Intra- and inter-observer reliability of MRI examination of intervertebral disc abnormalities in patients with cervical myelopathy

Purpose: Intervertebral cervical disc herniation (CDH) is a relatively common disorder that can coexist with degenerative changes to worsen cervicogenic myelopathy. Despite the frequent disc abnormalities found in asymptomatic populations, magnetic resonance imaging (MRI) is considered excellent at detecting cervical spine myelopathy (CSM) associated with disc abnormality. The objective of this study was to investigate the intra- and inter-observer reliability of MRI detection of CSM in subjects who also had co-existing intervertebral disc abnormalities. Materials and methods: Seven experienced radiologists reviewed twice the MRI of 10 patients with clinically and/or imaging determined myelopathy. MRI assessment was performed individually, with and without operational guidelines. A Fleiss Kappa statistic was used to evaluate the intra- and inter-observer agreement. Results: The study found high intra-observer percent agreement but relatively low Kappa values on selected variables. Inter-observer reliability was also low and neither observation was improved with operational guidelines. We believe that those low values may be associated with the base rate problem of Kappa. Conclusion: In conclusion, this study demonstrated high intra-observer percent agreement in MR examination for intervertebral disc abnormalities in patients with underlying cervical myelopathy, but differing levels of intra- and inter-observer Kappa agreement among seven radiologists. (c) 2007 Elsevier Ireland Ltd. All rights reserved.

Intra and inter-examiner reliability of the subacromial impingement index

The present study aimed to assess the reliability of intra and inter-examiner subacromial impingement index (SII) measures obtained from radiographs. Thirty-six individuals were enrolled and divided into two groups: control group, composed of 18 volunteers in good general health without shoulder problems, and a group of 18 patients with subacromial impingement syndrome (SIS). Radiographic images were taken with the dominant upper limb in neutral rotation, while the volunteers held their arm at 90A degrees of abduction in the frontal plane. The beam of radiation at 30A degrees craniocaudal inclination was used to provide an antero-posterior image view. Three blinded examiners each performed three measurements from the subacromial space (SS) and the anatomical neck of the humerus (NH). The SII was calculated as the ratio of the SS and the NH measures. The mean values of SII were compared using t-tests. The intra-class correlation coefficient (ICC) was used to assess intra- and inter-examiner reliability of the measures. The mean values of SII were greater for the control group (0.12) than for the SIS group (0.08; p = 0.0071). SII measurements showed excellent intra (0.96-0.99) and inter-examiner reliability (0.94) for both the control and SIS group. The results of this study show the potential use of the SII; a greater mean value for the control group compared to the SIS group and excellent reliability for intra- and inter-examiner measurement. Validation studies of the index should be conducted to correlate the index with clinical findings from subacromial impingement syndrome.

Evaluating Theobroma grandiflorum for comparative genomic studies with Theobroma cacao

The seeds of Theobroma cacao (cacao) are the source of cocoa, the raw material for the multi-billion dollar chocolate industry. Cacao`s two most important traits are its unique seed storage triglyceride (cocoa butter) and the flavor of its fermented beans (chocolate). The genome of T. cacao is being sequenced, and to expand the utility of the genome sequence to the improvement of cacao, we are evaluating Theobroma grandiflorum, the closest economically important species of Theobroma for its potential use in a comparative genomic study. T. grandiflorum differs from cacao in important agronomic traits such as flavor of the fermented beans, disease resistance to witches` broom and abscission of mature fruits. By comparing genomic sequences and analyzing viable inter-specific hybrids, we hope to identify the key genes that regulate cacao`s most important traits. We have investigated the utility in T. grandiflorum of three types of markers (microsatellite markers, single-strand conformational polymorphism markers and single nucleotide polymorphism (SNP) markers) developed in cacao. Through sequencing of amplicons of 12 diverse individuals of both cacao and T. grandiflorum, we have identified new intra- and inter-specific SNPs. Two markers which had no overlap of alleles between the species were used to genotype putative inter-specific hybrid seedlings. Sequence conservation was significant and species-specific differences numerous enough to suggest that comparative genomics of T. grandiflorum and T. cacao will be useful in elucidating the genetic differences that lead to a variety of important agronomic trait differences.

