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The emergence of consumer-generated data and the growing availability of Machine Learning (ML) techniques are revolutionizing marketing practices. Marketers and researchers are far from having a thorough understanding of the broad range of opportunities ML applications offer in creating and maintaining a competitive business advantage. In this paper, we propose a taxonomy of ML use cases in marketing based on a systematic review of academic and business literature. We have identified 11 recurring use cases, organized in 4 homogeneous families which correspond to the fundamentals leverage areas of ML in marketing, namely: shopper fundamentals, consumption experience, decision making, and financial impact. We discuss the recurring patterns identified in the taxonomy and provide a conceptual framework for its interpretation and extension, highlighting practical implications for marketers and researchers.

Glycosaminoglycans (GAGs) are long linear sulfated polysaccharides implicated in processes linked to disease development such as mucopolysaccharidosis, respiratory failure, cancer, and viral infections, thereby serving as potential biomarkers. A successful clinical translation of GAGs as biomarkers depends on the availability of standardized GAG measurements. However, owing to the analytical complexity associated with the quantification of GAG concentration and structural composition, a standardized method to simultaneously measure multiple GAGs is missing. In this study, we sought to characterize the analytical performance of a ultra-high-performance liquid chromatography coupled with triple-quadrupole tandem mass spectrometry (UHPLC-MS/MS)-based kit for the quantification of 17 GAG disaccharides. The kit showed acceptable linearity, selectivity and specificity, accuracy and precision, and analyte stability in the absolute quantification of 15 GAG disaccharides. In native human samples, here using urine as a reference matrix, the analytical performance of the kit was acceptable for the quantification of CS disaccharides. Intra- and inter-laboratory tests performed in an external laboratory demonstrated robust reproducibility of GAG measurements showing that the kit was acceptably standardized. In conclusion, these results indicated that the UHPLC-MS/MS kit was standardized for the simultaneous measurement of GAG disaccharides allowing for comparability of measurements and enabling translational research.SummaryAnalytical performance of a kit for standardized GAG measurements, based on an established UHPLC-MS/MS method

The type IIa Na(+)/P(i), cotransporter (NaPi-IIa) mediates electrogenic transport of three Na(+) and one divalent P(i) ion (and one net positive charge) across the cell membrane. Sequence comparison of electrogenic NaPi-IIa and IIb isoforms with the electroneutral NaPi-IIc isoform pointed to the third transmembrane domain (TMD-3) as a possibly significant determinant of substrate binding. To elucidate the role of TMD-3 in the topology and mechanism underlying NaPi-IIa function we subjected it to cysteine scanning mutagenesis. The constructs were expressed in Xenopus oocytes and P(i) transport kinetics were assayed by electrophysiology and radiotracer uptake. Cys substitution resulted in only marginally altered kinetics of P(i) transport in those mutants providing sufficient current for analysis. Only one site, at the extracellular end of TMD-3, appeared to be accessible to methanethiosulfonate reagents. However, additional mutations carried out at D224 (replaced by E, G or N) and N227 (replaced by D or Q) resulted in markedly altered voltage and substrate dependencies of the P(i)-dependent currents. Replacing Asp-224 (highly conserved in electrogenic a and b isoforms) with Gly (the residue found in the electroneutral c isoform) resulted in a mutant that mediated electroneutral Na(+)-dependent P(i) transport. Since electrogenic NaPi-II transports 3 Na(+)/transport cycle, whereas electroneutral NaPi-IIc only transports 2, we speculate that this loss of electrogenicity might result from the loss of one of the three Na(+) binding sites in NaPi-IIa.

