bluuebunny/crossref_metadata_2025 huggingface dataset:
WC/Co Tool Wear in Dry Turning of Commercially Pure Aluminium
Authors: Wang Xin , Kwon Patrick Y.
Published: January 2014

Dry turning of commercially pure aluminum was performed with carbide inserts to generate tool wear. Thus, the wear on the carbides tools were generated by purely interacting with aluminum and without any abrasive, which would be the baseline wear for all aluminum alloys. The flank wear was the main mode, which increased with the cutting speed and decreased as the grain size of the carbides increases. Two types of tool wear pattern have been observed with scanning electron microscopy (SEM) and laser scanning confocal microscope (LSCM): (1) the cavities left from the carbide grains which were dislodged by the adhered layer of the work material and (2) the abrasion on the flank surface caused by the dislodged carbide grains. The width of the scoring marks was correlated with the carbide grain size, which corroborates the abrasion by the dislodged carbide grains from the carbide tool. Energy-dispersive X-ray spectroscopy (EDX) showed that the concentration of the cobalt binder was reduced on the worn area of the insert. The preferential wear of the cobalt binder is believed to facilitate the carbide grain pull-out. Therefore, the wear mechanism in turning pure aluminum is a combination of adhesion and abrasion.
Experimental and Simulation of Friction Effects in an Open-Die Microforging/Extrusion Process
Authors: Ghassemali Ehsan , Tan Ming-Jen , Chao Voon Lim Samuel , Beng Wah Chua , Wollmar Jarfors Anders Eric
Published: January 2014

Friction effects during a progressive microforming process for production of micropins of various diameters were experimentally investigated and were analytically modeled, using a hybrid friction model. The response surface method and ANOVA analysis were used to generalize the findings for various pin diameters. Besides, it was shown that to get an accurate result in simulation, the friction model must be considered locally instead of a global friction model for the whole process. The effect of friction factor on the final micropart dimensions (the effect on the instantaneous location of the neutral plane) and the forming pressure were investigated. The results showed a reduction in the friction factor as die diameter increased. Following that, the optimum frictional condition to obtain the highest micropart aspect ratio was defined as the maximum friction on the interface between the die upper surface and the punch surface, together with a minimum friction inside the die orifice.
Study of Protein Facilitated Water and Nutrient Transport in Plant Phloem
Authors: Jackie Sze Tsun-kay , Dutta Prashanta , Liu Jin
Published: January 2014

Biological systems use transporter proteins to create concentration gradients for a variety of purposes. In plant, sucrose transporter proteins play a vital role in driving fluid flow through the phloem by generating chemical potential. In this study, we investigate these nanoscale phenomena of protein directed active transport in a microscale biological system. We presented a mathematical model for protein facilitated sucrose loading considering six different states of the sucrose transporter protein. In addition, we developed a quasi-one dimensional transport model to study protein facilitated pumping mechanisms in plant phloem. Here we specifically study the influence of transporter protein reaction rates, apoplast proton concentration, membrane electrical potential, and cell membrane hydraulic permeability on flow through the phloem. This study reveals that increasing companion cell side deprotonation rate significantly enhances the sieve tube sugar concentrations, which results in much higher water transport. Lower apoplast pH increases the transport rate, but the flow control is less noticeable for a pH less than 5. A more negative membrane electrical potential difference will significantly accelerate the transporter proteins' ability to pump water and nutrients. Higher companion cell and sieve element membrane hydraulic permeability also promotes flows through the phloem; however, the flow difference is less noticeable at higher permeabilities when near typical plant cell membrane ranges.
Design Work of a Compressor Stage Through High-To-Low Speed Compressor Transformation
Authors: Chenkai Zhang , Jun Hu , Zhiqiang Wang , Xiang Gao
Published: January 2014

Low-speed model testing has advantages such as great accuracy and low cost and risk, so it is widely used in the design procedure of the high pressure compressor (HPC) exit stage. The low-speed model testing project is conducted in Nanjing University of Aeronautics and Astronautics (NUAA) to represent aerodynamic load and flow field structure of the seventh stage of a high-performance ten-stage high-pressure compressor. This paper outlines the design work of the low speed four-stage axial compressor, the third stage of which is the testing stage. The first two stages and the last stage provide the compressor with entrance and exit conditions, respectively. The high-to-low speed transformation process involves both geometric and aerodynamic considerations. Accurate similarities demand the same Mach number and Reynolds number, which will not be maintained due to motor power/size and its low-speed feature. Compromises of constraints are obvious. Modeling principles are presented in high-to-low speed transformation. Design work was carried out based on these principles. Four main procedures were conducted successively in the general design, including establishment of low-speed modeling target, global parameter design of modeling stage, throughflow aerodynamic design, and blading design. In global parameter design procedure, rotational speed, shroud diameter, hub-tip ratio, midspan chord, and axial spacing between stages were determined by geometrical modeling principles. During the throughflow design process, radial distributions of aerodynamic parameters such as D-factor, pressure-rise coefficient, loss coefficients, stage reaction, and other parameters were obtained by determined aerodynamic modeling principles. Finally, rotor and stator blade profiles of the low speed research compressor (LSRC) at seven span locations were adjusted to make sure that blade surface pressure coefficients agree well with that of the HPC. Three-dimensional flow calculations were performed on the low-speed four-stage axial compressor, and the resultant flow field structures agree well with that of the HPC. It is worth noting that a large separation zone appears in both suction surfaces of LSRC and HPC. How to diminish it through 3D blading design in the LSRC test rig is our further work.
Fatigue Life of the Strain Hardened Austenitic Stainless Steel in Simulated Pressurized Water Reactor Primary Water
Authors: Huin Nicolas , Tsutsumi Kazuya , Couvant Thierry , Henaff Gilbert , Mendez Jose
Published: January 2014

