bluuebunny/crossref_metadata_2025_split huggingface dataset:
Novel System for Efficient Isolation of Clostridium Double-Crossover Allelic Exchange Mutants Enabling Markerless Chromosomal Gene Deletions and DNA Integration
Authors: Al-Hinai Mohab A. , Fast Alan G. , Papoutsakis Eleftherios T. 
Published: September 2012

ABSTRACT

Isolation of
Clostridium
mutants based on gene replacement via allelic exchange remains a major limitation for this important genus. Use of a heterologous counterselection marker can facilitate the identification of the generally rare allelic exchange events. We report on the development of an inducible counterselection marker and describe its utility and broad potential in quickly and efficiently generating markerless DNA deletions and integrations at any genomic locus without the need for auxotrophic mutants or the use of the mobile group II introns. This system is based on a codon-optimized
mazF
toxin gene from
Escherichia coli
under the control of a lactose-inducible promoter from
Clostridium perfringens
. This system is potentially applicable to almost all members of the genus
Clostridium
due to their similarly low genomic GC content and comparable codon usage. We isolated all allelic-exchange-based gene deletions (
ca_p0167
,
sigF
, and
sigK
) or disruptions (
ca_p0157
and
sigF
) we attempted and integrated a 3.6-kb heterologous DNA sequence (made up of a
Clostridium ljungdahlii
2.1-kb formate dehydrogenase [
fdh
] gene plus a FLP recombination target [FRT]-flanked thiamphenicol resistance marker) into the
Clostridium acetobutylicum
chromosome. Furthermore, we report on the development of a plasmid system with inducible segregational instability, thus enabling efficient deployment of the FLP-FRT system to generate markerless deletion or integration mutants. This enabled expeditious deletion of the thiamphenicol resistance marker from the
fdh
integrant strain as well as the
sigK
deletion strain. More generally, our system can potentially be applied to other organisms with underdeveloped genetic tools.
New Insight into the Ochratoxin A Biosynthetic Pathway through Deletion of a Nonribosomal Peptide Synthetase Gene in Aspergillus carbonarius
Authors: Gallo Antonia , Bruno Kenneth S. , Solfrizzo Michele , Perrone Giancarlo , Mulè Giuseppina , Visconti Angelo , Baker Scott E. 
Published: September 2012

ABSTRACT

Ochratoxin A (OTA), a mycotoxin produced by
Aspergillus
and
Penicillium
species, is composed of a dihydroisocoumarin ring linked to phenylalanine, and its biosynthetic pathway has not yet been completely elucidated. Most of the knowledge regarding the genetic and enzymatic aspects of OTA biosynthesis has been elucidated in
Penicillium
species. In
Aspergillus
species, only
pks
genes involved in the initial steps of the pathway have been partially characterized. In our study, the inactivation of a gene encoding a nonribosomal peptide synthetase (NRPS) in OTA-producing
A. carbonarius
ITEM 5010 has eliminated the ability of this fungus to produce OTA. This is the first report on the involvement of an
nrps
gene product in OTA biosynthetic pathway in an
Aspergillus
species. The absence of OTA and ochratoxin α, the isocoumaric derivative of OTA, and the concomitant increase of ochratoxin β, the dechloro analog of ochratoxin α, were observed in the liquid culture of transformed strain. The data provide the first evidence that the enzymatic step adding phenylalanine to polyketide dihydroisocoumarin precedes the chlorination step to form OTA in
A. carbonarius
and that ochratoxin α is a product of hydrolysis of OTA, giving an interesting new insight into the biosynthetic pathway of the toxin.
The C-Terminal Domain of the Virulence Factor MgtC Is a Divergent ACT Domain
Authors: Yang Yinshan , Labesse Gilles , Carrère-Kremer Séverine , Esteves Kevin , Kremer Laurent , Cohen-Gonsaud Martin , Blanc-Potard Anne-Béatrice 
Published: September 2012

