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Nonislet-cell tumor hypoglycemia (NICTH) is a rare paraneoplastic phenomenon well described in dogs and humans. Tumors associated with NICTH secrete incompletely processed forms of insulin-like growth factor-II (IGF-II), commonly named big IGF-II. These forms have increased bioavailability and interact with the insulin and IGF-I receptor causing hypoglycemia and growth-promoting effects. Immunoassays designed for human samples have been used to measure canine IGF-I and -II, but they possess some limitations. In addition, there are no validated methods for measurement of big IGF-II in dogs. In the present study, a targeted parallel reaction monitoring MS-based method previously developed for cats has been optimized and applied to simultaneously quantify the serum levels of IGF-I, IGF-II, and IGFBP-3, and for the first time, the levels of big IGF-II in dogs. This method allows the absolute quantification of IGF proteins using a mixture of QPrEST proteins previously designed for humans. The method possesses good linearity and repeatability and has been used to evaluate the IGF-system in a dog with NICTH syndrome. In this dog, the levels of big IGF-II decreased by 80% and the levels of IGF-I and IGFBP-3 increased approximately 20- and 4-times, respectively, after removal of the tumor.

Increased knowledge on serum protein profiles during early pregnancy in dogs would be valuable for several reasons, including animal welfare. Inflammatory changes during this period have been described. Today, mass spectrometry (MS) is a well-established technique to perform unbiased qualitative and quantitative studies of proteins in body fluids regardless of species. In the present study, a shotgun proteomic analysis based on nano-liquid chromatography-MS was performed to identify proteins of altered abundance during canine pregnancy, and, thereafter, a targeted parallel reaction monitoring (PRM)-method was developed and applied to absolutely quantify the concentrations of a selection of these proteins. Among the 32 proteins found altered between pregnant and non-pregnant dogs in the initial analysis, 12 were selected based on their changes in concentration and known biological importance, and these were analyzed using the PRM method. The PRM method showed good linearity, repeatability and sensitivity, and confirmed the higher concentration of Fibrinogen A, protein S alpha and C-reactive protein at early time points in pregnant bitches. In conclusion, the combination of both methods allowed the identification of several altered proteins, and the quantification and description of the concentration patterns for a selection of them during the early stage of dog pregnancy.

Significance: MS is a powerful technique that allows the investigation of protein variations in samples from different origin, such as serum from dogs. The application of a shotgun proteomic analysis as a screening method has revealed the alteration of several proteins after fifteen days of pregnancy in dogs. The complementary development of a PRM MS-based method for several of these proteins has enabled the absolute quantification of their concentrations at five different time points during early pregnancy. With the MS technique, a combination of proteins can be studied with lower limits of detection than with immunoassays. Care should be taken not to interpret the observed changes in pregnant dogs as signs of disease.

BACKGROUND: Insulin-like growth factor-I (IGF-I) is used to screen for acromegaly in diabetic cats. In humans, most circulating IGF-I forms ternary complexes (TC) with IGF-binding protein (IGFBP-3) and an acid-labile subunit. Compared to humans, the amount of TC in cats is more variable. Insulin-like growth factor-I concentrations are reported to increase during insulin treatment, more rapidly in cats achieving remission.

OBJECTIVES: To investigate (i) factors associated with circulating IGF-I concentrations, including IGFBP-profiles (ii) effect of insulin treatment on IGF-I concentrations and (iii) IGF-I as prognostic marker of diabetes mellitus remission.

ANIMALS: Thirty-one privately owned diabetic cats of which 24 were followed 1 year, and 13 healthy cats.

METHODS: Prospective study. Serum insulin, IGF-I, glucose, and fructosamine concentrations were measured. IGF-binding forms were determined by chromatography in 14 diabetic and 13 healthy cats; and IGF-I, IGF-II, IGFBP-3, and IGFBP-5 by mass spectrometry in 3 cats achieving remission.

RESULTS: Insulin-like growth factor-I median (interquartile range) before start of insulin treatment was 300 (160-556) ng/mL. Insulin-like growth factor-I was positively associated with TC (P < .0001) and endogenous insulin (P = .005) and negatively associated with fructosamine (P < .0001). Median IGF-I was higher 2-4 weeks after start of insulin treatment compared with baseline (300 versus 670 ng/mL, P = .0001) and predicted future remission (P = .046). In cats that went into remission, the amount of TC and IGFBP-3 increased, suggesting increase in IGF-I is dependent on TC formation.

