bluuebunny/crossref_metadata_2025 huggingface dataset:
Functional expression of the olfactory signaling system in the kidney
Authors: Pluznick Jennifer L. , Zou Dong-Jing , Zhang Xiaohong , Yan Qingshang , Rodriguez-Gil Diego J. , Eisner Christoph , Wells Erika , Greer Charles A. , Wang Tong , Firestein Stuart , Schnermann Jurgen , Caplan Michael J.
Published: January 2009

Olfactory-like chemosensory signaling occurs outside of the olfactory epithelium. We find that major components of olfaction, including olfactory receptors (ORs), olfactory-related adenylate cyclase (AC3) and the olfactory G protein (G
olf
), are expressed in the kidney. AC3 and G
olf
colocalize in renal tubules and in macula densa (MD) cells which modulate glomerular filtration rate (GFR). GFR is significantly reduced in AC3
−/−
mice, suggesting that AC3 participates in GFR regulation. Although tubuloglomerular feedback is normal in these animals, they exhibit significantly reduced plasma renin levels despite up-regulation of COX-2 expression and nNOS activity in the MD. Furthermore, at least one member of the renal repertoire of ORs is expressed in a MD cell line. Thus, key components of olfaction are expressed in the renal distal nephron and may play a sensory role in the MD to modulate both renin secretion and GFR.
Active-site architecture and catalytic mechanism of the lipid A deacylase LpxR of
Salmonella typhimurium
Authors: Rutten Lucy , Mannie Jean-Paul B. A. , Stead Christopher M. , Raetz Christian R. H. , Reynolds C. Michael , Bonvin Alexandre M. J. J. , Tommassen Jan P. , Egmond Maarten R. , Trent M. Stephen , Gros Piet
Published: January 2009

The lipid A portion of lipopolysaccharide, the major component of the outer leaflet of the outer membrane of Gram-negative bacteria, is toxic to humans. Modification of lipid A by enzymes often reduces its toxicity. The outer-membrane protein LpxR from
Salmonella typhimurium
is a lipid A-modifying enzyme. It removes the 3′-acyloxyacyl moiety of the lipid A portion of lipopolysaccharide in a Ca
2+
-dependent manner. Here, we present the crystal structure of
S. typhimurium
LpxR, crystallized in the presence of zinc ions. The structure, a 12-stranded β-barrel, reveals that the active site is located between the barrel wall and an α-helix formed by an extracellular loop. Based on site-directed mutagenesis and modeling of a substrate on the active site, we propose a catalytic mechanism similar to that of phospholipase A2, in which a Ca
2+
forms the oxyanion hole and a histidine activates a water molecule (or a cascade of two water molecules) that subsequently attacks the carbonyl oxygen of the scissile bond.
Fractures of the fifth metacarpal
Authors: Gudmundsen T. E. , Borgen L.
Published: January 2009

Background: The literature is limited concerning the epidemiological issues of fracture in the fifth metacarpal bone.

Purpose: To explore the incidence and types of this fracture, and how the fracture is related to age, gender, trauma mechanism, and at what day of the week the causing trauma occurred.

Material and Methods: All hand fractures registered at the Department of Radiology, Buskerud Hospital, Norway, in the period between 2004 and 2006 were reviewed.

Results: Of 1475 hand fractures, 271 (18.4%) were located in the fifth metacarpal bone. In 130 of these cases (121 male, nine female), the trauma had occurred through aggression, such as a fistfight or from punching a hard object. The majority of fractures were located in the neck of the metacarpal bone (boxer's fracture). The median age of this group was 22 (7–51) years. Concerning the time of trauma, there was a significant peak during weekends (95 patients). One hundred forty-one of the fifth metacarpal fractures were caused by accidental trauma (99 males, 42 females). In this group, the fractures were located approximately equally in the subcapital, diaphyseal, and basal part of the metacarpal. The median age was 34 (2–90) years. The accidental traumas occurred about as often during the weekdays as during the weekend.

Conclusion: Of all hand fractures, fractures of the fifth metacarpal bone are the most common. There are as many aggression-related fractures as accidental fractures in the fifth metacarpal bone. When the fracture is caused by aggression, the typical patient is a young male sustaining fracture during the weekend.
MARGINAL DISTRIBUTIONS FOR COSMIC VARIANCE LIMITED COSMIC MICROWAVE BACKGROUND POLARIZATION DATA
Authors: Eriksen H. K. , Wehus I. K.
Published: January 2009

We provide computationally convenient expressions for all marginal distributions of the polarization cosmic microwave background (CMB) power spectrum distribution P(C
ℓ|σℓ), where C
ℓ = {C
TT
ℓ, C
TE
ℓ, C
EE
ℓ, C
BB
ℓ} denotes the set of ensemble-averaged polarization CMB power spectra, and σℓ = {σTT
ℓ, σTE
ℓ, σEE
ℓ, σBB
ℓ} the set of the realization-specific polarization CMB power spectra. This distribution describes the CMB posterior power spectrum for cosmic variance limited data. The expressions derived here are general, and may be useful in a wide range of applications. Two specific applications are described in this paper. First, we employ the derived distributions within the CMB Gibbs sampling framework and demonstrate a new conditional CMB power spectrum sampling algorithm that allows for different binning schemes for each power spectrum. This is useful because most CMB experiments have very different signal-to-noise ratios for temperature and polarization. Second, we provide new Blackwell–Rao estimators for each of the marginal polarization distributions, which are relevant to power spectrum and likelihood estimation. Because these estimators represent marginals, they are not affected by the exponential behavior of the corresponding joint expression, but converge quickly.
A randomized trial of home telemonitoring in a typical elderly heart failure population in North West London: results of the Home‐HF study
Authors: Dar Owais , Riley Jillian , Chapman Callum , Dubrey Simon W. , Morris Stephen , Rosen Stuart D. , Roughton Michael , Cowie Martin R.
Published: January 2009

Aims

Heart failure chiefly affects the elderly, with frequent emergency admissions. Telemonitoring can identify worsening heart failure but previous randomized trials have enrolled selected patient populations. The Home‐HF study examined the impact of home telemonitoring on typical heart failure patients discharged from three acute hospitals in North West London, UK.

