bluuebunny/crossref_metadata_2025_split huggingface dataset:
Estimation of Intracellular Concentration of Stavudine Triphosphate in HIV-Infected Children Given a Reduced Dose of 0.5 Milligrams per Kilogram Twice Daily
Authors: Sy Sherwin K. B. , Innes Steve , Derendorf Hartmut , Cotton Mark F. , Rosenkranz Bernd 
Published: December 2013

ABSTRACTThe antiviral efficacy of stavudine depends on the trough concentration of its intracellular metabolite, stavudine-triphosphate (d4T-TP), while the degree of stavudine's mitochondrial toxicity depends on its peak concentration. Rates of mitochondrial toxicity are high when stavudine is used at the current standard pediatric dose (1 mg/kg twice daily [BID]). Evidence from adult work suggests that half of the original standard adult dose (i.e., 20 mg BID) may be equally effective, with markedly less mitochondrial toxicity. We present a population pharmacokinetic model to predict intracellular d4T-TP concentrations in pediatric HIV-infected patients administered a dose of 0.5 mg/kg BID. Our model predicted that the reduced pediatric dose would result in a trough intracellular d4T-TP concentration above that of the reduced 20-mg adult dose and a peak concentration below that of the 20-mg adult dose. The simulated pediatric intracellular d4T-TP at 0.5 mg/kg BID resulted in median peak and trough values of approximately 23.9 fmol/106cells (95% prediction interval [PI], 14.2 to 41 fmol/106cells) and 14.8 fmol/106cells (95% PI, 7.2 to 31 fmol/106cells), respectively. The peak and trough concentrations resulting from a 20-mg BID adult dose were 28.4 fmol/106cells (95% PI, 17.3 to 45.5 fmol/106cells) and 13 fmol/106cells (95% PI, 6.8 to 28.6 fmol/106cells), respectively. Halving the current standard pediatric dose should therefore not compromise antiviral efficacy, while markedly reducing mitochondrial toxicity.
Image-Based 384-Well High-Throughput Screening Method for the Discovery of Skyllamycins A to C as Biofilm Inhibitors and Inducers of Biofilm Detachment in Pseudomonas aeruginosa
Authors: Navarro Gabriel , Cheng Andrew T. , Peach Kelly C. , Bray Walter M. , Bernan Valerie S. , Yildiz Fitnat H. , Linington Roger G. 
Published: December 2013

ABSTRACT

To date, most antibiotics have primarily been developed to target bacteria in the planktonic state. However, biofilm formation allows bacteria to develop tolerance to antibiotics and provides a mechanism to evade innate immune systems. Therefore, there is a significant need to identify small molecules to prevent biofilm formation and, more importantly, to disperse or eradicate preattached biofilms, which are a major source of bacterial persistence in nosocomial infections. We now present a modular high-throughput 384-well image-based screening platform to identify
Pseudomonas aeruginosa
biofilm inhibitors and dispersal agents. Biofilm coverage measurements were accomplished using non-z-stack epifluorescence microscopy to image a constitutively expressing green fluorescent protein (GFP)-tagged strain of
P. aeruginosa
and quantified using an automated image analysis script. Using the redox-sensitive dye XTT, bacterial cellular metabolic activity was measured in conjunction with biofilm coverage to differentiate between classical antibiotics and nonantibiotic biofilm inhibitors/dispersers. By measuring biofilm coverage and cellular activity, this screen identifies compounds that eradicate biofilms through mechanisms that are disparate from traditional antibiotic-mediated biofilm clearance. Screening of 312 natural-product prefractions identified the cyclic depsipeptide natural products skyllamycins B and C as nonantibiotic biofilm inhibitors with 50% effective concentrations (EC
50
s) of 30 and 60 μM, respectively. Codosing experiments of skyllamycin B and azithromycin, an antibiotic unable to clear preattached biofilms, demonstrated that, in combination, these compounds were able to eliminate surface-associated biofilms and depress cellular metabolic activity. The skyllamycins represent the first known class of cyclic depsipeptide biofilm inhibitors/dispersers.
Functional Linkage between Genes That Regulate Osmotic Stress Responses and Multidrug Resistance Transporters: Challenges and Opportunities for Antibiotic Discovery
Authors: Cohen B. Eleazar 
Published: December 2013

