RESUMEN
Neuroligin-2 (Nlgn2) is a key synaptic adhesion protein at virtually all GABAergic synapses, which recruits GABAARs by promoting assembly of the postsynaptic gephyrin scaffold. Intriguingly, loss of Nlgn2 differentially affects subsets of GABAergic synapses, indicating that synapse-specific interactors and redundancies define its function, but the nature of these interactions remain poorly understood. Here we investigated how Nlgn2 function in hippocampal area CA1 is modulated by two proposed interaction partners, MDGA1 and MDGA2. We show that loss of MDGA1 expression, but not heterozygous deletion of MDGA2, ameliorates the abnormal cytosolic gephyrin aggregation, the reduction in inhibitory synaptic transmission and the exacerbated anxiety-related behaviour characterizing Nlgn2 knockout (KO) mice. Additionally, combined Nlgn2 and MDGA1 deletion causes an exacerbated layer-specific loss of gephyrin puncta. Given that both Nlgn2 and the MDGA1 have been correlated with many psychiatric disorders, our data support the notion that cytosolic gephyrin aggregation may represent an interesting target for novel therapeutic strategies.
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Proteínas Portadoras , Moléculas de Adhesión Celular Neuronal , Proteínas de la Membrana , Ratones Noqueados , Receptores de GABA-A , Sinapsis , Animales , Moléculas de Adhesión Celular Neuronal/metabolismo , Moléculas de Adhesión Celular Neuronal/genética , Proteínas de la Membrana/metabolismo , Proteínas de la Membrana/genética , Ratones , Proteínas Portadoras/metabolismo , Proteínas Portadoras/genética , Sinapsis/metabolismo , Receptores de GABA-A/metabolismo , Receptores de GABA-A/genética , Citosol/metabolismo , Masculino , Proteínas del Tejido Nervioso/metabolismo , Proteínas del Tejido Nervioso/genética , Transmisión Sináptica , Ratones Endogámicos C57BL , Región CA1 Hipocampal/metabolismoRESUMEN
Nucleostemin (NS) plays a role in liver regeneration, and aging reduces its expression in the baseline and regenerating livers following 70% partial hepatectomy (PHx). Here we interrogate the mechanism controlling NS expression during liver regeneration and aging. The NS promoter was analyzed by TRANSFAC. Functional studies were performed using cell-based luciferase assay, endogenous NS expression in Hep3B cells, mouse livers with a gain-of-function mutation of C/EBPα (S193D), and mouse livers with C/EBPα knockdown. We found a CAAT box with four C/EBPα binding sites (-1216 to -735) and a GC box with consensus binding sites for c-Myc, E2F1, and p300-associated protein complex (-633 to -1). Age-related changes in NS expression correlated positively with the expression of c-Myc, E2F1, and p300, and negatively with that of C/EBPα and C/EBPß. PHx upregulated NS expression at 1d, coinciding with an increase in E2F1 and a decrease in C/EBPα. C/EBPα bound to the consensus sequences found in the NS promoter in vitro and in vivo, inhibited its transactivational activity in a binding site-dependent manner, and decreased the expression of endogenous NS in Hep3B cells. In vivo activation of C/EBPα by the S193D mutation resulted in a 4th-day post-PHx reduction of NS, a feature shared by 16-m/o livers. Finally, C/EBPα knockdown increased its expression in aged (24-m/o) livers under both baseline and regeneration conditions. This study reports the C/EBPα suppression of NS expression in aged livers, providing a new perspective on the mechanistic orchestration of tissue homeostasis in aging.
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Envejecimiento , Proteínas de Unión al GTP , Regeneración Hepática , Proteínas Nucleares , Regiones Promotoras Genéticas , Proteínas Proto-Oncogénicas c-myc , Animales , Regeneración Hepática/genética , Regeneración Hepática/fisiología , Ratones , Envejecimiento/metabolismo , Envejecimiento/fisiología , Envejecimiento/genética , Humanos , Proteínas Nucleares/metabolismo , Proteínas Nucleares/genética , Proteínas de Unión al GTP/metabolismo , Proteínas de Unión al GTP/genética , Proteínas Proto-Oncogénicas c-myc/metabolismo , Proteínas Proto-Oncogénicas c-myc/genética , Proteína beta Potenciadora de Unión a CCAAT/metabolismo , Proteína beta Potenciadora de Unión a CCAAT/genética , Factor de Transcripción E2F1/metabolismo , Factor de Transcripción E2F1/genética , Hepatectomía , Sitios de Unión , Hígado/metabolismo , Proteína p300 Asociada a E1A/metabolismo , Regulación de la Expresión Génica , Transcripción Genética , Proteína alfa Potenciadora de Unión a CCAAT/metabolismo , Proteína alfa Potenciadora de Unión a CCAAT/genética , Masculino , Proteínas Portadoras/metabolismo , Proteínas Portadoras/genética , Ratones Endogámicos C57BL , Línea Celular Tumoral , Proteínas de Unión al ARNRESUMEN
INTRODUCTION: Short stature is one of the main reasons for consultation in outpatient clinics and paediatric endocrinology departments and is defined as height below the 3rd centile or less than -2 standard deviations (SDs). MATERIAL AND METHODS: The study's overarching aim was to analyse the PAPP-A2 gene at mutation sites described to date and at exons 3, 4, and 5, which encode the fragment of the catalytic domain with the active site of the pregnancy-associated plasma protein A2 (PAPP-A2) protein. The secondary aims of the study were clinical and auxological analysis of a group of patients with idiopathic short stature and biochemical analysis of growth hormone-insulin-like growth factor-1 (GH-IGF-1) axis parameters not assessed as part of the routine diagnosis of short stature, such as free IGF-1, insulin-like growth factor binding protein 5 (IGFBP-5), and acid-labile subunit (ALS) levels. Molecular analysis of the PAPP-A2 gene was performed using polymerase chain reaction (PCR) and direct sequencing. Biochemical analysis of free IGF-1, IGFBP-5, and ALS was performed by enzyme-linked immunosorbent assay (ELISA). RESULTS: The mean height standard deviation score (HSDS) in the study group was -2.95. None of the patients exhibited previously described mutations in the PAPP-A2 gene or mutations in exons 3, 4, and 5 encoding the fragment of catalytic domain with the active site of the PAPP-A2 protein. In 4 patients, the known, non-pathogenic, heterozygotic polymorphism c.2328C>T(rs10913241) in exon 5 was found. CONCLUSIONS: Free IGF-1 levels correlate better with height and HSDS than total IGF-1 levels. The previously described mutations in the PAPP-A2 gene and mutations in exons 3, 4, and 5 encoding the fragment of catalytic domain with the active site of the PAPP-A2 protein were not detected; only the known and non-pathogenic, heterozygotic polymorphism c.2328C>T(rs10913241) in exon 5 of the PAPP-A2 gene was observed.
