bluuebunny/crossref_metadata_2025_split huggingface dataset:
Identification of Differential Genes by Suppression Subtractive Hybridization: IV. Mirror Orientation Selection (MOS)
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. The major drawback of SSH is the presence of background clones that represent nondifferentially expressed DNA species in the subtracted libraries. In some cases, the number of background clones may considerably exceed the number of target clones. This protocol describes mirror orientation selection (MOS)--a simple procedure that substantially decreases the number of background clones in libraries generated by SSH. The MOS technique is based on the rationale that after PCR amplification, during SSH, background molecules will be present in one orientation only, relative to the adapter sequences. Genuine SSH clones will be present in both sequence orientations.
Identification of Differential Genes by Suppression Subtractive Hybridization: V. PCR-Based DNA Dot Blot
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. This protocol describes a method for the use of PCR-based DNA dot blots in the differential screening of arrayed subtracted DNA clones. For high-throughput screening, a 96-well or 384-well format PCR from one of several thermal cycler manufacturers is recommended. Alternatively, single tubes can be used.
Phenotypic Analysis of Arabidopsis Mutants: Flowering Time
Authors: Blázquez Miguel 
Published: July 2008

INTRODUCTIONFlowering in Arabidopsis thaliana is modulated by a number of environmental factors. It is therefore important to perform flowering-time measurements under controlled environmental conditions, including light quality and intensity, day length, temperature, watering, and spacing between plants. Ideally, pots of plants are rotated, and individual plants of different genotypes are placed randomly within the experimental setup. Because of the large influence of environmental effects, it is imperative that only plants grown at the same time and in the same place be compared. Flowering is also modulated by endogenous factors and can thus be affected by mutations in genes that are not primarily involved in the control of flowering time. The variation of flowering time between ecotypes is generally large. Therefore, only those mutants that are induced in the same background can be compared. This protocol describes a method for determining the flowering time of Arabidopsis plants.
Browsing HapMap Data 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. Research into the genetic contributions to a human disease commonly focuses on candidate genes identified from linkage and/or association studies, as well as from pathways suspected to be involved in a particular disease process. In studying candidate genes, a researcher will want to know whether there are any common SNPs in the immediate vicinity, what those SNPs’ alleles are, and the relative frequencies of the alleles in the population. The researcher will also be particularly interested in coding SNPs, whose alleles change the amino acid sequence of the gene product and therefore might represent functional variations. This protocol provides details on how to use the genome browser to navigate to and explore HapMap data for a gene or region of interest.
Retrieving HapMap Data via Bulk Download
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. Bulk downloads of chromosome- or genome-wide data provide text dumps of the entire HapMap data set. Although complete, such downloads do not provide any filtering or selection services. This protocol describes the retrieval of HapMap data via bulk download.
Bloodstream Infections Caused by Extended-Spectrum-β-Lactamase- Producing
Escherichia coli
: Risk Factors for Inadequate Initial Antimicrobial Therapy
Authors: Tumbarello Mario , Sali Michela , Trecarichi Enrico Maria , Leone Fiammetta , Rossi Marianna , Fiori Barbara , De Pascale Gennaro , D'Inzeo Tiziana , Sanguinetti Maurizio , Fadda Giovanni , Cauda Roberto , Spanu Teresa 
Published: July 2008

