Haem iron versus ferrous iron salts to treat iron deficiency anaemia in Gambian children: protocol for randomised controlled trial {1}.

BACKGROUND: The World Health Organization recommends universal iron supplementation for children aged 6-23 months in countries where anaemia is seen in over 40% of the population. Conventional ferrous salts have low efficacy due to low oral absorption in children with inflammation. Haem iron is more bioavailable, and its absorption may not be decreased by inflammation. This study aims to compare daily supplementation with haem iron versus ferrous sulphate on haemoglobin concentration and serum ferritin concentration after 12 weeks of supplementation. METHODS: This will be a two-arm, randomised controlled trial. Gambian children aged 6-12 months with anaemia will be recruited within a predefined geographical area and recruited by trained field workers. Eligible participants will be individually randomised using a 1:1 ratio within permuted blocks to daily supplementation for 12 weeks with either 10.0 mg of elemental iron as haem or ferrous sulphate. Safety outcomes such as diarrhoea and infection-related adverse events will be assessed daily by the clinical team (see Bah et al. Additional file 4_Adverse event eCRF). Linear regression will be used to analyse continuous outcomes, with log transformation to normalise residuals as needed. Binary outcomes will be analysed by binomial regression or logistic regression, Primary analysis will be by modified intention-to-treat (i.e., those randomised and who ingested at least one supplement dose of iron), with multiple imputations to replace missing data. Effect estimates will be adjusted for baseline covariates (C-reactive protein, alpha-1-acid glycoprotein, haemoglobin, ferritin, soluble transferrin receptor). DISCUSSION: This study will determine if therapeutic supplementation with haem iron is more efficacious than with conventional ferrous sulphate in enhancing haemoglobin and ferritin concentrations in anaemic children aged 6-12 months. TRIAL REGISTRATION: Pan African Clinical Trial Registry PACTR202210523178727.

CLPP-Null Eukaryotes with Excess Heme Biosynthesis Show Reduced L-arginine Levels, Probably via CLPX-Mediated OAT Activation.

The serine peptidase CLPP is conserved among bacteria, chloroplasts, and mitochondria. In humans and mice, its loss causes Perrault syndrome, which presents with growth deficits, infertility, deafness, and ataxia. In the filamentous fungus Podospora anserina, CLPP loss leads to longevity. CLPP substrates are selected by CLPX, an AAA+ unfoldase. CLPX is known to target delta-aminolevulinic acid synthase (ALAS) to promote pyridoxal phosphate (PLP) binding. CLPX may also influence cofactor association with other enzymes. Here, the evaluation of P. anserina metabolomics highlighted a reduction in arginine/histidine levels. In Mus musculus cerebellum, reductions in arginine/histidine and citrulline occurred with a concomitant accumulation of the heme precursor protoporphyrin IX. This suggests that the increased biosynthesis of 5-carbon (C5) chain deltaALA consumes not only C4 succinyl-CoA and C1 glycine but also specific C5 delta amino acids. As enzymes responsible for these effects, the elevated abundance of CLPX and ALAS is paralleled by increased OAT (PLP-dependent, ornithine delta-aminotransferase) levels. Possibly as a consequence of altered C1 metabolism, the proteome profiles of P. anserina CLPP-null cells showed strong accumulation of a methyltransferase and two mitoribosomal large subunit factors. The reduced histidine levels may explain the previously observed metal interaction problems. As the main nitrogen-storing metabolite, a deficiency in arginine would affect the urea cycle and polyamine synthesis. Supplementation of arginine and histidine might rescue the growth deficits of CLPP-mutant patients.

Single-Cell Hemoprotein Diet Changes Adipose Tissue Distributions and Re-Shapes Gut Microbiota in High-Fat Diet-Induced Obese Mice.

