Cholesterol-associated lysosomal disorder triggers cell death of hematological malignancy: Dynamic analysis on cytotoxic effects of LW-218

The integrity of lysosomes is of vital importance to survival of tumor cells. We demonstrated that LW-218, a synthetic flavonoid, induced rapid lysosomal enlargement accompanied with lysosomal membrane permeabilization in hematological malignancy. LW-218-induced lysosomal damage and lysosome-dependent cell death were mediated by cathepsin D, as the lysosomal damage and cell apoptosis could be suppressed by depletion of cathepsin D or lysosome alkalization agents, which can alter the activity of cathepsins. Lysophagy, was initiated for cell self-rescue after LW-218 treatment and correlated with calcium release and nuclei translocation of transcription factor EB. LW-218 treatment enhanced the expression of autophagy-related genes which could be inhibited by intracellular calcium chelator. Sustained exposure to LW-218 exhausted the lysosomal capacity so as to repress the normal autophagy. LW-218-induced enlargement and damage of lysosomes were triggered by abnormal cholesterol deposition on lysosome membrane which caused by interaction between LW-218 and NPC intracellular cholesterol transporter 1. Moreover, LW-218 inhibited the leukemia cell growth

A descrição matemática do desempenho eletroanalítico do compósito poli(alaranjado da acridina)-oxihidróxido de vanádio na detecção eletrorredutiva da entacapona / The mathematical description for the electroanalytical function of poly (acridine orange) - vanadium oxyhydroxide in electroreductive detection of entacapone

RESUMO Por meio de uma análise teórica, foi avaliado o desempenho do compósito do oxihidróxido do vanádio trivalente com o polímero do corante alaranjado da acridina, na detecção eletroquímica do fármaco antiparquinsônico entacapona. O processo eletroanalítico é baseado na eletrorredução do fármaco mencionado. Do desenvolvimento e da análise do modelo matemático correspondente, mediante a teoria de estabilidade linear e anáise de bifurcações, foi possível concluir que o compósito pode ser um modificador eficiente para a determinação da entacapona. Os comportamentos oscilatórios e monotônico, neste sistema, também são passíveis de realizar.

SUMMARY By means of a theoretical analysis, the function of trivalent vanadium oxyhydroxyde composite with the polymer of the acridine orange dye for electrochemical evaluation of entacapone antiparkinsonic drug has been evaluated. The electroanalytical process is based on the electrochemical reduction of the mentioned drug. From development and analysis of the correspondent mathematical model by means of linear stability theory and bifurcation analysis, it was possible to conclude that the composite is an efficient electrode modifier for entacapone determination. The oscillatory and monotonic instabilities in this system are also capable to realize.

Cardiotoxicity in Zebrafish Embryos of Euphorbiae Pekinensis Radix Before and After Processing with Vinegar / 中国实验方剂学杂志

Objective:Taking zebrafish embryos as research model, to investigate the toxic effect of different polar fractions of Euphorbiae Pekinensis Radix before and after processing with vinegar on heart. Method:Zebrafish embryos with normal development at 12 h after fertilization were treated with petroleum ether, dichloromethane and ethyl acetate extracts of Euphorbiae Pekinensis Radix before and after processing with vinegar for observation of cardiac development and function at 72 h. Result:Various polar fractions of Euphorbiae Pekinensis Radix before and after processing with vinegar had the cardiotoxicity on zebrafish embryos in a dose-dependent manner. In addition, the cardiotoxicity of different polar fractions was followed by petroleum ether, dichloromethane and ethyl acetate. The cardiotoxicity was mainly manifested as slow cardiac development, pericardial edema, decrease of heart rate and apoptosis of cardiac cells. Compared with the corresponding polar fraction of raw products, the cardiotoxicity of the same polar fraction of vinegar-processed products with similar doses decreased. Conclusion:Euphorbiae Pekinensis Radix has cardiotoxicity to zebrafish embryos and the cardiotoxicity is reduced after processing with vinegar, which can provide some experimental basis for further elucidation of the detoxication mechanism of Euphorbiae Pekinensis Radix processed with vinegar.

Mesoporous silica nanoparticles for drug and gene delivery

Mesoporous silica nanoparticles (MSNs) are attracting increasing interest for potential biomedical applications. With tailored mesoporous structure, huge surface area and pore volume, selective surface functionality, as well as morphology control, MSNs exhibit high loading capacity for therapeutic agents and controlled release properties if modified with stimuli-responsive groups, polymers or proteins. In this review article, the applications of MSNs in pharmaceutics to improve drug bioavailability, reduce drug toxicity, and deliver with cellular targetability are summarized. Particularly, the exciting progress in the development of MSNs-based effective delivery systems for poorly soluble drugs, anticancer agents, and therapeutic genes are highlighted.

