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Dietary fat content can reduce the methane production of dairy cows; however, the relevance fatty acid (FA) composition has towards this inhibitory effect is debatable. Furthermore, in-depth studies elucidating the effects of unsaturated fatty acids (UFA) on rumen function and the mechanism of reducing methane (CH4) production are lacking. This study exposed 10 Holstein cows with the same parity, similar milk yield to two total mixed rations: low unsaturated FA (LUFA) and high unsaturated FA (HUFA) with similar fat content. The LUFA group mainly added fat powder (C16:0 > 90%), and the HUFA group mainly replaced fat powder with extruded flaxseed. The experiment lasted 26 d, the last 5 d of which, gas exchange in respiratory chambers was conducted to measure gas emissions. We found that an increase in the UFA in diet did not affect milk production (P > 0.05) and could align the profile of milk FAs more closely with modern human nutritional requirements. Furthermore, we found that increasing the UFA content in the diet lead to a decrease in the abundance of Methanobrevibacterin the rumen (|linear discriminant analysis [LDA] score| > 2 and P < 0.05), which resulted in a decrease in the relative abundance of multiple enzymes (EC:1.2.7.12, EC:2.3.1.101, EC:3.5.4.27, EC:1.5.98.1, EC:1.5.98.2, EC:6.2.1.1, EC:2.1.1.86 and EC:2.8.4.1) during methanogenesis (P < 0.05). Compared with the LUFA group, the pathway of CH4metabolism was inhibited in the HUFA group (|LDA| > 2 and P < 0.05), which ultimately decreased CH4production (P < 0.05). Our results illustrated the mechanism involving decreased CH4production when fed a UFA diet in dairy cows. We believe that our study provides new evidence to explore CH4emission reduction measures for dairy cows.

A Co-catalyzed reductive cyclization of acrylate-containing 1,6-enynes is reported, providing an approach to construct five-membered carbocyclic and heterocyclic scaffolds containing enol ethers and all-carbon quaternary carbons. This novel process enables an E/Zmixture of 1,6-enynes to react with good functional group tolerance and good isolated yields, in an operationally simple manner.

The absolute configurations of the three unknown chiral centers in vinylamycin were predicted according to the structural comparison with microtermolide A and rakicidin A, and then total syntheses of vinylamycin were applied to determine the three unknown chiral centers as 14R, 15R, and 16S.

A metal-free intramolecular aminophosphination of sulfonamidoallenes with diarylphosphine oxides and Tf2O was developed. This method offers a general and practical procedure to construct valuable alkenylphosphine-substituted N-heterocycles via the bifunctionalization reaction of allenes in good yields under mild conditions.

Direct intramolecular aminoboration of sulfonamidoallenes was realized using BCl3as a boron source. The reactions benefited from the interaction between BCl3and sulfonamides and provided a variety of borylvinyl heterocycles in good isolated yields. When chiral substrates were involved in the reactions, high stereoselectivity was observed, as can be ascertained by single-crystal X-ray diffraction experiments. Derivatization of the thus-obtained borylvinyl compounds proceeded readily, and different functionalities could be obtained via oxidation, halogenation, and Suzuki coupling reactions.

A direct and convenient approach for the coupling of propargylic substrates with diphenylphosphine oxide in the presence of Tf2O and 2,6-lutidine has been developed. The method provides a general approach for the construction of attractive allenylphosphoryl skeletons with high atom and step economy under metal free conditions.

A catalyst-free method for the highly regioselective chloroboration of allenylsilanes is described. In the presence of BCl3and 2,6-lutidine, chloroboration of allenylsilanes proceeds smoothly without any catalyst, and the product could be treated with pinacol to afford the corresponding pinacol borates in one-pot reaction. This reaction provides a direct approach to construct valuable 2-silylallylboronate frameworks with operational simplicity and high atom-economy.

Erythropoiesis is the complex, dynamic, and tightly regulated process that generates all mature red blood cells. To understand this process, we mapped the developmental trajectories of progenitors from wild-type, erythropoietin-treated, and Flvcr1-deleted mice at single-cell resolution. Importantly, we linked the quantity of each cell’s surface proteins to its total transcriptome, which is a novel method. Deletion of Flvcr1 results in high levels of intracellular heme, allowing us to identify heme-regulated circuitry. Our studies demonstrate that in early erythroid cells (CD71+Ter119neg-lo), heme increases ribosomal protein transcripts, suggesting that heme, in addition to upregulating globin transcription and translation, guarantees ample ribosomes for globin synthesis. In later erythroid cells (CD71+Ter119lo-hi), heme decreases GATA1, GATA1-target gene, and mitotic spindle gene expression. These changes occur quickly. For example, in confirmatory studies using human marrow erythroid cells, ribosomal protein transcripts and proteins increase, and GATA1 transcript and protein decrease, within 15 to 30 minutes of amplifying endogenous heme synthesis with aminolevulinic acid. Because GATA1 initiates heme synthesis, GATA1 and heme together direct red cell maturation, and heme stops GATA1 synthesis, our observations reveal a GATA1–heme autoregulatory loop and implicate GATA1 and heme as the comaster regulators of the normal erythroid differentiation program. In addition, as excessive heme could amplify ribosomal protein imbalance, prematurely lower GATA1, and impede mitosis, these data may help explain the ineffective (early termination of) erythropoiesis in Diamond Blackfan anemia and del(5q) myelodysplasia, disorders with excessive heme in colony-forming unit-erythroid/proerythroblasts, explain why these anemias are macrocytic, and show why children with GATA1 mutations have DBA-like clinical phenotypes.

