bluuebunny/crossref_metadata_2025 huggingface dataset:
Tree growth and competition in an old‐growthPicea abiesforest of borealSweden: influence of tree spatial patterning
Authors: Fraver Shawn , D'Amato Anthony W. , Bradford John B. , Jonsson Bengt Gunnar , Jönsson Mari , Esseen Per‐Anders
Published: June 2013

Question

What factors best characterize tree competitive environments in this structurally diverse old‐growth forest, and do these factors vary spatially within and among stands?

Location

Old‐growthPicea abiesforest of borealSweden.

Methods

Using long‐term, mapped permanent plot data augmented with dendrochronological analyses, we evaluated the effect of neighbourhood competition on focal tree growth by means of standard competition indices, each modified to include various metrics of trees size, neighbour mortality weighting (for neighbours that died during the inventory period), and within‐neighbourhood tree clustering. Candidate models were evaluated using mixed‐model linear regression analyses, with mean basal area increment as the response variable. We then analysed stand‐level spatial patterns of competition indices and growth rates (viakriging) to determine if the relationship between these patterns could further elucidate factors influencing tree growth.

Results

Inter‐tree competition clearly affected growth rates, with crown volume being the size metric most strongly influencing the neighbourhood competitive environment. Including neighbour tree mortality weightings in models only slightly improved descriptions of competitive interactions. Although the within‐neighbourhood clustering index did not improve model predictions, competition intensity was influenced by the underlying stand‐level tree spatial arrangement: stand‐level clustering locally intensified competition and reduced tree growth, whereas in the absence of such clustering, inter‐tree competition played a lesser role in constraining tree growth.

Conclusions

Our findings demonstrate that competition continues to influence forest processes and structures in an old‐growth system that has not experienced major disturbances for at least two centuries. The finding that the underlying tree spatial pattern influenced the competitive environment suggests caution in interpreting traditional tree competition studies, in which tree spatial patterning is typically not taken into account. Our findings highlight the importance of forest structure – particularly the spatial arrangement of trees – in regulating inter‐tree competition and growth in structurally diverse forests, and they provide insight into the causes and consequences of heterogeneity in this old‐growth system.
Warming‐related growth responses at the southern limit distribution of mountain pine (Pinus mugo Turra subsp. mugo)
Authors: Palombo Caterina , Battipaglia Giovanna , Cherubini Paolo , Chirici Gherardo , Garfì Vittorio , Lasserre Bruno , Lombardi Fabio , Marchetti Marco , Tognetti Roberto
Published: June 2013

Questions

Could mountain pine (Pinus mugo Turra subsp. mugo) located above the timberline and at the southern limit of its potential geographic distribution be a promising species to monitor climatic change in specific environments such as the Mediterranean mountains? Does mountain pine krummholz upward expansion result from current climate change? Has the relationship between climate and tree ring growth of mountain pine changed? To what extent does climatic variability affect tree ring growth? Could mountain pine upward expansion contribute to loss of biodiversity in the undisturbed tree line ecotone on the Majella massif?

Location

Majella National Park, Apennines, Italy.

Methods

Dendroclimatological analysis was performed to examine the relationship between tree rings of mountain pine and climatic parameters at the tree line. Climate–growth relationships were analysed using correlation function (CF) analysis and moving CF (MCF).

Results

In the Majella massif, climate variables affected mountain pine tree growth at the tree line ecotone. Significant correlations were found with spring temperatures and summer precipitation. Essentially, three months played a key role in mountain pine growth: April and May of the current year and October of the previous year. MCF showed that mountain pine is a sensitive species, in terms of tree ring growth.

Conclusions

Growing above the limit of tree vegetation, mountain pine proved to be a promising species for monitoring climate‐ and land use‐driven influences over tree ring growth at high elevations. Our results show that mountain pine is particularly sensitive to climate at the beginning and at the end of the growing season.
Hepatitis C screening beyond CDC guidelines in an Egyptian immigrant community
Authors: Perumalswami Ponni V. , DeWolfe Miller F. , Orabee Hesham , Regab Amgad , Adams Mohamed , Kapelusznik Luciano , Aljibawi Faozia , Pagano William , Tong Virginia , Dieterich Douglas T.
Published: June 2013

Background & Aims

Many Egyptian‐born persons in the U.S. are at high risk of chronic hepatitis C virus (HCV) infection, yet are not aware of their infection and lack healthcare coverage or linkage to care. In this study, we target Egyptian‐born persons living in the New York City area for screening and link to care.

Methods

A unique partnership, the Hepatitis Outreach Network (HONE), combines the expertise and resources of the Mount Sinai School of Medicine, the NYC Department of Health and Mental Hygiene and community‐based organizations, to provide education, screening and link to care in communities with high prevalence of chronic viral hepatitis.

Results

Through four community‐based screening events, 192 Egyptian‐born persons were screened for HCV. Thirty (15.6%) persons were HCV positive. HCV antibody prevalence in those, whose national origin was Egypt, increased strongly with age and was associated with increasing number of years resident in Egypt and rural residents. Of the 30 Egyptian persons with HCV infection, 18 (60%) received a medical evaluation (2 with local providers and 16 at Mount Sinai). Of the HCV‐infected persons evaluated, treatment was recommended in four and begun in three (75%).

Conclusion

Egyptian‐born persons living in the New York City area have a high burden of HCV disease. HONE has successfully established targeted HCV screening in Egyptian‐born persons through use of several unique elements that effectively link them to care.
Dose escalation using helical tomotherapy improves local control in spine metastases from primary hepatic malignancies
Authors: Choi Yunseon , Kim Junwon , Lee Ikjae , Seong Jinsil
Published: June 2013

Background & Aims

This study was designed to reveal the prognostic significance of dose‐escalated radiotherapy with tomotherapy in local control for spine metastases of primary hepatic tumours.

