bluuebunny/crossref_metadata_2025 huggingface dataset:
Proteomic and phenotypic profiling of the amphibian pathogen Batrachochytrium dendrobatidis shows that genotype is linked to virulence
Authors: FISHER MATTHEW C. , BOSCH JAIME , YIN ZHIKANG , STEAD DAVID A. , WALKER JANET , SELWAY LAURA , BROWN ALISTAIR J. P. , WALKER LOUISE A. , GOW NEIL A. R. , STAJICH JASON E. , GARNER TRENTON W. J.
Published: January 2009

Population genetics of the amphibian pathogen Batrachochytrium dendrobatidis (Bd) show that isolates are highly related and globally homogenous, data that are consistent with the recent epidemic spread of a previously endemic organism. Highly related isolates are predicted to be functionally similar due to low levels of heritable genetic diversity. To test this hypothesis, we took a global panel of Bd isolates and measured (i) the genetic relatedness among isolates, (ii) proteomic profiles of isolates, (iii) the susceptibility of isolates to the antifungal drug caspofungin, (iv) the variation among isolates in growth and phenotypic characteristics, and (v) the virulence of isolates against the European common toad Bufo bufo. Our results show (i) genotypic differentiation among isolates, (ii) proteomic differentiation among isolates, (iii) no significant differences in susceptibility to caspofungin, (iv) differentiation in growth and phenotypic/morphological characters, and (v) differential virulence in B. bufo. Specifically, our data show that Bd isolates can be profiled by their genotypic and proteomic characteristics, as well as by the size of their sporangia. Bd genotypic and phenotypic distance matrices are significantly correlated, showing that less‐related isolates are more biologically unique. Mass spectrometry has identified a set of candidate genes associated with inter‐isolate variation. Our data show that, despite its rapid global emergence, Bd isolates are not identical and differ in several important characters that are linked to virulence. We argue that future studies need to clarify the mechanism(s) and rate at which Bd is evolving, and the impact that such variation has on the host–pathogen dynamic.
Inferring the past to predict the future: climate modelling predictions and phylogeography for the freshwater gastropod Radix balthica (Pulmonata, Basommatophora)
Authors: CORDELLIER M. , PFENNINGER M.
Published: January 2009

Understanding the impact of past climatic events on species may facilitate predictions of how species will respond to future climate change. To this end, we sampled populations of the common pond snail Radix balthica over the entire species range (northwestern Europe). Using a recently developed analytical framework that employs ecological niche modelling to obtain hypotheses that are subsequently tested with statistical phylogeography, we inferred the range dynamics of R. balthica over time. A Maxent modelling for present‐day conditions was performed to infer the climate envelope for the species, and the modelled niche was used to hindcast climatically suitable range at the last glacial maximum (LGM) c. 21 000 years ago. Ecological niche modelling predicted two suitable areas at the LGM within the present species range. Phylogeographic model selection on a COI mitochondrial DNA data set confirmed that R. balthica most likely spread from these two disjunct refuges after the LGM. The match observed between the potential range of the species at the LGM given its present climatic requirements and the phylogeographically inferred refugial areas was a clear argument in favour of niche conservatism in R. balthica, thus allowing to predict the future range. The subsequent projection of the potential range under a global change scenario predicts a moderate pole‐ward shift of the northern range limits, but a dramatic loss of areas currently occupied in France, western Great Britain and southern Germany.
Effects of dynamic landscape elements on fish dispersal: the example of creek chub (Semotilus atromaculatus)
Authors: BOIZARD J. , MAGNAN P. , ANGERS B.
Published: January 2009

Barriers along a watercourse and interconnections between drainage systems are dynamic landscape elements that are expected to play major roles in the dispersal and genetic structure of fish species. The objective of this study was to assess the role of these elements using creek chub (Semotilus atromaculatus) in the Mastigouche Wildlife Reserve (Québec, Canada) as model. Numerous impassable waterfalls and interconnections among drainage systems were inferred with geographic information systems and confirmed de visu. The analysis of 32 populations using seven nuclear microsatellites revealed the presence of three genetically distinct groups. Some groups were found upstream of impassable barriers and in adjacent portions of distinct drainage systems. Admixture among groups was also detected in some populations. Constraining phylogenetic procedures as well as Mantel correlation tests confirmed that the genetic structure is more likely to result from interconnections between the drainage systems than from the permanent network. This study indicates that landscape elements such as interconnections are of major importance for circumventing impassable barriers and colonizing lakes that are otherwise inaccessible. Such an approach could be relevant for determining the origins of fish species (i.e. native vs. introduced) in the context of conservation.
Fine‐ and regional‐scale genetic structure of the exotic ascidian Styela clava (Tunicata) in southwest England, 50 years after its introduction
Authors: DUPONT L. , VIARD F. , DOWELL M. J. , WOOD C. , BISHOP J. D. D.
Published: January 2009

Styela clava, an ascidian native to the northwest Pacific, was first recorded in the Atlantic at Plymouth, southwest England, in 1953. It now ranges in the northeast Atlantic from Portugal to northern Denmark, and has colonized the east coast of North America. Within the region of first introduction, we aimed to characterize current genetic diversity in the species, elucidate the respective roles of human‐aided vs. natural dispersal, and assess the extent of larval dispersal by looking for genetic differentiation at very small scales. Eight sites, mostly marinas, were studied along c. 200 km of coast in southwest England encompassing Plymouth. Five microsatellite loci were genotyped in 303 individuals to analyse gene flow at regional (among sites) and fine (within sites) scales. F‐statistics and assignment tests were used to investigate regional genetic structure. At the fine scale, deviation from mutation–drift equilibrium was tested, and isolation by distance and genetic clustering analyses were undertaken. Significant genetic differentiation existed between sites, unrelated to geographical separation; migration between geographically distant marinas was inferred, highlighting the likely importance of human‐mediated dispersal in range expansion and occupancy by S. clava. Fine‐scale population structure was present within at least four sites, which may be explained by the limited dispersal ability of this ascidian and recruitment from differentiated pools of larvae. Populations in enclosed marinas had higher self‐recruitment rates than those in open sites. Some marinas might therefore function as reservoirs of propagules for subsequent spread, whereas others might be sinks for migrants.
Antigenic protein modifications in Ehrlichia
Authors: THOMAS S. , THIRUMALAPURA N. , CROSSLEY E. C. , ISMAIL N. , WALKER D. H
Published: January 2009

