bluuebunny/crossref_metadata_2025_split huggingface dataset:
Robust effects of cloud superparameterization on simulated daily rainfall intensity statistics across multiple versions of the Community Earth System Model
Authors: Kooperman Gabriel J. , Pritchard Michael S. , Burt Melissa A. , Branson Mark D. , Randall David A. 
Published: January 2016

AbstractThis study evaluates several important statistics of daily rainfall based on frequency and amount distributions as simulated by a global climate model whose precipitation does not depend on convective parameterization—Super‐Parameterized Community Atmosphere Model (SPCAM). Three superparameterized and conventional versions of CAM, coupled within the Community Earth System Model (CESM1 and CCSM4), are compared against two modern rainfall products (GPCP 1DD and TRMM 3B42) to discriminate robust effects of superparameterization that emerge across multiple versions. The geographic pattern of annual‐mean rainfall is mostly insensitive to superparameterization, with only slight improvements in the double‐ITCZ bias. However, unfolding intensity distributions reveal several improvements in the character of rainfall simulated by SPCAM. The rainfall rate that delivers the most accumulated rain (i.e., amount mode) is systematically too weak in all versions of CAM relative to TRMM 3B42 and does not improve with horizontal resolution. It is improved by superparameterization though, with higher modes in regions of tropical wave, Madden‐Julian Oscillation, and monsoon activity. Superparameterization produces better representations of extreme rates compared to TRMM 3B42, without sensitivity to horizontal resolution seen in CAM. SPCAM produces more dry days over land and fewer over the ocean. Updates to CAM's low cloud parameterizations have narrowed the frequency peak of light rain, converging toward SPCAM. Poleward of 50°, where more rainfall is produced by resolved‐scale processes in CAM, few differences discriminate the rainfall properties of the two models. These results are discussed in light of their implication for future rainfall changes in response to climate forcing.
Plant protein 2‐Cys peroxiredoxin TaBAS1 alleviates oxidative and nitrosative stresses incurred during cryopreservation of mammalian cells
Authors: Chow‐shi‐yée Mélanie , Grondin Mélanie , Averill‐Bates Diana A. , Ouellet François 
Published: January 2016

ABSTRACTThere is increasing demand for cryopreserved cells such as liver and pancreatic cells for clinical applications. Cryopreservation at ultra‐low temperatures requires use of cryoprotectants (e.g., dimethyl sulfoxide (DMSO)) to maintain cell integrity during freezing and thawing processes. Standard cryoprotectants are cytotoxic and more effective cryopreservation technologies are urgently needed for long‐term storage of cells. As an alternative, soluble protein extracts (WPE) from winter wheat successfully replaced DMSO as a cryoprotectant for several mammalian cell types. To identify novel cryoactive proteins, the WPE was separated by chromatography and cryoactive fractions were analyzed by mass spectrometry. The wheat protein 2‐Cys peroxiredoxin BAS1 (renamed TaBAS1) was identified as a potential cryoactive candidate. Recombinant proteins were prepared and found to possess dual functions as a peroxidase antioxidant and molecular chaperone, and display cryoprotective properties for hepatocytes and insulin‐secreting INS832/13 cells. Following cryopreservation with TaBAS1, cells were plateable and showed high post‐thaw viability, good adhesion properties, and well‐maintained cell‐specific metabolic functions. The overall quality of these cell types was equivalent or improved compared to cells that were cryopreserved with DMSO. The antioxidant and chaperone functions of TaBAS1 likely explain its efficacy in reducing oxidative/nitrosative stresses in cryopreserved cells. The plant protein TaBAS1 could be a promising molecule to include in cryostorage protocols. Biotechnol. Bioeng. 2016;113: 1511–1521. © 2015 Wiley Periodicals, Inc.
Directed evolution increases desaturation of a cyanobacterial fatty acid desaturase in eukaryotic expression systems
Authors: Bai Shuangyi , Wallis James G. , Denolf Peter , Browse John 
Published: January 2016

