bluuebunny/crossref_metadata_2025_split huggingface dataset:
Biochemical Analysis of Recombinant AlkJ from Pseudomonas putida Reveals a Membrane-Associated, Flavin Adenine Dinucleotide-Dependent Dehydrogenase Suitable for the Biosynthetic Production of Aliphatic Aldehydes
Authors: Kirmair Ludwig , Skerra Arne 
Published: February 2014

ABSTRACT

The noncanonical alcohol dehydrogenase AlkJ is encoded on the alkane-metabolizing
alk
operon of the mesophilic bacterium
Pseudomonas putida
GPo1. To gain insight into the enzymology of AlkJ, we have produced the recombinant protein in
Escherichia coli
and purified it to homogeneity using His
6
tag affinity and size exclusion chromatography (SEC). Despite synthesis in the cytoplasm, AlkJ was associated with the bacterial cell membrane, and solubilization with
n
-dodecyl-β-
d
-maltoside was necessary to liberate the enzyme. SEC and spectrophotometric analysis revealed a dimeric quaternary structure with stoichiometrically bound reduced flavin adenine dinucleotide (FADH
2
). The holoenzyme showed thermal denaturation at moderate temperatures around 35°C, according to both activity assay and temperature-dependent circular dichroism spectroscopy. The tightly bound coenzyme was released only upon denaturation with SDS or treatment with urea-KBr and, after air oxidation, exhibited the characteristic absorption spectrum of FAD. The enzymatic activity of purified AlkJ for 1-butanol, 1-hexanol, and 1-octanol as well as the
n
-alkanol derivative ω-hydroxy lauric acid methyl ester (HLAMe) was quantified in the presence of the artificial electron acceptors phenazine methosulfate (PMS) and 2,6-dichlorophenolindophenol (DCPIP), indicating broad substrate specificity with the lowest activity on the shortest alcohol, 1-butanol. Furthermore, AlkJ was able to accept as cosubstrates/oxidants the ubiquinone derivatives Q
0
and Q
1
, also in conjunction with cytochrome
c
, which suggests coupling to the bacterial respiratory chain of this membrane-associated enzyme in its physiological environment. Using gas chromatographic analysis, we demonstrated specific biocatalytic conversion by AlkJ of the substrate HLAMe to the industrially relevant aldehyde, thus enabling the biotechnological production of 12-amino lauric acid methyl ester via subsequent enzymatic transamination.
A Conserved Streptococcal Membrane Protein, LsrS, Exhibits a Receptor-Like Function for Lantibiotics
Authors: Biswas Saswati , Biswas Indranil 
Published: February 2014

ABSTRACT

Streptococcus mutans
strain GS-5 produces a two-peptide lantibiotic, Smb, which displays inhibitory activity against a broad spectrum of bacteria, including other streptococci. For inhibition, lantibiotics must recognize specific receptor molecules present on the sensitive bacterial cells. However, so far no such receptor proteins have been identified for any lantibiotics. In this study, using a powerful transposon mutagenesis approach, we have identified in
Streptococcus pyogenes
a gene that exhibits a receptor-like function for Smb. The protein encoded by that gene, which we named LsrS, is a membrane protein belonging to the CAAX protease family. We also found that nisin, a monopeptide lantibiotic, requires LsrS for its optimum inhibitory activity. However, we found that LsrS is not required for inhibition by haloduracin and galolacticin, both of which are two-peptide lantibiotics closely related to Smb. LsrS appears to be a well-conserved protein that is present in many streptococci, including
S. mutans
. Inactivation of SMU.662, an LsrS homolog, in
S. mutans
strains UA159 and V403 rendered the cells refractory to Smb-mediated killing. Furthermore, overexpression of LsrS in
S. mutans
created cells more susceptible to Smb. Although LsrS and its homolog contain the CAAX protease domain, we demonstrate that inactivation of the putative active sites on the LsrS protein has no effect on its receptor-like function. This is the first report describing a highly conserved membrane protein that displays a receptor-like function for lantibiotics.
Conditional, Temperature-Induced Proteolytic Regulation of Cyanobacterial RNA Helicase Expression
Authors: Tarassova Oxana S. , Chamot Danuta , Owttrim George W. 
Published: February 2014

