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Chromosome segregation errors during cell divisions generate aneuploidies and micronuclei, which can undergo extensive chromosomal rearrangements such as chromothripsis1-5. Selective pressures then shape distinct aneuploidy and rearrangement patterns-for example, in cancer6,7-but it is unknown whether initial biases in segregation errors and micronucleation exist for particular chromosomes. Using single-cell DNA sequencing8 after an error-prone mitosis in untransformed, diploid cell lines and organoids, we show that chromosomes have different segregation error frequencies that result in non-random aneuploidy landscapes. Isolation and sequencing of single micronuclei from these cells showed that mis-segregating chromosomes frequently also preferentially become entrapped in micronuclei. A similar bias was found in naturally occurring micronuclei of two cancer cell lines. We find that segregation error frequencies of individual chromosomes correlate with their location in the interphase nucleus, and show that this is highest for peripheral chromosomes behind spindle poles. Randomization of chromosome positions, Cas9-mediated live tracking and forced repositioning of individual chromosomes showed that a greater distance from the nuclear centre directly increases the propensity to mis-segregate. Accordingly, chromothripsis in cancer genomes9 and aneuploidies in early development10 occur more frequently for larger chromosomes, which are preferentially located near the nuclear periphery. Our findings reveal a direct link between nuclear chromosome positions, segregation error frequencies and micronucleus content, with implications for our understanding of tumour genome evolution and the origins of specific aneuploidies during development.
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Aneuploidia , Posicionamento Cromossômico , Segregação de Cromossomos , Cromossomos , Proteína 9 Associada à CRISPR , Linhagem Celular , Linhagem Celular Tumoral , Segregação de Cromossomos/genética , Cromossomos/genética , Cromossomos/metabolismo , Cromotripsia , Crescimento e Desenvolvimento/genética , Humanos , Interfase , Micronúcleos com Defeito Cromossômico , Mitose , Neoplasias/genética , Neoplasias/patologia , Organoides/citologia , Organoides/metabolismo , Análise de Sequência de DNA , Análise de Célula ÚnicaRESUMO
Various cancer types exhibit characteristic and recurrent aneuploidy patterns. The origins of these cancer type-specific karyotypes are still unknown, partly because introducing or eliminating specific chromosomes in human cells still poses a challenge. Here, we describe a novel strategy to induce mis-segregation of specific chromosomes in different human cell types. We employed Tet repressor or nuclease-dead Cas9 to link a microtubule minus-end-directed kinesin (Kinesin14VIb) from Physcomitrella patens to integrated Tet operon repeats and chromosome-specific endogenous repeats, respectively. By live- and fixed-cell imaging, we observed poleward movement of the targeted loci during (pro)metaphase. Kinesin14VIb-mediated pulling forces on the targeted chromosome were counteracted by forces from kinetochore-attached microtubules. This tug-of-war resulted in chromosome-specific segregation errors during anaphase and revealed that spindle forces can heavily stretch chromosomal arms. By single-cell whole-genome sequencing, we established that kinesin-induced targeted mis-segregations predominantly result in chromosomal arm aneuploidies after a single cell division. Our kinesin-based strategy opens the possibility to investigate the immediate cellular responses to specific aneuploidies in different cell types; an important step toward understanding how tissue-specific aneuploidy patterns evolve.
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Cinesinas , Fuso Acromático , Humanos , Cinesinas/genética , Cinesinas/metabolismo , Fuso Acromático/genética , Fuso Acromático/metabolismo , Cinetocoros/metabolismo , Microtúbulos/metabolismo , Segregação de Cromossomos , Anáfase , AneuploidiaRESUMO
An abnormal chromosome number, or aneuploidy, underlies developmental disorders and is a common feature of cancer, with different cancer types exhibiting distinct patterns of chromosomal gains and losses. To understand how specific aneuploidies emerge in certain tissues and how they contribute to disease development, various methods have been developed to alter the karyotype of mammalian cells and mice. In this review, we provide an overview of both classic and novel strategies for inducing or selecting specific chromosomal gains and losses in human and murine cell systems. We highlight how these customized aneuploidy models helped expanding our knowledge of the consequences of specific aneuploidies to (cancer) cell physiology.
