bluuebunny/crossref_metadata_2025_split huggingface dataset:
Generating HapMap Data Text Reports Using the Genome Browser
Authors: Smith Albert Vernon 
Published: July 2008

INTRODUCTIONThe primary goal of the International Haplotype Map Project has been to develop a haplotype map of the human genome that describes the common patterns of genetic variation, in order to accelerate the search for the genetic causes of human disease. Within the project, ~3.9 million distinct single-nucleotide polymorphisms (SNPs) have been genotyped in 270 individuals from four worldwide populations. The project data are available for unrestricted public use at the HapMap website. This site, which is the primary portal to genotype data produced by the project, offers bulk downloads of the data set, as well as interactive data browsing and analysis tools that are not available elsewhere. In many cases, a researcher will be interested in downloading HapMap data from a region of interest for local analysis. This protocol describes the direct download of genotype, frequency, tag-SNPs, and other reports from the project website, using the genome browser.
Manipulating HapMap Data Using HaploView
Authors: Smith Albert Vernon 
Published: July 2008

INTRODUCTIONThe primary goal of the International Haplotype Map Project has been to develop a haplotype map of the human genome that describes the common patterns of genetic variation, in order to accelerate the search for the genetic causes of human disease. Within the project, ~3.9 million distinct single-nucleotide polymorphisms (SNPs) have been genotyped in 270 individuals from four worldwide populations. The project data are available for unrestricted public use at the HapMap website. This site, which is the primary portal to genotype data produced by the project, offers bulk downloads of the data set, as well as interactive data browsing and analysis tools that are not available elsewhere. Advanced users who wish to exercise fine control over the display of regions of high linkage disequilibrium (LD) or who wish to experiment with new algorithms for tag-SNP picking may wish to analyze HapMap data using the HaploView program. This program works well in combination with the HapMap website genome browser. A big advantage of HaploView over the genome browser is that it displays simultaneous high- and low-power views of regions of LD, and gives immediate feedback during scrolling and zooming operations. This protocol describes the use of HaploView to manipulate HapMap data.
Retrieving HapMap Data Using HapMart: Figure 1.
Authors: Smith Albert Vernon 
Published: July 2008

INTRODUCTIONThe primary goal of the International Haplotype Map Project has been to develop a haplotype map of the human genome that describes the common patterns of genetic variation, in order to accelerate the search for the genetic causes of human disease. Within the project, ~3.9 million distinct single-nucleotide polymorphisms (SNPs) have been genotyped in 270 individuals from four worldwide populations. The project data are available for unrestricted public use at the HapMap Web site. This site, which is the primary portal to genotype data produced by the project, offers bulk downloads of the data set, as well as interactive data browsing and analysis tools that are not available elsewhere. Because of performance considerations, interactive access to HapMap data via the genome browser on the HapMap Web site is limited to regions no more than 5 Mb wide. Researchers who wish to obtain data for chromosome- or genome-wide data have two choices: Bulk download or HapMart access. HapMart, described in this protocol, allows researchers to select SNPs using diverse criteria and to display just those aspects of the data set that they are interested in.
Generation of Driver and Reporter Constructs for the GAL4 Expression System in Drosophila: Figure 1.
Authors: Southall Tony D. , Brand Andrea H. 
Published: July 2008

INTRODUCTIONThe GAL4 system is a method for ectopic gene expression that allows the selective activation of any cloned gene in a wide variety of tissue- and cell-specific patterns. This protocol describes the generation of driver and reporter lines for use with the GAL4 system in Drosophila. A promoter-GAL4 fusion is constructed using a P-element transformable vector, and a GAL4-responsive target gene is created via generation of an upstream activation sequence (UAS)-reporter construct. An alternative strategy for integration using the phiC31 system is also provided. Transformant lines are generated using standard procedures for microinjection.
High-Throughput Paraffin Section In Situ Hybridization and Dual Immunohistochemistry on Mouse Tissues: Figure 1.
Authors: Rumballe Bree , Georgas Kylie , Little Melissa H. 
Published: July 2008

INTRODUCTIONSection in situ hybridization (SISH) is a high-resolution tool used to analyze gene expression patterns. This protocol utilizes the Tecan Freedom EVO150 platform to perform high-throughput SISH on paraffin sections to detect mRNA with a digoxigenin (DIG)-labeled probe. The slide is mounted and imaged before performing immunohistochemistry (IHC) on the same section. The dual reaction enables a marker of protein expression to be localized on the same section as the mRNA and facilitates more accurate annotation of the gene expression.
Identification of Differential Genes by Suppression Subtractive Hybridization: An Overview
Authors: Rebrikov Denis V. 
Published: July 2008

