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Mammalian cytosine DNA methylation (5mC) is associated with the integrity of the genome and the transcriptional status of nuclear DNA. Due to technical limitations, it has been less clear if mitochondrial DNA (mtDNA) is methylated and whether 5mC has a regulatory role in this context. Here, we used bisulfite-independent single-molecule sequencing of native human and mouse DNA to study mitochondrial 5mC across different biological conditions. We first validated the ability of long-read nanopore sequencing to detect 5mC in CpG (5mCpG) and non-CpG (5mCpH) context in nuclear DNA at expected genomic locations (i.e. promoters, gene bodies, enhancers, and cell type-specific transcription factor binding sites). Next, using high coverage nanopore sequencing we found low levels of mtDNA CpG and CpH methylation (with several exceptions) and little variation across biological processes: differentiation, oxidative stress, and cancer. 5mCpG and 5mCpH were overall higher in tissues compared to cell lines, with small additional variation between cell lines of different origin. Despite general low levels, global and single-base differences were found in cancer tissues compared to their adjacent counterparts, in particular for 5mCpG. In conclusion, nanopore sequencing is a useful tool for the detection of modified DNA bases on mitochondria that avoid the biases introduced by bisulfite and PCR amplification. Enhanced nanopore basecalling models will provide further resolution on the small size effects detected here, as well as rule out the presence of other DNA modifications such as oxidized forms of 5mC.

OBJECTIVES: To investigate the relationships between brush sign and cerebral collateral status on infarct growth after successful thrombectomy. METHODS: HIBISCUS-STROKE cohort includes acute ischemic stroke patients treated with thrombectomy after MRI triage and undergoing a day-6 MRI including FLAIR images to quantify final infarct volume (FIV). Successful reperfusion was defined as a modified thrombolysis in cerebral infarction score  ≥ 2B. Infarct growth was calculated by subtracting FIV from baseline ischemic core after co-registration and considered large (LIG) when  \\textgreater 11.6 mL. Brush sign was assessed on T2*-weighted-imaging and collaterals were assessed using the hypoperfusion intensity ratio, which is the volume of Time-To-Tmax (Tmax)  ≥ 10 s divided by the volume of Tmax  ≥  6 s. Good collaterals were defined by a hypoperfusion intensity ratio  \\textless 0.4. RESULTS: One hundred and twenty-nine patients were included, of whom 45 (34.9%) had a brush sign and 63 (48.8%) good collaterals. Brush sign was associated with greater infarct growth (p = 0.01) and larger FIV (p = 0.02). Good collaterals were associated with a smaller baseline ischemic core (p \\textless 0.001), larger penumbra (p = 0.04), and smaller FIV (p \\textless 0.001). Collateral status was not significantly associated with brush sign (p = 0.20) or with infarct growth (p = 0.67). Twenty-eight (22.5%) patients experienced LIG. Univariate regressions indicated that brush sign (odds ratio (OR) = 4.8; 95% confidence interval (CI): [1.9;13.3]; p = 0.004) and hemorrhagic transformation (OR = 1.7; 95%CI: [1.2;2.6]; p = 0.04) were predictive of LIG. In multivariate regression, only the brush sign remained predictive of LIG (OR = 5.2; 95%CI: [1.8-16.6], p = 0.006). CONCLUSIONS: Brush sign is a predictor of LIG after successful thrombectomy and cerebral collateral status is not. KEY POINTS: • Few predictors of ischemic growth are known in ischemic stroke patients achieving successful mechanical thrombectomy. • Our results suggest that the brush sign-a surrogate marker of severe hypoperfusion-is independently associated with large ischemic growth (\\textgreater 11.6 mL) after successful thrombectomy whereas cerebral collateral status does not.

Genetically encoded biosensors based on fluorescent proteins (FPs) are widely used to monitor dynamics and sub-cellular spatial distribution of calcium ion (Ca2+) fluxes and their role in intracellular signaling pathways. The development of different mutations in the Ca2+-sensitive elements of the cameleon probes has allowed sensitive range of Ca2+ measurements in almost all cellular compartments. Region of the endoplasmic reticulum (ER) tethered to mitochondria, named as the mitochondrial-associated membranes (MAMs), has received an extended attention since the last 5 years. Indeed, as MAMs are essential for calcium homeostasis and mitochondrial function, molecular tools have been developed to assess quantitatively Ca2+ levels in the MAMs. However, sensitivity of the first generation Ca2+ biosensors on the surface of the outer-mitochondrial membrane (OMM) do not allow to measure μM or sub-μM changes in Ca2+ concentration which prevents to measure the native activity (unstimulated exogenously) of endogenous channels. In this study, we assembled a new ratiometric highly sensitive Ca2+ biosensor expressed on the surface of the outer-mitochondrial membrane (OMM). It allows the detection of smaller differences than the previous biosensor in or at proximity of the MAMs. Noteworthy, we demonstrated that IP3-receptors have an endogenous activity which participate to the Ca2+ leak channel on the surface of the OMM during hypoxia or when SERCA activity is blocked.

