Structure-function studies of an engineered scaffold protein derived from Stefin A. II: Development and applications of the SQT variant

Constrained binding peptides (peptide aptamers) may serve as tools to explore protein conformations and disrupt protein-protein interactions. The quality of the protein scaffold, by which the binding peptide is constrained and presented, is of crucial importance. SQT (Stefin A Quadruple Mutant-Tracy) is our most recent development in the Stefin A-derived scaffold series. Stefin A naturally uses three surfaces to interact with its targets. SQT tolerates peptide insertions at all three positions. Peptide aptamers in the SQT scaffold can be expressed in bacterial, yeast and human cells, and displayed as a fusion to truncated pIII on phage. Peptides that bind to CDK2 can show improved binding in protein microarrays when presented by the SQT scaffold. Yeast two-hybrid libraries have been screened for binders to the POZ domain of BCL-6 and to a peptide derived from PBP2', specific to methicillin-resistant Staphylococcus aureus. Presentation of the Noxa BH3 helix by SQT allows specific interaction with Mcl-1 in human cells. Together, our results show that Stefin A-derived scaffolds, including SQT, can be used for a variety of applications in cellular and molecular biology. We will henceforth refer to Stefin A-derived engineered proteins as Scannins.     

Rosuvastatin Enhances the Catabolism of LDL apoB‐100 in Subjects with Combined Hyperlipidemia in a Dose Dependent Manner

Dose‐associated effects of rosuvastatin on the metabolism of apolipoprotein (apo) B‐100 in triacylglycerol rich lipoprotein (TRL, d < 1.019 g/ml) and low density lipoprotein (LDL) and of apoA‐I in high density lipoprotein (HDL) were assessed in subjects with combined hyperlipidemia. Our primary hypothesis was that maximal dose rosuvastatin would decrease the apoB‐100 production rate (PR), as well as increase apoB‐100 fractional catabolic rate (FCR). Eight subjects received placebo, rosuvastatin 5 mg/day, and rosuvastatin 40 mg/day for 8 weeks each in sequential order. The kinetics of apoB‐100 in TRL and LDL and apoA‐I in HDL were determined at the end of each phase using stable isotope methodology, gas chromatography‐mass spectrometry, and multicompartmental modeling. Rosuvastatin at 5 and 40 mg/day decreased LDL cholesterol by 44 and 54 % (both P < 0.0001), triacylglycerol by 14 % (ns) and 35 % (P < 0.01), apoB by 30 and 36 % (both P < 0.0001), respectively, and had no significant effects on HDL cholesterol or apoA‐I levels. Significant decreases in plasma markers of cholesterol synthesis and increases in cholesterol absorption markers were observed. Rosuvastatin 5 and 40 mg/day increased TRL apoB‐100 FCR by 36 and 46 % (both ns) and LDL apoB‐100 by 63 and 102 % (both P < 0.05), respectively. HDL apoA‐I PR increased with low dose rosuvastatin (12 %, P < 0.05) but not with maximal dose rosuvastatin. Neither rosuvastatin dose altered apoB‐100 PR or HDL apoA‐I FCR. Our data indicate that maximal dose rosuvastatin treatment in subjects with combined hyperlipidemia resulted in significant increases in the catabolism of LDL apoB‐100, with no significant effects on apoB‐100 production or HDL apoA‐I kinetics.     

Morphological,mechanical, and thermal characterization of biopolymer composites based on polylactide and nanographite platelets

This work focuses on development and optimization of polylactide (PLA) and nanographite platelets (NGP) based composites to display possible superior mechanical and improved thermal stability. Melt blending and dry mixing methods of fabrication were employed at temperature of 180°C. Different Loading fractions of NGP were incorporated into polymer matrix. Morphological evaluation techniques such as XRD and TEM were applied to determine the degree of dispersion of NGPs into PLA matrix. Mechanical properties were evaluated and correlated to structural morphologies of PLA/NGP composites. Thermal properties of composites were studied to examine possible changes in T_g, T_c, T_m, and percentage crystallinity of these composites. The effect of mixing was also explored through double extrusion of some samples. It was concluded that composites containing 3 wt% NGP showed optimum mechanical performance without any significant changes in the thermal characteristics. POLYM. COMPOS., 2012. © 2012 Society of Plastics Engineers     

