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Objective Our New York City Municipal Public Health System-based multisite ambulatory and school-based Gotham Health clinics offer waived point-of-care tests and provider-performed microscopy to the local communities. Our Gotham Health laboratory service conducts system-wide centralized implementation, monitoring, and oversight of the POCT operations. Laboratory staffing has always been an issue for us as there is a decades-long shortage of laboratory staff, primarily licensed medical technologists and technicians, in New York, like many other states. Our clinical laboratory operations team struggled to hire qualified people even before the COVID-19 pandemic onset. It has faced more significant challenges with the emergence of SARS-CoV-2 pandemic cases in New York City and across the country since mid-March 2020. Methods As staffing continues to be a struggle, it directly affected the POCT performances and a system-wide reduction in the test numbers during the pandemic. We investigated to identify the factors that made staffing more challenging. Results The impact on our POCT started after laboratory staff relocated to the acute care hospital laboratories to provide testing support during the pandemic’s peak. That caused significant delays or complete cessation of POCT operations in the clinics due to a lack of oversight support. We also experienced the risk of more vacated positions where staff already feel overworked, overwhelmed, and emotionally drained, causing professional burnout. The significant challenges identified are noncompliance with vaccine mandates resulting in job dismissal and voluntary resignations in exchange for higher-paying laboratories. Finally, the other challenges identified were frequent sick calls due to mental fatigue, retirement of seasoned staff, and inability to attract qualified technologists to meet the demands of increasing test-ordering patterns. Conclusions Determining the factors that culminated in the staffing issues becoming more challenging during the COVID-19 pandemic in our ambulatory care clinic laboratory operations will help us in future crisis planning and mitigation.

Erythropoiesis involves complex interrelated molecular signals influencing cell survival, differentiation, and enucleation. Diseases associated with ineffective erythropoiesis, such as β-thalassemias, exhibit erythroid expansion and defective enucleation. Clear mechanistic determinants of what make erythropoiesis effective are lacking. We previously demonstrated that exogenous transferrin ameliorates ineffective erythropoiesis in β-thalassemic mice. In the current work, we utilize transferrin treatment to elucidate a molecular signature of ineffective erythropoiesis in β-thalassemia. We hypothesize that compensatory mechanisms are required in β-thalassemic erythropoiesis to prevent apoptosis and enhance enucleation. We identify pleckstrin-2—a STAT5-dependent lipid binding protein downstream of erythropoietin—as an important regulatory node. We demonstrate that partial loss of pleckstrin-2 leads to worsening ineffective erythropoiesis and pleckstrin-2 knockout leads to embryonic lethality in β-thalassemic mice. In addition, the membrane-associated active form of pleckstrin-2 occurs at an earlier stage during β-thalassemic erythropoiesis. Furthermore, membrane-associated activated pleckstrin-2 decreases cofilin mitochondrial localization in β-thalassemic erythroblasts and pleckstrin-2 knockdown in vitro induces cofilin-mediated apoptosis in β-thalassemic erythroblasts. Lastly, pleckstrin-2 enhances enucleation by interacting with and activating RacGTPases in β-thalassemic erythroblasts. This data elucidates the important compensatory role of pleckstrin-2 in β-thalassemia and provides support for the development of targeted therapeutics in diseases of ineffective erythropoiesis., Maria Feola et al., elucidate the compensatory role of pleckstrin-2 in ineffective erythropoiesis in β-thalassemic mice by reducing cofilin-mediated apoptosis and enhancing enucleation by activating RacGTPases. These findings could support future therapeutic interventions.

