Search

Many animals, including fish, can utilize both vision and the chemical senses in intra-specific communication. However, the relative influence of these sensory modalities on behavioral and physiological responses in social interactions is not well understood. The aim of this study was therefore to investigate the relative effects of visual and chemical stimuli from dominant individuals on the behavioral and physiological responses of subordinate rainbow trout (Oncorhynchus mykiss). External electrodes were used to detect ECG signals from free-swimming fish. This method allowed the simultaneous recording of behavioral and physiological responses, and possible sex differences in these responses were also investigated. The results suggest that, in this context, visual cues are more important than chemical cues in settling the social hierarchy in rainbow trout because a combination of chemical and visual exposure generally yielded a response in focal fish that was similar to the response elicited by visual exposure alone. Both activity and physiological responses were most pronounced during the first ten seconds after exposure, with subordinate fish moving closer to the dominant, accompanied by a strong bradycardic response. Furthermore, females acted more boldly and moved closer to the dominant fish than males, but here the effect of the modes was additive, with a stronger effect of the combined visual and chemical exposure. Overall, the extra information furnished to the fish in the form of chemical cues did not change either the behavioral or the physiological response. This result suggests that visual cues are more important than chemically mediated ones for social communication and individual recognition in rainbow trout.

Background: The mechanisms that determine the duration of fecal shedding of norovirus in humans have not been described in detail., Objectives: We investigated serum inflammatory mediator levels in relation to the duration of viral shedding in norovirus infection., Study Design: A prospective cohort study of patients hospitalized with acute norovirus genogroup II infection. Rectal swab samples were obtained at inclusion and day 7, 14, 21 and 28. Serum levels of 42 inflammatory mediators were determined with a Luminex-based cytokine assay. Sera from 20 healthy blood donors served as controls., Results: Altogether, 28 patients (54% women, median age 83 years, median duration of symptoms 3 days) were included. Twelve subjects cleared the virus within 14 days and 16 were norovirus-RNA positive for >21 days, constituting the two study groups ("rapid" vs. "slow" clearance). Individuals with norovirus infection had higher levels of IL-18, CXCL9, CXCL10, soluble IL-2 receptor and macrophage migration inhibitory factor (MIF), compared to controls (p<0.05), with the highest median concentrations in the slow clearance group. In contrast, CCL5 levels were lower in the slow compared to the rapid clearance group (median 54 vs. 134 ng/mL, p<0.05), and lower in norovirus-infected patients than in controls., Conclusion: Low levels of CCL5 were associated with longer duration of viral shedding, suggesting that CCL5 may influence the clearance of norovirus., (Copyright © 2015 Elsevier B.V. All rights reserved.)

The aim of this systematic review was to synthesize the evidence for changes for proprioceptive variables consisting of movement and position sense in participants with glenohumeral musculoskeletal disorders. Five databases were searched until 13th August 2013. Methodological quality was assessed and meta-analyses were performed for active and passive joint reposition sense (AJPS and PJPS) and movement sense, determined with threshold to detection of passive motion (TTDPM). The search yielded 17 studies, four of which were classified as having high methodological quality, seven as moderate and six as low quality. For participants with post-traumatic glenohumeral instability, pooled findings indicate moderate evidence for higher TTDPM for involved shoulders compared to control groups and the contralateral uninvolved side, indicating decreased movement sense. For AJPS and PJPS there was moderate to limited evidence for significant increased errors for involved compared to uninvovled shoulders, but not when compared to the control groups. Limited evidence was found for decreased AJPS acuity for patients with chronic rotator cuff pain and for patients with unspecified shoulder pain compared to healthy controls. Movement sense is most likely to be impaired after shoulder injury involving post-traumatic instability when compared to the contralateral shoulder and to controls, while deficits for AJPS and PJPS are more likely to be evident compared to the contralateral shoulder in participants with glenohumeral musculoskeletal disorders., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Objective: Continuous infusion of levodopa carbidopa intestinal gel (LCIG) is associated with a significant improvement in the symptoms and quality of life of selected patients with advanced Parkinson's disease. Percutaneous endoscopic gastrostomy with jejunal extension (PEG/J) was first described in 1998 and has become the most common and standard technique for fixing the tubing in place for LCIG infusion., Material and Methods: A workshop was held in Stockholm, Sweden, to discuss the PEG/J placement for the delivery of LCIG in Parkinson's disease patients with the primary goal of providing guidance on best practice for the Nordic countries., Results: Suggested procedures for preparation of patients for PEG/J placement, aftercare, troubleshooting and redo-procedures for use in the Nordic region are described and discussed., Conclusions: LCIG treatment administered through PEG/J-tubes gives a significant increase in quality of life for selected patients with advanced Parkinson's disease. Although minor complications are common, serious complications are infrequent, and the tube insertion procedures have a good safety record. Further development of delivery systems and evaluation of approaches designed to reduce the demand for redo endoscopy are required.

Background: Women experience more sore throats than men after endotracheal intubation., Aim: The aim of this study was to investigate the incidence of self-rated sore throat immediately, and 2-4 hours postoperatively, in women after elective gynaecological surgery under general anaesthesia using an endotracheal tube (ETT) size 6.5 or 7.0mm in inner diameter., Method: Eighty-two female participants who had undergone elective gynaecological surgery participated, 44 and 38 were intubated with size 6.5 mm ETT and 7.0mm ETT respectively. They estimated the occurrence of sore throat preoperatively and postoperatively, according to a 4-point Likert scale. Statistical data were analysed using the Package for Social Science (SPSS) 19., Results: Sore throat was present in 29.5% of participants who were intubated with size 6.5 mm ETT immediately postoperatively and in 39.5% of those who were intubated with size 7.0 mm ETT., Conclusion: Nurses specialising in the supervision of daily care specific to the intubated patient should note and alleviate sore throat as part of their nursing care., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

The genetic diversity of 23 chickpea accessions representing Kyrgyz landraces and cultivars, ICARDA breeding lines, Spanish and Turkish cultivars was characterized using nine microsatellite (SSR) markers which generated a total of 122 alleles. The number of alleles (Na) per locus varied from 9 to 20. The observed heterozygosity (Ho) ranged between 0.05 and 0.43 (average 0.13) whereas both the expected heterozygosity (He) and polymorphic information content (PIC) ranged from 0.71 to 0.90 (average 0.83). Analysis of molecular variance (AMOVA) showed that 62% of the total genetic variation was found within accessions while the remaining 38% was found among accessions. Principal coordinate analysis (PCoA) indicated the presence of two groups. The two Kyrgyz cultivars were found apart from these groups. Cluster analysis generally confirmed the results of PCoA and also separated the Kyrgyz cultivars from the subcluster formed by Kyrgyz landraces and the subclusters formed by breeding lines from ICARDA along with landraces from Turkey and Spain. In addition, protein content and mineral concentration were determined. Protein content and mineral concentrations for Ca, S, Mg, P, K, Fe, Mn, Cu and Zn varied significantly among accessions. The results show that Kyrgyz germplasm provides a source of diversity for improvement of chickpea., (© 2014 The Authors.)

Key Message: Identification of novel resistance QTL against wheat aphids. First QTL-resistance report for R. padi in wheat and chromosome 2DL for S. graminum . These sources have potential use in wheat breeding. The aphids Rhopalosiphum padi and Schizaphis graminum are important pests of common wheat (Triticum aestivum L.). Characterization of the genetic bases of resistance sources is crucial to facilitate the development of resistant wheat cultivars to these insects. We examined 140 recombinant inbred lines (RILs) from the cross of Seri M82 wheat (susceptible) with the synthetic hexaploid wheat CWI76364 (resistant). RILs were phenotyped for R. padi antibiosis and tolerance traits. Phenotyping of S. graminum resistance was based on leaf chlorosis in a greenhouse screening and the number of S. graminum/tiller in the field. RILs were also scored for pubescence. Using a sequence-based genotyping method, we located genomic regions associated with these resistance traits. A quantitative trait locus (QTL) for R. padi antibiosis (QRp.slu.4BL) that explained 10.2 % of phenotypic variation was found in chromosome 4BL and located 14.6 cM apart from the pubescence locus. We found no association between plant pubescence and the resistance traits. We found two QTLs for R. padi tolerance (QRp.slu.5AL and QRp.slu.5BL) in chromosomes 5AL and 5BL, with an epistatic interaction between a locus in chromosome 3AL (EnQRp.slu.5AL) and QRp.slu.5AL. These genomic regions explained about 35 % of the phenotypic variation. We re-mapped a previously reported gene for S. graminum resistance (putatively Gba) in 7DL and found a novel QTL associated with the number of aphids/tiller (QGb.slu-2DL) in chromosome 2DL. This is the first report on the genetic mapping of R. padi resistance in wheat and the first report where chromosome 2DL is shown to be associated with S. graminum resistance.

