Melanoma/inmunología , Carcinoma Basocelular/genética , Carcinoma Basocelular/inmunología , Carcinoma de Células Escamosas/genética , Carcinoma de Células Escamosas/inmunología , Queratosis Actínica/genética , Queratosis Actínica/inmunología , Polimorfismo de Nucleótido SimpleRESUMEN
Topical tirbanibulin is a highly effective and well tolerated novel treatment option for actinic keratoses (AKs). This study aimed to characterize the mode of action of tirbanibulin in keratinocytes (NHEK) and cutaneous squamous cell carcinoma (cSCC) cell lines (A431, SCC-12) in vitro. Tirbanibulin significantly reduced proliferation in a dose-dependent manner in all investigated cell lines, inhibited migration, and induced G2/M-cell cycle arrest only in the cSCC cell lines analyzed, and induced apoptosis solely in A431, which showed the highest sensitivity to tirbanibulin. In general, we detected low basal expression of phosphorylated SRC in all cell lines analyzed, therefore, interference with SRC signaling does not appear to be the driving force regarding the observed effects of tirbanibulin. The most prominent tirbanibulin-mediated effect was on ß-tubulin-polymerization, which was especially impaired in A431. Additionally, tirbanibulin induced an increase of the proinflammatory cytokines IL-1α, bFGF and VEGF in A431. In conclusion, tirbanibulin mediated anti-tumor effects predominantly in A431, while healthy keratinocytes and more dedifferentiated SCC-12 were less influenced. These effects of tirbanibulin are most likely mediated via dysregulation of ß-tubulin-polymerization and may be supported by proinflammatory aspects.
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Apoptosis , Carcinoma de Células Escamosas , Movimiento Celular , Proliferación Celular , Queratinocitos , Neoplasias Cutáneas , Tubulina (Proteína) , Humanos , Carcinoma de Células Escamosas/tratamiento farmacológico , Carcinoma de Células Escamosas/patología , Carcinoma de Células Escamosas/metabolismo , Queratinocitos/efectos de los fármacos , Queratinocitos/metabolismo , Línea Celular Tumoral , Tubulina (Proteína)/metabolismo , Neoplasias Cutáneas/tratamiento farmacológico , Neoplasias Cutáneas/patología , Neoplasias Cutáneas/metabolismo , Apoptosis/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Movimiento Celular/efectos de los fármacos , Antineoplásicos/farmacología , Polimerizacion/efectos de los fármacos , Queratosis Actínica/tratamiento farmacológico , Queratosis Actínica/patología , Queratosis Actínica/metabolismo , Transducción de Señal/efectos de los fármacos , Acetamidas , Morfolinas , PiridinasRESUMEN
The first systematic investigation of germacrane-type sesquiterpenes from Pilea cavaleriei Levl. subsp. cavaleriei was conducted. Eleven undescribed germacrane analogues named cavalinols A-K were identified. Their planar structures were determined by extensive analysis of 1D and 2D NMR spectroscopic data, and the absolute configurations were further determined by X-ray single crystal diffraction, Mosher method, and time dependent density functional theory (TDDFT) electron circular dichroism (ECD) calculation, with the aid from DFT NMR calculation and NOESY experiment. Except for the common 10-memebered ring, ten new compounds contained a p-coumaroyl sidechain connected to C-8 of the nucleus skeleton. All the isolated compounds were screened for anti-inflammatory activity in LPS stimulated RAW 264.7 cells, and compounds 5 and 6 showed moderate activity.
