Knottin cyclization: impact on structure and dynamics

Background  

Present in various species, the knottins (also referred to as inhibitor cystine knots) constitute a group of extremely stable miniproteins with a plethora of biological activities. Owing to their small size and their high stability, knottins are considered as excellent leads or scaffolds in drug design. Two knottin families contain macrocyclic compounds, namely the cyclotides and the squash inhibitors. The cyclotide family nearly exclusively contains head-to-tail cyclized members. On the other hand, the squash family predominantly contains linear members. Head-to-tail cyclization is intuitively expected to improve bioactivities by increasing stability and lowering flexibility as well as sensitivity to proteolytic attack.     

Use of passive radio frequency identification technologies to monitor nest usage in the northern carmine bee‐eater (Merops n. nubicus)

The use of radio frequency identification (RFID) technology is common in animal‐monitoring applications in the wild and in zoological and agricultural settings. RFID is used to track animals and to collect information about movements and other behaviors, as well as to automate or improve husbandry. Disney's Animal Kingdom® uses passive RFID technology to monitor nest usage by a breeding colony of northern carmine bee‐eaters. We implemented RFID technologies in various equipment configurations, initially deploying low‐frequency (LF) 125 kHz RFID and later changing to high‐frequency (HF) 13.56 MHz RFID technology, to monitor breeding behavior in the flock. We installed antennas connected to RFID readers at the entrances of nest tunnels to detect RFID transponders attached to leg bands as birds entered and exited tunnels. Both LF‐RFID and HF‐RFID systems allowed the characterization of nest visitation, including the timing of nest activity, breeding pair formation, identification of egg‐laying females, participation by nonresidents, and detection of nest disruptions. However, we collected a substantially larger volume of data using the increased bandwidth and polling speed inherent with HF‐RFID, which permitted tag capture of multiple birds simultaneously and resulted in fewer missed nest visits in comparison to LF‐RFID. Herein, we describe the evolution of the RFID setups used to monitor nest usage for more than 7 years, the types of data that can be gained using RFID at nests, and how we used these data to gain insights into carmine bee‐eater breeding behavior and improve husbandry.     

On the use of adenovirus dodecahedron as a carrier for glycoconjugate vaccines

Glycoconjugate Journal - Virus-Like Particles (VLPs) have been used as immunogenic molecules in numerous recombinant vaccines. VLPs can also serve as vaccine platform to exogenous antigens, usually...     

DGGE fingerprinting of bacteria in soils from eight ecologically different sites around Casey Station,Antarctica

Bacterial community structures in soils collected from eight sites around Casey Station, Antarctica, were investigated using denaturing gradient gel electrophoresis (DGGE) of amplified 16S rRNA gene fragments. Higher bacterial diversity was found in soils from protected or relatively low human-impacted sites in comparison to highly impacted sites. However, the highest diversity was detected in samples from Wilkes Tip, a former waste disposal site that has been undisturbed for the last 50 years. Comparison of community structure based on non-metric multidimensional scaling plots revealed that all sites, except the hydrocarbon-contaminated (oil spill) site, were clustered with a 45% similarity. A total of 23 partial 16S rRNA gene sequences were obtained from the excised DGGE bands, with the majority of the sequences closely related to those of the Cytophaga–Flexibacter–Bacteroides group. No significant correlation was established between environmental variables, including soil pH, electrical conductivity, carbon, nitrogen, water content and heavy metals, with bacterial diversity across the eight study sites.     

Prey preference of Orius sauteri between Western Flower Thrips and spider mites

Prey preference of polyphagous predators plays an important role in the suppression of various species of pest insects. In this study, the prey preference of Orius sauteri (Poppius) (Heteroptera: Anthocoridae) between deutonymphs of Tetranychus urticae Koch (Acari: Tetranychidae) and second instars of Frankliniella occidentalis (Pergande) (Thysanoptera: Thripidae), and the influence of spider mite density on the preference were examined in the laboratory in three-dimensional set-ups at 25 ± 1 °C and 70 ± 5% r.h. The results showed that the predatory bug in the presence of equal densities of the two prey species had a clear preference for thrips (preference index β: 0.86 ± 0.02), consuming 7.2 thrips larvae and 1.5 spider mite deutonymphs during the experimental periods of 6 h. The number of thrips consumed by O. sauteri did not change when the density ratio of thrips to spider mites was decreased from 1:1 to 1:5. Predation on spider mites increased when their ratio to thrips increased, but the disproportionate predation (i.e., the functional response) of O. sauteri towards spider mites resulted in a linear increase in the preference for F. occidentalis . The possible implications of these findings for the suppression of spider mites by O. sauteri in relation to the recent invasion of F. occidentalis into China are discussed.     

