bluuebunny/crossref_metadata_2025 huggingface dataset:
Reduced dose measurement of absolute myocardial blood flow using dynamic SPECT imaging in a porcine model
Authors: Timmins Rachel , Klein Ran , Petryk Julia , Marvin Brian , Wei Lihui , deKemp Robert A. , Ruddy Terrence D. , Wells R. Glenn
Published: August 2015

Purpose:

Absolute myocardial blood flow (MBF) and myocardial flow reserve (MFR) measurements provide important additional information over traditional relative perfusion imaging. Recent advances in camera technology have made this possible with single‐photon emission tomography (SPECT). Low dose protocols are desirable to reduce the patient radiation risk; however, increased noise may reduce the accuracy of MBF measurements. The authors studied the effect of reducing dose on the accuracy of dynamic SPECT MBF measurements.

Methods:

Nineteen 30–40 kg pigs were injected with 370 + 1110 MBq of Tc‐99m sestamibi or tetrofosmin or 37 + 111 MBq of Tl‐201 at rest + stress. Microspheres were injected simultaneously to measure MBF. The pigs were imaged in list‐mode for 11 min starting at the time of injection using a Discovery NM 530c camera (GE Healthcare). Each list file was modified so that 3/4, 1/2, 1/4, 1/8, 1/16, and 1/32 of the original counts were included in the projections. Modified projections were reconstructed with CT‐based attenuation correction and an energy window‐based scatter correction and analyzed with FlowQuant kinetic modeling software using a 1‐compartment model. A modified Renkin‐Crone extraction function was used to convert the tracer uptake rate K1 to MBF values. The SPECT results were compared to those from microspheres.

Results:

Correlation between SPECT and microsphere MBF values for the full injected activity was r ≥ 0.75 for all 3 tracers and did not significantly degrade over all count levels. The mean MBF and MFR and the standard errors in the estimates were not significantly worse than the full‐count data at 1/4‐counts (Tc99m‐tracers) and 1/2‐counts (Tl‐201).

Conclusions:

Dynamic SPECT measurement of MBF and MFR in pigs can be performed with 1/4 (Tc99m‐tracers) or 1/2 (Tl‐201) of the standard injected activity without significantly reducing accuracy and precision.
Low tube voltage dual source computed tomography to reduce contrast media doses in adult abdomen examinations: A phantom study
Authors: Thor Daniel , Brismar Torkel B. , Fischer Michael A.
Published: August 2015

Purpose:

To evaluate the potential of low tube voltage dual source (DS) single energy (SE) and dual energy (DE) computed tomography (CT) to reduce contrast media (CM) dose in adult abdominal examinations of various sizes while maintaining soft tissue and iodine contrast‐to‐noise ratio (CNR).

Methods:

Four abdominal phantoms simulating a body mass index of 16 to 35 kg/m2 with four inserted syringes of 0, 2, 4, and 8 mgI/ml CM were scanned using a 64‐slice DS‐CT scanner. Six imaging protocols were used; one single source (SS) reference protocol (120 kV, 180 reference mAs), four low kV SE protocols (70 and 80 kV using both SS and DS), and one DE protocol at 80/140 kV. Potential CM reduction with unchanged CNRs relative to the 120 kV protocol was calculated along with the corresponding increase in radiation dose.

Results:

The potential contrast media reductions were determined to be approximately 53% for DS 70 kV, 51% for SS 70 kV, 44% for DS 80 kV, 40% for SS 80 kV, and 20% for DE (all differences were significant, P < 0.05). Constant CNR could be achieved by using DS 70 kV for small to medium phantom sizes (16–26 kg/m2) and for all sizes (16–35 kg/m2) when using DS 80 kV and DE. Corresponding radiation doses increased by 60%–107%, 23%–83%, and 6%–12%, respectively.

Conclusions:

DS single energy CT can be used to reduce CM dose by 44%–53% with maintained CNR in adult abdominal examinations at the cost of an increased radiation dose. DS dual‐energy CT allows reduction of CM dose by 20% at similar radiation dose as compared to a standard 120 kV single source.
Low frequency acoustic pulse propagation in temperate forests
Authors: Albert Donald G. , Swearingen Michelle E. , Perron Frank E. , Carbee David L.
Published: August 2015

Measurements of acoustic pulse propagation for a 30-m path were conducted in an open field and in seven different forest stands in the northeastern United States consisting of deciduous, evergreen, or mixed tree species. The waveforms recorded in forest generally show the pulse elongation characteristic of propagation over a highly porous ground surface, with high frequency scattered arrivals superimposed on the basic waveform shape. Waveform analysis conducted to determine ground properties resulted in acoustically determined layer thicknesses of 4–8 cm in summer, within 2 cm of the directly measured thickness of the litter layers. In winter the acoustic thicknesses correlated with the site-specific snow cover depths. Effective flow resistivity values of 50–88 kN s m−4 were derived for the forest sites in summer, while lower values typical for snow were found in winter. Reverberation times (T60) were typically around 2 s, but two stands (deciduous and pruned spruce planted on a square grid) had lower values of about 1.2 s. One site with a very rough ground surface had very low summer flow resistivity value and also had the longest reverberation time of about 3 s. These measurements can provide parameters useful for theoretical predictions of acoustic propagation within forests.
Improving clustering by imposing network information
Authors: Gerber Susanne , Horenko Illia
Published: August 2015

