Multiple recurrent de novo CNVs, including duplications of the 7q11.23 Williams syndrome region, are strongly associated with autism

We have undertaken a genome-wide analysis of rare copy-number variation (CNV) in 1124 autism spectrum disorder (ASD) families, each comprised of a single proband, unaffected parents, and, in most kindreds, an unaffected sibling. We find significant association of ASD with de novo duplications of 7q11.23, where the reciprocal deletion causes Williams-Beuren syndrome, characterized by a highly social personality. We identify rare recurrent de novo CNVs at five additional regions, including 16p13.2 (encompassing genes USP7 and C16orf72) and Cadherin 13, and implement a rigorous approach to evaluating the statistical significance of these observations. Overall, large de novo CNVs, particularly those encompassing multiple genes, confer substantial risks (OR = 5.6; CI = 2.6-12.0, p = 2.4 × 10(-7)). We estimate there are 130-234 ASD-related CNV regions in the human genome and present compelling evidence, based on cumulative data, for association of rare de novo events at 7q11.23, 15q11.2-13.1, 16p11.2, and Neurexin 1.     

Adverse Drug Events in Older Hospitalized Patients: Results and Reliability of a Comprehensive and Structured Identification Strategy

Background

Older patients are at high risk for experiencing Adverse Drug Events (ADEs) during hospitalization. To be able to reduce ADEs in these vulnerable patients, hospitals first need to measure the occurrence of ADEs, especially those that are preventable. However, data on preventable ADEs (pADEs) occurring during hospitalization in older patients are scarce, and no ‘gold standard’ for the identification of ADEs exists.

Methodology

The study was conducted in three hospitals in the Netherlands in 2007. ADEs were retrospectively identified by a team of experts using a comprehensive and structured patient chart review (PCR) combined with a trigger-tool as an aid. This ADE identification strategy was applied to a cohort of 250 older hospitalized patients. To estimate the intra- and inter-rater reliabilities, Cohen’s kappa values were calculated.

Principal Findings

In total, 118 ADEs were detected which occurred in 62 patients. This ADE yield was 1.1 to 2.7 times higher in comparison to other ADE studies in older hospitalized patients. Of the 118 ADEs, 83 (70.3%) were pADEs; 51 pADEs (43.2% of all ADEs identified) caused serious patient harm. Patient harm caused by ADEs resulted in various events. The overall intra-rater agreement of the developed strategy was substantial (κ = 0.74); the overall inter-rater agreement was only fair (κ = 0.24).

Conclusions/Significance

The ADE identification strategy provided a detailed insight into the scope of ADEs occurring in older hospitalized patients, and showed that the majority of (serious) ADEs can be prevented. Several strategy related aspects, as well as setting/study specific aspects, may have contributed to the results gained. These aspects should be considered whenever ADE measurements need to be conducted. The results regarding pADEs can be used to design tailored interventions to effectively reduce harm caused by medication errors. Improvement of the inter-rater reliability of a PCR remains challenging.     

Inhibition of complex I of the electron transport chain causes O2-{middle dot}-mediated mitochondrial outgrowth

Recent evidence indicates that oxidative stress is central to the pathogenesis of a wide variety of degenerative diseases, aging, and cancer. Oxidative stress occurs when the delicate balance between production and detoxification of reactive oxygen species is disturbed. Mammalian cells respond to this condition in several ways, among which is a change in mitochondrial morphology. In the present study, we have used rotenone, an inhibitor of complex I of the respiratory chain, which is thought to increase mitochondrial O₂^–· production, and mitoquinone (MitoQ), a mitochondria-targeted antioxidant, to investigate the relationship between mitochondrial O₂^–· production and morphology in human skin fibroblasts. Video-rate confocal microscopy of cells pulse loaded with the mitochondria-specific cation rhodamine 123, followed by automated analysis of mitochondrial morphology, revealed that chronic rotenone treatment (100 nM, 72 h) significantly increased mitochondrial length and branching without changing the number of mitochondria per cell. In addition, this treatment caused a twofold increase in lipid peroxidation as determined with C11-BODIPY^581/591. Finally, digital imaging microscopy of cells loaded with hydroethidine, which is oxidized by O₂^–· to yield fluorescent ethidium, revealed that chronic rotenone treatment caused a twofold increase in the rate of O₂^–· production. MitoQ (10 nM, 72 h) did not interfere with rotenone-induced ethidium formation but abolished rotenone-induced outgrowth and lipid peroxidation. These findings show that increased mitochondrial O₂^–· production as a consequence of, for instance, complex I inhibition leads to mitochondrial outgrowth and that MitoQ acts downstream of this O₂^–· to prevent alterations in mitochondrial morphology. rhodamine 123; video-rate confocal microscopy; superoxide; MitoQ     

