Microfluidic PCR-based target enrichment: A case study in two rapid radiations of Commiphora (Burseraceae) from Madagascar

Developing effective and cost-efficient multilocus nuclear datasets for angiosperm species is a continuing challenge to the systematics community. Here we describe the development and validation of a novel set of 91 nuclear markers for PCR-based target enrichment. Using microfluidic PCR and Illumina MiSeq, we generated nuclear, subgenomic libraries for 96 species simultaneously and sequenced them for a total cost of ca. $6000 USD. Approximately half of these costs include reusable reagents (primers, barcodes, and custom sequencing primers) and taxon sampling could be increased by an order of magnitude to maximize sequencing depth efficiency. The principle benefit of microfluidic PCR over alternative target enrichment strategies is that it bypasses costly library preparation. After sequencing, we evaluated the ability of the loci to resolve species level relationships within two recently radiated lineages of endemic Madagascan Commiphora Jacq. (Burseraceae) species. Our results demonstrate that (i) effective nuclear markers can be designed for non-model angiosperm taxa from these publicly available datasets; (ii) that microfluidic PCR amplification followed by high throughput sequencing can produce highly complete taxon by locus sequence data matrices with minimal resource investment; and (iii) that these numerous nuclear phylogenomic markers can improve our understanding of phylogenetic relationships withinCommiphora. We provide a synopsis of ongoing activities to enhance this microfluidic PCR-based target enrichment strategy through broader primer assays, multiplexing, and increased efficiency of sequencing depth.     

Interaction of bracken-fern extract with vitamin C in human submandibular gland and oral epithelium cell lines

he consumption of bracken-fern (Pteridium aquilinum) as food is associated with a high incidence of cancer in humans and animals. Thus far, the carcinogenic effects of bracken-fern consumption could be related to chromosome aberrations verified in animal and in human peripheral lymphocytes. We tested the in vitro effects of vitamin C (10 and 100 μg/ml) on the reversibility of DNA damage caused by bracken-fern on human submandibular gland (HSG) cells and on oral epithelium cells (OSCC-3) previously exposed to bracken-fern extract. DNA damage (i.e. nuclei with increased levels of DNA migration) was determined by comet assay, cell morphology was evaluated by light microscopy and cellular degeneration was assessed by the acridine orange/ethidium bromide fluorescent-dyeing test. Results showed that vitamin C alone did not reduce DNA damage caused by bracken-fern in HSG and OSSC-3 cells. However, at a higher concentration (100 μg/ml), vitamin C induced DNA damage in both cell lines. Moreover, vitamin C (10 and 100 μg/ml) together with bracken-fern extract showed synergistic effects on the frequency of DNA damage in HSG cells. In addition, cells treated with bracken-fern extract or vitamin C alone, or with their association, showed apoptosis morphological features, such as chromatin condensation, cytoplasmic volume loss, changes in membrane symmetry and the appearance of vacuoles; these alterations were observed in both cell lines. These results demonstrate that bracken-fern extract was cytotoxic to HSG and OSCC-3 cells, causing cell death by apoptosis, and that vitamin was not able to revert these effects.     

Mimicking the nicotinic receptor binding site by a single chain Fv selected by competitive panning from a synthetic phage library

We have developed a novel competitive method to select from a phage display library a single chain Fv which is able to mimic the alpha-bungarotoxin binding site of the muscle nicotinic receptor. The single chain Fv was selected from a large synthetic library using alpha-bungarotoxin-coated magnetic beads. Toxin-bound phages were then eluted by competition with affinity purified nicotinic receptor. Recognition of the toxin by the anti-alpha-bungarotoxin single chain Fv was very similar to that of the receptor, such as indicated by the epitope mapping of alpha-bungarotoxin through overlapping synthetic peptides. Moreover, several positively charged residues located in the toxin second loop and in the C-terminal region were found to be critical, to a similar extent, for toxin recognition by the single chain Fv and the receptor. However, although the anti-alpha-bungarotoxin single chain Fv seems to mimic the toxin binding site of the nicotinic receptor, it does not bind other nicotinic agonists or antagonists. Our results suggest that competitive selection of anti-ligand antibody phages can allow the production of receptor-mimicking molecules directly and exclusively targeted at one specific ligand. Since physiologically and pharmacologically different ligands can produce opposite effects on receptor functions, such selective ligand decoys can have important therapeutic applications.     

