A novel isolation method of Brucella species and molecular tracking of Brucella suis biovar 2 in domestic and wild animals

Brucella suis biovar 2 is the most common aetiological agent of porcine brucellosis in Europe. B. suis biovar 2 is considered to have low zoonotic potential, but is a causative agent of reproductive losses in pigs, and it is thus economically important. The multilocus variable-number of tandem repeats genotyping analysis of 16 loci (MLVA-16) has proven to be highly discriminatory and is the most suitable assay for simultaneously identifying B. suis and tracking infections. The aim of this study was to investigate the relatedness between isolates of B. suis biovar 2 obtained during a brucellosis outbreak in domestic pigs and isolates from wild boars and hares collected from proximal or remote geographical areas by MLVA-16. A cluster analysis of the MLVA-16 data revealed that most of the isolates obtained from Switzerland clustered together, with the exception of one isolate. The outbreak isolates constituted a unique subcluster (with a genetic similarity >93.8%) distinct from that of the isolates obtained from wild animals, suggesting that direct transmission of the bacterium from wild boars to domestic pigs did not occur in this outbreak. To obtain a representative number of isolates for MLVA-16, alternative methods of Brucella spp. isolation from tissue samples were compared with conventional direct cultivation on a Brucella-selective agar. We observed an enhanced sensitivity when mechanical homogenisation was followed by host cell lysis prior to cultivation on the Brucella-selective agar. This work demonstrates that MLVA-16 is an excellent tool for both monitoring brucellosis and investigating outbreaks. Additionally, we present efficient alternatives for the isolation of Brucella spp.

Core networks for visual-concrete and abstract thought content: a brain electric microstate analysis

Commonality of activation of spontaneously forming and stimulus-induced mental representations is an often made but rarely tested assumption in neuroscience. In a conjunction analysis of two earlier studies, brain electric activity during visual-concrete and abstract thoughts was studied. The conditions were: in study 1, spontaneous stimulus-independent thinking (post-hoc, visual imagery or abstract thought were identified); in study 2, reading of single nouns ranking high or low on a visual imagery scale. In both studies, subjects' tasks were similar: when prompted, they had to recall the last thought (study 1) or the last word (study 2). In both studies, subjects had no instruction to classify or to visually imagine their thoughts, and accordingly were not aware of the studies' aim. Brain electric data were analyzed into functional topographic brain images (using LORETA) of the last microstate before the prompt (study 1) and of the word-type discriminating event-related microstate after word onset (study 2). Conjunction analysis across the two studies yielded commonality of activation of core networks for abstract thought content in left anterior superior regions, and for visual-concrete thought content in right temporal-posterior inferior regions. The results suggest that two different core networks are automatedly activated when abstract or visual-concrete information, respectively, enters working memory, without a subject task or instruction about the two classes of information, and regardless of internal or external origin, and of input modality. These core machineries of working memory thus are invariant to source or modality of input when treating the two types of information.

Development of a rapid column-switching LC-MS/MS method for the quantification of THCCOOH and THCCOOH-glucuronide in whole blood for assessing cannabis consumption frequency

The concentration of 11-nor-9-carboxy-Δ(9)-tetrahydrocannabinol (THCCOOH) in whole blood is used as a parameter for assessing the consumption behavior of cannabis consumers. The blood level of THCCOOH-glucuronide might provide additional information about the frequency of cannabis use. To verify this assumption, a column-switching liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the rapid and direct quantification of free and glucuronidated THCCOOH in human whole blood was newly developed. The method comprised protein precipitation, followed by injection of the processed sample onto a trapping column and subsequent gradient elution to an analytical column for separation and detection. The total LC run time was 4.5 min. Detection of the analytes was accomplished by electrospray ionization in positive ion mode and selected reaction monitoring using a triple-stage quadrupole mass spectrometer. The method was fully validated by evaluating the following parameters: linearity, lower limit of quantification, accuracy and imprecision, selectivity, extraction efficiency, matrix effect, carry-over, dilution integrity, analyte stability, and re-injection reproducibility. All acceptance criteria were analyzed and the predefined criteria met. Linearity ranged from 5.0 to 500 μg/L for both analytes. The method was successfully applied to whole blood samples from a large collective of cannabis consumers, demonstrating its applicability in the forensic field.

