bluuebunny/crossref_metadata_2025_split huggingface dataset:
Phosphofructokinase and Malate Dehydrogenase Participate in the In Vitro Maturation of Porcine Oocytes
Authors: Breininger E , Vecchi Galenda BE , Alvarez GM , Gutnisky C , Cetica PD 
Published: October 2014

ContentsOocyte maturation depends on the metabolic activity of cumulus–oocyte complex (COC) that performs nutritive and regulatory functions during this process. In this work, the enzymes [phosphofructokinase (PFK) and malate dehydrogenase (MDH)] were tested to elucidate the metabolic profile of porcine COCs during the in vitro maturation (IVM). Enzymatic activity was expressed in U/COC and U/mg protein (specific activity) as mean ± SEM. In vitro maturation was performed with 2‐oxoglutarate (5, 10 and 20 mm) or hydroxymalonate (30, 60 and 100 mm) inhibitors of PFK and MDH, respectively. The PFK and MDH activities (U) remained constant during maturation. For PFK, the U were (2.48 ± 0.23) 10−5 and (2.54 ± 0.32) 10−5, and for MDH, the U were (4.72 ± 0.42) 10−5 and (4.38 ± 0.25) 10−5 for immature and in vitro matured COCs, respectively. The specific activities were significantly lower after IVM, for PFK (4.29 ± 0.48) 10−3 and (0.94 ± 0.12) 10−3, and for MDH (9.08 ± 0.93) 10−3 and (1.89 ± 0.10) 10−3 for immature and in vitro matured COCs, respectively. In vitro maturation percentages and enzymatic activity diminished with 20 mm 2‐oxoglutarate or 60 mm hydroxymalonate (p < 0.05). Viability was not affected by any concentration of the inhibitors evaluated. The U remained unchanged during IVM; however, the increase in the total protein content per COC provoked a decrease in the specific activity of both enzymes. Phosphofructokinase and MDH necessary for oocyte IVM would be already present in the immature oocyte. The presence of inhibitors of these enzymes impairs the meiotic maturation. Therefore, the participation of these enzymes in the energy metabolism of the porcine oocyte during IVM is confirmed in this study.
Use of Adaptive Laboratory Evolution To Discover Key Mutations Enabling Rapid Growth of Escherichia coli K-12 MG1655 on Glucose Minimal Medium
Authors: LaCroix Ryan A. , Sandberg Troy E. , O'Brien Edward J. , Utrilla Jose , Ebrahim Ali , Guzman Gabriela I. , Szubin Richard , Palsson Bernhard O. , Feist Adam M. 
Published: October 2014

ABSTRACT

Adaptive laboratory evolution (ALE) has emerged as an effective tool for scientific discovery and addressing biotechnological needs. Much of ALE's utility is derived from reproducibly obtained fitness increases. Identifying causal genetic changes and their combinatorial effects is challenging and time-consuming. Understanding how these genetic changes enable increased fitness can be difficult. A series of approaches that address these challenges was developed and demonstrated using
Escherichia coli
K-12 MG1655 on glucose minimal media at 37°C. By keeping
E. coli
in constant substrate excess and exponential growth, fitness increases up to 1.6-fold were obtained compared to the wild type. These increases are comparable to previously reported maximum growth rates in similar conditions but were obtained over a shorter time frame. Across the eight replicate ALE experiments performed, causal mutations were identified using three approaches: identifying mutations in the same gene/region across replicate experiments, sequencing strains before and after computationally determined fitness jumps, and allelic replacement coupled with targeted ALE of reconstructed strains. Three genetic regions were most often mutated: the global transcription gene
rpoB
, an 82-bp deletion between the metabolic
pyrE
gene and
rph
, and an IS element between the DNA structural gene
hns
and
tdk
. Model-derived classification of gene expression revealed a number of processes important for increased growth that were missed using a gene classification system alone. The methods described here represent a powerful combination of technologies to increase the speed and efficiency of ALE studies. The identified mutations can be examined as genetic parts for increasing growth rate in a desired strain and for understanding rapid growth phenotypes.
Improved Bacterial Detection Using Immobilized Acyl-Lysyl Oligomers
Authors: Marjieh Ibrahim , Meir Ohad , Zaknoon Fadia , Mor Amram 
Published: October 2014

