bluuebunny/crossref_metadata_2025 huggingface dataset:
Cholesterol Promotes Hemifusion and Pore Widening in Membrane Fusion Induced by Influenza Hemagglutinin
Authors: Biswas Subrata , Yin Shu-Rong , Blank Paul S. , Zimmerberg Joshua
Published: April 2008

Cholesterol-specific interactions that affect membrane fusion were tested for using insect cells; cells that have naturally low cholesterol levels (<4 mol %). Sf9 cells were engineered (HAS cells) to express the hemagglutinin (HA) of the influenza virus X-31 strain. Enrichment of HAS cells with cholesterol reduced the delay between triggering and lipid dye transfer between HAS cells and human red blood cells (RBC), indicating that cholesterol facilitates membrane lipid mixing prior to fusion pore opening. Increased cholesterol also increased aqueous content transfer between HAS cells and RBC over a broad range of HA expression levels, suggesting that cholesterol also favors fusion pore expansion. This interpretation was tested using both trans-cell dye diffusion and fusion pore conductivity measurements in cholesterol-enriched cells. The results of this study support the hypothesis that host cell cholesterol acts at two stages in membrane fusion: (1) early, prior to fusion pore opening, and (2) late, during fusion pore expansion.
Comment on ‘Comparison of runaway dynamics in LH and ECRH heated discharges in the Frascati Tokamak Upgrade’
Authors: Chen Z.Y. , Ju H.J. , Wan B.N. , Shi Y.J.
Published: April 2008

In their paper, the authors used the injected parallel refractive index of lower hybrid waves (LHWs) to evaluate the anomalous Doppler resonant interaction of runaway electrons with LHWs. This led to the conclusion of no wave–particle resonant effect in FTU. This is not true when the upshift effect in the LHW spectrum is considered. The wave–particle resonant energy for the upshifted LHW spectrum is consistent with the measured runaway energy during the lower hybrid current drive phase, while the predicted runaway energy based on the loop voltage is much higher than the measured runaway energy after the injection of LHW power. This indicates that the rapid decrease in runaway energy after the injection of LHW power possibly results from the anomalous Doppler resonant effect.
swissPIT: a novel approach for pipelined analysis of mass spectrometry data
Authors: Quandt Andreas , Hernandez Patricia , Masselot Alexandre , Hernandez Céline , Maffioletti Sergio , Pautasso Cesare , Appel Ron D. , Lisacek Frederique
Published: April 2008

The identification and characterization of peptides from tandem mass spectrometry (MS/MS) data represents a critical aspect of proteomics. Today, tandem MS analysis is often performed by only using a single identification program achieving identification rates between 10–50% (Elias and Gygi, 2007). Beside the development of new analysis tools, recent publications describe also the pipelining of different search programs to increase the identification rate (Hartler et al., 2007; Keller et al., 2005).

The Swiss Protein Identification Toolbox (swissPIT) follows this approach, but goes a step further by providing the user an expandable multi-tool platform capable of executing workflows to analyze tandem MS-based data. One of the major problems in proteomics is the absent of standardized workflows to analyze the produced data. This includes the pre-processing part as well as the final identification of peptides and proteins. The main idea of swissPIT is not only the usage of different identification tool in parallel, but also the meaningful concatenation of different identification strategies at the same time. The swissPIT is open source software but we also provide a user-friendly web platform, which demonstrates the capabilities of our software and which is available at http://swisspit.cscs.ch upon request for account.

Contact:  andreas.quandt@isb-sib.ch
The role of the dorsal striatum and dorsal hippocampus in probabilistic and deterministic odor discrimination tasks
Authors: Atallah Hisham E. , Rudy Jerry W. , O’Reilly Randall C.
Published: April 2008

Three experiments explored the contribution of the cortico-striatal system and the hippocampus system to the acquisition of solutions to simultaneous instrumental odor discriminations. Inactivation of the dorsal striatum after rats had reached criterion on a three problem probabilistic set of discriminations—A (80%) vs. B (20%), C (67%) vs. D (33%), E(67%) vs. F(33%)—impaired test performance and disrupted performance when the rats were tested with novel cue combinations (C vs. F and E vs. D), where control animals chose C and F. In contrast, inactivating the dorsal hippocampus enhanced performance on this task and on a deterministic discrimination A (100%) vs. B (0%). These results are consistent with the complementary learning systems view, which assumes that the cortico-striatal and hippocampal system capture information in parallel. How this information combines to influence task performance depends on the compatibility of the content captured by each system. These results suggest that the trial-specific information captured by the hippocampal system can be incompatible with the across-trial integration of trial outcomes captured by the cortico-striatal system.
Inverse temporal contributions of the dorsal hippocampus and medial prefrontal cortex to the expression of long-term fear memories
Authors: Quinn Jennifer J. , Ma Quang D. , Tinsley Matthew R. , Koch Christof , Fanselow Michael S.
Published: April 2008

