bluuebunny/crossref_metadata_2025_split huggingface dataset:
Preclinical Characterization of BMS-791325, an Allosteric Inhibitor of Hepatitis C Virus NS5B Polymerase
Authors: Lemm Julie A. , Liu Mengping , Gentles Robert G. , Ding Min , Voss Stacey , Pelosi Lenore A. , Wang Ying-Kai , Rigat Karen L. , Mosure Kathleen W. , Bender John A. , Knipe Jay O. , Colonno Richard , Meanwell Nicholas A. , Kadow John F. , Santone Kenneth S. , Roberts Susan B. , Gao Min 
Published: April 2014

ABSTRACT

BMS-791325 is an allosteric inhibitor that binds to thumb site 1 of the hepatitis C virus (HCV) NS5B RNA-dependent RNA polymerase. BMS-791325 inhibits recombinant NS5B proteins from HCV genotypes 1, 3, 4, and 5 at 50% inhibitory concentrations (IC
50
) below 28 nM. In cell culture, BMS-791325 inhibited replication of HCV subgenomic replicons representing genotypes 1a and 1b at 50% effective concentrations (EC
50
s) of 3 nM and 6 nM, respectively, with similar (3 to 18 nM) values for genotypes 3a, 4a, and 5a. Potency against genotype 6a showed more variability (9 to 125 nM), and activity was weaker against genotype 2 (EC
50
, 87 to 925 nM). Specificity was demonstrated by the absence of activity (EC
50
s of >4 μM) against a panel of mammalian viruses, and cytotoxic concentrations (50%) were >3,000-fold above the HCV EC
50
. Resistance substitutions selected by BMS-791325 in genotype 1 replicons mostly mapped to a single site, NS5B amino acid 495 (P495A/S/L/T). Additive or synergistic activity was observed in combination studies using BMS-791325 with alfa interferon plus ribavirin, inhibitors of NS3 protease or NS5A, and other classes of NS5B inhibitor (palm site 2-binding or nucleoside analogs). Plasma and liver exposures
in vivo
in several animal species indicated that BMS-791325 has a hepatotropic disposition (liver-to-plasma ratios ranging from 1.6- to 60-fold across species). Twenty-four hours postdose, liver exposures across all species tested were ≥10-fold above the inhibitor EC
50
s observed with HCV genotype 1 replicons. These findings support the evaluation of BMS-791325 in combination regimens for the treatment of HCV. Phase 3 studies are ongoing.
Randomized, Placebo-Controlled, Single-Ascending-Dose Study of BMS-791325, a Hepatitis C Virus (HCV) NS5B Polymerase Inhibitor, in HCV Genotype 1 Infection
Authors: Sims Karen D. , Lemm Julie , Eley Timothy , Liu Menping , Berglind Anna , Sherman Diane , Lawitz Eric , Vutikullird Apinya B. , Tebas Pablo , Gao Min , Pasquinelli Claudio , Grasela Dennis M. 
Published: April 2014

ABSTRACT

BMS-791325 is a nonnucleoside inhibitor of hepatitis C virus (HCV) NS5B polymerase with low-nanomolar potency against genotypes 1a (50% effective concentration [EC
50
], 3 nM) and 1b (EC
50
, 7 nM)
in vitro
. BMS-791325 safety, pharmacokinetics, and antiviral activity were evaluated in a double-blind, placebo-controlled, single-ascending-dose study in 24 patients (interferon naive and experienced) with chronic HCV genotype 1 infection, randomized (5:1) to receive a single dose of BMS-791325 (100, 300, 600, or 900 mg) or placebo. The prevalence and phenotype of HCV variants at baseline and specific posttreatment time points were assessed. Antiviral activity was observed in all cohorts, with a mean HCV RNA decline of ≈2.5 log
10
copies/ml observed 24 h after a single 300-mg dose. Mean plasma half-life among cohorts was 7 to 9 h; individual 24-hour levels exceeded the protein-adjusted EC
90
for genotype 1 at all doses. BMS-791325 was generally well tolerated, with no serious adverse events or discontinuations. Enrichment for resistance variants was not observed at 100 to 600 mg. At 900 mg, variants (P495L/S) associated with BMS-791325 resistance
in vitro
were transiently observed in one patient, concurrent with an observed HCV RNA decline of 3.4 log
10
IU/ml, but were replaced with wild type by 48 h. Single doses of BMS-791325 were well tolerated; demonstrated rapid, substantial, and exposure-related antiviral activity; displayed dose-related increases in exposure; and showed viral kinetic and pharmacokinetic profiles supportive of once- or twice-daily dosing. These results support its further development in combination with other direct-acting antivirals for HCV genotype 1 infection. (This trial has been registered at ClinicalTrials.gov under registration no. NCT00664625.)
Efficiency of Incorporation and Chain Termination Determines the Inhibition Potency of 2′-Modified Nucleotide Analogs against Hepatitis C Virus Polymerase
Authors: Fung Amy , Jin Zhinan , Dyatkina Natalia , Wang Guangyi , Beigelman Leo , Deval Jerome 
Published: April 2014

