The variability of urinary cotinine levels in young children: implications for measuring ETS exposure.

This study examined the within-subject variability of urinary cotinine levels in young children (aged = 0.6-7.2 years) of smoking parents to determine the number of urine samples needed to provide accurate estimates of exposure to environmental tobacco smoke (ETS) for different time intervals. Secondary analyses were conducted of five independent studies (N = 376), in which multiple urinary cotinine measures had been collected over time periods up to 13 months. Over measurement periods of 4-15 days, the within-subject cotinine levels varied 3-5 times more than would be expected based on measurement error alone. Over 7-13 months, the within-subject variability was 10-20 times higher than would be expected based on the measurement error. Findings indicated that cotinine measures from single urine samples provided highly accurate estimates of only recent exposure (i.e., 2-3 days; rho = 0.99). To achieve similarly precise estimates of the mean cotinine level of an individual child over 4-15 days, up to nine urine samples may be necessary. Up to 12 urine samples may be required to achieve similarly precise estimates of ETS exposure over a 4- to 13-month period. Epidemiologic and clinical research on ETS exposure in children can benefit from multiple urine samples (a) to accurately measure average exposure at the level of the individual child, (b) to describe temporal patterns, (c) to detect incidences of peak exposure that would remain underrecognized if monitoring is limited to a single time point, and (d) to establish stable baseline levels and endpoints based on urine samples collected over clinically relevant time periods.     

Response intensity in content-specific fear conditioning comparing 20% versus 13% CO?-enriched air as unconditioned stimuli.

This study examined the relation between the intensity of CO?-induced psychophysiological responses and content-specific fear conditioning. Sex-balanced groups of undergraduates (N?=?96) were assigned to 1 of 3 conditioned stimuli (CSs) differing in fear-relevance, and within each CS, to either 20% or 13% CO?-enriched air (unconditioned stimuli [UCS]). Several psychophysiological measures were assessed before, during, and following conditioning phases. Consistent with expectation, electrodermal and cardiac conditioned responses were larger and more resistant to extinction when associated with fear-relevant compared with fear-irrelevant stimuli, and this overall effect of fear-relevance was more robust to the more intense UCS. Severity and frequency of DSM-IV (Diagnostic and Statistical Manual of Mental Disorders, 4th ed ) panic symptoms also varied reliably with UCS intensity, and women reported more distress and symptoms than men. Overall, the findings suggest that content-specific fear conditioning is mediated, in part, by the intensity of the bodily response. The authors discuss clinical and theoretical implications for understanding fear onset in the absence of obvious environmental pain or trauma. (PsycINFO Database Record (c) 2010 APA, all rights reserved)     

Iterative multi-party agreement negotiation for establishing collaborations

Inter-domain collaborations suffer not only from technological obstacles that hinder interoperability, but also from diverting business objectives of the involved domains. Today, hand-crafted contracts define the terms and conditions for service interactions. Electronic negotiation can serve as the enabler of dynamic interdomain collaborations by providing a large degree of freedom for the automation of agreement formation and electronic contracting. Negotiation by electronic means has been an area of intensive research for many years now. However, most effort was put on the determination of prices and neglected that real-world agreements also consist of complex dependencies of interdependent obligations. In this paper we present agreement negotiation as a tool to establish ad hoc services collaborations. Our novel protocol allows for the discovery of complex agreement options and for the formation of multi-party agreements. The protocol works through an iterative exchange of requirements and offers. A major benefit over existing bilateral negotiation protocols is that our protocol is capable of discovering potential collaborations between different parties. It will leave each negotiating party with a complete agreement document after a successful negotiation. This comprehensive agreement document defines the interdependent obligations between all parties and is well suited for E-Contracting.     

Refining Genotypes and Phenotypes in KCNA2-Related Neurological Disorders

Pathogenic variants in KCNA2, encoding for the voltage-gated potassium channel K_v1.2, have been identified as the cause for an evolving spectrum of neurological disorders. Affected individuals show early-onset developmental and epileptic encephalopathy, intellectual disability, and movement disorders resulting from cerebellar dysfunction. In addition, individuals with a milder course of epilepsy, complicated hereditary spastic paraplegia, and episodic ataxia have been reported. By analyzing phenotypic, functional, and genetic data from published reports and novel cases, we refine and further delineate phenotypic as well as functional subgroups of KCNA2-associated disorders. Carriers of variants, leading to complex and mixed channel dysfunction that are associated with a gain- and loss-of-potassium conductance, more often show early developmental abnormalities and an earlier onset of epilepsy compared to individuals with variants resulting in loss- or gain-of-function. We describe seven additional individuals harboring three known and the novel KCNA2 variants p.(Pro407Ala) and p.(Tyr417Cys). The location of variants reported here highlights the importance of the proline(405)–valine(406)–proline(407) (PVP) motif in transmembrane domain S6 as a mutational hotspot. A novel case of self-limited infantile seizures suggests a continuous clinical spectrum of KCNA2-related disorders. Our study provides further insights into the clinical spectrum, genotype–phenotype correlation, variability, and predicted functional impact of KCNA2 variants.     

