bluuebunny/crossref_metadata_2025_split huggingface dataset:
Preparation of Baskets for Drosophila Egg Collections, Treatments, and Incubations
Authors: Shermoen Antony W. 
Published: September 2008

INTRODUCTIONThe key consideration in preparation of baskets for Drosophila embryo collection is basket size, since the collected embryos must be in approximately one monolayer (or more). This article describes two baskets (homemade and commercial) used by the author for large numbers of embryos.
Subbing Slides
Authors: Paul Carol Ann , Beltz Barbara , Berger-Sweeney Joanne 
Published: September 2008

INTRODUCTIONWhen mounting sections on a slide, it is essential that the slide be ultraclean. To ensure that the section sticks to the slide, slides can be covered with a thin layer of gelatin. The process of cleaning and gelatin-coating is called “subbing.” Several boxes of slides should be subbed at a time and stored, covered, in the refrigerator until they are needed. Subbed slides can also be purchased commercially.
Analyzing Glucose Phosphate Isomerase Isozymes in Chimeric Mouse Tissues by Electrophoresis
Authors: Nagy Andras , Gertsenstein Marina , Vintersten Kristina , Behringer Richard 
Published: September 2008

INTRODUCTIONMouse strains carry different alleles at the ubiquitously expressedGpi1(glucose phosphate isomerase) locus (Gpi1a,Gpi1b,Gpi1c), and this is the basis for a widely used method for determining the genotypic composition of different tissues in mouse chimeras. To determine chimerism by this method, it is necessary to separate the differently charged isozymes from tissue homogenates electrophoretically and to visualize them using a color reaction. Because GPI is a dimer, tissues that normally form by cell fusion (e.g., skeletal muscle) have a heterodimeric form of GPI in a chimera.
Phenotypic Analysis of Arabidopsis Mutants: Heavy Metal Stress
Authors: Lee David , Schroeder Julian 
Published: September 2008

INTRODUCTIONArsenic (As) is a major environmental contaminant, arising from both industrial and agricultural use, and is one of the metals given top priority for remediation by the U.S. Department of Energy on the basis of its toxicity and widespread contamination. However, many of the molecular and genetic mechanisms contributing to arsenic toxicity and detoxification remain unknown in plants. The toxicity of arsenic depends on the arsenic oxidation state. The main arsenic oxidation states are As(III) and As(V), both of which are highly toxic to plants and animals. In plants, As(III) disrupts the sulfhydryl groups of proteins and induces the production of hydrogen peroxide, which leads to membrane damage, whereas As(V) uncouples phosphorylation in mitochondria and at high levels can inhibit seed germination. As described in this protocol, resistance or sensitivity to arsenic can be easily scored by measuring germination and root bending in exposed seedlings.
Noninvasive Human Nuclear Transfer with Embryonic Stem Cells: Figure 1.
Authors: McElroy Sohyun L. , Reijo Pera Renee A. 
Published: September 2008

INTRODUCTIONIn somatic cell nuclear transfer (SCNT), the nucleus of a somatic cell is transferred to an enucleated oocyte for reprogramming to an embryonic cell state through the use of the endogenous machinery. SCNT technology has been used to produce offspring, establish embryonic stem cells, and study epigenetic reprogramming, as mediated by oocytes, in several animal species. In humans, there are ethical and practical issues that limit availability of oocytes donated by women of reproductive age specifically for research. Thus, there is a need to more exhaustively explore alternatives, including oocyte sources and different SCNT protocols. Nuclear transfer (NT) techniques are important factors that impact development of NT embryos. The procedures of enucleation of oocyte genetic material and introduction of the donor nucleus vary depending on species and laboratories. Hoechst staining has been used successfully for invasive enucleation in many animal studies, though it is known that Hoechst staining and ultraviolet (UV) light can damage oocyte mitochondrial DNA. More recently, noninvasive NT techniques that rely on polarized microscopic imaging systems have been used to visualize the meiotic spindle without DNA staining and UV illumination. This protocol describes a method for noninvasive human nuclear transfer by visualizing the oocyte spindle without DNA staining.
Preparation of Mouse Embryonic Fibroblast Feeder Cells for Human Embryonic Stem Cell Culture
Authors: McElroy Sohyun L. , Reijo Pera Renee A. 
Published: September 2008