ABSTRACTMgtC is a virulence factor of unknown function important for survival inside macrophages in several intracellular bacterial pathogens, includingMycobacterium tuberculosis. It is also involved in adaptation to Mg2+deprivation, but previous work suggested that MgtC is not a Mg2+transporter. In this study, we demonstrated that the amount of theM. tuberculosisMgtC protein is not significantly increased by Mg2+deprivation. Members of the MgtC protein family share a conserved membrane N-terminal domain and a more divergent cytoplasmic C-terminal domain. To get insights into MgtC functional and structural organization, we have determined the nuclear magnetic resonance (NMR) structure of the C-terminal domain ofM. tuberculosisMgtC. This structure is not affected by the Mg2+concentration, indicating that it does not bind Mg2+. The structure of the C-terminal domain forms a βαββαβ fold found in small molecule binding domains called ACT domains. However, theM. tuberculosisMgtC ACT domain differs from canonical ACT domains because it appears to lack the ability to dimerize and to bind small molecules. We have shown, using a bacterial two-hybrid system, that theM. tuberculosisMgtC protein can dimerize and that the C-terminal domain somehow facilitates this dimerization. Taken together, these results indicate thatM. tuberculosisMgtC does not have an intrinsic function related to Mg2+uptake or binding but could act as a regulatory factor based on protein-protein interaction that could be facilitated by its ACT domain.
Bacillus subtilis hlpB Encodes a Conserved Stand-Alone HNH Nuclease-Like Protein That Is Essential for Viability Unless the
hlpB
Deletion Is Accompanied by the Deletion of Genes Encoding the AddAB DNA Repair Complex
Authors: Pediaditakis Miriam , Kaufenstein Miriam , Graumann Peter L. 
Published: September 2012

ABSTRACT

The HNH domain is found in many different proteins in all phylogenetic kingdoms and in many cases confers nuclease activity. We have found that the
Bacillus subtilis
hlpB
(
yisB
) gene encodes a stand-alone HNH domain, homologs of which are present in several bacterial genomes. We show that the protein we term HlpB is essential for viability. The depletion of HlpB leads to growth arrest and to the generation of cells containing a single, decondensed nucleoid. This apparent condensation-segregation defect was cured by additional
hlpB
copies in
trans
. Purified HlpB showed cooperative binding to a variety of double-stranded and single-stranded DNA sequences, depending on the presence of zinc, nickel, or cobalt ions. Binding of HlpB was also influenced by pH and different metals, reminiscent of HNH domains. Lethality of the
hlpB
deletion was relieved in the absence of
addA
and of
addAB
, two genes encoding proteins forming a RecBCD-like end resection complex, but not of
recJ
, which is responsible for a second end-resectioning avenue. Like AddA-green fluorescent protein (AddA-GFP), functional HlpB-YFP or HlpB-FlAsH fusions were present throughout the cytosol in growing
B. subtilis
cells. Upon induction of DNA damage, HlpB-FlAsH formed a single focus on the nucleoid in a subset of cells, many of which colocalized with the replication machinery. Our data suggest that HlpB plays a role in DNA repair by rescuing AddAB-mediated recombination intermediates in
B. subtilis
and possibly also in many other bacteria.
Preparticipation cardiovascular screening among National Collegiate Athletic Association Division I Institutions
Authors: Coris Eric Emmanuel , Sahebzamani Frances , Curtis Anne , Jennings Jason , Walz Stephen M , Nugent Dylan , Reese Erika , Zwygart Kira K , Konin Jeff G , Pescasio Michele , Drezner Jonathan A 
Published: September 2012

BackgroundSudden cardiac arrest is the leading cause of death in competitive athletes during sport, and screening strategies for the prevention of sudden cardiac death are debated. The purpose of this study was to assess the incorporation of routine non-invasive cardiovascular screening (NICS), such as ECG or echocardiography, in Division I collegiate preparticipation examinations.MethodsCross-sectional survey of current screening practices sent to the head athletic trainer of all National Collegiate Athletic Association (NCAA) Division I football programmes listed in the National Athletic Trainers’ Association directory.ResultsSeventy-four of 116 (64%) programmes responded. Thirty-five of 74 (47%) of responding schools have incorporated routine NICS testing. ECG is the primary modality for NICS in 31 (42%) of schools, and 17 (49%) also utilise echocardiography. Sixty-four per cent of the programmes that do NICS routinely screen their athletes only once as incoming freshmen. Of institutions that do not conduct NICS, American Heart Association guidelines against routine NICS and cost were the most common reasons reported.ConclusionsWhile substantial debate exists regarding protocols for cardiovascular screening in athletes, nearly half of NCAA Division I football programmes in this study already incorporate NICS into their preparticipation screening programme. Additional research is needed to understand the impact of NICS in collegiate programmes.
‘Sedentary behaviour counselling’: the next step in lifestyle counselling in primary care; pilot findings from the Rapid Assessment Disuse Index (RADI) study
Authors: Shuval Kerem , DiPietro Loretta , Skinner Celette Sugg , Barlow Carolyn E , Morrow Jay , Goldsteen Robert , Kohl Harold W 
Published: September 2012

