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1.
Transpl Immunol ; 85: 102070, 2024 Jun 03.
Artigo em Inglês | MEDLINE | ID: mdl-38839020

RESUMO

BACKGROUND: Acute myocardial infarction (AMI) is a global health problem with high mortality. Early diagnosis can prevent the development of AMI and provide valuable information for subsequent treatment. Angiogenesis has been shown to be a critical factor in the development of infarction and targeting this process may be a potential protective strategy for preventing myocardial injury and improving the prognosis of AMI patients. This study aimed to screen and verify diagnostic markers related to angiogenesis in AMI and to investigate the molecular mechanisms of action associated with AMI in terms of immune cell infiltration. METHODS: The GSE66360 and the GSE60993 datasets were both downloaded from the GEO database and were used as the training cohort and the external validation cohort, respectively. Angiogenesis-related genes (ARGs) were downloaded from the MSigDB database. The hub ARGs were identified via LASSO, RF, and SVM-RFE algorithms. ROC curves were used to assess the accuracy of the hub ARGs. The potential mechanisms of the hub ARGs were analyzed by GSEA. The ssGSEA algorithm was used to determine differences in immune cell infiltration and immune function. The CIBERSORT algorithm was used for immune cell infiltration analysis. In addition, we constructed a ceRNA network map of differentially expressed ARGs. RESULTS: We identified the thrombomodulin (THBD) gene from ARGs as a potential diagnostic marker for AMI based on the LASSO, SVM-RFE, and RF algorithms. THBD was differentially expressed and had a potential diagnostic value (area under the curve [AUC] = 0.931 and 0.765 in the training and testing datasets, respectively). GSEA showed that the MAPK signaling pathway was more enriched in the high-expression group of THBD (P < 0.05). Immune cell infiltration analysis demonstrated that THBD was mainly positively correlated with monocytes (R = 0.48, P = 0.00055) and neutrophils (R = 0.36, P = 0.013). Finally, in the ceRNA regulatory network, THBD was closely associated with 9 miRNAs and 42 lncRNAs involved in AMI. CONCLUSION: THBD can be used as a potential diagnostic marker for AMI. This study provides new insights for future AMI diagnosis and molecular mechanism research. Moreover, immune cell infiltration plays an essential role in the occurrence and development of AMI.

2.
World J Gastrointest Oncol ; 16(4): 1192-1203, 2024 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-38660657

RESUMO

BACKGROUND: Indentifying predictive factors for postoperative recurrence of hepatocellular carcinoma (HCC) has great significance for patient prognosis. AIM: To explore the value of gadolinium ethoxybenzyl diethylenetriamine pentaacetic acid (Gd-EOB-DTPA) enhanced magnetic resonance imaging (MRI) combined with clinical features in predicting early recurrence of HCC after resection. METHODS: A total of 161 patients with pathologically confirmed HCC were enrolled. The patients were divided into early recurrence and non-early recurrence group based on the follow-up results. The clinical, laboratory, pathological results and Gd-EOB-DTPA enhanced MRI imaging features were analyzed. RESULTS: Of 161 patients, 73 had early recurrence and 88 were had non-early recurrence. Univariate analysis showed that patient age, gender, serum alpha-fetoprotein level, the Barcelona Clinic Liver Cancer stage, China liver cancer (CNLC) stage, microvascular invasion (MVI), pathological satellite focus, tumor size, tumor number, tumor boundary, tumor capsule, intratumoral necrosis, portal vein tumor thrombus, large vessel invasion, nonperipheral washout, peritumoral enhancement, hepatobiliary phase (HBP)/tumor signal intensity (SI)/peritumoral SI, HBP peritumoral low signal and peritumoral delay enhancement were significantly associated with early recurrence of HCC after operation. Multivariate logistic regression analysis showed that patient age, MVI, CNLC stage, tumor boundary and large vessel invasion were independent predictive factors. External data validation indicated that the area under the curve of the combined predictors was 0.861, suggesting that multivariate logistic regression was a reasonable predictive model for early recurrence of HCC. CONCLUSION: Gd-EOB-DTPA enhanced MRI combined with clinical features would help predicting the early recurrence of HCC after operation.

