Creating a completely “cell‐free” system for protein synthesis

Cell‐free protein synthesis is a promising tool to take biotechnology outside of the cell. A cell‐free approach provides distinct advantages over in vivo systems including open access to the reaction environment and direct control over all chemical components for facile optimization and synthetic biology integration. Promising applications of cell‐free systems include portable diagnostics, biotherapeutics expression, rational protein engineering, and biocatalyst production. The highest yielding and most economical cell‐free systems use an extract composed of the soluble component of lysed Escherichia coli. Although E. coli lysis can be highly efficient (>99.999%), one persistent challenge is that the extract remains contaminated with up to millions of cells per mL. In this work, we examine the potential of multiple decontamination strategies to further reduce or eliminate bacteria in cell‐free systems. Two strategies, sterile filtration and lyophilization, effectively eliminate contaminating cells while maintaining the systems’ protein synthesis capabilities. Lyophilization provides the additional benefit of long‐term stability at storage above freezing. Technologies for personalized, portable medicine and diagnostics can be expanded based on these foundational sterilized and completely “cell‐free” systems. © 2015 American Institute of Chemical Engineers Biotechnol. Prog., 31:1716–1719, 2015     

“Water‐in‐Salt” Electrolyte Makes Aqueous Sodium‐Ion Battery Safe,Green, and Long‐Lasting

Narrow electrochemical stability window (1.23 V) of aqueous electrolytes is always considered the key obstacle preventing aqueous sodium‐ion chemistry of practical energy density and cycle life. The sodium‐ion water‐in‐salt electrolyte (NaWiSE) eliminates this barrier by offering a 2.5 V window through suppressing hydrogen evolution on anode with the formation of a Na⁺‐conducting solid‐electrolyte interphase (SEI) and reducing the overall electrochemical activity of water on cathode. A full aqueous Na‐ion battery constructed on Na_0.66[Mn_0.66Ti_0.34]O₂ as cathode and NaTi₂(PO₄)₃ as anode exhibits superior performance at both low and high rates, as exemplified by extraordinarily high Coulombic efficiency (>99.2%) at a low rate (0.2 C) for >350 cycles, and excellent cycling stability with negligible capacity losses (0.006% per cycle) at a high rate (1 C) for >1200 cycles. Molecular modeling reveals some key differences between Li‐ion and Na‐ion WiSE, and identifies a more pronounced ion aggregation with frequent contacts between the sodium cation and fluorine of anion in the latter as one main factor responsible for the formation of a dense SEI at lower salt concentration than its Li cousin.     

Proinflammatory interleukins' production by adipose tissue‐derived mesenchymal stromal cells: the impact of cell culture conditions and cell‐to‐cell interaction

The impact of culture conditions and interaction with activated peripheral blood mononuclear cells on the interleukin (IL) gene expression profile and proinflammatory IL‐6 and IL‐8 production by adipose‐derived stromal cells (ASCs) was investigated. A microarray analysis revealed a wide range of IL genes either under standard (20%) or hypoxic (5%) O₂ concentrations, some highly up‐regulated at hypoxia. IL‐6 and IL‐8 production was inversely dependent on cell culture density. In early (first–third) passages, IL‐6 and IL‐8 concentration was higher at 20% O₂ and in late (8th‐12th) passages under 5% O₂. Interaction between ASCs and mononuclear cells in indirect setting was accompanied with a significant decrease of IL‐6 and did not result in the elevation of IL‐8 concentration. Thereby, the production of proinflammatory interleukins (IL‐6 and IL‐8) may be affected by the ASC intrinsic features (density in culture, and duration of expansion), as well as by microenvironmental factors, such as hypoxia and the presence of blood‐borne cells. These data are important for elucidating ASC paracrine activity regulation in vitro. They would also be on demand for optimisation of the cell therapy protocols, based on the application of ASC biologically active substances. Copyright © 2015 John Wiley & Sons, Ltd. SIGNIFICANCE PARAGRAPH Ex vivo expansion is widely used for increasing the number of adipose‐derived stromal cells (ASCs) and improving of their quality. The present study was designed to elucidate the particular factors influencing the interleukin production in ASCs. The presented data specified the parameters (i.e. cell density, duration of cultivation, hypoxia, etc.) that should be taken in mind when ASCs are intended to be used in protocols implying their paracrine activity. These data would be of considerable interest for researchers and clinicians working in the biomedical science.     