Transcriptome analysis of Taenia solium cysticerci using Open Reading Frame ESTs (ORESTES)

Background: Human infection by the pork tapeworm Taenia solium affects more than 50 million people worldwide, particularly in underdeveloped and developing countries. Cysticercosis which arises from larval encystation can be life threatening and difficult to treat. Here, we investigate for the first time the transcriptome of the clinically relevant cysticerci larval form. Results: Using Expressed Sequence Tags (ESTs) produced by the ORESTES method, a total of 1,520 high quality ESTs were generated from 20 ORESTES cDNA mini-libraries and its analysis revealed fragments of genes with promising applications including 51 ESTs matching antigens previously described in other species, as well as 113 sequences representing proteins with potential extracellular localization, with obvious applications for immune-diagnosis or vaccine development. Conclusion: The set of sequences described here will contribute to deciphering the expression profile of this important parasite and will be informative for the genome assembly and annotation, as well as for studies of intra- and inter-specific sequence variability. Genes of interest for developing new diagnostic and therapeutic tools are described and discussed.

High-throughput SNP genotyping in the highly heterozygous genome of Eucalyptus: assay success, polymorphism and transferability across species

Background: High-throughput SNP genotyping has become an essential requirement for molecular breeding and population genomics studies in plant species. Large scale SNP developments have been reported for several mainstream crops. A growing interest now exists to expand the speed and resolution of genetic analysis to outbred species with highly heterozygous genomes. When nucleotide diversity is high, a refined diagnosis of the target SNP sequence context is needed to convert queried SNPs into high-quality genotypes using the Golden Gate Genotyping Technology (GGGT). This issue becomes exacerbated when attempting to transfer SNPs across species, a scarcely explored topic in plants, and likely to become significant for population genomics and inter specific breeding applications in less domesticated and less funded plant genera. Results: We have successfully developed the first set of 768 SNPs assayed by the GGGT for the highly heterozygous genome of Eucalyptus from a mixed Sanger/454 database with 1,164,695 ESTs and the preliminary 4.5X draft genome sequence for E. grandis. A systematic assessment of in silico SNP filtering requirements showed that stringent constraints on the SNP surrounding sequences have a significant impact on SNP genotyping performance and polymorphism. SNP assay success was high for the 288 SNPs selected with more rigorous in silico constraints; 93% of them provided high quality genotype calls and 71% of them were polymorphic in a diverse panel of 96 individuals of five different species. SNP reliability was high across nine Eucalyptus species belonging to three sections within subgenus Symphomyrtus and still satisfactory across species of two additional subgenera, although polymorphism declined as phylogenetic distance increased. Conclusions: This study indicates that the GGGT performs well both within and across species of Eucalyptus notwithstanding its nucleotide diversity >= 2%. The development of a much larger array of informative SNPs across multiple Eucalyptus species is feasible, although strongly dependent on having a representative and sufficiently deep collection of sequences from many individuals of each target species. A higher density SNP platform will be instrumental to undertake genome-wide phylogenetic and population genomics studies and to implement molecular breeding by Genomic Selection in Eucalyptus.

Development and validation of a simple and rapid capillary zone electrophoresis method for determination of nnrti nevirapine in pharmaceutical formulations

A simple and fast capillary zone electrophoresis (CZE) method has been developed and validated for quantification of a non-nucleoside reverse transcriptase inhibitor (NNRTI) nevirapine, in pharmaceuticals. The analysis was optimized using 10 mmol L-1 sodium phosphate buffer pH 2.5, +25 kV applied voltage, hydrodynamic injection 0.5 psi for 5 s and direct UV detection at 200 µm. Diazepam (50.0 µg mL-1) was used as internal standard. Under these conditions, nevirapine was analyzed in approximately less than 2.5 min. The analytical curve presented a coefficient of correlation of 0.9994. Limits of detection and quantification were 1.4 µg mL-1 and 4.3 µg mL-1, respectively. Intra- and inter-day precision expressed as relative standard deviations were 1.4% and 1.3%, respectively and the mean recovery was 100.81%. The active pharmaceutical ingredient was subjected to hydrolysis (acid, basic and neutral) and oxidative stress conditions. No interference of degradation products and tablet excipients were observed. This method showed to be rapid, simple, precise, accurate and economical for determination of nevirapine in pharmaceuticals and it is suitable for routine quality control analysis since CE offers benefits in terms of quicker method development and significantly reduced operating costs.

