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1.
Genome Res ; 33(10): 1833-1847, 2023 10.
Artigo em Inglês | MEDLINE | ID: mdl-37914227

RESUMO

Structural variations have emerged as an important driving force for genome evolution and phenotypic variation in various organisms, yet their contributions to genetic diversity and adaptation in domesticated animals remain largely unknown. Here we constructed a pangenome based on 250 sequenced individuals from 32 pig breeds in Eurasia and systematically characterized coding sequence presence/absence variations (PAVs) within pigs. We identified 308.3-Mb nonreference sequences and 3438 novel genes absent from the current reference genome. Gene PAV analysis showed that 16.8% of the genes in the pangene catalog undergo PAV. A number of newly identified dispensable genes showed close associations with adaptation. For instance, several novel swine leukocyte antigen (SLA) genes discovered in nonreference sequences potentially participate in immune responses to productive and respiratory syndrome virus (PRRSV) infection. We delineated previously unidentified features of the pig mobilome that contained 490,480 transposable element insertion polymorphisms (TIPs) resulting from recent mobilization of 970 TE families, and investigated their population dynamics along with influences on population differentiation and gene expression. In addition, several candidate adaptive TE insertions were detected to be co-opted into genes responsible for responses to hypoxia, skeletal development, regulation of heart contraction, and neuronal cell development, likely contributing to local adaptation of Tibetan wild boars. These findings enhance our understanding on hidden layers of the genetic diversity in pigs and provide novel insights into the role of SVs in the evolutionary adaptation of mammals.


Assuntos
Cruzamento , Genoma , Humanos , Animais , Suínos , Variação Genética , Mamíferos
2.
Anal Chem ; 96(25): 10380-10390, 2024 Jun 25.
Artigo em Inglês | MEDLINE | ID: mdl-38860916

RESUMO

To reduce the risk of atherosclerotic disease, it is necessary to not only diagnose the presence of atherosclerotic plaques but also assess the vulnerability risk of plaques. Accurate detection of the reactive oxygen species (ROS) level at plaque sites represents a reliable way to assess the plaque vulnerability. Herein, through a simple one-pot reaction, two near-infrared (NIR) fluorescent dyes, one is ROS responsive and the other is inert to ROS, are coassembled in an amphiphilic amino acid-assembled nanoparticle. In the prepared NIR fluorescent amino acid nanoparticle (named FANP), the fluorescent properties and ROS-responsive behaviors of the two fluorescent dyes are well maintained. Surface camouflage through red blood cell membrane (RBCM) encapsulation endows the finally obtained FANP@RBCM nanoprobe with not only further reduced cytotoxicity and improved biocompatibility but also increased immune escape capability, prolonged blood circulation time, and thus enhanced accumulation at atherosclerotic plaque sites. In vitro and in vivo experiments demonstrate that FANP@RBCM not only works well in probing the occurrence of atherosclerotic plaques but also enables plaque vulnerability assessment through the accurate detection of the ROS level at plaque sites in a reliable ratiometric mode, thereby holding great promise as a versatile tool for the diagnosis and risk assessment of atherosclerotic disease.


Assuntos
Aminoácidos , Corantes Fluorescentes , Nanopartículas , Placa Aterosclerótica , Espécies Reativas de Oxigênio , Placa Aterosclerótica/diagnóstico por imagem , Animais , Espécies Reativas de Oxigênio/metabolismo , Corantes Fluorescentes/química , Nanopartículas/química , Camundongos , Aminoácidos/química , Humanos , Medição de Risco , Imagem Óptica , Raios Infravermelhos , Células RAW 264.7
3.
Anal Chem ; 96(6): 2692-2701, 2024 02 13.
Artigo em Inglês | MEDLINE | ID: mdl-38305871

RESUMO

In recent years, the CRISPR/Cas12a-based sensing strategy has shown significant potential for specific target detection due to its rapid and sensitive characteristics. However, the "always active" biosensors are often insufficient to manipulate nucleic acid sensing with high spatiotemporal control. It remains crucial to develop nucleic acid sensing devices that can be activated at the desired time and space by a remotely applied stimulus. Here, we integrated photoactivation with the CRISPR/Cas12a system for DNA and RNA detection, aiming to provide high spatiotemporal control for nucleic acid sensing. By rationally designing the target recognition sequence, this photoactivation CRISPR/Cas12a system could recognize HPV16 and survivin, respectively. We combined the lateral flow assay strip test with the CRISPR/Cas12a system to realize the visualization of nucleic acid cleavage signals, displaying potential instant test application capabilities. Additionally, we also successfully realized the temporary control of its fluorescent sensing activity for survivin by photoactivation in vivo, allowing rapid detection of target nucleic acids and avoiding the risk of contamination from premature leaks during storage. Our strategy suggests that the CRISPR/Cas12a platform can be triggered by photoactivation to sense various targets, expanding the technical toolbox for precise biological and medical analysis. This study represents a significant advancement in nucleic acid sensing and has potential applications in disease diagnosis and treatment.


