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The excellent features of non-invasive molecular imaging, its progressive technology (real-time, whole-body imaging and quantification), and global impact by a growing infrastructure for positron emission tomography (PET) scanners are encouraging prospects to investigate new concepts, which could transform clinical care of complex infectious diseases. Researchers are aiming towards the extension beyond the routinely available radiopharmaceuticals and are looking for more effective tools that interact directly with causative pathogens. We reviewed and critically evaluated (challenges or pitfalls) antibiotic-derived PET radiopharmaceutical development efforts aimed at infection imaging. We considered both radiotracer development for infection imaging and radio-antibiotic PET imaging supplementing other tools for pharmacologic drug characterization; overall, a total of 20 original PET radiotracers derived from eleven approved antibiotics.

The white rhinoceros is the largest of the five extant rhinoceros species. The population is declining rapidly because of intense poaching. However, normal anatomical descriptions in this species are lacking. The purpose of this study is to describe the osseous anatomy of the middle and inner ear of the southern white rhinoceros using micro-focus X-ray computed tomography imaging. Four temporal bones obtained from two 1-day old southern white rhinoceros preserved in 10% formalin were scanned. Tri-dimensional reconstructions were obtained and volumes of the middle ear ossicles and inner ear structures were calculated. Excellent high spatial resolution 3D images were obtained for all samples and virtual models of the auditory ossicles and bony labyrinth were generated. Visualization of the tympanic membrane, middle ear and inner ear structures was possible in all samples. Whereas the stapes and incus had a shape similar to their human or equine counterparts, the malleus showed a unique appearance with a long rostral branch projecting latero-distally to the manubrium. The cochlea described 2 turns rostro-laterally around its axis, with a medial direction of rotation. However, identification of the soft tissue structures of the middle ear was sometimes difficult and visualization of the small structures of the membranous labyrinth was not possible using this formalin fixation and alternative techniques should be investigated. Further investigations are needed in order to provide a complete virtual model including both soft and bone tissues of this difficultly accessible region.

OBJECTIVES To describe and evaluate the clinical application of temporomandibular joint injections using betamethasone and ropivacaine in German Shepherd dogs suffering from non-odontogenic orofacial pain due to temporomandibular dysplasia and/or osteoarthritis. MATERIALS AND METHODS Outcomes in dogs presented with clinical signs of non-odontogenic orofacial pain associated to temporomandibular joint dysplasia and/or arthritis and treated with a temporomandibular joint injection were retrospectively-prospectively evaluated. RESULTS The overall clinical signs free period ranged between 25 to 1579 days, with an average of 461 days. The clinical signs free period for temporomandibular joint osteoarthritis scores 1, 2 and 3 were on average 659 days (180-1579 days), 134 days (42-355 days) and 723 days (25-1377 days), respectively. Similarly the temporomandibular dysplasia scores 1, 2 and 3 were on average 306 days (26-1579 days), 1377 days and 669 days (25-1429 days) respectively. Those dogs in which only one side was injected the clinical signs free period average was 639 days (25-1578 days), compared with dogs in which both temporomandibular joints were injected showing a clinical signs free period average of 378 days (42-1377 days). CLINICAL SIGNIFICANCE The temporomandibular joint injection technique proved to be feasible with a decent outcome in dogs suffering from non-odontogenic orofacial pain associated with temporomandibular joint osteoarthritis and/or dysplasia. Further randomised studies are required to confirm the effectiveness of this intervention.

Summary An 11-year-old Thoroughbred mare was diagnosed with sialolithiasis of the right parotid salivary duct. A firm nonpainful subcutaneous mass was palpable in the right maxillary region adjacent to premolar 4 (tooth 108). Radiographic and ultrasonographic examinations identified a discrete mineralised ovoid mass that was hyperechoic and produced an acoustic shadow. Surgical excision of the mass was performed under standing sedation using a transcutaneous approach and the excised sialolith submitted for histopathological and mineral composition analyses. Histological examination found no evidence of a nidus at the sialolith's core. Mineral analysis of the sialolith revealed its composition to be 40% calcium phosphate (apatite) and 60% calcium carbonate. The mare recovered with no post-operative complications and was clinically unremarkable 2 years later.

