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1.
J Chromatogr A ; 1654: 462448, 2021 Sep 27.
Artigo em Inglês | MEDLINE | ID: mdl-34392123

RESUMO

Although, in general, the application of coated capillaries is recommended for the separation of intact proteins, bare silica capillary is still the most often used capillary due to its simplicity and cheapness. In this work, the performance of bare fused silica capillary for intact protein analysis was compared to that of different (dynamically coated polybrene (PB) and permanently coated linear polyacrylamide (LPA)) coated capillaries using capillary zone electrophoresis - mass spectrometry (CZE-MS). In cases where low pH (pH=1.8) was used in bare silica capillaries, good precision (0.56-0.78 RSD% and 1.7-6.5 RSD% for migration times and peak areas, respectively), minimal adsorption and separation efficiency (N= 27 000/m - 322 000/m) similar to or even better than those obtained with the coated capillaries (created by an intricate multi-step process) was achieved. The PB and the LPA capillaries demonstrated their slightly better resolving power in terms of separating the different forms/variants of the same protein (e.g., hemoglobin subunits). Among the studied capillaries the one with LPA coating showed the most stable separations in the long term (n=25: 0.18-0.49 RSD% and 3.1-4.9 RSD% for migration times and peak areas, respectively). For the separation of a few proteins or even a larger number of proteins in biological samples (e.g., snake venom) the application of the simple and cheap bare fused silica capillary can be considered as an efficient choice.


Assuntos
Técnicas de Química Analítica , Eletroforese Capilar , Espectrometria de Massas , Proteínas , Resinas Acrílicas/química , Técnicas de Química Analítica/instrumentação , Técnicas de Química Analítica/métodos , Eletroforese Capilar/instrumentação , Brometo de Hexadimetrina/química , Proteínas/química , Proteínas/isolamento & purificação
2.
Hematology ; 26(1): 365-370, 2021 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-33971806

RESUMO

OBJECTIVES: Treating red blood cells (RBCs) with dithiothreitol (DTT) is a wildly-recommended to overcome the interference of the daratumumab (DARA) with blood compatibility testing. Nevertheless, DTT can be hard to obtain in the clinical laboratory, while its use in routine practice may be time-consuming. In the following study, we explored the feasibility of using a commercial 2-mercaptoethanol (2-ME) working solution or the time-saving Polybrene method to mitigate DARA interference. METHODS: Antibody screening and cross-matching were performed using 2-ME or DTT-based indirect antiglobulin tests (IATs) and Polybrene method (with human IgG anti-E same IATs titer as DARA as positive control) on 37 samples. Most clinically important blood group antigens on RBCs were detected after treatment with 2-ME or DTT. RESULTS: Treating RBCs with 2-ME eliminates the DARA interference with the antibody screening or cross-matching; yet, K antigen is denatured during treatment. DARA does not interfere with antibody screening and cross-matching via Polybrene method, while 2+ agglutinations of anti-E antibody with the same titer (IATs method) as DARA could be observed in the positive controls via this method. CONCLUSION: 2-ME-based IATs or Polybrene method could replace DTT-based IATs to mitigate DARA interference.


Assuntos
Anticorpos Monoclonais/química , Tipagem e Reações Cruzadas Sanguíneas , Brometo de Hexadimetrina/química , Mercaptoetanol/química , Feminino , Humanos , Masculino
3.
Methods Mol Biol ; 2143: 55-62, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32524472

RESUMO

The manipulation of gene expression is an essential tool to study the function of genes or signaling pathways. Uniform and robust gene manipulation is crucial for successful assays. However, neuronal cells are generally difficult-to-transfect cells with conventional DNA/RNA transfection reagents. Therefore, virus-mediated gene delivery is a primary choice for the studies of gene functions in neurons. In this chapter, we will describe the methods for lentivirus-mediated gene expression or knockdown in DRG neurons.


Assuntos
Gânglios Espinais/citologia , Vetores Genéticos/genética , Lentivirus/genética , Células Receptoras Sensoriais/virologia , Transdução Genética , Animais , DNA Recombinante/genética , Genes Reporter , Vetores Genéticos/administração & dosagem , Células HEK293 , Brometo de Hexadimetrina/farmacologia , Humanos , Proteínas Luminescentes/genética , Células Receptoras Sensoriais/metabolismo , Células Receptoras Sensoriais/ultraestrutura , Ligação Viral/efeitos dos fármacos
4.
J Cardiovasc Pharmacol ; 75(6): 603-607, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32168154