A Minimally Invasive Technique for the Detection and Analysis of Pulmonary Fat Embolism: A Feasibility Study*

We investigated the feasibility of postmortem percutaneous needle biopsy (PNB) for obtaining pulmonary samples adequate for the study of pulmonary fat embolism (PFE). Samples of both lungs were obtained from 26 cadavers via two different methods: (i) PNB and (ii) the double-edged knife technique, the gold standard at our institute. After water storage and Sudan III staining, six forensic pathologists independently examined all samples for the presence and severity of PFE. The results were compared and analyzed in each case regarding the vitality of the PFE and its relationship to the cause of death. The results showed that PFE was almost identically diagnosed and graded on the samples obtained via both methods. The discrepancies between the two techniques did not affect the diagnoses of vitality or cause of death related to PFE. This study demonstrates the feasibility of the PNB sampling method for the diagnosis and interpretation of PFE in the postmortem setting.

Frog oocytes to unveil the structure and supramolecular organization of human transport proteins

Structural analyses of heterologously expressed mammalian membrane proteins remain a great challenge given that microgram to milligram amounts of correctly folded and highly purified proteins are required. Here, we present a novel method for the expression and affinity purification of recombinant mammalian and in particular human transport proteins in Xenopus laevis frog oocytes. The method was validated for four human and one murine transporter. Negative stain transmission electron microscopy (TEM) and single particle analysis (SPA) of two of these transporters, i.e., the potassium-chloride cotransporter 4 (KCC4) and the aquaporin-1 (AQP1) water channel, revealed the expected quaternary structures within homogeneous preparations, and thus correct protein folding and assembly. This is the first time a cation-chloride cotransporter (SLC12) family member is isolated, and its shape, dimensions, low-resolution structure and oligomeric state determined by TEM, i.e., by a direct method. Finally, we were able to grow 2D crystals of human AQP1. The ability of AQP1 to crystallize was a strong indicator for the structural integrity of the purified recombinant protein. This approach will open the way for the structure determination of many human membrane transporters taking full advantage of the Xenopus laevis oocyte expression system that generally yields robust functional expression.

A tool for the qualitative comparison of membrane-embedded and detergent-solubilized membrane protein structures in projection

The calculation of projection structures (PSs) from Protein Data Bank (PDB)-coordinate files of membrane proteins is not well-established. Reports on such attempts exist but are rare. In addition, the different procedures are barely described and thus difficult if not impossible to reproduce. Here we present a simple, fast and well-documented method for the calculation and visualization of PSs from PDB-coordinate files of membrane proteins: the projection structure visualization (PSV)-method. The PSV-method was successfully validated using the PS of aquaporin-1 (AQP1) from 2D crystals and cryo-transmission electron microscopy, and the PDB-coordinate file of AQP1 determined from 3D crystals and X-ray crystallography. Besides AQP1, which is a relatively rigid protein, we also studied a flexible membrane transport protein, i.e. the L-arginine/agmatine antiporter AdiC. Comparison of PSs calculated from the existing PDB-coordinate files of substrate-free and L-arginine-bound AdiC indicated that conformational changes are detected in projection. Importantly, structural differences were found between the PSV-method calculated PSs of the detergent-solubilized AdiC proteins and the PS from cryo-TEM of membrane-embedded AdiC. These differences are particularly exciting since they may reflect a different conformation of AdiC induced by the lateral pressure in the lipid bilayer.

Development and validation of a liquid chromatography-tandem mass spectrometry method for the simultaneous determination of nine corticosteroid residues in bovine liver samples

A liquid chromatography tandem mass spectrometry (LC-MS/MS) confirmatory method for the simultaneous determination of nine corticosteroids in liver, including the four MRL compounds listed in Council Regulation 37/2010, was developed. After an enzymatic deconjugation and a solvent extraction of the liver tissue, the resulting solution was cleaned up through an SPE Oasis HLB cartridge. The analytes were then detected by liquid chromatography-negative-ion electrospray tandem mass spectrometry, using deuterium-labelled internal standards. The procedure was validated as a quantitative confirmatory method according to the Commission Decision 2002/657/EC criteria. The results showed that the method was suitable for statutory residue testing regarding the following performance characteristics: instrumental linearity, specificity, precision (repeatability and intra-laboratory reproducibility), recovery, decision limit (CCα), detection capability (CCβ) and ruggedness. All the corticosteroids can be detected at a concentration around 1 μg kg(-1); the recoveries were above 62% for all the analytes. Repeatability and reproducibility (within-laboratory reproducibility) for all the analytes were below 7.65% and 15.5%, respectively.

