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1.
Methods Mol Biol ; 2519: 17-26, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-36066706

RESUMO

Cellular division is a fundamental process of cellular growth. First, cells replicate their DNA in S phase and then undergo mitosis which, under normal conditions, leads to complete cell division. Moreover, mitotic activity correlates to cellular growth activity. The simplest and classical method to measure mitotic activity (mitotic index (MI)), is the manual counting of mitotic cells among a given cell population of interest. The latter can be accomplished via phase contrast microscope observation. However, Giemsa staining may improve accuracy and consistency. Fluorescence immunostaining targeting specific phosphorylations of proteins at critical cell cycle steps will provide further improved analysis via high-throughput capacity of flow or imaging cytometer. Finally, time lapse image analysis provides quantitative and qualitative metrics delineating the process of cellular division including timing of division, duration of mitosis, and failure to procced through or complete mitosis.


Assuntos
Mitose , Ciclo Celular , Índice Mitótico , Fosforilação , Fase S
2.
Methods Mol Biol ; 2519: 73-82, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-36066711

RESUMO

Sister chromatid exchange (SCE) is the exchange event of genetic material between two identical sister chromatid. Elevation of SCE frequency is considered as a result of replication stress from genetic defects, ROS stress, and genomic damages. SCE staining needs extra processes compared to regular Giemsa staining. Usually two rounds of cell cycle progress are required to observe SCE under microscope. SCE can be visualized with the fluorescence plus Giemsa (FPG) staining method or fluorescence staining methods with immunocytochemistry to BrdU or Click reaction to EdU which provide more clear images of SCE. This chapter will provide the detailed method for the SCE staining and measurement for the traditional FPG staining, BrdU monoclonal antibody staining method, and newly developed EdU Click reaction staining method.


Assuntos
Cromátides , Troca de Cromátide Irmã , Bromodesoxiuridina/metabolismo , Ciclo Celular , Divisão Celular , Cromátides/genética , Cromátides/metabolismo
3.
Gene ; 850: 146961, 2023 Jan 20.
Artigo em Inglês | MEDLINE | ID: mdl-36220450

RESUMO

BACKGROUND: Gastric cancer is a common malignant tumor of digestive tract. The study aimed to identify candidate genes associated with the proliferation and survival of gastric cancer cell through CRISPR-cas9 screening data, which may provide new therapeutic targets for gastric cancer patients. METHODS: Candidate genes related to gastric cancer cell viability by CRISPR-cas9 screening from Depmap and genes differentially expressed between gastric cancer tissues and normal gastric tissues from TCGA were overlapped. WGCNA and KEGG analysis was conducted to performed to identify key pathways and genes. Using CMap, we identified small molecules that might reverse candidate gene expression of gastric cancer. LASSO regression was used to construct a signature to predict overall survival of gastric cancer patients. CCK8 assay was performed to assess the effects of candidate gene on gastric cancer cell proliferation. RESULTS: A total of 710 candidate genes related to gastric cancer cell viability in the DepMap were identified and overlapped with differentially expressed genes in TCGA database, which were enriched in the cell cycle pathway. CMap analysis suggested that molecule drug LY294002 might be a novel choice for gastric cancer treatment. Using Cox univariate analysis and Lasso analysis, we developed a prognostic model including 12 candidate genes, and conducted subgroup analysis and external validation. Moreover, knockdown of the key candidate gene CNIH4 inhibited the proliferation of gastric cancer cells. CONCLUSION: Cell cycle pathway and CNIH4, identified by CRISPR-cas9 screening, were a key pathway and gene that regulate cell viability in gastric cancer. CNIH4 has significant prognostic values and can serve as a new target for gastric cancer patient treatment.


