Growth and development of Encarsia formosa (Hymenoptera: Aphelinidae) in the greenhouse whitefly, Trialeurodes vaporariorum (Homoptera: Aleyrodidae): effect of host age

The tiny parasitoid wasp, Encarsia formosa, has been used successfully to control greenhouse whiteflies (GHWFs) in greenhouses in many countries throughout the world. Therefore, there has been considerable interest in developing methods for artificially rearing this wasp. However, little information is available concerning the regulation of its development including the host-parasitoid interactions that are required for the parasitoid to complete its life cycle. Here we confirm that parasitoid developmental rates differ significantly based upon the host instar parasitized. Development was faster when 3rd and 4th instar GHWFs were offered for parasitization than when 1st or 2nd instars were used. Our results show that it is primarily the embryo and the first two parasitoid instars that exhibit prolonged developmental times when 1st and 2nd instar whiteflies are parasitized. Although percent emergence was not affected by host age at the time of parasitization, adult longevity as well as adult emergence pattern varied greatly depending upon the instar parasitized. When 3rd and 4th instar GHWFs were selected for oviposition, adult wasps lived significantly longer than when 1st or 2nd instars were used; also, there was a sharp emergence peak on the 2nd day after emergence was first observed (reduced or absent when 1st or 2nd instar GHWFs were parasitized) and the emergence period was reduced from between 8 and 11 days to 5 days. In general, the younger the host instar parasitized, the less synchronous was parasitoid development. Previous reports that E. formosa will not molt to the 2nd instar until the host has reached its 4th instar were not confirmed. When 1st instar host nymphs were parasitized, 2nd instar parasitoids were detected in 3rd instar hosts. Importantly, however, no matter which instar was parasitized, the parasitoid never molted to its last instar until the host had reached Stage 5 of its last instar, a stage in which host pharate adult formation has been initiated. It appears, then, that a condition(s) associated with host pharate adult formation is required for the parasitoid's final larval molt. Results reported here should facilitate the development of in vitro rearing systems for E. formosa.     

GLUT12 functions as a basal and insulin-independent glucose transporter in the heart

Glucose uptake from the bloodstream is the rate-limiting step in whole body glucose utilization, and is regulated by a family of membrane proteins called glucose transporters (GLUTs). Although GLUT4 is the predominant isoform in insulin-sensitive tissues, there is recent evidence that GLUT12 could be a novel second insulin-sensitive GLUT. However, its physiological role in the heart is not elucidated and the regulation of insulin-stimulated myocardial GLUT12 translocation is unknown. In addition, the role of GLUT12 has not been investigated in the diabetic myocardium. Thus, we hypothesized that, as for GLUT4, insulin regulates GLUT12 translocation to the myocardial cell surface, which is impaired during diabetes. Active cell surface GLUT (-4 and -12) content was quantified (before and after insulin stimulation) by a biotinylated photolabeled assay in both intact perfused myocardium and isolated cardiac myocytes of healthy and type 1 diabetic rodents. GLUT localization was confirmed by immunofluorescent confocal microscopy, and total GLUT protein expression was measured by Western blotting. Insulin stimulation increased translocation of GLUT-4, but not -12, in the healthy myocardium. Total GLUT4 content of the heart was decreased during diabetes, while there was no difference in total GLUT12. Active cell surface GLUT12 content was increased in the diabetic myocardium, potentially as a compensatory mechanism for the observed downregulation of GLUT4. Collectively, our data suggest that, in contrast to GLUT4, insulin does not mediate GLUT12 translocation, which may function as a basal GLUT located primarily at the cell surface in the myocardium.     

Comparative study of hydrogel-immobilized l-glutamate oxidases for a novel thick-film biosensor and its application in food samples

Novel, thick-film biosensors have been developed for the determination of l-glutamate in foodstuffs. The sensors were prepared by immobilization of l-glutamate oxidase by using polycarbamylsulfonate-hydrogel on a thick-film sensor. l-Glutamate oxidases obtained from Streptomyces sp. with different degree of purification were compared with their characteristic response to l-glutamate at different conditions and for their specificity, inhibition, and storage properties. These sensors were applied to determine monosodium glutamate in soy sauce samples and show good correlation with colorimetric method.     

