bluuebunny/crossref_metadata_2025_split huggingface dataset:
Structural basis for dynamic mechanism of nitrate/nitrite antiport by NarK
Authors: Fukuda Masahiro , Takeda Hironori , Kato Hideaki E. , Doki Shintaro , Ito Koichi , Maturana Andrés D. , Ishitani Ryuichiro , Nureki Osamu 
Published: May 2015

AbstractNarK belongs to the nitrate/nitrite porter (NNP) family in the major facilitator superfamily (MFS) and plays a central role in nitrate uptake across the membrane in diverse organisms, including archaea, bacteria, fungi and plants. Although previous studies provided insight into the overall structure and the substrate recognition of NarK, its molecular mechanism, including the driving force for nitrate transport, remained elusive. Here we demonstrate that NarK is a nitrate/nitrite antiporter, using an in vitro reconstituted system. Furthermore, we present the high-resolution crystal structures of NarK from Escherichia coli in the nitrate-bound occluded, nitrate-bound inward-open and apo inward-open states. The integrated structural, functional and computational analyses reveal the nitrate/nitrite antiport mechanism of NarK, in which substrate recognition is coupled to the transport cycle by the concomitant movement of the transmembrane helices and the key tyrosine and arginine residues in the substrate-binding site.
Characterization of genome-wide ordered sequence-tagged Mycobacterium mutant libraries by Cartesian Pooling-Coordinate Sequencing
Authors: Vandewalle Kristof , Festjens Nele , Plets Evelyn , Vuylsteke Marnik , Saeys Yvan , Callewaert Nico 
Published: May 2015

AbstractReverse genetics research approaches require the availability of methods to rapidly generate specific mutants. Alternatively, where these methods are lacking, the construction of pre-characterized libraries of mutants can be extremely valuable. However, this can be complex, expensive and time consuming. Here, we describe a robust, easy to implement parallel sequencing-based method (Cartesian Pooling-Coordinate Sequencing or CP-CSeq) that reports both on the identity as well as on the location of sequence-tagged biological entities in well-plate archived clone collections. We demonstrate this approach using a transposon insertion mutant library of the Mycobacterium bovis BCG vaccine strain, providing the largest resource of mutants in any strain of the M. tuberculosis complex. The method is applicable to any entity for which sequence-tagged identification is possible.
STED nanoscopy with fluorescent quantum dots
Authors: Hanne Janina , Falk Henning J. , Görlitz Frederik , Hoyer Patrick , Engelhardt Johann , Sahl Steffen J. , Hell Stefan W. 
Published: May 2015

AbstractThe widely popular class of quantum-dot molecular labels could so far not be utilized as standard fluorescent probes in STED (stimulated emission depletion) nanoscopy. This is because broad quantum-dot excitation spectra extend deeply into the spectral bands used for STED, thus compromising the transient fluorescence silencing required for attaining super-resolution. Here we report the discovery that STED nanoscopy of several red-emitting commercially available quantum dots is in fact successfully realized by the increasingly popular 775 nm STED laser light. A resolution of presently ∼50 nm is demonstrated for single quantum dots, and sub-diffraction resolution is further shown for imaging of quantum-dot-labelled vimentin filaments in fibroblasts. The high quantum-dot photostability enables repeated STED recordings with >1,000 frames. In addition, we have evidence that the tendency of quantum-dot labels to blink is largely suppressed by combined action of excitation and STED beams. Quantum-dot STED significantly expands the realm of application of STED nanoscopy, and, given the high stability of these probes, holds promise for extended time-lapse imaging.
Change in ocean subsurface environment to suppress tropical cyclone intensification under global warming
Authors: Huang Ping , Lin I. -I , Chou Chia , Huang Rong-Hui 
Published: May 2015

AbstractTropical cyclones (TCs) are hazardous natural disasters. Because TC intensification is significantly controlled by atmosphere and ocean environments, changes in these environments may cause changes in TC intensity. Changes in surface and subsurface ocean conditions can both influence a TC’s intensification. Regarding global warming, minimal exploration of the subsurface ocean has been undertaken. Here we investigate future subsurface ocean environment changes projected by 22 state-of-the-art climate models and suggest a suppressive effect of subsurface oceans on the intensification of future TCs. Under global warming, the subsurface vertical temperature profile can be sharpened in important TC regions, which may contribute to a stronger ocean coupling (cooling) effect during the intensification of future TCs. Regarding a TC, future subsurface ocean environments may be more suppressive than the existing subsurface ocean environments. This suppressive effect is not spatially uniform and may be weak in certain local areas.
Highly reproductive Escherichia coli cells with no specific assignment to the UAG codon
Authors: Mukai Takahito , Hoshi Hiroko , Ohtake Kazumasa , Takahashi Mihoko , Yamaguchi Atsushi , Hayashi Akiko , Yokoyama Shigeyuki , Sakamoto Kensaku 
Published: May 2015