The rapid identification of bacteria and fungi directly from the blood of patients with suspected bloodstream infections aids in diagnosis and guides treatment decisions. The development of an automated, rapid, and sensitive molecular technology capable of detecting the diverse agents of such infections at low titers has been challenging, due in part to the high background of genomic DNA in blood. PCR followed by electrospray ionization mass spectrometry (PCR/ESI-MS) allows for the rapid and accurate identification of microorganisms but with a sensitivity of about 50% compared to that of culture when using 1-ml whole-blood specimens. Here, we describe a new integrated specimen preparation technology that substantially improves the sensitivity of PCR/ESI-MS analysis. An efficient lysis method and automated DNA purification system were designed for processing 5 ml of whole blood. In addition, PCR amplification formulations were optimized to tolerate high levels of human DNA. An analysis of 331 specimens collected from patients with suspected bloodstream infections resulted in 35 PCR/ESI-MS-positive specimens (10.6%) compared to 18 positive by culture (5.4%). PCR/ESI-MS was 83% sensitive and 94% specific compared to culture. Replicate PCR/ESI-MS testing from a second aliquot of the PCR/ESI-MS-positive/culture-negative specimens corroborated the initial findings in most cases, resulting in increased sensitivity (91%) and specificity (99%) when confirmed detections were considered true positives. The integrated solution described here has the potential to provide rapid detection and identification of organisms responsible for bloodstream infections.

Tropomodulin (Tmod) is a protein that binds and caps the pointed ends of actin filaments in erythroid and nonerythoid cell types. Targeted deletion of mouse tropomodulin3 (Tmod3) leads to embryonic lethality at E14.5-E18.5, with anemia due to defects in definitive erythropoiesis in the fetal liver. Erythroid burst-forming unit and colony-forming unit numbers are greatly reduced, indicating defects in progenitor populations. Flow cytometry of fetal liver erythroblasts shows that late-stage populations are also decreased, including reduced percentages of enucleated cells. Annexin V staining indicates increased apoptosis of Tmod3(-/-) erythroblasts, and cell-cycle analysis reveals that there are more Ter119(hi) cells in S-phase in Tmod3(-/-) embryos. Notably, enucleating Tmod3(-/-) erythroblasts are still in the process of proliferation, suggesting impaired cell-cycle exit during terminal differentiation. Tmod3(-/-) late erythroblasts often exhibit multilobular nuclear morphologies and aberrant F-actin assembly during enucleation. Furthermore, native erythroblastic island formation was impaired in Tmod3(-/-) fetal livers, with Tmod3 required in both erythroblasts and macrophages. In conclusion, disruption of Tmod3 leads to impaired definitive erythropoiesis due to reduced progenitors, impaired erythroblastic island formation, and defective erythroblast cell-cycle progression and enucleation. Tmod3-mediated actin remodeling may be required for erythroblast-macrophage adhesion, coordination of cell cycle with differentiation, and F-actin assembly and remodeling during erythroblast enucleation.

The putative first intracellular and third extracellular linkers are known to play important roles in defining the transport properties of the type IIa Na+-coupled phosphate cotransporter (Kohler, K., I.C. Forster, G. Stange, J. Biber, and H. Murer. 2002b. J. Gen. Physiol. 120:693–705). To investigate whether other stretches that link predicted transmembrane domains are also involved, the substituted cysteine accessibility method (SCAM) was applied to sites in the predicted first and fourth extracellular linkers (ECL-1 and ECL-4). Mutants based on the wild-type (WT) backbone, with substituted novel cysteines, were expressed in Xenopus oocytes, and their function was assayed by isotope uptake and electrophysiology. Functionally important sites were identified in both linkers by exposing cells to membrane permeant and impermeant methanethiosulfonate (MTS) reagents. The cysteine modification reaction rates for sites in ECL-1 were faster than those in ECL-4, which suggested that the latter were less accessible from the extracellular medium. Generally, a finite cotransport activity remained at the end of the modification reaction. The change in activity was due to altered voltage-dependent kinetics of the Pi-dependent current. For example, cys substitution at Gly-134 in ECL-1 resulted in rate-limiting, voltage-independent cotransport activity for V ≤ −80 mV, whereas the WT exhibited a linear voltage dependency. After cys modification, this mutant displayed a supralinear voltage dependency in the same voltage range. The opposite behavior was documented for cys substitution at Met-533 in ECL-4. Modification of cysteines at two other sites in ECL-1 (Ile-136 and Phe-137) also resulted in supralinear voltage dependencies for hyperpolarizing potentials. Taken together, these findings suggest that ECL-1 and ECL-4 may not directly form part of the transport pathway, but specific sites in these linkers can interact directly or indirectly with parts of NaPi-IIa that undergo voltage-dependent conformational changes and thereby influence the voltage dependency of cotransport.

