From linear to metric functional analysis.

This article presents the beginning of a metric functional analysis. A major notion is metric functionals which extends that of horofunctions in metric geometry. Applications of the main tools are found in a wide variety of subjects such as random walks on groups, complex dynamics, surface topology, deep learning, evolution equations, and game theory, thus branching well outside of pure mathematics. In several cases, linear notions fail to describe linear phenomena that are naturally captured by metric concepts. An extension of the mean ergodic theorem testifies to this. A general metric fixed-point theorem is also proved.

Discovery of Species-unique Peptide Biomarkers of Bacterial Pathogens by Tandem Mass Spectrometry-based Proteotyping.

Mass spectrometry (MS) and proteomics offer comprehensive characterization and identification of microorganisms and discovery of protein biomarkers that are applicable for diagnostics of infectious diseases. The use of biomarkers for diagnostics is widely applied in the clinic and the use of peptide biomarkers is increasingly being investigated for applications in the clinical laboratory. Respiratory-tract infections are a predominant cause for medical treatment, although, clinical assessments and standard clinical laboratory protocols are time-consuming and often inadequate for reliable diagnoses. Novel methods, preferably applied directly to clinical samples, excluding cultivation steps, are needed to improve diagnostics of infectious diseases, provide adequate treatment and reduce the use of antibiotics and associated development of antibiotic resistance. This study applied nano-liquid chromatography (LC) coupled with tandem MS, with a bioinformatics pipeline and an in-house database of curated high-quality reference genome sequences to identify species-unique peptides as potential biomarkers for four bacterial pathogens commonly found in respiratory tract infections (RTIs): Staphylococcus aureus; Moraxella catarrhalis; Haemophilus influenzae and Streptococcus pneumoniae The species-unique peptides were initially identified in pure cultures of bacterial reference strains, reflecting the genomic variation in the four species and, furthermore, in clinical respiratory tract samples, without prior cultivation, elucidating proteins expressed in clinical conditions of infection. For each of the four bacterial pathogens, the peptide biomarker candidates most predominantly found in clinical samples, are presented. Data are available via ProteomeXchange with identifier PXD014522. As proof-of-principle, the most promising species-unique peptides were applied in targeted tandem MS-analyses of clinical samples and their relevance for identifications of the pathogens, i.e. proteotyping, was validated, thus demonstrating their potential as peptide biomarker candidates for diagnostics of infectious diseases.

Identification of surface proteins in a clinical Staphylococcus haemolyticus isolate by bacterial surface shaving.

BACKGROUND: The skin commensal Staphylococcus haemolyticus is an emerging nosocomial pathogen. Despite its clinical relevance, published information about S. haemolyticus virulence factors is scarce. In this study, the adhesive and biofilm forming properties of ten clinical and ten commensal S. haemolyticus strains were examined using standard adhesion and biofilm assays. One of the clinical strains was used to identify expressed surface proteins using bacterial surface shaving. Protein abundance was examined by a comparative analysis between bacterial protein expression after human keratinocyte (HaCaT) colonization and growth in cell culture media supplemented with serum. Relative protein quantification was performed by labeling peptides with tandem mass tags (TMT) prior to Mass Spectrometry analysis. Surface proteins can be used as novel targets for antimicrobial treatment and in diagnostics. RESULTS: Adherence to fibronectin, collagen and plastic was low in all tested strains, but with significantly higher adhesion to fibronectin (p = 0.041) and collagen (p = 0.001) in the commensal strains. There was a trend towards higher degree of biofilm formation in the clinical strains (p = 0.059). By using surface shaving, 325 proteins were detected, of which 65 were classified as surface proteins. Analyses showed that the abundance of nineteen (5.8%) proteins were significantly changed following HaCaT colonization. The bacterial Toll/interleukin-1 like (TIRs) domain containing protein (p = 0.04), the transglycosylase SceD (p = 0.01), and the bifunctional autolysin Atl (p = 0.04) showed a 1.4, 1.6- and 1.5-fold increased abundance. The staphylococcal secretory antigen (SsaA) (p = 0.04) was significantly downregulated (- 1.5 fold change) following HaCaT colonization. Among the 65 surface proteins the elastin binding protein (Ebps), LPXAG and LPXSG domain containing proteins and five LPXTG domain containing proteins were identified; three Sdr-like proteins, the extracellular matrix binding protein Embp and a SasH-like protein. CONCLUSIONS: This study has provided novel knowledge about expression of S. haemolyticus surface proteins after direct contact with eukaryotic cells and in media supplemented with serum. We have identified surface proteins and immune evasive proteins previously only functionally described in other staphylococcal species. The identification of expressed proteins after host-microbe interaction offers a tool for the discovery and design of novel targets for antimicrobial treatment.