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Antiinflamatorios , Fitoquímicos , Sesquiterpenos de Germacrano , Ratones , Células RAW 264.7 , Animales , Estructura Molecular , Sesquiterpenos de Germacrano/aislamiento & purificación , Sesquiterpenos de Germacrano/farmacología , Sesquiterpenos de Germacrano/química , Fitoquímicos/aislamiento & purificación , Fitoquímicos/farmacología , Antiinflamatorios/aislamiento & purificación , Antiinflamatorios/farmacología , Antiinflamatorios/química , China , Sesquiterpenos/aislamiento & purificación , Sesquiterpenos/farmacología , Sesquiterpenos/químicaRESUMEN
Energy metabolism has always been a hot topic in cancer progression and targeted therapy, and exploring the role of genes in energy metabolic pathways in cancer cells has become key to address this issue. Eukaryotic translation initiation factor 2α kinase 2 (EIF2AK2) plays regulatory roles in cancer and disorders of energy metabolism. Indeed, the role of EIF2AK2 in energy metabolism has been underestimated. The aim of this study is to reveal the expression specificity of EIF2AK2 in gastric cancer (GC) progression and metastasis, and to demonstrate the role of EIF2AK2 in energy metabolism, cytoskeleton, proliferation, death and metastasis pathways in GC cells. Mechanistically, EIF2AK2 overexpression promoted cytoskeleton remodeling and ATP production, mediated cell proliferation and metastasis, upregulated OAS1 expression, decreases p-AMPK expression and inhibited apoptosis in GC cells. Conversely, knockdown of EIF2AK2 resulted in the opposite effect. However, overexpression of OAS1 mediated the upregulation of mitochondrial membrane potential and promoted ATP production and NAD+/NADH ratio, but knockdown of OAS1 inhibited the above effects. In addition, knockdown of OAS1 had no effect on EIF2AK2 expression, but inhibited AMPK and upregulated p-AMPK expression. In conclusion, our study identified EIF2AK2 and OAS1 as previously undescribed regulators of energy metabolism in GC cells. We hypothesized that EIF2AK2-OAS1 axis may regulate energy metabolism and inhibit cellular malignant behavior in cancer cells by affecting ATP production to induce AMPK phosphorylation, suggesting EIF2AK2 as a potential therapeutic target for cancer cell progression.
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Proteínas Quinasas Activadas por AMP , Adenosina Trifosfato , Neoplasias Gástricas , eIF-2 Quinasa , Neoplasias Gástricas/patología , Neoplasias Gástricas/metabolismo , Neoplasias Gástricas/genética , Humanos , Adenosina Trifosfato/metabolismo , Proteínas Quinasas Activadas por AMP/metabolismo , eIF-2 Quinasa/metabolismo , Fosforilación , Línea Celular Tumoral , Técnicas de Silenciamiento del GenRESUMEN
The 9p21.3 genomic locus is a hot spot for disease-associated single-nucleotide polymorphisms (SNPs), and its strongest associations are with coronary artery disease (CAD). The disease-associated SNPs are located within the sequence of a long noncoding RNA ANRIL, which potentially contributes to atherogenesis by regulating vascular cell stress and proliferation, but also affects pancreatic ß-cell proliferation. Altered expression of a neighboring gene, CDKN2B, has been also recognized to correlate with obesity and hepatic steatosis in people carrying the risk SNPs. In the present study, we investigated the impact of 9p21.3 on obesity accompanied by hyperlipidemia in mice carrying a deletion of the murine ortholog for the 9p21.3 (Chr4Δ70/Δ70) risk locus in hyperlipidemic Ldlr-/-ApoB100/100 background. The Chr4Δ70/Δ70 mice showed decreased mRNA expression of insulin receptors in white adipose tissue already at a young age, which developed into insulin resistance and obesity by aging. In addition, the Sirt1-Ppargc1a-Ucp2 pathway was downregulated together with the expression of Cdkn2b, specifically in the white adipose tissue in Chr4Δ70/Δ70 mice. These results suggest that the 9p21.3 locus, ANRIL lncRNA, and their murine orthologues may regulate the key energy metabolism pathways in a white adipose tissue-specific manner in the presence of hypercholesterolemia, thus contributing to the pathogenesis of metabolic syndrome.
