bluuebunny/crossref_metadata_2025_split huggingface dataset:
Amplification and Purification of T4-Like Escherichia coli Phages for Phage Therapy: from Laboratory to Pilot Scale
Authors: Bourdin Gilles , Schmitt Bertrand , Marvin Guy Laure , Germond Jacques-Edouard , Zuber Sophie , Michot Lise , Reuteler Gloria , Brüssow Harald 
Published: December 2013

ABSTRACT

We investigated the amplification and purification of phage preparations with respect to titer, contamination level, stability, and technical affordability. Using various production systems (wave bags, stirred-tank reactors, and Erlenmeyer flasks), we obtained peak titers of 10
9
to 10
10
PFU/ml for T4-like coliphages. Phage lysates could be sterilized through 0.22-μm membrane filters without titer loss. Phages concentrated by differential centrifugation were not contaminated with cellular debris or bacterial proteins, as assessed by electron microscopy and mass spectrometry, respectively. Titer losses occurred by high-speed pelleting of phages but could be decreased by sedimentation through a sucrose cushion. Alternative phage concentration methods are prolonged medium-speed centrifugation, strong anion-exchange chromatography, and ultrafiltration, but the latter still allowed elevated lipopolysaccharide contamination. T4-like phages could not be pasteurized but maintained their infectivity titer in the cold chain. In the presence of 10 mM magnesium ions, phages showed no loss of titer over 1 month at 30°C.
Enumeration of Salmonellae in Table Eggs, Pasteurized Egg Products, and Egg-Containing Dishes by Using Quantitative Real-Time PCR
Authors: Jakočiūnė Džiuginta , Pasquali Frédérique , da Silva Cristiana Soares , Löfström Charlotta , Hoorfar Jeffrey , Klein Günter , Manfreda Gerardo , Olsen John Elmerdahl 
Published: December 2013

ABSTRACT

Salmonellae are a major cause of food-borne outbreaks in Europe, with eggs and egg products being identified as major sources. Due to the low levels of salmonellae in eggs and egg products, direct quantification is difficult. In the present study, enrichment quantitative real-time PCR (qPCR) was employed for enumeration of salmonellae in different matrices: table eggs, pasteurized egg products, and egg-containing dishes.
Salmonella enterica
serovar Enteritidis and
S. enterica
serovar Tennessee were used to artificially contaminate these matrices. The results showed a linear regression between the numbers of salmonellae and the quantification cycle (

C
q

) values for all matrices used, with the exception of pasteurized egg white. Standard curves were constructed by using both stationary-phase cells and heat-stressed cells, with similar results. Finally, this method was used to evaluate the fate of salmonellae in two egg-containing dishes, long egg and tiramisu, at abused refrigeration temperatures, and results indicated the growth of bacteria over a 1-week period. In conclusion, enrichment qPCR was shown to be reliable for enumeration of salmonellae in different egg products.
Protein Engineering by Random Mutagenesis and Structure-Guided Consensus of Geobacillus stearothermophilus Lipase T6 for Enhanced Stability in Methanol
Authors: Dror Adi , Shemesh Einav , Dayan Natali , Fishman Ayelet 
Published: December 2013

ABSTRACT

The abilities of enzymes to catalyze reactions in nonnatural environments of organic solvents have opened new opportunities for enzyme-based industrial processes. However, the main drawback of such processes is that most enzymes have a limited stability in polar organic solvents. In this study, we employed protein engineering methods to generate a lipase for enhanced stability in methanol, which is important for biodiesel production. Two protein engineering approaches, random mutagenesis (error-prone PCR) and structure-guided consensus, were applied in parallel on an unexplored lipase gene from
Geobacillus stearothermophilus
T6. A high-throughput colorimetric screening assay was used to evaluate lipase activity after an incubation period in high methanol concentrations. Both protein engineering approaches were successful in producing variants with elevated half-life values in 70% methanol. The best variant of the random mutagenesis library, Q185L, exhibited 23-fold-improved stability, yet its methanolysis activity was decreased by one-half compared to the wild type. The best variant from the consensus library, H86Y/A269T, exhibited 66-fold-improved stability in methanol along with elevated thermostability (+4.3°C) and a 2-fold-higher fatty acid methyl ester yield from soybean oil. Based on
in silico
modeling, we suggest that the Q185L substitution facilitates a closed lid conformation that limits access for both the methanol and substrate excess into the active site. The enhanced stability of H86Y/A269T was a result of formation of new hydrogen bonds. These improved characteristics make this variant a potential biocatalyst for biodiesel production.
Biochemical Characterization of a Novel
l
-Asparaginase with Low Glutaminase Activity from Rhizomucor miehei and Its Application in Food Safety and Leukemia Treatment
Authors: Huang Linhua , Liu Yu , Sun Yan , Yan Qiaojuan , Jiang Zhengqiang 
Published: December 2013

