DDRT-PCR approaches applied for preeminent results in the isolation of DETs from fish brain tissues.

Differential Display (DD) is a technique widely used in studies of differential expression. Most of these analyses, especially those involving fish species, are restricted to species from North America and Europe or to commercial species, as salmonids. Studies related to South American fish species are underexplored. Thus, the present work aimed to describe DD technique modifications in order to improve outcomes related to the isolation of DETs (Differentially Expressed Transcripts), using Leporinus macrocephalus, a large commercially exploited South American species, as a fish design. Different DDRT-PCR approaches were applied to brain samples and the products of the reactions were analyzed on 6% polyacrylamide gels stained with 0.17% Silver Nitrate (AgNO3). The use of PCR reactions under high stringency conditions and longer oligonucleotides based on VNTR (Variable Number of Tandem Repeats) core sequences led to better results when compared to low stringency PCR conditions and the use of decamer oligonucleotides. The improved approach led to the isolation of differentially expressed transcripts on adult males and females of L. macrocephalus. This study indicates that some modifications on the DDRT-PCR method can ensure isolation of DETs from different fish tissues and the development of robust data related to this approach.

Bronchial collapse during bronchoalveolar lavage in horses is an indicator of lung inflammation.

REASONS FOR PERFORMING STUDY: The bronchoalveolar lavage (BAL) procedure can return variable volumes of fluid, possibly depending on the presence of bronchial collapse during fluid aspiration and on the severity of lung inflammation. OBJECTIVES: We tested the hypothesis that horses with bronchial collapse during BAL are at higher risk of having severe lung inflammation. STUDY DESIGN: Prospective field study. METHODS: Bronchial collapse was graded using a new simple scoring method (0, 1 or 2) during a standardised BAL procedure in the field on 131 horses with normal, mild/moderate or severe lower airway inflammation on cytology of BAL fluid. RESULTS: Of the 131 horses, 37 (28%), 55 (42%) and 39 (30%) horses had bronchial collapse scores of 0, 1 and 2, respectively. There was a difference in collapse scores between all the BAL inflammation categories (P<0.001). Severe collapse had a positive predictive value of 0.95 for both mild/moderate and severe BAL inflammation, with a prevalence of 63% and 20%, respectively. The BAL fluid return volume in the horses with severe collapse scores was lower than volumes in the partial (score 1/2) and no collapse (score 0/2) groups (P<0.001). The BAL fluid volume was negatively correlated with BAL neutrophil percentage (P<0.001). CONCLUSIONS: Airway collapse during BAL is associated with airway inflammation and neutrophilia. POTENTIAL RELEVANCE: During a standardised BAL procedure, clinicians can expect lung inflammation in horses that have bronchial collapse and bronchial collapse in horses with lung inflammation. Lung inflammation may be a contributing factor in the mechanism of bronchial collapse during BAL in horses.

Endoscopic assessment of airway inflammation in horses.

BACKGROUND: Comprehensive endoscopic scoring of the upper and lower airways for inflammation has not been critically assessed among a large population of horses. The relationship between upper and lower airways described in humans by the "one airway, one disease" concept might also apply to horses. HYPOTHESIS/OBJECTIVES: To evaluate if an association exists between endoscopic inflammatory scores and mucus scores of upper and lower airways and to investigate if endoscopic findings correlate with the lower airway inflammation measured by bronchoalveolar lavage (BAL) cytology. METHODS: Prospective field study. Pharyngitis, pharyngeal mucus, tracheal mucus, tracheal septum thickness, and bronchial mucus were scored using new and previously described scoring systems on a convenience sample of 128 horses with and without lung inflammation. Based on BAL fluid cytology, horses were categorized as having normal, moderate, or severe inflammation of the lower airways. RESULTS: All 5 endoscopy scores showed excellent interobserver agreement. Tracheal mucus (P < .001), tracheal septum thickness (P = .036), and bronchial mucus (P = .037) were significantly increased in horses with severe inflammation BALs and were correlated among themselves but not with upper airways scores. BAL neutrophils percentage was correlated with tracheal mucus (r(s) = 0.41, P < .001), bronchial mucus (r(s) = 0.27, P = .003), and had a weak negative correlation with pharyngitis (r(s) = -0.25, P = .004). CONCLUSIONS AND CLINICAL IMPORTANCE: Lower airway endoscopy scores are reflective of lower airway inflammation; however, upper and lower airways are independent in terms of severity of inflammation. Therefore, observing upper airway inflammation is not an indication to test for lower airway inflammation.

