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1.
Food Chem ; 372: 131218, 2022 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-34624783

RESUMO

Glucose is a primary source of energy used in most organisms. Thus, development of reliable approaches to measure intracellular glucose uptake is an important research issue. 2-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl) amino]-2-deoxy-d-glucose (2-NBDG), as a fluorescent glucose derivative, has been widely used to track intracellular glucose uptake by fluorescence imaging and measuring in mammalian cells. However, the avoid-less cross-interference of intrinsic autofluorescence background and tested fluorescent compounds limits its ability to provide trustworthy information on intracellular glucose uptake. By the extraction, separation and detection of 2-NBDG, a simple, sensitive and accurate HPLC-FLD method was established and validated for the measurement of intracellular glucose uptake in HepG2 cells. The developed method has been employed successfully to assess the glucose uptake activity of anti-diabetic drugs and fluorescent natural products. A fit-for-purpose partial validation was further performed for quantification and comparison of glucose uptake in AML12, LO2 hepatocytes, L6 myoblasts and 3T3-L1 preadipocytes.

2.
Food Funct ; 2021 Nov 17.
Artigo em Inglês | MEDLINE | ID: mdl-34787617

RESUMO

The therapeutic targets of berberine for hepatocellular carcinoma (HCC) and its detailed mechanisms remain unexplored. Here, an integration of network pharmacology, proteomic, bioinformatic and in vitro biochemical approach was proposed to reveal therapeutic targets and pathways underlying the antiproliferative activity of berberine against HepG2 cells. Results indicated that berberine caused the cytotoxicity and inhibited the growth of HepG2 cells with IC50 values ranging from 92 µM to 118 µM. Network pharmacology analysis revealed that targeting apoptosis and cell cycle pathways by berberine contributed to its antitumor efficacy against HCC. Proteomic analysis demonstrated that mitochondria-related apoptosis pathways were involved in the cytotoxic action of berberine, as evidenced by the expression of mitochondrial dysfunction-mediated proteins. Moreover, a total of 160 significantly altered proteins were screened, among which AKAP12 presented significantly increased levels under berberine treatment. Bioinformatic analysis of various public datasets showed that expression of AKAP12 in HCC liver tissues was downregulated, emphasizing its role as a tumor suppressor. Immunoblotting validated the increased levels of AKAP12, while co-immunoprecipitation identified its interaction with Cyclin D1. These data, together with flow cytometry analysis, suggested that AKAP12 mediated cell cycle arrest, thereby suppressing cell proliferation. Altogether, the antiproliferative action of berberine in HepG2 cells involves both apoptosis and cell cycle arrest. Regulating AKAP12 signalling by berberine might provide a promising strategy for HCC treatment.

3.
Biomater Sci ; 2021 Nov 16.
Artigo em Inglês | MEDLINE | ID: mdl-34783803

RESUMO

Bacterial infection is one of the most significant public health challenges due to the limited choices of antibiotics which can overcome antibiotic-resistant bacteria. The promising nonantibiotic therapeutic alternatives for antibiotic-resistant bacterial infection are urgently needed to reduce the disease burden. Herein, the water-soluble branched poly(amino ester) with inherently antibacterial (chemotherapy) and enhanced inflammatory response activity (immunotherapy) was prepared via Michael addition polymerization to combat bacterial infection. These polymers can not only damage bacteria walls, leading to the death of bacteria but also activate macrophages to low-output nitric oxide (NO), TNF-α and interleukin (IL)-1ß to kill and clean bacteria. Importantly, these polymers can efficiently inhibit aminoglycoside-resistant P. aeruginosa even at a low dose of 500 nmol L-1. Furthermore, these polymers can treat subcutaneous bacterial infections in vivo. In this study, we first report a water-soluble branched polymer to combat bacteria through the combination of chemotherapy and immunotherapy, which will open a new path to design promising potential therapeutic alternatives for bacterial infection.

