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1.
Anal Chim Acta ; 1182: 338968, 2021 Oct 16.
Artigo em Inglês | MEDLINE | ID: mdl-34602206

RESUMO

Optimal handling is the most important means to ensure adequate sample quality. We aimed to investigate whether pre-centrifugation delay time and temperature could be accurately predicted and to what extent variability induced by pre-centrifugation management can be adjusted for. We used untargeted liquid chromatography-mass spectrometry (LC-MS) metabolomics to predict and evaluate the influence of pre-centrifugation temperature and delayed time on plasma samples. Pre-centrifugation temperature (4, 25 and 37 °C; classification rate 87%) and time (5-210 min; Q2 = 0.82) were accurately predicted using Random Forest (RF). Metabolites uniquely reflecting temperature and temperature-time interactions were discovered using a combination of RF and generalized linear models. Time-related metabolite profiles suggested a perturbed stability of the metabolome at all temperatures in the investigated time period (5-210 min), and the variation at 4 °C was observed in particular before 90 min. Fourteen and eight metabolites were selected and validated for accurate prediction of pre-centrifugation temperature (classification rate 94%) and delay time (Q2 = 0.90), respectively. In summary, the metabolite profile was rapidly affected by pre-centrifugation delay at all temperatures and thus the pre-centrifugation delay should be as short as possible for metabolomics analysis. The metabolite panels provided accurate predictions of pre-centrifugation delay time and temperature in healthy individuals in a separate validation sample. Such predictions could potentially be useful for assessing legacy samples where relevant metadata is lacking. However, validation in larger populations and different phenotypes, including disease states, is needed.


Assuntos
Metaboloma , Espectrometria de Massas em Tandem , Centrifugação , Cromatografia Líquida , Humanos , Plasma
2.
Vet Clin Pathol ; 50(3): 354-358, 2021 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-34467535

RESUMO

BACKGROUND: The hematocrit (Hct) or packed cell volume (PCV) reflects the blood volume occupied by red blood cells. The development of point-of-care (PoC) instruments can accelerate the ease of measuring Hct/PCV compared with traditional capillary centrifugation (TCC) methods. However, no studies have compared Hct/PCV levels in cattle at high elevation with other measurement methods. OBJECTIVES: In this study, we aimed to compare methods to estimate Hcts/PCVs of rangeland cattle at high elevation. We specifically wanted to determine if Hct/PCV levels measured with a commercial PoC instrument (i-Stat with CHEM8+ cartridges [PoCi ]) were comparable to Hct/PCV levels measured with traditional laboratory methods. METHODS: We assessed the Hct/PCV of 94 mature beef cattle (black Angus; Bos taurus) at ~2195 m above sea level using paired analyses of the PoCi and TCC methods from each animal. We used paired samples t-tests to compare mean Hct/PCVs. Correlation analyses relative to the line of identity and Passing-Bablok regression were used to assess systematic and proportional differences, respectively, and Bland-Altman plots were used to assess agreement between the two methods. RESULTS: The PoCi estimated a Hct of 28.2% ± 0.7% (SE), which was lower than the TCC estimated PCV of 39.2% ± 0.5%. The Bland-Altman plot revealed poor agreement between the two methods in addition to a -11% bias for the PoCi . The Passing-Bablok regression revealed both systematic and proportional bias between the two methods. CONCLUSIONS: Point-of-care blood instruments were not comparable to TCC methods for quantifying Hct/PCVs of cattle living at high elevations.


Assuntos
Altitude , Sistemas Automatizados de Assistência Junto ao Leito , Animais , Bovinos , Centrifugação/veterinária , Contagem de Eritrócitos/veterinária , Hematócrito/veterinária
3.
Aerosp Med Hum Perform ; 92(8): 633-641, 2021 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-34503616

