bluuebunny/crossref_metadata_2025_split huggingface dataset:
A new method to customize protein expression vectors for fast, efficient and background free parallel cloning
Authors: Scholz Judith , Besir Hüseyin , Strasser Claudia , Suppmann Sabine 
Published: February 2013

Abstract

Background
Expression and purification of correctly folded proteins typically require screening of different parameters such as protein variants, solubility enhancing tags or expression hosts. Parallel vector series that cover all variations are available, but not without compromise. We have established a fast, efficient and absolutely background free cloning approach that can be applied to any selected vector.

Results
Here we describe a method to tailor selected expression vectors for parallel Sequence and Ligation Independent Cloning. SLIC cloning enables precise and sequence independent engineering and is based on joining vector and insert with 15–25 bp homologies on both DNA ends by homologous recombination. We modified expression vectors based on pET, pFastBac and pTT backbones for parallel PCR-based cloning and screening in E.coli, insect cells and HEK293E cells, respectively. We introduced the toxic ccdB gene under control of a strong constitutive promoter for counterselection of insert less vector. In contrast to DpnI treatment commonly used to reduce vector background, ccdB used in our vector series is 100% efficient in killing parental vector carrying cells and reduces vector background to zero. In addition, the 3’ end of ccdB functions as a primer binding site common to all vectors. The second shared primer binding site is provided by a HRV 3C protease cleavage site located downstream of purification and solubility enhancing tags for tag removal. We have so far generated more than 30 different parallel expression vectors, and successfully cloned and expressed more than 250 genes with this vector series. There is no size restriction for gene insertion, clone efficiency is > 95% with clone numbers up to 200. The procedure is simple, fast, efficient and cost-effective. All expression vectors showed efficient expression of eGFP and different target proteins requested to be produced and purified at our Core Facility services.

Conclusion
This new expression vector series allows efficient and cost-effective parallel cloning and thus screening of different protein constructs, tags and expression hosts.
Vasorelaxant effect of Prunus yedoensis bark
Authors: Lee Kyungjin , Ham Inhye , Yang Gabsik , Lee Mihwa , Bu Youngmin , Kim Hocheol , Choi Ho-Young 
Published: February 2013

Abstract

Background

Prunus yedoensis Matsum. is used as traditional medicine—‘Yaeng-Pi’ or ‘Hua-Pi’—in Japan and Korea. However, no studies have examined the pharmacological activities of the P. yedoensis bark. Only the antioxidant and antiviral activities of P. yedoensis fruit and the anti-hyperglycaemic effect of P. yedoensis leaf have been investigated. While studying the antihypertensive effects of several medicinal plants, we found that a methanol extract of P. yedoensis bark (MEPY) had distinct vasorelaxant effects on rat aortic rings.

Methods
The aortic rings were removed from Sprague–Dawley rats and suspended in organ chambers containing 10 ml Krebs-Henseleit solution. The aortic rings were placed between 2 tungsten stirrups and connected to an isometric force transducer. Changes in tension were recorded via isometric transducers connected to a data acquisition system.

Results
MEPY relaxed the contraction induced by phenylephrine (PE) both in endothelium-intact and endothelium-denuded aortic rings concentration dependently. However, the vasorelaxant effects of MEPY on endothelium-denuded aortic rings were lower than endothelium-intact aortic rings. The vasorelaxant effects of MEPY on endothelium-intact aortic rings were reduced by pre-treatment with l-NAME, methylene blue, or ODQ. However, pre-treatment with indomethacin, atropine, glibenclamide, tetraethylammonium, or 4-aminopyridine had no affection. In addition, MEPY inhibited the contraction induced by extracellular Ca2+ in endothelium-denuded rat thoracic aorta rings pre-contracted by PE (1 μM) or KCl (60 mM) in Ca2+-free solution.

Conclusions
Our results suggest that MEPY exerts its vasorelaxant effects via the activation of NO formation by means of l-Arg and NO-cGMP pathways and via the blockage of extracellular Ca2+ channels.
Src-mediated morphology transition of lung cancer cells in three-dimensional organotypic culture
Authors: Nguyen Hong T , Zhuang Yan , Sun Lichun , Kantrow Steven P , Kolls Jay K , You Zongbing , Zhuo Ying , Shan Bin 
Published: February 2013

