How the Environmental influences on Child Health Outcome (ECHO) Cohort can spur discoveries in environmental epidemiology.

NIH's Environmental influences on Child Health Outcome (ECHO) program is an innovative, large, collaborative research initiative whose mission is to enhance the health of children for generations to come. The goal of the ECHO Cohort is to examine effects of a broad array of early environmental exposures on child health and development. It includes longitudinal data and biospecimens from over 100,000 children and family members from diverse settings across the U.S. ECHO investigators have published collaborative analyses showing associations of environmental exposures--primarily in the developmentally sensitive pre-, peri-, and post-natal periods--with preterm birth and childhood asthma, obesity, neurodevelopment, and positive health. Investigators have addressed health disparities, joint effects of environmental and social determinants, and effects of mixtures of chemicals. The ECHO Cohort is now entering its second 7-year cycle (2023-2030), which will add the preconception period to its current focus on prenatal through adolescence. Through a controlled access public use database, ECHO makes its deidentified data available to the general scientific community. ECHO Cohort data provide opportunities to fill major knowledge gaps in in environmental epidemiology, and to inform policies, practices, and programs to enhance child health.

NIH workshop on human milk composition: summary and visions.

Nationally representative data from mother-child dyads that capture human milk composition (HMC) and associated health outcomes are important for advancing the evidence to inform federal nutrition and related health programs, policies, and consumer information across the governments in the United States and Canada as well as in nongovernment sectors. In response to identified gaps in knowledge, the National Institute of Diabetes and Digestive and Kidney Diseases of the NIH sponsored the "Workshop on Human Milk Composition-Biological, Environmental, Nutritional, and Methodological Considerations" held 16-17 November 2017 in Bethesda, Maryland. Through presentations and discussions, the workshop aimed to 1) share knowledge on the scientific need for data on HMC; 2) explore the current understanding of factors affecting HMC; 3) identify methodological challenges in human milk (HM) collection, storage, and analysis; and 4) develop a vision for a research program to develop an HMC data repository and database. The 4 workshop sessions included 1) perspectives from both federal agencies and nonfederal academic experts, articulating scientific needs for data on HMC that could lead to new research findings and programmatic advances to support public health; 2) information about the factors that influence lactation and/or HMC; 3) considerations for data quality, including addressing sampling strategies and the complexities in standardizing collection, storage, and analyses of HM; and 4) insights on how existing research programs and databases can inform potential visions for HMC initiatives. The general consensus from the workshop is that the limited scope of HM research initiatives has led to a lack of robust estimates of the composition and volume of HM consumed and, consequently, missed opportunities to improve maternal and infant health.

An analysis of the NIH-supported sickle cell disease research portfolio.

Sickle cell disease (SCD), an inherited blood disorder is due to a single amino acid substitution on the beta chain of hemoglobin, and is characterized by anemia, severe infections, acute and chronic pain, and multi-organ damage. The National Institutes of Health (NIH) is dedicated to support basic, translational and clinical science research to improve care and ultimately, to find a cure for SCD that causes such suffering. This report provides a detailed analysis of grants funded by the NIH for SCD research in Fiscal Years 2007 through 2013. During this period, the NIH supported 247 de novo grants totaling $272,210,367 that address various aspects of SCD. 83% of these funds supported research project grants investigating the following 5 scientific themes: Pathology of Sickle Red Blood Cells; Globin Gene Expression; Adhesion and Vascular Dysfunction; Neurological Complications and Organ-specific Dysfunction; and Pain Management and Intervention. The remaining 17% of total funds supported career development and training grants; Small Business Innovation Research (SBIR) and Small Business Technology Transfer (STTR) grants; large Center grants; and Conference grants. Further analysis showed that the National Heart, Lung, and Blood Institute (NHLBI) is the largest funder of SCD research within NIH with 67% of total grants, contributing 77% of total funds; followed by the National Institute for Digestive Diseases and Kidney (NIDDK) that is funding 19% of grants, contributing 13% of total funds. The remaining 14% of grants totaling 10% of the funds were supported by all other NIH Institutes/Centers (ICs) combined. In summary, the NIH is using multiple funding mechanisms to support a sickle cell disease research agenda that is intended to advance the detection, treatment, and cure of this debilitating genetic disease.