Methods and results

Patients hospitalized with heart failure were randomized to telemonitoring or usual specialist care. Primary outcome measures were days alive and out of hospital. Secondary outcome measures were number and duration of heart failure hospitalizations, clinic visits, and quality of life. We recruited 182 patients. There was no difference in the primary outcome measure in the two groups, but there were significantly fewer unplanned hospitalizations for heart failure decompensation, and a reduction in clinic and emergency room visits in the telemonitoring group. There was no statistically significant difference in the mean direct health service costs.

Conclusion

Home telemonitoring in a typical elderly population of heart failure patients produces a similar outcome to ‘usual’ specialist care, but reduces clinic and emergency room visits and unplanned heart failure rehospitalizations at little additional cost. This method of disease monitoring may allow specialist services to increase the number of patients under their care.
Essential and overlapping functions for mammalian Argonautes in microRNA silencing
Authors: Su Hong , Trombly Melanie I. , Chen Jian , Wang Xiaozhong
Published: January 2009

MicroRNA (miRNA) silencing fine-tunes protein output and regulates diverse biological processes. Argonaute (Ago) proteins are the core effectors of the miRNA pathway. In lower organisms, multiple Agos have evolved specialized functions for distinct RNA silencing pathways. However, the roles of mammalian Agos have not been well characterized. Here we show that mouse embryonic stem (ES) cells deficient for Ago1–4 are completely defective in miRNA silencing and undergo apoptosis. In miRNA silencing–defective ES cells, the proapoptotic protein Bim, a miRNA target, is increased, and up-regulation of Bim is sufficient to induce ES cell apoptosis. Expression of activated Akt inhibits Bim expression and partially rescues the growth defect in Ago-deficient ES cells. Furthermore, reintroduction of any single Ago into Ago-deficient cells is able to rescue the endogenous miRNA silencing defect and apoptosis. Consistent with this, each Ago is functionally equivalent with bulged miRNA duplexes for translational repression, whereas Ago1 and Ago2 appear to be more effective at utilizing perfectly matched siRNAs. Thus, our results demonstrate that mammalian Agos all contribute to miRNA silencing, and individual Agos have largely overlapping functions in this process.
The materials science X-ray beamline BL8 at the DELTA storage ring
Authors: Lützenkirchen-Hecht Dirk , Wagner Ralph , Haake Ulrich , Watenphul Anke , Frahm Ronald
Published: January 2009

The hard X-ray beamline BL8 at the superconducting asymmetric wiggler at the 1.5 GeV Dortmund Electron Accelerator DELTA is described. This beamline is dedicated to X-ray studies in the spectral range from ∼1 keV to ∼25 keV photon energy. The monochromator as well as the other optical components of the beamline are optimized accordingly. The endstation comprises a six-axis diffractometer that is capable of carrying heavy loads related to non-ambient sample environments such as, for example, ultrahigh-vacuum systems, high-pressure cells or liquid-helium cryostats. X-ray absorption spectra from several reference compounds illustrate the performance. Besides transmission measurements, fluorescence detection for dilute sample systems as well as surface-sensitive reflection-mode experiments have been performed. The results show that high-quality EXAFS data can be obtained in the quick-scanning EXAFS mode within a few seconds of acquisition time, enabling time-resolved in situ experiments using standard beamline equipment that is permanently available. The performance of the new beamline, especially in terms of the photon flux and energy resolution, is competitive with other insertion-device beamlines worldwide, and several sophisticated experiments including surface-sensitive EXAFS experiments are feasible.
Relationship between oxidative stress‐related biomarkers and antioxidant status with asthma and atopy in young adults: a population‐based study
Authors: García‐Larsen V. , Chinn S. , Rodrigo R. , Amigo H. , Bustos P. , Rona R. J.
Published: January 2009

Background and aim
Enhanced oxidative stress has been described in adults who suffer from symptoms of asthma and poor lung function. This study assessed the relation between markers of oxidative stress and antioxidant status and lung function, symptoms of asthma, atopy and bronchial hyperresponsiveness (BHR) in young adults.

Methods
A sub‐sample of 589 individuals aged 22–28 years, selected from a total of 1232 included in a survey assessing early and current risk factors for chronic diseases, participated in the study. Participants were from an agricultural area of Chile, responded to a Spanish version of the European Community Respiratory Health Survey questionnaire, were skin tested to eight allergens, and challenged with methacholine to assess BHR. Five hundred and eighty‐five individuals had measures of plasma biomarkers ferric reducing ability of plasma, uric acid, protein carbonyls and 564 had 8‐iso‐prostaglandin F2α (8‐iso‐PGF2α) assessed.

