Identification of Proteins Associated with the Formation of Oral Biofilms

ABSTRACT Objective: To identify proteins associated with the formation of Streptococcus gordonii and Fusobacterium nucleatum biofilms. Material and Methods: Biofilms composed of two bacterial species, S. gordonii and F. nucleatum, were cultured for 1, 4, 7, and 10 days. The presence of both species was confirmed via amplification of the srtA and radD genes using real-time PCR. The concentrations of proteins associated with the biofilms and individual species were quantified using Western blotting. Results: The protein profiles of S. gordonii and F. nucleatum from individual cultures determined using one-dimensional electrophoresis revealed proteins found in S. gordonii and in F. nucleatum. Ct and reciprocal Ct values were determined for the exposed S. gordonii and F. nucleatum biofilms. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) protein was detected in biofilms and F. nucleatum, whereas HSP40 protein was present only in biofilms after 7 and 10 days of formation. Conclusion: HSP40 was detected only in the formed biofilms; thus, HSP40 is an essential proteins for adhesion.

Correlación entre los microorganismos oxidantes del amonio y los factores ambientales durante el compostaje de estiércol de ganado / Correlation between ammonia-oxidizing microorganisms and environmental factors during cattle manure composting

Abstract Cattle manure composting was performed in an aerated vessel. Community structure and diversity of ammonia-oxidizing bacteria (AOB) and ammonia-oxidizing archaea (AOA) were investigated using polymerase chain reaction and denaturing gradient gel electrophoresis (PCR-DGGE) techniques targeting the ammonia monooxygenase alpha subunit (amoA) gene and the correlation between AOB and AOA communities and environmental factors was explored. Thirteen (13) AOB sequences were obtained, which were closely related to Nitrosomonas spp., Nitrosomonas eutropha, and Nitrosospira spp. and uncultured bacteria, among which Nitrosomonas spp. were predominant. Excessively high temperature and high ammonium concentration were not favorable for AOB growth. Five AOA sequences, belonging to Candidatus Nitrososphaera gargensis and to an uncultured archaeon, were obtained. During composting, community diversity of AOB and AOA fluctuated, with AOA showing a higher Shannon-Wiener index. The AOB community changed more dramatically in the mesophilic stage and the early thermophilic stage, whereas the most obvious AOA community succession occurred in the late thermophilic stage, the cooling stage and the maturity stage. Water content, total nitrogen (TN) and ammonium concentration were more relevant to the AOB community structure, while higher correlations were observed between ammonia, nitrate and TN and the AOA community. AOB community diversity was negatively correlated with pH (r = -0.938, p < 0.01) and water content (r = -0.765, p < 0.05), while positively correlated with TN (r = 0.894, p < 0.01). AOA community diversity was negatively correlated with ammonium concentration (r = -0.901, p < 0.01). Ammonium concentration played an important role in the succession of AOB and AOA communities during composting.

Resumen Se llevó a cabo un compostaje de estiércol de ganado en un recipiente aireado. Se investigó la estructura de la comunidad y la diversidad de bacterias oxidantes del amoníaco (AOB) y las arqueas oxidantes del amoníaco (AOA) mediante el uso de las técnicas de reacción en cadena de la polimerasa y la electroforesis en gel con gradiente de desnaturalización (PCR-DGGE) dirigidas al gen de la subunidad alfa de la amonio monooxigenasa (amoA), y se exploró la correlación entre las comunidades AOB, AOA y los factores ambientales. Se obtuvieron 13 secuencias de AOB, las cuales se relacionaron estrechamente con Nitrosomonas spp., Nitrosomonas eutropha y Nitrosospira spp., y bacterias no cultivadas, entre las cuales fueron predominantes las Nitrosomonas spp. La temperatura excesivamente alta y la concentración de amonio elevada no fueron favorables para el crecimiento de las AOB. Se obtuvieron 5 secuencias de AOA, pertenecientes a Candidatus Nitrososphaera gargensis y un Archaeon no cultivado. Durante el compostaje, la diversidad de AOB y AOA fluctuó y las AOA mostraron un índice de Shannon-Wiener más alto. La comunidad de AOB cambió significativamente en la etapa mesofílica y la etapa termofílica temprana, mientras que la sucesión más obvia de la comunidad AOA ocurrió en la etapa termofílica tardía y las etapas de enfriamiento y de maduración. El contenido de agua, el nitrógeno total (TN) y la concentración de amonio fueron más relevantes para la estructura de la comunidad AOB, mientras que se observaron correlaciones mayores entre amoníaco, nitrato y TN, y la comunidad AOA. La diversidad de la comunidad AOB se correlacionó negativamente con el pH (r= -0,938; p < 0,01) y el contenido de agua (r = -0,765; p < 0,05), mientras que se relacionó positivamente con TN (r = 0,894; p < 0,01). La diversidad de la comunidad AOA se correlacionó negativamente con la concentración de amonio (r = -0,901; p < 0,01). La concentración de amonio desempenó un papel importante en la sucesión de las comunidades AOB y AOA durante el compostaje.