Assuntos
Técnicas Biossensoriais , Ácidos Nucleicos , Sistemas CRISPR-Cas/genética , Survivina/genética , Biomarcadores , Testes Imediatos
4.
Anal Chem ; 96(16): 6426-6435, 2024 Apr 23.
Artigo em Inglês | MEDLINE | ID: mdl-38604773

RESUMO

Sensors designed based on the trans-cleavage activity of CRISPR/Cas12a systems have opened up a new era in the field of biosensing. The current design of CRISPR/Cas12-based sensors in the "on-off-on" mode mainly focuses on programming the activator strand (AS) to indirectly switch the trans-cleavage activity of Cas12a in response to target information. However, this design usually requires the help of additional auxiliary probes to keep the activator strand in an initially "blocked" state. The length design and dosage of the auxiliary probe need to be strictly optimized to ensure the lowest background and the best signal-to-noise ratio. This will inevitably increase the experiment complexity. To solve this problem, we propose using AS after the "RESET" effect to directly regulate the Cas12a enzymatic activity. Initially, the activator strand was rationally designed to be embedded in a hairpin structure to deprive its ability to activate the CRISPR/Cas12a system. When the target is present, target-mediated strand displacement causes the conformation change in the AS, the hairpin structure is opened, and the CRISPR/Cas12a system is reactivated; the switchable structure of AS can be used to regulate the degree of activation of Cas12a according to the target concentration. Due to the advantages of low background and stability, the CRISPR/Cas12a-based strategy can not only image endogenous biomarkers (miR-21) in living cells but also enable long-term and accurate imaging analysis of the process of exogenous virus invasion of cells. Release and replication of virus genome in host cells are indispensable hallmark events of cell infection by virus; sensitive monitoring of them is of great significance to revealing virus infection mechanism and defending against viral diseases.


Assuntos
Técnicas Biossensoriais , Sistemas CRISPR-Cas , MicroRNAs , Sistemas CRISPR-Cas/genética , Técnicas Biossensoriais/métodos , Humanos , MicroRNAs/análise , MicroRNAs/metabolismo , Regulação Alostérica , Proteínas Associadas a CRISPR/metabolismo , Endodesoxirribonucleases/metabolismo , Endodesoxirribonucleases/química , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Células HEK293
5.
Anal Chem ; 95(19): 7796-7803, 2023 05 16.
Artigo em Inglês | MEDLINE | ID: mdl-37129996

RESUMO

The sensitive and accurate detection of biomarkers plays an important role in clinical diagnosis and drug discovery. Currently, amplification-based methods for biomarker detection are widely explored. However, the key challenges of these methods are limited reproducibility and high background noise. To overcome these limitations, we develop a robust plasmonic nanoparticle-coupled single-molecule kinetic fingerprinting (PNP-SMKF) method to achieve ultrasensitive detection of protein kinase A (PKA). Transient binding of a short fluorescent probe with the genuine target produces a distinct kinetic signature that is completely different from that of the background signal, allowing us to recognize PKA sensitively. Importantly, integrating a plasmonic nanoparticle efficiently breaks the concentration limit of the imager strand for single-molecule imaging, thus achieving a much faster imaging speed. A limit of detection (LOD) of as low as 0.0005 U/mL is readily realized. This method holds great potential as a versatile platform for enzyme detection and inhibitor screening in the future.


Assuntos
Técnicas Biossensoriais , Nanopartículas , Reprodutibilidade dos Testes , Nanotecnologia , Biomarcadores , Corantes Fluorescentes/química , Limite de Detecção , Técnicas Biossensoriais/métodos
6.
Anal Chem ; 95(42): 15725-15735, 2023 10 24.
Artigo em Inglês | MEDLINE | ID: mdl-37819747

RESUMO

The trans-cleavage activity of CRISPR/Cas12a has been widely used in biosensing. However, many CRISPR/Cas12a-based biosensors, especially those that work in "on-off-on" mode, usually suffer from high background and thus impossible intracellular application. Herein, this problem is efficiently overcome by elaborately designing the activator strand (AS) of CRISPR/Cas12a using the "RESET" effect found by our group. The activation ability of the as-designed AS to CRISPR/Cas12a can be easily inhibited, thus assuring a low background for subsequent biosensing applications, which not only benefits the detection sensitivity improvement of CRISPR/Cas12a-based biosensors but also promotes their applications in live cells as well as makes it possible to design high-performance biosensors with greatly improved flexibility, thus achieving the analysis of a wide range of targets. As examples, by using different strategies such as strand displacement, strand cleavage, and aptamer-substrate interaction to reactivate the inhibited enzyme activity, several CRISPR/Cas12a-based biosensing systems are developed for the sensitive and specific detection of different targets, including nucleic acid (miR-21), biological small molecules (ATP), and enzymes (hOGG1), giving the detection limits of 0.96 pM, 8.6 µM, and 8.3 × 10-5 U/mL, respectively. Thanks to the low background, these biosensors are demonstrated to work well for the accurate imaging analysis of different biomolecules in live cells. Moreover, we also demonstrate that these sensing systems can be easily combined with lateral flow assay (LFA), thus holding great potential in point-of-care testing, especially in poorly equipped or nonlaboratory environments.


