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1.
Cells ; 10(3)2021 03 04.
Artigo em Inglês | MEDLINE | ID: mdl-33806297

RESUMO

Since the outbreak of the COVID-19 crisis, the handling of biological samples from confirmed or suspected SARS-CoV-2-positive individuals demanded the use of inactivation protocols to ensure laboratory operators' safety. While not standardized, these practices can be roughly divided into two categories, namely heat inactivation and solvent-detergent treatments. These routine procedures should also apply to samples intended for Extracellular Vesicles (EVs) analysis. Assessing the impact of virus-inactivating pre-treatments is therefore of pivotal importance, given the well-known variability introduced by different pre-analytical steps on downstream EVs isolation and analysis. Arguably, shared guidelines on inactivation protocols tailored to best address EVs-specific requirements will be needed among the analytical community, yet deep investigations in this direction have not yet been reported. We here provide insights into SARS-CoV-2 inactivation practices to be adopted prior to serum EVs analysis by comparing solvent/detergent treatment vs. heat inactivation. Our analysis entails the evaluation of EVs recovery and purity along with biochemical, biophysical and biomolecular profiling by means of a set of complementary analytical techniques: Nanoparticle Tracking Analysis, Western Blotting, Atomic Force Microscopy, miRNA content (digital droplet PCR) and tetraspanin assessment by microarrays. Our data suggest an increase in ultracentrifugation (UC) recovery following heat treatment; however, it is accompanied by a marked enrichment in EVs-associated contaminants. On the other hand, solvent/detergent treatment is promising for small EVs (<150 nm range), yet a depletion of larger vesicular entities was detected. This work represents a first step towards the identification of optimal serum inactivation protocols targeted to EVs analysis.


Assuntos
/sangue , Contenção de Riscos Biológicos/métodos , Vesículas Extracelulares/química , Inativação de Vírus , /virologia , Detergentes/farmacologia , Vesículas Extracelulares/efeitos dos fármacos , Vesículas Extracelulares/genética , Temperatura Alta , Humanos , MicroRNAs/análise , Análise em Microsséries , Microscopia de Força Atômica , Tetraspaninas/análise , Ultracentrifugação
2.
Nat Commun ; 12(1): 2202, 2021 04 13.
Artigo em Inglês | MEDLINE | ID: mdl-33850135

RESUMO

Artificial native-like lipid bilayer systems constructed from phospholipids assembling into unilamellar liposomes allow the reconstitution of detergent-solubilized transmembrane proteins into supramolecular lipid-protein assemblies called proteoliposomes, which mimic cellular membranes. Stabilization of these complexes remains challenging because of their chemical composition, the hydrophobicity and structural instability of membrane proteins, and the lability of interactions between protein, detergent, and lipids within micelles and lipid bilayers. In this work we demonstrate that metastable lipid, protein-detergent, and protein-lipid supramolecular complexes can be successfully generated and immobilized within zeolitic-imidazole framework (ZIF) to enhance their stability against chemical and physical stressors. Upon immobilization in ZIF bio-composites, blank liposomes, and model transmembrane metal transporters in detergent micelles or embedded in proteoliposomes resist elevated temperatures, exposure to chemical denaturants, aging, and mechanical stresses. Extensive morphological and functional characterization of the assemblies upon exfoliation reveal that all these complexes encapsulated within the framework maintain their native morphology, structure, and activity, which is otherwise lost rapidly without immobilization.


Assuntos
Detergentes/química , Exoesqueleto Energizado , Imobilização/métodos , Bicamadas Lipídicas/química , Proteínas de Membrana/química , Membrana Celular , ATPases Transportadoras de Cobre , Proteínas de Escherichia coli , Cinética , Bicamadas Lipídicas/metabolismo , Proteínas de Membrana/metabolismo , Micelas , Fosfolipídeos , Proteolipídeos , Espalhamento de Radiação , Lipossomas Unilamelares , Difração de Raios X
3.
Bioanalysis ; 13(5): 387-394, 2021 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-33661025

RESUMO

Aim: For oncolytic virus trials, regulatory agencies often require pharmaceutical industry to evaluate risks of released viruses from patients to environment. This study was to establish a real-time PCR method to assess viral shedding and viral stability in human urine. Results/methodology: Herein, we describe an incubation of viral drug product in human urine and use of real-time PCR as a simple, efficient and high throughput assay to assess the level and stability of a nonenveloped and single stranded RNA virus. The viral stability issue is critical to the collection, transport, storage and testing of clinical samples. Discussion/conclusion: In summary, this simple method provides useful viral stability information at various temperatures and detergents. A similar approach may apply to other RNA viruses (including SARS-CoV-2).