Modeling of ultrasonic processing

Curing of fiber‐reinforced thermoset polymer composites requires an elevated temperature to accelerate the crosslinking reaction and also hydrostatic pressure to consolidate the part and suppress the formation of voids. These processing conditions can be provided by autoclaves of appropriate size, but these are expensive and sometimes difficult to schedule. Ultrasonic debulking followed by oven cure is an attractive alternative to autoclave cure. In this technique a movable “horn” driven at ultrasonic frequency is applied to the surface of the uncured part. This generates pressure and at the same time produces heating by viscoelastic dissipation. The part can be debulked to net shape and staged through the action of the ultrasound. There are a large enough number of experimental parameters in ultrasonic debulking and staging to make purely empirical process optimization difficult, and this paper outlines numerical simulation methods useful in understanding and developing the process. © 2004 Wiley Periodicals, Inc. J Appl Polym Sci 93: 1609–1615, 2004     

Orientation and Feeding Responses of the Pollen Beetle, Meligethes aeneus, to Candytuft, Iberis amara

The pollen beetle, Meligethes aeneus, which is an important pest of oilseed rape, Brassica napus, and turnip rape, B. rapa var. campestris, does not oviposit in all species of the Brassicaceae. The relationship between M. aeneus and candytuft, Iberis amara (Brassicacae), was investigated as part of chemical ecological studies into the development of control methods employing non-host-derived repellents. In choice and nonchoice feeding tests, M. aeneus completely rejected I. amara. However, in a field experiment using traps baited with flowering racemes of I. amara and B. napus, M. aeneus was attracted to both species. Gas chromatographic (GC) and GC-electroantennogram (GC-EAG) analyses indicated that the profiles of the floral volatiles of the two species are different. At least 12 compounds among the I. amara floral volatiles were detected by the M. aeneus antenna, and, of these, hexanoic acid, (E)-4,8-dimethyl-1,3,7-nonatriene and alpha-cedrene were not found among B. napus flower volatiles. Since M. aeneus is stimulated by floral volatiles to approach I. amara, but rejects it near, or at, the plant surface, I. amara does not produce repellents that could be used to manipulate M. aeneus. However, it may contain feeding deterrent(s) that could be used in "push-pull" control techniques or in the development of resistant brassicaceous crops.     

Comparison of the bligh and dyer and folch methods for total lipid determination in a broad range of marine tissue

For many studies, it is important to measure the total lipid content of biological samples accurately. The Bligh and Dyer method of extraction was developed as a rapid but effective method for determining total lipid content in fish muscle. However, it is also widely used in studies measuring total lipid content of whole fish and other tissues. Although some investigators may have used modified Bligh and Dyer procedures, rarely have modifications been specified nor has their effectiveness been quantitatively evaluated. Thus, we compared this method with that of the classic Folch extraction in determining total lipid content of fish samples ranging from 0.5 to 26.6% lipid. We performed both methods as originally specified, i.e., using the chloroform/methanol/water ratios of 1:2:0.8 and 2:2:1.8 (before and after dilution, respectively) for Bligh and Dyer and of 8:4:3 for Folch, and with the initial solvent/sample ratios of (3+1):1 (Bligh and Dyer) and 20:1 (Folch). We also compared these with several other solvent/sample ratios. In samples containing <2% lipid, the results of the two methods did not differ. However, for samples containing >2% lipid, the Bligh and Dyer method produced significantly lower estimates of lipid content, and this underestimation increased significantly with increasing lipid content of the sample. In the highest lipid samples, lipid content was underestimated by up to 50% using the Bligh and Dyer method. However, we found a highly significant linear relationship between the two methods, which will permit the correction of reported lipid levels in samples previously analyzed using an unmodified Bligh and Dyer extraction. In the future, modifications to procedures and solvent/sample ratios should be described.     

