bluuebunny/crossref_metadata_2025_split huggingface dataset:
Development of a Real-Time Reverse Transcription-PCR Assay for Global Differentiation of Yellow Fever Virus Vaccine-Related Adverse Events from Natural Infections
Authors: Hughes Holly R. , Russell Brandy J. , Mossel Eric C. , Kayiwa John , Lutwama Julius , Lambert Amy J. 
Published: April 2018

ABSTRACT
Yellow fever (YF) is a reemerging public health threat, with frequent outbreaks prompting large vaccination campaigns in regions of endemicity in Africa and South America. Specific detection of vaccine-related adverse events is resource-intensive, time-consuming, and difficult to achieve during an outbreak. To address this, we have developed a highly transferable rapid yellow fever virus (YFV) vaccine-specific real-time reverse transcription-PCR (RT-PCR) assay that distinguishes vaccine from wild-type lineages. The assay utilizes a specific hydrolysis probe that includes locked nucleic acids to enhance specific discrimination of the YFV17D vaccine strain genome. Promisingly, sensitivity and specificity analyses reveal this assay to be highly specific to vaccine strain(s) when tested on clinical samples and YFV cell culture isolates of global origin. Taken together, our data suggest the utility of this assay for use in laboratories of varied capacity for the identification and differentiation of vaccine-related adverse events from wild-type infections of both African and South American origin.
Broad-Spectrum Regulation of Nonreceptor Tyrosine Kinases by the Bacterial ADP-Ribosyltransferase EspJ
Authors: Pollard Dominic J. , Berger Cedric N. , So Ernest C. , Yu Lu , Hadavizadeh Kate , Jennings Patricia , Tate Edward W. , Choudhary Jyoti S. , Frankel Gad 
Published: April 2018

ABSTRACT

Tyrosine phosphorylation is key for signal transduction from exogenous stimuli, including the defense against pathogens. Conversely, pathogens can subvert protein phosphorylation to control host immune responses and facilitate invasion and dissemination. The bacterial effectors EspJ and SeoC are injected into host cells through a type III secretion system by enteropathogenic and enterohemorrhagic
Escherichia coli
(EPEC and EHEC, respectively),
Citrobacter rodentium
, and
Salmonella enterica
, where they inhibit Src kinase by coupled amidation and ADP-ribosylation.
C. rodentium
, which is used to model EPEC and EHEC infections in humans, is a mouse pathogen triggering colonic crypt hyperplasia (CCH) and colitis. Enumeration of bacterial shedding and CCH confirmed that EspJ affects neither tolerance nor resistance to infection. However, comparison of the proteomes of intestinal epithelial cells isolated from mice infected with wild-type
C. rodentium
or
C. rodentium
encoding catalytically inactive EspJ revealed that EspJ-induced ADP-ribosylation regulates multiple nonreceptor tyrosine kinases
in vivo
. Investigation of the substrate repertoire of EspJ revealed that in HeLa and A549 cells, Src and Csk were significantly targeted; in polarized Caco2 cells, EspJ targeted Src and Csk and the Src family kinase (SFK) Yes1, while in differentiated Thp1 cells, EspJ modified Csk, the SFKs Hck and Lyn, the Tec family kinases Tec and Btk, and the adapter tyrosine kinase Syk. Furthermore, Abl (HeLa and Caco2) and Lyn (Caco2) were enriched specifically in the EspJ-containing samples. Biochemical assays revealed that EspJ, the only bacterial ADP-ribosyltransferase that targets mammalian kinases, controls immune responses and the Src/Csk signaling axis.

IMPORTANCE
Enteropathogenic and enterohemorrhagic
Escherichia coli
(EPEC and EHEC, respectively) strains cause significant mortality and morbidity worldwide.
Citrobacter rodentium
is a mouse pathogen used to model EPEC and EHEC pathogenesis
in vivo
. Diarrheal disease is triggered following injection of bacterial effectors, via a type III secretion system (T3SS), into intestinal epithelial cells (IECs). While insights into the role of the effectors were historically obtained from pathological, immunologic, or cell culture phenotypes, subtle roles of individual effectors
in vivo
are often masked. The aim of this study was to elucidate the role and specificity of the ADP-ribosyltransferase effector EspJ. For the first time, we show that the
in vivo
processes affected by a T3SS effector can be studied by comparing the proteomes of IECs extracted from mice infected with wild-type
C. rodentium
or an
espJ
catalytic mutant. We show that EspJ, the only bacterial ADP-ribosyltransferase that targets mammalian kinases, regulates the host immune response
in vivo
.
A Biofilm Matrix-Associated Protease Inhibitor Protects Pseudomonas aeruginosa from Proteolytic Attack
Authors: Tseng Boo Shan , Reichhardt Courtney , Merrihew Gennifer E. , Araujo-Hernandez Sophia A. , Harrison Joe J. , MacCoss Michael J. , Parsek Matthew R. 
Published: April 2018