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Proteína 5 de Unión a Factor de Crecimiento Similar a la Insulina , Factor I del Crecimiento Similar a la Insulina , Proteína Plasmática A Asociada al Embarazo , Humanos , Proteína Plasmática A Asociada al Embarazo/genética , Proteína Plasmática A Asociada al Embarazo/metabolismo , Proteína Plasmática A Asociada al Embarazo/análisis , Femenino , Factor I del Crecimiento Similar a la Insulina/genética , Factor I del Crecimiento Similar a la Insulina/análisis , Factor I del Crecimiento Similar a la Insulina/metabolismo , Masculino , Niño , Adolescente , Proteína 5 de Unión a Factor de Crecimiento Similar a la Insulina/genética , Proteínas Portadoras/genética , Glicoproteínas/genética , Glicoproteínas/sangre , Trastornos del Crecimiento/genética , Trastornos del Crecimiento/sangre , Mutación , PreescolarRESUMEN
miR-31 is a highly conserved microRNA that plays crucial roles in cell proliferation, migration and differentiation. We discovered that miR-31 and some of its validated targets are enriched on the mitotic spindle of the dividing sea urchin embryo and mammalian cells. Using the sea urchin embryo, we found that miR-31 inhibition led to developmental delay correlated with increased cytoskeletal and chromosomal defects. We identified miR-31 to directly suppress several actin remodeling transcripts, including ß-actin, Gelsolin, Rab35 and Fascin. De novo translation of Fascin occurs at the mitotic spindle of sea urchin embryos and mammalian cells. Importantly, miR-31 inhibition leads to a significant a increase of newly translated Fascin at the spindle of dividing sea urchin embryos. Forced ectopic localization of Fascin transcripts to the cell membrane and translation led to significant developmental and chromosomal segregation defects, highlighting the importance of the regulation of local translation by miR-31 at the mitotic spindle to ensure proper cell division. Furthermore, miR-31-mediated post-transcriptional regulation at the mitotic spindle may be an evolutionarily conserved regulatory paradigm of mitosis.
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MicroARNs , Biosíntesis de Proteínas , Huso Acromático , Animales , MicroARNs/metabolismo , MicroARNs/genética , Huso Acromático/metabolismo , Regulación del Desarrollo de la Expresión Génica , Humanos , Proteínas de Microfilamentos/metabolismo , Proteínas de Microfilamentos/genética , Mitosis/genética , Proteínas Portadoras/metabolismo , Proteínas Portadoras/genética , Desarrollo Embrionario/genética , Embrión no Mamífero/metabolismo , Segregación Cromosómica/genética , Actinas/metabolismo , Actinas/genética , Erizos de Mar/embriología , Erizos de Mar/genética , Erizos de Mar/metabolismoRESUMEN
The juvenile hormone binding protein (JHBP) and takeout (TO) genes, mediated by the juvenile hormone (JH), play a crucial role in regulating the reproductive physiology of insects. Our previous study revealed that spinosad-resistant Frankliniella occidentalis (NIL-R) exhibited reduced fecundity and significant changes in JHBP/TO family gene expression. We hypothesized that these genes were involved in regulating the fitness costs associated with resistance. In this study, 45 JHBP/TO genes were identified in F. occidentalis, among which FoTO2 and FoTO10 were duplicates. Additionally, eight genes exhibited significant down-regulation in the NIL-R population. Two genes (FoTO6 and FoTO24) that exhibited the most significant differential expression between the spinosad-susceptible (Ivf03) and NIL-R populations were selected to investigate their roles in resistance fitness using RNA interference (RNAi). Following interference with FoTO6, FoTO24, and their combination, the expression levels of vitellogenin (Vg) were downregulated by 3%-30%, 13%-28%, and 14%-32% from the 2nd day to the 5th day, respectively; Krüppel-homolog 1 (Kr-h1) expression was down-regulated by 3%-65%, 11%-34%, and 11%-39% from the 2nd day to the 5th day, respectively; ovariole length was shortened by approximately 18%, 21%, and 24%, respectively; and the average number of eggs decreased from 407 to 260, 148, and 106, respectively. Additionally, a JH supplementation experiment on the NIL-R population revealed that the expression levels of both FoTO6, FoTO24, Vg and Kr-h1 were significantly upregulated compared with those observed in the Ivf03 population, resulting in increased fecundity. These results suggest that FoTO6 and FoTO24 are involved in JH-mediated regulation of the reproductive fitness cost of resistance to spinosad. Further, FoTO6 and FoTO24 can be considered potential target genes for applying RNAi technology in the scientific management of F. occidentalis.
