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Phytogenic compounds may influence salivation or salivary properties. However, their effects on the bovine salivary proteome have not been evaluated. We investigated changes in the bovine salivary proteome due to transition from forage to high-concentrate diet, with and without supplementation with a phytogenic feed additive. Eight non-lactating cows were fed forage, then transitioned to a 65% concentrate diet (DM basis) over a week. Cows were control (n = 4, CON) or supplemented with a phytogenic feed additive (n = 4, PHY). Proteomic analysis was conducted using liquid chromatography coupled with mass spectrometry. We identified 1233 proteins; 878 were bovine proteins, 189 corresponded to bacteria, and 166 were plant proteins. Between forage and high-concentrate, 139 proteins were differentially abundant (P 0.05), with 48 proteins having a log2FC difference |2|. The salivary proteome reflected shifts in processes involving nutrient utilization, body tissue accretion, and immune response. Between PHY and CON, 195 proteins were differently abundant (P 0.05), with 37 having a log2FC difference |2|; 86 proteins were increased by PHY, including proteins involved in smell recognition. Many differentially abundant proteins correlated (r |0.70|) with salivary bicarbonate, total mucins or pH. Results provide novel insights into the bovine salivary proteome using a non-invasive approach, and the association of specific proteins with major salivary properties influencing rumen homeostasis. SIGNIFICANCE: Phytogenic compounds may stimulate salivation due to their olfactory properties, but their effects on the salivary proteome have not been investigated. We investigated the effect of high-concentrate diets and supplementation with a phytogenic additive on the salivary proteome of cows. We show that analysis of cows' saliva can be a non-invasive approach to detect effects occurring not only in the gut, but also systemically including indications for gut health and immune response. Thus, results provide unique insights into the bovine salivary proteome, and will have a crucial contribution to further understand animal response in terms of nutrient utilization and immune activity due to the change from forage to a high-energy diet. Additionally, our findings reveal changes due to supplementation with a phytogenic feed additive with regard to health and olfactory stimulation. Furthermore, findings suggest an association between salivary proteins and other components like bicarbonate content.

Knufia chersonesos is an ascomycotal representative of black fungi, a morphological group of polyextremotolerant melanotic fungi, whose ability to resort to recalcitrant carbon sources makes it an interesting candidate for degradation purposes. A secretome screening towards polyesterases was carried out for the fungus and its non-melanized mutant, grown in presence of the synthetic copolyester Polybutylene adipate terephthalate (PBAT) as additional or sole carbon source, and resulted in the identification of 37 esterolytic and lipolytic enzymes across the established cultivation conditions. Quantitative proteomics allowed to unveil 9 proteins being constitutively expressed at all conditions and 7 which were instead detected as up-regulated by PBAT exposure. Protein functional analysis and structure prediction indicated similarity of these enzymes to microbial polyesterases of known biotechnological use such as MHETase from Ideonella sakaiensis and CalA from Candida albicans. For both strains, PBAT hydrolysis was recorded at all cultivation conditions and primarily the corresponding monomers were released, which suggests degradation to the polymer's smallest building block. The work presented here aims to demonstrate how investigations of the secretome can provide new insights into the eco-physiology of polymer degrading fungi and ultimately aid the identification of novel enzymes with potential application in polymer processing, recycling and degradation.

Yersinia ruckeri is an important bacterial pathogen of fish, in particular salmonids, it has been associated with systemic infections worldwide and, like many enteric bacteria, it is a facultative intracellular pathogen. However, the effect of Y. ruckeri’s interactions with the host at the cellular level have received little investigation. In the present study, a culture of Chinook Salmon Embryo (CHSE) cell line was exposed to Y. ruckeri. Afterwards, the proteins were investigated and identified by mass spectrometry and compared to the content of unexposed cultures. The results of this comparison showed that 4.7% of the identified proteins were found at significantly altered concentrations following infection. Interestingly, infection with Y. ruckeri was associated with significant changes in the concentration of surface adhesion proteins, including a significantly decreased presence of β-integrins. These surface adhesion molecules are known to be the target for several adhesion molecules of Yersiniaceae. The concentration of several anti-apoptotic regulators (HSP90 and two DNAj molecules) appeared similarly downregulated. Taken together, these findings suggest that Y. ruckeri affects the proteome of infected cells in a notable manner and our results shed some light on the interaction between this important bacterial pathogen and its host.