Influence of inoculum age on hybridoma culture kinetics

To determine the influence of the inoculum age on the kinetics of hybridoma growth and metabolism, spinner flasks have been inoculated with cells previously propagated in T flasks for 43, 52, 62 and 71 hr respectively. Increasing the age of the inoculum is found to result in a longer lag phase, in a lower maximum specific growth rate and in a reduced maximal cell density. During the growth phase specific rates of glucose and glutamine uptake and of ammonia and lactate production are similar. However, with the older inoculum, much higher metabolic activities are observed during the lag phase. The production of antibodies is delayed with increasing inoculum age, but the final antibody concentrations are similar, which indicates a higher specific antibody production rate when inoculating with older cells.     

Structural and functional studies on the interaction of GspC and GspD in the type II secretion system

Type II secretion systems (T2SSs) are critical for secretion of many proteins from Gram-negative bacteria. In the T2SS, the outer membrane secretin GspD forms a multimeric pore for translocation of secreted proteins. GspD and the inner membrane protein GspC interact with each other via periplasmic domains. Three different crystal structures of the homology region domain of GspC (GspC(HR)) in complex with either two or three domains of the N-terminal region of GspD from enterotoxigenic Escherichia coli show that GspC(HR) adopts an all-β topology. N-terminal β-strands of GspC and the N0 domain of GspD are major components of the interface between these inner and outer membrane proteins from the T2SS. The biological relevance of the observed GspC-GspD interface is shown by analysis of variant proteins in two-hybrid studies and by the effect of mutations in homologous genes on extracellular secretion and subcellular distribution of GspC in Vibrio cholerae. Substitutions of interface residues of GspD have a dramatic effect on the focal distribution of GspC in V. cholerae. These studies indicate that the GspC(HR)-GspD(N0) interactions observed in the crystal structure are essential for T2SS function. Possible implications of our structures for the stoichiometry of the T2SS and exoprotein secretion are discussed.     

Sensitivity to low-dose/low-LET ionizing radiation in mammalian cells harboring mutations in succinate dehydrogenase subunit C is governed by mitochondria-derived reactive oxygen species

It has been hypothesized that ionizing radiation-induced disruptions in mitochondrial O? metabolism lead to persistent heritable increases in steady-state levels of intracellular superoxide (O?(?U+2212)) and hydrogen peroxide (H?O?) that contribute to the biological effects of radiation. Hamster fibroblasts (B9 cells) expressing a mutation in the gene coding for the mitochondrial electron transport chain protein succinate dehydrogenase subunit C (SDHC) demonstrate increases in steady-state levels of O??- and H?O?. When B9 cells were exposed to low-dose/low-LET radiation (5-50 cGy), they displayed significantly increased clonogenic cell killing compared with parental cells. Clones derived from B9 cells overexpressing a wild-type human SDHC (T4, T8) demonstrated significantly increased surviving fractions after exposure to 5-50 cGy relative to B9 vector controls. In addition, pretreatment with polyethylene glycol-conjugated CuZn superoxide dismutase and catalase as well as adenoviral-mediated overexpression of MnSOD and/or mitochondria-targeted catalase resulted in significantly increased survival of B9 cells exposed to 10 cGy ionizing radiation relative to vector controls. Adenoviral-mediated overexpression of either MnSOD or mitochondria-targeted catalase alone was equally as effective as when both were combined. These results show that mammalian cells over expressing mutations in SDHC demonstrate low-dose/low-LET radiation sensitization that is mediated by increased levels of O??- and H?O?. These results also support the hypothesis that mitochondrial O??- and H?O? originating from SDH are capable of playing a role in low-dose ionizing radiation-induced biological responses.     

Impact of hyperthermic intraperitoneal chemotherapy on Hsp27 protein expression in serum of patients with peritoneal carcinomatosis

Despite the strong rationale for combining cytoreductive surgery (CRS) with hyperthermic intraperitoneal chemotherapy (HIPEC) in patients with peritoneal carcinomatosis, thermotolerance and chemoresistance might result from heat shock protein overexpression. The aim of the present study was thus to determine whether the heat shock protein 27 (Hsp27), a potential factor in resistance to treatment, could have a higher level in serum from patients under this combined therapy. Patients receiving CRS plus HIPEC for peritoneal carcinomatosis (group 1), patients with cancer or a history of cancer undergoing abdominal surgery (group 2), and patients without malignancies undergoing abdominal surgery (group 3) were included. Hsp27 serum levels were determined before and at different times following CRS and HIPEC using enzyme-linked immunosorbent assay. In group 1 (n = 25), the high Hsp27 levels, observed at the end of surgery compared with before (p < 0.0001), decreased during HIPEC, but remained significantly higher than before surgery (p < 0.0005). In groups 2 (n = 11) and 3 (n = 15), surgery did not significantly increase Hsp27 levels. A targeted molecular strategy, inhibiting Hsp27 expression in tumor tissue, could significantly reduce resistance to the combined CRS plus HIPEC treatment. This approach should be further assessed in a clinical phase I trial.