Selection and Validation of Reference Genes for Gene Expression Studies in an Equine Adipose-Derived Mesenchymal Stem Cell Differentiation Model by Proteome Analysis and Reverse-Transcriptase Quantitative Real-Time PCR.

Adipose-derived stem cells (ADSCs) are used in tissue regeneration therapies. The objective of this study is to identify stable reference genes (RGs) for use in gene expression studies in a characterized equine adipose-derived mesenchymal stem cell (EADMSC) differentiation model. ADSCs were differentiated into adipocytes (ADs) or osteoblasts (OBs), and the proteomes from these cells were analyzed by liquid chromatography tandem mass spectrometry. Proteins that were stably expressed in all three cells types were identified, and the mRNA expression stabilities for their corresponding genes were validated by RT-qPCR. PPP6R1, CCDC97, and then either ACTB or EPHA2 demonstrated the most stable mRNA levels. Normalizing target gene Cq data with at least three of these RGs simultaneously, as per MIQE guidelines (PPP6R1 and CCDC97 with either ACTB or EPHA2), resulted in congruent conclusions. FABP5 expression was increased in ADs (5.99 and 8.00 fold, p = 0.00002 and p = 0.0003) and in OBs (5.18 and 5.91 fold, p = 0.0011 and p = 0.0023) relative to ADSCs. RUNX2 expression was slightly higher in ADs relative to ADSCs (1.97 and 2.65 fold, p = 0.04 and p = 0.01), but not in OBs (0.9 and 1.03 fold, p = 0.58 and p = 0.91).

KIT D816V is dimerization-independent and activates downstream pathways frequently perturbed in mastocytosis.

KIT, a type III tyrosine kinase receptor, plays a crucial role in haematopoietic development. The KIT receptor forms a dimer after ligand binding; this activates tyrosine kinase activity leading to downstream signal transduction. The D816V KIT mutation is extensively implicated in haematological malignancies, including mastocytosis and leukaemia. KIT D816V is constitutively active, but the molecular nuances that lead to constitutive tyrosine kinase activity are unclear. For the first time, we present experimental evidence that the KIT D816V mutant does not dimerize like KIT wild type. We further show evidence of decreased stabilization of the tyrosine kinase domain in the KIT D816V mutant, a phenomenon that might contribute to its constitutive activity. Since the mechanism of KIT D816V activation varies from that of the wild type, we explored downstream signal transduction events and found that even though KIT D816V targets similar signalling moieties, the signalling is amplified in the mutant compared to stem cell factor-activated wild type receptor. Uniquely, KIT D816V induces infection-related pathways and the spliceosome pathway, providing alternate options for selective as well as combinatorial therapeutic targeting.

Heat Shock Alters the Proteomic Profile of Equine Mesenchymal Stem Cells.

The aim of this research was to determine the impact of heat stress on cell differentiation in an equine mesenchymal stem cell model (EMSC) through the application of heat stress to primary EMSCs as they progressed through the cell specialization process. A proteomic analysis was performed using mass spectrometry to compare relative protein abundances among the proteomes of three cell types: progenitor EMSCs and differentiated osteoblasts and adipocytes, maintained at 37 °C and 42 °C during the process of cell differentiation. A cell-type and temperature-specific response to heat stress was observed, and many of the specific differentially expressed proteins were involved in cell-signaling pathways such as Notch and Wnt signaling, which are known to regulate cellular development. Furthermore, cytoskeletal proteins profilin, DSTN, SPECC1, and DAAM2 showed increased protein levels in osteoblasts differentiated at 42 °C as compared with 37 °C, and these cells, while they appeared to accumulate calcium, did not organize into a whorl agglomerate as is typically seen at physiological temperatures. This altered proteome composition observed suggests that heat stress could have long-term impacts on cellular development. We propose that this in vitro stem cell culture model of cell differentiation is useful for investigating molecular mechanisms that impact cell development in response to stressors.

