bluuebunny/crossref_metadata_2025_split huggingface dataset:
Retroviral Vector Production by Transient Transfection
Authors: Cornetta Kenneth , Pollok Karen E. , Miller A. Dusty 
Published: April 2008

INTRODUCTIONThis protocol describes vector production by transient transfection. The production of retroviral vectors requires a full-length copy of the vector RNA to be incorporated into virions. This is accomplished by coexpressing vector RNA and the viral proteins required for virion formation from expression plasmids. To avoid generation of replication-competent virus, the viral genes are carried by separate plasmids. Generally, the gag and pol genes are on one plasmid, and the viral envelope gene is on a second plasmid. The viral protein-coding regions can be expressed using various promoters to decrease homology and thereby decrease recombination. Because these plasmids do not contain the packaging (ψ) sequence, the viral genes are unlikely to be incorporated into virions.
Generation of Stable Vector-Producing Cells for Retroviral Vectors: Figure 1.
Authors: Cornetta Kenneth , Pollok Karen E. , Miller A. Dusty 
Published: April 2008

INTRODUCTIONThis procedure describes the generation of clonal vector-producing cells that will provide an unlimited amount of unrearranged retroviral vector. The procedure involves transfection of one packaging cell line to generate a vector that is used to transduce a second packaging cell line. The resultant vector-producing clones generally contain a single integrated copy of the retroviral vector, and virus produced from this integrated vector is as genetically homogeneous as possible. Although the vector produced by a given packaging cell line can sometimes be used to transduce the same cell line, the transduction rate is typically low because of receptor blockage by the Env protein made by the target packaging cells. Indeed, this procedure will select for target cells that express low Env protein levels and thus are less resistant to transduction, but at the same time will ultimately produce less vector because of low Env production. Therefore, to obtain the highest vector titers, it is important to use pairs of packaging cells such that receptor blockage is not an issue. In this example, we use PE501 ecotropic packaging cells for transfection and broad-host-range PT67 packaging cells to make stable vector-producing cells.
Transduction of Cell Lines by Retroviral Vectors
Authors: Cornetta Kenneth , Pollok Karen E. , Miller A. Dusty 
Published: April 2008

INTRODUCTIONThis protocol is suitable for transduction of many adherent cell lines. The number of target cells transduced can be varied as needed by maintaining the ratio of surface area to volume and using plates/flasks of various sizes. The protocol can also easily be adapted for non-adherent cells using similar vector-to-cell ratios.
Labeling the Nucleus with Fluorescent Dyes for Imaging: Figure 1.
Authors: Chazotte Brad 
Published: April 2008

INTRODUCTIONThe eukaryotic cell has evolved to compartmentalize its functions and transport various metabolites among cellular compartments. Therefore, in cell biology, the study of organization and structure/function relationships is of great importance. The nucleus contains almost all of the cell’s DNA and is bounded by a double membrane. Inside and adjacent to the inner membrane of the nuclear envelope is the nuclear lamina. It is composed of a fibrous meshwork comprising one or more of three major intermediate filament-like polypeptides: lamins A, B, and C. The outer nuclear membrane is contiguous with the endoplasmic reticulum. Several fluorescent stains are available that label DNA and allow easy visualization of the nucleus in interphase cells and chromosomes in mitotic cells. These stains include Hoechst and 4′,6-diamidino-2-phenylindole (DAPI), which are used in this article. Although not as bright as the vital Hoechst stains for DNA, DAPI has greater photostability. One advantage of Hoechst 33342 over DAPI is that the former is membrane-permeant and is, therefore, useful for imaging living cells, because it does not require cell fixation or permeabilization.
Desorption Electrospray Ionization (DESI) Analysis of Tryptic Digests/Peptides
Authors: Takats Zoltan , Wiseman Justin M. , Ifa Demian R. , Cooks R. Graham 
Published: April 2008