“Closer‐to‐home” strategy benefits juvenile survival in a long‐distance migratory bird

Human‐induced changes in the climate and environment that occur at an unprecedented speed are challenging the existence of migratory species. Faced with these new challenges, species with diverse and flexible migratory behaviors may suffer less from population decline, as they may be better at responding to these changes by altering their migratory behavior. At the individual level, variations in migratory behavior may lead to differences in fitness and subsequently influence the population's demographic dynamics. Using lifetime GPS bio‐logging data from 169 white storks (Ciconia ciconia), we explore whether the recently shortened migration distance of storks affects their survival during different stages of their juvenile life. We also explore how other variations in migratory decisions (i.e., time, destination), movement activity (measured using overall body dynamic acceleration), and early life conditions influence juvenile survival. We observed that their first autumn migration was the riskiest period for juvenile white storks. Individuals that migrated shorter distances and fledged earlier experienced lower mortality risks. In addition, higher movement activity and overwintering “closer‐to‐home” (with 84.21% of the tracked individuals stayed Europe or North Africa) were associated with higher survival. Our study shows how avian migrants can change life history decisions over only a few decades, and thus it helps us to understand and predict how migrants respond to the rapidly changing world.     

Rational Design of Hierarchical “Ceramic‐in‐Polymer” and “Polymer‐in‐Ceramic” Electrolytes for Dendrite‐Free Solid‐State Batteries

Solid polymer electrolytes as one of the promising solid‐state electrolytes have received extensive attention due to their excellent flexibility. However, the issues of lithium (Li) dendrite growth still hinder their practical applications in solid‐state batteries (SSBs). Herein, composite electrolytes from “ceramic‐in‐polymer” (CIP) to “polymer‐in‐ceramic” (PIC) with different sizes of garnet particles are investigated for their effectiveness in dendrite suppression. While the CIP electrolyte with 20 vol% 200 nm Li_6.4La₃Zr_1.4Ta_0.6O₁₂ (LLZTO) particles (CIP‐200 nm) exhibits the highest ionic conductivity of 1.6 × 10^?4 S cm^?1 at 30 °C and excellent flexibility, the PIC electrolyte with 80 vol% 5 µm LLZTO (PIC‐5 µm) shows the highest tensile strength of 12.7 MPa. A sandwich‐type composite electrolyte (SCE) with hierarchical garnet particles (a PIC‐5 µm interlayer sandwiched between two CIP‐200 nm thin layers) is constructed to simultaneously achieve dendrite suppression and excellent interfacial contact with Li metal. The SCE enables highly stable Li plating/stripping cycling for over 400 h at 0.2 mA cm^?2 at 30 °C. The LiFePO₄/SCE/Li cells also demonstrate excellent cycle performance at room temperature. Fabricating sandwich‐type composite electrolytes with hierarchical filler designs can be an effective strategy to achieve dendrite‐free SSBs with high performance and high safety at room temperature.     

Microporous cell‐laden hydrogels for engineered tissue constructs

In this article, we describe an approach to generate microporous cell‐laden hydrogels for fabricating biomimetic tissue engineered constructs. Micropores at different length scales were fabricated in cell‐laden hydrogels by micromolding fluidic channels and leaching sucrose crystals. Microengineered channels were created within cell‐laden hydrogel precursors containing agarose solution mixed with sucrose crystals. The rapid cooling of the agarose solution was used to gel the solution and form micropores in place of the sucrose crystals. The sucrose leaching process generated homogeneously distributed micropores within the gels, while enabling the direct immobilization of cells within the gels. We also characterized the physical, mechanical, and biological properties (i.e., microporosity, diffusivity, and cell viability) of cell‐laden agarose gels as a function of engineered porosity. The microporosity was controlled from 0% to 40% and the diffusivity of molecules in the porous agarose gels increased as compared to controls. Furthermore, the viability of human hepatic carcinoma cells that were cultured in microporous agarose gels corresponded to the diffusion profile generated away from the microchannels. Based on their enhanced diffusive properties, microporous cell‐laden hydrogels containing a microengineered fluidic channel can be a useful tool for generating tissue structures for regenerative medicine and drug discovery applications. Biotechnol. Bioeng. 2010; 106: 138–148. © 2010 Wiley Periodicals, Inc.     

