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In eukaryotic cells, intron lariats produced by the spliceosome contain a 2′5′ phosphodiester linkage. The RNA lariat debranching enzyme, Dbr1, is the only enzyme known to hydrolyze this bond. Dbr1 is a member of the metallophosphoesterase (MPE) family of enzymes, and recent X-ray crystal structures and biochemistry data demonstrate that Dbr1 from Entamoeba histolytica uses combinations of Mn2+, Zn2+, and Fe2+ as enzymatic cofactors. Here, we examine the kinetic properties and metal dependence of the Dbr1 homolog from Saccharomyces cerevisiae (yDbr1). Elemental analysis measured stoichiometric quantities of Fe and Zn in yDbr1 purified following heterologous expression E. coli. We analyzed the ability of Fe2+, Zn2+, and Mn2+ to reconstitute activity in metal-free apoenzyme. Purified yDbr1 was highly active, turning over substrate at 5.6 sec−1, and apo-yDbr1 reconstituted with Fe2+ was the most active species, turning over at 9.2 sec−1. We treated human lymphoblastoid cells with the iron-chelator deferoxamine and measured a twofold increase in cellular lariats. These data suggest that Fe is an important biological cofactor for Dbr1 enzymes.

Amniotic products are potent immunomodulators used clinically to repair tissue injury. Little information exists regarding the potential of cell-free human amniotic fluid (hAF) to treat cardiovascular disease. Herein, we sought to determine the influence and efficacy of acellular hAF on myocardial ischemia-reperfusion injury. Processed hAF was obtained from volunteer donors at the time of elective caesarean section and manufactured using proprietary methods. Left anterior descending coronary artery ligation was performed on rats for 60 min. Thirty minutes after release and reperfusion, either saline or hAF was injected intramyocardially. Serial echocardiography revealed that compared with saline-injected rats, hAF animals maintained their ejection fraction and did not adversely remodel through the 4-wk period. This preserved ventricular function correlated with decreased infarct size, less fibrosis, and reduced expression of cytokines and infiltrating inflammatory cells. Comparative arrays of different donor hAF lots confirmed the presence of a wide array of immunomodulatory and host-defense proteins. The observed functional cardioprotection was furthermore evident when given intravenously and across multiple hAF donors. In conclusion, our data demonstrate, for the first time, the cardioprotective effect of acellular hAF on myocardial injury. These observations spanned across diverse donors and likely result from the mixture of a plethora of naturally produced cytokines, chemokines, and immune-modulating proteins rather than a single, defined mechanistic culprit. The ubiquitous availability of hAF as a cell-free solution further suggests its potential for widespread adoption as a therapy for myocardial ischemia-reperfusion injury.

The Mendelian inheritance pattern of acute intermittent porphyria, hereditary coproporphyria, and variegate porphyria is autosomal dominant, but the clinical phenotype is heterogeneous. Within the general population, penetrance is low, but among first-degree relatives of a symptomatic proband, penetrance is higher. These observations suggest that genetic factors, in addition to mutation of the specific enzyme of the biosynthetic pathway of heme, contribute to the clinical phenotype. Recent studies by others suggested that the genotype of the transporter protein ABCB6 contribute to the porphyria phenotype. Identifying the molecule(s) that are transported by ABCB6 has been problematic and has led to uncertainty with respect to how or if variants/mutants contribute to phenotypic heterogeneity. Knockout mouse models of Abcb6 have not provided a direction for investigation as homozygous knockout animals do not have a discrete phenotype. To address the proposed link between ABC6 genotype and porphyria phenotype, a large cohort of patients with acute hepatic porphyria and erythropoietic protoporphyria was analyzed. Our studies showed that ABCB6 genotype did not correlate with disease severity. Therefore, genotyping of ABCB6 in patients with acute hepatic porphyria and erythropoietic protoporphyria is not warranted.

As part of the inflammatory response by macrophages, Irg1 is induced, resulting in millimolar quantities of itaconate being produced. This immunometabolite remodels the macrophage metabolome and acts as an antimicrobial agent when excreted. Itaconate is not synthesized within the erythron but instead may be acquired from central macrophages within the erythroid island. Previously, we reported that itaconate inhibits hemoglobinization of developing erythroid cells. Herein we show that this action is accomplished by inhibition of tetrapyrrole synthesis. In differentiating erythroid precursors, cellular heme and protoporphyrin IX synthesis are reduced by itaconate at an early step in the pathway. In addition, itaconate causes global alterations in cellular metabolite pools, resulting in elevated levels of succinate, 2-hydroxyglutarate, pyruvate, glyoxylate, and intermediates of glycolytic shunts. Itaconate taken up by the developing erythron can be converted to itaconyl–coenzyme A (CoA) by the enzyme succinyl-CoA:glutarate-CoA transferase. Propionyl-CoA, propionyl-carnitine, methylmalonic acid, heptadecanoic acid, and nonanoic acid, as well as the aliphatic amino acids threonine, valine, methionine, and isoleucine, are increased, likely due to the impact of endogenous itaconyl-CoA synthesis. We further show that itaconyl-CoA is a competitive inhibitor of the erythroid-specific 5-aminolevulinate synthase (ALAS2), the first and rate-limiting step in heme synthesis. These findings strongly support our hypothesis that the inhibition of heme synthesis observed in chronic inflammation is mediated not only by iron limitation but also by limitation of tetrapyrrole synthesis at the point of ALAS2 catalysis by itaconate. Thus, we propose that macrophage-derived itaconate promotes anemia during an inflammatory response in the erythroid compartment.