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Aneuploidia , Aberrações Cromossômicas , Humanos , Animais , Camundongos , Cariotipagem , Cariótipo , MamíferosRESUMO
The continued spread of COVID-19 poses significant threats to the safety of the community. Since it is still uncertain when the pandemic will end, it is vital to understand the factors contributing to new cases of COVID-19, especially from the transportation perspective. This paper examines the effect of the United States residents' daily trips by distances on the spread of COVID-19 in the community. The artificial neural network method is used to construct and test the predictive model using data collected from two sources: Bureau of Transportation Statistics and the COVID-19 Tracking Project. The dataset uses ten daily travel variables by distances and new tests from March to September 2020, with a sample size of 10,914. The results indicate the importance of daily trips at different distances in predicting the spread of COVID-19. More specifically, trips shorter than 3 mi and trips between 250 and 500 mi contribute most to predicting daily new cases of COVID-19. Additionally, daily new tests and trips between 10 and 25 mi are among the variables with the lowest effects. This study's findings can help governmental authorities evaluate the risk of COVID-19 infection based on residents' daily travel behaviors and form necessary strategies to mitigate the risks. The developed neural network can be used to predict the infection rate and construct various scenarios for risk assessment and control.
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OBJECTIVES: To assess inter-modality agreement and accuracy for medial temporal lobe atrophy (MTA) ratings across radiologists with varying clinical experience in a non-demented population. METHODS: Four raters (two junior radiologists and two senior neuroradiologists) rated MTA on CT and MRI scans using Scheltens' MTA scale. Ratings were compared to a consensus rating by two experienced neuroradiologists for estimation of true positive and negative rates (TPR and TNR) and over- and underestimation of MTA. Inter-modality agreement expressed as Cohen's κ (dichotomized data), Cohen's κw, and two-way mixed, single measures, consistency ICC (ordinal data) were determined. Adequate agreement was defined as κ/κw ≥ 0.80 and ICC ≥ 0.80 (significance level at 95% CI ≥ 0.65). RESULTS: Forty-nine subjects (median age 72 years, 27% abnormal MTA) with cognitive impairment were included. Only junior radiologists achieved adequate agreement expressed as Cohen's κ. All raters achieved adequate agreement expressed as Cohen's κw and ICC. True positive rates varied from 69 to 100% and TNR varied from 85 to 100%. No under- or overestimation of MTA was observed. Ratings did not differ between radiologists. CONCLUSION: We conclude that radiologists with varying experience achieve adequate inter-modality agreement and similar accuracy when Scheltens' MTA scale is used to rate MTA on a non-demented population. However, TPR varied between radiologists which could be attributed to rating style differences. KEY POINTS: ⢠Radiologists with varying experience achieve adequate inter-modality agreement with similar accuracy when Scheltens' MTA scale is used to rate MTA on a non-demented population. ⢠Differences in rating styles might affect accuracy, this was most evident for senior neuroradiologists, and only junior radiologists achieved adequate agreement on dichotomized (abnormal/normal) ratings. ⢠The use of an MTA scale template might compensate for varying clinical experience which could make it applicable for clinical use.
