bluuebunny/crossref_metadata_2025 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

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

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

MgtC 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

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

Background

Sudden 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.

Methods

Cross-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.

Results

Seventy-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.

Conclusions

While 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

Background

Accumulating 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.

Methods

A 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.

Results

A 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).

Conclusions

This 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

Objective

The 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.

Design

An online questionnaire based on the national guidelines for aetiological investigations for deafness was designed.

Setting

The 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.

Participants

The questionnaire was sent to 100 recipients; from this 52 responses were obtained.

Outcome measures

Variability in the investigations offered for hearing loss.

Results

There 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.

Conclusions

There 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

Objectives

In 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.

Design

An educational study.

Setting

Simulation centre in a medical school.

Participants

113 final-year medical students.

Primary and secondary outcomes

The 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.

Results

During 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.

Conclusions

We 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

Objectives

To 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.

Design

Prospective follow-up study.

Setting

In all, 400 primary healthcare centres and occupational healthcare clinics in Finland.

Participants

We 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.

Interventions

Lifestyle intervention (individual and/or group-based counselling).

Primary outcome measures

Incidence of T2D and fasting and 2 h glucose measured at baseline and follow-up.

Results

A 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).

Conclusions

The 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

Objectives

To 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).

Methods

The 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.

Results

There 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.

Conclusions

The 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

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

Objectives

To 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.

Methods

A 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.

Results

For 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.

Conclusions

A 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.
THE VLASOV–POISSON–BOLTZMANN SYSTEM FOR SOFT POTENTIALS
Authors: DUAN RENJUN , YANG TONG , ZHAO HUIJIANG
Published: September 2012

An important physical model describing the dynamics of dilute weakly ionized plasmas in the collisional kinetic theory is the Vlasov–Poisson–Boltzmann system for which the plasma responds strongly to the self-consistent electrostatic force. This paper is concerned with the electron dynamics of kinetic plasmas in the whole space when the positive charged ion flow provides a spatially uniform background. We establish the global existence and optimal convergence rates of solutions near a global Maxwellian to the Cauchy problem on the Vlasov–Poisson–Boltzmann system for angular cutoff soft potentials with -2 ≤ γ < 0. The main idea is to introduce a time-dependent weight function in the velocity variable to capture the singularity of the cross-section at zero relative velocity.
KINETIC LIMITS FOR PAIR-INTERACTION DRIVEN MASTER EQUATIONS AND BIOLOGICAL SWARM MODELS
Authors: CARLEN ERIC , DEGOND PIERRE , WENNBERG BERNT
Published: September 2012

We consider a class of stochastic processes modeling binary interactions in an N-particle system. Examples of such systems can be found in the modeling of biological swarms. They lead to the definition of a class of master equations that we call pair-interaction driven master equations. In the spatially homogeneous case, we prove a propagation of chaos result for this class of master equations which generalizes Mark Kac's well-known result for the Kac model in kinetic theory. We use this result to study kinetic limits for two biological swarm models. We show that propagation of chaos may be lost at large times and we exhibit an example where the invariant density is not chaotic.
MAXIMAL COMMUTATIVE SUBRINGS AND SIMPLICITY OF ORE EXTENSIONS
Authors: ÖINERT JOHAN , RICHTER JOHAN , SILVESTROV SERGEI D.
Published: September 2012

The aim of this paper is to describe necessary and sufficient conditions for simplicity of Ore extension rings, with an emphasis on differential polynomial rings. We show that a differential polynomial ring, R[x; id R, δ], is simple if and only if its center is a field and R is δ-simple. When R is commutative we note that the centralizer of R in R[x; σ, δ] is a maximal commutative subring containing R and, in the case when σ = id R, we show that it intersects every nonzero ideal of R[x; id R, δ] nontrivially. Using this we show that if R is δ-simple and maximal commutative in R[x; id R, δ], then R[x; id R, δ] is simple. We also show that under some conditions on R the converse holds.
STRATIFYING PAIRS OF SUBCATEGORIES FOR CPS-STRATIFIED ALGEBRAS
Authors: ÁGOSTON ISTVÁN , LUKÁCS ERZSÉBET
Published: September 2012

