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1.
Anim Sci J ; 92(1): e13608, 2021 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-34405491

RESUMO

This study aims to investigate the morphology and distribution of mitochondria, spindles, and chromosomes in oocytes of aged mice and examine the effects of SRT1720 on oocyte maturation. C57BL/6J mice were divided into young (4-8 weeks) and aged groups (48-52 weeks). In vitro maturation media contained (0.05, 0.1, and 1.0 µM) SRT1720 and 0.1-µM dimethyl sulfoxide (DMSO control). The rate of chromosome misalignment and spindle misorientation in oocytes of aged mice were significantly higher than that of young mice (P < 0.01). Fluorescence intensity of mitochondria from oocytes of aged mice was significantly lower than that of young mice (P < 0.01). SRT1720 at 0.1 µM significantly improved oocyte maturation, fertilization, and blastocyst formation in aged mice compared with young mice (P < 0.01). Additionally, immunofluorescence intensity of mitochondria, normal spindle morphology, and chromosome alignment were notably enhanced with SRT1720 when compared with the DSMO control group for metaphase II (MII)-stage oocytes matured in vitro (P < 0.01); 0.1-µM SRT1720 enhanced the expression level of SRIT1 in oocytes from aged mice. In summary, the aged mice oocytes showed increased nuclear and cytoplasmic defects, whereas SRT1720 enhanced oocyte maturation and quality. We concluded that 0.1-µM SRT1720 was an appropriate concentration for in vitro maturation media.


Assuntos
Envelhecimento/efeitos dos fármacos , Envelhecimento/patologia , Compostos Heterocíclicos de 4 ou mais Anéis/farmacologia , Técnicas de Maturação in Vitro de Oócitos , Oócitos/efeitos dos fármacos , Oócitos/crescimento & desenvolvimento , Animais , Blastocisto , Cromossomos/metabolismo , Relação Dose-Resposta a Droga , Feminino , Fertilização/efeitos dos fármacos , Camundongos Endogâmicos C57BL , Mitocôndrias/metabolismo , Mitocôndrias/patologia , Oócitos/citologia , Oócitos/ultraestrutura , Fuso Acromático/metabolismo , Fuso Acromático/patologia
2.
Cell Prolif ; 54(10): e13119, 2021 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-34435400

RESUMO

OBJECTIVES: Histone deacetylase 8 (HDAC8) is one of the class I HDAC family proteins, which participates in the neuronal disorders, parasitic/viral infections, tumorigenesis and many other biological processes. However, its potential function during female germ cell development has not yet been fully understood. MATERIALS AND METHODS: HDAC8-targeting siRNA was microinjected into GV oocytes to deplete HDAC8. PCI-34051 was used to inhibit the enzyme activity of HDAC8. Immunostaining, immunoblotting and fluorescence intensity quantification were applied to assess the effects of HDAC8 depletion or inhibition on the oocyte meiotic maturation, spindle/chromosome structure, γ-tubulin dynamics and acetylation level of α-tubulin. RESULTS: We observed that HDAC8 was localized in the nucleus at GV stage and then translocated to the spindle apparatus from GVBD to M II stages in porcine oocytes. Depletion of HDAC8 led to the oocyte meiotic failure by showing the reduced polar body extrusion rate. In addition, depletion of HDAC8 resulted in aberrant spindle morphologies and misaligned chromosomes due to the defective recruitment of γ-tubulin to the spindle poles. Notably, these meiotic defects were photocopied by inhibition of HDAC8 activity using its specific inhibitor PCI-34051. However, inhibition of HDAC8 did not affect microtubule stability as assessed by the acetylation level of α-tubulin. CONCLUSIONS: Collectively, our findings demonstrate that HDAC8 acts as a regulator of spindle assembly during porcine oocyte meiotic maturation.


