Condensed,Microtubule-coating Thin Organelles for Orthogonal Translation in Mammalian Cells

Membraneless organelles are capable of selectively performing complex tasks in living cells despite dynamically exchanging with their surroundings. This is an exquisite example how self-organization of proteins and RNAs can lead to more complex functionalities in living systems. Importantly, the absence of a membrane boundary can enable easier access to larger macromolecular complexes that can be challenging to be transported across a membrane. We previously formed orthogonally translating designer membraneless organelles by combining phase separation with kinesin motor proteins to highly enrich engineered translational factors in large organelles. We also showed that even submicron thick designer organelles can be formed, by mounting them onto membranes, which, presumable assisted by 2D condensation, leads to thin film-like condensates. In this study we show that orthogonal translation can also be built with fiber-like appearing organelles. Here, the microtubule-end binding protein EB1 was used to form fiber-like OT organelles along the microtubule cytoskeleton that perform highly selective and efficient orthogonal translation. We also show an improved simplified design of OT organelles. Together this extends OT organelle technology and demonstrates that the microtubule cytoskeleton is a powerful platform for advanced synthetic organelle engineering.     

Supercritical Water Oxidation of Wastewater and Sludges – Design Considerations

The SCWO process is a promising technology for the treatment of industrial wastewaters and sludges. The commercial or industrial development of this technology mainly goes through engineering considerations, such as reactor design, solids separation and equipment corrosion. In this paper, state of the art of these topics and the flow sheets and energy and mass balances for diluted wastewater and sludge treatment are presented. This plant simulation has been done using the software ASPEN PLUS and it shows that the SCWO process is an interesting alternative from the energetic point of view. The energy integration of a SCWO plant for 2 m³/h sludges with a heating value of 23000 kJ/kg can produce 420 kW as mechanical work and 2522 kg/h of process steam (0.3 MPa). For diluted wastewater the process is autothermal for feeds with an enthalpic content of 900 kJ/kg.     

Compatibility of neural stem cells with functionalized self-assembling peptide scaffold <Emphasis Type="Italic">in vitro</Emphasis>

In this study, we evaluated the behavior of neural stem cells (NSCs) using a new peptide hydrogel scaffold named IKVAVmx, which was made by mixing self-assembling peptide RADA16 and designer peptide RADA16-IKVAV solutions. NSCs derived from rat cerebral cortex were culture-expanded in neuorobasal medium and seeded on the RADA16 and IKVAVmx hydrogels. Cells could penetrate the hydrogels and form a 3D cellular network. Compared to pure RADA16 scaffold, we found that IKVAVmx scaffold significantly promoted cell proliferation and stimulated cell migration into the 3D scaffold. Moreover, Immunocytochemistry and Western blot analysis indicated that the differentiation ratio of neurons from NSCs in IKVAVmx scaffold was higher than that in pure RADA16 scaffold. These results suggested that this new hydrogel scaffold provided an ideal substrate for NSCs 3D culture and suggested its further application for neural tissue engineering.     

The Gift from Nature： Bio-Inspired Strategy for Developing Innovative Bridges

Biology has been a brilliant teacher and a precious textbook to man-made construction for thousands of years, because it allows one to learn and be inspired by nature＇s remarkable and efficient structural systems. However, the emerging biomimetic studies have been of increasing interest for civil engineering design only in the past two decades. Bridge design is one of aspects on structural engineering of biomimeties that offers an enormous potential for inspiration in various aspects, such as the ge- ometry, structure, mechanism, energy use and the intelligence. Recently built bridges and design proposals in which biological systems have produced a range of inspiration are reviewed in this paper. Multidisciplinary cooperation is discussed for the implementation of bio-inspired methods in future design. A case study about using bio-inspired strategy is trying to present a problem-solving approach, yet further cooperation is still needed to utilize biomimetie studies for design inspiration. This paper aims to call a close multidisciplinary collaboration that promotes engineers to build more sustainable and smart structural systems for bridges in the 21 st century.     