In vivo dehydroepiandrosterone restores age-associated defects in the protein kinase C signal transduction pathway and related functional responses

Elderly subjects are at increased risk of pneumonia, influenza, and tuberculosis. Besides the known age-related decrease in mechanisms for mechanical clearance of the lungs, impaired alveolar macrophage function contributes to the increased risk of illness in the elderly. We have previously shown that age-induced macrophage immunodeficiencies are associated with a defective system for anchoring protein kinase C. Castration of young male rats produces effects on alveolar macrophages similar to those of aging, suggesting a relationship between circulating sex hormones, particularly androgens, and the decreases in the receptor for activated C kinase (RACK-1) and macrophage function observed. The aging process in humans and rats is associated with a decline in the plasma concentrations of dehydroepiandrosterone (DHEA) and its sulfate, among other steroid hormones. We report here that in vitro and in vivo administration of DHEA to rats restores the age-decreased level of RACK-1 and the LPS-stimulated production of TNF-alpha in alveolar macrophages. DHEA in vivo also restores age-decreased spleen mitogenic responses and the level of RACK-1 expression. These findings suggest that the age-related loss in immunological responses, linked to defective pathways of signal transduction, are partially under hormonal control and can be restored by appropriate replacement therapy.     

Anthropogenic impact of mercury accumulation in fish from the Rio Madeira and Rio Negro rivers (Amazonia)

Fish is an important concentrator of mono-methyl mercury and the main route to human contamination. We compared fish Hg bioaccumulation (within similar weight ranges) in two Amazonian river habitats during high-water seasons. The Rio Madeira has been greatly impacted by agriculture, alluvial gold extraction, and a hydroelectric reservoir, whereas the Rio Negro is much less affected by these human activities. The species at the top of the food web, Hoplias malabaricus (piscivorous; 80-668 ng Hg/g) and Cichla spp. (piscivorous; 42–747 ng Hg/g) showed the highest range of Hg concentrations. Nonpiscivorous species with comparable weight range, such as Potamorhina latior (detritivorous; 20–157 ng Hg/g) and Myleus torquatus (herbivorous; 2–182 ng Hg/g), had lower Hg concentrations. Triportheus elongatus (omnivorous; 5–350 ng Hg/g), with the lowest weight range, also showed a low range of Hg concentrations. Despite the Rio Madeira's higher sediment load as well as environmental impacts (deforestation, agriculture, hydroelectric reservoir, and alluvial gold mining) on natural Hg release, fish Hg bioaccumulation was no different between the two river habitats for nonpiscivorous species. In this small observational study only the species at the top of the food web (M. torquatus, Cichla spp, T. elongatus) showed higher mean Hg concentrations in the Rio Madeira than the dominantly wilderness habitat of the Rio Negro.     

Arcobacter butzleri,Arcobacter cryaerophilus,and Arcobacter skirrowii Circulation in a Dairy Farm and Sources of Milk Contamination

Even though dairy cows are known carriers of Arcobacter species and raw or minimally processed foods are recognized as the main sources of human Arcobacter infections in industrialized countries, data on Arcobacter excretion patterns in cows and in milk are scant. This study aimed to identify potentially pathogenic Arcobacter species in a dairy herd and to investigate the routes of Arcobacter transmission among animals and the potential sources of cattle infection and milk contamination. A strategy of sampling the same 50 dairy animals, feed, water, and milk every month for a 10-month period, as well as the sampling of quarter milk, animal teats, the milking environment, and animals living on the farm (pigeons and cats), was used to evaluate, by pulsed-field gel electrophoresis (PFGE), the characteristic patterns in animals, their living environment, and the raw milk they produced. Of the 463 samples collected, 105 (22.6%) were positive for Arcobacter spp. by culture examination. All the matrices except quarter milk and pigeon gut samples were positive, with prevalences ranging from 15 to 83% depending on the sample. Only three Arcobacter species, Arcobacter cryaerophilus (54.2%), A. butzleri (34.2%), and A. skirrowii (32.3%), were detected. PFGE analysis of 370 isolates from positive samples provided strong evidence of Arcobacter circulation in the herd: cattle likely acquire the microorganisms by orofecal transmission, either by direct contact or from the environment, or both. Water appears to be a major source of animal infection. Raw milk produced by the farm and collected from a bulk tank was frequently contaminated (80%) by A. butzleri; our PFGE findings excluded primary contamination of milk, whereas teats and milking machine surfaces could be sources of Arcobacter milk contamination.     

