Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 3.312
Filtrar
1.
BMC Cancer ; 21(1): 1087, 2021 Oct 08.
Artigo em Inglês | MEDLINE | ID: mdl-34625031

RESUMO

BACKGROUND: Cancer remains one of the leading causes of death worldwide, despite the possibilities to detect early onset of the most common cancer types. The search for the optimal therapy is complicated by the cancer diversity within tumors and the unsynchronized development of cancerous cells. Therefore, it is necessary to characterize cancer cell populations after treatment has been applied, because cancer recurrence is not rare. In our research, we concentrated on small cancer cell subpopulation (microcells) that has a potential to be cancer resistance source. Previously made experiments has shown that these cells in small numbers form in specific circumstances after anticancer treatment. METHODS: In experiments described in this research, the anticancer agents' paclitaxel and doxorubicin were used to stimulate the induction of microcells in fibroblast, cervix adenocarcinoma, and melanoma cell lines. Mainly for the formation of microcells in melanoma cells. The drug-stimulated cells were then characterized in terms of their formation efficiency, morphology, and metabolic activity. RESULTS: We observed the development of cancer microcells and green fluorescent protein (GFP) transfection efficiency after stress. In the time-lapse experiment, we observed microcell formation through a renewal process and GFP expression in the microcells. Additionally, the microcells were viable after anticancer treatment, as indicated by the nicotinamide adenine dinucleotide hydrogen phosphate (NADPH) enzyme activity assay results. Taken together, these findings indicate that cancer microcells are viable and capable of resisting the stress induced by anticancer drugs, and these cells are prone to chemical substance uptake from the environment. CONCLUSION: Microcells are not only common to a specific cancer type, but can be found in any tumor type. This study could help to understand cancer emergence and recurrence. The appearance of microcells in the studied cancer cell population could be an indicator of the individual anticancer therapy effectiveness and patient survival.


Assuntos
Antineoplásicos/farmacologia , Resistencia a Medicamentos Antineoplásicos , Neoplasias/tratamento farmacológico , Neoplasias/patologia , Adenocarcinoma/metabolismo , Adenocarcinoma/patologia , Contagem de Células , Linhagem Celular Tumoral , Núcleo Celular/ultraestrutura , Autorrenovação Celular , Sobrevivência Celular/efeitos dos fármacos , Proteínas de Ligação a DNA/metabolismo , Doxorrubicina/farmacologia , Complexos Endossomais de Distribuição Requeridos para Transporte/metabolismo , Feminino , Fibroblastos/efeitos dos fármacos , Proteínas de Fluorescência Verde/metabolismo , Células HeLa , Humanos , Indicadores e Reagentes/farmacocinética , Melanoma/metabolismo , Melanoma/patologia , Microscopia Eletrônica , NADP/metabolismo , Recidiva Local de Neoplasia/metabolismo , Recidiva Local de Neoplasia/patologia , Neoplasias/metabolismo , Neoplasias/ultraestrutura , Vermelho Neutro/farmacocinética , Paclitaxel/farmacologia , Estresse Fisiológico , Imagem com Lapso de Tempo , Fatores de Transcrição/metabolismo , Transfecção , Neoplasias do Colo do Útero/tratamento farmacológico , Neoplasias do Colo do Útero/metabolismo , Neoplasias do Colo do Útero/patologia
2.
Nat Commun ; 12(1): 5903, 2021 10 08.
Artigo em Inglês | MEDLINE | ID: mdl-34625543

RESUMO

Circadian clocks are self-sustained and cell-autonomous oscillators. They respond to various extracellular cues depending on the time-of-day and the signal intensity. Phase Transition Curves (PTCs) are instrumental in uncovering the full repertoire of responses to a given signal. However, the current methodologies for reconstructing PTCs are low-throughput, laborious, and resource- and time-consuming. We report here the development of an efficient and high throughput assay, dubbed Circadian Single-Cell Oscillators PTC Extraction (Circa-SCOPE) for generating high-resolution PTCs. This methodology relies on continuous monitoring of single-cell oscillations to reconstruct a full PTC from a single culture, upon a one-time intervention. Using Circa-SCOPE, we characterize the effects of various pharmacological and blood-borne resetting cues, at high temporal resolution and a wide concentration range. Thus, Circa-SCOPE is a powerful tool for comprehensive analysis and screening for circadian clocks' resetting cues, and can be valuable for basic as well as translational research.