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Hipercolesterolemia , Resistencia a la Insulina , Obesidad , Animales , Obesidad/genética , Obesidad/metabolismo , Resistencia a la Insulina/genética , Hipercolesterolemia/genética , Hipercolesterolemia/metabolismo , Hipercolesterolemia/complicaciones , Ratones , Humanos , Cromosomas Humanos Par 9/genética , Masculino , Eliminación de Gen , Sitios Genéticos , Ratones Endogámicos C57BL , Tejido Adiposo Blanco/metabolismo , ARN Largo no Codificante/genética , ARN Largo no Codificante/metabolismoRESUMEN
Acute lung injury (ALI), a critical complication observed in various clinical disorders, is characterized by widespread inflammation, neutrophil infiltration, and proinflammatory cytokine production. This study showed that the recently identified non-coding RNA ISIR and its human homolog gene AK131315 played a role in regulating lipopolysaccharide (LPS)-induced inflammatory responses. ISIR and AK131315 increased the production of inflammatory cytokines in LPS-stimulated macrophages, and exogenous ISIR aggravated LPS-induced lung inflammation in an animal model of ALI. Mechanistically, ISIR promoted LPS-triggered NF-κB and MAPK signaling and the transcription of proinflammatory cytokines by enhancing TAK1 activation. Furthermore, a significant correlation was observed between AK131315 expression and pulmonary infectious caused by Gram-negative bacteria, suggesting that AK131315 plays an important role in bacterial infections. Altogether, these findings indicate that ISIR regulates LPS-induced inflammation and AK131315 is involved in the pathogenesis of bacterial infections.
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Lesión Pulmonar Aguda , Lipopolisacáridos , Quinasas Quinasa Quinasa PAM , FN-kappa B , Lipopolisacáridos/inmunología , Lesión Pulmonar Aguda/inducido químicamente , Lesión Pulmonar Aguda/genética , Lesión Pulmonar Aguda/inmunología , Quinasas Quinasa Quinasa PAM/metabolismo , Quinasas Quinasa Quinasa PAM/genética , FN-kappa B/metabolismo , Animales , Humanos , Ratones , Ratones Endogámicos C57BL , Masculino , Citocinas/metabolismo , Citocinas/genética , Células RAW 264.7 , Inflamación/genética , Inflamación/inducido químicamente , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Transducción de Señal , Modelos Animales de EnfermedadRESUMEN
The proportion of human isolates with reduced neuraminidase inhibitors (NAIs) susceptibility in highly pathogenic avian influenza (HPAI) H7N9 virus was high. These drug-resistant strains showed good replication capacity without serious loss of fitness. In the presence of oseltamivir, R229I substitution were found in HA1 region of the HPAI H7N9 virus before NA R292K appeared. HPAI H7N9 or H7N9/PR8 recombinant viruses were developed to study whether HA R229I could increase the fitness of the H7N9 virus bearing NA 292K. Replication efficiency was assessed in MDCK or A549 cells. Neuraminidase enzyme activity and receptor-binding ability were analyzed. Pathogenicity in C57 mice was evaluated. Antigenicity analysis was conducted through a two-way HI test, in which the antiserum was obtained from immunized ferrets. Transcriptomic analysis of MDCK infected with HPAI H7N9 24hpi was done. It turned out that HA R229I substitution from oseltamivir induction in HA1 region increased (1) replication ability in MDCK(P < 0.05) and A549(P < 0.05), (2) neuraminidase enzyme activity, (3) binding ability to both α2,3 and α2,6 receptor, (4) pathogenicity to mice(more weight loss; shorter mean survival day; viral titer in respiratory tract, P < 0.05; Pathological changes in pneumonia), (5) transcriptome response of MDCK, of the H7N9 virus bearing NA 292K. Besides, HA R229I substitution changed the antigenicity of H7N9/PR8 virus (>4-fold difference of HI titre). It indicated that through the fine-tuning of HA-NA balance, R229I increased the fitness and changed the antigenicity of H7N9 virus bearing NA 292K. Public health attention to this mechanism needs to be drawn.