Ultra high performance liquid chromatography method development for separation of omeprazole and related substances on core-shell columns using a Quality by Design approach.

An updated and improved method for analysis of omeprazole/esomeprazole and related substances on core-shell columns was developed using Fusion LC Method Development™. The method was optimized with respect to column type, column temperature, mobile phase pH level, and gradient time. Four different core-shell columns were examined to develop a method suitable for both high performance- and ultra-high performance liquid chromatography using a Quality by Design approach. The final method offers two alternative columns: Poroshell EC C18 (3.0 × 100 mm, 2.7 µm) or Poroshell HPH (3.0 × 100 mm, 2.7 µm) with the same gradient elution condition and mobile phase composition. Total run time is 18 min with 12 min of gradient elution. Phosphate buffer (15 mM, pH 7.8) is selected as the aqueous mobile phase and acetonitrile as the organic mobile phase. Column temperature is set at 40°C and ultraviolet detection at 302 nm. Furthermore, by studying parameters in a systematic way, an understanding of the effect of the input parameters enhances the method robustness and should allow for regulatory flexibility in terms of post-approval changes. Compared to the current United States Pharmacopeia method, the updated method is faster, more efficient and performs well above acceptance criteria.

Optimizing Column Length and Particle Size in Preparative Batch Chromatography Using Enantiomeric Separations of Omeprazole and Etiracetam as Models: Feasibility of Taguchi Empirical Optimization.

The overreaching purpose of this study is to evaluate new approaches for determining the optimal operational and column conditions in chromatography laboratories, i.e., how best to select a packing material of proper particle size and how to determine the proper length of the column bed after selecting particle size. As model compounds, we chose two chiral drugs for preparative separation: omeprazole and etiracetam. In each case, two maximum allowed pressure drops were assumed: 80 and 200 bar. The processes were numerically optimized (mechanistic modeling) with a general rate model using a global optimization method. The numerical predictions were experimentally verified at both analytical and pilot scales. The lower allowed pressure drop represents the use of standard equipment, while the higher allowed drop represents more modern equipment. For both compounds, maximum productivity was achieved using short columns packed with small-particle size packing materials. Increasing the allowed backpressure in the separation leads to an increased productivity and reduced solvent consumption. As advanced numerical calculations might not be available in the laboratory, we also investigated a statistically based approach, i.e., the Taguchi method (empirical modeling), for finding the optimal decision variables and compared it with advanced mechanistic modeling. The Taguchi method predicted that shorter columns packed with smaller particles would be preferred over longer columns packed with larger particles. We conclude that the simpler optimization tool, i.e., the Taguchi method, can be used to obtain "good enough" preparative separations, though for accurate processes, optimization, and to determine optimal operational conditions, classical numerical optimization is still necessary.

Proteomic analysis of enterotoxigenic Escherichia coli (ETEC) in neutral and alkaline conditions.