Background: To our knowledge no clinical criteria for the identification of community onset viral gastroenteritis in individual patients have been evaluated systematically with modern PCR-based diagnostic assays as gold standard., Objective: The aim of this study was to identify factors independently associated with the detection of virus by PCR in rectal swab samples from patients with acute community onset gastroenteritis., Methods: A prospective observational study was conducted from December 2010 through March 2011 at the emergency department (ED) of a large teaching hospital. All patients who reported vomiting and/or diarrhoea up to 48 h prior to their visit to the ED were asked to participate. A rectal swab sample was obtained from each patient. Symptoms, date of onset, and epidemiological data were recorded. Samples were analysed with a multiple real-time PCR targeting 6 viral agents (astrovirus, adenovirus, rotavirus, sapovirus, and norovirus GI and GII)., Results: Two hundred and five patients fulfilled the inclusion criteria, of whom 66 agreed to participate; their median (IQR) age was 65 (38-84) y and 43 (65%) were females. Thirty-one (47%) were positive by PCR for at least 1 of the agents examined (26 norovirus, 2 sapovirus, 2 rotavirus, and 1 adenovirus). Diarrhoea and a short duration of symptoms (≤ 2 days) were independently associated with a positive rectal swab sample, with odds ratios of 7.5 (95% confidence interval (CI) 2.0-28) and 10.4 (95% CI 1.9-56), respectively (p < 0.01 for both). A multivariate model including these 2 variables had a sensitivity of 81% (25/31) and a specificity of 69% (24/35)., Conclusions: Diarrhoea and a short duration of symptoms were the only anamnestic criteria independently associated with acute community onset viral gastroenteritis confirmed by PCR.

This study describes for the first time the expression levels of genes encoding membrane transporters and drug-metabolizing enzymes in the lungs of ex-smoking patients with chronic obstructive pulmonary disease (COPD). Membrane transporters and drug-metabolizing enzymes are key determinants of drug uptake, metabolism, and elimination for systemically administered as well as inhaled drugs, with consequent influence on clinical efficacy and patient safety. In this study, while no difference in gene expression was found between healthy and COPD subjects, we identified a significant regional difference in mRNA expression of both membrane transporters and drug-metabolizing enzymes between central and peripheral tissue in both healthy and COPD subjects. The majority of the differentially expressed genes were higher expressed in the central airways such as the transporters SLC2A1 (GLUT1), SLC28A3 (CNT3), and SLC22A4 (OCTN1) and the drug-metabolizing enzymes GSTZ1, GSTO2, and CYP2F1. Together, this increased knowledge of local pharmacokinetics in diseased and normal lung may improve modeling of clinical outcomes of new chemical entities intended for inhalation therapy delivered to COPD patients. In addition, based on the similarities between COPD and healthy subjects regarding gene expression of membrane transporters and drug-metabolizing enzymes, our results suggest that clinical pharmacological studies in healthy volunteers could be a valid model of COPD patients regarding drug disposition of inhaled drugs in terms of drug metabolism and drug transporters.

The use of intensive forestry on part of the forested area in Sweden increases the production of forest biomass and enables an increased use of such biomass to mitigate climate change. However, with increasing conflicting interests in forests and forestry, the success of such a strategy depends on the public acceptance. In this paper, the results of a mail survey show that although a majority of the general public in Sweden supports measures to increase forest growth, they oppose the use of intensive forestry practices such as the cultivation of exotic tree species, clones, and forest fertilization. The acceptance of such practices is mainly influenced by the perceptions of their environmental consequences. Public acceptance was highest for forest fertilization, whereas clone cultivation was the least accepted practice.

Several organic cations, such as guanidino compounds and polyamines, have been found to accumulate in plasma of patients with kidney failure due to inadequate renal clearance. Here, we studied the interaction of cationic uremic toxins with renal organic cation transport in a conditionally immortalized human proximal tubule epithelial cell line (ciPTEC). Transporter activity was measured and validated in cell suspensions by studying uptake of the fluorescent substrate 4-(4-(dimethylamino)styryl)-N-methylpyridinium-iodide (ASP(+)). Subsequently, the inhibitory potencies of the cationic uremic toxins, cadaverine, putrescine, spermine and spermidine (polyamines), acrolein (polyamine breakdown product), guanidine, and methylguanidine (guanidino compounds) were determined. Concentration-dependent inhibition of ASP(+) uptake by TPA, cimetidine, quinidine, and metformin confirmed functional endogenous organic cation transporter 2 (OCT2) expression in ciPTEC. All uremic toxins tested inhibited ASP(+) uptake, of which acrolein required the lowest concentration to provoke a half-maximal inhibition (IC50 = 44 ± 2 μM). A Dixon plot was constructed for acrolein using three independent inhibition curves with 10, 20, or 30 μM ASP(+), which demonstrated competitive or mixed type of interaction (K i = 93 ± 16 μM). Exposing the cells to a mixture of cationic uremic toxins resulted in a more potent and biphasic inhibitory response curve, indicating complex interactions between the toxins and ASP(+) uptake. In conclusion, ciPTEC proves a suitable model to study cationic xenobiotic interactions. Inhibition of cellular uptake transport was demonstrated for several uremic toxins, which might indicate a possible role in kidney disease progression during uremia.

Recently the clinical importance of human organic cation transporters 1 (hOCT1/SLC22A1) and 2 (hOCT2/SLC22A2) in drug disposition, for example, clearance, toxicity, and drug-drug interactions, have been highlighted [Annu. Rev. Pharmacol. Toxicol. 2012, 52, 249-273; Nat. Rev. Drug Discovery 2010, 9 (3), 215-236]. Consequently, there is an extensive need for experimental assessment of structure-transport relationships as well as tools to predict drug uptake by these transporters in ADMET (absorption, distribution, metabolism, excretion, toxicity) investigations. In the present study, we developed a robust assay for screening unlabeled compound uptake by hOCT1 and hOCT2 using transfected HEK293 cells. For the first time, an extensive data set comprising uptake of 354 compounds is presented. As expected, there was a large overlap in substrate specificity between the two organic cation transporters. However, several compounds selectively taken up by either hOCT1 or hOCT2 were identified. In particular, a chemical series of phenylthiophenecarboxamide ureas was identified as selective hOCT1 substrates. Moreover, the drivers for transport differed: molecular volume was the most important determinant of hOCT1 substrates, whereas H-bonding parameters like polar surface area (PSA) dominated for hOCT2.

The current studies were undertaken to gain new insights into the interplay and mechanism of membrane transporters involved in the permeability of estrone-3-sulfate (E1S) in Caco-2 cells cultured either on the bottom of multiwell plastic dishes or on filter support. We demonstrate that Caco-2 cells from the "Deutsche sammlung von mikroorganismen und zellkulturen" (DSMZ) exhibit extensive and consistent carrier-mediated uptake of [(3)H]-E1S after a culture period of 11-13 days. The kinetic characterization, the inhibitory profile and the pH dependence for the initial linear uptake permeability (PUP) of [(3)H]-E1S suggest that the organic anion transporting polypeptide (OATP) 2B1 is the main transporter involved in the apical E1S PUP in Caco-2 cells from the DSMZ. Furthermore, our results indicate that the efflux transporter breast cancer resistance protein (BCRP) affects E1S PUP, even when uptake is measured at the initial linear uptake phase. Although almost identical results were obtained for cells cultured on plastic dishes and on filter supports, the OATP2B1 stimulator dexamethasone did not affect the PUP for cells grown on dishes but increased [(3)H]-E1S PUP by more than 2-fold for filter grown cells. The basolateral PUP of [(3)H]-E1S of filter grown cells was inhibited by several inhibitors of the bidirectional transporter organic solute transporter α/β (OSTα/β). Efflux studies were performed by loading the cells with either [(3)H]-E1S or [(3)H]-taurocholic acid (TCA) and subsequently measuring the efflux of radio labeled substance in the absence or presence of BCRP or OST α/β inhibitors. Similar effluxes of [(3)H]-E1S was observed across the apical and basolateral membrane, and the apical efflux was greatly decreased in the presence of the BCRP inhibitor fumitremorgin C. In contrast, efflux of [(3)H]-TCA to the basolateral compartment was clearly larger than to the apical compartment. Trans-stimulation of basolateral [(3)H]-E1S efflux was observed in the presence of taurolithocholic acid (TLC), although none of the applied OSTα/β inhibitors were able to confirm the existence of carrier-mediated efflux at the basolateral membrane, neither for [(3)H]-E1S nor for [(3)H]-TCA. These results highlight the importance of transporter interplay for E1S and drug compounds in Caco-2 cells and emphasize the importance of identifying the basolateral transporters in these cells.

