Multisensor hyperspectral imaging approach for the microchemical analysis of ultramarine blue pigments.

This work presents a multisensor hyperspectral approach for the characterization of ultramarine blue, a valuable historical pigment, at the microscopic scale combining the information of four analytical techniques at the elemental and molecular levels. The hyperspectral images collected were combined in a single hypercube, where the pixels of the various spectral components are aligned on top of each other. Selected spectral descriptors have been defined to reduce data dimensionality before applying unsupervised chemometric data analysis approaches. Lazurite, responsible for the blue color of the pigment, was detected as the major mineral phase present in synthetic and good quality pigments. Impurities like pyrite were detected in lower quality samples, although the clear identification of other mineral phases with silicate basis was more difficult. There is no correlation between the spatial distribution of the bands arising in the Raman spectra of natural samples in the region 1200-1850 cm-1 and any of the transition metals or rare earth elements (REE). With this information, the previous hypothesis (based on bulk analysis) attributing these bands to luminescence emissions due to impurities of these elements must be revised. We propose the consideration of CO2 molecules trapped in the cages of the aluminosilicate structure of sodalite-type. Additionally, correlation between certain Raman features and the combined presence of Ca, P, and REE, in particular Nd, was detected for the lowest quality pigment. Our results highlight the usefulness of fusing chemical images obtained via different imaging techniques to obtain relevant information on chemical structure and properties.

Heteroresistant Bacteria Detected by an Extended Raman-Based Antibiotic Susceptibility Test.

Worldwide, multiresistant bacterial strains are emerging at unprecedented rates. This development seriously threatens the ability of humanity to treat even common infections, resulting in disability and death. Furthermore, this development endangers all medical achievements including cancer therapy or organ transplantations. Therefore, the World Health Organization has endorsed antimicrobial resistance as a great threat to humanity. To still allow effective treatment of patients, rapid, automated, and reliable antibiotic susceptibility testing (AST) of bacterial pathogens is essential. Thereby, speed and sensitivity of the AST results are crucial for improving patient care. Here, Raman spectroscopy as a nondestructive technique providing chemical-specific information is employed to monitor the deuterium uptake of metabolically active bacteria during antibiotic treatment, enabling fast and reliable AST. For this purpose, a bulk sample-preparation method was developed, allowing a high-throughput analysis of a significant number of cells. A protocol was developed for Gram-positive (Enterococcus faecalis) and Gram-negative (Escherichia coli) reference strains and was tested on 51 clinical isolates with well-characterized resistance phenotypes against ampicillin, ciprofloxacin, meropenem, and vancomycin. Borderline resistant and heteroresistant phenotypes were observed and further investigated. This is of critical importance as the sensitive detection of low-frequency heteroresistance in bacterial populations is a huge challenge. Such isolates seem susceptible but are resistant to treatment in vivo. Automatable analysis detects strong phenotypes within 3 h. On the basis of experimental and modeled data, heteroresistance is estimated to be detectable down to frequencies of 10-6 and investigated on clinical isolates as a proof-of-concept study, but requiring longer incubation time.

Apoptosis induced by the fungal pathogen gliotoxin requires a triple phosphorylation of Bim by JNK.

We previously reported that gliotoxin (GT), the major virulence factor of the mold Aspergillus fumigatus causing invasive aspergillosis (IA) in immunocompromised patients, induces apoptosis in a Bak-dependent manner. The signaling pathway leading to Bak activation and subsequent mitochondrial outer membrane permeabilization (MOMP) is elusive. Here, we show that GT and the supernatant of A. fumigatus (but not its GT-defective mutant) activate the JNK pathway and require a co-operative JNK-mediated BimEL phosphorylation at three sites (S100, T112 and S114) to induce apoptosis in mouse fibroblasts, human bronchial and mouse alveolar epithelial cells. Cells (i) treated with the JNK inhibitor SP600125, (ii) deleted or knocked down for JNK1/2 or Bim or (iii) carrying the BimEL triple phosphomutant S100A/T112A/S114A instead of wild-type BimEL are similarly resistant to GT-induced apoptosis. Triple-phosphorylated BimEL is more stable, redistributes from a cytoskeletal to a membrane fraction, better interacts with Bcl-2 and Bcl-xL and more effectively activates Bak than the unphosphorylated mutant. These data indicate that JNK-mediated BimEL phosphorylation at S100, T112 and S114 constitutes a novel regulatory mechanism to activate Bim in response to apoptotic stimuli.

