Binding and uptake of single and dual-opsonized targets by macrophages

Our current understanding of phagocytosis is largely derived from studies of individual receptor-ligand interactions and their downstream signaling pathways. Because phagocytes are exposed to a variety of ligands on heterogeneous target particles in vivo, it is important to observe the engagement of multiple receptors simultaneously and the triggered involvement of downstream signaling pathways. Potential crosstalk between the two well-characterized opsonic receptors, FcγR and CR3, was briefly explored in the early 1970s, where macrophages were challenged with dual-opsonized targets. However, subsequent studies on receptor crosstalk were primarily restricted to using single opsonins on different targets, typically at saturating opsonin conditions. Beyond validating these initial explorations on receptor crosstalk, we identify the early signaling mechanisms that underlie the binding and phagocytosis during the simultaneous activation of both opsonic receptors, through the presence of a dual-opsonized target (immunoglobulin G [IgG] and C3bi), compared with single receptor activation. For this purpose, we used signaling protein inhibitor studies as well as live cell brightfield and fluorescent imaging to fully understand the role of tyrosine kinases, F-actin dynamics and internalization kinetics for FcγR and CR3. Importantly, opsonic receptors were studied together and in isolation, in the context of sparsely opsonized targets. We observed enhanced particle binding and a synergistic effect on particle internalization during the simultaneous activation of FcγR and CR3 engaged with sparsely opsonized targets. Inhibition of early signaling and cytoskeletal molecules revealed a differential involvement of Src kinase for FcγR- vs CR3- and dual receptor-mediated phagocytosis. Src activity recruits Syk kinase and we observed intermediate levels of Syk phosphorylation in dual-opsonized particles compared with those opsonized with IgG or C3bi alone. These results likely explain the intermediate levels of F-actin that is recruited to sites of dual-opsonized particle uptake and the notoriously delayed internalization of C3bi-opsonized targets by macrophages.     

D1S80 single-locus discrimination among African populations

The highly polymorphic D1S80 locus has no known genetic function. However, this variable number of tandem repeats (VNTR) locus has been highly valuable in forensic identification. In this study we report the allele and genotype frequencies of five African populations (Benin, Cameroon, Egypt, Kenya, and Rwanda), which can be used as databases to help characterize populations and identify individuals. The allele frequencies were used to infer genetic associations through phylogenetic, principal component, and G test statistical analyses. Compliance with Hardy-Weinberg equilibrium expectations was determined as were F(ST) estimates, theta p values, and power of discrimination assessment for each population. Our analyses of 28 additional populations demonstrate that the D1S80 locus alone can be used to discriminate geographic and ethnic groups. We have generated databases useful for human identification and phylogenetic studies.     

Perspective on the host response to human metapneumovirus infection: what can we learn from respiratory syncytial virus infections?

Human metapneumovirus (HMPV) is a recently discovered pathogen first identified in respiratory specimens from young children suffering from clinical respiratory syndromes ranging from mild to severe lower respiratory tract illness. HMPV has worldwide prevalence, and is a leading cause of respiratory tract infection in the first years of life, with a spectrum of disease similar to respiratory syncytial virus (RSV). The disease burden associated with HMPV infection has not been fully elucidated; however, studies indicate that HMPV may cause upper or lower respiratory tract illness in patients between ages 2 months and 87 years, may co-circulate with RSV, and HMPV infection may be associated with asthma exacerbation. The mechanisms and effector pathways contributing to immunity or disease pathogenesis following infection are not fully understood; however, given the clinical significance of HMPV, there is a need for a fundamental understanding of the immune and pathophysiological processes that occur following infection to provide the foundation necessary for the development of effective vaccine or therapeutic intervention strategies. This review provides a current perspective on the processes associated with HMPV infection, immunity, and disease pathogenesis.     

Targeted molecular trait stacking in cotton through targeted double‐strand break induction

Recent developments of tools for targeted genome modification have led to new concepts in how multiple traits can be combined. Targeted genome modification is based on the use of nucleases with tailor‐made specificities to introduce a DNA double‐strand break (DSB) at specific target loci. A re‐engineered meganuclease was designed for specific cleavage of an endogenous target sequence adjacent to a transgenic insect control locus in cotton. The combination of targeted DNA cleavage and homologous recombination–mediated repair made precise targeted insertion of additional trait genes (hppd, epsps) feasible in cotton. Targeted insertion events were recovered at a frequency of about 2% of the independently transformed embryogenic callus lines. We further demonstrated that all trait genes were inherited as a single genetic unit, which will simplify future multiple‐trait introgression.     