ABSTRACT
The global need to improve bacterial detection in liquid media has motivated multidisciplinary research efforts toward developing new approaches that overcome the shortcomings of traditional techniques. We recently proposed the use of oligomers of acylated lysyls (OAKs) in their resin-linked form (ROAKs) for the efficient, robust, and inexpensive filtration of bacteria. Here, to investigate the potential for the use of ROAKs in downstream applications, we first examined the capacity of ROAKs to capture bacteria as a function of environmental conditions and structure-activity relationships (SARs). We next assessed their ability to release the captured bacteria and then combined both abilities to improve real-time PCR outcomes. ROAKs were able to deplete liquid samples of bacterial content after incubation or continuous flow, illustrating the efficient capture of different bacterial species under a wide range of ionic strength and pH conditions. We also show circumstances for the significant release of captured bacteria, live or dead, for further analysis. Finally, the SAR study revealed a shorter ROAK derivative exhibiting a capture capacity similar to that of the parent construct but the increased recovery of ROAK-bound bacteria, enabling improvement of the detection sensitivity by 20-fold. Collectively, the data support the potential usefulness of a simple, robust, and efficient approach for rapid capture/analysis of bacteria from tap water and, possibly, from more complex media.
Highly Diverse Endophytic and Soil Fusarium oxysporum Populations Associated with Field-Grown Tomato Plants
Authors: Demers Jill E. , Gugino Beth K. , Jiménez-Gasco María del Mar 
Published: October 2014

ABSTRACT

The diversity and genetic differentiation of populations of
Fusarium oxysporum
associated with tomato fields, both endophytes obtained from tomato plants and isolates obtained from soil surrounding the sampled plants, were investigated. A total of 609 isolates of
F. oxysporum
were obtained, 295 isolates from a total of 32 asymptomatic tomato plants in two fields and 314 isolates from eight soil cores sampled from the area surrounding the plants. Included in this total were 112 isolates from the stems of all 32 plants, a niche that has not been previously included in
F. oxysporum
population genetics studies. Isolates were characterized using the DNA sequence of the translation elongation factor 1α gene. A diverse population of 26 sequence types was found, although two sequence types represented nearly two-thirds of the isolates studied. The sequence types were placed in different phylogenetic clades within
F. oxysporum
, and endophytic isolates were not monophyletic. Multiple sequence types were found in all plants, with an average of 4.2 per plant. The population compositions differed between the two fields but not between soil samples within each field. A certain degree of differentiation was observed between populations associated with different tomato cultivars, suggesting that the host genotype may affect the composition of plant-associated
F. oxysporum
populations. No clear patterns of genetic differentiation were observed between endophyte populations and soil populations, suggesting a lack of specialization of endophytic isolates.
Recovery of Plasmid pEMB1, Whose Toxin-Antitoxin System Stabilizes an Ampicillin Resistance-Conferring β-Lactamase Gene in Escherichia coli, from Natural Environments
Authors: Lee Hyo Jung , Jin Hyun Mi , Park Moon Su , Park Woojun , Madsen Eugene L. , Jeon Che Ok 
Published: October 2014

ABSTRACT

Non-culture-based procedures were used to investigate plasmids showing ampicillin resistance properties in two different environments: remote mountain soil (Mt. Jeombong) and sludge (Tancheon wastewater treatment plant). Total DNA extracted from the environmental samples was directly transformed into
Escherichia coli
TOP10, and a single and three different plasmids were obtained from the mountain soil and sludge samples, respectively. Interestingly, the restriction fragment length polymorphism pattern of the plasmid from the mountain soil sample, designated pEMB1, was identical to the pattern of one of the three plasmids from the sludge sample. Complete DNA sequencing of plasmid pEMB1 (8,744 bp) showed the presence of six open reading frames, including a β-lactamase gene. Using BLASTX, the
orf5
and
orf6
genes were suggested to encode a CopG family transcriptional regulator and a plasmid stabilization system, respectively. Functional characterization of these genes using a knockout
orf5
plasmid (pEMB1Δ
parD
) and the cloning and expression of
orf6
(pET21b
parE
) indicated that these genes were antitoxin (
parD
) and toxin (
parE
) genes. Plasmid stability tests using pEMB1 and pEMB1Δ
parDE
in
E. coli
revealed that the
orf5
and
orf6
genes enhanced plasmid maintenance in the absence of ampicillin. Using a PCR-based survey, pEMB1-like plasmids were additionally detected in samples from other human-impacted sites (sludge samples) and two other remote mountain soil samples, suggesting that plasmids harboring a β-lactamase gene with a ParD-ParE toxin-antitoxin system occurs broadly in the environment. This study extends knowledge about the dissemination and persistence of antibiotic resistance genes in naturally occurring microbial populations.
Biochemical and Kinetic Characterization of the Recombinant ADP-Forming Acetyl Coenzyme A Synthetase from the Amitochondriate Protozoan Entamoeba histolytica
Authors: Jones Cheryl P. , Ingram-Smith Cheryl 
Published: October 2014

