Mitochondria are considered as the power‐generating units of the cell due to their key role in energy metabolism and cell signaling. However, mitochondrial components could be found in the ...extracellular space, as fragments or encapsulated in vesicles. In addition, this intact organelle has been recently reported to be released by platelets exclusively in specific conditions. Here, we demonstrate for the first time, that blood preparation with resting platelets, contains whole functional mitochondria in normal physiological state. Likewise, we show, that normal and tumor cultured cells are able to secrete their mitochondria. Using serial centrifugation or filtration followed by polymerase chain reaction‐based methods, and Whole Genome Sequencing, we detect extracellular full‐length mitochondrial DNA in particles over 0.22 µm holding specific mitochondrial membrane proteins. We identify these particles as intact cell‐free mitochondria using fluorescence‐activated cell sorting analysis, fluorescence microscopy, and transmission electron microscopy. Oxygen consumption analysis revealed that these mitochondria are respiratory competent. In view of previously described mitochondrial potential in intercellular transfer, this discovery could greatly widen the scope of cell‐cell communication biology. Further steps should be developed to investigate the potential role of mitochondria as a signaling organelle outside the cell and to determine whether these circulating units could be relevant for early detection and prognosis of various diseases.
In addition to their intracellular mobility, mitochondria and their components can exist outside the cells from which they originate. As a result, they are capable of acting on non-parental distant ...cells and mediate intercellular communication in physiological conditions and in a variety of pathologies. It has recently been demonstrated that this horizontal transfer governs a wide range of biological processes, such as tissue homeostasis, the rescue of injured recipient cells, and tumorigenesis. In addition, due to mitochondria’s bacterial ancestry, they and their components can be recognized as damage-associated molecular patterns (DAMPs) by the immune cells, leading to inflammation. Here, we provide an overview of the most current and significant findings concerning the different structures of extracellular mitochondria and their by-products and their functions in the physiological and pathological context. This account illustrates the ongoing expansion of our understanding of mitochondria’s biological role and functions in mammalian organisms.
Mitochondria can reside outside the cells from which they originate. Therefore, they can facilitate intercellular communication in both healthy and pathological conditions and modulate the immune response. This review focuses on the continual advancement of our knowledge regarding the biological function of mitochondria in mammalian species.
To our knowledge, this is the first comprehensive study on the influence of several pre-analytical and demographic parameters that could be a source of variability in the quantification of nuclear ...and mitochondrial circulating DNA (NcirDNA and McirDNA). We report data from a total of 222 subjects, 104 healthy individuals and 118 metastatic colorectal cancer (mCRC) patients. Approximately 50,000 and 3,000-fold more mitochondrial than nuclear genome copies were found in the plasma of healthy individuals and mCRC patients, respectively. In healthy individuals, NcirDNA concentration was statistically influenced by age (p = 0.009) and gender (p = 0.048). Multivariate analysis with logistic regression specified that age over 47 years-old was predictive to have higher NcirDNA concentration (OR = 2.41; p = 0.033). McirDNA concentration was independent of age and gender in healthy individuals. In mCRC patients, NcirDNA and McirDNA levels were independent of age, gender, delay between food intake and blood collection, and plasma aspect, either with univariate or multivariate analysis. Nonetheless, ad hoc study suggested that menopause and blood collection time might have tendency to influence cirDNA quantification. In addition, high significant statistical differences were found between mCRC patients and healthy individuals for NcirDNA (p < 0.0001), McirDNA (p < 0.0001) and McirDNA/NcirDNA ratio (p < 0.0001). NcirDNA and McirDNA levels do not vary in the same way with regards to cancer vs healthy status, pre-analytical and demographic factors.
We investigated the influence of hypoxia on the concentration of mitochondrial and nuclear cell-free DNA (McfDNA and NcfDNA, respectively).
By an ultra-sensitive quantitative PCR-based assay, McfDNA ...and NcfDNA were measured in the supernatants of different colorectal cell lines, and in the plasma of C57/Bl6 mice engrafted with TC1 tumour cells, in normoxic or hypoxic conditions.
Our data when setting cell culture conditions highlighted the higher stability of McfDNA as compared to NcfDNA and revealed that cancer cells released amounts of nuclear DNA equivalent to the mass of a chromosome over a 6-h duration of incubation. In cell model, hypoxia induced a great increase in NcfDNA and McfDNA concentrations within the first 24 h. After this period, cfDNA total concentrations remained stable in hypoxia consecutive to a decrease of nuclear DNA release, and noteworthy, to a complete inhibition of daily mitochondrial DNA release. In TC1-engrafted mice submitted to intermittent hypoxia, plasma NcfDNA levels are much higher than in mice bred in normoxia, unlike plasma McfDNA concentration that is not impacted by hypoxia.