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Doença de Alzheimer , Disfunção Cognitiva , Idoso , Doença de Alzheimer/patologia , Atrofia/patologia , Disfunção Cognitiva/patologia , Humanos , Imageamento por Ressonância Magnética , Radiologistas , Lobo Temporal/diagnóstico por imagem , Lobo Temporal/patologiaRESUMO
BACKGROUND: Carotid atherosclerotic plaques with intraplaque hemorrhage (IPH) are associated with elevated stroke risk. IPH is predominantly imaged based on paramagnetic properties of the upstream hemoglobin degradation product methemoglobin. This is an explorative observational study to test the feasibility of a spoiled gradient echo based T2* weighted MRI sequence (3D MEDIC) for carotid plaque imaging, and to compare signs suggestive of the downstream degradation product hemosiderin on 3D MEDIC with signs of methemoglobin on a T1wBB sequence. METHODS: Patients with recent TIA or stroke were selected based on the presence on non-calcified plaque components on CTA to promote an enriched prevalence of IPH in the material. Patients (n = 42) underwent 3T MRI with 3D MEDIC and 2D turbo spin echo T1w black blood (T1wBB). Images were independently evaluated by two neuroradiologists and Cohens Kappa was used for inter-reader agreement for each sequence. RESULTS: The technical feasibility for 3D MEDIC, was 34/42 patients (81%). Non-calcified plaque components with susceptibility effect without simultaneous T1-shortening-a combination suggestive of hemosiderin, was seen in 13/34 of the plaques. An equally large group display elevated T1w signal in combination with signal loss on 3D MEDIC, a combination suggestive of both hemosiderin and methemoglobin. Cohen's kappa for inter-reader agreement was 0.64 (CI 0.345-0.925) for 3D MEDIC and 0.94 (CI 0.81-1.00) for T1wBB. CONCLUSIONS: 3D MEDIC shows signal loss, without elevated T1w signal on T1wBB, in non-calcified tissue in many plaques in this group of patients. If further studies, including histological verification, confirm that the 3D MEDIC susceptibility effect is indeed caused by hemosiderin, 3D MEDIC could aid in the detection of IPH, beyond elevation of T1w signal.
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Artérias Carótidas/diagnóstico por imagem , Hemorragia/diagnóstico por imagem , Hemossiderina/análise , Imageamento por Ressonância Magnética/métodos , Metemoglobina/análise , Placa Aterosclerótica/diagnóstico por imagem , Adulto , Idoso , Idoso de 80 Anos ou mais , Estudos de Viabilidade , Feminino , Hemorragia/etiologia , Humanos , Ataque Isquêmico Transitório/complicações , Masculino , Pessoa de Meia-Idade , Placa Aterosclerótica/química , Placa Aterosclerótica/complicações , Acidente Vascular Cerebral/complicaçõesRESUMO
PURPOSE: Reports from 3-T vessel wall MRI imaging have shown contrast enhancement following thrombectomy for acute stroke, suggesting potential intimal damage. Comparisons have shown higher SNR and more lesions detected by vessel wall imaging when using 7 T compared with 3 T. The aim of this study was to investigate the vessel walls after stent retriever thrombectomy using high-resolution vessel wall imaging at 7 T. METHODS: Seven patients with acute stroke caused by occlusion of the distal internal carotid artery (T-occlusion), or proximal medial cerebral artery, and treated by stent retriever thrombectomy with complete recanalization were included and examined by 7-T MRI within 2 days. The MRI protocol included a high-resolution black blood sequence with prospective motion correction (iMOCO), acquired before and after contrast injection. Flow measurements were performed in the treated and untreated M1 segments. RESULTS: All subjects completed the MRI examination. Image quality was independently rated as excellent by two neuroradiologists for all cases, and the level of motion artifacts did not impair diagnostic quality, despite severe motion in some cases. Contrast enhancement correlated with the deployment location of the stent retrievers. Flow data showed complete restoration of flow after treatment. CONCLUSION: Vessel wall imaging with prospective motion correction can be performed in patients following thrombectomy with excellent imaging quality at 7 T. We show that vessel wall contrast enhancement is the normal post-operative state and corresponds to the deployment location of the stent retriever.
