bluuebunny/crossref_metadata_2025_split huggingface dataset:
In Vitro
Activity and Resistance Profile of Samatasvir, a Novel NS5A Replication Inhibitor of Hepatitis C Virus
Authors: Bilello J. P. , Lallos L. B. , McCarville J. F. , La Colla M. , Serra I. , Chapron C. , Gillum J. M. , Pierra C. , Standring D. N. , Seifer M. 
Published: May 2014

ABSTRACT

The hepatitis C virus (HCV) nonstructural 5A (NS5A) protein is a clinically validated target for drugs designed to treat chronic HCV infection. This study evaluated the
in vitro
activity, selectivity, and resistance profile of a novel anti-HCV compound, samatasvir (IDX719), alone and in combination with other antiviral agents. Samatasvir was effective and selective against infectious HCV and replicons, with 50% effective concentrations (EC
50
s) falling within a tight range of 2 to 24 pM in genotype 1 through 5 replicons and with a 10-fold EC
50
shift in the presence of 40% human serum in the genotype 1b replicon. The EC
90
/EC
50
ratio was low (2.6). A 50% cytotoxic concentration (CC
50
) of >100 μM provided a selectivity index of >5 × 10
7
. Resistance selection experiments (with genotype 1a replicons) and testing against replicons bearing site-directed mutations (with genotype 1a and 1b replicons) identified NS5A amino acids 28, 30, 31, 32, and 93 as potential resistance loci, suggesting that samatasvir affects NS5A function. Samatasvir demonstrated an overall additive effect when combined with interferon alfa (IFN-α), ribavirin, representative HCV protease, and nonnucleoside polymerase inhibitors or the nucleotide prodrug IDX184. Samatasvir retained full activity in the presence of HIV and hepatitis B virus (HBV) antivirals and was not cross-resistant with HCV protease, nucleotide, and nonnucleoside polymerase inhibitor classes. Thus, samatasvir is a selective low-picomolar inhibitor of HCV replication
in vitro
and is a promising candidate for future combination therapies with other direct-acting antiviral drugs in HCV-infected patients.
Flow Cytometry-Based Analysis of Artemisinin-Resistant Plasmodium falciparum in the Ring-Stage Survival Assay
Authors: Amaratunga Chanaki , Neal Aaron T. , Fairhurst Rick M. 
Published: May 2014

ABSTRACT

The ring-stage survival assay (RSA) is a powerful tool for phenotyping artemisinin-resistant
Plasmodium falciparum
but requires experienced microscopists to count viable parasites among 10,000 erythrocytes in Giemsa-stained thin blood smears. Here we describe a rapid flow cytometric assay that accurately counts viable parasites among 250,000 erythrocytes in suspension. This method performs as well as light microscopy and can be used to standardize the collection of RSA data between research groups in laboratory and field settings.
Decreased Ceftriaxone Susceptibility in Emerging (35B and 6C) and Persisting (19A) Streptococcus pneumoniae Serotypes in the United States, 2011-2012: Ceftaroline Remains Active
In Vitro
among β-Lactam Agents
Authors: Mendes Rodrigo E. , Biek Donald , Critchley Ian A. , Farrell David J. , Sader Helio S. , Jones Ronald N. 
Published: May 2014

ABSTRACT

Totals of 8.7% (103/1,190) and 21.0% (249/1,190) of the
Streptococcus pneumoniae
isolates recovered from specimens collected in the United States during the 2011-2012 AWARE (
A
ssessing
W
orldwide
A
ntimicrobial
R
esistance
E
valuation) Surveillance Program were ceftriaxone nonsusceptible according to the CLSI (≤1 μg/ml for susceptible) and EUCAST (≤0.5 μg/ml for susceptible) criteria, respectively. Decreased susceptibility to ceftriaxone (MIC, 1 μg/ml) was frequently observed among serotypes 19A (51.4%; 128/249) and 35B (29.7%; 74/249), which were most often observed in the East South Central and South Atlantic U.S. Census regions. Ceftaroline (MIC
50/90
, 0.12/0.25 μg/ml) remained active (≥96.8% susceptible) when tested against these less susceptible isolates.
Development and Characterization of a Long-Term Murine Model of Streptococcus pneumoniae Infection of the Lower Airways
Authors: Haste Louise , Hulland Kathryn , Bolton Sarah , Yesilkaya Hasan , McKechnie Kenneth , Andrew Peter W. 
Published: May 2014

