Concomitant cocaine use is a major problem in clinical practice in methadone maintenance treatment (MMT) and may interfere with successful treatment. Data from European methadone populations is sparse. This register-based study sought to explore the association between prescribed methadone dose and concomitant cocaine and heroin use in the methadone population of Basel City.
The study included 613 methadone patients between April 1, 2003 and March 31, 2004. Anonymized data was taken from the methadone register of Basel City. For analysis of the prescribed methadone dose distribution, the patient sample was split into three methadone dosage groups: a low dose group (LDG) (n = 200; < 60 mg/day), a medium dose group (MDG) (n = 273; 60 to 100 mg/day), and a high dose group (HDG) (n = 140; > 100 mg/day). Concomitant drug use was based on self-report.
Analysis showed a significant difference in self-reported cocaine use between groups (p < 0.001). Patients in the LDG reported significantly fewer cocaine consumption days compared to the MDG (p < 0.001) and the HDG (p < 0.05). Patients in the HDG reported significantly fewer heroin consumption days than those in the LDG (p < 0.01) and the MDG (p < 0.001). In logistic regression analysis, cocaine use was significantly associated with heroin use (OR 4.9).
Cocaine use in methadone patients may be associated with heroin use, which indicates the importance of prescribing appropriate methadone dosages in order to indirectly reduce cocaine use.
Cocaine use; Methadone dose; Methadone maintenance treatment; Opioid dependence
Genome-wide association studies (GWAS) have led to the identification of single nucleotide polymorphisms in or near several loci that are associated with the risk of obesity and nonalcoholic fatty liver disease (NAFLD). We hypothesized that missense variants in GWAS and related candidate genes may underlie cases of extreme obesity and NAFLD-related cirrhosis, an extreme manifestation of NAFLD.
We performed whole-exome sequencing on 6 Caucasian patients with extreme obesity [mean body mass index (BMI) 84.4] and 4 obese Caucasian patients (mean BMI 57.0) with NAFLD-related cirrhosis.
Sequence analysis was performed on 24 replicated GWAS and selected candidate obesity genes and 5 loci associated with NAFLD. No missense variants were identified in 19 of the 29 genes analyzed, although all patients carried at least 2 missense variants in the remaining genes without excess homozygosity. One patient with extreme obesity carried 2 novel damaging mutations in BBS1 and was homozygous for benign and damaging MC3R variants. In addition, 1 patient with NAFLD-related cirrhosis was compound heterozygous for rare damaging mutations in PNPLA3.
These results indicate that analyzing candidate loci previously identified by GWAS analyses using whole-exome sequencing is an effective strategy to identify potentially causative missense variants underlying extreme obesity and NAFLD-related cirrhosis.
Extreme obesity; Nonalcoholic fatty liver disease; Cirrhosis; Genome-wide association studies; Whole-exome sequencing
There is a need of guidance on how local irritancy data should be incorporated into risk assessment procedures, particularly with respect to the derivation of occupational exposure limits (OELs). Therefore, a board of experts from German committees in charge of the derivation of OELs discussed the major challenges of this particular end point for regulatory toxicology. As a result, this overview deals with the question of integrating results of local toxicity at the eyes and the upper respiratory tract (URT). Part 1 describes the morphology and physiology of the relevant target sites, i.e., the outer eye, nasal cavity, and larynx/pharynx in humans. Special emphasis is placed on sensory innervation, species differences between humans and rodents, and possible effects of obnoxious odor in humans. Based on this physiological basis, Part 2 describes a conceptual model for the causation of adverse health effects at these targets that is composed of two pathways. The first, “sensory irritation” pathway is initiated by the interaction of local irritants with receptors of the nervous system (e.g., trigeminal nerve endings) and a downstream cascade of reflexes and defense mechanisms (e.g., eyeblinks, coughing). While the first stages of this pathway are thought to be completely reversible, high or prolonged exposure can lead to neurogenic inflammation and subsequently tissue damage. The second, “tissue irritation” pathway starts with the interaction of the local irritant with the epithelial cell layers of the eyes and the URT. Adaptive changes are the first response on that pathway followed by inflammation and irreversible damages. Regardless of these initial steps, at high concentrations and prolonged exposures, the two pathways converge to the adverse effect of morphologically and biochemically ascertainable changes. Experimental exposure studies with human volunteers provide the empirical basis for effects along the sensory irritation pathway and thus, “sensory NOAEChuman” can be derived. In contrast, inhalation studies with rodents investigate the second pathway that yields an “irritative NOAECanimal.” Usually the data for both pathways is not available and extrapolation across species is necessary. Part 3 comprises an empirical approach for the derivation of a default factor for interspecies differences. Therefore, from those substances under discussion in German scientific and regulatory bodies, 19 substances were identified known to be human irritants with available human and animal data. The evaluation started with three substances: ethyl acrylate, formaldehyde, and methyl methacrylate. For these substances, appropriate chronic animal and a controlled human exposure studies were available. The comparison of the sensory NOAEChuman with the irritative NOAECanimal (chronic) resulted in an interspecies extrapolation factor (iEF) of 3 for extrapolating animal data concerning local sensory irritating effects. The adequacy of this iEF was confirmed by its application to additional substances with lower data density (acetaldehyde, ammonia, n-butyl acetate, hydrogen sulfide, and 2-ethylhexanol). Thus, extrapolating from animal studies, an iEF of 3 should be applied for local sensory irritants without reliable human data, unless individual data argue for a substance-specific approach.
