bluuebunny/crossref_metadata_2025_split huggingface dataset:
Isolation of Single Choanoflagellate Cells from Field Samples and Establishment of Clonal Cultures
Authors: King Nicole , Young Susan L. , Abedin Monika , Carr Martin , Leadbeater Barry S.C. 
Published: February 2009

INTRODUCTIONChoanoflagellates are heterotrophic nanoflagellates: small, colorless protozoa that are present in marine and freshwater environments as well as in hydrated soils. Because they are the closest living relatives of the metazoa, the study of their cell biology and genomes promises to provide new insights into metazoan ancestry and origins. This protocol describes how to enrich freshly collected field samples and isolate single choanoflagellate cells. Two methods are commonly used: isolation by micropipetting single cells and isolation by dilution. The two methods are complementary and each has its advantages and disadvantages. The micropipette technique requires considerable experience. However, provided a single cell has been isolated, any culture arising from it will be clonal. The dilution technique does not require the same level of skill, but single-cell isolation is not ensured. For the latter technique, repeating the isolation procedure using higher dilutions is advised to ensure that only one cell is the basis of an established culture.
Long-Term Frozen Storage of Choanoflagellate Cultures
Authors: King Nicole , Young Susan L. , Abedin Monika , Carr Martin , Leadbeater Barry S.C. 
Published: February 2009

INTRODUCTIONChoanoflagellates are heterotrophic nanoflagellates: small, colorless protozoa that are present in marine and freshwater environments as well as in hydrated soils. Because they are the closest living relatives of the metazoa, the study of their cell biology and genomes promises to provide new insights into metazoan ancestry and origins. Choanoflagellates can be stored under stable conditions by freezing them with liquid nitrogen, as described here. To prevent cross-contamination among vials stored in the liquid phase of a liquid nitrogen freezer, the use of CryoFlex tubing to fully seal the vials is encouraged.
Visualizing the Subcellular Localization of Actin, β-Tubulin, and DNA in Monosiga brevicollis
Authors: King Nicole , Young Susan L. , Abedin Monika , Carr Martin , Leadbeater Barry S.C. 
Published: February 2009

INTRODUCTIONChoanoflagellates are heterotrophic nanoflagellates: small, colorless protozoa that are present in marine and freshwater environments as well as in hydrated soils. Because they are the closest living relatives of the metazoa, the study of their cell biology and genomes promises to provide new insights into metazoan ancestry and origins. They occur as spherical to ovoid cells exhibiting morphological similarities to the choanocytes (feeding cells) of sponges. Specifically, they are characterized by a single anterior flagellum surrounded by a funnel-shaped collar composed of 30 or more actin-based tentacles or microvilli. These structures can be visualized by staining for actin and β-tubulin. In this protocol, Monosiga brevicollis cells are fixed in dilute formaldehyde, attached to coverslips, and then exposed to antibodies against β-tubulin and an actin-specific stain; a simple DNA stain highlights the nucleus. The central challenge of this protocol is to treat the cells gently so that they retain their ultrastructure and remain attached to the coverslips during processing.
Preparation of Total RNA from Monosiga brevicollis and Other Choanoflagellates
Authors: King Nicole , Young Susan L. , Abedin Monika , Carr Martin , Leadbeater Barry S.C. 
Published: February 2009

INTRODUCTIONChoanoflagellates are heterotrophic nanoflagellates: small, colorless protozoa that are present in marine and freshwater environments as well as in hydrated soils. Because they are the closest living relatives of the metazoa, the study of their cell biology and genomes promises to provide new insights into metazoan ancestry and origins. The preparation of RNA is an important prerequisite for characterizing the gene expression profile of choanoflagellates under different conditions. Although most standard protocols (e.g., those using TRIzol) are sufficient for many purposes, the approach described here minimizes RNA degradation and provides a high-quality template for downstream protocols, including real-time polymerase chain reaction.
Rapid Preparation of Genomic DNA from Monosiga brevicollis and Other Choanoflagellates
Authors: King Nicole , Young Susan L. , Abedin Monika , Carr Martin , Leadbeater Barry S.C. 
Published: February 2009

INTRODUCTIONChoanoflagellates are heterotrophic nanoflagellates: small, colorless protozoa that are present in marine and freshwater environments as well as in hydrated soils. Because they are the closest living relatives of the metazoa, the study of their cell biology and genomes promises to provide new insights into metazoan ancestry and origins. This protocol describes a straightforward but crude isolation strategy for genomic DNA that has been effective on all tested choanoflagellate species. The resulting genomic DNA provides a suitable template for a variety of polymerase chain reaction (PCR) techniques, but is too impure for procedures that require very high-quality DNA such as Southern blotting.
Preparation of High-Molecular-Weight Genomic DNA from Monosiga brevicollis and Other Choanoflagellates
Authors: King Nicole , Young Susan L. , Abedin Monika , Carr Martin , Leadbeater Barry S.C. 
Published: February 2009