ABSTRACTDirected evolution of a cyanobacterial Δ9 fatty acid desaturase (DSG) from Synechococcus elongatus, PCC6301 created new, more productive desaturases and revealed the importance of certain amino acid residues to increased desaturation. A codon‐optimized DSG open reading frame with an endoplasmic‐reticulum retention/retrieval signal appended was used as template for random mutagenesis. Increased desaturation was detected using a novel screen based on complementation of the unsaturated fatty acid auxotrophy of Saccharomyces cerevisiae mutant ole1Δ. Amino acid residues whose importance was discovered by the random processes were further examined by saturation mutation to determine the best amino acid at each identified location in the peptide chain and by combinatorial analysis. One frequently‐detected single amino acid change, Q240R, yielded a nearly 25‐fold increase in total desaturation in S. cerevisiae. Several other variants of the protein sequence with multiple amino acid changes increased total desaturation more than 60‐fold. Many changes leading to increased desaturation were in the vicinity of the canonical histidine‐rich regions known to be critical for electron transfer mediated by these di‐iron proteins. Expression of these evolved proteins in the seed of Arabidopsis thaliana altered the fatty acid composition, increasing monounsaturated fatty acids and decreasing the level of saturated fatty acid, suggesting a potential application of these desaturases in oilseed crops. Biotechnol. Bioeng. 2016;113: 1522–1530. © 2016 Wiley Periodicals, Inc.
Targeted transgene insertion into the CHO cell genome using Cre recombinase‐incorporating integrase‐defective retroviral vectors
Authors: Kawabe Yoshinori , Shimomura Takuya , Huang Shuohao , Imanishi Suguru , Ito Akira , Kamihira Masamichi 
Published: January 2016

ABSTRACTRetroviral vectors have served as efficient gene delivery tools in various biotechnology fields. However, viral DNA is randomly inserted into the genome, which can cause problems, such as insertional mutagenesis and gene silencing. Previously, we reported a site‐specific gene integration system, in which a transgene is integrated into a predetermined chromosomal locus of Chinese hamster ovary (CHO) cells using integrase‐defective retroviral vectors (IDRVs) and Cre recombinase. In this system, a Cre expression plasmid is transfected into founder cells before retroviral transduction. In practical applications of site‐specific gene modification such as for hard‐to‐transfect cells or for in vivo gene delivery, both the transgene and the Cre protein into retroviral virions should be encapsulate. Here, we generated novel hybrid IDRVs in which viral genome and enzymatically active Cre can be delivered (Cre‐IDRVs). Cre‐IDRVs encoding marker genes, neomycin resistance and enhanced green fluorescent protein (EGFP), flanked by wild‐type and mutated loxP sites were produced using an expression plasmid for a chimeric protein of Cre and retroviral gag‐pol. After analyzing the incorporation of the Cre protein into retroviral virions by Western blotting, the Cre‐IDRV was infected into founder CHO cells, in which marker genes (hygromycin resistance and red fluorescent protein) flanked with corresponding loxP sites are introduced into the genome. G418‐resistant colonies expressing GFP appeared and the site‐specific integration of the transgene into the expected chromosomal site was confirmed by PCR and sequencing of amplicons. Moreover, when Cre‐IDRV carried a gene expression unit for a recombinant antibody, the recombinant cells in which the antibody expression cassette was integrated in a site‐specific manner were generated and the cells produced the recombinant antibody. This method may provide a promising tool to perform site‐specific gene modification according to Cre‐based cell engineering. Biotechnol. Bioeng. 2016;113: 1600–1610. © 2016 Wiley Periodicals, Inc.
Mannose metabolism in recombinant CHO cells and its effect on IgG glycosylation
Authors: Slade Peter G. , Caspary R. Guy , Nargund Shilpa , Huang Chung‐Jr 
Published: January 2016

ABSTRACTUnderstanding the causes of high‐mannose (HM) glycosylation of recombinant IgG in CHO cells would facilitate the production of therapeutics. CHO cells grown with mannose as the major carbon source demonstrated a dramatic increase in total HM glycosylation in recombinant IgG, with no effect on cell growth, viability, or titer. Quantitative metabolomics and 13C flux analysis were used to explore the mechanism for increased HM glycosylation and understand the metabolism of mannose in CHO cells. It was demonstrated that mannose was a good carbon source for CHO cell growth and IgG production, readily entering both glycolysis and the TCA Cycle. Previous mechanisms for increased HM glycosylation during antibody production have been attributed to changes in pH, osmolality, increased specific productivity, and nutrient limitation. The results from this study propose a novel mechanism where an increased carbon flux in the GDP‐mannose synthetic pathway increased the intracellular concentration of mannose‐containing metabolites. The abnormally high concentration of mannose and mannose–metabolites were shown to inhibit α‐mannosidase activity and it was proposed that this inhibition in the ER and Golgi caused the production of IgG with increased high‐mannose glycosylation. Biotechnol. Bioeng. 2016;113: 1468–1480. © 2016 Wiley Periodicals, Inc.
Fully automatized high‐throughput enzyme library screening using a robotic platform
Authors: Dörr Mark , Fibinger Michael P.C. , Last Daniel , Schmidt Sandy , Santos‐Aberturas Javier , Böttcher Dominique , Hummel Anke , Vickers Clare , Voss Moritz , Bornscheuer Uwe T. 
Published: January 2016

