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1.
J Virol ; 96(9): e0033622, 2022 05 11.
Artigo em Inglês | MEDLINE | ID: mdl-35404082

RESUMO

Epstein-Barr virus (EBV), the first identified human tumor virus, is etiologically associated with various kinds of malignant and benign diseases, accounting for 265,000 cancer incident cases and 164,000 cancer deaths in 2017. EBV prophylactic vaccine development has been gp350 centered for several decades. However, clinical studies show that gp350-centered vaccines fail to prevent EBV infection. Advances in the EBV infection mechanisms shed light on gB and gHgL, the two key components of the infection apparatus. In this study, for the first time, we utilized recombinant vesicular stomatitis virus (VSV) to display EBV gB (VSV-ΔG-gB/gB-G) or gHgL (VSV-ΔG-gHgL). In vitro studies confirmed successful virion production and glycoprotein presentation on the virion surface. In mouse models, VSV-ΔG-gB/gB-G or VSV-ΔG-gHgL elicited potent humoral responses. Neutralizing antibodies elicited by VSV-ΔG-gB/gB-G were prone to prevent B cell infection, while those elicited by VSV-ΔG-gHgL were prone to prevent epithelial cell infection. Combinatorial vaccination yields an additive effect. The ratio of endpoint neutralizing antibody titers to the endpoint total IgG titers immunized with VSV-ΔG-gHgL was approximately 1. The ratio of IgG1/IgG2a after VSV-ΔG-gB/gB-G immunization was approximately 1 in a dose-dependent, adjuvant-independent manner. Taken together, VSV-based EBV vaccines can elicit a high ratio of epithelial and B lymphocyte neutralizing antibodies, implying their unique potential as EBV prophylactic vaccine candidates. IMPORTANCE Epstein-Barr virus (EBV), one of the most common human viruses and the first identified human oncogenic virus, accounted for 265,000 cancer incident cases and 164,000 cancer deaths in 2017 as well as millions of nonmalignant disease cases. So far, no prophylactic vaccine is available to prevent EBV infection. In this study, for the first time, we reported the VSV-based EBV vaccines presenting two key components of the EBV infection apparatus, gB and gHgL. We confirmed potent antigen-specific antibody generation; these antibodies prevented EBV from infecting epithelial cells and B cells, and the IgG1/IgG2a ratio indicated balanced humoral-cellular responses. Taken together, we suggest VSV-based EBV vaccines are potent prophylactic candidates for clinical studies and help eradicate numerous EBV-associated malignant and benign diseases.


Assuntos
Infecções por Vírus Epstein-Barr , Vesiculovirus , Vacinas Virais , Animais , Anticorpos Neutralizantes/sangue , Anticorpos Antivirais/sangue , Infecções por Vírus Epstein-Barr/prevenção & controle , Herpesvirus Humano 4/fisiologia , Imunidade Humoral , Imunoglobulina G/sangue , Camundongos , Vesiculovirus/genética , Vacinas Virais/imunologia
2.
Nano Lett ; 21(6): 2476-2486, 2021 03 24.
Artigo em Inglês | MEDLINE | ID: mdl-33683126

RESUMO

Epstein-Barr virus (EBV) infection is a global health concern infecting over 90% of the population. However, there is no currently available vaccine. EBV primarily infects B cells, where the major glycoprotein 350 (gp350) is the main target of neutralizing antibodies. Given the advancement of nanoparticle vaccines, we describe rationally designed vaccine modalities presenting 60 copies of gp350 on self-assembled nanoparticles in a repetitive array. In a mouse model, gp350s on lumazine synthase (LS) and I3-01 adjuvanted with MF59 or aluminum hydroxide (Alhydrogel) elicited over 65- to 133-fold higher neutralizing antibody titers than the corresponding gp350 monomer to EBV. Furthermore, immunization with gp350D123-LS and gp350D123-I3-01 vaccine induced a Th2-biased response. For the nonhuman primate model, gp350D123-LS in MF59 elicited higher titers of total IgG and neutralizing antibodies than the monomeric gp350D123. Overall, these results support gp350D123-based nanoparticle vaccine design as a promising vaccine candidate for potent protection against EBV infection.


