bluuebunny/crossref_metadata_2025_split huggingface dataset:
A volatile discourse – reviewing aspects of ammonia emissions, models and atmospheric concentrations in The Netherlands
Authors: Hanekamp J. C. , Briggs W. M. , Crok M. 
Published: June 2017

AbstractIn the Netherlands, there is a vigorous debate on ammonia emissions, atmospheric concentrations and deposition between stakeholders and research institutions. In this article, we scrutinise some aspects of the ammonia discourse. In particular, we want to improve the understanding of the methodology for handling experimentally determined ammonia emissions. We show that uncertainty in published results is substantial. This uncertainty is under‐ or even unreported, and as a result, data in national emission inventories are overconfident by a wide margin. Next, we demonstrate that the statistical handling of data on atmospheric ammonia concentrations to produce national yearly atmospheric averages is oversimplified and consequently atmospheric concentrations are substantially overestimated. Finally, we show that the much‐discussed ‘ammonia gap’ – either the discrepancy between calculated and measured atmospheric ammonia concentrations or the difference observed between estimated NH3 emission levels and those indicated by atmospheric measurements – is an expression of the widespread overconfidence placed in atmospheric modelling.
Genome‐wide analysis of specific alterations in transcript structure and accumulation caused by nutrient deficiencies in Arabidopsis thaliana
Authors: Nishida Sho , Kakei Yusuke , Shimada Yukihisa , Fujiwara Toru 
Published: June 2017

SummaryThe alteration of transcript structure contributes to transcriptome plasticity. In this study, we analyzed the genome‐wide response of exon combination patterns to deficiencies in 12 different nutrients in Arabidopsis thaliana roots. RNA sequencing analysis and bioinformatics using a simulation survey revealed more than 600 genes showing varying exon combinations. The overlap between genes showing differential expression (DE) and genes showing differential exon combination (DC) was notably low. Additionally, gene ontology analysis showed that gene functions were not shared between the DE and DC genes, suggesting that the genes showing DC had different roles than those showing DE. Most of the DC genes were nutrient specific. For example, two homologs of the MYB transcription factor genes MYB48 and MYB59 showed differential alternative splicing only in response to low levels of potassium. Alternative splicing of those MYB genes modulated DNA‐binding motifs, and MYB59 is reportedly involved in the inhibition of root elongation. Therefore, the increased abundance of MYB isoforms with an intact DNA‐binding motif under low potassium may be involved in the active inhibition of root elongation. Overall, we provide global and comprehensive data for DC genes affected by nutritional deficiencies, which contribute to elucidating an unknown mechanism involved in adaptation to nutrient deficiency.
Development and testing of a stepwise thaw and dilute protocol for cryopreserved umbilical cord blood units
Authors: Pasha Roya , Elmoazzen Heidi , Pineault Nicolas 
Published: June 2017

BACKGROUNDIt is clinically important to maintain high viability and potency of umbilical cord blood units (CBUs) for transplantation during thawing. In the absence of a standard thawing protocol, this study was designed to develop one based on the consensus practice of transplant centers and address the shortage of dextran 40 thawing solution.STUDY DESIGN AND METHODSFrozen CBU aliquots were thawed using dextran 40 thawing solution while manipulating temperature and volume of diluent and mode of dilution. The effects of these on CD45+ and CD34+ cell viability were measured through annexin V and SYTOX green staining. The developed protocol was then used to compare dextran 40 and PLASMA‐LYTE A thawing solutions and finally tested on whole CBUs.RESULTSStep‐by‐step investigations resulted in the development of a protocol that thaws and dilutes CBUs with room temperature diluent to five times the original volume using two sequential dilutions separated by equilibration times. PLASMA‐LYTE A diluent provided superior viability of CD45+ and CD34+ cells than dextran 40 and recovered more colony‐forming units. However, both diluents were equally effective in maintaining stability of the thawed CBU for 4 hours. Moreover, the stem cell–enriched CD34+CD38– subpopulations appeared more resistant to cryoinjuries than their CD34+CD38+ counterpart.CONCLUSIONThe developed thawing protocol recovers viable CD45+ and CD34+ cells above the standard thresholds and maintains CBU potency. PLASMA‐LYTE A for thawing solution proved to be an efficient alternative to dextran 40. Finally, greater dilution should be avoided to maintain the viability of CD45+ cells and maximize graft cell dose.
Peptide Inhibitors Targeting the Neisseria gonorrhoeae Pivotal Anaerobic Respiration Factor AniA
Authors: Sikora Aleksandra E. , Mills Robert H. , Weber Jacob V. , Hamza Adel , Passow Bryan W. , Romaine Andrew , Williamson Zachary A. , Reed Robert W. , Zielke Ryszard A. , Korotkov Konstantin V. 
Published: June 2017