Analysis of human serum and urine for tentative identification of potentially carcinogenic pesticide-associated N-nitroso compounds using high-resolution mass spectrometry.

Human serum and urine samples were analyzed for a suite of nitrosatable pesticides and potentially carcinogenic pesticide-associated N-nitroso (PANN) compounds. Formation of PANN compounds may occur in vivo after consumption of food or water containing trace amounts of nitrosatable pesticide residues and nitrate. Using a modified version of the Quick, Easy, Cheap, Effective, Rugged, and Safe (QuEChERS) method, nine nitrosatable pesticides and byproducts were extracted from serum and urine from 64 individuals from two different sample populations in Atlantic Canada: (i) Prince Edward Island, a region where nitrate and trace amounts of nitrosatable pesticides have been detected in groundwater; and (ii) Halifax, Nova Scotia, a non-agricultural urban area. Samples were then analyzed using ultra-high pressure liquid chromatography (UHPLC) coupled with high-resolution accurate mass (HRAM) single-stage orbitrap mass spectrometry (MS), which allows for semi-targeted analysis and tentative identification of a virtually limitless number of exposure biomarkers. Two nitrosatable target analytes, ethylenethiourea (ETU) and 3,5,6-trichloro-2-pyridinol (TCPy) were found in serum, while atrazine (ATR) and ETU were detected in urine. Five and six PANN compounds were tentatively identified in serum and urine, respectively. The two PANN compounds that were most frequently tentatively identified in serum were N-nitroso dimethoate (N-DIM) and N-nitroso omethoate (N-OME) with detection frequencies of 78% and 95%, respectively. This is the first biomonitoring study of its kind to investigate PANN compounds in human serum and urine.

N-linked glycosylation enzymes in the diatom Thalassiosira oceanica exhibit a diel cycle in transcript abundance and favor for NXT-type sites.

N-linked glycosylation is a posttranslational modification affecting protein folding and function. The N-linked glycosylation pathway in algae is poorly characterized, and further knowledge is needed to understand the cell biology of algae and the evolution of N-linked glycosylation. This study investigated the N-linked glycosylation pathway in Thalassiosira oceanica, an open ocean diatom adapted to survive at growth-limiting iron concentrations. Here we identified and annotated the genes coding for the essential enzymes involved in the N-linked glycosylation pathway of T. oceanica. Transcript levels for genes coding for calreticulin, oligosaccharyltransferase (OST), N-acetylglucosaminyltransferase (GnT1), and UDP-glucose glucosyltransferase (UGGT) under high- and low-iron growth conditions revealed diel transcription patterns with a significant decrease of calreticulin and OST transcripts under iron-limitation. Solid-phase extraction of N-linked glycosylated peptides (SPEG) revealed 118 N-linked glycosylated peptides from cells grown in high- and low-iron growth conditions. The identified peptides had 81% NXT-type motifs, with X being any amino acids except proline. The presence of N-linked glycosylation sites in the iron starvation-induced protein 1a (ISIP1a) confirmed its predicted topology, contributing to the biochemical characterization of ISIP1 proteins. Analysis of extensive oceanic gene databases showed a global distribution of calreticulin, OST, and UGGT, reinforcing the importance of glycosylation in microalgae.

TRPM8 as the rapid testosterone signaling receptor: Implications in the regulation of dimorphic sexual and social behaviors.

Testosterone regulates dimorphic sexual behaviors in all vertebrates. However, the molecular mechanism underlying these behaviors remains unclear. Here, we report that a newly identified rapid testosterone signaling receptor, Transient Receptor Potential Melastatin 8 (TRPM8), regulates dimorphic sexual and social behaviors in mice. We found that, along with higher steroid levels in the circulation, TRPM8-/- male mice exhibit increased mounting frequency indiscriminate of sex, delayed sexual satiety, and increased aggression compared to wild-type controls, while TRPM8-/- females display an increased olfaction-exploratory behavior. Furthermore, neuronal responses to acute testosterone application onto the amygdala were attenuated in TRPM8-/- males but remained unchanged in females. Moreover, activation of dopaminergic neurons in the ventral tegmental area following mating was impaired in TRPM8-/- males. Together, these results demonstrate that TRPM8 regulates dimorphic sexual and social behaviors, and potentially constitutes a signalosome for mediation of sex-reward mechanism in males. Thus, deficiency of TRPM8 might lead to a delayed sexual satiety phenomenon.

