Improvement of a Potential Anthrax Therapeutic by Computational Protein Design

Past anthrax attacks in the United States have highlighted the need for improved measures against bioweapons. The virulence of anthrax stems from the shielding properties of the Bacillus anthracis poly-γ-d-glutamic acid capsule. In the presence of excess CapD, a B. anthracis γ-glutamyl transpeptidase, the protective capsule is degraded, and the immune system can successfully combat infection. Although CapD shows promise as a next generation protein therapeutic against anthrax, improvements in production, stability, and therapeutic formulation are needed. In this study, we addressed several of these problems through computational protein engineering techniques. We show that circular permutation of CapD improved production properties and dramatically increased kinetic thermostability. At 45 °C, CapD was completely inactive after 5 min, but circularly permuted CapD remained almost entirely active after 30 min. In addition, we identify an amino acid substitution that dramatically decreased transpeptidation activity but not hydrolysis. Subsequently, we show that this mutant had a diminished capsule degradation activity, suggesting that CapD catalyzes capsule degradation through a transpeptidation reaction with endogenous amino acids and peptides in serum rather than hydrolysis.     

Seascape genetics of the stalked kelp Pterygophora californica and comparative population genetics in the Santa Barbara Channel

We conducted a population genetic analysis of the stalked kelp, Pterygophora californica, in the Santa Barbara Channel, California, USA. The results were compared with previous work on the genetic differentiation of giant kelp, Macrocystis pyrifera, in the same region. These two sympatric kelps not only share many life history and dispersal characteristics but also differ in that dislodged P. californica does not produce floating rafts with buoyant fertile sporophytes, commonly observed for M. pyrifera. We used a comparative population genetic approach with these two species to test the hypothesis that the ability to produce floating rafts increases the genetic connectivity among kelp patches in the Santa Barbara Channel. We quantified the association of habitat continuity and oceanographic distance with the genetic differentiation observed in stalked kelp, like previously conducted for giant kelp. We compared both overall (across all patches) and pairwise (between patches) genetic differentiation. We found that oceanographic transit time, habitat continuity, and geographic distance were all associated with genetic connectivity in P. californica, supporting similar previous findings for M. pyrifera. Controlling for differences in heterozygosity between kelp species using Jost's D_EST, we showed that global differentiation and pairwise differentiation were similar among patches between the two kelp species, indicating that they have similar dispersal capabilities despite their differences in rafting ability. These results suggest that rafting sporophytes do not play a significant role in effective dispersal of M. pyrifera at ecologically relevant spatial and temporal scales.     

Variability in the macrozooplankton community off the Antarctic Peninsula

Summary This paper presents an analysis of zooplankton net sampling surveys carried out during four expeditions to the Antarctic Peninsula region. Cluster analysis documented two to three site groupings for the epipelagic zone and one mesopelagic site cluster below 200 to 300 m depth. Analysis of species dominance, constancy, diversity and evenness indices did not allow clear designation and separation of communities in terms of these parameters.Computation of a rank correlation matrix for each season allowed the characterization of species groups. There were no perfect indicator species in the very strict sense. The main differences in the composition of the zooplankton between the site clusters were due mainly to changes in abundance rather than to presence or absence of particular species. However, the interpretation of the complex species and site groupings led to the conclusion that we can define three distinct communities: an oceanic, a neritic, and a mesopelagic community beneath 200 to 300 m. A so-called transitional cluster represents a mixing zone created by frequent occurrence of species from both the oceanic and neritic community. The location of the described oceanic and neritic community sites seem to be relatively stable with minor latitudinal changes during the seasons, while occurrence and abundance of most species changes with the time of the year. The usefulness of particular species (e.g. Euphausia superba) as indicator species also change during the year     

Polyribosome disaggregation and cell-free protein synthesis in preparations from cerebral cortex of hyperphenylalaninemic rats