Hemoglobinuria paroxística nocturna. el rol de la anhidrasa carbónica-i urinaria en la hemólisis intravascular / Peroxysmal nocturnal hemoglobinuria: the role of urinary carbonic anhydrase-I in intravascular haemolysis

La Hemoglobinuria Paroxística Nocturna (HPN) se caracteriza por hemólisis intravascular crónica mediada por complemento. Cuando se produce la hemolisis se libera a circulación Anhidrasa Carbónica- I (AC-I), una enzima que se halla en alta concentración en el eritrocito y por su bajo peso molecular filtra por el glomérulo. El objetivo del presente trabajo fue detectar la excreción de la AC-I en orina de pacientes con HPN por Electroforesis Bidimensional de Utilidad Clínica (2D UC), y compararla con otras causas de hemólisis, de origen renal y postrenal. Se evaluaron 8 pacientes con HPN sin tratamiento con eculizumab un inhibidor del C5 del complemento, y 5 de ellos postratamiento, 12 orinas de pacientes con nefritis lúpica y 10 orinas de pacientes con hemólisis postrenal. La AC-I puede estar presente en la orina, en los tres grupos, sin embargo la relación AC-I/Hemoglobina en la hemólisis intravascular está invertida en comparación con la hemolisis glomerular y post-renal. Los pacientes con HPN tratados con eculizumab no presentan AC-I, y sería de utilidad en el seguimiento de los pacientes tratados con el inhibidor del C5, para evidenciar posibles escapes hemolíticos.

Paroxysmal Nocturnal Hemoglobinuria (PNH) is characterized by chronic complement mediated haemolysis. In these conditions it might be expected that carbonic anhydrase-I (AC-I) would be liberated into the plasma and excreted in the urine, by its high concentration in the erythrocyte and low molecular weight. The objective of the present study was to detect the urinary excretion of AC-I from patients with PNH by wodimensional clinical utility electrophoresis (2D UC) and to compare it with other causes of renal and post-renal haemolysis. We evaluated 8 patients with PNH without eculizumab, a complement C5 inhibitor, 5 of them posttreatment, 12 urine of patients with lupus nephritis and 10 urine of patients with post-renal hemolysis. AC-I may be present in the urine, in all three groups, however, the AC-I/Haemoglobin ratio in intravascular haemolysis is reversed compared to glomerular and post-renal haemolysis. Patients with PNH treated with eculizumab do not have AC-I and would be useful in monitoring patients treated with the C5 inhibitor to evidence possible haemolytic leaks.

Evaluación de la zona gamma del proteinograma por electroforesis: correspondencia clínico-patológica / Evaluation of protein electrophoresis in the gamma zone: clinicopathological correspondence / Avaliação da área gamma do proteinograma por eletroforese: correspondência clínico-patológica

El proteinograma por electroforesis (PxE) sérico es solicitado para detectar modificaciones del perfil proteico. El objetivo del trabajo fue evaluar las alteraciones de la zona gammaglobulina y su correspondencia con distintos estados clínico-patológicos. Se incluyeron 7.259 pacientes (1-89 años) a los que en 2013 se les solicitó PxE. Según el trazado densitométrico, en la zona gammaglobulina se reconocieron diferentes grupos: hipogammaglobulinemia (<0,60 g/dL), hipergammaglobulinemia policlonal (≥1,80 g/dL), banda monoclonal (BM) y bandas oligoclonales. Prevaleció la hipergammaglobulinemia policlonal (4,2%), seguida por BM (1,4%) e hipogammaglobulinemia (0,8%). Hipergammaglobulinemia policlonal (>3 g/dL) se observó en: hepatitis autoinmune, cirrosis, síndrome de Sjögren, enfermedad mixta del tejido conectivo, HIV, hepatitis C y enfermedad de Castleman. El hallazgo de BM correspondió a 47% de pacientes con gammapatía monoclonal de significado incierto y 40% con mieloma múltiple; el 0,5% fueron casos nuevos. Con hipogammaglobulinemias, en adultos prevaleció la inmunosupresión terapéutica (55%), seguida por diabetes/síndrome metabólico/hipotiroidismo (23%); en niños, 22% por inmunosupresión y 78% con hipogammaglobulinemia no clasificada como inmunodeficiencia primaria. Se concluye que en 6,4% de los PxE se observó alteración de la zona gammaglobulina; prevaleció la hipergammaglobulinemia policlonal. En 1 de cada 200 PxE se pesquisó un paciente con BM. El hallazgo de hipergammaglobulinemia policlonal o BM se correspondió con distintos estados clínico-patológicos.

