bluuebunny/crossref_metadata_2025 huggingface dataset:
Protein kinase C‐dependent regulation of ClC‐1 channels in active human muscle and its effect on fast and slow gating
Authors: Riisager Anders , de Paoli Frank Vincenzo , Yu Wei‐Ping , Pedersen Thomas Holm , Chen Tsung‐Yu , Nielsen Ole Bækgaard
Published: February 2016

Key points

Regulation of ion channel function during repeated firing of action potentials is commonly observed in excitable cells. Recently it was shown that muscle activity is associated with rapid, protein kinase C (PKC)‐dependent ClC‐1 Cl− channel inhibition in rodent muscle.

While this PKC‐dependent ClC‐1 inhibition during muscle activity was shown to be important for the maintenance of contractile endurance in rat muscle it is unknown whether a similar regulation exists in human muscle.

Also, the molecular mechanisms underlying the observed PKC‐dependent ClC‐1 inhibition are unclear.

Here we present the first demonstration of ClC‐1 inhibition in active human muscle fibres, and we determine the changes in ClC‐1 gating that underlie the PKC‐dependent ClC‐1 inhibition in active muscle using human ClC‐1 expressed in Xenopus oocytes.

This activity‐induced ClC‐1 inhibition is suggested to represent a mechanism by which human muscle fibres maintain their excitability during sustained activity.

Repeated firing of action potentials (APs) is known to trigger rapid, protein kinase C (PKC)‐dependent inhibition of ClC‐1 Cl− ion channels in rodent muscle and this inhibition is important for contractile endurance. It is currently unknown whether similar regulation exists in human muscle, and the molecular mechanisms underlying PKC‐dependent ClC‐1 inhibition are unclear. This study first determined whether PKC‐dependent ClC‐1 inhibition exists in active human muscle, and second, it clarified how PKC alters the gating of human ClC‐1 expressed in Xenopus oocytes. In human abdominal and intercostal muscles, repeated AP firing was associated with 30–60% reduction of ClC‐1 function, which could be completely prevented by PKC inhibition (1 μm GF109203X). The role of the PKC‐dependent ClC‐1 inhibition was evaluated from rheobase currents before and after firing 1000 APs: while rheobase current was well maintained after activity under control conditions it rose dramatically if PKC‐dependent ClC‐1 inhibition had been prevented with the inhibitor. This demonstrates that the ClC‐1 inhibition is important for maintenance of excitability in active human muscle fibres. Oocyte experiments showed that PKC activation lowered the overall open probability of ClC‐1 in the voltage range relevant for AP initiation in muscle fibres. More detailed analysis of this reduction showed that PKC mostly affected the slow gate of ClC‐1. Indeed, there was no effect of PKC activation in C277S mutated ClC‐1 in which the slow gate is effectively locked open. It is concluded that regulation of excitability of active human muscle fibres relies on PKC‐dependent ClC‐1 inhibition via a gating mechanism.
Improved mass resolution and mass accuracy in TOF-SIMS spectra and images using argon gas cluster ion beams
Authors: Shon Hyun Kyong , Yoon Sohee , Moon Jeong Hee , Lee Tae Geol
Published: February 2016

The popularity of argon gas cluster ion beams (Ar-GCIB) as primary ion beams in time-of-flight secondary ion mass spectrometry (TOF-SIMS) has increased because the molecular ions of large organic- and biomolecules can be detected with less damage to the sample surfaces. However, Ar-GCIB is limited by poor mass resolution as well as poor mass accuracy. The inferior quality of the mass resolution in a TOF-SIMS spectrum obtained by using Ar-GCIB compared to the one obtained by a bismuth liquid metal cluster ion beam and others makes it difficult to identify unknown peaks because of the mass interference from the neighboring peaks. However, in this study, the authors demonstrate improved mass resolution in TOF-SIMS using Ar-GCIB through the delayed extraction of secondary ions, a method typically used in TOF mass spectrometry to increase mass resolution. As for poor mass accuracy, although mass calibration using internal peaks with low mass such as hydrogen and carbon is a common approach in TOF-SIMS, it is unsuited to the present study because of the disappearance of the low-mass peaks in the delayed extraction mode. To resolve this issue, external mass calibration, another regularly used method in TOF-MS, was adapted to enhance mass accuracy in the spectrum and image generated by TOF-SIMS using Ar-GCIB in the delayed extraction mode. By producing spectra analyses of a peptide mixture and bovine serum albumin protein digested with trypsin, along with image analyses of rat brain samples, the authors demonstrate for the first time the enhancement of mass resolution and mass accuracy for the purpose of analyzing large biomolecules in TOF-SIMS using Ar-GCIB through the use of delayed extraction and external mass calibration.
Sample size requirements for knowledge‐based treatment planning
Authors: Boutilier Justin J. , Craig Tim , Sharpe Michael B. , Chan Timothy C. Y.
Published: February 2016

Purpose:

To determine how training set size affects the accuracy of knowledge‐based treatment planning (KBP) models.

Methods:

The authors selected four models from three classes of KBP approaches, corresponding to three distinct quantities that KBP models may predict: dose–volume histogram (DVH) points, DVH curves, and objective function weights. DVH point prediction is done using the best plan from a database of similar clinical plans; DVH curve prediction employs principal component analysis and multiple linear regression; and objective function weights uses either logistic regression or K‐nearest neighbors. The authors trained each KBP model using training sets of sizes n = 10, 20, 30, 50, 75, 100, 150, and 200. The authors set aside 100 randomly selected patients from their cohort of 315 prostate cancer patients from Princess Margaret Cancer Center to serve as a validation set for all experiments. For each value of n, the authors randomly selected 100 different training sets with replacement from the remaining 215 patients. Each of the 100 training sets was used to train a model for each value of n and for each KBT approach. To evaluate the models, the authors predicted the KBP endpoints for each of the 100 patients in the validation set. To estimate the minimum required sample size, the authors used statistical testing to determine if the median error for each sample size from 10 to 150 is equal to the median error for the maximum sample size of 200.