Assuntos
Relógios Circadianos/fisiologia , Análise de Célula Única/métodos , Imagem com Lapso de Tempo/métodos , Animais , Ritmo Circadiano/fisiologia , Ensaios de Triagem em Larga Escala , Humanos , Camundongos , Células NIH 3T3 , Esteroides/sangue
3.
Hum Reprod ; 36(11): 2840-2847, 2021 10 18.
Artigo em Inglês | MEDLINE | ID: mdl-34536006

RESUMO

STUDY QUESTION: Using time-lapse data, can the current consensus for the timing of fertilisation assessment of oocytes, cultured in standard incubation, be optimised? SUMMARY ANSWER: The optimum time to perform fertilisation assessment for oocytes cultured in standard incubation is 16.5 ± 0.5 h post-insemination (hpi), and the current consensus requires modification in order to minimise the chance of fertilisation being missed. WHAT IS KNOWN ALREADY: Time-lapse incubation allows the embryologist to retrospectively review collated images of oocytes and embryos to capture important embryological observations that may otherwise be missed in standard incubation. According to expert consensus, the optimum time to perform the assessment of fertilisation is 17 ± 1 hpi. STUDY DESIGN, SIZE, DURATION: A retrospective, multicentre analysis utilised data obtained from 54 746 ICSI-derived embryos and 23 602 IVF-derived embryos cultured in time-lapse incubation between January 2011 and November 2019. PARTICIPANTS/MATERIALS, SETTING, METHODS: Using time-lapse imaging (TLI), the precise time of pronuclei appearance and disappearance was recorded, where applicable, and the number of oocytes with two pronuclei observable during 10 30-min intervals from 15 hpi to 20 hpi was determined. MAIN RESULTS AND THE ROLE OF CHANCE: Between 15 and 17.5 hpi, the average number of oocytes exhibiting normal fertilisation, elicited as two pronuclei, was 98.19% with the highest proportion of oocytes having visible pronuclei at 16-16.5 hpi (98.32%). At 18-18.5 hpi, the number of visible pronuclei reduced to 95.53% and continued to fall to 87.02% at 19.5-20 hpi. LIMITATIONS, REASONS FOR CAUTION: The authors' expectation is that these findings are transferable to other settings, however it is possible that, with alternative culture media and incubation environments, calibration of this timing may be required. As data cannot readily be recorded for pronuclear appearance for IVF-derived embryos, it is not possible to determine the optimum time to perform the fertilisation assessment for IVF-derived embryos. WIDER IMPLICATIONS OF THE FINDINGS: By fine-tuning the time at which fertilisation assessment takes place the accuracy of the assessment can be increased to maximise the number of fertilised oocytes identified, thereby increasing the number of usable embryos for the patient. Without TLI and following current consensus guidelines, over 11% (n = 3000) of oocytes would have been marked as unfertilised within this cohort. Further to this, depending on the time of a standard fertilisation assessment, up to 300 embryos which resulted in live births could have been categorised as unfertilised, as they presented no visible pronuclei at the conventional assessment time-point. STUDY FUNDING/COMPETING INTEREST(S): A.C. is a minor shareholder in CARE Fertility Limited. Validated algorithmic time-lapse embryo selection is offered to patients at CARE Fertility at an additional charge as an adjuvant treatment option. TRIAL REGISTRATION NUMBER: N/A.


Assuntos
Fertilização In Vitro , Oócitos , Feminino , Fertilização , Humanos , Estudos Retrospectivos , Imagem com Lapso de Tempo
4.
Nat Commun ; 12(1): 5660, 2021 09 27.
Artigo em Inglês | MEDLINE | ID: mdl-34580289

RESUMO

Small Open Reading Frames (smORFs) coding for peptides of less than 100 amino-acids are an enigmatic and pervasive gene class, found in the tens of thousands in metazoan genomes. Here we reveal a short 80 amino-acid peptide (Pegasus) which enhances Wingless/Wnt1 protein short-range diffusion and signalling. During Drosophila wing development, Wingless has sequential functions, including late induction of proneural gene expression and wing margin development. Pegasus mutants produce wing margin defects and proneural expression loss similar to those of Wingless. Pegasus is secreted, and co-localizes and co-immunoprecipitates with Wingless, suggesting their physical interaction. Finally, measurements of fixed and in-vivo Wingless gradients support that Pegasus increases Wingless diffusion in order to enhance its signalling. Our results unveil a new element in Wingless signalling and clarify the patterning role of Wingless diffusion, while corroborating the link between small open reading frame peptides, and regulation of known proteins with membrane-related functions.


