bluuebunny/crossref_metadata_2025_split huggingface dataset:
Sensitive, Efficient Quantitation of
13
C-Enriched Nucleic Acids via Ultrahigh-Performance Liquid Chromatography–Tandem Mass Spectrometry for Applications in Stable Isotope Probing
Authors: Wilhelm Roland , Szeitz András , Klassen Tara L. , Mohn William W. 
Published: September 2014

ABSTRACT

Stable isotope probing (SIP) of nucleic acids is a powerful tool for studying the functional traits of microbial populations within complex communities, but SIP involves a number of technical challenges. Many of the difficulties in DNA-SIP and RNA-SIP experiments can be effectively overcome with an efficient, sensitive method for quantitating the isotopic enrichment of nucleic acids. Here, we present a sensitive method for quantitating
13
C enrichment of nucleic acids, requiring a few nanograms of sample, and we demonstrate its utility in typical DNA-SIP and RNA-SIP experiments. All five nucleobases (adenine, guanine, cytosine, thymine, and uracil) were separated and detected by using ultrahigh-performance liquid chromatography–tandem mass spectrometry. We detected all isotopic species in samples with as low as 1.5 atom%
13
C above natural abundance, using 1-ng loadings. Quantitation was used to characterize the isotopic enrichment kinetics of cellulose- and lignin-based microcosm experiments and to optimize the recovery of enriched nucleic acids. Application of our method will minimize the quantity of expensive isotopically labeled substrates required and reduce the risk of failed experiments due to insufficient recovery of labeled nucleic acids for sequencing library preparation.
Effects of Nicotine on Streptococcus gordonii Growth, Biofilm Formation, and Cell Aggregation
Authors: Huang R. , Li M. , Ye M. , Yang K. , Xu X. , Gregory R. L. 
Published: September 2014

ABSTRACT

Streptococcus gordonii
is a commensal species of human oral flora. It initiates dental biofilm formation and provides binding sites for later colonizers to attach to and generate mature biofilm. Smoking is the second highest risk factor for periodontal disease, and cigarette smoke extract has been reported to facilitate
Porphyromonas gingivalis
-
S. gordonii
dual-species biofilm formation. Our hypothesis is that nicotine, one of the most important and active components of tobacco, stimulates
S. gordonii
multiplication and aggregation. In the present study,
S. gordonii
planktonic cell growth (kinetic absorbance and CFU), biofilm formation (crystal violet stain and confocal laser scanning microscopy [CLSM]), aggregation with/without sucrose, and 11 genes that encode binding proteins or regulators of gene expression were investigated. Results demonstrated planktonic cell growth was stimulated by 1 to 4 mg/ml nicotine treatment. Biofilm formation was increased at 0.5 to 4 mg/ml nicotine. CLSM indicated bacterial cell mass was increased by 2 and 4 mg/ml nicotine, but biofilm extracellular polysaccharide was not significantly affected by nicotine. Cell aggregation was upregulated by 4, 8, and 16 mg/ml nicotine with sucrose and by 16 mg/ml nicotine without sucrose. Quantitative reverse transcriptase PCR indicated
S. gordonii
abpA
,
scaA
,
ccpA
, and
srtA
were upregulated in planktonic cells by 2 mg/ml nicotine. In conclusion, nicotine stimulates
S. gordonii
planktonic cell growth, biofilm formation, aggregation, and gene expression of binding proteins. Those effects may promote later pathogen attachment to tooth surfaces, the accumulation of tooth calculus, and the development of periodontal disease in cigarette smokers.
High-Level Production of Plasmid DNA by Escherichia coli DH5α Ω
sacB
by Introducing
inc
Mutations
Authors: Trivedi Ram Narayan , Akhtar Parvez , Meade Jonathan , Bartlow Patrick , Ataai Mohammad M. , Khan Saleem A. , Domach Michael M. 
Published: September 2014

