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1.
Dev Psychobiol ; 66(6): e22523, 2024 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-38970242

RESUMO

The current literature suggests that relaxin-3/relaxin/insulin-like family peptide receptor 3 (RLN-3/RXFP-3) system is involved in the pathophysiology of affective disorders because the results of anatomical and pharmacological studies have shown that the RLN-3 signaling pathway plays a role in modulating the stress response, anxiety, arousal, depression-like behavior, and neuroendocrine homeostasis. The risk of developing mental illnesses in adulthood is increased by exposure to stress in early periods of life. The available data indicate that puberty is especially characterized by the development of the neural system and emotionality and is a "stress-sensitive" period. The presented study assessed the short-term changes in the expression of RLN-3 and RXFP-3 mRNA in the stress-dependent brain regions in male pubertal Wistar rats that had been subjected to acute stress. Three stressors were applied from 42 to 44 postnatal days (first day: a single forced swim; second day: stress on an elevated platform that was repeated three times; third day: restraint stress three times). Anxiety (open field, elevated plus maze test) and anhedonic-like behavior (sucrose preference test) were estimated during these tests. The corticosterone (CORT) levels and blood morphology were estimated. We found that the RXFP-3 mRNA expression decreased in the brainstem, whereas it increased in the hypothalamus 72 h after acute stress. These molecular changes were accompanied by the increased levels of CORT and anxiety-like behavior detected in the open field test that had been conducted earlier, that is, 24 h after the stress procedure. These findings shed new light on the neurochemical changes that are involved in the compensatory response to adverse events in pubertal male rats and support other data that suggest a regulatory interplay between the RLN-3 pathway and the hypothalamus-pituitary-adrenal axis activity in the mechanisms of anxiety-like behavior.


Assuntos
Ansiedade , Encéfalo , RNA Mensageiro , Ratos Wistar , Receptores Acoplados a Proteínas G , Estresse Psicológico , Animais , Masculino , Ratos , Estresse Psicológico/metabolismo , Estresse Psicológico/fisiopatologia , Ansiedade/metabolismo , Ansiedade/fisiopatologia , Receptores Acoplados a Proteínas G/metabolismo , Receptores Acoplados a Proteínas G/genética , Encéfalo/metabolismo , RNA Mensageiro/metabolismo , Comportamento Animal/fisiologia , Relaxina/metabolismo , Relaxina/genética , Receptores de Peptídeos/metabolismo , Receptores de Peptídeos/genética , Maturidade Sexual/fisiologia , Proteínas do Tecido Nervoso
2.
Hereditas ; 161(1): 20, 2024 Jul 02.
Artigo em Inglês | MEDLINE | ID: mdl-38956710

RESUMO

BACKGROUND: Nasopharyngeal carcinoma (NPC) is a malignant epithelial tumor of the nasopharyngeal mucosa with a high incidence rate all over the world. Methyltransferase-like 14 (METTL14) is a major RNA N6-adenosine methyltransferase implicated in tumor progression by regulating RNA function. This study is designed to explore the biological function and mechanism of METTL14 in NPC. METHODS: METTL14 and Amine oxidase copper containing 1 (AOC1) expression were detected by real-time quantitative polymerase chain reaction (RT-qPCR). The protein levels of METTL14, AOC1, Cyclin D1, B-cell lymphoma-2 (Bcl-2), and N-cadherin were measured using western blot. Cell proliferation, cycle progression, apoptosis, migration, and invasion were assessed using 5-ethynyl-2'-deoxyuridine (EdU), Colony formation, flow cytometry, wound scratch, and transwell assays. The interaction between METTL14 and AOC1 was verified using RNA immunoprecipitation (RIP), methylated RNA immunoprecipitation (MeRIP), and dual-luciferase reporter assays. The biological role of METTL14 on NPC tumor growth was examined by the xenograft tumor model in vivo. RESULTS: METTL14 and AOC1 were highly expressed in NPC tissues and cells. Moreover, METTL14 knockdown might block NPC cell proliferation, migration, invasion, and induce cell apoptosis in vitro. In mechanism, METTL14 might enhance the stability of AOC1 mRNA via m6A methylation. METTL14 silencing might repress NPC tumor growth in vivo. CONCLUSION: METTL14 might boosted the development of NPC cells partly by regulating the stability of AOC1 mRNA, which provided a promising therapeutic target for NPC treatment.


Assuntos
Proliferação de Células , Regulação Neoplásica da Expressão Gênica , Metiltransferases , Carcinoma Nasofaríngeo , Neoplasias Nasofaríngeas , Estabilidade de RNA , RNA Mensageiro , Humanos , Carcinoma Nasofaríngeo/genética , Carcinoma Nasofaríngeo/metabolismo , Metiltransferases/genética , Metiltransferases/metabolismo , Linhagem Celular Tumoral , Animais , Neoplasias Nasofaríngeas/genética , Neoplasias Nasofaríngeas/metabolismo , RNA Mensageiro/genética , Apoptose/genética , Camundongos , Movimento Celular , Progressão da Doença , Masculino , Feminino
3.
Sci Rep ; 14(1): 15313, 2024 07 03.
Artigo em Inglês | MEDLINE | ID: mdl-38961125

RESUMO

Epileptogenesis is the process by which a normal brain becomes hyperexcitable and capable of generating spontaneous recurrent seizures. The extensive dysregulation of gene expression associated with epileptogenesis is shaped, in part, by microRNAs (miRNAs) - short, non-coding RNAs that negatively regulate protein levels. Functional miRNA-mediated regulation can, however, be difficult to elucidate due to the complexity of miRNA-mRNA interactions. Here, we integrated miRNA and mRNA expression profiles sampled over multiple time-points during and after epileptogenesis in rats, and applied bi-clustering and Bayesian modelling to construct temporal miRNA-mRNA-mRNA interaction networks. Network analysis and enrichment of network inference with sequence- and human disease-specific information identified key regulatory miRNAs with the strongest influence on the mRNA landscape, and miRNA-mRNA interactions closely associated with epileptogenesis and subsequent epilepsy. Our findings underscore the complexity of miRNA-mRNA regulation, can be used to prioritise miRNA targets in specific systems, and offer insights into key regulatory processes in epileptogenesis with therapeutic potential for further investigation.


