Enhanced photodynamic therapy of TiO2/N-succinyl-chitosan composite for killing cancer cells

Abstract The aim of this study was to investigate the effect of TiO2/N-succinyl-chitosan composite (TiO2/ NSCS) photodynamic therapy (PDT), while considering the effects of various light sources on the activation of photosensitizer. The methyl thiazolyl tetrazolium assay was used to examine the cell survival rate of the cells. The results showed that glioma cell strain (U251) was the most sensitive cancer cell strain to TiO2/NSCS. When the concentration of TiO2/NSCS was between 0 and 800 μg·mL-1, there was no obvious cytotoxicity to normal liver cells (HL-7702) and U251 cells. During the PDT process, the photokilling effect of TiO2/NSCS on U251 cells under ultraviolet-A (UVA) light irradiation was stronger than that of pure TiO2, and its killing effects were positively correlated with concentration and irradiation time. In addition, both UVA and visible light could excite TiO2/ NSCS, which had significant killing effect on U251 cells. The results of acridine orange/ethidium bromide fluorescent double staining and Annexin V/propidium iodide double staining indicated that TiO2/NSCS under UVA and visible light irradiation could kill U251 cells by inducing apoptosis, and the apoptosis rate of TiO2/NSCS treatment groups was higher than that of TiO2 treatment groups. Therefore, TiO2/NSCS might be used as a potential photosensitizer in PDT.

In vitro evaluation of hydroxyapatite, chitosan, and carbon nanotube composite biomaterial to support bone healing / Avaliação in vitro do biomaterial compósito à base de hidroxiapatita, quitosana e nanotubo de carbono como adjuvante no reparo ósseo

Hydroxyapatite, chitosan, and carbon nanotube composite biomaterial were developed to improve bone healing. Previous studies suggested that a combination of biomaterials and mesenchymal stem cells (MSCs) can potentially help promote bone regeneration. In the present study, we first developed hydroxyapatite, chitosan, and carbon nanotube composite biomaterial. Then, the effect of different concentrations of the extract on the viability of Vero cells (ATCC CCL-81) and MSCs obtained from sheep bone marrow using methylthiazol tetrazolium (MTT) and propidium iodide (PI) assays were evaluated. The biomaterial group demonstrated an absence of cytotoxicity, similar to the control group. Samples with 50% and 10% biomaterial extract concentrations showed higher cell viability compared to samples from the control group (MTT assay). These results suggest that the presence of this composite biomaterial can be used with MSCs. This study also concluded that hydroxyapatite, chitosan, and carbon nanotube composite biomaterial were not cytotoxic. Therefore, these could be used for performing in vivo tests.(AU)

O compósito à base de hidroxiapatita, quitosana e nanotubo de carbono foi desenvolvido com o intuito de auxiliar na consolidação óssea. Estudos anteriores sugerem que a combinação de substitutos ósseos e células-tronco mesenquimais (CTM) podem auxiliar a potencializar e promover a regeneração óssea. No presente estudo, o biomaterial foi desenvolvido e a viabilidade e a citotoxicidade de células Vero (ATCC CCL-81) e CTM obtidas de medula óssea provenientes de ovinos utilizando ensaios metil-tiazol-tetrazólio, MTT e iodeto de propídeo (PI) foram avaliadas em diferentes concentrações de extrato desse compósito. O compósito demonstrou ausência de citotoxicidade com comportamento semelhante ao grupo controle. Amostras com 50% e 10% de concentração de extrato do compósito mostraram resultados maiores comparados ao grupo controle (ensaio MTT). Esses resultados também sugerem que a presença do biomaterial pode ser utilizada em associação a CTM. Assim, esse estudo conclui que o compósito apresentado de hidroxiapatita, quitosana e nanotubo de cabono não foi considerado citotóxico e pode ser utilizado em teste in vivo.(AU)

Luz, giberelinas y profundidad de siembra inciden sobre la germinación de semillas de Amaranthus hybridus L / Light, gibberellins and burial depth affect seed germination of Amaranthus hybridus L

RESUMEN Amaranthus hybridus L., conocido como bledo, es una planta maleza en una gran variedad de cultivos en el mundo, que se caracteriza por producir alto número de semillas latentes. Para generar estrategias de manejo sostenibles en esta especie es necesario comprender mejor la fisiología de sus semillas. Por tanto, el objetivo fue evaluar el efecto de la luz, de las giberelinas y la profundidad de la siembra sobre la germinación de semillas de bledo. En un primer experimento, se evaluaron cuatro tratamientos de luz (rojo, azul, sin luz y luz natural «testigo¼); en el segundo, se imbibieron semillas en diferentes concentraciones de ácido giberélico (0, 200, 400 y 800mg L-1 de GA3) y, en un último experimento, se evaluaron ocho profundidades de siembra (10, 20, 30, 40, 50, 60, 70 y 80mm), utilizando turba como sustrato. Se encontró mayor porcentaje de germinación en oscuridad, indicando que son semillas fotoblásticas negativas. Para el experimento de giberelinas, el mejor resultado se obtuvo con 400 y 800mg L-1 de ácido giberélico. En el tercer experimento, se observó alto porcentaje de emergencia de plántulas en las primeras profundidades (10mm a 30mm); a mayor profundidad hubo menor emergencia. La respuesta diferencial a la luz, a la aplicación de las giberelinas y el alto porcentaje de semillas viables que no germinaron permite concluir que semillas de bledo poseen latencia fisiológica.