Dystonia in neurodegeneration with brain iron accumulation: outcome of bilateral pallidal stimulation.

Neurodegeneration with brain iron accumulation encompasses a heterogeneous group of rare neurodegenerative disorders that are characterized by iron accumulation in the brain. Severe generalized dystonia is frequently a prominent symptom and can be very disabling, causing gait impairment, difficulty with speech and swallowing, pain and respiratory distress. Several case reports and one case series have been published concerning therapeutic outcome of pallidal deep brain stimulation in dystonia caused by neurodegeneration with brain iron degeneration, reporting mostly favourable outcomes. However, with case studies, there may be a reporting bias towards favourable outcome. Thus, we undertook this multi-centre retrospective study to gather worldwide experiences with bilateral pallidal deep brain stimulation in patients with neurodegeneration with brain iron accumulation. A total of 16 centres contributed 23 patients with confirmed neurodegeneration with brain iron accumulation and bilateral pallidal deep brain stimulation. Patient details including gender, age at onset, age at operation, genetic status, magnetic resonance imaging status, history and clinical findings were requested. Data on severity of dystonia (Burke Fahn Marsden Dystonia Rating Scale-Motor Scale, Barry Albright Dystonia Scale), disability (Burke Fahn Marsden Dystonia Rating Scale-Disability Scale), quality of life (subjective global rating from 1 to 10 obtained retrospectively from patient and caregiver) as well as data on supportive therapy, concurrent pharmacotherapy, stimulation settings, adverse events and side effects were collected. Data were collected once preoperatively and at 2-6 and 9-15 months postoperatively. The primary outcome measure was change in severity of dystonia. The mean improvement in severity of dystonia was 28.5% at 2-6 months and 25.7% at 9-15 months. At 9-15 months postoperatively, 66.7% of patients showed an improvement of 20% or more in severity of dystonia, and 31.3% showed an improvement of 20% or more in disability. Global quality of life ratings showed a median improvement of 83.3% at 9-15 months. Severity of dystonia preoperatively and disease duration predicted improvement in severity of dystonia at 2-6 months; this failed to reach significance at 9-15 months. The study confirms that dystonia in neurodegeneration with brain iron accumulation improves with bilateral pallidal deep brain stimulation, although this improvement is not as great as the benefit reported in patients with primary generalized dystonias or some other secondary dystonias. The patients with more severe dystonia seem to benefit more. A well-controlled, multi-centre prospective study is necessary to enable evidence-based therapeutic decisions and better predict therapeutic outcomes.

Basin-scale coherence in phenology of shrimps and phytoplankton in the North Atlantic Ocean.

Climate change could lead to mismatches between the reproductive cycles of marine organisms and their planktonic food. We tested this hypothesis by comparing shrimp (Pandalus borealis) egg hatching times and satellite-derived phytoplankton bloom dynamics throughout the North Atlantic. At large spatial and long temporal (10 years or longer) scales, hatching was correlated with the timing of the spring phytoplankton bloom. Annual egg development and hatching times were determined locally by bottom water temperature. We conclude that different populations of P. borealis have adapted to local temperatures and bloom timing, matching egg hatching to food availability under average conditions. This strategy is vulnerable to interannual oceanographic variability and long-term climatic changes.

Analytical modeling of symmetry breaking in extraordinary optoconductance.

Extraordinary optoconductance (EOC) devices with symmetric leads have been shown to have a symmetric positional dependence when exposed to focused illumination. While advantageous for a position sensitive detector (PSD), this symmetric positional dependence, when the device is uniformly illuminated, leads to a minimization of the output voltage. Here, with the aid of a previously employed point charge model, we address two ways to break the symmetry and recover the output signal. The first is to impose uniform illumination but only on half the sample. This method has practical limitations as the device is miniaturized to the nanoscale. The second is via asymmetric placement of the voltage probes in a four-probe measurement. Crucial to the discussion is the effect of the surface charge density. Several ways of modeling the induced surface charge density are presented. Utilizing the above described approach, optimal asymmetric lead positions are found.

Model-supported estimation of mortality rates in Baltic cod (Gadus morhua callarias L.) larvae: the varying impact of 'critical periods'.