Requirement for erythroblast-macrophage protein (Emp) in definitive erythropoiesis.

Emp, erythroblast-macrophage protein was initially identified as a mediator of erythroblast-macrophage interactions during erythroid differentiation. More recent studies have shown that targeted disruption of Emp leads to abnormal erythropoiesis in the fetal liver, and fetal demise. To further address the activity of Emp in the hematopoietic lineage in adult bone marrow, we conducted fetal liver HSC reconstitution assay. Emp null fetal liver cells were transplanted into lethally irradiated wild-type sibling mice, and assessed the erythropoietic activity. We found that Emp null cells rescued lethally irradiated mice with efficiency comparable to that of wild-type cells. However, the recipients of Emp null cells showed abnormal erythropoiesis as indicated by the presence of persistent anemia, extensive extramedullary erythropoiesis, and increased apoptosis of erythroid precursors. Extramedullary erythropoiesis suggests perturbed interactions between the Emp-deficient hematopoietic cells and the wild-type niche. Furthermore, in spleen colony-forming unit assays, proliferation rates of the Emp null cells were greater than those of the wild-type cells. Similarly, in vitro burst-forming unit-erythroid and colony-forming unit-erythroid assays showed increased erythroid colony numbers from Emp null livers. Morphologic examination showed that Emp null CFU-E-derived erythroblasts were immature compared to those derived from wild-type CFU-Es, suggesting that loss of Emp function in erythroid cells results in impaired proliferation and terminal differentiation. These results demonstrate that Emp plays a cell intrinsic role in the erythroid lineage.

Changing pattern of the subcellular distribution of erythroblast macrophage protein (Emp) during macrophage differentiation.

Erythroblast macrophage protein (Emp) mediates the attachment of erythroid cells to macrophages and is required for normal differentiation of both cell lineages. In erythroid cells, Emp is believed to be involved in nuclear extrusion, however, its role in macrophage differentiation is unknown. Information on the changes in the expression level and subcellular distribution of Emp in differentiating macrophages is essential for understanding the function of Emp. Macrophages of varying maturity were examined by immunofluorescence microscopy and biochemical methods. Our data show that Emp is expressed in all stages of maturation, but its localization pattern changes dramatically during maturation: in immature macrophages, a substantial fraction of Emp is associated with the nuclear matrix, whereas in more mature cells, Emp is expressed largely at cell surface. Pulse-chase experiments show that nascent Emp migrates intracellularly from the cytoplasm to the plasma membrane more efficiently in mature macrophages than in immature cells. Incubation of erythroid cells with macrophages in culture shows that erythroid cells attach to mature macrophages but not to immature macrophage precursors. Together, our data show that the temporal and spatial expression of Emp correlates with its role in erythroblastic island formation and suggest that Emp may be involved in multiple cellular functions.

Absence of erythroblast macrophage protein (Emp) leads to failure of erythroblast nuclear extrusion.