Assuntos
Infecções por Vírus Epstein-Barr , Nanopartículas , Vacinas , Animais , Anticorpos Neutralizantes , Anticorpos Antivirais , Infecções por Vírus Epstein-Barr/prevenção & controle , Herpesvirus Humano 4 , Imunização , Camundongos
3.
Mol Reprod Dev ; 83(11): 1027-1040, 2016 11.
Artigo em Inglês | MEDLINE | ID: mdl-27696585

RESUMO

Kinesin family member 1B (KIF1B) is an important microtubule-dependent monomeric motor in mammals, although little is known about its role in meiosis. We profiled KIF1B expression and localization during oocyte maturation and early embryonic development in mice, revealing a dynamic pattern throughout meiotic progression. Depletion or inhibition of KIF1B leads to abnormal polar body extrusion, disordered spindle dynamics, defects in chromosome congression, increased aneuploidy, and impaired embryonic development. Further, KIF1B depletion affects the distribution of mitochondria and abundance of ATP. Taken together, our study demonstrates that mouse KIF1B is important for spindle assembly, chromosome congression, and mitochondrial distribution during oocyte maturation and early embryonic development. Mol. Reprod. Dev. 83: 1027-1040, 2016 © 2016 Wiley Periodicals, Inc.


Assuntos
Embrião de Mamíferos/embriologia , Desenvolvimento Embrionário/fisiologia , Cinesinas/metabolismo , Meiose/fisiologia , Trifosfato de Adenosina/genética , Trifosfato de Adenosina/metabolismo , Animais , Cromossomos de Mamíferos/genética , Cromossomos de Mamíferos/metabolismo , Embrião de Mamíferos/citologia , Feminino , Cinesinas/genética , Masculino , Camundongos , Mitocôndrias/genética , Mitocôndrias/metabolismo , Oócitos , Corpos Polares/metabolismo , Fuso Acromático/genética , Fuso Acromático/metabolismo
4.
Cell Rep Med ; 5(5): 101573, 2024 May 21.
Artigo em Inglês | MEDLINE | ID: mdl-38776874

RESUMO

Epstein-Barr virus (EBV) is linked to various malignancies and autoimmune diseases, posing a significant global health challenge due to the lack of specific treatments or vaccines. Despite its crucial role in EBV infection in B cells, the mechanisms of the glycoprotein gp42 remain elusive. In this study, we construct an antibody phage library from 100 EBV-positive individuals, leading to the identification of two human monoclonal antibodies, 2B7 and 2C1. These antibodies effectively neutralize EBV infection in vitro and in vivo while preserving gp42's interaction with the human leukocyte antigen class II (HLA-II) receptor. Structural analysis unveils their distinct binding epitopes on gp42, different from the HLA-II binding site. Furthermore, both 2B7 and 2C1 demonstrate potent neutralization of EBV infection in HLA-II-positive epithelial cells, expanding our understanding of gp42's role. Overall, this study introduces two human anti-gp42 antibodies with potential implications for developing EBV vaccines targeting gp42 epitopes, addressing a critical gap in EBV research.


Assuntos
Anticorpos Monoclonais , Epitopos , Infecções por Vírus Epstein-Barr , Herpesvirus Humano 4 , Humanos , Herpesvirus Humano 4/imunologia , Infecções por Vírus Epstein-Barr/imunologia , Infecções por Vírus Epstein-Barr/virologia , Anticorpos Monoclonais/imunologia , Epitopos/imunologia , Animais , Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , Camundongos , Antígenos de Histocompatibilidade Classe II/imunologia , Antígenos de Histocompatibilidade Classe II/metabolismo , Proteínas Virais/imunologia , Linfócitos B/imunologia
5.
In Vitro Cell Dev Biol Anim ; 59(7): 550-563, 2023 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-37639049