AbstractEscherichia coli is a widely used host organism for recombinant technology and the bacterial incorporation of non-natural amino acids promises the efficient synthesis of proteins with novel structures and properties. In the present study, we developed E. coli strains in which the UAG codon was reserved for non-natural amino acids, without compromising the reproductive strength of the host cells. Ninety-five of the 273 UAG stop codons were replaced synonymously in the genome of E. coli BL21(DE3), by exploiting the oligonucleotide-mediated base-mismatch-repair mechanism. This genomic modification allowed the safe elimination of the UAG-recognizing cellular component (RF-1), thus leaving the remaining 178 UAG codons with no specific molecule recognizing them. The resulting strain B-95.ΔA grew as vigorously as BL21(DE3) in rich medium at 25–42°C and its derivative B-95.ΔAΔfabR was better adapted to low temperatures and minimal media than B-95.ΔA. UAG was reassigned to synthetic amino acids by expressing the specific pairs of UAG-reading tRNA and aminoacyl-tRNA synthetase. Due to the preserved growth vigor, the B-95.ΔA strains showed superior productivities for hirudin molecules sulfonated on a particular tyrosine residue and the Fab fragments of Herceptin containing multiple azido groups.
Brain structural alterations associated with young women with subthreshold depression
Authors: Li Haijiang , Wei Dongtao , Sun Jiangzhou , Chen Qunlin , Zhang Qinglin , Qiu Jiang 
Published: May 2015

AbstractNeuroanatomical abnormalities in patients with major depression disorder (MDD) have been attracted great research attention. However, the structural alterations associated with subthreshold depression (StD) remain unclear and, therefore, require further investigation. In this study, 42 young women with StD and 30 matched non-depressed controls (NCs) were identified based on two-time Beck Depression Inventory scores. Whole-brain voxel-based morphometry (VBM) and region of interest method were used to investigate altered gray matter volume (GMV) and white matter volume (WMV) among a non-clinical sample of young women with StD. VBM results indicated that young women with StD showed significantly decreased GMV in the right inferior parietal lobule than NCs; increased GMV in the amygdala, posterior cingulate cortex and precuneus; and increased WMV in the posterior cingulate cortex and precuneus. Together, structural alterations in specific brain regions, which are known to be involved in the fronto-limbic circuits implicated in depression may precede the occurrence of depressive episodes and influence the development of MDD.
Time-dependent mechanical-electrical coupled behavior in single crystal ZnO nanorods
Authors: Kim Yong-Jae , Yun Tae Gwang , Choi In-Chul , Kim Sungwoong , Park Won Il , Han Seung Min , Jang Jae-il 
Published: May 2015

AbstractNanoscale time-dependent mechanical-electrical coupled behavior of single crystal ZnO nanorods was systematically explored, which is essential for accessing the long-term reliability of the ZnO nanorod-based flexible devices. A series of compression creep tests combined with in-situ electrical measurement was performed on vertically-grown single crystal ZnO nanorods. Continuous measurement of the current (I)-voltage (V) curves before, during, after the creep tests revealed that I is non-negligibly increased as a result of the time-dependent deformation. Analysis of the I-V curves based on the thermionic emission-diffusion theory allowed extraction of nanorod resistance, which was shown to decrease as time-dependent deformation. Finally, based on the observations in this study, a simple analytical model for predicting the reduction in nanorod resistance as a function of creep strain that is induced from diffusional mechanisms is proposed and this model was demonstrated to be in an excellent agreement with the experimental results.
Chronic hypoxia in pregnancy affected vascular tone of renal interlobar arteries in the offspring
Authors: Tang Jiaqi , Zhu Zhoufeng , Xia Shuixiu , Li Na , Chen Ningjing , Gao Qinqin , Li Lingjun , Zhou Xiuwen , Li Dawei , Zhu Xiaolin , Tu Qing , Li Weisheng , Wu Chonglong , Li Jiayue , Zhong Yuan , Li Xiang , Mao Caiping , Xu Zhice 
Published: May 2015