Results:

The minimum required sample size was different for each model. The DVH point prediction method predicts two dose metrics for the bladder and two for the rectum. The authors found that more than 200 samples were required to achieve consistent model predictions for all four metrics. For DVH curve prediction, the authors found that at least 75 samples were needed to accurately predict the bladder DVH, while only 20 samples were needed to predict the rectum DVH. Finally, for objective function weight prediction, at least 10 samples were needed to train the logistic regression model, while at least 150 samples were required to train the K‐nearest neighbor methodology.

Conclusions:

In conclusion, the minimum required sample size needed to accurately train KBP models for prostate cancer depends on the specific model and endpoint to be predicted. The authors' results may provide a lower bound for more complicated tumor sites.
Comparing measurement errors for formants in synthetic and natural vowels
Authors: Shadle Christine H. , Nam Hosung , Whalen D. H.
Published: February 2016

The measurement of formant frequencies of vowels is among the most common measurements in speech studies, but measurements are known to be biased by the particular fundamental frequency (F0) exciting the formants. Approaches to reducing the errors were assessed in two experiments. In the first, synthetic vowels were constructed with five different first formant (F1) values and nine different F0 values; formant bandwidths, and higher formant frequencies, were constant. Input formant values were compared to manual measurements and automatic measures using the linear prediction coding-Burg algorithm, linear prediction closed-phase covariance, the weighted linear prediction-attenuated main excitation (WLP-AME) algorithm [Alku, Pohjalainen, Vainio, Laukkanen, and Story (2013). J. Acoust. Soc. Am. 134(2), 1295–1313], spectra smoothed cepstrally and by averaging repeated discrete Fourier transforms. Formants were also measured manually from pruned reassigned spectrograms (RSs) [Fulop (2011). Speech Spectrum Analysis (Springer, Berlin)]. All but WLP-AME and RS had large errors in the direction of the strongest harmonic; the smallest errors occur with WLP-AME and RS. In the second experiment, these methods were used on vowels in isolated words spoken by four speakers. Results for the natural speech show that F0 bias affects all automatic methods, including WLP-AME; only the formants measured manually from RS appeared to be accurate. In addition, RS coped better with weaker formants and glottal fry.
Prediction of break-out sound from a rectangular cavity via an elastically mounted panel
Authors: Wang Gang , Li Wen L. , Du Jingtao , Li Wanyou
Published: February 2016

The break-out sound from a cavity via an elastically mounted panel is predicted in this paper. The vibroacoustic system model is derived based on the so-called spectro-geometric method in which the solution over each sub-domain is invariably expressed as a modified Fourier series expansion. Unlike the traditional modal superposition methods, the continuity of the normal velocities is faithfully enforced on the interfaces between the flexible panel and the (interior and exterior) acoustic media. A fully coupled vibro-acoustic system is obtained by taking into account the strong coupling between the vibration of the elastic panel and the sound fields on the both sides. The typical time-consuming calculations of quadruple integrals encountered in determining the sound power radiation from a panel has been effectively avoided by reducing them, via discrete cosine transform, into a number of single integrals which are subsequently calculated analytically in a closed form. Several numerical examples are presented to validate the system model, understand the effects on the sound transmissions of panel mounting conditions, and demonstrate the dependence on the size of source room of the “measured” transmission loss.
Difference in Pacing Between Time- and Distance-Based Time Trials in Trained Cyclists
Authors: Abbiss Chris R. , Thompson Kevin G. , Lipski Marcin , Meyer Tim , Skorski Sabrina
Published: February 2016

The purpose of this study was to compare the pacing profiles between distance- and duration-based trials of short and long duration. Thirteen trained cyclists completed 2 time-based (6 and 30 min) and 2 distance-based (4 and 20 km) self-paced cycling time trials. Participants were instructed to complete each trial with the highest average power output. Ratings of perceived exertion (RPEs) were measured throughout the trials. Average power output was not different between the 4-km and 6-min trials (324 ± 46 vs 325 ± 45 W; P = .96) or between the 20-km and 30-min trials (271 ± 44 vs 267 ± 38 W; P = .24). Power output was greater on commencement of the distance-based trials when short and long trials were analyzed together. Furthermore, the rate of decline in power output over the 1st 40% of the trial was greater in the 20-km trial than in the 30-min trial (P = .01) but not different between the 4-km and the 6-min trials (P = .13). RPE was greater in the 4-km trial than in the 6-min trial but not different between the 20-km and 30-min trials. These findings indicate that athletes commenced distance-based time trials at relatively higher power outputs than a similar time-based trial. Such findings may result from discrete differences in our ability to judge or predict an exercise endpoint when performing time- and distance-based trials.
Phosphoproteomic analysis of interacting tumor and endothelial cells identifies regulatory mechanisms of transendothelial migration
Authors: Locard-Paulet Marie , Lim Lindsay , Veluscek Giulia , McMahon Kelly , Sinclair John , van Weverwijk Antoinette , Worboys Jonathan D. , Yuan Yinyin , Isacke Clare M. , Jørgensen Claus
Published: February 2016