Assuntos
Epilepsia , Perfilação da Expressão Gênica , Redes Reguladoras de Genes , MicroRNAs , RNA Mensageiro , Convulsões , Animais , MicroRNAs/genética , MicroRNAs/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ratos , Convulsões/genética , Convulsões/metabolismo , Epilepsia/genética , Epilepsia/metabolismo , Masculino , Regulação da Expressão Gênica , Teorema de Bayes , Modelos Animais de Doenças , Transcriptoma
4.
Sci Rep ; 14(1): 15304, 2024 07 03.
Artigo em Inglês | MEDLINE | ID: mdl-38961188

RESUMO

Inflammation, corticosteroids, and loading all affect tendon healing, with an interaction between them. However, underlying mechanisms behind the effect of corticosteroids and the interaction with loading remain unclear. The aim of this study was to investigate the role of dexamethasone during tendon healing, including specific effects on tendon cells. Rats (n = 36) were randomized to heavy loading or mild loading, the Achilles tendon was transected, and animals were treated with dexamethasone or saline. Gene and protein analyses of the healing tendon were performed for extracellular matrix-, inflammation-, and tendon cell markers. We further tested specific effects of dexamethasone on tendon cells in vitro. Dexamethasone increased mRNA levels of S100A4 and decreased levels of ACTA2/α-SMA, irrespective of load level. Heavy loading + dexamethasone reduced mRNA levels of FN1 and TenC (p < 0.05), while resolution-related genes were unaltered (p > 0.05). In contrast, mild loading + dexamethasone increased mRNA levels of resolution-related genes ANXA1, MRC1, PDPN, and PTGES (p < 0.03). Altered protein levels were confirmed in tendons with mild loading. Dexamethasone treatment in vitro prevented tendon construct formation, increased mRNA levels of S100A4 and decreased levels of SCX and collagens. Dexamethasone during tendon healing appears to act through immunomodulation by promoting resolution, but also through an effect on tendon cells.


Assuntos
Tendão do Calcâneo , Dexametasona , Traumatismos dos Tendões , Cicatrização , Dexametasona/farmacologia , Animais , Ratos , Cicatrização/efeitos dos fármacos , Traumatismos dos Tendões/tratamento farmacológico , Traumatismos dos Tendões/metabolismo , Tendão do Calcâneo/efeitos dos fármacos , Tendão do Calcâneo/metabolismo , Tendão do Calcâneo/lesões , Tendão do Calcâneo/patologia , Proteína A4 de Ligação a Cálcio da Família S100/metabolismo , Proteína A4 de Ligação a Cálcio da Família S100/genética , Masculino , Anexina A1/metabolismo , Anexina A1/genética , Actinas/metabolismo , Actinas/genética , Colágeno/metabolismo , Ratos Sprague-Dawley , Tendões/efeitos dos fármacos , Tendões/metabolismo , Matriz Extracelular/metabolismo , Matriz Extracelular/efeitos dos fármacos , RNA Mensageiro/metabolismo , RNA Mensageiro/genética , Fatores de Transcrição Hélice-Alça-Hélice Básicos
5.
Front Endocrinol (Lausanne) ; 15: 1385079, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38948517

RESUMO

Background: 177Lu-oxodotreotide peptide receptor therapy (LuPRRT) is an efficient treatment for midgut neuroendocrine tumors (NETs) of variable radiological response. Several clinical, biological, and imaging parameters may be used to establish a relative disease prognosis but none is able to predict early efficacy or toxicities. We investigated expression levels for mRNA and miRNA involved in radiosensitivity and tumor progression searching for correlations related to patient outcome during LuPRRT therapy. Methods: Thirty-five patients received LuPRRT for G1/G2 midgut NETs between May 2019 and September 2021. Peripheral blood samples were collected prior to irradiation, before and 48 h after the second and the fourth LuPRRT, and at 6-month follow-up. Multiple regression analyses and Pearson correlations were performed to identify the miRNA/mRNA signature that will best predict response to LuPRRT. Results: Focusing on four mRNAs and three miRNAs, we identified a miRNA/mRNA signature enabling the early identification of responders to LuPRRT with significant reduced miRNA/mRNA expression after the first LuPRRT administration for patients with progressive disease at 1 year (p < 0.001). The relevance of this signature was reinforced by studying its evolution up to 6 months post-LuPRRT. Moreover, nadir absolute lymphocyte count within the first 2 months after the first LuPRRT administration was significantly related to low miRNA/mRNA expression level (p < 0.05) for patients with progressive disease. Conclusion: We present a pilot study exploring a miRNA/mRNA signature that correlates with early hematologic toxicity and therapeutic response 12 months following LuPRRT. This signature will be tested prospectively in a larger series of patients.