Spirulina enhances the viability of Lactobacillus rhamnosus E/N after freeze-drying in a protective medium of sucrose and lactulose.

AIMS: Response surface methodology (RSM) was used to optimize a protective medium for enhancing the viability of Lactobacillus rhamnosus E/N cells during lyophilization. METHODS AND RESULTS: Spirulina, sucrose and lactulose were selected, on the basis of a Plackett-Burman factorial design, as important protectants having the following protective effects on cell viability: 102.025, 36.885 and -34.42, respectively. A full-factorial central composite design was applied to determine optimal levels of three used agents. CONCLUSION: The optimal protective medium composition was determined to be: Spirulina 1.304% (w/v), lactulose 5.48% (w/v), and sucrose 13.04% (w/v) (Polish Patent P-393189). The predictive value of cell viability in this medium was 89.619%, and experimental viability obtained during freeze-drying was 87.5%. SIGNIFICANCE AND IMPACT OF THE STUDY: In this study, Spirulina was used for the first time as the protective agent in freeze-drying medium, significantly increasing lactobacilli viability and giving synbiotic character of the final product.

Evaluation of a risk-screening questionnaire to detect equine lung inflammation: results of a large field study.

REASONS FOR PERFORMING STUDY: The diagnosis of equine recurrent airway obstruction (RAO) and inflammatory airway disease (IAD) is based on clinical signs and increased inflammatory cell percentages in the bronchoalveolar lavage (BAL) fluid. Since a BAL is an invasive procedure, a risk-screening questionnaire (RSQ) would be a valuable screening tool for lung inflammation. OBJECTIVE: To evaluate the accuracy of a RSQ to detect lower airway inflammation (LAI) in a large population of horses. METHODS: A standardised BAL was performed in the field on 167 horses in Alberta, Canada. Horses were separated into 3 categories: 1) BAL normal; 2) BAL mild to moderate LAI (MLAI), and 3) BAL severe LAI (SLAI). The horse owners were asked to complete a RSQ. The RSQ scores were compared to the BAL results to determine the likelihood of a horse having MLAI, SLAI or no LAI. RESULTS: Based on BAL cytology, 28 (17%) horses were normal and 139 (83%) were abnormal, with 110 (66%) showing MLAI and 29 (17%) SLAI. Horses with SLAI and MLAI had a mean RSQ score of 0.95 and 0.70, respectively, compared to 0.60 for normal BAL horses. Horses with SLAI showed more clinical signs than normal and MLAI horses. The sensitivity and negative predictive values of the RSQ for detecting SLAI using a cut-off score of 0.87, were excellent at 0.90 (95%CI 0.73-0.98) and 0.96 (95%CI 0.82-1.00). Questions on the clinical signs typically found in RAO cases differed significantly between horses with BAL SLAI and those with BAL normal. CONCLUSIONS: Prevalence of MLAI was high in this population. Although the RSQ did not allow differentiating normal horses from horses with MLAI, it has a high sensitivity to detect horses with SLAI and is therefore a good screening tool for SLAI.

The Plackett-Burman design in optimization of media components for biomass production of Lactobacillus rhamnosus OXY.

The central composite design was developed to search for an optimal medium for the growth of Lactobacillus rhamnosus OXY. The effect of various media components, such as carbon sources, simple and complex nitrogen sources, mineral agents, and growth factors (vitamins B, amino acids) was examined. The first-order model based on Plackett-Burman design showed that glucose, sodium pyruvate, meat extract and mineral salts significantly influenced the growth of the examined bacteria. The second-order polynomial regression confirmed that maximum biomass production could be achieved by the combination of glucose (12.38 g/l), sodium pyruvate (3.15 g/l), meat extract (4.08 g/l), potassium phosphate (1.46 g/l), sodium acetate (3.65 g/l) and ammonium citrate (1.46 g/l). The validation of the predicted model carried out in bioreactor conditions confirmed the usefulness of the new medium for the culture of L. rhamnosus OXY in large scale. The optimal medium makes the culture of the probiotic bacterium L. rhamnosus OXY more cost effective.

Differentiation of three Lactobacillus rhamnosus strains (E/N, Oxy and Pen) by SDS-PAGE and two-dimensional electrophoresis of surface-associated proteins.