Assuntos
Receptores Citoplasmáticos e Nucleares , Neoplasias Gástricas , Humanos , Ciclo Celular , Proliferação de Células/genética , Sistemas CRISPR-Cas , Detecção Precoce de Câncer/métodos , Receptores Citoplasmáticos e Nucleares/genética , Neoplasias Gástricas/diagnóstico , Neoplasias Gástricas/tratamento farmacológico , Neoplasias Gástricas/genética , Bases de Dados Genéticas
4.
Braz. j. biol ; 83: e248746, 2023. graf
Artigo em Inglês | LILACS, VETINDEX | ID: biblio-1339351

RESUMO

Abstract Colorectal cancer (CRC) is one of the most common cancers leading to comorbidities and mortalities globally. The rational of current study was to evaluate the combined epigallocatechin gallate and quercetin as a potent antitumor agent as commentary agent for therapeutic protocol. The present study investigated the effect of epigallocatechin Gallate (EGCG) (150mg) and quercetin (200mg) at different proportions on proliferation and induction of apoptosis in human colon cancer cells (HCT-116). Cell growth, colonogenic, Annexin V in addition cell cycle were detected in response to phytomolecules. Data obtained showed that, the colony formation was inhibited significantly in CRC starting from the lowest concentration tested of 10 µg/mL resulting in no colonies as visualized by a phase-contrast microscope. Data showed a significant elevation in the annexin V at 100 µg/mL EGCG(25.85%) and 150 µg/mL quercetin (48.35%). Moreover, cell cycle analysis showed that this combination caused cell cycle arrest at the G1 phase at concentration of 100 µg/mL (72.7%) and 150 µg/mL (75.25%). The combined effect of epigallocatechin Gallate and quercetin exert antiproliferative activity against CRC, it is promising in alternative conventional chemotherapeutic agent.


Resumo O câncer colorretal (CCR) é um dos cânceres mais comuns, levando a comorbidades e mortalidade em todo o mundo. O racional do presente estudo foi avaliar a combinação de galato de epigalocatequina e quercetina como um agente antitumoral potente como agente de comentário para protocolo terapêutico. O presente estudo investigou o efeito de galato de epigalocatequina (EGCG) (150 mg) e quercetina (200 mg) em diferentes proporções na proliferação e indução de apoptose em células de câncer de cólon humano (HCT-116). O crescimento celular, colonogênico, anexina V, além do ciclo celular foram detectados em resposta a fitomoléculas. Os dados obtidos mostraram que a formação de colônias foi inibida significativamente no CRC a partir da concentração mais baixa testada de 10 µg/mL, resultando em nenhuma colônia conforme visualizado por um microscópio de contraste de fase. Os dados mostraram uma elevação significativa na anexina V a 100 µg/mL de EGCG (25,85%) e 150 µg/mL de quercetina (48,35%). Além disso, a análise do ciclo celular mostrou que essa combinação causou parada do ciclo celular na fase G1 na concentração de 100 µg/mL (72,7%) e 150 µg/mL (75,25%). O efeito combinado da epigalocatequina galato e quercetina exerce atividade antiproliferativa contra o CCR, é promissor como agente quimioterápico alternativo convencional.


Assuntos
Humanos , Neoplasias Colorretais/tratamento farmacológico , Catequina/análogos & derivados , Catequina/farmacologia , Quercetina/farmacologia , Ciclo Celular , Anexina A5 , Linhagem Celular Tumoral , Proliferação de Células
5.
J Ethnopharmacol ; 300: 115729, 2023 Jan 10.
Artigo em Inglês | MEDLINE | ID: mdl-36162544