Results
All participants had detectable plasma 8‐iso‐PGF2α and carbonyl levels. There was no indication for an association between markers of antioxidant status or oxidative stress with any of the outcomes studied.

Conclusion
The levels of oxidative stress‐related biomarkers and antioxidant status in plasma may not be related to asthma in the general population in the absence of more severe symptoms or exacerbations.
Effect of levocetirizine on nasal provocation testing with adenosine monophosphate compared with allergen challenge in allergic rhinitis
Authors: Vaidyanathan S. , Nair A. , Barnes M. L. , Meldrum K. , Lipworth B. J.
Published: January 2009

Background
End‐organ hyperreactivity is an important feature of the allergic airway. There are no data directly comparing the responsiveness to treatment of different nasal provocation tests (NPT).

Objective
We compared the effect of levocetirizine on nasal adenosine 5′‐monophosphate (AMP) with specific allergen challenge in patients with intermittent and persistent allergic rhinitis (AR).

Methods
Patients with AR were randomized in double‐blind cross‐over fashion to receive single doses of levocetirizine 5 mg or identical placebo, with nasal challenge performed 12 h after dosing. Sixteen participants completed per protocol. Nasal AMP or allergen challenge was conducted on separate days with 1‐ and 2‐week washout periods in between, respectively. Measurements of peak nasal inspiratory flow (PNIF) were made over 60 min after each challenge. The primary end‐point was the provocative concentration of AMP or allergen causing a 20% drop in the PNIF (PC20).

Results
The time‐profile for PNIF recovery [area under the 60 min time–response curve as % PNIF change (min)] were significantly attenuated for AMP challenge, as mean difference [95% confidence interval (CI)]: 11.57 (3.87, 19.25), P=0.005 and for allergen challenge: 17.82 (0.11, 35.53), P=0.04. A highly significant correlation was shown between methods for the area under the curve: (R=0.86, P<0.001). A statistically significant correlation was also seen for the PC20: (R=0.94, P<0.001). PC20 improvement amounted to a 1.26 (95% CI 0.16, 2.35) and 0.16 (95% CI −0.41, 0.73) doubling‐dilution shifts for allergen and AMP challenges, respectively. Bland–Altman plots confirmed good agreement between methods.

Conclusion
A high correlation and statistical agreement has been demonstrated between AMP and allergen challenge for all outcome measures. In particular, the recovery profile after NPT is a sensitive and discriminatory measure of anti‐allergic treatment.
On the origin and specificity of antibodies to neuromuscular blocking (muscle relaxant) drugs: an immunochemical perspective
Authors: Baldo B. A. , Fisher M. M. , Pham N. H.
Published: January 2009

Following the demonstration 25 years ago that substituted ammonium groups on neuromuscular blocking drugs (NMBDs) are the main allergenic structures recognized by IgE antibodies in the sera of some patients who experience anaphylaxis during anaesthesia, immunoassays for these drugs were quickly applied to supplement skin tests in the diagnostic assessment of suspected adverse reactions to anaesthetic agents. Many subjects who react to an NMBD do so on first exposure and this led to the speculation that the origin of allergic sensitization is an environmental agent(s) or another drug containing an ammonium ion. Direct antibody binding and hapten inhibition studies revealed that morphine, which contains a tertiary amino group, was strongly recognized by IgE in sera from anaphylactic patients and a morphine‐solid phase immunoassay was found to be superior to NMBD‐based assays for the detection of NMBD‐reactive IgE antibodies. Extensive inhibition experiments indicate the likelihood of antibody combining site heterogeneity with recognition at the fine structural level of features additional, and adjacent to, ammonium ions. Further quantitative investigations are needed to identify these neighbouring groups on different NMBDs. Recent work has implicated the morphine analogue pholcodine as the sensitizing agent in Norway where, unlike Sweden, anaphylactic reactions to NMBDs are not uncommon and the medicament is available over‐the‐counter. This has led to the suggestion that allergenic sensitization to the ammonium group of pholcodine may account for the different incidences of anaphylaxis during anaesthesia in the two countries. This work is subjected to critical review and some alternative speculations on the nature and origin of the sensitizing agent(s) are presented.
Relevance of the hygiene hypothesis to early vs. late onset allergic rhinitis
Authors: Matheson M. C. , Walters E. H. , Simpson J. A. , Wharton C. L. , Ponsonby A.‐L. , Johns D. P. , Jenkins M. A. , Giles G. G. , Hopper J. L. , Abramson M. J. , Dharmage S. C.
Published: January 2009

Introduction
The hygiene hypothesis proposes that reduced exposure to infections in early life increases the risk of developing allergic conditions including allergic rhinitis. We examined the association between markers of the hygiene hypothesis and allergic rhinitis that developed before 7 years of age and allergic rhinitis that developed after 7 years of age.

Methods
The Tasmanian Longitudinal Health Study (TAHS) is a population‐based cohort (n=8583) study of respiratory disease. Participants have been followed from 7 to 44 years of age. Information on potential risk factors, allergies and respiratory symptoms was collected longitudinally. Using multi‐nomial logistic regression, exposure to siblings, infections, tonsillectomy and farm residence during childhood were examined as risk factors for allergic rhinitis that developed before or after 7 years of age. All analyses were adjusted for gender, maternal and paternal atopy, mother's age at participant's birth, paternal socio‐economic status in 1968 and personal socio‐economic status in 2004.