INTRODUCTIONChoanoflagellates are heterotrophic nanoflagellates: small, colorless protozoa that are present in marine and freshwater environments as well as in hydrated soils. Because they are the closest living relatives of the metazoa, the study of their cell biology and genomes promises to provide new insights into metazoan ancestry and origins. This protocol describes a simple genomic DNA isolation strategy that should be effective for almost all choanoflagellate species and will provide genomic templates suitable for polymerase chain reactions and Southern blot analysis.
Separation of Choanoflagellate and Bacterial Genomic DNA
Authors: King Nicole , Young Susan L. , Abedin Monika , Carr Martin , Leadbeater Barry S.C. 
Published: February 2009

INTRODUCTIONChoanoflagellates are heterotrophic nanoflagellates: small, colorless protozoa that are present in marine and freshwater environments as well as in hydrated soils. Because they are the closest living relatives of the metazoa, the study of their cell biology and genomes promises to provide new insights into metazoan ancestry and origins. However, the preparation of genomic DNA from cultures of Monosiga brevicollis or other choanoflagellates can be complicated by the presence of abundant bacterial DNA. Indeed, bacterial DNA can represent as much as 90% of the total yield from a standard genomic DNA preparation. This protocol describes the use of a CsCl gradient to separate choanoflagellate genomic DNA from the DNA of its bacterial prey. This strategy works only when the G+C content of the choanoflagellate genomic DNA is significantly different from that of its bacterial food. The final DNA sample from this protocol is expected to be 90%-95% enriched for choanoflagellate genomic DNA and to provide an adequate template for genome sequencing projects.
The OP9-DL1 System: Generation of T-Lymphocytes from Embryonic or Hematopoietic Stem Cells In Vitro
Authors: Holmes Roxanne , Zúñiga-Pflücker Juan Carlos 
Published: February 2009

INTRODUCTIONDifferentiation of mouse embryonic stem cells (ESCs) or hematopoietic stem cells (HSCs) from fetal liver or bone marrow into T-lymphocytes can be achieved in vitro with the support of OP9-DL1 cells, a bone-marrow-derived stromal cell line that ectopically expresses the Notch ligand, Delta-like 1 (Dll1). This approach provides a simple, versatile, and efficient culture system that allows for the commitment, differentiation, and proliferation of T-lineage cells from different sources of stem cells. This article contains a series of protocols, the first of which describes the establishment, maintenance, and storage of OP9 and OP9-DL1 cells. Subsequent protocols detail how to co-culture the OP9 and OP9-DL1 cells with either ESCs or HSCs from fetal liver or bone marrow, leading to in vitro differentiation of the stem cells into lymphocytes.
Patient safety incidents associated with airway devices in critical care: a review of reports to the UK National Patient Safety Agency
Authors: Thomas A. N. , McGrath B. A. 
Published: February 2009

SummaryWe used key words and letter sequences to identify airway‐associated patient safety incidents submitted to the UK National Patient Safety Agency from critical care units in England and Wales. We identified 1085 such airway incidents submitted in the two years from October 2005 to September 2007. Three hundred and twelve incidents (28.8%) involved neonates or babies. Of the total 1085 incidents, 200 (18.4%) were associated with tracheal intubation, 53 (4.9%) with tracheostomy and 893 (82.3%) were post‐procedure problems. One hundred and ten incidents (10.1%) were associated with more than temporary harm. Eighty‐eight intubation incidents were associated with equipment problems. Partial displacement of tubes resulted in more than temporary harm to the patient more frequently than complete tube displacement (15.7% vs 3.8%). Capnography was not described in any cases of displacement or blockage of tracheal or tracheostomy tubes. Recommendations concerning minimum standards for capnography, availability and checking of equipment and tracheostomy placement are made.
Influence of food intake on the bioavailability and efficacy of oral calcitonin
Authors: Karsdal Morten A. , Byrjalsen Inger , Azria Möise , Arnold Michel , Choi Les , Riis Bente J. , Christiansen Claus 
Published: February 2009

