Experiencia en el uso de los protocolos Biomed-2 para el estudio de reordenamientos de TCR e inmunoglobulinas en proliferaciones linfoides en el Instituto Nacional de Cancerología, Colombia / Experience with the BIOMED-2 standardized polymerase chain reaction protocol for immunoglobulin and T- cell receptor clonality analysis in the Instituto Nacional de Cancerología, Colombia

Introducción. El consorcio europeo BIOMED-2 se creó para determinar si una población linfoide de difícil clasificación patológica es clonal. En Colombia, la implementación de estas pruebas comenzó en el 2015 en el Instituto Nacional de Cancerología E.S.E. (Bogotá). Objetivos. Determinar el comportamiento de las pruebas de reordenamiento clonal o clonalidad linfoide. y determinar las dificultades de su uso en nuestro medio verificando su adaptación local y los resultados en una serie retrospectiva de casos y consecutiva de proliferaciones linfoides sometidas a los protocolos BIOMED-2. Materiales y métodos. A partir de las historias clínicas, se recolectaron los datos clínicos e histológicos y los resultados de los análisis de los reordenamientos en todos los casos de proliferaciones linfoides sometidas a los protocolos BIOMED-2, entre febrero de 2015 y mayo de 2019. Resultados. Se hallaron 132 casos, de los cuales 47 se clasificaron mediante los protocolos de Biomed-2 como hiperplasias linfoides reactivas, 62 como linfomas T, 19 como linfomas B y 3 como neoplasias linfoides de linaje no establecido. Solo en un caso falló la extracción de ADN. Según estos resultados, la mayor dificultad diagnóstica para el patólogo fue el análisis de los infiltrados linfoides T, la mayoría (44) de los cuales correspondía a lesiones cutáneas. Conclusiones. Las pruebas de clonalidad pueden usarse en tejidos de diversa calidad en nuestro medio como ayuda en el diagnóstico de proliferaciones linfoides de difícil clasificación. Es importante hacerlas e interpretarlas de manera multidisciplinaria y considerar cada caso por separado.

Introduction: The European BIOMED-2 consortium was created to evaluate clonality in lymphoproliferations that are difficult to diagnose. In Colombia, the implementation of these tests began in 2015 at the Instituto Nacional de Cancerología E.S.E., Bogotá. Objectives: To determine the behavior of the rearrangement tests for lymphoid clonality and the difficulties of its implementation in our field through a series of retrospective and consecutive cases of lymphoid proliferation subjected to the BIOMED-2 protocols. Materials and methods: Clinical and histological data and the results of the rearrangement analysis of all cases of lymphoid proliferation subjected to the BIOMED-2 protocols between February 2015 and May 2019 were collected from clinical histories. Results: We recovered 132 samples from which 47 corresponded to reactive lymphoid hyperplasias, 62 to T lymphomas, 19 to B lymphomas, and three to lymphoid neoplasms of unestablished lineage. Only in one case did DNA extraction fail. According to these results, the greatest diagnostic difficulty for the pathologist was the analysis of T lymphoid infiltrates, most of which (44) were skin lesions. Conclusions: Clonality tests can be used in tissues of different quality to help in the diagnosis of lymphoid proliferations that are difficult to classify. It is important to implement and interpret them in an interdisciplinary way considering each case separately.

Effects of flunixin meglumine, meloxicam, and firocoxib on the acute-phase proteins in horses following standing castration

ABSTRACT: Excessive infection and inflammation are the most common complications associated with castration. The objective of this study was to compare the efficacy of flunixin meglumine (FM), meloxicam (MX), or firocoxib (FX) for inflammation control after castration in horses using acute-phase proteins (APP) as markers of inflammation. Thirty healthy, unbroken, mixed-breed horses (body weight 358.62±45.57kg and age 4.99±2.63 years) were randomly (n=10 animals/group) allocated to receive one of three different post-castration anti-inflammatory medicines: Group 1 (FM 1.1mg/kg bwt, IV, s.i.d for 5 days); Group 2 (MX 0.6mg/kg bwt, IV, s.i.d for 5 days); and Group 3 (FX 0.1mg/kg bwt, IV, s.i.d for 5 days). All horses were castrated in standing position, using the open technique. Serum and peritoneal APP concentrations were measured by sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis (PAGE) and determined before castration (0), and 3, 5, 24, 48, 72, 120 and 168 hours after castration. The results were submitted to analysis of variance using the SAS statistical program, and means were compared by the Student-Newman-Keuls test (p 0.05). Three animals from the MX group developed hyperthermia (with rectal temperatures of 39.8, 39.3 and 38.9°C on day 4, 5 and 6, respectively) and showed local clinical signs of inflammation (inguinal and excessive scrotal edema) and reluctance to walk, as well as a rigid gait of the hind limbs. The same complications were observed in one FX horse. No complications were observed among the FM animals. The castration resulted in significant changes in serum and peritoneal values of total proteins, ceruloplasmin (Cp), transferrin (Tf), albumin (Alb), haptoglobin (Hp) and 1-acid glycoprotein (Gp) in animals of all experimental groups. However, the animals of the MX and FX groups presented more intense acute phase response compared to the animals of the FM group. Changes in the APP were associated with the surgical trauma of castration, but the differences between groups were associated with the ability of the nonsteroidal anti-inflammatory drug to control the inflammation. In conclusion, and based on the findings of acute phase proteins, flunixin is more efficient to control the magnitude of inflammation following castration as compared to meloxicam and firocoxib.

