bluuebunny/crossref_metadata_2025 huggingface dataset:
Forage chain arrangement for sustainable livestock systems in a Mediterranean area
Authors: Cosentino S. L. , Gresta F. , Testa G.
Published: October 2013

Mediterranean forage systems suffer from limited availability of fresh forage because of water deficits and extreme temperatures. Consequently, fresh forage is unavailable for at least 6–7 months a year, and farmers must buy feed to support livestock production. With the aim of overcoming these limitations, a 2‐year trial was conducted on three distinct sites in Sicily (at 10, 600 and 1200 m elevation) with thirty‐four varieties of forage species belonging to nine biennial/perennial and thirteen annual species. Results showed that by integrating grasses and legumes, species from environments with different climatic conditions enable the season of forage production to be extended from mid‐April to mid‐November. Quality traits of forage in different areas varied in relation to species and varieties. In general, the sown‐forage quality was better than in pastures and fallows in the same areas commonly used to feed animals. This also leads to a reduction in the use of supplementary feeds. Among the tested species, Lolium multiflorum and Medicago sativa emerged as the most promising for filling the forage‐deficit periods, and Trifolium spp. and Vicia sativa were found to be superior for increasing forage quality. The results are discussed in the context of adapting Mediterranean forage supplies for ensuring greater sustainability of livestock production in mountain, hill and plain areas. The proposed forage chain arrangement represents part of local potential adaptation to climate limitations and climate change.
The Interaction Between Nuclear Waste Glass and Ordinary Portland Cement
Authors: Ferrand Karine , Liu Sanheng , Lemmens Karel
Published: October 2013

The interaction between simulated reference waste glasses SON68 and SM539, and cement has been studied in suspensions of ordinary Portland cement and synthetic cement water with pH 13.5 at 30°C. The cement appears to trigger glass dissolution by consumption of glass matrix components. This leads to fast glass dissolution at a constant rate with formation of a porous gel layer on the glass. A glass dissolution model has been proposed considering that the silicon coming from the glass reacts with portlandite to form C‐S‐H phases. The transformation of C‐S‐H into C‐A‐S‐H phases is a second parallel driving force especially for the Al‐rich SM539 glass. After consumption of the portlandite, the glass alteration rate is expected to decrease.
New Insight into the Residual Rate of Borosilicate Glasses: Effect of S/V and Glass Composition
Authors: Gin Stéphane , Frugier Pierre , Jollivet Patrick , Bruguier Florence , Curti Enzo
Published: October 2013

We report and discuss initial and residual rate measurements performed on 6 borosilicate glasses to highlight (i) some glass composition effects (namely Zn and Fe), (ii) the role of dissolved silica, and (iii) the importance of the glass‐surface‐area‐to‐solution‐volume (S/V) ratio. We show that there is no apparent relation between the initial and the residual rate. As an example, the well‐known positive effect of Zn on short‐term glass dissolution becomes adverse under residual rate conditions. Dissolved silica as well as S/V strongly impact the transient rate drop regime, but have no or minor effects on the magnitude of the residual rate of the SON68 glass. Increasing S/V is indeed a good mean to explore higher reaction progresses. Additional simulations performed with the GRAAL model show that, in the case of SON68 glass altered in deionized water, the residual rate continuously diminishes despite the formation of secondary crystalline phases. This result can be used to derive conservative rate values. More generally, it is shown that the residual rate can fluctuate depending on the relative importance of two coupled mechanisms: the formation of a passivating layer and the transformation of this layer into more stable compounds.
Genetics and Morphology Characterize the Dinoflagellate Symbiodinium voratum, n. sp., (Dinophyceae) as the Sole Representative of Symbiodinium Clade E
Authors: Jeong Hae Jin , Lee Sung Yeon , Kang Nam Seon , Yoo Yeong Du , Lim An Suk , Lee Moo Joon , Kim Hyung Seop , Yih Wonho , Yamashita Hiroshi , LaJeunesse Todd C.
Published: October 2013

Dinoflagellates in the genus Symbiodinium are ubiquitous in shallow marine habitats where they commonly exist in symbiosis with cnidarians. Attempts to culture them often retrieve isolates that may not be symbiotic, but instead exist as free‐living species. In particular, cultures of Symbiodinium clade E obtained from temperate environments were recently shown to feed phagotrophically on bacteria and microalgae. Genetic, behavioral, and morphological evidence indicate that strains of clade E obtained from the northwestern, southwestern, and northeastern temperate Pacific Ocean as well as the Mediterranean Sea constitute a single species: Symbiodinium voratum n. sp. Chloroplast ribosomal 23S and mitochondrial cytochrome b nucleotide sequences were the same for all isolates. The D1/D2 domains of nuclear ribosomal DNA were identical among Western Pacific strains, but single nucleotide substitutions differentiated isolates from California (USA) and Spain. Phylogenetic analyses demonstrated that S. voratum is well‐separated evolutionarily from other Symbiodinium spp. The motile, or mastigote, cells from different cultures were morphologically similar when observed using light, scanning, and transmission electron microscopy; and the first complete Kofoidian plate formula for a Symbiodinium sp. was characterized. As the largest of known Symbiodinium spp., the average coccoid cell diameters measured among cultured isolates ranged between 12.2 (± 0.2 SE) and 13.3 (± 0.2 SE) μm. Unique among species in the genus, a high proportion (approximately 10–20%) of cells remain motile in culture during the dark cycle. Although S. voratum occurs on surfaces of various substrates and is potentially common in the plankton of coastal areas, it may be incapable of forming stable mutualistic symbioses.
Expression of astrocytic genes coding for proteins implicated in neural excitation and brain edema is altered after acute liver failure
Authors: Thumburu Kiran K. , Dhiman Radha K. , Vasishta Rakesh K. , Chakraborti Anuradha , Butterworth Roger F. , Beauchesne Elizabeth , Desjardins Paul , Goyal Sandeep , Sharma Navneet , Duseja Ajay , Chawla Yogesh
Published: October 2013