Acridine orange staining for fast identification ofthe spermatogenic cells during STA-PUT velocity sedimentation / 基础医学与临床

Objective To establish a fast and accurate technique of identifying the pachytene spermatocytes,round spermatids and elongating and condensing spermatids during STA-PUT velocity sedimentation.Methods Using STA-PUT velocity sedimentation method to isolate the pachytene spermatocytes, round spermatids and elongating and condensing spermatids from mouse testes.To determine the cell populations` distribution,each tube of cell fraction was then partially transfered to the 96 plate well,and each well was added with acridine orange dye.Then each well was analyzed using fluorescence microscopy.Results Three types of spermatogenic cells can be identified quickly and accurately by it`s specific cytoplasm/nucleus character using the acridine orange dye staining under fluorescence detection.Conclusions A rubust method to quickly and accurately determine the pachytene spermatocytes,round spermatids and elongating and condensing spermatids during STA-PUT velocity sedimentation is successfully developed.

Improvement of Bacillus strains by mutation for overproduction of exopolygalacturonases.

Pectinases, produced by microorganisms, have wide range application in food industry, textile processing, paper making, coffee and tea fermentation, etc. It accounts for 10% of the global industrial enzymes produced. The most important and widely used commercial pectinase polygalacturonase is produced by alkalophilic strains of Bacillus sp. and Streptomyces sp. Here, we explored 29 bacterial strains isolated from rotten mango samples for polygalacturonase production and selected 16 strains through preliminary screening by well-plate method for enzyme activity. The maximum zone of inhibition of pectin was observed up to 28 mm in diameter but one strain ZM11 was exhibiting no activity. Quantitative dinitrisalicylic acid (DNS) assay for polygalacturonase enzyme was also performed for the selected bacterial isolates. All the strains bestowed significant enzyme activity with the highest activity of 2.4 U/µL exhibited by strain ZM3 (P ≤0.05). Characterization of the isolates was performed using different biochemical tests which also confirmed the isolates as members of the genus Bacillus. Mutation was induced to the selected strains by UV light and acridine orange for different periods of time. Qualitative and quantitative assays of the mutant bacterial isolates showed that the enzyme activity increased to 4.62 U/µL which clearly indicated that induced mutation enhanced the ability of Bacillus strains to produce more polygalacturonase enzyme up to 3-fold as compared to the wild strains (P ≤0.05). Molecular characterization by 16S rRNA sequences further confirmed that the bacterial isolates belong to Bacillus subtilis and B. amyloliquefaciens.

Dental fluorosis: A histological study using Light and Confocal microscopy.

Aims: To observe and characterize the histological features of fluorosed teeth under light and confocal microscope (CFM). Materials and Methods: A total of 25 fluorosed teeth and 5 normal teeth were collected from dentists across Dindigul, a known endemic area of fluorosis in South India. Ground sections of respective teeth were observed under light microscope and the sections were subsequently stained with acridine orange and studied under CFM. Results: Histological changes were observed in the ground sections of fluorosed teeth as compared with the normal teeth. Depending on the degree of fluorosis, the affected teeth showed various features of hypomineralization in enamel and dentin. Conclusions: Fluoride interacts with both mineral phases and organic macromolecules by strong ionic and hydrogen bonds resulting in incomplete crystal growth at prism peripheries. This presents as hypomineralization of enamel and dentin.

Comparison of four diagnostic techniques for detection of Trichomonas vaginalis infection in females attending tertiary care hospital of North India.

Background: Trichomonas vaginalis causes a common sexually transmitted disease trichomoniasis, which may lead to increased risk of transmission of human immunodefi ciency virus infection and other pelvic infl ammatory diseases. Wet mount examination is the most common test for diagnosis, but it has low sensitivity. Acridine orange staining can be used for diagnosis, but it requires special microscopic facility. Culture is considered as the gold standard, but it takes a long time for diagnosis. OSOM Trichomonas Rapid Test is a recently introduced rapid method based on immunochromatographic assay of trichomonal protein antigens. Hence, the present study was done to compare these four diagnostic techniques for detection of trichomoniasis in females with vaginal discharge. Materials and Methods: Vaginal swabs were taken from 835 female patients and wet mount examination, acridine orange staining, culture in Kupferberg medium, and OSOM Trichomonas Rapid Test, were performed. Results: Out of 835 patients included in our study, 68 (8.1%) positive cases of trichomoniasis were detected by culture. OSOM Trichomonas Rapid Test detected 63 (7.5%) cases, acridine orange staining detected 53 (6.3%) cases, whereas, wet mount examination detected only 45 (5.4%) positive cases. OSOM Trichomonas Rapid Test performed well and showed high sensitivity and specifi city of 88.2% and 99.6%, respectively. Conclusion: As OSOM Trichomonas Rapid Test is a point of care test and gave better results than both wet mount examination and acridine orange staining; it can be used as a routine test in peripheral areas lacking laboratory facilities.

