bluuebunny/crossref_metadata_2025 huggingface dataset:
Heterologous Expression of the Clostridium carboxidivorans CO Dehydrogenase Alone or Together with the Acetyl Coenzyme A Synthase Enables both Reduction of CO
2
and Oxidation of CO by Clostridium acetobutylicum
Authors: Carlson Ellinor D. , Papoutsakis Eleftherios T.
Published: June 2017

With recent advances in synthetic biology, CO
2
could be utilized as a carbon feedstock by native or engineered organisms, assuming the availability of electrons. Two key enzymes used in autotrophic CO
2
fixation are the CO dehydrogenase (CODH) and acetyl coenzyme A (acetyl-CoA) synthase (ACS), which form a bifunctional heterotetrameric complex. The CODH/ACS complex can reversibly catalyze CO
2
to CO, effectively enabling a biological water-gas shift reaction at ambient temperatures and pressures. The CODH/ACS complex is part of the Wood-Ljungdahl pathway (WLP) used by acetogens to fix CO
2
, and it has been well characterized in native hosts. So far, only a few recombinant CODH/ACS complexes have been expressed in heterologous hosts, none of which demonstrated
in vivo
CO
2
reduction. Here, functional expression of the
Clostridium carboxidivorans
CODH/ACS complex is demonstrated in the solventogen
Clostridium acetobutylicum
, which was engineered to express CODH alone or together with the ACS. Both strains exhibited CO
2
reduction and CO oxidation activities. The CODH reactions were interrogated using isotopic labeling, thus verifying that CO was a direct product of CO
2
reduction, and vice versa. CODH apparently uses a native
C. acetobutylicum
ferredoxin as an electron carrier for CO
2
reduction. Heterologous CODH activity depended on actively growing cells and required the addition of nickel, which is inserted into CODH without the need to express the native Ni insertase protein. Increasing CO concentrations in the gas phase inhibited CODH activity and altered the metabolite profile of the CODH-expressing cells. This work provides the foundation for engineering a complete and functional WLP in nonnative host organisms.

IMPORTANCE
Functional expression of CO dehydrogenase (CODH) from
Clostridium carboxidivorans
was demonstrated in
C. acetobutylicum
, which is natively incapable of CO
2
fixation. The expression of CODH, alone or together with the
C. carboxidivorans
acetyl-CoA synthase (ACS), enabled
C. acetobutylicum
to catalyze both CO
2
reduction and CO oxidation. Importantly, CODH exhibited activity in both the presence and absence of ACS.
13
C-tracer studies confirmed that the engineered
C. acetobutylicum
strains can reduce CO
2
to CO and oxidize CO during growth on glucose.
The Irr and RirA Proteins Participate in a Complex Regulatory Circuit and Act in Concert To Modulate Bacterioferritin Expression in Ensifer meliloti 1021
Authors: Costa Daniela , Amarelle Vanesa , Valverde Claudio , O'Brian Mark R. , Fabiano Elena
Published: June 2017

In this work we found that the
bfr
gene of the rhizobial species
Ensifer meliloti
, encoding a bacterioferritin iron storage protein, is involved in iron homeostasis and the oxidative stress response. This gene is located downstream of and overlapping the
smc03787
open reading frame (ORF). No well-predicted RirA or Irr boxes were found in the region immediately upstream of the
bfr
gene although two presumptive RirA boxes and one presumptive Irr box were present in the putative promoter of
smc03787
. We demonstrate that
bfr
gene expression is enhanced under iron-sufficient conditions and that Irr and RirA modulate this expression. The pattern of
bfr
gene expression as well as the response to Irr and RirA is inversely correlated to that of
smc03787
. Moreover, our results suggest that the small RNA SmelC759 participates in RirA- and Irr-mediated regulation of
bfr
expression and that additional unknown factors are involved in iron-dependent regulation.

IMPORTANCE
E. meliloti
belongs to the
Alphaproteobacteria
, a group of bacteria that includes several species able to associate with eukaryotic hosts, from mammals to plants, in a symbiotic or pathogenic manner. Regulation of iron homeostasis in this group of bacteria differs from that found in the well-studied
Gammaproteobacteria
. In this work we analyzed the effect of
rirA
and
irr
mutations on
bfr
gene expression. We demonstrate the effect of an
irr
mutation on iron homeostasis in this bacterial genus. Moreover, results obtained indicate a complex regulatory circuit where multiple regulators, including RirA, Irr, the small RNA SmelC759, and still unknown factors, act in concert to balance
bfr
gene expression.
Different Amplicon Targets for Sequencing-Based Studies of Fungal Diversity
Authors: De Filippis Francesca , Laiola Manolo , Blaiotta Giuseppe , Ercolini Danilo
Published: June 2017

Target-gene amplicon sequencing is the most exploited high-throughput sequencing application in microbial ecology. The targets are taxonomically relevant genes, with 16S rRNA being the gold standard for bacteria. As for fungi, the most commonly used target is the internal transcribed spacer (ITS). However, the uneven ITS length among species may promote preferential amplification and sequencing and incorrect estimation of their abundance. Therefore, the use of different targets is desirable. We evaluated the use of three different target amplicons for the characterization of fungal diversity. After an
in silico
primer evaluation, we compared three amplicons (the ITS1-ITS2 region [ITS1-2], 18S ribosomal small subunit RNA, and the D1/D2 domain of the 26S ribosomal large subunit RNA), using biological samples and a mock community of common fungal species. All three targets allowed for accurate identification of the species present. Nevertheless, high heterogeneity in ITS1-2 length was found, and this caused an overestimation of the abundance of species with a shorter ITS, while both 18S and 26S amplicons allowed for more reliable quantification. We demonstrated that ITS1-2 amplicon sequencing, although widely used, may lead to an incorrect evaluation of fungal communities, and efforts should be made to promote the use of different targets in sequencing-based microbial ecology studies.