We have previously observed that feeding with single-cell hemoprotein (heme-SCP) in dogs (1 g/day for 6 days) and broiler chickens (1 ppm for 32 days) increased the proportion of lactic acid bacteria in the gut while reducing their body weights by approximately 1~2%. To define the roles of heme-SCP in modulating body weight and gut microbiota, obese C57BL/6N mice were administered varied heme-SCP concentrations (0, 0.05, and 0.5% heme-SCP in high fat diet) for 28 days. The heme-SCP diet seemed to restrain weight gain till day 14, but the mice gained weight again later, showing no significant differences in weight. However, the heme-SCP-fed mice had stiffer and oilier bodies compared with those of the control mice, which had flabby bodies and dull coats. When mice were dissected at day 10, the obese mice fed with heme-SCP exhibited a reduction in subcutaneous fat with an increase in muscle mass. The effect of heme-SCP on the obesity-associated dyslipidemia tended to be corroborated by the blood parameters (triglyceride, total cholesterol, and C-reactive protein) at day 10, though the correlation was not clear at day 28. Notably, the heme-SCP diet altered gut microbiota, leading to the proliferation of known anti-obesity biomarkers such as Akkermansia, Alistipes, Oscillibacter, Ruminococcus, Roseburia, and Faecalibacterium. This study suggests the potential of heme-SCP as an anti-obesity supplement, which modulates serum biochemistry and gut microbiota in high-fat diet-induced obese mice.

Combination Therapy with Gallium Protoporphyrin and Gallium Nitrate Exhibits Enhanced Antimicrobial Activity In Vitro and In Vivo against Methicillin-Resistant Staphylococcus aureus.

There is a major need for the development of new therapeutics to combat antibiotic-resistant Staphylococcus aureus. Recently, gallium (Ga)-based complexes have shown promising antimicrobial effects against various bacteria, including multidrug-resistant organisms, by targeting multiple heme/iron-dependent metabolic pathways. Among these, Ga protoporphyrin (GaPP) inhibits bacterial growth by targeting heme pathways, including aerobic respiration. Ga(NO3)3, an iron mimetic, disrupts elemental iron pathways. Here, we demonstrate the enhanced antimicrobial activity of the combination of GaPP and Ga(NO3)3 against methicillin-resistant S. aureus (MRSA) under iron-limited conditions, including small colony variants (SCV). This therapy demonstrated significant antimicrobial activity without inducing slow-growing SCV. We also observed that the combination of GaPP and Ga(NO3)3 inhibited the MRSA catalase but not above that seen with Ga(NO3)3 alone. Neither GaPP nor Ga(NO3)3 alone or their combination inhibited the dominant superoxide dismutase expressed (SodA) under the iron-limited conditions examined. Intranasal administration of the combination of the two compounds improved drug biodistribution in the lungs compared to intraperitoneal administration. In a murine MRSA lung infection model, we observed a significant increase in survival and decrease in MRSA lung CFUs in mice that received combination therapy with intranasal GaPP and Ga(NO3)3 compared to untreated control or mice receiving GaPP or Ga(NO3)3 alone. No drug-related toxicity was observed as assessed histologically in the spleen, lung, nasal cavity, and kidney for both single and repeated doses of 10 mg Ga /Kg of mice over 13 days. Our results strongly suggest that GaPP and Ga(NO3)3 in combination have excellent synergism and potential to be developed as a novel therapy for infections with S. aureus.

Engineering Therapeutics to Detoxify Hemoglobin, Heme, and Iron.

Hemolysis (i.e., red blood cell lysis) can increase circulatory levels of cell-free hemoglobin (Hb) and its degradation by-products, namely heme (h) and iron (Fe). Under homeostasis, minor increases in these three hemolytic by-products (Hb/h/Fe) are rapidly scavenged and cleared by natural plasma proteins. Under certain pathophysiological conditions, scavenging systems become overwhelmed, leading to the accumulation of Hb/h/Fe in the circulation. Unfortunately, these species cause various side effects such as vasoconstriction, hypertension, and oxidative organ damage. Therefore, various therapeutics strategies are in development, ranging from supplementation with depleted plasma scavenger proteins to engineered biomimetic protein constructs capable of scavenging multiple hemolytic species. In this review, we briefly describe hemolysis and the characteristics of the major plasma-derived protein scavengers of Hb/h/Fe. Finally, we present novel engineering approaches designed to address the toxicity of these hemolytic by-products.

Targeting hemoglobin receptors IsdH and IsdB of Staphylococcus aureus with a single VHH antibody inhibits bacterial growth.