CONCLUSIONS: Insulin treatment should be accounted for when interpreting IGF-I in diabetic cats. Insulin-like growth factor-I 2-4 weeks after initiation of insulin treatment shows promise as prognostic marker for remission in diabetic cats.

Objective: The progesterone receptor modulator (PRM) mifepristone holds the potential to be developed for regular contraception. However, long-term treatment can cause thickening of the endometrium and PRM-associated endometrial changes (PAEC). The objective of this study was to explore the molecular expression of endometrium displaying PAEC after mifepristone treatment in order to understand the future implications of PAEC and safety of long-term use. Study design: Endometrial biopsies were obtained from premenopausal women following 3 months of continuous mifepristone treatment. The biopsies were evaluated regarding occurrence of PAEC and followed up by a comparative analysis of gene expression in PAEC endometrium (n=7) with endometrium not displaying PAEC (n=4). Methods used included microarray analysis, Ingenuity Pathway Analysis (IPA) and real-time polymerase chain reaction. Results: Three genes relevant within endometrial function were up-regulated with PAEC: THY1 (p=.02), ADAM12 (p=.04) and TN-C (p=.04). The proliferation marker MKi67 was not altered (p=.31). None of the differentially regulated genes were involved in the endometrial cancer-signaling pathway (based on IPA knowledge database). Conclusion: The genes altered in endometrium displaying PAEC after 3 months of mifepristone exposure are mainly involved in the structural architecture of tissue. Implications: PAEC features may be explained by the altered genes and their networks affecting tissue architecture although not involved in endometrial cancer signaling pathways, and thus, treatment with mifepristone at this dosage does not show any adverse effect at endometrial level.

The discovery of oestrogen receptor beta (ER beta/ESR2) was a landmark discovery. Its reported expression and homology with breast cancer pharmacological target ER alpha (ESR1) raised hopes for improved endocrine therapies. After 20 years of intense research, this has not materialized. We here perform a rigorous validation of 13 anti-ER beta antibodies, using well-characterized controls and a panel of validation methods. We conclude that only one antibody, the rarely used monoclonal PPZ0506, specifically targets ER beta in immunohistochemistry. Applying this antibody for protein expression profiling in 44 normal and 21 malignant human tissues, we detect ER beta protein in testis, ovary, lymphoid cells, granulosa cell tumours, and a subset of malignant melanoma and thyroid cancers. We do not find evidence of expression in normal or cancerous human breast. This expression pattern aligns well with RNA-seq data, but contradicts a multitude of studies. Our study highlights how inadequately validated antibodies can lead an exciting field astray.

Functional biological materials are a growing research area with potential applicability in medicine and biotechnology. Using genetic engineering, the possibility to introduce additional functions into spider silk-based materials has been realized. Recently, a recombinant spider silk fusion protein, Z-4RepCT, was produced intracellularly in Escherichia coli and could after purification self-assemble into silk-like fibers with ability to bind antibodies via the IgG-binding Z domain. In this study, the use of the methylotrophic yeast Pichia pastoris for production of Z-4RepCT has been investigated. Temperature, pH and production time were influencing the amount of soluble Z-4RepCT retrieved from the extracellular fraction. Purification of secreted Z-4RepCT resulted in a mixture of full-length and degraded silk proteins that failed to self-assemble into fibers. A position in the C-terminal domain of 4RepCT was identified as being subjected to proteolytic cleavage by proteases in the Pichia culture supernatant. Moreover, the C-terminal domain was subjected to glycosylation during production in P. pastoris. These observed alterations of the CT domain are suggested to contribute to the failure in fiber assembly. As alternative approach, Z-4RepCT retrieved from the intracellular fraction, which was less degraded, was used and shown to retain ability to assemble into silk-like fibers after enzymatic deglycosylation.