Algorithmic framework imposing network information on clustering algorithms is introduced and applied to neuroscience data.
Intrinsic coupling modes reveal the functional architecture of cortico-tectal networks
Authors: Stitt Iain , Galindo-Leon Edgar , Pieper Florian , Engler Gerhard , Fiedler Eva , Stieglitz Thomas , Engel Andreas K.
Published: August 2015

Correlation of ongoing neural dynamics reveals the various carrier frequencies of cortico-tectal functional interaction.
Toxicology across scales: Cell population growth in vitro predicts reduced fish growth
Authors: Stadnicka-Michalak Julita , Schirmer Kristin , Ashauer Roman
Published: August 2015

Cells in vitro can save lives of experimental fish used in chemical safety testing.
Why do animal eyes have pupils of different shapes?
Authors: Banks Martin S. , Sprague William W. , Schmoll Jürgen , Parnell Jared A. Q. , Love Gordon D.
Published: August 2015

A novel explanation of why certain pupil shapes are visually advantageous for terrestrial animals in different ecological niches.
Replicating nucleosomes
Authors: Ramachandran Srinivas , Henikoff Steven
Published: August 2015

Reestablishment of the nucleosome landscape behind the replication fork is fundamental for epigenetic memory during development.
Viral Agents Causing Brown Cap Mushroom Disease of Agaricus bisporus
Authors: Eastwood Daniel , Green Julian , Grogan Helen , Burton Kerry
Published: August 2015

The symptoms of viral infections of fungi range from cryptic to severe, but there is little knowledge of the factors involved in this transition of fungal/viral interactions. Brown cap mushroom disease of the cultivated
Agaricus bisporus
is economically important and represents a model system to describe this transition. Differentially expressed transcript fragments between mushrooms showing the symptoms of brown cap mushroom disease and control white noninfected mushrooms have been identified and sequenced. Ten of these RNA fragments have been found to be upregulated over 1,000-fold between diseased and nondiseased tissue but are absent from the
Agaricus bisporus
genome sequence and hybridize to double-stranded RNAs extracted from diseased tissue. We hypothesize that these transcript fragments are viral and represent components of the disease-causing agent, a bipartite virus with similarities to the family
Partitiviridae
. The virus fragments were found at two distinct levels within infected mushrooms, at raised levels in infected, nonsymptomatic, white mushrooms and at much greater levels (3,500 to 87,000 times greater) in infected mushrooms exhibiting brown coloration. In addition, differential screening revealed 9 upregulated and 32 downregulated host
Agaricus bisporus
transcripts. Chromametric analysis was able to distinguish color differences between noninfected white mushrooms and white infected mushrooms at an early stage of mushroom growth. This method may be the basis for an “on-farm” disease detection assay.
Distribution and Genetic Diversity of Bacteriocin Gene Clusters in Rumen Microbial Genomes
Authors: Azevedo Analice C. , Bento Cláudia B. P. , Ruiz Jeronimo C. , Queiroz Marisa V. , Mantovani Hilário C.
Published: August 2015

Some species of ruminal bacteria are known to produce antimicrobial peptides, but the screening procedures have mostly been based on
in vitro
assays using standardized methods. Recent sequencing efforts have made available the genome sequences of hundreds of ruminal microorganisms. In this work, we performed genome mining of the complete and partial genome sequences of 224 ruminal bacteria and 5 ruminal archaea to determine the distribution and diversity of bacteriocin gene clusters. A total of 46 bacteriocin gene clusters were identified in 33 strains of ruminal bacteria. Twenty gene clusters were related to lanthipeptide biosynthesis, while 11 gene clusters were associated with sactipeptide production, 7 gene clusters were associated with class II bacteriocin production, and 8 gene clusters were associated with class III bacteriocin production. The frequency of strains whose genomes encode putative antimicrobial peptide precursors was 14.4%. Clusters related to the production of sactipeptides were identified for the first time among ruminal bacteria. BLAST analysis indicated that the majority of the gene clusters (88%) encoding putative lanthipeptides contained all the essential genes required for lanthipeptide biosynthesis. Most strains of
Streptococcus
(66.6%) harbored complete lanthipeptide gene clusters, in addition to an open reading frame encoding a putative class II bacteriocin. Albusin B-like proteins were found in 100% of the
Ruminococcus albus
strains screened in this study. The
in silico
analysis provided evidence of novel biosynthetic gene clusters in bacterial species not previously related to bacteriocin production, suggesting that the rumen microbiota represents an underexplored source of antimicrobial peptides.
Interactions between Cooccurring Lactic Acid Bacteria in Honey Bee Hives
Authors: Rokop Z. P. , Horton M. A. , Newton I. L. G.
Published: August 2015

In contrast to the honey bee gut, which is colonized by a few characteristic bacterial clades, the hive of the honey bee is home to a diverse array of microbes, including many lactic acid bacteria (LAB). In this study, we used culture, combined with sequencing, to sample the LAB communities found across hive environments. Specifically, we sought to use network analysis to identify microbial hubs sharing nearly identical operational taxonomic units, evidence which may indicate cooccurrence of bacteria between environments. In the process, we identified interactions between noncore bacterial members (FructobacillusandLactobacillaceae) and honey bee-specific “core” members. BothFructobacillusandLactobacillaceaecolonize brood cells, bee bread, and nectar and may serve the role of pioneering species, establishing an environment conducive to the inoculation by honey bee core bacteria. Coculture assays showed that these noncore bacterial members promote the growth of honey bee-specific bacterial species. Specifically,Fructobacillusby-products in spent medium supported the growth of the Firm-5 honey bee-specific cladein vitro. Metabolic characterization ofFructobacillususing carbohydrate utilization assays revealed that this strain is capable of utilizing the simple sugars fructose and glucose, as well as the complex plant carbohydrate lignin. We testedFructobacillusfor antibiotic sensitivity and found that this bacterium, which may be important for establishment of the microbiome, is sensitive to the commonly used antibiotic tetracycline. Our results point to the possible significance of “noncore” and environmental microbial community members in the modulation of honey bee microbiome dynamics and suggest that tetracycline use by beekeepers should be limited.
Changes in Microbial Biofilm Communities during Colonization of Sewer Systems
Authors: Auguet O. , Pijuan M. , Batista J. , Borrego C. M. , Gutierrez O.
Published: August 2015

