bluuebunny/crossref_metadata_2025 huggingface dataset:
Development and Experimental Investigation of a Tubular Combustor for Pyrolysis Oil Burning
Authors: Beran Martin , Axelsson Lars-Uno
Published: August 2014

The growing demand for more economical and environmentally friendly power generation forces the industry to search for fuels that can replace the conventional fossil fuels. This has led to significant developments in the production of alternative fuels during the last years, which have made them a reliable and relatively efficient source of energy. One example of these alternative fuels is the pyrolysis oil. However, higher viscosity, lower heat content, limited chemical stability, and its ability to create sediment make pyrolysis oil challenging for gas turbines. The OPRA OP16 gas turbine is an all radial single-shaft gas turbine rated at 1.9 MW. The all radial design, together with the lack of intricate cooling geometries in the hot section, makes this gas turbine suitable for operation on these fuels. This paper presents an experimental investigation of pyrolysis oil combustion in a tubular combustor developed, especially for low-calorific fuels. The experiments have been performed in an atmospheric combustion test rig, and the results have been compared to the results obtained from ethanol and diesel combustion. It was found that it was possible to burn pure pyrolysis oil in the load range between 70% and 100% with a combustion efficiency exceeding 99% and without creation of sediments on the combustor inner wall. It was found that the NOx emissions were similar for pyrolysis oil and diesel, whereas the CO emissions were twice as high for pyrolysis oil. A comparison between the air blast nozzle and the pressure nozzle was performed. The air blast nozzle was found to be more suitable due to its better performance over a wider operating range and that it is more resistant to erosion and abrasion. It was found that the maximum allowed droplet size of the pyrolysis oil spray should be about 50–70% of the droplet size for diesel fuel.
Ultrahigh NA, high aspect ratio interference lithography with resonant dielectric underlayers
Authors: Lowrey Sam , Bourke Levi , Ding Boyang , Blaikie Richard
Published: August 2014

High aspect ratio imaging for immersion interference lithography in the ultrahigh numerical aperture regime, where evanescent fields are responsible for the exposure, is demonstrated experimentally using a resonant dielectric underlayer system consisting of HfO stacked upon SiO2. Improvements in producing these high aspect ratio grating structures compared with previous work [P. Mehrotra, C. A. Mack, and R. J. Blaikie, Opt. Express 21, 13710 (2013)] are shown, which has allowed subsequent lift-off pattern transfer for ∼55 nm half-pitch gratings patterned using a 405 nm exposure wavelength, corresponding to better than λ/7 resolution. Resist structures with aspect ratios close to 3:1 (height to half-pitch) demonstrate the limitations of our lithography system and highlight necessary improvements for higher aspect ratio resist structures to be achieved. Model and preliminary experimental results are presented for a scheme to mitigate resist collapse with very high aspect ratio structures.
Influence of oxygen precursors and annealing on Fe3O4 films grown on GaN templates by metal organic chemical vapor deposition
Authors: Huang Shimin , Gu Shulin , Tang Kun , Ye Jiandong , Xu Zhonghua , Zhu Shunming , Zheng Youdou
Published: August 2014

O2 and N2O, popular oxygen precursors for oxide films growth, have been employed to grow Fe3O4 films on GaN templates via metal organic chemical vapor deposition (MOCVD). A (111)-oriented Fe3O4 film was preferably deposited when N2O was used as O precursor, while a Fe2O3 film was grown with O2 as O precursor. A high-temperature annealing has caused a phase transition from α-Fe2O3 to Fe3O4 for O2 case, but no obvious change occurred on the Fe3O4 film for N2O case. Thinner Fe3O4 layer was then grown on a GaN template with N2O as O precursor to form ferromagnetic material (FM)/nonmagnetic material (NM) heterostructure, which is critical for the realization of spin injection in GaN based wide band gap semiconductors. The Ga diffusion from the GaN template to the Fe3O4 layer has been controlled at a rather low level possibly due to the employed low-temperature growth, leading to a high-quality FM/NM heterostructure. An obvious enhancement on the Ga diffusion has been observed upon subsequent annealing for the thin Fe3O4 layer, which dispersed the FM/NM interface, improved the crystal quality, but weakened the magnetic properties. This study indicates that MOCVD is a suitable method to grow high-quality Fe3O4 films on GaN and should be a powerful tool for the realization of GaN based spin light-emitting diode.
Multiple‐frequency excitation wideband MRI (ME‐WMRI)
Authors: Wu Edzer L. , Chiueh Tzi‐Dar , Chen Jyh‐Horng
Published: August 2014

Purpose:

In this study multiple‐frequency excitation wideband MRI (ME‐WMRI), a technique designed to acquire simultaneously excited images without blurring was proposed.

Methods:

ME‐WMRI consists of (a) a coherent acquisition sequence that applies refocusing gradient during readout to mitigate signal dephasing, and (b) a signal enhancement procedure that further reduces image blurring. This two‐step method was tested on phantom imaging andin vivo imaging with up to 3.84 pixel blur.

Results:

Imaging results in both phantom andin vivo show that the lost details were successfully restored in the blur‐mitigated images, and the accelerated images were comparable to standard images.