Methods

From April 2006 to May 2012, 23 hepatocellular carcinoma patients and 7 intrahepatic cholangiocellular carcinoma patients (total 30 patients, 42 spinal lesions) were treated for metastatic spine lesions with helical tomotherapy (HT). The gross tumour volume (GTV) was defined as a tumour evident from computed tomography and magnetic resonance imaging. Median values were as follows: GTV total dose of 48 Gy (range 21–51), fraction size of 6 Gy (range 3–8) and eight fractions (range 3–17). Pain response was checked according to visual analogue scale (from 0 to 10).

Results

The median follow‐up was 5.6 months. Six events of local failure occurred, including five lesions in which spinal canals were involved at radiotherapy. Local control rate at 3 months was 86.6%. Biological equivalent dose (BED) was correlated with local control (AUC = 0.833). Higher BED (>56.0 Gy10) was associated with increased local control (P = 0.004). The median time to local progression in patients receiving ≤56.0 Gy10 and >56.0 Gy10 were 3 and 20.8 months respectively. Dose escalation (BED > 56.0 Gy10) was also associated with improved progression‐free survival (median 14.7 vs. 2.8 months, P = 0.010). Pain reduction was observed in 90.9% (20/22) of patients with painful bone metastases.

Conclusions

Dose‐escalated radiotherapy (BED > 56.0 Gy10) using HT improved local control in spinal metastases of hepatic malignancies.
Could pericytic mimicry represent another type of melanoma cell plasticity with embryonic properties?
Authors: Lugassy Claire , Péault Bruno , Wadehra Madhuri , Kleinman Hynda K. , Barnhill Raymond L.
Published: June 2013

We hypothesize that the interaction between angiotropic melanoma cells and the abluminal vascular surface can induce or sustain embryonic and/or stem cell migratory properties in these tumor cells. As a result, such angiotropic melanoma cells may migrate along the abluminal vascular surface, demonstrating pericytic mimicry. Through these cellular interactions, melanoma cells may migrate toward secondary sites.
Dietary Feeding of Opuntia Humifusa Inhibits UVB Radiation‐Induced Carcinogenesis by Reducing Inflammation and Proliferation in Hairless Mouse Model
Authors: Lee Jin‐A , Jung Bock‐Gie , Kim Tae‐Hoon , Lee Su‐Gil , Park Young‐Seok , Lee Bong‐Joo
Published: June 2013

It has been validated that ultraviolet B (UVB) irradiation induced both squamous and basal cell carcinomas, as a tumor initiator and promoter. Opuntia humifusa is a member of the Cactaceae family which has been demonstrated in our previous study to have a chemopreventive effect in 7, 12‐dimethylbenz[a]anthracene and 12‐O‐tetradecanoylphorbol‐13‐acetate induced skin carcinogenesis models. Therefore, this study was designed to determine the protective effects of O.humifusa against photocarcinogenesis. O. humifusa was administrated to mice as a dietary feeding, following exposure to UVB radiation (180 mJ/cm2) twice a week of 30 weeks for skin tumor development in hairless mice. Dietary O.humifusa inhibited UVB‐induced epidermal hyperplasia, infiltration of leukocytes, level of myeloperoxidase and the levels of proinflammatory cytokines, tumor necrosis factor‐ α (TNF‐α), interleukin‐1β (IL‐1β) and interleukin‐6 (IL‐6), in UVB exposed skin. Also, O.humifusa significantly inhibited both protein and mRNA expression level of cyclooxygenase‐2 (COX‐2), nitric oxide synthase (iNOS), proliferating cell nuclear antigen (PCNA) and cyclin D1 compared to the non‐O.humifusa treated group. Collectively, these results suggest that O.humifusa could inhibit photocarcinogenesis in mouse skin and that protective effect is associated with the inhibition of not only UVB‐induced inflammatory responses involving COX‐2, iNOS and proinflammatory cytokines, but also the down‐regulation of UVB‐induced cellular proliferation.
TheNSAIDs Indomethacin and Diflunisal as Scavengers of Photogenerated Reactive Oxygen Species
Authors: Purpora Rebeca , Massad Walter , Ferrari Gabriela , Reynoso Eugenia , Criado Susana , Miskoski Sandra , Pajares Adriana , García Norman A.
Published: June 2013

Diflunisal (DFNS) and Indomethacin (IMTC) are two profusely employed NSAIDs that provide anti‐inflammatory and analgesic effects in humans. The scavenging of reactive oxygen species (ROS) by both NSAIDs was systematically studied in pH 7 aqueous solution. The ROS O2(1Δg), O2•−and H2O2, generated by visible light irradiation of Riboflavin (Rf) in the presence of DFNS and IMTC, are deactivated by the NSAIDs. The ROS scavenging action by both NSAIDs constitutes an interesting result and adds one more positive aspect to the beneficial actions attributed to these drugs. Nevertheless it should be taken into account that several NSAIDs, in particular IMTC, have been connected to the pathogenesis of gastric mucosal lesions, which in some cases includes ROS generating‐ability. DFNS quenches ROS in a dominant physical fashion. It constitutes an excellent protective‐antioxidant provided that is practically not destroyed/oxidized after the ROS scavenging action. IMTC, being also an efficient interceptor of ROS, belong to the so‐called group ofsacrificial‐ROS quenchers: It is easily degraded by the oxidative species in the scavenging action. Although this property is negative in the context of prolonged ROS elimination, exhibits a promissory aspect for the degradation of pharmaceutical contaminants, such as NSAIDs, in waste waters.
Destabilization of the Oxygen Evolving Complex of Photosystem II by Al3+
Authors: Hasni Imed , Hamdani Saber , Carpentier Robert
Published: June 2013

