Ultra-stable peptide scaffolds for protein engineering-synthesis and folding of the circular cystine knotted cyclotide cycloviolacin O2

The cyclic cystine knot motif, as defined by the cyclotide peptide family, is an attractive scaffold for protein engineering. To date, however, the utilisation of this scaffold has been limited by the inability to synthesise members of the most diverse and biologically active subfamily, the bracelet cyclotides. This study describes the synthesis and first direct oxidative folding of a bracelet cyclotide-cycloviolacin O2-and thus provides an efficient method for exploring the most potent cyclic cystine knot peptides. The linear chain of cycloviolacin O2 was assembled by solid-phase Fmoc peptide synthesis and cyclised by thioester-mediated native chemical ligation, and the inherent difficulties of folding bracelet cyclotides were successfully overcome in a single-step reaction. The folding pathway was characterised and was found to include predominating fully oxidised intermediates that slowly converted to the native peptide structure.     

Redesign of the coenzyme specificity in L-Lactate dehydrogenase from Bacillus stearothermophilus using site-directed mutagenesis and media engineering

L-Lactate dehydrogenase (LDH) from Bacillus stearothermophilusis a redox enzyme which has a strong preference for NADH overNADPH as coenzyme. To exclude NADPH from the coenzyme-bindingpocket, LDH contains a conserved aspartate residue at position52. However, this residue is probably not solely responsiblefor the NADH specificity. In this report we examine the possibilitiesof altering the coenzyme specificity of LDH by introducing arange of different point mutations in the coenzyme-binding domain.Furthermore, after choosing the mutant with the highest selectivityfor NADPH, we also investigated the possibility of further alteringthe coenzyme specificity by adding an organic solvent to thereaction mixture. The LDH mutant, I51K:D52S, exhibited a 56-foldincreased specificity to NADPH over the wild-type LDH in a reactionmixture containing 15% methanol. Furthermore, the NADPH turnovernumber of this mutant was increased almost fourfold as comparedwith wild-type LDH. To explain the altered coenzyme specificityexhibited by the D52SI51K double mutant, molecular dynamicssimulations were performed.     

Dual-magnetron open field sputtering system for sideways deposition of thin films

A dual-magnetron system for deposition inside tubular substrates has been developed. The two magnetrons are facing each other and have opposing magnetic fields forcing electrons and thereby also ionized material to be transported radially towards the substrate. The depositions were made employing direct current magnetron sputtering (DCMS) and high power impulse magnetron sputtering (HiPIMS). To optimize the deposition rate, the system was characterized at different separation distances between the magnetrons under the same sputtering conditions. The deposition rate is found to increase with increasing separation distance independent of discharge technique. The emission spectrum from the HiPIMS plasma shows a highly ionized fraction of the sputtered material. The electron densities of the order of 10¹⁶ m^− 3 and 10¹⁸ m^− 3 have been determined in the DCMS and the HiPIMS plasma discharges respectively. The results demonstrate a successful implementation of the concept of sideways deposition of thin films providing a solution for coating complex shaped surfaces.     

Levels of Circulating PD-L1 Are Decreased in Patients with Resectable Cholangiocarcinoma

Tumor resection represents the only curative treatment option for patients with biliary tract cancers (BTCs), including intrahepatic cholangiocarcinoma (CCA), perihilar and extrahepatic CCA and gallbladder cancer. However, many patients develop early tumor recurrence and are unlikely to benefit from surgery. Therefore, markers to identify ideal surgical candidates are urgently needed. Circulating programmed cell death 1 ligand 1 (PD-L1) has recently been associated with different malignancies, including pancreatic cancer which closely resembles BTC in terms of patients’ prognosis and tumor biology. Here, we aim at evaluating a potential role of circulating PD-L1 as a novel biomarker for resectable BTC. Methods: Serum levels of PD-L1 were analyzed by ELISA in 73 BTC patients and 42 healthy controls. Results: Circulating levels of preoperative PD-L1 were significantly lower in patients with BTC compared to controls. Patients with low PD-L1 levels displayed a strong trend towards an impaired prognosis, and circulating PD-L1 was negatively correlated with experimental markers of promalignant tumor characteristics such as CCL1, CCL21, CCL25 and CCL26. For 37 out of 73 patients, postoperative PD-L1 levels were available. Interestingly, after tumor resection, circulating PD-L1 raised to almost normal levels. Notably, patients with further decreasing PD-L1 concentrations after surgery showed a trend towards an impaired postoperative outcome. Conclusion: Circulating PD-L1 levels were decreased in patients with resectable BTC. Lack of normalization of PD-L1 levels after surgery might identify patients at high risk for tumor recurrence or adverse outcome.     