Chemical Synthesis of A Pore‐Forming Antimicrobial Protein,Caenopore‐5, by Using Native Chemical Ligation at a Glu‐Cys Site

The 2014 report from the World Health Organization (WHO) on antimicrobial resistance revealed an alarming rise in antibiotic resistance all around the world. Unlike classical antibiotics, with the exception of a few species, no acquired resistance towards antimicrobial peptides (AMPs) has been reported. Therefore, AMPs represent leads for the development of novel antibiotics. Caenopore‐5 is constitutively expressed in the intestine of the nematode Caenorhabditis elegans and is a pore‐forming AMP. The protein (82 amino acids) was successfully synthesised by using Boc solid‐phase peptide synthesis and native chemical ligation. No γ‐linked by‐product was observed despite the use of a C‐terminal Glu‐thioester. The folding of the synthetic protein was confirmed by ¹H NMR spectroscopy and circular dichroism and compared with data recorded for recombinant caenopore‐5. The permeabilisation activities of the protein and of shortened analogues were evaluated.     

Effect of Visual Cues and a Mixture of Isothiocyanates on Trap Capture of Cabbage Seed Weevil, Ceutorhynchus assimilis

The effect of trap design, trap color, and a mixture of isothiocyanates on the capture of Ceutorhynchus assimilis (Paykull) was studied in a series of field experiments. Unbaited yellow water traps, a yellow sticky box trap, and a yellow sticky card trap, mounted vertically, or at 45° to the vertical with the yellow card facing upwards were effective for capturing seed weevils, but a horizontal yellow sticky card trap caught few. White, green, and black traps were unattractive. During migratory periods, trap catch could be enhanced two to four times with a lure consisting of a mixture of allyl, 3-butenyl, 4-pentenyl, and 2-phenylethyl isothiocyanates, but this bait did not attract in a trap with ineffective design. The sticky card trap, mounted at 45° to the vertical and baited with the isothiocyanate mixture, may be useful for monitoring movement of C. assimilis during migratory periods. However, during the colonization phase, lures of either a mixture of isothiocyanates or of allyl isothiocyanate alone were not attractive.     

Small‐Molecule APOBEC3G DNA Cytosine Deaminase Inhibitors Based on a 4‐Amino‐1,2,4‐triazole‐3‐thiol Scaffold

APOBEC3G (A3G) is a single‐stranded DNA cytosine deaminase that functions in innate immunity against retroviruses and retrotransposons. Although A3G can potently restrict Vif‐deficient HIV‐1 replication by catalyzing excessive levels of G→A hypermutation, sublethal levels of A3G‐catalyzed mutation may contribute to the high level of HIV‐1 fitness and its incurable prognosis. To chemically modulate A3G catalytic activity with the goal of decreasing the HIV‐1 genomic mutation rate, we synthesized and biochemically evaluated a class of 4‐amino‐1,2,4‐triazole‐3‐thiol small‐molecule inhibitors identified by high‐throughput screening. This class of compounds exhibits low‐micromolar (3.9–8.2 μM ) inhibitory potency and remarkable specificity for A3G versus the related cytosine deaminase, APOBEC3A. Chemical modification of inhibitors, A3G mutational screening, and thiol reactivity studies implicate C321, a residue proximal to the active site, as the critical A3G target for this class of molecules.     

Optical Properties and Associated Hygroscopicity of Clay Aerosols

Airborne mineral dust particles contribute a significant fraction to the total aerosol mass, thus they make a substantial contribution to the Earth's radiative budget by direct scattering and absorption of radiation. Quantifying their contribution is complicated by the variability of optical properties as a function of water uptake. To improve understanding, we directly measured the relative humidity (RH) dependence of extinction [fRH_ext(RH, Dry)] for three key silicate clay components (illite, kaolinite, and montmorillonite) of mineral dust aerosols through cavity ring-down spectroscopy at 532 nm. The three clays studied show significant differences in fRH_ext(RH, Dry) at three RH values, and reasons for this are explored. With 68% RH as an example, we used the fRH_ext(RH, Dry) and Mie theory to calculate a growth factor for comparison with other measurement techniques. Humidified tandem differential mobility analyzer and quartz crystal microbalance growth factors from the literature are larger than our optical measurements indicate. An apparent decrease in particle size calculated from optical measurements for illite and kaolinite was further investigated by determining the aerosol electrical mobility size distribution of 68% RH and dry clay particles at that indicated shrinkage of approximately 10% at elevated humidity. Direct optical measurement has advantages because the effects of irregular shape and internal voids are observed. Our calculated growth factors provide a lower limit and can be incorporated into climate models in conjunction with other results to reduce the uncertainty associated with the optical response to water uptake on clay aerosols.