The inhibitory effect of Al3+ on photosynthetic electron transport was investigated in isolated thylakoid membranes of spinach. A combination of oxygen evolution, chlorophyll fluorescence induction (FI) and decay and thermoluminescence measurements have been used to characterize photosystem II (PSII) electron transport in the presence of this toxic metal cation. Our results show that below 3 mm, Al3+ already caused a destabilization of the Mn4O5Ca cluster of the oxygen evolving complex (OEC). At these concentrations, an increase in the relative amplitude of the first phase (OJ) of FI curve and retardation of the fluorescence decay kinetics following excitation with a single turnover flash were also observed. A transmembrane structural modification of PSII polypeptides due to the interaction of Al3+ at the OEC is proposed to retard electron transfer between the quinones QA and QB. Above 3 mm, Al3+ strongly retarded fluorescence induction and significantly reduced Fv/Fm together with the maximal amplitude of chlorophyll fluorescence induced by a single turnover flash. This chlorophyll fluorescence quenching was attributed to the formation of P680+ due to inhibition of electron transfer between tyrosine 161 of D1 subunit and P680.
Effects of Vasectomy on Seminal Plasma Alkaline Phosphatase in Male Alpacas (Vicugña pacos)
Authors: Pearson LK , Campbell AJ , Sandoval S , Tibary A
Published: June 2013

Contents

Azoospermia is a common finding in male alpacas which present for infertility. The challenge is to differentiate azoospermia of testicular origin from non‐testicular origin. In several species, alkaline phosphatase (AP) concentrations in seminal plasma have been used as a diagnostic marker of contributions of the testis and epididymis to the ejaculate. The purpose of this study was to determine whetherAPassay could differentiate testicular from non‐testicular azoospermia in male alpacas. An experimental model of bilateral outflow obstruction (pre‐scrotal vasectomy) was used in 22 male alpacas, aged 2–9 years. No reproductive history was available. Animals were submitted for electroejaculation (EE) under general anaesthesia and vasectomy performed. Five weeks later, animals were submitted forEE. Vasectomy was not successful in one animal, which was removed from analysis.APlevels were compared in seminal plasma in the pre‐ and post‐vasectomy samples. The mean ± SEMconcentration ofAPin pre‐vasectomy seminal plasma was 504.29 ± 166.45 U/l (range 10–2910); the post‐vasectomy levels were 252.48 ± 81.77 U/l (range 0–1640; p = 0.06). In 71.4% of animals,APlevels decreased, varying from 18% to 100% reduction. Results of this study suggest thatAPis not produced exclusively by the testis and epididymis in alpacas and thatAPassay is not a valid diagnostic test for determination of origin of azoospermia; the gold standard for diagnosis of origin of azoospermia remains testicular biopsy.
Organic Geochemistry of the Cenomanian–Turonian Bahloul Formation Petroleum Source Rock, Central and Northern Tunisia
Authors: Affouri Hassene , Montacer Mabrouk , Disnar Jean‐Robert
Published: June 2013

Total organic carbon (TOC) determination, Rock‐Eval pyrolysis, extractable organic matter content (EOM) fractionation, gas chromatography (GC) and gas chromatography–mass spectrometry (GC–MS) analyses, were carried out on 79 samples from eleven outcrop cross sections of the Bahloul Formation in central and northern Tunisia. The TOC content varied between 0.23 to 35.6%, the highest average values (18.73%, 8.46% and 4.02%) being at the east of the study area (at Ain Zakkar, Oued Bahloul and Dyr Ouled Yahia localities, respectively). The Rock‐Eval maximum pyrolysis temperature (Tmax) values in the 424–453°C range delineated a general east‐west trend increase in the organic matter (OM) maturity. The disparity in hydrogen index (HI) values, in the range 114–824 mg hydrocarbons (HC) g−1 TOC, is relevant for the discrepancy in the level of OM preservation and maturity among localities and samples. The n‐alkane distributions, maximizing in the C17 to C20 range, are typical for a marine planktonic origin, whereas pristine/phytane (Pr/Ph) average values in the 1–2 range indicate an oxic to suboxic depositional environment. Pr/n‐C17 and Ph/n‐C18 ratios in the 0.38–6.2 and 0.68–3.25 range, respectively, are consistent with other maturity indicators and the contribution of specific bacteria to phytol as a precursor of isoprenoids. The thermal maturity varies between late diagenesis to main‐stage of petroleum generation based on the optic and the cis‐trans isomerisation of the C29 sterane [20S/(20S+20R) and 14β(H),17β(H)/(14β(H),17β(H)+14α(H),17α(H)), respectively] and the terpane [18α(H)22,29,30‐Trisnorneohopane/(18α(H)22,29,30‐Trisnorneohopane+17α(H)22,29,30‐Trisnorhopane): Ts/(Ts+Tm)] ratios. The Bahloul OM is represented by an open marine to estuarine algal facies with a specific bacterial contribution as revealed by the relative abundance of the ααα‐20R C27 (33–44%), C28 (22–28%) and C29 (34–41%) steranes and by the total terpanes/total steranes ratio (1.2–5.33). These results attested that the Bahloul OM richness was controlled both by an oxygen minimum zone induced by high productivity and restricted circulation in narrow half graben structures and around diapirs of the Triassic salt.
Phylogenetic relationships of Microdontinae (Diptera: Syrphidae) based on molecular and morphological characters
Authors: REEMER MENNO , STÅHLS GUNILLA
Published: June 2013

