bluuebunny/crossref_metadata_2025_split huggingface dataset:
Itaconic acid production from glycerol using Escherichia coli harboring a random synonymous codon‐substituted 5′‐coding region variant of the cadA gene
Authors: Jeon Ho‐Geun , Cheong Dae‐Eun , Han Yunjon , Song Jae Jun , Choi Jong Hyun 
Published: December 2015

ABSTRACTAspergillus terreus cadA, encoding cis‐aconitate decarboxylase, is an essential gene for itaconic acid (IA) biosynthesis, but it is primarily expressed as insoluble aggregates in most industrial hosts. This has been a hurdle for the development of recombinant strategies for IA production. Here, we created a library of synonymous codon variants (scv) of the cadA gene containing synonymous codons in the first 10 codons (except ATG) and screened it in Escherichia coli. Among positive clones, E. coli scvCadA_No8 showed more than 95% of expressed CadA in the soluble fraction, and in production runs, produced threefold more IA than wild‐type E. coli in Luria‐Bertani broth supplemented with 0.5% glucose. In M9 minimal media containing 0.85 g/L citrate and 1% glycerol, E. coli scvCadA_No8 produced 985.6 ± 33.4 mg/L IA during a 72‐h culture after induction with isopropyl β‐D‐1‐thiogalactopyranoside. In a 2‐L fed‐batch fermentation consisting of two stages (growth and nitrogen limitation conditions), we obtained 7.2 g/L IA by using E. coli by introducing only the scv_cadA gene and optimizing culture conditions for IA production. These results could be combined with metabolic engineering and generate an E. coli strain as an industrial IA producer. Biotechnol. Bioeng. 2016;113: 1504–1510. © 2015 Wiley Periodicals, Inc.
Modification of gellan gum with nanocrystalline hydroxyapatite facilitates cell expansion and spontaneous osteogenesis
Authors: Jamshidi Parastoo , Chouhan Gurpreet , Williams Richard L. , Cox Sophie C. , Grover Liam M. 
Published: December 2015

ABSTRACTNanocomposites composed of hydrogels and calcium phosphates are of great interest in the development of bone graft replacements since they may have a structural and compositional resemblance to bone. Culture beads formed from such materials could be used in stirred tank culture and thereby enable cell expansion in a sufficiently efficient manner to allow for the generation of enough large number of cells for large‐scale bone reconstruction. Although combinations of materials such as alginate, collagens, and various calcium phosphates have been investigated as culture beads, these materials are unsuitable for application since they have been shown to rapidly degrade in physiological conditions and enable relatively little tailoring of mechanical properties. In this study, gellan gum‐nano sized hydroxyapatite (nHA) composites, which have been shown to be resistant to degradation and easily modified with respect to modulus, were formulated and characterized as regards their ability to enable cell attachment and proliferation. It was shown that the addition of 5 wt% of nHA to the culture beads enabled cell attachment and that an increase in nHA concentration to up to 25 wt% enhanced the rate of cell proliferation. Most importantly, it was demonstrated that the addition of nHA to the cell culture beads enabled the formation of nodules in culture of MC3T3‐E1 cells and strikingly stimulated the osteogenic differentiation of bone marrow stromal cells in the absence of osteogenic media when compared with tissue culture plastic (TCP) with the same condition. Biotechnol. Bioeng. 2016;113: 1568–1576. © 2016 Wiley Periodicals, Inc.
Droplet digital PCR‐aided screening and characterization of Pichia pastoris multiple gene copy strains
Authors: Cámara Elena , Albiol Joan , Ferrer Pau 
Published: December 2015