This review encompasses the most important advances in liver functions and hepatotoxicity and analyzes which mechanisms can be studied in vitro. In a complex architecture of nested, zonated lobules, the liver consists of approximately 80 % hepatocytes and 20 % non-parenchymal cells, the latter being involved in a secondary phase that may dramatically aggravate the initial damage. Hepatotoxicity, as well as hepatic metabolism, is controlled by a set of nuclear receptors (including PXR, CAR, HNF-4α, FXR, LXR, SHP, VDR and PPAR) and signaling pathways. When isolating liver cells, some pathways are activated, e.g., the RAS/MEK/ERK pathway, whereas others are silenced (e.g. HNF-4α), resulting in up- and downregulation of hundreds of genes. An understanding of these changes is crucial for a correct interpretation of in vitro data. The possibilities and limitations of the most useful liver in vitro systems are summarized, including three-dimensional culture techniques, co-cultures with non-parenchymal cells, hepatospheres, precision cut liver slices and the isolated perfused liver. Also discussed is how closely hepatoma, stem cell and iPS cell-derived hepatocyte-like-cells resemble real hepatocytes. Finally, a summary is given of the state of the art of liver in vitro and mathematical modeling systems that are currently used in the pharmaceutical industry with an emphasis on drug metabolism, prediction of clearance, drug interaction, transporter studies and hepatotoxicity. One key message is that despite our enthusiasm for in vitro systems, we must never lose sight of the in vivo situation. Although hepatocytes have been isolated for decades, the hunt for relevant alternative systems has only just begun.

To improve our understanding of drug-target interactions, we explored the effect of introducing substituted amine residues with increased chain length in the P3 residue of the thrombin inhibitor melagatran. Inhibition, kinetic, and thermodynamic data obtained via stopped-flow spectroscopy (SF), isothermal microcalorimetry (ITC), and surface plasmon resonance (SPR) biosensor analysis were interpreted with the help of X-ray crystal structures of the enzyme-inhibitor complexes. The association rate became faster when the lipophilicity of the inhibitors was increased. This was coupled to an increased enthalpic component and a corresponding decreased entropic component. The dissociation rates were reduced with an increase in chain length, with only a smaller increase and a decrease in the enthalpic and entropic components, respectively. Overall, the affinity increased with an increase in chain length, with similar changes in the enthalpic and entropic components. ITC analysis confirmed the equilibrium data from SPR analysis, showing that the interaction of melagatran was the most enthalpy-driven interaction. Structural analysis of the thrombin-inhibitor complex showed that the orientation of the P1 and P2 parts of the molecules was very similar, but that there were significant differences in the interaction between the terminal part of the P3 side chain and the binding pocket. A combination of charge repulsion, H-bonds, and hydrophobic interactions could be used to explain the observed kinetic and thermodynamic profiles for the ligands. In conclusion, changes in the structure of a lead compound can have significant effects on its interaction with the target that translate directly into kinetic and thermodynamic effects. In contrast to what may be intuitively expected, hydrogen bond formation and breakage are not necessarily reflected in enthalpy gains and losses, respectively.

Background: Norovirus enteritis (NVE) can be fatal in frail patients. High blood lactate levels indicate hypoperfusion and predict mortality in many infectious diseases. The objective was to determine the frequency and association with mortality of elevated lactate levels in patients with community-onset NVE., Methods: A retrospective cohort study was performed. All hospitalized adult patients with community-onset NVE verified by polymerase chain reaction during the period August 2008 to June 2009 were included. Vital signs and venous lactate on arrival, co-morbid conditions, and time of death were registered. The outcome measure was 30-day all-cause mortality., Results: Eighty-two patients with a median age of 77 y (interquartile range (IQR) 53-86 y) were included, of whom 47 (57%) were female and 49 (60%) had at least 1 major co-morbid condition. Lactate levels were above the upper limit of normal (ULN; 1.6 mmol/l) in 45 patients (55%). The overall 30-day mortality rate was 7% (6/82). Mortality was 18% (5/28) with lactate ≥ 2.4 mmol/l (> 50% above the ULN) on admission compared to 2% (1/54) with lactate < 2.4 mmol/l (p < 0.05). Patients who died had a higher median lactate level compared to survivors: 4.5 (IQR 2.7-7.9) mmol/l vs 1.7 (IQR 1.3-2.5) mmol/l, respectively (p < 0.01). The adjusted odds ratio for death within 30 days for a 1 mmol/l increase in lactate was 2.5 (95% confidence interval 1.003-6.3, p = 0.049)., Conclusions: We observed a high proportion of patients with elevated lactate levels in community-onset NVE. Lactate elevation could predict mortality. Measurement of blood lactate may be a valuable tool in the clinical management of patients with a suspected norovirus infection.

When selecting a landfill leachate treatment method the contaminant composition of the leachate should be considered in order to obtain the most cost-effective treatment option. In this study the filter material pine bark was evaluated as a treatment for five landfill leachates originating from different cells of the same landfill in Sweden. The objective of the study was to determine the uptake, or release, of metals and dissolved organic carbon (DOC) during a leaching test using the pine bark filter material with the five different landfill leachates. Furthermore the change of toxicity after treatment was studied using a battery of aquatic bioassays assessing luminescent bacteria (Vibrio fischeri) acute toxicity (30-min Microtox®), immobility of the crustacean Daphnia magna, growth inhibition of the algae Pseudokirchneriella subcapitata and the aquatic plant Lemna minor; and genotoxicity with the bacterial Umu-C assay. The results from the toxicity tests and the chemical analysis were analyzed in a Principal Component Analysis and the toxicity of the samples before and after treatment was evaluated in a toxicity classification. The pine bark filter material reduced the concentrations of metal contaminants from the landfill leachates in the study, with some exceptions for Cu and Cd. The Zn uptake of the filter was high for heavily contaminated leachates (≥73%), although some desorption of zinc occurred in less contaminated waters. Some of the leachates may require further treatment due to discharge into a natural recipient in order to reduce the risk of possible biological effects. The difference in pH changes between the different leachates was probably due to variations in buffering capacity, affected by physicochemical properties of the leachate. The greatest desorption of phenol during filtration occurred in leachates with high conductivity or elevated levels of metals or salts. Generally, the toxicity classification of the leachates implies that although filter treatment with pine bark removes metal contaminants from the leachates effectively, it does not alter leachate toxicity noticeably. The leachates with the highest conductivity, pH and metal concentrations are most strongly correlated with an increased toxic response in the score plots of both untreated and treated leachates. This is in line with the toxicity classification of the leachate samples. The results from this study highlight the importance of evaluating treatment efficiency from the perspective of potential recipient effects, rather than in terms of residual concentrations of individual contaminants when treating waters with a complex contamination matrix, such as landfill leachates., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

The limit of detection of low-molecular weight compounds in tissue sections, analyzed by matrix assisted laser desorption ionization mass spectrometry imaging (MALDI-MSI), was significantly improved by employing sample washing using a pH-controlled buffer solution. The pH of the washing solutions were set at values whereby the target analytes would have low solubility. Washing the tissue sections in the buffered solution resulted in removal of endogenous soluble ionization-suppressing compounds and salts, while the target compound remained in situ with minor or no delocalization during the buffered washing procedure. Two pharmaceutical compounds (cimetidine and imipramine) and one new protease inhibitor compound were successfully used to evaluate the feasibility of the pH-controlled tissue washing protocol for MALDI-MSI. Enhancement in signal-to-noise ratio was achieved by a factor of up to 10.

