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Successful goal-directed actions require constant fine-tuning of the motor system. This fine-tuning is thought to rely on an implicit adaptation process that is driven by sensory prediction errors (e.g., where you see your hand after reaching vs. where you expected it to be). Individuals with low vision experience challenges with visuomotor control, but whether low vision disrupts motor adaptation is unknown. To explore this question, we assessed individuals with low vision and matched controls with normal vision on a visuomotor task designed to isolate implicit adaptation. We found that low vision was associated with attenuated implicit adaptation only for small visual errors, but not for large visual errors. This result highlights important constraints underlying how low-fidelity visual information is processed by the sensorimotor system to enable successful implicit adaptation.
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Baixa Visão , Humanos , MãosRESUMO
SIGNIFICANCE: Understanding longitudinal changes in why individuals frequent low-vision clinics is crucial for ensuring that patient care keeps current with changing technology and changing lifestyles. Among other findings, our results suggest that reading remains a prevailing patient complaint, with shifting priorities toward technology-related topics. PURPOSE: This study aimed to understand changes in patient priorities and patient care in low vision over the past decade. METHODS: We conducted a retrospective study of examination records (2009 to 2019, 3470 examinations) from two U.S. low-vision clinics. Automated word searches summarized two properties of the records: topics discussed during the case history and types of rehabilitative devices assessed. Logistic regression was used to model the effects of examination year, patient age, patient sex, and level of visual impairment. RESULTS: Collapsing across all years, the most common topic discussed was reading (78%), followed by light-related topics (71%) and technology (59%). Whereas the odds of discussing reading trended downward over the decade (odds ratio, 0.57; P = .03), technology, social interaction, mobility, and driving trended upward (odds ratios, 4.53, 3.31, 2.71, and 1.95; all P 's < 0.001). The most frequently assessed devices were tinted lenses (95%). Over time, video magnifier and spectacle assessments trended downward (odds ratios, 0.64 and 0.72; P = .004, 0.04), whereas assessments of other optical aids increased. The data indicate several consistent differences among patient demographics. CONCLUSIONS: Reading is likely to remain a prevailing patient complaint, but an increase in technology-related topics suggests shifting priorities, particularly in younger demographics. "Low-tech" optical aids have remained prominent in low-vision care even as "high-tech" assistive devices in the marketplace continue to advance.
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Síndrome da Imunodeficiência Adquirida , Baixa Visão , Humanos , Baixa Visão/epidemiologia , Estudos Retrospectivos , Acuidade Visual , Óculos , LeituraRESUMO
BACKGROUND: While vaccine development is itself a challenge; ensuring optimal vaccine uptake at population level can present an even more significant challenge. Therefore, this study aimed to assess the Pakistani population's attitude and preferences towards the Coronavirus disease 2019 (COVID-19) vaccine. METHOD: A cross-sectional study was carried out through an online self-administered questionnaire from 27 September 2020 to 11 October 2020. A total of 883 people responded to the survey. The questionnaire included the participants' socio-demographic variables, attitudes, beliefs towards the COVID-19 vaccine and acceptance and rejection of vaccination, and reasons for them. Logistic regression analysis was used to analyze the predictors for vaccine acceptance and willingness to pay for the vaccine. RESULTS: A majority (70.8%) of respondents will accept the COVID-19vaccine if available, and 66.8% showed a positive attitude towards vaccination. Monthly family income, education level, self-diagnosis of COVID-19 or a friend, family member, or colleague are significant factors influencing the acceptance of COVID-19 vaccination. The dogma of being naturally immune to COVID-19 was a key reason for the refusal of the vaccine. Less than half (48%) of those who refuse will vaccinate themselves if government officials have made it compulsory. A third (33.9%) of participants were willing to pay up to (7 USD) 1000 Pkr (Pakistani Rupees) for the vaccine. CONCLUSION: The population's positive attitude should be improved by increasing awareness and eradicating false myths about vaccines through large-scale campaigns.
