Monkeypox virus viral chemokine inhibitor (MPV vCCI), a potent inhibitor of rhesus macrophage inflammatory protein-1

Monkeypox virus (MPV) is an orthopoxvirus with considerable homology to variola major, the etiologic agent of smallpox. Although smallpox was eradicated in 1976, the outbreak of MPV in the U.S. highlights the health hazards associated with zoonotic infections. Like other orthopoxviruses, MPV encodes a secreted chemokine binding protein, vCCI that is abundantly expressed and secreted from MPV infected cells. EMSA data shows vCCI efficiently binds rhesus MIP-1α (rhMIP-1α) at near one to one stoichiometry. In vitro chemotaxis experiments demonstrate that vCCI completely inhibits rhMIP-1α mediated chemotaxis, while in vivo recruitment assays in rhesus macaques using chemokine-saturated implants show a decrease in the number of CD14⁺ cells responding to rhMIP-1α when vCCI is present, suggesting vCCI is effectively inhibiting chemokine function both in vitro and in vivo. More importantly, we demonstrate that vCCI can diminish the severity of the acute phase and completely inhibit the relapsing phase of experimental allergic encephalomyelitis (EAE) disease. These data represent the first in vitro and in vivo characterization of vCCI emphasizing its function as a potent inhibitor of rhMIP-1α. Furthermore, the ability of vCCI to inhibit relapsing EAE disease represents a novel therapeutic approach for treating chemokine-mediated diseases.     

Comparative proteomics of human monkeypox and vaccinia intracellular mature and extracellular enveloped virions.

Orthopoxviruses are among the largest and most complex of the animal viruses. In response to the recent emergence of monkeypox in Africa and the threat of smallpox bioterrorism, two orthopoxviruses with different pathogenic potentials, human monkeypox virus and vaccinia virus, were proteomically compared with the goal of identifying proteins required for pathogenesis. Orthopoxviruses were grown in HeLa cells to two different viral forms (intracellular mature virus and extracellular enveloped virus), purified by sucrose gradient ultracentrifugation, denatured using RapiGest surfactant, and digested with trypsin. Unfractionated samples and strong cation exchange HPLC fractions were analyzed by high-resolution reversed-phase nano-LC-MS/MS, and analyses of the MS/MS spectra using SEQUEST and X! Tandem resulted in the confident identification of hundreds of monkeypox, vaccinia, and copurified host-cell proteins. The unfractionated samples were additionally analyzed by LC-MS using an LTQ-Orbitrap, and the accurate mass and elution time tag approach was used to perform quantitative comparisons. Possible pathophysiological roles of differentially abundant Orthopoxvirus proteins are discussed. Data, processed results, and protocols are available at http://www.proteomicsresource.org/.     

The Influence of Physical Habitat,Pyrethroids, and Metals on Benthic Community Condition in an Urban and Residential Stream in California

This study was designed to characterize benthic communities and physical habitat in both an urban (Kirker Creek) and residential (Pleasant Grove Creek) stream in California in late spring of 2006 and 2007. Concurrent water quality evaluations, physical sediment parameters, pyrethroids, bulk metals, and SEM/AVS ratios were also measured during both years of this study. The relationship of various benthic metrics to physical habitat metrics, pyrethroids, and metals was evaluated for each stream using stepwise multiple linear regressions with both years combined for each stream, as well as both years and both streams combined, to increase the statistical power for determining significant relationships. Physical habitat was determined to be poor in each stream during both years of sampling. More than 100 benthic taxa were reported annually for both streams based on 2006 and 2007 sampling. A significant result from the stepwise regression analysis combining data for 2 years across both streams is that when habitat metrics and to a lesser degree metals are considered in the statistical models pyrethroids do not display any significant relationships to the benthic metrics. In summary, it is apparent from this analysis that the health of benthic communities in both streams is primarily affected by habitat metrics.     

FogBank: a single cell segmentation across multiple cell lines and image modalities

Background

Many cell lines currently used in medical research, such as cancer cells or stem cells, grow in confluent sheets or colonies. The biology of individual cells provide valuable information, thus the separation of touching cells in these microscopy images is critical for counting, identification and measurement of individual cells. Over-segmentation of single cells continues to be a major problem for methods based on morphological watershed due to the high level of noise in microscopy cell images. There is a need for a new segmentation method that is robust over a wide variety of biological images and can accurately separate individual cells even in challenging datasets such as confluent sheets or colonies.

Results

We present a new automated segmentation method called FogBank that accurately separates cells when confluent and touching each other. This technique is successfully applied to phase contrast, bright field, fluorescence microscopy and binary images. The method is based on morphological watershed principles with two new features to improve accuracy and minimize over-segmentation.First, FogBank uses histogram binning to quantize pixel intensities which minimizes the image noise that causes over-segmentation. Second, FogBank uses a geodesic distance mask derived from raw images to detect the shapes of individual cells, in contrast to the more linear cell edges that other watershed-like algorithms produce.We evaluated the segmentation accuracy against manually segmented datasets using two metrics. FogBank achieved segmentation accuracy on the order of 0.75 (1 being a perfect match). We compared our method with other available segmentation techniques in term of achieved performance over the reference data sets. FogBank outperformed all related algorithms. The accuracy has also been visually verified on data sets with 14 cell lines across 3 imaging modalities leading to 876 segmentation evaluation images.

Conclusions

FogBank produces single cell segmentation from confluent cell sheets with high accuracy. It can be applied to microscopy images of multiple cell lines and a variety of imaging modalities. The code for the segmentation method is available as open-source and includes a Graphical User Interface for user friendly execution.