The application of acridine orange fluorescent staining method in circulating tumor cell / 天津医药

Objective To establish the screening platform of circulating tumor cells (CTCs) using acridine orange fluo?rescent (AO-F) dyeing method, and to apply it in the screening of peripheral blood CTCs in patients with kidney cancer. Methods Twenty-seven patients with metastatic renal cell carcinoma was included in this study. Primitive tumor cells and kidney cancer cell line 769-P were cultured with different concentrations of fetal bovine serum. Smears were prepared and observed under fluorescence microscopy. The percentage of AO-F positive staining of 769-P cells under 5 random sights was calculated. The sensitivity of AO-F staining to cells was evaluated. The 5 mL morning fasting venous blood was obtained from 10 subjects with healthy check-up. The 1×106 cell suspension was prepared. The logarithmic phase of renal tumor cells was used to prepare tube containing 500, 200, 100, 50 and 10 tumor cell suspension, which were mixed with 1×106 nucleated cells to establish CTCs model of renal cancer. AO-F staining method was used to detect the expression of AO-F positive cells. The correlation between expression of AO-F positive cells and clinical parameters was analyzed. Results The prima?ry cells and cell line 769-P showed similar bright color and morphological characteristics. The percentage of AO-F positive staining in 769-P cells was 93%±3%under 5 random sights. The recovery rates (%) of four groups (500, 200, 100 and 50 tu?mor cell suspension) were 10.2±3.8, 9.2±2.3, 10.8±2.6 and 10.5±1.9, respectively. There were no significant differences in recovery rates between four groups (P>0.05). The group of 10 tumor cell suspension could find AO-F positive staining cells occasionally. Zero case was positive in controls. Nine of 27 patients were positive and the rate was 33.33%. There were no significant statistical differences in AO-F positive rates between gender, age, tumor size, pathological pattern, Furhman stage, metastasis of lung and presence of tumor (P>0.05). Conclusion It is confirmed that the method of CTCs staining with AO-F, which has high specificity and reproducibility, is feasible to detect CTCs and worthy of being studied. There is a certain reference value to predict tumor recurrence and metastasis.

Reticulocyte count: comparison among methods / Contagem de reticulócitos: comparação entre métodos

Introduction: The automated counting of reticulocytes has some advantages over the manual method routinely used in clinical laboratories. Technological innovations provide more statistically reliable results, while optimizing the time to perform this test. However, the cost for implementing the automated procedure in laboratory routines still constitutes a barrier to its use in small- and medium-size Brazilian laboratories. Objective: This study evaluated the performance of a new laboratory protocol for reticulocyte counting by flow cytometry using acridine orange (FC/AO), compared with the manual method and with another automated one by flow cytometry using the commercial kit BD Retic-Count (FC/RC) Conclusion: The results showed that, besides being comparable to the manual method, still considered standard, the evaluated new protocol is economically more advantageous than the automated methods currently available, and its cost is comparable to that of the manual method for laboratories that already have appropriate equipment and infrastructure. .

Introdução: A contagem automatizada de reticulócitos apresenta vantagens em relação ao método manual, rotineiramente utilizado em laboratórios clínicos. Inovações tecnológicas permitem resultados estatisticamente mais confiáveis, além de otimizarem o tempo para realização desse exame. No entanto, o custo para aplicação do procedimento automatizado em rotinas laboratoriais ainda constitui uma barreira para sua implementação em laboratórios brasileiros de pequeno e médio porte. Objetivo: O presente estudo avaliou o desempenho de um protocolo laboratorial para contagem de reticulócitos por citometria de fluxo utilizando acridine orange (CF/AO), comparando-o com o método manual e o automatizado por citometria de fluxo utilizando o kit comercial BD Retic-Count (CF/RC). Conclusão: Os resultados mostraram que, além de ser comparável com o método manual, ainda considerado padrão, o protocolo avaliado é economicamente mais vantajoso do que os métodos automatizados atualmente disponíveis, sendo o seu custo comparável com o do método manual para laboratórios que já apresentam aparelhagem e infraestrutura adequadas. .