Assuntos
Proteínas de Drosophila/metabolismo , Drosophila/fisiologia , Regulação da Expressão Gênica no Desenvolvimento , Peptídeos/metabolismo , Asas de Animais/crescimento & desenvolvimento , Proteína Wnt1/metabolismo , Animais , Animais Geneticamente Modificados , Microscopia Intravital , Peptídeos/genética , Imagem com Lapso de Tempo
5.
Nat Commun ; 12(1): 5700, 2021 09 29.
Artigo em Inglês | MEDLINE | ID: mdl-34588437

RESUMO

Bacterial biofilms are aggregates of surface-associated cells embedded in an extracellular polysaccharide (EPS) matrix, and are typically stationary. Studies of bacterial collective movement have largely focused on swarming motility mediated by flagella or pili, in the absence of a biofilm. Here, we describe a unique mode of collective movement by a self-propelled, surface-associated biofilm-like multicellular structure. Flavobacterium johnsoniae cells, which move by gliding motility, self-assemble into spherical microcolonies with EPS cores when observed by an under-oil open microfluidic system. Small microcolonies merge, creating larger ones. Microscopic analysis and computer simulation indicate that microcolonies move by cells at the base of the structure, attached to the surface by one pole of the cell. Biochemical and mutant analyses show that an active process drives microcolony self-assembly and motility, which depend on the bacterial gliding apparatus. We hypothesize that this mode of collective bacterial movement on solid surfaces may play potential roles in biofilm dynamics, bacterial cargo transport, or microbial adaptation. However, whether this collective motility occurs on plant roots or soil particles, the native environment for F. johnsoniae, is unknown.


Assuntos
Biofilmes , Flavobacterium/fisiologia , Locomoção , Simulação por Computador , Microscopia Intravital , Técnicas Analíticas Microfluídicas , Raízes de Plantas/microbiologia , Microbiologia do Solo , Imagem com Lapso de Tempo
6.
Neuron ; 109(18): 2884-2901.e7, 2021 09 15.
Artigo em Inglês | MEDLINE | ID: mdl-34534453

RESUMO

In non-neuronal cells, clathrin has established roles in endocytosis, with clathrin cages enclosing plasma membrane infoldings, followed by rapid disassembly and reuse of monomers. However, in neurons, clathrin is conveyed in slow axonal transport over days to weeks, and the underlying transport/targeting mechanisms, mobile cargo structures, and even its precise presynaptic localization and physiologic role are unclear. Combining live imaging, photobleaching/conversion, mass spectrometry, electron microscopy, and super-resolution imaging, we found that unlike in dendrites, where clathrin cages rapidly assemble and disassemble, in axons, clathrin and related proteins organize into stable "transport packets" that are unrelated to endocytosis and move intermittently on microtubules, generating an overall slow anterograde flow. At synapses, multiple clathrin packets abut synaptic vesicle (SV) clusters, and clathrin packets also exchange between synaptic boutons in a microtubule-dependent "superpool." Within synaptic boundaries, clathrin is surprisingly dynamic, continuously exchanging between local clathrin assemblies, and its depletion impairs SV recycling. Our data provide a conceptual framework for understanding clathrin trafficking and presynaptic targeting that has functional implications.


Assuntos
Transporte Axonal/fisiologia , Vesículas Revestidas por Clatrina/metabolismo , Clatrina/metabolismo , Hipocampo/metabolismo , Sinapses/metabolismo , Animais , Animais Recém-Nascidos , Células Cultivadas , Clatrina/química , Vesículas Revestidas por Clatrina/química , Hipocampo/química , Hipocampo/citologia , Camundongos , Transporte Proteico/fisiologia , Ratos , Ratos Wistar , Sinapses/química , Imagem com Lapso de Tempo/métodos
7.
Nat Methods ; 18(9): 1091-1102, 2021 09.
Artigo em Inglês | MEDLINE | ID: mdl-34413523

RESUMO

Mitochondria display complex morphology and movements, which complicates their segmentation and tracking in time-lapse images. Here, we introduce Mitometer, an algorithm for fast, unbiased, and automated segmentation and tracking of mitochondria in live-cell two-dimensional and three-dimensional time-lapse images. Mitometer requires only the pixel size and the time between frames to identify mitochondrial motion and morphology, including fusion and fission events. The segmentation algorithm isolates individual mitochondria via a shape- and size-preserving background removal process. The tracking algorithm links mitochondria via differences in morphological features and displacement, followed by a gap-closing scheme. Using Mitometer, we show that mitochondria of triple-negative breast cancer cells are faster, more directional, and more elongated than those in their receptor-positive counterparts. Furthermore, we show that mitochondrial motility and morphology in breast cancer, but not in normal breast epithelia, correlate with metabolic activity. Mitometer is an unbiased and user-friendly tool that will help resolve fundamental questions regarding mitochondrial form and function.


