bluuebunny/crossref_metadata_2025 huggingface dataset:
Bioprospecting for Trichothecene 3-
O
-Acetyltransferases in the Fungal Genus
Fusarium
Yields Functional Enzymes with Different Abilities To Modify the Mycotoxin Deoxynivalenol
Authors: Khatibi Piyum A. , Newmister Sean A. , Rayment Ivan , McCormick Susan P. , Alexander Nancy J. , Schmale David G.
Published: December 2010

The trichothecene mycotoxin deoxynivalenol (DON) is a common contaminant of small grains, such as wheat and barley, in the United States. New strategies to mitigate the threat of DON need to be developed and implemented. TRI101 and TRI201 are trichothecene 3-
O
-acetyltransferases that are able to modify DON and reduce its toxicity. Recent work has highlighted differences in the activities of TRI101 from two different species of
Fusarium
(
F. graminearum
and
F. sporotrichioides
), but little is known about the relative activities of TRI101/TRI201 enzymes produced by other species of
Fusarium
. We cloned
TRI101
or
TRI201
genes from seven different species of
Fusarium
and found genetic identity between sequences ranging from 66% to 98%.
In vitro
feeding studies using transformed yeast showed that all of the TRI101/TRI201 enzymes tested were able to acetylate DON; conversion of DON to 3-acetyl-deoxynivalenol (3ADON) ranged from 50.5% to 100.0%, depending on the
Fusarium
species from which the gene originated. A time course assay showed that the rate of acetylation varied from species to species, with the gene from
F. sporotrichioides
having the lowest rate. Steady-state kinetic assays using seven purified enzymes produced catalytic efficiencies for DON acetylation ranging from 6.8 × 10
4
M
−1
·s
−1
to 4.7 × 10
6
M
−1
·s
−1
. Thermostability measurements for the seven orthologs ranged from 37.1°C to 43.2°C. Extended sequence analysis of portions of
TRI101/TRI201
from 31 species of
Fusarium
(including known trichothecene producers and nonproducers) suggested that other members of the genus may contain functional
TRI101/TRI201
genes, some with the potential to outperform those evaluated in the present study.
High-Throughput Plasmid Content Analysis of
Borrelia burgdorferi
B31 by Using Luminex Multiplex Technology
Authors: Norris Steven J. , Howell Jerrilyn K. , Odeh Evelyn A. , Lin Tao , Gao Lihui , Edmondson Diane G.
Published: December 2010

Borrelia burgdorferi
, the causative agent of Lyme disease in North America, is an invasive pathogen that causes persistent multiorgan manifestations in humans and other mammals. Genetic studies of this bacterium are complicated by the presence of multiple plasmid replicons, many of which are readily lost during
in vitro
culture. The analysis of
B. burgdorferi
plasmid content by plasmid-specific PCR and agarose gel electrophoresis or other existing techniques is informative, but these techniques are cumbersome and challenging to perform in a high-throughput manner. In this study, a PCR-based Luminex assay was developed for determination of the plasmid content of the strain
B. burgdorferi
B31. This multiplex, high-throughput method allows simultaneous detection of the plasmid contents of many
B. burgdorferi
strains in a 96-well format. The procedure was used to evaluate the occurrence of plasmid loss in 44 low-passage
B. burgdorferi
B31 clones and in a library of over 4,000 signature-tagged mutagenesis (STM) transposon mutant clones. This analysis indicated that only 40% of the clones contained all plasmids, with (in order of decreasing frequency) lp5, lp56, lp28-1, lp25, cp9, lp28-4, lp28-2, and lp21 being the most commonly missing plasmids. These results further emphasize the need for careful plasmid analysis in Lyme disease
Borrelia
studies. Adaptations of this approach may also be useful in the evaluation of plasmid content and chromosomal gene variations in additional Lyme disease
Borrelia
strains and other organisms with variable genomes and in the correlation of these genetic differences with pathogenesis and other biological properties.
Imaging Hydrated Microbial Extracellular Polymers: Comparative Analysis by Electron Microscopy
Authors: Dohnalkova Alice C. , Marshall Matthew J. , Arey Bruce W. , Williams Kenneth H. , Buck Edgar C. , Fredrickson James K.
Published: December 2010

Microbe-mineral and -metal interactions represent a major intersection between the biosphere and geosphere but require high-resolution imaging and analytical tools for investigation of microscale associations. Electron microscopy has been used extensively for geomicrobial investigations, and although usedbona fide, the traditional methods of sample preparation do not preserve the native morphology of microbiological components, especially extracellular polymers. Herein, we present a direct comparative analysis of microbial interactions by conventional electron microscopy approaches with imaging at room temperature and a suite of cryogenic electron microscopy methods providing imaging in the close-to-natural hydrated state.In situ, we observed an irreversible transformation of the hydrated bacterial extracellular polymers during the traditional dehydration-based sample preparation that resulted in their collapse into filamentous structures. Dehydration-induced polymer collapse can lead to inaccurate spatial relationships and hence could subsequently affect conclusions regarding the nature of interactions between microbial extracellular polymers and their environment.
Natural and Engineered Hydroxyectoine Production Based on the
Pseudomonas stutzeri ectABCD-ask
Gene Cluster
Authors: Seip Britta , Galinski Erwin A. , Kurz Matthias
Published: December 2010

