bluuebunny/crossref_metadata_2025 huggingface dataset:
Sensor Kinase PA4398 Modulates Swarming Motility and Biofilm Formation in Pseudomonas aeruginosa PA14
Authors: Strehmel Janine , Neidig Anke , Nusser Michael , Geffers Robert , Brenner-Weiss Gerald , Overhage Joerg
Published: December 2014

Pseudomonas aeruginosa
is an opportunistic human pathogen that is able to sense and adapt to numerous environmental stimuli by the use of transcriptional regulators, including two-component regulatory systems. In this study, we demonstrate that the sensor kinase PA4398 is involved in the regulation of swarming motility and biofilm formation in
P. aeruginosa
PA14. A PA4398
−
mutant strain was considerably impaired in swarming motility, while biofilm formation was increased by approximately 2-fold. The PA4398
−
mutant showed no changes in growth rate, rhamnolipid synthesis, or the production of the Pel exopolysaccharide but exhibited levels of the intracellular second messenger cyclic dimeric GMP (c-di-GMP) 50% higher than those in wild-type cells. The role of PA4398 in gene regulation was investigated by comparing the PA4398
−
mutant to the wild-type strain by using microarray analysis, which demonstrated that 64 genes were up- or downregulated more than 1.5-fold (
P
< 0.05) under swarming conditions. In addition, more-sensitive real-time PCR studies were performed on genes known to be involved in c-di-GMP metabolism. Among the dysregulated genes were several involved in the synthesis and degradation of c-di-GMP or in the biosynthesis, transport, or function of the iron-scavenging siderophores pyoverdine and pyochelin, in agreement with the swarming phenotype observed. By analyzing additional mutants of selected pyoverdine- and pyochelin-related genes, we were able to show that not only
pvdQ
but also
pvdR
,
fptA
,
pchA
,
pchD
, and
pchH
are essential for the normal swarming behavior of
P. aeruginosa
PA14 and may also contribute to the swarming-deficient phenotype of the PA4398
−
mutant in addition to elevated c-di-GMP levels.
Prepartum and Postpartum Rumen Fluid Microbiomes: Characterization and Correlation with Production Traits in Dairy Cows
Authors: Lima Fabio S. , Oikonomou Georgios , Lima Svetlana F. , Bicalho Marcela L. S. , Ganda Erika K. , de Oliveira Filho Jose C. , Lorenzo Gustavo , Trojacanec Plamen , Bicalho Rodrigo C.
Published: December 2014

Microbes present in the rumen of dairy cows are essential for degradation of cellulosic and nonstructural carbohydrates of plant origin. The prepartum and postpartum diets of high-producing dairy cows are substantially different, but in what ways the rumen microbiome changes in response and how those changes may influence production traits are not well elucidated. Here, we sequenced the 16S and 18S rRNA genes using the MiSeq platform to characterize the prepartum and postpartum rumen fluid microbiomes in 115 high-producing dairy cows, including both primiparous and multiparous animals. Discriminant analysis identified differences between the microbiomes of prepartum and postpartum samples and between primiparous and multiparous cows. 18S rRNA sequencing revealed an overwhelming dominance of the protozoan class Litostomatea, with over 90% of the eukaryotic microbial population belonging to that group. Additionally, fungi were relatively more prevalent and Litostomatea relatively less prevalent in prepartum samples than in postpartum ones. The core rumen microbiome (common to all samples) consisted of 64 bacterial taxa, of which members of the genus
Prevotella
were the most prevalent. The Chao1 richness index was greater for prepartum multiparous cows than for postpartum multiparous cows. Multivariable models identified bacterial taxa associated with increased or reduced milk production, and general linear models revealed that a metagenomically based prediction of productivity is highly associated with production of actual milk and milk components. In conclusion, the structure of the rumen fluid microbiome shifts between the prepartum and first-week postpartum periods, and its profile within the context of this study could be used to accurately predict production traits.
Growth of Anaerobic Methane-Oxidizing Archaea and Sulfate-Reducing Bacteria in a High-Pressure Membrane Capsule Bioreactor
Authors: Timmers Peer H. A. , Gieteling Jarno , Widjaja-Greefkes H. C. Aura , Plugge Caroline M. , Stams Alfons J. M. , Lens Piet N. L. , Meulepas Roel J. W.
Published: December 2014

Communities of anaerobic methane-oxidizing archaea (ANME) and sulfate-reducing bacteria (SRB) grow slowly, which limits the ability to perform physiological studies. High methane partial pressure was previously successfully applied to stimulate growth, but it is not clear how different ANME subtypes and associated SRB are affected by it. Here, we report on the growth of ANME-SRB in a membrane capsule bioreactor inoculated with Eckernförde Bay sediment that combines high-pressure incubation (10.1 MPa methane) and thorough mixing (100 rpm) with complete cell retention by a 0.2-μm-pore-size membrane. The results were compared to previously obtained data from an ambient-pressure (0.101 MPa methane) bioreactor inoculated with the same sediment. The rates of oxidation of labeled methane were not higher at 10.1 MPa, likely because measurements were done at ambient pressure. The subtype ANME-2a/b was abundant in both reactors, but subtype ANME-2c was enriched only at 10.1 MPa. SRB at 10.1 MPa mainly belonged to the SEEP-SRB2 and Eel-1 groups and the
Desulfuromonadales
and not to the typically found SEEP-SRB1 group. The increase of ANME-2a/b occurred in parallel with the increase of SEEP-SRB2, which was previously found to be associated only with ANME-2c. Our results imply that the syntrophic association is flexible and that methane pressure and sulfide concentration influence the growth of different ANME-SRB consortia. We also studied the effect of elevated methane pressure on methane production and oxidation by a mixture of methanogenic and sulfate-reducing sludge. Here, methane oxidation rates decreased and were not coupled to sulfide production, indicating trace methane oxidation during net methanogenesis and not anaerobic methane oxidation, even at a high methane partial pressure.
Atmospheric Hydrogen Scavenging: from Enzymes to Ecosystems
Authors: Greening Chris , Constant Philippe , Hards Kiel , Morales Sergio E. , Oakeshott John G. , Russell Robyn J. , Taylor Matthew C. , Berney Michael , Conrad Ralf , Cook Gregory M.
Published: December 2014