Functionally important sites in the predicted first and fourth extracellular linkers of the type IIa Na+/Pi cotransporter (NaPi-IIa) were identified by cysteine scanning mutagenesis (Ehnes et al., 2004). Cysteine substitution or modification with impermeant and permeant methanethiosulfonate (MTS) reagents at certain sites resulted in changes to the steady-state voltage dependency of the cotransport mode (1 mM Pi, 100 mM Na+ at pH 7.4) of the mutants. At Gly-134 (ECL-1) and Met-533 (ECL-4), complementary behavior of the voltage dependency was documented with respect to the effect of cys-substitution and modification. G134C had a weak voltage dependency that became even stronger than that of the wild type (WT) after MTS incubation. M533C showed a WT-like voltage dependency that became markedly weaker after MTS incubation. To elucidate the underlying mechanism, the steady-state and presteady-state kinetics of these mutants were studied in detail. The apparent affinity constants for Pi and Na+ did not show large changes after MTS exposure. However, the dependency on external protons was changed in a complementary manner for each mutant. This suggested that cys substitution at Gly-134 or modification of Cys-533 had induced similar conformational changes to alter the proton modulation of transport kinetics. The changes in steady-state voltage dependency correlated with changes in the kinetics of presteady-state charge movements determined in the absence of Pi, which suggested that voltage-dependent transitions in the transport cycle were altered. The steady-state and presteady-state behavior was simulated using an eight-state kinetic model in which the transition rate constants of the empty carrier and translocation of the fully loaded carrier were found to be critical determinants of the transport kinetics. The simulations predict that cys substitution at Gly-134 or cys modification of Cys-533 alters the preferred orientation of the empty carrier from an inward to outward-facing conformation for hyperpolarizing voltages.

Acute myocardial infarction (MI), which involves the rupture of existing atheromatous plaque, remains highly unpredictable despite recent advances in the diagnosis and treatment of coronary artery disease. Accordingly, a clinical measurement that can predict an impending MI is desperately needed. Here, we characterize circulating endothelial cells (CECs) using an automated and clinically feasible CEC three-channel fluorescence microscopy assay in 50 consecutive patients with ST-segment elevation MI and 44 consecutive healthy controls. CEC counts were significantly elevated in MI cases versus controls, with median numbers of 19 and 4 cells/ml, respectively (P = 1.1 × 10(-10)). A receiver-operating characteristic (ROC) curve analysis demonstrated an area under the ROC curve of 0.95, suggesting near-dichotomization of MI cases versus controls. We observed no correlation between CECs and typical markers of myocardial necrosis (ρ = 0.02, creatine kinase-myocardial band; ρ = -0.03, troponin). Morphological analysis of the microscopy images of CECs revealed a 2.5-fold increase (P < 0.0001) in cellular area and a twofold increase (P < 0.0001) in nuclear area of MI CECs versus healthy controls, age-matched CECs, as well as CECs obtained from patients with preexisting peripheral vascular disease. The distribution of CEC images that contained from 2 to 10 nuclei demonstrates that MI patients were the only subject group to contain more than 3 nuclei per image, indicating that multicellular and multinuclear clusters are specific for acute MI. These data indicate that CEC counts may serve as a promising clinical measure for the prediction of atherosclerotic plaque rupture events.