ABSTRACT

A novel fungal gene encoding the
Rhizomucor miehei
l
-asparaginase (RmAsnase) was cloned and expressed in
Escherichia coli
. Its deduced amino acid sequence shared only 57% identity with the amino acid sequences of other reported
l
-asparaginases. The purified
l
-asparaginase homodimer had a molecular mass of 133.7 kDa, a high specific activity of 1,985 U/mg, and very low glutaminase activity. RmAsnase was optimally active at pH 7.0 and 45°C and was stable at this temperature for 30 min. The final level of acrylamide in biscuits and bread was decreased by about 81.6% and 94.2%, respectively, upon treatment with 10 U RmAsnase per mg flour. Moreover, this
l
-asparaginase was found to potentiate a lectin's induction of leukemic K562 cell apoptosis, allowing lowering of the drug dosage and shortening of the incubation time. Overall, our findings suggest that RmAsnase possesses a remarkable potential for the food industry and in chemotherapeutics for leukemia.
Age-Related Shifts in the Density and Distribution of Genetic Marker Water Quality Indicators in Cow and Calf Feces
Authors: Shanks Orin C. , Kelty Catherine A. , Peed Lindsay , Sivaganesan Mano , Mooney Thomas , Jenkins Michael 
Published: December 2013

ABSTRACT

Calves make up about 16% of the current bovine population in the United States and can excrete high levels of human pathogens in their feces. We describe the density and distribution of genetic markers from 9 PCR- and real-time quantitative PCR-based assays, including CF128, CF193, CowM2, CowM3, GenBac3, Entero1, EC23S857, CampF2, and ttr-6, commonly used to help assess ambient surface water quality. Each assay was tested against a collection of 381 individual bovine fecal samples representing 31 mother and calf pairings collected over a 10-month time period from time of birth through weaning. Genetic markers reported to be associated with ruminant and/or bovine fecal pollution were virtually undetected in calves for up to 115 days from birth, suggesting that physiological changes in calf ruminant function impact host-associated genetic marker shedding. In addition, general fecal indicator markers for
Bacteroidales
,
Escherichia coli
, and
Enterococcus
spp. exhibited three separate trends across time, indicating that these bacteria respond differently to age-related physiological and dietary changes during calf development. The results of this study suggest that currently available PCR-based water quality indicator technologies can under- or overestimate fecal pollution originating from calves and identify a need for novel calf-associated source identification methods.
Immune Evasion, Stress Resistance, and Efficient Nutrient Acquisition Are Crucial for Intracellular Survival of Candida glabrata within Macrophages
Authors: Seider Katja , Gerwien Franziska , Kasper Lydia , Allert Stefanie , Brunke Sascha , Jablonowski Nadja , Schwarzmüller Tobias , Barz Dagmar , Rupp Steffen , Kuchler Karl , Hube Bernhard 
Published: December 2013

ABSTRACT

Candida glabrata
is both a human fungal commensal and an opportunistic pathogen which can withstand activities of the immune system. For example,
C. glabrata
can survive phagocytosis and replicates within macrophages. However, the mechanisms underlying intracellular survival remain unclear. In this work, we used a functional genomic approach to identify
C. glabrata
determinants necessary for survival within human monocyte-derived macrophages by screening a set of 433 deletion mutants. We identified 23 genes which are required to resist killing by macrophages. Based on homologies to
Saccharomyces cerevisiae
orthologs, these genes are putatively involved in cell wall biosynthesis, calcium homeostasis, nutritional and stress response, protein glycosylation, or iron homeostasis. Mutants were further characterized using a series of
in vitro
assays to elucidate the genes' functions in survival. We investigated different parameters of
C. glabrata
-phagocyte interactions: uptake by macrophages, replication within macrophages, phagosomal pH, and recognition of mutant cells by macrophages as indicated by production of reactive oxygen species and tumor necrosis factor alpha (TNF-α). We further studied the cell surface integrity of mutant cells, their ability to grow under nutrient-limited conditions, and their susceptibility to stress conditions mirroring the harsh environment inside a phagosome. Additionally, resistance to killing by neutrophils was analyzed. Our data support the view that immune evasion is a key aspect of
C. glabrata
virulence and that increased immune recognition causes increased antifungal activities by macrophages. Furthermore, stress resistance and efficient nutrient acquisition, in particular, iron uptake, are crucial for intraphagosomal survival of
C. glabrata
.
Molecular Components of Nitrate and Nitrite Efflux in Yeast
Authors: Cabrera Elisa , González-Montelongo Rafaela , Giraldez Teresa , de la Rosa Diego Alvarez , Siverio José M. 
Published: December 2013