Objective Preterm infants frequently receive red cell transfusions; however, the effect of transfusions on cognition is unclear. We evaluated the relationship between transfusions and cognitive outcomes in preterm infants enrolled in a randomized trial of erythropoiesis stimulating agents (ESAs). Study design Preterm infants were randomized to ESAs or placebo during initial hospitalization, and transfusions recorded. Children were evaluated using standard developmental tests of cognition at 18-22 months (56 ESA, 24 placebo) and 3.5-4 years (39 ESA, 14 placebo). Results Cognitive scores at 18-22 months were inversely correlated with transfusion volume (p = 0.02). Among those receiving ≥1 transfusion, cognitive scores were significantly higher in the ESA-treated group (p = 0.003). At 3.5-4 years, transfusions were not correlated with cognitive scores. Conclusions In the placebo group, transfused children had lower cognitive scores than did non-transfused children at 18-22 months. In the ESA group, cognitive scores did not differ by transfusion status, suggesting ESAs might provide neuroprotection.

BACKGROUND AND AIMS The risk for hepatocellular carcinoma (HCC) is increased in acute hepatic porphyrias (AHP). The aim of this study was to explore the clinicopathologic characteristics, outcomes, and frequency of HCC in patients with AHP in the United States. APPROACH AND RESULTS This cross-sectional analysis evaluated patients with HCC in a multicenter, longitudinal study of AHP. Among 327 patients with AHP, 5 (1.5%) were diagnosed with HCC. Of the 5 HCC cases, 4 had acute intermittent porphyria and 1 had variegate porphyria, confirmed by biochemical and/or genetic testing. All patients were white females, with a median age of 27 years (range 21-75) at diagnosis. The median age at HCC diagnosis was 69 years (range 61-74). AHP was asymptomatic in 2 patients; 2 reported sporadic attacks; and 1 reported recurrent attacks (>4 attacks/year). All patients had a single HCC lesion on liver imaging that was 1.8-6.5 centimeters in diameter. Serum alpha fetoprotein levels were below 10 ng/mL in all 4 patients with available results. Four patients underwent liver resection, and 1 was treated with radioembolization. No significant inflammation or fibrosis was found in adjacent liver tissues of 3 patients who underwent liver resection. Two patients developed recurrence of HCC at 22 and 26 months following liver resection. All patients are alive with survival times from HCC diagnosis ranging from 26-153 months. CONCLUSION In this U.S. study, 1.5% of patients with AHP had HCC. HCC in AHP occurred in the absence of cirrhosis, which contrasts with other chronic liver diseases. Patients with AHP, regardless of clinical attacks, should be screened for HCC, beginning at age 50. The pathogenesis of hepatocarcinogenesis in AHP is unknown and needs further investigation.

Background: Acute Intermittent Porphyria (AIP) is a rare inborn error of heme biosynthesis characterized clinically by life-threatening acute neurovisceral attacks. Few studies have assessed quality of life (QoL) tools in the AIP population, and none are validated for use in AIP. In this study, the Patient Reported Outcomes Measurement Information System 57 (PROMIS-57) scales were assessed in AIP patients to determine the factor structure of PROMIS items and to compare this to previously proposed factor structures in the general population.Methods: Baseline data from the Porphyrias Consortium’s Longitudinal Study of the Porphyrias was obtained for 259 AIP patients. This included detailed disease and medical history information as well as PROMIS-57 data. Exploratory and confirmatory factor analyses (EFA and CFA, respectively) were conducted on baseline PROMIS-57 data. CFA was used to test the hypothesized three-factor structure from the PROMIS literature. Results: Internal consistency was high for all the PROMIS scales assessed. The EFA revealed a five-factor model, each consisting of a separate domain: pain interference, anxiety, depression, sleep disturbance, and fatigue. Model fit was good overall. A CFA of the hypothesized three-factor model did not converge suggesting an inappropriate structural model. Conclusion: The EFA showed a five-factor model that fit the data well, however a CFA of the three-factor model observed in the general population did not fit the data well. These findings, together with previous studies assessing correlations with clinical features, indicate that the PROMIS scales may be valid and reliable measures for AIP patients. However, further analyses are needed to confirm this. Further studies should assess correlations with other tools, whether the PROMIS scales are responsive to treatment, if they capture QoL longitudinally, and a CFA in a second sample.

Multidrug Resistance Proteins (MRPs) are transporters that play critical roles in cancer even though the physiological substrates of these enigmatic transporters are poorly elucidated. InCaenorhabditis elegans, MRP5/ABCC5 is an essential heme exporter asmrp-5mutants are unviable due to their inability to export heme from the intestine to extra-intestinal tissues. Heme supplementation restores viability of these mutants but fails to restore male reproductive deficits. Correspondingly, cell biological studies show that MRP5 regulates heme levels in the mammalian secretory pathway even though MRP5 knockout (KO) mice do not show reproductive phenotypes. The closest homolog of MRP5 is MRP9/ABCC12, which is absent inC. elegansraising the possibility that MRP9 may genetically compensate for MRP5. Here, we show that MRP5 and MRP9 double KO mice are viable but reveal significant male reproductive deficits. Although MRP9 is highly expressed in sperm, MRP9 KO mice show reproductive phenotypes only when MRP5 is absent. Both ABCC transporters localize to mitochondrial-associated membranes (MAMs), dynamic scaffolds that associate the mitochondria and endoplasmic reticulum. Consequently, DKO mice reveal abnormal sperm mitochondria with reduced mitochondrial membrane potential and fertilization rates. Metabolomics show striking differences in metabolite profiles in the DKO testes and RNA-seq show significant alterations in genes related to mitochondrial function and retinoic acid metabolism. Targeted functional metabolomics reveal lower retinoic acid levels in the DKO testes and higher levels of triglycerides in the mitochondria. These findings establish a model in which MRP5 and MRP9 play a concerted role in regulating male reproductive functions and mitochondrial sufficiency.Significance StatementMRPs are typically implicated in cancer biology. Here, we show that MRP9 and MRP5 localize to mitochondrial-associated membranes and play a concerted role in maintaining mitochondrial homeostasis and male reproductive fitness. Our work fills in significant gaps in our understanding of MRP9 and MRR5 with wider implications in male fertility. It is plausible that variants in these transporters are associated with male reproductive dysfunction.