ABSTRACT

Pseudomonas aeruginosa
produces an extracellular biofilm matrix that consists of nucleic acids, exopolysaccharides, lipid vesicles, and proteins. In general, the protein component of the biofilm matrix is poorly defined and understudied relative to the other major matrix constituents. While matrix proteins have been suggested to provide many functions to the biofilm, only proteins that play a structural role have been characterized thus far. Here we identify proteins enriched in the matrix of
P. aeruginosa
biofilms. We then focused on a candidate matrix protein, the serine protease inhibitor ecotin (PA2755). This protein is able to inhibit neutrophil elastase, a bactericidal enzyme produced by the host immune system during
P. aeruginosa
biofilm infections. We show that ecotin binds to the key biofilm matrix exopolysaccharide Psl and that it can inhibit neutrophil elastase when associated with Psl. Finally, we show that ecotin protects both planktonic and biofilm
P. aeruginosa
cells from neutrophil elastase-mediated killing. This may represent a novel mechanism of protection for biofilms to increase their tolerance against the innate immune response.

IMPORTANCE
Proteins associated with the extracellular matrix of bacterial aggregates called biofilms have long been suggested to provide many important functions to the community. To date, however, only proteins that provide structural roles have been described, and few matrix-associated proteins have been identified. We developed a method to identify matrix proteins and characterized one. We show that this protein, when associated with the biofilm matrix, can inhibit a bactericidal enzyme produced by the immune system during infection and protect biofilm cells from death induced by the enzyme. This may represent a novel mechanism of protection for biofilms, further increasing their tolerance against the immune response. Together, our results are the first to show a nonstructural function for a confirmed matrix-interacting protein.
Extracellular Electron Transfer Powers Enterococcus faecalis Biofilm Metabolism
Authors: Keogh Damien , Lam Ling Ning , Doyle Lucinda E. , Matysik Artur , Pavagadhi Shruti , Umashankar Shivshankar , Low Pui Man , Dale Jennifer L. , Song Yiyang , Ng Sean Pin , Boothroyd Chris B. , Dunny Gary M. , Swarup Sanjay , Williams Rohan B. H. , Marsili Enrico , Kline Kimberly A. 
Published: April 2018

ABSTRACT

Enterococci are important human commensals and significant opportunistic pathogens. Biofilm-related enterococcal infections, such as endocarditis, urinary tract infections, wound and surgical site infections, and medical device-associated infections, often become chronic upon the formation of biofilm. The biofilm matrix establishes properties that distinguish this state from free-living bacterial cells and increase tolerance to antimicrobial interventions. The metabolic versatility of the enterococci is reflected in the diversity and complexity of environments and communities in which they thrive. Understanding metabolic factors governing colonization and persistence in different host niches can reveal factors influencing the transition to biofilm pathogenicity. Here, we report a form of iron-dependent metabolism for
Enterococcus faecalis
where, in the absence of heme, extracellular electron transfer (EET) and increased ATP production augment biofilm growth. We observe alterations in biofilm matrix depth and composition during iron-augmented biofilm growth. We show that the
ldh
gene encoding
l
-lactate dehydrogenase is required for iron-augmented energy production and biofilm formation and promotes EET.