香姜,越南植物一新记录种

据已有文献记载,香姜(姜科)(Alpinia coriandriodora D. Fang)仅分布于广西,现首次在越南发现其分布。该种在形态上与竹叶山姜(A. bambusifolia D. Fang)相近,但其叶片椭圆状披针形、唇瓣浅黄色及带红褐色条纹和花药附属体三角状而与后者有别。提供该种详细的形态学描述以及图版,亦包括在越南的分布及生态的资料。凭证标本保存于越南国立自然博物馆(VNMN)和中国科学院华南植物园(IBSC)。     

Protein design for pathway engineering

Design and construction of biochemical pathways has increased the complexity of biosynthetically-produced compounds when compared to single enzyme biocatalysis. However, the coordination of multiple enzymes can introduce a complicated set of obstacles to overcome in order to achieve a high titer and yield of the desired compound. Metabolic engineering has made great strides in developing tools to optimize the flux through a target pathway, but the inherent characteristics of a particular enzyme within the pathway can still limit the productivity. Thus, judicious protein design is critical for metabolic and pathway engineering. This review will describe various strategies and examples of applying protein design to pathway engineering to optimize the flux through the pathway. The proteins can be engineered for altered substrate specificity/selectivity, increased catalytic activity, reduced mass transfer limitations through specific protein localization, and reduced substrate/product inhibition. Protein engineering can also be expanded to design biosensors to enable high through-put screening and to customize cell signaling networks. These strategies have successfully engineered pathways for significantly increased productivity of the desired product or in the production of novel compounds.     

An computer information system for genetic engineering

The article describes the vectors data base and the software for its use (the VECTOR-PC system). At present the prototype versions of data base and VECTOR-PC exist and are in test exploitation. The original data base entry format contains 17 main fields for specific genetic engineering information. The VECTOR-PC system includes programs for data base search and support, and also the "genetic engineering designer", which allows the user to design his own hypothetic structures from the objects of data base and to receive detailed information about them. The system is destined for IBM PC or compatible computers.     

CRISPR-interceded CHO cell line development approaches

For industrial production of recombinant protein biopharmaceuticals, Chinese hamster ovary (CHO) cells represent the most widely adopted host cell system, owing to their capacity to produce high-quality biologics with human-like posttranslational modifications. As opposed to random integration, targeted genome editing in genomic safe harbor sites has offered CHO cell line engineering a new perspective, ensuring production consistency in long-term culture and high biotherapeutic expression levels. Corresponding the remarkable advancements in knowledge of CRISPR-Cas systems, the use of CRISPR-Cas technology along with the donor design strategies has been pushed into increasing novel scenarios in cell line engineering, allowing scientists to modify mammalian genomes such as CHO cell line quickly, readily, and efficiently. Depending on the strategies and production requirements, the gene of interest can also be incorporated at single or multiple loci. This review will give a gist of all the most fundamental recent advancements in CHO cell line development, such as different cell line engineering approaches along with donor design strategies for targeted integration of the desired construct into genomic hot spots, which could ultimately lead to the fast-track product development process with consistent, improved product yield and quality.     

STEAM by another name: Transdisciplinary practice in art and design education

ABSTRACT

The recent movement to include art and design in Science, Technology, Engineering, and Mathematics (STEM) education has made Science, Technology, Engineering, Arts, and Mathematics (STEAM) an increasingly common acronym in the education lexicon. The STEAM movement builds on existing models of interdisciplinary curriculum, but what makes the union of art and design with the STEM disciplines so persuasive? In this article, I draw from research on interdisciplinary curricular projects that fit into the category of STEAM, but may also be considered inquiries into the role of art and design in the creative inquiry process, in order to sketch a transdisciplinary curriculum model that may be applied across disciplines.     

Finding the fittest fold: using the evolutionary record to design new proteins

For many years, the holy grail of protein engineering has been the design of artificial amino acid sequences that fold into stable proteins with desired functions. In the current issue of Nature, two papers from the Ranganathan group (Russ et al., 2005; Socolich et al., 2005) report remarkable success in the design of artificial WW domains. Their method, termed statistical coupling analysis (Lockless and Ranganathan, 1999), does not use structural or physicochemical information but instead extracts information about essential patterns of amino acids from the evolutionary record.     