We report on the presence of a functional hydroxyectoine biosynthesis gene cluster,
ectABCD-ask
, in
Pseudomonas stutzeri
DSM5190
T
and evaluate the suitability of
P. stutzeri
DSM5190
T
for hydroxyectoine production. Furthermore, we present information on heterologous
de novo
production of the compatible solute hydroxyectoine in
Escherichia coli
. In this host, the
P. stutzeri
gene cluster remained under the control of its salt-induced native promoters. We also noted the absence of trehalose when hydroxyectoine genes were expressed, as well as a remarkable inhibitory effect of externally applied betaine on hydroxyectoine synthesis. The specific heterologous production rate in
E. coli
under the conditions employed exceeded that of the natural producer
Pseudomonas stutzeri
and, for the first time, enabled effective hydroxyectoine production at low salinity (2%), with the added advantage of simple product processing due to the absence of other cosolutes.
Possible Implications of Chytrid Parasitism for Population Subdivision in Freshwater Cyanobacteria of the Genus
Planktothrix
Authors: Sønstebø Jørn Henrik , Rohrlack Thomas
Published: December 2010

Populations of the cyanobacterium
Planktothrix
comprise multiple coexisting oligopeptide chemotypes that can behave differently in nature. We tested whether this population subdivision can, in principle, be driven by parasitic chytrid fungi, which are almost neglected agents of
Planktothrix
mortality. Two chytrid strains, Chy-Lys2009 and Chy-Kol2008, were isolated from
Planktothrix
-dominated lakes in Norway. The two strains shared 98.2% and 86.2% of their 28S and internal transcribe spacer rRNA gene sequences, respectively. A phylogenetic analysis placed them in the order Rhizophydiales family Angulomycetaceae. Chy-Lys2009 and Chy-Kol2008 could completely lyse
Planktothrix
cultures within days, while they failed to infect other filamentous cyanobacteria. The effect on
Planktothrix
was chemotype dependent, and both chytrid strains showed distinct chemotype preferences. These findings identify chytrid fungi infecting
Planktothrix
as highly potent and specialized parasites which may exert strong selective pressure on their hosts. According to established hypotheses on host-parasite coevolution, parasitism with the above properties may result in subdivision of
Planktothrix
populations into coexisting chemotypes and periodic shifts in the relative
Planktothrix
chemotype composition. These predictions are in agreement with field observations. Moreover, a genetic analysis verified the co-occurrence of Chy-Lys2009 and Chy-Kol2008 or related chytrid strains along with distinct
Planktothrix
chemotypes in at least one water body. Our findings are consistent with a scenario where chytrid parasitism is one driving force of
Planktothrix
population subdivision, which in turn leads to polymorphism in parasitic chytrid fungi. Future studies should test the validity of this scenario under field conditions.
Inhibition of Cereulide Toxin Synthesis by Emetic
Bacillus cereus
via Long-Chain Polyphosphates
Authors: Frenzel Elrike , Letzel Thomas , Scherer Siegfried , Ehling-Schulz Monika
Published: December 2010

Severe intoxications caused by the
Bacillus cereus
emetic toxin cereulide can hardly be prevented due to the ubiquitous distribution and heat resistance of spores and the extreme thermal and chemical stability of cereulide. It would therefore be desirable to inhibit cereulide synthesis during food manufacturing processes or in prepared foods, which are stored under time-temperature abuse conditions. Toward this end, the impacts of three long-chain polyphosphate (polyP) formulations on growth and cereulide production were examined. The inhibition was dependent on the concentration and the type of the polyP blend, indicating that polyPs and not the orthophosphates were effective. Quantitative PCR (qPCR) monitoring at sublethal concentrations revealed that polyPs reduced the transcription of
ces
nonribosomal peptide synthetase (NRPS) genes by 3- to 4-fold along with a significantly reduced toxin production level. At lower concentrations, toxin synthesis was decreased, although the growth rate was not affected. These data indicate a differential effect on toxin synthesis independent of growth inhibition. The inhibition of toxin synthesis in food was also observed. Despite the growth of
B. cereus
, toxin synthesis was reduced by 70 to 100% in two model food systems (reconstituted infant food and oat milk), which were analyzed with HEp-2 cell culture assays and high-performance liquid chromatography (HPLC)/electrospray ionization-time of flight mass spectrometry (ESI-TOF-MS). Accordingly,
ces
promoter activity was strongly downregulated, as visualized by using a
lux
-based reporter strain. These data illustrate the potential of polyphosphate formulations to reduce the risk of cereulide synthesis in food and may contribute to targeted hurdle concepts.
Metagenomic Analyses: Past and Future Trends
Authors: Simon Carola , Daniel Rolf
Published: December 2010

Metagenomics has revolutionized microbiology by paving the way for a cultivation-independent assessment and exploitation of microbial communities present in complex ecosystems. Metagenomics comprising construction and screening of metagenomic DNA libraries has proven to be a powerful tool to isolate new enzymes and drugs of industrial importance. So far, the majority of the metagenomically exploited habitats comprised temperate environments, such as soil and marine environments. Recently, metagenomes of extreme environments have also been used as sources of novel biocatalysts. The employment of next-generation sequencing techniques for metagenomics resulted in the generation of large sequence data sets derived from various environments, such as soil, the human body, and ocean water. Analyses of these data sets opened a window into the enormous taxonomic and functional diversity of environmental microbial communities. To assess the functional dynamics of microbial communities, metatranscriptomics and metaproteomics have been developed. The combination of DNA-based, mRNA-based, and protein-based analyses of microbial communities present in different environments is a way to elucidate the compositions, functions, and interactions of microbial communities and to link these to environmental processes.
Systematic Screen of Schizosaccharomyces pombe Deletion Collection Uncovers Parallel Evolution of the Phosphate Signal Transduction Pathway in Yeasts
Authors: Henry Theresa C. , Power Juliette E. , Kerwin Christine L. , Mohammed Aishat , Weissman Jonathan S. , Cameron Dale M. , Wykoff Dennis D.
Published: December 2010