In vitro and in vivo studies have suggested that reduced astrocytic uptake of neuronally released glutamate, alterations in expression of glial fibrillary acidic protein (GFAP) and aquaporin‐4 (AQP‐4) contribute to brain edema in acute liver failure (ALF). However, there is no evidence to date to suggest that these alterations occur in patients with ALF. We analyzed the mRNA expression of excitatory amino acid transporters (EAAT‐1, EAAT‐2), GFAP, and AQP‐4 in the cerebral cortex obtained at autopsy from eight patients with ALF and from seven patients with no evidence of hepatic or neurological disorders by real‐time PCR, and protein expression was assessed using immunoblotting and immunohistochemistry. We demonstrated a significant decrease in GFAP mRNA and protein levels in ALF patients compared to controls. While the loss of EAAT‐2 protein in ALF samples was post‐translational in nature, EAAT‐1 protein remained within normal limits. Immunohistochemistry confirmed that, in all cases, the losses of EAAT‐2 and GFAP were uniquely astrocytic in their localization. AQP‐4 mRNA expression was significantly increased and its immunohistochemistry demonstrated increased AQP‐4 immunoreactivity in the glial end‐feet process surrounding the microvessels. These findings provide evidence of selective alterations in the expression of genes coding for key astrocytic proteins implicated in central nervous system (CNS) excitability and brain edema in human ALF.

image

We investigated the gene expression of astrocytic proteins involved in astrocyte swelling causing brain edema in autopsied brain tissues of patients with acute liver failure. This study demonstrated loss of GFAP expression and up‐regulation of AQP‐4 protein expression leading to cerebral edema, and loss of EAAT‐2 expression implicated in excitatory neurotransmission. These findings may provide new drug targets against CNS complications of acute liver failure.
An endurance‐enhancing effect of peanut meal protein hydrolysate in mice: possible involvement of a specific peanut peptide
Authors: Liu J. , Chen L. W. , Ji K. M. , Yu L. , Zhang Z. J.
Published: October 2013

To improve the functional properties of peanut meal protein for wide utilization, hydrolysis was conducted by alcalase. Compared with saline and peanut meal protein, intragastric administration of low molecular weight (<1 kD) peanut meal peptide (PPH I) could significantly prolong swimming time, increase levels of blood sugar, non‐esterified fatty acids (NEFA) and liver glycogen and decrease blood lactate content in mice. Levels of Pro, Leu, Val and His in low molecular weight peanut meal peptides were higher significantly than those in other peanut meal protein hydrolysates. Hydrophobic amino acids, such as Pro, Tyr and His, could perhaps capture free radical and increase antioxidant capacity of peanut peptide and retard fatigue induced by free radical. After separation by HPLC, a primary peptide P1, Pro‐Glu‐Ile‐Glu‐Val, was sequenced. Its N‐terminal was Val, and it was rich in antioxidant amino acid, Pro and Ile. Levels of plasma glucose, NEFA and liver glycogen in PPH I group were higher than those in mice intragastric administration with peptide P1, and the swimming time is longer in PPH I group than in P1 group. So, the high content of P1 was one of the reason why PPH I had high endurance‐enhancing capacity.
Therapeutic strategies to attenuate hemorrhagic transformation after tissue plasminogen activator treatment for acute ischemic stroke
Authors: Shimohata Takayoshi , Kanazawa Masato , Kawamura Kunio , Takahashi Tetsuya , Nishizawa Masatoyo
Published: October 2013

Here, we describe a therapeutic strategy for attenuating hemorrhagic transformation (HT) after tissue plasminogen activator (tPA) treatment for acute ischemic stroke. Recent studies have shown that tPA treatment is beneficial within 4.5 h of onset for patients with acute ischemic stroke. However, the risk of serious or fatal symptomatic hemorrhage increases with delayed initiation of treatment. HT is considered to be caused by ischemic/reperfusion injury, as well as the toxicity of tPA itself. Therapeutic strategies to attenuate HT after tPA treatment might involve (i) identification of risk factors for HT after tPA treatment and (ii) the development of thrombolytic drugs, which are less likely to cause bleeding, or drugs that can be concomitantly administered for vascular protection. Several studies have shown that matrix metalloproteinases and free radicals are potential therapeutic targets. In addition, we recently showed that inhibition of the vascular endothelial growth factor (VEGF) signaling pathway might be a promising therapeutic strategy for attenuating HT after tPA treatment. Further studies are required to link the results obtained in experimental animal models to human clinical trials.
Wheat syntenome unveils new evidences of contrasted evolutionary plasticity between paleo‐ and neoduplicated subgenomes
Authors: Pont Caroline , Murat Florent , Guizard Sébastien , Flores Raphael , Foucrier Séverine , Bidet Yannick , Quraishi Umar Masood , Alaux Michael , Doležel Jaroslav , Fahima Tzion , Budak Hikmet , Keller Beat , Salvi Silvio , Maccaferri Marco , Steinbach Delphine , Feuillet Catherine , Quesneville Hadi , Salse Jérôme
Published: October 2013