BackgroundAccumulating evidence emphasises a relationship between prolonged sitting and increased risk for cardiometabolic disorders and premature death irrespective of the protective effects of physical activity. Primary care physicians have the potential to play a key role in modifying patients’ sedentary behaviour alongside physical activity.MethodsA pilot study examining sedentary behaviour and physical activity counselling in a primary care clinic. A total of 157 patients completed a detailed survey related to lifestyle counselling received from their primary care physician. We analysed these responses to describe counselling practices within the 5A framework, and to examine correlates (ie, patients’ demographics, sedentary behaviour and physical activity and clinical variables) related to receiving counselling.ResultsA total of 10% received general advice to decrease sitting time, in comparison with 53% receiving general physical activity counselling. None, however, received a written plan pertaining to sedentary behaviour whereas 14% received a written physical activity prescription. Only 2% were provided with specific strategies for sedentary behaviour change in comparison with 10% for physical activity change. Multivariable analysis revealed that patients who were obese were more likely to receive counselling to decrease sitting (OR=7.0; 95% CI 1.4 to 35.2). In comparison, higher odds for receiving physical activity counselling were associated with being younger, aged 40–59 years (OR=2.4; 95% CI 1.1 to 5.4); and being a non-smoker (OR=6.1; 95% CI 1.3 to 28.4).ConclusionsThis study is the first to assess sedentary behaviour counselling practices in primary care and such practices appear to be infrequent. Future research should attempt to establish a ‘knowledge base’ to inform development of sedentary behaviour interventions, which should be followed by testing feasibility, efficacy, and subsequent effectiveness of these programmes in a clinical setting.
Deafness in children: a national survey of aetiological investigations
Authors: Rangan Shankar , Borgstein Bernie , Lowe Janet 
Published: September 2012

ObjectiveThe aim of this study was to obtain national data regarding adherence to national guidelines for aetiological investigations for hearing loss in children and highlight any variations in practice. Information was also collected on possible factors affecting lack of adherence.DesignAn online questionnaire based on the national guidelines for aetiological investigations for deafness was designed.SettingThe questionnaire was distributed to the leads of all the Newborn Hearing Screening Programme (NHSP) sites across England through the Medical Research Council Hearing & Communication Group.ParticipantsThe questionnaire was sent to 100 recipients; from this 52 responses were obtained.Outcome measuresVariability in the investigations offered for hearing loss.ResultsThere was a 52% response rate. Analysis of the responses showed that audiovestibular physicians and paediatricians in audiology were more likely than other specialists to request level 1 investigations (investigations that are recommended to be offered in all cases). Respondents from London and the North West were more likely to request level 1 investigations compared with those from other regions. In all, 14 of the 19 audiovestibular physicians and paediatricians in audiology requested level 1 investigations routinely, but only 11 of 33 from other specialties did likewise. Of the 20 respondents from London and the Northwest, 15 requested level 1 investigations routinely, whereas only 10 of the 32 respondents from the other regions did the same. The difference was statistically significant in both cases. The geographical variation was specially marked for family audiograms and MRI.ConclusionsThere is significant variation from the national guidelines in requesting aetiological investigations for permanent hearing impairment (PHI) in children, depending on the specialty of the clinician and the geographical region, these variations appear partly to be due to the availability of local resources but also due to lack of awareness of the importance of some investigations.
A metric-based analysis of structure and content of telephone consultations of final-year medical students in a high-fidelity emergency medicine simulation
Authors: Henn Patrick , Power David , Smith Simon D , Power Theresa , Hynes Helen , Gaffney Robert , McAdoo John D 
Published: September 2012