ABSTRACTChronic obstructive pulmonary disease (COPD) is characterized by long periods of stable symptoms, but exacerbations occur, which result in a permanent worsening of symptoms. Previous studies have shown a link between bacterial colonization of the lower airways of COPD sufferers and an increase in exacerbation frequency. One of the most frequent bacterial colonizers isStreptococcus pneumoniae. To mimic this aspect of COPD, a murine model of low-level pneumococcal colonization in the lung has been developed, in whichS. pneumoniaepersisted in the lungs for at least 28 days. From day 14 postinfection, bacterial numbers remained constant until at least 28 days postinfection, and animals showed no outward signs of disease. The bacterial presence correlated with a low-level inflammatory response that was localized to small foci across the left and inferior lobes of the lung. The cellular response was predominantly monocytic, and focal fibroplasia was observed at the airway transitional zones. Physiological changes in the lungs were investigated with a Forced Maneuvers system. This new model provides a means of study of a long-term pulmonary infection with a human pathogen in a rodent system. This is an excellent tool for the development of future models that mimic complex respiratory diseases such as COPD and asthma.
Proteome Analysis of Coinfection of Epithelial Cells with Filifactor alocis and Porphyromonas gingivalis Shows Modulation of Pathogen and Host Regulatory Pathways
Authors: Aruni A. Wilson , Zhang Kangling , Dou Yuetan , Fletcher Hansel 
Published: May 2014

ABSTRACT

Changes in periodontal status are associated with shifts in the composition of the bacterial community in the periodontal pocket. The relative abundances of several newly recognized microbial species, including
Filifactor alocis
, as-yet-unculturable organisms, and other fastidious organisms have raised questions on their impact on disease development. We have previously reported that the virulence attributes of
F. alocis
are enhanced in coculture with
Porphyromonas gingivalis
. We have evaluated the proteome of host cells and
F. alocis
during a polymicrobial infection. Coinfection of epithelial cells with
F. alocis
and
P. gingivalis
strains showed approximately 20% to 30% more proteins than a monoinfection. Unlike
F. alocis
ATCC 35896, the D-62D strain expressed more proteins during coculture with
P. gingivalis
W83 than with
P. gingivalis
33277. Proteins designated microbial surface component-recognizing adhesion matrix molecules (MSCRAMMs) and cell wall anchor proteins were highly upregulated during the polymicrobial infection. Ultrastructural analysis of the epithelial cells showed formation of membrane microdomains only during coinfection. The proteome profile of epithelial cells showed proteins related to cytoskeletal organization and gene expression and epigenetic modification to be in high abundance. Modulation of proteins involved in apoptotic and cell signaling pathways was noted during coinfection. The enhanced virulence potential of
F. alocis
may be related to the differential expression levels of several putative virulence factors and their effects on specific host cell pathways.
Proteomic Analysis of the Burkholderia pseudomallei Type II Secretome Reveals Hydrolytic Enzymes, Novel Proteins, and the Deubiquitinase TssM
Authors: Burtnick Mary N. , Brett Paul J. , DeShazer David 
Published: May 2014

ABSTRACT

Burkholderia pseudomallei
, the etiologic agent of melioidosis, is an opportunistic pathogen that harbors a wide array of secretion systems, including a type II secretion system (T2SS), three type III secretion systems (T3SS), and six type VI secretion systems (T6SS). The proteins exported by these systems provide
B. pseudomallei
with a growth advantage
in vitro
and
in vivo
, but relatively little is known about the full repertoire of exoproducts associated with each system. In this study, we constructed deletion mutations in
gspD
and
gspE
, T2SS genes encoding an outer membrane secretin and a cytoplasmic ATPase, respectively. The secretion profiles of
B. pseudomallei
MSHR668 and its T2SS mutants were noticeably different when analyzed by SDS-PAGE. We utilized liquid chromatography-tandem mass spectrometry (LC-MS/MS) to identify proteins present in the supernatants of
B. pseudomallei
MSHR668 and
B. pseudomallei
Δ
gspD
grown in rich and minimal media. The MSHR668 supernatants contained 48 proteins that were either absent or substantially reduced in the supernatants of Δ
gspD
strains. Many of these proteins were putative hydrolytic enzymes, including 12 proteases, two phospholipases, and a chitinase. Biochemical assays validated the LC-MS/MS results and demonstrated that the export of protease, phospholipase C, and chitinase activities is T2SS dependent. Previous studies had failed to identify the mechanism of secretion of TssM, a deubiquitinase that plays an integral role in regulating the innate immune response. Here we present evidence that TssM harbors an atypical signal sequence and that its secretion is mediated by the T2SS. This study provides the first in-depth characterization of the
B. pseudomallei
T2SS secretome.
Burkholderia pseudomallei Capsular Polysaccharide Conjugates Provide Protection against Acute Melioidosis
Authors: Scott Andrew E. , Burtnick Mary N. , Stokes Margaret G. M. , Whelan Adam O. , Williamson E. Diane , Atkins Timothy P. , Prior Joann L. , Brett Paul J. 
Published: May 2014