ABSTRACT

Some eukaryotes, such as plant and fungi, are capable of utilizing nitrate as the sole nitrogen source. Once transported into the cell, nitrate is reduced to ammonium by the consecutive action of nitrate and nitrite reductase. How nitrate assimilation is balanced with nitrate and nitrite efflux is unknown, as are the proteins involved. The nitrate assimilatory yeast
Hansenula polymorpha
was used as a model to dissect these efflux systems. We identified the sulfite transporters Ssu1 and Ssu2 as effective nitrate exporters, Ssu2 being quantitatively more important, and we characterize the Nar1 protein as a nitrate/nitrite exporter. The use of strains lacking either
SSU2
or
NAR1
along with the nitrate reductase gene
YNR1
showed that nitrate reductase activity is not required for net nitrate uptake. Growth test experiments indicated that Ssu2 and Nar1 exporters allow yeast to cope with nitrite toxicity. We also have shown that the well-known
Saccharomyces cerevisiae
sulfite efflux permease Ssu1 is also able to excrete nitrite and nitrate. These results characterize for the first time essential components of the nitrate/nitrite efflux system and their impact on net nitrate uptake and its regulation.
Role of Retrograde Trafficking in Stress Response, Host Cell Interactions, and Virulence of Candida albicans
Authors: Liu Yaoping , Solis Norma V. , Heilmann Clemens J. , Phan Quynh T. , Mitchell Aaron P. , Klis Frans M. , Filler Scott G. 
Published: December 2013

ABSTRACT

In
Saccharomyces cerevisiae
, the vacuolar protein sorting complexes Vps51/52/53/54 and Vps15/30/34/38 are essential for efficient endosome-to-Golgi complex retrograde transport. Here we investigated the function of Vps15 and Vps51, representative members of these complexes, in the stress resistance, host cell interactions, and virulence of
Candida albicans
. We found that
C. albicans
vps15
Δ/Δ and
vps51
Δ/Δ mutants had abnormal vacuolar morphology, impaired retrograde protein trafficking, and dramatically increased susceptibility to a variety of stressors. These mutants also had reduced capacity to invade and damage oral epithelial cells
in vitro
and attenuated virulence in the mouse model of oropharyngeal candidiasis. Proteomic analysis of the cell wall of the
vps51
Δ/Δ mutant revealed increased levels of the Crh11 and Utr2 transglycosylases, which are targets of the calcineurin signaling pathway. The transcript levels of the calcineurin pathway members
CHR11
,
UTR2
,
CRZ1
,
CNA1
, and
CNA2
were elevated in the
vps15
Δ/Δ and
vps51
Δ/Δ mutants. Furthermore, these strains were highly sensitive to the calcineurin-specific inhibitor FK506. Also, deletion of
CHR11
and
UTR2
further increased the stress susceptibility of these mutants. In contrast, overexpression of
CRH11
and
UTR2
partially rescued their defects in stress resistance, but not host cell interactions. Therefore, intact retrograde trafficking in
C. albicans
is essential for stress resistance, host cell interactions, and virulence. Aberrant retrograde trafficking stimulates the calcineurin signaling pathway, leading to the increased expression of Chr11 and Utr2, which enables
C. albicans
to withstand environmental stress.
Using Aspergillus nidulans To Identify Antifungal Drug Resistance Mutations
Authors: He Xiaoxiao , Li Shengnan , Kaminskyj Susan G. W. 
Published: December 2013