Erythropoiesis is the complex, dynamic, and tightly regulated process that generates all mature red blood cells. To understand this process, we mapped the developmental trajectories of progenitors from wild-type, erythropoietin-treated, and Flvcr1-deleted mice at single-cell resolution. Importantly, we linked the quantity of each cell's surface proteins to its total transcriptome, which is a novel method. Deletion of Flvcr1results in high levels of intracellular heme, allowing us to identify heme-regulated circuitry. Our studies demonstrate that in early erythroid cells (CD71+Ter119neg-lo), heme increases ribosomal protein transcripts, suggesting that heme, in addition to upregulating globin transcription and translation, guarantees ample ribosomes for globin synthesis. In later erythroid cells (CD71+Ter119lo-hi), heme decreases GATA1, GATA1-target gene, and mitotic spindle gene expression. These changes occur quickly. For example, in confirmatory studies using human marrow erythroid cells, ribosomal protein transcripts and proteins increase, and GATA1 transcript and protein decrease, within 15 to 30 minutes of amplifying endogenous heme synthesis with aminolevulinic acid. Because GATA1 initiates heme synthesis, GATA1 and heme together direct red cell maturation, and heme stops GATA1 synthesis, our observations reveal a GATA1–heme autoregulatory loop and implicate GATA1 and heme as the comaster regulators of the normal erythroid differentiation program. In addition, as excessive heme could amplify ribosomal protein imbalance, prematurely lower GATA1, and impede mitosis, these data may help explain the ineffective (early termination of) erythropoiesis in Diamond Blackfan anemia and del(5q) myelodysplasia, disorders with excessive heme in colony-forming unit-erythroid/proerythroblasts, explain why these anemias are macrocytic, and show why children with GATA1 mutations have DBA-like clinical phenotypes.

A Rh-catalyzed regioselective, stereoselective carbocyclization/borylation of acrylate-containing 1,6-enynes was described, which offers a general and practical method for constructing versatile and densely functionalized pyrrolidines with the (Z) geometry or the uncommon (E) geometry at the double bond with different substituents of the alkyne, with sterically hindered and conjugative aryl groups favoring the latter, featuring a tetrasubstituted vinyl boronate, containing an all-carbon quaternary stereocenter.

•A heme rheostat regulates normal erythropoiesis as well as the ineffective erythropoiesis of DBA and MDS-5q.•Marrow cells from patients with MDS-5q with or without the deletion of 5q share transcription profiles.

Absolute configurations of the three consecutive chiral centers in the cyclic depsipeptide microtermolide A have been tentatively assigned as 2‴R, 3‴R, and 4‴R. However, on the basis of a structural comparison with vinylamycin, another depsipeptide with a unique 4-amino-2,4-pentadienolate structure, the chiral centers could also be assigned as 2‴R, 3‴R, and 4‴S. Here, the first total synthesis of microtermolide A is reported and the configurations of the three consecutive chiral centers were confirmed to be 2‴R, 3‴R, and 4‴S. A similar approach was used to determine the analogous centers in microtermolide B as 2‴R, 3‴R, and 4‴S.

Congenital erythropoietic porphyria (CEP) is an autosomal recessive disorder of heme synthesis characterized by reduced activity of uroporphyrinogen III synthase and the accumulation of nonphysiologic isomer I porphyrin metabolites, resulting in ineffective erythropoiesis and devastating skin photosensitivity. Management of the disease primarily consists of supportive measures. Increased activity of 5-aminolevulinate synthase 2 (ALAS2) has been shown to adversely modify the disease phenotype. Herein, we present a patient with CEP who demonstrated a remarkable improvement in disease manifestations in the setting of iron deficiency. Hypothesizing that iron restriction improved her symptoms by decreasing ALAS2 activity and subsequent porphyrin production, we treated the patient with off-label use of deferasirox to maintain iron deficiency, with successful results. We confirmed the physiology of her response with marrow culture studies.

Congenital erythropoietic porphyria (CEP) is an autosomal recessive disorder of heme synthesis characterized by reduced activity of uroporphyrinogen III synthase and the accumulation of nonphysiologic isomer I porphyrin metabolites, resulting in ineffective erythropoiesis and devastating skin photosensitivity. Management of the disease primarily consists of supportive measures. Increased activity of 5-aminolevulinate synthase 2 (ALAS2) has been shown to adversely modify the disease phenotype. Herein, we present a patient with CEP who demonstrated a remarkable improvement in disease manifestations in the setting of iron deficiency. Hypothesizing that iron restriction improved her symptoms by decreasing ALAS2 activity and subsequent porphyrin production, we treated the patient with off-label use of deferasirox to maintain iron deficiency, with successful results. We confirmed the physiology of her response with marrow culture studies.

A Pd-catalyzed borylsilylative cyclization of 1,6-allenynes with PhMe2SiBpin was developed. This method provides a practical and general method to afford the carbocycles and heterocycles bearing silyl and boryl groups with excellent regioselectivities and stereoselectivities in high to excellent yields.

Lysosomal storage diseases are devastating illnesses, in large part because of their neurologic consequences. Because significant morbidity occurs prenatally, in utero (IU) therapy is an attractive therapeutic approach.

The matricellular angiogenesis inhibitor, thrombospondin (TSP) 2, has been shown to be an important modulator of wound healing and the foreign body response. Specifically, TSP2-null mice display improved healing with minimal scarring and form well-vascularized foreign body capsules. In this study we performed subcutaneous implantation of sponges and investigated the resulting angiogenic and fibrogenic responses. Histological and immunohistochemical analysis of sponges, excised at 7, 14, and 21 days after implantation, revealed significant differences between TSP2-null and wild-type mice. Most notably, TSP2-null mice exhibited increased angiogenesis and fibrotic encapsulation of the sponge. However, invasion of dense tissue was compromised, even though its overall density was increased. Furthermore, histomorphometry and biochemical assays demonstrated a significant increase in the extracellular distribution of matrix metalloproteinase (MMP) 2, but no change in the levels of active transforming growth factor-β1. The alterations in neovascularization, dense tissue invasion, and MMP2 in TSP2-null mice coincided with the deposition of TSP2 in the extracellular matrix of wild-type animals. These observations support the proposed role of TSP2 as a modulator of angiogenesis and matrix remodeling during tissue repair. In addition, they provide in vivoevidence for a newly proposed function of TSP2 as a modulator of extracellular MMP2 levels.