RESUMO: Infecção e inflamação excessivas são as complicações mais comuns associadas à castração. O objetivo deste estudo foi comparar a eficácia do flunixin meglumine (FM), meloxicam (MX) ou firocoxib (FX) no controle da inflamação após a castração em cavalos usando proteínas da fase aguda (APP) como marcadores de inflamação. Trinta equinos saudáveis (358,62±45,57kg; 4,99±2,63 anos) foram em função dos anti-inflamatórios utilizados após as castrações aleatoriamente (n= 10 animais/grupo) alocados em três diferentes grupos: Grupo 1 (FM 1,1mg/kg de peso, IV, sid por 5 dias); Grupo 2 (MX 0,6mg/kg de peso, IV, s.i.d por 5 dias); e Grupo 3 (FX 0,1mg/kg de peso, IV, s.i.d por 5 dias). Todos os cavalos foram castrados em posição quadrupedal, utilizando a técnica aberta. As concentrações de APP sérica e peritoneal foram separadas por eletroforese em gel de poliacrilamida (PAGE) com dodecil-sulfato de sódio (SDS) e determinadas no momento 0 (antes da castração) e com 3, 5, 24, 48, 72, 120 e 168 horas após a castração. Os resultados foram submetidos à análise de variância pelo programa estatístico SAS e as médias foram comparadas pelo teste de Student-Newman-Keuls (p 0,05). Três animais do grupo MX desenvolveram hipertermia (com temperatura retal de 39,8, 39,3 e 38,9° C nos dias 4, 5 e 6, respectivamente) e mostraram sinais clínicos locais de inflamação (edema inguinal e escrotal excessivo) e relutância em andar, bem como marcha rígida dos membros posteriores. As mesmas complicações foram observadas em um cavalo do FX. Não foram observadas complicações entre os animais do FM. Independente do grupo, a castração resultou em alterações significativas nos valores séricos e peritoneais de proteínas totais, ceruloplasmina (Cp), transferrina (Tf), albumina (Alb), haptoglobina (Hp) e glicoproteína ácida 1 (Gp). No entanto, os animais dos grupos MX e FX apresentaram resposta de fase aguda mais intensa quando comparados aos animais do FM. Alterações na resposta de fase aguda deveram-se ao trauma cirúrgico da castração, mas as diferenças entre os grupos foram associadas à capacidade do anti-inflamatório em controlar a inflamação. Em conclusão, baseado da resposta de fase aguda, o flunixin em comparação com o meloxicam e o firocoxib é mais eficiente no controle da inflamação após a castração em equinos.

Effects of flunixin meglumine, meloxicam, and firocoxib on the acute-phase proteins in horses following standing castration / Efeitos do flunixin meglumine, meloxicam e firocoxib na concentração de proteínas da fase aguda após em equinos após castração

Excessive infection and inflammation are the most common complications associated with castration. The objective of this study was to compare the efficacy of flunixin meglumine (FM), meloxicam (MX), or firocoxib (FX) for inflammation control after castration in horses using acute-phase proteins (APP) as markers of inflammation. Thirty healthy, unbroken, mixed-breed horses (body weight 358.62±45.57kg and age 4.99±2.63 years) were randomly (n=10 animals/group) allocated to receive one of three different post-castration anti-inflammatory medicines: Group 1 (FM 1.1mg/kg bwt, IV, s.i.d for 5 days); Group 2 (MX 0.6mg/kg bwt, IV, s.i.d for 5 days); and Group 3 (FX 0.1mg/kg bwt, IV, s.i.d for 5 days). All horses were castrated in standing position, using the open technique. Serum and peritoneal APP concentrations were measured by sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis (PAGE) and determined before castration (0), and 3, 5, 24, 48, 72, 120 and 168 hours after castration. The results were submitted to analysis of variance using the SAS statistical program, and means were compared by the Student-Newman-Keuls test (p<0.05). Three animals from the MX group developed hyperthermia (with rectal temperatures of 39.8, 39.3 and 38.9°C on day 4, 5 and 6, respectively) and showed local clinical signs of inflammation (inguinal and excessive scrotal edema) and reluctance to walk, as well as a rigid gait of the hind limbs. The same complications were observed in one FX horse. No complications were observed among the FM animals. The castration resulted in significant changes in serum and peritoneal values of total proteins, ceruloplasmin (Cp), transferrin (Tf), albumin (Alb), haptoglobin (Hp) and α1-acid glycoprotein (Gp) in animals of all experimental groups. However, the animals of the MX and FX groups presented more intense acute phase response compared to the animals of the FM group. Changes in the APP were associated with the surgical trauma of castration, but the differences between groups were associated with the ability of the nonsteroidal anti-inflammatory drug to control the inflammation. In conclusion, and based on the findings of acute phase proteins, flunixin is more efficient to control the magnitude of inflammation following castration as compared to meloxicam and firocoxib.(AU)

Infecção e inflamação excessivas são as complicações mais comuns associadas à castração. O objetivo deste estudo foi comparar a eficácia do flunixin meglumine (FM), meloxicam (MX) ou firocoxib (FX) no controle da inflamação após a castração em cavalos usando proteínas da fase aguda (APP) como marcadores de inflamação. Trinta equinos saudáveis (358,62±45,57kg; 4,99±2,63 anos) foram em função dos anti-inflamatórios utilizados após as castrações aleatoriamente (n= 10 animais/grupo) alocados em três diferentes grupos: Grupo 1 (FM 1,1mg/kg de peso, IV, sid por 5 dias); Grupo 2 (MX 0,6mg/kg de peso, IV, s.i.d por 5 dias); e Grupo 3 (FX 0,1mg/kg de peso, IV, s.i.d por 5 dias). Todos os cavalos foram castrados em posição quadrupedal, utilizando a técnica aberta. As concentrações de APP sérica e peritoneal foram separadas por eletroforese em gel de poliacrilamida (PAGE) com dodecil-sulfato de sódio (SDS) e determinadas no momento 0 (antes da castração) e com 3, 5, 24, 48, 72, 120 e 168 horas após a castração. Os resultados foram submetidos à análise de variância pelo programa estatístico SAS e as médias foram comparadas pelo teste de Student-Newman-Keuls (p<0,05). Três animais do grupo MX desenvolveram hipertermia (com temperatura retal de 39,8, 39,3 e 38,9° C nos dias 4, 5 e 6, respectivamente) e mostraram sinais clínicos locais de inflamação (edema inguinal e escrotal excessivo) e relutância em andar, bem como marcha rígida dos membros posteriores. As mesmas complicações foram observadas em um cavalo do FX. Não foram observadas complicações entre os animais do FM. Independente do grupo, a castração resultou em alterações significativas nos valores séricos e peritoneais de proteínas totais, ceruloplasmina (Cp), transferrina (Tf), albumina (Alb), haptoglobina (Hp) e glicoproteína ácida α1 (Gp). No entanto, os animais dos grupos MX e FX apresentaram resposta de fase aguda mais intensa quando comparados aos animais do FM. Alterações na resposta de fase aguda deveram-se ao trauma cirúrgico da castração, mas as diferenças entre os grupos foram associadas à capacidade do anti-inflamatório em controlar a inflamação. Em conclusão, baseado da resposta de fase aguda, o flunixin em comparação com o meloxicam e o firocoxib é mais eficiente no controle da inflamação após a castração em equinos.(AU)