Abstract
A fribotic tumor microenvironment promotes progression of cancer. In this study, we utilize a reconstituted basement membrane mimics Matrigel based three-dimensional organotypic culture (rBM 3-D) to investigate the mechanisms that mediate the tumor promoting effects of the fibrogenic mediators TGF-β1 and type I collagen (Col-1) on lung adenocarcinoma cells. Similar to normal alveolar epithelial cells, the well-differentiated lung adenocarcinoma cells in rBM 3-D culture undergo acinar morphogeneis that features polarized epithelial cell spheres with a single central lumen. Either TGF-β1 or Col-1 modestly distorts acinar morphogenesis. On the other hand, TGF-β1 and Col-1 synergistically induce a transition from acinar morphology into stellate morphology that is characteristic of invasive and metastatic cancer cells. Inhibition of the Src kinase activity abrogates induction of stellate morphology, activation of Akt and mTOR, and the expression of tumor promoting genes by TGF-β1 and Col-1. To a similar extent, pharmacological inhibition of mTOR abrogates the cellular responses to TGF-β1 and Col-1. In summary, we demonstrate that TGF-β1 and Col-1 promote stellate morphogenesis of lung cancer cells. Our findings further suggest that the Src-Akt-mTOR axis mediates stellate morphogenesis. These findings also indicate that rBM 3-D culture can serve as an ideal platform for swift and cost-effective screening of therapeutic candidates at the interface of the tumor and its microenvironment.
Improvement of left ventricular remodeling after myocardial infarction with eight weeks L-thyroxine treatment in rats
Authors: Chen Yue-Feng , Weltman Nathan Y , Li Xiang , Youmans Steven , Krause David , Gerdes Anthony Martin 
Published: February 2013

Abstract

Background
Left ventricular (LV) remodeling following large transmural myocardial infarction (MI) remains a pivotal clinical issue despite the advance of medical treatment over the past few decades. Identification of new medications to improve the remodeling process and prevent progression to heart failure after MI is critical. Thyroid hormones (THs) have been shown to improve LV function and remodeling in animals post-MI and in the human setting. However, changes in underlying cellular remodeling resulting from TH treatment are not clear.

Methods
MI was produced in adult female Sprague–Dawley rats by ligation of the left descending coronary artery. L-thyroxine (T4) pellet (3.3 mg, 60 days sustained release) was used to treat MI rats for 8 weeks. Isolated myocyte shape, arterioles, and collagen deposition in the non-infarcted area were measured at terminal study.

Results
T4 treatment improved LV ±dp/dt, normalized TAU, and increased myocyte cross-sectional area without further increasing myocyte length in MI rats. T4 treatment increased the total LV tissue area by 34%, increased the non-infarcted tissue area by 41%, and increased the thickness of non-infarcted area by 36% in MI rats. However, myocyte volume accounted for only ~1/3 of the increase in myocyte mass in the non-infarct area, indicating the presence of more myocytes with treatment. T4 treatment tended to increase the total length of smaller arterioles (5 to 15 μm) proportional to LV weight increase and also decreased collagen deposition in the LV non-infarcted area. A tendency for increased metalloproteinase-2 (MMP-2) expression and tissue inhibitor of metalloproteinases (TIMPs) -1 to −4 expression was also observed in T4 treated MI rats.

Conclusions
These results suggest that long-term T4 treatment after MI has beneficial effects on myocyte, arteriolar, and collagen matrix remodeling in the non-infarcted area. Most importantly, results suggest improved survival of myocytes in the peri-infarct area.
Characterization of antiviral resistance mutations among the Eastern Indian Hepatitis B virus infected population
Authors: Panigrahi Rajesh , Biswas Avik , De Binay Krishna , Chakrabarti Sekhar , Chakravarty Runu 
Published: February 2013

Abstract

Background
Antiviral therapy using nucleos(t)ide analogues (NAs) is an effective control measure of chronic hepatitis B virus (HBV) infection; however they need long term treatment. Presence of drug-resistance mutations may get in the way of the efficacy of antiviral therapy. Our study was aimed at defining the prevalence of HBV drug-resistance in HBVrt region in a population of 147 HBsAg positive patients.

Findings
HBV/D has shown multiple types of HBVrt mutations both among treatment naïve (65.0%, 13 of 20 HBV/D) and treated patients (56.2%, 9 of 16 HBV/D). In additional, several mutations, with a suggested role in drug resistance, were detected among the treatment naïve as well as the treated patients. The mutations reported to be involved in reduction of drug effectiveness, was common among non-responders to therapy as well as among the naïve patients. Notably, classical antiviral resistance mutations (rtL80I/V-rtI169T-rtV173L-rtL180M-rtA181T/V/S-rtT184A/S/G/C-rtA194T-rtS202C /G/I -rtM204V/I-rtN236T-rtM250V) were not detected.