ABSTRACT

Conditional proteolysis is a crucial process regulating the abundance of key regulatory proteins associated with the cell cycle, differentiation pathways, or cellular response to abiotic stress in eukaryotic and prokaryotic organisms. We provide evidence that conditional proteolysis is involved in the rapid and dramatic reduction in abundance of the cyanobacterial RNA helicase, CrhR, in response to a temperature upshift from 20 to 30°C. The proteolytic activity is not a general protein degradation response, since proteolysis is only present and/or functional in cells grown at 30°C and is only transiently active at 30°C. Degradation is also autoregulatory, since the CrhR proteolytic target is required for activation of the degradation machinery. This suggests that an autoregulatory feedback loop exists in which the target of the proteolytic machinery, CrhR, is required for activation of the system. Inhibition of translation revealed that only elongation is required for induction of the temperature-regulated proteolysis, suggesting that translation of an activating factor was already initiated at 20°C. The results indicate that
Synechocystis
responds to a temperature shift via two independent pathways: a CrhR-independent sensing and signal transduction pathway that regulates induction of
crhR
expression at low temperature and a CrhR-dependent conditional proteolytic pathway at elevated temperature. The data link the potential for CrhR RNA helicase alteration of RNA secondary structure with the autoregulatory induction of conditional proteolysis in the response of
Synechocystis
to temperature upshift.
Regulation of Expression of
abcA
and Its Response to Environmental Conditions
Authors: Villet Regis A. , Truong-Bolduc Que Chi , Wang Yin , Estabrooks Zoe , Medeiros Heidi , Hooper David C. 
Published: February 2014

ABSTRACT

The ATP-dependent transporter gene
abcA
in
Staphylococcus aureus
confers resistance to hydrophobic β-lactams. In strain ISP794,
abcA
is regulated by the transcriptional regulators MgrA and NorG and shares a 420-nucleotide intercistronic region with the divergently transcribed
pbp4
gene, which encodes the transpeptidase Pbp4. Exposure of exponentially growing cells to iron-limited media, oxidative stress, and acidic pH (5.5) for 0.5 to 2 h had no effect on
abcA
expression. In contrast, nutrient limitation produced a significant increase in
abcA
transcripts. We identified three additional regulators (SarA, SarZ, and Rot) that bind to the overlapping promoter region of
abcA
and
pbp4
in strain MW2 and investigated their role in the regulation of
abcA
expression. Expression of
abcA
is decreased by 10.0-fold
in vivo
in a subcutaneous abscess model.
In vitro
,
abcA
expression depends on
rot
and
sarZ
regulators. Moenomycin A exposure of strain MW2 produced an increase in
abcA
transcripts. Relative to MW2, the MIC of moenomycin was decreased 8-fold for MW2Δ
abcA
and increased 10-fold for the MW2
abcA
overexpresser, suggesting that moenomycin is a substrate of AbcA.
The
mthA
Mutation Conferring Low-Level Resistance to Streptomycin Enhances Antibiotic Production in Bacillus subtilis by Increasing the
S
-Adenosylmethionine Pool Size
Authors: Tojo Shigeo , Kim Ji-Yun , Tanaka Yukinori , Inaoka Takashi , Hiraga Yoshikazu , Ochi Kozo 
Published: February 2014

ABSTRACT

Certain Str
r
mutations that confer low-level streptomycin resistance result in the overproduction of antibiotics by
Bacillus subtilis
. Using comparative genome-sequencing analysis, we successfully identified this novel mutation in
B. subtilis
as being located in the
mthA
gene, which encodes
S
-adenosylhomocysteine/methylthioadenosine nucleosidase, an enzyme involved in the
S
-adenosylmethionine (SAM)-recycling pathways. Transformation experiments showed that this
mthA
mutation was responsible for the acquisition of low-level streptomycin resistance and overproduction of bacilysin. The
mthA
mutant had an elevated level of intracellular SAM, apparently acquired by arresting SAM-recycling pathways. This increase in the SAM level was directly responsible for bacilysin overproduction, as confirmed by forced expression of the
metK
gene encoding SAM synthetase. The
mthA
mutation fully exerted its effect on antibiotic overproduction in the genetic background of
rel

