Immobilized metal ion affinity chromatography: a review on its applications

After 35?years of development, immobilized metal ion affinity chromatography (IMAC) has evolved into a popular protein purification technique. This review starts with a discussion of its mechanism and advantages. It continues with its applications which include the purification of histidine-tagged proteins, natural metal-binding proteins, and antibodies. IMAC used in conjunction with mass spectroscopy for phosphoprotein fractionation and proteomics is also covered. Finally, this review addresses the developments, limitations, and considerations of IMAC in the biopharmaceutical industry.     

Hippo Signaling Regulates Pancreas Development through Inactivation of Yap

The mammalian pancreas is required for normal metabolism, with defects in this vital organ commonly observed in cancer and diabetes. Development must therefore be tightly controlled in order to produce a pancreas of correct size, cell type composition, and physiologic function. Through negative regulation of Yap-dependent proliferation, the Hippo kinase cascade is a critical regulator of organ growth. To investigate the role of Hippo signaling in pancreas biology, we deleted Hippo pathway components in the developing mouse pancreas. Unexpectedly, the pancreas from Hippo-deficient offspring was reduced in size, with defects evident throughout the organ. Increases in the dephosphorylated nuclear form of Yap are apparent throughout the exocrine compartment and correlate with increases in levels of cell proliferation. However, the mutant exocrine tissue displays extensive disorganization leading to pancreatitis-like autodigestion. Interestingly, our results suggest that Hippo signaling does not directly regulate the pancreas endocrine compartment as Yap expression is lost following endocrine specification through a Hippo-independent mechanism. Altogether, our results demonstrate that Hippo signaling plays a crucial role in pancreas development and provide novel routes to a better understanding of pathological conditions that affect this organ.     

Water quality response to the Angora Fire, Lake Tahoe, California

The Angora Fire (summer of 2007) was the largest and most severe wildfire in recent history within the Lake Tahoe basin of the Sierra Nevada. To determine the watershed response and to assess the potential for downstream impacts of nutrient and sediment delivery to Lake Tahoe, we monitored the post-fire hydrology and stream water chemistry for 2 years at four locations along the length of Angora Creek, a perennial stream draining the burned watershed. When compared with unburned streams, the hydrology of Angora Creek indicated an earlier and faster melting of the spring snowpack. Peak stream water concentrations of total N (TN) and ammonium occurred within the burned area, whereas peak concentrations of nitrate (NO₃ ^?), total P, soluble reactive P, total suspended solids, turbidity, electrical conductivity (EC), and dissolved organic C occurred below the burned area. In comparison to pre-fire data, TN, NO₃ ^?, TP, total dissolved P, EC, and turbidity increased following the fire, particularly in the wetter second year. Yields for subwatershed areas suggest that the burned urban subwatershed was the largest source of nutrients and sediments, whereas the wet meadow subwatershed downstream of the burned area retained materials. Erosion control efforts, below-average annual precipitation and the timing of its arrival (absence of summer and fall rainstorms), and the existence of a wet meadow below the burned watershed likely reduced the negative impacts that would have been expected from such a severe wildfire.     

Comparative injection-site pain and tolerability of subcutaneous serum-free formulation of interferonβ-1a versus subcutaneous interferonβ-1b: results of the randomized,multicenter, Phase IIIb REFORMS study

Background

In patients with relapsing–remitting multiple sclerosis (RRMS), subcutaneous (sc) interferon (IFN)β-1a and IFNβ-1b have been shown to reduce relapse rates. A formulation of IFNβ-1a has been produced without fetal bovine serum and without human serum albumin as an excipient (not currently approved for use in the US). The objectives of this study were to evaluate tolerability, injection-site redness, subject-reported satisfaction with therapy, and clinical safety and efficacy of the serum-free formulation of IFNβ-1a versus IFNβ-1b in IFNβ-treatment-naïve patients with RRMS. The objectives of the extension phase were to evaluate long-term safety and tolerability of IFNβ-1a.