IMPORTANCE
Amplicon-sequencing approaches for fungi may rely on different targets affecting the diversity and abundance of the fungal species. An increasing number of studies will address fungal diversity by high-throughput amplicon sequencing. The description of the communities must be accurate and reliable in order to draw useful insights and to address both ecological and biological questions. By analyzing a mock community and several biological samples, we demonstrate that using different amplicon targets may change the results of fungal microbiota analysis, and we highlight how a careful choice of the target is fundamental for a thorough description of the fungal communities.
Flow Cytometry Is a Powerful Tool for Assessment of the Viability of Fungal Conidia in Metalworking Fluids
Authors: Vanhauteghem D. , Demeyere K. , Callaert N. , Boelaert A. , Haesaert G. , Audenaert K. , Meyer E.
Published: June 2017

Fungal contamination of metalworking fluids (MWF) is a dual problem in automated processing plants because resulting fungal biofilms obstruct cutting, drilling, and polishing machines. Moreover, some fungal species of MWF comprise pathogens such as
Fusarium solani
. Therefore, the development of an accurate analytical tool to evaluate conidial viability in MWF is important. We developed a flow cytometric method to measure fungal viability in MWF using
F. solani
as the model organism. To validate this method, viable and dead conidia were mixed in several proportions and flow was cytometrically analyzed. Subsequently, we assessed the fungicidal activity of two commercial MWF using flow cytometry (FCM) and compared it with microscopic analyses and plating experiments. We evaluated the fungal growth in both MWF after 7 days using quantitative PCR (qPCR) to assess the predictive value of FCM. Our results showed that FCM distinguishes live from dead conidia as early as 5 h after exposure to MWF, whereas the microscopic germination approach detected conidial viability much later and less accurately. At 24 h, microscopic analyses of germinating conidia and live/dead analyses by FCM correlated well, although the former consistently underestimated the proportion of viable conidia. In addition, the reproducibility and sensitivity of the flow cytometric method were high and allowed assessment of the fungicidal properties of two commercial MWF. Importantly, the obtained flow cytometric results on viability of
F. solani
conidia at both early time points (5 h and 24 h) correlated well with fungal biomass measurements assessed via a qPCR methodology 7 days after the start of the experiment.

IMPORTANCE
This result shows the predictive power of flow cytometry (FCM) in assessing the fungicidal capacity of MWF formulations. It also implies that FCM can be implemented as a rapid detection tool to estimate the viable fungal load in an industrial processing matrix (MWF).
Novel Antibiotic Resistance Determinants from Agricultural Soil Exposed to Antibiotics Widely Used in Human Medicine and Animal Farming
Authors: Lau Calvin Ho-Fung , van Engelen Kalene , Gordon Stephen , Renaud Justin , Topp Edward
Published: June 2017

Antibiotic resistance has emerged globally as one of the biggest threats to human and animal health. Although the excessive use of antibiotics is recognized as accelerating the selection for resistance, there is a growing body of evidence suggesting that natural environments are “hot spots” for the development of both ancient and contemporary resistance mechanisms. Given that pharmaceuticals can be entrained onto agricultural land through anthropogenic activities, this could be a potential driver for the emergence and dissemination of resistance in soil bacteria. Using functional metagenomics, we interrogated the “resistome” of bacterial communities found in a collection of Canadian agricultural soil, some of which had been receiving antibiotics widely used in human medicine (macrolides) or food animal production (sulfamethazine, chlortetracycline, and tylosin) for up to 16 years. Of the 34 new antibiotic resistance genes (ARGs) recovered, the majority were predicted to encode (multi)drug efflux systems, while a few share little to no homology with established resistance determinants. We characterized several novel gene products, including putative enzymes that can confer high-level resistance against aminoglycosides, sulfonamides, and broad range of beta-lactams, with respect to their resistance mechanisms and clinical significance. By coupling high-resolution proteomics analysis with functional metagenomics, we discovered an unusual peptide, PPP
AZI 4
, encoded within an alternative open reading frame not predicted by bioinformatics tools. Expression of the proline-rich PPP
AZI 4
can promote resistance against different macrolides but not other ribosome-targeting antibiotics, implicating a new macrolide-specific resistance mechanism that could be fundamentally linked to the evolutionary design of this peptide.

IMPORTANCE
Antibiotic resistance is a clinical phenomenon with an evolutionary link to the microbial pangenome. Genes and protogenes encoding specialized and potential resistance mechanisms are abundant in natural environments, but understanding of their identity and genomic context remains limited. Our discovery of several previously unknown antibiotic resistance genes from uncultured soil microorganisms indicates that soil is a significant reservoir of resistance determinants, which, once acquired and “repurposed” by pathogenic bacteria, can have serious impacts on therapeutic outcomes. This study provides valuable insights into the diversity and identity of resistance within the soil microbiome. The finding of a novel peptide-mediated resistance mechanism involving an unpredicted gene product also highlights the usefulness of integrating proteomics analysis into metagenomics-driven gene discovery.
Indole-Induced Reversion of Intrinsic Multiantibiotic Resistance in Lysobacter enzymogenes
Authors: Han Yong , Wang Yan , Yu Yameng , Chen Haotong , Shen Yuemao , Du Liangcheng
Published: June 2017