ABSTRACT Amaranthus hybridus L., known as bledo, is a weed in a wide variety of crops worldwide, characterized by producing high number of seeds with dormancy. To generate sustainable management strategies of this species, it is necessary to better understand the physiology of its seeds. Therefore, the objective was to evaluate the effect of light, gibberellins, and seed burying depth on seed germination of bledo. In the first experiment, four light conditions were evaluated (red, blue, without light and natural light (control)), in the second experiment the seeds were imbibed in different concentrations of gibberellic acid (0, 200, 400 and 800mg L-1 of GA3), and in the last experiment, 8 planting depths were evaluated (10, 20, 30, 40, 50, 60, 70 or 80mm) using peat as a substrate. A higher percentage of seed germination in darkness was found, indicating that this species had negative photoblastic seeds. For the gibberellin experiment, the best result was obtained with treatment with 400 and 800mg L-1 of gibberellic acid. In the third experiment, a high percentage of seedling emergence was observed from the first depths (10mm to 30mm); the greater was the depth, the less emergence was found. The differential responses to light, the application of gibberellins, and the high percentage of non-germinated viable seeds allowed to conclude that bledo seeds possess physiological dormancy.

Morphological characteristics and seed viability of Schizolobium parahyba (Vell.) S.F. Blake / Características morfológicas y viabilidad de semillas de Schizolobium parahyba (Vell.) S.F. Blake

ABSTRACT Schizolobium parahyba is a fast-growing tropical tree, with various uses: forestry, agroforestry, landscaping, boat building, cabinetmaking and light packaging. Studies of morphometry and viability of forest species seeds are important for their identification, conservation and sustainable use. The objective of the study was to describe the morphometric and viability characteristics of the seeds of S. parahyba. Seeds of free pollination of plantations from three locations in Tierralta (Córdoba - Colombia) were used. In each plantation, five trees were randomly taken and, of each, 100 seeds to estimate the morphometric characteristics and seed weight. The morphological description was made with a sample of 10 seeds. The viability was determined by the tetrazolium test in an experiment under a completely randomized design, with six treatments and four replicates of 25 seeds each. The six treatments corresponded to the combinations of three concentrations of tetrazolium (0.5; 1.0 and 1.5%) and two staining times (2 and 3 hours). The width, length, and ratio width / length of the seed showed little variation, compared to the freshweight of a seed, freshweight of 100 seeds and number of seeds per kilogram. Tetrazolium and germination tests showed similar and reliable results. The concentration of 0.5% tetrazolium, with immersion of 2 hours, was sufficient to determine the viability of seeds of S. parahyba.

RESUMEN Schizolobium parahyba es un árbol tropical de crecimiento rápido, con diversos usos: forestería, agroforestería, paisajismo, construcción de embarcaciones, ebanistería y embalajes livianos. Los estudios de morfometría y viabilidad de semillas de especies forestales son importantes para la identificación, la conservación y el uso sostenible de éstas. El objetivo del estudio fue describir las características morfométricas y viabilidad de la semilla de S. parahyba. Se utilizó semilla sexual de polinizaciónlibre de plantaciones de tres localidades de Tierralta (Córdoba, Colombia). En cada plantación, se tomaron al azar cinco árboles y de cada uno 100 semillas, para la estimación de las características morfométricas y el peso de la semilla. La descripción morfológica, se hizo con una muestra de 10 semillas. La viabilidad, se determinó mediante la prueba de tetrazolio, en un experimento bajo un diseño completamente al azar, con seis tratamientos y cuatro repeticiones de 25 semillas cada una; los seis tratamientos correspondieron a las combinaciones de tres concentraciones de tetrazolio (0,5; 1,0 y 1,5%) y dos tiempos de tinción (2 y 3 horas). Las características de ancho, longitud y relación ancho/longitud de la semilla, acusaron poca variación, en comparación con el peso de una semilla, peso frescode 100 semillas y número de semilla por kilogramo. Las pruebas de tetrazolio y de germinaciónmostraron resultados similares y confiables. La concentración de 0,5% de tetrazolio, con inmersión de 2 horas, es suficiente para determinar la viabilidad de la semilla de S. parahyba.

Viability of Cyperus rotundus L. tubers after application of herbicide in pre- and postemergence / Viabilidade de tubérculos de Cyperus rotundus L. após a aplicação de herbicidas em pré e pós-emergência

Chemical management is the most widely adopted technique to control weeds in sugarcane crops. Purple nutsedge (Cyperus rotundus L.) is among the most difficult species to control. Herbicides need to be absorbed and translocated in a sufficient amount for the tuber chain to be effective. Thus, this study aimed to evaluate the effectiveness of the herbicides sulfentrazone, diclosulam, imazapic, imazapyr, halosulfuron, ethoxysulfuron, monosodium methyl arsenate (MSMA), and 2,4-dichlorophenoxyacetic acid (2,4-D) in controlling C. rotundus and reducing the viability of its tubers. The experiment was carried out in a greenhouse in a completely randomized design, with nine treatments and four replications. The herbicides sulfentrazone (800 g active ingredient ­ ai·ha?1), diclosulam (193.17 g ai·ha?1), imazapic (133 g ai·ha?1), and imazapyr (500 g acid equivalent ­ ae·ha?1) were applied in preemergence at five days after planting the tubers, while halosulfuron (112.5 g ai·ha?1), ethoxysulfuron (135 g ai·ha?1), MSMA (1,975 g ai·ha?1), and 2,4-D (1,340 g ae·ha?1) were applied in postemergence (4 to 5 leaves). The parameters visual control, shoot dry matter, number of tubers and bulbs, weight of tubers + bulbs, number of epigeal manifestations, and tuber viability were analyzed. The herbicides diclosulam, halosulfuron, and ethoxysulfuron provided 100% control of the shoot at 90 days after application (DAA). All herbicides reduced the number of bulbs, weight of tubers + bulbs, and shoot dry matter. The herbicides sulfentrazone, imazapic, halosulfuron, ethoxysulfuron, and MSMA provided the highest reduction in tuber viability.(AU)