Accumulation of protoporphyrin IX (PPIX) and Zn-PPIX, are the clinical hallmarks of protoporphyria. Phenotypic expression of protoporphyria is due to decreased activity of ferrochelatase (FECH) or to increased activity of aminolevulinic acid synthase (ALAS) in red blood cells. Other genetic defects have been shown to contribute to disease severity including loss of function mutations in the mitochondrial AAA-ATPase, CLPX and mutations in the Iron-responsive element binding protein 2 (IRP2), in mice. It is clear that multiple paths lead to a common phenotype of excess plasma PPIX that causes a phototoxic reaction on sun exposed areas. In this study we examined the association between mitochondrial iron acquisition and utilization with activity of FECH. Our data show that there is a metabolic link between the activity FECH and levels of MFRN1 mRNA. We examined the correlation between FECH activity and MFRN1 mRNA in cell lines established from patients with the classical protoporphyria, porphyria due to defects in ALAS2 mutations. Our data confirm MFRN1 message levels positively correlated with FECH enzymatic activity in all cell types.

Author(s): Naik, Hetanshi; Overbey, Jessica R; Desnick, Robert J; Anderson, Karl E; Bissell, D Montgomery; Bloomer, Joseph; Bonkovsky, Herbert L; Phillips, John D; Wang, Bruce; Singal, Ashwani; Balwani, Manisha | Abstract: BackgroundErythropoietic protoporphyria (EPP) and X-linked Protoporphyria (XLP) are rare photodermatoses presenting with severe phototoxicity. Although anecdotally, providers who treat EPP patients acknowledge their life-altering effects, tools that fully capture their impact on quality of life (QoL) are lacking.MethodsAdult patients with EPP/XLP were given four validated QoL tools: the Patient Reported Outcomes Measurement Information System 57 (PROMIS-57), the Hospital Anxiety and Depression Scale (HADS), the Illness Perception Questionnaire Revised (IPQR), and an EPP-Specific tool. All patients received the PROMIS-57 while the HADS, IPQR, and EPP-Specific tools were introduced at a later date. Associations between responses and clinical phenotypes were explored.ResultsTwo hundred and two patients were included; 193 completed PROMIS-57, 104 completed IPQR, 103 completed HADS, and 107 completed the EPP-Specific tool. The IPQR showed that patients strongly believed EPP/XLP had a negative impact on their lives. Mean scores in anxiety and depression domains of both HADS and PROMIS-57 were normal; however, anxiety scores from HADS were borderline/abnormal in 20% of patients. The EPP-Specific tool revealed a decreased QoL in most patients. The PROMIS-57 showed that 21.8% of patients have clinically significant pain interference. Several tool domains correlated with measures of disease severity, most being from the PROMIS-57.ConclusionsImpaired QoL is an important consequence of EPP/XLP. PROMIS-57 was most sensitive in evaluating impaired QoL in EPP/XLP. Further research is needed to compare the effectiveness of it for assessing response to treatment.

Erythropoietic protoporphyria (EPP), the most common porphyria of childhood and the third most common porphyria of adulthood, is characterized clinically by painful, non-blistering cutaneous photosensitivity. Two distinct inheritance patterns involving mutations affecting genes that encode enzymes of the heme biosynthetic pathway underlie the clinical phenotype. Aminolevulinic acid synthase 2 (ALAS2), the rate limiting enzyme of the heme pathway in the erythron, is a therapeutic target in EPP because inhibiting enzyme function would reduce downstream production of protoporphyrin IX (PPIX), preventing accumulation of the toxic molecule and thereby ameliorating symptoms. Isoniazid (INH) is widely used for treatment of latent and active M. tuberculosis (TB). Sideroblastic anemia is observed in some patients taking INH, and studies have shown that this process is a consequence of inhibition of ALAS2 by INH. Based on these observations, we postulated that INH might have therapeutic activity in patients with EPP. We challenged this hypothesis in a murine model of EPP and showed that, after 4 weeks of treatment with INH, both plasma PPIX and hepatic PPIX were significantly reduced. Next, we tested the effect of INH on patients with EPP. After eight weeks, no significant difference in plasma or red cell PPIX was observed among the 15 patients enrolled in the study. These results demonstrate that while INH can lower PPIX in an animal model of EPP, the standard dose used to treat TB is insufficient to affect levels in humans.

Protodunes emerge from a flat sand bed at the upwind margin of White Sands Dune Field, and, over several hundred meters, transition into fully developed dunes. Here, we investigate spatial and temporal changes in topography across this transition from 2007 to 2016 using lidar-derived topography, structure-from-motion-derived topography, and RTK GPS. We characterize the deposits present in 2015 using ground penetrating radar. Symmetric protodunes give way downwind to an asymmetric protodune at the transition to slipface development. Between 2007 and 2016, protodune amplitude increased from 0.2 m to 4.0 m, migration rate increased from 3.2 m/yr to 6.1 m/yr, and wavelength increased from 76 m to 122 m. Ground-penetrating radar surveys show strata between flat and 15° make up the stratigraphic architecture of the protodunes. Strata increase in steepness commensurate with an increase in amplitude. Decimeter accumulations of low-angle strata associated with initial protodune stages give way to 4 m of accumulation composed of sets up to 1 m thick prior to slipface development. Topsets present in the thickest sets indicate near critical angles of bedform climb. Growth and slipface development occur by aerodynamic sand trapping and protodune merging. Changes in asymmetry erase initial slipfaces prior to permanent slipface development, after which efficient sand trapping and scour promotes the transition to a dune across 20 m in 5 years. Protodune stratification has hallmarks of sandsheet stratification and can be appreciated within the greater suite of processes that create low-angle eolian stratification found in modern and ancient environments.