Contagem de reticulócitos por citometria de fluxo utilizando acridine orange: validação de um novo protocolo / Flow cytometry reticulocyte counting using acridine orange: validation of a new protocol

Introduction: Currently, the reticulocyte counting is a challenge for clinical laboratories in Brazil, mainly for the ordinary ones, which still use the manual method. This method has some limitations, since it consists of a laborious method, time consuming, with low accuracy. Objectives: This study has developed and evaluated the performance of a New Laboratory Protocol for flow cytometry (FC) reticulocytes counting using acridine orange (AO) as dye, aiming to standardize a more precise, easy, fast implementation, and low cost protocol. After standardization of the New Protocol (FC/AO), it was compared with the manual method. The results were analyzed according to the recommendations of the National Committee for Clinical Laboratory Standards (NCCLS), now known as Clinical and Laboratory Standards Institute (CLSI), to evaluate the interchangeability of methods in linear regression analysis and paired t test, besides other quality control tests. Conclusion: Based on these results concerning to the correlation between the methods and the tests related to quality control, we can admit that FC/AO for reticulocyte counting shows undeniable advantages when compared to the preexisting manual method...

Introdução: Atualmente, a contagem de reticulócitos representa um desafio para os laboratórios clínicos no Brasil, principalmente os de pequeno e médio porte, nos quais ainda se utiliza o método manual. Este método apresenta algumas limitações, classificando-se como tedioso, demorado e de baixa precisão. Objetivos: O presente estudo desenvolveu e avaliou o desempenho de um novo protocolo laboratorial para contagem de reticulócitos por citometria de fluxo (CF) utilizando acridine orange (AO) como corante, visando padronizar um protocolo preciso, de fácil e rápida execução e custo acessível. Após a padronização do novo protocolo desenvolvido (CF/AO), fez-se a comparação com o método manual. Os resultados foram analisados de acordo com recomendações do National Committee for Clinical Laboratory Standards (NCCLS), atualmente Clinical and Laboratory Standards Institute (CLSI), para avaliar a intercambialidade entre os métodos, por meio da análise de regressão linear e teste t pareado, além de outros testes de controle de qualidade. Conclusão: Diante dos resultados obtidos referentes à correlação entre os métodos e os testes voltados ao controle de qualidade, pode-se admitir que o CF/AO estabelecido para contagem de reticulócitos possui vantagens inegáveis quando comparado com o método manual...

Excretion product of shigella dysenteriae (sdyep) induced cell death in early larval stage of zebrafish (danio rerio): acridine orange and ethidium bromide (AO/EB) in vivo staining / Producto de excreción de shigella dysenteriae (pesdy) induce muerte celular en larvas de pez cebra (danio rerio): marcaje in vivo con naranja de acridina y bromuro de etidio (NA/BE)

In the field of studies of acute toxicity induced by bacterial agents, Shiga toxins have been relevant due to the severity of the extra-intestinal diseases they cause. Numerous studies have shown that Shiga toxin induced apoptosis in different cell types; however, this important process has been little studied in vivo experimental models. In this study, the effects of excretion products of Shigella dysenteriae, in which Shiga toxin is present, were investigated on early larval stages of Zebrafish, an animal model with many advantages over other in vivo experimental models traditionally used. Both the collection of eggs and larvae of Zebrafish, and the product from excretion from Shigella dysenteriae (SdyEP) were performed according to laboratory standards. Also, toxicity bioassay, larvae treatment with pure and diluted solution, 10-1, 10-2, 10-3 , 10-4 and 10-5, v/v SdyEP and cell death in vivo using Acridine Orange (AO) and Ethidium Bromide (EB) were applied. The excretion product of Shigella dysenteriae (SdyEP) effect was expressed in terms of larval mortality and dependent dilution rather than incubation time. The larval population surviving treatment with Shigella excretion product presents severe morphological effects. The larval population generally presents notable severe morphological damage, the necrosis state is represented by the opacity of the larvae after being treated for 24 h (b) compared to control. Other changes associated with larval anatomy were also observed; particularly the caudal end curvature was significant into 10%. The use of AO/EB revealed a distribution pattern from fluorescence into green and orange in surviving larvae SdyEP poisoning, there was a large population of dead cells around the anal and caudal region as evidenced by the presence of orange nuclei in greater numbers as controls in the larvae. The results support the application of coloring AO/EB in Zebrafish experimental models for the evaluation of the toxic action of new molecules and new products with therapeutic potential.

