Suitability of a CMV/EGFP cassette to monitor stable expression from human artificial chromosomes but not transient transfer in the cells forming viable clones

Human artificial chromosomes (HACs) were generated by transfer of telomerized PAC constructs containing alpha satellite DNA of various human chromosomes. To monitor which cells took up constructs and subsequently formed stable clones under blasticidin S (BS) selection, a CMV/EGFP expression cassette was inserted into a HAC construct based on chromosome 5 alpha satellite DNA (142 kb). Lipofection into HT1080 cells resulted in a small proportion of cells exhibiting bright green fluorescence on day 1. Areas containing such early green cells were marked, and plates monitored over 2 weeks. In only one out of 41 marked areas, a viable clone developed. In the remaining 40 areas, the green cells ceased division at 1-8 cells. In contrast, outside the marked areas, 16 stable clones formed which did not exhibit green fluorescence during the first cell divisions, but all cells of each became green around day 4 -6. Fluorescence in situ hybridization (FISH) analysis of isolated clonal lines demonstrated low copy HAC formation without integration. We conclude that transient expression of an EGFP marker on HAC DNA is not a suitable means for the identification of the proportion of transfected cells which are capable of forming viable clones. One explanation could be that the high copy number required to consistently detect transient EGFP expression (Schindelhauer and Laner, 2002) impairs viability and clone formation. Copyright (C) 2004 S. Karger AG, Basel

Ornithine Transcarbamylase Deficiency: If at First You Do Not Diagnose, Try and Try Again

Ornithine transcarbamylase (OTC) deficiency is well known for its diagnosis in the neonatal period. Presentation often occurs after protein feeding and manifests as poor oral intake, vomiting, lethargy progressing to seizure, respiratory difficulty, and eventually coma. Presentation at adulthood is rare (and likely underdiagnosed); however, OTC deficiency can be life-threatening and requires prompt investigation and treatment. Reports and guidelines are scarce due to its rarity. Here, we present a 59-year-old woman with a past history of irritable bowel syndrome who underwent a reparative operation for rectal prolapse and enterocele. Her postoperative course was complicated by a bowel perforation (which was repaired), prolonged mechanical ventilation, tracheostomy, critical illness myopathy, protein-caloric malnutrition, and altered mental status. After standard therapy for delirium failed, further investigation showed hyperammonemia and increased urine orotic acid, ultimately leading to the diagnosis of OTC deficiency. This case highlights the importance of considering OTC deficiency in hospitalized adults, especially during the diagnostic evaluation for altered mental status

Proximity-Induced Novel Ferromagnetism Accompanied with Resolute Metallicity in NdNiO3 Heterostructure

Employing X-ray magnetic circular dichroism (XMCD), angle-resolved photoemission spectroscopy (ARPES), and momentum-resolved density fluctuation (MRDF) theory, the magnetic and electronic properties of ultrathin NdNiO3 (NNO) film in proximity to ferromagnetic (FM) La0.67Sr0.33MnO3 (LSMO) layer are investigated. The experimental data shows the direct magnetic coupling between the nickelate film and the manganite layer which causes an unusual ferromagnetic (FM) phase in NNO. Moreover, it is shown the metal–insulator transition in the NNO layer, identified by an abrupt suppression of ARPES spectral weight near the Fermi level (EF), is absent. This observation suggests that the insulating AFM ground state is quenched in proximity to the FM layer. Combining the experimental data (XMCD and AREPS) with the momentum-resolved density fluctuation calculation (MRDF) reveals a direct link between the MIT and the magnetic orders in NNO systems. This work demonstrates that the proximity layer order can be broadly used to modify physical properties and enrich the phase diagram of RENiO3 (RE = rare-earth element)

Stereotypical Chronic Lymphocytic Leukemia B-Cell Receptors Recognize Survival Promoting Antigens on Stromal Cells

Chronic lymphocytic leukemia (CLL) is the most common leukemia in the Western world. Survival of CLL cells depends on their close contact with stromal cells in lymphatic tissues, bone marrow and blood. This microenvironmental regulation of CLL cell survival involves the stromal secretion of chemo- and cytokines as well as the expression of adhesion molecules. Since CLL survival may also be driven by antigenic stimulation through the B-cell antigen receptor (BCR), we explored the hypothesis that these processes may be linked to each other. We tested if stromal cells could serve as an antigen reservoir for CLL cells, thus promoting CLL cell survival by stimulation through the BCR. As a proof of principle, we found that two CLL BCRs with a common stereotyped heavy chain complementarity-determining region 3 (previously characterized as “subset 1”) recognize antigens highly expressed in stromal cells – vimentin and calreticulin. Both antigens are well-documented targets of autoantibodies in autoimmune disorders. We demonstrated that vimentin is displayed on the surface of viable stromal cells and that it is present and bound by the stereotyped CLL BCR in CLL-stroma co-culture supernatant. Blocking the vimentin antigen by recombinant soluble CLL BCR under CLL-stromal cell co-culture conditions reduces stroma-mediated anti-apoptotic effects by 20–45%. We therefore conclude that CLL BCR stimulation by stroma-derived antigens can contribute to the protective effect that the stroma exerts on CLL cells. This finding sheds a new light on the understanding of the pathobiology of this so far mostly incurable disease

