bluuebunny/crossref_metadata_2025_split huggingface dataset:
High-Throughput
Plasmodium falciparum
Growth Assay for Malaria Drug Discovery
Authors: Baniecki Mary Lynn , Wirth Dyann F. , Clardy Jon 
Published: November 2006

ABSTRACT

New therapeutic agents for the treatment of malaria, the world's most deadly parasitic disease, are urgently needed. Malaria afflicts 300 to 500 million people and results in 1 to 2 million deaths annually, and more than 85% of all malaria-related mortality involves young children and pregnant women in sub-Saharan Africa. The emergence of multidrug-resistant parasites, especially in
Plasmodium falciparum
, has eroded the efficacy of almost all currently available therapeutic agents. The discovery of new drugs, including drugs with novel cellular targets, could be accelerated with a whole-organism high-throughput screen (HTS) of structurally diverse small-molecule libraries. The standard whole-organism screen is based on incorporation of [
3
H]hypoxanthine and has liabilities, such as limited throughput, high cost, multiple labor-intensive steps, and disposal of radioactive waste. Recently, screens have been reported that do not use radioactive incorporation, but their reporter signal is not robust enough for HTS. We report a
P. falciparum
growth assay that is technically simple, robust, and compatible with the automation necessary for HTS. The assay monitors DNA content by addition of the fluorescent dye 4′,6-diamidino-2-phenylindole (DAPI) as a reporter of blood-stage parasite growth. This DAPI
P. falciparum
growth assay was used to measure the 50% inhibitory concentrations (IC
50
s) of a diverse set of known antimalarials. The resultant IC
50
s compared favorably with those obtained in the [
3
H]hypoxanthine incorporation assay. Over 79,000 small molecules have been tested for antiplasmodial activity using the DAPI
P. falciparum
growth assay, and 181 small molecules were identified as highly active against multidrug-resistant parasites.
Fitness Costs of Fluoroquinolone Resistance in
Streptococcus pneumoniae
Authors: Rozen Daniel E. , McGee Lesley , Levin Bruce R. , Klugman Keith P. 
Published: November 2006

ABSTRACT

The fitness cost of the genes responsible for resistance to fluoroquinolones in clinical isolates of
Streptococcus pneumoniae
were estimated in vitro in a common genetic background. Naturally occurring
parC
,
parE
, and
gyrA
loci containing mutations in the quinolone-resistance-determining regions were introduced by transformation into
S. pneumoniae
strain R6 individually and in combinations. The fitness of these transformants was estimated by pairwise competition experiments with a common R6 strain. On average, single
par
and
gyr
mutants responsible for low-level MIC resistance (first-step resistance) impose a fitness burden of approximately 8%. Some of these mutants engender no measurable cost, while one, a
parE
mutant, reduces the fitness of these bacteria by more than 40%. Most interestingly, the addition of the second
par
or
gyr
mutations required for clinically significant, high-MIC fluoroquinolone resistance does not increase the fitness burden imposed by these single genes and can even reduce it. We discuss the implications of these results for the epidemiology of fluoroquinolone resistance and the evolution of acquired resistance in treated patients.
Treatment and Outcomes of Infections by Methicillin-Resistant
Staphylococcus aureus
at an Ambulatory Clinic
Authors: Szumowski John D. , Cohen Daniel E. , Kanaya Fumihide , Mayer Kenneth H. 
Published: November 2006

ABSTRACT

Community-acquired methicillin-resistant
Staphylococcus aureus
(MRSA) skin and soft tissue infections (SSTI) have become increasingly common. This study's objectives were to describe the clinical spectrum of MRSA in a community health center and to determine whether the use of specific antimicrobials correlated with increased probability of clinical resolution of SSTI. A retrospective chart review of 399 sequential cases of culture-confirmed
S. aureus
SSTI, including 227 cases of MRSA SSTI, among outpatients at Fenway Community Health (Boston, MA) from 1998 to 2005 was done. The proportion of
S. aureus
SSTI due to MRSA increased significantly from 1998 to 2005 (
P
< 0.0001). Resistance to clindamycin was common (48.2% of isolates). At the beginning of the study period, most patients with MRSA SSTI empirically treated with antibiotics received a beta-lactam, whereas by 2005, 76% received trimethoprim-sulfamethoxazole (TMP-SMX) (
P
< 0.0001). Initially, few MRSA isolates were sensitive to the empirical antibiotic, but 77% were susceptible by 2005 (
P
< 0.0001). A significantly higher percentage of patients with MRSA isolates had clinical resolution on the empirical antibiotic by 2005 (
P
= 0.037). Use of an empirical antibiotic to which the clinical isolate was sensitive was associated with increased odds of clinical resolution on empirical therapy (odds ratio = 5.91), controlling for incision and drainage and HIV status. MRSA now accounts for the majority of SSTI due to
S. aureus
at Fenway, and improved rates of clinical resolution on empirical antibiotic therapy have paralleled increasing use of empirical TMP-SMX for these infections. TMP-SMX appears to be an appropriate empirical antibiotic for suspected MRSA SSTI, especially where clindamycin resistance is common.
Proinflammatory and Antimicrobial Nitric Oxide in Gingival Fluid of Diabetic Patients with Periodontal Disease
Authors: Skaleric Uros , Gaspirc Boris , McCartney-Francis Nancy , Masera Andrej , Wahl Sharon M. 
Published: November 2006