Local irritants; Chemosensory perception; Regulatory toxicology; Interspecies extrapolation
Auditory prostheses can partially restore speech comprehension when hearing fails. Sound coding with current prostheses is based on electrical stimulation of auditory neurons and has limited frequency resolution due to broad current spread within the cochlea. In contrast, optical stimulation can be spatially confined, which may improve frequency resolution. Here, we used animal models to characterize optogenetic stimulation, which is the optical stimulation of neurons genetically engineered to express the light-gated ion channel channelrhodopsin-2 (ChR2). Optogenetic stimulation of spiral ganglion neurons (SGNs) activated the auditory pathway, as demonstrated by recordings of single neuron and neuronal population responses. Furthermore, optogenetic stimulation of SGNs restored auditory activity in deaf mice. Approximation of the spatial spread of cochlear excitation by recording local field potentials (LFPs) in the inferior colliculus in response to suprathreshold optical, acoustic, and electrical stimuli indicated that optogenetic stimulation achieves better frequency resolution than monopolar electrical stimulation. Virus-mediated expression of a ChR2 variant with greater light sensitivity in SGNs reduced the amount of light required for responses and allowed neuronal spiking following stimulation up to 60 Hz. Our study demonstrates a strategy for optogenetic stimulation of the auditory pathway in rodents and lays the groundwork for future applications of cochlear optogenetics in auditory research and prosthetics.
Many untargeted LC–ESI–HRMS based metabolomics studies are still hampered by the large proportion of non-biological sample derived signals included in the generated raw data. Here, a novel, powerful stable isotope labelling (SIL)-based metabolomics workflow is presented, which facilitates global metabolome extraction, improved metabolite annotation and metabolome wide internal standardisation (IS). The general concept is exemplified with two different cultivation variants, (1) co-cultivation of the plant pathogenic fungi Fusarium graminearum on non-labelled and highly 13C enriched culture medium and (2) experimental cultivation under native conditions and use of globally U-13C labelled biological reference samples as exemplified with maize and wheat. Subsequent to LC–HRMS analysis of mixtures of labelled and non-labelled samples, two-dimensional data filtering of SIL specific isotopic patterns is performed to better extract truly biological derived signals together with the corresponding number of carbon atoms of each metabolite ion. Finally, feature pairs are convoluted to feature groups each representing a single metabolite. Moreover, the correction of unequal matrix effects in different sample types and the improvement of relative metabolite quantification with metabolome wide IS are demonstrated for the F. graminearum experiment. Data processing employing the presented workflow revealed about 300 SIL derived feature pairs corresponding to 87–135 metabolites in F. graminearum samples and around 800 feature pairs corresponding to roughly 350 metabolites in wheat samples. SIL assisted IS, by the use of globally U-13C labelled biological samples, reduced the median CV value from 7.1 to 3.6 % for technical replicates and from 15.1 to 10.8 % for biological replicates in the respective F. graminearum samples.