The glomerular basement membrane (GBM) and extra-cellular matrix (ECM) are essential to maintain a functional interaction between the glomerular podocytes and the fenestrated endothelial cells in the formation of the slit diaphragm for the filtration of blood. Dysregulation of ECM homeostasis can cause Focal segmental glomerulosclerosis (FSGS). Despite this central role, alterations in ECM composition during FSGS have not been analyzed in detail yet. Here, we characterized the ECM proteome changes in miR-193a-overexpressing mice, which suffer from FSGS due to suppression of Wilms’ tumor 1 (WT1). By mass spectrometry we identified a massive activation of the acute phase response, especially the complement and fibrinogen pathways. Several protease inhibitors (ITIH1, SERPINA1, SERPINA3) were also strongly increased. Complementary analysis of RNA expression data from both miR-193a mice and human FSGS patients identified additional candidate genes also mainly involved in the acute phase response. In total, we identified more than 60 dysregulated, ECM-associated genes with potential relevance for FSGS progression. Our comprehensive analysis of a murine FSGS model and translational comparison with human data offers novel targets for FSGS therapy.

Devil's claw is used for the treatment of inflammatory symptoms and degenerative disorders in horses since many years, but without the substantive pharmacokinetic data. The pharmacokinetic parameters of harpagoside, the main active constituent of Harpagophytum procumbens DC ex Meisn., were evaluated in equine plasma after administration of Harpagophytum extract FB 8858 in an open, single‐dose, two‐treatment, two‐period, randomized cross‐over design. Six horses received a single dose of Harpagophytum extract, corresponding to 5 mg/kg BM harpagoside, and after 7 days washout period, 10 mg/kg BM harpagoside via nasogastric tube. Plasma samples at certain time points (before and 0–24 hr after administration) were collected, cleaned up by solid‐phase extraction, and harpagoside concentrations were determined by LC‐MS/MS using apigenin‐7‐glucoside as internal standard. Plasma concentration‐time data and relevant parameters were described by noncompartmental model through PKSolver software. Harpagoside could be detected up to 9 hr after administration. C max was found at 25.59 and 55.46 ng/ml, t 1/2 at 2.53 and 2.32 hr, respectively, and t max at 1 hr in both trials. AUC 0–inf was 70.46 and 117.85 ng hr ml−1, respectively. A proportional relationship between dose, C max and AUC was observed. Distribution (V z/F) was 259.04 and 283.83 L/kg and clearance (CL/F) 70.96 and 84.86 L hr−1 kg−1, respectively. Treatment of horses with Harpagophytum extract did not cause any clinically detectable side effects.

A proteomic and biochemical approach was performed to assess the effects of an induced muscle injury on the haemolymph of bivalve molluscs. For this purpose, Mytilus galloprovincialis were exposed to puncture of adductor muscle for three consecutive days, and their haemolymph proteome was then compared to healthy animals using 2-dimensional electrophoresis (2-DE) to identify proteins that differed significantly in abundance. Those proteins were then subjected to tandem mass spectrometry and 6 proteins, namely myosin, tropomyosin, CuZn superoxide dismutase (SOD), triosephosphate isomerase, EP protein and small heat shock protein were identified. SOD and tropomyosin changes were verified by spectrophotometric measurements and western blotting, respectively. As some of the proteins identified are related to muscular damage and oxidative stress, other biomarkers associated with these processes that can be evaluated by automatic biochemical assays were measured including troponin, creatine kinase (CK), and aspartate aminotransferase (AST) for muscle damage, and SOD, trolox equivalent antioxidant capacity (TEAC) and esterase activity (EA) for oxidative stress. Significantly higher concentrations of troponin, CK, AST, and TEAC were observed in mussels after puncture, being also possible biomarkers of non-specific induced damage.

