bluuebunny/crossref_metadata_2025 huggingface dataset:
Biomarkers: refining diagnosis and expediting drug development – reality, aspiration and the role of open innovation
Authors: Salter H. , Holland R.
Published: March 2014

In the last decade, there have been intensive efforts to invent, qualify and use novel biomarkers as a means to improve success rates in drug discovery and development. The biomarkers field is maturing and this article considers whether these research efforts have brought about the expected benefits. The characteristics of a clinically useful biomarker are described and the impact this area of research has had is evaluated by reviewing a few, key examples of emerging biomarkers. There is evidence that the impact has been genuine and is increasing in both the drug and the diagnostic discovery and development processes. Beneficial impact on patient health outcomes seems relatively limited thus far, with the greatest impact in oncology (again, both in terms of novel drugs and in terms of more refined diagnoses and therefore more individualized treatment). However, the momentum of research would indicate that patient benefits are likely to increase substantially and to broaden across multiple therapeutic areas. Even though this research was originally driven by a desire to improve the drug discovery and development process, and was therefore funded with this aim in mind, it seems likely that the largest impact may actually come from more refined diagnosis. Refined diagnosis will facilitate both better allocation of healthcare resources and the use of treatment regimens which are optimized for the individual patient. This article also briefly reviews emerging technological approaches and how they relate to the challenges inherent in biomarker discovery and validation, and discusses the role of public/private partnerships in innovative biomarker research.
Fatty acids and sleep in UK children: subjective and pilot objective sleep results from the DOLAB study – a randomized controlled trial
Authors: Montgomery Paul , Burton Jennifer R. , Sewell Richard P. , Spreckelsen Thees F. , Richardson Alexandra J.
Published: March 2014

Sleep problems in children are associated with poor health, behavioural and cognitive problems, as are deficiencies of long‐chain omega‐3 fatty acids such as docosahexaenoic acid. Theory and some evidence support a role for these fatty acids in sleep regulation, but this issue has received little formal investigation. We examined associations between blood fatty acid concentrations (from fingerstick blood samples) and subjective sleep (using an age‐standardized parent questionnaire) in a large epidemiological sample of healthy children aged 7–9 years (n = 395) from mainstream UK schools. In a randomized controlled trial, we then explored whether 16‐week supplementation (600 mg day−1) with algal docosahexaenoic acid versus placebo might improve sleep in a subset of those children (n = 362) who were underperforming in reading. In a randomly selected subsample (n = 43), sleep was also assessed objectively via actigraphy. In 40% of the epidemiological sample, Child Sleep Habits Questionnaire scores indicated clinical‐level sleep problems. Furthermore, poorer total sleep disturbance scores were associated weakly but significantly with lower blood docosahexaenoic acid (std coeff. −0.105*) and a lower docosahexaenoic acid : arachidonic acid ratio (std coeff. −0.119**). The treatment trial showed no significant effects on subjective sleep measures. However, in the small actigraphy subsample, docosahexaenoic acid supplementation led on average to seven fewer wake episodes and 58 min more sleep per night. Cautiously, we conclude that higher blood levels of docosahexaenoic acid may relate to better child sleep, as rated by parents. Exploratory pilot objective evidence from actigraphy suggests that docosahexaenoic acid supplementation may improve children's sleep, but further investigations are needed.
Control of hypoglossal motoneurones during naturally occurring sleep and wakefulness in the intact, unanaesthetized cat: a field potential study
Authors: Fung Simon J. , Chase Michael H.
Published: March 2014

The present electrophysiological study was designed to determine the discharge threshold of hypoglossal motoneurones during naturally occurring states of sleep and wakefulness in the intact, unanaesthetized cat. The antidromic field potential, which reflects the net level of membrane excitability of motoneurones and therefore their discharge threshold, was recorded in the hypoglossal nucleus following stimulation of the hypoglossal nerve. The amplitude of the antidromic field potential was larger during wakefulness and non‐rapid eye movement (NREM) sleep compared with REM sleep. There was no significant difference in the amplitude of the field potential when wakefulness was compared with NREM sleep (P = 0.103, df = 3, t = 2.324). However, there was a 46% reduction in amplitude during REM sleep compared with NREM sleep (P < 0.001, df = 10, t = 6.421) or wakefulness (P < 0.01, df = 4, t = −4.598). These findings indicate that whereas the excitability of motoneurones that comprise the hypoglossal motor pool is relatively constant during wakefulness and NREM sleep, their excitability is significantly reduced during REM sleep. This state‐dependent pattern of control of hypoglossal motoneurones during REM sleep is similar to that reported for motoneurones in other motor nuclei at all levels of the neuraxis. The decrease in the evoked response of hypoglossal motoneurones, which reflects a significant increase in the discharge threshold of individual motoneurones, results in atonia of the lingual and related muscles during REM sleep.
A pharmacokinetic/clinical approach to postulate a local action of intra‐articular xylazine administration in the horse: a preliminary study
Authors: Di Salvo A. , Della Rocca G. , Bazzica C. , Giontella A. , Cagnardi P. , Nannarone S.
Published: March 2014