ABSTRACTA fully automatized robotic platform has been established to facilitate high‐throughput screening for protein engineering purposes. This platform enables proper monitoring and control of growth conditions in the microtiter plate format to ensure precise enzyme production for the interrogation of enzyme mutant libraries, protein stability tests and multiple assay screenings. The performance of this system has been exemplified for four enzyme classes important for biocatalysis such as Baeyer–Villiger monooxygenase, transaminase, dehalogenase and acylase in the high‐throughput screening of various mutant libraries. This allowed the identification of novel enzyme variants in a sophisticated and highly reliable manner. Furthermore, the detailed optimization protocols should enable other researchers to adapt and improve their methods. Biotechnol. Bioeng. 2016;113: 1421–1432. © 2016 Wiley Periodicals, Inc.
Identification of an important motif that controls the activity and specificity of sugar transporters
Authors: Wang Meng , Yu Chenzhao , Zhao Huimin 
Published: January 2016

ABSTRACTEfficient glucose–xylose co‐utilization is critical for economical biofuel production from lignocellulosic biomass. To enable glucose–xylose co‐utilization, a highly active xylose specific transporter without glucose inhibition is desirable. However, our understanding of the structure‐activity/specificity relationship of sugar transporters in general is limited, which hinders our ability to engineer xylose‐specific transporters. In this study, via homology modeling and analysis of hexose sugar transporter HXT14 mutants, we identified a highly conserved YYX(T/P) motif that plays an important role in controlling the activity and specificity of sugar transporters. We demonstrated that mutating the two tyrosine residues of the motif to phenylalanine, respectively, improved glucose transport capacity across several different sugar transporters. Furthermore, we illustrated that by engineering the fourth position in the YYX(T/P) motif, the sugar specificity of transporters was significantly altered or even reversed towards xylose. Finally, using the engineered sugar transporter, genuine glucose–xylose co‐fermentation was achieved. Biotechnol. Bioeng. 2016;113: 1460–1467. © 2016 Wiley Periodicals, Inc.
Combinatorial optimization of CO2 transport and fixation to improve succinate production by promoter engineering
Authors: Yu Jun‐Han , Zhu Li‐Wen , Xia Shi‐Tao , Li Hong‐Mei , Tang Ya‐Ling , Liang Xin‐Hua , Chen Tao , Tang Ya‐Jie 
Published: January 2016

ABSTRACTTo balance the flux of an engineered metabolic pathway to achieve high yield of target product is a major challenge in metabolic engineering. In previous work, the collaborative regulation of CO2 transport and fixation was investigated with co‐overexpressing exogenous genes regulating both CO2 transport (sbtA and bicA) and PEP carboxylation (phosphoenolpyruvate (PEP) carboxylase (ppc) and carboxykinase (pck)) under trc promoter in Escherichia coli for succinate biosynthesis. For balancing metabolic flux to maximize succinate titer, a combinatorial optimization strategy to fine‐tuning CO2 transport and fixation process was implemented by promoter engineering in this study. Firstly, based on the energy matrix a synthetic promoter library containing 20 rationally designed promoters with strengths ranging from 0.8% to 100% compared with the widely used trc promoter was generated. Evaluations of rfp and cat reporter genes provided evidence that the synthetic promoters were stably and had certain applicability. Secondly, four designed promoters with different strengths were used for combinatorial assembly of single CO2 transport gene (sbtA or bicA) and single CO2 fixation gene (ppc or pck) expression. Three combinations, such as Tang1519 (P4‐bicA + pP19‐pck), Tang1522 (P4‐sbtA + P4‐ppc), Tang1523 (P4‐sbtA + P17‐ppc) with a more than 10% increase in succinate production were screened in bioreactor. Finally, based on the above results, co‐expression of the four transport and fixation genes were further investigated. Co‐expression of sbtA, bicA, and ppc with weak promoter P4 and pck with strong promoter P19 (AFP111/pT‐P4‐bicA‐P4‐sbtA + pACYC‐P19‐pck‐P4‐ppc) provided the best succinate production among all the combinations. The highest succinate production of 89.4 g/L was 37.5% higher than that obtained with empty vector control. This work significantly enhanced succinate production through combinatorial optimization of CO2 transport and fixation. The promoter engineering and combinatorial optimization strategies used herein represents a powerful approach to tailor‐making metabolic pathways for the production of other industrially important chemicals. Biotechnol. Bioeng. 2016;113: 1531–1541. © 2016 Wiley Periodicals, Inc.
Tunable osteogenic differentiation of hMPCs in tubular perfusion system bioreactor
Authors: Nguyen Bao‐Ngoc B. , Ko Henry , Fisher John P. 
Published: January 2016