ABSTRACT
All cells need to protect themselves against the osmotic challenges of their environment by maintaining low permeability to ions across their cell membranes. This is a basic principle of cellular function, which is reflected in the interactions among ion transport and drug efflux genes that have arisen during cellular evolution. Thus, upon exposure to pore-forming antibiotics such as amphotericin B (AmB) or daptomycin (Dap), sensitive cells overexpress common resistance genes to protect themselves from added osmotic challenges. These genes share pathway interactions with the various types of multidrug resistance (MDR) transporter genes, which both preserve the native lipid membrane composition and at the same time eliminate disruptive hydrophobic molecules that partition excessively within the lipid bilayer. An increased understanding of the relationships between the genes (and their products) that regulate osmotic stress responses and MDR transporters will help to identify novel strategies and targets to overcome the current stalemate in drug discovery.
Assessment of an Anti-Alpha-Toxin Monoclonal Antibody for Prevention and Treatment of Staphylococcus aureus-Induced Pneumonia
Authors: Hua L. , Hilliard J. J. , Shi Y. , Tkaczyk C. , Cheng L. I. , Yu X. , Datta V. , Ren S. , Feng H. , Zinsou R. , Keller A. , O'Day T. , Du Q. , Cheng L. , Damschroder M. , Robbie G. , Suzich J. , Stover C. K. , Sellman B. R. 
Published: December 2013

ABSTRACTAlpha-toxin (AT) is a major virulence factor in the disease pathogenesis ofStaphylococcus aureus. We previously identified a monoclonal antibody (MAb) against AT that reduced disease severity in a mouse dermonecrosis model. Here, we evaluate the activity of an affinity-optimized variant, LC10, in a mouse model ofS. aureuspneumonia. Passive immunization with LC10 increased survival and reduced bacterial numbers in the lungs and kidneys of infected mice and showed protection against diverseS. aureusclinical isolates. The lungs ofS. aureus-infected mice exhibited bacterial pneumonia, including widespread inflammation, whereas the lungs of mice that received LC10 exhibited minimal inflammation and retained healthy architecture. Consistent with reduced immune cell infiltration, LC10-treated animals had significantly lower (P< 0.05) proinflammatory cytokine and chemokine levels in the bronchoalveolar lavage fluid than did those of the control animals. This reduction in inflammation and damage to the LC10-treated animals resulted in reduced vascular protein leakage and CO2levels in the blood. LC10 was also assessed for its therapeutic activity in combination with vancomycin or linezolid. Treatment with a combination of LC10 and vancomycin or linezolid resulted in a significant increase (P< 0.05) in survival relative to the monotherapies and was deemed additive to synergistic by isobologram analysis. Consistent with improved survival, the lungs of animals treated with antibiotic plus LC10 exhibited less inflammatory tissue damage than those that received monotherapy. These data provide insight into the mechanisms of protection provided by AT inhibition and support AT as a promising target for immunoprophylaxis or adjunctive therapy againstS. aureuspneumonia.
Pathogenicity of Clinical Acinetobacter baumannii Isolates in a Galleria mellonella Host Model According to
bla
OXA-40
Gene and Epidemiological Outbreak Status
Authors: Esterly John S. , McLaughlin Milena M. , Malczynski Michael , Qi Chao , Zembower Teresa R. , Scheetz Marc H. 
Published: December 2013

ABSTRACT

Clinical studies have suggested that
bla
OXA-40
-positive
Acinetobacter baumannii
isolates are associated with poor patient outcomes; however, reasons for unfavorable outcomes are difficult to discern in clinical studies. The objective of this study was to assess the virulence of carbapenem-resistant
A. baumannii
according to
bla
OXA-40
and epidemiological outbreak status in a
Galleria mellonella
model. Eight isolates of
A. baumannii
were studied. Nonoutbreak isolates and
bla
OXA-40
-negative isolates more rapidly killed infected
G. mellonella
(
P
< 0.01).
A Geobacter sulfurreducens Strain Expressing Pseudomonas aeruginosa Type IV Pili Localizes OmcS on Pili but Is Deficient in Fe(III) Oxide Reduction and Current Production
Authors: Liu Xing , Tremblay Pier-Luc , Malvankar Nikhil S. , Nevin Kelly P. , Lovley Derek R. , Vargas Madeline 
Published: December 2013