Conclusion
The prevalence of putative NAr mutations among non responders to therapy suggests that they might have role in reduced efficacy of currently available antivirals and requires further investigations.
A retrospective analysis of geriatric trauma patients: venous lactate is a better predictor of mortality than traditional vital signs
Authors: Salottolo Kristin M , Mains Charles W , Offner Patrick J , Bourg Pamela W , Bar-Or David 
Published: February 2013

AbstractBackgroundTraditional vital signs (TVS), including systolic blood pressure (SBP), heart rate (HR) and their composite, the shock index, may be poor prognostic indicators in geriatric trauma patients. The purpose of this study is to determine whether lactate predicts mortality better than TVS.MethodsWe studied a large cohort of trauma patients age ≥ 65 years admitted to a level 1 trauma center from 2009-01-01 - 2011-12-31. We defined abnormal TVS as hypotension (SBP < 90 mm Hg) and/or tachycardia (HR > 120 beats/min), an elevated shock index as HR/SBP ≥ 1, an elevated venous lactate as ≥ 2.5 mM, and occult hypoperfusion as elevated lactate with normal TVS. The association between these variables and in-hospital mortality was compared using Chi-square tests and multivariate logistic regression.ResultsThere were 1987 geriatric trauma patients included, with an overall mortality of 4.23% and an incidence of occult hypoperfusion of 20.03%. After adjustment for GCS, ISS, and advanced age, venous lactate significantly predicted mortality (OR: 2.62, p < 0.001), whereas abnormal TVS (OR: 1.71, p = 0.21) and SI ≥ 1 (OR: 1.18, p = 0.78) did not. Mortality was significantly greater in patients with occult hypoperfusion compared to patients with no sign of circulatory hemodynamic instability (10.67% versus 3.67%, p < 0.001), which continued after adjustment (OR: 2.12, p = 0.01).ConclusionsOur findings demonstrate that occult hypoperfusion was exceedingly common in geriatric trauma patients, and was associated with a two-fold increased odds of mortality. Venous lactate should be measured for all geriatric trauma patients to improve the identification of hemodynamic instability and optimize resuscitative efforts.
Profiling of hepatic clearance pathways of Pittsburgh compound B and human liver cytochrome p450 phenotyping
Authors: Van Vlaslaer Anne , Mortishire-Smith Russell J , Mackie Claire , Langlois Xavier , Schmidt Mark E 
Published: February 2013

Abstract

Background

11C-PiB has been developed as a positron-emission tomography (PET) ligand for evaluating fibrillar β-amyloid (Aβ) in the human brain. The ligand is rapidly metabolized, with approximately 10% of intact tracer remaining 30 min after injection. When 11C-PiB is used as a treatment endpoint in intervention studies for Alzheimer’s disease (AD), a concern is whether the clearance of the tracer changes from one scan to the next, increasing within subject variability in the PET signal. Subjects enrolled in AD trials may start or stop medications that inhibit or induce xenobiotic metabolizing enzymes such as the cytochrome P450 (CYP) isozymes.

Findings
We conducted CYP phenotyping in recombinantly expressed systems, and in human liver microsomes, to evaluate CYP isozyme contributions to the metabolism of PiB (carrier) and profiled microsomal and hepatocyte incubations for metabolites. The metabolism of PiB appears to be polyzymic, with direct conjugation via UDP-glucuronosyltransferases (UGTs) also occurring.

Conclusion
It is unlikely that CYP inhibition or induction will significantly influence the clearance of 11C-PiB.
Pervasive and dynamic protein binding sites of the mRNA transcriptome in Saccharomyces cerevisiae
Authors: Freeberg Mallory A , Han Ting , Moresco James J , Kong Andy , Yang Yu-Cheng , Lu Zhi John , Yates John R , Kim John K 
Published: February 2013

Abstract
Background
Protein-RNA interactions are integral components of nearly every aspect of biology, including regulation of gene expression, assembly of cellular architectures, and pathogenesis of human diseases. However, studies in the past few decades have only uncovered a small fraction of the vast landscape of the protein-RNA interactome in any organism, and even less is known about the dynamics of protein-RNA interactions under changing developmental and environmental conditions.

