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Corals especially the reef-building species are very important to marine ecosystems. Proteomics has been used for researches on coral diseases, bleaching and responses to the environment change. A robust and versatile protein extraction protocol is required for coral proteomics. However, a comparative evaluation of different protein extraction protocols is still not available for proteomic analysis of stony corals. In the present study, five protocols were compared for protein extraction from stony corals. The five protocols were TRIzol, phenol-based extraction (PBE), trichloroacetic acid (TCA)-acetone, glass bead-assisted extraction (GBAE) and a commercially available kit. PBE, TRIzol and the commercial kit were more robust for extracting proteins from stony corals. The protein extraction efficiency and repeatability, two dimensional electrophoresis (2-DE) and matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI TOF MS) were employed to evaluate the protocols. The results indicated that PBE protocol had the better protein extraction efficiency than the others. Protein extraction coverage varied among the procedures. Each protocol favored for certain proteins. Therefore, it is very important for coral proteomic analysis to select a suitable protein protocol upon the experimental design. In general, PBE protocol can be the first choice for extracting proteins from stony corals., (© 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Microalgae of the genus Prototheca (P.) are associated with rare but severe infections (protothecosis) and represent a potential zoonotic risk. Genotype (GT) 2 of P. zopfii has been established as pathogenic agent for humans, dogs and cattle, whereas GT1 is considered to be non-pathogenic. Since pathogenesis is poorly understood, the aim of this study was to determine immunogenic proteins and potential virulence factors of P. zopfii GT2. Therefore, 2D western blot analyses with sera and isolates of two dogs naturally infected with P. zopfii GT2 have been performed. Cross-reactivity was determined by including the type strains of P. zopfii GT2, P. zopfii GT1 and P. blaschkeae, a close relative of P. zopfii, which is known to cause subclinical forms of bovine mastitis. The sera showed a high strain-, genotype-, and species-cross-reactivity. A total of 198 immunogenic proteins have been analysed via MALDI- TOF MS. The majority of the 86 identified proteins are intracellularly located (e.g. malate dehydrogenase, oxidoreductase, 3-dehydroquinate synthase) but some antigens and potential virulence factors, known from other pathogens, have been found (e.g. phosphomannomutase, triosephosphate isomerase). One genotype-specific antigen could be identified as heat shock protein 70 (Hsp70), a well-known antigen of eukaryotic pathogens with immunological importance when located extracellularly. Both sera were reactive to glyceraldehyde-3-phosphate-dehydrogenase of all investigated strains. This house-keeping enzyme is found to be located on the surface of several pathogens as virulence factor. Flow-cytometric analysis revealed its presence on the surface of P. blaschkeae.

The olive flounder ( Paralichthys olivaceus ) is a cultivated marine species that is economically important in Korea and Japan. Several bacterial pathogens have caused severe mortalities in farmed olive flounder, especially Streptococcus parauberis . We collected 145 S. parauberis isolates from diseased olive flounders from 2003 to 2008 in Jeju Island, South Korea and characterized them by Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI TOF MS) and by serology. The serological analysis divided the isolates into serotype I (62.1%) and serotype II (36.6%) and the proteome analysis divided the isolates into cluster 1 (43.4%) and cluster 2 (56.6%). All cluster 1 isolates had serotype I, but cluster 2 consisted of serotype I (32.9%), serotype II (64.6%), and others (2.5%). Further detailed analysis of the mass spectra led to identification of several specific m /z peaks that enabled discrimination between cluster 1 and 2 and between serotype I and II within cluster 2. Our results suggest that MALDI TOF MS analysis has potential as an alternative method for the rapid and reliable identification of the fish pathogen S. parauberis . [ABSTRACT FROM AUTHOR]

Microalgae of the genus Prototheca (P.) are associated with rare but severe infections (protothecosis) and represent a potential zoonotic risk. Genotype (GT) 2 of P. zopfii has been established as pathogenic agent for humans, dogs, and cattle, whereas GT1 is considered to be non-pathogenic. Since pathogenesis is poorly understood, the aim of this study was to determine immunogenic proteins and potential virulence factors of P. zopfii GT2. Therefore, 2D western blot analyses with sera and isolates of two dogs naturally infected with P. zopfii GT2 have been performed. Cross-reactivity was determined by including the type strains of P. zopfii GT2, P. zopfii GT1, and P. blaschkeae, a close relative of P. zopfii, which is known to cause subclinical forms of bovine mastitis. The sera showed a high strain-, genotype-, and species-cross-reactivity. A total of 198 immunogenic proteins have been analyzed via MALDI-TOF MS. The majority of the 86 identified proteins are intracellularly located (e.g., malate dehydrogenase, oxidoreductase, 3-dehydroquinate synthase) but some antigens and potential virulence factors, known from other pathogens, have been found (e.g., phosphomannomutase, triosephosphate isomerase). One genotype-specific antigen could be identified as heat shock protein 70 (Hsp70), a well-known antigen of eukaryotic pathogens with immunological importance when located extracellularly. Both sera were reactive to glyceraldehyde-3-phosphate-dehydrogenase of all investigated strains. This house-keeping enzyme is found to be located on the surface of several pathogens as virulence factor. Flow-cytometric analysis revealed its presence on the surface of P. blaschkeae. [ABSTRACT FROM AUTHOR]