INTRODUCTIONEmbryonic stem cells (ESCs) are derived from the inner cell mass of day 5-6 blastocysts. ESCs are pluripotent, meaning that they are able to differentiate into all derivatives of the three primary germ layers (ectoderm, endoderm, and mesoderm). In order to maintain the undifferentiated status of human ESCs (hESCs), feeder cells are used to provide both a suitable attachment substrate and critical soluble factors. Since the first hESC lines were established on mouse embryonic fibroblasts (MEFs), mitotically inactivated MEFs have commonly been used for supporting the culture of undifferentiated hESCs. Some previous studies suggest that MEFs may support hESC growth better than the human feeder cells typically isolated from post-natal tissues. This protocol describes a method for isolation and irradiation of MEFs for use in hESC culture.
Method for Single-Cell Sorting and Expansion of Genetically Modified Human Embryonic Stem Cells
Authors: Nicholas Cory R. , Reijo Pera Renee A. 
Published: September 2008

INTRODUCTIONThe potential for human embryonic stem cells (hESCs) to differentiate into all three embryonic germ layers, and the germline, can be used as a tool to understand the mechanisms of cell lineage-specific differentiation and development in vitro and in vivo. Therefore, it is important to develop technologies for the manipulation of hESCs and their effective utilization in research. One such manipulation is genetic modification. Reliable methods for genetic modification of hESCs with ubiquitous and tissue-specific reporters will assist in the identification and isolation of specific cell types following hESC differentiation. This protocol describes a method for hESC transduction with a ubiquitous eGFP reporter construct using self-inactivating lentivirus, followed by single-cell isolation and expansion of homogenous genetically modified hESCs using fluorescence-activated cell sorting (FACS).
DNA Methylation Analysis of Human Imprinted Loci by Bisulfite Genomic Sequencing
Authors: Angeles Vanessa T. , Reijo Pera Renee A. 
Published: September 2008

INTRODUCTIONThe genomic imprinting mechanism regulates differential expression of paternally and maternally derived genes. During development, primordial germ cells (PGCs) have a unique methylation pattern which undergoes demethylation, both globally and at imprinted loci. Later, the methylation status of imprinted genes is re-established during gametogenesis by de novo methylation. Errors in the methylation patterns of imprinted genes have been associated with syndromes such as Beckwith-Wiedemann, Angelman, and Prader-Willi in humans. Human embryonic stem cells (hESCs) have unique methylation patterns compared to somatic stem cells and cancer cells. The status of allelic expression or methylation of human imprinted genes can be studied in detail in human embryos/blastocysts and hESCs to understand early development and differentiation. This protocol describes the bisulfite genomic sequencing method for analysis of methylation patterns of imprinted loci in human embryonic stem cells. Multiple imprinted loci can be analyzed by performing the following protocol with gene-specific primers.
Metaphase Spreads and Spectral Karyotyping of Human Embryonic Stem Cells
Authors: Nguyen Ha Nam , Reijo Pera Renee A. 
Published: September 2008

INTRODUCTIONSpectral karyotyping is a powerful technique for enumeration of chromosomes in plant, animal, and human cells with high precision. It is also used as a tool for detecting and diagnosing abnormalities in individual chromosomes, such as deletions, duplications, and translocations. There are three basic steps to obtaining good spectral karyotypes: (1) making metaphase spreads, (2) hybridizing probes to the DNA, and (3) analyzing the spectral characteristics of the chromosomes. There are two techniques for spectral karyotyping that are widely used: spectral karyotype (SKY) and multicolor fluorescence in situ hybridization (M-FISH). Both techniques use multicolored fluorescent probes which emit at different wavelengths (200-700 nm) upon light excitation to hybridize to the target DNA sequences. The methods differ in the way in which light is captured and analyzed by the corresponding software. In this protocol, methods for making metaphase spreads from human embryonic stem cells (hESCs) for spectral karyotyping (SKY) using the spectral imaging system and software from Applied Spectral Imaging are described.
Distinct difference of flaA genotypes of Legionella pneumophila between isolates from bath water and cooling tower water
Authors: Amemura‐Maekawa Junko , Kura Fumiaki , Chang Bin , Suzuki‐Hashimoto Atsuko , Ichinose Masayuki , Endo Takuro , Watanabe Haruo 
Published: September 2008