13C-labelling; Internal standardisation; Metabolomics; Fusarium; Wheat; Maize
The development of efficient strategies for generating fully human monoclonal antibodies with unique functional properties that are exploitable for tailored therapeutic interventions remains a major challenge in the antibody technology field. Here, we present a methodology for recovering such antibodies from antigen-encountered human B cell repertoires. As the source for variable antibody genes, we cloned immunoglobulin G (IgG)-derived B cell repertoires from lymph nodes of 20 individuals undergoing surgery for head and neck cancer. Sequence analysis of unselected “LYmph Node Derived Antibody Libraries” (LYNDAL) revealed a naturally occurring distribution pattern of rearranged antibody sequences, representing all known variable gene families and most functional germline sequences. To demonstrate the feasibility for selecting antibodies with therapeutic potential from these repertoires, seven LYNDAL from donors with high serum titers against herpes simplex virus (HSV) were panned on recombinant glycoprotein B of HSV-1. Screening for specific binders delivered 34 single-chain variable fragments (scFvs) with unique sequences. Sequence analysis revealed extensive somatic hypermutation of enriched clones as a result of affinity maturation. Binding of scFvs to common glycoprotein B variants from HSV-1 and HSV-2 strains was highly specific, and the majority of analyzed antibody fragments bound to the target antigen with nanomolar affinity. From eight scFvs with HSV-neutralizing capacity in vitro, the most potent antibody neutralized 50% HSV-2 at 4.5 nM as a dimeric (scFv)2. We anticipate our approach to be useful for recovering fully human antibodies with therapeutic potential.
fully human antibodies; combinatorial libraries; phage display; lymph nodes; herpes simplex virus; neutralizing antibodies; scFv; immune libraries
Renal dysfunction is common in clinical settings in which cardiac function is compromised such as heart failure, cardiac surgery or sepsis, and is associated with high morbidity and mortality. Levosimendan is a calcium sensitizer and potassium channel opener used in the treatment of acute heart failure. This review describes the effects of the inodilator levosimendan on renal function. A panel of 25 scientists and clinicians from 15 European countries (Austria, Finland, France, Hungary, Germany, Greece, Italy, Portugal, the Netherlands, Slovenia, Spain, Sweden, Turkey, the United Kingdom, and Ukraine) convened and reached a consensus on the current interpretation of the renal effects of levosimendan described both in non-clinical research and in clinical study reports. Most reports on the effect of levosimendan indicate an improvement of renal function in heart failure, sepsis and cardiac surgery settings. However, caution should be applied as study designs differed from randomized, controlled studies to uncontrolled ones. Importantly, in the largest HF study (REVIVE I and II) no significant changes in the renal function were detected. As it regards the mechanism of action, the opening of mitochondrial KATP channels by levosimendan is involved through a preconditioning effect. There is a strong rationale for randomized controlled trials seeking beneficial renal effects of levosimendan. As an example, a study is shortly to commence to assess the role of levosimendan for the prevention of acute organ dysfunction in sepsis (LeoPARDS).
Levosimendan; Renal dysfunction
The interactions between proteins and nucleic acids have a fundamental function in many biological processes, including gene transcription, RNA homeostasis, protein translation and pathogen sensing for innate immunity. While our knowledge of the ensemble of proteins that bind individual mRNAs in mammalian cells has been greatly augmented by recent surveys, no systematic study on the non-sequence-specific engagement of native human proteins with various types of nucleic acids has been reported.
We designed an experimental approach to achieve broad coverage of the non-sequence-specific RNA and DNA binding space, including methylated cytosine, and tested for interaction potential with the human proteome. We used 25 rationally designed nucleic acid probes in an affinity purification mass spectrometry and bioinformatics workflow to identify proteins from whole cell extracts of three different human cell lines. The proteins were profiled for their binding preferences to the different general types of nucleic acids. The study identified 746 high-confidence direct binders, 139 of which were novel and 237 devoid of previous experimental evidence. We could assign specific affinities for sub-types of nucleic acid probes to 219 distinct proteins and individual domains. The evolutionarily conserved protein YB-1, previously associated with cancer and drug resistance, was shown to bind methylated cytosine preferentially, potentially conferring upon YB-1 an epigenetics-related function.
The dataset described here represents a rich resource of experimentally determined nucleic acid-binding proteins, and our methodology has great potential for further exploration of the interface between the protein and nucleic acid realms.
Multimorbidity has a negative impact on health-related quality of life (HRQL). Previous studies included only a limited number of conditions. In this study, we analyse the impact of a large number of conditions on HRQL in multimorbid patients without preselecting particular diseases. We also explore the effects of these conditions on the specific dimensions of HRQL.