IMPORTANCE
Bacterial metabolic versatility can often influence the outcome of host-pathogen interactions, yet causes of metabolic shifts are difficult to resolve. The bacterial biofilm matrix provides the structural and functional support that distinguishes this state from free-living bacterial cells. Here, we show that the biofilm matrix can immobilize iron, providing access to this growth-promoting resource which is otherwise inaccessible in the planktonic state. Our data show that in the absence of heme,
Enterococcus faecalis
l
-lactate dehydrogenase promotes EET and uses matrix-associated iron to carry out EET. Therefore, the presence of iron within the biofilm matrix leads to enhanced biofilm growth.
Palladin Compensates for the Arp2/3 Complex and Supports Actin Structures during
Listeria
Infections
Authors: Dhanda Aaron S. , Vogl A. Wayne , Albraiki Sharifah E. , Otey Carol A. , Beck Moriah R. , Guttman Julian A. 
Published: April 2018

ABSTRACT

Palladin is an important component of motile actin-rich structures and nucleates branched actin filament arrays
in vitro
. Here we examine the role of palladin during
Listeria monocytogenes
infections in order to tease out novel functions of palladin. We show that palladin is co-opted by
L. monocytogenes
during its cellular entry and intracellular motility. Depletion of palladin resulted in shorter and misshapen comet tails, and when actin- or VASP-binding mutants of palladin were overexpressed in cells, comet tails disintegrated or became thinner. Comet tail thinning resulted in parallel actin bundles within the structures. To determine whether palladin could compensate for the Arp2/3 complex, we overexpressed palladin in cells treated with the Arp2/3 inhibitor CK-666. In treated cells, bacterial motility could be initiated and maintained when levels of palladin were increased. To confirm these findings, we utilized a cell line depleted of multiple Arp2/3 complex subunits. Within these cells,
L. monocytogenes
failed to generate comet tails. When palladin was overexpressed in this Arp2/3 functionally null cell line, the ability of
L. monocytogenes
to generate comet tails was restored. Using purified protein components, we demonstrate that
L. monocytogenes
actin clouds and comet tails can be generated (in a cell-free system) by palladin in the absence of the Arp2/3 complex. Collectively, our results demonstrate that palladin can functionally replace the Arp2/3 complex during bacterial actin-based motility.

IMPORTANCE
Structures containing branched actin filaments require the Arp2/3 complex. One of the most commonly used systems to study intracellular movement generated by Arp2/3-based actin motility exploits actin-rich comet tails made by
Listeria
. Using these infections together with live imaging and cell-free protein reconstitution experiments, we show that another protein, palladin, can be used in place of Arp2/3 to form actin-rich structures. Additionally, we show that palladin is needed for the structural integrity of comet tails as its depletion or mutation of critical regions causes dramatic changes to comet tail organization. These findings are the first to identify a protein that can functionally replace the Arp2/3 complex and have implications for all actin-based structures thought to exclusively use that complex.
An Evolutionarily Conserved Mechanism for Intrinsic and Transferable Polymyxin Resistance
Authors: Xu Yongchang , Wei Wenhui , Lei Sheng , Lin Jingxia , Srinivas Swaminath , Feng Youjun 
Published: April 2018

ABSTRACT

Polymyxins, a family of cationic antimicrobial cyclic peptides, act as a last line of defense against severe infections by Gram-negative pathogens with carbapenem resistance. In addition to the intrinsic resistance to polymyxin E (colistin) conferred by
Neisseria eptA
, the plasmid-borne mobilized colistin resistance gene
mcr-1
has been disseminated globally since the first discovery in Southern China, in late 2015. However, the molecular mechanisms for both intrinsic and transferable resistance to colistin remain largely unknown. Here, we aim to address this gap in the knowledge of these proteins. Structural and functional analyses of EptA and MCR-1 and -2 have defined a conserved 12-residue cavity that is required for the entry of the lipid substrate, phosphatidylethanolamine (PE). The
in vitro
and
in vivo
data together have allowed us to visualize the similarities in catalytic activity shared by EptA and MCR-1 and -2. The expression of either EptA or MCR-1 or -2 is shown to remodel the surface of enteric bacteria (e.g.,
Escherichia coli
,
Salmonella enterica
,
Klebsiella pneumoniae
, etc.), rendering them resistant to colistin. The parallels in the PE substrate-binding cavities among EptA, MCR-1, and MCR-2 provide a comprehensive understanding of both intrinsic and transferable colistin resistance. Domain swapping between EptA and MCR-1 and -2 reveals that the two domains (transmembrane [TM] region and
p
hospho
e
thanol
a
mine [PEA] transferase) are not functionally exchangeable. Taken together, the results represent a common mechanism for intrinsic and transferable PEA resistance to polymyxin, a last-resort antibiotic against multidrug-resistant pathogens.

