Lateral diffusion of redox components in the mitochondrial inner membrane is unaffected by inner membrane folding and matrix density

We report the first lateral diffusion measurements of redox components in normal-sized, matrix-containing, intact mitoplasts (inner membrane-matrix particles). The diffusion measurements were obtained by submicron beam fluorescence recovery after photobleaching measurements of individual, intact, rat liver mitoplasts bathed in different osmolarity media to control the matrix density and the extent of inner membrane folding. The data reveal that neither the extent of mitochondrial matrix density nor the complexity of the inner membrane folding have a significant effect on the mobility of inner membrane redox components. Diffusion coefficients for Complex I (NADH:ubiquinone oxidoreductase), Complex III (ubiquinol: cytochrome c oxidoreductase), Complex IV (cytochrome oxidase), ubiquinone, and phospholipid were found to be effectively invariant with the matrix density and/or membrane folding and essentially the same as values we reported previously for spherical, fused, ultralarge, matrix-free, inner membranes. Diffusion of proton-transporting Complex V (ATP synthase) appeared to be 2-3-fold slower at the greatest matrix density and degree of membrane folding. Consistent with a diffusion-coupled mechanism of electron transport, comparison of electron transport frequencies (productive collisions) with the theoretical, diffusion-controlled, collision frequencies (maximum collisions possible) revealed that there were consistently more calculated than productive collisions for all redox partners. Theoretical analyses of parameters for submicron fluorescence recovery after photobleaching measurements in intact mitoplasts support the finding of highly mobile redox components diffusing at the same rates as determined in conventional fluorescence recovery after photobleaching measurements in fused, ultralarge inner membranes. These findings support the Random Collision Model of Mitochondrial Electron Transport at the level of the intact mitoplast and suggest a similar conclusion for the intact mitochondrion

Development and internal validation of the multivariable CIPHER (Collaborative Integrated Pregnancy High-dependency Estimate of Risk) clinical risk prediction model

Background: Intensive care unit (ICU) outcome prediction models, such as Acute Physiology And Chronic Health Evaluation (APACHE), were designed in general critical care populations and their use in obstetric populations is contentious. The aim of the CIPHER (Collaborative Integrated Pregnancy High-dependency Estimate of Risk) study was to develop and internally validate a multivariable prognostic model calibrated specifically for pregnant or recently delivered women admitted for critical care.Methods: A retrospective observational cohort was created for this study from 13 tertiary facilities across five high-income and six low- or middle-income countries. Women admitted to an ICU for more than 24 h during pregnancy or less than 6 weeks post-partum from 2000 to 2012 were included in the cohort. A composite primary outcome was defined as maternal death or need for organ support for more than 7 days or acute life-saving intervention. Model development involved selection of candidate predictor variables based on prior evidence of effect, availability across study sites, and use of LASSO (Least Absolute Shrinkage and Selection Operator) model building after multiple imputation using chained equations to address missing data for variable selection. The final model was estimated using multivariable logistic regression. Internal validation was completed using bootstrapping to correct for optimism in model performance measures of discrimination and calibration.Results: Overall, 127 out of 769 (16.5%) women experienced an adverse outcome. Predictors included in the final CIPHER model were maternal age, surgery in the preceding 24 h, systolic blood pressure, Glasgow Coma Scale score, serum sodium, serum potassium, activated partial thromboplastin time, arterial blood gas (ABG) pH, serum creatinine, and serum bilirubin. After internal validation, the model maintained excellent discrimination (area under the curve of the receiver operating characteristic (AUROC) 0.82, 95% confidence interval (CI) 0.81 to 0.84) and good calibration (slope of 0.92, 95% CI 0.91 to 0.92 and intercept of −0.11, 95% CI −0.13 to −0.08).Conclusions: The CIPHER model has the potential to be a pragmatic risk prediction tool. CIPHER can identify critically ill pregnant women at highest risk for adverse outcomes, inform counseling of patients about risk, and facilitate bench-marking of outcomes between centers by adjusting for baseline risk

Brain ultrasonography findings in neonates with exposure to cocaine during pregnancy