Lysobacter
species are a group of environmental bacteria that are emerging as a new source of antibiotics. One characteristic of
Lysobacter
is intrinsic resistance to multiple antibiotics, which had not been studied. To understand the resistance mechanism, we tested the effect of blocking two-component regulatory systems (TCSs) on the antibiotic resistance of
Lysobacter enzymogenes
, a prolific producer of antibiotics. Upon treatment with LED209, an inhibitor of the widespread TCS QseC/QseB,
L. enzymogenes
produced a large amount of an unknown metabolite that was barely detectable in the untreated culture. Subsequent structural elucidation by nuclear magnetic resonance (NMR) unexpectedly revealed that the metabolite was indole. Indole production was also markedly induced by adrenaline, a known modulator of QseC/QseB. Next, we identified two TCS genes,
L. enzymogenes
qseC
(
Le-qseC
) and
Le-qseB
, in
L. enzymogenes
and found that mutations of
Le-qseC
and
Le-qseB
also led to a dramatic increase in indole production. We then chemically synthesized a fluorescent indole probe that could label the cells. While the
Le-qseB
(cytoplasmic response regulator) mutant was clearly labeled by the probe, the
Le-qseC
(membrane sensor) mutant was not labeled. It was reported previously that indole can enhance antibiotic resistance in bacteria. Therefore, we tested if the dramatic increase in the level of indole production in
L. enzymogenes
upon blocking of
Le-qseC
and
Le-qseB
would lead to enhanced antibiotic resistance. Surprisingly, we found that indole caused the intrinsically multiantibiotic-resistant bacterium
L. enzymogenes
to become susceptible. Point mutations at conserved amino acids in
Le
-QseC also led to antibiotic susceptibility. Because indole is known as an interspecies signal, these findings may have implications.

IMPORTANCE
The environmental bacterium
Lysobacter
is a new source of antibiotic compounds and exhibits intrinsic antibiotic resistance. Here, we found that the inactivation of a two-component regulatory system (TCS) by an inhibitor or by gene deletion led to a remarkable increase in the level of production of a metabolite in
L. enzymogenes
, and this metabolite was identified to be indole. We chemically synthesized a fluorescent indole probe and found that it could label the wild type and a mutant of the TCS cytoplasmic response regulator but not a mutant of the TCS membrane sensor. Indole treatment caused the intrinsically multidrug-resistant bacterium
L. enzymogenes
to be susceptible to antibiotics. Mutations of the TCS sensor also led to antibiotic susceptibility. Because indole is known as an interspecies signal between gut microbiota and mammalian hosts, the observation that indole could render intrinsically resistant
L. enzymogenes
susceptible to common antibiotics may have implications.
Functional Analysis of a Novel β-(1,3)-Glucanase from Corallococcus sp. Strain EGB Containing a Fascin-Like Module
Authors: Zhou Jie , Li Zhoukun , Wu Jiale , Li Lifeng , Li Ding , Ye Xianfeng , Luo Xue , Huang Yan , Cui Zhongli , Cao Hui
Published: June 2017

A novel β-(1,3)-glucanase gene designated
lamC
, cloned from
Corallococcus
sp. strain EGB, contains a fascin-like module and a glycoside hydrolase family 16 (GH16) catalytic module. LamC displays broad hydrolytic activity toward various polysaccharides. Analysis of the hydrolytic products revealed that LamC is an exo-acting enzyme on β-(1,3)(1,3)- and β-(1,6)-linked glucan substrates and an endo-acting enzyme on β-(1,4)-linked glucan and xylan substrates. Site-directed mutagenesis of conserved catalytic Glu residues (E304A and E309A) demonstrated that these activities were derived from the same active site. Excision of the fascin-like module resulted in decreased activity toward β-(1,3)(1,3)-linked glucans. The carbohydrate-binding assay showed that the fascin-like module was a novel β-(1,3)-linked glucan-binding module. The functional characterization of the fascin-like module and catalytic module will help us better understand these enzymes and modules.

IMPORTANCE
In this report of a bacterial β-(1,3)(1,3)-glucanase containing a fascin-like module, we reveal the β-(1,3)(1,3)-glucan-binding function of the fascin-like module present in the N terminus of LamC. LamC displays exo-β-(1,3)/(1,6)-glucanase and endo-β-(1,4)-glucanase/xylanase activities with a single catalytic domain. Thus, LamC was identified as a novel member of the GH16 family.
Periplasmic Nicotine Dehydrogenase NdhAB Utilizes Pseudoazurin as Its Physiological Electron Acceptor in Agrobacterium tumefaciens S33
Authors: Yu Wenjun , Wang Rongshui , Huang Haiyan , Xie Huijun , Wang Shuning
Published: June 2017