ABSTRACT

Entamoeba histolytica
, an amitochondriate protozoan parasite that relies on glycolysis as a key pathway for ATP generation, has developed a unique extended PP
i
-dependent glycolytic pathway in which ADP-forming acetyl-coenzyme A (CoA) synthetase (ACD; acetate:CoA ligase [ADP-forming]; EC 6.2.1.13) converts acetyl-CoA to acetate to produce additional ATP and recycle CoA. We characterized the recombinant
E. histolytica
ACD and found that the enzyme is bidirectional, allowing it to potentially play a role in ATP production or in utilization of acetate. In the acetate-forming direction, acetyl-CoA was the preferred substrate and propionyl-CoA was used with lower efficiency. In the acetyl-CoA-forming direction, acetate was the preferred substrate, with a lower efficiency observed with propionate. The enzyme can utilize both ADP/ATP and GDP/GTP in the respective directions of the reaction. ATP and PP
i
were found to inhibit the acetate-forming direction of the reaction, with 50% inhibitory concentrations of 0.81 ± 0.17 mM (mean ± standard deviation) and 0.75 ± 0.20 mM, respectively, which are both in the range of their physiological concentrations. ATP and PP
i
displayed mixed inhibition versus each of the three substrates, acetyl-CoA, ADP, and phosphate. This is the first example of regulation of ACD enzymatic activity, and possible roles for this regulation are discussed.
Tetrahymena Pot2 Is a Developmentally Regulated Paralog of Pot1 That Localizes to Chromosome Breakage Sites but Not to Telomeres
Authors: Cranert Stacey , Heyse Serena , Linger Benjamin R. , Lescasse Rachel , Price Carolyn 
Published: October 2014

ABSTRACT

Tetrahymena
telomeres are protected by a protein complex composed of Pot1, Tpt1, Pat1, and Pat2. Pot1 binds the 3′ overhang and serves multiple roles in telomere maintenance. Here we describe Pot2, a paralog of Pot1 which has evolved a novel function during
Tetrahymena
sexual reproduction. Pot2 is unnecessary for telomere maintenance during vegetative growth, as the telomere structure is unaffected by
POT2
macronuclear gene disruption. Pot2 is expressed only in mated cells, where it accumulates in developing macronuclei around the time of two chromosome processing events: internal eliminated sequence (IES) excision and chromosome breakage. Chromatin immunoprecipitation (ChIP) demonstrated Pot2 localization to regions of chromosome breakage but not to telomeres or IESs. Pot2 association with chromosome breakage sites (CBSs) occurs slightly before chromosome breakage. Pot2 did not bind CBSs or telomeric DNA
in vitro
, suggesting that it is recruited to CBSs by another factor. The telomere proteins Pot1, Pat1, and Tpt1 and the IES binding factor Pdd1 fail to colocalize with Pot2. Thus, Pot2 is the first protein found to associate specifically with CBSs. The selective association of Pot2 versus Pdd1 with CBSs or IESs indicates a mechanistic difference between the chromosome processing events at these two sites. Moreover, ChIP revealed that histone marks characteristic of IES processing, H3K9me3 and H3K27me3, are absent from CBSs. Thus, the mechanisms of chromosome breakage and IES excision must be fundamentally different. Our results lead to a model where Pot2 directs chromosome breakage by recruiting telomerase and/or the endonuclease responsible for DNA cleavage to CBSs.
WALK 2.0: Examining the effectiveness of Web 2.0 features to increase physical activity in a ‘real world’ setting: an ecological trial protocol
Authors: Caperchione Cristina M , Kolt Gregory S , Savage Trevor N , Rosenkranz Richard R , Maeder Anthony J , Vandelanotte Corneel , Duncan Mitch J , Van Itallie Anetta , Tague Rhys , Mummery W Kerry 
Published: October 2014