4.
Nucleic Acids Res ; 2021 Sep 27.
Artigo em Inglês | MEDLINE | ID: mdl-34570230

RESUMO

To date, only some cancer patients can benefit from chemotherapy and targeted therapy. Drug resistance continues to be a major and challenging problem facing current cancer research. Rapidly accumulated patient-derived clinical transcriptomic data with cancer drug response bring opportunities for exploring molecular determinants of drug response, but meanwhile pose challenges for data management, integration, and reuse. Here we present the Cancer Treatment Response gene signature DataBase (CTR-DB, http://ctrdb.ncpsb.org.cn/), a unique database for basic and clinical researchers to access, integrate, and reuse clinical transcriptomes with cancer drug response. CTR-DB has collected and uniformly reprocessed 83 patient-derived pre-treatment transcriptomic source datasets with manually curated cancer drug response information, involving 28 histological cancer types, 123 drugs, and 5139 patient samples. These data are browsable, searchable, and downloadable. Moreover, CTR-DB supports single-dataset exploration (including differential gene expression, receiver operating characteristic curve, functional enrichment, sensitizing drug search, and tumor microenvironment analyses), and multiple-dataset combination and comparison, as well as biomarker validation function, which provide insights into the drug resistance mechanism, predictive biomarker discovery and validation, drug combination, and resistance mechanism heterogeneity.

5.
Small ; 17(43): e2103127, 2021 10.
Artigo em Inglês | MEDLINE | ID: mdl-34510742

RESUMO

Conjugated polymers (CPs) are capable of coordinating the electron coupling phenomenon to bestow powerful optoelectronic features. The light-harvesting and light-amplifying properties of CPs are extensively used in figuring out the biomedical issues with special emphasis on accurate diagnosis, effective treatment, and precise theranostics. This review summarizes the recent progress of CP materials in bioimaging, cancer therapeutics, and introduces the design strategies by rationally tuning the optical properties. The recent advances of CPs in bioimaging applications are first summarized and the challenges to clear the future directions of CPs in the respective area are discussed. In the following sections, the focus is on the burgeoning applications of CPs in phototherapy of the tumor, and illustrates the underlying photo-transforming mechanism for further molecular designing. Besides, the recent progress in the CPs-assistant drug therapy, mainly including drug delivery, gene therapeutic, the optical-activated reversion of tumor resistance, and synergistic therapy has also been discussed elaborately. In the end, the potential challenges and future developments of CPs on cancer diagnosis and therapy are also illuminated for the improvement of optical functionalization and the promotion of clinical translation.


Assuntos
Nanopartículas , Neoplasias , Humanos , Neoplasias/diagnóstico por imagem , Neoplasias/terapia , Fototerapia , Polímeros , Nanomedicina Teranóstica
6.
J Mater Chem B ; 9(33): 6658-6667, 2021 Sep 07.
Artigo em Inglês | MEDLINE | ID: mdl-34378630

RESUMO

Biofilm infections present an enormous challenge in today's healthcare settings. Currently, pH-switchable antibacterial agents are being developed to eradicate biofilms. However, most pH-switchable antibacterial agents are less lethal to planktonic bacteria under neutral conditions, and cannot prevent the dispersed bacteria from seeding acute infection again. Herein, this work reports the applications of semiconducting polymer dots (Pdots) with a double adhesion mechanism in imaging and inhibiting bacteria inside (weak acidic conditions) and outside (neutral conditions) biofilms. Clew-like Pdots were prepared by covalently linking phenylboronic acid (PBA) and pH-responsive naphthalimide (NA) ramification in semiconducting polymers. Under neutral conditions, the Pdots combined with bacteria through the formation of boronate esters between PBA and diols. Under weakly acidic conditions, the partial borate bond fractured, and the Pdots adhered onto the bacterial surface through the positively charged NA in Pdots. Furthermore, the Pdots display negligible toxicity to mammalian cells and tissues. More importantly, the Pdots can selectively damage the bacterial membrane and inhibit bacteria in vivo. This work highlights the feasibility of using semiconducting Pdots to image and inhibit bacteria inside and outside biofilms, which represents a highly effective strategy to cope with biofilm infections.