Assuntos
Neoplasias Intestinais , MicroRNAs , Tumores Neuroendócrinos , RNA Mensageiro , Humanos , Tumores Neuroendócrinos/genética , Tumores Neuroendócrinos/sangue , Tumores Neuroendócrinos/terapia , Tumores Neuroendócrinos/radioterapia , Tumores Neuroendócrinos/patologia , Masculino , Feminino , MicroRNAs/sangue , MicroRNAs/genética , Pessoa de Meia-Idade , Neoplasias Intestinais/sangue , Neoplasias Intestinais/patologia , Neoplasias Intestinais/genética , Neoplasias Intestinais/tratamento farmacológico , RNA Mensageiro/genética , RNA Mensageiro/sangue , Idoso , Seguimentos , Adulto , Prognóstico , Biomarcadores Tumorais/sangue , Biomarcadores Tumorais/genética , Somatostatina/análogos & derivados , Somatostatina/uso terapêutico , Receptores de Peptídeos/genética , Compostos Radiofarmacêuticos/uso terapêutico , Compostos Radiofarmacêuticos/administração & dosagem , Lutécio , Radioisótopos
6.
Oncoimmunology ; 13(1): 2373526, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38948931

RESUMO

Prostate cancer (PCa) is characterized as a "cold tumor" with limited immune responses, rendering the tumor resistant to immune checkpoint inhibitors (ICI). Therapeutic messenger RNA (mRNA) vaccines have emerged as a promising strategy to overcome this challenge by enhancing immune reactivity and significantly boosting anti-tumor efficacy. In our study, we synthesized Tetra, an mRNA vaccine mixed with multiple tumor-associated antigens, and ImmunER, an immune-enhancing adjuvant, aiming to induce potent anti-tumor immunity. ImmunER exhibited the capacity to promote dendritic cells (DCs) maturation, enhance DCs migration, and improve antigen presentation at both cellular and animal levels. Moreover, Tetra, in combination with ImmunER, induced a transformation of bone marrow-derived dendritic cells (BMDCs) to cDC1-CCL22 and up-regulated the JAK-STAT1 pathway, promoting the release of IL-12, TNF-α, and other cytokines. This cascade led to enhanced proliferation and activation of T cells, resulting in effective killing of tumor cells. In vivo experiments further revealed that Tetra + ImmunER increased CD8+T cell infiltration and activation in RM-1-PSMA tumor tissues. In summary, our findings underscore the promising potential of the integrated Tetra and ImmunER mRNA-LNP therapy for robust anti-tumor immunity in PCa.


Assuntos
Adjuvantes Imunológicos , Antígenos de Neoplasias , Vacinas Anticâncer , Células Dendríticas , Neoplasias da Próstata , RNA Mensageiro , Animais , Masculino , Neoplasias da Próstata/imunologia , Neoplasias da Próstata/terapia , Neoplasias da Próstata/patologia , Neoplasias da Próstata/genética , Neoplasias da Próstata/tratamento farmacológico , Antígenos de Neoplasias/imunologia , Camundongos , Células Dendríticas/imunologia , Adjuvantes Imunológicos/farmacologia , Adjuvantes Imunológicos/administração & dosagem , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA Mensageiro/administração & dosagem , Vacinas Anticâncer/administração & dosagem , Vacinas Anticâncer/imunologia , Humanos , Camundongos Endogâmicos C57BL , Linhagem Celular Tumoral , Vacinas de mRNA , Linfócitos T CD8-Positivos/imunologia , Linfócitos T/imunologia , Linfócitos T/metabolismo , Imunoterapia/métodos , Ativação Linfocitária/efeitos dos fármacos
7.
BMC Vet Res ; 20(1): 286, 2024 Jul 03.
Artigo em Inglês | MEDLINE | ID: mdl-38961471

RESUMO

BACKGROUND: The milk's nutritional value is determined by its constituents, including fat, protein, carbohydrates, and minerals. The mammary gland's ability to produce milk is controlled by a complex network of genes. Thereby, the fat, protein, and lactose synthesis must be boost in milk to increase milk production efficiency. This can be accomplished by fusing genetic advancements with proper management practices. Therefore, this study aimed to investigate the association between the Lipoprotein lipase (LPL), kappa casein CSN3, and Glucose transporter 1 (GLUT1) genes expression levels and such milk components as fat, protein, and lactose in different dairy breeds during different stages of lactation. METHODS: To achieve such a purpose, 94 milk samples were collected (72 samples from 36 multiparous black-white and red-white Holstein-Friesian (HF) cows and 22 milk samples from 11 Egyptian buffaloes) during the early and peak lactation stages. The milk samples were utilized for milk analysis and genes expressions analyses using non- invasive approach in obtaining milk fat globules (MFGs) as a source of Ribonucleic acid (RNA). RESULTS: LPL and CSN3 genes expressions levels were found to be significantly higher in Egyptian buffalo than Holstein-Friesian (HF) cows as well as fat and protein percentages. On the other hand, GLUT1 gene expression level was shown to be significantly higher during peak lactation than early lactation. Moreover, lactose % showed a significant difference in peak lactation phase compared to early lactation phase. Also, fat and protein percentages were significantly higher in early lactation period than peak lactation period but lactose% showed the opposite pattern of Egyptian buffalo. CONCLUSION: Total RNA can be successfully obtained from MFGs. The results suggest that these genes play a role in glucose absorption and lactose synthesis in bovine mammary epithelial cells during lactation. Also, these results provide light on the differential expression of these genes among distinct Holstein-Friesian cow breeds and Egyptian buffalo subspecies throughout various lactation phases.