Retrograde amnesia following disruptions of hippocampal function is often temporally graded, with recent memories being more impaired. Evidence supports the existence of one or more neocortical long-term memory storage/retrieval site(s). Neurotoxic lesions of the medial prefrontal cortex (mPFC) or the dorsal hippocampus (DH) were made 1 day or 200 days following trace fear conditioning. Recently encoded trace fear memories were most disrupted by DH lesions, while remotely encoded trace and contextual memories were most disrupted by mPFC lesions. These data strongly support the consolidation theory of hippocampus function and implicate the mPFC as a site of long-term memory storage/retrieval.
L-type voltage-gated calcium channels in conditioned fear: A genetic and pharmacological analysis
Authors: McKinney Brandon C. , Sze Wilson , White Jessica A. , Murphy Geoffrey G.
Published: April 2008

Using pharmacological approaches, others have suggested that L-type voltage-gated calcium channels (L-VGCCs) mediate both consolidation and extinction of conditioned fear. In the absence of L-VGCC isoform-specific antagonists, we have begun to investigate the subtype-specific role of LVGCCs in consolidation and extinction of conditioned fear using a molecular genetics approach. Previously, we used this approach to demonstrate that the Cav1.3 isoform mediates consolidation, but not extinction, of contextually conditioned fear. Here, we used mice in which the gene for the L-VGCC pore-forming subunit Cav1.2 was conditionally deleted in forebrain excitatory neurons (Cav1.2cKO mice) to address the role of Cav1.2 in consolidation and extinction of conditioned fear. We demonstrate that Cav1.2cKO mice consolidate and extinguish conditioned fear as well as control littermates. These data suggest that Cav1.2 is not critical for these processes and together with our previous data argue against a role for either of the brain-expressed L-VGCCs (Cav1.2 or Cav1.3) in extinction of conditioned fear. Additionally, we present data demonstrating that the L-VGCC antagonist nifedipine, which has been used in previous conditioned fear extinction studies, impairs locomotion, and induces an aversive state. We further demonstrate that this aversive state can enter into associations with conditioned stimuli that are present at the time that it is experienced, suggesting that previous studies using nifedipine were likely confounded by drug toxicity. Taken together, our genetic and pharmacological data argue against a role for Cav1.2 in consolidation of conditioned fear as well as a role for L-VGCCs in extinction of conditioned fear.
Inactivation of the anterior cingulate cortex blocks expression of remote, but not recent, conditioned taste aversion memory
Authors: Ding Hoi Ki , Teixeira Cátia M. , Frankland Paul W.
Published: April 2008

Previous studies have shown that medial prefrontal cortical regions, such as the anterior cingulate cortex (ACC), play a key role in the expression of remote spatial and contextual memory. To evaluate whether this role is conserved in hippocampal-independent tasks we trained mice in the conditioned taste aversion (CTA) paradigm. Lidocaine-induced inactivation of the ACC blocked the expression of CTA tested one month (remote), but not one day (recent), after conditioning with either a weak or strong unconditioned stimulus (US). These data suggest that the ACC may play a conserved role in remote memory, regardless of memory strength or content.
Different involvement of type 1, 2, and 3 ryanodine receptors in memory processes
Authors: Galeotti Nicoletta , Quattrone Alessandro , Vivoli Elisa , Norcini Monica , Bartolini Alessandro , Ghelardini Carla
Published: April 2008

The administration of the ryanodine receptor (RyR) agonist 4-Cmc (0.003–9 nmol per mouse intracerebroventricularly [i.c.v.]) ameliorated memory functions, whereas the RyR antagonist ryanodine (0.0001–1 nmol per mouse i.c.v.) induced amnesia in the mouse passive avoidance test. The role of the type 1, 2, and 3 RyR isoforms in memory processes was then evaluated by inhibiting the expression of the three RyR proteins in the mouse brain. A selective knockdown of the RyR isoforms was obtained by the i.c.v. administration of antisense oligonucleotides (aODNs) complementary to the sequence of RyR1, RyR2 and RyR3 proteins, as demonstrated by immunoblotting experiments. RyR1 (5–9 nmol per mouse i.c.v.) knockdown mice did not show any memory dysfunction. Conversely, RyR2 (1–7 nmol per mouse i.c.v.) and RyR3 (1–7 nmol per mouse i.c.v.) knockdown animals showed an impairment of memory processes. This detrimental effect was temporary and reversible, disappearing 7 d after the end of the aODN treatment. At the highest effective doses, none of the compounds used impaired motor coordination, as revealed by the rota rod test, nor modified spontaneous mobility and inspection activity, as revealed by the hole-board test. In conclusion, the lack of any involvement of cerebral RyR1 was demonstrated. These findings also showed the involvement of type 2 and type 3 RyR in the modulation of memory functions identifying these cerebral RyR isoforms as critical targets underlying memory processes.
Plant compositional change over time increases with rainfall in Serengeti grasslands
Authors: Michael Anderson T.
Published: April 2008