Electronic supplementary material

The online version of this article (doi:10.1186/s12859-014-0431-x) contains supplementary material, which is available to authorized users.     

High-throughput screening for developability during early-stage antibody discovery using self-interaction nanoparticle spectroscopy

The discovery of monoclonal antibodies (mAbs) that bind to a particular molecular target is now regarded a routine exercise. However, the successful development of mAbs that (1) express well, (2) elicit a desirable biological effect upon binding, and (3) remain soluble and display low viscosity at high concentrations is often far more challenging. Therefore, high throughput screening assays that assess self-association and aggregation early in the selection process are likely to yield mAbs with superior biophysical properties. Here, we report an improved version of affinity-capture self-interaction nanoparticle spectroscopy (AC-SINS) that is capable of screening large panels of antibodies for their propensity to self-associate. AC-SINS is based on concentrating mAbs from dilute solutions around gold nanoparticles pre-coated with polyclonal capture (e.g., anti-Fc) antibodies. Interactions between immobilized mAbs lead to reduced inter-particle distances and increased plasmon wavelengths (wavelengths of maximum absorbance), which can be readily measured by optical means. This method is attractive because it is compatible with dilute and unpurified mAb solutions that are typical during early antibody discovery. In addition, we have improved multiple aspects of this assay for increased throughput and reproducibility. A data set comprising over 400 mAbs suggests that our modified assay yields self-interaction measurements that are well-correlated with other lower throughput assays such as cross-interaction chromatography. We expect that the simplicity and throughput of our improved AC-SINS method will lead to improved selection of mAbs with excellent biophysical properties during early antibody discovery.     

The detection and characterization of bacteria-sized protists in “Protist-free” filtrates and their potential impact on experimental marine ecology

Nuclepore filters of 0.6–1.0m pore size have been used to prepare protist-free water for a number of studies in microbial ecology. This procedure has been called into question by a recent study claiming that a significant portion of bacterial loss in filtrates could be due to uncharacterized predators passing through 0.6m filters. We were unable to directly observe protists in 0.6m filtrates using phase contrast, epifluorescence, or transmission electron microscopy. Using the culture techniques of rice grain enrichment and most probable number, however, we were able to observe and quantify several species of bacterivorous nanoflagellates that developed not only in 0.6m, but also in 0.4m seawater filtrates. The ability of predacious nanoflagellates to squeeze through bacteria-sized pores questions studies of bacterial production and chemical cycling that have assumed protist-free filtrates.     

Palmitylation of neuromodulin (GAP-43) is not required for phosphorylation by protein kinase C.

Neuromodulin (also designated GAP-43, B-50, and F-1) is a prominent protein kinase C substrate attached to the membranes of neuronal growth cones during development and to presynaptic membranes in discrete subsets of adult synapses. In this study, we have examined the relationship between the attachment of neuromodulin to membranes and its phosphorylation by protein kinase C. To address this issue, we have compared wild-type and mutant neuromodulins expressed in cells that normally lack the protein. Wild-type neuromodulin expressed in Chinese hamster ovary cells was associated with membranes, incorporated [3H]palmitic acid, and was phosphorylated in response to phorbol ester treatment. Substitution of serine 41, the in vitro protein kinase C site, abolished the phorbol ester response, indicating that serine 41 serves as the sole protein kinase C phosphorylation site in vivo. Substitution of the putative fatty acylation sites, cysteines 3 and 4, abolished membrane association as well as [3H]palmitic acid labeling of neuromodulin. Fatty acylation therefore appears to serve as the mechanism for anchoring neuromodulin to membranes. Surprisingly, the soluble cysteine substitution mutant was phosphorylated by protein kinase C at a rate indistinguishable from that of the wild-type protein. Therefore, membrane association may not be required for the phosphorylation of neuromodulin by protein kinase C.     

Some observations on the primitive and definitive erythrocytes of the developing chick

Summary The cytological changes in the primitive and definitive erythrocytes of the incubating chick have been followed. Observations have been made on the nucleoli, vital granules, mitochondria,Golgi apparatus, reticulum ofSinigaglia and the reticulation patterns of the basophilic substance. The cells of the primitive and definitive lines are ordinarily readily distinguished from one another. Data are included on the rate of disappearance of the primitive cells from the circulation. They may persist as long as two weeks after hatching. Giant primitive erythrocytes are common during the first week of incubation. The cells have one, two three or four nuclei. The nuclearplasma relationship is maintained somewhere near a constant. These atypical cells are due to aberrations in mitosis. Data on the percentage of mitosis in both types of erythrocytes are also included. The initial activity of the spleen and bone-marrow is reflected in the blood stream. There is a distinct rise in the proportion of young definitive erythrocytes. An attempt is made to correlate the findings ofHall (1934) on the changing affinity of the hemoglobin for oxygen with the changing blood picture. The primitive line does not persist long enough to account for the phenomenon. It is suggested, however, that the hemoglobin of the erythrocytes produced by the yolk sac may differ from that of the cells produced by the spleen and bone-marrow. With Plates I–III.     

BOOK REVIEWS

Book reviewed in this article:
CENTRAL AND SOUTH AMERICA: Preliminary Report on the Smithsonian Institution-Harvard University Archaeological Expedition to Northwestern Honduras, 1936 . W illiam D uncan S trong , A lfred K idder II and A. J. D rexel P aul , J r .
CENTRAL AND SOUTH AMERICA: An Archaeological Reconnaissance of Northwestern Honduras: A Report of the Work of the Tulane University-Danish National Museum Expedition to Central America 1935 . J ens Y de .