+

but not the
rel
mutant, as demonstrated using an
mthA relA
double mutant. Strikingly, the
mthA
mutation activated, at the transcription level, even the dormant ability to produce another antibiotic, neotrehalosadiamine, at concentrations of 150 to 200 μg/ml, an antibiotic not produced (<1 μg/ml) by the wild-type strain. These findings establish the significance of SAM in initiating bacterial secondary metabolism. They also suggest a feasible methodology to enhance or activate antibiotic production, by introducing either the
rsmG
mutation to
Streptomyces
or the
mthA
mutation to eubacteria, since many eubacteria have
mthA
homologues.
Effect of Bidirectional Rotation of An Acupuncture Needle at Li10 on Acupuncture Needle Sensation and Experimentally-Induced Contact Heat Pain in Healthy Human Volunteers
Authors: Benham Alex , Johnson Mark I 
Published: February 2014

Introduction There is insufficient evidence of a relationship between acupuncture needle sensations ( de qi) and hypoalgesia. The aim of this study was to investigate the effects of bidirectional needle rotation at LI10 on acupuncture needle sensations and heat pain thresholds. Methods Twenty-two healthy participants received one acupuncture needle at LI10 with bidirectional rotation of the needle in one experimental session and one acupuncture needle at LI10 with mock rotation in a separate session, in a randomised order. Measurements of heat pain thresholds were taken before needle insertion, during needle retention and 15 min after needle removal. At each measurement time point, participants rated needle sensations using the Massachusetts Acupuncture Sensation Scale (MASS) and a visual analogue scale (VAS) of overall intensity of needle sensation. Results Bidirectional needle rotation produced significantly higher scores for VAS, MASStotal, MASSpain and MASSsensation compared with mock rotation (all p<0.001). There were significantly higher pain thresholds relative to pre-intervention baseline during (p=0.014) and after (p<0.001) bidirectional needle rotation but not during (p=0.1) or after (p=0.62) mock bidirectional needle rotation. Bidirectional needle rotation increased the pain threshold relative to baseline 15 min after the needles were removed (p=0.009). A significant but low correlation between needle sensation and change in pain threshold after needling was only found when data from mock and rotation interventions were combined. Conclusions Needle rotation increases the magnitude of hypoalgesia. There is tentative evidence that needle sensation may be associated with the amount of change in pain threshold.
Engagement techniques and playing level impact the biomechanical demands on rugby forwards during machine-based scrummaging
Authors: Preatoni Ezio , Stokes Keith A , England Michael E , Trewartha Grant 
Published: February 2014

ObjectivesThis cross-sectional study investigated the factors that may influence the physical loading on rugby forwards performing a scrum by studying the biomechanics of machine-based scrummaging under different engagement techniques and playing levels.Methods34 forward packs from six playing levels performed repetitions of five different types of engagement techniques against an instrumented scrum machine under realistic training conditions. Applied forces and body movements were recorded in three orthogonal directions.ResultsThe modification of the engagement technique altered the load acting on players. These changes were in a similar direction and of similar magnitude irrespective of the playing level. Reducing the dynamics of the initial engagement through a fold-in procedure decreased the peak compression force, the peak downward force and the engagement speed in excess of 30%. For example, peak compression (horizontal) forces in the professional teams changed from 16.5 (baseline technique) to 8.6 kN (fold-in procedure). The fold-in technique also reduced the occurrence of combined high forces and head-trunk misalignment during the absorption of the impact, which was used as a measure of potential hazard, by more than 30%. Reducing the initial impact did not decrease the ability of the teams to produce sustained compression forces.ConclusionsDe-emphasising the initial impact against the scrum machine decreased the mechanical stresses acting on forward players and may benefit players’ welfare by reducing the hazard factors that may induce chronic degeneration of the spine.
Cost-effectiveness of initial stress cardiovascular MR, stress SPECT or stress echocardiography as a gate-keeper test, compared with upfront invasive coronary angiography in the investigation and management of patients with stable chest pain: mid-term outcomes from the CECaT randomised controlled trial
Authors: Thom Howard , West Nicholas E J , Hughes Vikki , Dyer Matthew , Buxton Martin , Sharples Linda D , Jackson Christopher H , Crean Andrew M ,   The CECaT study group
Published: February 2014