Assuntos
Técnicas Biossensoriais , Ácidos Nucleicos , Sistemas CRISPR-Cas/genética , Bioensaio , Processamento de Imagem Assistida por Computador , Oligonucleotídeos
7.
Drug Dev Res ; 83(2): 389-396, 2022 04.
Artigo em Inglês | MEDLINE | ID: mdl-34402084

RESUMO

Cataract is the number one cause of blindness in the world. Fibrosis of the lens is the main cause of cataract. Pathological epithelial-mesenchymal transition (EMT) plays an important role in the development of fibrotic cataract. Inhibition of EMT may be an effective treatment for fibrosis of lens epithelial cells. Naringin (NRG) is one of the major citrus flavonoids, which has many pharmacological properties, including anti-inflammatory and cardioprotective. However, the effect of NRG on cataract induced by abnormal fibrosis of LECs is not clear. Herein, we found NRG inhibited transforming growth factor ß2 (TGFß2)-induced SRA01/04 cell viability. Additionally, NRG inhibited TGFß2-induced cell migration and EMT. We further noticed that NRG inhibited autophagy and Smad2/3 phosphorylation in LECs. We therefore thought Naringenin inhibited autophagy and EMT of human LECs by regulating the Smad2/3 pathway. NRG could therefore serve as a promising drug for cataract treatment.


Assuntos
Catarata , Transição Epitelial-Mesenquimal , Autofagia , Catarata/tratamento farmacológico , Catarata/metabolismo , Catarata/patologia , Células Epiteliais , Fibrose , Flavanonas , Humanos , Transdução de Sinais , Proteína Smad2
8.
Anal Chem ; 92(20): 13936-13944, 2020 10 20.
Artigo em Inglês | MEDLINE | ID: mdl-32972135

RESUMO

The fat mass and obesity-associated enzyme (FTO) can catalyze the demethylation of N6-methyladenosine (m6A) residues in mRNA, regulates the cellular level of m6A modification, and plays a critical role in human obesity and cancers. Herein, we develop a single-quantum-dot (QD)-based fluorescence resonance energy transfer (FRET) sensor for the identification of specific FTO demethylase inhibitors. The FTO-mediated demethylation of m6A can induce the cleavage of demethylated DNA to generate the biotinylated DNA fragments, which may function as capture probes to assemble the Cy5-labeled reporter probes onto the QD surface, enabling the occurrence of FRET between the QD and Cy5. The presence of inhibitors can inhibit the FTO demethylation and consequently abolish FRET between the QD and Cy5. The inhibition effect of inhibitors upon FTO demethylation can be simply evaluated by monitoring the decrease of Cy5 counts. We use this nanosensor to screen several small-molecule inhibitors and identify diacerein as a highly selective inhibitor of FTO. Diacerein can inhibit the demethylation activity of endogenous FTO in HeLa cells. Interestingly, diacerein is neither a structural mimic of 2-oxoglutarate (2-OG) nor a chelator of metal ions, and it can selectively inhibit FTO demethylation by competitively binding the m6A-containing substrate.


Assuntos
Dioxigenase FTO Dependente de alfa-Cetoglutarato/antagonistas & inibidores , Técnicas Biossensoriais/métodos , Inibidores Enzimáticos/química , Transferência Ressonante de Energia de Fluorescência , Pontos Quânticos/química , Adenosina/análogos & derivados , Adenosina/química , Dioxigenase FTO Dependente de alfa-Cetoglutarato/genética , Dioxigenase FTO Dependente de alfa-Cetoglutarato/metabolismo , Antraquinonas/química , Antraquinonas/metabolismo , Sítios de Ligação , Carbocianinas/química , Domínio Catalítico , Desmetilação do DNA , Inibidores Enzimáticos/metabolismo , Células HeLa , Humanos , Simulação de Dinâmica Molecular , RNA Mensageiro/química , RNA Mensageiro/metabolismo , RNA Interferente Pequeno/metabolismo
9.
J Bone Miner Metab ; 37(5): 805-814, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-30706148

