HEK293T Cells with TFAM Disruption by CRISPR-Cas9 as a Model for Mitochondrial Regulation

The mitochondrial transcription factor A (TFAM) is considered a key factor in mitochondrial DNA (mtDNA) copy number. Given that the regulation of active copies of mtDNA is still not fully understood, we investigated the effects of CRISPR-Cas9 gene editing of TFAM in human embryonic kidney (HEK) 293T cells on mtDNA copy number. The aim of this study was to generate a new in vitro model by CRISPR-Cas9 system by editing the TFAM locus in HEK293T cells. Among the resulting single-cell clones, seven had high mutation rates (67–96%) and showed a decrease in mtDNA copy number compared to control. Cell staining with Mitotracker Red showed a reduction in fluorescence in the edited cells compared to the non-edited cells. Our findings suggest that the mtDNA copy number is directly related to TFAM control and its disruption results in interference with mitochondrial stability and maintenance.

Quantitative Analysis of Neuronal Mitochondrial Movement Reveals Patterns Resulting from Neurotoxicity of Rotenone and 6-hydroxydopamine

Alterations in mitochondrial dynamics, including their intracellular trafficking, are common early manifestations of neuronal degeneration. However, current methodologies used to study mitochondrial trafficking events rely on parameters that are mostly altered in later stages of neurodegeneration. Our objective was to establish a reliable computational methodology to detect early alterations in neuronal mitochondrial trafficking. We propose a novel quantitative analysis of mitochondria trajectories based on innovative movement descriptors, including straightness, efficiency, anisotropy, and kurtosis. Using biological data from differentiated SH-SY5Y cells treated with the mitochondrial toxicants 6-hydroxydopamine and rotenone, we evaluated time- and dose-dependent alterations in trajectory descriptors. MitoTracker Red CMXRos-labelled mitochondria movement was analyzed by total internal reflection fluorescence microscopy followed by computational modelling to describe the process. This innovative analysis of mitochondria trajectories, based on the aforementioned trajectory descriptors, provides insights into mitochondrial movement characteristics and can be a consistent and sensitive method to detect alterations in mitochondrial trafficking occurring in the earliest time points of neurodegeneration.

Difluoroboron β-diketonate polylactic acid oxygen nanosensors for intracellular neuronal imaging

Critical for metabolism, oxygen plays an essential role in maintaining the structure and function of neurons. Oxygen sensing is important in common neurological disorders such as strokes, seizures, or neonatal hypoxic–ischemic injuries, which result from an imbalance between metabolic demand and oxygen supply. Phosphorescence quenching by oxygen provides a non-invasive optical method to measure oxygen levels within cells and tissues. Difluoroboron β-diketonates are a family of luminophores with high quantum yields and tunable fluorescence and phosphorescence when embedded in certain rigid matrices such as poly (lactic acid) (PLA). Boron nanoparticles (BNPs) can be fabricated from dye-PLA materials for oxygen mapping in a variety of biological milieu. These dual-emissive nanoparticles have oxygen-insensitive fluorescence, oxygen-sensitive phosphorescence, and rigid matrix all in one, enabling real-time ratiometric oxygen sensing at micron-level spatial and millisecond-level temporal resolution. In this study, BNPs are applied in mouse brain slices to investigate oxygen distributions and neuronal activity. The optical properties and physical stability of BNPs in a biologically relevant buffer were stable. Primary neuronal cultures were labeled by BNPs and the mitochondria membrane probe MitoTracker Red FM. BNPs were taken up by neuronal cell bodies, at dendrites, and at synapses, and the localization of BNPs was consistent with that of MitoTracker Red FM. The brain slices were stained with the BNPs, and the BNPs did not significantly affect the electrophysiological properties of neurons. Oxygen maps were generated in living brain slices where oxygen is found to be mostly consumed by mitochondria near synapses. Finally, the BNPs exhibited excellent response when the conditions varied from normoxic to hypoxic and when the neuronal activity was increased by increasing K+ concentration. This work demonstrates the capability of BNPs as a non-invasive tool in oxygen sensing and could provide fundamental insight into neuronal mechanisms and excitability research.