We have known for 40 years that soils can consume the trace amounts of molecular hydrogen (H
2
) found in the Earth's atmosphere. This process is predicted to be the most significant term in the global hydrogen cycle. However, the organisms and enzymes responsible for this process were only recently identified. Pure culture experiments demonstrated that several species of
Actinobacteria
, including streptomycetes and mycobacteria, can couple the oxidation of atmospheric H
2
to the reduction of ambient O
2
. A combination of genetic, biochemical, and phenotypic studies suggest that these organisms primarily use this fuel source to sustain electron input into the respiratory chain during energy starvation. This process is mediated by a specialized enzyme, the group 5 [NiFe]-hydrogenase, which is unusual for its high affinity, oxygen insensitivity, and thermostability. Atmospheric hydrogen scavenging is a particularly dependable mode of energy generation, given both the ubiquity of the substrate and the stress tolerance of its catalyst. This minireview summarizes the recent progress in understanding how and why certain organisms scavenge atmospheric H
2
. In addition, it provides insight into the wider significance of hydrogen scavenging in global H
2
cycling and soil microbial ecology.
New Mode of Energy Metabolism in the Seventh Order of Methanogens as Revealed by Comparative Genome Analysis of “Candidatus Methanoplasma termitum”
Authors: Lang Kristina , Schuldes Jörg , Klingl Andreas , Poehlein Anja , Daniel Rolf , Brune Andreas
Published: December 2014

The recently discovered seventh order of methanogens, the
Methanomassiliicoccales
(previously referred to as “
Methanoplasmatales
”), so far consists exclusively of obligately hydrogen-dependent methylotrophs. We sequenced the complete genome of “
Candidatus
Methanoplasma termitum” from a highly enriched culture obtained from the intestinal tract of termites and compared it with the previously published genomes of three other strains from the human gut, including the first isolate of the order. Like all other strains, “
Ca
. Methanoplasma termitum” lacks the entire pathway for CO
2
reduction to methyl coenzyme M and produces methane by hydrogen-dependent reduction of methanol or methylamines, which is consistent with additional physiological data. However, the shared absence of cytochromes and an energy-converting hydrogenase for the reoxidation of the ferredoxin produced by the soluble heterodisulfide reductase indicates that
Methanomassiliicoccales
employ a new mode of energy metabolism, which differs from that proposed for the obligately methylotrophic
Methanosphaera stadtmanae
. Instead, all strains possess a novel complex that is related to the F
420
:methanophenazine oxidoreductase (Fpo) of
Methanosarcinales
but lacks an F
420
-oxidizing module, resembling the apparently ferredoxin-dependent Fpo-like homolog in
Methanosaeta thermophila
. Since all
Methanomassiliicoccales
also lack the subunit E of the membrane-bound heterodisulfide reductase (HdrDE), we propose that the Fpo-like complex interacts directly with subunit D, forming an energy-converting ferredoxin:heterodisulfide oxidoreductase. The dual function of heterodisulfide in
Methanomassiliicoccales
, which serves both in electron bifurcation and as terminal acceptor in a membrane-associated redox process, may be a unique characteristic of the novel order.
Individual Predisposition to Staphylococcus aureus Colonization in Pigs on the Basis of Quantification, Carriage Dynamics, and Serological Profiles
Authors: Espinosa-Gongora Carmen , Dahl Jan , Elvstrøm Anders , van Wamel Willem J. , Guardabassi Luca
Published: December 2014

Previous research on
Staphylococcus aureus
in pigs focused on livestock-associated methicillin-resistant
S. aureus
(MRSA) and had a qualitative cross-sectional design. This study aimed to elucidate the frequency, load, and stability of
S. aureus
nasal carriage in pigs over time and investigated possible associations between carriage and immune response. Nasal swabs were collected three times weekly from 480 tagged adult pigs in 20 Danish production farms.
S. aureus
and MRSA were quantified on selective media by the most-probable-number method. The levels of IgG against 10
S. aureus
antigens in serum were quantified in selected pigs by a Luminex assay. All the farms were positive for
S. aureus
and 15 for MRSA, leading to overall prevalences of persistent and intermittent carriers and noncarriers of 24, 52, and 23%, respectively. Carriage frequency and nasal loads were significantly higher on MRSA-positive farms. Logistic-regression modeling revealed the presence of individual pigs characterized by high nasal loads (≥10,000 CFU per swab) and stable carriage regardless of farm- and pen-associated factors. On the other hand, the humoral response was strongly influenced by these environmental factors. The existence of a minority of shedders contributing to maintenance of
S. aureus
within farms opens up new perspectives on the control of MRSA in pig farming.
Novel Phage Group Infecting Lactobacillus delbrueckii subsp. lactis, as Revealed by Genomic and Proteomic Analysis of Bacteriophage Ldl1
Authors: Casey Eoghan , Mahony Jennifer , Neve Horst , Noben Jean-Paul , Dal Bello Fabio , van Sinderen Douwe
Published: December 2014