SDS-PAGE of extracted surface-associated proteins of Lactobacillus rhamnosus strains E/N, Oxy, and Pen, was performed. The obtained protein patterns allowed differentiation of the examined strains, which was not accomplished by the commonly used RAPD genotypic method. The differentiation by the SDS-PAGE method proved to be a useful tool for strain-specific identification, which was further confirmed by 2DE analysis. Therefore, it can be used as an alternative or complementary method for both conventional and genotypic identification procedures, especially when closely related lactobacilli isolates are identified.

Identification of sexually dimorphic gene expression in brain tissue of the fish Leporinus macrocephalus through mRNA differential display and real time PCR analyses.

Differentially expressed genes in males and females of vertebrate species generally have been investigated in gonads and, to a lesser extent, in other tissues. Therefore, we attempted to identify sexually dimorphic gene expression in the brains of adult males and females of Leporinus macrocephalus, a gonochoristic fish species that presents a ZZ/ZW sex determination system, throughout a comparative analysis using differential display reverse transcriptase-PCR and real-time PCR. Four cDNA fragments were characterized, representing candidate genes with differential expression between the samples. Two of these fragments presented no significant identity with previously reported gene sequences. The other two fragments, isolated from male specimens, were associated to the gene that codes for the protein APBA2 (amyloid beta (A4) precursor protein-binding, family A, member 2) and to the Rab 37 gene, a member of the Ras oncogene family. The overexpression of these genes has been associated to a greater production of the beta-amyloid protein which, in turns, is the major factor that leads to Alzheimer's disease, and to the development of brain-tumors, respectively. Quantitative RT-PCR analyses revealed a higher Apba2 gene expression in males, thus validating the previous data on differential display. L. macrocephalus may represent an interesting animal model to the understanding of the function of several vertebrate genes, including those involved in neurodegenerative and cancer diseases.

Palifermin for prevention of oral mucositis after haematopoietic stem cell transplantation- single centre experience.

PURPOSE: Oral mucositis (OM) is one of the most debilitating and common side effects in patients treated with high-dose chemotherapy supported by haematopoietic stem cell transplantation (HSCT). We tested the effectiveness of palifermin to avoid oral mucosal injury induced by the conditioning regimen. PATIENTS AND METHODS: Twenty patients with haematological malignancies were treated with palifermin for prevention of OM during HSCT procedures. Nine patients received allogeneic haematopoietic stem cells, and in 11 autologous HSCT was performed. The control group was composed of patients who had been treated with HSCT previously, before the palifermin era. The source of graft was peripheral blood. RESULTS: Among patients treated with palifermin no grade 2-4 OM was observed. No patient had to receive opioid analgesics or total parenteral nutrition. 30% of the patients developed grade 1 OM of 4-5 days' duration. In the control group OM was observed in all cases, with 50% of the patients developing grade 3-4 OM. Median duration of OM was 10 and 12 days for auto- and allogeneic patients, respectively. In comparison with the control group, treatment with palifermin was associated with significant reduction of grade 2-4 OM, shorter duration of OM, less analgesics intake, and reduced number of days with antibiotic treatment. Additionally, allogeneic patients treated with palifermin had shorter time to platelet engraftment. CONCLUSION: Palifermin reduces incidence, severity and duration of OM, and decreases the number of days with analgesics and antibiotics. For allogeneic patients it can shorten the time to platelet engraftment, but this observation needs further studies.

Cytogenetic analyses of two Curimatidae species (Pisces; Characiformes) from the Paranapanema and Tietê Rivers.

Cytogenetic analyses were performed in two Curimatidae species (Steindachnerina insculpta and Cyphocharax modesta) from the Paranapanema and Tietê Rivers (São Paulo State, Brazil), showing a karyotype composed of 54 meta-submetacentric chromosomes in both species. Silver- and chromomycyn-staining and fluorescent in situ hybridization (FISH) using a 18S rDNA probe indicated that the nucleolar organizer regions (NORs) of both species are localized in the terminal region of the long arm of two metacentric chromosomes. Although a single NOR system was evidenced in both analyzed species, S. insculpta and C. modesta presented the nucleolar organizer regions in distinct chromosome pairs, indicating that these cistrons can be considered cytogenetic markers. Variation on the amount and distribution of the constitutive heterochromatin (C-bands) could also be detected between the two species - while S. insculpta presented few heterochromatic blocks, intensely stained C-bands were evidenced in C. modesta specially in the terminal region of the long arm of the NOR-bearing chromosomes. Although most Curimatidae species have been characterized by homogeneous karyotypes, isolated populations could be established under different environmental conditions leading to karyotype micro-structure variations specially related to the NORs localization and C-banding distribution. The obtained data were useful for the cytogenetic characterization and differentiation of S. insculpta and C. modesta and could be used in evolutionary inferences in the Curimatidae group.