En el campo de los estudios de toxicidad aguda inducida por agentes bacterianos, las toxinas Shiga resultan relevantes debido a la severidad de las enfermedades extra-intestinales que causan. Numerosos estudios han demostrado que la toxina Shiga induce la apoptosis en diferentes tipos de células, sin embargo, este importante proceso ha sido poco estudiado en modelos experimentales in vivo. En este estudio, fueron evaluados los efectos del productos de excreción de Shigella dysenteriae (PESdy), sobre estadios larvarios de pez cebra (Danio rerio), un modelo animal con muchas ventajas sobre otros modelos experimentales in vivo utilizados tradicionalmente. Tanto la recolección de los huevos y larvas de pez cebra, así como la obtención del producto de la excreción, se realizaron de acuerdo a los estándares de laboratorio. Poblaciones larvarias, fueron tratadas con distintas soluciones; pura y diluidas, 101, 10-2, 10-3, 10-4 y 10-5, v/v de PESdy. La muerte celular in vivo, usando naranja de acridina (NA) y bromuro de etidio (BE) fue evaluada. El efecto del SdyEP, se expresó como dependiente de la concentracion y del tiempo de exposición. La población de larvas sobrevivientes, presentaron curvatura troncal en un 10%, en relación a los controles. La necrosis se puso en evidencia a través de la opacidad de las larvas después de 24 h. El uso de NA/BE reveló un patrón de distribución de la fluorescencia en verde y naranja en larvas sobrevivientes al tratamiento. Una granpoblación de células muertas, alrededor de la región anal y caudal, se ponen en evidencia por la presencia de núcleos de naranja en mayor número que en los controles. Los resultados apoyan la aplicación de la coloración NA/BE en modelo experimental de pez cebra, para la evaluación de la acción tóxica de nuevas moléculas y nuevos productos de excreción bacterial con potencial terapéutico.

Excretion product of shigella dysenteriae (sdyep) induced cell death in early larval stage of zebrafish (danio rerio): acridine orange and ethidium bromide (ao/eb) in vivo staining / El producto de excreción de la shigella dysenteriae (pesdy) Induce muerte celular en larvas de pez cebra (danio rerio): marcaje in vivo con naranja de acridina y bromuro de etidio (na/be)

In the field of studies of acute toxicity induced by bacterial agents, Shiga toxins have been relevant due to the severity of the extra-intestinal diseases they cause. Numerous studies have shown that Shiga toxin induced apoptosis in different cell types; however, this important process has been little studied in vivo experimental models. In this study, the effects of excretion products of Shigella dysenteriae, in which Shiga toxin is present, we investigated on early larval stages of Zebrafish, an animal model with many advantages over other in vivo experimental models traditionally used. Both the collection of eggs and larvae of Zebrafish, and the product from excretion from Shigella dysenteriae (SdyEP) were performed according to laboratory standards. Also, toxicity bioassay, larvae treatment with pure and diluted solution, 10-1, 10-2, 10-3 , 10-4 and 10-5 v/v SdyEP and cell death in vivo using Acridine Orange (AO) and Ethidium Bromide (EB) were applied. The excretion product of Shigella dysenteriae (SdyEP) effect was expressed in terms of larval mortality and dependent dilution rather than incubation time. The larval population surviving treatment with Shigella excretion product presents severe morphological effects. The larval population generally presents notable severe morphological damage, the necrosis state is represented by the opacity of the larvae after being treated for 24 h (b) compared to control. Other changes associated with larval anatomy were also observed; particularly the caudal end curvature was significant into 10%. The use of AO/EB revealed a distribution pattern from fluorescence into green and orange in surviving larvae SdyEP poisoning, there was a large population of dead cells around the anal and caudal region as evidenced by the presence of orange nuclei in greater numbers as controls in the larvae. The results support the application of coloring AO/EB in Zebrafish experimental...