Background: Viral agents, especially norovirus, are the most common cause of nosocomial spread of epidemic gastroenteritis (GE). Rapid and reliable detection of these agents could reduce the risk of outbreaks., Objective: To evaluate the diagnostic performance of rectal swab samples compared to standard stool samples for detection of agents causing viral GE by PCR., Study Design: Complete pairs of rectal swab and stool samples, obtained simultaneously from patients with symptoms of acute onset GE, were analysed with a multiple real-time PCR targeting six different gastroenteritis agents (astro-, adeno-, rota-, sapo- and norovirus GI and II). Cycle threshold (Ct) values were registered for positive samples. A positive PCR result in either sample for any virus was considered gold standard., Results: 69 sample pairs were included of which 29 were negative in both sample types and 38 were positive in both sample types. One pair was positive in the stool sample only and another pair was positive in the rectal swab sample only. Sensitivity for both sample types was 97.5% (39/40)., Conclusion: Rectal swab samples are as reliable as stool samples for PCR-based diagnosis of viral gastroenteritis in patients with a short duration of symptoms and may be used as a complement to stool samples, especially when immediate sampling is desirable., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Drug metabolism in liver is the major pathway for xenobiotic elimination from the body. Access to intracellular metabolising enzymes is possible through passive diffusion of lipophilic drugs through cell membrane or active uptake of more polar drugs by specific uptake transporters. Organic Anion Transporting Polypeptides (OATP/SLCO) and Organic Cation Transporters (OCT/SLC22A) are among the most important transporters involved in xenobiotic transport into hepatocytes. Isolated hepatocytes are the model of choice for drug metabolism and drug transport investigations. These primary cells are used either as fresh directly after isolation from liver biopsies, or after subsequent cryopreservation in liquid nitrogen. While cryopreserved hepatocytes are a more convenient and flexible tool for in vitro investigations, information on the functionality of transporter activity after cryopreservation is still sparse. The present study investigated the effect of cryopreservation of human hepatocytes on the uptake of [(3)H]-estradiol-17β-glucuronide (E(2)17βG, substrate of OATP1B1/3/SLCO1B1/3) and [(3)H]-1-methyl-4-phenylpyridinium (MPP+, substrate of OCT1/SLC22A1) into hepatocytes from 6 and 5 human donors, respectively. The results showed that cryopreserved human hepatocytes display carrier-mediated uptake of E(2)17βG and MPP+. While the affinity of E(2)17βG for OATP1B1/3/SLCO1B1/3 was not affected by cryopreservation (Km unchanged, the Wilcoxon signed pair t test gave p=1), V(max) and CL(uptake) values decreased in average by 47% (p=0.06). The passive diffusion of E(2)17βG decreased significantly after cryopreservation (p=0.03). Cryopreservation did not affect Km, V(max) or the passive diffusion of MPP+ in human hepatocytes. In conclusion, the present study showed that cryopreserved human hepatocytes are useful tool to investigate hepatic uptake mediated by OATP1B1/3/SLCO1B1/3 or OCT1/SLC22A1, two of the most important hepatic uptake transporters., (Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.)

Dosimetry, safety and the efficacy of drugs in the lungs are critical factors in the development of inhaled medicines. This article considers the challenges in each of these areas with reference to current industry practices for developing inhaled products, and suggests collaborative scientific approaches to address these challenges. The portfolio of molecules requiring delivery by inhalation has expanded rapidly to include novel drugs for lung disease, combination therapies, biopharmaceuticals and candidates for systemic delivery via the lung. For these drugs to be developed as inhaled medicines, a better understanding of their fate in the lungs and how this might be modified is required. Harmonized approaches based on 'best practice' are advocated for dosimetry and safety studies; this would provide coherent data to help product developers and regulatory agencies differentiate new inhaled drug products. To date, there are limited reports describing full temporal relationships between pharmacokinetic (PK) and pharmacodynamic (PD) measurements. A better understanding of pulmonary PK and PK/PD relationships would help mitigate the risk of not engaging successfully or persistently with the drug target as well as identifying the potential for drug accumulation in the lung or excessive systemic exposure. Recommendations are made for (i) better industry-academia-regulatory co-operation, (ii) sharing of pre-competitive data, and (iii) open innovation through collaborative research in key topics such as lung deposition, drug solubility and dissolution in lung fluid, adaptive responses in safety studies, biomarker development and validation, the role of transporters in pulmonary drug disposition, target localisation within the lung and the determinants of local efficacy following inhaled drug administration., (Copyright © 2010 Elsevier B.V. All rights reserved.)

Low polymorphism in cultivated watermelon has been reported in previous studies, based mainly on US Plant Introductions and watermelon cultivars, most of which were linked to breeding programmes associated with disease resistance. Since germplasm sampled in a putative centre of origin in southern Africa may harbour considerably higher variability, DNA marker-based diversity was estimated among 81 seedlings from eight accessions of watermelon collected in Zimbabwe; five accessions of cow-melons (Citrullus lanatus var. citroides) and three of sweet watermelons (C. lanatus var. lanatus). Two molecular marker methods were used, random amplified polymorphic DNA (RAPD) and simple sequence repeats (SSR) also known as microsatellite DNA. Ten RAPD primers produced 138 markers of which 122 were polymorphic. Nine SSR primer pairs detected a total of 43 alleles with an average of 4.8 alleles per locus. The polymorphic information content (PIC) ranged from 0.47 to 0.77 for the RAPD primers and from 0.39 to 0.97 for the SSR loci. Similarity matrices obtained with SSR and RAPD, respectively, were highly correlated but only RAPD was able to provide each sample with an individual-specific DNA profile. Dendrograms and multidimensional scaling (MDS) produced two major clusters; one with the five cow-melon accessions and the other with the three sweet watermelon accessions. One of the most variable cow-melon accessions took an intermediate position in the MDS analysis, indicating the occurrence of gene flow between the two subspecies. Analysis of molecular variation (AMOVA) attributed most of the variability to within-accessions, and contrary to previous reports, sweet watermelon accessions apparently contain diversity of the same magnitude as the cow-melons.

Readouts that define the physiological distributions of drugs in tissues are an unmet challenge and at best imprecise, but are needed in order to understand both the pharmacokinetic and pharmacodynamic properties associated with efficacy. Here we demonstrate that it is feasible to follow the in vivo transport of unlabeled drugs within specific organ and tissue compartments on a platform that applies MALDI imaging mass spectrometry to tissue sections characterized with high definition histology. We have tracked and quantified the distribution of an inhaled reference compound, tiotropium, within the lungs of dosed rats, using systematic point by point MS and MS/MS sampling at 200 microm intervals. By comparing drug ion distribution patterns in adjacent tissue sections, we observed that within 15 min following exposure, tiotropium parent MS ions (mass-to-charge; m/z 392.1) and fragmented daughter MS/MS ions (m/z 170.1 and 152.1) were dispersed in a concentration gradient (80 fmol-5 pmol) away from the central airways into the lung parenchyma and pleura. These drug levels agreed well with amounts detected in lung compartments by chemical extraction. Moreover, the simultaneous global definition of molecular ion signatures localized within 2-D tissue space provides accurate assignment of ion identities within histological landmarks, providing context to dynamic biological processes occurring at sites of drug presence. Our results highlight an important emerging technology allowing specific high resolution identification of unlabeled drugs at sites of in vivo uptake and retention.

The ultimate goal of MALDI-Imaging Mass Spectrometry (MALDI-IMS) is to achieve spatial localization of analytes in tissue sections down to individual tissue compartments or even at the level of a few cells. With compound tissue imaging, it is possible to track the transportation of an unlabelled, inhaled reference compound within lung tissue, through the application of MALDI-IMS. The procedure for isolation and preparation of lung tissues is found to be crucial in order to preserve the anatomy and structure of the pulmonary compartments. To avoid delocalization of analytes within lung tissue compartments we have applied an in-house designed nano-spotter, based on a microdispenser mounted on an XY table, of which movement and spotting functionality were fully computer controlled. We demonstrate the usefulness of this platform in lung tissue sections isolated from rodent in vivo model, applied to compound tissue imaging as exemplified with the determination of the spatial distribution of (1alpha,2beta,4beta,7beta)-7-[(hydroxidi-2-thienylacetyl)oxy]-9,9-dimethyl-3-oxa-9-azoniatricyclo[3.3.1.0(2,4)]nonane, also known as tiotropium. We provide details on tissue preparation protocols and sample spotting technology for successful identification of drug in mouse lung tissue by using MALDI-Orbitrap instrumentation., (Copyright 2010 Elsevier B.V. All rights reserved.)