O manejo químico é o mais adotado para o controle de plantas daninhas na cultura da cana-de-açúcar. Entre as espécies de difícil controle está a tiririca (Cyperus rotundus L.). Os herbicidas utilizados precisam ser absorvidos e transportados para a cadeia de tubérculos em quantidades suficientes para serem eficazes. Assim, objetivou-se com este estudo avaliar a eficácia dos herbicidas sulfentrazona, diclosulam, imazapique, imazapir, halossulfurom, etoxissulfurom, metano arseniato ácido monossodico (MSMA) e ácido diclorofenoxiacético (2,4-D) no controle de C. rotundus e na redução da viabilidade de seus tubérculos. O experimento foi realizado em casa de vegetação, em delineamento inteiramente randomizado, com nove tratamentos e quatro repetições. Os herbicidas sulfentrazone (800 g ingrediente ativo ­ i.a.·ha-1), diclosulam (193,17 g i.a.·ha-1), imazapique (133 g i.a.·ha-1) e imazapir (500 g equivalente ­ e.a.·ha-1) foram aplicados cinco dias após o plantio dos tubérculos em pré-emergência, enquanto que o halossulfurom (112,5 g i.a.·ha-1), etoxissulfurom (135 g i.a.·ha-1), MSMA (1.975 g i.a.·ha-1) e 2,4-D (1.340 g e.a.·ha-1) foram aplicados em pós-emergência (4 a 5 folhas). Foram feitas avaliações visuais de controle, massa seca da parte aérea, número de tubérculos e bulbos, peso de tubérculos + bulbos, número de manifestações epígeas e viabilidade de tubérculos. Aos 90 dias após a aplicação (DAA) os herbicidas diclosulam, halossulfurom e etoxissulfurom propiciaram 100% de controle da parte aérea. Todos os herbicidas reduziram o número de bulbos, peso de tubérculos + bulbos e massa seca da parte aérea. Sulfentrazona, imazapic, halossulfurom, etoxissulfurom e MSMA foram os herbicidas que mais reduziram a viabilidade dos tubérculos.(AU)

Vigor tests in seeds creole of Phaseolus lunatus L / Testes de vigor em sementes crioulas de Phaseolus lunatus L

Seeds with a good physiological quality are essential to high productivity. However, for some seeds, Phaseolus lunatus L., the viability and vigor analyze show deficiency in quality. This work aimed to evaluate the physiological quality of Phaseolus lunatus L. seeds using several vigor tests. The study was realized at the Laboratory of Seed Analysis of the Agricultural Sciences Center of the Federal University of Paraíba, in Areia ­ PB, Brazil. We used seeds from five cultivars of P. lunatus (Branca, Orelha de Vó, Cearense, Rosinha, and Roxinha). To determine the physiological quality, we measured the water content, the viability and vigor test (germination, emergence, electrical conductivity, tetrazolium, first germination count and emergence, seedling length and dry mass). We used a completely randomized design for seeds evaluation, and the field test was performed in randomized blocks. The water content of P. lunatus did not differ among the cultivars, indicating a uniformity in the percentage of moisture. Also, the percentage and speed of emergency and the dry mass of seedlings did not differ among the varieties. The tetrazolium and electrical conductivity tests indicated that Rosinha cultivar had the worst viability concerning the other cultivars, showing a low percentage of living tissue and lower electrical conductivity. The seeds of Roxinha cultivar had the better physiological quality, and the Cearense cultivar had the worst quality evaluation. We recommend the tetrazolium test but not the electrical conductivity test to evaluate the vigor of P. lunatus.

Em qualquer cultura, para se ter uma elevada produtividade é indispensável à aquisição de sementes com alta qualidade fisiológica, mas para sementes de Phaseolus lunatus L., ainda ocorre deficiência em análises de viabilidade e vigor. Dessa forma, o objetivo neste trabalho foi avaliar a qualidade fisiológica das sementes de cultivares de Phaseolus lunatus por diferentes testes de vigor. A pesquisa foi realizada no Laboratório de Análise de Sementes do Centro de Ciências Agrárias da Universidade Federal da Paraíba, em Areia - PB, com sementes de cinco cultivares de Phaseolus lunatus (Branca, Orelha de Vó, Cearense, Rosinha e Roxinha). Para avaliação da qualidade fisiológica das sementes determinou-se o teor de água e realizou-se testes de viabilidade e vigor (germinação, emergência, condutividade elétrica, tetrazólio, primeira contagem de germinação e emergência, comprimento e massa seca de plântulas). O delineamento utilizado foi o inteiramente casualizado, com exceção dos testes realizados em campo, que foi em blocos ao acaso. O teor de água das sementes dos cultivares de feijão-fava não diferiu estatisticamente entre si, indicando que houve uniformidade na porcentagem de umidade. Não houve diferença significativa entre as sementes dos cultivares de feijão-fava quando avaliou-se a porcentagem e velocidade de emergência, bem como a massa seca de plântulas. Os resultados dos testes de tetrazólio e condutividade elétrica constatou-se maior viabilidade para as sementes do cultivar Roxinha, Branca, Cearense e Orelha de Vó quando comparadas com as da Rosinha, uma vez que houve um maior percentual de tecidos vivos e uma menor condutividade elétrica, respectivamente. As sementes do cultivar Roxinha são de melhor qualidade fisiológica e, o cultivar Cearense de pior qualidade; o teste de tetrazólio é recomendado para avaliação do vigor de sementes de cultivares de Phaseolus lunatus, enquanto, o teste de condutividade elétrica não é recomendado para avaliar o vigor das sementes dos cultivares de P. lunatus.