To develop effective vaccination strategies against Ehrlichia, we have previously reported developing an animal model of cross‐protection in which C57BL/6 mice primed with E. muris were resistant to lethal infection with Ixodes ovatus ehrlichia (IOE). Polyclonal antibody produced in mice after priming with E. muris and later injected with IOE‐detected antigenic proteins in E. muris and IOE cell lysates. Cross‐reaction of antigenic proteins was observed when we probed both the E. muris and IOE cell lysates with IOE and E. muris‐specific polyclonal antibody. Analysis of the total proteins of E. muris and IOE by two dimensional electrophoresis showed that both E. muris and IOE have the same antigenic proteins. Finally, studies on post‐translational protein modifications using a novel technique, Eastern blotting, showed that E. muris proteins are more lipoylated and glycosylated than those of IOE.
Neogene exhumation of the Marrakech High Atlas (Morocco) recorded by apatite fission‐track analysis
Authors: Balestrieri Maria Laura , Moratti Giovanna , Bigazzi Giulio , Algouti Ahmed
Published: January 2009

Apatite fission‐track (AFT) data have been obtained along a traverse across the Marrakech High Atlas to constrain its tectono‐thermal evolution. AFT ages vary between 212 ± 15 Ma and 20 ± 4 Ma. An Early Miocene AFT age accompanied by long mean track length from the central part of the chain has been interpreted as the timing of the main inversion of this region with the creation of relief because of the shortening induced by the interplay between the African and Eurasian plates. Thermal modelling of samples collected south of the South Atlas Fault Zone indicates a Middle‐Late Miocene or even later cooling that has been attributed to the component of the uplift of the chain related to the thermal anomaly present beneath the Atlas Mountains.
Identification of the novel protein QQS as a component of the starch metabolic network in Arabidopsis leaves
Authors: Li Ling , Foster Carol M. , Gan Qinglei , Nettleton Dan , James Martha G. , Myers Alan M. , Wurtele Eve Syrkin
Published: January 2009

Little is known about the role of proteins that lack primary sequence homology with any known motifs (proteins with unknown functions, PUFs); these comprise more than 10% of all proteins. This paper offers a generalized experimental strategy for identifying the functions of such proteins, particularly in relation to metabolism. Using this strategy, we have identified a novel regulatory function for Arabidopsis locus At3g30720 (which we term QQS for qua‐quine starch). QQS expression, revealed through global mRNA profiling, is up‐regulated in an Arabidopsis Atss3 mutant that lacks starch synthase III and has increased leaf starch content. Analysis of public microarray data using MetaOmGraph (metnetdb.org), in combination with transgenic Arabidopsis lines containing QQS promoter–GUS transgenes, indicated that QQS expression responds to a variety of developmental/genetic/environmental perturbations. In addition to the increase in the Atss3 mutant, QQS is up‐regulated in the carbohydrate mutants mex1 and sis8. A 586 nt sequence for the QQS mRNA was identified by 5′ and 3′ RACE experiments. The QQS transcript is predicted to encode a protein of 59 amino acids, whose expression was confirmed by immunological Western blot analysis. The QQS gene is recognizable in sequenced Arabidopsis ecotypes, but is not identifiable in any other sequenced species, including the closely related Brassica napus. Transgenic RNA interference lines in which QQS expression is reduced show excess leaf starch content at the end of the illumination phase of a diurnal cycle. Taken together, the data identify QQS as a potential novel regulator of starch biosynthesis.
The ABC transporter BcatrB from Botrytis cinerea exports camalexin and is a virulence factor on Arabidopsis thaliana
Authors: Stefanato Francesca L. , Abou‐Mansour Eliane , Buchala Antony , Kretschmer Matthias , Mosbach Andreas , Hahn Matthias , Bochet Christian G. , Métraux Jean‐Pierre , Schoonbeek Henk‐jan
Published: January 2009

Arabidopsis thaliana is known to produce the phytoalexin camalexin in response to abiotic and biotic stress. Here we studied the mechanisms of tolerance to camalexin in the fungus Botrytis cinerea, a necrotrophic pathogen of A. thaliana. Exposure of B. cinerea to camalexin induces expression of BcatrB, an ABC transporter that functions in the efflux of fungitoxic compounds. B. cinerea inoculated on wild‐type A. thaliana plants yields smaller lesions than on camalexin‐deficient A. thaliana mutants. A B. cinerea strain lacking functional BcatrB is more sensitive to camalexin in vitro and less virulent on wild‐type plants, but is still fully virulent on camalexin‐deficient mutants. Pre‐treatment of A. thaliana with UV‐C leads to increased camalexin accumulation and substantial resistance to B. cinerea. UV‐C‐induced resistance was not seen in the camalexin‐deficient mutants cyp79B2/B3, cyp71A13, pad3 or pad2, and was strongly reduced in ups1. Here we demonstrate that an ABC transporter is a virulence factor that increases tolerance of the pathogen towards a phytoalexin, and the complete restoration of virulence on host plants lacking this phytoalexin.
A microRNA–transcription factor module regulates lateral organ size and patterning in Arabidopsis
Authors: Larue Clayton T. , Wen Jiangqi , Walker John C.
Published: January 2009