The intrasubfamilial classification of Microdontinae Rondani (Diptera: Syrphidae) has been a challenge: until recently more than 300 out of more than 400 valid species names were classified in Microdon Meigen. We present phylogenetic analyses of molecular and morphological characters (both separate and combined) of Microdontinae. The morphological dataset contains 174 characters, scored for 189 taxa (9 outgroup), representing all 43 presently recognized genera and several subgenera and species groups. The molecular dataset, representing 90 ingroup species of 28 genera, comprises sequences of five partitions in total from the mitochondrial gene COI and the nuclear ribosomal genes 18S and 28S. We test the sister‐group relationship of Spheginobaccha with the other Microdontinae, attempt to elucidate phylogenetic relationships within the Microdontinae and discuss uncertainties in the classification of Microdontinae. Trees based on molecular characters alone are poorly resolved, but combined data are better resolved. Support for many deeper nodes is low, and placement of such nodes differs between parsimony and Bayesian analyses. However, Spheginobaccha is recovered as highly supported sister group in both. Both analyses agree on the early branching of Mixogaster, Schizoceratomyia, Afromicrodon and Paramicrodon. The taxonomical rank in relation to the other Syrphidae is discussed briefly. An additional analysis based on morphological characters only, including all 189 taxa, used implied weighting. A range of weighting strengths (k‐values) is applied, chosen such that values of character fit of the resulting trees are divided into regular intervals. Results of this analysis are used for discussing the phylogenetic relationships of genera unrepresented in the molecular dataset.
Association of IL‐10 and CCL5 single nucleotide polymorphisms with tuberculosis in the Sudanese population
Authors: Mhmoud Najwa , Fahal Ahmed , Wendy van de Sande W. J.
Published: June 2013

Objective

To investigate the genetic determinants for developing tuberculosis in Sudan.

Methods

Case study of 232 patients with tuberculosis and 206 healthy matched controls from Sudan. In the study population, three single nucleotide polymorphisms (SNPs) in the promoter regions of CCL5 and two in the promoter region of IL‐10 were genotyped using polymerase chain reaction and restriction fragment length polymorphism (PCR‐RFLP). These five SNPs influence the expression of these genes.

Results

There were significant differences in allele distribution for CCL5 ‐28 C/G (rs 2280788) and IL‐10 ‐592 A/C (rs1800872) in patients with tuberculosis compared with healthy controls.

Conclusion

This indicates that the genotypes obtained for CCL5 and IL‐10 are associated with an increased risk of developing active TB.
Preload‐adjusted left ventricular dP/dtmax: a sensitive, continuous, load‐independent contractility index
Authors: Blaudszun Grégoire , Licker Marc J. , Morel Denis R.
Published: June 2013

New findings

What is the central question of this study?

The preload‐adjusted maximal change in pressure over time (PAdP/dtmax) accurately reflects systolic performance in varying conditions of inotropy and afterload. The objective of this study was to address whether PAdP/dtmax is a valid indicator of left ventricular function in the setting of large variations in preload.

What is the main finding and its importance?

In a model of acute preload reduction, PAdP/dtmax properly reflected left ventricular systolic function. Given its simplicity, this real‐time index derived from single beat analysis should be tested further in clinical settings.

The classical indicators of left ventricular (LV) performances have been derived from pressure–volume (PV) and stroke work–volume plots obtained during various loading or pharmacological interventions. More recently, the preload‐adjusted maximal change in pressure over time (PAdP/dtmax), derived from single beat PV analysis, has been shown to reflect the LV systolic performance accurately in varying conditions of inotropy and afterload. The objective of this study was to address whether PAdP/dtmax is a valid indicator of LV function in the setting of large preload variations, compared with the classical dP/dtmax–end‐diastolic volume (EDV) and stroke work–EDV (preload recruitable stroke work) relationships. Nine anaesthetized and mechanically ventilated rats were instrumented with a ventricular conductance catheter. Stepwise preload reduction was achieved by repeated blood withdrawals (up to a total of 5 ml). Steady‐state and dynamic PV loops were recorded during brief occlusion of the inferior vena cava, and LV function parameters were derived from these recordings. Our results demonstrate that PAdP/dtmax behaved in a similar manner to preload recruitable stroke work, reflecting well‐maintained LV contractility during controlled haemorrhage until mean arterial pressure decreased below 40 mmHg. In contrast, dP/dtmax–EDV increased significantly and exhibited a curvilinear response that was associated with a large inter‐ and intra‐animal variability. In a model of acute preload reduction, PAdP/dtmax was found to be the best indicator of systolic LV function. Given its simplicity, this real‐time index derived from single beat analysis should be tested further in clinical settings.
Direct Assessment of Viral Diversity in Soils by Random PCR Amplification of Polymorphic DNA
Authors: Srinivasiah Sharath , Lovett Jacqueline , Polson Shawn , Bhavsar Jaysheel , Ghosh Dhritiman , Roy Krishnakali , Fuhrmann Jeffry J. , Radosevich Mark , Wommack K. Eric
Published: June 2013