Two special types of module subcategories are defined over stratified algebras of Cline, Parshall and Scott. We show that for every stratified algebra there exists a (not necessarily unique) cotorsion pair of subcategories which describe to a large extent the stratification structure of the algebra. These subcategories generalize the notion of modules with standard and costandard filtration for standardly stratified and quasi-hereditary algebras.
AN INTRODUCTION TO DSmT FOR INFORMATION FUSION
Authors: ONERA JEAN DEZERT , SMARANDACHE FLORENTIN
Published: September 2012

The management and combination of uncertain, imprecise, fuzzy and even paradoxical or highly conflicting sources of information has always been, and still remains today, of primal importance for the development of reliable modern information systems involving artificial and approximate reasoning. In this short paper, we present an introduction of our recent theory of plausible and paradoxical reasoning, known as Dezert-Smarandache Theory (DSmT), developed to deal with imprecise, uncertain and conflicting sources of information. We focus our presentation on the foundations of DSmT and on its most important rules of combination, rather than on browsing specific applications of DSmT available in literature. Several simple examples are given throughout this presentation to show the efficiency and the generality of this new theory.
Genetics ofBorrelia burgdorferi
Authors: Brisson Dustin , Drecktrah Dan , Eggers Christian H. , Samuels D. Scott
Published: September 2012

The spirochetes in the Borrelia burgdorferi sensu lato genospecies group cycle in nature between tick vectors and vertebrate hosts. The current assemblage of B. burgdorferi sensu lato, of which three species cause Lyme disease in humans, originated from a rapid species radiation that occurred near the origin of the clade. All of these species share a unique genome structure that is highly segmented and predominantly composed of linear replicons. One of the circular plasmids is a prophage that exists as several isoforms in each cell and can be transduced to other cells, likely contributing to an otherwise relatively anemic level of horizontal gene transfer, which nevertheless appears to be adequate to permit strong natural selection and adaptation in populations of B. burgdorferi. Although the molecular genetic toolbox is meager, several antibiotic-resistant mutants have been isolated, and the resistance alleles, as well as some exogenous genes, have been fashioned into markers to dissect gene function. Genetic studies have probed the role of the outer membrane lipoprotein OspC, which is maintained in nature by multiple niche polymorphisms and negative frequency-dependent selection. One of the most intriguing genetic systems in B. burgdorferi is vls recombination, which generates antigenic variation during infection of mammalian hosts.
RNA as a Structural and Regulatory Component of the Centromere
Authors: Gent Jonathan I. , Dawe R. Kelly
Published: September 2012

Despite many challenges, great progress has been made in identifying kinetochore proteins and understanding their overall functions relative to spindles and centromeric DNA. In contrast, less is known about the specialized centromeric chromatin environment and how it may be involved in regulating the assembly of kinetochore proteins. Multiple independent lines of evidence have implicated transcription and the resulting RNA as an important part of this process. Here, we summarize recent literature demonstrating the roles of centromeric RNA in regulating kinetochore assembly and maintenance. We also review literature suggesting that the process of centromeric transcription may be as important as the resulting RNA and that such transcription may be involved in recruiting the centromeric histone variant CENH3.
Dosage Compensation of the Sex Chromosomes
Authors: Disteche Christine M.
Published: September 2012

Differentiated sex chromosomes evolved because of suppressed recombination once sex became genetically controlled. In XX/XY and ZZ/ZW systems, the heterogametic sex became partially aneuploid after degeneration of the Y or W. Often, aneuploidy causes abnormal levels of gene expression throughout the entire genome. Dosage compensation mechanisms evolved to restore balanced expression of the genome. These mechanisms include upregulation of the heterogametic chromosome as well as repression in the homogametic sex. Remarkably, strategies for dosage compensation differ between species. In organisms where more is known about molecular mechanisms of dosage compensation, specific protein complexes containing noncoding RNAs are targeted to the X chromosome. In addition, the dosage-regulated chromosome often occupies a specific nuclear compartment. Some genes escape dosage compensation, potentially resulting in sex-specific differences in gene expression. This review focuses on dosage compensation in mammals, with comparisons to fruit flies, nematodes, and birds.
Regulation of Insulin Secretion in Human Pancreatic Islets
Authors: Rorsman Patrik , Braun Matthias
Published: September 2012