ABSTRACT

Ribonucleotide analog inhibitors of the RNA-dependent RNA polymerase of hepatitis C virus (HCV) represent one of the most exciting recent developments in HCV antiviral therapy. Although it is well established that these molecules cause chain termination by competing at the triphosphate level with natural nucleotides for incorporation into elongating RNA, strategies to rationally optimize antiviral potency based on enzyme kinetics remain elusive. In this study, we used the isolated HCV polymerase elongation complex to determine the pre-steady-state kinetics of incorporation of 2′F-2′C-Me-UTP, the active metabolite of the anti-HCV drug sofosbuvir. 2′F-2′C-Me-UTP was efficiently incorporated by HCV polymerase with apparent

K
d

(equilibrium constant) and
k
pol
(rate of nucleotide incorporation at saturating nucleotide concentration) values of 113 ± 28 μM and 0.67 ± 0.05 s
−1
, respectively, giving an overall substrate efficiency (
k
pol
/

K
d

) of 0.0059 ± 0.0015 μM
−1
s
−1
. We also measured the substrate efficiency of other UTP analogs and found that substitutions at the 2′ position on the ribose can greatly affect their level of incorporation, with a rank order of OH > F > NH
2
> F-C-Me > C-Me > N
3
> ara. However, the efficiency of chain termination following the incorporation of UMP analogs followed a different order, with only 2′F-2′C-Me-, 2′C-Me-, and 2′ara-UTP causing complete and immediate chain termination. The chain termination profile of the 2′-modified nucleotides explains the apparent lack of correlation observed across all molecules between substrate efficiency at the single-nucleotide level and their overall inhibition potency. To our knowledge, these results provide the first attempt to use pre-steady-state kinetics to uncover the mechanism of action of 2′-modified NTP analogs against HCV polymerase.
Phase 1B Study of the Pharmacokinetics and Safety of Posaconazole Intravenous Solution in Patients at Risk for Invasive Fungal Disease
Authors: Maertens Johan , Cornely Oliver A. , Ullmann Andrew J. , Heinz Werner J. , Krishna Gopal , Patino Hernando , Caceres Maria , Kartsonis Nicholas , Waskin Hetty , Robertson Michael N. 
Published: April 2014

ABSTRACT

This was a phase 1B, dose-ranging, multicenter, pharmacokinetics, and safety study of cyclodextrin-based posaconazole intravenous (i.v.) solution administered through a central line to subjects at high risk for invasive fungal disease (part 1 of a 2-part study [phase 1B/3]). Initially, the safety and tolerability of single-dose posaconazole i.v. 200 mg (
n
= 10) were compared with those of a placebo (
n
= 11). Subsequently, 2 doses were evaluated, posaconazole i.v. 200 mg once daily (q.d.) (
n
= 21) and 300 mg q.d. (
n
= 24). The subjects received twice-daily (b.i.d.) posaconazole i.v. on day 1, followed by 13 days of posaconazole i.v. q.d., then 14 days of posaconazole oral suspension 400 mg b.i.d. The steady-state (day 14) exposure target (average concentration [areas under concentration-time curve {AUCs}/24 h, average concentrations at steady state {
C
avg
s}], of ≥500 to ≤2,500 ng/ml in ≥90% of the subjects) was achieved by 94% of the subjects for 200 mg posaconazole q.d. and by 95% of subjects for 300 mg posaconazole q.d. The desired exposure target (mean steady-state
C
avg
, ∼1,200 ng/ml) was 1,180 ng/ml in the 200-mg dosing cohort and was exceeded in the 300-mg dosing cohort (1,430 ng/ml). Posaconazole i.v. was well tolerated. Posaconazole i.v. 300 mg q.d. was selected for the phase 3 study segment. (This study has been registered at ClinicalTrials.gov under registration no. NCT01075984.)
Lysine Acetylation in Sexual Stage Malaria Parasites Is a Target for Antimalarial Small Molecules
Authors: Trenholme Katharine , Marek Linda , Duffy Sandra , Pradel Gabriele , Fisher Gillian , Hansen Finn K. , Skinner-Adams Tina S. , Butterworth Alice , Ngwa Che Julius , Moecking Jonas , Goodman Christopher D. , McFadden Geoffrey I. , Sumanadasa Subathdrage D. M. , Fairlie David P. , Avery Vicky M. , Kurz Thomas , Andrews Katherine T. 
Published: April 2014

