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1.
Theranostics ; 11(20): 9967-9987, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34815798

RESUMO

Background: BRCA1 plays critical roles in mammary gland development and mammary tumorigenesis. And loss of BRCA1 induces mammary tumors in a stochastic manner. These tumors present great heterogeneity at both intertumor and intratumor levels. Methods: To comprehensively elucidate the heterogeneity of BRCA1 deficient mammary tumors and the underlying mechanisms for tumor initiation and progression, we conducted bulk and single cell RNA sequencing (scRNA-seq) on both mammary gland cells and mammary tumor cells isolated from Brca1 knockout mice. Results: We found the BRCA1 deficient tumors could be classified into four subtypes with distinct molecular features and different sensitivities to anti-cancer drugs at the intertumor level. Whereas within the tumors, heterogeneous subgroups were classified mainly due to the different activities of cell proliferation, DNA damage response/repair and epithelial-to-mesenchymal transition (EMT). Besides, we reconstructed the BRCA1 related mammary tumorigenesis to uncover the transcriptomes alterations during this process via pseudo-temporal analysis of the scRNA-seq data. Furthermore, from candidate markers for BRCA1 mutant tumors, we discovered and validated one oncogene Mrc2, whose loss could reduce mammary tumor growth in vitro and in vivo. Conclusion: Our study provides a useful resource for better understanding of mammary tumorigenesis induced by BRCA1 deficiency.

2.
Lab Chip ; 2021 Nov 11.
Artigo em Inglês | MEDLINE | ID: mdl-34761772

RESUMO

Microfluidics has been the most promising platform for drug screening with a limited number of cells. However, convenient on-chip preparation of a wide range of drug concentrations remains a large challenge and has restricted wide acceptance of microfluidics in precision medicine. In this paper, we report a digital microfluidic system with an innovative control structure and chip design for on-chip drug dispensing to generate concentrations that span three to four orders of magnitude, enabling single drug or combinatorial multi-drug screening with simple electronic control. Specifically, we utilize droplet ejection from a drug drop sitting on a special electrode, named a drug dispenser, under high-voltage pulse actuation to deliver the desired amount of drugs to be picked up by a cell suspension drop driven by low-voltage sine wave actuation. Our proof-of-principle validation for this technique as a convenient single and multi-drug screening involved testing of the drug toxicity of two chemotherapeutics, cisplatin (Cis) and epirubicin (EP), towards MDA-MB-231 breast cancer cells and MCF-10A normal breast cells. The results are consistent with those screened based on traditional 96-well plates. These findings demonstrate the reliability of the drug screening system with an on-chip drug dispenser. This system with fewer cancer cells, less drug consumption, a small footprint, and high scalability with regard to concentration could pave the way for drug screening on biopsied primary tumor cells for precision medicine or any concentration-related research.

3.
Nanotechnology ; 33(7)2021 Nov 24.
Artigo em Inglês | MEDLINE | ID: mdl-34749351

RESUMO

Carbon nanotube (CNT) field-emission x-ray source has great potential in x-ray communication (XCOM) because of its controllable emission and instantaneous response. A novel voltage loading mode was proposed in this work to achieve high-frequency pulse x-ray emission. The characteristics of cathode current and pulse x-ray versus voltage, frequency, and pulse amplitude were studied, and XCOM data transmission experiment was carried out. Results showed that the CNT cold cathode x-ray source, as a communication signal source, could work in 1.05 MHz pulse emission frequency. When the grid voltage was higher than 470 V, the pulse x-ray waveform amplitude achieved peak, and the shape exhibited a pseudo square wave. The duty cycle of the x-ray waveform exceeded 50%, reaching 56% when the pulse frequency reached 1 MHz. In the XCOM data transmission experiment, the pulsed x-ray waveform was well consistent with the loading data signal voltage waveform under different pulse-emission frequencies. This work realized the x-ray high-frequency pulse emission of CNT cold cathode x-ray source and lays a foundation for the development and application of CNT cold cathode x-ray source in XCOM.