Assuntos
Neoplasias da Mama/patologia , Imageamento Tridimensional/métodos , Mitocôndrias , Software , Imagem com Lapso de Tempo/métodos , Algoritmos , Neoplasias da Mama/metabolismo , Células Cultivadas , Feminino , Humanos , Glândulas Mamárias Humanas/citologia , Mitocôndrias/metabolismo , NAD/metabolismo , Reprodutibilidade dos Testes , Neoplasias de Mama Triplo Negativas/patologia
8.
Andrologia ; 53(11): e14211, 2021 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-34437729

RESUMO

The objective of this study was to investigate the impact of male age, semen quality and days of ejaculatory abstinence on embryo morphokinetics. A total of 1,220 zygotes obtained from 139 couples in a private in vitro fertilisation centre were analysed. The timing of specific events from the point of insemination, such as timings to pronuclei appearance and fading, to two, three, four, five, six, seven and eight cells and to blastulation were recorded. Multivariate linear regression analysis was used to evaluate the influence of paternal factors on embryo morphokinetic events. Paternal age was positively correlated with delayed cell cleavage and blastulation, and negatively associated with implantation rate, and clinical pregnancy and live-birth chances. The ejaculatory abstinence was inversely correlated with the implantation rate. Inverse relationships were observed between semen parameters (sperm count, progressive sperm motility, total motile sperm count and morphology) and the timing of specific events during embryo development. Sperm morphology was also positively associated with implantation rate and pregnancy and live-birth chances. Increased paternal age and ejaculatory abstinence, and poor semen quality correlate with delayed cell cleavage and blastulation and negatively impact intracytoplasmic sperm injection outcomes.


Assuntos
Análise do Sêmen , Motilidade Espermática , Divisão Celular , Desenvolvimento Embrionário , Feminino , Fertilização In Vitro , Humanos , Masculino , Gravidez , Taxa de Gravidez , Estudos Retrospectivos , Injeções de Esperma Intracitoplásmicas , Imagem com Lapso de Tempo
9.
Nat Commun ; 12(1): 4693, 2021 08 03.
Artigo em Inglês | MEDLINE | ID: mdl-34344862

RESUMO

Many cellular processes, including cell division, development, and cell migration require spatially and temporally coordinated forces transduced by cell-surface receptors. Nucleic acid-based molecular tension probes allow one to visualize the piconewton (pN) forces applied by these receptors. Building on this technology, we recently developed molecular force microscopy (MFM) which uses fluorescence polarization to map receptor force orientation with diffraction-limited resolution (~250 nm). Here, we show that structured illumination microscopy (SIM), a super-resolution technique, can be used to perform super-resolution MFM. Using SIM-MFM, we generate the highest resolution maps of both the magnitude and orientation of the pN traction forces applied by cells. We apply SIM-MFM to map platelet and fibroblast integrin forces, as well as T cell receptor forces. Using SIM-MFM, we show that platelet traction force alignment occurs on a longer timescale than adhesion. Importantly, SIM-MFM can be implemented on any standard SIM microscope without hardware modifications.


Assuntos
Microscopia de Fluorescência/métodos , Receptores de Superfície Celular/metabolismo , Animais , Fenômenos Biomecânicos , Plaquetas/metabolismo , Linfócitos T CD8-Positivos , Corantes Fluorescentes/metabolismo , Humanos , Integrinas/metabolismo , Camundongos , Sondas Moleculares/metabolismo , Células NIH 3T3 , Paxilina/metabolismo , Receptores de Antígenos de Linfócitos T/metabolismo , Imagem com Lapso de Tempo
10.
Neuron ; 109(18): 2847-2863.e11, 2021 09 15.
Artigo em Inglês | MEDLINE | ID: mdl-34407390

RESUMO

Asymmetric neuronal expansion is thought to drive evolutionary transitions between lissencephalic and gyrencephalic cerebral cortices. We report that Neurog2 and Ascl1 proneural genes together sustain neurogenic continuity and lissencephaly in rodent cortices. Using transgenic reporter mice and human cerebral organoids, we found that Neurog2 and Ascl1 expression defines a continuum of four lineage-biased neural progenitor cell (NPC) pools. Double+ NPCs, at the hierarchical apex, are least lineage restricted due to Neurog2-Ascl1 cross-repression and display unique features of multipotency (more open chromatin, complex gene regulatory network, G2 pausing). Strikingly, selectively eliminating double+ NPCs by crossing Neurog2-Ascl1 split-Cre mice with diphtheria toxin-dependent "deleter" strains locally disrupts Notch signaling, perturbs neurogenic symmetry, and triggers cortical folding. In support of our discovery that double+ NPCs are Notch-ligand-expressing "niche" cells that control neurogenic periodicity and cortical folding, NEUROG2, ASCL1, and HES1 transcript distribution is modular (adjacent high/low zones) in gyrencephalic macaque cortices, prefiguring future folds.