RESUMO

Adenoviral vectors are useful tools in manipulating a gene of interest in vitro and in vivo, including in the vascular system. The transduction efficiencies of adenoviral vectors in vascular cells such as endothelial cells (ECs) and vascular smooth muscle cells (VSMCs) are known to be lower than those in epithelial cell types. The effective entry for adenoviral vectors is primarily mediated through the coxsackievirus and adenovirus receptor (CAR), which has been shown to be expressed in both cell types. Cationic liposomes have been used to enhance adenovirus transduction efficiency in nonepithelial cells. Accordingly, the aim of this study is to obtain new information regarding differences in transduction efficiencies, cationic liposome sensitivity, and CAR expression between ECs and VSMCs. Using cultured rat aortic ECs and VSMCs, here, we have compared transduction efficiency of adenoviruses with or without inclusion of liposomes and CAR expression. A significant increase in basal transduction efficiency was observed in ECs compared with VSMCs. Cationic liposome polybrene enhanced transduction efficiency in VSMCs, whereas decreased efficiency was observed in ECs. Western blotting demonstrated expression of the CAR in ECs but not in VSMCs. Proteomic analysis and mouse aorta immunostaining further suggests significant expression of the CAR in ECs but not in VSMCs. In conclusion, adenoviruses can effectively transduce the gene of interest in aortic ECs likely because of abundant expression of the CAR, whereas cationic liposomes such as polybrene enhance the transduction efficiency in VSMCs lacking CAR expression.


Assuntos
Adenoviridae/genética , Proteína de Membrana Semelhante a Receptor de Coxsackie e Adenovirus/metabolismo , Células Endoteliais/metabolismo , Vetores Genéticos , Músculo Liso Vascular/metabolismo , Miócitos de Músculo Liso/metabolismo , Transdução Genética , Proteína ADAM17/genética , Proteína ADAM17/metabolismo , Animais , Células Cultivadas , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Brometo de Hexadimetrina/química , Lipossomos , Masculino , Camundongos Endogâmicos C57BL , Ratos , Ratos Sprague-Dawley , Receptor Tipo 1 de Angiotensina/genética , Receptor Tipo 1 de Angiotensina/metabolismo
5.
J Chromatogr A ; 1613: 460625, 2020 Feb 22.
Artigo em Inglês | MEDLINE | ID: mdl-31668999

RESUMO

Jumonji domain-containing proteins (JMJDs) play an important role in the epigenetic regulation of gene expression. Aberrant regulation of histone modification has been observed in the progression of a variety of diseases, such as neurological disorders and cancer. Therefore, discovery of selective modulators of JMJDs is very attractive in new drug discovery. Herein, a simple capillary electrophoresis (CE) method was developed for screening of inhibitors against JMJD3. A known JMJD3 inhibitor GSK-J1, 5-carboxyfluorescein labeled substrate peptide with an amino acid sequence of KAPRKQLATKAARK(me3)SAPATGG (truncated from histone H3), as well as a small chemical library composed of 37 purified natural compounds and 30 natural extracts were used for method development and validation. The separation of substrate from its demethylated product was achieved by addition of polycation hexadimethrine bromide (HDB) in the running buffer. The enzyme activity was thus assayed accurately through separating the demethylated product from the substrate and then measuring the peak area of the product. The enzyme inhibition can be read out by comparing the peak area of the demethylated product obtained in the present of inhibitors and that of the negative control in the absence of any inhibitor. The merit of the method is proved by discovering two new JMJD3 inhibitors: salvianic acid A and puerarin 6''-O-xyloside.


Assuntos
Eletroforese Capilar/métodos , Inibidores Enzimáticos/farmacologia , Histona Desmetilases com o Domínio Jumonji/antagonistas & inibidores , Desmetilação , Brometo de Hexadimetrina/química , Bibliotecas de Moléculas Pequenas
7.
J Chromatogr A ; 1601: 375-384, 2019 Sep 13.
Artigo em Inglês | MEDLINE | ID: mdl-31160095

RESUMO

Therapeutic monoclonal antibodies (mAbs) are complex glycoproteins and ensuring their safety, efficacy and quality is still challenging. Indeed, during their manufacturing process, they are exposed to several stresses that can lead to their denaturation, misfolding or dimerization. We report here a new method based on capillary electrophoresis coupled to native mass spectrometry (MS) with a sheath liquid interface to analyze an intact therapeutic mAb, Infliximab, under non-denaturing conditions that preserve its conformational heterogeneity as well as self-association without inducing further unfolding / denaturation. For capillary zone electrophoresis (CZE) separation, a triple layer coating using polybrene-dextran sulfate-polybrene was employed. A sheath liquid composed of isopropanol - water - acetic acid with a flow rate of 10 µL min-1 and mild MS conditions allowed optimal signal intensities. A specific mass spectrum was obtained for each Infliximab conformation in a "stressed" formulated preparation. This is the first time that within a single analysis different conformational states, i.e. native and unfolded monomers as well as dimers are simultaneously detected. The results and the lack of analytical bias arising from the CZE-MS conditions were confirmed by using atomic force microscopy (AFM) as an orthogonal technique. A middle-up approach combined to CZE-MS analysis of the stressed samples suggested that the dimer formation involved mostly Fab-Fab interactions.