BACKGROUND: Changes in the survival-rate during the larval phase may strongly influence the recruitment level in marine fish species. During the larval phase different 'critical periods' are discussed, e.g. the hatching period and the first-feeding period. No such information was available for the Baltic cod stock, a commercially important stock showing reproduction failure during the last years. We calculated field-based mortality rates for larval Baltic cod during these phases using basin-wide abundance estimates from two consecutive surveys. Survey information was corrected by three dimensional hydrodynamic model runs. RESULTS: The corrections applied for transport were of variable impact, depending on the prevailing circulation patterns. Especially at high wind forcing scenarios, abundance estimates have the potential to be biased without accounting for transport processes. In May 1988 mortality between hatch and first feeding amounted to approximately 20% per day. Mortality rates during the onset of feeding were considerably lower with only 7% per day. In August 1991 the situation was vice versa: Extremely low mortality rates of 0.08% per day were calculated between hatch and first feeding, while the period between the onset of feeding to the state of an established feeder was more critical with mortality rates of 22% per day. CONCLUSIONS: Mortality rates during the different proposed 'critical periods' were found to be highly variable. Survival rates of Baltic cod are not only influenced by a single 'critical period', but can be limited at different points during the larval phase, depending on several biotic and abiotic factors.

Constitutive activity of histamine h(3) receptors stably expressed in SK-N-MC cells: display of agonism and inverse agonism by H(3) antagonists.

Agonist-independent activity of G-protein-coupled receptor, also referred to as constitutive activity, is a well-documented phenomenon and has been reported recently for both the histamine H(1) and H(2) receptors. Using SK-N-MC cell lines stably expressing the human and rat H(3) receptors at physiological receptor densities (500-600 fmol/mg of protein), we show that both the rat and human H(3) receptors show a high degree of constitutive activity. The forskolin-mediated cAMP production in SK-N-MC cells is inhibited strongly upon expression of the G(i)-coupled H(3) receptor. The cAMP production can be further inhibited upon agonist stimulation of the H(3) receptor and can be enhanced by a variety of H(3) antagonists acting as inverse agonists at the H(3) receptor. Thioperamide, clobenpropit, and iodophenpropit raise the cAMP levels in SK-N-MC cells with potencies that match their receptor binding affinities. Surprisingly, impentamine and burimamide act as effective H(3) agonists. Modification of the amine group of impentamine dramatically affected the pharmacological activity of the ligand. Receptor affinity was reduced slightly for most impentamine analogs, but the functional activity of the ligands varied from agonist to neutral antagonist and inverse agonist, indicating that subtle changes in the chemical structures of impentamine analogs have major impact on the (de)activation steps of the H(3) receptor. In conclusion, upon stable expression of the rat and human H(3) receptor in SK-N-MC cells constitutive receptor activity is detected. In this experimental system, H(3) receptors ligands, previously identified as H(3) antagonists, cover the whole spectrum of pharmacological activities, ranging from full inverse agonists to agonists.

Regulation of Staphylococcus aureus type 5 and type 8 capsular polysaccharides by CO(2).

Staphylococcus aureus expression of capsular polysaccharide type 5 (CP5) has been shown to be downregulated by CO(2). Here we show that CO(2) reduces CP5 expression at the transcriptional level and that CO(2) regulates CP8 expression depending on the genetic background of the strains. Growth in the presence of air supplemented with 5% CO(2) caused a significant decrease in CP8 expression in four S. aureus strains, a marginal effect in four strains, and higher CP8 expression in strain Becker. Absolute CP8 expression in the nine S. aureus strains differed largely from strain to strain. Four groups of strains were established due to sequence variations in the promoter region of cap5 and cap8. To test whether these sequence variations are responsible for the different responses to CO(2), promoter regions from selected strains were fused to the reporter gene xylE in pLC4, and the plasmids were electrotransformed into strains Becker and Newman. XylE activity was negatively regulated by CO(2) in all derivatives of strain Newman and was always positively regulated by CO(2) in all derivatives of strain Becker. Differences in promoter sequences did not influence the pattern of CP8 expression. Therefore, the genetic background of the strains rather than differences in the promoter sequence determines the CO(2) response. trans-acting regulatory molecules may be differentially expressed in strain Becker versus strain Newman. The strain dependency of the CP8 expression established in vitro was also seen in lung tissue sections of patients with cystic fibrosis infected with CP8-positive S. aureus strains.

Development of a pharmacophore model for histamine H3 receptor antagonists, using the newly developed molecular modeling program SLATE.