ABSTRACT

Therapies to prevent transmission of malaria parasites to the mosquito vector are a vital part of the global malaria elimination agenda. Primaquine is currently the only drug with such activity; however, its use is limited by side effects. The development of transmission-blocking strategies requires an understanding of sexual stage malaria parasite (gametocyte) biology and the identification of new drug leads. Lysine acetylation is an important posttranslational modification involved in regulating eukaryotic gene expression and other essential processes. Interfering with this process with histone deacetylase (HDAC) inhibitors is a validated strategy for cancer and other diseases, including asexual stage malaria parasites. Here we confirm the expression of at least one HDAC protein in
Plasmodium falciparum
gametocytes and show that histone and nonhistone protein acetylation occurs in this life cycle stage. The activity of the canonical HDAC inhibitors trichostatin A (TSA) and suberoylanilide hydroxamic acid (SAHA; Vorinostat) and a panel of novel HDAC inhibitors on early/late-stage gametocytes and on gamete formation was examined. Several compounds displayed early/late-stage gametocytocidal activity, with TSA being the most potent (50% inhibitory concentration, 70 to 90 nM). In contrast, no inhibitory activity was observed in
P. falciparum
gametocyte exflagellation experiments. Gametocytocidal HDAC inhibitors caused hyperacetylation of gametocyte histones, consistent with a mode of action targeting HDAC activity. Our data identify HDAC inhibitors as being among a limited number of compounds that target both asexual and sexual stage malaria parasites, making them a potential new starting point for gametocytocidal drug leads and valuable tools for dissecting gametocyte biology.
Identification of a Diguanylate Cyclase and Its Role in Porphyromonas gingivalis Virulence
Authors: Chaudhuri Swarnava , Pratap Siddharth , Paromov Victor , Li Zhijun , Mantri Chinmay K. , Xie Hua 
Published: April 2014

ABSTRACT

Porphyromonas gingivalis
is a Gram-negative obligate anaerobic bacterium and is considered a keystone pathogen in the initiation of periodontitis, one of the most widespread infectious diseases. Bacterial bis-(3′-5′) cyclic GMP (cyclic di-GMP [c-di-GMP]) serves as a second messenger and is involved in modulating virulence factors in numerous bacteria. However, the role of this second messenger has not been investigated in
P. gingivalis
, mainly due to a lack of an annotation regarding diguanylate cyclases (DGCs) in this bacterium. Using bioinformatics tools, we found a protein, PGN_1932, containing a GGDEF domain. A deletion mutation in the
pgn_1932
gene had a significant effect on the intracellular c-di-GMP level in
P. gingivalis
. Genetic analysis showed that expression of the
fimA
and
rgpA
genes, encoding the major protein subunit of fimbriae and an arginine-specific proteinase, respectively, was downregulated in the
pgn_1932
mutant. Correspondingly, FimA protein production and the fimbrial display on the mutant were significantly reduced. Mutations in the
pgn_1932
gene also had a significant impact on the adhesive and invasive capabilities of
P. gingivalis
, which are required for its pathogenicity. These findings provide evidence that the PGN_1932 protein is both responsible for synthesizing c-di-GMP and involved in biofilm formation and host cell invasion by
P. gingivalis
by controlling the expression and biosynthesis of FimA.
Antiapoptotic Activity of Coxiella burnetii Effector Protein AnkG Is Controlled by p32-Dependent Trafficking
Authors: Eckart Rita A. , Bisle Stephanie , Schulze-Luehrmann Jan , Wittmann Irene , Jantsch Jonathan , Schmid Benedikt , Berens Christian , Lührmann Anja 
Published: April 2014

ABSTRACTIntracellular bacterial pathogens frequently inhibit host cell apoptosis to ensure survival of their host, thereby allowing bacterial propagation. The obligate intracellular pathogenCoxiella burnetiidisplays antiapoptotic activity which depends on a functional type IV secretion system (T4SS). Accordingly, antiapoptotic T4SS effector proteins, like AnkG, have been identified. AnkG inhibits pathogen-induced apoptosis, possibly by binding to the host cell mitochondrial protein p32 (gC1qR). However, the molecular mechanism of AnkG activity remains unknown. Here, we demonstrate that ectopically expressed AnkG associates with mitochondria and traffics into the nucleus after apoptosis induction, although AnkG lacks a predicted nuclear localization signal. We identified the p32 interaction region in AnkG and constructed an AnkG mutant (AnkGR22/23S) unable to bind to p32. By using this mutant, we found that intracellular localization and trafficking of AnkG into the nucleus are dependent on binding to p32. Furthermore, we demonstrated that nuclear localization of AnkG but not binding to p32 is required for apoptosis inhibition. Thus, the antiapoptotic activity of AnkG is controlled by p32-mediated intracellular trafficking, which, in turn, seems to be regulated by host cell processes that sense stress.
Epidemic Clostridium difficile Strains Demonstrate Increased Competitive Fitness Compared to Nonepidemic Isolates
Authors: Robinson Catherine D. , Auchtung Jennifer M. , Collins James , Britton Robert A. 
Published: April 2014