Assuntos
Anticorpos Monoclonais/análise , Eletroforese Capilar , Espectrometria de Massas , Controle de Qualidade , Sulfato de Dextrana/química , Brometo de Hexadimetrina/química , Infliximab/análise
8.
Acta bioquím. clín. latinoam ; 53(1): 37-42, mar. 2019. graf, tab
Artigo em Espanhol | LILACS | ID: biblio-1001076

RESUMO

El ácido siálico tiene importantes funciones biológicas, muchas de las cuales determinan su participación en la respuesta inmune. El objetivo del trabajo fue comparar el efecto de Trichinella spiralis y Trichinella patagoniensis n.sp. sobre la desialización eritrocitaria. Se trabajó con 10 concentrados de larvas musculares de T. spiralis y 10 de T. patagoniensis de la misma concentración larval. Se realizó el tratamiento incubando el sedimento de eritrocitos frescos con igual volumen de concentrado larval (37 ºC), tomando muestra a los 30, 60 y 90 minutos. Los controles fueron incubados de la misma forma con solución salina. Se aplicó el método de Titulación de la Agregación por Polibrene y se determinó el CexpST. Los resultados mostraron que el valor medio del CexpST en los eritrocitos tratados con T. spiralis fue significativamente menor que en los glóbulos tratados con T. patagoniensis, para todos los tiempos estudiados. El aumento del tiempo de tratamiento también disminuyó significativamente el valor medio del CexpST para las dos especies. Éste fue significativamente menor a los 90 minutos de incubación que a los 60 minutos y éstos a su vez menores que a los 30 minutos. Se concluye que T. spiralis provocó mayor desialización eritrocitaria que T. patagoniensis en las condiciones experimentales estudiadas.


Sialic acid has important biological functions, many of which determine its participation in the immune response. The objective of this paper was to compare the effect of Trichinella spiralis and Trichinella patagoniensis n.sp. on erythrocyte desialization. Work was performed on 10 larval concentrates of muscle larvae of T. spiralis and 10 of T. patagoniensis of the same larval concentration. The treatment was carried out incubating the sediment of fresh erythrocytes with an equal volume of larval concentrate (37 °C), taking samples at 30, 60 and 90 minutes. The controls were incubated in the same way treated with saline solution. Titration of aggregation by Polybrene Method was applied and the CexpST was determined. The results showed that the mean value of CexpST in erythrocytes with T. spiralis was significantly lower than in the globules treated with T. patagoniensis, for all the studied times. The increase in treatment time also significantly decreased the mean value of CexpST for the two species, being significantly lower at 90 minutes of incubation than at 60 minutes and these in turn lower than at 30 minutes. It is concluded that T. spiralis caused greater erythrocyte desialization than T. patagoniensis in the experimental conditions studied.


O ácido siálico tem importantes funções biológicas, muitas das quais determinam sua participação na resposta imune. O objetivo foi comparar o efeito de Trichinella spiralis e Trichinella patagoniensis n.sp. sobre a dessialização eritrocitária. Trabalhou-se com 10 concentrados de larvas musculares de T. spiralis e 10 de T. patagoniensis da mesma concentração larval. Realizou-se o tratamento incubando o sedimento de eritrócitos frescos com igual volume de concentrado larval (37 ºC), tomando amostra aos 30, 60 e 90 minutos. Os controles foram incubados da mesma forma com solução salina. Foi aplicado o método de Titulação da Agregação por Polibrene e se determinouo CexpST. Os resultados mostraram que o valor médio do CexpST nos eritrócitos Tratados com T. spiralis foi significativamente menor que nos glóbulos tratados com T. patagoniensis, para todos os tempos estudados. O aumento do tempo de tratamento também diminuiu significativamente o valor médio do CexpST para as duas espécies, sendo significativamente menor aos 90 minutos de incubação que aos 60 minutos e eles por sua vez menores que aos 30 minutos. Conclui-se que T. spiralis provocou maior dessialização eritrocitária que T. patagoniensis nas condições experimentais estudadas.