New molecular modeling tools were developed to construct a qualitative pharmacophore model for histamine H3 receptor antagonists. The program SLATE superposes ligands assuming optimum hydrogen bond geometry. One or two ligands are allowed to flex in the procedure, thereby enabling the determination of the bioactive conformation of flexible H3 antagonists. In the derived model, four hydrogen-bonding site points and two hydrophobic pockets available for binding antagonists are revealed. The model results in a better understanding of the structure-activity relationships of H3 antagonists. To validate the model, a series of new antagonists was synthesized. The compounds were designed to interact with all four hydrogen-bonding site points and the two hydrophobic pockets simultaneously. These ligands have high H3 receptor affinity, thereby illustrating how the model can be used in the design of new classes of H3 antagonists.

Identification of rat H3 receptor isoforms with different brain expression and signaling properties.

We identified the cDNAs of three functional rat H3 receptor isoforms (H3A, H3B, and H3C) and one nonfunctional truncated H3 receptor (H3T). The H3A, H3B, and H3C receptor isoforms vary in the length of their third intracellular loop; the H3B and H3C receptor lack 32 and 48 amino acids, respectively. Transient expression of the H3A, H3B, and H3C receptors in COS-7 cells results in high affinity binding for the H3 antagonist [125I]iodophenpropit, which is displaced by selective H3 agonists and antagonists. The three isoforms differentially couple to the Gi protein-dependent inhibition of adenylate cyclase or stimulation of p44/p42 mitogen activated protein kinase (MAPK), a new signaling pathway for the H3 receptor. Whereas the H3A receptor was less effective in inhibiting forskolin-induced cAMP production compared with the H3B or H3C receptor, this isoform was more effective in the stimulation of p44/p42 MAPK. The H3 receptor isoforms also displayed differential CNS expression in key areas involved in regulation of sensory, endocrine, and cognitive functions. A differential H3 receptor isoform expression was seen in, for example, hippocampus, where a characteristic dorsoventral distribution was revealed. Differential H3 receptor expression was also characteristic for the cerebellum, indicating possible histaminergic regulation of motor functions. The identification of these new H3 receptor isoforms and their specific signaling properties adds a new level of complexity to our understanding of the role of histamine, and the H3 receptor in brain function. The heterogeneous distribution of the isoforms suggests that H3 receptor isoform-specific regulation is important in several brain functions.

Constitutive activity of the histamine H(1) receptor reveals inverse agonism of histamine H(1) receptor antagonists.

Transient expression of the wild-type human histamine H(1) receptor in SV40-immortalised African green monkey kidney cells resulted in an agonist-independent elevation of the basal levels of the second messenger inositoltrisphospate. Several histamine H(1) receptor antagonists, including the therapeutically used anti-allergics cetirizine, loratadine and epinastine reduced this constitutive histamine H(1) receptor activity. Inverse agonism, i.e., stabilisation of an inactive conformation of the human histamine H(1) receptor, may therefore be a key component of the anti-allergic mechanism of action of clinically used antihistamines.

Mutational analysis of the antagonist-binding site of the histamine H(1) receptor.

We combined in a previously derived three-dimensional model of the histamine H(1) receptor (Ter Laak, A. M., Timmerman, H., Leurs, H., Nederkoorn, P. H. J., Smit, M. J., and Donne-Op den Kelder, G. M. (1995) J. Comp. Aid. Mol. Design. 9, 319-330) a pharmacophore for the H(1) antagonist binding site (Ter Laak, A. M., Venhorst, J., Timmerman, H., and Donné-Op de Kelder, G. M. (1994) J. Med. Chem. 38, 3351-3360) with the known interacting amino acid residue Asp(116) (in transmembrane domain III) of the H(1) receptor and verified the predicted receptor-ligand interactions by site-directed mutagenesis. This resulted in the identification of the aromatic amino acids Trp(167), Phe(433), and Phe(436) in transmembrane domains IV and VI of the H(1) receptor as probable interaction points for the trans-aromatic ring of the H(1) antagonists. Subsequently, a specific interaction of carboxylate moieties of two therapeutically important, zwitterionic H(1) antagonists with Lys(200) in transmembrane domain V was predicted. A Lys(200) --> Ala mutation results in a 50- (acrivastine) to 8-fold (d-cetirizine) loss of affinity of these zwitterionic antagonists. In contrast, the affinities of structural analogs of acrivastine and cetirizine lacking the carboxylate group, triprolidine and meclozine, respectively, are unaffected by the Lys(200) --> Ala mutation. These data strongly suggest that Lys(200), unique for the H(1) receptor, acts as a specific anchor point for these "second generation" H(1) antagonists.

Quality assessment and improvement in a university-based anticoagulation management service.

