Lack of induction of rat liver mixed-function oxidases after chronic administration of high brotizolam doses to rats

Rats were treated with 10, 200 or 400 mg/kg brotizolam (Lendormin) for 4 weeks, then liver microsomes were prepared and the in vitro transformation of several model substances studied. Furthermore, after similar treatment of rats, the metabolite pattern in the plasma was studied using [14C]brotizolam as a marker. Finally the same investigations were performed after pretreating the rats with the enzyme inducers, phenobarbital or 3-methylchol-anthrene, for 3 days instead of brotizolam. The amount of microsomal protein in the rat liver was increased after all 3 doses of brotizolam, the liver weight after the highest dose only. Activity of the flavoenzyme NADPH cytochrome-c reductase was the only enzyme activity increased after 200 and 400 mg/kg brotizolam, whereas cytochrome P-450 content decreased after 400 mg/kg brotizolam. Activities of the mixed-function oxidases studied were not changed at all. Marked changes after brotizolam administration were seen in the metabolite pattern. The higher doses led to reduced amounts of both of the very polar metabolites. Simultaneously metabolite We 964 (= brotizolam hydroxylated at the methyl group) and the unchanged brotizolam increased several-fold. Treatment of rats with phenobarbital or 3-methylcholanthrene showed the typical but different changes in enzyme activities. The metabolite pattern of brotizolam, however, was not changed. From the results it is concluded that a 4-week treatment of rats with up to 400 mg/kg brotizolam causes no induction of mixed-function oxidases in the liver. The changes of the metabolite pattern described can be discussed as an effect of liver enzyme saturation.     

Material Extrusion of Structural Polymer–Aluminum Joints—Examining Shear Strength,Wetting, Polymer Melt Rheology and Aging

Generating polymer–metal structures by means of additive manufacturing offers huge potential for customized, sustainable and lightweight solutions. However, challenges exist, primarily with regard to reliability and reproducibility of the additively generated joints. In this study, the polymers ABS, PETG and PLA, which are common in material extrusion, were joined to grit-blasted aluminum substrates. Temperature dependence of polymer melt rheology, wetting and tensile single-lap-shear strength were examined in order to obtain appropriate thermal processing conditions. Joints with high adhesive strength in the fresh state were aged for up to 100 days in two different moderate environments. For the given conditions, PETG was most suitable for generating structural joints. Contrary to PETG, ABS–aluminum joints in the fresh state as well as PLA–aluminum joints in the aged state did not meet the demands of a structural joint. For the considered polymers and processing conditions, this study implies that the suitability of a polymer and a thermal processing condition to form a polymer–aluminum joint by material extrusion can be evaluated based on the polymer’s rheological properties. Moreover, wetting experiments improved estimation of the resulting tensile single-lap-shear strength.     

Clinical impairment in premanifest and early Huntington's disease is associated with regionally specific atrophy

TRACK‐HD is a multicentre longitudinal observational study investigating the use of clinical assessments and 3‐Tesla magnetic resonance imaging as potential biomarkers for future therapeutic trials in Huntington's disease (HD). The cross‐sectional data from this large well‐characterized dataset provide the opportunity to improve our knowledge of how the underlying neuropathology of HD may contribute to the clinical manifestations of the disease across the spectrum of premanifest (PreHD) and early HD. Two hundred and thirty nine gene‐positive subjects (120 PreHD and 119 early HD) from the TRACK‐HD study were included. Using voxel‐based morphometry (VBM), grey and white matter volumes were correlated with performance in four domains: quantitative motor (tongue force, metronome tapping, and gait); oculomotor [anti‐saccade error rate (ASE)]; cognition (negative emotion recognition, spot the change and the University of Pennsylvania smell identification test) and neuropsychiatric measures (apathy, affect and irritability). After adjusting for estimated disease severity, regionally specific associations between structural loss and task performance were found (familywise error corrected, P < 0.05); impairment in tongue force, metronome tapping and ASE were all associated with striatal loss. Additionally, tongue force deficits and ASE were associated with volume reduction in the occipital lobe. Impaired recognition of negative emotions was associated with volumetric reductions in the precuneus and cuneus. Our study reveals specific associations between atrophy and decline in a range of clinical modalities, demonstrating the utility of VBM correlation analysis for investigating these relationships in HD. Hum Brain Mapp, 2013. © 2011 Wiley Periodicals, Inc.     

Diagnosing breast cancer using diffuse reflectance spectroscopy and intrinsic fluorescence spectroscopy

Using diffuse reflectance spectroscopy and intrinsic fluorescence spectroscopy, we have developed an algorithm that successfully classifies normal breast tissue, fibrocystic change, fibroadenoma, and infiltrating ductal carcinoma in terms of physically meaningful parameters. We acquire 202 spectra from 104 sites in freshly excised breast biopsies from 17 patients within 30 min of surgical excision. The broadband diffuse reflectance and fluorescence spectra are collected via a portable clinical spectrometer and specially designed optical fiber probe. The diffuse reflectance spectra are fit using modified diffusion theory to extract absorption and scattering tissue parameters. Intrinsic fluorescence spectra are extracted from the combined fluorescence and diffuse reflectance spectra and analyzed using multivariate curve resolution. Spectroscopy results are compared to pathology diagnoses, and diagnostic algorithms are developed based on parameters obtained via logistic regression with cross-validation. The sensitivity, specificity, positive predictive value, negative predictive value, and overall diagnostic accuracy (total efficiency) of the algorithm are 100, 96, 69, 100, and 91%, respectively. All invasive breast cancer specimens are correctly diagnosed. The combination of diffuse reflectance spectroscopy and intrinsic fluorescence spectroscopy yields promising results for discrimination of breast cancer from benign breast lesions and warrants a prospective clinical study.     

