Use of Ultra-short Columns for Therapeutic Protein Separations, Part 2: Designing the Optimal Column Dimension for Reversed-Phase Liquid Chromatography.

In the first part of the series, it was demonstrated that very fast (<30 s) separations of therapeutic protein species are feasible using ultra-short (5 × 2.1 mm) columns. In the second part, our purpose was to find the appropriate column length; therefore, a systematic study was performed using various custom-made prototype reversed-phase liquid chromatography (RPLC) columns ranging from 2 to 50 mm lengths. It was found that on a low dispersion ultrahigh-pressure liquid chromatography instrument, columns between 10 and 20 mm were most effective when made with 2.1 mm i.d. tubing. However, with the same LC instrument, 3 mm i.d. columns as short as ∼5 to 10 mm could be effectively used. In both cases, it has been found to be best to keep injection volumes below 0.6 µL, which presents a potential limit to further decreasing column length, given the current capabilities of autosampler instrumentation. The additional volume of the column hardware outside of the packed bed (extra-bed volume) of very small columns is also a limiting factor to decrease the column length. For columns shorter than 10 mm, columns' extra-bed volume was seen to make considerable contributions to band broadening. However, the use of ultra-short columns seemed to be a very useful approach for RPLC of large proteins (>25 kDa) and could also work well for ∼12 kDa as the lowest limit of molecular mass. In summary, a renewed interest in the use of ultra-short columns is warranted, and additional method development will be to the benefit of the biopharmaceutical industry as there is an ever-increasing demand for faster, yet accurate assays (e.g., high-throughput screening) of proteins.

Atlas of Ohio Aquatic Insects: Volume II, Plecoptera.

BACKGROUND: We provide volume II of a distributional atlas of aquatic insects for the eastern USA state of Ohio. This treatment of stoneflies (Plecoptera) is companion to Armitage et al. (2011) on caddisflies (Trichoptera). We build on a recent analysis of Ohio stonefly diversity patterns based on large drainages (DeWalt et al. 2012), but add 3717 new records to the data set. We base most analyses on the United States Geological Survey Hierarchical Unit Code eight (HUC8) drainage scale. In addition to distributional maps for each species, we provide analyses of species richness versus HUC8 drainage area and the number of unique locations in a HUC8 drainage, species richness versus Ohio counties, analyze adult presence phenology throughout the year, and demonstrate stream size range affiliation for each species. NEW INFORMATION: This work is based on a total of 7797 specimen records gathered from 21 regional museums, agency data, personal collections, and from the literature Table 1. To our knowledge this is the largest stonefly data set available for a similarly sized geopolitical area anywhere in the world. These data are made available as a Darwin Core Archive supported by the Pensoft Integrated Publishing Toolkit (DeWalt et al. 2016b). All known published papers reporting stoneflies from Ohio are detailed in Suppl. material 1. We recovered 102 species from Ohio, including all nine Nearctic families Table 2​. Two species were removed from the DeWalt et al. (2012) list and two new state records added. Perlidae (32 spp.) was most speciose, compared to the low diversity Pteronarcyidae (2 spp.) and Peltoperlidae (1 sp.). The richest HUC8 drainages occurred in northeastern, south-central, and southern regions of the state where drainages were heavily forested, had the highest slopes, and were contained within or adjacent to the unglaciated Allegheny and Appalachian Plateaus. Species poor drainages occurred mainly in the northwestern region where Wisconsinan aged lake plains climaxed to an expansive wooded wetland, the Black Swamp. The unglaciated Lower Scioto drainage (72 spp.) in south-central Ohio supported the greatest species richness. There was no relationship between species richness and HUC8 drainage size, but the number of unique locations in a drainage strongly related to species richness. All Ohio counties were represented in the data set with Hocking County (59 spp.) of the Lower Scioto drainage being the richest and most heavily sampled. Adult presence phenology was influenced by phylogenetic relationships such that the superfamily Nemouroidea (Capniidae, Leuctridae, Nemouridae, and Taeniopterygidae) generally emerged in winter and spring while the superfamilies Pteronarcyoidea (Pteronarcyidae, Peltoperlidae) and Perloidea (Chloroperlidae, Perlidae, Perlodidae) emerged later, some species continuing emergence through summer months. Species often occupied specific stream size ranges, while others were generalists. Two species once histrorically abundant in the western Lake Erie Bass Islands no longer reside there. Each of the 102 species is discussed in detail, including several that require additional collecting efforts to confirm their identities, presence, and distribution in Ohio.