Cytogenetic analyses of two Curimatidae species (Pisces; Characiformes) from the Paranapanema and Tietê Rivers

Cytogenetic analyses were performed in two Curimatidae species (Steindachnerina insculpta and Cyphocharax modesta) from the Paranapanema and Tietê Rivers (São Paulo State, Brazil), showing a karyotype composed of 54 meta-submetacentric chromosomes in both species. Silver- and chromomycyn-staining and fluorescent in situ hybridization (FISH) using a 18S rDNA probe indicated that the nucleolar organizer regions (NORs) of both species are localized in the terminal region of the long arm of two metacentric chromosomes. Although a single NOR system was evidenced in both analyzed species, S. insculpta and C. modesta presented the nucleolar organizer regions in distinct chromosome pairs, indicating that these cistrons can be considered cytogenetic markers. Variation on the amount and distribution of the constitutive heterochromatin (C-bands) could also be detected between the two species - while S. insculpta presented few heterochromatic blocks, intensely stained C-bands were evidenced in C. modesta specially in the terminal region of the long arm of the NOR-bearing chromosomes. Although most Curimatidae species have been characterized by homogeneous karyotypes, isolated populations could be established under different environmental conditions leading to karyotype micro-structure variations specially related to the NORs localization and C-banding distribution. The obtained data were useful for the cytogenetic characterization and differentiation of S. insculpta and C. modesta and could be used in evolutionary inferences in the Curimatidae group.

Análises citogenéticas foram realizadas em duas espécies de Curimatidae (Steindachnerina insculpta e Cyphocharax modestus) provenientes dos rios Paranapanema e Tietê (Estado de São Paulo, Brasil), evidenciando um cariótipo composto por 54 cromossomos meta-submetacêntricos em ambas as espécies. Coloração com nitrato de prata e cromomicina e hibridação in situ fluorescente (FISH), utilizando uma sonda de DNAr 18S, mostraram que as regiões organizadoras de nucléolos (RONs) de ambas as espécies estão localizadas na região terminal do braço longo de dois cromossomos metacêntricos. Embora as espécies analisadas tenham apresentado um sistema de RONs simples, S. insculpta e C. modesta apresentaram as regiões organizadoras de nucléolos em distintos pares de cromossomos, indicando que estes cístrons podem ser considerados marcadores citogenéticos. Variação na quantidade e distribuição de heterocromatina constitutiva (bandas C) também pôde ser detectada entre as duas espécies - enquanto S. insculpta apresentou poucos blocos heterocromáticos, bandas C intensamente coradas foram evidenciadas em C. modesta especialmente na região terminal do braço longo dos cromossomos portadores de RONs. Embora a maioria das espécies de Curimatidae seja caracterizada por cariótipos homogêneos, populações isoladas podem ter se estabelecido sob condições ambientais distintas, levando à ocorrência de variações na micro-estrutura cariotípica especialmente relacionadas à localização das RONs e à distribuição das bandas C. Os dados obtidos mostraram-se úteis para caracterização e diferenciação citogenética de S. insculpta e C. modesta e podem ser utilizados em inferências evolutivas no grupo Curimatidae.

Genomic organization and evolution of the 5S ribosomal DNA in Tilapiini fishes.

5S rDNA sequences present an intense dynamism and have proved to be valuable as genetic markers to distinguish closed related species and also in the understanding of the evolutionary dynamic of repetitive sequences in the genomes. In order to identify patterns of 5S rDNA organization and their evolution in the genome of fish species, such genomic segment was investigated in the tilapias Oreochromis niloticus and Tilapia rendalli, and in the hybrid O. urolepis hornorum x O. mossambicus. A dual 5S rDNA system was identified in the three analyzed tilapia samples. Although each 5S rDNA class was conserved among the three samples, a distinct 5S rDNA genome organization pattern could be evidenced for each sample. The presence of a dual 5S rDNA system seems to be a general trait among non-related teleost fish orders, suggesting that evolutionary events of duplication have occurred before the divergence of the main groups of teleost fishes.