RESUMO

ETHNOPHARMACOLOGICAL RELEVANCE: The North-eastern parts of India have immense therapeutic floras, Ottelia alismoides is an aquatic plant that has been in use for a long time in traditional medicine for treating diseases like cancer, tuberculosis, diabetes, febrifuge, hemorrhoids, and rubefacient. In lung and skin carcinoma cells with a high rate of proliferation and metastasis including drug resistance and non-specific target activity, generates important challenges towards their treatment strategy. Thus, finding novel therapeutic targets to treat lung and skin cancer progression is essential to enhance the patients' survival with treatment. AIM OF THE STUDY: The purpose of this study was to evaluate the apoptotic potential of acetone extract of O. alismoides (L.) Pers. (OA-AC) and to identify the compounds responsible for this effect, HRLC-MS-QTOF analysis of the extract has been undertaken along with in-silico molecular docking analysis of the identified compounds. MATERIALS AND METHODS: A549 and A431 cells were treated with acetone extract of O. alismoides (OA-AC) at 24 h and 48 h exposure and cell cycle phase distribution was evaluated and also apoptosis induction activity was evaluated by OA-EtBr staining and Mitochondrial outer membrane potential assay. Western blotting was performed for the evaluation of apoptotic protein expression. At last, the HR-LCMS of OA-AC was analyzed to identify the compounds responsible for the apoptotic activity of the extract. RESULTS: The cell cycle phase distribution analysis in A549 and A431 cells at 24hrs exposure with 10 µg/mL and 25 µg/mL of OA-AC showed a potent arrest or blockage at the G2/M phase of the cell cycle with reduced expression of cyclin B and p-Cdc2. At 48 h exposure, apoptosis was observed in these cancer cells with elevated expression of Bax, p21 and cleaved caspase 3 and reduced expression of the Bcl2. CONCLUSION: AO-EtBr staining of these cancer cells reveals that the death induced by OA-AC was apoptotic in nature with depolarization of mitochondrial membrane due to loss or damage of the mitochondrial membrane. The HRLC-MS-QTOF analysis of OA-AC depicted 14 major isolable compounds and molecular docking analysis displayed 4 compounds that might act as an inhibitor of cyclin B for G2/M phase arrest that leads to apoptotic induction in the cells.


Assuntos
Carcinoma , Hydrocharitaceae , Acetona , Apoptose , Carcinoma/tratamento farmacológico , Caspase 3 , Ciclo Celular , Pontos de Checagem do Ciclo Celular , Linhagem Celular Tumoral , Pontos de Checagem da Fase G2 do Ciclo Celular , Humanos , Hydrocharitaceae/metabolismo , Irritantes , Simulação de Acoplamento Molecular , Extratos Vegetais/farmacologia , Extratos Vegetais/uso terapêutico , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Proteína X Associada a bcl-2
6.
J Pharm Biomed Anal ; 223: 115166, 2023 Jan 20.
Artigo em Inglês | MEDLINE | ID: mdl-36403346

RESUMO

Tyrosine kinases have been intensively investigated as drug targets for several decades, since they regulate many cellular processes including cell growth, differentiation, and proliferation. Indeed, the deregulation of tyrosine kinases has been confirmed to play a vital role in the pathophysiology of many diseases. During the last few years, varieties of techniques have been developed to search for new tyrosine kinase inhibitors for cancer therapy, such as traditional filtration binding assay, scintillation proximity assay and some high-throughput screening methods. In this review, we describe the basic rules, merits and demerits, and application of a number of general and advanced technologies. The purpose of this review is to provide an insight into the numerous assays to achieve the exploration of new tyrosine kinase inhibitors.


Assuntos
Bioensaio , Ensaios de Triagem em Larga Escala , Ciclo Celular , Proteínas Tirosina Quinases , Inibidores de Proteínas Quinases/farmacologia , Tirosina
7.
Gene ; 851: 147029, 2023 Jan 30.
Artigo em Inglês | MEDLINE | ID: mdl-36356905

RESUMO

The DNA polymerase delta (Pol δ), a heterotetramer of four subunits (Pol δ4), plays a pivotal role in DNA replication, as well as in DNA damage repair. Pold4, as the smallest subunit of Pol δ, is degraded in response to DNA damage or when entering into S-phase. This leads to the conversion of Pol δ4 to the trimeric complex Pol δ3. However, the contribution of Pold4 has not been fully elucidated in mammals. Cdm1, the Pold4 ortholog in Schizosaccharomyces pombe, is dispensable for cell growth and DNA damage repair, and there are no Pold4 orthologs in Saccharomyces cerevisiae. We previously generated a knockout mouse model of Pold3 and revealed its essential role in genome stability. Unexpectedly, we here found that Pold4 knockout mice are viable and fertile. In addition, Pold4 knockout mice do not exhibit any pathologic changes in the lung and spleen, tissues with the most abundant expression of Pold4. Moreover, Pold4 knockout mouse tail tip fibroblasts (TTF) exhibited normal cell growth, cell cycle, DNA replication, DNA damage and DNA repair capacity. These results suggested that Pol δ3 but not Pol δ4 may be responsible for these processes in normal cells. Interestingly, 19-month-old wild-type (WT) mice had tumors in the liver, while Pold4 knockout mice did not, and Pold4 knockout mice showed increased longevity. In further, this provided evidence suggested that Pold4 could be a potential novel target for lung carcinoma because its depletion does not affect normal cells but does affect cancer cells.