RESUMO

AbstractBACKGROUND: Members of the public will soon be taking commercial suborbital spaceflights with significant Gx (chest-to-back) acceleration potentially reaching up to 6 Gx. Pulmonary physiology is gravity-dependent and is likely to be affected, which may have clinical implications for medically susceptible individuals.METHODS: During 2-min centrifuge exposures ranging up to 6 Gx, 11 healthy subjects were studied using advanced respiratory techniques. These sustained exposures were intended to allow characterization of the underlying pulmonary response and did not replicate actual suborbital G profiles. Regional distribution of ventilation in the lungs was determined using electrical impedance tomography. Neural respiratory drive (from diaphragm electromyography) and work of breathing (from transdiaphragmatic pressures) were obtained via nasoesophageal catheters. Arterial blood gases were measured in a subset of subjects. Measurements were conducted while breathing air and breathing 15 oxygen to simulate anticipated cabin pressurization conditions.RESULTS: Acceleration caused hypoxemia that worsened with increasing magnitude and duration of Gx. Minimum arterial oxygen saturation at 6 Gx was 86 1 breathing air and 79 1 breathing 15 oxygen. With increasing Gx the alveolar-arterial (A-a) oxygen gradient widened progressively and the relative distribution of ventilation reversed from posterior to anterior lung regions with substantial gas-trapping anteriorly. Severe breathlessness accompanied large progressive increases in work of breathing and neural respiratory drive.DISCUSSION: Sustained high-G acceleration at magnitudes relevant to suborbital flight profoundly affects respiratory physiology. These effects may become clinically important in the most medically susceptible passengers, in whom the potential role of centrifuge-based preflight evaluation requires further investigation.Pollock RD, Jolley CJ, Abid N, Couper JH, Estrada-Petrocelli L, Hodkinson PD, Leonhardt S, Mago-Elliott S, Menden T, Rafferty G, Richmond G, Robbins PA, Ritchie GAD, Segal MJ, Stevenson AT, Tank HD, Smith TG. Pulmonary effects of sustained periods of high-G acceleration relevant to suborbital spaceflight. Aerosp Med Hum Perform. 2021; 92(7):633641.


Assuntos
Medicina Aeroespacial , Voo Espacial , Aceleração , Centrifugação , Gravitação , Humanos
4.
Aerosp Med Hum Perform ; 92(7): 579-587, 2021 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-34503632

RESUMO

INTRODUCTION: The paragliding reserve parachute system is safety-critical but underused, unstandardized, and known to fail. This study aimed to characterize reserve parachute deployment under radial acceleration to make recommendations for system design and paraglider pilot training.METHODS: There were 88 licensed amateur paraglider pilots who were filmed deploying their reserve parachutes from a centrifuge. Of those, 43 traveled forward at 4 G simulating a spiral dive, and 45 traveled backward at 3 G simulating a rotational maneuver known as SAT. Tests incorporated ecologically valid body, hand, and gaze positions, and cognitive loading and switching akin to real deployment. The footage was reviewed by subject matter experts and compared to previous work in linear acceleration.RESULTS: Of the pilots, 2.3 failed to extract the reserve container from the harness. SAT appeared more cognitively demanding than spiral, despite lower G. Participants located the reserve handle by touch not sight. The direction of travel influenced their initial contact with the harness: 82.9 searched first on their hip in spiral, 63.4 searched first on their thigh in SAT. Search patterns followed skeletal landmarks. Participants had little directional control over their throw.CONCLUSIONS: Paraglider pilots are part of the reserve system. Maladaptive behaviors observed under stress highlighted that components must work in harmony with pilots natural responses, with minimal cognitive demands or need for innovation or problem-solving. Recommendations include positioning prominent, tactile reserve handles overlying the pilots hip; deployment bags extractable with any angle of pull; deployment in a single sweeping backward action; and significantly increasing reserve deployment drills.Wilkes M, Long G, Charles R, Massey H, Eglin C, Tipton MJ. Paraglider reserve parachute deployment under radial acceleration. Aerosp Med Hum Perform. 2021; 92(7):579587.