WHAT IS ALREADY KNOWN ABOUT THIS SUBJECT • No orally bioavailable peptides are currently approved by the US Food and Drug Administration or the European Medicines Agency.• Attempts have been made to develop an oral formulation of salmon calcitonin (sCT).• However, the effect of food intake on the bioavailability and efficacy of oral peptide formulations has not been systematically investigated. WHAT THIS STUDY ADDS • We provide evidence that oral doses of sCT may be optimally received in the fasting state or prior to meal intake.• Fasting or preprandial dosing led to improved bioavailability and effect on pharmacodynamic biomarkers of efficacy.• The results may aid other researchers involved in the formulation of oral peptides.AIMS To investigate the influence of food intake on the bioavailability and pharmacodynamic effects of salmon calcitonin (sCT).METHODS A single‐blind, randomized, partly placebo‐controlled study was conducted in 36 healthy postmenopausal female volunteers aged 62–74 years. The influence of food intake on oral dosing with 0.8 mg of sCT at 22.00 h was evaluated for a (i) predose meal at 18.00 h, (ii) predose meal at 20.00 h, (iii) predose meal at 21.00 h, (iv) postdose meal at 22.10 h, (v) no meal, and (vi) meal at 20.00 h and placebo at 22.00 h. Study biomarkers were plasma sCT levels and changes in the bone resorption marker CTX‐I (C‐terminal telopeptide of collagen type I).RESULTS The predose meal at 18.00 and 21.00 h significantly decreased relative oral bioavailability of sCT to 26% [95% confidence interval (CI) 0.09, 0.73 and 0.09, 0.75, P= 0.009 and P= 0.01]. The meal consumed 10 min after dosing decreased the oral bioavailability of sCT to 59% (95% CI 0.21, 1.68), although nonsignificant (P= 0.48). This decreased bioavailability led to lower relative suppression of serum CTX‐I, with an AUC of the 4‐h efficacy response of −91%–×–hours for those receiving a meal at 18.00 h, compared with −238%–×–hours for fasting subjects. The Dunnett‐adjusted difference between these two treatment sequences was 147%–×–hours (95% CI 68, 225) (P= 0.0003). The AUC was comparable among fasting subjects and those consuming a meal 10 min after dosing.CONCLUSIONS Postprandial dosing may limit the bioavailability of orally administered sCT. Maximal benefit can be achieved by dosing at least 10 min prior to meal time.
Metformin increases plasma ghrelin in Type 2 diabetes
Authors: Doogue Matthew P. , Begg Evan J. , Moore M. Peter , Lunt Helen , Pemberton Chris J. , Zhang Mei 
Published: February 2009

WHAT IS ALREADY KNOWN ABOUT THIS SUBJECT • Metformin, unlike the other major antihyperglycaemic drugs, is not associated with weight gain.• Ghrelin is an appetite‐stimulating hormone whose concentrations vary in relation to food, obesity and diabetes control.• Reports are conflicting about how metformin affects ghrelin concentrations, and this study was aimed at resolving this issue in patients with Type 2 diabetes. WHAT THIS STUDY ADDS • In this study an increase in ghrelin concentrations was seen in response to metformin treatment in patients with Type 2 diabetes.• This effect was opposite to what might be expected if the effect of metformin on weight control was mediated via suppression of ghrelin.• It is likely that the ghrelin response was secondary to improved glycaemic control.• Meal time changes in appetite and satiety did not correlate with changes in ghrelin, which suggests ghrelin may not be important in meal initiation.AIMS Metformin treatment of Type 2 diabetes is not usually associated with weight gain, and may assist with weight reduction. Plasma ghrelin concentrations are inversely associated with obesity and food intake. Metformin might therefore affect ghrelin concentrations, although previous studies have shown variable results in this regard. The primary aim of this study was to determine the effect of metformin on plasma ghrelin, appetite and satiety in patients with Type 2 diabetes.METHODS Eighteen patients with Type 2 diabetes were studied before and after 6 weeks of metformin treatment, which was titrated to 1 g b.d. On the study days patients were fed standard meals of 390 kcal at 08.00 and 12.30 h, plasma samples were collected at 15‐ and 30‐min intervals, and appetite and satiety were measured on visual analogue scales. Changes in the area under the concentration–time curves (AUCs) of plasma ghrelin, insulin, glucose, appetite and satiety were assessed and examined for correlations with metformin AUCs. Changes in fasting adiponectin and leptin were also measured.RESULTS Treatment with metformin increased the mean AUC (07.30–16.30 h) of plasma ghrelin by 24% (P= 0.003), while decreasing those of glucose by 19% (P < 0.001) and insulin by 19% (P= 0.001). No changes were detected in hunger and satiety, or in fasting adiponectin or leptin concentrations. There were no clear correlations between metformin plasma concentrations (AUC) and changes in plasma glucose, insulin or ghrelin.CONCLUSIONS Treatment of Type 2 diabetes with metformin was associated with increased plasma ghrelin concentrations, without associated changes in hunger and satiety.
Pharmacokinetics and pharmacodynamics of LC15‐0444, a novel dipeptidyl peptidase IV inhibitor, after multiple dosing in healthy volunteers
Authors: Lim Kyoung Soo , Cho Joo‐Youn , Kim Bo‐Hyung , Kim Jung‐Ryul , Kim Hwa‐Sook , Kim Dong‐Kyu , Kim Sung‐Ho , Yim Hyeon Joo , Lee Sung‐Hack , Shin Sang‐Goo , Jang In‐Jin , Yu Kyung‐Sang 
Published: February 2009