Conclusions:

The proposed ME‐WMRI method can effectively undo the image blurring, and thus removes the limitation on fast simultaneous multislice MRI systems with a large acceleration factor.
Feasibility of vibro‐acoustography with a quasi‐2D ultrasound array transducer for detection and localizing of permanent prostate brachytherapy seeds: A pilot ex vivo study
Authors: Mehrmohammadi Mohammad , Alizad Azra , Kinnick Randall R. , Davis Brian J. , Fatemi Mostafa
Published: August 2014

Purpose:

Effective permanent prostate brachytherapy (PPB) requires precise placement of radioactive seeds in and around the prostate. The impetus for this research is to examine a new ultrasound‐based imaging modality, vibro‐acoustography (VA), which may serve to provide a high rate of PPB seed detection while also effecting enhanced prostate imaging. The authors investigate the ability of VA, implemented on a clinical ultrasound (US) scanner and equipped with a quasi‐2D (Q2D) array US transducer, to detect and localize PPB seeds in excised prostate specimens.

Methods:

Nonradioactive brachytherapy seeds were implanted into four excised cadaver prostates. A clinical US scanner equipped with a Q2D array US transducer was customized to acquire both US and C‐scan VA images at various depths. The VA images were then used to detect and localize the implanted seeds in prostate tissue. To validate the VA results, computed tomography (CT) images of the same tissue samples were obtained to serve as the reference by which to evaluate the performance of VA in PPB seed detection.

Results:

The results indicate that VA is capable of accurately identifying the presence and distribution of PPB seeds with a high imaging contrast. Moreover, a large ratio of the PPB seeds implanted into prostate tissue samples could be detected through acquired VA images. Using CT‐based seed identification as the standard, VA was capable of detecting 74%–92% of the implanted seeds. Additionally, the angular independency of VA in detecting PPB seeds was demonstrated through a well‐controlled phantom experiment.

Conclusions:

Q2DVA detected a substantial portion of the seeds by using a 2D array US transducer in excised prostate tissue specimens. While VA has inherent advantages associated with conventional US imaging, it has the additional advantage of permitting detection of PPB seeds independent of their orientation. These results suggest the potential of VA as a method for PPB imaging that ultimately may allow US‐based real‐time intraoperative dosimetry.
An image‐guidance system for dynamic dose calculation in prostate brachytherapy using ultrasound and fluoroscopy
Authors: Kuo Nathanael , Dehghan Ehsan , Deguet Anton , Mian Omar Y. , Le Yi , Burdette E. Clif , Fichtinger Gabor , Prince Jerry L. , Song Danny Y. , Lee Junghoon
Published: August 2014

Purpose:

Brachytherapy is a standard option of care for prostate cancer patients but may be improved by dynamic dose calculation based on localized seed positions. The American Brachytherapy Society states that the major current limitation of intraoperative treatment planning is the inability to localize the seeds in relation to the prostate. An image‐guidance system was therefore developed to localize seeds for dynamic dose calculation.

Methods:

The proposed system is based on transrectal ultrasound (TRUS) and mobile C‐arm fluoroscopy, while using a simple fiducial with seed‐like markers to compute pose from the nonencoded C‐arm. Three or more fluoroscopic images and an ultrasound volume are acquired and processed by a pipeline of algorithms: (1) seed segmentation, (2) fiducial detection with pose estimation, (3) seed matching with reconstruction, and (4) fluoroscopy‐to‐TRUS registration.

Results:

The system was evaluated on ten phantom cases, resulting in an overall mean error of 1.3 mm. The system was also tested on 37 patients and each algorithm was evaluated. Seed segmentation resulted in a 1% false negative rate and 2% false positive rate. Fiducial detection with pose estimation resulted in a 98% detection rate. Seed matching with reconstruction had a mean error of 0.4 mm. Fluoroscopy‐to‐TRUS registration had a mean error of 1.3 mm. Moreover, a comparison of dose calculations between the authors’ intraoperative method and an independent postoperative method shows a small difference of 7% and 2% forD90 and V100, respectively. Finally, the system demonstrated the ability to detect cold spots and required a total processing time of approximately 1 min.

Conclusions:

The proposed image‐guidance system is the first practical approach to dynamic dose calculation, outperforming earlier solutions in terms of robustness, ease of use, and functional completeness.
Feasibility study of using statistical process control to customized quality assurance in proton therapy
Authors: Rah Jeong‐Eun , Shin Dongho , Oh Do Hoon , Kim Tae Hyun , Kim Gwe‐Ya
Published: August 2014

Purpose:

To evaluate and improve the reliability of proton quality assurance (QA) processes and, to provide an optimal customized tolerance level using the statistical process control (SPC) methodology.

Methods:

The authors investigated the consistency check of dose per monitor unit (D/MU) and range in proton beams to see whether it was within the tolerance level of the daily QA process. This study analyzed the difference between the measured and calculated ranges along the central axis to improve the patient‐specific QA process in proton beams by using process capability indices.

Results:

The authors established a customized tolerance level of ±2% for D/MU and ±0.5 mm for beam range in the daily proton QA process. In the authors’ analysis of the process capability indices, the patient‐specific range measurements were capable of a specification limit of ±2% in clinical plans.

Conclusions:

SPC methodology is a useful tool for customizing the optimal QA tolerance levels and improving the quality of proton machine maintenance, treatment delivery, and ultimately patient safety.
Coordination of Swarming Motility, Biosurfactant Synthesis, and Biofilm Matrix Exopolysaccharide Production in Pseudomonas aeruginosa
Authors: Wang Shiwei , Yu Shan , Zhang Zhenyin , Wei Qing , Yan Lu , Ai Guomin , Liu Hongsheng , Ma Luyan Z.
Published: August 2014