Bread wheat derives from a grass ancestor structured in seven protochromosomes followed by a paleotetraploidization to reach a 12 chromosomes intermediate and a neohexaploidization (involving subgenomes A, B and D) event that finally shaped the 21 modern chromosomes. Insights into wheat syntenome in sequencing conserved orthologous set (COS) genes unravelled differences in genomic structure (such as gene conservation and diversity) and genetical landscape (such as recombination pattern) between ancestral as well as recent duplicated blocks. Contrasted evolutionary plasticity is observed where the B subgenome appears more sensitive (i.e. plastic) in contrast to A as dominant (i.e. stable) in response to the neotetraploidization and D subgenome as supra‐dominant (i.e. pivotal) in response to the neohexaploidization event. Finally, the wheat syntenome, delivered through a public web interface PlantSyntenyViewer at http://urgi.versailles.inra.fr/synteny-wheat, can be considered as a guide for accelerated dissection of major agronomical traits in wheat.
Precise marker excision system using an animal‐derived piggyBac transposon in plants
Authors: Nishizawa‐Yokoi Ayako , Endo Masaki , Osakabe Keishi , Saika Hiroaki , Toki Seiichi
Published: October 2013

Accurate and effective positive marker excision is indispensable for the introduction of desired mutations into the plant genome via gene targeting (GT) using a positive/negative counter selection system. In mammals, the moth‐derived piggyBac transposon system has been exploited successfully to eliminate a selectable marker from a GT locus without leaving a footprint. Here, we present evidence that the piggyBac transposon also functions in plant cells. To demonstrate the use of the piggyBac transposon for effective marker excision in plants, we designed a transposition assay system that allows the piggyBac transposition to be visualized as emerald luciferase (Eluc) luminescence in rice cells. The Eluc signal derived from piggyBac excision was observed in hyperactive piggyBac transposase‐expressing rice calli. Polymerase chain reaction, Southern blot analyses and sequencing revealed the efficient and precise transposition of piggyBac in these calli. Furthermore, we have demonstrated the excision of a selection marker from a reporter locus in T0 plants without concomitant re‐integration of the transposon and at a high frequency (44.0% of excision events), even in the absence of negative selection.
Differential effect of sympathetic activation on tissue oxygenation in gastrocnemius and soleus muscles during exercise in humans
Authors: Horiuchi Masahiro , Fadel Paul J. , Ogoh Shigehiko
Published: October 2013

New Findings

What is the central question of this study?

The normal ability of sympathetic nerves to cause vasoconstriction is blunted in exercising skeletal muscle, a phenomenon termed ‘functional sympatholysis’. Animal studies suggest that functional sympatholysis appears to occur preferentially in fast‐twitch type II glycolytic compared with slow‐twitch type I oxidative skeletal muscle. We asked whether these findings can be extended to humans.

What is the main finding and its importance?

We show that skeletal muscles composed largely of fast‐twitch type II fibres may also be more sensitive to functional sympatholysis in humans, particularly at lower exercise intensities. Additionally, independent of muscle fibre type composition, the magnitude of sympatholysis is strongly related to exercise‐induced increases in metabolic demand.

Animal studies suggest that functional sympatholysis appears to occur preferentially in glycolytic (largely type II) compared with oxidative (largely type I) skeletal muscle. Whether these findings can be extended to humans currently remains unclear. In 12 healthy male subjects, vasoconstrictor responses in gastrocnemius (i.e. primarily type II) and soleus muscles (i.e. primarily type I) were measured using near‐infrared spectroscopy to detect decreases in muscle oxygenation (HbO2) in response to sympathetic activation evoked by a cold pressor test (CPT). The HbO2 responses to a CPT at rest were compared with responses during steady‐state plantar flexion exercise (30 repetitions min−1) performed at 10, 20 and 40% maximal voluntary contraction (MVC) for 6 min. In resting conditions, HbO2 at the gastrocnemius (−14 ± 1%) and soleus muscles (−16 ± 1%) decreased significantly during CPT, with no differences between muscles. During planter flexion at 20% MVC, the change in HbO2 in response to the CPT was blunted in gastrocnemius but not soleus, whereas during 40% MVC both muscles exhibited a significant attenuation to sympathetic activation. The decreases in HbO2 in response to the CPT during exercise were significantly correlated with the metabolic demands of exercise (the decreases in HbO2 in response to steady‐state plantar flexion) in both gastrocnemius and soleus muscles. Collectively, these results suggest that skeletal muscles composed mainly of glycolytic type II fibres are more sensitive to functional sympatholysis, particularly at lower intensities of exercise. Moreover, the blunting of sympathetic vasoconstriction during exercise is strongly related to metabolic demand; an effect that appears independent of fibre type composition.
Influence of locomotor muscle afferent inhibition on the ventilatory response to exercise in heart failure
Authors: Olson Thomas P. , Joyner Michael J. , Eisenach John H. , Curry Timothy B. , Johnson Bruce D.
Published: October 2013

New Findings

What is the central question of this study?

Patients with heart failure often develop ventilatory abnormalities at rest and during exercise, but the mechanisms underlying these abnormalities remain unclear. This study investigated the influence of inhibiting afferent neural feedback from locomotor muscles on the ventilatory response during exercise in heart failure patients.

What is the main finding and its importance?

Our results suggest that inhibiting afferent feedback from locomotor muscle via intrathecal opioid administration significantly reduces the ventilatory response to exercise in heart failure patients.