The coexistence of sulfate-reducing bacteria (SRB) and methanogenic archaea (MA) in anaerobic biofilms developed in sewer inner pipe surfaces favors the accumulation of sulfide (H
2
S) and methane (CH
4
) as metabolic end products, causing severe impacts on sewerage systems. In this study, we investigated the time course of H
2
S and CH
4
production and emission rates during different stages of biofilm development in relation to changes in the composition of microbial biofilm communities. The study was carried out in a laboratory sewer pilot plant that mimics a full-scale anaerobic rising sewer using a combination of process data and molecular techniques (e.g., quantitative PCR [qPCR], denaturing gradient gel electrophoresis [DGGE], and 16S rRNA gene pyrotag sequencing). After 2 weeks of biofilm growth, H
2
S emission was notably high (290.7 ± 72.3 mg S-H
2
S liter
−1
day
−1
), whereas emissions of CH
4
remained low (17.9 ± 15.9 mg COD-CH
4
liter
−1
day
−1
). This contrasting trend coincided with a stable SRB community and an archaeal community composed solely of methanogens derived from the human gut (i.e.,
Methanobrevibacter
and
Methanosphaera
). In turn, CH
4
emissions increased after 1 year of biofilm growth (327.6 ± 16.6 mg COD-CH
4
liter
−1
day
−1
), coinciding with the replacement of methanogenic colonizers by species more adapted to sewer conditions (i.e.,
Methanosaeta
spp.). Our study provides data that confirm the capacity of our laboratory experimental system to mimic the functioning of full-scale sewers both microbiologically and operationally in terms of sulfide and methane production, gaining insight into the complex dynamics of key microbial groups during biofilm development.
Gene Deletion Strategy To Examine the Involvement of the Two Chondroitin Lyases in Flavobacterium columnare Virulence
Authors: Li Nan , Qin Ting , Zhang Xiao Lin , Huang Bei , Liu Zhi Xin , Xie Hai Xia , Zhang Jin , McBride Mark J. , Nie Pin
Published: August 2015

Flavobacterium columnare
is an important bacterial pathogen of freshwater fish that causes high mortality of infected fish and heavy economic losses in aquaculture. The pathogenesis of this bacterium is poorly understood, in part due to the lack of efficient methods for genetic manipulation. In this study, a gene deletion strategy was developed and used to determine the relationship between the production of chondroitin lyases and virulence. The
F. johnsoniae
ompA
promoter (P
ompA
) was fused to
sacB
to construct a counterselectable marker for
F. columnare
.
F. columnare
carrying P
ompA-sacB
failed to grow on media containing 10% sucrose. A suicide vector carrying P
ompA-sacB
was constructed, and a gene deletion strategy was developed. Using this approach, the chondroitin lyase-encoding genes,
cslA
and
cslB
, were deleted. The Δ
cslA
and Δ
cslB
mutants were both partially deficient in digestion of chondroitin sulfate A, whereas a double mutant (Δ
cslA
Δ
cslB
) was completely deficient in chondroitin lyase activity. Cells of
F. columnare
wild-type strain G
4
and of the chondroitin lyase-deficient Δ
cslA
Δ
cslB
mutant exhibited similar levels of virulence toward grass carp in single-strain infections. Coinfections, however, revealed a competitive advantage for the wild type over the chondroitin lyase mutant. The results indicate that chondroitin lyases are not essential virulence factors of
F. columnare
but may contribute to the ability of the pathogen to compete and cause disease in natural infections. The gene deletion method developed in this study may be employed to investigate the virulence factors of this bacterium and may have wide application in many other members of the phylum
Bacteroidetes
.
Comparative Analysis of Extremely Thermophilic Caldicellulosiruptor Species Reveals Common and Unique Cellular Strategies for Plant Biomass Utilization
Authors: Zurawski Jeffrey V. , Conway Jonathan M. , Lee Laura L. , Simpson Hunter J. , Izquierdo Javier A. , Blumer-Schuette Sara , Nookaew Intawat , Adams Michael W. W. , Kelly Robert M.
Published: August 2015