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Antivirales , Subtipo H7N9 del Virus de la Influenza A , Neuraminidasa , Infecciones por Orthomyxoviridae , Oseltamivir , Replicación Viral , Animales , Oseltamivir/farmacología , Subtipo H7N9 del Virus de la Influenza A/genética , Subtipo H7N9 del Virus de la Influenza A/efectos de los fármacos , Subtipo H7N9 del Virus de la Influenza A/patogenicidad , Subtipo H7N9 del Virus de la Influenza A/inmunología , Subtipo H7N9 del Virus de la Influenza A/fisiología , Neuraminidasa/genética , Neuraminidasa/metabolismo , Perros , Replicación Viral/efectos de los fármacos , Antivirales/farmacología , Humanos , Ratones , Infecciones por Orthomyxoviridae/virología , Células de Riñón Canino Madin Darby , Células A549 , Ratones Endogámicos C57BL , Farmacorresistencia Viral/genética , Sustitución de Aminoácidos , Gripe Humana/virología , Hurones , Glicoproteínas Hemaglutininas del Virus de la Influenza/genética , Glicoproteínas Hemaglutininas del Virus de la Influenza/inmunología , Glicoproteínas Hemaglutininas del Virus de la Influenza/metabolismo , Femenino , Proteínas Virales/genética , Proteínas Virales/metabolismoRESUMEN
Mutations in Prkra gene, which encodes PACT/RAX cause early onset primary dystonia DYT-PRKRA, a movement disorder that disrupts coordinated muscle movements. PACT/RAX activates protein kinase R (PKR, aka EIF2AK2) by a direct interaction in response to cellular stressors to mediate phosphorylation of the α subunit of the eukaryotic translation initiation factor 2 (eIF2α). Mice homozygous for a naturally arisen, recessively inherited frameshift mutation, Prkra lear-5J exhibit progressive dystonia. In the present study, we investigate the biochemical and developmental consequences of the Prkra lear-5J mutation. Our results indicate that the truncated PACT/RAX protein retains its ability to interact with PKR, however, it inhibits PKR activation. Furthermore, mice homozygous for the mutation have abnormalities in the cerebellar development as well as a severe lack of dendritic arborization of Purkinje neurons. Additionally, reduced eIF2α phosphorylation is noted in the cerebellums and Purkinje neurons of the homozygous Prkra lear-5J mice. These results indicate that PACT/RAX mediated regulation of PKR activity and eIF2α phosphorylation plays a role in cerebellar development and contributes to the dystonia phenotype resulting from this mutation.
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BACKGROUND: Polycystic ovary syndrome (PCOS) is a prevalent endocrine disorder affecting women of reproductive ages. Our previous study has implicated a possible link between RNA editing and PCOS, yet the actual role of RNA editing, its association with clinical features, and the underlying mechanisms remain unclear. METHODS: Ten RNA-Seq datasets containing 269 samples of multiple tissue types, including granulosa cells, T helper cells, placenta, oocyte, endometrial stromal cells, endometrium, and adipose tissues, were retrieved from public databases. Peripheral blood samples were collected from twelve PCOS and ten controls and subjected to RNA-Seq. Transcriptome-wide RNA-Seq data analysis was conducted to identify differential RNA editing (DRE) between PCOS and controls. The functional significance of DRE was evaluated by luciferase reporter assays and overexpression in human HEK293T cells. Dehydroepiandrosterone and lipopolysaccharide were used to stimulate human KGN granulosa cells to evaluate gene expression. RESULTS: RNA editing dysregulations across multiple tissues were found to be associated with PCOS in public datasets. Peripheral blood transcriptome analysis revealed 798 DRE events associated with PCOS. Through weighted gene co-expression network analysis, our results revealed a set of hub DRE events in PCOS blood. A DRE event in the eukaryotic translation initiation factor 2-alpha kinase 2 (EIF2AK2:chr2:37,100,559) was associated with PCOS clinical features such as luteinizing hormone (LH) and the ratio of LH over follicle-stimulating hormone. Luciferase assays, overexpression, and knockout of RNA editing enzyme adenosine deaminase RNA specific (ADAR) showed that the ADAR-mediated editing cis-regulated EIF2AK2 expression. EIAF2AK2 showed a higher expression after dehydroepiandrosterone and lipopolysaccharide stimulation, triggering changes in the downstrean MAPK pathway. CONCLUSIONS: Our study presented the first evidence of cross-tissue RNA editing dysregulation in PCOS and its clinical associations. The dysregulation of RNA editing mediated by ADAR and the disrupted target EIF2AK2 may contribute to PCOS development via the MPAK pathway, underlining such epigenetic mechanisms in the disease.