4.
Theranostics ; 11(19): 9415-9430, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34646378

RESUMO

The feasibility of personalized medicine for cancer treatment is largely hampered by costly, labor-intensive and time-consuming models for drug discovery. Herein, establishing new pre-clinical models to tackle these issues for personalized medicine is urgently demanded. Methods: We established a three-dimensional tumor slice culture (3D-TSC) platform incorporating label-free techniques for time-course experiments to predict anti-cancer drug efficacy and validated the 3D-TSC model by multiphoton fluorescence microscopy, RNA sequence analysis, histochemical and histological analysis. Results: Using time-lapse imaging of the apoptotic reporter sensor C3 (C3), we performed cell-based high-throughput drug screening and shortlisted high-efficacy drugs to screen murine and human 3D-TSCs, which validate effective candidates within 7 days of surgery. Histological and RNA sequence analyses demonstrated that 3D-TSCs accurately preserved immune components of the original tumor, which enables the successful achievement of immune checkpoint blockade assays with antibodies against PD-1 and/or PD-L1. Label-free multiphoton fluorescence imaging revealed that 3D-TSCs exhibit lipofuscin autofluorescence features in the time-course monitoring of drug response and efficacy. Conclusion: This technology accelerates precision anti-cancer therapy by providing a cheap, fast, and easy platform for anti-cancer drug discovery.

5.
Adv Sci (Weinh) ; 8(22): e2101176, 2021 11.
Artigo em Inglês | MEDLINE | ID: mdl-34605222

RESUMO

Most breast cancers at an advanced stage exhibit an aggressive nature, and there is a lack of effective anticancer options. Herein, the development of patient-derived organoids (PDOs) is described as a real-time platform to explore the feasibility of tailored treatment for refractory breast cancers. PDOs are successfully generated from breast cancer tissues, including heavily treated specimens. The microtubule-targeting drug-sensitive response signatures of PDOs predict improved distant relapse-free survival for invasive breast cancers treated with adjuvant chemotherapy. It is further demonstrated that PDO pharmaco-phenotyping reflects the previous treatment responses of the corresponding patients. Finally, as clinical case studies, all patients who receive at least one drug predicate to be sensitive by PDOs achieve good responses. Altogether, the PDO model is developed as an effective platform for evaluating patient-specific drug sensitivity in vitro, which can guide personal treatment decisions for breast cancer patients at terminal stage.

6.
Adv Sci (Weinh) ; 8(21): e2100974, 2021 11.
Artigo em Inglês | MEDLINE | ID: mdl-34514747

RESUMO

Fibroblast growth factor receptor 2 (FGFR2) is a membrane-spanning tyrosine kinase that mediates FGF signaling. Various FGFR2 alterations are detected in breast cancer, yet it remains unclear if activation of FGFR2 signaling initiates tumor formation. In an attempt to answer this question, a mouse model berrying an activation mutation of FGFR2 (FGFR2-S252W) in the mammary gland is generated. It is found that FGF/FGFR2 signaling drives the development of triple-negative breast cancer accompanied by epithelial-mesenchymal transition that is regulated by FGFR2-STAT3 signaling. It is demonstrated that FGFR2 suppresses BRCA1 via the ERK-YY1 axis and promotes tumor progression. BRCA1 knockout in the mammary gland of the FGFR2-S252W mice significantly accelerated tumorigenesis. It is also shown that FGFR2 positively regulates PD-L1 and that a combination of FGFR2 inhibition and immune checkpoint blockade kills cancer cells. These data suggest that the mouse models mimic human breast cancers and can be used to identify actionable therapeutic targets.

7.
Oxid Med Cell Longev ; 2021: 5551338, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34055194

RESUMO

Introduction: Reactive oxygen species (ROS) induced by extracellular cytokines trigger the expression of inflammatory mediators in osteoarthritis (OA) chondrocyte. Peroxisome proliferator-activated receptor gamma (PPARγ) exerts an anti-inflammatory effect. The aim of this study was to elucidate the role of PPARγ in interleukin-1ß- (IL-1ß-) induced cyclooxygenase-2 (COX-2) and prostaglandin E2 (PGE2) expression through ROS generation in OA chondrocytes. Methods: IL-1ß-induced ROS generation and chondrocyte apoptosis were determined by flow cytometry. Contents of NADPH oxidase (NOX), caspase-3, and caspase-9 were evaluated by biochemical detection. The involvement of NOX2 and mitogen-activated protein kinases (MAPKs) in IL-1ß-induced COX-2 and PGE2 expression was investigated using pharmacologic inhibitors and further analyzed by western blotting. Activation of PPARγ was performed by using a pharmacologic agonist and was analyzed by western blotting. Results: IL-1ß-induced COX-2 and PGE2 expression was mediated through NOX2 activation/ROS production, which could be attenuated by N-acetylcysteine (NAC; a scavenger of ROS), GW1929 (PPARγ agonist), DPI (diphenyleneiodonium chloride, NOX2 inhibitor), SB203580 (p38MAPK inhibitor), PD98059 (extracellular signal-regulated kinase, ERK inhibitor), and SP600125 (c-Jun N-terminal kinase, JNK inhibitor). ROS activated p38MAPK to enter the nucleus, which was attenuated by PPARγ. Conclusion: In OA chondrocytes, IL-1ß induced COX-2 and PGE2 expression via activation of NOX2, which led to ROS production and MAPK activation. The activation of PPARγ exerted protective roles in the pathogenesis of OA.