Microbiological, genomic and transcriptomic analyses were used to examine three species from the bacterial genus
Caldicellulosiruptor
with respect to their capacity to convert the carbohydrate content of lignocellulosic biomass at 70°C to simple sugars, acetate, lactate, CO
2
, and H
2
.
Caldicellulosiruptor bescii
,
C. kronotskyensis
, and
C. saccharolyticus
solubilized 38%, 36%, and 29% (by weight) of unpretreated switchgrass (
Panicum virgatum
) (5 g/liter), respectively, which was about half of the amount of crystalline cellulose (Avicel; 5 g/liter) that was solubilized under the same conditions. The lower yields with
C. saccharolyticus
, not appreciably greater than the thermal control for switchgrass, were unexpected, given that its genome encodes the same glycoside hydrolase 9 (GH9)-GH48 multidomain cellulase (CelA) found in the other two species. However, the genome of
C. saccharolyticus
lacks two other cellulases with GH48 domains, which could be responsible for its lower levels of solubilization. Transcriptomes for growth of each species comparing cellulose to switchgrass showed that many carbohydrate ABC transporters and multidomain extracellular glycoside hydrolases were differentially regulated, reflecting the heterogeneity of lignocellulose. However, significant differences in transcription levels for conserved genes among the three species were noted, indicating unexpectedly diverse regulatory strategies for deconstruction for these closely related bacteria. Genes encoding the Che-type chemotaxis system and flagellum biosynthesis were upregulated in
C. kronotskyensis
and
C. bescii
during growth on cellulose, implicating motility in substrate utilization. The results here show that capacity for plant biomass deconstruction varies across
Caldicellulosiruptor
species and depends in a complex way on GH genome inventory, substrate composition, and gene regulation.
Identification of the Gene Cluster for the Anaerobic Degradation of 3,5-Dihydroxybenzoate (α-Resorcylate) in Thauera aromatica Strain AR-1
Authors: Molina-Fuentes Águeda , Pacheco Daniel , Marín Patricia , Philipp Bodo , Schink Bernhard , Marqués Silvia
Published: August 2015

Thauera aromatica
strain AR-1 degrades 3,5-dihydroxybenzoate (3,5-DHB) with nitrate as an electron acceptor. Previous biochemical studies have shown that this strain converts 3,5-DHB to hydroxyhydroquinone (1,2,4-trihydroxybenzene) through water-dependent hydroxylation of the aromatic ring and subsequent decarboxylation, and they suggest a pathway homologous to that described for the anaerobic degradation of 1,3-dihydroxybenzene (resorcinol) by
Azoarcus anaerobius
. Southern hybridization of a
T. aromatica
strain AR-1 gene library identified a 25-kb chromosome region based on its homology with
A. anaerobius
main pathway genes. Sequence analysis defined 20 open reading frames. Knockout mutations of the most relevant genes in the pathway were generated by reverse genetics. Physiological and biochemical analyses identified the genes for the three main steps in the pathway which were homologous to those described in
A. anaerobius
and suggested the function of several auxiliary genes possibly involved in enzyme maturation and intermediate stabilization. However,
T. aromatica
strain AR-1 had an additional enzyme to metabolize hydroxyhydroquinone, a putative cytoplasmic quinone oxidoreductase. In addition, a specific tripartite ATP-independent periplasmic (TRAP) transport system was required for efficient growth on 3,5-DHB. Reverse transcription-PCR (RT-PCR) analysis showed that the pathway genes were organized in five 3,5-DHB-inducible operons, three of which have been shown to be under the control of a single LysR-type transcriptional regulator, DbdR. Despite sequence homology, the genetic organizations of the clusters in
T. aromatica
strain AR-1 and
A. anaerobius
differed substantially.
The Hybrid Pre-CTXΦ-RS1 Prophage Genome and Its Regulatory Function in Environmental Vibrio cholerae O1 Strains
Authors: Wang Hongxia , Pang Bo , Xiong Lifeng , Wang Duochun , Wang Xiaomei , Zhang Lijuan , Kan Biao
Published: August 2015

The cholera toxin genes of
Vibrio cholerae
are encoded by CTXΦ, a lysogenic bacteriophage. Infection with this phage plays a determinant role in toxigenicity conversion and the emergence of new clones of pathogenic
V. cholerae
. Multiple phage alleles, defined by sequence types of the repressor gene
rstR
, have been found, showing the divergence of phage genomes. Pre-CTXΦ, which is characterized by the absence of toxin genes, is predicted to be the precursor of CTXΦ. We have found a new pre-CTXΦ prophage genome (named pre-CTX
ZJ
Φ for its novel
rstR
allele) in nontoxigenic
V. cholerae
O1 isolates that were obtained during surveillance of the estuary water of the Zhujiang River. A novel hybrid genome of the helper phage RS1 was identified in an environmental strain carrying pre-CTX
ZJ
Φ in this study. The chromosomal integration and genomic arrangement of pre-CTX
ZJ
Φ and RS1 were determined. The RS2 of pre-CTX
ZJ
Φ was shown to have a function in replication, but it seemed to have lost its ability to integrate. The RstR of pre-CTX
ZJ
Φ exerted the highest repression of its own
rstA
promoter compared to other RstRs, suggesting
rstR
-specific phage superinfection immunity and potential coinfection with other pre-CTXΦ/CTXΦ alleles. The environmental strain carrying pre-CTX
ZJ
Φ could still be infected by CTX
ET
Φ, the most common phage allele in the strains of the seventh cholera pandemic, suggesting that this nontoxigenic clone could potentially undergo toxigenicity conversion by CTXΦ infection and become a new toxigenic clone despite already containing the pre-CTXΦ prophage.
Biochemical Characterization of the Lactobacillus reuteri Glycoside Hydrolase Family 70 GTFB Type of 4,6-α-Glucanotransferase Enzymes That Synthesize Soluble Dietary Starch Fibers
Authors: Bai Yuxiang , van der Kaaij Rachel Maria , Leemhuis Hans , Pijning Tjaard , van Leeuwen Sander Sebastiaan , Jin Zhengyu , Dijkhuizen Lubbert
Published: August 2015