Ldl1 is a virulent phage infecting the dairy starter
Lactobacillus delbrueckii
subsp.
lactis
LdlS. Electron microscopy analysis revealed that this phage exhibits a large head and a long tail and bears little resemblance to other characterized phages infecting
Lactobacillus delbrueckii
.
In vitro
propagation of this phage revealed a latent period of 30 to 40 min and a burst size of 59.9 ± 1.9 phage particles. Comparative genomic and proteomic analyses showed remarkable similarity between the genome of Ldl1 and that of
Lactobacillus plantarum
phage ATCC 8014-B2. The genomic and proteomic characteristics of Ldl1 demonstrate that this phage does not belong to any of the four previously recognized
L. delbrueckii
phage groups, necessitating the creation of a new group, called group e, thus adding to the knowledge on the diversity of phages targeting strains of this industrially important lactic acid bacterial species.
Transcriptomic Analysis of Temperature Responses of Aspergillus kawachii during Barley Koji Production
Authors: Futagami Taiki , Mori Kazuki , Wada Shotaro , Ida Hiroko , Kajiwara Yasuhiro , Takashita Hideharu , Tashiro Kosuke , Yamada Osamu , Omori Toshiro , Kuhara Satoru , Goto Masatoshi
Published: December 2014

The koji mold
Aspergillus kawachii
is used for making the Japanese distilled spirit shochu. During shochu production,
A. kawachii
is grown in solid-state culture (koji) on steamed grains, such as rice or barley, to convert the grain starch to glucose and produce citric acid. During this process, the cultivation temperature of
A. kawachii
is gradually increased to 40°C and is then lowered to 30°C. This temperature modulation is important for stimulating amylase activity and the accumulation of citric acid. However, the effects of temperature on
A. kawachii
at the gene expression level have not been elucidated. In this study, we investigated the effect of solid-state cultivation temperature on gene expression for
A. kawachii
grown on barley. The results of DNA microarray and gene ontology analyses showed that the expression of genes involved in the glycerol, trehalose, and pentose phosphate metabolic pathways, which function downstream of glycolysis, was downregulated by shifting the cultivation temperature from 40 to 30°C. In addition, significantly reduced expression of genes related to heat shock responses and increased expression of genes related with amino acid transport were also observed. These results suggest that solid-state cultivation at 40°C is stressful for
A. kawachii
and that heat adaptation leads to reduced citric acid accumulation through activation of pathways branching from glycolysis. The gene expression profile of
A. kawachii
elucidated in this study is expected to contribute to the understanding of gene regulation during koji production and optimization of the industrially desirable characteristics of
A. kawachii
.
Detection and Genomic Characterization of Motility in Lactobacillus curvatus: Confirmation of Motility in a Species outside the Lactobacillus salivarius Clade
Authors: Cousin Fabien J. , Lynch Shónagh M. , Harris Hugh M. B. , McCann Angela , Lynch Denise B. , Neville B. Anne , Irisawa Tomohiro , Okada Sanae , Endo Akihito , O'Toole Paul W.
Published: December 2014

Lactobacillusis the largest genus within the lactic acid bacteria (LAB), with almost 180 species currently identified. Motility has been reported for at least 13Lactobacillusspecies, all belonging to theLactobacillus salivariusclade. Motility in lactobacilli is poorly characterized. It probably confers competitive advantages, such as superior nutrient acquisition and niche colonization, but it could also play an important role in innate immune system activation through flagellin–Toll-like receptor 5 (TLR5) interaction. We now report strong evidence of motility in a species outside theL. salivariusclade,Lactobacillus curvatus(strain NRIC 0822). The motility ofL. curvatusNRIC 0822 was revealed by phase-contrast microscopy and soft-agar motility assays. Strain NRIC 0822 was motile at temperatures between 15°C and 37°C, with a range of different carbohydrates, and under varying atmospheric conditions. We sequenced theL. curvatusNRIC 0822 genome, which revealed that the motility genes are organized in a single operon and that the products are very similar (>98.5% amino acid similarity over >11,000 amino acids) to those encoded by the motility operon ofLactobacillusacidipiscisKCTC 13900 (shown for the first time to be motile also). Moreover, the presence of a large number of mobile genetic elements within and flanking the motility operon ofL. curvatussuggests recent horizontal transfer between members of two distinctLactobacillusclades:L. acidipiscisin theL. salivariusclade andL. curvatusin theL. sakeiclade. This study provides novel phenotypic, genetic, and phylogenetic insights into flagellum-mediated motility in lactobacilli.
Global Transcriptional Response of Clostridium difficile Carrying the ϕCD38-2 Prophage
Authors: Sekulovic Ognjen , Fortier Louis-Charles
Published: December 2014