Over the last 20 years or so, many studies have revealed the deleterious effect of the environment on fatigue life of austenitic stainless steels in primary water reactor (PWR) primary water. The fatigue life correlation factor, so-called Fen, which corresponds to the ratio of fatigue life in air at room temperature to that in water under reactor operating conditions, has been standardized to consider the effect on fatigue life evaluation, and the formulations are function of strain rate and temperature due to their noticeable negative effect compared with other factors (Chopra and Shack, 2007, “Effect of LWR Coolant Environments on the Fatigue Life of Reactor Materials,” Final Report, Report No. NUREG/CR-6909, ANL-06/08; Codes for Nuclear Power Generation Facilities, 2009, "Environmental Fatigue Evaluation Method for Nuclear Power Plants," JSME S NF1-2009, The Japan Society of Mechanical Engineers, Tokyo, Japan). However, mechanism causing fatigue life reduction remains to be cleared. As one of the possible approaches to examine the underlying mechanism of environmental effect, the authors focused on the effect of plastic strain, because it could lead microstructural evolution on the material. In addition, in the case of stress corrosion cracking (SCC), it is well known that the strain-hardening prior to exposure to the primary water can lead to remarkable increase of the susceptibility to cracking (Vaillant et al., 2009, “Stress Corrosion Cracking Propagation of Cold-Worked Austenitic Stainless Steels in PWR Environment,” 14th International Conference on Environmental Degradation of Materials in Nuclear Power Systems; Couvant et al., 2009, "Development of Understanding of the Interaction Between Localized Deformation and SCC of Austenitic Stainless Steels Exposed to Primary PWR Environment," 14th International Conference on Environmental Degradation of Materials in Nuclear Power Systems). However, its effect on fatigue life has not necessarily been cleared yet. The main effort in this study addressed the effect of the prior strain-hardening on low cycle fatigue life in the primary water. A plate of 304LSS was strain hardened by cold rolling or tension prior to fatigue testing. The tests were performed under axial strain control at 300 °C in primary water including B/Li and hydrogen, and in air. The effect on environmental fatigue life was investigated through a comparison of Fen in experiments and in regulations, and also the effect on the fatigue limit defined at 106 cycles was discussed.
Effect of the Membrane Thermal Conductivity on the Performance of a Polymer Electrolyte Membrane Fuel Cell
Authors: Iranzo A. , Salva A. , Tapia E. , Rosa F.
Published: January 2014

The thermal conductivity of the polymer electrolyte membrane (PEM) of fuel cells is an important property affecting the overall cell performance. However, very few studies or fuel cell models include the dependence of this property on temperature and humidification conditions. In addition, no detailed studies have been reported for the quantitative understanding of how this property influences important aspects of the cell such as performance, water management, and membrane durability. This work presents results of a sensibility study performed for different membrane thermal conductivities, analyzing the influence of this parameter on the main cell response variables. The work has been performed with the aid of a computational fluid dynamics (CFD) model developed for a 50 cm2 fuel cell with serpentine flow field bipolar plates, previously validated against experimental measurements. The results show to what extent the cell performance, water management, and durability issues such as MEA temperature gradients are influenced by the membrane thermal conductivity, especially at high current densities, leading up to a 50% increase in the cell electric power at 1000 mA/cm2 when the thermal conductivity of the membrane is set to 0.26 W/(m K) instead of to the base value of 0.13 W/(m K).
Performance Analysis of a Direct Methanol Fuel Cell Stack With Bipolar Plate Incorporated With Innovative Flow-Field Combination
Authors: Shen Chia-Chieh , Jung Guo-Bin , Weng Feng-Bor , Yeh Chia-Chen , Lee Chih-Hung , Su Yi-Ju
Published: January 2014

Increasing interest in utilizing direct methanol fuel cells for portable applications has prompted the need for understanding of their operating characteristics. Approximately 80% of a direct methanol fuel cell stack's volume and weight arise from the bipolar plates. The bipolar plates have grooved anode and cathode flow fields, and have a critical influence on the cell stack performance and stability. However, there is little published data regarding design expansion from single cell to stack, and literature regarding the fuel/oxidant distribution in each cell is especially scant. Hence, this topic is the subject of the present study, which reports the design of a complete direct methanol fuel cell consisting of five single cells including a graphite bipolar plate, as well as an innovative anode and cathode flow channel design. By observing variations in operating parameters, such as applied load and the flow of methanol solution and air, the impact of each parameter on the output performance and stability of the stack was investigated.
Effect of Thermal Deformation on Part Errors in Metal Powder Based Additive Manufacturing Processes
Authors: Paul Ratnadeep , Anand Sam , Gerner Frank
Published: January 2014

