Screening ethnically diverse human embryonic stem cells identifies a chromosome 20 minimal amplicon conferring growth advantage

The International Stem Cell Initiative analyzed 125 human embryonic stem (ES) cell lines and 11 induced pluripotent stem (iPS) cell lines, from 38 laboratories worldwide, for genetic changes occurring during culture. Most lines were analyzed at an early and late passage. Single-nucleotide polymorphism (SNP) analysis revealed that they included representatives of most major ethnic groups. Most lines remained karyotypically normal, but there was a progressive tendency to acquire changes on prolonged culture, commonly affecting chromosomes 1, 12, 17 and 20. DNA methylation patterns changed haphazardly with no link to time in culture. Structural variants, determined from the SNP arrays, also appeared sporadically. No common variants related to culture were observed on chromosomes 1, 12 and 17, but a minimal amplicon in chromosome 20q11.21, including three genes expressed in human ES cells, ID1, BCL2L1 and HM13, occurred in >20% of the lines. Of these genes, BCL2L1 is a strong candidate for driving culture adaptation of ES cells.     

The effect of an mGluR5 inhibitor on procedural memory and avoidance discrimination impairments in Fmr1 KO mice

Fragile X syndrome (FXS) is the most common inherited form of intellectual disability. Patients with FXS do not only suffer from cognitive problems, but also from abnormalities/deficits in procedural memory formation. It has been proposed that a lack of fragile X mental retardation protein (FMRP) leads to altered long-term plasticity by deregulation of various translational processes at the synapses, and that part of these impairments might be rescued by the inhibition of type I metabotropic glutamate receptors (mGluRs). We recently developed the Erasmus Ladder, which allows us to test, without any invasive approaches, simultaneously, both procedural memory formation and avoidance behavior during unperturbed and perturbed locomotion in mice. Here, we investigated the impact of a potent and selective mGluR5 inhibitor (Fenobam) on the behavior of Fmr1 KO mice during the Erasmus Ladder task. Fmr1 KO mice showed deficits in associative motor learning as well as avoidance behavior, both of which were rescued by intraperitoneal administration of Fenobam. While the Fmr1 KO mice did benefit from the treatment, control littermates suffered from a significant negative side effect in that their motor learning skills, but not their avoidance behavior, were significantly affected. On the basis of these studies in the FXS animal model, it may be worthwhile to investigate the effects of mGluR inhibitors on both the cognitive functions and procedural skills in FXS patients. However, the use of mGluR inhibitors appears to be strongly contraindicated in healthy controls or non-FXS patients with intellectual disability.     

A comprehensive characterization of genome-wide copy number aberrations in colorectal cancer reveals novel oncogenes and patterns of alterations

To develop a comprehensive overview of copy number aberrations (CNAs) in stage-II/III colorectal cancer (CRC), we characterized 302 tumors from the PETACC-3 clinical trial. Microsatellite-stable (MSS) samples (n = 269) had 66 minimal common CNA regions, with frequent gains on 20 q (72.5%), 7 (41.8%), 8 q (33.1%) and 13 q (51.0%) and losses on 18 (58.6%), 4 q (26%) and 21 q (21.6%). MSS tumors have significantly more CNAs than microsatellite-instable (MSI) tumors: within the MSI tumors a novel deletion of the tumor suppressor WWOX at 16 q23.1 was identified (p<0.01). Focal aberrations identified by the GISTIC method confirmed amplifications of oncogenes including EGFR, ERBB2, CCND1, MET, and MYC, and deletions of tumor suppressors including TP53, APC, and SMAD4, and gene expression was highly concordant with copy number aberration for these genes. Novel amplicons included putative oncogenes such as WNK1 and HNF4A, which also showed high concordance between copy number and expression. Survival analysis associated a specific patient segment featured by chromosome 20 q gains to an improved overall survival, which might be due to higher expression of genes such as EEF1B2 and PTK6. The CNA clustering also grouped tumors characterized by a poor prognosis BRAF-mutant-like signature derived from mRNA data from this cohort. We further revealed non-random correlation between CNAs among unlinked loci, including positive correlation between 20 q gain and 8 q gain, and 20 q gain and chromosome 18 loss, consistent with co-selection of these CNAs. These results reinforce the non-random nature of somatic CNAs in stage-II/III CRC and highlight loci and genes that may play an important role in driving the development and outcome of this disease.     