The l2 minor capsid protein of human papillomavirus type 16 interacts with a network of nuclear import receptors

The L2 minor capsid proteins enter the nucleus twice during viral infection: in the initial phase after virion disassembly and in the productive phase when, together with the L1 major capsid proteins, they assemble the replicated viral DNA into virions. In this study we investigated the interactions between the L2 protein of high-risk human papillomavirus type 16 (HPV16) and nuclear import receptors. We discovered that HPV16 L2 interacts directly with both Kapbeta(2) and Kapbeta(3). Moreover, binding of Ran-GTP to either Kapbeta(2) or Kapbeta(3) inhibits its interaction with L2, suggesting that the Kapbeta/L2 complex is import competent. In addition, we found that L2 forms a complex with the Kapalpha(2)beta(1) heterodimer via interaction with the Kapalpha(2) adapter. In agreement with the binding data, nuclear import of L2 in digitonin-permeabilized cells could be mediated by either Kapalpha(2)beta(1) heterodimers, Kapbeta(2), or Kapbeta(3). Mapping studies revealed that HPV16 L2 contains two nuclear localization signals (NLSs), in the N terminus (nNLS) and C terminus (cNLS), that could mediate its nuclear import. Together the data suggest that HPV16 L2 interacts via its NLSs with a network of karyopherins and can enter the nucleus via several import pathways mediated by Kapalpha(2)beta(1) heterodimers, Kapbeta(2), and Kapbeta(3).     

Characterisation of neurons expressing calbindin immunoreactivity in the ileum of the unweaned and mature sheep

We have identified the enteric neuron types expressing immunoreactivity for the calcium-binding protein calbindin D28k (CALB) in cryostat sections and whole-mount preparations of myenteric (MP) and submucosal (SMP) plexuses of sheep ileum. We wished to determine whether CALB-IR in the sheep enteric nervous system was expressed in Dogiel type II cells, as in guinea-pig and rat ileum, and could therefore be used as a marker for intrinsic primary afferent neurons. The neurochemical coding of CALB-containing myenteric and submucosal neurons in ileum of unweaned lamb and mature sheep and its co-localisation with various neural markers was studied immunohistochemically. An antiserum against neuronal nuclear protein (NeuN) failed to detect the entire neuronal population; it was expressed only in 48% of neuron-specific enolase (NSE)-immunoreactive (NSE-IR) neurons. Human neuronal protein appeared to occur in the large majority or all neurons. Almost all CALB-IR neurons were: (1) radially multidendritic; (2) eccentric multidendritic; (3) Dogiel type II. CALB-IR occurred in 20–25% of myenteric and 65–75% of submucosal neurons in lamb and mature sheep, with higher values in mature sheep. Nearly all CALB-IR neurons were common choline acetyltransferase (cChAT)-IR, whereas only about 20% of cChAT-IR somata were CALB-IR. In lamb and mature sheep, 90% of MP CALB-IR neurons were peripheral choline acetyltransferase (pChAT)-IR. In lamb SMP, 80±13% of CALB-IR cells were also pChAT-IR, whereas all those in mature SMP were pChAT-IR. Fewer myenteric CALB-IR neurons exhibited tachykinin (TK) in mature sheep (49%) than in lamb (88%). This was also the case for submucosal ganglia (mature sheep, 63%; lamb, 89%). In lamb MP, 77±7% of CALB-IR cells were NeuN-positive. In mature sheep, 73±10% of CALB-IR somata were NeuN-IR, but NeuN failed to stain SMP neurons. In the MP of suckling and mature sheep, Dogiel type II CALB-IR neurons were calcitonin gene-related peptide (CGRP)-IR. In the SMP at both stages, Dogiel type II CALB-IR somata (about 50% of CALB-IR neurons) were also CGRP-IR. Only small proportions of CALB-IR neurons showed immunoreactivity for calretinin or nitric oxide synthase (NOS), although large populations of CALB and NOS neurons occurred in the ganglia. Thus, CALB is a marker of most Dogiel type II neurons in the sheep but is not confined to Dogiel II neurons. CGRP is a more selective marker of Dogiel type II neurons, being only found in this neuron type.This work was supported by a grant from the Ministero dellIstruzione, dellUniversità e della Ricerca (MIUR)     

In Saccharomyces cerevisiae a short amino acid sequence facilitates excretion in the growth medium of periplasmic proteins

In Saccharomyces cerevisiae the cell wall is a barrier to excretion of proteins in the growth medium. Although small proteins are more easily released than bigger ones, other factors besides molecular sieving may play a role in partitioning of periplasmic proteins. By using several complementary approaches including enzyme-activity assays, quantitative immunoblotting on subcellular fractions and growth media, as well as a novel approach involving the use of flow cytometry and specific antibodies, we show that residues 1–8 of mature glucoamylase greatly enhance excretion of both glucoamylase and β-galactosidase in vivo and facilitate extraction of periplasmic proteins in vitro . Immunological data obtained by flow cytometry on whole cells indicate that this amino acid sequence increases the fraction of enzyme reaching the outer cell-wall layers. This amino acid sequence may define a novel type of topogenic sequence, facilitating the crossing of the yeast cell wall in vivo and facilitating extraction of periplasmic proteins by non-disruptive means in vitro .