The study aims to evaluate whether the analgesic effect of intra‐articular (IA) route of xylazine administered to horses following arthroscopic surgery is due to a local or a systemic action. Two connected studies were performed. In the first, 1 mg/kg b.w. of xylazine was injected IA, and blood samples were taken to assess drug systemic absorption. In addition, systemic effects of the drug (sedation, ataxia or reduction of respiratory and cardiac rate) were registered. Control horses injected with saline IA were included in the study to exclude the influence of anaesthesia in the occurrence of these manifestations. In the second study, 1 mg/kg b.w. of xylazine was administered intravenously (i.v.) in healthy horses. Blood samples were collected to determine the concentrations of xylazine, and the same signs of systemic effects of the drug were recorded. By correlating these parameters, a systemic ‘no effect’ concentration was defined. Pharmacokinetic data after IA administration resulted in some xylazine absorption (bioavailability equal to 58.12%) with values above the systemic ‘no effect’ concentration. The occurrence of some signs related to systemic effects in horses receiving IA xylazine was significant compared with horses receiving saline. In conclusion, a systemic action of the drug after IA administration cannot be excluded.
A plea for simultaneously considering matrix quality and local environmental conditions when analysing landscape impacts on effective dispersal
Authors: Pflüger Femke J. , Balkenhol Niko
Published: March 2014

Landscape genetics has tremendous potential for enhancing our understanding about landscape effects on effective dispersal and resulting genetic structures. However, the vast majority of landscape genetic studies focus on effects of the landscape among sampling locations on dispersal (i.e. matrix quality), while effects of local environmental conditions are rather neglected. Such local environmental conditions include patch size, habitat type or resource availability and are commonly used in (meta‐) population ecology and population genetics. In our opinion, landscape genetic studies would greatly benefit from simultaneously incorporating both matrix quality and local environmental conditions when assessing landscape effects on effective dispersal. To illustrate this point, we first outline the various ways in which environmental heterogeneity can influence different stages of the dispersal process. We then propose a three‐step approach for assessing local and matrix effects on effective dispersal and review how both types of effects can be considered in landscape genetic analyses. Using simulated data, we show that it is possible to correctly disentangle the relative importance of matrix quality vs. local environmental conditions for effective dispersal. We argue that differentiating local and matrix effects in such a way is crucial for predicting future species distribution and persistence, and for optimal conservation decisions that are based on landscape genetics. In sum, we think it is timely to move beyond purely statistical, pattern‐oriented analyses in landscape genetics and towards process‐oriented approaches that consider the full range of possible landscape effects on dispersal behaviour and resulting gene flow.
Postoperative hypoalbuminemia as a risk factor for surgical site infection after oral cancer surgery
Authors: Lee J‐I , Kwon M , Roh J‐L , Choi JW , Choi S‐H , Nam SY , Kim SY
Published: March 2014

Objectives

Postoperative surgical site infection (SSI) is a frequent postoperative complication in patients with oral cancer and significantly affects patient recovery and medical expenses. The aim of this study was to examine the predictors of SSI in patients undergoing major surgery for oral or oropharyngeal squamous cell carcinoma (OSCC) and to determine the relationship between perioperative albumin and the development of SSI.

Subjects and methods

In 337 consecutive patients who underwent clean‐contaminated surgery for OSCC, serum albumin, glucose, and hemoglobin levels were perioperatively measured. Differences between the groups were examined using Fisher's exact test, Mann–Whitney U‐test, and multiple logistic regression analysis.

Results

Surgical site infection was detected in 88 (26.1%) patients with median time to development of 10 (2–25) days. Multiple logistic regression analysis showed that only postoperative serum albumin < 2.5 g dl−1 was an independent variable predictive of SSI (P = 0.003). The duration of hospital stay was negatively correlated with postoperative albumin (R2 = –0.302, P < 0.001).

Conclusion

Early postoperative hypoalbuminemia <2.5 g dl−1 is an independent risk factor for the development of SSI in patients undergoing oral cancer surgery. Clinicians should be aware of the implications of postoperative hypoalbuminemia and consider more intensive postoperative care in these patients.
Characterization of mesenchymal progenitor cells in crown and root pulp from human mesiodentes
Authors: Sato M , Toriumi T , Watanabe N , Watanabe E , Akita D , Mashimo T , Akiyama Y , Isokawa K , Shirakawa T , Honda MJ
Published: March 2014

Objective

Mesiodentes are usually found in the central position of the upper or lower jaw as supernumerary teeth. Here, we obtained 10 mesiodentes and three permanent teeth (PT) and separated the dental pulp (DP) from these into crown and root portions. We then characterized and compared the isolated crown portion‐derived cells (crown cells) with root portion‐derived cells (root cells) using a range of in vitro assays.

Materials and methods

Crown cells and root cells were examined for cell surface marker expression, colony‐forming unit‐fibroblast (CFU‐F), cell proliferation, cell cycle characteristics and markers, and osteogenic and adipogenic differentiation.

Results

The proportion of CD105‐positive cells (CD105+ cells) in the crown cells vs the root cells varied among the mesiodentes, but not among the PT. When there were more CD105+ cells in the root cells than in the crown cells, the root cells showed higher CFU‐F, proliferation capacity, and osteogenic differentiation capacity. In contrast, when the crown cells contained more CD105+ cells than the root cells, the crown cells showed the higher CFU‐F, proliferation capacity, and osteogenic differentiation capacity. In addition, the sorted CD105+ cells showed higher CFU‐F and proliferation capacity than the sorted CD105− cells.