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Proteínas de Insectos , Resistencia a los Insecticidas , Thysanoptera , Animales , Resistencia a los Insecticidas/genética , Proteínas de Insectos/genética , Proteínas de Insectos/metabolismo , Thysanoptera/genética , Thysanoptera/fisiología , Thysanoptera/efectos de los fármacos , Insecticidas/farmacología , Femenino , Reproducción/genética , Macrólidos/farmacología , Vitelogeninas/genética , Vitelogeninas/metabolismo , Combinación de Medicamentos , Hormonas Juveniles/metabolismo , Hormonas Juveniles/farmacología , Interferencia de ARN , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Aptitud GenéticaRESUMEN
Gene therapy holds promise for treatment of inherited retinal dystrophies, a group of rare genetic disorders characterized by severe loss of vision. Here, we report up to 3-year pre-specified interim safety and efficacy results of an open-label first-in-human dose-escalation phase 1/2 gene therapy clinical trial in 12 patients with retinal dystrophy caused by biallelic mutations in the retinaldehyde-binding protein 1 (RLBP1) gene of the visual cycle. The primary endpoints were systemic and ocular safety and recovery of dark adaptation. Secondary endpoints included microperimetry, visual field sensitivity, dominant eye test and patient-reported outcomes. Subretinal delivery of an adeno-associated viral vector (AAV8-RLBP1) was well tolerated with dose-dependent intraocular inflammation which responded to corticosteroid treatment, and focal atrophy of the retinal pigment epithelium as the dose limiting toxicity. Dark adaptation kinetics, the primary efficacy endpoint, improved significantly in all dose-cohorts. Treatment with AAV8-RLBP1 resulted in the resolution of disease-related retinal deposits, suggestive of successful restoration of the visual cycle. In conclusion, to date, AAV8-RLBP1 has shown preliminary safety and efficacy in patients with RLBP1-associated retinal dystrophy. Trial number: NCT03374657.
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Dependovirus , Terapia Genética , Distrofias Retinianas , Humanos , Terapia Genética/métodos , Distrofias Retinianas/genética , Distrofias Retinianas/terapia , Masculino , Adulto , Femenino , Dependovirus/genética , Proteínas Portadoras/genética , Persona de Mediana Edad , Vectores Genéticos/administración & dosificación , Vectores Genéticos/genética , Adulto Joven , Resultado del Tratamiento , Mutación , AdolescenteRESUMEN
Osteoarthritis (OA) is the most common form of arthritic disease, and phenotypic modification of chondrocytes is an important mechanism that contributes to the loss of cartilage homeostasis. This study identified that Fascin actin-bundling protein 1 (FSCN1) plays a pivotal role in regulating chondrocytes phenotype and maintaining cartilage homeostasis. Proteome-wide screening revealed markedly upregulated FSCN1 protein expression in human OA cartilage. FSCN1 accumulation was confirmed in the superficial layer of OA cartilage from humans and mice, primarily in dedifferentiated-like chondrocytes, associated with enhanced actin stress fiber formation and upregulated type I and III collagens. FSCN1-inducible knockout mice exhibited delayed cartilage degeneration following experimental OA surgery. Mechanistically, FSCN1 promoted actin polymerization and disrupted the inhibition of Decorin on TGF-ß1, leading to excessive TGF-ß1 production and ALK1/Smad1/5 signaling activation, thus, accelerated chondrocyte dedifferentiation. Intra-articular injection of FSCN1-overexpressing adeno-associated virus exacerbated OA progression in mice, which was mitigated by an ALK1 inhibitor. Moreover, FSCN1 inhibitor NP-G2-044 effectively reduced extracellular matrix degradation in OA mice, cultured human OA chondrocytes, and cartilage explants by suppressing ALK1/Smad1/5 signaling. These findings suggest that targeting FSCN1 represents a promising therapeutic approach for OA.
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Proteínas Portadoras , Condrocitos , Proteínas de Microfilamentos , Osteoartritis , Animales , Humanos , Masculino , Ratones , Proteínas Portadoras/metabolismo , Proteínas Portadoras/genética , Cartílago Articular/metabolismo , Cartílago Articular/patología , Condrocitos/metabolismo , Condrocitos/patología , Ratones Endogámicos C57BL , Ratones Noqueados , Proteínas de Microfilamentos/genética , Proteínas de Microfilamentos/metabolismo , Osteoartritis/patología , Osteoartritis/metabolismo , Osteoartritis/genética , Fenotipo , Receptores Odorantes , Transducción de SeñalRESUMEN
BACKGROUND: Spermatogonial stem cells (SSCs) are essential for the maintenance and initiation of male spermatogenesis. Despite the advances in understanding SSC biology in mouse models, the mechanisms underlying human SSC development remain elusive. RESULTS: Here, we analyzed the signaling pathways involved in SSC regulation by testicular somatic cells using single-cell sequencing data (GEO datasets: GSE149512 and GSE112013) and identified that Leydig cells communicate with SSCs through pleiotrophin (PTN) and its receptor syndecan-2 (SDC2). Immunofluorescence, STRING prediction, and protein immunoprecipitation assays confirmed the interaction between PTN and SDC2 in spermatogonia, but their co-localization was observed only in approximately 50% of the cells. The knockdown of SDC2 in human SSC lines impaired cell proliferation, DNA synthesis, and the expression of PLZF, a key marker for SSC self-renewal. Transcriptome analysis revealed that SDC2 knockdown downregulated the expression of GFRA1, a crucial factor for SSC proliferation and self-renewal, and inhibited the HIF-1 signaling pathway. Exogenous PTN rescued the proliferation and GFRA1 expression in SDC2 knockdown SSC lines. In addition, we found downregulation of PTN and SDC2 as well as altered localization in non-obstructive azoospermia (NOA) patients, suggesting that downregulation of PTN and SDC2 may be associated with impaired spermatogenesis. CONCLUSIONS: Our results uncover a novel mechanism of human SSC regulation by the testicular microenvironment and suggest a potential therapeutic target for male infertility.