A glycopeptide fraction (GPF) from internal organs of green sea urchins (Strongylocentrotus droebachiensis M&uuml, ller, Strongylocentrotidae) has been reported to be an effective bronchitis treatment. In this study, we evaluated the pharmacokinetic and tissue distribution of GPF, following single and repeated intranasal (i/n) administration over the course of seven days in rats. The method measuring lactate dehydrogenase as biomarker was used to analyse the plasma and tissue concentrations of GPF. GPF appears in the plasma 15 min after single i/n administration (100 &micro, g/kg) and reaches its maximum at 45 min. The area under the curve (AUC)0&ndash, 24 and Cmax were similar using both i/n and intravenous administration, while mean residence time (MRT) and T1/2 after i/n administration were significantly higher compared with intravenous (i/v) administration. The absolute bioavailability of GPF after i/n administration was 89%. The values of tissue availability (ft) provided evidence about the highest concentration of GPF in the nose mucosa (ft = 34.9), followed by spleen (ft = 4.1), adrenal glands (ft = 3.8), striated muscle (ft = 1.8), kidneys (ft = 0.5), and liver (ft = 0.3). After repeated dose administration, GPF exhibited significantly higher AUC0&ndash, 24 and MRT, indicating its accumulation in the plasma.

We investigated the regulation of chemical signals of house mice living in seminatural social conditions. We found that male mice more than doubled the excretion of major urinary proteins (MUPs) after they acquired a territory and become socially dominant. MUPs bind and stabilize the release of volatile pheromone ligands, and some MUPs exhibit pheromonal properties themselves. We conducted olfactory assays and found that female mice were more attracted to the scent of dominant than subordinate males when they were in estrus. Yet, when male status was controlled, females were not attracted to urine with high MUP concentration, despite being comparable to levels of dominant males. To determine which compounds influence female attraction, we conducted additional analyses and found that dominant males differentially upregulated the excretion of particular MUPs, including the pheromone MUP20 (darcin), and a volatile pheromone that influences female reproductive physiology and behavior. Our findings show that once male house mice become territorial and socially dominant, they upregulate the amount and types of excreted MUPs, which increases the intensities of volatiles and the attractiveness of their urinary scent to sexually receptive females.

The downstream processing of enveloped virus-like particles is very challenging because of the biophysical and structural similarity between correctly assembled particles and contaminating vesicular particles present in the feedstock. We used hydroxyl-functionalized polymethacrylate monoliths, providing hydrophobic and electrostatic binding contributions, for the purification of HIV-1 gag virus-like particles. The clarified culture supernatant was conditioned with ammonium sulfate and after membrane filtration loaded onto a 1 mL monolith. The binding capacity was 2 × 1012 /mL monolith and was only limited by the pressure drop. By applying either a linear or a step gradient elution, to decrease the ammonium sulfate concentration, the majority of double-stranded DNA (88-90%) and host cell protein impurities (39-61%) could be removed while the particles could be separated into two fractions. Proteomic analysis and evaluation of the p24 concentration showed that one fraction contained majority of the HIV-1 gag and the other fraction was less contaminated with proteins originated from intracellular compartments. We were able to process up to 92 bed volumes of conditioned loading material within 3 h and eluted in average 7.3 × 1011 particles per particle fraction, which is equivalent to 730 vaccination doses of 1 × 109 particles.

The analysis of glycoproteins in body fluids represents a central task in the study of vital processes. Herein, we assessed the combined use of Concanavalin A and Wheat Germ Agglutinin as ligands to fractionate and enrich glycoproteins from oviductal fluid (OF), which is a source of molecules involved in fertilization. First, the selectivity was corroborated by a gel-based approach using glycoprotein staining and enzymatic deglycosylation. Nanoliquid chromatography-tandem mass spectrometry (nLC-ESI-MS/MS) further allowed the reliable identification of 134 nonbound as well as 130 lectin-bound OF proteins. Enrichment analysis revealed that 77% of the annotated proteins in the lectin-bound fraction were known glycoproteins (p-value [FDR] = 1.45E-31). The low variance of the number of peptide spectrum matches for each protein within replicates indicated a consistent reproducibility of the whole workflow (median CV 17.3% for technical replicates and 20.7% for biological replicates). Taken together, this study highlights the applicability of a lectin-based workflow for the comprehensive analysis of OF proteins and gives for the first time an insight into the broad glycoprotein content of OF.