Screening and characterization of siderophore producing endophytic bacteria from Cicer arietinum and Pisum sativum plants

Siderophores are low molecular weight iron chelating secondary metabolites synthesized by various groupsof microorganisms help in scavenging iron-limited conditions. Siderophores produced by endophytic bacteriafacilitate the plant growth by providing iron to plants. The objective of this study was to isolate and screenthe siderophore producing endophytes from nodules and roots of Cicer arietinum and Pisum sativum plants.Out of total 84 isolates, only 14 endophytes produced siderophore and quantitative analysis was also done.Ten best siderophore producers (above 65% siderophore units) were characterized for the type of siderophoreproduced. Most of them were producing hydroxamate and carboxylate type of siderophores. These 10 isolateswere evaluated for other plant growth promoting (PGP) traits in vitro. All of them were producing ammoniaand indole-3-acetic acid (IAA). Isolate CPFR10 was found to be positive for all the PGP traits viz. ammonia,organic acid, HCN, and IAA production. Diversity analysis of these 10 isolates using Amplified rDNARestriction Analysis profile revealed nine genotypes at 90% similarity

Efeitos citotóxicos do aripiprazol nas linhagens celulares MKN45 e NIH3T3 e efeitos genotóxicos nos linfócitos sanguíneos periféricos humanos / Cytotoxic effects of aripiprazole on mkn45 and nih3t3 cell lines and genotoxic effects on human peripheral blood lymphocytes

ABSTRACT BACKGROUND: Gastric cancer is known as the fourth most common cancer. Current treatments for cancer have damaged the sensitive tissues of the healthy body, and in many cases, cancer will be recurrent. Therefore, need for treatments that are more effective is well felt. Researchers have recently shifted their attention towards antipsychotic dopamine antagonists to treat cancer. The anticancer activities of aripiprazole remain unknown. OBJECTIVE: This study aimed to evaluate the efficacy and safety of aripiprazole on gastric cancer and normal cell lines. METHODS: In this regard, the cytotoxicity and genotoxicity of aripiprazole were investigated in MKN45 and NIH3T3 cell lines by methyl tetrazolium assay and on peripheral blood lymphocytes by micronucleus assay. For this purpose, cells were cultured in 96 wells plate. Stock solutions of aripiprazole and cisplatin were prepared. After cell incubation with different concentrations of aripiprazole (1, 10, 25, 50, 100 and 200 μL), methyl tetrazolium solution was added. For micronucleus assay fresh blood was added to RPMI culture medium 1640 supplemented, and different concentrations of aripiprazole (50, 100 and 200 μL) were added. RESULTS: The finding of present study showed that the IC50 of aripiprazole in the cancer cell line (21.36 μg/mL) was lower than that in the normal cell line (54.17 μg/mL). Moreover, the micronucleus assay showed that the frequency of micronuclei of aripiprazole at concentrations below 200 μM was much less than cisplatin. CONCLUSION: Aripiprazole can be a good cytotoxic compound and good candidate for further studies of cancer therapy.

RESUMO CONTEXTO: O câncer gástrico é conhecido como o quarto câncer mais comum. Os tratamentos atuais para o câncer danificaram os tecidos sensíveis do corpo saudável e, em muitos casos, o cancro será recorrente. Portanto, a necessidade de tratamentos que são mais eficazes é desejada. Recentemente, os pesquisadores mudaram sua atenção para os antagonistas antipsicóticos da dopamina para tratar o câncer. As atividades anticâncer de aripiprazol permanecem desconhecidas. OBJETIVO: Este estudo objetivou avaliar a eficácia e a segurança do aripiprazol no câncer gástrico e nas linhagens celulares normais. MÉTODOS: A este respeito, a citotoxicidade e a genotoxicidade do aripiprazol foram investigadas em linhas celulares MKN45 e NIH3T3 por ensaio de metil tetrazólio e em linfócitos periféricos de sangue por ensaio de micronúcleos. Para este efeito, as células foram cultivadas em 96 placas. As soluções de estoque de aripiprazol e cisplatina foram preparadas. Após incubação celular com diferentes concentrações de aripiprazol (1, 10, 25, 50, 100 e 200 μL), a solução de metil tetrazólio foi adicionada. Para o ensaio do micronúcleo o sangue fresco foi adicionado ao meio de cultura RPMI 1640 suplementado, e as concentrações diferentes de aripiprazole (50, 100 e 200 μL) foram adicionadas. RESULTADOS: O presente estudo mostrou que o IC50 de aripiprazol na linhagem celular cancerosa (21,36 μg/mL) foi menor do que na linha celular normal (54,17 μg/ mL). Além disso, o ensaio de micronúcleos demonstrou que a frequência de micronúcleos de aripiprazol em concentrações inferiores a 200 μM foi muito inferior à cisplatina. CONCLUSÃO: O aripiprazol pode ser um bom composto citotóxico e bom candidato para estudos adicionais da terapia do câncer.

Chronic granulomatous disease presenting with small bone osteomyelitis in a young child: A case report

Chronic granulomatous disease (CGD) is a life threatening inherited disorder with varied clinical presentations often characterized by recurrent bacterial and fungal infections along with widespread granulomatous tissue response. The disease results from phagocytic defects characterized by deficiencies in oxidative burst of neutrophils. Nitroblue tetrazolium reduction test (NBT) and Dihydrorhodamine (DHR) with PMA stimulation by flow cytometry are quick, simple, sensitive and specific laboratory tests that help establish early and reliable diagnosis of CGD with an overall improvement in survival and disease prognosis. We report a case of 2-year old child who presented with small bone osteomyelitis involving bilateral feet and was later diagnosed to have autosomal recessive CGD due to mutation in NCF1 gene.

Toxicity of N-Succinyl-Chitosan to Bovine Hemoglobin and Human Liver Cells / 中国药学杂志

OBJECTIVE: To investigate the toxicity of N-succinyl-chitosan (NSCS) to bovine hemoglobin (BHb) and human liver cells (HL-7702). METHODS: BHb was used as a research object and the toxic effect of NSCS was investigated by UV-Vis absorption spectroscopy, fluorescence spectroscopy and synchrotron spectroscopy under the simulative human physiological condition. At the same time, human HL-7702 cells was used as a research object and methyl thiazolyl tetrazolium (MTT) assay was employed to examine the cytotoxicity of NSCS. RESULTS: The results of UV-Vis absorption spectroscopy and MTT showed that the toxicity of NSCS was weak, and substitution degree had little effect on it. The result of fluorescence spectroscopy demonstrated that the intrinsic fluorescence of BHb was quenched by NSCS and the quenching effect slightly increased with the increase of substitution degree. The quenching mechanism was mainly dynamic quenching, and the major driving forces were hydrophobic and electrostatic force. CONCLUSION: The result of synchronous fluorescence spectroscopy reveals that NSCS has almost no influence on the conformation of BHb. The toxicity of NSCS to BHb and HL-7702 is weak.