WHAT IS ALREADY KNOWN ABOUT THIS SUBJECT • The importance of efficient drug development using biomarkers has been increasingly emphasized, from preclinical studies to clinical trials.• However, as yet few ‘validated’ or ‘qualified’ biomarkers are used in early‐stage drug development in terms of clinical pharmacology and disease pathophysiology. WHAT THIS STUDY ADDS • This first‐time‐in‐human study provides evidence of the pharmacological activity of LC15‐0444 in humans, by using dipeptidyl peptidase IV activity and active glucagon‐like peptide‐1 concentrations.• LC15‐0444 possesses pharmacokinetic and pharmacodynamic characteristics that support a once‐daily dosing regimen.AIMS LC15‐0444 is a selective and competitive inhibitor of dipeptidyl peptidase (DPP) IV with potential for the treatment of Type 2 diabetes. The aim was to investigate the pharmacokinetic (PK) and pharmacodynamic (PD) profiles after multiple oral ascending doses of LC15‐0444 in healthy male subjects.METHODS A dose block‐randomized, double‐blind, placebo‐controlled, parallel group study was performed in three groups with 10 subjects (eight for active drug; two for placebo) per group; each group received 200, 400 or 600 mg of LC15‐0444 once daily for 10 days. Blood and urine samples were collected up to 24 h after the first dosing and up to 72 h after the last dosing.RESULTS The LC15‐0444 concentration–time profiles exhibited characteristics of multicompartment disposition. No dose‐ or time‐dependent change in PK parameters was observed. Mean elimination half‐life was in a range 16.6–20.1 h in the dose groups. Mean renal clearance and fraction of unchanged drug excreted in urine was 18.6–21.9 and 0.40–0.48 l h−1, respectively. In the steady state, mean accumulation ratios by dose groups were between 1.22 and 1.31. More than 80% inhibition of DPP IV activity from baseline was sustained for >24 h in all dose groups.CONCLUSIONS This study provides evidence of the pharmacological activity of LC15‐0444 in humans. LC15‐0444 possesses PK and PD characteristics that support a once‐daily dosing regimen.
Short‐term effects of inhaled salbutamol on autonomic cardiovascular control in healthy subjects: a placebo‐controlled study
Authors: Cekici Leyla , Valipour Arschang , Kohansal Robab , Burghuber Otto Chris 
Published: February 2009

WHAT IS ALREADY KNOWN ABOUT THIS SUBJECT • Previous reports have demonstrated a link between inhaled β2‐agonist use and cardiovascular morbidity and mortality.• The underlying mechanism for this relationship, however, remains controversial. WHAT THIS STUDY ADDS • Inhalation of a therapeutic dose of salbutamol resulted in significant haemodynamic changes, which were accompanied by a shift in cardiovascular autonomic tone towards increased sympathetic outflow in the absence of baroreceptor activation.• The observed changes in cardiac autonomic function may contribute to an increased cardiac risk associated with inhaled β2‐agonist treatment.AIMS To investigate short‐term effects of inhaled salbutamol on haemodynamic changes and cardiovascular autonomic control.METHODS A randomized, single‐blinded, placebo‐controlled study of 0.2 mg of inhaled salbutamol was conducted on 12 healthy nonsmoking volunteers with a mean age of 24 ± 2 years at two different testing sessions. Non‐invasively obtained continuous haemodynamic measurements of cardiac output, beat‐to‐beat arterial blood pressure, and total peripheral resistance were recorded prior to and for a total of 120 min after inhalation of the respective study drug. Continuous cardiovascular autonomic tone was recorded using power spectral analysis of heart rate and blood pressure variability. Spontaneous baroreceptor activity was assessed by the sequence method.RESULTS There were no significant changes in any of the baseline parameters between the different testing sessions. Inhalation of salbutamol caused a significant increase in cardiac output from 6.7 ± 1.3 to 7.7 ± 1.4 l min−1 (P < 0.05), and a decrease in total peripheral resistance from 1076 ± 192 to 905 ± 172 dyne s−1 cm−5 (P < 0.05) within 15 min after inhalation. Moreover, salbutamol significantly increased sympathetically mediated low‐frequency heart rate variability (P < 0.01), whereas parasympathetically mediated high‐frequency heart rate variability decreased (P < 0.01). All changes persisted for approximately 30 min and were fully reversible at 120 min. There were no significant changes in systolic blood pressure variability or spontaneous baroreceptor activity.CONCLUSIONS Inhalation of therapeutic doses of salbutamol in healthy subjects resulted in significant haemodynamic changes and a shift of sympathovagal balance towards increased sympathetic tone in the absence of baroreceptor activation.
Determinants of the drug utilization profile in the paediatric population in Italy's Lombardy Region
Authors: Clavenna Antonio , Sequi Marco , Bortolotti Angela , Merlino Luca , Fortino Ida , Bonati Maurizio 
Published: February 2009