Assuntos
RNA Viral/metabolismo , Reação em Cadeia da Polimerase em Tempo Real , Viroses/diagnóstico , /diagnóstico , Detergentes/química , Humanos , Estabilidade de RNA , RNA Viral/sangue , RNA Viral/urina , /isolamento & purificação , Temperatura , Viroses/virologia
4.
J Dairy Sci ; 104(5): 5631-5642, 2021 May.
Artigo em Inglês | MEDLINE | ID: mdl-33663818

RESUMO

Subacute ruminal acidosis (SARA) continues to be a common and costly metabolic disorder in high-producing dairy cows worldwide. The objective of this study was to evaluate if increasing the concentration of physically effective neutral detergent fiber (peNDF) in diets can reduce the risk of SARA in cows fed a high-concentrate diet. Thirty second-parity Holstein cows in mid lactation (131 ± 8.3 d in milk) were randomly allocated to 3 dietary treatments (10 dairy cows per group): high (11.3%, high peNDF8.0), medium (10.6%, medium peNDF8.0), or low (9.0%, low peNDF8.0) concentration of peNDF8.0. The diets were prepared by mixing the same total mixed ration (57% concentrate and 43% roughages) for 10, 18, or 60 min, respectively. The treatments were fed for 36 d with 21 d for adaptation and 15 d for sampling. The peNDF8.0 intake was positively correlated with the peNDF8.0 concentration. Chewing and ruminating times adjusted for dry matter intake and NDF intake were linearly increased with the increased dietary peNDF8.0 concentration. The high peNDF8.0 diet decreased the number of meals per day. The increased dietary peNDF8.0 concentration linearly increased the rumen fluid pH, the molar percentage of acetate and isobutyrate, acetate-to-propionate ratio, and ammonia nitrogen concentration, but linearly decreased the molar percentages of propionate and valerate. The total VFA concentration and the molar percentages of butyrate and isovalerate remained unchanged. Meanwhile, the increase in the peNDF8.0 concentration of the diet linearly increased the activities of carboxymethyl cellulase, avicelase, ß-glucanase, and ferulic acid esterase in rumen fluid, but did not affect the activities of xylanase. Total plasma antioxidant capacity, γ-glutamyl transpeptidase activity, and plasma concentrations of total protein, albumin, creatinine, and malondialdehyde were linearly decreased by the increased dietary peNDF8.0 concentration. The increase in peNDF8.0 concentration raised the plasma concentrations of glucose, triglyceride, cholesterol, and blood urea nitrogen. Somatic cell counts in the milk were positively correlated with the dietary peNDF8.0 concentration. The feed and milk energy efficiencies were unaffected by the treatments. Shortening the total mixed ration mixing time may be a practical strategy to increase the peNDF8.0 concentration and reduce the risk of SARA in dairy cows fed high-concentrate diets.


Assuntos
Lactação , Rúmen , Animais , Bovinos , Detergentes/metabolismo , Dieta/veterinária , Fibras na Dieta/metabolismo , Digestão , Feminino , Fermentação , Concentração de Íons de Hidrogênio , Mastigação , Leite , Plasma , Gravidez , Rúmen/metabolismo
5.
Cochrane Database Syst Rev ; 3: CD011675, 2021 03 10.
Artigo em Inglês | MEDLINE | ID: mdl-33734426