Acute intermittent porphyria (AIP) is an inborn error of heme biosynthesis due to the deficiency of hydroxymethylbilane synthase (HMBS) activity. Human AIP heterozygotes have episodic acute neurovisceral attacks that typically start after puberty, whereas patients with homozygous dominant AIP (HD-AIP) have early-onset chronic neurological impairment, including ataxia and psychomotor retardation. To investigate the dramatically different manifestations, knock-in mice with human HD-AIP missense mutations c.500G>A (p.Arg167Glu) or c.518_519GC>AG (p.Arg173Glu), designated R167Q or R173Q mice, respectively, were generated and compared with the previously established T1/T2 mice with ~30% residual HMBS activity and the heterozygous AIP phenotype. Homozygous R173Q mice were embryonic lethal, while R167Q homozygous mice (R167Q(+/+)) had ~5% of normal HMBS activity, constitutively elevated plasma and urinary 5-aminolevulinic acid (ALA) and porphobilinogen (PBG), profound early-onset ataxia, delayed motor development and markedly impaired rotarod performance. Central nervous system (CNS) histology was grossly intact, but CNS myelination was delayed and overall myelin volume was decreased. Heme concentrations in liver and brain were similar to those of T1/T2 mice. Notably, ALA and PBG concentrations in the cerebral spinal fluid and CNS regions were markedly elevated in R167Q(+/+) mice compared with T1/T2 mice. When the T1/T2 mice were administered phenobarbital, ALA and PBG markedly accumulated in their liver and plasma, but not in the CNS, indicating that ALA and PBG do not readily cross the blood–brain barrier. Taken together, these studies suggest that the severe HD-AIP neurological phenotype results from decreased myelination and the accumulation of locally produced neurotoxic porphyrin precursors within the CNS.

Acute intermittent porphyria (AIP), an autosomal dominant disorder due to the half-normal activity of hydroxymethylbilane synthase (HMBS), is characterized by acute neurovisceral attacks that are precipitated by factors that induce heme biosynthesis. Molecular diagnosis is the most sensitive and specific diagnostic test for AIP, and importantly, it permits the identification of asymptomatic family members for genetic counseling and avoidance of precipitating factors. Here, we report the identification of 40 novel HMBS mutations, including 11 missense, four nonsense, 16 small insertions or deletions, eight consensus splice site mutations, and a complex insertion-deletion mutation in unrelated individuals with AIP. Prokaryotic expression of the missense mutations demonstrated that all mutants had ≤5% of expressed wildtype activity, except for c.1039G>C (p.A347P), which had 51% residual HMBS activity but was markedly thermolabile. Of note, the mutation c.612G>T (p.Q204H) altered the last nucleotide of exon 10, which resulted in an alternative HMBS transcript with an in-frame nine base-pair deletion at the 3'-terminus of exon 10 (encoding protein Q204HΔ3). When expressed, Q204HΔ3 and an in-frame three base-pair deletion (c.639_641delTGC) had no detectable HMBS activity. Western blot analyses and mapping of the missense mutations on the human HMBS crystal structure revealed that mutations near the active site or at the dimerization interface resulted in stably expressed proteins, while most that altered surface residues resulted in unstable proteins, presumably due to improper protein folding. These studies identified novel pathogenic HMBS mutations and expanded the molecular heterogeneity of AIP.

Erythropoietin (EPO) signaling is critical to many processes essential to terminal erythropoiesis. Despite the centrality of iron metabolism to erythropoiesis, the mechanisms by which EPO regulates iron status are not well-understood. To this end, here we profiled gene expression in EPO-treated 32D pro-B cells and developing fetal liver erythroid cells to identify additional iron regulatory genes. We determined that FAM210B, a mitochondrial inner-membrane protein, is essential for hemoglobinization, proliferation, and enucleation during terminal erythroid maturation. Fam210b deficiency led to defects in mitochondrial iron uptake, heme synthesis, and iron-sulfur cluster formation. These defects were corrected with a lipid-soluble, small-molecule iron transporter, hinokitiol, in Fam210b-deficient murine erythroid cells and zebrafish morphants. Genetic complementation experiments revealed that FAM210B is not a mitochondrial iron transporter but is required for adequate mitochondrial iron import to sustain heme synthesis and iron-sulfur cluster formation during erythroid differentiation. FAM210B was also required for maximal ferrochelatase activity in differentiating erythroid cells. We propose that FAM210B functions as an adaptor protein that facilitates the formation of an oligomeric mitochondrial iron transport complex, required for the increase in iron acquisition for heme synthesis during terminal erythropoiesis. Collectively, our results reveal a critical mechanism by which EPO signaling regulates terminal erythropoiesis and iron metabolism.