Clostridium difficile
is one of the most dangerous pathogens in hospital settings. Most strains of
C. difficile
carry one or more prophages, and some of them, like ϕCD38-2 and ϕCD119, can influence the expression of toxin genes. However, little is known about the global host response in the presence of a given prophage. In order to fill this knowledge gap, we used high-throughput RNA sequencing (RNA-seq) to conduct a genome-wide transcriptomic analysis of the epidemic
C. difficile
strain R20291 carrying the ϕCD38-2 prophage. A total of 39 bacterial genes were differentially expressed in the R20291 lysogen, 26 of them being downregulated. Several of the regulated genes encode transcriptional regulators and phosphotransferase system (PTS) subunits involved in glucose, fructose, and glucitol/sorbitol uptake and metabolism. ϕCD38-2 also upregulated the expression of a group of regulatory genes located in phi-027, a resident prophage common to most ribotype 027 isolates. The most differentially expressed gene was that encoding the conserved phase-variable cell wall protein CwpV, which was upregulated ∼20-fold in the lysogen. Quantitative PCR and immunofluorescence showed that the increased
cwpV
expression results from a greater proportion of cells actively transcribing the gene. Indeed, ∼95% of lysogenic cells express
cwpV
, as opposed to only ∼5% of wild-type cells. Furthermore, the higher proportion of cells expressing
cwpV
results from a higher frequency of recombination of the genetic switch controlling phase variation, which we confirmed to be dependent on the host-encoded recombinase RecV. In summary, ϕCD38-2 interferes with phase variation of the surface protein CwpV and the expression of metabolic genes.
Metatranscriptomic Analyses of Plant Cell Wall Polysaccharide Degradation by Microorganisms in the Cow Rumen
Authors: Dai Xin , Tian Yan , Li Jinting , Su Xiaoyun , Wang Xuewei , Zhao Shengguo , Liu Li , Luo Yingfeng , Liu Di , Zheng Huajun , Wang Jiaqi , Dong Zhiyang , Hu Songnian , Huang Li
Published: December 2014

The bovine rumen represents a highly specialized bioreactor where plant cell wall polysaccharides (PCWPs) are efficiently deconstructed via numerous enzymes produced by resident microorganisms. Although a large number of fibrolytic genes from rumen microorganisms have been identified, it remains unclear how they are expressed in a coordinated manner to efficiently degrade PCWPs. In this study, we performed a metatranscriptomic analysis of the rumen microbiomes of adult Holstein cows fed a fiber diet and obtained a total of 1,107,083 high-quality non-rRNA reads with an average length of 483 nucleotides. Transcripts encoding glycoside hydrolases (GHs) and carbohydrate binding modules (CBMs) accounted for ∼1% and ∼0.1% of the total non-rRNAs, respectively. The majority (∼98%) of the putative cellulases belonged to four GH families (i.e., GH5, GH9, GH45, and GH48) and were primarily synthesized by
Ruminococcus
and
Fibrobacter
. Notably, transcripts for GH48 cellobiohydrolases were relatively abundant compared to the abundance of transcripts for other cellulases. Two-thirds of the putative hemicellulases were of the GH10, GH11, and GH26 types and were produced by members of the genera
Ruminococcus
,
Prevotella
, and
Fibrobacter
. Most (∼82%) predicted oligosaccharide-degrading enzymes were GH1, GH2, GH3, and GH43 proteins and were from a diverse group of microorganisms. Transcripts for CBM10 and dockerin, key components of the cellulosome, were also relatively abundant. Our results provide metatranscriptomic evidence in support of the notion that members of the genera
Ruminococcus
,
Fibrobacter
, and
Prevotella
are predominant PCWP degraders and point to the significant contribution of GH48 cellobiohydrolases and cellulosome-like structures to efficient PCWP degradation in the cow rumen.
Emergence Shapes the Structure of the Seed Microbiota
Authors: Barret Matthieu , Briand Martial , Bonneau Sophie , Préveaux Anne , Valière Sophie , Bouchez Olivier , Hunault Gilles , Simoneau Philippe , Jacques Marie-Agnès
Published: December 2014

Seeds carry complex microbial communities, which may exert beneficial or deleterious effects on plant growth and plant health. To date, the composition of microbial communities associated with seeds has been explored mainly through culture-based diversity studies and therefore remains largely unknown. In this work, we analyzed the structures of the seed microbiotas of different plants from the family Brassicaceae and their dynamics during germination and emergence through sequencing of three molecular markers: the ITS1 region of the fungal internal transcribed spacer, the V4 region of 16S rRNA gene, and a species-specific bacterial marker based on a fragment ofgyrB. Sequence analyses revealed important variations in microbial community composition between seed samples. Moreover, we found that emergence strongly influences the structure of the microbiota, with a marked reduction of bacterial and fungal diversity. This shift in the microbial community composition is mostly due to an increase in the relative abundance of some bacterial and fungal taxa possessing fast-growing abilities. Altogether, our results provide an estimation of the role of the seed as a source of inoculum for the seedling, which is crucial for practical applications in developing new strategies of inoculation for disease prevention.
Complex Population Structure of Borrelia burgdorferi in Southeastern and South Central Canada as Revealed by Phylogeographic Analysis
Authors: Mechai S. , Margos G. , Feil E. J. , Lindsay L. R. , Ogden N. H.
Published: December 2014

Lyme disease, caused by the bacterium
Borrelia burgdorferi
sensu stricto
, is an emerging zoonotic disease in Canada and is vectored by the blacklegged tick,
Ixodes scapularis
. Here we used Bayesian analyses of sequence types (STs), determined by multilocus sequence typing (MLST), to investigate the phylogeography of
B. burgdorferi
populations in southern Canada and the United States by analyzing MLST data from 564
B. burgdorferi
-positive samples collected during surveillance. A total of 107 Canadian samples from field sites were characterized as part of this study, and these data were combined with existing MLST data for samples from the United States and Canada. Only 17% of STs were common between both countries, while 49% occurred only in the United States, and 34% occurred only in Canada. However, STs in southeastern Ontario and southwestern Quebec were typically identical to those in the northeastern United States, suggesting a recent introduction into this region from the United States. In contrast, STs in other locations in Canada (the Maritimes; Long Point, Ontario; and southeastern Manitoba) were frequently unique to those locations but were putative descendants of STs previously found in the United States. The picture in Canada is consistent with relatively recent introductions from multiple refugial populations in the United States. These data thus point to a geographic pattern of populations of
B. burgdorferi
in North America that may be more complex than simply comprising northeastern, midwestern, and Californian groups. We speculate that this reflects the complex ecology and spatial distribution of key reservoir hosts.
Influenza Gain-of-Function Experiments: Their Role in Vaccine Virus Recommendation and Pandemic Preparedness
Authors: Schultz-Cherry S. , Webby R. J. , Webster R. G. , Kelso A. , Barr I. G. , McCauley J. W. , Daniels R. S. , Wang D. , Shu Y. , Nobusawa E. , Itamura S. , Tashiro M. , Harada Y. , Watanabe S. , Odagiri T. , Ye Z. , Grohmann G. , Harvey R. , Engelhardt O. , Smith D. , Hamilton K. , Claes F. , Dauphin G.
Published: December 2014