In metal additive manufacturing (AM) processes, parts are manufactured in layers by sintering or melting metal or metal alloy powder under the effect of a powerful laser or an electron beam. As the laser/electron beam scans the powder bed, it melts the powder in successive tracks which overlap each other. This overlap, called the hatch overlap, results in a continuous cycle of rapid melting and resolidification of the metal. The melting of the metal from powder to liquid and subsequent solidification causes anisotropic shrinkage in the layers. The thermal strains caused by the thermal gradients existing between the different layers and between the layers and the substrate leads to considerable thermal stresses in the part. As a result, stress gradients develop in the different directions of the part which lead to distortion and warpage in AM parts. The deformations due to shrinkage and thermal stresses have a significant effect on the dimensional inaccuracies of the final part. A three-dimensional thermomechanical finite element (FE) model has been developed in this paper which calculates the thermal deformation in AM parts based on slice thickness, part orientation, scanning speed, and material properties. The FE model has been validated and benchmarked with results already available in literature. The thermal deformation model is then superimposed with a geometric virtual manufacturing model of the AM process to calculate the form and runout errors in AM parts. Finally, the errors in the critical features of the AM parts calculated using the combined thermal deformation and geometric model are correlated with part orientation and slice thickness.
“Entransy,” and Its Lack of Content in Physics
Authors: Bejan Adrian
Published: January 2014

Here, I show that “entransy” has no meaning in physics, because, at bottom, it rests on the false claim that in order to transfer heat to a solid body of thermodynamic temperature T, the heat transfer must be proportional to T. Entransy “dissipation” is a number proportional to well known measures of irreversibility such as entropy generation and lost exergy (destroyed available work). Furthermore, the “principle of entransy dissipation minimization” adds nothing to existing work based on minimum entropy generation, minimum thermal resistance, and constructal law. The broader trend illustrated by the entransy hoax is that it is becoming easy to take an existing idea, change the keywords, and publish it as new.
Effectiveness of Diathermy in Comparison With Ultrasound or Corticosteroids in Patients With Tendinopathy: A Critically Appraised Topic
Authors: Szlosek Philip A. , Taggart John , Cavallario Julie M. , Hoch Johanna M.
Published: January 2014

Clinical Scenario:

Many therapeutic modalities have been used to treat the pain and inflammation commonly associated with tendinopathies. One modality that has been used to treat patients with tendinopathies is diathermy.

Focused Clinical Question:

Is there evidence to suggest that diathermy is more or equally as effective at reducing pain in patients with tendinopathy when compared with ultrasound or corticosteroid treatments?

Summary of Search, “Best Evidence” Appraised, and Key Findings:

The literature was searched for randomized control trials (RCTs) that investigated the effects of diathermy treatments in comparison with ultrasound or corticosteroid treatments on pain in patients with tendinopathy. Three RCTs were selected from the search results and included in this critically appraised topic.

Clinical Bottom Line:

There is moderate evidence to support that diathermy is more effective at reducing pain in patients with tendinopathy than ultrasound and equally as effective as corticosteroid treatments.

Strength of Recommendation:

There is grade B evidence to support that diathermy is more effective at reducing pain in patients with tendinopathy than ultrasound and equally effective at reducing pain as corticosteroid treatments.
Association of Ankle Kinematics and Performance on the Y-Balance Test With Inclinometer Measurements on the Weight-Bearing-Lunge Test
Authors: Kang Min-Hyeok , Lee Dong-Kyu , Park Kyung-Hee , Oh Jae-Seop
Published: January 2014

Context:

Ankle-dorsiflexion range of motion has often been measured in the weight-bearing condition in the clinical setting; however, little is known about the relationship between the weight-bearing-lunge test (WBLT) and both ankle kinematics and performance on dynamic postural-control tests.

Objective:

To examine whether ankle kinematics and performance on the Lower Quarter Y-Balance Test (YBT-LQ) are correlated with results of the WBLT using an inclinometer and tape measure.

Design:

Cross-sectional.

Setting:

University motionanalysis laboratory.

Participants:

30 physically active participants.

Interventions:

None.

Main Outcome Measures:

The WBLT was evaluated using an inclinometer and a tape measure. The reach distances in the anterior, posteromedial, and posterolateral directions on the YBT-LQ were normalized by limb length. Ankle dorsiflexion during the YBT-LQ was recorded using a 3-dimensional motion-analysis system. Simple linear regression was used to examine the relationship between the WBLT results and both ankle dorsiflexion and the normalized reach distance in each direction on the YBT-LQ.

Results:

The WBLT results were significantly correlated with ankle dorsiflexion in all directions on the YBT-LQ (P < .05). A strong correlation was found between the inclinometer measurement of the WBLT and ankle dorsiflexion (r = .74, r2 = .55), whereas the tape-measure results on the WBLT were moderately correlated with ankle dorsiflexion (r = .64, r2 = .40) during the anterior reach on the YBT-LQ. Only the normalized anterior reach distance was significantly correlated with the results for the inclinometer (r = .68, r2 = .46) and the tape measure (r = .64, r2 = .41) on the WBLT.