For many years, polyketide natural products have provided the scientific community with a rich source of novel molecular architectures, many of which have become important therapeutics for clinical use.[1] In 1993, the polyketide rhizopodin was isolated from the myxobacterium Myxococcus stipitatus.[2] It was shown to display an interesting array of biological properties, including potent anti-tumor activity against a range of cancer cell lines in the low nanomolar range and the ability to inhibit the polymerization of actin.[2,3] Despite its original structural assignment as the 19-membered monomeric lactone 1a (monorhizopodin), recent studies revealed dimeric structure 2 to be the correct architecture of rhizopodin (Figure 1).[4] These molecules have started to attract attention from the synthetic community, although no total syntheses have been reported to date.[5]

The temperature dependence of the transport kinetics of flounder Na(+)-coupled inorganic phosphate (P(i)) cotransporters (NaPi-IIb) expressed in Xenopus oocytes was investigated using radiotracer and electrophysiological assays. (32)P(i) uptake was strongly temperature-dependent and decreased by approximately 80% at a temperature change from 25 degrees C to 5 degrees C. The corresponding activation energy (E (a)) was approximately 14 kcal mol(-1) for the cotransport mode. The temperature dependence of the cotransport and leak modes was determined from electrogenic responses to 1 mM P(i) and phosphonoformic acid (PFA), respectively, under voltage clamp. The magnitude of the P(i)- and PFA-induced changes in holding current decreased with temperature. E (a) at -100 mV for the cotransport and leak modes was approximately 16 kcal mol(-1) and approximately 11 kcal mol(-1), respectively, which suggested that the leak is mediated by a carrier, rather than a channel, mechanism. Moreover, E (a) for cotransport was voltage-independent, suggesting that a major conformational change in the transport cycle is electroneutral. To identify partial reactions that confer temperature dependence, we acquired presteady-state currents at different temperatures with 0 mM P(i) over a range of external Na(+). The relaxation time constants increased, and the peak time constant shifted toward more positive potentials with decreasing temperature. Likewise, there was a depolarizing shift of the charge distribution, whereas the total available charge and apparent valency predicted from single Boltzmann fits were temperature-independent. These effects were explained by an increased temperature sensitivity of the Na(+)-debinding rate compared with the other voltage-dependent rate constants.

Renal type IIa Na + -coupled inorganic phosphate (P i ) cotransporters (NaPi-IIa) mediate divalent P i transport in an electrogenic manner, whereas the renal type IIc isoform (NaPi-IIc) is electroneutral, yet it shows high sequence identity with NaPi-IIa. Dual uptake ( 32 P i / 22 Na) assays confirmed that NaPi-IIc displayed Na + -coupled P i cotransport with a 2:1 (Na + :P i ) stoichiometry compared with 3:1 established for NaPi-IIa. This finding suggested that the electrogenicity of NaPi-IIa arises from the interaction of an additional Na + ion compared with NaPi-IIc. To identify the molecular elements responsible for the functional difference between isoforms, we used chimera and amino acid replacement approaches. Transport activity of chimeras constructed with NaPi-IIa and NaPi-IIc indicated that residues within the first six transmembrane domains were essential for the electrogenicity of NaPi-IIa. Sequence comparison between electrogenic and electroneutral isoforms revealed differences in the charge and polarity of residues clustered in three areas, one of which included part of the predicted third transmembrane domain. Here, substitution of three residues with their NaPi-IIa equivalents in NaPi-IIc (S189A, S191A, and G195D) resulted in a transporter that displayed a 1:1 charge/P i coupling, a 3:1 Na + :P i stoichiometry, and transient currents that resembled pre-steady-state relaxations. The mutant's weaker voltage dependency and 10-fold lower apparent P i affinity compared with NaPi-IIa indicated that other residues important for the NaPi-IIa kinetic fingerprint exist. Our findings demonstrate that, through a minimal number of side chain substitutions, we can effect a switch from electroneutral to electrogenic cotransporter function, concomitant with the appearance of a cosubstrate interaction site.