Assuntos
Proteínas de Schizosaccharomyces pombe , Schizosaccharomyces , Camundongos , Animais , Replicação do DNA/genética , Reparo do DNA/genética , DNA Polimerase III/genética , Dano ao DNA , Ciclo Celular , Camundongos Knockout , Saccharomyces cerevisiae , Mamíferos
8.
Methods Mol Biol ; 2589: 225-239, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-36255628

RESUMO

In eukaryotes, the organization of DNA wrapped around histones regulates DNA-dependent processes. Changes in epigenetic modifications modulate the compaction of DNA into chromatin and, thus, regulate DNA metabolism in time and space. Hence, to catalog the spatiotemporal epigenetic information and its relation to the dynamic nuclear landscape is of paramount importance. Here, we present a method, based on FiJi and the statistical image analysis tool nucim(R), to classify in 3D the nuclear DNA compaction in single interphase cells. We, furthermore, mapped the distribution of (epi)genetic marks and nuclear proteins/processes to the compaction classes along with their dynamics over the cell cycle. These techniques allow to catalog and quantify the dynamic changes in the epigenome in space and time and in single cells.


Assuntos
Código das Histonas , Histonas , Histonas/metabolismo , Cromatina/genética , DNA/genética , Epigênese Genética , Ciclo Celular
9.
Semin Cell Dev Biol ; 136: 49-63, 2023 Feb 28.
Artigo em Inglês | MEDLINE | ID: mdl-35422389

RESUMO

Ribosomes are macromolecular machines that are globally required for the translation of all proteins in all cells. Ribosome biogenesis, which is essential for cell growth, proliferation and survival, commences with transcription of a variety of RNAs by RNA Polymerases I and III. RNA Polymerase I (Pol I) transcribes ribosomal RNA (rRNA), while RNA Polymerase III (Pol III) transcribes 5S ribosomal RNA and transfer RNAs (tRNA) in addition to a wide variety of small non-coding RNAs. Interestingly, despite their global importance, disruptions in Pol I and Pol III function result in tissue-specific developmental disorders, with craniofacial anomalies and leukodystrophy/neurodegenerative disease being among the most prevalent. Furthermore, pathogenic variants in genes encoding subunits shared between Pol I and Pol III give rise to distinct syndromes depending on whether Pol I or Pol III function is disrupted. In this review, we discuss the global roles of Pol I and III transcription, the consequences of disruptions in Pol I and III transcription, disorders arising from pathogenic variants in Pol I and Pol III subunits, and mechanisms underpinning their tissue-specific phenotypes.


Assuntos
Doenças Neurodegenerativas , RNA Polimerase I , Humanos , RNA Polimerase I/genética , RNA Polimerase I/metabolismo , Doenças Neurodegenerativas/metabolismo , RNA Polimerase III/genética , RNA Polimerase III/metabolismo , Ribossomos/metabolismo , Ciclo Celular , Transcrição Genética
10.
J Inorg Biochem ; 238: 112028, 2023 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-36274479