ABSTRACTPichia (syn. Komagataella) pastoris is a widely used yeast platform for heterologous protein production. Expression cassettes are usually stably integrated into the genome of this host via homologous recombination. Although increasing gene dosage is a powerful strategy to improve recombinant protein production, an excess in the number of gene copies often leads to decreased product yields and increased metabolic burden, particularly for secreted proteins. We have constructed a series of strains harboring different copy numbers of a Rhizopus oryzae lipase gene (ROL), aiming to find the optimum gene dosage for secreted Rol production. In order to accurately determine ROL gene dosage, we implemented a novel protocol based on droplet digital PCR (ddPCR), and cross validated it with conventional real‐time PCR. Gene copy number determination based on ddPCR allowed for an accurate ranking of transformants according to their ROL gene dosage. Results indicated that ddPCR was particularly superior at lower gene dosages (one to five copies) over quantitative real‐time PCR (qPCR). This facilitated the determination of the optimal ROL gene dosage as low as two copies. The ranking of ROL gene dosage versus Rol yield was consistent at both small scale and bioreactor chemostat cultures, thereby easing clone characterization in terms of gene dosage dependent physiological effects, which could be discriminated even among strains differing by only one ROL copy. A selected two‐copy strain showed twofold increase in Rol specific production in a chemostat culture over the single copy strain. Conversely, strains harboring more than two copies of the ROL gene showed decreased product and biomass yields, as well as altered substrate consumption specific rates, compared to the reference (one‐copy) strain. Biotechnol. Bioeng. 2016;113: 1542–1551. © 2015 Wiley Periodicals, Inc.
Kinetics of tRNAPyl‐mediated amber suppression in Escherichia coli translation reveals unexpected limiting steps and competing reactions
Authors: Wang Jinfan , Kwiatkowski Marek , Forster Anthony C. 
Published: December 2015

ABSTRACTThe utility of ribosomal incorporation of unnatural amino acids (AAs) in vivo is generally restricted by low efficiencies, even with the most widely used suppressor tRNAPyl. Because of the difficulties of studying incorporation in vivo, almost nothing is known about the limiting steps after tRNA charging. Here, we measured the kinetics of all subsequent steps using a purified Escherichia coli translation system. Dipeptide formation from initiator fMet‐tRNAfMet and tRNAPyl charged with allylglycine or methylserine displayed unexpectedly sluggish biphasic kinetics, ∼30‐fold slower than for native substrates. The amplitude of the fast phases increased with increasing EF‐Tu concentration, allowing measurement of Kd values of EF‐Tu binding, both of which were ∼25‐fold weaker than normal. However, binding could be increased ∼30‐fold by lowering temperature. The fast phase rates were limited by the surprisingly ∼10‐fold less efficient binding of EF‐Tu:GTP:AA‐tRNAPyl ternary complex to the ribosomes, not GTP hydrolysis or peptide bond formation. Furthermore, processivity was unexpectedly impaired as ∼40% of the dipeptidyl‐tRNAPyl could not be elongated to tripeptide. Dipeptide formation was slow enough that termination due to misreading the UAG codon by non‐cognate RF2 became very significant. This new understanding provides a framework for improving unnatural AA incorporation by amber suppression. Biotechnol. Bioeng. 2016;113: 1552–1559. © 2015 Wiley Periodicals, Inc.
The effect of PVDF‐TrFE scaffolds on stem cell derived cardiovascular cells
Authors: Hitscherich Pamela , Wu Siliang , Gordan Richard , Xie Lai‐Hua , Arinzeh Treena , Lee Eun Jung 
Published: December 2015

ABSTRACTRecently, electrospun polyvinylidene fluoride (PVDF) and polyvinylidene fluoride‐trifluoroethylene (PVDF‐TrFE) scaffolds have been developed for tissue engineering applications. These materials have piezoelectric activity, wherein they can generate electric charge with minute mechanical deformations. Since the myocardium is an electroactive tissue, the unique feature of a piezoelectric scaffold is attractive for cardiovascular tissue engineering applications. In this study, we examined the cytocompatibility and function of pluripotent stem cell derived cardiovascular cells including mouse embryonic stem cell‐derived cardiomyocytes (mES‐CM) and endothelial cells (mES‐EC) on PVDF‐TrFE scaffolds. MES‐CM and mES‐EC adhered well to PVDF‐TrFE and became highly aligned along the fibers. When cultured on scaffolds, mES‐CM spontaneously contracted, exhibited well‐registered sarcomeres and expressed classic cardiac specific markers such as myosin heavy chain, cardiac troponin T, and connexin43. Moreover, mES‐CM cultured on PVDF‐TrFE scaffolds responded to exogenous electrical pacing and exhibited intracellular calcium handling behavior similar to that of mES‐CM cultured in 2D. Similar to cardiomyocytes, mES‐EC also demonstrated high viability and maintained a mature phenotype through uptake of low‐density lipoprotein and expression of classic endothelial cell markers including platelet endothelial cell adhesion molecule, endothelial nitric oxide synthase, and the arterial specific marker, Notch‐1. This study demonstrates the feasibility of PVDF‐TrFE scaffold as a candidate material for developing engineered cardiovascular tissues utilizing stem cell‐derived cells. Biotechnol. Bioeng. 2016;113: 1577–1585. © 2015 Wiley Periodicals, Inc.
Combining 3D human in vitro methods for a 3Rs evaluation of novel titanium surfaces in orthopaedic applications
Authors: Stevenson G. , Rehman S. , Draper E. , Hernández‐Nava E. , Hunt J. , Haycock J.W. 
Published: December 2015