Differential regulatory T cell activity in HIV type 1-exposed seronegative individuals.

The potential role of conventional and regulatory T cells (Tregs) in protection from HIV-1 infection remains unclear. To address this question, we analyzed samples from 129 HIV-1-exposed seronegative individuals (HESN) from an HIV-1-serodiscordant couples cohort. To assess the presence of HIV-specific T cell responses and Treg function, we measured the proliferation of T cells in response to HIV-1 peptide pools in peripheral blood mononuclear cells (PBMCs) and PBMCs depleted of Tregs. We identified HIV-specific CD4(+) and CD8(+) T cell responses and, surprisingly, the overall CD4(+) and CD8(+) T cell response rate was not increased when Tregs were removed from cell preparations. Of the 20 individuals that had HIV-1-specific CD4(+) T cell responses, only eight had Tregs that could suppress this proliferation. When compared with individuals whose Tregs could suppress HIV-1-specific CD4(+) T cell proliferation, individuals with Tregs unable to suppress showed a trend toward increased T cell activation and Treg frequency and a significant increase in HIV-1-specific production of microphage inflammatory protein-1ß (MIP-1ß) by CD4(+) T cells, autocrine production of which has been shown to be protective in terms of HIV-1 infection of CD4(+) T cells.

Sequence similarity between the erythrocyte binding domain of the Plasmodium vivax Duffy binding protein and the V3 loop of HIV-1 strain MN reveals a functional heparin binding motif involved in binding to the Duffy antigen receptor for chemokines.

BACKGROUND: The HIV surface glycoprotein gp120 (SU, gp120) and the Plasmodium vivax Duffy binding protein (PvDBP) bind to chemokine receptors during infection and have a site of amino acid sequence similarity in their binding domains that often includes a heparin binding motif (HBM). Infection by either pathogen has been found to be inhibited by polyanions. RESULTS: Specific polyanions that inhibit HIV infection and bind to the V3 loop of X4 strains also inhibited DBP-mediated infection of erythrocytes and DBP binding to the Duffy Antigen Receptor for Chemokines (DARC). A peptide including the HBM of PvDBP had similar affinity for heparin as RANTES and V3 loop peptides, and could be specifically inhibited from heparin binding by the same polyanions that inhibit DBP binding to DARC. However, some V3 peptides can competitively inhibit RANTES binding to heparin, but not the PvDBP HBM peptide. Three other members of the DBP family have an HBM sequence that is necessary for erythrocyte binding, however only the protein which binds to DARC, the P. knowlesi alpha protein, is inhibited by heparin from binding to erythrocytes. Heparitinase digestion does not affect the binding of DBP to erythrocytes. CONCLUSION: The HBMs of DBPs that bind to DARC have similar heparin binding affinities as some V3 loop peptides and chemokines, are responsible for specific sulfated polysaccharide inhibition of parasite binding and invasion of red blood cells, and are more likely to bind to negative charges on the receptor than cell surface glycosaminoglycans.

Sequence similarity between the erythrocyte binding domain 1 of the Plasmodium vivax Duffy binding protein and the V3 loop of HIV-1 strain MN reveals binding residues for the Duffy Antigen Receptor for Chemokines.

BACKGROUND: The surface glycoprotein (SU, gp120) of the human immunodeficiency virus (HIV) must bind to a chemokine receptor, CCR5 or CXCR4, to invade CD4+ cells. Plasmodium vivax uses the Duffy Binding Protein (DBP) to bind the Duffy Antigen Receptor for Chemokines (DARC) and invade reticulocytes. RESULTS: Variable loop 3 (V3) of HIV-1 SU and domain 1 of the Plasmodium vivax DBP share a sequence similarity. The site of amino acid sequence similarity was necessary, but not sufficient, for DARC binding and contained a consensus heparin binding site essential for DARC binding. Both HIV-1 and P. vivax can be blocked from binding to their chemokine receptors by the chemokine, RANTES and its analog AOP-RANTES. Site directed mutagenesis of the heparin binding motif in members of the DBP family, the P. knowlesi alpha, beta and gamma proteins abrogated their binding to erythrocytes. Positively charged residues within domain 1 are required for binding of P. vivax and P. knowlesi erythrocyte binding proteins. CONCLUSION: A heparin binding site motif in members of the DBP family may form part of a conserved erythrocyte receptor binding pocket.