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Background Previously, ~ 1.4 of mitochondrial DNA (mtDNA) molecules in a single nucleoid was reported. Such a minimum number would reflect a minimum nucloid division. Results We applied 3D-double-color direct stochastic optical reconstruction microscopy (dSTORM), i.e. nanoscopy with ~ 25 nm x,y-resolution, together with our novel method of Delaunay tessellation for the identification of unbiased 3D-overlaps. Noncoding D-loops were recognized in HeLa cells by mtDNA fluorescence in situ hybridization (mtFISH) 7S-DNA 250-bp probe, containing biotin, visualized by anti-biotin/Cy3B-conjugated antibodies. Other mtFISH probes with biotin or Alexa Fluor 647 (A647) against ATP6-COX3 gene overlaps (1,100 bp) were also used. Nucleoids were imaged by anti-DNA/(A647-)-Cy3B-conjugated antibodies. Resulting histograms counting mtFISH-loci/nucleoid overlaps demonstrated that 45–70% of visualized nucleoids contained two or more D-loops or ATP6-COX3-loci, indicating two or more mtDNA molecules per nucleoid. With increasing number of mtDNA per nucleoid, diameters were larger and their distribution histograms peaked at ~ 300 nm. A wide nucleoid diameter distribution was obtained also using 2D-STED for their imaging by anti-DNA/A647. At unchanged mtDNA copy number in osteosarcoma 143B cells, TFAM expression increased nucleoid spatial density 1.67-fold, indicating expansion of existing mtDNA and its redistribution into more nucleoids upon the higher TFAM/mtDNA stoichiometry. Validation of nucleoid imaging was also done with two TFAM mutants unable to bend or dimerize, respectively, which reduced both copy number and nucleoid spatial density by 20%. Conclusions We conclude that frequently more than one mtDNA molecule exists within a single nucleoid and that mitochondrial nucleoids do exist in a non-uniform size range.

Aims: Expression of the HER2 oncogene in breast cancer is associated with resistance to treatment, and Her2 may regulate bioenergetics. Therefore, we investigated whether disruption of the electron transport chain (ETC) is a viable strategy to eliminate Her2high disease. Results: We demonstrate that Her2high cells and tumors have increased assembly of respiratory supercomplexes (SCs) and increased complex I-driven respiration in vitro and in vivo. They are also highly sensitive to MitoTam, a novel mitochondrial-targeted derivative of tamoxifen. Unlike tamoxifen, MitoTam efficiently suppresses experimental Her2high tumors without systemic toxicity. Mechanistically, MitoTam inhibits complex I-driven respiration and disrupts respiratory SCs in Her2high background in vitro and in vivo, leading to elevated reactive oxygen species production and cell death. Intriguingly, higher sensitivity of Her2high cells to MitoTam is dependent on the mitochondrial fraction of Her2. Innovation: Oncogenes such as HER...

This paper describes the Workflow Designer — a prototype web-based application allowing drag-and-drop creating, editing, and running workflows from a predefined library of methods. Adding a new method requires a minimal effort from users. Moreover, any workflow can be exported or imported in JSON format to ensure reusability and local execution of exported JSON configurations. The Workflow Designer can be applied to any general computation if the custom method library is available. The application has been successfully tested using electroencephalographic signal processing workflows. Článek popisuje Workflow Designer - prototypovou webovou aplikaci, která umožňuje snadné vytváření, úpravy a spouštění pracovních postupů z předdefinované knihovny metod. Přidání nové metody vyžaduje od uživatelů minimální úsilí. Kromě toho lze libovolný pracovní postup exportovat nebo importovat ve formátu JSON, aby se zajistilo opětovné použití a lokální vykonání exportovaných konfigurací JSON. Workflow designer lze použít na jakýkoli obecný výpočet, pokud je k dispozici knihovna vlastních metod. Aplikace byla úspěšně otestována pomocí workflow na zpracování elektroencefalografických signálů.