ABSTRACT

Clostridium difficile
infection is the most common cause of severe cases of antibiotic-associated diarrhea (AAD) and is a significant health burden. Recent increases in the rate of
C. difficile
infection have paralleled the emergence of a specific phylogenetic clade of
C. difficile
strains (ribotype 027; North American pulsed-field electrophoresis 1 [NAP1]; restriction endonuclease analysis [REA] group BI). Initial reports indicated that ribotype 027 strains were associated with increased morbidity and mortality and might be hypervirulent. Although subsequent work has raised some doubt as to whether ribotype 027 strains are hypervirulent, the strains are considered epidemic isolates that have caused severe outbreaks across the globe. We hypothesized that one factor that could lead to the increased prevalence of ribotype 027 strains would be if these strains had increased competitive fitness compared to strains of other ribotypes. We developed a moderate-throughput
in vitro
model of
C. difficile
infection and used it to test competition between four ribotype 027 clinical isolates and clinical isolates of four other ribotypes (001, 002, 014, and 053). We found that ribotype 027 strains outcompeted the strains of other ribotypes. A similar competitive advantage was observed when two ribotype pairs were competed in a mouse model of
C. difficile
infection. Based upon these results, we conclude that one possible mechanism through which ribotype 027 strains have caused outbreaks worldwide is their increased ability to compete in the presence of a complex microbiota.
Role of Intracellular Carbon Metabolism Pathways in Shigella flexneri Virulence
Authors: Waligora E. A. , Fisher C. R. , Hanovice N. J. , Rodou A. , Wyckoff E. E. , Payne S. M. 
Published: April 2014

ABSTRACT

Shigella flexneri
, which replicates in the cytoplasm of intestinal epithelial cells, can use the Embden-Meyerhof-Parnas, Entner-Doudoroff, or pentose phosphate pathway for glycolytic carbon metabolism. To determine which of these pathways is used by intracellular
S. flexneri
, mutants were constructed and tested in a plaque assay for the ability to invade, replicate intracellularly, and spread to adjacent epithelial cells. Mutants blocked in the Embden-Meyerhof-Parnas pathway (
pfkAB
and
pykAF
mutants) invaded the cells but formed very small plaques. Loss of the Entner-Doudoroff pathway gene
eda
resulted in small plaques, but the double
eda edd
mutant formed normal-size plaques. This suggested that the plaque defect of the
eda
mutant was due to buildup of the toxic intermediate 2-keto-3-deoxy-6-phosphogluconic acid rather than a specific requirement for this pathway. Loss of the pentose phosphate pathway had no effect on plaque formation, indicating that it is not critical for intracellular
S. flexneri
. Supplementation of the epithelial cell culture medium with pyruvate allowed the glycolysis mutants to form larger plaques than those observed with unsupplemented medium, consistent with data from phenotypic microarrays (Biolog) indicating that pyruvate metabolism was not disrupted in these mutants. Interestingly, the wild-type
S. flexneri
also formed larger plaques in the presence of supplemental pyruvate or glucose, with pyruvate yielding the largest plaques. Analysis of the metabolites in the cultured cells showed increased intracellular levels of the added compound. Pyruvate increased the growth rate of
S. flexneri
in vitro
, suggesting that it may be a preferred carbon source inside host cells.
Chlamydia trachomatis Polymorphic Membrane Protein D Is a Virulence Factor Involved in Early Host-Cell Interactions
Authors: Kari Laszlo , Southern Timothy R. , Downey Carey J. , Watkins Heather S. , Randall Linnell B. , Taylor Lacey D. , Sturdevant Gail L. , Whitmire William M. , Caldwell Harlan D. 
Published: April 2014