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Vacinas contra COVID-19 , COVID-19 , Atitude , Estudos Transversais , Humanos , Paquistão , SARS-CoV-2 , VacinaçãoRESUMO
Focal adhesions (FAs) are micron-sized protein assemblies that coordinate cell adhesion, migration, and mechanotransduction. How the many proteins within FAs are organized into force sensing and transmitting structures is poorly understood. We combined fluorescent molecular tension sensors with super-resolution light microscopy to visualize traction forces within FAs with <100 nm spatial resolution. We find that αvß3 integrin selectively localizes to high force regions. Paxillin, which is not generally considered to play a direct role in force transmission, shows a higher degree of spatial correlation with force than vinculin, talin, or α-actinin, proteins with hypothesized roles as force transducers. These observations suggest that αvß3 integrin and paxillin may play important roles in mechanotransduction.
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Proteínas do Citoesqueleto/metabolismo , Adesões Focais/fisiologia , Mecanotransdução Celular/fisiologia , Proteínas de Membrana/metabolismo , Microscopia de Fluorescência/métodos , Imagem Molecular/métodos , Adesividade , Animais , Células Cultivadas , Transferência Ressonante de Energia de Fluorescência/métodos , Humanos , Aumento da Imagem/métodos , Camundongos , Estresse Mecânico , Resistência à Tração/fisiologiaRESUMO
Sample homogeneity dictates whether analyzing a test portion of an entire sample can provide representative information about incurred mycotoxins. In this study, we evaluated particle-size-distribution-based homogeneity of laboratory mycotoxin samples using laser diffraction particle size analysis and International Organization for Standardization (ISO) Guide 35: 2017. Incurred whole corn, compound feed, peanut butter, and wheat flour (500 g each) were comminuted using wet, cryogenic, or dry milling. We used a sample dividing (riffling) device to obtain representative subsamples (25 g each) and developed a laser diffraction particle size analysis procedure by optimizing key parameters such as the refractive index, absorption, and stirring rate. The homogeneity of the particle size distribution within laboratory subsamples was characterized using the optimized laser diffraction procedure. An assessment of homogeneity was also performed for individual mycotoxins in each incurred matrix sample following the procedure described in ISO Guide 35. The concentrations of the incurred mycotoxins were determined using liquid chromatography-mass spectrometry (LC-MS). Within- and between-subsample variances of incurred aflatoxin B1 in peanut butter; deoxynivalenol in corn, compound feed, and wheat flour; and fumonisins in compound feed corroborated that when the particle size measurements were less than 850 µm, mycotoxins concentrations were consistent across independent test portions, which was confirmed using an analysis of variance (F-test). This study highlights the benefits of laser diffraction particle size analysis and suggests its use as a test procedure to evaluate homogeneity in new sample commodities.
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Micotoxinas , Micotoxinas/análise , Tamanho da Partícula , Farinha/análise , Contaminação de Alimentos/análise , Triticum , Zea maysRESUMO
Objectives Preoperative fasting plays a pivotal role in adequately preparing patients for anaesthesia and surgical procedures. However, it is imperative to consider not only the medical aspects but also patients' overall comfort, as this can significantly contribute to improved surgical outcome. The primary objective of this quality improvement project (QIP) is to provide healthcare professionals, including anaesthetists, surgeons, nurses, and stakeholders with information regarding insights required to embrace the concept of preoperative snack prescription as a strategy for enhancing patient-centred care. Methods This QIP was conducted in the vascular surgery department of a district general hospital in Wales, United Kingdom. A prospective analysis was conducted in two cycles, i.e., the pre-intervention group (PrIG) and post-intervention group (PoIG), with preoperative snacks such as biscuits, chips, or cakes, being prescribed to the PoIG. A total of 40 patients who met the inclusion criteria were enrolled in this study, with 20 patients participating in each cycle. The timing of preoperative meals, i.e., the closest preoperative breakfast, lunch, or dinner, preoperative snacks (for the PoIG), anaesthesia commencement, and surgical commencement were collected. Data analysis was performed using IBM SPSS Statistics for Windows, Version 26.0 (Released 2019; IBM Corp., Armonk, New York, United States), in conjunction with Microsoft Excel (Microsoft Corporation, Redmond, Washington, United States). Results In our QIP, the PrIG and PoIG comprised 40% (8 out of 20) and 35% (7 out of 20) female patients, respectively, with mean ages of 74 years (range, 61-86 years) and 61.3 years (range, 36-81 years). Within the PrIG, the mean duration from the preoperative meal to anaesthesia and surgery commencement was 17.8 hours (range, 14.6-22.5 hours) and 18.5 hours (range, 16.0-23.3 hours), respectively. In the PoIG, following the initiation of preoperative snack prescription, the mean time intervals between preoperative snack prescription and anaesthesia and surgery commencement were 10.9 hours (range, 6.5-16.0 hours) and 12.0 hours (range, 7.5-16.5 hours), respectively. Conclusions In summary, our QIP has successfully integrated preoperative snack prescription into the local hospital's preoperative care policy, prioritising the balance between patient safety and comfort. Based on our single-centre experience, we observed a significant reduction in the time interval between preoperative fasting and the initiation of anaesthesia, decreasing from 18.3 hours to 10.9 hours post-implementation of preoperative snacks. This QIP holds relevance for healthcare professionals as it underscores the benefits of shorter fasting periods, which contribute to heightened patient satisfaction and comfort.