5.
Food Chem ; 451: 139442, 2024 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-38688099

RESUMO

Enshi Yulu green tea (ESYL) is the most representative traditional steamed green tea in Enshi, Hubei. Different ESYL grades exhibit distinct flavors, tastes, and prices. In this study, a visual sensor based on 4-MPBA Au@AgNPs was developed for the rapid and accurate identification of ESYL grades. The recognition mechanism involved the binding of 4-MPBA Au@AgNPs with polyphenolic compounds in ESYL to form borate esters and the conversion of Ag+ to Ag0, with the generated Ag0 depositing on the surface of 4-MPBA Au@AgNPs. The results showed that the sensor can amplify the color differences of different grades of ESYL. The visual results were also validated by the partial least squares discriminant analysis model, demonstrating an enhancement in recognition accuracy from 68.2 % to 95.5 % compared to the original extraction solution. The colorimetric sensor developed in this study is expected to provide a new approach for traceability research of other foods.


Assuntos
Colorimetria , Ouro , Prata , Chá , Colorimetria/métodos , Chá/química , Prata/química , Ouro/química , Nanopartículas Metálicas/química , Camellia sinensis/química
6.
Nat Chem Biol ; 2024 Mar 29.
Artigo em Inglês | MEDLINE | ID: mdl-38553609

RESUMO

Cytosine base editors (CBEs) are effective tools for introducing C-to-T base conversions, but their clinical applications are limited by off-target and bystander effects. Through structure-guided engineering of human APOBEC3A (A3A) deaminase, we developed highly accurate A3A-CBE (haA3A-CBE) variants that efficiently generate C-to-T conversion with a narrow editing window and near-background level of DNA and RNA off-target activity, irrespective of methylation status and sequence context. The engineered deaminase domains are compatible with PAM-relaxed SpCas9-NG variant, enabling accurate correction of pathogenic mutations in homopolymeric cytosine sites through flexible positioning of the single-guide RNAs. Dual adeno-associated virus delivery of one haA3A-CBE variant to a mouse model of tyrosinemia induced up to 58.1% editing in liver tissues with minimal bystander editing, which was further reduced through single dose of lipid nanoparticle-based messenger RNA delivery of haA3A-CBEs. These results highlight the tremendous promise of haA3A-CBEs for precise genome editing to treat human diseases.

7.
Nat Biotechnol ; 42(4): 638-650, 2024 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-37322276

RESUMO

Base editors have substantial promise in basic research and as therapeutic agents for the correction of pathogenic mutations. The development of adenine transversion editors has posed a particular challenge. Here we report a class of base editors that enable efficient adenine transversion, including precise A•T-to-C•G editing. We found that a fusion of mouse alkyladenine DNA glycosylase (mAAG) with nickase Cas9 and deaminase TadA-8e catalyzed adenosine transversion in specific sequence contexts. Laboratory evolution of mAAG significantly increased A-to-C/T conversion efficiency up to 73% and expanded the targeting scope. Further engineering yielded adenine-to-cytosine base editors (ACBEs), including a high-accuracy ACBE-Q variant, that precisely install A-to-C transversions with minimal Cas9-independent off-targeting effects. ACBEs mediated high-efficiency installation or correction of five pathogenic mutations in mouse embryos and human cell lines. Founder mice showed 44-56% average A-to-C edits and allelic frequencies of up to 100%. Adenosine transversion editors substantially expand the capabilities and possible applications of base editing technology.