RESUMO

Conservation of genetic resources is an important way to protect endangered species. At present, mesenchymal stem cells (MSCs) have been isolated from the bone marrow and umbilical cords of giant pandas. However, the types and quantities of preserved cell resources were rare and limited, and none of MSCs was derived from female reproductive organs. Here, we first isolated MSCs from the endometrium of giant panda. These cells showed fibroblast morphology and expressed Sox2, Klf4, Thy1, CD73, CD105, CD44, CD49f, and CD105. Endometrium mesenchymal stem cells (eMSCs) of giant panda could induce differentiation into three germ layers in vitro. RNA-seq analysis showed that 833 genes were upregulated and 716 genes were downregulated in eMSCs compared with skin fibroblast cells. The results of GO and the KEGG analysis of differentially expressed genes (DEGs) were mainly focused on transporter activity, signal transducer activity, pathways regulating pluripotency of stem cells, MAPK signaling pathway, and PI3K-Akt signaling pathway. The genes PLCG2, FRK, JAK3, LYN, PIK3CB, JAK2, CBLB, and MET were identified as hub genes by PPI network analysis. In addition, the exosomes of eMSCs were also isolated and identified. The average diameter of exosomes was 74.26 ± 13.75 nm and highly expressed TSG101 and CD9 but did not express CALNEXIN. A total of 277 miRNAs were detected in the exosomes; the highest expression of miRNA was the has-miR-21-5p. A total of 14461 target genes of the whole miRNAs were predicted and proceeded with functional analysis. In conclusion, we successfully isolated and characterized the giant panda eMSCs and their exosomes, and analyzed their functions through bioinformatics techniques. It not only enriched the conservation types of giant panda cell resources and promoted the protection of genetic diversity, but also laid a foundation for the application of eMSCs and exosomes in the disease treatment of giant pandas.


Assuntos
Exossomos , Células-Tronco Mesenquimais , MicroRNAs , Ursidae , Feminino , Animais , Ursidae/genética , Exossomos/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Células-Tronco Mesenquimais/metabolismo , MicroRNAs/genética , Endométrio/metabolismo
6.
Adv Sci (Weinh) ; 10(35): e2302116, 2023 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-37890462

RESUMO

Epstein-Barr virus (EBV) is associated with various malignancies and infects >90% of the global population. EBV latent proteins are expressed in numerous EBV-associated cancers and contribute to carcinogenesis, making them critical therapeutic targets for these cancers. Thus, this study aims to develop mRNA-based therapeutic vaccines that express the T-cell-epitope-rich domain of truncated latent proteins of EBV, including truncatedlatent membrane protein 2A (Trunc-LMP2A), truncated EBV nuclear antigen 1 (Trunc-EBNA1), and Trunc-EBNA3A. The vaccines effectively activate both cellular and humoral immunity in mice and show promising results in suppressing tumor progression and improving survival time in tumor-bearing mice. Furthermore, it is observed that the truncated forms of the antigens, Trunc-LMP2A, Trunc-EBNA1, and Trunc-EBNA3A, are more effective than full-length antigens in activating antigen-specific immune responses. In summary, the findings demonstrate the effectiveness of mRNA-based therapeutic vaccines targeting the T-cell-epitope-rich domain of EBV latent proteins and providing new treatment options for EBV-associated cancers.


Assuntos
Infecções por Vírus Epstein-Barr , Neoplasias , Camundongos , Animais , Herpesvirus Humano 4/genética , Infecções por Vírus Epstein-Barr/terapia , Epitopos de Linfócito T , Vacinas de mRNA , Proteínas de Membrana , RNA Mensageiro/genética
7.
Cell Host Microbe ; 31(11): 1882-1897.e10, 2023 11 08.
Artigo em Inglês | MEDLINE | ID: mdl-37848029

RESUMO

Epstein-Barr virus (EBV) is a global public health concern, as it is known to cause multiple diseases while also being etiologically associated with a wide range of epithelial and lymphoid malignancies. Currently, there is no available prophylactic vaccine against EBV. gB is the EBV fusion protein that mediates viral membrane fusion and participates in host recognition, making it critical for EBV infection in both B cells and epithelial cells. Here, we present a gB nanoparticle, gB-I53-50 NP, that displays multiple copies of gB. Compared with the gB trimer, gB-I53-50 NP shows improved structural integrity and stability, as well as enhanced immunogenicity in mice and non-human primate (NHP) preclinical models. Immunization and passive transfer demonstrate a robust and durable protective antibody response that protects humanized mice against lethal EBV challenge. This vaccine candidate demonstrates significant potential in preventing EBV infection, providing a possible platform for developing prophylactic vaccines for EBV.