Assuntos
Histona Desacetilases/metabolismo , Meiose/fisiologia , Oócitos/metabolismo , Fuso Acromático/metabolismo , Acetilação/efeitos dos fármacos , Animais , Fenômenos Biológicos/efeitos dos fármacos , Cromossomos/efeitos dos fármacos , Cromossomos/metabolismo , Cromossomos/fisiologia , Feminino , Ácidos Hidroxâmicos/farmacologia , Indóis/farmacologia , Meiose/efeitos dos fármacos , Microtúbulos/efeitos dos fármacos , Microtúbulos/metabolismo , Microtúbulos/fisiologia , Oócitos/efeitos dos fármacos , Oócitos/fisiologia , Fuso Acromático/efeitos dos fármacos , Fuso Acromático/fisiologia , Suínos , Tubulina (Proteína)/metabolismo
3.
Nat Commun ; 12(1): 4369, 2021 07 16.
Artigo em Inglês | MEDLINE | ID: mdl-34272403

RESUMO

There is a strong demand for methods that can efficiently reconstruct valid super-resolution intact genome 3D structures from sparse and noise single-cell Hi-C data. Here, we develop Single-Cell Chromosome Conformation Calculator (Si-C) within the Bayesian theory framework and apply this approach to reconstruct intact genome 3D structures from single-cell Hi-C data of eight G1-phase haploid mouse ES cells. The inferred 100-kb and 10-kb structures consistently reproduce the known conserved features of chromatin organization revealed by independent imaging experiments. The analysis of the 10-kb resolution 3D structures reveals cell-to-cell varying domain structures in individual cells and hyperfine structures in domains, such as loops. An average of 0.2 contact reads per divided bin is sufficient for Si-C to obtain reliable structures. The valid super-resolution structures constructed by Si-C demonstrate the potential for visualizing and investigating interactions between all chromatin loci at the genome scale in individual cells.


Assuntos
Cromatina/metabolismo , Cromossomos/metabolismo , Células-Tronco Embrionárias/metabolismo , Genoma , Análise de Célula Única/métodos , Animais , Teorema de Bayes , Cromatina/química , Cromatina/genética , Cromossomos/química , Cromossomos/genética , Fase G1 , Haploidia , Hibridização in Situ Fluorescente , Camundongos , Conformação Molecular
4.
Exp Cell Res ; 405(2): 112657, 2021 08 15.
Artigo em Inglês | MEDLINE | ID: mdl-34081985

RESUMO

Checkpoint kinases (Chk) 1/2 are known for DNA damage checkpoint and cell cycle control in somatic cells. According to recent findings, the involvement of Chk1 in oocyte meiotic resumption and Chk2 is regarded as an essential regulator for progression at the post metaphase I stage (MI). In this study, AZD7762 (Chk1/2 inhibitor) and SB218078 (Chk1 inhibitor) were used to uncover the joint roles of Chk1/2 and differentiate the importance of Chk1 and Chk2 during oocyte meiotic maturation. Inhibition of Chk1/2 or Chk1 alone had no significant effect on germinal vesicle breakdown (GVBD) but significantly inhibited the first polar body (PB1). Interestingly, inhibition of Chk1 alone could not increase or completely block the extrusion of PB1 like Chk1/2 inhibition. Also, Chk1/2 inhibition resulted in defective meiotic spindle organization and chromosome condensation both in MI and metaphase II (MII) stages of oocytes. The location of γ-tubulin and Securin were abnormal or missing, while P38 MAPK was activated by Chk1/2 inhibition. Meanwhile, Chk1/2 inhibition reduced the percentage of the second polar body extrusion and pronuclear formation. In conclusion, our results further understand the functions and regulatory mechanism of Chk1/2 during oocyte meiotic maturation.


Assuntos
Cromossomos/metabolismo , Meiose/fisiologia , Metáfase/fisiologia , Oócitos/metabolismo , Animais , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Feminino , Camundongos , Securina/metabolismo , Tubulina (Proteína)/metabolismo
5.
Nat Commun ; 12(1): 3708, 2021 06 17.
Artigo em Inglês | MEDLINE | ID: mdl-34140506

RESUMO

3D genome alternations can dysregulate gene expression by rewiring enhancer-promoter interactions and lead to diseases. We report integrated analyses of 3D genome alterations and differential gene expressions in 18 newly diagnosed T-lineage acute lymphoblastic leukemia (T-ALL) patients and 4 healthy controls. 3D genome organizations at the levels of compartment, topologically associated domains and loop could hierarchically classify different subtypes of T-ALL according to T cell differentiation trajectory, similar to gene expressions-based classification. Thirty-four previously unrecognized translocations and 44 translocation-mediated neo-loops are mapped by Hi-C analysis. We find that neo-loops formed in the non-coding region of the genome could potentially regulate ectopic expressions of TLX3, TAL2 and HOXA transcription factors via enhancer hijacking. Importantly, both translocation-mediated neo-loops and NUP98-related fusions are associated with HOXA13 ectopic expressions. Patients with HOXA11-A13 expressions, but not other genes in the HOXA cluster, have immature immunophenotype and poor outcomes. Here, we highlight the potentially important roles of 3D genome alterations in the etiology and prognosis of T-ALL.