Materials and Methods
This analysis is based on a multicenter, prospective cohort study of 3189 multimorbid primary care patients aged 65 to 85. The impact of 45 conditions on HRQL was analysed. The severity of the conditions was rated. The EQ-5D, consisting of 5 dimensions and a visual-analogue-scale (EQ VAS), was employed. Data were analysed using multiple ordinary least squares and multiple logistic regressions. Multimorbidity measured by a weighted count score was significantly associated with lower overall HRQL (EQ VAS), b = −1.02 (SE: 0.06). Parkinson’s disease had the most pronounced negative effect on overall HRQL (EQ VAS), b = −12.29 (SE: 2.18), followed by rheumatism, depression, and obesity. With regard to the individual EQ-5D dimensions, depression (OR = 1.39 to 3.3) and obesity (OR = 1.44 to 1.95) affected all five dimensions of the EQ-5D negatively except for the dimension anxiety/depression. Obesity had a positive effect on this dimension, OR = 0.78 (SE: 0.07). The dimensions “self-care”, OR = 4.52 (SE: 1.37) and “usual activities”, OR = 3.59 (SE: 1.0), were most strongly affected by Parkinson’s disease. As a limitation our sample may only represent patients with at most moderate disease severity.
The overall HRQL of multimorbid patients decreases with an increasing count and severity of conditions. Parkinson’s disease, depression and obesity have the strongest impact on HRQL. Further studies should address the impact of disease combinations which require very large sample sizes as well as advanced statistical methods.
► We developed a technology for dual-color single-molecule sensitive analysis of microarrays. ► The platform is well suitable for amplification-free expression profiling of minute samples. ► Expression changes found in a rare subpopulation of multiple myeloma CD138 negative progenitor cells could be validated using qPCR.
We present a highly sensitive bioanalytical microarray assay that enables the analysis of small genomic sample material. By combining an optimized cDNA purification step with single molecule cDNA detection on the microarray, the platform has improved sensitivity compared to conventional systems, allowing amplification-free determination of expression profiles with as little as 600 ng total RNA. Total RNA from cells was reverse transcribed into fluorescently labeled cDNA and purified employing a precipitation method that minimizes loss of cDNA material. The microarray was scanned on a fluorescence chip-reader with single molecule sensitivity. Using the newly developed platform we were able to analyze the RNA expression profile of a subpopulation of rare multiple myeloma CD138 negative progenitor (MM CD138neg) cells. The high-sensitivity microarray approach led to the identification of a set of 20 genes differentially expressed in MM CD138neg cells. Our work demonstrates the applicability of a straight-forward single-molecule DNA array technology to current topics of molecular and cellular cancer research, which are otherwise difficult to address due to the limited amount of sample material.
Expression profiling; Microarray; Subpopulation analysis; Single molecule detection; Fluorescence microscopy
In a world where resources are scarce and urban areas consume the vast majority of these resources, it is vital to make cities greener and more sustainable. Advanced systems to improve and automate processes within a city will play a leading role in smart cities. From smart design of buildings, which capture rain water for later use, to intelligent control systems, which can monitor infrastructures autonomously, the possible improvements enabled by sensing technologies are immense. Ubiquitous sensing poses numerous challenges, which are of a technological or social nature. This paper presents an overview of the state of the art with regards to sensing in smart cities. Topics include sensing applications in smart cities, sensing platforms and technical challenges associated with these technologies. In an effort to provide a holistic view of how sensing technologies play a role in smart cities, a range of applications and technical challenges associated with these applications are discussed. As some of these applications and technologies belong to different disciplines, the material presented in this paper attempts to bridge these to provide a broad overview, which can be of help to researchers and developers in understanding how advanced sensing can play a role in smart cities.
advanced sensing; sensor networks; smart cities; internet of things
The scientific literature in the area of laser acupuncture is rather large; however, the actual mechanisms and effects have not yet been proven in detail. Since the early days of laser acupuncture, there are still many open questions concerning technical parameters of this innovative technique. In this paper, we report about the most important technical parameters (wavelength, output power, power density, energy density, dose range, and continuous or pulsed laser) for laser acupuncture and present quantitative results for optimal laser stimulation, which allow eliciting reproducible effects in the periphery and in the brain. There are several position statements on laser acupuncture and also several review articles in scientific literature concerning clinical effectiveness of laser acupuncture. For example, the Australian Medical Acupuncture College stated recently that “the optimal energy density for laser acupuncture and biostimulation, based on current clinical experience, is 4 J/cm2”. However, our results of previous research studies and of this paper clearly show that dose must be adjusted according to the individual responses.