RESUMO

This study evaluated the preventative effects of metformin (Met) on glucocorticoid (GC)-induced osteoporosis in a rat model, compared with alendronate (Aln). Twenty-eight 3-month-old female Sprague-Dawley rats were randomly assigned into four groups: normal control (Ctr), methylprednisolone (MP, 13 mg/kg/day, sc, 5 days per week), MP plus Aln orally (1 mg/kg/day), and MP plus Met orally (200 mg/kg/day). After 9 weeks, serum bone metabolic biochemistry, bone densitometry and histomorphometry were performed. The GC-induced osteoporosis model was characterized by decreased osteocalcin, increased tartrate-resistant acid phosphatase-5b (TRAP-5b), and decreased bone mineral density (BMD) in the femur and fifth lumbar vertebra (L5). Histomorphometrically, MP significantly decreased trabecular bone volume, decreased bone formation and increased bone resorption in proximal metaphysis, compared with the controls. Aln and Met increased the BMDs of femur (0.305 ± 0.011 vs. 0.280 ± 0.012, P < 0.05; 0.304 ± 0.019 vs. 0.280 ± 0.012, P < 0.05) and L5 (0.399 ± 0.029 vs. 0.358 ± 0.022, P < 0.05; 0.397 ± 0.022 vs. 0.358 ± 0.022, P < 0.05), compared with the model group. Met increased osteocalcin and decreased TRAP-5b, but Aln only decreased TRAP-5b, compared with model group. In histomorphometry of tibial proximal metaphysis, Aln and Met increased trabecular bone volume (39.21 ± 2.46 vs. 30.98 ± 5.83, P < 0.05; 38.97 ± 5.56 vs. 30.98 ± 5.83, P < 0.05), while Met increased the bone formation dynamic parameters and decreased bone resorption dynamic parameters, but Aln just decreased bone resorption dynamic parameters, compared with model group significantly. These findings suggest that metformin prevents GC-induced bone loss by suppressing bone resorption and stimulating bone formation in trabecular bone. The action mode of metformin was different from alendronate, which only suppressed bone resorption.


Assuntos
Glucocorticoides/efeitos adversos , Metformina/uso terapêutico , Osteoporose/induzido quimicamente , Osteoporose/tratamento farmacológico , Substâncias Protetoras/uso terapêutico , Alendronato/farmacologia , Animais , Glicemia/metabolismo , Peso Corporal/efeitos dos fármacos , Densidade Óssea/efeitos dos fármacos , Feminino , Fêmur/efeitos dos fármacos , Fêmur/fisiologia , Fêmur/fisiopatologia , Lipídeos/sangue , Vértebras Lombares/efeitos dos fármacos , Vértebras Lombares/patologia , Vértebras Lombares/fisiopatologia , Metformina/farmacologia , Metilprednisolona/farmacologia , Osteocalcina/sangue , Osteoporose/sangue , Osteoporose/fisiopatologia , Substâncias Protetoras/farmacologia , Ratos Sprague-Dawley , Fosfatase Ácida Resistente a Tartarato/sangue
10.
J Cell Mol Med ; 21(7): 1361-1372, 2017 07.
Artigo em Inglês | MEDLINE | ID: mdl-28158928

RESUMO

Activation of osteoblasts in bone formation and osteoclasts in bone resorption is important during the bone fracture healing process. There has been a long interest in identifying and developing a natural therapy for bone fracture healing. In this study, we investigated the regulation of osteoclast differentiation by baicalin, which is a natural molecule extracted from Eucommiaulmoides (small tree native to China). It was determined that baicalin enhanced osteoclast maturation and bone resorption activity in a dose-dependent manner. Moreover, this involves the activation of MAPK, increased Mitf nuclear translocation and up-regulation of downstream osteoclast-related target genes expression. The baicalin-induced effect on osteoclast differentiation can be mimicked by specific inhibitors of p-ERK (U0126) and the Mitf-specific siRNA, respectively. Protein-ligand docking prediction identified that baicalin might bind to RANK, which is the upstream receptor of p-ERK/Mitf signalling in osteoclasts. This indicated that RANK might be the binding target of baicalin. In sum, our findings revealed baicalin increased osteoclast maturation and function via p-ERK/Mitf signalling. In addition, the results suggest that baicalin can potentially be used as a natural product for the treatment of bone fracture.


Assuntos
Flavonoides/administração & dosagem , Fraturas Ósseas/tratamento farmacológico , MAP Quinase Quinase 1/genética , Fator de Transcrição Associado à Microftalmia/genética , Receptor Ativador de Fator Nuclear kappa-B/genética , Animais , Reabsorção Óssea/genética , Reabsorção Óssea/fisiopatologia , Butadienos/administração & dosagem , Diferenciação Celular/efeitos dos fármacos , Fraturas Ósseas/genética , Fraturas Ósseas/fisiopatologia , Humanos , MAP Quinase Quinase 1/antagonistas & inibidores , Camundongos , Fator de Transcrição Associado à Microftalmia/antagonistas & inibidores , Nitrilas/administração & dosagem , Osteoclastos/efeitos dos fármacos , Osteoclastos/metabolismo , Osteogênese/genética , Ligação Proteica , Células RAW 264.7 , Transdução de Sinais/efeitos dos fármacos
11.
J Asian Nat Prod Res ; 19(2): 176-187, 2017 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-28024413