Methicillin-resistant Staphylococcus aureus, or MRSA, is one of the major causative agents of hospital-acquired infections worldwide. Novel antimicrobial strategies efficient against antibiotic-resistant strains are necessary and not only against S. aureus. Among those, strategies that aim at blocking or dismantling proteins involved in the acquisition of essential nutrients, helping the bacteria to colonize the host, are intensively studied. A major route for S. aureus to acquire iron from the host organism is the Isd (iron surface determinant) system. In particular, the hemoglobin receptors IsdH and IsdB located on the surface of the bacterium are necessary to acquire the heme moiety containing iron, making them a plausible antibacterial target. Herein, we obtained an antibody of camelid origin that blocked heme acquisition. We determined that the antibody recognized the heme-binding pocket of both IsdH and IsdB with nanomolar order affinity through its second and third complementary-determining regions. The mechanism explaining the inhibition of acquisition of heme in vitro could be described as a competitive process in which the complementary-determining region 3 from the antibody blocked the acquisition of heme by the bacterial receptor. Moreover, this antibody markedly reduced the growth of three different pathogenic strains of MRSA. Collectively, our results highlight a mechanism for inhibiting nutrient uptake as an antibacterial strategy against MRSA.

Identification of Multiple Iron Uptake Mechanisms in Enterococcus faecalis and Their Relationship to Virulence.

Among the unfavorable conditions bacteria encounter within the host is restricted access to essential trace metals such as iron. To overcome iron deficiency, bacteria deploy multiple strategies to scavenge iron from host tissues, with abundant examples of iron acquisition systems being implicated in bacterial pathogenesis. Yet the mechanisms utilized by the major nosocomial pathogen Enterococcus faecalis to maintain intracellular iron balance are poorly understood. In this study, we conducted a systematic investigation to identify and characterize the iron acquisition mechanisms of E. faecalis and to determine their contribution to virulence. Bioinformatic analysis and literature surveys revealed that E. faecalis possesses three conserved iron uptake systems. Through transcriptomics, we discovered two novel ABC-type transporters that mediate iron uptake. While inactivation of a single transporter had minimal impact on the ability of E. faecalis to maintain iron homeostasis, inactivation of all five systems (Δ5Fe strain) disrupted intracellular iron homeostasis and considerably impaired cell growth under iron deficiency. Virulence of the Δ5Fe strain was generally impaired in different animal models but showed niche-specific variations in mouse models, leading us to suspect that heme can serve as an iron source to E. faecalis during mammalian infections. Indeed, heme supplementation restored growth of Δ5Fe under iron depletion and virulence in an invertebrate infection model. This study revealed that the collective contribution of five iron transporters promotes E. faecalis virulence and that the ability to acquire and utilize heme as an iron source is critical to the systemic dissemination of E. faecalis.

Depletion assisted hemin affinity (DAsHA) proteomics reveals an expanded landscape of heme-binding proteins in the human proteome.

Heme b (iron protoporphyrin IX) plays important roles in biology as a metallocofactor and signaling molecule. However, the targets of heme signaling and the network of proteins that mediate the exchange of heme from sites of synthesis or uptake to heme dependent or regulated proteins are poorly understood. Herein, we describe a quantitative mass spectrometry (MS)-based chemoproteomics strategy to identify exchange labile hemoproteins in human embryonic kidney HEK293 cells that may be relevant to heme signaling and trafficking. The strategy involves depleting endogenous heme with the heme biosynthetic inhibitor succinylacetone (SA), leaving putative heme-binding proteins in their apo-state, followed by the capture of those proteins using hemin-agarose resin, and finally elution and identification by MS. By identifying only those proteins that interact with high specificity to hemin-agarose relative to control beaded agarose in an SA-dependent manner, we have expanded the number of proteins and ontologies that may be involved in binding and buffering labile heme or are targets of heme signaling. Notably, these include proteins involved in chromatin remodeling, DNA damage response, RNA splicing, cytoskeletal organization, and vesicular trafficking, many of which have been associated with heme through complementary studies published recently. Taken together, these results provide support for the emerging role of heme in an expanded set of cellular processes from genome integrity to protein trafficking and beyond.

Lead exposure represses mitochondrial metabolism by activation of heme-binding protein BACH1 in differentiated SH-SY5Y cell.

