Studies of the anatomy of the extrahepatic biliary tract in Mammalia.

v.22:no.6(1940)

New histochemical and morphological findings in the female genital tract of Boophilus microplus (Acari, Ixodidae): an attempt toward the elucidation of fertilization in ticks

At present not only is the site of fertilization in ticks still unknown but it is also unclear as to how this mystery can be solved. Signs of fertilization can be observed throughout the female genital tract and these can be clues for the elucidation of the unsolved questions relating to ticks fertilization. In Boophilus microplus (Canestrini, 1887) the most important signs are the following: the final eversion of the acrosomal canal in females ready for oviposition; the presence of small tubules, resembling the subplasmalemal process of the spermatozoon between the oviduct cells; budding nuclei throughout the female genital tract; and the two Feulgen and DAPI positive areas in the oocyte at vitelogenesis. These morphological characteristics suggest that fertilization takes place in the internal cylinder which extends from the uterus to the ovary itself.

Buccal apparatus and gastrointestinal tract dimensions associated to the diet of early life stages of Centropomus undecimalis (Centropomidae, Actinopterygii)

The present study aims to compare the buccal apparatus and gastrointestinal tract of early life stages of Centropomus undecimalis (Bloch, 1792), and relate them to its diet. A total of 190 individuals collected with a channel net in the Catuama estuary (07º40'9.9''S, 34º50'36.7''W), northern coast of the state of Pernambuco, were examined. Morphometrical and meristic data were analyzed for the two initial developmental periods (larval and juvenile). Their digestive tube was morphologically characterized and its content identified. The longest transverse axis of food items was measured, and compared to the standard length (SL) and mouth gape size (D) of the individuals. Body measurement regressions differed significantly (p<0.001) between larvae and juveniles. The stomachs with food content (n=118 individuals) presented a proportion of 62% full and 30% empty (being 8% damaged). They differed in relation to the fullness level and presented a coiled shape when empty. The number of food items in relation to SL and D did not present an evident correlation. Larvae (SL<10 mm) feed on small copepods, while juveniles (SL=11.1 to 64.7 mm) ingest larvae of various decapod species, showing a distinct diet between these initial developmental stages.

Hydrographical and biological investigations in Norwegian fiords / by O. Nordgaard. The protist plankton and the diatoms in bottom samples / by E. Jørgensen. With 21 plates and 10 figures in the text.

chapters 3-4

Estimulació beta-adrenèrgica en la síndrome del destret respiratori agut (SDRA)

Estudi elaborat a partir d’una estada al Royal Brompton Hospital, Londres, Regne Unit, durant octubre i novembre del 2006.Els beneficis de la estimulació beta-adrenèrgica en pacients amb lesió pulmonar aguda (LPA) són coneguts, però no es disposa de dades sobre el possible efecte antiinflamatori. El condensat d'aire exhalat (CAE) és una tècnica no-invasiva de recollida de mostres del tracte respiratori inferior, podent ser útil en la monitorització de patologies respiratòries. S’ha usat marcadors biològics en el CAE de pacients ventilats mecànicament amb LPA per estudiar el possible efecte antiinflamatori que el salbutamol hi podria exercir. El CAE va ser recollit abans i després de l'administració de salbutamol inahalat. Inmediatament després es va mesurar la conductivitat i el pH abans i després de la desgasificació amb heli. Es va mesurar la concentració de nitrits i nitrats. Les mostres varen ser liofilitzades i guardades a -80ºC. La concentració de leucotriè B4 es va mesurar després de la reconstitució de la mostra. Els resultats s'expressen com a mitjana (error estàndard de la mostra). No s'han detectat diferències entre els valors de CAE basals dels pacients amb LPA i els de referència de la població sana de Barcelona. Es conclou doncs que el CAE és una tècnica no invasiva que pot ser usada en la monitorització de paceints ventilats mecànicament. El salbutamol inhalat incrementa de manera significativa el pH del CAE dels paceints amb LPA, tot i que un efecte directe de la inhalació de slabutamol no pot ser desestimat.