Delphinidin Increases the Sensitivity of Ovarian Cancer Cell Lines to 3-bromopyruvate

3-Bromopyruvic acid (3-BP) is a promising anticancer compound. Two ovary cancer (OC) cell lines, PEO1 and SKOV3, showed relatively high sensitivity to 3-BP (half maximal inhibitory concentration (IC50) of 18.7 and 40.5 µM, respectively). However, the further sensitization of OC cells to 3-BP would be desirable. Delphinidin (D) has been reported to be cytotoxic for cancer cell lines. We found that D was the most toxic for PEO1 and SKOV3 cells from among several flavonoids tested. The combined action of 3-BP and D was mostly synergistic in PEO1 cells and mostly weakly antagonistic in SKOV3 cells. The viability of MRC-5 fibroblasts was not affected by both compounds at concentrations of up to 100 µM. The combined action of 3-BP and D decreased the level of ATP and of dihydroethidium (DHE)-detectable reactive oxygen species (ROS), cellular mobility and cell staining with phalloidin and Mitotracker Red in both cell lines but increased the 2’,7’-dichlorofluorescein (DCFDA)-detectable ROS level and decreased the mitochondrial membrane potential and mitochondrial mass only in PEO1 cells. The glutathione level was increased by 3-BP+D only in SKOV3 cells. These differences may contribute to the lower sensitivity of SKOV3 cells to 3-BP+D. Our results point to the possibility of sensitization of at least some OC cells to 3-BP by D.

THE INFLUENCE OF AND PEPTIDES ON MITOCHONDRIES STAIN AND L7A RIBOSOMES PROTEIN EXPRESSION DURING HUMAN PINEAL GLAND AND THYMUS CELL SENESCENCE

Методом конфокальной лазерной сканирующей микроскопии верифицированы новые молекулярные мишени действия геропротекторных пептидов AEDG (эпиталона) и KE (вилона). Показано, что при старении клеток эпифиза и тимуса in vitro окраска митохондрий MitoTracker Red снижается, а синтез рибосомального белка L 7 A компенсаторно возрастает. Пептид AEDG в 1,5 раза повышал площадь окрашивания митохондрий MitoTracker Red и на 22 % снижал экспрессию белка рибосом L 7 A в культурах клеток эпифиза человека при их репликативном старении. Пептид KE в 1,5 раза повышал площадь окрашивания митохондрий MitoTracker Red и на 15 % снижал экспрессию белка рибосом L 7 A в культурах клеток тимуса человека при их репликативном старении. Можно предположить, что пептиды AEDG и KE обладают тканеспецифическим свойством, нормализующим функции митохондрий и рибосом пинеалоцитов и тимоцитов. It was verified new molecular targets of geroprotective activity of AEDG (epitalon) and KE (vilon) peptides by the method of confocal laser scanning microscopy. It was shown that the MitoTracker Red mitochondries staining decreased and L 7 A ribosomal protein synthesis compensatory increased during pineal and thymic cell senescence in vitro . AEDG peptide increases in 1,5 times the square of MitoTracker Red mitochondries staining and decreases on 22% the expression of ribosomal protein L 7 A in cultures of human pineal gland cells during its senescence. KE peptide increases in 1,5 times the square of MitoTracker Red mitochondries staining and decreases on 15% the expression of ribosomal protein L 7 A in cultures of human thymic cells during its senescence. The square of MitoTracker Red mitochondries staining decreases and the expression of L 7 A ribosomal protein compensatory increases during pineal gland and thymic cells senescence. We can suppose that AEDG and KE peptides have a tissue-specific effect that normalizes the functions of mitochondria and ribosomes of pinealocytes and thymocytes.

Ram semen quality can be assessed by flow cytometry several hours after post-fixation

Summary Ram spermatozoa are very sensitive to any cold shock or oxidative damage, therefore making them unsuitable for prolonged storage or distant transport to specialized laboratories for flow-cytometric analysis. The aim of this study was to stain ram semen samples with several fluorescent markers and analyse their stability during formaldehyde fixation. Briefly, freshly collected semen samples were stained for apoptosis (annexin V-FITC, YO-PRO™-1 and FLICA), acrosomal damage (PNA-AF488 and FITC-conjugated antibody against GAPDHS), mitochondrial activity (Mitotracker probes), oxidative damage [dihydroethidium (DHE) and CellROX™ Green] and cell viability (live/dead fixable viability dyes). Next, samples were fixed in buffer containing formaldehyde and then washed. Stained sample were analyzed using flow cytometer before fixation, immediately after fixation, and at 5 h and 20 h post-fixation. Fluorescent signals and the proportion of positively stained spermatozoa were compared statistically in fresh and post-fixed samples. All examined markers, except YO-PRO-1 (decreased significantly, P < 0.05), retained their fluorescence intensities after fixation. In conclusion, several tested markers were able to withstand formaldehyde fixation of ram semen samples as follows: annexin V and FLICA for apoptosis; PNA for acrosomal status; MitoTracker Red CMXRos for mitochondrial activity; and CellROX Green for oxidative status in combination with a suitable live/dead fixable viability dye. This optimized methodology could help to comprehensively analyse the quality of ram semen from local farms countrywide.

