The ubiquitous mitochondrial protein unfoldase CLPX regulates erythroid heme synthesis by control of iron utilization and heme synthesis enzyme activation and turnover.

Heme plays a critical role in catalyzing life-essential redox reactions in all cells, and its synthesis must be tightly balanced with cellular requirements. Heme synthesis in eukaryotes is tightly regulated by the mitochondrial AAA+ unfoldase CLPX (caseinolytic mitochondrial matrix peptidase chaperone subunit X), which promotes heme synthesis by activation of δ-aminolevulinate synthase (ALAS/Hem1) in yeast and regulates turnover of ALAS1 in human cells. However, the specific mechanisms by which CLPX regulates heme synthesis are unclear. In this study, we interrogated the mechanisms by which CLPX regulates heme synthesis in erythroid cells. Quantitation of enzyme activity and protein degradation showed that ALAS2 stability and activity were both increased in the absence of CLPX, suggesting that CLPX primarily regulates ALAS2 by control of its turnover, rather than its activation. However, we also showed that CLPX is required for PPOX (protoporphyrinogen IX oxidase) activity and maintenance of FECH (ferrochelatase) levels, which are the terminal enzymes in heme synthesis, likely accounting for the heme deficiency and porphyrin accumulation observed in Clpx-/- cells. Lastly, CLPX is required for iron utilization for hemoglobin synthesis during erythroid differentiation. Collectively, our data show that the role of CLPX in yeast ALAS/Hem1 activation is not conserved in vertebrates as vertebrates rely on CLPX to regulate ALAS turnover as well as PPOX and FECH activity. Our studies reveal that CLPX mutations may cause anemia and porphyria via dysregulation of ALAS, FECH, and PPOX activities, as well as of iron metabolism.

Modeling Cataract Surgery in Mice.

Cataract surgery (CS) is an effective treatment for cataracts, a major cause of visual disability worldwide. However, CS leads to ocular inflammation, and in the long term, it can result in posterior capsular opacification (PCO) and/or lens dislocation driven by the post-surgical overgrowth of lens epithelial cells (LECs) and their conversion to myofibroblasts and/or aberrant fiber cells. However, the molecular mechanisms by which CS results in inflammation and PCO are still obscure because most in vitro models do not recapitulate the wound healing response of LECs seen in vivo, while traditional animal models of cataract surgery, such as rabbits, do not allow the genetic manipulation of gene expression to test mechanisms. Recently, our laboratory and others have successfully used genetically modified mice to study the molecular mechanisms that drive the induction of proinflammatory signaling and LEC epithelial to mesenchymal transition, leading to new insight into PCO pathogenesis. Here, we report the established protocol for modeling cataract surgery in mice, which allows for robust transcriptional profiling of the response of LECs to lens fiber cell removal via RNAseq, the evaluation of protein expression by semi-quantitative immunofluorescence, and the use of modern mouse genetics tools to test the function of genes that are hypothesized to participate in the pathogenesis of acute sequelae like inflammation as well as the later conversion of LECs to myofibroblasts and/or aberrant lens fiber cells.

Implication of Vaginal and Cesarean Section Delivery Method in Black-White Differentials in Infant Mortality in the United States: Linked Birth/Infant Death Records, 2007-2016.

Racial/ethnic disparities in infant mortality (IM) continue to persist in the United States, with Black/African Americans (AA) being disproportionally affected with a three-fold increase in mortality compared to Whites. Epidemiological data have identified maternal characteristics in IM risk such as preeclampsia, eclampsia, maternal education, smoking, maternal weight, maternal socioeconomic status (SES), and family structure. Understanding the social gradient in health including implicit bias, as inherent in the method of labor and delivery and the racial heterogeneity, may facilitate intervention mapping in narrowing the Black-White IM risk differences. We aimed to assess the temporal/racial trends and the methods of delivery, mainly vaginal vs. cesarean section (C-section) as an exposure function of IM. The United States linked birth/infant death records (2007-2016) were used with a cross-sectional ecological design. The analysis involved chi squared statistic, incidence rate estimation by binomial regression model, and period percent change. Of the 40,445,070 births between 2007 and 2016, cumulative mortality incidence was 249,135 (1.16 per 1000). The IM rate was highest among Black/AA (11.41 per 1000), intermediate among Whites (5.19 per 1000), and lowest among Asian /Pacific Islanders (4.24 per 1000). The cumulative incidence rate difference, comparing vaginal to cesarean procedure was 1.73 per 1000 infants, implying excess IM with C-section. Compared to C-section, there was a 31% decreased risk of IM among mothers with vaginal delivery, rate ratio (RR) = 0.69, 95% confidence interval (CI): 0.64-0.74. Racial disparities were observed in the method of delivery associated with IM. Black/AA mothers with vaginal delivery had a 6% decreased risk of IM compared to C-section, RR = 0.94, 95% CI: 0.92-0.95, while Whites with vaginal delivery had a 38% decrease risk of IM relative to C-section, RR= 0.68, 95% CI: 0.67-0.69, p < 0.001. Infant mortality varied by race, with Black/AA disproportionally affected, which is explained in part by labor and delivery procedures, suggestive of reliable and equitable intrapartum assessment of Black/AA mothers during labor, as well as implicit bias marginalization in the healthcare system.