In mammals, the functional unit for definitive erythropoiesis is the erythroblastic island, a multicellular structure composed of a central macrophage surrounded by developing erythroblasts. Erythroblast-macrophage interactions play a central role in the terminal maturation of erythroblasts, including enucleation. One possible mediator of this cell-cell interaction is the protein Emp (erythroblast macrophage protein). We used targeted gene inactivation to define the function of Emp during hematopoiesis. Emp null embryos die perinatally and show profound alterations in the hematopoietic system. A dramatic increase in the number of nucleated, immature erythrocytes is seen in the peripheral blood of Emp null fetuses. In the fetal liver virtually no erythroblastic islands are observed, and the number of F4/80-positive macrophages is substantially reduced. Those present lack cytoplasmic projections and are unable to interact with erythroblasts. Interestingly, wild type macrophages can bind Emp-deficient erythroblasts, but these erythroblasts do not extrude their nuclei, suggesting that Emp impacts enucleation in a cell autonomous fashion. Previous studies have implicated the actin cytoskeleton and its reorganization in both erythroblast enucleation as well as in macrophage development. We demonstrate that Emp associates with F-actin and that this interaction is important in the normal distribution of F-actin in both erythroblasts and macrophages. Thus, Emp appears to be required for erythroblast enucleation and in the development of the mature macrophages. The availability of an Emp null model provides a unique experimental system to study the enucleation process and to evaluate the function of macrophages in definitive erythropoiesis.

Novel peptidomimetic cysteine protease inhibitors as potential antimalarial agents.

The synthesis of a new class of peptidomimetics 1a-j, based on a 1,4-benzodiazepine scaffold and on a C-terminal aspartyl aldehyde building block, is described. Compounds 1a-j provided significant inhibitory activity against falcipains 2A and 2B (FP-2A and FP-2B), two cysteine proteases from Plasmodium falciparum.

Cloning and characterization of Plasmodium falciparum cysteine protease, falcipain-2B.

The gene for malaria parasite cysteine protease falcipain-2B has been isolated from the Plasmodium falciparum genomic DNA. Falcipain-2B gene is located adjacent to the falcipain-2A gene on chromosome 11, and the two enzymes show extensive sequence identity at the amino acid level. Using reverse transcribed polymerase chain reaction (RT-PCR), the transcript of falcipain-2B was detected at the trophozoite stage of P. falciparum in human erythrocytes. Recombinant falcipain-2B protein expressed in bacteria exhibits protease activity as established by the cleavage of fluorescent peptide substrate as well as in-gel gelatin zymography. Importantly, the recombinant falcipain-2B cleaved host ankyrin but not protein 4.1 as assessed by the erythrocyte inside-out-vesicle assay in vitro. Notwithstanding its predicted hemoglobinase function, the P. falciparum falcipain-2B may contribute and orchestrate selective proteolytic events during the exit of malaria parasite from human red blood cells.

Emp is a component of the nuclear matrix of mammalian cells and undergoes dynamic rearrangements during cell division.

Emp, originally detected in erythroblastic islands, is expressed in numerous cell types and tissues suggesting a functionality not limited to hematopoiesis. To study the function of Emp in non-hematopoietic cells, an epitope-tagged recombinant human Emp was expressed in HEK cells. Preliminary studies revealed that Emp partitioned into both the nuclear and Triton X-100-insoluble cytoskeletal fractions in approximately a 4:1 ratio. In this study, we report investigations of Emp in the nucleus. Sequential extractions of interphase nuclei showed that recombinant Emp was present predominantly in the nuclear matrix. Immunofluorescence microscopy showed that Emp was present in typical nuclear speckles enriched with the spliceosome assembly factor SC35 and partially co-localized with actin staining. Coimmunoprecipitation and GST-pull-down assays confirmed the apparent close association of Emp with nuclear actin. During mitosis, Emp was detected at the mitotic spindle/spindle poles, as well as in the contractile ring during cytokinesis. These results suggest that Emp undergoes dynamic rearrangements within the nuclear architecture that are correlated with cell division.

Characterization of events preceding the release of malaria parasite from the host red blood cell.