ABSTRACTTo investigate the genetic difference of Legionella pneumophila in human‐made environments, we collected isolates of L. pneumophila from bath water (n = 167) and cooling tower water (n = 128) primarily in the Kanto region in 2001 and 2005. The environmental isolates were serogrouped and sequenced for a target region of flaA. A total of 14 types of flaA genotypes were found: 10 from cooling tower water and nine from bath water. The flaA genotypes of isolates from cooling tower water were quite different from those of bath water.
Overexpression of carboxylesterase‐2 results in enhanced efficacy of topoisomerase I inhibitor, irinotecan (CPT‐11), for multiple myeloma
Authors: Yano Hiroki , Kayukawa Satoshi , Iida Shinsuke , Nakagawa Chiharu , Oguri Tetsuya , Sanda Takaomi , Ding Jianming , Mori Fumiko , Ito Asahi , Ri Masaki , Inagaki Atsushi , Kusumoto Shigeru , Ishida Takashi , Komatsu Hirokazu , Inagaki Hiroshi , Suzuki Atsushi , Ueda Ryuzo 
Published: September 2008

Multiple myeloma (MM) remains an incurable disease and further development of novel agents is needed. Because constitutive expression of topoisomerase I (TopoI) in MM cells and the efficacy of SN‐38, an active metabolite of irinotecan (CPT‐11), have been reported, we investigated the therapeutic potential of CPT‐11. Of the eight MM cell lines analyzed, four showed 50% inhibitory concentration values of less than 2 µg/mL for CPT‐11 and less than 2 ng/mL for SN‐38. This efficacy was partly explained by the high expression level of human carboxylesterase‐2 (hCE‐2) in MM cells. Interestingly, high expression of hCE‐2 represented the nature of normal plasma cells, suggesting that hCE‐2 could efficiently generate SN‐38 within the plasma cells. As expected, higher sensitivity to CPT‐11 was observed in hCE‐2‐overexpressing U266 cells than mock U266 cells. On the other hand, the expression levels of hCE‐1, TopoI, UGT1A and ABCG2 did not seem to be associated with the sensitivity of MM cells to CPT‐11. In a murine xenograft model inoculated s.c. with RPMI8226 cells, administration of CPT‐11 alone significantly reduced the tumor volume. When a combination of CPT‐11 and bortezomib was administered, the subcutaneous tumors completely disappeared. Thus, clinical trials on CPT‐11 in patients with relapsed or refractory MM are warranted. (Cancer Sci 2008; 99: 2309–2314)
The influence of labour on the pharmacokinetics of intravenously administered amoxicillin in pregnant women
Authors: Muller Anouk E. , Dörr P. Joep , Mouton Johan W. , De Jongh Joost , Oostvogel Paul M. , Steegers Eric A. P. , Voskuyl Rob A. , Danhof Meindert 
Published: August 2008

WHAT IS ALREADY KNOWN ABOUT THIS SUBJECT • Examples exist that pharmacokinetics of drugs in pregnant women can differ from that in non‐pregnant individuals.• In pregnant women before the onset of labour, the pharmacokinetics of amoxicillin is similar to that in non‐pregnant individuals, but for women during labour this is unknown. WHAT THIS STUDY ADDS • Labour influences the pharmacokinetics of amoxicillin.• During labour and even more in the immediate postpartum period, the peripheral volume of distribution was decreased compared with pregnant women before the onset of labour.• The volume of distribution increases with an increasing amount of oedema.AIMSMany physiological changes take place during pregnancy and labour. These might change the pharmacokinetics of amoxicillin, necessitating adjustment of the dose for prevention of neonatal infections. We investigated the influence of labour on the pharmacokinetics of amoxicillin.METHODSPregnant women before and during labour were recruited and treated with amoxicillin intravenously. A postpartum dose was offered. Blood samples were obtained and amoxicillin concentrations were determined using high‐pressure liquid chromatography. The pharmacokinetics were characterized by nonlinear mixed‐effects modelling using NONMEM.RESULTSThe pharmacokinetics of amoxicillin in 34 patients was best described by a three‐compartment model. Moderate interindividual variability was identified in CL, central and peripheral volumes of distribution. The volume of distribution (V) increased with an increasing amount of oedema. Labour influenced the parameter estimate of peripheral volume of distribution (V2). V2 was decreased during labour, and even more in the immediate postpartum period. For all patients the population estimates (mean ± SE) for CL and V were 21.1 ± 4.1 l h−1 (CL), 8.7 ± 6.6 l (V1), 11.8 ± 7.7 l (V2) and 20.5 ± 15.4 l (V3) respectively.CONCLUSIONSThe peripheral distribution volume of amoxicillin in pregnant women during labour and immediately postpartum is decreased. However, these changes are not clinically relevant and do not warrant deviations from the recommended dosing regimen for amoxicillin during labour in healthy pregnant patients.
New treatments in type 2 diabetes: a focus on the incretin‐based therapies
Authors: Barnett Anthony H. 
Published: September 2008