Detection of hypothalamic infarction in intraluminal middle cerebral artery occlusion model rat by DWI scanning / 上海交通大学学报（医学版）

Objective: To detect and evaluate the hypothalamic infarction in middle cerebral artery occlusion (MCAO) model rat. Methods: For 15 Sprague-Dawley rats weighed 200-250 g, aged 6-8 months, their right middle cerebral artery was occluded for 90 min by a silicon-coated 4-0 nylon filament and reperfused. Sprague-Dawley rats underwent diffusion weighted MR imaging (DWI) scanning (at 1 h and 24 h after reperfusion) and 2, 3, 5-triphenyl tetrazolium chloride (TTC) staining (at 24 h after reperfusion) to determine the hypothalamic and cerebral infarct volume. The relationship between hypothalamic infarct volume and cerebral infarction volume was analyzed by DWI scanning. The results of TTC staining were compared with those of 24 h DWI scanning. Results: Fifteen Sprague-Dawley rats successfully received intraluminal MCAO/reperfusion procedures. The incidences of hypothalamic infarction on brain DWI scanning and TTC staining were 100% and 40% at 24 h after reperfusion, respectively. Therefore, DWI scanning was more sensitive than TTC staining to detect hypothalamic injury (P=0.001). The hypothalamic infarct volume on DWI scanning was (8.59±2.89) mm3 and (11.65±3.19) mm3 at 1 h and 24 h after reperfusion, respectively. On DWI scanning, hypothalamic and cerebral infarct volume at 24 h after reperfusion were correlated with each other significantly (r=0.573, P=0.025), so were the increases of hypothalamic and cerebral infarct volume (r=0.554, P=0.032) from 1 h to 24 h. Conclusion: DWI scanning was more sensitive than TTC staining to detect hypothalamic injury in intraluminal transient MCAO model. Hypothalamic and cerebral infarct volume were correlated with each other.

Viabilidad y germinación de semillas de Hechtiaperotensis (Bromeliaceae) / Viability and germination of Hechtia perotensis (Bromeliaceae) seed

ResumenPoblaciones endémicas de Hechtiaperotensis se han descrito en Puebla y Veracruz, México. La recolección de semillas de buena calidad permite su uso en conservación, investigación y restauración ecológica. Algunos compuestos para evaluar la calidad de las semillas silvestres y endémicas, como nitrato de potasio (KNO3) y ácido giberílico (AG3), se utilizan para incrementar la germinación de las semillas y disminuir la latencia. La prueba de cloruro de trifeniltetrazolio (tetrazolio) se correlaciona con la viabilidad de las semillas porque se basa en la actividad de las deshidrogenasas de tejidos vivos que catalizan la respiración mitocondrial. El objetivo de este estudio fue conocer el tamaño y el peso de las cápsulas y las semillas y la germinación y viabilidad de las semillas de H. perotensis recolectadas en Veracruz en 2012 y 2015. Las hipótesis fueron (1) que la germinación y la viabilidad de las semillas son independientes del año de recolecta, (2) que hay una concentración adecuada de tetrazolio para identificar la viabilidad de las semillas y (3) que el pretratamiento con KNO3 o AG3 incrementa la germinación de las semillas. La germinación se evaluó con un diseño completamente al azar con tres tratamientos (testigo y promotores de la germinación KNO3 0.2 % y AG3 500 mg/L), cuatro tratamientos para la prueba de viabilidad (testigo, 0.2, 0.5 y 1.0 % de tetrazolio) y seis repeticiones de cada tratamiento. La germinación se evaluó en 100 semillas y la viabilidad en 25. Los resultados entre y dentro de años se analizaron con ANDEVA y prueba de comparación múltiple de medias de Tukey. La proporción de semillas no germinadas se cuantificó junto con el número de plántulas normales y anormales, semillas con embrión viable o sin él, y con viabilidad baja o sin viabilidad. En promedio la muestra recolectada en 2012 tuvo 36 % de semillas con embrión viable, 7 con viabilidad baja, 24 % no viables y 33 % sin embrión. Este resultado fue significativamente diferente al de 2015, que presentó 87 % de embriones viables, 10 % con viabilidad baja, 0 % no viables y 3 % sin embrión. La germinación también fue significativamente diferente entre los años (22 y 92 %). Los tratamientos pregerminativos no modificaron la germinación. La germinación y la viabilidad de las semillas de H. perotensis varían significativamente entre los años de recolecta.