RESUMO

BACKGROUND: Leg ulcers are open skin wounds that occur below the knee but above the foot. The majority of leg ulcers are venous in origin, occurring as a result of venous insufficiency, where the flow of blood through the veins is impaired; they commonly arise due to blood clots and varicose veins. Compression therapy, using bandages or stockings, is the primary treatment for venous leg ulcers. Wound cleansing can be used to remove surface contaminants, bacteria, dead tissue and excess wound fluid from the wound bed and surrounding skin, however, there is uncertainty regarding the effectiveness of cleansing and the best method or solution to use. OBJECTIVES: To assess the effects of wound cleansing, wound cleansing solutions and wound cleansing techniques for treating venous leg ulcers. SEARCH METHODS: In September 2019 we searched the Cochrane Wounds Specialised Register; the Cochrane Central Register of Controlled Trials (CENTRAL); Ovid MEDLINE (including In-Process & Other Non-Indexed Citations); Ovid Embase and EBSCO CINAHL Plus. We also searched clinical trials registries for ongoing and unpublished studies, and scanned reference lists of relevant included studies as well as reviews, meta-analyses and health technology reports to identify additional studies. There were no restrictions with respect to language, date of publication or study setting. SELECTION CRITERIA: We considered randomised controlled trials (RCTs) comparing wound cleansing with no wound cleansing, or RCTs comparing different wound cleansing solutions, or different wound cleansing techniques. DATA COLLECTION AND ANALYSIS: We screened studies for their appropriateness for inclusion, assessed their risk of bias using the Cochrane 'Risk of bias' tool, and used GRADE methodology to determine the certainty of evidence. Two review authors undertook these tasks independently, using predetermined criteria. We contacted study authors for missing data where possible. MAIN RESULTS: We included four studies with a total of 254 participants. All studies included comparisons between different types of cleansing solutions, and three of these reported our primary outcomes of complete wound healing or change in ulcer size over time, or both. Two studies reported the secondary outcome, pain. One study (27 participants), which compared polyhexamethylene biguanide (PHMB) solution with saline solution for cleansing venous leg ulcers, did not report any of the review's primary or secondary outcomes. We did not identify any studies that compared cleansing with no cleansing, or that explored comparisons between different cleansing techniques. One study (61 participants) compared aqueous oxygen peroxide with sterile water. We are uncertain whether aqueous oxygen peroxide makes any difference to the number of wounds completely healed after 12 months of follow-up (risk ratio (RR) 1.88, 95% confidence interval (CI) 1.10 to 3.20). Similarly, we are uncertain whether aqueous oxygen peroxide makes any difference to change in ulcer size after eight weeks of follow-up (mean difference (MD) -1.38 cm2, 95% CI -4.35 to 1.59 cm2). Finally, we are uncertain whether aqueous oxygen peroxide makes any difference to pain reduction, assessed after eight weeks of follow-up using a 0 to 100 pain rating, (MD 3.80, 95% CI -10.83 to 18.43). The evidence for these outcomes is of very low certainty (we downgraded for study limitations and imprecision; for the pain outcome we also downgraded for indirectness). Another study (40 participants) compared propyl betaine and polihexanide with a saline solution. The authors did not present the raw data in the study report so we were unable to conduct independent statistical analysis of the data. We are uncertain whether propyl betaine and polihexanide make any difference to the number of wounds completely healed, change in ulcer size over time, or wound pain reduction. The evidence is of very low certainty (we downgraded for study limitations and imprecision). The final study (126 participants) compared octenidine dihydrochloride/phenoxyethanol (OHP) with Ringer's solution. We are uncertain whether OHP makes any difference to the number of wounds healed (RR 0.96, 95% CI 0.53 to 1.72) or to the change in ulcer size over time (we were unable to conduct independent statistical analysis of available data). The evidence is of very low certainty (we downgraded for study limitations and imprecision). None of the studies reported patient preference, ease of use of the method of cleansing, cost or health-related quality of life. In one study comparing propyl betaine and polihexanide with saline solution the authors do not report any adverse events occurring. We are uncertain whether OHP makes any difference to the number of adverse events compared with Ringer's solution (RR 0.58, 95% CI 0.29 to 1.14). The evidence is of very low certainty (we downgraded for study limitations and imprecision). AUTHORS' CONCLUSIONS: There is currently a lack of RCT evidence to guide decision making about the effectiveness of wound cleansing compared with no cleansing and the optimal approaches to cleansing of venous leg ulcers. From the four studies identified, there is insufficient evidence to demonstrate whether the use of PHMB solution compared with saline solution; aqueous oxygen peroxide compared with sterile water; propyl betaine and polihexanide compared with a saline solution; or OHP compared with Ringer's solution makes any difference in the treatment of venous leg ulcers. Evidence from three of the studies is of very low certainty, due to study limitations and imprecision. One study did not present data for the primary or secondary outcomes. Further well-designed studies that address important clinical, quality of life and economic outcomes may be important, based on the clinical and patient priority of this uncertainty.


Assuntos
Desinfetantes/uso terapêutico , Úlcera Varicosa/terapia , Cicatrização/efeitos dos fármacos , Idoso , Anti-Infecciosos Locais/uso terapêutico , Betaína/uso terapêutico , Viés , Biguanidas/uso terapêutico , Intervalos de Confiança , Detergentes/uso terapêutico , Etilenoglicóis/uso terapêutico , Feminino , Humanos , Peróxido de Hidrogênio/uso terapêutico , Masculino , Pessoa de Meia-Idade , Medição da Dor/métodos , Piridinas/uso terapêutico , Ensaios Clínicos Controlados Aleatórios como Assunto , Solução de Ringer/uso terapêutico , Solução Salina/uso terapêutico , Úlcera Varicosa/patologia
6.
Nature ; 590(7846): 509-514, 2021 02.
Artigo em Inglês | MEDLINE | ID: mdl-33568813

RESUMO

Mechanosensitive channels sense mechanical forces in cell membranes and underlie many biological sensing processes1-3. However, how exactly they sense mechanical force remains under investigation4. The bacterial mechanosensitive channel of small conductance, MscS, is one of the most extensively studied mechanosensitive channels4-8, but how it is regulated by membrane tension remains unclear, even though the structures are known for its open and closed states9-11. Here we used cryo-electron microscopy to determine the structure of MscS in different membrane environments, including one that mimics a membrane under tension. We present the structures of MscS in the subconducting and desensitized states, and demonstrate that the conformation of MscS in a lipid bilayer in the open state is dynamic. Several associated lipids have distinct roles in MscS mechanosensation. Pore lipids are necessary to prevent ion conduction in the closed state. Gatekeeper lipids stabilize the closed conformation and dissociate with membrane tension, allowing the channel to open. Pocket lipids in a solvent-exposed pocket between subunits are pulled out under sustained tension, allowing the channel to transition to the subconducting state and then to the desensitized state. Our results provide a mechanistic underpinning and expand on the 'force-from-lipids' model for MscS mechanosensation4,11.