ABSTRACT

Neisseria gonorrhoeae
causes the sexually transmitted infection gonorrhea, which is highly prevalent worldwide and has a major impact on reproductive and neonatal health. The superbug status of
N. gonorrhoeae
necessitates the development of drugs with different mechanisms of action. Here, we focused on targeting the nitrite reductase AniA, which is a pivotal component of
N. gonorrhoeae
anaerobic respiration and biofilm formation. Our studies showed that gonococci expressing AniA containing the altered catalytic residues D137A and H280A failed to grow under anaerobic conditions, demonstrating that the nitrite reductase function is essential. To facilitate the pharmacological targeting of AniA, new crystal structures of AniA were refined to 1.90-Å and 2.35-Å resolutions, and a phage display approach with libraries expressing randomized linear dodecameric peptides or heptameric peptides flanked by a pair of cysteine residues was utilized. Biopanning experiments led to the identification of 29 unique peptides, with 1 of them, C7-3, being identified multiple times. Evaluation of their ability to interact with AniA using enzyme-linked immunosorbent assay and computational docking studies revealed that C7-3 was the most promising inhibitor, binding near the type 2 copper site of the enzyme, which is responsible for interaction with nitrite. Subsequent enzymatic assays and biolayer interferometry with a synthetic C7-3 and its derivatives, C7-3m1 and C7-3m2, demonstrated potent inhibition of AniA. Finally, the MIC
50
value of C7-3 and C7-3m2 against anaerobically grown
N. gonorrhoeae
was 0.6 mM. We present the first peptide inhibitors of AniA, an enzyme that should be further exploited for antigonococcal drug development.
Role of AxyZ Transcriptional Regulator in Overproduction of AxyXY-OprZ Multidrug Efflux System in Achromobacter Species Mutants Selected by Tobramycin
Authors: Bador Julien , Neuwirth Catherine , Grangier Nadège , Muniz Marie , Germé Leslie , Bonnet Jérémy , Pillay Vignesh-Guru , Llanes Catherine , de Curraize Claire , Amoureux Lucie 
Published: June 2017

ABSTRACT

AxyXY-OprZ is an RND-type efflux system that confers innate aminoglycoside resistance to
Achromobacter
spp. We investigated here a putative TetR family transcriptional regulator encoded by the
axyZ
gene located upstream of
axyXY-oprZ
. An in-frame
axyZ
gene deletion assay led to increased MICs of antibiotic substrates of the efflux system, including aminoglycosides, cefepime, fluoroquinolones, tetracyclines, and erythromycin, indicating that the product of
axyZ
negatively regulates expression of
axyXY-oprZ
. Moreover, we identified an amino acid substitution at position 29 of AxyZ (V29G) in a clinical
Achromobacter
strain that occurred during the course of chronic respiratory tract colonization in a cystic fibrosis (CF) patient. This substitution, also detected in three other strains exposed
in vitro
to tobramycin, led to an increase in the
axyY
transcription level (5- to 17-fold) together with an increase in antibiotic resistance level. This overproduction of AxyXY-OprZ is the first description of antibiotic resistance acquisition due to modification of a chromosomally encoded mechanism in
Achromobacter
and might have an impact on the management of infected CF patients. Indeed, tobramycin is widely used for aerosol therapy within this population, and we have demonstrated that it easily selects mutants with increased MICs of not only aminoglycosides but also fluoroquinolones, cefepime, and tetracyclines.
Multimechanistic Monoclonal Antibodies (MAbs) Targeting Staphylococcus aureus Alpha-Toxin and Clumping Factor A: Activity and Efficacy Comparisons of a MAb Combination and an Engineered Bispecific Antibody Approach
Authors: Tkaczyk C. , Kasturirangan S. , Minola A. , Jones-Nelson O. , Gunter V. , Shi Y. Y. , Rosenthal K. , Aleti V. , Semenova E. , Warrener P. , Tabor D. , Stover C. K. , Corti D. , Rainey G. , Sellman B. R. 
Published: June 2017