ABSTRACT

For small-copy-number pUC-type plasmids, the
inc1
and
inc2
mutations, which deregulate replication, were previously found to increase the plasmid copy number 6- to 7-fold. Because plasmids can exert a growth burden, it was not clear if further amplification of copy number would occur due to
inc
mutations when the starting point for plasmid copy number was orders of magnitude higher. To investigate further the effects of the
inc
mutations and the possible limits of plasmid synthesis, the parent plasmid pNTC8485 was used as a starting point. It lacks an antibiotic resistance gene and has a copy number of ∼1,200 per chromosome. During early stationary-phase growth in LB broth at 37°C,
inc2
mutants of pNTC8485 exhibited a copy number of ∼7,000 per chromosome. In minimal medium at late log growth, the copy number was found to be significantly increased, to approximately 15,000. In an attempt to further increase the plasmid titer (plasmid mass/culture volume), enzymatic hydrolysis of the selection agent, sucrose, at late log growth extended growth and tripled the total plasmid amount such that an approximately 80-fold gain in total plasmid was obtained compared to the value for typical pUC-type vectors. Finally, when grown in minimal medium, no detectable impact on the exponential growth rate or the fidelity of genomic or plasmid DNA replication was found in cells with deregulated plasmid replication. The use of
inc
mutations and the sucrose degradation method presents a simplified way for attaining high titers of plasmid DNA for various applications.
The Facultative Symbiont Rickettsia Protects an Invasive Whitefly against Entomopathogenic Pseudomonas syringae Strains
Authors: Hendry Tory A. , Hunter Martha S. , Baltrus David A. 
Published: September 2014

ABSTRACT

Facultative endosymbionts can benefit insect hosts in a variety of ways, including context-dependent roles, such as providing defense against pathogens. The role of some symbionts in defense may be overlooked, however, when pathogen infection is transient, sporadic, or asymptomatic. The facultative endosymbiont
Rickettsia
increases the fitness of the sweet potato whitefly (
Bemisia tabaci
) in some populations through mechanisms that are not yet understood. In this study, we investigated the role of
Rickettsia
in mediating the interaction between the sweet potato whitefly and
Pseudomonas syringae
, a common environmental bacterium, some strains of which are pathogenic to aphids. Our results show that
P. syringae
multiplies within whiteflies, leading to host death, and that whiteflies infected with
Rickettsia
show a decreased rate of death due to
P. syringae
. Experiments using plants coated with
P. syringae
confirmed that whiteflies can acquire the bacteria at a low rate while feeding, leading to increased mortality, particularly when the whiteflies are not infected with
Rickettsia
. These results suggest that
P. syringae
may affect whitefly populations in nature and that
Rickettsia
can ameliorate this effect. This study highlights the possible importance of interactions among opportunistic environmental pathogens and endosymbionts of insects.
Exposing the Secrets of Two Well-Known Lactobacillus casei Phages, J-1 and PL-1, by Genomic and Structural Analysis
Authors: Dieterle Maria Eugenia , Bowman Charles , Batthyany Carlos , Lanzarotti Esteban , Turjanski Adrián , Hatfull Graham , Piuri Mariana 
Published: September 2014

ABSTRACT

Bacteriophage J-1 was isolated in 1965 from an abnormal fermentation of Yakult using
Lactobacillus casei
strain Shirota, and a related phage, PL-1, was subsequently recovered from a strain resistant to J-1. Complete genome sequencing shows that J-1 and PL-1 are almost identical, but PL-1 has a deletion of 1.9 kbp relative to J-1, resulting in the loss of four predicted gene products involved in immunity regulation. The structural proteins were identified by mass spectrometry analysis. Similarly to phage A2, two capsid proteins are generated by a translational frameshift and undergo proteolytic processing. The structure of gene product 16 (gp16), a putative tail protein, was modeled based on the crystal structure of baseplate distal tail proteins (Dit) that form the baseplate hub in other
Siphoviridae
. However, two regions of the C terminus of gp16 could not be modeled using this template. The first region accounts for the differences between J-1 and PL-1 gp16 and showed sequence similarity to carbohydrate-binding modules (CBMs). J-1 and PL-1 GFP-gp16 fusions bind specifically to
Lactobacillus casei/paracasei
cells, and the addition of
l
-rhamnose inhibits binding. J-1 gp16 exhibited a higher affinity than PL-1 gp16 for cell walls of
L. casei
ATCC 27139 in phage adsorption inhibition assays, in agreement with differential adsorption kinetics observed for both phages in this strain. The data presented here provide insights into how
Lactobacillus
phages interact with their hosts at the first steps of infection.
Mutational Analysis of Essential Septins Reveals a Role for Septin-Mediated Signaling in Filamentation
Authors: Blankenship Jill R. , Cheng Shaoji , Woolford Carol A. , Xu Wenjie , Johnson Tanner M. , Rogers P. David , Fanning Saranna , Nguyen M. Hong , Clancy Cornelius J. , Mitchell Aaron P. 
Published: September 2014