ObjectivesIn this study we aimed to analyse the structure and content of telephone consultations of final-year medical students in a high-fidelity emergency medicine simulation. The purpose was to identify any areas of deficiency within structure and content in the effective transfer of clinical information via the telephone of final-year medical students.DesignAn educational study.SettingSimulation centre in a medical school.Participants113 final-year medical students.Primary and secondary outcomesThe primary outcome was to analyse the structure and content of telephone consultations of final-year medical students in a high-fidelity emergency medicine simulation. The secondary outcome was to identify any areas of deficiency within structure and content in the effective transfer of clinical information via the telephone of final-year medical students.ResultsDuring phone calls to a senior colleague 30% of students did not positively identify themselves, 29% did not identify their role, 32% did not positively identify the recipient of the phone call, 59% failed to positively identify the patient, 49% did not read back the recommendations of their senior colleague and 97% did not write down the recommendations of their senior colleague.ConclusionsWe identified a deficiency in our students skills to communicate relevant information via the telephone, particularly failure to repeat back and write down instructions. We suggest that this reflects a paucity of opportunities to practice this skill in context during the undergraduate years. The assumption that this skill will be acquired following qualification constitutes a latent error within the healthcare system. The function of undergraduate medical education is to produce graduates who are fit for purpose at the point of graduation.
Do statins interfere with lifestyle intervention in the prevention of diabetes in primary healthcare? One-year follow-up of the FIN-D2D project
Authors: Rautio Nina , Jokelainen Jari , Oksa Heikki , Saaristo Timo , Peltonen Markku , Vanhala Mauno , Puolijoki Hannu , Moilanen Leena , Tuomilehto Jaakko , Keinänen-Kiukaanniemi Sirkka , Uusitupa Matti 
Published: September 2012

ObjectivesTo examine whether the use of statins is associated with the incidence of type 2 diabetes (T2D) and changes in glucose metabolism among individuals at high risk for T2D participating in 1-year lifestyle intervention in primary healthcare setting.DesignProspective follow-up study.SettingIn all, 400 primary healthcare centres and occupational healthcare clinics in Finland.ParticipantsWe screened altogether 10 149 individuals at increased risk for T2D; of these, 2798 non-diabetic individuals verified by a 2 h glucose tolerance test participated in the 1-year follow-up.InterventionsLifestyle intervention (individual and/or group-based counselling).Primary outcome measuresIncidence of T2D and fasting and 2 h glucose measured at baseline and follow-up.ResultsA total of 484 individuals (17.3%) used statins at the baseline. Of them 31 (7.5%) developed T2D during the follow-up, compared to 126 (6.5%) of statin non-users (OR 1.17, 95% CI 0.78 to 1.76, p=0.442). Interestingly, fasting glucose increased by 0.08 mmol/l in statin users, but remained unchanged in non-users, the difference being 0.074 mmol/l (95% CI 0.014 to 0.134) and remained significant even after adjustment for age, sex, baseline fasting glucose, the presence of cardiovascular disease (CVD), use of antihypertensive and/or coronary artery disease medication, weight and 1-year weight change (adjusted p=0.042).ConclusionsThe incidence of T2D did not differ between the statin users and non-users. The finding that fasting glucose slightly increased in statin users in spite of lifestyle interventions suggests the view that the use of statins might have unfavourable effects on glucose metabolism and that statins might hamper beneficial effects of lifestyle intervention in people at high risk of T2D.
Effectiveness of a five-level Paediatric Triage System: an analysis of resource utilisation in the emergency department in Taiwan
Authors: Chang Yu-Che , Ng Chip-Jin , Wu Chang-Teng , Chen Li-Chin , Chen Jih-Chang , Hsu Kuang-Hung 
Published: September 2012