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Estenose das Carótidas/cirurgia , Angiografia Cerebral/métodos , Infarto da Artéria Cerebral Média/cirurgia , Angiografia por Ressonância Magnética , Imagem de Perfusão/métodos , Acidente Vascular Cerebral/cirurgia , Trombectomia , Doença Aguda , Idoso , Idoso de 80 Anos ou mais , Artéria Carótida Interna/diagnóstico por imagem , Artéria Carótida Interna/cirurgia , Estenose das Carótidas/diagnóstico por imagem , Feminino , Humanos , Aumento da Imagem , Imageamento Tridimensional/métodos , Infarto da Artéria Cerebral Média/diagnóstico por imagem , Arteriosclerose Intracraniana/diagnóstico por imagem , Arteriosclerose Intracraniana/cirurgia , Masculino , Pessoa de Meia-Idade , Complicações Pós-Operatórias/diagnóstico por imagem , Acidente Vascular Cerebral/diagnóstico por imagemRESUMO
INTRODUCTION: The mandibular foramen (MnF) and the mental foramen (MF) are essential anatomical landmarks that should be considered before any surgical procedures in the mandible. This study aimed to investigate the characteristics of the MnF and MF in relation to adjacent anatomical structures, as well as age and gender differences, using cone beam computed tomography (CBCT) projections. METHODS: The study was conducted from August 2023 to January 2024 at the Can Tho University of Medicine and Pharmacy Hospital, Vietnam. In this retrospective study, 50 CBCT images of Vietnamese patients were randomly taken for various clinical purposes. Furthermore, relevant data, such as gender and age groups, were selected to evaluate the correlations, along with specific inclusion criteria. Patients within the age range of 18-69 with a symmetrical mandible were included. RESULTS: The distance of the MnF-MN was 29.6±5.0 mm (right) and 30.1±4.6 mm (left) in males and 25.0±4.2 mm (right) and 26.3±5.0 mm (left) in females. The distance of the MnF-posterior border of the ramus (P) was 16.2±3.6 mm (right) and 15.0±2.3 mm (left) in males. For females, it was 17.1±2.9 mm (right) and 13.8±1.7 mm (left). The distance of the MF-body mandible (MB) was 15.4±2.4 mm (right) and 15.6±2.0 mm (left) in males and 14.0±2.1 mm (right) and 14.3±1.6 mm (left) in females. The distance of the MF-mandibular midline (MM) was 27.0±2.6 mm (right) and 27.0±2.9 mm (left) in males and 25.3±2.0 mm (right) and 25.1±2.2 mm (left) in females. These distances showed statistically significant differences depending on gender (P<0.05). CONCLUSION: It can be said that CBCT provides comprehensive information about the MnF and the MF for dentists in research and clinical practice.
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Neurorradiografia , Radiologia Intervencionista , Congressos como Assunto , Humanos , SuéciaRESUMO
Introduction: Breast cancer causes the most cancer-related death in women and is the costliest cancer in the US regarding medical service and prescription drug expenses. Breast cancer screening is recommended by health authorities in the US, but current screening efforts are often compromised by high false positive rates. Liquid biopsy based on circulating tumor DNA (ctDNA) has emerged as a potential approach to screen for cancer. However, the detection of breast cancer, particularly in early stages, is challenging due to the low amount of ctDNA and heterogeneity of molecular subtypes. Methods: Here, we employed a multimodal approach, namely Screen for the Presence of Tumor by DNA Methylation and Size (SPOT-MAS), to simultaneously analyze multiple signatures of cell free DNA (cfDNA) in plasma samples of 239 nonmetastatic breast cancer patients and 278 healthy subjects. Results: We identified distinct profiles of genome-wide methylation changes (GWM), copy number alterations (CNA), and 4-nucleotide oligomer (4-mer) end motifs (EM) in cfDNA of breast cancer patients. We further used all three signatures to construct a multi-featured machine learning model and showed that the combination model outperformed base models built from individual features, achieving an AUC of 0.91 (95% CI: 0.87-0.95), a sensitivity of 65% at 96% specificity. Discussion: Our findings showed that a multimodal liquid biopsy assay based on analysis of cfDNA methylation, CNA and EM could enhance the accuracy for the detection of early- stage breast cancer.