Agrobacterium tumefaciens
S33 can grow with nicotine as the sole source of carbon, nitrogen, and energy via a novel hybrid of the pyridine pathway and the pyrrolidine pathway. Characterization of the enzymes involved in the hybrid pathway is important for understanding its biochemical mechanism. Here, we report that the molybdenum-containing nicotine dehydrogenase (NdhAB), which catalyzes the initial step of nicotine degradation, is located in the periplasm of strain S33, while the 6-hydroxynicotine oxidase and 6-hydroxypseudooxynicoine oxidase are in the cytoplasm. This is consistent with the fact that NdhA has a Tat signal peptide. Interestingly, an open reading frame (ORF) adjacent to the
ndhAB
gene was verified to encode a copper-containing electron carrier, pseudoazurin (Paz), which has a signal peptide typical of bacterial Paz proteins. Both were transported into the periplasm after being produced in the cytoplasm. We purified NdhAB from the periplasmic fraction of strain S33 and found that with Paz as the physiological electron acceptor, NdhAB catalyzed the hydroxylation of nicotine at a specific rate of 110.52 ± 8.09 μmol · min
−1
· mg of protein
−1
, where the oxygen atom in the hydroxyl group of the product 6-hydroxynicotine was derived from H
2
O. The apparent

K
m

values for nicotine and Paz were 1.64 ± 0.07 μM and 3.61 ± 0.23 μM, respectively. NAD(P)
+
, O
2
, and ferredoxin could not serve as electron acceptors. Disruption of the
paz
gene disabled the strain for nicotine degradation, indicating that Paz is required for nicotine catabolism in the strain. These findings help our understanding of electron transfer during nicotine degradation in bacteria.

IMPORTANCE
Nicotine is a toxic and addictive
N
-heterocyclic aromatic alkaloid produced in tobacco. Its catabolism in organisms and degradation in tobacco wastes have become major concerns for human health and the environment. Bacteria usually decompose nicotine using the classical strategy of hydroxylating the pyridine ring with the help of activated oxygen by nicotine dehydrogenase, which binds one molybdopterin, two [2Fe2S] clusters, and usually one flavin adenine dinucleotide (FAD) as well. However, the physiological electron acceptor for the reaction is still unknown. In this study, we found that the two-component nicotine dehydrogenase from
Agrobacterium tumefaciens
S33, naturally lacking an FAD-binding domain, is located in the periplasmic space and uses a copper-containing electron carrier, pseudoazurin, as its physiological electron acceptor. We report here the role of pseudoazurin in a reaction catalyzed by a molybdopterin-containing hydroxylase occurring in the periplasmic space. These results provide new biochemical knowledge on microbial degradation of
N
-heterocyclic aromatic compounds.
Natural DNA Transformation Is Functional in Lactococcus lactis subsp. cremoris KW2
Authors: David Blandine , Radziejwoski Amandine , Toussaint Frédéric , Fontaine Laetitia , de Frahan Marie Henry , Patout Cédric , van Dillen Sabine , Boyaval Patrick , Horvath Philippe , Fremaux Christophe , Hols Pascal
Published: June 2017

Lactococcus lactis
is one of the most commonly used lactic acid bacteria in the dairy industry. Activation of competence for natural DNA transformation in this species would greatly improve the selection of novel strains with desired genetic traits. Here, we investigated the activation of natural transformation in
L. lactis
subsp.
cremoris
KW2, a strain of plant origin whose genome encodes the master competence regulator ComX and the complete set of proteins usually required for natural transformation. In the absence of knowledge about competence regulation in this species, we constitutively overproduced ComX in a reporter strain of late competence phase activation and showed, by transcriptomic analyses, a ComX-dependent induction of all key competence genes. We further demonstrated that natural DNA transformation is functional in this strain and requires the competence DNA uptake machinery. Since constitutive ComX overproduction is unstable, we alternatively expressed
comX
under the control of an endogenous xylose-inducible promoter. This regulated system was used to successfully inactivate the adaptor protein MecA and subunits of the Clp proteolytic complex, which were previously shown to be involved in ComX degradation in streptococci. In the presence of a small amount of ComX, the deletion of
mecA
,
clpC
, or
clpP
genes markedly increased the activation of the late competence phase and transformability. Altogether, our results report the functionality of natural DNA transformation in
L. lactis
and pave the way for the identification of signaling mechanisms that trigger the competence state in this species.

IMPORTANCE
Lactococcus lactis
is a lactic acid bacterium of major importance, which is used as a starter species for milk fermentation, a host for heterologous protein production, and a delivery platform for therapeutic molecules. Here, we report the functionality of natural transformation in
L. lactis
subsp.
cremoris
KW2 by the overproduction of the master competence regulator ComX. The developed procedure enables a flexible approach to modify the chromosome with single point mutation, sequence insertion, or sequence replacement. These results represent an important step for the genetic engineering of
L. lactis
that will facilitate the design of strains optimized for industrial applications. This will also help to discover natural regulatory mechanisms controlling competence in the genus
Lactococcus
.
Strain-Level Metagenomic Analysis of the Fermented Dairy Beverage Nunu Highlights Potential Food Safety Risks
Authors: Walsh Aaron M. , Crispie Fiona , Daari Kareem , O'Sullivan Orla , Martin Jennifer C. , Arthur Cornelius T. , Claesson Marcus J. , Scott Karen P. , Cotter Paul D.
Published: June 2017

The rapid detection of pathogenic strains in food products is essential for the prevention of disease outbreaks. It has already been demonstrated that whole-metagenome shotgun sequencing can be used to detect pathogens in food but, until recently, strain-level detection of pathogens has relied on whole-metagenome assembly, which is a computationally demanding process. Here we demonstrated that three short-read-alignment-based methods, i.e., MetaMLST, PanPhlAn, and StrainPhlAn, could accurately and rapidly identify pathogenic strains in spinach metagenomes that had been intentionally spiked with Shiga toxin-producing
Escherichia coli
in a previous study. Subsequently, we employed the methods, in combination with other metagenomics approaches, to assess the safety of nunu, a traditional Ghanaian fermented milk product that is produced by the spontaneous fermentation of raw cow milk. We showed that nunu samples were frequently contaminated with bacteria associated with the bovine gut and, worryingly, we detected putatively pathogenic
E. coli
and
Klebsiella pneumoniae
strains in a subset of nunu samples. Ultimately, our work establishes that short-read-alignment-based bioinformatics approaches are suitable food safety tools, and we describe a real-life example of their utilization.