This study suggests that hypoxia negatively modulates nuclear and, particularly, mitochondrial DNA releases in long-term hypoxia, and revealed that the underlying mechanisms are differently regulated.
To unequivocally address their unresolved intimate structures in blood, we scrutinized the size distribution of circulating cell-free DNA (cfDNA) using whole-genome sequencing (WGS) from both double- ...and single-strand DNA library preparations (DSP and SSP, n = 7) and using quantitative PCR (Q-PCR, n = 116). The size profile in healthy individuals was remarkably homogenous when using DSP sequencing or SSP sequencing. CfDNA size profile had a characteristic nucleosome fragmentation pattern. Overall, our data indicate that the proportion of cfDNA inserted in mono-nucleosomes, di-nucleosomes, and chromatin of higher molecular size (>1000 bp) can be estimated as 67.5% to 80%, 9.4% to 11.5%, and 8.5% to 21.0%, respectively. Although DNA on single chromatosomes or mono-nucleosomes is detectable, our data revealed that cfDNA is highly nicked (97%-98%) on those structures, which appear to be subjected to continuous nuclease activity in the bloodstream. Fragments analysis allows the distinction of cfDNA of different origins: first, cfDNA size profile analysis may be useful in cfDNA extract quality control; second, subtle but reliable differences between metastatic colorectal cancer patients and healthy individuals vary with the proportion of malignant cell-derived cfDNA in plasma extracts, pointing to a higher degree of cfDNA fragmentation and nuclease activity in samples with high malignant cell cfDNA content.
Ketogenic diets are low in carbohydrates and high in fat, which forces cells to rely more heavily upon mitochondrial oxidation of fatty acids for energy. Relative to normal cells, cancer cells are ...believed to exist under a condition of chronic mitochondrial oxidative stress that is compensated for by increases in glucose metabolism to generate reducing equivalents. In this study we tested the hypothesis that a ketogenic diet concurrent with radiation and chemotherapy would be clinically tolerable in locally advanced non-small cell lung cancer (NSCLC) and pancreatic cancer and could potentially exploit cancer cell oxidative metabolism to improve therapeutic outcomes. Mice bearing MIA PaCa-2 pancreatic cancer xenografts were fed either a ketogenic diet or standard rodent chow, treated with conventionally fractionated radiation (2 Gy/fraction), and tumor growth rates were assessed daily. Tumors were assessed for immunoreactive 4-hydroxy-2-nonenal-(4HNE)-modfied proteins as a marker of oxidative stress. Based on this and another previously published preclinical study, phase 1 clinical trials in locally advanced NSCLC and pancreatic cancer were initiated, combining standard radiation and chemotherapy with a ketogenic diet for six weeks (NSCLC) or five weeks (pancreatic cancer). The xenograft experiments demonstrated prolonged survival and increased 4HNE-modfied proteins in animals consuming a ketogenic diet combined with radiation compared to radiation alone. In the phase 1 clinical trial, over a period of three years, seven NSCLC patients enrolled in the study. Of these, four were unable to comply with the diet and withdrew, two completed the study and one was withdrawn due to a dose-limiting toxicity. Over the same time period, two pancreatic cancer patients enrolled in the trial. Of these, one completed the study and the other was withdrawn due to a dose-limiting toxicity. The preclinical experiments demonstrate that a ketogenic diet increases radiation sensitivity in a pancreatic cancer xenograft model. However, patients with locally advanced NSCLC and pancreatic cancer receiving concurrent radiotherapy and chemotherapy had suboptimal compliance to the oral ketogenic diet and thus, poor tolerance.
The function, origin and structural features of circulating nuclear DNA (cir-nDNA) and mitochondrial DNA (cir-mtDNA) are poorly known, even though they have been investigated in numerous clinical ...studies, and are involved in a number of routine clinical applications. Based on our previous report disproving the conventional plasma isolation used for cirDNA analysis, this work enables a direct topological comparison of the circulating structures associated with nuclear DNA and mitochondrial cell-free DNA.
We used a Q-PCR and low-pass whole genome sequencing (LP-WGS) combination approach of cir-nDNA and cir-mtDNA, extracted using a procedure that eliminates platelet activation during the plasma isolation process to prevent mitochondria release in the extracellular milieu. Various physical procedures, such as filtration and differential centrifugation, were employed to infer their circulating structures.