Precise regulatory mechanisms are necessary to properly control the enlargement and patterning of plant lateral organs. However, our understanding of the regulatory modules that govern both of these processes is limited. An emerging theme in plant development is microRNA (miRNA)‐mediated gene regulation of transcription factors, including several NAC domain family members such as CUP‐SHAPED COTYLEDON2 (CUC2). We uncovered a novel allele of CUC2, cuc2‐1D, that revealed important functions of miRNAs and CUC2 in a regulatory module governing lateral organ enlargement and patterning. cuc2‐1D carried a single point mutation in the CUC2 miRNA target site, disrupting miRNA targeting. Disruption of the tight balance between CUC2 and its targeting miRNA, miRNA164, led to over‐accumulation of CUC2 mRNA and expansion of the CUC2 expression domain. cuc2‐1D plants had enlarged vegetative and reproductive lateral organs relative to wild‐type plants. Mechanistically, these enlarged organs resulted from an increase in cell proliferation that occurred over a longer developmental time frame relative to wild‐type. This organ enlargement was dependent on the receptor‐like kinase, ERECTA (ER). This and lateral organ patterning phenotypes in cuc2‐1D suggest that miRNA164 and CUC2 are critical regulators of both processes. Therefore, we propose that miRNA164 and CUC2 form a central regulatory module that acts as a governor of lateral organ patterning and expansion.
The vacuolar processing enzyme OsVPE1 is required for efficient glutelin processing in rice
Authors: Wang Yihua , Zhu Susong , Liu Shijia , Jiang Ling , Chen Liangming , Ren Yulong , Han Xiaohua , Liu Feng , Ji Sulan , Liu Xi , Wan Jianmin
Published: January 2009

Rice (Oryza sativa L.) accumulates prolamines and glutelins as its major storage proteins. Glutelins are synthesized on rough endoplasmic reticulum as 57‐kDa precursors; they are then sorted into protein storage vacuoles where they are processed into acidic and basic subunits. We report a novel rice glutelin mutant, W379, which accumulates higher levels of the 57‐kDa glutelin precursor. Genetic analysis revealed that the W379 phenotype is controlled by a single recessive nuclear gene. Using a map‐based cloning strategy, we identified this gene, OsVPE1, which is a homolog of the Arabidopsis βVPE gene. OsVPE1 encodes a 497‐amino‐acid polypeptide. Nucleotide sequence analysis revealed a missense mutation in W379 that changes Cys269 to Gly. Like the wild‐type protein, the mutant protein is sorted into vacuoles; however, the enzymatic activity of the mutant OsVPE1 is almost completely eliminated. Further, we show that OsVPE1 is incorrectly cleaved, resulting in a mature protein that is smaller than the wild‐type mature protein. Taken together, these results demonstrate that OsVPE1 is a cysteine protease that plays a crucial role in the maturation of rice glutelins. Further, OsVPE1 Cys269 is a key residue for maintaining the Asn‐specific cleavage activity of OsVPE1.
A soil bacterium regulates plant acquisition of iron via deficiency‐inducible mechanisms
Authors: Zhang Huiming , Sun Yan , Xie Xitao , Kim Mi‐Seong , Dowd Scot E. , Paré Paul W.
Published: January 2009

Despite the abundance of iron in nature, it is the third most limiting nutrient for plants due to its minimal solubility in most soils. While certain soil microbes produce chelating agents that enhance the solubility of iron, the effectiveness of such siderophores in the assimilation of iron by plants is debated. With an increasing understanding that select soil microbes play a signaling role in activating growth and stress responses in plants, the question arises as to whether such symbionts regulate iron assimilation. Here we report a previously unidentified mechanism in which the growth‐promoting bacterium Bacillus subtilis GB03 activates the plant’s own iron acquisition machinery to increase assimilation of metal ions in Arabidopsis. Mechanistic studies reveal that GB03 transcriptionally up‐regulates the Fe‐deficiency‐induced transcription factor 1 (FIT1), which is necessary for GB03‐induction of ferric reductase FRO2 and the iron transporter IRT1. In addition, GB03 causes acidification of the rhizosphere by enhancing root proton release and by direct bacterial acidification, thereby facilitating iron mobility. As a result, GB03‐exposed plants have elevated endogenous iron levels as well as increased photosynthetic capacity compared with water‐treated controls. In contrast, loss‐of‐function fit1‐2 mutants are compromised in terms of enhanced iron assimilation and photosynthetic efficiency triggered by GB03. In all studies reported herein, a physical partition separating roots from bacterial media precludes non‐volatile microbial siderophores from contributing to GB03‐stimulated iron acquisition. These results demonstrate the potential of microbes to control iron acquisition in plants and emphasize the sophisticated integration of microbial signaling in photosynthetic regulation.
Community intervention to promote rational treatment of acute respiratory infection in rural Nepal
Authors: Holloway Kathleen A , Karkee Shiba B , Tamang Ashalal , Gurung Yam Bahadur , Kafle Kumud K , Pradhan Ramesh , Reeves Barnaby C
Published: January 2009

Objective  To evaluate a community education program about treatment of acute respiratory infection (ARI).

Methods  First, community case definitions for severe and mild ARI were developed. The intervention was then evaluated using a controlled before‐and‐after design. Household surveys collected data about ARI treatment in 20 clusters, each based around a school and health facility. Treatment indicators included percentages of cases attending health facilities and receiving antibiotics. The intervention consisted of an education program in schools culminating in street theater performances, discussions with mothers after performances and training for community leaders and drug retailers by paramedics. The intervention was conducted in mid‐2003. Indicators were measured before the intervention in Nov/Dec 2002 and again in Dec 2003/Jan 2004.