ABSTRACTBurkholderia pseudomallei, the etiologic agent of melioidosis, is a CDC tier 1 select agent that causes severe disease in both humans and animals. Diagnosis and treatment of melioidosis can be challenging, and in the absence of optimal chemotherapeutic intervention, acute disease is frequently fatal. Melioidosis is an emerging infectious disease for which there are currently no licensed vaccines. Due to the potential malicious use ofB. pseudomalleias well as its impact on public health in regions where the disease is endemic, there is significant interest in developing vaccines for immunization against this disease. In the present study, type A O-polysaccharide (OPS) andmanno-heptose capsular polysaccharide (CPS) antigens were isolated from nonpathogenic, select-agent-excluded strains ofB. pseudomalleiand covalently linked to carrier proteins. By using these conjugates (OPS2B1 and CPS2B1, respectively), it was shown that although high-titer IgG responses against the OPS or CPS component of the glycoconjugates could be raised in BALB/c mice, only those animals immunized with CPS2B1 were protected against intraperitoneal challenge withB. pseudomallei. Extending upon these studies, it was also demonstrated that when the mice were immunized with a combination of CPS2B1 and recombinantB. pseudomalleiLolC, rather than with CPS2B1 or LolC individually, they exhibited higher survival rates when challenged with a lethal dose ofB. pseudomallei. Collectively, these results suggest that CPS-based glycoconjugates are promising candidates for the development of subunit vaccines for immunization against melioidosis.
Host Defense Peptide Resistance Contributes to Colonization and Maximal Intestinal Pathology by Crohn's Disease-Associated Adherent-Invasive Escherichia coli
Authors: McPhee Joseph B. , Small Cherrie L. , Reid-Yu Sarah A. , Brannon John R. , Le Moual Hervé , Coombes Brian K. 
Published: May 2014

ABSTRACT

Host defense peptides secreted by colonocytes and Paneth cells play a key role in innate host defenses in the gut. In Crohn's disease, the burden of tissue-associated
Escherichia coli
commonly increases at epithelial surfaces where host defense peptides concentrate, suggesting that this bacterial population might actively resist this mechanism of bacterial killing. Adherent-invasive
E. coli
(AIEC) is associated with Crohn's disease; however, the colonization determinants of AIEC in the inflamed gut are undefined. Here, we establish that host defense peptide resistance contributes to host colonization by Crohn's-associated AIEC. We identified a plasmid-encoded genomic island (called PI-6) in AIEC strain NRG857c that confers high-level resistance to α-helical cationic peptides and α- and β-defensins. Deletion of PI-6 sensitized strain NRG857c to these host defense molecules, reduced its competitive fitness in a mouse model of infection, and attenuated its ability to induce cecal pathology. This phenotype is due to two genes in PI-6,
arlA
, which encodes a Mig-14 family protein implicated in defensin resistance, and
arlC
, an OmpT family outer membrane protease. Implicit in these findings are new bacterial targets whose inhibition might limit AIEC burden and disease in the gut.
Francisella tularensis LVS Induction of Prostaglandin Biosynthesis by Infected Macrophages Requires Specific Host Phospholipases and Lipid Phosphatases
Authors: Navratil Aaron R. , Brummett Ashley M. , Bryan Joshua D. , Woolard Matthew D. 
Published: May 2014