INTRODUCTIONThe analytical utility of desorption electrospray ionization (DESI) is such that it can be applied to qualitative proteomics research in the same way as matrix-assisted laser desorption/ionization (MALDI) and electrospray ionization (ESI) methods, although little work has yet been reported in this regard. Because DESI is a surface analysis technique and easily automated, it can be implemented for high-throughput applications, which include the analysis of chromatographic fractions of digested proteins. The analysis of tryptic peptides follows the same protocols as in typical MALDI or ESI methods, except that the mixture is spotted directly onto an insulating surface, allowed to dry, and analyzed directly without adding matrix compounds (as in the case of MALDI methods). The spectral characteristics are similar to those of ESI in that both singly and multiply charged analyte ions are detected. Spectra are highly similar to electrospray spectra of tryptic digests with regard to the overwhelming presence of multiply charged ions of peptides. DESI-mass spectrometry (DESI-MS) is an emerging technique with great promise, but its application range is still being investigated. Therefore, the protocol for DESI-MS analysis of tryptic digests/peptides presented here provides general procedures used for the applications that have been investigated so far. Optimal ion source parameters and surface types may vary, depending on the application.
Maximum Parsimony Method for Phylogenetic Prediction
Authors: Mount David W. 
Published: April 2008

INTRODUCTIONMaximum parsimony predicts the evolutionary tree or trees that minimize the number of steps required to generate the observed variation in the sequences from common ancestral sequences. For this reason, the method is also sometimes referred to as the minimum evolution method. A multiple sequence alignment (msa) is required to predict which sequence positions are likely to correspond. These positions will appear in vertical columns in the msa. For each aligned position, phylogenetic trees that require the smallest number of evolutionary changes to produce the observed sequence changes from ancestral sequences are identified. This analysis is continued for every position in the sequence alignment. Finally, those trees that produce the smallest number of changes overall for all sequence positions are identified. This method is best suited for sequences that are quite similar and is limited to small numbers of sequences.
Distance Methods for Phylogenetic Prediction
Authors: Mount David W. 
Published: April 2008

INTRODUCTIONPhylogenetic analysis of a multiple sequence alignment (msa) can be performed using distance methods, which are based on genetic distances between sequence pairs in an msa. The genetic distance between two sequences is the fraction of aligned positions in which the sequence has been changed. In contrast, sequence identity is the fraction of the aligned positions that are identical. Gaps may be ignored in distance calculations or treated like substitutions. A scoring or substitution matrix may also be used, making the calculation slightly more complicated, although the principle is the same. Sequence pairs that have the smallest distances are “neighbors.” On a tree, these sequences share a node or common ancestor position and are each joined to that node by a branch.
Desorption Electrospray Ionization: Proteomics Studies by a Method That Bridges ESI and MALDI
Authors: Takats Zoltan , Wiseman Justin M. , Ifa Demian R. , Cooks R. Graham 
Published: April 2008

INTRODUCTIONDesorption electrospray ionization (DESI) is a desorption ionization (DI) method by nature, and, like matrix-assisted laser desorption/ionization (MALDI), it is used for the analysis of material present on a surface. DESI includes features reminiscent of electrospray ionization (ESI) in respect to both its instrumental and mechanistic aspects. However, the analyte in the DESI experiment is not in solution as in ESI. Instead, a microelectrospray ion source is used to produce charged droplets, ionic clusters, and/or gas-phase ions (depending on chosen experimental conditions), and these are directed at the sample surface. The sample is present in the ambient environment. An electrical potential of several kilovolts (kV) is applied to the spray solution, and pneumatic nebulization is used to assist in desolvation. Ionization of molecules present on the sample surface occurs upon the impact of the ESI-originated, charged particles with the surface. Surfaces include deposited samples on sample holder targets as well as surfaces of natural objects such as biological tissues or minerals.
Improvement of the breaking strength of wound by combined treatment with recombinant human G‐CSF, recombinant human M‐CSF, and a TGF‐β1 receptor kinase inhibitor in rat skin
Authors: Sugiyama Kenji , Ishii Genichiro , Ochiai Atsushi , Esumi Hiroyasu 
Published: April 2008