Results of An International Study of Quantitative BCR/ABL Assays Using Defined RNA Samples

Molecular monitoring of BCR/ABL transcripts by real time quantitative reverse transcription PCR (qRT-PCR) is an essential technique for clinical management of patients with BCR/ABL-positive CML and ALL. Though quantitative BCR/ABL assays are performed in hundreds of laboratories worldwide, results among these laboratories cannot be reliably compared due to heterogeneity in test methods, data analysis, reporting, and lack of quantitative standards. Recent efforts towards standardization have been limited in scope. Aliquots of RNA were sent to clinical test centers worldwide in order to evaluate methods and reporting for e1a2, b2a2, and b3a2 transcript levels using their own qRT-PCR assays. Total RNA was isolated from tissue culture cells that expressed each of the different BCR/ABL transcripts. Serial log dilutions were prepared, ranging from 100 to 10-5, in RNA isolated from HL60 cells. Laboratories performed 5 independent qRT-PCR reactions for each sample type at each dilution. In addition, 15 qRT-PCR reactions of the 10-3 b3a2 RNA dilution were run to assess reproducibility within and between laboratories. Participants were asked to run the samples following their standard protocols and to report cycle threshold (Ct), quantitative values for BCR/ABL and housekeeping genes, and ratios of BCR/ABL to housekeeping genes for each sample RNA. Thirty-seven (n=37) participants have submitted qRT-PCR results for analysis (36, 37, and 34 labs generated data for b2a2, b3a2, and e1a2, respectively). The limit of detection for this study was defined as the lowest dilution that a Ct value could be detected for all 5 replicates. For b2a2, 15, 16, 4, and 1 lab(s) showed a limit of detection at the 10-5, 10-4, 10-3, and 10-2 dilutions, respectively. For b3a2, 20, 13, and 4 labs showed a limit of detection at the 10-5, 10-4, and 10-3 dilutions, respectively. For e1a2, 10, 21, 2, and 1 lab(s) showed a limit of detection at the 10-5, 10-4, 10-3, and 10-2 dilutions, respectively. Log %BCR/ABL ratio values provided a method for comparing results between the different laboratories for each BCR/ABL dilution series. Linear regression analysis revealed concordance among the majority of participant data over the 10-1 to 10-4 dilutions. The overall slope values showed comparable results among the majority of b2a2 (mean=0.939; median=0.9627; range (0.399 - 1.1872)), b3a2 (mean=0.925; median=0.922; range (0.625 - 1.140)), and e1a2 (mean=0.897; median=0.909; range (0.5174 - 1.138)) laboratory results (Fig. 1-3)). Thirty-four (n=34) out of the 37 laboratories reported Ct values for all 15 replicates and only those with a complete data set were included in the inter-lab calculations. Eleven laboratories either did not report their copy number data or used other reporting units such as nanograms or cell numbers; therefore, only 26 laboratories were included in the overall analysis of copy numbers. The median copy number was 348.4, with a range from 15.6 to 547,000 copies (approximately a 4.5 log difference); the median intra-lab %CV was 19.2% with a range from 4.2% to 82.6%. While our international performance evaluation using serially diluted RNA samples has reinforced the fact that heterogeneity exists among clinical laboratories, it has also demonstrated that performance within a laboratory is overall very consistent. Accordingly, the availability of defined BCR/ABL RNAs may facilitate the validation of all phases of quantitative BCR/ABL analysis and may be extremely useful as a tool for monitoring assay performance. Ongoing analyses of these materials, along with the development of additional control materials, may solidify consensus around their application in routine laboratory testing and possible integration in worldwide efforts to standardize quantitative BCR/ABL testing.

Ultra high performance supercritical fluid chromatography coupled with tandem mass spectrometry for screening of doping agents. II: Analysis of biological samples.

The potential and applicability of UHPSFC-MS/MS for anti-doping screening in urine samples were tested for the first time. For this purpose, a group of 110 doping agents with diverse physicochemical properties was analyzed using two separation techniques, namely UHPLC-MS/MS and UHPSFC-MS/MS in both ESI+ and ESI- modes. The two approaches were compared in terms of selectivity, sensitivity, linearity and matrix effects. As expected, very diverse retentions and selectivities were obtained in UHPLC and UHPSFC, proving a good complementarity of these analytical strategies. In both conditions, acceptable peak shapes and MS detection capabilities were obtained within 7min analysis time, enabling the application of these two methods for screening purposes. Method sensitivity was found comparable for 46% of tested compounds, while higher sensitivity was observed for 21% of tested compounds in UHPLC-MS/MS and for 32% in UHPSFC-MS/MS. The latter demonstrated a lower susceptibility to matrix effects, which were mostly observed as signal suppression. In the case of UHPLC-MS/MS, more serious matrix effects were observed, leading typically to signal enhancement and the matrix effect was also concentration dependent, i.e., more significant matrix effects occurred at the lowest concentrations.