Histones are abundant cellular proteins but, if not incorporated into chromatin, they are usually bound by histone chaperones. Here, we identify Arabidopsis NASP as a chaperone for histones H3.1 and H3.3. NASP interacts in vitro with monomeric H3.1 and H3.3 as well as with histone H3.1-H4 and H3.3-H4 dimers. However, NASP does not bind to monomeric H4. NASP shifts the equilibrium between histone dimers and tetramers towards tetramers but does not interact with tetramers in vitro. Arabidopsis NASP promotes [H3-H4]2 tetrasome formation, possibly by providing preassembled histone tetramers. However, NASP does not promote disassembly of in vitro preassembled tetrasomes. In contrast to its mammalian homolog, Arabidopsis NASP is a predominantly nuclear protein. In vivo, NASP binds mainly monomeric H3.1 and H3.3. Pulldown experiments indicated that NASP may also interact with the histone chaperone MSI1 and a HSC70 heat shock protein.

MSI1 belongs to a family of histone binding WD40-repeat proteins. Arabidopsis thaliana contains five genes encoding MSI1-like proteins, but their functions in diverse chromatin-associated complexes are poorly understood. Here, we show that MSI1 is part of a histone deacetylase complex. We copurified HISTONE DEACETYLASE19 (HDA19) with MSI1 and transcriptional regulatory SIN3-like proteins and provide evidence that MSI1 and HDA19 associate into the same complex in vivo. These data suggest that MSI1, HDA19, and HISTONE DEACETYLATION COMPLEX1 protein form a core complex that can integrate various SIN3-like proteins. We found that reduction of MSI1 or HDA19 causes upregulation of abscisic acid (ABA) receptor genes and hypersensitivity of ABA-responsive genes. The MSI1-HDA19 complex fine-tunes ABA signaling by binding to the chromatin of ABA receptor genes and by maintaining low levels of acetylation of histone H3 at lysine 9, thereby affecting the expression levels of ABA receptor genes. Reduced MSI1 or HDA19 levels led to increased tolerance to salt stress corresponding to the increased ABA sensitivity of gene expression. Together, our results reveal the presence of an MSI1-HDA19 complex that fine-tunes ABA signaling in Arabidopsis.

As the number of fully sequenced animal genomes and the performance of advanced mass spectrometry-based proteomics techniques are continuously improving, there is now a great opportunity to increase the knowledge of various animal proteomes. This research area is further stimulated by a growing interest from veterinary medicine and the pharmaceutical industry. Cerebrospinal fluid (CSF) is a good source for better understanding of diseases related to the central nervous system, both in humans and other animals. In this study, four high-abundant protein depletion columns, developed for human or rat serum, were evaluated for dog CSF. For the analysis, a shotgun proteomics approach, based on nanoLC-LTQ Orbitrap MS/MS, was applied. All the selected approaches were shown to deplete dog CSF with different success. It was demonstrated that the columns significantly improved the coverage of the detected dog CSF proteome. An antibody-based column showed the best performance, in terms of efficiency, repeatability and the number of proteins detected in the sample. In total 983 proteins were detected. Of those, 801 proteins were stated as uncharacterized in the UniProt database. To the best of our knowledge, this is the so far largest number of proteins reported for dog CSF in one single study.

Glycosaminoglycans (GAGs) are linear polysaccharides, consisting of repeated disaccharide units, attached to core proteins in all multicellular organisms. Chondroitin sulfate (CS) and dermatan sulfate (DS) constitute a subgroup of sulfated GAGs for which the degree of sulfation varies between species and tissues. One major goal in GAG characterization is to correlate structure to function. A common approach is to exhaustively degrade the GAG chains and thereafter determine the amount of component disaccharide units. In large-scale studies, there is a need for high-throughput screening methods since existing methods are either very time- or samples consuming. Here, we present a new strategy applying MALDI-TOF MS in positive ion mode for semi-qualitative and quantitative analysis of CS/DS derived disaccharide units. Only a few picomoles of sample are required per analysis and 10 samples can be analyzed in 25&#xa0;min, which makes this approach an attractive alternative to many established assay methods. The total CS/DS concentration in 19 samples derived from Caenorhabditis elegans and mammalian tissues and cells was determined. The obtained results were well in accordance with concentrations determined by a standard liquid chromatography-based method, demonstrating the applicability of the method for samples from various biological matrices containing CS/DS of different sulfation degrees.