4,6-α-Glucanotransferase (4,6-α-GTase) enzymes, such as GTFB and GTFW of
Lactobacillus reuteri
strains, constitute a new reaction specificity in glycoside hydrolase family 70 (GH70) and are novel enzymes that convert starch or starch hydrolysates into isomalto/maltopolysaccharides (IMMPs). These IMMPs still have linear chains with some α1→4 linkages but mostly (relatively long) linear chains with α1→6 linkages and are soluble dietary starch fibers. 4,6-α-GTase enzymes and their products have significant potential for industrial applications. Here we report that an N-terminal truncation (amino acids 1 to 733) strongly enhances the soluble expression level of fully active GTFB-ΔN (approximately 75-fold compared to full-length wild type GTFB) in
Escherichia coli
. In addition, quantitative assays based on amylose V as the substrate are described; these assays allow accurate determination of both hydrolysis (minor) activity (glucose release, reducing power) and total activity (iodine staining) and calculation of the transferase (major) activity of these 4,6-α-GTase enzymes. The data show that GTFB-ΔN is clearly less hydrolytic than GTFW, which is also supported by nuclear magnetic resonance (NMR) analysis of their final products. From these assays, the biochemical properties of GTFB-ΔN were characterized in detail, including determination of kinetic parameters and acceptor substrate specificity. The GTFB enzyme displayed high conversion yields at relatively high substrate concentrations, a promising feature for industrial application.
Dynamic Mechanisms of the Bactericidal Action of an Al
2
O
3
-TiO
2
-Ag Granular Material on an Escherichia coli Strain
Authors: Tartanson Marie-Anne , Soussan Laurence , Rivallin Matthieu , Pecastaings Sophie , Chis Cristian V. , Penaranda Diego , Roques Christine , Faur Catherine
Published: August 2015

The bactericidal activity of an Al
2
O
3
-TiO
2
-Ag granular material against an
Escherichia coli
strain was confirmed by a culture-based method. In particular, 100% of microorganisms were permanently inactivated in 30 to 45 min. The present work aimed to investigate the mechanisms of the bactericidal action of this material and their dynamics on
Escherichia coli
using different techniques. Observations by transmission electron microscopy (TEM) at different times of disinfection revealed morphological changes in the bacteria as soon as they were put in contact with the material. Notably highlighted were cell membrane damage; cytoplasm detachment; formation of vacuoles, possibly due to DNA condensation, in association with regions exhibiting different levels of electron density; and membrane lysis. PCR and flow cytometry analyses were used to confirm and quantify the observations of cell integrity. The direct exposure of cells to silver, combined with the oxidative stress induced by the reactive oxygen species (ROS) generated, was identified to be responsible for these morphological alterations. From the first 5 min of treatment with the Al
2
O
3
-TiO
2
-Ag material, 98% of
E. coli
isolates were lysed. From 30 min, cell viability decreased to reach total inactivation, although approximately 1% of permeable
E. coli
cells and 1% of intact cells (10
5
genomic units · ml
−1
) were evidenced. This study demonstrates that the bactericidal effect of the material results from a synergic action of desorbed and supported silver. Supported silver was shown to generate the ROS evidenced.
Scarless Genome Editing and Stable Inducible Expression Vectors for Geobacter sulfurreducens
Authors: Chan Chi Ho , Levar Caleb E. , Zacharoff Lori , Badalamenti Jonathan P. , Bond Daniel R.
Published: August 2015

Metal reduction by members of the
Geobacteraceae
is encoded by multiple gene clusters, and the study of extracellular electron transfer often requires biofilm development on surfaces. Genetic tools that utilize polar antibiotic cassette insertions limit mutant construction and complementation. In addition, unstable plasmids create metabolic burdens that slow growth, and the presence of antibiotics such as kanamycin can interfere with the rate and extent of
Geobacter
biofilm growth. We report here genetic system improvements for the model anaerobic metal-reducing bacterium
Geobacter sulfurreducens
. A motile strain of
G. sulfurreducens
was constructed by precise removal of a transposon interrupting the
fgrM
flagellar regulator gene using SacB/sucrose counterselection, and Fe(III) citrate reduction was eliminated by deletion of the gene encoding the inner membrane cytochrome
imcH
. We also show that RK2-based plasmids were maintained in
G. sulfurreducens
for over 15 generations in the absence of antibiotic selection in contrast to unstable pBBR1 plasmids. Therefore, we engineered a series of new RK2 vectors containing native constitutive
Geobacter
promoters, and modified one of these promoters for VanR-dependent induction by the small aromatic carboxylic acid vanillate. Inducible plasmids fully complemented Δ
imcH
mutants for Fe(III) reduction, Mn(IV) oxide reduction, and growth on poised electrodes. A real-time, high-throughput Fe(III) citrate reduction assay is described that can screen numerous
G. sulfurreducens
strain constructs simultaneously and shows the sensitivity of
imcH
expression by the vanillate system. These tools will enable more sophisticated genetic studies in
G. sulfurreducens
without polar insertion effects or need for multiple antibiotics.
Effects of Formulation on Microbicide Potency and Mitigation of the Development of Bacterial Insusceptibility
Authors: Cowley Nicola L. , Forbes Sarah , Amézquita Alejandro , McClure Peter , Humphreys Gavin J. , McBain Andrew J.
Published: August 2015