Experimental and Established Oximes as Pretreatment before Acute Exposure to Azinphos-Methyl

Poisoning with organophosphorus compounds (OPCs) represents an ongoing threat to civilians and rescue personal. We have previously shown that oximes, when administered prophylactically before exposure to the OPC paraoxon, are able to protect from its toxic effects. In the present study, we have assessed to what degree experimental (K-27; K-48; K-53; K-74; K-75) or established oximes (pralidoxime, obidoxime), when given as pretreatment at an equitoxic dosage of 25% of LD₀₁, are able to reduce mortality induced by the OPC azinphos-methyl. Their efficacy was compared with that of pyridostigmine, the only FDA-approved substance for such prophylaxis. Efficacy was quantified in rats by Cox analysis, calculating the relative risk of death (RR), with RR=1 for the reference group given only azinphos-methyl, but no prophylaxis. All tested compounds significantly (p ≤ 0.05) reduced azinphos-methyl-induced mortality. In addition, the efficacy of all tested experimental and established oximes except K-53 was significantly superior to the FDA-approved compound pyridostigmine. Best protection was observed for the oximes K-48 (RR = 0.20), K-27 (RR = 0.23), and obidoxime (RR = 0.21), which were significantly more efficacious than pralidoxime and pyridostigmine. The second-best group of prophylactic compounds consisted of K-74 (RR = 0.26), K-75 (RR = 0.35) and pralidoxime (RR = 0.37), which were significantly more efficacious than pyridostigmine. Pretreatment with K-53 (RR = 0.37) and pyridostigmine (RR = 0.52) was the least efficacious. Our present data, together with previous results on other OPCs, indicate that the experimental oximes K-27 and K-48 are very promising pretreatment compounds. When penetration into the brain is undesirable, obidoxime is the most efficacious prophylactic agent already approved for clinical use.     

MS Binding Assays for Glycine Transporter 2 (GlyT2) Employing Org25543 as Reporter Ligand

This study describes the first binding assay for glycine transporter 2 (GlyT2) following the concept of MS Binding Assays. The selective GlyT2 inhibitor Org25543 was employed as a reporter ligand and it was quantified with a highly sensitive and rapid LC-ESI-MS/MS method. Binding of Org25543 at GlyT2 was characterized in kinetic and saturation experiments with an off-rate of 7.07×10⁻³ s⁻¹, an on-rate of 1.01×10⁶ M⁻¹ s⁻¹, and an equilibrium dissociation constant of 7.45 nM. Furthermore, the inhibitory constants of 19 GlyT ligands were determined in competition experiments. The validity of the GlyT2 affinities determined with the binding assay was examined by a comparison with published inhibitory potencies from various functional assays. With the capability for affinity determination towards GlyT2 the developed MS Binding Assays provide the first tool for affinity profiling of potential ligands and it represents a valuable new alternative to functional assays addressing GlyT2.     

Elucidation of the Clustered Nano-Architecture of Radiation-Induced DNA Damage Sites and Surrounding Chromatin in Cancer Cells: A Single Molecule Localization Microscopy Approach