Conclusions:

Inclinometer measurements on the WBLT can be an appropriate tool for predicting the amount of ankle dorsiflexion during the YBT-LQ. Furthermore, WBLT should be measured in those who demonstrate poor dynamic balance.
ATP Receptor in
Arabidopsis
Authors: Hines Pamela J.
Published: January 2014

An
Arabidopsis
lectin receptor kinase, DORN1, is the plant receptor for extracellular adenosine triphosphate.
Lymphoma Gets NIK’d
Authors: Foley John F.
Published: January 2014

Some mantle cell lymphomas are addicted to alternative nuclear factor κB signaling, which is dependent on the kinase NIK.
Metabolic Reprogramming with a Long Noncoding RNA
Authors: Gough Nancy R.
Published: January 2014

HIF-1α and lincRNA-p21 form a positive feedback loop that reprograms metabolism in response to hypoxia.
Aquatic Hyphomycete Species Are Screened by the Hyporheic Zone of Woodland Streams
Authors: Cornut Julien , Chauvet Eric , Mermillod-Blondin Florian , Assemat Fiona , Elger Arnaud
Published: January 2014

Aquatic hyphomycetes strongly contribute to organic matter dynamics in streams, but their abilities to colonize leaf litter buried in streambed sediments remain unexplored. Here, we conducted field and laboratory experiments (slow-filtration columns and stream-simulating microcosms) to test the following hypotheses: (i) that the hyporheic habitat acting as a physical sieve for spores filters out unsuccessful strategists from a potential species pool, (ii) that decreased pore size in sediments reduces species dispersal efficiency in the interstitial water, and (iii) that the physicochemical conditions prevailing in the hyporheic habitat will influence fungal community structure. Our field study showed that spore abundance and species diversity were consistently reduced in the interstitial water compared with surface water within three differing streams. Significant differences occurred among aquatic hyphomycetes, with dispersal efficiency of filiform-spore species being much higher than those with compact or branched/tetraradiate spores. This pattern was remarkably consistent with those found in laboratory experiments that tested the influence of sediment pore size on spore dispersal in microcosms. Furthermore, leaves inoculated in a stream and incubated in slow-filtration columns exhibited a fungal assemblage dominated by only two species, while five species were codominant on leaves from the stream-simulating microcosms. Results of this study highlight that the hyporheic zone exerts two types of selection pressure on the aquatic hyphomycete community, a physiological stress and a physical screening of the benthic spore pool, both leading to drastic changes in the structure of fungal community.
Genes Conferring Copper Resistance in
Sinorhizobium meliloti
CCNWSX0020 Also Promote the Growth of
Medicago lupulina
in Copper-Contaminated Soil
Authors: Li Zhefei , Ma Zhanqiang , Hao Xiuli , Rensing Christopher , Wei Gehong
Published: January 2014

Sinorhizobium meliloti
CCNWSX0020, isolated from root nodules of
Medicago lupulina
growing in gold mine tailings in the northwest of China, displayed both copper resistance and growth promotion of leguminous plants in copper-contaminated soil. Nevertheless, the genetic and biochemical mechanisms responsible for copper resistance in
S. meliloti
CCNWSX0020 remained uncharacterized. To investigate genes involved in copper resistance, an
S. meliloti
CCNWSX0020 Tn
5
insertion library of 14,000 mutants was created. Five copper-sensitive mutants, named SXa-1, SXa-2, SXc-1, SXc-2, and SXn, were isolated, and the disrupted regions involved were identified by inverse PCR and subsequent sequencing. Both SXa-1 and SXa-2 carried a transposon insertion in
lpxXL
(
SM0020_18047
), encoding the LpxXL C-28 acyltransferase; SXc-1 and SXc-2 carried a transposon insertion in
merR
(
SM0020_29390
), encoding the regulatory activator; SXn contained a transposon insertion in
omp
(
SM0020_18792
), encoding a hypothetical outer membrane protein. The results of reverse transcriptase PCR (RT-PCR) combined with transposon gene disruptions revealed that
SM0020_05862
, encoding an unusual P-type ATPase, was regulated by the MerR protein. Analysis of the genome sequence showed that this P-type ATPase did not contain an N-terminal metal-binding domain or a CPC motif but rather TPCP compared with CopA from
Escherichia coli
. Pot experiments were carried out to determine whether growth and copper accumulation of the host plant
M. lupulina
were affected in the presence of the wild type or the different mutants. Soil samples were subjected to three levels of copper contamination, namely, the uncontaminated control and 47.36 and 142.08 mg/kg, and three replicates were conducted for each treatment. The results showed that the wild-type
S. meliloti
CCNWSX0020 enabled the host plant to grow better and accumulate copper ions. The plant dry weight and copper content of
M. lupulina
inoculated with the 5 copper-sensitive mutants significantly decreased in the presence of CuSO
4
.
Cold Air Plasma To Decontaminate Inanimate Surfaces of the Hospital Environment
Authors: Cahill Orla J. , Claro Tânia , O'Connor Niall , Cafolla Anthony A. , Stevens Niall T. , Daniels Stephen , Humphreys Hilary
Published: January 2014

The hospital environment harbors bacteria that may cause health care-associated infections. Microorganisms, such as multiresistant bacteria, can spread around the patient's inanimate environment. Some recently introduced biodecontamination approaches in hospitals have significant limitations due to the toxic nature of the gases and the length of time required for aeration. This study evaluated the
in vitro
use of cold air plasma as an efficient alternative to traditional methods of biodecontamination of hospital surfaces. Cultures of methicillin-resistant
Staphylococcus aureus
(MRSA), vancomycin-resistant enterococci (VRE), extended-spectrum-β-lactamase (ESBL)-producing
Escherichia coli
, and
Acinetobacter baumannii
were applied to different materials similar to those found in the hospital environment. Artificially contaminated sections of marmoleum, mattress, polypropylene, powder-coated mild steel, and stainless steel were then exposed to a cold air pressure plasma single jet for 30 s, 60 s, and 90 s, operating at approximately 25 W and 12 liters/min flow rate. Direct plasma exposure successfully reduced the bacterial load by log 3 for MRSA, log 2.7 for VRE, log 2 for ESBL-producing
E. coli
, and log 1.7 for
A. baumannii
. The present report confirms the efficient antibacterial activity of a cold air plasma single-jet plume on nosocomial bacterially contaminated surfaces over a short period of time and highlights its potential for routine biodecontamination in the clinical environment.
Identification of Genetic Bases of
Vibrio fluvialis
Species-Specific Biochemical Pathways and Potential Virulence Factors by Comparative Genomic Analysis
Authors: Lu Xin , Liang Weili , Wang Yunduan , Xu Jialiang , Zhu Jun , Kan Biao
Published: January 2014