BACKGROUND: Enterotoxigenic Escherichia coli (ETEC) is a major cause of diarrhea in children and travelers to endemic areas. Secretion of the heat labile AB5 toxin (LT) is induced by alkaline conditions. In this study, we determined the surface proteome of ETEC exposed to alkaline conditions (pH 9) as compared to neutral conditions (pH 7) using a LPI Hexalane FlowCell combined with quantitative proteomics. Relative quantitation with isobaric labeling (TMT) was used to compare peptide abundance and their corresponding proteins in multiple samples at MS/MS level. For protein identification and quantification samples were analyzed using either a 1D-LCMS or a 2D-LCMS approach. RESULTS: Strong up-regulation of the ATP synthase operon encoding F1Fo ATP synthase and down-regulation of proton pumping proteins NuoF, NuoG, Ndh and WrbA were detected among proteins involved in regulating the proton and electron transport under alkaline conditions. Reduced expression of proteins involved in osmotic stress was found at alkaline conditions while the Sec-dependent transport over the inner membrane and outer membrane protein proteins such as OmpA and the ß-Barrel Assembly Machinery (BAM) complex were up-regulated. CONCLUSIONS: ETEC exposed to alkaline environments express a specific proteome profile characterized by up-regulation of membrane proteins and secretion of LT toxin. Alkaline microenvironments have been reported close to the intestinal epithelium and the alkaline proteome may hence represent a better view of ETEC during infection.

Core-shell column Tanaka characterization and additional tests using active pharmaceutical ingredients.

In the last decade, core-shell particles have gained more and more attention in fast liquid chromatography separations due to their comparable performance with fully porous sub-2 µm particles and their significantly lower back pressure. Core-shell particles are made of a solid core surrounded by a shell of classic fully porous material. To embrace the developed core-shell column market and use these columns in pharmaceutical analytical applications, 17 core-shell C18 columns purchased from various vendors with various dimensions (50 mm × 2.1 mm to 100 mm × 3 mm) and particle sizes (1.6-2.7 µm) were characterized using Tanaka test protocols. Furthermore, four selected active pharmaceutical ingredients were chosen as test probes to investigate the batch to batch reproducibility for core-shell columns of particle size 2.6-2.7 µm, with dimension of 100 × 3 mm and columns of particle size 1.6 µm, with dimension 100 × 2.1 mm under isocratic elution. Columns of particle size 2.6-2.7 µm were also tested under gradient elution conditions. To confirm the claimed comparable efficiency of 2.6 µm core-shell particles as sub-2 µm fully porous particles, column performances of the selected core-shell columns were compared with BEH C18 , 1.7 µm, a fully porous column material as well.

Combining Chemometric Models with Adsorption Isotherm Measurements to Study Omeprazole in RP-LC.

The adsorption of the proton-pump inhibitor omeprazole was investigated using RP-LC with chemometric models combined with adsorption isotherm modelling to study the effect of pH and type of organic modifier (i.e., acetonitrile or methanol). The chemometric approach revealed that omeprazole was tailing with methanol and fronting with acetonitrile along with increased fronting at higher pH. The increased fronting with higher pH for acetonitrile was explored using a pH-dependent adsorption isotherm model that was determined using the inverse method and it agreed well with the experimental data. The model indicated that the peaks exhibit more fronting at high pH due to a larger fraction of charged omeprazole molecules. This model could accurately predict the shape of elution profiles at arbitrary pH levels in the studied interval. Using a two-layer adsorption isotherm model, the difference between acetonitrile and methanol was studied at the lowest pH at which almost all omeprazole molecules are neutral. Omeprazole had adsorbate-adsorbate interactions that were similar in strength for the acetonitrile and methanol mobile phases, while the solute-adsorbent interactions were almost twice as strong with methanol. The difference in the relative strengths of these two interactions likely explains the different peak asymmetries (i.e., tailing/fronting) in methanol and acetonitrile. In conclusion, thermodynamic modelling can complement chemometric modeling in HPLC method development and increase the understanding of the separation.

Preoperative MRI of the breast (POMB) influences primary treatment in breast cancer: a prospective, randomized, multicenter study.