ABSTRACT

Systemic fungal infections contribute to at least 10% of deaths in hospital settings. Most antifungal drugs target ergosterol (polyenes) or its biosynthetic pathway (azoles and allylamines), or beta-glucan synthesis (echinocandins). Antifungal drugs that target proteins are prone to the emergence of resistant strains. Identification of genes whose mutations lead to targeted resistance can provide new information on those pathways. We used
Aspergillus nidulans
as a model system to exploit its tractable sexual cycle and calcofluor white as a model antifungal agent to cross-reference our results with other studies. Within 2 weeks from inoculation on sublethal doses of calcofluor white, we isolated 24
A. nidulans
adaptive strains from sectoring colonies. Meiotic analysis showed that these strains had single-gene mutations. In each case, the resistance was specific to calcofluor white, since there was no cross-resistance to caspofungin (echinocandin). Mutation sites were identified in two mutants by next-generation sequencing. These were confirmed by reengineering the mutation in a wild-type strain using a gene replacement strategy. One of these mutated genes was related to cell wall synthesis, and the other one was related to drug metabolism. Our strategy has wide application for many fungal species, for antifungal compounds used in agriculture as well as health care, and potentially during protracted drug therapy once drug resistance arises. We suggest that our strategy will be useful for keeping ahead in the drug resistance arms race.
Relative bioequivalence of amoxicillin dissolved in breast milk
Authors: Yazdani-Brojeni Parvaneh , Garcia-Bournissen Facundo , Fujii Hisaki , Tanoshima Reo , Ito Shinya 
Published: December 2013

BackgroundOral antibiotics use in infants in developing countries is challenging because liquid formulations are often unavailable. However, dissolving solid formulation of drugs in water poses a risk of gastrointestinal infection. Although mother's milk may be a potential vehicle, no evidence exists to indicate that antibiotics dissolved in human milk are bioequivalent to those dissolved in water. Therefore, we compared pharmacokinetic parameters of an orally administered antibiotic, amoxicillin, dissolved in human milk, to those of water-dissolved amoxicillin.MethodsA pharmacokinetic study was conducted in 16 healthy adult volunteers in a randomised crossover design. Marketed amoxicillin powder for suspension was dissolved in either human milk or water at a final concentration of 50 mg/mL, and 10 mL was given orally in a fasting state. Timed blood samples were obtained and plasma amoxicillin was quantified using liquid chromatography-mass spectrometry.FindingsResults showed that pharmacokinetic parameters, including area-under-the-curve, Cmax and half-life of the water-based and milk-based amoxicillin administration were not significantly different. 90% CIs of the ratios of these parameters in concomitant breast milk administration to those of water were within 89% and 116%, suggesting they are bioequivalent (defined as a range between 80% and 125%).InterpretationWe conclude that oral administration of amoxicillin dissolved in human milk at 50 mg/mL results in pharmacokinetics profiles comparable to amoxicillin dissolved in water. Pharmaceutical interactions between amoxicillin and breast milk are unlikely, suggesting no need to modify dosing schedules.
What are effective approaches to increasing rates of organ donor registration among ethnic minority populations: a systematic review
Authors: Deedat Sarah , Kenten Charlotte , Morgan Myfanwy 
Published: December 2013

ObjectivesTo identify effective interventions to increase organ donor registration and improve knowledge about organ donation among ethnic minorities in North America and the UK.DesignSystematic review.Data SourcesMEDLINE, EMBASE, PsycINFO, CINAHL and Cochrane Central searched up to November 2012, together with four trials databases and the grey literature.Review methodsA systematic search followed by assessment of eligibility and quality. An interpretive and thematic approach to synthesis was undertaken. This examined the nature and delivery of interventions in relation to a range of outcomes: verified registration, changing knowledge and a measured shift towards greater readiness.Results18 studies were included in the review, comprising educational and mass media interventions. Mass media interventions alone reported no significant change in the intention or willingness to register. Educational interventions either alone or combined with mass media approaches were more effective in increasing registration rates, with a strong interpersonal component and an immediate opportunity to register identified as important characteristics in successful change.ConclusionsEffective interventions need to be matched to the populations’ stage of readiness to register. Measured outcomes should include registration and shifts along the pathway towards this behavioural outcome.
Diagnostic workup for fever of unknown origin: a multicenter collaborative retrospective study
Authors: Naito Toshio , Mizooka Masafumi , Mitsumoto Fujiko , Kanazawa Kenji , Torikai Keito , Ohno Shiro , Morita Hiroyuki , Ukimura Akira , Mishima Nobuhiko , Otsuka Fumio , Ohyama Yoshio , Nara Noriko , Murakami Kazunari , Mashiba Kouichi , Akazawa Kenichiro , Yamamoto Koji , Senda Shoichi , Yamanouchi Masashi , Tazuma Susumu , Hayashi Jun 
Published: December 2013