Los patrones electroforéticos de proteínas salivales permiten diferenciar los grupos transandino y cisandino de las especies de Rhodnius de Colombia / Salivary proteins electrophoretic patterns enabled differentiating Colombian Rhodnius Trans-Andean and Cis-Andean groups

Introducción. Las especies Rhodnius (Hemiptera: Reduviidae: Triatominae) están conformadas por insectos hematófagos vectores de Trypanosoma cruzi, agente etiológico de la enfermedad de Chagas, y T. rangeli, parásito infectivo pero no patógeno para el vertebrado. El estudio de la diversidad proteica de la saliva de estos insectos permite la obtención de perfiles electroforéticos unidimensionales característicos de algunas especies de triatominos. Sin embargo, el reporte de los patrones electroforéticos de proteínas salivales de las especies de Rhodnius ha sido escaso. Objetivo. Hacer un análisis comparativo de los perfiles electroforéticos unidimensionales de las proteínas salivales de R. colombiensis, R. pallescens, R. pictipes, R. prolixus y R. robustus. Materiales y métodos. Se obtuvieron los perfiles electroforéticos de la saliva de las especies en estudio mediante electroforesis en gel de poliacrilamida con dodecilsulfato sódico (Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis, SDS-PAGE) y se construyó un fenograma mediante el método UPGMA (Unweighted Pair Group Method Using Arithmetic Averages). Resultados. Los perfiles electroforéticos de las proteínas solubles de saliva presentaron bandas en un rango de masa aproximado de 15 a 45 kDa, los cuales permitieron diferenciar las cinco especies estudiadas. El fenograma reveló la existencia de dos grupos principales: uno conformado por los grupos cisandinos Pictipes y Prolixus y otro constituido por el grupo transandino Pallescens. Conclusiones. Existen diferencias en los perfiles electroforéticos de las proteínas salivales entre R. colombiensis, R. pallescens, R. pictipes, R. prolixus y R. robustus, cuya variabilidad permitió construir un fenograma congruente con los grupos del género Rhodnius.

Introduction: Rhodnius (Hemiptera: Reduviidae: Triatominae) species are made up of haematophagous insect vectors for Trypanosoma cruzi (Chagas' disease aetiological agent) and T. rangeli, an infective parasite that is not pathogenic for vertebrate hosts. The study of their salivary protein diversity enables the obtention of characteristic one-dimensional electrophoretic profiles of some triatomine species; however, few reports have dealt with Rhodnius species salivary proteins electrophoretic patterns. Objective: To compare R. colombiensis, R. pallescens, R. pictipes, R. prolixus, and R. robustus' salivary proteins one-dimensional electrophoretic profiles. Materials and methods: SDS-PAGE was used for obtaining electrophoretic profiles of saliva from the species under study. The unweighted pair group method with arithmetic mean (UPGMA) was used for constructing a phenogram. Results: Electrophoretic profiles of soluble saliva had protein bands ranging from 15 to 45 kDa, thereby enabling the five species studied to be differentiated. The phenogram revealed two main groups, one formed by the Pictipes and Prolixus cis-Andean groups and another consisting of the Pallescens trans-Andean group. Conclusion: Differences were revealed regarding R. colombiensis, R. pallescens, R. pictipes, R. prolixus, and R. robustus electrophoretic profiles of salivary proteins; their variability facilitated constructing a phenogram which was taxonomically congruent with the groups from the genus Rhodnius.

A simple and efficient method for removal of phenolic contaminants in wastewater using polyacrylamide entrapped mushroom tyrosinase

Phenolic compounds are priority pollutants with high toxicity even at low concentrations. Ever-increasingburden of pollutants in major rivers and other water bodies along with stringent environmental legislationand focus on adaptation to eco-friendly treatment approaches have necessitated the need for the removal ofthese phenolics before being discharged to rivers and other freshwater bodies. Compared to physicochemicaltreatment, enzymatic treatment has proven to be the best way to treat various phenolic compounds undermild conditions with different enzymes such as peroxidases, laccases, and tyrosinases. In this study, we havedesigned a simple and efficient method for removal of phenols from effluent wastewater using an immobilizedpreparation of mushroom tyrosinase. The enzyme was isolated from Agaricus bisporus (button mushroom)and partially purified, and subsequently, various immobilization matrices were evaluated for their efficiencyof immobilization, reproducibility, rate of degradation of phenolics, stability, and reusability. Experimentsshowed that the in situ polymerization of acrylamide monomer along with the enzyme gave the most effectiveentrapment with high reproducibility among the tested methods. Immobilized tyrosinase was much more stablethan the free tyrosinase in storage and that the immobilized tyrosinase could even retain about most of itsoriginal activity after repeated use of 10 times in a batch system. This method could provide and an economicaland stabilized immobilized-enzyme method for the removal of phenol in wastewater.

