Pharmacist-based antihypertensive medication review and assignment of morning versus evening dosing of once-daily antihypertensive medications: A pilot study to assess feasibility and efficacy in chronic kidney disease patients.

Evening dosing of antihypertensive medications lowers nighttime blood pressure, and in one large randomized trial, it reduced the risk for cardiovascular outcomes. However, the feasibility of nighttime dosing in routine clinical practice is unknown. The purpose of this pilot study was to evaluate the effect of a brief pharmacist intervention to assign patients to take antihypertensive medications at specific times of the day. In this pilot, randomized controlled trial, 79 patients with moderate to severe chronic kidney disease (CKD) taking one or more antihypertensive medications once daily were randomized to take one once-daily antihypertensive either in the morning or in the evening. A total of 79 patients were randomized (39 to morning dosing, 40 to evening dosing). Average (SD) age was 56.5 (14) years, 68% were male, and average (SD) estimated glomerular filtration rate (eGFR) was 36.6 (8.9) mL/min/1.73m2. Adherence, defined as taking the once-daily medication at the time indicated six or seven times in the last 7 days and not taking it at any other time during the day, was 91% in the morning arm and 95% in the evening arm (p = 0.57). This pilot demonstrates the feasibility and efficacy of a pharmacist-physician collaborative to assign once-daily antihypertensive medications to either morning or evening dosing.

Label-free proteomic analysis of the hydrophobic membrane protein complement in articular chondrocytes: a technique for identification of membrane biomarkers.

CONTEXT: There is insufficient knowledge about the chondrocyte membranome and its molecular composition. OBJECTIVE: To develop a Triton X-114 based separation technique using nanoLC-MS/MS combined with shotgun proteomics to identify chondrocyte membrane proteins. MATERIALS AND METHODS: Articular chondrocytes from equine metacarpophalangeal joints were separated into hydrophobic and hydrophilic fractions; trypsin-digested proteins were analysed by nanoLC-MS/MS. RESULTS: A total of 315 proteins were identified. The phase extraction method yielded a high proportion of membrane proteins (56%) including CD276, S100-A6 and three VDAC isoforms. DISCUSSION: Defining the chondrocyte membranome is likely to reveal new biomarker targets for conventional and biological drug discovery.

Analysis of mass spectrometry data from the secretome of an explant model of articular cartilage exposed to pro-inflammatory and anti-inflammatory stimuli using machine learning.

BACKGROUND: Osteoarthritis (OA) is an inflammatory disease of synovial joints involving the loss and degeneration of articular cartilage. The gold standard for evaluating cartilage loss in OA is the measurement of joint space width on standard radiographs. However, in most cases the diagnosis is made well after the onset of the disease, when the symptoms are well established. Identification of early biomarkers of OA can facilitate earlier diagnosis, improve disease monitoring and predict responses to therapeutic interventions. METHODS: This study describes the bioinformatic analysis of data generated from high throughput proteomics for identification of potential biomarkers of OA. The mass spectrometry data was generated using a canine explant model of articular cartilage treated with the pro-inflammatory cytokine interleukin 1 ß (IL-1ß). The bioinformatics analysis involved the application of machine learning and network analysis to the proteomic mass spectrometry data. A rule based machine learning technique, BioHEL, was used to create a model that classified the samples into their relevant treatment groups by identifying those proteins that separated samples into their respective groups. The proteins identified were considered to be potential biomarkers. Protein networks were also generated; from these networks, proteins pivotal to the classification were identified. RESULTS: BioHEL correctly classified eighteen out of twenty-three samples, giving a classification accuracy of 78.3% for the dataset. The dataset included the four classes of control, IL-1ß, carprofen, and IL-1ß and carprofen together. This exceeded the other machine learners that were used for a comparison, on the same dataset, with the exception of another rule-based method, JRip, which performed equally well. The proteins that were most frequently used in rules generated by BioHEL were found to include a number of relevant proteins including matrix metalloproteinase 3, interleukin 8 and matrix gla protein. CONCLUSIONS: Using this protocol, combining an in vitro model of OA with bioinformatics analysis, a number of relevant extracellular matrix proteins were identified, thereby supporting the application of these bioinformatics tools for analysis of proteomic data from in vitro models of cartilage degradation.

Patients with bipolar disorder show a selective deficit in the episodic simulation of future events.

A substantial body of evidence suggests that autobiographical recollection and simulation of future happenings activate a shared neural network. Many of the neural regions implicated in this network are affected in patients with bipolar disorder (BD), showing altered metabolic functioning and/or structural volume abnormalities. Studies of autobiographical recall in BD reveal overgeneralization, where autobiographical memory comprises primarily factual or repeated information as opposed to details specific in time and in place and definitive of re-experiencing. To date, no study has examined whether these deficits extend to future event simulation. We examined the ability of patients with BD and controls to imagine positive, negative and neutral future events using a modified version of the Autobiographical Interview (Levine, Svoboda, Hay, Winocur, & Moscovitch, 2002) that allowed for separation of episodic and non-episodic details. Patients were selectively impaired in imagining future positive, negative, and neutral episodic details; simulation of non-episodic details was equivalent across groups.