ABSTRACT

Extended-spectrum-β-lactamase (ESBL)-producing strains of
Escherichia coli
are a significant cause of bloodstream infections (BSI) in hospitalized and nonhospitalized patients. We previously showed that delaying effective antimicrobial therapy in BSI caused by ESBL producers significantly increases mortality. The aim of this retrospective 7-year analysis was to identify risk factors for inadequate initial antimicrobial therapy (IIAT) (i.e., empirical treatment based on a drug to which the isolate had displayed in vitro resistance) for inpatients with BSI caused by ESBL-producing
E. coli
. Of the 129 patients considered, 56 (43.4%) received IIAT for 48 to 120 h (mean, 72 h). Independent risk factors for IIAT include an unknown BSI source (odds ratios [OR], 4.86; 95% confidence interval [CI], 1.98 to 11.91;
P
= 0.001), isolate coresistance to ≥3 antimicrobials (OR, 3.73; 95% CI, 1.58 to 8.83;
P
= 0.003), hospitalization during the 12 months preceding BSI onset (OR, 3.33; 95% CI, 1.42 to 7.79;
P
= 0.005), and antimicrobial therapy during the 3 months preceding BSI onset (OR, 2.65; 95% CI, 1.11 to 6.29;
P
= 0.02). IIAT was the strongest risk factor for 21-day mortality and significantly increased the length of hospitalization after BSI onset. Our results underscore the need for a systematic approach to the management of patients with serious infections by ESBL-producing
E. coli
. Such an approach should be based on sound, updated knowledge of local infectious-disease epidemiology, detailed analysis of the patient's history with emphasis on recent contact with the health care system, and aggressive attempts to identify the infectious focus that has given rise to the BSI.
Mutations in the
fks1
Gene in
Candida albicans
,
C. tropicalis
, and
C. krusei
Correlate with Elevated Caspofungin MICs Uncovered in AM3 Medium Using the Method of the European Committee on Antibiotic Susceptibility Testing
Authors: Desnos-Ollivier Marie , Bretagne Stéphane , Raoux Dorothée , Hoinard Damien , Dromer Françoise , Dannaoui Eric 
Published: July 2008

ABSTRACT

Mutations in two specific regions of the Fks1 subunit of 1,3-β-
d
-glucan synthase are known to confer decreased caspofungin susceptibility on
Candida
spp. Clinical isolates of
Candida
spp. (404
Candida albicans
, 62
C. tropicalis
, and 21
C. krusei
isolates) sent to the French National Reference Center were prospectively screened for susceptibility to caspofungin in vitro by the broth microdilution reference method of the Antifungal Susceptibility Testing Subcommittee of the European Committee on Antibiotic Susceptibility Testing (AFST-EUCAST). Twenty-eight isolates (25
C. albicans
, 2
C. tropicalis
, and 1
C. krusei
isolate) for which the caspofungin MIC was above the MIC that inhibited 90% of the isolates of the corresponding species (MIC
90
) were subjected to molecular analysis in order to identify mutations in the
fks1
gene. Substitutions in the deduced protein sequence of Fks1 were found for 8 isolates, and 20 isolates had the wild-type sequence. Among the six
C. albicans
isolates harboring mutations, six patterns were observed involving amino acid changes at positions 641, 645, 649, and 1358. For
C. tropicalis
, one isolate showed an L644W mutation, and for one
C. krusei
isolate, two mutations, L658W and L701M, were found. Two media, RPMI medium and AM3, were tested for their abilities to distinguish between isolates with wild-type Fks1 and those with mutant Fks1. In RPMI medium, caspofungin MICs ranged from 0.25 to 2 μg/ml for wild-type isolates and from 1 to 8 μg/ml for mutant isolates. A sharper difference was observed in AM3: all wild-type isolates were inhibited by 0.25 μg/ml of caspofungin, while caspofungin MICs for all mutant isolates were ≥0.5 μg/ml. These data demonstrate that clinical isolates of
C. albicans
,
C. tropicalis
, and
C. krusei
with decreased susceptibility to caspofungin in vitro have diverse mutations in the
fks1
gene and that AM3 is potentially a better medium than RPMI for distinguishing between mutant and wild-type isolates using the AFST-EUCAST method.
Relationship between Vancomycin MIC and Failure among Patients with Methicillin-Resistant
Staphylococcus aureus
Bacteremia Treated with Vancomycin
Authors: Lodise T. P. , Graves J. , Evans A. , Graffunder E. , Helmecke M. , Lomaestro B. M. , Stellrecht K. 
Published: July 2008