Post-collision fires in road vehicles between 2002 and 2015

The loss of human lives and body injuries due to post-crash fires, either by smoke inhalation or due to burn injuries, are unfortunately not uncommon. The literature indicates that fire events related to crashes are still a significant problem. The increased combustible load in newer vehicles is an important factor to be taken into account for the fire safety, as well as their potential to release toxic fumes while burning. Trends indicate that the survivable collision energy will continue to increase, and, at the same time, the probability of post-crash fires rises with the collision energy. This means that the occupants of a vehicle may probably survive a high-energy collision but might sustain severe injuries or death due to a post-collision fire. This work reports a literature and interview study about post-crash fires including statistics on the causes and dynamics of post-crash fires in road vehicles based on the literature, crash and incident reports, as well as on interviews with medicine specialists. Results from this study indicate that fires in vehicles which originated by a collision event are a problem that remains to be solved.     

Epitopes Displayed in a Cyclic Peptide Scaffold Bind SARS-COV-2 Antibodies

The SARS-CoV-2 virus that causes COVID-19 is a global health issue. The spread of the virus has resulted in seven million deaths to date. The emergence of new viral strains highlights the importance of continuous surveillance of the SARS-CoV-2 virus by using timely and accurate diagnostic tools. Here, we used a stable cyclic peptide scaffolds to present antigenic sequences derived from the spike protein that are reactive to SARS-CoV-2 antibodies. Using peptide sequences from different domains of SARS-CoV-2 spike proteins, we grafted epitopes on the peptide scaffold sunflower trypsin inhibitor 1 (SFTI-1). These scaffold peptides were then used to develop an ELISA to detect SARS-CoV-2 antibodies in serum. We show that displaying epitopes on the scaffold improves reactivity overall. One of the scaffold peptides (S2_1146-1161_c) has reactivity equal to that of commercial assays, and shows diagnostic potential.     

Color of pigmented plastics—Measurements and predictions

The effect of the concentration of a colorant (masterbatch) and the surface texture on the color of an opaque injection‐molded plaque is experimentally described by means of the spectral reflectance and the CIELAB coordinates. It was noted that the surface texture had a marked influence on the experimental results and on the perceived color. The relation between the color of these pigmented plastic plaques and intrinsic physical material properties, such as light absorption and scattering coefficients, is described by means of the Kubelka‐Munk equation for pigment/paste mixtures. The concentration dependence of this Kubelka‐Munk equation for different masterbatch‐polymer mixtures can be modeled in terms of the color of the individual constituents using a fitting parameter. Further experiments were carried out which indicated that the fitting parameter had a physical meaning. The effect of the surface texture on the optical appearance of the plaques can also be accounted for by this parameter.     

Ethenolysis of esters of vegetable oils: Effect of B2O3 addition to Re2O7/SiO2.Al2O3-SnBu4 and CH3ReO3/SiO2.Al2O3 metathesis catalysts

Impregnation of SiO₂.Al₂O₃ (24.3% Al₂O₃ with 6% B₂O₃ increases the activity of supported Re₂O₇ in the ethenolysis of methyl oleate, but reduces the activity of CH₃ReO₃ on the same support. Re₂O₇/SiO₂.Al₂O₃/B₂O₃-SnBu₄ is shown to be an effective catalyst for the ethenolysis of methyl linoleate and of the methyl esters of olive oil, and can be recycled after calcination at least five times without loss of activity.