Assuntos
Trichinella , Trichinella spiralis , Ácidos Siálicos , Glóbulos , Ácido N-Acetilneuramínico , Alergia e Imunologia , Eritrócitos , Solução Salina , Brometo de Hexadimetrina , Sistema Imunitário , Larva , Métodos
9.
Electrophoresis ; 40(7): 1034-1040, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30578636

RESUMO

Electrokinetic motion of a micro oil droplet beneath a glass slide was experimentally investigated in this paper. The micro oil droplets were released under the glass slide in an aqueous solution and the motion along the glass slide was measured by a microscope. The experimental results indicate that while the electrokinetic mobility increases with the applied electric field, it decreases with the oil droplet size and the ionic concentration of the aqueous solution, respectively. By changing the zeta potential of the glass-liquid interface using polybrene coating from negative to positive, the direction of the electrokinetic mobility is reversed and the absolute value of the electrokinetic mobility increases significantly. Finally, pH effects were also investigated, and it was found that the electrokinetic mobility of the droplets reaches a maximum at pH = 6∼8.


Assuntos
Vidro , Gotículas Lipídicas/química , Campos Eletromagnéticos , Brometo de Hexadimetrina/química , Concentração de Íons de Hidrogênio , Cinética , Movimento (Física) , Propriedades de Superfície
10.
PLoS One ; 13(12): e0209606, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30586456

RESUMO

The unique capacity of mesenchymal stem cells (MSCs) to migrate to the sites of damage, following intravenous transplantation, along with their proliferation and differentiation abilities make them promising candidates for MSC-based gene therapy. This therapeutic approach requires high efficacy delivery of stable transgenes to ensure their adequate expression in MSCs. One of the methods to deliver transgenes is via the viral transduction of MSCs. However, due to low transduction efficiency of MSCs, various polications are used to promote the association of viral particles with membranes of target cells. Among these polications polybrene is the most widely used one. Unfortunately, viral infection in presence of polybrene was shown to negatively affect proliferation rate of stem cells. The molecular mechanism underlying this effect is not yet uncovered. Therefore, the present study aimed to elucidate the mechanism of this phenomenon as well as to develop an effective approach to overcome the negative impact of polybrene on the properties of human endometrium-derived mesenchymal stem cells (hMESCs) during lentiviral infection. We found that the negative effect on proliferation observed during the viral infection in presence of polybrene is mediated by the polycation itself. Furthermore, we revealed that the treatment with polybrene alone led to the p38 MAPK-dependent premature senescence of hMESCs. These findings allowed us to develop an effective strategy to attenuate the negative polybrene impact on the hMESCs properties during lentiviral infection by inhibiting the activity of p38 MAPK. Importantly, the proposed approach did not attenuate the transduction efficiency of hMESCs, yet prevented polybrene-induced senescence and thereby restored the proliferation of the infected cells. These results provide the plausible means to reduce side effects of polybrene during the viral infection of primary cells, particularly MSCs.


Assuntos
Senescência Celular/genética , Terapia Genética , Células-Tronco Mesenquimais/virologia , Proteínas Quinases p38 Ativadas por Mitógeno/genética , Apoptose/efeitos dos fármacos , Diferenciação Celular/genética , Proliferação de Células/genética , Endométrio/citologia , Endométrio/efeitos dos fármacos , Endométrio/virologia , Feminino , Citometria de Fluxo , Regulação da Expressão Gênica , Vetores Genéticos/genética , Brometo de Hexadimetrina/farmacologia , Humanos , Lentivirus/genética , Transplante de Células-Tronco Mesenquimais , Fosforilação , Espécies Reativas de Oxigênio , Transdução de Sinais/efeitos dos fármacos , Transdução Genética
11.
Cell Death Dis ; 9(10): 966, 2018 09 20.
Artigo em Inglês | MEDLINE | ID: mdl-30237514

RESUMO

Hexadimethrine bromide (Polybrene) was once used clinically as a heparin neutralizer and has recently found use as a promoter in virus-mediated gene therapy trials and gene transfer in research. However, the potential for tissue-specific toxicity of polybrene at low doses has been ignored so far. Here, we found that after intracerebroventricular (ICV) polybrene injection, mice showed disability of movement accompanied neural death and gliosis in brain, and in human neurons, polybrene induces concentration-dependent neuritic beading and fragmentation. Mechanistically, polybrene induces a rapid voltage-dependent calcium channel (VDCC)-mediated influx of extracellular Ca2+. The elevated cytoplasmic Ca2+ activates DRP1, which leads to mitochondrial fragmentation and metabolic dysfunction. At the same time, Ca2+ influx induces endoplasmic reticulum (ER) fragmentation and tightened associations between ER and mitochondria, which makes mitochondria prone to Ca2+ overloading and ensuing permeability transition. These results reveal an unexpected neuronal toxicity of polybrene, wherein Ca2+ influx serves as a regulator for both mitochondrial dynamics and ER-mitochondrial remodeling.