Effective doses of ionizing radiation during preoperative radiotherapy occasionally cause wound complications after subsequent surgery. The authors attempted to accelerate radiation‐impaired wound healing in animal models. Recombinant human granulocyte colony‐stimulating factor (rhG‐CSF), recombinant human macrophage colony‐stimulating factor (rhM‐CSF), and an inhibitor of transforming growth factor (TGF)‐β1 receptor kinase, SB431542, were injected s.c. into a full‐thickness incisional wound site in the dorsal skin of rats after local irradiation of X‐ray (30 Gy). Wound healing of irradiated skin was assessed using the breaking strength of the wound and histological analyses. The impaired wound healing in irradiated skin was found to be associated with impaired mobilization of bone marrow‐derived cells and enhanced expression of TGF‐β1 mRNA. The breaking strength of the wound in the irradiated skin was approximately one‐eighth of that in the non‐irradiated skin; however, following combined treatment with the above three compounds the breaking strength increased to approximately one‐half of that in the non‐irradiated skin. Histological analysis of the wounded skin revealed an increase in formation of collagen fibers and the panniculus carnosus following the combined treatment. Moreover, the increased breaking strength was associated with an increase in a subpopulation of fibrocytes (collagen I/ED1 double positive cells). These findings suggested that a combined treatment with rhG‐CSF, rhM‐CSF, and SB431542 is promising as a means of improving radiation‐impaired wound healing. (Cancer Sci 2008; 99: 1021–1028)
CYP2A6and the plasma level of 5‐chloro‐2, 4‐dihydroxypyridine are determinants of the pharmacokinetic variability of tegafur and 5‐fluorouracil, respectively, in Japanese patients with cancer given S‐1
Authors: Fujita Ken‐ichi , Yamamoto Wataru , Endo Shoji , Endo Hisashi , Nagashima Fumio , Ichikawa Wataru , Tanaka Ryuhei , Miya Toshimichi , Araki Kazuhiro , Kodama Keiji , Sunakawa Yu , Narabayashi Masaru , Miwa Keisuke , Ando Yuichi , Akiyama Yuko , Kawara Kaori , Kamataki Tetsuya , Sasaki Yasutsuna 
Published: April 2008

S‐1 is an oral anticancer agent composed of tegafur (FT), 5‐chloro‐2,4‐dihydroxypyridine (CDHP), and potassium oxonate. CDHP is added to prevent degradation of 5‐fluorouracil (5‐FU) by inhibiting dihydropyrimidine dehydrogenase. CYP2A6 is involved in the biotransformation of FT to 5‐FU. Thus, we prospectively analyzed the effects of theCYP2A6genotype, plasma level of CDHP, and patient characteristics on the pharmacokinetic (PK) variability of FT and 5‐FU. Fifty‐four Japanese patients with metastatic or recurrent cancers who received S‐1 were enrolled. TheCYP2A6polymorphisms (*4A, *7, and *9) with deficient or reduced activity were analyzed. All subjects were classified into three groups according to theirCYP2A6genotype: wild type (*1/*1), one‐variant allele (*1/any), or two‐variant alleles (combination other than *1). The PK of FT, 5‐FU, and CDHP were measured on day 1 of treatment. Multivariate regression analysis revealed that oral clearance of FT was associated with theCYP2A6genotype (analysis of variance [ANOVA],P = 0.000838). The oral clearance of FT seen in patients with the two‐variant alleles was significantly lower than those in wild type and the one‐variant allele (95% confidence intervals 0.75–2.41 and 0.41–1.82, respectively; Tukey‐Kramer test). The area under the time–concentration curve (AUC) of 5‐FU was significantly correlated with the AUC of CDHP (ANOVA,P = 0.00126). The AUC of 5‐FU and CDHP were inversely correlated with creatinine clearance (ANOVA,P = 0.0164 andP = 0.000762, respectively). Although theCYP2A6variants are the cause of the PK variability of FT, the AUC of CDHP affected by renal function is the key determinant of the variability in the PK of 5‐FU. (Cancer Sci2008; 99: 1049–1054)
Nuclear factor‐κB dependency of doxorubicin sensitivity in gastric cancer cells is determined by manganese superoxide dismutase expression
Authors: Cho Sung Jin , Park Jong‐Wan , Kang Jae Seung , Kim Woo Ho , Juhnn Yong‐Sung , Lee Jae‐Seon , Kim Young‐Hoon , Ko Young San , Nam Seon Young , Lee Byung Lan 
Published: April 2008