Developing tools for the study of protein carbohydrate interactions is an important goal in glycobiology. Cholera toxin inhibition is an interesting target in this context, as its inhibition may help the fight against cholera. For the study of novel ligands an affinity capillary electrophoresis (ACE) method was optimised and applied. The method uses unlabeled cholera toxin B-subunit (CTB) and unlabeled carbohydrate ligands based on ganglioside GM1-oligosaccharides (GM1os). In an optimized method at pH 4, adsorption of the protein to the capillary walls was prevented by a polybrene-dextran sulfate-polybrene (PB-DS-PB) coating. Different concentrations of the ligands were added to the background electrolyte. CTB binding was observed by a mobility shift that could be used for dissociation constant (Kd) determination. The Kd values of two GM1 derivatives differed by close to an order of magnitude (600 ± 20 nM and 90 ± 50 nM) which was in good agreement with the differences in their reported nanomolar IC50 values of an ELISA-type assay. Moreover, the selectivity of GM1os towards CTB was demonstrated using Influenza hemagglutinin (H5) as a binding competitor. The developed method can be an important platform for preclinical development of drugs targeting pathogen-induced secretory diarrhea. This article is protected by copyright. All rights reserved

Contains fulltext : 190759.pdf (Publisher’s version ) (Closed access) The complexity associated with how interventions result-or fail to result-in outcomes and how context matters is increasingly recognised. Logic models provide an important tool for handling complexity, with contrasting uses in programme evaluation and evidence synthesis. To reconcile these, we developed an approach that combines the strengths of both traditions, propose a taxonomy of logic models, and provide guidance on how to choose between approaches and types of logic models in systematic reviews and health technology assessments (HTA). The taxonomy distinguishes 3 approaches (a priori, staged, and iterative) and 2 types (systems-based and process-orientated) of logic models. An a priori logic model is specified at the start of the systematic review/HTA and remains unchanged. With a staged logic model, the reviewer prespecifies several points, at which major data inputs require a subsequent version. An iterative logic model is continuously modified throughout the systematic review/HTA process. System-based logic models describe the system, in which the interaction between participants, intervention, and context takes place; process-orientated models display the causal pathways leading from the intervention to multiple outcomes. The proposed taxonomy of logic models offers an improved understanding of the advantages and limitations of logic models across the spectrum from a priori to fully iterative approaches. Choice of logic model should be informed by scope of evidence synthesis, presence/absence of clearly defined population, intervention, comparison, outcome (PICO) elements, and feasibility considerations. Applications across distinct interventions and methodological approaches will deliver good practice case studies and offer further insights on the choice and implementation of logic modelling approaches.

Two springboks were diagnosed with chronic osteomyelitis of the metatarsus of the left hindlimb. The springboks had both suffered open fractures of the metatarsus of the left hindlimb 12 weeks and one week prior to presentation. External coaptation had initially been used to give stability and encourage fracture healing. This had proved unsuccessful in both cases and an infection and osteomyelitis had developed at the fracture sites. Two amputations had been subsequently performed in case 1 but had failed to resolve infection of the skin and subcutaneous tissues. An osteomyelitis of the underlying metatarsus had developed. No treatment apart from external coaptation had been performed before referral in case 2. Surgical amputation of the metatarsus and tarsus was performed as salvage procedures in both cases. The springboks adjusted to the loss of the left tarsus and metatarsus with no orthopaedic disorders at re-examinations 12 months after surgery.

CE-MS can be considered a useful analytical technique for the global profiling of (highly) polar and charged metabolites in various samples. Over the past few years, significant advancements have been made in CE-MS approaches for metabolomics studies. In this paper, which is a follow-up of a previous review paper covering the years 2012-2014 (Electrophoresis 2015, 36, 212-224), recent CE-MS strategies developed for metabolomics covering the literature from July 2014 to June 2016 are outlined. Attention will be paid to new CE-MS approaches for the profiling of anionic metabolites and the potential of SPE coupled to CE-MS is also demonstrated. Representative examples illustrate the applicability of CE-MS in the fields of biomedical, clinical, microbial, plant, and food metabolomics. A complete overview of recent CE-MS-based metabolomics studies is given in a table, which provides information on sample type and pretreatment, capillary coatings, and MS detection mode. Finally, general conclusions and perspectives are given.