7.
PLoS Genet ; 17(7): e1009369, 2021 07.
Artigo em Inglês | MEDLINE | ID: mdl-34237055

RESUMO

Spermatogonial stem cells (SSC), the foundation of spermatogenesis and male fertility, possess lifelong self-renewal activity. Aging leads to the decline in stem cell function and increased risk of paternal age-related genetic diseases. In the present study, we performed a comparative genomic analysis of mouse SSC-enriched undifferentiated spermatogonia (Oct4-GFP+/KIT-) and differentiating progenitors (Oct4-GFP+/KIT+) isolated from young and aged testes. Our transcriptome data revealed enormous complexity of expressed coding and non-coding RNAs and alternative splicing regulation during SSC differentiation. Further comparison between young and aged undifferentiated spermatogonia suggested these differentiation programs were affected by aging. We identified aberrant expression of genes associated with meiosis and TGF-ß signaling, alteration in alternative splicing regulation and differential expression of specific lncRNAs such as Fendrr. Epigenetic profiling revealed reduced H3K27me3 deposition at numerous pro-differentiation genes during SSC differentiation as well as aberrant H3K27me3 distribution at genes in Wnt and TGF-ß signaling upon aging. Finally, aged undifferentiated spermatogonia exhibited gene body hypomethylation, which is accompanied by an elevated 5hmC level. We believe this in-depth molecular analysis will serve as a reference for future analysis of SSC aging.


Assuntos
Células-Tronco Germinativas Adultas/citologia , Células-Tronco Germinativas Adultas/fisiologia , Envelhecimento/fisiologia , Epigenoma , 5-Metilcitosina/metabolismo , Envelhecimento/genética , Processamento Alternativo , Animais , Diferenciação Celular , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Lisina/genética , Lisina/metabolismo , Masculino , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , RNA Longo não Codificante/genética , Testículo/citologia
8.
Dalton Trans ; 50(31): 10806-10810, 2021 Aug 21.
Artigo em Inglês | MEDLINE | ID: mdl-34291266

RESUMO

Fluoro(dialkyl)stannylenoid 2 exhibits unique reactivity towards ethynes with acetylenic hydrogen and those with trimethylsilyl groups, though the corresponding free dialkylstannylene 1 is inactive against those ethynes. Stannylenoid 2 reacts smoothly with gaseous ethyne and phenylethyne at room temperature, giving the corresponding diethynylstannanes, di(phenylethynyl)stannane 3 and diethynylstannane 6, respectively, in good yields with the concomitant evolution of H2. Trimethylsilyl-substituted ethynes such as 1-trimethylsilyl-(2-phenyl)ethyne and 1,2-bis(trimethylsilyl)ethyne react similarly to give 3 and bis(trimethylsilylethynyl)stannane 8, respectively. Rather unexpectedly, the reaction of 2 with (trimethylsilyl)ethyne affords 1,2-bis(ethenylstannyl)ethyne 7 in a good yield. The reactions of 2 with methyl and ethyl propynoates give the same products 4 and 5 as those obtained during the reaction of dialkylstannylene 1 without CsF. Pathways involving the nucleophilic attack of cesium acetylide to an ethyne-complexed stannylene were proposed, while the detailed mechanisms remain unknown. The structure of 7 was studied by single crystal X-ray diffraction analysis.

9.
ChemSusChem ; 14(18): 3821-3824, 2021 Sep 20.
Artigo em Inglês | MEDLINE | ID: mdl-34291587

RESUMO

The use of polymeric carbon nitride (PCN) for photoredox catalysis is innovating and promoting toward sustainable energy economy. One of the drawbacks of this metal-free photocatalyst is its insufficient charge separation and transfer. Herein, a metal-free system was achieved by anchoring PCN on conductive carbon cloth (CCC). CCC in this system facilitated the charge separation and transport of the photoexcitation charges when PCN films were illuminated. Both photoelectrochemical water oxidation and photocatalytic overall water splitting were achieved, and the performances were improved two-fold with respect to the powder PCN.