Functionally important sites in the predicted first and fourth extracellular linkers of the type IIa Na+/Pi cotransporter (NaPi-IIa) were identified by cysteine scanning mutagenesis (Ehnes et al., 2004). Cysteine substitution or modification with impermeant and permeant methanethiosulfonate (MTS) reagents at certain sites resulted in changes to the steady-state voltage dependency of the cotransport mode (1 mM Pi, 100 mM Na+ at pH 7.4) of the mutants. At Gly-134 (ECL-1) and Met-533 (ECL-4), complementary behavior of the voltage dependency was documented with respect to the effect of cys-substitution and modification. G134C had a weak voltage dependency that became even stronger than that of the wild type (WT) after MTS incubation. M533C showed a WT-like voltage dependency that became markedly weaker after MTS incubation. To elucidate the underlying mechanism, the steady-state and presteady-state kinetics of these mutants were studied in detail. The apparent affinity constants for Pi and Na+ did not show large changes after MTS exposure. However, the dependency on external protons was changed in a complementary manner for each mutant. This suggested that cys substitution at Gly-134 or modification of Cys-533 had induced similar conformational changes to alter the proton modulation of transport kinetics. The changes in steady-state voltage dependency correlated with changes in the kinetics of presteady-state charge movements determined in the absence of Pi, which suggested that voltage-dependent transitions in the transport cycle were altered. The steady-state and presteady-state behavior was simulated using an eight-state kinetic model in which the transition rate constants of the empty carrier and translocation of the fully loaded carrier were found to be critical determinants of the transport kinetics. The simulations predict that cys substitution at Gly-134 or cys modification of Cys-533 alters the preferred orientation of the empty carrier from an inward to outward-facing conformation for hyperpolarizing voltages.

The putative first intracellular and third extracellular linkers are known to play important roles in defining the transport properties of the type IIa Na+-coupled phosphate cotransporter (Kohler, K., I.C. Forster, G. Stange, J. Biber, and H. Murer. 2002b. J. Gen. Physiol. 120:693-705). To investigate whether other stretches that link predicted transmembrane domains are also involved, the substituted cysteine accessibility method (SCAM) was applied to sites in the predicted first and fourth extracellular linkers (ECL-1 and ECL-4). Mutants based on the wild-type (WT) backbone, with substituted novel cysteines, were expressed in Xenopus oocytes, and their function was assayed by isotope uptake and electrophysiology. Functionally important sites were identified in both linkers by exposing cells to membrane permeant and impermeant methanethiosulfonate (MTS) reagents. The cysteine modification reaction rates for sites in ECL-1 were faster than those in ECL-4, which suggested that the latter were less accessible from the extracellular medium. Generally, a finite cotransport activity remained at the end of the modification reaction. The change in activity was due to altered voltage-dependent kinetics of the Pi-dependent current. For example, cys substitution at Gly-134 in ECL-1 resulted in rate-limiting, voltage-independent cotransport activity for Vor = -80 mV, whereas the WT exhibited a linear voltage dependency. After cys modification, this mutant displayed a supralinear voltage dependency in the same voltage range. The opposite behavior was documented for cys substitution at Met-533 in ECL-4. Modification of cysteines at two other sites in ECL-1 (Ile-136 and Phe-137) also resulted in supralinear voltage dependencies for hyperpolarizing potentials. Taken together, these findings suggest that ECL-1 and ECL-4 may not directly form part of the transport pathway, but specific sites in these linkers can interact directly or indirectly with parts of NaPi-IIa that undergo voltage-dependent conformational changes and thereby influence the voltage dependency of cotransport.

The Caenorhabditis elegans genome encodes nine homologues of mammalian glycoprotein-associated amino acid transporters. Two of these C. elegans proteins (AAT-1 and AAT-3) have been shown to function as catalytic subunits (light chains) of heteromeric amino acid transporters. These proteins need to associate with a glycoprotein heavy chain subunit (ATG-2) to reach the cell surface in a manner similar to that of their mammalian homologues. AAT-1 and AAT-3 contain a cysteine residue in the second putative extracellular loop through which a disulfide bridge can form with a heavy chain. In contrast, six C. elegans members of this family (AAT-4 to AAT-9) lack such a cysteine residue. We show here that one of these transporter proteins, AAT-9, reaches the cell surface in Xenopus oocytes without an exogenous heavy chain and that it functions as an exchanger of aromatic amino acids. Two-electrode voltage clamp experiments demonstrate that AAT-9 displays a substrate-activated conductance. Immunofluorescence shows that it is expressed close to the pharyngeal bulbs within C. elegans neurons. The selective expression of an aat-9 promoter-green fluorescent protein construct in several neurons of this region and in wall muscle cells around the mouth supports and extends these localization data. Taken together, the results show that AAT-9 is expressed in excitable cells of the nematode head and pharynx in which it may provide a pathway for aromatic amino acid transport.