ABSTRACT

There is growing concern that vancomycin has diminished activity for methicillin-resistant
Staphylococcus aureus
(MRSA) infections, with vancomycin MICs at the high end of the CLSI susceptibility range. Despite this growing concern, there are limited clinical data to support this notion. To better elucidate this, a retrospective cohort study was conducted among patients with MRSA bloodstream infections who were treated with vancomycin between January 2005 and May 2007. The inclusion criteria were as follows: at least 18 years old, nonneutropenic, with an MRSA culture that met the CDC criteria for bloodstream infection, had received vancomycin therapy within 48 h of the index blood culture, and survived >24 h after vancomycin administration. Failure was defined as 30-day mortality, bacteremia ≥10 days on vancomycin therapy, or a recurrence of MRSA bacteremia within 60 days of vancomycin discontinuation. Classification and regression tree (CART) analysis identified the vancomycin MIC breakpoint associated with an increased probability of failure. During the study period, 92 patients met the inclusion criteria. The vancomycin MIC breakpoint derived by CART analysis was ≥1.5 mg/liter. The 66 patients with vancomycin MICs of ≥1.5 mg/liter had a 2.4-fold increase in failure compared to patients with MICs of ≤1.0 mg/liter (36.4% and 15.4%, respectively;
P
= 0.049). In the Poisson regression, a vancomycin MIC of ≥1.5 mg/liter was independently associated with failure (adjusted risk ratio, 2.6; 95% confidence interval, 1.3 to 5.4;
P
= 0.01). These data strongly suggest that patients with MRSA bloodstream infections with vancomycin MICs of ≥1.5 mg/liter respond poorly to vancomycin. Alternative anti-MRSA therapies should be considered for these patients.
Association of Fluconazole Pharmacodynamics with Mortality in Patients with Candidemia
Authors: Baddley John W. , Patel Mukesh , Bhavnani Sujata M. , Moser Stephen A. , Andes David R. 
Published: July 2008

ABSTRACT

Recent studies of nonneutropenic patients with candidemia or candidiasis suggest that fluconazole pharmacodynamic parameters correlate with clinical outcomes; however, additional data of correlation to mortality in patients with candidemia would be valuable. We assessed the impact of MICs for
Candida
, fluconazole pharmacodynamics, and patient characteristics on all-cause mortality with use of a prospective cohort of 96 hospitalized patients with candidemia. Among 84 patients for whom
Candida
isolates were available for testing, the most frequent
Candida
species isolated were
Candida albicans
(44%), followed by
Candida parapsilosis
(20.2%), and
Candida glabrata
(20.2%). Fluconazole resistance (MIC of ≥64 μg/ml) was present in 7 (8.3%) to 10 (11.9%) of 84 isolates, depending on the MIC endpoint determination method (50% or 80% inhibition read at 24 or 48 h). Overall mortality occurred in 27 (28.1%) of 96 patients, and nonsurvivors were more likely to have fluconazole-resistant isolates (25% versus 6.7%;
P
= 0.02). Multivariable analysis demonstrated an association between fluconazole resistance and mortality, but it did not reach statistical significance (odds ratio, 5.3; 95% confidence interval, 0.8 to 33.4;
P
= 0.08). By pharmacodynamic analysis, a fluconazole area under the concentration-time curve/MIC of <11.5 or MIC of ≥64 was associated with increased patient mortality (
P
≤ 0.09). These data support previous findings of an antifungal exposure-response relationship to mortality in patients with candidemia. In addition, similar MICs were obtained using a 24- or 48-h MIC endpoint determination, thus providing the opportunity to assess earlier the impact of isolate susceptibility on therapy.
Role of Plasma Proteins in Pharmacokinetics of Micafungin, an Antifungal Antibiotic, in Analbuminemic Rats
Authors: Abe Fumie , Ueyama Jun , Kawasumi Noriyo , Nadai Masayuki , Hayashi Tamon , Kato Miki , Ohnishi Masafumi , Saito Hiroko , Takeyama Naoshi , Hasegawa Takaaki 
Published: July 2008