Iron availability for erythropoiesis and its dysregulation in β-thalassemia are incompletely understood. We previously demonstrated that exogenous apotransferrin leads to more effective erythropoiesis, decreasing erythroferrone (ERFE) and derepressing hepcidin in β-thalassemic mice. Transferrin-bound iron binding to transferrin receptor 1 (TfR1) is essential for cellular iron delivery during erythropoiesis. We hypothesize that apotransferrin's effect is mediated via decreased TfR1 expression and evaluate TfR1 expression in β-thalassemic mice in vivo and in vitro with and without added apotransferrin. Our findings demonstrate that β-thalassemic erythroid precursors overexpress TfR1, an effect that can be reversed by the administration of exogenous apotransferrin. In vitro experiments demonstrate that apotransferrin inhibits TfR1 expression independent of erythropoietin- and iron-related signaling, decreases TfR1 partitioning to reticulocytes during enucleation, and enhances enucleation of defective β-thalassemic erythroid precursors. These findings strongly suggest that overexpressed TfR1 may play a regulatory role contributing to iron overload and anemia in β-thalassemic mice. To evaluate further, we crossed TfR1+/- mice, themselves exhibiting iron-restricted erythropoiesis with increased hepcidin, with β-thalassemic mice. Resultant double-heterozygote mice demonstrate long-term improvement in ineffective erythropoiesis, hepcidin derepression, and increased erythroid enucleation in relation to β-thalassemic mice. Our data demonstrate for the first time that TfR1+/- haploinsufficiency reverses iron overload specifically in β-thalassemic erythroid precursors. Taken together, decreasing TfR1 expression during β-thalassemic erythropoiesis, either directly via induced haploinsufficiency or via exogenous apotransferrin, decreases ineffective erythropoiesis and provides an endogenous mechanism to upregulate hepcidin, leading to sustained iron-restricted erythropoiesis and preventing systemic iron overload in β-thalassemic mice.

The matrix (MA) domain of the HIV-1 structural precursor Gag (PrGag) protein targets PrGag proteins to membrane assembly sites, and facilitates incorporation of envelope proteins into virions. To evaluate the specific requirements for the MA membrane-binding domain (MBD) in HIV-1 assembly and replication, we examined viruses in which MA was replaced by alternative MBDs. Results demonstrated that the pleckstrin homology domains of AKT protein kinase and phospholipase C δ1 efficiently directed the assembly and release of virus-like particles (VLPs) from cells expressing chimeric proteins. VLP assembly and release also were mediated in a phorbol ester-dependent fashion by the cysteine-rich binding domain of phosphokinase Cγ. Although alternative MBDs promoted VLP assembly and release, the viruses were not infectious. Notably, PrGag processing was reduced, while cleavage of GagPol precursors resulted in the accumulation of Pol-derived intermediates within virions. Our results indicate that the HIV-1 assembly machinery is flexible with regard to its means of membrane association, but that alternative MBDs can interfere with the elaboration of infectious virus cores.

Iron overload results in significant morbidity and mortality in β-thalassemic patients. Insufficient hepcidin is implicated in parenchymal iron overload in β-thalassemia and approaches to increase hepcidin have therapeutic potential. We have previously shown that exogenous apo-transferrin markedly ameliorates ineffective erythropoiesis and increases hepcidin expression in Hbb(th1/th1) (thalassemic) mice. We utilize in vivo and in vitro systems to investigate effects of exogenous apo-transferrin on Smad and ERK1/2 signaling, pathways that participate in hepcidin regulation. Our results demonstrate that apo-transferrin increases hepcidin expression in vivo despite decreased circulating and parenchymal iron concentrations and unchanged liver Bmp6 mRNA expression in thalassemic mice. Hepatocytes from apo-transferrin-treated mice demonstrate decreased ERK1/2 pathway and increased serum BMP2 concentration and hepatocyte BMP2 expression. Furthermore, hepatocyte ERK1/2 phosphorylation is enhanced by neutralizing anti-BMP2/4 antibodies and suppressed in vitro in a dose-dependent manner by BMP2, resulting in converse effects on hepcidin expression, and hepatocytes treated with MEK/ERK1/2 inhibitor U0126 in combination with BMP2 exhibit an additive increase in hepcidin expression. Lastly, bone marrow erythroferrone expression is normalized in apo-transferrin treated thalassemic mice but increased in apo-transferrin injected wild-type mice. These findings suggest that increased hepcidin expression after exogenous apo-transferrin is in part independent of erythroferrone and support a model in which apo-transferrin treatment in thalassemic mice increases BMP2 expression in the liver and other organs, decreases hepatocellular ERK1/2 activation, and increases nuclear Smad to increase hepcidin expression in hepatocytes.