Mass spectrometry reveals an EPHA2-based mechanism for the regulation of metastatic cancer cell extravasation.
Notch for nucleotide sensing
Authors: VanHook Annalisa M.
Published: February 2016

Notch signaling connects nucleotide availability to germline development in
C
.
elegans
.
Screening of a Library of FDA-Approved Drugs Identifies Several Enterovirus Replication Inhibitors That Target Viral Protein 2C
Authors: Ulferts Rachel , de Boer S. Matthijn , van der Linden Lonneke , Bauer Lisa , Lyoo Hey Rhyoung , Maté Maria J. , Lichière Julie , Canard Bruno , Lelieveld Daphne , Omta Wienand , Egan David , Coutard Bruno , van Kuppeveld Frank J. M.
Published: February 2016

Enteroviruses (EVs) represent many important pathogens of humans. Unfortunately, no antiviral compounds currently exist to treat infections with these viruses. We screened the Prestwick Chemical Library, a library of approved drugs, for inhibitors of coxsackievirus B3, identified pirlindole as a potent novel inhibitor, and confirmed the inhibitory action of dibucaine, zuclopenthixol, fluoxetine, and formoterol. Upon testing of viruses of several EV species, we found that dibucaine and pirlindole inhibited EV-B and EV-D and that dibucaine also inhibited EV-A, but none of them inhibited EV-C or rhinoviruses (RVs). In contrast, formoterol inhibited all enteroviruses and rhinoviruses tested. All compounds acted through the inhibition of genome replication. Mutations in the coding sequence of the coxsackievirus B3 (CV-B3) 2C protein conferred resistance to dibucaine, pirlindole, and zuclopenthixol but not formoterol, suggesting that 2C is the target for this set of compounds. Importantly, dibucaine bound to CV-B3 protein 2C
in vitro
, whereas binding to a 2C protein carrying the resistance mutations was reduced, providing an explanation for how resistance is acquired.
Novel Structures and Temporal Changes of Arginine Catabolic Mobile Elements in Methicillin-Resistant Staphylococcus aureus Genotypes ST5-MRSA-II and ST764-MRSA-II in Japan
Authors: Urushibara Noriko , Kawaguchiya Mitsuyo , Onishi Mayumi , Mise Keiji , Aung Meiji Soe , Kobayashi Nobumichi
Published: February 2016

Twenty-two of 1,103 methicillin-resistant
Staphylococcus aureus
(MRSA) isolates containing the type II staphylococcal cassette chromosome
mec
element (SCC
mec
) (collected in Hokkaido, Japan, from 2008 to 2011) harbored the arginine catabolic mobile element (ACME). Five genetic variations were identified in the ACME-staphylococcal cassette chromosome
mec
composite islands, 66 to 79 kb in size. The percentage of ACME carriage temporally increased from 0.85% to 4.5% in parallel with the emergence of shorter variants (66 to 72 kb). Shorter variants may have a selective advantage and accelerate the dissemination of ACME in Japanese MRSA.
Antimicrobial Resistance in Salmonella in the United States from 1948 to 1995
Authors: Tadesse Daniel A. , Singh Aparna , Zhao Shaohua , Bartholomew Mary , Womack Niketta , Ayers Sherry , Fields Patricia I. , McDermott Patrick F.
Published: February 2016

We conducted a retrospective study of 2,149 clinical
Salmonella
strains to help document the historical emergence of antimicrobial resistance. There were significant increases in resistance to older drugs, including ampicillin, chloramphenicol, streptomycin, sulfamethoxazole, and tetracycline, which were most common in
Salmonella enterica
serotype Typhimurium. An increase in multidrug resistance was observed for each decade since the 1950s. These data help show how
Salmonella
evolved over the past 6 decades, after the introduction of new antimicrobial agents.
Multicenter Prospective Cohort Study of Renal Failure in Patients Treated with Colistin versus Polymyxin B
Authors: Rigatto Maria Helena , Oliveira Maura S. , Perdigão-Neto Lauro V. , Levin Anna S. , Carrilho Claudia M. , Tanita Marcos Toshiyuki , Tuon Felipe F. , Cardoso Douglas E. , Lopes Natane T. , Falci Diego R. , Zavascki Alexandre P.
Published: February 2016

Nephrotoxicity is the main adverse effect of colistin and polymyxin B (PMB). It is not clear whether these two antibiotics are associated with different nephrotoxicity rates. We compared the incidences of renal failure (RF) in patients treated with colistimethate sodium (CMS) or PMB for ≥48 h. A multicenter prospective cohort study was performed that included patients aged ≥18 years. The primary outcome was renal failure (RF) according to Risk, Injury, Failure, Loss, and End-stage renal disease (RIFLE) criteria. Multivariate analysis with a Cox regression model was performed. A total of 491 patients were included: 81 in the CMS group and 410 in the PMB group. The mean daily doses in milligrams per kilogram of body weight were 4.2 ± 1.3 and 2.4 ± 0.73 of colistin base activity and PMB, respectively. The overall incidence of RF was 16.9% (83 patients): 38.3% and 12.7% in the CMS and PMB groups, respectively (
P
< 0.001). In multivariate analysis, CMS therapy was an independent risk factor for RF (hazard ratio, 3.35; 95% confidence interval, 2.05 to 5.48;
P
< 0.001) along with intensive care unit admission, higher weight, older age, and bloodstream and intraabdominal infections. CMS was also independently associated with a higher risk of RF in various subgroup analyses. The incidence of RF was higher in the CMS group regardless of the patient baseline creatinine clearance. The development of RF during therapy was not associated with 30-day mortality in multivariate analysis. CMS was associated with significantly higher rates of RF than those of PMB. Further studies are required to confirm our findings in other patient populations.
Comprehensive Screening for Naturally Occurring Hepatitis C Virus Resistance to Direct-Acting Antivirals in the NS3, NS5A, and NS5B Genes in Worldwide Isolates of Viral Genotypes 1 to 6
Authors: Patiño-Galindo Juan Ángel , Salvatierra Karina , González-Candelas Fernando , López-Labrador F. Xavier
Published: February 2016