ABSTRACT

Secreted alpha-toxin and surface-localized clumping factor A (ClfA) are key virulence determinants in
Staphylococcus aureus
bloodstream infections. We previously demonstrated that prophylaxis with a multimechanistic monoclonal antibody (MAb) combination against alpha-toxin (MEDI4893*) and ClfA (11H10) provided greater strain coverage and improved efficacy in an
S. aureus
lethal bacteremia model. Subsequently, 11H10 was found to exhibit reduced affinity and impaired inhibition of fibrinogen binding to ClfA002 expressed by members of a predominant hospital-associated methicillin-resistant
S. aureus
(MRSA) clone, ST5. Consequently, we identified another anti-ClfA MAb (SAR114) from human tonsillar B cells with >100-fold increased affinity for three prominent ClfA variants, including ClfA002, and potent inhibition of bacterial agglutination by 112 diverse clinical isolates. We next constructed bispecific Abs (BiSAbs) comprised of 11H10 or SAR114 as IgG scaffolds and grafted anti-alpha-toxin (MEDI4893*) single-chain variable fragment to the amino or carboxy terminus of the anti-ClfA heavy chains. Although the BiSAbs exhibited
in vitro
potencies similar to those of the parental MAbs, only 11H10-BiSAb, but not SAR114-BiSAb, showed protective activity in murine infection models comparable to the respective MAb combination.
In vivo
activity with SAR114-BiSAb was observed in infection models with
S. aureus
lacking ClfA. Our data suggest that high-affinity binding to ClfA sequesters the SAR114-BiSAb to the bacterial surface, thereby reducing both alpha-toxin neutralization and protection
in vivo
. These results indicate that a MAb combination targeting ClfA and alpha-toxin is more promising for future development than the corresponding BiSAb.
Linezolid Dose That Maximizes Sterilizing Effect While Minimizing Toxicity and Resistance Emergence for Tuberculosis
Authors: Srivastava Shashikant , Magombedze Gesham , Koeuth Thearith , Sherman Carleton , Pasipanodya Jotam G. , Raj Prithvi , Wakeland Edward , Deshpande Devyani , Gumbo Tawanda 
Published: June 2017

ABSTRACT

Linezolid has an excellent sterilizing effect in tuberculosis patients but high adverse event rates. The dose that would maximize efficacy and minimize toxicity is unknown. We performed linezolid dose-effect and dose-scheduling studies in the hollow fiber system model of tuberculosis (HFS-TB) for sterilizing effect. HFS-TB units were treated with several doses to mimic human-like linezolid intrapulmonary pharmacokinetics and repetitively sampled for drug concentration, total bacterial burden, linezolid-resistant subpopulations, and RNA sequencing over 2 months. Linezolid-resistant isolates underwent whole-genome sequencing. The expression of genes encoding efflux pumps in the first 1 to 2 weeks revealed the same exposure-response patterns as the linezolid-resistant subpopulation. Linezolid-resistant isolates from the 2nd month of therapy revealed mutations in several efflux pump/transporter genes and a LuxR-family transcriptional regulator. Linezolid sterilizing effect was linked to the ratio of unbound 0- to 24-h area under the concentration-time curve (AUC
0–24
) to MIC. Optimal microbial kill was achieved at an AUC
0–24
/MIC ratio of 119. The optimal sterilizing effect dose for clinical use was identified using Monte Carlo simulations. Clinical doses of 300 and 600 mg/day (or double the dose every other day) achieved this target in 87% and >99% of 10,000 patients, respectively. The susceptibility breakpoint identified was 2 mg/liter. The simulations identified that a 300-mg/day dose did not achieve AUC
0–24
s associated with linezolid toxicity, while 600 mg/day achieved those AUC
0–24
s in <20% of subjects. The linezolid dose of 300 mg/day performed well and should be compared to 600 mg/day or 1,200 mg every other day in clinical trials.
Use of β-Lactam/β-Lactamase Inhibitors for Extended-Spectrum-β-Lactamase Infections: Defining the Right Patient Population
Authors: Tamma Pranita D. , Villegas Maria Virginia 
Published: June 2017