Assuntos
Infecções por Vírus Epstein-Barr , Vacinas , Cricetinae , Animais , Camundongos , Herpesvirus Humano 4 , Infecções por Vírus Epstein-Barr/prevenção & controle , Formação de Anticorpos , Células CHO , Anticorpos Neutralizantes , Anticorpos Antivirais
8.
Signal Transduct Target Ther ; 7(1): 42, 2022 02 08.
Artigo em Inglês | MEDLINE | ID: mdl-35136040

RESUMO

SARS-CoV-2 variants have evolved a variety of critical mutations, leading to antigenicity changes and immune escape. The recent emerging SARS-CoV-2 Omicron variant attracted global attention due to its significant resistance to current antibody therapies and vaccines. Here, we profiled the mutations of Omicron and other various circulating SARS-CoV-2 variants in parallel by computational interface analysis and in vitro experimental assays. We identified critical mutations that lead to antigenicity changes and diminished neutralization efficiency of a panel of 14 antibodies due to diverse molecular mechanisms influencing the antigen-antibody interaction. Our study identified that Omicron exhibited extraordinary potency in immune escape compared to the other variants of concern, and explores the application of computational interface analysis in SARS-CoV-2 mutation surveillance and demonstrates its potential for the early identification of concerning variants, providing preliminary guidance for neutralizing antibody therapy.


Assuntos
Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , Antígenos Virais , COVID-19 , Evasão da Resposta Imune , SARS-CoV-2 , Antígenos Virais/genética , Antígenos Virais/imunologia , COVID-19/genética , COVID-19/imunologia , Células HEK293 , Humanos , SARS-CoV-2/genética , SARS-CoV-2/imunologia
9.
ACS Nano ; 15(2): 2738-2752, 2021 02 23.
Artigo em Inglês | MEDLINE | ID: mdl-33464829

RESUMO

The coronavirus disease pandemic of 2019 (COVID-19) caused by the novel SARS-CoV-2 coronavirus resulted in economic losses and threatened human health worldwide. The pandemic highlights an urgent need for a stable, easily produced, and effective vaccine. SARS-CoV-2 uses the spike protein receptor-binding domain (RBD) to bind its cognate receptor, angiotensin-converting enzyme 2 (ACE2), and initiate membrane fusion. Thus, the RBD is an ideal target for vaccine development. In this study, we designed three different RBD-conjugated nanoparticle vaccine candidates, namely, RBD-Ferritin (24-mer), RBD-mi3 (60-mer), and RBD-I53-50 (120-mer), via covalent conjugation using the SpyTag-SpyCatcher system. When mice were immunized with the RBD-conjugated nanoparticles (NPs) in conjunction with the AddaVax or Sigma Adjuvant System, the resulting antisera exhibited 8- to 120-fold greater neutralizing activity against both a pseudovirus and the authentic virus than those of mice immunized with monomeric RBD. Most importantly, sera from mice immunized with RBD-conjugated NPs more efficiently blocked the binding of RBD to ACE2 in vitro, further corroborating the promising immunization effect. Additionally, the vaccine has distinct advantages in terms of a relatively simple scale-up and flexible assembly. These results illustrate that the SARS-CoV-2 RBD-conjugated nanoparticles developed in this study are a competitive vaccine candidate and that the carrier nanoparticles could be adopted as a universal platform for a future vaccine development.


Assuntos
Enzima de Conversão de Angiotensina 2/metabolismo , Vacinas contra COVID-19/uso terapêutico , COVID-19/prevenção & controle , Nanopartículas/uso terapêutico , SARS-CoV-2/fisiologia , Glicoproteína da Espícula de Coronavírus/metabolismo , Animais , COVID-19/metabolismo , Vacinas contra COVID-19/farmacologia , Chlorocebus aethiops , Feminino , Células HEK293 , Interações Hospedeiro-Patógeno , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Modelos Moleculares , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Glicoproteína da Espícula de Coronavírus/química , Células Vero
10.
mSphere ; 5(6)2020 12 02.
Artigo em Inglês | MEDLINE | ID: mdl-33268566