Patients with heart failure (HF) develop ventilatory abnormalities at rest and during exercise, but the mechanism(s) underlying these abnormalities remain unclear. We examined whether the inhibition of afferent neural feedback from locomotor muscles during exercise reduces exercise ventilation in HF patients. In a randomized, placebo‐controlled design, nine HF patients (age, 60 ± 2 years; ejection fraction, 27 ± 2%; New York Heart Association class 2 ± 1) and nine control subjects (age, 63 ± 2 years) underwent constant‐work submaximal cycling (65% peak power) with intrathecal fentanyl (impairing the cephalad projection of opioid receptor‐sensitive afferents) or sham injection. The hypercapnic ventilatory response was measured to determine whether cephalad migration of fentanyl occurred. There were no differences in hypercapnic ventilatory response within or between groups in either condition. Despite a lack of change in ventilation, tidal volume or respiratory rate, HF patients had a mild increase in arterial carbon dioxide () and a decrease in oxygen (; P < 0.05 for both) at rest. The control subjects demonstrated no change in , , ventilation, tidal volume or respiratory rate at rest. In response to fentanyl during exercise, HF patients had a reduction in ventilation (63 ± 6 versus 44 ± 3 l min−1, P < 0.05) due to a lower respiratory rate (30 ± 1 versus 26 ± 2 breaths min−1, P < 0.05). The reduced ventilation resulted in lower (97.6 ± 2.5 versus 79.5 ± 3.0 mmHg, P < 0.05) and increased (37.3 ± 0.9 versus 43.5 ± 1.1 mmHg, P < 0.05), with significant improvement in ventilatory efficiency (reduction in the ventilatory equivalent for carbon dioxide; P < 0.05 for all). The control subjects had no change in ventilation or measures of arterial blood gases. These data suggest that inhibition of afferent feedback from locomotor muscle significantly reduces the ventilatory response to exercise in HF patients.
Mother's exercise during pregnancy programmes vasomotor function in adult offspring
Authors: Bahls Martin , Sheldon Ryan D. , Taheripour Pardis , Clifford Kerry A. , Foust Kallie B. , Breslin Emily D. , Marchant‐Forde Jeremy N. , Cabot Ryan A. , Harold Laughlin M. , Bidwell Christopher A. , Newcomer Sean C.
Published: October 2013

New Findings

What is the central question of this study?

Adverse maternal behaviour during pregnancy is well known to have lifelong health consequences for offspring. We asked whether a positive maternal health behaviour may have positive long‐term effects for offspring. Specifically, we investigated whether aerobic maternal exercise improves nitric oxide signalling in femoral arteries of adult swine.

What is the main finding and its importance?

For the first time, a fetal programming outcome due to gestational exercise is reported in adult offspring. Second, contrary to previous speculation regarding the impact of in utero alterations on nitric oxide signalling, our data suggest differential myosin phosphorylation in vascular smooth muscle.

The intrauterine environment is influenced by maternal behaviour and programmes atherosclerotic disease susceptibility in offspring. The aim of this investigation was to test the hypothesis that mothers’ exercise during pregnancy improves endothelial function in 3‐, 5‐ and 9‐month‐old porcine offspring. The pregnant sows in the exercise group ran for an average of 39.35 ± 0.75 min at 4.81 ± 0.35 km h−1 each day for 5 days per week for all but the last week of gestation. This induced a significant reduction in resting heart rate (exercised group, 89.3 ± 3.5 beats min−1; sedentary group, 102.1 ± 3.1 beats min−1; P < 0.05) but no significant differences in gestational weight gain (65.8 ± 2.1 versus 63.3 ± 1.9%). No significant effect on bradykinin‐induced vasorelaxation with and without l‐NAME was observed. A significant main effect was identified on sodium nitroprusside‐induced vasorelaxation (P= 0.01), manifested by a reduced response in femoral arteries of all age groups from exercised‐trained swine. Nitric oxide signalling was not affected by maternal exercise. Protein expression of MYPT1 was reduced in femoral arteries from 3‐month‐old offspring of exercised animals. A significant interaction was observed for PPP1R14A (P < 0.05) transcript abundance and its protein product CPI‐17. In conclusion, pregnant swine are able to complete an exercise‐training protocol that matches the current recommendations for pregnant women. Gestational exercise is a potent stimulus for programming vascular smooth muscle relaxation in adult offspring. Specifically, exercise training for the finite duration of pregnancy decreases vascular smooth muscle responsiveness in adult offspring to an exogenous nitric oxide donor.
Numerical Simulation of GaN Growth in a Metalorganic Chemical Vapor Deposition Process
Authors: Meng Jiandong , Jaluria Yogesh
Published: October 2013

A detailed mathematical model for the growth of gallium nitride in a vertical impinging metalorganic chemical vapor deposition (MOCVD) reactor is developed first, and the complete chemical mechanisms are introduced. Then, one validation study is conducted to ensure its accuracy. After that, the flow, temperature and concentration profiles are predicted by numerical modeling. The dependence of the growth rate and uniformity of the deposited layers on operating conditions, such as reactor operating pressure, susceptor temperature, inlet velocity and concentration ratio of the precursors, is investigated to gain greater insight into the reactor performance and characteristics. Based on the simulation results, discussion is presented in this paper to offer the possibility of better control of the GaN film growth process and to ultimately lead to an optimization of the process, with respect to production rate and film quality.
Grinding Wheel Loading Evaluation Using Digital Image Processing
Authors: Adibi Hamed , Rezaei S. M. , Sarhan Ahmed A. D.
Published: October 2013