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Background Stroke is a prevalent ailment that impacts a substantial number of individuals globally, resulting in both physical impairment and mortality. One of its major causes is carotid artery stenosis. The symptoms and degree of stenosis are key indications for carotid endarterectomy (CEA). In this study, we highlight the indications and outcomes of carotid endarterectomy in our center. Methods This is a descriptive, retrospective, observational study. Data of patients who underwent CEA at Glan Clwyd Hospital from January 2018 to January 2023 was retrieved. The study sample consisted of patients diagnosed with symptomatic carotid artery stenosis who had CEA at Glan Clwyd Hospital. The data was analyzed using statistical software SPSS (IBM Corp. Released 2012. IBM SPSS Statistics for Windows, Version 21.0. Armonk, NY: IBM Corp). Results A total of 150 patients were enrolled in the study. A majority of the patients were male, accounting for 69.3% (n = 104), and had a mean age of 71.1 ± 9.9 standard deviation. A majority of the patients were smokers (48.7%) and had additional medical conditions, including hypertension (34%), ischemic heart disease (17.3%), chronic obstructive pulmonary disease (73.3%), and diabetes (46.7%). Nevertheless, the remaining comorbidities were less common. The outcome of the CEA among the patients was cardiac event 3.3% (n = 5); transient ischemic attack (TIA) 3.3% (n = 5); stroke 0.6% (n = 1); hemorrhage 2.6% (n = 4); surgical site infection 2% (n = 3); perioperative mortality 1.3% (n = 2); and cranial nerve injury 1.3% (n = 2). However, no complications were reported in most of the patients, 85.6% (n = 128). Conclusion An endarterectomy is quite advantageous for treating symptomatic stenosis. The findings can be applied to patients who are physically suitable for surgery. The efficacy of endarterectomy is contingent upon not only the severity of carotid stenosis but also various other parameters, such as the time elapsed between the presenting event and the surgical intervention, as well as the patient's overall medical condition. However, the CEA is the gold standard in surgical management for symptomatic carotid disease.
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Background Abdominal aortic aneurysm (AAA) is a dangerous disorder characterised by abnormal enlargement of the abdominal aorta. The severity of the aneurysm and the presence of symptoms determine the necessary monitoring or treatment to prevent potential fatalities. The objective of this study is to estimate the perioperative mortality and long-term outcome of endovascular abdominal aneurysm repair (EVAR). Patients and methods This is a descriptive, retrospective, observational study. We retrieved the data of the AAA patients who underwent EVAR at Glan Clwyd Hospital from January 2015 to January 2023. The study sample consisted of patients diagnosed with isolated AAA, with or without iliac branch involvement, who were deemed suitable for EVAR based on factors such as advanced age, presence of comorbidities, the complexity of the condition, history of prior surgery, fulfillment of indication criteria, and patient desire. The data was analysed using SPSS statistical software, version 21.0 (IBM Corp., Armonk, NY). Results Two hundred and twenty-two patients were studied. The outcome of the EVAR among the patients was endo-leak 28.4% (n = 63); migration 1.4% (n = 3); blockage 0.5% (n = 1); infolding 0.5% (n = 1); perioperative mortality 1.4% (3); and other complications like access site or acute kidney injury were 1.4% (n = 3). However, no complications were reported in most of the patients, 66.7% (n = 148). Upon evaluating the variables that could affect the outcome, we observed that the ASA grade, comorbidities, and the indication of the intervention had a significant effect on the outcome (P values = 0.000, 0.048, and 0.014, respectively). Conclusion The findings demonstrate that when EVAR is performed by a skilled team adhering to proper criteria, the results are optimal. The mortality rate during the perioperative period was 1.4%. Furthermore, we have shown a satisfactory rate of complications when compared to international data.