In recent years, controversy has arisen regarding the risks and benefits of certain types of gain-of-function (GOF) studies involving avian influenza viruses. In this article, we provide specific examples of how different types of data, including information garnered from GOF studies, have helped to shape the influenza vaccine production process—from selection of candidate vaccine viruses (CVVs) to the manufacture and stockpiling of safe, high-yield prepandemic vaccines for the global community. The article is not written to support a specific pro- or anti-GOF stance but rather to inform the scientific community about factors involved in vaccine virus selection and the preparation of prepandemic influenza vaccines and the impact that some GOF information has had on this process.
Socioeconomic inequalities in dental caries and their determinants in adolescents in New Delhi, India
Authors: Mathur Manu Raj , Tsakos Georgios , Millett Christopher , Arora Monika , Watt Richard
Published: December 2014

Objectives

To determine whether socioeconomic inequalities are correlated to dental caries experience and decayed teeth of Indian adolescents, and assess whether behavioural and psychosocial factors mediate this association.

Methods

Cross-sectional study of 1386 adolescents living in three diverse areas of New Delhi. Caries experience and number of decayed teeth were assessed clinically and a questionnaire was used to gather sociodemographic and psychosocial data. Zero Inflated Negative Binomial regression models were used to assess the relationship between the outcomes (caries experience and decayed teeth) and area of residence, adjusting for covariates.

Results

Significant inequalities in caries experience and number of decayed teeth were observed. Odds of an adolescent being caries free decreased by 66% (OR 0.34, 95% CI 0.23 to 0.49) and 70% (OR 0.30, 95% CI 0.21 to 0.43) in adolescents living in resettlement communities or urban slums, respectively, when compared with the middle class group. No difference was observed among those with caries experience/decayed teeth. Adjusting for covariates did not affect the inequalities.

Conclusions

Area of residence appears to be a very strong and significant determinant for an adolescent to be caries/decay free in India. Psychosocial and behavioural factors do not mediate the association between area of residence and oral health.
Cell cycle marker expression in benign and malignant intraductal papillary lesions of the breast
Authors: Loh Seow Foong , Cooper Caroline , Selinger Christina I , Barnes Elizabeth H , Chan Charles , Carmalt Hugh , West Richard , Gluch Laurence , Beith Jane M , Caldon C Elizabeth , O'Toole Sandra
Published: December 2014

Aims

The diagnosis of intraductal papillary lesions of the breast on core biopsy remains challenging in pathology, with most patients requiring formal surgical excision for a definitive diagnosis. The aim of this study was to determine whether a representative panel of proliferative cell cycle immunohistochemical markers (cyclin A2, cyclin B1 and cyclin D1) could improve the specificity of pathological diagnosis of these lesions.

Methods

A series of 68 surgically excised intraductal papillary lesion cases were retrospectively selected, and immunohistochemistry for cyclin A2, cyclin B1 and cyclin D1 was performed.

Results

Cyclin B1 (OR 1.80, 95% CI 1.01 to 3.2, p=0.046) and cyclin D1 (OR 1.13, 95% CI 1.05 to 1.22, p=0.002) expression was independently associated with a diagnosis of malignancy in papillary lesions, although expression was frequently heterogeneous and only focal. Cyclin A2 expression (OR 0.76, 95% CI 0.41 to 1.4, p=0.38) was not associated with a malignant diagnosis in multivariable logistic regression models. All three cyclins displayed high sensitivity (80%–95%) for a diagnosis of malignancy, although cyclin B1 showed a superior specificity of 72.7% compared with the low specificity of cyclins A2 and D1.

Conclusions

Our study has identified for the first time that the expression of key cell cycle markers differs between benign and malignant papillary breast lesions and identified changes to the mitotic marker, cyclin B1, as particularly significant. However, given the low level and heterogeneous nature of expression of these markers, there remains a significant risk of undersampling in core biopsies and thus they are unlikely to be useful in routine clinical practice.
Changes in referral, treatment and outcomes in patients with systemic lupus erythematosus in Germany in the 1990s and the 2000s
Authors: Albrecht Katinka , Huscher Dörte , Richter Jutta , Backhaus Marina , Bischoff Sascha , Kötter Ina , Thiele Katja , Zink Angela
Published: December 2014

Objective

To evaluate trends in the referral, treatment and outcome of patients with systemic lupus erythematosus (SLE) in Germany over two decades.

Methods

From 1993 to 2012, ∼1200 patients with SLE were recorded annually in the national database of the German Collaborative Arthritis Centres. Treatment patterns, healthcare use and outcomes, such as disease activity, function and work participation, were evaluated over time. Furthermore, two distinct cohorts of patients (enrolment 1994–1998, n=467; and 2004–2008, n=376) observed over 5 years were assessed for changes in outcomes.