Erythropoiesis involves stem cell differentiation to mature red blood cells (RBCs). Erythropoietin (Epo) is essential for erythroipoiesis, and Epo binding to Epo receptor triggers a complicated and incompletely understood set of potentially related molecular signals influencing cell survival, differentiation, and enucleation. Although Epo is associated with increased survival of erythroid precursors, it induces reactive oxygen species (ROS), and high Epo concentration has an anti-enucleation effect in vitro.i Furthermore, diseases of ineffective erythropoiesis, e.g. β-thalassemia, are associated with increased Epo and ROS concentrations implicated in the expansion of and damage to erythroid precursors, respectively. Treating erythroblasts with low dose ROS scavenger promotes enucleation, but high dose ROS scavenger leads to cell death,i suggesting that an optimal ROS concentration is integral to effective erythropoiesis. We and others have shown that ROS is increased in β-thalassemic erythroid precursors, but despite increased ROS, erythroid precursor apoptosis is not increased. We hypothesize that compensatory mechanisms prevent the ill-effects of increased ROS on erythroid precursors. Our prior experiments demonstrate disordered erythropoiesis in β-thalassemic (th1/th1) mice, restored in transferrin-treated th1/th1 mice,ii despite which, ROS remained increased in erythroid precursor from transferrin-treated th1/th1 mice. To identify mechanisms responsible for transferrin's effect, we performed RNA seq analysis of erythroblasts from wild type (WT), th1/th1, and transferrin-treated th1/th1 mice. We identified increased pleckstrin-2 (plek2) in th1/th1 relative to WT mice, normalized in transferrin-treated th1/th1 mice. We hypothesize that plek2 activation counteracts the ill effects of ROS and promotes enucleation in β-thalassemia. Using confocal microscopy, we demonstrate that 1) plek2 co-localizes with actin on the cell membrane after the pro-erythroblast stage but is in the nucleus throughout terminal erythropoiesis in WT mice; 2) membrane-associated plek2 is present earlier, in pro-erythroblasts, and remains membrane-associated until orthochromatophilic stage in th1/th1 mice; and 3) plek2 localization is normalized in transferrin-treated th1/th1 mice. Because plek2 activation leads to its association with the cell membrane and plek2 activation is increased in th1/th1 erythroblasts, we set out to explore the role of plek2 activation on ineffective erythropoiesis in transferrin-treated th1/th1 mice. Prior publications propose that plek2 interacts with and results in the phosphorylation of cofilin, preventing cofilin's translocation to the mitochondria as part of the apoptosis pathway in response to increased ROS.iii We demonstrate decreased in mitochondria cofilin localization and increased cellular p-cofilin in th1/th1 erythroblasts, normalized after transferrin treatment. These data suggest that despite an increase in ROS, plek2 and its induction of p-cofilin inhibit apoptosis in β-thalassemic erythroid precursors. Furthermore, in light of a prior report of an anti-enucleation effect of plek2 in vitroiii and the known regulation of enucleation by RacGTPasesiv, we hypothesize that plek2 activation triggers RacGTPase and prevents enucleation in th1/th1 mice. We demonstrate that in addition to changes in erythroblast RacGTPase concentration, membrane co-localization between plek2 and RacGTPase is enhanced and occurs earlier in th1/th1 erythroid differentiation relative to WT, normalized after transferrin treatment. Lastly, cleavage of Rho-associated kinase, Rock1, associated with enucleation,v is also decreased in th1/th1 erythroblasts, enhanced after transferrin treatment. Taken together, we speculate that plek2 haplo-insufficiency benefits β-thalassemic mice by enabling apoptosis of ineffective erythroblasts, or result in worsening, possibly lethal, phenotype in light of the direct or indirect (through effects on RacGTPase or Rock1) role of plek2 in enucleation. Presently, we are testing this hypothesis by generating plek2+/- and plek-/- β-thalassemic mice. In conclusion, we demonstrate the important compensatory role of plek2 in β-thalassemic erythropoiesis. i Zhao Exp Hematol 2016 ii Liu Blood 2013 iii Zhao haematol 2014 iv Ji Nat Cell Bio 2008 v Gabet Cell Death Diff 2011 Disclosures No relevant conflicts of interest to declare.