Serum protein electrophoresis (PEP) is requested to screen changes in the protein profile. The aim of this study was to evaluate alterations in the gamma globulin zone and correspondence with various clinical and pathological states. 7259 patients were included (1-89 years of age) who had been requested a PEP in 2013. According to the densitometric tracing, in the gamma globulin zone different groups were recognized: hypogammaglobulinemia (<0.60 g/dL), polyclonal hypergammaglobulinemia (≥1,80 g/dL), monoclonal band (MB) and oligoclonal band. The polyclonal hypergammaglobulinemia prevailed (4.2%), followed by MB (1.4%) and hypogammaglobulinemia (0.8%). Polyclonal hypergammaglobulinemia (>3 g/dL) was observed in autoimmune hepatitis, alcoholic cirrhosis, Sjögren's syndrome, mixed connective tissue disease, HIV, hepatitis C and Castleman's disease. The MB finding corresponded to a 47% of patients with monoclonal gammopathy of undetermined significance and 40% with multiple myeloma; 0.5% were new cases. In adults, hipogammaglobulinemias prevailed in therapeutic immunosuppression cases (55%), followed by patients with diabetes/ metabolic syndrome/ hypothyroidism (23%); in children, 22% with immunosuppression and 78% corresponded to hipogammaglobulinemias not classified as primary immunodeficiency. To conclude, an alteration in the gamma globulin zone was observed in 6.4% of PEP. In 1 out of 200 PEP MB was found. The finding of polyclonal hypergammaglobulinemia or MB corresponded to different clinicopathological states.

O proteinograma por eletroforese (PXE) sérico é solicitado para detectar modificações no perfil proteíco. O objetivo do trabalho foi avaliar as alterações da área gammaglobulina e sua correspondência com diversos estados clínico-patológicos. Incluíram-se 7259 pacientes (1-89 anos) aos quais, em 2013, foi solicitado um PxE. De acordo com o traçado densitométrico, na área gammaglobulina, diferente grupos foram reconhecidos: hipogammaglobulinemia (<0,60 g/dL), hipergammaglobulinemia policlonal (≥1,80 g/dL), banda monoclonal (BM) e bandas oligoclonais. Prevaleceu a hipergammaglobulinemia policlonal (4,2%), seguida por BM (1,4%) e hipogammaglobulinemia (0,8%). Hipergammaglobulinemia policlonal (>3 g/dL) foi observada em: Hepatite autoimune, cirrose, síndrome de Sjögren, doença mista do tecido conjuntivo, HIV, hepatite C e doença de Castleman. O achado de BM correspondeu a 47% de pacientes com gammapatia monoclonal de significado indeterminado e 40% com mieloma múltiplo; 0,5% eram casos novos. Com hipogammaglobulinemias em adultos prevaleceu a imunossupressão terapêutica (55%), seguida por diabete/síndrome metabólica/hipotireoidismo (23%); em crianças, 22% por imunossupressão e 78% com hipogammaglobulinemia não classificados como imunodeficiência primária. Conclui-se que em 6,4% dos PxE foi observada alteração da área gammaglobulina; prevaleceu a hipergammaglobulinemia policlonal. Em 1 de cada 200 PxE foi encontrado um paciente com BM. O achado de hipergammaglobulinemia policlonal ou BM se correspondeu com diferentes estados clínico-patológicos.

Use of a commercial agarose gel for analysis of urinary glycosaminoglycans in mucopolysaccharidoses

ABSTRACT Mucopolysaccharidoses (MPS) are a group of inherited metabolic disorders caused by deficiency of enzymes that degrade glycosaminoglycans (GAGs). Urinary excretion of GAGs is a common feature of MPS, and is considered their major biomarker. We aimed to adapt the GAG electrophoresis method to a commercial agarose gel which would be able to separate urinary GAGs in a simpler way with good sensitivity and reproducibility. Urine samples from patients previously diagnosed with MPS I, IV, and VI were used as electrophoretic standards. Samples from patients on enzyme replacement therapy (ERT) were also assessed. Commercial agarose gel electrophoresis was effective, showing proper definition and separation of GAG bands. Detection sensitivity exceeded 0.1 µg and band reproducibility were consistent. GAG bands quantified in urine samples from patients on ERT correlated very strongly (correlation coefficient = 0.98) with total GAG concentrations. This application of gel electrophoresis demonstrates the possibility of monitoring patients with MPS treated with ERT by analyzing separately the GAGs excreted in urine. We suggest this process should be applied to MPS screening as well as to follow-up of patients on treatment.