SummaryThe demonstration that the incretin hormone glucagon‐like peptide 1 can improve glycaemic control in patients with type 2 diabetes has led to the rapid development during the last decade of promising new classes of agent for the management of type 2 diabetes. These agents possess a range of physiological effects that are associated with improved glycaemic control in diabetes including stimulation of glucose‐dependent insulin secretion, suppression of glucagon secretion, slowing of gastric emptying, and reduction of food intake. In addition, preclinical studies suggest that incretin‐based therapies may improve β‐cell function via enhancement of β‐cell mass and induction of genes important for differentiated β‐cell function. Exenatide, and the dipeptidyl peptidase‐4 inhibitors, sitagliptin and vildagliptin are already approved, and liraglutide is currently completing Phase 3 trials. As these agents and standard oral therapies for type 2 diabetes lower glucose levels through different, but potentially complementary mechanisms, their use in combination should provide effective, potentially additive, glycaemic control. The incretin‐based therapies also offer other advantages such as weight loss with exenatide and liraglutide, a reduced risk of hypoglycaemia, and as suggested by preclinical studies, a potential β‐cell preserving effect. Long‐term outcome and safety data are not available for these agents, but they appear generally well‐tolerated in comparison with existing therapies for type 2 diabetes. The multiple underlying glucose‐lowering actions of the incretin‐based therapies, as well as a lack of weight gain or even weight loss, make these important new additions to available antidiabetic agents expanding the treatment options available for patients.
Adjustment of L‐T4 substitutive therapy in pregnant women with subclinical, overt or post‐ablative hypothyroidism
Authors: Verga Uberta , Bergamaschi Silvia , Cortelazzi Donatella , Ronzoni Stefania , Marconi Anna Maria , Beck‐Peccoz Paolo 
Published: September 2008

SummaryObjective  Maternal hyperthyrotropinaemia is associated with an increased risk of adverse maternal and neonatal outcomes. Physiological changes during pregnancy require an increased production of thyroid hormones (or an increase in daily substitutive doses of L‐T4 in hypothyroid patients) to meet the maternal and foetal needs. The aim of the study was to evaluate variations of substitutive L‐T4 doses that are able to maintain serum TSH between 0·5 and 2·5 mU/l in pregnant women with subclinical‐ (SH), overt‐ (OH) and post‐ablative (PH) hypothyroidism.Design  This was a retrospective study on hypothyroid pregnant women referred to the out‐patient department between January 2004 and December 2006.Patients and measurements  A total of 185 pregnant women were studied during gestation; 155 patients (76 SH, 52 OH, 27 PH) were already on L‐T4 before conception and 30 (SH) started L‐T4 therapy during gestation. Thyroid function and body weight were evaluated every 4–6 weeks.Results  In the group of patients already treated before conception, 134 (86·5%) increased L‐T4 doses during gestation one or more times, eight (6%) reached a definitive therapeutic dosage within the 12th week of pregnancy, 64 (47·8%) within the 20th week and 62 (46·2%) within the 31st week. This initial L‐T4 increase at the first evaluation during pregnancy was 22·9 ± 9·8 µg/day. The final L‐T4 doses were significantly different depending on the aetiology, being 101·0 ± 24·6 µg/day in SH, 136·8 ± 30·4 µg/day in OH and 159·0 ± 24·6 µg/day in PH. The per cent increase of L‐T4, expressed as Δ% of absolute dose, was +70% in SH, +45% in OH and +49% in PH as compared to baseline dose. In SH patients diagnosed during gestation, the starting L‐T4 dose was higher than L‐T4 dose before pregnancy of SH patients already treated (75·4 ± 14·5 and 63·2 ± 20·1 µg/day, respectively), whereas the final doses were similar. L‐T4 dose was increased one or more times in 24 patients (80%), 8 reached the definitive dosage within the second trimester (33·3%) and 16 within the third trimester (66·7%).Conclusions  Serum TSH and FT4 measurements are mandatory in pregnant patients and the optimal timing for increasing L‐T4 is the first trimester of pregnancy, though many patients require adjustments also during the second and third trimester. The aetiology of hypothyroidism influences the adjustment of L‐T4 therapy and SH patients needed a larger increase than OH and PH. Close monitoring during pregnancy appears to be mandatory in hypothyroid women.
Identification of the most active interleukin‐32 isoform
Authors: Choi Ji‐Da , Bae Su‐Young , Hong Jae‐Woo , Azam Tania , Dinarello Charles A. , Her Erk , Choi Whan‐Soo , Kim Bo‐Kyung , Lee Chang‐Kwon , Yoon Do‐Young , Kim Sun‐Jong , Kim Soo‐Hyun 
Published: September 2008