AbstractHypoxia during pregnancy could affect development of fetuses as well as cardiovascular systems in the offspring. This study was the first to demonstrate the influence and related mechanisms of prenatal hypoxia (PH) on renal interlobar arteries (RIA) in the 5-month-old male rat offspring. Following chronic hypoxia during pregnancy, phenylephrine induced significantly higher pressor responses and greater vasoconstrictions in the offspring. Nitric oxide mediated vessel relaxation was altered in the RIA. Phenylephrine-stimulated free intracellular calcium was significantly higher in the RIA of the PH group. The activity and expression of L-type calcium channel (Cav1.2), not T-type calcium channel (Cav3.2), was up-regulated. The whole-cell currents of calcium channels and the currents of Cav1.2 were increased compared with the control. In addition, the whole-cell K+ currents were decreased in the offspring exposed to prenatal hypoxia. Activity of large-conductance Ca2+-activated K+ channels and the expression of MaxiKα was decreased in the PH group. The results provide new information regarding the influence of prenatal hypoxia on the development of the renal vascular system and possible underlying cellular and ion channel mechanisms involved.
DNA immunoprecipitation semiconductor sequencing (DIP-SC-seq) as a rapid method to generate genome wide epigenetic signatures
Authors: Thomson John P. , Fawkes Angie , Ottaviano Raffaele , Hunter Jennifer M. , Shukla Ruchi , Mjoseng Heidi K. , Clark Richard , Coutts Audrey , Murphy Lee , Meehan Richard R. 
Published: May 2015

AbstractModification of DNA resulting in 5-methylcytosine (5 mC) or 5-hydroxymethylcytosine (5hmC) has been shown to influence the local chromatin environment and affect transcription. Although recent advances in next generation sequencing technology allow researchers to map epigenetic modifications across the genome, such experiments are often time-consuming and cost prohibitive. Here we present a rapid and cost effective method of generating genome wide DNA modification maps utilising commercially available semiconductor based technology (DNA immunoprecipitation semiconductor sequencing; “DIP-SC-seq”) on the Ion Proton sequencer. Focussing on the 5hmC mark we demonstrate, by directly comparing with alternative sequencing strategies, that this platform can successfully generate genome wide 5hmC patterns from as little as 500 ng of genomic DNA in less than 4 days. Such a method can therefore facilitate the rapid generation of multiple genome wide epigenetic datasets.
The Smc5-Smc6 heterodimer associates with DNA through several independent binding domains
Authors: Roy Marc-André , Dhanaraman Thillaivillalan , D’Amours Damien 
Published: May 2015

AbstractThe Smc5-6 complex is required for the maintenance of genome integrity through its functions in DNA repair and chromosome biogenesis. However, the specific mode of action of Smc5 and Smc6 in these processes remains largely unknown. We previously showed that individual components of the Smc5-Smc6 complex bind strongly to DNA as monomers, despite the absence of a canonical DNA-binding domain (DBD) in these proteins. How heterodimerization of Smc5-6 affects its binding to DNA and which parts of the SMC molecules confer DNA-binding activity is not known at present. To address this knowledge gap, we characterized the functional domains of the Smc5-6 heterodimer and identify two DBDs in each SMC molecule. The first DBD is located within the SMC hinge region and its adjacent coiled-coil arms, while the second is found in the conserved ATPase head domain. These DBDs can independently recapitulate the substrate preference of the full-length Smc5 and Smc6 proteins. We also show that heterodimerization of full-length proteins specifically increases the affinity of the resulting complex for double-stranded DNA substrates. Collectively, our findings provide critical insights into the structural requirements for effective binding of the Smc5-6 complex to DNA repair substrates in vitro and in live cells.
Highly efficient and stable organic light-emitting diodes with a greatly reduced amount of phosphorescent emitter
Authors: Fukagawa Hirohiko , Shimizu Takahisa , Kamada Taisuke , Yui Shota , Hasegawa Munehiro , Morii Katsuyuki , Yamamoto Toshihiro 
Published: May 2015

AbstractOrganic light-emitting diodes (OLEDs) have been intensively studied as a key technology for next-generation displays and lighting. The efficiency of OLEDs has improved markedly in the last 15 years by employing phosphorescent emitters. However, there are two main issues in the practical application of phosphorescent OLEDs (PHOLEDs): the relatively short operational lifetime and the relatively high cost owing to the costly emitter with a concentration of about 10% in the emitting layer. Here, we report on our success in resolving these issues by the utilization of thermally activated delayed fluorescent materials, which have been developed in the past few years, as the host material for the phosphorescent emitter. Our newly developed PHOLED employing only 1 wt% phosphorescent emitter exhibits an external quantum efficiency of over 20% and a long operational lifetime of about 20 times that of an OLED consisting of a conventional host material and 1 wt% phosphorescent emitter.
Comparative analysis of potassium deficiency-responsive transcriptomes in low potassium susceptible and tolerant wheat (Triticum aestivum L.)
Authors: Ruan Li , Zhang Jiabao , Xin Xiuli , Zhang Congzhi , Ma Donghao , Chen Lin , Zhao Bingzi 
Published: May 2015