Demonstration of beta-tropomyosin (Tpm2) and duplication of the alpha-slow tropomyosin gene (TPM3) in Atlantic salmon Salmo salar.

Beta tropomyosin (Tpm2) is demonstrated for the first time at the protein level in a fish species, using a combination of electrophoresis, mass spectrometric peptide mapping and end-group analysis. Tpm2 accounts for 50% of the total tropomyosin in slow trunk muscle of the adult Atlantic salmon as determined by quantitative carboxypeptidase digestion and is also present in the head and pectoral fin. It is absent in the fast skeletal (lighter-toned) trunk muscle, the most abundant muscle, which is composed solely of an alpha-fast (Tpm1) isoform. In contrast to the mammalian homologues, salmon Tpm2 migrates faster than salmon Tpm1 in the presence of anionic detergent. Other distinguishing characteristics are a reduced content of cysteine (one per chain) and tyrosine (five per chain) and a unique carboxyl-terminal region (residues 276-284). Two isoforms (paralogs) of alpha-slow tropomyosin (Tpm3) having different contents of methionine and histidine exist in slow trunk muscle indicating duplication of the TPM3 gene. Minor skeletal muscles, surveyed for the first time, contain a mix of at least two tropomyosins - Tpm2 (~ 50% of total) in pectoral fin, jaw and tongue and another isoform, either Tpm1 (pectoral fin) or alpha-1-like Tpm (jaw and tongue). Cheek muscle contains Tpm1 and alpha 1-like Tpm in varying proportion depending upon the section (light or dark). Of the two tropomyosins in tongue, Tpm2 displays comparatively weaker affinity for troponin-Sepharose. A feature of the major sarcomeric tropomyosins in Atlantic salmon is a pair of neighbouring glycines situated between residues 20-90.

Heat stress response in the closest algal relatives of land plants reveals conserved stress signaling circuits.

All land plants (embryophytes) share a common ancestor that likely evolved from a filamentous freshwater alga. Elucidating the transition from algae to embryophytes - and the eventual conquering of Earth's surface - is one of the most fundamental questions in plant evolutionary biology. Here, we investigated one of the organismal properties that might have enabled this transition: resistance to drastic temperature shifts. We explored the effect of heat stress in Mougeotia and Spirogyra, two representatives of Zygnematophyceae - the closest known algal sister lineage to land plants. Heat stress induced pronounced phenotypic alterations in their plastids, and high-performance liquid chromatography-tandem mass spectroscopy-based profiling of 565 transitions for the analysis of main central metabolites revealed significant shifts in 43 compounds. We also analyzed the global differential gene expression responses triggered by heat, generating 92.8 Gbp of sequence data and assembling a combined set of 8905 well-expressed genes. Each organism had its own distinct gene expression profile; less than one-half of their shared genes showed concordant gene expression trends. We nevertheless detected common signature responses to heat such as elevated transcript levels for molecular chaperones, thylakoid components, and - corroborating our metabolomic data - amino acid metabolism. We also uncovered the heat-stress responsiveness of genes for phosphorelay-based signal transduction that links environmental cues, calcium signatures and plastid biology. Our data allow us to infer the molecular heat stress response that the earliest land plants might have used when facing the rapidly shifting temperature conditions of the terrestrial habitat.

Therapy-Induced MHC I Ligands Shape Neo-Antitumor CD8 T Cell Responses during Oncolytic Virus-Based Cancer Immunotherapy.

