Heterologous expression of the Halothiobacillus neapolitanus carboxysomal gene cluster in Corynebacterium glutamicum

Compartmentalization represents a ubiquitous principle used by living organisms to optimize metabolic flux and to avoid detrimental interactions within the cytoplasm. Proteinaceous bacterial microcompartments (BMCs) have therefore created strong interest for the encapsulation of heterologous pathways in microbial model organisms. However, attempts were so far mostly restricted to Escherichia coli. Here, we introduced the carboxysomal gene cluster of Halothiobacillus neapolitanus into the biotechnological platform species Corynebacterium gluta-micum. Transmission electron microscopy, fluorescence microscopy and single molecule localization microscopy suggested the formation of BMC-like structures in cells expressing the complete carboxysome operon or only the shell proteins. Purified carboxysomes consisted of the expected protein components as verified by mass spectrometry. Enzymatic assays revealed the functional production of RuBisCO in C. glutamicum both in the presence and absence of carboxysomal shell proteins. Furthermore, we could show that eYFP is targeted to the carboxysomes by fusion to the large RuBisCO subunit. Overall, this study represents the first transfer of an α‐carboxysomal gene cluster into a Gram-positive model species supporting the modularity and orthogonality of these microcompartments, but also identified important challenges which need to be addressed on the way towards biotechnological application

Akbari–Ganji Method for Solving Equations of Euler–Bernoulli Beam with Quintic Nonlinearity

In many real word applications, beam has nonlinear transversely vibrations. Solving nonlinear beam systems is complicated because of the high dependency of the system variables and boundary conditions. It is important to have an accurate parametric analysis for understanding the nonlinear vibration characteristics. This paper presents an approximate solution of a nonlinear transversely vibrating beam with odd and even nonlinear terms using the Akbari–Ganji Method (AGM). This method is an effective approach to solve nonlinear differential equations. AGM is already used in the heat transfer science for solving differential equations, and in this research for the first time, it is applied to find the approximate solution of a nonlinear transversely vibrating beam. The advantage of creating new boundary conditions in this method in additional to predefined boundary conditions is checked for the proposed nonlinear case. To illustrate the applicability and accuracy of the AGM, the governing equation of transversely vibrating nonlinear beams is treated with different initial conditions. Since simply supported and clamped-clamped structures can be encountered in many engineering applications, these two boundary conditions are considered. The periodic response curves and the natural frequency are obtained by AGM and contrasted with the energy balance method (EBM) and the numerical solution. The results show that the present method has excellent agreements in contrast with numerical and EBM calculations. In most cases, AGM is applied straightforwardly to obtain the nonlinear frequency– amplitude relationship for dynamic behaviour of vibrating beams. The natural frequencies tested for various values of amplitude are clearly stated the AGM is an applicable method for the proposed nonlinear system. It is demonstrated that this technique saves computational time without compromising the accuracy of the solution. This approach can be easily extended to other nonlinear systems and is therefore widely applicable in engineering and other sciences

The Atg8 Family of Proteins—Modulating Shape and Functionality of Autophagic Membranes

Aging is a multifactorial process involving an accumulation of alterations on various organizational levels, which finally compromises viability and limits the lifespan of organisms. It is now well-established that many aspects of aging can be positively affected by (macro)autophagy, a mechanism of self-digestion found in virtually all eukaryotic cells. A comprehensive understanding of autophagy is thus expected to not only deepen our insight into the mechanisms of aging but to also open up new avenues toward increasing the healthy lifespan in humans. In this review, we focus on the Atg8 family of ubiquitin-like proteins, which play a crucial role in the autophagy process by virtue of their unique mode of reversible membrane association

Platoon-Based Assessment of Two-Way Two-Lane Roads Performance Measure: A Classification Method

Two-way two-lane roads have a significant impact on road transportation infrastructure. Platoon formation on two-lane roads is one of the factors that affect the quality of traffic flow on two-lane roads. More specifically, the creation of a platoon increases the density of vehicles and the number of overtaking maneuvers and decreases the traffic performance of two-lane roads. The present study made an effort to investigate the effect of the platoon characteristics on the traffic flow of vehicles on two-lane roads. Moreover, it strived to develop the nonlinear regression model with a new approach to capacity calculation. Finally, a method for estimating the level of service (LOS) based on the number of followers per capacity (NFPC) and LOS classification using the KNN method was presented. Considering these aims, first, the relevant variables (which were related to platoon) including time headway (Ht), average travel speed (ATS), platoon size (PS), average platoon speed (APS), percentage of heavy vehicles (HV), percent time spent following (PTSF), number of overtaking (NO), density (ρ), and traffic flow were investigated on the examined roads. The results showed that the speed and the Ht were the most effective and the least effective platoon characteristics, respectively. Moreover, it was accompanied by the increase in PTSF which resulted in the increase in overtaking maneuver. Finally, the results regarding the developed model showed that the NFPC measure was able to predict the traffic flow of two-lane roads in a more satisfactory way compared to the two criteria, namely, PTSF and ATS in the Highway Capacity Manual (2016) since it increased ATS by 65% and decreased the capacity of two-lane roads by 21%

Autophagy-Related Proteins GABARAP and LC3B Label Structures of Similar Size but Different Shape in Super-Resolution Imaging

Subcellular structures containing autophagy-related proteins of the Atg8 protein family have been investigated with conventional wide-field fluorescence and single molecule localisation microscopy. Fusion proteins of GABARAP and LC3B, respectively, with EYFP were overexpressed in HEK293 cells. While size distributions of structures labelled by the two proteins were found to be similar, shape distributions appeared quite disparate, with EYFP-GABARAP favouring circular structures and elliptical structures being dominant for EYFP-LC3B. The latter also featured a nearly doubled fraction of U-shape structures. The experimental results point towards highly differential localisation of the two proteins, which appear to label structures representing distinct stages or even specific channels of vesicular trafficking pathways. Our data also demonstrate that the application of super-resolution techniques expands the possibilities of fluorescence-based methods in autophagy studies and in some cases can rectify conclusions obtained from conventional fluorescence microscopy with diffraction-limited resolution

Deficiency of GABARAP but not its Paralogs Causes Enhanced EGF-induced EGFR Degradation

The γ-aminobutyric acid type A receptor-associated protein (GABARAP) and its close paralogs GABARAPL1 and GABARAPL2 constitute a subfamily of the autophagy-related 8 (Atg8) protein family. Being associated with a variety of dynamic membranous structures of autophagic and non-autophagic origin, Atg8 proteins functionalize membranes by either serving as docking sites for other proteins or by acting as membrane tethers or adhesion factors. In this study, we describe that deficiency for GABARAP alone, but not for its close paralogs, is sufficient for accelerated EGF receptor (EGFR) degradation in response to EGF, which is accompanied by the downregulation of EGFR-mediated MAPK signaling, altered target gene expression, EGF uptake, and EGF vesicle composition over time. We further show that GABARAP and EGFR converge in the same distinct compartments at endogenous GABARAP expression levels in response to EGF stimulation. Furthermore, GABARAP associates with EGFR in living cells and binds to synthetic peptides that are derived from the EGFR cytoplasmic tail in vitro. Thus, our data strongly indicate a unique and novel role for GABARAP during EGFR trafficking