IMPORTANCE
Foodborne pathogens are responsible for millions of illnesses each year. Here we demonstrate that short-read-alignment-based bioinformatics tools can accurately and rapidly detect pathogenic strains in food products by using shotgun metagenomics data. The methods used here are considerably faster than both traditional culturing methods and alternative bioinformatics approaches that rely on metagenome assembly; therefore, they can potentially be used for more high-throughput food safety testing. Overall, our results suggest that whole-metagenome sequencing can be used as a practical food safety tool to prevent diseases or to link outbreaks to specific food products.
Prevalence, incidence and aetiologies of pulmonary hypertension in Africa: a systematic review and meta-analysis protocol
Authors: Bigna Jean Joel , Noubiap Jean Jacques , Nansseu Jobert Richie , Aminde Leopold Ndemnge
Published: June 2017

Introduction

There are no data summarising the epidemiology of pulmonary hypertension (PH) among adults residing in Africa. Establishment of accurate epidemiological data on PH in this region may guide decision-making toward interventions to curb the burden of PH in Africa. The aim of this systematic review is to determine the prevalence, incidence and aetiologies of PH among people residing in Africa.

Methods and analysis

This systematic review and meta-analysis will follow the MOOSE guidelines for reporting. Relevant abstracts published until 30 September 2016 will be searched in PubMed/Medline, EMBASE (Excerpta Medica Database), African Journals Online and Africa Index Medicus. Full texts of eligible studies will then be accessed through PubMed, Google Scholar, HINARI and the respective journals’ websites. Relevant unpublished papers and conference proceedings will also be checked. Data will be analysed using STATA version 13 software. The study-specific estimates will be pooled through a random-effects meta-analysis model to obtain an overall summary estimate of the prevalence/incidence and aetiologies of PH across studies. Heterogeneity of studies will be evaluated by the χ2test on Cochrane’s Q statistic. Funnel plot analysis and Egger’s test will be done to detect publication bias. Results will be presented by geographical region (central, eastern, northern, southern and western Africa).

Ethics and dissemination

The current study is based on published data; ethical approval is, therefore, not required. This review will guide policy, practice and research by providing information on the magnitude of PH among people residing in Africa. Findings will be presented in evidence tables of individual studies as well as in summary tables. The final report of this systematic review, in the form of a scientific paper, will be published in a peer-reviewed journal. Furthermore, findings will be presented at conferences and submitted to relevant health authorities.

Review registration number

PROSPERO CRD42016049351.
GPs' attitudes, beliefs and behaviours regarding exercise for chronic knee pain: a questionnaire survey
Authors: Cottrell Elizabeth , Foster Nadine E , Porcheret Mark , Rathod Trishna , Roddy Edward
Published: June 2017

Objectives

The aim of this study was to investigate general practitioners’ (GPs) attitudes, beliefs and behaviours regarding the use of exercise for patients with chronic knee pain (CKP) attributable to osteoarthritis.

Setting

Primary care GPs in the UK.

Participants

5000 GPs, randomly selected from Binley’s database, were mailed a cross-sectional questionnaire survey.

Outcome measures

GPs’ attitudes and beliefs were investigated using attitude statements, and reported behaviours were identified using vignette-based questions. GPs were invited to report barriers experienced when initiating exercise with patients with CKP

Results

835 (17%) GPs responded. Overall, GPs were positive about general exercise for CKP. 729 (87%) reported using exercise, of which, 538 (74%) reported that they would use both general and local (lower limb) exercises. However, only 92 (11% of all responding) GPs reported initiating exercise in ways aligning with best-evidence recommendations. 815 (98%) GPs reported barriers in using exercise for patients with CKP, most commonly, insufficient time in consultations (n=419; 51%) and insufficient expertise (n=337; 41%).

Conclusions

While GPs’ attitudes and beliefs regarding exercise for CKP were generally positive, initiation of exercise was often poorly aligned with current recommendations, and barriers and uncertainties were reported. GPs’ use of exercise may be improved by addressing the key barriers of time and expertise, by developing a pragmatic approach that supports GPs to initiate individualised exercise, and/or by other professionals taking on this role.
How do smoking cessation medicines compare with respect to their neuropsychiatric safety? A protocol for a systematic review, network meta-analysis and cost-effectiveness analysis
Authors: Thomas Kyla H , Caldwell Deborah , Dalili Michael N , Gunnell David , Munafò Marcus R , Stevenson Matt , Welton Nicky J
Published: June 2017

Introduction

Cigarette smoking is one of the leading causes of early death in the UK and worldwide. Public health guidance recommends the use of varenicline, bupropion and nicotine replacement therapy (NRT) as smoking cessation aids in the UK. Additionally, the first electronic cigarette has been licensed for use as a smoking cessation medicine. However, there are ongoing concerns about the safety of these medicines. We present a protocol for a systematic review and network meta-analysis (NMA) to determine how these smoking cessation medicines compare to each other with respect to their neuropsychiatric safety in adult smokers. Secondary aims include updating the evidence regarding the effectiveness and cardiovascular safety of these medicines for use in a cost-effectiveness analysis.