Assuntos
Diferenciação Celular/fisiologia , Neocórtex/embriologia , Neocórtex/fisiologia , Neurogênese/fisiologia , Neurônios/fisiologia , Animais , Células Cultivadas , Feminino , Humanos , Macaca fascicularis , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Células NIH 3T3 , Neocórtex/citologia , Gravidez , Imagem com Lapso de Tempo/métodos
11.
J Neurosci ; 41(40): 8279-8296, 2021 10 06.
Artigo em Inglês | MEDLINE | ID: mdl-34413209

RESUMO

Experience-dependent formation and removal of inhibitory synapses are essential throughout life. For instance, GABAergic synapses are removed to facilitate learning, and strong excitatory activity is accompanied by the formation of inhibitory synapses to maintain coordination between excitation and inhibition. We recently discovered that active dendrites trigger the growth of inhibitory synapses via CB1 receptor-mediated endocannabinoid signaling, but the underlying mechanism remained unclear. Using two-photon microscopy to monitor the formation of individual inhibitory boutons in hippocampal organotypic slices from mice (both sexes), we found that CB1 receptor activation mediated the formation of inhibitory boutons and promoted their subsequent stabilization. Inhibitory bouton formation did not require neuronal activity and was independent of Gi/o-protein signaling, but was directly induced by elevating cAMP levels using forskolin and by activating Gs-proteins using DREADDs. Blocking PKA activity prevented CB1 receptor-mediated inhibitory bouton formation. Our findings reveal that axonal CB1 receptors signal via unconventional downstream pathways and that inhibitory bouton formation is triggered by an increase in axonal cAMP levels. Our results demonstrate an unexpected role for axonal CB1 receptors in axon-specific, and context-dependent, inhibitory synapse formation.SIGNIFICANCE STATEMENT Coordination between excitation and inhibition is required for proper brain function throughout life. It was previously shown that new inhibitory synapses can be formed in response to strong excitation to maintain this coordination, and this was mediated by endocannabinoid signaling via CB1 receptors. As activation of CB1 receptors generally results in the suppression of synaptic transmission, it remained unclear how CB1 receptors can mediate the formation of inhibitory synapses. Here we show that CB1 receptors on inhibitory axons signal via unconventional intracellular pathways and that inhibitory bouton formation is triggered by an increase in axonal cAMP levels and requires PKA activity. Our findings point to a central role for axonal cAMP signaling in activity-dependent inhibitory synapse formation.


Assuntos
Axônios/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , AMP Cíclico/metabolismo , Inibição Neural/fisiologia , Terminações Pré-Sinápticas/metabolismo , Receptor CB1 de Canabinoide/metabolismo , Animais , Axônios/química , AMP Cíclico/genética , Proteínas Quinases Dependentes de AMP Cíclico/genética , Feminino , Hipocampo/química , Hipocampo/metabolismo , Masculino , Camundongos , Camundongos Transgênicos , Microscopia de Fluorescência por Excitação Multifotônica/métodos , Técnicas de Cultura de Órgãos , Terminações Pré-Sinápticas/química , Receptor CB1 de Canabinoide/genética , Imagem com Lapso de Tempo/métodos
12.
Artif Intell Med ; 118: 102129, 2021 08.
Artigo em Inglês | MEDLINE | ID: mdl-34412846

RESUMO

Leukocytes are key cellular elements of the innate immune system in all vertebrates, which play a crucial role in defending organisms against invading pathogens. Tracking these highly migratory and amorphous cells in in vivo models such as zebrafish embryos is a challenging task in cellular immunology. As temporal and special analysis of these imaging datasets by a human operator is quite laborious, developing an automated cell tracking method is highly in demand. Despite the remarkable advances in cell detection, this field still lacks powerful algorithms to accurately associate the detected cell across time frames. The cell association challenge is mostly related to the amorphous nature of cells, and their complicated motion profile through their migratory paths. To tackle the cell association challenge, we proposed a novel deep-learning-based object linkage method. For this aim, we trained the 3D cell association learning network (3D-CALN) with enough manually labelled paired 3D images of single fluorescent zebrafish's neutrophils from two consecutive frames. Our experiment results prove that deep learning is significantly applicable in cell linkage and particularly for tracking highly mobile and amorphous leukocytes. A comparison of our tracking accuracy with other available tracking algorithms shows that our approach performs well in relation to addressing cell tracking problems.