Assuntos
Cromossomos/metabolismo , Proteínas de Homeodomínio/genética , Leucemia-Linfoma de Células T do Adulto/genética , Conformação Molecular , Leucemia-Linfoma Linfoblástico de Células T Precursoras/genética , Linfócitos T/metabolismo , Translocação Genética , Acetilação , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Linhagem Celular Tumoral , Linhagem da Célula , Criança , Sequenciamento de Cromatina por Imunoprecipitação , Cromossomos/genética , Progressão da Doença , Elementos Facilitadores Genéticos , Perfilação da Expressão Gênica , Regulação Leucêmica da Expressão Gênica/genética , Regulação Leucêmica da Expressão Gênica/imunologia , Ontologia Genética , Hematopoese/genética , Histonas/metabolismo , Proteínas de Homeodomínio/metabolismo , Humanos , Imunofenotipagem , Leucemia-Linfoma de Células T do Adulto/metabolismo , Leucemia-Linfoma de Células T do Adulto/mortalidade , Leucemia-Linfoma de Células T do Adulto/patologia , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Complexo de Proteínas Formadoras de Poros Nucleares/genética , Complexo de Proteínas Formadoras de Poros Nucleares/metabolismo , Leucemia-Linfoma Linfoblástico de Células T Precursoras/metabolismo , Leucemia-Linfoma Linfoblástico de Células T Precursoras/mortalidade , Leucemia-Linfoma Linfoblástico de Células T Precursoras/patologia , Prognóstico , Linfócitos T/patologia , Adulto Jovem
6.
Nat Commun ; 12(1): 3551, 2021 06 10.
Artigo em Inglês | MEDLINE | ID: mdl-34112792

RESUMO

Species harbor extensive structural variation underpinning recent adaptive evolution. However, the causality between genomic features and the induction of new rearrangements is poorly established. Here, we analyze a global set of telomere-to-telomere genome assemblies of a fungal pathogen of wheat to establish a nucleotide-level map of structural variation. We show that the recent emergence of pesticide resistance has been disproportionally driven by rearrangements. We use machine learning to train a model on structural variation events based on 30 chromosomal sequence features. We show that base composition and gene density are the major determinants of structural variation. Retrotransposons explain most inversion, indel and duplication events. We apply our model to Arabidopsis thaliana and show that our approach extends to more complex genomes. Finally, we analyze complete genomes of haploid offspring in a four-generation pedigree. Meiotic crossover locations are enriched for new rearrangements consistent with crossovers being mutational hotspots. The model trained on species-wide structural variation accurately predicts the position of >74% of newly generated variants along the pedigree. The predictive power highlights causality between specific sequence features and the induction of chromosomal rearrangements. Our work demonstrates that training sequence-derived models can accurately identify regions of intrinsic DNA instability in eukaryotic genomes.


Assuntos
Ascomicetos/genética , Ascomicetos/patogenicidade , Cromossomos/genética , Variação Genética , Genoma , Genômica/métodos , Aprendizado de Máquina , Meiose/genética , Arabidopsis/genética , Cromossomos/metabolismo , Simulação por Computador , Troca Genética , Eucariotos/genética , Evolução Molecular , Genes Duplicados , Estudo de Associação Genômica Ampla , Mutação INDEL , Modelos Genéticos , Linhagem , Filogenia , Polimorfismo Genético , Polimorfismo de Nucleotídeo Único , Retroelementos/genética , Inversão de Sequência
7.
Nat Commun ; 12(1): 3531, 2021 06 10.
Artigo em Inglês | MEDLINE | ID: mdl-34112794

RESUMO

Camptothecin and its derivatives are widely used for treating malignant tumors. Previous studies revealed only a limited number of candidate genes for camptothecin biosynthesis in Camptotheca acuminata, and it is still poorly understood how its biosynthesis of camptothecin has evolved. Here, we report a high-quality, chromosome-level C. acuminata genome assembly. We find that C. acuminata experiences an independent whole-genome duplication and numerous genes derive from it are related to camptothecin biosynthesis. Comparing with Catharanthus roseus, the loganic acid O-methyltransferase (LAMT) in C. acuminata fails to convert loganic acid into loganin. Instead, two secologanic acid synthases (SLASs) convert loganic acid to secologanic acid. The functional divergence of the LAMT gene and positive evolution of two SLAS genes, therefore, both contribute greatly to the camptothecin biosynthesis in C. acuminata. Our results emphasize the importance of high-quality genome assembly in identifying genetic changes in the evolutionary origin of a secondary metabolite.