Assuntos
Adenina , Edição de Genes , Animais , Camundongos , Humanos , Adenina/metabolismo , Mutação , Citosina/metabolismo , Adenosina , Sistemas CRISPR-Cas/genética , Mamíferos/genética
8.
Cell Death Dis ; 14(12): 818, 2023 12 12.
Artigo em Inglês | MEDLINE | ID: mdl-38086848

RESUMO

Numerous studies have proven the critical role of macrophages in the renal fibrosis process. Notably, G Protein-coupled Estrogen Receptor 1 (GPER1), a novel estrogen receptor, has been shown to play a ubiquitous role in regulating macrophage activities and proinflammatory pathways. However, the precise role of GPER1 in macrophage-mediated renal fibrosis is unknown. In this study, we aimed to investigate the function of macrophage GPER1 in the UUO-induced renal fibrosis model. Compared to vehicle-treated ovariectomized (OVX) female and male unilateral ureteral obstruction (UUO) models, we observed that G-1 (GPER1 agonist)-treated OVX female and male UUO mice had fewer renal fibrotic lesions and less M1 and M2 macrophage infiltration in the kidney tissues. Conversely, Gper1 deletion in male UUO mice accelerated renal fibrosis and increased inflammation. In vitro studies also revealed that GPER1 activation reduced M0 macrophage polarization towards M1 or M2 phenotypes. The RNA-sequencing analysis and immunoblotting indicated that GPER1 activation was primarily involved in downregulating immune pathways activation and inactivating MAPK pathways. Tubular epithelial cells co-cultured with G-1-pretreated M1 macrophages exhibited fewer injuries and immune activation. In addition, fibroblasts co-cultured with G-1-pretreated M2 macrophages showed downregulated extracellular matrix expression. Overall, this is the first study to demonstrate the effect of GPER1 on macrophage-mediated renal fibrosis via inhibition of M1 and M2 macrophage activation. These findings indicate that GPER1 may be a promising therapeutic target for treating renal fibrosis.


Assuntos
Nefropatias , Obstrução Ureteral , Feminino , Masculino , Camundongos , Animais , Obstrução Ureteral/metabolismo , Transdução de Sinais , Nefropatias/patologia , Macrófagos/metabolismo , Receptores de Estrogênio/metabolismo , Fibrose , Rim/patologia , Camundongos Endogâmicos C57BL
9.
World J Gastrointest Surg ; 15(10): 2320-2330, 2023 Oct 27.
Artigo em Inglês | MEDLINE | ID: mdl-37969709

RESUMO

BACKGROUND: Intra-abdominal infections (IAIs) is the most common type of surgical infection, with high associated morbidity and mortality rates. In recent years, due to the use of antibiotics, various drug-resistant bacteria have emerged, making the treatment of abdominal infections more challenging. Early surgical exploration can reduce the mortality of patients with abdominal infection and the occurrence of complications. However, available evidence regarding the optimal timing of IAI surgery is still weak. In study, we compared the effects of operation time on patients with abdominal cavity infection and tried to confirm the best timing of surgery. AIM: To assess the efficacy of early vs delayed surgical exploration in the treatment of IAI, in terms of overall mortality. METHODS: A systematic literature search was performed using PubMed, EMBASE, Cochrane Central Register of Controlled Trials, Ovid, and ScienceDirect. The systematic review was conducted using the Preferred Reporting Items for Systematic Reviews and Meta-analyses method. Based on the timing of the surgical operation, we divided the literature into two groups: Early surgery and delayed surgery. For the early and delayed surgery groups, the intervention was performed with and after 12 h of the initial surgical intervention, respectively. The main outcome measure was the mortality rate. The literature search was performed from May 5 to 20, 2021. We also searched the World Health Organization International Clinical Trials Registry Platform search portal and ClinicalTrials.gov on May 20, 2021, for ongoing trials. This study was registered with the International Prospective Register of Systematic Reviews. RESULTS: We identified nine eligible trial comparisons. Early surgical exploration of patients with IAIs (performed within 12 h) has significantly reduced the mortality and complications of patients, improved the survival rate, and shortened the hospital stay. CONCLUSION: Early surgical exploration within 12 h may be more effective for the treatment of IAIs relative to a delayed operation.