Innovative method for carbon dioxide determination in human postmortem cardiac gas samples using headspace-gas chromatography-mass spectrometry and stable labeled isotope as internal standard.

A novel approach to measure carbon dioxide (CO2) in gaseous samples, based on a precise and accurate quantification by (13)CO2 internal standard generated in situ is presented. The main goal of this study was to provide an innovative headspace-gas chromatography-mass spectrometry (HS-GC-MS) method applicable in the routine determination of CO2. The main drawback of the GC methods discussed in the literature for CO2 measurement is the lack of a specific internal standard necessary to perform quantification. CO2 measurement is still quantified by external calibration without taking into account analytical problems which can often occur considering gaseous samples. To avoid the manipulation of a stable isotope-labeled gas, we have chosen to generate in situ an internal labeled standard gas ((13)CO2) on the basis of the stoichiometric formation of CO2 by the reaction of hydrochloric acid (HCl) with sodium hydrogen carbonate (NaH(13)CO3). This method allows a precise measurement of CO2 concentration and was validated on various human postmortem gas samples in order to study its efficiency.

Distinctions between promastigotes of Leishmania species developing in the digestive tract of laboratory reared Lutzomyia longipalpis

Female Lutzomya longipalpis were exposed to infection by three different species/strains of Leishmania. When the insects were dissected four days after exposure, stained preparations were made of the flagellates contained in the digestive tract. Using traditional morphometric methods, L. amazonensis, L. guyanensis and an unnamed species of the mexicana complex could be distinguished from one another.

Validation and long-term evaluation of a modified on-line chiral analytical method for therapeutic drug monitoring of (R,S)-methadone in clinical samples.

Matrix effects, which represent an important issue in liquid chromatography coupled to mass spectrometry or tandem mass spectrometry detection, should be closely assessed during method development. In the case of quantitative analysis, the use of stable isotope-labelled internal standard with physico-chemical properties and ionization behaviour similar to the analyte is recommended. In this paper, an example of the choice of a co-eluting deuterated internal standard to compensate for short-term and long-term matrix effect in the case of chiral (R,S)-methadone plasma quantification is reported. The method was fully validated over a concentration range of 5-800 ng/mL for each methadone enantiomer with satisfactory relative bias (-1.0 to 1.0%), repeatability (0.9-4.9%) and intermediate precision (1.4-12.0%). From the results obtained during validation, a control chart process during 52 series of routine analysis was established using both intermediate precision standard deviation and FDA acceptance criteria. The results of routine quality control samples were generally included in the +/-15% variability around the target value and mainly in the two standard deviation interval illustrating the long-term stability of the method. The intermediate precision variability estimated in method validation was found to be coherent with the routine use of the method. During this period, 257 trough concentration and 54 peak concentration plasma samples of patients undergoing (R,S)-methadone treatment were successfully analysed for routine therapeutic drug monitoring.

Species identification in mammals from mixed biological samples based on mitochondrial DNA control region length polymorphism.

The ability to identify the species origin of an unknown biological sample is relevant in the fields of human and wildlife forensics. However, the detection of several species mixed in the same sample still remains a challenge. We developed and tested a new approach for mammal DNA identification in mixtures of two or three species, based on the analysis of mitochondrial DNA control region interspecific length polymorphism followed by direct sequencing. Contrary to other published methods dealing with species mixtures, our protocol requires a single universal primer pair and is not based on a pre-defined panel of species. Amplicons can be separated either on agarose gels or using CE. The advantages and limitations of the assay are discussed under different conditions, such as variable template concentration, amplicon sizes and size difference among the amplicons present in the mixture. For the first time, this protocol provides a simple, reliable and flexible method for simultaneous identification of multiple mammalian species from mixtures, without any prior knowledge of the species involved.