BACKGROUND: Breast magnetic resonance imaging (MRI) has shown high sensitivity in determining tumor extent, multifocality, and occult contralateral breast cancer. Low specificity, unnecessary mastectomies, and costs are arguments against MRI. The purpose of this study was to determine whether preoperative breast MRI would affect primary surgical management, reduce reexcision/reoperation procedures, and influence the choice of neoadjuvant treatment in patients with newly diagnosed breast cancer. METHODS: This prospective, randomized, multicenter study included 440 breast cancer patients younger than aged 56 years from three, Swedish, large-volume breast units. Patients were randomly allocated on a 1:1 basis to either preoperative staging with breast MRI (n = 220) or no breast MRI (n = 220) (control group). Treatment planning of all patients was discussed at multidisciplinary team conferences. RESULTS: In patients randomized to the MRI group, who had an observed higher percentage of planned breast-conserving surgery (BCS) compared with the control group, a change from suggested breast conservation to mastectomy occurred in 23 of 153 (15 %) patients. Breast MRI provided additional information in 83 of 220 (38 %) patients, which caused a change in treatment plan in 40 (18 %). The breast reoperation rate was significantly lower in the MRI group: 11 of 220 (5 %) versus 33 of 220 (15 %) in the control group (p < 0.001). The number of mastectomies, axillary reoperations, and the number of patients receiving neoadjuvant chemotherapy after definitive treatment did not differ significantly between the groups. CONCLUSIONS: Preoperative staging with breast MRI in women younger than age 56 years altered the treatment plan in 18 % of the patients. Although a higher MRI-related conversion rate from breast conservation to mastectomy was found, the final numbers of mastectomies did not differ between the two groups. The breast reoperation rate in the MRI group was significantly reduced.

Psychiatric disorders and comorbidity in women with Turner Syndrome: a retrospective national cohort study.

Turner syndrome (TS) is a genetic condition characterized by partial or complete monosomy X. A reduced life expectancy has been shown in TS, depending on an increased risk of aortic dissection, and ischemic heart disease. Studies covering the occurrence of psychiatric conditions are sparse within TS. Several case reports describe concomitant TS and neuropsychiatric abnormalities that may represent a pathogenetic link to genetics, as well as feature correlates of TS. The aim of this study was to determine the presence, and the frequency of psychiatric diagnosis in women with TS in a Swedish cohort followed during 25 years' time. Statistics from the entire female population in Sweden of corresponding age was used as reference. Data were retrieved from clinical examinations and validated from the National Board of Health and Welfare registries for women with TS (n = 487), aged 16 to 84 years, with respect to mental health disorders. The most common diagnoses in TS were mood and anxiety disorders. There was no increase in psychiatric diagnosis within the group with time, nor correlation to specific karyotype or somatic comorbidity as congenital heart disease and hypothyroidism, hormonal treatment, or childbirth. In addition, the frequency of psychiatric diagnosis in TS was lower than in the population-based data. Further investigations are needed in the view of the fact that women with Turner syndrome should not be burdened with more severe diagnoses.

Surface-Shaving of Staphylococcus aureus Strains and Quantitative Proteomic Analysis Reveal Differences in Protein Abundance of the Surfaceome.

Staphylococcus aureus is a pathogen known to cause a wide range of infections. To find new targets for identification and to understand host-pathogen interactions, many studies have focused on surface proteins. We performed bacterial-cell surface-shaving, followed by tandem mass tag for quantitative mass spectrometry proteomics, to examine the surfaceome of S. aureus. Two steps were performed, the first step including surface protein-deficient mutants of S. aureus Newman strain lacking important virulence genes (clfA and spa, important for adhesion and immune evasion and srtAsrtB, linking surface-associated virulence factors to the surface) and the second step including isolates of different clinical origin. All strains were compared to the Newman strain. In Step 1, altogether, 7880 peptides were identified, corresponding to 1290 proteins. In Step 2, 4949 peptides were identified, corresponding to 919 proteins and for each strain, approximately 20 proteins showed differential expression compared to the Newman strain. The identified surface proteins were related to host-cell-adherence and immune-system-evasion, biofilm formation, and survival under harsh conditions. The results indicate that surface-shaving of intact S. aureus bacterial strains in combination with quantitative proteomics is a useful tool to distinguish differences in protein abundance of the surfaceome, including the expression of virulence factors.

Autoimmune Disease in Turner Syndrome in Sweden: An up to 25 Years' Controlled Follow-up Study.

