Cell-free Mitochondrial DNA Research Articles

The landscape of cell-free mitochondrial DNA in liquid biopsy for cancer detection

BackgroundExisting methods to detect tumor signal in liquid biopsy have focused on the analysis of nuclear cell-free DNA (cfDNA). However, non-nuclear cfDNA and in particular mitochondrial DNA (mtDNA) has been understudied. We hypothesize that an increase in mtDNA in plasma could reflect the presence of cancer, and that leveraging cell-free mtDNA could enhance cancer detection.ResultsWe survey 203 healthy and 664 cancer plasma samples from three collection centers covering 12 cancer types with whole genome sequencing to catalogue the plasma mtDNA fraction. The mtDNA fraction is increased in individuals with cholangiocarcinoma, colorectal, liver, pancreatic, or prostate cancer, in comparison to that in healthy individuals. We detect almost no increase of mtDNA fraction in individuals with other cancer types. The mtDNA fraction in plasma correlates with the cfDNA tumor fraction as determined by somatic mutations and/or copy number aberrations. However, the mtDNA fraction is also elevated in a fraction of patients without an apparent increase in tumor-derived cfDNA. A predictive model integrating mtDNA and copy number analysis increases the area under the curve (AUC) from 0.73 when using copy number alterations alone to an AUC of 0.81.ConclusionsThe mtDNA signal retrieved by whole genome sequencing has the potential to boost the detection of cancer when combined with other tumor-derived signals in liquid biopsies.

Comparison of plasma mitochondrial DNA copy number in asymptomatic and symptomatic COVID-19 patients.

Since the beginning of the COVID-19 pandemic, a wide clinical spectrum, from asymptomatic infection to mild or severe disease and death, have been reported in COVID-19 patients. Studies have suggested several possible factors, which may affect the clinical outcome of COVID-19. A pro-inflammatory state and impaired antiviral response have been suggested as major contributing factors in severe COVID-19. Considering that mitochondria have an important role in regulating the immune responses to pathogens, pro-inflammatory signaling, and cell death, it has received much attention in SARS-CoV-2 infection. Recent studies have demonstrated that high levels of cell-free mitochondrial DNA (cf-mtDNA) are associated with an increased risk of COVID-19 intensive care unit (ICU) admission and mortality. However, there have been few studies on cf-mtDNA in SARS-CoV-2 infection, mainly focusing on critically ill COVID-19 cases. In the present study, we investigated cf-mtDNA copy number in COVID-19 patients and compared between asymptomatic and symptomatic cases, and assessed the clinical values. We also determined the cf-nuclear DNA (cf-nDNA) copy number and mitochondrial transcription factor A (TFAM) mRNA level in the studied groups. Plasma and buffy coat samples were collected from 37 COVID-19 patients and 33 controls. Briefly, after total DNA extraction, plasma cf-mtDNA, and cf-nDNA copy numbers were measured by absolute qPCR using a standard curve method. Furthermore, after total RNA extraction from buffy coat and cDNA synthesis, TFAM mRNA levels were evaluated by qPCR. The results showed that cf-mtDNA levels in asymptomatic COVID-19 patients were statistically significantly higher than in symptomatic cases (p value = 0.01). However, cf-nDNA levels were higher in symptomatic patients than in asymptomatic cases (p value = 0.00). There was no significant difference between TFAM levels in the buffy coat of these two groups (p value > 0.05). Also, cf-mtDNA levels showed good diagnostic potential in COVID-19 subgroups. cf-mtDNA is probably important in the outcome of SARS-CoV-2 infection due to its role in inflammation and immune response. It can also be a promising candidate biomarker for the diagnosis of COVID-19 subgroups. Further investigation will help understanding the COVID-19 pathophysiology and effective diagnostic and therapeutic strategies.