10.
Food Funct ; 14(13): 6128-6141, 2023 Jul 03.
Artigo em Inglês | MEDLINE | ID: mdl-37334479

RESUMO

Black rice was fermented with Neurospora crassa, after which the dietary fiber (DF) extracted from it was characterized and evaluated for its cholesterol-lowering effect in mice. The findings demonstrated that fermentation increased the level of soluble DF from 17.27% ± 0.12 to 29.69% ± 0.26 and increased the adsorption capacity of DF for water, oil, cholesterol, glucose and sodium cholate. The fermented DF had a more loose and porous structure than that extracted from unfermented rice. Additionally, feeding with DF from the fermented black rice significantly reduced body weight, lowered total cholesterol levels and improved the lipid profile in mice gavaged with a high dose (5 g per kg bw) or a low dose (2.5 g per kg·bw). ELISA showed that the hepatic expression of typical proteins and enzymes that are involved in cholesterol metabolism was regulated by the fermented rice DF, leading to reduced cholesterol production and increased cholesterol clearance. The fermented DF also modified the gut microbiota composition (e.g. Firmicutes reduced and Akkermansia increased), which promoted the production of short-chain fatty acids. In conclusion, fermentation can modify the structure and function of DF in black rice and the fermented dietary fiber has excellent cholesterol lowering effects possibly by cholesterol adsorption, cholesterol metabolism modulation, and intestinal microflora regulation.


Assuntos
Oryza , Camundongos , Animais , Oryza/metabolismo , Colesterol/metabolismo , Fibras na Dieta/análise , Fígado/metabolismo , Fermentação
11.
Food Chem ; 424: 136406, 2023 Oct 30.
Artigo em Inglês | MEDLINE | ID: mdl-37216781

RESUMO

In this work, a rapid and accurate strategy for classification of Chinese traditional cereal vinegars (CTCV) and antioxidant property predication was proposed by using the combination fluorescence spectroscopy and machine learning. Three characteristic fluorescent components were extracted by parallel factor analysis (PARAFAC), which have correlations greater than 0.8 with antioxidant activity of CTCV obtained by Pearson correlation analysis. Machine learning methods, including linear discriminant analysis (LDA), partial least squares-discriminant analysis (PLS-DA) and N-way partial least squares discriminant analysis (N-PLS-DA), were used for the classification of different types of CTCV, and the correct classification rates was higher than 97%. The antioxidant property of CTCV were further quantified by using optimized variable-weighted least-squares support vector machine based on particle swarm optimization (PSO-VWLS-SVM). The proposed strategy provides a basis for further research on antioxidant active ingredients and antioxidant mechanisms of CTCV, and enable the continued exploration and application of CTCV from different types.


Assuntos
Ácido Acético , Antioxidantes , Ácido Acético/análise , Antioxidantes/análise , Análise Discriminante , Grão Comestível/química , Análise dos Mínimos Quadrados , Espectrometria de Fluorescência/métodos , Máquina de Vetores de Suporte , China
12.
Int J Biol Macromol ; 239: 124277, 2023 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-37011747

RESUMO

Herein, the manganese-oxides-coated magnetic microcrystalline cellulose (MnOx@Fe3O4@MCC) was prepared by coprecipitation and subsequently modified with KMnO4 solution at room temperature, which was in turn applied for the removal of Pb(II) from wastewater. The adsorption properties of Pb(II) on MnOx@Fe3O4@MCC were investigated. The kinetics and isothermal data of Pb(II) were described well by the Pseudo-second-order model and the Langmuir isotherm model, respectively. At pH = 5, 318 K, the Langmuir maximum Pb(II) adsorption capacity of MnOx@Fe3O4@MCC was 446.43 mg/g, which is higher than many documented bio-based adsorbents. The results of Fourier transform infra-red and X-ray photoelectron spectroscopy indicated that the adsorption mechanisms for Pb(II) mainly involved surface complexation, ion exchange, electrostatic interaction and precipitation. Interestingly, the increased amount of carboxyl group on the surface of microcrystalline cellulose modified by KMnO4 was one of the important reasons for the high Pb(II) adsorption performance of MnOx@Fe3O4@MCC. Furthermore, MnOx@Fe3O4@MCC exhibited excellent activity (70.6 %) after five consecutive regeneration cycles, indicating its high stability and reusability. Endorsing to the cost-effectiveness, environmentally friendliness, and reusable nature, MnOx@Fe3O4@MCC can be counted as a great alternative contender for the remediation of Pb(II) from industrial wastewater.


