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.     

Breeding biology of Frances's Sparrowhawk Accipiter francesii in a lowland rainforest of northeastern Madagascar

Lily-Arison, R.de R. 2000. Breeding biology of Frances's Sparrowhawk Accipiter francesii in a lowland rainforest of northeastem Madagascar. Ostrich 71 (1 & 2): 332–335.

Frances's Sparrowhawks Accipiter fmncesii were studied during the breeding seasons 1994 and 1995 on Masoala Peninsula of northeastern Madagascar. Breeding coincides with the dry season, nest building in October, egg laying in November, hatching in December and fledging in January (beginning of the wet season). All but one of 10 pairs built new nests in 1995 and the mean distance of nest site from 1994 and 1995 nests was 105± 100 m (n = 6, ranging from 0 to 250m). Nests in primary forest were incubated for 79% of the observation time by females, 3% by males and eggs were unattended for 18% of the observation time. On a numerical basis the diet was composed of lizards (56%) and birds (23%), making up more than 79% of the identified prey items. A total of 33 eggs was laid in 13 nests (average clutch size 2.5). In 13 breeding attempts 29 (88%) hatched and all of those hatchlings fledged. Of the 14 My-documented breeding attempts 2.1 young fledged per breeding attempt and overall nesting success was 93%.

L'Épervier de France Accipiter francesii a été étudié pendant la saison de reproduction en 1994 et 1995 dans la Presqu'Ile Masoala, au partie Nord-Est de Madagascar. La saison de reproduction de cette espkce coincide avec la saison sèche: la construction du nid en Octobre, la ponte en Novembre, I'élosion en Déembre et l'envol du poussin en Janvier (début de la saison pluviale). Tous, sauf une des 10 paires ont construit des nouveaw nids en 1995 et la distance moyenne de I'endroit du nid de I'anné 1994 et 1995 est de 105 + 100 m (n = 6, variant de 0 à 250m). Dans la for[ebar]t primaire, les oeufs ont éeté incubés par la femelle pendant 79% du temps d'observation, 3% par le mile et non incubés pendant 18% du temps. Les proies sont principalement composées de lkzards (56%) et oiseaux (23%), atteignant plus de 79% des proies identifiés. 33 oeufs ont été pondus dans 13 nids (en moyenne 2.5 oeufs par nid). Pour ces 13 nids, 29 oeufs (88%) ont été éclos et tous ces poussins (100%) sont capables de voler. Sur 14 nids observés, 2.1 poussins par nid s'envolent et le tam de succés est de 93%.     

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.     

Constitutive expression of fluorescent protein by Aspergillus var. niger and Aspergillus carbonarius to monitor fungal colonization in maize plants

Aspergillus niger and Aspergillus carbonarius are two species in the Aspergillus section Nigri (black-spored aspergilli) frequently associated with peanut (Arachis hypogea), maize (Zea mays), and other plants as pathogens. These infections are symptomless and as such are major concerns since some black aspergilli produce important mycotoxins, ochratoxins A, and the fumonisins. To facilitate the study of the black aspergilli–maize interactions with maize during the early stages of infections, we developed a method that used the enhanced yellow fluorescent protein (eYFP) and the monomeric red fluorescent protein (mRFP₁) to transform A. niger and A. carbonarius, respectively. The results were constitutive expressions of the fluorescent genes that were stable in the cytoplasms of hyphae and conidia under natural environmental conditions. The hyphal in planta distribution in 21-day-old seedlings of maize were similar wild type and transformants of A. niger and A. carbonarius. The in planta studies indicated that both wild type and transformants internally colonized leaf, stem and root tissues of maize seedlings, without any visible disease symptoms. Yellow and red fluorescent strains were capable of invading epidermal cells of maize roots intercellularly within the first 3 days after inoculation, but intracellular hyphal growth was more evident after 7 days of inoculation. We also tested the capacity of fluorescent transformants to produce ochratoxin A and the results with A. carbonarius showed that this transgenic strain produced similar concentrations of this secondary metabolite. This is the first report on the in planta expression of fluorescent proteins that should be useful to study the internal plant colonization patterns of two ochratoxigenic species in the Aspergillus section Nigri.     

Reduction of palladium and production of nano-catalyst by Geobacter sulfurreducens

The present study is the first report on the ability of Geobacter sulfurreducens PCA to reduce Pd(II) and produce Pd(0) nano-catalyst, using acetate as electron donor at neutral pH (7.0?±?0.1) and 30 °C. The microbial production of Pd(0) nanoparticles (NPs) was greatly enhanced by the presence of the redox mediator, anthraquinone-2,6-disulfonate (AQDS) when compared with controls lacking AQDS and cell-free controls. A cell dry weight (CDW) concentration of 800 mg/L provided a larger surface area for Pd(0) NPs deposition than a CDW concentration of 400 mg/L. Sample analysis by transmission electron microscopy revealed the formation of extracellular Pd(0) NPs ranging from 5 to 15 nm and X-ray diffraction confirmed the Pd(0) nature of the nano-catalyst produced. The present findings open the possibility for a new alternative to synthesize Pd(0) nano-catalyst and the potential application for microbial metal recovery from metal-containing waste streams.     

