Improving Developmental Screening and Supporting Families with Paid Parent Partners.

OBJECTIVE: The primary goals of the project were to improve rates of developmental screening and appropriate referral for atypical screens for children from birth to 3 years in participating practices and to integrate parent partners (PPs) into primary care. METHOD: Parents' Place of Maryland, in collaboration with the Maryland Department of Health, developed a quality improvement project to improve rates of developmental screening, referral, and follow-up. Nine practices from across the state completed the project. Participating practices received training and technical assistance for screening, referral, and data collection as well as a paid part-time PP to assist with care coordination. Families of children with atypical developmental screening results were offered a referral to receive support in following through with referrals, including needed social services to address health care barriers. RESULTS: Developmental screening rates across practices increased from 65% to 95% during the 9-month project, and autism-specific screening increased from 75% to 94%. Appropriate referral rates tripled from 6% to 20% of children. After an initial learning curve, practices gradually increased referrals to PPs, and they served 292 individual families (total of 544 contacts) over 9 months. PPs helped families identify a variety of community services to meet their needs. CONCLUSION: While developmental screening rates can be improved using quality improvement processes, supporting families to connect to services requires additional resources. Integrating trained PPs into pediatric practices improves access to community-based services in a cost-efficient manner. More research is needed to identify the most efficient models for providing this service on a broader scale.

Locomotor and geotactic behavior of Drosophila melanogaster over-expressing neprilysin 2.

The neprilysin (M13) family of zinc-metallopeptidases has been implicated in a variety of physiological processes, but principally the control of neuropeptide levels in a range of animal species. The over-expression of the amyloid-degrading enzyme, neprilysin, as a therapeutic strategy for Alzheimer's disease is a concept that is gaining in popularity. Here we utilize the GAL4/UAS system to over-express the Drosophila melanogaster Nep2 gene, a close homologue of neprilysin, in flies yielding an increase in NEP2 protein that is detectable by both immunoblotting and enzyme activity. This increase in NEP2 caused a behavioral phenotype manifested in abnormal climbing behavior. Wild type flies climb in a linear, vertical path, but NEP2 over-expressing (Nep2(OEX)) flies tend to climb in a spiral pattern and display an increase in grooming behavior during frequent stationary periods. Nep2(OEX) flies also perform poorly in a geotaxis maze, taking ten times as long to complete the course compared to wild type Drosophila. We hypothesize that the poor performance of the Nep2(OEX) flies in locomotor assays is due to perturbation of neuropeptide signaling and provides evidence of detrimental effects of neprilysin over-expression.

Bioinformatic analysis of the neprilysin (M13) family of peptidases reveals complex evolutionary and functional relationships.

BACKGROUND: The neprilysin (M13) family of endopeptidases are zinc-metalloenzymes, the majority of which are type II integral membrane proteins. The best characterised of this family is neprilysin, which has important roles in inactivating signalling peptides involved in modulating neuronal activity, blood pressure and the immune system. Other family members include the endothelin converting enzymes (ECE-1 and ECE-2), which are responsible for the final step in the synthesis of potent vasoconstrictor endothelins. The ECEs, as well as neprilysin, are considered valuable therapeutic targets for treating cardiovascular disease. Other members of the M13 family have not been functionally characterised, but are also likely to have biological roles regulating peptide signalling. The recent sequencing of animal genomes has greatly increased the number of M13 family members in protein databases, information which can be used to reveal evolutionary relationships and to gain insight into conserved biological roles. RESULTS: The phylogenetic analysis successfully resolved vertebrate M13 peptidases into seven classes, one of which appears to be specific to mammals, and insect genes into five functional classes and a series of expansions, which may include inactive peptidases. Nematode genes primarily resolved into groups containing no other taxa, bar the two nematode genes associated with Drosophila DmeNEP1 and DmeNEP4. This analysis reconstructed only one relationship between chordate and invertebrate clusters, that of the ECE sub-group and the DmeNEP3 related genes. Analysis of amino acid utilisation in the active site of M13 peptidases reveals a basis for their biochemical properties. A relatively invariant S1' subsite gives the majority of M13 peptidases their strong preference for hydrophobic residues in P1' position. The greater variation in the S2' subsite may be instrumental in determining the specificity of M13 peptidases for their substrates and thus allows M13 peptidases to fulfil a broad range of physiological roles. CONCLUSION: The M13 family of peptidases have diversified extensively in all species examined, indicating wide ranging roles in numerous physiological processes. It is predicted that differences in the S2' subsite are fundamental to determining the substrate specificities that facilitate this functional diversity.