ABSTRACTFrancisella tularensisinduces the synthesis of prostaglandin E2(PGE2) by infected macrophages to alter host immune responses, thus providing a survival advantage to the bacterium. We previously demonstrated that PGE2synthesis byF. tularensis-infected macrophages requires cytosolic phospholipase A2 (cPLA2), cyclooxygenase 2 (COX-2), and microsomal prostaglandin E synthase 1 (mPGES1). During inducible PGE2synthesis, cPLA2hydrolyzes arachidonic acid (AA) from cellular phospholipids to be converted to PGE2. However, inF. tularensis-infected macrophages we observed a temporal disconnect between Ser505-cPLA2phosphorylation (a marker of activation) and PGE2synthesis. These results suggested to us that cPLA2is not responsible for the liberation of AA to be converted into PGE2byF. tularensis-infected macrophages. Utilizing small-molecule inhibitors, we demonstrated that phospholipase D and diacylglycerol lipase were required for providing AA for PGE2biosynthesis. cPLA2, on the other hand, was required for macrophage cytokine responses toF. tularensis. We also demonstrated for the first time that lipin-1 and PAP2a contribute to macrophage inflammation in response toF. tularensis. Our results identify both an alternative pathway for inducible PGE2synthesis and a role for lipid-modifying enzymes in the regulation of macrophage inflammatory function.
Deep Sequencing Analysis Reveals Temporal Microbiota Changes Associated with Development of Bovine Digital Dermatitis
Authors: Krull Adam C. , Shearer Jan K. , Gorden Patrick J. , Cooper Vickie L. , Phillips Gregory J. , Plummer Paul J. 
Published: May 2014

ABSTRACT

Bovine digital dermatitis (DD) is a leading cause of lameness in dairy cattle throughout the world. Despite 35 years of research, the definitive etiologic agent associated with the disease process is still unknown. Previous studies have demonstrated that multiple bacterial species are associated with lesions, with spirochetes being the most reliably identified organism. This study details the deep sequencing-based metagenomic evaluation of 48 staged DD biopsy specimens collected during a 3-year longitudinal study of disease progression. Over 175 million sequences were evaluated by utilizing both shotgun and 16S metagenomic techniques. Based on the shotgun sequencing results, there was no evidence of a fungal or DNA viral etiology. The bacterial microbiota of biopsy specimens progresses through a systematic series of changes that correlate with the novel morphological lesion scoring system developed as part of this project. This scoring system was validated, as the microbiota of each stage was statistically significantly different from those of other stages (
P
< 0.001). The microbiota of control biopsy specimens were the most diverse and became less diverse as lesions developed. Although
Treponema
spp. predominated in the advanced lesions, they were in relatively low abundance in the newly described early lesions that are associated with the initiation of the disease process. The consortium of
Treponema
spp. identified at the onset of disease changes considerably as the lesions progress through the morphological stages identified. The results of this study support the hypothesis that DD is a polybacterial disease process and provide unique insights into the temporal changes in bacterial populations throughout lesion development.
Virus-Encoded MicroRNAs Facilitate Gammaherpesvirus Latency and PathogenesisIn Vivo
Authors: Feldman Emily R. , Kara Mehmet , Coleman Carrie B. , Grau Katrina R. , Oko Lauren M. , Krueger Brian J. , Renne Rolf , van Dyk Linda F. , Tibbetts Scott A. 
Published: May 2014