10.
Chin Med ; 16(1): 30, 2021 Mar 19.
Artigo em Inglês | MEDLINE | ID: mdl-33741031

RESUMO

BACKGROUND: Jinqi Jiangtang (JQJT) has been widely used in clinical practice to prevent and treat type 2 diabetes. However, little research has been done to identify and classify its quality markers (Q-markers) associated with anti-diabetes bioactivity. In this study, a strategy combining mass spectrometry-based untargeted metabolomics with backpropagation artificial neural network (BP-ANN)-based machine learning approach was proposed to screen Q-markers from JQJT preparation. METHODS: This strategy mainly involved chemical profiling of herbal medicines, statistic processing of metabolomic datasets, detection of different anti-diabetes activities and establishment of BP-ANN model. The chemical features of seventy-eight batches of JQJT extracts were first profiled by using the untargeted UPLC-LTQ-Orbitrap metabolomic approach. The chemical features obtained which were associated with different anti-diabetes activities based on three modes of action were normalized, ranked, and then pre-selected by using ReliefF feature selection. BP-ANN model was then established and optimized to screen Q-markers based on mean impact value (MIV). RESULTS: Optimized BP-ANN architecture was established with high accuracy of R > 0.9983 and relative low error of MSE < 0.0014, which showed better performance than that of partial least square (PLS) model (R2 < 0.5). Meanwhile, the BP-ANN model was subsequently applied to further screen potential bioactive components from the pre-selected chemical features by calculating their MIVs. With this machine learning model, 10 potential Q-markers with bioactivity were discovered from JQJT. The tested anti-diabetes bioactivities of 78 batches of JQJT could be accurately predicted. CONCLUSIONS: This proposed artificial intelligence approach is desirable for quick and easy identification of Q-markers with bioactivity from JQJT preparation.

11.
Mitochondrial DNA B Resour ; 6(2): 358-360, 2021 Feb 08.
Artigo em Inglês | MEDLINE | ID: mdl-33659676

RESUMO

In this study, the complete mitochondrial genome of Cottiusculus nihonkaiensis was presented, and we also discussed its mitochondrial characteristics. The full length of the mitochondrial genome was 16,612 bp, containing 13 protein-coding genes (PCGs), two ribosomal RNAs (12S and 16S), 22 transfer RNA genes (tRNA), one non-coding control region (CR) and one origin of replication on the light-strand. Overall base composition of the complete mitochondrial DNA was 26.4% A, 17.4% G, 31.5% C, 24.7% T. The phylogenetic tree suggested that C. nihonkaiensis shared the most recent common ancestor with Gymnocanthus herzensteini, Gymnocanthus intermedius and Gymnocanthus tricuspis.

12.
ACS Omega ; 6(8): 5335-5347, 2021 Mar 02.
Artigo em Inglês | MEDLINE | ID: mdl-33681573

RESUMO

Natural gas hydrates are an ideal alternative clean energy source. Many countries are currently attempting the trial production of gas hydrates. Japan became the first country to achieve offshore hydrate trial production in 2013, and China conducted 60 days of continuous trial exploitation in 2017. This study analyzes the changes in the internal stress of the hydrate zone and hydrate saturation of the soil throughout the monitoring period and calculates the failure stress of the hydrate deposit layer. The Mohr-Coulomb model is used to simulate Japan's test exploitation conditions to verify the feasibility of the method. Finally, the hydrate decomposition range, the difference in the soil dip angle in the test exploitation area, and the bearing capacity of the hydrate reservoir are numerically simulated to evaluate the stability of the soil. Through the sensitivity analysis of the hydrate decomposition range and the inclination angle of the hydrate sediment layer, it can be found that the hydrate decomposition range has the greatest impact on the deformation, and the soil around the decomposition area may be sheared and collapsed. Within 1 week of decompression and exploitation, the hydrate decomposition radius is approximately 3 m. When the inclination angle increases from 3° to 9°, the sediment deformation increases by 12 times. Therefore, it is necessary to pay attention to the critical value of the decomposition range during the exploitation process.