Transferrin-bound iron binding to transferrin receptor 1 (TfR1) is essential for erythropoiesis, and TfR1 is found in highest concentrations on erythroid precursors due to high iron requirement for hemoglobin (Hb) synthesis. Diseases of ineffective erythropoiesis such as β-thalassemia, are characterized by anemia, expanded and extramedullary erythropoiesis, and iron overload. Iron overload results from insufficient hepcidin, a peptide hormone secreted by hepatocytes in response to iron load. In β-thalassemia, hepcidin is relatively suppressed as a consequence of erythroid expansion. Erythroferrone (ERFE), a recently described erythroid-derived hepcidin suppressor, has been proposed as the mechanism and found in higher concentration in bone marrow of β-thalassemic mice. We previous demonstrate that exogenous transferrin (Tf) ameliorates anemia in β-thalassemic mice, reversing splenomegaly, hepcidin suppression, and iron overload and recently confirmed a decrease in Erfe expression in erythroid precursors from Tf-treated β-thalassemic mice. We observed that although Tf-treated β-thalassemic mice exhibit a further decrease in MCV and MCH, suggesting a relatively more iron restricted erythropoiesis, TfR1 expression is decreased. We hypothesize that TfR1 is central to Tf's effect on erythropoiesis in β-thalassemic mice. Last year, we presented our analysis of th3/+ TfR1+/- double heterozygote mice which exhibit reversal of all erythropoiesis- and iron-related pathology in th3/+ mice, confirming our observations in Tf-treated β-thalassemic mice and further supporting our hypothesis. To evaluate the mechanism involved, we observed that despite suppressed TfR1 concentration in reticulocyte (P=0.006) and sorted bone marrow erythroid precursors (P=0.0004) from Tf-treated th1/th1 mice, cell surface TfR1 expression decreased on reticulocytes (P=0.003) but was surprisingly increased on late stage erythroid precursors (P=0.007) (Figure 1A), suggesting that exogenous Tf influences erythroid precursor enucleation. Because we previously demonstrate decreased serum soluble TfR1 in Tf-treated th1/th1 mice [Liu J Blood 2013], we hypothesize that exogenous Tf alters TfR1 shedding from erythroid precursor membranes, promoting enucleation and improved terminal differentiation. We observed decreased enucleation using syto60 in flow cytometry of fetal liver cells (FLC) from th3/+ relative to wild type (WT) embryos (35 vs. 51%, P=0.03) which is normalized by exposure of th3/+ FLCs to Tf in vitro (58 vs. 41%, P=0.001) (Figure 1B). Tf-treated th3/+ FLCs shed more TfR1 to the nuclear fraction relative to reticulocyte during enucleation (P=0.0001) (Figure 1C). Furthermore, enucleation isdecreased in vivo in th3/+ relative to WT FLCs and peripheral blood at E14.5 and normalized in th3/+ TfR1+/- double heterozygote mice (45 vs. 35%, P=0.002) (Figure 1D). Interestingly, we analyzed iron status in TfR1+/- mice revealing that serum hepcidin is increased relative to WT (323 vs. 190 ng/ml, P=0.04) despite minimally decreased serum and liver iron concentrations (no statistically significant differences) and increased Erfe expression in erythroid precursors (5-fold, P=0.04). Relative to th3/+ mice, double heterozygote mice exhibit decreased serum iron (94 vs. 133 ug/dl), non-heme liver iron (0.31 vs. 0.74 mg iron/g dry weight, P=0.02), and Erfe expression (0.3-fold, P=0.04). Although no difference is observed between double heterozygote mice and th3/+, serum hepcidin is significantly increased in double heterozygote mice compare to WT (392 vs. 190 ng/ml, P=0.01), suggesting a more appropriate hepcidin response to iron overload (Figure 1E). Taken together, we postulate that decreased TfR1 expression plays a critical role in reversing ineffective erythropoiesis by increasing enucleation and influences hepcidin regulation in an ERFE independent manner. Disclosures No relevant conflicts of interest to declare.