Roborovskin, a lipocalin in the urine of the Roborovski hamster, Phodopus roborovskii.

Many rodents are now known to exhibit an obligate proteinuria that delivers urine-mediated chemosignals. In this paper, we explore the urinary proteins of the Roborovski hamster (Phodopus roborovskii). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of urine from individual male and female Roborovski hamsters revealed 2 proteins, with approximate masses of 6 and 17 kDa, the expression pattern of which showed little variation between individuals or between sexes. Peptide mass fingerprints obtained from these 2 proteins revealed a number of features: 1) the proteins of a given mass were the same in all individuals regardless of sex, 2) the 6 kDa protein was not a fragment of the 21 kDa protein, and 3) neither protein was a fragment of a larger, conserved protein such as serum albumin. Electrospray mass spectrometry of purified protein preparations established the mass of the larger protein as invariant, at 17144 ± 2 Da in all samples. This protein has been termed roborovskin. The primary structure of roborovskin was determined by tandem mass spectrometry of peptides derived from independent and overlapping digestion with 3 proteases, supported by Edman degradation of the protein N-terminus. Roborovskin shared significant homology with olfactory-binding proteins from Myodes glareolus (bank vole) and with aphrodisin and submandibular protein from the golden hamster Mesocricetus auratus, all of which belong to the lipocalin superfamily. Lower levels of homology were also indicated between a variety of other lipocalins including the major urinary proteins from house mice and Norway rats. A model of the tertiary structure of roborovskin was constructed from the primary sequence by homology modeling. This model structure resembled other 8-stranded beta barrel lipocalins. Thus, the Roborovski hamster may demonstrate another variant of urinary lipocalin expression, as for the animals studied here, there appears to be no polymorphism in expression either between sexes or individuals.

Alterations in the chondrocyte surfaceome in response to pro-inflammatory cytokines.

BACKGROUND: Chondrocytes are exposed to an inflammatory micro-environment in the extracellular matrix (ECM) of articular cartilage in joint diseases such as osteoarthritis (OA) and rheumatoid arthritis (RA). In OA, degenerative changes and low-grade inflammation within the joint transform the behaviour and metabolism of chondrocytes, disturb the balance between ECM synthesis and degradation, and alter the osmolality and ionic composition of the micro-environment. We hypothesize that chondrocytes adjust their physiology to the inflammatory microenvironment by modulating the expression of cell surface proteins, collectively referred to as the 'surfaceome'. Therefore, the aim of this study was to characterize the surfaceome of primary equine chondrocytes isolated from healthy joints following exposure to the pro-inflammatory cytokines interleukin-1-beta (IL-1ß) and tumour necrosis factor-alpha (TNF-α). We employed combined methodology that we recently developed for investigating the surfaceome in stem cells. Membrane proteins were isolated using an aminooxy-biotinylation technique and analysed by mass spectrometry using high throughput shotgun proteomics. Selected proteins were validated by western blotting. RESULTS: Amongst the 431 unique cell surface proteins identified, a high percentage of low-abundance proteins, such as ion channels, receptors and transporter molecules were detected. Data are available via ProteomeXchange with identifier PXD014773. A high number of proteins exhibited different expression patterns following chondrocyte stimulation with pro-inflammatory cytokines. Low density lipoprotein related protein 1 (LPR-1), thrombospondin-1 (TSP-1), voltage dependent anion channel (VDAC) 1-2 and annexin A1 were considered to be of special interest and were analysed further by western blotting. CONCLUSIONS: Our results provide, for the first time, a repository for proteomic data on differentially expressed low-abundance membrane proteins on the surface of chondrocytes in response to pro-inflammatory stimuli.

Relative quantification of peptide phosphorylation in a complex mixture using 18O labeling.

We have developed a method to determine the degree of phosphorylation of a peptide in a complex mixture without enrichment or operation of the mass spectrometer in negative ion mode. Yeast lysate containing known amounts of synthetic peptides (VPQLEIVPNSAEERLHSMK and VPQLEIVPN[pS]AEERLHSMK) was labeled with (16)O and (18)O during hydrolysis. After treatment of one sample with a cocktail of phosphatases, the two samples were pooled. The intensity of the dephosphorylated peptide peaks was used to infer the degree of phosphorylation present before treatment. The linear dynamic range of this method is >10-fold before either of the peptide envelopes becomes indistinguishable from the surrounding noise. Since both the site of posttranslational modification and the proportion of the protein population that is modified are vital in protein function, the employment of this technique will provide a valuable tool for the analysis of the functional implications of protein phosphorylation.