ABSTRACTAbnormal nitric oxide (NO) synthesis has been implicated in the pathogenesis of both periodontal disease and diabetes mellitus. In diabetic patients, increased inducible NO synthase in inflamed gingiva correlated with NO in gingival crevicular fluid. Although increased NO reflected more-severe inflammation, it was associated with reductions in CFU ofPrevotella intermedia, a major periodontopathogen, highlighting dual roles for NO.
Toll-Like Receptors Differentially Regulate CC and CXC Chemokines in Skeletal Muscle via NF-κB and Calcineurin
Authors: Boyd John H. , Divangahi Maziar , Yahiaoui Linda , Gvozdic Dusanka , Qureshi Salman , Petrof Basil J. 
Published: November 2006

ABSTRACT
Immunologically active molecules such as cytokines and chemokines have been implicated in skeletal muscle weakness during sepsis as well as recovery from muscle injury. In sepsis, Toll-like receptors (TLRs) act as key sentinel molecules of the innate immune system. Here we determined skeletal muscle cell responses of two prototypical CC and CXC chemokine genes (monocyte chemoattractant protein 1 [MCP-1] and KC, respectively), to stimulation with specific TLR ligands. In addition, we examined whether NF-κB and calcineurin signaling are involved in these responses. Differentiated myotubes and intact whole muscles expressed TLR2, TLR4, TLR5, and TLR9. Stimulation with ligands for TLR2 (peptidoglycan) or TLR4 (LPS) elicited robust and equivalent levels of MCP-1 and KC mRNA expression, whereas stimulation of TLR5 (by flagellin) required gamma interferon priming to induce similar effects. Although both TLR2 and TLR4 ligands activated the NF-κB pathway, NF-κB reporter activity was approximately 20-fold greater after TLR4 stimulation than after TLR2 stimulation. Inhibitory effects of NF-κB blockade on TLR-mediated chemokine gene expression, by either pharmacological (pyrrolidine dithiocarbamate) or molecular (IKKβ dominant-negative transfection) methods, were also more pronounced during TLR4 stimulation. In contrast, inhibitory effects on TLR-mediated chemokine expression of calcineurin blockade (by FK506) were greater for TLR2 than for TLR4 stimulation. MCP-1 and KC mRNA levels also demonstrated differential responses to NF-κB and calcineurin blockade during stimulation with specific TLR ligands. We conclude that skeletal muscle cells differentially utilize the NF-κB and calcineurin pathways in a TLR-specific manner to enable complex regulation of CC and CXC chemokine gene expression.
Clustering of
Helicobacter pylori
VacA in Lipid Rafts, Mediated by Its Receptor, Receptor-Like Protein Tyrosine Phosphatase β, Is Required for Intoxication in AZ-521 Cells
Authors: Nakayama Masaaki , Hisatsune Jyunzo , Yamasaki Eiki , Nishi Yoshito , Wada Akihiro , Kurazono Hisao , Sap Jan , Yahiro Kinnosuke , Moss Joel , Hirayama Toshiya 
Published: November 2006

