Understanding ageing - the role of mitochondria in determination of caenorhabditis elegans life span

Mitochondria are organelles found in eukaryotic cells. They are involved in many vital cellular functions. Consequently, mitochondrial dysfunction leads to a variety of human disorders. Many studies of the last 50 years showed that mitochondria are involved in the regulation of physiological ageing. However, the underlying mechanisms are still unknown. We aimed to analyze the mitochondrial role in ageing in Caenorhabditis elegans model system. Its short life cycle, powerful genetic tools and known fates of all 959 post-mitotic somatic cells make this nematode an excellent model system for ageing studies. Besides numerous advantages, the small body size of the worm brings along certain technical limitations. We developed a toolkit to analyze mitochondrial morphology, metabolic profile and electron transport chain (ETC) activities on a single-tissue level. In addition, we adapted a method for analysis of mtDNA copy number for use on individual animals. Each mitochondrion has its own genome that is maintained by mitochondrial DNA polymerase gamma (POLG). By analyzing polg-1 mutant worms that are deficient in the sole mitochondrial DNA polymerase, we showed that C. elegans mtDNA replication mainly takes place in the gonad, the only proliferative tissue in adult worms. Thus mtDNA depletion leads to marked dysfunction of this organ. Severe mtDNA depletion leads to embryonic arrest, whereas mild depletion does not affect development. We showed that mtDNA replication does not take place during embryogenesis; it starts during the L3 larval stage, correlating with germline proliferation. Taken together, mtDNA copies in the somatic tissues mainly stem from the oocyte and stay relatively unchanged during development and early adulthood. Remarkably, somatic tissues are not severely affected in polg-1 deficient animals despite the marked overall mtDNA depletion in contrast to other model systems, namely flies and mice. Furthermore, we showed that mtDNA copy number exhibits substantial plasticity upon environmental stress. Mitochondria are the major source of ATP, which they form by oxidative phosphorylation (OXPHOS). Defective OXPHOS often results in severe phenotypes or premature death in several animal models. However, studies in C. elegans showed that dysfunction in the mitochondrial respiratory chain is not necessarily lethal. It can rather result in lifespan prolongation in the so-called “Mit mitochondrial) mutants”. We analyzed molecular mechanisms that underlie the longevity induced by mitochondrial dysfunction. It has been shown that different mechanisms can affect the longevity of Mit mutants. We found that succinate dehydrogenase activity of electron transport chain (ETC) complex II (CII) influences the lifespan of Mit mutants independently of the insulin-like/IGF-1 pathway. We showed that mitochondrial unfolding protein response (UPRmt) is up-regulated in both short- and long-lived Mit mutants. Furthermore, our results suggest that respiration rate is not necessarily linked to longevity. Analysis of several metabolic pathways in Mit mutants revealed that dysfunction of the mitochondrial respiratory chain leads to a common response characterized by up-regulation of the citric acid cycle, glycolysis, and some anaerobic pathways, accompanied by increase in neutral fat storage

New polyene macrolide family produced by submerged culture of Streptomyces durmitorensis

A new polyene macrolide family, closely related to the pentaene macrolide antibiotic roflamycoin, was isolated from the both fermentation broth and biomass of Streptomyces durmitorensis wild-type strain MS405. The main compound was identified by NMR and Fourier transform ion cyclotron resonance mass spectrometry as 32,33-didehydroroflamycoin (1; DDHR). Additional four structurally related compounds were determined solely by MS analysis. DDHR induces cell death by apoptosis in various cancer cell lines as demonstrated by DNA fragmentation. Striking feature of DDHR is its internal fluorescence allowing visualization of labeled plasma membranes and internal membrane structures. The Journal of Antibiotics (2011) 64, 717-722; doi:10.1038/ja.2011.81; published online 14 September 201

Cell-Free DNA Genomic Profiling and Its Clinical Implementation in Advanced Prostate Cancer.

