Metabolic modulation and cellular therapy of cardiac dysfunction and failure

At present the prevalence of heart failure rises along with aging of the population. Current heart failure therapeutic options are directed towards disease prevention via neurohormonal antagonism (β-blockers, angiotensin converting enzyme inhibitors and/or angiotensin receptor blockers and aldosterone antagonists), symptomatic treatment with diuretics and digitalis and use of biventricular pacing and defibrillators in a special subset of patients. Despite these therapies and device interventions heart failure remains a progressive disease with high mortality and morbidity rates. The number of patients who survive to develop advanced heart failure is increasing. These patients require new therapeutic strategies. In this review two of emerging therapies in the treatment of heart failure are discussed: metabolic modulation and cellular therapy. Metabolic modulation aims to optimize the myocardial energy utilization via shifting the substrate utilization from free fatty acids to glucose. Cellular therapy on the other hand has the goal to achieve true cardiac regeneration. We review the experimental data that support these strategies as well as the available pharmacological agents for metabolic modulation and clinical application of cellular therapy.     

RNA‐Interference Approach to Study Functions of NADPH : Cytochrome P450 Oxidoreductase in Human Hepatocytes

Human NADPH : cytochrome P450 oxidoreductase (POR) is encoded by a single gene on chromosome 7q11.2. This flavoprotein donates electrons derived from NADPH to a variety of acceptor proteins, including squalene monooxygenase, heme oxygenase, cytochrome b₅, and many microsomal cytochromes P450 (CYPs), which are involved in oxidative drug metabolism, steroidogenesis, and other functions. Numerous aspects related to cellular POR expression have not been systematically investigated. Interestingly, POR expression is lower compared to CYPs and may thus be limiting for monooxygenase activities, but conversely, POR knock‐out in mice resulted in compensatory upregulation of CYPs. POR may also influence intracellular cholesterol and lipid homeostasis. To systematically investigate such effects, we developed specific POR gene silencing in cell lines and primary human hepatocytes by RNA interference using small interfering RNAs (siRNAs). In HepG2 cells, POR mRNA could be reduced by 95% over 4 days accompanied by reduced protein content and activity. In primary human hepatocytes, POR mRNA knock‐down was less effective and more variable. Analysis of CYPs indicated induction of CYP3A4 but not CYP1A2 or CYP2D6. These results demonstrate that POR can be efficiently and almost completely silenced in HepG2 cells and, at least partially, in primary human hepatocytes. This will allow systematic studies of various consequences of POR variability in human cells.     

Discovering collectively informative descriptors from high-throughput experiments

Background  

Improvements in high-throughput technology and its increasing use have led to the generation of many highly complex datasets that often address similar biological questions. Combining information from these studies can increase the reliability and generalizability of results and also yield new insights that guide future research.     

1H, 13C and 15N backbone and side chain resonance assignments of the C-terminal domain of CdnL from Myxococcus xanthus

CdnL, a 164-residue protein essential for Myxococcus xanthus viability, is a member of a large family of bacterial proteins of unknown structure and function. Here, we report the ¹H, ¹³C and ¹⁵N backbone and side chain assignments for the stable C-terminal domain of CdnL identified by limited proteolysis.     

A Fish Eye Out of Water: Ten Visual Opsins in the Four-Eyed Fish,Anableps anableps

The “four-eyed” fish Anableps anableps has numerous morphological adaptations that enable above and below-water vision. Here, as the first step in our efforts to identify molecular adaptations for aerial and aquatic vision in this species, we describe the A. anableps visual opsin repertoire. We used PCR, cloning, and sequencing to survey cDNA using unique primers designed to amplify eight sequences from five visual opsin gene subfamilies, SWS1, SWS2, RH1, RH2, and LWS. We also used Southern blotting to count opsin loci in genomic DNA digested with EcoR1 and BamH1. Phylogenetic analyses confirmed the identity of all opsin sequences and allowed us to map gene duplication and divergence events onto a tree of teleost fish. Each of the gene-specific primer sets produced an amplicon from cDNA, indicating that A. anableps possessed and expressed at least eight opsin genes. A second PCR-based survey of genomic and cDNA uncovered two additional LWS genes. Thus, A. anableps has at least ten visual opsins and all but one were expressed in the eyes of the single adult surveyed. Among these ten visual opsins, two have key site haplotypes not found in other fish. Of particular interest is the A. anableps-specific opsin in the LWS subfamily, S180γ, with a SHYAA five key site haplotype. Although A. anableps has a visual opsin gene repertoire similar to that found in other fishes in the suborder Cyprinodontoidei, the LWS opsin subfamily has two loci not found in close relatives, including one with a key site haplotype not found in any other fish species. A. anableps opsin sequence data will be used to design in situ probes allowing us to test the hypothesis that opsin gene expression differs in the distinct ventral and dorsal retinas found in this species.     

DNA from KI,WU and Merkel Cell Polyomaviruses Is Not Detected in Childhood Central Nervous System Tumours or Neuroblastomas

Background

BK and JC polyomaviruses (BKV and JCV) are potentially oncogenic and have in the past inconclusively been associated with tumours of the central nervous system (CNS), while BKV has been hinted, but not confirmed to be associated with neuroblastomas. Recently three new polyomaviruses (KIPyV, WUPyV and MCPyV) were identified in humans. So far KIPyV and WUPyV have not been associated to human diseases, while MCPyV was discovered in Merkel Cell carcinomas and may have neuroepithelial cell tropism. However, all three viruses can be potentially oncogenic and this compelled us to investigate for their presence in childhood CNS and neuroblastomas.

Methodology

The presence of KI, WU and MCPyV DNA was analysed, by a joint WU and KI specific PCR (covering part of VP1) and by a MCPyV specific regular and real time quantitative PCR (covering part of Large T) in 25 CNS tumour biopsies and 31 neuroblastoma biopsies from the Karolinska University Hospital, Sweden. None of the three new human polyomaviruses were found to be associated with any of the tumours, despite the presence of PCR amplifiable DNA assayed by a S14 housekeeping gene PCR.

Conclusion

In this pilot study, the presence of MCPyV, KI and WU was not observed in childhood CNS tumours and neuroblastomas. Nonetheless, we suggest that additional data are warranted in tumours of the central and peripheral nervous systems and we do not exclude that other still not yet detected polyomaviruses could be present in these tumours.