In cancer therapy, the application of (fractionated) harsh radiation treatment is state of the art for many types of tumors. However, ionizing radiation is a “double-edged sword”—it can kill the tumor but can also promote the selection of radioresistant tumor cell clones or even initiate carcinogenesis in the normal irradiated tissue. Individualized radiotherapy would reduce these risks and boost the treatment, but its development requires a deep understanding of DNA damage and repair processes and the corresponding control mechanisms. DNA double strand breaks (DSBs) and their repair play a critical role in the cellular response to radiation. In previous years, it has become apparent that, beyond genetic and epigenetic determinants, the structural aspects of damaged chromatin (i.e., not only of DSBs themselves but also of the whole damage-surrounding chromatin domains) form another layer of complex DSB regulation. In the present article, we summarize the application of super-resolution single molecule localization microscopy (SMLM) for investigations of these structural aspects with emphasis on the relationship between the nano-architecture of radiation-induced repair foci (IRIFs), represented here by γH2AX foci, and their chromatin environment. Using irradiated HeLa cell cultures as an example, we show repair-dependent rearrangements of damaged chromatin and analyze the architecture of γH2AX repair clusters according to topological similarities. Although HeLa cells are known to have highly aberrant genomes, the topological similarity of γH2AX was high, indicating a functional, presumptively genome type-independent relevance of structural aspects in DSB repair. Remarkably, nano-scaled chromatin rearrangements during repair depended both on the chromatin domain type and the treatment. Based on these results, we demonstrate how the nano-architecture and topology of IRIFs and chromatin can be determined, point to the methodological relevance of SMLM, and discuss the consequences of the observed phenomena for the DSB repair network regulation or, for instance, radiation treatment outcomes.     

Liver Lipids of Patients with Hepatitis B and C and Associated Hepatocellular Carcinoma

Hepatocellular carcinoma (HCC) still remains a difficult to cure malignancy. In recent years, the focus has shifted to lipid metabolism for the treatment of HCC. Very little is known about hepatitis B virus (HBV) and C virus (HCV)-related hepatic lipid disturbances in non-malignant and cancer tissues. The present study showed that triacylglycerol and cholesterol concentrations were similar in tumor adjacent HBV and HCV liver, and were not induced in the HCC tissues. Higher levels of free cholesterol, polyunsaturated phospholipids and diacylglycerol species were noted in non-tumorous HBV compared to HCV liver. Moreover, polyunsaturated phospholipids and diacylglycerols, and ceramides declined in tumors of HBV infected patients. All of these lipids remained unchanged in HCV-related HCC. In HCV tumors, polyunsaturated phosphatidylinositol levels were even induced. There were no associations of these lipid classes in non-tumor tissues with hepatic inflammation and fibrosis scores. Moreover, these lipids did not correlate with tumor grade or T-stage in HCC tissues. Lipid reprogramming of the three analysed HBV/HCV related tumors mostly resembled HBV-HCC. Indeed, lipid composition of non-tumorous HCV tissue, HCV tumors, HBV tumors and HBV/HCV tumors was highly similar. The tumor suppressor protein p53 regulates lipid metabolism. The p53 and p53S392 protein levels were induced in the tumors of HBV, HCV and double infected patients, and this was significant in HBV infection. Negative correlation of tumor p53 protein with free cholesterol indicates a role of p53 in cholesterol metabolism. In summary, the current study suggests that therapeutic strategies to target lipid metabolism in chronic viral hepatitis and associated cancers have to consider disease etiology.     

An Engineered sgsh Mutant Zebrafish Recapitulates Molecular and Behavioural Pathobiology of Sanfilippo Syndrome A/MPS IIIA

Mucopolysaccharidosis IIIA (MPS IIIA, Sanfilippo syndrome type A), a paediatric neurological lysosomal storage disease, is caused by impaired function of the enzyme N-sulfoglucosamine sulfohydrolase (SGSH) resulting in impaired catabolism of heparan sulfate glycosaminoglycan (HS GAG) and its accumulation in tissues. MPS IIIA represents a significant proportion of childhood dementias. This condition generally leads to patient death in the teenage years, yet no effective therapy exists for MPS IIIA and a complete understanding of the mechanisms of MPS IIIA pathogenesis is lacking. Here, we employ targeted CRISPR/Cas9 mutagenesis to generate a model of MPS IIIA in the zebrafish, a model organism with strong genetic tractability and amenity for high-throughput screening. The sgsh^Δex5−6 zebrafish mutant exhibits a complete absence of Sgsh enzymatic activity, leading to progressive accumulation of HS degradation products with age. sgsh^Δex5−6 zebrafish faithfully recapitulate diverse CNS-specific features of MPS IIIA, including neuronal lysosomal overabundance, complex behavioural phenotypes, and profound, lifelong neuroinflammation. We further demonstrate that neuroinflammation in sgsh^Δex5−6 zebrafish is largely dependent on interleukin-1β and can be attenuated via the pharmacological inhibition of Caspase-1, which partially rescues behavioural abnormalities in sgsh^Δex5−6 mutant larvae in a context-dependent manner. We expect the sgsh^Δex5−6 zebrafish mutant to be a valuable resource in gaining a better understanding of MPS IIIA pathobiology towards the development of timely and effective therapeutic interventions.