ABSTRACT

In a multicenter, multinational observational study that included neutropenic patients with bloodstream infections by extended-spectrum-β-lactamase-producing species, Gudiol et al. (Antimicrob. Agents Chemother. 61:e00164-17, 2017,
https://doi.org/10.1128/AAC.00164-17
) demonstrated that β-lactam/β-lactamase inhibitors are effective treatment options. A review of this work, however, reminds us that some lingering questions remain for specific high-risk subgroups.
PA3225 Is a Transcriptional Repressor of Antibiotic Resistance Mechanisms in Pseudomonas aeruginosa
Authors: Hall Clayton W. , Zhang Li , Mah Thien-Fah 
Published: June 2017

ABSTRACT

The
tssABC1
locus is part of the Hcp secretion island I (HSI-I) type VI secretion system (T6SS) in
Pseudomonas aeruginosa
. Previous work implicated the
tssC1
gene in
P. aeruginosa
biofilm-specific antibiotic resistance, and
tssC1
is preferentially expressed in biofilms compared to planktonic cells. Using a DNA-dependent protein pulldown approach, we discovered that PA3225, an uncharacterized LysR-type transcriptional regulator, specifically bound to the
tssABC1
upstream regulatory region. The deletion of PA3225 led to a 2-fold decrease in
tssA1
expression levels in planktonic cells compared to the wild type, and
tssA1
expression was slightly reduced in ΔPA3225 biofilms compared to wild-type biofilms. Intriguingly, further investigations revealed that the ΔPA3225 mutant was less susceptible to multiple, structurally unrelated antibiotics with various mechanisms of action when grown planktonically. The ΔPA3225 mutant was additionally more resistant to ciprofloxacin when grown in a biofilm. The decreased antibiotic susceptibility of the ΔPA3225 strain was linked to the transcriptional upregulation of the MexAB-OprM efflux pump. By using transcriptome sequencing (RNA-seq), other PA3225-regulated genes were identified, and the products of these genes, such as the putative ABC transporter PA3228, may also contribute to antibiotic resistance.
Safety, Tolerability, Systemic Exposure, and Metabolism of CRS3123, a Methionyl-tRNA Synthetase Inhibitor Developed for Treatment of Clostridium difficile, in a Phase 1 Study
Authors: Nayak Seema U. , Griffiss J. McLeod , Blumer Jeffrey , O'Riordan Mary Ann , Gray Wesley , McKenzie Robin , Jurao Robert A. , An Amanda T. , Le Melissa , Bell Stacie J. , Ochsner Urs A. , Jarvis Thale C. , Janjic Nebojsa , Zenilman Jonathan M. 
Published: June 2017

ABSTRACT

Clostridium difficile
causes antibiotic-associated diarrhea and is a major public health concern. Current therapies disrupt the protective intestinal flora, do not reliably prevent recurrent infections, and will be decreasingly effective should less susceptible strains emerge. CRS3123 is an oral agent that inhibits bacterial methionyl-tRNA synthetase and has potent activity against
C. difficile
and aerobic Gram-positive bacteria but little activity against Gram-negative bacteria, including anaerobes. This first-in-human, double-blind, placebo-controlled, dose escalation study evaluated the safety and systemic exposure of CRS3123 after a single oral dose in healthy adults. Five cohorts of eight subjects each received CRS3123 or placebo in a 3:1 ratio. Doses for the respective active arms were 100 mg, 200 mg, 400 mg, 800 mg, and 1,200 mg. Blood and urine were collected for pharmacokinetic analysis. CRS3123 concentrations were measured with validated LC-MS/MS techniques. There were no serious adverse events or immediate allergic reactions during administration of CRS3123. In the CRS3123-treated groups, the most frequent adverse events were decreased hemoglobin, headache, and abnormal urine analysis; all adverse events in the active-treatment groups were mild to moderate, and their frequency did not increase with dose. Although CRS3123 systemic exposure increased at higher doses, the increase was less than dose proportional. The absorbed drug was glucuronidated at reactive amino groups on the molecule, which precluded accurate pharmacokinetic analysis of the parent drug. Overall, CRS3123 was well tolerated over this wide range of doses. This safety profile supports further investigation of CRS3123 as a treatment for
C. difficile
infections. (This study has been registered at ClinicalTrials.gov under identifier NCT01551004.)
Chlamydia muridarum with Mutations in Chromosomal Genes
tc0237
and/or
tc0668
Is Deficient in Colonizing the Mouse Gastrointestinal Tract
Authors: Shao Lili , Zhang Tianyuan , Liu Quanzhong , Wang Jie , Zhong Guangming 
Published: June 2017