IntroductionLow levels of health-enhancing physical activity require novel approaches that have the potential to reach broad populations. Web-based interventions are a popular approach for behaviour change given their wide reach and accessibility. However, challenges with participant engagement and retention reduce the long-term maintenance of behaviour change. Web 2.0 features present a new and innovative online environment supporting greater interactivity, with the potential to increase engagement and retention. In order to understand the applicability of these innovative interventions for the broader population, ‘real-world’ interventions implemented under ‘everyday conditions’ are required. The aim of this study is to investigate the difference in physical activity behaviour between individuals using a traditional Web 1.0 website with those using a novel Web 2.0 website.Methods and analysisIn this study we will aim to recruit 2894 participants. Participants will be recruited from individuals who register with a pre-existing health promotion website that currently provides Web 1.0 features (http://www.10000steps.org.au). Eligible participants who provide informed consent will be randomly assigned to one of the two trial conditions: the pre-existing 10 000 Steps website (with Web 1.0 features) or the newly developed WALK 2.0 website (with Web 2.0 features). Primary and secondary outcome measures will be assessed by self-report at baseline, 3 months and 12 months, and include: physical activity behaviour, height and weight, Internet self-efficacy, website usability, website usage and quality of life.Ethics and disseminationThis study has received ethics approval from the University of Western Sydney Human Research Ethics Committee (Reference Number H8767) and has been funded by the National Health and Medical Research Council (Reference Number 589903). Study findings will be disseminated widely through peer-reviewed publications, academic conferences and local community-based presentations.Trial registration numberAustralian New Zealand Clinical Trials Registry Number: ACTRN12611000253909, WHO Universal Trial Number: U111-1119-1755
Migrant status and child and adolescent psychological well-being: evidence from Hong Kong's ‘Children of 1997’ birth cohort
Authors: Leung Cherry Y , Leung Gabriel M , Schooling C Mary 
Published: October 2014

BackgroundIn Western settings, migration is associated with psychological well-being, but studies inevitably focus on culturally distinct ethnic minorities, making it difficult to distinguish migration from cultural assimilation. Many children in Hong Kong, a developed non-Western setting, have migrant parents with the same Chinese ethnicity. This study examined the association of migration with the child's psychological well-being in Hong Kong.MethodsMultivariable linear regression was used in Hong Kong's ‘Children of 1997’ Chinese birth cohort to examine the adjusted associations of migration (both parents Hong Kong born n=4285, both parents migrant n=1921, mother-only migrant n=462, father-only migrant n=1110) with a parent-reported Rutter score for child behaviour at ∼7 (n=6294, 80% follow-up) and ∼11 years (n=5598, 71% follow-up), self-reported Culture-Free Self-Esteem Inventory score at ∼11 years (n=6937, 88% follow-up) and self-reported Patient Health Questionnaire-9 (PHQ-9) depressive symptom score at ∼13 years (n=5797, 73% follow-up), adjusted for sex, highest parental education and occupation, household income, maternal and paternal age at birth, age of assessment and survey mode (PHQ-9 only).ResultsMigration was unrelated to the overall self-esteem or depressive symptoms, but both parents migrant was associated with better behaviour (lower Rutter scores) at ∼7 years (β-coefficient (β) −1.07, 95% CI –1.48 to −0.66) and ∼11 years (−0.89, 95% CI −1.33 to −0.45).ConclusionsIn a non-Western context, migration appeared to be protective for childhood behaviour.
The detrimental clinical impact of severe angiographic vasospasm may be diminished by maximal medical therapy and intensive endovascular treatment
Authors: Mortimer Alex Mark , Steinfort Brendan , Faulder Ken , Bradford Celia , Finfer Simon , Assaad Nazih , Harrington Timothy 
Published: October 2014