Results  Two thousand and seven hundred and nineteen households were surveyed and 3654 under‐fives were identified, of whom 377 had severe ARI. After implementing the intervention, health post (HP) attendance rose by 13% in under‐fives with severe ARI and fell by 9% in under‐fives with mild ARI (test of interaction, P = 0.01). Use of prescribed antibiotics increased in under‐fives with severe ARI by 21% but only by 1% in under‐fives with mild ARI (test of interaction, P = 0.38). Irrespective of ARI severity, the use of non‐prescribed antibiotics dropped by 5% (P = 0.002), and consultation with female community health volunteers (FCHVs)and use of safe home remedies increased by 6.7% (P not estimated) and 5.7% (P = 0.008) respectively.

Conclusion  The intervention was implemented using local structures and in difficult circumstances, yet had a moderate impact. Thus it has the potential to effect large scale changes in behaviour and merits replication elsewhere.
Differential expression of the interleukin 5 receptor α isoforms in blood and tissue eosinophils of nasal polyp patients
Authors: Gevaert P. , Hellman C. , Lundblad L. , Lundahl J. , Holtappels G. , Van Cauwenberge P. , Tavernier J. , Bachert C.
Published: January 2009

Background:  Given the key role of interleukin‐5 (IL‐5) in eosinophil function, we investigated the regulated expression of the membrane‐anchored (TM‐IL‐5Rα) isoform, or a secreted (SOL IL‐5Rα) isoform, on both protein and transcript level in vitro and in vivo.

Methods:  A real‐time PCR, FACS and ELISA were established to determine IL‐5Rα isoform expression in peripheral blood and nasal tissue from control subjects and nasal polyp (NP) patients with or without asthma. Human peripheral blood eosinophils were incubated with IL‐5 and were analyzed for SOL‐IL‐5Rα and TM‐IL‐5Rα mRNA and protein levels in comparison with CD‐69 expression.

Results:  SOL‐IL‐5Rα and TM‐IL‐5Rα mRNA and protein expression was significantly increased in NP vs controls. In polyp tissue, SOL‐IL‐5Rα expression correlated to disease severity and eosinophils counts, whereas TM‐IL‐5Rα levels were inversely correlated to eosinophils counts and SOL‐IL‐5Rα expression. FACS analysis revealed increased CD‐69 and decreased TM‐IL‐5Rα expression in NP tissue eosinophils vs blood eosinophils. Incubation of blood eosinophils with IL‐5 caused up‐regulation of CD‐69 and down‐regulation of TM‐IL‐5Rα after 2 and 24 h.

Conclusion:  The expression of SOL‐IL‐5Rα and TM‐IL‐5Rα differs according to the eosinophil activation state and localization in the body (blood vs tissue) and may therefore be involved in the fine‐tuning of the eosinophil homeostasis. Exposure of eosinophils to IL‐5 reduces their responsiveness to IL‐5 by regulated expression of the IL‐5Rα isoforms. Since, TM‐IL‐5Rα is down‐regulated and SOL‐IL‐5Rα (antagonistic) is upregulated in NP tissue, our findings are important to understand the clinical trials with anti‐IL‐5 in humans.
Exposure to maternal smoking in the first year of life interferes in breast‐feeding protective effect against the onset of respiratory allergy from birth to 5 yr
Authors: Guedes H. T. V. , Souza L. S. F.
Published: January 2009

The aim of this study was to evaluate the interaction between the exclusive breast‐feeding protective effect and the exposure to tobacco smoke at domicile in the first year of life, on the onset of respiratory allergy (asthma and rhinitis) in children until 5 yr of age. This is prospective cohort study, observational, institutional based. Three hundred children born in a public hospital of Salvador‐Bahia (Brazil) were followed from birth to 5 yr of age. Data from 268 children at 60 months of life were analyzed. Occurrence of allergic symptoms were studied and correlated with gender, allergic relatives in first degree, exclusive breast‐feeding duration, smoking mother, and presence of other smoker at home, considering the first year of life. Exclusive breast‐feeding for at least 6 months showed a protection effect against the onset of respiratory allergy in children from birth to 5 yr (p < 0.05); odds ratio (OR): 0.33 (95% CI: 0.18–0.59). Breast‐fed children for less then 6 months compared with those breast‐fed for 6 months or more, presented a higher risk (OR: 2.34–95% CI: 1.4–3.74) for developing allergic respiratory symptoms just to 5 yr. The protective effect of exclusive prolonged breast‐feeding on the onset of respiratory allergy in children from birth until 5 yr was lost when their mothers were smokers (OR: 2.50–95% CI 1.19–5.19). Therefore, the protective effect of breast‐feeding in the first year of life on the onset of allergic symptoms until the age of 5 yr was confirmed. This study proposes a confounding effect of maternal smoking on this protection, exposed by a higher risk for present allergic symptoms until the age of 5 yr, in children exclusively breast‐fed for 6 months or more, when their mothers smoked.
Teamwork and patient safety in dynamic domains of healthcare: a review of the literature
Authors: MANSER T.
Published: January 2009

Aims/Background: This review examines current research on teamwork in highly dynamic domains of healthcare such as operating rooms, intensive care, emergency medicine, or trauma and resuscitation teams with a focus on aspects relevant to the quality and safety of patient care.

Results: Evidence from three main areas of research supports the relationship between teamwork and patient safety: (1) Studies investigating the factors contributing to critical incidents and adverse events have shown that teamwork plays an important role in the causation and prevention of adverse events. (2) Research focusing on healthcare providers' perceptions of teamwork demonstrated that (a) staff's perceptions of teamwork and attitudes toward safety‐relevant team behavior were related to the quality and safety of patient care and (b) perceptions of teamwork and leadership style are associated with staff well‐being, which may impact clinician' ability to provide safe patient care. (3) Observational studies on teamwork behaviors related to high clinical performance have identified patterns of communication, coordination, and leadership that support effective teamwork.