Results

The mean disease duration at the first visit to a rheumatologist decreased from 2.6 (1994) to 1.5 (2012) years. Glucocorticoids (69%), antimalarials (56%), azathioprine (22%), non-steroidal anti-inflammatory drugs (23%) and mycophenolate mofetil (15%) were the most frequently used treatments in 2012. A significant increase was observed in the use of antimalarials and mycophenolate mofetil. The use of glucocorticoids at >7.5 mg/day decreased from 27% (1994) to 10% (2012). The average length of sick leave taken due to SLE declined from 9 weeks (1997) to 6 weeks (2012). When comparing the two longitudinal cohorts, in the cohort from the 2000s, the intraindividual decline of disease activity was significantly stronger (p<0.001), and fewer patients retired early (36% vs 46%).

Conclusions

The disease activity and resource use declined considerably over the observation period, and more patients remained in the labour force. Earlier treatment onset, faster modification of the treatment regimen and more intensive use of anti-inflammatory therapy may account for the improved outcomes in patients with SLE across the years.
Monocytes regulate systemic coagulation and inflammation in abdominal sepsis
Authors: Wang Yongzhi , Braun Oscar Ö. , Zhang Su , Norström Eva , Thorlacius Henrik
Published: December 2014

Abdominal sepsis is associated with significant changes in systemic inflammation and coagulation. The purpose of the present study was to examine the role of peripheral blood monocytes for systemic coagulation, including thrombin generation and consumption of coagulation factors. Abdominal sepsis was induced by cecal ligation and puncture (CLP) in C57BL/6 mice. Plasma and lung levels of IL-6 and C-X-C motif (CXC) chemokines [chemokine CXC ligand (CXCL)1, CXCL2, and CXCL5], pulmonary activity of myeloperoxidase, thrombin generation, and coagulation factors were determined 6 h after CLP induction. Administration of clodronate liposomes decreased circulating levels of monocytes by 96%. Time to peak thrombin formation was increased and peak and total thrombin generation was decreased in plasma from CLP animals. Monocyte depletion decreased time to peak formation of thrombin and increased peak and total generation of thrombin in septic animals. In addition, monocyte depletion decreased the CLP-induced increase in the levels of thrombin-antithrombin complexes in plasma. Depletion of monocytes increased plasma levels of prothrombin, factor V, factor X, and protein C in septic mice. Moreover, depletion of monocytes decreased CLP-induced levels of IL-6 and CXC chemokines in the plasma and lung by >59% and 20%, respectively. CLP-induced myeloperoxidase activity in the lung was attenuated by 44% in animals depleted of monocytes. Taken together, our findings show, for the first time, that peripheral blood monocytes regulate systemic coagulation. The results of our study improve our understanding of the pathophysiology of sepsis and encourage further attempts to target innate immune cell functions in abdominal sepsis.
Mechanosignaling through YAP and TAZ drives fibroblast activation and fibrosis
Authors: Liu Fei , Lagares David , Choi Kyoung Moo , Stopfer Lauren , Marinković Aleksandar , Vrbanac Vladimir , Probst Clemens K. , Hiemer Samantha E. , Sisson Thomas H. , Horowitz Jeffrey C. , Rosas Ivan O. , Fredenburgh Laura E. , Feghali-Bostwick Carol , Varelas Xaralabos , Tager Andrew M. , Tschumperlin Daniel J.
Published: December 2014

Pathological fibrosis is driven by a feedback loop in which the fibrotic extracellular matrix is both a cause and consequence of fibroblast activation. However, the molecular mechanisms underlying this process remain poorly understood. Here we identify yes-associated protein (YAP) (homolog of drosophila Yki) and transcriptional coactivator with PDZ-binding motif (TAZ) (also known as Wwtr1), transcriptional effectors of the Hippo pathway, as key matrix stiffness-regulated coordinators of fibroblast activation and matrix synthesis. YAP and TAZ are prominently expressed in fibrotic but not healthy lung tissue, with particularly pronounced nuclear expression of TAZ in spindle-shaped fibroblastic cells. In culture, both YAP and TAZ accumulate in the nuclei of fibroblasts grown on pathologically stiff matrices but not physiologically compliant matrices. Knockdown of YAP and TAZ together in vitro attenuates key fibroblast functions, including matrix synthesis, contraction, and proliferation, and does so exclusively on pathologically stiff matrices. Profibrotic effects of YAP and TAZ operate, in part, through their transcriptional target plasminogen activator inhibitor-1, which is regulated by matrix stiffness independent of transforming growth factor-β signaling. Immortalized fibroblasts conditionally expressing active YAP or TAZ mutant proteins overcome soft matrix limitations on growth and promote fibrosis when adoptively transferred to the murine lung, demonstrating the ability of fibroblast YAP/TAZ activation to drive a profibrotic response in vivo. Together, these results identify YAP and TAZ as mechanoactivated coordinators of the matrix-driven feedback loop that amplifies and sustains fibrosis.
Distinct macrophage phenotypes in allergic and nonallergic lung inflammation
Authors: Robbe Patricia , Draijer Christina , Borg Thiago R. , Luinge Marjan , Timens Wim , Wouters Inge M. , Melgert Barbro N. , Hylkema Machteld N.
Published: December 2014