Vibrio fluvialis
is an important food-borne pathogen that causes diarrheal illness and sometimes extraintestinal infections in humans. In this study, we sequenced the genome of a clinical
V. fluvialis
strain and determined its phylogenetic relationships with other
Vibrio
species by comparative genomic analysis. We found that the closest relationship was between
V. fluvialis
and
V. furnissii
, followed by those with
V. cholerae
and
V. mimicus
. Moreover, based on genome comparisons and gene complementation experiments, we revealed genetic mechanisms of the biochemical tests that differentiate
V. fluvialis
from closely related species. Importantly, we identified a variety of genes encoding potential virulence factors, including multiple hemolysins, transcriptional regulators, and environmental survival and adaptation apparatuses, and the type VI secretion system, which is indicative of complex regulatory pathways modulating pathogenesis in this organism. The availability of
V. fluvialis
genome sequences may promote our understanding of pathogenic mechanisms for this emerging pathogen.
A Rice Gene for Microbial Symbiosis,
Oryza sativa
CCaMK
, Reduces CH
4
Flux in a Paddy Field with Low Nitrogen Input
Authors: Bao Zhihua , Watanabe Aya , Sasaki Kazuhiro , Okubo Takashi , Tokida Takeshi , Liu Dongyan , Ikeda Seishi , Imaizumi-Anraku Haruko , Asakawa Susumu , Sato Tadashi , Mitsui Hisayuki , Minamisawa Kiwamu
Published: January 2014

Plants have mutualistic symbiotic relationships with rhizobia and fungi by the common symbiosis pathway, of which Ca
2+
/calmodulin-dependent protein kinase (encoded by
CCaMK
) is a central component. Although
Oryza sativa
CCaMK
(
OsCCaMK
) is required for fungal accommodation in rice roots, little is known about the role of
OsCCaMK
in rice symbiosis with bacteria. Here, we report the effect of a
Tos17
-induced
OsCCaMK
mutant (NE1115) on CH
4
flux in low-nitrogen (LN) and standard-nitrogen (SN) paddy fields compared with wild-type (WT) Nipponbare. The growth of NE1115 was significantly decreased compared with that of the WT, especially in the LN field. The CH
4
flux of NE1115 in the LN field was significantly greater (156 to 407% in 2011 and 170 to 816% in 2012) than that of the WT, although no difference was observed in the SN field. The copy number of
pmoA
(encodes methane monooxygenase in methanotrophs) was significantly higher in the roots and rhizosphere soil of the WT than in those of NE1115. However, the
mcrA
(encodes methyl coenzyme M reductase in methanogens) copy number did not differ between the WT and NE1115. These results were supported by a
13
C-labeled CH
4
-feeding experiment. In addition, the natural abundance of
15
N in WT shoots (3.05‰) was significantly lower than in NE1115 shoots (3.45‰), suggesting greater N
2
fixation in the WT because of dilution with atmospheric N
2
(0.00‰). Thus, CH
4
oxidation and N
2
fixation were simultaneously activated in the root zone of WT rice in the LN field and both processes are likely controlled by
OsCCaMK
.
Temporal Alterations in the Secretome of the Selective Ligninolytic Fungus Ceriporiopsis subvermispora during Growth on Aspen Wood Reveal This Organism's Strategy for Degrading Lignocellulose
Authors: Hori Chiaki , Gaskell Jill , Igarashi Kiyohiko , Kersten Phil , Mozuch Michael , Samejima Masahiro , Cullen Dan
Published: January 2014

The white-rot basidiomycetes efficiently degrade all wood cell wall polymers. Generally, these fungi simultaneously degrade cellulose and lignin, but certain organisms, such asCeriporiopsis subvermispora, selectively remove lignin in advance of cellulose degradation. However, relatively little is known about the mechanism of selective ligninolysis. To address this issue,C. subvermisporawas grown in liquid medium containing ball-milled aspen, and nano-liquid chromatography-tandem mass spectrometry was used to identify and estimate extracellular protein abundance over time. Several manganese peroxidases and an aryl alcohol oxidase, both associated with lignin degradation, were identified after 3 days of incubation. A glycoside hydrolase (GH) family 51 arabinofuranosidase was also identified after 3 days but then successively decreased in later samples. Several enzymes related to cellulose and xylan degradation, such as GH10 endoxylanase, GH5_5 endoglucanase, and GH7 cellobiohydrolase, were detected after 5 days. Peptides corresponding to potential cellulose-degrading enzymes GH12, GH45, lytic polysaccharide monooxygenase, and cellobiose dehydrogenase were most abundant after 7 days. This sequential production of enzymes provides a mechanism consistent with selective ligninolysis byC. subvermispora.
The Global Response Regulator ExpA Controls Virulence Gene Expression through RsmA-Mediated and RsmA-Independent Pathways in
Pectobacterium wasabiae
SCC3193
Authors: Broberg M. , Lee G. W. , Nykyri J. , Lee Y. H. , Pirhonen M. , Palva E. T.
Published: January 2014