Assuntos
Aceleração , Pilotos , Centrifugação , Humanos
5.
Exp Appl Acarol ; 85(1): 41-48, 2021 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-34557977

RESUMO

Among the skin disorders of dogs, scabies is notable for its high occurrence rate and the need for veterinary interventions. There are two obstacles to making this diagnosis through direct investigation under a microscope (DIM): the continual need to make new slides when the results are negative and the long time needed for reading these slides. Thus, the objective of the present study was to compare efficacy between DIM and the technique of centrifugation-flotation in sucrose solution (CFSS) in samples from dogs in the semiarid region of the state of Paraíba, Brazil. Samples from 136 dogs were used, and three slides were made for each examination (DIM and CFSS). The readings were halted in cases of positivity. Positive samples were obtained from 56.6% of the dogs (77/136), of which 76.6% (59/77) were positive through both techniques, 13% (10/77) only through DIM and 10.4% (8/77) only through CFSS. The positivity rate did not differ statistically between the techniques. CFSS showed higher quality of readings, due to the considerably fewer artifacts on the slides, thereby optimizing the reading time. Sensitivity (85.6%), specificity (88.1%), accuracy (86.8%), positive predictive value (88.1%) and negative predictive value (85.1%) were obtained and the kappa coefficient (0.73) was considered substantial. It was concluded that CFSS showed high diagnostic capacity for scabies, similar to that of DIM, with optimized reading time, fewer artifacts and better display of mites.


Assuntos
Doenças do Cão , Escabiose , Animais , Centrifugação , Técnicas de Laboratório Clínico , Doenças do Cão/diagnóstico , Cães , Sarcoptes scabiei , Sacarose
6.
Rev Assoc Med Bras (1992) ; 67(4): 536-541, 2021 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-34495057

RESUMO

OBJECTIVE: Clinical studies claim that platelet-rich plasma (PRP) accelerates tissue healing due to its high concentration of growth factors and that the combination with leukocytes improves the antimicrobial effect of the concentrate. Most of these studies obtained PRP using different separation systems, and few analyzed the content of the PRP used for treatment. This study characterized the composition of PRP and white blood cells (WBC) from a single donor produced by three commercially available PRP separation systems and two anticoagulated general analytical tubes. METHODS: Five patients donated 50 mL of blood, which was processed to produce PRP and WBC using three PRP concentration systems (i.e., Easy PRP Kit, GloPRP, and Wego) and two tubes for general analysis anticoagulated with ethylenediaminetetraacetic acid (EDTA) and citrate. Platelets and WBC in combination with their concentrates were analyzed by automated systems in a clinical laboratory. RESULTS: There were no significant differences in the average concentrations of PRP platelets and WBC between GloPRP and the tubes for general analysis with EDTA and citrate; however, the Easy PRP Kit gave results much superior to the rest of the methods, especially comparing it with the Wego Kit, whose concentrates were especially low, even nonexistent for WBC. CONCLUSIONS: The Easy PRP Kit concentrates WBC-rich PRP, resulting in increased WBC concentrations, compared with low WBC-low PRP of GloPRP and general tube methods for EDTA and citrate analysis and the even lower concentration of PRP from the Wego Kit, with the absence of leukocytes.


Assuntos
Plasma Rico em Plaquetas , Separação Celular , Centrifugação , Estudos Transversais , Humanos , Contagem de Leucócitos
7.
Gen Dent ; 69(5): 14-19, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34424206

RESUMO

The use of autogenous materials to promote tissue regeneration has guided the direction of modern dentistry, and platelet-rich fibrin (PRF) is a promising biomaterial for tissue engineering. This in vitro immunohistochemical study aimed to analyze the presence of factors of endothelial growth and cell differentiation in PRF membranes by using the CD31 (endothelial cells) and CD163 (monocytes) markers. Five men and 5 women, aged between 25 and 60 years and without systemic health problems, were enrolled in the study. Blood samples were collected, submitted to a centrifugation protocol, and fixed in 4% formaldehyde, and then immunohistochemical analysis was performed. The histologic analysis of the slides showed that the fibrin clot was formed by a dense fiber network and cells trapped in its structure. One sample was excluded from the markers testing due to poor quality. All 9 of the valid samples were positive for the CD31 and CD163 markers, with reactivity ranging from 5% to 30% and 10% to 40% of cells, respectively. The immunohistochemical analysis showed the presence of CD31 and CD163 in the PRF membranes, indicating the potential for vascular neoformation and the significant presence of monocytes, which play an important role in tissue remodeling via their differentiation into macrophages.