The growth promoting effect of supplementing animal feed with antibiotics like tetracycline has traditionally been attributed to their antibiotic character. However, more evidence has been accumulated on their direct anti-inflammatory effect during the last two decades. Here we used a pig model to explore the systemic molecular effect of feed supplementation with sub therapeutic levels of oxytetracycline (OTC) by analysis of serum proteome changes. Results showed that OTC promoted growth, coinciding with a significant down regulation of different serum proteins related to inflammation, oxidation and lipid metabolism, confirming the anti-inflammatory mechanism of OTC. Interestingly, apart from the classic acute phase reactants also down regulation was seen of a hibernation associated plasma protein (HP-27), which is to our knowledge the first description in pigs. Although the exact function in non-hibernators is unclear, down regulation of HP-27 could be consistent with increased appetite, which is possibly linked to the anti-inflammatory action of OTC. Given that pigs are good models for human medicine due to their genetic and physiologic resemblance, the present results might also be used for rational intervention in human diseases in which inflammation plays an important role such as obesity, type 2 diabetes and cardiovascular diseases.

Oviductal fluid (ODF) proteins modulate and support reproductive processes in the oviduct. In the present study, proteins involved in the biological events that precede fertilization have been identified in the rabbit ODF proteome, isolated from the ampulla and isthmus of the oviduct at different time points within 8 h after intrauterine insemination. A workflow is used that integrates lectin affinity capture with stable-isotope dimethyl labeling prior to nanoLC-MS/MS analysis. In total, over 400 ODF proteins, including 214 lectin enriched glycoproteins, are identified and quantified. Selected data are validated by Western blot analysis. Spatiotemporal alterations in the abundance of ODF proteins in response to insemination are detected by global analysis. A subset of 63 potentially biologically relevant ODF proteins is identified, including extracellular matrix components, chaperones, oxidoreductases, and immunity proteins. Functional enrichment analysis reveals an altered peptidase regulator activity upon insemination. In addition to protein identification and abundance changes, N-glycopeptide analysis further identifies 281 glycosites on 199 proteins. Taken together, these results show, for the first time, the evolving oviductal milieu early upon insemination. The identified proteins are likely those that modulate in vitro processes, including spermatozoa function.

The head kidney and spleen are major lymphoid organs of the teleost fish. The authors identify proteome profiles of head kidney and spleen of rainbow trout (Oncorhynchus mykiss) using a shotgun proteomic approach. Gene ontology annotation of proteins is predicted using bioinformatic tools. This study represents detailed proteome profiles of head kidney and spleen of rainbow trout, with a total of 3241 and 2542 proteins identified, respectively. It is found that lymphoid organs are equipped with a variety of functional proteins related to defense, receptor, signal transduction, antioxidant, cytoskeleton, transport, binding, and metabolic processes. The identified proteome profiles will serve as a template for understanding lymphoid organ functions in salmonids and will increase the amount of spectra information of rainbow trout proteins in the public data repository PRIDE. This data can be accessed via ProteomeXchange with identifiers PXD008473 and PXD008478.