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To monitor co-exposure to toxic mycotoxins in dried fruits, it is advantageous to simultaneously determine multiple mycotoxins using a single extraction and liquid chromatography with tandem mass spectrometry (LC-MS/MS) analysis. In this study, we applied a stable isotope dilution and LC-MS/MS method to multi-mycotoxin analysis in dried fruits, selecting raisins, plums, figs, and cranberries for matrix extension. Samples were prepared using cryogenic grinding, followed by the fortification of carbon-13 (13C) uniformly labeled internal standards for twelve mycotoxins, and extraction using 50% acetonitrile. Homogeneity of prepared samples, defined as particle size Dv90 < 850 µm for the tested matrices, was characterized using a laser diffraction particle size analyzer, and reached using cryogenic grinding procedures. The majority of recoveries in the four matrices for aflatoxins and ochratoxin A spiked at 1−100 ng/g; fumonisins, T-2 toxin, HT-2 toxin, and zearalenone spiked at 10−1000 ng/g, ranged from 80 to 120% with relative standard deviations (RSDs) of <20%. Deoxynivalenol was not detected at 10 and 100 ng/g in plums, and additional troubleshooting procedures using liquid-liquid extraction (LLE), solid phase extraction (SPE), and elution gradient were evaluated to improve the detectability of the mycotoxin. Furthermore, we confirmed the identity of detected mycotoxins, ochratoxin A and deoxynivalenol, in incurred samples using enhanced product ion scans and spectral library matching.
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Coabalamin-dependent O-demethylase in Blautia sp. strain MRG-PMF1 was found to catalyze the unprecedented allyl aryl ether cleavage reaction. To expand the potential biotechnological applications, the reaction mechanism of the allyl aryl ether C-O bond cleavage, proposed to utilize the reactive Co(I) supernucleophile species, was studied further from the anaerobic whole-cell biotransformation. Various allyl naphthyl ether derivatives were reacted with Blautia sp. MRG-PMF1 O-demethylase, and stereoisomers of allyl naphthyl ethers, including prenyl and but-2-enyl naphthyl ethers, were converted to the corresponding naphthol in a stereoselective manner. The allyl aryl ether cleavage reaction was regioselective, and 2-naphthyl ethers were converted faster than the corresponding 1-naphthyl ethers. However, MRG-PMF1 cocorrinoid O-demethylase was not able to convert (2-methylallyl) naphthyl ether substrates, and the conversion of propargyl naphthyl ether was extremely slow. From the results, it was proposed that the allyl ether cleavage reaction follows the nucleophilic conjugate substitution (SN2') mechanism. The reactivity and mechanism of the new allyl ether cleavage reaction by cobalamin-dependent O-demethylase would facilitate the application of Blautia sp. MRG-PMF1 O-demethylase in the area of green biotechnology. IMPORTANCE Biodegradation of environmental pollutants and valorization of biomaterials in a greener way is of great interest. Cobalamin-dependent O-demethylase in Blautia sp. MRG-PMF1 exclusively involves anaerobic C1 metabolism by cleaving the C-O bond of aromatic methoxy group and also produces various aryl alcohols by metabolizing allyl aryl ether compounds. Whereas methyl ether cleavage reaction is known to follow the SN2' mechanism, the reaction pattern and mechanism of the new allyl ether cleavage reaction by cobalamin-dependent O-demethylase have never been studied. For the first time, stereoselectivity and the SN2' mechanism of allyl aryl ether cleavage reaction by Blautia sp. MRG-PMF1 O-demethylase is reported, and the results would facilitate the application of Blautia sp. MRG-PMF1 O-demethylase in the area of green biotechnology.