Assuntos
Caseínas , Glicolipídeos , Glicoproteínas , Lactação , Gotículas Lipídicas , Glândulas Mamárias Animais , Leite , RNA Mensageiro , Animais , Bovinos/genética , Lactação/genética , Feminino , Gotículas Lipídicas/metabolismo , Leite/química , Leite/metabolismo , Glicolipídeos/metabolismo , Caseínas/genética , Caseínas/metabolismo , Glicoproteínas/genética , Glicoproteínas/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Glândulas Mamárias Animais/metabolismo , Lipase Lipoproteica/genética , Lipase Lipoproteica/metabolismo , Transportador de Glucose Tipo 1/genética , Transportador de Glucose Tipo 1/metabolismo , Búfalos/genética , Búfalos/metabolismo , Lactose/metabolismo , Lactose/análise , Proteínas do Leite/análise , Proteínas do Leite/metabolismo , Proteínas do Leite/genética , Regulação da Expressão Gênica
8.
J Extracell Vesicles ; 13(7): e12464, 2024 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-38961538

RESUMO

MPS IIIC is a lysosomal storage disease caused by mutations in heparan-α-glucosaminide N-acetyltransferase (HGSNAT), for which no treatment is available. Because HGSNAT is a trans-lysosomal-membrane protein, gene therapy for MPS IIIC needs to transduce as many cells as possible for maximal benefits. All cells continuously release extracellular vesicles (EVs) and communicate by exchanging biomolecules via EV trafficking. To address the unmet need, we developed a rAAV-hHGSNATEV vector with an EV-mRNA-packaging signal in the 3'UTR to facilitate bystander effects, and tested it in an in vitro MPS IIIC model. In human MPS IIIC cells, rAAV-hHGSNATEV enhanced HGSNAT mRNA and protein expression, EV-hHGSNAT-mRNA packaging, and cleared GAG storage. Importantly, incubation with EVs led to hHGSNAT protein expression and GAG contents clearance in recipient MPS IIIC cells. Further, rAAV-hHGSNATEV transduction led to the reduction of pathological EVs in MPS IIIC cells to normal levels, suggesting broader therapeutic benefits. These data demonstrate that incorporating the EV-mRNA-packaging signal into a rAAV-hHGSNAT vector enhances EV packaging of hHGSNAT-mRNA, which can be transported to non-transduced cells and translated into functional rHGSNAT protein, facilitating cross-correction of disease pathology. This study supports the therapeutic potential of rAAVEV for MPS IIIC, and broad diseases, without having to transduce every cell.


Assuntos
Efeito Espectador , Dependovirus , Vesículas Extracelulares , Terapia Genética , RNA Mensageiro , Humanos , Terapia Genética/métodos , Dependovirus/genética , RNA Mensageiro/metabolismo , RNA Mensageiro/genética , Vesículas Extracelulares/metabolismo , Mucopolissacaridose III/terapia , Mucopolissacaridose III/metabolismo , Mucopolissacaridose III/genética , Vetores Genéticos , Acetiltransferases/metabolismo , Acetiltransferases/genética
9.
Med Sci (Paris) ; 40(6-7): 525-533, 2024.
Artigo em Francês | MEDLINE | ID: mdl-38986097

RESUMO

Many diseases originate from either the absence or defective expression of a given protein. For some of them, the lacking protein is secreted or can be taken up by cells when delivered exogenously. In such cases, therapies initially involved administering the physiological protein extracted from human tissues. Subsequently, genetic engineering enabled the production of proteins through cell fermentation after introducing the corresponding gene. For many other pathologies, the deficient protein cannot be delivered exogenously. Thus, an endogenous production of the therapeutic protein by the cells themselves is necessary. Messenger RNA (mRNA) technology, like its predecessor DNA, aims to supplement the genetic information needed to produce the therapeutic protein within the cells. However, unlike DNA-based therapies, mRNA transfer allows for transient expression of the protein of interest, which offers an advantage in numerous pathologies. Nonetheless, mastering the quantity, quality, and spatio-temporal regulation of protein production encoded by therapeutic mRNA remains a significant challenge for the development of this approach.


Title: « ReNAissance ¼1 des biothérapies par ARN. Abstract: Nombre de maladies ont pour origine une absence d'expression ou une expression défectueuse d'une protéine donnée. Pour certaines d'entre elles, la protéine faisant défaut est circulante et peut être captée par les cellules lorsqu'elle est délivrée de façon exogène. Dans ce cas, les thérapies ont d'abord consisté en l'administration de la protéine thérapeutique extraite de tissus humains. Par la suite, le génie génétique a permis la production des protéines par fermentation de cellules après y avoir introduit le gène correspondant. Pour beaucoup d'autres maladies, la protéine faisant défaut ne peut être délivrée de façon exogène. Une production endogène de la protéine thérapeutique, par les cellules elles-mêmes est donc nécessaire. La technologie de l'ARN messager (ARNm), comme celle la précédant de l'ADN, se propose de supplémenter, au cœur des cellules, l'information génétique nécessaire pour produire elles-mêmes la protéine thérapeutique. Cependant, contrairement aux thérapies utilisant l'ADN, le transfert d'ARNm permet une expression transitoire de la protéine d'intérêt ce qui constitue un avantage dans nombre de maladies. La maîtrise de la quantité, de la qualité et de la régulation spatio-temporelle de la production d'une protéine codée par l'ARNm thérapeutique représente, néanmoins, un défi important pour le développement de cette approche.


Assuntos
RNA Mensageiro , Humanos , RNA Mensageiro/genética , Terapia Genética/métodos , Terapia Genética/tendências , Animais , Terapia Biológica/métodos , Terapia Biológica/tendências , RNA/genética
10.
Eur J Pharm Biopharm ; 201: 114380, 2024 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-38960290

RESUMO

We have used pulsed field gradient (PFG)-NMR diffusion experiments, also known as DOSY, in combination with small angle X-ray scattering measurements to investigate structure and molecular exchange dynamics between pharmaceutical lipid nanoparticles and the bulk phase. Using liposomes and lipoplexes formed after complexation of the liposomes with messenger mRNA as test systems, information on dynamics of encapsulated water molecules, lipids and excipients was obtained. The encapsulated fraction, having a diffusivity similar to that of the liposomes, could be clearly identified and quantified by the NMR diffusion measurements. The unilamellar liposome membranes allowed a fast exchange of water molecules, while sucrose, used as an osmolyte and model solute, showed very slow exchange. Upon interactions with mRNA a topological transition from a vesicular to a lamellar organization took place, where the mRNA was inserted in repeating lipid bilayer stacks. In the lipoplexes, a small fraction of tightly bound water molecules was present, with a diffusivity that was influenced by the additional presence of sucrose. This extended information on dynamic coherencies inside pharmaceutical nanoparticle products, provided by the combined application of SAXS and PFG-NMR diffusion measurements, can be valuable for evaluation of quality and comparability of nanoscaled pharmaceuticals.