ABSTRACT

Chlamydia trachomatis
is an obligate intracellular mucosotropic pathogen of significant medical importance. It is the etiological agent of blinding trachoma and bacterial sexually transmitted diseases, infections that afflict hundreds of millions of people globally. The
C. trachomatis
polymorphic membrane protein D (PmpD) is a highly conserved autotransporter and the target of broadly cross-reactive neutralizing antibodies; however, its role in host-pathogen interactions is unknown. Here we employed a targeted reverse genetics approach to generate a
pmpD
null mutant that was used to define the role of PmpD in the pathogenesis of chlamydial infection. We show that
pmpD
is not an essential chlamydial gene and the
pmpD
null mutant has no detectable deficiency in cultured murine cells or in a murine mucosal infection model. Notably, however, the
pmpD
null mutant was significantly attenuated for macaque eyes and cultured human cells. A reduction in
pmpD
null infection of human endocervical cells was associated with a deficiency in chlamydial attachment to cells. Collectively, our results show that PmpD is a chlamydial virulence factor that functions in early host-cell interactions. This study is the first of its kind using reverse genetics to evaluate the contribution of a
C. trachomatis
gene to disease pathogenesis.
Republished: Antineutrophil cytoplasmic antibody-associated vasculitides: is it time to split up the group?
Authors: Millet Arnaud , Pederzoli-Ribeil Magali , Guillevin Loïc , Witko-Sarsat Véronique , Mouthon Luc 
Published: April 2014

Abstract
Antineutrophil cytoplasmic antibody (ANCA)-associated vasculitides are a heterogeneous group of diseases corresponding to necrotising inflammation of small vessels with a wide range of clinical presentations. At least two of the diseases are believed to exhibit a common ground of pathophysiological mechanisms. These are granulomatosis with polyangiitis (GPA, formerly known as Wegener’s granulomatosis) and microscopic polyangiitis (MPA). ANCA directed against proteinase 3 (PR3) are preferentially associated with GPA, and anti-myeloperoxidase (MPO) ANCA are associated mainly with MPA and eosinophilic GPA (formerly known as Churg-Strauss syndrome). Anti-MPO and anti-PR3 antibodies can activate neutrophils in vitro. In vivo data are available for humans and mice on the pathogenicity of anti-MPO but it is more controversial for PR3-ANCA. A recent genome-wide association study of patients with ANCA-associated vasculitides confirmed the genetic contribution to the pathogenesis of these conditions, with significant association of PR3-ANCA and human leukocyte antigen-DP and the genes encoding α1-antitrypsin and PR3. MPO-ANCA were significantly associated with human leukocyte antigen-DQ. Thus, recent results from epidemiological studies, genome-wide association study and therapeutic trials have suggested that these entities are, in fact, distinct. We have summarised these results and discuss the idea that these two entities should be studied separately as the nature of the two auto-antigens suggests at a molecular level despite shared ANCA involvement.
Physicians, Clinics, and Neighborhoods: Multiple Levels of Influence on Colorectal Cancer Screening
Authors: Pruitt Sandi L. , Leonard Tammy , Zhang Song , Schootman Mario , Halm Ethan A. , Gupta Samir 
Published: April 2014

Abstract
Background: We (i) described variability in colorectal cancer (CRC) test use across multiple levels, including physician, clinic, and neighborhood; and (ii) compared the performance of novel cross-classified models versus traditional hierarchical models.
Methods: We examined multilevel variation in CRC test use among patients not up-to-date with screening in a large, urban safety net health system (2011–2012). Outcomes included: (i) fecal occult blood test (FOBT) or (ii) colonoscopy and were ascertained using claims data during a 1-year follow-up. We compared Bayesian (i) cross-classified four-level logistic models nesting patients within separate, nonoverlapping “levels” (physicians, clinics, and census tracts) versus (ii) three hierarchical two-level models using deviance information criterion. Models were adjusted for covariates (patient sociodemographic factors, driving time to clinic, and census tract poverty rate).
Results: Of 3,195 patients, 157 (4.9%) completed FOBT and 292 (9.1%) completed colonoscopy during the study year. Patients attended 19 clinics, saw 177 physicians, and resided in 332 census tracts. Significant variability was observed across all levels in both hierarchical and cross-classified models that was unexplained by measured covariates. For colonoscopy, variance was similar across all levels. For FOBT, physicians, followed by clinics, demonstrated the largest variability. Model fit using cross-classified models was superior or similar to 2-level hierarchical models.
Conclusions: Significant and substantial variability was observed across neighborhood, physician, and clinic levels in CRC test use, suggesting the importance of factors at each of these levels on CRC testing.
Impact: Future multilevel research and intervention should consider the simultaneous influences of multiple levels, including clinic, physician, and neighborhood. Cancer Epidemiol Biomarkers Prev; 23(7); 1346–55. ©2014 AACR.