Assuntos
Chumbo , Poluentes Químicos da Água , Adsorção , Manganês , Águas Residuárias , Óxidos/química , Fenômenos Magnéticos , Poluentes Químicos da Água/química , Cinética , Concentração de Íons de Hidrogênio
13.
Spectrochim Acta A Mol Biomol Spectrosc ; 294: 122517, 2023 Jun 05.
Artigo em Inglês | MEDLINE | ID: mdl-36868024

RESUMO

Copper (Cu2+), as a heavy metal, accumulates in the human body to a certain extent, which can induce various diseases and endanger human health. Rapid and sensitive detection of Cu2+ is highly desired. In present work, a glutathione modified quantum dot (GSH-CdTe QDs) was synthesized and applied in a "turn-off" fluorescence probe to detect Cu2+. The fluorescence of GSH-CdTe QDs could be rapidly quenched in the presence of Cu2+ through aggregation-caused quenching (ACQ), resulting from the interaction between the surface functional groups of GSH-CdTe QDs and Cu2+ and the electrostatic attraction. In the range of 20-1100 nM, the Cu2+ concentration showed a good linear relationship with the fluorescence decline of the sensor, and the LOD is 10.12 nM, which was lower than the U.S. Environmental Protection Agency (EPA) defined limit (20 µM). Moreover, aiming to attain visual analysis, colorimetric method was also used for rapidly detecting Cu2+ by capturing the change in fluorescence color. Interestingly, the proposed approach has successfully been applied for the detection of Cu2+ in real samples (i.e., environment water, food and traditional Chinese medicine) with satisfactory results, which provides a promising strategy for the detection of Cu2+ in practical application with the merits of being rapid, simple and sensitive.


Assuntos
Compostos de Cádmio , Pontos Quânticos , Humanos , Cobre/análise , Limite de Detecção , Telúrio , Espectrometria de Fluorescência/métodos , Corantes Fluorescentes , Glutationa , Íons
14.
Methods Mol Biol ; 2606: 63-72, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-36592308

RESUMO

Base editors, mostly cytidine base editors (CBEs) and adenine base editors (ABEs), are powerful tools for precise base editing. However, current base editors can only edit either adenines or cytosines. Thus, our lab has developed a dual base editor (A&C-BEmax) through the fusion of cytidine and adenosine deaminases to Cas9n to achieve both C•G to T•A and A•T to G•C mutations, which enables A/C simultaneous conversion in the same allele (up to 30%) in human cells. Here, we described a protocol for the usage of A&C-BEmax in human cells. This protocol includes standard dual base editing experiments in HEK293T cells and data analysis of dual base editing outcomes using BE-analyzer. All the workflow of experiments can be completed within 2-3 weeks.


