bluuebunny/crossref_metadata_2025 huggingface dataset:
Lack of Clinically Significant Pharmacokinetic Interaction between the Thrombopoietin Receptor Agonist Eltrombopag and Hepatitis C Virus Protease Inhibitors Boceprevir and Telaprevir
Authors: Wire Mary Beth , Fang Lei , Hussaini Azra , Kleha Joseph F. , Theodore Dickens
Published: August 2014

Eltrombopag is an orally bioavailable thrombopoietin receptor agonist approved for the treatment of thrombocytopenia associated with chronic immune (idiopathic) thrombocytopenic purpura and chronic hepatitis C virus (HCV) infection. This study evaluated the potential drug-drug interactions between eltrombopag and the HCV protease inhibitors boceprevir and telaprevir. In this open-label, 3-period, single-sequence, and crossover study, 56 healthy adult subjects were randomized 1:1 to cohort 1 (boceprevir) or 2 (telaprevir). The dosing was as follows: period 1, single 200-mg dose of eltrombopag; period 2, 800 mg boceprevir or 750 mg telaprevir every 8 hours (q8h) for 10 days; and period 3, single 200-mg dose of eltrombopag with either 800 mg boceprevir or 750 mg telaprevir q8h (3 doses). All doses were administered with food, and eltrombopag was administered specifically with low-calcium food. There was a 3-day washout between periods 1 and 2 and no washout between periods 2 and 3. Serial pharmacokinetic samples were collected for 72 h in periods 1 and 3 and for 8 h in period 2. The coadministration of eltrombopag increased the rate of boceprevir absorption, resulting in a 20% increase in the maximum concentration in plasma (
C
max
), a 1-h-earlier time to
C
max
(
T
max
) for boceprevir, a 32% decrease in the concentration at the end of the dosing interval (
C
τ
), and no change in the area under the concentration-time curve over the dosing interval (AUC
0-τ
). The coadministration of eltrombopag did not alter telaprevir pharmacokinetics, and the coadministration of boceprevir or telaprevir did not alter eltrombopag pharmacokinetics. Dysgeusia, headache, and somnolence occurred in ≥2 subjects. One subject withdrew because of nausea, headache, dizziness, sinus pressure, and vomiting. There were no severe or serious adverse events. Dose adjustment is not required when eltrombopag is coadministered with boceprevir or telaprevir given the lack of clinically significant pharmacokinetic interaction.
The 6-Aminoquinolone WC5 Inhibits Different Functions of the Immediate-Early 2 (IE2) Protein of Human Cytomegalovirus That Are Essential for Viral Replication
Authors: Mercorelli Beatrice , Luganini Anna , Muratore Giulia , Massari Serena , Terlizzi Maria Elena , Tabarrini Oriana , Gribaudo Giorgio , Palù Giorgio , Loregian Arianna
Published: August 2014

The human cytomegalovirus (HCMV) immediate-early 2 (IE2) protein is a multifunctional factor essential for viral replication. IE2 modulates both viral and host gene expression, deregulates cell cycle progression, acts as an immunomodulator, and antagonizes cellular antiviral responses. Based on these facts, IE2 has been proposed as an important target for the development of innovative antiviral approaches. We previously identified the 6-aminoquinolone WC5 as a promising inhibitor of HCMV replication, and here, we report the dissection of its mechanism of action against the viral IE2 protein. Using glutathione
S
-transferase (GST) pulldown assays, mutagenesis, cell-based assays, and electrophoretic mobility shift assays, we demonstrated that WC5 does not interfere with IE2 dimerization, its interaction with TATA-binding protein (TBP), and the expression of a set of cellular genes that are stimulated by IE2. On the contrary, WC5 targets the regulatory activity exerted by IE2 on different responsive viral promoters. Indeed, WC5 blocked the IE2-dependent negative regulation of the major immediate-early promoter by preventing IE2 binding to the
crs
element. Moreover, WC5 reduced the IE2-dependent transactivation of a series of indicator constructs driven by different portions of the early
UL54
gene promoter, and it also inhibited the transactivation of the murine CMV early
E1
promoter by the IE3 protein, the murine cytomegalovirus (MCMV) IE2 homolog. In conclusion, our results indicate that the overall anti-HCMV activity of WC5 depends on its ability to specifically interfere with the IE2-dependent regulation of viral promoters. Importantly, our results suggest that this mechanism is conserved in murine CMV, thus paving the way for further preclinical evaluation in an animal model.
BCX4430, a Novel Nucleoside Analog, Effectively Treats Yellow Fever in a Hamster Model
Authors: Julander Justin G. , Bantia Shanta , Taubenheim Brian R. , Minning Dena M. , Kotian Pravin , Morrey John D. , Smee Donald F. , Sheridan William P. , Babu Yarlagadda S.
Published: August 2014

No effective antiviral therapies are currently available to treat disease after infection with yellow fever virus (YFV). A Syrian golden hamster model of yellow fever (YF) was used to characterize the effect of treatment with BCX4430, a novel adenosine nucleoside analog. Significant improvement in survival was observed after treatment with BCX4430 at 4 mg/kg of body weight per day dosed intraperitoneally (i.p.) twice daily (BID). Treatment with BCX4430 at 12.5 mg/kg/day administered i.p. BID for 7 days offered complete protection from mortality and also resulted in significant improvement of other YF disease parameters, including weight loss, serum alanine aminotransferase levels (6 days postinfection [dpi]), and viremia (4 dpi). In uninfected hamsters, BCX4430 at 200 mg/kg/day administered i.p. BID for 7 days was well tolerated and did not result in mortality or weight loss, suggesting a potentially wide therapeutic index. Treatment with BCX4430 at 12 mg/kg/day i.p. remained effective when administered once daily and for only 4 days. Moreover, BCX4430 dosed at 200 mg/kg/day i.p. BID for 7 days effectively treated YF, even when treatment was delayed up to 4 days after virus challenge, corresponding with peak viral titers in the liver and serum. BCX4430 treatment did not preclude a protective antibody response, as higher neutralizing antibody (nAb) concentrations corresponded with increasing delays of treatment initiation, and greater nAb responses resulted in the protection of animals from a secondary challenge with YFV. In summary, BCX4430 is highly active in a hamster model of YF, even when treatment is initiated at the peak of viral replication.
Safety and Pharmacokinetic Profiles of Phosphorodiamidate Morpholino Oligomers with Activity against Ebola Virus and Marburg Virus: Results of Two Single-Ascending-Dose Studies
Authors: Heald Alison E. , Iversen Patrick L. , Saoud Jay B. , Sazani Peter , Charleston Jay S. , Axtelle Tim , Wong Michael , Smith William B. , Vutikullird Apinya , Kaye Edward
Published: August 2014