An overview of the design and application of coupled solid-phase extraction-capillary electrophoresis (SPE-CE) systems reported in the literature between July 2013 and June 2015 is provided in this paper. The present article is a continuation of our previous review papers on this topic which covered the time period 2000-2013 (Electrophoresis 2008, 29, 108-128; Electrophoresis 2010, 31, 44-54; Electrophoresis 2012, 33, 243-250; Electrophoresis 2014, 35, 128-137). The use of in-line and on-line SPE-CE approaches is treated and outlined in this review. Recent advancements, such as, for example, the use of aptamers as affinity material for in-line SPE-CE, the use of a bead string design for in-line fritless SPE-CE, and new interfacing techniques for the on-line coupling of SPE to CE, are outlined. Selected examples demonstrate the applicability of the coupled SPE-CE systems for biomedical, pharmaceutical, environmental, and food studies. A complete overview of the recent SPE-CE studies is given in table format, providing information on sample type, SPE sorbent, coupling mode, detection mode, and LOD. Finally, some general conclusions and perspectives are provided.

RATIONALE, AIMS AND OBJECTIVES: The physician assistant (PA) is trained to perform clinical tasks traditionally performed by medical doctors (MDs). Previous research showed no difference in the level of clinical skills of PAs compared with MDs in a specific niche, that is the specialty in which they are employed. However, MDs as well as PAs working within a specialty have to be able to recognize medical problems in the full scope of medicine. The objective is to examine PA students' level of general clinical skills across the breadth of clinical cases. METHOD: A cross-sectional study was conducted. PA students and recently graduated MDs in the Netherlands were observed on their clinical skills by means of an objective structured clinical examination comprising five stations with common medical cases. The level of mastering history taking, physical examination, communication and clinical reasoning of PA students and MDs were described in means and standard deviation. Cohen's d was used to present effect sizes. RESULTS: PA students and MDs score about equal on history taking (PA 5.8 +/- 0.8 vs. MD 5.7 +/- 0.7), physical examination (PA 4.8 +/- 1.3 vs. MD 5.4 +/- 0.8) and communication (PA: 8.2 +/- 0.8 vs. MD: 8.6 +/- 0.5) in the full scope of medicine. In the quality of the report, including the patient management plan, PA students scored a mean of 6.0 +/- 0.6 and MDs 6.8 +/- 0.6. CONCLUSIONS: In this setting in the Netherlands, PA students and MDs score about equal in the appraisal of common cases in medical practice. The slightly lower scores of PA students' clinical reasoning in the full scope of clinical care may have raise attention to medical teams working with PAs and PA training programmes.

Objectives To assess outcomes for patients undergoing transcatheter aortic valve implantation (TAVI) versus surgical aortic valve replacement but with less than high risk. Background While there is abundant data for high risk patients there is insufficient data for reduced risk. Methods Patients undergoing TAVI or SAVR between 2007 and 2012 in Karlsruhe were considered. They were assessed by cardiac computed tomography, transoesophageal echocardiogram, and logistic EuroSCORE I (ES) and groups compared using Propensity Score Matching. Results The mean ES was 10.1±2.8 in the TAVI group (n = 419) and 5.7 ± 3.2 in the SAVR group (n = 722; P

The alleged traditional medicinal properties of rhinoceros horn resulted in a dramatic escalation in rhinoceros poaching in South Africa. Despite the listing of all species of rhinoceros in the International Union for Conservation of Nature Red List of threatened animals, their numbers are still declining rapidly. Based on the assumption that rhinoceros' horn consists of a collection of hollow tubules and intertubular hollow spaces, which allow internal fluid distribution, a horn devaluation procedure through infusion of chemicals and dyes was recently introduced. This procedure is costly and has a mortality risk. This study study provides the first detailed description of the development and resultant structure of the rhinoceros horn. The unique solid structure which consists of a large number of tightly packed filaments is the result of the cellular orientation of squamous epithelium corneocytes. What was previously thought to be microtubules is an optical illusion created by the orientation of the corneocytes in the solid filaments. We contest the scientific basis for infusing chemicals into the rhinoceros horn as a deterrent for human use.