Assuntos
Camptotheca/metabolismo , Camptotecina/metabolismo , Cromossomos/metabolismo , Genoma de Planta , Metabolismo Secundário/genética , Camptotheca/enzimologia , Camptotheca/genética , Camptotecina/biossíntese , Cromossomos/genética , Sistema Enzimático do Citocromo P-450 , Evolução Molecular , Regulação da Expressão Gênica de Plantas/genética , Genes Duplicados , Genômica , Iridoides/metabolismo , Oxirredutases atuantes sobre Doadores de Grupo CH-CH , Filogenia , Proteína O-Metiltransferase/genética , Proteína O-Metiltransferase/metabolismo , RNA-Seq , Vimblastina/metabolismo
9.
Nat Rev Mol Cell Biol ; 22(8): 511-528, 2021 08.
Artigo em Inglês | MEDLINE | ID: mdl-33953379

RESUMO

Understanding how chromatin is folded in the nucleus is fundamental to understanding its function. Although 3D genome organization has been historically difficult to study owing to a lack of relevant methodologies, major technological breakthroughs in genome-wide mapping of chromatin contacts and advances in imaging technologies in the twenty-first century considerably improved our understanding of chromosome conformation and nuclear architecture. In this Review, we discuss methods of 3D genome organization analysis, including sequencing-based techniques, such as Hi-C and its derivatives, Micro-C, DamID and others; microscopy-based techniques, such as super-resolution imaging coupled with fluorescence in situ hybridization (FISH), multiplex FISH, in situ genome sequencing and live microscopy methods; and computational and modelling approaches. We describe the most commonly used techniques and their contribution to our current knowledge of nuclear architecture and, finally, we provide a perspective on up-and-coming methods that open possibilities for future major discoveries.


Assuntos
Cromatina/química , Genoma , Cromatina/genética , Cromatina/metabolismo , Mapeamento Cromossômico , Cromossomos/química , Cromossomos/genética , Cromossomos/metabolismo , Biologia Computacional , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Microscopia , Modelos Moleculares , Análise de Sequência de DNA
10.
Mutat Res ; 822: 111743, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33975127

RESUMO

DNA double-strand breaks (DSBs) are one of the most dangerous assaults on the genome, and yet their natural and programmed production are inherent to life. When DSBs arise close together they are particularly deleterious, and their repair may require an altered form of the DNA damage response. Our understanding of how clustered DSBs are repaired in the germline is unknown. Using laser microirradiation, we examine early events in the repair of clustered DSBs in germ cells within Caenorhabditis elegans. We use precise temporal resolution to show how the recruitment of MRE-11 to complex damage is regulated, and that clustered DNA damage can recruit proteins from various repair pathways. Abrogation of non-homologous end joining or COM-1 attenuates the recruitment of MRE-11 through distinct mechanisms. The synaptonemal complex plays both positive and negative regulatory roles in these mutant contexts. These findings indicate that MRE-11 is regulated by modifying its accessibility to chromosomes.


Assuntos
Proteínas de Caenorhabditis elegans , Caenorhabditis elegans , Cromossomos , Quebras de DNA de Cadeia Dupla , Reparo do DNA por Junção de Extremidades , DNA de Helmintos , Meiose , Animais , Caenorhabditis elegans/genética , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Cromossomos/genética , Cromossomos/metabolismo , DNA de Helmintos/genética , DNA de Helmintos/metabolismo
11.
PLoS Genet ; 17(5): e1009567, 2021 05.
Artigo em Inglês | MEDLINE | ID: mdl-34014923