13.
Cell Prolif ; 54(5): e13000, 2021 May.
Artigo em Inglês | MEDLINE | ID: mdl-33666296

RESUMO

OBJECTIVES: Mammalian spermatogenesis is a biological process of male gamete formation. Gonocytes are the only precursors of spermatogonial stem cells (SSCs) which develop into mature spermatozoa. DDX5 is one of DEAD-box RNA helicases and expresses in male germ cells, suggesting that Ddx5 plays important functions during spermatogenesis. Here, we explore the functions of Ddx5 in regulating the specification of gonocytes. MATERIALS AND METHODS: Germ cell-specific Ddx5 knockout (Ddx5-/- ) mice were generated. The morphology of testes and epididymides and fertility in both wild-type and Ddx5-/- mice were analysed. Single-cell RNA sequencing (scRNA-seq) was used to profile the transcriptome in testes from wild-type and Ddx5-/- mice at postnatal day (P) 2. Dysregulated genes were validated by single-cell qRT-PCR and immunofluorescent staining. RESULTS: In male mice, Ddx5 was expressed in germ cells at different stages of development. Germ cell-specific Ddx5 knockout adult male mice were sterile due to completely devoid of germ cells. Male germ cells gradually disappeared in Ddx5-/- mice from E18.5 to P6. Single-cell transcriptome analysis showed that genes involved in cell cycle and glial cell line-derived neurotrophic factor (GDNF) pathway were significantly decreased in Ddx5-deficient gonocytes. Notably, Ddx5 ablation impeded the proliferation of gonocytes. CONCLUSIONS: Our study reveals the critical roles of Ddx5 in fate determination of gonocytes, offering a novel insight into the pathogenesis of male sterility.


Assuntos
RNA Helicases DEAD-box/metabolismo , Células Germinativas/metabolismo , Animais , Animais Recém-Nascidos , RNA Helicases DEAD-box/genética , Regulação da Expressão Gênica no Desenvolvimento , Genótipo , Células Germinativas/citologia , Fator Neurotrófico Derivado de Linhagem de Célula Glial/metabolismo , Infertilidade/metabolismo , Infertilidade/patologia , Masculino , Camundongos , Camundongos Knockout , Análise de Sequência de RNA , Análise de Célula Única , Testículo/metabolismo , Testículo/patologia
14.
Mitochondrial DNA B Resour ; 6(1): 236-238, 2021 Jan 20.
Artigo em Inglês | MEDLINE | ID: mdl-33553633

RESUMO

In this study, the complete mitochondrial genome of Ilisha striatula is presented, and we also discussed its mitochondrial characteristics. The full length of the mitochondrial genome was 16,847 bp, including 13 protein-coding genes (PCGs), 2 ribosomal RNAs, 22 transfer RNA genes, one non-coding control region (CR), and one origin of replication on the light-strand. The total nucleotide composition of the mitochondrial genome was 31.11% A, 24.62% T, 29.10% C, 15.16% G, and AT was 55.73%. The phylogenetic tree based on the ML method supported the placement of I. striatula in a close relationship with I. elongata. These data contribute to a better understanding of the systematics of the Pristigasteridae.

15.
ACS Omega ; 5(48): 31244-31253, 2020 Dec 08.
Artigo em Inglês | MEDLINE | ID: mdl-33324834

RESUMO

During the exploration of hydrates or oil and gas exploitation through the hydrate layer, heat transfer causes hydrates to decompose. The gas and water generated by this decomposition increase the pressure of the gas in the decomposition zone, resulting in excessive pore pressure. The seepage of gas and water and the decomposition of hydrates lead to soil deformation, which may be caused by soil softening. This may lead to geological disasters, such as ocean landslides, seabed subsidence, and even gas explosions. The natural phenomena of soil eruption caused by hydrate decomposition currently include Siberian pits and Bermuda craters. From these two natural phenomena, climate change is considered to affect hydrate decomposition, causing ocean acidification and dissolved oxygen consumption, which may have more serious consequences than global warming alone. Therefore, it is extremely important to study how hydrate decomposition causes soil to erupt and release gas into the ocean and the atmosphere. This paper is primarily based on on-site data collected from the Siberian pit in the case of hydrate decomposition resulting in increased pore pressure, resulting in soil eruption. The relationship between the thickness of the upper cladding layer, the pressure causing the destruction of the upper cladding layer, and the destruction length of the upper cladding layer was obtained through numerical simulation.