Opistocystidae Cernosvitov, 1936 is a largely Neotropical oligochaete taxon containing seven species. Its familial status has never been formally challenged, although possible close relationships with Naididae and Phreodrilidae have been noted. Mitochondrial 12S and 16S rDNA, and nuclear 18S rDNA, of a range of aquatic oligochaete taxa, including Trieminentia corderoi (Opistocystidae), were analysed by Bayesian inference. This showed that T. corderoi is a derived lineage within Naididae, closely related to Pristina and its monotypic subfamily Pristininae. Opistocystidae as a whole (with its three genera, Opistocysta, Trieminentia, and Crustipellis) is thus likely to be a group within Naididae.

The liver-specific organic cation transport protein (OCT1; SLC22A1) transports several cationic drugs including the antidiabetic drug metformin and the anticancer agents oxaliplatin and imatinib. In this study, we explored the chemical space of registered oral drugs with the aim of studying the inhibition pattern of OCT1 and of developing predictive computational models of OCT1 inhibition. In total, 191 structurally diverse compounds were examined in HEK293-OCT1 cells. The assay identified 47 novel inhibitors and confirmed 15 previously known inhibitors. The enrichment of OCT1 inhibitors was seen in several drug classes including antidepressants. High lipophilicity and a positive net charge were found to be the key physicochemical properties for OCT1 inhibition, whereas a high molecular dipole moment and many hydrogen bonds were negatively correlated to OCT1 inhibition. The data were used to generate OPLS-DA models for OCT1 inhibitors; the final model correctly predicted 82% of the inhibitors and 88% of the noninhibitors of the test set.

Trees of 68 apple cultivars, aimed for preservation by the 'National Program for diversity of cultivated plants' as mandate cultivars, were analysed using a set of 10 SSR (simple sequence repeat) primer pairs and the self-incompatibility (S-)locus to evaluate genetic diversity and reveal inter-cultivar relationships. The 12 polymorphic SSR loci exhibited 2 to 15 alleles, with expected heterozygozity (H(e)) ranging from 0.36 to 0.88 and a mean of 0.74. Numerous alleles were classified as rare or unique (35% and 18% respectively). For the S-locus, a total of 14 alleles were identified in this study. Five alleles, S1-S3, S5 and S7 had frequencies ranging from 11 to 18%, whereas the remaining 9 alleles were below 6%. All sexually obtained cultivars could be distinguished with the set of SSR loci. Sports were identical with their progenitors in two cases, but differed in one SSR allele in a third case. An SSR-based dendrogram, based on Roger's genetic distances, did not reveal any clear pattern of clustering. The genetic distances were, however, correlated with a corresponding matrix obtained in a previously conducted RAPD-based study of the same cultivars. Non-mandate parents of Swedish mandate cultivars together with some other reference cultivars were included in this study to check the accuracy of allele scoring, verify parentage and compare the results of this study with those presented in previously published studies. Some discrepancies in allele sizing were revealed and the possibilities of avoiding this problem are discussed.

Chronic immune stimulation such as Helicobacter pylori (hp) infection, Sjögren's syndrome or coeliac disease may initiate non-Hodgkin lymphoma (NHL). The opposite (appearance of autoimmunity) has also been reported. The aim of this study was to describe the pattern of these immune markers in patients with lymphoid malignancies. Sera from 96 patients with NHL (median age 72, range 38-88, F/M 41/55) were analysed with ELISA to determine the frequency of antibodies against guinea pig (gp) and human recombinant (hr) transglutaminase type 2 (Tg2), and hr factor XIII subunit a* (part of the Tg-family), extractable nuclear antigen (ENA), and hp. As hp antibodies decrease in younger age cohorts a sex- and age-matched control group of 768 persons was used. The control population for transglutaminase antibodies consisted of 59 blood donors, (median 42 years, range 19-65) was analysed with a commercial kit. Gp-Tg2-IgG positivity was documented in 72% and hr-Tg2-IgG positivity in 15% (5% positive controls for both; P < 0.001 and ns, respectively). For IgA 3% had gp-Tg2 and 4% hr-Tg2 (5% in controls: ns for both). Anti-FXIII-IgA positivity was found in 22% (5% in controls; P = 0.03). Unspecific anti-ENA-IgG positivity was found in 24% (P < 0.001), while only 2% had specific ENA autoantibodies. Moreover, 36% were positive for anti-hp-IgG, while controls were positive in 54% (P < 0.001). The frequency of unspecific autoantibodies was increased. No differences could be noted in specific autoantibodies (hr-Tg2-IgA). In contrast, fewer than expected were anti-hp-positive. A defective immune response, similar to that in autoimmune diseases, could contribute to the pathogenesis of lymphoid malignancies.

A solid-phase microextraction (SPME) method using gas chromatography-electron-capture negative ionisation mass spectrometry (GC-ECNI-MS) and isotope dilution quantification for the analysis of nitroaromatic compounds in complex, water based samples has been optimised. For ionisation, ECNI was the most sensitive and selective method. SPME was compared to solid-phase extraction (SPE) and found to be more sensitive for these small volume samples. LODs were in the range 0.02-38ngL(-1) for SPME and 6-184ngL(-1) for SPE, respectively. The SPME method was applied on samples in the ngL(-1) level from artificial reed beds treated with sludge containing residues from explosives and pharmaceuticals.

Increasing evidence suggests a key role of transport proteins in the pharmacokinetics of drugs. Within the solute carrier (SLC) family, various organic cation transporters (OCTs), organic anion transporters (OATs), and organic anion transporting polypeptides (OATPs) that interact with drug molecules have been identified. Traditionally, cellular uptake assays require multiple steps and provide low experimental throughput. We here demonstrate the use of a scintillation proximity approach to detect substrate uptake by human drug transporters in real time. HEK293 cells stably transfected with hOCT1, hOATP1B1, or hOAT3 were grown directly in Cytostar-T scintillating microplates. Confluent cell monolayers were incubated with 14C- or 3H-labeled transporter substrates. Cellular uptake brings the radioisotopes into proximity with the scintillation plate base. The resulting light emission signals were recorded on-line in a microplate scintillation counter. Results show time- and concentration-dependent uptake of 14C-tetraethylammonium, 3H-methylphenylpyridinium (HEK-hOCT1), 3H-estradiol-17beta-D-glucuronide (HEK-hOATP1B1), and 3H-estrone-3-sulfate (HEK-hOAT3), while no respective uptake was detected in empty vector-transfected cells. Km of 14C-tetraethylammonium and 3H-estrone-3-sulfate uptake and hOAT3 inhibition by ibuprofen and furosemide were similar to conventional dish uptake studies. The scintillation proximity approach is high throughput, amenable to automation and allows for identification of SLC transporter substrates and inhibitors in a convenient and reliable fashion, suggesting its broad applicability in drug discovery.

This paper briefly describes waste treatment trends in Sweden and describes an example of state-of-the-art treatment in a local integrated waste treatment system (Borås). The focus is on treatment of household waste, to illustrate what can be done to establish a sustainable waste treatment system with high recovery of materials and energy. The various processes have resulted in a low amount of landfilled household waste, and high proportions of recovered energy and materials. Heat, electricity, vehicle fuel and fertilizers, metals and construction materials are recovered from the local waste in Borås, with less than 4% of domestic waste being sent to landfill.

Screening of new chemical entities for metabolic stability using hepatocytes is routinely used in drug discovery. To enhance compound throughput, an optimized automated microassay for determination of intrinsic clearance was developed. Dulbecco's modified Eagle's medium, Hanks' balanced salt solution, and Leibovitz L-15 medium (L-15) were tested for their ability to maintain cell viability during incubation in 96-well plates. L-15 was found to keep pH within 0.1 units and maintain high viability during several hours of incubation. Moreover, two different thawing protocols for cryopreserved hepatocytes were compared. Protocol 2 resulted in a nearly 100% increase in post-thaw yield, whereas no difference was observed in cell viability. The microassay was validated using human cryopreserved hepatocytes and 19 reference compounds covering the most important phase I and II liver metabolizing enzymes ranging from low to medium and high clearance compounds. The day-to-day variation was determined, revealing an overall good precision of the assay. In vitro-in vivo correlations, for both fresh rat and cryopreserved human hepatocytes, were calculated. For 86% (human) and 77% (rat) of the compounds, calculated hepatic clearance was within twofold observed clearance in vivo. Using the validation data, variance component analysis was applied to determine within and between-experiment variability, enabling estimation of variation and detection limit for any combination of repeated experiments and replicate samples. Based on the precision desired, this provides a tool to select the most optimal and cost-effective assay approach for different compounds considering the actual phase in the drug discovery program.