Results
Here, we describe the gPAR-CLIP (global photoactivatable-ribonucleoside-enhanced crosslinking and immunopurification) approach for capturing regions of the untranslated, polyadenylated transcriptome bound by RNA-binding proteins (RBPs) in budding yeast. We report over 13,000 RBP crosslinking sites in untranslated regions (UTRs) covering 72% of protein-coding transcripts encoded in the genome, confirming 3' UTRs as major sites for RBP interaction. Comparative genomic analyses reveal that RBP crosslinking sites are highly conserved, and RNA folding predictions indicate that secondary structural elements are constrained by protein binding and may serve as generalizable modes of RNA recognition. Finally, 38% of 3' UTR crosslinking sites show changes in RBP occupancy upon glucose or nitrogen deprivation, with major impacts on metabolic pathways as well as mitochondrial and ribosomal gene expression.

Conclusions
Our study offers an unprecedented view of the pervasiveness and dynamics of protein-RNA interactions in vivo.
Follistatin-like 3 (FSTL3) Mediated Silencing of Transforming Growth Factor β (TGFβ) Signaling Is Essential for Testicular Aging and Regulating Testis Size
Authors: Oldknow Karla J. , Seebacher Jan , Goswami Tapasree , Villen Judit , Pitsillides Andrew A. , O'Shaughnessy Peter J. , Gygi Steven P. , Schneyer Alan L. , Mukherjee Abir 
Published: February 2013

Abstract
Follistatin-like 3 (FSTL3) is a glycoprotein that binds and inhibits the action of TGFβ ligands such as activin. The roles played by FSTL3 and activin signaling in organ development and homeostasis are not fully understood. The authors show mice deficient in FSTL3 develop markedly enlarged testes that are also delayed in their age-related regression. These FSTL3 knockout mice exhibit increased Sertoli cell numbers, allowing for increased spermatogenesis but otherwise showing normal testicular function. The data show that FSTL3 deletion leads to increased AKT signaling and SIRT1 expression in the testis. This demonstrates a cross-talk between TGFβ ligand and AKT signaling and leads to a potential mechanism for increased cellular survival and antiaging. The findings identify crucial roles for FSTL3 in limiting testis organ size and promoting age-related testicular regression.
The pro-Forms of Insulin-Like Growth Factor I (IGF-I) Are Predominant in Skeletal Muscle and Alter IGF-I Receptor Activation
Authors: Durzyńska Julia , Philippou Anastassios , Brisson Becky K. , Nguyen-McCarty Michelle , Barton Elisabeth R. 
Published: February 2013

Abstract
IGF-I is a key regulator of muscle development and growth. The pre-pro-peptide produced by the Igf1gene undergoes several posttranslational processing steps to result in a secreted mature protein, which is thought to be the obligate ligand for the IGF-I receptor (IGF-IR). The goals of this study were to determine what forms of IGF-I exist in skeletal muscle, and whether the mature IGF-I protein was the only form able to activate the IGF-IR. We measured the proportion of IGF-I species in murine skeletal muscle and found that the predominant forms were nonglycosylated pro-IGF-I and glycosylated pro-IGF-I, which retained the C-terminal E peptide extension, instead of mature IGF-I. These forms were validated using samples subjected to viral expression of IGF-I combined with furin and glycosidase digestion. To determine whether the larger molecular weight IGF-I forms were also ligands for the IGF-IR, we generated each specific form through transient transfection of 3T3 cells and used the enriched media to perform kinase receptor activation assays. Compared with mature IGF-I, nonglycosylated pro-IGF-I had similar ability to activate the IGF-IR, whereas glycosylation of pro-IGF-I significantly reduced receptor activation. Thus, it is important to understand not only the quantity, but also the proportion of IGF-I forms produced, to evaluate the true biological activity of this growth factor.
Quantitative parametric MRI of articular cartilage: a review of progress and open challenges
Authors: Binks D A , Hodgson R J , Ries M E , Foster R J , Smye S W , McGonagle D , Radjenovic A 
Published: February 2013

With increasing life expectancies and the desire to maintain active lifestyles well into old age, the impact of the debilitating disease osteoarthritis (OA) and its burden on healthcare services is mounting. Emerging regenerative therapies could deliver significant advances in the effective treatment of OA but rely upon the ability to identify the initial signs of tissue damage and will also benefit from quantitative assessment of tissue repair in vivo. Continued development in the field of quantitative MRI in recent years has seen the emergence of techniques able to probe the earliest biochemical changes linked with the onset of OA. Quantitative MRI measurements including T 1, T 2 and T 1ρ relaxometry, diffusion weighted imaging and magnetisation transfer have been studied and linked to the macromolecular structure of cartilage. Delayed gadolinium-enhanced MRI of cartilage, sodium MRI and glycosaminoglycan chemical exchange saturation transfer techniques are sensitive to depletion of cartilage glycosaminoglycans and may allow detection of the earliest stages of OA. We review these current and emerging techniques for the diagnosis of early OA, evaluate the progress that has been made towards their implementation in the clinic and identify future challenges in the field.
Zum Verkauf der Stralsunder Gymnasialbibliothek
Authors: Schlechter Armin 
Published: February 2013