DSP-S cir-mtDNA mean size profiles distributed on a slightly shorter range than SSP-S. SSP-S detected 40-fold more low-sized cir-mtDNA fragments (<90 bp/nt) and three-fold less long-sized fragments (>200 bp/nt) than DSP-S. The ratio of the fragment number below 90 bp over the fragment number above 200 bp was very homogenous among both DSP-S and SSP-S profiles, being 134-fold lower with DSP-S than with SSP-S. Cir-mtDNA and cir-nDNA DSP-S and SSP-S mean size profiles of healthy individuals ranged in different intervals with periodic sub-peaks only detectable with cir-nDNA. The very low amount of cir-mtDNA fragments of short size observed suggested that most of the cir-mtDNA is poorly fragmented and appearing longer than ∼1,000 bp, the readout limit of this LP-WGS method. Data suggested that cir-nDNA is, among DNA extracted in plasma, associated with ∼8.6% of large structures (apoptotic bodies, large extracellular vesicles (EVs), cell debris…), ∼27.7% in chromatin and small EVs and ∼63.7% mainly in oligo- and mono-nucleosomes. By contrast, cir-mtDNA appeared to be preponderantly (75.7%) associated with extracellular mitochondria, either in its free form or with large EVs; to a lesser extent, it was also associated with other structures: small EVs (∼18.4%), and exosomes or protein complexes (∼5.9%).
This is the first study to directly compare the structural features of cir-nDNA and cir-mtDNA. The significant differences revealed between both are due to the DNA topological structure contained in the nucleus (chromatin) and in the mitochondria (plasmid) that determine their biological stability in blood. Although cir-nDNA and cir-mtDNA are principally associated with mono-nucleosomes and cell-free mitochondria, our study highlights the diversity of the circulating structures associated with cell-free DNA. They consequently have different pharmacokinetics as well as physiological functions. Thus, any accurate evaluation of their biological or diagnostic individual properties must relies on appropriate pre-analytics, and optimally on the isolation or enrichment of one category of their cirDNA associated structures.
SARS-CoV-2 entry into host cells is mediated by the Spike (S) protein of the viral envelope. The S protein is composed of two subunits: S1 that induces binding to the host cell via its interaction ...with the ACE2 receptor of the cell surface and S2 that triggers fusion between viral and cellular membranes. Fusion by S2 depends on its heptad repeat domains that bring membranes close together and its fusion peptide (FP) that interacts with and perturbs the membrane structure to trigger fusion. Recent studies have suggested that cholesterol and ceramide lipids from the cell surface may facilitate SARS-CoV-2 entry into host cells, but their exact mode of action remains unknown. We have used a combination of in vitro liposome–liposome and in situ cell–cell fusion assays to study the lipid determinants of S-mediated membrane fusion. Our findings reveal that both cholesterol and ceramide lipids facilitate fusion, suggesting that targeting these lipids could be effective against SARS-CoV-2. As a proof of concept, we examined the effect of chlorpromazine (CPZ), an antipsychotic drug known to perturb membrane structure. Our results show that CPZ effectively inhibits S-mediated membrane fusion, thereby potentially impeding SARS-CoV-2 entry into the host cell.
Background. There is little data regarding the effectiveness of palliative radiation with conventional fractionation for metastatic renal cell carcinoma (RCC), which has been described as ...radioresistant. We conducted a retrospective analysis of patients with metastatic bony disease from RCC treated with radiation therapy at our institution. Methods. Forty patients with histologically confirmed RCC with a total of 53 treatment courses were included. Pain response after radiotherapy was recorded and freedom from progression was generated using posttreatment radiographs. Patient data was analyzed to assess influence on local control. Results. Patients had a median age of 63. Median follow-up was 9.3 months. The most common radiation dose was 30 Gy in 10 fractions. Pain control after radiotherapy was achieved in 73.6% of patients. Increasing age was associated with nonresponse at the initial pain assessment post-RT (p=0.02). In lesions with initial pain response, nonclear cell histology was associated with increased pain recurrence (p=0.01) and a shorter duration to pain recurrence (p=0.01). Radiographic control at 1 year was 62%. Conclusions. Pain response and control rates for osseous metastatic disease in RCC are comparable to other histologies when treated with conventional fractionation. These appear to be inferior to reported control rates from stereotactic treatments.
Abstract The purpose of this study was to examine cerebral blood flow (CBF) as measured by arterial spin labeling (ASL) in tissue classified as white matter hyperintensities (WMH), normal appearing ...white matter, and grey matter. Seventeen healthy older adults received structural and ASL MRI. Cerebral blood flow was derived for three tissue types: WMH, normal appearing white matter, and grey matter. Cerebral blood flow was lower in WMH areas relative to normal appearing white matter, which in turn, was lower than grey matter. Regions with consistently lower CBF across individuals were more likely to appear as WMH. Results are consistent with an emerging literature linking diminished regional perfusion with the risk of developing WMH.
PDF