Purification and characterization of extracellular alkaline protease from Streptomyces sp. LCJ12A isolated from Pichavaram mangroves

Extracellular protease from culture supernatants of Streptomyces sp. LCJ12A was purified and characterized in thisstudy. The collected supernatant of Streptomyces sp. LCJ12A was purified by ammonium sulfate precipitation anddiethylaminoethyl cellulose chromatography. The molecular weight of the enzyme was 20.1 kDa. Sodium dodecylsulfate-polyacrylamide gel electrophoresis was used to determine the molecular weight of the enzyme. The culture filtrate,partially purified enzyme, and ammonium sulfate precipitate displayed catalysis and stability over pH 3–11.5 and showedoptimal activity at pH 10. The culture filtrate, ammonium sulfate precipitate, and partially purified enzyme were goodbetween 30 and 40°C and the optimum temperature was 35°C. The purified protease exhibited high catalytic activityand stability under different pH and temperature. The partially purified protease from Streptomyces sp. LCJ12A showeda substantial relative activity of 78% with bovine serum albumin (BSA), 43% with gelatin, and 96% relative activitywith casein. Lineweaver–Burk plot was used to calculate the Km and Vmax values for the partially purified protease.The Km values of Streptomyces sp. LCJ12A were 73.5 mM for casein, 44.54 mM for BSA, and 43.45 mM for gelatin.The Vmax values were 500 mM min−1 for casein, 303.03 mM min−1 for BSA, and 270.27 mM min−1 for gelatin fromStreptomyces sp. LCJ12A. The statistical analysis confirmed that compared to the other substrates, casein was significant.

ISSR Analysis of Genetic Relationship and Population Structure of Turpinia arguta Fresh Leaves in Jiangxi Province / 中国实验方剂学杂志

Objective:To explore genetic relationship and population structure of Turpinia arguta in six locations of Jiangxi province by inter-simple sequence repeat （ISSR） molecular marker technique， and to provide theoretical basis for the protection and utilization of this medicinal material resource. Method:A total of 22 samples from six locations in four counties in Jiangxi province were collected， and genomic DNA was extracted by kit method. Polymerase chain reaction （PCR） amplification was performed using sixty-four universal ISSR molecular marker primers， and the products were detected with polyacrylamide gel electrophoresis （PAGE）. NTsys 2.10e software was selected to calculate the genetic similarity coefficient by unweighted pair group method with arithmetic mean （UPGMA） and cluster analysis. Population genetic structure was analyzed by Structure 2.1 software. Result:A total of forty-eight ISSR primers were amplified to obtain the product， the percent of polymorphic bands ranged from 45.45% to 100%. UPGMA cluster analysis showed that these plant individuals could not be clustered according to their respective executive locations. Analysis of population genetic structure showed that 22 samples of T. arguta could be divided into three populations. Conclusion:There is gene exchange among the populations of T. arguta in Jiangxi province， and it can affect the genetic structure of germplasm resources from different geographical sources.

Diagnostic potential of saliva proteome analysis: a review and guide to clinical practice

Abstract: Proteomic techniques have become popular in medicine and dentistry because of their widespread use in analyzing bodily fluids such as blood, saliva, urine, and gingival crevicular fluids as well as hard tissues such as enamel, dentine, and cementum. This review is a guide to proteomic techniques in general dentistry, summarizing techniques and their clinical application in understanding and diagnosing diseases and their use in identifying biomarkers of various diseases.

Discrimination of Rana dybowskii,Its Analogues and Counterfeits Based on Polyacrylamide Gel Electrophoretograms Combined with Cluster Analysis and Multivariate Statistical Analysis / 中国实验方剂学杂志

Objective:To establish an effective classification and identification method for sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) maps of Rana dybowskii,its analogues and counterfeits based on cluster analysis and multivariate statistical analysis. Method:SDS-PAGE maps of 18 batches of R. dybowskii,its analogues and 2 counterfeits were obtained by SDS-PAGE method. SDS-PAGE maps were transformed into data matrix. NTSYSpc 2.10e statistical analysis software was used for cluster analysis,and SMICA-P 14.1 software was used for multivariate statistical analysis. Unsupervised Principal Component Analysis (PCA),Supervised Partial Least Squares Discriminant Analysis (PLS-DA) and Orthogonal Partial Least Squares Discriminant Analysis (OPLS-DA) were performed for multivariate analysis and evaluation. Result:SDS-PAGE maps technology combined with cluster analysis and multivariate statistical analysis could accurately classify and identify R. dybowskii,its analogues and counterfeits. Cluster analysis could cluster four kinds of medicinal materials into four branches except No.1 medicinal materials. PCA results were superior to cluster analysis. Supervised PLS-DA and OPLS-DA results in multivariate statistical analysis were superior to unsupervised PCA. The classification and identification efficiencies of OPLS-DA were better than those of unsupervised PCA. OPLS-DA aggregated R. dybowskii,its analogues and 2 counterfeits into four groups. Six different protein components were obtained by comprehensive analysis of variable importance in projection (VIP) value, and OPLS-DA Bi load diagram,with relative molecular weights were 51.363,35.838,14.565,17.563,15.358 and 21.696 kDa,respectively. Conclusion:SDS-PAGE maps combined with cluster analysis and multivariate statistical analysis can be used as an effective method to classify and identify R. dybowskii,its analogues and counterfeits. This study provides a reference for the quality evaluation and screening of R. dybowskii.