Oncolytic viruses (OVs), known for their cancer-killing characteristics, also overturn tumor-associated defects in antigen presentation through the MHC class I pathway and induce protective neo-antitumor CD8 T cell responses. Nonetheless, whether OVs shape the tumor MHC-I ligandome remains unknown. Here, we investigated if an OV induces the presentation of novel MHC I-bound tumor antigens (termed tumor MHC-I ligands). Using comparative mass spectrometry (MS)-based MHC-I ligandomics, we determined differential tumor MHC-I ligand expression following treatment with oncolytic reovirus in a murine ovarian cancer model. In vitro, we found that reovirus changes the tumor ligandome of cancer cells. Concurrent multiplexed quantitative proteomics revealed that the reovirus-induced changes in tumor MHC-I ligand presentation were mostly independent of their source proteins. In an in vivo model, tumor MHC-I ligands induced by reovirus were detectable not only in tumor tissues but also the spleens (a source of antigen-presenting cells) of tumor-bearing mice. Most importantly, therapy-induced MHC-I ligands stimulated antigen-specific IFNγ responses in antitumor CD8 T cells from mice treated with reovirus. These data show that therapy-induced MHC-I ligands may shape underlying neo-antitumor CD8 T cell responses. As such, they should be considered in strategies promoting the efficacy of OV-based cancer immunotherapies.

Knockdown of the small heat-shock protein p26 by RNA interference modifies the diapause proteome of Artemia franciscana.

Embryos of the crustacean Artemia franciscana may arrest as gastrulae, forming cysts that enter diapause, which is a state of reduced metabolism and enhanced stress tolerance. Diapausing cysts survive physiological stresses for years due, in part, to molecular chaperones. p26, a small heat-shock protein, is an abundant diapause-specific molecular chaperone in cysts, and it affects embryo development and stress tolerance. p26 is therefore thought to influence many proteins in cysts, and this study was undertaken to determine how the loss of p26 by RNA interference (RNAi) affects the diapause proteome of A. franciscana. The proteome was analyzed by shot-gun proteomics coupled to differential isotopic labeling and tandem mass spectrometry. Proteins in the diapause proteome included metabolic enzymes, antioxidants, binding proteins, structural proteins, transporters, translation factors, receptors, and signal transducers. Proteins within the diapause proteome either disappeared or were reduced in amount when p26 was knocked down, or conversely, proteins appeared or increased in amount. Those proteins that disappeared may be p26 substrates, whereas the synthesis of those proteins that appeared or increased may be regulated by p26. This study provides the first global characterization of the diapause proteome of A. franciscana and demonstrates that the sHsp p26 influences proteome composition.

Regulation of cell surface protease receptor S100A10 by retinoic acid therapy in acute promyelocytic leukemia (APL)☆.

S100A10 (p11), a member of the S100 family of small dimeric EF-hand-type Ca2+-binding proteins, plays a role in a variety of both intracellular and extracellular processes. Previous studies have suggested that p11 is intrinsically unstable and requires binding to annexin A2 (p36) to prevent its rapid ubiquitylation and degradation. Our laboratory has shown that p11 levels are stimulated by the expression of the oncoprotein, PML/RARα. Furthermore, treatment of the APL cell line, NB4 with all-trans retinoic acid (ATRA) causes the rapid loss of p36 and p11 protein. However, the mechanism by which ATRA regulates p11 levels has not been established. Here, we show that the proteasomal inhibitor, lactacystin reversed the ATRA-dependent loss of p11, but did not cause an accumulation of ubiquitylated forms of p11, suggesting that ATRA promotes the proteasomal degradation of p11 in an ubiquitin-independent manner. ATRA treatment of MCF-7 breast cancer cells reduced p11 but not p36 transcript and protein levels, thus indicating that ATRA can regulate p11 levels independently of PML/RARα and p36. Overexpression of p36 upregulated p11 protein but not mRNA levels, indicating that p36 affects p11 post translationally. The forced expression of ubiquitin and p11 in 293 T cells resulted in ubiquitylation of p11 that was blocked by mutagenesis of lysine 57. This study highlights the complex regulation of p11 by retinoid signaling and challenges the hypothesis that ubiquitin-mediated proteasomal degradation of p11 represents a universal mechanism of regulation of this protein.