Assuntos
Microscopia Crioeletrônica , Proteínas de Escherichia coli/metabolismo , Proteínas de Escherichia coli/ultraestrutura , Escherichia coli/química , Canais Iônicos/metabolismo , Canais Iônicos/ultraestrutura , Membranas Artificiais , Fosfatidilcolinas/metabolismo , Detergentes/farmacologia , Escherichia coli/ultraestrutura , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Interações Hidrofóbicas e Hidrofílicas , Canais Iônicos/química , Canais Iônicos/genética , Bicamadas Lipídicas/química , Bicamadas Lipídicas/metabolismo , Mecanotransdução Celular/efeitos dos fármacos , Modelos Moleculares , Mutação , Nanoestruturas/química , Nanoestruturas/ultraestrutura , Fosfatidilcolinas/química , Fosfatidilcolinas/farmacologia , Conformação Proteica/efeitos dos fármacos , beta-Ciclodextrinas/farmacologia
7.
J Virol Methods ; 289: 114062, 2021 03.
Artigo em Inglês | MEDLINE | ID: mdl-33428990

RESUMO

BACKGROUND: Diagnostic real time reverse transcription PCR (rRT-PCR) is usually done using nucleic acid (NA) purified from the sample. In the SARS-CoV-2 pandemic reagents and utensils for NA purification has been in short supply. This has generated interest in methods that eliminate the need for NA purification. OBJECTIVES: To investigate if addition of detergent to rRT-PCR master mix (MM) enabled in-well direct lysis and detection of SARS-CoV-2 in clinical eSwab specimens. STUDY DESIGN: IGEPAL-CA-630 (IGEPAL) was added to SARS-CoV-2 MM to 0.3 % final concentration and crude sample was added directly to the PCR well containing MM. Cycle of positivity (Cp) and categorical agreement was compared in samples tested in standard rRT-PCR after NA purification and in in-well lysis, direct rRT-PCR. RESULTS: In-well lysis direct rRT-PCR detected SARS-CoV-2 in 27/30 previously SARS-CoV-2+ samples with an average bias of 3.26 cycles (95 %CI: 0.08-6.43 cycles). All 30 previously test negative samples remained negative when tested in in-well lysis, direct PCR. CONCLUSIONS: Supplementation of detergent to MM was shown to be useful for the detection of SARS CoV-2 in eSwab specimens (COPAN) by direct rRT-PCR without prior NA purification.


Assuntos
/métodos , RNA Viral/isolamento & purificação , Manejo de Espécimes/métodos , Detergentes/química , Humanos
8.
Int J Biol Macromol ; 171: 382-388, 2021 Feb 28.
Artigo em Inglês | MEDLINE | ID: mdl-33434547

RESUMO

The current study heterologously expressed a cutinase from Fusarium verticillioides by Pichia pastoris and investigated its properties and effects on the hydrolysis of rice straw. The optimal pH and temperature for F. verticillioides cutinase were 8.0 and 50 °C, respectively. F. verticillioides cutinase had poor thermal stability and could be inhibited by some metal ions, inhibitors, and detergents (5 mM), including Ni2+, Zn2+, Cu2+, Ca2+, Mn2+, sodium dodecyl sulfate, EDTA, and Tween-20. F. verticillioides cutinase could tolerate 15% methanol and dimethyl sulfoxide but was significantly repressed by 15% ethanol and acetone with 48% and 63% residual activity, respectively. F. verticillioides cutinase could degrade the cuticle of rice straw with palmitic acid and stearic acid as the main products. However, the dissolving sugars released from the rice straw treated with F. verticillioides cutinase were significantly reduced by 29.2 µg/mL compared with the control (107.9 µg/mL). Similarly, the reducing sugars produced from the cellulase hydrolysis of rice straw pretreated with F. verticillioides cutinase were reduced by 63.5 µg/mL relative to the control (253.6 µg/mL). Scanning electron microscopy results showed that numerous tuberculate or warty protrusions were present nearly everywhere on the surface of rice straw treated with F. verticillioides cutinase, and some protrusions even covered and blocked the stomata of the rice straw surface. Current limited data indicate that F. verticillioides cutinase might not be an appropriate choice for improving the utilization of agricultural straws.


Assuntos
Hidrolases de Éster Carboxílico/farmacologia , Proteínas Fúngicas/farmacologia , Fusarium/enzimologia , Oryza , Caules de Planta/efeitos dos fármacos , Hidrolases de Éster Carboxílico/biossíntese , Hidrolases de Éster Carboxílico/genética , Celulase/farmacologia , Detergentes/farmacologia , Ácidos Graxos/isolamento & purificação , Fermentação , Proteínas Fúngicas/biossíntese , Proteínas Fúngicas/genética , Concentração de Íons de Hidrogênio , Hidrólise , Microbiologia Industrial/métodos , Metais/farmacologia , Oryza/química , Caules de Planta/química , Proteínas Recombinantes/farmacologia , Solventes/farmacologia , Açúcares/isolamento & purificação
9.
Methods Mol Biol ; 2187: 1-25, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-32770498