ABSTRACTThere were no significant differences in the pharmacokinetics of micafungin and expression of hepatic multidrug resistance-associated protein 2 (ABCC2/Mrp2) between analbuminemic and Sprague-Dawley rats. Micafungin bound strongly to high-density lipoprotein (HDL) and moderately to gamma globulin. These results suggest that HDL and gamma globulin contribute to the pharmacokinetics of micafungin.
Combinations of Cyclophilin Inhibitor NIM811 with Hepatitis C Virus NS3-4A Protease or NS5B Polymerase Inhibitors Enhance Antiviral Activity and Suppress the Emergence of Resistance
Authors: Mathy Joanna E. , Ma Sue , Compton Teresa , Lin Kai 
Published: July 2008

ABSTRACT
Chronic hepatitis C virus (HCV) infection remains a major global health burden while current interferon-based therapy is suboptimal. Efforts to develop more effective antiviral agents mainly focus on two viral targets: NS3-4A protease and NS5B polymerase. However, resistant mutants against these viral specific inhibitors emerge quickly both in vitro and in patients, particularly in the case of monotherapy. An alternative and complementary strategy is to target host factors such as cyclophilins that are also essential for viral replication. Future HCV therapies will most likely be combinations of multiple drugs of different mechanisms to maximize antiviral activity and to suppress the emergence of resistance. Here, the effects of combining a host cyclophilin inhibitor NIM811 with other viral specific inhibitors were investigated in vitro using HCV replicon. All of the combinations led to more pronounced antiviral effects than any single agent, with no significant increase of cytotoxicity. Moreover, the combination of NIM811 with a nucleoside (NM107) or a non-nucleoside (thiophene-2-carboxylic acid) polymerase inhibitor was synergistic, while the combination with a protease inhibitor (BILN2061) was additive. Resistant clones were selected in vitro with these inhibitors. Interestingly, it was much more difficult to develop resistance against NIM811 than viral specific inhibitors. No cross-resistance was observed among these inhibitors. Most notably, NIM811 was highly effective in blocking the emergence of resistance when used in combination with viral protease or polymerase inhibitors. Taken together, these results illustrate the significant advantages of combining inhibitors targeting both viral and host factors as key components of future HCV therapies.
Complete Sequence of p07-406, a 24,179-Base-Pair Plasmid Harboring theblaVIM-7Metallo-β-Lactamase Gene in aPseudomonas aeruginosaIsolate from the United States
Authors: Li Hongyang , Toleman Mark A. , Bennett Peter M. , Jones Ronald N. , Walsh Timothy R. 
Published: July 2008

ABSTRACTAn outbreak involving aPseudomonas aeruginosastrain that was resistant to all tested antimicrobials except polymyxin B occurred in a hospital in Houston, TX. Previous studies on this strain showed that it possesses a novel mobile metallo-β-lactamase (MBL) gene, designatedblaVIM-7, located on a plasmid (p07-406). Here, we report the complete sequence, annotation, and functional characterization of this plasmid. p07-406 is 24,179 bp in length, and 29 open reading frames were identified related to known or putatively recognized proteins. Analysis of this plasmid showed it to be comprised of four distinct regions: (i) a region of 5,200 bp having a Tn501-like mercuric resistance (mer) transposon upstream of the replication region; (ii) a Tn3-like transposon carrying a truncated integron with ablaVIM-7gene and an insertion sequence inserted at the other end of this transposon; (iii) a region of four genes, upstream of the Tn3-like transposon, possessing very high similarity to plasmid pXcB fromXanthomonas campestrispv.citricommonly associated with plants; (iv) a backbone sequence similar to the backbone structure of the IncP group plasmid Rms149, pB10, and R751. This is the first plasmid to be sequenced carrying an MBL gene and highlights the amelioration of DNA segments from disparate origins, most noticeably from plant pathogens.
Effects of Subinhibitory Concentrations of Antibiotics on SOS and DNA Repair Gene Expression in
Staphylococcus aureus
Authors: Mesak Lili Rosana , Miao Vivian , Davies Julian 
Published: July 2008