Hierarchically Porous,Ultrathick, “Breathable” Wood‐Derived Cathode for Lithium‐Oxygen Batteries

In this work, a hierarchically porous and ultrathick “breathable” wood‐based cathode for high‐performance Li‐O₂ batteries is developed. The 3D carbon matrix obtained from the carbonized and activated wood (denoted as CA‐wood) serves as a superconductive current collector and an ideal porous host for accommodating catalysts. The ruthenium (Ru) nanoparticles are uniformly anchored on the porous wall of the aligned microchannels (denoted as CA‐wood/Ru). The aligned open microchannels inside the carbon matrix contribute to unimpeded oxygen gas diffusion. Moreover, the hierarchical pores on the microchannel walls can be facilely impregnated by electrolyte, forming a continuous supply of electrolyte. As a result, numerous ideal triphase active sites are formed where electrolyte, oxygen, and catalyst accumulate on the porous walls of microchannels. Benefiting from the numerous well‐balanced triple‐phase active sites, the assembled Li‐O₂ battery with the CA‐wood/Ru cathode (thickness: ≈700 µm) shows a high specific area capacity of 8.58 mA h cm^?2 at 0.1 mA cm^?2. Moreover, the areal capacity can be further increased to 56.0 mA h cm^?2 by using an ultrathick CA‐wood/Ru cathode with a thickness of ≈3.4 mm. The facile ultrathick wood‐based cathodes can be applied to other cathodes to achieve a super high areal capacity without sacrificing the electrochemical performance.     

Polymer‐in‐“Quasi‐Ionic Liquid” Electrolytes for High‐Voltage Lithium Metal Batteries

Due to the limited oxidation stability (<4 V) of ether oxygen in its polymer structure, polyethylene oxide (PEO)‐based polymer electrolytes are not compatible with high‐voltage (>4 V) cathodes, thus hinder further increases in the energy density of lithium (Li) metal batteries (LMBs). Here, a new type of polymer‐in‐“quasi‐ionic liquid” electrolyte is designed, which reduces the electron density on ethereal oxygens in PEO and ether solvent molecules, induces the formation of stable interfacial layers on both surfaces of the LiNi_1/3Mn_1/3Co_1/3O₂ (NMC) cathode and the Li metal anode in Li||NMC batteries, and results in a capacity retention of 88.4%, 86.7%, and 79.2% after 300 cycles with a charge cutoff voltage of 4.2, 4.3, and 4.4 V for the LMBs, respectively. Therefore, the use of “quasi‐ionic liquids” is a promising approach to design new polymer electrolytes for high‐voltage and high‐specific‐energy LMBs.     

Exploring cellular adhesion and differentiation in a micro‐/nano‐hybrid polymer scaffold

Polymer scaffolds play an important role in three dimensional (3‐D) cell culture and tissue engineering. To best mimic the archiecture of natural extracellular matrix (ECM), a nano‐fibrous and micro‐porous combined (NFMP) scaffold was fabricated by combining phase separation and particulate leaching techniques. The NFMP scaffold possesses architectural features at two levels, including the micro‐scale pores and nano‐scale fibers. To evaluate the advantages of micro/nano combination, control scaffolds with only micro‐pores or nano‐fibers were fabricated. Cell grown in NFMP and control scaffolds were characterized with respect to morphology, proliferation rate, diffentiation and adhesion. The NFMP scaffold combined the advantages of micro‐ and nano‐scale structures. The NFMP scaffold nano‐fibers promoted neural differentiation and induced “3‐D matrix adhesion”, while the NFMP scaffold micro‐pores facilitated cell infiltration. This study represents a systematic comparison of cellular activities on micro‐only, nano‐only and micro/nano combined scaffolds, and demonstrates the unique advantages of the later. © 2010 American Institute of Chemical Engineers Biotechnol. Prog., 2010     