Wheel loading entails chip accumulation in porosities between grains or welding to the top of cutting grains. It is considered one of the most prevalent problems in grinding Nickel-based super alloys. Identification of wheel loading is an important issue for optimizing the dressing intervals, but it can be a time consuming and an expensive process. A novel technique based on digital image processing to determine the loading areas over the surface of CBN vitrified grinding wheels using the toolbox of MATLAB is presented in this paper. The optical characteristics of the metal chips, the abrasive grains and wheel bond are considered. Experiments were performed to examine the repeatability of the proposed technique. The results were verified by the use of a scanning electron microscope. Based on the proposed technique, the effects of cutting parameters on the loaded area to wheel surface ratio in relation to grinding performance were studied empirically.
Selman A. Waksman, Winner of the 1952 Nobel Prize for Physiology or Medicine
Authors: Woodruff H. Boyd
Published: October 2013

The history of the discovery and development of streptomycin is reviewed here from the personal standpoint of a member of Dr. Selman Waksman's antibiotic screening research team. The team approach of eight individuals illustrates how the gradual enhancement of the screening methodology was developed. I illustrate three study periods with key aspects in the development of streptomycin which led to a Nobel Prize being granted to Professor Waksman. One item not previously emphasized is the employment of a submerged culture technique for large-scale production of streptomycin, thus enabling rapid animal testing and human clinical trials with
Mycobacterium tuberculosis
. Another is that purified streptomycin was shown by Dr. Waksman to be distinctly different from the substances called natural products, which are no longer patentable in the United States; therefore, streptomycin was found to be patentable. A third item not previously emphasized is his emphasis on the screening of actinomycetes, including the newly named
Streptomyces
genus. All of these factors contributed to the success of streptomycin in the treatment of tuberculosis. In combination, their successes led to Dr. Waksman's department becoming a new pharmacological research area, specializing in drug discovery. These unique accomplishments all burnish the prior rationales used by the Karolinska Institute in granting Dr. Waksman alone the 1952 Nobel Prize for Physiology or Medicine.
Primer Modification Improves Rapid and Sensitive
In Vitro
and Field-Deployable Assays for Detection of High Plains Virus Variants
Authors: Arif M. , Aguilar-Moreno G. S. , Wayadande A. , Fletcher J. , Ochoa-Corona F. M.
Published: October 2013

A high consequence pathogen,
High plains virus
(HPV) causes considerable damage to wheat if the crop is infected during early stages of development. Methods for the early, accurate, and sensitive detection of HPV in plant tissues are needed for the management of disease outbreaks and reservoir hosts. In this study, the effectiveness of five methods—real-time SYBR green and TaqMan reverse transcription-quantitative PCR (RT-qPCR), endpoint RT-PCR, RT-helicase dependent amplification (RT-HDA) and the Razor Ex BioDetection System (Razor Ex)—for the broad-range detection of HPV variants was evaluated. Specific PCR primer sets and probes were designed to target the HPV nucleoprotein gene. Primer set HPV6F and HPV4R, which amplifies a product of 96 bp, was validated
in silico
against published sequences and
in vitro
against an inclusivity panel of infected plant samples and an exclusivity panel of near-neighbor viruses. The primers were modified by adding a customized 22 nucleotide long tail at the 5′ terminus, raising the primers' melting temperature (

T
m

; ca. 10°C) to make them compatible with RT-HDA (required optimal

T
m

= 68°C), in which the use of primers lacking such tails gave no amplification. All of the methods allowed the detection of as little as 1 fg of either plasmid DNA carrying the target gene sequence or of infected plant samples. The described
in vitro
and in-field assays are accurate, rapid, sensitive, and useful for pathogen detection and disease diagnosis, microbial quantification, and certification and breeding programs, as well as for biosecurity and microbial forensics applications.
Detection of Extended-Spectrum Beta-Lactamase (ESBL)-Producing Escherichia coli on Flies at Poultry Farms
Authors: Blaak Hetty , Hamidjaja Raditijo A. , van Hoek Angela H. A. M. , de Heer Lianne , de Roda Husman Ana Maria , Schets Franciska M.
Published: October 2013

In the Netherlands, extended-spectrum beta-lactamase (ESBL)-producing
Escherichia coli
bacteria are highly prevalent in poultry, and chicken meat has been implicated as a source of ESBL-producing
E. coli
present in the human population. The current study describes the isolation of ESBL-producing
E. coli
from house flies and blow flies caught at two poultry farms, offering a potential alternative route of transmission of ESBL-producing
E. coli
from poultry to humans. Overall, 87 flies were analyzed in 19 pools. ESBL-producing
E. coli
bacteria were detected in two fly pools (10.5%): a pool of three blow flies from a broiler farm and a pool of eight house flies from a laying-hen farm. From each positive fly pool, six isolates were characterized and compared with isolates obtained from manure (
n
= 53) sampled at both farms and rinse water (
n
= 10) from the broiler farm. Among six fly isolates from the broiler farm, four different types were detected with respect to phylogenetic group, sequence type (ST), and ESBL genotype: A
0
/ST3519/SHV-12, A
1
/ST10/SHV-12, A
1
/ST58/SHV-12, and B1/ST448/CTX-M-1. These types, as well as six additional types, were also present in manure and/or rinse water at the same farm. At the laying-hen farm, all fly and manure isolates were identical, carrying
bla
TEM-52
in an A
1
/ST48 genetic background. The data imply that flies acquire ESBL-producing
E. coli
at poultry farms, warranting further evaluation of the contribution of flies to dissemination of ESBL-producing
E. coli
in the community.
Osmoadaptation Strategy of the Most Halophilic Fungus, Wallemia ichthyophaga, Growing Optimally at Salinities above 15% NaCl
Authors: Zajc Janja , Kogej Tina , Galinski Erwin A. , Ramos José , Gunde-Cimerman Nina
Published: October 2013