Zusammenfassung:
Der von der Stadt Stralsund im Juni 2012 beschlossene Verkauf der bis dahin im Archiv aufbewahrten, historischen Gymnasialbibliothek an einen Antiquar hat zu großen Protesten geführt. Die Stadt hat nach der Einschaltung von Gutachtern den Verkauf rückabgewickelt, so dass der größte Teil der Sammlung, die aufgrund mangelnder moderner Erschließung nicht benutzt werden konnte, gerettet wurde. Im gleichen Zusammenhang wurde die Stadt auf den katastrophalen
Erhaltungszustand der Bestände des Stadtarchivs aufmerksam.
Assessing Psychological Violence and Harassment at Work: Reliability and Validity of the Japanese Version of the Inventory of Violence and Psychological Harassment (IVAPT) Comparing NAQ‐R and LIPT
Authors: Moreno Manuel Pando , Beltrán Carolina Aranda , Tsuno Kanami , Inoue Akiomi , Kawakami Norito 
Published: February 2013

Assessing Psychological Violence and Harassment at Work: Reliability and Validity of the Japanese Version of the Inventory of Violence and Psychological Harassment (IVAPT) Comparing NAQ‐R and LIPT: Manuel PANDO MORENO, et al. Institute of Research on Occupational Health, University of Guadalajara, Mexico—ObjectiveThe objective of this study was to examine reliability and validity of the Japanese version of the Inventory of Violence and Psychological Harassment (IVAPT) (Pando, 2006), a 22‐item measure of psychological harassment at work and presence and intensity of psychological violence widely used in Latin American countries.MethodsThe IVAPT was translated into Japanese, and the translation was amended through a small pretest and a back‐translation and finalized. A total of 1,810 out of 4,072 civil servants completed a questionnaire including the IVAPT.ResultsCronbach's alpha of the scale was 0.97 for psychological violence and 0.94 for psychological harassment at work. An exploratory factor analysis showed that the first factor explained 64.5% of the total variance. Data did not well fit to previously reported one‐or three‐factor structures. Psychological violence and harassment at work were more frequent among older respondents. Intensity of psychological violence was well concordant with other scales of workplace bullying, i.e., Leymann Inventory of Psychological Terrorization (LIPT) and Negative Acts Questionnaire‐Revised (NAQ‐R), and psychological harassment at work was well concordant with the NAQ‐R.ConclusionsThe Japanese version of the IVAPT showed high internal consistency reliability. While the first factor explained a large proportion of the variance, the IVAPT seems to have a unique factor structure in the Japanese sample. Concurrent validity of the IVAPT was supported by the comparison with the other scales.
A Growth Model of Stress Voids in Integrated Circuit Metallization
Authors: Kordic S. , Collart E.J.H. 
Published: February 2013

ABSTRACTStress voiding is caused by the difference in the thermal expansion coefficients of the metallization lines and the surrounding passivation. Volumes of individual stress voids are measured as a function of stress time at 200°C, 180°C, and 150°C in 1-μm-wide AlCu(1%wt) integrated circuit metallization lines. The time needed for void growth to saturate, and the total void volume depend on the stress temperature. If the void growth is regarded as an isothermal phase transformation in which voids are formed as precipitates at the void nucleation sites, the void volume growth is accurately described by the Avrami equation. Depending on the temperature, the time power n ranges between 0.5 and 1.5. First principle calculations are in excellent agreement with the measurements. The impact of the above results on electromigration testing is discussed.
Influence of Voed Geometry on Electromigration Failure in Via-Line Structures
Authors: Miner B. , Sriram T. S. , Pelillo A. , Bill S. A. 
Published: February 2013

ABSTRACTWe present in this work a series of detailed failure analyses of via-line structures to correlate void geometry and resistance behavior during accelerated electromigration testing. We find that while the void volume is dependent only on the time under stress, the resistance change is affected very strongly by void geometry and location. The primary effect of void location and geometry is to determine the availability of alternate current paths and how soon during the test these come into effect. The microstructure and crystallography of the connecting Al lines appears to play a secondary role in determining which samples fail early. The processing of small geometry vias can lead to high resistance interfaces between the via and underlying metal which can lead to large variations in electromigration lifetime depending on void geometry and location.