Structure and Specificity of the Bacterial Cysteine Methyltransferase Effector NleE Suggests a Novel Substrate in Human DNA Repair Pathway

Enteropathogenic E. coli (EPEC) and related enterobacteria rely on a type III secretion system (T3SS) effector NleE to block host NF-κB signaling. NleE is a first in class, novel S-adenosyl-L-methionine (SAM)-dependent methyltransferase that methylates a zinc-coordinating cysteine in the Npl4-like Zinc Finger (NZF) domains in TAB2/3 adaptors in the NF-κB pathway, but its mechanism of action and other human substrates are unknown. Here we solve crystal structure of NleE-SAM complex, which reveals a methyltransferase fold different from those of known ones. The SAM, cradled snugly at the bottom of a deep and narrow cavity, adopts a unique conformation ready for nucleophilic attack by the methyl acceptor. The substrate NZF domain can be well docked into the cavity, and molecular dynamic simulation indicates that Cys673 in TAB2-NZF is spatially and energetically favorable for attacking the SAM. We further identify a new NleE substrate, ZRANB3, that functions in PCNA binding and remodeling of stalled replication forks at the DNA damage sites. Specific inactivation of the NZF domain in ZRANB3 by NleE offers a unique opportunity to suggest that ZRANB3-NZF domain functions in DNA repair processes other than ZRANB3 recruitment to DNA damage sites. Our analyses suggest a novel and unexpected link between EPEC infection, virulence proteins and genome integrity.     

Osteoclast Derivation from Mouse Bone Marrow

Osteoclasts are highly specialized cells that are derived from the monocyte/macrophage lineage of the bone marrow. Their unique ability to resorb both the organic and inorganic matrices of bone means that they play a key role in regulating skeletal remodeling. Together, osteoblasts and osteoclasts are responsible for the dynamic coupling process that involves both bone resorption and bone formation acting together to maintain the normal skeleton during health and disease.As the principal bone-resorbing cell in the body, changes in osteoclast differentiation or function can result in profound effects in the body. Diseases associated with altered osteoclast function can range in severity from lethal neonatal disease due to failure to form a marrow space for hematopoiesis, to more commonly observed pathologies such as osteoporosis, in which excessive osteoclastic bone resorption predisposes to fracture formation.An ability to isolate osteoclasts in high numbers in vitro has allowed for significant advances in the understanding of the bone remodeling cycle and has paved the way for the discovery of novel therapeutic strategies that combat these diseases. Here, we describe a protocol to isolate and cultivate osteoclasts from mouse bone marrow that will yield large numbers of osteoclasts.     

Production and interaction of oxygen and nitric oxide free radicals in PMA stimulated macrophages during the respiratory burst

The activity of nitric oxide synthase (NOS) during the respiratory burst in phorbol-1,2-myristate-1,3-acetate (PMA) stimulated macrophages has been the topic of much debate in the literature. To help clarify the role of NOS, we have examined the chemiluminescence arising from peroxynitrite production, nitrite/nitrate and nitric oxide production, and oxygen consumption during the respiratory burst in PMA-stimulated macrophages. The Griess reaction was used to measure nitrite/nitrate, spin trapping with N-methyl D-glucamine dithiocarbamate (MGD)2-Fe2+ was used to quantify nitric oxide, and the spin probe 2,2,6,6-tetramethylpiperidine-N-oxyl-4-ol (TEMPOL) was used to measure oxygen consumption. Oxygen free radical production (hydroxyl and superoxide free radicals) was also investigated using the spin trap 5,5-dimethyl-1-pyroline-1-oxide (DMPO). The chemiluminescence emitted by the PMA-stimulated macrophages and nitrite/nitrate in the culture system were both found to increase. However, the rate of nitric oxide release remained constant, indicating that the activity of NOS is not enhanced during the respiratory burst in PMA stimulated macrophages.     

Accurate,Fully-Automated NMR Spectral Profiling for Metabolomics

Many diseases cause significant changes to the concentrations of small molecules (a.k.a. metabolites) that appear in a person’s biofluids, which means such diseases can often be readily detected from a person’s “metabolic profile"—i.e., the list of concentrations of those metabolites. This information can be extracted from a biofluids Nuclear Magnetic Resonance (NMR) spectrum. However, due to its complexity, NMR spectral profiling has remained manual, resulting in slow, expensive and error-prone procedures that have hindered clinical and industrial adoption of metabolomics via NMR. This paper presents a system, BAYESIL, which can quickly, accurately, and autonomously produce a person’s metabolic profile. Given a 1D ¹H NMR spectrum of a complex biofluid (specifically serum or cerebrospinal fluid), BAYESIL can automatically determine the metabolic profile. This requires first performing several spectral processing steps, then matching the resulting spectrum against a reference compound library, which contains the “signatures” of each relevant metabolite. BAYESIL views spectral matching as an inference problem within a probabilistic graphical model that rapidly approximates the most probable metabolic profile. Our extensive studies on a diverse set of complex mixtures including real biological samples (serum and CSF), defined mixtures and realistic computer generated spectra; involving > 50 compounds, show that BAYESIL can autonomously find the concentration of NMR-detectable metabolites accurately (~ 90% correct identification and ~ 10% quantification error), in less than 5 minutes on a single CPU. These results demonstrate that BAYESIL is the first fully-automatic publicly-accessible system that provides quantitative NMR spectral profiling effectively—with an accuracy on these biofluids that meets or exceeds the performance of trained experts. We anticipate this tool will usher in high-throughput metabolomics and enable a wealth of new applications of NMR in clinical settings. BAYESIL is accessible at http://www.bayesil.ca.