Wallemia ichthyophaga
is a fungus from the ancient basidiomycetous genus
Wallemia
(Wallemiales, Wallemiomycetes) that grows only at salinities between 10% (wt/vol) NaCl and saturated NaCl solution. This obligate halophily is unique among fungi. The main goal of this study was to determine the optimal salinity range for growth of the halophilic
W. ichthyophaga
and to unravel its osmoadaptation strategy. Our results showed that growth on solid growth media was extremely slow and resulted in small colonies. On the other hand, in the liquid batch cultures, the specific growth rates of
W. ichthyophaga
were higher, and the biomass production increased with increasing salinities. The optimum salinity range for growth of
W. ichthyophaga
was between 15 and 20% (wt/vol) NaCl. At 10% NaCl, the biomass production and the growth rate were by far the lowest among all tested salinities. Furthermore, the cell wall content in the dry biomass was extremely high at salinities above 10%. Our results also showed that glycerol was the major osmotically regulated solute, since its accumulation increased with salinity and was diminished by hypo-osmotic shock. Besides glycerol, smaller amounts of arabitol and trace amounts of mannitol were also detected. In addition,
W. ichthyophaga
maintained relatively small intracellular amounts of potassium and sodium at constant salinities, but during hyperosmotic shock, the amounts of both cations increased significantly. Given our results and the recent availability of the genome sequence,
W. ichthyophaga
should become well established as a novel model organism for studies of halophily in eukaryotes.
A Distinct Model of Synergism between a Processive Endocellulase (TfCel9A) and an Exocellulase (TfCel48A) from Thermobifida fusca
Authors: Kostylev Maxim , Wilson David
Published: October 2013

Lignocellulosic biomass is digested in nature by the synergistic activities of enzymes with complementary properties, and understanding synergistic interactions will improve the efficiency of industrial biomass use for sustainable fuels and chemicals. Cel9A and Cel48A from a model bacterium,
Thermobifida fusca
(TfCel9A and TfCel48A, respectively), are two cellulases with different properties and have previously been shown to synergize well with each other. TfCel9A is a processive endocellulase with relatively high activity on crystalline cellulose. TfCel48A is a reducing end-directed exocellulase with very low activity on crystalline cellulose. Neither enzyme fits its respective role in the classical synergism model of enzymatic cellulose digestion. Using the results of time course, endpoint, and sequential addition activity assays, we propose a model of synergistic cooperation between the two cellulases. TfCel9A is most effective on fresh bacterial cellulose with a presumably uniform surface at the molecular level. Its processive activity likely erodes the surface and thus reduces its own activity. TfCel48A is able to hydrolyze the TfCel9A-modified substrate efficiently and replenish the uniform surface required by TfCel9A, creating a feedback mechanism. The model of synergistic interactions is comparable to an earlier proposed model for
Trichoderma reesei
Cel7A and Cel7B, but the roles of endo- and exocellulases are reversed, a finding which suggests that bacteria and fungi may have evolved different approaches to efficient biomass degradation.
CRISPRs: Molecular Signatures Used for Pathogen Subtyping
Authors: Shariat Nikki , Dudley Edward G.
Published: October 2013

Rapid and accurate strain identification is paramount in the battle against microbial outbreaks, and several subtyping approaches have been developed. One such method uses
c
lustered
r
egular
i
nterspaced
s
hort
p
alindromic
r
epeats (CRISPRs), DNA repeat elements that are present in approximately half of all bacteria. Though their signature function is as an adaptive immune system against invading DNA such as bacteriophages and plasmids, CRISPRs also provide an excellent framework for pathogen tracking and evolutionary studies. Analysis of the spacer DNA sequences that reside between the repeats has been tremendously useful for bacterial subtyping during molecular epidemiological investigations. Subtyping, or strain identification, using CRISPRs has been employed in diverse Gram-positive and Gram-negative bacteria, including
Mycobacterium tuberculosis
,
Salmonella enterica
, and the plant pathogen
Erwinia amylovora
. This review discusses the several ways in which CRISPR sequences are exploited for subtyping. This includes the well-established spoligotyping methodologies that have been used for 2 decades to type
Mycobacterium
species, as well as in-depth consideration of newer, higher-throughput CRISPR-based protocols.
Critical Assessment of Glyco- and Phospholipid Separation by Using Silica Chromatography
Authors: Heinzelmann Sandra M. , Bale Nicole J. , Hopmans Ellen C. , Sinninghe Damsté Jaap S. , Schouten Stefan , van der Meer Marcel T. J.
Published: October 2013

Phospholipid-derived fatty acids (PLFAs) are commonly used to characterize microbial communities
in situ
and the phylogenetic positions of newly isolated microorganisms. PLFAs are obtained through separation of phospholipids from glycolipids and neutral lipids using silica column chromatography. We evaluated the performance of this separation method for the first time using direct detection of intact polar lipids (IPLs) with high-performance liquid chromatography–mass spectrometry (HPLC-MS). We show that under either standard or modified conditions, the phospholipid fraction contains not only phospholipids but also other lipid classes such as glycolipids, betaine lipids, and sulfoquinovosyldiacylglycerols. Thus, commonly reported PLFA compositions likely are not derived purely from phospholipids and perhaps may not be representative of fatty acids present in living microbes.
Temporal Variability of the Bioaerosol Background at a Subway Station: Concentration Level, Size Distribution, and Diversity of Airborne Bacteria
Authors: Dybwad Marius , Skogan Gunnar , Blatny Janet Martha
Published: October 2013