Intra and interannual variability in the Madeira River water chemistry and sediment load

Concentrations of cations (Na(+), Ca(2+), Mg(2+), K(+), NH(4) (+)), anions (HCO(3) (-), Cl(-), NO(3) (-), SO(4) (2-), PO(4) (3-)) and suspended sediments in the Madeira River water were determined near the city of Porto Velho (RO), in order to assess variation in water chemistry from 2004 to 2007. Calcium and bicarbonate were the dominant cation and anion, respectively. Significant seasonal differences were found, with highest concentrations occurring during the dry season, as expected from the drainage of Andean carbonate-rich substratum. Interannual variations were also observed, but became significant only when annual average discharge was 25% less than normal. Under this atypical discharge condition, bicarbonate was replaced by sulfate, and higher suspended sediment concentrations and loads were also observed. Compared to previously published studies, it appears that no significant changes in water chemistry have occurred during the last 20-30 years, although differences in approaches and sampling designs among this and previous studies may not allow detection of modest changes. The calculated suspended sediment load reported here is close to the values presented elsewhere, reinforcing the relative importance of this river as a sediment supplier for the Amazon Basin. Seasonality has a significant control on the chemistry of Madeira River waters, and severe decrease in discharge due to anthropogenic changes, such as construction of reservoirs or the occurrence of drier years-a plausible consequence of global climate change-may lead to modification in the chemical composition as well in the sediment deliver to the Amazon River.

Effects of recovery type after a judo match on blood lactate and performance in specific and non-specific judo tasks

The objective of the present study was to verify if active recovery (AR) applied after a judo match resulted in a better performance when compared to passive recovery (PR) in three tasks varying in specificity to the judo and in measurement of work performed: four upper-body Wingate tests (WT); special judo fitness test (SJFT); another match. For this purpose, three studies were conducted. Sixteen highly trained judo athletes took part in study 1, 9 in study 2, and 12 in study 3. During AR judokas ran (15 min) at the velocity corresponding to 70% of 4 mmol l(-1) blood lactate intensity (similar to 50% (V) over dotO(2) peak), while during PR they stayed seated at the competition area. The results indicated that the minimal recovery time reported in judo competitions (15 min) is long enough for sufficient recovery of WT performance and in a specific high-intensity test (SJFT). However, the odds ratio of winning a match increased ten times when a judoka performed AR and his opponent performed PR, but the cause of this phenomenon cannot be explained by changes in number of actions performed or by changes in match`s time structure.

Peptidase activities in the semen from the ductus deferens and uterus of the neotropical rattlesnake Crotalus durissus terrificus

To understand the role of peptidases in seminal physiology of Crotalus durissus terrificus, intra- and inter-seasonal activity levels of acid (APA), basic (APB), puromycin-sensitive (APN-PS) and puromycin-insensitive neutral (APN-PI), cystyl (CAP), dipeptidyl-IV (DPPIV), type-1 pyroglutamyl (PAP-I) and prolyl-imino (PIP) aminopeptidases as well as prolyl endopeptidase (POP) were evaluated in soluble (SF) and/or membrane-bound (MF) fractions of semen collected from the ductus deferens of the male reproductive tract and from the posterior portion of the uterus. Seminal APB, PIP and POP were detected in SF, while other peptidases were detected in SF and MF. Only the convoluted posterior uterus in winter and autumn had semen. Relative to other examined peptidases, in general, APN-PI, APN-PS and APB activities were predominant in the semen from the uterus and throughout the year in the semen from the ductus deferens, suggesting their great relevance in the seminal physiology of C. d. terrificus. The levels of peptidase activities in the ductus deferens semen varied seasonally and were different from those of semen in the uterus, suggesting that their modulatory actions on susceptible peptides are integrated to the male reproductive cycle events and spermatozoa viability of this snake.