RESUMO

Lipid rafts are membrane areas characterized by the clustering of selected membrane lipids, as the result of their phase separation forming a liquid-ordered phase floating in the lipid-disordered bulk membrane. van Meer and Simons hypothesized the existence of lipid rafts to explain the differential composition of the apical and basolateral domains of polarized epithelial cells and proposed that association of given proteins with lipid rafts along the traffic route might represent an important mechanism for protein sorting. However, great attention was paid to the lipid raft theory after Simons and Ikonen highlighted the enrichment of several proteins involved in signal transduction in "detergent-insoluble, glycolipid-enriched complexes," and postulated that lipid rafts might serve as hubs in regulating intracellular signaling. Most notably, the feature of detergent-insolubility was incorporated in the definition of lipid rafts used in 1997 by these authors. "Lipid rafts" and "detergent-resistant membranes" became almost synonymous after the publication, in 1992, of the seminal paper by Brown and Rose, describing the separation of a low-density, Triton X-100-insoluble fraction from epithelial cells, enriched in GSL and apical GPI-anchored proteins and depleted of basolateral membrane marker proteins. This paper provided a working definition of lipid rafts and a putative biochemical method for their separation. More than 2000 papers have been published using "the Triton method." Evidences obtained by the use of alternative biochemical methods for the isolation of lipid rafts and of methods enabling to analyze the dynamics of lipid rafts in intact cells highlighted the several limitations of the Triton X-100 method. On the other hand, the main findings obtained by this method have not been confuted, and the method is still widely used.In this chapter, we will discuss the most relevant methodological aspects related to the preparation of detergent-resistant membrane fractions, with a special focus on neural cells and tissues.


Assuntos
Lipídeos de Membrana/química , Microdomínios da Membrana/química , Neurônios/química , Animais , Biomarcadores/química , Bovinos , Membrana Celular/química , Detergentes/química , Células Epiteliais/química , Camundongos , Octoxinol/química , Transporte Proteico/fisiologia , Ratos , Transdução de Sinais/fisiologia , Solubilidade
10.
Methods Mol Biol ; 2187: 27-35, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-32770499

RESUMO

Lipid rafts are microdomains on plasma membrane that contain high levels of cholesterol and sphingolipids. Because of the detergent-resistant property of lipid rafts, lipid rafts isolated by methods that use detergents frequently yield different results. Artifacts can also be introduced through the use of detergents. These limitations could be overcome with a detergent-free method which eliminates possible artificial influences. Importantly, lipid rafts prepared with a detergent-free method is more compatible to mass spectrometric analysis since the ion suppression effect is largely reduced.This chapter describes a detergent-free two-step method for preparation of lipid rafts. Firstly, a purified plasma membrane fraction is prepared from cells by sedimentation of the postnuclear supernatant (PNS) in a Percoll gradient. Secondly, the as-prepared plasma membranes are sonicated to release lipid rafts which are further isolated by flotation in a continuous gradient of Optiprep solution. Then, we introduce a typical shotgun lipidomics workflow that can be used as a cost-effective and relatively high throughput method to determine the lipidomes of lipid rafts.The method also makes an easy start for lipidomics studies.


Assuntos
Detergentes/química , Lipidômica/métodos , Microdomínios da Membrana/química , Fracionamento Celular/métodos , Colesterol/química , Espectrometria de Massas/métodos , Esfingolipídeos/química
11.
Methods Mol Biol ; 2187: 99-112, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-32770503

RESUMO

Lipid rafts (LRs) represent cellular microdomains enriched in sphingolipids and cholesterol which may fuse to form platforms in which signaling molecules can be organized and regulated (Simons and Ikonen, Nature 387:569-572, 1997; Pike, Biochem J 378:281-292, 2004; Grassme et al., J Immunol 168: 300-307, 2002; Cheng et al., J Exp Med 190:1549-1550, 1999; Kilkus et al., J Neurosci Res 72(1) 62-75, 2003). In a proposed Model 1 (Cheng et al., J Exp Med 190:1549-1550, 1999) the LR has a well-ordered central core composed mainly of cholesterol and sphingolipids that is surrounded by a zone of decreasing lipid order. Detergents such as Triton X-100 can solubilize the core (and a significant amount of phosphoglyceride), but the LRs will be insoluble at 4 °C and be enriched in a well-characterized set of biomarkers. Model 2 proposes that the LRs are homogeneous, but there is selectivity in the lipids (and proteins) extracted by the 1% Triton X-100. Model 3 proposes LRs with distinct lipid compositions are highly structured and can be destroyed by binding molecules such as beta-methylcyclodextrin or filipin. These may be Caveolin in some cell types but not in brain. Since it is unlikely that two LR preparations will be exactly the same this review will concentrate on LRs defined as "small (50 nm) membranous particles which are insoluble in 1% Triton X-100 at 4 °C and have a low buoyant density (Simons and Ikonen, Nature 387:569-572, 1997; Pike, Biochem J 378:281-292, 2004; Grassme et al., J Immunol 168: 300-307, 2002; Cheng et al., J Exp Med 190:1549-1550, 1999; Kilkus et al., J Neurosci Res 72(1):62-75, 2003; Testai et al., J Neurochem 89:636-644, 2004). We will present a generic method for isolating LRs for both lipidomic, proteomic, and cellular signaling analysis [1-6].