Dynamics of 5S rDNA in the tilapia (Oreochromis niloticus) genome: repeat units, inverted sequences, pseudogenes and chromosome loci.

In higher eukaryotes, the 5S ribosomal DNA (5S rDNA) is organized in tandem arrays with repeat units composed of a coding region and a non-transcribed spacer sequence (NTS). These tandem arrays can be found on either one or more chromosome pairs. 5S rDNA copies from the tilapia fish, Oreochromis niloticus, were cloned and the nucleotide sequences of the coding region and of the non-transcribed spacer were determined. Moreover, the genomic organization of the 5S rDNA tandem repeats was investigated by fluorescence IN SITU hybridization (FISH) and Southern blot hybridization. Two 5S rDNA classes, one consisting of 1.4-kb repeats and another one with 0.5-kb repeats were identified and designated 5S rDNA type I and type II, respectively. An inverted 5S rRNA gene and a 5S rRNA putative pseudogene were also identified inside the tandem repeats of 5S rDNA type I. FISH permitted the visualization of the 5S rRNA genes at three chromosome loci, one of them consisting of arrays of the 5S rDNA type I, and the two others corresponding to arrays of the 5S rDNA type II. The two classes of the 5S rDNA, the presence of pseudogenes, and the inverted genes observed in the O. niloticus genome might be a consequence of the intense dynamics of the evolution of these tandem repeat elements.

Molecular organization of 5S rDNA in fishes of the genus Brycon.

There are few reports on the genomic organization of 5S rDNA in fish species. To characterize the 5S rDNA nucleotide sequence and chromosomal localization in the Neotropical fishes of the genus Brycon, 5S rDNA copies from seven species were generated by PCR. The nucleotide sequences of the coding region (5S rRNA gene) and the nontranscribed spacer (NTS) were determined, revealing that the 5S rRNA genes were highly conserved, while the NTSs were widely variable among the species analyzed. Moreover, two classes of NTS were detected in each species, characterized by base substitutions and insertions-deletions. Using fluorescence in situ hybridization (FISH), two 5S rDNA chromosome loci that could be related to the two 5S rDNA NTS classes were observed in at least one of the species studied. 5S rDNA sequencing and chromosomal localization permitted the characterization of Brycon spp. and suggest a higher similarity among some of them. The data obtained indicate that the 5S rDNA can be an useful genetic marker for species identification and evolutionary studies.

Nucleotide sequence of 5S rDNA and localization of the ribosomal RNA genes to metaphase chromosomes of the Tilapiine cichlid fish, Oreochromis niloticus.

In this study, we report the cloning and nucleotide sequence of PCR-generated 5S rDNA from the Tilapiine cichlid fish, Oreochromis niloticus. Two types of 5S rDNA were detected that differed by insertions and/or deletions and base substitutions within the non-transcribed spacer (NTS). Two 5S rDNA loci were observed by fluorescent in situ hybridization (FISH) in metaphase spreads of tilapia chromosomes. FISH using an 18S rDNA probe and silver nitrate sequential staining of 5S-FISH slides showed three 18S rDNA loci that are not syntenic to the 5S rDNA loci.

[The organization of the database and data flow in mass screening for cervical cancer]. / Organizacja bazy danych oraz przeplywu danych w centralnie sterowanym skryningu cytologicznym.

Mass screening, because of very many potential patients, requires storing and processing a great deal of medical and population information. That is why it should be supported not only by human resources but by computer techniques as well. The example of a computer science application in medicine is Populations Database System (PDB) which was designed and implemented in the Department of Institute of Mother and Child in Bialystok. The aim of this work is to evaluate PDB System's effectiveness in mass screening for cervical cancer. Population database contains several standard database files (DBF) and indexes. All the data is organized as a relational database. Every data relationship is at least in 1NF (first normal form). Functional dependency holds for the structures of database. Because of great variety of stored data it was essential to design how to enter information and how to combine database files to avoid redundancy. It has particular importance for the special functions of system, for example printing and sending individual invitation for an examination. In addition the system can realize all standard database functions and some statistical analysis. Special attention was paid to the problem of data security which is particularly important for medical information. Thanks to PDB system we could realize mass and active screening for cervical cancer in Bialystok. Without computer techniques it would be impossible to store, process and interpret so much data.