RESUMO

While Epstein-Barr virus (EBV) is the major cause of nasopharyngeal carcinoma (NPC), the value of the humoral immune response to EBV glycoproteins and NPC development remains unclear. Correlation between antiglycoprotein antibody levels, neutralization of EBV infectivity, and the risk of NPC requires systematic study. Here, we applied a cytometry-based method and enzyme-linked immunosorbent assay to measure neutralization of infectivity and antibody response to EBV glycoproteins (gH/gL, gB, gp350, and gp42) of plasma samples from 20 NPC cases and 20 high-risk and 20 low-risk healthy controls nested within a screening cohort in Sihui, southern China. We found that NPC cases have similar plasma neutralizing activity in both B cells and epithelial cells and EBV glycoprotein-specific IgA and IgG antibody levels compared with those of healthy controls. Significant correlations were observed between gH/gL IgG and gB IgG and the neutralizing ability against EBV infection of epithelial cells and B cells. These results indicate that a high level of glycoprotein antibodies may favor protection against primary EBV infection, instead of being low-risk biomarkers for NPC in long-term EBV-infected adults. In conclusion, this study provides novel insights into the humoral immune response to EBV infection and NPC development, providing valuable leads for future research that is important for prevention and treatment of EBV-related diseases.IMPORTANCE Epstein-Barr virus (EBV) is a human oncogenic gammaherpesvirus that infects over 90% of humans in the world and is causally associated with a spectrum of epithelial and B-cell malignancies such as nasopharyngeal carcinoma (NPC). A prophylactic vaccine against EBV is called for, but no approved vaccine is available yet. Therefore, EBV remains a major public health concern. To facilitate novel vaccines and therapeutics for NPC, it is of great importance to explore the impact of humoral immune response to EBV glycoproteins before the development of NPC. Therefore, in this study, we systematically assessed the correlation between antiglycoprotein antibody levels, neutralization of EBV infectivity, and the risk of NPC development. These results provide valuable information that will contribute to designing effective prevention and treatment strategies for EBV-related diseases such as NPC.


Assuntos
Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , Linfócitos B/imunologia , Infecções por Vírus Epstein-Barr/imunologia , Antígenos Nucleares do Vírus Epstein-Barr/imunologia , Herpesvirus Humano 4/imunologia , Neoplasias Nasofaríngeas/imunologia , Anticorpos Neutralizantes/sangue , Anticorpos Antivirais/sangue , Biomarcadores/sangue , China , Células Epiteliais/imunologia , Células Epiteliais/virologia , Infecções por Vírus Epstein-Barr/sangue , Infecções por Vírus Epstein-Barr/virologia , Feminino , Glicoproteínas/imunologia , Humanos , Imunoglobulina A/sangue , Imunoglobulina A/imunologia , Imunoglobulina G/sangue , Imunoglobulina G/imunologia , Masculino , Pessoa de Meia-Idade , Neoplasias Nasofaríngeas/sangue , Neoplasias Nasofaríngeas/virologia
11.
FEBS J ; 287(23): 5130-5147, 2020 12.
Artigo em Inglês | MEDLINE | ID: mdl-32562308

RESUMO

In contrast to the homeokinesis of mitosis, asymmetric division of cytoplasm is the conspicuous feature of meiosis in mammalian oocytes. Protein regulator of cytokinesis 1 (PRC1) is an important regulator during mitotic spindle assembly and cytoplasmic division, but its functions in oocyte meiosis and early embryo development have not been fully elucidated. In this study, we detected PRC1 expression and localization and revealed a nuclear, spindle midzone-related dynamic pattern throughout meiotic and mitotic progressions. Treatment of oocytes with the reagents taxol or nocodazole disturbed the distribution of PRC1 in metaphase II oocytes. Further, PRC1 depletion led to failure of first polar body (PB1) extrusion and spindle migration, aneuploidy and defective kinetochore-microtubule attachment and spindle assembly. Overexpression of PRC1 resulted in PB1 extrusion failure, aneuploidy and serious defects of spindle assembly. To investigate PRC1 function in early embryos, we injected Prc1 morpholino into zygotes and 2-cell stage embryos. Depletion of PRC1 in zygotes impaired 4-cell, morula and blastocyst formation. Loss of PRC1 in single or double blastomeres in 2-cell stage embryos significantly impaired cell division, indicating its indispensable role in early embryo development. Co-immunoprecipitation showed that PRC1 interacts with polo-like kinase 1 (PLK1), and functional knockdown and rescue experiments demonstrated that PRC1 recruits PLK1 to the spindle midzone to regulate cytoplasmic division during meiosis. Finally, kinesin family member 4 knockdown downregulates PRC1 expression and leads to PRC1 localization failure. Taken together, our data suggest PRC1 plays an important role during oocyte maturation and early embryonic development by regulating chromosome dynamics and cytoplasmic division.