Intermittent androgen suppression (IAS) therapy for prostate cancer patients attempts to maintain the hormone dependence of the tumor cells by cycles alternating between androgen suppression (AS) and treatment cessation till a certain prostate-specific antigen (PSA) threshold is reached. Side effects are expected to be reduced, compared to standard continuous androgen suppression (CAS) therapy. The present study examined the effect of IAS on bone metabolism by determinations of serum procollagen I N-terminal peptide (PINP), a biochemical marker of collagen synthesis. A total of 105 treatment cycles of 58 patients with prostate cancer stages ≥pT2 was studied assessing testosterone, PSA and PINP levels at monthly intervals. During phases of AS lasting for up to nine months PSA levels were reversibly reduced, indicating apoptotic regression of the prostatic tumors. Within the first cycle PINP increased at the end of the AS period and peaked in the treatment cessation phase. During the following two cycles a similar pattern was observed for PINP, except a break in collagen synthesis as indicated by low PINP levels in the first months off treatment. Therefore, measurements of the serum PINP concentration indicated increased bone matrix synthesis in response to >6 months of AS, which uninterruptedly continued into the first treatment cessation phase, with a break into each of the following two pauses. In summary, synthesis of bone matrix collagen increases while degradation decreases during off-treatment phases in patients undergoing IAS. Although a direct relationship between bone matrix turnover and risk of fractures is difficult to establish, IAS for treatment of biochemical progression of prostate tumors is expected to reduce osteoporosis in elderly men often at high risk for bone fractures representing a highly suitable patient population for this kind of therapy.
intermittent androgen suppression; prostate cancer; prostate-specific antigen; bone turnover; PINP
A common goal of outdoor augmented reality (AR) is the presentation of annotations that are registered to anchor points in the real world. We present an enhanced approach for registering and tracking such anchor points, which is suitable for current generation mobile phones and can also successfully deal with the wide variety of viewing conditions encountered in real life outdoor use. The approach is based on on-the-fly generation of panoramic images by sweeping the camera over the scene. The panoramas are then used for stable orientation tracking, while the user is performing only rotational movements. This basic approach is improved by several new techniques for the re-detection and tracking of anchor points. For the re-detection, specifically after temporal variations, we first compute a panoramic image with extended dynamic range, which can better represent varying illumination conditions. The panorama is then searched for known anchor points, while orientation tracking continues uninterrupted. We then use information from an internal orientation sensor to prime an active search scheme for the anchor points, which improves matching results. Finally, global consistency is enhanced by statistical estimation of a global rotation that minimizes the overall position error of anchor points when transforming them from the source panorama in which they were created, to the current view represented by a new panorama. Once the anchor points are redetected, we track the user's movement using a novel 3-degree-of-freedom orientation tracking approach that combines vision tracking with the absolute orientation from inertial and magnetic sensors. We tested our system using an AR campus guide as an example application and provide detailed results for our approach using an off-the-shelf smartphone. Results show that the re-detection rate is improved by a factor of 2 compared to previous work and reaches almost 90% for a wide variety of test cases while still keeping the ability to run at interactive frame rates.
► Enhanced approach to register and track annotations' anchor points. ► Panoramic image with extended dynamic range to increase image quality. ► Use of internal orientation sensor to prime an active search for the anchor points. ► Minimizing position error of anchor points by aligning panoramas. ► Tracking using a 3-degree-of-freedom orientation tracking based on sensor fusion.
Augmented reality; Annotation; Tracking; Mobile phone
Age-related gene expression patterns of Homo sapiens as well as of model organisms such as Mus musculus, Saccharomyces cerevisiae, Caenorhabditis elegans and Drosophila melanogaster are a basis for understanding the genetic mechanisms of ageing. For an effective analysis and interpretation of expression profiles it is necessary to store and manage huge amounts of data in an organized way, so that these data can be accessed and processed easily.
GiSAO.db (Genes involved in senescence, apoptosis and oxidative stress database) is a web-based database system for storing and retrieving ageing-related experimental data. Expression data of genes and miRNAs, annotation data like gene identifiers and GO terms, orthologs data and data of follow-up experiments are stored in the database. A user-friendly web application provides access to the stored data. KEGG pathways were incorporated and links to external databases augment the information in GiSAO.db. Search functions facilitate retrieval of data which can also be exported for further processing.
We have developed a centralized database that is very well suited for the management of data for ageing research. The database can be accessed at https://gisao.genome.tugraz.at and all the stored data can be viewed with a guest account.
Reports on clinical measurements of bone mineral density (BMD) in prostate cancer patients undergoing intermittent androgen suppression therapy (IAS) that allows for hormonal recovery between treatment cycles indicate decreased osteoporosis compared to continuous androgen suppression therapy (CAS). In the present study the effect of IAS on bone metabolism by determinations of CrossLaps, a biochemical marker of collagen degradation, were examined.