IntroductionHuman amniotic fluid (hAF) has been shown to reduce inflammation in multiple experimental models. hAF has previously been approved by the US Food and Drug Administration (FDA) as a human cellular and tissue product for tissue injury for human administration, and used safely in thousands of patients as a therapeutic treatment for diverse conditions. Given the profound inflammatory response observed in patients with COVID-19, and the successful completion of 10-patient pilot study of intravenous hAF, we present a trial design for a larger clinical trial of intravenous hAF for the treatment of COVID-19.Methods and analysisThis paper describes the methodology of a phase I/II randomised, double-blinded, placebo-controlled clinical trial to determine the safety and feasibility of using acellular sterile filtered amniotic fluid as a treatment for patients with COVID-19. Primary outcome will be the change in C-reactive protein. Secondary outcomes include safety, biomarker inflammatory levels and clinically relevant outcomes at 30 days, including mortality, ventilator-free days and hospital and intensive care unit length of stay. Exploratory outcomes of health-related quality-of-life patient-reported outcomes will be collected. Hospitalised patients with laboratory-confirmed COVID-19 will be recruited.Ethics and disseminationThis study was approved by the University of Utah Institutional Review Board (IRB_0013292), approved by the US FDA under Investigational New Drug (No 23369) and is registered on ClinicalTrials.gov. Results will be disseminated via peer-reviewed publications and conference presentations.Trial registration numberNCT04497389; Pre-results.

Background Immature reticulocytes (IRC) are the first cells to respond to changes in erythropoiesis. For antidoping applications, measurement of IRC may improve detection of blood doping practices. Unfortunately, this small cell population has limited stability in liquid blood samples and is difficult to measure with optimal precision. We developed a method to measure 3 IRC membrane proteins in dried blood spots (DBS) to monitor changes in erythropoiesis. Methods DBS spots were washed with buffers to remove soluble proteins, membrane proteins remaining in the spot were digested with trypsin, and one peptide for each protein was measured by LC-MS/MS. IRC protein concentration was determined using a DBS single point calibrator. Results Intraassay precision for IRC proteins was between 5%–15%. IRC proteins were stable in DBS for 29 days at room temperature. In a longitudinal study of 25 volunteers, the mean intraindividual variation for 3 IRC proteins was 17%, 20%, and 24% from capillary blood DBS. In comparison, the mean longitudinal variation for IRC counts measured on an automated hematology analyzer was 38%. IRC protein concentration from capillary blood DBS correlated well with venous blood DBS protein concentrations. Conclusions Measurement of IRC proteins in DBS samples provides a method to measure changes in erythropoiesis with improved analytical sensitivity, stability, and precision. When combined with the inherent advantages of capillary blood collection in the field, this method may substantially improve the detection of blood doping practices.

International audience; Mitochondrial iron import is essential for iron-sulfur cluster formation and heme biosynthesis. Two nuclear-encoded vertebrate mitochondrial high-affinity iron importers, mitoferrin1 (Mfrn1) and Mfrn2, have been identified in mammals. In mice, the gene encoding Mfrn1, solute carrier family 25 member 37 (Slc25a37), is highly expressed in sites of erythropoiesis, and whole-body Slc25a37 deletion leads to lethality. Here, we report that mice with a deletion of Slc25a28 (encoding Mfrn2) are born at expected Mendelian ratios, but show decreased male fertility due to reduced sperm numbers and sperm motility. Mfrn2 -/- mice placed on a low-iron diet exhibited reduced mitochondrial manganese, cobalt, and zinc levels, but not reduced iron. Hepatocyte-specific loss of Slc25a37 (encoding Mfrn1) in Mfrn2 -/- mice did not affect animal viability, but resulted in a 40% reduction in mitochondrial iron and reduced levels of oxidative phosphorylation proteins. Placing animals on a low-iron diet exaggerated the reduction in mitochondrial iron observed in liver-specific Mfrn1/2-knockout animals. Mfrn1 -/-/Mfrn2 -/- bone marrow-derived macrophages or skin fibroblasts in vitro were unable to proliferate, and overexpression of Mfrn1-GFP or Mfrn2-GFP prevented this proliferation defect. Loss of both mitoferrins in hepatocytes dramatically reduced regeneration in the adult mouse liver, further supporting the notion that both mitoferrins transport iron and that their absence limits proliferative capacity of mammalian cells. We conclude that Mfrn1 and Mfrn2 contribute to mitochondrial iron homeostasis and are required for high-affinity iron import during active proliferation of mammalian cells.

Acute hepatic porphyria comprises a group of rare, genetic diseases caused by mutations in genes involved in heme biosynthesis. Patients can experience acute neurovisceral attacks, debilitating chronic symptoms, and long-term complications. There is a lack of multinational, prospective data characterizing the disease and current treatment practices in severely affected patients. EXPLORE is a prospective, multinational, natural history study characterizing disease activity and clinical management in patients with acute hepatic porphyria who experience recurrent attacks. Eligible patients had a confirmed acute hepatic porphyria diagnosis and had experienced ≥3 attacks in the prior 12 months or were receiving prophylactic treatment. A total of 112 patients were enrolled and followed for at least 6 months. In the 12 months prior to the study, patients reported a median (range) of 6 (0-52) acute attacks, with 52 (46%) patients receiving hemin prophylaxis. Chronic symptoms were reported by 73 (65%) patients, with 52 (46%) patients experiencing these daily. During the study, 98 (88%) patients experienced a total of 483 attacks, 77% of which required treatment at a healthcare facility and/or hemin administration (median [range] annualized attack rate 2.0 [0.0-37.0]). Elevated levels of hepatic δ-aminolevulinic acid synthase 1 messenger ribonucleic acid levels, δ-aminolevulinic acid, and porphobilinogen compared with the upper limit of normal in healthy individuals were observed at baseline and increased further during attacks. Patients had impaired quality of life and increased healthcare utilization. Conclusions: Patients experienced attacks often requiring treatment in a healthcare facility and/or with hemin, as well as chronic symptoms that adversely influence day-to-day functioning. In this patient group, the high disease burden and diminished quality of life highlight the need for novel therapies. This article is protected by copyright. All rights reserved.