In the field of metabolomics, CE-MS is now regarded as a useful complementary analytical technique for the profiling of (highly) polar ionogenic metabolites in biological samples. Over the past few years, significant advancements have been made in CE-MS approaches for metabolic profiling studies. This paper, which is a follow-up of three previous review papers covering the years 2000-2012 [Electrophoresis 2009, 30, 276-291; Electrophoresis 2011, 32, 52-65; Electrophoresis 2013, 34, 86-98], provides an update of these developments covering the scientific literature from July 2012 to June 2014. Attention will be paid to novel interfacing techniques for coupling CE to MS and their implications for metabolomics studies. The potential of CEC-MS and MEKC-MS are also considered, and CE-MS systems for high-throughput metabolic profiling are discussed. The applicability of CE-MS for metabolomics studies is demonstrated by representative examples in the fields of biomedical, clinical, microbial, plant, environmental, and food metabolomics. An overview of recent CE-MS-based metabolomics studies is given in a table, which provides information on sample type and pretreatment, capillary coatings, and MS detection mode. Finally, general conclusions and perspectives are given.

UNLABELLED A systematic review and meta-analysis were performed to determine the effect of therapeutic hypothermia using low-technology methods, in settings with facilities for intensive care, in term or near-term infants with hypoxic-ischaemic encephalopathy on mortality, neurological morbidity at discharge and neurological morbidity at 6-24 months. CONCLUSION Meta-analysis of three randomised controlled studies showed that low-technology therapeutic hypothermia in an intensive care setting significantly reduced the mortality and the neurological morbidity in survivors at discharge.

Background Evidence synthesis has seen major methodological advances in reducing uncertainty and estimating the sizes of the effects. Much less is known about how to assess the relative value of different outcomes. Objective To identify studies that assessed preferences for outcomes in health conditions. Methods Search strategy: we searched MEDLINE, EMBASE, PsycINFO and the Cochrane Library in February 2014. Inclusion criteria: eligible studies investigated preferences of patients, family members, the general population or healthcare professionals for health outcomes. The intention of this review was to include studies which focus on theoretical alternatives; studies which assessed preferences for distinct treatments were excluded. Data extraction: study characteristics as study objective, health condition, participants, elicitation method, and outcomes assessed in the study were extracted. Main results One hundred and twenty-four studies were identified and categorized into four groups: (1) multi criteria decision analysis (MCDA) (n = 71), (2) rating or ranking (n = 25), (3) utility eliciting (n = 5) and (4) studies comparing different methods (n = 23). The number of outcomes assessed by method group varied. The comparison of different methods or subgroups within one study often resulted in different hierarchies of outcomes. Conclusions A dominant method most suitable for application in evidence syntheses was not identified. As preferences of patients differ from those of other stakeholders (especially medical professionals), the choice of the group to be questioned is consequential. Further research needs to focus on validity and applicability of the identified methods.

Over the last decade, extensive numerical analyses have been conducted on axially loaded uniplanar K gap, T and X joints made of circular hollow sections (CHS), considering a wide range of geometrical parameters and different types of chord load. Based on the large amount of numerical data, in 2007, new joint capacity and chord stress equations were derived for CHS joints. This article presents detailed comparisons made between the (mean) strength equations for K gap joints and the experimental data available. These design equations were first discussed and approved by Sub-commission XV-E of the International Institute of Welding (IIW), subsequently published in the CIDECT Design Guide for CHS joints [1] and adopted in the 3rd edition of the IIW Static Design Procedure for Welded Hollow Section Joints [2], which formed the basis for the ISO 14346 recommendations [3] published recently.

This paper presents a summary, with background information, of the design recommendations developed recently for axially loaded X and T joints between elliptical hollow sections (EHS). These recommendations are based on experimental tests, finite element (FE) calibration and parametric studies carried out in a collaborative research programme between the universities of Singapore, Toronto and Delft. The strength equations are presented in a similar manner to those currently adopted for circular hollow section (CHS) joints and rectangular hollow section (RHS) joints in ISO 14346 (2013). The ISO recommendations are based on, and are similar to, those of the International Institute of Welding, IIW-XV-E, which were also published by the Comite International pour le Developpement et l’Etude de la Construction Tubulaire (CIDECT) in recent editions of their Design Guides.