Abstract 84 Tropomodulin1 (Tmod1) binds tropomyosin and caps the pointed ends of the short actin filaments in the spectrin-actin network of red blood cells (RBCs). Tmod1-null mice display a mild sphero-elliptocytic anemia due to mis-regulation of actin filament lengths and a disrupted membrane skeleton. The mild phenotype may be explained by the compensation of Tmod3, which is not found in wild-type RBCs but exists in Tmod1-null RBCs (one-fifth level of Tmod1). Our experiments with human erythroblasts show that the expression of Tmod1 increases during terminal differentiation while the level of Tmod3 is relatively constant, only decreasing at a very late stage. To investigate the role of Tmod3 in RBCs, we created a Tmod3 knockout mouse from ES cells (#RRF004, BayGenomics) with a gene-trap vector insertion into intron 1 of Tmod3. Both RT-PCR and western-blot results show that the expression of Tmod3 is abolished in Tmod3−/− mice. Tmod3+/− mice are viable and fertile, while Tmod3−/− animals are embryonic lethal, with most nulls dying between E16.5-E17.5. Tmod3−/− embryos are pale and anemic with a smaller fetal liver, suggesting that the lethality might be due to defective definitive erythropoiesis. This is supported by analysis of peripheral blood, which shows fewer definitive enucleated erythroblasts in Tmod3-null embryos. Flow-cytometry of fetal liver erythroblasts labeled with Ter119 and CD71 indicates that the late stage R3 population is reduced by about one-third in absence of Tmod3, while R1-R2 populations are somewhat increased. In addition, Annexin V staining shows a two-fold increase in apoptotic cells in the fetal liver, most of which are in the R1 population. Measurement of enucleation frequency in R populations shows a marked reduction of enucleated cells as the erythroblasts mature through the R3-R5 populations. These data indicate that definitive erythropoiesis is defective due to impaired erythroblast terminal differentiation in absence of Tmod3. To determine the underlying mechanisms, we used histology and confocal fluorescence microscopy to investigate the morphology and actin cytoskeleton of erythroblasts in process of enucleation. These experiments show abnormal nuclear morphology in orthochromatic Tmod3-null fetal liver erythroblasts, as well as defective F-actin contractile ring assembly in Tmod3−/− erythroblasts in process of nuclear expulsion, suggesting a role for Tmod3 in enucleation. Since macrophages are required for production of definitive erythroblasts and enucleation in vivo, we explored the role of macrophages in the Tmod3−/− phenotype. Immunofluorescence staining of fetal liver cryosections with F4/80, Ter119 and Hoechst reveals that macrophages display strikingly less dendritic morphologies in the Tmod3−/− mice, with macrophages sometimes containing Ter119-positive nucleated erythroblasts. Isolation of native erythroblast-macrophage islands from fetal liver demonstrates that islands isolated from Tmod3−/− fetal livers contain fewer erythroblasts compared with those from wild-type fetal liver. Further, reconstitution experiments indicate that erythroblasts from Tmod3−/− fetal liver are unable to form normal islands, indicating that Tmod3 function is required in erythroblasts. In conclusion, our study shows that knockout of Tmod3 leads to defective definitive erythropoiesis and embryonic lethality in mice, due to defects in island formation and abnormal enucleation. These data suggest that Tmod3-mediated actin remodeling may be required for erythroblast-macrophage adhesion as well as contractile ring assembly during erythroblast enucleation. Disclosures: No relevant conflicts of interest to declare.