Conclusion

These results indicated that proportion of CD105+ cells is an effective means of characterizing DP‐derived cells in mesiodentes.
The Arabidopsis U11/U12‐65K is an indispensible component of minor spliceosome and plays a crucial role in U12 intron splicing and plant development
Authors: Jung Hyun Ju , Kang Hunseung
Published: March 2014

The U12‐dependent introns have been identified in a wide range of eukaryotes and are removed from precursor‐mRNAs by U12 intron‐specific minor spliceosome. Although several proteins unique to minor spliceosome have been identified, the nature of their effect on U12 intron splicing as well as plant growth and development remain largely unknown. Here, we characterized the functional role of an U12‐type spliceosomal protein, U11/U12‐65K in Arabidopsis thaliana. The transgenic knockdown plants generated by artificial miRNA‐mediated silencing strategy exhibited severe defect in growth and development, such as severely arrested primary inflorescence stems, serrated leaves, and the formation of many rosette leaves after bolting. RNA sequencing and reverse transcription polymerase chain reaction (RT‐PCR) analyses revealed that splicing of 198 out of the 234 previously predicted U12 intron‐containing genes and 32 previously unidentified U12 introns was impaired in u11/u12‐65k mutant. Moreover, the U11/U12‐65K mutation affected alternative splicing, as well as U12 intron splicing, of many introns. Microarray analysis revealed that the genes involved in cell wall biogenesis and function, plant development, and metabolic processes are differentially expressed in the mutant plants. U11/U12‐65K protein bound specifically to U12 small nuclear RNA (snRNA), which is necessary for branch‐point site recognition. Taken together, these results provide clear evidence that U11/U12‐65K is an indispensible component of minor spliceosome and involved in U12 intron splicing and alternative splicing of many introns, which is crucial for plant development.
Aid to the Services Sector: Does it Affect Manufacturing Exports?
Authors: Ferro Esteban , Portugal‐Perez Alberto , Wilson John S.
Published: March 2014

The authors evaluated the impact of foreign aid to five services sectors (transportation, information and communications technologies (ICT), energy, banking/financial services, and business services) on exports of downstream manufacturing sectors in developing countries. To address the reverse causality between aid and exports, they relied on an original identification strategy that exploits (i) the variation of aid flows to services sectors and (ii) the variation of service intensities across industrial sectors and countries using input–output data. They found a positive effect of aid to services, in general, on downstream manufacturing exports of developing countries across regions and income‐level groups.
Differential modulation of sympathetic and respiratory activities by cholinergic mechanisms in the nucleus of the solitary tract in rats
Authors: Furuya Werner I. , Bassi Mirian , Menani José V. , Colombari Eduardo , Zoccal Daniel B. , Colombari Débora S. A.
Published: March 2014

New Findings

What is the central question of this study?

Is sympathorespiratory activity affected in a different manner by cholinergic mechanisms in the intermediate (iNTS) and commissural nucleus of the solitary tract (cNTS) and are cholinergic mechanisms involved in baro‐ and chemoreflexes?

What is the main finding and its importance?

Acetylcholine (ACh) injected into the iNTS promotes sympatho‐inhibition and reduces the phrenic frequency, whereas ACh injected into the cNTS increases phrenic frequency and affects sympathetic–respiratory coupling, without changing the sympathetic activity. These responses are abolished by mecamylamine (nicotinic antagonist) in the NTS. Mecamylamine in the cNTS also reduces peripheral chemoreflex‐induced tachypnoea.

The contribution of cholinergic mechanisms of the nucleus of the solitary tract (NTS) to cardiorespiratory control is not completely clear. In the present study, we investigated the involvement of the cholinergic mechanisms in the intermediate NTS (iNTS) and commissural NTS (cNTS) on the control of sympathetic (SNA) and phrenic nerve activity (PNA). Decorticated, arterially perfused in situ preparations of male juvenile rats (60–100 g) were used. Acetylcholine (10 mm, 60 nl) injected into the iNTS reduced SNA (−54 ± 4%, versus vehicle −5 ± 3%; P < 0.001) and PNA (−30 ± 4%, versus vehicle −5 ± 6%; P < 0.001), whereas injections of ACh into the cNTS increased PNA (30 ± 6%, versus vehicle 5 ± 3%; P < 0.001), without changing SNA. Pretreatment with mecamylamine (nicotinic antagonist; 5 mm) abolished all the effects of ACh injected into the iNTS or the cNTS, whereas atropine (muscarinic antagonist; 5 mm) reduced only the effects of ACh injected into the cNTS. Mecamylamine injected into the cNTS also reduced the tachypnoea in response to peripheral chemoreflex activation. The baroreflex was unaltered by injections of atropine or mecamylamine into the NTS. The results suggest that ACh and mainly nicotinic receptors in the NTS are involved in the modulation of SNA and PNA, with distinct functions between the iNTS and the cNTS. An involvement of the nicotinic receptors in the cNTS in the tachypnoea in response to peripheral chemoreflex activation is also suggested.
Enhanced field emission from hydrogenated SnO2 nanoparticles embedded in TiO2 film on fluorinated tin oxide substrate
Authors: Zhang Xu-Qiang , Chen Jian-Biao , Zhu Wei-Dong , Wang Cheng-Wei
Published: March 2014

Hydrogenated SnO2 nanoparticles (H:SNPs) were prepared on fluorinated tin oxide covered glass using reduction and hydrogenation technologies with TiO2 sol. By adjusting the hydrogenated temperature over the range of 400–550 °C, the H:SNPs were uniformly embedded in the TiO2 film, exhibiting the ability to precisely control their density and size using this method. Simultaneously, their band structures were modified, resulting in a reduced work function and an increased electrical conductivity. Hence, the optimized H:SNPs prepared at 500 °C showed excellent field emission (FE) performances, with both a low turn-on field of 3.81 V/μm and remarkable FE stability over a 480-min period.
Tungsten anode spectral model using interpolating cubic splines: Unfiltered x‐ray spectra from 20 kV to 640 kV
Authors: Hernandez Andrew M. , Boone John M.
Published: March 2014

Purpose:

Monte Carlo methods were used to generate lightly filtered high resolution x‐ray spectra spanning from 20 kV to 640 kV.

