Establishing a Klebsiella pneumoniae-Based Cell-Free Protein Synthesis System.

Cell-free protein synthesis (CFPS) systems are emerging as powerful platforms for in vitro protein production, which leads to the development of new CFPS systems for different applications. To expand the current CFPS toolkit, here we develop a novel CFPS system derived from a chassis microorganism Klebsiella pneumoniae, an important industrial host for heterologous protein expression and the production of many useful chemicals. First, we engineered the K. pneumoniae strain by deleting a capsule formation-associated wzy gene. This capsule-deficient strain enabled easy collection of the cell biomass for preparing cell extracts. Then, we optimized the procedure of cell extract preparation and the reaction conditions for CFPS. Finally, the optimized CFPS system was able to synthesize a reporter protein (superfolder green fluorescent protein, sfGFP) with a maximum yield of 253 ± 15.79 µg/mL. Looking forward, our K. pneumoniae-based CFPS system will not only expand the toolkit for protein synthesis, but also provide a new platform for constructing in vitro metabolic pathways for the synthesis of high-value chemicals.

Effects of Dislocation Density Evolution on Mechanical Behavior of OFHC Copper during High-Speed Machining.

This paper aims at investigating the change in material behavior induced by microstructure evolution during high-speed machining processes. Recently, high-speed machining has attracted quite a lot of interest from researchers due to its high efficiency and surface quality in machining large-scale components. However, the material behavior could change significantly at high-cutting speeds compared to the conventional cutting conditions, including their microstructure and t mechanical response. This is due to the basic physics of material at microscopic levels with high strain, high strain rates, and high temperatures. In this study, the dislocation density-related microstructure evolution process and mechanical behavior of OFHC (Oxygen-free high-conductivity) copper in high-speed machining with speeds ranging from 750 m/min to 3000 m/min are investigated. SEM (Scanning Electron Microscope) and advanced EBSD (Electron Backscattered Diffraction) techniques are used to obtain high-quality images of the microstructures and analyze the dislocation density and grain size evolution with different cutting speeds. Moreover, as material plasticity is induced by the motion of dislocations at micro-scales, a dislocation-density based (DDB) model is applied to predict strain-stress and microstructure information during the cutting process. The distributions of dislocation densities, both statistically stored dislocations (SSDs) and geometrically necessary dislocations (GNDs), are obtained through simulation and experimentation, respectively. The results show that the fluctuation in the cutting forces at high cutting speeds is induced by the specific evolution and distribution of the dislocation density under high strain-rates, and the periodical distribution of sub-surface and fracture behavior during chip separation, which are also found to be influenced by the evolution of the dislocation density.

Present and future prospects for polypeptide multilayer nanofilms in biotechnology and medicine.

Polypeptide multilayer nanofilms are a promising nanotechnology for commercial product development because the processes used to prepare them are simple, flexible, reliable, automatable, and scalable. Moreover, these materials can display a remarkable diversity of physical, chemical, and biological properties. Furthermore, the constituents of these nanofilms, in most cases the nanofilms themselves, and the fabrication process are environmentally benign. Nanofilm structure and function can be tailored to address two Grand Challenges of the US National Nanotechnology Initiative.

Phase report of medical treatment in Sichuan Province, China after the Wenchuan earthquake.

OBJECTIVE: To retrospectively analyze medical treatment during the Wenchuan earthquake and evaluate the emergency medical response. METHOD: On the basis of the data reported to the provincial disaster relief headquarters by cities and counties in Sichuan Province, we established a database for data processing by using SPSS v11.0. Descriptive statistical analysis was performed. RESULTS: The emergency response of Sichuan health system was quick, effective, and powerful. CONCLUSION: In the face of extraordinary disaster, Sichuan Province effectively completed emergency medical treatment despite the enormous workload and associated difficulties achieving initial success in earthquake relief.

Trans-province transfer of 10,373 patients injured in Wenchuan earthquake.

OBJECTIVE: A retrospective summary of the planning, organization, and implementation of the trans-province transfer of patients injured in Wenchuan Earthquake with an emphasis on experiences that may be helpful in future emergency rescue and patient transfer. METHODS: We collected the daily reports of a patient transfer team attached to the Sichuan Rescue Headquarters from May 12 through May 31, 2008. RESULTS: Under the guidance of policy made by the coordinating group of the Ministry of Health, and with the close cooperation of the railway and airline departments the transferring group transferred 10,373 patients in the period studied. The transfers were from 11 disaster areas, including Chengdu, Mianyang, and Deyang, to 20 cities and provinces, including Chongqing, Jiangsu, and Zhejiang. There were no casualties during transfer, and thus the biggest peacetime government-organized trans-province patient-transfer in China's history achieved success. CONCLUSION: Trans-province patient transfer is an effective measure to compensate for inadequate medical materials, relieve pressure on medical rescue, and guarantee quality of treatment. In the future, emergency plans for different types of disasters will be established, the information platform will be improved, and transfer procedures will be specified.