Assuntos
Lipossomos , Espectroscopia de Ressonância Magnética , Nanopartículas , RNA Mensageiro , Espalhamento a Baixo Ângulo , Difração de Raios X , Nanopartículas/química , Espectroscopia de Ressonância Magnética/métodos , Difusão , Cinética , Difração de Raios X/métodos , Sacarose/química , Lipídeos/química , Água/química , Excipientes/química , Bicamadas Lipídicas/química
11.
Transl Vis Sci Technol ; 13(7): 7, 2024 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-38980261

RESUMO

Purpose: Lipid nanoparticles (LNPs) show promise in their ability to introduce mRNA to drive protein expression in specific cell types of the mammalian eye. Here, we examined the ability of mRNA encapsulated in LNPs with two distinct formulations to drive gene expression in mouse and human retina and other ocular tissues. Methods: We introduced mRNA-carrying LNPs into two biological systems. Intravitreal injections were tested to deliver LNPs into the mouse eye. Human retinal pigment epithelium (RPE) and retinal explants were used to assess mRNA expression in human tissue. We analyzed specificity of expression using histology, immunofluorescence, and imaging. Results: In mice, mRNAs encoding GFP and ciliary neurotrophic factor (CNTF) were specifically expressed by Müller glia and RPE. Acute inflammatory changes measured by microglia distribution (Iba-1) or interleukin-6 (IL-6) expression were not observed 6 hours post-injection. Human RPE also expressed high levels of GFP. Human retinal explants expressed GFP in cells with apical and basal processes consistent with Müller glia and in perivascular cells consistent with macrophages. Conclusions: We demonstrated the ability to reliably transfect subpopulations of retinal cells in mouse eye tissues in vivo and in human ocular tissues. Of significance, intravitreal injections were sufficient to transfect the RPE in mice. To our knowledge, we demonstrate delivery of mRNA using LNPs in human ocular tissues for the first time. Translational Relevance: Ocular gene-replacement therapies using non-viral vector methods are a promising alternative to adeno-associated virus (AAV) vectors. Our studies show that mRNA LNP delivery can be used to transfect retinal cells in both mouse and human tissues without inducing significant inflammation. This methodology could be used to transfect retinal cell lines, tissue explants, mice, or potentially as gene-replacement therapy in a clinical setting in the future.


Assuntos
Injeções Intravítreas , Nanopartículas , RNA Mensageiro , Epitélio Pigmentado da Retina , Animais , Humanos , RNA Mensageiro/administração & dosagem , RNA Mensageiro/metabolismo , Camundongos , Epitélio Pigmentado da Retina/metabolismo , Nanopartículas/química , Camundongos Endogâmicos C57BL , Fator Neurotrófico Ciliar/genética , Fator Neurotrófico Ciliar/metabolismo , Fator Neurotrófico Ciliar/administração & dosagem , Retina/metabolismo , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Células Ependimogliais/metabolismo , Técnicas de Transferência de Genes , Lipossomos
12.
J Med Virol ; 96(7): e29793, 2024 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-39023111

RESUMO

Various vaccine platforms were developed and deployed against the COVID-19 disease. The Fc-mediated functions of IgG antibodies are essential in the adaptive immune response elicited by vaccines. However, the long-term changes of protein subunit vaccines and their combinations with messenger RNA (mRNA) vaccines are unknown. A total of 272 serum and plasma samples were collected from individuals who received first to third doses of the protein subunit Medigen, the mRNA (BNT, Moderna), or the adenovector AstraZeneca vaccines. The IgG subclass level was measured using enzyme-linked immunosorbent assay, and Fc-N glycosylation was measured using liquid chromatography coupled to tandem mass spectrometry. Antibody-dependent-cellular-phagocytosis (ADCP) and complement deposition (ADCD) of anti-spike (S) IgG antibodies were measured by flow cytometry. IgG1 and 3 reached the highest anti-S IgG subclass level. IgG1, 2, and 4 subclass levels significantly increased in mRNA- and Medigen-vaccinated individuals. Fc-glycosylation was stable, except in female BNT vaccinees, who showed increased bisection and decreased galactosylation. Female BNT vaccinees had a higher anti-S IgG titer than that of males. ADCP declined in all groups. ADCD was significantly lower in AstraZeneca-vaccinated individuals. Each vaccine produced specific long-term changes in Fc structure and function. This finding is critical when selecting a vaccine platform or combination to achieve the desired immune response.


