The contribution of CLIP2 haploinsufficiency to the clinical manifestations of the Williams-Beuren syndrome

Williams-Beuren syndrome is a rare contiguous gene syndrome, characterized by intellectual disability, facial dysmorphisms, connective-tissue abnormalities, cardiac defects, structural brain abnormalities, and transient infantile hypercalcemia. Genes lying telomeric to RFC2, including CLIP2, GTF2I and GTF2IRD1, are currently thought to be the most likely major contributors to the typical Williams syndrome cognitive profile, characterized by a better-than-expected auditory rote-memory ability, a relative sparing of language capabilities, and a severe visual-spatial constructive impairment. Atypical deletions in the region have helped to establish genotype-phenotype correlations. So far, however, hardly any deletions affecting only a single gene in the disease region have been described. We present here two healthy siblings with a pure, hemizygous deletion of CLIP2. A putative role in the cognitive and behavioral abnormalities seen in Williams-Beuren patients has been suggested for this gene on the basis of observations in a knock-out mouse model. The presented siblings did not show any of the clinical features associated with the syndrome. Cognitive testing showed an average IQ for both and no indication of the Williams syndrome cognitive profile. This shows that CLIP2 haploinsufficiency by itself does not lead to the physical or cognitive characteristics of the Williams-Beuren syndrome, nor does it lead to the Williams syndrome cognitive profile. Although contribution of CLIP2 to the phenotype cannot be excluded when it is deleted in combination with other genes, our results support the hypothesis that GTF2IRD1 and GTF2I are the main genes causing the cognitive defects associated with Williams-Beuren syndrome.     

Genetic evidence for population expansion in Hydrotaea irritans (Fallèn) (Diptera: Muscidae)

Allozyme variation in the sheep headfly Hydrotaea irritans was studied on two spatial scales. Geographic variation among seven Danish and one Dutch population revealed significant but rather low genetic differentiation with F _ST = 0.01 over all loci. The Dutch population was on average not more different from the Danish populations than the Danish populations from each other. Allele frequencies were very skewed with the most common allele always exceeding 0.85 and usually 0.9 in frequency, but with many rare alleles at some loci. Tests for neutrality of the variation at the nine polymorphic loci revealed highly significant deviations from expected homozygosity in this species, which was not found in a comparative analysis of allozyme variation at similar loci of seven other Hydrotaea species. To explain the peculiar observed pattern of allozyme variation in H. irritans , it is suggested that this species has successfully expanded its range and spread through northern and central Europe in the recent past. Alternatively, H. irritans may have recently invaded a new niche, resulting in increased abundance of the species and subsequent dispersal to former areas of the species distribution.     

Genome-wide copy number profiling of single cells in S-phase reveals DNA-replication domains

Single-cell genomics is revolutionizing basic genome research and clinical genetic diagnosis. However, none of the current research or clinical methods for single-cell analysis distinguishes between the analysis of a cell in G1-, S- or G2/M-phase of the cell cycle. Here, we demonstrate by means of array comparative genomic hybridization that charting the DNA copy number landscape of a cell in S-phase requires conceptually different approaches to that of a cell in G1- or G2/M-phase. Remarkably, despite single-cell whole-genome amplification artifacts, the log2 intensity ratios of single S-phase cells oscillate according to early and late replication domains, which in turn leads to the detection of significantly more DNA imbalances when compared with a cell in G1- or G2/M-phase. Although these DNA imbalances may, on the one hand, be falsely interpreted as genuine structural aberrations in the S-phase cell’s copy number profile and hence lead to misdiagnosis, on the other hand, the ability to detect replication domains genome wide in one cell has important applications in DNA-replication research. Genome-wide cell-type-specific early and late replicating domains have been identified by analyses of DNA from populations of cells, but cell-to-cell differences in DNA replication may be important in genome stability, disease aetiology and various other cellular processes.     

Gas chromatography time-of-flight mass spectrometry based metabolomic approach to evaluating toxicity of triptolide