Yersinia ruckeri is the causative agent of enteric redmouth disease in salmonids. Head kidney and spleen are major lymphoid organs of the teleost fish where antigen presentation and immune defense against microbes take place. We investigated proteome alteration in head kidney and spleen of the rainbow trout following Y. ruckeri strains infection. Organs were analyzed after 3, 9 and 28 days post exposure with a shotgun proteomic approach. GO annotation and protein-protein interaction were predicted using bioinformatic tools. Thirty four proteins from head kidney and 85 proteins from spleen were found to be differentially expressed in rainbow trout during the Y. ruckeri infection process. These included lysosomal, antioxidant, metalloproteinase, cytoskeleton, tetraspanin, cathepsin B and c-type lectin receptor proteins. The findings of this study regarding the immune response at the protein level offer new insight into the systemic response to Y. ruckeri infection in rainbow trout. This proteomic data facilitate a better understanding of host-pathogen interactions and response of fish against Y. ruckeri biotype 1 and 2 strains. Protein-protein interaction analysis predicts carbon metabolism, ribosome and phagosome pathways in spleen of infected fish, which might be useful in understanding biological processes and further studies in the direction of pathways.

Apolipoprotein A-I (Apo A-I) is a major protein in lipid/lipoprotein metabolism and decreased serum levels have been observed in many species in response to inflammatory and infectious challenges. Little is known about the porcine homologue, therefore in this work we have characterized it through biochemical and proteomic techniques. In 2DE, porcine serum Apo A-I is found as three spots, the two more acidic ones corresponding to the mature protein, the more basic spot to the protein precursor. Despite high sequence coverage in LC-MS/MS, we did not find a sequence or PTM difference between the two mature protein species. Besides this biochemical characterization, we measured overall levels and relative species abundance of serum Apo A-I in four different viral and bacterial porcine infectious diseases. Lower overall amounts of Apo A-I were observed in Salmonella typhimurium and Escherichia coli infections. In the 2DE protein pattern, an increase of the protein precursor together with a lower level of mature protein species were detected in the porcine circovirus type 2-systemic disease and S. typhimurium infection. These results reveal that both the porcine serum Apo A-I concentration and the species pattern are influenced by the nature of the infectious disease.

The oviductal epithelium is crucial for the integrity of the female organ. Previously we got evidence that the surface proteome of oviductal epithelial cells (Oecs) is promptly altered in response to insemination and thus suggested that this early phase plays a notable regulatory role in maintaining cellular function. This study further aimed to assess the effect of semen on the cellular and molecular mechanisms in rabbit Oecs. A quantitative gel-based proteomic approach was applied to analyze changes at three time points (0 h, 1 h, 2 h) after intrauterine insemination (IUI) compared to time matched controls. Within two hours the abundance of 22 protein species was evidently altered in the intracellular fraction. Functional analysis revealed that the proteins were primarily involved in proteostasis as well as metabolic processes. The analysis of phosphoproteins specified a role of mitogen-activated protein kinase (MAPK) signaling molecules. Concurrently, semen increased oviduct-specific glycoprotein (OVGP1) secretion. A correlation between OVGP1 abundance and microtubule-associated proteins 1A/1B–light chain 3 lipidation was observed. The localization and changes in abundance of selected proteins were corroborated by antibody-based methods. These results clearly show that the early phase of interaction acts as a trigger for cellular adaptation to meet an altered demand in the female organ. Significance The oviductal epithelium and its secreted proteins exert a pivotal role in reproductive processes, including the final maturation of male gametes. Thereby, the regulation and subsequently the functionality of the oviductal epithelial cell layer are important factors for the establishment of the appropriate milieu in the female reproductive tract. Notably, male gametes themselves have been shown to be an extrinsic modulatory factor of the oviductal epithelium. Accordingly a comprehensive knowledge about the underlying cellular and molecular mechanisms in the epithelial cells is of interest, also with regard to in vitro purposes. So far, the role of the early phase of interaction in the female organ has not been considered in detail. To get a further insight into the underlying cellular and molecular mechanisms, herein we analyzed the effect of semen on oviductal epithelial cells (Oecs) on the intracellular proteome level within the first two hours after insemination. The present study revealed a directed response of Oecs in vivo and disclosed intracellular pathways that are affected by the interplay between semen and the female reproductive tract. The prompt adaptation of the secretory activity and remodeling of the oviductal epithelium was accompanied by the concerted alterations of protein species that are primarily involved in the maintenance of cellular homeostasis. Besides emphasizing the importance of the early interaction phase for subsequent reproductive processes, the gained knowledge might further be implemented for in vitro applications as well.