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Proteínas Portadoras , Proliferación Celular , Citocinas , Receptores del Factor Neurotrófico Derivado de la Línea Celular Glial , Células Intersticiales del Testículo , Sindecano-2 , Masculino , Humanos , Proliferación Celular/fisiología , Células Intersticiales del Testículo/metabolismo , Citocinas/metabolismo , Receptores del Factor Neurotrófico Derivado de la Línea Celular Glial/metabolismo , Receptores del Factor Neurotrófico Derivado de la Línea Celular Glial/genética , Sindecano-2/metabolismo , Sindecano-2/genética , Proteínas Portadoras/metabolismo , Proteínas Portadoras/genética , Supervivencia Celular/fisiología , Espermatogonias/metabolismo , Transducción de Señal/fisiología , Células Madre Germinales Adultas/metabolismo , Células Madre Germinales Adultas/fisiologíaRESUMEN
Gut microbiome dysbiosis has been widely implicated in cognitive impairment, but the identity of the specific bacterial taxa and mechanisms are not fully elucidated. Brain glucose hypometabolism coincides with the cognitive decline. This study explored the link among cognition, gut microbiota and glucose uptake based on the fecal microbiota transplantation from mild cognitive impairment individuals (MCI-FMT) and investigated whether similar mechanisms were involved in 27-hydroxycholesterol (27-OHC)-induced cognitive decline. Our results showed that the MCI-FMT mice exhibited learning and memory decline and morphological lesions in the brain and colon tissues. There were reduced 18F-fluorodeoxyglucose uptake, downregulated expression of glucose transporters (GLUT1,3,4) and upregulated negative regulator of glucose uptake (TXNIP) in the brain. MCI-FMT altered the bacterial composition and diversity of the recipient mice, and the microbial signatures highlighted by the increased abundance of Bacteroides recapitulated the negative effects of MCI bacterial colonization. However, inhibiting Bacteroidetes or TXNIP increased the expression of GLUT1 and GLUT4, significantly improving brain glucose uptake and cognitive performance in 27-OHC-treated mice. Our study verified that cognitive decline and abnormal cerebral glucose uptake were associated with gut microbiota dysbiosis; we also revealed the involvement of Bacteroidetes and molecular mechanisms of TXNIP-related glucose uptake in cognitive deficits caused by 27-OHC.
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Bacteroidetes , Encéfalo , Cognición , Disfunción Cognitiva , Disbiosis , Trasplante de Microbiota Fecal , Microbioma Gastrointestinal , Glucosa , Transducción de Señal , Animales , Disfunción Cognitiva/metabolismo , Disfunción Cognitiva/microbiología , Ratones , Glucosa/metabolismo , Encéfalo/metabolismo , Bacteroidetes/metabolismo , Disbiosis/microbiología , Disbiosis/metabolismo , Masculino , Humanos , Ratones Endogámicos C57BL , Proteínas Portadoras/metabolismo , Proteínas Portadoras/genética , Proteínas Facilitadoras del Transporte de la Glucosa/metabolismo , Proteínas Facilitadoras del Transporte de la Glucosa/genética , TiorredoxinasRESUMEN
Psoriasis is a chronic inflammatory skin condition with numerous causes, including genetic, immunological and infectious factors. The course of psoriasis is long and recurrence is common; pathogenesis is not completely understood. However, there is an association between advancement of psoriasis and aberrant microRNA (miR or miRNA)155 expression. Through bioinformatics, the present study aimed to analyze the differentially expressed genes and miRNAs in psoriasis and its biological mechanism and function psoriatic inflammation. First of all, differentially expressed genes (DEGs) and miRNAs (DEMs) in patients with psoriasis were identified using GEO2R interactive web application. A psoriasis inflammatory model was established using lipopolysaccharide (LPS)treated HaCaT keratinocytes, which were transfected with miR155 mimic or inhibitor. Cell Counting Kit8 was used for the assessment of cell viability and proliferation, and changes in the cell cycle were examined using flow cytometry. ELISA and reverse transcriptionquantitative PCR (RTqPCR) were used to detect the expression levels of the inflammatory factors IL1ß and IL6. The dualluciferase reporter assay was used to verify the targeting association between miR1555p and IFN regulatory factor 2 binding protein 2 (IRF2BP2). To verify the targeting association of miR155 and the IRF2BP2/kruppellike factor 2 (KLF2)/NFκB signaling pathway, expression levels of IRF2BP2, KLF2 and p65 were identified by RTqPCR and western blotting. IRF2BP2 levels were also confirmed by immunofluorescence, in conjunction with bioinformatics database analysis. Overexpression of miR155 inhibited proliferation of HaCaT cells and increased the number of cells in S phase and decreasing number of cells in G1 and G2 phase. In the LPSinduced inflammatory state, miR155 overexpression heightened the inflammatory response of HaCaT cells while inhibition of miR155 lessened it. Suppression of inflammatory cytokine expression by miR1555p inhibitor was reversed by knockdown of IRF2BP2. miR155 was shown to interact with IRF2BP2 to negatively regulate its expression, leading to decreased KLF2 expression and increased p65 expression and secretion of inflammatory factors, intensifying the inflammatory response of HaCaT cells. Therefore, miR155 may contribute to development of psoriasis by inducing tissue and cell damage by increasing the inflammatory response of HaCaT cells via the IRF2BP2/KLF2/NFκB pathway. In conclusion, the results of the present study offer novel perspectives on the role of miR155 in the onset and progression of psoriasis.