Chronic exposure to farm environments is a risk factor for nonallergic lung disease. In contrast to allergic asthma, in which type 2 helper T cell (Th2) activation is dominant, exposure to farm dust extracts (FDE) induces Th1/Th17 lung inflammation, associated with neutrophil infiltration. Macrophage influx is a common feature of both types of lung inflammation, allergic and nonallergic. However, macrophage functions and phenotypes may vary according to their polarized state, which is dependent on the cytokine environment. In this study, we aimed to characterize and quantify the lung macrophage populations in two established murine models of allergic and nonallergic lung inflammation by means of fluorescence-activated cell sorting and immunohistochemistry. We demonstrated that, whereas in allergic asthma M2-dominant macrophages predominated in the lungs, in nonallergic inflammation M1-dominant macrophages were more prevalent. This was confirmed in vitro using a macrophage cell line, where FDE exerted a direct effect on macrophages, inducing M1-dominant polarization. The polarization of macrophages diverged depending on the exposure and inflammatory status of the tissue. Interfering with this polarization could be a target for treatment of different types of lung inflammation.
α-Tubulin Acetylation Elevated in Metastatic and Basal-like Breast Cancer Cells Promotes Microtentacle Formation, Adhesion, and Invasive Migration
Authors: Boggs Amanda E. , Vitolo Michele I. , Whipple Rebecca A. , Charpentier Monica S. , Goloubeva Olga G. , Ioffe Olga B. , Tuttle Kimberly C. , Slovic Jana , Lu Yiling , Mills Gordon B. , Martin Stuart S.
Published: December 2014

Metastatic cases of breast cancer pose the primary challenge in clinical management of this disease, demanding the identification of effective therapeutic strategies that remain wanting. In this study, we report that elevated levels of α-tubulin acetylation are a sufficient cause of metastatic potential in breast cancer. In suspended cell culture conditions, metastatic breast cancer cells exhibited high α-tubulin acetylation levels that extended along microtentacle (McTN) protrusions. Mutation of the acetylation site on α-tubulin and enzymatic modulation of this posttranslational modification exerted a significant impact on McTN frequency and the reattachment of suspended tumor cells. Reducing α-tubulin acetylation significantly inhibited migration but did not affect proliferation. In an analysis of more than 140 matched primary and metastatic tumors from patients, we found that acetylation was maintained and in many cases increased in lymph node metastases compared with primary tumors. Proteomic analysis of an independent cohort of more than 390 patient specimens further documented the relationship between increased α-tubulin acetylation and the aggressive behaviors of basal-like breast cancers, with a trend toward increased risk of disease progression and death in patients with high-intensity α-tubulin acetylation in primary tumors. Taken together, our results identify a tight correlation between acetylated α-tubulin levels and aggressive metastatic behavior in breast cancer, with potential implications for the definition of a simple prognostic biomarker in patients with breast cancer. Cancer Res; 75(1); 203–15. ©2014 AACR.
Epigenetic Silencing of miR-490-3p Reactivates the Chromatin Remodeler SMARCD1 to PromoteHelicobacter pylori–Induced Gastric Carcinogenesis
Authors: Shen Jing , Xiao Zhangang , Wu William K.K. , Wang Maggie H. , To Ka F. , Chen Yangchao , Yang Weiqin , Li May S.M. , Shin Vivian Y. , Tong Joanna H. , Kang Wei , Zhang Lin , Li Minxing , Wang Lin , Lu Lan , Chan Ruby L.Y. , Wong Sunny H. , Yu Jun , Chan Matthew T.V. , Chan Francis K.L. , Sung Joseph J.Y. , Cheng Alfred S.L. , Cho Chi H.
Published: December 2014

Chromatin remodeling has emerged as a hallmark of gastric cancer, but the regulation of chromatin regulators other than genetic change is unknown. Helicobacter pylori causes epigenetic dysregulation to promote gastric carcinogenesis, but the roles and functions of microRNAs (miRNA) in this multistage cascade are not fully explored. In this study, miRNA expression in preneoplastic and neoplastic lesions in murine stomachs induced by H. pylori and N-methyl-N-nitrosourea (MNU) was profiled by miRNA expression array. miR-490-3p exhibited progressive downregulation in gastritis, intestinal metaplasia, and adenocarcinoma during H. pylori and MNU-induced gastric carcinogenesis. Significant downregulation of miR-490-3p was confirmed in human gastric cancer tissues in which its regulatory region was found to be hypermethylated. miR-490-3p exerted growth- and metastasis-suppressive effects on gastric cancer cells through directly targeting SMARCD1, a SWItch/Sucrose NonFermentable (SWI/SNF) chromatin remodeling complex subunit. Knockdown of SMARCD1 significantly attenuated the protumorigenic effects of miR-490-3p inhibitor, whereas enforced expression of SMARCD1 promoted in vitro and in vivo oncogenic phenotypes of gastric cancer cells. SMARCD1 was markedly upregulated in gastric cancer in which its high expression was associated with shortened patients' survival independent of TNM staging. In conclusion, hypermethylation-mediated silencing of miR-490-3p reactivates SMARCD1 to confer malignant phenotypes, mechanistically linking H. pylori, chromatin remodeling, and gastric carcinogenesis. Cancer Res; 75(4); 754–65. ©2014 AACR.
Acute Tissue Injury Activates Satellite Cells and Promotes Sarcoma Formation via the HGF/c-MET Signaling Pathway
Authors: Van Mater David , Añó Leonor , Blum Jordan M. , Webster Micah T. , Huang WeiQiao , Williams Nerissa , Ma Yan , Cardona Diana M. , Fan Chen-Ming , Kirsch David G.
Published: December 2014

