Functional annotation of heart enriched mitochondrial genes GBAS and CHCHD10 through guilt by association

Despite the mitochondria ubiquitous nature many of their components display divergences in their expression profile across different tissues. Using the bioinformatics-approach of guilt by association (GBA) we exploited these variations to predict the function of two so far poorly annotated genes: Coiled-coil-helix-coiled-coil-helix domain containing 10 (CHCHD10) and glioblastoma amplified sequence (GBAS). We predicted both genes to be involved in oxidative phosphorylation. Through in vitro experiments using gene-knockdown we could indeed confirm this and furthermore we asserted CHCHD 10 to play a role in complex IV activity

Diagnostic Value of Monitoring Human Cytomegalovirus Late pp67 mRNA Expression in Renal-Allograft Recipients by Nucleic Acid Sequence-Based Amplification

The diagnostic value of monitoring human cytomegalovirus (HCMV) late pp67 mRNA expression by nucleic acid sequence-based amplification (NASBA) after renal-allograft transplantation was evaluated. RNAs were isolated from 489 whole-blood specimens of 42 patients for the specific amplification of the late pp67 (UL65) mRNA. NASBA results were compared to results from the pp65 antigenemia assay, virus isolation by cell culture, and serology. The sensitivity value for NASBA proved to be higher than that for the antigenemia assay (50 versus 35%) for the detection of HCMV infection, while the sensitivity values of cell culture and NASBA were comparable (54 and 50%, respectively). NASBA detected the onset of HCMV infection simultaneously with cell culture and the antigenemia assay. Both the antigenemia assay and NASBA are very specific (100%) and highly predictive (100%) for the onset of HCMV infection. Antiviral therapy with ganciclovir resulted in negative results for cell culture, the antigenemia assay, and NASBA. In conclusion, monitoring HCMV pp67 mRNA expression by NASBA is a highly specific method for the detection of HCMV infection in renal-allograft recipients and is more sensitive than the antigenemia assay. Furthermore, NASBA can be used to monitor the progression of HCMV infections and the effect of antiviral therapy on viral activity

Electrical signals affect the cardiomyocyte transcriptome independently of contraction

Martherus RS, Vanherle SJ, Timmer ED, Zeijlemaker VA, Broers JL, Smeets HJ, Geraedts JP, Ayoubi TA. Electrical signals affect the cardiomyocyte transcriptome independently of contraction. Physiol Genomics 42A: 283-289, 2010. First published September 21, 2010; doi:10.1152/physiolgenomics.00182.2009.-Cardiomyocytes in vivo are continuously subjected to electrical signals that evoke contractions and instigate drastic changes in the cells' morphology and function. Studies on how electrical stimulation affects the cardiac transcriptome have remained limited to a small number of heart-specific genes. Furthermore, these studies have ignored the interplay between the electrical excitation and the subsequent contractions. We carried out a genomewide assessment of the effects of electrical signaling on gene expression, while distinguishing between the effects deriving from the electrical pulses themselves and the effects instigated by the evoked contractions. Changes in gene expression in primary cultures of neonatal ventricular cardiomyocytes from Lewis Rattus norvegicus were investigated with microarrays and RT-quantitative PCR (QPCR). A series of experiments was included in which the culture medium was supplemented with the contraction inhibitor blebbistatin to allow for electrical stimulation in the absence of contraction. Electrical stimulation was shown to directly enhance calcium handling and induce cardiomyocyte differentiation by arresting cell division and activating key cardiac transcription factors as well as additional differentiation mechanisms such as wnt signaling. Several genes involved in metabolism were also directly activated by electrical stimulation. Furthermore, our data suggest that contraction exerts negative feedback on the transcription of various genes. Together, these observations indicate that intercellular electric currents between adjacent cardiomyocytes have an important role in cardiomyocyte development. They act at least partially through a pulse-specific gene expression program that is activated independently from the evoked contractions

Replication Errors Made During Oogenesis Lead to Detectable De Novo mtDNA Mutations in Zebrafish Oocytes with a Low mtDNA Copy Number.

Of all pathogenic mitochondrial DNA (mtDNA) mutations in humans, ~25% is de novo, although the occurrence in oocytes has never been directly assessed. We used next generation sequencing to detect point mutations directly in the mtDNA of 3-15 individual mature oocytes and three somatic tissues from eight zebrafish females. Various statistical and biological filters allowed reliable detection of de novo variants with heteroplasmy >/=1.5%. In total, we detected 38 de novo base substitutions, but no insertions or deletions. These 38 de novo mutations were present in 19 of 103 mature oocytes, indicating that ~20% of the mature oocytes carry at least one de novo mutation with heteroplasmy >/=1.5%. This frequency of de novo mutations is close to that deducted from the reported error rate of polymerase gamma, the mitochondrial replication enzyme, implying that mtDNA replication errors made during oogenesis are a likely explanation. Substantial variation in the mutation prevalence among mature oocytes can be explained by the highly variable mtDNA copy number, since we previously reported that ~20% of the primordial germ cells have a mtDNA copy number of </=73 and would lead to detectable mutation loads. In conclusion, replication errors made during oogenesis are an important source of de novo mtDNA base substitutions and their location and heteroplasmy level determine their significance

Patient-derived fibroblasts indicate oxidative stress status and may justify antioxidant therapy in OXPHOS disorders

Oxidative phosphorylation disorders are often associated with increased oxidative stress and antioxidant therapy is frequently given as treatment. However, the role of oxidative stress in oxidative phosphorylation disorders or patients is far from clear and consequently the preventive or therapeutic effect of antioxidants is highly anecdotic. Therefore, we performed a systematic study of a panel of oxidative stress parameters (reactive oxygen species levels, damage and defense) in fibroblasts of twelve well-characterized oxidative phosphorylation patients with a defect in the POLG1 gene, in the mitochondrial DNA-encoded tRNA-Leu gene (m.3243A>G or m.3302A>G) and in one of the mitochondrial DNA-encoded NADH dehydrogenase complex 1 (Cl) subunits. All except two cell lines (one POLG1 and one tRNA-Leu) showed increased reactive oxygen species levels compared with controls, but only four (two Cl and two tRNA-Leu) cell lines provided evidence for increased oxidative protein damage. The absence of a correlation between reactive oxygen species levels and oxidative protein damage implies differences in damage prevention or correction. This was investigated by gene expression studies, which showed adaptive and compensating changes involving antioxidants and the unfolded protein response, especially in the POLG1 group. This study indicated that patients display individual responses and that detailed analysis of fibroblasts enables the identification of patients that potentially benefit from antioxidant therapy. Furthermore, the fibroblast model can also be used to search for and test novel, more specific antioxidants or explore ways to stimulate compensatory mechanisms