We have recently shown that the adhesive defect observed in dermal fibroblasts derived from thrombospondin 2 (TSP2)-null mice results from an increase in matrix metalloproteinase 2 (MMP2) levels (Yang, Z., Kyriakides, T. R., and Bornstein, P. (2000) Mol. Biol. Cell11, 3353–3364). Adhesion was restored by replacement of TSP2 and by inhibitors of MMP2 activity. In pursuing the observation that TSP2 and MMP2 interact, we now demonstrate that this interaction is required for optimal clearance of extracellular MMP2 by fibroblasts. Since TSP2 is known to be endocytosed by the scavenger receptor, low density lipoprotein receptor-related protein (LRP), we determined whether interference with LRP function affected fibroblast adhesion and/or extracellular MMP2 levels. Addition of heparin, which competes for the binding of TSP2 to LRP coreceptor proteoglycans, inhibited adhesion of control but not TSP2-null cells, and a blocking antibody to LRP as well as the LRP inhibitor, receptor-associated protein, also inhibited adhesion and increased MMP2 levels only in control fibroblasts. TSP2 did not inhibit active MMP2 directly and did not inhibit the activation of pro-MMP2. Finally, the internalization of 125I-MMP2 was reduced in TSP2-null compared with control fibroblasts. We propose that clearance of MMP2-TSP2 complexes by LRP is an important mechanism for the regulation of extracellular MMP2 levels in fibroblasts, and perhaps in other cells. Thus, some features of the phenotype of TSP2-null mice, such as abnormal collagen fibrillogenesis, accelerated wound healing, and increased angiogenesis, could result in part from increased MMP2 activity.

Thrombospondin 2 (TSP2)-null mice, generated by targeted disruption of the Thbs2 gene, display a complex phenotype that is characterized, in part, by a variety of connective tissue abnormalities and increased vascular density in skin and subcutaneous tissues. In this paper we summarize the evidence that TSP2 functions as a matricellular protein to influence cell function by modulating cell–matrix interactions, rather than acting as an integral component of the matrix. Thus, the structurally abnormal collagen fibrils detected in skin appear to be the consequence of the defective adhesion demonstrated by dermal fibroblasts in culture that, in turn, result from increased matrix metalloproteinase 2 (MMP2, gelatinase A) production by these cells. Corroborating evidence for such a mode of action comes from transmission electron microscopic images of developing flexor muscle tendons that show distinct abnormalities in fibroblast–collagen fibril interactions in TSP2-null tissue. The increased vascular density seen in skin of TSP2-null mice can be reproduced in a number of models of injury, including subcutaneous implantation of polyvinyl alcohol sponges and silicone rubber discs, and excisional skin wounds. Experiments are proposed to distinguish between a primarily endothelial cell versus an extracellular matrix origin for the increased angiogenesis in TSP2-null mice.Journal of Investigative Dermatology Symposium Proceedings (2000) 5, 61–66; doi:10.1046/j.1087-0024.2000.00005.x

Thrombospondin 2 (TSP2)-null mice, generated by disruption of theThbs2gene, display a variety of connective tissue abnormalities, including fragile skin and the presence of abnormally large collagen fibrils with irregular contours in skin and tendon. In this study we demonstrate that TSP2-null skin fibroblasts show a defect in attachment to a number of matrix proteins, and a reduction in cell spreading. To investigate the molecular mechanisms responsible for these abnormal cell–matrix interactions, we compared the levels of matrix metalloproteinases (MMPs) in wild-type and mutant fibroblasts. Isolation and analysis of gelatinases from conditioned media by gelatin-agarose affinity chromatography and gelatinolytic assays demonstrated that TSP2-null fibroblasts produce a 2-fold increase in gelatinase A (MMP2) compared with wild-type cells. The adhesive defect was corrected by treatment of TSP2-null fibroblasts with soluble TSP2, with the MMP inhibitors BB94 and tissue inhibitor of metalloproteinase-2, and with a neutralizing antibody to MMP2. Moreover, stable transfection of TSP2-null fibroblasts with mouse TSP2 cDNA corrected both the adhesive defect and the altered expression of MMP2. Finally, MMP2 was shown to interact with TSP2 in a direct-binding plate assay. We conclude that TSP2 plays an important role in cell–matrix interactions, and that a deficiency in the protein results in increased levels of MMP2 that contribute to the adhesive defect in TSP2-null fibroblasts and could play a role in the complex phenotype of TSP2-null mice.

Cell migration is a fundamental process of wound repair in biological systems. In an attempt to identify plasma membrane glycoconjugates which mediate cell migration, migrating and nonmigrating rabbit corneal epithelia were analyzed for reactivity with monoclonal antibodies (mAbs) specific for unsubstituted N-acetyllactosamine (mAb 1B2), Lex(mAbs 7A and MMA), and sialyl Lex(mAb CSLEX1) carbohydrate chains of neolactoglycoconjugates. Immunohistochemical analyses indicated that regardless of whether the epithelia analyzed were from corneas of animals in vivo, corneas in organ culture, or cells in tissue culture, migrating cells stained intensely with mAb 1B2, whereas nonmigrating cells either did not stain or stained only weakly. mAbs MMA and 7A stained migrating epithelium as well as basal and middle cell layers of normal, nonmigrating epithelium. mAb CSLEX1 did not stain wounded corneas but stained the superficial cell layer of normal corneal epithelium. Biochemical analyses by TLC immunostaining revealed the presence of three mAb 1B2-reactive glycosphingolipids (GSL), neolactotetraosyl-(nLc4, paragloboside), neolactohexaosyl- (nLc6), and neolacto-octaosylceramide (nLc8) in migrating epithelia. In contrast, nonmigrating epithelia contained only trace amounts of these glycolipids. Exogenous addition of nLc4, but not various other GSLs including a Lex-GSL (SSEA-1), stimulated re-epithelialization of wounds in an experimental model of corneal epithelial wound healing. Moreover, re-epithelialization of wounds was significantly inhibited by mAb 1B2 but not by mAb MMA. The data suggest that neolacto-GSLs of corneal epithelium may be among the molecules which mediate healing of corneal epithelial wounds by influencing cell migration.

A metal-free intramolecular borylative cyclization of 1,6-allenynes driven by BCl3was developed. This method provides a general and practical strategy to construct valuable pyrrolidines containing all-carbon quaternary centers or 3,5-dihydroazepine derivatives depending on the substituents of the allene, with conjugative and sterically hindered phenyl groups favoring the latter.

No relevant conflicts of interest to declare.