Tertiary motifs as building blocks for the design of protein‐binding peptides

Despite advances in protein engineering, the de novo design of small proteins or peptides that bind to a desired target remains a difficult task. Most computational methods search for binder structures in a library of candidate scaffolds, which can lead to designs with poor target complementarity and low success rates. Instead of choosing from pre‐defined scaffolds, we propose that custom peptide structures can be constructed to complement a target surface. Our method mines tertiary motifs (TERMs) from known structures to identify surface‐complementing fragments or “seeds.” We combine seeds that satisfy geometric overlap criteria to generate peptide backbones and score the backbones to identify the most likely binding structures. We found that TERM‐based seeds can describe known binding structures with high resolution: the vast majority of peptide binders from 486 peptide‐protein complexes can be covered by seeds generated from single‐chain structures. Furthermore, we demonstrate that known peptide structures can be reconstructed with high accuracy from peptide‐covering seeds. As a proof of concept, we used our method to design 100 peptide binders of TRAF6, seven of which were predicted by Rosetta to form higher‐quality interfaces than a native binder. The designed peptides interact with distinct sites on TRAF6, including the native peptide‐binding site. These results demonstrate that known peptide‐binding structures can be constructed from TERMs in single‐chain structures and suggest that TERM information can be applied to efficiently design novel target‐complementing binders.     

Degree of chromatin fragmentation and frequency of nuclear pyknosis in Percoll-fractionated thymocytes of irradiated rats

The relationship between nuclear chromatin degradation to polydeoxyribonucleotides (PDN) and other features of interphase death were studied using thymocytes of normal and X-irradiated rats. Fractionation of the thymic cells in Percoll gradients was performed in order to separate dead from intact cells. The degree of radiation-induced chromatin fragmentation, as assessed by electrophoresis, was similar for PDN from all Percoll bands. Following irradiation 87-98 per cent of 'heavy' thymocytes were pyknotic and almost devoid of receptors to autologous erythrocytes thus comprising a dead cell population. A direct relationship between PDN content and nuclear pyknosis was noted throughout the gradient. The loss of autologous rosette-forming ability was directly related to other indices of interphase death. The possibility of PDN originating from pyknosis-prone cells and the capacity of radiosensitive thymocytes to form autologous rosettes are discussed.     

Constraint-based strain design using continuous modifications (CosMos) of flux bounds finds new strategies for metabolic engineering

In recent years, a growing number of metabolic engineering strain design techniques have employed constraint-based modeling to determine metabolic and regulatory network changes which are needed to improve chemical production. These methods use systems-level analysis of metabolism to help guide experimental efforts by identifying deletions, additions, downregulations, and upregulations of metabolic genes that will increase biological production of a desired metabolic product. In this work, we propose a new strain design method with continuous modifications (CosMos) that provides strategies for deletions, downregulations, and upregulations of fluxes that will lead to the production of the desired products. The method is conceptually simple and easy to implement, and can provide additional strategies over current approaches. We found that the method was able to find strain design strategies that required fewer modifications and had larger predicted yields than strategies from previous methods in example and genome-scale networks. Using CosMos, we identified modification strategies for producing a variety of metabolic products, compared strategies derived from Escherichia coli and Saccharomyces cerevisiae metabolic models, and examined how imperfect implementation may affect experimental outcomes. This study gives a powerful and flexible technique for strain engineering and examines some of the unexpected outcomes that may arise when strategies are implemented experimentally.     

Microscopy: an art?

There can be no doubt that the finest creator of beauty is Mother Nature. And in many ways, science is the exploration of this beauty and of the mechanisms that have created it. Microscopy, as a technique in scientific investigation, has had a key role in uncovering nature's beauty, which has led some to propose that microscopy could be described as an art or even an art form. But is this claim justified?     

Exploiting mAb structure characteristics for a directed QbD implementation in early process development

In today’s biopharmaceutical industries, the lead time to develop and produce a new monoclonal antibody takes years before it can be launched commercially. The reasons lie in the complexity of the monoclonal antibodies and the need for high product quality to ensure clinical safety which has a significant impact on the process development time. Frameworks such as quality by design are becoming widely used by the pharmaceutical industries as they introduce a systematic approach for building quality into the product. However, full implementation of quality by design has still not been achieved due to attrition mainly from limited risk assessment of product properties as well as the large number of process factors affecting product quality that needs to be investigated during the process development. This has introduced a need for better methods and tools that can be used for early risk assessment and predictions of critical product properties and process factors to enhance process development and reduce costs. In this review, we investigate how the quantitative structure–activity relationships framework can be applied to an existing process development framework such as quality by design in order to increase product understanding based on the protein structure of monoclonal antibodies. Compared to quality by design, where the effect of process parameters on the drug product are explored, quantitative structure–activity relationships gives a reversed perspective which investigates how the protein structure can affect the performance in different unit operations. This provides valuable information that can be used during the early process development of new drug products where limited process understanding is available. Thus, quantitative structure–activity relationships methodology is explored and explained in detail and we investigate the means of directly linking the structural properties of monoclonal antibodies to process data. The resulting information as a decision tool can help to enhance the risk assessment to better aid process development and thereby overcome some of the limitations and challenges present in QbD implementation today.     