Methods:

X‐ray spectra were simulated for a conventional tungsten anode. The Monte Carlo N‐Particle eXtended radiation transport code (MCNPX 2.6.0) was used to produce 35 spectra over the tube potential range from 20 kV to 640 kV, and cubic spline interpolation procedures were used to create piecewise polynomials characterizing the photon fluence per energy bin as a function of x‐ray tube potential. Using these basis spectra and the cubic spline interpolation, 621 spectra were generated at 1 kV intervals from 20 to 640 kV. The tungsten anode spectral model using interpolating cubic splines (TASMICS) produces minimally filtered (0.8 mm Be) x‐ray spectra with 1 keV energy resolution. The TASMICS spectra were compared mathematically with other, previously reported spectra.

Results:

Using pairedt‐test analyses, no statistically significant difference (i.e., p > 0.05) was observed between compared spectra over energy bins above 1% of peak bremsstrahlung fluence. For all energy bins, the correlation of determination (R2) demonstrated good correlation for all spectral comparisons. The mean overall difference (MOD) and mean absolute difference (MAD) were computed over energy bins (above 1% of peak bremsstrahlung fluence) and over all the kV permutations compared. MOD and MAD comparisons with previously reported spectra were 2.7% and 9.7%, respectively (TASMIP), 0.1% and 12.0%, respectively [R. Birch and M. Marshall, “Computation of bremsstrahlung x‐ray spectra and comparison with spectra measured with a Ge(Li) detector,” Phys. Med. Biol. 24, – (1979)], 0.4% and 8.1%, respectively (Poludniowski), and 0.4% and 8.1%, respectively (AAPM TG 195). The effective energy of TASMICS spectra with 2.5 mm of added Al filtration ranged from 17 keV (at 20 kV) to 138 keV (at 640 kV); with 0.2 mm of added Cu filtration the effective energy was 9 keV at 20 kV and 169 keV at 640 kV.

Conclusions:

Ranging from 20 kV to 640 kV, 621 x‐ray spectra were produced and are available at 1 kV tube potential intervals. The spectra are tabulated at 1 keV intervals. TASMICS spectra were shown to be largely equivalent to published spectral models and are available in spreadsheet format for interested users by emailing the corresponding author (JMB).
Measurements and simulations of focused beam for orthovoltage therapy
Authors: Abbas Hassan , Mahato Dip N. , Satti Jahangir , MacDonald C. A.
Published: March 2014

Purpose:

Megavoltage photon beams are typically used for therapy because of their skin‐sparing effect. However, a focused low‐energy x‐ray beam would also be skin sparing, and would have a higher dose concentration at the focal spot. Such a beam can be produced with polycapillary optics. MCNP5 was used to model dose profiles for a scanned focused beam, using measured beam parameters. The potential of low energy focused x‐ray beams for radiation therapy was assessed.

Methods:

A polycapillary optic was used to focus the x‐ray beam from a tungsten source. The optic was characterized and measurements were performed at 50 kV. PMMA blocks of varying thicknesses were placed between optic and the focal spot to observe any variation in the focusing of the beam after passing through the tissue‐equivalent material. The measured energy spectrum was used to model the focused beam in MCNP5. A source card (SDEF) in MCNP5 was used to simulate the converging x‐ray beam. Dose calculations were performed inside a breast tissue phantom.

Results:

The measured focal spot size for the polycapillary optic was 0.2 mm with a depth of field of 5 mm. The measured focal spot remained unchanged through 40 mm of phantom thickness. The calculated depth dose curve inside the breast tissue showed a dose peak several centimeters below the skin with a sharp dose fall off around the focus. The percent dose falls below 10% within 5 mm of the focus. It was shown that rotating the optic during scanning would preserve the skin‐sparing effect of the focused beam.

Conclusions:

Low energy focused x‐ray beams could be used to irradiate tumors inside soft tissue within 5 cm of the surface.
Microbiota-Dependent Crosstalk Between Macrophages and ILC3 Promotes Intestinal Homeostasis
Authors: Mortha Arthur , Chudnovskiy Aleksey , Hashimoto Daigo , Bogunovic Milena , Spencer Sean P. , Belkaid Yasmine , Merad Miriam
Published: March 2014

Gut Immune Tolerance

With the constant assault of food antigens and its billions of resident microbes, the gut is an important site of immune tolerance. By studying specific intestinal immune cell populations in genetically modified mice,

Mortha
et al.