The phosphate signal transduction (PHO) pathway, which regulates genes in response to phosphate starvation, is well defined in
Saccharomyces cerevisiae
. We asked whether the PHO pathway was the same in the distantly related fission yeast
Schizosaccharomyces pombe
. We screened a deletion collection for mutants aberrant in phosphatase activity, which is primarily a consequence of
pho1
+
transcription. We identified a novel zinc finger-containing protein (encoded by
spbc27b12.11c
+
), which we have named
pho7
+
, that is essential for
pho1
+
transcriptional induction during phosphate starvation. Few of the
S. cerevisiae
genes involved in the PHO pathway appear to be involved in the regulation of the phosphate starvation response in
S. pombe
. Only the most upstream genes in the PHO pathway in
S. cerevisiae
(
ADO1
,
DDP1
, and
PPN1
) share a similar role in both yeasts. Because
ADO1
and
DDP1
regulate ATP and IP
7
levels, we hypothesize that the ancestor of these yeasts must have sensed similar metabolites in response to phosphate starvation but have evolved distinct mechanisms in parallel to sense these metabolites and induce phosphate starvation genes.
Biochemical and Genetic Evidence for the Presence of Multiple Phosphatidylinositol- and Phosphatidylinositol 4,5-Bisphosphate-Specific Phospholipases C in Tetrahymena
Authors: Leondaritis George , Sarri Theoni , Dafnis Ioannis , Efstathiou Antonia , Galanopoulou Dia
Published: December 2010

Eukaryotic phosphoinositide-specific phospholipases C (PI-PLC) specifically hydrolyze phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2], produce the Ca2+-mobilizing agent inositol 1,4,5-trisphosphate, and regulate signaling in multicellular organisms. Bacterial PtdIns-specific PLCs, also present in trypanosomes, hydrolyze PtdIns and glycosyl-PtdIns, and they are considered important virulence factors. All unicellular eukaryotes studied so far contain a single PI-PLC-like gene. In this report, we show that ciliates are an exception, since we provide evidence thatTetrahymenaspecies contain two sets of functional genes coding for both bacterial and eukaryotic PLCs. Biochemical characterization revealed two PLC activities that differ in their phosphoinositide substrate utilization, subcellular localization, secretion to extracellular space, and sensitivity to Ca2+. One of these activities was identified as a typical membrane-associated PI-PLC activated by low-micromolar Ca2+, modestly activated by GTPγSin vitro, and inhibited by the compound U73122 [1-(6-{[17β-3-methoxyestra-1,3,5(10)-trien-17-yl]amino}hexyl)-1H-pyrrole-2,5-dione]. Importantly, inhibition of PI-PLCin vivoresulted in rapid upregulation of PtdIns(4,5)P2levels, suggesting its functional importance in regulating phosphoinositide turnover inTetrahymena. Byin silicoand molecular analysis, we identified two PLC genes that exhibit significant similarity to bacterial but not trypanosomal PLC genes and three eukaryotic PI-PLC genes, one of which is a novel inactive PLC similar to proteins identified only in metazoa. Comparative studies of expression patterns and PI-PLC activities in threeT. thermophilastrains showed a correlation between expression levels and activity, suggesting that the three eukaryotic PI-PLC genes are functionally nonredundant. Our findings imply the presence of a conserved and elaborate PI-PLC-Ins(1,4,5)P3-Ca2+regulatoryaxis in ciliates.
Identification of an Extracellular Polysaccharide Network Essential for Cytochrome Anchoring and Biofilm Formation in
Geobacter sulfurreducens
Authors: Rollefson Janet B. , Stephen Camille S. , Tien Ming , Bond Daniel R.
Published: December 2010

Transposon insertions in
Geobacter sulfurreducens
GSU1501, part of an ATP-dependent exporter within an operon of polysaccharide biosynthesis genes, were previously shown to eliminate insoluble Fe(III) reduction and use of an electrode as an electron acceptor. Replacement of GSU1501 with a kanamycin resistance cassette produced a similarly defective mutant, which could be partially complemented by expression of GSU1500 to GSU1505 in
trans
. The Δ1501 mutant demonstrated limited cell-cell agglutination, enhanced attachment to negatively charged surfaces, and poor attachment to positively charged poly-
d
-lysine- or Fe(III)-coated surfaces. Wild-type and mutant cells attached to graphite electrodes, but when electrodes were poised at an oxidizing potential inducing a positive surface charge (+0.24 V versus the standard hydrogen electrode [SHE]), Δ1501 mutant cells detached. Scanning electron microscopy revealed fibrils surrounding wild-type
G. sulfurreducens
which were absent from the Δ1501 mutant. Similar amounts of type IV pili and pilus-associated cytochromes were detected on both cell types, but shearing released a stable matrix of
c
-type cytochromes and other proteins bound to polysaccharides. The matrix from the mutant contained 60% less sugar and was nearly devoid of
c
-type cytochromes such as OmcZ. The addition of wild-type extracellular matrix to Δ1501 cultures restored agglutination and Fe(III) reduction. The polysaccharide binding dye Congo red preferentially bound wild-type cells and extracellular matrix material over mutant cells, and Congo red inhibited agglutination and Fe(III) reduction by wild-type cells. These results demonstrate a crucial role for the
xap
(extracellular anchoring polysaccharide) locus in metal oxide attachment, cell-cell agglutination, and localization of essential cytochromes beyond the
Geobacter
outer membrane.
Identification of Missing Genes and Enzymes for Autotrophic Carbon Fixation in
Crenarchaeota
Authors: Ramos-Vera W. Hugo , Weiss Michael , Strittmatter Eric , Kockelkorn Daniel , Fuchs Georg
Published: December 2010