This article presents an overview of the design and application of coupled SPE-CE systems that have been reported in the literature between January 2011 and June 2013. The present paper is an update of three previous review papers covering the years 2000-2011 (Electrophoresis 2008, 29, 108-128; Electrophoresis 2010, 31, 44-54; Electrophoresis 2012, 33, 243-250). The use of in-line and on-line SPE-CE approaches is described in this review. Emerging technological developments, such as the use of in-line frit-free SPE and chip-based SPE for extraction of sample components prior to CE analysis, are outlined. Selected examples illustrate the applicability of SPE-CE in biomedical, pharmaceutical, and environmental analysis. A complete overview of recent SPE-CE studies is given in table format, providing information on sample type, SPE sorbent, coupling mode, detection mode, and LOD. Finally, some general conclusions and future perspectives are provided.

A new MEKC-ESI-MS method for the analysis of amino acids (AAs) in human urine was developed employing ammonium perfluorooctanoate (APFO) as volatile surfactant. The influence of APFO on the MS signal of AAs was evaluated by infusion experiments, which showed that APFO hardly affects analyte responses and presents significantly less ion suppression than equal concentrations of ammonium acetate. In order to obtain efficient separation of AAs, MEKC parameters such as the pH and APFO concentration of the BGE, were optimized. Optimum AA resolution, including baseline separation of leucine and isoleucine, was obtained using 150 mM APFO (pH 9.0) as BGE, representing a considerable selectivity improvement over CE using 50 mM ammonium acetate (pH 9.0). Optimization of CE-MS parameters, such as sheath liquid composition and flow rate, and ESI and MS settings, led to LODs ranging from 9 to 26 ng/mL for the 20 tested AAs, which is highly favorable for an MEKC-MS method. Good linearity (r(2) > 0.99) and repeatability were obtained for all AAs tested with RSD values of 3.0-6.7% for peak area and

Loss of the caudal maxilla and ventral orbit after tumor resections can have negative functional and esthetic influences on the eye involved. This article reports on a case of a caudal maxillary acanthomatous ameloblastoma involving the ventral orbit that was resected and stabilized with a masseter muscle flap. The masseter muscle flap was generated from the superficial belly of the masseter muscle in order to close a defect in the orbital rim, created by a caudal maxillectomy. None of the published complications such as enophthalmos, excessive lacrimation, globe deviation, or strabismus were noted, 8 months following the procedure. The only clinical sign present at the time of re-evaluation was mild lacrimation. The authors propose the use of a masseter muscle flap as a viable technique in stabilizing the ventral orbit after caudal maxillectomy and ventral orbitectomy, preventing the complications associated with this surgery.

CE-MS has emerged as a powerful technique for the profiling of (highly) polar and charged metabolites in biological samples. This review provides an update of the most recent developments in CE-MS for metabolomics covering the scientific literature from July 2010 to June 2012. The present paper is an update of two previous review papers covering the years 2000-2010 (Electrophoresis 2009, 30, 276-291; Electrophoresis 2011, 32, 52-65). Emerging technological developments used in CE-MS for metabolomics are discussed, such as the use of novel interfacing techniques for coupling CE to MS. Representative examples illustrate the applicability of CE-MS in the fields of biomedical, clinical, microbial, plant, environmental and food metabolomics. Concerning targeted and non-targeted approaches, a comprehensive overview of recent CE-MS-based metabolomics studies is given in a table. Information on sample type and pretreatment, capillary coatings and MS detection mode is provided. Finally, general conclusions and perspectives are provided. © 2012.

Since its introduction in 1987, CE-MS has become an increasingly important technique for the analysis of biomolecules. Since our previous update on CE-MS methods within the field of intact protein analysis (Electrophoresis 2011, 32, 66-82), a variety of interesting methodological improvements and applications have been reported in literature. Therefore, this article presents an overview of the development and application of CE-MS for intact protein analysis as published between June 2010 and June 2012. The article is divided in sections that treat CE coupled to MS through ESI, MALDI, and ICP ionization, respectively. In the section about CE-ESI-MS, technological developments with respect to CE-MS interfacing, prevention of protein adsorption, and chip-based CE-MS are treated in more detail. Novel interfacing strategies and the development of improved capillary coating strategies appeared to be the major developments. Furthermore, in all sections, the applicability of CE-MS for intact protein analysis is demonstrated by representative examples, including important developments in the fields of biopharmaceutical characterization and the analysis of proteins in biological samples. Finally, some general conclusions and future perspectives are given. © 2012.