RESUMO

Ferulic acid (FA) is an active component of the traditional Chinese herb Angelica sinensis. Numerous health benefits have been attributed to FA, but few studies have investigated the effects of FA on osteoblasts (Obs). Our work studied the effects of FA on proliferation, differentiation, and mineralization of rat calvarial Obs and examined the signaling pathways involved. Cell proliferation and differentiation were evaluated by Cell Counting Kit-8 (CCK-8) and alkaline phosphatase (ALP) assay kit, respectively. Cyclic guanosine monophosphate (cGMP)-dependent protein kinase II (PKGII) expression was silenced by small interfering RNA (siRNA). The mRNA expression was investigated by semi-quantitative PCR. FA (40-2560 µM) promoted Ob proliferation and differentiation; at 40-640 µM, FA stimulated calcified nodule formation and increased the expression of osteogenic genes encoding osteopontin and collagen-l. FA (40-2560 µM) increased cGMP levels in Obs and upregulated the expression of PKGII, EnaCα, and ENaCγ mRNAs. Downregulated ENaCα mRNA expression in Obs transfected with the siRNA for PKGII was reversed when FA was introduced into Obs. These results demonstrated that FA promoted proliferation, differentiation, and mineralization of Obs in vitro, and enhanced osteogenic genes expression partly through the cGMP-PKGII-ENaC signaling pathway.


Assuntos
Proliferação de Células/efeitos dos fármacos , Ácidos Cumáricos/farmacologia , Osteoblastos/efeitos dos fármacos , Fosfatase Alcalina/metabolismo , Animais , Diferenciação Celular , GMP Cíclico/metabolismo , Estrutura Molecular , Osteogênese/efeitos dos fármacos , Osteopontina , RNA Mensageiro/metabolismo , RNA Interferente Pequeno/genética , Ratos , Transdução de Sinais/efeitos dos fármacos
12.
Zhongguo Zhong Yao Za Zhi ; 42(2): 280-284, 2017 Jan.
Artigo em Chinês | MEDLINE | ID: mdl-28948731

RESUMO

To determine the optimum aqueous extract protocol for Yugubao traditional Chinese medicines formula by using orthogonal experiment design. Through serum pharmacology research, L9(34) orthogonal design with single factor investigation was used to optimize the aqueous extract protocol for Yugubao formula. The effect of water extraction on activity of alkaline phosphatase (ALP) in osteoblast was referred as the evaluation index for investigating four factors: water consumption (A), heating time (B), soaking time (C), and number of decocting (D), analyzing the optimum extraction conditions, and verifying the effectiveness of this process. The optimum aqueous extract protocol for Yugubao was as follows: adding 8 times water into Chinese medical materials, heating for 60 min, soaking for 30 min, and decocting for 1 time. The drug serum of this aqueous extract of Yugubao could significantly up-regulate the osteogenic genes expression. The optimum aqueous extract protocol for Yugubao formula was established in this experiment, providing evidence for the development and utilization of Yugubao traditional Chinese medicines formula.


Assuntos
Fracionamento Químico/métodos , Medicamentos de Ervas Chinesas/química , Extratos Vegetais/química , Medicina Tradicional Chinesa , Projetos de Pesquisa , Tecnologia Farmacêutica , Água
13.
Mol Biol Rep ; 41(4): 2161-9, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-24481877

RESUMO

The amiloride-sensitive epithelial sodium channel (ENaC) is a major contributor to intracellular sodium homeostasis. In addition to epithelial cells, osteoblasts (Obs) express functional ENaCs. Moreover, a correlation between bone Na content and bone disease has been reported, suggesting that ENaC-mediated Na(+) regulation may influence osteogenesis. Obs were isolated and cultured by enzyme digestion. Cell proliferation and differentiation were evaluated by WST-8 assay kit and AKP assay kit respectively. PKGII expression was silenced by siRNA. The mRNA expression was investigated by semi-quantitative PCR and the protein expression was determined by Western-blot. The cell-permeable cGMP analog 8-(4-chlorophenylthio)-cGMP (8-pCPT-cGMP) increased α-ENaC channel expression in primary rat Obs as indicated by RT-PCR. In addition, 8-pCPT-cGMP stimulation enhanced expression of the mRNA encoding cGMP-dependent protein kinases II (PKGII). The cGMP analog also promoted osteoblast proliferation, differentiation and induced the expression of several osteogenic genes, including core binding factor al, osteocalcin, alkaline phosphatase, collagen type I, and osteopontin. Furthermore, the expression of α-ENaC, the main functional subunit of ENaC, was reduced when a small interfering RNA specific for PKGII was introduced into Obs. Treatment with 8-pCPT-cGMP in cells transfected with the siRNA for PKGII partially reversed downregulated α-ENaC mRNA expression. Our results suggest that 8-pCPT-cGMP stimulates proliferation, differentiation, and osteogenic gene expression in Obs through cGMP/PKGII-dependent regulation of ENaC channel expression. The cGMP/PKGII signaling pathway is a potential target for pharmaceutical interventions to treat metabolic bone diseases.