Защитные свойства соединения АИКАР in vivo при радиационном воздействии

Purpose: To study the effect of 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR) on the survival rate of mice and proportion of polychromatophilic erythrocytes (PCE) in the bone marrow cells with micronuclei (MN), as well as post-irradiation urinary excretion of cell-free nuclear DNA (cf-nDNA) and mitochondrial DNA (cf-mtDNA) in rats. Material and methods: Male Balb/c mice aged 2 months and Fisher-344 male rats aged 3 months were used. To determine the survival rate of mice, X-irradiation was performed at a dose of 8 Gy, and to analysis the proportion of PCE in the bone marrow cells with MN, at a dose of 2 Gy. Rats were X-irradiated at a dose of 5 Gy. AICAR was administered to animals intraperitoneally at a dose of 400 mg/kg. The drug was administered 30 min before and 20 min after irradiation of the animals. The DNA content was measured by real-time PCR. Results: The results of the study showed that the introduction of AICAR causes a statistically significant increase in the survival rate of irradiated animals. The greatest effect was shown in the group of mice treated with AICAR 20 min after their irradiation at a lethal dose. The introduction of AICAR before irradiation reduces the proportion of PCE with MN by 30 %, and after irradiation ‒ by 70 %, in comparison to the control. AICAR promoted enhanced urinary excretion of cf-nDNA and cf-mtDNA fragments in rats after irradiation. Conclusion: The results show that AICAR acts as a radiomitigation effector and promotes active DNA excretion of damaged cell from animal tissues in the post-radiation period.

Circulating Cell-Free Mitochondrial DNA and Depressive Symptoms Among Low-Active Adults Who Smoke.

Mitochondrial dysfunction is implicated in the pathophysiology of psychiatric disorders. Levels of circulating cell-free mitochondrial DNA (cf-mtDNA) are observed to be altered in depression. However, the few studies that have measured cf-mtDNA in depression have reported conflicting findings. This study examined cf-mtDNA and depressive symptoms in low-active adults who smoke. Participants were adults 18 to 65 years old ( N = 109; 76% female) with low baseline physical activity and depressive symptoms recruited for a smoking cessation study. Self-report measures assessed depression severity, positive and negative affect, and behavioral activation. Blood was collected and analyzed for cf-mtDNA. Relationships between depressive symptoms and cf-mtDNA were examined with correlations and linear regression. Levels of cf-mtDNA were associated with categorically defined depression (Center for Epidemiologic Studies Depression Scale score >15), lower positive affect, and decreased behavioral activation ( p < .05). Relationships remained significant after adjustment for age, sex, and nicotine dependence. In a linear regression model including all depressive symptom measures as predictors, Center for Epidemiologic Studies Depression Scale group and lower positive affect remained significant. This work suggests that mitochondrial changes are associated with depressive symptoms in low-active adults who smoke. Higher levels of cf-mtDNA in association with depression and with lower positive affect and decreased behavioral activation are consistent with a possible role for mitochondrial function in depressive symptoms.

Clinicopathological and circulating cell-free DNA profile in myositis associated with anti-mitochondrial antibody.

Anti-mitochondrial antibodies (AMAs) are associated with idiopathic inflammatory myopathies (IIMs). We aimed to summarize the clinicopathological characteristics, assess circulating cell-free mitochondrial DNA (ccf-mtDNA), and circulating cell-free nuclear DNA (ccf-nDNA) in AMA-associated IIMs. Medical records of 37 IIMs patients with AMAs were reviewed. Circulating cell-free mtDNA and ccf-nDNA levels in sera from IIMs patients with AMAs (n = 21), disease controls (n = 66) and healthy controls (HCs) (n = 23) were measured and compared. Twenty-eight immune-mediated necrotizing myopathy (IMNM) patients, 23 dermatomyositis (DM) patients, and 15 anti-synthetase syndrome (ASS) patients were enrolled as disease controls. Correlations between variables were analyzed. Limb weakness was observed in 75.7% and neck weakness in 56.8% of patients. Cardiac involvement occurred in 51.4% of patients. Muscle pathology revealed 81.1% of IMNM, 5.4% polymyositis, and 13.5% nonspecific myositis. Microinfarction was observed in 8.1% of patients. Serum ccf-mtDNA levels in AMA-associated IIMs were significantly higher than those in HCs (p < 0.001), but no significant differences between AMA-associated IIMs and IMNM, DM, or ASS. Serum ccf-nDNA levels in AMA-associated IIMs were significantly higher than those in HCs (p = 0.02), and significantly lower than those in DM (p = 0.02). Serum ccf-nDNA levels correlated negatively with MMT8 total scores (rs = -0.458, p = 0.037) and positively with mRS scores (rs = 0.486, p = 0.025). Serum ccf-nDNA levels were significantly higher in the non-remission group (p < 0.01). AMA-associated IIMs exhibit distinct clinicopathological features. Serum ccf-nDNA may serve as a potential marker for disease severity and prognosis in AMA-associated IIMs.