Quantifying antimicrobial resistance at veal calf farms

This study was performed to determine a sampling strategy to quantify the prevalence of antimicrobial resistance on veal calf farms, based on the variation in antimicrobial resistance within and between calves on five farms. Faecal samples from 50 healthy calves (10 calves/farm) were collected. From each individual sample and one pooled faecal sample per farm, 90 selected Escherichia coli isolates were tested for their resistance against 25 mg/L amoxicillin, 25 mg/L tetracycline, 0.5 mg/L cefotaxime, 0.125 mg/L ciprofloxacin and 8/152 mg/L trimethoprim/sulfamethoxazole (tmp/s) by replica plating. From each faecal sample another 10 selected E. coli isolates were tested for their resistance by broth microdilution as a reference. Logistic regression analysis was performed to compare the odds of testing an isolate resistant between both test methods (replica plating vs. broth microdilution) and to evaluate the effect of pooling faecal samples. Bootstrap analysis was used to investigate the precision of the estimated prevalence of resistance to each antimicrobial obtained by several simulated sampling strategies. Replica plating showed similar odds of E. coli isolates tested resistant compared to broth microdilution, except for ciprofloxacin (OR 0.29, p≤0.05). Pooled samples showed in general lower odds of an isolate being resistant compared to individual samples, although these differences were not significant. Bootstrap analysis showed that within each antimicrobial the various compositions of a pooled sample provided consistent estimates for the mean proportion of resistant isolates. Sampling strategies should be based on the variation in resistance among isolates within faecal samples and between faecal samples, which may vary by antimicrobial. In our study, the optimal sampling strategy from the perspective of precision of the estimated levels of resistance and practicality consists of a pooled faecal sample from 20 individual animals, of which 90 isolates are tested for their susceptibility by replica plating.     

Impact of ivermectin on onchocerciasis transmission: assessing the empirical evidence that repeated ivermectin mass treatments may lead to elimination/eradication in West-Africa

BACKGROUND: The Onchocerciasis Control Program (OCP) in West Africa has been closed down at the end of 2002. All subsequent control will be transferred to the participating countries and will almost entirely be based on periodic mass treatment with ivermectin. This makes the question whether elimination of infection or eradication of onchocerciasis can be achieved using this strategy of critical importance. This study was undertaken to explore this issue. METHODS: An empirical approach was adopted in which a comprehensive analysis was undertaken of available data on the impact of more than a decade of ivermectin treatment on onchocerciasis infection and transmission. Relevant entomological and epidemiological data from 14 river basins in the OCP and one basin in Cameroon were reviewed. Areas were distinguished by frequency of treatment (6-monthly or annually), endemicity level and additional control measures such as vector control. Assessment of results were in terms of epidemiological and entomological parameters, and as a measure of inputs, therapeutic and geographical coverage rates were used. RESULTS: In all of the river basins studied, ivermectin treatment sharply reduced prevalence and intensity of infection. Significant transmission, however, is still ongoing in some basins after 10-12 years of ivermectin treatment. In other basins, transmission may have been interrupted, but this needs to be confirmed by in-depth evaluations. In one mesoendemic basin, where 20 rounds of four-monthly treatment reduced prevalence of infection to levels as low as 2-3%, there was significant recrudescence of infection within a few years after interruption of treatment. CONCLUSIONS: Ivermectin treatment has been very successful in eliminating onchocerciasis as a public health problem. However, the results presented in this paper make it almost certain that repeated ivermectin mass treatment will not lead to the elimination of transmission of onchocerciasis from West Africa. Data on 6-monthly treatments are not sufficient to draw definitive conclusions.     