Strategic Management of an Invasive Ant‐scale Mutualism Enables Recovery of a Threatened Tropical Tree Species

Mutualisms between invasive ants and honeydew‐producing insects can have widespread negative effects on natural ecosystems. This is becoming an increasingly serious problem worldwide, causing certain ecosystems to change radically. Management of these abundant and influential mutualistic species is essential if the host ecosystem is to recover to its former non‐invaded status. This negative effect is particularly prevalent on some tropical islands, including Cousine Island, Seychelles. On this island, the invasive ant Pheidole megacephala has caused serious indirect damage to the threatened native Pisonia grandis trees via a mutualism with an invasive scale insect, Pulvinaria urbicola. We aimed to suppress the ant, thereby decoupling the mutualism and enabling recovery of the Pisonia trees. We treated all areas where ant pressure was high with a selective formicidal bait, which was deployed in custom‐made bait stations designed to avoid risk of treatment to endemic fauna. In the treated area, ant foraging activity was reduced by 93 percent and was followed by a 100 percent reduction in scale insect density. Abundance of endemic herbivorous insects and herbivorous activity increased significantly, however, after the decline in mutualistic species densities. Despite the native herbivore increase, there was considerable overall improvement in Pisonia shoot condition and an observed increase in foliage density. Our results demonstrate the benefit of strategic management of highly mutualistic alien species to the native Pisonia trees. It also supports the idea that area‐wide suppression is a feasible alternative to eradication for achieving positive conservation management at the level of the forest ecosystem.     

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 Himalayas: Barrier and conduit for gene flow

The Himalayan mountain range is strategically located at the crossroads of the major cultural centers in Asia, the Middle East and Europe. Although previous Y‐chromosome studies indicate that the Himalayas served as a natural barrier for gene flow from the south to the Tibetan plateau, this region is believed to have played an important role as a corridor for human migrations between East and West Eurasia along the ancient Silk Road. To evaluate the effects of the Himalayan mountain range in shaping the maternal lineages of populations residing on either side of the cordillera, we analyzed mitochondrial DNA variation in 344 samples from three Nepalese collections (Newar, Kathmandu and Tamang) and a general population of Tibet. Our results revealed a predominantly East Asian‐specific component in Tibet and Tamang, whereas Newar and Kathmandu are both characterized by a combination of East and South Central Asian lineages. Interestingly, Newar and Kathmandu harbor several deep‐rooted Indian lineages, including M2, R5, and U2, whose coalescent times from this study (U2, >40 kya) and previous reports (M2 and R5, >50 kya) suggest that Nepal was inhabited during the initial peopling of South Central Asia. Comparisons with our previous Y‐chromosome data indicate sex‐biased migrations in Tamang and a founder effect and/or genetic drift in Tamang and Newar. Altogether, our results confirm that while the Himalayas acted as a geographic barrier for human movement from the Indian subcontinent to the Tibetan highland, it also served as a conduit for gene flow between Central and East Asia. Am J Phys Anthropol 151:169–182, 2013. © 2013 Wiley Periodicals, Inc.     

Deep Whole-Genome Sequencing of 100 Southeast Asian Malays

Whole-genome sequencing across multiple samples in a population provides an unprecedented opportunity for comprehensively characterizing the polymorphic variants in the population. Although the 1000 Genomes Project (1KGP) has offered brief insights into the value of population-level sequencing, the low coverage has compromised the ability to confidently detect rare and low-frequency variants. In addition, the composition of populations in the 1KGP is not complete, despite the fact that the study design has been extended to more than 2,500 samples from more than 20 population groups. The Malays are one of the Austronesian groups predominantly present in Southeast Asia and Oceania, and the Singapore Sequencing Malay Project (SSMP) aims to perform deep whole-genome sequencing of 100 healthy Malays. By sequencing at a minimum of 30× coverage, we have illustrated the higher sensitivity at detecting low-frequency and rare variants and the ability to investigate the presence of hotspots of functional mutations. Compared to the low-pass sequencing in the 1KGP, the deeper coverage allows more functional variants to be identified for each person. A comparison of the fidelity of genotype imputation of Malays indicated that a population-specific reference panel, such as the SSMP, outperforms a cosmopolitan panel with larger number of individuals for common SNPs. For lower-frequency (<5%) markers, a larger number of individuals might have to be whole-genome sequenced so that the accuracy currently afforded by the 1KGP can be achieved. The SSMP data are expected to be the benchmark for evaluating the value of deep population-level sequencing versus low-pass sequencing, especially in populations that are poorly represented in population-genetics studies.