The quality improvement process in a university-based anticoagulation management service is described. A clinic overview describes the reporting structure, patient population, clinic operations, and patient care processes. Quality improvement is an inherent component to any patient care process. The Anticoagulation Clinic is addressing improvements by implementing point-of-care testing, adjusting the full-time equivalent mix, developing a credentialing process for anticoagulation practitioners, and creating a new consult form. By obtaining a new computer tracking program, improved clinical outcome tracking (i.e., thromboembolic and hemorrhagic rates) will occur. Enhancing the format of patient education and additional patient and physician satisfaction surveys are components of educational and psychological outcome improvements.

Involvement of Asn-293 in stereospecific agonist recognition and in activation of the beta 2-adrenergic receptor.

To investigate the molecular mechanism for stereospecific binding of agonists to beta 2-adrenergic receptors we used receptor models to identify potential binding sites for the beta-OH-group of the ligand, which defines the chiral center. Ser-165, located in transmembrane helix IV, and Asn-293, situated in the upper half of transmembrane helix VI, were identified as potential binding sites. Mutation of Ser-165 to Ala did not change the binding of either isoproterenol isomer as revealed after transient expression in human embryonic kidney (HEK)-293 cells. In contrast, a receptor mutant in which Asn-293 was replaced by Leu showed substantial loss of stereospecific isoproterenol binding. Adenylyl cyclase stimulation by this mutant after stable expression in CHO cells confirmed the substantial loss of stereospecificity for isoproterenol. In a series of agonists the loss of affinity in the Leu-293 mutant receptor was strongly correlated with the intrinsic activity of the compounds. Full agonists showed a 10-30-fold affinity loss, whereas partial agonists had almost the same affinity for both receptors. Stereospecific recognition of antagonists was unaltered in the Leu-293 mutant receptor. These data indicate a relationship between stereospecificity and intrinsic activity of agonists and suggest that Asn-293 is important for both properties of the agonist-receptor interaction.

The integration of employees with disabilities in Germany and the importance of workplace design.

This paper reviews the occupational integration of employees with disabilities in Germany. A short sketch of historical trends is followed by a presentation of the current tasks and goals of rehabilitation, as well as the legal and institutional framing conditions for the integration and reintegration of people with disabilities into the work process. It is shown that one important guarantee for the success of integration is an ergonomic workplace design that takes the needs of employees with disabilities into account.

A promoter-screening plasmid and xylose-inducible, glucose-repressible expression vectors for Staphylococcus carnosus.

We describe a series of plasmid vectors for DNA cloning in staphylococci. pPS11 is a promoter probe plasmid containing a promoterless lipase (Lip)-encoding gene (lip). Insertion of a promoter-bearing DNA fragment at the single BamHI site turns on lip expression. Lip activity can be easily determined to estimate the strength of the inserted promoter. pPS11 served also as a basis for the construction of vectors which allow xylose-inducible gene expression in Staphylococcus carnosus (Sc). Using plasmid pCX15, we studied xylose-inducible lip expression in Sc. The lip expression is under transcriptional control of the repressor, XylR. The xylR gene, the XylR target sequence and the xylA promoter/operator sequence with the cis-acting catabolite-responsive element (cre) are derived from the xyl operon of S. xylosus. The single BamHI site in front of the lip ribosome-binding site (RBS) also makes it possible to put other promoterless genes under transcriptional control of XylR. To facilitate the controlled expression of genes which merely start with the start codon and have no RBS, or to insert genes with their own RBS, pCX26 and pCX26 delta lip were constructed. The influence of xylose and glucose on lip expression was studied both in a batch culture and in a fermentor under controlled pH conditions. With pCX15, the presence of xylose led to a 40-fold increase in extracellular Lip activity, while the presence of glucose caused a repression of lip expression. The results suggest that the xylA promoter is subject to two different regulatory mechanisms, one of which involves the repression of the xylA promoter by XylR in the absence of xylose, and the other involves a glucose-mediated catabolite repression which dominates over the xylose induction.

Combined factor VIII/factor XI deficiency may cause intra-familial clinical variability in haemophilia A among Ashkenazi Jews.

Heterozygous factor XI deficiency occurs very frequently among Ashkenazi Jews. To investigate the potential influence of a co-inherited factor XI deficiency state on the clinical phenotype of mild/moderate haemophilia A, 28 unrelated haemophiliacs of Jewish origin were screened for the two most common factor XI gene mutations. Gene lesions were identified in two out of 14 patients of Ashkenazi origin. In the one family analysed further, co-inheritance of both factor XI and factor VIII deficiencies was associated with a bleeding tendency that was more severe than that associated with either deficiency alone.