RESUMO

The widely conserved kinase Aurora B regulates important events during cell division. Surprisingly, recent work has uncovered a few functions of Aurora-family kinases that do not require kinase activity. Thus, understanding this important class of cell cycle regulators will require strategies to distinguish kinase-dependent from independent functions. Here, we address this need in C. elegans by combining germline-specific, auxin-induced Aurora B (AIR-2) degradation with the transgenic expression of kinase-inactive AIR-2. Through this approach, we find that kinase activity is essential for AIR-2's major meiotic functions and also for mitotic chromosome segregation. Moreover, our analysis revealed insight into the assembly of the ring complex (RC), a structure that is essential for chromosome congression in C. elegans oocytes. AIR-2 localizes to chromosomes and recruits other components to form the RC. However, we found that while kinase-dead AIR-2 could load onto chromosomes, other components were not recruited. This failure in RC assembly appeared to be due to a loss of RC SUMOylation, suggesting that there is crosstalk between SUMOylation and phosphorylation in building the RC and implicating AIR-2 in regulating the SUMO pathway in oocytes. Similar conditional depletion approaches may reveal new insights into other cell cycle regulators.


Assuntos
Aurora Quinase B/metabolismo , Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/citologia , Caenorhabditis elegans/enzimologia , Segregação de Cromossomos , Oócitos/enzimologia , Animais , Caenorhabditis elegans/genética , Cromossomos/metabolismo , Meiose/genética , Mitose/genética , Oócitos/citologia , Fosforilação , Reprodutibilidade dos Testes , Fuso Acromático/enzimologia , Sumoilação
12.
Biophys J ; 120(12): 2521-2531, 2021 06 15.
Artigo em Inglês | MEDLINE | ID: mdl-34023297

RESUMO

DNA looping plays an important role in cells in both regulating and protecting the genome. Often, studies of looping focus on looping by prokaryotic transcription factors like lac repressor or by structural maintenance of chromosomes proteins such as condensin. Here, however, we are interested in a different looping method whereby condensing agents (charge ≥+3) such as protamine proteins neutralize the DNA, causing it to form loops and toroids. We considered two previously proposed mechanisms for DNA looping by protamine. In the first mechanism, protamine stabilizes spontaneous DNA fluctuations, forming randomly distributed loops along the DNA. In the second mechanism, protamine binds and bends the DNA to form a loop, creating a distribution of loops that is biased by protamine binding. To differentiate between these mechanisms, we imaged both spontaneous and protamine-induced loops on short-length (≤1 µm) DNA fragments using atomic force microscopy. We then compared the spatial distribution of the loops to several model distributions. A random looping model, which describes the mechanism of spontaneous DNA folding, fit the distribution of spontaneous loops, but it did not fit the distribution of protamine-induced loops. Specifically, it failed to predict a peak in the spatial distribution of loops at an intermediate location along the DNA. An electrostatic multibinding model, which was created to mimic the bind-and-bend mechanism of protamine, was a better fit of the distribution of protamine-induced loops. In this model, multiple protamines bind to the DNA electrostatically within a particular region along the DNA to coordinate the formation of a loop. We speculate that these findings will impact our understanding of protamine's in vivo role for looping DNA into toroids and the mechanism of DNA condensation by condensing agents more broadly.


Assuntos
DNA , Protaminas , Cromossomos/metabolismo , DNA/genética , Repressores Lac/metabolismo , Conformação de Ácido Nucleico
13.
Nat Commun ; 12(1): 2981, 2021 05 20.
Artigo em Inglês | MEDLINE | ID: mdl-34016985

RESUMO

The spatial folding of chromosomes inside the nucleus has regulatory effects on gene expression, yet the impact of genome reshuffling on this organization remains unclear. Here, we take advantage of chromosome conformation capture in combination with single-nucleotide polymorphism (SNP) genotyping and analysis of crossover events to study how the higher-order chromatin organization and recombination landscapes are affected by chromosomal fusions in the mammalian germ line. We demonstrate that chromosomal fusions alter the nuclear architecture during meiosis, including an increased rate of heterologous interactions in primary spermatocytes, and alterations in both chromosome synapsis and axis length. These disturbances in topology were associated with changes in genomic landscapes of recombination, resulting in detectable genomic footprints. Overall, we show that chromosomal fusions impact the dynamic genome topology of germ cells in two ways: (i) altering chromosomal nuclear occupancy and synapsis, and (ii) reshaping landscapes of recombination.