Assuntos
Condrócitos/metabolismo , Interleucina-1beta/metabolismo , Osteoartrite/genética , PPAR gama/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Animais , Apoptose , Humanos , Osteoartrite/patologia , Ratos , Espécies Reativas de Oxigênio
8.
Nat Commun ; 12(1): 3046, 2021 05 24.
Artigo em Inglês | MEDLINE | ID: mdl-34031426

RESUMO

Nasopharyngeal carcinoma (NPC) is a malignant head and neck cancer type with high morbidity in Southeast Asia, however the pathogenic mechanism of this disease is poorly understood. Using integrative pharmacogenomics, we find that NPC subtypes maintain distinct molecular features, drug responsiveness, and graded radiation sensitivity. The epithelial carcinoma (EC) subtype is characterized by activations of microtubule polymerization and defective mitotic spindle checkpoint related genes, whereas sarcomatoid carcinoma (SC) and mixed sarcomatoid-epithelial carcinoma (MSEC) subtypes exhibit enriched epithelial-mesenchymal transition (EMT) and invasion promoting genes, which are well correlated with their morphological features. Furthermore, patient-derived organoid (PDO)-based drug test identifies potential subtype-specific treatment regimens, in that SC and MSEC subtypes are sensitive to microtubule inhibitors, whereas EC subtype is more responsive to EGFR inhibitors, which is synergistically enhanced by combining with radiotherapy. Through combinational chemoradiotherapy (CRT) screening, effective CRT regimens are also suggested for patients showing less sensitivity to radiation. Altogether, our study provides an example of applying integrative pharmacogenomics to establish a personalized precision oncology for NPC subtype-guided therapies.


Assuntos
Carcinoma Nasofaríngeo/tratamento farmacológico , Carcinoma Nasofaríngeo/genética , Neoplasias Nasofaríngeas/tratamento farmacológico , Neoplasias Nasofaríngeas/genética , Farmacogenética/métodos , Avaliação Pré-Clínica de Medicamentos/métodos , Transição Epitelial-Mesenquimal , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Técnicas de Inativação de Genes , Humanos , Pessoa de Meia-Idade , Carcinoma Nasofaríngeo/patologia , Neoplasias Nasofaríngeas/patologia , Medicina de Precisão , Transcriptoma , Sequenciamento Completo do Exoma
9.
Theranostics ; 11(10): 5010-5027, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33754041

RESUMO

Background: Autophagy has been implicated as a crucial component in spermatogenesis, and autophagy dysfunction can lead to reproductive disorders in animal models, including yeast, C. elegans and mice. However, the sophisticated transcriptional networks of autophagic genes throughout human spermatogenesis and their biological significance remain largely uncharacterized. Methods: We profiled the transcriptional signatures of autophagy-related genes during human spermatogenesis by assessing specimens from nine fertile controls (including two normal persons and seven obstructive azoospermia (OA) patients) and one nonobstructive azoospermia (NOA) patient using single-cell RNA sequencing (scRNA-seq) analysis. Dysregulation of autophagy was confirmed in two additional NOA patients by immunofluorescence staining. Gene knockdown was used to identify the role of Cst3 in autophagy during spermatogenesis. Results: Our data uncovered a unique, global stage-specific enrichment of autophagy-related genes. Human-mouse comparison analysis revealed that the stage-specific expression pattern of autophagy-related genes was highly conserved in mammals. More importantly, dysregulation of some clusters of autophagy-related genes was observed in NOA patients, suggesting the association of autophagy with male infertility. Cst3, a human-mouse conserved and autophagy-related gene that is actively expressed in spermatogonia and early spermatocytes, was found to regulate spermatogonial stem cell (SSC) maintenance and subsequent male germ cell development. Knockdown of Cst3 increased autophagic activity in mouse SSCs and subsequently suppressed the transcription of SSC core factors such as Oct4, Id1, and Nanos3, which could be efficiently rescued by manipulating autophagic activity. Conclusions: Our study provides comprehensive insights into the global transcriptional signatures of autophagy-related genes and confirms the importance of autophagy homeostasis in SSC maintenance and normal spermatogenesis, opening new avenues for further dissecting the significance of the autophagy regulatory network in spermatogenesis as well as male infertility.