16.
Plant Signal Behav ; 15(9): 1789818, 2020 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-32649276

RESUMO

Anionic phospholipid phosphatidic acid (PA) behaves as an important second messenger involved in many cellular processes, such as development, cytoskeletal dynamics, vesicle trafficking, and stress response. Recently, it was reported that PA can directly bind with the rice Shaker K+ channel OsAKT2 to inhibit its channel activity. Two adjacent arginine residues (R644 and R645) in ANK domain were identified as a PA-binding site essential to the PA-mediated inhibition of OsAKT2. However, there may be still other PA-binding sites unidentified in OsAKT2. Here, using a PA biosensor (PAleon), we found that the exogenous PA treatment significantly increased the PA level at the plasma membrane of Xenopus oocytes which were used to express OsAKT2 for electrophysiological assays. As reported previously, exogenous PA markedly inhibited OsAKT2 K+ currents. Replacement of two adjacent basic residues (R190 and K191) in the S4 voltage sensor by glycine completely abolished the time-dependent K+ currents of OsAKT2, but this variant was insensitive to PA treatment. In addition, we also identified other two adjacent arginines (R755 and R756) located in the cytosolic domain as a PA-binding site, which were also essential to the PA-mediated inhibition of OsAKT2. These results provide a more comprehensive understanding of the PA-K+ channel interaction mechanism. Combining the findings here with the previous study, we propose that multiple basic residues (R190/K191, R644/R645, and R755/R756) in different domains of OsAKT2 contribute to PA-mediated regulation of OsAKT2.


Assuntos
Oryza/metabolismo , Ácidos Fosfatídicos/farmacologia , Proteínas de Plantas/metabolismo , Canais de Potássio/metabolismo , Membrana Celular/metabolismo , Eletrofisiologia , Glicina/metabolismo , Ácidos Fosfatídicos/metabolismo , Fosfolipídeos/metabolismo , Proteínas de Plantas/genética , Canais de Potássio/genética
17.
Plant J ; 102(4): 649-665, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-32128922

RESUMO

The plant Shaker K+ channel AtAKT2 has been identified as a weakly rectifying channel that can stabilize membrane potentials to promote photoassimilate phloem loading and translocation. Thus, studies on functional characterization and regulatory mechanisms of AtAKT2-like channels in crops are highly important for improving crop production. Here, we identified the rice OsAKT2 as the ortholog of Arabidopsis AtAKT2, which is primarily expressed in the shoot phloem and localized at the plasma membrane. Using an electrophysiological assay, we found that OsAKT2 operated as a weakly rectifying K+ channel, preventing H+ /sucrose-symport-induced membrane depolarization. Three critical amino acid residues (K193, N206, and S326) are essential to the phosphorylation-mediated gating change of OsAKT2, consistent with the roles of the corresponding sites in AtAKT2. Disruption of OsAKT2 results in delayed growth of rice seedlings under short-day conditions. Interestingly, the lipid second messenger phosphatidic acid (PA) inhibits OsAKT2-mediated currents (both instantaneous and time-dependent components). Lipid dot-blot assay and liposome-protein binding analysis revealed that PA directly bound with two adjacent arginine residues in the ANK domain of OsAKT2, which is essential to PA-mediated inhibition of OsAKT2. Electrophysiological and phenotypic analyses also showed the PA-mediated inhibition of AtAKT2 and the negative correlation between intrinsic PA level and Arabidopsis growth, suggesting that PA may inhibit AKT2 function to affect plant growth and development. Our results functionally characterize the Shaker K+ channel OsAKT2 and reveal a direct link between phospholipid signaling and plant K+ channel modulation.


Assuntos
Arabidopsis/genética , Oryza/genética , Ácidos Fosfatídicos/metabolismo , Canais de Potássio/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Membrana Celular/metabolismo , Oryza/metabolismo , Canais de Potássio/genética , Plântula/genética , Plântula/metabolismo
18.
Development ; 147(2)2020 01 22.
Artigo em Inglês | MEDLINE | ID: mdl-31969357

RESUMO

The development and maintenance of the correct morphology of sperm is important for their functions. Cellular morphogenesis of sperm occurs during the post-meiotic developmental stage; however, little is known about what coordinates this process. In the present study, we investigated the role of A-kinase anchoring protein 3 (AKAP3) during mouse spermiogenesis, using both mouse genetics and proteomics. It was found that AKAP3 is essential for the formation of the specific subcellular structure of the sperm flagellum, motility of sperm and male fertility. Additionally, lack of AKAP3 caused global changes of the sperm proteome and mislocalization of sperm proteins, including accumulation of RNA metabolism and translation factors and displacement of PKA subunits in mature sperm, which may underlie misregulated PKA activity and immotility in sperm. Interestingly, sperm lacking a complete fibrous sheath from both Akap3 and Akap4 null mice accumulated F-actin filaments and morphological defects during post-testicular maturation in the epididymis. These results suggest that the subcellular structures of sperm could be formed via independent pathways, and elucidate the roles of AKAP3 during the coordinated synthesis and organization of the sperm proteome and sperm morphology.