Assuntos
Fibrina Rica em Plaquetas , Adulto , Plaquetas , Centrifugação , Células Endoteliais , Feminino , Fibrina , Humanos , Masculino , Pessoa de Meia-Idade
8.
Artigo em Inglês | MEDLINE | ID: mdl-34444580

RESUMO

Military aircrew are occupationally exposed to a high-G environment. A tolerance test and surveillance is necessary for military aircrew before flight training. A cardiac force index (CFI) has been developed to assess long-distance running by health technology. We added the parameter CFI to the G tolerance test and elucidated the relationship between the CFI and G tolerance. A noninvasive device, BioHarness 3.0, was used to measure heart rate (HR) and activity while resting and walking on the ground. The formula for calculating cardiac function was CFI = weight × activity/HR. Cardiac force ratio (CFR) was calculated by walking CFI (WCFI)/resting CFI (RCFI). G tolerance included relaxed G tolerance (RGT) and straining G tolerance (SGT) tested in the centrifuge. Among 92 male participants, the average of RCFI, WCFI, and CFR were 0.02 ± 0.04, 0.15 ± 0.04, and 10.77 ± 4.11, respectively. Each 100-unit increase in the WCFI increased the RGT by 0.14 G and the SGT by 0.17 G. There was an increased chance of RGT values higher than 5 G and SGT values higher than 8 G according to the WCFI increase. Results suggested that WCFI is positively correlated with G tolerance and has the potential for G tolerance surveillance and programs of G tolerance improvement among male military aircrew.


Assuntos
Militares , Centrifugação , Testes Diagnósticos de Rotina , Humanos , Masculino , Caminhada
9.
Se Pu ; 39(7): 679-685, 2021 Jul 08.
Artigo em Chinês | MEDLINE | ID: mdl-34227364

RESUMO

The novel coronavirus disease 2019 (COVID-19) outbreak has brought to light unprecedented challenges to global public health security. Researchers have devoted their efforts to in-depth research on severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) to bring the epidemic under control as rapidly as possible. Among the many areas of burgeoning SARS-CoV-2 related research, various analytical technologies have been applied to the advancement of virus detection, and development of vaccines and innovative therapies. Separation technologies with the merits of simple operation, high separation efficiency, and high selectivity, have become widely used and are key to progress in life science, medicine, pharmaceutical discovery and development, and other fields. Separation technologies have played an irreplaceable role in the isolation, detection, diagnosis, treatment, and prevention of this novel coronavirus. In this review, an overview of the relevant literature is presented from ISI Web of Science spanning Jan. 1st, 2020-Dec. 31, 2020, using "SARS-CoV-2" or "COVID-19" as keywords. The top 20 research directions are summarized, based on papers published in high impact international journals (e. g. Nature, Science, and Cell). Incorporating the impact of published papers, this review summarizes the primary separation technologies applied in these coronavirus studies, and discusses contributions of the following six technologies: affinity chromatography and size exclusion chromatography, liquid chromatography, magnetic bead separation technology, centrifugal technology, micro/nano-separation technology, and electrophoresis. First, affinity chromatography and size exclusion chromatography are discussed, which are the most frequently used protein purification techniques in Nature, Science, and Cell. The SARS-CoV-2 related proteins purified by affinity chromatography and size exclusion chromatography are summarized, and their applications in coronavirus transmission, infection mechanisms, and drug screening are introduced. Subsequently, high performance liquid chromatography (HPLC) is introduced, which is mainly employed for assessing the purity of candidate drugs. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) incorporates the strengths of HPLC and MS, offering both high separation efficiency and structural analysis capabilities with extended applications. LC-MS/MS has been applied to characterization of the binding of SARS-CoV-2 related proteins to potential inhibitors, and to metabolic analyses of candidate drugs. In SARS-CoV-2 nucleic acid tests, magnetic bead separation technology plays a crucial role in the separation of novel coronaviruses. In combination with other analytical techniques, magnetic bead separation technology can be applied to cytological analyses and immunological detection by functionalization of bead surfaces. Centrifugal technology is undoubtedly the most basic separation technology. It has been employed in almost all SARS-CoV-2 related researches. By controlling centrifugation speed, centrifugal technology can rapidly isolate virus particles or cultured cells from complex samples. Micro-nano separation technologies, such as microfluidics, offer advantages including small size, low sample consumption, rapid diffusion, and large surface area. In general, microfluidic technologies are often used in combination with other technologies to realize highly sensitive detection of SARS-CoV-2 related proteins. Finally, the applications of electrophoresis are introduced, which commonly engages in the analysis of polymerase chain reaction (PCR) products. In novel coronavirus studies, the application of electrophoresis has been relatively limited but has potential with further development to contribute significantly to future research. In conclusion, this review summarizes the contributions of six primary separation technologies to novel coronavirus studies, including epidemic detection and prevention, analyzes the main problems facing coronavirus detection efforts, and discusses the role of separation technologies in addressing these problems, with the aim of providing references for broader application of separation technologies.