(
10.1126/science.1249288
, published online 13 March; see the Perspective by

Aychek and Jung

) found that gut macrophages produce the cytokine interleukin-1 (IL-1) in response to signals derived from the microbiota. IL-1 acts on type 3 innate lymphoid cells in the intestine, which then produce the cytokine, colony-stimulating factor 2 (Csf2). Csf-2, in turn, induces myeloid cells (including dendritic cells and macrophages) to produce regulatory factors like retinoic acid and interleukin-10, which support the conversion and expansion of regulatory T cells, a population of cells known to be critical for maintaining immune tolerance in the gut.
Hydrologic Regulation of Chemical Weathering and the Geologic Carbon Cycle
Authors: Maher K. , Chamberlain C. P.
Published: March 2014

Hydrologic Thermostat

When the silicate-rich rocks and minerals in Earth's interior are uplifted and exposed to Earth's surface, they dissolve. On a geologic time scale, this chemical weathering process ultimately creates a sink for CO
2
, thereby influencing global temperatures.
Maher and Chamberlain
(p.
1502
, published online 13 March) developed a theoretical framework for understanding the fundamental relationship between weathering, tectonics, and the geological carbon cycle. The analysis suggests that temperature plays less of a role in regulating chemical weathering—which is dependent on the balance of tectonic uplift and erosion—than runoff on continents and the time that silicate minerals are exposed to fluids. Plateaus in weathering fluxes with increasing runoff or temperature allows for the stabilization of atmospheric CO
2
despite high rates of uplift or erosion.
Transcriptional and Phenotypic Responses of Listeria monocytogenes to Chlorine Dioxide
Authors: Pleitner Aaron M. , Trinetta Valentina , Morgan Mark T. , Linton Richard L. , Oliver Haley F.
Published: March 2014

Significant food-borne disease outbreaks have occurred from consumption of ready-to-eat foods, including produce, contaminated withListeria monocytogenes. Challenging food matrices (e.g., cantaloupe, sprouts) with limited processing steps postharvest to reduce pathogen loads have underscored a need for new mitigation strategies. Chlorine dioxide (ClO2) is increasingly being used in produce and other food systems to reduce food-borne pathogen levels. The goal of this study was to characterize the transcriptional response and survival ofL. monocytogenes10403S exposed to ClO2. The transcriptional profile of log-phase cells exposed to 300 mg/liter ClO2for 15 min was defined by whole-genome microarray. A total of 340 genes were significantly differentially expressed. Among the differentially expressed genes, 223 were upregulated (fold change ≥ 1.5; adjustedPvalue < 0.05) in role categories responsible for protein fate, cellular processes, and energy metabolism. There were 113 and 16 genes differentially expressed belonging to regulatory networks of σBand CtsR, respectively. We assessedL. monocytogenes10403S survival after exposure to 100, 300, and 500 mg/liter aqueous ClO2in brain heart infusion (BHI) broth; there was a significant difference between cells exposed to 500 mg/liter ClO2and those exposed to all other conditions over time (Pvalue < 0.05). Isogenic ΔsigBand ΔctsRmutants exposed to 300 mg/liter ClO2were more sensitive to ClO2than the wild type under the same conditions. These results provide an initial insight into the mechanisms thatL. monocytogenesemploys to survive sublethal ClO2and further our understanding of the inactivation mechanisms of this increasingly used sanitizer.
Nitrogen Utilization and Metabolism in Ruminococcus albus 8
Authors: Kim Jong Nam , Henriksen Emily DeCrescenzo , Cann Isaac K. O. , Mackie Roderick I.
Published: March 2014

The model rumen
Firmicutes
organism
Ruminococcus albus
8 was grown using ammonia, urea, or peptides as the sole nitrogen source; growth was not observed with amino acids as the sole nitrogen source. Growth of
R. albus
8 on ammonia and urea showed the same growth rate (0.08 h
−1
) and similar maximum cell densities (for ammonia, the optical density at 600 nm [OD
600
] was 1.01; and for urea, the OD
600
was 0.99); however, growth on peptides resulted in a nearly identical growth rate (0.09 h
−1
) and a lower maximum cell density (OD
600
= 0.58). To identify differences in gene expression and enzyme activities, the transcript abundances of 10 different genes involved in nitrogen metabolism and specific enzyme activities were analyzed by harvesting mRNA and crude protein from cells at the mid- and late exponential phases of growth on the different N sources. Transcript abundances and enzyme activities varied according to nitrogen source, ammonia concentration, and growth phase. Growth of
R. albus
8 on ammonia and urea was similar, with the only observed difference being an increase in urease transcript abundance and enzyme activity in urea-grown cultures. Growth of
R. albus
8 on peptides showed a different nitrogen metabolism pattern, with higher gene transcript abundance levels of
gdhA
,
glnA
,
gltB
,
amtB
,
glnK
, and
ureC
, as well as higher activities of glutamate dehydrogenase and urease. These results demonstrate that ammonia, urea, and peptides can all serve as nitrogen sources for
R. albus
and that nitrogen metabolism genes and enzyme activities of
R. albus
8 are regulated by nitrogen source and the level of ammonia in the growth medium.
Genomic and Transcriptomic Analyses of the Facultative Methanotroph Methylocystis sp. Strain SB2 Grown on Methane or Ethanol
Authors: Vorobev Alexey , Jagadevan Sheeja , Jain Sunit , Anantharaman Karthik , Dick Gregory J. , Vuilleumier Stéphane , Semrau Jeremy D.
Published: March 2014