Goal, Scope and Background: Sweden has prohibited the deposition of organic waste since January, 2005. Since 1 million tons of sludge is produced every year in Sweden and the capacity for incineration does not fill the demands, other methods of sludge management have to be introduced to a larger degree. One common method in the USA and parts of Europe is the use of wetlands to treat wastewater and sewage sludge. The capacity of reed beds to affect the toxicity of a complex mixture of nitroaromatics in sludge, however, is not fully elucidated. In this study, an industrial sludge containing explosives and pharmaceutical residues was therefore treated in artificial reed beds and the change in toxicity was studied. Nitroaromatic compounds, which are the main ingredients of many pharmaceuticals and explosives, are well known to cause cytotoxicity and genotoxicity. Recently performed studies have also showed that embryos of zebrafish (Danio rerio) are sensitive to nitroaromatic compounds. Therefore, we tested the sludge passing through constructed wetlands in order to detect any changes in levels of embryotoxicity, genotoxicity and dioxin-like activity (AhR-agonists). We also compared unplanted and planted systems in order to examine the impact of the root system on the fate of the toxicants., Methods: An industrial sludge containing a complex mixture of nitroaromatics was added daily to small-scale constructed wetlands (vertical flow), both unplanted and planted with Phragmites australis. Sludge with an average dry weight of 1.25%, was added with an average hydraulic loading rate of 1.2 L/day. Outgoing water was collected daily and stored at -20 degrees C. The artificial wetland sediment was Soxhlet extracted, followed by clean-up with multi-layer silica, or extracted by ultrasonic treatment, yielding one organic extract and one water extract of the same sample. Genotoxicity of the extracts was measured according to the ISO protocol for the umu-C genotoxicity assay (ISO/TC 147/SC 5/ WG9 N8), using Salmonella typhimurium TA1535/pSK1002 as test organism. Embryotoxicity and teratogenicity were studied using the fish egg assay with zebrafish (Danio rerio) and the dioxin-like activity was measured using the DR-CALUX assay. Chemical analyses of nitroaromatic compounds were performed using Solid Phase Micro Extraction (SPME) and GC-MS., Results: Organic extracts of the bed material showed toxic potential in all three toxicity tests after two years of sludge loading. There was a difference between the planted and the unplanted beds, where the toxicity of organic extracts overall was higher in the bed material from the planted beds. The higher toxicity of the planted beds could have been caused by the higher levels of total carbon in the planted beds, which binds organic toxicants, and by enrichment caused by lower volumes of outgoing water from the planted beds., Discussion: Developmental disorders were observed in zebrafish exposed directly in contact to bed material from unplanted beds, but not in fish exposed to bed material from planted beds. Hatching rates were slightly lower in zebrafish exposed to outgoing water from unplanted beds than in embryos exposed to outgoing water from planted beds. Genotoxicity in the outgoing water was below detection limit for both planted and unplanted beds. Most of the added toxicants via the sludge were unaccounted for in the outgoing water, suggesting that the beds had toxicant removal potential, although the mechanisms behind this remain unknown., Conclusions: During the experimental period, the beds received a sludge volume (dry weight) of around three times their own volume. In spite of this, the toxicity in the bed material was lower than in the sludge. Thus, the beds were probably able to actually decrease the toxicity of the added, sludge-associated toxicants. When testing the acetone extracts of the bed material, the planted bed showed a higher toxicity than the unplanted beds in all three toxicity tests. The toxicity of water extracts from the unplanted beds, detected by the fish egg assay, were higher than the water extracts from the planted beds. No genotoxicity was detected in outgoing water from either planted or unplanted beds. All this together indicates that the planted reed beds retained semi-lipophilic acetone-soluble toxic compounds from the sludge better than the unplanted beds, which tended to leak out more of the water soluble toxic compounds in the outgoing water. The compounds identified by SPME/GC in the outgoing water were not in sufficient concentrations to have caused induction in the genotoxicity test., Recommendations and Perspectives: This study has pointed out the benefits of using constructed wetlands receiving an industrial sludge containing a complex mixture of nitroaromatics to reduce toxicity in the outgoing water. The water from planted, constructed wetlands could therefore be directed to a recipient without further cleaning. The bed material should be investigated over a longer period of time in order to evaluate potential accumulation and leakage prior to proper usage or storage. The plants should be investigated in order to examine uptake and possible release when the plant biomass is degraded.

This review brings you up-to-date with the hepatocyte research on: 1) in vitro-in vivo correlations of metabolism and clearance; 2) CYP enzyme induction, regulation, and cross-talk using human hepatocytes and hepatocyte-like cell lines; 3) the function and regulation of hepatic transporters and models used to elucidate their role in drug clearance; 4) mechanisms and examples of idiosyncratic and intrinsic hepatotoxicity; and 5) alternative cell systems to primary human hepatocytes. We also report pharmaceutical perspectives of these topics and compare methods and interpretations for the drug development process.

An industrial sludge containing a complex mixture of nitroaromatic compounds was treated in industrial large-scale aerobic and anaerobic biodegradation processes, performed in compost sacs. The goal was to study changes in genotoxicity during the two different oxygen regimes using the umuC genotoxicity assay. The composting sac was actively aerated during 3 months and allowed to mature for another 3 months. The anaerobic sac was not aerated for 5 months and aerated during the last month in order to enhance degradation of remaining organic carbon. The sludge was obtained from the wastewater treatment plant at an industrial area in Karlskoga, Sweden. The biodegradation study was performed at a commercial waste treatment plant in Stockholm, according to the company routine procedure when treating household waste in sealed sacs. The material from the non-aerated system showed increased genotoxicity in the acetone-soluble fraction after treatment, as did the water-soluble fraction. The subsequent aeration period did not decrease the toxicity below the genotoxicity limit. The increase in the water-soluble genotoxic compounds may pose an environmental problem during secondary storage or use of sludge treated this way, since leakage of water-dissolved genotoxic compounds may occur. The composting process also generated genotoxicity, but this was restricted to acetone-soluble compounds, while the water-soluble compounds remained low in genotoxicity. The aerated process therefore seems more favorable in term of risk reduction of this industrial sludge, although it is necessary to optimize the aerated process in order to achieve non-toxic levels of potential genotoxic compounds extractable by organic solvents.

In this study we explore the biochemical consequences of alginate encapsulation on betaTC3 cells. (13)C NMR spectroscopy and isotopomer analysis were used to investigate the effects of encapsulation on several enzymatic processes associated with the TCA cycle. Our data show statistically significant differences in various enzymatic fluxes related to the TCA cycle and insulin secretion between monolayer and alginate-encapsulated cultures. The principal cause for these effects was the process of trypsinization. Embedding the trypsinized cells in alginate beads did not have a compounded effect on the enzymatic fluxes of entrapped cells. However, an additional small but statistically significant decrease in insulin secretion was measured in encapsulated cells. Finally, differences in either enzymatic fluxes or glucose consumption as a function of bead diameter were not observed. However, differences in T(2), assessed by (1)H NMR microimaging, were observed as a function of bead diameter, suggesting that smaller beads became more organized with time in culture, while larger beads displayed a looser organization.

"Save the brain" is a network for thrombolysis of stroke in the Uppsala County, Sweden. The network involves the ambulance services, the emergency ward, and the departments of medicine and neurology. The county has 298,655 inhabitants. During one year, 2001-2002, 184 "save the brain" transports were performed. After an initial clinical examination at the emergency ward, 67 of these patients (36%) were investigated with hyperacute CT. After a second clinical examination, 13 patients (19%) received intravenous thrombolysis. The time lag from onset of symptoms to thrombolysis varied from one and a half hour to four hours.