Thirty percent of Diamond Blackfan anemia (DBA) cases result from haploinsufficiency of ribosomal protein S19 and ~40% from haploinsufficiencies of 15 other ribosomal proteins. The macrocytic anemia of myelodysplasia with deletion of chromosome 5q (del(5q)MDS), which results from the acquisition of RPS14 haploinsufficiency, has a similar clinical phenotype. Although these mutations disrupt ribosome assembly and impair protein translation, how this causes macrocytic anemia remains uncertain and controversial. Since 95% of the protein content of red cells is globin, we hypothesized that any germline or somatic mutation that slows protein synthesis would impair globin production relative to heme production. This is because the synthesis of heme, a chemical chelate, depends on only small amounts of protein (enzymes) and the rate limiting enzyme, ALAS2, is an early GATA1 target. Studies of marrow cells from patients with DBA and del(5q) MDS show that heme synthesis indeed progresses normally, while globin synthesis is delayed. This results in excess heme in CFU-E/proerythroblasts, excessive ROS and cell death (Sci Transl Med8:338RA67, 2016). Similar results are seen in a murine model of heme excess (Flvcr1-deletion) (J Clin Invest125:4681, 2015) and murine models of DBA. As slowing heme synthesis improves the coordination of heme and globin and improves red cell production, a phase 2 study is underway (PI Bart Scott) to determine the efficacy of aggressive iron chelation to slow heme synthesis in patients with very low to intermediate risk MDS and anemia. More recently, we have quantitated the cell surface expression of CD71, CD44 and Ter119 on individual murine erythroid cells from normal, Flvcr1-deleted mice with macrocytic anemia, and erythropoietin-treated mice. We then barcoded and assessed the cell's total transcriptome. By linking these datasets, we uncovered a GATA1-heme autoregulatory loop which regulates normal erythropoiesis and contributes to the failed erythropoiesis of ribosomal protein haploinsufficiency. We show that heme normally upregulates ribosome protein transcription in early erythroid cells. Thus, in addition to increasing globin transcription and translation (via Bach1 and HRI), heme assures adequate ribosomes for globin synthesis. In later erythroid cells, heme decreases GATA1, GATA1 target genes and mitotic spindle gene expression, assuring that red cell differentiation appropriately terminates and cell division ceases. In human marrow CD36+GlyA- or CD36+GlyA+ cells, these changes occur within 15 minutes of inducing endogenous heme synthesis with ALA (bypasses ALAS2) and iron. As excess heme would increase ROS, increase ribosomal protein imbalance to intensify P53 expression, prematurely lower GATA1, and impede mitosis, our data explain the ineffective (early termination of) erythropoiesis in DBA and del(5q) MDS, help explain why these anemias are macrocytic, and reconcile the disparate observations of others.

No relevant conflicts of interest to declare.

When heme synthesis exceeds globin synthesis, red blood cell differentiation fails, resulting in severe anemia.

GATA1 promotes the transcription of ALAS2, the first and rate limiting step of heme synthesis, and the transcription of many other erythroid-specific genes. It also increases its own transcription while silencing proliferation genes active in early progenitors and thus assures that erythroid differentiation correctly initiates. Heme then transcriptionally and translationally upregulates globin to guarantee adequate hemoglobin production in each cell as it matures. In mice lacking the heme exporter, FLVCR1, excess heme and ROS accumulate and erythropoiesis fails at the CFU-E/proerythroblast stage, resulting in a severe macrocytic anemia (HGB 4.4±0.97 vs 14.8±0.57 g/dL; MCV 66.9±6.2 vs 48.4±0.65 fL). To determine how excess heme causes ineffective erythropoiesis and whether heme is key to terminating differentiation in normal erythroid cells, we performed RNA sequencing of single early erythroid cells (BFU-E to basophilic erythroblasts) from wildtype control and Flvcr1-deleted mice and linked this transcription data to the total quantity of Ter119 on that cell. Principal component analysis (PCA) identified 4 transcriptionally unique clusters A, B, C, & D, which contained cells with negative, low, intermediate, and high Ter119 levels respectively. α- and β-globin transcription were highly correlated (r=0.975), occurred in all cells, increased as Ter119 expression increased, and upregulated in Flvcr1-deleted cells. Gene set enrichment analysis (GSEA) comparing control cells to Flvcr1-deleted cells revealed excess heme results in significant downregulation of the hallmark heme metabolism pathway genes (heme biosynthesis and erythroid differentiation genes), upregulation of the ribosome pathway genes, and no alteration of the P53 pathway genes. All eight heme biosynthetic enzyme genes were expressed equivalently in cluster A cells from control and Flvcr1-deleted mice; however expression in Flvcr1-deleted cells was significantly reduced in clusters B-D. Of the 181 erythroid differentiation genes in the hallmark heme pathway, Gata1had the greatest reduction (67%) in Flvcr1-deleted cells. Coupled two-way clustering analysis (CTWC) identified 150 genes co-regulated with Gata1including 106 known GATA1 target genes which were all poorly upregulated in Flvcr1-deleted cells in clusters B-D. Independent microarray analysis of mRNA from control and Flvcr1-deleted CD71+erythroid cells confirmed low Gata1mRNA and low GATA1-dependent gene expression in the Flvcr1-deleted cells. To determine if excess heme was directly responsible for Gata1downregulation, we treated K562, HEL-R, and primary human erythroid marrow cells with aminolevulinic acid (ALA) and iron to increase endogenous heme synthesis. In the primary cells, GATA1 protein decreased by 30-43% (p=0.03) within 15 minutes and 66% by 90 minutes (similar decreases observed in cell lines), suggesting that heme disrupts GATA1 protein function resulting in the loss of autoregulation and reduced GATA1mRNA. Of 88 genes in the ribosome pathway, 73 were significantly upregulated in Flvcr1-deleted cells, including 16 of the 17 ribosomal protein genes linked to Diamond-Blackfan anemia (DBA) or del(5q) myelodysplastic syndrome (MDS). When heme synthesis was induced in primary human erythroid marrow cells with ALA and iron, the transcription of ribosome protein genes such as Rps19, Rps14, and Rpl35 increased, further supporting the concept that heme assures sufficient ribosome production for globin protein synthesis. While P53 activation is a key factor in ineffective erythropoiesis caused by ribosomal protein imbalance (i.e., DBA and del(5q) MDS), GSEA did not reveal any increased activation of the P53 pathway in Flvcr1-deleted cells. To confirm that P53 was not involved in the ineffective erythropoiesis caused by excess heme, we generated mice lacking both P53 and FLVCR1. These double mutant mice had severe macrocytic anemia (HGB 2.4±0.70 g/dL; MCV 56.5±4.3 fL) comparable to mice lacking just FLVCR1. Thus, GATA1 turns on heme synthesis and initiates the erythroid differentiation program. GATA1 with heme assure each cell’s appropriate progression. Then heme turns off GATA1 to end differentiation. By linking excess heme to prematurely low GATA1, our data may also explain the ineffective (early termination of) erythropoiesis in DBA and reconcile the observations of Sci Transl Med8:338ra67, 2016 and Nat Med20:748, 2014.