Gentiopicroside, a Secoiridoid Glycoside from Gentiana rigescens Franch, Extends the Lifespan of Yeast via Inducing Mitophagy and Antioxidative Stress

Gentiopicroside (GPS), an antiaging secoiridoid glycoside, was isolated from Gentiana rigescens Franch, a traditional Chinese medicine. It prolonged the replicative and chronological lifespans of yeast. Autophagy, especially mitophagy, and antioxidative stress were examined to clarify the mechanism of action of this compound. The free green fluorescent protein (GFP) signal from the cleavage of GFP-Atg8 and the colocation signal of MitoTracker Red CMXRos and GFP were increased upon the treatment of GPS. The free GFP in the cytoplasm and free GFP and ubiquitin of mitochondria were significantly increased at the protein levels in the GPS-treated group. GPS increased the expression of an essential autophagy gene, ATG32 gene, but failed to extend the replicative and chronological lifespans of ATG32 yeast mutants. GPS increased the survival rate of yeast under oxidative stress condition; enhanced the activities of catalase, superoxide dismutase, and glutathione peroxidase; and decreased the levels of reactive oxygen species and malondialdehyde. The replicative lifespans of Δsod1, Δsod2, Δuth1, and Δskn7 were not affected by GPS. These results indicated that autophagy, especially mitophagy, and antioxidative stress are involved in the antiaging effect of GPS.

The Dynamic Influence of Olorofim (F901318) on the Cell Morphology and Organization of Living Cells of Aspergillus fumigatus

The first characterized antifungal in the orotomide class is olorofim. It targets the de novo pyrimidine biosynthesis pathway by inhibiting dihydroorotate dehydrogenase (DHODH). The pyrimidines uracil, thymine and cytosine are the building blocks of DNA and RNA; thus, inhibition of their synthesis is likely to have multiple effects, including affecting cell cycle regulation and protein synthesis. Additionally, uridine-5′-triphosphate (UTP) is required for the formation of uridine-diphosphate glucose (UDP-glucose), which is an important precursor for several cell wall components. In this study, the dynamic effects of olorofim treatment on the morphology and organization of Aspergillus fumigatus hyphae were analyzed microscopically using confocal live-cell imaging. Treatment with olorofim led to increased chitin content in the cell wall, increased septation, enlargement of vacuoles and inhibition of mitosis. Furthermore, vesicle-like structures, which could not be stained or visualized with a range of membrane- or vacuole-selective dyes, were found in treated hyphae. A colocalization study of DHODH and MitoTracker Red FM confirmed for the first time that A. fumigatus DHODH is localized in the mitochondria. Overall, olorofim treatment was found to significantly influence the dynamic structure and organization of A. fumigatus hyphae.