Assuntos
COVID-19/diagnóstico , Epidemias/prevenção & controle , SARS-CoV-2/isolamento & purificação , Tecnologia/tendências , COVID-19/epidemiologia , Centrifugação , Cromatografia Líquida , Humanos , Magnetismo , Espectrometria de Massas em Tandem
10.
Int J Mol Sci ; 22(13)2021 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-34281153

RESUMO

Cytocentrifugation is a common technique for the capture of cells on microscopic slides. It usually requires a special cytocentrifuge or cytorotor and cassettes. In the study presented here, we tested the new concept of cytocentrifugation based on the threaded connection of the lid and the sample holder to ensure an adjustable flow of solutions through the filters and the collection of the filtered solutions in the reservoir during centrifugation. To test this concept, we developed a device for the preparation of cell samples on circular coverslips. The device was tested for the capture and sample processing of both eukaryotic and prokaryotic cells, cell nuclei, and mitochondria for microscopy analysis including image cytometry. Moreover, an efficient procedure was developed for capturing formaldehyde-fixed cells on non-treated coverslips without cell drying. The results showed that the tested arrangement enables the effective capture and processing of all of the tested samples and the developed device represents an inexpensive alternative to common cytocentrifuges, as only the paper filter is consumed during sample processing, and no special centrifuge, cytorotor, or cassette is necessary. As no additional system of solution removal is required during sample staining, the tested concept also facilitates the eventual automation of the staining procedure.


Assuntos
Centrifugação/instrumentação , Técnicas Citológicas/instrumentação , Técnicas Citológicas/métodos , Animais , Centrifugação/métodos , Humanos , Microscopia/métodos , Manejo de Espécimes/métodos , Coloração e Rotulagem/métodos
11.
Nucleic Acids Res ; 49(14): 8007-8023, 2021 08 20.
Artigo em Inglês | MEDLINE | ID: mdl-34233004

RESUMO

The 'open' and 'compact' regions of chromatin are considered to be regions of active and silent transcription, respectively. However, individual genes produce transcripts at different levels, suggesting that transcription output does not depend on the simple open-compact conversion of chromatin, but on structural variations in chromatin itself, which so far have remained elusive. In this study, weakly crosslinked chromatin was subjected to sedimentation velocity centrifugation, which fractionated the chromatin according to its degree of compaction. Open chromatin remained in upper fractions, while compact chromatin sedimented to lower fractions depending on the level of nucleosome assembly. Although nucleosomes were evenly detected in all fractions, histone H1 was more highly enriched in the lower fractions. H1 was found to self-associate and crosslinked to histone H3, suggesting that H1 bound to H3 interacts with another H1 in an adjacent nucleosome to form compact chromatin. Genome-wide analyses revealed that nearly the entire genome consists of compact chromatin without differences in compaction between repeat and non-repeat sequences; however, active transcription start sites (TSSs) were rarely found in compact chromatin. Considering the inverse correlation between chromatin compaction and RNA polymerase binding at TSSs, it appears that local states of chromatin compaction determine transcription levels.