CONTEXT: Turner syndrome (TS) is the most common chromosomal aberration in women; it is the result of structural or numeric abnormalities in the X chromosome. Autoimmune hypothyroidism has been recognized as one of the more prominent disorders associated with TS. OBJECTIVE: This work aimed to study the prevalence of autoimmune diseases in TS. METHODS: A cross-sectional, longitudinal, 25-year follow-up study was conducted of patients from adult Turner centers at the University Hospitals, Sweden. During 1994 to 2020, a total of 503 women aged 16 to 71 years with TS were evaluated consecutively every fifth year according to national guidelines. A random population sample of women, n = 401, aged 25 to 44 years, from the World Health Organization Monitoring of Trends and Determinants for Cardiovascular Disease (MONICA) project served as controls. Serum thyrotropin, free thyroxine, vitamin B12, antithyroid peroxidase (anti-TPO), and antitransglutaminase antibodies were measured. RESULTS: Mean follow-up time (years) was 16 ± 7 for patients and 13 ± 1 for controls. From study start, the prevalence increased in TS for hypothyroidism 40% to 58%, vitamin B12 deficiency 5% to 12%, celiac disease 4% to 7%, positive anti-TPO 26% to 41%, and antitransglutaminase antibodies 6% to 8% (P < .0001 vs controls). Type 1 diabetes and Addison disease were rare. The only interrelationship was between hypothyroidism and vitamin B12 deficiency, both in TS and controls. No association between autoimmune disease and karyotype, antecedent growth hormone treatment, or ongoing estrogen hormone replacement, was seen in TS. CONCLUSION: In women with TS up to older than 80 years, more than half developed hypothyroidism, mainly autoimmune, during follow-up. Awareness of vitamin B12 deficiency and celiac disease throughout life is also recommended in women with TS.

Combining phenotypic and proteomic approaches to identify membrane targets in a 'triple negative' breast cancer cell type.

BACKGROUND: The continued discovery of therapeutic antibodies, which address unmet medical needs, requires the continued discovery of tractable antibody targets. Multiple protein-level target discovery approaches are available and these can be used in combination to extensively survey relevant cell membranomes. In this study, the MDA-MB-231 cell line was selected for membranome survey as it is a 'triple negative' breast cancer cell line, which represents a cancer subtype that is aggressive and has few treatment options. METHODS: The MDA-MB-231 breast carcinoma cell line was used to explore three membranome target discovery approaches, which were used in parallel to cross-validate the significance of identified antigens. A proteomic approach, which used membrane protein enrichment followed by protein identification by mass spectrometry, was used alongside two phenotypic antibody screening approaches. The first phenotypic screening approach was based on hybridoma technology and the second was based on phage display technology. Antibodies isolated by the phenotypic approaches were tested for cell specificity as well as internalisation and the targets identified were compared to each other as well as those identified by the proteomic approach. An anti-CD73 antibody derived from the phage display-based phenotypic approach was tested for binding to other 'triple negative' breast cancer cell lines and tested for tumour growth inhibitory activity in a MDA-MB-231 xenograft model. RESULTS: All of the approaches identified multiple cell surface markers, including integrins, CD44, EGFR, CD71, galectin-3, CD73 and BCAM, some of which had been previously confirmed as being tractable to antibody therapy. In total, 40 cell surface markers were identified for further study. In addition to cell surface marker identification, the phenotypic antibody screening approaches provided reagent antibodies for target validation studies. This is illustrated using the anti-CD73 antibody, which bound other 'triple negative' breast cancer cell lines and produced significant tumour growth inhibitory activity in a MDA-MB-231 xenograft model. CONCLUSIONS: This study has demonstrated that multiple methods are required to successfully analyse the membranome of a desired cell type. It has also successfully demonstrated that phenotypic antibody screening provides a mechanism for rapidly discovering and evaluating antibody tractable targets, which can significantly accelerate the therapeutic discovery process.

Absorption and scattering of light from ensembles of randomly oriented aggregates.