In total 100 IAS treatment cycles of 75 patients with prostate cancer stages ≥ pT2 were studied. Clinical data and monthly laboratory tests (testosterone, prostate-specific antigen; PSA) of these patients were monitored together with measurements of C-terminal telopeptide collagen fragments using CrossLaps® ELISA assays.
During phases of androgen suppression (AS) lasting for 9 months serum testosterone (<1 ng/mL) and PSA (<2 ng/mL) levels were reversibly reduced, indicating partial growth arrest and apoptotic regression of the prostatic tumors. Serum CrossLaps concentrations peaked at the last 2 months of the AS phases (0.91 ± 0.25 μg/L; mean ± SEM) and were reduced below initial values (0.21 ± 0.43 versus baseline of 0.43 ± 0.06 μg/L) during therapy cessation periods until tumor progression-related increases.
Measurements of the serum concentration of CrossLaps in prostate cancer patients receiving IAS indicated that treatment cessation phases rapidly reversed increased bone degradation associated with AS phases, in strong agreement with the clinical observations reporting reduced loss of BMD in IAS when compared to CAS. In terms of clinical outcomes, IAS seems to be as effective as CAS while showing reduced side effects, as demonstrated here by the reduction of androgen-induced bone matrix degradation.
intermittent androgen suppression; prostate cancer; prostate-specific antigen; testosterone; bone turnover; CrossLaps
AIM: To determine free and conjugated serum bile acid (BA) levels in inflammatory bowel disease (IBD) subgroups with defined clinical manifestations.
METHODS: Comprehensive serum BA profiling was performed in 358 IBD patients and 310 healthy controls by liquid chromatography coupled to electrospray ionization tandem mass spectrometry.
RESULTS: Serum levels of hyodeoxycholic acid, the CYP3A4-mediated detoxification product of the secondary BA lithocholic acid (LCA), was increased significantly in Crohn’s disease (CD) and ulcerative colitis (UC), while most other serum BA species were decreased significantly. Total BA, total BA conjugate, and total BA glycoconjugate levels were decreased only in CD, whereas total unconjugated BA levels were decreased only in UC. In UC patients with hepatobiliary manifestations, the conjugated primary BAs glycocholic acid, taurocholic acid, and glycochenodeoxycholic acid were as significantly increased as the secondary BAs LCA, ursodeoxycholic acid, and tauroursodeoxycholic acid compared to UC patients without hepatobiliary manifestations. Finally, we found that in ileocecal resected CD patients, the unconjugated primary BAs, cholic acid and chenodeoxycholic acid, were increased significantly compared to controls and patients without surgical interventions.
CONCLUSION: Serum BA profiling in IBD patients that indicates impaired intestinal barrier function and increased detoxification is suitable for advanced diagnostic characterization and differentiation of IBD subgroups with defined clinical manifestations.
Bile acids; Liquid chromatography; Tandem mass spectrometry; Inflammatory bowel disease; Crohn’s disease; Ulcerative colitis
Platinum(IV) compounds like oxoplatin (cis, cis, trans-diammine-dichlorido-dihydroxido-platinum(IV)) show increased stability and therefore can be applied orally. In a panel of 38 human cancer cell lines this drug induced S-phase arrest and cell death with IC50 values 2.5-fold higher than cisplatin. Oxoplatin may be converted to cisplatin by intracellular reducing agents, however, exposure to 0.1 M HCl mimicking gastric acid yielded cis-diammine-tetrachlorido-platinum(IV) exhibiting twofold increased activity. Similar results were obtained for another platinum(IV) compound, JM 149 (ammine-dichlorido-(cyclohexylamine)-dihydroxido-platinum(IV)), but not for its parent drug JM 216/satraplatin. Genome-wide expression profiling of H526 small cell lung cancer cells treated with these platinum species revealed clear differences in the expression pattern of affected genes between oxoplatin and cisplatin. In conclusion, oxoplatin constitutes a potent oral agent that is either reduced or converted to distinct active compounds, for example, by gastric acid or acidic areas prevailing in solid tumors, in dependence of the respective pharmaceutical formulation.
AIM: To test the hypothesis that hydrolysis of sphingomyelin to ceramide changes the composition of tight junctions (TJs) with increasing permeability of the intestinal epithelium.
METHODS: Monolayers of Caco-2 cells were used as an in vitro model for the intestinal barrier. Permeability was determined by quantification of transepithelial flux and transepithelial resistance. Sphingolipid-rich membrane microdomains were isolated by a discontinuous sucrose gradient and characterized by Western-blot. Lipid content of microdomains was analysed by tandem mass spectrometry. Ceramide was subcellularly localized by immunofluorescent staining.