Previous work from the Plains of Serengeti National Park (SNP) suggested that plant species composition (i.e. the composite measure of species’ identities and abundances) is unchanged on the temporal scale of decades (Belsky 1985), raising the possibility that African grazing ecosystems may be resistant to climate change. In this study, grassland plant composition was measured three times between 2000 and 2007 at eight permanent sites spanning the SNP environmental gradient. Confirming previous work, plots in the Serengeti Plains were relatively stable with respect to plant composition through time. In contrast, sites in all other regions of the ecosystem showed greater change in plant species composition during the study. Three factors, annual rainfall, dry season rainfall, and fire frequency, were linearly correlated (all r>0.73) with the amount of composition change at the sites. A path model, which accounted for the strong correlation between annual and dry season rainfall and for the known effect of annual rainfall on fire frequency, was used to elucidate which factors were associated with composition change and which were spuriously correlated. The path model demonstrated that change in plant composition was positively related to annual rainfall, but not dry season rainfall or fire frequency. However, dry season rainfall was positively associated with changes in plant species identity, one component of compositional change, across sites over time. Because climate models predict both increased wet season and decreased dry season rainfall in East Africa, these findings raise uncertainty about how these grasslands will respond to future changes. This study and others (Belsky 1985) suggests that plant assemblages adapted to low rainfall in Serengeti may exhibit compositionally stability, while the same may not be true in regions of higher average rainfall and larger species pools.
A novel PRKAR1A mutation associated with hepatocellular carcinoma in a young patient and a variable Carney complex phenotype in affected subjects in older generations
Authors: Gennari Monia , Stratakis Constantine A. , Hovarth Anelia , Pirazzoli Piero , Cicognani Alessandro
Published: April 2008

Context  Carney complex (CNC) is an autosomal dominant multiple endocrine neoplasia syndrome (OMIM 160980). About 70% of cases are familiar; most have mutations of the PRKAR1A gene on chromosome 17q22–24. There is little phenotype–genotype correlation known to date.

Objective  To study the genotype–phenotype correlation in a family with newly diagnosed CNC and three generations of subjects bearing the same PRKAR1A mutation. The proband was diagnosed with hepatocellular carcinoma, a tumour that appears to be associated with CNC.

Design  The study consisted of clinical and genetic analysis of a total of 10 individuals belonging to a large Italian family.

Patients  The index case was referred for PRKAR1A gene mutation analysis because he met the diagnostic criteria for a clinical diagnosis of CNC.

Results  The PRKAR1A‐inactivating mutation c.502 +1G > A in the intron 5 splice‐donor site was detected after bidirectional sequencing of germline DNA. The mutation causes a frameshift in the transcribed sequence and a nonsense mRNA that was shown to be degraded; this leads to PRKAR1A haploinsufficiency in all tissues. All available relatives were screened first by DNA testing and, if the latter was positive, by clinical, biochemical and imaging means.

Conclusions  A novel PRKAR1A mutation with an apparently low penetrance and variable expression is reported; the same mutation is also associated with a hepatocellular carcinoma. This is the first time a PRKAR1A mutation is reported in individuals who were diagnosed with CNC after retrospective family screening and following the identification of a proband; the finding has implications for genetic counselling on PRKAR1A and/or CNC.
Mixed‐lineage eosinophil/basophil crisis in MDS: a rare form of progression
Authors: Wimazal F. , Baumgartner C. , Sonneck K. , Zauner C. , Geissler P. , Schur S. , Samorapoompichit P. , Müllauer L. , Födinger M. , Agis H. , Sperr W. R. , Valent P.
Published: April 2008

Background   Basophilic crisis and eosinophilia are well recognized features of advanced chronic myeloid leukaemia. In other myeloid neoplasms, however, transformation with marked basophilia and eosinophilia is considered unusual.

Design   We examined the long‐term follow‐up of 322 patients with de novo myelodysplastic syndromes (MDS) to define the frequency of basophilic, eosinophilic and mixed lineage (basophilic and eosinophilic) transformation.

Results   Of all patients, only one developed mixed lineage crisis (≥ 20% basophils and ≥ 20% eosinophils). In this patient, who initially suffered from chronic myelomonocytic leukaemia, basophils increased to 48% and eosinophils up to 31% at the time of progression. Mixed lineage crisis was not accompanied by an increase in blast cells or organomegaly. The presence of BCR/ABL and other relevant fusion gene products (FIP1L1/PDGFRA, AML1/ETO, PML/RARα, CBFβ/MYH11) were excluded by PCR. Myelomastocytic transformation/myelomastocytic leukaemia and primary mast cell disease were excluded by histology, KIT mutation analysis, electron microscopy and immunophenotyping. Basophils were thus found to be CD123+, CD203c+, BB1+, KIT‐ cells, and to express a functional IgE‐receptor. Among the other patients with MDS examined, 4(1·2%) were found to have marked basophilia (≥ 20%) and 7(2·1%) were found to have massive eosinophilia ( ≥ 20%), whereas mixed‐lineage crisis was detected in none of them.

Conclusions   Mixed basophil/eosinophil crisis may develop in patients with MDS but is an extremely rare event.
Respiratory infection recurrence and passive smoking in early atherosclerosis in children and adolescents with type 1 diabetes
Authors: Odermarsky M. , Andersson S. , Pesonen E. , Sjöblad S. , Ylä‐Herttuala S. , Liuba P.
Published: April 2008

Background  Optimal glucose control in juvenile type 1 diabetes mellitus is necessary but not sufficient to reduce the burden of cardiovascular events in later life. This emphasizes the importance of searching for other possible risk factors associated with diabetes. We investigated whether recurrent episodes of acute respiratory infections and exposure to tobacco smoke could influence vascular phenotypes for early atherosclerosis in children and adolescents with type 1 diabetes.

Materials and methods  Common carotid artery elasticity and intima‐media thickness along with circulating markers of lipid, inflammatory and glycaemic profiles were investigated in up to 98 children and adolescents with type 1 diabetes. The number of clinically manifest acute respiratory tract infections (RTI) during the past year, and the degree of exposure to environmental tobacco smoke (ETS), were assessed by separate questionnaires.