Two autotrophic carbon fixation cycles have been identified in
Crenarchaeota
. The dicarboxylate/4-hydroxybutyrate cycle functions in anaerobic or microaerobic autotrophic members of the
Thermoproteales
and
Desulfurococcales
. The 3-hydroxypropionate/4-hydroxybutyrate cycle occurs in aerobic autotrophic
Sulfolobales
; a similar cycle may operate in autotrophic aerobic marine
Crenarchaeota
. Both cycles form succinyl-coenzyme A (CoA) from acetyl-CoA and two molecules of inorganic carbon, but they use different means. Both cycles have in common the (re)generation of acetyl-CoA from succinyl-CoA via identical intermediates. Here, we identified several missing enzymes/genes involved in the seven-step conversion of succinyl-CoA to two molecules of acetyl-CoA in
Thermoproteus neutrophilus
(
Thermoproteales
),
Ignicoccus hospitalis
(
Desulfurococcales
), and
Metallosphaera sedula
(
Sulfolobales
). The identified enzymes/genes include succinyl-CoA reductase, succinic semialdehyde reductase, 4-hydroxybutyrate-CoA ligase, bifunctional crotonyl-CoA hydratase/(
S
)-3-hydroxybutyryl-CoA dehydrogenase, and beta-ketothiolase. 4-Hydroxybutyryl-CoA dehydratase, which catalyzes a mechanistically intriguing elimination of water, is well conserved and rightly can be considered the key enzyme of these two cycles. In contrast, several of the other enzymes evolved from quite different sources, making functional predictions based solely on genome interpretation difficult, if not questionable.
Pyruvate Formate Lyase Acts as a Formate Supplier for Metabolic Processes during Anaerobiosis in
Staphylococcus aureus
Authors: Leibig Martina , Liebeke Manuel , Mader Diana , Lalk Michael , Peschel Andreas , Götz Friedrich
Published: December 2010

Previous studies demonstrated an upregulation of pyruvate formate lyase (Pfl) and NAD-dependent formate dehydrogenase (Fdh) in
Staphylococcus aureus
biofilms. To investigate their physiological role, we constructed
fdh
and
pfl
deletion mutants (Δ
fdh
and Δ
pfl
). Although formate dehydrogenase activity in the
fdh
mutant was lost, it showed little phenotypic alterations under oxygen-limited conditions. In contrast, the
pfl
mutant displayed pleiotropic effects and revealed the importance of formate production for anabolic metabolism. In the
pfl
mutant, no formate was produced, glucose consumption was delayed, and ethanol production was decreased, whereas acetate and lactate production were unaffected. All metabolic alterations could be restored by addition of formate or complementation of the Δ
pfl
mutant. In compensation reactions, serine and threonine were consumed better by the Δ
pfl
mutant than by the wild type, suggesting that their catabolism contributes to the refilling of formyl-tetrahydrofolate, which acts as a donor of formyl groups in, e.g., purine and protein biosynthesis. This notion was supported by reduced production of formylated peptides by the Δ
pfl
mutant compared to that of the parental strain, as demonstrated by weaker formyl-peptide receptor 1 (FPR1)-mediated activation of leukocytes with the mutant. FPR1 stimulation could also be restored either by addition of formate or by complementation of the mutation. Furthermore, arginine consumption and
arc
operon transcription were increased in the Δ
pfl
mutant. Unlike what occurred with the investigated anaerobic conditions, a biofilm is distinguished by nutrient, oxygen, and pH gradients, and we thus assume that Pfl plays a significant role in the anaerobic layer of a biofilm. Fdh might be critical in (micro)aerobic layers, as formate oxidation is correlated with the generation of NADH/H
+
, whose regeneration requires respiration.
Unexpected Coregulator Range for the Global Regulator Lrp of
Escherichia coli
and
Proteus mirabilis
Authors: Hart Benjamin R. , Blumenthal Robert M.
Published: December 2010

The Lrp/AsnC family of transcription factors links gene regulation to metabolism in bacteria and archaea. Members of this family, collectively, respond to a wide range of amino acids as coregulators. In
Escherichia coli
, Lrp regulates over 200 genes directly and is well known to respond to leucine and, to a somewhat lesser extent, alanine. We focused on Lrp from
Proteus mirabilis
and
E. coli
, orthologs with 98% identity overall and identical helix-turn-helix motifs, for which a previous study nevertheless found functional differences. Sequence differences between these orthologs, within and adjacent to the amino acid-responsive RAM domain, led us to test for differential sensitivity to coregulatory amino acids. In the course of this investigation, we found, via
in vivo
reporter fusion assays and
in vitro
electrophoretic mobility shift experiments, that
E. coli
Lrp itself responded to a broader range of amino acids than was previously appreciated. In particular, for both the
E. coli
and
P. mirabilis
orthologs, Lrp responsiveness to methionine was similar in magnitude to that to leucine. Both Lrp orthologs are also fairly sensitive to Ile, His, and Thr. These observations suggest that Lrp ties gene expression in the
Enterobacteriaceae
rather extensively to physiological status, as reflected in amino acid pools. These findings also have substantial implications for attempts to model regulatory architecture from transcriptome measurements or to infer such architecture from genome sequences, and they suggest that even well-studied regulators deserve ongoing exploration.
H-NS Binding and Repression of the
ctx
Promoter in
Vibrio cholerae
Authors: Stonehouse Emily A. , Hulbert Robin R. , Nye Melinda B. , Skorupski Karen , Taylor Ronald K.
Published: December 2010