Metabolomics allows high-throughput analysis of low-molecular-weight compounds in biofluids that reflect the physiological status and biochemical metabolism of living systems. Hence it has the potential to evaluate toxicity and clarifies the metabolism-related toxic mechanisms. In this study a promising candidate drug parent, triptolide, was given to Sprague?CDawley rats as a model toxicant at a single dose of 0.6, or 2.4?mg/kg, i.g. Both routine biochemical assays and histopathological inspection showed time-dependent hepatic toxicity at the higher dose, but no obvious toxicity at the lower dose. Meanwhile, serum metabolome was profiled using the non-targeted metabolomic tool, gas chromatography time-of-flight mass spectrometry. Based on the acquired metabolomic data, mathematical models were calculated and the metabolic patterns of serum were evaluated using projection to latent structure-discriminant analysis. The relative distance of each treated group from the normal control was calculated to provide a measure of toxicity. Treatment with triptolide at either the higher or lower dose caused deviations in the metabolic pattern and resulted in perturbation of taurine, creatinine, free fatty acids, ??-hydroxybutyrate, tricarboxylic acid cycle intermediates, and amino acids. This finding indicates the dysfunction of ??-oxidation of free fatty acids and impairment of the mitochondria and confirms the hepatic toxicity of triptolide. The identified toxic markers and the calculated relative distance values quantitatively demonstrated dose- and time-dependent toxicity, whereas the scores plot of the model provided the qualitative information. The metabolomic approach was non-invasive and more sensitive than routine toxic assessment, and the results of both methods correlated well.     

Characterization of upper lamina propria interstitial cells in bladders from patients with neurogenic detrusor overactivity and bladder pain syndrome

The upper lamina propria (ULP) area of interstitial cells (IC) in bladder has been studied for more than a decade in several species including human beings. Nevertheless there is still lack of uniformity in terminology of this cell layer. The aim of the present study was to add new data to the morphological and immunohistochemical phenotype of these cells and to find out whether this phenotype is changed in bladders from patients with neurogenic detrusor overactivity (NDO) and bladder pain syndrome (BPS). Bladder tissue was obtained from a control group and from patients with NDO and BPS. Samples were processed for morphology, electron microscopy and immunohistochemistry. A morphological and immunohistochemical phenotype for the ULP IC was assessed and changes in this phenotype were looked for in samples from patients with NDO and BPS. The ULP IC were characterized ultrastructurally by the presence of actin filaments with densifications, many caveolae and abundant rough endoplasmic reticulum (RER); on immunohistochemistry ULP IC were immunoreactive for α-sma, vimentin, CD10 and podoplanin and categorized as interstitial Cajal-like cells (ICLC). In NDO and BPS bladders we found a phenotypical shift towards a fibroblastic phenotype which was even more pronounced in the NDO group. In both groups there was also an increased presence in ULP lymphocytes. The ULP area in the human bladder contains a population of ICLC with distinct ultrastructural morphology and immunohistochemical phenotype. Their unique α-sma(+) /desmin(-) /CD34(-) phenotype allows studying this population in various bladder disorders. In bladders form patients with BPS and NDO, we observed these ULP ICLC to shift towards a fibroblast phenotype.     

Prediction of the pharmacokinetic parameters of triptolide in rats based on endogenous molecules in pre-dose baseline serum

Background

Individual variances usually affect drug metabolism and disposition, and hence result in either ineffectiveness or toxicity of a drug. In addition to genetic polymorphism, the multiple confounding factors of lifestyles, such as dietary preferences, contribute partially to individual variances. However, the difficulty of quantifying individual diversity greatly challenges the realization of individualized drug therapy. This study aims at quantitative evaluating the association between individual variances and the pharmacokinetics.

Methodology/Principal Findings

Molecules in pre-dose baseline serum were profiled using gas chromatography mass spectrometry to represent the individual variances of the model rats provided with high fat diets (HFD), routine chows and calorie restricted (CR) chows. Triptolide and its metabolites were determined using high performance liquid chromatography mass spectrometry. Metabonomic and pharmacokinetic data revealed that rats treated with the varied diets had distinctly different metabolic patterns and showed differential C_max values, AUC and drug metabolism after oral administration of triptolide. Rats with fatty chows had the lowest C_max and AUC values and the highest percentage of triptolide metabolic transformation, while rats with CR chows had the highest C_max and AUC values and the least percentage of triptolide transformation. Multivariate linear regression revealed that in baseline serum, the concentrations of creatinine and glutamic acid, which is the precursor of GSH, were linearly negatively correlated to C_max and AUC values. The glutamic acid and creatinine in baseline serum were suggested as the potential markers to represent individual diversity and as predictors of the disposal and pharmacokinetics of triptolide.

Conclusions/Significance

These results highlight the robust potential of metabonomics in characterizing individual variances and identifying relevant markers that have the potential to facilitate individualized drug therapy.     