Assuntos
Anticorpos Antivirais , Vacinas contra COVID-19 , COVID-19 , Imunoglobulina G , SARS-CoV-2 , Vacinas de Subunidades Antigênicas , Vacinas de mRNA , Humanos , Imunoglobulina G/sangue , Feminino , Anticorpos Antivirais/sangue , Masculino , COVID-19/prevenção & controle , COVID-19/imunologia , SARS-CoV-2/imunologia , SARS-CoV-2/genética , Adulto , Pessoa de Meia-Idade , Vacinas contra COVID-19/imunologia , Vacinas de Subunidades Antigênicas/imunologia , Vacinas de Subunidades Antigênicas/genética , Vacinas de Subunidades Antigênicas/administração & dosagem , Glicosilação , Glicoproteína da Espícula de Coronavírus/imunologia , Glicoproteína da Espícula de Coronavírus/genética , Idoso , RNA Mensageiro/genética , Adulto Jovem , Vacinas de Subunidades Proteicas
13.
Front Immunol ; 15: 1344995, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-39011034

RESUMO

Background: Allergic rhinitis (AR) is a complex disease in which gene-environment interactions contribute to its pathogenesis. Epigenetic modifications, such as N6-methyladenosine (m6A) modification of mRNA, play important roles in regulating gene expression in multiple physiological and pathological processes. However, the function of m6A modification in AR and the inflammatory response is poorly understood. Methods: We used the ovalbumin (OVA) and aluminum hydroxide to induce an AR mouse model. Nasal symptoms, histopathology, and serum cytokines were examined. We performed combined m6A and RNA sequencing to analyze changes in m6A modification profiles. Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and methylated RNA immunoprecipitation sequencing qPCR (MeRIP-qPCR) were used to verify differential methylation of mRNAs and the m6A methylation level. Knockdown or inhibition of Alkbh5 in nasal mucosa of mice was mediated by lentiviral infection or IOX1 treatment. Results: We showed that m6A was enriched in a group of genes involved in MAPK signaling pathway. Moreover, we identified a MAPK pathway involving Map3k8, Erk2, and Nfκb1 that may play a role in the disrupted inflammatory response associated with nasal inflammation. The m6A eraser, Alkbh5, was highly expressed in the nasal mucosa of AR model mice. Furthermore, knockdown of Alkbh5 expression by lentiviral infection resulted in high MAPK pathway activity and a significant nasal mucosa inflammatory response. Our findings indicate that ALKBH5-mediated m6A dysregulation likely contributes to a nasal inflammatory response via the MAPK pathway. Conclusion: Together, our data show that m6A dysregulation mediated by ALKBH5, is likely to contribute to inflammation of the nasal mucosa via the MAPK signaling pathway, suggesting that ALKBH5 is a potential biomarker for AR treatment.


Assuntos
Adenosina , Homólogo AlkB 5 da RNA Desmetilase , Modelos Animais de Doenças , Sistema de Sinalização das MAP Quinases , Mucosa Nasal , RNA Mensageiro , Rinite Alérgica , Animais , Mucosa Nasal/imunologia , Mucosa Nasal/metabolismo , Mucosa Nasal/patologia , Rinite Alérgica/imunologia , Rinite Alérgica/metabolismo , Rinite Alérgica/genética , Camundongos , Adenosina/análogos & derivados , Adenosina/metabolismo , Metilação , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Homólogo AlkB 5 da RNA Desmetilase/metabolismo , Homólogo AlkB 5 da RNA Desmetilase/genética , Feminino , Camundongos Endogâmicos BALB C , Inflamação/genética , Inflamação/imunologia , Citocinas/metabolismo
14.
Front Immunol ; 15: 1384442, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38947333

RESUMO

The One Health approach, which integrates the health of humans, animals, plants, and ecosystems at various levels, is crucial for addressing interconnected health threats. This is complemented by the advent of mRNA vaccines, which have revolutionized disease prevention. They offer broad-spectrum effectiveness and can be rapidly customized to target specific pathogens. Their utility extends beyond human medicine, showing potential in veterinary practices to control diseases and reduce the risk of zoonotic transmissions. This review place mRNA vaccines and One Health in the context of tick-borne diseases. The potential of these vaccines to confer cross-species immunity is significant, potentially disrupting zoonotic disease transmission cycles and protecting the health of both humans and animals, while reducing tick populations, infestations and circulation of pathogens. The development and application of mRNA vaccines for tick and tick-borne pathogens represent a comprehensive strategy in global health, fostering a healthier ecosystem for all species in our interconnected world.


Assuntos
Saúde Única , Doenças Transmitidas por Carrapatos , Carrapatos , Vacinas de mRNA , Animais , Humanos , Doenças Transmitidas por Carrapatos/prevenção & controle , Doenças Transmitidas por Carrapatos/imunologia , Doenças Transmitidas por Carrapatos/transmissão , Carrapatos/microbiologia , Carrapatos/imunologia , Zoonoses/prevenção & controle , RNA Mensageiro/genética , RNA Mensageiro/imunologia , Vacinas Sintéticas/imunologia
15.
Sichuan Da Xue Xue Bao Yi Xue Ban ; 55(3): 527-534, 2024 May 20.
Artigo em Chinês | MEDLINE | ID: mdl-38948273