Mutation analysis of NANOS3 in Brazilian women with primary ovarian failure

OBJECTIVES: Primary ovarian failure is a rare disorder, and approximately 90% of cases are of unknown etiology. The aim of this study was to search for mutations in NANOS3, a gene that was recently related to the etiology of primary ovarian failure, in a group of Brazilian women. METHODS: We screened for NANOS3 DNA variants in 30 consecutive women who were previously diagnosed with primary ovarian failure, of unknown etiology and compared the results with those from 185 women with normal fertility. The NANOS3 gene was amplified by polymerase chain reaction using pairs of specific primers and then sequenced. The resulting sequences were compared with control sequences available in the National Center for Biotechnology and Information database. RESULTS: No mutations in NANOS3 were found in primary ovarian failure patients, but four previously described polymorphisms were identified at a similar frequency in the control and primary ovarian failure groups. CONCLUSIONS: Mutations in NANOS3 were not associated with primary ovarian failure in the present cohort.

Proteinuria en gammapatías monoclonales / Proteinuria in monoclonal gammopathies

La presencia de proteínas en la orina se denomina proteinuria, en adultos se define clínicamente por una excreción urinaria de proteínas superior a 150 mg en 24 horas y se produce por una alteración en la barrera de filtración glomerular, que permite el escape de las proteínas en la orina. La proteinuria es frecuente en diferentes enfermedades, particularmente en las gammapatías monoclonales. Los criterios para el diagnóstico de estas entidades incluyen la presencia de células plasmáticas anormales en la médula ósea, una proteína monoclonal en suero aumentada, una proteína monoclonal en orina o lesiones osteolíticas. Para el diagnóstico, evaluación y monitoreo del tratamiento de las gammapatías monoclonales se realizan los estudios electroforéticos de proteínas plasmáticas o proteinogramas séricos y urinarios, técnica que permite la separación de proteínas en función de su migración diferencial al ser sometidas a un campo eléctrico(AU)

The presence of protein in urine is called proteinuria in adults is clinically defined by a urinary excretion of more than 150 mg in 24 hours and proteins occurs by a change in the glomerular filtration barrier, which allows the escape of proteins in urine. Proteinuria is common in various diseases, particularly monoclonal gammopathies. The criteria for diagnosis of these entities include the presence of abnormal plasma cells in the bone marrow, a monoclonal protein increased serum, a monoclonal protein in urine or osteolytic lesions. For diagnosis, evaluation and treatment monitoring of monoclonal gammopathies, electrophoretic studies proteins plasma or serum and urinary proteinograms technique that allows separation of proteins based on their differential migration when subjected to an electric field is performed(AU)

Salivary protein characteristics from saliva of carious lesion-free and high caries adults

It has been argued that specific salivary proteins could have a protective effect against caries, but data from the many available studies are rather contradictory. The purpose of this study was to analyze whether there is a relationship between protein concentration, electrophoretic profile and concentration of salivary IgA and the presence or absence of caries in adults. Adults with high caries activity (HC) and without caries lesions (CF), assessed by ICDAS criteria, were asked to provide unstimulated saliva samples. Protein concentration (μg/mL) was determined using the Bradford method. Western blotting was used to detect IgA. Data were compared using Student’s t test at p<0.05. Total protein concentration in CF was higher (50.65±7.5 μg/mL) than in HC individuals (26.80±2.5 μg/mL) (p=0.001). More protein bands were visualized in the gels from CF than the HC group (p=0.001). CF subjects showed higher salivary IgA concentration (11.27±0.5 μg) than HC individuals (1.71±0.2μg) (p=0.001).Salivary composition in high caries experience and cariesfree young adults seems to differ in terms of the type and amount of proteins. Further research is needed to expand these findings.

Se ha descrito que proteínas salivales específicas podrían tener un efecto protector sobre la caries, sin embargo, los datos de los numerosos estudios disponibles son contradictorios. El propósito de este trabajo fue analizar si existe una relación entre la concentración total de proteínas, perfil electroforético y la concentración de IgA salival y la presencia o ausencia de lesiones de caries en adultos. Se obtuvieron muestras de flujo salival no estimulado de adultos con alta actividad de caries (HC) y sin lesiones de caries (CF), evaluados según criterios ICDAS. La concentración total de proteínas (mg / ml) se determinó utilizando el método de Bradford. Para detección de IgA se empleó Western Blot. Los datos se compararon mediante la prueba t student, estableciendo diferencias significativas si p<0,05. La concentración total de proteínas en CF fue mayor (50,65 ± 7,5 mg / ml) que en individuos HC (26,80 ± 2,5 mg / ml) (p=0,001). En los geles, se visualizó un mayor número de bandas de proteínas en CF que en el grupo HC (p=0,001). Los Sujetos CF mostraron mayor concentración de IgA salival (11,27 ± 0,5 μg) que los individuos HC (1,71 ± 0,2 μg) (p=0,001). La composición salival de sujetos adultos jóvenes con alta experiencia y libres de caries, parece ser diferente en función del tipo y la cantidad de proteínas. Se requiere de más investigación para profundizar estos resultados.