No relevant conflicts of interest to declare.

Twenty-five percent of Diamond Blackfan anemia (DBA) cases result from haploinsufficiency of RPS19 and ~30% from haploinsufficiencies of other ribosomal proteins. The macrocytic anemia of del(5q) MDS results from RPS14 haploinsufficiency. When tested, these mutations disrupt ribosome assembly, yet how this causes macrocytic anemia remains uncertain. The dominant hypothesis is that P53 pathway activation leads to cell apoptosis, however, why erythropoiesis is severely impacted, while other hematopoietic as well as non-hematopoietic cells function appropriately, is difficult to explain. These observations led us to predict that DBA and del(5q) MDS share a pathogenesis that reflects the rapid kinetics of red cell and hemoglobin production, and specifically that poor ribosome assembly leads to an inability to quickly translate mRNA to protein, delayed globin synthesis in early erythroid precursors, and excess heme. Since free heme is toxic, cell death ensues. To explore this, we studied marrow from DBA (n=3) and del(5q) MDS (n=6) patients in cultures that optimize erythroid differentiation and showed that (a) cell numbers significantly decrease between stages I (lin-CD71+CD36-GlyA-)(BFU-E/early CFU-E) and II, (lin-CD71+CD36+GlyA-)(CFU-E/early proerythroblasts; Li et al. Blood2014;124:3636); and (b) the cells that survive this insult differentiate normally to stages III (lin-CD36+GlyA+) and IV (lin-CD36-GlyA+). Using marrow from the index DBA patient, a 28-year old transfusion-dependent female with inherited RPS19 haploinsufficiency, and (as available) marrow from other DBA and del(5q) MDS patients (n=2-7 patients per study), we demonstrated that heme synthesis (assessed by ALAS2mRNA and protein) initiates normally while globin production (α- and β-globin mRNA and protein) is slowed, and that stage I and II cells have excess heme. Next, to discern the cell-specific characteristics that allow a DBA or del(5q) MDS cell to survive and fully mature, we compared those cells able to advance to stages III and IV in patient and concurrent control cultures. Stage III-IV GlyA+DBA cells expressed 2.4-fold less ALAS2 mRNAand 2-fold more FLVCR mRNA than GlyA+control cells (p values<0.05). Since the levels of ALAS2 and FLVCR mRNA were similar in stage I DBA and control cells, the time when heme synthesis initiates, these data imply that DBA cells capable of down-regulating their heme synthesis (have less ALAS2) or content (have moreFLVCR and increased heme export) preferentially survive. Data from del(5q) MDS patients are similar - cells progressing to stages III and IV have 2.9-fold less ALAS2 mRNA (p<0.05) and 9.4-fold higher FLVCR mRNA (p<0.01) than control cells. That heme synthesis proceeds normally, while globin production initiates slowly, was confirmed by sequential sampling of marrow cultures at days 3, 7, 10, 13, and 17; and as expected, DBA and del(5q) MDS cells at day 7 had increased reactive oxygen species (ROS). Consistent with these observations, treatment with 10 μM succinylacetone, a specific inhibitor of heme synthesis, improved the erythroid cell output of DBA and del(5q) MDS cultures by 68-95% (p values<0.05), while the erythroid cell output of concurrent control cultures decreased by 4-13%. Cell death appears to result both from apoptosis (not shown) and ferroptosis, a ROS-induced process previously characterized in kidney epithelial cells and cancer cell lines (Dixon et al. Cell2012;149:1060). Recent data showing P53 sensitizes cells to ferroptosis by impeding the transcription of SCL7A11, a cysteine-glutamate antiporter, leading to less cysteine uptake, less GSH synthesis, less GPX4, and less antioxidants (Jiang et al. Nature2015;520:57) link our observations to work implicating P53 activation in DBA and del(5q) MDS anemia. In support of this connection, DBA marrow cells at culture day 10 have 57% less SLC7A11 mRNA than control cells in preliminary experiments. Together, our studies demonstrate that erythropoiesis fails when heme exceeds globin. The data also suggest that decreasing heme synthesis, either directly or by chelating iron, may improve these anemias. They further argue that lenalidomide (destabilizes P53) and therapies decreasing heme should be synergistic. Since diverse mutations could impede brisk protein transcription or translation, and hence brisk globin production, heme toxicity might also contribute to anemia in MDS patients without del(5q).

CEP is a rare, devastating disorder resulting from an autosomal recessive inheritance of mutations in the uroporphyrinogen III synthase (UROS) gene, encoding the fourth enzyme in the heme synthetic pathway. The disease is characterized by photosensitivity with blistering, skin fragility and disfiguration, as well as ineffective erythropoiesis and chronic hemolysis. The severity of disease is variable and directly related to the reduction in enzyme activity with different mutations in UROS. Other factors also modulate disease severity, including the activity of 5-aminolevulinate synthase 2 (ALAS2), the rate-limiting enzyme in erythroid heme production (To-Figueras et al, Blood 118:1443, 2011). Management of CEP is largely comprised of sun avoidance and supportive measures, though hematopoietic stem cell transplantation has been effective in some patients. We present a patient with severe CEP who demonstrated a two-year sustained improvement in hemolysis and photosensitivity with iron deficiency induced by chelation therapy and spontaneous iron losses, underscoring the hypothesis that clinical interventions that decrease heme synthesis may dramatically improve the disease phenotype.

No relevant conflicts of interest to declare.

No relevant conflicts of interest to declare.