Conclusion: In recent years, research using diverse methodological approaches has led to significant progress in team research in healthcare. The challenge for future research is to further develop and validate instruments for team performance assessment and to develop sound theoretical models of team performance in dynamic medical domains integrating evidence from all three areas of team research identified in this review. This will help to improve team training efforts and aid the design of clinical work systems supporting effective teamwork and safe patient care.
Beneficial effects of high positive end‐expiratory pressure in lung respiratory mechanics during laparoscopic surgery
Authors: MARACAJÁ‐NETO L. F. , VERÇOSA N. , RONCALLY A. C. , GIANNELLA A. , BOZZA F. A. , LESSA M. A.
Published: January 2009

Background: The effect of neuromuscular blockade (NMB) and positive end‐expiratory pressure (PEEP) on the elastic properties of the respiratory system during pneumoperitoneum (PnP) remains a controversial subject. The main objective of the present study was to evaluate the effects of NMB and PEEP on respiratory mechanics.

Methods: We performed a dynamic analysis of respiratory mechanics in patients subjected to PnP. Twenty‐one patients underwent cholecystectomy videolaparoscopy and total intravenous anesthesia. The respiratory system resistance (RRS), pulmonary elastance (EP), chest wall elastance (ECW), and respiratory system elastance (ERS) were computed via the least squares fit technique using an equation describing the motion of the respiratory system, which uses primary signs such as airway pressure, tidal volume, air flow, and esophageal pressures. Measurements were taken after tracheal intubation, PnP, NMB, establishment of PEEP (10 cmH2O), and PEEP withdrawal [zero end‐expiratory pressure (ZEEP)].

Results: PnP significantly increased ERS by 27%; both EP and ECW increased 21.3 and 64.1%, respectively (P<0.001). NMB did not alter the respiratory mechanic properties. Setting PEEP reduced ERS by 8.6% (P<0.05), with a reduction of 10.9% in EP (P<0.01) and a significant decline of 15.7% in RRS (P<0.05). These transitory changes in elastance disappeared after ZEEP.

Conclusions: We concluded that the 10 cmH2O of PEEP attenuates the effects of PnP in respiratory mechanics, lowering RRS, EP, and ERS. These effects may be useful in the ventilatory approach for patients experiencing a non‐physiological increase in IAP owing to PnP in laparoscopic procedures.
Additives in intravenous anesthesia modulates pulmonary inflammation in a model of LPS‐induced respiratory distress
Authors: HAITSMA J. J. , LACHMANN B. , PAPADAKOS P. J.
Published: January 2009

Background: It has been suggested that propofol with ethylenediaminetetraacetic acid (EDTA) can modulate the systemic inflammatory response. Prolonged higher levels of pulmonary inflammation are associated with poor outcome of patients with acute lung injury. In the present study, we hypothesized that pulmonary inflammation could be modulated by propofol with EDTA compared with propofol with sulfite.

Methods: Respiratory distress was induced in rats (n=25) by intratracheal nebulization of lipopolysaccharide (LPS). After 24 h, animals were randomized to either propofol with EDTA (PropofolEDTA), propofol with sulfite (Propofolsulfite) or ketamine/midazolam (Ket/Mid); control animals received saline (n=30). Animals were ventilated for 4 h and blood gases were measured hourly. Bronchoalveolar lavage (BAL) was performed for cytokine analysis of: tumor necrosis factor (TNF), interleukin (IL)‐6 and macrophage inflammatory protein (MIP)‐2.

Results: LPS led to increased pulmonary inflammation in all groups compared with the control groups. Gas exchange deteriorated over time only in the LPS Propofolsulfite group and was significantly lower than the Ket/Mid group. Only IL‐6 was significantly higher in the LPS Propofolsulfite group compared with both the Ket/Mid group and the PropofolEDTA group.

Conclusion: Pulmonary IL‐6 can be modulated by additives in systemic anesthesia.

Implication Statement: This study demonstrates that pulmonary inflammation caused by direct lung injury can be modulated by intravenous anesthesia used in critically ill patients.
The successful use of peripheral nerve blocks for femoral amputation
Authors: BECH B. , MELCHIORS J. , BØRGLUM J. , JENSEN K.
Published: January 2009

We present a case report of four patients with severe cardiac insufficiency where peripheral nerve blocks guided by either nerve stimulation or ultrasonography were the sole anaesthetic for above‐knee amputation. The patients were breathing spontaneously and remained haemodynamically stable during surgery. Thus, use of peripheral nerve blocks for femoral amputation in high‐risk patients seems to be the technique of choice that can lower perioperative risk.
Microparticle sizing by dynamic light scattering in fresh‐frozen plasma
Authors: Lawrie A. S. , Albanyan A. , Cardigan R. A. , Mackie I. J. , Harrison P.
Published: January 2009

Background   We have previously shown that fresh‐frozen plasma (FFP) contains red blood cell‐derived procoagulant microparticles (MPs) that are removable by 0·2 µm filtration. Given the limitations of current methods for accurately sizing MPs, we have applied the novel approach of dynamic light scattering (DLS) to characterize the size distributions of these MPs within FFP.

Methods   Fresh‐frozen plasma was prepared from blood Group A and O donations (n = 10 of each) after an overnight hold of whole blood at 4°C. On the day of analysis, plasma was thawed to 37°C and daughter aliquots were studied pre‐ and post‐filtration (0·2 µm filtration device, Ceveron® MFU‐500, Technoclone). MP size and dispersity was assessed using a Zetasizer Nano S (Malvern Instruments Ltd), which employs a 173° backscatter detector and an N5 Submicron Particle Size Analyser (Beckman Coulter) using multi‐angle measurements (30·1°, 62·6° and 90°). The analysers presented MP size distribution graphically as intensity plots, mean size, standard deviation and polydispersity index.