WHAT IS ALREADY KNOWN ABOUT THIS SUBJECT • Drug utilization studies performed in the Italian paediatric population have found a prescription profile that appears to be specific to Italy.• Quantitative and qualitative differences with other European countries have been found.• The prevalence of drug prescription is higher in southern Italy compared with northern Italy. WHAT THIS STUDY ADDS • This is the first study to evaluate the geographical differences in the pattern of drug prescribing to children and adolescents in a large Italian region.• Large differences in the prescription prevalence rate were found between local health units.• Living in the eastern part of the Lombardy region is associated with the highest risk of drug exposure.AIMS To evaluate the intraregional differences in drug prescribing to children and adolescents.METHODS Prescriptions reimbursed by the National Health System, involving 1543 203 children and adolescents <18 years old and dispensed during 2005 by the retail pharmacies of 15 local health units (LHU) in the Lombardy Region, were analysed. Logistic regression analysis was performed to evaluate the association between drug prescription and age, gender, prescriber, and setting.RESULTS A total of 747 790 youths (48%) received at least one drug prescription. The prescription prevalence rate was highest in children 1–5 years old (65%), decreased with increasing age to 38% in adolescents, and was slightly higher in boys than in girls. Antibiotics and anti‐asthmatics were the most prescribed therapeutic classes. Amoxicillin + clavulanic acid was the most prescribed drug (18% of children; 20% of packages). Large differences were found in the drug prescription prevalence rate between the different LHUs. The rate ranged between 38.4 and 54.8%, and was not correlated to hospitalization rate in the paediatric population. Being 1–5 years old [odds ratio (OR) 4.51, 95% confidence interval (CI) 4.43, 4.58] and living in the eastern part of the region (OR 2.06, 95% CI 1.99, 2.13) were the factors associated with the highest risk of drug exposure.CONCLUSIONS The results resemble the profiles observed in other Italian contexts, in particular concerning the wide use of antibiotics and anti‐asthmatics. However, large differences were found between LHUs, highlighting the need for more detailed investigations on therapeutic needs, drug use, and related variables in different geographic contexts.
Novel mechanism of U18666A-induced tumour necrosis factor-α production in RAW 264·7 macrophage cells
Authors: Iftakhar-E-Khuda I , Koide N , Hassan F , Noman A S M , Dagvadorj J , Tumurkhuu G , Naiki Y , Komatsu T , Yoshida T , Yokochi T 
Published: February 2009

Summary
U18666A is a cholesterol transport-inhibiting agent that is used widely to mimic Niemann–Pick type C disease. The effect of U18666A on tumour necrosis factor (TNF)-α production in mouse macrophage cell line, RAW 264·7 cells and peritoneal macrophages was examined. U18666A induced TNF-α mRNA expression 48 h after the treatment, and TNF-α production 48 and 72 h after stimulation in RAW 264·7 cells. U18666A accumulated intracellular free cholesterol in the culture of normal medium but not cholesterol-free medium. U18666A also induced reactive oxygen species (ROS) generation in normal medium but much less in cholesterol-free medium. Anti-oxidant N-acetyl-L-cysteine (NAC) abolished U18666A-induced TNF-α production. U18666A led to the phosphorylation of p38 mitogen-activated protein kinase 24 and 48 h after the stimulation and the p38 activation was inhibited in presence of cholesterol-free medium or NAC. A p38 inhibitor reduced U18666A-induced TNF-α production. Taken together, U18666A was suggested to induce TNF-α production in RAW 264·7 cells via free cholesterol accumulation-mediated ROS generation.