Engineering Pyrrolysyl-tRNA Synthetase for the Incorporation of Non-Canonical Amino Acids with Smaller Side Chains

Site-specific incorporation of non-canonical amino acids (ncAAs) into proteins has emerged as a universal tool for systems bioengineering at the interface of chemistry, biology, and technology. The diversification of the repertoire of the genetic code has been achieved for amino acids with long and/or bulky side chains equipped with various bioorthogonal tags and useful spectral probes. Although ncAAs with relatively small side chains and similar properties are of great interest to biophysics, cell biology, and biomaterial science, they can rarely be incorporated into proteins. To address this gap, we report the engineering of PylRS variants capable of incorporating an entire library of aliphatic “small-tag” ncAAs. In particular, we performed mutational studies of a specific PylRS, designed to incorporate the shortest non-bulky ncAA (S-allyl-l-cysteine) possible to date and based on this knowledge incorporated aliphatic ncAA derivatives. In this way, we have not only increased the number of translationally active “small-tag” ncAAs, but also determined key residues responsible for maintaining orthogonality, while engineering the PylRS for these interesting substrates. Based on the known plasticity of PylRS toward different substrates, our approach further expands the reassignment capacities of this enzyme toward aliphatic amino acids with smaller side chains endowed with valuable functionalities.

Small Residues Inhibit Homo-Dimerization of the Human Carbonic Anhydrase XII Transmembrane Domain

Amino acids with small side chains and motifs of small residues in a distance of four are rather abundant in human single-span transmembrane helices. While interaction of such helices appears to be common, the role of the small residues in mediating and/or stabilizing transmembrane helix oligomers remains mostly elusive. Yet, the mere existence of (small)xxx(small) motifs in transmembrane helices is frequently used to model dimeric TM helix structures. The single transmembrane helix of the human carbonic anhydrases XII contains a large number of amino acids with small side chains, and critical involvement of these small amino acids in dimerization of the transmembrane domain has been suggested. Using the GALLEX assay, we show here that the transmembrane domain indeed forms a strong transmembrane helix oligomer within a biological membrane. However, single or multiple mutations of small residue(s) to isoleucine almost always increased, rather than decreased, the interaction propensities. Reduction of helix flexibility and of protein–lipid contacts caused by a reduced lipid accessible surface area likely results in stabilization of helix–helix interactions within the membrane.

The Increase in VO2 Max and Passing Using Exercise Small-Sided Game 3 vs 3 and 6 vs 6 On Football Players

The purpose of this study was to determine the increase in vo2max and passing skills using small-sided games 3 vs 3 and 6 vs 6 exercises in soccer players. This study uses a quantitative approach, the type of research is experimental research, the sample used is 51 football players in Trenggalek under-14 who were selected randomly, this research was carried out in Trenggalek district, namely the Siwalan field, Sumbergedong field, and Sumberagung field, the instrument in the study This study uses a multistage fitness test and a passing test in football, data analysis uses the Manova test. The results showed that there was a difference between the small-sided games 3 vs 3 exercise group and the 6 vs 6 small-sided games group to increase vo2max with a sig value. 0.031 < 0.05, and there is a difference between the small-sided games 3 vs 3 practice group and the 6 vs 6 small-sided games group to improve passing with a sig value of 0.027 < 0.05. It can be concluded that overall small-side game exercises 3 vs 3 and 6 vs 6 can increase vo2max and passing for football players.

How TGfU Influence on Students’ Motivational Outcomes in Physical Education? A Study in Elementary School Context

The purpose of this study was to implement a comprehensive teaching program based on the principles of Teaching Games for Understanding (TGfU) model and questioning, and to assess its consequences for students’ satisfaction of basic psychological needs, motivation, perceptions of ability and intention to be physically active during Physical Education lessons in primary education. A quasi-experimental design was utilized. Participants were 111 students from two different groups of fifth and sixth graders, all enrolled in one primary school. Participants were divided into experimental and control group. Experimental group experienced a TGfU unit, according to small side games and the questioning. Control group experienced a small side games unit, without questioning. Within-group results showed that experimental group students reported significantly higher mean scores in all dependents variables of the study, in both genders. Results showed that control group only reported significantly higher mean scores in intention to be physically active variable, also in both genders. The results demonstrate the need to implement didactic units under comprehensive pedagogical approaches to improve motivation and the intention to develop healthy lifestyle habits in female and male students. More researches are needed to support this evidence.

Double kissing inflation outside the stent secures the patency of small side branch without rewiring

Background The jailed balloon technique is widely used for coronary bifurcation lesions, but a residual risk of SB occlusion remains, necessitating SB rewiring and further interventions, including balloon inflation or stenting, which may result in failure and SB loss. This study introduced a novel modified technique of small side branch (SB) protection, namely, double kissing inflation outside the stent (DKo) technique, for coronary bifurcations without the need for SB rewiring. Methods We performed the DKo technique in consecutive patients in our center from 1/2019 to 12/2019. The procedure was as follows. We inserted a guide wire into both branches followed by proper preparation. The SB balloon was simultaneously inflated with main vessel (MV) stenting. The SB balloon remained in situ until it was kissing inflated with postdilation of the bifurcation core, which is different from traditional strategies. The proximal optimization technique was performed with a short noncompliant balloon strictly not exceeding the bifurcation. Rates of SB loss and in-hospital outcomes were evaluated. Results The technique was successfully performed in all 117 enrolled patients without any rewiring or SB loss. The mean lesion lengths of the MV and SB were 38.3 ± 19.9 mm and 11.7 ± 7.1 mm, respectively. On average, 1.5 ± 0.6 stents were used per patient, while the mean pressure of the SB balloon was 7.4 ± 3.1 atm. DKo achieved excellent procedural success in the proximal and distal MVs: increased minimal lumen diameter (0.64 ± 0.58 mm to 3.05 ± 0.38 mm, p < 0.001; 0.57 ± 0.63 mm to 2.67 ± 0.35 mm, p < 0.001) and low residual stenosis (11.4 ± 3.4%; 7.2 ± 4.6%). DKo secured the patency of the SB without any rewiring and improved the SB stenosis with minimal lumen diameter (0.59 ± 0.48 mm to 1.20 ± 0.42 mm, p < 0.001) and stenosis (71.9 ± 19.4% to 42.2 ± 14.0%, p < 0.001). No MACE was noted in the hospital. Conclusions DKo for bifurcation lesions was shown to be acceptable with high procedural success and excellent SB protection.