Results   Carotid artery compliance (CAC) was decreased in patients with high (≥ 4/year; n = 22) recurrence of RTI compared to the remaining patients (n = 40; P < 0·05). In a multivariate analysis, the number of RTI during the past year and HbA1C were independently associated with decreased CAC (P < 0·05 for both). The inverse relationship between RTI recurrence and CAC was strengthened by frequent exposure to ETS.

Conclusions   High recurrence of respiratory infections in young type 1 diabetics is associated with increased stiffening of the carotid artery particularly in those often exposed to tobacco smoke.
Applicability and safety of recombinant activated factor VII to control non‐haemophilic haemorrhage: investigational experience in 265 children
Authors: HERBERTSON M. , KENET G.
Published: April 2008

Summary.  Experience of recombinant activated factor VII (rFVIIa, NovoSeven®; Novo Nordisk A/S, Bagsvaerd, Denmark) to control haemorrhage in non‐haemophilic children is limited. The object of this study was to examine the applicability and safety of rFVIIa amongst a group of non‐haemophilic paediatric subjects. Details of all non‐haemophilic children ≤16 years receiving rFVIIa whose data were recorded in the investigational, internet‐based registry, haemostasis.com were analysed. A total of 265 children (mean age 7.7 years) were treated with rFVIIa; the median dose administered was 78.4 μg kg−1 body weight (range 9.0–393.4) and the median total dose received 100.0 μg kg−1 body weight (range 10.9–1341.2). Therapeutic areas included surgery (34.5%), coagulopathy (including thrombocytopenia; 29.0%), spontaneous bleeding (17.2%), trauma (8.4%) and intracranial haemorrhage (4.5%). Two patients experienced thromboembolic events following administration of rFVIIa. Thirty‐nine patients died on account of haemorrhage or complications relating to their underlying condition; neither the thromboembolic events nor the deaths were related to rFVIIa administration. Bleeding stopped in 118/237 (49.8%), markedly decreased in 54/237 (22.8%), decreased in 51/237 (21.5%), remained unchanged in 13/237 (5.5%) and increased in 1/237 (0.4%) patients. These results suggest that rFVIIa is safe and widely applicable in children to control non‐haemophilic haemorrhage.
Diet and dental erosion in young people in south‐east Brazil
Authors: WATERHOUSE PAULA J. , AUAD SHEYLA M. , NUNN JUNE H. , STEEN IAN N. , MOYNIHAN PAULA J.
Published: April 2008

Background.  The regular consumption of acidic foods and drinks may be associated with dental erosion, and soft drink consumption appears to be increasing both in developed and developing countries. Dentists are aware that an acidic diet can contribute to the development of erosion; however, there may be confusion within the profession concerning the general health message of eating five portions of fruits and vegetables each day.

Objective.  The aim of this study was to investigate associations between dental erosion and the consumption of acidic foods and beverages in schoolchildren in south‐east Brazil. The objective was to gather information, by means of a dietary questionnaire, on frequency of intake and patterns of consumption of acidic foods and drinks in a group of schoolchildren. The hypothesis was that the experience of dental erosion among the study sample was associated with the frequency and pattern of consumption of soft drinks, fruit juices, fruits, and yogurt.

Methods.  A cross‐sectional study was conducted in Três Corações, south‐east Brazil. A sample of 458 schoolchildren, mean age 13.8 (SD 0.39) years, completed the study. Information about potential dietary risk factors for dental erosion was collected through a questionnaire survey completed by the schoolchildren. For the dental examinations, the subjects were examined for dental erosion in a school room. Associations between dental erosion and the variables under study were investigated through processes of bivariate and multivariate analyses. The statistical significance level was set at 5%.

Results.  Analysis of the questionnaire surveys showed that the frequency of consumption of sugared carbonated drinks was the only variable independently associated with the erosive process, with subjects who had a daily consumption of such drinks having a greater likelihood of having erosion (P = 0.015, odds ratio 1.752, 95% confidence interval 1.116–2.750).

Conclusions.  Of all tested factors in this sample of schoolchildren the consumption of sugared carbonated drinks is most associated with dental erosion.
ORIGINAL ARTICLE: Cycads in the insular South‐west Pacific: dispersal or vicariance?
Authors: Keppel Gunnar , Hodgskiss Paul D. , Plunkett Gregory M.
Published: April 2008

Aim  Cycads constitute an ancient plant group that is generally believed to disperse poorly. However, one group of cycads (subsection Rumphiae) is thought to have dispersed relatively recently from a Malesian source area westwards to East Africa and eastwards into the Pacific, using a floatation‐facilitating layer in their seeds. We use morphological and allozyme characters to investigate the relationships among the species within this group and to deduce whether the wide distribution was achieved by recent dispersal (as evidenced by high genetic similarity) or more distant vicariance events (high genetic differentiation).

Location  We examined specimens collected throughout the range of subsection Rumphiae, from East Africa through Southeast Asia to Tonga in the South‐west Pacific.

Methods  We investigated relationships within subsection Rumphiae of the genus Cycas by analysing 18 variable (11 informative) morphological characters and 22 allozyme loci for seven of the 10 species currently assigned to this taxon.