Abstract:Endemic populations of Hechtiaperotensis have been described in Puebla and Veracruz, Mexico. Good quality seed collections can be used in conservation, research and ecological restoration. To evaluate seed quality of wild and endemic species, some compounds are used as effective promoters of germination, such as potassium nitrate (KNO3) and gibberellic acid (AG3), because they increase seed germination capacity and reduce latency. The triphenyl tetrazolium chloride (tetrazolium) test correlates seed viability because it is based on the activity of dehydrogenases in live tissues that catalyze mitochondrial respiration. The objective of this study was to obtain information on size and weight of capsules and seeds and seed germination and viability of H.perotensis, collected in Veracruz in the year 2012 and 2015. The hypotheses were 1) that seed germination and viability are independent of the year of collection, 2) that there is a tetrazolium concentration that can identify seed viability better than others, and 3) that pretreatment with KNO3 or AG3 improves seed germination. Seed germination was assessed using a completely randomized design with three treatments (control and the germination promoters 0.2 % KNO3 and 500 mg/L AG3), four treatments for the viability test (control, 0.2, 0.5 and 1.0 % of tetrazolium) and six replicates for each treatment. A total of one hundred seeds for germination experiments, and 25 seeds for the viability test were used. The results between and within years were analyzed with ANOVA and multiple comparison with the Tukey test. The proportion of non-germinated seeds was quantified along with the number of normal and abnormal seedlings, seeds with viable embryo, seeds without embryo, and seeds with low or no viability. On average, for the 2012 collected sample, 36 % had viable embryos, 7 % had low viability, 24 % were not viable and 33 % had no embryo. This result was significantly different from the 2015 sample, for which 87 % of seed showed viable embryos, 10 % had low viability, 0 % was not viable and 3 % had no embryo. Seed germination was also significantly different between years (22 and 92 %) Pregerminative treatments did not improve germination. Seed germination and viability of H. perotensis significantly varied between years of seed collection. Rev. Biol. Trop. 65 (1): 153-165. Epub 2017 March 01.

Alteration of metabolic characteristics on the masseter muscle fiber of unilateral chewing rats and its adenosine monophosphate activated protein kinase regulatory mechanism / 华西口腔医学杂志

<p><b>OBJECTIVE</b>This study aims to determine the influence of unilateral chewing on metabolic characteristics of masseter muscle fibers in rats and the regulatory effect of an adenosine monophosphate activated protein kinase (AMPK) signal pathway on metabolism.</p><p><b>METHODS</b>Rats were submitted to exodontia of all the right maxillary molars and divided into 2, 4, 6, and 8 weeks groups, and corresponding control groups were set as well. Sections were stained by nicotine adenine dinucleotide tetrazolim reductase(NADH-TRase) to demonstrate the types, proportion, and density of masseter muscle fibers. AMPKα1 and p-AMPK(Thr172) levels in bilateral masseter muscles were detected by Western blot.</p><p><b>RESULTS</b>In the 2-week group, the percentage of dark fibers augmented in the ipsilateral side, whereas the percentage of intermediary fibers in the contralateral side was increased accompanied by a decrease of light fibers, compared with the control group (P<0.05). The percentage of dark fibers was increased in the bilateral sides, whereas the percentage of dark fiber in the ipsilateral sides surpassed that of the contralateral sides in the 4, 6, and 8-week groups. The percentage of intermediary fibers was decreased in the bilateral sides in the 6 and 8-week groups (P<0.05). The percentage of light fibers was reduced in the ipsilateral sides in the 8-week group, whereas no alteration was observed in contralateral sides (P>0.05). In the ipsilateral sides, p-AMPK (Thr172)/AMPKα1 levels were increased in the 2 and 4-week groups (P<0.05), whereas no change was observed in the contralateral sides in either group (P>0.05).</p><p><b>CONCLUSIONS</b>Unilateral chewing increases the oxidative metabolic ability in bilateral masseter muscle fibers especially in the non-working side accompanied with change of muscle fiber types. The improvement of aerobic metabolism ability is related to the AMPK signal pathway.
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Effect of Valerian Ligusticum Pill on angiogenesis after injury of cerebral ischemia reperfusion in rats / 药学学报

This study was designed to detect the impact of Valerian Ligusticum Pill (VLP) on cerebral ischemia reperfusion injury in rats, and explore the mechanism of angiogenesis. Sixty SD male rats were randomly divided into five groups, including sham operation group, model group, VLP-low (30mg·kg-1) group, VLP-high (50mg·kg-1) group and nimodipine (10mg·kg-1) group. The ischemia reperfusion injury model was induced by occlusion of middle cerebral artery with suture embolus, reperfusion after 30 minutes' ischemia. When the rats were awake, the first neurological function scores was determined with modified neurological severity score (mNSS). The rats were given VLP (30mg·kg-1, 50mg·kg-1) and nimodipine (10mg·kg-1) through intragastric administration at 2 mL, once a day for a total of 7 days, while an equal amount of distilled water was used in the sham operation group and model control group. After 7 days, the rats were given second neurological function scores, and improvement of neurological function=[the first score]-[the second score]. The rats were sacrificed to investigate the infarction volume percentage with 2,3,5-triphenyl tetrazolium chloride method; do the qualitative and half quantitative analyses for protein vascular endothelial growth factor receptor 2 (VEGFR2) in the tissue of cortex infarction around by Western blot; detect the new blood vessels of cortex infarction around by ki67/lectin immunofluorescence double staining method. Results suggest that VLP could significantly improve the neurological function, reduce the percentage of infarct volume, increase the expression of VEGFR2 and number of new blood vessels in the cortex infarction around compared with model group. In conclusion, VLP may relive the acute cerebral ischemia reperfusion injury in rats by inducing angiogenesis.

The experimental study of 3-day-old newborn rats with hypoxic-ischemic brain damage in MRI / 实用放射学杂志

Objective To explore the changes of MR diffusion imaging (DWI) appearance in newborn rats with hypoxic‐ischemic brain damage(HIBD) ,and its relationship with the changes of Triphenyl tetrazolium chloride (TTC) staining .Methods Using liga‐tion of the left carotid artery method to establish three different degrees of HIBD animal models ;DWI was performed at each time point(6-12 h ,12-24 h ,3 d ,7 d);Fresh brain tissue taken from another model groups of newborn rats in 12 h ,24 h ,3 d ,7 d were staining in TTC ,then we observed its relationship with DWI .Results The lesion location of three model groups mainly distributed in the left side of cortex and subcortical region ,with prolonged hypoxia time ,hippocampus ,lateral side white matter ,thalamus were also have varying degrees of involvement .The right side of the cortex and subcortical in some cases involved .TTC staining showed posi‐tive results in 3 d ,its loss stained area were consistent with DWI abnormal signal area .Conclusion DWI can be evaluation of HIBD lesions early .The early lesions of HIBD mainly distributed in the left side of cortex and subcortical region .