Dogs are relevant to biomedical research in connection both to veterinary medicine for their role as pets and to basic investigations for their use as animal models in pathology, pharmacology and toxicology studies. Proteomic analysis of biological fluids is less advanced for dogs than for other animal species but a wealth of information has already been gathered, which we summarize in this review. As a remarkable feature, we also assemble here for due reference a number of 2-DE serum/plasma or urine patterns in health and disease; some of them correspond to unpublished data from the University of Veterinary Medicine Vienna.

The microcolonial black fungus Cryomyces antarcticus is an extremophile organism growing on and in rock in the Antarctic desert. Ecological plasticity and stress tolerance make it a perfect model organism for astrobiology. 2D-gel electrophoresis and MALDI-TOF/TOF mass spectrometry were performed to explore the protein repertoire, which allows the fungus to survive in the harsh environment. Only a limited number of proteins could be identified by using sequence homologies in public databases. Due to the rather low identification rate by sequence homology, this study reveals that a major part of the proteome of C. antarcticus varies significantly from other fungal species.

Aflatoxin (AF) B1, B2, G1 and G2 were determined in 120 samples of selected Thai commodities including unpolished rice, unpolished glutinous rice, chilli powder, whole dried chilli pods and raw peanut. The mean concentrations of the total AFs for analysed samples were 0.16, 25.43, 14.18, 6.62 and 1.43 µg kg(-1) with positive incidences of 4%, 20%, 97%, 37% and 30%, respectively. Quantitative analysis was performed using HPLC equipped with post-column derivatisation and fluorescence detection. Sample clean-up was carried out using immunoaffinity columns for selective enrichment of AFs. The method was validated by using certified reference material, which showed recoveries over 85%. The limit of detections (LODs) and limit of quantifications (LOQs) were in a range between 0.01-0.11 µg kg(-1) and 0.03-0.38 µg kg(-1), respectively. The results clearly demonstrated that AFs were detectable in different matrices. Chilli powder was found to have the highest level of AFs contamination followed by chilli pods, peanut and rice, respectively. However, among the selected commodities, unpolished rice contained only trace levels of AFB1 and AFB2. With regard to the fact that AFs are a natural contaminant in commodities, this report calls to attention the regular monitoring and effective control of food commodities to prevent health hazards.

Polyclonal immunoglobulin (Ig) concentrates are important biological medicinal products and the assurance of their quality and safety is crucial. In our present approach we used proteomic methods to check the purity of commercial Ig products of different origin. The experimental setup included nonreducing 2DE or DIGE combined with MALDI-TOF and the thrombin generation assay, a routine safety test for pharmaceutical Ig preparations, and was complemented by a specific immunoassay. 2DE patterns displayed contaminations with trace amounts of human apolipoprotein H (Apo-H), transferrin, albumin, and its fragments. In contrast to the latter, Apo-H is a protein that is active in the coagulation cascade, and thus a potential involvement in thromboembolic events in vivo cannot be excluded. It was found by 2DE and MALDI-TOF to be a contaminant of several Ig preparations. Spiking experiments of Ig preparations with pure Apo-H demonstrated an Apo-H concentration dependent increase in thrombin generation assay values. Traces of Apo-H are possibly also contributing to unwanted side effects, as already known for factor XIa. The significance of Apo-H contaminations for these side effects might be verified by detailed analyses of pharmacovigilance data.