Results  Distinctive morphological characters are few and fail to resolve relationships within the group. Allozyme data show that species within this subsection are closely related and suggest that there are two groups within the subsection, one comprising Cycas thouarsii (East Africa) and C. edentata (the Philippines), and the other the remaining species (from Malesia and the Pacific). The Australian species C. silvestris is sister to subsection Rumphiae in the morphological analysis but is closely allied to C. rumphii (nested within the subsection) in the allozyme analysis, suggesting that Rumphiae may be paraphyletic and that characters thought to be taxonomically important may need to be re‐evaluated.

Main conclusions  Cycads within subsection Rumphiae are closely related, and the wide distribution of this group was probably achieved through relatively recent oceanic dispersal events. Separate events probably account for the dispersal of these cycads into the Pacific and to Africa. The origin and distribution of C. silvestris (Australia) could be explained by a dispersal event from New Guinea or may have resulted from a former land connection between Australia and New Guinea.
Multi‐scale determinants of dung beetle assemblage structure across abiotic gradients of the Kalahari–Nama Karoo ecotone, South Africa
Authors: Davis Adrian L.V. , Scholtz Clarke H. , Deschodt Christian
Published: April 2008

Aim Changing conditions across spatial gradients are primary determinants of biotic regions, local habitats, and distributional edges. We investigate how a climatic gradient and edaphic mosaic interact as multi‐scale drivers of spatial patterns in scarabaeine dung beetles. The patterns are tested for congruency with ecoregion and floral boundaries over the same gradient, as responses to physical factors often differ among higher taxa.

Location Southern Africa and the Nama Karoo–Kalahari ecotone, Northern Cape, South Africa.

Methods Data consisted of the climatic distributions of 104 species and their abundances at 223 sites in two ecoregions/floral biomes, four bioregions, and 13 vegetation units. Factor analyses determined the biogeographical composition of the species, and regional‐ to local‐scale patterns in species abundance structure. Hierarchical analysis of oblique factors determined the proportional contribution of spatial variance to patterns. One‐wayanovawas used to test for significant separation of patterns along factor axes. Stepwise multiple regression was used to determine correlations of five physical attributes with species richness, Shannon‐Wiener diversity, and factor loadings for the study sites.

Results Four biogeographical influences overlap in the study region, although rank contribution declines from south‐west arid through north‐east savanna to widespread and south‐east highland taxa. Species abundance structure comprises five subregional patterns, two centred to the north‐east (Kalahari, Isolated Kalahari Dune) dominated by Kalahari influence, and three to the south‐west (Nama Karoo subdivisions: Bushmanland, ‘Upper’, ‘Stony Prieska’) dominated by south‐west arid influence. Kalahari deep sands are characterized especially by a warmer, moister climate, whereas the Nama Karoo mosaic of deep or stony soils is characterized especially by north‐west aridity (Bushmanland), south‐east cooler temperatures (‘Upper’), or excessively stony soils (‘Stony Prieska’). Four of the subregional patterns each comprised three localized patterns related primarily to relative stoniness, edge effects from geographical position, or incidence of rainfall. Species richness and diversity declined with decreasing rainfall and increasing stoniness.

Main conclusions Climatic and edaphic factors are important multi‐scale determinants of spatial patterns in dung beetle assemblage structure, with edaphic factors becoming more important at local spatial scales. The patterns are roughly congruent with the Kalahari Savanna–Nama Karoo ecotone at the floral biome or ecoregion scale, but show limited coincidence with finer‐scale floral classification.
Constraint factor graph cut–based active contour method for automated cellular image segmentation in RNAi screening
Authors: CHEN C. , LI H. , ZHOU X. , WONG S. T. C.
Published: April 2008

Image‐based, high throughput genome‐wide RNA interference (RNAi) experiments are increasingly carried out to facilitate the understanding of gene functions in intricate biological processes. Automated screening of such experiments generates a large number of images with great variations in image quality, which makes manual analysis unreasonably time‐consuming. Therefore, effective techniques for automatic image analysis are urgently needed, in which segmentation is one of the most important steps. This paper proposes a fully automatic method for cells segmentation in genome‐wide RNAi screening images. The method consists of two steps: nuclei and cytoplasm segmentation. Nuclei are extracted and labelled to initialize cytoplasm segmentation. Since the quality of RNAi image is rather poor, a novel scale‐adaptive steerable filter is designed to enhance the image in order to extract long and thin protrusions on the spiky cells. Then, constraint factor GCBAC method and morphological algorithms are combined to be an integrated method to segment tight clustered cells. Compared with the results obtained by using seeded watershed and the ground truth, that is, manual labelling results by experts in RNAi screening data, our method achieves higher accuracy. Compared with active contour methods, our method consumes much less time. The positive results indicate that the proposed method can be applied in automatic image analysis of multi‐channel image screening data.
Cluster identification in AA5754 aluminium sheets using mathematical morphology analysis
Authors: TEWARI A. , TIWARI S. , BISWAS P. , MISHRA R. K.
Published: April 2008