Specification of Bacteriophage Isolated Against Clinical Methicillin-Resistant Staphylococcus Aureus

OBJECTIVES: The emergence of resistant bacteria is being increasingly reported around the world, potentially threatening millions of lives. Amongst resistant bacteria, methicillin-resistant Staphylococcus aureus (MRSA) is the most challenging to treat. This is due to emergent MRSA strains and less effective traditional antibiotic therapies to Staphylococcal infections. The use of bacteriophages (phages) against MRSA is a new, potential alternate therapy. In this study, morphology, genetic and protein structure of lytic phages against MRSA have been analysed. METHODS: Isolation of livestock and sewage bacteriophages were performed using 0.4 μm membrane filters. Plaque assays were used to determine phage quantification by double layer agar method. Pure plaques were then amplified for further characterization. Sulfate-polyacrylamide gel electrophoresis and random amplification of polymorphic DNA were run for protein evaluation, and genotyping respectively. Transmission electron microscope was also used to detect the structure and taxonomic classification of phage visually. RESULTS: Head and tail morphology of bacteriophages against MRSA were identified by transmission electron microscopy and assigned to the Siphoviridae family and the Caudovirales order. CONCLUSION: Bacteriophages are the most abundant microorganism on Earth and coexist with the bacterial population. They can destroy bacterial cells successfully and effectively. They cannot enter mammalian cells which saves the eukaryotic cells from lytic phage activity. In conclusion, phage therapy may have many potential applications in microbiology and human medicine with no side effect on eukaryotic cells.

Acute phase proteins in serum and cerebrospinal fluid in healthy cattle: possible use for assessment of neurological diseases / Proteínas de fase aguda no soro e no líquido cefalorraquidiano de bovinos hígidos: possíveis usos na avaliação de doenças neurológicas

Use of acute-phase proteins (APPs) for assessment of health and disease in animals has increased greatly within the last decade. The objective was to determine the normal concentration of APPs in the serum and cerebrospinal fluid (CSF) of healthy cattle by polyacrylamide gel electrophoresis. Fifty crossbred animals (350±70kg of BW and 18±1.2 months of age), 25 heifers and 25 steers were used. CSF samples were collected from atlanto-occipital (AO) site and blood samples were obtained from the jugular vein. CSF and serum protein electrophoresis were performed by means of sodium dodecyl sulphate-polyacrylamide gel electrophoresis. Thirty-seven proteins with molecular weights ranging from 7 and 37kDa were identified in CSF of all animals. These eight were nominally identified with immunoglobulin A and G, celuloplasmin, transferrin, albumin, α1-antitripsin, acidic glycoprotein, and haptoglobin. All protein fractions in CSF did not differ between heifers and steers. In sera, 34 proteins with molecular weights between 7 and 244kDa were identified in heifers and steers. Similar proteins were nominally identified in the sera, but only the CSF presented α1-antitripsin. The serum values of acidic glycoprotein and immunoglobulin G were significantly higher in steers compared with heifers. In conclusion, measurement of CSF acute phase protein concentrations can be useful in diagnosing and monitoring the progression of bovine neurological diseases, perhaps even to guide therapeutic procedures. The CSF electrophoretic profile of healthy cattle does not change depending on gender.(AU)

O uso de proteínas de fase aguda (PFAs) para a avaliação da saúde e da doença em animais de produção tem aumentado consideravelmente na última década. O objetivo deste estudo foi determinar a concentração normal de PAFs no soro e no líquido cefalorraquidiano (LCR) de bovinos sadios por meio da eletroforese em gel de poliacrilamida. Foram avaliados cinquenta animais mestiços (350±70kg de PV e 18±1,2 meses de idade), 25 novilhas e 25 novilhos. As amostras de LCR foram colhidas no espaço atlanto-occipital (AO) e as amostras de sangue obtidas da veia jugular. As PAFs do soro e do LCR foram determinadas através da eletroforese em gel poliacrilamida. Trinta e sete proteínas com pesos moleculares que variaram entre 7 e 37kDa foram identificadas no LCR de todos os animais, independente do sexo. Estas oito proteínas foram nominalmente identificadas como imunoglobulina A e G, ceruloplasmina, transferrina, albumina, α1-antitripsina, glicoproteína ácida, e haptoglobina. As frações de proteínas presentes no LCR não diferiram entre novilhas e novilhos. No soro de machos e fêmeas, 34 proteínas com pesos moleculares entre 7 e 244 kDa foram identificadas. As proteínas do soro foram similarmente identificadas, entretanto a α1-antitripsina foi identificada somente no LCR. Os valores séricos de glicoproteína ácida e imunoglobulina G foram significativamente mais elevados nas novilhas em comparação aos novilhos. Em conclusão, a determinação das concentrações de proteínas de fase aguda presentes do LCR pode ser útil no diagnóstico e monitoramento da progressão de doenças neurológicas bovinas, talvez possa ainda direcionar procedimentos terapêuticos. O perfil eletroforético do LCR de bovinos hígidos não se altera em função do sexo.(AU)

Removal of polyacrylamide hydrogel injections from breast: a retrospective analysis / 中华医学美学美容杂志

Objective To explore the features of magnetic resonance imaging (MRI) and removal procedures of the polyacrylamide hydrogel (PAAG) injections from the breast.Methods From January 2015 to December 2016,113 continuous cases of breast PAAG implants were involved into this study.Features of MRI,characteristics of PAAG and tissue changes around PAAG were recorded and analyzed.Results The PAAG was demonstrated high T2 signals on MRI that was consistent with the surgical findings.In total of 226 breasts,the PAAG injected implants were removed with peri-areolar incision approach,and PAAG in free state was squeezed and capsular or infiltrated tissues were dissected.In 15 breasts,capsules were thin with no infiltrated tissue.In other 211 breasts,a lot of PAAG infiltrated tissues were observed and these infiltrated tissues included gland,fat tissue,pectoralis major muscle,pectoralis minor muscle or intercostal muscles.Conclusions In these cases,PAAG has been injected into breasts for 7 to 20 years.For such long period,PAAG infiltrated tissues present a lot of difficulties and more complicated to remove.Dissection is the strategy we recommend.