ABSTRACTGammaherpesviruses, including Epstein-Barr virus (EBV), Kaposi sarcoma-associated herpesvirus (KSHV, or HHV-8), and murine gammaherpesvirus 68 (MHV68, γHV68, or MuHV-4), are B cell-tropic pathogens that each encode at least 12 microRNAs (miRNAs). It is predicted that these regulatory RNAs facilitate infection by suppressing host target genes involved in a wide range of key cellular pathways. However, the precise contribution that gammaherpesvirus miRNAs make to viral life cycle and pathogenesisin vivois unknown. MHV68 infection of mice provides a highly useful system to dissect the function of specific viral elements in the context of both asymptomatic infection and disease. Here, we report (i) analysis ofin vitroandin vivoMHV68 miRNA expression, (ii) generation of an MHV68 miRNA mutant with reduced expression of all 14 pre-miRNA stem-loops, and (iii) comprehensive phenotypic characterization of the miRNA mutant virusin vivo. The profile of MHV68 miRNAs detected in infected cell lines varied with cell type and did not fully recapitulate the profile from cells latently infectedin vivo. The miRNA mutant virus, MHV68.Zt6, underwent normal lytic replicationin vitroandin vivo, demonstrating that the MHV68 miRNAs are dispensable for acute replication. During chronic infection, MHV68.Zt6 was attenuated for latency establishment, including a specific defect in memory B cells. Finally, MHV68.Zt6 displayed a striking attenuation in the development of lethal pneumonia in mice deficient in IFN-γ. These data indicate that the MHV68 miRNAs may facilitate virus-driven maturation of infected B cells and implicate the miRNAs as a critical determinant of gammaherpesvirus-associated disease.IMPORTANCEGammaherpesviruses such as EBV and KSHV are widespread pathogens that establish lifelong infections and are associated with the development of numerous types of diseases, including cancer. Gammaherpesviruses encode many small noncoding RNAs called microRNAs (miRNAs). It is predicted that gammaherpesvirus miRNAs facilitate infection and disease by suppressing host target transcripts involved in a wide range of key cellular pathways; however, the precise contribution that these regulatory RNAs make toin vivovirus infection and pathogenesis is unknown. Here, we generated a mutated form of murine gammaherpesvirus (MHV68) to dissect the function of gammaherpesvirus miRNAsin vivo. We demonstrate that the MHV68 miRNAs were dispensable for short-term virus replication but were important for establishment of lifelong infection in the key virus reservoir of memory B cells. Moreover, the MHV68 miRNAs were essential for the development of virus-associated pneumonia, implicating them as a critical component of gammaherpesvirus-associated disease.
Peptidoglycan Synthesis Machinery in Agrobacterium tumefaciens During Unipolar Growth and Cell Division
Authors: Cameron Todd A. , Anderson-Furgeson James , Zupan John R. , Zik Justin J. , Zambryski Patricia C. 
Published: May 2014

ABSTRACT

The synthesis of peptidoglycan (PG) in bacteria is a crucial process controlling cell shape and vitality. In contrast to bacteria such as
Escherichia coli
that grow by dispersed lateral insertion of PG, little is known of the processes that direct polar PG synthesis in other bacteria such as the
Rhizobiales
. To better understand polar growth in the
Rhizobiales
Agrobacterium tumefaciens
, we first surveyed its genome to identify homologs of (~70) well-known PG synthesis components. Since most of the canonical cell elongation components are absent from
A. tumefaciens
, we made fluorescent protein fusions to other putative PG synthesis components to assay their subcellular localization patterns. The cell division scaffolds FtsZ and FtsA, PBP1a, and a
Rhizobiales
- and
Rhodobacterales
-specific
l
,
d
-transpeptidase (LDT) all associate with the elongating cell pole. All four proteins also localize to the septum during cell division. Examination of the dimensions of growing cells revealed that new cell compartments gradually increase in width as they grow in length. This increase in cell width is coincident with an expanded region of LDT-mediated PG synthesis activity, as measured directly through incorporation of exogenous
d
-amino acids. Thus, unipolar growth in the
Rhizobiales
is surprisingly dynamic and represents a significant departure from the canonical growth mechanism of
E. coli
and other well-studied bacilli.

IMPORTANCE
Many rod-shaped bacteria, including pathogens such as
Brucella
and
Mycobacteriu
, grow by adding new material to their cell poles, and yet the proteins and mechanisms contributing to this process are not yet well defined. The polarly growing plant pathogen
Agrobacterium tumefaciens
was used as a model bacterium to explore these polar growth mechanisms. The results obtained indicate that polar growth in this organism is facilitated by repurposed cell division components and an otherwise obscure class of alternative peptidoglycan transpeptidases (
l
,
d
-transpeptidases). This growth results in dynamically changing cell widths as the poles expand to maturity and contrasts with the tightly regulated cell widths characteristic of canonical rod-shaped growth. Furthermore, the abundance and/or activity of
l
,
d
-transpeptidases appears to associate with polar growth strategies, suggesting that these enzymes may serve as attractive targets for specifically inhibiting growth of
Rhizobiales
,
Actinomycetales
, and other polarly growing bacterial pathogens.
A case of left main coronary artery embolus further embolising to the left anterior descending artery
Authors: Giri Smith , Hwang Inyong , Alsafwah Shadwan 
Published: May 2014

SummaryCoronary embolism is an uncommon cause of myocardial infarction. The usual source of a coronary embolus is an intracardiac thrombus or vegetation. Embolisation to the left main coronary artery is an extremely rare event and is usually fatal. We present a case of a 38-year-old woman with embolisation to the left main coronary artery which further embolised distally to the left anterior descending artery leading to a non-ST elevation myocardial infarction. The non-occlusive nature of the left main coronary artery embolus might have led to a favourable prognosis in our patient.