Low/intermediate risk myelodysplastic syndromes (MDS) is associated with relatively longer survival and high transfusion requirements, resulting in secondary iron overload. Iron overload is an independent variable associated with poor prognosis, impacting survival, and iron chelation therapy is associated with prolonged survival in transfusion-dependent MDS patients. We have previously shown that exogenous transferrin (Tf) results in more circulating red blood cells (RBCs), increased hemoglobin (Hb), reversal of splenomegaly, and improvement in ineffective erythropoiesis in β-thalassemic mice (Li Nat Med 2010) and recently demonstrate that erythroferrone (ERFE), a newly described physiologic erythroid regulator of hepcidin, is normalized in Tf-treated β-thalassemic mice (Chen, manuscript in review). We postulate that ineffective erythropoiesis in β-thalassemia shares many characteristics with that of low risk MDS, such as defective erythroid differentiation, anemia, splenomegaly, and systemic iron overload, suggesting that similar effects of exogenous Tf may be relevant also in MDS. Partial deletion of chromosome 5, 5q-syndrome, is the most common cytogenetic abnormality in low/intermediate risk MDS. We characterize a mouse model of 5q-syndrome, Cd74+/lox Nid67+/lox, Lmo2Cre+ mice (5q-mice) (Barlow Nat Med 2010). Our findings reveal that 5q-mice exhibit macrocytic anemia (Hb 6 vs. 12 g/dL, P Disclosures No relevant conflicts of interest to declare.

Erythropoiesis is a process during which multipotent hematopoietic stem cells proliferate, differentiate and ultimately produce enucleated reticulocytes. Terminal erythroid differentiation begins at the morphologically recognizable pro-erythroblast (pro-E) stage and is completed when orthochromatic erythroblasts (ortho-E) expel their nuclei to produce reticulocytes. Progressive differentiation between these stages occurs in homologous cell division progressively doubling proportions of pro-E, basophilic (baso-E), polychromatophilic (poly-E), and ortho-E, and multiple signaling pathways are involved in the generation of enucleated erythroid cells, including multiple steps requiring actin cytoskeleton reorganization. We have previously shown that β-thalassemic mice (th1/th1) demonstrate a disordered progression from pro-E to baso-E and that exogenous transferrin therapy restores normal proportion of early stage erythroid precursors in th1/th1 mice (Liu Blood 2013). To identify genes that play novel function in different stages of terminal erythropoiesis, we performed RNA seq analysis of sorted bone marrow pro-E from WT, th1/th1, and transferrin-treated th1/th1 mice. We identify pleckstrin-2 (plek2) as a gene of interest with a 15-fold increase in plek2 mRNA expression in th1/th1 relative to WT mice, normalized in transferrin-treated th1/th1 mice. Plek2 is an actin binding protein, like pleckstrin-1, contains a central DEP domain known to bind RacGTPase, is Epo dependent, and is expressed in all stages of terminal erythropoiesis. We evaluate plek2 mRNA and protein expression in sorted bone marrow erythroid precursors from WT, th1/th1, and transferrin-treated th1/th1 mice. Our data demonstrates a statistically significant increase in plek2 mRNA in th1/th1 relative to WT mice, with the highest expression of plek2 in poly-E, normalized in transferrin-treated th1/th1 mice (Figure 1A). A similar pattern of increased protein concentration in th1/th1 relative to WT mice and normalization in transferrin-treated th1/th1 mice is evident in sorted bone marrow samples (Figure 1B). Prior in vitro studies demonstrate that membrane localization of plek2 is required for erythroid differentiation. Thus, we performed sub-cellular fractionation in bone marrow erythroid precursors and determined for the first time that in sorted erythroblasts from WT bone marrow, plek2 is found exclusively in the cytoplasm in pro-E and in both cytoplasm and membrane from baso-E to ortho-E (Figure 2), co-localized with actin filaments in the membrane (data not shown). In contrast, sorted erythroblasts from th1/th1 bone marrow reveal membrane-associated plek2 starting from pro-E, demonstrating earlier co-localization with actin filaments (data not shown) and suggesting an earlier activation of plek2 and consequent actin cytoskeleton reorganization during erythroid differentiation in th1/th1 mice, normalized in transferrin-treated th1/th1 mice (Figure 2). Erythropoiesis involves a complicated and incompletely understood set of potentially related molecular signals influencing cell survival, differentiation, enucleation, and release into the circulation. For example, although Epo increases survival, Epo signaling also activates RacGTPases, inhibiting enucleation. Recent in vitro data demonstrates that knockdown of plek2 affected enucleation with significantly lower reticulocyte count. Although the involvement of RacGTPase in plek2-mediated erythroid differentiation has not been explored, we hypothesize that plek2 activation triggers RacGTPase and prevents enucleation in th1/th1 mice. Our data demonstrates that RacGTPase concentration is increased in sorted bone marrow erythroid precursors from th1/th1 relative to WT mice and normalized in transferrin-treated th1/th1 mice (Figure 1B). These results suggest that plek2 plays an important role in erythropoiesis likely as a key factor in the improved enucleation of transferrin-treated th1/th1 mice. Disclosures No relevant conflicts of interest to declare.