Expression of the
ctx
and
tcp
genes, which encode cholera toxin and the toxin coregulated pilus, the
Vibrio cholerae
O1 virulence determinants having the largest contribution to cholera disease, is repressed by the nucleoid-associated protein H-NS and activated by the AraC-like transcriptional regulator ToxT. To elucidate the molecular mechanism by which H-NS controls transcription of the
ctxAB
operon, H-NS repression and binding were characterized by using a promoter truncation series, gel mobility shift assays, and DNase I footprinting. Promoter regions found to be important for H-NS repression correlated with
in vitro
binding. Four main H-NS binding regions are present at
ctx
. One region overlaps the high-affinity ToxT binding site and extends upstream, another overlaps the ToxT low-affinity binding site around the −35 element, and the remaining two are located adjacent to one another downstream of the transcriptional start site. Competition for binding to the overlapping H-NS/ToxT binding sites was observed in gel mobility shift assays, where ToxT was found to displace H-NS from the
ctx
promoter region. In addition, regulatory differences between the
ctx
and
tcpA
promoters were examined. H-NS was found to have a higher relative binding affinity for the
ctx
promoter than for the
tcpA
promoter
in vitro
. In contrast to ToxT-dependent activation of the
tcpA
promoter, ToxT activation of
ctx
did not require the C-terminal domain of the α-subunit of RNA polymerase. These findings demonstrate that transcriptional regulation of
ctx
and
tcpA
by H-NS and ToxT is mechanistically distinct, and this may lead to important differences in the expression of these coregulated genes.
Preliminary study on the correlation between grading and histology of solitary pulmonary nodules and contrast enhancement and [18F]fluorodeoxyglucose standardised uptake value after evaluation by dynamic multiphase CT and PET/CT
Authors: Cappabianca Salvatore , Porto Annamaria , Petrillo Mario , Greco Barbara , Reginelli Alfonso , Ronza Francesco , Setola Francesca , Rossi Giovanni , Di Matteo Andrea , Muto Roberto , De Rimini Maria Luisa , Piccolo Sergio , Catalano Mara , Muto Pietro , De Rosa Nicoletta , Barra Enrica , De Rosa Ilaria , Antinolfi Francesca , Antinolfi Giuseppe , Caputi Mario , Brunese Luca , Grassi Roberto , Rotondo Antonio
Published: December 2010

Aim

To evaluate whether the histology and grading of solitary pulmonary nodules (SPNs) correlated with the results of dynamic multiphase multidetector CT (MDCT) and the [18F]fluorodeoxyglucose standardised uptake value (SUV) in 30 patients.

Methods

Chest x-rays of 270 patients with incidentally detected SPNs were retrospectively evaluated. Thirty patients with histologically proven SPNs were enrolled. On MDCT and positron emission tomography (PET)/CT images, two experts measured the density of nodules in all perfusion phases and the SUV. Net enhancement (NE) was calculated by subtracting peak pre-contrast density from peak post-contrast density. The Pearson test was used to correlate nodule NE, SUV, grading, histology and diameter.

Results

Of the 30 malignant SPNs, six were classified as G1 (median NE, 31.5 Hounsfield units (HU); median SUV, 4.8 units), 15 were classified as G2 (median NE, 49 HU; median SUV, 6 units), and nine were classified as G3 (median NE, 32 HU; median SUV, 4.5 units). A highly negative correlation was found in G3 SPNs between NE and the corresponding diameters (r=−0.834; p=0.00524). NE increased with the increase in diameter (r=0.982; p=0.284). SUV increased as the SPN diameter increased (r=0.789; p=0.421). NE and SUV were higher in G2 than G1 SPNs, and lower in G2 than G3 SPNs (r=0.97; p=0.137).

Conclusions

The significant correlation in dedifferentiated (G3) SPNs between NE and diameter (r=−0.834; p=0.00524) supports the theory that stroma and neoangiogenesis are fundamental in SPN growth. The highly negative correlation between NE and diameter demonstrates a net decrease in perfusion despite an increase in dimension. The multidisciplinary approach used herein may result in a more precise prognosis and consequently a better therapeutic outcome, particularly in patients with undifferentiated lung cancer.
MLH1 function is context dependent in colorectal cancers
Authors: Jackson Thomas , Ahmed Mohamed A H , Seth Rashmi , Jackson Darryl , Ilyas Mohammad
Published: December 2010

Background and aims

Loss of mismatch repair (MMR) function in sporadic colorectal cancer occurs most commonly because of inactivation of MLH1, and it causes an increase in mutation rate. However, it is uncertain whether loss of MMR alters any other cellular function. The aim of this study was to investigate the role of MMR in regulating cell numbers and apoptosis.

Methods

MLH1 protein levels were manipulated by (a) cloning and forcibly expressing MLH1 in HCT116 (a cell line with MLH1 mutation) and RKO (a cell line with MLH1 silencing), and (b) knockdown of MLH1 in SW480 (a cell line with normal MMR function). Cell number and apoptotic bodies were measured in standard and ‘high stress’ (ie, after staurosporine exposure) conditions.

Results

Restoration of MLH1 function in HCT116 and RKO resulted in increased cell number (p<0.001 for both cell lines) and decreased numbers of floating apoptotic bodies (p<0.01 in HCT116) in standard culture conditions. However, on induction of apoptotic stress, restoration of MLH1 resulted in reduced cell numbers (p<0.005). Knockdown of MLH1 in SW480 had no effect on cell numbers or apoptosis.

Conclusions

MLH1 function may be context dependent: in ‘low stress’ conditions it may act to inhibit apoptosis, while in ‘high stress’ conditions it may induce apoptosis. However, within the context of chromosomal instability, the effect of MLH1 on cell numbers is limited.
The use of gadolinium chelates as an alternative radiographic contrast agent for use with flat panel detector angiography
Authors: Hui Ferdinand K , Tong Frank C , Dion Jacques E
Published: December 2010

Gadolinium chelates can be utilized as a fluoroscopic contrast agent when iodinated contrast is contraindicated. However, the nephrotoxicity of gadolinium at radiographic doses limits its role as fluoroscopic contrast. Gadolinium chelate-enhanced digital subtraction angiography (DSA) is particularly limited by the kV of DSA, where gadolinium chelates are relatively more radiolucent. Flat panel detectors improve conspicuity of gadolinium chelates during DSA and should be employed where possible.
Blood pressure measurement in the artery proximal and distal to an intra-arterial embolus during thrombolytic therapy
Authors: Sorimachi Takatoshi , Morita Kenichi , Ito Yasushi , Fujii Yukihiko
Published: December 2010

Background and aim

When a cerebral embolism occurs, pressure gradients in the arteries between sites proximal and distal to the embolus exert hydromechanical forces that push the embolus distally, and potentially affect successful revascularization during clot removal. We investigated the relationships between blood pressures that occur immediately distal and proximal to the embolus, systemic blood pressure and clinical variables.