Results
Greater cumulative exposure to siblings before the age of 2 years was strongly inversely associated with early onset allergic rhinitis (<1 year sib exposure: OR=0.6, 95% CI 0.3–1.0; 1–3 years sib exposure: OR=0.6, 95% CI 0.4–0.9; >3 years sib exposure: OR=0.4, 95% CI 0.3–0.8) less so with later onset allergic rhinitis. The risk of early onset allergic rhinitis decreased with increasing viral infections (OR=0.7, 95% CI 0.5–0.9) during childhood. Having a tonsillectomy before 7 years of age increased the risk of early onset allergic rhinitis (OR=1.7, 95% CI 1.2–2.5). None of these factors was associated with later onset allergic rhinitis.

Conclusions
Exposures relevant to the hygiene hypothesis were important predictors for the development of early onset but less so for later onset allergic rhinitis. The exact mechanisms by which siblings and infections protect against allergic rhinitis are unclear. The stronger findings for earlier onset allergic rhinitis suggest that family structure and infections have most impact on disease risk in early life. Further research should focus on early onset allergic rhinitis when exploring causal explanations for any sibling effect.
Interannual spatial variability of krill (Euphausia superba) influences seabird foraging behavior near Elephant Island, Antarctica
Authors: SANTORA JARROD A. , REISS CHRISTIAN S. , COSSIO ANTHONY M. , VEIT RICHARD R.
Published: January 2009

We investigate the influence of krill (principally Euphausia superba) patchiness on the foraging distributions of seabirds to understand how variation in krill influences patch dynamics between krill and birds. At sea‐surveys were conducted near Elephant Island, Antarctica, for 3 yr (2004–2006) during the annual U.S. Antarctic Marine Living Resources (AMLR) program. Standardized strip‐transect surveys were used to map seabirds, and a combination of acoustic and net surveys was used to map krill. We measured patch size of krill and seabirds and elucidated how krill patch dynamics influence foraging seabirds. The spatial association between krill and predators was influenced by the size and arrangement of krill patches. We found a negative relationship between abundance and patchiness of krill and predators, indicating that when krill is less abundant, its predators are less abundant and concentrated. We conclude that annual patch dynamics of krill strongly influences the local abundance and distribution of seabirds. Such information should be used to interpret potential interactions between seabirds and krill fisheries operating near Elephant Island.
Application of environmental scanning electron microscopy to determine biological surface structure
Authors: KIRK S.E. , SKEPPER J.N. , DONALD A.M.
Published: January 2009

The use of environmental scanning electron microscopy in biology is growing as more becomes understood about the advantages and limitations of the technique. These are discussed and we include new evidence about the effect of environmental scanning electron microscopy imaging on the viability of mammalian cells. We show that although specimen preparation for high‐vacuum scanning electron microscopy introduces some artefacts, there are also challenges in the use of environmental scanning electron microscopy, particularly at higher resolutions. This suggests the two technologies are best used in combination. We have used human monocyte‐derived macrophages as a test sample, imaging their complicated and delicate membrane ruffles and protrusions. We have also explored the possibility of using environmental scanning electron microscopy for dynamic experiments, finding that mammalian cells cannot be imaged and kept alive in the environmental scanning electron microscopy. The dehydration step in which the cell surface is exposed causes irreversible damage, probably via loss of membrane integrity during liquid removal in the specimen chamber. Therefore, mammalian cells should be imaged after fixation where possible to protect against damage as a result of chamber conditions.
A practical calibration procedure for fluorescence colocalization at the single organelle level
Authors: ANLAUF E. , DEROUICHE A.
Published: January 2009

We have determined practical requirements for antigen colocalization on subcellular structures. The calibration standards used were 175‐nm fluorescent microspheres and microtubules (∼255  nm) in cultured astrocytes. The colocalization problem became apparent with detection of anti‐α‐tubulin labelling in three colour channels, when images were not identical. Complete superimposition could only be achieved by shifting the single colour image stacks relative to one another in three dimension, and images in the xy plane. The errors could be traced in chromatic aberration (100 nm each between blue and green, and green and red), and to a lateral pixel shift (∼150 nm). For such colocalization, it was essential to apply image projection on the camera chip at high magnification (200×), focus steps (100 nm) smaller than required by the Nyquist criterion and very narrow band filter sets, in addition to high‐aperture achromat lenses. Several steps in the deconvolution settings and in 3D reconstruction had to be standardized. As a result, the 175‐nm microspheres, which displayed roughly symmetric diffraction patterns above and below the object plane, were reconstructed as spherical objects. Several neighbouring microtubules could be resolved with a limit <200 nm. Also, three‐colour colocalization on a single tubule was validated. Applying this setup, it was possible to colocalize several antigens in astrocytes, at the level of organelles, presumptive exocytosis vesicles. We colocalized glutamate and 14‐3‐3 protein as well as synaptophysin and 14‐3‐3 protein, which may be involved in early steps of exocytosis. The practical parameters validate colocalization on subcellular structures at a resolution limit below conventional light microscopy.
An atomic force microscope operating at hypergravity for in situ measurement of cellular mechano‐response
Authors: VAN LOON J.J.W.A. , VAN LAAR M.C. , KORTERIK J.P. , SEGERINK F.B. , WUBBELS R.J. , DE JONG H.A.A. , VAN HULST N.F.
Published: January 2009