ExpA (GacA) is a global response regulator that controls the expression of major virulence genes, such as those encoding plant cell wall-degrading enzymes (PCWDEs) in the model soft rot phytopathogen
Pectobacterium wasabiae
SCC3193. Several studies with pectobacteria as well as related phytopathogenic gammaproteobacteria, such as
Dickeya
and
Pseudomonas
, suggest that the control of virulence by ExpA and its homologues is executed partly by modulating the activity of RsmA, an RNA-binding posttranscriptional regulator. To elucidate the extent of the overlap between the ExpA and RsmA regulons in
P. wasabiae
, we characterized both regulons by microarray analysis. To do this, we compared the transcriptomes of the wild-type strain, an
expA
mutant, an
rsmA
mutant, and an
expA rsmA
double mutant. The microarray data for selected virulence-related genes were confirmed through quantitative reverse transcription (qRT-PCR). Subsequently, assays were performed to link the observed transcriptome differences to changes in bacterial phenotypes such as growth, motility, PCWDE production, and virulence
in planta
. An extensive overlap between the ExpA and RsmA regulons was observed, suggesting that a substantial portion of ExpA regulation appears to be mediated through RsmA. However, a number of genes involved in the electron transport chain and oligogalacturonide metabolism, among other processes, were identified as being regulated by ExpA independently of RsmA. These results suggest that ExpA may only partially impact fitness and virulence via RsmA.
Animal Rennets as Sources of Dairy Lactic Acid Bacteria
Authors: Cruciata Margherita , Sannino Ciro , Ercolini Danilo , Scatassa Maria L. , De Filippis Francesca , Mancuso Isabella , La Storia Antonietta , Moschetti Giancarlo , Settanni Luca
Published: January 2014

The microbial composition of artisan and industrial animal rennet pastes was studied by using both culture-dependent and -independent approaches. Pyrosequencing targeting the 16S rRNA gene allowed to identify 361 operational taxonomic units (OTUs) to the genus/species level. Among lactic acid bacteria (LAB),
Streptococcus thermophilus
and some lactobacilli, mainly
Lactobacillus crispatus
and
Lactobacillus reuteri
, were the most abundant species, with differences among the samples. Twelve groups of microorganisms were targeted by viable plate counts revealing a dominance of mesophilic cocci. All rennets were able to acidify ultrahigh-temperature-processed (UHT) milk as shown by pH and total titratable acidity (TTA). Presumptive LAB isolated at the highest dilutions of acidified milks were phenotypically characterized, grouped, differentiated at the strain level by randomly amplified polymorphic DNA (RAPD)-PCR analysis, and subjected to 16S rRNA gene sequencing. Only 18 strains were clearly identified at the species level, as
Enterococcus casseliflavus
,
Enterococcus faecium
,
Enterococcus faecalis
,
Enterococcus lactis
,
Lactobacillus delbrueckii
, and
Streptococcus thermophilus
, while the other strains, all belonging to the genus
Enterococcus
, could not be allotted into any previously described species. The phylogenetic analysis showed that these strains might represent different unknown species. All strains were evaluated for their dairy technological performances. All isolates produced diacetyl, and 10 of them produced a rapid pH drop in milk, but only 3 isolates were also autolytic. This work showed that animal rennet pastes can be sources of LAB, mainly enterococci, that might contribute to the microbial diversity associated with dairy productions.
Taxonomic Identification of Commensal Bacteria Associated with the Mucosa and Digesta throughout the Gastrointestinal Tracts of Preweaned Calves
Authors: Malmuthuge Nilusha , Griebel Philip J. , Guan Le Luo
Published: January 2014

Bacterial colonization in the gastrointestinal tracts (GIT) of preweaned calves is very important, since it can influence early development and postweaning performance and health. This study investigated the composition of the bacteria along the GIT (rumen, jejunum, ileum, cecum, and colon) of preweaned bull calves (3 weeks old) using pyrosequencing to understand the segregation of bacteria between the mucosal surface and digesta. Phylogenetic analysis revealed that a total of 83 genera belonging to 13 phyla were distributed throughout the GIT of preweaned calves, with the
Firmicutes
,
Bacteroidetes
, and
Proteobacteria
predominating. Quantitative PCR (qPCR) analysis of selected abundant bacterial genera (
Prevotella
,
Bacteroides
,
Lactobacillus
, and
Faecalibacterium
) revealed that their prevalence was significantly different among the GIT regions and between mucosa- and digesta-associated communities. Rumens contained the most diverse bacterial population, consisting of 47 genera, including 16 rumen-specific genera, followed by the large intestine and then the small intestine. Bacterial species richness was higher at the mucosal surface than in the local digesta, with the exception of the rumen. The majority of bacteria found on the rumen epithelial surface and within the small intestine could not be identified due to a lack of known genus-level information. Thus, future studies will be required to fully characterize the microbiome during the development of the rumens and the mucosal immune systems of newborn calves. This is the first study to analyze in depth the bacterial composition of the GIT microbiome in preweaned calves, which extends previous findings regarding early rumen colonization and bacterial segregation between mucosa- and digesta-associated microbial communities.
Occurrence of Diarrheagenic Virulence Genes and Genetic Diversity in
Escherichia coli
Isolates from Fecal Material of Various Avian Hosts in British Columbia, Canada
Authors: Chandran Abhirosh , Mazumder Asit
Published: January 2014