INTRODUCTIONSuppression subtractive hybridization (SSH) is one of the most powerful and popular methods for generating subtracted cDNA or genomic DNA libraries. This technique can be used to compare two mRNA populations and obtain cDNAs representing genes that are either overexpressed or exclusively expressed in one population as compared to another. It can also be used for comparison of genomic DNA populations. We have used SSH in studies of regeneration and development on various types of model organisms (including freshwater planaria regeneration, Xenopus laevis development, and mammalian brain cortex development). We also use SSH for the analysis of strain-specific genes in bacteria with different characteristics. During these studies, a large number of differentially regulated and differentially presented genes have been identified, including transcriptional regulation factors and restriction modification enzymes. This article describes the SSH method and considerations for its use.
The GAL4 System: A Versatile Toolkit for Gene Expression in Drosophila
Authors: Southall Tony D. , Elliott David A. , Brand Andrea H. 
Published: July 2008

INTRODUCTIONThe generation of gain-of-function phenotypes by ectopic expression of known genes provides a powerful complement to the genetic approach, in which genes are studied or identified through mutations that generally reduce or eliminate gene function. The GAL4 system is a method for ectopic gene expression that allows the selective activation of any cloned gene in a wide variety of tissue- and cell-specific patterns. A key advantage of the system is the separation of the GAL4 protein from its target gene in distinct transgenic lines, which ensures that the target gene is silent until the introduction of GAL4. Recent modifications and adaptations of the GAL4 system to make the system inducible have further expanded its scope, enabling greater temporal control over the activity of GAL4. There are now large resources for the community, including thousands of GAL4 lines and a wide selection of reporter lines. Here we present an overview of the GAL4 system, highlighting recent developments and discussing methods for generating and analyzing transgenic flies for GAL4-mediated ectopic expression.
Nuclear Stains
Authors: Kiernan John Alan 
Published: July 2008

INTRODUCTIONFor the study of microscopic anatomy and of pathological material, it is usual to stain sections of tissue in such a way as to impart a dark color to the nuclei of cells and a lighter, contrasting color to the cytoplasm and extracellular structures. Nuclear stains, including cationic, anionic, and metal complexing dyes, are considered in this article. The rationales of the techniques are discussed, but methods of higher chemical specificity (e.g., for nucleic acids, carbohydrates, and functional groups of proteins) are not covered.
Anionic Counterstains
Authors: Kiernan John Alan 
Published: July 2008

INTRODUCTIONFor the study of microscopic anatomy and of pathological material, it is usual to stain sections of tissue in such a way as to impart a dark color to the nuclei of cells and a lighter, contrasting color to the cytoplasm and extracellular structures. In most of the general “oversight” methods used in histology, a blue, purple, or black nuclear stain is followed by a paler, usually pink, counterstain, which colors all the other components of the tissue. Simple counterstaining procedures using anionic dyes are considered in this article. The rationales of the techniques are discussed, but methods of higher chemical specificity (e.g., for nucleic acids, carbohydrates, and functional groups of proteins) are not covered.
Mutation ofArabidopsis BARD1Causes Meristem Defects by Failing to ConfineWUSCHELExpression to the Organizing Center
Authors: Han Pei , Li Qing , Zhu Yu-Xian 
Published: July 2008

AbstractStem cell fate in the Arabidopsis thaliana shoot apical meristem (SAM) is controlled by WUSCHEL (WUS) and CLAVATA. Here, we examine BARD1 (for BRCA1-associated RING domain 1), which had previously been implicated in DNA repair functions; we find that it also regulates WUS expression. We observed severe SAM defects in the knockout mutant bard1-3. WUS transcripts accumulated &gt;238-fold in bard1-3 compared with the wild type and were located mainly in the outermost cell layers instead of the usual organizing center. A specific WUS promoter region was recognized by nuclear protein extracts obtained from wild-type plants, and this protein-DNA complex was recognized by antibodies against BARD1. The double mutant (wus-1 bard1-3) showed prematurely terminated SAM structures identical to those of wus-1, indicating that BARD1 functions through regulation of WUS. BARD1 overexpression resulted in reduced WUS transcript levels, giving a wus-1–like phenotype. Either full-length BARD1 or a clone that encoded the C-terminal domain (BARD1:C-ter;bard1-3) was sufficient to complement the bard1-3 phenotype, indicating that BARD1 functions through its C-terminal domain. Our data suggest that BARD1 regulates SAM organization and maintenance by limiting WUS expression to the organizing center.
Genotyping as a Tool for Antibiotic Resistance Surveillance of
Neisseria gonorrhoeae
in New Caledonia: Evidence of a Novel Genotype Associated with Reduced Penicillin Susceptibility
Authors: Vernel-Pauillac Frédérique , Nandi Sobhan , Nicholas Robert A. , Goarant Cyrille 
Published: July 2008