The deterioration of sleep in the older population is a prevalent feature that contributes to a decrease in quality of life. Inappropriate entrainment of the circadian clock by light is considered to contribute to the alteration of sleep structure and circadian rhythms in the elderly. The present study investigates the effects of aging on non-visual spectral sensitivity to light and tests the hypothesis that circadian disturbances are related to a decreased light transmittance. In a within-subject design, eight aged and five young subjects were exposed at night to 60 minute monochromatic light stimulations at 9 different wavelengths (420–620 nm). Individual sensitivity spectra were derived from measures of melatonin suppression. Lens density was assessed using a validated psychophysical technique. Although lens transmittance was decreased for short wavelength light in the older participants, melatonin suppression was not reduced. Peak of non-visual sensitivity was, however, shifted to longer wavelengths in the aged participants (494 nm) compared to young (484 nm). Our results indicate that increased lens filtering does not necessarily lead to a decreased non-visual sensitivity to light. The lack of age-related decrease in non-visual sensitivity to light may involve as yet undefined adaptive mechanisms.

Excessive inflammatory response has been implicated in severe respiratory forms of coronavirus disease 2019 (COVID-19). Trace elements such as zinc, selenium, and copper are known to modulate inflammation and immunity. This study aimed to assess the relationships between antioxidant vitamins and mineral trace elements levels as well as COVID-19 severity in older adults hospitalized. In this observational retrospective cohort study, the levels of zinc, selenium, copper, vitamin A, β-carotene, and vitamin E were measured in 94 patients within the first 15 days of hospitalization. The outcomes were in-hospital mortality secondary to COVID-19 or severe COVID-19. A logistic regression analysis was conducted to test whether the levels of vitamins and minerals were independently associated with severity. In this cohort (average age of 78 years), severe forms (46%) were associated with lower zinc (p = 0.012) and β-carotene (p < 0.001) concentrations, and in-hospital mortality (15%) was associated with lower zinc (p = 0.009), selenium (p = 0.014), vitamin A (p = 0.001), and β-carotene (p = 0.002) concentrations. In regression analysis, severe forms remained independently associated with lower zinc (aOR 2.13, p = 0.018) concentrations, and death was associated with lower vitamin A (aOR = 0.165, p = 0.021) concentrations. Low plasma concentrations of zinc and vitamin A were associated with poor prognosis in older people hospitalized with COVID-19.

Disturbances of the daily sleep/wake cycle are common non-motor symptoms of Parkinson's disease (PD). However, the impact of dopamine (DA) depletion on circadian rhythms in PD patients or non-human primate (NHP) models of the disorder have not been investigated. We evaluated alterations of circadian rhythms in NHP following MPTP lesion of the dopaminergic nigro-striatal system. DA degeneration was assessed by in vivo PET ([(11)C]-PE2I) and post-mortem TH and DAT quantification. In a light∶dark cycle, control and MPTP-treated NHP both exhibit rest-wake locomotor rhythms, although DA-depleted NHP show reduced amplitude, decreased stability and increased fragmentation. In all animals, 6-sulphatoxymelatonin peaks at night and cortisol in early morning. When the circadian system is challenged by exposure to constant light, controls retain locomotor rest-wake and hormonal rhythms that free-run with stable phase relationships whereas in the DA-depleted NHP, locomotor rhythms are severely disturbed or completely abolished. The amplitude and phase relations of hormonal rhythms nevertheless remain unaltered. Use of a light-dark masking paradigm shows that expression of daily rest-wake activity in MPTP monkeys requires the stimulatory and inhibitory effects of light and darkness. These results suggest that following DA lesion, the central clock in the SCN remains intact but, in the absence of environmental timing cues, is unable to drive downstream rhythmic processes of striatal clock gene and dopaminergic functions that control locomotor output. These findings suggest that the circadian component of the sleep-wake disturbances in PD is more profoundly affected than previously assumed.