Contamination of surface water by fecal microorganisms originating from human and nonhuman sources is a public health concern. In the present study,
Escherichia coli
isolates (
n
= 412) from the feces of various avian host sources were screened for various virulence genes:
stx
1
and
stx
2
(Shiga toxin-producing
E. coli
[STEC]),
eae
(enteropathogenic
E. coli
[EPEC]),
est
-h,
est
-p, and
elt
(encoding heat-stable toxin [ST] variants STh and STp and heat-labile toxin [LT], respectively) (enterotoxigenic
E. coli
[ETEC]), and
ipaH
(enteroinvasive
E. coli
[EIEC]). None of the isolates were found to be positive for
stx
1
, while 23% (
n
= 93) were positive for only
stx
2
, representing STEC, and 15% (
n
= 63) were positive for only
eae
, representing EPEC. In addition, five strains obtained from pheasant were positive for both
stx
2
and
eae
and were confirmed as non-O157 by using an
E. coli
O157
rfb
(
rfb
O157
) TaqMan assay. Isolates positive for the virulence genes associated with ETEC and EIEC were not detected in any of the hosts. The repetitive element palindromic PCR (rep-PCR) fingerprint analysis identified 143 unique fingerprints, with an overall Shannon diversity index of 2.36. Multivariate analysis of variance (MANOVA) showed that the majority of the STEC and EPEC isolates were genotypically distinct from nonpathogenic
E. coli
and clustered independently. MANOVA analysis also revealed spatial variation among the
E. coli
isolates, since the majority of the isolates clustered according to the sampling locations. Although the presence of virulence genes alone cannot be used to determine the pathogenicity of strains, results from this study show that potentially pathogenic STEC and EPEC strains can be found in some of the avian hosts studied and may contaminate surface water and potentially impact human health.
Hot-Alkaline DNA Extraction Method for Deep-Subseafloor Archaeal Communities
Authors: Morono Yuki , Terada Takeshi , Hoshino Tatsuhiko , Inagaki Fumio
Published: January 2014

A prerequisite for DNA-based microbial community analysis is even and effective cell disruption for DNA extraction. With a commonly used DNA extraction kit, roughly two-thirds of subseafloor sediment microbial cells remain intact on average (i.e., the cells are not disrupted), indicating that microbial community analyses may be biased at the DNA extraction step, prior to subsequent molecular analyses. To address this issue, we standardized a new DNA extraction method using alkaline treatment and heating. Upon treatment with 1 M NaOH at 98°C for 20 min, over 98% of microbial cells in subseafloor sediment samples collected at different depths were disrupted. However, DNA integrity tests showed that such strong alkaline and heat treatment also cleaved DNA molecules into short fragments that could not be amplified by PCR. Subsequently, we optimized the alkaline and temperature conditions to minimize DNA fragmentation and retain high cell disruption efficiency. The best conditions produced a cell disruption rate of 50 to 80% in subseafloor sediment samples from various depths and retained sufficient DNA integrity for amplification of the complete 16S rRNA gene (i.e., ∼1,500 bp). The optimized method also yielded higher DNA concentrations in all samples tested compared with extractions using a conventional kit-based approach. Comparative molecular analysis using real-time PCR and pyrosequencing of bacterial and archaeal 16S rRNA genes showed that the new method produced an increase in archaeal DNA and its diversity, suggesting that it provides better analytical coverage of subseafloor microbial communities than conventional methods.
Calcineurin Controls Hyphal Growth, Virulence, and Drug Tolerance of Candida tropicalis
Authors: Chen Ying-Lien , Yu Shang-Jie , Huang Hsin-Yu , Chang Ya-Lin , Lehman Virginia N. , Silao Fitz Gerald S. , Bigol Ursela G. , Bungay Alice Alma C. , Averette Anna , Heitman Joseph
Published: January 2014

Candida tropicalis
, a species closely related to
Candida albicans
, is an emerging fungal pathogen associated with high mortality rates of 40 to 70%. Like
C. albicans
and
Candida dubliniensis
,
C. tropicalis
is able to form germ tubes, pseudohyphae, and hyphae, but the genes involved in hyphal growth machinery and virulence remain unclear in
C. tropicalis
. Recently, echinocandin- and azole-resistant
C. tropicalis
isolates have frequently been isolated from various patients around the world, making treatment difficult. However, studies of the
C. tropicalis
genes involved in drug tolerance are limited. Here, we investigated the roles of calcineurin and its potential target, Crz1, for core stress responses and pathogenesis in
C. tropicalis
. We demonstrate that calcineurin and Crz1 are required for hyphal growth, micafungin tolerance, and virulence in a murine systemic infection model, while calcineurin but not Crz1 is essential for tolerance of azoles, caspofungin, anidulafungin, and cell wall-perturbing agents, suggesting that calcineurin has both Crz1-dependent and -independent functions in
C. tropicalis
. In addition, we found that calcineurin and Crz1 have opposite roles in controlling calcium tolerance. Calcineurin serves as a negative regulator, while Crz1 plays a positive role for calcium tolerance in
C. tropicalis
.
High-Throughput Chemical Screening for Antivirulence Developmental Phenotypes in Trypanosoma brucei
Authors: MacGregor Paula , Ivens Alasdair , Shave Steven , Collie Iain , Gray David , Auer Manfred , Matthews Keith R.
Published: January 2014