A minority of methanotrophs are able to utilize multicarbon compounds as growth substrates in addition to methane. The pathways utilized by these microorganisms for assimilation of multicarbon compounds, however, have not been explicitly examined. Here, we report the draft genome of the facultative methanotroph
Methylocystis
sp. strain SB2 and perform a detailed transcriptomic analysis of cultures grown with either methane or ethanol. Evidence for use of the canonical methane oxidation pathway and the serine cycle for carbon assimilation from methane was obtained, as well as for operation of the complete tricarboxylic acid (TCA) cycle and the ethylmalonyl-coenzyme A (EMC) pathway. Experiments with
Methylocystis
sp. strain SB2 grown on methane revealed that genes responsible for the first step of methane oxidation, the conversion of methane to methanol, were expressed at a significantly higher level than those for downstream oxidative transformations, suggesting that this step may be rate limiting for growth of this strain with methane. Further, transcriptomic analyses of
Methylocystis
sp. strain SB2 grown with ethanol compared to methane revealed that on ethanol (i) expression of the pathway of methane oxidation and the serine cycle was significantly reduced, (ii) expression of the TCA cycle dramatically increased, and (iii) expression of the EMC pathway was similar. Based on these data, it appears that
Methylocystis
sp. strain SB2 converts ethanol to acetyl-coenzyme A, which is then funneled into the TCA cycle for energy generation or incorporated into biomass via the EMC pathway. This suggests that some methanotrophs have greater metabolic flexibility than previously thought and that operation of multiple pathways in these microorganisms is highly controlled and integrated.
Biochar Enhances Aspergillus niger Rock Phosphate Solubilization by Increasing Organic Acid Production and Alleviating Fluoride Toxicity
Authors: Mendes Gilberto de Oliveira , Zafra David Lopez , Vassilev Nikolay Bojkov , Silva Ivo Ribeiro , Ribeiro José Ivo , Costa Maurício Dutra
Published: March 2014

During fungal rock phosphate (RP) solubilization, a significant quantity of fluoride (F
−
) is released together with phosphorus (P), strongly inhibiting the process. In the present study, the effect of two F
−
adsorbents [activated alumina (Al
2
O
3
) and biochar] on RP solubilization by
Aspergillus niger
was examined. Al
2
O
3
adsorbed part of the F
−
released but also adsorbed soluble P, which makes it inappropriate for microbial RP solubilization systems. In contrast, biochar adsorbed only F
−
while enhancing phosphate solubilization 3-fold, leading to the accumulation of up to 160 mg of P per liter. By comparing the values of F
−
measured in solution at the end of incubation and those from a predictive model, it was estimated that up to 19 mg of F
−
per liter can be removed from solution by biochar when added at 3 g liter
−1
to the culture medium. Thus, biochar acted as an F
−
sink during RP solubilization and led to an F
−
concentration in solution that was less inhibitory to the process. In the presence of biochar,
A. niger
produced larger amounts of citric, gluconic, and oxalic acids, whether RP was present or not. Our results show that biochar enhances RP solubilization through two interrelated processes: partial removal of the released F
−
and increased organic acid production. Given the importance of organic acids for P solubilization and that most of the RPs contain high concentrations of F
−
, the proposed solubilization system offers an important technological improvement for the microbial production of soluble P fertilizers from RP.
Microbiota of Human Breast Tissue
Authors: Urbaniak Camilla , Cummins Joanne , Brackstone Muriel , Macklaim Jean M. , Gloor Gregory B. , Baban Chwanrow K. , Scott Leslie , O'Hanlon Deidre M. , Burton Jeremy P. , Francis Kevin P. , Tangney Mark , Reid Gregor
Published: March 2014

In recent years, a greater appreciation for the microbes inhabiting human body sites has emerged. In the female mammary gland, milk has been shown to contain bacterial species, ostensibly reaching the ducts from the skin. We decided to investigate whether there is a microbiome within the mammary tissue. Using 16S rRNA sequencing and culture, we analyzed breast tissue from 81 women with and without cancer in Canada and Ireland. A diverse population of bacteria was detected within tissue collected from sites all around the breast in women aged 18 to 90, not all of whom had a history of lactation. The principal phylum was
Proteobacteria
. The most abundant taxa in the Canadian samples were
Bacillus
(11.4%),
Acinetobacter
(10.0%),
Enterobacteriaceae
(8.3%),
Pseudomonas
(6.5%),
Staphylococcus
(6.5%),
Propionibacterium
(5.8%),
Comamonadaceae
(5.7%),
Gammaproteobacteria
(5.0%), and
Prevotella
(5.0%). In the Irish samples the most abundant taxa were
Enterobacteriaceae
(30.8%),
Staphylococcus
(12.7%),
Listeria welshimeri
(12.1%),
Propionibacterium
(10.1%), and
Pseudomonas
(5.3%). None of the subjects had signs or symptoms of infection, but the presence of viable bacteria was confirmed in some samples by culture. The extent to which these organisms play a role in health or disease remains to be determined.
In Vivo
Self-Assembly of Stable Green Fluorescent Protein Fusion Particles and Their Uses in Enzyme Immobilization
Authors: Venning-Slater Mark , Hooks David O. , Rehm Bernd H. A.
Published: March 2014