Risk assessments of the potential for microbicides to select for reduced bacterial susceptibility have been based largely on data generated through the exposure of bacteria to microbicides in aqueous solution. Since microbicides are normally formulated with multiple excipients, we have investigated the effect of formulation on antimicrobial activity and the induction of bacterial insusceptibility. We tested 8 species of bacteria (7 genera) before and after repeated exposure (14 passages), using a previously validated gradient plating system, for their susceptibilities to the microbicides benzalkonium chloride, benzisothiozolinone, chlorhexidine, didecyldimethyl ammonium chloride, DMDM-hydantoin, polyhexamethylene biguanide, thymol, and triclosan in aqueous solution (nonformulated) and in formulation with excipients often deployed in consumer products. Susceptibilities were also assessed following an additional 14 passages without microbicide to determine the stability of any susceptibility changes. MICs and minimum bactericidal concentrations (MBC) were on average 11-fold lower for formulated microbicides than for nonformulated microbicides. After exposure to the antimicrobial compounds, of 72 combinations of microbicide and bacterium there were 19 ≥4-fold (mean, 8-fold) increases in MIC for nonformulated and 8 ≥4-fold (mean, 2-fold) increases in MIC for formulated microbicides. Furthermore, there were 20 ≥4-fold increases in MBC (mean, 8-fold) for nonformulated and 10 ≥4-fold (mean, 2-fold) increases in MBC for formulated microbicides. Susceptibility decreases fully or partially reverted back to preexposure values for 49% of MICs and 72% of MBCs after further passage. In summary, formulated microbicides exhibited greater antibacterial potency than unformulated actives and susceptibility decreases after repeated exposure were lower in frequency and extent.
Red Fluorescent Proteins for Gene Expression and Protein Localization Studies in Streptococcus pneumoniae and Efficient Transformation with DNA Assembled via the Gibson Assembly Method
Authors: Beilharz Katrin , van Raaphorst Renske , Kjos Morten , Veening Jan-Willem
Published: August 2015

During the last decades, a wide range of fluorescent proteins (FPs) have been developed and improved. This has had a great impact on the possibilities in biological imaging and the investigation of cellular processes at the single-cell level. Recently, we have benchmarked a set of green fluorescent proteins (GFPs) and generated a codon-optimized superfolder GFP for efficient use in the important human pathogen
Streptococcus pneumoniae
and other low-GC Gram-positive bacteria. In the present work, we constructed and compared four red fluorescent proteins (RFPs) in
S. pneumoniae
. Two orange-red variants, mOrange2 and TagRFP, and two far-red FPs, mKate2 and mCherry, were codon optimized and examined by fluorescence microscopy and plate reader assays. Notably, protein fusions of the RFPs to FtsZ were constructed by direct transformation of linear Gibson assembly (isothermal assembly) products, a method that speeds up the strain construction process significantly. Our data show that mCherry is the fastest-maturing RFP in
S. pneumoniae
and is best suited for studying gene expression, while mKate2 and TagRFP are more stable and are the preferred choices for protein localization studies. The RFPs described here will be useful for cell biology studies that require multicolor labeling in
S. pneumoniae
and related organisms.
A Model of Extracellular Enzymes in Free-Living Microbes: Which Strategy Pays Off?
Authors: Traving Sachia J. , Thygesen Uffe H. , Riemann Lasse , Stedmon Colin A.
Published: August 2015

An initial modeling approach was applied to analyze how a single, nonmotile, free-living, heterotrophic bacterial cell may optimize the deployment of its extracellular enzymes. Free-living cells live in a dilute and complex substrate field, and to gain enough substrate, their extracellular enzymes must be utilized efficiently. The model revealed that surface-attached and free enzymes generate unique enzyme and substrate fields, and each deployment strategy has distinctive advantages. For a solitary cell, surface-attached enzymes are suggested to be the most cost-efficient strategy. This strategy entails potential substrates being reduced to very low concentrations. Free enzymes, on the other hand, generate a radically different substrate field, which suggests significant benefits for the strategy if free cells engage in social foraging or experience high substrate concentrations. Swimming has a slight positive effect for the attached-enzyme strategy, while the effect is negative for the free-enzyme strategy. The results of this study suggest that specific dissolved organic compounds in the ocean likely persist below a threshold concentration impervious to biological utilization. This could help explain the persistence and apparent refractory state of oceanic dissolved organic matter (DOM). Microbial extracellular enzyme strategies, therefore, have important implications for larger-scale processes, such as shaping the role of DOM in ocean carbon sequestration.
Semirational Directed Evolution of Loop Regions in Aspergillus japonicus β-Fructofuranosidase for Improved Fructooligosaccharide Production
Authors: Trollope K. M. , Görgens J. F. , Volschenk H.
Published: August 2015

The
Aspergillus japonicus
β-fructofuranosidase catalyzes the industrially important biotransformation of sucrose to fructooligosaccharides. Operating at high substrate loading and temperatures between 50 and 60°C, the enzyme activity is negatively influenced by glucose product inhibition and thermal instability. To address these limitations, the solvent-exposed loop regions of the β-fructofuranosidase were engineered using a combined crystal structure- and evolutionary-guided approach. This semirational approach yielded a functionally enriched first-round library of 36 single-amino-acid-substitution variants with 58% retaining activity, and of these, 71% displayed improved activities compared to the parent. The substitutions yielding the five most improved variants subsequently were exhaustively combined and evaluated. A four-substitution combination variant was identified as the most improved and reduced the time to completion of an efficient industrial-like reaction by 22%. Characterization of the top five combination variants by isothermal denaturation assays indicated that these variants displayed improved thermostability, with the most thermostable variant displaying a 5.7°C increased melting temperature. The variants displayed uniquely altered, concentration-dependent substrate and product binding as determined by differential scanning fluorimetry. The altered catalytic activity was evidenced by increased specific activities of all five variants, with the most improved variant doubling that of the parent. Variant homology modeling and computational analyses were used to rationalize the effects of amino acid changes lacking direct interaction with substrates. Data indicated that targeting substitutions to loop regions resulted in improved enzyme thermostability, specific activity, and relief from product inhibition.
Impacts of Long-Term Irrigation of Domestic Treated Wastewater on Soil Biogeochemistry and Bacterial Community Structure
Authors: Wafula Denis , White John R. , Canion Andy , Jagoe Charles , Pathak Ashish , Chauhan Ashvini
Published: August 2015