Mitochondrial health index correlates with plasma circulating cell-free mitochondrial DNA in bipolar disorder.

Although mitochondrial dysfunction is known to play an essential role in the pathophysiology of bipolar disorder (BD), there is a glaring gap in our understanding of how mitochondrial dysfunction can modulate clinical phenotypes. An emerging paradigm suggests mitochondria play an important non-energetic role in adaptation to stress, impacting cellular resilience and acting as a source of systemic allostatic load. Known as mitochondrial allostatic load, this (phenomenon) occurs when mitochondria are unable to recalibrate and maintain cell homeostasis. This study aimed to evaluate the composite mitochondrial health index (MHI) in BD subjects and non-psychiatry controls. We will also explore whether lower MIH will be related to higher cell-free mtDNA (ccf-mtDNA) levels and poor clinical outcomes. In this study, 14 BD-I patients and 16 age- and sex-matched non-psychiatry controls were enrolled. Peripheral blood mononuclear cells (PBMCs) were used to measure the enzymatic activities of citrate synthase and complexes I, II, and IV and mtDNA copy number. Ccf-mtDNA was evaluated by qPCR in plasma. Mitochondrial quality control (MQC) proteins were evaluated by western blotting. After adjusting for confounding variables, such as age, sex, body mass index (BMI), and smoking status, patients with BD presented lower MHI compared to non-psychiatry controls, as well as higher ccf-mtDNA levels that negatively correlated with MHI. Because the MQC network is essential to maintain mitochondrial health, MHI and ccf-mtDNA were also examined in relation to several MQC-related proteins, such as Fis-1, Opa-1, and LC3. Our results showed that MHI correlated negatively with Fis-1 and positively with Opa-1 and LC3. Accordingly, ccf-mtDNA had a positive correlation with Fis-1 and a negative correlation with Opa-1 and LC3. Furthermore, we found a noteworthy inverse correlation between illness severity and MHI, with lower MHI and higher ccf-mtDNA levels in subjects with a longer illness duration, worse functional status, and higher depressive symptoms. Our findings indicate that mitochondrial allostatic load contributes to BD, suggesting mitochondria represent a potential biological intersection point that could contribute to impaired cellular resilience and increased vulnerability to stress and mood episodes. Ultimately, by linking mitochondrial dysfunction to disease progression and poor outcomes, we might be able to build a predictive marker that explains how mitochondrial function and its regulation contribute to BD development and that may eventually serve as a treatment guide for both old and new therapeutic targets.

Effect of Bariatric Surgery on Plasma Cell-Free Mitochondrial DNA, Insulin Sensitivity and Metabolic Changes in Obese Patients.

While improvement of mitochondrial function after bariatric surgery has been demonstrated, there is limited evidence about the effects of bariatric surgery on circulatory cell-free (cf) mitochondrial DNA (mtDNA) and intracellular mtDNA abundance. Plasma and peripheral blood mononuclear (PBM) cells were isolated from healthy controls (HC) and bariatric surgery patients before surgery and 2 weeks, 3 months, and 6 months after surgery. At baseline, the plasma level of short cf-mtDNA (ND6, ~100 bp) fragments was significantly higher in obese patients compared to HC. But there was no significant variation in mean ND6 values post-surgery. A significant positive correlation was observed between preop plasma ND6 levels and HgbA1c, ND6 and HOMA-IR 2 weeks post-surgery, and mtDNA content 6 months post-surgery. Interestingly, plasma from both HC and obese groups at all time points post-surgery contains long (~8 kb) cf-mtDNA fragments, suggesting the presence of near-intact and/or whole mitochondrial genomes. No significant variation was observed in mtDNA content post-surgery compared to baseline data in both PBM and skeletal muscle samples. Overall, bariatric surgery improved insulin sensitivity and other metabolic parameters without significant changes in plasma short cf-mtDNA levels or cellular mtDNA content. Our study provides novel insights about possible molecular mechanisms underlying the metabolic effects of bariatric surgery and suggests the development of new generalized approaches to characterize cf-mtDNA.