Assuntos
Proteínas de Ciclo Celular/metabolismo , Cromossomos , Citoplasma/fisiologia , Desenvolvimento Embrionário , Meiose , Oócitos/fisiologia , Fuso Acromático/fisiologia , Animais , Proteínas de Ciclo Celular/genética , Feminino , Cinesinas/genética , Cinesinas/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos ICR , Oócitos/citologia , Oogênese , Gravidez , Espermatozoides/citologia , Espermatozoides/fisiologia , Zigoto/citologia , Zigoto/fisiologia
12.
Math Biosci Eng ; 16(5): 5584-5594, 2019 06 17.
Artigo em Inglês | MEDLINE | ID: mdl-31499726

RESUMO

The authenticity of the image is crucial to many cases. The efficient detection of the JPEG compression history of bitmap image could reveal the possibility of tampering on the image. In this paper, we propose a lightweight but reliable JPEG compression detection method based on image information loss. An efficient feature of the decreasing percentage of zero coefficient is proposed to detect the JPEG compression history of an image, due to the increasing JPEG compression quality factor. In our method, estimated original images are first created. Then the given image and its estimated counterpart are compressed to get the JPEG coefficient. After that, the image information loss will be calculated. Through the analysis, the goal of the compression history detection can be achieved. Extensive experimental results have demonstrated that the proposed method outperforms the existing methods.

13.
Theriogenology ; 113: 92-101, 2018 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-29477014

RESUMO

Paracrine factors such as glial cell line-derived neurotrophic factor (GDNF), which was originally derived from the supernatants of a rat glioma cell line, play pivotal roles in oocyte maturation and early embryo development in mammals, such as mice, rats, pigs, sheep, and even humans. However, whether GDNF facilitates in vitro oocyte maturation or early embryo development in bovines is not yet known. We show for the first time that GDNF and its receptor, GDNF family receptor alpha-1 (GFRA1), are presented in ovarian follicles at different stages as well as during oocyte maturation and early embryo development. Immunostaining results revealed the subcellular localizations of GDNF and GFRA1 in oocytes throughout follicle development, first in germinal vesicles and during blastocyst embryo stages. The ability of exogenously applied GDNF to promote oocyte maturation and early embryo development was evaluated in culture, where we found that an optimal concentration of 50 ng/mL promotes the maturation of cumulus-oocyte complexes and the nuclei of denuded oocytes as well as the development of embryos after IVF. To further investigate the potential mechanism by which GDNF promotes oocyte maturation, bovine oocytes were treated with morpholinos targeting Gfra1. The suppression of GFRA1 presence blocked endogenous and exogenous GDNF functions, indicating that the effects of GDNF that are essential and beneficial for bovine oocyte maturation and early embryo development occur through this receptor. Furthermore, we show that supplementation with GDNF improves the efficiency of bovine IVF embryo production.