Two identical single-ascending-dose studies evaluated the safety and pharmacokinetics (PK) of AVI-6002 and AVI-6003, two experimental combinations of phosphorodiamidate morpholino oligomers with positive charges (PMO
plus
) that target viral mRNA encoding Ebola virus and Marburg virus proteins, respectively. Both AVI-6002 and AVI-6003 were found to suppress disease in virus-infected nonhuman primates in previous studies. AVI-6002 (a combination of AVI-7537 and AVI-7539) or AVI-6003 (a combination of AVI-7287 and AVI-7288) were administered as sequential intravenous (i.v.) infusions of a 1:1 fixed dose ratio of the two subcomponents. In each study, 30 healthy male and female subjects between 18 and 50 years of age were enrolled in six-dose escalation cohorts of five subjects each and received a single i.v. infusion of active study drug (0.005, 0.05, 0.5, 1.5, 3, and 4.5 mg/kg per component) or placebo in a 4:1 ratio. Both AVI-6002 and AVI-6003 were safe and well tolerated at the doses studied. A maximum tolerated dose was not observed in either study. The four chemically similar PMO
plus
components exhibited generally similar PK profiles. The mean peak plasma concentration and area under the concentration-time curve values of the four components exhibited dose-proportional PK. The estimated plasma half-life of all four components was 2 to 5 h. The safety of the two combinations and the PK of the four components were similar, regardless of the target RNA sequence.
Respiratory Flexibility in Response to Inhibition of Cytochrome
c
Oxidase in Mycobacterium tuberculosis
Authors: Arora Kriti , Ochoa-Montaño Bernardo , Tsang Patricia S. , Blundell Tom L. , Dawes Stephanie S. , Mizrahi Valerie , Bayliss Tracy , Mackenzie Claire J. , Cleghorn Laura A. T. , Ray Peter C. , Wyatt Paul G. , Uh Eugene , Lee Jinwoo , Barry Clifton E. , Boshoff Helena I.
Published: August 2014

We report here a series of five chemically diverse scaffolds that have
in vitro
activities on replicating and hypoxic nonreplicating bacilli by targeting the respiratory
bc
1
complex in
Mycobacterium tuberculosis
in a strain-dependent manner. Deletion of the cytochrome
bd
oxidase generated a hypersusceptible mutant in which resistance was acquired by a mutation in
qcrB
. These results highlight the promiscuity of the
bc
1
complex and the risk of targeting energy metabolism with new drugs.
The Production of Reactive Oxygen Species Is a Universal Action Mechanism of Amphotericin B against Pathogenic Yeasts and Contributes to the Fungicidal Effect of This Drug
Authors: Mesa-Arango Ana Cecilia , Trevijano-Contador Nuria , Román Elvira , Sánchez-Fresneda Ruth , Casas Celia , Herrero Enrique , Argüelles Juan Carlos , Pla Jesús , Cuenca-Estrella Manuel , Zaragoza Oscar
Published: August 2014

Amphotericin B (AMB) is an antifungal drug that binds to ergosterol and forms pores at the cell membrane, causing the loss of ions. In addition, AMB induces the accumulation of reactive oxygen species (ROS), and although these molecules have multiple deleterious effects on fungal cells, their specific role in the action mechanism of AMB remains unknown. In this work, we studied the role of ROS in the action mechanism of AMB. We determined the intracellular induction of ROS in 44 isolates of different pathogenic yeast species (
Candida albicans
,
Candida parapsilosis
,
Candida glabrata
,
Candida tropicalis
,
Candida krusei
,
Cryptococcus neoformans
, and
Cryptococcus gattii
). We also characterized the production of ROS in AMB-resistant isolates. We found that AMB induces the formation of ROS in all the species tested. The inhibition of the mitochondrial respiratory chain by rotenone blocked the induction of ROS by AMB and provided protection from the killing action of the antifungal. Moreover, this phenomenon was absent in strains that displayed resistance to AMB. These strains showed an alteration in the respiration rate and mitochondrial membrane potential and also had higher catalase activity than that of the AMB-susceptible strains. Consistently, AMB failed to induce protein carbonylation in the resistant strains. Our data demonstrate that the production of ROS by AMB is a universal and important action mechanism that is correlated with the fungicidal effect and might explain the low rate of resistance to the molecule. Finally, these data provide an opportunity to design new strategies to improve the efficacy of this antifungal.
Maternal Intravenous Administration of Azithromycin Results in Significant Fetal Uptake in a Sheep Model of Second Trimester Pregnancy
Authors: Kemp Matthew W. , Miura Yuichiro , Payne Matthew S. , Jobe Alan H. , Kallapur Suhas G. , Saito Masatoshi , Stock Sarah J. , Spiller O. Brad , Ireland Demelza J. , Yaegashi Nobuo , Clarke Michael , Hahne Dorothee , Rodger Jennifer , Keelan Jeffrey A. , Newnham John P.
Published: August 2014