We have identified sultam thioureas as novel inhibitors of West Nile virus (WNV) replication. One such compound inhibited WNV, with a 50% effective concentration of 0.7 μM, and reduced reporter expression from cells that harbored a WNV-based replicon. Our results demonstrate that sultam thioureas can block a postentry, preassembly step of WNV replication.

β-thalassemia is an inherited blood disorder caused by reduced or absence of β-globin expression which results in imbalanced globin synthesis, ineffective erythropoiesis, and anemia. How the imbalance between α- and β-globin results in ineffective erythropoiesis, if apoptosis or dysfunctional differentiation of erythroid precursors results in ineffective erythropoiesis, and whether disrupted iron regulation and / or iron overload in β-thalassemia is directly involved in the pathophysiology of ineffective erythropoiesis is incompletely understood. Iron is critical for hemoglobin synthesis and erythropoiesis is dependent on transferrin (Tf) bound iron. Tf functions as the main iron transporter in circulation, where it exists in three forms: as iron-free apo-transferrin (apoTf), monoferric Tf, or diferric Tf (holoTf). Typically, iron is bound to 30% of all Tf binding sites in circulation at which point monoferric Tf is found in the highest concentration relative to holoTf. . We have previously shown that exogenous apoTf ameliorates anemia in a mouse model of β-thalassemia intermedia (th1/th1), resulting in reduced splenomegaly, reticulocytosis, and α-globin precipitation on circulatory red blood cells (RBC). We also observe a decrease in mean corpuscular hemoglobin (MCH) and mean corpuscular volume (MCV), serum iron, Tf saturation, together suggesting that relative iron deficiency improves iron metabolism and ineffective erythropoiesis in apoTf-treated th1/th1 mice. We hypothesize that exogenous apoTf decreases cytosolic iron and heme as a consequence of increased monoferric Tf which results in less iron entering cells via Tf:TfR1 binding. Our current data reveals that in vitro incubation of purified apoTf and holoTf at 37C results in the formation of monoferric Tf, and injection of wild-type (WT) mice with a single intraperitoneal dose of apoTf (10mg) decreases holoTf (P=0.01) and increases monoferric Tf (P=0.02) in the serum 6 hours after injection. Using both calcium mobilization and anti-Tf antibodies in flow cytometry, we demonstrate that apoTf results in no TfR1 binding relative to holoTf in CHO cells (P Disclosures No relevant conflicts of interest to declare.

Iron overload causes morbidity and mortality in patients with β-thalassemia. Transfusion independent patients develop iron overload from increased dietary iron absorption, implicating inappropriately low hepcidin and supporting the therapeutic potential of approaches to increase hepcidin. Relatively low liver hepcidin mRNA expression is also characteristic of mouse models of β-thalassemia, and a recently identified “erythroid factor,” erythroferrone, has been implicated in hepcidin suppression in thalassemia. We have previously shown that exogenous apo-transferrin injections result in relatively iron restricted erythropoiesis, ameliorate ineffective erythropoiesis, and increase hepcidin expression in Hbbth1/th1 β-thalassemia intermedia (thalassemic) mice. We now explore the effect of exogenous apo-transferrin on signaling pathways (i.e. Smad and Erk) and circulating parameters thought to participate in hepcidin regulation in vivo and in vitro in wild type (WT) and thalassemic mice. Our results demonstrate that apo-transferrin injection increase both serum hepcidin concentration (603 vs. 306 ng/ml, n=13-24 per group, P Figure 1 Figure 1. Figure 2 Figure 2. Figure 3 Figure 3. Disclosures Westerman: Intrinsic Lifesciences LTD: Employment, Equity Ownership.

