Dissecting the stability determinants of a challenging de novo protein fold using massively parallel design and experimentation

Designing entirely new protein structures remains challenging because we do not fully understand the biophysical determinants of folding stability. Yet some protein folds are easier to design than others. Previous work identified the 43-residue αββ&#945 fold as especially challenging: the best designs had only a 2% success rate, compared to 39-87% success for other simple folds (1). This suggested the αββ&#945 fold would be a useful model system for gaining a deeper understanding of folding stability determinants and for testing new protein design methods. Here, we designed over ten thousand new αββ&#945 proteins and found over three thousand of them to fold into stable structures using a high-throughput protease-based assay. Nuclear magnetic resonance, hydrogen-deuterium exchange, circular dichroism, deep mutational scanning, and scrambled sequence control experiments indicated that our stable designs fold into their designed αββ&#945 structures with exceptional stability for their small size. Our large dataset enabled us to quantify the influence of universal stability determinants including nonpolar burial, helix capping, and buried unsatisfied polar atoms, as well as stability determinants unique to the αββ&#945 topology. Our work demonstrates how large-scale design and test cycles can solve challenging design problems while illuminating the biophysical determinants of folding.

In Vitro Activity of WCK 5222 (Cefepime-Zidebactam) against Worldwide Collected Gram-Negative Bacilli Not Susceptible to Carbapenems

ABSTRACT WCK 5222 (cefepime-zidebactam, 2 g + 1g, every 8 h [q8h]) is in clinical development for the treatment of infections caused by carbapenem-resistant and multidrug-resistant (MDR) Gram-negative bacilli. We determined the in vitro susceptibility of 1,385 clinical isolates of non-carbapenem-susceptible Enterobacterales, MDR Pseudomonas aeruginosa (also non-carbapenem susceptible), Stenotrophomonas maltophilia, and Burkholderia spp. collected worldwide (49 countries) from 2014 to 2016 to cefepime-zidebactam (1:1 ratio), ceftazidime-avibactam, imipenem-relebactam, ceftolozane-tazobactam, and colistin using the CLSI broth microdilution method. Cefepime-zidebactam inhibited 98.5% of non-carbapenem-susceptible Enterobacterales (n = 1,018) at ≤8 μg/ml (provisional cefepime-zidebactam-susceptible MIC breakpoint). Against the subset of metallo-β-lactamase (MBL)-positive Enterobacterales (n = 214), cefepime-zidebactam inhibited 94.9% of isolates at ≤8 μg/ml. Further, it inhibited 99.6% of MDR P. aeruginosa (n = 262) isolates at ≤32 μg/ml (proposed cefepime-zidebactam-susceptible pharmacokinetic/pharmacodynamic MIC breakpoint), including all MBL-positive isolates (n = 94). Moreover, cefepime-zidebactam was active against the majority of isolates of Enterobacterales (≥95%) and P. aeruginosa (99%) that were not susceptible to ceftazidime-avibactam, ceftolozane-tazobactam, imipenem-relebactam, and colistin. Most isolates (99%) of S. maltophilia (n = 101; MIC50, 8 μg/ml; MIC90, 32 μg/ml) and Burkholderia spp. (n = 4; MIC range, 16 to 32 μg/ml) were also inhibited by cefepime-zidebactam at ≤32 μg/ml. The activity of cefepime-zidebactam against carbapenem-resistant Gram-negative bacteria is ascribed to its β-lactam enhancer mechanism of action (i.e., zidebactam binding to penicillin binding protein 2 [PBP2] and its universal stability to both serine β-lactamases and MBLs). The results from this study support the continued development of cefepime-zidebactam as a potential therapy for infections caused by Enterobacterales, P. aeruginosa, and other nonfermentative Gram-negative bacilli where resistance to marketed antimicrobial agents is a limiting factor.

Una reflexión sobre el origen de la importancia de la música en la antigua Mesopotamia a partir de los cilindros de Gudea A y B

Resumen: Diversos textos nos muestran a la Música como una realidad especialmente considerada en Mesopotamia, aunque ninguno de esos textos nos dice por qué tenía esa importancia. No obstante, un análisis del relato sobre la construcción del Templo de Ninĝirsu, contenido en los Cilindros de Gudea, podría indicarnos que la importancia de la Música residiría en definir a las divinidades como gobernantes sabios y prudentes del Universo, importante conducta a la hora de mantener la tan ansiada estabilidad del Universo en Mesopotamia.Abstract: Several texts show the Music as a specially considered reality in Mesopotamia, but none of those texts say why the Music had this importance. However, an analysis of the tale about the building of Ninĝirsu’s Temple, contained in the Cylinders of Gudea, could indicate us that the Music was important because it defined to the deities as wise and prudent governors of the Universe, an important conduct in order to maintain the so eagered Universal stability in Mesopotamia.Palabras clave: Música, Gudea de Lagaš, Ninĝirsu, Sabiduría.Key words: Music, Gudea of Lagaš, Ninĝirsu, Wisdom.

Nonlinear Perfect Tracking Control for a Robot Arm with Uncertainties Using Operator-Based Robust Right Coprime Factorization Approach

<div class=""abs_img""><img src=""[disp_template_path]/JRM/abst-image/00270001/06.jpg"" width=""300"" />Plant uncertainties compensation</div> In this paper, a robust nonlinear perfect tracking control for a robot arm with uncertainties is proposed by using operator-based robust right coprime factorization approach. In general, there exist unknown modelling errors in measuring structural parameters of the robot arm and external disturbances in real situations. In the present control system design, the effect of the modelling errors and disturbances on the system performance is considered to be uncertainties of the robot arm dynamics. Considering the uncertainties, a robust nonlinear perfect tracking control using operator-based robust right coprime factorization is investigated. That is, first, considering the unknown uncertain plant generates limitations in obtaining the so-called universal stability and tracking conditions, the effect of uncertain plant is compensated by designed operator-based feedback control scheme. Second, a new perfect tracking condition is proposed for improving the trajectory of the robot arm. Finally, the effectiveness of the designed system is confirmed by simulation results. </span>