Assuntos
Bovinos/embriologia , Técnicas de Cultura Embrionária , Fator Neurotrófico Derivado de Linhagem de Célula Glial/farmacologia , Técnicas de Maturação in Vitro de Oócitos/veterinária , Oócitos/fisiologia , Animais , Linhagem Celular , Feminino , Regulação da Expressão Gênica/efeitos dos fármacos , Receptores de Fator Neurotrófico Derivado de Linhagem de Célula Glial/genética , Receptores de Fator Neurotrófico Derivado de Linhagem de Célula Glial/metabolismo , Folículo Ovariano/metabolismo
14.
Oncotarget ; 8(11): 17491-17503, 2017 Mar 14.
Artigo em Inglês | MEDLINE | ID: mdl-28177917

RESUMO

Mammalian oocyte meiotic maturation is the precondition of early embryo development. Lots of microtubules (MT)-associated proteins participate in oocyte maturation process. Cytoskeleton-associated protein 5 (CKAP5) is a member of the XMAP215 family that regulates microtubule dynamics during mitosis. However, its role in meiosis has not been fully studied. Here, we investigated the function of CKAP5 in mouse oocyte meiotic maturation and early embryo development. Western blot showed that CKAP5 expression increased from GVBD, maintaining at high level at metaphase, and decreased after late 1-cell stage. Confocal microscopy showed there is no specific accumulation of CKAP5 at interphase (GV, PN or 2-cell stage). However, once cells enter into meiotic or mitotic division, CKAP5 was localized at the whole spindle apparatus. Treatment of oocytes with the tubulin-disturbing reagents nocodazole (induces MTs depolymerization) or taxol (prevents MTs depolymerization) did not affect CKAP5 expression but led to a rearrangement of CKAP5. Further, knock-down of CKAP5 resulted in a failure of first polar body extrusion, serious defects in spindle assembly, and failure of chromosome alignment. Loss of CKAP5 also decreased early embryo development potential. Furthermore, co-immunoprecipitation showed that CKAP5 bound to clathrin heavy chain 1 (CLTC). Taken together, our results demonstrate that CKAP5 is important in oocyte maturation and early embryo development, and CKAP5 might work together with CLTC in mouse oocyte maturation.


Assuntos
Cadeias Pesadas de Clatrina/metabolismo , Desenvolvimento Embrionário/fisiologia , Proteínas Associadas aos Microtúbulos/metabolismo , Oócitos/metabolismo , Fuso Acromático/metabolismo , Animais , Western Blotting , Feminino , Imunofluorescência , Imunoprecipitação , Meiose/fisiologia , Camundongos , Microscopia Confocal
15.
Sci Rep ; 6: 19465, 2016 Jan 19.
Artigo em Inglês | MEDLINE | ID: mdl-26781721

RESUMO

The method of vitrification has been widely used for cryopreservation. However, the effectiveness of this method for mammalian oocytes could be improved by optimizing each step of the process. In the present study, we tested the effects of varying several key parameters to determine the most effective protocol for mouse oocyte vitrification. We found that cryoprotectant containing ethylene glycol and dimethylsulfoxide plus 20% fetal calf serum produced the highest rates of oocyte survival, fertilization, and blastocyst formation. The duration and temperature of oocyte exposure to vitrification and thawing solutions influenced survival rate. The presence of cumulus cells surrounding oocytes and the incubation of thawed oocytes in Toyoda-Yokoyama-Hosoki medium also increased oocyte survival. Open pulled straw and nylon loop methods were more effective than the mini-drop method. Finally, the combination of these improved methods resulted in better spindle morphology when compared to the unimproved methods. These results demonstrate that the outcomes of mouse oocyte vitrification can be improved by a suitable combination of cryopreservation methods, which could be applied to future clinical research with human oocytes.


Assuntos
Sobrevivência Celular/efeitos dos fármacos , Criopreservação/métodos , Crioprotetores/farmacologia , Oócitos/citologia , Animais , Blastocisto/efeitos dos fármacos , Blastocisto/fisiologia , Sobrevivência Celular/fisiologia , Dimetil Sulfóxido/farmacologia , Desenvolvimento Embrionário/efeitos dos fármacos , Desenvolvimento Embrionário/fisiologia , Etilenoglicol/farmacologia , Feminino , Fertilização/efeitos dos fármacos , Fertilização/fisiologia , Fertilização in vitro/efeitos dos fármacos , Fertilização in vitro/métodos , Masculino , Camundongos , Vitrificação
16.
PeerJ ; 4: e1761, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-26966678