Abstract 514

Feline Leukemia Virus, Subgroup C (FLVCR) is a newly found cytoplasmic heme exporter. Retroviral interference with FLVCR display results in a loss of erythroid cells after the CFU-E/proerythroblast stage of differentiation and severe anemia in cats. We studied expression of FLVCR in thalassemia with relatively redundant free cytoplasmic heme, evaluated the correlation of FLVCR expression with clinical parameters, and explore the importance of FLVCR for erythropoiesis in human cells. Samples of bone marrow from 50 children with thalassemia (n=21), other hemolytic anemia(AIHA, G6PD Deficiency, n=17) and non-anemia controls (n=12) were collected. Expression of FLVCR protein and mRNA in mononuclear red cells was quantitated in CFU-E/proerythroblasts (CD71 bright, CD117+) and later erythroid precursors (CD71 bright, CD117 negative cells) by 3 color flow cytometry and fluorescence quantitative Real-time PCR. Expression of FLVCR is higher in early precursors than later erythroid cells: Specifically, the percentage of specimens with high levels of FLVCR protein expression in proerythroblasts and the mean fluorescence intensities(MFI) of thalassemia, other hemolytic anemia and non-anemias were 78.9%, 84.2%,74.85% and 306,347, 291.5 respectively; while these values in later erythroid precursors were 8.8%,10.6%,6.9% and 150,122,119 respectively, P<0.01. Expression of FLVCR tended to be higher in proerythroblasts of thalassemia and other hemolytic anemia:Positive rate and MFI of FLVCR protein expression in thess two groups were 78.9%,306 and 84.2%,347 respectively, as compared with cells of non-anemias (74.85%,291.5), with no statistical differences, P>0.05. Expression of FLVCR is significantly higher in later erythroid precursors of thalassemia:Positive rate and MFI of FLVCR protein expression in later erythroid precursors of thalassemia were 8.8%, and 150 respectively, with significantly higher levels of expression compared with non-anemias (6.9%,119) P<0.05. Expression of FLVCR mRNA in proerythroblasts of thalassemia, other hemolytic anemia and non-anemias were 3.6,4.85,3.7 respectively; while in later erythroblast were 4.13,6.06,3.92 respectively, P<0.01. Hemolytic anemia patients with higher expression of FLVCR had higher reticulocyte count, higher percentage of later erythroblast and lower LDH, P<0.05. Thalassemic patients with higher expression of FLVCR had higher percentage of sideroblasts and serum ferritin, P<0.05. Expression of FLVCR protein was remarkably upregulated in proerythroblasts, combined with previous studies in vitro and identification of FLVCR as a human heme exporter, supporting FLVCR plays an important role in developing erythroid cells to protect them from heme toxicity. There was no significantly up-regulation of FLVCR expression in proerythroblasts of thalassemia, indicating probably other pathways exist in regulating heme metabolic balance combined with FLVCR; Expression of FLVCR in later erythroid precursors of thalassemia was higher than that of non-anemias, which implies FLVCR up-regulates with compensation, this may help counteract the ineffectiveness of late erythropoiesis that results from the poor coordination of heme and globin synthesis. The disparity between FLVCR protein and mRNA may reflect posttranscriptional regulation of protein level. Clinical results also support effect of FLVCR on erythropoiesis. FLVCR expression has close relation with iron metabolic parameters.

FLVCR is a heme export protein that is required for proerythroblast survival (Cell 18:757, 2004), macrophage heme-iron recycling, and systemic iron homeostasis (ASH abstract, 2007). However, the mechanism and regulation of export through this and related major facilitator superfamily (MFS) members (e.g., Glut4, GlpT) is unknown. To gain insights into the structure and function of FLVCR, we studied the export of zinc mesoporphyrin (ZnMP), a fluorescent heme analog that traffics like heme and blocks heme oxygenase (HO) activity. NRK cells engineered to express huFLVCR were loaded with ZnMP for 30 min, then incubated at 37°C × 90 min with no carrier protein, albumin, or hemopexin (HPXN). In the absence of carrier protein, no export was seen. Export was near complete in the presence of 152 μM (1%) albumin, but less extensive with 15.2 μM albumin and absent with 1.52 μM albumin. Interestingly, HPXN appeared 100 fold more efficacious in inducing ZnMP export, as the residual ZnMP fluorescence in the presence of 0.152 μM HPXN was equivalent to residual intensity in the presence of 15.2 μM albumin. Comparable results were obtained in studies of human blood macrophages. Heme trafficking was next quantitated using 55Fe-heme and a similar study design (ZnMP was added to block HO activity). In the presence of 1.52 μM albumin, 6% of the 55Fe-heme load was exported, while 52% was exported in the presence of 1.52 μM HPXN. When the albumin concentration was increased 100 fold to 152 μM, 67% was exported. As Kds are ∼10 nM and <1 pM, for albumin and HPXN, respectively, our data argue that the rate of heme export depends on the avidity of this binding. Carrier proteins coordinate the heme-iron through two histidines and stabilize this interaction with multiple aromatic amino acids lining the heme-binding pocket. Of note, there are three conserved histidine residues in FLVCR, two of which (aa 145 and 198) are on adjacent extracellular loops, and based upon known MFS family member structures (Protein Sci. 13:1832, 2004), are potentially optimally located to coordinate the heme-iron exiting the channel. Adjacent transmembrane regions contain several aromatic residues capable of lining a heme pocket. These histidine residues are conserved in human, cat, and mouse FLVCR, but are not conserved in closely related paralogs (Gene 286:203, 2002), which lack heme export function. We hypothesize they act as a temporary docking site for heme after it exits through the transport channel and prior to being picked up by HPXN in the circulation, and thus as a structural regulator of export efficiency. We anticipate that when heme binds to this site, the channel is held in the open-out position preventing additional heme from being transported. We are currently using site-directed mutagenesis to generate a mutant FLVCR lacking these two histidines and will test its export function. To our knowledge, comparable regulatory mechanisms have not been previously described for other MFS family members or for transport proteins more broadly. Since HPXN is present in a high concentration in human serum (17–25 μM, similar to transferrin, 22–31 μM), it may function physiologically to facilitate the recycling of heme-iron from macrophages after they ingest senescent red cells, in addition to its role as a scavenger protein for protection from internal bleeding or extravascular hemolysis. By extension, modulation of the serum HPXN concentration might ameliorate the anemia of chronic inflammation.