ABSTRACTIn this study, we report on a group of complementary human osteoblast in vitro test methods for the preclinical evaluation of 3D porous titanium surfaces. The surfaces were prepared by additive manufacturing (electron beam melting [EBM]) and plasma spraying, allowing the creation of complex lattice surface geometries. Physical properties of the surfaces were characterized by SEM and profilometry and 3D in vitro cell culture using human osteoblasts. Primary human osteoblast cells were found to elicit greater differences between titanium sample surfaces than an MG63 osteoblast‐like cell line, particularly in terms of cell survival. Surface morphology was associated with higher osteoblast metabolic activity and mineralization on rougher titanium plasma spray coated surfaces than smoother surfaces. Differences in osteoblast survival and metabolic activity on titanium lattice structures were also found, despite analogous surface morphology at the cellular level. 3D confocal microscopy identified osteoblast organization within complex titanium surface geometries, adhesion, spreading, and alignment to the biomaterial strut geometries. Mineralized nodule formation throughout the lattice structures was also observed, and indicative of early markers of bone in‐growth on such materials. Testing methods such as those presented are not traditionally considered by medical device manufacturers, but we suggest have value as an increasingly vital tool in efficiently translating pre‐clinical studies, especially in balance with current regulatory practice, commercial demands, the 3Rs, and the relative merits of in vitro and in vivo studies. Biotechnol. Bioeng. 2016;113: 1586–1599. © 2015 The Authors. Biotechnology and Bioengineering Published by Wiley Periodicals, Inc.
TAP‐mediated processing of exoerythrocytic antigens is essential for protection induced with radiation‐attenuated Plasmodium sporozoites
Authors: Pichugin Alexander , Steers Nick , De La Vega Patricia , Zarling Stasya , Chalom Isaac , Krzych Urszula 
Published: December 2015

MHC class I dependent CD8+ T cells are essential for protection induced by radiation‐attenuated Plasmodium sporozoites (RAS) in murine malaria models. Apart from the mechanism of activation of CD8+ T cells specific for the circumsporozoite protein, the major sporozoite antigen (Ag), CD8+ T cells specific for other exoerythrocytic Ags that have been shown to mediate protection have not been thoroughly investigated. Specifically, mechanisms of processing and presentation of exoerythrocytic Ags, which includes liver stage (LS) Ags, remain poorly understood. We hypothesize that as exogenous proteins, LS Ags are processed by mechanisms involving either the TAP‐dependent phagosomal‐to‐cytosol or TAP‐independent vacuolar pathway of cross‐presentation. We used TAP‐deficient mice to investigate whether LS Ag mediated induction of naïve CD8+ T cells and their recall during sporozoite challenge occur by the TAP‐dependent or TAP‐independent pathways. On the basis of functional attributes, CD8+ T cells were activated via the TAP‐independent pathway during immunizations with Plasmodium berghei RAS; however, IFN‐γ+CD8+ T cells previously induced by P. berghei RAS in TAP‐deficient mice failed to be recalled against sporozoite challenge and the mice became parasitemic. On the basis of these observations, we propose that TAP‐associated Ag processing is indispensable for sterile protection induced with P. berghei RAS.
A stabilized Nitsche‐type extended embedding mesh approach for 3D low‐ and high‐Reynolds‐number flows
Authors: Schott B. , Shahmiri S. , Kruse R. , Wall W.A. 
Published: December 2015

