Identification of "pathologs" (disease-related genes) from the RIKEN mouse cDNA dataset using human curation plus FACTS, a new biological information extraction system

Background: A major goal in the post-genomic era is to identify and characterise disease susceptibility genes and to apply this knowledge to disease prevention and treatment. Rodents and humans have remarkably similar genomes and share closely related biochemical, physiological and pathological pathways. In this work we utilised the latest information on the mouse transcriptome as revealed by the RIKEN FANTOM2 project to identify novel human disease-related candidate genes. We define a new term patholog to mean a homolog of a human disease-related gene encoding a product ( transcript, anti-sense or protein) potentially relevant to disease. Rather than just focus on Mendelian inheritance, we applied the analysis to all potential pathologs regardless of their inheritance pattern. Results: Bioinformatic analysis and human curation of 60,770 RIKEN full-length mouse cDNA clones produced 2,578 sequences that showed similarity ( 70 - 85% identity) to known human-disease genes. Using a newly developed biological information extraction and annotation tool ( FACTS) in parallel with human expert analysis of 17,051 MEDLINE scientific abstracts we identified 182 novel potential pathologs. Of these, 36 were identified by computational tools only, 49 by human expert analysis only and 97 by both methods. These pathologs were related to neoplastic ( 53%), hereditary ( 24%), immunological ( 5%), cardio-vascular (4%), or other (14%), disorders. Conclusions: Large scale genome projects continue to produce a vast amount of data with potential application to the study of human disease. For this potential to be realised we need intelligent strategies for data categorisation and the ability to link sequence data with relevant literature. This paper demonstrates the power of combining human expert annotation with FACTS, a newly developed bioinformatics tool, to identify novel pathologs from within large-scale mouse transcript datasets.

Quantification of carbamazepine, carbamazepine-10,11-epoxide, phenytoin and phenobarbital in plasma samples by stir bar-sorptive extraction and liquid chromatography

A sensitive and reproducible stir bar-sorptive extraction and high-performance liquid chromatography-UV detection (SBSE/HPLC-UV) method for therapeutic drug monitoring of carbamazepine, carbamazepine-10,11-epoxide, phenytoin and phenobarbital in plasma samples is described and compared with a liquid:liquid extraction (LLE/HPLC-UV) method. Important factors in the optimization of SBSE efficiency such as pH, extraction time and desorption conditions (solvents, mode magnetic stir, mode ultrasonic stir, time and number of steps) assured recoveries ranging from 72 to 86%, except for phenytoin (62%). Separation was obtained using a reverse phase C-18 column with UV detection (210 nm). The mobile phase consisted of water: acetonitrile (78:22, v/v). The SBSE/HPLC-UV method was linear over a working range of 0.08-40.0 mu g mL(-1) for carbamazepine, carbamazepine-10,11-epoxide and phenobarbital and 0.125-40.0 mu g mL(-1) for phenytoin, The intra-assay and inter-assay precision and accuracy were studied at three concentrations (1.0, 4.0 and 20.0 mu g mL(-1)). The intra-assay coefficients of variation (CVs) for all compounds were less than 8.8% and all inter-CVs were less than 10%. Limits of quantification were 0.08 mu g mL(-1) for carbamazepine, carbamazepine-10,11-epoxide and phenobarbital and 0.125 mu g mL(-1) for phenytoin. No interference of the drugs normally associated with antiepileptic drugs was observed. Based on figures of merit results, the SBSE/HPLC-UV proved adequate for antiepileptic drugs analyses from therapeutic levels. This method was successfully applied to the analysis of real samples and was as effective as the LLE/HPLC-UV method. (c) 2008 Elsevier B.V. All rights reserved.

Simultaneous analysis of parabens in cosmetic products by stir bar sorptive extraction and liquid chromatography

A sensitive and precise stir bar sorptive extraction (SBSE) combined with LC (SBSE/LC) analysis is described for simultaneous determination of methyl, ethyl, propyl, and butyl parabens in commercial cosmetic products in agreement with the European Union Cosmetics Directive 76/768/EEC. Important factors in the optimization of SB SE efficiency are discussed, such as time and temperature of extraction, pH, and ionic strength of the sample, matrix effects, and liquid desorption conditions by different modes (magnetic stirring, ultrasonic). The LOQs of the SBSE/LC method ranged from 30 to 200 ng/mg, with linear response over a dynamic range, from the LOQ to 2.5 mu g/mg, with a coefficient of determination higher than 0.993. The interday precision of the SBSE/LC method presented a coefficient of variation lower than 5%. The effectiveness of the proposed method was proven for analysis of commercial cosmetic products such as body creams, antiperspirant creams, and sunscreens.