Treatment of intrauterine infection is likely key to preventing a significant proportion of preterm deliveries before 32 weeks of gestation. Azithromycin (AZ) may be an effective antimicrobial in pregnancy; however, few gestation age-approriate data are available to inform the design of AZ-based treatment regimens in early pregnancy. We aimed to determine whether a single intra-amniotic AZ dose or repeated maternal intravenous (i.v.) AZ doses would safely yield therapeutic levels of AZ in an 80-day-gestation (term is 150 days) ovine fetus. Fifty sheep carrying single pregnancies at 80 days gestation were randomized to receive either: (i) a single intra-amniotic AZ administration or (ii) maternal intravenous AZ administration every 12 h. Amniotic fluid, maternal plasma, and fetal AZ concentrations were determined over a 5-day treatment regimen. Markers of liver injury and amniotic fluid inflammation were measured to assess fetal injury in response to drug exposure. A single intra-amniotic administration yielded significant AZ accumulation in the amniotic fluid and fetal lung. In contrast, repeated maternal intravenous administrations achieved high levels of AZ accumulation in the fetal lung and liver and a statistically significant increase in the fetal plasma drug concentration at 120 h. There was no evidence of fetal injury in response to drug exposure. These data suggest that (i) repeated maternal i.v. AZ dosing yields substantial fetal tissue uptake, although fetal plasma drug levels remain low; (ii) transfer of AZ from the amniotic fluid is less than transplacental transfer; and (iii) exposure to high concentrations of AZ did not elicit overt changes in fetal white blood cell counts, amniotic fluid monocyte chemoattractant protein 1 concentrations, or hepatotoxicity, all consistent with an absence of fetal injury.
Characterization of Urinary Tract Infection-Associated Shiga Toxin-Producing Escherichia coli
Authors: Toval Francisco , Schiller Roswitha , Meisen Iris , Putze Johannes , Kouzel Ivan U. , Zhang Wenlan , Karch Helge , Bielaszewska Martina , Mormann Michael , Müthing Johannes , Dobrindt Ulrich
Published: August 2014

Enterohemorrhagic
Escherichia coli
(EHEC), a subgroup of Shiga toxin (Stx)-producing
E. coli
(STEC), is a leading cause of diarrhea and hemolytic-uremic syndrome (HUS) in humans. However, urinary tract infections (UTIs) caused by this microorganism but not associated with diarrhea have occasionally been reported. We geno- and phenotypically characterized three EHEC isolates obtained from the urine of hospitalized patients suffering from UTIs. These isolates carried typical EHEC virulence markers and belonged to HUS-associated
E. coli
(HUSEC) clones, but they lacked virulence markers typical of uropathogenic
E. coli
. One isolate exhibited a localized adherence (LA)-like pattern on T24 urinary bladder epithelial cells. Since the glycosphingolipids (GSLs) globotriaosylceramide (Gb3Cer) and globotetraosylceramide (Gb4Cer) are well-known receptors for Stx but also for P fimbriae, a major virulence factor of extraintestinal pathogenic
E. coli
(ExPEC), the expression of Gb3Cer and Gb4Cer by T24 cells and in murine urinary bladder tissue was examined by thin-layer chromatography and mass spectrometry. We provide data indicating that Stxs released by the EHEC isolates bind to Gb3Cer and Gb4Cer isolated from T24 cells, which were susceptible to Stx. All three EHEC isolates expressed
stx
genes upon growth in urine. Two strains were able to cause UTI in a murine infection model and could not be outcompeted in urine
in vitro
by typical uropathogenic
E. coli
isolates. Our results indicate that despite the lack of ExPEC virulence markers, EHEC variants may exhibit in certain suitable hosts, e.g., in hospital patients, a uropathogenic potential. The contribution of EHEC virulence factors to uropathogenesis remains to be further investigated.
Influenza A Virus Alters Pneumococcal Nasal Colonization and Middle Ear Infection Independently of Phase Variation
Authors: Wren John T. , Blevins Lance K. , Pang Bing , King Lauren B. , Perez Antonia C. , Murrah Kyle A. , Reimche Jennifer L. , Alexander-Miller Martha A. , Swords W. Edward
Published: August 2014

Streptococcus pneumoniae
(pneumococcus) is both a widespread nasal colonizer and a leading cause of otitis media, one of the most common diseases of childhood. Pneumococcal phase variation influences both colonization and disease and thus has been linked to the bacteria's transition from colonizer to otopathogen. Further contributing to this transition, coinfection with influenza A virus has been strongly associated epidemiologically with the dissemination of pneumococci from the nasopharynx to the middle ear. Using a mouse infection model, we demonstrated that coinfection with influenza virus and pneumococci enhanced both colonization and inflammatory responses within the nasopharynx and middle ear chamber. Coinfection studies were also performed using pneumococcal populations enriched for opaque or transparent phase variants. As shown previously, opaque variants were less able to colonize the nasopharynx.
In vitro
, this phase also demonstrated diminished biofilm viability and epithelial adherence. However, coinfection with influenza virus ameliorated this colonization defect
in vivo
. Further, viral coinfection ultimately induced a similar magnitude of middle ear infection by both phase variants. These data indicate that despite inherent differences in colonization, the influenza A virus exacerbation of experimental middle ear infection is independent of the pneumococcal phase. These findings provide new insights into the synergistic link between pneumococcus and influenza virus in the context of otitis media.
Localization of Burkholderia cepacia Complex Bacteria in Cystic Fibrosis Lungs and Interactions with Pseudomonas aeruginosa in Hypoxic Mucus
Authors: Schwab Ute , Abdullah Lubna H. , Perlmutt Olivia S. , Albert Daniel , Davis C. William , Arnold Roland R. , Yankaskas James R. , Gilligan Peter , Neubauer Heiner , Randell Scott H. , Boucher Richard C.
Published: August 2014