Comparação entre duas metodologias para a realização do teste de redução do tetrazólio nitroazul (NBT) em caprinos / Comparison among two methodologys at realization nitroblue tetrazolium reduction test (NBT) in caprines

O teste de redução do tetrazólio nitroazul (NBT) é um dos métodos mais utilizados para a avaliação do metabolismo oxidativo dos neutrófilos. Porém, o custo por amostra e o tempo de utilização do corante são desvantagens do método. O objetivo deste trabalho foi comparar duas metodologias para realização do teste do NBT a fim de maximizar a sua utilização e diminuir o custo por amostra processada. Foram utilizadas 10 cabras, adultas clinicamente sadias e colhidas amostras de sangue por meio de venipunção jugular em tubos de 10mL sendo retirada uma alíquota de 500µL em tubo "eppendorff" contendo 2µL de heparina. Através desta alíquota foram realizadas três metodologias para o teste do NBT: A) técnica original Park & Good (1970); B) redução de 50% do volume do corante; C) redução de 50% e armazenamento do corante a -20°C por 180 dias. Foram confeccionados e corados esfregaços sanguíneos (May Grünwald-Giemsa) das amostras com as metodologias propostas e realizada contagem de 100 neutrófilos em microscopia óptica para determinar o percentual de neutrófilos reativos ao NBT. A análise estatística pelo método de Análise de Medidas Repetida (ANOVA) demonstrou diferenças significativas (P<0,05) entre os dois métodos e o original (A=14,4±4,6; B=1,9±1,4; C=1,1±0,9), porém apresentou alta correlação pelo teste de Spearman entre os métodos (rs=0,82 (AxB), rs=0,75 (BxC) e rs=0,93 (AxC)). Conclui-se que o teste de Redução do Tetrazólio Nitroazul (NBT) segundo a metodologia descrita por Park & Good (1970) e a redução do corante em 50% do seu volume e a sua redução associada ao congelamento por 180 dias a -20ºC podem ser realizadas em caprinos, porém os valores devem ser comparados com as respectivas metodologias utilizadas.(AU)

The nitroblue tetrazolium reduction test (NBT) is one of the most used methods to evaluate the oxidative metabolism of neutrophils. However, the cost for each sample and dye's life-time are disadvantages of the method. This paper aim was to compare two NBT test methodologies in order to maximize its use and decrease the cost for each sample. It was made using 10 adult and healthy goats and blood samples were taken through venipuncion in jugular vein, using 10mL tubes, and then a 500µL sample was taken in eppendoff tubes with 2µL of heparin. Using this sample were made three methodologies for the NBT test: A) Park & Good (1970) original technique; B) dye reduction in 50% of the volume; C) 50% reduction and dye storage at -20°C for 180 days. It was made and dyed the blood smear (May Grünwald-Giemsa) from the samples, using the proposed methodologies and then made the counting of 100 neutrophils in optic microscopy, to determinate the percentage of NBT reactive neutrophils. The statistic analysis by the method of Repeated Measuring Analysis (ANOVA) evinced significant differences (P<0,05) between the two methods and the original (A=14.4±4.6; B=1.9±1.4; C=1.1±0.9), however it showed high correlation by the Spearman test between the methods (rs=0,82 (AxB), rs=0,75 (BxC) e rs=0.93 (AxC)). It was concluded that the original Park & Good (1970) method and two methodologies for dye reduction in 50% and it's reduction associated with NBT dye freezing for 180 days can be used in caprines, but the values should be compared with their respective methodologies.(AU)

Comparação entre duas metodologias para a realização do teste de redução do tetrazólio nitroazul (NBT) em caprinos

Abstract: The nitroblue tetrazolium reduction test (NBT) is one of the most used methods to evaluate the oxidative metabolism of neutrophils. However, the cost for each sample and dye's life-time are disadvantages of the method. This paper aim was to compare two NBT test methodologies in order to maximize its use and decrease the cost for each sample. It was made using 10 adult and healthy goats and blood samples were taken through venipuncion in jugular vein, using 10mL tubes, and then a 500L sample was taken in eppendoff tubes with 2L of heparin. Using this sample were made three methodologies for the NBT test: A) Park & Good (1970) original technique; B) dye reduction in 50% of the volume; C) 50% reduction and dye storage at -20°C for 180 days. It was made and dyed the blood smear (May Grünwald-Giemsa) from the samples, using the proposed methodologies and then made the counting of 100 neutrophils in optic microscopy, to determinate the percentage of NBT reactive neutrophils. The statistic analysis by the method of Repeated Measuring Analysis (ANOVA) evinced significant differences (P 0,05) between the two methods and the original (A=14.4±4.6; B=1.9±1.4; C=1.1±0.9), however it showed high correlation by the Spearman test between the methods (rs=0,82 (AxB), rs=0,75 (BxC) e rs=0.93 (AxC)). It was concluded that the original Park & Good (1970) method and two methodologies for dye reduction in 50% and it's reduction associated with NBT dye freezing for 180 days can be used in caprines, but the values should be compared with their respective methodologies.