Bacterial inclusion bodies are aggregations of mostly inactive and misfolded proteins. However, previously the
in vivo
self-assembly of green fluorescent protein (GFP) fusions into fluorescent particles which displayed specific binding sites suitable for applications in bioseparation and diagnostics was demonstrated. Here, the suitability of GFP particles for enzyme immobilization was assessed. The enzymes tested were a thermostable α-amylase from
Bacillus licheniformis
,
N
-acetyl-
d
-neuraminic acid aldolase (NanA) from
Escherichia coli
, and organophosphohydrolase (OpdA) from
Agrobacterium radiobacter
. Respective GFP particles were isolated and could be stably maintained outside the cell. These enzyme-bearing GFP particles exhibited considerable stability across a range of temperature, pH, and storage conditions and could be recycled. The α-amylase-bearing particles retained activity after treatments at 4 to 85°C and at pHs 4 to 10, were stable for 3 months at 4°C, and could be recycled up to three times. OpdA-bearing particles retained degradation activity after treatments at 4 to 45°C and at pHs 5 to 10 and were able to be recycled up to four times. In contrast, the performance of NanA-bearing particles rapidly declined (>50% loss) after each recycling step and 3 months storage at 4°C. However, they were still able to convert
N
-acetylmannosamine and pyruvate to
N
-acetylneuraminic acid after treatment at 4 to 85°C and at pHs 4 to 11. Fluorescent GFP fusion particles represent a novel method for the immobilization and display of enzymes. Potential applications include diagnostic assays, biomass conversion, pharmaceutical production, and bioremediation.
Soil Microbial Responses to Increased Moisture and Organic Resources along a Salinity Gradient in a Polar Desert
Authors: Van Horn David J. , Okie Jordan G. , Buelow Heather N. , Gooseff Michael N. , Barrett John E. , Takacs-Vesbach Cristina D.
Published: March 2014

Microbial communities in extreme environments often have low diversity and specialized physiologies suggesting a limited resistance to change. The McMurdo Dry Valleys (MDV) are a microbially dominated, extreme ecosystem currently undergoing climate change-induced disturbances, including the melting of massive buried ice, cutting through of permafrost by streams, and warming events. These processes are increasing moisture across the landscape, altering conditions for soil communities by mobilizing nutrients and salts and stimulating autotrophic carbon inputs to soils. The goal of this study was to determine the effects of resource addition (water/organic matter) on the composition and function of microbial communities in the MDV along a natural salinity gradient representing an additional gradient of stress in an already extreme environment. Soil respiration and the activity of carbon-acquiring extracellular enzymes increased significantly (
P
< 0.05) with the addition of resources at the low- and moderate-salinity sites but not the high-salinity site. The bacterial community composition was altered, with an increase in
Proteobacteria
and
Firmicutes
with water and organic matter additions at the low- and moderate-salinity sites and a near dominance of
Firmicutes
at the high-salinity site. Principal coordinate analyses of all samples using a phylogenetically informed distance matrix (UniFrac) demonstrated discrete clustering among sites (analysis of similarity [ANOSIM],
P
< 0.05 and
R
> 0.40) and among most treatments within sites. The results from this experimental work suggest that microbial communities in this environment will undergo rapid change in response to the altered resources resulting from climate change impacts occurring in this region.
High-Resolution Melt Analysis for Rapid Comparison of Bacterial Community Compositions
Authors: Hjelmsø Mathis Hjort , Hansen Lars Hestbjerg , Bælum Jacob , Feld Louise , Holben William E. , Jacobsen Carsten Suhr
Published: March 2014

In the study of bacterial community composition, 16S rRNA gene amplicon sequencing is today among the preferred methods of analysis. The cost of nucleotide sequence analysis, including requisite computational and bioinformatic steps, however, takes up a large part of many research budgets. High-resolution melt (HRM) analysis is the study of the melt behavior of specific PCR products. Here we describe a novel high-throughput approach in which we used HRM analysis targeting the 16S rRNA gene to rapidly screen multiple complex samples for differences in bacterial community composition. We hypothesized that HRM analysis of amplified 16S rRNA genes from a soil ecosystem could be used as a screening tool to identify changes in bacterial community structure. This hypothesis was tested using a soil microcosm setup exposed to a total of six treatments representing different combinations of pesticide and fertilization treatments. The HRM analysis identified a shift in the bacterial community composition in two of the treatments, both including the soil fumigant Basamid GR. These results were confirmed with both denaturing gradient gel electrophoresis (DGGE) analysis and 454-based 16S rRNA gene amplicon sequencing. HRM analysis was shown to be a fast, high-throughput technique that can serve as an effective alternative to gel-based screening methods to monitor microbial community composition.
Large-Scale Intersubspecific Recombination in the Plant-Pathogenic Bacterium Xylella fastidiosa Is Associated with the Host Shift to Mulberry
Authors: Nunney Leonard , Schuenzel Erin L. , Scally Mark , Bromley Robin E. , Stouthamer Richard
Published: March 2014

Homologous recombination plays an important role in the structuring of genetic variation of many bacteria; however, its importance in adaptive evolution is not well established. We investigated the association of intersubspecific homologous recombination (IHR) with the shift to a novel host (mulberry) by the plant-pathogenic bacterium
Xylella fastidiosa
. Mulberry leaf scorch was identified about 25 years ago in native red mulberry in the eastern United States and has spread to introduced white mulberry in California. Comparing a sequence of 8 genes (4,706 bp) from 21 mulberry-type isolates to published data (352 isolates representing all subspecies), we confirmed previous indications that the mulberry isolates define a group distinct from the 4 subspecies, and we propose naming the taxon
X. fastidiosa
subsp.
morus
. The ancestry of its gene sequences was mixed, with 4 derived from
X. fastidiosa
subsp.
fastidiosa
(introduced from Central America), 3 from
X. fastidiosa
subsp.
multiplex
(considered native to the United States), and 1 chimeric, demonstrating that this group originated by large-scale IHR. The very low within-type genetic variation (0.08% site polymorphism), plus the apparent inability of native
X. fastidiosa
subsp.
multiplex
to infect mulberry, suggests that this host shift was achieved after strong selection acted on genetic variants created by IHR. Sequence data indicate that a single ancestral IHR event gave rise not only to
X. fastidiosa
subsp.
morus
but also to the
X. fastidiosa
subsp.
multiplex
recombinant group which infects several hosts but is the only type naturally infecting blueberry, thus implicating this IHR in the invasion of at least two novel native hosts, mulberry and blueberry.
Improved HF183 Quantitative Real-Time PCR Assay for Characterization of Human Fecal Pollution in Ambient Surface Water Samples
Authors: Green Hyatt C. , Haugland Richard A. , Varma Manju , Millen Hana T. , Borchardt Mark A. , Field Katharine G. , Walters William A. , Knight R. , Sivaganesan Mano , Kelty Catherine A. , Shanks Orin C.
Published: March 2014