The potential of CE with native fluorescence detection (Flu) for the profiling of the therapeutic protein erythropoietin (EPO) was studied. EPO is a highly heterogeneous glycoprotein comprising a large number of isoforms. CE was applied to induce separation among the various glycoforms. Native Flu of EPO provided high detection selectivity yielding good signal-to-noise ratios and stable baselines, particularly when compared to conventional UV absorbance detection. In order to enhance EPO isoform resolution, CE was performed using a capillary with a neutral coating in combination with a simple BGE of 2.0 M acetic acid (pH 2.1). CE-Flu analysis of the EPO biological reference preparation of the European Pharmacopeia resulted in a highly detailed glycoform profile. Migration time RSDs for selected EPO isoforms were less than 0.22% and 0.80% for intraday and interday repeatability, respectively. RSDs for relative peak intensity of the major EPO isoforms were less than 3%. The achieved resolution, migration time stability, and sensitivity allowed discrimination of different EPO products (EPO-α and EPO-β) based on the recorded glycoform pattern. The developed CE-Flu method is relatively straightforward, and shows potential for quality control in biopharmaceutical production.

SUMMARY Variation in masticatory induced stress, caused by shape changes in the human skull, is quantified in this article. A comparison on masticatory induced stress is presented subject to a variation in human skull shape. Non-rigid registration is employed to obtain appropriate computational domain representations. This procedure allows the isolation of shape from other variations that could affect the results. An added benefit, revealed through the use of non-rigid registration to acquire appropriate domain representation, is the possibility of direct and objective comparison and manipulation. The effect of mapping uncertainty on the direct comparison is also quantified. As shown in this study, exact difference values are not necessarily obtained, but a non-rigid map between subject shapes and numerical results gives an objective indication on the location of differences. Copyright © 2012 John Wiley & Sons, Ltd.

BACKGROUND AND OBJECTIVES: Treatment of dilutional coagulopathy by transfusing fresh frozen plasma (FFP) remains sub-optimal. We hypothesized that partial replacement of transfused FFP by fibrinogen concentrate results in improved coagulant activity and haemostasis. This was tested in a controlled clinical intervention trial with patients experiencing massive bleeding during major surgery. METHODS: Patients undergoing major elective surgery were treated according to current protocols. When transfusion with FFP was required, patients were randomized as follows: group A received 4 units FFP and group B received 2 units FFP plus 2 g fibrinogen concentrate. Blood samples were taken before and after the intervention. Analysts were blinded to the treatment type. RESULTS: Group A (B) consisted of 21 (22) patients, in 16 (17) of whom bleeding stopped after intervention. Plasma fibrinogen increased significantly more in group B (0.57 g/l) than in group A (0.05 g/l). However, levels of prothrombin and factors VIII, IX and X increased more in group A than in group B. Rotational thromboelastometry (ROTEM) of whole blood and plasma revealed improved fibrin clot formation in group B but not in group A. Thrombin generation [calibrated automated thrombogram (CAT)] in plasma increased more in group A. Principal parameters determining whole-blood thromboelastometry were the fibrinogen level and platelet count. In vitro addition of fibrinogen and prothrombin complex concentrate to pre-intervention samples restored both ROTEM and CAT parameters. CONCLUSIONS: Partial replacement of transfused FFP by fibrinogen increases fibrin clot formation at the expense of less improved thrombin generation. Coagulation factors other than fibrinogen alone are required for full restoration of haemostasis.

A goal in proteomics is the analysis of proteins by LC-MS. The proteins are enzymatically digested and the resulting peptides are chromatographically separated and introduced into a tandem MS. The obtained MS data are used for a search in sequence databases, providing identification scores for the proteins. A method to improve that score is to increase the chromatographic separation and peak capacity. In this study, the chromatographic conditions were optimized for a relatively large gradient time by varying the flow rate and gradient composition. The influence of the monolithic column length (15 and 64 cm) and particle diameter (1.8 μm; 15-cm length) on the sample peak capacity, productivity and identification score was studied. For comparison of gradient systems, a scaling factor was introduced to normalize the properties/performance of columns for material, diameter and length. As model proteins/digests, a simple (myoglobin) and a larger (BSA) protein were used. The smallest peak width, highest identification scores (54 and 89% for BSA and myoglobin, respectively) and productivity (5.0 and 4.0, respectively) were obtained for the 15-cm particulate column. The study also demonstrates that a further increase in the chromatographic performance is beneficial for BSA but hardly increases the identification score for the relatively small myoglobin.