Resumo: O teste de redução do tetrazólio nitroazul (NBT) é um dos métodos mais utilizados para a avaliação do metabolismo oxidativo dos neutrófilos. Porém, o custo por amostra e o tempo de utilização do corante são desvantagens do método. O objetivo deste trabalho foi comparar duas metodologias para realização do teste do NBT a fim de maximizar a sua utilização e diminuir o custo por amostra processada. Foram utilizadas 10 cabras, adultas clinicamente sadias e colhidas amostras de sangue por meio de venipunção jugular em tubos de 10mL sendo retirada uma alíquota de 500L em tubo "eppendorff" contendo 2L de heparina. Através desta alíquota foram realizadas três metodologias para o teste do NBT: A) técnica original Park & Good (1970); B) redução de 50% do volume do corante; C) redução de 50% e armazenamento do corante a -20°C por 180 dias. Foram confeccionados e corados esfregaços sanguíneos (May Grünwald-Giemsa) das amostras com as metodologias propostas e realizada contagem de 100 neutrófilos em microscopia óptica para determinar o percentual de neutrófilos reativos ao NBT. A análise estatística pelo método de Análise de Medidas Repetida (ANOVA) demonstrou diferenças significativas (P 0,05) entre os dois métodos e o original (A=14,4±4,6; B=1,9±1,4; C=1,1±0,9), porém apresentou alta correlação pelo teste de Spearman entre os métodos (rs=0,82 (AxB), rs=0,75 (BxC) e rs=0,93 (AxC)). Conclui-se que o teste de Redução do Tetrazólio Nitroazul (NBT) segundo a metodologia descrita por Park & Good (1970) e a redução do corante em 50% do seu volume e a sua redução associada ao congelamento por 180 dias a -20ºC podem ser realizadas em caprinos, porém os valores devem ser comparados com as respectivas metodologias utilizadas.

The Unreliability of MTT Assay in the Cytotoxic Test of Primary Cultured Glioblastoma Cells

MTT assay is commonly used to assess the cellular cytotoxicity caused by anticancer drugs in glioblastomas. However, there have been some reports insisting that MTT assay exhibited non-specific intracellular reduction of tetrazolium which led to underestimated results of cytotoxicity. Here, we examine whether or not MTT assay can lead to incorrect information regarding alcohol-induced cytotoxicity on immortalized and primary glioblastoma cells. MTT assay was applied to assess the ethanol-induced cytotoxicity at various ethanol concentrations. The cellular cytotoxicity induced by different doses of ethanol was analyzed and compared through several cytotoxic assays. Ethanol-induced cytotoxicity observed through MTT assay on both cell types was shown to be ethanol dose-dependent below a 3% concentration. However, the cytotoxicity was shown to be markedly underestimated only in primary cells at a 5% concentration. RT-PCR and Western Blot showed increased expressions of pro-apoptotic proteins and decreased expressions of anti-apoptotic proteins in an ethanol dose-dependent manner in both cell types. Furthermore, we present a possible mechanism for the unreliable result of MTT assay. A high concentration of ethanol induces more severe membrane damage and increased intracellular concentration of NADH in primary cells which enhances the nonspecific reduction of tetrazolium salt. Together, our findings demonstrate that the cytotoxicity on primary cells could inaccurately be assessed when detected through MTT assay. Therefore, a careful interpretation is needed when one would analyze the cytotoxic results of MTT assay, and it is suggested that other assays must be accompanied to produce more reliable and accurate cytotoxic results on primary glioblastoma cells.

Anti-Mycobacterium tuberculosis activity and cytotoxicity of Calophyllum brasiliense Cambess (Clusiaceae)

We evaluated the in vitro anti-Mycobacterium tuberculosis activity and the cytotoxicity of dichloromethane extract and pure compounds from the leaves of Calophyllum brasiliense. Purification of the dichloromethane extract yielded the pure compounds (-) mammea A/BB (1), (-) mammea B/BB (2) and amentoflavone (3). The compound structures were elucidated on the basis of spectroscopic and spectrometric data. The contents of bioactive compounds in the extracts were quantified using high performance liquid chromatography coupled to an ultraviolet detector. The anti-M. tuberculosis activity of the extracts and the pure compounds was evaluated using a resazurin microtitre assay plate. The cytotoxicity assay was performed in J774G.8 macrophages using the 3-(4,5-dimethyl thiazol-2-yl)-2,5-diphenyl tetrazolium bromide colourimetric method. The quantification of the dichloromethane extract showed (1) and (2) at concentrations of 31.86 ± 2.6 and 8.24 ± 1.1 µg/mg of extract, respectively. The dichloromethane and aqueous extracts showed anti-M. tuberculosis H37Rv activity of 62.5 and 125 µg/mL, respectively. Coumarins (1) and (2) showed minimal inhibitory concentration ranges of 31.2 and 62.5 µg/mL against M. tuberculosis H37Rv and clinical isolates. Compound (3) showed no activity against M. tuberculosis H37Rv. The selectivity index ranged from 0.59-1.06. We report the activity of the extracts and coumarins from the leaves of C. brasiliense against M. tuberculosis.

Morphological characteristics and cell viability of coffee plants calli / Características morfológicas e viabilidade celular de calos de cafeeiro

The aim of this research was to characterize and compare two types of calli from leaf explants of Coffea arabica (cultivar Catiguá). Cells of different types of callus were successfully characterized regarding viability and internal and external morphological characteristics. It was obtained two morphologically distinct types of callus: (i) yellow friable and (ii) transparent watery. The yellow friable calli showed higher cell viability and embryogenic characteristics. Scanning and transmission electron microscopy showed embryogenic characteristics in cells of the yellow friable calli evidenced by the presence of small and isodiametric cells, while transparent watery calli showed elongated cells and large cytoplasm vacuolization.

O objetivo deste trabalho foi caracterizar e comparar dois tipos de calos de explantes foliares de Coffea arabica (cultivar Catiguá). Células de diferentes tipos de calos foram caracterizadas quanto a viabilidade e características morfológicas externas e internas. Foram obtidos dois tipos de calos morfologicamente distintos: (a) amarelo friável e (b) transparente aquoso. Os calos amarelos friáveis apresentaram maior viabilidade celular e características embriogênicas. Microscopia eletrônica de varredura e transmissão mostraram características embriogênicas em calos amarelos friáveis evidenciadas pela presença de células pequenas e isodiamétricas. Os calos transparentes aquosos apresentaram células alongadas e vacuolizadas.