Pyrrole‐coupled salicylimine‐based fluorescence “turn on” probe for highly selective recognition of Zn2+ ions in mixed aqueous media: Application in living cell imaging

Cation sensing behaviour of a pyrrole‐based derivative (2‐hydroxyl 3 methyl 6 isopropyl benzaldehyde}‐3,4‐dimethyl‐1H‐pyrrole‐2‐carbohydrazide (receptor 3) has been explored and is found to be selective towards Zn²⁺ over a variety of tested cations. The receptor 3 has shown high selectivity and sensitivity towards Zn²⁺ over the other alkali, alkaline earth and transition metal ions. In the presence of Zn²⁺, absorption band of receptor 3 has shown the red shift. The sensing behaviour has been suggested to continue via enhancement process which has further been supported by UV‐vis absorption and theoretical density functional theory (DFT) calculations indicating the formation of a 1:1 complex between the pyrrole based receptor 3 and Zn²⁺. The present work is presenting a highly selective dual channel colorimetric sensor for zinc with great sensitivity. The developed sensor was successfully applied to image intracellular Zn²⁺ in living cells. Copyright © 2015 John Wiley & Sons, Ltd.     

Osteogenic differentiation and osteochondral tissue engineering using human adipose‐derived stem cells

Osteogenesis and the production of composite osteochondral tissues were investigated using human adult adipose‐derived stem cells and polyglycolic acid (PGA) mesh scaffolds under dynamic culture conditions. For osteogenesis, cells were expanded with or without osteoinduction factors and cultured in control or osteogenic medium for 2 weeks. Osteogenic medium enhanced osteopontin and osteocalcin gene expression when applied after but not during cell expansion. Osteogenesis was induced and mineralized deposits were present in tissues produced using PGA culture in osteogenic medium. For development of osteochondral constructs, scaffolds seeded with stem cells were precultured in either chondrogenic or osteogenic medium, sutured together, and cultured in dual‐chamber stirred bioreactors containing chondrogenic and osteogenic media in separate compartments. After 2 weeks, total collagen synthesis was 2.1‐fold greater in the chondroinduced sections of the composite tissues compared with the osteoinduced sections; differentiation markers for cartilage and bone were produced in both sections of the constructs. The results from the dual‐chamber bioreactor highlight the challenges associated with achieving simultaneous chondrogenic and osteogenic differentiation in tissue engineering applications using a single stem‐cell source. © 2012 American Institute of Chemical Engineers Biotechnol. Prog., 2013     

Strong “bottom‐up” influences on small mammal populations: State‐space model analyses from long‐term studies

“Bottom‐up” influences, that is, masting, plus population density and climate, commonly influence woodland rodent demography. However, “top‐down” influences (predation) also intervene. Here, we assess the impacts of masting, climate, and density on rodent populations placed in the context of what is known about “top‐down” influences. To explain between‐year variations in bank vole Myodes glareolus and wood mouse Apodemus sylvaticus population demography, we applied a state‐space model to 33 years of catch‐mark‐release live‐trapping, winter temperature, and precise mast‐collection data. Experimental mast additions aided interpretation. Rodent numbers in European ash Fraxinus excelsior woodland were estimated (May/June, November/December). December–March mean minimum daily temperature represented winter severity. Total marked adult mice/voles (and juveniles in May/June) provided density indices validated against a model‐generated population estimate; this allowed estimation of the structure of a time‐series model and the demographic impacts of the climatic/biological variables. During two winters of insignificant fruit‐fall, 6.79 g/m² sterilized ash seed (as fruit) was distributed over an equivalent woodland similarly live‐trapped. September–March fruit‐fall strongly increased bank vole spring reproductive rate and winter and summer population growth rates; colder winters weakly reduced winter population growth. September–March fruit‐fall and warmer winters marginally increased wood mouse spring reproductive rate and September–December fruit‐fall weakly elevated summer population growth. Density dependence significantly reduced both species' population growth. Fruit‐fall impacts on demography still appeared after a year. Experimental ash fruit addition confirmed its positive influence on bank vole winter population growth with probable moderation by colder temperatures. The models show the strong impact of masting as a “bottom‐up” influence on rodent demography, emphasizing independent masting and weather influences; delayed effects of masting; and the importance of density dependence and its interaction with masting. We conclude that these rodents show strong “bottom‐up” and density‐dependent influences on demography moderated by winter temperature. “Top‐down” influences appear weak and need further investigation.     