The role of nuclear factor‐κB (NF‐κB) activation in cancer cell apoptosis appears to be tailored specifically for each cell type and the type of NF‐κB inducer. The present study aimed to determine whether or not NF‐κB activation is associated with chemosensitivity to doxorubicin (DOX) using the DOX‐sensitive SNU‐601 and DOX‐resistant SNU‐216 gastric cancer cell lines. The effect of NF‐κB activation on DOX (1 µg/mL) sensitivity was analyzed after the suppression of NF‐κB activation using transfection of the super‐suppressive mutant form of IκBα (mIκBα) or pretreatment with pyrrolidine dithiocarbamate. In addition, the association between NF‐κB and manganese superoxide dismutase (MnSOD) in relation to DOX sensitivity was analyzed after the modulation of MnSOD expression. The NF‐κB activity was much higher in DOX‐resistant SNU‐216 cells than in DOX‐sensitive SNU‐601 cells before and after DOX treatment. Overexpression of mIκBα or pyrrolidine dithiocarbamate pretreatment decreased the DOX resistance in SNU‐601 cells with low MnSOD expression, but not in SNU‐216 cells with high MnSOD expression. In comparison, the overexpression of MnSOD, which also suppressed NF‐κB activation in both cell lines, increased DOX resistance in SNU‐601 cells. Blocking of MnSOD expression using RNA interference techniques increased DOX sensitivity in SNU‐216 cells, which was further augmented by the additional inhibition of NF‐κB activity. Our results showed that whether NF‐κB contributes to DOX sensitivity in gastric cancer cells is determined by the level of MnSOD expression. Thus, targeting both MnSOD and NF‐κB may be helpful for increasing the efficacy of DOX treatment of DOX‐resistant SNU gastric cancer cells. (Cancer Sci 2008; 99: 1117–1124)
Evidence of the in vivo esterification of budesonide in human airways
Authors: Van Den Brink Karen I. Maassen , Boorsma Martin , Staal‐van den Brekel A. Jeske , Edsbäcker Staffam , Wouters Emiel F. , Thorsson Lars 
Published: April 2008

WHAT IS ALREADY KNOWN ABOUT THIS SUBJECT • In vitro studies with human bronchial epithelial cells have shown that budesonide undergoes rapid, extensive and reversible intracellular esterification, a finding that is believed to contribute to retention and prolonged duration of action.• A case report has suggested retention of budesonide in the lungs of patients undergoing surgical resection.• This study evaluated, for the first time, the esterification (and distribution) of inhaled budesonide and fluticasone propionate in vivo in human lung. WHAT THIS STUDY ADDS • This study has unequivocally shown that budesonide undergoes esterification in human lungs in vivo.• Budesonide was detectable in central and peripheral lung tissue for 10 h, and budesonide oleate up to 43 h after inhalation.• The sustained retention of budesonide esters in the lungs probably contributes to the prolonged duration of action and once‐daily efficacy of budesonide.AIMSBudesonide, unlike fluticasone propionate, undergoes fatty acid esterification in the lungs, and there is a need to characterize fully the distribution and fate of the two drugs after inhalation in humans.METHODSThis open‐label, randomized study was performed in adults undergoing whole lung or lobar resection resulting from lung cancer. Patients were given single 1000‐μg doses of both budesonide and fluticasone propionate via dry powder inhalers before surgery. Tissue samples from peripheral and central lung, an ex vivo bronchial brush sample and intercostal muscle, together with plasma samples, were taken during surgery and analysed by liquid chromatography plus tandem mass spectrometry.RESULTSLung tissue samples were obtained from 22 patients at surgery, 1–43 h after drug dosing. Budesonide was detectable from earliest sampling in central and peripheral lung tissue up to 10 h (in six of 22 samples), fluticasone propionate up to 22 h after inhalation (in 16 of 22 samples), and budesonide oleate up to 43 h after inhalation (in 21 of 22 samples). Budesonide, but not fluticasone propionate, was detected in intercostal muscle for up to 10 h after inhalation. Bronchial brush samples showed the presence of fluticasone propionate for up to 18 h, suggesting the presence of undissolved drug powder particles in the airway lumen.CONCLUSIONSustained retention of esterified budesonide in the lungs supports the prolonged duration of action of budesonide and suitability for once‐daily administration.
Eosinophils: Biological Properties and Role in Health and Disease
Authors: HOGAN SIMON P. , ROSENBERG HELENE F. , MOQBEL REDWAN , PHIPPS SIMON , FOSTER PAUL S. , LACY PAIGE , KAY A. BARRY , ROTHENBERG MARC E. 
Published: April 2008

SUMMARYEosinophils are pleiotropic multifunctional leukocytes involved in initiation and propagation of diverse inflammatory responses, as well as modulators of innate and adaptive immunity. In this review, the biology of eosinophils is summarized, focusing on transcriptional regulation of eosinophil differentiation, characterization of the growing properties of eosinophil granule proteins, surface proteins and pleiotropic mediators, and molecular mechanisms of eosinophil degranulation. New views on the role of eosinophils in homeostatic function are examined, including developmental biology and innate and adaptive immunity (as well as their interaction with mast cells and T cells) and their proposed role in disease processes including infections, asthma, and gastrointestinal disorders. Finally, strategies for targeted therapeutic intervention in eosinophil‐mediated mucosal diseases are conceptualized.
Zinc speciation and isotopic exchangeability in soils polluted with heavy metals
Authors: Diesing W. E. , Sinaj S. , Sarret G. , Manceau A. , Flura T. , Demaria P. , Siegenthaler A. , Sappin‐Didier V. , Frossard E. 
Published: April 2008