Assuntos
Cálcio/metabolismo , Retículo Endoplasmático/metabolismo , Brometo de Hexadimetrina/toxicidade , Mitocôndrias/metabolismo , Degeneração Neural/induzido quimicamente , Neurônios/citologia , Neurônios/efeitos dos fármacos , Animais , Células Cultivadas , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Dinâmica Mitocondrial , Espécies Reativas de Oxigênio/metabolismo
12.
J Chin Med Assoc ; 81(9): 830-836, 2018 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-29941298

RESUMO

BACKGROUNDS: The plasticity of retinal stem cells (RSCs), a type of cells that can differentiate into neuron cells and photoreceptor cells, endows them with potential therapeutic properties that can be applied to regenerative medicine. Gene modification of these stem cells before trans-differentiation and transplantation enhances their survival and increases their therapeutic function. The different ways to effectively deliver gene into RSCs are still discussed. This study aimed to use the acoustic waves to improve the efficacy of gene delivery for RSCs. METHODS: RSCs were obtained from non-fetal human ocular pigmented ciliary margin tissues. The enhanced green fluorescent protein-encoded murine stem cell retroviruses (MSCV) were prepared and used to infect RSCs. Glass chambers containing RSCs, retroviruses, and various concentrations of polybrene (0, 0.8, 2, 4 and 8 µg/mL) were exposed under 20 or 25 Vp-p ultrasonic standing wave fields (USWF) for 5 min. The percentage of green fluorescent protein positive cells in each sample was calculated and compared to test the efficacy of gene transduction. RESULTS: Our results showed that the efficiency of gene transduction by MSCV infection was enhanced following the concentration of polybrene and the energy of USWF. The percentage of green fluorescent protein positive cells was significantly higher in chambers that contained 8 µg/mL of polybrene and was exposed to 20Vp-p of USWF for 5 min. In addition, the percentage increased in chambers contained 2, 4 and 8 µg/mL of polybrene when they were exposed to 25Vp-p of USWF. Comparing to those did not treated with ultrasound, the efficiency of retroviral transduction to RSCs increased 4-fold after exposed to USWF for 5 min. CONCLUSION: We demonstrated the ability of ultrasound standing waves to improve retroviral transduction into RSCs. We believe that this may be applied to the experimental designs of future studies and may have possible therapeutic uses.


Assuntos
Retroviridae/genética , Som , Células-Tronco/metabolismo , Transdução Genética/métodos , Adulto , Idoso , Agregação Celular , Separação Celular , Células Cultivadas , Brometo de Hexadimetrina/farmacologia , Humanos , Lactente , Retina
13.
Acta bioquím. clín. latinoam ; 52(2): 235-240, jun. 2018. graf, tab
Artigo em Espanhol | LILACS | ID: biblio-949337

RESUMO

Trichinella spiralis es la especie que causa la mayoría de los casos de infección humana en todo el mundo. Se comunicó que el contacto de los eritrocitos con concentrados de larvas recién nacidas (LRN) no viables provoca la disminución de ácido siálico globular. El objetivo del trabajo fue estudiar la desialización eritrocitaria producida por LRN mantenidas en cultivo. Se realizaron 2 experiencias en las que se incubaron 80 larvas con 100 μL de eritrocitos en 1 mL de medio RPMI suplementado durante 1, 2, 3, 4 y 24 horas a 37 ºC. Se aplicó el Método de Titulación de la Agregación por Polibrene y se calculó Título, Score Total y CexpCASP en los eritrocitos control e incubados con LRN. Los resultados mostraron que en la primera y segunda hora la captación de ácido siálico fue moderada. A las 3 horas el título disminuyó significativamente en relación al del control y el CexpCASP (0,14±0,014) indicó la pérdida casi total de ácido siálico globular. Ambos valores se mantuvieron a las 4 y 24 horas. Al comparar con estudios similares realizados con larvas infectantes, se sugiere que, in vivo, las LRN captarían más rápidamente el ácido siálico que las larvas musculares.


Trichinella spiralis is the species that causes most human cases of infection in the world. It was reported that contact of erythrocytes with concentrates of non-viable newborn larvae (NL) causes the decrease in erythrocyte sialic acid. The objective was to study erythrocyte desialylation produced by NL maintained in culture. Two experiments were conducted, in which 80 larvae were incubated with 100 μL of erythrocytes in 1 mL of supplemented RPMI medium for 1, 2, 3, 4 and 24 hours at 37 ºC. Titration of Aggregation by Polybrene Method was used and Title, Total Score and CexpCASP were calculated in Control erythrocytes and erythrocytes incubated with NL. The results showed that the sialic acid capture was moderated in the first and second hour. At three hours of incubation, the Title decreased significantly in relation to Control and CexpCASP (0.14±0.014) indicated almost total loss of erythrocyte sialic acid. Both values were maintained at 4 and 24 hours. When compared to similar studies conducted with infective larvae, it is suggested that, in vivo, NL would capture sialic acid faster than muscle larvae.