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Inflamación , Factores de Transcripción de Tipo Kruppel , MicroARNs , FN-kappa B , Psoriasis , Transducción de Señal , Humanos , MicroARNs/genética , MicroARNs/metabolismo , Factores de Transcripción de Tipo Kruppel/genética , Factores de Transcripción de Tipo Kruppel/metabolismo , Psoriasis/genética , Psoriasis/metabolismo , Psoriasis/patología , FN-kappa B/metabolismo , Transducción de Señal/genética , Inflamación/genética , Inflamación/metabolismo , Inflamación/patología , Células HaCaT , Proliferación Celular/genética , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Regulación de la Expresión Génica , Queratinocitos/metabolismo , Lipopolisacáridos/farmacología , Proteínas de Unión al ADN , Factores de TranscripciónRESUMEN
BACKGROUND: Group A Streptococcus (Strep A) causes both uncomplicated and severe invasive infections, as well as the post-infection complications acute rheumatic fever and rheumatic heart disease. Despite the high global burden of disease resulting from Strep A infections, there is not a licensed vaccine. A 30-valent M protein-based vaccine has previously been shown to be immunogenic in animal models and in a Phase I clinical trial (NCT02564237). Here, we assessed the immunogenicity of a 30-valent messenger (m)RNA vaccine designed to express the same M peptide targets as the 30-valent protein vaccine and compared it with the protein vaccine. METHODS: Female New Zealand white rabbits were immunized with one of four vaccine formulations (3 doses of each formulation at days 1, 28, and 56): soluble mRNA (100 µg/animal), C-terminal transmembrane mRNA (100 µg/animal), protein vaccine (400 µg/animal), or a non-translatable RNA control (100 µg/animal). Serum was collected one day prior to the first dose and on days 42 and 70. Rabbit serum samples were assayed for antibody levels against synthetic M peptides by ELISA. HL-60 opsonophagocytic killing (OPK) assays were performed to assess functional antibody levels. RESULTS: Serum IgG levels were similar for the mRNA and protein vaccines. The CtTM version of the mRNA vaccine elicited slightly higher antibody levels than the mRNA designed to express soluble proteins. OPK activity was similar for the mRNA and protein vaccines, regardless of M type. CONCLUSIONS: The total antibody responses and functional antibody levels elicited by the 30-valent mRNA Strep A vaccines were similar to those observed following immunization with the analogous protein vaccine. The mRNA vaccine platform provides potential advantages to protein-based vaccines including inherent adjuvant activity, increased production efficiency, lower cost, and the potential to rapidly change epitopes/peptides, all of which are important considerations related to multivalent Strep A vaccine development.
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Anticuerpos Antibacterianos , Antígenos Bacterianos , Infecciones Estreptocócicas , Vacunas Estreptocócicas , Streptococcus pyogenes , Animales , Conejos , Femenino , Vacunas Estreptocócicas/inmunología , Vacunas Estreptocócicas/administración & dosificación , Vacunas Estreptocócicas/genética , Anticuerpos Antibacterianos/sangre , Anticuerpos Antibacterianos/inmunología , Infecciones Estreptocócicas/prevención & control , Infecciones Estreptocócicas/inmunología , Antígenos Bacterianos/inmunología , Antígenos Bacterianos/genética , Streptococcus pyogenes/inmunología , Streptococcus pyogenes/genética , Inmunogenicidad Vacunal , Proteínas de la Membrana Bacteriana Externa/inmunología , Proteínas de la Membrana Bacteriana Externa/genética , Proteínas Portadoras/inmunología , Proteínas Portadoras/genética , Vacunas Sintéticas/inmunología , Vacunas Sintéticas/administración & dosificación , ARN Mensajero/genética , ARN Mensajero/inmunologíaRESUMEN
ETHNOPHARMACOLOGICAL RELEVANCE: Sanqi oral solution (SQ) is a traditional Chinese patent medicine, widely used to treat chronic kidney diseases (CKD) in the clinic in China. Previous studies have confirmed its anti-renal fibrosis effect, but the specific pharmacological mechanism is still unclear. AIM OF THE STUDY: Focusing on energy metabolism in fibroblasts, the renoprotective mechanism of SQ was investigated in vitro and in vivo. METHODS: Firstly, the fingerprint of SQ was constructed and its elementary chemical composition was analyzed. In the 5/6Nx rats experiment, the efficacy of SQ on the kidney was evaluated by detecting serum and urine biochemical indexes and pathological staining of renal tissues. Lactic acid and pyruvic acid levels in serum and renal tissues were detected. PCNA protein expression in kidney tissue was detected by immunofluorescence assay and Western blot. Expression levels of HIF-1α, PKM2 and HK2 were determined by immunohistochemistry, Western blot or RT-qPCR assay. In addition, the effect of SQ intervention on cell proliferation and glycolysis was evaluated in TGF-ß1-induced NRK-49F cells, and the role of SQ exposure and HIF-1α/PKM2/glycolysis pathway were further investigated by silencing and overexpressing HIF-1α gene in NRK-49F cells. RESULTS: In 5/6 Nx rats, SQ effectively improved renal function and treated renal injury. It reduced the levels of lactic acid and pyruvic acid in kidney homogenates from CKD rats and decreased the expression levels of HIF-1α, PKM2, HK2, α-SMA, vimentin, collagen I and PCNA in kidney tissues. Similar results were observed in vitro. SQ inhibited NRK-49F cell proliferation, glycolysis and the expression levels of HIF-1α, PKM2 induced by TGF-ß1. Furthermore, we established NRK-49F cells transfected with siRNA or pDNA to silence or overexpress the HIF-1α gene. Overexpression of HIF-1α promoted cellular secretion of lactic acid and pyruvic acid in TGF-ß1-induced NRK-49F cells, however, this change was reversed by intervention with SQ or silencing the HIF-1α gene. Overexpression of HIF-1α can further induce increased PKM2 expression, while SQ intervention can reduce PKM2 expression. Moreover, PKM2 expression was also inhibited after silencing HIF-1α gene, and SQ was not effective even when given. CONCLUSION: The mechanism of action of SQ was explored from the perspective of energy metabolism, and it was found to regulate PKM2-activated glycolysis, inhibit fibroblast activation, and further ameliorate renal fibrosis in CKD by targeting HIF-1α.