Background: Cocaine exposure during pregnancy has been reported to have detrimental effects on the fetus. Objective: To describe the findings on cranial ultrasonography (CUS) as part of a neonatal screening programme for exposed neonates. Materials and methods: The study was a semiprospective analysis of a 12-year cohort of neonates born to mothers who had used cocaine during their pregnancy and who had follow-up according to a strict clinical protocol. Results: In total, 154 neonates (78 boys, 76 girls) were included, of whom 29 (19%) were born preterm, and 125 (81%) were born full-term. Abnormalities on CUS were seen in 37 neonates (24%; 95% CI 18-31%). The abnormalities were classified as minor in 20 (13%; 95% CI 9-19%) and mildly abnormal in 17 (11%; 95% CI 7-17%). None of the infants showed severe abnormalities. The abnormalities were not associated with the duration or maximum amount of cocaine use during pregnancy. Conclusion: None of the infants had severe abnormalities. Detected abnormalities were not correlated with the duration or maximum amount of cocaine use. Given these findings, we feel that routine cranial ultrasonography in this population is not warranted

Three-dimensional super-resolution microscopy of the inactive X chromosome territory reveals a collapse of its active nuclear compartment harboring distinct Xist RNA foci

Background: A Xist RNA decorated Barr body is the structural hallmark of the compacted inactive X territory in female mammals. Using super resolution three-dimensional structured illumination microscopy (3D-SIM) and quantitative image analysis, we compared its ultrastructure with active chromosome territories (CTs) in human and mouse somatic cells, and explored the spatio-temporal process of Barr body formation at onset of inactivation in early differentiating mouse embryonic stem cells (ESCs). Results: We demonstrate that all CTs are composed of structurally linked chromatin domain clusters (CDCs). In active CTs the periphery of CDCs harbors low-density chromatin enriched with transcriptionally competent markers, called the perichromatin region (PR). The PR borders on a contiguous channel system, the interchromatin compartment (IC), which starts at nuclear pores and pervades CTs. We propose that the PR and macromolecular complexes in IC channels together form the transcriptionally permissive active nuclear compartment (ANC). The Barr body differs from active CTs by a partially collapsed ANC with CDCs coming significantly closer together, although a rudimentary IC channel system connected to nuclear pores is maintained. Distinct Xist RNA foci, closely adjacent to the nuclear matrix scaffold attachment factor-A (SAF-A) localize throughout Xi along the rudimentary ANC. In early differentiating ESCs initial Xist RNA spreading precedes Barr body formation, which occurs concurrent with the subsequent exclusion of RNA polymerase II (RNAP II). Induction of a transgenic autosomal Xist RNA in a male ESC triggers the formation of an `autosomal Barr body' with less compacted chromatin and incomplete RNAP II exclusion. Conclusions: 3D-SIM provides experimental evidence for profound differences between the functional architecture of transcriptionally active CTs and the Barr body. Basic structural features of CT organization such as CDCs and IC channels are however still recognized, arguing against a uniform compaction of the Barr body at the nucleosome level. The localization of distinct Xist RNA foci at boundaries of the rudimentary ANC may be considered as snap-shots of a dynamic interaction with silenced genes. Enrichment of SAF-A within Xi territories and its close spatial association with Xist RNA suggests their cooperative function for structural organization of Xi

Integrated high-content quantification of intracellular ROS levels and mitochondrial morphofunction

Oxidative stress arises from an imbalance between the production of reactive oxygen species (ROS) and their removal by cellular antioxidant systems. Especially under pathological conditions, mitochondria constitute a relevant source of cellular ROS. These organelles harbor the electron transport chain, bringing electrons in close vicinity to molecular oxygen. Although a full understanding is still lacking, intracellular ROS generation and mitochondrial function are also linked to changes in mitochondrial morphology. To study the intricate relationships between the different factors that govern cellular redox balance in living cells, we have developed a high-contentmicroscopy-based strategy for simultaneous quantification of intracellular ROS levels and mitochondrial morphofunction. Here, we summarize the principles of intracellular ROS generation and removal, and we explain the major considerations for performing quantitative microscopy analyses of ROS and mitochondrial morphofunction in living cells. Next, we describe our workflow, and finally, we illustrate that a multiparametric readout enables the unambiguous classification of chemically perturbed cells as well as laminopathy patient cells