O-Antigen-Negative Salmonella enterica Serovar Typhimurium Is Attenuated in Intestinal Colonization but Elicits Colitis in Streptomycin-Treated Mice

Lipopolysaccharide (LPS) is a major constituent of the outer membrane and an important virulence factor of Salmonella enterica subspecies 1 serovar Typhimurium (serovar Typhimurium). To evaluate the role of LPS in eliciting intestinal inflammation in streptomycin-treated mice, we constructed an O-antigen-deficient serovar Typhimurium strain through deletion of the wbaP gene. The resulting strain was highly susceptible to human complement activity and the antimicrobial peptide mimic polymyxin B. Furthermore, it showed a severe defect in motility and an attenuated phenotype in a competitive mouse infection experiment, where the ΔwbaP strain (SKI12) was directly compared to wild-type Salmonella. Nevertheless, the ΔwbaP strain (SKI12) efficiently invaded HeLa cells in vitro and elicited acute intestinal inflammation in streptomycin-pretreated mice. Our experiments prove that the presence of complete LPS is not essential for in vitro invasion or for triggering acute colitis.Salmonella spp. are a common cause of bacterial food-borne infections. Diseases caused by Salmonella spp. range from gastrointestinal symptoms such as fever, diarrhea, abdominal pain, and nausea to severe systemic infections. Salmonella enterica subspecies 1 serovar Typhimurium (serovar Typhimurium) is one of the most frequent enteropathogens, causing large numbers of diarrheal infections worldwide by colonizing the gut and triggering mucosal inflammation (33). The type III secretion system 1 (TTSS-1) and TTSS-2 encoded on Salmonella pathogenicity island 1 (SPI1) and SPI2 on the Salmonella genome are employed by the pathogen for mediating bacterial entry into the gut mucosa (SPI1) as well as the intracellular survival followed by systemic spread of the bacteria (SPI2) (9). Acute enteric serovar Typhimurium infection and the mechanisms leading to intestinal inflammation can be analyzed using a well-defined mouse model for Salmonella colitis: streptomycin-pretreated, naïve mice develop a vigorous local inflammation of the large intestine upon intragastric infection with serovar Typhimurium (3).Besides the SPI1- and SPI2-encoded TTSSs, serovar Typhimurium requires numerous additional virulence factors for colonizing the host, resisting host immune defense, and finally, triggering disease. One key virulence factor for serovar Typhimurium is lipopolysaccharide (LPS), a major surface component (42). It contributes to the stability of the outer membrane, serves as a permeability barrier, and protects the bacterium against environmental challenges (34). LPS is composed of three domains. The lipid A part, also known as endotoxin, anchors LPS molecules in the outer membrane with its fatty acid chains. It is connected through the inner core consisting of heptoses and Kdo (3-deoxy-d-manno-octulosonic acid), with the outer core containing hexoses and N-acetylhexoses. Linked to the last glucose of the outer core is the polymeric O-antigen region. This region is composed of 16 to >100 repeats of an oligosaccharide structure containing four to six monosaccharides (27).The endotoxic properties of LPS are mediated by the lipid A moiety, which can be recognized by Toll-like receptor 4 and thus triggers an innate immune response (16, 32). The O antigen, in combination with the inner and outer cores, serves as protection against complement antimicrobial peptides, detergents, and certain antibiotics. Furthermore, the O-antigen region is a key determinant for recognition by the adaptive immune response (40).A number of studies have established an important role for O-antigen side chains in Salmonella virulence. A signature-tagged mutagenesis screening by Morgan and coworkers proved that mutations in genes for enzymes involved in the biosynthesis of O-antigen side chains attenuated bacteria in their ability to colonize chick and calf intestines (25). Interestingly, a mutant in wbaP, the phosphogalactosyltransferase starting O-antigen biosynthesis, was able to colonize calves but showed an attenuated phenotype in chicks (25). Moreover, screening for Salmonella genes required for long-term systemic infection after intraperitoneal injection showed negative selection for mutants in O-antigen biosynthesis (21). Coinfection experiments by Nevola et al. show that mutants lacking O antigen are still able to colonize the murine intestine but are attenuated in competitive infection experiments (30). Furthermore, a recent in vitro study with Salmonella enterica serovar Typhi showed that O-antigen side chains are not necessary for adhesion to and invasion of epithelial cells. However, mutants lacking the complete outer core are severely attenuated (14). In general, the loss of core structures seems more detrimental than the loss of O-antigen side chains. However, it had remained unclear whether the O-antigen side chains are required for triggering intestinal inflammation.We wanted to analyze the role of O-antigen side chains in a well-established mouse model for enteric infections (3) and in an in vitro cellular invasion assay (36). Thus, we deleted the gene encoding the phosphogalactosyltransferase WbaP. This enzyme adds phosphogalactose to undecaprenylphosphate, the first step in O-antigen side chain biosynthesis in the cytoplasm of serovar Typhimurium (35, 43, 44). Streptomycin-pretreated mice were orally infected with the wbaP mutant strain (SKI12), and in line with published work, we found that the ΔwbaP mutant strain (SKI12) was significantly attenuated in a competitive infection assay. In spite of this, the wbaP mutant alone was able to trigger acute colitis. This demonstrates that serovar Typhimurium permits substantial manipulation of the O-antigen structure without losing its ability to trigger mucosal inflammation.