Biofilm formation is a complex process in which many factors are involved. Bacterial swarming motility and exopolysaccharides both contribute to biofilm formation, yet it is unclear how bacteria coordinate swarming motility and exopolysaccharide production. Psl and Pel are two key biofilm matrix exopolysaccharides in
Pseudomonas aeruginosa
. This opportunistic pathogen has three types of motility, swimming, twitching, and swarming. In this study, we found that elevated Psl and/or Pel production reduced the swarming motility of
P. aeruginosa
but had little effect on swimming and twitching. The reduction was due to decreased rhamnolipid production with no relation to the transcription of
rhlAB
, two key genes involved in the biosynthesis of rhamnolipids. Rhamnolipid-negative
rhlR
and
rhlAB
mutants synthesized more Psl, whereas exopolysaccharide-deficient strains exhibited a hyperswarming phenotype. These results suggest that competition for common sugar precursors catalyzed by AlgC could be a tactic for
P. aeruginosa
to balance the synthesis of exopolysaccharides and rhamnolipids and to control bacterial motility and biofilm formation inversely because the biosynthesis of rhamnolipids, Psl, and Pel requires AlgC to provide the sugar precursors and an additional
algC
gene enhances the biosynthesis of Psl and rhamnolipids. In addition, our data indicate that the increase in RhlI/RhlR expression attenuated Psl production. This implied that the quorum-sensing signals could regulate exopolysaccharide biosynthesis indirectly in bacterial communities. In summary, this study represents a mechanism that bacteria utilize to coordinate swarming motility, biosurfactant synthesis, and biofilm matrix exopolysaccharide production, which is critical for biofilm formation and bacterial survival in the environment.
High-Level Diversity of Tailed Phages, Eukaryote-Associated Viruses, and Virophage-Like Elements in the Metaviromes of Antarctic Soils
Authors: Zablocki Olivier , van Zyl Lonnie , Adriaenssens Evelien M. , Rubagotti Enrico , Tuffin Marla , Cary Stephen Craig , Cowan Don
Published: August 2014

The metaviromes of two distinct Antarctic hyperarid desert soil communities have been characterized. Hypolithic communities, cyanobacterium-dominated assemblages situated on the ventral surfaces of quartz pebbles embedded in the desert pavement, showed higher virus diversity than surface soils, which correlated with previous bacterial community studies. Prokaryotic viruses (i.e., phages) represented the largest viral component (particularly
Mycobacterium
phages) in both habitats, with an identical hierarchical sequence abundance of families of tailed phages (
Siphoviridae
>
Myoviridae
>
Podoviridae
). No archaeal viruses were found. Unexpectedly, cyanophages were poorly represented in both metaviromes and were phylogenetically distant from currently characterized cyanophages. Putative phage genomes were assembled and showed a high level of unaffiliated genes, mostly from hypolithic viruses. Moreover, unusual gene arrangements in which eukaryotic and prokaryotic virus-derived genes were found within identical genome segments were observed.
Phycodnaviridae
and
Mimiviridae
viruses were the second-most-abundant taxa and more numerous within open soil. Novel virophage-like sequences (within the Sputnik clade) were identified. These findings highlight high-level virus diversity and novel species discovery potential within Antarctic hyperarid soils and may serve as a starting point for future studies targeting specific viral groups.
Biosynthetic Pathway for the Cyanide-Free Production of Phenylacetonitrile in Escherichia coli by Utilizing Plant Cytochrome P450 79A2 and Bacterial Aldoxime Dehydratase
Authors: Miki Yuta , Asano Yasuhisa
Published: August 2014

The biosynthetic pathway for the production of phenylacetonitrile (PAN), which has a wide variety of uses in chemical and pharmaceutical industries, was constructed in
Escherichia coli
utilizing enzymes from the plant glucosinolate-biosynthetic and bacterial aldoxime-nitrile pathways. First, the single-step reaction to produce
E
,
Z
-phenylacetaldoxime (PAOx) from
l
-Phe was constructed in
E. coli
by introducing the genes encoding cytochrome P450 (CYP) 79A2 and CYP reductase from
Arabidopsis thaliana
, yielding the
E
,
Z
-PAOx-producing transformant. Second, this step was expanded to the production of PAN by further introducing the aldoxime dehydratase (Oxd) gene from
Bacillus
sp. strain OxB-1, yielding the PAN-producing transformant. The
E
,
Z
-PAOx-producing transformant also produced phenethyl alcohol and PAN as by-products, which were suggested to be the metabolites of
E
,
Z
-PAOx produced by
E. coli
enzymes, while the PAN-producing transformant accumulated only PAN in the culture broth, which suggested that the CYP79A2 reaction (the conversion of
l
-Phe to
E
,
Z
-PAOx) was a potential bottleneck in the PAN production pathway. Expression of active CYP79A2 and concentration of biomass were improved by the combination of the autoinduction method, coexpression of
groE
, encoding the heat shock protein GroEL/GroES, N-terminal truncation of CYP79A2, and optimization of the culture conditions, yielding a >60-fold concentration of
E
,
Z
-PAOx (up to 2.9 mM). The concentration of PAN was 4.9 mM under the optimized conditions. These achievements show the potential of this bioprocess to produce nitriles and nitrile derivatives in the absence of toxic chemicals.
Illuminating Microbial Dark Matter in Meromictic Sakinaw Lake
Authors: Gies Esther A. , Konwar Kishori M. , Beatty J. Thomas , Hallam Steven J.
Published: August 2014