Most men with prostate cancer (PCa), despite potentially curable localized disease at initial diagnosis, progress to metastatic disease. Despite numerous treatment options, choosing the optimal treatment for individual patients remains challenging. Biomarkers guiding treatment sequences in an advanced setting are lacking. To estimate the diagnostic potential of liquid biopsies in guiding personalized treatment of PCa, we evaluated the utility of a custom-targeted next-generation sequencing (NGS) panel based on the AmpliSeq HD Technology. Ultra-deep sequencing on plasma circulating free DNA (cfDNA) samples of 40 metastatic castration-resistant PCa (mCRPC) and 28 metastatic hormone-naive PCa (mCSPC) was performed. CfDNA somatic mutations were detected in 48/68 (71%) patients. Of those 68 patients, 42 had matched tumor and cfDNA samples. In 21/42 (50%) patients, mutations from the primary tumor tissue were detected in the plasma cfDNA. In 7/42 (17%) patients, mutations found in the primary tumor were not detected in the cfDNA. Mutations from primary tumors were detected in all tested mCRPC patients (17/17), but only in 4/11 with mCSPC. AR amplifications were detected in 12/39 (31%) mCRPC patients. These results indicate that our targeted NGS approach has high sensitivity and specificity for detecting clinically relevant mutations in PCa

Mitochondrial DNA level, but not active replicase, is essential for Caenorhabditis elegans development

A number of studies showed that the development and the lifespan of Caenorhabditis elegans is dependent on mitochondrial function. In this study, we addressed the role of mitochondrial DNA levels and mtDNA maintenance in development of C. elegans by analyzing deletion mutants for mitochondrial polymerase gamma (polg-1(ok1548)). Surprisingly, even though previous studies in other model organisms showed necessity of polymerase gamma for embryonic development, homozygous polg-1(ok1548) mutants had normal development and reached adulthood without any morphological defects. However, polg-1 deficient animals have a seriously compromised gonadal function as a result of severe mitochondrial depletion, leading to sterility and shortened lifespan. Our results indicate that the gonad is the primary site of mtDNA replication, whilst the mtDNA of adult somatic tissues mainly stems from the developing embryo. Furthermore, we show that the mtDNA copy number shows great plasticity as it can be almost tripled as a response to the environmental stimuli. Finally, we show that the mtDNA copy number is an essential limiting factor for the worm development and therefore, a number of mechanisms set to maintain mtDNA levels exist, ensuring a normal development of C. elegans even in the absence of the mitochondrial replicase

Analysis of AR/ARV7 Expression in Isolated Circulating Tumor Cells of Patients with Metastatic Castration-Resistant Prostate Cancer (SAKK 08/14 IMPROVE Trial)

Despite several treatment options and an initial high response rate to androgen deprivation therapy, the majority of prostate cancers will eventually become castration-resistant in the metastatic stage (mCRPC). Androgen receptor splice variant 7 (ARV7) is one of the best-characterized androgen receptor (AR) variants whose expression in circulating tumor cells (CTCs) has been associated with enzalutamide resistance. ARV7 expression analysis before and during enzalutamide treatment could identify patients requiring alternative systemic therapies. However, a robust test for the assessment of the ARV7 status in patient samples is still missing. Here, we implemented an RT-qPCR-based assay for detection of AR full length (ARFL)/ARV7 expression in CTCs for clinical use. Additionally, as a proof-of-principle, we validated a cohort of 95 mCRPC patients initiating first line treatment with enzalutamide or enzalutamide/metformin within a clinical trial. A total of 95 mCRPC patients were analyzed at baseline of whom 27.3% (26/95) had ARFL+ARV7+, 23.1% (22/95) had ARFL+ARV7−, 23.1% (22/95) had ARFL−ARV7−, and 1.1% (1/95) had ARFL−ARV7+ CTCs. In 11.6% (11/95), no CTCs could be isolated. A total of 25/95 patients had another CTC analysis at progressive disease, of whom 48% (12/25) were ARV7+. Of those, 50% (6/12) were ARV7− and 50% (6/12) were ARV7+ at baseline. Our results show that mRNA analysis of isolated CTCs in mCRPC is feasible and allows for longitudinal endocrine agent response monitoring and hence could contribute to treatment optimization in mCRPC

Changes in the muscular outputs of young judoists during resistance exercises performed on unstable equipment: A case study