Methods

36 patients with embolism of the internal carotid artery (13 patients) or the proximal middle cerebral artery (23 patients) were treated using intra-arterial thrombolysis. The mean blood pressure (MBP) at the sites proximal (proximal MBP) and distal (distal MBP) to the embolus was measured with a microcatheter. Simultaneously, the systemic MBP was recorded by cuff measurement.

Results

Proximal, distal and systemic MBPs (mean±SD) were 95.2±13.2 mm Hg, 35.9±13.5 mm Hg and 98.2±12.2 mm Hg, respectively. The proximal MBP was significantly higher in the patients with systemic MBP≥100 mm Hg than those with systemic MBP<100 mm Hg (p<0.05). The distal MBP showed no difference between the high and normal systemic MBP groups.

Conclusions

Higher systemic blood pressure produced stronger hydromechanical forces on the clot, forming a proximal–distal blood pressure gradient across the clot. The stronger hydromechanical force could result in higher compaction of the clot, thus making it more difficult to remove.
Preoperative direct percutaneous embolization of spinal metastasis from renal cell carcinoma
Authors: Ammirati Mario , Spiliopoulos Konstantinos , Epstein Clara Raquel , Gabriel Josue , Bourekas Eric C
Published: December 2010

Preoperative direct percutaneous embolization has been very rarely used in hypervascular metastatic spinal tumors to decrease blood loss during the surgery. A patient is presented with solitary spinal metastasis due to renal cell carcinoma who underwent a two-stage spondylectomy. Transarterial tumor embolization with polyvinyl alcohol (PVA) particles and liquid coil placement, and percutaneous tumor embolization with PVA particles were used before the first and the second stage, respectively.
Trigeminocardiac reflex during the percutaneous injection of ethylene vinyl alcohol copolymer (Onyx) into a juvenile nasopharyngeal angiofibroma: a report of two cases: Figure 1
Authors: Potti Tommy A , Gemmete Joseph J , Pandey Aditya S , Chaudhary Neeraj
Published: December 2010

The trigeminocardiac reflex (TCR) is a rare but well-described phenomenon encountered during invasive procedures involving the face, orbit, and cranial base. The reflex is characterized by the abrupt onset of hypotension, bradycardia, asystole, and dysrhythmias. With temporary cessation of the surgical procedure, vital signs typically stabilize without the need for further investigation, though anticholinergic drugs are often used to prevent prolonged hypotension and bradycardia. Two separate cases of the TCR were encountered during the percutaneous embolization of a juvenile nasopharyngeal angiofibroma with dimethylsulfoxide (DMSO) before the injection of ethylene vinyl alcohol copolymer (Onyx, ev3, Irvine, California, USA). In both cases, the injection of DMSO precipitated approximately 30 s of bradycardia/asystole, which then resolved after halting the procedure and administering anticholinergic drugs. There were no additional occurrences afterward and the patients underwent tumor excision with good recovery.
Use of a gonadotrophin‐releasing hormone vaccine in headshaking horses
Authors: Pickles K. J. , Berger J. , Davies R. , Roser J. , Madigan J. E.
Published: December 2010

The aim of this study was to investigate the use of a gonadotrophin‐releasing hormone (GnRH) vaccine in the treatment of headshaking in horses. Fifteen geldings received two doses of the GnRH vaccine four weeks apart. Serum was collected before and after vaccination to measure concentrations of luteinising hormone (LH) (10 horses) and follicle‐stimulating hormone (FSH) (six horses). Owners recorded the frequency of seven common headshaking behaviours using a visual analogue scale (VAS) before vaccination and at two, four, eight, 12, 16 and 20 weeks after vaccination. Serum LH (P=0.008) and FSH (P=0.03) concentrations decreased significantly following vaccination. Although approximately one‐third of the owners reported a subjective improvement in headshaking, serial scoring did not indicate a reduction in headshaking behaviours following vaccination with a commercial GnRH vaccine. Vaccination reactions were observed in four of 15 horses (27 per cent), including one case of severe, presumed immune‐mediated, myositis.
New formula for the estimation of shear resistance of fibre reinforced beams
Authors: Nguyen-Minh Long , Rovňák Marián
Published: December 2010

The paper presents an improved, fracture-mechanics-based formula for estimation of the ultimate shear resistance of steel fibre reinforced concrete beams. The proposed formula was evaluated using 312 published and 28 new test results and its accuracy was compared with 12 already known formulas. Twenty eight tests on reinforced concrete beams subjected to non-symmetrically placed concentrated loads were carried out, in which two different types of steel fibres: short straight and long hooked-end were used. The influence of the volume of fibres on shear resistance and cracking behaviour of the beams for each type of fibre as well as their combination was investigated. The obtained results indicate the validity of the new formula for a wide range of beam and material parameters and prove its applicability for a reasonably precise estimation of the ultimate shear resistance of steel fibre reinforced concrete beams.
Effectiveness of liposomal paclitaxel against MCF-7 breast cancer cells
Authors: Heney Melanie , Alipour Misagh , Vergidis Dimitrios , Omri Abdelwahab , Mugabe Clement , Th’ng John , Suntres Zacharias
Published: December 2010