Despite recent advances in metagenomic and single-cell genomic sequencing to investigate uncultivated microbial diversity and metabolic potential, fundamental questions related to population structure, interactions, and biogeochemical roles of candidate divisions remain. Numerous molecular surveys suggest that stratified ecosystems manifesting anoxic, sulfidic, and/or methane-rich conditions are enriched in these enigmatic microbes. Here we describe diversity, abundance, and cooccurrence patterns of uncultivated microbial communities inhabiting the permanently stratified waters of meromictic Sakinaw Lake, British Columbia, Canada, using 454 sequencing of the small-subunit rRNA gene with three-domain resolution. Operational taxonomic units (OTUs) were affiliated with 64 phyla, including more than 25 candidate divisions. Pronounced trends in community structure were observed for all three domains with eukaryotic sequences vanishing almost completely below the mixolimnion, followed by a rapid and sustained increase in methanogen-affiliated (∼10%) and unassigned (∼60%) archaeal sequences as well as bacterial OTUs affiliated with
Chloroflexi
(∼22%) and candidate divisions (∼28%). Network analysis revealed highly correlated, depth-dependent cooccurrence patterns between
Chloroflexi
, candidate divisions WWE1, OP9/JS1, OP8, and OD1, methanogens, and unassigned archaeal OTUs indicating niche partitioning and putative syntrophic growth modes. Indeed, pathway reconstruction using recently published Sakinaw Lake single-cell genomes affiliated with OP9/JS1 and OP8 revealed complete coverage of the Wood-Ljungdahl pathway with potential to drive syntrophic acetate oxidation to hydrogen and carbon dioxide under methanogenic conditions. Taken together, these observations point to previously unrecognized syntrophic networks in meromictic lake ecosystems with the potential to inform design and operation of anaerobic methanogenic bioreactors.
Expanding the Verrucomicrobial Methanotrophic World: Description of Three Novel Species of Methylacidimicrobium gen. nov
Authors: van Teeseling Muriel C. F. , Pol Arjan , Harhangi Harry R. , van der Zwart Sietse , Jetten Mike S. M. , Op den Camp Huub J. M. , van Niftrik Laura
Published: August 2014

Methanotrophic
Verrucomicrobia
have been found in geothermal environments characterized by high temperatures and low pH values. However, it has recently been hypothesized that methanotrophic
Verrucomicrobia
could be present under a broader range of environmental conditions. Here we describe the isolation and characterization of three new species of mesophilic acidophilic verrucomicrobial methanotrophs from a volcanic soil in Italy. The three new species showed 97% to 98% 16S rRNA gene identity to each other but were related only distantly (89% to 90% on the 16S rRNA level) to the thermophilic genus
Methylacidiphilum
. We propose the new genus
Methylacidimicrobium
, including the novel species
Methylacidimicrobium
fagopyrum
,
Methylacidimicrobium
tartarophylax
, and
Methylacidimicrobium
cyclopophantes
. These mesophilic
Methylacidimicrobium
spp. were more acid tolerant than their thermophilic relatives; the most tolerant species,
M. tartarophylax
, still grew at pH 0.5. The variation in growth temperature optima (35 to 44°C) and maximum growth rates (µmax; 0.013 to 0.040 h
−1
) suggested that all species were adapted to a specific niche within the geothermal environment. All three species grew autotrophically using the Calvin cycle. The cells of all species contained glycogen particles and electron-dense particles in their cytoplasm as visualized by electron microscopy. In addition, the cells of one of the species (
M. fagopyrum
) contained intracytoplasmic membrane stacks. The discovery of these three new species and their growth characteristics expands the known diversity of verrucomicrobial methanotrophs and shows that they are present in many more ecosystems than previously assumed.
WblAch, a Pivotal Activator of Natamycin Biosynthesis and Morphological Differentiation in Streptomyces chattanoogensis L10, Is Positively Regulated by AdpAch
Authors: Yu Pin , Liu Shui-Ping , Bu Qing-Ting , Zhou Zhen-Xing , Zhu Zhen-Hong , Huang Fang-Liang , Li Yong-Quan
Published: August 2014

Detailed mechanisms ofWhiB-like (Wbl) proteins involved in antibiotic biosynthesis and morphological differentiation are poorly understood. Here, we characterize the role of WblAch, aStreptomyces chattanoogensisL10 protein belonging to this superfamily. Based on DNA microarray data and verified by real-time quantitative PCR (qRT-PCR), the expression ofwblAchwas shown to be positively regulated by AdpAch. Gel retardation assays and DNase I footprinting experiments showed that AdpAchhas specific DNA-binding activity for the promoter region ofwblAch. Gene disruption and genetic complementation revealed that WblAchacts in a positive manner to regulate natamycin production. WhenwblAchwas overexpressed in the wild-type strain, the natamycin yield was increased by ∼30%. This provides a strategy to generate improved strains for natamycin production. Moreover, transcriptional analysis showed that the expression levels ofwhigenes (includingwhiA,whiB,whiH, andwhiI) were severely depressed in the ΔwblAchmutant, suggesting that WblAchplays a part in morphological differentiation by influencing the expression of thewhigenes.
Heterologous Expression and Characterization of the Manganese-Oxidizing Protein from Erythrobacter sp. Strain SD21
Authors: Nakama Katherine , Medina Michael , Lien Ahn , Ruggieri Jordan , Collins Krystle , Johnson Hope A.
Published: August 2014

The manganese (Mn)-oxidizing protein (MopA) fromErythrobactersp. strain SD21 is part of a unique enzymatic family that is capable of oxidizing soluble Mn(II). This enzyme contains two domains, an animal heme peroxidase domain, which contains the catalytic site, followed by a C-terminal calcium binding domain. Different from the bacterial Mn-oxidizing multicopper oxidase enzymes, little is known about MopA. To gain a better understanding of MopA and its role in Mn(II) oxidation, the 238-kDa full-length protein and a 105-kDa truncated protein containing only the animal heme peroxidase domain were cloned and heterologously expressed inEscherichia coli. Despite having sequence similarity to a peroxidase, hydrogen peroxide did not stimulate activity, nor was activity significantly decreased in the presence of catalase. Both pyrroloquinoline quinone (PQQ) and hemin increased Mn-oxidizing activity, and calcium was required. TheKmfor Mn(II) of the full-length protein in cell extract was similar to that of the natively expressed protein, but theKmvalue for the truncated protein in cell extract was approximately 6-fold higher than that of the full-length protein, suggesting that the calcium binding domain may aid in binding Mn(II). Characterization of the heterologously expressed MopA has provided additional insight into the mechanism of bacterial Mn(II) oxidation, which will aid in understanding the role of MopA and Mn oxidation in bioremediation and biogeochemical cycling.
Flocculation Causes Inhibitor Tolerance in Saccharomyces cerevisiae for Second-Generation Bioethanol Production
Authors: Westman Johan O. , Mapelli Valeria , Taherzadeh Mohammad J. , Franzén Carl Johan
Published: August 2014