ABSTRACTThe use of bioreactors for bone tissue engineering has been widely investigated. While the benefits of shear stress on osteogenic differentiation are well known, the underlying effects of dynamic culture on subpopulations within a bioreactor are less evident. In this work, we explore the influence of applied flow in the tubular perfusion system (TPS) bioreactor on the osteogenic differentiation of human mesenchymal progenitor cells (hMPCs), specifically analyzing the effects of axial position along the growth chamber. TPS bioreactor experiments conducted with unidirectional flow demonstrated enhanced expression of osteogenic markers in cells cultured downstream from the inlet flow. We utilized computational fluid dynamic modeling to confirm uniform shear stress distribution on the surface of the scaffolds and along the length of the growth chamber. The concept of paracrine signaling between cell populations was validated with the use of alternating flow, which diminished the differences in osteogenic differentiation between cells cultured at the inlet and outlet of the growth chamber. After the addition of controlled release of bone morphogenic protein‐2 (BMP‐2) into the system, osteogenic differentiation among subpopulations along the growth chamber was augmented, yet remained homogenous. These results allow for greater understanding of axial bioreactor cultures, their microenvironment, and how well‐established parameters of osteogenic differentiation affect bone tissue development. With this work, we have demonstrated the capability of tuning osteogenic differentiation of hMPCs through the application of fluid flow and the addition of exogenous growth factors. Such precise control allows for the culture of distinct subpopulation within one dynamic system for the use of complex engineered tissue constructs. Biotechnol. Bioeng. 2016;113: 1805–1813. © 2016 Wiley Periodicals, Inc.
A droplet microfluidics platform for rapid microalgal growth and oil production analysis
Authors: Kim Hyun Soo , Guzman Adrian R. , Thapa Hem R. , Devarenne Timothy P. , Han Arum 
Published: January 2016

ABSTRACTMicroalgae have emerged as a promising source for producing future renewable biofuels. Developing better microalgal strains with faster growth and higher oil production rates is one of the major routes towards economically viable microalgal biofuel production. In this work, we present a droplet microfluidics‐based microalgae analysis platform capable of measuring growth and oil content of various microalgal strains with single‐cell resolution in a high‐throughput manner. The platform allows for encapsulating a single microalgal cell into a water‐in‐oil emulsion droplet and tracking the growth and division of the encapsulated cell over time, followed by on‐chip oil quantification. The key feature of the developed platform is its capability to fluorescently stain microalgae within microdroplets for oil content quantification. The performance of the developed platform was characterized using the unicellular microalga Chlamydomonas reinhardtii and the colonial microalga Botryococcus braunii. The application of the platform in quantifying growth and oil accumulation was successfully confirmed using C. reinhardtii under different culture conditions, namely nitrogen‐replete and nitrogen‐limited conditions. These results demonstrate the capability of this platform as a rapid screening tool that can be applied to a wide range of microalgal strains for analyzing growth and oil accumulation characteristics relevant to biofuel strain selection and development. Biotechnol. Bioeng. 2016;113: 1691–1701. © 2016 Wiley Periodicals, Inc.
Synthetic and natural consensus design for engineering charge within an affibody targeting epidermal growth factor receptor
Authors: Case Brett A. , Hackel Benjamin J. 
Published: January 2016