RESUMO

Infertility affects an estimated 10 to 15 percent of couples worldwide, with approximately half of the cases attributed to male-related issues. Most men diagnosed with infertility exhibit symptoms such as oligospermia, asthenospermia, azoospermia, and compromised sperm quality. Spermatogenesis is a complex and tightly coordinated process of germ cell differentiation, precisely regulated at transcriptional, posttranscriptional, and translational levels to ensure stage-specific gene expression during the development of spermatogenic cells and normal spermiogenesis. N6-methyladenosine (m6A) stands out as the most prevalent modification on eukaryotic mRNA, playing pivotal roles in various biological processes, including mRNA splicing, transportation, and translation. RNA methylation modification is a dynamic and reversible process primarily mediated by "writers", removed by "erasers", and recognized by "readers". In mammals, the aberrant methylation modification of m6A on mRNA is associated with a variety of diseases, including male infertility. However, the precise involvement of disrupted m6A modification in the pathogenesis of human male infertility remains unresolved. Intriguingly, a significant correlation has been found between the expression levels of m6A regulators in the testis and the severity of sperm concentration, motility, and morphology. Aberrant expression patterns of m6A regulatory proteins have been detected in anomalous human semen samples, including those of oligospermia, asthenozoospermia, and azoospermia. Furthermore, the examination of both sperm samples and testicular tissues revealed abnormal mRNA m6A modification, leading to reduced sperm motility and concentration in infertile men. Consequently, it is hypothesized that dysregulation of m6A modification might serve as an integral link in the mechanism of male infertility. This paper presents a comprehensive review of the recent discoveries regarding the spatial and temporal expression dynamics of m6A regulators in testicular tissues and the correlation between deregulated m6A regulators and human male infertility. Previous studies predominantly utilized constitutive or conditional knockout animal models for testicular phenotypic investigations. However, gene suppression in additional tissues could potentially influence the testis in constitutive knockout models. Furthermore, considering the compromised spermatogenesis observed in constitutive animals, distinguishing between the indirect effects of gene depletion on testicular development and its direct impact on the spermatogenic process is challenging, due to their intricate relationship. Such confounding factors might compromise the validity of the findings. To address this challenge, an inducible and conditional gene knockout model may serve as a superior approach. To date, nearly all reported studies have concentrated solely on the level changes of m6A and its regulators in germs cells, while the understanding of the function of m6A modification in testicular somatic cells remains limited. Testicular somatic cells, including peritubular myoid cells, Sertoli cells, and Leydig cells, play indispensable roles during spermatogenesis. Hence, comprehensive exploration of m6A modification within these cells as an additional crucial regulatory mechanism is warranted. In addition, exploration into the presence of unique methylation mechanisms or m6A regulatory factors within the testes is warranted. To elucidate the role of m6A modification in germ cells and testicular somatic cells, detailed experimental strategies need to be implemented. Among them, manipulation of the levels of key enzymes involved in m6A methylation and demethylation might be the most effective approach. Moreover, comprehensive analysis of the gene expression profiles involved in various signaling pathways, such as Wnt/ß-catenin, Ras/MAPK, and Hippo, in m6A-modified germ cells and testicular somatic cells can provide more insight into its regulatory role in the spermatogenesis process. Further research in this area could provide valuable insights for developing innovative strategies to treat male infertility. Finally, considering the mitigation impact of m6A imbalance regulation on disease, investigation concerning whether restoring the equilibrium of m6A modification regulation can restore normal spermatogenesis function is essential, potentially elucidating the pivotal clinical significance of m6A modulation in male infertility.


Assuntos
Adenosina , Infertilidade Masculina , Espermatogênese , Masculino , Humanos , Adenosina/análogos & derivados , Adenosina/metabolismo , Espermatogênese/genética , Infertilidade Masculina/genética , Infertilidade Masculina/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Metilação , Animais , Metiltransferases/metabolismo , Metiltransferases/genética , Espermatozoides/metabolismo , Testículo/metabolismo
16.
PLoS One ; 19(7): e0305222, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38959243

RESUMO

BACKGROUND: Currently, there are few treatment-predictive and prognostic biomarkers in triple-negative breast cancer (TNBC). Caveolin-1 (CAV1) is linked to chemoresistance and several important processes involved in tumor progression and metastasis, such as epithelial-mesenchymal transition (EMT). Herein, we report that high CAV1 gene expression is an independent factor of poor prognosis in TNBC. METHODS: CAV1 gene expression was compared across different molecular features (e.g., PAM50 subtypes). CAV1 expression was assessed in relation to clinical outcomes using Cox regression adjusted for clinicopathological predictors. Differential gene expression and gene set enrichment analyses were applied to compare high- and low-expressing CAV1 tumors. Tumor microenvironment composition of high- and low-expressing CAV1 tumors was estimated using ECOTYPER. Tumor tissue microarrays were used to evaluate CAV1 protein levels in stromal and malignant cells. RESULTS: In the SCAN-B (n = 525) and GSE31519 (n = 327) cohorts, patients with CAV1-high tumors had an increased incidence of early recurrence adjusted HR 1.78 (95% CI 1.12-2.81) and 2.20 (95% CI 1.39-3.47), respectively. In further analysis, high CAV1 gene expression was associated with a molecular profile indicating altered metabolism, neovascularization, chemoresistance, EMT, suppressed immune response, and active tumor microenvironment. Protein levels of CAV1 in malignant and stromal cells were not correlated with CAV1 gene expression. CONCLUSION: CAV1 gene expression in TNBC is a biomarker that merits further investigation in clinical trials and as a therapeutic target.


Assuntos
Caveolina 1 , Resistencia a Medicamentos Antineoplásicos , Neoplasias de Mama Triplo Negativas , Microambiente Tumoral , Humanos , Caveolina 1/genética , Caveolina 1/metabolismo , Neoplasias de Mama Triplo Negativas/genética , Neoplasias de Mama Triplo Negativas/patologia , Neoplasias de Mama Triplo Negativas/metabolismo , Microambiente Tumoral/genética , Feminino , Resistencia a Medicamentos Antineoplásicos/genética , Pessoa de Meia-Idade , Regulação Neoplásica da Expressão Gênica , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Prognóstico , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/metabolismo , Transição Epitelial-Mesenquimal/genética , Idoso
17.
Nat Commun ; 15(1): 5550, 2024 Jul 02.
Artigo em Inglês | MEDLINE | ID: mdl-38956014

RESUMO

Oocyte in vitro maturation is a technique in assisted reproductive technology. Thousands of genes show abnormally high expression in in vitro maturated metaphase II (MII) oocytes compared to those matured in vivo in bovines, mice, and humans. The mechanisms underlying this phenomenon are poorly understood. Here, we use poly(A) inclusive RNA isoform sequencing (PAIso-seq) for profiling the transcriptome-wide poly(A) tails in both in vivo and in vitro matured mouse and human oocytes. Our results demonstrate that the observed increase in maternal mRNA abundance is caused by impaired deadenylation in in vitro MII oocytes. Moreover, the cytoplasmic polyadenylation of dormant Btg4 and Cnot7 mRNAs, which encode key components of deadenylation machinery, is impaired in in vitro MII oocytes, contributing to reduced translation of these deadenylase machinery components and subsequently impaired global maternal mRNA deadenylation. Our findings highlight impaired maternal mRNA deadenylation as a distinct molecular defect in in vitro MII oocytes.