SummaryThis paper presents a stabilized extended finite element method (XFEM) based fluid formulation to embed arbitrary fluid patches into a fixed background fluid mesh. The new approach is highly beneficial when it comes to computational grid generation for complex domains, as it allows locally increased resolutions independent from size and structure of the background mesh. Motivating applications for such a domain decomposition technique are complex fluid‐structure interaction problems, where an additional boundary layer mesh is used to accurately capture the flow around the structure. The objective of this work is to provide an accurate and robust XFEM‐based coupling for low‐ as well as high‐Reynolds‐number flows. Our formulation is built from the following essential ingredients: Coupling conditions on the embedded interface are imposed weakly using Nitsche's method supported by extra terms to guarantee mass conservation and to control the convective mass transport across the interface for transient viscous‐dominated and convection‐dominated flows. Residual‐based fluid stabilizations in the interior of the fluid subdomains and accompanying face‐oriented fluid and ghost‐penalty stabilizations in the interface zone stabilize the formulation in the entire fluid domain. A detailed numerical study of our stabilized embedded fluid formulation, including an investigation of variants of Nitsche's method for viscous flows, shows optimal error convergence for viscous‐dominated and convection‐dominated flow problems independent of the interface position. Challenging two‐dimensional and three‐dimensional numerical examples highlight the robustness of our approach in all flow regimes: benchmark computations for laminar flow around a cylinder, a turbulent driven cavity flow at Re = 10000 and the flow interacting with a three‐dimensional flexible wall. Copyright © 2016 John Wiley & Sons, Ltd.
Dynamic thalamus parcellation from resting‐state fMRI data
Authors: Ji Bing , Li Zhihao , Li Kaiming , Li Longchuan , Langley Jason , Shen Hui , Nie Shengdong , Zhang Renjie , Hu Xiaoping 
Published: December 2015

AbstractThe thalamus is a relay center between various subcortical brain areas and the cerebral cortex with delineation of its constituent nuclei being of particular interest in many applications. While previous studies have demonstrated efficacy of connectivity‐based thalamus segmentation, they used approaches that do not consider the dynamic nature of thalamo‐cortical interactions. In this study, we explicitly exploited the dynamic variation of thalamo‐cortical connections to identify different states of functional connectivity and performed state‐specific thalamus parcellation. With normalized spectral clustering successively applied in temporal and spatial domains, nine thalamo‐cortical connectivity states were identified and the dynamic thalamus parcellation revealed finer thalamic structures with improved atlas correspondence. The present results extend our understanding of thalamo‐cortical connectivity and provide a more comprehensive view of the thalamo‐cortical interaction. Hum Brain Mapp 37:954–967, 2016. © 2015 Wiley Periodicals, Inc.
Intensity‐modulated radiotherapy for head and neck surgeons
Authors: Gutiontov Stanley I. , Shin Edward J. , Lok Benjamin , Lee Nancy Y. , Cabanillas Ruben 
Published: December 2015

AbstractThe development of intensity‐modulated radiotherapy (IMRT) has played a major role in improving outcomes and decreasing morbidity in patients with head and neck cancer. This review addresses this vital modality with a focus on the important role of the head and neck surgeon. The technique as well as its benefits and points of caution are outlined, the definitions of tumor and treatment volumes are discussed, and the dose and fractionation are detailed. Following this are several sections dedicated to the role of the head and neck surgeon in the planning of both definitive and postoperative radiotherapy to the primary site and neck. There is a focus throughout on anatomic and surgical considerations; commonly encountered situations are illustrated. With a deeper understanding of this technique and their own pivotal contribution to target delineation, head and neck surgeons will be poised to expand their role and improve cancer care for their patients. © 2015 Wiley Periodicals, Inc. Head Neck 38: E2368–E2373, 2016
Intensity‐modulated proton therapy for nasopharyngeal carcinoma: Decreased radiation dose to normal structures and encouraging clinical outcomes
Authors: Lewis Gary D. , Holliday Emma B. , Kocak–Uzel Esengul , Hernandez Mike , Garden Adam S. , Rosenthal David I. , Frank Steven J. 
Published: December 2015