Osteostimulation scaffolds of stem cells: BMP‐7‐derived peptide‐decorated alginate porous scaffolds promote the aggregation and osteo‐differentiation of human mesenchymal stem cells

The scaffolds for stem cell‐based bone tissue engineering should hold the ability to guide stem cells osteo‐differentiating. Otherwise, stem cells will differentiate into unwanted cell types or will form tumors in vivo. Alginate, a natural polysaccharide with great biocompatibility, was widely used in biomedical applications. However, the limited bioactivity and poor osteogenesis capability of pristine alginate hampered its further application in tissue engineering. In this work, a bone forming peptide‐1 (BFP‐1), derived from bone morphogenetic protein‐7, was grafted to alginate polymer chains to prepare peptide‐decorated alginate porous scaffolds (pep‐APS) for promoting osteo‐differentiation of human mesenchymal stem cells (hMSCs). SEM images of pep‐APS exhibited porous structure with about 90% porosity (pore size 100–300 μm), which was appropriate for hMSCs ingrowth. The adhesion, proliferation and aggregation of hMSCs grown on pep‐APS were enhanced in vitro. Moreover, pep‐APS promoted the alkaline phosphatase (ALP) activity of hMSCs, and the osteo‐related genes expression was obviously up‐regulated. The immunochemical staining and western blot analysis results showed high expression level of OCN and Col1a1 in the hMSCs grown on pep‐APS. This work provided a facile and valid strategy to endow the alginate polymers themselves with specific bioactivity and prepare osteopromoting scaffold with enhanced osteogenesis ability, possessing potential applications in stem cell therapy and regenerative medicine.     

The bifunctional SDF‐1‐AnxA5 fusion protein protects cardiac function after myocardial infarction

Stromal cell‐derived factor‐1 (SDF‐1) is a well‐characterized cytokine that protects heart from ischaemic injury. However, the beneficial effects of native SDF‐1, in terms of promoting myocardial repair, are limited by its low concentration in the ischaemic myocardium. Annexin V (AnxA5) can precisely detect dead cells in vivo. As massive cardiomyocytes die after MI, we hypothesize that AnxA5 can be used as an anchor to carry SDF‐1 to the ischaemic myocardium. In this study, we constructed a fusion protein consisting of SDF‐1 and AnxA5 domains. The receptor competition assay revealed that SDF‐1‐AnxA5 had high binding affinity to SDF‐1 receptor CXCR4. The treatment of SDF‐1‐AnxA5 could significantly promote phosphorylation of AKT and ERK and induce chemotactic response, angiogenesis and cell survival in vitro. The binding membrane assay and immunofluorescence revealed that AnxA5 domain had the ability to specifically recognize and bind to cells injured by hypoxia. Furthermore, SDF‐1‐AnxA5 administered via peripheral vein could accumulate at the infarcted myocardium in vivo. The treatment with SDF‐1‐AnxA5 attenuated cell apoptosis, enhanced angiogenesis, reduced infarcted size and improved cardiac function after mouse myocardial infarction. Our results suggest that the bifunctional SDF‐1‐AnxA5 can specifically bind to dead cells. The systemic administration of bifunctional SDF‐1‐AnxA5 effectively provides cardioprotection after myocardial infarction.