Assuntos
Cromatina/metabolismo , Cromossomos/metabolismo , Recombinação Genética , Espermatócitos/metabolismo , Animais , Evolução Biológica , Núcleo Celular/genética , Núcleo Celular/metabolismo , Células Cultivadas , Cromatina/genética , Pareamento Cromossômico/genética , Segregação de Cromossomos , Cromossomos/genética , Europa (Continente) , Fertilidade/genética , Técnicas de Genotipagem/métodos , Masculino , Camundongos , Polimorfismo de Nucleotídeo Único , Cultura Primária de Células , Análise do Sêmen , Espermatócitos/citologia
14.
Nucleic Acids Res ; 49(10): 5654-5670, 2021 06 04.
Artigo em Inglês | MEDLINE | ID: mdl-34048565

RESUMO

Integrons confer a rapid adaptation capability to bacteria. Integron integrases are able to capture and shuffle novel functions embedded in cassettes. Here, we investigated cassette recruitment in the Vibrio cholerae chromosomal integron during horizontal transfer. We demonstrated that the endogenous integrase expression is sufficiently triggered, after SOS response induction mediated by the entry of cassettes during conjugation and natural transformation, to mediate significant cassette insertions. These insertions preferentially occur at the attIA site, despite the presence of about 180 attC sites in the integron array. Thanks to the presence of a promoter in the attIA site vicinity, all these newly inserted cassettes are expressed and prone to selection. We also showed that the RecA protein is critical for cassette recruitment in the V. cholerae chromosomal integron but not in mobile integrons. Moreover, unlike the mobile integron integrases, that of V. cholerae is not active in other bacteria. Mobile integrons might have evolved from the chromosomal ones by overcoming host factors, explaining their large dissemination in bacteria and their role in antibioresistance expansion.


Assuntos
Cromossomos/metabolismo , Transferência Genética Horizontal/genética , Integrases/metabolismo , Integrons/genética , Vibrio cholerae/metabolismo , Cromossomos/genética , Escherichia coli/metabolismo , Regulação Bacteriana da Expressão Gênica/genética , Integrases/genética , Recombinases Rec A/genética , Recombinases Rec A/metabolismo , Recombinação Genética/genética , Vibrio cholerae/genética
15.
J Cancer Res Clin Oncol ; 147(8): 2187-2198, 2021 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-34047821

RESUMO

BACKGROUND: Ensuring genetic integrity is essential during the cell cycle to avoid aneuploidy, one of the underlying causes of malignancies. Aurora kinases are serine/threonine kinase that play a vital role in maintaining the genomic integrity of the cells. There are three forms of aurora kinases in the mammalian cells, which are highly conserved and act together with several other proteins to control chromosome alignment and its equal distribution to daughter cells in mitosis and meiosis. METHODS: We provide here a detailed analysis of Aurora B kinase (ABK) in terms of its expression, structure, function, disease association and potential therapeutic implications. RESULTS: ABK plays an instrumental in mitotic entry, chromosome condensation, spindle assembly, cytokinesis, and abscission. Small-molecule inhibitors of ABK are designed and synthesized to control cancer progression. A detailed understanding of ABK pathophysiology in different cancers is of great significance in designing and developing effective therapeutic strategies. CONCLUSION: In this review, we have discussed the physiological significance of ABK followed by its role in cancer progression. We further highlighted available small-molecule inhibitors to control the tumor proliferation and their mechanistic insights.


Assuntos
Aurora Quinase B/fisiologia , Terapia de Alvo Molecular , Neoplasias/terapia , Animais , Antineoplásicos/uso terapêutico , Aurora Quinase B/antagonistas & inibidores , Aurora Quinase B/genética , Ciclo Celular/genética , Cromossomos/genética , Cromossomos/metabolismo , Progressão da Doença , Humanos , Mitose/genética , Terapia de Alvo Molecular/métodos , Terapia de Alvo Molecular/tendências , Neoplasias/etiologia , Neoplasias/patologia
16.
Mol Cell Biol ; 41(7): e0004421, 2021 06 23.
Artigo em Inglês | MEDLINE | ID: mdl-33941618