ABSTRACT

Helicobacter pylori
vacuolating cytotoxin, VacA, induces multiple effects on epithelial cells through different cellular events: one involves pore formation, leading to vacuolation, mitochondrial damage, and apoptosis, and the second involves cell signaling, resulting in stimulation of proinflammatory responses and cell detachment. Our recent data demonstrated that VacA uses receptor-like protein tyrosine phosphatase β (RPTPβ) as a receptor, of which five residues (QTTQP) at positions 747 to 751 are involved in binding. In AZ-521 cells, which mainly express RPTPβ, VacA, after binding to RPTPβ in non-lipid raft microdomains on the cell surface, is localized with RPTPβ in lipid rafts in a temperature- and VacA concentration-dependent process. Methyl-β-cyclodextrin (MCD) did not block binding to RPTPβ but inhibited translocation of VacA with RPTPβ to lipid rafts and all subsequent events. On the other hand, 5-nitro-2-(3-phenylpropylamino)-benzoic acid (NPPB), which disrupts anion channels, did not inhibit translocation of VacA to lipid rafts or VacA-induced activation of p38 mitogen-activated protein (MAP) kinase, but inhibited VacA internalization followed by vacuolation. Thus, p38 MAP kinase activation did not appear to be required for internalization. In contrast, phosphatidylinositol-specific phospholipase C (PI-PLC) inhibited translocation, as well as p38 MAP kinase/ATF-2 activation, internalization, and VacA-induced vacuolation. Neither NPPB nor PI-PLC affected VacA binding to cells and to its receptor, RPTPβ. Thus, receptor-dependent translocation of VacA to lipid rafts is critical for signaling pathways leading to p38 MAP kinase/ATF-2 activation and vacuolation.
Signal Transduction and Nuclear Responses in
Staphylococcus aureus-
Induced Expression of Human β-Defensin 3 in Skin Keratinocytes
Authors: Menzies Barbara E. , Kenoyer Aimee 
Published: November 2006

ABSTRACT

The human β-defensin 3 (hBD-3) is an inducible epithelial peptide antibiotic that has potent antistaphylococcal activity. Infection of skin epithelial cells with viable
Staphylococcus aureus
, a common skin pathogen, induces increased gene expression of hBD-3 and other antimicrobial peptides. The aim of this study was to identify signaling pathways and nuclear responses that contribute to the gene expression of hBD-3 in primary human keratinocytes upon contact with
S. aureus
. Increased hBD-3 peptide was observed by immunofluorescence microscopy in keratinocytes exposed to
S. aureus
and to lipoteichoic acid (LTA). Both are ligands for the cell surface Toll-like receptor 2 (TLR2), and thus the contribution of TLR2 signaling in hBD-3 expression was examined. Functional inhibition of TLR2 prior to
S. aureus
stimulation significantly decreased hBD-3 mRNA levels by 37%, attesting to the involvement of this surface receptor in the initial recognition and downstream signaling for hBD-3 expression. Treatment of keratinocytes with a p38 mitogen-activated protein kinase (MAPK) inhibitor prior to either
S. aureus
or LTA stimulation was associated with reduced hBD-3 mRNA transcripts and peptide. We also propose a role for the MAPK-regulated transcriptional activating protein 1 in
S. aureus
-induced hBD-3 gene expression. Combined, these studies indicate a role for TLR2 signaling and MAPK activation in the upregulation of hBD-3 and demonstrate the innate immune capacity of skin keratinocytes under conditions of
S. aureus
challenge to enhance the local expression of this antistaphylococcal peptide antibiotic.
Immunogenicity of aYersinia pestisVaccine Antigen Monomerized by Circular Permutation
Authors: Chalton David A. , Musson Julie A. , Flick-Smith Helen , Walker Nicola , McGregor Alistair , Lamb Heather K. , Williamson E. Diane , Miller Julie , Robinson John H. , Lakey Jeremy H. 
Published: November 2006

ABSTRACTCaf1, a chaperone-usher protein fromYersinia pestis, is a major protective antigen in the development of subunit vaccines against plague. However, recombinant Caf1 forms polymers of indeterminate size. We report the conversion of Caf1 from a polymer to a monomer by circular permutation of the gene. Biophysical evaluation confirmed that the engineered Caf1 was a folded monomer. We compared the immunogenicity of the engineered monomer with polymeric Caf1 in antigen presentation assays to CD4 T-cell hybridomas in vitro, as well as in the induction of antibody responses and protection against subcutaneous challenge withY. pestisin vivo. In C57BL/6 mice, for which the majorH-2b-restricted immunodominant CD4 T-cell epitopes were intact in the engineered monomer, immunogenicity and protective efficacy were preserved, although antibody titers were decreased 10-fold. Disruption of anH-2d-restricted immunodominant CD4 T-cell epitope during circular permutation resulted in a compromised T-cell response, a low postvaccination antibody titer, and a lack of protection of BALB/c mice. The use of circular permutation in vaccine design has not been reported previously.
Up-Regulation of Gamma Interferon Receptor Expression Due toChlamydia-Toll-Like Receptor Interaction Does Not Enhance Signal Transducer and Activator of Transcription 1 Signaling
Authors: Shirey Kari Ann , Jung Joo-Yong , Carlin Joseph M. 
Published: November 2006