Human specific anti-type IV collagen monoclonal antibodies, characterization and immunohistochemical application

This paper describes two new monoclonal antibodies reactive with human specific type IV collagen epitopes in frozen as well as routinely fixed and processed tissue sections. The antibodies (1042 and 1043) were raised against human placental type IV collagen and were shown by immunoblotting and ELISA tests to react exclusively with type IV collagen determinants. Extensive immunohistochemical survey studies on panels of tissues from various species, using unfixed cryostat sections, demonstrated that antibody 1042 reacted only with human type IV collagen whereas antibody 1043 in addition reacted with rabbit type IV collagen. All tissues showed homogeneous staining of the basement membrane, indicating that the detected epitopes did not show organ-specific distribution. Tissue processing protocols for using these monoclonal antibodies on routinely processed paraffin embedded tissues were developed. It was found that whereas polyclonal anti-type IV collage antisera required pepsin digestion, our monoclonal antibodies required pronase or papain digestion to restore type IV collagen immunoreactivity in paraffin sections. It is concluded that these monoclonal anti-type IV collagen antibodies detect species specific epitopes which can be detected in routinely processed paraffin embedded tissues after appropriate enzyme pretreatment.     

Proliferative activity of gastric and duodenal endocrine cells in the rat

Summary The replicative activity and migration of gastrin, somatostatin and serotonin cells in rat stomach and doudenum was studied using combined immunocytochemistry and autoradiography after ³H thymidine pulse-labeling. Our results show that a small proportion of gastrin, somatostatin and serotonin immunoreactive cells displays proliferative activity. The overall labeling index ranged from 1.3% for gastric endocrine cells to 3.2% for duodenal endocrine cells.In a pulse chase experiment, labeling indices of immunoreactive cells were estimated at several time intervals after ³H thymidine administration. Significant differences in labeling index were not found. Migration of ³H thymidine labeled endocrine cells towards the luminal surface was not found in the stomach nor in the doudenum.It is concluded that 1) these endocrine cells have replicating activity; 2) the replicative activity of endocrine cells is higher in the duodenum than in the stomach; 3) the various cell types do not show significant differences in replicating activity and 4) endocrine cells did not seem to migrate to the luminal surface of the mucosa along with the other epithelial cells.     

Erythrocyte aging: a comparison of model systems for simulating cellular aging in vitro

Senescent cell antigen (SCANT) is a "neo antigen" that appears on the surface of normal old cells and initiates IgG binding and cellular removal. To investigate the mechanism by which SCANT is generated from its parent molecule, band 3, we subjected intact human erythrocytes to treatments that have been reported to result in changes in band 3 and/or to mimick aging in vitro. The validity of these treatments as model systems for erythrocyte aging was evaluated using a "red cell aging panel" that provides a biochemical profile of a senescent red cell. Treatments were assessed for their ability to induce in vitro the following changes observed in normal erythrocytes aged in vivo: 1 increased breakdown of band 3 as detected by immunoblotting, 2 decrease in anion transport efficiency as detected with a sulfate self-exchange assay, 3 decrease in total glyceraldehyde 3-phosphate dehydrogenase activity with an increase in membrane-bound activity, and 4 increase in the binding of autologous IgG as detected with a protein A binding assay. Neither incubation with the free radical-generating xanthine oxidase/xanthine system, nor treatment with malondialdehyde, and end product of free radical-initiated lipid (per)oxidation, results in age-specific changes. Loading of the cells with calcium and oxidation with iodate results in increased breakdown of band 3, but does not lead to increased binding of autologous IgG. Only erythrocytes that have been stored for 3-4 weeks show the same structural and functional changes as observed during aging in vivo.     

Comparison of blood flow parameters measured by Doppler ultrasound and radionuclide techniques in the baboon (Papio ursinus)

Ascending aortic blood velocity was measured in the baboon (Papio ursinus) by using continuous wave Doppler ultrasound. The blood flow parameters thus obtained were compared to those by the standardized radionuclide technique. It appears that, due to the anatomical position of the ascending aorta and brachiocephalic trunks in relation to the ultrasound beam, Doppler ultrasound does not provide an accurate method of measuring aortic blood velocity in the baboon, which could be the reason for the poor correlation of the results from the two techniques.