ABSTRACT

The conductive pili of
Geobacter
species play an important role in electron transfer to Fe(III) oxides, in long-range electron transport through current-producing biofilms, and in direct interspecies electron transfer. Although multiple lines of evidence have indicated that the pili of
Geobacter sulfurreducens
have a metal-like conductivity, independent of the presence of
c
-type cytochromes, this claim is still controversial. In order to further investigate this phenomenon, a strain of
G. sulfurreducens
, designated strain PA, was constructed in which the gene for the native PilA, the structural pilin protein, was replaced with the PilA gene of
Pseudomonas aeruginosa
PAO1. Strain PA expressed and properly assembled
P. aeruginosa
PilA subunits into pili and exhibited a profile of outer surface
c
-type cytochromes similar to that of a control strain expressing the
G. sulfurreducens
PilA. Surprisingly, the strain PA pili were decorated with the
c
-type cytochrome OmcS in a manner similar to the control strain. However, the strain PA pili were 14-fold less conductive than the pili of the control strain, and strain PA was severely impaired in Fe(III) oxide reduction and current production. These results demonstrate that the presence of OmcS on pili is not sufficient to confer conductivity to pili and suggest that there are unique structural features of the
G. sulfurreducens
PilA that are necessary for conductivity.
An Oleaginous Bacterium That Intrinsically Accumulates Long-Chain Free Fatty Acids in its Cytoplasm
Authors: Katayama Taiki , Kanno Manabu , Morita Naoki , Hori Tomoyuki , Narihiro Takashi , Mitani Yasuo , Kamagata Yoichi 
Published: December 2013

ABSTRACT

Medium- and long-chain fatty acids are present in organisms in esterified forms that serve as cell membrane constituents and storage compounds. A large number of organisms are known to accumulate lipophilic materials as a source of energy and carbon. We found a bacterium, designated GK12, that intrinsically accumulates free fatty acids (FFAs) as intracellular droplets without exhibiting cytotoxicity. GK12 is an obligatory anaerobic, mesophilic lactic acid bacterium that was isolated from a methanogenic reactor. Phylogenetic analysis based on 16S rRNA gene sequences showed that GK12 is affiliated with the family
Erysipelotrichaceae
in the phylum
Firmicutes
but is distantly related to type species in this family (less than 92% similarity in 16S rRNA gene sequence). Saturated fatty acids with carbon chain lengths of 14, 16, 18, and 20 were produced from glucose under stress conditions, including higher-than-optimum temperatures and the presence of organic solvents that affect cell membrane integrity. FFAs were produced at levels corresponding to up to 25% (wt/wt) of the dry cell mass. Our data suggest that FFA accumulation is a result of an imbalance between excess membrane fatty acid biosynthesis due to homeoviscous adaptation and limited β-oxidation activity due to anaerobic growth involving lactic acid fermentation. FFA droplets were not further utilized as an energy and carbon source, even under conditions of starvation. A naturally occurring bacterium that accumulates significant amounts of long-chain FFAs with noncytotoxicity would provide useful strategies for microbial biodiesel production.
Novel Maltotriose-Hydrolyzing Thermoacidophilic Type III Pullulan Hydrolase from Thermococcus kodakarensis
Authors: Ahmad Nasir , Rashid Naeem , Haider Muhammad Saleem , Akram Mehwish , Akhtar Muhammad 
Published: December 2013

ABSTRACT

A novel thermoacidophilic pullulan-hydrolyzing enzyme (PUL) from hyperthermophilic archaeon
Thermococcus kodakarensis
(TK-PUL) that efficiently hydrolyzes starch under industrial conditions in the absence of any additional metal ions was cloned and characterized. TK-PUL possessed both pullulanase and α-amylase activities. The highest activities were observed at 95 to 100°C. Although the enzyme was active over a broad pH range (3.0 to 8.5), the pH optima for both activities were 3.5 in acetate buffer and 4.2 in citrate buffer. TK-PUL was stable for several hours at 90°C. Its half-life at 100°C was 45 min when incubated either at pH 6.5 or 8.5. The