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Despite their promise, circulating tumor DNA (ctDNA)-based assays for multi-cancer early detection face challenges in test performance, due mostly to the limited abundance of ctDNA and its inherent variability. To address these challenges, published assays to date demanded a very high-depth sequencing, resulting in an elevated price of test. Herein, we developed a multimodal assay called SPOT-MAS (screening for the presence of tumor by methylation and size) to simultaneously profile methylomics, fragmentomics, copy number, and end motifs in a single workflow using targeted and shallow genome-wide sequencing (~0.55×) of cell-free DNA. We applied SPOT-MAS to 738 non-metastatic patients with breast, colorectal, gastric, lung, and liver cancer, and 1550 healthy controls. We then employed machine learning to extract multiple cancer and tissue-specific signatures for detecting and locating cancer. SPOT-MAS successfully detected the five cancer types with a sensitivity of 72.4% at 97.0% specificity. The sensitivities for detecting early-stage cancers were 73.9% and 62.3% for stages I and II, respectively, increasing to 88.3% for non-metastatic stage IIIA. For tumor-of-origin, our assay achieved an accuracy of 0.7. Our study demonstrates comparable performance to other ctDNA-based assays while requiring significantly lower sequencing depth, making it economically feasible for population-wide screening.
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DNA Tumoral Circulante , Detecção Precoce de Câncer , Neoplasias , Humanos , Biomarcadores Tumorais/sangue , Biomarcadores Tumorais/genética , Ácidos Nucleicos Livres/sangue , Ácidos Nucleicos Livres/genética , DNA Tumoral Circulante/sangue , DNA Tumoral Circulante/genética , DNA de Neoplasias/sangue , DNA de Neoplasias/genética , Detecção Precoce de Câncer/métodos , Neoplasias Hepáticas , Neoplasias/sangue , Neoplasias/diagnóstico , Neoplasias/genéticaRESUMO
Histology is a long standing and well-established gold standard for pathological characterizations. In recent years however, synchrotron radiation-based micro-computed tomography (SRµCT) has become a tool for extending the imaging of two-dimensional thin sections into three-dimensional imaging of tissue blocks, enabling so-called virtual histology with arbitrary clipping planes, volumetric rendering and automatic segmentation. In this study, we present a thorough characterization of human carotid plaques after endarterectomy of patients with stroke or transient ischemic attack (TIA), investigating several different pathologic structures using both SRµCT and histology. Phase-contrast SRµCT was performed with two different magnifications (voxel sizes 6.5 µm and 0.65 µm, respectively), and histology was performed with multiple different stainings (Alpha-actin, Glycophorin A, von Kossa, Movat, CD68). The 0.65 µm high-resolution SRµCT was performed on selected areas with plaque typical relevant morphology, identified on the 6.5 µm low-resolution SRµCT. The tomography datasets were reconstructed with additional 3D volume rendering and compared to histology. In total, nine different regions with typical pathologic structures were identified and imaged with high-resolution SRµCT. The results show many characteristics typical for advanced atherosclerotic plaques, clinically relevant, namely ruptures with thrombosis, neo-vascularization, inflammatory infiltrates in shoulder regions, lipid rich necrotic cores (LRNC), thin fibrous cap, calcifications, lumen irregularities, and changes in vessel wall structures such as the internal elastic membrane. This method's non-destructive nature renders details of micro-structures with an excellent visual likeness to histology, with the additional strength of multiplanar and 3D visualization and the possibility of multiple re-scans.