SummaryCorrect characterization of heavy metal availability is a prerequisite for the management of polluted soils. Our objective was to describe zinc (Zn) availability in polluted soils by measuring the isotopic exchangeability of Zn in soil/solution (E value) and in soil/plant systems (L value), by assessing the transfer of Zn and 65Zn in the fractions of a six‐step selective sequential extraction (SSE) in incubated soils and by identifying Zn forms in soils by means of extended X‐ray absorption fine structure (EXAFS) spectroscopy. We distinguished three pools of exchangeable Zn: the pool of Zn exchangeable within 1 minute, which is observed in all soils, Zn exchangeable in the medium term, and the slowly and not exchangeable Zn. The amount of Zn present in the first two pools was similar to the L value measured with Thlaspi caerulescens. The first three steps of the SSE solubilized the first pool and a fraction of the second pool. Most of the second pool and a fraction of the third pool were extracted in the fourth step of the SSE, while the rest of the third pool was extracted in the final steps of the SSE. The EXAFS study conducted on two soils showed that more than half of the Zn was present in species weakly bound to organic compounds and/or outer sphere inorganic and organic complexes. Other species included strongly sorbed Zn species and Zn species in crystalline minerals. The EXAFS study of selected SSE residues showed that the specificity and the efficiency of the extractions depended on the properties of the soil studied.
Molar incisor hypomineralization: a study of aetiological factors in a group of UK children*
Authors: WHATLING ROSEMARY , FEARNE JANICE M 
Published: April 2008

Objectives.  The objectives were to investigate the aetiology of molar incisor hypomineralization (MIH), and to discuss previously published studies.Subjects and methods.  One hundred and nine children attending the Department of Paediatric Dentistry at the Royal London Hospital were included in the study: 57 children with MIH and 52 controls. Their mothers completed a medical history interview.Results.  No significant associations were found with MIH and delivery and birth complications, breastfeeding, immunization history, other illnesses and allergies, general anaesthetics, fluoride history, and trauma or abscesses affecting the primary predecessors. A family history of enamel defects was more commonly reported for MIH children, but the association was not statistically significant. However, MIH was significantly more common among those whose mothers had experienced problems during pregnancy (P = 0.025), those who had chickenpox between the ages of 3 and 3.99 (P = 0.047), and those for whom amoxycillin was the only antibiotic they had received (P = 0.028).Conclusions.  The aetiology of MIH remains unclear, and this study questions whether it is because of a lone aetiological insult. This study recommends further research looking at the links with chickenpox occurring around the third year of life and amoxycillin.
Clinical performance of a diode laser fluorescence device for the detection of occlusal caries in primary teeth
Authors: KAVVADIA KATERINA , LAGOUVARDOS PANAGIOTIS 
Published: April 2008

Objectives.  To correlate the DIAGNOdentTM readings (LF) with those of direct visual (DV) examination, indirect visual (IDV) examination, bitewing radiography (BWR), and pit and fissure opening (PFO) for the detection of occlusal caries in primary teeth; to determine the validity of this device using PFO as reference; and to evaluate its reliability.Methods.  Two calibrated operators examined 130 teeth with 405 examination sites using DV, IDV and radiographic examinations, and LF. Of the 405 sites, 155 were visually scored for caries after pit and fissure opening.Results.  Pit and fissure opening was found to significantly correlate with all methods (rhoLF = 0.48, rhoBWX = 0.48, rhoDV = 0.44, rhoIDV = 0.41). For enamel lesions, higher sensitivity (0.76) was found with DV, while higher specificity (0.88) with the LF. For lesions into dentin, however, higher sensitivity (0.78) was found with the LF, while higher specificity (0.98) with the BWR. The device's accuracy was found to be 0.61 for enamel lesions, while for lesions into dentin 0.70, and its reliability was excellent (ICC = 0.97).Conclusion.  The LF device presented high reliability in the detection of occlusal caries in primary teeth and its performance was similar to DV and radiographic examinations.