K
m

value toward pullulan was 2 mg ml
−1
, with a
V
max
of 109 U mg
−1
. Metal ions were not required for the activity and stability of recombinant TK-PUL. The enzyme was able to hydrolyze both α-1,6 and α-1,4 glycosidic linkages in pullulan. The most preferred substrate, after pullulan, was γ-cyclodextrin, which is a novel feature for this type of enzyme. Additionally, the enzyme hydrolyzed a variety of polysaccharides, including starch, glycogen, dextrin, amylose, amylopectin, and cyclodextrins (α, β, and γ), mainly into maltose. A unique feature of TK-PUL was the ability to hydrolyze maltotriose into maltose and glucose.
Intimin Gene (
eae
) Subtype-Based Real-Time PCR Strategy for Specific Detection of Shiga Toxin-Producing Escherichia coli Serotypes O157:H7, O26:H11, O103:H2, O111:H8, and O145:H28 in Cattle Feces
Authors: Bibbal Delphine , Loukiadis Estelle , Kérourédan Monique , Peytavin de Garam Carine , Ferré Franck , Cartier Philippe , Gay Emilie , Oswald Eric , Auvray Frédéric , Brugère Hubert 
Published: December 2013

ABSTRACT

Shiga toxin-producing
Escherichia coli
(STEC) strains belonging to serotypes O157:H7, O26:H11, O103:H2, O111:H8, and O145:H28 are known to be associated with particular subtypes of the intimin gene (
eae
), namely, γ1, β1, ε, θ, and γ1, respectively. This study aimed at evaluating the usefulness of their detection for the specific detection of these five main pathogenic STEC serotypes in cattle feces. Using real-time PCR assays, 58.7% of 150 fecal samples were found positive for at least one of the four targeted
eae
subtypes. The simultaneous presence of
stx
,
eae
, and one of the five O group markers was found in 58.0% of the samples, and the five targeted
stx
plus
eae
plus O genetic combinations were detected 143 times. However, taking into consideration the association between
eae
subtypes and O group markers, the resulting
stx
plus
eae
subtype plus O combinations were detected only 46 times. The 46 isolation assays performed allowed recovery of 22
E. coli
strains belonging to one of the five targeted STEC serogroups. In contrast, only 2 of 39 isolation assays performed on samples that were positive for
stx
,
eae
and an O group marker, but that were negative for the corresponding
eae
subtype, were successful. Characterization of the 24
E. coli
isolates showed that 6 were STEC, including 1 O157:H7, 3 O26:H11, and 2 O145:H28. The remaining 18 strains corresponded to atypical enteropathogenic
E. coli
(aEPEC). Finally, the more discriminating
eae
subtype-based PCR strategy described here may be helpful for the specific screening of the five major STEC in cattle feces.
An Unbiased Method for Clustering Bacterial Effectors Using Host Cellular Phenotypes
Authors: Dowling Andrea J. , Hodgson David J. 
Published: December 2013

ABSTRACTWe present a novel method implementing unbiased high-content morphometric cell analysis to classify bacterial effector phenotypes. This clustering methodology represents a significant advance over more qualitative visual approaches and can also be used to classify, and therefore predict the likely function of, unknown effector genes from any microbial genome. As a proof of concept, we use this approach to investigate 23 genetic regions predicted to encode antimacrophage effectors located across the genome of the insect and human pathogenPhotorhabdus asymbiotica. Statistical cluster analysis using multiple cellular measures categorized treated macrophage phenotypes into three major groups relating to their putative functionality: (i) adhesins, (ii) cytolethal toxins, and (iii) cytomodulating toxins. Further investigation into their effects on phagocytosis revealed that several effectors also modulate this function and that the nature of this modulation (increased or decreased phagocytosis) is linked to the phenotype cluster group. Categorizing potential functionalities in this way allows rapid functional follow-up of key candidates for more-directed cell biological or biochemical investigation. Such an unbiased approach to the classification of candidate effectors will be useful for describing virulence-related regions in a wide range of genomes and will be useful in assigning putative functions to the growing number of microbial genes whose function remains unclear from homology searching.
Motility Characteristics Are Altered for Rickettsia bellii Transformed To Overexpress a Heterologous
rickA
Gene
Authors: Oliver Jonathan D. , Burkhardt Nicole Y. , Felsheim Roderick F. , Kurtti Timothy J. , Munderloh Ulrike G. 
Published: December 2013