Results   Of the instruments used, only the N5 utilizing a 30·1° angle of measurement could detect MPs of the expected size distribution and demonstrate their removal by filtration. MPs (range of mean particle diameters: pre, 101–464 nm; post, 21–182 nm filtration) were significantly smaller post‐filtration (P < 0·0001), but polydispersity index (median: pre, 0·746, post, 0·769) exhibited no significant change. There was no significant difference between the size of MPs from blood Group O (pre, 247 nm) and Group A (pre, 289 nm) samples (P = 0·44).

Conclusion   Our data demonstrates that DLS offers a novel approach to assessing MP size and distribution, a technique that could be easily adopted as a means of assessing MPs within either FFP or other blood products.
Establishment of a cell line expressing recombinant factor VII and its subsequent conversion to active form FVIIa through hepsin by genetic engineering method
Authors: Halabian R. , Roudkenar M. H. , Esmaeili N. S. , Masroori N. , Roushandeh A. M. , Najafabadi A. J.
Published: January 2009

Background  Factor VII (FVII) is a plasma glycoprotein that participates in the coagulation process leading to generation of fibrin. It is converted to factor VIIa that plays an important role in the coagulation cascade. The aim of this study was isolating and cloning the genes of human factor VII and hepsin and subsequent co‐transfection of the constructs to Chinese hamster ovary (CHO) cell line to obtain rFVIIa.

Methods  Factor VII and hepsin cDNAs were isolated from HepG2 cell line and cloned into pcDNA3·1 (+) vector. The constructs were co‐trasfected to CHO cell line. A cell line that permanently expressed recombinant factor VII (rFVII) and hepsin was established. The expression of rFVII was confirmed by reverse transcriptase–polymerase chain reaction, enzyme‐linked immunosorbent assay and Western blot analysis. Biological activity of rFVII was evaluated by prothrombin time assay.

Results  The results showed that the genes of FVII and hepsin were successfully cloned and expressed. Stable CHO cells co‐transfected with pcNDA3·1‐FVII and pcNDA3·1‐hepsin expressed FVII and hepsin mRNA, but there was no expression in the CHO cells transfected with insert free pcDNA3·1. FVIIa protein was secreted to medium of CHO cells co‐transfected with pcNDA3·1‐FVII and pcNDA3·1‐hepsin. The expected band of rFVII was detected in Western blot analysis. A three‐ to fourfold decrease in clotting time was observed when human FVII‐depleted plasma was used in combination with human thromboplastin in the presence of rFVII, confirming the biological activity of rFVII.

Conclusion  As we are aware, this is the first report of establishing a cell line expressing FVIIa using genetic engineering methods.
IMAGING LUNG AERATION AND LUNG LIQUID CLEARANCE AT BIRTH USING PHASE CONTRAST X‐RAY IMAGING
Authors: Hooper Stuart B , Kitchen Marcus J , Siew Melissa LL , Lewis Robert A , Fouras Andreas , B te Pas Arjan , Siu Karen KW , Yagi Naoto , Uesugi Kentaro , Wallace Megan J
Published: January 2009

The transition to extra‐uterine life at birth is critically dependent on airway liquid clearance to allow the entry of air and the onset of gaseous ventilation. We have used phase contrast X‐ray imaging to identify factors that regulate lung aeration at birth in spontaneously breathing term and mechanically ventilated preterm rabbit pups.

Phase contrast X‐ray imaging exploits the difference in refractive index between air and water to enhance image contrast, enabling the smallest air‐filled structures of the lung (alveoli; < 100 µm) to be resolved. Using this technique, the lungs become visible as they aerate, allowing the air–liquid interface to be observed as it moves distally during lung aeration.

Spontaneously breathing term rabbit pups rapidly aerate their lungs, with most fully recruiting their functional residual capacity (FRC) within the first few breaths. The increase in FRC occurs mainly during individual breaths, demonstrating that airway liquid clearance and lung aeration is closely associated with inspiration. We suggest that transpulmonary pressures generated by inspiration provide a hydrostatic pressure gradient for the movement of water out of the airways and into the surrounding lung tissue after birth.

In mechanically ventilated preterm pups, lung aeration is closely associated with lung inflation and a positive end‐expiratory pressure is required to generate and maintain FRC after birth.

In summary, phase contrast X‐ray imaging can image the air‐filled lung with high temporal and spatial resolution and is ideal for identifying factors that regulate lung aeration at birth in both spontaneously breathing term and mechanically ventilated preterm neonates.
EFFECTS OF EMULSIFIED ISOFLURANE ON HAEMODYNAMICS AND CARDIOMYOCYTE APOPTOSIS IN RATS WITH MYOCARDIAL ISCHAEMIA
Authors: Hu Zhao‐Yang , Liu Jin
Published: January 2009

It has been shown that inhaled isoflurane limits the size of myocardial infarcts. The aim of the present study was to examine the effects of emulsified isoflurane on cardiac function and myocardial apoptosis in an ischaemia model of myocardial injury.

In the first study, 48 rats were randomly allocated to six groups (n = 8 in each): control (saline); emulsified isoflurane (EIso) at 1, 2 or 4 mL/kg; 30% intralipid (vehicle for EIso); and sham operated. Rats received isovolumetric intravenous infusions for 30 min and then, 30 min after cessation of the infusion, 90 min coronary occlusion. Haemodynamics and myocardial infarct size were measured. In the second study, another 48 rats were randomized into six groups (n = 8 in each). After 90 min ischaemia, rats were killed for histopathological study, immunohistochemical evaluation and apoptosis measurement.