AbstractPotassium (K+) deficiency as a common abiotic stress can inhibit the growth of plants and thus reduce the agricultural yields. Nevertheless, scarcely any development has been promoted in wheat transcriptional changes under K+ deficiency. Here we investigated root transcriptional changes in two wheat genotypes, namely, low-K+ tolerant “Tongzhou916” and low-K+ susceptible “Shiluan02-1”. There were totally 2713 and 2485 probe sets displayed expression changes more than 1.5-fold in Tongzhou916 and Shiluan02-1, respectively. Low-K+ responsive genes mainly belonged to the categories as follows: metabolic process, cation binding, transferase activity, ion transporters and so forth. We made a comparison of gene expression differences between the two wheat genotypes. There were 1321 and 1177 up-regulated genes in Tongzhou916 and Shiluan02-1, respectively. This result indicated that more genes took part in acclimating to low-K+ stress in Tongzhou916. In addition, there were more genes associated with jasmonic acid, defense response and potassium transporter up-regulated in Tongzhou916. Moreover, totally 19 genes encoding vacuolar H+-pyrophosphatase, ethylene-related, auxin response, anatomical structure development and nutrient reservoir were uniquely up-regulated in Tongzhou916. For their important role in root architecture, K+ uptake and nutrient storage, unique genes above may make a great contribution to the strong low-K+ tolerance in Tongzhou916.
Single cell resolution in vivo imaging of DNA damage following PARP inhibition
Authors: Yang Katherine S. , Kohler Rainer H. , Landon Matthieu , Giedt Randy , Weissleder Ralph 
Published: May 2015

AbstractTargeting DNA repair pathways is a powerful strategy to treat cancers. To gauge efficacy in vivo, typical response markers include late stage effects such as tumor shrinkage, progression free survival, or invasive repeat biopsies. These approaches are often difficult to answer critical questions such as how a given drug affects single cell populations as a function of dose and time, distance from microvessels or how drug concentration (pharmacokinetics) correlates with DNA damage (pharmacodynamics). Here, we established a single-cell in vivo pharmacodynamic imaging read-out based on a truncated 53BP1 double-strand break reporter to determine whether or not poly(ADP-ribose) polymerase (PARP) inhibitor treatment leads to accumulation of DNA damage. Using this reporter, we show that not all PARP inhibitor treated tumors incur an increase in DNA damage. The method provides a framework for single cell analysis of cancer therapeutics in vivo.
SMAD3 deficiency promotes vessel wall remodeling, collagen fiber reorganization and leukocyte infiltration in an inflammatory abdominal aortic aneurysm mouse model
Authors: Dai Xiaohua , Shen Jianbin , Priyanka Annam Neeraja , Jiang Hong , Levi Edi , Schworer Charles M. , Tromp Gerard , Arora Anandita , Higgins Mary , Wang Xiao-Fan , Yang Maozhou , Li Hui J. , Zhang Kezhong , Kuivaniemi Helena , Li Li 
Published: May 2015

AbstractTGF-β signaling plays critical roles in the pathogenesis of aneurysms; however, it is still unclear whether its role is protective or destructive. In this study, we investigate the role of SMAD3 in the pathogenesis of calcium chloride (CaCl2)-induced abdominal aortic aneurysms (AAA) in Smad3−/−, Smad3+/− and Smad3+/+ mice. We find that loss of SMAD3 drastically increases wall thickening of the abdominal aorta. Histological analyses show significant vessel wall remodeling with elastic fiber fragmentation. Remarkably, under polarized light, collagen fibers in the hyperplastic adventitia of Smad3−/− mice show extensive reorganization accompanied by loosely packed thin and radial collagen fibers. The expressions of matrix metalloproteinases including MMP2, MMP9 and MMP12 and infiltration of macrophage/T cells are drastically enhanced in the vascular wall of Smad3−/− mice. We also observe marked increase of NF-κB and ERK1/2 signaling as well as the expression of nuclear Smad2, Smad4 and TGF-β1 in the vessel wall of Smad3−/− mice. In addition, we find that SMAD3 expression is reduced in the dedifferentiated medial smooth muscle-like cells of human AAA patients. These findings provide direct in vivo evidence to support the essential roles of SMAD3 in protecting vessel wall integrity and suppressing inflammation in the pathogenesis of AAAs.
Circulating mRNA Profiling in Esophageal Squamous Cell Carcinoma Identifies FAM84B As A Biomarker In Predicting Pathological Response to Neoadjuvant Chemoradiation
Authors: Hsu Feng-Ming , Chia-Hsien Cheng Jason , Chang Yih-Leong , Lee Jang-Ming , Koong Albert C. , Chuang Eric Y. 
Published: May 2015