227 Investigating the Use of Flavin Mononucleotide &amp; Cell Free Mitochondrial DNA as a Biomarkers of Ischaemic Damage to Livers &amp; Kidneys Before Transplant Surgery

Abstract Aim Before accepting an organ for transplantation, it is important to ensure it is safe to use. Flavin mononucleotide (FMN) has been demonstrated to be an accurate predictor of 90-day graft loss in liver transplant surgery. Previous studies revealed that cell free mitochondrial DNA (cf mtDNA) is predictive of ischemic acute kidney injury in kidney transplantation. There is no validated scoring system or biomarker in clinical use to aid the decision-making process before transplantation. Consequently, transplant surgeons accept or decline organs based on their clinical judgement. There is a need for an objective method to assess organ quality before transplantation. Method Patient consent and demographics were noted prior to transplant surgery. Perfusion solution was flushed through donor livers or kidneys. FMN concentration was measured by fluorescence spectroscopy and cf mtDNA concentration was measured by digital droplet polymerase chain reaction. The concentrations of FMN and cf mtDNA were correlated with donor age &amp; BMI and total length of recipient intensive treatment unit (ITU) stay. Results Amongst 8 transplanted livers, the study showed a negative trend between donor BMI and both FMN &amp; cf mtDNA. There was a strong correlation between FMN concentration and ITU duration (r = 0.7353, p = 0.0376). Amongst 14 transplanted kidneys, MT-ND6 subunit concentration strongly correlated with donor BMI (r = 0.8473, p = 0.0003). Conclusions Despite more research being required to consolidate the role of these biomarkers, the study reinforces the idea that FMN and cf mtDNA can play a role as point of care tests in assessing organ quality prior to transplantation.

Mitochondrial health, NLRP3 inflammasome activation, and white matter integrity in adolescent mood disorders: A pilot study

Adolescence is a particularly important period for brain development and is also when mood disorders typically emerge. Several psychiatric illnesses exhibit mitochondrial dysfunction, elevated inflammation, and impaired white matter integrity. This study explored the intersection of mitochondrial health, NLRP3 inflammasome activation, and white matter integrity in a small cohort of 29 adolescent patients with mood disorders (bipolar disorder (BD): n = 11, major depressive disorder (MDD): n = 19) and 19 healthy controls. In this sample, adolescents with mood disorders showed lower fractional anisotropy of the ventral cingulum bundle than healthy controls. Across all adolescents, we demonstrated a significant relationship between mitochondrial electron transport chain gene expression, and NLRP3 inflammasome gene expression and activation. Furthermore, circulating cell free mitochondrial DNA was associated with lower white matter integrity in the anterior thalamic radiation. Exploratory subgroup analyses revealed that adolescents with bipolar disorder exhibited lower levels of mitochondrial gene expression and volume, along with increased sensitivity to NLRP3 inflammasome activation compared to adolescents with unipolar depression. Overall, our results reveal relationships between peripherally-measured endpoints of mitochondrial health and NLRP3 inflammasome activation, and centrally measured endpoints of white matter integrity in adolescents. Together with subtle patterns of aberrant neural and biological structure and function in association with mood disorder diagnoses, these results may shed light on the pathophysiology of disease in this early phase of illness.

Activation of cell-free mtDNA-TLR9 signaling mediates chronic stress-induced social behavior deficits

Inflammation and social behavior deficits are associated with a number of neuropsychiatric disorders. Chronic stress, a major risk factor for depression and other mental health conditions is known to increase inflammatory responses and social behavior impairments. Disturbances in mitochondria function have been found in chronic stress conditions, however the mechanisms that link mitochondrial dysfunction to stress-induced social behavior deficits are not well understood. In this study, we found that chronic restraint stress (RS) induces significant increases in serum cell-free mitochondrial DNA (cf-mtDNA) levels in mice, and systemic Deoxyribonuclease I (DNase I) treatment attenuated RS-induced social behavioral deficits. Our findings revealed potential roles of mitophagy and Mitochondrial antiviral-signaling protein (MAVS) in mediating chronic stress-induced changes in cf-mtDNA levels and social behavior. Furthermore, we showed that inhibition of Toll-like receptor 9 (TLR9) attenuates mtDNA-induced social behavior deficits. Together, these findings show that cf-mtDNA-TLR9 signaling is critical in mediating stress-induced social behavior deficits.