Exposure to lead (Pb), a known toxin causing developmental neurotoxicity, can impair neurogenesis and oxidative phosphorylation (OXPHOS), but the mechanism is not clarified. In the current study, we aim to explore the effects of Pb on the differentiation of SH-SY5Y cells and investigate the role of heme and heme-binding protein BACH1 during differentiation. We found that Pb exposure caused a shift from OXPHOS to glycolysis, resulting in neurogenesis impairment by decreasing neurite growth and downregulation of PSD95 and Synapsin-1 in differentiated SH-SY5Y cells. Heme reduction mediated this mitochondria metabolism repression caused by Pb depending on BACH1 activation. Hemin supplement alleviated Pb-induced OXPHOS damage and adenosine triphosphate (ATP) reduction in differentiated SH-SY5Y cells, and further protected for Pb-induced damage of synapse. Heme binding factor BACH1 was negatively regulated by heme content and BACH1 knockout rescued the Pb-induced transcription and expression decline of genes related to OXPHOS and abrogated Pb-induced growth inhibition of axon promotion and synapse formation. Collectively, the present study demonstrates that heme deficiency mediates OXPHOS damage caused by Pb through BACH1 activation, resulting in neurogenesis impairment.

HNOXPred: a web tool for the prediction of gas-sensing H-NOX proteins from amino acid sequence.

SUMMARY: HNOXPred is a webserver for the prediction of gas-sensing heme-nitric oxide/oxygen (H-NOX) proteins from amino acid sequence. H-NOX proteins are gas-sensing hemoproteins found in diverse organisms ranging from bacteria to eukaryotes. Recently, gas-sensing complex multi-functional proteins containing only the conserved amino acids at the heme centers of H-NOX proteins, have been identified through a motif-based approach. Based on experimental data and H-NOX candidates reported in the literature, HNOXPred is created to automate and facilitate the identification of similar H-NOX centers across systems. The server features HNOXSCORES scaled from 0 to 1 that consider in its calculation, the physicochemical properties of amino acids constituting the heme center in H-NOX in addition to the conserved amino acids within the center. From user input amino acid sequence, the server returns positive hits and their calculated HNOXSCORES ordered from high to low confidence which are accompanied by interpretation guides and recommendations. The utility of this server is demonstrated using the human proteome as an example. AVAILABILITY AND IMPLEMENTATION: The HNOXPred server is available at https://www.hnoxpred.com. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Hemolysis, free hemoglobin toxicity, and scavenger protein therapeutics.

During hemolysis, erythrophagocytes dispose damaged red blood cells. This prevents the extracellular release of hemoglobin, detoxifies heme, and recycles iron in a linked metabolic pathway. Complementary to this process, haptoglobin and hemopexin scavenge and shuttle the red blood cell toxins hemoglobin and heme to cellular clearance. Pathological hemolysis outpaces macrophage capacity and scavenger synthesis across a diversity of diseases. This imbalance leads to hemoglobin-driven disease progression. To meet a void in treatment options, scavenger protein-based therapeutics are in clinical development.

Hypochlorous acid facilitates inducible nitric oxide synthase subunit dissociation: The link between heme destruction, disturbance of the zinc-tetrathiolate center, and the prevention by melatonin.

Inducible nitric oxide synthase (iNOS) is a zinc-containing hemoprotein composed of two identical subunits, each containing a reductase and an oxygenase domain. The reductase domain contains binding sites for NADPH, FAD, FMN, and tightly bound calmodulin and the oxygenase domain contains binding sites for heme, tetrahydrobiopterin (H4B), and l-arginine. The enzyme converts l-arginine into nitric oxide (NO) and citrulline in the presence of O2. It has previously been demonstrated that myeloperoxidase (MPO), which catalyzes formation of hypochlorous acid (HOCl) from hydrogen peroxide (H2O2) and chloride (Cl-), is enhanced in inflammatory diseases and could be a potent scavenger of NO. Using absorbance spectroscopy and gel filtration chromatography, we investigated the role of increasing concentrations of HOCl in mediating iNOS heme destruction and subsequent subunit dissociation and unfolding. The results showed that dimer iNOS dissociation between 15 and 100 µM HOCl was accompanied by loss of heme content and NO synthesis activity. The dissociated subunits-maintained cytochrome c and ferricyanide reductase activities. There was partial unfolding of the subunits at 300 µM HOCl and above, and the subunit unfolding transition was accompanied by loss of reductase activities. These events can be prevented when the enzyme is preincubated with melatonin prior to HOCl addition. Melatonin supplementation to patients experiencing low NO levels due to inflammatory diseases may be helpful to restore physiological NO functions.