Quantitative image analysis of particle distribution in the microstructure of continuous cast (CC) and direct chill cast (DC) AA5754 aluminium alloy sheets have been conducted. This information can be used as an input for modelling mechanical deformation and instability in these materials. The quantitative analysis reveals that there are significant differences in the microstructure of the two materials even though the total content of second‐phase particles is statistically similar. Qualitative observation shows the second‐phase particles to be arranged in the form of streaks parallel to the rolling direction in the CC sheets and in a uniform random manner in the DC sheets. The main difference in the geometric microstructure of the CC and DC material is the spatial arrangement of the second‐phase particles. A new mathematical technique called proximity analysis is developed to identify clusters and group of particles belonging to a cluster. Quantification through proximity analysis reveals that the particle clusters in CC sheet are in the form of long clusters (streaks) parallel to the rolling direction and are significantly longer than those in DC sheets (with the largest cluster in CC being four times larger than DC), and also have anisotropic angular orientation parallel to the rolling direction. The lower value of fracture strain observed in the CC sheets compared to DC sheets is attributed to a combination of large sizes of clusters and their preferential alignment along the rolling direction in the CC microstructure.
Correction for non‐perpendicularity of beam and tilt axis in tomographic reconstructions with the IMOD package
Authors: MASTRONARDE D. N.
Published: April 2008

A correction for non‐perpendicularity between the beam axis and the tilt axis in electron tomographic tilt series has been implemented in the IMOD software package and its value and limitations have been explored. Correction for this effect can provide a significant improvement in the alignment error and the reconstruction quality in some cases. However, when the projection model being fit includes an anisotropic shrinkage (i.e. stretch) in the plane of the specimen, adding a variable for the beam tilt does not produce a lower alignment error; it is thus not possible to distinguish between the effects of stretch and beam tilt. Test reconstructions indicate that an alignment solution that includes stretch will adequately correct for the effects of a beam tilt. For specimens subject to deformation under the beam, an alignment solution that accounts for stretch is preferable to one that accounts for beam tilt instead, provided that the markers used for alignment are sufficiently well distributed. Otherwise, a correction for beam tilt should be used.
Atomic force microscopy to characterize the molecular size of prion protein
Authors: KUNZE S. , LEMKE K. , METZE J. , BLOUKAS G. , KOTTA K. , PANAGIOTIDIS C. H. , SKLAVIADIS T. , BODEMER W.
Published: April 2008

The conformational transition of α‐helix‐rich cellular prion protein (PrPC) to an isomer with high β‐sheet content is associated with transmissible spongiform encephalopathies. With the ultimate long‐term goal of using imaging techniques to study PrP aggregation, we report the results of initial experiments to determine whether PrP molecules could be visualized as single molecules, and if the observed size corresponded to the calculated size for PrP. The investigation of single molecules, and not those embedded into larger aggregates, was the key in our experimental approach. Using atomic force microscopy (AFM) as an imaging method, the immobilization of recombinant histidine (His)10‐tagged PrP on mica was performed in the presence of different heavy metal ions. The addition of Cu2+ resulted in an enhanced PrP immobilization, whereas Ni2+ reduced coverage of the surface by PrP. High‐resolution data from dried PrP preparations provided a first approximation to geometrical parameters of PrP precipitates, which indicated that the volume of a single PrP molecule was 30 nm3. Molecular dynamics simulations performed to complement the structural aspects of the AFM investigation yielded a calculated molecular volume of 33 nm3 for PrP. These experimentally observed and theoretically expected values provide basic knowledge for further studies on the size and composition of larger amyloidal PrP aggregates, PrP isoforms or mutants such as PrP molecules without octarepeats.
Evolution of residual stress and crack morphologies during 3D FIB tomographic analysis of alumina
Authors: ELFALLAGH F. , INKSON B. J.
Published: April 2008

Three‐dimensional focused ion beam (FIB) tomography is increasingly being used for 3D characterization of microstructures in the 50 nm–20 μm range. FIB tomography is a destructive, invasive process, and microstructural changes may potentially occur during the analysis process. Here residual stress and crack morphologies in single‐crystal sapphire samples have been concurrently analyzed using Cr3+ fluorescence spectroscopy and FIB tomography. Specifically, maps of surface residual stress have been obtained from optically polished single‐crystal alumina [surface orientation (1 ī 0 2)], from FIB milled surface trenches, from Vickers micro‐indentation sites (loads 50 g–300 g), and from Vickers micro‐indentation sites during FIB serial sectioning. The residual stress maps clearly show that FIB sputtering generates residual stress changes. For the case of the Vickers micro‐indentations, FIB sputtering causes significant changes in residual stress during the FIB tomographic serial sectioning. 3D reconstruction of the crack distribution around micro‐indentation sites shows that the cracks observed are influenced by the location of the FIB milled surface trenches due to localized stress changes.
Pseudovacuoles – immobilized by high‐pressure freezing – are associated with blebbing in walker carcinosarcoma cells
Authors: VANHECKE D. , BELLMANN R. , BAUM O. , GRABER W. , EGGLI P. , KELLER H. , STUDER D.
Published: April 2008