Viruses are the most abundant and diverse biological entities within soils, yet their ecological impact is largely unknown. Defining how soil viral communities change with perturbation or across environments will contribute to understanding the larger ecological significance of soil viruses. A new approach to examining the composition of soil viral communities based on random PCR amplification of polymorphic DNA (RAPD-PCR) was developed. A key methodological improvement was the use of viral metagenomic sequence data for the design of RAPD-PCR primers. This metagenomically informed approach to primer design enabled the optimization of RAPD-PCR sensitivity for examining changes in soil viral communities. Initial application of RAPD-PCR viral fingerprinting to soil viral communities demonstrated that the composition of autochthonous soil viral assemblages noticeably changed over a distance of meters along a transect of Antarctic soils and across soils subjected to different land uses. For Antarctic soils, viral assemblages segregated upslope from the edge of dry valley lakes. In the case of temperate soils at the Kellogg Biological Station, viral communities clustered according to land use treatment. In both environments, soil viral communities changed along with environmental factors known to shape the composition of bacterial host communities. Overall, this work demonstrates that RAPD-PCR fingerprinting is an inexpensive, high-throughput means for addressing first-order questions of viral community dynamics within environmental samples and thus fills a methodological gap between narrow single-gene approaches and comprehensive shotgun metagenomic sequencing for the analysis of viral community diversity.
Bacterially Induced Weathering of Ultramafic Rock and Its Implications for Phytoextraction
Authors: Becerra-Castro Cristina , Kidd Petra , Kuffner Melanie , Prieto-Fernández Ángeles , Hann Stephan , Monterroso Carmela , Sessitsch Angela , Wenzel Walter , Puschenreiter Markus
Published: June 2013

The bioavailability of metals in soil is often cited as a limiting factor of phytoextraction (or phytomining). Bacterial metabolites, such as organic acids, siderophores, or biosurfactants, have been shown to mobilize metals, and their use to improve metal extraction has been proposed. In this study, the weathering capacities of, and Ni mobilization by, bacterial strains were evaluated. Minimal medium containing ground ultramafic rock was inoculated with either of two
Arthrobacter
strains: LA44 (indole acetic acid [IAA] producer) or SBA82 (siderophore producer, PO
4
solubilizer, and IAA producer). Trace elements and organic compounds were determined in aliquots taken at different time intervals after inoculation. Trace metal fractionation was carried out on the remaining rock at the end of the experiment. The results suggest that the strains act upon different mineral phases. LA44 is a more efficient Ni mobilizer, apparently solubilizing Ni associated with Mn oxides, and this appeared to be related to oxalate production. SBA82 also leads to release of Ni and Mn, albeit to a much lower extent. In this case, the concurrent mobilization of Fe and Si indicates preferential weathering of Fe oxides and serpentine minerals, possibly related to the siderophore production capacity of the strain. The same bacterial strains were tested in a soil-plant system: the Ni hyperaccumulator
Alyssum serpyllifolium
subsp.
malacitanum
was grown in ultramafic soil in a rhizobox system and inoculated with each bacterial strain. At harvest, biomass production and shoot Ni concentrations were higher in plants from inoculated pots than from noninoculated pots. Ni yield was significantly enhanced in plants inoculated with LA44. These results suggest that Ni-mobilizing inoculants could be useful for improving Ni uptake by hyperaccumulator plants.
Roles of the Yap1 Transcription Factor and Antioxidants in Saccharomyces cerevisiae's Tolerance to Furfural and 5-Hydroxymethylfurfural, Which Function as Thiol-Reactive Electrophiles Generating Oxidative Stress
Authors: Kim Daehee , Hahn Ji-Sook
Published: June 2013

Development of the tolerance of
Saccharomyces cerevisiae
strains to furfural and 5-hydroxymethylfurfural (HMF) is an important issue for cellulosic ethanol production. Although furfural and HMF are known to induce oxidative stress, the underlying mechanisms are largely unknown. In this study, we show that both furfural and HMF act as thiol-reactive electrophiles, thus directly activating the Yap1 transcription factor via the H
2
O
2
-independent pathway, depleting cellular glutathione (GSH) levels, and accumulating reactive oxygen species in
Saccharomyces cerevisiae
. However, furfural showed higher reactivity than did HMF toward GSH
in vitro
and
in vivo
. In line with such toxic mechanisms, overexpression of
YAP1

C620F

, a constitutively active mutant of
YAP1
, and Yap1 target genes encoding catalases (
CTA1
and
CTT1
) increased tolerance to furfural and HMF. However, increasing GSH levels by overexpression of genes for GSH biosynthesis (
GSH1
and
GLR1
) or by the exogenous addition of GSH to the culture medium enhanced tolerance to furfural but not to HMF.
Facility-Specific “House” Microbiome Drives Microbial Landscapes of Artisan Cheesemaking Plants
Authors: Bokulich Nicholas A. , Mills David A.
Published: June 2013

Cheese fermentations involve the growth of complex microbial consortia, which often originate in the processing environment and drive the development of regional product qualities. However, the microbial milieus of cheesemaking facilities are largely unexplored and the true nature of the fermentation-facility relationship remains nebulous. Thus, a high-throughput sequencing approach was employed to investigate the microbial ecosystems of two artisanal cheesemaking plants, with the goal of elucidating how the processing environment influences microbial community assemblages. Results demonstrate that fermentation-associated microbes dominated most surfaces, primarily
Debaryomyces
and
Lactococcus
, indicating that establishment of these organisms on processing surfaces may play an important role in microbial transfer, beneficially directing the course of sequential fermentations. Environmental organisms detected in processing environments dominated the surface microbiota of washed-rind cheeses maturing in both facilities, demonstrating the importance of the processing environment for populating cheese microbial communities, even in inoculated cheeses. Spatial diversification within both facilities reflects the functional adaptations of microbial communities inhabiting different surfaces and the existence of facility-specific “house” microbiota, which may play a role in shaping site-specific product characteristics.
Adjustment of Trehalose Metabolism in Wine Saccharomyces cerevisiae Strains To Modify Ethanol Yields
Authors: Rossouw D. , Heyns E. H. , Setati M. E. , Bosch S. , Bauer F. F.
Published: June 2013