ABSTRACT

Antibiotic resistance in
Neisseria gonorrhoeae
continues to be a major concern in public health. Resistance of
N. gonorrhoeae
bacteria to penicillin G is widespread in most developed countries, which has necessitated a change to newer drugs for treatment of gonococcal infections. Recent reports indicate that resistance to these newer drugs is increasing, highlighting the need for accurate therapeutic recommendations. In some countries or communities, however,
N. gonorrhoeae
isolates are still susceptible to penicillin, so the use of this antibiotic for single-dose treatments of medically under-resourced patients is beneficial. In order to evaluate the adequacy and sustainability of this treatment approach, we explored the presence and prevalence of chromosomally mediated resistance determinants in
N. gonorrhoeae
isolates collected from 2005 to 2007 in New Caledonia. We developed two new real-time PCR assays targeting the
penB
and
mtrR
determinants, to be used together with a previously described duplex assay targeting the
penA
and
ponA
determinants. The results of this study provided evidence that neither the most-common
mtrR
determinants nor the most-resistance-associated
penB
alleles are currently circulating in New Caledonia, suggesting that penicillin should still be considered a valuable treatment strategy. Additionally, using our genotyping assay, we observed an unexpected
penB
genotype at a relatively high frequency that was associated with a decreased susceptibility to penicillin (average MIC, 0.15 μg/ml). Sequencing revealed that this genotype corresponded to an A102S mutation in the
penB
gene. The molecular tools developed in this study can be used successfully for prospective epidemiological monitoring and surveillance of penicillin susceptibility.
Evaluation of Daptomycin Pharmacodynamics and Resistance at Various Dosage Regimens against
Staphylococcus aureus
Isolates with Reduced Susceptibilities to Daptomycin in an In Vitro Pharmacodynamic Model with Simulated Endocardial Vegetations
Authors: Rose Warren E. , Leonard Steven N. , Rybak Michael J. 
Published: July 2008

ABSTRACT

The need to investigate novel dosing regimens and combinations is essential in combating poor treatment outcomes for
Staphylococcus aureus
bacteremia and endocarditis. We evaluated the impact of simulated standard- and high-dose daptomycin in combination with gentamicin or rifampin against daptomycin-susceptible and nonsusceptible matched strains of
S. aureus.
These strains were collected from the daptomycin bacteremia and endocarditis clinical trial and consisted of three susceptible strains (MIC, 0.25 mg/liter) and four nonsusceptible isolates (MICs, 2 to 4 mg/liter). Daptomycin regimens of 6 and 10 mg/kg of body weight daily alone and in combination with gentamicin at 5 mg/kg daily or rifampin at 300 mg every 8 h were evaluated using an in vitro model with simulated endocardial vegetations over 96 h. Rapid bactericidal activity, identified by time to 99.9% kill, was displayed in all regimens with the daptomycin-susceptible strains. Concentration-dependent activity was noted by more-rapid killing with the 10-mg/kg/day dose. The addition of gentamicin improved activity in the majority of susceptible isolates. Daptomycin 6-mg/kg/day monotherapy displayed bactericidal activity for only one of the nonsusceptible isolates and for only two isolates with increased doses of 10 mg/kg/day. Combination regimens demonstrated improvement with some but not all nonsusceptible isolates. Three isolates developed a reduction in daptomycin susceptibility with 6-mg/kg/day monotherapy, but this was suppressed with both combination therapy and high-dose daptomycin. These results suggest that high-dose daptomycin therapy and combination therapy may be reasonable treatment options for susceptible isolates; however, more investigations are needed to confirm the variability of these regimens with nonsusceptible isolates.
Intracellular Time Course, Pharmacokinetics, and Biodistribution of Isoniazid and Rifabutin following Pulmonary Delivery of Inhalable Microparticles to Mice
Authors: Verma Rahul Kumar , Kaur Jatinder , Kumar Kaushlendra , Yadav Awadh Bihari , Misra Amit 
Published: July 2008