Pancreatic β cells secrete insulin, the body's only hormone capable of lowering plasma glucose levels. Impaired or insufficient insulin secretion results in diabetes mellitus. The β cell is electrically excitable; in response to an elevation of glucose, it depolarizes and starts generating action potentials. The electrophysiology of mouse β cells and the cell's role in insulin secretion have been extensively investigated. More recently, similar studies have been performed on human β cells. These studies have revealed numerous and important differences between human and rodent β cells. Here we discuss the properties of human pancreatic β cells: their glucose sensing, the ion channel complement underlying glucose-induced electrical activity that culminates in exocytotic release of insulin, the cellular control of exocytosis, and the modulation of insulin secretion by circulating hormones and locally released neurotransmitters. Finally, we consider the pathophysiology of insulin secretion and the interactions between genetics and environmental factors that may explain the current diabetes epidemic.
IL-1β stimulation of CCD-18co myofibroblasts enhances repair of epithelial monolayers through Wnt-5a
Authors: Raymond Meera , Marchbank Tania , Moyer Mary P. , Playford Raymond J. , Sanderson Ian R. , Kruidenier Laurens
Published: September 2012

Subepithelial myofibroblasts are involved in the initiation and coordination of intestinal epithelial repair, but the molecular signaling pathways are largely unknown. The cellular adaptations that occur during repair range from dedifferentiation and migration to proliferation and redifferentiation, in a way that is strongly reminiscent of normal crypt-to-villus epithelial maturation. We therefore hypothesized that Wnt/β-catenin signaling may have a pivotal role in intestinal epithelial wound repair. We used the established scratch wound method in Caco-2 cells and in nontransformed NCM460 cells to monitor the effects of IL-1β-stimulated colonic myofibroblasts (CCD-18co) on intestinal epithelial repair, with immunoblotting and immunodepletion to examine the conditioned media. Conditioned media from IL-1β-stimulated, but not -untreated, myofibroblasts increased Caco-2 wound closure twofold over 24 h. IL-1β-stimulated myofibroblasts downregulated the differentiation marker sucrase-isomaltase in the Caco-2 cells, whereas the proliferation marker c-myc was upregulated. Array expression profiling identified Wnt-5a as the Wnt-related gene that was most upregulated (28-fold) by IL-1β stimulation of CCDs. Recombinant Wnt-5a enhanced proliferation of Caco-2 and NCM460 cells. In scratch assays, it increased migration of the leading edge in both cell lines. Wnt-5a immunodepletion of the IL-1β-CCD conditioned media abrogated the ability to enhance the repair. Wnt-5a often acts through a noncanonical signal transduction pathway. Further experiments supported this pathway in epithelial wound healing: IL-1β-CCD-mediated repair was not affected by the addition of the canonical Wnt antagonist Dickkopf-1. Furthermore, media from stimulated myofibroblasts (but not Wnt-5a-depleted media) increased c-jun in Caco-2 cell nuclear extracts. Myofibroblast-mediated noncanonical Wnt-5a signaling is therefore important in the dedifferentiation and migration stages of epithelial wound repair.
Cardiomyocyte ATP release through pannexin 1 aids in early fibroblast activation
Authors: Dolmatova Elena , Spagnol Gaelle , Boassa Daniela , Baum Jennifer R. , Keith Kimberly , Ambrosi Cinzia , Kontaridis Maria I. , Sorgen Paul L. , Sosinsky Gina E. , Duffy Heather S.
Published: September 2012