A Novel Method for the Homogenization of Daily Temperature Series and Its Relevance for Climate Change Analysis

Instrumental daily series of temperature are often affected by inhomogeneities. Several methods are available for their correction at monthly and annual scales, whereas few exist for daily data. Here, an improved version of the higher-order moments (HOM) method, the higher-order moments for autocorrelated data (HOMAD), is proposed. HOMAD addresses the main weaknesses of HOM, namely, data autocorrelation and the subjective choice of regression parameters. Simulated series are used for the comparison of both methodologies. The results highlight and reveal that HOMAD outperforms HOM for small samples. Additionally, three daily temperature time series from stations in the eastern Mediterranean are used to show the impact of homogenization procedures on trend estimation and the assessment of extremes. HOMAD provides an improved correction of daily temperature time series and further supports the use of corrected daily temperature time series prior to climate change assessment.

Development and validation of a liquid chromatography-tandem mass spectrometry method for the quantitation of synthetic cannabinoids of the aminoalkylindole type and methanandamide in serum and its application to forensic samples

After the discovery of synthetic cannabimimetic substances in 'Spice'-like herbal mixtures marketed as 'incense' or 'plant fertilizer' the active compounds have been declared as controlled substances in several European countries. As expected, a monitoring of new herbal mixtures which continue to appear on the market revealed that shortly after control measures have been taken by legal authorities, other compounds were added to existing mixtures and to new products. Several compounds of the aminoalkylindole type have been detected so far in herbal mixtures but still their consumption cannot be detected by commonly used drug-screening procedures, encouraging drug users to substitute cannabis with those products. There is a increasing demand on the part of police authorities, hospitals and psychiatrists for detection and quantification of synthetic cannabinoids in biological samples originating from psychiatric inpatients, emergency units or assessment of fitness to drive. Therefore, a liquid chromatography-tandem mass spectrometry method after liquid-liquid extraction for the quantitation of JWH-015, JWH-018, JWH-073, JWH-081, JWH 200, JWH-250, WIN 55,212-2 and methanandamide and the detection of JWH-019 and JWH-020 in human serum has been developed and fully validated according to guidelines for forensic toxicological analyses. The method was successfully applied to 101 serum samples from 80 subjects provided by hospitals, detoxification and therapy centers, forensic psychiatric centers and police authorities. Fifty-seven samples or 56.4% were found positive for at least one aminoalkylindole. JWH-019, JWH-020, JWH-200, WIN 55,212-2 and methanandamide were not detected in any of the analyzed samples.

Radiological anatomy of the obturator nerve and its articular branches: basis to develop a method of radiofrequency denervation for hip joint pain

OBJECTIVE: A previous study of radiofrequency neurotomy of the articular branches of the obturator nerve for hip joint pain produced modest results. Based on an anatomical and radiological study, we sought to define a potentially more effective radiofrequency method. DESIGN: Ten cadavers were studied, four of them bilaterally. The obturator nerve and its articular branches were marked by wires. Their radiological relationship to the bone structures on fluoroscopy was imaged and analyzed. A magnetic resonance imaging (MRI) study was undertaken on 20 patients to determine the structures that would be encountered by the radiofrequency electrode during different possible percutaneous approaches. RESULTS: The articular branches of the obturator nerve vary in location over a wide area. The previously described method of denervating the hip joint did not take this variation into account. Moreover, it approached the nerves perpendicularly. Because optimal coagulation requires electrodes to lie parallel to the nerves, a perpendicular approach probably produced only a minimal lesion. In addition, MRI demonstrated that a perpendicular approach is likely to puncture femoral vessels. Vessel puncture can be avoided if an oblique pass is used. Such an approach minimizes the angle between the target nerves and the electrode, and increases the likelihood of the nerve being captured by the lesion made. Multiple lesions need to be made in order to accommodate the variability in location of the articular nerves. CONCLUSIONS: The method that we described has the potential to produce complete and reliable nerve coagulation. Moreover, it minimizes the risk of penetrating the great vessels. The efficacy of this approach should be tested in clinical trials.