Asunto(s)
Síndrome del Ovario Poliquístico , Edición de ARN , eIF-2 Quinasa , Humanos , Síndrome del Ovario Poliquístico/genética , Femenino , Edición de ARN/genética , eIF-2 Quinasa/genética , Adulto , Células HEK293 , Perfilación de la Expresión Génica , Relevancia ClínicaRESUMEN
The complexities of energy transfer mechanisms in the flagella of mammalian sperm flagella have been intensively investigated and demonstrate significant diversity across species. Enzymatic shuttles, particularly adenylate kinase (AK) and creatine kinase (CK), are pivotal in the efficient transfer of intracellular ATP, showing distinct tissue- and species-specificity. Here, the expression profiles of AK and CK were investigated in mice and found to fall into four subgroups, of which Subgroup III AKs were observed to be unique to the male reproductive system and conserved across chordates. Both AK8 and AK9 were found to be indispensable to male reproduction after analysis of an infertile male cohort. Knockout mouse models showed that AK8 and AK9 were central to promoting sperm motility. Immunoprecipitation combined with mass spectrometry revealed that AK8 and AK9 interact with the radial spoke (RS) of the axoneme. Examination of various human and mouse sperm samples with substructural damage, including the presence of multiple RS subunits, showed that the head of radial spoke 3 acts as an adapter for AK9 in the flagellar axoneme. Using an ATP probe together with metabolomic analysis, it was found that AK8 and AK9 cooperatively regulated ATP transfer in the axoneme, and were concentrated at sites associated with energy consumption in the flagellum. These findings indicate a novel function for RS beyond its structural role, namely, the regulation of ATP transfer. In conclusion, the results expand the functional spectrum of AK proteins and suggest a fresh model regarding ATP transfer within mammalian flagella.
Asunto(s)
Adenosina Trifosfato , Adenilato Quinasa , Axonema , Ratones Noqueados , Motilidad Espermática , Cola del Espermatozoide , Animales , Adenilato Quinasa/metabolismo , Masculino , Ratones , Axonema/metabolismo , Motilidad Espermática/fisiología , Cola del Espermatozoide/metabolismo , Adenosina Trifosfato/metabolismo , Humanos , Metabolismo Energético , Espermatozoides/metabolismo , Flagelos/metabolismo , Creatina Quinasa/metabolismo , Infertilidad Masculina/metabolismo , Infertilidad Masculina/genéticaRESUMEN
Pulmonary arterial hypertension (PAH) is a disorder with a large genetic component. Biallelic mutations of EIF2AK4, which encodes the kinase GCN2, are causal in two ultra-rare subtypes of PAH, pulmonary veno-occlusive disease and pulmonary capillary haemangiomatosis. EIF2AK4 variants of unknown significance have also been identified in patients with classical PAH, though their relationship to disease remains unclear. To provide patients with diagnostic information and enable family testing, the functional consequences of such rare variants must be determined, but existing computational methods are imperfect. We applied a suite of bioinformatic and experimental approaches to sixteen EIF2AK4 variants that had been identified in patients. By experimentally testing the functional integrity of the integrated stress response (ISR) downstream of GCN2, we determined that existing computational tools have insufficient sensitivity to reliably predict impaired kinase function. We determined experimentally that several EIF2AK4 variants identified in patients with classical PAH had preserved function and are therefore likely to be non-pathogenic. The dysfunctional variants of GCN2 that we identified could be subclassified into three groups: misfolded, kinase-dead, and hypomorphic. Intriguingly, members of the hypomorphic group were amenable to paradoxical activation by a type-1½ GCN2 kinase inhibitor. This experiment approach may aid in the clinical stratification of EIF2AK4 variants and potentially identify hypomorophic alleles receptive to pharmacological activation.