Carprofen inhibits the release of matrix metalloproteinases 1, 3, and 13 in the secretome of an explant model of articular cartilage stimulated with interleukin 1ß.

INTRODUCTION: Arthritic diseases are characterized by the degradation of collagenous and noncollagenous extracellular matrix (ECM) components in articular cartilage. The increased expression and activity of matrix metalloproteinases (MMPs) is partly responsible for cartilage degradation. This study used proteomics to identify inflammatory proteins and catabolic enzymes released in a serum-free explant model of articular cartilage stimulated with the pro-inflammatory cytokine interleukin 1ß (IL-1ß). Western blotting was used to quantify the release of selected proteins in the presence or absence of the cyclooxygenase-2 specific nonsteroidal pro-inflammatory drug carprofen. METHODS: Cartilage explant cultures were established by using metacarpophalangeal joints from horses euthanized for purposes other than research. Samples were treated as follows: no treatment (control), IL-1ß (10 ng/ml), carprofen (100 µg/ml), and carprofen (100 µg/ml) + IL-1ß (10 ng/ml). Explants were incubated (37°C, 5% CO2) over twelve day time courses. High-throughput nano liquid chromatography/mass spectrometry/mass spectrometry uncovered candidate proteins for quantitative western blot analysis. Proteoglycan loss was assessed by using the dimethylmethylene blue (DMMB) assay, which measures the release of sulfated glycosaminoglycans (GAGs). RESULTS: Mass spectrometry identified MMP-1, -3, -13, and the ECM constituents thrombospondin-1 (TSP-1) and fibronectin-1 (FN1). IL-1ß stimulation increased the release of all three MMPs. IL-1ß also stimulated the fragmentation of FN1 and increased chondrocyte cell death (as assessed by ß-actin release). Addition of carprofen significantly decreased MMP release and the appearance of a 60 kDa fragment of FN1 without causing any detectable cytotoxicity to chondrocytes. DMMB assays suggested that carprofen initially inhibited IL-1ß-induced GAG release, but this effect was transient. Overall, during the two time courses, GAG release was 58.67% ± 10.91% (SD) for IL-1ß versus 52.91% ± 9.35% (SD) with carprofen + IL-1ß. CONCLUSIONS: Carprofen exhibits beneficial anti-inflammatory and anti-catabolic effects in vitro without causing any detectable cytotoxicity. Combining proteomics with this explant model provides a sensitive screening system for anti-inflammatory compounds.

Impaired episodic memory for events encoded during mania in patients with bipolar disorder.

To date, very few studies have focused on autobiographical memory in patients with bipolar disorder. We examined whether mood state at the time of event encoding (i.e., manic, depressed, euthymic) influences subsequent recollection in these patients. We administered the Autobiographical Interview, a method that allowed us to dissociate episodic and semantic aspects of autobiographical memory. We also compared the memory perspective from which patients recollected these events. Patients were selectively impaired in recollecting episodic details of events encoded during mania but not depression or euthymia. No significant differences emerged between patients and controls for recollection of non-episodic details, regardless of mood state. Patients with bipolar disorder were also more likely than matched controls to recall memories from an observer perspective. These preliminary findings indicate a moderating influence of mood state at the time of event encoding on the subsequent recollection of autobiographical events in patients with bipolar disorder.

Applications of proteomics in cartilage biology and osteoarthritis research.

In osteoarthritis (OA) the turnover of extracellular matrix (ECM) macromolecules is disrupted by catabolic changes that lead to the production of a range of inflammatory mediators and the loss and fragmentation of proteoglycans, fibrillar and non-fibrillar collagens. These events result in the degradation and release of ECM fragments, which are potential biomarkers that can be detected in synovial fluid, blood and urine. Proteomics is increasingly applied in cartilage research and has the potential to advance our understanding of the biology of this tissue. It can also provide mechanistic insight into disease pathogenesis and progression and facilitate biomarker discovery. Here we review the area of cartilage and chondrocyte proteomics and published studies relevant to arthritis and OA biomarkers, highlighting areas of current and future research and development. Markers of tissue turnover in joints have the capacity to reflect disease-relevant biological activity potentially enabling a more rational approach to healthcare management. Therefore proteomic studies of cartilage, chondrocytes and their subcellular fractions and other joint cells and tissues may be particularly relevant in diagnostic orthopedics and therapeutic research.

High throughput proteomic analysis of the secretome in an explant model of articular cartilage inflammation.