Balneotherapy is a potential risk factor for Pseudomonas aeruginosa colonization

ABSTRACT The practice of immersion in burn patient has been abandoned in many parts of the world but in Brazil it is still common. The aim of this study was to ascertain if balneotherapy is a risk factor for Pseudomonas aeruginosa colonization in thermally injured patients. Eighteen patients from a Burn Center were studied for 14 weeks for Pseudomonas aeruginosa. Samples were collected by swabbing the exudate of wounds, before and after giving bath to the patients and from balneotherapy table. Pulsed-field gel electrophoresis was used to determine bacterial genetic relatedness. Thirty-seven P. aeruginosa isolates were detected from 292 swabs collected from patients' burn surface area and from the balneotherapy table. Profile analysis of P. aeruginosa DNA fragmentation showed 10 clones among the 37 strains analyzed. Type A is the most prevalent clone, with 23 strains distributed into eight subtypes. These were present in the swabs collected, before and after the patients' bath, from the surface of the bath table, suggesting that there was cross-contamination between the patients in different ways. This work demonstrates that balneotherapy is a risk factor in the Burn Center studied, because the same clone was found among P. aeruginosa isolates collected at various points and times.

RESUMO A prática de balneotarapia em paciente queimado foi abandonada em muitas partes do mundo, mas no Brasil ainda é comum. O objetivo deste estudo foi verificar se a balneoterapia é um fator de risco para a colonização por Pseudomonas aeruginosa em pacientes queimados. Dezoito pacientes internados em um Centro de Queimadura (CQ) foram acompanhados por 14 semanas. Amostras foram coletadas do exsudato de feridas, antes e depois do banho dos pacientes e também da mesa onde a balneoterapia foi realizada. A relação genética entre as cepas de P. aeruginosa foi determinada pela electroforese em gel de campo pulsado. Trinta e sete cepas foram detectadas a partir de 292 swabs coletados de área de superfície das feridas dos pacientes e da mesa de balneoterapia. Análise de fragmentação do DNA das 37 P. aeruginosa mostrou a existência de 10 clones. O tipo A foi o clone mais prevalente, com 23 cepas distribuídas em oito subtipos. Estas estavam presentes nas lesões dos pacientes antes e após o banho e na mesa onde o banho foi realizado, sugerindo contaminação cruzada inter e intra-pacientes e pacientes e mesa de banho. Este trabalho mostra que a balneoterapia é um fator de risco para colonização por P. aeruginosa, no CQ estudado, pois um mesmo clone da bactéria foi encontrado nos isolados coletados em vários pontos e épocas diferentes.

Evaluation of different protein extraction methods for banana (Musa spp.) root proteome analysis by two-dimensional electrophoresis.

Four protocols viz., the trichloroacetic acid-acetone (TCA), phenol-ammonium acetate (PAA), phenol/SDS-ammonium acetate (PSA) and trisbase-acetone (TBA) were evaluated with modifications for protein extraction from banana (Grand Naine) roots, considered as recalcitrant tissues for proteomic analysis. The two-dimensional electrophoresis (2-DE) separated proteins were compared based on protein yield, number of resolved proteins, sum of spot quantity, average spot intensity and proteins resolved in 4-7 pI range. The PAA protocol yielded more proteins (0.89 mg/g of tissues) and protein spots (584) in 2-DE gel than TCA and other protocols. Also, the PAA protocol was superior in terms of sum of total spot quantity and average spot intensity than TCA and other protocols, suggesting phenol as extractant and ammonium acetate as precipitant of proteins were the most suitable for banana rooteomics analysis by 2-DE. In addition, 1:3 ratios of root tissue to extraction buffer and overnight protein precipitation were most efficient to obtain maximum protein yield.

The Milk Borne Pathogens of Raw Milk from Some Egyptian Farms in Different Seasons.

Milk has good quality protein and is a unique substance in that it is consumed as fluid milk with minimal processing and also it is the raw material used to manufacture a wide variety of products. Milk is susceptible to contamination by many pathogenic microorganisms, which result in infection and threat to consumer’s health. The aim of this study was to determinate occurrence of pathogenic microorganisms in raw milk in four seasons from different locations in Egypt, the obtained counts results showed that the samples gave the lowest Total Plate Count (TPC) of 3x105cfu/ml in winter’s samples. While, the summer's sample showed the highest TPC of 5.8x107cfu/ml. E. coli count ranged from 2x102cfu/ml to 5.8x 105cfu/ml which the lowest count was noticed in winter’s samples. Staphylococcal count ranged from 2.7 x 103cfu/ml (winter sample) to1.28 x 106cfu/ml (another sample in the same season). These results indicated poor hygienic standard of raw milk from uncontrolled environments and the increased public health risk of those consuming raw milk from such uncontrolled sources and all these tests consume time but with Cultureindependent methods that are based on protocols where total DNA (or RNA) is directly extracted from the substrate it can save time. Coupled with a global analysis, these methods make it possible to study the total diversity from the bulk extract in a single step.