73 Cytokine addition does not increase developmental competence of invitro-produced bovine embryos

Recently in porcine, the addition of 3 cytokines (FGF2, LIF, and IGF1) improved oocyte maturation, quadrupling the number of piglets born per oocyte collected (Yuan et al. 2017 Proc. Natl. Acad. Sci. USA 114, E5796-E5804). We hypothesised that in the bovine, addition of these cytokines to maturation (MCyt) and culture media (CCyt) would lower lipid content and increase mitochondrial activity, representing an improved developmental competence when compared with standard maturation (MCon) and culture (CCon) conditions. The experimental design was a 2 (MCon and MCyt)×2 (CCon and CCyt) factorial in 8 replicates, testing the interactions of each maturation medium with each culture medium. Invitro-produced embryos produced aspirating cumulus-oocyte complexes (COCs) from 2 to 8mm follicles of abattoir ovaries. The COCs (n=2156) were matured for 23h in MCon or MCyt media at 6% CO2 in air, fertilised with semen from one of two bulls, and cultured in CCon or CCyt media at 38.5°C, 6% CO2, 5% O2, and 89% N2. On Day 7.5 post-fertilisation, blastocyst rates were evaluated and embryos (n=4/replicate/group) were stained with 1µgmL−1 Nile Red for lipid quantification or 300 nM MitoTracker Red CMX-Rosamine for mitochondrial polarity. Images were obtained with confocal microscopy and fluorescent intensity (AFU) was measured by Image J software (National Institutes for Health) adjusted per cell number. Data were analysed by GLM using ANOVA and l.s.d. with SAS (SAS Institute Inc.). Results (Table 1) indicated similar cleavage and blastocyst rates between all groups (P&gt;0.05). The combination of MCon×CCon resulted in higher mitochondrial activity than any other combination (P&lt;0.05). The MCon×CCyt showed the highest lipid levels, whereas MCyt×CCyt showed the lowest lipid levels (P&lt;0.05). Results suggest that the combination of MCyt×CCyt media produces the lowest lipid levels, whereas the MCon×CCon media lead to the highest mitochondrial activity. The addition of cytokines to both maturation and culture media maintains competence and lowers lipid content; however, it also seems to lower mitochondrial activity. Cryopreservation studies and evaluation of pregnancy rates are ongoing. Table 1.Oocyte and embryo developmental competence matured and cultured in control and cytokine-added media Treatment Oocytes (n) Cleavage (%) Blastocysts per oocyte (%) Nile Red per cell (AFU) MitoTracker per cell (AFU) Maturation main effects MCon 1000 96.8±0.4 29.9±2.7 36.1±2.1a 385.1±65.8a MCyt 1156 96.0±0.4 26.2±2.7 30.0±2.1b 209.1±65.8b Culture main effects CCon 1036 96.2±0.4 28.0±2.7 33.1±2.1 392.0±65.8a CCyt 1120 97.7±0.4 28.1±2.7 33.0±2.1 202.2±65.8b Interactions MCon×CCon 461 96.6±0.8 27.1±3.7 33.6±3.0ab 559.0±91.1a MCon×CCyt 539 95.3±0.9 29.5±2.7 38.6±3.0a 211.2±91.1b MCyt×CCon 575 95.4±0.6 27.4±2.1 32.7±3.0bc 224.9±91.1b MCyt×CCyt 581 95.6±0.9 23.2±2.9 27.4±3.0c 193.1±91.1b a-cValues with different superscript in the same column differ (P&lt;0.05).

Sirt5 Attenuates Cisplatin-Induced Acute Kidney Injury through Regulation of Nrf2/HO-1 and Bcl-2

Cisplatin- (CDDP) induced acute kidney injury (AKI) limits the clinical use of cisplatin. Several sirtuin (SIRT) family proteins are involved in AKI, while the roles of Sirt5 in cisplatin-induced AKI remain unknown. In the present study, we characterized the role and mechanism of Sirt5 in cisplatin-induced apoptosis using the human kidney 2 (HK-2) cell line. CDDP treatment decreased Sirt5 expression of HK-2 cells in a dose-dependent manner. In addition, Sirt5 overexpression enhanced the metabolic activity in CDDP-treated HK-2 cells while Sirt5 siRNA attenuated it. Forced expression of Sirt5 inhibited CDDP-induced apoptosis while Sirt5 siRNA showed the opposite effects. Accordingly, Sirt5 overexpression inhibited the level of caspase 3 cleavage and cytochrome c levels. Furthermore, we found that Sirt5 increased mitochondrial membrane potentials and ameliorated intracellular ROS production. Mitotracker Red staining indicated that Sirt5 overexpression was able to maintain the mitochondrial density during CDDP treatment. We also investigated possible downstream targets of Sirt5 and found that Sirt5 increased Nrf2, HO-1, and Bcl-2 while it decreased Bax protein expression. Sirt5 siRNA showed the opposite effect on these proteins. The levels of Nrf2, HO-1, and Bcl-2 proteins in HK-2 cells were also decreased after CDDP treatment. Moreover, Nrf2 and Bcl-2 siRNA partly abolished the protecting effect of Sirt5 on CDDP-induced apoptosis and cytochrome c release. Catalase inhibitor 3-AT also abolished the cytoprotective effect of Sirt5. Together, the results demonstrated that Sirt5 attenuated cisplatin-induced apoptosis and mitochondrial injury in human kidney HK-2 cells, possibly through the regulation of Nrf2/HO-1 and Bcl-2.