En el campo de los estudios de toxicidad aguda inducida por agentes bacterianos, las toxinas Shiga resultan relevantes debido a la severidad de las enfermedades extra-intestinales que causan. Numerosos estudios han demostrado que la toxina Shiga induce la apoptosis en diferentes tipos de células, sin embargo, este importante proceso ha sido poco estudiado en modelos experimentales in vivo. En este estudio, fueron evaluados los efectos del productos de excreción de Shigella dysenteriae (PESdy), sobre estadios larvarios de pez cebra (Danio rerio), un modelo animal con muchas ventajas sobre otros modelos experimentales in vivo utilizados tradicionalmente. Tanto la recolección de los huevos y larvas de pez cebra, así como la obtención del producto de la excreción, se realizaron de acuerdo a los estándares de laboratorio. Poblaciones larvarias, fueron tratadas con distintas soluciones; pura y diluidas, 10-1, 10-2, 10-3 , 10-4 and 10-5 v/v de PESdy. La muerte celular in vivo, usando naranja de acridina (NA) y bromuro de etidio (BE) fue evaluada. El efecto del PESdy, se expresó como dependiente de la concentración y del tiempo de exposición. La población de larvas sobrevivientes, presentaron curvatura troncal en un 10 por ciento, en relación a los controles. La necrosis se puso en evidencia a través de la opacidad de las larvas después de 24 h. El uso de NA/BE reveló un patrón de distribución de la fluorescencia en verde y naranja en larvas sobrevivientes al tratamiento. Una gran población de células muertas, alrededor de la región anal y caudal, se ponen en evidencia por la presencia de núcleos de naranja en mayor número que en los controles. Los resultados apoyan la aplicación de la coloración NA/BE en modelo experimental...

Acridine orange stain of Mycobacterium leprae / 나학회지

The Ziehl-Neelson's AFB staining method was mainly used for the AFB observation of the diagnosis of leprosy. However, the fluorescent stain performs better and allows the detection of more positive smears. The limitation for its widespread use has been the high cost for fluorescent microscopes. Novel light-emitting diodes (LED) are inexpensive solutions for fluorescent microscopes, and thus fluorescent stain may be a cost-effective step to improve the diagnosis of leprosy in resource-poor countries. And the comparison of auramine and acridine orange for staining of acid-fast bacteria was showed significantly more acid-fast rods after using acridine orange and the number of "false positive" results was somewhat higher on auramine staining. So acridine orange offers a good alternative to auramine which is considered carcinogenic. This study evaluated the comparison of the Ziehl-Neelson's AFB stain and the acridine orange stain in the skin smear based on PCR. As PCR results were taken as gold standard, results of the study revealed that the sensitivity of Ziehl-Neelson's AFB stain was 50% and that of acridine orange stain was 92.2%. This study confirmed that the fluorescence stain method is more sensitive than the Ziehl-Neelsen's staining method. It is suggested that the training of laboratory technicians on fluorescence microscopy should be scaled up for increased disease control.

Protection effect of hepatocyte growth factor on tumor cells from apoptosis and its mechanism / 中国药学杂志

OBJECTIVE: To investigate the protection of hepatocyte growth factor (HGF) on 5 kinds of tumor cells (B cell lymphoma cell line Raji, human acute myeloid leukemia cell line HL-60, cervical cancer cell line HeLa, prostate cancer cell line PC-3, and non-small cell lung cancer cell line A549) from apoptosis induced by etoposide (VP-16). METHODS: Normal control group, drug group, and HGF protection group were set. CCK-8 assay was used to measure proliferation inhibition on 5 kinds of tumor cells by VP-16. Quantitative and qualitative analysis on 5 kinds of tumor cells was performed through acridine orange (AO) fluorescent staining, flow cytometry, HE staining, and transmission electron microscopy. RESULTS: CCK-8 assay revealed that the concentrations of VP-16, which can significantly inhibit the proliferation of Raji, HL-60, PC-3, HeLa, and A549 cell lines, were 100, 1,400, 200, and 200 μg·mL-1, respectively. The typical morphologic changes of cell apoptosis were observed under transmission electronic microscope. Flow cytometry showed that the apoptotic rates of tumor cells in the drug groups were significantly higher than those in normal control group (P>0.01, P>0.05) and in HGF protection groups (P>0.01, P>0.05). AO and HE staining revealed that cells in normal control group appeared to have regular cell morphology, but the cells apoptotic rates in the drug groups were significantly higher than those in normal control groups (P>0.01, P>0.05) and in HGF protection groups (P>0.01, P>0.05). CONCLUSION: HGF can significantly protect 5 kinds of tumor cells from apoptosis induced by VP-16. The mechanism need further investigation.

Reliability of acridine orange fluorescence microscopy in oral cytodiagnosis.