Myocardial infarction (MI) is a serious acute cardiovascular syndrome that causes myocardial injury due to blood flow obstruction to a specific myocardial area. Under ischemic–reperfusion settings, a burst of reactive oxygen species is generated, leading to redox imbalance that could be attributed to several molecules, including myoglobin. Myoglobin is dynamic and exhibits various oxidation–reduction states that have been an early subject of attention in the food industry, specifically for meat consumers. However, rarely if ever have the myoglobin optical properties been used to measure the severity of MI. In the current study, we develop a novel imaging pipeline that integrates tissue clearing, confocal and light sheet fluorescence microscopy, combined with imaging analysis, and processing tools to investigate and characterize the oxidation–reduction states of myoglobin in the ischemic area of the cleared myocardium post-MI. Using spectral imaging, we have characterized the endogenous fluorescence of the myocardium and demonstrated that it is partly composed by fluorescence of myoglobin. Under ischemia–reperfusion experimental settings, we report that the infarcted myocardium spectral signature is similar to that of oxidized myoglobin signal that peaks 3 h post-reperfusion and decreases with cardioprotection. The infarct size assessed by oxidation–reduction imaging at 3 h post-reperfusion was correlated to the one estimated with late gadolinium enhancement MRI at 24 h post-reperfusion. In conclusion, this original work suggests that the redox state of myoglobin can be used as a promising imaging biomarker for characterizing and estimating the size of the MI during early phases of reperfusion.

Studies involving the associations between vitamin D supplementation taken before the onset of COVID-19 infection and the clinical outcomes are still scarce and this issue remains controversial. This study aimed to assess the relationships between vitamin D (VitD) status and supplementation and coronavirus disease 2019 (COVID-19) severity in older adults (average age of 78 years) hospitalized for COVID-19. We conducted an observational retrospective cohort study with 228 older hospitalized patients during the first wave of the COVID-19 pandemic. The outcomes were in-hospital mortality secondary to COVID-19 or critically severe COVID-19. A logistic regression analysis was conducted to test whether pre-hospital VitD supplementation was independently associated with severity. In this study, 46% of patients developed a severe form and the overall in-hospital mortality was 15%. Sixty-six (29%) patients received a VitD supplement during the 3 months preceding the infection onset. Additionally, a VitD supplement was associated with fewer severe COVID-19 forms (OR = 0.426, p = 0.0135) and intensive care unit (ICU) admissions (OR = 0.341, p = 0.0076). As expected, age > 70 years, male gender and BMI ≥ 35 kg/m2 were independent risk factors for severe forms of COVID-19. No relationship between serum 25(OH)D levels and the severity of the COVID-19 was identified. VitD supplementation taken during the 3 months preceding the infection onset may have a protective effect on the development of severe COVID-19 forms in older adults. Randomized controlled trials and large-scale cohort studies are necessary to strengthen this observation.

The Ca2+ release in microdomains formed by intercompartmental contacts, such as mitochondria-associated endoplasmic reticulum membranes (MAMs), encodes a signal that contributes to Ca2+ homeostasis and cell fate control. However, the composition and function of MAMs remain to be fully defined. Here, we focused on the transient receptor potential vanilloid 1 (TRPV1), a Ca2+-permeable ion channel and a polymodal nociceptor. We found TRPV1 channels in the reticular membrane, including some at MAMs, in a rat cardiomyoblast cell line (SV40-transformed H9c2) by Western blotting, immunostaining, cell fractionation, and proximity ligation assay. We used chemical and genetic probes to perform Ca2+ imaging in four cellular compartments: the endoplasmic reticulum (ER), cytoplasm, mitochondrial matrix, and mitochondrial surface. Our results showed that the ER Ca2+ released through TRPV1 channels is detected at the mitochondrial outer membrane and transferred to the mitochondria. Finally, we observed that prolonged TRPV1 modulation for 30 min alters the intracellular Ca2+ equilibrium and influences the MAM structure or the hypoxia/reoxygenation-induced cell death. Thus, our study provides the first evidence that TRPV1 channels contribute to MAM Ca2+ exchanges.

Primary Carnitine Deficiency (PCD) is a fatty acid oxidation disorder that will be included in the expansion of the French newborn screening (NBS) program at the beginning of 2023. This disease is of high complexity to screen, due to its pathophysiology and wide clinical spectrum. To date, few countries screen newborns for PCD and struggle with high false positive rates. Some have even removed PCD from their screening programs. To understand the risks and pitfalls of implementing PCD to the newborn screening program, we reviewed and analyzed the literature to identify hurdles and benefits from the experiences of countries already screening this inborn error of metabolism. In this study, we therefore, present the main pitfalls encountered and a worldwide overview of current practices in PCD newborn screening. In addition, we address the optimized screening algorithm that has been determined in France for the implementation of this new condition.