Self-Propelled Proteomotors with Active Cell-Free mtDNA Clearance for Enhanced Therapy of Sepsis-Associated Acute Lung Injury.

Acute lung injury (ALI) is a frequent and serious complication of sepsis with limited therapeutic options. Gaining insights into the inflammatory dysregulation that causes sepsis-associated ALI can help develop new therapeutic strategies. Herein, the crucial role of cell-free mitochondrial DNA (cf-mtDNA) in the regulation of alveolar macrophage activation during sepsis-associated ALI is identified. Most importantly, a biocompatible hybrid protein nanomotor (NM) composed of recombinant deoxyribonuclease I (DNase-I) and human serum albumin (HSA) via glutaraldehyde-mediated crosslinking is prepared to obtain an inhalable nanotherapeutic platform targeting pulmonary cf-mtDNA clearance. The synthesized DNase-I/HSA NMs are endowed with self-propulsive capability and demonstrate superior performances in stability, DNA hydrolysis, and biosafety. Pulmonary delivery of DNase-I/HSA NMs effectively eliminates cf-mtDNAs in the lungs, and also improves sepsis survival by attenuating pulmonary inflammation and lung injury. Therefore, pulmonary cf-mtDNA clearance strategy using DNase-I/HSA NMs is considered to be an attractive approach for sepsis-associated ALI.

The Ratio of cf-mtDNA vs. cf-nDNA in the Follicular Fluid of Women Undergoing IVF Is Positively Correlated with Age.

Age-related mitochondrial markers may facilitate the prognosis of artificial reproductive technology outcomes. In this report, we present our study concerning the ratio of cf-mtDNA/cf-nDNA, namely the amount of cell-free mitochondrial DNA relative to cell-free nuclear DNA, in the follicular fluid (FF) of women undergoing IVF, aiming to generate a molecular fingerprint of oocyte quality. The values of this ratio were measured and compared among three groups of women (101 in total): (A) 31 women with polycystic ovary syndrome (PCOS), (B) 34 women younger than 36 years, and (C) 36 women older than 35 years of age. Real-time quantitative PCR (qPCR) was performed to quantify the ratio by using nuclear- and mitochondrial-specific primers and analyzed for potential correlation with age and pregnancy rate. Our analysis showed that the level of FF-cf-mtDNA was lower in the group of advanced-age women than in the groups of PCOS and non-PCOS women. Moreover, a significant positive correlation between FF-cf-mtDNA and the number of mature (MII) oocytes was observed. Collectively, the data show that the relative ratio of cf- mtDNA to cf-nDNA content in human FF can be an effective predictor for assessing the corresponding oocyte's age-related performance in IVF.

Liquid Biopsy in Neurological Diseases.

The most recent and non-invasive approach for studying early-stage biomarkers is liquid biopsy. This implies the extraction and analysis of non-solid biological tissues (serum, plasma, saliva, urine, and cerebrospinal fluid) without undergoing invasive procedures to determine disease prognosis. Liquid biopsy can be used for the screening of several components, such as extracellular vesicles, microRNAs, cell-free DNA, cell-free mitochondrial and nuclear DNA, circulating tumour cells, circulating tumour DNA, transfer RNA, and circular DNA or RNA derived from body fluids. Its application includes early disease diagnosis, the surveillance of disease activity, and treatment response monitoring, with growing evidence for validating this methodology in cancer, liver disease, and central nervous system (CNS) disorders. This review will provide an overview of mentioned liquid biopsy components, which could serve as valuable biomarkers for the evaluation of complex neurological conditions, including Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis, multiple sclerosis, epilepsy, stroke, traumatic brain injury, CNS tumours, and neuroinfectious diseases. Furthermore, this review highlights the future directions and potential limitations associated with liquid biopsy.