Assuntos
Proteína Quinase Dependente de GMP Cíclico Tipo II/metabolismo , GMP Cíclico/metabolismo , Canais Epiteliais de Sódio/metabolismo , Osteoblastos/metabolismo , Osteogênese/fisiologia , Transdução de Sinais , Animais , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/genética , Proliferação de Células/efeitos dos fármacos , GMP Cíclico/análogos & derivados , GMP Cíclico/farmacologia , Proteína Quinase Dependente de GMP Cíclico Tipo II/genética , Canais Epiteliais de Sódio/genética , Regulação da Expressão Gênica/efeitos dos fármacos , Camundongos Knockout , Osteoblastos/citologia , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , Interferência de RNA , RNA Interferente Pequeno/genética , Ratos , Tionucleotídeos/farmacologia
14.
ACS Nano ; 2024 Jan 17.
Artigo em Inglês | MEDLINE | ID: mdl-38231016

RESUMO

The spacing between cells has a significant impact on cell-cell interactions, which are critical to the fate and function of both individual cells and multicellular organisms. However, accurately measuring the distance between cell membranes and the variations between different membranes has proven to be a challenging task. In this study, we employ metal-induced energy transfer (MIET) imaging/spectroscopy to determine and track the intermembrane distance and variations with nanometer precision. We have developed a DNA-based molecular adhesive called the DNA nanobrush, which serves as a cellular adhesive for connecting the plasma membranes of different cells. By manipulating the number of base pairs within the DNA nanobrush, we can modify various aspects of membrane-membrane interactions such as adhesive directionality, distance, and forces. We demonstrate that such nanometer-level changes can be detected with MIET imaging/spectroscopy. Moreover, we successfully employed MIET to measure distance variations between a cellular plasma membrane and a model membrane. This experiment not only showcases the effectiveness of MIET as a powerful tool for accurately quantifying membrane-membrane interactions but also validates the potential of DNA nanobrushes as cellular adhesives. This innovative method holds significant implications for advancing the study of multicellular interactions.

15.
Molecules ; 18(2): 1693-703, 2013 Jan 28.
Artigo em Inglês | MEDLINE | ID: mdl-23358326

RESUMO

Alzheimer's disease (AD) pathogenesis involves an imbalance between free radical formation and destruction. In order to obtain a novel preclinical anti-AD drug candidate, we synthesized a series of novel hydroxyl chalcone analogs which possessed anti-free radical activity, and screened their effects on scavenging 2,2-diphenyl-1-picrylhydrazyl (DPPH) and OH free radicals in vitro. Compound C7, 4,2'-dihydroxy-3,5-dimethoxychalcone was found to have potent activity in these anti-free radical activity tests. Further research revealed that C7 could elevate glutathione peroxidase (GSH-PX) and super oxide dismutase (SOD) levels and lower malonaldehyde (MDA) level in vivo in the Alzheimer's model. The indication of C7's effect on AD needs further study.


Assuntos
Doença de Alzheimer/tratamento farmacológico , Chalcona/síntese química , Chalcona/uso terapêutico , Radicais Livres/metabolismo , Doença de Alzheimer/enzimologia , Doença de Alzheimer/patologia , Animais , Benzofenonas/química , Compostos de Bifenilo/metabolismo , Encéfalo/efeitos dos fármacos , Encéfalo/enzimologia , Encéfalo/patologia , Chalcona/análogos & derivados , Chalcona/química , Modelos Animais de Doenças , Sequestradores de Radicais Livres/farmacologia , Glutationa Peroxidase/metabolismo , Hidroxilação/efeitos dos fármacos , Concentração Inibidora 50 , Espectroscopia de Ressonância Magnética , Malondialdeído/metabolismo , Camundongos , Oxirredução/efeitos dos fármacos , Picratos/metabolismo , Escopolamina , Superóxido Dismutase/metabolismo
16.
J Mol Neurosci ; 73(1): 28-38, 2023 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-36542318