Assuntos
Aprendizagem por Associação , Peixe-Zebra , Algoritmos , Animais , Humanos , Leucócitos , Imagem com Lapso de Tempo
13.
Nat Commun ; 12(1): 4883, 2021 08 12.
Artigo em Inglês | MEDLINE | ID: mdl-34385449

RESUMO

Pure organic room-temperature phosphorescent (RTP) materials have been suggested to be promising bioimaging materials due to their good biocompatibility and long emission lifetime. Herein, we report a class of RTP materials. These materials are developed through the simple introduction of an aromatic carbonyl to a tetraphenylpyrrole molecule and also exhibit aggregation-induced emission (AIE) properties. These molecules show non-emission in solution and purely phosphorescent emission in the aggregated state, which are desirable properties for biological imaging. Highly crystalline nanoparticles can be easily fabricated with a long emission lifetime (20 µs), which eliminate background fluorescence interference from cells and tissues. The prepared nanoparticles demonstrate two-photon absorption characteristics and can be excited by near infrared (NIR) light, making them promising materials for deep-tissue optical imaging. This integrated aggregation-induced phosphorescence (AIP) strategy diversifies the existing pool of bioimaging agents to inspire the development of bioprobes in the future.


Assuntos
Corantes Fluorescentes/química , Luminescência , Microscopia de Fluorescência por Excitação Multifotônica/métodos , Nanopartículas/química , Pirróis/química , Imagem com Lapso de Tempo/métodos , Animais , Células HeLa , Humanos , Camundongos Endogâmicos BALB C , Camundongos Nus , Microscopia Confocal/métodos , Microscopia Eletrônica de Transmissão/métodos , Nanopartículas/ultraestrutura , Tamanho da Partícula
14.
Sci Rep ; 11(1): 16959, 2021 08 20.
Artigo em Inglês | MEDLINE | ID: mdl-34417510

RESUMO

Babesia parasite invades exclusively red blood cell (RBC) in mammalian host and induces alterations to host cell for survival. Despite the importance of Babesia in livestock industry and emerging cases in humans, their basic biology is hampered by lack of suitable biological tools. In this study, we aimed to develop a synchronization method for Babesia bovis which causes the most pathogenic form of bovine babesiosis. Initially, we used compound 2 (C2), a specific inhibitor of cyclic GMP-dependent protein kinase (PKG), and a derivative of C2, ML10. While both inhibitors were able to prevent B. bovis egress from RBC and increased percentage of binary forms, removal of inhibitors from culture did not result in a synchronized egress of parasites. Because using PKG inhibitors alone was not efficient to induce a synchronized culture, we isolated viable and invasive B. bovis merozoites and showed dynamics of merozoite invasion and development in RBCs. Using isolated merozoites we showed that BbVEAP, VESA1-export associated protein, is essential for parasite development in the RBC while has no significant role in invasion. Given the importance of invasion for the establishment of infection, this study paves the way for finding novel antigens to be used in control strategies against bovine babesiosis.


Assuntos
Babesia bovis/fisiologia , Merozoítos/fisiologia , Parasitos/fisiologia , Animais , Babesia bovis/efeitos dos fármacos , Proteínas Quinases Dependentes de GMP Cíclico/antagonistas & inibidores , Proteínas Quinases Dependentes de GMP Cíclico/metabolismo , Eritrócitos/efeitos dos fármacos , Eritrócitos/parasitologia , Cinética , Merozoítos/efeitos dos fármacos , Parasitos/efeitos dos fármacos , Inibidores de Proteínas Quinases/farmacologia , Imagem com Lapso de Tempo
15.
Cells ; 10(8)2021 08 06.
Artigo em Inglês | MEDLINE | ID: mdl-34440781

RESUMO

Nuclear movements during meiotic prophase, driven by cytoskeleton forces, are a broadly conserved mechanism in opisthokonts and plants to promote pairing between homologous chromosomes. These forces are transmitted to the chromosomes by specific associations between telomeres and the nuclear envelope during meiotic prophase. Defective chromosome movements (CMs) harm pairing and recombination dynamics between homologues, thereby affecting faithful gametogenesis. For this reason, modelling the behaviour of CMs and their possible microvariations as a result of mutations or physico-chemical stress is important to understand this crucial stage of meiosis. Current developments in high-throughput imaging and image processing are yielding large CM datasets that are suitable for data mining approaches. To facilitate adoption of data mining pipelines, we present ChroMo, an interactive, unsupervised cloud application specifically designed for exploring CM datasets from live imaging. ChroMo contains a wide selection of algorithms and visualizations for time-series segmentation, motif discovery, and assessment of causality networks. Using ChroMo to analyse meiotic CMs in fission yeast, we found previously undiscovered features of CMs and causality relationships between chromosome morphology and trajectory. ChroMo will be a useful tool for understanding the behaviour of meiotic CMs in yeast and other model organisms.