Simultaneous determination of econazole nitrate, main impurities and preservatives in cream formulation by high performance liquid chromatography

A reversed-phase high performance liquid chromatographic (RP-HPLC) method for determination of econazole nitrate, preservatives (methylparaben and propylparaben) and its main impurities (4-chlorobenzl alcohol and alpha-(2,4-dicholorophenyl)-1H-imidazole-1-ethanol) in cream formulations, has been developed and validated. Separation was achieved on a column Bondclone (R) C18 (300 mm x 3.9 mm i.d., 10 mu m) using a gradient method with mobile phase composed of methanol and water. The flow rate was 1.4 mL min(-1), temperature of the column was 25 C and the detection was made at 220 nm. Miconazole nitrate was used as an internal standard. The total run time was less than 15 min, The analytical curves presented coefficient of correlation upper to 0.99 and detection and quantitation limits were calculated for all molecules. Excellent accuracy and precision were obtained for econazole nitrate. Recoveries varied from 97.9 to 102.3% and intra- and inter-day precisions, calculated as relative standard deviation (R.S.D), were lower than 2.2%. Specificity, robustness and assay for econazole nitrate were also determined. The method allowed the quantitative determination of econazole nitrate, its impurities and preservatives and could be applied as a stability-indicating method for econazole nitrate in cream formulations. (C) 2008 Elsevier B.V. All rights reserved.

Development and validation of a method for quantitative determination of econazole nitrate in cream formulation by capillary zone electrophoresis

A simple, fast, inexpensive and reliable capillary zone electrophoresis (CZE) method for the determination of econazole nitrate in cream formulations has been developed and validated. Optimum conditions comprised a pH 2.5 phosphate buffer at 20 mmol L(-1) concentration, +30 kV applied voltage in a 31.5 cm x 50 mu m I.D. capillary. Direct UV detection at 200 nm led to an adequate sensitivity without interference from sample excipients. A single extraction step of the cream sample in hydrochloric acid was performed prior to injection. Imidazole (100 mu g mL(-1)) was used as internal standard. Econazole nitrate migrates in approximately 1.2 min. The analytical curve presented a coefficient of correlation of 0.9995. Detection and quantitation limits were 1.85 and 5.62 mu g mL(-1), respectively. Excellent accuracy and precision were obtained. Recoveries varied from 98.1 to 102.5% and intra- and inter-day precisions, calculated as relative standard deviation (RSD), were better than 2.0%. The proposed CZE method presented advantageous performance characteristics and it can be considered suitable for the quality control of econazole nitrate cream formulations. (c) 2008 Elsevier B.V. All rights reserved.

UV-Derivative Spectrophotometric and Stability-Indicating High-Performance Liquid Chromatographic Methods for Determination of Simvastatin in Tablets

A stability-indicating high-performance liquid chromatographic (HPLC) and a second-order derivative spectrophotometric (UVDS) analytical methods were validated and compared for determination of simvastatin in tablets. The HPLC method was performed with isocratic elution using a C18 column and a mobile phase composed of methanol:acetonitrile:water (60:20:20, v/v/v) at a flow rate of 1.0 ml/min. The detection was made at 239 nm. In UVDS method, methanol and water were used in first dilution and distilled water was used in consecutive dilutions and as background. The second-order derivative signal measurement was taken at 255 nm. Analytical curves showed correlation coefficients > 0.999 for both methods. The quantitation limits (QL) were 2.41 mu g/ml for HPLC and 0.45 mu g/ml for UVDS, respectively. Intra and inter-day relative standard deviations were < 2.0 %. Statistical analysis with t- and F-tests are not exceeding their critical values demonstrating that there is no significant difference between the two methods at 95 % confidence level.