Effect of remote ischemic conditioning on atrial fibrillation and outcome after coronary artery bypass grafting (RICO-trial)

Background: Pre- and postconditioning describe mechanisms whereby short ischemic periods protect an organ against a longer period of ischemia. Interestingly, short ischemic periods of a limb, in itself harmless, may increase the ischemia tolerance of remote organs, e.g. the heart (remote conditioning, RC). Although several studies have shown reduced biomarker release by RC, a reduction of complications and improvement of patient outcome still has to be demonstrated. Atrial fibrillation (AF) is one of the most common complications after coronary artery bypass graft surgery (CABG), affecting 27-46% of patients. It is associated with increased mortality, adverse cardiovascular events, and prolonged in-hospital stay. We hypothesize that remote ischemic pre- and/or post-conditioning reduce the incidence of AF following CABG, and improve patient outcome.Methods/design: This study is a randomized, controlled, patient and investigator blinded multicenter trial. Elective CABG patients are randomized to one of the following four groups: 1) control, 2) remote ischemic preconditioning, 3) remote ischemic postconditioning, or 4) remote ischemic pre- and postconditioning. Remote conditio

Blood cell gene expression associated with cellular stress defense is modulated by antioxidant-rich food in a randomised controlled clinical trial of male smokers

Background Plant-based diets rich in fruit and vegetables can prevent development of several chronic age-related diseases. However, the mechanisms behind this protective effect are not elucidated. We have tested the hypothesis that intake of antioxidant-rich foods can affect groups of genes associated with cellular stress defence in human blood cells. Trial registration number: NCT00520819 http://clinicaltrials.gov. Methods In an 8-week dietary intervention study, 102 healthy male smokers were randomised to either a diet rich in various antioxidant-rich foods, a kiwifruit diet (three kiwifruits/d added to the regular diet) or a control group. Blood cell gene expression profiles were obtained from 10 randomly selected individuals of each group. Diet-induced changes on gene expression were compared to controls using a novel application of the gene set enrichment analysis (GSEA) on transcription profiles obtained using Affymetrix HG-U133-Plus 2.0 whole genome arrays. Results Changes were observed in the blood cell gene expression profiles in both intervention groups when compared to the control group. Groups of genes involved in regulation of cellular stress defence, such as DNA repair, apoptosis and hypoxia, were significantly upregulated (GSEA, FDR q-values < 5%) by both diets compared to the control group. Genes with common regulatory motifs for aryl hydrocarbon receptor (AhR) and AhR nuclear translocator (AhR/ARNT) were upregulated by both interventions (FDR q-values < 5%). Plasma antioxidant biomarkers (polyphenols/carotenoids) increased in both groups. Conclusions The observed changes in the blood cell gene expression profiles suggest that the beneficial effects of a plant-based diet on human health may be mediated through optimization of defence processes

Ornithine Transcarbamylase Deficiency: If at First You Do Not Diagnose, Try and Try Again

Ornithine transcarbamylase (OTC) deficiency is well known for its diagnosis in the neonatal period. Presentation often occurs after protein feeding and manifests as poor oral intake, vomiting, lethargy progressing to seizure, respiratory difficulty, and eventually coma. Presentation at adulthood is rare (and likely underdiagnosed); however, OTC deficiency can be life-threatening and requires prompt investigation and treatment. Reports and guidelines are scarce due to its rarity. Here, we present a 59-year-old woman with a past history of irritable bowel syndrome who underwent a reparative operation for rectal prolapse and enterocele. Her postoperative course was complicated by a bowel perforation (which was repaired), prolonged mechanical ventilation, tracheostomy, critical illness myopathy, protein-caloric malnutrition, and altered mental status. After standard therapy for delirium failed, further investigation showed hyperammonemia and increased urine orotic acid, ultimately leading to the diagnosis of OTC deficiency. This case highlights the importance of considering OTC deficiency in hospitalized adults, especially during the diagnostic evaluation for altered mental status