Assuntos
Algoritmos , Segregação de Cromossomos , Cromossomos Fúngicos , Interpretação de Imagem Assistida por Computador , Meiose , Microscopia de Fluorescência , Schizosaccharomyces/crescimento & desenvolvimento , Imagem com Lapso de Tempo , Automação Laboratorial , Computação em Nuvem , Ensaios de Triagem em Larga Escala , Schizosaccharomyces/genética , Fatores de Tempo
16.
IEEE Trans Nanobioscience ; 20(4): 497-506, 2021 10.
Artigo em Inglês | MEDLINE | ID: mdl-34398761

RESUMO

Studying the dynamics of nanostructures in the intracellular space is important because it allows gaining insights into the mechanism of complex biological functions of organelles. Understanding such dynamical processes can contribute to the development of nanomedicine for the diagnosis and treatment of many diseases caused by the interaction of multiple genes and environmental factors. Here a quantitative measure of spatial-temporal dynamics of nanostructures within a cell line in the context of nonlinear dynamics is introduced, where early endosomes, late endosomes, and lysosomes recorded by time-lapse confocal imaging are examined. The mathematical derivation of the proposed technique is based on the concept of recurrence dynamics and sequential rate of change over time. The quantification introduced as fuzzy recurrence exponents can be generalized for characterizing the dynamics of experimental evolutions in other nanostructures of living cells captured under the optical microscope.


Assuntos
Endossomos , Nanoestruturas , Lisossomos , Dinâmica não Linear , Imagem com Lapso de Tempo
17.
Cells ; 10(7)2021 07 08.
Artigo em Inglês | MEDLINE | ID: mdl-34359896

RESUMO

BACKGROUND: Glioblastoma multiforme (GBM) is characterized by several genetic abnormalities, leading to cell cycle deregulation and abnormal mitosis caused by a defective checkpoint. We previously demonstrated that arecaidine propargyl ester (APE), an orthosteric agonist of M2 muscarinic acetylcholine receptors (mAChRs), arrests the cell cycle of glioblastoma (GB) cells, reducing their survival. The aim of this work was to better characterize the molecular mechanisms responsible for this cell cycle arrest. METHODS: The arrest of cell proliferation was evaluated by flow cytometry analysis. Using immunocytochemistry and time-lapse analysis, the percentage of abnormal mitosis and aberrant mitotic spindles were assessed in both cell lines. Western blot analysis was used to evaluate the modulation of Sirtuin2 and acetylated tubulin-factors involved in the control of cell cycle progression. RESULTS: APE treatment caused arrest in the M phase, as indicated by the increase in p-HH3 (ser10)-positive cells. By immunocytochemistry, we found a significant increase in abnormal mitoses and multipolar mitotic spindle formation after APE treatment. Time-lapse analysis confirmed that the APE-treated GB cells were unable to correctly complete the mitosis. The modulated expression of SIRT2 and acetylated tubulin in APE-treated cells provides new insights into the mechanisms of altered mitotic progression in both GB cell lines. CONCLUSIONS: Our data show that the M2 agonist increases aberrant mitosis in GB cell lines. These results strengthen the idea of considering M2 acetylcholine receptors a novel promising therapeutic target for the glioblastoma treatment.


Assuntos
Glioblastoma/metabolismo , Glioblastoma/patologia , Mitose , Receptor Muscarínico M2/metabolismo , Fuso Acromático/metabolismo , Acetilação/efeitos dos fármacos , Arecolina/análogos & derivados , Arecolina/farmacologia , Linhagem Celular Tumoral , Núcleo Celular/efeitos dos fármacos , Núcleo Celular/metabolismo , Humanos , Metáfase/efeitos dos fármacos , Sirtuína 2/metabolismo , Imagem com Lapso de Tempo , Tubulina (Proteína)/metabolismo
18.
Radiat Res ; 196(3): 261-271, 2021 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-34237141