RESUMO

Three series of cis- and trans-[bis(benzimidazol-2-ylidene)dichlorido]platinum(II) and cis-[(benzimidazol-2-ylidene)(DMSO)dichlorido]platinum(II) complexes were synthesised and screened for cytotoxicity against six human cancer cell lines. Depending on their N-alkyl and 5-alkoxycarbonyl substituents, two-digit nanomolar to single-digit micromolar IC50 values against cancer cell lines intrinsically resistant to or ill-responding to cisplatin were reached by both cis- and trans-configured complexes. The stability of the complexes under aqueous biotest conditions was shown via 1H and 195Pt NMR monitoring to be dependent on their configuration and their N-substituents. Localisation studies employing click reactions with 1-alkyne- or cyclopropene-tagged derivatives revealed that the cis-complexes accumulated in the cell nuclei and the trans-complexes in the mitochondria. While the most active cis-complexes showed modes of action akin to those of cisplatin, the most active trans-complexes differed from cisplatin by much lower rates of cellular uptake and ROS production, and by their non-interaction with the cell cycle and the DNA of cancer cells. Thus, we identified structural key elements for the synthesis of optimised trans-configured NHC platinum(II) complexes with high activity also against cisplatin-refractory cancer cells.


Assuntos
Antineoplásicos , Neoplasias , Humanos , Cisplatino/farmacologia , Platina/farmacologia , Platina/química , Antineoplásicos/química , Ciclo Celular
11.
Mol Cell Proteomics ; 21(1): 100169, 2022 01.
Artigo em Inglês | MEDLINE | ID: mdl-34742921

RESUMO

Comprehensive proteome analysis of rare cell phenotypes remains a significant challenge. We report a method for low cell number MS-based proteomics using protease digestion of mildly formaldehyde-fixed cells in cellulo, which we call the "in-cell digest." We combined this with averaged MS1 precursor library matching to quantitatively characterize proteomes from low cell numbers of human lymphoblasts. About 4500 proteins were detected from 2000 cells, and 2500 proteins were quantitated from 200 lymphoblasts. The ease of sample processing and high sensitivity makes this method exceptionally suited for the proteomic analysis of rare cell states, including immune cell subsets and cell cycle subphases. To demonstrate the method, we characterized the proteome changes across 16 cell cycle states (CCSs) isolated from an asynchronous TK6 cells, avoiding synchronization. States included late mitotic cells present at extremely low frequency. We identified 119 pseudoperiodic proteins that vary across the cell cycle. Clustering of the pseudoperiodic proteins showed abundance patterns consistent with "waves" of protein degradation in late S, at the G2&M border, midmitosis, and at mitotic exit. These clusters were distinguished by significant differences in predicted nuclear localization and interaction with the anaphase-promoting complex/cyclosome. The dataset also identifies putative anaphase-promoting complex/cyclosome substrates in mitosis and the temporal order in which they are targeted for degradation. We demonstrate that a protein signature made of these 119 high-confidence cell cycle-regulated proteins can be used to perform unbiased classification of proteomes into CCSs. We applied this signature to 296 proteomes that encompass a range of quantitation methods, cell types, and experimental conditions. The analysis confidently assigns a CCS for 49 proteomes, including correct classification for proteomes from synchronized cells. We anticipate that this robust cell cycle protein signature will be crucial for classifying cell states in single-cell proteomes.


Assuntos
Peptídeo Hidrolases , Proteômica , Contagem de Células , Ciclo Celular , Proteínas de Ciclo Celular/metabolismo , Mitose , Proteômica/métodos
12.
In Vitro Cell Dev Biol Anim ; 58(2): 169-178, 2022 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-35194763

RESUMO

Cell adhesion to extracellular matrix proteins mediates resistance to radio- and chemotherapy by activating integrin signaling. In addition, mutual and cooperative interactions between integrin and growth factor receptor signaling contribute to the cellular radiation response. Here, we investigate to which extend the crosstalk between ß1 integrins and growth factor receptor signaling determines the cellular radiation response of fibroblasts by assessing clonogenic survival and cell cycling. By utilizing growth factor signaling competent and either ß1 integrin wildtype GD25ß1A fibroblasts or ß1 integrin mutant, signaling incompetent GD25ß1B fibroblasts, we show basal clonogenic survival to depend on growth factor receptor but not integrin signaling. Our data further suggest the cooperation between ß1 integrins and growth factor receptors to be critical for enhancing the radiation-induced G2/M cell cycle block leading to improved clonogenic radiation survival. By pharmacological inhibition of EGFR and PI3K, we additionally show that the essential contribution of EGFR signaling to radiogenic G2/M cell cycle arrest depends on the co-activation of the ß1 integrin signaling axis, but occurs independent of PI3K. Taken together, elucidation of the signaling circuitry underlying the EGFR/ß1 integrin crosstalk may support the development of advanced molecular targeted therapies for radiation oncology.