Assuntos
Sistemas CRISPR-Cas , Edição de Genes , Humanos , Edição de Genes/métodos , Células HEK293 , Mutação , Citosina , Adenina
15.
J Cardiothorac Vasc Anesth ; 37(5): 788-800, 2023 05.
Artigo em Inglês | MEDLINE | ID: mdl-36435722

RESUMO

The effects of the transversus thoracic muscle plane (TTP) block on postoperative pain have become increasingly controversial. This meta-analysis compared the effects of the TTP block versus no block on postoperative analgesia and side effects to determine whether this new technique is a reliable alternative for pain management. PubMed, Cochrane Library, Embase, Web of Science, ClinicalTrials.gov, China National Knowledge Infrastructure, Chongqing VIP information, and Wanfang Data were searched for clinical studies investigating the analgesic effect of the TTP block compared to controls. The primary outcomes included the postoperative pain scores at rest and during movement, morphine consumption in 24 hours, and the rate of postoperative nausea and vomiting (PONV). Eleven randomized controlled trials (RCTs), including 682 patients, were reviewed. The meta-analysis showed that the TTP block significantly could reduce the pain scores at 0 (at rest: mean difference [MD], -2.28; 95% CI: -2.67 to -1.90) (during movement: MD: -2.09, 95% CI: -2.62 to -1.56) and 12 hours (at rest: -1.42, 95% CI: -2.03 to -0.82) (during movement: MD: -2.13, 95% CI: -2.80 to -1.46) after surgery, 24-hour postoperative analgesic consumption (MD: -23.18, 95% CI: -33.71 to -12.66), and the incidence of PONV (odds ratio, 0.36, 95% CI: 0.15-0.88). Furthermore, the trial sequence analysis confirmed the result of less 24-hour postoperative analgesic consumption in the TTP block group. As a novel technique, the TTP block exhibited a superior postoperative analgesic effect during the early postoperative period. Nevertheless, additional well-designed RCTs are needed.


Assuntos
Analgésicos Opioides , Bloqueio Nervoso , Humanos , Analgésicos Opioides/efeitos adversos , Náusea e Vômito Pós-Operatórios/epidemiologia , Náusea e Vômito Pós-Operatórios/prevenção & controle , Náusea e Vômito Pós-Operatórios/induzido quimicamente , Bloqueio Nervoso/efeitos adversos , Bloqueio Nervoso/métodos , Ensaios Clínicos Controlados Aleatórios como Assunto , Analgésicos , Dor Pós-Operatória/etiologia , Dor Pós-Operatória/prevenção & controle , Ultrassonografia de Intervenção/métodos , Músculos , Músculos Abdominais/diagnóstico por imagem
16.
Mol Ther ; 31(3): 744-759, 2023 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-36457249

RESUMO

Editing efficiency is pivotal for the efficacies of CRISPR-based gene therapies. We found that fusing an HMG-D domain to the N terminus of SpCas9 (named efficiency-enhanced Cas9 [eeCas9]) significantly increased editing efficiency by 1.4-fold on average. The HMG-D domain also enhanced the activities of non-NGG PAM Cas9 variants, high-fidelity Cas9 variants, smaller Cas9 orthologs, Cas9-based epigenetic regulators, and base editors in cell lines. Furthermore, we discovered that eeCas9 exhibits comparable off-targeting effects with Cas9, and its specificity could be increased through ribonucleoprotein delivery or using hairpin single-guide RNAs and high-fidelity Cas9s. The entire eeCas9 could be packaged into an adeno-associated virus vector and exhibited a 1.7- to 2.6-fold increase in editing efficiency targeting the Pcsk9 gene in mice, leading to a greater reduction of serum cholesterol levels. Moreover, the efficiency of eeA3A-BE3 also surpasses that of A3A-BE3 in targeting the promoter region of γ-globin genes or BCL11A enhancer in human hematopoietic stem cells to reactivate γ-globin expression for the treatment of ß-hemoglobinopathy. Together, eeCas9 and its derivatives are promising editing tools that exhibit higher activity and therapeutic efficacy for both in vivo and ex vivo therapeutics.