Rifampicin determination in plasma by stir bar-sorptive extraction and liquid chromatography

A sensitive and reproducible stir bar-sorptive extraction and high performance liquid chromatography-UV detection (SBSE/HPLC-UV) method for therapeutic drug monitoring of rifampicin in plasma samples is described and compared with a liquid:liquid extraction (LLE/HPLC-UV) method. This miniaturized method can result in faster analysis, higher sample throughput, lower solvent consumption and less workload per sample while maintaining or even improving sensitivity. Important factors in the optimization of SBSE efficiency such as pH, temperature, extraction time and desorption conditions (solvents, mode magnetic stir, mode ultrasonic stir, time and number of steps) were optimized recoveries ranging from 75 to 80%. Separation was obtained using a reverse phase C(8) column with UV detection (254 nm). The mobile phase consisted of methanol:0.25 N sodium acetate buffer, pH 5.0 (58:42, v/v). The SBSE/HPLC-UV method was linear over a working range of 0.125-50.0 mu g mL(-1). The intra-assay and inter-assay precision and accuracy were studied at three concentrations (1.25, 6.25 and 25.0 mu g mL(-1)). The intra-assay coefficients of variation (CVs) for all compounds were less than 10% and all inter-CVs were less than 10%. Limits of quantification were 0.125 mu g mL(-1). Stability studies showed rifampicin was stable in plasma for 12 h after thawing; the samples were also stable for 24 h after preparation. Based on the figures of merit results, the SBSE/HPLC-UV proved to be adequate to the rifampicin analyses from therapeutic to toxic levels. This method was successfully applied to the analysis of real samples and was as effective as the LLE/HPLC-UV method. (C) 2009 Elsevier B.V. All rights reserved.

Chemical treatment of Escherichia coli .1. Extraction of intracellular protein from uninduced cells

Extraction of intracellular protein from Escherichia coli is traditionally achieved by mechanical disruption. A chemical treatment that destroys the integrity of the bacterial cell wall and could provide an alternative technique is examined in this study. Treatment with a combination of the chelating agent ethylenediaminetetraacetate (EDTA) (greater than 0.3 mM) and the chaotropic agent urea (6 M) is highly effective at releasing protein from uninduced E. coli. The 6 M urea in the presence of 3 mM EDTA can release cytoplasmic protein from both logarithmic-phase and stationary-phase E. coli cells at levels equivalent to mechanical disruption. The concentrations of the two chemical agents were the major variables affecting the maximum levels of protein release. Several minor variables and interactions were also identified. The kinetics of protein release is first order. For 2, 4, and 6 M urea with 3 mM EDTA, the time constant is approximately 2.5 min independent of urea concentration. Kinetics for 3 mM EDTA without urea is considerably slower, with a time constant of 12.3 min. (C) 1997 John Wiley & Sons, Inc.

Extraction of electrical characteristics from pixels of multifrequency EIT images