MHC-I Ligand Discovery Using Targeted Database Searches of Mass Spectrometry Data: Implications for T-Cell Immunotherapies.

Class I major histocompatibility complex (MHC-I)-bound peptide ligands dictate the activation and specificity of CD8+ T cells and thus are important for devising T-cell immunotherapies. In recent times, advances in mass spectrometry (MS) have enabled the precise identification of these MHC-I peptides, wherein MS spectra are compared against a reference proteome. Unfortunately, matching these spectra to reference proteome databases is hindered by inflated search spaces attributed to a lack of enzyme restriction in the searches, limiting the efficiency with which MHC ligands are discovered. Here we offer a solution to this problem whereby we developed a targeted database search approach and accompanying tool SpectMHC, that is based on a priori-predicted MHC-I peptides. We first validated the approach using MS data from two different allotype-specific immunoprecipitates for the C57BL/6 mouse background. We then developed allotype-specific HLA databases to search previously published MS data sets of human peripheral blood mononuclear cells (PBMCs). This targeted search strategy improved peptide identifications for both mouse and human ligandomes by greater than 2-fold and is superior to traditional "no enzyme" searches of reference proteomes. Our targeted database search promises to uncover otherwise missed novel T-cell epitopes of therapeutic potential.

Determination of Flow Rates in Capillary Liquid Chromatography Coupled to a Nanoelectrospray Source using Droplet Image Analysis Software.

Liquid chromatography coupled to electrospray tandem mass spectrometry (LC-ESI-MS/MS) is widely used in proteomic and metabolomic workflows. Considerable analytical improvements have been observed when the components of LC systems are scaled down. Currently, nano-ESI is typically done at capillary LC flow rates ranging from 200 to 300 nL/min. At these flow rates, trouble shooting and leak detection of LC systems has become increasingly challenging. In this paper we present a novel proof-of-concept approach to measure flow rates at the tip of electrospray emitters when the ionization voltage is turned off. This was achieved by estimating the changes in the droplet volume over time using digital image analysis. The results are comparable with the traditional methods of measuring flow rates, with the potential advantages of being fully automatable and nondisruptive.

Iodination on tyrosine residues during oxidation with sodium periodate in solid phase extraction of N-linked glycopeptides.

Solid-phase extraction of N-linked glycopeptides (SPEG) using hydrazide-modified supports has become a common sample preparation procedure in glycoproteomic experiments. We demonstrate that iodination of tyrosine residues occur in SPEG as a side reaction during an oxidation step with sodium periodate. MS/MS analysis of oxidized bovine serum albumin and carbonic anhydrase digests revealed a characteristic shift of m/z 125.9 on all y and b fragment ions containing the modified tyrosine residues. Selected reaction monitoring (SRM) measurements showed that the peak intensity from of the iodinated peptides increased during the course of oxidation. After an hour of oxidation, SRM analysis revealed that the strongest signal from an iodinated peptide was approximately one-tenth of the intensity of the corresponding unmodified peptide. Iodinated tyrosine residues were also identified in serum samples subjected to SPEG and analyzed by LC-ESI-MS/MS. We recommend assessing this side reaction by including iodotyrosine as a variable modification when performing database searches on SPEG experiments. For SRM-based acquisitions, we encourage the avoidance of tyrosine-containing glycopeptides or, if this is not practical, monitoring transitions that contain the potential modified iodinated tyrosine residue to monitor the presence of the iodinated form of the glycopeptide.

Structural investigation of bacterial lipopolysaccharides by mass spectrometry and tandem mass spectrometry.