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Ataque Isquêmico Transitório , Placa Aterosclerótica , Artérias Carótidas/diagnóstico por imagem , Artérias Carótidas/patologia , Humanos , Ataque Isquêmico Transitório/patologia , Placa Aterosclerótica/diagnóstico por imagem , Placa Aterosclerótica/patologia , Síncrotrons , Microtomografia por Raio-X/métodosRESUMO
Understanding the future development of COVID-19 is the key to contain the spreading of the coronavirus. The purpose of this paper is to explore a potential relationship between United States residents' daily trips by distance and the COVID-19 infections in the near future. The study used the daily travel data from the Bureau of Transportation Statistics (BTS) and the COVID-19 data from the Centers for Disease Control and Prevention (CDC) in the United States. Time-series forecast models using Autoregressive Moving Average (ARIMA) method were constructed to project future trends of United States residents' daily trips by distance at the national level from November 30, 2020, to February 28, 2021. A comparative trend analysis was conducted to detect the patterns of daily trips and the spread of COVID-19 during that period. The results revealed a closed loop scenario, in which the residents' travel behavior dynamically changes based on their risk perception of COVID-19 in an infinite loop. A detected lag in the travel behavior between short trips and long trips further worsens the situation and creates more difficulties in finding an effective solution to break the loop. The study shed new light on efforts to contain and control the spread of the coronavirus. The loop can only be broken with proper and prompt mitigation strategies to reduce the burden on hospitals and healthcare systems and save more lives.
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BACKGROUND AND AIMS: Histopathology is the gold standard for analysis of atherosclerotic plaques but has drawbacks due to the destructive nature of the method. Ex vivo MRI is a non-destructive method to image whole plaques. Our aim was to use quantitative high field ex vivo MRI to classify plaque components, with histology as gold standard. METHODS: Surgically resected carotid plaques from 12 patients with recent TIA or stroke were imaged at 11.7 T MRI. Quantitative T1/T2* mapping sequences and qualitative T1/T2* gradient echo sequences with voxel size of 30 × 30 × 60 µm3 were obtained prior to histological preparation, sectioning and staining for lipids, inflammation, hemorrhage, and fibrous tissue. Regions of interest (ROI) were selected based on the histological staining at multiple levels matched between histology and MRI. The MRI parameters of each ROI were then analyzed with quadratic discriminant analysis (QDA) for classification. RESULTS: A total of 965 ROIs, at 70 levels matched between histology and MRI, were registered based on histological staining. In the nine plaques where three or more plaque components were possible to co-localize with MRI, the mean degree of misclassification by QDA was 16.5 %. One of the plaques contained mostly fibrous tissue and lipids and had no misclassifications, and two plaques mostly contained fibrous tissue. QDA generally showed good classification for fibrous tissue and lipids, whereas plaques with hemorrhage and inflammation had more misclassifications. CONCLUSION: 11.7 T ex vivo high field MRI shows good visual agreement with histology in carotid plaques. T1/T2* maps analyzed with QDA is a promising non-destructive method to classify plaque components, but with a higher degree of misclassifications in plaques with hemorrhage or inflammation.
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Aurora B kinase is essential for faithful chromosome segregation during mitosis. During (pro)metaphase, Aurora B is concentrated at the inner centromere by the kinases Haspin and Bub1. However, how Haspin and Bub1 collaborate to control Aurora B activity at centromeres remains unclear. Here, we show that either Haspin or Bub1 activity is sufficient to recruit Aurora B to a distinct chromosomal locus. Moreover, we identified a small, Bub1 kinase-dependent Aurora B pool that supported faithful chromosome segregation in otherwise unchallenged cells. Joined inhibition of Haspin and Bub1 activities fully abolished Aurora B accumulation at centromeres. While this impaired the correction of erroneous KT-MT attachments, it did not compromise the mitotic checkpoint, nor the phosphorylation of the Aurora B kinetochore substrates Hec1, Dsn1, and Knl1. This suggests that Aurora B substrates at the kinetochore are not phosphorylated by centromere-localized pools of Aurora B, and calls for a reevaluation of the current spatial models for how tension affects Aurora B-dependent kinetochore phosphorylation.