ABSTRACT

Reporter clones of
Staphylococcus aureus
with different SOS response- and DNA repair-associated promoter-
lux
gene fusion constructs were constructed to study the effects of sub-MICs of antibiotics on the transcription of the SOS and methyl mismatch repair (MMR) genes. Fluoroquinolones (FQs) upmodulated both the SOS and the MMR genes. The patterns of antibiotic-induced transcriptional modulation were altered in FQ-resistant mutants.
Correlation of Antimicrobial Resistance with β-Lactamases, the OmpA-Like Porin, and Efflux Pumps in Clinical Isolates of
Acinetobacter baumannii
Endemic to New York City
Authors: Bratu Simona , Landman David , Martin Don Antonio , Georgescu Claudiu , Quale John 
Published: July 2008

ABSTRACT

Acinetobacter baumannii
strains resistant to all β-lactams, aminoglycosides, and fluoroquinolones have emerged in many medical centers. Potential mechanisms contributing to antimicrobial resistance were investigated in 40 clinical isolates endemic to New York City. The isolates were examined for the presence of various β-lactamases, aminoglycoside-modifying enzymes, and mutations in
gyrA
and
parC
. Expression of the genes encoding the β-lactamase AmpC, the efflux systems AdeABC and AbeM, and the OmpA-like porin was also examined by real-time reverse transcription-PCR. No VIM, IMP, KPC, OXA-23-type, OXA-24-type, or OXA-58 β-lactamases were detected, although several isolates had acquired
bla
SHV-5
. Most cephalosporin-resistant isolates had increased levels of expression of
ampC
and/or had acquired
bla
SHV-5
; however, isolates without these features still had reduced susceptibility to cefepime that was mediated by the AdeABC efflux system. Although most isolates with IS
Aba1
upstream of the
bla
OXA-51
-like carbapenemase gene were resistant to meropenem, several remained susceptible to imipenem. The presence of aminoglycoside-modifying enzymes and gyrase mutations accounted for aminoglycoside and fluoroquinolone resistance, respectively. The increased expression of
adeABC
was not an important contributor to aminoglycoside or fluoroquinolone resistance but did correlate with reduced susceptibility to tigecycline. The expression of
abeM
and
ompA
and phenotypic changes in OmpA did not correlate with antimicrobial resistance.
A. baumannii
has become a well-equipped nosocomial pathogen; defining the relative contribution of these and other mechanisms of antimicrobial resistance will require further investigation.
Inhibition of Malaria Parasite Development by a Cyclic Peptide That Targets the Vital Parasite Protein SERA5
Authors: Fairlie W. Douglas , Spurck Tim P. , McCoubrie Joanne E. , Gilson Paul R. , Miller Susanne K. , McFadden Geoffrey I. , Malby Robyn , Crabb Brendan S. , Hodder Anthony N. 
Published: July 2008

ABSTRACTThe serine repeat antigen (SERA) proteins of the malaria parasitesPlasmodiumspp. contain a putative enzyme domain similar to that of papain family cysteine proteases. InPlasmodium falciparumparasites, more than half of the SERA family proteins, including the most abundantly expressed form, SERA5, have a cysteine-to-serine substitution within the putative catalytic triad of the active site. Although SERA5 is required for blood-stage parasite survival, the occurrence of a noncanonical catalytic triad casts doubt on the importance of the enzyme domain in this function. We used phage display to identify a small (14-residue) disulfide-bonded cyclic peptide (SBP1) that targets the enzyme domain of SERA5. Biochemical characterization of the interaction shows that it is dependent on the conformation of both the peptide and protein. Addition of this peptide to parasite cultures compromised development of late-stage parasites compared to that of control parasites or those incubated with equivalent amounts of the carboxymethylated peptide. This effect was similar in two different strains ofP. falciparumas well as in a transgenic strain where the gene encoding the related serine-type parasitophorous vacuole protein SERA4 was deleted. In compromised parasites, the SBP1 peptide crosses both the erythrocyte and parasitophorous vacuole membranes and accumulates within the parasitophorous vacuole. In addition, both SBP1 and SERA5 were identified in the parasite cytosol, indicating that the plasma membrane of the parasite was compromised as a result of SBP1 treatment. These data implicate an important role for SERA5 in the regulation of the intraerythrocytic development of late-stage parasites and as a target for drug development.