Exploration of swapping enzymatic function between two proteins: A simulation study of chorismate mutase and isochorismate pyruvate lyase

The enzyme chorismate mutase EcCM from Escherichia coli catalyzes one of the few pericyclic reactions in biology, the transformation of chorismate to prephenate. The isochorismate pyruvate lyase PchB from Pseudomonas aeroginosa catalyzes another pericyclic reaction, the isochorismate to salicylate transformation. Interestingly, PchB possesses weak chorismate mutase activity as well thus being able to catalyze two distinct pericyclic reactions in a single active site. EcCM and PchB possess very similar folds, despite their low sequence identity. Using molecular dynamics simulations of four combinations of the two enzymes (EcCM and PchB) with the two substrates (chorismate and isochorismate) we show that the electrostatic field due to EcCM at atoms of chorismate favors the chorismate to prephenate transition and that, analogously, the electrostatic field due to PchB at atoms of isochorismate favors the isochorismate to salicylate transition. The largest differences between EcCM and PchB in electrostatic field strengths at atoms of the substrates are found to be due to residue side chains at distances between 0.6 and 0.8 nm from particular substrate atoms. Both enzymes tend to bring their non‐native substrate in the same conformation as their native substrate. EcCM and to a lower extent PchB fail in influencing the forces on and conformations of the substrate such as to favor the other chemical reaction (isochorismate pyruvate lyase activity for EcCM and chorismate mutase activity for PchB). These observations might explain the difficulty of engineering isochorismate pyruvate lyase activity in EcCM by solely mutating active site residues.     

Design,construction, and characterization of a second‐generation DARPin library with reduced hydrophobicity

Designed ankyrin repeat proteins (DARPins) are well‐established binding molecules based on a highly stable nonantibody scaffold. Building on 13 crystal structures of DARPin‐target complexes and stability measurements of DARPin mutants, we have generated a new DARPin library containing an extended randomized surface. To counteract the enrichment of unspecific hydrophobic binders during selections against difficult targets containing hydrophobic surfaces such as membrane proteins, the frequency of apolar residues at diversified positions was drastically reduced and substituted by an increased number of tyrosines. Ribosome display selections against two human caspases and membrane transporter AcrB yielded highly enriched pools of unique and strong DARPin binders which were mainly monomeric. We noted a prominent enrichment of tryptophan residues during binder selections. A crystal structure of a representative of this library in complex with caspase‐7 visualizes the key roles of both tryptophans and tyrosines in providing target contacts. These aromatic and polar side chains thus substitute the apolar residues valine, leucine, isoleucine, methionine, and phenylalanine of the original DARPins. Our work describes biophysical and structural analyses required to extend existing binder scaffolds and simplifies an existing protocol for the assembly of highly diverse synthetic binder libraries.     

酒精发酵过程中酿酒酵母耐受环境胁迫的研究进展

提高酿酒酵母细胞耐受环境胁迫（高渗透压、高浓度酒精和高温）的能力对酒精工业生产具有重要的意义。对提高酿酒酵母耐受性的研究方法和策略的发展历程进行了综述。基因组学、转录组学和蛋白质组学等现代组学技术在这一领域的研究获得了广泛的应用。这些技术将提供期待的信息,去理性改造并获得更加耐受胁迫的工业酵母菌株。     

胰岛素对糖尿病兔牙槽骨缺损修复的影响

目的：探讨胰岛素对糖尿病兔牙槽骨缺损修复治疗的效果,为糖尿病所致的牙周炎提供临床治疗的依据,方法：40只大耳白兔随机分为4组：A组制备健康兔的牙槽骨缺损;B组为胰岛素组,制备健康兔牙槽骨缺损后,用胰岛素治疗;C组为糖尿病组,制备糖尿病兔牙槽骨缺损;D组为糖尿病胰岛素治疗组,制备糖尿病兔牙槽骨缺损后,用胰岛素治疗。每组10只,缺损制备后4、8周各处死5只,对各组成骨情况进行组织学观察及测定。结果：组织学观察A、B、D组修复区可见大量新骨形成,以B组为显著;C组仅见少许成骨,多为纤维组织。新生骨面积比和成骨细胞数在4、8周时均为D组大于c组,B组大于A组,组间差异有统计学意义（P〈0．05）。证明应用胰岛素促进糖尿病兔缺损牙槽骨形成新骨的效果明显。结论：胰岛素能够促进糖尿病兔牙槽骨缺损的戍骨,为,临床上治疗糖尿病并发牙周炎提供一种新的手段。     

Acoustic detection of cell adhesion to a coated quartz crystal microbalance – implications for studying the biocompatibility of polymers

Biocompatibility of polymers is an important parameter for the successful application of polymers in tissue engineering. In this work, quartz crystal microbalance (QCM) devices were used to follow the adhesion of NIH 3T3 fibroblasts to QCM surfaces modified with fibronectin (FN) and poly-D -lysine (PDL). The variations in sensor resonant frequency (Δf) and motional resistance (ΔR), monitored as the sensor signal, revealed that cell adhesion was favored in the PDL-coated QCMs. Fluorescence microscopy images of seeded cells showed more highly spread cells on the PDL substrate, which is consistent with the results of the QCM signals. The sensor signal was shown to be sensitive to extracellular matrix (ECM)-binding motifs. Ethylenediaminetetraacetic acid (EDTA) and soluble Gly-Arg-Gly-Asp-Ser (GRGDS) peptides were used to interfere with cell-ECM binding motifs onto FN-coated QCMs. The acquired acoustic signals successfully showed that in the presence of 30 mM EDTA or 1 mM GRGDS, cell adhesion is almost completely abolished due to the inhibition/blocking of integrin function by these compounds. The results presented here demonstrate the potential of the QCM sensor to study cell adhesion, to monitor the biocompatibility of polymers and materials, and to assess the effect of adhesion modulators. QCM sensors have great potential in tissue engineering applications, as QCM sensors are able to analyze the biocompatibility of surfaces and it has the added advantage of being able to evaluate, in situ and in real time, the effect of specific drugs/treatments on cells.     