Methods

This randomized, parallel-group, open-label study was conducted at 27 clinical sites in the US. Eligible patients aged 18–60 years were randomized to receive either IFNβ-1a, titrated to 44 μg sc three times weekly (tiw) (n = 65), or IFNβ-1b, titrated to 250 μg sc every other day (n = 64) over 12 weeks. Following this, all patients received IFNβ-1a 44 μg tiw for 82–112 weeks. Primary endpoint was mean change in patient-reported pain, as assessed by visual analog scale (VAS) diary pain score (from 0 mm [no pain] to 100 mm [worst possible pain]) at the injection site, from pre-injection to 30 min post-injection over the first 21 full-dose injections. Secondary assessments included proportion of patients pain-free as recorded by VAS diary and the Short-Form McGill Pain questionnaire VAS.

Results

A total of 129 patients were included in the intent-to-treat analysis. Mean (standard deviation) change in VAS diary pain score was not significantly different between groups, although numerically lower with IFNβ-1a versus IFNβ-1b from pre-injection to immediately post-injection (1.46 [2.93] vs. 4.63 [10.57] mm), 10 min post-injection (0.70 [1.89] vs. 1.89 [5.75] mm), and 30 min post-injection (0.67 [2.32] vs. 1.14 [4.94] mm). Proportion of patients pain-free at all time periods post-injection was also not significantly different between groups. Adverse events were consistent with the known safety profiles of these treatments.

Conclusions

In IFNβ-treatment-naïve patients with RRMS, both the serum-free formulation of IFNβ-1a and IFNβ-1b treatments were generally accompanied by low-level injection-site pain and were well tolerated.

Trial registration

ClinicalTrials.gov NCT00428584

     

Identification of a General O-linked Protein Glycosylation System in Acinetobacter baumannii and Its Role in Virulence and Biofilm Formation

Acinetobacter baumannii is an emerging cause of nosocomial infections. The isolation of strains resistant to multiple antibiotics is increasing at alarming rates. Although A. baumannii is considered as one of the more threatening “superbugs” for our healthcare system, little is known about the factors contributing to its pathogenesis. In this work we show that A. baumannii ATCC 17978 possesses an O-glycosylation system responsible for the glycosylation of multiple proteins. 2D-DIGE and mass spectrometry methods identified seven A. baumannii glycoproteins, of yet unknown function. The glycan structure was determined using a combination of MS and NMR techniques and consists of a branched pentasaccharide containing N-acetylgalactosamine, glucose, galactose, N-acetylglucosamine, and a derivative of glucuronic acid. A glycosylation deficient strain was generated by homologous recombination. This strain did not show any growth defects, but exhibited a severely diminished capacity to generate biofilms. Disruption of the glycosylation machinery also resulted in reduced virulence in two infection models, the amoebae Dictyostelium discoideum and the larvae of the insect Galleria mellonella, and reduced in vivo fitness in a mouse model of peritoneal sepsis. Despite A. baumannii genome plasticity, the O-glycosylation machinery appears to be present in all clinical isolates tested as well as in all of the genomes sequenced. This suggests the existence of a strong evolutionary pressure to retain this system. These results together indicate that O-glycosylation in A. baumannii is required for full virulence and therefore represents a novel target for the development of new antibiotics.     

Unique arthropod communities on different host-plant genotypes results in greater arthropod diversity

Studies on the effect of plant-species diversity on various ecological processes has led to the study of the effects of plant-genetic diversity in the context of community genetics. Arthropod diversity can increase with plant-species or plant-genetic diversity (Wimp et al. in Ecol Lett 7:776–780, 2004). Plant diversity effects can be difficult to separate from other ecological processes, for example, complementarity. We asked three basic questions: (1) Are arthropod communities unique on different host-plant genotypes? (2) Is arthropod diversity greater when associated with greater plant-genetic diversity? (3) Are arthropod communities more closely associated with host-plant genetics than the plant neighborhood? We studied canopy arthropods on Populus fremontii trees randomly planted in a common garden. All trees were planted in a homogeneous matrix, which helped to reduce P. fremontii neighborhood effects. One sample was comprised of few P. fremontii genotypes with many clones. A second sample was comprised of many P. fremontii genotypes with few clones. A second data set was used to examine the relationships between the arthropod community with P. fremontii genetic composition and the neighborhood composition of the focal host plant. Unique arthropod communities were associated with different P. fremontii genotypes, and arthropod community diversity was greater in the sample with greater P. fremontii genotypic diversity. Arthropod community similarity was negatively correlated with P. fremontii genetic distance, but arthropod community similarity was not related to the neighborhood of the P. fremontii host plant.     