This paper presents one approach to the absorption and scattering of light from aggregates of primary particles. The primary particles are sphere-like and small compared to the wavelength, whereas the aggregate can be large compared to the wavelength. This situation applies to when soot particles formed in flames are measured using methods based on laser light. The method presented in this work, called generalized Rayleigh-Debye-Gans, leads to closed-form expressions for the scattered intensity and the absorbed power of an ensemble of aggregates with random positions and orientations. The expressions ensure a fast and accurate numerical evaluation of the scattering and absorption from ensembles of aggregates. The numerical results are compared with the ones obtained from the T-matrix method and the discrete dipole approximation method.

Sustainable Delicacy: Variation in Quality and Sensory Aspects in Wild Boar (Sus scrofa) Meat and Comparison to Pork Meat-A Case Study.

The aim of this study was to evaluate quality and sensory variation in wild boar meat in comparison to pork. Meat quality in wild boar is expected to vary more compared to pork due to different feeding environment, age and gender. In order to be able to promote wild boar meat as a sustainable high-quality product, there is a need to evaluate the variation in meat quality attributes, including technological, compositional and sensory/texture aspects. We evaluated carcass characteristics, pH, colour, lipid profile and sensory aspects of wild boar meat of different age and gender and compared them with pork. Wild boars had lower carcass weight (p = <0.0001) and higher ultimate pH (p = 0.0063) compared to domestic pigs. Intramuscular fat content had a tendency to be higher in wild boar meat (p = 0.1010), as well as the proportion of nutritional valuable n-3 FA (p = 0.0029). The colour of pork was more pink (p = 0.0276) and pale (p = <0.0001) compared to meat from wild boar. Meat from wild boar gilts received the highest sensory scores. Based on these findings, we suggest that meat from younger animals could be sold in different cuts directly while meat from older animals might be more suitable for the production of sausage.

Metataxonomic and metaproteomic profiling of the oral microbiome in oral lichen planus - a pilot study.

Background: A growing body of evidence demonstrates a different bacterial composition in the oral cavity of patients with oral lichen planus (OLP). Patients and methods: Buccal swab samples were collected from affected and non-affected sites of six patients with reticular OLP and the healthy oral mucosa of six control subjects. 16S rRNA gene MiSeq sequencing and mass spectrometry-based proteomics were utilised to identify the metataxonomic and metaproteomic profiles of the oral microbiome in both groups. Results: From the metataxonomic analysis, the most abundant species in the three subgroups were Streptococcus oralis and Pseudomonas aeruginosa, accounting for up to 70% of the total population. Principal Coordinates Analysis showed differential clustering of samples from the healthy and OLP groups. ANCOM-BC compositional analysis revealed multiple species (including P. aeruginosa and several species of Veillonella, Prevotella, Streptococcus and Neisseria) significantly over-represented in the control group and several (including Granulicatella elegans, Gemella haemolysans and G. parahaemolysans) in patients with OLP. The metaproteomic data were generally congruent and revealed that several Gemella haemolysans-belonging peptidases and other proteins with inflammatory and virulence potential were present in OLP lesions. Conclusion: Our data suggest that several bacterial species are associated with OLP. Future studies with larger cohorts should be conducted to determine their role in the aetiology of OLP and evaluate their potential as disease biomarkers.

Strain-level typing and identification of bacteria using mass spectrometry-based proteomics.

Because of the alarming expansion in the diversity and occurrence of bacteria displaying virulence and resistance to antimicrobial agents, it is increasingly important to be able to detect these microorganisms and to differentiate and identify closely related species, as well as different strains of a given species. In this study, a mass spectrometry proteomics approach is applied, exploiting lipid-based protein immobilization (LPI), wherein intact bacterial cells are bound, via membrane-gold interactions, within a FlowCell. The bound cells are subjected to enzymatic digestion for the generation of peptides, which are subsequently identified, using LC-MS. Following database matching, strain-specific peptides are used for subspecies-level discrimination. The method is shown to enable a reliable typing and identification of closely related strains of the same bacterial species, herein illustrated for Helicobacter pylori .

Microfluidic flow cell for sequential digestion of immobilized proteoliposomes.