Serum levels of proteins and acute-phase proteins in captive emus (Dromaius novaehllandieae) of different ages / Teores séricos de proteínas, inclusive proteínas de fase aguda, em emus (Dromaius novaehollandiae) de diferentes faixas etárias e criados em cativeiro

Protein electrophoresis is a relatively simple technique that allows separating serum protein fractions, and provides important information in the investigation and diagnosis of several diseases. This study determined the levels of acute-phase proteins in the serum of healthy, captive emus (Dromaius novaehollandiae). Animals were divided into two groups (n=11 in each) based on age, with 1-year-old and 4-year-old emus. Acute-phase proteins were separated by SDS-PAGE. Ceruloplasmin, transferrin, albumin, haptoglobin, acidic glycoprotein, IgA, and IgG were detected in the serum of all animals. Protein profiles varied significantly with age (P<0.05). Individuals in the 4-year-old emus group had higher values of ceruloplasmin, transferrin, albumin, haptoglobin, and acidic glycoprotein, compared with the group with 1-year-old animals, showing the role of age in the protein profile of this species. Reference values for acute-phase proteins in healthy emus may be useful in the evaluation of health status and in the diagnosis of diseases affecting the species.(AU)

A eletroforese de proteínas é um método relativamente simples, que permite a separação das proteínas do plasma em frações. Sua interpretação fornece informações importantes para a investigação e o diagnóstico de inúmeras doenças. O objetivo deste estudo foi o de determinar a concentração das proteínas de fase aguda no soro de emus (Dromaius novaehollandiae) hígidos e criados em cativeiro. As aves foram separadas em dois grupos: grupo 1: (n=11), aves com um ano de idade; grupo 2: (n=11), aves com quatro anos de idade. As proteínas de fase aguda foram separadas por eletroforese em gel de poliacrilamida (SDS-PAGE). Identificaram-se as proteínas ceruloplasmina, transferrina, albumina, IgG, haptoglobina, glicoproteína ácida, IgA e IgG no soro de todos os emus. Houve diferença (P<0.05) entre os traçados eletroforéticos em função da faixa etária. As aves do grupo 2 apresentaram valores superiores de ceruloplasmina, transferrina, albumina, haptoglobina e glicoproteína ácida quando comparadas às aves do grupo 1. Conclui-se que o perfil eletroforético de emus sofre alterações conforme a idade analisada. O estabelecimento de valores de referência para as proteínas de fase aguda de emus hígidos poderá auxiliar estudos futuros na avaliação da saúde assim como no diagnóstico de doenças em emus.(AU)

Construction and expression of anti IL-4R single antibody of prokaryotic expression vector / 天津医药

Objective To construct anti-IL-4R murine anti-human single-chain variable fragment (scFvs) antibodies through BL21 (DE3) prokaryotic expression system. Methods The anti-IL-4R scFv sequence was optimizated on the basis of previous findings. The optimized scFv sequence was analyzed. The recombinant plasmid pET-32a-scFv was constructed. The recombinant plasmid was detected through enzyme identification, and was turned into BL21 (DE3) prokaryotic expression bacteria to express the pET-32a-scFv recombinant protein in E.coli BL21 (DE3). The purification and renaturation were researched, and SDS-PAGE analysis was studied. The molecular weight of ScFv against IL-4R was analyzed by SDS-PAGE. The expression of the fusion protein was detected by Western-blot assay. Results The length of fusion gene scFv-MLT sequence was 761 bp. The molecular weight of the recombinant expression of proteins of anti-IL-4R single antibody was approximately 45 ku. The recombinant proteins showed high specificity with anti-6 × His-tag antibody. Conclusion This experiment successfully constructs pET-32a-scFv prokaryotic expression system of recombinant protein with high immune reactivity, which provides the basis for further study of anti-IL-4R single chain antibody as drug target.

Kinetics of the expressions of radiation-induced plasma proteins of the cardiac territory in electrophoresis / Cinética das expressões de proteínas plasmáticas radioinduzidas do território cardíaco em eletroforese

ABSTRACT Introduction: Radiation induces acute and late alterations in blood proteins. Objective: The present study aims at analyzing in time kinetics the electrophoretic profiles of expression bands of blood protein, in the molecular weight range greater than and equal to that of albumin, modulated by ionizing radiation in the cardiac territory, in animal model. Material and methods: Animals were exposed to a whole-body dose of 5 Gy ionizing radiation (Co-60). Serum samples were collected from isogenic Wistar rats (control and irradiated groups). At a time kinetics of 12, 24, 48, 72, 96 hours and 35 days post-irradiation, thoracolaparotomies were performed with anesthesia, and 0.3 ml of blood was collected between the left ventricle and the pulmonary artery. The samples were held in heparin and their components were separated by centrifugation. Protein bands with similar molecular weight were identified by vertical 10% electrophoresis, silver staining. Results: The findings indicate a systemic acute altered expression of proteins with molecular weight greater than or equal to that of albumin in acrylamide gel, presenting suppression and increased expression due to modulation of preexisting bands, identified in time kinetics. Conclusion: These findings point out to acute alterations of protein expression modulated in time, but also to a late modulation of gene expression.

RESUMO Introdução: A radiação de corpo inteiro induz alterações agudas e tardias no perfil proteico sanguíneo. Objetivo: O presente estudo tem como escopo analisar em cinética de tempo o perfil eletroforético de bandas de expressão das proteínas solúveis do sangue, na faixa de peso molecular superior e igual à albumina, moduladas por radiação ionizante no território cardíaco em modelo animal. Materiais e métodos: Animais foram expostos à radiação de Co-60, em dose de 5 Gy corpo inteiro. Foram coletadas amostras de soro sanguíneo em ratos Wistar isogênicos (no grupo-controle e no irradiado). Em uma cinética de tempo de 12, 24, 48, 72 e 96 horas e 35 dias pós-exposição, foram realizadas toracolaparotomias com anestesia profunda e, posteriormente, foi coletado 0,3 ml de sangue entre ventrículo esquerdo e artéria pulmonar. As amostras foram heparinizadas, sendo, em seguida, separados seus componentes por centrifugação. As bandas proteicas de peso molecular similar foram identificadas por eletroforese vertical a 10%, coradas com prata. Resultados: Os achados indicam alteração aguda sistêmica da expressão das proteínas de peso molecular superior ou igual à albumina em gel de acrilamida, representando supressão e aumento de expressões por meio da modulação de bandas preexistentes identificadas em cinética de tempo. Conclusão: Esses achados apontam tanto para uma alteração aguda modulada no tempo da expressão proteica quanto para uma alteração moderada tardia da expressão gênica.