Assuntos
Cromatina/ultraestrutura , Nucleossomos/genética , Sítio de Iniciação de Transcrição , Transcrição Genética , Centrifugação , Cromatina/genética , Montagem e Desmontagem da Cromatina/genética , Genoma Humano/genética , Histonas/genética , Humanos , Nucleossomos/ultraestrutura , Ligação Proteica/genética , Fatores de Transcrição/genética
12.
Biomech Model Mechanobiol ; 20(5): 1809-1817, 2021 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-34138382

RESUMO

Coronavirus and its spread all over the world have been the most challenging crisis in 2020. Hospitals are categorized among the most vulnerable centers due to their presumably highest traffic of this virus. In this study, centrifugal isolation of coronavirus is successfully deployed for purifying hospitals' air using air conditioners and ducts, suggesting an efficient setup. Numerical simulations have been used to testify the proposed setup due to the complexities of using experimental investigation such as high cost and clinical hazards of the airborne SARS-CoV-2 in the air. Results show that a 20-cm pipe with an inlet velocity of 4 m/s constitutes the best choice for the separation and purification of air from the virus. The proposed scalable method also efficiently separates larger particles, but it can separate smaller particles too. Numerical results also suggest installing the air purifying system on the floor of the hospitals' room for maximum efficiency.


Assuntos
Filtros de Ar , Simulação por Computador , Hospitais , Análise Numérica Assistida por Computador , SARS-CoV-2/isolamento & purificação , Aerossóis , Centrifugação , Estudos de Viabilidade , Humanos
13.
Methods Mol Biol ; 2310: 17-31, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34095995

RESUMO

Mitochondria possess a genome that codes for proteins, in the same fashion as the nuclear genome. However, the small, circular mitochondrial DNA (mtDNA) molecule has a reduced base pair content, for it can only code for 2 rRNA, 22 tRNA molecules, and 13 proteins, all of them part of the mitochondrial respiratory chain. As such, all of the other mitochondrial components derive from nuclear genome. This separation leads to a requirement for a well-tuned coordination between both genomes, in order to produce fully functional mitochondria. A vast number of pathologies have been demonstrated to involve, to some extent, alterations in mitochondrial function that, no doubt, can be caused by alterations to the respiratory chain activity. As such, several methods and techniques have been developed to assess both content and function of mitochondrial proteins, in order to help understand mitochondrial involvement on the pathogenesis of disease. In this chapter, we will address some of these methods, with the main focus being on isolated mitochondria.


Assuntos
Complexo de Proteínas da Cadeia de Transporte de Elétrons/metabolismo , Mitocôndrias Hepáticas/enzimologia , Proteínas Mitocondriais/metabolismo , Fosforilação Oxidativa , Animais , Fracionamento Celular , Centrifugação , Camundongos , Ratos
14.
Methods Mol Biol ; 2310: 79-89, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34096000

RESUMO

Mitochondria are the organelles where the most fundamental processes of energy transformation within the cell are located. They are also involved in several processes like apoptosis and autophagy, reactive oxygen species formation, and calcium signaling, which are crucial for proper cell functioning. In addition, mitochondrial genome hosts genes encoding important proteins incorporated in respiratory chain complexes and indispensable for the oxidative phosphorylation. Studying isolated mitochondria is, therefore, crucial for better understanding of cell physiology. The presented protocol describes a relatively simple and handy method for crude mitochondrial fraction isolation from different mammalian cell lines. It includes mechanical cells disruption (homogenization) and differential centrifugation. In addition, this chapter presents two basic ways to assess mitochondrial functionality: by measuring mitochondrial inner membrane potential and coupled respiration.