RESUMO

The impacts of lysine demethylase 1B (KDM1B) have been probed in multiple diseases, but the effects of KDM1B on SS remained obscure. The study aimed to unravel the efficiency of KDM1B on SS progression via the paired box 6 (PAX6)/clusterin (CLU) axis. NODB10. H2b mice were selected to establish the SS model. KDM1B, Pax6, and CLU expression in SS mice was assessed. Adeno-associated viruses carrying KDM1B, Pax6, and CLU were injected into the SS mice to detect tear secretion, epithelium corneal fluorescein staining scores, and levels of specific markers of lacrimal gland epithelial cells, neurotransmitter receptors that induce secretion from the lacrimal gland, and genes encoding normal tear components. The relation among KDM1B, Pax6, and CLU was examined. The rescue experiments were conducted for verifying the interaction among KDM1B, Pax6, and CLU. KDM1B expression was elevated, while Pax6 and CLU levels were decreased in the lacrimal gland tissues of SS mouse models. KDM1B decrement and Pax6 augmentation improved tear secretion, reduced corneal fluorescein staining score, decreased levels of specific markers of lacrimal gland epithelial cells, and increased levels of neurotransmitter receptors that induce secretion from the lacrimal gland and genes encoding normal tear components. KDM1D suppressed Pax6 expression by mediating H3K4me2 demethylation. Pax6 promoted the expression of CLU at the transcriptional level by binding to the CLU promoter. Silencing of Pax6 or CLU could reverse the effects of KDM1B reduction on improving the tear secretion disorder of SS mice. Silencing KDM1B mitigates the tear secretion disorder of SS mice via modulating the Pax6/CLU axis.


Assuntos
Síndrome de Sjogren , Animais , Camundongos , Síndrome de Sjogren/diagnóstico , Síndrome de Sjogren/genética , Síndrome de Sjogren/metabolismo , Lisina , Clusterina , Camundongos Endogâmicos NOD , Fluoresceínas , Fator de Transcrição PAX6/genética
17.
Indian J Ophthalmol ; 71(4): 1304-1315, 2023 04.
Artigo em Inglês | MEDLINE | ID: mdl-37026262

RESUMO

Dry eye disease (DED) is a common multi-factorial disease that is characterized by tear film instability. Diquafosol tetrasodium (DQS), an ophthalmic solution, has been shown to be beneficial in the treatment of DED. The goal of this study was to provide an update on the safety and efficacy of topical 3% DQS in treating DED patients. A thorough search for all the published randomized controlled trials (RCTs) up to March 31, 2022 in CENTRAL, PubMed, Scopus, and Google Scholar databases was performed. Data were reported as standardized mean difference (SMD) with 95% confidence interval (CI). Modified Jadad scale was used for sensitivity analysis. Funnel plot and Egger's regression test assessed the publication bias. Fourteen RCTs evaluating the safety and efficacy of topical 3% DQS treatment in DED patients were included. Eight included RCTs reported data on the DED after cataract surgery. Overall findings suggest that 3% DQS treatment in DED patients was associated with significantly better improvement at 4 weeks in tear breakup time, Schirmer test, fluorescein staining scores, and Rose Bengal staining score as compared to patients treated with others eye drops including artificial tears or 01% sodium hyaluronate. However, no significant difference in ocular surface disease index was observed. Our findings suggest that 3% DQS treatment is safer and had a superior efficacy compared to artificial tears or sodium hyaluronate for treating DED in general and DED after cataract surgery.


Assuntos
Síndromes do Olho Seco , Soluções Oftálmicas , Polifosfatos , Humanos , Síndromes do Olho Seco/tratamento farmacológico , Ácido Hialurônico , Lubrificantes Oftálmicos , Soluções Oftálmicas/uso terapêutico , Ensaios Clínicos Controlados Aleatórios como Assunto , Lágrimas , Extração de Catarata , Polifosfatos/uso terapêutico , Nucleotídeos de Uracila/uso terapêutico
18.
Front Microbiol ; 14: 1274925, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-38098666

RESUMO

Ongoing extensive research in the field of gut microbiota (GM) has highlighted the crucial role of gut-dwelling microbes in human health. These microbes possess 100 times more genes than the human genome and offer significant biochemical advantages to the host in nutrient and drug absorption, metabolism, and excretion. It is increasingly clear that GM modulates the efficacy and toxicity of drugs, especially those taken orally. In addition, intra-individual variability of GM has been shown to contribute to drug response biases for certain therapeutics. For instance, the efficacy of cyclophosphamide depends on the presence of Enterococcus hirae and Barnesiella intestinihominis in the host intestine. Conversely, the presence of inappropriate or unwanted gut bacteria can inactivate a drug. For example, dehydroxylase of Enterococcus faecalis and Eggerthella lenta A2 can metabolize L-dopa before it converts into the active form (dopamine) and crosses the blood-brain barrier to treat Parkinson's disease patients. Moreover, GM is emerging as a new player in personalized medicine, and various methods are being developed to treat diseases by remodeling patients' GM composition, such as prebiotic and probiotic interventions, microbiota transplants, and the introduction of synthetic GM. This review aims to highlight how the host's GM can improve drug efficacy and discuss how an unwanted bug can cause the inactivation of medicine.