ABSTRACT

Intracellular concentrations of isoniazid and rifabutin resulting from administration of inhalable microparticles of these drugs to phorbol-differentiated THP-1 cells and the pharmacokinetics and biodistribution of these drugs upon inhalation of microparticles or intravenous administration of free drugs to mice were investigated. In cultured cells, both microparticles and dissolved drugs established peak concentrations of isoniazid (∼1.4 and 1.1 μg/10
6
cells) and rifabutin (∼2 μg/ml and ∼1.4 μg/10
6
cells) within 10 min. Microparticles maintained the intracellular concentration of isoniazid for 24 h and rifabutin for 96 h, whereas dissolved drugs did not. The following pharmacokinetic parameters were calculated using WinNonlin from samples obtained after inhalation using an in-house apparatus (figures in parentheses refer to parameters obtained after intravenous administration of an equivalent amount, i.e., 100 μg of either drug, to parallel groups): isoniazid, serum half-life (
t
1/2
) = 18.63 ± 5.89 h (3.91 ± 1.06 h), maximum concentration in serum (
C
max
) = 2.37 ± 0.23 μg·ml
−1
(3.24 ± 0.57 μg·ml
−1
), area under the concentration-time curve from 0 to 24 h (AUC
0-24
) = 55.34 ± 13.72 μg/ml
−1
h
−1
(16.64 ± 1.80 μg/ml
−1
h
−1
), and clearance (CL) = 63.90 ± 13.32 ml·h
−1
(4.43 ± 1.85 ml·h
−1
); rifabutin,
t
1/2
= 119.49 ± 29.62 h (20.18 ± 4.02 h),
C
max
= 1.59 ± 0.01 μg·ml
−1
(3.47 ± 0.33 μg·ml
−1
), AUC
0-96
= 109.35 ± 14.78 μg/ml
−1
h
−1
(90.82 ± 7.46 μg/ml
−1
h
−1
), and CL = 11.68 ± 7.00 ml·h
−1
(1.03 ± 0.11 ml·h
−1
). Drug targeting to the lungs in general and alveolar macrophages in particular was observed. It was concluded that inhaled microparticles can reduce dose frequency and improve the pharmacologic index of the drug combination.
Efficacy of Pyrvinium Pamoate against
Cryptosporidium parvum
Infection In Vitro and in a Neonatal Mouse Model
Authors: Downey Autumn S. , Chong Curtis R. , Graczyk Thaddeus K. , Sullivan David J. 
Published: July 2008

ABSTRACT

No effective approved drug therapy exists for
Cryptosporidium
infection of immunocompromised patients. Here we investigated the nonabsorbed anthelmintic drug pyrvinium pamoate for inhibition of the growth of the intestinal protozoan parasite
Cryptosporidium parvum
. The concentration of pyrvinium that effected 50% growth inhibition in human enterocytic HCT-8 cells by a quantitative alkaline phosphatase immunoassay was 354 nM. For comparison, in the same assay, 50% growth inhibition was obtained with 711 μM paromomycin or 27 μM chloroquine. We used a neonatal mouse model to measure the anti-
Cryptosporidium
activity of pyrvinium pamoate in vivo. Beginning 3 days after infection, pyrvinium at 5 or 12.5 mg/kg of body weight/day was administered to the treatment group mice for 4 or 6 consecutive days. Nine days after infection, the mice were sacrificed, and drug efficacy was determined by comparing the numbers of oocysts in the fecal smears of treated versus untreated mice. The intensities of trophozoite infection in the ileocecal intestinal regions were also compared using hematoxylin-and-eosin-stained histological slides. We observed a >90% reduction in infection intensity in pyrvinium-treated mice relative to that in untreated controls, along with a substantial reduction in tissue pathology. Based on these results, pyrvinium pamoate is a potential drug candidate for the treatment of cryptosporidiosis in both immunocompetent and immunocompromised individuals.
Lung Microdialysis Study of Levofloxacin in Rats following Intravenous Infusion at Steady State
Authors: Marchand Sandrine , Frasca Denis , Dahyot-Fizelier Claire , Breheret Céline , Mimoz Olivier , Couet William 
Published: July 2008