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Poluentes Ambientais , Éteres Metílicos , Éter , Oxirredutases O-Desmetilantes , Naftóis , Éteres/química , Éteres/metabolismo , Etil-Éteres , Vitamina B 12 , Materiais BiocompatíveisRESUMO
Enzyme-linked immunosorbent assays (ELISAs) are a cornerstone of modern molecular detection, but the technique still faces notable challenges. One of the biggest problems is discriminating true signal generated by target molecules versus non-specific background. Here, we developed a Single-Molecule Colocalization Assay (SiMCA) that overcomes this problem by employing total internal reflection fluorescence microscopy to quantify target proteins based on the colocalization of fluorescent signal from orthogonally labeled capture and detection antibodies. By specifically counting colocalized signals, we can eliminate the effects of background produced by non-specific binding of detection antibodies. Using TNF-α, we show that SiMCA achieves a three-fold lower limit of detection compared to conventional single-color assays and exhibits consistent performance for assays performed in complex specimens such as serum and blood. Our results help define the pernicious effects of non-specific background in immunoassays and demonstrate the diagnostic gains that can be achieved by eliminating those effects.
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Anticorpos , Proteínas , Ensaio de Imunoadsorção Enzimática/métodos , Imunoensaio/métodos , Sensibilidade e EspecificidadeRESUMO
Pseudomonas syringae pv. tomato, the causal agent of bacterial speck of tomato, has recently become an increasing problem in California due to the evolution and prevalence of highly aggressive race 1 strains. In resistant plant genotypes, the type III effectors AvrPto and AvrPtoB are recognized by the tomato proteins Pto and Prf. We investigated the prevalence of avrPto and avrPtoB in strains collected over the last 13 years in California. All race 1 strains retained avrPtoB but did not express AvrPtoB protein at detectable levels in vitro. However, deletion of avrPtoB indicated that this effector protein is still expressed at low levels in race 1 during infection. avrPto was detected in four race 1 strains but a key amino acid polymorphism prevents this new protein from interacting with and eliciting Pto-mediated resistance. Growth curve analyses demonstrate that this new avrPto allele is still functional and can enhance P. syringae virulence on tomato. Multilocus sequence typing was used to resolve phylogenetic relationships and revealed that the majority of race 0 and 1 strains were most closely related to P. syringae T1. Collectively, these data support the hypothesis that existing P. syringae populations evolved to overcome genetic resistance by altering the expression and sequence of avrPto and avrPtoB effectors.
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Doenças das Plantas/microbiologia , Pseudomonas syringae/genética , Pseudomonas syringae/metabolismo , Sequência de Aminoácidos , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , California , Predisposição Genética para Doença , Filogenia , Doenças das Plantas/genética , Folhas de Planta/microbiologia , Pseudomonas syringae/classificação , Pseudomonas syringae/patogenicidade , VirulênciaRESUMO
Gut metabolism of natural products is of great interest due to the altered biological activity of the metabolites. To study the gut metabolism of the dietary furanocoumarins, the biotransformation of Angelica dahurica was studied with human gut microbiota. The major components of Avenula dahurica, including xanthotoxin (1), bergapten (2), imperatorin (3), isoimperatorin (4), oxypeucedanin (5), and byakangelicol (6), were all metabolized by the human fecal sample, and each furanocoumarin was also biotransformed by Blautia sp. MRG-PMF1 responsible for intestinal O-demethylation. Oxypeucedanin (5) and byakangelicol (6) were converted to oxypeucedanin hydrate (9) and desmethylbyakangelicin (12), respectively. The gut microbial conversion of xanthotoxin (1) and bergapten (2) with the MRG-PMF1 strain resulted in the production of xanthotoxol (7) and bergaptol (8), respectively, due to the methyl aryl ether cleavage by O-methyltransferase. Unexpectedly, the biotransformation of prenylated furanocoumarins, imperatorin (3), and isoimperatorin (4) resulted in the corresponding deprenylated furanocoumarins of xanthotoxol (7) and bergaptol (8), respectively. The cleavage of the prenyl aryl ether group by gut microbiota was unprecedented metabolism. Our data presented the first deprenylation of prenylated natural products, presumably by the anaerobic prenyl aryl ether cleavage reaction catalyzed by Co-corrinoid enzyme.