We present a novel atomic force microscope (AFM) system, operational in liquid at variable gravity, dedicated to image cell shape changes of cells in vitro under hypergravity conditions. The hypergravity AFM is realized by mounting a stand‐alone AFM into a large‐diameter centrifuge. The balance between mechanical forces, both intra‐ and extracellular, determines both cell shape and integrity. Gravity seems to be an insignificant force at the level of a single cell, in contrast to the effect of gravity on a complete (multicellular) organism, where for instance bones and muscles are highly unloaded under near weightless (microgravity) conditions. However, past space flights and ground based cell biological studies, under both hypogravity and hypergravity conditions have shown changes in cell behaviour (signal transduction), cell architecture (cytoskeleton) and proliferation. Thus the role of direct or indirect gravity effects at the level of cells has remained unclear. Here we aim to address the role of gravity on cell shape. We concentrate on the validation of the novel AFM for use under hypergravity conditions. We find indications that a single cell exposed to 2 to 3 × g reduces some 30–50% in average height, as monitored with AFM. Indeed, in situ measurements of the effects of changing gravitational load on cell shape are well feasible by means of AFM in liquid. The combination provides a promising technique to measure, online, the temporal characteristics of the cellular mechano‐response during exposure to inertial forces.
Three‐dimensional reconstruction of the guinea pig inner ear, comparison of OPFOS and light microscopy, applications of 3D reconstruction
Authors: HOFMAN R. , SEGENHOUT J.M. , WIT H.P.
Published: January 2009

Three‐dimensional (3D) reconstruction of anatomical structures can give additional insight into the morphology and function of these structures. We compare 3D reconstructions of the guinea pig inner ear, using light microscopy and orthogonal plane fluorescence optical sectioning microscopy. Applications of 3D reconstruction of the inner ear are further explored. For each method two bullas were prepared for 3D reconstruction. Both methods are explained. In general, the 3D reconstructions using orthogonal plane fluorescence optical sectioning microscopy are superior to light microscopy. The exact spiral shape of the cochlea could be reconstructed using orthogonal plane fluorescence optical sectioning microscopy and the length of the basilar membrane measured. When a resolution of 20 μm is sufficient, orthogonal plane fluorescence optical sectioning microscopy is a superior technique for 3D reconstruction of inner ear structures in animals.
Microwave‐assisted rapid plant sample preparation for transmission electron microscopy
Authors: ZECHMANN B. , ZELLNIG G.
Published: January 2009

The preparation of plant leaf material for transmission electron microscopical investigations can be a very time‐ and labour‐consuming task as the reagents infiltrate the samples quite slowly and as usually most steps have to be performed manually. Fixation, buffer washes, dehydration, resin infiltration and polymerization of the resin‐infiltrated leaf samples can take several days before the specimen can be cut ultrathin and used for ultrastructural investigations. In this study, we present a microwave‐assisted automated sample preparation procedure that reduces preparation time from at least 3 days to about 5 h – with only a few steps that have to be performed manually – until the plant sample can be ultrathin sectioned and observed with the transmission electron microscope. For studying the efficiency of this method we have compared the ultrastructure of different leaf material (Arabidopsis thaliana, Nicotiana tabacum and Picea abies) which was prepared with a conventional, well‐established chemical fixation and embedding protocol and a commercially available automated microwave tissue processor. Despite the massive reduction in sample preparation time no negative effects on cutting properties of the blocks, stability of the sections in the electron beam, contrast and ultrastructure of the cells were observed under the transmission electron microscope when samples were prepared with the microwave‐assisted protocol. Additionally, no negative effects were detected on the dimensions of fine structures of grana stacks (including membranes, inter‐ and intrathylakoidal spaces), the nuclear envelope and the plasma membrane as the diameter of these structural components did not differ between leaf samples (of the same species) that were processed with the automated microwave tissue processor or by conventional fixation and embedding at room temperature.
Stroboscopic near‐field imaging for the analysis of contraction of muscle cells
Authors: MORIGUCHI C. , EGAMI C. , KAWATA Y. , TERAKAWA S. , TSUCHIMORI M. , WATANABE O.
Published: January 2009

We have developed a stroboscopic near‐field optical microscope for observation of biological specimens and observed glycerinated muscles before and after muscle contraction with the developed system. In the system, the optical field distribution localized near the specimen is recorded as the surface topographic distribution of a photosensitive film surface. Our system is very useful for observing living biological specimens with high resolutions, because it is possible to get stroboscopic image by using a photosensitive film as detecting optical distributions instead of a scanning of probes. We have succeeded in observing inner structures of muscle cells with sub‐wavelength resolution and achieved higher contrast than an ordinary optical microscope.
Visualization of flagellar interactions on bacterial carpets
Authors: HESSE W.R. , KIM M.J.
Published: January 2009

Methods for the in‐depth study of the physics of microscale actuation of microfluidics environments by flagellated bacteria ‘teamsters’ have been developed. These methods, which include single and multi‐colour fluorescent labelling and electron microscopy allow for the analysis of the effect that individual flagellar filaments have on bacterially driven microstructures, and allow for the investigation of the interaction and coordination of flagellar filaments of neighbouring bacteria on densely packed monolayers of bacteria, ‘bacterial carpets’. We show that the flagella of bacteria that are immobilized on a surface often interact with each other, and that the flagella of these bacteria do not often form multi‐flagella bundles that are aligned with the cell body.
Comparison of different preparation methods of biological samples for FIB milling and SEM investigation
Authors: LEŠER V. , DROBNE D. , PIPAN Ž. , MILANI M. , TATTI F.
Published: January 2009