Methods and analysis

We will include randomised controlled trials and observational studies with control groups comparing monotherapy with varenicline, bupropion, NRT or electronic cigarette and combination therapies to each other, placebo or usual care. The primary composite safety outcome will be serious adverse events, defined as events that resulted in death, were life threatening, required hospitalisation or resulted in significant disability or congenital/birth defect. The preferred effectiveness outcome will be sustained smoking cessation defined as abstinence for a minimum of 6 months as determined by biochemical validation. We will include trials identified by previous reviews and search relevant databases for newly published trials as well as contacting study authors to identify unpublished information. We will conduct fixed-effect and random-effect meta-analyses for each pairwise comparison of treatments and outcome; where these estimates differ, we will consider reasons for heterogeneity, quantified using the between-study variance (τ2). For each outcome, we will construct a NMA in a Bayesian framework which will be compared with the pair-wise results, allowing us to rank treatments. The effectiveness estimates from the NMA will be entered into a probabilistic economic model.

Ethics and dissemination

Ethics approval is not required for this evidence synthesis study as it involves analysis of secondary data from randomised controlled trials and observational studies. The review will make an important contribution to the knowledge base around the effectiveness, safety and cost-effectiveness of smoking cessation medicines. Results will be disseminated to the general public, healthcare practitioners and clinicians, academics, industry and policy makers.

PROSPERO registration number

CRD42016041302.
Factors influencing psychological, social and health outcomes after major burn injuries in adults: cohort study protocol
Authors: Druery Martha , Newcombe Peter A , Cameron Cate M , Lipman Jeffrey
Published: June 2017

Introduction

The goal of burn care is that ‘the quality of the outcome must be worth the pain of survival’. More research is needed to understand how best to deliver care for patients with burns to achieve this aim. Loss of independence, function as well as loss of income for patients with burns and carers cause a significant burden at both individual and societal levels. Much is being done to advance knowledge in the clinical care field; however, there has been a paucity of research exploring psychosocial outcomes. This paper describes the study background and methods, as implemented in an Australian cohort study of psychosocial outcomes after major burn injuries.

Methods and analysis

In this inception cohort study, a target sample of 230 participants, aged 18 years or over, admitted to a single statewide burns centre with a burn injury are identified by hospital staff for inclusion. Baseline survey data are collected either in person or by telephone within 28 days of the injury and participants then followed up with telephone interviews at 3, 6 and 12 months postburn. Injury and burns treatment information is collected from medical records. Social support is measured as a predictor variable using the Multidimensional Scale of Perceived Social Support. Outcome data are collected via standardised measures in the domains of Quality of Life (SF-12, EQ-5D, BSHS-B), depression (PHQ-9), post-traumatic stress disorder (PCL-C, PAS), community integration (CIQ-R) and Quality-Adjusted Life Years (EQ-5D). Additional survey questions measure life satisfaction, return to work and public services utilisation at 12 months postinjury. Data analysis methods will include analysis of variance, Pearson correlation and hierarchical multiple regression analyses.

Ethics and dissemination

Hospital-based and University of Queensland Human Research Ethics Committees have approved the protocol. Results from the study will be disseminated at national and international conferences, in peer-reviewed journals and in a doctoral thesis.

Trial registration number

Australia New Zealand Clinical Trials Registry (ACTRN12616000828426). Retrospectively registered on 23 June 2016; pre-results.
Reducing the number of unnecessary liver function tests requested on the Paediatric Intensive Care Unit
Authors: Sinitsky Lynn , Brierley Joe
Published: June 2017

Between January and October 2014, Great Ormond Street Hospital Paediatric Intensive Care Unit (PICU) was spending an average £23,392 on blood tests per month. Blood tests should be requested based on previous results and the patient's clinical condition, medication and nutritional status. However, more blood tests were being ordered than clinically indicated: an audit in October 2014 showed liver function tests (LFTs) were requested daily on most patients, even with previous normal results.

A driver diagram identified three primary drivers for blood test requesting: decision-making, situational awareness and computer-based ordering. Decision-making for routine blood tests was the responsibility of the bedside nurses on each night shift. The communication between the nurses and doctors was an identified secondary driver. The project's primary aim was to reduce unnecessary LFTs requests on PICU over 6 months by implementing a blood test request form, a table of common investigations to facilitate and document discussion between the nursing and medical teams. The secondary aims were to reduce other unnecessary blood test requests, including full blood counts (FBC), coagulation screens and CRP.

This project was conducted in three phases: construction, testing and implementation of the blood test form. PDSA cycles were used within each phase. Two PICU nurse champions were engaged to provide bedside support, education and feedback.

In the 8-month period following implementation, there was a significant sustained reduction in LFTs requests. A similar pattern of sustained reduction also occurred for FBC, coagulation screens and CRP requests. This sustained reduction in blood tests requested equated to a saving in excess of £36,000.

This project was successful: the reduction in the number of inappropriate blood tests had clear financial benefit for PICU and reduced blood loss for patients. Early engagement and support from key stakeholders avoided conflict, guaranteed data sharing and aided engagement of bedside nurses.
Gender, race and the presentation of acute coronary syndrome and serious cardiopulmonary diagnoses in ED patients with chest pain
Authors: Allabban Ahmed , Hollander Judd E , Pines Jesse M
Published: June 2017

Objective

To assess the relationship between reported chest pain symptoms and a diagnosis of acute coronary syndrome (ACS) and serious cardiopulmonary diagnoses (SCPD) in black males, white males, black females and white females.