Goals, Scope and Background: From 2005, deposition of organic waste will be banned in Sweden. Likewise, in Germany and Austria, similar bans are being planned, and further countries will probably follow. Thus, there is a need to develop new methods and to refine established techniques for sludge management in the whole of the European Union. For this end, there is also an urgent need for appropriate ecotoxicological approaches to elucidate and assess the hazard potential of sewage sludge. Therefore, the present study was designed to assess the capacity of various established sludge treatment methods using different oxygen regimes to degrade recalcitrant nitro-substituted organic compounds and reduce their toxicity. Sewage sludge samples from a wastewater treatment plant in Sweden (Cambrex Karlskoga AB, industrial area Björkborn) receiving wastewater from industries manufacturing pharmaceutical substances, chemical intermediates and explosives were processed with different sludge treatment methods. Among other treatment methods, bioreactors (for anaerobic and aerobic sludge treatment) were used. In the present investigation, a battery of in vitro bioassays was employed to compare the cytotoxic and genotoxic potentials of different fractions of sludge samples in order to elucidate whether the treatments were suitable to reduce the toxicity of the sludge., Methods: In order to investigate the cytotoxicity of the extracts of treated and untreated sludge samples, the acute cytotoxicity test with the permanent cell line RTL-W1 was used. Genotoxicity was tested by means of the comet assay (single cell gel electrophoresis) with RTL-W1 cells, and mutagenicity was assessed with the Ames test using the Salmonella typhimurium strains TA98, TA98NR and TA100. Sludge toxicity was tested in different fractions of organic extracts produced by acetone and hexane extractions. The subsequent clean-up procedure (silica gel chromatography and elution with hexane and dichloromethane) resulted in two fractions, a lipophilic hexane-fraction and a semi-lipophilic dichloromethane-fraction. For the genotoxicity and mutagenicity tests, these fractions were reunited at equal ratios., Results and Discussion: The acute cytotoxicity test with RTL-W1 cells revealed a high cytotoxic potential for the semi-lipophilic DM-fractions of all sludge samples with NR50 values (= effective concentration for 50% cell death in the neutral red test) from 8.9 up to 20 mg sludge d.w./ml medium. A low cytotoxic potential for the hexane fractions of the untreated sludge samples (NR50 400 to > 400 mg sludge d.w./ml medium) was observed, whereas the hexane fractions of the treated sludge samples showed elevated cytotoxicity increasing further with treatment in the bioreactors. The comet assay indicated that three out of eight of the reunited fractions had a significant genotoxic potential. Whereas the genotoxic potential of one sample treated anaerobically was very high with an induction factor of 11.6, a similar sample (taken from the same anaerobic reactor four months later) and one untreated sample showed lower potentials. The samples treated in another anaerobic bioreactor as well as the samples treated aerobically showed no genotoxic potential. Results indicate that aerobic treatment was basically adequate for reducing the genotoxicity of the sludge, whereas anaerobic treatment was only partly useful for reduction of genotoxicity. The Ames test revealed a very high mutagenic potential for the reunited fractions of the untreated sludge samples with strain TA98 (maximum induction factors (IFmax) up to 45) and a relatively high potential for one of the samples treated aerobically (S2, IFmax = 18 (TA98, S9-)), thus documenting the suitability of both anaerobic and aerobic treatments to reduce the mutagenicity of the samples, however, with the aerobic treatment being less effective., Conclusions: Overall, none of the microbiological treatments for wastewater sludge in bioreactors was found to be ideal for general toxicity reduction of the sludge samples. Whereas cytotoxicity of the sludge increased or levelled off in most cases following either treatment, genotoxicity both increased or decreased after anaerobic treatment, depending on the specific sample. However, mutagenicity could generally be reduced by anaerobic treatment and, to a lesser degree, by aerobic treatment., Recommendations and Perspectives: The complex modification of the diverse damage potentials of sludge sample extracts by use of an in vitro biotest battery following treatment for toxicity reduction in bioreactors showed that considerations of different toxicological endpoints is essential for an adequate hazard assessment. Whereas in the case of cytotoxicity reduction, the reactors proved ineffective, mutagenicity could be reduced significantly at least in some cases in this case study.

Sesquiterpene lactones (SLs) are natural products responsible for the anti-inflammatory activity of a variety of medicinal plants, mainly from the Asteraceae family. Here, we investigated whether they also influence the process of exocytosis of pro-inflammatory enzymes, such as the human neutrophil elastase (HNE). Altogether, eight structurally different SLs from the eudesmanolide, guaianolide, pseudoguaianolide, and germacranolide type were studied. Neutrophils were isolated from fresh human blood. After pre-incubation with different concentrations of the respective SL and cytochalasin B, the exocytosis of elastase was initiated either by platelet activating factor or N-formyl-methionyl-leucyl-phenylalanine. Inhibition of HNE release was measured by p-nitroaniline formation. The SLs exhibited an inhibitory effect on elastase release from neutrophils challenged either by platelet activating factor or N-formyl-methionyl-leucyl-phenylalanine. Concentration-response curves were recorded and the IC(50) values ranged from 2 to 30 microM. Studies on isolated HNE showed that a selective direct inhibition on HNE can be excluded. Interestingly, the inhibitory activity did not correlate with the number of alpha,beta-unsaturated carbonyl functions. The structure-activity relationship and the molecular mechanism are discussed.

The aim of this study was to investigate the involvement of calmodulin in phospholipase D activation in SH-SY5Y cells. Cells prelabelled with [3H]-palmitic acid were incubated with calmodulin antagonists and/or other compounds. Phosphatidylethanol, a specific marker for phospholipase D activity, and phosphatidic acid were analysed. The calmodulin antagonists, calmidazolium and trifluoperazine, induced an extensive increase in phosphatidylethanol formation, and thus increased basal phospholipase D activity, in a dose- and time-dependent manner. The effect of calmidazolium on carbachol-induced activation of muscarinic receptors was also studied. Calmidazolium did not significantly affect the amount of phosphatidylethanol formed following carbachol addition. However, taking into account the increase in basal activity observed after calmidazolium addition, calmidazolium probably inhibits the muscarinic receptor-induced phospholipase D activation. In addition to phosphatidylethanol, basal phosphatidic acid levels were also increased after calmidazolium and trifluoperazine addition. Incubation with calmidazolium (10 microM) for 10 min induced a two-fold increase in phosphatidic acid. The calmidazolium-induced increase in basal phospholipase D activity was not affected by the protein kinase inhibitors H7 and staurosporine. On the other hand tyrosine kinase inhibitors abolished the calmidazolium-induced activation of phospholipase D. Calmidazolium also induced tyrosine phosphorylation in parallel to the phospholipase D activation. In conclusion, our data indicate that calmodulin antagonists induce phospholipase D activity in SH-SY5Y cells via a tyrosine kinase dependent pathway. This may point to a negative control of phospholipase D by calmodulin although a calmodulin-independent mechanism cannot be excluded. Calmodulin antagonists may be useful tools to further elucidate the mechanisms of phospholipase D regulation.

The binding of a series of low-molecular-mass, active-site-directed thrombin inhibitors (399-575 Da) to human alpha-thrombin was investigated by surface plasmon resonance technology (BIACORE), stopped-flow spectrophotometry, and isothermal titration microcalorimetry (ITC). The equilibrium constants K(D) (nM to microM range) at 25 degrees C obtained from the BIACORE analysis correlated well with the inhibition constants K(i) in a chromogenic inhibition assay. The interactions between thrombin and three potent inhibitors, melagatran, inogatran, and CH-248, were further investigated at temperatures between 278 and 310K. A one-to-one binding stoichiometry found with ITC was supported by BIACORE data. K(i) and K(D) values increased with the temperature, mainly due to higher values for dissociation rate constants. The changes in enthalpy, DeltaH, and entropy, DeltaS, determined from the linear van't Hoff plots (R coefficient > 0.99), were linearly correlated by chemical compensation. Both techniques indicated clear differences in DeltaS for the three inhibitors, with a strong correlation to the number of rotational bonds. Immobilization of thrombin increased the binding stability at higher temperature and reduced the DeltaH by 20 kJ mol(-1). DeltaH values obtained from the inhibition kinetics and BIACORE were thus not identical, but correlated well with ITC data obtained at 37 degrees C. The two thermodynamic techniques allowed further differentiation between compounds of similar affinity; furthermore, kinetic analysis, hence analysis of the transition state, is complementary to ITC. A direct BIACORE binding assay might be a useful alternative to more elaborate inhibition studies., ((c)2001 Elsevier Science.)