Samples from large (100–200 tons) batches of palm kernel cake (PKC, n = 20) and copra meal (CM, n = 13) were collected at production facilities of four Indonesian feed mill manufacturers and analysed for aflatoxin B1 (AFB1) by ELISA. Recoveries using spiked samples ranged from 86 to 113 %, with relative standard deviations of

Animals with different health status have been studied in order to extend the knowledge about protein composition of porcine saliva samples and to discover potential salivary markers for systemic disease in porcine production. Clinical examination of animals was performed at farm level where 10 healthy pigs and 10 animals with evident clinical signs of disease were randomly selected. Saliva and blood samples were obtained and afterwards animals were humanely sacrificed to perform a complete necropsy. Levels of two acute phase proteins, haptoglobin and C-reactive protein, were used to identify possible active infections of the animals. Moreover, serological analysis, to the main porcine infectious diseases in the area, was performed. Salivary proteins were separated by two-dimensional gel electrophoresis followed by mass spectrometry for the identification of specific proteins. A total of 58 spots out of 75 were successfully identified by MS, which correspond to 20 unique proteins. Two different approaches were used to perform a statistical comparison of saliva protein patterns from healthy and diseased animals using the relative spot volume (% spot volume/total volume of all spot in the gel, approach "A") or taking also into account the total protein content of each saliva sample (μg of spot/mL of saliva, approach "B"). Both analyses showed three proteins in common that are differentially regulated between states. However, approach B was selected for biomarker searching since it gave an estimation of protein concentration and showed differential expression of proteins between both health states in a total of 10 proteins, which were up-regulated in disease. Mass spectrometric analysis identified those proteins as salivary lipocalin, lipocalin 1, double headed protease inhibitor protein, adenosine deaminase, haptoglobin, albumin fragments, S100-A8, S100-A9, S100-A12 and pancreatic alpha amylase. These proteins could be considered as potential salivary markers of disease.

Plant proteomes are complex and therefore their analyses represent major technical challenges. In fact, proteome analyses depend on several crucial steps, such as the amount of proteins, extraction, separation, visualization, identification, quantification, and the interaction between proteins and other molecules in a plant tissue at a given time.

Major urinary proteins (MUPs) are often suggested to be highly polymorphic, and thereby provide unique chemical signatures used for individual and genetic kin recognition; however, studies on MUP variability have been lacking. We surveyed populations of wild house mice (Mus musculus musculus), and examined variation of MUP genes and proteins. We sequenced several Mup genes (9 to 11 loci) and unexpectedly found no inter-individual variation. We also found that microsatellite markers inside the MUP cluster show remarkably low levels of allelic diversity, and significantly lower than the diversity of markers flanking the cluster or other markers in the genome. We found low individual variation in the number and types of MUP proteins using a shotgun proteomic approach, even among mice with variable MUP electrophoretic profiles. We identified gel bands and spots using high-resolution mass spectrometry and discovered that gel-based methods do not separate MUP proteins, and therefore do not provide measures of MUP diversity, as generally assumed. The low diversity and high homology of Mup genes are likely maintained by purifying selection and gene conversion, and our results indicate that the type of selection on MUPs and their adaptive functions need to be re-evaluated.

Salivary glycoprotein profiles, obtained after boronic acid enrichment, were studied for the first time in pigs in order to search for specific overall alterations related to acute inflammatory condition. Five healthy pigs and five pigs suffering from rectal prolapse were used, and the levels of acute phase proteins were measured to determine the degree of inflammation of the animals. The enriched glycoprotein profiles, achieved by two-dimensional gel electrophoresis (2DE) were statistically evaluated and spots that appeared differentially regulated between states were subjected to MS analysis for protein identification. Spots from three unique proteins were identified: carbonic anhydrase VI (CA VI), α-1-antichymotrypsin and haptoglobin (Hp). CA VI appeared as two adjacent horizontal spot trains in the glycoprotein profile of healthy animals in its regular isoelectric points (pI). One spot of α-1-antichymotrypsin was found in saliva from pigs with rectal prolapse in an unusual basic pI, and was considered as a breakdown product. Hp was identified as several spot trains in saliva from pigs with rectal prolapse in an unusual alkaline pI and was consequently further investigated. SDS-PAGE and 2DE of paired serum and saliva samples combined with Western blot analysis showed that the unusual Hp position observed in saliva samples was absent in serum. Furthermore, N-glycans from serum and saliva Hp glycopatterns were evaluated from SDS-PAGE Hp bands and showed that the serum N-glycan distribution in Hp β-chain was comparable in quantity and quality in both groups of animals. In saliva, no Hp β-chain derived N-glycans could unambiguously be identified from this sample set, thus needing further detailed investigations in the future.