By applying high pressure freezing and freeze‐substitution, we observed large inclusions of homogeneous appearance in the front of locomoting Walker carcinosarcoma cells that have not been described earlier. Live cell imaging revealed that these inclusions were poor in lipids and nucleic acids but had a high lysine (and hence protein) content. Usually one such structure 2–5 μm in size was present at the front of motile Walker cells, predominantly in the immediate vicinity of newly forming blebs. By correlating the lysine‐rich areas in fixed and embedded cells with electron microscopic pictures, inclusions could be assigned to confined, faintly stained cytoplasmic areas that lacked a surrounding membrane; they were therefore called pseudovacuoles. After high‐pressure freezing and freeze substitution, pseudovacuoles appeared to be filled with 20 nm large electron‐transparent patches surrounded by 12 and 15 nm large particles. The heat shock protein Hsp90 was identified by peptide sequencing as a major fluorescent band on SDS‐PAGE of lysine‐labelled Walker cell extracts. By immunofluorescence, Hsp90 was found to be enriched in pseudovacuoles. Colocalization of the lysine with a potassium‐specific dye in living cells revealed that pseudovacuoles act as K+ stores in the vicinity of forming blebs. We propose that pseudovacuoles might support blebbing by locally regulating the intracellular hydrostatic pressure.
Electron microscopy of the early Caenorhabditis elegans embryo
Authors: MÜLLER‐REICHERT T. , MÄNTLER J. , SRAYKO M. , O'TOOLE E.
Published: April 2008

The early Caenorhabditis elegans embryo is currently a popular model system to study centrosome assembly, kinetochore organization, spindle formation, and cellular polarization. Here, we present and review methods for routine electron microscopy and 3D analysis of the early C. elegans embryo. The first method uses laser‐induced chemical fixation to preserve the fine structure of isolated embryos. This approach takes advantage of time‐resolved fixation to arrest development at specific stages. The second method uses high‐pressure freezing of whole worms followed by freeze‐substitution (HPF‐FS) for ultrastructural analysis. This technique allows staging of developing early embryos within the worm uterus, and has the advantage of superior sample preservation required for high‐resolution 3D reconstruction. The third method uses a correlative approach to stage isolated, single embryos by light microscopy followed by HPF‐FS and electron tomography. This procedure combines the advantages of time‐resolved fixation and superior ultrastructural preservation by high‐pressure freezing and allows a higher throughput electron microscopic analysis. The advantages and disadvantages of these methods for different applications are discussed.
Controlled microaspiration for high‐pressure freezing: a new method for ultrastructural preservation of fragile and sparse tissues for TEM and electron tomography
Authors: TRIFFO W. J. , PALSDOTTIR H. , McDONALD K. L. , LEE J. K. , INMAN J. L. , BISSELL M. J. , RAPHAEL R. M. , AUER M.
Published: April 2008

High‐pressure freezing is the preferred method to prepare thick biological specimens for ultrastructural studies. However, the advantages obtained by this method often prove unattainable for samples that are difficult to handle during the freezing and substitution protocols. Delicate and sparse samples are difficult to manipulate and maintain intact throughout the sequence of freezing, infiltration, embedding and final orientation for sectioning and subsequent transmission electron microscopy. An established approach to surmount these difficulties is the use of cellulose microdialysis tubing to transport the sample. With an inner diameter of 200 μm, the tubing protects small and fragile samples within the thickness constraints of high‐pressure freezing, and the tube ends can be sealed to avoid loss of sample. Importantly, the transparency of the tubing allows optical study of the specimen at different steps in the process. Here, we describe the use of a micromanipulator and microinjection apparatus to handle and position delicate specimens within the tubing. We report two biologically significant examples that benefit from this approach, 3D cultures of mammary epithelial cells and cochlear outer hair cells. We illustrate the potential for correlative light and electron microscopy as well as electron tomography.
Melatonin Activates Brain Dopaminergic Systems in the Eel with an Inhibitory Impact on Reproductive Function
Authors: Sébert M.‐E. , Legros C. , Weltzien F.‐A. , Malpaux B. , Chemineau P. , Dufour S.
Published: April 2008

In the eel, a deficit in gonadotrophin‐releasing hormone (GnRH) and a strong dopaminergic (DA) inhibition are responsible for the blockade of gonad development if silver eels are prevented from their reproductive migration. Environmental factors that eels encounter during their oceanic reproductive migration are thought to play an important role in the stimulation of eel pubertal development. We investigated the potential role of melatonin, a known mediator of the effects of external factors on reproductive function in vertebrates. We demonstrated that a long‐term melatonin treatment increased brain tyrosine hydroxylase (TH, the rate limiting enzyme of DA synthesis) mRNA expression in a region‐dependent way. Melatonin stimulated the dopaminergic system of the preoptic area, which is involved in the inhibitory control of gonadotrophin [luteinising hormone (LH) and follicle‐stimulating hormone (FSH)] synthesis and release. Moreover, we showed that the increased TH expression appeared to be consistent with melatonin binding site distribution as shown by 2[125I]‐melatonin labelling studies. On the other hand, melatonin had no effects on the two eel native forms of GnRH (mGnRH and cGnRH‐II) mRNA expression. Concerning the pituitary–gonad axis, we showed that melatonin treatment decreased both gonadotrophin β‐subunit (LHβ, FSHβ) mRNA expression and reduced sexual steroid (11‐ketotestosterone, oestradiol) plasma levels. This indicates that melatonin treatment had a negative effect on eel reproductive function. To our knowledge, the results of the present study provide the first evidence that melatonin enhances TH expression in specific brain regions in a non‐mammalian species. By this mechanism melatonin could represent one pathway by which environmental factors could modulate reproductive function in the eel.
Regulation of Hypophysiotrophic Corticotrophin‐Releasing Hormone‐ and Thyrotrophin‐Releasing Hormone‐Synthesising Neurones by Brainstem Catecholaminergic Neurones
Authors: Wittmann G.
Published: April 2008