Transferrin receptor 1 (TfR1) is found in highest concentrations on erythroid precursors due to the disproportionately high iron requirement for hemoglobin synthesis, making transferrin-bound iron binding to TfR1 essential for erythropoiesis. Recent data reveals that TfR1 mRNA expression (6.48±2.23 vs. 1.0±0.25 relative to GAPDH, P=0.04 in sorted basophilic erythroblasts), whole cell protein concentration measured using ImageJ (11496±1783 vs. 1620±1448, P=0.0001 in reticulocytes), and cell surface concentration measured using flow cytometry (mean fluorescence index 17314±2370 vs. 11930±2530, P=0.002 in bone marrow basophilic erythroblasts) are increased in β-thalassemic (th1/th1) relative to wild type (WT) mice. We hypothesized that a relative decrease in TfR1 expression would improve the phenotype in β-thalassemic mice and crossed TfR1+/- (TfR1 heterozygote) mice [Levy JE Nat Gen 1999] with th3/+ mice, another commonly used mouse model of β-thalassemia. Of the 50 pups born, 13 had th3 genotype, 12 (92%) of which also contained the mutant TfR1, suggesting a strong survival advantage of TfR1 heterozygote th3/+ (compound heterozygotes) relative to th3/+ mice. Analysis of 3-4 month old compound heterozygotes revealed a significant decrease in splenomegaly (0.007±0.001 vs. 0.016±0.0041 g spleen/g body weight, P=0.0009), reticulocytosis (1019±186 vs. 1672±218 x 10^9 cells/uL, P=0.001), and α-globin precipitation on circulating RBCs (Figure 1) relative to th3/+ mice. Furthermore, compound heterozygotes exhibit improvement in circulating RBCs (12±0.1 vs. 9±0.6 x 10^6 cells/uL, P We previously demonstrate that exogenous apo-transferrin (apoTf) injections result in more circulating RBCs, increased hemoglobin, and reversal of splenomegaly in th1/th1 mice [Li H Nat Med 2010]. We hypothesize that ineffective erythropoiesis in th1/th1 mice is TfR1-mediated and involves excess iron delivery to erythroid precursors. To further explore the role of TfR1 in erythropoiesis, we evaluate apoTf-treated th1/th1 mice. TfR1 mRNA expression is unchanged in apoTf-treated relative to untreated th1/th1 mice despite more iron restricted erythropoiesis (MCH 24.56±0.72 vs. 33.98±1.67 pg, P Taken together, our data suggest that TfR1 plays a critical role in erythropoiesis, both in an iron-dependent and possibly independent capacity. We postulate that a defect in TfR1 trafficking, perhaps with a delayed or incomplete removal of TfR1 during erythroid differentiation, occurs in β-thalassemia, that reduction of TfR1 in β-thalassemic mice partially reverses ineffective erythropoiesis, and that exogenous apoTf decreases TfR1 expression and exosomal release while increasing membrane and endosomal cycling. Figure 1 Figure 1. Figure 2 Figure 2. Figure 3 Figure 3. Disclosures No relevant conflicts of interest to declare.

The nucleocapsid (NC) domains of retrovirus precursor Gag (PrGag) proteins play an essential role in virus assembly. Evidence suggests that NC binding to viral RNA promotes dimerization of PrGag capsid (CA) domains, which triggers assembly of CA N-terminal domains (NTDs) into hexamer rings that are interconnected by CA C-terminal domains. To examine the influence of dimerization on human immunodeficiency virus type 1 (HIV-1) Gag protein assembly in vitro, we analyzed the assembly properties of Gag proteins in which NC domains were replaced with cysteine residues that could be linked via chemical treatment. In accordance with the model that Gag protein pairing triggers assembly, we found that cysteine cross-linking or oxidation reagents induced the assembly of virus-like particles. However, efficient assembly also was observed to be temperature dependent or required the tethering of NTDs. Our results suggest a multistep pathway for HIV-1 Gag protein assembly. In the first step, Gag protein pairing through NC-RNA interactions or C-terminal cysteine linkage fosters dimerization. Next, a conformational change converts assembly-restricted dimers or small oligomers into assembly-competent ones. At the final stage, final particle assembly occurs, possibly through a set of larger intermediates.