Typing of Streptococcus mutans strains isolated from caries free and susceptible subjects by multilocus enzyme electrophoresis

This study was evaluated the clonal diversity of Streptococcus mutans in caries-free and caries-active subjects using MLEE. Strains from caries-free subjects were grouped in a single taxon. Unrooted dendrogram showed that different strains clustered in four different clades, also showed that more than one clonal type can be found in a same individual.

Analysis of hemoglobin electrophoresis results and physicians investigative practices in Saudi Arabia.

BACKGROUND AND OBJECTIVES: Riyadh and central province falls in a moderate prevalent zone of hemoglobinopathies in Saudi Arabia. However, it has been observed that the physicians working in Saudi Arabia invariably advise all cases of anemia for hemoglobin electrophoresis (HE). The present work was carried out to study the yield of the HE in Riyadh and the investigative practices of the physicians advising HE. SETTINGS AND DESIGN: The study was carried out in the hospitals of King Saud University from 2009 to 2011 in order to assess the yield of HE in referred cases of clinical anemia. MATERIALS AND METHODS: A total of 1073 cases divided in two groups of males and females had undergone complete blood count and red blood cell morphology. Cellulose acetate HE was performed and all the positive results were reconfirmed on the high performance liquid chromatography (HPLC). The results were analyzed for the type of hemoglobinopathies. For statistical analysis Statistical Package for Social Sciences 15 version (SPSS Inc., Chicago, IL, USA) was used. RESULTS: A total of 405 males and 668 females blood samples were included in the present study. 116 (28.5%) males and 167 (25%) females showed an abnormal pattern on HE. The incidence of beta thalassemia trait was higher in females while sickle cell trait was predominantly seen in males. Red cell indices were reduced considerably in thalassemias, but were unaffected in sickle cell disorders, except those which had concurrent alpha trait. The total yield of HE was 26.6% which was much less than expected. CONCLUSION: The physicians are advised to rule out iron deficiency and other common causes of anemia before investigating the cases for hemoglobinopathies, which employs time consuming and expensive tests of HE and HPLC.

Various methods available for detection of apoptotic cells- A review.

Apoptosis is a process of programmed cell death occurring in multicellular organisms in whom development, maintenance and sculpturing organs and tissues. Taken together, apoptotic processes are of widespread biological significance; being involved in e.g. development, differentiation, proliferation/homoeostasis, regulation and function of the immune system and in the removal of defected harmful cells. Dys regulation of apoptosis can play a primary or secondary role leading to cancer whereas excessive apoptosis contributes to neuro degeneration, autoimmunity, AIDS, and ischemia. Gaining insight into the techniques for detecting apoptotic cells will allow the development of more effective, higher specific and therefore better-tolerable therapeutic approaches. The goal of this review article is to provide a general overview of current knowledge, on the various technical approaches for detecting apoptotic cells.

Distribución de tipos de paraproteínas en una muestra de enfermos con mieloma múltiple / Distribution of types of paraproteins in a sample of patients with multiple myeloma

Introducción: el mieloma múltiple (MM) es una enfermedad caracterizada por una proliferación monoclonal de inmunoglobulinas que representa aproximadamente el 15 por ciento de las hemopatías malignas. Métodos: se realizó un estudio de la distribución de las clases, sub clases y tipos de cadenas ligeras de inmunoglobulinas en 285 enfermos con el diagnóstico de MM. Se emplearon tres métodos: electroforesis de proteínas en suero para la detección de la inmunoglobulina monoclonal o paraproteína, electroforesis de inmunofijación y doble inmunodifusión para identificar las clases, sub clases y tipo de cadenas ligeras. Resultados: se encontraron 206 enfermos (72.28 por ciento) con MM IgG; 73 (25.62 por ciento) con MM IgA y 6 (2.1 por ciento) con MM IgM. La distribución de sub clases de IgG fue: 130 casos (63.11 por ciento) IgG1, 43 (20.87 por ciento) IgG2, 21 (10.19 porciento) IgG3 y 12 (5.83 por ciento) IgG4; y la de sub clases de IgA fue de 59 enfermos (80.82 por ciento) IgA1 y 14 (19.18 por ciento) IgA2. Del total de enfermos 187 (65.61 por ciento) mostraron cadenas ligeras tipo kappa y 98 (34.38 por ciento) tipo lambda. Conclusiones: los datos obtenidos en nuestro estudio permitieron identificar la frecuencia de distribución de las clases, subclases y cadenas ligeras en una muestra de enfermos con MM