Assuntos
Detergentes/química , Exossomos/metabolismo , Lipídeos/isolamento & purificação , Microdomínios da Membrana/metabolismo , Animais , Biomarcadores/metabolismo , Encéfalo/metabolismo , Caveolinas/metabolismo , Linhagem Celular Tumoral , Colesterol/metabolismo , Filipina/metabolismo , Humanos , Camundongos , Octoxinol/química , Proteômica/métodos , Transdução de Sinais/fisiologia , Esfingolipídeos/metabolismo , beta-Ciclodextrinas/metabolismo
12.
Methods Mol Biol ; 2187: 131-145, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-32770505

RESUMO

The traditional methods to study lipid rafts and their association with membrane proteins are based mainly on the isolation of a detergent-resistant membrane by biochemical fractionation. However, the use of detergents may induce lipid segregation and/or redistribution of membrane proteins during the process of sample preparation. Here, we describe a detergent-free method to study the glycolipid and growth factor receptor interaction and their association with lipid rafts. This method combines the biochemical and immunoblotting tools with confocal microscopic imaging, which allows for evaluation and verification of the membrane protein interaction and association with the lipid rafts components in a multifaceted manner.


Assuntos
Glicolipídeos/metabolismo , Lipídeos de Membrana/metabolismo , Microdomínios da Membrana/metabolismo , Receptores de Fatores de Crescimento/metabolismo , Animais , Fracionamento Celular/métodos , Células Cultivadas , Detergentes/metabolismo , Proteínas de Membrana/metabolismo , Camundongos
13.
Methods Mol Biol ; 2187: 313-325, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-32770515

RESUMO

The analysis of protein enrichment in the detergent-resistant membranes (DRMs) isolated from immune cells enables us to analyze a link between the membrane lipid dynamics and cell activation. Here, we describe the fractionation of detergent-resistant membranes and the correlative analysis of the enrichment of T cell receptor (TCR) and ω-azido-modified synthetic ceramide in those fractions upon TCR stimulation.


Assuntos
Membrana Celular/metabolismo , Esfingolipídeos/metabolismo , Fracionamento Celular/métodos , Linhagem Celular Tumoral , Células Cultivadas , Ceramidas/metabolismo , Detergentes/metabolismo , Humanos , Células Jurkat , Lipídeos de Membrana/metabolismo , Proteínas de Membrana/metabolismo , Receptores de Antígenos de Linfócitos T/metabolismo
14.
J Dairy Sci ; 104(3): 2966-2978, 2021 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-33358799

RESUMO

Choline is usually supplemented as ruminally protected choline chloride to prevent its degradation in the rumen, but the effects of unprotected choline on ruminal fermentation are unclear. Some research indicates a possible role of dietary fiber on microbial degradation of choline; therefore we aimed to evaluate the effects of unprotected choline chloride on ruminal fermentation and to investigate whether those effects depend on dietary neutral detergent fiber (NDF) concentration. Our hypothesis was that dietary NDF concentration would influence choline chloride effects on microbial ruminal fermentation. We used 8 fermentors in a duplicated 4 × 4 Latin square with a 2 × 2 factorial arrangement, combining 2 factors: (1) dietary NDF concentration and (2) unprotected choline chloride supplementation. Resulting treatments are (1) 30%NDF/Ctrl [30% NDF control diet without supplemental choline (Cho)]; (2) 30%NDF/Cho [30% NDF diet plus 1.9 g of choline ion per kg of dry matter (DM)]; (3) 40%NDF/Ctrl (40% NDF control diet without supplemental choline); and (4) 40%NDF/Cho (40% NDF diet plus 1.9 g of choline ion per kg of DM). Four 10-d periods were completed, each consisting of 7 d for adaptation and 3 d for collection of samples for estimation of nutrient disappearance and daily average concentrations of volatile fatty acids and NH3-N. In addition, kinetics of pH, acetate, and propionate were evaluated at 0, 1, 2, 4, 6, and 8 h after morning feeding. On the last day of each period, bacteria pellets were harvested for 15N analysis and N metabolism. Fixed effects of dietary NDF concentration, unprotected choline chloride supplementation, and their interaction (NDF × Cho) were tested using the MIXED procedure of SAS version 9.4 (SAS Institute Inc., Cary, NC). Choline tended to increase total volatile fatty acid concentrations and decreased acetate molar proportion regardless of dietary NDF concentration, but it increased propionate molar proportion and decreased acetate to propionate ratio only with the 30% NDF diet. Supplementing choline decreased NDF disappearance regardless of dietary NDF; however, organic matter disappearance tended to be reduced only when choline was added to 40% NDF. Our data indicate that unprotected choline chloride effects on ruminal fermentation depend on dietary NDF concentration, allowing for a greater propionate synthesis without decreasing organic matter disappearance when fed with a 30% NDF diet.