Assuntos
Autofagia/genética , Azoospermia/genética , Cistatina C/genética , Espermatogênese/genética , Adulto , Células-Tronco Germinativas Adultas/metabolismo , Perfilação da Expressão Gênica , Técnicas de Silenciamento de Genes , Redes Reguladoras de Genes , Humanos , Masculino , Meiose/genética , Pessoa de Meia-Idade , RNA-Seq , Análise de Célula Única , Ducto Deferente
10.
Adv Sci (Weinh) ; 7(23): 2001914, 2020 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-33304752

RESUMO

Resistance to therapeutic drugs occurs in virtually all types of cancers, and the tolerance to one drug frequently becomes broad therapy resistance; however, the underlying mechanism remains elusive. Combining a whole whole-genome-wide RNA interference screening and an evolutionary drug pressure model with MDA-MB-231 cells, it is found that enhanced protein damage clearance and reduced mitochondrial respiratory activity are responsible for cisplatin resistance. Screening drug-resistant cancer cells and human patient-derived organoids for breast and colon cancers with many anticancer drugs indicates that activation of mitochondrion protein import surveillance system enhances proteasome activity and minimizes caspase activation, leading to broad drug resistance that can be overcome by co-treatment with a proteasome inhibitor, bortezomib. It is further demonstrated that cisplatin and bortezomib encapsulated into nanoparticle further enhance their therapeutic efficacy and alleviate side effects induced by drug combination treatment. These data demonstrate a feasibility for eliminating broad drug resistance by targeting its common mechanism to achieve effective therapy for multiple cancers.

11.
Int J Biol Sci ; 16(15): 3085-3099, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-33061820

RESUMO

Well-orchestrated epigenetic modifications during early development are essential for embryonic survival and postnatal growth. Erroneous epigenetic modifications due to environmental perturbations such as manipulation and culture of embryos during in vitro fertilization (IVF) are linked to various short- or long-term consequences. Among these, DNA methylation defects are of great concern. Despite the critical role of DNA methylation in determining embryonic development potential, the mechanisms underlying IVF-associated DNA methylation defects, however, remains largely elusive. We reported herein that repression of fibroblast growth factor (FGF) signaling as the main reason for IVF-associated DNA methylation defects. Comparative methylome analysis by postimplantation stage suggested that IVF mouse embryos undergo impaired de novo DNA methylation during implantation stage. Further analyses indicated that Dnmt3b, the main de novo DNA methyltransferase, was consistently inhibited during the transition from the blastocyst to postimplantation stage (Embryonic day 7.5, E7.5). Using blastocysts and embryonic stem cells (ESCs) as the model, we showed repression of FGF signaling is responsible for Dnmt3b inhibition and global hypomethylation during early development, and MEK/ERK-SP1 pathway plays an essential mediating role in FGF signaling-induced transcriptional activation of Dnmt3b. Supplementation of FGF2, which was exclusively produced in the maternal oviduct, into embryo culture medium significantly rescued Dnmt3b inhibition. Our study, using mouse embryos as the model, not only identifies FGF signaling as the main target for correcting IVF-associated epigenetic errors, but also highlights the importance of oviductal paracrine factors in supporting early embryonic development and improving in vitro culture system.

12.
Nat Commun ; 11(1): 4875, 2020 09 25.
Artigo em Inglês | MEDLINE | ID: mdl-32978388

RESUMO

Single-cell whole-exome sequencing (scWES) is a powerful approach for deciphering intratumor heterogeneity and identifying cancer drivers. So far, however, simultaneous analysis of single nucleotide variants (SNVs) and copy number variations (CNVs) of a single cell has been challenging. By analyzing SNVs and CNVs simultaneously in bulk and single cells of premalignant tissues and tumors from mouse and human BRCA1-associated breast cancers, we discover an evolution process through which the tumors initiate from cells with SNVs affecting driver genes in the premalignant stage and malignantly progress later via CNVs acquired in chromosome regions with cancer driver genes. These events occur randomly and hit many putative cancer drivers besides p53 to generate unique genetic and pathological features for each tumor. Upon this, we finally identify a tumor metastasis suppressor Plekha5, whose deficiency promotes cancer metastasis to the liver and/or lung.