PurposeAcute intermittent porphyria (AIP) is a rare inborn error of heme biosynthesis characterized by life-threatening acute attacks. Few studies have assessed quality of life (QoL) in AIP and those that have had small sample sizes and used tools that may not have captured important domains.MethodsBaseline data from the Porphyrias Consortium's Longitudinal Study were obtained for 259 patients, including detailed disease and medical history data, and the following Patient-Reported Outcomes Measurement Information System (PROMIS) scales: anxiety, depression, pain interference, fatigue, sleep disturbance, physical function, and satisfaction with social roles. Relationships between PROMIS scores and clinical and biochemical AIP features were explored.ResultsPROMIS scores were significantly worse than the general population across all domains, except depression. Each domain discriminated well between asymptomatic and symptomatic patients with symptomatic patients having worse scores. Many important clinical variables like symptom frequency were significantly associated with domain scores in univariate analyses, showing responsiveness of the scales, specifically pain interference and fatigue. However, most regression models only explained ~20% of the variability observed in domain scores.ConclusionPain interference and fatigue were the most responsive scales in measuring QoL in this AIP cohort. Future studies should assess whether these scales capture longitudinal disease progression and treatment response.

During erythroid differentiation, the erythron must remodel its protein constituents so that the mature red cell contains hemoglobin as the chief cytoplasmic protein component. For this, ∼109 molecules of heme must be synthesized, consuming 1010 molecules of succinyl-CoA. It has long been assumed that the source of succinyl-coenzyme A (CoA) for heme synthesis in all cell types is the tricarboxylic acid (TCA) cycle. Based upon the observation that 1 subunit of succinyl-CoA synthetase (SCS) physically interacts with the first enzyme of heme synthesis (5-aminolevulinate synthase 2, ALAS2) in erythroid cells, it has been posited that succinyl-CoA for ALA synthesis is provided by the adenosine triphosphate-dependent reverse SCS reaction. We have now demonstrated that this is not the manner by which developing erythroid cells provide succinyl-CoA for ALA synthesis. Instead, during late stages of erythropoiesis, cellular metabolism is remodeled so that glutamine is the precursor for ALA following deamination to α-ketoglutarate and conversion to succinyl-CoA by α-ketoglutarate dehydrogenase (KDH) without equilibration or passage through the TCA cycle. This may be facilitated by a direct interaction between ALAS2 and KDH. Succinate is not an effective precursor for heme, indicating that the SCS reverse reaction does not play a role in providing succinyl-CoA for heme synthesis. Inhibition of succinate dehydrogenase by itaconate, which has been shown in macrophages to dramatically increase the concentration of intracellular succinate, does not stimulate heme synthesis as might be anticipated, but actually inhibits hemoglobinization during late erythropoiesis.

PEX34, encoding a peroxisomal protein implicated in regulating peroxisome numbers, was identified as a high copy suppressor, capable of bypassing impaired acetate utilization of agc1∆ yeast. However, improved growth of agc1∆ yeast on acetate is not mediated through peroxisome proliferation. Instead, stress to the endoplasmic reticulum and mitochondria from PEX34 overexpression appears to contribute to enhanced acetate utilization of agc1∆ yeast. The citrate/2-oxoglutarate carrier Yhm2p is required for PEX34 stimulated growth of agc1∆ yeast on acetate medium, suggesting that the suppressor effect is mediated through increased activity of a redox shuttle involving mitochondrial citrate export. Metabolomic analysis also revealed redirection of acetyl-coenzyme A (CoA) from synthetic reactions for amino acids in PEX34 overexpressing yeast. We propose a model in which increased formation of products from the glyoxylate shunt, together with enhanced utilization of acetyl-CoA, promotes the activity of an alternative mitochondrial redox shuttle, partially substituting for loss of yeast AGC1.

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.

ATP-binding cassette subfamily B member 10 (Abcb10) is a mitochondrial ATP-binding cassette (ABC) transporter that complexes with mitoferrin1 and ferrochelatase to enhance heme biosynthesis in developing red blood cells. Reductions in Abcb10 levels have been shown to reduce mitoferrin1 protein levels and iron import into mitochondria, resulting in reduced heme biosynthesis. As an ABC transporter, Abcb10 binds and hydrolyzes ATP, but its transported substrate is unknown. Here, we determined that decreases in Abcb10 did not result in protoporphyrin IX accumulation in morphant-treated zebrafish embryos or in differentiated Abcb10-specific shRNA murine Friend erythroleukemia (MEL) cells in which Abcb10 was specifically silenced with shRNA. We also found that the ATPase activity of Abcb10 is necessary for hemoglobinization in MEL cells, suggesting that the substrate transported by Abcb10 is important in mediating increased heme biosynthesis during erythroid development. Inhibition of 5-aminolevulinic acid dehydratase (EC 4.2.1.24) with succinylacetone resulted in both 5-aminolevulinic acid (ALA) accumulation in control and Abcb10-specific shRNA MEL cells, demonstrating that reductions in Abcb10 do not affect ALA export from mitochondria and indicating that Abcb10 does not transport ALA. Abcb10 silencing resulted in an alteration in the heme biosynthesis transcriptional profile due to repression by the transcriptional regulator Bach1, which could be partially rescued by overexpression of Alas2 or Gata1, providing a mechanistic explanation for why Abcb10 shRNA MEL cells exhibit reduced hemoglobinization. In conclusion, our findings rule out that Abcb10 transports ALA and indicate that Abcb10's ATP-hydrolysis activity is critical for hemoglobinization and that the substrate transported by Abcb10 provides a signal that optimizes hemoglobinization.