Structural determinants of species-selective substrate recognition in human and Drosophila serotonin transporters revealed through computational docking studies

To identify potential determinants of substrate selectivity in serotonin (5-HT) transporters (SERT), models of human and Drosophila serotonin transporters (hSERT, dSERT) were built based on the leucine transporter (LeuT(Aa)) structure reported by Yamashita et al. (Nature 2005;437:215-223), PBDID 2A65. Although the overall amino acid identity between SERTs and the LeuT(Aa) is only 17%, it increases to above 50% in the first shell of the putative 5-HT binding site, allowing de novo computational docking of tryptamine derivatives in atomic detail. Comparison of hSERT and dSERT complexed with substrates pinpoints likely structural determinants for substrate binding. Forgoing the use of experimental transport and binding data of tryptamine derivatives for construction of these models enables us to critically assess and validate their predictive power: A single 5-HT binding mode was identified that retains the amine placement observed in the LeuT(Aa) structure, matches site-directed mutagenesis and substituted cysteine accessibility method (SCAM) data, complies with support vector machine derived relations activity relations, and predicts computational binding energies for 5-HT analogs with a significant correlation coefficient (R = 0.72). This binding mode places 5-HT deep in the binding pocket of the SERT with the 5-position near residue hSERT A169/dSERT D164 in transmembrane helix 3, the indole nitrogen next to residue Y176/Y171, and the ethylamine tail under residues F335/F327 and S336/S328 within 4 A of residue D98. Our studies identify a number of potential contacts whose contribution to substrate binding and transport was previously unsuspected.     

Isolation and characterization of a protective bacterial culture isolated from honey active against American Foulbrood disease

The cell surface of mycobacteria is quite rich in lipids. Glycopeptidolipids, surface-exposed lipids that typify some mycobacterial species, have been associated with a phenotypic switch between rough and smooth colony morphotypes. This conversion in Mycobacterium smegmatis is correlated with the absence/presence of glycopeptidolipids on the cell surface and is due to insertion sequence mobility. Here, we show that the occurrence of a high amount of glycopeptidolipids in the smooth variant leads to lower invasion abilities and lower internalization by macrophages. We further show that the high production of glycopeptidolipids on the cell surface can confer a selective advantage to the smooth variant when grown in vitro . This higher fitness under the laboratory condition might explain the selection of smooth variants in several independent laboratories. The implications of these findings are discussed.     

Risk Assessment as a Decision-Making Tool for Treatment of Emissions at a New Aluminum Smelter in Iceland: 3. Ecological Assessment

Predictive ecological risk assessment was used to determine whether any risk-reduction benefit would result from the installation and operation of wet scrubbers at an aluminum smelter in East Iceland. Sulfur dioxide and hydrogen fluoride exposure will not result in any appreciable risk to mosses, lichens, lodgepole pine, and heather/heath grassland communities beyond the dilution zone without scrubbers. With scrubbers, exceedances of plant criteria may occur beyond the dilution zone. Critical concentrations of polycyclic aromatic hydrocarbons (PAHs) and hydrogen fluoride (HF) in terrestrial wildlife food items were converted to critical air concentrations, which were then compared to modeled air concentrations. Terrestrial wildlife species are not expected to be exposed to PAH concentrations that result in appreciable risk. If wildlife species are assumed to consume only grasses, predicted HF exposures will not result in exceedance of toxicity thresholds. However, if wildlife species are assumed to consume only heather, thresholds are exceeded at some locations for the rock ptarmigan and wood mouse. Results of population modeling indicate no potential for population impacts to rock ptarmigan outside the facility boundary. Impacts to wood mouse carrying capacity are expected to extend beyond the dilution zone with scrubbers, but not without.