Assuntos
Proteína 9 Associada à CRISPR , Sistemas CRISPR-Cas , Animais , Humanos , Camundongos , Proteína 9 Associada à CRISPR/genética , Proteína 9 Associada à CRISPR/metabolismo , Edição de Genes , Pró-Proteína Convertase 9/genética , Pró-Proteína Convertase 9/metabolismo , gama-Globinas/genética , Terapia Genética
17.
J Am Soc Nephrol ; 34(4): 572-589, 2023 04 01.
Artigo em Inglês | MEDLINE | ID: mdl-36522157

RESUMO

SIGNIFICANCE STATEMENT: Epigenetic changes have been proposed to mediate nephron endowment during development, a critical determinant of future renal disease development. Hydroxymethyl cytosine, an epigenetic modification important for gene regulation, is abundant in the human kidney, but its physiologic role and the role of DNA demethylase enzymes encoded by the Tet1 , Tet2 , or Tet3 , which mediate cytosine hydroxymethylation, are unclear. By genetically deleting Tet1 , Tet2 , or Tet3 in nephron progenitors in mice, the authors showed that combined Tet2 and Tet3 loss in nephron progenitors cause defective kidney development, leading to kidney failure and perinatal death. Tet2 and Tet3 deletion also caused an alteration in demethylation and expression of genes critical for nephron formation. These findings establish that Tet2- and Tet3 -mediated cytosine hydroxymethylation in nephron progenitors plays a critical role in nephron endowment. BACKGROUND: Nephron endowment is a key determinant of hypertension and renal disease in later life. Epigenetic changes have been proposed to mediate fetal programming and nephron number. DNA cytosine methylation, which plays a critical role in gene regulation, is affected by proteins encoded by the ten-eleven translocation (TET) DNA demethylase gene family ( Tet1 , Tet2 , and Tet3 ), but the roles of TET proteins in kidney development and nephron endowment have not been characterized . METHODS: To study whether epigenetic changes-specifically, active DNA hydroxymethylation mediated by Tet1 , Tet2 , and Tet3- are necessary for nephron progenitor differentiation and nephron endowment, we generated mice with deletion of Tet1 , Tet2 , or Tet3 in Six2-positive nephron progenitors cells (NPCs). We then performed unbiased omics profiling, including whole-genome bisulfite sequencing on isolated Six2-positive NPCs and single-cell RNA sequencing on kidneys from newborn mice. RESULTS: We did not observe changes in kidney development or function in mice with NPC-specific deletion of Tet1 , Tet2 , Tet3 or Tet1 / Tet2 , or Tet1 / Tet3 . On the other hand, mice with combined Tet2 and Tet3 loss in Six2-positive NPCs failed to form nephrons, leading to kidney failure and perinatal death. Tet2 and Tet3 loss in Six2 -positive NPCs resulted in defective mesenchymal to epithelial transition and renal vesicle differentiation. Whole-genome bisulfite sequencing, single-cell RNA sequencing, and gene and protein expression analysis identified a defect in expression in multiple genes, including the WNT- ß -catenin signaling pathway, due to a failure in demethylation of these loci in the absence of Tet2 and Tet3 . CONCLUSIONS: These findings suggest that Tet2- and Tet3 -mediated active cytosine hydroxymethylation in NPCs play a key role in kidney development and nephron endowment.


Assuntos
Dioxigenases , Morte Perinatal , Insuficiência Renal , Gravidez , Feminino , Camundongos , Humanos , Animais , Citosina/metabolismo , Dioxigenases/metabolismo , Néfrons/metabolismo , Diferenciação Celular/genética , Células-Tronco/fisiologia , Metilação de DNA , Insuficiência Renal/genética , Oxigenases de Função Mista/metabolismo , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/metabolismo , Proteínas do Tecido Nervoso/genética , Proteínas de Homeodomínio/genética
18.
Nat Biotechnol ; 41(5): 663-672, 2023 05.
Artigo em Inglês | MEDLINE | ID: mdl-36357717