RESUMO

To investigate the repairability of X-ray induced DNA damage, particularly non-double-strand breaks in living cells, enhanced green fluorescent protein (EGFP)-expressing plasmids X-ray irradiated and then transfected into nonirradiated human cells, MCF7 and MCF10A. Live-cell imaging of EGFP fluorescence was performed to measure the efficiency of plasmid repair in cells. The number of EGFP-expressing cells significantly decreased with increasing X-ray dose for both cell lines. The obtained kinetic curves of EGFP expression indicating plasmid repair were quantitatively compared against algebraically calculated ones based on the values of the transfected plasmids that had been treated with nicking or restriction enzymes. Then, assuming a Poisson distribution of single-strand breaks (SSBs), the number of cells carrying these nicked plasmids that could express EGFP were estimated. Our experimental results revealed considerably fewer cells expressing EGFP compared to the expected values we had calculated. These results suggest that the lower proportion of cells expressing EGFP as a measure of plasmid repair was due not only to the complex chemical structures of termini created by SSBs compared to those created by enzyme treatments, but also that base lesions or AP sites proximately arising at the strand-break termini might compromise EGFP expression. These results emphasize that radiation-induced DNA breaks are less repairable than enzymatically induced DNA breaks, which is not apparent when using conventional gel electrophoresis assays of plasmid DNA.


Assuntos
Genes Reporter/efeitos da radiação , Proteínas de Fluorescência Verde/genética , Plasmídeos/efeitos da radiação , Linhagem Celular , Dano ao DNA , Reparo do DNA , DNA Recombinante/efeitos da radiação , Células Epiteliais/efeitos da radiação , Genes BRCA1 , Proteínas de Fluorescência Verde/biossíntese , Humanos , Microscopia Intravital , Células MCF-7 , Microscopia de Fluorescência , Conformação de Ácido Nucleico/efeitos da radiação , Plasmídeos/genética , Imagem com Lapso de Tempo , Transfecção
19.
Curr Protoc ; 1(7): e194, 2021 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-34242490

RESUMO

Candida albicans biofilm formation in the presence of drugs can be examined through time-lapse microscopy. In many cases, the images are used qualitatively, which limits their utility for hypothesis testing. We employed a machine-learning algorithm implemented in the Orbit Image Analysis program to detect the percent area covered by cells from each image. This is combined with custom R scripts to determine the growth rate, growth asymptote, and time to reach the asymptote as quantitative proxies for biofilm formation. We describe step-by-step protocols that go from sample preparation for time-lapse microscopy through image analysis parameterization and visualization of the model fit. © 2021 Wiley Periodicals LLC. Basic Protocol 1: Sample preparation Basic Protocol 2: Time-lapse microscopy: Evos protocol Basic Protocol 3: Batch file renaming Basic Protocol 4: Machine learning analysis of Evos images with Orbit Basic Protocol 5: Parametrization of Orbit output in R Basic Protocol 6: Visualization of logistic fits in R.


Assuntos
Candida albicans , Microscopia , Biofilmes , Processamento de Imagem Assistida por Computador , Imagem com Lapso de Tempo
20.
Methods Mol Biol ; 2328: 253-259, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34251631

RESUMO

Enhancers are one of the main classes of cis-regulatory elements (CREs) in the regulation of plant gene expression. Plant enhancers can be predicted based on genomic signatures associated with open chromatin. However, predicted enhancers need to be validated experimentally. We developed an experimental system for rapid enhancer validation. Predicted enhancer candidates are cloned into a vector containing a minimal 35S promoter and a luciferase reporter gene. The construct is then agroinfiltrated into Nicotiana benthamiana leaves followed by bioluminescence signal detection and analysis. Positive bioluminescence signals indicate the enhancer function of each candidate, and the relative signal strength from different enhancers can be quantitatively measured and compared. In summary, we have developed an efficient and rapid plant enhancer validation assay based on a bioluminescent luciferase reporter and agroinfiltration-based N. benthamiana leaf transient expression. This assay can be used for the initial screening of candidate enhancers that are active in leaf tissue. The system can potentially be used to examine the activity of candidate enhancers under different environmental conditions.


Assuntos
Elementos Facilitadores Genéticos , Genes Reporter , Medições Luminescentes/métodos , Tabaco/metabolismo , Agrobacterium/metabolismo , Regulação da Expressão Gênica de Plantas/genética , Vetores Genéticos , Luciferases/genética , Folhas de Planta/metabolismo , Folhas de Planta/efeitos da radiação , Imagem com Lapso de Tempo , Tabaco/genética , Tabaco/crescimento & desenvolvimento , Transformação Genética
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA
...