The localization of
Burkholderia cepacia
complex (Bcc) bacteria in cystic fibrosis (CF) lungs, alone or during coinfection with
Pseudomonas aeruginosa
, is poorly understood. We performed immunohistochemistry for Bcc and
P. aeruginosa
bacteria on 21 coinfected or singly infected CF lungs obtained at transplantation or autopsy. Parallel
in vitro
experiments examined the growth of two Bcc species,
Burkholderia cenocepacia
and
Burkholderia multivorans
, in environments similar to those occupied by
P. aeruginosa
in the CF lung. Bcc bacteria were predominantly identified in the CF lung as single cells or small clusters within phagocytes and mucus but not as “biofilm-like structures.” In contrast,
P. aeruginosa
was identified in biofilm-like masses, but densities appeared to be reduced during coinfection with Bcc bacteria. Based on chemical analyses of CF and non-CF respiratory secretions, a test medium was defined to study Bcc growth and interactions with
P. aeruginosa
in an environment mimicking the CF lung. When test medium was supplemented with alternative electron acceptors under anaerobic conditions,
B. cenocepacia
and
B. multivorans
used fermentation rather than anaerobic respiration to gain energy, consistent with the identification of fermentation products by high-performance liquid chromatography (HPLC). Both Bcc species also expressed mucinases that produced carbon sources from mucins for growth. In the presence of
P. aeruginosa
in vitro
, both Bcc species grew anaerobically but not aerobically. We propose that Bcc bacteria (i) invade a
P. aeruginosa
-infected CF lung when the airway lumen is anaerobic, (ii) inhibit
P. aeruginosa
biofilm-like growth, and (iii) expand the host bacterial niche from mucus to also include macrophages.
NOD2 Stimulation by Staphylococcus aureus-Derived Peptidoglycan Is Boosted by Toll-Like Receptor 2 Costimulation with Lipoproteins in Dendritic Cells
Authors: Schäffler Holger , Demircioglu Dogan Doruk , Kühner Daniel , Menz Sarah , Bender Annika , Autenrieth Ingo B. , Bodammer Peggy , Lamprecht Georg , Götz Friedrich , Frick Julia-Stefanie
Published: August 2014

Mutations in the nucleotide-binding oligomerization domain-containing protein 2 (NOD2) play an important role in the pathogenesis of Crohn's disease. NOD2 is an intracellular pattern recognition receptor (PRR) that senses bacterial peptidoglycan (PGN) structures, e.g., muramyl dipeptide (MDP). Here we focused on the effect of more-cross-linked, polymeric PGN fragments (PGNpol) in the activation of the innate immune system. In this study, the effect of combined NOD2 and Toll-like receptor 2 (TLR2) stimulation was examined compared to single stimulation of the NOD2 receptor alone. PGNpol species derived from a lipoprotein-containing
Staphylococcus aureus
strain (SA113) and a lipoprotein-deficient strain (SA113 Δ
lgt
) were isolated. While PGNpol constitutes a combined NOD2 and TLR2 ligand, lipoprotein-deficient PGNpolΔ
lgt
leads to activation of the immune system only via the NOD2 receptor. Murine bone marrow-derived dendritic cells (BMDCs), J774 cells, and Mono Mac 6 (MM6) cells were stimulated with these ligands. Cytokines (interleukin-6 [IL-6], IL-12p40, and tumor necrosis factor alpha [TNF-α]) as well as DC activation and maturation parameters were measured. Stimulation with PGNpolΔ
lgt
did not lead to enhanced cytokine secretion or DC activation and maturation. However, stimulation with PGNpol led to strong cytokine secretion and subsequent DC maturation. These results were confirmed in MM6 and J774 cells. We showed that the NOD2-mediated activation of DCs with PGNpol was dependent on TLR2 costimulation. Therefore, signaling via both receptors leads to a more potent activation of the immune system than that with stimulation via each receptor alone.
Use of Immunodampening To Overcome Diversity in the Malarial Vaccine Candidate Apical Membrane Antigen 1
Authors: Harris Karen S. , Adda Christopher G. , Khore Madhavi , Drew Damien R. , Valentini-Gatt Antonina , Fowkes Freya J. I. , Beeson James G. , Dutta Sheetij , Anders Robin F. , Foley Michael
Published: August 2014

Apical membrane antigen 1 (AMA1) is a leading malarial vaccine candidate; however, its polymorphic nature may limit its success in the field. This study aimed to circumvent AMA1 diversity by dampening the antibody response to the highly polymorphic loop Id, previously identified as a major target of strain-specific, invasion-inhibitory antibodies. To achieve this, five polymorphic residues within this loop were mutated to alanine, glycine, or serine in AMA1 of the 3D7 and FVO
Plasmodium falciparum
strains. Initially, the corresponding antigens were displayed on the surface of bacteriophage, where the alanine and serine but not glycine mutants folded correctly. The alanine and serine AMA1 mutants were expressed in
Escherichia coli
, refolded
in vitro
, and used to immunize rabbits. Serological analyses indicated that immunization with a single mutated form of 3D7 AMA1 was sufficient to increase the cross-reactive antibody response. Targeting the corresponding residues in an FVO backbone did not achieve this outcome. The inclusion of at least one engineered form of AMA1 in a biallelic formulation resulted in an antibody response with broader reactivity against different AMA1 alleles than combining the wild-type forms of 3D7 and FVO AMA1 alleles. For one combination, this extended to an enhanced relative growth inhibition of a heterologous parasite line, although this was at the cost of reduced overall inhibitory activity. These results suggest that targeted mutagenesis of AMA1 is a promising strategy for overcoming antigenic diversity in AMA1 and reducing the number of variants required to induce an antibody response that protects against a broad range of
Plasmodium falciparum
AMA1 genotypes. However, optimization of the immunization regime and mutation strategy will be required for this potential to be realized.
Damage to the Blood-Brain Barrier during Experimental Cerebral Malaria Results from Synergistic Effects of CD8+T Cells with Different Specificities
Authors: Poh Chek Meng , Howland Shanshan W. , Grotenbreg Gijsbert M. , Rénia Laurent
Published: August 2014