The taxonomic position of Saccharomyces boulardii as evaluated by sequence analysis of the D1/D2 domain of 26S rDNA, the ITS1-5.8S rDNA-ITS2 region and the mitochondrial cytochrome-c oxidase II gene

A taxonomic study was carried out on eight strains of Saccharomyces boulardii. Morphological and physiological characteristics were consistent with those of Saccharomyces cerevisiae. Sequences of the D1/D2 domain of the 26S rDNA were identical for all strains examined and had a similarity value of 100% compared to sequences of the type strain of S. cerevisiae (CBS 1171T) and strain S288c. For all S. boulardii isolates was found the exact same ITS1-5.8S rDNA-ITS2 sequence, which displayed a close resemblance with the sequences published for S288c (99.9%), CBS 1171(T) (99.3%) and other S. cerevisiae strains. Sequence analysis of the mitochondrial cytochrome-c oxidase II gene (COX2) also resulted in identical sequences for the S. boulardii isolates and comparisons with available nucleotide sequences revealed close relatedness to strains of S. cerevisiae including S288c (99.5%) and CBS 1171(T) (96.6%). The electrophoretic karyotypes of the S. boulardii strains appeared quite uniform and although very typical of S. cerevisiae, they formed a cluster separate from strains of this species. The results of the present study strongly indicate a close relatedness of S. boulardii to S. cerevisiae and thereby support the recognition of S. boulardii as a member of S. cerevisiae and not as a separate species.     

Benthic decomposition of Ulva lactuca: A controlled laboratory experiment

The degradation of an Ulva lactuca mat (0.2 kg dw m⁻²) was studied in a controlled flow-through mesocosm for 31 d. Sediment chambers without U. lactuca served as controls. Fluxes of ∑CO₂, O₂, inorganic nitrogen, and urea were determined during the incubation period in addition to sulfate reduction rates, POC and PON content, enumeration of specific bacterial populations and evaluation of the physiological state of the added U. lactuca thalli. After U. lactuca addition to the chambers, there was an immediate increase in the efflux of ∑CO₂ from 11 to 27 mmol-C m⁻² d⁻¹ and a concomitant increase in O₂ uptake from 11 to 23 mmol m⁻² d⁻¹. These effluxes remained elevated throughout the incubation period. In contrast, the NH₄⁺ efflux increased from 0.1 to 1.8 mmol NH₄⁺ m⁻² d⁻¹ during the first 3 d of incubation, followed by 6 d with a constant efflux rate, after which time it decreased gradually to 0.3 mmol NH₄⁺ m⁻² d⁻¹ by the end of the experiment. In total, NH₄⁺accounted for 83% of the total nitrogen efflux after addition of U. lactuca. During the 31 d incubation period there was a continuous colonization of the thalli by bacteria. Sulfate reducers associated with the thalli accounted for 3% of the carbon oxidation on day 31. The molar C:N ratio in mineralization products (the ratio between the efflux of ∑CO₂ and NH₄⁺ + NO₂⁻ + NO₃⁻) increased from 15 mol mol⁻¹ at day 11 after U. lactuca addition to >80 mol mol⁻¹ by the end of the incubation. Since the C:N ratio in the mineralization products was much higher than the original thallus material (8.9 mol mol⁻¹) it is probable that a preferential incorporation of NH₄⁺ into the increasing bacterial biomass occurred. The nitrogen for bacterial growth was most likely obtained from degradation of U. lactuca thalli as there was no stimulation of urea-N turnover in the sediment during incubation. The net increase in bacteria cell number in the 18-mm thick thallus layer was estimated to be 7.6 × 10⁹ to 2.4 × 10¹⁰ bacterial cells cm⁻³. In contrast, the bacterial cell number remained constant in the −Ulva incubations.     

Cytokeratin 5 and cytokeratin 20 inversely correlate with tumour grading in Ta non-muscle-invasive bladder cancer

Cytokeratin 5 is a marker of basal molecular subtypes of muscle-invasive bladder cancer (MIBC), which correlates with worse overall survival compared to luminal subtypes. Our observations have not confirmed CK5 as a marker of high-grade (HG) disease in Ta non-muscle-invasive bladder cancer (NMIBC). Therefore, to understand the basal-luminal immunohistochemistry profile in Ta NMIBC, we performed immunohistochemistry for CK5, P40, P63 (basal), GATA3 and CK20 (luminal) and studied the correlation with HG and clinical outcome in 109 patients with Ta NMIBC. HG and low-grade (LG) diseases were scored in each patient. Four different CK5 patterns were evaluated: absent (median 41.3%), normal (72.5%), rising (84.4%) and full thickness (23.9%). The median percentage of GATA3 was 100%. HG disease and CK5 expression and rising CK5 pattern had a significant inverse correlation, whereas HG disease and CK20 expression had a significant positive correlation. We also found a significant inverse correlation between CK5 expression and CK20 expression. Quantitative PCR confirmed that the presence of CK5 correlated with up-regulation of CK5 RNA. None of the markers could differentiate patients with regard to clinical outcome. Our results suggest a role for CK5 and CK20 in differentiating between LG and HG disease in Ta NMIBC.