Pretreatment with 2 and 4 mL/kg EIso significantly attenuated decreases in left ventricular systolic pressure and dP/dtmax, and increases in left ventricular end‐diastolic pressure and –dP/dpmax, and alleviated myocardial injury compared with the control, intralipid and 1 mL/kg EIso groups (P < 0.05). Infusion of 1 mL/kg EIso and intralipid had no effect on haemodynamics, infarct size or histological variables.

Expression of Bcl‐2 was increased, whereas expression of Bax and caspase 3 was decreased, after preconditioning with 2 and 4 mL/kg EIso (P < 0.05). The apoptotic index in the 2 and 4 mL/kg Eiso‐treated groups was reduced compared with that in the control and intralipid groups (P < 0.01).

In conclusion, EIso ameliorates cardiac dysfunction, attenuates myocardial damage and inhibits apoptosis after ischaemia, which may be attributed, in part, to diminished expression of apoptosis‐related protein.
ACUTE EFFECTS OF NICOTINE ON ARTERIAL STIFFNESS AND WAVE REFLECTION IN HEALTHY YOUNG NON‐SMOKERS
Authors: Adamopoulos Dionysios , Argacha Jean‐François , Gujic Marko , Preumont Nicolas , Degaute Jean‐Paul , Van De Borne Philippe
Published: January 2009

Recently, we have demonstrated that cigarette smoke exposure proportionally increases plasma nicotine levels and arterial wave reflection to the aorta. However, the exact contribution of nicotine to the smoke‐induced enhancement of wave reflection and the potential underlying mechanisms have not been fully investigated.

The present study was a prospective study in 15 healthy male non‐smokers. All received a placebo and a 2 mg nicotine tablet, according to a randomized double‐blind cross‐over study design. Each subject underwent repeated measurements at baseline and for 1 h after nicotine or placebo intake, using carotid–femoral pulse wave velocity (PWV) to assess arterial compliance. Concurrently, aortic pressures and the augmentation index were evaluated using applanation tonometry.

Plasma nicotine concentrations achieved 1 h after intake of the nicotine tablet reached comparable levels to those achieved after 1 h exposure to passive smoke (3.6 ± 0.4vs3.2 ± 0.4 ng/mL, respectively;P = 0.4).

Nicotine enhanced arterial wave reflection to the aorta, as assessed by the augmentation index corrected for heart rate (4.2 ± 1.3vs–0.7 ± 0.8% with placebo;P = 0.001). In addition, a progressive increase in carotid–femoral PWV was noted after nicotine administration (0.3 ± 0.1vs–0.02 ± 0.1 m/s with placebo;P = 0.04). This remained significant even after adjustment for changes in mean blood pressure and heart rate (P = 0.01).

Plasma nicotine concentrations comparable to those achieved after exposure to passive smoke enhance arterial wave reflection to the aorta. This is accompanied by an increase in carotid–femoral PWV, denoting a deterioration of arterial compliance by nicotine.
CILOSTAMIDE PRODUCES HYPERPOLARIZATION ASSOCIATED WITH KATP CHANNEL ACTIVATION, BUT DOES NOT AUGMENT ENDOTHELIUM‐DERIVED HYPERPOLARIZATION IN RAT MESENTERIC ARTERIES
Authors: Kansui Yasuo , Goto Kenichi , Fujii Koji , Oniki Hideyuki , Matsumura Kiyoshi , Iida Mitsuo
Published: January 2009

Non‐nitric oxide/prostaglandin‐mediated endothelium‐derived hyperpolarization (EDH) is considered to be mediated, in part, by gap junctions and it has been suggested that cAMP increases endothelium‐derived hyperpolarizing factor (EDHF)‐mediated relaxation through the modulation of gap junctions. Cilostamide, which inhibits phosphodiesterase III, has been suggested to augment EDHF‐type relaxation by increasing the concentration of cAMP.

In the present study, we investigated the effect of cilostamide on EDH per se in mesenteric arteries of Wistar rats using a conventional microelectrode technique.

The resting membrane potential of the mesenteric arteries was significantly more negative in the presence of 10−6 mol/L cilostamide compared with control conditions. Furthermore, EDH in response to 10−6 mol/L acetylcholine (ACh) in the presence of 10−5 mol/L indomethacin and 10−4 mol/L NG‐nitro‐L‐arginine was decreased in the presence of 10−6 mol/L cilostamide by approximately 5 and 3.5 mV in proximal and distal arteries, respectively.

Glibenclamide (10−5 mol/L), an ATP‐sensitive potassium channel (KATP) inhibitor, abolished the hyperpolarization to 10−6 mol/L cilostamide. Furthermore, in the presence of glibenclamide, ACh‐induced EDH was unaffected by cilostamide, suggesting that the inhibition of ACh‐induced hyperpolarization by cilostamide in the absence of glibenclamide may be due to the smaller driving force for hyperpolarization because of the more negative membrane potential under such conditions.

The findings of the present study suggest that cilostamide produces hyperpolarization by activating KATP channels, presumably by increasing cAMP. However, cilostamide alone may not directly affect EDH.
MAGNOLOL DEPRESSES UROTENSIN‐II‐INDUCED CELL PROLIFERATION IN RAT CARDIAC FIBROBLASTS
Authors: Liou Jer‐Young , Chen Yen‐Ling , Loh Shih‐Hurng , Chen Po‐Yuan , Hong Chuang‐Ye , Chen Jin‐Jer , Cheng Tzu‐Hurng , Liu Ju‐Chi
Published: January 2009

Accumulating evidence suggests that oxidative stress plays a key role in the development of cardiac fibrosis. Urotensin‐II (U‐II) has been reported to play an important role in cardiac remodelling and fibrosis. Recently, we demonstrated the involvement of reactive oxygen species (ROS) production in U‐II‐induced cardiac fibroblast proliferation. Magnolol is an anti‐oxidant compound extracted from the cortices of Magnolia officinalis. Thus, it is feasible that magnolol may attenuate cardiac fibroblast proliferation by inhibiting ROS production. Therefore, the aims of the present study were to determine whether magnolol alters U‐II‐induced cell proliferation and to identify the putative underlying signalling pathways in rat cardiac fibroblasts.