ObjectiveFever of unknown origin (FUO) can be caused by many diseases, and varies depending on region and time period. Research on FUO in Japan has been limited to single medical institution or region, and no nationwide study has been conducted. We identified diseases that should be considered and useful diagnostic testing in patients with FUO.DesignA nationwide retrospective study.Setting17 hospitals affiliated with the Japanese Society of Hospital General Medicine.ParticipantsThis study included patients ≥18 years diagnosed with ‘classical fever of unknown origin’ (axillary temperature ≥38°C at least twice over a ≥3-week period without elucidation of a cause at three outpatient visits or during 3 days of hospitalisation) between January and December 2011.ResultsA total of 121 patients with FUO were enrolled. The median age was 59 years (range 19–94 years). Causative diseases were infectious disease in 28 patients (23.1%), non-infectious inflammatory disease in 37 (30.6%), malignancy in 13 (10.7%), other in 15 (12.4%) and unknown in 28 (23.1%). The median interval from fever onset to evaluation at each hospital was 28 days. The longest time required for diagnosis involved a case of familial Mediterranean fever. Tests performed included blood cultures in 86.8%, serum procalcitonin in 43.8% and positron emission tomography in 29.8% of patients.ConclusionsWith the widespread use of CT, FUO due to deep-seated abscess or solid tumour is decreasing markedly. Owing to the influence of the ageing population, polymyalgia rheumatica was the most frequent cause (9 patients). Four patients had FUO associated with HIV/AIDS, an important cause of FUO in Japan. In a relatively small number of cases, cause remained unclear. This may have been due to bias inherent in a retrospective study. This study identified diseases that should be considered in the differential diagnosis of FUO.
Targeted antibiotic treatment of lame sheep with footrot using either oxytetracycline or gamithromycin
Authors: Strobel H. , Lauseker M. , Forbes A. B. 
Published: December 2013

A study on parenteral antibiotic treatment in sheep footrot was conducted on 10 farms in southern Germany to obtain information on the efficacy of gamithromycin under practical use conditions compared with a positive control. On each farm, 20 (10 on one farm) lame sheep were clinically evaluated and divided into two groups. On day 1, sheep were treated once according to group with either long‐acting oxytetracycline (OTC) at 20 mg/kg bodyweight or gamithromycin at 6 mg/kg; clinical responses were assessed 21 days later. When compared with day 1, both treatments reduced clinical lameness, as reflected in the reduction in the number of footrot‐affected feet (OTC: 79.3 per cent; gamithromycin: 93.7 per cent) and in the severity of the lesions. The difference between the two treatments was significant (P<0.01) with an OR of 6.1 in favour of gamithromycin. Of the 33 sheep that were still lame on day 21, nine mildly affected animals were not re‐treated and the remaining 24 sheep were re‐treated with gamithromycin. On day 42, all but two (on the same farm) of the 33 sheep were cured, giving an overall response rate in this study to one or two parenteral antibiotic treatments of 99 per cent.
Tumor Suppressor VHL Functions in the Control of Mitotic Fidelity
Authors: Hell Michael P. , Duda Maria , Weber Thomas C. , Moch Holger , Krek Wilhelm 
Published: December 2013

Abstract
The von Hippel–Lindau (VHL) tumor suppressor protein pVHL is commonly mutated in clear cell renal cell carcinoma (ccRCC) and has been implicated in the control of multiple cellular processes that might be linked to tumor suppression, including promoting proper spindle orientation and chromosomal stability. However, it is unclear whether pVHL exerts these mitotic regulatory functions in vivo as well. Here, we applied ischemic kidney injury to stimulate cell division in otherwise quiescent mouse adult kidneys. We show that in the short term (5.5 days after surgery), Vhl-deficient kidney cells demonstrate both spindle misorientation and aneuploidy. The spindle misorientation phenotype encompassed changes in directed cell division, which may manifest in the development of cystic lesions, whereas the aneuploidy phenotype involved the occurrence of lagging chromosomes but not chromosome bridges, indicative of mitotic checkpoint impairment. Intriguingly, in the long term (4 months after the ischemic insult), Vhl-deficient kidneys displayed a heterogeneous pattern of ccRCC precursor lesions, including cysts, clear cell–type cells, and dysplasia. Together, these data provide direct evidence for a key role of pVHL in mediating oriented cell division and faithful mitotic checkpoint function in the renal epithelium, emphasizing the importance of pVHL as a controller of mitotic fidelity in vivo. Cancer Res; 74(9); 2422–31. ©2013 AACR.