RESULTS: Exogenous sphingomyelinase increased transepithelial permeability and decreased transepithelial resistance at concentrations as low as 0.01 U/mL. Lipid analysis showed rapid accumulation of ceramide in the membrane fractions containing occludin and claudin-4, representing TJs. In these fractions we observed a concomitant decrease of sphingomyelin and cholesterol with increasing concentrations of ceramide. Immunofluorescent staining confirmed clustering of ceramide at the sites of cell-cell contacts. Neutralization of surface ceramide prevented the permeability-increase induced by platelet activating factor.
CONCLUSION: Our findings indicate that changes in lipid composition of TJs impair epithelial barrier functions. Generation of ceramide by sphingomyelinases might contribute to disturbed barrier function seen in diseases such as inflammatory, infectious, toxic or radiogenic bowel disease.
Ceramide; Cholesterol; Tight-junction; Caco-2 cells; Permeability; Inflammatory bowel disease
12R-lipoxygenase (12R-LOX) and the epidermal LOX-3 (eLOX-3) constitute a novel LOX pathway involved in terminal differentiation in skin. This view is supported by recent studies showing that inactivating mutations in 12R-LOX and eLOX-3 are linked to the development of autosomal recessive congenital ichthyosis. We show that 12R-LOX deficiency in mice results in a severe impairment of skin barrier function. Loss of barrier function occurs without alterations in proliferation and stratified organization of the keratinocytes, but is associated with ultrastructural anomalies in the upper granular layer, suggesting perturbance of the assembly/extrusion of lamellar bodies. Cornified envelopes from skin of 12R-LOX–deficient mice show increased fragility. Lipid analysis demonstrates a disordered composition of ceramides, in particular a decrease of ester-bound ceramide species. Moreover, processing of profilaggrin to monomeric filaggrin is impaired.
This study indicates that the 12R-LOX–eLOX-3 pathway plays a key role in the process of epidermal barrier acquisition by affecting lipid metabolism, as well as protein processing.
During August 2006, a protracted outbreak of Salmonella (S.) Enteritidis infections in a large Hamburg nursing home was investigated.
A site visit of the home was conducted and food suppliers' premises tested for Salmonella. Among nursing home residents a cohort study was carried out focusing on foods consumed in the three days before the first part of the outbreak. Instead of relying on residents' memory, data from the home's patient food ordering system was used as exposure data. S. Enteritidis isolates from patients and suspected food vehicles were phage typed and compared.
Within a population of 822 nursing home residents, 94 case patients among residents (1 fatality) and 17 among staff members were counted 6 through 29 August. The outbreak peaked 7 through 9 August, two days after a spell of very warm summer weather. S. Enteritidis was consistently recovered from patients' stools throughout the outbreak. Among the food items served during 5 through 7 August, the cohort study pointed to afternoon cake on all three days as potential risk factors for disease. Investigation of the bakery supplying the cake yielded S. Enteritidis from cakes sampled 31 August. Comparison of the isolates by phage typing demonstrated both isolates from patients and the cake to be the exceedingly rare phage type 21c.
Cake (various types served on various days) contaminated with S. Enteritidis were the likely vehicle of the outbreak in the nursing home. While the cakes were probably contaminated with low pathogen dose throughout the outbreak period, high ambient summer temperatures and failure to keep the cake refrigerated led to high pathogen dose in cake on some days and in some of the housing units. This would explain the initial peak of cases, but also the drawn out nature of the outbreak with cases until the end of August. Suggestions are made to nursing homes, aiding in outbreak prevention. Early outbreak detection is crucial, such that counter measures can be swift and drawn-out outbreaks of nosocomial food-borne infections avoided.
In the search for novel bioactive compounds from sponge-derived microorganisms, we have recently identified two structurally and biosynthetically unprecedented fungal metabolites, the novel-type alkaloids sorbicillactone A and sorbicillactone B. Sorbicillactone A is active against leukemia cells without showing notable cytotoxicity. Therefore, we have developed an efficient process for its biotechnological production and isolation on a large scale supplying sufficient material for the ongoing preclinical investigations and structure-activity relationship (SAR) studies.
sorbicillactone A; anti-leukemic activity; large-scale production; BIOTECmarin
It is commonly accepted that embryonic segmentation of vertebrates is regulated by a segmentation clock, which is induced by the cycling genes Hes1 and Hes7. Their products form dimers that bind to the regulatory regions and thereby repress the transcription of their own encoding genes. An increase of the half-life of Hes7 protein causes irregular somite formation. This was shown in recent experiments by Hirata et al. In the same work, numerical simulations from a delay differential equations model, originally invented by Lewis, gave additional support. For a longer half-life of the Hes7 protein, these simulations exhibited strongly damped oscillations with, after few periods, severely attenuated the amplitudes. In these simulations, the Hill coefficient, a crucial model parameter, was set to 2 indicating that Hes7 has only one binding site in its promoter. On the other hand, Bessho et al. established three regulatory elements in the promoter region.