Yeast has long been considered the microorganism of choice for second-generation bioethanol production due to its fermentative capacity and ethanol tolerance. However, tolerance toward inhibitors derived from lignocellulosic materials is still an issue. Flocculating yeast strains often perform relatively well in inhibitory media, but inhibitor tolerance has never been clearly linked to the actual flocculation ability
per se
. In this study, variants of the flocculation gene
FLO1
were transformed into the genome of the nonflocculating laboratory yeast strain
Saccharomyces cerevisiae
CEN.PK 113-7D. Three mutants with distinct differences in flocculation properties were isolated and characterized. The degree of flocculation and hydrophobicity of the cells were correlated to the length of the gene variant. The effect of different strength of flocculation on the fermentation performance of the strains was studied in defined medium with or without fermentation inhibitors, as well as in media based on dilute acid spruce hydrolysate. Strong flocculation aided against the readily convertible inhibitor furfural but not against less convertible inhibitors such as carboxylic acids. During fermentation of dilute acid spruce hydrolysate, the most strongly flocculating mutant with dense cell flocs showed significantly faster sugar consumption. The modified strain with the weakest flocculation showed a hexose consumption profile similar to the untransformed strain. These findings may explain why flocculation has evolved as a stress response and can find application in fermentation-based biorefinery processes on lignocellulosic raw materials.
Simultaneous Quantification of Active Carbon- and Nitrogen-Fixing Communities and Estimation of Fixation Rates Using Fluorescence
In Situ
Hybridization and Flow Cytometry
Authors: McInnes Allison S. , Shepard Alicia K. , Raes Eric J. , Waite Anya M. , Quigg Antonietta
Published: August 2014

Understanding the interconnectivity of oceanic carbon and nitrogen cycles, specifically carbon and nitrogen fixation, is essential in elucidating the fate and distribution of carbon in the ocean. Traditional techniques measure either organism abundance or biochemical rates. As such, measurements are performed on separate samples and on different time scales. Here, we developed a method to simultaneously quantify organisms while estimating rates of fixation across time and space for both carbon and nitrogen. Tyramide signal amplification fluorescence
in situ
hybridization (TSA-FISH) of mRNA for functionally specific oligonucleotide probes for
rbcL
(ribulose-1,5-bisphosphate carboxylase/oxygenase; carbon fixation) and
nifH
(nitrogenase; nitrogen fixation) was combined with flow cytometry to measure abundance and estimate activity. Cultured samples representing a diversity of phytoplankton (cyanobacteria, coccolithophores, chlorophytes, diatoms, and dinoflagellates), as well as environmental samples from the open ocean (Gulf of Mexico, USA, and southeastern Indian Ocean, Australia) and an estuary (Galveston Bay, Texas, USA), were successfully hybridized. Strong correlations between positively tagged community abundance and
14
C/
15
N measurements are presented. We propose that these methods can be used to estimate carbon and nitrogen fixation in environmental communities. The utilization of mRNA TSA-FISH to detect multiple active microbial functions within the same sample will offer increased understanding of important biogeochemical cycles in the ocean.
Detection of Pathogenic Viruses in Sewage Provided Early Warnings of Hepatitis A Virus and Norovirus Outbreaks
Authors: Hellmér Maria , Paxéus Nicklas , Magnius Lars , Enache Lucica , Arnholm Birgitta , Johansson Annette , Bergström Tomas , Norder Heléne
Published: August 2014

Most persons infected with enterically transmitted viruses shed large amounts of virus in feces for days or weeks, both before and after onset of symptoms. Therefore, viruses causing gastroenteritis may be detected in wastewater, even if only a few persons are infected. In this study, the presence of eight pathogenic viruses (norovirus, astrovirus, rotavirus, adenovirus, Aichi virus, parechovirus, hepatitis A virus [HAV], and hepatitis E virus) was investigated in sewage to explore whether their identification could be used as an early warning of outbreaks. Samples of the untreated sewage were collected in proportion to flow at Ryaverket, Gothenburg, Sweden. Daily samples collected during every second week between January and May 2013 were pooled and analyzed for detection of viruses by concentration through adsorption to milk proteins and PCR. The largest amount of noroviruses was detected in sewage 2 to 3 weeks before most patients were diagnosed with this infection in Gothenburg. The other viruses were detected at lower levels. HAV was detected between weeks 5 and 13, and partial sequencing of the structural VP1protein identified three different strains. Two strains were involved in an ongoing outbreak in Scandinavia and were also identified in samples from patients with acute hepatitis A in Gothenburg during spring of 2013. The third strain was unique and was not detected in any patient sample. The method used may thus be a tool to detect incipient outbreaks of these viruses and provide early warning before the causative pathogens have been recognized in health care.
Dynamics of Metabolic Activities and Gene Expression in the Roseobacter Clade Bacterium Phaeobacter sp. Strain MED193 during Growth with Thiosulfate
Authors: Muthusamy Saraladevi , Baltar Federico , González José M. , Pinhassi Jarone
Published: August 2014