In the bloodstream of mammalian hosts, the sleeping sickness parasite,
Trypanosoma brucei
, exists as a proliferative slender form or a nonproliferative, transmissible, stumpy form. The transition between these developmental forms is controlled by a density-dependent mechanism that is important for the parasite's infection dynamics, immune evasion via ordered antigenic variation, and disease transmissibility. However, stumpy formation has been lost in most laboratory-adapted trypanosome lines, generating monomorphic parasites that proliferate uncontrolled as slender forms
in vitro
and
in vivo
. Nonetheless, these forms are readily amenable to cell culture and high-throughput screening for trypanocidal lead compounds. Here, we have developed and exploited a high-throughput screen for developmental phenotypes using a transgenic monomorphic cell line expressing a reporter under the regulation of gene control signals from the stumpy-specific molecule PAD1. Using a whole-cell fluorescence-based assay to screen over 6,000 small molecules from a kinase-focused compound library, small molecules able to activate stumpy-specific gene expression and proliferation arrest were assayed in a rapid assay format. Independent follow-up validation identified one hit able to induce modest, yet specific, changes in mRNA expression indicative of a partial differentiation to stumpy forms in monomorphs. Further, in pleomorphs this compound induced a stumpy-like phenotype, entailing growth arrest, morphological changes, PAD1 expression, and enhanced differentiation to procyclic forms. This not only provides a potential tool compound for the further understanding of stumpy formation but also demonstrates the use of high-throughput screening in the identification of compounds able to induce specific phenotypes, such as differentiation, in African trypanosomes.
Regulatory and Functional Diversity of Methylmercaptopropionate Coenzyme A Ligases from the Dimethylsulfoniopropionate Demethylation Pathway in Ruegeria pomeroyi DSS-3 and Other Proteobacteria
Authors: Bullock Hannah A. , Reisch Chris R. , Burns Andrew S. , Moran Mary Ann , Whitman William B.
Published: January 2014

The organosulfur compound dimethylsulfoniopropionate (DMSP) is produced by phytoplankton and is ubiquitous in the surface ocean. Once released from phytoplankton, marine bacteria degrade DMSP by either the cleavage pathway to form the volatile gas dimethylsulfide (DMS) or the demethylation pathway, yielding methanethiol (MeSH), which is readily assimilated or oxidized. The enzyme DmdB, a methylmercaptopropionate (MMPA)-coenzyme A (CoA) ligase, catalyzes the second step in the demethylation pathway and is a major regulatory point. The two forms of DmdB present in the marine roseobacter
Ruegeria pomeroyi
DSS-3, RPO_DmdB1 and RPO_DmdB2, and the single form in the SAR11 clade bacterium “
Candidatus
Pelagibacter ubique” HTCC1062, PU_DmdB1, were characterized in detail. DmdB enzymes were also examined from
Ruegeria lacuscaerulensis
ITI-1157,
Pseudomonas aeruginosa
PAO1, and
Burkholderia thailandensis
E264. The DmdB enzymes separated into two phylogenetic clades. All enzymes had activity with MMPA and were sensitive to inhibition by salts, but there was no correlation between the clades and substrate specificity or salt sensitivity. All
Ruegeria
species enzymes were inhibited by physiological concentrations (70 mM) of DMSP. However, ADP reversed the inhibition of RPO_DmdB1, suggesting that this enzyme was responsive to cellular energy charge. MMPA reversed the inhibition of RPO_DmdB2 as well as both
R. lacuscaerulensis
ITI-1157 DmdB enzymes, suggesting that a complex regulatory system exists in marine bacteria. In contrast, the DmdBs of the non-DMSP-metabolizing
P. aeruginosa
PAO1 and
B. thailandensis
E264 were not inhibited by DMSP, suggesting that DMSP inhibition is a specific adaptation of DmdBs from marine bacteria.
Salinity-Dependent Impacts of ProQ, Prc, and Spr Deficiencies on Escherichia coli Cell Structure
Authors: Kerr Craig H. , Culham Doreen E. , Marom David , Wood Janet M.
Published: January 2014

ProQ is a cytoplasmic protein with RNA chaperone activities that reside in FinO- and Hfq-like domains. Lesions at
proQ
decrease the level of the osmoregulatory glycine betaine transporter ProP. Lesions at
proQ
eliminated ProQ and Prc, the periplasmic protease encoded by the downstream gene
prc
. They dramatically slowed the growth of
Escherichia coli
populations and altered the morphologies of
E. coli
cells in high-salinity medium. ProQ and Prc deficiencies were associated with different phenotypes. ProQ-deficient bacteria were elongated unless glycine betaine was provided. High-salinity cultures of Prc-deficient bacteria included spherical cells with an enlarged periplasm and an eccentric nucleoid. The nucleoid-containing compartment was bounded by the cytoplasmic membrane and peptidoglycan. This phenotype was not evident in bacteria cultivated at low or moderate salinity, nor was it associated with murein lipoprotein (Lpp) deficiency, and it differed from those elicited by the MreB inhibitor A-22 or the FtsI inhibitor aztreonam at low or high salinity. It was suppressed by deletion of
spr
, which encodes one of three murein hydrolases that are redundantly essential for enlargement of the murein sacculus. Prc deficiency may alter bacterial morphology by impairing control of Spr activity at high salinity. ProQ and Prc deficiencies lowered the ProP activity of bacteria cultivated at moderate salinity by approximately 70% and 30%, respectively, but did not affect other osmoregulatory functions. The effects of ProQ and Prc deficiencies on ProP activity are indirect, reflecting their roles in the maintenance of cell structure.