Paclitaxel is an effective chemotherapeutic agent that is widely used for the treatment of several cancers, including breast, ovarian, and non-small-cell lung cancer. Due to its high lipophilicity, paclitaxel is difficult to administer and requires solubilization with Cremophor EL (polyethoxylated castor oil) and ethanol, which often lead to adverse side effects, including life-threatening anaphylaxis. Incorporation of paclitaxel in dimyristoylphosphatidylcholine:dimyristoylphosphatidylglycerol (DPPC:DMPG) liposomes can facilitate its delivery to cancer cells and eliminate the adverse reactions associated with the Cremophor EL vehicle. Accordingly, the effectiveness of liposomal paclitaxel on MCF-7 breast cancer cells was examined. The results from this study showed that (i) the lipid components of the liposomal formulation were nontoxic, (ii) the cytotoxic effects of liposomal paclitaxel were improved when compared with those seen with conventional paclitaxel, and (iii) the intracellular paclitaxel levels were higher in MCF-7 cells treated with the liposomal paclitaxel formulation. The results of these studies showed that delivery of paclitaxel as a liposomal formulation could be a promising strategy for enhancing its chemotherapeutic effects.
Cyclic stretch stimulates vascular smooth muscle cell alignment by redox-dependent activation of Notch3
Authors: Zhu Jian-Hong , Chen Chun-Lin , Flavahan Sheila , Harr Jennifer , Su Baogen , Flavahan Nicholas A.
Published: December 2010

Mice deficient in Notch3 have defects in arterial vascular smooth muscle cell (VSMC) mechanosensitivity, including impaired myogenic responses and autoregulation, and inappropriate VMSC orientation. Experiments were performed to determine if Notch3 is activated by mechanical stimulation and contributes to mechanosensitive responses of VSMCs, including cell realignment. Cyclic, uniaxial stretch (10%, 1 Hz) of human VSMCs caused Notch3 activation, demonstrated by a stretch-induced increase in hairy and enhancer of split 1/hairy-related transcription factor-1 expression, translocation of Notch3 to the nucleus, and a decrease in the Notch3 extracellular domain. These effects were prevented by inhibiting the expression [small interfering (si)RNA] or proteolytic activation of Notch3 { N-( R)-[2-(hydroxyaminocarbonyl)methyl]−4-methylpentanoyl-l-naphthylalanyl-l-alanine-2-aminoethyl amide (TAPI-1; 50 μmol/l) to inhibit TNF-α-converting enzyme (TACE) or N-[ N-(3,5-difluorophenacetyl-l-alanyl)]- S-phenylglycine t-butyl ester (DAPT; 20 μmol/l) to inhibit γ-secretase}. Stretch increased the activity of ROS within VSMCs, determined using dichlorodihydrofluorescein fluorescence. Catalase (1,200 U/ml), which degrades H2O2, inhibited the stretch-induced activation of Notch3, whereas in nonstretched cells, increasing H2O2activity [H2O2or manganese(III) tetrakis(1-methyl-4-pyridyl)porphyrin] caused activation of Notch3. Stretch increased the activity of TACE, which was prevented by catalase. Stretch-induced activation of p38 MAPK in VSMCs was inhibited either by catalase or by inhibiting Notch3 expression (siRNA). Stretch caused VSMCs to realign perpendicular to the direction of the mechanical stimulus, which was significantly inhibited by catalase or by inhibiting the expression (siRNA) or activation of Notch3 (TAPI-1 or DAPT). Therefore, cyclic uniaxial stretch activates Notch3 signaling through a ROS-mediated mechanism, and the presence of Notch3 is necessary for proper stretch-induced cell alignment in VSMCs. This mechanism may contribute to the physiological role of Notch3 in mediating developmental maturation of VSMCs.
Accelerated epithelial cell senescence in IPF and the inhibitory role of SIRT6 in TGF-β-induced senescence of human bronchial epithelial cells
Authors: Minagawa Shunsuke , Araya Jun , Numata Takanori , Nojiri Satoko , Hara Hiromichi , Yumino Yoko , Kawaishi Makoto , Odaka Makoto , Morikawa Toshiaki , Nishimura Stephen L. , Nakayama Katsutoshi , Kuwano Kazuyoshi
Published: December 2010

Reepithelialization of remodeled air spaces with bronchial epithelial cells is a prominent pathological finding in idiopathic pulmonary fibrosis (IPF) and is implicated in IPF pathogenesis. Recent studies suggest that epithelial senescence is a risk factor for development of IPF, indicating such reepithelialization may be influenced by the acceleration of cellular senescence. Among the sirtuin (SIRT) family, SIRT6, a class III histone deacetylase, has been demonstrated to antagonize senescence. We evaluated the senescence of bronchiolization in association with SIRT6 expression in IPF lung. Senescence-associated β-galactosidase staining and immunohistochemical detection of p21 were performed to evaluate cellular senescence. As a model for transforming growth factor (TGF)-β-induced senescence of abnormal reepithelialization, we used primary human bronchial epithelial cells (HBEC). The changes of SIRT6, p21, and interleukin (IL)-1β expression levels in HBEC, as well as type I collagen expression levels in fibroblasts, were evaluated. In IPF lung samples, an increase in markers of senescence and SIRT6 expression was found in the bronchial epithelial cells lining cystically remodeled air spaces. We found that TGF-β induced senescence in primary HBEC by increasing p21 expression, and, whereas TGF-β also induced SIRT6, it was not sufficient to inhibit cellular senescence. However, overexpression of SIRT6 efficiently inhibited TGF-β-induced senescence via proteasomal degradation of p21. TGF-β-induced senescent HBEC secreted increased amounts of IL-1β, which was sufficient to induce myofibroblast differentiation in fibroblasts. These findings suggest that accelerated epithelial senescence plays a role in IPF pathogenesis through perpetuating abnormal epithelial-mesenchymal interactions, which can be antagonized by SIRT6.
Epithelial EGF receptor signaling mediates airway hyperreactivity and remodeling in a mouse model of chronic asthma
Authors: Le Cras Timothy D. , Acciani Thomas H. , Mushaben Elizabeth M. , Kramer Elizabeth L. , Pastura Patricia A. , Hardie William D. , Korfhagen Thomas R. , Sivaprasad Umasundari , Ericksen Mark , Gibson Aaron M. , Holtzman Michael J. , Whitsett Jeffrey A. , Khurana Hershey Gurjit K.
Published: December 2010