ObjectivesTo compare outcomes and cost-effectiveness of various initial imaging strategies in the management of stable chest pain in a long-term prospective randomised trial.SettingRegional cardiothoracic referral centre in the east of England.Participants898 patients (69% man) entered the study with 869 alive at 2 years of follow-up. Patients were included if they presented for assessment of stable chest pain with a positive exercise test and no prior history of ischaemic heart disease. Exclusion criteria were recent infarction, unstable symptoms or any contraindication to stress MRI.Primary outcome measuresThe primary outcomes of this follow-up study were survival up to a minimum of 2 years post-treatment, quality-adjusted survival and cost-utility of each strategy.Results898 patients were randomised. Compared with angiography, mortality was marginally higher in the groups randomised to cardiac MR (HR 2.6, 95% CI 1.1 to 6.2), but similar in the single photon emission CT-methoxyisobutylisonitrile (SPECT-MIBI; HR 1.0, 95% CI 0.4 to 2.9) and ECHO groups (HR 1.6, 95% CI 0.6 to 4.0). Although SPECT-MIBI was marginally superior to other non-invasive tests there were no other significant differences between the groups in mortality, quality-adjusted survival or costs.ConclusionsNon-invasive cardiac imaging can be used safely as the initial diagnostic test to diagnose coronary artery disease without adverse effects on patient outcomes or increased costs, relative to angiography. These results should be interpreted in the context of recent advances in imaging technology.Trial registrationISRCTN 47108462, UKCRN 3696.
Validity of instruction leaflets for parents to measure their child's weight and height at home: results obtained from a randomised controlled trial
Authors: Huybrechts Inge , Beirlaen Celine , De Vriendt Tineke , Slimani Nadia , Pisa Pedro T , Schouppe Elien , De Coene Anja , De Bacquer Dirk , De Henauw Stefaan , Himes John H 
Published: February 2014

ObjectivesTo compare the validity of parent-reported height, weight and body mass index (BMI) values of children (aged 4–10 years), when measured at home by means of newly developed instruction leaflets in comparison with simple estimated parental reports.DesignRandomised controlled trial with control and intervention group using simple randomisation.SettingBelgian children and their parents recruited via schools (multistage cluster sampling design).Participants164 Belgian children (53% male; participation rate 62%).InterventionParents completed a questionnaire including questions about the height and weight of their child. Parents in the intervention group received instruction leaflets to measure their child's weight and height. Classes were randomly allocated to the intervention and control groups. Nurses measured height and weight following standardised procedures up to 2 weeks after parental reports.Outcome measuresWeight, height and BMI category of the child were derived from the index measurements and the parental reports.ResultsMean parent-reported weight was slightly more underestimated in the intervention group than in the control group relative to the index weights. However, for all three parameters (weight, height and BMI), correlations between parental reports and nurse measurements were higher in the intervention group. Sensitivity for underweight and overweight/obesity was respectively, 75% and 60% in the intervention group, and 67% and 43% in the control group. Weighed κ for classifying children in the correct BMI category was 0.30 in the control group and was 0.51 in the intervention group.ConclusionsAlthough mean parent-reported weight was slightly more underestimated in the intervention than in the control group, correlations were higher and there was considerably less misclassification into valid BMI categories for the intervention group. This pattern suggests that most of the parental deviations from the index measurements were probably due to random errors of measurement and that diagnostic measures could improve by encouraging parents to measure their children's weight and height at home by means of instruction leaflets.
Sick leave patterns as predictors of disability pension or long-term sick leave: a 6.75-year follow-up study in municipal eldercare workers
Authors: Stapelfeldt Christina Malmose , Nielsen Claus Vinther , Andersen Niels Trolle , Krane Line , Borg Vilhelm , Fleten Nils , Jensen Chris 
Published: February 2014