Assuntos
Detergentes , Rúmen , Ração Animal/análise , Animais , Colina/metabolismo , Detergentes/metabolismo , Dieta/veterinária , Fibras na Dieta/metabolismo , Digestão , Fermentação , Rúmen/metabolismo
15.
Int J Biol Macromol ; 169: 39-50, 2021 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-33316342

RESUMO

The Nocardiopsis alba strain OM-5 showed maximum protease production in submerged culture. The OM-5 protease was purified by hydrophobic interaction chromatography. The purified protease of 68 kDa showed maximum activity (3312 ± 1.64 U/mL) at 70 °C and was quite stable at 80 °C up to 4 M NaCl (w/v) at pH 9. The purified protease showed significant activity and stability in different cations, denaturing agents, metal ions, and osmolytes. The thermodynamic parameters including deactivation rate constant (Kd) and half lives (t1/2) at 50-80 °C were in the range of 2.50 × 10-3 to 5.50 × 10-3 and 277.25-111.25 min respectively at 0-4 M NaCl. The structural stability of the OM-5 protease under various harsh conditions was elucidated by circular dichroism (CD) spectroscopy followed by K2D3 analysis revealed that the native structure of OM-5 protease was stable even in sodium dodecyl sulfate and Tween 20 indicated by increased α-helices content assisted with decreased ß-sheets content.


Assuntos
Serina Endopeptidases/química , Serina Endopeptidases/isolamento & purificação , Actinobacteria/química , Proteínas de Bactérias/química , Proteínas de Bactérias/isolamento & purificação , Detergentes , Endopeptidases/química , Endopeptidases/isolamento & purificação , Estabilidade Enzimática/fisiologia , Concentração de Íons de Hidrogênio , Cinética , /metabolismo , Serina/química , Serina Proteases/isolamento & purificação , Tensoativos , Temperatura , Termodinâmica
16.
Biochim Biophys Acta Biomembr ; 1863(1): 183441, 2021 01 01.
Artigo em Inglês | MEDLINE | ID: mdl-32810489

RESUMO

G protein coupled receptors (GPCRs) function as guanine nucleotide exchange factors (GEFs) at heterotrimeric G proteins, and conduct this role embedded in a lipid bilayer. Detergents are widely used to solubilise GPCRs for structural and biophysical analysis, but are poor mimics of the lipid bilayer and may be deleterious to protein function. Amphipathic polymers have emerged as promising alternatives to detergents, which maintain a lipid environment around a membrane protein during purification. Of these polymers, the polymethacrylate (PMA) polymers have potential advantages over the most popular styrene maleic acid (SMA) polymer, but to date have not been applied to purification of membrane proteins. Here we use a class A GPCR, neurotensin receptor 1 (NTSR1), to explore detergent-free purification using PMA. By using an NTSR1-eGFP fusion protein expressed in Sf9 cells, a range of solubilisation conditions were screened, demonstrating the importance of solubilisation temperature, pH, NaCl concentration and the relative amounts of polymer and membrane sample. PMA-solubilised NTSR1 displayed compatibility with standard purification protocols and millimolar divalent cation concentrations. Moreover, the receptor in PMA discs showed stimulation of both Gq and Gi1 heterotrimers to an extent that was greater than that for the detergent-solubilised receptor. PMA therefore represents a viable alternative to SMA for membrane protein purification and has a potentially broad utility in studying GPCRs and other membrane proteins.


Assuntos
Ácidos Polimetacrílicos/química , Receptores de Neurotensina , Detergentes/química , Humanos , Receptores de Neurotensina/biossíntese , Receptores de Neurotensina/química , Receptores de Neurotensina/isolamento & purificação , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , Solubilidade
17.
Methods Mol Biol ; 2247: 257-267, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33301122

RESUMO

Membrane proteins (MPs) are stable in their native lipid environment. To enable structural and functional investigations, MPs need to be extracted from the membrane. This is a critical step that represents the main obstacle for MP biochemistry and structural biology. Here we describe detergent solubilization screening of MPs using dot-blot and Western-blot analyses. Good solubilization conditions are ranked for their best capacity to stabilize MPs using thermal shift assay. The protein functionality is evaluated by radioligand binding (for G-protein-coupled receptor) and ATPase activity (ABC Transporter) and finally the aggregation status as well as protein homogeneity are assessed by Native-polyacrylamide gel, chemical cross-linking, and size exclusion chromatography.


Assuntos
Descoberta de Drogas , Proteínas de Membrana/química , Adenosina Trifosfatases/química , Cromatografia em Gel , Reagentes para Ligações Cruzadas , Detergentes/química , Descoberta de Drogas/métodos , Ativação Enzimática , Ligantes , Eletroforese em Gel de Poliacrilamida Nativa , Estabilidade Proteica , Solubilidade , Soluções
18.
Exp Biol Med (Maywood) ; 246(6): 740-748, 2021 03.
Artigo em Inglês | MEDLINE | ID: mdl-33325749