Context and Aims: The oral cavity is the most predominant location in the head and neck region for primary malignant epithelial tumors. Oral cancer is estimated to be the sixth most common malignancy. Early recognition is imperative for successful treatment and good prognosis. Exfoliative cytology is a simple and reasonably effective technique for rapid initial evaluation of a suspicious oral lesion. The present study was conducted to determine the reliability of acridine orange fluorescence microscopy for cytodiagnosis as a more rapid and easier method for the final evaluation of the cytological specimen. Materials and Methods: Smears were collected from 20 individuals with oral lesions suspicious of malignancy, oral lesions not suggestive of malignancy and normal buccal mucosa. One smear was stained with Papanicolaou stain and another one with acridine orange stain. The differences in the study group and control group were compared by means of the χ2 (Chi-square) test. The results were considered statistically significant whenever P was <0.05. Results: The acridine orange fluorescence stain reliably demonstrated malignant cells based on the differential fluorescence - a cytochemical criterion. The efficacy of the stain was higher than the conventional Papanicolaou stain in screening of oral lesions suspicious of malignancy. However, the acridine orange fluorescence stain did not differentiate effectively between malignant cells and rapidly proliferating cells, as the technique is based on the nucleic acid content. Conclusion: The fluorescent acridine orange method can be used reliably for the screening of carcinomas and it is especially helpful in the follow-up detection of recurrent carcinoma in previously treated cases.

Blood culture gram stain, acridine orange stain and direct sensitivity-based antimicrobial therapy of bloodstream infection in patients with trauma.

Purpose: The purpose of this study was to ascertain if the simple practice of Gram stain, acridine orange stain and direct sensitivity determination of positive blood culture bottles could be used to guide early and appropriate treatment in trauma patients with clinical suspicion of sepsis. The study also aimed to evaluate the error in interpreting antimicrobial sensitivity by direct method when compared to standard method and find out if specific antibiotic-organism combination had more discrepancies. Findings from consecutive episodes of blood stream infection at an Apex Trauma centre over a 12-month period are summarized. Materials and Methods: A total of 509 consecutive positive blood cultures were subjected to Gram staining. AO staining was done in BacT/ALERT-positive Gram-stain negative blood cultures. Direct sensitivity was performed from 369 blood culture broths, showing single type of growth in Gram and acridine orange staining. Results of direct sensitivity were compared to conventional sensitivity for errors. Results: No 'very major' discrepancy was found in this study. About 5.2 and 1.8% minor error rates were noted in gram-positive and gram-negative bacteria, respectively, while comparing the two methods. Most of the discrepancies in gram-negative bacteria were noted in β lactam - β lactamase inhibitor combinations. Direct sensitivity testing was not reliable for reporting of methicillin and vancomycin resistance in Staphylococci. Conclusions: Gram stain result together with direct sensitivity testing is required for optimizing initial antimicrobial therapy in trauma patients with clinical suspicion of sepsis. Gram staining and AO staining proved particularly helpful in the early detection of candidaemia.

Alterações de cromatina em espermatozóides de ovinos e caprinos avaliadas por azul de toluidina e alaranjado de acridina / Chromatin alterations in ram and goat spermatozoa evaluated by toluidine blue and acridine orange

Reprodutores com espermograma normal podem se comportar como subférteis ou passarem por períodos de subfertilidade. As alterações na cromatina dos espermatozóides são possíveis explicações encontradas para tais comportamentos. Conduziu-se este trabalho, com o objetivo de testar a eficiência de azul de toluidina (AT) e do alaranjado de acridina (AA) na identificação de alterações na compactação de cromatina em espermatozóides de ovinos e caprinos, além de avaliar a correlação entre essas alterações e as de morfologia espermática. Para tal, foram avaliadas amostras de sêmen de 15 ovinos e de 15 caprinos, com dez repetições para cada método por animal. Calcularam-se a média, o desvio padrão (DP) e o coeficiente de variação (CV) para cada técnica e animal. Utilizou-se o teste t-Student para avaliar diferença entre as médias obtidas nos dois métodos. Também foram calculados a correlação de Pearson e os coeficientes kappa ponderado e não ponderado para avaliar a concordância entre os métodos com AT e AA. Foi verificado que nem sempre as anomalias morfológicas de cabeça são acompanhadas por alterações na cromatina identificáveis pelos métodos utilizados neste trabalho. O método AT é mais estável e possui maior sensibilidade do que AA para ambas as espécies, sendo o mais indicado para caprinos. Contudo, em razão de apresentar repetibilidade muito baixa, ambos os métodos não são indicados para avaliação espermática em ovinos.