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Fibroblastos , Fibrosis , Glucólisis , Subunidad alfa del Factor 1 Inducible por Hipoxia , Riñón , Ratas Sprague-Dawley , Insuficiencia Renal Crónica , Proteínas de Unión a Hormona Tiroide , Animales , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Subunidad alfa del Factor 1 Inducible por Hipoxia/genética , Insuficiencia Renal Crónica/tratamiento farmacológico , Insuficiencia Renal Crónica/metabolismo , Fibrosis/tratamiento farmacológico , Fibroblastos/efectos de los fármacos , Fibroblastos/metabolismo , Masculino , Glucólisis/efectos de los fármacos , Ratas , Riñón/efectos de los fármacos , Riñón/patología , Riñón/metabolismo , Medicamentos Herbarios Chinos/farmacología , Línea Celular , Piruvato Quinasa/metabolismo , Piruvato Quinasa/genética , Proteínas de la Membrana/metabolismo , Proteínas de la Membrana/genética , Hormonas Tiroideas , Administración Oral , Proliferación Celular/efectos de los fármacos , Transducción de Señal/efectos de los fármacos , Proteínas Portadoras/metabolismo , Proteínas Portadoras/genética , Factor de Crecimiento Transformador beta1/metabolismo , Factor de Crecimiento Transformador beta1/genéticaRESUMEN
Bone homeostasis relies on the delicate balance between osteoblast-mediated bone formation and osteoclast-mediated bone resorption. The casein kinase 2 interacting protein-1 (CKIP-1), a specific CK2α subunit-interacting protein, has been documented as one of the crucial negative regulators of bone formation. CKIP-1 siRNA therapy has constraints that limit its use in clinical applications. Therefore, it is necessary to explore effective targeting strategies for CKIP-1. In this study, we observed an upregulation of CKIP-1 protein expression in the microgravity environment, while its ubiquitination levels decreased. We further investigated the interaction between CKIP-1 and VHL and found that VHL enhanced CKIP-1 degradation through the ubiquitylation-proteasome system (UPS). Additionally, we discovered a small molecule ligand, named C77, through DNA-encoded library (DEL) screening, which binds to CKIP-1 both in vivo and in vitro, as confirmed by Surface Plasmon Resonance (SPR) and the Cellular Thermal shift assay (CETSA), respectively. Our findings demonstrated the potential of VHL and C77 as guiding factors in the development of CKIP-1-based Proteolysis-Targeting Chimeras (PROTACs), which could be future therapeutic interventions in disuse osteoporosis.
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Osteoporosis , Proteína Supresora de Tumores del Síndrome de Von Hippel-Lindau , Humanos , Ligandos , Osteoporosis/metabolismo , Osteoporosis/tratamiento farmacológico , Osteoporosis/terapia , Proteína Supresora de Tumores del Síndrome de Von Hippel-Lindau/metabolismo , Proteína Supresora de Tumores del Síndrome de Von Hippel-Lindau/genética , Ubiquitinación , Proteínas Portadoras/metabolismo , Proteínas Portadoras/genética , Proteolisis , Animales , Complejo de la Endopetidasa Proteasomal/metabolismo , Unión Proteica , Ratones , Péptidos y Proteínas de Señalización IntracelularRESUMEN
The TRESK (K2P18.1, KCNK18) background potassium channel is expressed in primary sensory neurons and has been reported to contribute to the regulation of pain sensations. In the present study, we examined the interaction of TRESK with NDFIP1 (Nedd4 family-interacting protein 1) in the Xenopus oocyte expression system by two-electrode voltage clamp and biochemical methods. We showed that the coexpression of NDFIP1 abolished the TRESK current under the condition where the other K+ channels were not affected. Mutations in the three PPxY motifs of NDFIP1, which are responsible for the interaction with the Nedd4 ubiquitin ligase, prevented a reduction in the TRESK current. Furthermore, the overexpression of a dominant-negative Nedd4 construct in the oocytes coexpressing TRESK with NDFIP1 partially reversed the down-modulating effect of the adaptor protein on the K+ current. The biochemical data were also consistent with the functional results. An interaction between epitope-tagged versions of TRESK and NDFIP1 was verified by co-immunoprecipitation experiments. The coexpression of NDFIP1 with TRESK induced the ubiquitination of the channel protein. Altogether, the results suggest that TRESK is directly controlled by and highly sensitive to the activation of the NDFIP1-Nedd4 system. The NDFIP1-mediated reduction in the TRESK component may induce depolarization, increase excitability, and attenuate the calcium dependence of the membrane potential by reducing the calcineurin-activated fraction in the ensemble background K+ current.