RESUMO

Prions are self-perpetuating, alternative protein conformations associated with neurological diseases and normal cellular functions. Saccharomyces cerevisiae contains many endogenous prions, providing a powerful system to study prionization. Previously, we demonstrated that Swi1, a component of the SWI/SNF chromatin-remodeling complex, can form the prion [SWI+]. A small region, Swi11-38, with a unique amino acid composition of low complexity, acts as a prion domain and supports [SWI+] propagation. Here, we further examine Swi11-38 through site-directed mutagenesis. We found that mutations of the two phenylalanine residues or the threonine tract inhibit Swi11-38 aggregation. In addition, mutating both phenylalanines can abolish de novo prion formation by Swi11-38, whereas mutating only one phenylalanine does not. Replacement of half of or the entire eight-threonine tract with alanines has the same effect, possibly disrupting a core region of Swi11-38 aggregates. We also show that Swi11-38 and its prion-fold-maintaining mutants form high-molecular-weight, SDS-resistant aggregates, whereas the double-phenylalanine mutants eliminate these protein species. These results indicate the necessity of the large hydrophobic residues and threonine tract in Swi11-38 in prionogenesis, possibly acting as important aggregable regions. Our findings thus highlight the importance of specific amino acid residues in the Swi1 prion domain in prion formation and maintenance.


Assuntos
Proteínas Cromossômicas não Histona/genética , Regulação Fúngica da Expressão Gênica/genética , Mutação/genética , Príons/genética , Proteínas de Saccharomyces cerevisiae/genética , Fatores de Transcrição/genética , Montagem e Desmontagem da Cromatina , Proteínas Cromossômicas não Histona/metabolismo , Cromossomos/metabolismo , Príons/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Fatores de Transcrição/metabolismo
17.
Food Chem Toxicol ; 152: 112199, 2021 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-33865936

RESUMO

Lactobacillus curvatus WiKim 38 (LCW), isolated from kimchi, has shown novel immunomodulatory and anti-inflammatory properties. In the present study, to obtain data on the safety of LCW, we performed three genotoxicity (bacterial reverse mutation, chromosome aberration, and micronucleus) and two general toxicity (single-dosing and 13-week repeated-dosing) studies. In the genotoxicity assessment, LCW showed no increased reverse mutation for 4 strains of Salmonella typhimurium and a strain of Escherichia coli. In addition, LCW did not induce chromosome aberrations at concentrations up to 5000 µg/mL in cultured Chinese hamster lung (CHL) cells and did not induce an increased frequency of micronuclei in the bone marrow cells of rats at concentrations up to 2000 mg/kg. In the acute toxicity study using Sprague-Dawley (SD) rats, the approximate lethal dose of LCW was determined to be over 5000 mg/kg body weight (b.w.) in both sexes. Finally, in the subchronic toxicity study, no LCW-related adverse effects were observed at concentrations up to 5000 mg/kg b.w./day. Consequently, LCW is considered not to have mutagenic effects, and its no-observed-adverse-effect-level (NOAEL) is 5000 mg/kg b.w., equivalent to approximately 4.71 × 109 CFU/kg b.w., suggesting the LCW could be a potential probiotic for humans based on its safety profile.


Assuntos
Lactobacillus/patogenicidade , Probióticos/toxicidade , Animais , Células da Medula Óssea/metabolismo , Cromossomos/metabolismo , Escherichia coli/genética , Feminino , Masculino , Testes para Micronúcleos , Nível de Efeito Adverso não Observado , Ratos Sprague-Dawley , Salmonella typhimurium/genética , Testes de Toxicidade Aguda , Testes de Toxicidade Subcrônica
18.
Nucleic Acids Res ; 49(8): 4493-4505, 2021 05 07.
Artigo em Inglês | MEDLINE | ID: mdl-33872375

RESUMO

An essential questions of gene regulation is how large number of enhancers and promoters organize into gene regulatory loops. Using transcription-factor binding enrichment as an indicator of enhancer strength, we identified a portion of H3K27ac peaks as potentially strong enhancers and found a universal pattern of promoter and enhancer distribution: At actively transcribed regions of length of ∼200-300 kb, the numbers of active promoters and enhancers are inversely related. Enhancer clusters are associated with isolated active promoters, regardless of the gene's cell-type specificity. As the number of nearby active promoters increases, the number of enhancers decreases. At regions where multiple active genes are closely located, there are few distant enhancers. With Hi-C analysis, we demonstrate that the interactions among the regulatory elements (active promoters and enhancers) occur predominantly in clusters and multiway among linearly close elements and the distance between adjacent elements shows a preference of ∼30 kb. We propose a simple rule of spatial organization of active promoters and enhancers: Gene transcriptions and regulations mainly occur at local active transcription hubs contributed dynamically by multiple elements from linearly close enhancers and/or active promoters. The hub model can be represented with a flower-shaped structure and implies an enhancer-like role of active promoters.