Introduction: multiple mieloma (MM) is a disease characterized by a monoclonal proliferation of immunoglobulins representing approximately 15 percent of malignant hemopathies. Methods: the distribution of classes, subclasses and light chains of monoclonal immunoglobulins was studied in 285 patients with MM. Three methods were used: serum protein electrophoresis for the detection of monoclonal immunoglobulins or paraproteins, immunofixation electrophoresis and double immunodiffusion to identify classes, subclasses and light chain types. Results: 206 patients (72.28 percent) with IgG MM, 73 (25,62 percent) with IgA MM, and 6 (2,1 percent) with IgM MM were found. The distribution of IgG subclasses was: 130 cases (63,11 percent) IgG1; 43 (20,87 percent) IgG2; 21 (10,19 percent) IgG3: and 12 (5,83 percent) IgG4. Distribution of IgA subclasses was: 59 patients (80,82 percent) IgA1 and 14 (19,18 percent) IgA2; 187 patients (65,62 percent) showed kappa light chains and 98 (34,38 percent) were lambda. Conclusions: the data obtained in our study allowed us to identify the frequency of distribution of classes, subclasses and light chains in a sample of patients with MM

Zimography is an effective method for detection of matrix metalloproteinase 2 (MMP-2) activity in cultured human fibroblasts

PURPOSE: To describe a method to characterize the gelatinase activity of cultured human periodontal fibroblasts stimulated with Pam3Cys and E. coli LPS, ligands of TLR2 and TLR4 respectively, and by centrifugation of the cultures, simulating an orthodontic force. METHODS: To study MMP-2 activity, primary cultures of human periodontal fibroblasts were stimulated with the addition of TLRs 2 and 4 ligands and the application of mechanical force by centrifugation at 141 x g for 30 min. Supernatant media was collected 24 hours later to perform protein quantification and zymography. RESULTS: MMP-2 activity suffered an increase in cultures co-stimulated with TLRs 2 and 4 ligands alone or with the presence of mechanical force application compared to basal levels. CONCLUSION: Zymography, one of the several methods to study MMPs activities, is a simple, qualitative and efficient method based on electrophoresis of bis-acrylamide gels copolymerized with a protein substrate.

Técnicas electroforéticas en el HYDRASYS 2. Utilidad diagnóstica en diferentes enfermedades / Electrophoretic techniques in HYDRASYS 2. Diagnostic usability in different diseases

Se presentan los resultados de la estandarización de las técnicas de electroforesis de hemoglobina (Hb), isoenzimas de la deshidrogenasa láctica (LDH) y proteinuria en el equipo Hydrasys 2, así como el estudio de pacientes atendidos en el Instituto de Hematología e Inmunología y en otros centros hospitalarios del país. Se realizó el diagnóstico de 149 portadores de hemoglobinopatías (AS, AC, b talasemia heterocigótica, variante rápida), 60 enfermos (SS, SC, CC), 24 pacientes con a talasemia o deficiencia de hierro y se cuantificó la hemoglobina fetal a 93 casos con hemoglobinopatía S. Se determinaron los valores normales de actividad e isoenzimas de LDH en la población mediante el estudio de 50 donantes de sangre. En los pacientes con anemia drepanocítica se encontró un aumento significativo de la isoenzima 1 (p= 0,000) y disminución de isoenzimas 3 (p= 0,002). Se realizó el estudio de proteínas en orina a 8 pacientes con enfermedades hematológicas que presentaron microalbuminuria al menos en 2 ocasiones, con concentraciones ³ 0,04 g/L. En 2 pacientes el resultado fue normal; en 2 se encontraron proteínas de origen tubular; y en otros 2, proteínas de origen glomerular

We present the results of the standardization of the techniques of electrophoresis of hemoglobin (Hb), lactate dehydrogenase isoenzymes (LDH) and proteinuria in HYDRASYS 2 equipment, and the study of patients treated at the Institute of Hematology and Immunology and other hospitals in the country. 149 hemoglobinopathies carriers were diagnosed (AS, AC, b thalassemia heterozygous fast variant), 60 patients (SS, SC, CC), 24 patients with athalassemia or iron deficiency. Fetal hemoglobin was quantified in 93 cases with hemoglobinopaty S. Normal values of activity and LDH isoenzymes were determined in the population through the study of 50 blood donors. In patients with sickle cell anemia we found a significant increase in isoenzyme 1 (p=0.000) and isozyme 3 decreased (p=0.002). We performed the study of proteins in urine in 8 patients with hematologic malignancies who had microalbuminuria at least 2 times, with concentrations ³ 0.04 g / L. In 2 patients the results were normal, in 2 proteins were tubular origin, and in 2, proteins of glomerular origin

Comparação entre teste bioquímico clássico e o método da reação em cadeia da polimerase (PCR) para identificação de estirpes de Enterococcus Faecalis isoladas da cavidade oral / Comparison between biochemist classic test and the Polymerase Chain Reaction (PCR) for identification of Enterococcus faecalis from the oral cavity