An examination of the cuticle of six aquatic oligochaete species using transmission electron microscopy revealed a larger morphological variation than previously known. Three freshwater species, Aulodrilus pluriseta, Spirosperma ferox (both Tubificidae), and Pristina breviseta (Naididae), and three marine species, Clitellio arenarius, Heterochaeta costata (both Tubificidae), and Paranais litoralis (Naididae), were investigated. The arrangement of the collagen fibers in the cuticle differs among the studied species. Only S. ferox shows an "orthogonal grid," i.e., layers of parallel fibers perpendicular to each other, as earlier described for lumbricids and enchytraeids. Clitellio arenarius and H. costata have fibers arranged in layers, while A. pluriseta and P. litoralis have irregularly distributed fibers. Pristina breviseta lacks cuticular fibers. The matrix surrounding the collagen fibers (when present) continues outside the fiber layer, making up a thin epicuticle, which has a unique banding in each of the studied species. The external surface of the epicuticle is covered with epicuticular projections. Their number, shape, and attachment to the epicuticle vary among the studied species. Furthermore, a distinctive internal substructure of the projections was observed in H. costata, A. pluriseta, S. ferox, and P. breviseta. Microvilli, extensions from the epidermal cells, penetrate the cuticle and terminate at its outer surface. In three species microvilli were observed to pinch off the epicuticular projections. The size, number, and shape of the latter vary; no typical microvilli were observed in S. ferox.

The male genital duct in Tubificidae consists of a funnel, a vas deferens, an atrium, and, frequently, a copulatory structure. There may also be a diffuse or compact prostate gland in association with the duct. The morphogenesis of this duct is described for Rhyacodrilus coccineus and Monopylephorus rubroniveus (Rhyacodrilinae). The funnel and vas deferens in both species originate from peritoneal (mesodermal) cells in the posterior septum in the testis segment. The atrium in R. coccineus develops from a primary epidermal (ectodermal) invagination. A typical atrium is not formed in M. rubroniveus; the entire duct is of mesodermal origin. In the latter species, a shallow epidermal invagination occurs, into which both male ducts open, but it bears resemblance to a copulatory structure, which usually forms from a secondary invagination, rather than to a proper atrium. We therefore conclude that M. rubroniveus lacks an atrium. The copulatory structure is termed the male bursa. Both species have diffuse prostate glands that differentiate from peritoneal (mesodermal) cells surrounding the male duct. In R. coccineus the cells cover the atrium, whereas in M. rubroniveus they cover only a part of the vas deferens. The development of the spermathecae and female ducts is also examined. The spermatheca is of ectodermal origin in both studied species, i.e., it forms as an invagination of the epidermis. The female duct develops from peritoneal (mesodermal) cells in the posterior septum of the ovary segment. However, in M. rubroniveus the first sign of the duct disappears and a proper duct never develops., (Copyright 1999 Wiley-Liss, Inc.)

As interest in scuba diving is increasing in both sexes, doctors need to be aware of the risks encountered when diving and about gender-related differences in these risks. Individuals prone to panic attacks, claustrophobia or reckless risk-taking should avoid diving. In tolerating cold, muscle mass is more important than the amount of subcutaneous fat. The risk of decompression disease seems to be slightly greater among women, probably due to their fat distribution. Pregnant women are recommended not to dive, because the risk of birth defects seems to be greater among those who do, and there is a serious risk of fetal decompression disease. All participants in the sport must be responsible for their own diving safety.

A recent study carried out by our group demonstrated that exogenous dopamine increases the somatostatin (SS) receptor-effector system in the rat striatum. The present study examined the participation of the D1- and D2-dopaminergic systems in the modulation of the rat striatal SS receptor-effector system by use of the D1-receptor agonist and antagonist SKF 38393 and SCH 23390, respectively, and the D2-receptor agonist and antagonist bromocriptine and raclopride, respectively. In view of the rapid onset of dopamine action, the effect of dopaminergic agents on the SS mechanism of action were studied 3 h after their administration. SKF 38393 (4 mg/kg i.p.) or bromocriptine (2 mg/kg i.p.) administered to male Wistar rats increased the number of 125I-Tyr3-SMS receptors in the striatum (52 and 30%, respectively) without changing the affinity constant. The effect of SKF 38393 on 125I-Tyr3-SMS binding was antagonized by the D1-specific antagonist SCH 23390 (0.25 mg/kg i.p.) whereas the effect of bromocriptine was abolished by the D2-specific antagonist raclopride (5 mg/kg i.p.). No change in binding was produced when SKF 38393 or bromocriptine were added directly to the incubation medium. The acute systemic administration of SCH 23390 or raclopride alone had no effect on the binding of 125I-Tyr3-SMS to its receptors. The increase of the number of 125I-Tyr3-SMS receptor induced by SKF 38393 or bromocriptine was accompanied by an increase in the capacity of SMS 201-995 to inhibit basal and forskolin (FK)-stimulated adenylyl cyclase (AC) activity when compared to the control groups. In addition, the effect of SMS 201-995 on the mass accumulation of inositol 1,4,5-trisphosphate (IP3) was investigated. SKF 38393 as well as bromocriptine increased the capacity of SMS 201-995 to accumulate IP3 in the rat striatum although this effect was only statistically significant in the case of SKF 38393. These results suggest that the activation of D1 and D2 receptors increases the activity of the SS receptor-effector system, the effect being greater in the case of D1 receptors. These findings are consistent with a functional interaction between dopamine and SS in the rat striatum.

The cholinergic regulation of phospholipase D activity was studied in SH-SY5Y human neuroblastoma cells with phosphatidylethanol formation as a specific marker for the enzyme activity. The muscarinic antagonists, hexahydrosiladifenidol and pirenzepine, inhibited carbachol-induced phosphatidylethanol formation in a concentration-dependent manner and the inhibitory constants indicated that muscarinic M1 receptors are responsible for the major part of the phospholipase D activation. The mechanism of receptor-mediated phospholipase D activation varies between different cell types and receptors. In SH-SY5Y cells, the carbachol-induced phospholipase D activity was inhibited by protein kinase C inhibitors. Since both phospholipases D and C are activated by muscarinic stimulation in SH-SY5Y cells, most of the phospholipase D activation is probably secondary to the protein kinase C activation that follows phospholipase C-mediated increase in diacylglycerols. Other kinases may be involved in the regulation since also a tyrosine kinase inhibitor decreased the phosphatidylethanol formation. Stimulation of G-protein(s) and increase in the intracellular Ca2+ concentration activated phospholipase D and may be additional mechanisms for the muscarinic regulation of phospholipase D in SH-SY5Y cells. Propranolol, an inhibitor of phosphatidic acid phosphohydrolase, increased the carbachol-induced formation of phosphatidic acid at the expense of 1,2-diacylglycerol. This indicates that phospholipase D contributes to the formation of 1,2-diacylglycerol after carbachol stimulation in SH-SY5Y cells.

Phosphatidylethanol (PEth) is formed only in the presence of ethanol, via the action of phospholipase D. We studied PEth in blood as a possible marker of alcohol abuse in 15 male alcoholics admitted for detoxification. Blood was drawn on the first day after admission and up to 28 days thereafter. PEth in whole blood was 13.2 +/- 2.2 mumol liter-1 (mean +/- SE) at first sampling and remained detectable up to 14 days after admission. Blood ethanol was 0 on the morning after admission. The time courses of PEth disappearance varied among individuals. No PEth could be found in blood of control persons who had abstained from ethanol for 4 days. Levels of PEth and carbohydrate-deficient transferrin or gamma-glutamyltranspeptidase did not correlate. Its high specificity and prolonged detectability suggest PEth in blood as a marker of recent alcohol abuse.

The use of magnetic resonance imaging (MRI) has resulted in the detection of an increasing number of children with an apparently leukodystrophic white matter. Laboratory tests and the clinical presentation, however, often do not correspond to any known entity and the course is sometimes not progressively deteriorating. Such children with white-matter changes and no known diagnosis were the subject of this Swedish multicentre study, in which MRI findings and clinical data from 100 children considered to have white-matter abnormalities were assessed during the period 1992-1995. At re-evaluation of MR images by an established "white-matter group" of neuroradiologists, paediatric neurologists, neurologists and neurochemists, the MRI signal of the white matter was considered normal in eleven children and eleven had mainly a grey matter affection. Of the remaining 78 children with white matter abnormalities, a diagnosis was found in 32, but in 46 children no diagnosis could be established. A progressive downhill course characterised 17, probably representing hitherto undefined types of leukodystrophies. Five children had a relapsing-remitting course, and in 11 it was difficult to establish whether the course was progressive or stationary. The disease was non-progressive in 13. This group of non-leukodystrophic white-matter changes obviously represents maldevelopments of myelin formation, thus dys- or hypomyelination rather than demyelination.