The process of merozoite release involves proteolysis of both the parasitophorous vacuole membrane (PVM) and red blood cell membrane (RBCM), but the precise temporal sequence remains controversial. Using immunofluorescence microscopy and Western blotting of parasite-infected RBCs, we observed that the intraerythrocytic parasite was enclosed in a continuous ring of PVM at early stages of parasite development while at the segmented schizont stage, the PVM appeared to be integrated in the cluster of newly formed merozoites. Subsequently, such clusters were detected extraerythrocytically together with single merozoites devoid of the PVM at low frequency, suggesting a primary rupture of RBCM, followed by PVM rupture and release of invasive merozoites. Secondly, since cysteine proteases are implicated in the process of parasite release, antimalarial effects of 4 cysteine protease inhibitors (leupeptin, E64, E64d, and MDL) were tested at the late schizont stage and correlated with the integrity of PVM and RBCM. We observed that leupeptin and E64 treatment produced extraerythrocytic clusters of merozoites associated with PVM suggesting inhibition of PVM lysis but not RBCM lysis. Merozoites in these clusters developed into rings upon removal of the inhibitors. In contrast, E64d and MDL caused an irreversible parasite death blocking further development. Future characterization of the mechanism(s) of inhibition may facilitate the design of novel antimalarial inhibitors.

Effect of antibiotics on growth and laccase production from Cyathus bulleri and Pycnoporus cinnabarinus.

The effect of nine different antibiotics (chloramphenicol, ampicillin trihydrate, kanamycin A monosulfate, neomycin sulfate, erythromycin, thiostrepton, tetracycline, apramycin sulfate and streptomycin sulfate) on growth and laccase production from Cyathus bulleri and Pycnoporus cinnabarinus has been investigated. All the antibiotics tested at a concentration of 200 mg/l affected the fungal growth, release of protein and laccase production to different extent. Inhibition in fungal growth was found to be positively correlated with increase in laccase production. Interestingly, apramycin sulfate inhibited biomass production (14.9-26.2%), nevertheless, it stimulated maximum laccase production (18.2 U/ml) in both the fungi. Increasing concentrations of apramycin sulfate enhanced laccase production from P. cinnabarinus but not from C. bulleri.

Activation of transglutaminase in mu-calpain null erythrocytes.

Intracellular transglutaminases (protein-glutamine: amine gamma-glutamyltransferase, EC 2.3.2.13) are calcium-dependent thiol enzymes that catalyze the covalent cross-linking of proteins, including those in the erythrocyte membrane. Several studies suggest that the activation of some transglutaminases is positively regulated by the calcium-dependent cysteine protease, mu-calpain. Using mu-calpain null (Capn1(-/-)) mouse erythrocytes, we demonstrate that the activation of soluble as well as membrane-bound forms of transglutaminase (TG2) in mouse erythrocytes was independent of mu-calpain. Also, the absence of mu-calpain or any detectable cysteine protease did not affect the transglutaminase activity in the erythrocyte lysate. Our studies also identify physiological substrates of mu-calpain in the erythrocyte membrane and show that their cleavage has no discernible effect on the transglutaminase mediated cross-linking of membrane proteins. Taken together, these data suggest the existence of a calpain-independent mechanism for the activation of transglutaminase 2 by calcium ions in the mouse erythrocytes and presumably also in non-erythroid cells.

Ankyrin peptide blocks falcipain-2-mediated malaria parasite release from red blood cells.

Falcipain-2 (FP-2) is a dual-function protease that cleaves hemoglobin at the early trophozoite stage and erythrocyte membrane ankyrin and protein 4.1 at the late stages of parasite development. FP-2-mediated cleavage of ankyrin and protein 4.1 is postulated to cause membrane instability facilitating parasite release in vivo. To test this hypothesis, here we have determined the precise peptide sequence at the hydrolysis site of ankyrin to develop specific inhibitor(s) of FP-2. Mass spectrometric analysis of the hydrolysis products showed that FP-2-mediated cleavage of ankyrin occurred immediately after arginine 1,210. A 10-mer peptide (ankyrin peptide, AnkP) containing the cleavage site completely inhibited the FP-2 enzyme activity in vitro and abolished all of the known functions of FP-2. To determine the effect of this peptide on the growth and development of P. falciparum, the peptide was delivered into intact parasite-infected red blood cells (RBCs) via the Antennapedia homeoprotein internalization domain. Growth and maturation of trophozoites and schizonts was markedly inhibited in the presence of the fused AnkP peptide. <10% of new ring-stage parasites were detected compared with the control sample. Together, our results identify a specific peptide derived from the spectrin-binding domain of ankyrin that blocks late-stage malaria parasite development in RBCs. Confocal microscopy with FP-2-specific antibodies demonstrated the proximity of the enzyme in apposition with the RBC membrane, further corroborating the proposed function of FP-2 in the cleavage of RBC skeletal proteins.