When a new approach in microscopy is introduced, broad interest is attracted only when the sample preparation procedure is elaborated and the results compared with the outcome of the existing methods. In the work presented here we tested different preparation procedures for focused ion beam (FIB) milling and scanning electron microscopy (SEM) of biological samples. The digestive gland epithelium of a terrestrial crustacean was prepared in a parallel for FIB/SEM and transmission electron microscope (TEM). All samples were aldehyde‐fixed but followed by different further preparation steps. The results demonstrate that the FIB/SEM samples prepared for conventional scanning electron microscopy (dried) is suited for characterization of those intracellular morphological features, which have membranous/lamellar appearance and structures with composition of different density as the rest of the cell. The FIB/SEM of dried samples did not allow unambiguous recognition of cellular organelles. However, cellular organelles can be recognized by FIB/SEM when samples are embedded in plastic as for TEM and imaged by backscattered electrons. The best results in terms of topographical contrast on FIB milled dried samples were obtained when samples were aldehyde‐fixed and conductively stained with the OTOTO method (osmium tetroxide/thiocarbohydrazide/osmium tetroxide/thiocarbohydrazide/osmium tetroxide). In the work presented here we provide evidence that FIB/SEM enables both, detailed recognition of cell ultrastructure, when samples are plastic embedded as for TEM or investigation of sample surface morphology and subcellular composition, when samples are dried as for conventional SEM.
Regulation of COP1 nuclear localization by the COP9 signalosome via direct interaction with CSN1
Authors: Wang Xiping , Li Wenjun , Piqueras Raquel , Cao Kaiming , Deng Xing Wang , Wei Ning
Published: January 2009

COP1 and COP9 signalosome (CSN) are key regulators of plant light responses and development. Deficiency in either COP1 or CSN causes a constitutive photomorphogenic phenotype. Through coordinated actions of nuclear‐ and cytoplasmic‐localization signals, COP1 can respond to light signals by differentially partitions between nuclear and cytoplasmic compartments. Previous genetic analysis in Arabidopsis indicated that the nuclear localization of COP1 requires CSN, an eight‐subunit heteromeric complex. However the mechanism underlying the functional relationship between COP1 and CSN is unknown. We report here that COP1 weakly associates with CSN in vivo. Furthermore, we report on the direct interaction involving the coiled‐coil domain of COP1 and the N‐terminal domain of the CSN1 subunit. In onion epidermal cells, expression of CSN1 can stimulate nuclear localization of GUS‐COP1, and the N‐terminal domain of CSN1 is necessary and sufficient for this function. Moreover, CSN1‐induced COP1 nuclear localization requires the nuclear‐localization sequences of COP1, as well as its coiled‐coil domain, which contains both the cytoplasmic localization sequences and the CSN1 interacting domain. We also provide genetic evidence that the CSN1 N‐terminal domain is specifically required for COP1 nuclear localization in Arabidopsis hypocotyl cells. This study advances our understanding of COP1 localization, and the molecular interactions between COP1 and CSN.
Rapid analysis of poplar lignin monomer composition by a streamlined thioacidolysis procedure and near‐infrared reflectance‐based prediction modeling
Authors: Robinson Andrew R. , Mansfield Shawn D.
Published: January 2009

Determination of the physico‐chemical attributes of plant cell walls, such as lignin content and composition, is of paramount importance in germplasm screening and for evaluating the results of plant breeding and genetic engineering. There are escalating needs for analyses to be robust, reproducible, accurate, and efficient. We have recently modified an established protocol for discrimination of lignin monomers, thioacidolysis, with the goal of increasing sample throughput while maintaining accuracy and reducing equipment load and consumption of reagents. Numerous methodological changes related to volume scaling, selection of the processing vessel, and sample handling were addressed. The revised protocol permitted rapid processing of some 50 or more samples per person per day. A direct comparison between methods using hybrid poplar (Populus alba × tremula) wood samples, resulted in quantities of p‐hydroxyphenyl (H), guaiacyl (G), and syringyl (S) lignin monomers that were equivalent to those derived from the original protocol. The revised methodology was then applied to quickly generate phenotypic trait data from 267 hybrid poplar trees (including wild type and eight C4H::F5H transgenic lines), for the development of a near‐infrared‐based model for predicting the proportion of lignin monomers across a broad phenotypic range of S:G. The resulting partial least squares regression model performed well under full cross‐validation, giving strong, linear relationships between actual and predicted monomer proportions, and very high predictive accuracy for the predominant G and S monomers. This research brings considerable refinement to the thioacidolysis procedure, and establishes a method for rapidly and accurately quantifying cell‐wall lignin composition that could effectively be employed in routine phenotypic screening platforms.
Respiratory virus induction of alpha‐, beta‐ and lambda‐interferons in bronchial epithelial cells and peripheral blood mononuclear cells
Authors: Khaitov M. R. , Laza‐Stanca V. , Edwards M. R. , Walton R. P. , Rohde G. , Contoli M. , Papi A. , Stanciu L. A. , Kotenko S. V. , Johnston S. L.
Published: January 2009

Background:  Respiratory viruses, predominantly rhinoviruses are the major cause of asthma exacerbations. Impaired production of interferon‐β in rhinovirus infected bronchial epithelial cells (BECs) and of the newly discovered interferon‐λs in both BECs and bronchoalveolar lavage cells, is implicated in asthma exacerbation pathogenesis. Thus replacement of deficient interferon is a candidate new therapy for asthma exacerbations. Rhinoviruses and other respiratory viruses infect both BECs and macrophages, but their relative capacities for α‐, β‐ and λ‐interferon production are unknown.