ABSTRACT

The rickettsial protein RickA activates host cell factors associated with the eukaryotic actin cytoskeleton and is likely involved with rickettsial host cell binding and infection and the actin-based motility of spotted fever group rickettsiae. The
rickA
gene sequence and protein vary substantially between
Rickettsia
species, as do observed motility-associated phenotypes. To help elucidate the function of RickA and determine the effects of species-specific RickA variations, we compared extracellular binding, intracellular motility, and intercellular spread phenotypes of three
Rickettsia bellii
variants. These included two shuttle vector-transformed
R. bellii
strains and the wild-type isolate from which they were derived,
R. bellii
RML 369C. Both plasmid shuttle vectors carried spectinomycin resistance and a GFPuv reporter; one contained
Rickettsia monacensis
-derived
rickA
, and the other lacked the
rickA
gene.
Rickettsia bellii
transformed to express
R. monacensis rickA
highly overexpressed this transcript in comparison to its native
rickA
. These rickettsiae also moved at higher velocities and followed a more curved path than the negative-control transformants. A lower proportion of
R. monacensis rickA
-expressing bacteria ever became motile, however, and they formed smaller plaques.
Novel Firmicutes Group Implicated in the Dechlorination of Two Chlorinated Xanthones, Analogues of Natural Organochlorines
Authors: Krzmarzick Mark J. , Miller Hanna R. , Yan Tao , Novak Paige J. 
Published: December 2013

ABSTRACT

Although the abundance and diversity of natural organochlorines are well established, much is still unknown about the degradation of these compounds. Triplicate microcosms were used to determine whether, and which, bacterial communities could dechlorinate two chlorinated xanthones (2,7-dichloroxanthone and 5,7-dichloro-1,3-dihydroxylxanthone), analogues of a diverse class of natural organochlorines. According to quantitative-PCR (qPCR) results, several known dechlorinating genera were either not present or not enriched during dechlorination of the xanthones. Denaturing gradient gel electrophoresis, however, indicated that several
Firmicutes
were enriched in the dechlorinating cultures compared to triplicate controls amended with nonchlorinated xanthones. One such group, herein referred to as the Gopher group, was further studied with a novel qPCR method that confirmed enrichment of Gopher group 16S rRNA genes in the dechlorinating cultures. The enrichment of the Gopher group was again tested with two new sets of triplicate microcosms. Enrichment was observed during chlorinated xanthone dechlorination in one set of these triplicate microcosms. In the other set, two microcosms showed clear enrichment while a third did not. The Gopher group is a previously unidentified group of
Firmicutes
, distinct from but related to the
Dehalobacter
and
Desulfitobacterium
genera; this group also contains clones from at least four unique cultures capable of dechlorinating anthropogenic organochlorines that have been previously described in the literature. This study suggests that natural chlorinated xanthones may be effective biostimulants to enhance the remediation of pollutants and highlights the idea that novel genera of dechlorinators likely exist and may be active in bioremediation and the natural cycling of chlorine.
Protein Kinase A Is Part of a Mechanism That Regulates Nuclear Reimport of the Nuclear tRNA Export Receptors Los1p and Msn5p
Authors: Pierce Jacqueline B. , van der Merwe George , Mangroo Dev 
Published: December 2013

ABSTRACT

The two main signal transduction mechanisms that allow eukaryotes to sense and respond to changes in glucose availability in the environment are the cyclic AMP (cAMP)/protein kinase A (PKA) and AMP-activated protein kinase (AMPK)/Snf1 kinase-dependent pathways. Previous studies have shown that the nuclear tRNA export process is inhibited in
Saccharomyces cerevisiae
deprived of glucose. However, the signal transduction pathway involved and the mechanism by which glucose availability regulates nuclear-cytoplasmic tRNA trafficking are not understood. Here, we show that inhibition of nuclear tRNA export is caused by a block in nuclear reimport of the tRNA export receptors during glucose deprivation. Cytoplasmic accumulation of the tRNA export receptors during glucose deprivation is not caused by activation of Snf1p. Evidence obtained suggests that PKA is part of the mechanism that regulates nuclear reimport of the tRNA export receptors in response to glucose availability. This mechanism does not appear to involve phosphorylation of the nuclear tRNA export receptors by PKA. The block in nuclear reimport of the tRNA export receptors appears to be caused by activation of an unidentified mechanism when PKA is turned off during glucose deprivation. Taken together, the data suggest that PKA facilitates return of the tRNA export receptors to the nucleus by inhibiting an unidentified activity that facilitates cytoplasmic accumulation of the tRNA export receptors when glucose in the environment is limiting. A PKA-independent mechanism was also found to regulate nuclear tRNA export in response to glucose availability. This mechanism, however, does not regulate nuclear reimport of the tRNA export receptors.