Metagenomic analyses of surface seawater reveal that genes for sulfur oxidation are widespread in bacterioplankton communities. However, little is known about the metabolic processes used to exploit the energy potentially gained from inorganic sulfur oxidation in oxic seawater. We therefore studied the
sox
gene system containing
Roseobacter
clade isolate
Phaeobacter
sp. strain MED193 in acetate minimal medium with and without thiosulfate. The addition of thiosulfate enhanced the bacterial growth yields up to 40% in this strain. Concomitantly,
soxB
and
soxY
gene expression increased about 8-fold with thiosulfate and remained 11-fold higher than that in controls through stationary phase. At stationary phase, thiosulfate stimulated protein synthesis and anaplerotic CO
2
fixation rates up to 5- and 35-fold, respectively. Several genes involved in anaplerotic CO
2
fixation (i.e., pyruvate carboxylase, propionyl coenzyme A [CoA], and crotonyl-CoA carboxylase) were highly expressed during active growth, coinciding with high CO
2
fixation rates. The high expression of key genes in the ethylmalonyl-CoA pathway suggests that this is an important pathway for the utilization of two-carbon compounds in
Phaeobacter
sp. MED193. Overall, our findings imply that
Roseobacter
clade bacteria carrying
sox
genes can use their lithotrophic potential to gain additional energy from sulfur oxidation for both increasing their growth capacity and improving their long-term survival.
AmiE, a Novel
N
-Acylhomoserine Lactone Acylase Belonging to the Amidase Family, from the Activated-Sludge Isolate Acinetobacter sp. Strain Ooi24
Authors: Ochiai Seiji , Yasumoto Sera , Morohoshi Tomohiro , Ikeda Tsukasa
Published: August 2014

Many Gram-negative bacteria use
N
-acyl-
l
-homoserine lactones (AHLs) as quorum-sensing signal molecules. We have reported that
Acinetobacter
strains isolated from activated sludge have AHL-degrading activity. In this study, we cloned the
amiE
gene as an AHL-degradative gene from the genomic library of
Acinetobacter
sp. strain Ooi24. High-performance liquid chromatography analysis revealed that AmiE functions as an AHL acylase, which hydrolyzes the amide bond of AHL. AmiE showed a high level of degrading activity against AHLs with long acyl chains but no activity against AHLs with acyl chains shorter than eight carbons. AmiE showed homology with a member of the amidases (EC 3.5.1.4) but not with any known AHL acylase enzymes. An amino acid sequence of AmiE from Ooi24 showed greater than 99% identities with uncharacterized proteins from
Acinetobacter ursingii
CIP 107286 and
Acinetobacter
sp. strain CIP 102129, but it was not found in the draft or complete genome sequences of other
Acinetobacter
strains. The presence of transposase-like genes around the
amiE
genes of these three
Acinetobacter
strains suggests that
amiE
is transferred by a putative transposon. Furthermore, the expression of AmiE in
Pseudomonas aeruginosa
PAO1 reduced AHL accumulation and elastase activity, which were regulated by AHL-mediated quorum sensing.
Diversity of Salmonella Isolates from Central Florida Surface Waters
Authors: McEgan Rachel , Chandler Jeffrey C. , Goodridge Lawrence D. , Danyluk Michelle D.
Published: August 2014

Identification of
Salmonella
serotypes is important for understanding the environmental diversity of the genus
Salmonella
. This study evaluates the diversity of
Salmonella
isolates recovered from 165 of 202 Central Florida surface water samples and investigates whether the serotype of the environmental
Salmonella
isolates can be predicted by a previously published multiplex PCR assay (S. Kim, J. G. Frye, J. Hu, P. J. Fedorka-Cray, R. Gautom, and D. S. Boyle, J. Clin. Microbiol. 44:3608–3615, 2006,
http://dx.doi.org/10.1128/JCM.00701-06
). Multiplex PCR was performed on 562
Salmonella
isolates (as many as 36 isolates per water sample) to predict serotypes. Kauffmann-White serogrouping was used to confirm multiplex PCR pattern groupings before isolates were serotyped, analyzed by pulsed-field gel electrophoresis, and assayed for antimicrobial susceptibility. In 41.2% of the
Salmonella
-positive water samples, all
Salmonella
isolates had identical multiplex PCR patterns; in the remaining 58.8%, two or more multiplex PCR patterns were identified. Within each sample, isolates with matching multiplex PCR patterns had matching serogroups. The multiplex patterns of 495 isolates (88.1%) did not match any previously reported pattern. The remaining 68 isolates matched reported patterns but did not match the serotypes for those patterns. The use of the multiplex PCR allowed the number of isolates requiring further analysis to be reduced to 223. Thirty-three
Salmonella enterica
serotypes were identified; the most frequent included serotypes Muenchen, Rubislaw, Anatum, Gaminara, and IV_50:z4,z23:−. A majority (141/223) of
Salmonella
isolates clustered into one genotypic group.
Salmonella
isolates in Central Florida surface waters are serotypically, genotypically, and phenotypically (in terms of antimicrobial susceptibility) diverse. While isolates could be grouped as different or potentially the same using multiplex PCR, the multiplex PCR pattern did not predict the
Salmonella
serotype.
Indoor-Air Microbiome in an Urban Subway Network: Diversity and Dynamics
Authors: Leung Marcus H. Y. , Wilkins David , Li Ellen K. T. , Kong Fred K. F. , Lee Patrick K. H.
Published: August 2014