Hyperuricemia influences tryptophan metabolism via inhibition of multidrug resistance protein 4 (MRP4) and breast cancer resistance protein (BCRP)

Hyperuricemia is related to a variety of pathologies, including chronic kidney disease (CKD). However, the pathophysiological mechanisms underlying disease development are not yet fully elucidated. Here, we studied the effect of hyperuricemia on tryptophan metabolism and the potential role herein of two important uric acid efflux transporters, multidrug resistance protein 4 (MRP4) and breast cancer resistance protein (BCRP). Hyperuricemia was induced in mice by treatment with the uricase inhibitor oxonic acid, confirmed by the presence of urate crystals in the urine of treated animals. A transport assay, using membrane vesicles of cells overexpressing the transporters, revealed that uric acid inhibited substrate-specific transport by BCRP at clinically relevant concentrations (calculated IC₅₀ value: 365 ± 13 μM), as was previously reported for MRP4. Moreover, we identified kynurenic acid as a novel substrate for MRP4 and BCRP. This finding was corroborated by increased plasma levels of kynurenic acid observed in Mrp4^?/? (107 ± 19 nM; P = 0.145) and Bcrp^?/? mice (133 ± 10 nM; P = 0.0007) compared to wild type animals (71 ± 11 nM). Hyperuricemia was associated with > 1.5 fold increase in plasma kynurenine levels in all strains. Moreover, hyperuricemia led to elevated plasma kynurenic acid levels (128 ± 13 nM, P = 0.005) in wild type mice but did not further increase kynurenic acid levels in knockout mice. Based on our results, we postulate that elevated uric acid levels hamper MRP4 and BCRP functioning, thereby promoting the retention of other potentially toxic substrates, including kynurenic acid, which could contribute to the development of CKD.     

Paraoxonase Genotype,LDL-oxidation and Carotid Atherosclerosis in Male Life-long Smokers

Paraoxonase (PON-1) is a high-density lipoprotein (HDL) associated enzyme that hydrolyzes lipid peroxides in vitro, which may therefore protect against the onset of atherosclerosis. Heavy smokers are more exposed to oxidative stress and hence at high-risk for oxidative modification of LDL.

Our hypothesis is that the anti-oxidative properties of PON-1 inhibit LDL oxidation, especially in populations exposed to high oxidative stress.

We have studied the effects of PON-1 genotype and smoking to variation in oxidative status parameters and intima-media thickness (IMT), a surrogate marker of atherosclerosis. The contribution of two common polymorphisms in the PON-1 gene (Q192R and L55M) to LDL oxidizability, autoantibodies directed against oxLDL and IMT were studied in 207 male life-long smokers. Smokers were classified into average, heavy and excessive smokers based on pack years of cigarettes smoked.

PON-1 genotype was not associated with autoantibodies to oxLDL, LDL oxidizability or IMT. Smoking was associated with IMT in subgroups with the high levels of LDL, but not in the population at large.

The lack of association of PON-1 genotype with oxidative status parameters and IMT suggests that PON-1 is not a major inhibitor of LDL oxidation in a population of life-long smokers.     

A dynamic phosphate budget model for a eutrophic lake

The relations between the external nutrient loading of lakes, recycling through sediments and the resulting productivity are complicated by feed-back mechanisms, seasonal variations and trends. Simulation is a useful tool for the identification of controlling factors and the assessment of the effects of management measures, supplementary to experimental research. The model variables in our dynamic phosphate budget model include inorganic and organic particulate phosphate and dissolved o-phosphate, in both sediments and overlying water. Sediments may be aerobic or anaerobic, depending on topography, temperature and composition. The major processes described are primary production, mineralisation, sedimentation, adsorption and diffusion. Several model parameters have been estimated directly for Lake Brielle (Netherlands). The sediment dilution rate, the extent of anaerobic conditions and the number and character of adsorption sites are important controlling factors.     