We aimed to define the taxonomic status of 40 haemolytic and/or proteolytic strains of the genus Acinetobacter which were previously classified into five putative species termed as genomic species 14BJ (n=9), genomic species 17 (n=9), taxon 18 (n=7), taxon 19 (n=6) and taxon 20 (n=9). The strains were recovered mostly from human clinical specimens or soil and water ecosystems and were highly diverse in geographical origin and time of isolation. Comparative analysis of the rpoB and gyrB gene sequences of all strains, and the whole-genome sequences of selected strains, showed that these putative species formed five respective, well-supported clusters within a distinct clade of the genus Acinetobacter which typically, although not exclusively, encompasses strains with strong haemolytic activity. The whole-genome-based average nucleotide identity (ANIb) values supported the species status of each of these clusters. Moreover, the distinctness and coherence of the clusters were supported by whole-cell profiling based on MALDI-TOF MS. Congruent with these findings were the results of metabolic and physiological testing. We conclude that the five putative taxa represent respective novel species, for which the names Acinetobacter courvalinii sp. nov. (type strain ANC 3623(T) =CCUG 67960(T) =CIP 110480(T) =CCM 8635(T)), Acinetobacter dispersus sp. nov. (type strain ANC 4105(T) =CCUG 67961(T) =CIP 110500(T) =CCM 8636(T)), Acinetobacter modestus sp. nov. (type strain NIPH 236(T) =CCUG 67964(T) =CIP 110444(T) =CCM 8639(T)), Acinetobacter proteolyticus sp. nov. (type strain NIPH 809(T) =CCUG 67965(T) =CIP 110482(T) =CCM 8640(T)) and Acinetobacter vivianii sp. nov. (type strain NIPH 2168(T) =CCUG 67967(T) =CIP 110483(T) =CCM 8642(T)) are proposed.

Diabetes is a silent disease. It is the 8th most common cause of death that does not hurt until it is too late and the disease has developed. Technology plays a vital role in managing diabetes and educating patients about importance of the treatment. The patient must be able to manage his blood glucose level. However, blood glucose level is measured sporadically as it causes important discomfort to the patient. Measuring glucose level in subcutaneous tissue is minimally invasive technique and thus considerably comfortable, but this level may be different from blood glucose level. We implemented a recently proposed method of blood glucose level calculation from the continuously measured subcutaneous tissue glucose level. Then, we developed a web portal that makes this method accessible to any doctor's office and any diabetic patient. To the best of our knowledge, we are the very first web portal that does this. In this paper, we describe the portal.

Expression of the HER2 oncogene in breast cancer is associated with resistance to treatment, and Her2 may regulate bioenergetics. Therefore, we investigated whether disruption of the electron transport chain (ETC) is a viable strategy to eliminate Her2We demonstrate that Her2Oncogenes such as HER2 can restructure ETC, creating a previously unrecognized therapeutic vulnerability exploitable by SC-disrupting agents such as MitoTam.We propose that the ETC is a suitable therapeutic target in Her2

We aimed to study contribution of flavin IF, or ubiquinone IQ and IIIQ sites of superoxide formation by the respiratory chain (RC) complexes 1 and 3 in vivo in cancer (HepG2) and non-cancer cells (insulinoma INS-1E) using novel site-specific S1QEL and S3QEL suppressors1. Rates of superoxide release into the mitochondrial matrix (Jm) were semiquantified by MitoSOX Red fluorescence using confocal microscopy (for feasibility see2). Reflecting the IQ superoxide formation, S1QEL nearly completely suppressed rotenone-induced superoxide formation in glycolytic HepG2 cells but was inactive when these cells were forced to oxidative phosphorylation by aglycemia. Similarly, S1QEL was inefficient in INS-1E independently of the glucose dose. In contrast, with the decreasing glucose in INS-1E cells, the S3QEL suppressor indicated a lower superoxide fraction ascribed to the IIIQ site and higher remaining complex 1 IF site contribution, which correlated with higher NADH/NAD+. The export of redox equivalents by three mitochondrial shuttles was responsible for the observed NAD+ increase at higher glucose.

Calcium-independent phospholipases (iPLA2s) are a family of enzymes participating in cellular signaling by simultaneously producing free fatty acids and lysophospholipids. Phospholipase iPLA2γ (PNPLA8) is targeted to mitochondria and has been shown to augment GPR40-dependent insulin secretion in β-cell model insulinoma INS1-E cells [1]. Here, we investigated the participation of iPLA2γ on the blood glucose homeostasis and pancreatic β-cell insulin secretion using iPLA2γ-KO mice. Wild type (wt) controls and iPLA2γ-KO mice showed similar cholesterol, triglyceride and basal glucose levels after 6 hours of starving. However, the uric acid levels were increased in iPLA2γ-KOmice. Following the intraperitoneal injection of Intralipid, iPLA2γ-KO mice exhibited a prolonged hyperglycemic state compared to wt mice. Glucose-stimulated insulin release in isolated pancreatic islets (PI) was moderately decreased in iPLA2γ-KO PI. Physiologically relevant concentrations of palmitic acid stimulated insulin secretion in PI from wt mice, and this stimulation was absent in iPLA2γ-KO PI. In conclusion, the data are consistent with iPLA2γ ablation causing impairment of GPR40-dependent insulin secretion, resulting in prolonged glycemia. Thus, our results support the role of iPLA2γ in regulating insulin secretion in vivo.