Relationship between circulating mitochondrial DNA and microRNA in patients with major depression

BackgroundMicroRNAs (miRNAs) and circulating cell-free mitochondrial DNA (ccf-mtDNA) have attracted interest as biological markers of affective disorders. In response to stress, it is known that miRNAs in mitochondria diffuse out of the cytoplasm alongside mtDNA; however, this process has not yet been identified. We hypothesized that miRNAs derived from specific cell nuclei cause mitochondrial damage and mtDNA fragmentation under MDD-associated stress conditions. MethodsA comprehensive analysis of the plasma miRNA levels and quantification of the plasma ccf-mtDNA copy number were performed in 69 patients with depression to determine correlations and identify genes and pathways interacting with miRNAs. The patients were randomly assigned to receive either selective serotonin reuptake inhibitors (SSRI) or mirtazapine. Their therapeutic efficacy over four weeks was evaluated in relation to miRNAs correlated with ccf-mtDNA copy number. ResultsThe expression levels of the five miRNAs showed a significant positive correlation with the ccf-mtDNA copy number after correcting for multiple testing. These miRNAs are involved in gene expression related to thyroid hormone synthesis, the Hippo signaling pathway, vasopressin-regulated water reabsorption, and lysine degradation. Of these five miRNAs, miR-6068 and miR-4708-3p were significantly associated with the SSRI and mirtazapine treatment outcomes, respectively. LimitationsThis study did not show comparison with a healthy group. ConclusionsThe expression levels of specific miRNAs were associated with ccf-mtDNA copy number in untreated depressed patients; moreover, these miRNAs were linked to antidepressant treatment outcomes. These findings are expected to lead to the elucidation of new pathological mechanism of depression.

Next-generation sequencing reveals mitogenome diversity in plasma extracellular vesicles from colorectal cancer patients

BackgroundRecent reports have demonstrated that the entire mitochondrial genome can be secreted in extracellular vesicles (EVs), but the biological attributes of this cell-free mitochondrial DNA (mtDNA) remain insufficiently understood. We used next-generation sequencing to compare plasma EV-derived mtDNA to that of whole blood (WB), peripheral blood mononuclear cells (PBMCs), and formalin-fixed paraffin-embedded (FFPE) tumor tissue from eight rectal cancer patients and WB and fresh-frozen (FF) tumor tissue from eight colon cancer patients.MethodsTotal DNA was isolated before the mtDNA was enriched by PCR with either two primer sets generating two long products or multiple primer sets (for the FFPE tumors), prior to the sequencing. mtDNA diversity was assessed as the total variant number, level of heteroplasmy (mutant mtDNA copies mixed with wild-type copies), variant distribution within the protein-coding genes, and the predicted functional effect of the variants in the different sample types. Differences between groups were compared by paired Student’s t-test or ANOVA with Dunnett’s multiple comparison tests when comparing matched samples from patients. Mann–Whitney U test was used when comparing differences between the cancer types and patient groups. Pearson correlation analysis was performed.ResultsIn both cancer types, EV mtDNA presented twice as many variants and had significantly more low-level heteroplasmy than WB mtDNA. The EV mtDNA variants were clustered in the coding regions, and the proportion of EV mtDNA variants that were missense mutations (i.e., estimated to moderately affect the mitochondrial protein function) was significantly higher than in WB and tumor tissues. Nonsense mutations (i.e., estimated to highly affect the mitochondrial protein function) were only observed in the tumor tissues and EVs.ConclusionTaken together, plasma EV mtDNA in CRC patients exhibits a high degree of diversity.Trial registrationClinicalTrials.gov: NCT01816607. Registered 22 March 2013.