The ability ofSaccharomyces cerevisiaeto efficiently produce high levels of ethanol through glycolysis has been the focus of much scientific and industrial activity. Despite the accumulated knowledge regarding glycolysis, the modification of flux through this pathway to modify ethanol yields has proved difficult. Here, we report on the systematic screening of 66 strains with deletion mutations of genes encoding enzymes involved in central carbohydrate metabolism for altered ethanol yields. Five of these strains showing the most prominent changes in carbon flux were selected for further investigation. The genes were representative of trehalose biosynthesis (TPS1, encoding trehalose-6-phosphate synthase), central glycolysis (TDH3, encoding glyceraldehyde-3-phosphate dehydrogenase), the oxidative pentose phosphate pathway (ZWF1, encoding glucose-6-phosphate dehydrogenase), and the tricarboxylic acid (TCA) cycle (ACO1andACO2, encoding aconitase isoforms 1 and 2). Two strains exhibited lower ethanol yields than the wild type (tps1Δ andtdh3Δ), while the remaining three showed higher ethanol yields. To validate these findings in an industrial yeast strain, theTPS1gene was selected as a good candidate for genetic modification to alter flux to ethanol during alcoholic fermentation in wine. Using low-strength promoters active at different stages of fermentation, the expression of theTPS1gene was slightly upregulated, resulting in a decrease in ethanol production and an increase in trehalose biosynthesis during fermentation. Thus, the mutant screening approach was successful in terms of identifying target genes for genetic modification in commercial yeast strains with the aim of producing lower-ethanol wines.
Application of Denaturing High-Performance Liquid Chromatography for Monitoring Sulfate-Reducing Bacteria in Oil Fields
Authors: Priha Outi , Nyyssönen Mari , Bomberg Malin , Laitila Arja , Simell Jaakko , Kapanen Anu , Juvonen Riikka
Published: June 2013

Sulfate-reducing bacteria (SRB) participate in microbially induced corrosion (MIC) of equipment and H
2
S-driven reservoir souring in oil field sites. Successful management of industrial processes requires methods that allow robust monitoring of microbial communities. This study investigated the applicability of denaturing high-performance liquid chromatography (DHPLC) targeting the dissimilatory sulfite reductase ß-subunit (
dsrB
) gene for monitoring SRB communities in oil field samples from the North Sea, the United States, and Brazil. Fifteen of the 28 screened samples gave a positive result in real-time PCR assays, containing 9 × 10
1
to 6 × 10
5
dsrB
gene copies ml
−1
. DHPLC and denaturing gradient gel electrophoresis (DGGE) community profiles of the PCR-positive samples shared an overall similarity; both methods revealed the same samples to have the lowest and highest diversity. The SRB communities were diverse, and different
dsrB
compositions were detected at different geographical locations. The identified
dsrB
gene sequences belonged to several phylogenetic groups, such as
Desulfovibrio
,
Desulfococcus
,
Desulfomicrobium
,
Desulfobulbus
,
Desulfotignum
,
Desulfonatronovibrio
, and
Desulfonauticus
. DHPLC showed an advantage over DGGE in that the community profiles were very reproducible from run to run, and the resolved gene fragments could be collected using an automated fraction collector and sequenced without a further purification step. DGGE, on the other hand, included casting of gradient gels, and several rounds of rerunning, excising, and reamplification of bands were needed for successful sequencing. In summary, DHPLC proved to be a suitable tool for routine monitoring of the diversity of SRB communities in oil field samples.
Substantial within-Animal Diversity of Salmonella Isolates from Lymph Nodes, Feces, and Hides of Cattle at Slaughter
Authors: Gragg Sara E. , Loneragan Guy H. , Nightingale Kendra K. , Brichta-Harhay Dayna M. , Ruiz Henry , Elder Jacob R. , Garcia Lyda G. , Miller Markus F. , Echeverry Alejandro , Ramírez Porras Rosa G. , Brashears Mindy M.
Published: June 2013

Lymph nodes (mandibular, mesenteric, mediastinal, and subiliac;
n
= 68) and fecal (
n
= 68) and hide (
n
= 35) samples were collected from beef carcasses harvested in an abattoir in Mexico. Samples were analyzed for
Salmonella
, and presumptive colonies were subjected to latex agglutination. Of the isolates recovered, a subset of 91 was characterized by serotyping, pulsed-field gel electrophoresis (PFGE), and antimicrobial susceptibility phenotyping.
Salmonella
was isolated from 100% (hide), 94.1% (feces), 91.2% (mesenteric), 76.5% (subiliac), 55.9% (mandibular), and 7.4% (mediastinal) of samples. From the 87 typeable isolates, eight
Salmonella enterica
serotypes, including Kentucky (32.2%), Anatum (29.9%), Reading (17.2%), Meleagridis (12.6%), Cerro (4.6%), Muenster (1.1%), Give (1.1%), and Mbandaka (1.1%), were identified.
S
. Meleagridis was more likely (
P
= 0.03) to be recovered from lymph nodes than from feces or hides, whereas
S
. Kentucky was more likely (
P
= 0.02) to be recovered from feces and hides than from lymph nodes. The majority (59.3%) of the
Salmonella
isolates were pansusceptible; however, multidrug resistance was observed in 13.2% of isolates. Typing by PFGE revealed that
Salmonella
strains generally clustered by serotype, but some serotypes (Anatum, Kentucky, Meleagridis, and Reading) were comprised of multiple PFGE subtypes. Indistinguishable PFGE subtypes and, therefore, serotypes were isolated from multiple sample types, and multiple PFGE subtypes were commonly observed within an animal. Given the overrepresentation of some serotypes within lymph nodes, we hypothesize that certain
Salmonella
strains may be better at entering the bovine host than other
Salmonella
strains or that some may be better adapted for survival within lymph nodes. Our data provide insight into the ecology of
Salmonella
within cohorts of cattle and offer direction for intervention opportunities.
Development of a Dual-Index Sequencing Strategy and Curation Pipeline for Analyzing Amplicon Sequence Data on the MiSeq Illumina Sequencing Platform
Authors: Kozich James J. , Westcott Sarah L. , Baxter Nielson T. , Highlander Sarah K. , Schloss Patrick D.
Published: June 2013