IMPORTANCE
EptA and MCR-1 and -2 remodel the outer membrane, rendering bacteria resistant to colistin, a final resort against carbapenem-resistant pathogens. Structural and functional analyses of EptA and MCR-1 and -2 reveal parallel PE lipid substrate-recognizing cavities, which explains intrinsic and transferable colistin resistance in gut bacteria. A similar mechanism is proposed for the catalytic activities of EptA and MCR-1 and -2. Together, they constitute a common mechanism for intrinsic and transferable polymyxin resistance.
Strengthening health district management competencies in Ghana, Tanzania and Uganda: lessons from using action research to improve health workforce performance
Authors: Martineau Tim , Raven Joanna , Aikins Moses , Alonso-Garbayo Alvaro , Baine Sebastian , Huss Reinhard , Maluka Stephen , Wyss Kaspar 
Published: April 2018

BackgroundTo achieve Universal Health Coverage (UHC), more health workers are needed; also critical is supporting optimal performance of existing staff. Integrated human resource management (HRM) strategies, complemented by other health systems strategies, are needed to improve health workforce performance, which is possible at district level in decentralised contexts. To strengthen the capacity of district management teams to develop and implement workplans containing integrated strategies for workforce performance improvement, we introduced an action-research-based management strengthening intervention (MSI). This consisted of two workshops, follow-up by facilitators and meetings between participating districts. Although often used in the health sector, there is little evaluation of this approach in middle-income and low-income country contexts. The MSI was tested in three districts in Ghana, Tanzania and Uganda. This paper reports on the appropriateness of the MSI to the contexts and its effects.MethodsDocumentary evidence (workshop reports, workplans, diaries, follow-up visit reports) was collected throughout the implementation of the MSI in each district and interviews (50) and focus-group discussions (6) were conducted with managers at the end of the MSI. The findings were analysed using Kirkpatrick’s evaluation framework to identify effects at different levels.FindingsThe MSI was appropriate to the needs and work patterns of District Health Management Teams (DHMTs) in all contexts. DHMT members improved management competencies for problem analysis, prioritisation and integrated HRM and health systems strategy development. They learnt how to refine plans as more information became available and the importance of monitoring implementation. The MSI produced changes in team behaviours and confidence. There were positive results regarding workforce performance or service delivery; these would increase with repetition of the MSI.ConclusionsThe MSI is appropriate to the contexts where tested and can improve staff performance. However, for significant impact on service delivery and UHC, a method of scaling up and sustaining the MSI is required.
Sepsis recognition tools in acute ambulatory care: associations with process of care and clinical outcomes in a service evaluation of an Emergency Multidisciplinary Unit in Oxfordshire
Authors: Camm Christian Fielder , Hayward Gail , Elias Tania C N , Bowen Jordan S T , Hassanzadeh Roya , Fanshawe Thomas , Pendlebury Sarah T , Lasserson Daniel S 
Published: April 2018

ObjectiveTo assess the performance of currently available sepsis recognition tools in patients referred to a community-based acute ambulatory care unit.DesignService evaluation of consecutive patients over a 4-month period.SettingCommunity-based acute ambulatory care unit.Data collection and outcome measuresObservations, blood results and outcome data were analysed from patients with a suspected infection. Clinical features at first assessment were used to populate sepsis recognition tools including: systemic inflammatory response syndrome (SIRS) criteria, National Early Warning Score (NEWS), quick Sequential Organ Failure Assessment (qSOFA) and National Institute for Health and Care Excellence (NICE) criteria. Scores were assessed against the clinical need for escalated care (use of intravenous antibiotics, fluids, ongoing ambulatory care or hospital treatment) and poor clinical outcome (all-cause mortality and readmission at 30 days after index assessment).ResultsOf 533 patients (median age 81 years), 316 had suspected infection with 120 patients requiring care escalated beyond simple community care. SIRS had the highest positive predictive value (50.9%, 95% CI 41.6% to 60.3%) and negative predictive value (68.9%, 95% CI 62.6% to 75.3%) for the need for escalated care. Both NEWS and SIRS were better at predicting the need for escalated care than qSOFA and NICE criteria in patients with suspected infection (all P<0.001). While new-onset confusion predicted the need for escalated care for infection in patients ≥85 years old (n=114), 23.7% of patients ≥85 years had new-onset confusion without evidence for infection.ConclusionsAcute ambulatory care clinicians should use caution in applying the new NICE endorsed criteria for determining the need for intravenous therapy and hospital-based location of care. NICE criteria have poorer performance when compared against NEWS and SIRS and new-onset confusion was prevalent in patients aged ≥85 years without infection.
Systematic assessment of the growth plates of the wrist in young gymnasts: development and validation of the Amsterdam MRI assessment of the Physis (AMPHYS) protocol
Authors: Kox Laura S , Kraan Rik B J , van Dijke Kees F , Hemke Robert , Jens Sjoerd , de Jonge Milko C , Oei Edwin H G , Smithuis Frank F , Terra Maaike P , Maas Mario 
Published: April 2018