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Aurora Quinase B/metabolismo , Segregação de Cromossomos , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Cinetocoros/enzimologia , Microtúbulos/enzimologia , Mitose , Proteínas Serina-Treonina Quinases/metabolismo , Aurora Quinase B/genética , Proteínas Cromossômicas não Histona/genética , Proteínas Cromossômicas não Histona/metabolismo , Proteínas do Citoesqueleto/genética , Proteínas do Citoesqueleto/metabolismo , Células HCT116 , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Cinesinas/genética , Cinesinas/metabolismo , Pontos de Checagem da Fase M do Ciclo Celular , Proteínas Associadas aos Microtúbulos/genética , Proteínas Associadas aos Microtúbulos/metabolismo , Microtúbulos/genética , Fosforilação , Proteínas Serina-Treonina Quinases/genética , Transdução de Sinais , Fatores de TempoRESUMO
The CRISPR/Cas9 system is a highly effective tool for genome editing. Key to robust genome editing is the efficient delivery of the CRISPR/Cas9 machinery. Viral delivery systems are efficient vehicles for the transduction of foreign genes but commonly used viral vectors suffer from a limited capacity in the genetic information they can carry. Baculovirus however is capable of carrying large exogenous DNA fragments. Here we investigate the use of baculoviral vectors as a delivery vehicle for CRISPR/Cas9 based genome-editing tools. We demonstrate transduction of a panel of cell lines with Cas9 and an sgRNA sequence, which results in efficient knockout of all four targeted subunits of the chromosomal passenger complex (CPC). We further show that introduction of a homology directed repair template into the same CRISPR/Cas9 baculovirus facilitates introduction of specific point mutations and endogenous gene tags. Tagging of the CPC recruitment factor Haspin with the fluorescent reporter YFP allowed us to study its native localization as well as recruitment to the cohesin subunit Pds5B.
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Baculoviridae/genética , Sistemas CRISPR-Cas/genética , Edição de Genes/métodos , Linhagem Celular Tumoral , Técnicas de Inativação de Genes , Genoma Humano/genética , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Mutação Puntual , Proteínas Serina-Treonina Quinases/genéticaAssuntos
Proteínas de Drosophila/química , Engenharia de Proteínas , Sequência de Aminoácidos , Animais , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Concentração de Íons de Hidrogênio , Proteínas de Insetos/química , Luz , Dados de Sequência Molecular , Estrutura Secundária de Proteína , Espalhamento de RadiaçãoRESUMO
It was hypothesized that a mycotoxin binder, Grainsure E, would inhibit adverse effects of a mixture of fumonisin B1, deoxynivalenol, and zearalenone in rats. For 14 and 28 days, 8-10 Sprague-Dawley rats were fed control diet, Grainsure E (0.5%), toxins (7 µg fumonisin B1/g, 8 µg of deoxynivalenol/g and 0.2 µg of zearalenone/g), toxins (12 µg of fumonisin B1/g, 9 µg of deoxynivalenol/g, and 0.2 µg of zearalenone/g + Grainsure E), or pair-fed to control for food intake of toxin-fed rats. After 28 days, decreased body weight gain was prevented by Grainsure E in toxin-fed female rats, indicating partial protection against deoxynivalenol and fumonisin B1. Two effects of fumonisin B1 were partly prevented by Grainsure E in toxin-fed rats, increased plasma alanine transaminase (ALT) and urinary sphinganine/sphingosine, but sphinganine/sphingosine increase was not prevented in females at the latter time point. Grainsure E prevented some effects of fumonisin B1 and deoxynivalenol in rats.