Methods:  To provide guidance regarding which interferon type is the best candidate for development for treatment/prevention of asthma exacerbations we investigated respiratory virus induction of α‐, β‐ and λ‐interferons in BECs and peripheral blood mononuclear cells (PBMCs) by reverse transferase‐polymerase chain reaction and enzyme‐linked immunosorbent assay.

Results:  Rhinovirus infection of BEAS‐2B BECs induced interferon‐α mRNA expression transiently at 8 h and interferon‐β later at 24 h while induction of interferon‐λ was strongly induced at both time points. At 24 h, interferon‐α protein was not detected, interferon‐β was weakly induced while interferon‐λ was strongly induced. Similar patterns of mRNA induction were observed in primary BECs, in response to both rhinovirus and influenza A virus infection, though protein levels were below assay detection limits. In PBMCs interferon‐α, interferon‐β and interferon‐λ mRNAs were all strongly induced by rhinovirus at both 8 and 24 h and proteins were induced: interferon‐α>‐β>‐λ. Thus respiratory viruses induced expression of α‐, β‐ and λ‐interferons in BECs and PBMCs. In PBMCs interferon‐α>‐β>‐λ while in BECs, interferon‐λ>‐β>‐α.

Conclusions:  We conclude that interferon‐λs are likely the principal interferons produced during innate responses to respiratory viruses in BECs and interferon‐αs in PBMCs, while interferon‐β is produced by both cell types.
Inhibition of allergic responses by CD40 gene silencing
Authors: Suzuki M. , Zheng X. , Zhang X. , Ichim T. E. , Sun H. , Kubo N. , Beduhn M. , Shunnar A. , Garcia B. , Min W.‐P.
Published: January 2009

Background:  Gene silencing using small interfering RNA (siRNA) is a potent method of specifically knocking down molecular targets. Small interfering RNA is therapeutically promising, however, treatment of allergic diseases with siRNA has not been explored in vivo. The aim of this study was to evaluate therapeutic effects of CD40 siRNA on inhibition of allergic responses.

Methods:  Mice sensitized with ovalbumin (OVA) and alum were treated with CD40 siRNA, scrambled siRNA, or phosphate buffer saline (PBS) alone, and then challenged intranasally with OVA.

Results:  A significant reduction in nasal allergic symptoms was observed in the CD40 siRNA treated OVA‐allergic mice compared to the controls of scrambled siRNA and PBS alone, which is correlated with the decrease of local eosinophil accumulation. CD40 siRNA treatment knocked down CD40 expression on dendritic cells (DCs) in vivo and impaired their antigen presenting function. Treatment with CD40 siRNA resulted in inhibition of OVA‐specific T cell response and decrease of interleukin‐4 (IL‐4), IL‐5, and interferon‐γ production from T cells stimulated with OVA. Administration of CD40 siRNA also suppressed CD40 expression on B cells, resulting in down‐regulation of OVA‐specific immunoglobulin E (IgE), IgG1, and IgG2a levels. Additionally, increased regulatory T cells were observed in the CD40 siRNA treated mice.

Conclusions:  The present study demonstrates a novel therapeutic use for siRNA in allergy. CD40 siRNA attenuated allergy through inhibition of DC and B cell functions and generation of regulatory T (Treg) cells.
A quantitative genetic analysis of intermediate asthma phenotypes
Authors: Thomsen S. F. , Ferreira M. A. R. , Kyvik K. O. , Fenger M. , Backer V.
Published: January 2009

Aim:  To study the relative contribution of genetic and environmental factors to the correlation between exhaled nitric oxide (FeNO), airway responsiveness, airway obstruction, and serum total immunoglobulin E (IgE).

Methods:  Within a sampling frame of 21 162 twin subjects, 20–49 years of age, from the Danish Twin Registry, a total of 575 subjects (256 intact pairs and 63 single twins) who either themselves and/or their co‐twins reported a history of asthma at a nationwide questionnaire survey, were clinically examined. Traits were measured using standard techniques. Latent factor models were fitted to the observed data using maximum likelihood methods.

Results:  Additive genetic factors explained 67% of the variation in FeNO, 43% in airway responsiveness, 22% in airway obstruction, and 81% in serum total IgE. In general, traits had genetically and environmentally distinct variance structures. The most substantial genetic similarity was observed between FeNO and serum total IgE, genetic correlation (ρA) = 0.37, whereas the strongest environmental resemblance was observed between airway responsiveness and airway obstruction, specific environmental correlation (ρE) = −0.46, and between FeNO and airway responsiveness, ρE = 0.34.

Conclusions:  Asthma is a complex disease characterized by a set of etiologically heterogeneous biomarkers, which likely constitute diverse targets of intervention.
Chronic urticaria: a patient survey on quality‐of‐life, treatment usage and doctor–patient relation
Authors: Maurer M. , Ortonne J ‐P. , Zuberbier T.
Published: January 2009

Background:  Chronic urticaria (CU) is a common skin disorder characterized by recurrent spontaneous outbreaks of itchy wheals and/or angioedema. It has been shown to have substantial impact on patient quality‐of‐life, but little else is known about patient perspectives on CU and its treatment.