ObjectivesTo develop and validate a protocol for MRI assessment of the distal radial and ulnar periphyseal area in gymnasts and non-gymnasts.MethodsTwenty-four gymnasts with wrist pain, 18 asymptomatic gymnasts and 24 non-gymnastic controls (33 girls) underwent MRI of the wrist on a 3T scanner. Sequences included coronal proton density-weighted images with and without fat saturation, and three-dimensional water-selective cartilage scan and T2 Dixon series. Skeletal age was determined using hand radiographs. Three experienced musculoskeletal radiologists established a checklist of possible (peri)physeal abnormalities based on literature and clinical experience. Five other musculoskeletal radiologists and residents evaluated 30 MRI scans (10 from each group) using this checklist and reliability was determined using the intraclass correlation coefficient (ICC) and Fleiss’ kappa. A final evaluation protocol was established containing only items with fair to excellent reliability.ResultsTwenty-seven items were assessed for reliability. Intra-rater and inter-rater agreement was good to excellent (respective ICCs 0.60–0.91 and 0.60–0.78) for four epiphyseal bone marrow oedema-related items, physeal signal intensity, metaphyseal junction and depth of metaphyseal intrusions. For physeal thickness, thickness compared with proximal physis of first metacarpal, metaphyseal intrusions, physeal connection of intrusions and metaphyseal bone marrow signal intensity, intra-rater agreement was fair to excellent (ICC/kappa 0.55–0.85) and inter-rater agreement was fair (ICC/kappa 0.41–0.59). Twelve items were included in the final protocol.ConclusionThe Amsterdam MRI assessment of the Physis protocol facilitates patient-friendly and reliable assessment of the (peri)physeal area in the radius and ulna.
In vivobioassay to test the pathogenicity of missense humanAIPvariants
Authors: Aflorei Elena Daniela , Klapholz Benjamin , Chen Chenghao , Radian Serban , Dragu Anca Neluta , Moderau Nina , Prodromou Chrisostomos , Ribeiro Paulo S , Stanewsky Ralf , Korbonits Márta 
Published: April 2018

BackgroundHeterozygous germline loss-of-function mutations in the aryl hydrocarbon receptor-interacting protein gene (AIP) predispose to childhood-onset pituitary tumours. The pathogenicity of missense variants may pose difficulties for genetic counselling and family follow-up.ObjectiveTo develop anin vivo system to test the pathogenicity of humanAIPmutations using the fruit flyDrosophila melanogaster.MethodsWe generated a null mutant of theDrosophila AIPorthologue,CG1847,a gene located on the Xchromosome, which displayed lethality at larval stage in hemizygous knockout male mutants (CG1847exon1_3). We tested human missense variants of ‘unknown significance’, with ‘pathogenic’ variants as positive control.ResultsWe found that humanAIPcan functionally substitute forCG1847, as heterologous overexpression of humanAIPrescued maleCG1847exon1_3lethality, while a truncated version ofAIPdid not restore viability. Flies harbouring patient-specific missenseAIPvariants (p.C238Y, p.I13N, p.W73R and p.G272D) failed to rescueCG1847exon1_3mutants, while seven variants (p.R16H, p.Q164R, p.E293V, p.A299V, p.R304Q, p.R314W and p.R325Q) showed rescue, supporting a non-pathogenic role for these latter variants corresponding to prevalence and clinical data.ConclusionOurin vivomodel represents a valuable tool to characterise putative disease-causing humanAIPvariants and assist the genetic counselling and management of families carryingAIPvariants.