Rapid advances in sequencing technology have changed the experimental landscape of microbial ecology. In the last 10 years, the field has moved from sequencing hundreds of 16S rRNA gene fragments per study using clone libraries to the sequencing of millions of fragments per study using next-generation sequencing technologies from 454 and Illumina. As these technologies advance, it is critical to assess the strengths, weaknesses, and overall suitability of these platforms for the interrogation of microbial communities. Here, we present an improved method for sequencing variable regions within the 16S rRNA gene using Illumina's MiSeq platform, which is currently capable of producing paired 250-nucleotide reads. We evaluated three overlapping regions of the 16S rRNA gene that vary in length (i.e., V34, V4, and V45) by resequencing a mock community and natural samples from human feces, mouse feces, and soil. By titrating the concentration of 16S rRNA gene amplicons applied to the flow cell and using a quality score-based approach to correct discrepancies between reads used to construct contigs, we were able to reduce error rates by as much as two orders of magnitude. Finally, we reprocessed samples from a previous study to demonstrate that large numbers of samples could be multiplexed and sequenced in parallel with shotgun metagenomes. These analyses demonstrate that our approach can provide data that are at least as good as that generated by the 454 platform while providing considerably higher sequencing coverage for a fraction of the cost.
Increases in the Amounts of Vibrio spp. in Oysters upon Addition of Exogenous Bacteria
Authors: Froelich Brett , Oliver James
Published: June 2013

The bacterial pathogen
Vibrio vulnificus
is found naturally in brackish coastal waters but can be greatly concentrated by filter-feeding organisms such as shellfish. Numerous experiments in which exogenous
V. vulnificus
cells are added to oysters in an attempt to measure uptake and depuration have been performed. In nearly all cases, results have shown that laboratory-grown bacteria are rapidly taken up by the oysters but ultimately eliminated, while naturally present
Vibrio
populations in oysters are resistant to depuration. In this study, oysters harvested during winter months, with low culturable
Vibrio
concentrations, were incubated in aquaria supplemented with strains of
V. vulnificus
that were either genotypically or phenotypically distinct from the background bacteria. These exogenous cells were eliminated from the oysters, as previously seen, but other vibrios already inhabiting the oysters responded to the
V. vulnificus
inoculum by rapidly increasing in number and maintaining a large stable population. The presence of such an oyster-adapted
Vibrio
population would be expected to prevent colonization by exogenous
V. vulnificus
cells, thus explaining the rapid depuration of these added bacteria.
Relationship between Presence of Cows with Milk Positive for Mycobacterium avium subsp. paratuberculosis-Specific Antibody by Enzyme-Linked Immunosorbent Assay and Viable M. avium subsp. paratuberculosis in Dust in Cattle Barns
Authors: Eisenberg Susanne W. F. , Chuchaisangrat Ruj , Nielen Mirjam , Koets Ad P.
Published: June 2013

Paratuberculosis, or Johne's disease, in cattle is caused byMycobacterium aviumsubsp.paratuberculosis, which has recently been suspected to be transmitted through dust. This longitudinal study on eight commercialM. aviumsubsp.paratuberculosis-positive dairy farms studied the relationship between the number of cows withM. aviumsubsp.paratuberculosisantibody-positive milk and the presence of viableM. aviumsubsp.paratuberculosisin settled-dust samples, including their temporal relationship. Milk and dust samples were collected in parallel monthly for 2 years.M. aviumsubsp.paratuberculosisantibodies in milk were measured by enzyme-linked immunosorbent assay (ELISA) and used as a proxy forM. aviumsubsp.paratuberculosisshedding. Settled-dust samples were collected by using electrostatic dust collectors (EDCs) at six locations in housing for dairy cattle and young stock. The presence of viableM. aviumsubsp.paratuberculosiswas identified by liquid culture and PCR. The results showed a positive relationship (odds ratio [OR], 1.2) between the number of cows with ELISA-positive milk and the odds of having positive EDCs in the same airspace as the adult dairy cattle. Moreover, the total number of lactating cows also showed an OR slightly above 1. This relationship remained the same for settled-dust samples collected up to 2 months before or after the time of milk sampling. The results suggest that removal of adult cows with milk positive forM. aviumsubsp.paratuberculosis-specific antibody by ELISA might result in a decrease in the presence of viableM. aviumsubsp.paratuberculosisin dust and therefore in the environment. However, this decrease is likely delayed by several weeks at least. In addition, the data support the notion thatM. aviumsubsp.paratuberculosisexposure of young stock is reduced by separate housing.
Saccharification of Cellulose by Recombinant Rhodococcus opacus PD630 Strains
Authors: Hetzler Stephan , Bröker Daniel , Steinbüchel Alexander
Published: June 2013