Context dependence of the assembly, structure, and stability of polypeptide multilayer nanofilms.

Polyelectrolyte multilayer nanofilms and nanocomposites have shown considerable promise for the rational development of multifunctional materials with wide-ranging properties. Polypeptides are a distinctive and largely unexplored class of polyelectrolytes in this context. Methods now exist for the synthesis of peptides with control at the level of the amino acid sequence, and for the preparation of these polymers in massive quantities. Here, we analyze the roles of six designed 32mer peptides in the fabrication, structure, and stability of multilayer nanofilms prepared by layer-by-layer self-assembly. The data show that amino acid sequence and the specific combination of anionic and cationic peptides together have a marked impact on nanofilm growth behavior, secondary structure content, and density in experimental studies. The same factors determine physical properties of the corresponding interpolypeptide complexes in molecular dynamics simulations.

Quantal self-assembly of polymer layers in polypeptide multilayer nanofilms.

Simple molecular models predict key aspects of the "microscopic" assembly behavior of various peptide systems in the fabrication of multilayer films. Such films show substantial differences in density for different peptide systems. The data suggest that exponential film growth is possible in the absence of polymer diffusion and that "macroscopic" assembly behavior is more a function of peptide-peptide interactions than peptide sequence alone.

A molecular dynamics study of the physical basis of stability of polypeptide multilayer nanofilms.

Electrostatic layer-by-layer assembly (LBL) is a versatile method of fabricating ultrathin multilayer films, coatings, and microcapsules from materials in solution, notably, oppositely charged polyelectrolytes in water. Polypeptides, a special type of polyelectrolyte, have recently shown promise for a range of applications in biotechnology and medicine, for example, artificial cells, drug delivery systems, cell/tissue scaffolds, artificial viruses, and implantable device coatings. Poly(L-lysine) (PLL) and poly(L-glutamic acid) (PLGA) at neutral pH are model oppositely charged polypeptides. Experimental studies have shown that PLL/PLGA multilayer films contain a substantial amount of beta-sheets. Here, we present findings of a molecular dynamics (MD) study of the physical basis of interaction between PLL and PLGA in multilayer film models. Simulations have been carried out to study structural and dynamical properties of PLL/PLGA aggregates in beta-sheet conformation. The results suggest that hydrophobic interactions, in addition to electrostatics interactions, play a significant role in PLL/PLGA multilayers. The preferred orientation of peptides in the beta-sheet structures is antiparallel within sheets and parallel between sheets. Intersheet hydrogen-bond formation is more likely the result of peptide association than the cause. The approach provides a general means to understand better how various types of noncovalent interactions contribute to the structure and stability of polypeptide multilayer films.

Protein-inspired multilayer nanofilms: science, technology and medicine.

The field of polypeptide multilayer nanofilm research flourishes where study of protein structure and function shares a border with development of polyelectrolyte multilayers. The soil is fertile for creative input and promises a harvest of interesting results: the structure of a film can be predetermined on a layer-by-layer (LBL) basis, a huge variety of polypeptide sequences can be realized in large quantities by modern methods of synthesis, and the fabrication process is environmentally benign. In electrostatic LBL assembly, multilayer film assembly is driven primarily by coulombic interactions, but hydrophobic interactions and hydrogen bonds also contribute to film formation and stability, the amount depending on polypeptide design. Most peptides suitable for LBL assembly form films with a large percentage of beta-sheet at neutral pH; it would appear that beta-sheet is favored over alpha-helix in this context by the contribution to entropy of the number of ways of forming a beta-sheet from a single polypeptide chain. Film thickness and roughness depend rather substantially on amino acid composition. Promising applications of the polypeptide multilayer film platform technology include coatings for medical implant devices, scaffolds for tissue engineering, coatings for targeted drug delivery, artificial cells for oxygen therapeutics, and artificial viruses for immunization. In each case peptide structure is tailored to the application. Here we summarize recent results of experimental studies and computational work from our laboratory, showing how the study of protein structure has inspired the design of polypeptide films and pointing out new opportunities for technology development. This work also provides a brief introduction to polypeptide structure and multilayer films.

Polypeptide multilayer films.

Research on polypeptide multilayer films, coatings, and microcapsules is located at the intersection of several disciplines: synthetic polymer chemistry and physics, biomaterials science, and nanoscale engineering. The past few years have witnessed considerable growth in each of these areas. Unexplored territory has been found at the borders, and new possibilities for technology development are taking form from technological advances in polypeptide production, sequencing of the human genome, and the nature of peptides themselves. Most envisioned applications of polypeptide multilayers have a biomedical bent. Prospects seem no less positive, however, in fields ranging from food technology to environmental science. This review of the present state of polypeptide multilayer film research covers key points of polypeptides as materials, means of polymer production and film preparation, film characterization methods, focal points of current research in basic science, and the outlook for a few specific applications. In addition, it discusses how the study of polypeptide multilayer films could help to clarify the physical basis of assembly and stability of polyelectrolyte multilayers, and mention is made of similarities to protein folding studies.