RESUMO

Cumulus cells are a group of closely associated granulosa cells that surround and nourish oocytes. Previous studies have shown that cumulus cells contribute to oocyte maturation and fertilization through gap junction communication. However, it is not known how this gap junction signaling affects in vivo versus in vitro maturation of oocytes, and their subsequent fertilization and embryonic development following insemination. Therefore, in our study, we performed mouse oocyte maturation and insemination using in vivo- or in vitro-matured oocyte-cumulus complexes (OCCs, which retain gap junctions between the cumulus cells and the oocytes), in vitro-matured, denuded oocytes co-cultured with cumulus cells (DCs, which lack gap junctions between the cumulus cells and the oocytes), and in vitro-matured, denuded oocytes without cumulus cells (DOs). Using these models, we were able to analyze the effects of gap junction signaling on oocyte maturation, fertilization, and early embryo development. We found that gap junctions were necessary for both in vivo and in vitro oocyte maturation. In addition, for oocytes matured in vivo, the presence of cumulus cells during insemination improved fertilization and blastocyst formation, and this improvement was strengthened by gap junctions. Moreover, for oocytes matured in vitro, the presence of cumulus cells during insemination improved fertilization, but not blastocyst formation, and this improvement was independent of gap junctions. Our results demonstrate, for the first time, that the beneficial effect of gap junction signaling from cumulus cells depends on oocyte maturation and fertilization methods.

17.
IEEE Trans Neural Netw Learn Syst ; 24(1): 35-46, 2013 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-24808205

RESUMO

This paper proposes a novel nonnegative sparse representation approach, called two-stage sparse representation (TSR), for robust face recognition on a large-scale database. Based on the divide and conquer strategy, TSR decomposes the procedure of robust face recognition into outlier detection stage and recognition stage. In the first stage, we propose a general multisubspace framework to learn a robust metric in which noise and outliers in image pixels are detected. Potential loss functions, including L1 , L2,1, and correntropy are studied. In the second stage, based on the learned metric and collaborative representation, we propose an efficient nonnegative sparse representation algorithm to find an approximation solution of sparse representation. According to the L1 ball theory in sparse representation, the approximated solution is unique and can be optimized efficiently. Then a filtering strategy is developed to avoid the computation of the sparse representation on the whole large-scale dataset. Moreover, theoretical analysis also gives the necessary condition for nonnegative least squares technique to find a sparse solution. Extensive experiments on several public databases have demonstrated that the proposed TSR approach, in general, achieves better classification accuracy than the state-of-the-art sparse representation methods. More importantly, a significant reduction of computational costs is reached in comparison with sparse representation classifier; this enables the TSR to be more suitable for robust face recognition on a large-scale dataset.


Assuntos
Algoritmos , Inteligência Artificial/normas , Face/anatomia & histologia , Aumento da Imagem/métodos , Interpretação de Imagem Assistida por Computador/métodos , Reconhecimento Automatizado de Padrão/normas
18.
IEEE Trans Image Process ; 20(6): 1485-94, 2011 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-21216713

RESUMO

Principal component analysis (PCA) minimizes the mean square error (MSE) and is sensitive to outliers. In this paper, we present a new rotational-invariant PCA based on maximum correntropy criterion (MCC). A half-quadratic optimization algorithm is adopted to compute the correntropy objective. At each iteration, the complex optimization problem is reduced to a quadratic problem that can be efficiently solved by a standard optimization method. The proposed method exhibits the following benefits: 1) it is robust to outliers through the mechanism of MCC which can be more theoretically solid than a heuristic rule based on MSE; 2) it requires no assumption about the zero-mean of data for processing and can estimate data mean during optimization; and 3) its optimal solution consists of principal eigenvectors of a robust covariance matrix corresponding to the largest eigenvalues. In addition, kernel techniques are further introduced in the proposed method to deal with nonlinearly distributed data. Numerical results demonstrate that the proposed method can outperform robust rotational-invariant PCAs based on L(1) norm when outliers occur.


Assuntos
Algoritmos , Interpretação Estatística de Dados , Aumento da Imagem/métodos , Interpretação de Imagem Assistida por Computador/métodos , Reconhecimento Automatizado de Padrão/métodos , Análise de Componente Principal , Entropia , Modelos Estatísticos , Reprodutibilidade dos Testes , Sensibilidade e Especificidade
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