ABSTRACT

Septin proteins are conserved structural proteins that often demarcate regions of cell division. The essential nature of the septin ring, composed of several septin proteins, complicates investigation of the functions of the ring, although careful analysis in the model yeast
Saccharomyces cerevisiae
has elucidated the role that septins play in the cell cycle. Mutation analysis of nonessential septins in the pathogenic fungus
Candida albicans
has shown that septins also have vital roles in
c
ell
w
all
r
egulation (CWR), hyphal formation, and pathogenesis. While mutations in nonessential septins have been useful in establishing phenotypes, the septin defect is so slight that identifying causative associations between septins and downstream effectors has been difficult. In this work, we describe decreased abundance by mRNA perturbation (DAmP) alleles of essential septins, which display a septin defect more severe than the defect observed in deletions of nonessential septins. The septin DAmP alleles have allowed us to genetically separate the roles of septins in hyphal growth and CWR and to identify the cyclic AMP pathway as a pathway that likely acts in a parallel manner with septins in hyphal morphogenesis.
Isoprenoid Biosynthesis in Plasmodium falciparum
Authors: Guggisberg Ann M. , Amthor Rachel E. , Odom Audrey R. 
Published: September 2014

ABSTRACT

Malaria kills nearly 1 million people each year, and the protozoan parasite
Plasmodium falciparum
has become increasingly resistant to current therapies. Isoprenoid synthesis via the methylerythritol phosphate (MEP) pathway represents an attractive target for the development of new antimalarials. The phosphonic acid antibiotic fosmidomycin is a specific inhibitor of isoprenoid synthesis and has been a helpful tool to outline the essential functions of isoprenoid biosynthesis in
P. falciparum
. Isoprenoids are a large, diverse class of hydrocarbons that function in a variety of essential cellular processes in eukaryotes. In
P. falciparum
, isoprenoids are used for tRNA isopentenylation and protein prenylation, as well as the synthesis of vitamin E, carotenoids, ubiquinone, and dolichols. Recently, isoprenoid synthesis in
P. falciparum
has been shown to be regulated by a sugar phosphatase. We outline what is known about isoprenoid function and the regulation of isoprenoid synthesis in
P. falciparum
, in order to identify valuable directions for future research.
The Contractile Vacuole as a Key Regulator of Cellular Water Flow in Chlamydomonas reinhardtii
Authors: Komsic-Buchmann Karin , Wöstehoff Luisa , Becker Burkhard 
Published: September 2014

ABSTRACT

Most freshwater flagellates use contractile vacuoles (CVs) to expel excess water. We have used
Chlamydomonas reinhardtii
as a green model system to investigate CV function during adaptation to osmotic changes in culture medium. We show that the contractile vacuole in
Chlamydomonas
is regulated in two different ways. The size of the contractile vacuoles increases during cell growth, with the contraction interval strongly depending on the osmotic strength of the medium. In contrast, there are only small fluctuations in cytosolic osmolarity and plasma membrane permeability. Modeling of the CV membrane permeability indicates that only a small osmotic gradient is necessary for water flux into the CV, which most likely is facilitated by the aquaporin major intrinsic protein 1 (MIP1). We show that MIP1 is localized to the contractile vacuole, and that the expression rate and protein level of MIP1 exhibit only minor fluctuations under different osmotic conditions. In contrast, SEC6, a protein of the exocyst complex that is required for the water expulsion step, and a dynamin-like protein are upregulated under strong hypotonic conditions. The overexpression of a CreMIP1-GFP construct did not change the physiology of the CV. The functional implications of these results are discussed.
Off-Target Effects of the Septin Drug Forchlorfenuron on Nonplant Eukaryotes
Authors: Heasley Lydia R. , Garcia Galo , McMurray Michael A. 
Published: September 2014