Assuntos
Proteína BRCA1/genética , Proteína BRCA1/metabolismo , Predisposição Genética para Doença/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Lesões Pré-Cancerosas/genética , Animais , Neoplasias da Mama/genética , Linhagem Celular Tumoral , Variações do Número de Cópias de DNA , Modelos Animais de Doenças , Heterogeneidade Genética , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Fígado/patologia , Pulmão/patologia , Camundongos , Camundongos Knockout , Mutação , Lesões Pré-Cancerosas/patologia , Transcriptoma
13.
Nat Commun ; 11(1): 3256, 2020 06 26.
Artigo em Inglês | MEDLINE | ID: mdl-32591500

RESUMO

BRCA1 mutation carriers have a higher risk of developing triple-negative breast cancer (TNBC), which is a refractory disease due to its non-responsiveness to current clinical targeted therapies. Using the Sleeping Beauty transposon system in Brca1-deficient mice, we identified 169 putative cancer drivers, among which Notch1 is a top candidate for accelerating TNBC by promoting the epithelial-mesenchymal transition (EMT) and regulating the cell cycle. Activation of NOTCH1 suppresses mitotic catastrophe caused by BRCA1 deficiency by restoring S/G2 and G2/M cell cycle checkpoints, which may through activation of ATR-CHK1 signalling pathway. Consistently, analysis of human breast cancer tissue demonstrates NOTCH1 is highly expressed in TNBCs, and the activated form of NOTCH1 correlates positively with increased phosphorylation of ATR. Additionally, we demonstrate that inhibition of the NOTCH1-ATR-CHK1 cascade together with cisplatin synergistically kills TNBC by targeting the cell cycle checkpoint, DNA damage and EMT, providing a potent clinical option for this fatal disease.


Assuntos
Proteína BRCA1/deficiência , Carcinogênese/patologia , Receptor Notch1/metabolismo , Neoplasias de Mama Triplo Negativas/metabolismo , Neoplasias de Mama Triplo Negativas/patologia , Alelos , Animais , Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Proteína BRCA1/metabolismo , Morte Celular , Linhagem Celular Tumoral , Quinase 1 do Ponto de Checagem/metabolismo , Elementos de DNA Transponíveis/genética , Progressão da Doença , Transição Epitelial-Mesenquimal , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Camundongos Knockout , Mitose , Mutação/genética , Transdução de Sinais , Neoplasias de Mama Triplo Negativas/genética
14.
Theranostics ; 10(7): 2897-2917, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32194843

RESUMO

Highly plastic macrophages are pivotal players in the body's homeostasis and pathogenesis. Grasping the molecular or cellular factors that drive and support the macrophage activation will help to develop diagnostics and manipulate their functions in these contexts. However, the lack of in vivo characterization methods to reveal the dynamic activation of macrophages impedes these studies in various disease contexts. Methods: Here, in vitro bone marrow-derived macrophages (BMDMs) and in vivo Matrigel plug were used to evaluate how mitochondria dynamics supports cellular activation and functions. We conducted macrophage repolarization in vitro to track mitochondria dynamics during the shift of activation status. For in vivo diagnosis, a novel MitoTracker-loaded liposome was first developed to label macrophage mitochondria in mice before/after inflammatory stimulation. Results: Based on the typical activation of in vitro BMDMs, we found glycolysis based macrophages have punctate and discrete mitochondria, while OXPHOS active macrophages have elongated and interconnected mitochondria. M1, M2a, M2b, and M2c activated BMDMs showed clustered and differentiable features in mitochondrial morphology. These features also hold for Matrigel plug-recruited macrophages in mice. Furthermore, with the interventions on M2a macrophages in vitro, we demonstrated that mitochondria morphology could be a metabolic index to evaluate macrophage activation status under drug manipulation. Using the MitoTracker-loaded liposomes, we further achieved subcellular imaging of macrophage mitochondria in vivo. Their organization dynamics revealed the dynamic change from anti-inflammatory macrophages to inflammatory ones in vivo under the lipopolysaccharide (LPS) challenge. Conclusion: These results reveal that subcellular imaging of mitochondria organization can characterize the activation status of macrophage in vitro and in vivo at a single-cell level, which is critical for the studies of noninvasive diagnosis and therapeutic drug monitoring.