ABSTRACTGamma interferon (IFN-γ)-induced indoleamine dioxygenase (IDO), which inhibits chlamydial replication by reducing the availability of tryptophan, is up-regulated by interleukin-1β (IL-1β) and tumor necrosis factor alpha (TNF-α). The mechanisms by which this occurs include an increase in the synthesis of interferon regulatory factor-1 as well as a nuclear factor-κB (NF-κB)-dependent increase in the expression of IFN-γ receptors (IFN-γR). AlthoughChlamydiais susceptible to IDO, it up-regulates IFN-γR expression to a greater degree than either IL-1β or TNF-α, perhaps through interaction with Toll-like receptors (TLR). The purpose of this study was to determine the mechanism by whichChlamydia psittaciup-regulates IFN-γR expression and evaluate this effect on IDO induction. Infection of HEK 293 cells withC. psittaciincreased IFN-γR expression only in cells expressing either TLR2 or TLR4 and the adaptor protein MD-2. In addition, up-regulation of IFN-γR expression inChlamydia-infected HeLa cells could be blocked either by neutralizing TLRs with anti-TLR2 and/or anti-TLR4 or by inhibiting NF-κB transactivation with a proteasome inhibitor. Although the newly expressed IFN-γR inChlamydia-infected cells were capable of binding IFN-γ, they did not enhance IFN-γ-induced IDO activity in a manner similar to those observed for IL-1β and TNF-α. Instead, IDO activation inChlamydia-infected cells was no different than that induced in uninfected cells, despite the increase in IFN-γR expression. Furthermore, the amount of IFN-γ-induced signal transducer and activator of transcription 1 (STAT-1) activation in infected cells paralleled that observed in uninfected cells, suggesting that STAT-1 activation by these newly expressed receptors was impaired.
Differences in the Growth of Paired Ugandan Isolates ofMycobacterium tuberculosiswithin Human Mononuclear Phagocytes Correlate with Epidemiological Evidence of Strain Virulence
Authors: Theus Sue A. , Cave M. Donald , Eisenach Kathleen , Walrath Jessica , Lee Hung , Mackay Wilma , Whalen Christopher , Silver Richard F. 
Published: November 2006

ABSTRACTPrevious studies have suggested that isolates ofMycobacterium tuberculosisresponsible for tuberculosis outbreaks grow more rapidly within human mononuclear phagocytes than do other isolates. Clinical scenarios suggesting virulence of specificM. tuberculosisisolates are readily identified. Determination of appropriate “control” isolates for these studies is more problematic, but equally important for validating these assays and, ultimately, for identifying biologic differences betweenM. tuberculosisstrains that contribute to virulence. We utilized the database from a study of Ugandan tuberculosis patients and their household (HH) contacts to identifyM. tuberculosisisolates transmitted within HH and nontransmitted control isolates. Isolate pairs were evaluated from matched HH in each of three clinical scenarios: (i) coprevalent disease and no disease, (ii) incident disease and no disease, and (iii)M. tuberculosisinfection (purified protein derivative [PPD] positive) and no infection (PPD negative). Intracellular growth of paired organisms was determined in a blinded fashion using two models of intracellular infection in which we have previously demonstrated correlation between intracellular growth and strain virulence, primary human monocytes (MN) and THP-1 human macrophage-like cells. In both models, transmitted isolates from coprevalent disease HH displayed more rapid growth than nontransmitted control isolates. In the THP-1 model, this was also true of transmitted isolates from HH with incident disease and their controls. Differences in production of tumor necrosis factor alpha and interleukin-10 by matched isolates showed correlation with growth patterns in the THP-1 cells but not in MN. Paired isolates characterized in this manner may be of particular interest for further investigations of the virulence ofM. tuberculosis.
Expression ofPseudomonas aeruginosaToxin ExoS Effectively Induces Apoptosis in Host Cells
Authors: Jia Jinghua , Wang Yanping , Zhou Lei , Jin Shouguang 
Published: November 2006