Assuntos
Integrina beta1 , Transdução de Sinais , Animais , Ciclo Celular , Fibroblastos/metabolismo , Integrina beta1/genética , Integrina beta1/metabolismo , Receptores de Fatores de Crescimento/metabolismo
13.
Proc Natl Acad Sci U S A ; 119(49): e2113504119, 2022 Dec 06.
Artigo em Inglês | MEDLINE | ID: mdl-36454750

RESUMO

Alternative polyadenylation (APA) plays an important role in posttranscriptional gene regulation such as transcript stability and translation efficiency. However, our knowledge about APA dynamics at the single-cell level is largely unexplored. Here, we developed single-cell polyadenylation sequencing, a strand-specific approach for sequencing the 3' end of transcripts, to investigate the landscape of APA at the single-cell level. By analyzing several cell lines, we found many genes using multiple polyA sites in bulk data are prone to use only one polyA site in each single cell. Interestingly, cell cycle genes were significantly enriched in genes with high variation in polyA site usages. Furthermore, the 414 genes showing a polyA site usage switch after cell synchronization enriched cell cycle genes, while the differentially expressed genes after cell synchronization did not enrich cell cycle genes. We further identified 812 genes showing polyA site usage changes between neighboring cell cycles, which were grouped into six clusters, with cell phase-specific functional categories enriched in each cluster. Deletion of one polyA site in MSL1 and SCCPDH results in slower and faster cell cycle progression, respectively, supporting polyA site usage switch played an important role in cell cycle. These results indicate that APA is an important layer for cell cycle regulation.


Assuntos
Poli A , Poliadenilação , Poliadenilação/genética , Genes cdc , Ciclo Celular/genética , Divisão Celular
14.
Sci Adv ; 8(48): eabo0876, 2022 Dec 02.
Artigo em Inglês | MEDLINE | ID: mdl-36459552

RESUMO

MacroH2A variants have been linked to inhibition of metastasis through incompletely understood mechanisms. Here, we reveal that solitary dormant disseminated cancer cells (DCCs) display increased levels of macroH2A variants in head and neck squamous cell carcinoma PDX in vivo models and patient samples compared to proliferating primary or metastatic lesions. We demonstrate that dormancy-inducing transforming growth factor-ß2 and p38α/ß pathways up-regulate macroH2A expression and that macroH2A variant overexpression is sufficient to induce DCC dormancy and suppress metastasis in vivo. Notably, inducible expression of the macroH2A2 variant in vivo suppresses metastasis via a reversible growth arrest of DCCs. This state does not require the dormancy-regulating transcription factors DEC2 and NR2F1; instead, transcriptomic analysis reveals that macroH2A2 overexpression inhibits cell cycle and oncogenic signaling programs, while up-regulating dormancy and senescence-associated inflammatory cytokines. We conclude that the macroH2A2-enforced dormant phenotype results from tapping into transcriptional programs of both quiescence and senescence to limit metastatic outgrowth.


Assuntos
Neoplasias de Cabeça e Pescoço , Histonas , Humanos , Carcinogênese , Divisão Celular , Ciclo Celular , Neoplasias de Cabeça e Pescoço/genética
15.
Nat Commun ; 13(1): 7444, 2022 Dec 02.
Artigo em Inglês | MEDLINE | ID: mdl-36460641

RESUMO

Mechanisms by which specific histone modifications regulate distinct gene networks remain little understood. We investigated how H3K79me2, a modification catalyzed by DOT1L and previously considered a general transcriptional activation mark, regulates gene expression during cardiogenesis. Embryonic cardiomyocyte ablation of Dot1l revealed that H3K79me2 does not act as a general transcriptional activator, but rather regulates highly specific transcriptional networks at two critical cardiogenic junctures: embryonic cardiogenesis, where it was particularly important for left ventricle-specific genes, and postnatal cardiomyocyte cell cycle withdrawal, with Dot1L mutants having more mononuclear cardiomyocytes and prolonged cardiomyocyte cell cycle activity. Mechanistic analyses revealed that H3K79me2 in two distinct domains, gene bodies and regulatory elements, synergized to promote expression of genes activated by DOT1L. Surprisingly, H3K79me2 in specific regulatory elements also contributed to silencing genes usually not expressed in cardiomyocytes. These results reveal mechanisms by which DOT1L successively regulates left ventricle specification and cardiomyocyte cell cycle withdrawal.