ObjectivesThe aim was to study whether a workplace-registered frequent short-term sick leave spell pattern was an early indicator of future disability pension or future long-term sick leave among municipal eldercare workers.SettingThe municipal healthcare sector in the city of Aarhus, which is the second largest city in Denmark.ParticipantsAll elder care employees who worked the entire year of 2004 in the municipality of Aarhus, Denmark (N=2774). The employees’ sick leave days during 2004 were categorised into: 0–2 and 3–17 short (1–7 days) spells, 2–13 mixed short and long (8+ days) spells and long spells only. Student workers (n=180), employees who were absent due to maternal/paternal leave (n=536) and employees who did not work the entire year of 2004 (n=1218) were not included.Primary outcomeDisability pension and long-term sick leave (≥8 weeks) were subsequently identified in a National register. The cumulative incidence proportion as a function of follow-up weeks was estimated using the Kaplan-Meier curve. The relative cumulative incidence (RR) of experiencing events within 352 weeks was analysed in a generalised linear regression model using the pseudo values method adjusted for age, occupation, unfavourable work factors and sick leave length.ResultsA frequent short-term and a mixed sick leave pattern showed RRs of being granted a disability pension of 2.08 (95% CI 1.00 to 4.35) and 2.61 (95% CI 1.33 to 5.12) compared with 0–2 short spells. The risk of long-term sick leave was significantly increased for all sick leave patterns compared with 0–2 short spells. Adding sick leave length to the models attenuated all RRs and they became non-significant.ConclusionsSick leave length was a better indicator of future workability than spell frequency. Preventive actions should target employees engaged in homecare. The more sick leave days the greater the preventive potential seems, irrespective of spell frequency.
Case definitions for chronic fatigue syndrome/myalgic encephalomyelitis (CFS/ME): a systematic review
Authors: Brurberg Kjetil Gundro , Fønhus Marita Sporstøl , Larun Lillebeth , Flottorp Signe , Malterud Kirsti 
Published: February 2014

ObjectiveTo identify case definitions for chronic fatigue syndrome/myalgic encephalomyelitis (CFS/ME), and explore how the validity of case definitions can be evaluated in the absence of a reference standard.DesignSystematic review.SettingInternational.ParticipantsA literature search, updated as of November 2013, led to the identification of 20 case definitions and inclusion of 38 validation studies.Primary and secondary outcome measureValidation studies were assessed for risk of bias and categorised according to three validation models: (1) independent application of several case definitions on the same population, (2) sequential application of different case definitions on patients diagnosed with CFS/ME with one set of diagnostic criteria or (3) comparison of prevalence estimates from different case definitions applied on different populations.ResultsA total of 38 studies contributed data of sufficient quality and consistency for evaluation of validity, with CDC-1994/Fukuda as the most frequently applied case definition. No study rigorously assessed the reproducibility or feasibility of case definitions. Validation studies were small with methodological weaknesses and inconsistent results. No empirical data indicated that any case definition specifically identified patients with a neuroimmunological condition.ConclusionsClassification of patients according to severity and symptom patterns, aiming to predict prognosis or effectiveness of therapy, seems useful. Development of further case definitions of CFS/ME should be given a low priority. Consistency in research can be achieved by applying diagnostic criteria that have been subjected to systematic evaluation.
Intracranial haemorrhage in thrombocytopenic haematology patients—a nested case–control study: the InCiTe study protocol
Authors: Estcourt Lise J , Stanworth Simon J , Collett Dave , Murphy Mike F 
Published: February 2014

IntroductionIntracranial haemorrhage (ICH) is one of the most serious side-effects of severe thrombocytopenia in haematology patients. ICH is rare, but can have devastating consequences (death or major morbidity). It is unknown why some patients with severe thrombocytopenia bleed and others do not.Study aimsPrimary aim was to identify risk factors for ICH in patients with haematological malignancies. Secondary aims were to identify short-term outcomes for these patients at 30 days (major morbidity and mortality) and produce a more accurate estimate of ICH incidence in this population. This information is key to identifying means to improve treatment and quality of care.Methods/analysisThis is a UK-wide case–control study of ICH nested within a 4-year prospective surveillance study set up specifically for the case–control study. Each case will be matched to one control. Cases will be adult haematology patients (≥16 years) who have had any type or severity of ICH who are receiving, about to receive or have just received myeloablative chemotherapy (defined as chemotherapy expected to cause a significant thrombocytopenia <50×109/L for >5 days) or a haemopoietic stem cell transplant. Only patients being treated with curative intent will be included. Controls will be patients who fulfil the same inclusion criteria as cases (apart from ICH) and were treated at the same hospital immediately before the index case. Cases and controls will be matched to type of treatment (myeloablative chemotherapy or haemopoietic stem cell transplant). Hospitals across the UK will participate in a monthly email reporting strategy (started June 2011), as to whether a case of ICH occurred during the preceding calendar month. Case and control forms will be sent to any hospital reporting an eligible case. Conditional logistic regression will be used to calculate ORs. Denominator data for incidence estimates will use national registry data.Study RegistrationISRCTN05026912 (prospective registration). NIHR Portfolio (UKCRN ID 10712).