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Integrin-based adhesion complexes link the cytoskeleton to the extracellular matrix (ECM) and are central to the construction of multicellular animal tissues. How biological function emerges from the tens to thousands of proteins present within a single adhesion complex remains unclear. We used fluorescent molecular tension sensors to visualize force transmission by individual integrins in living cells. These measurements revealed an underlying functional modularity in which integrin class controlled adhesion size and ECM ligand specificity, while the number and type of connections between integrins and F-actin determined the force per individual integrin. In addition, we found that most integrins existed in a state of near-mechanical equilibrium, a result not predicted by existing models of cytoskeletal force transduction. A revised model that includes reversible cross-links within the F-actin network can account for this result and suggests one means by which cellular mechanical homeostasis can arise at the molecular level.
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Photopolymerizable hydrogels derived from naturally occurring polymers have attracted significant interest in tissue-engineering applications due to their excellent biocompatibility, hydrophilic nature favourable for cell ingrowth and ability to be cured in situ through a minimally invasive procedure. In this study, we developed a composite hydrogel consisting of photocrosslinkable methacrylated glycol chitosan (MeGC) and semi-interpenetrating collagen (Col) with a riboflavin photoinitiator under blue light. The incorporation of Col in MeGC hydrogels enhanced the compressive modulus and slowed the degradation rate of the hydrogels. MeGC-Col composite hydrogels significantly enhanced cellular attachment, spreading, proliferation and osteogenic differentiation of mouse bone marrow stromal cells (BMSCs) seeded on the hydrogels compared with pure MeGC hydrogels, as observed by upregulated alkaline phosphatase (ALP) activity as well as increased mineralization. Similarly, when cells were encapsulated within hydrogels, BMSCs exhibited greater proliferation, ALP activity and mineral deposits in the presence of Col. These findings demonstrate that MeGC-Col composite hydrogels may be useful in promoting bone regeneration. Copyright © 2014 John Wiley & Sons, Ltd.
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Osso e Ossos/química , Quitosana/química , Colágeno/química , Hidrogéis/química , Engenharia Tecidual/métodos , Fosfatase Alcalina/química , Animais , Diferenciação Celular , Proliferação de Células , Células Cultivadas , Luz , Células-Tronco Mesenquimais/citologia , Camundongos , Osteogênese , Fotoquímica , Polímeros/químicaRESUMO
Autofocusing and feature detection are two essential processes for performing automated biological cell manipulation tasks. In this paper, we have introduced a technique capable of focusing on a holding pipette and a mammalian cell under a bright-field microscope automatically, and a technique that can detect and track the presence and orientation of the polar body of an oocyte that is rotated at the tip of a micropipette. Both algorithms were evaluated by using mouse oocytes. Experimental results show that both algorithms achieve very high success rates: 100% and 96%. As robust and accurate image processing methods, they can be widely applied to perform various automated biological cell manipulations.