Subway systems are indispensable for urban societies, but microbiological characteristics of subway aerosols are relatively unknown. Previous studies investigating microbial compositions in subways employed methodologies that underestimated the diversity of microbial exposure for commuters, with little focus on factors governing subway air microbiology, which may have public health implications. Here, a culture-independent approach unraveling the bacterial diversity within the urban subway network in Hong Kong is presented. Aerosol samples from multiple subway lines and outdoor locations were collected. Targeting the 16S rRNA gene V4 region, extensive taxonomic diversity was found, with the most common bacterial genera in the subway environment among those associated with skin. Overall, subway lines harbored different phylogenetic communities based on α- and β-diversity comparisons, and closer inspection suggests that each community within a line is dependent on architectural characteristics, nearby outdoor microbiomes, and connectedness with other lines. Microbial diversities and assemblages also varied depending on the day sampled, as well as the time of day, and changes in microbial communities between peak and nonpeak commuting hours were attributed largely to increases in skin-associated genera in peak samples. Microbial diversities within the subway were influenced by temperature and relative humidity, while carbon dioxide levels showed a positive correlation with abundances of commuter-associated genera. This Hong Kong data set and communities from previous studies conducted in the United States formed distinct community clusters, indicating that additional work is required to unravel the mechanisms that shape subway microbiomes around the globe.
Circular Bacteriocins: Biosynthesis and Mode of Action
Authors: Gabrielsen Christina , Brede Dag A. , Nes Ingolf F. , Diep Dzung B.
Published: August 2014

Circular bacteriocins are a group of N-to-C-terminally linked antimicrobial peptides, produced by Gram-positive bacteria of the phylum
Firmicutes
. Circular bacteriocins generally exhibit broad-spectrum antimicrobial activity, including against common food-borne pathogens, such as
Clostridium
and
Listeria
spp. These peptides are further known for their high pH and thermal stability, as well as for resistance to many proteolytic enzymes, properties which make this group of bacteriocins highly promising for potential industrial applications and their biosynthesis of particular interest as a possible model system for the synthesis of highly stable bioactive peptides. In this review, we summarize the current knowledge on this group of bacteriocins, with emphasis on the recent progress in understanding circular bacteriocin genetics, biosynthesis, and mode of action; in addition, we highlight the current challenges and future perspectives for the application of these peptides.
Impact of Fertilizing with Raw or Anaerobically Digested Sewage Sludge on the Abundance of Antibiotic-Resistant Coliforms, Antibiotic Resistance Genes, and Pathogenic Bacteria in Soil and on Vegetables at Harvest
Authors: Rahube Teddie O. , Marti Romain , Scott Andrew , Tien Yuan-Ching , Murray Roger , Sabourin Lyne , Zhang Yun , Duenk Peter , Lapen David R. , Topp Edward
Published: August 2014

The consumption of crops fertilized with human waste represents a potential route of exposure to antibiotic-resistant fecal bacteria. The present study evaluated the abundance of bacteria and antibiotic resistance genes by using both culture-dependent and molecular methods. Various vegetables (lettuce, carrots, radish, and tomatoes) were sown into field plots fertilized inorganically or with class B biosolids or untreated municipal sewage sludge and harvested when of marketable quality. Analysis of viable pathogenic bacteria or antibiotic-resistant coliform bacteria by plate counts did not reveal significant treatment effects of fertilization with class B biosolids or untreated sewage sludge on the vegetables. Numerous targeted genes associated with antibiotic resistance and mobile genetic elements were detected by PCR in soil and on vegetables at harvest from plots that received no organic amendment. However, in the season of application, vegetables harvested from plots treated with either material carried gene targets not detected in the absence of amendment. Several gene targets evaluated by using quantitative PCR (qPCR) were considerably more abundant on vegetables harvested from sewage sludge-treated plots than on vegetables from control plots in the season of application, whereas vegetables harvested the following year revealed no treatment effect. Overall, the results of the present study suggest that producing vegetable crops in ground fertilized with human waste without appropriate delay or pretreatment will result in an additional burden of antibiotic resistance genes on harvested crops. Managing human exposure to antibiotic resistance genes carried in human waste must be undertaken through judicious agricultural practice.
Mutation of the
Erwinia amylovora argD
Gene Causes Arginine Auxotrophy, Nonpathogenicity in Apples, and Reduced Virulence in Pears
Authors: Ramos Laura S. , Lehman Brian L. , Peter Kari A. , McNellis Timothy W.
Published: August 2014

Fire blight is caused by
Erwinia amylovora
and is the most destructive bacterial disease of apples and pears worldwide. In this study, we found that
E. amylovora
argD
(
1000
)::Tn
5
, an
argD
Tn
5
transposon mutant that has the Tn
5
transposon inserted after nucleotide 999 in the
argD
gene-coding region, was an arginine auxotroph that did not cause fire blight in apple and had reduced virulence in immature pear fruits. The
E. amylovora
argD
gene encodes a predicted
N
-acetylornithine aminotransferase enzyme, which is involved in the production of the amino acid arginine. A plasmid-borne copy of the wild-type
argD
gene complemented both the nonpathogenic and the arginine auxotrophic phenotypes of the
argD
(
1000
)::Tn
5
mutant. However, even when mixed with virulent
E. amylovora
cells and inoculated onto immature apple fruit, the
argD
(
1000
)::Tn
5
mutant still failed to grow, while the virulent strain grew and caused disease. Furthermore, the pCR2.1-
argD
complementation plasmid was stably maintained in the
argD
(
1000
)::Tn
5
mutant growing in host tissues without any antibiotic selection. Therefore, the pCR2.1-
argD
complementation plasmid could be useful for the expression of genes, markers, and reporters in
E. amylovora
growing
in planta
, without concern about losing the plasmid over time. The ArgD protein cannot be considered an
E. amylovora
virulence factor because the
argD
(
1000
)::Tn
5
mutant was auxotrophic and had a primary metabolism defect. Nevertheless, these results are informative about the parasitic nature of the fire blight disease interaction, since they indicate that
E. amylovora
cannot obtain sufficient arginine from apple and pear fruit tissues or from apple vegetative tissues, either at the beginning of the infection process or after the infection has progressed to an advanced state.
Acquisition and Role of Molybdate in Pseudomonas aeruginosa
Authors: Pederick Victoria G. , Eijkelkamp Bart A. , Ween Miranda P. , Begg Stephanie L. , Paton James C. , McDevitt Christopher A.
Published: August 2014