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Proteínas Portadoras , Oocitos , Canales de Potasio , Ubiquitinación , Animales , Canales de Potasio/metabolismo , Canales de Potasio/genética , Oocitos/metabolismo , Proteínas Portadoras/metabolismo , Proteínas Portadoras/genética , Humanos , Proteínas de la Membrana/metabolismo , Proteínas de la Membrana/genética , Xenopus laevis , Ubiquitina-Proteína Ligasas Nedd4/metabolismo , Ubiquitina-Proteína Ligasas Nedd4/genética , Ubiquitina-Proteína Ligasas/metabolismo , Ubiquitina-Proteína Ligasas/genética , Unión Proteica , Potasio/metabolismo , Proteínas de XenopusRESUMEN
Multiple myeloma is the second most hematological cancer. RUVBL1 and RUVBL2 form a subcomplex of many chromatin remodeling complexes implicated in cancer progression. As an inhibitor specific to the RUVBL1/2 complex, CB-6644 exhibits remarkable anti-tumor activity in xenograft models of Burkitt's lymphoma and multiple myeloma (MM). In this work, we defined transcriptional signatures corresponding to CB-6644 treatment in MM cells and determined underlying epigenetic changes in terms of chromatin accessibility. CB-6644 upregulated biological processes related to interferon response and downregulated those linked to cell proliferation in MM cells. Transcriptional regulator inference identified E2Fs as regulators for downregulated genes and MED1 and MYC as regulators for upregulated genes. CB-6644-induced changes in chromatin accessibility occurred mostly in non-promoter regions. Footprinting analysis identified transcription factors implied in modulating chromatin accessibility in response to CB-6644 treatment, including ATF4/CEBP and IRF4. Lastly, integrative analysis of transcription responses to various chemical compounds of the molecular signature genes from public gene expression data identified CB-5083, a p97 inhibitor, as a synergistic candidate with CB-6644 in MM cells, but experimental validation refuted this hypothesis.
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ATPasas Asociadas con Actividades Celulares Diversas , ADN Helicasas , Regulación Neoplásica de la Expresión Génica , Mieloma Múltiple , Humanos , Mieloma Múltiple/genética , Mieloma Múltiple/tratamiento farmacológico , Mieloma Múltiple/metabolismo , Mieloma Múltiple/patología , ADN Helicasas/genética , ADN Helicasas/metabolismo , Línea Celular Tumoral , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , ATPasas Asociadas con Actividades Celulares Diversas/genética , ATPasas Asociadas con Actividades Celulares Diversas/metabolismo , ATPasas Asociadas con Actividades Celulares Diversas/antagonistas & inhibidores , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Proliferación Celular/efectos de los fármacos , Ensamble y Desensamble de Cromatina/efectos de los fármacos , Epigénesis Genética/efectos de los fármacos , Factores de Transcripción/metabolismo , Factores de Transcripción/genética , Factores de Transcripción/antagonistas & inhibidores , Antineoplásicos/farmacología , Antineoplásicos/uso terapéuticoRESUMEN
SGIP1 encodes a protein Src homology 3-domain growth factor receptor-bound 2-like endophilin interacting protein 1. It is involved in the regulation of clathrin-mediated endocytosis along with having a role in energy homeostasis in neuronal systems. We generated an isogenic human induced pluripotent stem cell (iPSC) line with a biallelic frameshift variant in SGIP1. This exon has been shown to be subject to alternative splicing, leading to an isoform lacking 24 amino acids that are present in the longest SGIP isoform. The newly generated iPSC line will be helpful to dissect the differential properties of the two SGIP isoforms.
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Sistemas CRISPR-Cas , Exones , Mutación del Sistema de Lectura , Células Madre Pluripotentes Inducidas , Células Madre Pluripotentes Inducidas/metabolismo , Humanos , Línea Celular , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Diferenciación CelularRESUMEN
BACKGROUND: Hermansky-Pudlak Syndrome (HPS), a rare autosomal recessive disorder, is characterized by oculocutaneous albinism, bleeding diathesis, and sometimes severe lung problems and inflammatory bowel disease. Symptoms include skin and hair pigmentation variations, along with visual impairments. Variants in eleven genes encoding protein complexes essential for membrane trafficking and intracellular endosomal transport pathways underlie various recognized HPS subtypes. This study focuses on HPS-9, a subtype of Hermansky-Pudlak Syndrome caused by a variant in the BLOC1S6 gene, which is a subunit of the BLOC1 complex. In this study, a novel Copy Number Variation (CNV) in the aforementioned gene in an Iranian family is reported. The study aims to better understand the etiology of HPS-9 symptoms by identifying and confirming the variant and determining whether the gene is expressed despite the deletion. There have only been five reports of this syndrome in the literature thus far. Our novel CNV represents a significant contribution to understanding the genetic basis of HPS-9. RESULTS: This study investigates a male patient presenting with albinism. Whole Exome Sequencing (WES) identified a homozygous deletion of approximately 350 bp using CNV analysis. The deletion affects the intronic region of the BLOC1S6 gene, causing uncertainties in defining the exact boundaries due to WES limitations. Primer walking and GAP-PCR techniques were used to define the deletion boundaries. Subsequent assessments of this variant across other family members helped identify homozygous affected members and heterozygous carriers. The absence of BLOC1S6 expression in the affected individual was confirmed through Real-time PCR experiments. These findings underscore the importance of understanding the implications for the patient's healthcare and potential therapeutic approaches. CONCLUSION: This study introduces a case of Hermansky-Pudlak Syndrome Type 9 (HPS-9) caused by a homozygous deletion in the BLOC1S6 gene. We identified an approximately 7-kb deletion encompassing exon 1 and the intronic region of the gene. The absence of BLOC1S6 expression, confirmed via Real-time PCR, highlights the importance of studying the pathogenicity of the deletion and its impact on the patient's health. Our findings contribute to the sparse knowledge on HPS-9 and underscore the need for further exploration into the genetic causes of this rare disorder.