Rhizobial HmuSpSym as a heme-binding factor is required for optimal symbiosis between Mesorhizobium amorphae CCNWGS0123 and Robinia pseudoacacia.

Nitrogen-fixing root nodules are formed by symbiotic association of legume hosts with rhizobia in nitrogen-deprived soils. Successful symbiosis is regulated by signals from both legume hosts and their rhizobial partners. HmuS is a heme degrading factor widely distributed in bacteria, but little is known about the role of rhizobial hmuS in symbiosis with legumes. Here, we found that inactivation of hmuSpSym in the symbiotic plasmid of Mesorhizobium amorphae CCNWGS0123 disrupted rhizobial infection, primordium formation, and nitrogen fixation in symbiosis with Robinia pseudoacacia. Although there was no difference in bacteroids differentiation, infected plant cells were shrunken and bacteroids were disintegrated in nodules of plants infected by the ΔhmuSpSym mutant strain. The balance of defence reaction was also impaired in ΔhmuSpSym strain-infected root nodules. hmuSpSym was strongly expressed in the nitrogen-fixation zone of mature nodules. Furthermore, the HmuSpSym protein could bind to heme but not degrade it. Inactivation of hmuSpSym led to significantly decreased expression levels of oxygen-sensing related genes in nodules. In summary, hmuSpSym of M. amorphae CCNWGS0123 plays an essential role in nodule development and maintenance of bacteroid survival within R. pseudoacacia cells, possibly through heme-binding in symbiosis.

Iron overload and impaired iron handling contribute to the dystrophic pathology in models of Duchenne muscular dystrophy.

BACKGROUND: Oxidative stress is implicated in the pathophysiology of Duchenne muscular dystrophy (DMD, caused by mutations in the dystrophin gene), which is the most common and severe of the muscular dystrophies. To our knowledge, the distribution of iron, an important modulator of oxidative stress, has not been assessed in DMD. We tested the hypotheses that iron accumulation occurs in mouse models of DMD and that modulation of iron through the diet or chelation could modify disease severity. METHODS: We assessed iron distribution and total elemental iron using LA-ICP-MS on skeletal muscle cross-sections of 8-week-old Bl10 control mice and dystrophic mdx mice (with moderate dystrophy) and dystrophin/utrophin-null mice (dko, with severe dystrophy). In addition, mdx mice (4 weeks) were treated with either an iron chelator (deferiprone 150 mg/kg/day) or iron-enriched feed (containing 1% added iron as carbonyl iron). Immunoblotting was used to determine the abundance of iron- and mitochondria-related proteins. (Immuno)histochemical and mRNA assessments of fibrosis and inflammation were also performed. RESULTS: We observed a significant increase in total elemental iron in hindlimb muscles of dko mice (+50%, P < 0.05) and in the diaphragm of mdx mice (+80%, P < 0.05), with both tissues exhibiting severe pathology. Iron dyshomeostasis was further evidenced by an increase in the storage protein ferritin (dko: +39%, P < 0.05) and ferroportin compared with Bl10 control mice (mdx: +152% and dko: +175%, P < 0.05). Despite having features of iron overload, dystrophic muscles had lower protein expression of ALAS-1, the rate-limiting enzyme for haem synthesis (dko -44%, P < 0.05), and the haem-containing protein myoglobin (dko -54%, P < 0.05). Deferiprone treatment tended to decrease muscle iron levels in mdx mice (-30%, P < 0.1), which was associated with lower oxidative stress and fibrosis, but suppressed haem-containing proteins and mitochondrial content. Increasing iron via dietary intervention elevated total muscle iron (+25%, P < 0.05) but did not aggravate the pathology. CONCLUSIONS: Muscles from dystrophic mice have increased iron levels and dysregulated iron-related proteins that are associated with dystrophic pathology. Muscle iron levels were manipulated by iron chelation and iron enriched feed. Iron chelation reduced fibrosis and reactive oxygen species (ROS) but also suppressed haem-containing proteins and mitochondrial activity. Conversely, iron supplementation increased ferritin and haem-containing proteins but did not alter ROS, fibrosis, or mitochondrial activity. Further studies are required to investigate the contribution of impaired ferritin breakdown in the dysregulation of iron homeostasis in DMD.