Increases in the epidermal growth factor receptor (EGFR) have been associated with the severity of airway thickening in chronic asthmatic subjects, and EGFR signaling is induced by asthma-related cytokines and inflammation. The goal of this study was to determine the role of EGFR signaling in a chronic allergic model of asthma and specifically in epithelial cells, which are increasingly recognized as playing an important role in asthma. EGFR activation was assessed in mice treated with intranasal house dust mite (HDM) for 3 wk. EGFR signaling was inhibited in mice treated with HDM for 6 wk, by using either the drug erlotinib or a genetic approach that utilizes transgenic mice expressing a mutant dominant negative epidermal growth factor receptor in the lung epithelium (EGFR-M mice). Airway hyperreactivity (AHR) was assessed by use of a flexiVent system after increasing doses of nebulized methacholine. Airway smooth muscle (ASM) thickening was measured by morphometric analysis. Sensitization to HDM (IgG and IgE), inflammatory cells, and goblet cell changes were also assessed. Increased EGFR activation was detected in HDM-treated mice, including in bronchiolar epithelial cells. In mice exposed to HDM for 6 wk, AHR and ASM thickening were reduced after erlotinib treatment and in EGFR-M mice. Sensitization to HDM and inflammatory cell counts were similar in all groups, except neutrophil counts, which were lower in the EGFR-M mice. Goblet cell metaplasia with HDM treatment was reduced by erlotinib, but not in EGFR-M transgenic mice. This study demonstrates that EGFR signaling, especially in the airway epithelium, plays an important role in mediating AHR and remodeling in a chronic allergic asthma model.
FOXA1 plays a role in regulating secretoglobin 1a1 expression in the absence of CCAAT/enhancer binding protein activities in lung in vivo
Authors: Kido Taketomo , Tomita Takeshi , Okamoto Minoru , Cai Yan , Matsumoto Yoshimi , Vinson Charles , Maru Yoshiro , Kimura Shioko
Published: December 2010

Secretoglobin (SCGB) 1A1, also called Clara cell secretor protein (CCSP) or Clara cell-specific 10-kDa protein (CC10), is a small molecular weight secreted protein mainly expressed in lung, with anti-inflammatory/immunomodulatory properties. Previous in vitro studies demonstrated that CCAAT/enhancer-binding proteins (C/EBPs) are the major transcription factors for the regulation of Scbg1a1 gene expression, whereas FOXA1 had a minimum effect on the transcription. To determine the in vivo role of C/EBPs in the regulation of SCGB1A1 expression, experiments were performed in which A-C/EBP, a dominant-negative form of C/EBP that interferes with DNA binding activities of all C/EBPs, was specifically expressed in lung. Surprisingly, despite the in vitro findings, expression of SCGB1A1 mRNA was not decreased in vivo in the absence of C/EBPs. This may be due to a compensatory role assumed by FOXA1 in the regulation of Scgb1a1 gene expression in lung in the absence of active C/EBPs. This disconnect between in vitro and in vivo results underscores the importance of studies using animal models to determine the role of specific transcription factors in the regulation of gene expression in intact multicellular complex organs such as lung.
FUS/TLS Is a Novel Mediator of Androgen-Dependent Cell-Cycle Progression and Prostate Cancer Growth
Authors: Brooke Greg N. , Culley Rachel L. , Dart D. Alwyn , Mann David J. , Gaughan Luke , McCracken Stuart R. , Robson Craig N. , Spencer-Dene Bradley , Gamble Simon C. , Powell Sue M. , Wait Robin , Waxman Jonathan , Walker Marjorie M. , Bevan Charlotte L.
Published: December 2010

Progression of prostate cancer is highly dependent upon the androgen receptor pathway, such that knowledge of androgen-regulated proteins is vital to understand and combat this disease. Using a proteomic screen, we found the RNA-binding protein FUS/TLS (Fused in Ewing's Sarcoma/Translocated in Liposarcoma) to be downregulated in response to androgen. FUS has recently been shown to be recruited by noncoding RNAs to the regulatory regions of target genes such as cyclin D1, in which it represses transcription by disrupting complex formation. Here we show that FUS has some characteristics of a putative tumor suppressor, as its overexpression promoted growth inhibition and apoptosis of prostate cancer cells, whereas its knockdown increased cell proliferation. This effect was reproducible in vivo, such that increasing FUS levels in tumor xenografts led to dramatic tumor regression. Furthermore, FUS promoted conditions that favored cell-cycle arrest by reducing the levels of proliferative factors such as cyclin D1 and Cdk6 and by increasing levels of the antiproliferative Cdk inhibitor p27. Immunohistochemical analysis revealed that FUS expression is inversely correlated with Gleason grade, demonstrating that patients with high levels of FUS survived longer and were less likely to have bone metastases, suggesting that loss of FUS expression may contribute to cancer progression. Taken together, our results address the question of how androgens regulate cell-cycle progression, by demonstrating that FUS is a key link between androgen receptor signaling and cell-cycle progression in prostate cancer. Cancer Res; 71(3); 914–24. ©2010 AACR.