ABSTRACTProtein ligand charge can impact physiological delivery with charge reduction often benefiting performance. Yet neutralizing mutations can be detrimental to protein function. Herein, three approaches are evaluated to introduce charged‐to‐neutral mutations of three cations and three anions within an affibody engineered to bind epidermal growth factor receptor. These approaches—combinatorial library sorting or consensus design, based on natural homologs or library‐sorted mutants—are used to identify mutations with favorable affinity, stability, and recombinant yield. Consensus design, based on 942 affibody homologs, yielded a mutant of modest function (Kd = 11 ±4 nM, Tm = 62°C, and yield = 4.0 ± 0.8 mg/L as compared to 5.3 ± 1.7 nM, 71°C, and 3.5 ± 0.3 mg/L for the parental affibody). Extension of consensus design to 10 additional mutants exhibited varied performance including a substantially improved mutant (Kd = 6.9 ± 1.4 nM, Tm = 71°C, and 12.7 ± 0.9 mg/L yield). Sorting a homolog‐based combinatorial library of 7 × 105 mutants generated a distribution of mutants with lower stability and yield, but did identify one strongly binding variant (Kd = 1.2 ± 0.3 nM, Tm = 69°C, and 6.0 ± 0.4 mg/L yield). Synthetic consensus design, based on the amino acid distribution in functional library mutants, yielded higher affinities (P = 0.05) with comparable stabilities and yields. The best of four analyzed clones had Kd = 1.7 ± 0.5 nM, Tm = 68°C, and 7.0 ± 0.5 mg/L yield. While all three approaches were effective in creating targeted affibodies with six charged‐to‐neutral mutations, synthetic consensus design proved to be the most robust. Synthetic consensus design provides a valuable tool for ligand engineering, particularly in the context of charge manipulation. Biotechnol. Bioeng. 2016;113: 1628–1638. © 2016 Wiley Periodicals, Inc.
Redistribution of connexin 43 during cell division
Authors: Vanderpuye Oluseyi A. , Bell Cheryl L. , Murray Sandra A. 
Published: January 2016

AbstractGap junction channels, once clustered into gap junction plaques, allow communication of essential metabolites between cells. Gap junction plaques have been reported to be lost from the cell surface during cell division. The mechanism involved in this loss of gap junction plaques during mitosis is unclear, but we hypothesize that an endoexocytotic mechanism that results in cytoplasmic double‐membraned annular gap junction vesicles is involved. In this study, gap junction plaque changes in dividing cells were examined in SW‐13 adrenocortical tumor cells. Endogenous gap junction protein, connexin 43 (Cx43), was detected with immunofluorescence, and live cell imaging was used to monitor green fluorescent protein‐tagged Cx43 (Cx43‐GFP). Mitotic stages were identified by Hoechst chromosomal staining. During interphase, large gap junction plaques were detected; however, the presence of these plaques decreased, whereas cytoplasmic puncta increased beginning with prophase. The cytoplasmic puncta were demonstrated with immunoelectron microscopy to be Cx43‐ positive annular gap junction vesicles. As gap junction plaques reformed at cleavage furrows between daughter cells, the number of annular gap junctions decreased during cytokinesis. The data are consistent with the mechanism of gap junction plaque loss during mitosis relying on an endoexocytotic process that results in annular gap junction vesicles formation. The rapid formation of gap junction plaques during cytokinesis points to the intriguing possibility of connexin recycling from annular gap junction vesicles to form gap junction plaques as mitosis is completed.
A new breakable stent for recoarctation in early infancy: Preliminary Clinical Experience
Authors: Grohmann Jochen , Sigler Matthias , Siepe Matthias , Stiller Brigitte 
Published: January 2016

ObjectivesTranscatheter treatment of aortic coarctation (CoA) via stent implantation has become an established treatment option depending on the patient's age and CoA type.BackgroundThe Osypka BabyStent® is a low‐profiled, balloon‐expandable cobalt‐chrome stent to treat aortic CoA in infants, which is breakable to permit unrestricted growth. We hereby evaluated our initial clinical experience to demonstrate the feasibility and efficacy of this new nonlicensed device, which we have occasionally implanted in critically ill patients or when redo‐surgery would entail an excessively high risk.MethodsRetrospective single‐center analysis of all available data during and after treatment with a BabyStent implanted in infants with considerable re‐CoA or reobstruction of the aortic arch after former surgery. All interventions took place under fluoroscopy and conscious sedation with local anesthesia or general anesthesia.ResultsFive BabyStents were implanted in four infants with technical success in all of them—median age 10 weeks (range 5–21), median bodyweight 3.8 kg (range 2.7–4.5). Aortic diameters enlarged from median 2.25 mm (range 1.5–3.3) to median 5.3 mm (range 4.6–6.0). The follow‐up period lasting up to 26 months (median 8.5, range 2–26) was uneventful concerning stent‐related complications.ConclusionsBabyStent® implantation for recoarctation was effective. However, our initial experience with the device is limited to short‐ and midterm follow‐up only. None of the stents was subsequently overdilated with the intention to break due to our patients’ limited somatic growth so far. A multicenter survey has been initiated to justify device approval. © 2016 Wiley Periodicals, Inc.