AbstractBackgroundIntensity‐modulated proton therapy (IMPT) has the potential to spare dose to organs at risk (OAR) when compared to intensity‐modulated radiotherapy (IMRT) while maintaining excellent clinical outcomes.MethodsTen patients with nasopharyngeal carcinoma (NPC) were identified for whom IMPT was planned; 9 patients also had a comparison photon‐based IMRT plan generated. Dosimetric comparison of mean radiation dose to 29 adjacent OAR was performed. Disease control, survival, and toxicity outcomes were collected from the medical records.ResultsThere were significant differences in mean doses in 15 of the 29 OAR; 13 OAR received lower mean dose with proton‐based plans. Median follow‐up was 24.5 months (range, 19–32 months). Two‐year locoregional control was 100% and the 2‐year overall survival was 88.9%.ConclusionWe observed dosimetric advantages conferred by IMPT compared to IMRT. Further study is needed to determine if these translate into reduced toxicity and/or improved disease control. © 2015 Wiley Periodicals, Inc. Head Neck 38: E1886–E1895, 2016
Learning history and cholinergic modulation in the dorsal hippocampus are necessary for rats to infer the status of a hidden event
Authors: Fast Cynthia D. , Flesher M. Melissa , Nocera Nathanial A. , Fanselow Michael S. , Blaisdell Aaron P. 
Published: December 2015

ABSTRACTIdentifying statistical patterns between environmental stimuli enables organisms to respond adaptively when cues are later observed. However, stimuli are often obscured from detection, necessitating behavior under conditions of ambiguity. Considerable evidence indicates decisions under ambiguity rely on inference processes that draw on past experiences to generate predictions under novel conditions. Despite the high demand for this process and the observation that it deteriorates disproportionately with age, the underlying mechanisms remain unknown. We developed a rodent model of decision‐making during ambiguity to examine features of experience that contribute to inference. Rats learned either a simple (positive patterning) or complex (negative patterning) instrumental discrimination between the illumination of one or two lights. During test, only one light was lit while the other relevant light was blocked from physical detection (covered by an opaque shield, rendering its status ambiguous). We found experience with the complex negative patterning discrimination was necessary for rats to behave sensitively to the ambiguous test situation. These rats behaved as if they inferred the presence of the hidden light, responding differently than when the light was explicitly absent (uncovered and unlit). Differential expression profiles of the immediate early gene cFos indicated hippocampal involvement in the inference process while localized microinfusions of the muscarinic antagonist, scopolamine, into the dorsal hippocampus caused rats to behave as if only one light was present. That is, blocking cholinergic modulation prevented the rat from inferring the presence of the hidden light. Collectively, these results suggest cholinergic modulation mediates recruitment of hippocampal processes related to past experiences and transfer of these processes to make decisions during ambiguous situations. Our results correspond with correlations observed between human brain function and inference abilities, suggesting our experiments may inform interventions to alleviate or prevent cognitive dysfunction. © 2015 Wiley Periodicals, Inc.
Association of Serum Asymmetric Dimethylarginine With Muscle Strength and Gait Speed: A Cross-Sectional Study of the HEIJO-KYO Cohort
Authors: Obayashi Kenji , Saeki Keigo , Maegawa Taeko , Sakai Takahide , Kitagawa Maiko , Otaki Naoto , Kataoka Hiroshi , Kurumatani Norio 
Published: December 2015

ABSTRACT

Muscle strength and gait speed are related with functional limitations and disabilities and also predict cardiovascular and all-cause mortality. Nitric oxide (NO) plays an important role in regulating physiological process in skeletal muscles; however, the association between serum asymmetric dimethylarginine (ADMA) level, an endogenous competitive inhibitor of NO synthesis, and physical performance has not yet been studied. We investigated the associations of serum ADMA level with muscle strength and usual gait speed in a cross-sectional study of 550 elderly individuals (mean age, 71.2 ± 6.6 years). Mean ADMA level was 0.45 ± 0.06 µmol/L; mean grip and quadriceps strengths were 27.7 ± 8.4 kg and 165.1 ± 81.6 Nm, respectively; and mean gait speed was 1.37 ± 0.30 m/s. In multivariate linear regression analysis adjusted for potential confounding factors (age, gender, body weight, smoking and drinking status, household income, hypertension, diabetes, renal function, and physical activity), higher serum ADMA level was significantly associated with lower grip and quadriceps strengths and slower gait speed (grip strength: β, –1.257; 95% confidence interval [CI], –1.990 to –0.525; p = 0.001; quadriceps strength: β, –11.730; 95% CI, –20.924 to –2.536; p = 0.012; gait speed: β, –0.065; 95% CI, –0.108 to –0.022; p = 0.003). Our findings indicate the significant association between serum ADMA level and physical performance among elderly individuals, which was independent of the important potential confounders. © 2015 American Society for Bone and Mineral Research.