This study employed a targeted high-throughput proteomic approach to identify the major proteins present in the secretome of articular cartilage. Explants from equine metacarpophalangeal joints were incubated alone or with interleukin-1beta (IL-1ß, 10ng/ml), with or without carprofen, a non-steroidal anti-inflammatory drug, for six days. After tryptic digestion of culture medium supernatants, resulting peptides were separated by HPLC and detected in a Bruker amaZon ion trap instrument. The five most abundant peptides in each MS scan were fragmented and the fragmentation patterns compared to mammalian entries in the Swiss-Prot database, using the Mascot search engine. Tryptic peptides originating from aggrecan core protein, cartilage oligomeric matrix protein (COMP), fibronectin, fibromodulin, thrombospondin-1 (TSP-1), clusterin (CLU), cartilage intermediate layer protein-1 (CILP-1), chondroadherin (CHAD) and matrix metalloproteinases MMP-1 and MMP-3 were detected. Quantitative western blotting confirmed the presence of CILP-1, CLU, MMP-1, MMP-3 and TSP-1. Treatment with IL-1ß increased MMP-1, MMP-3 and TSP-1 and decreased the CLU precursor but did not affect CILP-1 and CLU levels. Many of the proteins identified have well-established extracellular matrix functions and are involved in early repair/stress responses in cartilage. This high throughput approach may be used to study the changes that occur in the early stages of osteoarthritis.

A comparison of 2-D chromatography separations using UV and (18)O quantification of proteins in similar proteomes.

Proteins present in two mitochondria preparations were separated by 2-D chromatography using the ProteomeLab PF-2D Protein Fractional System, protein fractionation in two dimensions (PF-2D). The proteins in each first-dimension fraction were determined by trypsinization and LC-MS/MS. Chromatography peaks were quantified by UV detection using the "Mapping Tools" software (Beckman). The proteins present in UV detected peaks were trypsinized and identified by automated MS/MS sequencing. Relative amounts of the proteins present in the equivalent peak for each sample were assessed by comparison of the intensities of the constituent peptides and a predicted PF-2D value was calculated from the total ion count (TIC) for each peptide. Relative quantification for (18)O labeled peptides was performed using the ZoomQuant (v1.43b) software [1, 2]. We found that the chromatography peaks detected by UV generally contained several proteins. Using (18)O labeling we determined that in each peak the ratios of the constituent proteins were different. When these ratios were normalized using the TIC to account for abundance, the resulting ratio corresponded to that determined by UV. The predicted value for the PF-2D score corresponded to the observed value for each peak irrespective of the number or proteins detected.

Differential expression of cardiac mitochondrial proteins.

Mitochondria were isolated from whole hearts of Dahl salt sensitive (SS) and chromosome 13 consomic control (SS.13BN/Mcwi) rats using a mechanical homogenization process followed by density centrifugation. The proteins present in the two mitochondria preparations were quantified; equal amounts of protein from each sample were taken and trypsinized in the presence of either 16O or 18O before pooling. Incorporation of one or two 18O atoms at the C-terminus of the peptide cleaved by trypsin allows the distinction between the two samples. The proteins were identified by automated MS/MS sequencing and relative amounts of each protein assessed by comparison of the intensities of the constituent peptides. Relative quantification was performed using the ZoomQuant (v1.24) software. Nine proteins were found to be differentially expressed. Electron transfer flavoprotein alpha (P13803, ETFA) protein expression was two-fold lower in the SS compared to the SS.13BN. This was confirmed by Western blot and 2-DE gel quantification. Potential functional implications of this differential expression include an impaired capacity of the heart to oxidize fatty acids in the SS strain compared to the control. Mathematical modeling of mitochondrial electron transport predicted that the observed change in ETFA expression may result in decreased activity of the electron transport chain.

Isolation of signal transduction complexes using biotin and crosslinking methodologies.

We have developed a strategy to preferentially label the N-terminal alpha-amino groups of intact proteins allowing the internal epsilon-amino groups to remain free to react with chemical crosslinking reagents. The convergence of these methodologies allows biotinylated ligands to bind to their receptors within the cell membrane followed by removal of the crosslinked complex from cell lysate. This technique allows for the isolation of protein complexes in an MS-compatible system, thus providing a tool for furthering our understanding of signal transduction.

On the determinants of induction in responding for sucrose when food pellet reinforcement is upcoming.

Rats' rates of leverpressing for low-concentration liquid-sucrose reinforcers in the first half of an experimental session increase when food pellet, rather than sucrose, reinforcers will be available in the second half. Experiment 1 determined that this induction effect was the outcome of food pellet reinforcement's increasing response rates, not of continued sucrose reinforcement's decreasing them. Experiments 2 and 3 showed that induction was primarily controlled by the conditions of reinforcement in the current session, not by those in the previous one. Experiment 4 showed little evidence that the induction was the outcome of Pavlovian processes. These results suggest that induction may occur because of processes operating at the level of the entire session. They also provide a link to a seemingly related area of study: contrast effects. Some of the results are consistent with what is known about contrast effects, but there are also several, yet unexplained differences.