FLVCR is a heme export protein that is required for proerythroblast survival (Cell18:757, 2004), macrophage heme-iron recycling, and systemic iron homeostasis (ASH abstract, 2007). However, the mechanism and regulation of export through this and related major facilitator superfamily (MFS) members (e.g., Glut4, GlpT) is unknown. To gain insights into the structure and function of FLVCR, we studied the export of zinc mesoporphyrin (ZnMP), a fluorescent heme analog that traffics like heme and blocks heme oxygenase (HO) activity. NRK cells engineered to express huFLVCR were loaded with ZnMP for 30 min, then incubated at 37°C × 90 min with no carrier protein, albumin, or hemopexin (HPXN). In the absence of carrier protein, no export was seen. Export was near complete in the presence of 152 μM (1%) albumin, but less extensive with 15.2 μM albumin and absent with 1.52 μM albumin. Interestingly, HPXN appeared 100 fold more efficacious in inducing ZnMP export, as the residual ZnMP fluorescence in the presence of 0.152 μM HPXN was equivalent to residual intensity in the presence of 15.2 μM albumin. Comparable results were obtained in studies of human blood macrophages. Heme trafficking was next quantitated using 55Fe-heme and a similar study design (ZnMP was added to block HO activity). In the presence of 1.52 μM albumin, 6% of the 55Fe-heme load was exported, while 52% was exported in the presence of 1.52 μM HPXN. When the albumin concentration was increased 100 fold to 152 μM, 67% was exported. As Kds are ∼10 nM and <1 pM, for albumin and HPXN, respectively, our data argue that the rate of heme export depends on the avidity of this binding. Carrier proteins coordinate the heme-iron through two histidines and stabilize this interaction with multiple aromatic amino acids lining the heme-binding pocket. Of note, there are three conserved histidine residues in FLVCR, two of which (aa 145 and 198) are on adjacent extracellular loops, and based upon known MFS family member structures (Protein Sci.13:1832, 2004), are potentially optimally located to coordinate the heme-iron exiting the channel. Adjacent transmembrane regions contain several aromatic residues capable of lining a heme pocket. These histidine residues are conserved in human, cat, and mouse FLVCR, but are not conserved in closely related paralogs (Gene286:203, 2002), which lack heme export function. We hypothesize they act as a temporary docking site for heme after it exits through the transport channel and prior to being picked up by HPXN in the circulation, and thus as a structural regulator of export efficiency. We anticipate that when heme binds to this site, the channel is held in the open-out position preventing additional heme from being transported. We are currently using site-directed mutagenesis to generate a mutant FLVCR lacking these two histidines and will test its export function. To our knowledge, comparable regulatory mechanisms have not been previously described for other MFS family members or for transport proteins more broadly. Since HPXN is present in a high concentration in human serum (17–25 μM, similar to transferrin, 22–31 μM), it may function physiologically to facilitate the recycling of heme-iron from macrophages after they ingest senescent red cells, in addition to its role as a scavenger protein for protection from internal bleeding or extravascular hemolysis. By extension, modulation of the serum HPXN concentration might ameliorate the anemia of chronic inflammation.

Feline Leukemia Virus, Subgroup C (FLVCR) is a newly found cytoplasmic heme exporter. Retroviral interference with FLVCR display results in a loss of erythroid cells after the CFU-E/proerythroblast stage of differentiation and severe anemia in cats. We studied expression of FLVCR in thalassemia with relatively redundant free cytoplasmic heme, evaluated the correlation of FLVCR expression with clinical parameters, and explore the importance of FLVCR for erythropoiesis in human cells. Samples of bone marrow from 50 children with thalassemia (n=21), other hemolytic anemia(AIHA, G6PD Deficiency, n=17) and non-anemia controls (n=12) were collected. Expression of FLVCR protein and mRNA in mononuclear red cells was quantitated in CFU-E/proerythroblasts (CD71 bright, CD117+) and later erythroid precursors (CD71 bright, CD117 negative cells) by 3 color flow cytometry and fluorescence quantitative Real-time PCR. Expression of FLVCR is higher in early precursors than later erythroid cells: Specifically, the percentage of specimens with high levels of FLVCR protein expression in proerythroblasts and the mean fluorescence intensities(MFI) of thalassemia, other hemolytic anemia and non-anemias were 78.9%, 84.2%,74.85% and 306,347, 291.5 respectively; while these values in later erythroid precursors were 8.8%,10.6%,6.9% and 150,122,119 respectively, P<0.01. Expression of FLVCR tended to be higher in proerythroblasts of thalassemia and other hemolytic anemia:Positive rate and MFI of FLVCR protein expression in thess two groups were 78.9%,306 and 84.2%,347 respectively, as compared with cells of non-anemias (74.85%,291.5), with no statistical differences, P>0.05. Expression of FLVCR is significantly higher in later erythroid precursors of thalassemia:Positive rate and MFI of FLVCR protein expression in later erythroid precursors of thalassemia were 8.8%, and 150 respectively, with significantly higher levels of expression compared with non-anemias (6.9%,119) P<0.05. Expression of FLVCR mRNA in proerythroblasts of thalassemia, other hemolytic anemia and non-anemias were 3.6,4.85,3.7 respectively; while in later erythroblast were 4.13,6.06,3.92 respectively, P<0.01. Hemolytic anemia patients with higher expression of FLVCR had higher reticulocyte count, higher percentage of later erythroblast and lower LDH, P<0.05. Thalassemic patients with higher expression of FLVCR had higher percentage of sideroblasts and serum ferritin, P<0.05. Expression of FLVCR protein was remarkably upregulated in proerythroblasts, combined with previous studies in vitro and identification of FLVCR as a human heme exporter, supporting FLVCR plays an important role in developing erythroid cells to protect them from heme toxicity. There was no significantly up-regulation of FLVCR expression in proerythroblasts of thalassemia, indicating probably other pathways exist in regulating heme metabolic balance combined with FLVCR; Expression of FLVCR in later erythroid precursors of thalassemia was higher than that of non-anemias, which implies FLVCR up-regulates with compensation, this may help counteract the ineffectiveness of late erythropoiesis that results from the poor coordination of heme and globin synthesis. The disparity between FLVCR protein and mRNA may reflect posttranscriptional regulation of protein level. Clinical results also support effect of FLVCR on erythropoiesis. FLVCR expression has close relation with iron metabolic parameters.