MRP5 and MRP9 play a concerted role in male reproduction and mitochondrial function.

Multidrug Resistance Proteins (MRPs) are transporters that play critical roles in cancer even though the physiological substrates of these enigmatic transporters are poorly elucidated. In Caenorhabditis elegans, MRP5/ABCC5 is an essential heme exporter because mrp-5 mutants are unviable due to their inability to export heme from the intestine to extraintestinal 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 in C. elegans, raising the possibility that MRP9 may genetically compensate for MRP5. Here, we show that MRP5 and MRP9 double KO (DKO) 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, 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 sequencing shows 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.

Hemozoin (beta-hematin) Formation Inhibitors: Promising Target for the Development of New Antimalarials: Current Update and Future Prospect.

BACKGROUND: Malaria is responsible for social and economic burden in most lowincome malaria-affected countries. Thus, newer antimalarials are needed to tackle morbidities and mortalities associated with the drug-resistant malarial strains. Haemoglobin digestion inside the food vacuole of malarial parasite would lead to producing redox-active and toxic-free heme. The detoxification process adopted by Plasmodium sp. would give rise to hemozoin (Hz) (betahematin) formation. Targeting the pathway of hemozoin formation is considered a validated target for the discovery of newer antimalarials. OBJECTIVE: This study aims to collect detailed information about aspects of hemozoin (Hz) (betahematin) inhibitors. METHODS: A systemic search has been carried out using PubMed, Google Scholar, CNKI, etc., for relevant studies having the keyword, 'hemozoin or beta-hematin' for almost the last 2 decades (2000-2021). RESULTS: This review tries to summarize all the recent advancements made for the developments of synthetic, natural isolated phytoconstituents and plant extracts inhibiting the hemozoin (betahematin) formation. CONCLUSION: Thus they would act as promising antimalarial candidates in the near future.

Excess heme upregulates heme oxygenase 1 and promotes cardiac ferroptosis in mice with sickle cell disease.

Sickle cell disease (SCD) is characterized by increased hemolysis, which results in plasma heme overload and ultimately cardiovascular complications. Here, we hypothesized that increased heme in SCD causes upregulation of heme oxygenase 1 (Hmox1), which consequently drives cardiomyopathy through ferroptosis, an iron-dependent non-apoptotic form of cell death. First, we demonstrated that the Townes SCD mice had higher levels of hemopexin-free heme in the serum and increased cardiomyopathy, which was corrected by hemopexin supplementation. Cardiomyopathy in SCD mice was associated with upregulation of cardiac Hmox1, and inhibition or induction of Hmox1 improved or worsened cardiac damage, respectively. Because free iron, a product of heme degradation through Hmox1, has been implicated in toxicities including ferroptosis, we evaluated the downstream effects of elevated heme in SCD. Consistent with Hmox1 upregulation and iron overload, levels of lipid peroxidation and ferroptotic markers increased in SCD mice, which were corrected by hemopexin administration. Moreover, ferroptosis inhibitors decreased cardiomyopathy, whereas a ferroptosis inducer erastin exacerbated cardiac damage in SCD and induced cardiac ferroptosis in nonsickling mice. Finally, inhibition or induction of Hmox1 decreased or increased cardiac ferroptosis in SCD mice, respectively. Together, our results identify ferroptosis as a key mechanism of cardiomyopathy in SCD.

Molecular insights on the conformational dynamics of a P76C mutant of human cytochrome c and the enhancement on its peroxidase activity.

In apoptotic pathway, the interaction of Cytochrome c (Cytc) with cardiolipin in vivo is a key process to induce peroxidase activity of Cytc and trigger the release of Cytc in the inner mitochondria into cytosol. The peroxidase active form of Cytc occurs due to local conformational changes that support the opening of the heme crevice and the loss of an axial ligand between Met80 and heme Fe. Structural adjustments at the Ω-loop segments of Cytc are required for such process. To study the role of the distal Ω-loop segments comprising residues 71-85 in human Cytc (hCytc), we investigated a cysteine mutation at Pro76, one of the highly conserved residues in this loop. The effect of P76C mutant was explored by the combination of experimental characterizations and molecular dynamics (MD) simulations. The peroxidase activity of the P76C mutant was found to be significantly increased by ∼13 folds relative to the wild type. Experimental data on global denaturation, alkaline transition, heme bleaching, and spin-labeling Electron Spin Resonance were in good agreement with the enhancement of peroxidase activity. The MD results of hCytc in the hexacoordinate form suggest the important changes in P76C mutant occurred due to the unfolding at the central Ω-loop (residues 40-57), and the weakening of H-bond between Tyr67 and Met80. Whereas the experimental data implied that the P76C mutant tend to be in equilibrium between the pentacoordinate and hexacoordinate forms, the MD and experimental information are complementary and were used to support the mechanisms of peroxidase active form of hCytc.