Assuntos
Fracionamento Celular , Metabolismo Energético , Mitocôndrias/metabolismo , Animais , Respiração Celular , Centrifugação , Fluorometria , Células HeLa , Humanos , Células Jurkat , Potencial da Membrana Mitocondrial , Camundongos , Consumo de Oxigênio , Ratos
15.
Methods Mol Biol ; 2310: 161-178, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34096003

RESUMO

Mitochondria play a key role in cell death and its regulation. The permeabilization of the outer mitochondrial membrane, which is mainly controlled by proteins of the BCL-2 family, is a key event that can be directly induced by different signaling pathways, including p53-mediated, and results in the release of proapoptotic factors to the cytosol, such as cytochrome c, second mitochondria-derived activator of caspases/direct inhibitor-of-apoptosis (IAP) binding protein with low pI (SMAC/Diablo), Omi serine protease (Omi/HtrA2), apoptosis-inducing factor (AIF), or endonuclease G (Endo-G). Hence, the determination of subcellular localization of these proteins is extremely important to predict cell fate and elucidate the specific mechanism of apoptosis. Here we describe experimental protocols that can be used to study the subcellular location of different proapoptotic proteins to be used in basic cell biology and toxicology studies.


Assuntos
Proteínas Reguladoras de Apoptose/metabolismo , Apoptose , Western Blotting , Imunofluorescência , Imunoprecipitação , Mitocôndrias/metabolismo , Animais , Fracionamento Celular , Linhagem Celular , Centrifugação , Humanos , Microscopia de Fluorescência , Mitocôndrias/patologia , Transporte Proteico
16.
Nat Methods ; 18(6): 631-634, 2021 06.
Artigo em Inglês | MEDLINE | ID: mdl-34092791

RESUMO

L1CAM is a transmembrane protein expressed on neurons that was presumed to be found on neuron-derived extracellular vesicles (NDEVs) in human biofluids. We developed a panel of single-molecule array assays to evaluate the use of L1CAM for NDEV isolation. We demonstrate that L1CAM is not associated with extracellular vesicles in human plasma or cerebrospinal fluid and therefore recommend against its use as a marker in NDEV isolation protocols.


Assuntos
Vesículas Extracelulares/metabolismo , Molécula L1 de Adesão de Célula Nervosa/metabolismo , Biomarcadores/metabolismo , Centrifugação , Cromatografia em Gel , Meios de Cultivo Condicionados , Humanos , Molécula L1 de Adesão de Célula Nervosa/sangue , Molécula L1 de Adesão de Célula Nervosa/líquido cefalorraquidiano , Neurônios/metabolismo
17.
Methods Mol Biol ; 2276: 31-39, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34060030

RESUMO

As the powerhouse of the cell, mitochondria, plays a crucial role in many aspects of life, whereby mitochondrial dysfunctions are associated with pathogenesis of many diseases, like neurodegenerative diseases, obesity, cancer, and metabolic as well as cardiovascular disorders. Mitochondria analysis frequently starts with isolation and enrichment procedures, which have become increasingly important in biomedical research. Unfortunately, isolation procedures can easily cause changes in the structural integrity of mitochondria during in vitro handling having impact on their function. This carries the risk that conclusions about isolated mitochondria may be drawn on the basis of experimental artifacts. Here we critically review a commonly used isolation procedure for mitochondria utilizing differential (gradient) centrifugation and depict major challenges to achieve "functional" mitochondria as basis for comprehensive physiological studies.


Assuntos
Fracionamento Celular/métodos , Centrifugação/métodos , Microscopia Eletrônica/métodos , Mitocôndrias/metabolismo , Animais , Artefatos , Humanos
18.
Molecules ; 26(10)2021 May 14.
Artigo em Inglês | MEDLINE | ID: mdl-34068851

RESUMO

Solubilization of carbon nanotubes (CNTs) is a fundamental technique for the use of CNTs and their conjugates as nanodevices and nanobiodevices. In this work, we demonstrate the preparation of CNT suspensions with "green" detergents made from coconuts and bamboo as fundamental research in CNT nanotechnology. Single-walled CNTs (SWNTs) with a few carboxylic acid groups (3-5%) and pristine multi-walled CNTs (MWNTs) were mixed in each detergent solution and sonicated with a bath-type sonicator. The prepared suspensions were characterized using absorbance spectroscopy, scanning electron microscopy, and Raman spectroscopy. Among the eight combinations of CNTs and detergents (two types of CNTs and four detergents, including sodium dodecyl sulfate (SDS) as the standard), SWNTs/MWNTs were well dispersed in all combinations except the combination of the MWNTs and the bamboo detergent. The stability of the suspensions prepared with coconut detergents was better than that prepared with SDS. Because the efficiency of the bamboo detergents against the MWNTs differed significantly from that against the SWNTs, the natural detergent might be useful for separating CNTs. Our results revealed that the use of the "green" detergents had the advantage of dispersing CNTs as well as SDS.