Trichinella spiralis é a espécie que causa a maioria dos casos de infecção humana em todo o mundo. Comunicou-se que o contato dos eritrócitos com concentrados de larvas recém-nascidas (LRN), não viáveis, provoca a diminuição de ácido siálico globular. O objetivo do trabalho foi estudar a dessialização eritrocitária produzida por LRN mantidas em cultivo. Foram realizadas duas experiências nas quais se incubaram 80 larvas com 100 μL de eritrócitos em 1 mL de meio RPMI suplementado durante 1, 2, 3, 4 e 24 horas a 37 °C. Foi aplicado o Método de Titulação da Agregação por Polibreno e se calculou Título, Pontuação Total (ST) e CexpCASP nos eritrócitos. Os resultados mostraram que na primeira e segunda hora a captação de ácido siálico foi moderada. Às 3 horas o título diminuiu significativamente em relação ao do controle e o CexpCASP (0,14±0,014) indicou a perda quase total de ácido siálico globular. Ambos os valores se mantiveram às 4 e 24 horas. Ao comparar com estudos similares realizados com larvas infetantes, sugere-se que, in vivo, as LRN captariam mais rapidamente o ácido siálico que as larvas musculares.


Assuntos
Trichinella spiralis/patogenicidade , Técnicas In Vitro , Luto , Trichinella spiralis , Ácido N-Acetilneuramínico , Brometo de Hexadimetrina , Infecções , Larva , Métodos
14.
Front Immunol ; 9: 891, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29867926

RESUMO

Trauma is a leading cause of death worldwide with 5.8 million deaths occurring yearly. Almost 40% of trauma deaths are due to bleeding and occur in the first few hours after injury. Of the remaining severely injured patients up to 25% develop a dysregulated immune response leading to multiple organ dysfunction syndrome (MODS). Despite improvements in trauma care, the morbidity and mortality of this condition remains very high. Massive traumatic injury can overwhelm endogenous homeostatic mechanisms even with prompt treatment. The underlying mechanisms driving MODS are also not fully elucidated. As a result, successful therapies for trauma-related MODS are lacking. Trauma causes tissue damage that releases a large number of endogenous damage-associated molecular patterns (DAMPs). Mitochondrial DAMPs released in trauma, such as mitochondrial DNA (mtDNA), could help to explain part of the immune response in trauma given the structural similarities between mitochondria and bacteria. MtDNA, like bacterial DNA, contains an abundance of highly stimulatory unmethylated CpG DNA motifs that signal through toll-like receptor-9 to produce inflammation. MtDNA has been shown to be highly damaging when injected into healthy animals causing acute organ injury to develop. Elevated circulating levels of mtDNA have been reported in trauma patients but an association with clinically meaningful outcomes has not been established in a large cohort. We aimed to determine whether mtDNA released after clinical trauma hemorrhage is sufficient for the development of MODS. Secondly, we aimed to determine the extent of mtDNA release with varying degrees of tissue injury and hemorrhagic shock in a clinically relevant rodent model. Our final aim was to determine whether neutralizing mtDNA with the nucleic acid scavenging polymer, hexadimethrine bromide (HDMBr), at a clinically relevant time point in vivo would reduce the severity of organ injury in this model. CONCLUSIONS: We have shown that the release of mtDNA is sufficient for the development of multiple organ injury. MtDNA concentrations likely peak at different points in the early postinjury phase dependent on the degree of isolated trauma vs combined trauma and hemorrhagic shock. HDMBr scavenging of circulating mtDNA (and nuclear DNA, nDNA) is associated with rescue from severe multiple organ injury in the animal model. This suggests that HDMBr could have utility in rescue from human trauma-induced MODS.


Assuntos
DNA Bacteriano/imunologia , DNA Mitocondrial/imunologia , Brometo de Hexadimetrina/uso terapêutico , Insuficiência de Múltiplos Órgãos/tratamento farmacológico , Traumatismo Múltiplo/tratamento farmacológico , Choque Hemorrágico/tratamento farmacológico , Adulto , Idoso , Alarminas/imunologia , Alarminas/metabolismo , Animais , Estudos de Coortes , DNA Bacteriano/sangue , DNA Mitocondrial/sangue , Modelos Animais de Doenças , Feminino , Brometo de Hexadimetrina/farmacologia , Humanos , Masculino , Pessoa de Meia-Idade , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/imunologia , Mitocôndrias/metabolismo , Mitocôndrias/patologia , Insuficiência de Múltiplos Órgãos/imunologia , Insuficiência de Múltiplos Órgãos/mortalidade , Insuficiência de Múltiplos Órgãos/patologia , Traumatismo Múltiplo/imunologia , Traumatismo Múltiplo/mortalidade , Traumatismo Múltiplo/patologia , Estudos Prospectivos , Ratos Wistar , Choque Hemorrágico/imunologia , Choque Hemorrágico/mortalidade , Choque Hemorrágico/patologia , Índices de Gravidade do Trauma , Resultado do Tratamento , Adulto Jovem
15.
Methods Mol Biol ; 1730: 295-304, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29363083