Assuntos
Redes Reguladoras de Genes , Miócitos Cardíacos , Divisão Celular , Ciclo Celular/genética , Ventrículos do Coração
16.
Food Res Int ; 162(Pt A): 111991, 2022 12.
Artigo em Inglês | MEDLINE | ID: mdl-36461230

RESUMO

Individual-cell heterogeneity is a major source of variability in biological systems affecting importantly, among others, microbial behavior. Characterization of cell populations of pathogenic bacterial strains in their entirety, ignoring the phenotypic variability of single cells, may result in erroneous safety risk estimates. The objective of the present study was the evaluation and comparison of the heterogeneity in the individual-cell growth dynamics of different strains of Salmonella enterica. The stochasticity in the growth of single cells of five S. enterica ser. Typhimurium strains was quantitatively described using time-lapse microscopy, and the existence of a strain effect was statistically assessed. In total, 831 growing microcolonies originating from single cells were monitored and analyzed, and the growth kinetic parameters of lag time (λ) and maximum specific growth rate (µmax) for each one of them were estimated. An extensive heterogeneity in individual-cell growth kinetics was recorded, while significant inter-strain differences in their heterogeneity were evident based on simultaneous Bonferroni confidence intervals and Levene's tests. The Logistic and LogLogistic probability distribution provided the best fitting for µmax and λ data, respectively for all the tested strains. The strain effect on the above distributions was also demonstrated with pairwise comparisons of the decile differences. The impact of strain-dependent heterogeneity on microbial growth was visualized by comparing stochastic growth curves of different strains using Monte Carlo simulation. In conclusion, the individual-cell growth dynamics of S. enterica are heterogeneous, with the magnitude of the observed heterogeneity appearing to be an inherent characteristic of bacterial strains.


Assuntos
Salmonella enterica , Ciclo Celular , Proliferação de Células , Simulação por Computador , Cinética
17.
Pestic Biochem Physiol ; 188: 105231, 2022 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-36464350

RESUMO

Viruses arrest the host cell cycle and using multiple functions of host cells is an important approach for their replication. Baculovirus arrests infected insect cells at both the late S and G2/M phase, but the strategy employed by baculovirus is not clearly understood. Our research suggests that the Bombyx mori nucleopolyhedrovirus (BmNPV) could arrest the cell cycle in the G2/M phase to promote virus replication, and also that the viral protein LEF-11 could inhibit host cell proliferation and arrest the cell cycle by inhibiting the cell cycle checkpoint proteins BmCyclinB and BmCDK1. Furthermore, we found that LEF-11 interacts with BmIMPI to regulate cell proliferation, but not by direct interaction with BmCyclinB or BmCDK1. In addition, our findings showed that BmIMPI was important and necessary for LEF-11 induced cell cycle arrest in the G2/M phase. Moreover, BmIMPI was found to interact with BmCyclinB and BmCDK1, and down-regulate the expression of BmCyclinB and BmCDK1 to compromise the cell cycle and cell proliferation. Taken together, the data presented demonstrated that baculovirus LEF-11 regulates BmIMPI to inhibit host cell proliferation and provide a new insight into the molecular mechanisms employed by viruses to induce cell cycle arrest.