CD8+T cells play a pathogenic role in the development of murine experimental cerebral malaria (ECM) induced byPlasmodium bergheiANKA (PbA) infection in C57BL/6 mice. Only a limited number of CD8+epitopes have been described. Here, we report the identification of a new epitope from the bergheilysin protein recognized by PbA-specific CD8+T cells. Induction and functionality of these specific CD8+T cells were investigated in parallel with previously reported epitopes, using new tools such as tetramers and reporter cell lines that were developed for this study. We demonstrate that CD8+T cells of diverse specificities induced during PbA infection share many characteristics. They express cytolytic markers (gamma interferon [IFN-γ], granzyme B) and chemokine receptors (CXCR3, CCR5) and damage the blood-brain barrierin vivo. Our earlier finding that brain microvessels in mice infected with PbA, but not with non-ECM-causing strains, cross-presented a shared epitope was generalizable to these additional epitopes. Suppressing the induction of specific CD8+T cells through tolerization with a high-dose peptide injection was unable to confer protection against ECM, suggesting that CD8+T cells of other specificities participate in this process. The tools that we developed can be used to further investigate the heterogeneity of CD8+T cell responses that are involved in ECM.
Role of the Oligopeptide Permease ABC Transporter of Moraxella catarrhalis in Nutrient Acquisition and Persistence in the Respiratory Tract
Authors: Jones Megan M. , Johnson Antoinette , Koszelak-Rosenblum Mary , Kirkham Charmaine , Brauer Aimee L. , Malkowski Michael G. , Murphy Timothy F.
Published: August 2014

Moraxella catarrhalis
is a strict human pathogen that causes otitis media in children and exacerbations of chronic obstructive pulmonary disease in adults, resulting in significant worldwide morbidity and mortality.
M. catarrhalis
has a growth requirement for arginine; thus, acquiring arginine is important for fitness and survival.
M. catarrhalis
has a putative oligopeptide permease ABC transport operon (
opp
) consisting of five genes (
oppB
,
oppC
,
oppD
,
oppF
, and
oppA
), encoding two permeases, two ATPases, and a substrate binding protein. Thermal shift assays showed that the purified recombinant substrate binding protein OppA binds to peptides 3 to 16 amino acid residues in length regardless of the amino acid composition. A mutant in which the
oppBCDFA
gene cluster is knocked out showed impaired growth in minimal medium where the only source of arginine came from a peptide 5 to 10 amino acid residues in length. Whether methylated arginine supports growth of
M. catarrhalis
is important in understanding fitness in the respiratory tract because methylated arginine is abundant in host tissues. No growth of wild-type
M. catarrhalis
was observed in minimal medium in which arginine was present only in methylated form, indicating that the bacterium requires
l
-arginine. An
oppA
knockout mutant showed marked impairment in its capacity to persist in the respiratory tract compared to the wild type in a mouse pulmonary clearance model. We conclude that the Opp system mediates both uptake of peptides and fitness in the respiratory tract.
Hydrogen Peroxide Production from Glycerol Metabolism Is Dispensable for Virulence of Mycoplasma gallisepticum in the Tracheas of Chickens
Authors: Szczepanek S. M. , Boccaccio M. , Pflaum K. , Liao X. , Geary S. J.
Published: August 2014

Hydrogen peroxide (H
2
O
2
) is a by-product of glycerol metabolism in mycoplasmas and has been shown to cause cytotoxicity for cocultured eukaryotic cells. There appears to be selective pressure for mycoplasmas to retain the genes needed for glycerol metabolism. This has generated interest and speculation as to their function during infection. However, the actual effects of glycerol metabolism and H
2
O
2
production on virulence
in vivo
have never been assessed in any
Mycoplasma
species. To this end, we determined that the wild-type (WT) R
low
strain of the avian pathogen
Mycoplasma gallisepticum
is capable of producing H
2
O
2
when grown in glycerol and is cytotoxic to eukaryotic cells in culture. Transposon mutants with mutations in the genes present in the glycerol transport and utilization pathway, namely,
glpO
,
glpK
, and
glpF
, were identified. All mutants assessed were incapable of producing H
2
O
2
and were not cytotoxic when grown in glycerol. We also determined that vaccine strains ts-11 and 6/85 produce little to no H
2
O
2
when grown in glycerol, while the naturally attenuated F strain does produce H
2
O
2
. Chickens were infected with one of two
glpO
mutants, a
glpK
mutant, R
low
, or growth medium, and tracheal mucosal thickness and lesion scores were assessed. Interestingly, all
glp
mutants were reproducibly virulent in the respiratory tracts of the chickens. Thus, there appears to be no link between glycerol metabolism/H
2
O
2
production/cytotoxicity and virulence for this
Mycoplasma
species in its natural host. However, it is possible that glycerol metabolism is required by
M. gallisepticum
in a niche that we have yet to study.
Inhibition of Local Immune Responses by the Frog-Killing Fungus Batrachochytrium dendrobatidis
Authors: Fites J. Scott , Reinert Laura K. , Chappell Timothy M. , Rollins-Smith Louise A.
Published: August 2014