Background GNPAT p.D519G positivity is significantly increased in HFE p.C282Y homozygotes with markedly increased iron stores. We sought to determine associations of p.D519G and iron-related variables with iron stores in p.C282Y homozygotes. Methods We defined markedly increased iron stores as serum ferritin > 2247 pmol/L (> 1000 μg/L) and either hepatic iron > 236 μmol/g dry weight or iron > 10 g by induction phlebotomy (men and women). We defined normal or mildly elevated iron stores as serum ferritin < 674.1 pmol/L (< 300 μg/L) or either age ≥ 40 y with iron ≤ 2.5 g iron by induction phlebotomy or age ≥ 50 y with ≤ 3.0 g iron by induction phlebotomy (men only). We compared participant subgroups using univariate methods. Using multivariable logistic regression, we evaluated associations of markedly increased iron stores with these variables: age; iron supplement use (dichotomous); whole blood units donated; erythrocyte units received as transfusion; daily alcohol consumption, g; and p.D519G positivity (heterozygosity or homozygosity). Results The mean age of 56 participants (94.6% men) was 55 ± 10 (SD) y; 41 had markedly increased iron stores. Prevalences of swollen/tender 2nd/3rd metacarpophalangeal joints and elevated aspartate or alanine aminotransferase were significantly greater in participants with markedly increased iron stores. Only participants with markedly increased iron stores had cirrhosis. In multivariable analyses, p.D519G positivity was the only exposure variable significantly associated with markedly increased iron stores (odds ratio 9.9, 95% CI [1.6, 60.3], p = 0.0126). Conclusions GNPAT p.D519G is strongly associated with markedly increased iron stores in p.C282Y homozygotes after correction for age, iron-related variables, and alcohol consumption.

Free heme is cytotoxic as exemplified by hemolytic diseases and genetic deficiencies in heme recycling and detoxifying pathways. Thus, intracellular accumulation of heme has not been observed in mammalian cells to date. Here we show that mice deficient for the heme transporter SLC48A1 (also known as HRG1) accumulate over ten-fold excess heme in reticuloendothelial macrophage lysosomes that are 10 to 100 times larger than normal. Macrophages tolerate these high concentrations of heme by crystallizing them into hemozoin, which heretofore has only been found in blood-feeding organisms. SLC48A1 deficiency results in impaired erythroid maturation and an inability to systemically respond to iron deficiency. Complete heme tolerance requires a fully-operational heme degradation pathway as haplo insufficiency of HMOX1 combined with SLC48A1 inactivation causes perinatal lethality demonstrating synthetic lethal interactions between heme transport and degradation. Our studies establish the formation of hemozoin by mammals as a previously unsuspected heme tolerance pathway., eLife digest Specialized cells, known as red blood cells, are responsible for transporting oxygen to various organs in the body. Each red blood cell contains over a billion molecules of heme which make up the iron containing portion of the hemoglobin protein that binds and transports oxygen. When red blood cells reach the end of their life, they are degraded, and the heme and iron inside them is recycled to produce new red blood cells. Heme, however, is highly toxic to cells, and can cause severe tissue damage if not properly removed. Scavenger cells called macrophages perform this recycling role in the spleen, liver and bone marrow. Collectively, macrophages can process around five million red blood cells every second or about 100 trillion heme molecules. But, it is unclear how they are able to handle such enormous volumes. Macrophages isolated from human and mice have been shown to transport heme from damaged red blood cells using a protein called HRG1. To investigate the role HRG1 plays in heme-iron recycling, Pek et al. used a gene editing tool known an CRISPR/Cas9 to remove the gene for HRG1 from the macrophages of mice. If HRG1 is a major part of this process, removing the gene should result in a build-up of toxic heme and eventual death of the mouse. But, rather than dying of heme-iron overload as expected, these mutant mice managed to survive. Pek et al. found that despite being unable to recycle heme, these mice were still able to make new red blood cells as long as they had a diet that was rich in iron. However, the darkening color of the spleen, bone marrow, and liver in these HRG1 deficient mice indicated that these mice were still accumulating high levels of heme. Further experiments revealed that these mice protected themselves from toxicity by converting the excess heme into crystals called hemozoin. This method of detoxification is commonly seen in blood-feeding parasites, and this is the first time it has been observed in a mammal. These crystals invite new questions about how mammals recycle heme and what happens when this process goes wrong. The next step is to ask whether humans also start to make hemozoin if the gene for HRG1 is faulty. If so, this could open a new avenue of exploration into treatments for red blood cell diseases like anemia and iron overload.

Porphyrias, is a general term for a group of metabolic diseases that are genetic in nature. In each specific porphyria the activity of specific enzymes in the heme biosynthetic pathway is defective and leads to accumulation of pathway intermediates. Phenotypically, each disease leads to either neurologic and/or photo-cutaneous symptoms based on the metabolic intermediate that accumulates. In each porphyria the distinct patterns of these substances in plasma, erythrocytes, urine and feces are the basis for diagnostically defining the metabolic defect underlying the clinical observations. Porphyrias may also be classified as either erythropoietic or hepatic, depending on the principal site of accumulation of pathway intermediates. The erythropoietic porphyrias are congenital erythropoietic porphyria (CEP), and erythropoietic protoporphyria (EPP). The acute hepatic porphyrias include ALA dehydratase deficiency porphyria, acute intermittent porphyria (AIP), hereditary coproporphyria (HCP) and variegate porphyria (VP). Porphyria cutanea tarda (PCT) is the only porphyria that has both genetic and/or environmental factors that lead to reduced activity of uroporphyrinogen decarboxylase in the liver. Each of the 8 enzymes in the heme biosynthetic pathway have been associated with a specific porphyria (Table 1). Mutations affecting the erythroid form of ALA synthase (ALAS2) are most commonly associated with X-linked sideroblastic anemia, however, gain-of-function mutations of ALAS2 have also been associated with a variant form of EPP. This overview does not describe the full clinical spectrum of the porphyrias, but is meant to be an overview of the biochemical steps that are required to make heme in both erythroid and non-erythroid cells.