The noncellulolytic actinomycete
Rhodococcus opacus
strain PD630 is the model oleaginous prokaryote with regard to the accumulation and biosynthesis of lipids, which serve as carbon and energy storage compounds and can account for as much as 87% of the dry mass of the cell in this strain. In order to establish cellulose degradation in
R. opacus
PD630, we engineered strains that episomally expressed six different cellulase genes from
Cellulomonas fimi
ATCC 484 (
cenABC
,
cex
,
cbhA
) and
Thermobifida fusca
DSM43792 (
cel6A
), thereby enabling
R. opacus
PD630 to degrade cellulosic substrates to cellobiose. Of all the enzymes tested, five exhibited a cellulase activity toward carboxymethyl cellulose (CMC) and/or microcrystalline cellulose (MCC) as high as 0.313 ± 0.01 U · ml
−1
, but recombinant strains also hydrolyzed cotton, birch cellulose, copy paper, and wheat straw. Cocultivations of recombinant strains expressing different cellulase genes with MCC as the substrate were carried out to identify an appropriate set of cellulases for efficient hydrolysis of cellulose by
R. opacus
. Based on these experiments, the multicellulase gene expression plasmid pCellulose was constructed, which enabled
R. opacus
PD630 to hydrolyze as much as 9.3% ± 0.6% (wt/vol) of the cellulose provided. For the direct production of lipids from birch cellulose, a two-step cocultivation experiment was carried out. In the first step, 20% (wt/vol) of the substrate was hydrolyzed by recombinant strains expressing the whole set of cellulase genes. The second step was performed by a recombinant cellobiose-utilizing strain of
R. opacus
PD630, which accumulated 15.1% (wt/wt) fatty acids from the cellobiose formed in the first step.
Serotypes, Virulence Factors, and Antimicrobial Susceptibilities of Vaginal and Fecal Isolates of Escherichia coli from Giant Pandas
Authors: Wang Xin , Yan Qigui , Xia Xiaodong , Zhang Yanming , Li Desheng , Wang Chengdong , Chen Shijie , Hou Rong
Published: June 2013

Although
Escherichia coli
typically colonizes the intestinal tract and vagina of giant pandas, it has caused enteric and systemic disease in giant pandas and greatly impacts the health and survival of this endangered species. In order to understand the distribution and characteristics of
E. coli
from giant pandas, 67 fecal and 30 vaginal
E. coli
isolates from 21 giant pandas were characterized for O serogroups, phylogenetic groups, antimicrobial susceptibilities, and pulsed-field gel electrophoresis (PFGE) profiles. In addition, these isolates were tested for the presence of extraintestinal pathogenic
E. coli
(ExPEC) and diarrheagenic
E. coli
(DEC) by multiplex PCR detection of specific virulence genes. The most prevalent serogroups for all
E. coli
isolates were O88, O18, O167, O4, and O158. ExPEC isolates were detected mostly in vaginal samples, and DEC isolates were detected only in fecal samples. Phylogenetic group B1 predominated in fecal isolates, while groups B2 and D were frequently detected in vaginal isolates. Resistance to trimethoprim-sulfamethoxazole was most frequently observed, followed by resistance to nalidixic acid and tetracycline. All except five isolates were typeable by using XbaI and were categorized into 74 PFGE patterns. Our findings indicate that panda
E. coli
isolates exhibited antimicrobial resistance, and potentially pathogenic
E. coli
isolates were present in giant pandas. In addition, these
E. coli
isolates were genetically diverse. This study may provide helpful information for developing strategies in the future to control
E. coli
infections of giant pandas.
Haloarchaeal-Type β-Ketothiolases Involved in Poly(3-Hydroxybutyrate-
co
-3-Hydroxyvalerate) Synthesis in Haloferax mediterranei
Authors: Hou Jing , Feng Bo , Han Jing , Liu Hailong , Zhao Dahe , Zhou Jian , Xiang Hua
Published: June 2013

The key enzymes for poly(3-hydroxybutyrate-
co
-3-hydroxyvalerate) (PHBV) biosynthesis in haloarchaea have been identified except the β-ketothiolase(s), which condense two acetyl coenzyme A (acetyl-CoA) molecules to acetoacetyl-CoA, or one acetyl-CoA and one propionyl-CoA to 3-ketovaleryl-CoA. Whole-genome analysis has revealed eight potential β-ketothiolase genes in the haloarchaeon
Haloferax mediterranei
, among which the PHBV-specific BktB and PhaA were identified by gene knockout and complementation analysis. Unlike all known bacterial counterparts encoded by a single gene, the haloarchaeal PhaA that was involved in acetoacetyl-CoA generation, was composed of two different types of subunits (PhaAα and PhaAβ) and encoded by the cotranscribed HFX_1023 (
phaA
α) and HFX_1022 (
phaA
β) genes. Similarly, the BktB that was involved in generation of acetoacetyl-CoA and 3-ketovaleryl-CoA, was also composed of two different types of subunits (BktBα and BktBβ) and encoded by cotranscribed HFX_6004 (
bktB
α) and HFX_6003 (
bktB
β). BktBα and PhaAα were the catalytic subunits and determined substrate specificities of BktB and PhaA, respectively. Their catalytic triad “Ser-His-His” was distinct from the bacterial “Cys-His-Cys.” BktBβ and PhaAβ both contained an oligosaccharide-binding fold domain, which was essential for the β-ketothiolase activity. Interestingly, BktBβ and PhaAβ were functionally interchangeable, although PhaAβ preferred functioning with PhaAα. In addition, BktB showed biotechnological potential for the production of PHBV with the desired 3-hydroxyvalerate fraction in haloarchaea. This is the first report of the haloarchaeal type of PHBV-specific β-ketothiolases, which are distinct from their bacterial counterparts in both subunit composition and catalytic residues.