Assuntos
Ativação de Macrófagos , Macrófagos , Mitocôndrias , Análise de Célula Única/métodos , Animais , Biomarcadores/metabolismo , Células Cultivadas , Macrófagos/metabolismo , Macrófagos/ultraestrutura , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Mitocôndrias/metabolismo , Mitocôndrias/ultraestrutura
15.
Adv Sci (Weinh) ; 7(6): 1903616, 2020 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-32195105

RESUMO

The breast cancer susceptibility gene 1 (BRCA1) is a major tumor suppressor gene and is most frequently mutated in hereditary breast cancer. BRCA1 plays a critical role in many biological processes, especially maintaining genomic stability in the nucleus, yet its role in the cytoplasm remains elusive. Here, it is revealed that BRCA1 maintains a healthy mitochondrial network through regulating mitochondrial dynamics, including fission and fusion. BRCA1 deficiency causes dysfunctional mitochondrial dynamics through increased expression of mitofusin1/2. With mitochondrial stress, BRCA1 is recruited to the mitochondrial outer membrane, where it plays an essential role in maintaining a healthy mitochondrial network. Consequently, BRCA1 deficiency impairs stress-induced mitophagy through blocking ataxia-telangiectasia mutated (ATM)-AMP-activated protein kinase (AMPK)-Dynamin-related protein 1 (DRP1)-mediated mitochondrial fission and triggers NLRP3 inflammasome activation, which creates a tumor-associated microenvironment, thereby facilitating tumor proliferation and metastasis. It is further shown that inflammasome inhibition can prevent tumor recurrence and metastasis. This study uncovers an important role of BRCA1 in regulating mitophagy and suggests a therapeutic approach for fighting this deadly disease.

16.
Int J Biol Sci ; 16(8): 1450-1462, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32210732

RESUMO

Keratinocyte is the predominant cell type in the epidermis of skin, and it provides the protective barrier function for the body. Various signaling pathways have been implicated in keratinocyte differentiation in animal models; However, their temporal regulation and interactions are still to be explored in pluripotent stem cell models. In this report, we use human embryonic stem cells to demonstrate that epidermal ectoderm and subsequent keratinocyte cell fate can be determined step by step under the regulation of defined factors. The inhibition of TGFß initiates ectodermal lineage differentiation, and the activation of BMP pathway drives epidermal TP63 expression. Meanwhile, the timely activation of WNT pathway suppresses extraembryonic lineage, and promotes epidermal cell fate. With further specification by NOTCH inhibition, more than 90% of cells become TP63-positive stage Ⅱ keratinocytes. Finally, stage Ⅲ keratinocytes are produced under defined hypo-calcium keratinocyte culture conditions, and are further matured in mouse xenograft model. This study not only establishes an in vitro platform to study keratinocyte cell fate determination, but also provides an efficient protocol to produce keratinocytes for disease models and clinical applications.


Assuntos
Técnicas de Cultura de Células , Células-Tronco Embrionárias/citologia , Queratinócitos/citologia , Transdução de Sinais , Animais , Diferenciação Celular , Linhagem da Célula , Ectoderma , Epiderme , Humanos , Masculino , Camundongos , Camundongos SCID , Técnicas de Cultura de Órgãos , Pele , Fator de Crescimento Transformador beta/metabolismo , Proteínas Wnt/metabolismo
17.
Int J Mol Sci ; 21(3)2020 Feb 03.
Artigo em Inglês | MEDLINE | ID: mdl-32028587

RESUMO

Gamma-aminobutyric acid (GABA) is widely distributed in nature and considered a potent bioactive compound with numerous and important physiological functions, such as anti-hypertensive and antidepressant activities. There is an ever-growing demand for GABA production in recent years. Lactic acid bacteria (LAB) are one of the most important GABA producers because of their food-grade nature and potential of producing GABA-rich functional foods directly. In this paper, the GABA-producing LAB species, the biosynthesis pathway of GABA by LAB, and the research progress of glutamate decarboxylase (GAD), the key enzyme of GABA biosynthesis, were reviewed. Furthermore, GABA production enhancement strategies are reviewed, from optimization of culture conditions and genetic engineering to physiology-oriented engineering approaches and co-culture methods. The advances in both the molecular mechanisms of GABA biosynthesis and the technologies of synthetic biology and genetic engineering will promote GABA production of LAB to meet people's demand for GABA. The aim of the review is to provide an insight of microbial engineering for improved production of GABA by LAB in the future.