ABSTRACT

Chlamydiae colonize the gastrointestinal tracts of both animals and humans. However, their medical significance remains unknown. We have previously shown that wild-type
Chlamydia muridarum
spreads to and establishes stable colonization of the gastrointestinal tract following intravaginal inoculation. In the present study, we found that
C. muridarum
with mutations in chromosomal genes
tc0237
and/or
tc0668
was defective in spreading to the mouse gastrointestinal tract, which correlated with its attenuated pathogenicity in the upper genital tract. This correlation was more consistent than that of chlamydial pathogenicity with ascending infection in the genital tract, since attenuated
C. muridarum
spread significantly less to the gastrointestinal tract but maintained robust ascending infection of the upper genital tract. Transcervical inoculation further confirmed the correlation between
C. muridarum
spreading to the gastrointestinal tract and its pathogenicity in the upper genital tract. Finally, defective spreading of
C. muridarum
mutants was due to their inability to colonize the gastrointestinal tract since intragastric inoculation did not rescue the mutants' colonization. Thus, promoting
C. muridarum
colonization of the gastrointestinal tract may represent a primary function of the TC0237 and TC0668 proteins. Correlation of chlamydial colonization of the gastrointestinal tract with chlamydial pathogenicity in the upper genital tract suggests a potential role for gastrointestinal chlamydiae in genital tract pathogenicity.
Cyclic AMP-Independent Control of Twitching Motility in Pseudomonas aeruginosa
Authors: Buensuceso Ryan N. C. , Daniel-Ivad Martin , Kilmury Sara L. N. , Leighton Tiffany L. , Harvey Hanjeong , Howell P. Lynne , Burrows Lori L. 
Published: June 2017

ABSTRACT

FimV is a
Pseudomonas aeruginosa
inner membrane hub protein that modulates levels of the second messenger, cyclic AMP (cAMP), through the activation of adenylate cyclase CyaB. Although type IVa pilus (T4aP)-dependent twitching motility is modulated by cAMP levels, mutants lacking FimV are twitching impaired, even when exogenous cAMP is provided. Here we further define FimV's cAMP-dependent and -independent regulation of twitching. We confirmed that the response regulator of the T4aP-associated Chp chemotaxis system, PilG, requires both FimV and the CyaB regulator, FimL, to activate CyaB. However, in cAMP-replete backgrounds—lacking the cAMP phosphodiesterase CpdA or the CheY-like protein PilH or expressing constitutively active CyaB—
pilG
and
fimV
mutants failed to twitch. Both cytoplasmic and periplasmic domains of FimV were important for its cAMP-dependent and -independent roles, while its septal peptidoglycan-targeting LysM motif was required only for twitching motility. Polar localization of the sensor kinase PilS, a key regulator of transcription of the major pilin, was FimV dependent. However, unlike its homologues in other species that localize flagellar system components, FimV was not required for swimming motility. These data provide further evidence to support FimV's role as a key hub protein that coordinates the polar localization and function of multiple structural and regulatory proteins involved in
P. aeruginosa
twitching motility.

IMPORTANCE
Pseudomonas aeruginosa
is a serious opportunistic pathogen. Type IVa pili (T4aP) are important for its virulence, because they mediate dissemination and invasion via twitching motility and are involved in surface sensing, which modulates pathogenicity via changes in cAMP levels. Here we show that the hub protein FimV and the response regulator of the Chp system, PilG, regulate twitching independently of their roles in the modulation of cAMP synthesis. These functions do not require the putative scaffold protein FimL, proposed to link PilG with FimV. PilG may regulate asymmetric functioning of the T4aP system to allow for directional movement, while FimV appears to localize both structural and regulatory elements—including the PilSR two-component system—to cell poles for optimal function.