There is no comprehensive study available on the natural hepatitis C virus (HCV) polymorphism in sites associated with resistance including all viral genotypes which may present variable susceptibilities to particular direct-acting antivirals (DAAs). This study aimed to analyze the frequencies, genetic barriers, and evolutionary histories of naturally occurring resistance-associated variants (RAVs) in the six main HCV genotypes. A comprehensive analysis of up to 103 RAVs was performed in 2,901, 2,216, and 1,344 HCV isolates for the NS3, NS5A, and NS5B genes, respectively. We report significant intergenotypic differences in the frequencies of natural RAVs for these three HCV genes. In addition, we found a low genetic barrier for the generation of new RAVs, irrespective of the viral genotype. Furthermore, in 1,126 HCV genomes, including sequences spanning the three genes, haplotype analysis revealed a remarkably high frequency of viruses carrying more than one natural RAV to DAAs (53% of HCV-1a, 28.5% of HCV-1b, 67.1% of HCV-6, and 100% of genotype 2, 3, 4, and 5 haplotypes). With the exception of HCV-1a, the most prevalent haplotypes showed RAVs in at least two different viral genes. Finally, evolutionary analyses revealed that, while most natural RAVs appeared recently, others have been efficiently transmitted over time and cluster in well-supported clades. In summary, and despite the observed high efficacy of DAA-based regimens, we show that naturally occurring RAVs are common in all HCV genotypes and that there is an overall low genetic barrier for the selection of resistance mutations. There is a need for natural DAA resistance profiling specific for each HCV genotype.
Antimicrobial Activities of Ceftaroline and Comparator Agents against Bacterial Organisms Causing Bacteremia in Patients with Skin and Skin Structure Infections in U.S. Medical Centers, 2008 to 2014
Authors: Sader Helio S. , Flamm Robert K. , Mendes Rodrigo E. , Farrell David J. , Jones Ronald N.
Published: February 2016

We evaluated the antimicrobial susceptibility of 1,454 organisms consecutively collected from patients with bacteremia associated with skin and skin structure infections. The most common organisms obtained were
Staphylococcus aureus
(670 organisms [46.1%]),
Escherichia coli
(200 organisms [13.8%]), β-hemolytic streptococci (βHS) (138 organisms [9.5%]), and
Klebsiella pneumoniae
(109 organisms [7.5%]). The susceptibility rates for ceftaroline were 97.9% for
S. aureus
(95.9% among methicillin-resistant
S. aureus
[MRSA]), 100.0% for βHS, 86.5% for
E. coli
, and 89.0% for
K. pneumoniae
. Ceftaroline and tigecycline provided the best overall coverage.
Novel Engineered Peptides of a Phage Lysin as Effective Antimicrobials against Multidrug-Resistant Acinetobacter baumannii
Authors: Thandar Mya , Lood Rolf , Winer Benjamin Y. , Deutsch Douglas R. , Euler Chad W. , Fischetti Vincent A.
Published: February 2016

Acinetobacter baumannii
is a Gram-negative bacterial pathogen responsible for a range of nosocomial infections. The recent rise and spread of multidrug-resistant
A. baumannii
clones has fueled a search for alternative therapies, including bacteriophage endolysins with potent antibacterial activities. A common feature of these lysins is the presence of a highly positively charged C-terminal domain with a likely role in promoting outer membrane penetration. In the present study, we show that the C-terminal amino acids 108 to 138 of phage lysin PlyF307, named P307, alone were sufficient to kill
A. baumannii
(>3 logs). Furthermore, P307 could be engineered for improved activity, the most active derivative being P307
SQ-8C
(>5-log kill). Both P307 and P307
SQ-8C
showed high
in vitro
activity against
A. baumannii
in biofilms. Moreover, P307
SQ-8C
exhibited MICs comparable to those of levofloxacin and ceftazidime and acted synergistically with polymyxin B. Although the peptides were shown to kill by disrupting the bacterial cytoplasmic membrane, they did not lyse human red blood cells or B cells; however, serum was found to be inhibitory to lytic activity. In a murine model of
A. baumannii
skin infection, P307
SQ-8C
reduced the bacterial burden by ∼2 logs in 2 h. This study demonstrates the prospect of using peptide derivatives from bacteriophage lysins to treat topical infections and remove biofilms caused by Gram-negative pathogens.
Molecular Characterization of Escherichia coli Strains Isolated from Retail Meat That Harbor
bla
CTX-M
and
fosA3
Genes
Authors: Xie Miaomiao , Lin Dachuan , Chen Kaichao , Chan Edward Wai Chi , Yao Wen , Chen Sheng
Published: February 2016