Background: Mastocytosis is an uncommon disease resulting from proliferation of abnormal mast cells infiltrating skin, bone marrow, liver, and other tissues. The aim of this study was to find differences in gene expression in peripheral blood cells of patients with indolent systemic mastocytosis compared to healthy controls. The second aim was to define a specific gene expression profile in patients with mastocytosis. Methods: Twenty-two patients with indolent systemic mastocytosis and 43 healthy controls were studied. Whole genome gene expression analysis was performed on RNA samples isolated from the peripheral blood. For amplification and labelling of the RNA, the Illumina TotalPrep 96 RNA Amplification Kit was used. Human HT-12_V3_expression arrays were processed. Data analysis was performed using GeneSpring, Genecodis, and Transcriptional System Regulators. Results: Comparison of gene expression between patients and controls revealed a significant difference (P 2 in gene expression in 2303 of the 48.794 analysed transcripts. Functional annotation indicated that the main pathways in which the differently expressed genes were involved are ubiquitin-mediated proteolysis, MAPK signalling pathway, pathways in cancer, and Jak-STAT signalling. The expression distributions for both groups did not overlap at all, indicating that many genes are highly differentially expressed in both groups. Conclusion: We were able to find abnormalities in gene expression in peripheral blood cells of patients with indolent systemic mastocytosis and to construct a gene expression profile which may be useful in clinical practice to predict the presence of mastocytosis and in further research of novel drugs.

A lamp-based fluorescence detection (Flu) system for CE was extended with a wavelength-resolved (WR) detector to allow recording of full protein emission spectra. WRFlu was achieved using a fluorescence cell that employs optical fibres to lead excitation light from a Xe-Hg lamp to the capillary window and protein fluorescence emission to a spectrograph equipped with a CCD. A 280 nm band pass filter etc. together with a 300 nm short pass cut-off filter was used for excitation. A capillary cartridge was modified to hold the detection cell in a commercial CE instrument enabling WRFlu in routine CE. The performance of the WRFlu detection was evaluated and optimised using lysozyme as model protein. Based on reference spectral data, a signal-intensity adjustment was introduced to correct for transmission losses in the detector optics that occurred for lower protein emission wavelengths. CE-WRFlu of lysozyme was performed using BGEs of 50 mM sodium phosphate (pH 6.5 or 3.0) and a charged-polymer coated capillary. Using the 3-D data set, signal averaging over time and emission-wavelength intervals was carried out to improve the S/N of emission spectra and electropherograms. The detection limit for lysozyme was 21 nM, providing sufficient sensitivity to obtain spectral information on protein impurities.

Capillary electrophoresis/mass spectrometry (CE/MS) is predominantly carried out using electrospray ionization (ESI). Recently, atmospheric pressure chemical ionization (APCI) and atmospheric pressure photoionization (APPI) have become available for CE/MS. With the VUV lamp turned off, the APPI source may also be used for CE/MS by thermospray ionization (TSI). In the present study the suitability of ESI, APCI, APPI and TSI for drug impurity profiling by CE/MS in the positive ion mode is evaluated. The drugs carbachol, lidocaine and proguanil and their potential impurities were used as test compounds, representing different molecular polarities. A background electrolyte of 100 mM acetic acid (pH 4.5) provided baseline separation of nearly all impurities from the respective drugs. APPI yielded both even- and odd-electron ions, whereas the other ionization techniques produced even-electron ions only. In-source fragmentation was more pronounced with APCI and APPI than with ESI and TSI, which was most obvious for proguanil and its impurities. In general, ESI and TSI appeared the most efficient ionization techniques for impurities that are charged in solution achieving detection limits of 100 ng/mL (full-scan mode). APPI and APCI showed a lower efficiency, but allowed ionization of low and high polarity analytes, although quaternary ammonium compounds (e.g. carbachol) could not be detected. Largely neutral compounds, such as the lidocaine impurity 2,6-dimethylaniline, could not be detected by TSI, and yielded similar detection limits (500 ng/mL) for ESI, APPI and APCI. In many cases, impurity detection at the 0.1% (w/w) level was possible when 1 mg/mL of parent drug was injected with at least one of the CE/MS systems. Overall, the tested CE/MS systems provide complementary information as illustrated by the detection and identification of an unknown impurity in carbachol.