Bioreactors in tissue engineering—principles,applications and commercial constraints

Bioreactor technology is vital for tissue engineering. Usually, bioreactors are used to provide a tissue-specific physiological in vitro environment during tissue maturation. In addition to this most obvious application, bioreactors have the potential to improve the efficiency of the overall tissue-engineering concept. To date, a variety of bioreactor systems for tissue-specific applications have been developed. Of these, some systems are already commercially available. With bioreactor technology, various functional tissues of different types were generated and cultured in vitro. Nevertheless, these efforts and achievements alone have not yet led to many clinically successful tissue-engineered implants. We review possible applications for bioreactor systems within a tissue-engineering process and present basic principles and requirements for bioreactor development. Moreover, the use of bioreactor systems for the expansion of clinically relevant cell types is addressed. In contrast to cell expansion, for the generation of functional three-dimensional tissue equivalents, additional physical cues must be provided. Therefore, bioreactors for musculoskeletal tissue engineering are discussed. Finally, bioreactor technology is reviewed in the context of commercial constraints.     

Deriving metabolic engineering strategies from genome-scale modeling with flux ratio constraints

Optimized production of bio-based fuels and chemicals from microbial cell factories is a central goal of systems metabolic engineering. To achieve this goal, a new computational method of using flux balance analysis with flux ratios (FBrAtio) was further developed in this research and applied to five case studies to evaluate and design metabolic engineering strategies. The approach was implemented using publicly available genome-scale metabolic flux models. Synthetic pathways were added to these models along with flux ratio constraints by FBrAtio to achieve increased (i) cellulose production from Arabidopsis thaliana; (ii) isobutanol production from Saccharomyces cerevisiae; (iii) acetone production from Synechocystis sp. PCC6803; (iv) H₂ production from Escherichia coli MG1655; and (v) isopropanol, butanol, and ethanol (IBE) production from engineered Clostridium acetobutylicum. The FBrAtio approach was applied to each case to simulate a metabolic engineering strategy already implemented experimentally, and flux ratios were continually adjusted to find (i) the end-limit of increased production using the existing strategy, (ii) new potential strategies to increase production, and (iii) the impact of these metabolic engineering strategies on product yield and culture growth. The FBrAtio approach has the potential to design “fine-tuned” metabolic engineering strategies in silico that can be implemented directly with available genomic tools.     

Protein design in systems metabolic engineering for industrial strain development

Accelerating the process of industrial bacterial host strain development, aimed at increasing productivity, generating new bio-products or utilizing alternative feedstocks, requires the integration of complementary approaches to manipulate cellular metabolism and regulatory networks. Systems metabolic engineering extends the concept of classical metabolic engineering to the systems level by incorporating the techniques used in systems biology and synthetic biology, and offers a framework for the development of the next generation of industrial strains. As one of the most useful tools of systems metabolic engineering, protein design allows us to design and optimize cellular metabolism at a molecular level. Here, we review the current strategies of protein design for engineering cellular synthetic pathways, metabolic control systems and signaling pathways, and highlight the challenges of this subfield within the context of systems metabolic engineering.     

Breaking the 10% Efficiency Barrier in Organic Photovoltaics: Morphology and Device Optimization of Well‐Known PBDTTT Polymers

With the advances in organic photovoltaics (OPVs), the invention of model polymers with superior properties and wide applicability is of vital importance to both the academic and industrial communities. The recent inspiring advances in OPV research have included the emergence of poly(benzodithiophene‐co‐thieno[3,4‐b]thiophene) (PBDTTT)‐based materials. Through the combined efforts on PBDTTT polymers, over 10% efficiencies have been realized recently in various types of OPV devices. This review attempts to critically summarize the recent advances with respect to five well‐known PBDTTT polymers and their design considerations, basic properties, photovoltaic performance, as well as device application in conventional, inverted, tandem solar cells. These PBDTTT polymers also make great contributions to the rapid advances in the field of emerging ternary blends and fullerene‐free OPVs with top performances. Addtionally, new challenges in developing novel photovoltaic polymers with more superior properties are prospected. More importantly, the research of highly efficient PBDTTT‐based polymers provides useful insights and builds fundamentals for new types of OPV applications with various architectures.     

Interactions of donor sources and media influence the histo‐morphological quality of full‐thickness skin models

Human artificial skin models are increasingly employed as non‐animal test platforms for research and medical purposes. However, the overall histopathological quality of such models may vary significantly. Therefore, the effects of manufacturing protocols and donor sources on the quality of skin models built‐up from fibroblasts and keratinocytes derived from juvenile foreskins is studied. Histo‐morphological parameters such as epidermal thickness, number of epidermal cell layers, dermal thickness, dermo‐epidermal adhesion and absence of cellular nuclei in the corneal layer are obtained and scored accordingly. In total, 144 full‐thickness skin models derived from 16 different donors, built‐up in triplicates using three different culture conditions were successfully generated. In univariate analysis both media and donor age affected the quality of skin models significantly. Both parameters remained statistically significant in multivariate analyses. Performing general linear model analyses we could show that individual medium‐donor‐interactions influence the quality. These observations suggest that the optimal choice of media may differ from donor to donor and coincides with findings where significant inter‐individual variations of growth rates in keratinocytes and fibroblasts have been described. Thus, the consideration of individual medium‐donor‐interactions may improve the overall quality of human organ models thereby forming a reproducible test platform for sophisticated clinical research.