A total of 55 cefotaxime-resistant
Escherichia coli
isolates were obtained from retail meat products purchased in Shenzhen, China, during the period November 2012 to May 2013. Thirty-seven of these 55 isolates were found to harbor a
bla
CTX-M
gene, with the
bla
CTX-M-1
group being the most common type.
bla
CMY-2
was detected in 16 isolates, alone or in combination with other extended-spectrum β-lactamase (ESBL) determinants. Importantly, the
fosA3
gene, which encodes fosfomycin resistance, was detected in 12 isolates, with several being found to reside in the conjugative plasmid that harbored the
bla
CTX-M
gene. The insertion sequence IS
26
was observed upstream of some of the
bla
CTX-M-55
and
fosA3
genes. Conjugation experiments showed that
bla
CTX-M
genes from 15 isolates were transferrable, with Inc I1 and Inc FII being the most prevalent replicons. High clonal diversity was observed among the
bla
CTX-M
producers, suggesting that horizontal transfer of the
bla
CTX-M
genes among
E. coli
strains in retail meats is a common event and that such strains may constitute an important reservoir of
bla
CTX-M
genes, which may be readily disseminated to other potential human pathogens.
Removal of Dolutegravir by Hemodialysis in HIV-Infected Patients with End-Stage Renal Disease
Authors: Moltó José , Graterol Fredzzia , Miranda Cristina , Khoo Saye , Bancu Ioana , Amara Alieu , Bonjoch Anna , Clotet Bonaventura
Published: February 2016

Data on dolutegravir removal by hemodialysis are lacking. To study this, we measured dolutegravir plasma concentrations in samples of blood entering and leaving the dialyzer and of the resulting dialysate from 5 HIV-infected patients with end-stage renal disease. The median dolutegravir hemodialysis extraction ratio was 7%. The dolutegravir concentrations after the dialysis session remained far above the protein-binding-adjusted inhibitory concentration. Our results show minimal dolutegravir removal by hemodialysis, with no specific dolutegravir dosage adjustments required in this setting. (This study is registered at ClinicalTrials.gov under registration number NCT02487706.)
Differences in Host Cell Invasion and Salmonella Pathogenicity Island 1 Expression between Salmonella enterica Serovar Paratyphi A and Nontyphoidal
S
. Typhimurium
Authors: Elhadad Dana , Desai Prerak , Grassl Guntram A. , McClelland Michael , Rahav Galia , Gal-Mor Ohad
Published: February 2016

Active invasion into nonphagocytic host cells is central to
Salmonella enterica
pathogenicity and dependent on multiple genes within
Salmonella
pathogenicity island 1 (SPI-1). Here, we explored the invasion phenotype and the expression of SPI-1 in the typhoidal serovar
S
. Paratyphi A compared to that of the nontyphoidal serovar
S
. Typhimurium. We demonstrate that while
S
. Typhimurium is equally invasive under both aerobic and microaerobic conditions,
S
. Paratyphi A invades only following growth under microaerobic conditions. Transcriptome sequencing (RNA-Seq), reverse transcription-PCR (RT-PCR), Western blot, and secretome analyses established that
S
. Paratyphi A expresses much lower levels of SPI-1 genes and secretes lesser amounts of SPI-1 effector proteins than
S
. Typhimurium, especially under aerobic growth. Bypassing the native SPI-1 regulation by inducible expression of the SPI-1 activator, HilA, considerably elevated SPI-1 gene expression, host cell invasion, disruption of epithelial integrity, and induction of proinflammatory cytokine secretion by
S
. Paratyphi A but not by
S
. Typhimurium, suggesting that SPI-1 expression is naturally downregulated in
S
. Paratyphi A. Using streptomycin-treated mice, we were able to establish substantial intestinal colonization by
S
. Paratyphi A and showed moderately higher pathology and intestinal inflammation in mice infected with
S
. Paratyphi A overexpressing
hilA
. Collectively, our results reveal unexpected differences in SPI-1 expression between
S
. Paratyphi A and
S
. Typhimurium, indicate that
S
. Paratyphi A host cell invasion is suppressed under aerobic conditions, and suggest that lower invasion in aerobic sites and suppressed expression of immunogenic SPI-1 components contributes to the restrained inflammatory infection elicited by
S
. Paratyphi A.
XerC Contributes to Diverse Forms of Staphylococcus aureus Infection via
agr
-Dependent and
agr
-Independent Pathways
Authors: Atwood Danielle N. , Beenken Karen E. , Loughran Allister J. , Meeker Daniel G. , Lantz Tamara L. , Graham Justin W. , Spencer Horace J. , Smeltzer Mark S.
Published: February 2016

We demonstrate that mutation of
xerC
, which reportedly encodes a homologue of an
Escherichia coli
recombinase, limits biofilm formation in the methicillin-resistant
Staphylococcus aureus
strain LAC and the methicillin-sensitive strain UAMS-1. This was not due to the decreased production of the polysaccharide intracellular adhesin (PIA) in either strain because the amount of PIA was increased in a UAMS-1
xerC
mutant and undetectable in both LAC and its isogenic
xerC
mutant. Mutation of
xerC
also resulted in the increased production of extracellular proteases and nucleases in both LAC and UAMS-1, and limiting the production of either class of enzymes increased biofilm formation in the isogenic
xerC
mutants. More importantly, the limited capacity to form a biofilm was correlated with increased antibiotic susceptibility in both strains in the context of an established biofilm
in vivo
. Mutation of
xerC
also attenuated virulence in a murine bacteremia model, as assessed on the basis of the bacterial loads in internal organs and overall lethality. It also resulted in the decreased accumulation of alpha toxin and the increased accumulation of protein A. These findings suggest that
xerC
may impact the functional status of
agr
. This was confirmed by demonstrating the reduced accumulation of RNAIII and AgrA in LAC and UAMS-1
xerC
mutants. However, this cannot account for the biofilm-deficient phenotype of
xerC
mutants because mutation of
agr
did not limit biofilm formation in either strain. These results demonstrate that
xerC
contributes to biofilm-associated infections and acute bacteremia and that this is likely due to
agr
-independent and -dependent pathways, respectively.
Distinct Contributions of Enzymic Functional Groups to the 2′,3′-Cyclic Phosphodiesterase, 3′-Phosphate Guanylylation, and 3′-ppG/5′-OH Ligation Steps of the Escherichia coli RtcB Nucleic Acid Splicing Pathway
Authors: Maughan William P. , Shuman Stewart
Published: February 2016