Fibrosis following myocardial infarction is associated with increases in arrhythmias and sudden cardiac death. Initial steps in the development of fibrosis are not clear; however, it is likely that cardiac fibroblasts play an important role. In immune cells, ATP release from pannexin 1 (Panx1) channels acts as a paracrine signal initiating activation of innate immunity. ATP has been shown in noncardiac systems to initiate fibroblast activation. Therefore, we propose that ATP release through Panx1 channels and subsequent fibroblast activation in the heart drives the development of fibrosis in the heart following myocardial infarction. We identified for the first time that Panx1 is localized within sarcolemmal membranes of canine cardiac myocytes where it directly interacts with the postsynaptic density 95/Drosophila disk large/zonula occludens-1-containing scaffolding protein synapse-associated protein 97 via its carboxyl terminal domain (amino acids 300–357). Induced ischemia rapidly increased glycosylation of Panx1, resulting in increased trafficking to the plasma membrane as well as increased interaction with synapse-associated protein 97. Cellular stress enhanced ATP release from myocyte Panx1 channels, which, in turn, causes fibroblast transformation to the activated myofibroblast phenotype via activation of the MAPK and p53 pathways, both of which are involved in the development of cardiac fibrosis. ATP release through Panx1 channels in cardiac myocytes during ischemia may be an early paracrine event leading to profibrotic responses to ischemic cardiac injury.
Genetic disruption of protein kinase Cδ reduces endotoxin-induced lung injury
Authors: Chichger Havovi , Grinnell Katie L. , Casserly Brian , Chung Chun-Shiang , Braza Julie , Lomas-Neira Joanne , Ayala Alfred , Rounds Sharon , Klinger James R. , Harrington Elizabeth O.
Published: September 2012

The pathogenesis of acute lung injury and acute respiratory distress syndrome is characterized by sequestration of leukocytes in lung tissue, disruption of capillary integrity, and pulmonary edema. PKCδ plays a critical role in RhoA-mediated endothelial barrier function and inflammatory responses. We used mice with genetic deletion of PKCδ (PKCδ−/−) to assess the role of PKCδ in susceptibility to LPS-induced lung injury and pulmonary edema. Under baseline conditions or in settings of increased capillary hydrostatic pressures, no differences were noted in the filtration coefficients ( kf) or wet-to-dry weight ratios between PKCδ+/+ and PKCδ−/− mice. However, at 24 h after exposure to LPS, the kf values were significantly higher in lungs isolated from PKCδ+/+ than PKCδ−/− mice. In addition, bronchoalveolar lavage fluid obtained from LPS-exposed PKCδ+/+ mice displayed increased protein and cell content compared with LPS-exposed PKCδ−/− mice, but similar changes in inflammatory cytokines were measured. Histology indicated elevated LPS-induced cellularity and inflammation within PKCδ+/+ mouse lung parenchyma relative to PKCδ−/− mouse lungs. Transient overexpression of catalytically inactive PKCδ cDNA in the endothelium significantly attenuated LPS-induced endothelial barrier dysfunction in vitro and increased kf lung values in PKCδ+/+ mice. However, transient overexpression of wild-type PKCδ cDNA in PKCδ−/− mouse lung vasculature did not alter the protective effects of PKCδ deficiency against LPS-induced acute lung injury. We conclude that PKCδ plays a role in the pathological progression of endotoxin-induced lung injury, likely mediated through modulation of inflammatory signaling and pulmonary vascular barrier function.
Maternal allergen exposure reprograms the developmental lung transcriptome in atopic and normoresponsive rat pups
Authors: Carpe Nicole , Mandeville Isabel , Kho Alvin T. , Qiu Weiliang , Martin James G. , Tantisira Kelan G. , Raby Benjamin A. , Weiss Scott T. , Kaplan Feige
Published: September 2012