Objectives: To investigate salivary proteins with proteomic technologies to evaluate protein composition differences between samples with cleft lip and palate and healthy controls. Design, Participants: Matrix-assisted laser desorptionhionization tandem time-of-flight (MALDI TOF/TOF) mass spectrometry was used as a high-throughput analytical technique for identification of nonsyndromic cleft lip and palate stimulated salivary proteins. The samples consisted of two groups: 31 cleft lip and palate patients and a control group with 20 healthy volunteers. Results: The presence of cleft lip and palate stimulated the expression of several proteins, included adaptor-related protein complex 3, dermokine, nidogen 1 precursor, transforming growth factor-β3, and a zinc finger RAN-binding domain containing 2. Conclusions: The salivary proteome of cleft lip and palate patients differs from the protein composition of healthy control saliva samples. Several common secreted proteins such as actins, salivary cystatins, and keratins were upregulated by cleft; increased levels of TGF-β3 and dermokine were detected in the pathologic samples. The current proteomic results suggest keratinocyte activation among patients with cleft lip and palate. The score of our preliminary results suggests the hypothesis that identified salivary proteins are of vital clinical importance in tissue regeneration and the molecular repair mechanism seen in patients with cleft lip and palate. [ABSTRACT FROM AUTHOR]

Comparative proteomic analysis was performed to identify proteins in the midgut of Takifugu rubripes ( Fugu) in response to excessive fluoride. Sixteen fish were randomly divided into a control group and an experimental group. The control group was raised in soft water alone ( F−− == 0.4 mg/L), whereas the experimental group was raised in the soft water with sodium fluoride at a high concentration of 35 mg/L. After 3 days, proteins were extracted from the fish midgut and then subjected to two-dimensional (2-D) PAGE analysis. The matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI TOF/TOF MS) was applied to identify the differential expressed proteins between the two groups. Among 377 and 528 proteins detected in the control and the treated groups, respectively, 17 proteins were up-regulated and 218 were down-regulated ( P < 0.01) in the fluoride-treated group, compared with the control group. We further analyzed 17 up-regulated proteins by MALDI TOF/TOF MS and identified 12 of them by MASCOT, of which eight were known proteins. Consistent with their annotated functions, these proteins seem to be involved in apoptosis and other functions related to fluorosis. Our results provide initial insights into the effects of excessive fluoride exposure on physiological and biochemical functions of Fugu midgut as well as on the toxicological mechanism of fluoride in both fish and human. [ABSTRACT FROM AUTHOR]

To investigate comparative proteomics of the pufferfish kidney exposed to excessive fluoride, the authors randomly put 16 fish into the control and treated groups that were raised in softwater alone (F− = 0.4 mg/L) or with sodium fluoride of 35 mg/L for 3 days, respectively. Then proteins of the fish kidneys were profiled by two-dimensional electrophoresis, and the matrix assisted laser desorption ionization time of flight mass spectrometry (MALDI TOF MS) was applied to identify the spots on the gel with altered densities. On average, 547 and 516 protein spots were detected in the control and the treated groups, respectively. Among them, 32 protein spots showed significant alteration ( p < 0.05) between the fluoride-treated and the control groups, and 22 differentially expressed protein spots were identified by MALDI TOF-TOF MS. Consistent with their previously annotated functions, these proteins appear to be involved in the biological functions associated with fluorosis. These results will greatly advance one’s understanding of the effects of fluoride exposure on the physiological and biochemical functions of takifugu kidney as well as the toxicological mechanism of fluoride-causing fluorosis in both fish and humans. [ABSTRACT FROM AUTHOR]