RESUMO

Shortages of N95 respirators for use by medical personnel have driven consideration of novel conservation strategies, including decontamination for reuse and extended use. Decontamination methods listed as promising by the Centers for Disease Control and Prevention (CDC) (vaporous hydrogen peroxide (VHP), wet heat, ultraviolet irradiation (UVI)) and several methods considered for low resource environments (bleach, isopropyl alcohol and detergent/soap) were studied for two commonly used surgical N95 respirators (3M™ 1860 and 1870+ Aura™). Although N95 filtration performance depends on the electrostatically charged electret filtration layer, the impact of decontamination on this layer is largely unexplored. As such, respirator performance following decontamination was assessed based on the fit, filtration efficiency, and pressure drop, along with the relationship between (1) surface charge of the electret layer, and (2) elastic properties of the straps. Decontamination with VHP, wet heat, UVI, and bleach did not degrade fit and filtration performance or electret charge. Isopropyl alcohol and soap significantly degraded fit, filtration performance, and electret charge. Pressure drop across the respirators was unchanged. Modest degradation of N95 strap elasticity was observed in mechanical fatigue testing, a model for repeated donnings and doffings. CDC recommended decontamination methods including VHP, wet heat, and UV light did not degrade N95 respirator fit or filtration performance in these tests. Extended use of N95 respirators may degrade strap elasticity, but a loss of face seal integrity should be apparent during user seal checks. NIOSH recommends performing user seal checks after every donning to detect loss of appropriate fit. Decontamination methods which degrade electret charge such as alcohols or detergents should not be used on N95 respirators. The loss of N95 performance due to electret degradation would not be apparent to a respirator user or evident during a negative pressure user seal check.


Assuntos
/prevenção & controle , Descontaminação/métodos , /provisão & distribução , 2-Propanol/farmacologia , Detergentes/farmacologia , Humanos , Peróxido de Hidrogênio/farmacologia , Hipoclorito de Sódio/farmacologia , Eletricidade Estática , Raios Ultravioleta
19.
PLoS One ; 15(12): e0243922, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-33373373

RESUMO

Habits such as smoking and alcohol drinking and existing esophageal malfunction are considered the main risk factors for esophageal carcinogenesis. Caustic ingestion of acidic or alkaline agents or strong irritants can induce severe esophageal corrosive injury and increase esophageal cancer risk. We studied the relationship between esophageal carcinoma and acute detergent or pesticide poisoning by using nationwide health insurance data. Methodology/Principle findings: We compared a pesticide/detergent intoxication cohort (N = 21,840) and an age- and gender-matched control cohort (N = 21,840) identified from the National Health Insurance Research Database between 2000 and 2011. We used the multivariable Cox proportional model to determine esophageal carcinoma risk. The overall incidence density of esophageal cancer was 1.66 per 10,000 person-years in the comparison cohort and 4.36 per 10,000 person-years in the pesticide/detergent intoxication cohort. The corresponding adjusted hazard ratio (HR) for esophageal cancer was 2.33 (95% confidence interval [CI] = 1.41-3.86) in the pesticide/detergent intoxication cohort compared with the control cohort. Patients with corrosive and detergent intoxication did not have a higher risk of esophageal cancer (adjusted HR = 0.98, 95% CI = 0.29-3.33) than those without pesticide/detergent intoxication. However, patients with pesticide intoxication had a significantly higher risk of esophageal cancer (adjusted HR = 2.52, 95% CI = 1.52-4.18) than those without pesticide/detergent intoxication. Conclusion: In the present study, after adjusting for conventional risk factors, we observed that pesticide intoxication could exert substantial effects through increased esophageal cancer risk. However, patients with detergent intoxication may not have an increased risk of esophageal cancer.


Assuntos
Carcinoma/epidemiologia , Cáusticos/envenenamento , Neoplasias Esofágicas/epidemiologia , Praguicidas/envenenamento , Adulto , Idoso , Carcinoma/induzido quimicamente , Carcinoma/patologia , Estudos de Coortes , Detergentes/envenenamento , Ingestão de Alimentos/efeitos dos fármacos , Neoplasias Esofágicas/induzido quimicamente , Neoplasias Esofágicas/patologia , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Modelos de Riscos Proporcionais , Fatores de Risco
20.
Biomed Khim ; 66(6): 469-476, 2020 Nov.
Artigo em Russo | MEDLINE | ID: mdl-33372905

RESUMO

Using electrospray ionization tandem mass spectrometry, a comparative analysis of the HaCaT keratinocyte proteins encoded by the 18th chromosome was performed before and after exposure to sodium dodecyl sulfate (25 mg/ml) and to Triton X-100 (12.5 mg/ml) in a subtoxic dose for 48 hours. Proteins were identified using the SearchGUI platform (X!Tandem and MS-GF+ search engines). In total, 1284 proteins were found in immortalized human HaCaT keratinocytes and about 75% of them were identified by two or more peptides. Were identified, that 26 proteins were encoded by genes of chromosome 18. Among these proteins, 17 were common for control cells and HaCaT cells treated with SDS. Proteins MARE2 and CTIF were identified only in control keratinocytes. Seven identified proteins encoded by genes of chromosome 18 were found only in detergent-treated keratinocytes: LMAN1, NDUV2, SPB3, VPS4B, KDSR, ROCK1 and RHG28.


Assuntos
Queratinócitos , Linhagem Celular , Cromossomos Humanos Par 18/genética , Detergentes/farmacologia , Humanos , Lectinas de Ligação a Manose , Proteínas de Membrana , Proteoma/genética , Dodecilsulfato de Sódio/farmacologia , Quinases Associadas a rho
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