Some patients with soft-tissue sarcoma (STS) report a history of injury at the site of their tumor. Although this phenomenon is widely reported, there are relatively few experimental systems that have directly assessed the role of injury in sarcoma formation. We recently described a mouse model of STS whereby p53 is deleted and oncogenic Kras is activated in muscle satellite cells via a Pax7CreER driver following intraperitoneal injection with tamoxifen. Here, we report that after systemic injection of tamoxifen, the vast majority of Pax7-expressing cells remain quiescent despite mutation of p53 and Kras. The fate of these muscle progenitors is dramatically altered by tissue injury, which leads to faster kinetics of sarcoma formation. In adult muscle, quiescent satellite cells will transition into an active state in response to hepatocyte growth factor (HGF). We show that modulating satellite cell quiescence via intramuscular injection of HGF increases the penetrance of sarcoma formation at the site of injection, which is dependent on its cognate receptor c-MET. Unexpectedly, the tumor-promoting effect of tissue injury also requires c-Met. These results reveal a mechanism by which HGF/c-MET signaling promotes tumor formation after tissue injury in a mouse model of primary STS, and they may explain why some patients develop a STS at the site of injury. Cancer Res; 75(3); 605–14. ©2014 AACR.
Real-time, Near-Infrared Fluorescence Imaging with an Optimized Dye/Light Source/Camera Combination for Surgical Guidance of Prostate Cancer
Authors: Neuman Brian P. , Eifler John B. , Castanares Mark , Chowdhury Wasim H. , Chen Ying , Mease Ronnie C. , Ma Rong , Mukherjee Amarnath , Lupold Shawn E. , Pomper Martin G. , Rodriguez Ronald
Published: December 2014

Purpose: The prostate-specific membrane antigen (PSMA) is a surface glycoprotein overexpressed on malignant prostate cells, as well as in the neovasculature of many tumors. Recent efforts to target PSMA for imaging prostate cancer rely on suitably functionalized low-molecular-weight agents. YC-27 is a low-molecular-weight, urea-based agent that enables near-infrared (NIR) imaging of PSMA in vivo.

Experimental Design: We have developed and validated a laparoscopic imaging system (including an optimized light source, LumiNIR) that is capable of imaging small tumor burdens with minimal background fluorescence in real-time laparoscopic extirpative surgery of small prostate tumor xenografts in murine and porcine models.

Results: In a mouse model, we demonstrate the feasibility of using real-time NIR laparoscopic imaging to detect and surgically remove PSMA-positive xenografts. We then validate the use of our laparoscopic real-time NIR imaging system in a large animal model. Our novel light source, which is optimized for YC-27, is capable of detecting as little as 12.4 pg/mL of the compound (2.48-pg YC-27 in 200-μL agarose). Finally, in a mouse xenograft model, we demonstrate that the use of real-time NIR imaging can reduce positive surgical margins (PSM).

Conclusions: These data indicate that a NIR-emitting fluorophore targeted to PSMA may allow improved surgical treatment of human prostate cancer, reduce the rate of PSMs, and alleviate the need for adjuvant radiotherapy postoperatively. Clin Cancer Res; 21(4); 771–80. ©2014 AACR.
Immune Escape Mechanisms as a Guide for Cancer Immunotherapy
Authors: Beatty Gregory L. , Gladney Whitney L.
Published: December 2014

Immunotherapy has demonstrated impressive outcomes for some patients with cancer. However, selecting patients who are most likely to respond to immunotherapy remains a clinical challenge. Here, we discuss immune escape mechanisms exploited by cancer and present strategies for applying this knowledge to improving the efficacy of cancer immunotherapy. Clin Cancer Res; 21(4); 687–92. ©2014 AACR.
Vaccine-Induced Tumor Necrosis Factor–Producing T Cells Synergize with Cisplatin to Promote Tumor Cell Death
Authors: van der Sluis Tetje C. , van Duikeren Suzanne , Huppelschoten Suzanna , Jordanova Ekaterina S. , Beyranvand Nejad Elham , Sloots Arjen , Boon Louis , Smit Vincent T.H.B.M. , Welters Marij J.P. , Ossendorp Ferry , van de Water Bob , Arens Ramon , van der Burg Sjoerd H. , Melief Cornelis J.M.
Published: December 2014

Purpose: Cancer immunotherapy, such as vaccination, is an increasingly successful treatment modality, but its interaction with chemotherapy remains largely undefined. Therefore, we explored the mechanism of synergy between vaccination with synthetic long peptides (SLP) of human papillomavirus type 16 (HPV16) and cisplatin in a preclinical tumor model for HPV16.

Experimental Design: SLP vaccination in this preclinical tumor model allowed the elucidation of novel mechanisms of synergy between chemo- and immunotherapy. By analyzing the tumor immune infiltrate, we focused on the local intratumoral effects of chemotherapy, vaccination, or the combination.

Results: Of several chemotherapeutic agents, cisplatin synergized best with SLP vaccination in tumor eradication, without requirement for the maximum-tolerated dose (MTD). Upon SLP vaccination, tumors were highly infiltrated with HPV-specific, tumor necrosis factor-α (TNFα)- and interferon-γ (IFNγ)–producing T cells. Upon combined treatment, tumor cell proliferation was significantly decreased compared with single treated and untreated tumors. Furthermore, we showed that TNFα strongly enhanced cisplatin-induced apoptotic tumor cell death in a JNK-dependent manner. This is consistent with upregulation of proapoptotic molecules and with enhanced cell death in vivo upon combined SLP vaccination and cisplatin treatment. In vivo neutralization of TNFα significantly reduced the antitumor responses induced by the combined treatment.

Conclusion: Taken together, our data show that peptide vaccination with cisplatin treatment leads to decreased tumor cell proliferation and TNFα-induced enhanced cisplatin-mediated killing of tumor cells, together resulting in superior tumor eradication. Clin Cancer Res; 21(4); 781–94. ©2014 AACR.