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Separação Celular/métodos , Rastreamento de Células/métodos , Interpretação de Imagem Assistida por Computador/métodos , Micromanipulação/métodos , Corpos Polares/citologia , Robótica/métodos , Animais , Células Cultivadas , Aumento da Imagem/métodos , Camundongos , Reprodutibilidade dos Testes , Sensibilidade e EspecificidadeRESUMO
Metastasis requires tumor cells to navigate through a stiff stroma and squeeze through confined microenvironments. Whether tumors exploit unique biophysical properties to metastasize remains unclear. Data show that invading mammary tumor cells, when cultured in a stiffened three-dimensional extracellular matrix that recapitulates the primary tumor stroma, adopt a basal-like phenotype. Metastatic tumor cells and basal-like tumor cells exert higher integrin-mediated traction forces at the bulk and molecular levels, consistent with a motor-clutch model in which motors and clutches are both increased. Basal-like nonmalignant mammary epithelial cells also display an altered integrin adhesion molecular organization at the nanoscale and recruit a suite of paxillin-associated proteins implicated in invasion and metastasis. Phosphorylation of paxillin by Src family kinases, which regulates adhesion turnover, is similarly enhanced in the metastatic and basal-like tumor cells, fostered by a stiff matrix, and critical for tumor cell invasion in our assays. Bioinformatics reveals an unappreciated relationship between Src kinases, paxillin, and survival of breast cancer patients. Thus adoption of the basal-like adhesion phenotype may favor the recruitment of molecules that facilitate tumor metastasis to integrin-based adhesions. Analysis of the physical properties of tumor cells and integrin adhesion composition in biopsies may be predictive of patient outcome.
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Adesão Celular/fisiologia , Integrinas/metabolismo , Paxilina/metabolismo , Mama/metabolismo , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Matriz Extracelular/metabolismo , Feminino , Proteína-Tirosina Quinases de Adesão Focal/metabolismo , Humanos , Metástase Neoplásica/fisiopatologia , Fosforilação , Transdução de SinaisRESUMO
Integrin-based adhesions play critical roles in cell migration. Talin activates integrins and flexibly connects integrins to the actomyosin cytoskeleton, thereby serving as a 'molecular clutch' that transmits forces to the extracellular matrix to drive cell migration. Here we identify the evolutionarily conserved Kank protein family as novel components of focal adhesions (FAs). Kank proteins accumulate at the lateral border of FAs, which we term the FA belt, and in central sliding adhesions, where they directly bind the talin rod domain through the Kank amino-terminal (KN) motif and induce talin and integrin activation. In addition, Kank proteins diminish the talin-actomyosin linkage, which curbs force transmission across integrins, leading to reduced integrin-ligand bond strength, slippage between integrin and ligand, central adhesion formation and sliding, and reduced cell migration speed. Our data identify Kank proteins as talin activators that decrease the grip between the integrin-talin complex and actomyosin to regulate cell migration velocity.
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Proteínas de Transporte/metabolismo , Adesões Focais/metabolismo , Integrinas/metabolismo , Talina/metabolismo , Proteínas Adaptadoras de Transdução de Sinal , Animais , Adesão Celular , Membrana Celular/metabolismo , Movimento Celular , Células Cultivadas , Proteínas do Citoesqueleto , Citoesqueleto/metabolismo , Matriz Extracelular/metabolismo , Camundongos , Vinculina/metabolismoRESUMO
Three-dimensional (3-D) positioning and orientation of embryos/oocytes is necessary to facilitate micromanipulation tasks such as cell injection and cellular structural biopsy commonly performed under a microscope. Conventional cell orientation is performed manually by using a vacuum equipped micropipette to aspirate and release the cell, which is a trial-and-error approach. The conventional method relies heavily on the skill of the operator; it also suffers from low precision, low success rate and low controllability. These drawbacks illustrate the need for a systematic 3-D cell rotational system to automate the cell orientation process. In this paper, we present a noninvasive single cell rotation system that can automatically orientate a zebrafish embryo to a desired position when both the cytoplasm and the yolk are in the focal plane. A three-point-contact model for cell rotation that involves a custom-designed rotational stage is introduced to provide precise rotational position control. A vision recognition algorithm is also proposed to enable the visual servoing function of the system. Experimental results show that the proposed system can achieve high success rates of 92.5% (x-axis rotation with 40 trails) and 97.5% (about the z-axis with 80 trails). The system can also successfully complete 3-D cell orientation at an average speed of 31 s/cell with a high in-plane rotation accuracy of 0.3 (°) . As a high precise, high controllable and deterministic cell manipulating system, it provides a starting point for automated cell manipulation for intracytoplasmic sperm injection and embryo biopsy for preimplantation genetic diagnosis.