Hypophysiotrophic corticotrophin‐releasing hormone (CRH)‐ and thyrotrophin‐releasing hormone (TRH)‐synthesising neurones are the principal hypothalamic regulators of glucocorticoid and thyroid hormone secretion, respectively. These two neuroendocrine cell populations are closely situated in the hypothalamic paraventricular nucleus and are targets of neuronal afferent pathways that convey important signals for adapting the neurosecretory activity of CRH and TRH neurones to actual demands. The catecholaminergic afferents of CRH and TRH neurones originate from both noradrenaline‐ and adrenaline‐synthesising cell groups located in the brainstem, and collectively represent one of the most well studied neural inputs of these neurones. The present review summarises the data obtained in recent years concerning the functional significance of the catecholaminergic innervation of hypophysiotrophic CRH and TRH neurones in rats.
Reconstruction of the Sudden Oak Death epidemic in California through microsatellite analysis of the pathogen Phytophthora ramorum
Authors: MASCHERETTI S. , CROUCHER P. J. P. , VETTRAINO A. , PROSPERO S. , GARBELOTTO M.
Published: April 2008

The genetic structure of the clonally reproducing Sudden Oak Death (SOD) pathogen in California was investigated using seven variable microsatellites. A total of 35 multilocus genotypes were identified among 292 samples representative of populations from 14 forest sites and of the nursery trade. amova indicated significant genetic variability both within (44.34%) and among populations (55.66%). Spatial autocorrelation analyses indicated that Moran's index of similarity reached a minimum of 0.1 at 350 m, increased to 0.4 at 1500 m and then decreased to zero at 10 km. These results suggest a bimodal pattern of spread, with medium range dispersal (1500–10 000 m) putatively attributed to the presence of strong winds. Lack of genetic structure was identified for three groups of populations. One group notably included the nurseries’ population and two forest populations, both linked to early reports of the pathogen. A neighbour‐joining analysis based on pairwise ΦST values indicated that the clade inclusive of the nurseries’ populations is basal to all California populations. A network analysis identified three common genotypes as the likely founders of the California infestation and proposes a stepwise model for local evolution of novel genotypes. This was supported by the identification in the same locations of novel genotypes and of their 1‐ or 2‐step parents. We hypothesize that the few undifferentiated population groups indicate historical human spread of the pathogen, while the general presence of genetically structured populations indicates that new infestations are currently generated by rare medium or long‐range natural movement of the pathogen, followed by local generation of new genotypes.
Review: Familial Parkinson's disease – genetics, clinical phenotype and neuropathology in relation to the common sporadic form of the disease
Authors: Schiesling Carola , Kieper Nicole , Seidel Kay , Krüger Rejko
Published: April 2008

The identification of the first gene in familial Parkinson's disease (PD) only 10 years ago was a major step in the understanding of the molecular mechanisms in neurodegeneration. Alpha‐synuclein aggregation was not only recognized as a key event in neurodegeneration in patients carrying mutations in this gene, but it turned out to be the most consistent marker to define Lewy body pathology also in non‐heritable idiopathic PD (IPD). Subsequent comprehensive pathoanatomical studies of IPD brains led to a novel concept of an ascending pathological process in variable stages that are reflected by alpha‐synuclein aggregation at specific predilection sites. To date, more than seven genes are known to cause familial PD. The fact that these genetic forms of Parkinsonism present with clinical features indistinguishable from IPD, but may display neuropathological features that are not consistent with IPD, underscores the need of a more differentiated approach to familial and sporadic forms of Parkinsonism. Indeed, in distinct populations, mutations in one single gene were found to cause the disease in up to 40% of patients formerly described as ‘idiopathic’ cases. These findings indicate that IPD, as defined by a late‐onset disorder with no (apparent) genetic contribution, is part of a clinical syndrome that becomes more and more heterogeneous in terms of aetiology, with overlapping clinical and pathoanatomical features. Thus in the present review, we discuss clues from familial PD to our understanding of the molecular pathogenesis of neurodegeneration with special consideration of the variable clinical and neuropathological aspects.
Secondarily reduced foreleg armature in Perochaeta dikowi sp.n. (Diptera: Cyclorrhapha: Sepsidae) due to a novel mounting technique
Authors: ANG YUCHEN , PUNIAMOORTHY NALINI , MEIER RUDOLF
Published: April 2008

Abstract The males of almost all sepsid species have strongly modified forelegs that are used to clamp the female’s wingbase during mounting. Here, we describe a new species in the genus Perochaeta whose males have unmodified forelegs. We use DNA sequence data for ten genes to reconstruct the position of Perochaeta on the phylogenetic tree for Sepsidae, and reveal that the lack of foreleg armature in Perochaeta dikowi sp.n. is secondary. Through the study of the mating behaviour of the new species, we demonstrate that the loss of armature is correlated with a new mounting technique during which the males of P. dikowi do not use the foreleg to clamp the female’s wingbase. Instead, the male approaches the female from behind and bends his abdomen forwards in order to establish genital contact. Our study shows how data from morphology, phylogenetics, and behavioural biology can complement each other to yield a deeper understanding of how changes in morphology and behaviour are correlated.