A Novel Amplification-Refractory Mutation System-PCR Strategy to Screen MT-TL1 Pathogenic Variants in Patient Repositories.

Aim: Pathogenic variants within mitochondrial tRNA and rRNA genes negatively affect protein synthesis function and cause oxidative phosphorylation defects. The majority of mitochondrial cytopathies are caused by pathogenic point variants within the mitochondrial tRNA gene for leucine (MT-TL1). This study was designed to evaluate a novel amplification-refractory mutation system (ARMS)-PCR based assay to screen patient samples with a clinical diagnosis of mitochondrial cytopathies. Methods: Tissue DNA samples from 219 affected individuals were screened for the pathogenic variants m.3271T>C, m.3291Ty >C, m.3303C>T, m.3256C>T, and m.3260A>G along with the most frequent m.3243A>G mutation in the MT-TL1 gene. The assay included a "High Resolution Melt curve analysis" to enhance detection limits. The precision of the assay was verified using synthetic controls with variant heteroplasmy ratios. Results: The screening identified the second reported m.3303C>T case as well as two patients with m.3243A>G variants and a rare variant exhibiting m.3290T>C. Conclusion: ARMS-PCR is superior to Sanger sequencing for the detection of variations exhibiting low heteroplasmy. These results provide "proof of concepts" for the implementation of this application for future screening of rare mtDNA variations in sample repositories.

In Vitro Comparison of Adipokine Export Signals.

Mammalian cells are widely used for recombinant protein production in research and biotechnology. Utilization of export signals significantly facilitates production and purification processes. 35 years after the discovery of the mammalian export machinery, there still are obscurities regarding the efficiency of the export signals. The aim of this study was the comparative evaluation of the efficiency of selected export signals using adipocytes as a cell model. Adipocytes have a large capacity for protein secretion including several enzymes, adipokines, and other signaling molecules, providing a valid system for a quantitative evaluation. Constructs that expressed N-terminal fusion export signals were generated to express Enhanced Green Fluorescence Protein (EGFP) as a reporter for quantitative and qualitative evaluation. Furthermore, fluorescent microscopy was used to trace the intracellular traffic of the reporter. The export efficiency of six selected proteins secreted from adipocytes was evaluated. Quantitative comparison of intracellular and exported fractions of the recombinant constructs demonstrated a similar efficiency among the studied sequences with minor variations. The export signal of Retinol Binding Protein (RBP4) exhibited the highest efficiency. This study presents the first quantitative data showing variations among export signals, in adipocytes which will help optimization of recombinant protein distribution.

Tenotomy immobilization as a model to investigate skeletal muscle fibrosis (with emphasis on Secreted frizzled-related protein 2).

The pathological endpoint of congenital and senile myopathies is chronic muscle degeneration characterized by the atrophy of contractile elements, accompanied by fibrosis and fatty infiltration of the interstitium. Tenotomy is the release of preload that causes abrupt shortening of the muscle and models atrophy and fibrosis without prominent inflammatory response. Fibrosis in the skeletal muscle is known to be triggered by transforming growth factor (TGF)-ß, which is activated by inflammatory events. As these were lacking, tenotomy provided an opportunity to investigate transcriptional events on a background without inflammation. An unbiased look at the transcriptome of tenotomy-immobilized soleus muscle revealed that the majority of the transcriptional changes took place in the first 4 wk. Regarding atrophy, proteasomal and lysosomal pathways were actively involved in accompanying cathepsins and calpains in the breakdown of the macromolecular contractile machinery. The transcriptome provided clear-cut evidence for the upregulation of collagens and several extracellular matrix components that define fibrotic remodeling of the skeletal muscle architecture as well as activation of the fibro-adipogenic precursors. Concomitantly, Sfrp2, a Wnt antagonist as well as a procollagen processor, accompanied fibrosis in skeletal muscle with an expression that was stringently confined to the slow-twitch fibers. An interpreted mechanistic scenario construed the kinetic events initiated through the abnormal shortening of the muscle fibers as enough to trigger the resident latent TGF-ß in the extracellular matrix, leading to the activation of fibroadipogenic precursors. As in the heart, Sfrp2 shows itself to be a therapeutic target for the prevention of irreversible fibrosis in degenerative skeletal muscle conditions.

Changes in the expression of selenoproteins in mesial temporal lobe epilepsy patients.

Selenoproteins are enzymes containing selenium in their structure and are involved in cellular processes such as defense against oxidative stress and cell survival. The aim of this study is to investigate the expression of four selenoproteins (GPX1, TRXR1, SELP and SELW) in the hippocampus of intractable mesial temporal lobe epilepsy (MTLE) patients who underwent curative surgery. The selenoproteins is investigated at the mRNA level via RT-PCR and in situ hybridization and by immunostaining at the protein level. The expression of SELW exhibited a relative induction of more than tenfold, and immunostaining findings provided evidence that this upregulation is confined to neurons. GPX1 was also upregulated 2.3-fold, and TRXR1 was downregulated between 70 and 20% in MTLE patients. The profound induction of SELW has been accompanied by GPX1 and displayed a strong correlation with BCL2 expression, suggesting a protective role for these selenoproteins, and may be an indicator of a defense mechanism in surviving neurons.

Liposome-mediated intraperitoneal interleukin 10 gene transfer increases survival in cecal litigation and puncture model of sepsis.

Interleukin 10 (IL-10) has been considered to alleviate the inflammatory cytokine response in various models of sepsis. Although being regarded as a key immunomodulator molecule to be beneficial for the treatment of sepsis, recombinant IL-10 treatment is limited by efficacy and tolerability. We tested a novel approach and conducted i.p. liposomal IL-10 gene transfer 24 h before the cecal ligation and puncture in mice and observed 75% mortality at the end of the 7th day. The mortality was 100% in the group where the gene transfer was not performed. The transgene expression is observed mainly in the endothelium in all vital organs. The results demonstrate the advantageous role of de novo IL-10 synthesis in early stages of sepsis and suggest the beneficial impact of gene transfer approach to recombinant protein infusions.

Four novel thymidine phosphorylase gene mutations in mitochondrial neurogastrointestinal encephalomyopathy syndrome (MNGIE) patients.

Mitochondrial neurogastrointestinal encephalomyopathy syndrome (MNGIE) is a rare autosomal recessive neurologic disorder characterised by multiple mitochondrial DNA deletions. In this study, five Turkish MNGIE patients are investigated for mtDNA deletions and TP gene mutations. The probands presented all the clinical criteria of the typical MNGIE phenotype; the muscle biopsy specimens also confirmed the diagnosis with ragged red fibres and cytochrome C oxidase (COX) negative fibres. The mitochondrial DNA analysis revealed no deletions in the probands' skeletal muscle samples. We have identified four novel mutations in the TP gene while one of the patients also harboured a nucleotide change, which was previously reported as a mutation.