Escherichia coli
RtcB is a founding member of a family of manganese-dependent RNA repair enzymes that join RNA 2′,3′-cyclic phosphate (RNA>p) or RNA 3′-phosphate (RNAp) ends to 5′-OH RNA (
HO
RNA) ends in a multistep pathway whereby RtcB (i) hydrolyzes RNA>p to RNAp, (ii) transfers GMP from GTP to RNAp to form to RNAppG, and (iii) directs the attack of 5′-OH on RNAppG to form a 3′-5′ phosphodiester splice junction. The crystal structure of the homologous archaeal RtcB enzyme revealed an active site with two closely spaced manganese ions, Mn1 and Mn2, that interact with the GTP phosphates. By studying the reactions of wild-type
E. coli
RtcB and RtcB alanine mutants with 3′-phosphate-, 2′,3′-cyclic phosphate-, and 3′-ppG-terminated substrates, we found that enzymic constituents of the two metal coordination complexes (Cys78, His185, and His281 for Mn1 and Asp75, Cys78, and His168 for Mn2 in
E. coli
RtcB) play distinct catalytic roles. For example, whereas the C78A mutation abolished all steps assayed, the D75A mutation allowed cyclic phosphodiester hydrolysis but crippled 3′-phosphate guanylylation, and the H281A mutant was impaired in overall
HO
RNAp and
HO
RNA>p ligation but was able to seal a preguanylylated substrate. The archaeal counterpart of
E. coli
RtcB Arg189 coordinates a sulfate anion construed to mimic the position of an RNA phosphate. We propose that Arg189 coordinates a phosphodiester at the 5′-OH end, based on our findings that the R189A mutation slowed the step of RNAppG/
HO
RNA sealing by a factor of 200 compared to that with wild-type RtcB while decreasing the rate of RNAppG formation by only 3-fold.

IMPORTANCE
RtcB enzymes comprise a widely distributed family of manganese- and GTP-dependent RNA repair enzymes that ligate 2′,3′-cyclic phosphate ends to 5′-OH ends via RNA 3′-phosphate and RNA(3′)pp(5′)G intermediates. The RtcB active site includes two adjacent manganese ions that engage the GTP phosphates. Alanine scanning of
Escherichia coli
RtcB reveals distinct contributions of metal-binding residues Cys78, Asp75, and His281 at different steps of the RtcB pathway. The RNA contacts of RtcB are uncharted. Mutagenesis implicates Arg189 in engaging the 5′-OH RNA end.
Lack of the H-NS Protein Results in Extended and Aberrantly Positioned DNA during Chromosome Replication and Segregation in Escherichia coli
Authors: Helgesen Emily , Fossum-Raunehaug Solveig , Skarstad Kirsten
Published: February 2016

The architectural protein H-NS binds nonspecifically to hundreds of sites throughout the chromosome and can multimerize to stiffen segments of DNA as well as to form DNA-protein-DNA bridges. H-NS has been suggested to contribute to the orderly folding of the
Escherichia coli
chromosome in the highly compacted nucleoid. In this study, we investigated the positioning and dynamics of the origins, the replisomes, and the SeqA structures trailing the replication forks in cells lacking the H-NS protein. In H-NS mutant cells, foci of SeqA, replisomes, and origins were irregularly positioned in the cell. Further analysis showed that the average distance between the SeqA structures and the replisome was increased by ∼100 nm compared to that in wild-type cells, whereas the colocalization of SeqA-bound sister DNA behind replication forks was not affected. This result may suggest that H-NS contributes to the folding of DNA along adjacent segments. H-NS mutant cells were found to be incapable of adopting the distinct and condensed nucleoid structures characteristic of
E. coli
cells growing rapidly in rich medium. It appears as if H-NS mutant cells adopt a “slow-growth” type of chromosome organization under nutrient-rich conditions, which leads to a decreased cellular DNA content.

IMPORTANCE
It is not fully understood how and to what extent nucleoid-associated proteins contribute to chromosome folding and organization during replication and segregation in
Escherichia coli
. In this work, we find
in vivo
indications that cells lacking the nucleoid-associated protein H-NS have a lower degree of DNA condensation than wild-type cells. Our work suggests that H-NS is involved in condensing the DNA along adjacent segments on the chromosome and is not likely to tether newly replicated strands of sister DNA. We also find indications that H-NS is required for rapid growth with high DNA content and for the formation of a highly condensed nucleoid structure under such conditions.
Quality gaps identified through mortality review
Authors: Kobewka Daniel M , van Walraven Carl , Turnbull Jeffrey , Worthington James , Calder Lisa , Forster Alan
Published: February 2016

Background

Hospital mortality rate is a common measure of healthcare quality. Morbidity and mortality meetings are common but there are few reports of hospital-wide mortality-review processes to provide understanding of quality-of-care problems associated with patient deaths.

Objective

To describe the implementation and results from an institution-wide mortality-review process.

Design

A nurse and a physician independently reviewed every death that occurred at our multisite teaching institution over a 3-month period. Deaths judged by either reviewer to be unanticipated or to have any opportunity for improvement were reviewed by a multidisciplinary committee. We report characteristics of patients with unanticipated death or opportunity for improved care and summarise the opportunities for improved care.