The Effect of the Interleukin‐1 Cytokine Family Members IL‐1F6 and IL‐1F8 on Adipocyte Differentiation

Obesity is characterized by chronic low‐grade inflammation originating from expanding adipose tissue. In the present study, we examined the adipogenic expression levels of IL‐1F6 and IL‐1F8, both members of the IL‐1 family of cytokines, and their effects on adipose tissue gene expression. Although IL‐1F6 is primarily present in adipose tissue resident macrophages and induced by inflammation, IL‐1F8 is absent. IL‐1F6, but not IL‐1F8, reduces adipocyte differentiation, as shown by a significant decrease in PPARγ gene expression. Finally, both IL‐1F6 and IL‐1F8 are able to induce inflammatory gene expression in mature adipocytes. In conclusion, we demonstrate for the first time that IL‐1F6 is present in adipose tissue and that IL‐1F6 and IL‐1F8 are involved in the regulation of adipose tissue gene expression. Importantly, IL‐1F6 inhibits PPARγ expression which may lead to reduced adipocyte differentiation suggesting metabolic effects of this cytokine.     

Increasing the transglycosylation activity of alpha-galactosidase from Bifidobacterium adolescentis DSM 20083 by site-directed mutagenesis

The alpha-galactosidase (AGA) from Bifidobacterium adolescentis DSM 20083 has a high transglycosylation activity. The optimal conditions for this activity are pH 8, and 37 degrees C. At high melibiose concentration (600 mM), approximately 64% of the enzyme-substrate encounters resulted in transglycosylation. Examination of the acceptor specificity showed that AGA required a hydroxyl group at C-6 for transglycosylation. Pentoses, hexuronic acids, deoxyhexoses, and alditols did not serve as acceptor molecules. Disaccharides were found to be good acceptors. A putative 3D-structure of the catalytic site of AGA was obtained by homology modeling. Based on this structure and amino acid sequence alignments, site-directed mutagenesis was performed to increase the transglycosylation efficiency of the enzyme, which resulted in four positive mutants. The positive single mutations were combined, resulting in six double mutants. The mutant H497M had an increase in transglycosylation of 16%, whereas most of the single mutations showed an increase of 2%-5% compared to the wild-type AGA. The double mutants G382C-Y500L, and H497M-Y500L had an increase in transglycosylation activity of 10%-16%, compared to the wild-type enzyme, whereas the increase for the other double mutants was low (4%-7%). The results show that with a single mutation (H497M) the transglycosylation efficiency can be increased from 64% to 75% of all enzyme-substrate encounters. Combining successful single mutants in double mutations did not necessarily result in an extra increase in transglycosylation efficiency. The donor and acceptor specificity did not change in the mutants, whereas the thermostability of the mutants with G382C decreased drastically.     

Structural rearrangements of sucrose phosphorylase from Bifidobacterium adolescentis during sucrose conversion

The reaction mechanism of sucrose phosphorylase from Bifidobacterium adolescentis (BiSP) was studied by site-directed mutagenesis and x-ray crystallography. An inactive mutant of BiSP (E232Q) was co-crystallized with sucrose. The structure revealed a substrate-binding mode comparable with that seen in other related sucrose-acting enzymes. Wild-type BiSP was also crystallized in the presence of sucrose. In the dimeric structure, a covalent glucosyl intermediate was formed in one molecule of the BiSP dimer, and after hydrolysis of the glucosyl intermediate, a beta-D-glucose product complex was formed in the other molecule. Although the overall structure of the BiSP-glucosyl intermediate complex is similar to that of the BiSP(E232Q)-sucrose complex, the glucose complex discloses major differences in loop conformations. Two loops (residues 336-344 and 132-137) in the proximity of the active site move up to 16 and 4 A, respectively. On the basis of these findings, we have suggested a reaction cycle that takes into account the large movements in the active-site entrance loops.     

Synthesis of heparosan oligosaccharides by Pasteurella multocida PmHS2 single-action transferases

Pasteurella multocida heparosan synthase PmHS2 is a dual action glycosyltransferase that catalyzes the polymerization of heparosan polymers in a non-processive manner. The two PmHS2 single-action transferases, obtained previously by site-directed mutagenesis, have been immobilized on Ni(II)-nitrilotriacetic acid agarose during the purification step. A detailed study of the polymerization process in the presence of non-equal amounts of PmHS2 single-action transferases revealed that the glucuronyl transferase (PmHS2-GlcUA(+)) is the limiting catalyst in the polymerization process. Using experimental design, it was determined that the N-acetylglucosaminyl transferase (PmHS2-GlcNAc(+)) plays an important role in the control of heparosan chain elongation depending on the number of heparosan chains and the UDP-sugar concentrations present in the reaction mixture. Furthermore, for the first time, the synthesis of heparosan oligosaccharides alternately using PmHS2-GlcUA(+) and PmHS2-GlcNAc(+) is reported. It was shown that the synthesis of heparosan oligosaccharides by PmHS2 single-action transferases do not require the presence of template molecules in the reaction mixture.