Amphibians are suffering unprecedented global declines. A leading cause is the infectious disease chytridiomycosis caused by the chytrid fungus
Batrachochytrium dendrobatidis
. Chytridiomycosis is a skin disease which disrupts transport of essential ions leading to death. Soluble factors produced by
B. dendrobatidis
impair amphibian and mammalian lymphocytes
in vitro
, but previous studies have not shown the effects of these inhibitory factors
in vivo
. To demonstrate
in vivo
inhibition of immunity by
B. dendrobatidis
, a modified delayed-type-hypersensitivity (DTH) protocol was developed to induce innate and adaptive inflammatory swelling in the feet of
Xenopus laevis
by injection of killed bacteria or phytohemagglutinin (PHA). Compared to previous protocols for PHA injection in amphibians, this method induced up to 20-fold greater inflammatory swelling. Using this new protocol, we measured DTH responses induced by killed bacteria or PHA in the presence of
B. dendrobatidis
supernatants. Swelling induced by single injection of PHA or killed bacteria was not significantly affected by
B. dendrobatidis
supernatants. However, swelling caused by a secondary injection of PHA, was significantly reduced by
B. dendrobatidis
supernatants. As previously described
in vitro
, factors from
B. dendrobatidis
appear to inhibit lymphocyte-mediated inflammatory swelling but not swelling caused by an inducer of innate leukocytes. This suggests that
B. dendrobatidis
is capable of inhibiting lymphocytes in a localized response to prevent adaptive immune responses in the skin. The modified protocol used to induce inflammatory swelling in the present study may be more effective than previous methods to investigate amphibian immune competence, particularly in nonmodel species.
Genetic Examination and Mass Balance Analysis of Pyruvate/Amino Acid Oxidation Pathways in the Hyperthermophilic Archaeon Thermococcus kodakarensis
Authors: Nohara Kenta , Orita Izumi , Nakamura Satoshi , Imanaka Tadayuki , Fukui Toshiaki
Published: August 2014

The present study investigated the simultaneous oxidation of pyruvate and amino acids during H
2
-evolving growth of the hyperthermophilic archaeon
Thermococcus kodakarensis
. The comparison of mass balance between a cytosolic hydrogenase (HYH)-deficient strain (the Δ
hyhBGSL
strain) and the parent strain indicated that NADPH generated via H
2
uptake by HYH was consumed by reductive amination of 2-oxoglutarate catalyzed by glutamate dehydrogenase. Further examinations were done to elucidate functions of three enzymes potentially involved in pyruvate oxidation: pyruvate formate-lyase (PFL), pyruvate:ferredoxin oxidoreductase (POR), and 2-oxoisovalerate:ferredoxin oxidoreductase (VOR) under the HYH-deficient background in
T. kodakarensis
. No significant change was observed by deletion of
pflDA
, suggesting that PFL had no critical role in pyruvate oxidation. The growth properties and mass balances of Δ
porDAB
and Δ
vorDAB
strains indicated that POR and VOR specifically functioned in oxidation of pyruvate and branched-chain amino acids, respectively, and the lack of POR or VOR was compensated for by promoting the oxidation of another substrate driven by the remaining oxidoreductase. The H
2
yields from the consumed pyruvate and amino acids were increased from 31% by the parent strain to 67% and 82% by the deletion of
hyhBGSL
and double deletion of
hyhBGSL
and
vorDAB
, respectively. Significant discrepancies in the mass balances were observed in excess formation of acetate and NH
3
, suggesting the presence of unknown metabolisms in
T. kodakarensis
grown in the rich medium containing pyruvate.
Listeria monocytogenes Is Resistant to Lysozyme through the Regulation, Not the Acquisition, of Cell Wall-Modifying Enzymes
Authors: Burke Thomas P. , Loukitcheva Anastasia , Zemansky Jason , Wheeler Richard , Boneca Ivo G. , Portnoy Daniel A.
Published: August 2014

Listeria monocytogenes
is a Gram-positive facultative intracellular pathogen that is highly resistant to lysozyme, a ubiquitous enzyme of the innate immune system that degrades cell wall peptidoglycan. Two peptidoglycan-modifying enzymes, PgdA and OatA, confer lysozyme resistance on
L. monocytogenes
; however, these enzymes are also conserved among lysozyme-sensitive nonpathogens. We sought to identify additional factors responsible for lysozyme resistance in
L. monocytogenes
. A forward genetic screen for lysozyme-sensitive mutants led to the identification of 174 transposon insertion mutations that mapped to 13 individual genes. Four mutants were killed exclusively by lysozyme and not other cell wall-targeting molecules, including the peptidoglycan deacetylase encoded by
pgdA
, the putative carboxypeptidase encoded by
pbpX
, the orphan response regulator encoded by
degU
, and the highly abundant noncoding RNA encoded by
rli31
. Both
degU
and
rli31
mutants had reduced expression of
pbpX
and
pgdA
, yet DegU and Rli31 did not regulate each other. Since
pbpX
and
pgdA
are also present in lysozyme-sensitive bacteria, this suggested that the acquisition of novel enzymes was not responsible for lysozyme resistance, but rather, the regulation of conserved enzymes by DegU and Rli31 conferred high lysozyme resistance. Each lysozyme-sensitive mutant exhibited attenuated virulence in mice, and a time course of infection revealed that the most lysozyme-sensitive strain was killed within 30 min of intravenous infection, a phenotype that was recapitulated in purified blood. Collectively, these data indicate that the genes required for lysozyme resistance are highly upregulated determinants of
L. monocytogenes
pathogenesis that are required for avoiding the enzymatic activity of lysozyme in the blood.
CcpA-Mediated Catabolite Activation of the Bacillus subtilis ilv-leu Operon and Its Negation by Either CodY- or TnrA-Mediated Negative Regulation
Authors: Fujita Yasutaro , Satomura Takenori , Tojo Shigeo , Hirooka Kazutake
Published: August 2014