EB1 Levels Are Elevated in Ascorbic Acid (AA)-stimulated Osteoblasts and Mediate Cell-Cell Adhesion-induced Osteoblast Differentiation

Osteoblasts are differentiated mesenchymal cells that function as the major bone-producing cells of the body. Differentiation cues including ascorbic acid (AA) stimulation provoke intracellular changes in osteoblasts leading to the synthesis of the organic portion of the bone, which includes collagen type I α1, proteoglycans, and matrix proteins, such as osteocalcin. During our microarray analysis of AA-stimulated osteoblasts, we observed a significant up-regulation of the microtubule (MT) plus-end binding protein, EB1, compared with undifferentiated osteoblasts. EB1 knockdown significantly impaired AA-induced osteoblast differentiation, as detected by reduced expression of osteoblast differentiation marker genes. Intracellular examination of AA-stimulated osteoblasts treated with EB1 siRNA revealed a reduction in MT stability with a concomitant loss of β-catenin distribution at the cell cortex and within the nucleus. Diminished β-catenin levels in EB1 siRNA-treated osteoblasts paralleled an increase in phospho-β-catenin and active glycogen synthase kinase 3β, a kinase known to target β-catenin to the proteasome. EB1 siRNA treatment also reduced the expression of the β-catenin gene targets, cyclin D1 and Runx2. Live immunofluorescent imaging of differentiated osteoblasts revealed a cortical association of EB1-mcherry with β-catenin-GFP. Immunoprecipitation analysis confirmed an interaction between EB1 and β-catenin. We also determined that cell-cell contacts and cortically associated EB1/β-catenin interactions are necessary for osteoblast differentiation. Finally, using functional blocking antibodies, we identified E-cadherin as a major contributor to the cell-cell contact-induced osteoblast differentiation.     

On the origins,rapid expansion and genetic diversity of native Americans from hunting‐gatherers to agriculturalists

Given the importance of Y‐chromosome haplogroup Q to better understand the source populations of contemporary Native Americans, we studied 8 biallelic and 17 microsatellite polymorphisms on the background of 128 Q Y‐chromosomes from geographically targeted populations. The populations examined in this study include three from the Tuva Republic in Central Asia (Bai‐Tai, Kungurtug, and Toora‐Hem, n = 146), two from the northeastern tip of Siberia (New Chaplino and Chukchi, n = 32), and two from Mesoamerica (Mayans from Yucatan, Mexico n = 72, and Mayans from the Guatemalan Highlands, n = 43). We also see evidence of a dramatic Mesoamerican post‐migration population growth in the ubiquitous and diverse Y‐STR profiles of the Mayan and other Mesoamerican populations. In the case of the Mayans, this demographic growth was most likely fueled by the agricultural‐ and trade‐based subsistence adopted during the Pre‐Classic, Classic and Post‐Classic periods of their empire. The limited diversity levels observed in the Altaian and Tuvinian regions of Central Asia, the lowest of all populations examined, may be the consequence of bottleneck events fostered by the spatial isolation and low effective population size characteristic of a nomadic lifestyle. Furthermore, our data illustrate how a sociocultural characteristic such as mode of subsistence may be of impact on the genetic structure of populations. We analyzed our genetic data using Multidimensional Scaling Analysis of populations, Principal Component Analysis of individuals, Median‐joining networks of M242, M346, L54, and M3 individuals, age estimations based on microsatellite variation utilizing genealogical and evolutionary mutation rates/generation times and estimation of Y‐ STR average gene diversity indices. Am J Phys Anthropol, 2013. © 2013 Wiley Periodicals, Inc.     

The Role of Geography in the Assessment of Quality: Evidence from the Medicare Advantage Program

The Affordable Care Act set in motion a renewed emphasis on quality of care evaluation. However, the evaluation strategies of quality by the Centers for Medicare and Medicaid Services do not consider geography when comparisons are made among plans. Using an overall measure of a plan’s quality in the public sector—the Medicare Advantage (MA) star ratings—we explored the impact of geography in these ratings. We identified 2,872 U.S counties in 2010. The geographic factor predicted a larger fraction of the MA ratings’ compared to socio-demographic factors which explained less. Also, after the risk adjustments, almost half of the U.S. states changed their ranked position in the star ratings. Further, lower MA star ratings were identified in the Southeastern region. These findings suggest that the geographic component effect on the ratings is not trivial and should be considered in future adjustments of the metric, which may enhance the transparency, accountability, and importantly level the playing field more effectively when comparing quality across health plans.