N-acetylcysteine modulates rotenone-induced mitochondrial Complex I dysfunction in THP-1 cells

Mitochondrial Complex I dysfunction and oxidative stress have been part of the pathophysiology of several diseases ranging from mitochondrial disease to chronic diseases such as diabetes, mood disorders and Parkinson’s Disease. Nonetheless, to investigate the potential of mitochondria-targeted therapeutic strategies for these conditions, there is a need further our understanding on how cells respond and adapt in the presence of Complex I dysfunction. In this study, we used low doses of rotenone, a classical inhibitor of mitochondrial complex I, to mimic peripheral mitochondrial dysfunction in THP-1 cells, a human monocytic cell line, and explored the effects of N-acetylcysteine on preventing this rotenone-induced mitochondrial dysfunction. Our results show that in THP-1 cells, rotenone exposure led to increases in mitochondrial superoxide, levels of cell-free mitochondrial DNA, and protein levels of the NDUFS7 subunit. N-acetylcysteine (NAC) pre-treatment ameliorated the rotenone-induced increase of cell-free mitochondrial DNA and NDUFS7 protein levels, but not mitochondrial superoxide. Furthermore, rotenone exposure did not affect protein levels of the NDUFV1 subunit but induced NDUFV1 glutathionylation. In summary, NAC may help to mitigate the effects of rotenone on Complex I and preserve the normal function of mitochondria in THP-1 cells.

Circadian disruption and psychostimulants dysregulates plasma acute-phase proteins and circulating cell-free mitochondrial DNA

BackgroundPrevious studies have indicated a close link between the inflammatory response, exacerbated by circadian disruption and psychostimulants such as cocaine and methamphetamine (METH). Indicators of this inflammation include cortisol and acute-phase proteins (APPs) like C-reactive protein (CRP), complement C3 (C3), and serum amyloid A (SAA). The connection between these inflammation markers and circulating mitochondrial DNA (mtDNA) has been gaining attention. However, the specific influence of cocaine and METH on APP, cortisol, and mtDNA levels in mice with disturbed circadian rhythm has yet to be explored, which is the main aim of this research. MethodsIn our study, we employed 10–12-week-old male C57BL/6J mice, which underwent an imposed 6-h phase advance every six days for a total of eight cycles. This process led to the formation of mice with disrupted circadian rhythm and sleep disorders (CRSD). We administered 11 dosages of cocaine and METH 15 mg/kg and 20 mg/kg, respectively to these CRSD mice over the course of 22 days. Quantitative assessments of CRP, C3, SAA, cortisol, and cell-free circulating mtDNA were conducted using enzyme-linked immunosorbent assay (ELISA), Western Blot, and quantitative real-time polymerase chain reaction (qRT-PCR) techniques. ResultsThe experiment revealed that disruption in circadian rhythm alone or cocaine or METH on their own increased CRP, C3, SAA, and cortisol levels in comparison with the control group. CRSD mice, exposed to cocaine and METH, showed a significant rise in CRP, C3, and SAA, while those without exposure remained stable. We also found a reduction in circulating cell-free mtDNA in all CRSD mice, regardless of cocaine and METH exposure. ConclusionsThe findings of our study affirm that the levels of CRP, C3, SAA, and cortisol, which reflect inflammation, are enhanced by circadian disruption, cocaine, and METH, and these levels show a strong correlation with the content of circulating cell-free mtDNA. Furthermore, it also shows the potential link between the disruption of the circadian clock and the inflammatory response triggered by cocaine and METH.

Cell-Free Mitochondrial DNA as a Novel biomarker for stress-related conditions - Meta-Analysis

Mitochondria is a central organelle that regulates various physiological functions of the cell, like Reactive oxygen species (ROS) generation, apoptosis, and energy metabolism; its dysfunction due to various stress factors will result in compromised O2 regulation, mitochondrial-related diseases and other conditions. Few studies have shown that Circulating cell-free mitochondrial DNA(CFmt-DNA) content in body fluids will change many stress, environmental and exercise needs. In this study, we aim to understand the relationship between CFmt-DNA and various stress conditions like Alzheimer's disease, Acute Appendicitis, strenuous exercise, major depressive disorder etc. This meta-analysis is done by PRISMA standards, and a specified methodology was used to conduct the study. We looked for relevant studies up to August 2022 in PubMed, Web of Science, and Science direct ELSEVIER. Meta Mar web tool was used to analyse the data. Additionally, research on publication bias was also done. After the initial search, a total of 832 articles were identified. Six articles were chosen for our meta-analysis after filtering out meta-analyses or reviews (165 publications), erroneous data (591 publications) and studies that lacked access to raw data (73 publications). Data from six studies were combined to compare the normalised CFmt-DNA copy numbers between the experimental group under various stress conditions and the control group. The copy number of CFmt-DNA did not differ significantly in comparison between the control and the experimental; the Standardized Mean Difference (SMD) was −1.38 (95% CI, −3.56 to 0.79), P = 0.1699 (P > 0.05) in the random effect model. The results of the random effects analysis showed that the study has heterogeneity in the research (I2 = 96%, P < 0.01). In the subgroup analysis, the sample collected from Cerebrospinal fluid (CSF), Tissue and Ex vivo lung perfusion (EVLPP) were statistically significant (P < 0.05). In conclusion, CFmt-DNA can be used as a potential biomarker even if there are variations in copy number. The copy number of CFmt-DNA is influenced by the stress situation and the biological source of the sample.