Background Diabetic Goto Kakizaki (GK) rats represent an established model of type 2 diabetes that exhibit an onset of pancreatic islet (PI) pathology characterized by islet hypertrophy with a decreased number of insulin-secreting β-cells. Among the remaining β-cells, oxidative phosphorylation (OXPHOS) and consequently glucose-stimulated insulin secretion (GSIS) are impaired, perhaps owing to a deficit in mitochondrial DNA (mtDNA). We sought to identify this abnormality. Methods β-Cells were obtained from Accutase-dissolved PI isolated from GK or Wistar rats and sorted based on the positive Zn2+ signal of Newport Green. The mtDNA copy number per cell was quantified as the amplicon ratio by polymerase chain reaction using specific primers against the rat ND5 mt gene and UCP2 nuclear gene. Results The 12-month-old GK rats exhibited drastically reduced copy numbers per remaining β-cell, from 7,400 ± 600 in 12-month old Wistar rats (100%) to 24 ± 4%; mtDNA content in heart and liver was 70 ± 25% and 60 ± 20%, respectively. Versus age-paired Wistar rats, 6- and 4-month-old GK rats showed reductions to 60 ± 15% and 50 ± 20%, respectively. Conclusions OXPHOS of remnant β-cells in diabetic GK was drastically impaired due to the lack of sufficient mtDNA levels. We suggest the use of mtDNA quantification to quickly assess PI quality before transplantation.

Mitochondrial calcium-independent phospholipase A2γ (iPLA2γ) belongs to a family of enzymes that participate in cellular signaling by simultaneously producing free fatty acids and lysophospholipids. We have shown previously that the activity of iPLA2γ is increased following the addition of oxidants, such as tert-butyl hydroperoxide (tBHP) or H2O2. We have also shown that iPLA2γ-liberated fatty acids induce H+ transport mediated by mitochondrial uncoupling proteins and mitochondrial adenine nucleotide translocase (ANT) in heart, lung and spleen mitochondria, leading to a decrease in the mitochondrial protonmotive force and subsequent decrease in mitochondrial superoxide production [1,2]. Here we tested the hypothesis that iPLA2γ serves similar antioxidant function in brain. Using brain mitochondria isolated from wild-type (WT) mice, we found that low micromolar concentrations of tBHP induced increase in respiration and a parallel decrease in mitochondrial superoxide and H2O2 production. These effects of tBHP were fully prevented by bovine serum albumin and inhibited by R-bromoenol lactone, a selective inhibitor of iPLA2γ, and were absent in brain mitochondria isolated from iPLA2γ-KO mice. Moreover, the tBHP-induced effects were fully inhibited by carboxyatractyloside, indicating the participation of ANT. In addition, we observed decrease in mRNA levels of selected antioxidant enzymes and increase in lipid peroxidation products in brain tissues homogenates of iPLA2γ-KO mice compared to the WT controls. Our results support the hypothesis of an antioxidant role of iPLA2γ in brain, consistent with iPLA2γ-dependent, ANT-mediated attenuation of mitochondrial superoxide production. Supported by LTAUSA17174

Mitochondria of pancreatic β-cells act as the perfect glucose sensor substantiating glucose-stimulated insulin secretion (GSIS). Despite controversies, whether this leads or not to the increased superoxide release into the matrix, it has been recently indicated that H2O2 degradation is favored in the matrix upon GSIS [1]. We attempted to study this in model β-cells, INS-1E cells [2], using confocal microscopy time-resolved monitoring with Mito-HyPer. We found a substantial negative drift in fluorescence F485/F405 ratio, indicating degradation and possible slow H2O2 efflux in INS-1E cells pre-incubated with 3 mM or 11 mM glucose. After glucose addition to 25 mM, the H2O2 decrease was enhanced by several folds, which was prevented by inhibition of mitochondrial redox shuttles exporting NADPH to the cell cytosol on the expense of matrix NADH. We conclude that pancreatic β-cells do not create mitochondrial oxidative stress upon GSIS.