We have developed a microfluidic flow cell where stepwise enzymatic digestion is performed on immobilized proteoliposomes and the resulting cleaved peptides are analyzed with liquid chromatography-tandem mass spectrometry (LC-MS/MS). The flow cell channels consist of two parallel gold surfaces mounted face to face with a thin spacer and feature an inlet and an outlet port. Proteoliposomes (50-150 nm in diameter) obtained from red blood cells (RBC), or Chinese hamster ovary (CHO) cells, were immobilized on the inside of the flow cell channel, thus forming a stationary phase of proteoliposomes. The rate of proteoliposome immobilization was determined using a quartz crystal microbalance with dissipation monitoring (QCM-D) which showed that 95% of the proteoliposomes bind within 5 min. The flow cell was found to bind a maximum of 1 µg proteoliposomes/cm(2), and a minimum proteoliposome concentration required for saturation of the flow cell was determined to be 500 µg/mL. Atomic force microscopy (AFM) studies showed an even distribution of immobilized proteoliposomes on the surface. The liquid encapsulated between the surfaces has a large surface-to-volume ratio, providing rapid material transfer rates between the liquid phase and the stationary phase. We characterized the hydrodynamic properties of the flow cell, and the force acting on the proteoliposomes during flow cell operation was estimated to be in the range of 0.1-1 pN, too small to cause any proteoliposome deformation or rupture. A sequential proteolytic protocol, repeatedly exposing proteoliposomes to a digestive enzyme, trypsin, was developed and compared with a single-digest protocol. The sequential protocol was found to detect ~65% more unique membrane-associated protein (p < 0.001, n = 6) based on peptide analysis with LC-MS/MS, compared to a single-digest protocol. Thus, the flow cell described herein is a suitable tool for shotgun proteomics on proteoliposomes, enabling more detailed characterization of complex protein samples.

Mechanical properties of plant cell walls probed by relaxation spectra.

Transformants and mutants with altered cell wall composition are expected to display a biomechanical phenotype due to the structural role of the cell wall. It is often quite difficult, however, to distinguish the mechanical behavior of a mutant's or transformant's cell walls from that of the wild type. This may be due to the plant's ability to compensate for the wall modification or because the biophysical method that is often employed, determination of simple elastic modulus and breakstrength, lacks the resolving power necessary for detecting subtle mechanical phenotypes. Here, we apply a method, determination of relaxation spectra, which probes, and can separate, the viscoelastic properties of different cell wall components (i.e. those properties that depend on the elastic behavior of load-bearing wall polymers combined with viscous interactions between them). A computer program, BayesRelax, that deduces relaxation spectra from appropriate rheological measurements is presented and made accessible through a Web interface. BayesRelax models the cell wall as a continuum of relaxing elements, and the ability of the method to resolve small differences in cell wall mechanical properties is demonstrated using tuber tissue from wild-type and transgenic potatoes (Solanum tuberosum) that differ in rhamnogalacturonan I side chain structure.

Pre-anaesthetic metomidate sedation delays the stress response after caudal artery cannulation in Atlantic cod (Gadus morhua).

Recovery from caudal artery cannulation with and without pre-anaesthesia metomidate sedation was assessed in Atlantic cod (Gadus morhua). The levels of plasma cortisol, glucose, electrolytes and acid-base parameters were compared between sedated and unsedated cod and to those in uncannulated individuals, where the samples were obtained by sacrificial sampling (reference level). Metomidate sedation delayed the stress response, causing sedated cod plasma cortisol to return to the reference level more slowly [day 4 post surgery (PS)] than in unsedated cod (day 2 PS). Plasma glucose was elevated in both sedated and unsedated cod up to and including day 5 PS. Plasma K(+) was lower and pH was higher in cannulated cod than in the reference from 24 h PS until the end of experimentation, indicating a stress effect of sacrificial sampling on plasma K(+) and pH that was likely caused by an acute stress response. Metomidate sedation delayed the stress response following CA cannulation and should therefore not be used as a pre-anaesthetic sedation in Atlantic cod. The caudal artery cannulation can be a useful tool in obtaining repeated blood samples from Atlantic cod given an adequate recovery time, which was determined to be 6 days irrespective of pre-anaesthesia sedation status.