Expression of Raf kinase inhibitor protein in human hepatoma tissues by two‑dimensional gel electrophoresis and matrix‑assisted laser desorption/ionization time‑of‑flight methods.

PURPOSE: Hepatocellular carcinoma (HCC) is the most common malignant liver tumor. To reduce the mortality and improve the effectiveness of therapy, it is important to search for changes in tumor‑specific biomarkers whose function may involve in disease progression and which may be useful as potential therapeutic targets. MATERIALS AND METHODS: In this study, we use two‑dimensional polyacrylamide gel electrophoresis (2‑DE) and matrix‑assisted laser desorption/ionization time‑of‑flight mass spectrometry to observe proteome alterations of 12 tissue pairs isolated from HCC patients: Normal and tumorous tissue. Comparing the tissue types with each other, 40 protein spots corresponding to fifteen differentially expressed between normal and cancer part of HCC patients. RESULTS: Raf kinase inhibitor protein (RKIP), an inhibitor of Raf‑mediated activation of mitogen‑activated protein kinase/extracellular signal‑regulated kinase, may play an important role in cancer metastasis and cell proliferation and migration of human hepatoma cells. RKIP may be considered as a marker for HCC, because its expression level changes considerably in HCC compared with normal tissue. In addition, we used the methods of Western blotting and real time‑polymerase chain reaction to analysis the protein expression and gene expression of RKIP. The result showed RKIP protein and gene expression in tumor part liver tissues of HCC patient is lower than peritumorous non‑neoplastic liver tissue of the corresponding HCC samples. CONCLUSION: These results strongly suggest that RKIP may be considered to be a marker for HCC and RKIP are down‑regulated in liver cancer cell.

Differential proteomic analysis of transgenic Bt rice and its non-transgenic parental rice / 国际药学研究杂志

Objective: Under the methodology of differential proteomics and bioinformatics, the impact of the exogenous gene on the expression of rice in the proteome is discussed, aiming to explore into the study of genetically modified rice in the proteomics. Methods: The total protein was extracted from genetically modified rice Huahui No.1(HH1) and non-transgenic rice Minghui 63(MH63), the method of two-dimensional gel electrophoresis was applied to generate corresponding proteome two-dimensional polyacrylamide gel(2D-PAGE) electrophoresis spectrum; then, the mass spectrometry and bioinformatics analysis were conducted after the selection of protein spots with significant differences. Results: The comparing and matching of protein spots between transgenic Bt(cry1Ab/1Ac) rice and non-transgenic rice 2D-PAGE profiles identified 28 protein spots with significant differences. With non-transgenic rice as a reference, transgenic Bt rice held 18 relatively high and 10 relatively low expressions; mass spectrometry and bioinformatics retrieval were made on the different protein spots. It was found that the differentiated protein was mainly involved in energy metabolism, protein synthesis, redox stress response and other biological processes. Conclusion: Differences exist between transgenic Bt HH1 and its parental rice MH63 on the expression of proteome; however, there are neither anti-nutritional and allergenic protein, nor new or toxic proteins among these differentiated proteins.

Differential proteomic analysis of transgenic Bt rice and its non-transgenic parental rice / 国际药学研究杂志

Objective Under the methodology of differential proteomics and bioinformatics,the impact of the exogenous gene on the expression of rice in the proteome is discussed,aiming to explore into the study of genetically modified rice in the proteomics. Methods The total protein was extracted from genetically modified rice Huahui No.1(HH1) and non-transgenic rice Minghui 63 (MH63),the method of two-dimensional gel electrophoresis was applied to generate corresponding proteome two-dimensional poly?acrylamide gel(2D-PAGE)electrophoresis spectrum;then,the mass spectrometry and bioinformatics analysis were conducted after the selection of protein spots with significant differences. Results The comparing and matching of protein spots between transgenic Bt (cry1Ab/1Ac)rice and non-transgenic rice 2D-PAGE profiles identified 28 protein spots with significant differences. With non-trans?genic rice as a reference,transgenic Bt rice held 18 relatively high and 10 relatively low expressions;mass spectrometry and bioinfor?matics retrieval were made on the different protein spots. It was found that the differentiated protein was mainly involved in energy me?tabolism,protein synthesis,redox stress response and other biological processes. Conclusion Differences exist between transgenic Bt HH1 and its parental rice MH63 on the expression of proteome;however,there are neither anti-nutritional and allergenic protein, nor new or toxic proteins among these differentiated proteins.

Electroforesis horizontal en geles de poliacrilamida en la detección y tipificación del virus de papiloma humano / Horizontal electrophoresis in polyacrylamide gels in the detection and typing of human papillomavirus