We show that – with the same half life – the delay system is highly sensitive to changes in the Hill coefficient. A small increase changes the qualitative behaviour of the solutions drastically. There is sustained oscillation and hence the model can no longer explain the disruption of the segmentation clock. On the other hand, the Hill coefficient is correlated with the number of active binding sites, and with the way in which dimers bind to them. In this paper, we adopt response functions in order to estimate Hill coefficients for a variable number of active binding sites. It turns out that three active transcription factor binding sites increase the Hill coefficient by at least 20% as compared to one single active site.
Our findings lead to the following crucial dichotomy: either Hirata's model is correct for the Hes7 oscillator, in which case at most two binding sites are active in its promoter region; or at least three binding sites are active, in which case Hirata's delay system does not explain the experimental results. Recent experiments by Chen et al. seem to support the former hypothesis, but the discussion is still open.
The quantification of single nucleotide polymorphism (SNP) allele frequencies in pooled DNA samples using real time PCR is a promising approach for large-scale diagnostics and genotyping. The limits of detection (LOD) and limits of quantification (LOQ) for mutant SNP alleles are of particular importance for determination of the working range, which, in the case of allele-specific real time PCR, can be limited by the variance of calibration data from serially diluted mutant allele samples as well as by the variance of the 100% wild-type allele samples (blank values). In this study, 3σ and 10σ criteria were applied for the calculation of LOD and LOQ values. Alternatively, LOQ was derived from a 20% threshold for the relative standard deviation (%RSD) of measurements by fitting a curve for the relationship between %RSD and copy numbers of the mutant alleles. We found that detection and quantification of mutant alleles were exclusively limited by the variance of calibration data since the estimated LODcalibration (696 in 30 000 000 copies, 0.0023%), LOQ20%RSD (1470, 0.0049%) and LOQcalibration (2319, 0.0077) values were significantly higher than the LODblank (130, 0.0004%) and LOQblank (265, 0.0009%) values derived from measurements of wild-type allele samples. No significant matrix effects of the genomic background DNA on the estimation of LOD and LOQ were found. Furthermore, the impact of large genome sizes and the general application of the procedure for the estimation of LOD and LOQ in quantitative real time PCR diagnostics are discussed.
Using the standing droplet technique in the renal proximal convolution and simultaneous microperfusion of the peritubular capillaries, the zero net flux transtubular concentration difference of taurocholate (ΔCTC−) at 45 s was determined as a measure of active bile acid reabsorption in vivo. Starting with 0.1 mmol/liter taurocholate in both perfusates the control ΔCTC− of 0.042 mmol/liter fell to 0.006 mmol/liter (P < 0.001) when the Na+ concentration in the perfusates was reduced to zero. Removal of bicarbonate from the perfusates to alter pH had no influence on ΔCTC−. When glycocholate was added to the perfusates ΔCTC− was decreased, while probenecid increased ΔCTC−.
These observations were extended by studies performed with brush border membrane vesicles derived from renal cortex. The initial (20 s) uptake of 0.01 mmol/liter taurocholate in the presence of a Nao+ > Nai+ gradient was stimulated twofold compared with its uptake in the absence of a Na+ gradient. Uptake of taurocholate was osmotically and temperature sensitive. Membranes preloaded with unlabeled glycocholate showed accelerated entry of labeled taurocholate (trans-stimulation) only in the presence of Na+. Replacement of Na+ in the media with K+, Li+, and choline+ decreased initial taurocholate uptake by 49, 53, and 62%, respectively. Stimulation of taurocholate transport by cation gradient diffusion potentials was unlikely inasmuch as the addition of valinomycin under K+ gradient conditions had no effect. A transmembrane pH gradient (pHo < pHi) did not influence initial uptake of taurocholate. Finally, in the presence of Na+ taurocholate transport showed cis-inhibition with unlabeled bile acids and saturation kinetics with respect to increasing taurocholate concentrations. The micropuncture and vesicle data indicate that the net transport of taurocholate in the proximal tubule is the result of an electroneutral Na+-taurocholate cotransport across the brush border membrane.