Computer modelling has shown that electrical characteristics of individual pixels may be extracted from within multiple-frequency electrical impedance tomography (MFEIT) images formed using a reference data set obtained from a purely resistive, homogeneous medium. In some applications it is desirable to extract the electrical characteristics of individual pixels from images where a purely resistive, homogeneous reference data set is not available. One such application of the technique of MFEIT is to allow the acquisition of in vivo images using reference data sets obtained from a non-homogeneous medium with a reactive component. However, the reactive component of the reference data set introduces difficulties with the extraction of the true electrical characteristics from the image pixels. This study was a preliminary investigation of a technique to extract electrical parameters from multifrequency images when the reference data set has a reactive component. Unlike the situation in which a homogenous, resistive data set is available, it is not possible to obtain the impedance and phase information directly from the image pixel values of the MFEIT images data set, as the phase of the reactive reference is not known. The method reported here to extract the electrical characteristics (the Cole-Cole plot) initially assumes that this phase angle is zero. With this assumption, an impedance spectrum can be directly extracted from the image set. To obtain the true Cole-Cole plot a correction must be applied to account for the inherent rotation of the extracted impedance spectrum about the origin, which is a result of the assumption. This work shows that the angle of rotation associated with the reactive component of the reference data set may be determined using a priori knowledge of the distribution of frequencies of the Cole-Cole plot. Using this angle of rotation, the true Cole-Cole plot can be obtained from the impedance spectrum extracted from the MFEIT image data set. The method was investigated using simulated data, both with and without noise, and also for image data obtained in vitro. The in vitro studies involved 32 logarithmically spaced frequencies from 4 kHz up to 1 MHz and demonstrated that differences between the true characteristics and those of the impedance spectrum were reduced significantly after application of the correction technique. The differences between the extracted parameters and the true values prior to correction were in the range from 16% to 70%. Following application of the correction technique the differences were reduced to less than 5%. The parameters obtained from the Cole-Cole plot may be useful as a characterization of the nature and health of the imaged tissues.

Evaluating SVM and MLDA in the extraction of discriminant regions for mental state prediction

Pattern recognition methods have been successfully applied in several functional neuroimaging studies. These methods can be used to infer cognitive states, so-called brain decoding. Using such approaches, it is possible to predict the mental state of a subject or a stimulus class by analyzing the spatial distribution of neural responses. In addition it is possible to identify the regions of the brain containing the information that underlies the classification. The Support Vector Machine (SVM) is one of the most popular methods used to carry out this type of analysis. The aim of the current study is the evaluation of SVM and Maximum uncertainty Linear Discrimination Analysis (MLDA) in extracting the voxels containing discriminative information for the prediction of mental states. The comparison has been carried out using fMRI data from 41 healthy control subjects who participated in two experiments, one involving visual-auditory stimulation and the other based on bimanual fingertapping sequences. The results suggest that MLDA uses significantly more voxels containing discriminative information (related to different experimental conditions) to classify the data. On the other hand, SVM is more parsimonious and uses less voxels to achieve similar classification accuracies. In conclusion, MLDA is mostly focused on extracting all discriminative information available, while SVM extracts the information which is sufficient for classification. (C) 2009 Elsevier Inc. All rights reserved.

Organ-Sparing Microsurgical Resection of Incidental Testicular Tumors Plus Microdissection for Sperm Extraction and Cryopreservation in Azoospermic Patients: Surgical Aspects and Technical Refinements

INTRODUCTION The management of nonpalpable testicular masses is a challenging task, and coexisting infertility can further complicate the treatment decisions. We present our technique for microsurgical organ-sparing resection of incidental nonpalpable testicular nodules combined with microdissection for testicular sperm extraction and tissue cryopreservation in azoospermic patients. TECHNICAL CONSIDERATIONS Five infertile patients with azoospermia presented with nonpalpable hypoechoic testicular masses that were detected by Ultrasonography and underwent organ-sparing surgery. The testis was delivered through an inguinal incision, and the blood circulation was interrupted with a vascular clamp placed on the spermatic cord. Sludged ice was used to prevent warm ischemia, and a temperature probe was used to control the temperature at 12 degrees-15 degrees C. Real-time reflex ultrasonography was used to locate the tumor, and a stereotaxic hook-shaped needle was inserted under ultrasound guidance. The needle was placed adjacent to the tumor to guide the microsurgical resection. The tunica albuginea was incised over the tumor, which was dissected and removed, along with the adjoining parenchymal tissue. Frozen section studies were performed and, if malignancy was confirmed, biopsies of the tumor cavity margins and remaining parenchyma were obtained to ensure the absence of residual tumor. Microdissection was performed for excision of selected enlarged tubules that were processed and cryopreserved. CONCLUSIONS We present a technique for microsurgical organ-sparing resection of testicular tumor and sperm extraction that can be used in selected infertile patients with azoospermia in whom incidental masses have been diagnosed by ultrasonography. This conservative approach should be especially considered for patients with a solitary testis or bilateral tumors. UROLOGY 73: 887-892, 2009. (C) 2009 Elsevier Inc.