Plasmodium falciparum cysteine protease falcipain-2 cleaves erythrocyte membrane skeletal proteins at late stages of parasite development.

Plasmodium falciparum-derived cysteine protease falcipain-2 cleaves host erythrocyte hemoglobin at acidic pH and specific components of the membrane skeleton at neutral pH. Analysis of stage-specific expression of these 2 proteolytic activities of falcipain-2 shows that hemoglobin-hydrolyzing activity is maximum in early trophozoites and declines rapidly at late stages, whereas the membrane skeletal protein hydrolyzing activity is markedly increased at the late trophozoite and schizont stages. Among the erythrocyte membrane skeletal proteins, ankyrin and protein 4.1 are cleaved by native and recombinant falcipain-2 near their C-termini. To identify the precise peptide sequence at the hydrolysis site of protein 4.1, we used a recombinant construct of protein 4.1 as substrate followed by MALDI-MS analysis of the cleaved product. We show that falcipain-2-mediated cleavage of protein 4.1 occurs immediately after lysine 437, which lies within a region of the spectrin-actin-binding domain critical for erythrocyte membrane stability. A 16-mer peptide containing the cleavage site completely inhibited the enzyme activity and blocked falcipain-2-induced fragmentation of erythrocyte ghosts. Based on these results, we propose that falcipain-2 cleaves hemoglobin in the acidic food vacuole at the early trophozoite stage, whereas it cleaves specific components of the red cell skeleton at the late trophozoite and schizont stages. It is the proteolysis of skeletal proteins that causes membrane instability, which, in turn, facilitates parasite release in vivo.

Dematin interacts with the Ras-guanine nucleotide exchange factor Ras-GRF2 and modulates mitogen-activated protein kinase pathways.

Erythroid dematin is a major component of red blood cell junctional complexes that link the spectrin-actin cytoskeleton to the overlying plasma membrane. Transcripts of dematin are widely distributed including human brain, heart, lung, skeletal muscle, and kidney. In vitro, dematin binds and bundles actin filaments in a phosphorylation-dependent manner. The primary structure of dematin consists of a C-terminal domain homologous to the 'headpiece' domain of villin, an actin-binding protein of the brush border cytoskeleton. Except filamentous actin, no other binding partners of dematin have been identified. To investigate the physiological function of dematin, we employed the yeast two-hybrid assay to identify dematin-interacting proteins in the adult human brain. Here, we show that dematin interacts with the guanine nucleotide exchange factor Ras-GRF2 by yeast two-hybrid assay, and this interaction is further confirmed by blot overlay, surface plasmon resonance, co-transfection, and co-immunoprecipitation assays. Human Ras-GRF2 is expressed in a variety of tissues and, similar to other guanine nucleotide exchange factors (GEFs), displays anchorage independent growth in soft agar. Co-transfection and immunoblotting experiments revealed that dematin blocks transcriptional activation of Jun by Ras-GRF2 and activates ERK1 via a Ras-GRF2 independent pathway. Because much of the present evidence has centered on the identification of the Rho family of GTPases as key regulators of the actin cytoskeleton, the direct association between dematin and Ras-GRF2 may provide an alternate mechanism for regulating the activation of Rac and Ras GTPases via the actin cytoskeleton.