ObjectivesTo examine the effectiveness of a five-level Paediatric Triage and Acuity System (Ped-TTAS) by comparing the reliability of patient prioritisation and resource utilisation with the four-level Paediatric Taiwan Triage System (Ped-TTS) among non-trauma paediatric patients in the emergency department (ED).MethodsThe study design used was a retrospective longitudinal analysis based on medical chart review and a computer database. Except for a shorter list of complaints and some abnormal vital sign criteria modifications, the structure and triage process for applying Ped-TTAS was similar to that of the Paediatric Canadian Emergency Triage and Acuity Scale. Non-trauma paediatric patients presenting to the ED were triaged by well-trained triage nurses using the four-level Ped-TTS in 2008 and five-level Ped-TTAS in 2010. Hospitalisation rates and medical resource utilisation were analysed by acuity levels between the contrasting study groups.ResultsThere was a significant difference in patient prioritisation between the four-level Ped-TTS and five-level Ped-TTAS. Improved differentiation was observed with the five-level Ped-TTAS in predicting hospitalisation rates and medical costs.ConclusionsThe five-level Ped-TTAS is better able to discriminate paediatric patients by triage acuity in the ED and is also more precise in predicting resource utilisation. The introduction of a more accurate acuity and triage system for use in paediatric emergency care should provide greater patient safety and more timely utilisation of appropriate ED resources.
Aortic intramural haematoma: pathogenesis, clinical features and imaging evaluation
Authors: Hoey Edward T D , Wai Debbie , Ganeshan Arul , Watkin Richard W 
Published: September 2012

Abstract
Intramural haematoma (IMH) is a localised haemorrhage within the aortic wall. Imaging plays a central role in diagnosing IMH, differentiating it from aortic dissection (AD) and assessing for complications. Imaging is also important for prognostication and to help guide clinical decision making as a number of imaging characteristics have been correlated with increased mortality rates including location, mural thickness and aortic diameter. Multidetector CT is the leading technique for diagnosis and classification of IMH owing to speed of image acquisition, multiplanar capabilities and excellent spatial resolution. MRI is rarely used to investigate the initial presentation of IMH but is frequently used for serial follow-up studies. The clinical outcome of IMH may be favourable, with spontaneous regression over time, or it may be complicated by pericardial tamponade, aortic regurgitation and development of AD. Early surgical management is the treatment of choice for patients with Stanford type A IMH whereas most patients with Stanford type B IMH have a good short-term outcome with aggressive control of hypertension. This article reviews the pathogenesis, clinical features and complications of IMH as well as the role of advanced imaging techniques in its evaluation.
Comparative effectiveness of a rapid point-of-care test for detection ofChlamydia trachomatisamong women in a clinical setting
Authors: Huang Wei , Gaydos Charlotte A , Barnes Mathilda R , Jett-Goheen Mary , Blake Diane R 
Published: September 2012

ObjectivesTo compare the effectiveness and cost-effectiveness of a promising new point-of-care (POC) chlamydia test with traditional nucleic acid amplification testing (NAAT), and to determine the characteristics that would make a POC test most cost-effective.MethodsA decision tree was constructed to model chlamydia screening visits to a sexually transmitted disease clinic by a hypothetical cohort of 10 000 women. The model incorporated programmatic screening costs, treatment costs and medical costs averted through prevention of pelvic inflammatory disease (PID) and its sequelae. Parameter values and costs were estimated for each node in the decision tree based on primary data, published data and unpublished health data.ResultsFor the base-case scenario (POC sensitivity 92.9%; 47.5% of women willing to wait 40 min for test results; test cost $33.48), POC was estimated to save US$5050 for each case of PID averted compared with NAAT. One-way sensitivity analyses indicated that POC would dominate NAAT if the POC test cost is <US$41.52 or if POC sensitivity is ≥87.1%. In a probabilistic sensitivity analysis (Monte Carlo simulations, 10 000 iterations), 10.8% of iterations indicated that the POC strategy dominated the NAAT strategy. The mean incremental cost-effectiveness ratio indicated that the POC strategy would save US$28 in total, and avert 14 PID cases.ConclusionsA promising new chlamydia POC test is likely to be cost-effective compared with traditional NAAT. The POC test sensitivity, cost and proportion of women willing to wait for the POC test result are key elements to determining the cost-effectiveness of any new POC test strategy.