Freshwater scarcity and regulations on wastewater disposal have necessitated the reuse of treated wastewater (TWW) for soil irrigation, which has several environmental and economic benefits. However, TWW irrigation can cause nutrient loading to the receiving environments. We assessed bacterial community structure and associated biogeochemical changes in soil plots irrigated with nitrate-rich TWW (referred to as pivots) for periods ranging from 13 to 30 years. Soil cores (0 to 40 cm) were collected in summer and winter from five irrigated pivots and three adjacently located nonirrigated plots. Total bacterial and denitrifier gene abundances were estimated by quantitative PCR (qPCR), and community structure was assessed by 454 massively parallel tag sequencing (MPTS) of small-subunit (SSU) rRNA genes along with terminal restriction fragment length polymorphism (T-RFLP) analysis ofnirK,nirS, andnosZfunctional genes responsible for denitrification of the TWW-associated nitrate. Soil physicochemical analyses showed that, regardless of the seasons, pH and moisture contents (MC) were higher in the irrigated (IR) pivots than in the nonirrigated (NIR) plots; organic matter (OM) and microbial biomass carbon (MBC) were higher as a function of season but not of irrigation treatment. MPTS analysis showed that TWW loading resulted in the following: (i) an increase in the relative abundance ofProteobacteria, especiallyBetaproteobacteriaandGammaproteobacteria; (ii) a decrease in the relative abundance ofActinobacteria; (iii) shifts in the communities of acidobacterial groups, along with a shift in thenirKandnirSdenitrifier guilds as shown by T-RFLP analysis. Additionally, bacterial biomass estimated by genus/group-specific real-time qPCR analyses revealed that higher numbers of total bacteria,Acidobacteria,Actinobacteria,Alphaproteobacteria, and thenirSdenitrifier guilds were present in the IR pivots than in the NIR plots. Identification of thenirK-containing microbiota as a proxy for the denitrifier community indicated that bacteria belonged to alphaproteobacteria from theRhizobiaceaefamily within the agroecosystem studied. Multivariate statistical analyses further confirmed some of the above soil physicochemical and bacterial community structure changes as a function of long-term TWW application within this agroecosystem.
Prophage Lysin Ply30 Protects Mice from Streptococcus suis and Streptococcus equi subsp. zooepidemicus Infections
Authors: Tang Fang , Li Dezhi , Wang Haojin , Ma Zhe , Lu Chengping , Dai Jianjun
Published: August 2015

Streptococcus suis
and
Streptococcus equi
subsp.
zooepidemicus
are capable of infecting humans and various animals, causing significant problems for the worldwide swine industry. As antibiotic resistance has increased, lysosomal enzymes encoded by phages have shown potential for use against pathogenic bacteria. In this study, a novel bacteriophage lysin, Ply30, encoded by the
S. suis
prophage phi30c, was recombinantly expressed and purified. Ply30 showed high bacteriolysis activity on
S. suis
and
S. equi
subsp.
zooepidemicus in vitro
. The ratio of the optical density at 600 nm (OD
600
) with treatment versus the OD
600
with no treatment for most tested
S. suis
and
S. equi
subsp.
zooepidemicus
strains decreased from 1 to <0.3 and <0.5, respectively, within 1 h. The results of plate viability assays showed that treated bacteria suffered a 1- to 2-log decrease in CFU within 1 h. The optimal concentration of Ply30 was 50 μg/ml, and the optimal pH was 7. Moreover, Ply30 maintained high activity over a wide pH range (pH 6 to 10). The MICs of Ply30 against
Streptococcus
strains ranged from 16 to 512 μg/ml.
In vivo
, a 2-mg dose of Ply30 protected 90% (9/10 mice) of mice from infection with
S. equi
subsp.
zooepidemicus
and 80% (8/10 mice) of mice from infection with
S. suis
. Seven days after lysin Ply30 treatment, bacterial loads were significantly decreased in all tested organs and blood compared with those at 1 h postinfection without Ply30 treatment. Ply30 showed
in vitro
and
in vivo
antimicrobial efficiency and protected mice against two kinds of bacterial infections, indicating that Ply30 may be an effective therapeutic against streptococci.
Eisosome Ultrastructure and Evolution in Fungi, Microalgae, and Lichens
Authors: Lee Jae-Hyeok , Heuser John E. , Roth Robyn , Goodenough Ursula
Published: August 2015