Assuntos
Proteínas de Ancoragem à Quinase A/metabolismo , Infertilidade Masculina/metabolismo , Espermatozoides/metabolismo , Citoesqueleto de Actina/metabolismo , Actinas/metabolismo , Animais , Sequência de Bases , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Epididimo/metabolismo , Deleção de Genes , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout , Mutação/genética , Proteoma/metabolismo , Transdução de Sinais , Espermatozoides/anormalidades , Espermatozoides/patologia , Espermatozoides/ultraestrutura , Frações Subcelulares/metabolismo
19.
Sci Rep ; 9(1): 18877, 2019 12 11.
Artigo em Inglês | MEDLINE | ID: mdl-31827197

RESUMO

CRISPR/Cas9 technology has been widely used for targeted genome modification both in vivo and in vitro. However, an effective method for evaluating genome editing efficiency and screening single-cell clones for desired modification is still lacking. Here, we developed this real time PCR method based on the sensitivity of Taq DNA polymerase to nucleotide mismatch at primer 3' end during initiating DNA replication. Applications to CRISPR gRNAs targeting EMX1, DYRK1A and HOXB13 genes in Lenti-X 293 T cells exhibited comprehensive advantages. Just in one-round qPCR analysis using genomic DNA from cells underwent CRISPR/Cas9 or BE4 treatments, the genome editing efficiency could be determined accurately and quickly, for indel, HDR as well as base editing. When applied to single-cell clone screening, the genotype of each cell colony could also be determined accurately. This method defined a rigorous and practical way in quantify genome editing events.


Assuntos
Sistemas CRISPR-Cas , Edição de Genes/métodos , RNA Guia/genética , Reação em Cadeia da Polimerase em Tempo Real , Genoma , Células HEK293 , Humanos
20.
Plant Sci ; 287: 110190, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31481213

RESUMO

Phosphatidic acid (PA) is a lipid secondary messenger involved in intracellular signaling in eukaryotes. It has been confirmed that PA mediates salt stress signaling by promoting activation of Mitogen-activated Protein Kinase 6 (MPK6) which phosphorylates Na+/H+ antiporter SOS1. However, the MPK6-upstream kinases and their relationship to PA remain unclear. Here, we found that, among the six tested Arabidopsis Mitogen-activated Protein Kinase Kinases (MKKs), PA specifically bound to MKK7 and MKK9 which phosphorylate MPK6, and promoted the activation of MKK7/MKK9. Based on phenotypic and physiological analyses, we found that MKK7 and MKK9 positively regulate Arabidopsis salt tolerance and are functionally redundant. NaCl treatment can induce significant increase in MKK7/MKK9 activities, and this depends, in part, on the Phospholipase Dα1 (PLDα1). MKK7 and MKK9 also mediate the NaCl-induced activation of MPK6. Furthermore, PA or NaCl treatment could induce translocation of MKK7/MKK9 to the plasma membrane, whereas this translocation disappeared in pldα1. These results indicate that PA binds to MKK7 and MKK9, increases their kinase activity and plasma membrane localization during Arabidopsis response to salt stress. Together with the PA-MPK6-SOS1 pathway identified previously, this mechanism may maximize the signal transduction efficiency, providing novel insights into the link between lipid signaling and MAPK cascade.


Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/fisiologia , MAP Quinase Quinase 7/metabolismo , Sistema de Sinalização das MAP Quinases/genética , Quinases de Proteína Quinase Ativadas por Mitógeno/metabolismo , Ácidos Fosfatídicos/farmacologia , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Membrana Celular/metabolismo , MAP Quinase Quinase 7/genética , Quinases de Proteína Quinase Ativadas por Mitógeno/genética , Proteínas Quinases Ativadas por Mitógeno/genética , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Fosforilação , Estresse Salino , Tolerância ao Sal/genética
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