In the present study, the use of a robust and sensitive mass spectrometry based protein profiling analysis was tested as diagnostic tools for women with an ovarian tumor. The potential additional diagnostic value of serum protein profiles independent of the information provided by CA125 were also investigated. Protein profiles of 113 serum samples from women with an ovarian tumor (54 malign and 59 benign) were generated using MALDI-TOF MS. A total of 98 peaks with a significant difference (p< 0.01) in intensity between women with benign tumors/cysts and malignant ovarian tumors were identified. After average linkage clustering, a profile of 46 statistical significant mass peaks was identified to distinguish malignant tumors and benign tumors/cysts. In the subgroup of women with normal CA125 values (&les; 35 U/mL) (62 patients) 36 of the 504 mass peaks showed significant (p< 0.05) differences in intensity between benign and malignant disease. After average linkage clustering, 25 statistical significant mass values were identified in this clinical difficult and important subgroup presenting with normal CA125 values. The current study demonstrates the potential of mass spectrometry based serum protein profiling as a diagnostic tool in discrimination between benign ovarian tumors/cysts and malignant ovarian tumors. Additionally, the method provided diagnostic information independent of CA125. [ABSTRACT FROM AUTHOR]

Comparative proteomics was performed to identify proteins in the cardiac muscle of Takifugu rubripes in initial response to excessive fluoride. Sixteen fish were randomly divided into a control group and an experimental group. The control group was raised in softwater alone ( F− = 0.4 mg/L), and the experimental group was raised in the same water with sodium fluoride at a high concentration of 35 mg/L. After 3 days, proteins were extracted from the fish cardiac muscle and then were subjected to 2D PAGE analysis. The matrix assisted laser desorption ionization time of flight mass spectrometry (Maldi TOF MS) was applied to identify the proteins that were differentially expressed from the two groups of fish. Average of 318 and 275 proteins detected in the control and the experimental group, respectively, 167 spots were matched and 21 highly differentially expressed proteins were further analyzed by Maldi TOF-TOF MS, and 15 were identified by MASCOT. These 15 proteins include Telomerase reverse transcriptase, 4SNc-Tudor domain protein, protein disulfide isomerase ER-60, Tuba1 protein, mitogen-activated protein kinase 10, and SMC4 protein. Consistent with their previously known functions, these identified proteins seem to be involved in apoptosis and other functions associated with fluorosis. These results will contribute to our understanding for the effects of fluoride exposure on the physiological and biochemical functions of takifugu, and the toxicological mechanism of fluoride causing fluorosis in both fish and human. [ABSTRACT FROM AUTHOR]

Abstract: The Dauer larva is a non-feeding alternative larval stage of some nematodes specialized for long-term survival and dispersal. In this study we compared proteome maps obtained from Dauer larvae with those from the corresponding third larval stage (L3) of the feeding life cycle of C. elegans wild-type strain N2. We demonstrate at the protein level that altered metabolism may participate in longevity determination of Dauers. We detected huge amounts of alcohol dehydrogenase (CE12212) and aldehyde dehydrogenase (CE29809) in Dauer animals, indicating highly active fermentative pathways. Inorganic pyrophosphatase (CE05448) that enables to metabolize pyrophosphate as a high-energy source was over-expressed in Dauers. An interesting differentially expressed protein was phosphatidylethanolamine-binding protein (CE38516) that was found in high abundance in samples from Dauer larvae. Protein synthesis may be lowered in Dauer animals by the reduced expression of splicing factor rsp-3 (CE31089) and methionyl-tRNA synthase (CE34219). We observed significantly lower amounts of the pepsin-like aspartyl protease 1 (CE21681) in non-feeding Dauers, which is in agreement with reduced nutrient digestion. Finally, the hypothetical protein R08E5.2 (CE33294) was present in high abundance in L3 animals. [Copyright &y& Elsevier]

Smad3 is an important component of transforming growth factor-β (TGFβ) intracellular signalling. To identify novel interacting proteins of Smad3, we performed pull-down assays with Smad3 constructs fused to glutathione-S-transferase. Proteins which formed complexes with these constructs were analyzed by two-dimensional gel electrophoresis, and were identified by matrix-assisted laser desorption-ionization time-of-flight mass spectrometry. We identified 14 proteins interacting with the Smad3 construct lacking the N-terminal Mad homology domain 1 (MH1), and 12 proteins interacting with the construct lacking the C-terminal MH2 domain. Proteins involved in signalling processes, in metabolism regulation, novel proteins, and components of cytoskeleton form four groups of interacting proteins. Interactions of AGP7, sex-determining region Y protein, actin β and sterol regulatory element binding protein-2 (SREBP-2) proteins with Smad3 constructs were confirmed by immunoblotting with specific antibodies. Interaction of Smad3 with SREBP-2 was also confirmed by co-immunoprecipitation of myc-Smad3 and Flag-SREBP-2 upon expression in mammalian cells. We found that SREBP-2 inhibited the transcriptional activity of Smad3 in luciferase reporter assays. [Copyright &y& Elsevier]