In microaerophilic or anaerobic environments,
Pseudomonas aeruginosa
utilizes nitrate reduction for energy production, a process dependent on the availability of the oxyanionic form of molybdenum, molybdate (MoO
4
2−
). Here, we show that molybdate acquisition in
P. aeruginosa
occurs via a high-affinity ATP-binding cassette permease (ModABC). ModA is a cluster D-III solute binding protein capable of interacting with molybdate or tungstate oxyanions. Deletion of the
modA
gene reduces cellular molybdate concentrations and results in inhibition of anaerobic growth and nitrate reduction. Further, we show that conditions that permit nitrate reduction also cause inhibition of biofilm formation and an alteration in fatty acid composition of
P. aeruginosa
. Collectively, these data highlight the importance of molybdate for anaerobic growth of
P. aeruginosa
and reveal novel consequences of nitrate reduction on biofilm formation and cell membrane composition.
PCR and Culture Identification of Pathogenic Leptospira spp. from Coastal Soil in Leyte, Philippines, after a Storm Surge during Super Typhoon Haiyan (Yolanda)
Authors: Saito Mitsumasa , Miyahara Satoshi , Villanueva Sharon Y. A. M. , Aramaki Natsumi , Ikejiri Mami , Kobayashi Yoshie , Guevarra Jonathan P. , Masuzawa Toshiyuki , Gloriani Nina G. , Yanagihara Yasutake , Yoshida Shin-ichi
Published: August 2014

Leptospirosis is a zoonosis caused by pathogenic
Leptospira
spp. Most of the outbreaks of leptospirosis occur after floods caused by heavy rain in countries where
Leptospira
spp. are endemic. It has been believed that the overflow of seawater rarely causes outbreaks of leptospirosis because the leptospires are killed by salt water. On 8 November 2013, a storm surge caused by Super Typhoon Haiyan (Yolanda) inundated the entire coastal areas of Tacloban and Palo in Leyte, Philippines. The present study was carried out in order to determine whether the environmental leptospires in soil were able to survive after the storm surge in the affected areas. We collected 23 wet soil samples along the coastal areas of Tacloban and Palo 2 months after the storm surge. The samples were suspended in HEPES buffer, and the supernatants were cultured in liquid or semisolid Korthof's medium supplemented with five antimicrobial agents to inhibit the growth of contaminants. Leptospires were isolated from primary cultures of 22 out of 23 samples. The DNA of pathogenic
Leptospira
species was detected in 11 samples (47.8%) by analysis of
flaB
by nested PCR. Eventually, two pathogenic
Leptospira
strains were isolated and showed the highest 16S rRNA gene sequence similarity to
Leptospira kmetyi
. When these isolates were experimentally mixed with soil, they were found to survive in seawater for 4 days. These results show the possibility that leptospires living in soil survived after the storm surge. Our findings may serve as a warning that when seawater inundates the land during a storm surge or a tsunami, an outbreak of leptospirosis could occur in the disaster-stricken area.
Functional Implications and Ubiquitin-Dependent Degradation of the Peptide Transporter Ptr2 in Saccharomyces cerevisiae
Authors: Kawai Ken , Moriya Atsuto , Uemura Satoshi , Abe Fumiyoshi
Published: August 2014

The peptide transporter Ptr2 plays a central role in di- or tripeptide import in
Saccharomyces cerevisiae
. Although
PTR2
transcription has been extensively analyzed in terms of upregulation by the Ubr1-Cup9 circuit, the structural and functional information for this transporter is limited. Here we identified 14 amino acid residues required for peptide import through Ptr2 based on the crystallographic information of
Streptococcus thermophilus
peptide transporter PepT
st
and based on the conservation of primary sequences among the proton-dependent oligopeptide transporters (POTs). Expression of Ptr2 carrying one of the 14 mutations of which the corresponding residues of PepT
st
are involved in peptide recognition, salt bridge interaction, or peptide translocation failed to enable
ptr2
Δ
trp1
cell growth in alanyl-tryptophan (Ala-Trp) medium. We observed that Ptr2 underwent rapid degradation after cycloheximide treatment (half-life, approximately 1 h), and this degradation depended on Rsp5 ubiquitin ligase. The ubiquitination of Ptr2 most likely occurs at the N-terminal lysines 16, 27, and 34. Simultaneous substitution of arginine for the three lysines fully prevented Ptr2 degradation. Ptr2 mutants of the presumed peptide-binding site (E92Q, R93K, K205R, W362L, and E480D) exhibited severe defects in peptide import and were subjected to Rsp5-dependent degradation when cells were moved to Ala-Trp medium, whereas, similar to what occurs in the wild-type Ptr2, mutant proteins of the intracellular gate were upregulated. These results suggest that Ptr2 undergoes quality control and the defects in peptide binding and the concomitant conformational change render Ptr2 subject to efficient ubiquitination and subsequent degradation.