Assuntos
Cromossomos/metabolismo , Elementos Facilitadores Genéticos , Regulação da Expressão Gênica/genética , Histonas/metabolismo , Regiões Promotoras Genéticas , Acetilação , Sequenciamento de Cromatina por Imunoprecipitação , Cromossomos/genética , Bases de Dados Genéticas , Genoma Humano , Humanos , Modelos Genéticos , Família Multigênica , Vírus da Hepatite Murina , RNA-Seq , Ativação Transcricional/genética
19.
Front Cell Infect Microbiol ; 11: 638087, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33816340

RESUMO

Pseudomonas aeruginosa is noted for its intrinsic antibiotic resistance and capacity of acquiring additional resistance genes. In this study, the genomes of nine clinical P. aeruginosa isolates were fully sequenced. An extensive genetic comparison was applied to 18 P. aeruginosa accessory genetic elements (AGEs; 13 of them were sequenced in this study and located within P. aeruginosa chromosomes) that were divided into four groups: five related integrative and conjugative elements (ICEs), four related integrative and mobilizable elements (IMEs), five related unit transposons, and two related IMEs and their two derivatives. At least 45 resistance genes, involved in resistance to 10 different categories of antibiotics and heavy metals, were identified from these 18 AGEs. A total of 10 ß-lactamase genes were identified from 10 AGEs sequenced herein, and nine of them were captured within class 1 integrons, which were further integrated into ICEs and IMEs with intercellular mobility, and also unit transposons with intracellular mobility. Through this study, we identified for the first time 20 novel MGEs, including four ICEs Tn6584, Tn6585, Tn6586, and Tn6587; three IMEs Tn6853, Tn6854, and Tn6878; five unit transposons Tn6846, Tn6847, Tn6848, Tn6849, and Tn6883; and eight integrons In1795, In1778, In1820, In1784, In1775, In1774, In1789, and In1799. This was also the first report of two resistance gene variants bla CARB-53 and catB3s, and a novel ST3405 isolate of P. aeruginosa. The data presented here denoted that complex transposition and homologous recombination promoted the assembly and integration of AGEs with mosaic structures into P. aeruginosa chromosomes.


Assuntos
Antibacterianos , Pseudomonas aeruginosa , Cromossomos/metabolismo , Integrons , Pseudomonas aeruginosa/genética , beta-Lactamases/genética
20.
Front Cell Infect Microbiol ; 11: 660702, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33829001

RESUMO

Livestock-associated methicillin-resistant Staphylococcus aureus (LA-MRSA) of clonal complex CC398 typically carry various antimicrobial resistance genes, many of them located on plasmids. In the bovine LA-MRSA isolate Rd11, we previously identified plasmid pAFS11 in which resistance genes are co-localized with a novel ica-like gene cluster, harboring genes required for polysaccharide intercellular adhesin (PIA)-mediated biofilm formation. The ica genes on pAFS11 were acquired in addition to a pre-existing ica locus on the S. aureus Rd11 chromosomal DNA. Both loci consist of an icaADBC operon and icaR, encoding a corresponding icaADBC repressor. Despite carrying two biofilm gene copies, strain Rd11 did not produce PIA and transformation of pAFS11 into another S. aureus strain even slightly diminished PIA-mediated biofilm formation. By focusing on the molecular background of the biofilm-negative phenotype of pAFS11-carrying S. aureus, we identified the pAFS11-borne ica locus copy as functionally fully active. However, transcription of both plasmid- and core genome-derived icaADBC operons were efficiently suppressed involving IcaR. Surprisingly, although being different on the amino acid sequence level, the two IcaR repressor proteins are mutually replaceable and are able to interact with the icaA promoter region of the other copy. We speculate that this regulatory crosstalk causes the biofilm-negative phenotype in S. aureus Rd11. The data shed light on an unexpected regulatory interplay between pre-existing and newly acquired DNA traits in S. aureus. This also raises interesting general questions regarding functional consequences of gene transfer events and their putative implications for the adaptation and evolution of bacterial pathogens.


Assuntos
Staphylococcus aureus Resistente à Meticilina , Infecções Estafilocócicas , Animais , Proteínas de Bactérias/genética , Biofilmes , Bovinos , Cromossomos/metabolismo , Regulação Bacteriana da Expressão Gênica , Plasmídeos , Polissacarídeos Bacterianos , Staphylococcus aureus/genética , Staphylococcus epidermidis/genética
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