The
Bacillus subtilis ilv-leu
operon functions in the biosynthesis of branched-chain amino acids. It undergoes catabolite activation involving a promoter-proximal
cre
which is mediated by the complex of CcpA and P-Ser-HPr. This activation of
ilv-leu
expression is negatively regulated through CodY binding to a high-affinity site in the promoter region under amino acid-rich growth conditions, and it is negatively regulated through TnrA binding to the TnrA box under nitrogen-limited growth conditions. The CcpA-mediated catabolite activation of
ilv-leu
required a helix face-dependent interaction of the complex of CcpA and P-Ser-HPr with RNA polymerase and needed a 19-nucleotide region upstream of
cre
for full activation. DNase I footprinting indicated that CodY binding to the high-affinity site competitively prevented the binding of the complex of CcpA and P-Ser-HPr to
cre
. This CodY binding not only negated catabolite activation but also likely inhibited transcription initiation from the
ilv-leu
promoter. The footprinting also indicated that TnrA and the complex of CcpA and P-Ser-HPr simultaneously bound to the TnrA box and the
cre
site, respectively, which are 112 nucleotides apart; TnrA binding to its box was likely to induce DNA bending. This implied that interaction of TnrA bound to its box with the complex of CcpA and P-Ser-HPr bound to
cre
might negate catabolite activation, but TnrA bound to its box did not inhibit transcription initiation from the
ilv-leu
promoter. Moreover, this negation of catabolite activation by TnrA required a 26-nucleotide region downstream of the TnrA box.
Hydrogen Formation and Its Regulation in Ruminococcus albus: Involvement of an Electron-Bifurcating [FeFe]-Hydrogenase, of a Non-Electron-Bifurcating [FeFe]-Hydrogenase, and of a Putative Hydrogen-Sensing [FeFe]-Hydrogenase
Authors: Zheng Yanning , Kahnt Jörg , Kwon In Hyuk , Mackie Roderick I. , Thauer Rudolf K.
Published: August 2014

Ruminococcus albus
7 has played a key role in the development of the concept of interspecies hydrogen transfer. The rumen bacterium ferments glucose to 1.3 acetate, 0.7 ethanol, 2 CO
2
, and 2.6 H
2
when growing in batch culture and to 2 acetate, 2 CO
2
, and 4 H
2
when growing in continuous culture in syntrophic association with H
2
-consuming microorganisms that keep the H
2
partial pressure low. The organism uses NAD
+
and ferredoxin for glucose oxidation to acetyl coenzyme A (acetyl-CoA) and CO
2
, NADH for the reduction of acetyl-CoA to ethanol, and NADH and reduced ferredoxin for the reduction of protons to H
2
. Of all the enzymes involved, only the enzyme catalyzing the formation of H
2
from NADH remained unknown. Here, we report that
R. albus
7 grown in batch culture on glucose contained, besides a ferredoxin-dependent [FeFe]-hydrogenase (HydA2), a ferredoxin- and NAD-dependent electron-bifurcating [FeFe]-hydrogenase (HydABC) that couples the endergonic formation of H
2
from NADH to the exergonic formation of H
2
from reduced ferredoxin. Interestingly,
hydA2
is adjacent to the
hydS
gene, which is predicted to encode an [FeFe]-hydrogenase with a C-terminal PAS domain. We showed that
hydS
and
hydA2
are part of a larger transcriptional unit also harboring putative genes for a bifunctional acetaldehyde/ethanol dehydrogenase (Aad), serine/threonine protein kinase, serine/threonine protein phosphatase, and a redox-sensing transcriptional repressor. Since HydA2 and Aad are required only when
R. albus
grows at high H
2
partial pressures, HydS could be a H
2
-sensing [FeFe]-hydrogenase involved in the regulation of their biosynthesis.
Cell Division Resets Polarity and Motility for the Bacterium Myxococcus xanthus
Authors: Harvey Cameron W. , Madukoma Chinedu S. , Mahserejian Shant , Alber Mark S. , Shrout Joshua D.
Published: August 2014

Links between cell division and other cellular processes are poorly understood. It is difficult to simultaneously examine division and function in most cell types. Most of the research probing aspects of cell division has experimented with stationary or immobilized cells or distinctly asymmetrical cells. Here we took an alternative approach by examining cell division events within motile groups of cells growing on solid medium by time-lapse microscopy. A total of 558 cell divisions were identified among approximately 12,000 cells. We found an interconnection of division, motility, and polarity in the bacterium
Myxococcus xanthus
. For every division event, motile cells stop moving to divide. Progeny cells of binary fission subsequently move in opposing directions. This behavior involves
M. xanthus
Frz proteins that regulate
M. xanthus
motility reversals but is independent of type IV pilus “S motility.” The inheritance of opposing polarity is correlated with the distribution of the G protein RomR within these dividing cells. The constriction at the point of division limits the intracellular distribution of RomR. Thus, the asymmetric distribution of RomR at the parent cell poles becomes mirrored at new poles initiated at the site of division.
Use of probiotics to correct dysbiosis of normal microbiota following disease or disruptive events: a systematic review
Authors: McFarland Lynne V
Published: August 2014

Objective

To assess the evidence for the claim probiotics can correct dysbiosis of the normal microbiota resulting from disease or disruptive events.

Setting

Systematic review of published clinical trials of patients receiving a probiotic intervention for the prevention or treatment of various diseases.

Data sources

Sources searched (1985–2013): PubMed, EMBASE, Cochrane Database of Systematic Reviews, CINAHL, AMED and ISI Web of Science. Three on-line clinical trial registries were searched: Cochrane Central Register of Controlled trials, MetaRegister of Controlled Trials and National Institutes of Health.

Review methods

Included studies were randomised clinical trials of probiotic interventions having microbiological assays. Studies were evaluated following Preferred Reporting Items for Systematic Reviews and Meta-Analyses guidelines for specific probiotic strains. A standard data extraction form was used to collect the raw data.

Outcome measures

The primary outcome is the degree of microbiota correction by specific probiotic strains. Secondary outcome was the association between the degree of dysbiosis correction and clinical efficacy.