Prospective randomized comparison of human oocyte cryopreservation with slow-rate freezing or vitrification

Objective: To compare cryopreservation of mature human oocytes with slow-rate freezing and vitrification and determine which is most efficient at establishing a pregnancy. Design: Prospective randomized. Setting: Academically affiliated, private fertility center. Patient(s): Consenting patients with concerns about embryo cryopreservation and more than nine mature oocytes at retrieval were randomized to slow-rate freezing or vitrification of supernumerary (more than nine) oocytes. Intervention(s): Oocytes were frozen or vitrified, and upon request oocytes were thawed or warmed, respectively. Main Outcome Measure(s): Oocyte survival, fertilization, embryo development, and clinical pregnancy. Result(s): Patient use has resulted in 30 thaws and 48 warmings. Women`s age at time of cryopreservation was similar. Oocyte survival was significantly higher following vitrification/warming (81%) compared with freezing/thawing (67%). Fertilization was more successful in oocytes vitrified/warmed compared with frozen/thawed. Fertilized oocytes from vitrification/warming had significantly better cleavage rates (84%) compared with freezing/thawing (71%) and resulted in embryos with significantly better morphology. Although similar numbers of embryos were transferred, embryos resulting from vitrified oocytes had significantly enhanced clinical (38%) pregnancy rates compared with embryos resulting from frozen oocyte (13%). Miscarriage and/or spontaneous abortion rates were similar. Conclusion(s): Our results suggest that vitrification/warming is currently the most efficient means of oocyte cryopreservation in relation to subsequent success in establishing pregnancy. (Fertil Steril (R) 2010; 94: 2088-95. (C) 2010 by American Society for Reproductive Medicine.)

Changes in slow and fast alpha bands in subjects submitted to different amounts of functional electrostimulation

Objective: To examine the changes in slow (8-10 Hz)and fast (10-12 Hz) alpha bands of EEG in three groups of subjects submitted to different amounts of functional electrostimulation (FES). Our hypothesis is that different amounts of electrostimulation may cause different patterns of activation in the sensorimotor cortex. In particular, we expect to see an increase in alpha power due to habituation effects. We examine the two bands comprised by alpha rhythm (i.e., slow and fast alpha), since these two sub-rhythms are related to distinct aspects: general energy demands and specific motor aspects, respectively. Methods: The sample was composed of 27 students, both sexes, aging between 25 and 40 years old. The subjects were randomly distributed in three groups: control (n = 9), G24 (n = 9) and G36 (n = 9). A FES equipment (Neuro Compact-2462) was used to stimulate the right index finger extension. Simultaneously, the electroencephalographic signal was acquired. We investigated the absolute power in slow and fast alpha bands in the sensorimotor cortex. Results: The G36 indicated a significant increasing in absolute power values in lower and higher alpha components, respectively, when compared with the control group. Particularly, in the following regions: pre-motor cortex and primary motor cortex. Discussion: FES seems to promote cortical adaptations that are similar to those observed when someone learns a procedural task. FES application in the G36 was more effective in promoting such neural changes. The lower and higher components of alpha rhythms behave differently in their topographical distribution during FES application. These results suggest a somatotopic organization in primary motor cortex which can be represented by the fast alpha component. (C) 2008 Elsevier Ireland Ltd. All rights reserved.

An easy method for handling the genus Phoneutria (Araneae, Ctenidae) for venom extraction

This paper describes an easy, cheap, and safe method of capturing and handling the medically important spider Phoneutria for venom extraction. The method does not injure or kill the spider and allows the extraction of pure venom.

Histological and histomorphometrical alterations of the periodontal ligament in gerbils submitted to teeth extraction

This study verified the effect of unilateral teeth extraction on the periodontal ligament in gerbils (Meriones unguiculatus). Ten adult male gerbils weighing about 50 g had induced occlusal alterations by upper left molar extractions while the other ten animals, only submitted to surgical stress, were considered as controls. The periodontal ligament was characterized by qualitative and quantitative analysis, histological description and histomorphometric quantification. Significant alterations were observed on the left side of the experimental group (P < 0.05), the hypofunctional region, when it was compared with the contralateral side and the corresponding region of the control group. Two months after occlusal alterations induced by unilateral teeth extraction, atrophic histological alterations and a decrease in the periodontal space on the ipsilateral side characterized the periodontal ligament. In this study it was possible to conclude that the gerbil can be used in experimental models attempting to correlate the periodontium`s biological response to various mechanical stresses, as the periodontal ligament was shown to be highly sensitive to occlusal alterations.