Quantitative real-time PCR (qPCR) assays that target the human-associated HF183 bacterial cluster within members of the genus
Bacteroides
are among the most widely used methods for the characterization of human fecal pollution in ambient surface waters. In this study, we show that a current TaqMan HF183 qPCR assay (HF183/BFDrev) routinely forms nonspecific amplification products and introduce a modified TaqMan assay (HF183/BacR287) that alleviates this problem. The performance of each qPCR assay was compared in head-to-head experiments investigating limits of detection, analytical precision, predicted hybridization to 16S rRNA gene sequences from a reference database, and relative marker concentrations in fecal and sewage samples. The performance of the modified HF183/BacR287 assay is equal to or improves upon that of the original HF183/BFDrev assay. In addition, a qPCR chemistry designed to combat amplification inhibition and a multiplexed internal amplification control are included. In light of the expanding use of PCR-based methods that rely on the detection of extremely low concentrations of DNA template, such as qPCR and digital PCR, the new TaqMan HF183/BacR287 assay should provide more accurate estimations of human-derived fecal contaminants in ambient surface waters.
Examining the Role of Membrane Lipid Composition in Determining the Ethanol Tolerance of Saccharomyces cerevisiae
Authors: Henderson Clark M. , Block David E.
Published: March 2014

Yeast (Saccharomyces cerevisiae) has an innate ability to withstand high levels of ethanol that would prove lethal to or severely impair the physiology of other organisms. Significant efforts have been undertaken to elucidate the biochemical and biophysical mechanisms of how ethanol interacts with lipid bilayers and cellular membranes. This research has implicated the yeast cellular membrane as the primary target of the toxic effects of ethanol. Analysis of model membrane systems exposed to ethanol has demonstrated ethanol's perturbing effect on lipid bilayers, and altering the lipid composition of these model bilayers can mitigate the effect of ethanol. In addition, cell membrane composition has been correlated with the ethanol tolerance of yeast cells. However, the physical phenomena behind this correlation are likely to be complex. Previous work based on often divergent experimental conditions and time-consuming low-resolution methodologies that limit large-scale analysis of yeast fermentations has fallen short of revealing shared mechanisms of alcohol tolerance inSaccharomyces cerevisiae. Lipidomics, a modern mass spectrometry-based approach to analyze the complex physiological regulation of lipid composition in yeast and other organisms, has helped to uncover potential mechanisms for alcohol tolerance in yeast. Recent experimental work utilizing lipidomics methodologies has provided a more detailed molecular picture of the relationship between lipid composition and ethanol tolerance. While it has become clear that the yeast cell membrane composition affects its ability to tolerate ethanol, the molecular mechanisms of yeast alcohol tolerance remain to be elucidated.
Carbon Flux Analysis by
13
C Nuclear Magnetic Resonance To Determine the Effect of CO
2
on Anaerobic Succinate Production by Corynebacterium glutamicum
Authors: Radoš Dušica , Turner David L. , Fonseca Luís L. , Carvalho Ana Lúcia , Blombach Bastian , Eikmanns Bernhard J. , Neves Ana Rute , Santos Helena
Published: March 2014

Wild-type
Corynebacterium glutamicum
produces a mixture of lactic, succinic, and acetic acids from glucose under oxygen deprivation. We investigated the effect of CO
2
on the production of organic acids in a two-stage process: cells were grown aerobically in glucose, and subsequently, organic acid production by nongrowing cells was studied under anaerobic conditions. The presence of CO
2
caused up to a 3-fold increase in the succinate yield (1 mol per mol of glucose) and about 2-fold increase in acetate, both at the expense of
l
-lactate production; moreover, dihydroxyacetone formation was abolished. The redistribution of carbon fluxes in response to CO
2
was estimated by using
13
C-labeled glucose and
13
C nuclear magnetic resonance (NMR) analysis of the labeling patterns in end products. The flux analysis showed that 97% of succinate was produced via the reductive part of the tricarboxylic acid cycle, with the low activity of the oxidative branch being sufficient to provide the reducing equivalents needed for the redox balance. The flux via the pentose phosphate pathway was low (∼5%) regardless of the presence or absence of CO
2
. Moreover, there was significant channeling of carbon to storage compounds (glycogen and trehalose) and concomitant catabolism of these reserves. The intracellular and extracellular pools of lactate and succinate were measured by
in vivo
NMR, and the stoichiometry (H
+
:organic acid) of the respective exporters was calculated. This study shows that it is feasible to take advantage of natural cellular regulation mechanisms to obtain high yields of succinate with
C. glutamicum
without genetic manipulation.