Expression of Autophagy and Mitophagy Markers in Breast Cancer Tissues.

Mitochondria play important roles in regulating cell bioenergetics status and reactive oxygen species (ROS) generation. ROS-induced mitochondrial damage is among the main intracellular signal inducers of autophagy. Autophagy is a cellular catabolic process that regulates protein and organelle turnover, while a selective form of autophagy, mitophagy, specifically targets dysfunctional mitochondrial degradation. This study aims to measure the levels of autophagy, mitophagy, oxidative stress, and apoptosis in invasive breast carcinoma tissues using immunohistochemistry (IHC). Tissue microarrays of 76 patients with breast cancer were stained with six IHC markers (MnSOD, Beclin-1, LC3, BNIP3, Parkin, and cleaved caspase 3). The expression intensity was determined for each tumor tissue and the adjacent tumor-matched control tissues. Intermediate and strong staining scores of MnSOD, Beclin-1, LC-3, BNIP-3, and Parkin were significantly higher in tumor tissues compared to the adjacent matched control. The scoring intensity was further classified into tissues with negative staining and positive staining, which showed that positive scores of Beclin-1 and Parkin were significantly high in tumor tissues compared to other markers. Positive association was also noted between BNIP-3 and Beclin-1 as well as LC-3 and cleaved caspase-3 immunostaining. To our knowledge, this is one of the first studies that measure both mitophagy and autophagy in the same breast cancer tissues and the adjacent matched control. The findings from this study will be of great potential in identifying new cancer biomarkers and inspire significant interest in applying anti-autophagy therapies as a possible treatment for breast cancer.

Pathogenic Mitochondria DNA Mutations: Current Detection Tools and Interventions.

Mitochondria are best known for their role in energy production, and they are the only mammalian organelles that contain their own genomes. The mitochondrial genome mutation rate is reported to be 10-17 times higher compared to nuclear genomes as a result of oxidative damage caused by reactive oxygen species during oxidative phosphorylation. Pathogenic mitochondrial DNA mutations result in mitochondrial DNA disorders, which are among the most common inherited human diseases. Interventions of mitochondrial DNA disorders involve either the transfer of viable isolated mitochondria to recipient cells or genetically modifying the mitochondrial genome to improve therapeutic outcome. This review outlines the common mitochondrial DNA disorders and the key advances in the past decade necessary to improve the current knowledge on mitochondrial disease intervention. Although it is now 31 years since the first description of patients with pathogenic mitochondrial DNA was reported, the treatment for mitochondrial disease is often inadequate and mostly palliative. Advancements in diagnostic technology improved the molecular diagnosis of previously unresolved cases, and they provide new insight into the pathogenesis and genetic changes in mitochondrial DNA diseases.

Timing of The First Zygotic Cleavage Affects Post-Vitrification Viability of Murine Embryos Produced In Vivo.

BACKGROUND: Timing of the first zygotic cleavage is an accurate predictor of embryo quality. Embryos that cleaved early (EC) have been shown to exhibit higher develop- mental viability compared to those that cleaved at a later period (LC). However, the vi- ability of EC embryos in comparison to LC embryos after vitrification is unknown. The present study aims to investigate the post-vitrification developmental viability of murine EC versus LC embryos. MATERIALS AND METHODS: In this experimental study, female ICR mice (6-8 weeks old) were superovulated and cohabited with fertile males for 24 hours. Afterwards, their ovi- ducts were excised and embryos harvested. Embryos at the 2-cell stage were catego- rized as EC embryos, while zygotes with two pronuclei were categorized as LC embryos. Embryos were cultured in M16 medium supplemented with 3% bovine serum albumin (BSA) in a humidified 5% CO2atmosphere. Control embryos were cultured until the blastocyst stage without vitrification. Experimental embryos at the 2-cell stage were vitri- fied for one hour using 40% v/v ethylene glycol, 18% w/v Ficoll-70 and 0.5 M sucrose as the cryoprotectant. We recorded the numbers of surviving embryos from the control and experimental groups and their development until the blastocyst stage. Results were analyzed using the chi-square test. RESULTS: A significantly higher proportion of EC embryos (96.7%) from the control group developed to the blastocyst stage compared with LC embryos (57.5%, P<0.0001). Similarly, in the experimental group, a significantly higher percentage of vitrified EC embryos (69.4%) reached the blastocyst stage compared to vitrified LC embryos (27.1%, P<0.0001). CONCLUSION: Vitrified EC embryos are more vitrification tolerant than LC embryos. Prese- lection of EC embryos may be used as a tool for selection of embryos that exhibit higher developmental competence after vitrification.

Cytoskeletal alterations in different developmental stages of in vivo cryopreserved preimplantation murine embryos.

BACKGROUND: This study aimed to investigate the effects of vitrification and slow freezing on actin, tubulin, and nuclei of in vivo preimplantation murine embryos at various developmental stages using a Confocal Laser Scanning Microscope (CLSM). MATERIAL/METHODS: Fifty female mice, aged 4-6 weeks, were used in this study. Animals were superovulated, cohabitated overnight, and sacrificed. Fallopian tubes were excised and flushed. Embryos at the 2-cell stage were collected and cultured to obtain 4- and 8-cell stages before being cryopreserved using vitrification and slow freezing. Fixed embryos were stained with fluorescence-labelled antibodies against actin and tubulin, as well as DAPI for staining the nucleus. Labelled embryos were scanned using CLSM and images were analyzed with Q-Win software V3. RESULTS: The fluorescence intensity of both vitrified and slow-frozen embryos was significantly lower for tubulin, actin, and nucleus as compared to non-cryopreserved embryos (p<0.001). Intensities of tubulin, actin, and nucleus in each stage were also decreased in vitrified and slow-frozen groups as compared to non-cryopreserved embryos. CONCLUSIONS: Cryopreservation of mouse embryos by slow freezing had a more detrimental effect on the actin, tubulin, and nucleus structure of the embryos compared to vitrification. Vitrification is therefore superior to slow freezing in terms of embryonic cryotolerance.