Eisosomes are among the few remaining eukaryotic cellular differentations that lack a defined function(s). These trough-shaped invaginations of the plasma membrane have largely been studied in
Saccharomyces cerevisiae
, in which their associated proteins, including two BAR domain proteins, have been identified, and homologues have been found throughout the fungal radiation. Using quick-freeze deep-etch electron microscopy to generate high-resolution replicas of membrane fracture faces without the use of chemical fixation, we report that eisosomes are also present in a subset of red and green microalgae as well as in the cysts of the ciliate
Euplotes
. Eisosome assembly is closely correlated with both the presence and the nature of cell walls. Microalgal eisosomes vary extensively in topology and internal organization. Unlike fungi, their convex fracture faces can carry lineage-specific arrays of intramembranous particles, and their concave fracture faces usually display fine striations, also seen in fungi, that are pitched at lineage-specific angles and, in some cases, adopt a broad-banded patterning. The conserved genes that encode fungal eisosome-associated proteins are not found in sequenced algal genomes, but we identified genes encoding two algal lineage-specific families of predicted BAR domain proteins, called Green-BAR and Red-BAR, that are candidate eisosome organizers. We propose a model for eisosome formation wherein (i) positively charged recognition patches first establish contact with target membrane regions and (ii) a (partial) unwinding of the coiled-coil conformation of the BAR domains then allows interactions between the hydrophobic faces of their amphipathic helices and the lipid phase of the inner membrane leaflet, generating the striated patterns.
Raising the topic of weight in general practice: perspectives of GPs and primary care nurses
Authors: Blackburn Maxine , Stathi Afroditi , Keogh Edmund , Eccleston Christopher
Published: August 2015

Objective

To explore general practitioners’ (GPs) and primary care nurses’ perceived barriers to raising the topic of weight in general practice.

Design

A qualitative study using the Theoretical Domains Framework (TDF). 34 semistructured interviews were conducted to explore views, opinions and experiences of initiating a discussion about weight. Content and thematic analyses were used to analyse the interview transcripts.

Setting

General practices located in one primary care trust in the South West of England.

Participants

17 GPs and 17 nurses aged between 32 and 66 years. The modal age range for GPs was 30–39 years and for nurses, 40–49 years.

Results

Barriers were synthesised into three main themes: (1) limited understanding about obesity care, (2) concern about negative consequences, and (3) having time and resources to raise a sensitive topic. Most barriers were related to raising the topic in more routine settings, rather than when dealing with an associated medical condition. GPs were particularly worried about damaging their relationship with patients and emphasised the need to follow their patient's agenda.

Conclusions

Uncertainty about obesity, concerns about alienating patients and feeling unable to raise the topic within the constraints of a 10 min consultation, is adding to the reluctance of GPs and nurses to broach the topic of weight. Addressing these concerns through training or by providing evidence of effective interventions that are feasible to deliver within consultations may lead to greater practitioner engagement and willingness to raise the topic.
Physician and other healthcare personnel responses to hospital stroke quality of care performance feedback: a qualitative study
Authors: Ross Joseph S , Williams Linda , Damush Teresa M , Matthias Marianne
Published: August 2015

Background

Understanding how physicians and other healthcare personnel respond to hospital performance feedback initiatives may have important implications for quality improvement efforts. Our objective was to explore responses to the inaugural feedback of hospital performance on stroke quality of care measures among relevant physicians and personnel at the US Department of Veterans Health Administration (VHA) hospitals.

Methods

Qualitative interviews with hospital administrators, physicians, nurses and quality managers at 12 VHA hospitals in the USA after the inaugural national release of the report on quality of acute stroke care processes. Interview transcripts were analysed using an immersion/crystallisation approach to identify recurrent themes.

Results

Interviews were completed with 41 individuals at 12 VHA hospitals from diverse regions of the USA; the majority were clinicians, either physicians or nurses, and nearly all had 20 years of experience or more. Interviewees described general perceptions of internal performance feedback that were both positive and negative, such as the notion that performance feedback could provide value to clinicians and hospitals, but at the same time voiced concerns about being inundated with such data. Interviewees also expressed scepticism about public reporting of performance data, citing numerous concerns and limitations. However, when interviewees described specific experiences with performance feedback, nearly all reactions were positive, including excitement, interest and feeling validated about a job well done.

Discussion

Physicians and other healthcare personnel described hospital performance feedback on stroke quality of care measures to be broadly valuable but identified areas of concern related to the measurement process and public reporting.
Prognostic significance of TRAIL signalling molecules in cervical squamous cell carcinoma
Authors: Yao Qian , Du Juan , Lin Jie , Luo Yiming , Wang Yuxiang , Liu Yan , Zhang Bo , Ren Caixia , Liu Congrong
Published: August 2015

Aim

Tumour necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) is a member of the TNF superfamily that preferentially induces apoptosis in cancer cells while exhibiting little or no toxicity in normal cells. In this study, we evaluated the clinicopathological significance of TRAIL signalling members’ expression profiles in cervical squamous cell carcinoma (CSCC).

Methods

TRAIL, DR5, caspase-8 and cellular FLICE-inhibitory protein (c-FLIP) protein expression was investigated in 72 stage IA2-IIIA CSCC patients using immunohistochemistry. Correlation between protein expression and clinicopathological features, radiotherapy response and survival was statistically analysed.

Results

Positive c-FLIP expression was an independent negative indicator for disease-free survival (DFS) (p=0.015) in multivariate Cox regression analysis. The DR5 nuclear positive group (p=0.069 by log rank test) showed some advantage of radiotherapy for overall survival (OS) compared with the DR5 nuclear negative cohort (p=0.568 by log rank test). In addition, loss of TRAIL expression was associated with worse differentiation (p=0.004), while absence of caspase-8 staining was more frequently observed in cases with lymphovascular invasion (p=0.035).

Conclusions

High c-FLIP expression is shown to be an independent prognostic variable, DR5 nuclear expression may serve as a predictive biomarker for radiotherapy, and TRAIL as well as caspase-8 loss may be associated with malignant progression.