Slow crack growth and reliability of dental ceramics

Objective. To determine the slow crack growth (SCG) and Weibull parameters of five dental ceramics: a vitreous porcelain (V), a leucite-based porcelain (D), a leucite-based glass-ceramic (E1), a lithium disilicate glass-ceramic (E2) and a glass-infiltrated alumina composite (IC). Methods. Eighty disks (empty set 12mm x 1.1mm thick) of each material were constructed according to manufacturers` recommendations and polished. The stress corrosion susceptibility coefficient (n) was obtained by dynamic fatigue test, and specimens were tested in biaxial flexure at five stress rates immersed in artificial saliva at 37 degrees C. Weibull parameters were calculated for the 30 specimens tested at 1MPa/s in artificial saliva at 37 degrees C. The 80 specimens were distributed as follows: 10 for each stress rate (10(-2), 10(-1), 10(1), 10(2) MPa/s), 10 for inert strength (10(2) MPa/s, silicon oil) and 30 for 10(0) MPa/s. Fractographic analysis was also performed to investigate the fracture origin. Results. E2 showed the lowest slow crack growth susceptibility coefficient (17.2), followed by D (20.4) and V (26.3). E1 and IC presented the highest n values (30.1 and 31.1, respectively). Porcelain V presented the lowest Weibull modulus (5.2). All other materials showed similar Weibull modulus values, ranging from 9.4 to 11.7. Fractographic analysis indicated that for porcelain D, glass-ceramics E1 and E2, and composite IC crack deflection was the main toughening mechanism. Significance. This study provides a detailed microstructural and slow crack growth characterization of widely used dental ceramics. This is important from a clinical standpoint to assist the clinician in choosing the best ceramic material for each situation as well as predicting its clinical longevity. It also can be helpful in developing new materials for dental prostheses. (c) 2010 Academy of Dental Materials. Published by Elsevier Ltd. All rights reserved.

Effect of ion exchange on strength and slow crack growth of a dental porcelain

Objectives. To determine the effect of ion exchange on slow crack growth (SCG) parameters (n, stress corrosion susceptibility coefficient, and sigma(f0), scaling parameter) and Weibull parameters (m, Weibull modulus, and sigma(0), characteristic strength) of a dental porcelain. Methods. 160 porcelain discs were fabricated according to manufacturer`s instructions, polished through 1 mu m and divided into two groups: GC (control) and GI (submitted to an ion exchange procedure using a KNO(3) paste at 470 degrees C for 15 min). SCG parameters were determined by biaxial flexural strength test in artificial saliva at 37 degrees C using five constant stress rates (n =10). 20 specimens of each group were tested at 1 MPa/s to determine Weibull parameters. The SPT diagram was constructed using the least-squares fit of the strength data versus probability of failure. Results. Mean values of m and sigma(0) (95% confidence interval), n and sigma(f0) (standard deviation) were, respectively: 13.8 (10.1-18.8) and 60.4 (58.5 - 62.2), 24.1 (2.5) and 58.1 (0.01) for GC and 7.4 (5.3 -10.0) and 136.8 (129.1-144.7), 36.7 (7.3) and 127.9 (0.01) for GI. Fracture stresses (MPa) calculated using the SPT diagram for lifetimes of 1 day, 1 year and 10 years (at a 5% failure probability) were, respectively, 31.8, 24.9 and 22.7 for GC and 71.2, 60.6 and 56.9 for GI. Significance. For the porcelain tested, the ion exchange process improved strength and resistance to SCG, however, the material`s reliability decreased. The predicted fracture stress at 5% failure probability for a lifetime of 10 years was also higher for the ion treated group. (C) 009 Academy of Dental Materials. Published by Elsevier Ltd. All rights reserved.