Naturally occurring bioaerosol environments may present a challenge to biological detection-identification-monitoring (BIODIM) systems aiming at rapid and reliable warning of bioterrorism incidents. One way to improve the operational performance of BIODIM systems is to increase our understanding of relevant bioaerosol backgrounds. Subway stations are enclosed public environments which may be regarded as potential bioterrorism targets. This study provides novel information concerning the temporal variability of the concentration level, size distribution, and diversity of airborne bacteria in a Norwegian subway station. Three different air samplers were used during a 72-h sampling campaign in February 2011. The results suggested that the airborne bacterial environment was stable between days and seasons, while the intraday variability was found to be substantial, although often following a consistent diurnal pattern. The bacterial levels ranged from not detected to 10
3
CFU m
−3
and generally showed increased levels during the daytime compared to the nighttime levels, as well as during rush hours compared to non-rush hours. The airborne bacterial levels showed rapid temporal variation (up to 270-fold) on some occasions, both consistent and inconsistent with the diurnal profile. Airborne bacterium-containing particles were distributed between different sizes for particles of >1.1 μm, although ∼50% were between 1.1 and 3.3 μm. Anthropogenic activities (mainly passengers) were demonstrated as major sources of airborne bacteria and predominantly contributed 1.1- to 3.3-μm bacterium-containing particles. Our findings contribute to the development of realistic testing and evaluation schemes for BIODIM equipment by providing information that may be used to simulate operational bioaerosol backgrounds during controlled aerosol chamber-based challenge tests with biological threat agents.
Predominant Acidilobus-Like Populations from Geothermal Environments in Yellowstone National Park Exhibit Similar Metabolic Potential in Different Hypoxic Microbial Communities
Authors: Jay Z. J. , Rusch D. B. , Tringe S. G. , Bailey C. , Jennings R. M. , Inskeep W. P.
Published: October 2013

High-temperature (>70°C) ecosystems in Yellowstone National Park (YNP) provide an unparalleled opportunity to study chemotrophic archaea and their role in microbial community structure and function under highly constrained geochemical conditions.
Acidilobus
spp. (order
Desulfurococcales
) comprise one of the dominant phylotypes in hypoxic geothermal sulfur sediment and Fe(III)-oxide environments along with members of the
Thermoproteales
and
Sulfolobales
. Consequently, the primary goals of the current study were to analyze and compare replicate
de novo
sequence assemblies of
Acidilobus
-like populations from four different mildly acidic (pH 3.3 to 6.1) high-temperature (72°C to 82°C) environments and to identify metabolic pathways and/or protein-encoding genes that provide a detailed foundation of the potential functional role of these populations
in situ. De novo
assemblies of the highly similar
Acidilobus
-like populations (>99% 16S rRNA gene identity) represent near-complete consensus genomes based on an inventory of single-copy genes, deduced metabolic potential, and assembly statistics generated across sites. Functional analysis of coding sequences and confirmation of gene transcription by
Acidilobus
-like populations provide evidence that they are primarily chemoorganoheterotrophs, generating acetyl coenzyme A (acetyl-CoA) via the degradation of carbohydrates, lipids, and proteins, and auxotrophic with respect to several external vitamins, cofactors, and metabolites. No obvious pathways or protein-encoding genes responsible for the dissimilatory reduction of sulfur were identified. The presence of a formate dehydrogenase (Fdh) and other protein-encoding genes involved in mixed-acid fermentation supports the hypothesis that
Acidilobus
spp. function as degraders of complex organic constituents in high-temperature, mildly acidic, hypoxic geothermal systems.
Monitoring Rates and Heterogeneity of High-Pressure Germination of Bacillus Spores by Phase-Contrast Microscopy of Individual Spores
Authors: Kong Lingbo , Doona Christopher J. , Setlow Peter , Li Yong-qing
Published: October 2013

Germination of
Bacillus
spores with a high pressure (HP) of ∼150 MPa is via activation of spores' germinant receptors (GRs). The HP germination of multiple individual
Bacillus subtilis
spores in a diamond anvil cell (DAC) was monitored with phase-contrast microscopy. Major conclusions were that (i) >95% of wild-type spores germinated in 40 min in a DAC at ∼150 MPa and 37°C but individual spores' germination kinetics were heterogeneous; (ii) individual spores' HP germination kinetic parameters were similar to those of nutrient-triggered germination with a variable lag time (
T
lag
) prior to a period of the rapid release (Δ
T
release
) of the spores' dipicolinic acid in a 1:1 chelate with Ca
2+
(CaDPA); (iii) spore germination at 50 MPa had longer average
T
lag
values than that at ∼150 MPa, but the Δ
T
release
values at the two pressures were identical and HPs of <10 MPa did not induce germination; (iv)
B. subtilis
spores that lacked the cortex-lytic enzyme CwlJ and that were germinated with an HP of 150 MPa exhibited average Δ
T
release
values ∼15-fold longer than those for wild-type spores, but the two types of spores exhibited similar average
T
lag
values; and (v) the germination of wild-type spores given a ≥30-s 140-MPa HP pulse followed by a constant pressure of 1 MPa was the same as that of spores exposed to a constant pressure of 140 MPa that was continued for ≥35 min; (vi) however, after short 150-MPa HP pulses and incubation at 0.1 MPa (ambient pressure), spore germination stopped 5 to 10 min after the HP was released. These results suggest that an HP of ∼150 MPa for ≤30 s is sufficient to fully activate spores' GRs, which remain activated at 1 MPa but can deactivate at ambient pressure.
Glycine Betaine as a Direct Substrate for Methanogens (Methanococcoides spp.)
Authors: Watkins Andrew J. , Roussel Erwan G. , Parkes R. John , Sass Henrik
Published: October 2013