Methods

This was a secondary analysis of a prospective cohort study of 4162 ED patients with chest pain enrolled between 1999 and 2008. We used logistic regression, adjusting for age and cardiovascular comorbidities to test the association between 24 chest pain symptoms and 30-day ACS for the primary outcome and SCPD as the secondary outcome.

Result

In black males, diaphoresis was associated with ACS (OR 1.47; 95% CI 1.02 to 2.13), while in white males, left arm radiation, pressure/tightness and substernal pain were associated with ACS (OR 1.73, 95% CI 1.16 to 2.59; OR 1.65, 95% CI 1.16 to 2.59; OR 1.51, 95% CI 1.07 to 2.11, respectively). In black females, diaphoresis, palpitations and left arm radiation were associated with ACS (OR 1.66, 95% CI 1.17 to 2.35; 1.66, 95% CI 1.13 to 2.45; 1.44, 95% CI 1.02 to 2.03, respectively) while pleuritic pain, and left anterior chest pain lowered ACS risk (OR 0.69, 95% CI 0.5 to 0.96; 0.54, 95% CI 0.35 to 0.84). No symptoms predicted ACS or SCPD in white females. Fewer but similar symptoms predicted SCPD in white males and black females. No symptoms predicted SCPD in black males.

Conclusion

Chest pain symptoms are important predictors of ACS and SCPD in certain combinations of race and gender but less so in others. These differences might explain difficulties using symptoms to identify patients at higher or lower risk of ACS and SCPD in practice.
A homozygous founder mutation inTRAPPC6Bassociates with a neurodevelopmental disorder characterised by microcephaly, epilepsy and autistic features
Authors: Marin-Valencia Isaac , Novarino Gaia , Johansen Anide , Rosti Basak , Issa Mahmoud Y , Musaev Damir , Bhat Gifty , Scott Eric , Silhavy Jennifer L , Stanley Valentina , Rosti Rasim O , Gleeson Jeremy W , Imam Farhad B , Zaki Maha S , Gleeson Joseph G
Published: June 2017

Background

Transport protein particle (TRAPP) is a multisubunit complex that regulates membrane trafficking through the Golgi apparatus. The clinical phenotype associated with mutations in various TRAPP subunits has allowed elucidation of their functions in specific tissues. The role of some subunits in human disease, however, has not been fully established, and their functions remain uncertain.

Objective

We aimed to expand the range of neurodevelopmental disorders associated with mutations in TRAPP subunits by exome sequencing of consanguineous families.

Methods

Linkage and homozygosity mapping and candidate gene analysis were used to identify homozygous mutations in families. Patient fibroblasts were used to study splicing defect and zebrafish to model the disease.

Results

We identified six individuals from three unrelated families with a founder homozygous splice mutation inTRAPPC6B, encoding a core subunit of the complex TRAPP I. Patients manifested a neurodevelopmental disorder characterised by microcephaly, epilepsy and autistic features, and showed splicing defect. Zebrafishtrappc6bmorphants replicated the human phenotype, displaying decreased head size and neuronal hyperexcitability, leading to a lower seizure threshold.

Conclusion

This study provides clinical and functional evidence of the role ofTRAPPC6Bin brain development and function.
The Gebra–Magia Complex: mass-transport processes reworking trough-mouth fans in the Central Bransfield Basin (Antarctica)
Authors: Casas D. , García M. , Bohoyo F. , Maldonado A. , Ercilla G.
Published: June 2017

The Gebra–Magia Complex is an important example of a submarine mass-movement composite located on the lower continental slope of the Antarctic Peninsula (Central Bransfield Basin). Continuous instability dynamics over time is inferred to have affected the palaeo-trough-mouth fans present in the study area.

The depositional architecture and the outstanding relief of the Gebra Valley, which is the most striking morphological feature in the area, determine the asymmetrical morphology of the complex. This complex is characterized, from east to west, by an open-slope margin flanking the sidewall of the Gebra Valley, the Gebra Valley itself and a SW margin that is connected to the Magia area by a large scar approximately 7.8 km to the SW.

The Gebra Valley is a Quaternary debris valley resulting from repeated large-scale mass-transport and cut-and-fill processes. In contrast, the Magia area is dominated by unchannelized sedimentary instability processes, resulting in a different sedimentary architecture and morphology. The near-surface sediments in the Gebra–Magia Complex document the continuous occurrence of recent mass movements, as also evidenced by flows transported downslope as unchannelized or channelized flows.

Climate and tectonic activity are considered the primary factors controlling the development of the complex.
5-HT causes splanchnic venodilation
Authors: Seitz Bridget M. , Orer Hakan S. , Krieger-Burke Teresa , Darios Emma S. , Thompson Janice M. , Fink Gregory D. , Watts Stephanie W.
Published: June 2017