Cultured rat cardiac fibroblasts were pretreated with magnolol (1, 3 and 10 µmol/L) for 30 min, followed by exposure to U‐II (30 nmol/L) for 24 h, after which cell proliferation and endothelin‐1 (ET‐1) protein secretion was examined. The effects of magnolol on U‐II‐induced ROS formation and extracellular signal‐regulated kinase (ERK) phosphorylation were examined to elucidate the intracellular mechanisms by which magnolol affects cell proliferation and ET‐1 expression.

Urotensin‐II (30 nmol/L) stimulated cell proliferation, ET‐1 protein secretion and ERK phosphorylation, all of which were inhibited by magnolol (10 µmol/L). Pretreatment of cardiac fibroblasts with N‐acetylcysteine (5 mmol/L) for 30 min prior to exposure to U‐II resulted in inhibition of U‐II increased ROS formation. Similar effects were observed with 10 µmol/L magnolol.

In conclusion, the results suggest that magnolol inhibits cardiac fibroblast proliferation by interfering with ROS generation. Thus, the present study provides important new insights into the molecular pathways involved, which may contribute to our understanding of the effects of magnolol on the cardiovascular system.
HIGH‐CHOLESTEROL DIET AUGMENTS ENDOTHELIAL DYSFUNCTION VIA ELEVATED OXIDATIVE STRESS AND REDUCED TETRAHYDROBIOPTERIN IN INS2AKITA MICE, AN AUTOSOMAL DOMINANT MUTANT TYPE 1 DIABETIC MODEL
Authors: Yang Xiang‐Qun , Chen Alex F
Published: January 2009

Oxidative stress contributes to endothelial dysfunction and atherogenesis in diabetes. The present study tested the hypothesis that a high‐cholesterol diet accelerates endothelial dysfunction in Ins2Akita mice, a Type 1 diabetic model with a spontaneous autosomal preproinsulin gene (Ins2 gene) mutation, through further increase of superoxide production.

The Ins2Akita diabetic mice were fed a high‐cholesterol diet (1.25% cholesterol) for 4 months. Some Ins2Akita mice were also treated for 4 months with the selective NADPH oxidase inhibitor apocynin (4 mg/kg per day in drinking water). Oxidative stress markers, tetrahydrobiopterin (BH4) levels, GTP cyclohydrolase I activity and endothelial function were determined in serum or arteries afterwards.

Serum lipid peroxidation and arterial superoxide levels were increased, whereas arterial BH4 levels and GTP cyclohydrolase I activity were decreased, in Ins2Akita mice on a high‐cholesterol diet, resulting in impaired endothelium‐dependent nitric oxide‐mediated relaxation in response to acetylcholine.

In vivo treatment with apocynin not only blunted serum lipid peroxidation and arterial superoxide levels, but also increased BH4 levels and GTP cyclohydrolase I activity, resulting in improved endothelium‐dependent relaxation.

These results suggest that NADPH oxidase may play a potential role in oxidative stress‐induced arterial BH4 and GTP cyclohydrolase I deficiency, resulting in endothelial dysfunction in Ins2Akita Type 1 diabetic mice fed a high‐cholesterol diet.
ISCHAEMIC POST‐CONDITIONING PROTECTS BOTH ADULT AND AGED SPRAGUE‐DAWLEY RAT HEART FROM ISCHAEMIA–REPERFUSION INJURY THROUGH THE PHOSPHATIDYLINOSITOL 3‐KINASE–AKT AND GLYCOGEN SYNTHASE KINASE‐3β PATHWAYS
Authors: Yin Zhong , Gao Haokao , Wang Haichang , Li Lansun , Di Chunxia , Luan Ronghua , Tao Ling
Published: January 2009

Numerous studies have demonstrated that ischaemic post‐conditioning (IPoC) protects adult rats from myocardial ischaemia‐reperfusion (I/R) injury. Recent evidence suggests compromised cardioprotection by IPoC in aged mice. The present study was designed to test the hypothesis that IPoC protects against I/R injury in aged hearts, potentially through a phosphatidylinositol 3‐kinase (PI3‐K)–Akt‐ and glycogen synthase kinase (GSK)‐3β‐dependent mechanism.

Hearts from adult (3–4 months) or aged (16–18 months) Sprague‐Dawley rats were subjected in vivo to 30 min ischaemia followed by 3 h reperfusion. Ischaemic post‐conditioning (four cycles of 10 s reperfusion–10 s ischaemia) was applied at the beginning of reperfusion, either alone or in combination with the PI3‐K inhibitor LY294002 (0.3 mg/kg). Infarct size and the phosphorylation of Akt and GSK‐3β were determined.

Ischaemic post‐conditioning reduced infarct size in both adult and aged rat hearts. This protection was accompanied by a significant increase in phosphorylation of Akt and GSK‐3β. LY294002 abolished the IPoC‐induced phosphorylation of Akt and GSK‐3β, as well as the infarct‐limiting effect of IPoC in adult and aged rats. In addition, IPoC significantly attenuated plasma concentrations of creatine kinase and lactate dehydrogenase after reperfusion in both adult and aged rats.

In conclusion, IPoC, at the onset of reperfusion, reduces myocardial infarct size in both adult and aged rat hearts, potentially through a PI3‐K‐, Akt‐ and GSK‐3β‐dependent mechanism.