Assuntos
Detergentes/química , Nanotubos de Carbono/química , Centrifugação , Nanotubos de Carbono/ultraestrutura , Suspensões
19.
Molecules ; 26(10)2021 May 14.
Artigo em Inglês | MEDLINE | ID: mdl-34069021

RESUMO

The present work investigated the effect of Polylactic acid (PLA) fibers produced by centrifugal spinning with incorporated BaTiO3 particles to improve their bacteriostatic behavior. The PLA matrix and three composites, presenting three different amounts of fillers, were subjected to UV/O3 treatment monitoring the possible modifications that occurred over time. The morphological and physical properties of the surfaces were characterized by different microscopic techniques, contact angle, and surface potential measurements. Subsequently, the samples were tested in vitro with human dermal fibroblasts (HDF) to verify the cytotoxicity of the substrates. No significant differences between the PLA matrix and composites emerged; the high hydrophobicity of the fibers, derived by the polymer structure, represented an obstacle limiting the fibroblast attachment. Samples underwent bacterial exposure (Staphylococcus epidermidis) for 12 and 24 h. Increasing the concentration of BT, the number of living bacteria and their distribution decreased in comparison with the PLA matrix suggesting an effect of the inorganic filler, which generates a neutralization effect leading to reactive oxygen species (ROS) generation and subsequently to bacterial damages. These results suggest that the barium titanate (BT) fillers clearly improve the antibacterial properties of PLA fibers after aging tests made before bacterial exposure, representing a potential candidate in the creation of composites for medical applications.


Assuntos
Antibacterianos/farmacologia , Compostos de Bário/farmacologia , Poliésteres/farmacologia , Titânio/farmacologia , Contagem de Células , Sobrevivência Celular/efeitos dos fármacos , Centrifugação , Derme/citologia , Fibroblastos/citologia , Fibroblastos/efeitos dos fármacos , Humanos , Testes de Sensibilidade Microbiana , Viabilidade Microbiana/efeitos dos fármacos , Espectrometria por Raios X , Staphylococcus epidermidis/efeitos dos fármacos , Água/química
20.
Soft Matter ; 17(26): 6326-6336, 2021 Jul 07.
Artigo em Inglês | MEDLINE | ID: mdl-34136896

RESUMO

Interactions between colloidal-scale structures govern the physical properties of soft and biological materials, and knowledge of the forces associated with these interactions is critical for understanding and controlling these materials. A common approach to quantify colloidal interactions is to measure the interaction forces between colloids and a fixed surface. The centrifuge force microscope (CFM), a miniaturized microscope inside a centrifuge, is capable of performing hundreds of force measurements in parallel over a wide force range (10-2 to 104 pN), but CFM instruments are not widely used to measure colloid-surface interaction forces. In addition, current CFM instruments rely on brightfield illumination and are not capable of fluorescence microscopy. Here we present a fluorescence CFM (F-CFM) that combines both fluorescence and brightfield microscopy and demonstrate its use for measuring microscale colloidal-surface interaction forces. The F-CFM operates at speeds up to 5000 RPM, 2.5× faster than those previously reported, yielding a 6.25× greater maximum force than previous instruments. A battery-powered GoPro video camera enables real-time viewing of the microscopy video on a mobile device, and frequency analysis of the audio signal correlates centrifuge rotational speed with the video signal. To demonstrate the capability of the F-CFM, we measure the force required to detach hundreds of electrostatically stabilized colloidal microspheres attached to a charged glass surface as a function of ionic strength and compare the resulting force distributions with an approximated DLVO theory. The F-CFM will enable microscale force measurements to be correlated with fluorescence imaging in soft and biological systems.


Assuntos
Coloides , Vidro , Centrifugação , Microscopia de Força Atômica , Microscopia de Fluorescência
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