The “fetal origins hypothesis” argued that physiological changes consequent to in utero exposures ultimately contribute to disease susceptibility in later life. The dramatic increase in asthma prevalence is attributed to early exposures acting on preexisting asthma-susceptible genotypes. We showed previously that distinct transcriptome signatures distinguish the developmental respiratory phenotype of atopic (Brown Norway, BN) and normoresponsive (Lewis) rats. We aimed to determine whether maternal allergen exposure would influence asthma pathogenesis by reprogramming primary patterns of developmental lung gene expression. Postnatal offspring of dams sensitized to ovalbumin before mating and challenged during pregnancy were assessed for lung function, inflammatory biomarkers, and respiratory gene expression. Although maternal ovalbumin exposure resulted in characteristic features of an allergic response (bronchoalveolar lavage neutrophils, IgE, methacholine-induced lung resistance) in offspring of both strains, substantial strain-specific differences were observed in respiratory gene expression. Of 799 probes representing the top 5% of transcriptomic variation, only 112 (14%) were affected in both strains. Strain-specific gene signatures also exhibited marked differences in enrichment for gene ontologies, with immune regulation and cell proliferation being prominent in the BN strain, cell cycle and microtubule assembly gene sets in the Lewis strain. Multiple ovalbumin-specific probes in both strains were also differentially expressed in lymphoblastoid cell lines from human asthmatic vs. nonasthmatic sibling pairs. Our data point to the existence of distinct, genetically programmed responses to maternal exposures in developing lung. These different response patterns, if recapitulated in human fetal development, can contribute to long-term pulmonary health including interindividual susceptibility to asthma.
Contributions of ocular vestibular evoked myogenic potentials and the electrooculogram to periocular potentials produced by whole-body vibration
Authors: Todd Neil P. M. , Bell Steven L. , Paillard Aurore C. , Griffin Michael J.
Published: September 2012

In this paper we report the results of an experiment to investigate the emergence of ocular vestibular evoked myogenic potentials (OVEMPs) during the linear vestibular ocular reflex (LVOR) evoked by whole-body vibration (WBV). OVEMP and electrooculogram (EOG) montages were employed to record periocular potentials (POPs) from six subjects during WBV in the nasooccipital (NO) axis over a range of frequencies from 0.5 to 64 Hz with approximately constant peak head acceleration of 1.0 ms−2 (i.e., 0.1 g). Measurements were made in two context conditions: a fixation context to examine the effect of gaze eccentricity (0 vs. 20°), and a visual context, where a target was either head-fixed or earth-fixed. The principal results are that from 0.5 to 2 Hz POP magnitude in the earth-fixed condition is related to head displacement, so with constant acceleration at all frequencies it reduces with increasing frequency, but at frequencies greater than 2 Hz both POP magnitude and POP gain, defined as the ratio of POP magnitude at 20 and 0°, increase with increasing frequency. By exhibiting this high-pass characteristic, a property shared with the LVOR, the results are consistent with the hypothesis that the OVEMP, as commonly employed in the clinical setting, is a high-frequency manifestation of the LVOR. However, we also observed low-frequency acceleration following POPs in head-fixed conditions, consistent with a low-frequency OVEMP, and found evidence of a high-frequency visual context effect, which is also consistent with the OVEMP being a manifestation of the LVOR.
Simvastatin attenuates sympathetic hyperinnervation to prevent atrial fibrillation during the postmyocardial infarction remodeling process
Authors: Yu Tao , Zhu Wei , Gu Beiyin , Li Shuai , Wang Fabing , Liu Mingya , Wei Meng , Li Jingbo
Published: September 2012