Abstract 1036 Tropomodulin1 binds tropomyosin and caps the pointed ends of the short actin filaments in the spectrin-actin network of red blood cells (RBCs). Tmod1-null mice display a mild compensated, sphero-elliptocytic anemia with fragile RBCs, due to mis-regulation of actin filament lengths and a disrupted membrane skeleton lattice (Moyer et al. 2010. Blood 116:2590–2599). The mild phenotype may be explained by the appearance of Tmod3, which is not found in wild-type RBCs but is in Tmod1-null RBCs, albeit at only 1/5th of normal Tmod1 levels. Tmod3 is ubiquitously expressed, including in erythroblasts, and thus the presence of Tmod3 in Tmod1-null RBCs is likely due to aberrant persistence of Tmod3 during terminal differentiation and maturation. To investigate the role of Tmod3 in RBCs, we created a Tmod3 knockout mouse from ES cells (#RRF004, BayGenomics) in which intron 1 of the Tmod3 locus was disrupted by retroviral-mediated insertion of a gene-trap vector. The insertion creates a novel fusion transcript joining sequences from exons 5' to the insertion site to the beta-galactosidase marker. Tmod3+/− mice are viable and fertile, but Tmod3−/− animals are embryonic lethal, with 94% of nulls dying between E13.5 – E15.5 (175 total embryos, E12.5-E18.5). Tmod3−/− embryos are pale and anemic starting at E13.5, with much smaller livers. Tmod3 mRNA and protein are reduced in Tmod3+/− and absent in Tmod3−/−, demonstrating that this is a true null. Tmod1 mRNA and protein are also reduced, consistent with anemia, fewer erythroblasts and RBCs, indicating that Tmod1 expression does not compensate for absence of Tmod3. The peripheral blood of wild-type E13.5-E15.5 embryos contains abundant large primitive and smaller definitive RBCs, but few or no definitive enucleated RBCs are observed in Tmod3-null embryos. Fetal livers from Tmod3−/− embryos were about ½ the size of wild-type livers, and histology showed altered cellularity. However, the proliferative potential of erythroid progenitors appears not to be impaired based on similar numbers of BFU-E and CFU-E colonies from Tmod3+/+ and Tmod3−/− fetal livers. Cytospins of dissociated fetal liver cells from E13.5-E14.5 embryos reveal the presence of erythroblast/macrophage islands in both genotypes, but with some macrophages containing abundant ingested cellular debris in the Tmod3−/− cytospins. High magnification images show increased erythroblast blebbing in absence of Tmod3, suggesting a weaker cortex, and/or apoptosis of erythroblasts. Flow cytometry of E14.5 fetal liver erythroblasts labeled with Ter119 and CD71 indicate that the late stage R3 population is reduced by about 40–50% in absence of Tmod3, while R1-R2 populations are somewhat increased (possibly as a compensatory response to fetal anemia). In addition, Annexin V staining shows a 2-fold increase in apoptotic cells in the fetal liver, most of which are in the R1 population. Measurement of enucleation frequency in R populations shows a marked reduction of enucleated cells as the erythroblasts mature through the R3-R5 populations. To explore the role of macrophages in the Tmod3−/− phenotype, we performed immunofluorescence staining and confocal microscopy of fetal liver cryosections stained with F4/80 (macrophages), Ter119 (erythroblasts) and Hoechst (nuclei). Macrophages displayed strikingly less dendritic morphologies in the Tmod3−/−, with macrophages sometimes containing Ter119-positive nucleated erythroblasts. Our data show that definitive erythropoiesis is impaired during terminal differentiation in absence of Tmod3, and we speculate that this is due to defective erythroblast-macrophage interactions during terminal differentiation. Experiments are in progress to determine whether Tmod3 function is required in erythroblasts or macrophages, and to identify the molecular pathway in which Tmod3 functions in erythropoiesis. Supported by NIH/NHLBI grant R01HL083464 to V.M.F. Disclosures: No relevant conflicts of interest to declare.