Abstract 906 Hemoglobin (Hb) synthesis during terminal erythroid differentiation is iron dependent and iron delivery requires transferrin (Tf) to transferrin receptor 1 (TfR1) binding. After binding of Tf to TfR1, the ligand-receptor complex is endocytosed through a clathrin dependent mechanism, results in iron delivery via the endosomal compartment, and ends with TfR1 recycling back to the plasma membrane. During erythroid differentiation, TfR1 expression is downregulated and is completely absent from the mature red blood cells (RBCs). Reticulocyte TfR1 is rerouted from the endosomal recycling pathway by sorting to exosomes where it is shed from the cell. Exosomes are small membrane vesicles originating from the fusion of a multi-vesicular endosome compartment with the plasma membrane, leading to the secretion of intraluminal vesicles into circulation. Cell surface TfR1 expression is proportional to the concentration of soluble TfR1 found in circulation as a consequence of exosomal secretion and is increased both in iron deficiency and expanded erythropoiesis. Increased soluble TfR1 is observed in disease of expanded and ineffective erythropoiesis (IE) such as beta-thalassemia, a disease associated with anemia, extramedullary hematopoiesis (EMH), and splenomegaly. We previously demonstrated that apoTf-treated beta-thalassemic mice have more circulating RBCs, increased Hb, reversed splenomegaly and EMH, with fewer reticulocytes and erythroid precursors in the bone marrow and spleen. Furthermore, in both beta-thalassemic and C57BL/6 mice treated with apoTf, we observed a significant reduction in mean cellular hemoglobin (MCH). We hypothesize that TfR1 trafficking is impaired in beta-thalassemia and that exogenous apoTf reduces cellular iron uptake and normalizes TfR1 trafficking pathways, resulting in reduced heme synthesis and a lower MCH observed in apoTf-treated mice. To test this hypothesis, we evaluate 1) cellular iron uptake in cell culture and 2) TfR1 mRNA expression, cell surface expression, and endosomal/exosomal trafficking pathways in C57BL/6 and beta-thalassemic mice. Cell culture experiments were performed using K562 cells +/− 50- and 250-fold excess apoTf relative to holoTf. Mice were evaluated after 20 days of 10 mg human apoTf IP injections (compared with PBS injection). Using a calcein fluorescence quenching approach, we demonstrate that exogenous apoTf decreases iron uptake in culture in a dose response manner. Furthermore, in mouse bone marrow samples sorted using CD44/TER119, we show that TfR1 mRNA expression is higher in beta-thalassemic relative to C57BL/6 mice and increases further in apoTf treated mice in all stages of terminal erythroid differentiation. In addition, although western blot experiments show an increase in cellular TfR1 in beta-thalassemic relative to C57BL/6 mice, cell fractionation experiments demonstrate a proportional shift from the plasma membrane to the endosomal compartment in apoTf-treated mice and reticulocytes of apoTf treated beta-thalassemic mice exhibit significantly reduced TfR1 expression per cell. Finally, reticulocyte TfR1 sorting into exosomes is impaired in beta-thalassemic relative to C57BL/6 mice with a proportional increase in exosomal TfR1 clearance and a reduction in soluble TfR1 in the serum in apoTf treated beta-thalassemic mice. Taken together, our findings demonstrate for the first time that TfR1 trafficking is important for RBC physiology separately from its role in iron uptake and that exogenous apoTf enhances TfR1 endosomal trafficking, normalizes TfR1 sorting into exosomes, and reduces cellular iron uptake. Finally, our results elucidate mechanisms by which MCH is reduced in apoTf-treated mice and provide evidence for multiple consequences of Tf:TfR1 binding on erythroid differentiation and proliferation that characterize diseases of IE. Disclosures: No relevant conflicts of interest to declare.