282-LB: Analysis of Cell-Free Mitochondrial DNA in Obese Patients With and Without Type 2 Diabetes after Bariatric Surgery

While the improvement of mitochondrial function after bariatric surgery was shown, there is limited evidence about the metabolic effects of bariatric surgery on circulatory cell-free (cf) mitochondrial DNA (mtDNA). Therefore, the goal of this study was to compare plasma levels of cf-mtDNA in the association to the metabolic changes before and after surgery in obese patients. Plasma was isolated from 13 morbidly obese bariatric surgery patients with and without type 2 diabetes (T2D) and 8 healthy controls (HC). From each obese patient, blood was collected at baseline, 2 weeks, 3 and 6 months after surgery. QRT-PCR was used to quantify short cf-mtDNA (~100 bp) in isolated plasma within the ND6 region. At baseline, plasma level of short cf-mtDNA fragments was significantly higher in obese patients compared to HC. Additionally, when obese patients were grouped by presence or absence of T2D and compared to HC patients, plasma ND6 levels were significantly higher in obese T2D but not in non-T2D group. Bariatric surgery reduced BMI and improved insulin sensitivity and other whole-body metabolic parameters in all obese patients. No significant variation in mean ND6 values over time was observed post-surgery in all obese patients or when we plotted the data according to the T2D and non-T2D. Importantly, significant positive correlation was observed between plasma ND6 level and HOMA-IR but only 2 weeks post-surgery. Interestingly, plasma from both HC and obese groups at all time points post-surgery contain long (~8 kb) cf-mtDNA fragments, suggesting the presence of near intact and/or whole mitochondrial genomes. Overall, bariatric surgery decreased weight and improved insulin sensitivity and other metabolic parameters in obese patients without significant change in plasma cf-mtDNA levels. Further studies need to test potential determinants, characteristics and origin of cf-mtDNA associated with postoperative metabolic improvements following surgery. Disclosure L. Yuzefovych: None. V. M. Pastukh: None. W. Richards: Research Support; Ethicon, Inc. L. Rachek: None. Funding University of South Alabama (807 to L.R., W.R.)

Cell-free DNA as a potential biomarker of differentiation and toxicity in cardiac organoids

Cell-free DNA (cfDNA) present in the bloodstream or other bodily fluids holds potential as a non-invasive diagnostic for early disease detection. However, it remains unclear what cfDNA markers might be produced in response to specific tissue-level events. Organoid systems present a tractable and efficient method for screening cfDNA markers. However, research investigating the release of cfDNA from organoids is limited. Here, we present a scalable method for high-throughput screening of cfDNA from cardiac organoids. We demonstrate that cfDNA is recoverable from cardiac organoids, and that cfDNA release is highest early in differentiation. Intriguingly, we observed that the fraction of cell-free mitochondrial DNA appeared to decrease as the organoids developed, suggesting a possible signature of cardiac organoid maturation, or other cardiac growth-related tissue-level events. We also observe alterations in the prevalence of specific genomic regions in cardiac organoid-derived cfDNA at different timepoints during growth. In addition, we identify cfDNA markers that were increased upon addition of cardiotoxic drugs, prior to the onset of tissue demise. Together, these results indicate that cardiac organoids may be a useful system towards the identification of candidate predictive cfDNA markers of cardiac tissue development and demise.