Serotonin [5-hydroxytryptamine (5-HT)] causes relaxation of the isolated superior mesenteric vein, a splanchnic blood vessel, through activation of the 5-HT7receptor. As part of studies designed to identify the mechanism(s) through which chronic (≥24 h) infusion of 5-HT lowers blood pressure, we tested the hypothesis that 5-HT causes in vitro and in vivo splanchnic venodilation that is 5-HT7receptor dependent. In tissue baths for measurement of isometric contraction, the portal vein and abdominal inferior vena cava relaxed to 5-HT and the 5-HT1/7receptor agonist 5-carboxamidotryptamine; relaxation was abolished by the 5-HT7receptor antagonist SB-269970. Western blot analyses showed that the abdominal inferior vena cava and portal vein express 5-HT7receptor protein. In contrast, the thoracic vena cava, outside the splanchnic circulation, did not relax to serotonergic agonists and exhibited minimal expression of the 5-HT7receptor. Male Sprague-Dawley rats with chronically implanted radiotelemetry transmitters underwent repeated ultrasound imaging of abdominal vessels. After baseline imaging, minipumps containing vehicle (saline) or 5-HT (25 μg·kg−1·min−1) were implanted. Twenty-four hours later, venous diameters were increased in rats with 5-HT-infusion (percent increase from baseline: superior mesenteric vein, 17.5 ± 1.9; portal vein, 17.7 ± 1.8; and abdominal inferior vena cava, 46.9 ± 8.0) while arterial pressure was decreased (~13 mmHg). Measures returned to baseline after infusion termination. In a separate group of animals, treatment with SB-269970 (3 mg/kg iv) prevented the splanchnic venodilation and fall in blood pressure during 24 h of 5-HT infusion. Thus, 5-HT causes 5-HT7receptor-dependent splanchnic venous dilation associated with a fall in blood pressure.

NEW & NOTEWORTHY This research is noteworthy because it combines and links, through the 5-HT7receptor, an in vitro observation (venorelaxation) with in vivo events (venodilation and fall in blood pressure). This supports the idea that splanchnic venodilation plays a role in blood pressure regulation.
Microarray analysis of aging-associated immune system alterations in the rostral ventrolateral medulla of F344 rats
Authors: Balivada Sivasai , Ganta Chanran K. , Zhang Yongqing , Pawar Hitesh N. , Ortiz Richard J. , Becker Kevin G. , Khan Arshad M. , Kenney Michael J.
Published: June 2017

The rostral ventrolateral medulla (RVLM) is an area of the brain stem that contains diverse neural substrates that are involved in systems critical for physiological function. There is evidence that aging affects some neural substrates within the RVLM, although age-related changes in RVLM molecular mechanisms are not well established. The goal of the present study was to characterize the transcriptomic profile of the aging RVLM and to test the hypothesis that aging is associated with altered gene expression in the RVLM, with an emphasis on immune system associated gene transcripts. RVLM tissue punches from young, middle-aged, and aged F344 rats were analyzed with Agilent’s whole rat genome microarray. The RVLM gene expression profile varied with age, and an association between chronological age and specific RVLM gene expression patterns was observed [ P < 0.05, false discovery rate (FDR) < 0.3]. Functional analysis of RVLM microarray data via gene ontology profiling and pathway analysis identified upregulation of genes associated with immune- and stress-related responses and downregulation of genes associated with lipid biosynthesis and neurotransmission in aged compared with middle-aged and young rats. Differentially expressed genes associated with the complement system and microglial cells were further validated by quantitative PCR with separate RVLM samples ( P < 0.05, FDR < 0.1). The present results have identified age-related changes in the transcriptomic profile of the RVLM, modifications that may provide the molecular backdrop for understanding age-dependent changes in physiological regulation.
Effects of Nitrogen Rates and Time of Application on Yield of Maize: Rainfall Variability Influenced Time of N Application
Authors: Abebe Zerihun , Feyisa Hailu
Published: June 2017

Despite the fact that maize productivity is relatively better than other major cereal crops, its current productivity is still far below its potential productivity. N rate and time of application are among the major abiotic factors limiting the productivity of the crop. Because of such gaps, the experiment was conducted at Bako Agricultural Research Center in 2013 and 2014 cropping seasons to determine optimum N rate and time of application. Four levels of N rates (46, 69, 92, and 115 N kg ha−1) and four levels (T1, T2, T3, and T4) of different time of N application were arranged in factorial combinations. Moreover, previously recommended N and the control were arranged in a randomized complete block design with three replications. In 2013, the highest significant biomass yield (21.2 tha−1) was obtained at 115 N kg ha−1 and T4 followed by 69 N kg ha−1 at T1 and T2 and 92 N kg ha−1 at T2. In contrast, the highest grain yield in 2013 was obtained at 92 N kg ha−1 at T2 followed by 115 N kg ha−1 at either T2 or T4 and 69 N kg ha−1 at either T1 or T3 application time. Interestingly, a significant yield increase by 37% was obtained when 92 N kg ha−1 at the time of T2 was applied compared to previous recommended 110 N kg ha−1 rate and time of application. In 2014, however, the highest yield was recorded when 92 N kg ha−1 at T1 was used. Application of 46 N kg ha−1 at T2 showed statistically similar yield performance when compared with previous N recommendation. The lowest yield was recorded from the control plot in both years. In 2013, the maximum net profit and acceptable marginal rate of return (MMR) were obtained when 92 N kg ha−1 at T2 was used for maize production during erratic and heavy rainfall distribution, particularly at a time of N application. However, the maximum net benefit (30743 ETB ha−1) and acceptable MRR could be obtained when 92 N kg ha−1 at T1 was used if the rainfall amount and distribution are relatively uniform. In conclusion, application of 92 N kg ha−1 at T1 (10–15 DAP and 35–40 DAP) is the best N rate and time of application in good rainy seasons and hence recommended for the end users. However, in the case of erratic and heavy rainy seasons, application of 92 N kg ha−1 at three times application regimes (1/3 N at 10–15 days after planting (DAP), 1/3 N at 35–40 DAP and 55–60 DAP) should be used to get maximum profit and acceptable MRR.