Assuntos
Engenharia Genética/métodos , Lactobacillales/metabolismo , Ácido gama-Aminobutírico/metabolismo , Lactobacillales/genética , Lactobacillales/crescimento & desenvolvimento
18.
Artigo em Inglês | MEDLINE | ID: mdl-31956410

RESUMO

Background: Implantation failure limits the success of in vitro fertilization and embryo transfer (IVF-ET). Well-organized embryo-maternal crosstalk is essential for successful implantation. Previous studies mainly focused on the aberrant development of in vitro fertilized (IVF) embryos. In contrast, the mechanism of IVF-induced aberrant embryo-maternal crosstalk is not well defined. Results: In the present study, using ewes as the model, we profiled the proteome that features aberrant IVF embryo-maternal crosstalk following IVF-ET. By comparing in vivo (IVO) and IVF conceptuses, as well as matched endometrial caruncular (C) and intercaruncular (IC) areas, we filtered out 207, 295, and 403 differentially expressed proteins (DEPs) in each comparison. Proteome functional analysis showed that the IVF conceptuses were characterized by the increased abundance of energy metabolism and proliferation-related proteins, and the decreased abundance of methyl metabolism-related proteins. In addition, IVF endometrial C areas showed the decreased abundance of endometrial remodeling and redox homeostasis-related proteins; while IC areas displayed the aberrant abundance of protein homeostasis and extracellular matrix (ECM) interaction-related proteins. Based on these observations, we propose a model depicting the disrupted embryo-maternal crosstalk following IVF-ET: Aberrant energy metabolism and redox homeostasis of IVF embryos, might lead to an aberrant endometrial response to conceptus-derived pregnancy signals, thus impairing maternal receptivity. In turn, the suboptimal uterine environment might stimulate a compensation effect of the IVF conceptuses, which was revealed as enhanced energy metabolism and over-proliferation. Conclusion: Systematic proteomic profiling provides insights to understand the mechanisms that underlie the aberrant IVF embryo-maternal crosstalk. This might be helpful to develop practical strategies to prevent implantation failure following IVF-ET.

19.
Chem Commun (Camb) ; 55(95): 14255-14258, 2019 Nov 26.
Artigo em Inglês | MEDLINE | ID: mdl-31657388
20.
Oncol Lett ; 18(4): 3581-3590, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31516572

RESUMO

The sensitivity and utility of liquid biopsy in clinical practice requires some improvement. The aim of the present study was to improve the detection of epidermal growth factor (EGFR) and cellular tumor antigen p53 (TP53) mutations in liquid biopsies from patients with advanced non-small cell lung cancer (NSCLC) by combining plasma, sputum and urine samples under the same sequencing platform. Plasma, sputum and urine samples, and tumor tissues were obtained from 50 patients with NSCLC and were analyzed using next-generation sequencing. The sensitivity of EGFR-sensitive mutation detection was 84% in plasma, 63% in sputum, 28% in urine, and 91% when combining the three liquid samples (P<0.001). The sensitivity of TP53 mutation detection increased from 87% in plasma to 94% when the three samples were combined (P<0.001). The sensitivity of EGFR or TP53 mutations detection was higher in patients with multiple metastatic sites compared with patients ≤1 metastatic site. In addition, the progression free survival (PFS) rates obtained following analysis of the three samples independently in patients with EGFR sensitizing mutations were similar, and were 9.0 months in the tissue sample, 7.5 months in plasma, 7.9 months in the sputum and 7.3 months in urine (P=0.721). The PFS of patients with TP53 mutations was shorter compared with patients without TP53 mutations and was as follows: Tissue, 8.2 months compared with 10.2 months (P=0.412); plasma, 8.4 months compared with 10.2 months (P=0.466); sputum, 8.3 months compared with 9.1 months (P=0.904); and when combined, 8.8 months compared with 10.3 months (P=0.599). The combination of plasma, sputum and urine increased the detection of EGFR or TP53 mutation with higher sensitivity, and may improve the predictive value of personalized treatment.

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