RESUMO

In the field of metabolomics, capillary electrophoresis-mass spectrometry (CE-MS) can be considered a very useful analytical tool for the profiling of polar and charged metabolites. However, variability of migration time is an important issue in CE. An elegant way to minimize this problem is the use of non-covalently coated capillaries that is dynamic coating of the bare fused-silica capillary with solutions of charged polymers. In this protocol, an improved strategy for the profiling of cationic metabolites in urine by CE-MS using multilayered non-covalent capillary coatings is presented. Capillaries are coated with a bilayer of polybrene (PB) and poly(vinyl sulfonate) (PVS) or with a triple layer of PB, dextran sulfate (DS), and PB. The bilayer- and triple-layer-coated capillaries have a negative and positive outside layer, respectively. It is shown that the use of such capillaries provides very repeatable migration times.


Assuntos
Metabolômica/instrumentação , Urina/química , Sulfato de Dextrana/química , Eletroforese Capilar/instrumentação , Eletroforese Capilar/métodos , Brometo de Hexadimetrina/química , Humanos , Espectrometria de Massas/instrumentação , Espectrometria de Massas/métodos , Metabolômica/métodos , Polivinil/química , Ácidos Sulfônicos/química , Propriedades de Superfície
16.
Talanta ; 176: 69-76, 2018 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-28917807

RESUMO

A novel non-aqueous capillary electrophoresis - tandem mass spectrometry method for the simultaneous separation, identification and quantification of nine designer benzodiazepines (bentazepam, etizolam, deschloroetizolam, diclazepam, flubromazepam, flubromazolam, nimetazepam, phenazepam, and pyrazolam) was developed. A non-aqueous running electrolyte consisting of 25mM ammonium acetate with 100mM trifluoroacetic acid in acetonitrile was used. The separation was carried out using a semipermanent coated capillary (successive multiple ionic-polymer coating) with a strong anodic electroosmotic flow at a negative separation voltage within twelve minutes. Electrospray ionization with a triple quadrupole mass spectrometry was utilized for the identification and quantification of selected designer benzodiazepines in a positive ionization mode. The developed method was validated and applied on the analysis of spiked serum sample following a simple liquid-liquid extraction. The LODs of the designer benzodiazepines were between 1.5 and 15.0ngmL-1.


Assuntos
Benzodiazepinas/sangue , Drogas Desenhadas/análise , Eletroforese Capilar , Brometo de Hexadimetrina/química , Humanos , Limite de Detecção , Extração Líquido-Líquido , Espectrometria de Massas por Ionização por Electrospray , Espectrometria de Massas em Tandem
17.
Tissue Eng Part A ; 23(21-22): 1274-1282, 2017 11.
Artigo em Inglês | MEDLINE | ID: mdl-28586292

RESUMO

Extracellular vesicles (EVs) are nanometer-scale particles that are secreted by cells and mediate intercellular communication by transferring biomolecules between cells. Harnessing this mechanism for therapeutic biomolecule delivery represents a promising frontier for regenerative medicine and other clinical applications. One challenge to realizing this goal is that to date, our understanding of which factors affect EV uptake by recipient cells remains incomplete. In this study, we systematically investigated such delivery questions in the context of breast cancer cells, which are one of the most well-studied cell types with respect to EV delivery and therefore comprise a facile model system for this investigation. By displaying various targeting peptides on the EV surface, we observed that although displaying GE11 on EVs modestly increased uptake by MCF-7 cells, neuropeptide Y (NPY) display had no effect on uptake by the same cells. In contrast, neurotensin (NTS) and urokinase plasminogen activator (uPA) display reduced EV uptake by MDA-MB-231 cells. Interestingly, EV uptake rate did not depend on the source of the EVs; breast cancer cells demonstrated no increase in uptake on administration of breast cancer-derived EVs in comparison to HEK293FT-derived EVs. Moreover, EV uptake was greatly enhanced by delivery in the presence of polybrene and spinoculation, suggesting that maximal EV uptake rates are much greater than those observed under basal conditions in cell culture. By investigating how the cell's environment might provide cues that impact EV uptake, we also observed that culturing cells on soft matrices significantly enhanced EV uptake, compared to culturing on stiff tissue culture polystyrene. Each of these observations provides insights into the factors impacting EV uptake by breast cancer cells, while also providing a basis of comparison for systematically evaluating and perhaps enhancing EV uptake by various cell types.