ObjectiveSevere angiographic vasospasm (aVSP) is a risk factor for poor functional outcome following subarachnoid hemorrhage. We investigated the impact of angiographic surveillance and intensive endovascular treatment using transluminal balloon angioplasty (TBA) and/or verapamil infusion for severe aVSP through comparison of clinical outcomes in patients of similar presenting grade but with no/mild vasospasm.MethodsThis was an analysis of prospectively acquired clinical trial data. World Federation of Neurosurgical Societies (WFNS) grade 1–2 patients presenting within 72 h were included. Angiographic screening for vasospasm was undertaken at days 5–7 or in response to clinical deterioration. Severe aVSP was defined as >50% luminal narrowing on digital subtraction angiography. Treatment was instituted on the basis of radiographic findings and/or clinical deterioration. Discharge destination and favorable clinical outcomes (discharge Glasgow Outcome Score (GOS) 4–5, 90 day modified Rankin Scale (mRS) score 0–2, and GOS 4–5) for patients with severe aVSP were compared with those without significant vasospasm. Statistical analysis was undertaken using Fisher's exact test.Results63 WFNS grade 1–2 patients with minimal vasospasm were compared with 17 WFNS grade 1–2 patients with severe aVSP treated with induced hypertension and endovascular therapy. Results were available in 62 and 16 patients, respectively. Rates of favorable outcome did not differ significantly between the two groups. For patients with treated severe vasospasm, 90 day mRS 0–2 was seen in 15/17 (88.2%) and GOS 4–5 was achieved in 16/17 (94.1%).ConclusionsAn intensive endovascular approach of TBA and/or intra-arterial verapamil in combination with induced hypertension for severe aVSP may result in comparable clinical outcomes to those without vasospasm.
Astrocyte Elevated Gene-1 Interacts with Akt Isoform 2 to Control Glioma Growth, Survival, and Pathogenesis
Authors: Hu Bin , Emdad Luni , Bacolod Manny D. , Kegelman Timothy P. , Shen Xue-Ning , Alzubi Mohammad A. , Das Swadesh K. , Sarkar Devanand , Fisher Paul B. 
Published: October 2014

Abstract
The oncogene astrocyte elevated gene-1 (AEG-1; MTDH) is highly expressed in glioblastoma multiforme (GBM) and many other types of cancer, where it activates multiple signaling pathways that drive proliferation, invasion, angiogenesis, chemoresistance, radioresistance, and metastasis. AEG-1 activates the Akt signaling pathway and Akt and c-Myc are positive regulators of AEG-1 transcription, generating a positive feedback loop between AEG-1 and Akt in regulating tumorigenesis. Here, we describe in GBM cells a direct interaction between an internal domain of AEG-1 and the PH domain of Akt2, a major driver in GBM. Expression and interaction of AEG-1 and Akt2 are elevated in GBM and contribute to tumor cell survival, proliferation, and invasion. Clinically, in silico gene expression and immunohistochemical analyses of patient specimens showed that AEG-1 and Akt2 expression correlated with GBM progression and reduced patient survival. AEG-1–Akt2 interaction prolonged stabilization of Akt2 phosphorylation at S474, regulating downstream signaling cascades that enable cell proliferation and survival. Disrupting AEG-1–Akt2 interaction by competitive binding of the Akt2-PH domain led to reduced cell viability and invasion. When combined with AEG-1 silencing, conditional expression of Akt2-PH markedly increased survival in an orthotopic mouse model of human GBM. Our study uncovers a novel molecular mechanism by which AEG-1 augments glioma progression and offers a rationale to block AEG-1–Akt2 signaling function as a novel GBM treatment. Cancer Res; 74(24); 7321–32. ©2014 AACR.
Long-Chain Fatty Acid Analogues Suppress Breast Tumorigenesis and Progression
Authors: Gluschnaider Udi , Hertz Rachel , Ohayon Sarit , Smeir Elia , Smets Martha , Pikarsky Eli , Bar-Tana Jacob 
Published: October 2014

Abstract
Obesity and type 2 diabetes (T2D) are associated with increased breast cancer incidence and mortality, whereas carbohydrate-restricted ketogenic diets ameliorate T2D and suppress breast cancer. These observations suggest an inherent efficacy of nonesterified long-chain fatty acids (LCFA) in suppressing T2D and breast tumorigenesis. In this study, we investigated novel antidiabetic MEDICA analogues consisting of methyl-substituted LCFA that are neither β-oxidized nor esterified to generate lipids, prompting interest in their potential efficacy as antitumor agents in the context of breast cancer. In the MMTV-PyMT oncomouse model of breast cancer, in which we confirmed that tumor growth could be suppressed by a carbohydrate-restricted ketogenic diet, MEDICA treatment suppressed tumor growth, and lung metastasis, promoting a differentiated phenotype while suppressing mesenchymal markers. In human breast cancer cells, MEDICA treatment attenuated signaling through the STAT3 and c-Src transduction pathways. Mechanistic investigations suggested that MEDICA suppressed c-Src–transforming activity by elevating reactive oxygen species production, resulting in c-Src oxidation and oligomerization. Our findings suggest that MEDICA analogues may offer therapeutic potential in breast cancer and overcome the poor compliance of patients to dietary carbohydrate restriction. Cancer Res; 74(23); 6991–7002. ©2014 AACR.