ABSTRACT

The septins are a family of GTP-binding proteins that form cytoskeletal filaments. Septins are highly conserved and evolutionarily ancient but are absent from land plants. The synthetic plant cytokinin forchlorfenuron (FCF) was shown previously to inhibit budding yeast cell division and induce ectopic septin structures (M. Iwase, S. Okada, T. Oguchi, and A. Toh-e, Genes Genet. Syst. 79:199–206, 2004,
http://dx.doi.org/10.1266/ggs.79.199
). Subsequent studies in a wide range of eukaryotes have concluded that FCF exclusively inhibits septin function, yet the mechanism of FCF action in nonplant cells remains poorly understood. Here, we report that the cellular effects of FCF are far more complex than previously described. The reported growth arrest of budding yeast cells treated with 1 mM FCF partly reflects sensitization caused by a
bud4
mutation present in the W303 strain background. In wild-type (
BUD4
+
) budding yeast, growth was inhibited at FCF concentrations that had no detectable effect on septin structure or function. Moreover, FCF severely inhibited the proliferation of fission yeast cells, in which septin function is nonessential. FCF induced fragmentation of budding yeast mitochondrial reticula and the loss of mitochondrial membrane potential. Mitochondria also fragmented in cultured mammalian cells treated with concentrations of FCF that previously were assumed to target septins only. Finally, FCF potently inhibited ciliation and motility and induced mitochondrial disorganization in
Tetrahymena thermophila
without apparent alterations in septin structure. None of these effects was consistent with the inhibition of septin function. Our findings point to nonseptin targets as major concerns when using FCF.
Developing Objective Metrics for Unit Staffing (DOMUS) study
Authors: Siddiqui I , Whittingham B , Meadowcroft K , Richardson M , Cooper J C , Belcher J , Morris E , Ismail K M K 
Published: September 2014

ObjectiveSafe midwifery staffing levels on delivery suites is a priority area for any maternity service. Escalation policies are tools that provide an operational response to emergency pressures. The aim of this study was to assess the feasibility of using a scoring system to contemporaneously assess the required staffing level based on demand and use this to determine delivery suite escalation level and utilise the information generated regarding clinical activity (Demand) and staffing levels (Capacity) to generate unit-specific calculation for the actual number of midwifery staff required.SettingA maternity unit of a university-affiliated tertiary referral hospital.DesignOver a 12-month period, specifically designed scoring sheets were completed by delivery suite shift co-ordinators four times a day (04:00, 10:00, 16:00 and 22:00). Based on the dependency score (Demand) and the number of midwifery staff available (Capacity), an escalation level was determined for each shift. The 80th centile of the demand was used to determine optimal capacity.ResultsA total of 1160 scoring sheets were completed. Average staff number throughout the year on any shift was 7 (range 3–11). Average dependency score was 7 (range 1–14). The 80th centile for demand was calculated to be 11.ConclusionsThis study stresses the importance and usefulness of a simple tool that can be used to determine the level of escalation on delivery suite based on an objective scoring system and can also be used to determine the appropriate staffing on delivery suite.
Reducing non-attendance rates in a community mental health team
Authors: Filippidou Maria , Lingwood Susie , Mirza Ilyas 
Published: September 2014

AbstractThe project aimed to improve productivity of psychiatric out patient clinic using quality improvement techniques through "Listening Into Action", a national programme designed to engage and support front-line clinicians to make improvements to patient care. We identified reasons as to why our patients missed appointments and then introduced a system to reduce "did not attend" (DNA) rates.Non-attendance at appointments results in a waste of resources and increases waiting times. It has been reported that DNA rates in mental health are higher compared to other settings. Therefore, reducing DNA rates are a priority for mental health care providers.We collected DNA rates over a period of months over May 2013 to September 2013. We conducted a patient survey to inquire why the patients missed their appointments. The aim of the project and results from the survey were presented and discussed at the multi-disciplinary team meeting to generate ideas for improvement and engage the team with the project. As the most frequent response from the survey was ‘forgetting the appointment’, we decided to introduce text messaging as an intervention to remind patients of their appointments. We also ensured that staff updated the mobile phone records for the patients at each appointment. We monitored the DNA rates after introducing this change on a monthly basis.Following our intervention, there was an overall reduction in DNA rates for all disciplines from 11.4% to 10.62% with the greatest change for medical DNA’s from 17.7% to 11.8 %.Results from a patient survey showed that the reasons for non-attendance are multi-factorial and require a complex approach. Our intervention was a simple one but still it demonstrated some effectiveness. Reducing DNA rates requires interventions to be regularly monitored so that their effect is sustained over a period of time.
Improving surgical weekend handover
Authors: Culwick Caroline , Devine Chris , Coombs Catherine 
Published: September 2014