Results

Over a 3-month period, we reviewed all 427 deaths in our hospital in detail; 33 deaths (7.7%) were deemed unanticipated and 100 (23.4%) were deemed to be associated with an opportunity for improvement. We identified 97 opportunities to improve care. The most common gap in care was: ‘goals of care not discussed or the discussion was inadequate’ (n=25 (25.8%)) and ‘delay or failure to achieve a timely diagnosis’ (n=8 (8.3%)). Patients who had opportunities for improvement had longer length of stay and a lower baseline predicted risk of death in hospital. Nurse and physician reviewers spent approximately 142 h reviewing cases outside of committee meetings.

Conclusions

Our institution-wide mortality review found many quality gaps among decedents, in particular inadequate discussion of goals of care.
Raising the issue of DNAR orders in vascular surgery patients
Authors: McIntosh Rachel , Webb Hannah , Hartley Matthew , Brooks Marcus
Published: February 2016

The Tracey Report has recently raised the status of Do Not Attempt Cardio Pulmonary Resuscitation (DNACPR) orders in the hospital setting.[1] Guidelines are in place both nationally and locally to provide advice to clinicians on when to discuss DNACPR, and the approach to be taken. There was concern that on a busy regional vascular surgery unit, discussion of resuscitation status was not regular practice. Consequently, some patients were at risk of being inappropriately resuscitated, particularly out of hours.

The North Bristol Somerset and Gloucester DNAR decision tree[2] was the tool used to decide whether a patient should have a documented discussion and/or a DNACPR form completed. We correlated the outcome of the decision tree with the presence of a DNACPR form or documented resuscitation discussion. Baseline measurements from all vascular inpatients on the vascular surgery unit demonstrated that only 27% had a DNACPR form or documented discussion in concordance with the DNACPR Decision Tree outcome.

The aim of this project was to increase the proportion of patients with concordance of the DNACPR decision tree outcome with documented discussion or DNACPR form.

The following three simple interventions raised concordance from 27% to 64% of patients on the vascular surgery unit.

1. Including resuscitation status of each patient as a column in the doctors daily handover.

2. Posters in staff only areas to highlight the meaning of DNACPR and raise awareness of the DNACPR decision tree.

3. Educational meeting surrounding DNACPR with the vascular surgery consultants, led by a care of the elderly consultant .

This project has highlighted how raising awareness around DNACPR increases discussion amongst the clinical team surrounding resuscitation status of a patient. Consequently, this enables discussion to be had with patient and their family.
Low stimulus environments: reducing noise levels in continuing care
Authors: Brown Juliette , Fawzi Waleed , Shah Amar , Joyce Margaret , Holt Genevieve , McCarthy Cathy , Stevenson Carmel , Marange Rosca , Shakes Joy , Solomon-Ayeh Kwesi
Published: February 2016

In the low stimulus environment project, we aimed to reduce the levels of intrusive background noise on an older adult mental health ward, combining a very straightforward measure on decibel levels with a downstream measure of reduced distress and agitation as expressed in incidents of violence. This project on reducing background noise levels on older adult wards stemmed from work the team had done on reducing levels of violence and aggression.

We approached the problem using quality improvement methods. Reducing harm to patients and staff is a strategic aim of our Trust and in our efforts we were supported by the Trust’s extensive programme of quality improvement, including training and support provided by the Institute for Healthcare Improvement and the trust’s own Quality Improvement team.

Prior to the project we were running a weekly multi-disciplinary quality improvement group on the ward. We established from this a sub-group to address the specific problem of noise levels and invited carers of people with dementia on our ward to the group. The project was led by nursing staff. We used a noise meter app readily downloadable from the internet to monitor background noise levels on the ward and establish a baseline measure. As a group we used a driver diagram to identify an overall aim and a clear understanding of the major factors that would drive improvements. We also used a staff and carer survey to identify further areas to work on. Change ideas that came from staff and carers included the use of the noise meter to track and report back on noise levels, the use of posters to remind staff about noise levels, the introduction of a visual indication of current noise levels (the Yacker Tracker), the addition of relaxing background music, and adaptations to furniture and environment. We tested many of these over the course of nine months in 2015, using the iterative learning gained from multiple PDSA cycles.

The specific aim was a decrease from above 60dB to below 50dB in background noise on the wards. Following our interventions, we have managed to decrease noise levels on the ward to 53dB on average.

The success of this project to date has relied on the involvement of ward staff and carers - those most affected by the problem - in generating workable local solutions. As many of the change ideas amounted to harm free interventions it was easier for us to make a case to test them out in the real-life setting. Nevertheless we were surprised at how effective such seemingly simple ideas have been in improving the environment on the ward. We have incorporated the change ideas into routine practice and are advising other wards on similar projects.
Improving residents' handovers through just-in-time training for structured communication
Authors: Rourke Liam , Amin Aditi , Boyington Curtiss , Ao Peter , Frolova Natalia
Published: February 2016

In a recent quality assurance project we learned that nearly half of the handovers we examined were characterized as unsatisfactory by our residents, who provided examples in which their anxiety had been piqued and patient care had been affected. These reports substantiated a growing body of literature on the relationship between the quality of handover and the quality of patient care, so we sought to improve the quality and consistency of the in-hosptial handovers undertaken by our internal medicine residents. Senior residents attended morning report for three consecutive month long blocks and evaluated the quality of the handovers using an observational protocol comprised of 16 aspects of effective handover.