Assuntos
Neoplasias da Mama/metabolismo , Vesículas Extracelulares/metabolismo , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Vesículas Extracelulares/efeitos dos fármacos , Feminino , Células HEK293 , Brometo de Hexadimetrina/farmacologia , Humanos , Biblioteca de Peptídeos , Receptores de Superfície Celular/metabolismo , Regulação para Cima/efeitos dos fármacos
18.
Methods Mol Biol ; 1470: 103-19, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-27581288

RESUMO

Short hairpin RNA (shRNA)-pooled screening is a valuable and cost-effective tool for assaying the contribution of individual genes to cell viability and proliferation on a genomic scale. Here we describe the key considerations for the design and execution of a pooled shRNA screen to identify determinants of radiosensitivity.


Assuntos
Genômica/métodos , RNA Interferente Pequeno/efeitos da radiação , Tolerância a Radiação/genética , Antibacterianos/farmacologia , Contagem de Células , Biblioteca Gênica , Células HEK293 , Brometo de Hexadimetrina/farmacologia , Humanos , Reação em Cadeia da Polimerase
19.
Methods Mol Biol ; 1466: 93-105, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-27473484

RESUMO

Purity determination of somatropin as a recombinant protein is important to ensure its safety and quality. This is carried out by capillary zone electrophoresis in double-injection mode using polybrene/chondroitin sulfate A double-coated capillaries. Modification of the capillary wall eliminates protein-wall interactions which results in improved accuracy and precision of the determinations. In the double-injection mode two somatropin samples are analyzed within a single electrophoretic run. Prior to the second injection, the first injected plug is electrophoresed for a predetermined time period in order to adjust the inter-plug distance. Here, the principle for the separation of somatropin charge variants is described.


Assuntos
Eletroforese Capilar/métodos , Hormônio do Crescimento Humano/isolamento & purificação , Sulfatos de Condroitina/química , Brometo de Hexadimetrina/química , Hormônio do Crescimento Humano/química , Humanos , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação
20.
Anal Bioanal Chem ; 408(22): 6123-32, 2016 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-27372716

RESUMO

A capillary electrophoresis-mass spectrometry (CE-MS) method was developed for the characterization and integrity assessment of the Mycobacterium tuberculosis (MTB) antigens TB10.4 and Ag85B and their chemically produced glycoconjugates, which are glycovaccine candidates against tuberculosis (TB). In order to prevent protein adsorption to the inner capillary wall and to achieve efficient separation of the antigen proteoforms, a polyionic multilayer coating of polybrene-dextran sulfate-polybrene (PB-DS-PB) was used in combination with 1.5 M acetic acid as background electrolyte (BGE). Coupling of CE to high-resolution time-of-flight MS was achieved by a coaxial interface employing a sheath liquid of isopropanol-water (50:50, v/v) containing 0.1 % formic acid. The MTB antigens were exposed to experimental conditions used for chemical glycosylation (but no activated saccharide was added) in order to investigate their stability during glycovaccine production. CE-MS analysis revealed the presence of several closely related degradation products, including truncated, oxidized and conformational variants, which were assigned by accurate mass. Analysis of synthesized mannose conjugates of TB10.4 and Ag85B allowed the determination of the glycoform composition of the neo-glycoproteins next to the characterization of degradation products which were shown to be partly glycoconjugated. Moreover, the selectivity of CE-MS allowed specific detection of deamidated species (protein mass change of 1.0 Da only), indicating that chemical glycosylation increased susceptibility to deamidation. Overall, the results show that CE-MS represents a useful analytical tool for the detailed characterization and optimization of neo-glycoconjugate products. Graphical Abstract Flowchart illustrating Mycobacterium tuberculosis (MTB) antigen glycosylation, glycoconjugate variant and degradation product separation by capillary electrophoresis (CE) and their characterization by intact mass spectrometry (MS).


Assuntos
Aciltransferases/química , Antígenos de Bactérias/química , Proteínas de Bactérias/química , Eletroforese Capilar/métodos , Glicoconjugados/química , Mycobacterium tuberculosis/química , Vacinas contra a Tuberculose/química , Aciltransferases/imunologia , Adsorção , Antígenos de Bactérias/imunologia , Proteínas de Bactérias/imunologia , Glicoconjugados/imunologia , Glicosilação , Brometo de Hexadimetrina/química , Humanos , Espectrometria de Massas/métodos , Modelos Moleculares , Mycobacterium tuberculosis/imunologia , Tuberculose/imunologia , Tuberculose/microbiologia , Tuberculose/prevenção & controle , Vacinas contra a Tuberculose/imunologia
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