Results

The review of the literature found three distinct study designs: model A (restoration) assayed patients enrolled with a healthy, undisturbed microbiota and then assayed postdisruptive event and probiotic therapy; model B (alteration) assayed patients with pre-existing disrupted microbiota and then postprobiotic therapy; model C (no dysbiosis) assayed volunteers with no disruptive event prebiotic and postprobiotic. From a total of 63 trials, 83% of the probiotic products using model A restored the microbiota, 56% using model B improved the microbiota and only 21% using model C had any effect on microbiota. Clinical efficacy was more commonly associated with strains capable of restoration of the normal microbiota.

Conclusions

The ability to assess the degree of dysbiosis improvement is dependent on the enrolled population and the timing of microbiological assays. The functional claim for correcting dysbiosis is poorly supported for most probiotic strains and requires further research.

Trial registration number

PROSPERO (CRD42014007224).
A systematic review of behavioural marker systems in healthcare: what do we know about their attributes, validity and application?
Authors: Dietz Aaron S , Pronovost Peter J , Benson Kari N , Mendez-Tellez Pedro Alejandro , Dwyer Cynthia , Wyskiel Rhonda , Rosen Michael A
Published: August 2014

Objective

Behavioural marker systems are advocated as a method for providing accurate assessments, directing feedback and determining the impact of teamwork improvement initiatives. The present article reports on the state of quality surrounding their use in healthcare and discusses the implications of these findings for future research, development and application. In doing so, this article provides a practical resource where marker systems can be selected and evaluated based on their strengths and limitations.

Methods

Four research questions framed this review: what are the attributes of behavioural marker systems? What evidence of reliability and validity exists? What skills and expertise are required for their use? How have they been applied to investigate the relationship between teamwork and other constructs?

Results

Behavioural markers systems are generally designed for specific work domains or tasks. They often cover similar content with inconsistent terminology, which complicates the comparison of research findings across clinical domains. Although several approaches were used to establish the reliability and validity of marker systems, the marker system literature, as a whole, requires more robust reliability and validity evidence. The impact of rater training on rater proficiency was mixed, but evidence suggests that improvements can be made over time.

Conclusions

A consensus of definitions for teamwork constructs must be reached to ensure that the meaning behind behavioural measurement is understood across disciplines, work domains and task types. Future development efforts should focus on the cost effectiveness and feasibility of measurement tools including time spent training raters. Further, standards for the testing and reporting of psychometric evidence must be established. Last, a library of tools should be generated around whether the instrument measures general or domain-specific behaviours.
Hsp25 and Hsp72 content in rat skeletal muscle following controlled shortening and lengthening contractions
Authors: Holwerda Andrew M. , Locke Marius
Published: August 2014

The cytoprotective proteins, Hsp25 and Hsp72, are increased in skeletal muscle after nondamaging, shortening contractions, but the temporal pattern of expression and stimulatory mechanisms remain unclear. Thus, we sought to define the in vivo temporal patterns of expression for Hsp25 and Hsp72 after 2 opposing contractions types. To do this, male Sprague–Dawley rats had 1 tibialis anterior (TA) muscle electrically stimulated (5 sets of 20 repetitions) while being either forcibly lengthened (LC) or shortened (SC). At 2, 8, 24, 48, 72, or 168 h after the contractions both the stimulated and the nonstimulated (contra-lateral control) TA muscles were removed and processed to examine muscle damage (hemotoxylin and eosin staining) and Hsp content (Western blot analyses). Cross-sections from TA muscles subjected to LCs showed muscle fibre damage at 8 h and thereafter. In contrast, no muscle fibre damage was observed at any time point following SCs. When normalized to contra-lateral controls, Hsp25 and Hsp72 content were significantly (P < 0.01) increased at 24 h (3.1- and 3.8-fold, respectively) and thereafter. There were no significant increases in Hsp25 or Hsp72 content at any time point following SC. These data suggest that LCs, but not SCs, result in Hsp accumulation and that the fibre/cellular damage sustained from LCs may be the stimulus for elevating Hsp content.
Acid-base transport in pancreatic cancer: Molecular mechanisms and clinical potential
Authors: Kong Su Chii , Gianuzzo Andrea , Novak Ivana , Pedersen Stine Falsig
Published: August 2014

Solid tumors are characterized by a microenvironment that is highly acidic, while intracellular pH (pHi) is normal or even elevated. This is the result of elevated metabolic rates in the highly proliferative cancer cells, in conjunction with often greatly increased rates of net cellular acid extrusion. Studies in various cancers have suggested that while the acid extrusion mechanisms employed are generally the same as those in healthy cells, the specific transporters upregulated vary with the cancer type. The main such transporters include Na+/H+exchangers, various HCO3−transporters, H+pumps, and lactate-H+cotransporters. The mechanisms leading to their dysregulation in cancer are incompletely understood but include changes in transporter expression levels, trafficking and membrane localization, and posttranslational modifications. In turn, accumulating evidence has revealed that in addition to supporting their elevated metabolic rate, their increased acid efflux capacity endows the cancer cells with increased capacity for invasiveness, proliferation, and chemotherapy resistance. The pancreatic duct exhibits an enormous capacity for acid-base transport, rendering pHidysregulation a potentially very important topic in pancreatic ductal adenocarcinoma (PDAC). PDAC — accounting for about 90% of all pancreatic cancers — has one of the highest cancer mortality rates known, and new diagnostic and treatment options are highly needed. However, very little is known about whether pH regulation is altered in PDAC and, if so, the possible role of this in cancer development. Here, we review current models for pancreatic acid-base transport and pH homeostasis and summarize current views on acid-base dysregulation in cancer, focusing where possible on the few studies to date in PDAC. Finally, we present new data-mining analyses of acid-base transporter expression changes in PDAC and discuss essential directions for future work.