Background and Study Aim: Resistance exercises under unstable conditions have gained popularity among athletes. The aim of the study was to investigate the changes in muscular outputs (peak power and velocity of movement) during bench presses and squats under unstable conditions in comparison to the outputs under stable conditions. Material/Methods: A total of 20 participants, divided into two groups, took part in the study The first group consisted of 9 top-level young male judoists, while the second group consisted of 11 students. All of the exercises were performed once 70% of one-repetition maximum (1RM) was determined. Results: The analysis showed a significant reduction in muscular outputs during squats under unstable conditions (the BOSU ball) in comparison to stable conditions for the group of judoists. However, there was no significant reduction in muscular outputs during the bench presses under unstable conditions (the Swiss ball) compared to the stable flat bench. For the group of students, the analysis showed a significant reduction in muscular outputs during the bench press under unstable conditions when compared to the stable flat bench. In addition, for this group the muscular outputs were significantly lower during squats under unstable in comparison to stable conditions. Conclusions: The bench press as a resistance exercise performed on a Swiss ball with reduced training load cannot be recommended as an effective training model for judoists. Nevertheless, the squat with reduced training load under unstable conditions provided sufficient challenges to the neuromuscular system and could be incorporated into training programs. © ARCHIVES OF BUDO|SCIENCE OF MARTIAL ARTS

Analysis of AR/ARV7 Expression in Isolated Circulating Tumor Cells of Patients with Metastatic Castration-Resistant Prostate Cancer (SAKK 08/14 IMPROVE Trial)

Despite several treatment options and an initial high response rate to androgen deprivation therapy, the majority of prostate cancers will eventually become castration-resistant in the metastatic stage (mCRPC). Androgen receptor splice variant 7 (ARV7) is one of the best-characterized androgen receptor (AR) variants whose expression in circulating tumor cells (CTCs) has been associated with enzalutamide resistance. ARV7 expression analysis before and during enzalutamide treatment could identify patients requiring alternative systemic therapies. However, a robust test for the assessment of the ARV7 status in patient samples is still missing. Here, we implemented an RT-qPCR-based assay for detection of AR full length (ARFL)/ARV7 expression in CTCs for clinical use. Additionally, as a proof-of-principle, we validated a cohort of 95 mCRPC patients initiating first line treatment with enzalutamide or enzalutamide/metformin within a clinical trial. A total of 95 mCRPC patients were analyzed at baseline of whom 27.3% (26/95) had ARFL+ARV7+, 23.1% (22/95) had ARFL+ARV7-, 23.1% (22/95) had ARFL-ARV7-, and 1.1% (1/95) had ARFL-ARV7+ CTCs. In 11.6% (11/95), no CTCs could be isolated. A total of 25/95 patients had another CTC analysis at progressive disease, of whom 48% (12/25) were ARV7+. Of those, 50% (6/12) were ARV7- and 50% (6/12) were ARV7+ at baseline. Our results show that mRNA analysis of isolated CTCs in mCRPC is feasible and allows for longitudinal endocrine agent response monitoring and hence could contribute to treatment optimization in mCRPC

Succinate Dehydrogenase Upregulation Destabilize Complex I and Limits the Lifespan of <i>gas-1</i> Mutant

<div><p>Many <i>Caenorhabditis elegans</i> mutants with dysfunctional mitochondrial electron transport chain are surprisingly long lived. Both short-lived (<i>gas-1(fc21)</i>) and long-lived (<i>nuo-6(qm200)</i>) mutants of mitochondrial complex I have been identified. However, it is not clear what are the pathways determining the difference in longevity. We show that even in a short-lived <i>gas-1(fc21)</i> mutant, many longevity assurance pathways, shown to be important for lifespan prolongation in long-lived mutants, are active. Beside similar dependence on alternative metabolic pathways, short-lived <i>gas-1(fc21)</i> mutants and long-lived <i>nuo-6(qm200)</i> mutants also activate hypoxia-inducible factor –1α (HIF-1α) stress pathway and mitochondrial unfolded protein response (UPR^mt). The major difference that we detected between mutants of different longevity, is in the massive loss of complex I accompanied by upregulation of complex II levels, only in short-lived, <i>gas-1(fc21)</i> mutant. We show that high levels of complex II negatively regulate longevity in <i>gas-1(fc21)</i> mutant by decreasing the stability of complex I. Furthermore, our results demonstrate that increase in complex I stability, improves mitochondrial function and decreases mitochondrial stress, putting it inside a “window” of mitochondrial dysfunction that allows lifespan prolongation.</p> </div