OBJETIVO: estandarizar el uso de electroforesis horizontal en poliacrilamida como un método rápido de fácil implementación y de alta sensibilidad para la detección y tipificación del virus del papiloma humano por reacción en cadena de la polimerasa-polimorfismos de longitud de fragmentos de restricción con la enzima HpyCH4V como única enzima de restricción. MÉTODOS: Se utilizó el ácido desoxirribonucleico de 17 tipos de virus del papiloma humano, clonados en la colección del Laboratorio de Biología y Medicina Experimental (LABIOMEX-ULA), para la amplificación de la región flanqueada por los oligonucleótidos MY09/ MY11, los amplificados obtenidos se sometieron a digestión enzimática con la enzima HpyCH4V. El producto se corrió en geles de poliacrilamida de rápida polimerización en equipos de electroforesis horizontales. El ácido desoxirribonucleico se tiñó con bromuro de etidio y fueron fotografiados con la utilización de un trans-iluminador de luz ultravioleta. RESULTADOS: Se corrieron muestras de 17 tipos diferentes de virus del papiloma humano en geles de poliacrilamida en electroforesis horizontal sudmarina. Se observaron patrones de digestión, con la enzima de restricción HpyCH4V, bien definidos y distintos para 15 tipos diferentes. Se presentó una coincidencia de patrón polimorfismos de longitud de fragmentos de restricción para los tipos 45 y 52 ambos de alto riesgo. Se obtuvo una excelente resolución en corridas de 2,5 cm de longitud para cualquier patrón polimorfismos de longitud de fragmentos de restricción de los 17 tipos de virus del papiloma humano analizados. CONCLUSIÓN: El análisis de las corridas permitió una eficiente caracterización de los 17 tipos virales. La comparación del índice de movilidad relativa con polimorfismos de longitud de fragmentos de restricción virtual de los fragmentos amplificados presentó diferencias mínimas, el análisis de la movilidad en agarosa y poliacrilamida se ajustaron perfectamente permitiendo la sustitución de la agarosa por la poliacrilamida.

OBJECTIVE: to standardize the use of horizontal electrophoresis in polyacrylamide as a quick method of easy implementation and high sensitivity for the detection and typing of Human Papillomavirus by PCR-FRLP with HpyCH4V enzyme as unique restriction enzyme. METHODS: DNA from 17 Human Papillomavirus types, cloned in the collection of the Laboratory of Experimental Biology and Medicine, for amplification of the region flanked by the MY09/ MY11 oligonucleotides were used, the amplified obtained were subjected to enzymatic digestion with the enzyme HpyCH4V. The product was run on polyacrylamide gels rapid polymerization horizontal electrophoresis equipment. Stained with ethidium bromide and were photographed with the use of a trans-illuminating UV. RESULTS: Samples of 17 different Human Papillomavirus types polyacrylamide gel electrophoresis were run horizontally. Digestion patterns were observed with the restriction enzyme HpyCH4V, well-defined and different for different types 15. A pattern match for RFLP types 45 and 52 both high risk presented. Excellent resolution on runs of 2.5 cm in length for any RFLP pattern of the 17 Human Papillomavirus types analyzed was obtained. CONCLUSION: The analysis of the runs allows efficient characterization of the 17 Human Papillomavirus types. Comparing the relative mobility rate with the virtual RFLP of amplified fragments present minimal differences, analyzing mobility in agarose and polyacrylamide perfectly adjusted allowing for substitution of agarose by polyacrylamide.

Alterations in Calcium-Binding Properties of Sarcoplasmic Reticulum Membrane Proteins Following Cardiac Injury.

The objective of present work is to investigate metabolic alterations associated with heart failure, particularly one of its manifestations, a sustained hypocalcemia that causes hemodynamic changes contributed to subsequent myocardial injury. Comparative study was carried out using experimental models of pancreatic necrosis (PN) and crush syndrome (CS) accompanied by cardiac damage down to myocardial infarction. Study design: Wistar adult male rats randomly divided into groups (n=12/group). The controls are healthy intact animals. The pancreatic necrosis (PN) and crush syndrome (CS) groups were then randomly subdivided: PN group- into 3, 24 and 72 h groups concerning hemorrhage, early and late pancreatic necrosis respectively; CS group – into 2, 4, 24, and 48 h decompression stages. The rats were sacrificed to analyze spectra and calcium-binding properties of the membrane proteins isolated from the cardiomyocyte sarcoplasmic reticulum (SR). Development of pathological changes in the heart and pancreas were also monitored. Place and Duration of Study: Department of Pathological Biochemistry and Radioisotope Methods, H. Buniatyan Institute of Biochemistry of Natl. Acad. Sci (NAS), Republic of Armenia (RA). Experiments conducted between May 2011 and October 2013. Methodology: To study pathogenesis of hypocalcemia underlying myocardial damage a translocation of radioactive 45CaCI2 into cardiomyocytes and its intracellular distribution was examined. Binding of 45Ca2+ to the SR membrane proteins was measured after proteins separation by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDSPAGE) and radioactivity from the gel plates was counted by a gas-flow meter Berthold–II. Isoelectric focusing of the protein isolated from the SR of cardiomyocyte was performed. Results: Statistically significant changes in mean radio labeled calcium incorporation into a total protein fraction of the cardiomyocyte SR from control (13682±271) were determined by3h of PN (23055±168, P<.001), 24 h of PN (22876±240, P<.01), and by 72 h (3851±271,P<.01), P vs. control. Similarly, these parameters were detected following CS by 2h decompression (24179±225, P<.01), 4-24 hours decompression (21666±124, P<.001) and 48 h decompression (2941±189, P<.001), P vs. control. We demonstrate that drop in the binding calcium level observed was partially due to impaired affinity to calcium of the cardiomyocyte SR calcium-binding proteins during development of both PN and CS despite a simultaneous manifestation of affinity to calcium of the SR 32-kDa protein. Conclusion: In the present study we have clearly shown that both experimental acute pancreatitis and long-term compression injury may cause similar changes,а loss the calcium-binding properties of the cardiomyocyte proteins, particularly those of SR serving as a main calcium depot under physiological circumstances and appear to be involved in common cellular and molecular mechanisms of myocardial injury contributing to hypocalcemia. Simultaneously, both PN and/or CS cause similar manifestations of the new calcium-binding properties of the cardiomyocyte SR 32-kDa membrane protein, and mirrored dynamic changes in its calcium affinity suggested by Scatchard plot analysis indicating a common mechanism that would be a transient attempt of certain heart cells to compensate hypocalcemia, and thus emerge from an otherwise pathological outcome. Thus, the above mentioned changes could be used to identify patients at high risk of cardiovascular disease in different pathologies.