Inhibition of heme detoxification pathway in malaria parasite by 3-hydroxy-11-keto-ß-boswellic acid isolated from Boswellia serrata.

Malaria eradication is still a major global health problem in developing countries, which has been of more concern ever since the malaria parasite has developed resistance against frontline antimalarial drugs. Historical evidence proves that the plants possess a major resource for the development of novel anti-malarial drugs. In the present study, the bioactivity guided fractionation of the oleogum-resin of Boswellia serrata Roxb. yielded the optimum activity in the ethyl acetate fraction with an IC50 of 22 ± 3.9 µg/mL and 26.5 ± 4.5 µg/mL against chloroquine sensitive (NF54) and resistant (K1) strains of Plasmodium falciparum respectively. Further, upon fractionation, the ethyl acetate fraction yielded four major compounds, of which 3-Hydroxy-11-keto-ß-boswellic acid (KBA) was found to be the most potent with IC50 values 4.5 ± 0.60 µg/mL and 6.25 ± 1.02 µg/mL against sensitive and resistant strains respectively. KBA was found to inhibit heme detoxification pathways, one of the most common therapeutic targets, which probably lead to an increase in reactive oxygen species (ROS) and nitric oxide (NO) detrimental to P. falciparum. Further, the induced intracellular oxidative stress affected the macromolecules in terms of DNA damage, increased lipid peroxidation, protein carbonylation as well as loss of mitochondrial membrane potential. However, it did not exhibit any cytotoxic effect in VERO cells. Under in vivo conditions, KBA exhibited a significant reduction in parasitemia, retarding the development of anaemia, resulting in an enhancement of the mean survival time in Plasmodium yoelii nigeriensis (chloroquine-resistant) infected mice. Further, KBA did not exhibit any abnormality in serum biochemistry of animals that underwent acute oral toxicity studies at 2000 mg/kg body weight.

Comparison of two markers of iron sufficiency and neurodevelopmental outcomes.

BACKGROUND: Iron deficiency during critical windows of brain development is associated with suboptimal neurodevelopmental outcomes. Identifying markers of neonatal iron status that best correlate with neurodevelopmental outcome is critical for optimal management of iron supplementation of neonates. AIMS: We aimed to evaluate two markers of iron sufficiency, ferritin and zinc protoporphyrin-to-heme ratios (ZnPP/H), with neurodevelopmental outcomes. STUDY DESIGN: This is a retrospective cohort study. SUBJECTS: All infants with concurrent ferritin and ZnPP/H measurements obtained between October 2014 and April 2017 and Bayley Scales of Infant Development, 3rd Edition (BSID-III) evaluated at 24 months corrected age were included. OUTCOME MEASURES: Associations between iron markers (minimum, maximum and median ferritin and ZnPP/H) and BSID-III score at 24 months were assessed. RESULTS: 223 lab measurements from 62 infants were assessed. Mean gestational age was 28.1 weeks (SD = 2.6) with a mean birth weight of 1.1 kg (SD = 0.4). Significant associations between maximum and median ZnPP/H and motor score, and between median ZnPP/H and cognitive score were observed. Trends were also seen with higher minimum, median and maximum ZnPP/H associated with lower BSID-III scores, but did not reach statistical significance (p > 0.05). The associations between ferritin values and BSID scores were less consistent. CONCLUSIONS: A positive association was seen between ZnPP/H values and BSID-III scores. Trends between ferritin and BSID values were less consistent, potentially because ferritin is more affected by inflammation. Consideration should be given to using ZnPP/H preferentially to adjust iron supplementation in the NICU to improve neurodevelopmental outcomes.