Mass spectrometric studies are now playing a leading role in the elucidation of lipopolysaccharide (LPS) structures through the characterization of antigenic polysaccharides, core oligosaccharides and lipid A components including LPS genetic modifications. The conventional MS and MS/MS analyses together with CID fragmentation provide additional structural information complementary to the previous analytical experiments, and thus contribute to an integrated strategy for the simultaneous characterization and correct sequencing of the carbohydrate moiety.

Quantification of Greenland halibut serum vitellogenin: a trip from the deep sea to the mass spectrometer.

This paper focuses on the sequential steps involved in developing a technique for quantifying Greenland halibut vitellogenin, a serum protein biomarker, using a comprehensive mass spectrometric approach. In the first phase of this study, in-gel trypsin digestions of serum proteins separated by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) were analyzed by matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS). A characteristic band around a molecular mass of 185 kDa, present in the mature female specimens, but absent in the male samples, was identified as vitellognin according to the peptide mass fingerprint obtained by MALDI-MS. Subsequently, MALDI and electrospray ionization tandem mass spectrometry (ESI-MS/MS) analyses were performed on the digest of the vitellogenin band for de novo sequencing. From these studies, a characteristic 'signature' peptide (sequence: FFGQEIAFANIDK) was selected from a list of candidate peptides as a surrogate analytical standard used for quantification purposes. Sample preparation for vitellogenin quantification consisted of a simple one-step overnight trypsin digestion. Samples were spiked with an isotopologue signature peptide standard and analyzed by high-performance liquid chromatography (HPLC) coupled in-line to an electrospray quadrupole-hexapole-quadrupole tandem mass spectrometer, operated in selective reaction monitoring mode. Transitions [(m/z 750.0 --> 1020.4 and 750.0 --> 1205.4) and (754.8 --> 1028.6 and 754.8 --> 1213.2)] were monitored for the signature peptide and the internal standard, respectively. Samples obtained from the field showed that vitellogenin levels were in accordance with fish maturity determined by macroscopic examination of the gonad, proving this technique suitable for measuring vitellogenin as a serum protein biomarker for reproductive maturity in female fish.

Structural determination of the novel fragmentation routes of zwitteronic morphine opiate antagonists naloxonazine and naloxone hydrochlorides using electrospray ionization tandem mass spectrometry.

Electrospray ionization quadrupole time-of-flight (ESI-QqToF) mass spectra of the zwitteronic salts naloxonazine dihydrochloride 1 and naloxone hydrochloride 2, a common series of morphine opiate receptor antagonists, were recorded using different declustering potentials. The singly charged ion [M+H-2HCl](+) at m/z 651.3170 and the doubly charged ion [M+2H-2HCl](2+) at m/z 326.1700 were noted for naloxonazine dihydrochloride 1; and the singly charged ion [M+H-HCl](+) at m/z 328.1541 was observed for naloxone hydrochloride 2. Low-energy collision-induced dissociation tandem mass spectrometry (CID-MS/MS) experiments established the fragmentation routes of these compounds. In addition to the characteristic diagnostic product ions obtained, we noticed the formation of a series of radical product ions for the zwitteronic compounds 1 and 2, and also the formation of a distonic ion product formed from the singly charged ion [M+H-HCl](+) of naloxone hydrochloride 2. Confirmation of the various established fragmentation routes was effected by conducting a series of ESI-CID-QqTof-MS/MS product ion scans, which were initiated by CID in the atmospheric pressure/vacuum interface using a higher declustering potential. Deuterium labeling was also performed on the zwitteronic salts 1 and 2, in which the hydrogen atoms of the OH and NH groups were exchanged with deuterium atoms. Low-energy CID-QqTof-MS/MS product ion scans of the singly charged and doubly charged deuteriated molecules confirmed the initial fragmentation patterns proposed for the protonated molecules. Precursor ion scan analyses were also performed with a conventional quadrupole-hexapole-quadrupole tandem mass spectrometer and allowed the confirmation of the genesis of some diagnostic ions.

Absolute quantification of Atlantic salmon and rainbow trout vitellogenin by the 'signature peptide' approach using electrospray ionization QqToF tandem mass spectrometry.