Background The influence of two different sample treatments comprising the enrichment of glycoproteins by boronic acid and dynamic range compression by hexapeptide libraries, on the detection of stress markers in saliva of pigs was evaluated in this study. For this purpose, saliva samples collected before and after the application of an acute stress model consisting of nasal restraining in pigs were processed without any treatment and with the two different treatments mentioned above. Protein separation by two-dimensional gel electrophoresis (2-DE) followed by identification of proteins using MALDI-TOF/TOF mass spectrometry (MS) was used as proteomic technique. Results The application of each of the two different sample treatment protocols allowed the identification of unique proteins that could be potential salivary acute stress markers in pigs: lipocalin 1, protein S100-A8 and immunoglobulin M by enrichment of glycoproteins; protein S100-A9, double headed protease inhibitor submandibular gland, and haemoglobin by dynamic range compression; and protein S100-A12 by both protocols. Salivary lipocalin, prolactin inducible protein, light chain of immunoglobulins, adenosine deaminase and carbonic anhydrase VI were identified as potential markers in untreated saliva as well as one of the other treatments. Conclusion The use of different procedures allowed the detection of different potential stress markers. Although from a practical point of view, the use of saliva without further treatment as well as the enrichment of glycoproteins are less expensive and easy to do procedures.

A study on adsorption of AFB1 in corn (kernel and grained) on natural zeolite and bentonite has been investigated. The first work was adsorption in a batch system of standard AFB1 solution on adsorbents. Some factors such as contact time, concentration of AFB1 and particle size of adsorbent were evaluated. The amount of AFB1 adsorbed was calculated based on the difference of AFB1 concentration before and after adsorption determined by high performance liquid chromatography (HPLC) method. Adsorption of AFB1 in corn sample was emphasized by mixing aqueous suspension of sample with adsorbent. Concentration of AFB1 in suspension was analyzed by enzyme-linked immuno-sorbent assay (ELISA) method. Result shows that adsorption of AFB1 on adsorbents of natural zeolite and bentonite is very fast. Within 15 min 99% of AFB1 (200 ng/mL) has been adsorbed by 25 mg of bentonite and 96% by zeolite. The particle size higher than 200 mesh did not give significant effect on the AFB1 adsorption capability. Effectiveness of zeolite in adsorbing AFB1 is lower than that of bentonite. Capability in reducing AFB1 contamination in corn samples (kernel and meal) for both adsorbents is lower than that in standard solution.

Glycans play key roles in host-pathogen interactions; thus, knowing the N-glycomic repertoire of a pathogen can be helpful in deciphering its methods of establishing and sustaining a disease. Therefore, we sought to elucidate the glycomic potential of the facultative amoebal parasite Acanthamoeba. This is the first study of its asparagine-linked glycans, for which we applied biochemical tools and various approaches of mass spectrometry. An initial glycomic screen of eight strains from five genotypes of this human pathogen suggested, in addition to the common eukaryotic oligomannose structures, the presence of pentose and deoxyhexose residues on their N-glycans. A more detailed analysis was performed on the N-glycans of a genotype T11 strain (4RE); fractionation by HPLC and tandem mass spectrometric analyses indicated the presence of a novel mannosylfucosyl modification of the reducing terminal core as well as phosphorylation of mannose residues, methylation of hexose and various forms of pentosylation. The largest N-glycan in the 4RE strain contained two N-acetylhexosamine, thirteen hexose, one fucose, one methyl, and two pentose residues; however, in this and most other strains analyzed, glycans with compositions of Hex(8-9)HexNAc(2)Pnt(0-1) tended to dominate in terms of abundance. Although no correlation between pathogenicity and N-glycan structure can be proposed, highly unusual structures in this facultative parasite can be found which are potential virulence factors or therapeutic targets.