ABSTRACT

Lung microdialysis has been used with rats to investigate antibiotic distribution after single-dose administration. However, conducting such experiments after intravenous infusion at steady state would constitute a more convenient alternative, which was evaluated here, using levofloxacin (LVX) as a test compound. Microdialysis probes were inserted in blood and muscle, used as a comparator, between 9:00 a.m. and 11:00 a.m. Intravenous LVX infusion was started 6 h later and maintained until the end of the experiment at a rate of 1.0 mg·h
−1
. Lung microdialysis probes were inserted on the morning of the next day. Rats were kept anesthetized during dialysate collection. In vivo probe recoveries were estimated by retrodialysis using a calibrator method, with ciprofloxacin (CIP) as the calibrator. LVX and CIP were analyzed in dialysates by high-performance liquid chromatography. The steady-state tissue-to-blood unbound-drug concentration ratios were 1.00 ± 0.15 in muscle tissues and 1.06 ± 0.40 in lungs, suggesting passive distribution of LVX in tissue. Although providing no information on rate of distribution, microdialysis investigations following drug infusion at steady state appear to be an interesting approach for characterization of antibiotic distribution in rat lungs.
Mechanisms of Human Immunodeficiency Virus Type 1 Concerted Integration Related to Strand Transfer Inhibition and Drug Resistance
Authors: Zahm Jacob A. , Bera Sibes , Pandey Krishan K. , Vora Ajaykumar , Stillmock Kara , Hazuda Daria , Grandgenett Duane P. 
Published: July 2008

ABSTRACTThe “strand transfer inhibitors” of human immunodeficiency virus type-1 (HIV-1) integrase (IN), so named because of their pronounced selectivity for inhibiting strand transfer over 3′ OH processing, block virus replication in vivo and ex vivo and prevent concerted integration in vitro. We explored the kinetics of product formation and strand transfer inhibition within reconstituted synaptic complexes capable of concerted integration. Synaptic complexes were formed with viral DNA donors containing either two blunt ends, two 3′-OH-processed ends, or one of each. We determined that one blunt end within a synaptic complex is a sufficient condition for low-nanomolar-range strand transfer inhibition with naphthyridine carboxamide inhibitors L-870,810 and L-870,812. We further explored the catalytic properties and drug resistance profiles of a set of clinically relevant strand transfer inhibitor-resistant HIV-1 IN mutants. The diketo acids and naphthyridine carboxamides, mechanistically similar but structurally distinct strand transfer inhibitors, each select for a distinct set of drug resistance mutations ex vivo. The S153Y and N155S IN resistance mutants were selected with the diketo acid L-841,411, and the N155H mutant was selected with L-810,812. Each mutant exhibited some degree of catalytic impairment relative to the activity of wild type IN, although the N155H mutant displayed near-wild-type IN activities. The resistance profiles indicated that the S153Y mutation potentiates susceptibility to L-870,810 and L-870,812, while the N155S mutation confers resistance to L-870,810 and L-870,812. The N155H mutation confers resistance to L-870,810 and potentiates susceptibility to L-841,411. This study illuminates the interrelated mechanisms of concerted integration, strand transfer inhibition, and resistance to strand transfer inhibitors.
Convergent Evolutionary Analysis Identifies Significant Mutations in Drug Resistance Targets of
Mycobacterium tuberculosis
Authors: Hazbón Manzour Hernando , Motiwala Alifiya S. , Cavatore Magali , Brimacombe Michael , Whittam Thomas S. , Alland David 
Published: July 2008

ABSTRACT

Mycobacterium tuberculosis
adapts to the environment by selecting for advantageous single-nucleotide polymorphisms (SNPs). We studied whether advantageous SNPs could be distinguished from neutral mutations within genes associated with drug resistance. A total of 1,003 clinical isolates of
M. tuberculosis
were related phylogenetically and tested for the distribution of SNPs in putative drug resistance genes. Drug resistance-associated versus non-drug-resistance-associated SNPs in putative drug resistance genes were compared for associations with single versus multiple-branch outcomes using the chi-square and Fisher exact tests. All 286 (100%) isolates containing isoniazid (INH) resistance-associated SNPs had multibranch distributions, suggestive of multiple ancestry and convergent evolution. In contrast, all 327 (100%) isolates containing non-drug-resistance-associated SNPs were monophyletic and thus showed no evidence of convergent evolution (
P
< 0.001). Convergence testing was then applied to SNPs at position 481 of the
iniA
(Rv0342) gene and position 306 of the
embB
gene, both potential drug resistance targets for INH and/or ethambutol. Mutant
embB306
alleles showed multibranch distributions, suggestive of convergent evolution; however, all 44
iniA
(
H481Q
) mutations were monophyletic. In conclusion, this study validates convergence analysis as a tool for identifying mutations that cause INH resistance and explores mutations in other genes. Our results suggest that
embB306
mutations are likely to confer drug resistance, while
iniA
(
H481Q
) mutations are not. This approach may be applied on a genome-wide scale to identify SNPs that impact antibiotic resistance and other types of biological fitness.