Males with normal spermogram can behave as subfertile or pass for periods of subfertility. Chromatin alterations of spermatozoa can account for such behavior. The objective of the present work was to test the efficacy of toluidine blue (TB) and acridine orange (AO) in the identification of alterations in chromatin compaction in spermatozoa from rams and goats, in addition to evaluate the correlation between those alterations and the ones of spermatic morphology. In order to do that, samples of semen from 15 rams and 15 goats were evaluated with 10 replications for each method. Mean, standard deviation and coefficient of variation were calculated for each animal and technique. Student's t-test was used to evaluate differences between the averages of two methods. Pearson correlation coefficient, weighted and non weighted kappa coefficient were also calculated to evaluate the agreement between methods with TB and AO. It was verified that morphological alterations of head not always are accompanied by alterations in chromatin identified by the methods used in this work. The TB method is more stable and sensitive than AO method for both species and this is most appropriate for goats. However, due to the very low repeatability, both methods are not indicated for evaluating ram spermatozoa.

Interactions of bacterial polysaccharides with cationic dyes: Physicochemical studies.

Capsular polysaccharides (SPS) are an integral component of gram-negative bacteria, and also have potential use as vaccine. In this paper, interactions of SPS isolated from Klebsiella strains K20 and K51 with cationic dyes pinacyanol chloride (PCYN) and acridine orange (AO) were studied by absorbance and fluorescence measurements. Both the polysaccharides having glucuronic acid as the potential anionic site induced strong metachromasy (blue shift ~100 nm) in the PCYN. The spectral changes were studied at different polymer/dye molar ratios (P/D = 0-40). A complete reversal of metachromasy was observed upon addition of co-solvents, suggesting the breakaway of dye molecules from the biopolymer matrix. Binding constant, changes in free energy, enthalpy and entropy of the dye polymer complex were also computed from the spectral data at different temperatures to reveal the nature of the interaction. Quenching of fluorescence of AO by the polymers and the incorporated mechanisms were also explored.

Evaluación de la coloración diferencial de fluorescencia modificada en Pseudomonas spp. aisladas de suelo / Evaluation of the modified flourescence staining differential on Pseudomonas spp. isolated from soil

Se evaluó la coloración diferencial de fluorescencia modificada en Pseudomonas spp. aisladas de suelos de cultivos agrícolas del estado Sucre, a fin de observar eventos microscópicos relacionados con el ciclo celular. Cada especie de Pseudomonas identificada bioquímicamente se sembró en caldos incubados a temperatura ambiente, aerobiosis, durante 15, 20, 30 y 45 minutos, y 1, 24, 48 y 72 horas; luego, se elaboraron y colorearon los extendidos. En las 24 cepas de Pseudomonas identificadas, P. mendocina (41,67 por ciento), P. aeruginosa (37,50 por ciento) y P. putida (20,83 por ciento), se observaron variaciones de tinción en los diferentes tiempos de incubación como verde, amarilla y anaranjada, fluorescentes y de baja fluorescencia. La coloración emplea naranja de acridina que se intercala al ADN, provocando fluorescencia verde, e interactúa con el ARN provocando fluorescencia anaranjada; el decolorante remueve el naranja de acridina no unido al material genético y la fluoresceína de sodio produce color amarillo en bacterias que retienen suficiente cantidad de naranja de acridina. Las variaciones de tinción citoplasmática en Pseudomonas spp., están asociadas a la cantidad de ARN y ADN presente en la célula de acuerdo a la fase de su ciclo celular.

The modified fluorescence staining differential was evaluated using Pseudomonas spp. isolated from cultivated agricultural soils in the State of Sucre, in order to observe microscopic events related to the cellular cycle. Each species of biochemically identified Pseudomonas was inoculated into a broth and incubated at room temperature, aerobiosis, for 15, 20, 30 and 45 minutes and 1, 24, 48 and 72 hours; then, slides were made and stained. For the 24 identified strains of Pseudomonas, P. mendocina (41.67 percent), P. aeruginosa (37.50 percent) and P. putida (20.83 percent), staining variations such as green, yellow and orange, fluorescent and low fluorescence were observed for the different incubation times. The stain uses acridine orange that interacts with DNA by intercalation, causing green fluorescence; it interacts with RNA by electrostatic attraction causing orange fluorescence; the alcohol-acetone decolorant removes the acridine orange not united with the genetic material and sodium fluorescein produces a yellow color in bacteria that retain a sufficient amount of acridine orange. Cytoplasmatic staining variations in Pseudomonas spp., are associated with the amount of RNA and DNA present in the cells according to the phase of their cellular cycle.