With the advent of precision and genomic medicine, a critical issue is whether a disease gene variant is pathogenic or benign. Such is the case for the three autosomal dominant acute hepatic porphyrias (AHPs), including acute intermittent porphyria, hereditary coproporphyria, and variegate porphyria, each resulting from the half-normal enzymatic activities of hydroxymethylbilane synthase, coproporphyrinogen oxidase, and protoporphyrinogen oxidase, respectively. To date, there is no public database that documents the likely pathogenicity of variants causing the porphyrias, and more specifically, the AHPs with biochemically and clinically verified information. Therefore, an international collaborative with the European Porphyria Network and the National Institutes of Health/National Center for Advancing Translational Sciences/National Institute of Diabetes and Digestive and Kidney Diseases (NIH/NCATS/NIDDK)-sponsored Porphyrias Consortium of porphyria diagnostic experts is establishing an online database that will collate biochemical and clinical evidence verifying the pathogenicity of the published and newly identified variants in the AHP-causing genes. The overall goal of the International Porphyria Molecular Diagnostic Collaborative is to determine the pathogenic and benign variants for all eight porphyrias. Here we describe the overall objectives and the initial efforts to validate pathogenic and benign variants in the respective heme biosynthetic genes causing the AHPs.

Porphyria is a group of metabolic disorders due to altered enzyme activities within the heme biosynthetic pathway. It is a systemic disease with multiple potential contributions to mitochondrial dysfunction and oxidative stress. Recently, it has become possible to measure mitochondrial function from cells isolated from peripheral blood (cellular bioenergetics) using the XF96 analyzer (Seahorse Bioscience). Mitochondrial respiration in these cells is measured with the addition of activators and inhibitors of respiration. The output is measured as the O2 consumption rate (OCR) at basal conditions, ATP linked, proton leak, maximal, reserve capacity, non-mitochondrial, and oxidative burst. We performed cellular bioenergetics on 22 porphyria (12 porphyria cutanea tarda (PCT), seven acute hepatic porphyria (AHP), and three erythropoietic protoporphyria (EPP)) patients and 18 age and gender matched healthy controls. Of porphyria cases, eight were active (2 PCT, 1 EPP, and 5 AHP) and 14 in biochemical remission. The OCR were decreased in patients compared to healthy controls. The bioenergetic profile was significantly lower when measuring proton leak and the non-mitochondrial associated OCR in the eight active porphyria patients when compared to 18 healthy controls. In conclusion, we demonstrate that the bioenergetic profile and mitochondrial activities assessed in porphyria patients and is different than in healthy control individuals. Further, our novel preliminary findings suggest the existence of a mitochondrial dysfunction in porphyria and this may be used as potential non-invasive biomarker for disease activity. This needs to be assessed with a systematic examination in a larger patient cohort. Studies are also suggested to examine mitochondrial metabolism as basis to understand mechanisms of these findings and deriving mitochondrial based therapies for porphyria. Keywords: PCT, AIP, AHP, Protoporphyria, Mitochondrial

Physician-scientists and scientists in all the health professions are vital members of the U.S. biomedical workforce, but their numbers at academic health centers are declining. Mentorship has been identified as a key component in retention of faculty members at academic health centers. Effective mentoring may promote the retention of clinician-scientists in the biomedical workforce. The authors describe a holistic institutional mentoring program to support junior faculty members engaged in clinical and translational science at the University of Utah. The clinical and translational scholars (CATS) program leverages the resources of the institution, including the Center for Clinical and Translational Science, to augment departmental resources to support junior faculty investigators and uses a multilevel mentoring matrix that includes self, senior, scientific, peer, and staff mentorship. Begun in the Department of Pediatrics, the program was expanded in 2013 to include all departments in the school of medicine and the health sciences. During the two-year program, scholars learn management essentials and have leadership training designed to develop principal investigators. Of the 86 program participants since fiscal year 2008, 92% have received extramural awards, 99% remain in academic medicine, and 95% remain at the University of Utah. The CATS program has also been associated with increased inclusion of women and underrepresented minorities in the institutional research enterprise. The CATS program manifests institutional collaboration and coordination of resources, which have benefited faculty members and the institution. The model can be applied to other academic health centers to support and sustain the biomedical workforce.

BACKGROUNDLaboratory diagnosis of erythropoietic protoporphyria (EPP) requires a marked increase in total erythrocyte protoporphyrin (300–5000 μg/dL erythrocytes, reference interval CONTENTIn studying more than 180 patients with EPP and XLP, the Porphyrias Consortium found that erythrocyte protoporphyrin concentrations for some patients were much higher (4.3- to 46.7-fold) than indicated by previous reports provided by these patients. The discrepant earlier reports, which sometimes caused the diagnosis to be missed initially, were from laboratories that measure protoporphyrin only by hematofluorometry, which is intended primarily to screen for lead poisoning. However, the instrument can calculate results on the basis of assumed hematocrits and reports results as “free” and “zinc” protoporphyrin (with different reference intervals), implying separate measurements of metal-free and zinc protoporphyrin. Such misleading reports impair diagnosis and monitoring of patients with protoporphyria.SUMMARYWe suggest that laboratories should prioritize testing for EPP and XLP, because accurate measurement of erythrocyte total and metal-free protoporphyrin is essential for diagnosis and monitoring of these conditions, but less important for other disorders. Terms and abbreviations used in reporting erythrocyte protoporphyrin results should be accurately defined.

In an iron deficient child, oral iron repeatedly failed to improve the condition. Whole exome sequencing identified one previously reported plus two novel mutation in the TMPRSS6 gene, with no mutations in other iron-associated genes. We propose that these mutations result in a novel variety of iron-refractory iron deficiency anemia.