RESUMO

Cytosine base editors (CBEs) efficiently generate precise C·G-to-T·A base conversions, but the activation-induced cytidine deaminase/apolipoprotein B mRNA-editing enzyme catalytic polypeptide-like (AID/APOBEC) protein family deaminase component induces considerable off-target effects and indels. To explore unnatural cytosine deaminases, we repurpose the adenine deaminase TadA-8e for cytosine conversion. The introduction of an N46L variant in TadA-8e eliminates its adenine deaminase activity and results in a TadA-8e-derived C-to-G base editor (Td-CGBE) capable of highly efficient and precise C·G-to-G·C editing. Through fusion with uracil glycosylase inhibitors and further introduction of additional variants, a series of Td-CBEs was obtained either with a high activity similar to that of BE4max or with higher precision compared to other reported accurate CBEs. Td-CGBE/Td-CBEs show very low indel effects and a background level of Cas9-dependent or Cas9-independent DNA/RNA off-target editing. Moreover, Td-CGBE/Td-CBEs are more efficient in generating accurate edits in homopolymeric cytosine sites in cells or mouse embryos, suggesting their accuracy and safety for gene therapy and other applications.


Assuntos
Citosina , Edição de Genes , Camundongos , Animais , Edição de Genes/métodos , Citosina/metabolismo , Aminoidrolases/metabolismo , RNA , Sistemas CRISPR-Cas/genética , Citidina Desaminase/genética , Citidina Desaminase/metabolismo
19.
Adv Sci (Weinh) ; 9(34): e2201352, 2022 12.
Artigo em Inglês | MEDLINE | ID: mdl-36266974

RESUMO

Optogenetic genome engineering is a powerful technology for high-resolution spatiotemporal genetic manipulation, especially for in vivo studies. It is difficult to generate stable transgenic animals carrying a tightly regulated optogenetic system, as its long-term expression induces high background activity. Here, the generation of an enhanced photoactivatable Cre recombinase (ePA-Cre) transgenic mouse strain with stringent light responsiveness and high recombination efficiency is reported. Through serial optimization, ePA-Cre is developed to generate a transgenic mouse line that exhibits 175-fold induction upon illumination. Efficient light-dependent recombination is detected in embryos and various adult tissues of ePA-Cre mice crossed with the Ai14 tdTomato reporter. Importantly, no significant background Cre activity is detected in the tested tissues except the skin. Moreover, efficient light-inducible cell ablation is achieved in ePA-Cre mice crossed with Rosa26-LSL-DTA mice. In conclusion, ePA-Cre mice offer a tightly inducible, highly efficient, and spatiotemporal-specific genome engineering tool for multiple applications.


Assuntos
Camundongos Transgênicos , Camundongos , Animais
20.
Drug Des Devel Ther ; 16: 2161-2175, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35821701

RESUMO

Dexmedetomidine, a specific α2 adrenergic receptor agonist, is highly frequently used in the perioperatively for its favorable pharmacology, such as mitigating postoperative cognitive dysfunction. Increasing attention has been recently focused on the effect of whether dexmedetomidine influences cancer recurrence, which urges the discussion of the role of dexmedetomidine in tumor-progressive factors. The pharmacologic characteristics of dexmedetomidine, the tumor-progressive factors in the perioperative period, and the relationships between dexmedetomidine and tumor-progressive factors were described in this review. Available evidence suggests that dexmedetomidine could reduce the degree of immune function suppression, such as keeping the number of CD3+ cells, NK cells, CD4+/CD8+ ratio, and Th1/Th2 ratio stable and decreasing the level of proinflammatory cytokine (interleukin 6 and tumor necrosis factor-alpha) during cancer operations. However, dexmedetomidine exhibits different roles in cell biological behavior depending on cancer cell types. The conclusions on whether dexmedetomidine would influence cancer recurrence could not be currently drawn for the lack of strong clinical evidence. Therefore, this is still a new area that needs further exploration.


Assuntos
Dexmedetomidina , Neoplasias , Agonistas de Receptores Adrenérgicos alfa 2/farmacologia , Citocinas , Dexmedetomidina/farmacologia , Humanos , Neoplasias/tratamento farmacológico , Período Perioperatório
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