SummaryCytokines are crucial in host defence against pathogens such as bacteria, viruses, fungi and parasites. A newly described cytokine, interleukin‐32 (IL‐32), induces various proinflammatory cytokines (tumour necrosis factor‐α, IL‐1β, IL‐6) and chemokines in both human and mouse cells through the nuclear factor‐κB and p38 mitogen‐activated protein kinase inflammatory signal pathway. The IL‐32 primarily acts on monocytic cells rather than T cells. In an attempt to isolate the IL‐32 soluble receptor, we used an IL‐32 ligand‐affinity column to purify neutrophil proteinase 3, which is a serine proteinase involved in many inflammatory diseases. IL‐32 has biological activity associated with Mycobacterium tuberculosis and chronic proinflammatory diseases such as rheumatoid arthritis. IL‐32 is transcribed as six alternative splice variants and the biological activity of each individual isoform remains unknown. Here, we cloned the complementary DNA of the four IL‐32 isoforms (α, β, γ and δ) that are the most representative IL‐32 transcripts. To produce recombinant protein with a high yield, the amino acids of two cysteine residues were mutated to serine residues, because serine residues are not conserved among different species. The multi‐step purified recombinant IL‐32 isoform proteins were assessed for their biological activities with different cytokine assays. The γ isoform of IL‐32 was the most active, although all isoforms were biologically active. The present study will provide a specific target to neutralize endogenous IL‐32, which may contribute to basic and clinical immunology.
Evidence for multiple introductions of Centaurea stoebe micranthos (spotted knapweed, Asteraceae) to North America
Authors: MARRS R. A. , SFORZA R. , HUFBAUER R. A. 
Published: September 2008

AbstractInvasive species’ success may depend strongly on the genetic resources they maintain through the invasion process. We ask how many introductions have occurred in the North American weed Centaurea stoebe micranthos (Asteraceae), and explore whether genetic diversity and population structure have changed as a result of introduction. We surveyed individuals from 15 European native range sites and 11 North American introduced range sites at six polymorphic microsatellite loci. No significant difference existed in the total number of alleles or in the number of private alleles found in each range. Shannon–Weaver diversity of phenotype frequencies was also not significantly different between the ranges, while expected heterozygosity was significantly higher in the invasive range. Population structure was similar between the native range and the invasive range, and isolation by distance was not significant in either range. Traditional assignment methods did not allocate any North American individuals to the sampled European populations, while Bayesian assignment methods grouped individuals into nine genetic clusters, with three of them shared between North America and Europe. Invasive individuals tended to have genetically admixed profiles, while natives tended to assign more strongly to a single cluster. Many North American individuals share assignment with Romania and Bulgaria, suggesting two separate invasions that have undergone gene flow in North America. Samples from three other invasive range sites were genetically distinct, possibly representing three other unique introductions. Multiple introductions and the maintenance of high genetic diversity through the introduction process may be partially responsible for the invasive success of C. stoebe micranthos.