AbstractEsophageal cancer patients with pathological complete response (pCR) to neoadjuvant chemoradiation (CRT) have favorable outcomes. Currently, there was no reliable biomarker predicting the response to CRT. Perioperative circulating mRNA may be associated with prognosis, but its application for predicting treatment response is unclear. We prospectively assessed the value of circulating messenger RNA (mRNA) profiling in predicting pCR for esophageal squamous cell carcinoma (ESCC). Patients with ESCC completing CRT followed by surgery were enrolled for analysis. Venous peripheral blood was obtained before and after CRT and total RNA was extracted for hybridization-based whole genome expression analysis and quantitative RT-PCR. We found circulating expression profiling was significantly altered after CRT. Altered FAM84B expression was significantly predictive of pCR. The decrease of serum FAM84B protein level after CRT was also associated with pCR. Immunohistochemistry and western blot confirmed that FAM84B protein was overexpressed in the majority of patients and ESCC cell lines. Furthermore, knockdown of FAM84B delayed tumor growth in ectopic xenografts. We demonstrated the decreased of circulating FAM84B mRNA and protein after neoadjuvant CRT may predict pCR and FAM84B protein is overexpressed in ESCC. The potential of FAM84B as a novel predictive biomarker and its biological functions deserve further investigation.
Modulation of Igβ is essential for the B cell selection in germinal center
Authors: Todo Kagefumi , Koga Orie , Nishikawa Miwako , Hikida Masaki 
Published: May 2015

AbstractThe positive and negative selection of antigen-reactive B cells take place in the germinal center (GC) during an immune responses. However, the precise molecular mechanisms underlying these selection machineries, including the involvement of antigen receptor signaling molecules, remain to be elucidated. We found that expression levels of Igα and Igβ, which are the essential components of B cell antigen-receptor complex, were differentially regulated in GC B cells and that the expression of Igβ was more prominently down-regulated in a portion of GC B cells. The suppression of Igβ down-regulation reduced the number of GL7+GC B cells and the affinity maturation in T-dependent responses was markedly impaired. In addition, the disease phenotypes in autoimmune-prone mice were ameliorated by blocking of Igβ down-regulation. These results suggest that Igβ down-regulation is involved in the normal positive selection in GC and the accumulation of autoreactive B cells in autoimmune-prone mice.
The cytosolic tail of the tumor marker protein Trop2 - a structural switch triggered by phosphorylation
Authors: Pavšič Miha , Ilc Gregor , Vidmar Tilen , Plavec Janez , Lenarčič Brigita 
Published: May 2015

AbstractTrop2 is a transmembrane signaling glycoprotein upregulated in stem and carcinoma cells. Proliferation-enhancing signaling involves regulated intramembrane proteolytic release of a short cytoplasmic fragment, which is later engaged in a cytosolic signaling complex. We propose that Trop2 function is modulated by phosphorylation of a specific serine residue within this cytosolic region (Ser303) and by proximity effects exerted on the cytosolic tail by Trop2 dimerization. Structural characterization of both the transmembrane (Trop2TM) and cytosolic regions (Trop2IC) support this hypothesis and shows that the central region of Trop2IC forms an α-helix. Comparison of NMR structures of non-phosphorylated and phosphorylated forms suggest that phosphorylation of Trop2IC triggers salt bridge reshuffling, resulting in significant conformational changes including ordering of the C-terminal tail. In addition, we demonstrate that the cytosolic regions of two Trop2 subunits can be brought into close proximity via transmembrane part dimerization. Finally, we show that Ser303-phosphorylation significantly affects the structure and accessibility of functionally important regions of the cytosolic tail. These observed structural features of Trop2 at the membrane-cytosol interface could be important for regulation of Trop2 signaling activity.