Introdução: O gênero Enterococcus são habitantes normais do trato gastrintestinal e em menor proporção da vagina e uretra masculina. Tornaram-se importantes agentes de doenças humanas devido principalmente à sua elevada resistência aos agentes antimicrobianos e seus inúmeros fatores de virulência. Objetivo: comparar dois métodos laboratoriais utilizados na identificação de 60 estirpes de Enterococcus coletados da cavidade bucal. Foi realizada a identificação das linhagens por um esquema bioquímico clássico baseado nas características fenotípicas e pela técnica da Reação em Cadeia da Polimerase (PCR). Assim, colônias com características de isolamento próprias de Enterococcus sobre a superfície do M-Enterococcus ágar, foram submetidas aos seguintes testes bioquímicos: produção de catalase, hidrólise da esculina, tolerância ao cloreto de sódio a 6,5 por cento, fermentação do manitol, fermentação da arabinose, fermentação do sorbitol, desaminação da arginina, verificação da motilidade e produção de pigmento. Resultados e Discussão: os resultados dos testes foram interpretados e comparados com características fenotípicas de identificação de Enterococcus. A técnica de PCR foi realizada nessa mesma população de microrganismos. Pode-se observar que os testes bioquímicos clássicos identificaram todas as estirpes isoladas como sendo da espécie E. faecalis, enquanto a técnica de PCR revelou que 10 estirpes não pertenciam a espécie pesquisada. A análise estatística pelo método de Fisher revelou diferença significante entre a série bioquímica e o método da PCR (p<0,05). Conclusões: a coleção de linhagens de Enterococcus isoladas foram identificadas como E. faecalis através de um esquema bioquímico tradicional, baseando-se em suas características fenotípicas. Pela técnica da PCR 10 estirpes da coleção não foram identificadas como E. faecalis, revelando diferenças em suas características genotípicas.

Introduction: The genus Enterococcus are normal inhabitants of the gastro intestinal tract and to a lesser extent the vagina and male urethra. They become important agents of human diseases mainly due to its high resistance to antimicrobial agents and its many virulence factors Objective: The aim of this study was to compare two laboratory methods used to identify 60 strains of Enterococcus collected from the oral cavity. The strains were identified through a conventional biochemical scheme based on phenotypic properties and by the Polymerase Chain Reaction (PCR) technique. Colonies with insulation characteristics pertaining to Enterococcus on the surface of M-Enterococcus agar, were subjected to the following biochemical tests: production of catalase, esculin hydrolysis, 6.5 per cent sodium chloride tolerance, mannitol fermentation, arabinose fermentation, sorbitol fermentation, arginine deamination, motility assay and pigment production. Results and Discussion: The results were interpreted and compared with phenotypic identification of Enterococcus. The PCR technique was performed in this same population of microorganisms. The conventional biochemical tests identified all the isolates as being E. faecalis, while the PCR technique showed that 10 strains did not belong to the species studied. After statistical analysis using the Fisher's method, the research showed, in a significant way, that the conventional biochemical tests were more efficient for the identification of E. faecalis compared to the PCR(p <0.05). Conclusions: the collection of strains of Enterococcus isolates were identified as E. faecalis through a traditional biochemical scheme, based on the phenotypic characteristics. By PCR test collection of 10 strains were not identified as E. faecalis revealing differences in their genotypic characteristics.

A comparative study of hematological parameters of α and β thalassemias in a high prevalence zone: Saudi Arabia.

BACKGROUND AND AIMS: Saudi Arabia falls in the high prevalent zone of αα and β thalassemias. Early screening for the type of thalassemia is essential for further investigations and management. The study was carried out to differentiate the type of thalassemia based on red cell indices and other hematological parameters. MATERIALS AND METHODS: The study was carried out on 991 clinically suspected cases of thalassemias in Riyadh, Saudi Arabia. The hematological parameters were studied on Coulter STKS. Cellulose acetate hemoglobin electrophoresis and high-performance liquid chromatography (HPLC) were performed on all the blood samples. Gene deletion studies were carried out by restriction fragment length polymorphism (RFLP) technique using the restriction endonucleases Bam HI. STATISTICAL ANALYSIS: Statistical analysis was performed on SPSS 11.5 version. RESULTS: The hemoglobin electrophoresis and gene studies revealed that there were 406 (40.96%) and 59 (5.95 %) cases of β thalassemia trait and β thalassemia major respectively including adults and children. 426 cases of various deletion forms of α thalassemias were seen. Microcytosis was a common feature in β thalassemias trait and (-α/-α) and (--/αα) types of α thalassemias. MCH was a more significant distinguishing feature among thalassemias. β thalassemia major and α thalassemia (-α/αα) had almost normal hematological parameters. CONCLUSION: MCV and RBC counts are not statistically significant features for discriminating between α and β thalassemias. There is need for development of a discrimination index to differentiate between α and β thalassemias traits on the lines of discriminatory Indices available for distinguishing β thalassemias trait from iron deficiency anemia.