Nine marine methanogenic
Methanococcoides
strains, including the type strains of
Methanococcoides methylutens
,
M. burtonii
, and
M. alaskense
, were tested for the utilization of
N
-methylated glycines. Three strains (NM1, PM2, and MKM1) used glycine betaine (
N
,
N
,
N
-trimethylglycine) as a substrate for methanogenesis, partially demethylating it to
N
,
N
-dimethylglycine, whereas none of the strains used
N
,
N
-dimethylglycine or sarcosine (
N
-methylglycine). Growth rates and growth yields per mole of substrate with glycine betaine (3.96 g [dry weight] per mol) were similar to those with trimethylamine (4.11 g [dry weight] per mol). However, as glycine betaine is only partially demethylated, the yield per methyl group was significantly higher than with trimethylamine. If glycine betaine and trimethylamine are provided together, trimethylamine is demethylated to dimethyl- and methylamine with limited glycine betaine utilization. After trimethylamine is depleted, dimethylamine and glycine betaine are consumed rapidly, before methylamine. Glycine betaine extends the range of substrates that can be directly utilized by some methanogens, allowing them to gain energy from the substrate without the need for syntrophic partners.
The Cold-Induced Two-Component System CBO0366/CBO0365 Regulates Metabolic Pathways with Novel Roles in Group I Clostridium botulinum ATCC 3502 Cold Tolerance
Authors: Dahlsten Elias , Zhang Zhen , Somervuo Panu , Minton Nigel P. , Lindström Miia , Korkeala Hannu
Published: October 2013

The two-component system CBO0366/CBO0365 was recently demonstrated to have a role in cold tolerance of group IClostridium botulinumATCC 3502. The mechanisms under its control, ultimately resulting in increased sensitivity to low temperature, are unknown. A transcriptomic analysis with DNA microarrays was performed to identify the differences in global gene expression patterns of the wild-type ATCC 3502 and a derivative mutant with insertionally inactivatedcbo0365at 37 and 15°C. Altogether, 150 or 141 chromosomal coding sequences (CDSs) were found to be differently expressed in thecbo0365mutant at 37 or 15°C, respectively, and thus considered to be under the direct or indirect transcriptional control of the response regulator CBO0365. Of the differentially expressed CDSs, expression of 141 CDSs was similarly affected at both temperatures investigated, suggesting that the putative CBO0365 regulon was practically not affected by temperature. The regulon involved genes related to acetone-butanol-ethanol (ABE) fermentation, motility, arsenic resistance, and phosphate uptake and transport. Deteriorated growth at 17°C was observed for mutants with disrupted ABE fermentation pathway components (crt,bcd,bdh, andctfA), arsenic detoxifying machinery components (arsCandarsR), or phosphate uptake mechanism components (phoT), suggesting roles for these mechanisms in cold tolerance of group IC. botulinum. Electrophoretic mobility shift assays showed recombinant CBO0365 to bind to the promoter regions ofcrt,arsR, andphoT, as well as to the promoter region of its own operon, suggesting direct DNA-binding transcriptional activation or repression as a means for CBO0365 in regulating these operons. The results provide insight to the mechanisms group IC. botulinumutilizes in coping with cold.
Effects of Electroacupuncture on Bone Mineral Density, Oestradiol Level and Osteoprotegerin Ligand Expression in Ovariectomised Rabbits
Authors: He Jing , Yang Lin , Qing Yuxi , He Chengqi
Published: October 2013

Objectives

To investigate the effects of electroacupuncture (EA) on the oestradiol level, bone mineral density and osteoprotegerin ligand (OPGL) expression, and to explore whether EA might be a complementary method to prevent and treat osteoporosis.

Methods

A total of 21 New Zealand rabbits were randomly divided into three groups: a normal control (NC) group undergoing no surgery or EA; an ovariectomised (OVX) group, in which rabbits were ovariectomised but did not receive EA; an EA group, in which rabbits were ovariectomised and treated with EA. Acupuncture was applied at ST35, BL20 and BL23 points bilaterally. EA (10 Hz, 2 mA) was applied bilaterally at BL20 and BL23 for 30 min a day for 14 days. After 14 days, all animals were killed. OPGL expression level was determined by immunohistochemistry. Blood serum levels of oestradiol were measured by ELISA and bone mineral density was detected by dual-energy x-ray absorptiometry.

Results

After ovariectomy, the bone mineral density and oestradiol level decreased significantly in the OVX group compared with the NC group (p=0.001), whereas the OPGL expression level increased. After EA, the bone mineral density and oestradiol level increased compared with the OVX group (p=0.049 and p=0.012, respectively). The OPGL level OPGL level in the EA group was lower than that in the OVX group (p=0.022).

Conclusions

EA restored bone mineral density towards normal and was associated with increased plasma oestradiol level and reduced OPGL expression in an ovariectomised rabbit model of osteoporosis.