Heme is a component of cytochromes, catalases, glutathione peroxidase, hydroxylases, and nitric oxide synthase, as well as myoglobin and hemoglobin, and thus is critical to all aerobic cells. It is also a transcriptional and translational initiator of globin synthesis. However, free heme is toxic so that cells must balance synthesis with use. We recently determined that the cell surface receptor for Feline Leukemia Virus, subgroup C, (FLVCR), which causes pure red cell aplasia in cats, is a heme export protein (Cell; 118:6, 2004). To understand its physiologic significance, we generated constitutive (Flvcr+/-) and inducible (Flvcrflox/flox;Mx-cre) FLVCR null mice by targeted deletion of exon 3, and confirmed this produced a nonfunctional protein by zinc mesoporphyrin (ZnMP) and 55Fe-heme export studies. Intercrossing Flvcr+/- mice produced 0 null animals among 109 progeny genotyped. FLVCR null embryos die either before E8.5 or at E14.5, suggesting two separate mechanisms for their demise. In situ hybridization studies in normal mouse embryos show highest Flvcr transcript levels in the ectoplacental cone which forms placenta (E8.5), the placenta (E9.5), and high levels in the fetal liver (E12.5). These data plus studies with Flvcrflox/flox x Meox2-Cre mice (Cre inactivates Flvcr in all embryonic tissue, but not extraembryonic visceral endoderm and placenta) suggest that the early lethality of null mice is due to impaired maternal-embryo heme-iron transport. At E14.5, residual null mice were small with deformed limb buds and craniofacial abnormalities resembling the congenital anomalies of Diamond-Blackfan anemia. Flow cytometric analyses of fetal liver cells from deleted animals with Ter119 and CD71 demonstrated a block in erythroid differentiation at the CFU-E/proerythroblast stage, implying that their death results from a stage-specific failure in definitive erythropoiesis. Heterozygous null animals (Flvcr+/-) were viable with normal hematologic parameters. These mice had 30.9% of normal Flvcr mRNA by q RT-PCR, however, they demonstrated normal protein by western blot analysis and normal heme export function by ZnMP studies. Thus, heterozygous null mice compensate at the protein level, confirming FLVCR is critical in vivo. We next utilized our inducible mutant line (Flvcrflox/flox;Mx-cre) to generate a viable null mouse for study. Flvcr deleted mice developed pure red cell aplasia characterized by macrocytic anemia (deleted: HCT 13.8±1.2, MCV 68.35± 1.6; control: HCT 50.24 ± 2.0, MCV 51.3±1.5), reticulocytopenia, and reactive thrombocytosis. Marrow and spleen had excess cells with a proerythroblast morphology and lacked maturing red cells. Flow cytometry confirmed maturation arrest at the CFU-E/proerythroblast stage. CFU-E from FLVCR null mice were undetectable in culture. Deleted mice also demonstrated iron loading of hepatocytes. FLVCR is highly expressed in multiple human tissues with high heme-flux by western blot analyses, including liver, placenta, duodenum, and uterus, in addition to the bone marrow. Our studies prove that FLVCR is required for the survival or differentiation of proerythroblasts (that stage where heme synthesis intensifies). These results, plus additional data not shown, suggest that FLVCR may also protect nonerythroid tissues with high free heme exposure (uterus, duodenum), may mediate maternal-embryo heme-iron transport, and may mediate the transport of heme from hepatocyte to bile which would imply a novel mechanism for iron export from the body.

Heme is a component of cytochromes, catalases, glutathione peroxidase, hydroxylases, and nitric oxide synthase, as well as myoglobin and hemoglobin, and thus is critical to all aerobic cells. It is also a transcriptional and translational initiator of globin synthesis. However, free heme is toxic so that cells must balance synthesis with use. We recently determined that the cell surface receptor for Feline Leukemia Virus, subgroup C, (FLVCR), which causes pure red cell aplasia in cats, is a heme export protein (Cell; 118:6, 2004). To understand its physiologic significance, we generated constitutive (Flvcr+/−) and inducible (Flvcrflox/flox;Mx-cre) FLVCR null mice by targeted deletion of exon 3, and confirmed this produced a nonfunctional protein by zinc mesoporphyrin (ZnMP) and 55Fe-heme export studies. Intercrossing Flvcr+/−mice produced 0 null animals among 109 progeny genotyped. FLVCR null embryos die either before E8.5 or at E14.5, suggesting two separate mechanisms for their demise. In situ hybridization studies in normal mouse embryos show highest Flvcrtranscript levels in the ectoplacental cone which forms placenta (E8.5), the placenta (E9.5), and high levels in the fetal liver (E12.5). These data plus studies with Flvcrflox/floxx Meox2-Cremice (Cre inactivates Flvcrin all embryonic tissue, but not extraembryonic visceral endoderm and placenta) suggest that the early lethality of null mice is due to impaired maternal-embryo heme-iron transport. At E14.5, residual null mice were small with deformed limb buds and craniofacial abnormalities resembling the congenital anomalies of Diamond-Blackfan anemia. Flow cytometric analyses of fetal liver cells from deleted animals with Ter119 and CD71 demonstrated a block in erythroid differentiation at the CFU-E/proerythroblast stage, implying that their death results from a stage-specific failure in definitive erythropoiesis. Heterozygous null animals (Flvcr+/−) were viable with normal hematologic parameters. These mice had 30.9% of normal FlvcrmRNA by q RT-PCR, however, they demonstrated normal protein by western blot analysis and normal heme export function by ZnMP studies. Thus, heterozygous null mice compensate at the protein level, confirming FLVCR is critical in vivo. We next utilized our inducible mutant line (Flvcrflox/flox;Mx-cre) to generate a viable null mouse for study. Flvcrdeleted mice developed pure red cell aplasia characterized by macrocytic anemia (deleted: HCT 13.8±1.2, MCV 68.35± 1.6; control: HCT 50.24 ± 2.0, MCV 51.3±1.5), reticulocytopenia, and reactive thrombocytosis. Marrow and spleen had excess cells with a proerythroblast morphology and lacked maturing red cells. Flow cytometry confirmed maturation arrest at the CFU-E/proerythroblast stage. CFU-E from FLVCR null mice were undetectable in culture. Deleted mice also demonstrated iron loading of hepatocytes. FLVCR is highly expressed in multiple human tissues with high heme-flux by western blot analyses, including liver, placenta, duodenum, and uterus, in addition to the bone marrow. Our studies prove that FLVCR is required for the survival or differentiation of proerythroblasts (that stage where heme synthesis intensifies). These results, plus additional data not shown, suggest that FLVCR may also protect nonerythroid tissues with high free heme exposure (uterus, duodenum), may mediate maternal-embryo heme-iron transport, and may mediate the transport of heme from hepatocyte to bile which would imply a novel mechanism for iron export from the body.