Assuntos
Oócitos , Poliadenilação , Oócitos/metabolismo , Animais , Humanos , Feminino , Camundongos , Poli A/metabolismo , Técnicas de Maturação in Vitro de Oócitos , RNA Mensageiro/metabolismo , RNA Mensageiro/genética , Transcriptoma , RNA Mensageiro Estocado/metabolismo , RNA Mensageiro Estocado/genética , Metáfase , Exorribonucleases , Proteínas Repressoras , Proteínas de Ciclo Celular
18.
J Transl Med ; 22(1): 614, 2024 Jul 02.
Artigo em Inglês | MEDLINE | ID: mdl-38956643

RESUMO

Mesenchymal stem and stromal cells (MSCs) hold potential to treat a broad range of clinical indications, but clinical translation has been limited to date due in part to challenges with batch-to-batch reproducibility of potential critical quality attributes (pCQAs) that can predict potency/efficacy. Here, we designed and implemented a microcarrier-microbioreactor approach to cell therapy manufacturing, specific to anchorage-dependent cells such as MSCs. We sought to assess whether increased control of the biochemical and biophysical environment had the potential to create product with consistent presentation and elevated expression of pCQAs relative to established manufacturing approaches in tissue culture polystyrene (TCPS) flasks. First, we evaluated total cell yield harvested from dissolvable, gelatin microcarriers within a microbioreactor cassette (Mobius Breez) or a flask control with matched initial cell seeding density and culture duration. Next, we identified 24 genes implicated in a therapeutic role for a specific motivating indication, acute respiratory distress syndrome (ARDS); expression of these genes served as our pCQAs for initial in vitro evaluation of product potency. We evaluated mRNA expression for three distinct donors to assess inter-donor repeatability, as well as for one donor in three distinct batches to assess within-donor, inter-batch variability. Finally, we assessed gene expression at the protein level for a subset of the panel to confirm successful translation. Our results indicated that MSCs expanded with this microcarrier-microbioreactor approach exhibited reasonable donor-to-donor repeatability and reliable batch-to-batch reproducibility of pCQAs. Interestingly, the baseline conditions of this microcarrier-microbioreactor approach also significantly improved expression of several key pCQAs at the gene and protein expression levels and reduced total media consumption relative to TCPS culture. This proof-of-concept study illustrates key benefits of this approach to therapeutic cell process development for MSCs and other anchorage-dependent cells that are candidates for cell therapies.


Assuntos
Reatores Biológicos , Células-Tronco Mesenquimais , Síndrome do Desconforto Respiratório , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/metabolismo , Humanos , Síndrome do Desconforto Respiratório/terapia , Regulação da Expressão Gênica , Técnicas de Cultura de Células/métodos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Reprodutibilidade dos Testes
20.
J Cell Mol Med ; 28(13): e18496, 2024 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-38984939

RESUMO

Hepatocellular carcinoma (HCC), a prevalent malignancy worldwide, poses significant challenges in terms of prognosis, necessitating innovative therapeutic approaches. Ferroptosis offers notable advantages over apoptosis, holding promise as a novel therapeutic approach for HCC complexities. Moreover, while the interaction between long non-coding RNAs (lncRNAs) and mRNAs is pivotal in various physiological and pathological processes, their involvement in ferroptosis remains relatively unexplored. In this study, we constructed a ferroptosis-related lncRNA-mRNA correlation network in HCC using Pearson correlation analysis. Notably, the SLC7A11-AS1/SLC7A11 pair, exhibiting high correlation, was identified. Bioinformatics analysis revealed a significant correlation between the expression levels of this pair and key clinical characteristics of HCC patients, including gender, pathology, Ishak scores and tumour size. And poor prognosis was associated with high expression of this pair. Functional experiments demonstrated that SLC7A11-AS1, by binding to the 3'UTR region of SLC7A11 mRNA, enhanced its stability, thereby promoting HCC cell growth and resistance to erastin- induced ferroptosis. Additionally, in vivo studies confirmed that SLC7A11-AS1 knockdown potentiated the inhibitory effects of erastin on tumour growth. Overall, our findings suggest that targeting the SLC7A11-AS1/SLC7A11 pair holds promise as a potential therapeutic strategy for HCC patients.


Assuntos
Sistema y+ de Transporte de Aminoácidos , Carcinoma Hepatocelular , Ferroptose , Regulação Neoplásica da Expressão Gênica , Neoplasias Hepáticas , RNA Longo não Codificante , Ferroptose/genética , Humanos , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/patologia , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/tratamento farmacológico , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/patologia , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/tratamento farmacológico , Sistema y+ de Transporte de Aminoácidos/genética , Sistema y+ de Transporte de Aminoácidos/metabolismo , RNA Longo não Codificante/genética , RNA Longo não Codificante/metabolismo , Animais , Linhagem Celular Tumoral , Masculino , Feminino , Camundongos , Prognóstico , Proliferação de Células/genética , Camundongos Nus , Pessoa de Meia-Idade , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Piperazinas/farmacologia
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