19.
Cell Biol Toxicol ; 28(5): 279-89, 2012 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-22752439

RESUMO

PURPOSE: Glucocorticoids (GCs) can facilitate bone formation, but prolonged GCs exposure in vivo can lead to osteoporosis. The mechanisms underlying these reciprocal effects have not been elucidated. METHODS: The epithelial sodium channel (ENaC) is a possible regulator of osteoblast proliferation and differentiation, so we examined whether ENaC was involved in mediating the effects of dexamethasone (Dex) on osteoblast. RESULT: Expression of the functional α-ENaC subunit was upregulated by 10(-8)M and 10(-6)M Dex, but decreased by 10(-4)M Dex. Furthermore, Dex had similar dose-dependent effects on the expression of three osteogenic genes, Cbfa1, OPN, and OC, with low concentrations enhancing expression and higher concentrations suppressing expression. The effects of Dex on osteoblast proliferation, differentiation, and mineralization were examined in the presence and absence of the ENaC specific antagonist amiloride. Dex at 10(-8)M and 10(-6)M markedly increased osteoblast proliferation, alkaline phosphatase activity (an index of differentiation), and calcium nodule formation, while 10(-4)M had no effect or suppressed all these responses. Amiloride blocked the Dex-induced, osteoblast differentiation and mineralization but had no effect on osteoblast differentiation and mineralization when applied alone. But such changes did not show in osteoblast proliferation. However, the Dex-induced α-ENaC expression was not blocked by RU486, a GC receptor antagonist. CONCLUSION: These results suggest that changes in ENaC activity may involved in Dex-induced differentiation and mineralization of osteoblast. But the Dex-induced effect on ENaC did not mediated by the GC genomic mechanism in osteoblast at this study.


Assuntos
Calcificação Fisiológica/efeitos dos fármacos , Dexametasona/farmacologia , Canais Epiteliais de Sódio/metabolismo , Glucocorticoides/farmacologia , Osteoblastos/efeitos dos fármacos , Subunidades Proteicas/metabolismo , Fosfatase Alcalina/metabolismo , Amilorida/farmacologia , Animais , Animais Recém-Nascidos , Biomarcadores/metabolismo , Diferenciação Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Subunidade alfa 1 de Fator de Ligação ao Core/genética , Subunidade alfa 1 de Fator de Ligação ao Core/metabolismo , Relação Dose-Resposta a Droga , Bloqueadores do Canal de Sódio Epitelial/farmacologia , Canais Epiteliais de Sódio/genética , Expressão Gênica/efeitos dos fármacos , Antagonistas de Hormônios/farmacologia , Mifepristona/farmacologia , Osteoblastos/citologia , Osteoblastos/metabolismo , Cultura Primária de Células , Subunidades Proteicas/genética , Ratos , Regulação para Cima/efeitos dos fármacos
20.
Zhong Yao Cai ; 35(11): 1820-4, 2012 Nov.
Artigo em Chinês | MEDLINE | ID: mdl-23627098

RESUMO

OBJECTIVE: To study the influence of Plantaginis Semen on cell proliferation, differentiation and function of rat osteoblasts, and investigate the regulation effects of rat osteoblast epithelial sodium channel (ENaC) on bone formation. METHODS: The animal serum was prepared by serum pharmacology means. The cells were got by separating and inducing the SD neonatal rat's skull bone. Cell proliferation and differentiation were evaluated by CCK-8 assay kit and AKP assay kit respectively. Regulation effects on mRNA expression of ENaC and osteogenesis gene were investigated by semi-quantitative PCR. RESULTS: Plantaginis Semen stimulated the osteoblasts proliferation and differentiation,the difference between treatment group and control group had statistical significance (P < 0.01) in a dose-dependent manner. The effects of Plantaginis Semen serum on alpha-ENaC gene expression paralleled those on osteogenic gene (OC, ALP, OP) expression level. CONCLUSION: Plantaginis Semen stimulates proliferation, differentiation and the mRNA expression of ENaC and osteogenesis gene in rat osteoblasts. Our results suggest that ENaC participate in the effects of Plantaginis Semen serum on osteoblast bone formation. Regulation of ENaC channel expression and function may provide a new clue for research on treatment of osteoporosis with traditional Chinese medicine.


Assuntos
Medicamentos de Ervas Chinesas/farmacologia , Canais Epiteliais de Sódio/metabolismo , Osteoblastos/efeitos dos fármacos , Osteogênese , Plantago/química , Fosfatase Alcalina/genética , Fosfatase Alcalina/metabolismo , Animais , Diferenciação Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Relação Dose-Resposta a Droga , Medicamentos de Ervas Chinesas/isolamento & purificação , Canais Epiteliais de Sódio/genética , Regulação da Expressão Gênica/efeitos dos fármacos , Osteoblastos/citologia , Osteoblastos/metabolismo , Reação em Cadeia da Polimerase/métodos , RNA Mensageiro/metabolismo , Ratos , Sementes/química , Soro , Crânio/citologia
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