In this work, we present a simple method for absolute quantification of plasma vitellogenin from both rainbow trout and Atlantic salmon. Plasma samples obtained from control and beta-estradiol induced fish were digested with trypsin. A characteristic 'signature peptide' was selected and analyzed by high performance liquid chromatography (HPLC) coupled to an electrospray quadrupole-time-of-flight tandem mass spectrometer, using a deuterated homolog peptide as an internal standard. The hybrid tandem mass spectrometer was operated in a 'pseudo' selected reaction-monitoring mode in which three diagnostic product ions were monitored for identification and quantification purposes. The reproducibility (coefficient of variation approximately 5%) and sensitivity (limit of quantification (LOQ) of 0.009 mg/ml) achieved by this simple assay allow it to be considered as an alternative to immunological assays.

'De novo' sequencing of Atlantic cod vitellogenin tryptic peptides by matrix-assisted laser desorption/ionization quadrupole time-of-flight tandem mass spectrometry: similarities with haddock vitellogenin.

Vitellogenin is a protein produced by the liver of oviparous animals in response to circulating estrogens. The amino acid sequence of vitellogenin from Atlantic cod (Gadus morhua) has not yet been determined. In this study Atlantic cod vitellogenin was characterized using a 'bottom-up' mass spectrometric approach. Vitellogenin synthesis was induced 'in vivo' with beta-estradiol, and subjected to trypsin digestion for characterization by matrix-assisted laser desorption/ionization quadrupole time-of-flight tandem mass spectrometry. A peptide mass fingerprint was obtained and 'de novo' sequencing of the most abundant tryptic peptides was performed by low-energy collision-induced dissociation tandem mass spectrometry. As a result of these experiments, the sequences of various tryptic peptides have been elucidated. The database search has shown that Atlantic cod vitellogenin shares a series of common peptides with the two different known vitellogenin sequences of haddock, a closely related species. These findings allow us to propose that Atlantic cod might also co-express at least two distinct forms of vitellogenin.

Potential utility of the peripheral analgesic properties of morphine in stomatitis-related pain: a pilot study.

To determine the potential clinical utility of peripheral opioid action using a clinical model of cancer treatment-induced inflammation and pain that allowed for topical application of morphine in the damaged tissue (oral mucosa). This pilot study followed a two blocks design. Ten patients with painful oral mucositis were enrolled in the first block (dose-response relationship finding) and randomized in two groups to receive oral rinses with 15 ml of either 1 per thousand or 2 per thousand morphine solution. Twenty-two patients were enrolled into the second block (efficacy and safety determination). Additionally, serum concentrations of morphine were measured in five representative patients. In the first block (n=10) a dose-response relationship for topical morphine was found. Rinses with 2 per thousand -morphine solution showed better pain relief (median 80%, range 70-80%) than those with 1 per thousand (median 60%, range 55-70%; P=0.0238). Therefore, subsequent patients enrolled for the second block (n=22) received oral rinses with 2 per thousand -morphine solution. In these patients the time to good (>or=50%) or to complete (100%) pain relief was 28 (+/-12)min after the first mouthwash, and the duration of relief was on average 216 (+/-25)min. Twenty patients (90%) received the successive mouthwashes every 3 h and 10% of them every 2 h. The duration of severe pain at the moment of swallowing was 5.17 (+/-1.47) days. Only six patients needed supplementary analgesia, and the time elapsed before the first supplemental analgesic was 1.18 (+/-0.8) days. The duration of severe functional impairment was 1.52 (+/-1.31) days, thus allowing us to feed the patient by mouth with liquid-food supplementation. During our experiment no systemically active detectable concentrations of morphine were found (GC-MS analysis). The most important side effect attributable to morphine mouthwashes was burning/itching sensation (very mild to mild intensity). Patients with painful chemoradiotherapy-induced stomatitis could be alleviated using topical morphine mouthwashes.