AbstractEffective handovers are vital to patient safety and continuity of care, and this is recognised by several national bodies including the GMC. The existing model at Great Western Hospital (GWH) involved three general surgical teams and a urology team placing their printed patient lists, complete with weekend jobs, in a folder for the on-call team to collect at the weekend. We recognised a need to reduce time searching for patients, jobs and reviews, and to streamline weekend ward rounds.A unified weekend list ordering all surgical patients by ward and bed number was introduced. Discrepancies in the layout of each team’s weekday list necessitated the design of a new weekday list to match the weekend list to facilitate the easy transfer of information between the two lists. A colour coding system was also used to highlight specific jobs.Prior to this improvement project only 7.1% of those polled were satisfied with the existing system, after a series of interventions satisfaction increased to 85.7%. The significant increase in overall satisfaction with surgical handover following the introduction of the unified weekend list is promising. Locating patients and identifying jobs is easier and weekend ward rounds can conducted in a more logical and timely fashion. It has also helped facilitate the transition to consultant ward rounds of all surgical inpatients at the weekends with promising feedback from a recent consultants meeting.
Improving the frequency of visual infusion phlebitis (VIP) scoring on an oncology ward
Authors: Tzolos Evangelos , Salawu Abdulazeez 
Published: September 2014

AbstractPhlebitis from peripheral intravenous infusions is an important potential source of oncology patient morbidity. Important factors found to determine phlebitis incidence include the kind of infusion and dwell time of intravenous cannula. Early studies showed incidence rates of between 25-70% worldwide, and association with up to 10% of S. aureus bacteraemia. The introduction of the visual infusion phlebitis (VIP) score tool for assessment of the early signs of phlebitis, along with prompt removal of peripheral intravenous cannulas, has been very successful in reducing the incidence below the acceptable rate of 5%. However, achieving this goal depends on strict compliance with guidelines for cannula insertion, documentation, and assessment using the VIP tool.This study aimed to increase the use of VIP scoring tool to 100% on an oncology ward during a four to six month period in order to maximise its utility in phlebitis prevention. Three plan-do-study-act (PDSA) cycles were carried out, during which two major interventions were introduced. The first cycle aimed to improve junior doctors’ awareness of VIP scoring using presentations in induction meetings and posters. The second cycle ensured that ready access to the VIP tool was provided in the form of bedside intentional rounding charts. Proportions of intravenous cannulas with proper documentation and VIP assessment were measured before intervention and at nine subsequent bi-weekly time points.Pre-intervention, under 30% of cannulas were properly documented and assessed. This proportion rose to around 80% by the end of the second PDSA cycle and achieved 100% by the end of the third cycle.
HEART score and clinical gestalt have similar diagnostic accuracy for diagnosing ACS in an unselected population of patients with chest pain presenting in the ED
Authors: Visser Anniek , Wolthuis Albert , Breedveld Rob , ter Avest Ewoud 
Published: September 2014

BackgroundAcute coronary syndrome (ACS) can be a diagnostic challenge in the emergency department (ED). Recently, the HEART score was developed, a simple bedside scoring system that quantifies risk of ischaemic events in patients with undifferentiated chest pain presenting in the ED.ObjectiveIn this prospective cohort study, we compared the diagnostic accuracy of HEART score and clinical gestalt (clinical judgement) for diagnosing ACS in an unselected population of patients with chest pain presenting to the ED.MethodsHEART score (0–10) and clinical gestalt (low risk, intermediate risk or high risk of ACS) were prospectively determined in the ED in 255 patients presenting with chest pain by the treating physician. The reference standard was the presence of ACS, which was defined as either acute myocardial infarction (AMI) or the occurrence of a major adverse cardiac event within 6 weeks after presentation in the ED.Results75 out of 255 patients (29%) had an ACS. A HEART score ≤3 had a lower negative likelihood ratio (0.15 (0.06–0.36)) for ACS than a low risk based on clinical gestalt (0.35 (0.19–0.64)), whereas a high HEART score ≥7 had a higher positive likelihood ratio (5.2 (3.2–8.5) vs 3.1 (2.2–4.4)). However, c-statistic of HEART score was not significantly different from clinical gestalt (0.81 (0.76–0.86) vs 0.79 (0.73–0.84), p=0.13).ConclusionsOur study demonstrates that HEART score and clinical gestalt have similar diagnostic accuracy for diagnosing ACS in an unselected population of patients with chest pain presenting in the ED.