Abstract— Seven-day-old rats were injected intraperitoneally with l -phenylalanine (1 g/kg) and the time course of brain polyribosome disaggregation and changes in brain levels of phenylalanine, tryptophan and tyrosine were determined. Disaggregation of brain polyribosomes preceded the increase in levels of phenylalanine in brain, and followed the same time course as depletion of tryptophan from brain. The effects of several metabolites of phenylalanine (which are formed in phenylketonuria) on protein synthesis in vitro was determined for brain and liver systems. None of the compounds tested was inhibitory at concentrations below 10 mM and in all cases hepatic protein synthesis was more sensitive to inhibition than was the corresponding system from brain. Ribosomal dimers, formed in brain after injection of phenylalanine, were incapable of supporting high levels of protein synthesis in vitro, a finding that suggested that the inhibition of protein synthesis in vitro in cell-free systems of brain tissue after injection of phenylalanine into young rats was mediated by disaggregation of brain polyribosomes associated with tryptophan deficiency in brain.     

Chemotactic peptide, calcium and guanine nucleotide regulation of phospholipase C activity in membranes from DMSO-differentiated HL60 cells

Membranes prepared from DMSO-differentiated HL60 cells labeled with [3H]inositol hydrolyze polyphosphoinositides in a Ca2+-dependent manner, generating inositol 1,4-bisphosphate (IP2) and inositol 1,4,5-trisphosphate (IP3). Incubation of membranes with GTP or GTP gamma S reduces the concentration of Ca2+ required for activation. This nucleotide effect is potentiated by formyl-Met-Leu-Phe (FMLP). Pertussis toxin inhibits FMLP-induced augmentation, but not the induction of IP2/IP3 formation by GTP or GTP gamma S. These results suggest that differentiated HL60 cells contain a membrane-associated phospholipase C that degrades polyphosphoinositides and that activation of this enzyme is mediated by at least two guanine nucleotide binding proteins, one of which is linked to FMLP receptors and is pertussis toxin sensitive.     

Synthesis and SAR of 2-aryl-3-aminomethylquinolines as agonists of the bile acid receptor TGR5

Optimization of a screening hit from uHTS led to the discovery of TGR5 agonist 32, which was shown to have activity in a rodent model for diabetes.     

CURRICULUM GUIDE FOR RESEARCH ETHICS WORKSHOPS FOR COUNTRIES IN THE MIDDLE EAST

To help ensure the ethical conduct of research, many have recommended educational efforts in research ethics to investigators and members of research ethics committees (RECs). One type of education activity involves multi‐day workshops in research ethics. To be effective, such workshops should contain the appropriate content and teaching techniques geared towards the learning styles of the targeted audiences. To ensure consistency in content and quality, we describe the development of a curriculum guide, core competencies and associated learning objectives and activities to help educators organize research ethics workshops in their respective institutions. The curriculum guide is divided into modular units to enable planners to develop workshops of different lengths and choose content materials that match the needs, abilities, and prior experiences of the target audiences. The content material in the curriculum guide is relevant for audiences in the Middle East, because individuals from the Middle East who participated in a Certificate Program in research ethics selected and developed the training materials (e.g., articles, powerpoint slides, case studies, protocols). Also, many of the activities incorporate active‐learning methods, consisting of group work activities analyzing case studies and reviewing protocols. The development of such a workshop training curriculum guide represents a sustainable educational resource to enhance research ethics capacity in the Middle East.     

Soluble polyvinylpyrrolidine and bovine serum albumin adsorb polyphenols from soybean suspension cultures

Soybean (Glycine max [L.] Merrill, cv. Dare) suspension cultures grown in Gamborg B5 medium became discolored and the cells began aggregating after 1 week in culture, especially in the absence of 2,4-dichlorophenoxyacetic acid (2,4-D). The addition of either soluble polyvinylpyrrolidine (PVP) or bovine serum albumin (BSA) to cultures grown in Gamborg B5 medium with 2,4-D prevents discoloration and cell aggregation by adsorbing excess polyphenols from the cells. Transfer of the PVP-treated cultures to fresh medium without 2,4-D stimulated the recurrence of excess polyphenols. Cultures pretreated with BSA did not develop excess polyphenols when transferred to fresh 2,4-D-free medium. Addition of either PVP or BSA to cultures grown in the absence of 2,4-D was found to inhibit growth.