This paper describes the hyphenation of cIEF and MALDI-TOF MS via a fractionation or spotting device. After focusing in cIEF the compounds are hydrodynamically mobilized and deposited on a MALDI target plate using a sheath liquid interface, which provides the catholyte solution and the electrical ground. From previous experiments, sample conditions that resulted in a high resolution in cIEF and acceptable protein signal intensity in MS were selected [Silvertand et al., Electrophoresis, 2008, 29, 1985-1996]. Besides the mixture of test proteins, the sample solution contains 1% Pharmalyte, 0.3% hydroxyethyl cellulose and 0.1% Tween 20 and is used for both optimization as well as characterization of the cIEF-MALDI-TOF MS system. Hyphenation problems encountered are mainly due to transfer of the liquid from the needle to the MALDI target plate and are solved by choosing the proper sheath catholyte (200 mM NH4OH in 50% methanol with 0.1% Tween20). MS electropherograms were reconstructed by plotting the intensities of the m/z values corresponding to the proteins versus migration time (related to spot number). Reproducibility, peak width and signal intensity for different focusing and spotting (fractionation) times were calculated using these reconstructed MS electropherograms as well as the UV electropherograms. The best results were obtained with focusing time of 75 min (no under- or overfocusing) and a spotting time of 5 s (highest protein signal intensity in MS). The applicability of the system is demonstrated by the analysis of a biopharmaceutical (glucagon) and its deamidation product.

The gain in separation efficiency for protein digests using long monolithic columns has been evaluated for a LC-MS system with capillary monolithic columns of different lengths (150 and 750 mm). A mixture of BSA, alpha-casein and beta-casein tryptic digests was used as a test sample. Peak capacity and productivity (peak capacity per unit time) were determined from base peak chromatograms and MS/MS data were used for protein identification by MASCOT database searching. Peak capacity and protein identification scores were higher for the long column. Analyses with similar gradient slope for the two columns produced ratios of the peak capacities that were slightly higher than the expected value of the square root of the column length ratio. Peak capacity ratios varied from 2.7 to 4.0 for four different gradient slopes, while protein identification scores were 2-4 times higher for the long column. Similar values were obtained for the productivity of both columns and the highest productivity was obtained at gradient times of 45 and 75 min for the short and long column, respectively. The use of long monolithic columns improves peptide separation and increases reliability of protein identification for complex digests, especially if longer gradients are chosen.

Non-aqueous electrokinetic chromatography (NAEKC) using cationic cyclodextrins (CDs) was coupled to electrospray ionization mass spectrometry (ESI-MS). A methanolic background electrolyte (BGE) was used which contained the hydrochloride salts of the single-isomer derivative cyclodextrins 6-monodeoxy-6-mono(2-hydroxy)propylamino-β-cyclodextrin (IPA-β-CD) or 6-monodeoxy-6-mono(3-hydroxy)propylamino-β-cyclodextrin (PA-β-CD). Applying a reversed capillary electrophoresis (CE) polarity (−30 kV), efficient separation of negatively charged compounds was achieved with plate numbers of up to 190 000. PA-β-CD appeared to be the most suitable for the separation of various acidic drugs while also providing a high chiral selectivity. Analyte detection was achieved by ESI-MS in the negative-ion mode using a sheath-liquid interface. In order to prevent current drops caused by the cathodic electroosmotic flow, a pressure of 15 mbar was applied on the inlet vial during NAEKC/MS analysis. The effect of the cationic CDs on the MS signal intensities of acidic test drugs was thoroughly studied. When a voltage is applied across the CE capillary, the overall mobility of the cationic CDs is towards the inlet vial so that no CD molecules enter the ion source. The chloride counter ions of the CDs, which migrated towards the capillary outlet, were found to cause ionization suppression, although significant analyte signals could still be detected. Depending on the CD concentration in the BGE, limits of detection for acidic drugs were in the 50–400 ng/mL range in full-scan mode. Copyright © 2008 John Wiley & Sons, Ltd.