Expression of NEP2, a soluble neprilysin-like endopeptidase, during embryogenesis in Drosophila melanogaster.

Members of the neprilysin family of neutral endopeptidases (M13) are typically membrane-bound enzymes known to be involved in the extra-cellular metabolism of signalling peptides and have important roles during mammalian embryogenesis. In this study we show that membranes prepared from embryos of Drosophila melanogaster possess neprilysin-like activity that is inhibited by phosphoramidon and thiorphan, both inhibitors of mammalian neprilysin. Unexpectedly, we also found strong neprilysin-like neutral endopeptidase activity in a soluble embryo fraction, which we identify as NEP2 by Western blot and immunoprecipitation experiments using NEP2 specific antibodies. NEP2 is a soluble secreted member of the neprilysin family that has been shown previously to be expressed in larval and adult Malpighian tubules and in the testes of adult males. In situ hybridization studies reveal expression at stage 10-11 in a pattern similar to that previously described for stellate cell progenitors of the caudal visceral mesoderm. In later stages of embryogenesis, some of these cells appear to migrate into the growing Malpighian tubule. Recombinant NEP2 protein is N-glycosylated and displays optimum endopeptidase activity at neutral pH, consistent with a role as an extracellular peptidase. The recombinant enzyme hydrolyses Drosophila tachykinin peptides (DTK) at peptide bonds N-terminal to hydrophobic residues. DTK2, like Locusta tachykinin-1, was cleaved at the penultimate peptide bond (Gly(7)-Leu(8)), whereas the other Drosophila peptides were cleaved centrally at Xxx-Phe bonds. However, the rates of hydrolysis of the latter substrates were much slower than the hydrolysis rates of DTK2 and Locusta tachykinin-1, suggesting that the interaction of the bulky side-chain of phenylalanine at the S'(1) sub-site is less favorable for peptide bond hydrolysis. The secretion of NEP2 from tissues during embryogenesis suggests a possible developmental role for this endopeptidase in peptide signalling in D. melanogaster.

Drosophila melanogaster NEP2 is a new soluble member of the neprilysin family of endopeptidases with implications for reproduction and renal function.

The mammalian neprilysin (NEP) family members are typically type II membrane endopeptidases responsible for the activation/inactivation of neuropeptides and peptide hormones. Differences in substrate specificity and subcellular localization of the seven mammalian NEPs contribute to their functional diversity. The sequencing of the Drosophila melanogaster genome has revealed a large expansion of this gene family, resulting in over 20 fly NEP-like genes, suggesting even greater diversity in structure and function than seen in mammals. We now report that one of these genes (Nep2) codes for a secreted endopeptidase with a highly restricted pattern of expression. D. melanogaster NEP2 is expressed in the specialized stellate cells of the renal tubules and in the cyst cells that surround the elongating spermatid bundles in adult testis, suggesting roles for the peptidase in renal function and in spermatogenesis. D. melanogaster NEP2 was found in vesicle-like structures in the syncytial cytoplasm of the spermatid bundles, suggesting that the protein was acquired by endocytosis of protein secreted from the cyst cells. Expression of NEP2 cDNA in D. melanogaster S2 cells confirmed that the peptidase is secreted and is only weakly inhibited by thiorphan, a potent inhibitor of human NEP. D. melanogaster NEP2 also differs from human NEP in the manner in which the peptidase cleaves the tachykinin, GPSGFYGVR-amide. Molecular modelling suggests that there are important structural differences between D. melanogaster NEP2 and human NEP in the S1' and S2' ligand-binding subsites, which might explain the observed differences in inhibitor and substrate specificities. A soluble isoform of a mouse NEP-like peptidase is strongly expressed in spermatids, suggesting an evolutionarily conserved role for a soluble endopeptidase in spermatogenesis.