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Proteínas Portadoras , Variaciones en el Número de Copia de ADN , Síndrome de Hermanski-Pudlak , Lectinas , Femenino , Humanos , Masculino , Secuenciación del Exoma , Síndrome de Hermanski-Pudlak/genética , Irán , Linaje , Eliminación de Secuencia , Proteínas Portadoras/genética , Lectinas/genéticaRESUMEN
Liquid-liquid phase separation (LLPS) mediated by G3BP1/2 proteins and non-translating mRNAs mediates stress granule (SG) assembly. We investigated the phylogenetic evolution of G3BP orthologs from unicellular yeast to mammals and identified both conserved and divergent features. The modular domain organization of G3BP orthologs is generally conserved. However, invertebrate orthologs displayed reduced capacity for SG assembly in human cells compared to vertebrate orthologs. We demonstrated that the protein-interaction network facilitated by the NTF2L domain is a crucial determinant of this specificity. The evolution of the G3BP1 network coincided with its exploitation by certain viruses, as evident from the interaction between viral proteins and G3BP orthologs in insects and vertebrates. We revealed the importance and divergence of the G3BP interaction network in human SG formation. Leveraging this network, we established a 7-component in vitro SG reconstitution system for quantitative studies. These findings highlight the significance of G3BP network divergence in the evolution of biological processes.
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ADN Helicasas , Proteínas de Unión a Poli-ADP-Ribosa , Mapas de Interacción de Proteínas , ARN Helicasas , Proteínas con Motivos de Reconocimiento de ARN , Gránulos de Estrés , Humanos , Proteínas con Motivos de Reconocimiento de ARN/metabolismo , Proteínas con Motivos de Reconocimiento de ARN/genética , Proteínas de Unión a Poli-ADP-Ribosa/metabolismo , Proteínas de Unión a Poli-ADP-Ribosa/genética , ARN Helicasas/metabolismo , ARN Helicasas/genética , Gránulos de Estrés/metabolismo , Animales , ADN Helicasas/metabolismo , ADN Helicasas/genética , Filogenia , Células HeLa , Proteínas Portadoras/metabolismo , Proteínas Portadoras/genética , Proteínas de Unión al ARN , Proteínas Adaptadoras Transductoras de SeñalesRESUMEN
Hypertrophic cardiomyopathy (HCM) is a genetic disease characterized by unexplained left ventricular hypertrophy (LVH), diastolic dysfunction, and increased sudden-death risk. Early detection of the phenotypic expression of the disease in genetic carriers without LVH (Gen+/Phen-) is crucial for emerging therapies. This clinical study aims to identify echocardiographic predictors of phenotypic development in Gen+/Phen-. Sixteen Gen+/Phen- (one subject with troponin T, six with myosin heavy chain-7, and nine with myosin-binding protein C3 mutations), represented the study population. At first and last visit we performed comprehensive 2D speckle-tracking strain echocardiography. During a follow-up of 8 ± 5 years, five carriers developed LVH (LVH+). At baseline, these patients were older than those who did not develop LVH (LVH-) (30 ± 8 vs. 15 ± 8 years, p = 0.005). LVH+ had reduced peak global strain rate during the isovolumic relaxation period (SRIVR) (0.28 ± 0.05 vs. 0.40 ± 0.11 1/s, p = 0.048) and lower global longitudinal strain (GLS) (-19.8 ± 0.4 vs. -22.3 ± 1.1%; p < 0.0001) than LVH- at baseline. SRIVR and GLS were not correlated with age (overall, p > 0.08). This is the first HCM study investigating subjects before they manifest clinically significant or relevant disease burden or symptomatology, comparing at baseline HCM Gen+/Phen- subjects who will develop LVH with those who will not. Furthermore, we identified highly sensitive, easily obtainable, age- and load-independent echocardiographic predictors of phenotype development in HCM gene carriers who may undergo early preventive treatment.
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Cardiomiopatía Hipertrófica , Ecocardiografía , Hipertrofia Ventricular Izquierda , Mutación , Humanos , Masculino , Femenino , Ecocardiografía/métodos , Hipertrofia Ventricular Izquierda/genética , Hipertrofia Ventricular Izquierda/diagnóstico por imagen , Hipertrofia Ventricular Izquierda/etiología , Adulto , Cardiomiopatía Hipertrófica/genética , Cardiomiopatía Hipertrófica/diagnóstico por imagen , Persona de Mediana Edad , Adolescente , Cadenas Pesadas de Miosina/genética , Troponina T/genética , Heterocigoto , Proteínas Portadoras/genética , Adulto Joven , Fenotipo , Miosinas Cardíacas/genéticaRESUMEN
Defects in organellar acidification indicate compromised or infected compartments. Recruitment of the autophagy-related ATG16L1 complex to pathologically neutralized organelles targets ubiquitin-like ATG8 molecules to perturbed membranes. How this process is coupled to proton gradient disruption is unclear. Here, we reveal that the V1H subunit of the vacuolar ATPase (V-ATPase) proton pump binds directly to ATG16L1. The V1H/ATG16L1 interaction only occurs within fully assembled V-ATPases, allowing ATG16L1 recruitment to be coupled to increased V-ATPase assembly following organelle neutralization. Cells lacking V1H fail to target ATG8s during influenza infection or after activation of the immune receptor stimulator of interferon genes (STING). We identify a loop within V1H that mediates ATG16L1 binding. A neuronal V1H isoform lacks this loop and is associated with attenuated ATG8 targeting in response to ionophores in primary murine and human iPSC-derived neurons. Thus, V1H controls ATG16L1 recruitment following proton gradient dissipation, suggesting that the V-ATPase acts as a cell-intrinsic damage sensor.