Statin, as a 3-hydroxy-3-methyl-glutaryl-CoA reductase inhibitor, has been shown to prevent atrial fibrillation (AF) due to its anti-inflammatory and antioxidant effects. However, it is still not known whether statin can improve autonomic remodeling to prevent AF. In the present study, using an in vivo rat myocardial infarction (MI) model, we aimed to test whether simvastatin can attenuate nerve sprouting and sympathetic hyperinnervation to prevent AF during the post-MI remodeling process. Our data demonstrate that simvastatin, delivered 3 days after MI for 4 wk, can result in significant decreases in plasma levels of both TNF-α (239 ± 23 pg/ml) and IL-1β (123 ± 11 pg/ml) compared with MI rats without therapy (TNF-α, 728 ± 57 pg/ml; IL-1β, 213 ± 21 pg/ml; P < 0.05), which, however, were still higher than sham-operated rats (TNF-α, 194 ± 20 pg/ml; IL-1β, 75 ± 8 pg/ml; P < 0.05). The similar pattern of changes in inflammation responses was also observed in TNF-α and IL-1β protein expression in the left atrium free wall. The suppressed inflammation responses were associated with reduced superoxide and malondialdehyde generation in the atrium. These changes account for decreases in neural growth factor expression at levels of both mRNA (1.2 ± 0.09 AU vs. MI group, 1.78 ± 0.16 AU) and protein (1.57 ± 0.17 AU vs. MI group, 2.24 ± 0.19 AU; P < 0.05), thus resulting in reduced nerve sprouting and sympathetic hyperinnervation. Accordingly, the rate adaptation of the atrial effective refractory period also recovered, leading to the decreased inducibility of AF. These data suggest that simvastatin administration after MI can prevent AF through reduced sympathetic hyperinnervation.
The brain renin-angiotensin system and cardiovascular responses to stress: insights from transgenic rats with low brain angiotensinogen
Authors: Arnold Amy C. , Sakima Atsushi , Kasper Sherry O. , Vinsant Sherry , Garcia-Espinosa Maria Antonia , Diz Debra I.
Published: September 2012

The renin-angiotensin system (RAS) has been identified as an attractive target for the treatment of stress-induced cardiovascular disorders. The effects of angiotensin (ANG) peptides during stress responses likely result from an integration of actions by circulating peptides and brain peptides derived from neuronal and glial sources. The present review focuses on the contribution of endogenous brain ANG peptides to pathways involved in cardiovascular responses to stressors. During a variety of forms of stress, neuronal pathways in forebrain areas containing ANG II or ANG-(1–7) are activated to stimulate descending angiotensinergic pathways that increase sympathetic outflow to increase blood pressure. We provide evidence that glia-derived ANG peptides influence brain AT1receptors. This appears to result in modulation of the responsiveness of the neuronal pathways activated during stressors that elevate circulating ANG peptides to activate brain pathways involving descending hypothalamic projections. It is well established that increased cardiovascular reactivity to stress is a significant predictor of hypertension and other cardiovascular diseases. This review highlights the importance of understanding the impact of RAS components from the circulation, neurons, and glia on the integration of cardiovascular responses to stressors.
Maximal strength training and increased work efficiency: contribution from the trained muscle bed
Authors: Barrett-O'Keefe Zachary , Helgerud Jan , Wagner Peter D. , Richardson Russell S.
Published: September 2012

Maximal strength training (MST) reduces pulmonary oxygen uptake (V̇o2) at a given submaximal exercise work rate (i.e., efficiency). However, whether the increase in efficiency originates in the trained skeletal muscle, and therefore the impact of this adaptation on muscle blood flow and arterial-venous oxygen difference (a-vO2diff), is unknown. Thus five trained subjects partook in an 8-wk MST intervention consisting of half-squats with an emphasis on the rate of force development during the concentric phase of the movement. Pre- and posttraining measurements of pulmonary V̇o2(indirect calorimetry), single-leg blood flow (thermodilution), and single-leg a-vO2diff(blood gases) were performed, to allow the assessment of skeletal muscle V̇o2during submaximal cycling [237 ± 23 W; ∼60% of their peak pulmonary V̇o2(V̇o2peak)]. Pulmonary V̇o2peak(∼4.05 l/min) and peak work rate (∼355 W), assessed during a graded exercise test, were unaffected by MST. As expected, following MST there was a significant reduction in pulmonary V̇o2during steady-state submaximal cycling (∼237 W: 3.2 ± 0.1 to 2.9 ± 0.1 l/min). This was accompanied by a significant reduction in single-leg V̇o2(1,101 ± 105 to 935 ± 93 ml/min) and single-leg blood flow (6,670 ± 700 to 5,649 ± 641 ml/min), but no change in single-leg a-vO2diff(16.7 ± 0.8 to 16.8 ±0.4 ml/dl). These data confirm an MST-induced reduction in pulmonary V̇o2during submaximal exercise and identify that this change in efficiency originates solely in skeletal muscle, reducing muscle blood flow, but not altering muscle a-vO2diff.