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Dieta , Fumonisinas/antagonistas & inibidores , Micotoxinas , Tricotecenos/antagonistas & inibidores , Zearalenona/antagonistas & inibidores , Animais , Doença Hepática Induzida por Substâncias e Drogas/prevenção & controle , Feminino , Fumonisinas/administração & dosagem , Fumonisinas/toxicidade , Nefropatias/induzido quimicamente , Nefropatias/prevenção & controle , Hepatopatias , Masculino , Ratos , Ratos Sprague-Dawley , Tricotecenos/administração & dosagem , Tricotecenos/toxicidade , Zearalenona/administração & dosagem , Zearalenona/toxicidadeRESUMO
The outstanding rubber-like elasticity of resilin and resilin-mimetic proteins depends critically on the level of hydration. In this investigation, water vapor sorption and the role of hydration on the molecular chain dynamics and viscoelastic properties of resilin-mimetic protein, rec1-resilin is investigated in detail. The dynamic and equilibrium swelling behavior of the crosslinked protein hydrogels with different crosslink density are reported under various controlled environments. We propose three different stages of hydration; involving non-crystallizable water, followed by condensation or clustering of water around the already hydrated sites, and finally crystallizable water. The kinetics of water sorption for this engineering protein is observed to be comparable to hydrophilic polymers with a diffusion coefficient in the range of 10(-7) cm(2) s(-1). From the comparison between the absorption and desorption isotherms at a constant water activity, it has been observed that rec1-resilin exhibits sorption hysteresis only for the tightly bound water. Investigation of molecular mobility using differential scanning calorimetry, indicates that dehydrated crosslinked rec1-resilin is brittle with a glass transition temperature (T(g)) of >180 °C, which dramatically decreases with increasing hydration; and above a critical level of hydration rec1-resilin exhibits rubber-like elasticity. Nanoindentation studies show that even with little hydration (<10%), the mechanical properties of rec1-resilin gels change dramatically. Rheological investigations confirm that the equilibrium-swollen crosslinked rec1-resilin hydrogel exhibits outstanding elasticity and resilience of â¼ 92%, which exceeds that of any other synthetic polymer and biopolymer hydrogels.
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Elasticidade , Proteínas de Insetos/química , Viscosidade , Água/química , Sequência de Aminoácidos , Varredura Diferencial de Calorimetria , Cristalização , Cinética , Dados de Sequência Molecular , Homologia de Sequência de Aminoácidos , TermogravimetriaRESUMO
In this investigation, for the first time we report the effects of pH on the molecular orientation, packing density, structural properties, adsorption characteristics and viscoelastic behaviour of resilin-mimetic protein rec1-resilin at the solid-liquid interface using quartz crystal microbalance with dissipation monitoring (QCM-D) and surface plasmon resonance (SPR) spectroscopy. QCM-D and SPR data confirm that the binding ability of rec1-resilin on a substrate is strongly pH-dependent the protein packing density on a gold surface is calculated to be 4.45 x 10(13) per cm(2) at the isoelectric point (IEP approximately 4.9), 8.79 x 10(11) per cm(2) at pH 2 and 9.90 x 10(11) per cm(2) at pH 12, respectively. Our findings based on the thickness, dissipation and viscoelasticity of the rec1-resilin adlayer also indicate that it is adsorbed onto the gold substrate with different orientation depending on pH, such as back-on adsorption at acidic pH of 2, compact end-on bilayer adsorption at the IEP and side-on at high alkaline pH of 12. When rec1-resilin is 'pinned' to the substrate at IEP and subsequently exposed to an electrolyte solution adjusted to different pH, it switches from a compact globular conformation of the bio-macromolecule at the IEP to a coil conformation at pH between IEP to IED (IED = pKa value of tyrosine amino acid residue) and an extended coil conformation at pH > IED. This transformation from globule to coil to extended coil conformation is kinetically fast, robust and completely reversible. Such responsive surfaces created using 'smart' biomimetic rec1-resilin have the potential to find applications in many areas including biotechnology, medicine, sensors, controlled drug delivery systems and engineering.