Methods:  An internet survey was conducted with 321 randomly selected, representative adults in Germany and France who were diagnosed with CU. The survey included the Skindex‐29 questionnaire on quality‐of‐life and questions about treatment usage and patients’ relation to their physician. Regression analyses were used to identify predictors of quality‐of‐life, use of prescription medication and various aspects of the doctor–patient relation.

Results:  The survey confirmed that CU has substantial impact on quality‐of‐life, with median Skindex scores of 68 for symptoms, 50 for functioning and 53 for emotions. Only two in three respondents were taking prescription medication for their CU. Older respondents, French respondents and fully employed respondents were significantly (P < 0.01) more likely to be taking prescription medication. Only three in five respondents under a physician’s care reported that their physician had discussed the emotional impact of CU on them. Patients whose physicians had discussed this emotional impact were significantly (P < 0.001) more satisfied with treatment and more trusting of their physician.

Conclusions:  CU has a heavy impact on quality‐of‐life. Physicians need to be aware that many patients are not taking second generation anti‐histamines and counsel them better on this point. Physicians should also discuss the emotional impact of CU with patients, because it improves their satisfaction and trust.
Sensitization, asthma and allergic disease in young soccer players
Authors: Ventura M. T. , Cannone A. , Sinesi D. , Buquicchio R. , Carbonara M. , Di Leo E. , Bonini M. , Dagnello M. , Bonini S.
Published: January 2009

Background:  The aim of this study was to identify the prevalence of allergic disease in young soccer players compared to age‐matched students and to evaluate if this prevalence changes as the intensity of training increases.

Methods:  A modified ECRHS questionnaire was administered to 194 soccer players divided by age as Beginners (8–11 years), Juniors (12–16 years) and Under 21 (17–20 years) to evaluate the prevalence of allergic diseases and symptoms as well as drug consumption. Subjects with a positive personal history of allergic diseases underwent skin prick and/or patch tests. Age‐matched students (n = 136) were used as a control group.

Results:  The prevalence of allergic diseases was 34.5% in soccer players and 31.6% in control subjects (n.s.). Skin sensitization to inhalant allergens was detected in 14.4% of symptomatic soccer players and in 19.2% of control students (n.s.). Patch tests were positive in 35.7% of soccer players and 23.0% of controls with allergic dermatitis (n.s.). The prevalence of allergic diseases did not significantly change in relation to the intensity of training. Although the relative prevalence of sensitization to perennial allergens and asthma was less frequent in soccer players than in controls, and the occurrence of exercise‐induced bronchoconstriction was similar in the two groups, soccer players used twice as many anti‐allergic and anti‐asthmatic drugs as control students.

Conclusions:  An increasingly intensive training programme is not associated with greater risk of allergic disease in soccer players. Therapy regimens of allergic athletes and exercisers should be monitored more closely to guarantee adequate treatment yet avoid inappropriate drug use and doping practices.
Genetic structure of coastal and inland populations of the annual halophyteSuaeda maritima(L.)dumort.in Central Europe, inferred from amplified fragment length polymorphism markers
Authors: Prinz K. , Weising K. , Hensen I.
Published: January 2009

Naturally occurring inland salt habitats are highly threatened due to increasing fragmentation and area reduction, while the surroundings of former potash mining dumps have experienced a massive invasion by halophytes over the last 20 years. We reconstructed colonisation patterns of these purely anthropogenic inland salt sites using molecular markers in the obligate halophyteSuaeda maritima(L.)dumort.(Chenopodiaceae), a typical plant in such areas. In the present study, 120 individual plants from 40 coastal and inland populations in Central Europe were subjected to AFLP analysis with nine primer combinations. A total of 243 AFLP band positions were scored as presence/absence characters. Genetic diversity values were not significantly different in populations from natural and anthropogenic inland salt sites as compared to coastal habitats. Results from principal coordinate analysis, neighbour‐joining analysis and analysis of molecular variance (amova) all indicated that most of the genetic variation is preserved within populations, while genetic differentiation among populations is comparatively low. We conclude thatS. maritimahas repeatedly and independently colonised the surroundings of former potash mining dumps in Central Germany. However, the absence of founder effects and the lack of phylogeographic structure prevented us from identifying putative donor populations.
Evaluation of the waxy endosperm trait in proso millet (Panicum miliaceum)*
Authors: Graybosch R. A. , Baltensperger D. D.
Published: January 2009

The entire USDA‐ARS maintained collection of 650 accessions of proso millet (Panicum miliaceum L.) was evaluated for the presence of accessions with waxy (amylose‐free) endosperm starch. Six accessions, five of which derived from mainland China, were identified. Segregation ratios for waxy endosperm were evaluated in F2 and F3 populations derived from crosses between two waxy accessions, PI 436625 (Lung Shu 16) and PI 436626 (Lung Shu 18), and several wild‐type accessions. The waxy trait was found to be under the control of duplicate recessive alleles at two loci, herein designated wx‐1 and wx‐2. Wild‐type alleles at these loci were designated Wx‐1 and Wx‐2. Iodine‐binding revealed a mean grain‐starch amylose concentration of 3.5% in waxy lines and 25.3% in wild‐type proso. Expression of the granule‐bound starch synthase (waxy protein) in waxy lines was reduced to approximately one‐tenth that of wild‐type accessions. The waxy accessions identified now are available for the introgression of this trait into breeding lines adapted to the Great Plains of North America.