Assuntos
Baculoviridae , Replicação Viral , Divisão Celular , Pontos de Checagem do Ciclo Celular , Ciclo Celular
18.
BMC Plant Biol ; 22(1): 553, 2022 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-36456926

RESUMO

BACKGROUND: Spur, a structure capable of producing and storing nectar, not only plays a vital role in the pollination process but also promotes the rapid diversification of some plant lineages, which is considered a key innovation in plants. Spur is the focus of many studies, such as evolution and ecological hypothesis, but the current understanding of spur development is limited. High-throughput sequencing of Impatiens uliginosa was carried out to study the molecular mechanism of its spur development, which is believed to provide some insights into the spur development of Impatiens. RESULTS: Transcriptomic sequencing and analysis were performed on spurs and limbs of I. uliginosa at three developmental stages. A total of 47.83 Gb of clean data were obtained, and 49,716 unigene genes were assembled. After comparison with NR, Swiss-Prot, Pfam, COG, GO and KEGG databases, a total of 27,686 genes were annotated successfully. Through comparative analysis, 19,356 differentially expressed genes were found and enriched into 208 GO terms and 146 KEGG pathways, among which plant hormone signal transduction was the most significantly enriched pathway. One thousand thirty-two transcription factors were identified, which belonged to 33 TF families such as MYB, bHLH and TCP. Twenty candidate genes that may be involved in spur development were screened and verified by qPCR, such as SBP, IAA and ABP. CONCLUSIONS: Transcriptome data of different developmental stages of spurs were obtained, and a series of candidate genes related to spur development were identified. The importance of genes related to cell cycle, cell division, cell elongation and hormones in spur development was clarified. This study provided valuable information and resources for understanding the molecular mechanism of spur development in Impatiens.


Assuntos
Impatiens , Transcriptoma , Sequenciamento Completo do Exoma , Ciclo Celular , Bases de Dados de Proteínas
19.
Biochem Biophys Res Commun ; 637: 286-293, 2022 Dec 31.
Artigo em Inglês | MEDLINE | ID: mdl-36410278

RESUMO

Auger electrons can induce nanoscale physiochemical damage to DNA. The present study reports a sequential and systematic evaluation of the relationship between DNA damage such as double-strand breaks (DSBs) and the cell cycle for the Auger electron-emitting agent radiolabeled cisplatin with DNA binding ability. For dynamic imaging analysis, we used U2OS-derived cancer cells expressing two fluorescent fusion proteins: tumor-suppressor p53 binding protein 1 with a green fluorescent protein (53BP1-EGFP) and proliferating cell nuclear antigen with a red fluorescent protein (PCNA-DsRed). Time-lapse images of the cells were quantitatively analyzed using the ImageJ software with the deepImageJ plugin and the Google Colaboratory platform. From the middle-to-late G1 phase, around the G1-to-S phase transition, we found increased 53BP1 foci in cells treated with the radio-cisplatin. The radio-cisplatin caused significantly more DSBs than the nonradioactive cisplatin and saline in the G1 phase but not in the other phases. These results indicate that Auger electron-induced DNA damage, including DSBs, depends on the cell cycle. The G1 phase, which is associated with low DNA repair capacity and high radiosensitivity, is a promising target; thus, combining radiolabeled cisplatin with agents that arrest cells in the G1 phase could improve the DNA-damaging effect of Auger electrons and their therapeutic efficacy.


Assuntos
Cisplatino , Elétrons , Cisplatino/farmacologia , Divisão Celular , Ciclo Celular , Dano ao DNA
20.
Results Probl Cell Differ ; 70: 375-396, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-36348115

RESUMO

The cell cycle is governed by stringent epigenetic mechanisms that, in response to intrinsic and extrinsic regulatory cues, support fidelity of DNA replication and cell division. We will focus on (1) the complex and interdependent processes that are obligatory for control of proliferation and compromised in cancer, (2) epigenetic and topological domains that are associated with distinct phases of the cell cycle that may be altered in cancer initiation and progression, and (3) the requirement for mitotic bookmarking to maintain intranuclear localization of transcriptional regulatory machinery to reinforce cell identity throughout the cell cycle to prevent malignant transformation.


Assuntos
Epigênese Genética , Neoplasias , Humanos , Ciclo Celular/genética , Divisão Celular , Neoplasias/genética , Neoplasias/patologia , Cromatina , Regulação da Expressão Gênica
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