ABSTRACTPseudomonas aeruginosais an opportunistic bacterial pathogen that primarily infects immunocompromised individuals and patients with cystic fibrosis. Invasive strains ofP. aeruginosaare known to induce apoptosis at a high frequency in HeLa cells and in many other cell lines, a process that is dependent on the ADP-ribosylation (ADPRT) activity of a type III secreted protein ExoS. In our previous report, it was proposed thatP. aeruginosasecreting ExoS, upon infection, shuts down host cell survival signal pathways by inhibiting ERK1/2 and p38 activation, and it activates proapoptotic pathways through activation of JNK1/2, leading ultimately to cytochromecrelease and activation of caspases. In this study, we demonstrate that the expression of ExoS in HeLa cells by eukaryotic expression vector effectively caused apoptosis in an ADPRT activity-dependent manner, indicating that ExoS alone is sufficient to trigger apoptotic death of host cells independent of any other bacterial factors. By expressing an EGFP-ExoS fusion protein, we were able to directly correlate the death of HeLa cells with the presence of intracellular ExoS and further proved the dependence of this process on both JNK activation and mitochondrial proapoptotic event. The cellular pathway responsible for the ExoS-induced cytotoxicity appears to be well conserved, since the expression of the ADPRT-competent ExoS also induced rapid cell death in theDrosophila melanogasterS2 cell lines. The presented study not only highlights the ability of ExoS ADPRT to modulate host cell signaling, eventually leading to apoptosis, but also establishes ExoS as a valuable tool, in principle, for the elucidation of apoptosis mechanisms.
GliZ, a Transcriptional Regulator of Gliotoxin Biosynthesis, Contributes toAspergillus fumigatusVirulence
Authors: Bok Jin Woo , Chung DaWoon , Balajee S. Arunmozhi , Marr Kieren A. , Andes David , Nielsen Kristian Fog , Frisvad Jens C. , Kirby Katharine A. , Keller Nancy P. 
Published: November 2006

ABSTRACTGliotoxin is a nonribosomal peptide produced byAspergillus fumigatus. This compound has been proposed as anA. fumigatusvirulence factor due to its cytotoxic, genotoxic, and apoptotic properties. Recent identification of the gliotoxin gene cluster identified several genes (gligenes) likely involved in gliotoxin production, includinggliZ, encoding a putative Zn2Cys6binuclear transcription factor. Replacement ofgliZwith a marker gene (ΔgliZ) resulted in no detectable gliotoxin production and loss of gene expression of otherglicluster genes. Placement of multiple copies ofgliZin the genome increased gliotoxin production. Using endpoint survival data, theΔgliZand a multiple-copygliZstrain were not statistically different from the wild type in a murine pulmonary model; however, both the wild-type and the multiple-copygliZstrain were more virulent thanΔlaeA(a mutant reduced in production of gliotoxin and other toxins). A flow-cytometric analysis of polymorphonuclear leukocytes (PMNs) exposed to supernatants from wild-type,ΔgliZ, complementedΔgliZ, andΔlaeAstrains supported a role for gliotoxin in apoptotic but not necrotic PMN cell death. This may indicate that several secondary metabolites are involved inA. fumigatusvirulence.
Both Influenza-Induced Neutrophil Dysfunction and Neutrophil-Independent Mechanisms Contribute to Increased Susceptibility to a SecondaryStreptococcus pneumoniaeInfection
Authors: McNamee Lynnelle A. , Harmsen Allen G. 
Published: November 2006

ABSTRACTSince secondaryStreptococcus pneumoniaeinfections greatly increase the mortality of influenza infections, we determined the relative roles of neutrophil-dependent and -independent mechanisms in increased susceptibility toS. pneumoniaeduring influenza infection. Mice infected with influenza for 6 days, but not 3 days, showed a significant increase in susceptibility toS. pneumoniaeinfection compared to mice not infected with influenza. There was significant neutrophil accumulation in the lungs ofS. pneumoniae-infected mice regardless of whether or not they were infected with influenza for 3 or 6 days. Depletion of neutrophils in these mice resulted in increased susceptibility toS. pneumoniaein both the non-influenza-infected mice and mice infected with influenza for 3 days but not in the mice infected with influenza for 6 days, indicating that a prior influenza infection of 6 days may compromise neutrophil function, resulting in increased susceptibility to aS. pneumoniaeinfection. Neutrophils from the lungs of mice infected with influenza for 3 or 6 days exhibited functional impairment in the form of decreased phagocytosis and intracellular reactive oxygen species generation in response toS. pneumoniae. In addition, neutrophil-depleted mice infected with influenza for 6 days were more susceptible toS. pneumoniaethan neutrophil-depleted mice not infected with influenza, indicating that neutrophil-independent mechanisms also contribute to influenza-induced increased susceptibility toS. pneumoniae. Pulmonary interleukin-10 levels were increased in coinfected mice infected with influenza for 6 days but not 3 days. Thus, an influenza infection of 6 days increases susceptibility toS. pneumoniaeby both suppression of neutrophil function and by neutrophil-independent mechanisms such as enhanced cytokine production.