Experimental Design and Statistical Inference for Cluster Point Processes – with Applications to the Fruit Dispersion of Anemochorous Forest Trees

This paper deals with experimental design and statistical inference for cluster point processes. The results are applied to fruit dispersion models of forest trees where the corresponding design of experiments is given by the positions of the traps containing the collected fruits. It is shown that consideration of anisotropic behaviour can lead to more realistic models. Modelling interactivity effects between trees seems to be of great interest. It is shown that an approach based on ordered weighted averages yields an notable improvement of model quality. The mathematical background of such models (Choquet integral, fuzzy measures) is sketched in the appendix. Finally, results for choosing a D‐optimal sub‐design are presented.     

Design and structure of two new protein cages illustrate successes and ongoing challenges in protein engineering

In recent years, new protein engineering methods have produced more than a dozen symmetric, self‐assembling protein cages whose structures have been validated to match their design models with near‐atomic accuracy. However, many protein cage designs that are tested in the lab do not form the desired assembly, and improving the success rate of design has been a point of recent emphasis. Here we present two protein structures solved by X‐ray crystallography of designed protein oligomers that form two‐component cages with tetrahedral symmetry. To improve on the past tendency toward poorly soluble protein, we used a computational protocol that favors the formation of hydrogen‐bonding networks over exclusively hydrophobic interactions to stabilize the designed protein–protein interfaces. Preliminary characterization showed highly soluble expression, and solution studies indicated successful cage formation by both designed proteins. For one of the designs, a crystal structure confirmed at high resolution that the intended tetrahedral cage was formed, though several flipped amino acid side chain rotamers resulted in an interface that deviates from the precise hydrogen‐bonding pattern that was intended. A structure of the other designed cage showed that, under the conditions where crystals were obtained, a noncage structure was formed wherein a porous 3D protein network in space group I2₁3 is generated by an off‐target twofold homomeric interface. These results illustrate some of the ongoing challenges of developing computational methods for polar interface design, and add two potentially valuable new entries to the growing list of engineered protein materials for downstream applications.     

Hyaluronic acid and dextran-based semi-IPN hydrogels as biomaterials for bioprinting

Bioprinting is a recent technology in tissue engineering used for the design of porous constructs through layer-by-layer deposition of cell-laden material. This technology would benefit from new biomaterials that can fulfill specific requirements for the fabrication of well-defined 3D constructs, such as the preservation of cell viability and adequate mechanical properties. We evaluated the suitability of a novel semi-interpenetrating network (semi-IPN), based on hyaluronic acid and hydroxyethyl-methacrylate-derivatized dextran (dex-HEMA), to form 3D hydrogel bioprinted constructs. The rheological properties of the solutions allowed proper handling during bioprinting, whereas photopolymerization led to stable constructs of which their mechanical properties matched the wide range of mechanical strengths of natural tissues. Importantly, excellent viability was observed for encapsulated chondrocytes. The results demonstrate the suitability of hyaluronic acid/dex-HEMA semi-IPNs to manufacture bioprinted constructs for tissue engineering.     

Biodegradation of trichloroethylene by an endophyte of hybrid poplar

We isolated and characterized a novel endophyte from hybrid poplar. This unique endophyte, identified as Enterobacter sp. strain PDN3, showed high tolerance to trichloroethylene (TCE). Without the addition of inducers, such as toluene or phenol, PDN3 rapidly reduced TCE levels in medium from 72.4 μM to 30.1 μM in 24 h with a concurrent release of 127 μM chloride ion, and nearly 80% of TCE (55.3 μM) was dechlorinated by PDN3 in 5 days with 166 μM chloride ion production, suggesting TCE degradation.