During the first block, the resident observed a median of eight of the 16 practices occurring across the 46 handovers, and a large amount of variability. At the beginning of the subsequent block we presented a concise introduction to a structured handover procedure (SBARR). The median quality of the subsequent 33 handovers rose to 11, and the variability decreased considerably. In the next block we refined the SBARR orientation to focus on the errors observed in the previous blocks, and the improvement in the quality and variability was sustained. The minor change, which requires few resources to sustain, had a favourable impact on the quality of our residents' in-hospital handovers.
Reducing the overuse of βhCG measurements in the emergency gynaecology clinic
Authors: Frost Lucy
Published: February 2016

Serial βhCG testing can be a helpful tool in deciding how to manage pregnancy of unknown location. Its use in emergency gynaecology clinics can prevent unnecessary admission and intervention. However, despite NICE Guidelines on when it is safe to opt for conservative management, it was identified that there was a problem with over-testing of βhCG when patients could be discharged with instructions to repeat a urinary pregnancy test in two weeks. Two PDSA cycles were undertaken to improve the awareness of NICE guidelines: the first involved formal and informal educational sessions and the second involved the inclusion of a guideline summary on the front of patients’ notes when they were having serial βhCG tests for doctors to refer to.

Case notes were reviewed for 157 women who had βhCG tests at baseline and 48 hours. Of these, 139 were suitable for serial βhCG testing, and 83 of these were suitable for discharge after 48 hours. Of the 83 patients that were eligible for discharge, there were 31 unnecessary βhCG tests done, 23 of which were prior to intervention. A significant improvement was noted, with between 4-10 unnecessary βhCG tests per fortnight prior to intervention, 0-3 following the first intervention, and 0-2 following the second. Reduction in unnecessary βhCG testing has positive implications for patients, who do not have to take unnecessary time off work, prolong an already very distressing period, and have unnecessary blood tests. There are also cost and time saving implications for the hospital.
Empathy and affect: what can empathied bodies do?
Authors: Marshall George Robert Ellison , Hooker Claire
Published: February 2016

While there has been much interest in the apparent benefits of empathy in improving outcomes of medical care, there is continuing concern over the philosophical nature of empathy. We suggest that part of the difficulty in coming to terms with empathy is due to the modernist dichotomies that have structured Western medical discourse, such that doctor and patient, knower and known, cognitive and emotional, subject and object are situated in oppositional terms, with the result that such accounts cannot coherently encompass an emotional doctor, or a patient as knower, or empathy as other than a possession or a trait. This paper explores what, by contrast, a radical critique of the Cartesian world view, in the form of a Deleuzean theoretical framework, would open up in new perspectives on empathy. We extend the framework of emotional geography to askwhat happenswhen people areaffectedby empathy. We suggest that doctors and patients might be more productively understood as embodied subjects that areconfigured in their capacitiesby how they are affected by singular ‘events’ of empathy. We sketch out how the Deleuzean framework would make sense of these contentions and identify some possible implications for medical education and practice.
A fatal complication arising during pulmonic balloon valvuloplasty in a seven‐month‐old female bulldog
Authors: Thompson Kate , Pedro Brigite
Published: February 2016

A seven‐month‐old female bulldog with severe valvular pulmonic stenosis and ventricular septal defect presented for general anaesthesia for repeat balloon valvuloplasty. The first balloon valvuloplasty procedure was not successful. The second balloon valvuloplasty was effective but was associated with cardiovascular collapse, which was unresponsive to atropine and epinephrine administration and progressed to asystole. Cardiopulmonary resuscitation was unsuccessful. Anaesthetic management of dogs undergoing balloon valvuloplasty is challenging. Selection of anaesthetic technique must consider existing cardiac pathology, common intraprocedural and postprocedural complications and their management options. This case report describes a fatal intraoperative complication of balloon valvuloplasty unresponsive to anticholinergic and sympathomimetic treatment and discusses potential aetiologies of the complication and reasons for treatment failure.
Prey size and availability limits maximum size of rainbow trout in a large tailwater: insights from a drift-foraging bioenergetics model
Authors: Dodrill Michael J. , Yackulic Charles B. , Kennedy Theodore A. , Hayes John W.
Published: February 2016

The cold and clear water conditions present below many large dams create ideal conditions for the development of economically important salmonid fisheries. Many of these tailwater fisheries have experienced declines in the abundance and condition of large trout species, yet the causes of these declines remain uncertain. Here, we develop, assess, and apply a drift-foraging bioenergetics model to identify the factors limiting rainbow trout (Oncorhynchus mykiss) growth in a large tailwater. We explored the relative importance of temperature, prey quantity, and prey size by constructing scenarios where these variables, both singly and in combination, were altered. Predicted growth matched empirical mass-at-age estimates, particularly for younger ages, demonstrating that the model accurately describes how current temperature and prey conditions interact to determine rainbow trout growth. Modeling scenarios that artificially inflated prey size and abundance demonstrate that rainbow trout growth is limited by the scarcity of large prey items and overall prey availability. For example, shifting 10% of the prey biomass to the 13 mm (large) length class, without increasing overall prey biomass, increased lifetime maximum mass of rainbow trout by 88%. Additionally, warmer temperatures resulted in lower predicted growth at current and lower levels of prey availability; however, growth was similar across all temperatures at higher levels of prey availability. Climate change will likely alter flow and temperature regimes in large rivers with corresponding changes to invertebrate prey resources used by fish. Broader application of drift-foraging bioenergetics models to build a mechanistic understanding of how changes to habitat conditions and prey resources affect growth of salmonids will benefit management of tailwater fisheries.