Enantioselective Analysis of Fluoxetine and Norfluoxetine by LC in Culture Medium for Application in Biotransformation Studies Employing Fungi

A liquid chromatography method is described for the analysis of fluoxetine and norfluoxetine enantiomers in fungi cultures. The analytes were separated simultaneously by LC employing a serial system. The resolution was performed using a mobile phase of ethanol: 15 mM ammonium acetate buffer solution, pH 5.9: acetonitrile (77.5:17.5:5, v/v/v). UV detection was at 227 nm. Hexane: isoamyl alcohol (98:2, v/v) was used as extractor solvent. The calibration curves were linear over the concentration range of 12.5-3,750 ng mL(-1) (r a parts per thousand yen 0.996). The values for intra- and inter-day precision and accuracy were a parts per thousand currency sign10% for all analytes. The validated method was used to evaluate fluoxetine biotransformation to its mammalian metabolite, norfluoxetine, by selected endophytic fungi. Although the desired biotransformation was not observed in the conditions used here, the method could be used to evaluate the biotransformation of fluoxetine by other fungi or to be extended to other matrices with adequate procedures for sample preparation.

Intra- and extracellular osmotic regulation in the hololimnetic Caridea and Anomura: a phylogenetic perspective on the conquest of fresh water by the decapod Crustacea

We investigate extra- and intracellular osmoregulatory capability in two species of hololimnetic Caridea and Anomura: Macrobrachium brasiliense, a palaemonid shrimp, and Aegla franca, an aeglid anomuran, both restricted to continental waters. We also appraise the sharing of physiological characteristics by the hololimnetic Decapoda, and their origins and role in the conquest of fresh water. Both species survive salinity exposure well. While overall hyperosmoregulatory capability is weak in A. franca and moderate in M. brasiliense, both species strongly hyporegulate hemolymph [Cl(-)] but not osmolality. Muscle total free amino acids (FAA) increase slowly but markedly in response to the rapid rise in hemolymph osmolality consequent to hyperosmotic challenge: 3.5-fold in A. franca and 1.9-fold in M. brasiliense. Glycine, taurine, arginine, alanine and proline constitute a parts per thousand 85% of muscle FAA pools in fresh water; taurine, arginine, alanine each contribute a parts per thousand 22% in A. franca, while glycine predominates (70%) in M. brasiliense. These FAA also show the greatest increases on salinity challenge. Muscle FAA titers correlate strongly (R = 0.82) with hemolymph osmolalities across the main decapod sub/infraorders, revealing that marine species with high hemolymph osmolalities achieve isosmoticity of the intra- and extracellular fluids partly through elevated intracellular FAA concentrations; freshwater species show low hemolymph osmolalities and exhibit reduced intracellular FAA titers, consistent with isosmoticity at a far lower external osmolality. Given the decapod phylogeny adopted here and their multiple, independent invasions of fresh water, particularly by the Caridea and Anomura, our findings suggest that homoplastic strategies underlie osmotic and ionic homeostasis in the extant freshwater Decapoda.

Intra- and interspecific divergence in the nuclear sequences of the clock gene period in species of the Drosophila buzzatii cluster

We have measured nucleotide variation in the CLOCK/CYCLE heterodimer inhibition domain (CCID) of the clock X-linked gene period in seven species belonging to the Drosophila buzzatii cluster, namely D. buzzatii, Drosophila koepferae, Drosophila antonietae, Drosophila serido, Drosophila gouveai, Drosophila seriema and Drosophila borborema. We detected that the purifying selection is the main force driving the sequence evolution in period, in agreement with the important role of CCID in clock machinery. Our survey revealed that period provides valuable phylogenetic information that allowed to resolve phylogenetic relationships among D. gouveai, D. borborema and D. seriema, which composed a polytomic clade in preliminary studies. The analysis of patterns of intraspecific variation revealed two different lineages of period in D. koepferae, probably reflecting introgressive hybridization from D. buzzatii, in concordance with previous molecular data.